Novel resistance genes associated with disease resistance in soybean

Abstract

Methods and compositions for identifying, selecting and / or producing pathogen resistant plants, plant cells or seeds (e.g., legume or soybean plants, plant cells or seeds) are provided. Polynucleotides and polypeptides are provided that when expressed in a plant increase disease and / or pathogen resistance (e.g., resistance to Asian soybean rust and / or powdery mildew) in a plant. Plants, plant cells, and seeds comprising such polypeptides and polynucleotides are provided, as well as methods of their use.

Description

Technical Field

[0001] The present invention relates to compositions and methods for identifying, selecting, and generating enhanced disease and / or pathogen resistant plants using novel resistance genes.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 383,609, filed on November 14, 2022, U.S. Provisional Patent Application No. 63 / 426,524, filed on November 18, 2022, and U.S. Provisional Patent Application No. 63 / 509,586, filed on June 22, 2023, the content of each of these applications is hereby incorporated by reference in its entirety.

[0004] Statement Regarding Electronic Submission of Sequence Listing

[0005] A sequence listing in XML format is provided as an alternative to a paper copy. The sequence listing was submitted in accordance with 37 C.F.R. §§ 1.831 - 1.835, is approximately 140 kb in size, has the title 82721_PCT.xml, was generated on October 30, 2023, and was submitted via EFS-Web. This sequence listing is hereby incorporated by reference in its entirety into this specification. Background Art

[0006] It is known that plant pathogens cause considerable damage to important crops, which results in significant agricultural losses, along with widespread consequences for the food supply and other industries that rely on plant materials. Similarly, the applicant desires to reduce the incidence and / or impact of agricultural pathogens on crops.

[0007] Several pathogens are associated with damage to soybeans, which has the potential to cause significant yield losses both individually and collectively in the United States and worldwide. Exemplary pathogens include, but are not limited to, fungi (e.g., Phytophthora and Phakopsora pachyrhizi, the causal agent of Asian soybean rust), nematodes (e.g., Meloidogyne, particularly Meloidogyne javanica), and bacteria (e.g., Pseudomonas syringae). Given the significant threat these pathogens pose to the global food supply and the time and expense associated with treating soybean crops to prevent yield losses, there is a need for new methods for generating pathogen-resistant soybean cultivars. What is needed are novel resistance genes (herein, "R genes") that can be introduced into plants to control pathogens. Summary of the Invention

[0008] Compositions and methods are provided for enhancing disease resistance and / or pathogen resistance in plants, particularly leguminous plants, and more particularly soybean plants. The present disclosure provides TIRA and TIRB polypeptides, variants and active fragments of the TIRA and TIRB polypeptides, and fusion proteins of the TIRA and TIRB polypeptides capable of enhancing disease resistance. The present disclosure also provides TIRA and / or TIRB polypeptides modified to reduce their NADase activity, which are capable of enhancing disease resistance. The present disclosure further provides nucleic acids encoding polypeptides that enhance disease resistance, and plants expressing the polypeptides.

[0009] Methods are also provided for enhancing disease resistance by providing a plant with a nucleic acid encoding the disclosed polypeptides. The present disclosure further relates to methods for enhancing disease resistance in plants and improving the agronomic performance of plants by engineering genomic modifications to rebalance the relative levels of TIRA and TIRB polypeptides in the plants. Methods are also provided for genetically modifying the TIRA and / or TIRB polypeptides such that their expression in plants is balanced, thereby providing enhanced disease resistance and enhanced agronomic performance to the plants.

[0010] The foregoing and other objects and aspects of the invention will be explained in detail in the accompanying drawings and description set forth below. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Illustrates that a transgenic soybean event containing a pair of dumbbell-shaped TIR proteins confers enhanced ASR resistance. Leaves from a T0 event containing a pair of TIR polypeptides exhibit strong ASR resistance.

[0012] Figure 2 Is a bar graph showing that constructs containing two Tir genes (TirA and TirB) have a lower transformation efficiency compared to constructs containing a single Tir gene.

[0013] Figure 3 Depicts the negative growth phenotype of plants grown from an event containing two Tir genes (TirA and TirB). Plants grown from an event containing two Tir genes exhibit delayed germination, poor growth and / or death at the seedling stage.

[0014] Figure 4 Depicts the negative growth phenotype of plants grown from an event containing two Tir genes (specifically, the event generated by construct 24217). T1 plants at the mature stage (sub-panel A: 10 days after planting (DAP); sub-panel B: approximately 70 DAP) exhibit stunted plants and severe sterility compared to control plants (containing null events).

[0015] Figure 5Is a graph showing balanced and similar transcriptional levels of TIRA and TIRB polypeptides in wild soybean species (Glycine spp.). (Arrows indicate the donor line).

[0016] Figure 6 Graphically depicts unbalanced transcriptional levels of TIRA and TIRB polypeptides in transgenic events. As estimated using qRT-PCR, the transgenic events showed that the transcriptional level of TIRB was >20-fold higher compared to TIRA, while the transcriptional levels in the corresponding controls were comparable.

[0017] Figure 7 Describes leaves of events generated from constructs containing modified promoter combinations, modified gene coding sequences, and modified orientations. All events exhibited improved rust resistance relative to the control. Events generated from construct 24953 (sub-panel B), which expresses TirA and TirB in the direction of inserting TirA upstream of TirB, showed strong rust resistance compared to construct 24217 (sub-panel A) used as a control. Events generated from construct 24915, in which multiple introns in the coding sequences of the TirA and TirB genes were modified (sub-panel C), also showed strong rust resistance relative to the control. Events generated from construct 25337, in which the expression of the TirB gene was driven by a rust-responsive promoter while the expression of the TirA gene was driven by a constitutive Medicago promoter (sub-panel D), also showed strong rust resistance relative to the control.

[0018] Figure 8 Depicts leaves of events generated from constructs containing the TirA and TirB genes fused together and transcribed via a common promoter and terminator. All events exhibited strong rust resistance. Events generated from constructs 25046 (sub-panel A), 25047 (sub-panel B), and 25135 (sub-panel C), which express a fusion protein of TIRA-TIRB, all exhibited strong rust resistance. Events generated from these constructs had the expression of the TirA and TirB genes driven by a common Medicago promoter and terminator.

[0019] Figure 9 Is a graph depicting the relative expression of Tir polypeptides in T1 plants generated from construct 25046. These events showed that the amount of TIRB polypeptide expressed was approximately twice the amount of TIRA polypeptide expressed.

[0020] Figure 10Depicts T1 plants generated from construct 25337 expressing the TIRATIRB fusion protein. The plants grew normally and did not exhibit any adverse growth phenotypes at any growth stage. Subfigures A, B, and C show normal growth of T1 events from seedlings to the R7+ stage (subfigure A at DAP 7, subfigure B at DAP 16). Subfigures D and E show that the growth of T1 events from seedlings to the seedset stage was indistinguishable from null events, and the number of seeds produced per plant was normal (subfigure D: plants approximately 70 days old). Subfigure F shows that in T1 homozygous events, the transcriptional levels of TIRA polypeptide and TIRB polypeptide were comparable.

[0021] Figure 11 Depicts leaves of T1 homozygous events generated from construct 25337 expressing the TIRATIRB fusion protein. These plants showed higher resistance to multiple rust isolates (BRS, SUL, and RTP1) compared to the control.

[0022] Figure 12 Illustrates the amino acid alignment of TIRA1, TIRA2, TIRB1, and TIRB2 domains. This alignment indicates that the TIRB2 NAD enzyme catalytic residue (Glu, E) is essential for soybean resistance. Three of the four TIR domains (TIRA1, TIRA2, TIRB1, and TIRB2, highlighted in gray) contain glutamate (E) when aligned with known TIR domains (AtRBA1, AtRPS4_TIR, AtRRS1_TIR, and L6_TIR), highlighted by black rectangles. TIRA2 contains valine (V) instead of the E residue, as indicated by the red arrow within the black rectangle.

[0023] Figure 13 Depicts leaves of events generated from constructs containing one or more mutations in the NAD enzyme site of the TIR domain. These events exhibited different levels of rust resistance. Subfigure A: The event generated from construct 25313 containing a single mutant E87A in the TIRB2 domain showed strong rust resistance compared to the susceptible control. Subfigure B: The event generated from construct 25312 (subfigure B) containing a single gain-of-function mutant V251E in the TIRA1 domain showed strong rust resistance compared to the susceptible control. Subfigure C: The event generated from construct 25315 containing a triple mutation (E87A in the TIRB1 domain, E85A in the TIRA1 domain, and E257A in the TIRB2 domain) showed a susceptibility level comparable to the susceptible control. Subfigure D: The event generated from construct 25988 containing a double mutation (E87A in the TIRB1 domain and E85A in the TIRA1 domain) showed strong rust resistance compared to the susceptible control.

[0024] Figure 14 Depicts a leaf of an event produced by a construct with one or more mutations in the NAD enzyme site containing the TIRA1 and TIRB1 domains. The mutations do not appear to negatively impact rust resistance in the event when compared to a susceptible control (S). Events produced by construct 25988, which contains a double mutation (E87A in the TIRB1 domain and E85A in the TIRA1 domain), exhibit a higher level of resistance to soybean rust 21BR08 (subfigure A), soybean rust 21BRM (subfigure C), and soybean rust RTP1 (subfigure E) than events produced by construct 25046 (the corresponding control). The corresponding bar graphs of the measured tubulin mRNA levels also show that events of construct 25988 exhibit lower fungal biomass. The bar graphs show the relative expression (y-axis) of the soybean rust β-tubulin gene of the indicated events 14 days after inoculation with rust populations 21BR08 (subfigure B), 21BRM (subfigure D), and RTP1 (subfigure F). Resistance levels were measured at the molecular level for the events via qRT-PCR using fungal β-tubulin. The quantitative measurements are consistent with the phenotypic observations of the event resistance levels ( Figure 13 ).

[0025] Figure 15 Shows a leaf of an event produced by a construct expressing a wild-type TIR protein. These events exhibit resistance to powdery mildew (when constructs 24953, 25046, or 25047 are expressed), while constructs expressing mutant TIR proteins are susceptible to powdery mildew (when constructs 25311, 25313, or 25988 are expressed).

[0026] Figure 16Shows a rating table of multiple T1 homozygous events generated by constructs 24192, 24205, and 24217. The T0 and T1 generation soybean transgenic events generated by constructs 24192, 24205, and 24217 are characterized by their resistance to soybean rust. Leaves from primary events containing a single Tir gene and molecular stacks were placed in a petri dish on a wet paper towel and then inoculated with a spore suspension of a soybean rust isolate. Leaves from null events served as negative controls. The results were scored using a standard soybean rust rating scale, where the red-brown (RB) type indicates resistance, while the tan rating indicates susceptibility. The number after the RB rating is based on a combination of lesion density or lesion size (where resistance ranges from high to moderate, grades 1 - 4), and an indication of no spore formation (NSP) or very few spores formed (SPL). The number after the tan rating is based on a combination of pustule density and spore formation level, where spore formation ranges from low to high, grades 1 - 5. T0 and T1 soybean transgenic events expressing only the TIRA polypeptide (24205) or the TIRB polypeptide (24192) did not confer resistance to soybean rust. However, soybean resistance was observed when the TIRA and TIRB polypeptides were co-expressed (24217).

[0027] Figure 17 Shows a soybean resistance rating table of multiple T1 homozygous events generated by constructs, where TirB expression is driven by a rust-inducible promoter (construct 25337).

[0028] Figure 18 Shows a soybean resistance rating table of multiple T1 homozygous events generated by molecular stacks (constructs 24915, 24953, and 25337) containing different promoter combinations and the orientation of the TirA and TirB genes.

[0029] Figure 19 Shows a soybean resistance rating table of multiple T1 homozygous events generated by expression constructs (constructs 25046, 25047, and 25135) containing different fusions of the TirA and TirB gene products.

[0030] Figure 20 Shows the phenotypes of multiple T1 homozygous events generated by constructs containing alternative promoters (construct 25337), alternative Tir gene orientations (constructs 25943 and 24915), and fusion proteins (constructs 25046 and 25047), including severity of agronomic performance, rust resistance, and transformation efficiency. Comparison was made with a control containing construct 24217.

[0031] Figure 21Shows the phenotypes of multiple T1 homozygous events generated by constructs containing alternative promoters (construct 25337), alternative Tir gene orientations (constructs 25943 and 24915), and fusion proteins (constructs 25046 and 25047), including severity of agronomic performance, rust resistance, and transformation efficiency. Compared with the control containing construct 24217.

[0032] Figure 22 Shows the soybean resistance rating table of multiple T1 homozygous events generated by constructs (constructs 25046, 25311, 25313, 25314, 25315, and 25988) containing different mutations in the TIR domain. The ratings show that the active NADase residues in the TIRB2 domain are essential for soybean rust resistance.

[0033] Figure 23 Illustrates the expression of the novel bidirectional promoter (SEQ ID NO:49). Comparing the GUS expression and the corresponding staining patterns of the expression driven by the bidirectional promoter sequences in the relative sense and antisense strands indicates that these two sequences have the same GUS staining compared to the strong constitutive promoter (soybean ubiquitin in this article; SEQ ID NO:50). The data confirm the bidirectional activity of the promoter and indicate that the bidirectional promoter can be used to drive the expression of TIRA and TIRB polypeptides to confer disease resistance.

[0034] Figure 24 Compares the resistance characteristics of wild-type TIRA and TIRB proteins with mutants containing mutations in the NADase sites of the TIRA1 domain (mutation E85A), TIRA2 (mutation V253E), TIRB1 (mutation E87A), and / or TIRB2 (mutation E257A) domains. The resistance against three different pathogens was evaluated: Asian soybean rust (ASR), powdery mildew (PM), and Pseudomonas. (Nt = not tested).

[0035] Figure 25Depicts the leaves of events generated by constructs with mutations in the NAD enzyme sites of each containing the TIRA1 and TIRB1 domains and compares them to a susceptible control (S) and events containing wild-type TIR proteins. An increase in the level of resistance was observed in the double mutants. Events generated by construct 25988 containing the double mutation (E87A in the TIRB1 domain and E85A in the TIRA1 domain) showed a higher level of resistance to soybean rust 21BR08 (subfigure F), soybean rust 21BRM (subfigure D), and soybean rust RTP1 (subfigure B) than events generated by construct 25046 (the corresponding control). The corresponding bar graphs of the measured tubulin mRNA levels also showed that events of construct 25988 exhibited lower fungal biomass. The bar graphs show the relative expression (y-axis) of the soybean rust β-tubulin gene of the indicated events 14 days after inoculation with rust populations 21BR08 (subfigure E), 21BRM (subfigure C), and RTP1 (subfigure A). Resistance levels were measured at the molecular level for the events via qRT-PCR using fungal β-tubulin. The quantitative measurements were consistent with the phenotypic observations of the event resistance levels.

[0036] Figure 26 and 27 A - B compares the ASR resistance characteristics of wild-type TIRA and TIRB proteins with mutants containing mutations in the cNMP synthase sites of the TIRA1 domain (mutation C82A), TIRA2 (mutation C248A), TIRB1 (mutation C84A), or TIRB2 (mutation C254A) domains. Resistance ratings: R - resistant; S - susceptible; IR - moderately resistant; IS - moderately susceptible. Construct 28587 containing the mutation in TIRA1 had the highest ASR resistance and the lowest disease rating. Four replicates were performed using four samples per event, with two samples infected with rust isolate 21BR08 (BR_A / B) and two leaves infected with isolate RTP22 (RTP_A / B). Disease symptoms on the sampled leaves were scored on a scale of 1 to 9.5, with lower scores indicating lower disease levels. Each replicate included the susceptible control 06KG, which had the highest disease rating ( Figure 27 , subfigure A) and the highest average tubulin level ( Figure 27 , subfigure B). Among the constructs evaluated, construct 28587 containing the mutation in TIRA1 had the highest ASR resistance and the lowest disease rating in all replicates. The other constructs showed varying levels of moderate resistance and increased tubulin levels, but significantly less disease and less tubulin compared to the susceptible control.

[0037] Figure 28Described is the assay of the interaction between ASR effector proteins and R proteins in a heterologous assay system. The presence of the interaction is confirmed by detecting the hypersensitive response in plant cells. Agrobacterium cultures containing a control construct, an R gene construct, an effector protein construct, or a combination thereof are inoculated into young tobacco leaves using a blunt syringe. Cell death is observed 3 - 5 h after infiltration. A slightly discolored circle at the inoculation site indicates no obvious cell death. An obviously discolored circle at the inoculation site corresponds to a strong cell death response. Agrobacterium strains containing the R genes TIRA and / or TIRB or a given soybean rust effector are cultured on LB (Luria - Bertani) agar plates supplemented with 50 μg / mL spectinomycin and 50 μg / mL kanamycin and incubated overnight at 22 °C in a dark room. The cultures are scooped up with toothpicks and diluted in induction medium (5 mM MgSO4, 2 mg / ml MES, and 10 μM acetosyringone, adjusted to pH 5.6 with HCl). The bacterial concentration is measured and adjusted to OD 600 = 0.1 (for construct 25046) or 0.8 (for effector and GUS constructs). The resulting cultures are pre - induced at room temperature for 2 to 3 h. For co - infiltration, cultures carrying individual constructs are induced separately and mixed at a 1:1 ratio before infiltration. The photographs show the interaction of soybean rust effectors SPE - 87, 130, 196 (upper row) and SPE - 248 and 335 (lower row) expressed in Nicotiana tabacum with TIRA and TIRB in an Agrobacterium - mediated transient assay. The reference figure shows the sites inoculated with constructs containing only TIRA - TIRB (R gene + GUS, top of the leaf), only effector (effector + GUS, middle of the leaf), or co - infiltration of TIRA - TIRB protein and effector (R gene + effector, bottom of the leaf). When each of SPE - 87, 130, 196, 335, and 248 is co - infiltrated with construct 25046, they specifically trigger a hypersensitive local cell death response at the inoculation site. The assay is repeated 3 times. 6 / 6 means that all infiltration points show HR. Co - infiltration of only the effector protein or only the R protein with a control construct expressing GUS does not trigger a hypersensitive local cell death response at the inoculation site.

[0038] Figure 29Described is the Pseudomonas effector and R protein interaction assay in a heterologous assay system. The presence of the interaction was confirmed by detecting the hypersensitive response in plant cells. Agrobacterium strains containing the R genes TIRA and / or TIRB or a given soybean rust effector were cultured on LB (Luria-Bertani) agar plates supplemented with 50 μg / mL spectinomycin and 50 μg / mL kanamycin and incubated overnight at 22 °C in the dark. The cultures were scooped up with toothpicks and diluted in induction medium (5 mM MgSO4, 2 mg / ml MES, and 10 μM acetosyringone, adjusted to pH 5.6 with HCl). The bacterial concentration was measured and adjusted with the induction medium. For co-infiltration, cultures carrying individual constructs were induced separately and mixed at a 1:1 ratio prior to infiltration. The photographs show the interaction of the HopT1 family effectors HopT1-1 and HopT1-2 from Pseudomonas syringae pv. syringae in Nicotiana tabacum leaves expressing TIRA and TIRB in an Agrobacterium-mediated transient assay. The reference figure shows the sites inoculated with constructs containing only TIRA-TIRB (R gene + GUS, top of the leaf), only the effector (effector + GUS, middle of the leaf), or co-infiltration of the TIRA-TIRB protein and the effector (R gene + effector, bottom of the leaf). When each of HopT1-1 and HopT1-2 was co-infiltrated with construct 25046, they each specifically triggered a hypersensitive local cell death response at the inoculation site. Co-infiltration of only the effector protein or only the R protein with a control construct expressing GUS did not trigger a hypersensitive local cell death response at the inoculation site.

[0039] Figure 30 Demonstrated is that the HopT1 effector triggers TIRA- and TIRB-dependent HR in transgenic soybean leaves. The photographs show that when infiltrated into GM soybean leaves expressing TIRA and TIRB, the HR response indicates the interaction of the HopT1 family effector conjugated to Pseudomonas syringae pv. glycinea. No HR response was observed when Pseudomonas syringae pv. glycinea (which does not contain the HopT1 effector itself) was infiltrated (no conjugated effector).

[0040] Figure 31 Demonstrated is the bacterial growth of Pseudomonas syringae pv. glycinea infiltrated on leaves of GM events expressing TIRA and TIRB (as previously Figure 30As shown. When Pseudomonas syringae pv. glycinea carrying the HopT1 effector and conjugated with the HopT1 effector infiltrates on the leaves (NegCK) of the GM event expressing TIRA and TIRB, almost no bacterial growth is seen. In contrast, the growth of Pseudomonas syringae pv. glycinea is observed to be nearly 100 times higher on wild-type soybeans without TIRA and TIRB or on Pseudomonas syringae pv. glycinea without the HopT1 effector. Figure 32 Shows the disease progression (0-dpi vs. 4-dpi) of the construct.

[0041] Figure 33 Illustrates that TIRA and TIRB confer resistance to Pseudomonas syringae pv. syringae via interaction with the HopT1 effector. No HR response is observed in wild-type soybean leaves that do not express TIRA and TIRB proteins. When Pseudomonas syringae pv. syringae is co-infiltrated with HopT1-2, a strong HR response is observed in soybean leaves expressing TIRA and TIRB. The reference figure shows the site of infiltration.

[0042] Figure 34 Illustrates the interaction assay of powdery mildew effector proteins and R proteins in a heterologous assay system. The photograph shows the interaction of two powdery mildew effectors from Erysiphe pisi, EPCSEP-66 and 99, expressed in Nicotiana tabacum with TIRA and TIRB via Agrobacterium-mediated transient assay. The reference figure shows the site of inoculation of constructs containing only TIRA-TIRB (R gene + GUS, top of the leaf), only effector (effector + GUS, middle of the leaf), or co-infiltration of TIRA-TIRB protein and effector (R gene + effector, bottom of the leaf). When each of EPCSEP-66 and 99 is co-infiltrated with construct 25046, they specifically trigger a hypersensitive local cell death response at the inoculation site. Co-infiltration of only effector protein or only R protein with the control construct expressing GUS does not trigger a hypersensitive local cell death response at the inoculation site.

[0043] Figure 35Describes the oomycete effector and R protein interaction assays in a heterologous assay system. The photograph shows the interaction of two oomycete effectors from Plasmopara halstedii, DMCEP-46 and 84, expressed in Nicotiana tabacum with TIRA and TIRB in an Agrobacterium-mediated transient assay. The reference figure shows the sites inoculated with constructs containing only TIRA-TIRB (R gene + GUS, top of the leaf), only effector (effector + GUS, middle of the leaf), or co-infiltration of TIRA-TIRB protein and effector (R gene + effector, bottom of the leaf). When each of DMCEP-46 and 84 was co-infiltrated with construct 25046, they specifically triggered a hypersensitive local cell death response at the inoculation site. Co-infiltration of only the effector protein or only the R protein with the control construct expressing GUS did not trigger a hypersensitive local cell death response at the inoculation site.

[0044] Figure 36 Describes the soybean cyst nematode effector and R protein interaction assays in a heterologous assay system. The photograph shows the interaction of two oomycete effectors from soybean cyst nematode, SSNE-5, 18, and 28, expressed in Nicotiana tabacum with TIRA and TIRB in an Agrobacterium-mediated transient assay. The reference figure shows the sites inoculated with constructs containing only TIRA-TIRB (R gene + GUS, top of the leaf), only effector (effector + GUS, middle of the leaf), or co-infiltration of TIRA-TIRB protein and effector (R gene + effector, bottom of the leaf). When each of SSNE-5, 18, and 28 was co-infiltrated with construct 25046, they specifically triggered a hypersensitive local cell death response at the inoculation site. Co-infiltration of only the effector protein or only the R protein with the control construct expressing GUS did not trigger a hypersensitive local cell death response at the inoculation site.

[0045] Figure 37 Shows a comparison of effector recognition by TIRA and TIRB and other distantly functional homologs. The effector recognition of multiple effector proteins including soybean rust effector, cyst nematode effector, and Pseudomonas syringae ice nucleation effector was compared. The results showed that the recognition profiles of TIRA and TIRB and their functional homologs from Cajanus cajun were the same.

[0046] Brief description of the sequence listing

[0047] SEQ ID NO:1 is the amino acid sequence of the TIRA polypeptide of the TirA gene derived from Glycine canescens.

[0048] SEQ ID NO:2 is the amino acid sequence of the TIRB polypeptide of the TirA gene derived from Glycine cyrtoloba.

[0049] SEQ ID NO:3 is the genomic sequence of the TirA gene. SEQ ID NO:4 is the cDNA sequence of the TirA gene. SEQ ID NO:5 is the intronless version of the genomic sequence of the TirA gene. Each of SEQ ID NOs: 3-5 encodes the protein of SEQ ID NO:1.

[0050] SEQ ID NO:6 is the genomic sequence of the TirB gene. SEQ ID NO:7 is the cDNA sequence of the TirB gene. SEQ ID NO:8 is the intron-modified version of the genomic sequence of the TirB gene. Each of SEQ ID NOs: 6-8 encodes the protein of SEQ ID NO:2.

[0051] SEQ ID NOs: 9-12 are the amino acid sequences of the TIRATIRB fusion protein (comprising the TIRA and TIRB polypeptides).

[0052] SEQ ID NOs: 13-16 are the coding sequences of the TIRATIRB fusion protein of SEQ ID NOs: 9-12.

[0053] SEQ ID NO:17 is the coding sequence of the TIRATIRB fusion protein containing a loss-of-function E85A mutation at position 85 corresponding to the TIRA polypeptide (SEQ ID NO:1).

[0054] SEQ ID NO:18 is the coding sequence of the TIRATIRB fusion protein containing a gain-of-function V251E mutation at position 251 corresponding to the TIRA polypeptide (SEQ ID NO:1).

[0055] SEQ ID NO:19 is the coding sequence of the TIRATIRB fusion protein containing a loss-of-function E87A mutation at position 87 corresponding to the TIRB polypeptide (SEQ ID NO:2).

[0056] SEQ ID NO:20 is the coding sequence of the TIRATIRB fusion protein containing a loss-of-function E85A mutation at position 85 corresponding to the TIRA polypeptide (SEQ ID NO:1), and loss-of-function E87A and E257A mutations at positions 87 and 257 corresponding to the TIRB polypeptide (SEQ ID NO:2).

[0057] SEQ ID NO:21 is the coding sequence of a TIRATIRB fusion protein containing a loss-of-function E85A mutation at the position corresponding to position 85 of the TIRA polypeptide (SEQ ID NO:1) and a loss-of-function E87A mutation at the position corresponding to position 87 of the TIRB polypeptide (SEQ ID NO:2).

[0058] SEQ ID NO:22 is the plant-active constitutive promoter prMt12344 derived from Medicago truncatula.

[0059] SEQ ID NO:23 is the plant-active constitutive promoter prMt51866 derived from Medicago truncatula.

[0060] SEQ ID NO:24 is the plant-active constitutive promoter prMt15303 derived from Medicago truncatula.

[0061] SEQ ID NO:25 is the plant-active rust-inducible promoter prLuFIS1 derived from flaxseed.

[0062] SEQ ID NO:26 is the plant-active native promoter prGcaRG3a of the TirA gene from Glycine canescens.

[0063] SEQ ID NO:27 is the plant-active native promoter prGcaRG3b of the TirB gene from Glycine canescens.

[0064] SEQ ID NO:28 is the native terminator tGcaRG3a of the TirA gene from Glycine canescens.

[0065] SEQ ID NO:29 is the native terminator tGcaRG3b of the TirB gene from Glycine canescens.

[0066] SEQ ID NO:30 is the plant-active terminator tMt12344 derived from Medicago truncatula.

[0067] SEQ ID NO:31 is the plant-active terminator tMt51866 derived from Medicago truncatula.

[0068] SEQ ID NO:32 is the first intron iAtBAF60 of the Arabidopsis thaliana gene (AtBAF60), which is similar to the human Brahma-related protein BAF60.

[0069] SEQ ID NO:33 is the nucleotide sequence of the synthetic linker peptide (x linker). SEQ ID NO:36 is the linker peptide encoded by SEQ ID NO.33.

[0070] SEQ ID NO:34 is the nucleotide sequence of the self-cleaving peptide linker (xT2A linker-04). SEQ ID NO:37 is the linker peptide encoded by SEQ ID NO:34.

[0071] SEQ ID NO:35 is the nucleotide sequence of the self-cleaving peptide linker (xT2A linker-03). SEQ ID NO:38 is the linker peptide encoded by SEQ ID NO:35.

[0072] SEQ ID NO:39-40 are the genomic sequences of the first set of allelic variants (RG6a and RG6b) of the TirA and TirB genes, respectively. SEQ ID NO:41-42 are the genomic sequences of the second set of allelic variants (RG7a and RG7b) of the TirA and TirB genes, respectively. SEQ ID NO:43-44 are the genomic sequences of the third set of allelic variants (RG8a and RG8b) of the TirA and TirB genes, respectively. All allelic variants are derived from Glycine clandestina.

[0073] SEQ ID NO:45 is the nucleotide sequence of the TirA gene ortholog derived from Cajanus cajan. SEQ ID NO:45 encodes the TirA polypeptide ortholog of SEQ ID NO:46.

[0074] SEQ ID NO:47 is the nucleotide sequence of the TirB gene ortholog derived from Cajanus cajan. SEQ ID NO:48 encodes the TirB polypeptide ortholog of SEQ ID NO:47.

[0075] SEQ ID NO:49 is the nucleotide sequence of the bidirectional promoter derived from the genomic locus containing the R-protein genes Rg32 and Rg34.

[0076] SEQ ID NO:50 is the nucleotide sequence of the plant active constitutive promoter prUBQ3 of the ubiquitin 3 gene derived from Arabidopsis thaliana.

[0077] SEQ ID NO:51 is the polynucleotide sequence of the effector protein SPE-335 from Phakopsora pachyrhi z i).

[0078] SEQ ID NO:52 is the polynucleotide sequence of effector protein SPE-087 from Phakopsora pachyrhizi.

[0079] SEQ ID NO:53 is the polynucleotide sequence of effector protein SPE-130 from Phakopsora pachyrhizi.

[0080] SEQ ID NO:54 is the polynucleotide sequence of effector protein SPE-196 from Phakopsora pachyrhizi.

[0081] SEQ ID NO:55 is the polynucleotide sequence of effector protein SPE-248 from Phakopsora pachyrhizi.

[0082] SEQ ID NO:56 is the polynucleotide sequence of effector protein SSNE-05 from plant parasitic nematodes.

[0083] SEQ ID NO:57 is the polynucleotide sequence of effector protein SSNE-18 from plant parasitic nematodes.

[0084] SEQ ID NO:58 is the polynucleotide sequence of effector protein SSNE-28 from plant parasitic nematodes.

[0085] SEQ ID NO:59 is the polynucleotide sequence of effector protein HopT1-1 from Pseudomonas syringae pv. tomato DC3000. SEQ ID NO:61 is the amino acid sequence of the effector.

[0086] SEQ ID NO:60 is the polynucleotide sequence of effector protein HopT1-2 from Pseudomonas syringae pv. tomato DC3000. SEQ ID NO:62 is the amino acid sequence of the effector.

[0087] SEQ ID NO:63 is the polynucleotide sequence of effector protein EPCSEP-66 from Erysiphe pisi. SEQ IDNO:64 is the amino acid sequence of the effector.

[0088] SEQ ID NO:65 is the polynucleotide sequence of effector protein EPCSEP-99 from Erysiphe pisi. SEQ IDNO:66 is the amino acid sequence of the effector.

[0089] SEQ ID NO:67 is the polynucleotide sequence of oomycete effector DMCEP-46. SEQ ID NO:68 is the amino acid sequence of the effector.

[0090] SEQ ID NO:69 is the polynucleotide sequence of oomycete effector DMCEP-84. SEQ ID NO:70 is the amino acid sequence of the effector.

[0091] SEQ ID NO:71 is the polynucleotide sequence of an expression cassette (29466) that drives the endogenous expression of TIRA and TIRB.

[0092] SEQ ID NO:72 is the polynucleotide sequence of an expression cassette (VC30523) that drives the endogenous expression of TIRA and TIRB. Detailed Description

[0093] 1. Definitions

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this disclosure belongs.

[0095] Although the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are presented to facilitate understanding of the subject matter of this disclosure.

[0096] All references listed below, as well as all references cited in this disclosure, including but not limited to all patents, patent applications and their publications, articles in scientific journals, and database entries (e.g., database entries and all annotations available therein) are hereby incorporated by reference in their entirety to the extent that they supplement, explain, provide background for, or teach the methods, techniques, and / or compositions employed herein.

[0097] The nucleotide sequences provided herein are represented from left to right in the 5' to 3' direction and are represented using the standard codes for nucleotide bases as shown in 37 CFR §§ 1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25, e.g.: adenine (A), cytosine (C), thymine (T), and guanine (G).

[0098] Amino acids are also indicated using WIPO Standard ST.25, e.g.: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0099] The singular forms “a / an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0100] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, along with the absence of combinations when interpreted in the alternative (“or”).

[0101] As used herein, the term “about” when referring to a measurable value such as a dose, rate of administration, or period of time, etc. is intended to cover variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. As used herein, phrases such as “between about X and Y” mean “between about X and about Y”, and phrases such as “from about X to Y” mean “from about X to about Y”.

[0102] Unless the context indicates otherwise, phrases such as “between about X and Y”, “between about X and about Y”, “from X to Y”, and “from about X to about Y” (and similar phrases) as used herein shall be interpreted to include X and Y.

[0103] As used herein, “agronomic performance” or “enhanced agronomic performance” refers to the phenotypes (and potential genetic elements) present in a given plant that contribute to yield during the growing season. Agronomic performance includes emergence vigor, germination rate, nutrient potential, stress tolerance, plant height, height, stem or stalk width, disease resistance, branching, flowering, fruiting, seed size, seed density, lodging resistance, threshability, etc. Plants that exhibit enhanced agronomic performance have genetic elements that result in one or more or all of increased germination rate, higher plant height.

[0104] As used herein, “coding sequence” or “CDS” is a nucleic acid sequence that is transcribed into RNA (such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA). In an embodiment, the RNA is subsequently translated to produce a protein. In an exemplary embodiment, the CDS is derived from a cDNA sequence and includes the spliced exon sequences of the transcript in the DNA annotation and does not include any introns or 5′ or 3′-untranslated regions (UTRs). In other exemplary embodiments, the CDS is derived from a genomic DNA sequence and includes the spliced exon sequences of the transcript in the DNA annotation as well as one or more introns, and 5′ and / or 3′-untranslated regions (UTRs).

[0105] As used herein, a "codon-optimized" nucleotide sequence means the nucleotide sequence of a recombinant, transgenic, or synthetic polynucleotide, wherein the codons are selected to reflect the particular codon preference that a host cell or organism may have. This is typically done in such a way as to maintain the amino acid sequence of the polypeptide encoded by the codon-optimized nucleotide sequence. In certain embodiments, the nucleotide sequence is codon-optimized for the cell in which the construct is to be expressed (e.g., an animal, plant, fungal, or bacterial cell). For example, a construct to be expressed in a plant cell may have all or part of its sequence codon-optimized for expression in plants. See, e.g., U.S. Patent No. 6,121,014. In embodiments, the polynucleotides provided herein are codon-optimized for expression in a plant cell (e.g., a dicotyledonous plant cell, a monocotyledonous plant cell, a soybean cell) or a bacterial cell.

[0106] The terms "comprise," "comprises," and "comprising" as used in this specification indicate the presence of the stated feature, integer, step, operation, element, or component, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0107] As used herein, the transitional phrase "consisting essentially of" (and grammatical variants) means that the scope of a claim is to be interpreted to cover the specified materials or steps recited in the claim and those that do not materially affect one or more of the basic and novel features of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising."

[0108] As used herein, "expression cassette" means a nucleic acid molecule capable of directing the expression of at least one polynucleotide of interest (such as a polynucleotide encoding a TIRA polypeptide and / or a TIRB polypeptide and / or a TIRATIRB fusion protein, or an active variant or fragment thereof) in a suitable host cell, and comprising a promoter operably linked to the polynucleotide of interest (which is operably linked to a termination signal). The "expression cassette" may comprise additional polynucleotides to facilitate the proper translation of the polynucleotide of interest. The expression cassette may comprise other polynucleotides that are not related to the expression of the polynucleotide of interest but are present due to convenient restriction sites used to remove the expression cassette from an expression vector. In embodiments, at least one component in the expression cassette may be heterologous (i.e., foreign or modified from its native form at the composition and / or genomic locus) relative to at least one other component (such as a heterologous promoter, terminator, intron, and / or any regulatory element operably associated with the polynucleotide of interest). In other embodiments, the expression cassette may be naturally occurring and comprise natural regulatory elements, natural introns, and natural genomic DNA to permit the expression of TIRA and / or TIRB polypeptides and / or TIRATIRB fusion proteins, or active variants or fragments thereof. The expression cassette may be heterologous to the host, i.e., the expression cassette (or even the polynucleotide of interest) is not naturally present in the host cell and has been introduced into the host cell by transformation methods or breeding methods.

[0109] In embodiments of the invention, expression cassettes are provided that can direct the expression of only the TIRA polypeptide (or an active variant or fragment thereof), only the TIRB polypeptide (or an active variant or fragment thereof), or each of the TIRA and TIRB polypeptides (or an active variant or fragment of either polypeptide). In exemplary embodiments, a first expression cassette is provided for expressing the TIRA polypeptide (or an active variant or fragment thereof), the expression of the TIRA polypeptide being driven by a first heterologous promoter (such as a plant-active promoter), while a second expression cassette is provided for expressing the TIRB polypeptide (or an active variant or fragment thereof), the expression of the TIRB polypeptide being driven by a different second heterologous promoter (such as another plant-active promoter). In another exemplary embodiment, a single expression cassette is provided for expressing each of the TIRA polypeptide (or an active variant or fragment thereof) and the TIRB polypeptide (or an active variant or fragment thereof), the expression of the TIR polypeptide being driven by a common promoter (such as a plant-active promoter).

[0110] In an embodiment, a single expression cassette is provided for expressing TIRA and TIRB polypeptides that are linked together as a fusion protein, wherein the expression of the TIRA polypeptide (or an active variant or fragment thereof) and the TIRB polypeptide (or an active variant or fragment thereof) is driven by a common promoter (e.g., a plant-active promoter). Based on whether any linker sequence is present between the nucleotide sequences, the polypeptides can be expressed as a single fusion protein or as distinct proteins. In an exemplary embodiment, an expression cassette is provided that can direct the expression of a fusion protein comprising a TIRA polypeptide linked via a linker to a TIRB polypeptide, wherein the expression of the two polypeptides in the fusion protein is driven by a common single heterologous promoter and terminator. In an embodiment where the linker is self-cleaving, after the expression of the fusion protein, the protein can be cleaved into the constitutive TIRA and TIRB polypeptides. Based on the order of the nucleotide sequences encoding the TIRA polypeptide and the TIRB polypeptide in the expression cassette, the fusion protein can comprise the TIRA polypeptide at the N-terminus of the fusion protein (e.g., the nucleotide sequence encoding the TIRA polypeptide is upstream of the nucleotide sequence encoding the TIRB polypeptide), or the fusion protein can comprise the TIRB polypeptide at the N-terminus of the fusion protein (e.g., the nucleotide sequence encoding the TIRB polypeptide is upstream of the nucleotide sequence encoding the TIRA polypeptide).

[0111] In an embodiment of the invention, cells are provided that comprise a TIRA polypeptide (or an active variant or fragment thereof), a TIRB polypeptide (or an active variant or fragment thereof), or each of the TIRA and TIRB polypeptides (or an active variant or fragment of either polypeptide). In a particular embodiment, the cell is a plant cell, wherein the polynucleotide is stably integrated into the genome of the cell. In a specific embodiment, the plant cell has an increased polypeptide expression level compared to a control plant cell and the plant cell has increased disease resistance.

[0112] The term "introduced / introducing" defines the process of altering the content of a cell or plant by using traditional breeding or recombinant transformation techniques. Any method can be used to introduce a polynucleotide into a cell or a plant cell, including methods that result in stable transformation, transient transformation, or gene editing. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, PEG, electroporation, sonication methods (e.g., sonoporation), liposomes, microinjection, naked DNA, plasmid vectors, viral vectors (including episomal and integrative), gene infiltration, transgenesis, clustered regularly interspaced short palindromic repeats modification (CRISPR), transcription activator-like effector nucleases (TALEN) (Feng et al. 2013, Joung & Sander [Journal of Sandwich Structures and Materials] 2013), meganucleases, or zinc finger nucleases (ZFN) and any other well-known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell.

[0113] As used herein, the term "Glycine" refers to any of the perennial Glycine species, such as Glycine canescens, Glycine argyrea, Glycine clandestina, Glycine latrobeana, Glycine albicans, Glycine aphyonota, Glycine arenaria, Glycine curvata, Glycine cyrtoloba, Glycine dolichocarpa, Glycine falcata, Glycine gracei, Glycine hirticaulis, Glycine lactovirens, Glycine latifolia, Glycine microphylla, Glycine montis-douglas, Glycine peratosa, Glycine pescadrensis, Glycine pindanica, Glycine pullenii, Glycine rubiginosa, Glycine stenophita, Glycine syndetika, or Glycine tomentella.

[0114] As used herein, the term "allele" refers to one of two or more different nucleotides or nucleotide sequences occurring at a particular locus.

[0115] A marker is "associated with" a trait when the marker is linked to the trait and the presence of the marker indicates whether and / or to what extent the desired trait or trait form will occur in a plant / germplasm containing the marker. Similarly, a marker is "associated with" an allele when the marker is linked to the allele and the presence of the marker indicates whether the allele is present in a plant / germplasm containing the marker. For example, a "marker associated with enhanced pathogen resistance" or "enhanced disease resistance" is a marker whose presence or absence can be used to predict whether and / or to what extent a plant will exhibit a pathogen resistance or disease resistance phenotype.

[0116] Markers can be, but are not limited to, alleles, genes, haplotypes, restriction fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs), randomly amplified polymorphic DNAs (RAPDs), cleaved amplified polymorphic sequences (CAPS) (Rafalski and Tingey, Trends in Genetics 9:275 (1993)), amplified fragment length polymorphisms (AFLPs) (Vos et al., Nucleic Acids Res. 23:4407 (1995)), single nucleotide polymorphisms (SNPs) (Brookes, Gene 234:177 (1993)), sequence characterized amplified regions (SCARs) (Paran and Michelmore, Theor. Appl. Genet. 85:985 (1993)), sequence tagged sites (STSs) (Onozaki et al., Euphytica 138:255 (2004)), single-strand conformation polymorphisms (SSCPs) (Orita et al., Proc. Natl. Acad. Sci. USA 86:2766 (1989)), inter-simple sequence repeats (ISSRs) (Blair et al., Theor. Appl. Genet. 98:780 (1999)), inter-retrotransposon amplified polymorphisms (IRAPs), retrotransposon microsatellite amplified polymorphisms (REMAPs) (Kalendar et al., Theor. Appl. Genet. 98:704 (1999)), chromosomal intervals or RNA cleavage products (e.g., Lynx tags). Markers can be present in genomic nucleic acids or expressed nucleic acids (e.g., ESTs). The term marker can also refer to nucleic acids that are used as probes or primers (e.g., primer pairs) for amplifying, hybridizing, and / or detecting nucleic acid molecules according to methods well known in the art (e.g., using PCR).

[0117] As used herein, the terms "backcross" and "backcrossed" refer to a method by which a progeny plant is repeatedly backcrossed to one of its parents. In a backcrossing scheme, the "donor" parent is the parent plant having the desired gene or locus for introgression. The "recipient" parent (used once or more) or "recurrent" parent (used twice or more) is the parent plant into which the gene or locus is being introgressed. See, e.g., Ragot, M. et al., Marker-assisted Backcrossing: A Practical Example, Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backcross Breeding, Proceedings of the Symposium "Analysis of Molecular Marker Data", pp. 41-53 (1994). The initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on.

[0118] A centimorgan ("cM") is a measure of the recombination frequency. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus by crossing over in a single generation.

[0119] As used herein, the terms "cross" or "crossed" refer to the fusion of gametes by pollination to produce progeny (e.g., cells, seeds or plants). The term encompasses both sexual crosses (one plant pollinated by another) and selfing (self-pollination, e.g., when the pollen and ovules are from the same plant). The term "crossing" refers to the act of causing gametes to fuse by pollination to produce progeny.

[0120] As used herein, the terms "cultivar" and "variety" refer to a group of similar plants that can be distinguished from other varieties within the same species by structural or genetic characteristics and / or performance.

[0121] As used herein, the terms "desired allele", "favorable allele" and "allele of interest" are used interchangeably to refer to an allele associated with a desired trait (e.g., ASR resistance).

[0122] As used herein, a "disease resistance gene" or "resistance gene" or "R gene" refers to a nucleic acid having a nucleotide sequence (e.g., a DNA sequence) encoding a polypeptide, an R protein, or a resistance protein, which, when expressed in a plant cell, is capable of enhancing or improving or increasing the defense or immune response in the plant cell, thereby conferring upon the plant an increased resistance to one or more plant pathogens. In certain embodiments, the TirA and TirB genes of the present invention are disease resistance genes or R genes that encode polypeptides that confer enhanced pathogen resistance when co-expressed in a plant cell. The encoded TIRA and TIRB polypeptides, or active variants or fragments thereof, can be expressed in plants to enhance pathogen resistance to plant pathogens (e.g., fungal pathogens, bacterial pathogens, nematodes or sucking pests). As a non-limiting example, the R genes of the present invention and the polypeptides encoded thereby can be used to enhance resistance to fungal pathogens (Phytophthora, Asian soybean rust). In other examples, the R genes of the present invention and the polypeptides encoded thereby can be used to enhance resistance to other fungal pathogens (e.g., fungal pathogens causing powdery mildew) as well as nematodes (e.g., soybean cyst nematode (SCN) and root-knot nematode). In addition, the R genes of the present invention and the polypeptides encoded thereby can be used to enhance resistance to bacterial pathogens (e.g., Pseudomonas syringae pv. syringae).

[0123] In still other instances, the R genes of the present invention and the polypeptides they encode can be used to enhance resistance to piercing-sucking pests (including but not limited to aphids, bed bugs, whiteflies, etc.). Piercing-sucking pests are known in the art. For example, see Vleeshouwers and Oliver, 2014, Effectors as tools in disease resistance breeding against biotrophic, hemibiotrophic, and necrotrophic plant pathogens [Effectors as tools in disease resistance breeding against biotrophic, hemibiotrophic, and necrotrophic plant pathogens], MPMI [Molecular Plant-Microbe Interactions] Vol. 27, No. 3, (2014), pp. 196-206; Jayaraman et al. Effector-assisted breeding for bacterial wilt resistance in horticultural crops. [Effector-assisted breeding for bacterial wilt resistance in horticultural crops.] Hortic. Environ. Biotechnol. [Journal of Horticultural Environment and Biotechnology] 57, 415-423 (2016); Carolan et al., Predicted Effector Molecules in the Salivary Secretome of the Pea Aphid (Acyrthosiphon pisum): A Dual Transcriptomic / Proteomic Approach [Predicted Effector Molecules in the Salivary Secretome of the Pea Aphid (Acyrthosiphon pisum): A Dual Transcriptomic / Proteomic Approach], J.of Proteome Res[Journal of Proteome Research], 10, 4 (2011) pp. 1505 - 1518; Shan et al., Asalivary secretory protein from Riptortus pedestris facilitates pest infestation and soybean staygreen syndrome[Molecular Plant Pathology], (2023); Luo et al., Molecular Advances in Breeding for Durable Resistance against Pests and Diseases in Wheat: Opportunities and Challenges[Agronomy], 13, 3, p. 628 (2023); Fu et al., Two salivary proteins Sm10 and SmC002 from grain aphid Sitobion miscanthi modulate wheat defense and enhance aphid performance[Frontiers in Plant Science], 14, (2023); Xu et al., A salivary effector enables whitefly to feed on host plants by eliciting salicylic acid-signaling pathway[PNAS], 116(2) 490 - 495 (2018); and Naalden et al., Spotlight on the Roles of Whitefly Effectors in Insect–Plant Interactions[Front. Plant Sci.], July 2 (2021); and Sec.Plant Pathogen Interaction, Volume 12 (2021), the content of each of which is incorporated herein by reference in its entirety.

[0124] Examples of R genes can include one or more motifs associated with one or more domains of the corresponding R protein. Examples of R genes (such as the TirA and TirB genes of the present disclosure) encode polypeptides containing a Toll / interleukin-1 receptor (TIR) motif containing a Toll / interleukin-1 receptor domain. In other embodiments, the R gene can encode a polypeptide having a TNL motif that includes a Toll / interleukin-1 receptor (TIR) domain, a nucleotide binding site (NBS), and a leucine-rich repeat (LRR) domain. In still other embodiments, the R gene can encode a polypeptide having a CNL motif that includes a coiled-coil (CC) domain, a nucleotide binding site (NBS), and a leucine-rich repeat (LRR) domain. In further embodiments, the R gene can encode a polypeptide having one or more additional domains and motifs (such as a kinase domain and a WRKY domain). In other embodiments, the R gene can alternatively or additionally encode a polypeptide having a domain of unknown function, the function of which was unknown at that time. In still further embodiments, the R gene can alternatively or additionally encode a polypeptide having a putative effector binding domain and / or one or more putative transmembrane helices.

[0125] In embodiments, the nucleic acid sequence of the R gene is derived from a wild plant exhibiting increased resistance to a pathogen and includes at least one coding sequence encoding a polypeptide conferring resistance. The nucleic acid sequence of the R gene can further include nucleic acid sequences corresponding to one or more native regulatory elements (such as native introns, native promoters, native UTRs), one or more heterologous regulatory elements (such as heterologous promoters and introns), and combinations thereof. Inserting the R gene into a plant with reduced resistance to a pathogen (such as no resistance or partial or complete susceptibility) at a chromosomal location (such as stably integrated into the plant genome) or an extrachromosomal location (such as on a vector or plasmid) can confer wild plant-derived pathogen resistance to the recipient plant. For example, in a representative embodiment, the R gene of the present invention is derived from Glycine tomentella, Glycine cyrtoloba, or Glycine penghuensis, and can be inserted into a soybean (Glycine max) plant to confer or enhance the resistance of the soybean plant to Asian soybean rust and / or powdery mildew.

[0126] As used herein, "variants" of a polynucleotide sequence encoding a disease resistance R gene and / or "variants" of a polypeptide sequence encoding the corresponding R protein conferring disease resistance include (as non-limiting examples) annotated and splicing variants. As used herein, an annotated variant refers to sequences that differ from each other due to differences in regulatory sequence annotation, including but not limited to the position of the transcription start site, the position of the start ATG codon, the position of the splice site, the position of introns and / or exons, etc. In an embodiment, due to the choice of the upstream start codon, the first annotated variant of a polypeptide may be longer than the second annotated variant (e.g., the upstream methionine is annotated as the start codon in the first annotated variant, while the downstream methionine is annotated as the start codon in the second annotated variant). Annotated variants have the same activity as the corresponding R protein, including the ability to confer increased disease resistance.

[0127] As used herein, a "splicing variant" refers to a sequence that differs from a reference sequence due to DNA sequence changes that occur during splicing at the splice site. As used herein, "splicing" refers to the process by which a pre-mRNA transcript is converted into an mRNA molecule that can be translated into a protein. Typically, this is achieved by removing introns and splicing the exons of the transcript together. However, genetic changes in the sequence may occur at the boundaries of exons and introns, i.e., at the splice site. During splicing, the addition of one or more introns, or the removal of one or more exons, can result in different mRNA molecules being produced from the same gene, and in turn, different protein sequences being expressed from a single gene. These different sequences at the DNA, RNA, and / or protein levels are referred to herein as "alternative splicing variants". Splicing variant polypeptides encoded by alternative splicing of a gene sequence can include variants with more or fewer amino acids in the sequence and / or variants with alternative amino acids at specific positions within the sequence. In an embodiment, alternative splicing variants of a gene can be predicted based on different annotations of gene sequence regions as exons or introns (e.g., via analysis of the sequence using sequence prediction software). Splicing variants have the same activity as the corresponding R protein, including the ability to confer increased disease resistance.

[0128] As used herein, the terms "disease tolerance", "disease resistance", "having disease tolerance", or "having disease resistance" refer to the ability of a plant to tolerate and / or reproduce despite infection by the corresponding disease. Thus, "disease tolerance" or "disease resistance" means that, when compared to an appropriate control plant, the disease tolerance of the plant caused by a plant pathogen is statistically significantly increased and / or one or more disease symptoms are statistically significantly reduced or absent. In some embodiments, the increase in disease tolerance or resistance can be measured by (1) the ability of the plant to tolerate and / or reproduce despite infection by the corresponding disease; (2) by an infected disease-resistant leguminous crop or soybean plant having the same (or nearly the same) yield as an uninfected leguminous crop or soybean plant; or (3) by the delay or prevention of pathogen (e.g., fungus) proliferation (including the delay or prevention of disease-related symptoms). In still other embodiments, a plant or germplasm can be labeled "having disease resistance" if it exhibits "enhanced or increased pathogen resistance" compared to a control plant.

[0129] As used herein, the terms "enhanced pathogen resistance", "enhanced disease resistance", "increased resistance to a pathogen", or "confer pathogen resistance" refer to an improvement, enhancement, or increase in the ability of a plant to tolerate and / or reproduce despite infection by a pathogen or disease (e.g., Asian soybean rust). Enhanced disease resistance includes a reduction in the infection symptoms of diseases such as Asian soybean rust ("ASR"), soybean cyst nematode, Phytophthora, root-knot nematode, bacterial diseases, or sucking pests. Enhanced plant pathogen resistance can include any statistically significant increase in resistance to a plant pathogen, including, for example, an increase of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or higher. Conferring or enhancing or increasing resistance can include a reduction (partial or complete) in symptoms or phenotypic characteristics associated with pathogen susceptibility and / or an increase in phenotypic characteristics associated with pathogen resistance. In exemplary embodiments, conferring or increasing resistance to Asian soybean rust can include a statistically significant reduction in the number, size, and / or density of lesions, a change in lesion color (e.g., from tan coloring to reddish-brown coloring), a reduction in the number and density of pustule formations, a reduction in spore formation, a reduction in defoliation, a reduction in yield loss, or any combination thereof. In further embodiments, enhanced pathogen resistance can include a statistically significant reduction in the number, size, and / or density of cysts. Additionally, enhanced pathogen resistance can include the prevention or delay of pathogen (e.g., fungus) proliferation in the plant.

[0130] "Control" or "control plant" or "control plant cell" provides a reference point for measuring changes in the phenotype of a subject plant or plant cell. A control plant or plant cell can comprise, for example: (a) a wild-type plant or cell, i.e., having the same genotype as the starting material used to effect the genetic alteration of the subject plant or cell; (b) a plant or plant cell having the same genotype as the starting material but which has been transformed with a null construct (i.e., a construct having no known effect on the trait of interest, such as a construct containing a marker gene); (c) a plant or plant cell that is a non-transformed isolate among the progeny of the subject plant or plant cell; (d) a plant or plant cell that is genetically identical to the subject plant or plant cell but has not been exposed to the conditions or stimuli that would induce the expression of the gene of interest; or (e) the subject plant or plant cell itself under conditions where the gene of interest is not expressed.

[0131] "elite line" or "elite strain" is an agronomically superior line that has been produced from many cycles of breeding for superior agronomic performance. Numerous elite lines are available and are known to the ordinary skilled person in the field of soybean breeding. "elite population" is a class of elite individuals or lines that can be used to represent the prior art in terms of agronomically superior genotypes in a given crop species, such as soybean. Similarly, "elite germplasm" or an elite strain of germplasm is agronomically superior germplasm, typically derived from and / or capable of producing plants having superior agronomic performance, such as existing or newly developed elite soybean lines.

[0132] As used herein, "cisgenic" or "cisgenesis" relates to the optional insertion of one or more genes from the same or a related species or from a crossable donor into a genome (e.g., a plant genome). As used herein, "cisgenic construct" is a recombinant nucleic acid sequence that is present in a cell and optionally integrated into the cell genome, wherein the recombinant nucleic acid sequence comprises regulatory elements operably linked to a nucleic acid sequence of a gene of interest, wherein the regulatory elements and the gene of interest are native to the plant, or from a related species, or from a crossable donor, and are operably linked in the native cell at a genomic location different from the genomic location where they are integrated as a cisgenic construct. The introduction of specific alleles / genes existing in a gene pool via homologous inheritance without altering any DNA sequence can accelerate the breeding of species with long breeding cycles and no linkage drag.

[0133] "Superior" plants are any plants from superior lines, and thus superior plants are representative plants from superior varieties. Non-limiting examples of superior soybean varieties commercially available to farmers or soybean breeders include: AG00802, A0868, AG0902, A1923, AG2403, A2824, A3704, A4324, A5404, AG5903, AG6202, AG0934; AG1435; AG2031; AG2035; AG2433; AG2733; AG2933; AG3334; AG3832; AG4135; AG4632; AG4934; AG5831; AG6534; and AG7231 (Asgrow Seeds, Des Moines, Iowa, USA); BPR0144RR, BPR 4077NRR, and BPR 4390NRR (Bio Plant Research, Camp Point, Illinois, USA); DKB17-51 and DKB37-51 (DeKalb Genetics, DeKalb, Illinois, USA); DP 4546RR, and DP 7870RR (Delta & Pine Land Company, Lubbock, Texas, USA); JG03R501, JG 32R606CADD, and JG 55R503C (JGL Inc.), Greencastle, Indiana, USA); NKS13-K2 (NK Division of Syngenta Seeds, Golden Valley, Minnesota, USA); 90M01, 91M30, 92M33, 93M11, 94M30, 95M30, 97B52, P008T22R2; P16T17R2; P22T69R; P25T51R; P34T07R2; P35T58R; P39T67R; P47T36R; P46T21R; and P56T03R2 (Pioneer Hi-Bred International, Johnston, Iowa, USA); SG4771NRR and SG5161NRR / STS (Soygenetics, LLC, Lafayette, Indiana, USA); S00-K5, S11-L2, S28-Y2, S43-B1, S53-A1, S76-L9, S78-G6, S0009-M2; S007-Y4; S04-D3; S14-A6; S20-T6; S21-M7; S26-P3; S28-N6; S30-V6; S35-C3; S36-Y6; S39-C4; S47-K5; S48-D9; S52-Y2; S58-Z4; S67-R6; S73-S8; and S78-G6 (Syngenta Seeds, Henderson, Kentucky, USA); Richer (Northstar Seed Ltd., Alberta, Canada); 14RD62 (Stine Seed Co., Iowa, USA); or Armor4744 (Armor Seed, LLC, Alaska, USA).

[0134] As used herein, the term "agronomically superior" means a genotype having a number of distinguishable traits (such as emergence, vigor, nutritional vigor, disease resistance, seed set, standability, yield, and threshability) that allows a producer to harvest a commercially significant product.

[0135] A "natural" or "wild-type" nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence. Thus, for example, a "wild-type mRNA" is an mRNA that occurs naturally in an organism or is endogenous to the organism.

[0136] The terms "nucleic acid", "nucleic acid molecule", "nucleotide sequence", "oligonucleotide", "polynucleic acid", and "polynucleotide" may be used interchangeably herein, unless the context indicates otherwise, and refer to a polymer of nucleotides. These terms include, but are not limited to, DNA and RNA molecules, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and RNA, plasmid DNA, mRNA, antisense RNA, and RNA / DNA hybrids, any of which may be linear or branched, single-stranded or double-stranded, or a combination thereof. When dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and others may also be used for antisense, dsRNA, and ribozyme pairing interactions. For example, polynucleotides containing C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications may also be made, such as modifying the phosphodiester backbone or the 2'-hydroxyl group in the ribose moiety of RNA.

[0137] As used herein, "operably linked" or "operably associated" means that the indicated elements are functionally related to each other and are also generally physically related. Thus, as used herein, the terms "operably linked" or "operably associated" refer to nucleotide sequences on a single nucleic acid molecule that are functionally related. Thus, a first nucleotide sequence that is operably linked to a second nucleotide sequence refers to the situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For example, a promoter is operably associated with a nucleotide sequence if the promoter affects the transcription or expression of the nucleotide sequence. Those skilled in the art will understand that control sequences (e.g., promoters, introns, terminators, enhancers) need not be contiguous with the nucleotide sequence to which they are operably linked, so long as the control sequence is capable of performing its function of directing expression. Thus, for example, intervening untranslated, transcribed sequences may be present between a promoter and a nucleotide sequence, and the promoter may still be considered to be "operably linked to" or "operably associated with" the nucleotide sequence.

[0138] As used herein, the term "endogenous" refers to a substance that originates within an organism or cell. "Exogenous" refers to a substance that originates outside of an organism or cell. This typically applies to nucleic acid molecules used in the production of transformed or transgenic host cells and plants. For example, a nucleic acid molecule encoding a TIRA polypeptide or an active variant or fragment thereof, or a nucleic acid molecule encoding a TIRB polypeptide or an active variant or fragment thereof, or a nucleic acid molecule encoding two TIR polypeptides or a fusion protein comprising two TIR polypeptides is an exogenous nucleic acid for conferring or enhancing pathogen resistance in a plant cell transformed with the nucleic acid molecule.

[0139] As used herein, the terms "exotic", "exotic line" and "exotic germplasm" refer to any plant, line or germplasm that is not elite. Generally, exotic plants / germplasm are not derived from any known elite plants or germplasm, but are selected to introduce one or more desired genetic elements into a breeding program (e.g., to introduce novel alleles into a breeding program).

[0140] As used herein, the term "genome" when applied to a plant cell includes not only chromosomal DNA found within the nucleus, but also organelle DNA found within subcellular components of the cell.

[0141] The term "gene" means a nucleic acid comprising chromosomal DNA, genomic DNA, plasmid DNA, cDNA, artificial DNA polynucleotide or other DNA encoding a polypeptide of interest. In particular embodiments, the nucleic acid sequence of the gene encodes a protein that, when expressed, is at least partially responsible for a particular characteristic or trait. In embodiments, the gene can be natural, modified (e.g., by directed recombination or site-directed mutagenesis) or synthetic. In exemplary embodiments, the gene is transcribed in a cell into an RNA molecule (e.g., mRNA), where the RNA can encode a peptide, polypeptide or protein of interest and, in some instances, can also encode genetic elements flanking the coding sequence that are involved in regulating the expression of the mRNA or polypeptide of the invention. Thus, a gene can comprise several operably linked sequences, such as a promoter sequence, a 5′ leader sequence (including, for example, sequences involved in translation initiation), a (protein) coding region (comprising cDNA or genomic DNA), a 3′ untranslated sequence (comprising, for example, transcription termination sequence sites, introns (e.g., one or more natural, exotic or modified introns)). In exemplary embodiments, the nucleic acid sequence of an isolated gene can include introns, exons, 5′ or 3′-untranslated regions (UTRs) and natural regulatory elements (e.g., natural promoters). In other exemplary embodiments, the gene comprises the coding sequence of a polypeptide of interest but does not contain any regulatory elements. Thus, a nucleic acid encoding a TIRA polypeptide, a TIRB polypeptide or an active variant or fragment thereof can lack all natural or exotic / heterologous introns, can have one, two, three or more or all natural introns replaced by exotic or modified introns, or can have one or more natural regulatory elements (promoter, 5′UTR, 3′UTR and / or terminator) replaced by exotic or modified regulatory elements (promoter, 5′UTR, 3′UTR and / or terminator), or any combination thereof.

[0142] As used herein, "heterologous" with respect to a polypeptide or polynucleotide sequence is a sequence that is derived from a foreign species, or, if derived from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. Thus, a heterologous sequence is in a configuration not found in nature.

[0143] As used herein, the term "hybrid" refers to seeds and / or plants produced when at least two genetically distinct parents are crossed.

[0144] As used herein, the term "inbred" refers to a plant or variety that is substantially homozygous. The term can refer to a plant or variety that is substantially homozygous across the genome or a plant or plant variety that is substantially homozygous with respect to a particular genomic portion of interest.

[0145] As used herein, the terms "introgression", "introgressing", and "introgressed" refer to the natural and artificial transfer of desired alleles or combinations of desired alleles at one or more genetic loci from one genetic background to another. For example, desired alleles at a designated locus can be transmitted to at least one progeny by sexual crossing between two parents of the same species, where at least one of these parents has the desired allele within its genome. Alternatively, for example, the transfer of alleles can occur by recombination between two donor genomes, such as in fused protoplasts, where at least one donor protoplast has the desired allele within its genome. The desired alleles can be selected alleles that are tagged, QTLs, transgenes, etc. The progeny containing the desired alleles can be repeatedly backcrossed to a line having a desired genetic background and selected for the desired alleles, where the result is that the desired alleles become fixed in a desired genetic background. For example, a TIRA polypeptide, a TIRB polypeptide, two TIR polypeptides, a TIRATIRB fusion protein, or an active variant or fragment thereof, or a marker associated with enhanced ASR tolerance or resistance can be introgressed from a donor into a recurrent parent that does not have disease resistance. The resulting progeny can then be repeatedly backcrossed and selected until the progeny have one or more ASR tolerance alleles in the recurrent parent background.

[0146] As used herein, a "separated" nucleic acid molecule or gene is substantially separated from other nucleic acids or gene sequences that are normally associated with the nucleic acid, for example, separated from the chromosomal or extrachromosomal DNA of the cell in which the nucleic acid or gene naturally occurs. When a nucleic acid molecule contains a transgene or a part of a transgene present in the genome of another organism, it is a separated nucleic acid molecule. The term also includes nucleic acids that have been biochemically purified to substantially remove contaminating nucleic acids and other cellular components.

[0147] A polypeptide is referred to as "separated" if it is separated from the cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that naturally accompany it, or is chemically synthesized or recombinant. When a polypeptide molecule is expressed from a transgene in another organism, the polypeptide molecule is a separated polypeptide molecule. A monomeric polypeptide is separated when at least 60% by weight, preferably 90% by weight or more, more preferably 95% by weight or more, and most preferably more than 99% by weight of the sample consists of the monomeric polypeptide. Protein purity or homogeneity is indicated, for example, by polyacrylamide gel electrophoresis of a protein sample followed by visualization of individual polypeptide bands after staining of the polyacrylamide gel; high performance liquid chromatography; or other conventional methods. Proteins can be purified by any method known in the art, such as those described in Guide to Protein Purification, edited by Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer, New York, 1982.

[0148] A "locus" is the position on a chromosome where a gene, marker, or allele is located. In some embodiments, a locus can encompass one or more nucleotides.

[0149] "Non-naturally occurring legume or soybean variety" means any variety of legume or soybean that does not occur naturally in nature. "Non-naturally occurring legume or soybean variety" can be produced by any method known in the art, including but not limited to transforming legume or soybean plants or germplasm, transfecting legume or soybean plants or germplasm, and crossing naturally occurring legume or soybean varieties with non-naturally occurring soybean varieties. In some embodiments, a "non-naturally occurring legume or soybean variety" can contain one or more heterologous nucleotide sequences. In some embodiments, a "non-naturally occurring soybean variety" can contain a non-natural combination of two or more naturally occurring nucleotide sequences (i.e., two or more naturally occurring genes that do not occur naturally in the same soybean, such as genes not found in a soybean line (e.g., polynucleotides from a wild Glycine species)).

[0150] As used herein, the terms "phenotype", "phenotypic trait", or "trait" refer to one or more characteristics and / or manifestations of an organism. A phenotype is a manifestation that can be observed with the naked eye or by any other means of evaluation known in the art (e.g., microscopy, biochemical analysis, or electromechanical determination). In some cases, a phenotype or trait is directly controlled by a single gene or genetic locus, i.e., a "monogenic trait". In other cases, a phenotype or trait is the result of multiple genes.

[0151] As used herein, the term "plant" can refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, unless otherwise specified, the term "plant" can refer to any of the following: a whole plant, a plant component or organ (e.g., roots, stems, leaves, buds, flowers, pods, etc.), plant tissue, seeds, and / or plant cells. A plant cell is a plant cell obtained from a plant or a plant cell derived by culturing from a cell taken from a plant. Thus, the term "soybean plant" can refer to an entire soybean plant, one or more parts of a soybean plant (e.g., roots, root tips, stems, leaves, buds, flowers, pods, seeds, cotyledons, etc.), soybean plant cells, soybean plant protoplasts, and / or soybean plant callus.

[0152] A "plant cell" is the structural and physiological unit of a plant, containing a protoplast and a cell wall. A plant cell can be in the form of isolated single cells or cultured cells, or as part of a higher organizational unit such as, for example, a plant tissue, a plant organ, or an entire plant. In embodiments, the plant cells are non-reproductive and / or unable to regenerate an entire plant.

[0153] "Plant cell culture" means a culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0154] "Plant material" or "plant part" means a leaf, stem, root, flower or part of a flower, fruit, pollen, egg cell, zygote, seed, cutting, cell or tissue culture, or any other part or product of a plant. In an embodiment, the plant part may refer to an entire plant.

[0155] "Plant organ" is a distinct and visibly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0156] As used herein, the term "plant part" includes, but is not limited to, single cells and tissues from embryos, pollen, ovules, egg cells, seeds, leaves, flowers, flower parts, shoots, fruits, stems, stalks, roots, root tips, anthers, cuttings and seeds, fertilized eggs, anthers, buds, scions, rhizomes and / or plant cells (including intact plant cells in plants and / or parts of plants), plant protoplasts, plant tissues, plant cell tissue cultures, plant calluses, plant clumps, etc. In some embodiments, the plant part or plant cell can be regenerated into a plant, while in other embodiments, the plant part or plant cell cannot be regenerated into a plant.

[0157] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. It includes any plant tissue in a plant or in a culture. This term includes, but is not limited to: whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural or functional units. The combined or separate application of this term with any specific type of plant tissue listed above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[0158] "Plant pathogen" as used herein means a pathogen capable of infecting a plant and causing a plant disease. In an embodiment, the plant pathogen is a fungal plant pathogen. In a particular embodiment, the fungal pathogen is from the genus Phakopsora, including Phakopsora pachyrhizi and Phakopsora meibomiae species. These species are known to cause ASR in plants. In other particular embodiments, the plant pathogen is an ascomycete fungal pathogen that causes powdery mildew. In still other embodiments, the plant pathogen is a nematode, such as soybean cyst nematode (SCN) or root-knot nematode (RKN). In another embodiment, the plant pathogen is a bacterial plant pathogen such as Pseudomonas syringae pv. syringae. In still another embodiment, the plant pathogen is a sucking pest such as an aphid, stink bug or whitefly.

[0159] "Polyadenylation signal" or "polyA signal" refers to a nucleic acid sequence located at the 3' of a coding region, which causes the addition of adenylic acid nucleotides at the 3' end of the mRNA transcribed from the coding region.

[0160] "Polymerase Chain Reaction (PCR)" refers to a DNA amplification method that uses enzymatic techniques to create multiple copies of a nucleic acid sequence (amplicon). Copies of the DNA molecule are prepared by shuttling DNA polymerase between two amplicons. The basis of this amplification method is multiple cycles of temperature changes to denature, then re-anneal the amplicons (DNA primer molecules), followed by extension to synthesize new DNA strands in the region between the flanking amplicons. Nucleic acid amplification can be accomplished by any of a variety of nucleic acid amplification methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, in particular, in U.S. Patent Nos. 4,683,195 and 4,683,202 and PCR Protocols: A Guide to Methods and Applications, edited by Innis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify up to 22 kb of genomic DNA and up to 42 kb of phage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods, as well as other methods known in the field of DNA amplification, can be used in the practice of the present invention.

[0161] As used herein, the term "primer" refers to an oligonucleotide that can anneal to a nucleic acid target and serve as a starting point for DNA synthesis when placed under conditions that induce the synthesis of a primer extension product (e.g., in the presence of nucleotides and reagents for polymerization such as DNA polymerase and at suitable temperature and pH). For maximum efficiency in extension and / or amplification, in some embodiments, the primer (which is an extension primer in some embodiments and an amplification primer in some embodiments) is single-stranded. In some embodiments, the primer is an oligodeoxyribonucleotide. The primer is typically long enough to initiate the synthesis of an extension and / or amplification product in the presence of a reagent for polymerization. The minimum length of the primer can depend on a number of factors, including but not limited to the temperature and composition of the primer (A / T vs. G / C content). In the case of amplification primers, these amplification primers are typically provided as a pair of bidirectional primers consisting of one forward and one reverse primer, or as a pair of forward primers commonly used in the field of DNA amplification (such as in PCR amplification). Thus, it should be understood that as used herein, the term "primer" can refer to more than one primer, particularly in cases where there is some ambiguity in the information regarding one or more terminal sequences of the target region to be amplified. Thus, a "primer" can include a collection of primer oligonucleotides containing sequences representing possible variations in the sequence, or can include nucleotides that allow typical base pairing. Primers can be prepared by any suitable method known in the art. Methods for preparing oligonucleotides of specific sequences include, for example, cloning and restriction of the appropriate sequence and direct chemical synthesis. Chemical synthesis methods can include, for example, the phosphodiester or triester method, the diethylamino phosphate method, and the solid support method disclosed in U.S. Patent No. 4,458,066. If desired, the primer can be labeled by incorporating a detectable moiety, such as a spectroscopic moiety, a fluorescent moiety, a photochemical moiety, a biochemical moiety, an immunochemical moiety, or a chemical moiety. Primers can be created for the diagnosis of ASR resistance (i.e., capable of identification or selection based on the presence of an ASR resistance allele) for any advantageous SNP. PCR methods have been well described in manuals and are known to those skilled in the art. After PCR amplification, the target polynucleotide can be detected by hybridization to a probe polynucleotide that forms a stable hybrid with the target sequence under stringent to moderately stringent hybridization and washing conditions. If the probe is expected to be substantially completely complementary to the target sequence (i.e., about 99% or more), then stringent conditions can be used. If some mismatches are expected, for example, if a variant variety is expected to result in incomplete complementarity of the probe, then the stringency of hybridization can be reduced. In some embodiments, conditions are selected to exclude non-specific / fortuitous binding. Conditions affecting hybridization and conditions selected for non-specific binding are known in the art and are described, for example, in Sambrook and Russell (2001).Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. Generally, lower salt concentrations and higher temperatures for hybridization and / or washing increase the stringency of the hybridization conditions.

[0162] As used herein, the terms "progeny" and "progeny plant" refer to plants produced from one or more parent plants by asexual or sexual reproduction. Progeny plants can be obtained by cloning or selfing a single parent plant (i.e., the same plant serves as the donor of both male and female gametes) or by crossing two parent plants. The one or more progeny can be, for example, F1, F2, or any subsequent generation.

[0163] The term "promoter" or "promoter region" refers to a polynucleic acid molecule that functions as a regulatory element, typically found upstream (5') of a coding sequence, which controls the expression of the coding sequence by providing a recognition site for RNA polymerase and / or other factors necessary to initiate transcription at the correct site. As envisioned herein, the promoter or promoter region includes variations of the promoter derived by ligation to various regulatory sequences, random or controlled mutagenesis, and addition or duplication of enhancer sequences. The promoter regions disclosed herein and their biological function equivalents are responsible for driving the transcription of the coding sequence under their control when introduced into a host as part of a suitable recombinant DNA construct, as evidenced by their ability to produce mRNA. In some embodiments, the vector construct or expression construct or nucleic acid sequence disclosed herein contains a promoter that is heterologous to the nucleic acid sequence encoding the TIRA or TIRB polypeptide or TIRATIRB fusion protein or an active variant or fragment thereof. In other instances, the vector construct, expression construct, or nucleic acid sequence contains a promoter that is native or endogenous to the nucleic acid sequence encoding the TIRA or TIRB polypeptide, or an active variant or fragment thereof.

[0164] In still other instances, the vector construct, expression construct or nucleic acid sequence comprises a promoter that is native or endogenous to a nucleic acid sequence encoding an R protein that is different from the TIR polypeptide, or an active variant or fragment thereof. In certain embodiments, the vector construct, expression construct or nucleic acid sequence comprises a native bidirectional promoter derived from a genomic locus comprising the Rg32 and Rg34 genes (disclosed as SEQ ID NO:7 in U.S. Provisional Applications 6,342,652 and 6,350,958 and incorporated herein by reference in its entirety). The native bidirectional promoter is capable of driving gene expression in both directions, i.e., via the promoter sequence in the sense direction and the promoter sequence in the antisense direction or via reverse complementary sequences. In particular embodiments, the bidirectional promoter is operably coupled to each of a nucleic acid sequence encoding a TIRA polypeptide and a nucleic acid sequence encoding a TIRB polypeptide. In one particular exemplary embodiment, when the sequence is viewed from the sense direction, the nucleic acid sequence encoding the TIRA polypeptide is operably coupled downstream of the bidirectional promoter, while the nucleic acid sequence encoding the TIRB polypeptide is operably coupled upstream of the bidirectional promoter. In another particular exemplary embodiment, when the sequence is viewed from the sense direction, the nucleic acid sequence encoding the TIRA polypeptide is operably coupled upstream of the bidirectional promoter, while the nucleic acid sequence encoding the TIRB polypeptide is operably coupled upstream of the bidirectional promoter.

[0165] As used herein, the term "recombinant" refers to DNA, proteins, cells, seeds or organisms that are not naturally occurring and are the result of genetic engineering and thus are not typically found in nature. A "recombinant DNA molecule" is a DNA molecule that contains DNA sequences that are not naturally occurring in nature and is thus the result of human intervention, e.g., a DNA molecule composed of at least two DNA molecules that are heterologous to each other. An example of a recombinant DNA molecule is the DNA molecule provided herein encoding a TIRA and / or TIRB polypeptide, or a TIRATIRB fusion protein, or an active variant or fragment thereof, which is operably linked to heterologous regulatory elements, such as a heterologous promoter, a heterologous terminator or contains one or more heterologous introns or lacks one or more native introns. An example of a recombinant DNA molecule is the DNA molecule provided herein encoding a TIRA and / or TIRB polypeptide, or a TIRATIRB fusion protein, or an active variant or fragment thereof, which is operably linked to heterologous regulatory elements, such as a heterologous promoter, a heterologous terminator or contains one or more heterologous introns or lacks one or more native introns. A "recombinant protein" is a protein that contains an amino acid sequence that is not naturally occurring and is thus the result of human intervention, e.g., an engineered protein or a chimeric protein. A recombinant cell, seed or organism is a cell, seed or organism that contains transgenic DNA, e.g., a transgenic cell, seed, plant or plant part that contains a recombinant DNA molecule and is thus produced as a result of plant transformation.

[0166] In the context of two nucleic acid or two amino acid sequences, the phrase "substantially identical" refers to two or more sequences or subsequences that have at least about 50% nucleotide or amino acid residue identity (as measured using a sequence comparison algorithm or by visual inspection) when compared and aligned for maximum correspondence. In certain embodiments, substantially identical sequences have at least about 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity at the nucleotide or amino acid level. In certain embodiments, relative to a protein sequence or the nucleotide sequence encoding it, substantial identity exists in regions of the sequence of at least about 50 amino acid residues, 100 amino acid residues, 150 amino acid residues, 200 amino acid residues, 250 amino acid residues, 300 amino acid residues, 350 amino acid residues, 400 amino acid residues, 450 amino acid residues, 500 amino acid residues, 525 amino acid residues, 526 amino acid residues, 527 amino acid residues, 528 amino acid residues, 529 amino acid residues, 530 amino acid residues, 531 amino acid residues, 532 amino acid residues, 533 amino acid residues, 534 amino acid residues, 535 amino acid residues, 536 amino acid residues.

[0167] In the case of two nucleic acid or amino acid sequences, the terms "identity" or "identical" refer to the percentage of identical nucleotides or amino acids in the linear polynucleotide or amino acid sequence of a reference ("query") sequence (or its complementary strand) when the two sequences are aligned globally, as compared to a test ("subject") sequence. Unless otherwise stated, sequence identity as used herein refers to the value obtained using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) implemented in the EMBOSS Needle alignment tool, using the default matrix file EBLOSUM62 (for proteins) and default parameters (gap open = 10, gap extend = 0.5, end gap penalty = false, end gap open = 10, end gap extend = 0.5) or DNAfull (for nucleic acids) and default parameters (gap open = 10, gap extend = 0.5, end gap penalty = false, end gap open = 10, end gap extend = 0.5); or any equivalent program. EMBOSS Needle can be obtained, for example, from EMBL-EBI, such as at the website: ebi.ac.uk / Tools / psa / emboss_needle / and as described in the following publication: "The EMBL-EBI search and sequence analysis tools APIs in 2019. [EMBL-EBI Search and Sequence Analysis Tools APIs in 2019]" Madeira et al. Nucleic Acids Research [Nucleic Acids Research], June 2019, 47(W1):W636-W641. As used herein, the term "equivalent program" refers to any sequence comparison program that, for any two sequences under discussion, generates an alignment having the same nucleotide or amino acid residue matches and the same percentage of sequence identity as the corresponding alignment generated by EMBOSS Needle. In some embodiments, substantially identical nucleic acid or amino acid sequences can perform substantially the same function.

[0168] Two nucleotide sequences can also be considered to be substantially identical when they hybridize to each other under stringent conditions. In a representative embodiment, two nucleotide sequences that are considered to be substantially identical hybridize to each other under high stringency conditions.

[0169] The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a nucleic acid will hybridize to a target sequence selectively to a degree significantly higher than other sequences (e.g., at least 2-fold over non-target sequences), and optionally may substantially exclude binding to non-target sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target sequences that may be up to 100% complementary to a reference nucleotide sequence can be identified. Alternatively, conditions of medium or even low stringency may be used to allow some mismatches in the sequence, thereby detecting a lower degree of sequence similarity. For example, those skilled in the art will understand that in order to function as a primer or probe, a nucleic acid sequence only needs to be sufficiently complementary to a target sequence under the conditions employed to bind to it substantially, thereby forming a stable double-stranded structure. Thus, primers or probes can be used under high, medium or even low stringency conditions. Similarly, low or medium stringency conditions can be advantageous for detecting homolog, ortholog and / or paralog sequences with a degree of sequence identity lower than that which can be identified under high stringency conditions.

[0170] As used herein, the terms "complementary" or "complementarity" (and like terms) refer to the natural binding of polynucleotides through base pairing under permissive salt and temperature conditions. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be partial, where only some of the nucleotides bind, or it may be complete when there is full complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between the molecules. As used herein, the term "substantially complementary" (and like terms) means that two nucleic acid sequences are at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more complementary. Alternatively, the term "substantially complementary" (and like terms) may mean that two nucleic acid sequences can hybridize together under high stringency conditions as described herein.

[0171] As used herein, "specifically" or "selectively" hybridize (and like terms) refers to the binding, duplexing or hybridization of a molecule to a specific nucleic acid target sequence under stringent conditions (when the sequence is present in a complex mixture such as total cellular DNA or RNA), to substantially exclude non-target nucleic acids, or even to have no detectable binding, duplexing or hybridization with non-target sequences. Specifically or selectively hybridizing sequences are typically at least about 40% complementary, and optionally are substantially complementary or even fully complementary (i.e., 100% identical).

[0172] For DNA-DNA hybrids, T mcan be estimated from the equation of Meinkoth and Wahl, Anal. Biochem. [Analytical Biochemistry] 138:267-84 (1984): T m = 81.5 °C + 16.6 (log M) + 0.41 (GC%) - 0.61 (formamide%) - 500 / L; where M is the molarity of monovalent cations, GC% is the percentage of guanosine and cytosine nucleotides in the DNA, formamide% is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T m is the temperature at which 50% of the complementary target sequence hybridizes to a perfectly matched probe (at defined ionic strength and pH). For every 1% mismatch, T m is reduced by approximately 1 °C; thus, T m , hybridization, and / or wash conditions can be adjusted to hybridize to sequences with a desired degree of identity. For example, if a sequence with >90% identity is sought, T m can be reduced by 10 °C. Typically, stringent conditions are selected to be about 5 °C lower than the thermal melting point (T m ) of the particular sequence and its complement at defined ionic strength and pH. However, high stringency conditions can be utilized at the thermal melting point (T m ) or 1 °C, 2 °C, 3 °C, or 4 °C lower than the thermal melting point (T m ) for hybridization and / or washing; moderate stringency conditions can be utilized at 6 °C, 7 °C, 8 °C, 9 °C, or 10 °C lower than the thermal melting point (T m ) for hybridization and / or washing; low stringency conditions can be utilized at 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, or 20 °C lower than the thermal melting point (T m ) for hybridization and / or washing. If the desired degree of mismatch results in T mIf below 45 °C (aqueous solution) or 32 °C (formamide solution), the SSC concentration can optionally be increased so that higher temperatures can be used. Extensive guidance on nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993); Current Protocols in Molecular Biology, Chapter 2, edited by Ausubel et al., Greene Publishing and Wiley-Interscience, New York (1995); and Green and Sambrook, In: Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012).

[0173] Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M Na ions at about pH 7.0 to pH 8.3, typically about 0.01 M to 1.0 M Na ion concentration (or other salts), and the temperature is at least about 30 °C (for short probes, e.g., 10 to 50 nucleotides) and at least about 60 °C (for long probes, e.g., greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizers such as formamide or Denhardt's (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml water). Exemplary low stringency conditions include hybridization in a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 °C and washing in 1X to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50 °C to 55 °C. Exemplary medium stringency conditions include hybridization in 40% to 45% formamide, 1 M NaCl, 1% SDS at 37 °C and washing in 0.5X to 1X SSC at 55 °C to 60 °C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 °C and washing in 0.1X SSC at 60 °C to 65 °C. Another non-limiting example of high stringency conditions includes hybridization in 4X SSC, 5X Denhardt's, 0.1 mg / ml boiled salmon sperm DNA and 25 mM sodium phosphate at 65 °C and washing in 0.1X SSC, 0.1% SDS at 65 °C. Another illustration of high stringency hybridization conditions includes hybridization in 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50 °C and washing in 2X SSC, 0.1% SDS at 50 °C, alternatively washing in 1X SSC, 0.1% SDS at 50 °C, alternatively washing in 0.5X SSC, 0.1% SDS at 50 °C, or alternatively washing in 0.1X SSC, 0.1% SDS at 50 °C, or even washing in 0.1X SSC, 0.1% SDS at 65 °C. Those skilled in the art will understand that specificity typically depends on the washing after hybridization, and the relevant factors are the ionic strength and temperature of the final wash solution.

[0174] If the proteins encoded by nucleic acids that do not hybridize to each other under stringent conditions are substantially the same, then they are still substantially the same (e.g., due to the degeneracy of the genetic code).

[0175] Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid cross-reacts immunologically with the protein encoded by the second nucleic acid. Thus, one protein is typically substantially identical to a second protein, for example, where the difference between the two proteins is only a conservative substitution.

[0176] As used herein, the term "transgene" refers to a DNA molecule that has been artificially incorporated into the genome of an organism due to human intervention (such as a plant transformation method). As used herein, the term "transgene" means a transgene-containing entity, for example, a "transgenic plant" refers to a plant that contains a transgene in its genome, and a "transgenic trait" refers to a characteristic or phenotype that is conferred or conveyed by the presence of a transgene integrated into the plant genome. Due to this genomic alteration, transgenic plants are significantly different from the related wild-type plants, and transgenic traits are traits that do not naturally occur in wild-type plants. Transgenic plants can contain the recombinant DNA molecules and engineered proteins provided herein.

[0177] As used herein, the term "transgenic" and its grammatical variants refer to a plant in which a heterologous nucleic acid has been integrated into the genome, including any part derived from a plant, such as a cell, tissue, or organ. In certain embodiments, the heterologous nucleic acid is a recombinant construct, vector, or expression cassette containing one or more nucleic acids.

[0178] The term "vector" refers to a composition for transferring, delivering, or introducing one or more nucleic acids into a cell. A vector contains a nucleic acid molecule that contains one or more nucleotide sequences to be transferred, delivered, or introduced.

[0179] 2. Polynucleotides and polypeptides conferring increased disease resistance, and compositions thereof

[0180] Compositions, polypeptides, fusion proteins, polynucleotides, and active fragments and variants thereof that confer increased disease resistance are provided.

[0181] I. TIRA polypeptides and polynucleotides encoding TIRA polypeptides

[0182] TIRA polypeptides comprising SEQ ID NO:1 or an active fragment or variant of SEQ ID NO:1 are provided. Further provided are polynucleotides comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:1 or an active variant or fragment thereof; and polynucleotides comprising any one of SEQ ID NOs: 3-5 or an active fragment or variant thereof.

[0183] The TIRA polypeptide (SEQ ID NO:1) encodes a resistance protein that is 340 aa in length (also referred to herein as the R protein). The TIRA polypeptide (SEQ ID NO:1) contains several conserved domains, including two Toll / interleukin-1 receptor (TIR) domains (pfam accession number: 01582). The two conserved TIR domains of the TIRA polypeptide form a dumbbell structure, in which the first conserved TIR domain (referred to herein as the TIRA1 domain (amino acids 12 to 174 of SEQ ID NO:1)) is separated from the second conserved TIR domain (referred to herein as the TIRA2 domain (amino acids 183 - 339 of SEQ ID NO:1)). The TIRA1 domain is separated from the TIRA2 domain by approximately 10 amino acids. The positions of the conserved TIR domains (i.e., the TIRA1 and TIRA2 domains) within the TIRA polypeptide are listed in Table 1.

[0184] The TIR domains of some R proteins are known to function as NAD+ lyases, thereby triggering local cell death, also known as the hypersensitive response (see PNAS [Proceedings of the National Academy of Sciences of the United States of America] (2017) 114(10) E2053 - 2062; and Science 2019 August: 799 - 803). The TIRA1 domain of the TIRA polypeptide of SEQ ID NO:1 has NADase activity. As used herein, NADase activity means the ability of a polypeptide or its active fragment or variant or its domain to cleave the metabolic cofactor nicotinamide adenine dinucleotide (NAD+) into nicotinamide (Nam) and ADP - ribose (ADPR). In particular embodiments, the TIRA1 domain of the TIRA polypeptide (SEQ ID NO:1) or its active variant or fragment contains a conserved glutamate residue (e.g., amino acid 85 of SEQ ID NO:1 or its corresponding position), which is at least partially responsible for the catalytic cleavage that confers NADase activity to the TIRA1 domain. In particular embodiments, the TIRA2 domain of the TIRA polypeptide (SEQ ID NO:1) or its active variant or fragment contains a non - glutamate residue, such as a valine residue (e.g., amino acid 251 of SEQ ID NO:1 or its corresponding position), and thus the TIRA2 domain does not have NADase activity. In an embodiment, for example, the non - glutamate residue can be mutated to a glutamate residue to confer NADase activity to the TIRA2 domain. In other embodiments, a variant of the TIRA polypeptide (SEQ ID NO:1) contains a glutamate residue (e.g., at amino acid 251 of SEQ ID NO:1 or its corresponding position), which is responsible for the catalytic cleavage that confers NADase activity to the TIRA2 domain of the variant.

[0185] Provided are polynucleotides comprising a coding sequence encoding a TIRA polypeptide (SEQ ID NO:1) or an active variant or fragment thereof. In particular embodiments, the polynucleotide encoding a TIRA polypeptide (SEQ ID NO:1) or an active variant or fragment thereof has a coding sequence comprising or derived from the genomic sequence of the TirA gene, such as a polynucleotide comprising SEQ ID NO:3 or 5 or an active fragment or variant thereof. In other specific embodiments, the polynucleotide encoding a TIRA polypeptide (SEQ ID NO:1) or an active variant or fragment thereof has a coding sequence comprising or derived from the cDNA sequence of the TirA gene, such as a polynucleotide comprising SEQ ID NO:4 or an active fragment or variant thereof.

[0186] When co-expressed with a TIRB polypeptide (SEQ ID NO:2) or an active fragment or variant of the TIRB polypeptide, the TIRA polypeptide of SEQ ID NO:1 or an active fragment or variant of SEQ ID NO:1 confers disease resistance to plants, plant parts or seeds (such as leguminous plants, leguminous plant parts or leguminous seeds). In particular embodiments, when co-expressed with the TIRB polypeptide of SEQ ID NO:2 or an active fragment or variant of SEQ ID NO:2, the expression of the TIRA polypeptide of SEQ ID NO:1 or an active fragment or variant of SEQ ID NO:1 confers ASR and / or powdery mildew resistance to soybean plants, plant parts or seeds. In other particular embodiments, when co-expressed with the TIRB polypeptide of SEQ ID NO:2 or an active fragment or variant of SEQ ID NO:2, the expression of the TIRA polypeptide of SEQ ID NO:1 or an active fragment or variant of SEQ ID NO:1 confers increased nematode resistance (such as root knot nematode and / or soybean cyst nematode resistance), bacterial pathogen resistance (such as resistance to Pseudomonas syringae pv. syringae) and / or sucking pest resistance to soybean plants, plant parts or seeds.

[0187] Similarly, when co-expressed with a polynucleotide encoding a TIRB polypeptide of SEQ ID NO:2 or an active fragment or variant of SEQ ID NO:2 (e.g., a polynucleotide comprising any one of SEQ ID NO:6-8), a polynucleotide encoding a TIRA polypeptide of SEQ ID NO:1 or an active fragment or variant of SEQ ID NO:1 (e.g., a polynucleotide comprising any one of SEQ ID NO:3-5 or an active fragment or variant thereof) confers disease resistance to a plant, plant part or seed (e.g., a leguminous plant, leguminous plant part or leguminous seed). In particular embodiments, when co-expressed or co-transcribed with any one of SEQ ID NO:6-8 or a polynucleotide of an active fragment or variant of any one of SEQ ID NO:6-8, transcription and expression of any one of SEQ ID NO:3-5 or an active fragment or variant of any one of SEQ ID NO:3-5 confers ASR and / or powdery mildew resistance to a soybean plant, plant part or seed. In other particular embodiments, when co-expressed or co-transcribed with any one of SEQ ID NO:6-8 or a polynucleotide of an active fragment or variant of any one of SEQ ID NO:6-8, transcription and expression of any one of SEQ ID NO:3-5 or an active fragment or variant of any one of SEQ ID NO:3-5 confers increased nematode resistance (e.g., root-knot nematode and / or soybean cyst nematode resistance), bacterial pathogen resistance (e.g., resistance to Pseudomonas syringae pv. syringae) and / or sucking pest resistance to a soybean plant, plant part or seed.

[0188] As described below, when co-expressed with a polynucleotide encoding a TIRB polypeptide of SEQ ID NO:2 or an active fragment or variant of SEQ ID NO:2, active fragments and variants of the TIRA polypeptide of SEQ ID NO:1 include polypeptides comprising one or more mutations, truncations, insertions and / or deletions to SEQ ID NO:1, retaining the ability of the polypeptide to confer disease resistance to a plant, plant part or seed. In particular embodiments, the active variant or fragment may comprise one or more mutations to the NAD enzyme region of the TIRA polypeptide, e.g., one or more mutations in the TIRA1 domain and / or the TIRA2 domain, which results in a decrease in the NAD enzyme activity of the polypeptide while still retaining the ability of the polypeptide to confer disease resistance to a plant, plant part or seed when co-expressed with the TIRB polypeptide.

[0189] II. TIRB Polypeptides and Polynucleotides Encoding TIRB Polypeptides

[0190] Provided are TIRB polypeptides comprising SEQ ID NO:2 or an active fragment or variant thereof. Further provided are polynucleotides comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO:2 or an active variant or fragment thereof; and polynucleotides comprising any one of SEQ ID NOs:6-8 or an active fragment or variant thereof.

[0191] The TIRB polypeptide (SEQ ID NO:2) encodes a resistance protein (also referred to herein as the R protein) that is 367 aa in length. The TIRB polypeptide (SEQ ID NO:2) contains several conserved domains, which include two Toll / interleukin-1 receptor (TIR) domains (pfam accession number: 01582). The two conserved TIR domains of the TIRB polypeptide form a dumbbell structure, wherein the first conserved TIR domain (referred to herein as the TIRB1 domain (amino acids 8 to 174 of SEQ ID NO:2)) is separated from the second conserved TIR domain (referred to herein as the TIRB2 domain (amino acids 183-344 of SEQ ID NO:2)). The TIRB1 domain is separated from the TIRB2 domain by approximately 10 amino acids. The positions of the conserved TIR domains (i.e., the TIRB1 and TIRB2 domains) within the TIRB polypeptide are detailed in Table 1.

[0192] The TIR domains of the R protein are known to function as NAD+ lyases, thereby triggering local cell death, also known as the hypersensitive response (see PNAS [Proceedings of the National Academy of Sciences of the United States of America] (2017) 114(10) E2053-2062; and Science 2019 August: 799-803). Each of the TIRB1 and TIRB2 domains has NADase activity. As used herein, NADase activity means the ability of a polypeptide or an active fragment or variant thereof or its domain to cleave the metabolic cofactor nicotinamide adenine dinucleotide (NAD+) into nicotinamide (Nam) and ADP-ribose (ADPR). In particular embodiments, the TIRB1 domain of the TIRB polypeptide (SEQ ID NO:2) or an active variant or fragment thereof contains a conserved glutamate residue (e.g., amino acid 87 of SEQ ID NO:2 or its corresponding position), which is at least partially responsible for the catalytic cleavage conferring NADase activity to the TIRB1 domain. Similarly, the TIRB2 domain of the TIRB polypeptide (SEQ ID NO:2) or an active variant or fragment thereof contains a conserved glutamate residue (e.g., amino acid 257 of SEQ ID NO:2 or its corresponding position), which is at least partially responsible for the catalytic cleavage conferring NADase activity to the TIRB2 domain.

[0193] Provided are polynucleotides comprising a coding sequence encoding a TIRB polypeptide (SEQ ID NO:2) or an active variant or fragment thereof. In particular embodiments, the polynucleotide encoding a TIRB polypeptide (SEQ ID NO:2) or an active variant or fragment thereof has a coding sequence that comprises or is derived from the genomic sequence of the TirB gene, such as a polynucleotide comprising any one of SEQ ID NO:6 and 8 or an active fragment or variant thereof. In other specific embodiments, the polynucleotide encoding a TIRB polypeptide (SEQ ID NO:2) or an active variant or fragment thereof has a coding sequence that comprises or is derived from the cDNA sequence of the TirB gene, such as a polynucleotide comprising SEQ ID NO:7 or an active fragment or variant thereof.

[0194] When co-expressed with a TIRA polypeptide (SEQ ID NO:1) or an active fragment or variant of a TIRA polypeptide, the TIRB polypeptide of SEQ ID NO:2 or an active fragment or variant of SEQ ID NO:2 confers disease resistance to a plant, a plant part, or a seed (such as a leguminous plant, a leguminous plant part, or a leguminous seed). In particular embodiments, when co-expressed with the TIRA polypeptide of SEQ ID NO:1 or an active fragment or variant of SEQ ID NO:1, the expression of the TIRB polypeptide of SEQ ID NO:2 or an active fragment or variant of SEQ ID NO:2 confers ASR and / or powdery mildew resistance to a soybean plant, a plant part, or a seed. In other particular embodiments, when co-expressed with the TIRA polypeptide of SEQ ID NO:1 or an active fragment or variant of SEQ ID NO:1, the expression of the TIRB polypeptide of SEQ ID NO:2 or an active fragment or variant of SEQ ID NO:2 confers increased nematode resistance (such as root-knot nematode and / or soybean cyst nematode resistance), bacterial pathogen resistance (such as resistance to Pseudomonas syringae pv. syringae), and / or sucking pest resistance to a soybean plant, a plant part, or a seed.

[0195] Similarly, when co-expressed with a polynucleotide encoding the TIRA polypeptide of SEQ ID NO:1 or an active fragment or variant of SEQ ID NO:1 (e.g., a polynucleotide comprising any one of SEQ ID NOs: 3-5), a polynucleotide encoding the TIRB polypeptide of SEQ ID NO:2 or an active fragment or variant of SEQ ID NO:2 (e.g., a polynucleotide comprising any one of SEQ ID NOs: 6-8 or an active fragment or variant thereof) confers disease resistance to a plant, plant part or seed (e.g., a leguminous plant, leguminous plant part or leguminous seed). In particular embodiments, when co-expressed or co-transcribed with a polynucleotide of any one of SEQ ID NOs: 3-5 or an active fragment or variant of any one of SEQ ID NOs: 3-5, transcription and expression of a polynucleotide of any one of SEQ ID NOs: 6-8 or an active fragment or variant of any one of SEQ ID NOs: 6-8 confers ASR and / or powdery mildew resistance to a soybean plant, plant part or seed. In other particular embodiments, when co-expressed or co-transcribed with a polynucleotide of any one of SEQ ID NOs: 3-5 or an active fragment or variant of any one of SEQ ID NOs: 3-5, transcription and expression of a polynucleotide of any one of SEQ ID NOs: 6-8 or an active fragment or variant of any one of SEQ ID NOs: 6-8 confers increased nematode resistance (e.g., root-knot nematode and / or soybean cyst nematode resistance), bacterial pathogen resistance (e.g., resistance to Pseudomonas syringae pv. syringae) and / or sucking pest resistance to a soybean plant, plant part or seed.

[0196] As described below, when co-expressed with a polynucleotide encoding the TIRA polypeptide of SEQ ID NO:1 or an active fragment or variant of SEQ ID NO:1, active fragments and variants of the TIRB polypeptide of SEQ ID NO:2 include polypeptides comprising one or more mutations, truncations, insertions and / or deletions to SEQ ID NO:2, retaining the ability of the polypeptide to confer disease resistance to a plant, plant part or seed. In particular embodiments, the active variant or fragment may comprise one or more mutations to the NADase region of the TIRB polypeptide, e.g., one or more mutations in the TIRB1 domain and / or the TIRB2 domain, which results in a decrease in the NADase activity of the polypeptide while still retaining the ability of the polypeptide to confer disease resistance to a plant, plant part or seed when co-expressed with the TIRA polypeptide.

[0197] The functional domains of the TIRA and TIRB polypeptides are described and summarized in Table 1 below.

[0198] An example of an algorithm suitable for identifying conserved domains (such as the PFAM domains listed in Table 1) is the Conserved Domain Algorithm and related databases, which are described in: Marchler-Bauer et al. (Nucleic Acids Res. [Nucleic Acids Research] (2015) 43(D) 222-226; Nucleic Acids Res. [Nucleic Acids Research] (2017) 45(D) 200-203) and Shennan Lu et al. (Nucleic Acids Res. [Nucleic Acids Research] (2020) 48(D1) 265-268). Software for identifying conserved domains is available to the public through the National Center for Biotechnology Information, U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, USA.

[0199] Table 1A: Functional annotations of TIRA and TIRB polypeptides

[0200]

[0201] III. Fragments and variants of TIR polypeptides and polynucleotides

[0202] Also provided are active fragments and variants of the TIRA polypeptide (SEQ ID NO:1) and / or active fragments or variants of the TIRB polypeptide (SEQ ID NO:2). Further provided are polynucleotides comprising a nucleotide sequence encoding an active fragment or variant of the polypeptide of either SEQ ID NO:1 or 2; and polynucleotides comprising any active variant or fragment of any of SEQ ID NOs: 3-8.

[0203] In certain embodiments, the polynucleotide sequences (SEQ ID NOs: 3-5) and polypeptide sequences (SEQ ID NO: 1) of TIRA, and their active variants and fragments, increase disease resistance in plants when co-expressed with the polynucleotide sequences (SEQ ID NOs: 6-8) and polypeptide sequences (SEQ ID NO: 2) of TIRB, and their active variants and fragments, in plants, plant parts, or seeds. In particular embodiments, when compared to appropriate control plants, the polynucleotide sequences (SEQ ID NOs: 3-5) and polypeptide sequences (SEQ ID NO: 1) of the TIRA polypeptide and its active variants or fragments, and the polynucleotide sequences (SEQ ID NOs: 6-8) and polypeptide sequences (SEQ ID NO: 2) of the TIRB polypeptide and its active variants or fragments, increase disease resistance in plants when co-expressed in plants, plant parts, or seeds. Various methods by which such increased disease resistance can be measured are provided in the examples and discussed elsewhere herein.

[0204] Fragments of the TIRA polypeptide that increase disease resistance when expressed in plants, plants parts, or seeds include TIRA polypeptide fragments that are shorter than the full-length sequence and can include truncations or internal deletions at the N- or C-terminus. Active fragments of the TIRA polypeptide can be, for example, polypeptides of SEQ ID NO: 1 that are 10, 25, 50, 100, 150, 200, 250 or more amino acids in length when expressed in plants. Such bioactive portions can be prepared by recombinant techniques and evaluated for activity to confer increased resistance when co-expressed with the TIRB polypeptide or its active variants or fragments. As used herein, a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 1. Examples of active fragments of the TIRA polypeptide include TIRA polypeptide fragments having increased NADase activity. Such fragments will have increased NADase activity while retaining the ability of the TIRA polypeptide to (i) elicit an immune response when expressed in plants, and (ii) increase disease resistance in plants, plant parts, or seeds when co-expressed with the TIRB polypeptide or its active fragments or variants. Still other examples of active fragments of the TIRA polypeptide include TIRA polypeptide fragments having decreased NADase activity. Such fragments will have decreased NADase activity while retaining the ability of the TIRA polypeptide to (i) elicit an immune response when expressed in plants, and (ii) increase disease resistance in plants, plant parts, or seeds when co-expressed with the TIRB polypeptide or its active fragments or variants.

[0205] Fragments of the TIRB polypeptide that increase disease resistance when expressed in plants, plant parts, or seeds include TIRB polypeptide fragments that are shorter than the full-length sequence and can include truncations or internal deletions at the N- or C-terminus. Active fragments of the TIRB polypeptide can be, for example, polypeptides of lengths 10, 25, 50, 100, 150, 200, 250, or more amino acids of SEQ ID NO:2 when expressed in plants. Such bioactive portions can be prepared by recombinant techniques and evaluated for activity that confers increased resistance when co-expressed with the TIRA polypeptide or its active variants or fragments. As used herein, a fragment comprises at least 8 contiguous amino acids of SEQ ID NO:2. Examples of active fragments of the TIRB polypeptide include TIRB polypeptide fragments having NADase activity. Still other examples of active fragments of the TIRB polypeptide include TIRB polypeptide fragments having reduced NADase activity. Such fragments will have reduced NADase activity while retaining the ability of the TIRB polypeptide to (i) elicit an immune response when expressed in plants, and (ii) increase disease resistance in plants when co-expressed with the TIRA polypeptide or its active fragments or variants in plants, plant parts, or seeds.

[0206] Variant TIRA polypeptides comprise an amino acid sequence having at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the amino acid sequence of SEQ ID NO:1. Such active variants will increase disease resistance in plants when co-expressed with the TIRB polypeptide or its active variants or fragments in plants, plant parts, or seeds.

[0207] In some embodiments, variant polypeptides comprise a deletion and / or addition of one or more amino acids at one or more internal sites within a native polypeptide and / or a substitution (e.g., conservative substitution) of one or more amino acids at one or more sites of a native polypeptide. Particular embodiments of variants of the TIRA polypeptide include TIRA polypeptide variants having one or more mutations at one or more NADase sites in the TIRA polypeptide. As used herein, a "NADase site" refers to one or more positions within a polypeptide (e.g., within the conserved TIR domain of a TIR polypeptide (e.g., SEQ ID NO:1 or 2)) that are responsible for the NADase function of the polypeptide. In one exemplary embodiment, the variant TIRA polypeptide comprises a mutation at the NADase site of the TIRA1 and / or TIRA2 domain (e.g., at the position corresponding to position 85 of SEQ ID NO:1 and / or at the position corresponding to position 251 of SEQ ID NO:1). In particular embodiments, the variant includes a single glutamate-to-alanine mutation in the TIRA1 domain at the position corresponding to position 85 of SEQ ID NO:1 (also referred to herein as E85A) or a single valine-to-glutamate mutation in the TIRA2 domain at the position corresponding to position 251 of SEQ ID NO:1 (also referred to herein as V251E). In other particular embodiments, the variant includes a double mutation of a glutamate-to-alanine mutation in the TIRA1 domain at the position corresponding to position 85 of SEQ ID NO:1 and a valine-to-glutamate mutation in the TIRA2 domain at the position corresponding to position 251 of SEQ ID NO:1 (also referred to herein as the E85A+V251E mutant). Such variants will have altered NADase activity (e.g., loss-of-function mutations result in decreased NADase activity, gain-of-function mutations result in increased NADase activity), while retaining the ability of the TIRA polypeptide to (i) elicit an immune response when expressed in a plant, and (ii) enhance plant disease resistance when co-expressed with a TIRB polypeptide or an active fragment or variant thereof in a plant, a plant part, or a seed.

[0208] Variant TIRB polypeptides comprise an amino acid sequence having at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the amino acid sequence of SEQ ID NO:2. Such active variants will increase plant disease resistance when co-expressed with a TIRA polypeptide or an active variant or fragment thereof in a plant, a plant part, or a seed.

[0209] In some embodiments, the variant polypeptide comprises a deletion and / or addition of one or more amino acids at one or more internal sites within the native polypeptide and / or a substitution (e.g., conservative substitution) of one or more amino acids at one or more sites of the native polypeptide. In an exemplary embodiment, the variant TIRA polypeptide comprises a deletion and / or addition of one or more amino acids at one or more internal sites within the native polypeptide of SEQ ID NO:1 and / or a substitution (e.g., conservative substitution) of one or more amino acids at one or more sites of the native polypeptide of SEQ ID NO:1. Similarly, the variant TIRB polypeptide comprises a deletion and / or addition of one or more amino acids at one or more internal sites within the native polypeptide of SEQ ID NO:2 and / or a substitution (e.g., conservative substitution) of one or more amino acids at one or more sites of the native polypeptide of SEQ ID NO:2.

[0210] Particular embodiments of variants of the TIRB polypeptide include TIRB polypeptide variants having one or more mutations at one or more NAD enzymatic sites of the TIRB polypeptide. In one exemplary embodiment, the variant TIRB polypeptide comprises a loss-of-function mutation in the NAD enzymatic site of the TIRB1 domain, e.g., at the position corresponding to position 87 of SEQ ID NO:2. In a particular embodiment, the variant TIRB polypeptide comprises a glutamate-to-alanine mutation (also referred to herein as E87A) in the TIRB1 domain at the position corresponding to position 87 of SEQ ID NO:2. In particular embodiments, the variant TIRB polypeptide comprises a loss-of-function mutation of the NAD enzymatic site of the TIRB1 domain while retaining the functional NAD enzymatic site of the TIRB2 domain, e.g., a glutamate-to-alanine mutation (also referred to herein as E87A) at the position corresponding to position 87 of SEQ ID NO:2 in the TIRB1 domain, while retaining the glutamate residue at the position corresponding to position 257 of SEQ ID NO:2 in the TIRB2 domain. Such variants will have reduced NAD enzymatic activity while retaining the ability of the TIRB polypeptide to (i) elicit an immune response when expressed in plants, and (ii) increase plant disease resistance when co-expressed with the TIRA polypeptide or its active fragment or variant in plants, plant parts, or seeds.

[0211] In other cases, the TIRA polypeptide variant and / or the TIRB polypeptide variant comprises a tag, such as a His tag. In still other cases, the polypeptide variant comprises a detectable label, such as a detectable peptide label.

[0212] In some embodiments, variant TIRA polypeptides include annotated variants of the TIRA polypeptide that contain a different (e.g., more or fewer) number of amino acids relative to the polypeptide of SEQ ID NO:1. These annotated variants may arise from annotation of the TirA gene sequence, genomic sequence, or the translated gene sequence or genomic sequence using an alternative start codon. In an exemplary embodiment, annotation of the translated gene sequence with a relatively upstream start codon results in a variant having an additional number of amino acids, while annotation of the translated gene sequence with a relatively downstream start codon results in a variant having a fewer number of amino acids. In an embodiment, an annotated variant of SEQ ID NO:1 can contain an amino acid sequence having at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence of SEQ ID NO:1.

[0213] In some embodiments, variant TIRB polypeptides include annotated variants of the TIRB polypeptide that contain a different (e.g., more or fewer) number of amino acids relative to the polypeptide of SEQ ID NO:2. These annotated variants may arise from annotation of the TirB gene sequence, genomic sequence, or the translated gene sequence or genomic sequence using an alternative start codon. In an exemplary embodiment, annotation of the translated gene sequence with a relatively upstream start codon results in a variant having an additional number of amino acids, while annotation of the translated gene sequence with a relatively downstream start codon results in a variant having a fewer number of amino acids. In an embodiment, an annotated variant of SEQ ID NO:2 can contain an amino acid sequence having at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 9%1, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence of SEQ ID NO:2.

[0214] In still other embodiments, variant TIRA polypeptides include alternative splice variants of the TIRA polypeptide (or simply "splice variants") that contain a different (e.g., more or fewer) number of amino acids relative to the polypeptide of SEQ ID NO:1, and / or contain one or more substitutions relative to the polypeptide of SEQ ID NO:1. These splice variants may occur due to alternative splicing of exons and introns of the TirA gene or genomic sequence, resulting in variant mRNA transcripts that, upon expression, form variant protein sequences. In an embodiment, a splice variant of SEQ ID NO:1 may contain an amino acid sequence having at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the amino acid sequence of SEQ ID NO:1.

[0215] In still other embodiments, variant TIRB polypeptides include alternative splice variants of the TIRB polypeptide (or simply "splice variants") that contain a different (e.g., more or fewer) number of amino acids relative to the polypeptide of SEQ ID NO:2, and / or contain one or more substitutions relative to the polypeptide of SEQ ID NO:2. These splice variants may occur due to alternative splicing of exons and introns of the TirB gene or genomic sequence, resulting in variant mRNA transcripts that, upon expression, form variant protein sequences. In an embodiment, a splice variant of SEQ ID NO:2 may contain an amino acid sequence having at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the amino acid sequence of SEQ ID NO:2.

[0216] Fragments and variants of the nucleotide sequence can encode protein fragments that retain the biological activity of the native protein and have the ability to increase disease resistance. Alternatively, fragments or variants of the nucleotide sequence that are used as hybridization probes or in recombinant DNA constructs for gene editing do not necessarily encode protein fragments that retain biological activity. Thus, fragments of the nucleotide sequence can range from at least about 15, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 750, 900, 950, 1000 nucleotides, or less than the full-length nucleotide sequence encoding the proteins disclosed herein (i.e., any of SEQ ID NOs: 3-5 and 6-8).

[0217] Variants of the nucleotide sequence have at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to any one of the nucleotide sequences of SEQ ID NOs: 3-5 and 6-8. In certain embodiments, the variant nucleotide sequence encodes an active polypeptide of the invention. In other embodiments, the variant polynucleotide need not encode an active variant polypeptide and can be used as a component of a gene editing construct or as a probe or primer or for other tools for generating the plants and seeds provided herein.

[0218] In some embodiments, fragments and variants of the polypeptides disclosed herein each comprise one or more conserved domains of the canonical polypeptide. In some embodiments, an active variant or fragment can comprise a polypeptide having at least 40%, 50%, 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to one or more conserved domains in the canonical polypeptide sequence.

[0219] In one example, variants or fragments of the TIRA polypeptide (SEQ ID NO: 1) can comprise one or more of the conserved TIRA1 domain (aa 12 to 174 of SEQ ID NO: 1) and the TIRA2 domain (aa 183 to 339 of SEQ ID NO: 1). For example, active variants are provided wherein the amino acid sequence shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the full length of SEQ ID NO: 1 and further comprises a region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the consensus sequence of the PFAM domain of a given SEQ ID as shown in Table 1A. In other embodiments, active variants are provided wherein the amino acid sequence shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the full length of SEQ ID NO: 1 and further comprises a region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding region of SEQ ID NO: 1, which corresponds to the amino acid positions of the consensus sequence of the PFAM domain of the SEQ ID as shown in Table 1A.

[0220] In one example, a variant or fragment of the TIRB polypeptide (SEQ ID NO:2) can include one or more of the conserved TIRB1 domain (aa 8 to 174 of SEQ ID NO:2) and the TIRB2 domain (aa 183 to 344 of SEQ ID NO:2). For example, active variants are provided, wherein the amino acid sequence shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the full length of SEQ ID NO:2, and further includes a region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the consensus sequence of the PFAM domain as shown in Table 1A. In other embodiments, active variants are provided, wherein the amino acid sequence shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the full length of SEQ ID NO:2, and further includes a region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the corresponding region of SEQ ID NO:2, which corresponds to the amino acid positions of the consensus sequence of the PFAM domain as shown in Table 1A.

[0221] In the context of nucleic acid sequences, the term "corresponds to" means that when the nucleic acid sequences of certain sequences are aligned with each other, the nucleic acids that "correspond to" certain enumerated positions in the present invention are those that align with these positions in the reference sequence, but do not necessarily lie in these exact numerical positions with respect to the specific nucleic acid sequences of the present invention. The best alignment of the sequences for comparison can be performed by a computerized implementation of known algorithms or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are the Basic Local Alignment Search Tool (BLAST), which is available on the Internet (e.g., the website of EMBL-EBI), and the ClustalW / ClustalW2 / Clustal Omega programs, respectively. Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity, and FASTA, which are part of the Accelrys GCG software package available from Accelrys, Inc. (San Diego, Calif., USA). See also Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; Ausubel et al., 1988; and Sambrook and Russell, 2001, through the computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package of the Genetics Computer Group, 575 Science Dr., Madison, Wis., USA), or by visual inspection.

[0222] An example of an algorithm suitable for determining percent sequence identity and sequence similarity and for aligning sequences is the BLAST algorithm, which is described in the following references: Altschul et al., J. Mol. Biol. [Journal of Molecular Biology] 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990).

[0223] These initial neighborhood word hits act as seeds for initiating a search to find longer HSPs containing them. These word hits are then extended in both directions along each sequence until the cumulative alignment score can no longer increase. For nucleotide sequences, parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatch residue; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of these word hits in each direction is stopped when the cumulative alignment score drops from its maximum achieved value by an amount X; when the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation value (E) of 10, a cutoff of 100, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:10915 (1989)).

[0224] In addition to calculating the percent sequence identity, the BLAST algorithm performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, if the minimum sum probability in a comparison of a test nucleic acid sequence to a reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the test nucleic acid sequence is considered similar to the reference sequence.

[0225] The variants and fragments disclosed herein can be altered, e.g., by including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are known in the art. For example, amino acid sequence variants and fragments of the TIR polypeptide can be prepared by mutagenesis of the corresponding polynucleotide sequence. Methods for mutagenesis and polynucleotide alteration are known in the art.

[0226] Variant polynucleotides and polypeptides also encompass sequences and polypeptides derived from mutagenesis or recombination procedures, including but not limited to procedures such as DNA shuffling. Strategies for such DNA shuffling are known in the art.

[0227] Variants can be generated by introducing random mutations in the TIR polypeptide sequence. In other embodiments, variants can be designed specifically. In the case of designing mutants, variants with biological activity similar to the original polypeptide can be generated when amino acid identity is maintained in regions of the polypeptide that determine the biological activity of the polypeptide or are involved in determining the three-dimensional conformation of the polypeptide responsible for the biological activity. Biological activity can also be retained if conservative substitutions are made, where an amino acid of a given class is replaced with another amino acid of the same class. Thus, known amino acids can be grouped into one of the following categories: aliphatic or cyclic (glycine, alanine, valine, leucine, isoleucine, proline), aromatic (phenylalanine, tyrosine, tryptophan), acidic (aspartic acid, glutamic acid, asparagine, glutamine), basic (histidine, lysine, arginine), and sulfur- or hydroxyl-containing groups (serine, cysteine, methionine, threonine). A conservative substitution refers to the replacement of one amino acid of a class with another amino acid of the same type, and such replacement is least likely to substantially alter the biological activity of the variant.

[0228] Variants of the polypeptides and polynucleotides also include sequences from other organisms, particularly other plants, that are isolated based on their sequence identity to the polypeptide and polynucleotide sequences disclosed herein. Such sequences include sequences that are orthologs of the disclosed sequences. The term "ortholog" refers to genes that are derived from a common ancestral gene and are found in different species due to speciation. Genes found in different species are considered orthologs when their nucleotide sequences and / or the polypeptide sequences they encode share at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher sequence identity. The functions of orthologs tend to be highly conserved across species. Accordingly, the present disclosure encompasses isolated polynucleotides that encode a TIRA or TIRB polypeptide that confers or enhances disease resistance and that hybridize to the sequences disclosed herein or variants or fragments thereof. The present disclosure also encompasses isolated polynucleotides that encode a polypeptide that has a conserved TIR domain at corresponding positions when aligned with the polypeptides disclosed herein or variants or fragments thereof. Exemplary orthologs of the TIRA and TIRB polypeptides are described in Example 5 herein, including the polypeptides encoded by the CcRpp2-R1 (SEQ ID NO: 45-46) and CcRpp2-R3 (SEQ ID NO: 47-48) genes from Cajanus cajan. Additional orthologs that can be identified based on sequence identity can be found in Tables 3-4 of WO 2022 / 140257, the contents of which are incorporated herein by reference in their entirety.

[0229] Variants of the polypeptides and polynucleotides also include annotated variants and splice variants of orthologous sequences from other organisms, particularly other plants, that are isolated based on their sequence identity to the polypeptide and polynucleotide sequences disclosed herein.

[0230] Variants of polypeptides and polynucleotides also include sequences from the same organism that are present at different genomic locations (e.g., on different chromosomes) and that are isolated based on their sequence identity to the polypeptide and polynucleotide sequences disclosed herein. Such sequences include sequences that are paralogs of the disclosed sequences. The term "paralog" refers to a copy of a gene that is generated by a duplication event on the same genome. Genes found on the same genome are considered paralogs when their nucleotide sequences and / or the polypeptide sequences they encode share at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher sequence identity. The function of paralogs may be retained, although they may be expressed only under different selective pressures. Due to the lack of selective pressure on duplicate copies of a gene, paralogs may also develop different functions.

[0231] Variants of polypeptides and polynucleotides also contain sequences that are allelic variants of the disclosed sequences. The term "allelic variant" refers to different variants of the same gene at a single locus that can result in the same or similar phenotypic expression (e.g., increased disease resistance). These include genes from the same locus in the same organism, as well as genes from the same locus in organisms belonging to the same genus. Such allelic variants are isolated based on their sequence identity to the polypeptide and polynucleotide sequences disclosed herein. Genes found at the same locus are considered allelic variants when their nucleotide sequences and / or the polypeptide sequences they encode share at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher sequence identity. The function of allelic variants may be retained. Exemplary allelic variants of the TirA gene are described in Example 6 herein and include the RG6a gene (SEQ ID NO:39) from Glycine max (L.) Merr. var. penghuensis and the RG7a gene (SEQ ID NO:41) and RG8a gene (SEQ ID NO:43) from Glycine tomentella Hayata. Exemplary allelic variants of the TirB gene are described in Example 6 herein and include the RG6b gene (SEQ ID NO:40) from Glycine max (L.) Merr. var. penghuensis and the RG7b gene (SEQ ID NO:42) and RG8b gene (SEQ ID NO:44) from Glycine tomentella Hayata.

[0232] Variants of the polypeptides and polynucleotides also include sequences from other organisms, particularly other plants, which are identified based on their sequence identity to the polypeptide and polynucleotide sequences disclosed herein and which have functional identity to the polypeptide and polynucleotide sequences disclosed herein. Such variants having "functional identity" are referred to herein as "functionally identical variants". As used herein, "functional identity" refers to the presence of common functional activity, such as common enzyme activity or common mode of action. Polypeptides having functional identity may have low sequence identity; for example, their nucleotide sequences and / or the polypeptide sequences they encode may share about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher sequence identity. However, they may share specificity or preference for the same substrate, share kinetic parameters, etc. In one example, variant polypeptides and polynucleotides of the TIRA and TIRB polypeptides and polynucleotide sequences encoding such polypeptides include proteins that interact with the same set of effector proteins to produce a hypersensitive response in plant cells. Variants of the polypeptides and polynucleotides also include annotated variants and / or splice variants of sequences having functional identity to a reference sequence and which may interact with the same set of effector proteins to produce a hypersensitive response in plant cells.

[0233] As used herein, "effector" or "effector protein" or "plant pathogen effector" refers to a polypeptide molecule secreted by a pathogen that is used to counteract the plant's endogenous defense system. Effector proteins interact (e.g., physically interact) with plant-based resistance proteins or polypeptides encoded by plant-based resistance genes (e.g., R proteins such as the TIRA protein, the TIRB protein, or variants or active fragments thereof) to trigger a local immune response, including a hypersensitive programmed cell death response in infected plant cells / tissues. In one embodiment, "hypersensitive response" includes an increase in electrolyte leakage at the site of infection. In another embodiment, "hypersensitive response" includes an increase (e.g., a partial increase or a complete increase) in phenotypic characteristics associated with plant pathogen-specific protease-dependent cell death.

[0234] Typically, each R protein interacts with a different set of effector proteins, thereby defining a unique mode of action or site of action that confers disease resistance. In one exemplary embodiment, the TIRA polypeptide and the TIRB polypeptide are R proteins that interact with a different set of effector proteins to trigger a hypersensitive response in plant cells. As used herein, the terms "site of action" or "mode of action" refer to a specific interaction between an effector polypeptide from a pathogen and a plant-based disease resistance protein (R protein).

[0235] In particular embodiments, variants of the polypeptides and polynucleotides comprise sequences from other organisms, particularly other plants, which, when expressed in plants and assayed, interact with most or all of the effector proteins recognized by the TIRA and / or TIRB polypeptides to produce a local hypersensitive response. Section 9, Example 8 herein discloses assays for identifying the interaction of an expressed R protein or its variants or fragments with effector proteins and for identifying the presence of a local hypersensitive response.

[0236] As detailed below, variants of the polypeptides can also comprise fusion proteins produced by expressing the TIRA polypeptide linked to the TIRB polypeptide. In particular embodiments, the fusion protein comprises the TIRB polypeptide at the N-terminus of the fusion protein, such as the fusion proteins of SEQ ID NOs: 9-11. In other embodiments, the fusion protein comprises the TIRA polypeptide at the N-terminus of the fusion protein, such as the fusion protein of SEQ ID NO: 12. In embodiments, the polypeptides can be linked to each other in the fusion protein by a cleavable linker that results in the separation of the TIRA polypeptide from the TIRB polypeptide after transcription and expression of the fusion protein. Exemplary embodiments of fusion proteins comprising a self-cleaving linker include the fusion protein of SEQ ID NO: 10. Variant polynucleotides comprise nucleotides having a nucleotide sequence encoding such a fusion protein, e.g., a polynucleotide having a nucleotide sequence encoding the fusion protein of any one of SEQ ID NOs: 9-12 or an active variant or fragment thereof, or a polynucleotide having a nucleotide sequence of any one of SEQ ID NOs: 13-16 or an active variant or fragment thereof.

[0237] IV. Fusion proteins comprising the TIRA and TIRB polypeptides, and polynucleotides encoding such fusion proteins

[0238] The present invention also provides fusion proteins comprising the TIRA polypeptide (SEQ ID NO: 1) or an active fragment or variant thereof operably conjugated to the TIRB polypeptide (SEQ ID NO: 2) or an active fragment or variant thereof. In particular embodiments, the fusion protein comprises the polypeptide sequence of any one of SEQ ID NOs: 9-12 or an active variant or fragment thereof. Further provided are polynucleotides encoding fusion proteins comprising SEQ ID NO: 1 or an active variant or fragment thereof linked to SEQ ID NO: 2 or an active variant or fragment thereof; and polynucleotides comprising SEQ ID NO: 3-5 or an active variant or fragment thereof operably linked to SEQ ID NO: 6-8 or an active variant or fragment thereof. In particular embodiments, provided are polynucleotides encoding fusion proteins comprising any one of SEQ ID NOs: 9-12 or an active variant or fragment thereof, and polynucleotides comprising any one of SEQ ID NOs: 13-16 or variants and fragments thereof.

[0239] As used herein, "fusion protein", "fusion polypeptide", "TIRATIRB fusion protein / polypeptide" or "TIRATIRB protein / polypeptide" refers to a polypeptide sequence that at least when expressed contains a sequence of a TIRA polypeptide (or an active variant or fragment thereof) in frame with a sequence of a TIRB polypeptide (or an active variant or fragment thereof) in the same protein, such as where the sequence of the TIRA polypeptide (or an active variant or fragment thereof) is linked via a linker to the sequence of the TIRB polypeptide (or an active variant or fragment thereof). Such a fusion protein is encoded by a polynucleotide having a nucleotide sequence encoding a TIRA polypeptide (or an active variant or fragment thereof) that is arranged in transcriptional and translational frame with a nucleotide sequence encoding a TIRB polypeptide (or an active variant or fragment thereof).

[0240] In an exemplary embodiment, the fusion proteins provided herein comprise:

[0241] (i) a sequence of any TIRA polypeptide provided herein, including (a) the TIRA polypeptide of SEQ ID NO:1, or (b) a variant or fragment of the TIRA polypeptide of SEQ ID NO:1, such as the TIRA polypeptide of SEQ ID NO:1 further comprising a loss-of-function mutation in the NAD enzyme site of the TIRA1 domain or a gain-of-function mutation in the NAD enzyme site of the TIRA2 domain, or (c) an ortholog of the TIRA polypeptide of SEQ ID NO:1 (such as the ccRpp2-R1 polypeptide from Cajanus cajan provided in SEQ ID NO:46, or any TIRA ortholog provided in Tables 3-4 of WO2022 / 140257, the content of which is incorporated herein by reference in its entirety), or (d) an allelic variant of the TIRA polypeptide of SEQ ID NO:1 (e.g., any allelic variant encoded by the polynucleotides of SEQ ID NOs: 38, 40, and 42);

[0242] linked to:

[0243] (ii) The sequences of any TIRB polypeptides provided herein, including (a) the TIRB polypeptide of SEQ ID NO:2, or (b) variants or fragments of the TIRB polypeptide of SEQ ID NO:2, such as the TIRB polypeptide of SEQ ID NO:2 further comprising a loss-of-function mutation in the NAD enzyme site of the TIRB1 domain, or (c) orthologs of the TIRA polypeptide of SEQ ID NO:1 (such as the ccRpp2-R1 polypeptide from Cajanus cajan provided in SEQ ID NO:48, or any TIRB ortholog provided in Tables 3-4 of WO 2022 / 140257, the content of which is incorporated herein by reference in its entirety), or (d) allelic variants of the TIRB polypeptide of SEQ ID NO:2 (e.g., any allelic variant encoded by the polynucleotides of SEQ ID NO:39, 41, and 43). In a particular embodiment, the polypeptide of (i) is linked to the polypeptide of (ii) via any linker sequence disclosed herein.

[0244] "Fusion protein" also refers to a polypeptide sequence in which the sequence of a TIRA polypeptide (or an active variant or fragment thereof) is linked via a cleavable linker to the sequence of a TIRB polypeptide (or an active variant or fragment thereof). Such a fusion protein undergoes cleavage (e.g., self-cleavage) after fusion protein expression, resulting in the separation of the constituent polypeptides.

[0245] In an exemplary embodiment, the TIRA polypeptide or an active variant or fragment thereof is at the N-terminus of the fusion protein, and the TIRB polypeptide or an active variant or fragment thereof is at the C-terminus of the fusion protein. In a particular embodiment, the fusion protein comprising an N-terminal TIRA polypeptide and a C-terminal TIRB polypeptide comprises SEQ ID NO:12. In other exemplary embodiments, the TIRA polypeptide or an active variant or fragment thereof is at the C-terminus of the fusion protein, and the TIRB polypeptide or an active variant or fragment thereof is at the N-terminus of the fusion protein. In a particular embodiment, the fusion protein comprising an N-terminal TIRB polypeptide and a C-terminal TIRA polypeptide comprises any one of SEQ ID NO:9-11.

[0246] In embodiments, the TIRA polypeptide in the fusion protein is conjugated to the TIRB polypeptide via a linker sequence comprising one or more amino acids (aa) (e.g., comprising at least 1 aa, 2 aa, 3 aa, 5 aa, 10 aa, 30 aa, or at least 50 aa). In particular embodiments, the TIRA polypeptide or an active variant or fragment thereof is linked to the TIRB polypeptide or an active variant or fragment thereof via a linker sequence comprising a peptide sequence of any one of SEQ ID NOs: 36 - 38. In particular embodiments, the TIRA polynucleotide or an active variant or fragment thereof is linked to the TIRB polynucleotide or an active variant or fragment thereof via a linker sequence comprising a nucleotide sequence of any one of SEQ ID NOs: 33 - 35.

[0247] The linker sequence can include a non-cleavable linker sequence or a cleavable linker sequence. In particular embodiments, the TIRA polypeptide or an active variant or fragment thereof in the fusion protein (e.g., the fusion protein of SEQ ID NO: 9 or 12) is linked to the TIRB polypeptide or an active variant or fragment thereof via a non-cleavable linker sequence (x-linker-01 peptide; SEQ ID NO: 36), and / or the TIRA polynucleotide or an active variant or fragment thereof in the fusion protein (e.g., the fusion protein of SEQ ID NO: 9 or 12) is linked to the TIRB polynucleotide or an active variant or fragment thereof via a non-cleavable linker sequence (x-linker-01; SEQ ID NO: 33). In other particular embodiments, the TIRA polypeptide or an active variant or fragment thereof in the fusion protein (e.g., the fusion protein of SEQ ID NO: 10 or 11) is linked to the TIRB polypeptide or an active variant or fragment thereof via a cleavable linker sequence (e.g., the self-cleaving linker sequence of xT2A linker-03 peptide; SEQ ID NO: 38 or xT2A linker-04 peptide; SEQ ID NO: 37), and / or the TIRA polynucleotide or an active variant or fragment thereof in the fusion protein (e.g., the fusion protein of SEQ ID NO: 10 or 11) is linked to the TIRB polynucleotide or an active variant or fragment thereof via a cleavable linker sequence (e.g., the self-cleaving linker sequence of xT2A linker-03; SEQ ID NO: 35 or xT2A linker-04; SEQ ID NO: 34). Still other cleavable or non-cleavable linker sequences can also be used.

[0248] In embodiments of the TIRA-TIRB fusion protein comprising a cleavable linker sequence, after the fusion protein is expressed (i.e., after transcription and translation of the polynucleotide sequence encoding the fusion protein comprising the cleavable linker sequence), the fusion protein is cleaved (e.g., self-cleaved) to release the constitutive TIRA and TIRB polypeptides (or active variants or fragments thereof) in a form not linked to each other.

[0249] In other embodiments, the fusion protein comprises a tag or marker sequence fused to the N-terminus or C-terminus of the protein. The polynucleotide encoding the fusion protein comprising a linker sequence having one or more amino acids comprises a linker nucleotide sequence that is a multiple of 3 nucleotides such that the nucleotide sequence encoding the TIRA polypeptide (or an active variant or fragment thereof) is in-frame translationally with the nucleotide sequence encoding the TIRB polypeptide (or an active variant or fragment thereof).

[0250] The fusion protein is prepared by methods known in the art. In an exemplary embodiment, the fusion protein of the present disclosure is made by operably linking a single promoter to a nucleic acid that comprises (i) a polynucleotide encoding a TIRA polypeptide or an active variant or fragment thereof, which is arranged in-frame transcriptionally with (ii) a polynucleotide encoding a TIRB polypeptide or an active variant or fragment thereof. As a result, a single transcript is produced that, upon translation, yields a protein comprising a TIRA polypeptide fused to a TIRB polypeptide. In embodiments where the fusion protein comprises a cleavable linker sequence, after translation, the fusion protein is cleaved to form the constitutive TIRA and TIRB polypeptides. In a particular embodiment, the fusion protein of the present disclosure is made by operably linking a single promoter to a nucleic acid that comprises (i) a polynucleotide comprising any one of SEQ ID NOs: 3-5 or an active variant or fragment thereof, which is arranged in-frame transcriptionally with (ii) a polynucleotide comprising any one of SEQ ID NOs: 6-8 or an active variant or fragment thereof.

[0251] A TIRATIRB polypeptide encoding a fusion resistance protein of any one of SEQ ID NOs: 9 - 12, which comprises conserved domains of the previously disclosed TIRA and TIRB polypeptides. In particular, the fusion proteins of SEQ ID NOs: 9 - 12 comprise a TIRA1 domain, a TIRA2 domain, a TIRB1 domain, and a TIRB2 domain. The TIRATIRB fusion polypeptides of any one of SEQ ID NOs: 9 - 12, their active variants and fragments, when expressed in a plant, a plant part or a seed, confer or increase disease resistance in the plant, the plant part or the seed (such as a leguminous plant, a leguminous plant part or a leguminous seed). In particular embodiments, when expressed in a plant, a plant part or a seed, the expression of the TIRATIRB fusion protein of SEQ ID NOs: 9 - 12 or an active fragment or variant of any one of SEQ ID NOs: 9 - 12 confers or increases ASR and / or powdery mildew resistance in a soybean plant, a plant part or a seed. In other particular embodiments, when expressed in a plant, a plant part or a seed, the expression of the RG32RG34 fusion protein or an active fragment or variant thereof confers or increases resistance in a soybean plant, a plant part or a seed to soybean cyst nematode, root - knot nematode, bacterial pathogens (such as Pseudomonas syringae pv. syringae) and / or sucking pests (such as aphids, stink bugs, and whiteflies). Various methods for measuring such increased disease resistance are provided in the examples and discussed elsewhere herein.

[0252] Active fragments and variants of the TIRATIRB fusion proteins (any one of SEQ ID NOs: 9 - 12) are also provided. Polynucleotides comprising nucleotide sequences encoding active fragments or variants of the fusion proteins of any one of SEQ ID NOs: 9 - 12 are further provided; and polynucleotides comprising any one of SEQ ID NOs: 13 - 16 or an active variant or fragment thereof.

[0253] Fragments of the TIRATIRB fusion proteins that increase disease resistance when expressed in a plant, a plant part or a seed include TIRATIRB fusion protein fragments that are shorter than the full - length sequence and may comprise N - or C - terminal truncations or internal deletions. Active fragments of the TIRATIRB polypeptide can be polypeptides of lengths of 10, 25, 50, 100, 150, 200, 250 or more amino acids of any one of SEQ ID NOs: 9 - 12 when expressed in a plant. Such bioactive portions can be prepared by recombinant techniques and evaluated for activity to confer increased resistance when expressed in a plant, a plant part or a seed.

[0254] Variant TIRATIRB polypeptides comprise an amino acid sequence having at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence of any one of SEQ ID NOs: 9 - 12. When expressed in plants, plant parts or seeds, such active variants will increase the disease resistance of the plants. In some embodiments, the variant polypeptide comprises a deletion and / or addition of one or more amino acids at one or more internal sites within the TIRA polypeptide and / or TIRB polypeptide portion of a fusion protein and / or a substitution of one or more amino acids at one or more sites within the TIRA and / or TIRB portion of the fusion polypeptide.

[0255] Particular embodiments of the TIRATIRB fusion polypeptide variants include variants having one or more mutations (e.g., gain-of-function or loss-of-function mutations) at one or more NADase sites of the TIRA polypeptide and / or the TIRB polypeptide, resulting in a change (e.g., increase or decrease) in the NADase activity of the fusion protein or the constitutive TIR polypeptide. Example embodiments include variant fusion proteins comprising: a loss-of-function mutation at the NADase site of the TIRA1 domain of the fusion protein (e.g., at the position corresponding to position 85 of SEQ ID NO:1 (e.g., corresponding to the E85A mutation)), a gain-of-function mutation at the NADase site of the TIRA2 domain of the fusion protein (e.g., at the position corresponding to position 251 of SEQ ID NO:1 (e.g., corresponding to the V251E mutation)), a loss-of-function mutation at the NADase site of the TIRB1 domain of the fusion protein (e.g., at the position corresponding to position 87 of SEQ ID NO:2 (e.g., corresponding to the E87A mutation)) or any combination thereof, including but not limited to single mutants (e.g., E85A, E87A or V251E), double mutants (e.g., E85A+V251E; E85A+E87A; V251E+E87A) or triple mutants (e.g., E85A+V251E+E87A). Such variants will have modified (e.g., reduced) NADase activity while retaining the ability of the fusion protein to (i) elicit an immune response and (ii) increase disease resistance when expressed in a plant, plant part or seed. In embodiments where the variant fusion protein comprising one or more mutations in the NADase site comprises a cleavable linker sequence, after expression of the fusion protein, the protein is cleaved into constitutive TIR polypeptides comprising the corresponding NADase site mutations. In embodiments where the constitutive TIRA polypeptide comprises the E85A mutation (or corresponding mutation) and / or the constitutive TIRB polypeptide comprises the E87A mutation (or corresponding mutation), the resulting TIR polypeptide has reduced NADase activity relative to the unmutated version. In embodiments where the constitutive TIRA polypeptide comprises the V251E mutation (or corresponding mutation), the resulting TIRA polypeptide has increased NADase activity relative to the unmutated version.

[0256] Variant TIRATIRB polypeptides include fusion proteins that comprise an ortholog, paralog, or allelic variant of a TIRA polypeptide having an amino acid sequence with at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the amino acid sequence of SEQ ID NO:1, which is linked to an ortholog, paralog, or allelic variant of a TIRB polypeptide having an amino acid sequence with at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the amino acid sequence of SEQ ID NO:2. When expressed in a plant, plant part, or seed, such active variants will increase the disease resistance of the plant.

[0257] Variant polynucleotide sequences encoding the variant fusion proteins are also provided. In an exemplary embodiment, the variant fusion protein is encoded by a variant polynucleotide comprising any one of SEQ ID NO:17 - 19 and 21.

[0258] In other cases, the fusion protein variant comprises a tag, such as a His tag. In still other cases, the fusion protein variant comprises a detectable label.

[0259] Fragments and variants of the nucleotide sequence can encode fusion protein fragments that retain the biological activity of the original fusion protein and have the ability to increase disease resistance. Alternatively, fragments or variants of the nucleotide sequence that are used as hybridization probes or in recombinant DNA constructs for gene editing do not necessarily encode protein fragments that retain biological activity. Thus, fragments of the nucleotide sequence can be in the range of at least about 15, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 750, 900, 950, 1000 nucleotides, or less than the full - length nucleotide sequence encoding the proteins disclosed herein (i.e., any one of SEQ ID NO:17 - 19 and 21).

[0260] Variants of the nucleotide sequence have at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of the nucleotide sequences of SEQ ID NO: 17 - 19 and 21. In certain embodiments, the variant nucleotide sequence encodes an active fusion protein of the invention. In other embodiments, the variant polynucleotide need not encode an active variant fusion protein and can be used as a component of a gene editing construct or as a probe or primer or for other tools for generating the plants and seeds provided herein.

[0261] In some embodiments, fragments and variants of the fusion proteins disclosed herein each comprise one or more conserved domains of the canonical fusion protein. In some embodiments, an active variant or fragment can comprise a polypeptide having at least 40%, 50%, 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to one or more conserved domains in the canonical fusion protein sequence.

[0262] In one example, a variant or fragment of a fusion protein (any one of SEQ ID NO: 9 - 11) can comprise one or more of the conserved TIR domains. For example, active variants are provided wherein the amino acid sequence shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the full length of any one of SEQ ID NO: 9 - 11 and further comprises a region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the TIR domain (PFAM01582) shown in Table 1.

[0263] The variants and fragments disclosed herein can be altered, e.g., by including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are known in the art. For example, amino acid sequence variants and fragments of the fusion protein can be prepared by mutation of the corresponding polynucleotide sequence. Methods for mutagenesis and polynucleotide alteration are known in the art.

[0264] A polynucleotide encoding a fusion protein of any one of SEQ ID NOs: 9-12 and its active variants and fragments, and any one of SEQ ID NOs: 13-19 and 21 or its active variants and fragments can be included in an expression cassette operably linked to a heterologous plant active promoter. In an exemplary embodiment, the expression of the TIRATIRB fusion protein or its active variant or fragment is driven by a constitutive promoter or an endogenous promoter. In other exemplary embodiments, the expression of the TIRATIRB fusion protein or its active variant or fragment is driven by an inducible promoter (e.g., a rust-inducible promoter).

[0265] 3. Expression cassettes and regulatory elements

[0266] The polynucleotides provided herein can be provided in an expression cassette (also referred to herein as a "DNA construct") for expression in a target organism. The expression cassette will include 5' and 3' regulatory sequences operably linked to a polynucleotide encoding a TIRA polypeptide, a TIRB polypeptide, or a TIRATIRB fusion protein, or an active variant or fragment of a TIRA polypeptide, a TIRB polypeptide, or a TIRATIRB fusion protein, thereby allowing the polynucleotide to be expressed. The cassette can additionally contain at least one additional gene or genetic element to be co-transformed into the organism. In the case of including additional genes or elements, these components are operably linked. Alternatively, one or more additional genes or elements can be provided on multiple expression cassettes. Such expression cassettes are provided with multiple restriction sites and / or recombination sites to enable the insertion of the polynucleotide under the transcriptional regulation of the regulatory elements or regions. The expression cassette can additionally contain a selectable marker gene.

[0267] A "DNA construct" refers to genetic elements that are operably linked to each other to form a recombinant DNA molecule and can include elements that provide for the expression of a DNA polynucleotide molecule in a host cell and elements that provide for the maintenance of the construct in the host cell. The various genetic elements within the DNA construct can be native to the polynucleotide encoding the polypeptide or heterologous to the native polynucleotide encoding the polypeptide.

[0268] DNA constructs, vectors, and expression cassettes can be prepared incorporating nucleotide sequences encoding a TIRA polypeptide, a TIRB polypeptide, or a TIRATIRB fusion protein, or active variants or fragments of a TIRA polypeptide, a TIRB polypeptide, or a TIRATIRB fusion protein, for directing expression of the sequences directly from a host plant cell. Examples and methods of such constructs suitable for this purpose are generally described, for example, in Svab et al., Proc. Natl. Acad. Sci. USA 87:8526 - 8530, (1990) and Svab et al., Proc. Natl. Acad. Sci. USA 90:913 - 917 (1993) and U.S. Patent No. 5,693,507.

[0269] A plant expression cassette comprises an operable linkage of genetic elements that provides for the expression of a desired gene product upon introduction into a plant cell. A "plant expression cassette" refers to a DNA construct that contains regulatory elements operably linked to provide for the expression of a desired nucleic acid in a plant. A promoter, a leader sequence, an intron, a polynucleotide encoding a transit peptide, and a 3' transcriptional termination region are genetic elements that can be operably linked by one skilled in the art of plant molecular biology to provide a desired level of expression or function to a TIRA polypeptide and / or a TIRB polypeptide or a TIRATIRB fusion protein, or any active variant or fragment thereof.

[0270] A DNA construct can comprise one or more plant expression cassettes for expressing the DNA molecules of the present invention or other DNA molecules used in the genetic engineering of crop plants. An example of a DNA construct useful for expressing a TIRA polypeptide or an active variant or fragment thereof is a vector having a nucleic acid sequence encoding a TIRA polypeptide or an active fragment or variant thereof. An example of a DNA construct useful for expressing a TIRB polypeptide or an active variant or fragment thereof is a vector having a nucleic acid sequence encoding a TIRB polypeptide or an active fragment or variant thereof. An example of a DNA construct useful for expressing a TIRATIRB fusion polypeptide or an active variant or fragment thereof is a vector having a nucleic acid sequence encoding an active fragment or variant of each or any one of the TIRA and TIRB polypeptides.

[0271] In an embodiment, a DNA construct comprising a polynucleotide encoding a TIRA polypeptide of SEQ ID NO:1 or an active variant or fragment thereof comprises one or more of the native introns of the genomic sequence (SEQ ID NO:3) encoding the TIRA polypeptide. As a non-limiting example, the DNA construct can comprise at least 1 or all of the native introns of the genomic sequence encoding the TIRA polypeptide. In a particular embodiment, a DNA construct comprising a polynucleotide encoding a TIRA polypeptide of SEQ ID NO:1 or an active variant or fragment thereof comprises one or more of the following: (i) a first native intron (intron_1, positions 8021 to 8369 of SEQ ID NO:71 or positions 1231 to 1579 of SEQ ID NO:72) and (ii) a second native intron (intron_2, positions 8883 to 9159 of SEQ ID NO:71 or positions 2093 to 2369 of SEQ ID NO:72). In still further embodiments, one or more of the native introns can be replaced by other introns. Additionally, a DNA construct comprising one or more native introns of the genomic sequence encoding the TIRA polypeptide can comprise one or more additional introns from other sources that enhance the expression of the polypeptide of interest.

[0272] In an embodiment, a DNA construct comprising a polynucleotide encoding a TIRB polypeptide of SEQ ID NO:2 or an active variant or fragment thereof comprises one or more of the native introns of the genomic sequence (SEQ ID NO:6) encoding the TIRA polypeptide. As a non-limiting example, the DNA construct can comprise at least 1 or all of the native introns of the genomic sequence encoding the TIRB polypeptide. In a particular embodiment, a DNA construct comprising a polynucleotide encoding a TIRB polypeptide of SEQ ID NO:2 or 17 or an active variant or fragment thereof comprises one or more of the following: (i) a first native intron (intron_1, positions 8021 to 8369 of SEQ ID NO:71 or positions 1231 to 1579 of SEQ ID NO:72) and (ii) a second native intron (intron_2, positions 8883 to 9159 of SEQ ID NO:71 or positions 2093 to 2369 of SEQ ID NO:72). In still further embodiments, one or more of the native introns can be replaced by other introns. Additionally, a DNA construct comprising one or more native introns of the genomic sequence encoding the TIRB polypeptide can comprise one or more additional introns from other sources that enhance the expression of the polypeptide of interest.

[0273] In some embodiments, the vector may comprise multiple expression cassettes, such as when co-expression of the TIRA polypeptide and the TIRB polypeptide or active fragments or variants thereof is desired. An example of a DNA construct that can be used for co-expressing the TIRA polypeptide or an active variant or fragment thereof with the TIRB polypeptide or an active variant or fragment thereof is a vector having a nucleic acid sequence that comprises (i) a first polynucleotide encoding the TIRA polypeptide or an active fragment or variant thereof; and (ii) a second polynucleotide encoding the TIRB polynucleotide or an active fragment or variant thereof. In embodiments, the nucleic acid sequence is operably linked to a heterologous regulatory element. In a particular example, the vector comprises a first expression cassette containing a first polynucleotide encoding the TIRA polypeptide or an active fragment or variant thereof, the first polynucleotide being operably coupled to a first heterologous regulatory element, such as a first plant-active promoter that drives the expression of the TIRA polypeptide or an active fragment or variant thereof in a plant, plant part, or seed, and the vector further comprises a second expression cassette containing a second polynucleotide encoding the TIRB polypeptide or an active fragment or variant thereof, the second polynucleotide being operably coupled to a different second heterologous regulatory element, such as a different second plant-active promoter that drives the co-expression of the TIRB polypeptide or an active fragment or variant thereof in a plant, plant part, or seed.

[0274] In another exemplary embodiment, the vector comprises an expression cassette that contains a polynucleotide having a nucleotide sequence that comprises the genomic loci of each of the TirA and TirB genes and encodes the TIRA and TIRB polypeptides coupled to a common heterologous regulatory element, such as a single native plant-active promoter that drives the co-expression of the TIRA and TIRB polypeptides. In yet another embodiment, the vector comprises an expression cassette that contains a polynucleotide having a genomic or endogenous sequence of the TirA gene encoding the TIRA polypeptide and a polynucleotide having a genomic or endogenous sequence of the TirB gene encoding the TIRB polypeptide, the polynucleotides being driven by a common bidirectional native promoter. In a particular exemplary embodiment, the polynucleotide having the genomic sequence of the TirA gene (SEQ ID NO:3) and the polynucleotide having the genomic sequence of the TirB gene (SEQ ID NO:6) are driven by a common bidirectional native promoter (SEQ ID NO:49) derived from the genomic loci of the Rg32 and Rg34 genes.

[0275] An example of a DNA construct that can be used to express a TIRATIRB fusion protein or an active variant or fragment thereof is a vector having a nucleic acid sequence encoding a TIRATIRB fusion protein or an active fragment or variant thereof. An example of a DNA construct that can be used to express a TIRATIRB fusion protein or an active variant or fragment thereof is a vector having a nucleic acid sequence that comprises (i) a first polynucleotide encoding a TIRA polypeptide or an active fragment or variant thereof; and (ii) a second polynucleotide encoding a TIRB polynucleotide or an active fragment or variant thereof, wherein the nucleic acid sequence is operably linked to a common heterologous regulatory element. In a particular example, the vector comprises an expression cassette that contains a first polynucleotide encoding a TIRA polypeptide or an active fragment or variant thereof and a second polynucleotide encoding a TIRA polypeptide or an active fragment or variant thereof, each polynucleotide being operably coupled to a common heterologous regulatory element, such as a single plant active promoter that drives the expression of the two polypeptides fused together as a single fusion protein. In another particular example, the vector comprises an expression cassette that contains a polynucleotide that comprises a nucleotide sequence containing the genomic locus of each of the TirA and TIrB genes and encodes TIRA and TIRB polypeptides that are coupled to a common heterologous regulatory element (such as a single native plant active promoter (e.g., the bidirectional promoter of SEQ ID NO:49) that drives the expression of the two polypeptides as a fusion protein).

[0276] Expression of the DNA constructs, expression cassettes, and vectors of the present disclosure in plants, plant parts, or seeds confers plant disease resistance. In particular embodiments, a TIRA polypeptide (or an active fragment and variant thereof) and a TIRB polypeptide (or an active variant and fragment thereof) are co-expressed by expressing a nucleic acid molecule that comprises a nucleotide sequence that contains a first polynucleotide encoding a TIRA polypeptide and a second polynucleotide encoding a TIRB polypeptide, which confers disease resistance (such as fungal pathogen resistance, ASR resistance, powdery mildew resistance, nematode resistance, SCN resistance, RKN resistance, bacterial pathogen resistance, Pseudomonas syringae pv. syringae resistance, sucking pest resistance, aphid resistance, stink bug resistance, and / or whitefly resistance) to leguminous plants, plant parts, or seeds (such as soybean plants, plant parts, or seeds).

[0277] A translation leader sequence refers to a DNA molecule located between the gene promoter and the coding sequence. The translation leader sequence is present upstream of the translation initiation sequence in a fully processed mRNA. The translation leader sequence can affect the processing of the primary transcript to mRNA, mRNA stability, or translation efficiency. Examples of translation leader sequences include the maize and petunia heat shock protein leader sequences, plant virus coat protein leader sequences, the ribulose bisphosphate carboxylase (rubisco) gene leader sequences of plants, etc. (Turner and Foster, Molecular Biotechnology 3:225, 1995).

[0278] The "3′ untranslated sequence" (or 3′-UTR) means a DNA sequence located downstream of the structural polynucleotide sequence and includes sequences encoding polyadenylation and other regulatory signals capable of affecting mRNA processing or gene expression. The role of the polyadenylation signal in plants is to cause the addition of multiple adenosine nucleotides to the 3′ end of the mRNA precursor. The polyadenylation sequence can be derived from a natural gene, from various plant genes, or from T-DNA.

[0279] An example of a polyadenylation sequence is the nopaline synthase 3′ sequence (nos 3′; Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803-4807, 1983). The use of different 3′ untranslated sequences is illustrated in Ingelbrecht et al., Plant Cell 1:671-680, 1989.

[0280] In particular embodiments, the 3′-UTR of the TirA gene can be included, wherein the 3′-UTR of the TirA gene is derived from the genomic sequence of SEQ ID NO:3 and can include at least 500 bp, 1000 bp, or 2000 bp regions located immediately downstream of the stop codon. In other particular embodiments, the 3′-UTR of the TirB gene can be included, wherein the 3′-UTR of the TirB gene is derived from the genomic sequence of SEQ ID NO:5 and can include at least 500 bp, 100 bp, or 2000 bp regions located immediately downstream of the stop codon.

[0281] In certain embodiments, various 3′-UTRs disclosed in WO 2019103918, WO 2021000878, WO 2021022022, WO2022173659, WO 2021260673, WO 2021263249, or U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, or 63 / 383609 may be used; each of which is incorporated herein by reference in its entirety, including, for example, those disclosed as SEQ ID NO:20 in WO 2022173659.

[0282] A variety of transcription terminators can be used in expression cassettes. These transcription terminators are responsible for transcription termination outside of the transgene and proper mRNA polyadenylation. The termination region can be naturally associated with the transcription initiation region, can be naturally associated with the operably linked DNA sequence of interest, can be naturally associated with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, DNA sequence of interest, plant host, or any combination thereof). Suitable transcription terminators are those known to function in plants and include the CAMV 35S terminator, the tml terminator, the nopaline synthase terminator, and the pea rbcs E9 terminator. These terminators can be used in both monocotyledonous and dicotyledonous plants. In addition, the natural transcription terminator of the gene can be used. In particular embodiments, the natural terminator of the TirA gene (tGcaRG3a; SEQ ID NO:28) and / or the natural terminator of the TirB gene (tGcaRG3b; SEQ ID NO:29) can be used. The termination region used in the expression cassette can be obtained from, for example, the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0283] In certain embodiments, various terminators disclosed in WO 2019103918, WO 2021000878, WO 2021022022, WO2022173659, WO 2021260673, WO202163249, or U.S. Provisional Applications 63 / 481627, 63 / 426524 or 63 / 509586 can be used, including, for example, those disclosed in WO 2019103918, including SEQ ID NO:8 (RG1 terminator), SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:32; those disclosed in WO 2022173659, including SEQ IDNO:18 (RG30 terminator); or those disclosed in WO 2021022022 as SEQ ID NO:9 (TirA terminator) or SEQ ID NO:15 (TirB terminator); those disclosed in U.S. Provisional Application No. 63 / 481627 as SEQ ID NO:9 (RG31 terminator) or SEQ ID NO:11 (RG35 terminator); or those disclosed in U.S. Provisional Application Nos. 63 / 426524 and 63 / 509586 as SEQ ID NO:9 (RG32 terminator) or SEQ ID NO:12 or 13 (RG34 terminator); each of which is incorporated herein by reference in its entirety. In particular embodiments, the native terminator of the TirA gene (tGcaRG3a; SEQ IDNO:28) or the native terminator of the TirB gene (tGcaRG3b; SEQ ID NO:29) can be used.

[0284] "5' untranslated sequence" (or 5' untranslated region or 5'-UTR) means a DNA sequence located upstream of the start codon of a structural polynucleotide sequence and includes sequences capable of affecting the translation of an mRNA sequence. The 5'-UTR sequence is also referred to as a leader sequence. In different organisms, the 5'-UTR can remain untranslated and form complex secondary structures to regulate the translation of downstream sequences. The leader sequence can be derived from a native gene or from various plant genes. In particular embodiments, the 5'-UTR of the TirA gene can be included, wherein the 5'-UTR of the TirA gene is derived from the genomic sequence of SEQ ID NO:3 and can include at least 500 bp, 1000 bp, or 2000 bp region immediately upstream of the start codon or a 500 bp region immediately downstream of the transcription start site. In other particular embodiments, the 5'-UTR of the TirB gene can be included, wherein the 5'-UTR of the TirB gene is derived from the genomic sequence of SEQ ID NO:6 and can include at least 500 bp, 1000 bp, or 2000 bp region immediately upstream of the start codon or a 500 bp region immediately downstream of the transcription start site.

[0285] In certain embodiments, various 5'-UTRs disclosed in WO 2019103918, WO 2021000878, WO 2021022022, WO2022173659, WO 2021260673, WO 2021263249, or U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, or 63 / 383609 can be used; each of which is incorporated herein by reference in its entirety, including, for example, those disclosed as SEQ ID NO:19 in WO 2022173659.

[0286] It is also known that a variety of virus-derived untranslated leader sequences enhance expression, and these sequences are particularly effective in dicotyledonous plant cells. The expression cassette can contain one or more such leader sequences. In particular, leader sequences from Tobacco Mosaic Virus (TMV, "W sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g., Gallie et al. Nucl. Acids Res. [Nucleic Acids Research] 15:8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. [Plant Molecular Biology] 15:65-79 (1990)). Other leader sequences known in the art include, but are not limited to: picornavirus leader sequences, e.g., the EMCV leader sequence (encephalomyocarditis 5' non-coding region) (Elroy-Stein, O., Fuerst, T.R. and Moss, B. PNAS USA [Proceedings of the National Academy of Sciences of the United States of America] 86:6126-6130 (1989)); Potato Virus Y leader sequences, e.g., the Tobacco Etch Virus (TEV) leader sequence (Allison et al., 1986); Maize Dwarf Mosaic Virus (MDMV) leader sequence; (Virology [Virology] 154:9-20); human immunoglobulin heavy chain binding protein (BiP) leader sequence (Macejak, D.G. and Samow, P., Nature [Nature] 353:90-94 (1991)); the untranslated leader sequence of the coat protein mRNA from Alfalfa Mosaic Virus (AMV RNA 4) (Jobling, S.A. and Gehrke, L., Nature [Nature] 325:622-625 (1987)); Tobacco Mosaic Virus leader sequence (TMV) (Gallie, D.R. et al., Molecular Biology of RNA [Molecular Biology of RNA], 237-256 (1989)); and Maize Chlorotic Mottle Virus leader sequence (MCMV) (Lommel, S.A. et al., Virology [Virology] 81:382-385 (1991)). See also Della-Cioppa et al., Plant Physiology [Plant Physiology] 84:965-968 (1987).

[0287] Additional regulatory signals include, but are not limited to, transcription start sites, operons, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, termination signals, etc. See, e.g., U.S. Patent Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, edited by Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter referred to as "Sambrook 11"; Davis et al., eds., (1980).

[0288] Expression cassettes can also contain selectable marker genes for the selection of transformed cells. Selectable marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or acetolactate synthase (ALS). Selectable markers commonly used in transformation include the nptII gene, which confers resistance to kanamycin and related antibiotics (Messing and Vierra Gene 19:259-268 (1982); Bevan et al., Nature 304:184-187 (1983)); the pat and bar genes, which confer resistance to the herbicide glufosinate (also known as phosphinothricin; (see White et al., Nucl. Acids Res 18:1062 (1990), Spencer et al. Theor. Appl. Genet 79:625-631 (1990) and U.S. Patent Nos. 5,561,236 and 5,276,268); the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger and Diggelmann, Mol. Cell Biol. 4:2929-2931) and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2(7):1099-1104 (1983)); the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642); the glyphosate N-acetyltransferase (GAT) gene, which also confers resistance to glyphosate (Castle et al. (2004) Science, 304:1151-1154; U.S. Patent Application Publication Nos. 20070004912, 20050246798 and 20050060767); and the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629).

[0289] A. Promoter

[0290] A variety of promoters can be used in the various methods and compositions disclosed herein. The promoter can be selected based on the desired outcome. Nucleic acids can be used in combination with constitutive, inducible, tissue-preferred, or other promoters for expression in a target organism. See, for example, the promoters shown in the following: WO 99 / 43838 and U.S. Patent Nos.: 8,575,425; 7,790,846; 8,147,856; 8,586832; 7,772,369; 7,534,939; 6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; which are incorporated herein by reference.

[0291] For expression in plants, constitutive promoters can be used. Non-limiting examples of constitutive promoters include the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730). Inducible promoters include those that drive the expression of pathogenesis-related proteins (PR proteins), which are induced after pathogen infection. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116; and WO 99 / 43819, which are incorporated herein by reference.Promoters that are locally expressed at or near the site of pathogen infection can also be used (Marineau et al. (1987) Plant Mol. Biol. 9:335 - 342; Matton et al. (1989) Molecular Plant - Microbe Interactions 2:325 - 331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427 - 2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93 - 98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972 - 14977; Chen et al. (1996) Plant J. 10:955 - 966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91:2507 - 2511; Warner et al. (1993) Plant J. 3:191 - 201; Siebertz et al. (1989) Plant Cell 1:961 - 968; Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189 - 200; U.S. Patent No. 5,750,386 (nematode - inducible); and the references cited therein).

[0292] In particular embodiments, constitutive promoters for expressing the polypeptides of the present invention in plants include the soybean ubiquitin promoter (prGmUbi1), the CMV 35S promoter (pr35S), the promoter of the ubiquitin gene from Glycine soja accession number PI_599400 contig000104F (prGaUbiPI599400), the promoter of the ubiquitin gene from Arabidopsis thaliana (prUBQ3), or the alfalfa promoters (prMt12344 (SEQ ID NO:22) or prMt51186 (SEQ ID NO:23) or prMt15303 (SEQ ID NO:24).

[0293] In certain embodiments, various constitutive promoters disclosed in WO 2019103918, WO 2021000878, WO 2021022022, WO2022173659, WO 2021260673, WO 2021263249, or U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, or 63 / 383609 may be used; each of which is incorporated herein by reference in its entirety.

[0294] Wound-inducible promoters can be used in the constructs of the present invention. Such wound-inducible promoters include the pin II promoter (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Ouan et al. (1996) Nature Biotechnology 14:494-498); wunl and wun2 (U.S. Patent No. 5,428,148); winl and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like, all of which are incorporated herein by reference).

[0295] Still other inducible promoters can be used to express the polypeptides of the present invention in the constructs of the present invention. In embodiments, the inducible promoter is a rust-responsive or rust-inducible promoter. As used herein, a "rust-inducible promoter" is a plant promoter that is induced or activated in response to rust exposure or rust infection of a plant. In particular embodiments, a rust-responsive or rust-inducible promoter of the Fis1 gene from flax (Linum usitatissimum) (e.g., prLuFIS1; SEQ ID NO: 25) can be used, such as disclosed as SEQ ID NO: 22 in WO 2021022022 and as SEQ ID NO: 25 in U.S. Provisional Application No. 63 / 383609, the contents of which are incorporated herein by reference in their entirety. In other specific embodiments, a rust-responsive promoter derived from the ACO3 gene (Glyma.02G268200) from soybean (Glycine max) (e.g., prGmACO3) can be used, such as disclosed as SEQ ID NO: 14 in U.S. Provisional Application No. 63 / 481627; a rust-responsive promoter of the MYB gene (Glyma.19G164600) from soybean (Glycine max) (e.g., prGmMYB) can be used, such as disclosed as SEQ ID NO: 15 in U.S. Provisional Application No. 63 / 481627, the contents of which are incorporated herein by reference in their entirety.

[0296] Tissue-preferred promoters useful in the present invention include those shown in the following references: Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascim et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.

[0297] Leaf-preferred promoters include those shown in the following references: Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0298] Root-preferred promoters are known and include those shown in the following: Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (cytosolic glutamine synthetase (GS)); Bogusz et al. (1990) Plant Cell 2(7):633-641; Leach and Aoyagi (1991) Plant Science (Limerick) 79(1):69-76 (rolC and rolD); Teeri et al. (1989) EMBO J. 8(2):343-350; Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772 (VfENOD-GRP3 gene promoter); and Capana et al. (1994) Plant Mol. Biol. 25(4):681-691 (rolB promoter). See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

[0299] "Seed-preferred" promoters include "seed-specific" promoters (promoters that are active during seed development, such as promoters of seed storage proteins) and "seed germination" promoters (promoters that are active during seed germination). See Thompson et al. (1989) BioEssays 10:108. Seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase) (see WO 00 / 11177 and U.S. Patent No. 6,225,529). γ-zein is an endosperm-specific promoter. Globulin 1 (Gib-1) is a representative embryo-specific promoter. For dicotyledonous plants, seed-specific promoters include, but are not limited to, bean β-phaseolin, rapeseed albumin (napin), β-conglycinin, soybean lectin, cruciferin, etc. For monocotyledonous plants, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, γ-zein, waxy protein, shrunken-1, shrunken-2, globulin 1, etc. See also WO 00 / 12733, which discloses seed-preferred promoters from the endl and end2 genes.

[0300] In some embodiments, promoters that control the expression of resistance genes can be used to express a polynucleotide of interest. Such promoters include, but are not limited to, the various native R gene promoters shown in WO 2019103918, WO 2021000878, WO 2021022022, WO 2022173659, WO 2021260673, WO 2021263249, or U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, or 63 / 383609; each of which is incorporated herein by reference in its entirety, including those disclosed as SEQ ID NO:7 (RG1 promoter), SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, or SEQ ID NO:31 in WO2019103918; those disclosed as SEQ ID NO:7 (Rpp6907 promoter) in WO 2021000878; those disclosed as SEQ ID NO:9 (TirA promoter) or SEQ ID NO:14 (TirB promoter) in WO 2021022022; those disclosed as SEQ ID NO:15 (RG30 promoter) in WO 2022173659; those disclosed as SEQ ID NO:7 (RG21 promoter) in WO 2021263249 or WO 2021260673; and those disclosed as SEQ ID NO:7-8 (RG31 promoter) or SEQ ID NO:10 (RG35 promoter) in U.S. Patent Application 63 / 481627, or those disclosed as SEQ ID NO:8 (RG32 promoter) or SEQ ID NO:10 or 11 (RG34 promoter) in U.S. Patent Applications 63 / 426524 and 63 / 509586. Still other native promoters include the native TirA promoter (prGcaRG3a as shown in SEQ ID NO:26 herein) and the native TirB promoter (prGcaRG3b as shown in SEQ ID NO:27 herein), as well as modified versions of the native promoters.

[0301] In a further embodiment, the native promoter can be a promoter sequence derived from a genomic locus (each containing the Rg32 and Rg34 genes), disclosed as SEQ ID NO:18 in U.S. Provisional Applications 63 / 426524 and 63 / 509586. In a particular exemplary embodiment, the native promoter derived from a genomic locus contains the sequence shown as SEQ ID NO:49 herein, which drives bidirectional expression of a gene operably coupled thereto (e.g., bidirectional expression of the Rg32 and Rg34 genes; or bidirectional expression of the TirA and TirB genes). As detailed in Example 9, bidirectional expression of the TIR polypeptide can be obtained when the sense strand and the reverse complementary strand of the bidirectional promoter sequence of SEQ ID NO:49 are used to drive Tir gene expression.

[0302] For expression in a bacterial host, promoters that function in bacteria are known in the art. Such promoters include any known crystal protein gene promoter, including the promoter of any protein of the present invention, and promoters specific for the Bacillus thuringiensis σ factor. Alternatively, mutagenized or recombinant crystal protein-encoding gene promoters can be engineered recombinantly and used to promote the expression of the novel gene segments disclosed herein.

[0303] B. Native Regulatory Elements

[0304] Compositions are provided that contain novel regulatory elements. In one embodiment, polynucleotides are provided that contain a regulatory element operably linked to a polynucleotide of interest. Such regulatory elements include promoters and contain the nucleotide sequence shown as SEQ ID NO:26 or 27 or an active variant or fragment thereof. An active variant or fragment of a promoter will retain the ability to direct the expression of an operably linked polynucleotide sequence. Thus, an active variant of the promoter sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:26 or 27 and retains the ability to direct the expression of an operably linked nucleotide sequence. Fragments of such promoter sequences are also provided, which can contain at least 100, 200, 250, 300, 350, 400 or more nucleotides of the sequence shown in SEQ ID NO:26 or 27. Fragments of such promoters can be active fragments and retain the ability to direct the expression of an operably linked nucleotide sequence.

[0305] Regulatory elements are provided that contain a terminator sequence shown in SEQ ID NO:28 or 29 or an active variant or fragment thereof. An active variant or fragment of the terminator sequence will retain the ability to regulate the expression of an operably linked polynucleotide sequence. Thus, an active variant of the terminator sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:28 or 29 and retains the ability to direct the expression of an operably linked nucleotide sequence. Fragments of such terminator sequences are also provided, which may comprise at least 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more nucleotides of the sequences shown in SEQ ID NO:28 or 29. Fragments of such terminators can be active fragments and retain the ability to regulate the expression of an operably linked nucleotide sequence.

[0306] Regulatory elements are provided that contain an intron sequence or an active variant or fragment thereof shown at the following positions: positions 8021 to 8369 of SEQ ID NO:71 (intron_1); positions 8883 to 9159 of SEQ ID NO:71 (intron_2); positions 1231 to 1579 of SEQ ID NO:72 (intron_1); and / or positions 2093 to 2369 of SEQ ID NO:72 (intron_2). An active variant or fragment of the intron sequence will retain the ability to regulate the expression of an operably linked polynucleotide sequence. Thus, an active variant of the intron sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the intron sequences listed above and retains the ability to direct the expression of an operably linked nucleotide sequence. Fragments of such intron sequences are also provided, which may comprise at least 100, 200, 250, 300, 350, 400 or more nucleotides of any of the sequences shown below: positions 8021 to 8369 of SEQ ID NO:71 (intron_1); positions 8883 to 9159 of SEQ ID NO:71 (intron_2); positions 1231 to 1579 of SEQ ID NO:72 (intron_1) and positions 2093 to 2369 of SEQ ID NO:72 (intron_2). Fragments of such introns can be active fragments and retain the ability to regulate the expression of an operably linked nucleotide sequence.

[0307] In some aspects, the present disclosure provides expression cassettes. In some embodiments, the expression cassette comprises a nucleotide sequence comprising any one of SEQ ID NO: 3-5 and / or any one of SEQ ID NO: 6-8, wherein the nucleotide sequence is operably linked to a heterologous nucleotide sequence. In some embodiments, the expression cassette further comprises a selectable marker.

[0308] In some embodiments, the expression cassette is an endogenous expression cassette and comprises a nucleotide sequence comprising any one of SEQ ID NO: 73-74.

[0309] In some embodiments, the heterologous sequence of interest is a nucleic acid of interest encoding an RNA or protein of interest. In some embodiments, the RNA or protein of interest can confer desired characteristics to a plant, such as antibiotic resistance, virus resistance, insect resistance, disease resistance, resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in industrial processes, or altered reproductive capacity. In some embodiments, the RNA or protein of interest comprises a genome editing agent, such as a CRISPR / Cas agent (e.g., Cas protein and / or guide RNA), TALEN, DNA-guided nuclease, meganuclease, recombinase, or zinc finger nuclease. In some embodiments, the heterologous nucleotide sequence encodes a selectable marker.

[0310] The heterologous nucleotide sequence of interest may comprise a sequence encoding a polypeptide of interest, and in more particular embodiments, the heterologous nucleotide sequence of interest encodes a protein that increases plant disease resistance (e.g., increases resistance to fungal pathogens such as ASR resistance or powdery mildew resistance, increases resistance to nematode pathogens, increases resistance to bacterial pathogens, and / or resistance to sucking pests). Such sequences include, but are not limited to, polynucleotides encoding proteins that confer increased ASR resistance, as described in U.S. Patent Application No. US20200354739, and PCT Application Nos. WO 2019103918, WO 2021000878, WO 2021154632A1, WO2021022022, WO 2021022026, WO 2021022101, WO 2022173659, WO 2021260673, WO2021263249, and U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, and 63 / 383609, each of which is incorporated herein by reference in its entirety.

[0311] In some embodiments, the expression cassette is located in a vector, such as a plasmid, virus, or Agrobacterium. In some embodiments, the expression cassette is located in a plant cell, as discussed elsewhere herein.

[0312] 4. Plants, plant cells, and plant parts

[0313] The present invention provides plants, plant parts, plant cells and seeds that contain in their genome a nucleic acid sequence operably linked to a promoter active in a plant, wherein the nucleic acid sequence comprises polynucleotides encoding (i) a TIRA polypeptide as shown in SEQ ID NO:1 or an active variant or fragment thereof, and (ii) a TIRB polypeptide as shown in SEQ ID NO:2 or an active variant or fragment thereof. In particular embodiments, the plants, plant parts, plant cells and seeds co-express the TIRA and TIRB polypeptides or their active variants and fragments in their genome. In other particular embodiments, a plant, plant part, plant cell or seed expresses in its genome a nucleic acid sequence operably linked to a promoter active in a plant, wherein the nucleic acid sequence comprises a polynucleotide encoding the TIRA and TIRB polynucleotides (as a TIRATIRB fusion protein as shown in any one of SEQ ID NO:9-12 or an active variant or fragment of the fusion protein). In still further embodiments, plants, plant parts, plant cells and seeds are provided that contain in their genome a heterologous nucleic acid sequence that comprises a polynucleotide as shown in any one of SEQ ID NO:3-5, 6-8, 13-19 and 21 or an active variant and fragment thereof. Such heterologous polynucleotides can be transiently expressed or stably integrated into the genome.

[0314] Although soybean plants are used throughout the application to illustrate the compositions and methods, the polynucleotides provided herein can be introduced into any plant species (including but not limited to monocotyledonous and dicotyledonous plants). Examples of target plants include but are not limited to maize (corn), sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley and oilseed rape, Brassica, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, nectarine, fig, guava, mango, olive, papaya, cashew, macadamia, apricot, oats, vegetables, ornamental plants and conifers.

[0315] In certain embodiments, the plant is a legume. Examples of legumes include, but are not limited to, Phaseolus (e.g., French bean, green bean, climbing bean (Phaseolus vulgaris), Lima bean (Phaseolus lunatus)), Tepary bean (Phaseolus acutifolius), runner bean (Phaseolus coccineus); Glycine (e.g., wild soybean, soybean (Glycine max (L.))); pea (Pisum (e.g., shelled pea (sometimes called smooth or round-seeded pea; Pisum sativum)); marrowfat pea (Pisum sativum), sugar pea (Pisum sativum), also known as Dutch pea, edible-pod pea or mangetout (Pisum granda)); peanut (Arachis hypogaea), clover (Trifolium spp.), alfalfa (Medicago), kudzu (Pueraria lobata), common alfalfa, alfalfa (Medicago sativa), chickpea (Cicer), lentil (Lens culinaris), and lupine (Lupinus); vetch (Vicia), field bean, broad bean (Vicia faba), vetchling (Lathyrus) (e.g., chickling pea (Lathyrus sativus), heath pea (Lathyrus tuberosus));Genus Vigna (such as moth bean (Vigna aconiti folia), adzuki bean (Vigna angularis)), urad bean (Vigna mungo), mung bean (Vigna radiata), bambara groundnut (Vigna subterrane), rice bean (Vigna umbellata), wild cowpea (Vigna vexillata), cowpea (Vigna unguiculata) (also known as asparagus bean, cowpea); pigeon pea (Cajanus cajari); Cajanus cajan; genus Macrotyloma (such as geocarpagroundnut (Macrotyloma geocarpum)), horse bean (Macrotyloma uniflorum); goa bean (Psophocarpus tetragonolobus), African yam bean (Sphenostylis stenocarpa), Egyptian blackbean, lablab bean (Lablab purpureus), yam bean (Pachyrhizus erosus), guar bean (Cyamopsis tetragonolobus); and / or genus Canavalia (such as jack bean (Canavalia ensiformis)), sword bean (Canavalia gladiata).;

[0316] In one embodiment, the leguminous plant is soybean, and more particularly Glycine max.

[0317] The genus Glycine (soybean or soya bean) is a genus of the legume family of the soybean family. The Glycine plants provided herein can be Glycine arenaria, Glycine argyrea, Glycine cyrtoloba, Glycine canescens, Glycine penghuensis, Glycine curvata, Glycine falcata, Glycine latifolia, Glycine microphylla, Glycine pescadrensis, Glycine tabacina var. albopilosa, Glycine syndetica, Glycine soja Sieb. Et Zucc., Glycine max (L.) Merrill., Glycine tabacina, or Glycine tomentella.

[0318] In some embodiments, the plants (leguminous plants or soybean plants) provided herein are elite plants, elite germplasms, or derived from elite lines or elite germplasms. Numerous elite lines are available and are known to those of ordinary skill in the soybean breeding art and are discussed in further detail elsewhere herein.

[0319] In some embodiments, the plants provided herein can comprise one or more additional polynucleotides encoding additional polypeptides that increase plant disease resistance. Such combinations are described in more detail elsewhere herein.

[0320] In certain embodiments, plants, plant parts, or seeds having the heterologous polynucleotides or polypeptides or active variants and fragments thereof disclosed herein can have increased expression of the polynucleotide or polypeptide. In other embodiments, plants, plant parts, or seeds having the heterologous polynucleotides or polypeptides or active variants and fragments thereof disclosed herein can have increased activity levels of the polypeptide. Methods for generating such increased expression or activity levels are disclosed elsewhere herein and include, but are not limited to, breeding, gene editing, and transgenic techniques.

[0321] The plants produced as described above can be propagated to produce progeny plants, and progeny plants in which the polynucleotide conferring increased protein content and / or altered seed composition has been stably incorporated into their genomes can be selected and, if desired, further propagated.

[0322] In some embodiments, plant cells, seeds, or plant parts or harvest products can be obtained from the plants produced as above, and the plant cells, seeds, or plant parts can be screened using the methods disclosed above to demonstrate stable incorporation of the polynucleotide. The term "stable incorporation" refers to the integration of a nucleic acid sequence into the genome of a plant and the ability of the nucleic acid sequence to be inherited by its progeny.

[0323] In some embodiments, plant products can be harvested from the plants disclosed above and processed to produce processed products such as flour, soybean meal, oil, starch, etc. These processed products are also within the scope of the present invention provided that they contain the polynucleotides or polypeptides or variants thereof disclosed herein. Other soybean plant products include but are not limited to protein concentrates, protein isolates, soybean hulls, meal, flowers, oil, and whole soybeans themselves.

[0324] A seed lot is provided that includes a population of seeds containing in their genomes a heterologous nucleic acid sequence that includes a polynucleotide encoding a TIRA polypeptide shown in SEQ ID NO:1 or an active variant or fragment thereof, and a TIRB polypeptide shown in SEQ ID NO:2 or an active variant or fragment thereof (e.g., a polynucleotide encoding a TIRA polypeptide co-expressed with a TIRB polypeptide), or a polynucleotide encoding a TIRBTIRB fusion protein shown in any one of SEQ ID NO:9-12; and the seed lot has increased disease resistance. In other embodiments, the seed lot includes a population of seeds containing in their genomes a heterologous nucleic acid sequence that includes a polynucleotide shown in any one of SEQ ID NO:8-13 and 15-22 or variants and fragments thereof.

[0325] Such seeds can be from any plant, including but not limited to dicotyledonous crop plants, legumes, or soybeans. Methods of making a seed lot include harvesting seeds from plants having increased resistance to plant pathogens. Such a seed lot can contain at least 50, 100, 1000, 100000, or more seeds of the present invention.

[0326] A plant collection is further provided that produces seeds having increased resistance to the plant pathogens described herein. Such a plant collection has a heterologous nucleic acid sequence stably integrated into its genome, the heterologous nucleic acid sequence comprising polynucleotides encoding (i) a TIRA polypeptide shown in SEQ ID NO:1 or an active variant or fragment thereof, and (ii) a TIRB polypeptide shown in SEQ ID NO:2 or an active variant or fragment thereof, and such a plant collection has increased disease resistance. In other embodiments, the plant collection has a heterologous nucleic acid sequence stably integrated into its genome, the heterologous nucleic acid sequence comprising a polynucleotide encoding a fusion protein comprising (i) a TIRA polypeptide shown in SEQ ID NO:1 or an active variant or fragment thereof, and (ii) a TIRB polypeptide shown in SEQ ID NO:2 or an active variant or fragment thereof, and the plant collection has increased disease resistance, such as a fusion protein of any one of SEQ ID NOs: 9-12 or an active variant or fragment thereof. In other embodiments, a seed lot comprises a population of seeds that contain in their genome a heterologous nucleic acid sequence comprising a polynucleotide shown in any one of SEQ ID NOs: 3-8, 13-19, and 21 or a variant and fragment thereof. The term collection encompasses any set of plants joined together by proximity, such as plants in a field, greenhouse, or tray. The plant collection comprises at least 50, 100, 1000, 10000, 100000 or more plants of the invention.

[0327] I. Plants with rebalanced relative expression levels of TIRA and TIRB polypeptides

[0328] In embodiments, the plants, plant parts, plant cells, and seeds disclosed herein contain in their genome a polynucleotide encoding a TIRA polypeptide that is co-expressed with a TIRB polypeptide (or an active variant or fragment of any TIR polypeptide) or a fusion protein thereof, and the expression level of the TIRA polypeptide is altered relative to the relative expression level of the polypeptide in a control plant. In particular embodiments, the plants, plant parts, and seeds contain stably integrated in their genome (i) a polynucleotide encoding the TIRA polypeptide as shown in SEQ ID NO:1 or an active variant or fragment thereof, and a polynucleotide encoding the TIRB polypeptide as shown in SEQ ID NO:2 or an active variant or fragment thereof, or (ii) a polynucleotide encoding a fusion protein comprising the TIRA polypeptide as shown in SEQ ID NO:1 or an active variant or fragment thereof, and a polynucleotide encoding the TIRB polypeptide as shown in SEQ ID NO:2 (e.g., a fusion protein as shown in any of SEQ ID NO:9-12 or an active variant or fragment thereof, or a polynucleotide as shown in any of SEQ ID NO:13-19 and 21), the polynucleotide being coupled to a heterologous promoter, and the expression level of the TIRA polypeptide or an active variant or fragment thereof is rebalanced relative to the expression level of the TIRB polypeptide, or an active variant or fragment thereof, in the plant, plant part, or seed, compared to the relative expression level of the polypeptide in a control plant.

[0329] As used herein, "expression level" or "relative expression level" refers to the amount of translatable TIRA transcript (e.g., primary transcript or mRNA), or the amount of TIRA polypeptide produced by translation of the transcript, relative to the amount of translatable TIRB transcript (e.g., primary transcript or mRNA), or the amount of TIRB polypeptide produced by translation of the transcript.

[0330] As used herein, "imbalanced expression level" means that one TIR transcript or polypeptide is present in excess relative to another TIR transcript or polypeptide in a plant cell. In an exemplary embodiment, an imbalanced expression level means that the amount of TIRA transcript (i.e., the primary transcript or mRNA encoding the TIRA polypeptide or an active variant or fragment thereof) in a plant cell is in excess relative to the amount of TIRB transcript (i.e., the primary transcript or mRNA encoding the TIRB polypeptide or an active variant or fragment thereof). In other embodiments, an imbalanced expression level means that the amount of TIRB transcript (i.e., the primary transcript or mRNA encoding the TIRB polypeptide or an active variant or fragment thereof) in a plant cell is in excess relative to the amount of TIRA transcript (i.e., the primary transcript or mRNA encoding the TIRA polypeptide or an active variant or fragment thereof). In a particular embodiment, in a control plant or plant cell, the ratio of the imbalanced expression level of the TIRB polypeptide relative to the TIRA polypeptide is from 5:1 to 30:1, such as a ratio of 20:1.

[0331] As used herein, "rebalanced expression level" means that in a plant cell, after introduction of the polynucleotides of the invention encoding the TIRA polypeptide and the TIRB polypeptide, the amount of TIRA transcript (i.e., the primary transcript or mRNA encoding the TIRA polypeptide or an active variant or fragment thereof) is comparable to the amount of TIRB transcript (i.e., the primary transcript or mRNA encoding the TIRB polypeptide or an active variant or fragment thereof). In a particular embodiment, introduction of the polynucleotides of the invention encoding the TIRA polypeptide and the TIRB polypeptide results in an expression level of the TIRB polypeptide relative to the TIRA polypeptide of a ratio of from 0.5:1 to 2:1, such as a ratio of 1:1 or 1.5:1. In other particular embodiments, introduction of the polynucleotides of the invention encoding the TIRA polypeptide and the TIRB polypeptide results in an expression level of the TIRA polypeptide relative to the TIRB polypeptide of a ratio of from 0.5:1 to 2:1, such as a ratio of 1:1 or 1.5:1. In a particular embodiment, introduction of the polynucleotides of the invention results in a reduction in the transcription level of the TIRB polypeptide from being about 20-fold that of the TIRA polypeptide to being about 1.5-fold that of the TIRA polypeptide.

[0332] Plants, plant parts, plant cells, and seeds comprising TIRA polypeptides with unbalanced expression levels relative to TIRB polypeptides are capable of increasing the disease resistance of the plants. However, in some embodiments, such plants may exhibit reduced agronomic performance and / or an adverse growth phenotype, including but not limited to delayed germination, stunted plants, poor growth and development, and premature death, as disclosed in the examples herein. In contrast, embodiments of plants, plant parts, plant cells, and seeds comprising TIRA polypeptides with rebalanced expression levels relative to TIRB polypeptides show a reduction in negative growth phenotypes, including but not limited to improved germination rate, improved plant height, and higher vigor and / or enhanced agronomic performance, as disclosed in the examples herein.

[0333] In embodiments, plants, plant parts, plant cells, or seeds having TIR polypeptides with rebalanced expression levels relative to TIRB polypeptides, enhanced agronomic performance, and increased disease resistance have differential expression of the TIR polypeptides in the plants, plant parts, plant cells, or seeds via different regulatory elements (e.g., different promoters and / or terminators). In an exemplary embodiment, rebalancing is achieved by driving constitutive expression of the TIRA polypeptide and / or the TIRB polypeptide via a native promoter and optionally a native terminator. In a particular embodiment, plants, plant parts, plant cells, or seeds having rebalanced expression levels have constitutive expression of the TIRA polypeptide via the native promoter of the TIRA gene (prGcaRG3a; SEQ ID NO:26) and optionally the corresponding native terminator (e.g., prGcaRG3a; SEQ ID NO:28); constitutive expression of the TIRB polypeptide via the native promoter of the TIRB gene (prGcaRG3b; SEQ ID NO:27) and optionally the corresponding native terminator (tGcaRG3b; SEQ ID NO:29).

[0334] In another embodiment, a plant, plant part, plant cell, or seed having a TIRA polypeptide with a rebalanced expression level relative to a TIRB polypeptide, enhanced agronomic performance, and increased disease resistance has constitutive expression of the TIRA polypeptide driven by a constitutive plant-active promoter in the plant, plant part, plant cell, or seed and selective expression of the TIRB polypeptide driven by an inducible promoter in the plant, plant part, plant cell, or seed. In a particular embodiment, in a plant, plant part, plant cell, or seed, the constitutive expression of the TIRA polypeptide is driven by a constitutive plant-active promoter (e.g., prMt12344; SEQ ID NO:22, or prMt51186; SEQ ID NO:23; or prMt15303; SEQ ID NO:24, all from Medicago truntula), while the expression of the TIRB polypeptide is driven by an inducible promoter (e.g., the rust-inducible promoter prLuFIS1; SEQ ID NO:25).

[0335] In other embodiments, a plant, plant part, plant cell, or seed having a TIRA polypeptide with a rebalanced expression level relative to a TIRB polypeptide, enhanced agronomic performance, and increased disease resistance comprises a nucleic acid having a nucleotide sequence that expresses the TIRA polypeptide upstream of the transcription of the TIRB polypeptide. In a particular embodiment, a plant, plant part, plant cell, or seed having a rebalanced expression level of a TIR polypeptide comprises a polynucleotide comprising (i) a first expression cassette having a nucleotide sequence that drives the expression of the TIRA polypeptide or an active variant or fragment thereof via a first promoter, and (ii) a second expression cassette having a nucleotide sequence that drives the expression of the TIRB polypeptide or an active variant or fragment thereof via a different second promoter, wherein the first expression cassette is located upstream of the transcription of the second expression cassette on the polynucleotide.

[0336] In yet another particular embodiment, plants, plant parts, plant cells or seeds having a rebalanced expression level of a TIRA polypeptide relative to a TIRB polypeptide, enhanced agronomic performance and increased disease resistance comprise a nucleic acid having a nucleotide sequence that expresses a TIRA polypeptide and a nucleotide sequence that expresses a TIRB polypeptide, each nucleotide sequence operably coupled to a bidirectional promoter. In one particular embodiment, the bidirectional promoter is the promoter of SEQ ID NO:49. In one specific embodiment, the nucleotide sequence that expresses the TIRA polypeptide is operably coupled upstream of the bidirectional promoter, and the nucleotide sequence that expresses the TIRB polypeptide is operably coupled downstream of the bidirectional promoter. Thus, the expression of one TIR polypeptide is driven by the sense strand sequence of the bidirectional promoter, and the expression of the other TIR polypeptide is driven by the antisense or reverse complementary sequence of the bidirectional promoter. In another specific embodiment, the nucleotide sequence that expresses the TIRB polypeptide is operably coupled upstream of the bidirectional promoter, and the nucleotide sequence that expresses the TIRA polypeptide is operably coupled downstream of the bidirectional promoter.

[0337] In still other embodiments, rebalancing of the expression level of a TIRA polypeptide relative to a TIRB polypeptide in plants, plant parts, plant cells or seeds, as well as enhancement of agronomic performance and improvement of disease resistance, is achieved by expressing the TIRA polypeptide as a fusion protein with the TIRB polypeptide (e.g., having the TIRA polypeptide at the N-terminus or C-terminus of the fusion protein). In one particular embodiment, plants, plant parts, plant cells or seeds having a rebalanced expression level of a TIR polypeptide comprise a polynucleotide having a single expression cassette that contains a nucleotide sequence encoding a fusion protein of a TIRA polypeptide or an active variant or fragment thereof linked to a TIRB polypeptide or an active variant or fragment thereof, the expression of the fusion protein being driven by a common promoter. In specific embodiments, the plant comprises a fusion protein of any one of SEQ ID NOs:9-12 or an active variant or fragment thereof, or the plant comprises a polynucleotide encoding a fusion protein of any one of SEQ ID NOs:9-12 or an active variant or fragment thereof, or the plant comprises a polynucleotide of any one of SEQ ID NOs:13-19 and 21. In a particular embodiment, the fusion protein comprises a cleavable linker sequence between the TIRA polypeptide and the TIRB polypeptide of the fusion protein, wherein upon fusion expression, cleavage of the fusion protein at the linker sequence results in the release of the individual constitutive TIR polypeptides at balanced levels.

[0338] II. Plants with TIRA and TIRB Polypeptides Having Modified NAD Enzyme Activity Levels

[0339] In embodiments, the plants, plant parts, plant cells, and seeds disclosed herein contain, in their genome, a polynucleotide encoding a TIRA polypeptide (or an active variant or fragment of either) co-expressed with a TIRB polypeptide, or a fusion protein thereof, and may have a modified NADase activity level. In particular embodiments, the NADase activity of the TIRA and / or TIRB polypeptide is reduced by introducing loss-of-function mutations in the TIRA1 and / or TIRBA2 and / or TIRB1 domains. In other particular embodiments, the NADase activity of the TIRA polypeptide is increased by introducing gain-of-function mutations in the TIRA1 domain and / or the TIRB1 domain. In an exemplary embodiment, plants, plant parts, and seeds are provided that contain, in their genome, a polynucleotide encoding a TIRA polypeptide as shown in SEQ ID NO:1 or an active variant or fragment thereof, the polynucleotide further containing a loss-of-function mutation that reduces the NADase activity of the TIRA polypeptide or an active variant or fragment thereof, while maintaining the ability of the mutated TIRA polypeptide to confer disease resistance to the plant when co-expressed with a TIRB polypeptide or an active variant or fragment thereof. In particular embodiments, the plant contains a polynucleotide encoding a TIRA polypeptide that has a loss-of-function mutation that reduces the NADase activity of the TIRA polypeptide, the polynucleotide including a mutation in the TIRA1 domain of the TIRA polypeptide, such as a mutation from glutamate (E) to alanine (A) (or a conservative variant thereof) at the position corresponding to position 85 of SEQ ID NO:1. In particular embodiments, the plant additionally or alternatively contains a polynucleotide encoding a TIRA polypeptide that has a gain-of-function mutation that increases the NADase activity of the TIRA polypeptide, the polynucleotide including a mutation in the TIRA2 domain of the TIRA polypeptide, such as a mutation from valine (V) to glutamate (E) (or a conservative variant thereof) at the position corresponding to position 251 of SEQ ID NO:1.

[0340] In particular embodiments, a plant containing a fusion protein contains a polynucleotide of any one of SEQ ID NOs: 17, 18, and 21, the fusion protein comprising a TIRA polypeptide having a modified NADase activity.

[0341] In other exemplary embodiments, plants, plant parts, and seeds are provided that contain in their genome a polynucleotide encoding a TIRB polypeptide as shown in SEQ ID NO:2 or an active variant or fragment thereof, the polynucleotide further comprising a loss-of-function mutation that reduces the NAD enzymatic activity of the TIRB polypeptide or an active variant or fragment thereof, while maintaining the ability of the mutant TIRB polypeptide to confer disease resistance to the plant when co-expressed with a TIRA polypeptide or an active variant or fragment thereof. In particular embodiments, the plant contains a polynucleotide encoding a TIRB polypeptide that has a loss-of-function mutation that reduces the NAD enzymatic activity of the TIRB polypeptide, wherein the polynucleotide contains a mutation in the TIRB1 domain of the TIRB polypeptide, such as a glutamate (E) to alanine (A) mutation (or a conservative variant thereof) at the position corresponding to position 87 of SEQ ID NO:2, and no mutation in the TIRB2 domain of the TIRB polypeptide.

[0342] In particular embodiments, a plant containing a fusion protein contains a polynucleotide of either SEQ ID NO:19 or 21, the fusion protein comprising a TIRB polypeptide having modified NAD enzymatic activity.

[0343] In still other exemplary embodiments, plants, plant parts, and seeds are provided that contain in their genome a polynucleotide encoding an active variant or fragment of a TIRA polypeptide as shown in SEQ ID NO:1 and a TIRB polypeptide as shown in SEQ ID NO:2 or one of the two, the polynucleotide further having a loss-of-function mutation that reduces the NAD enzymatic activity of each of the TIRA and TIRB polypeptides or their active variants or fragments, while maintaining the ability of the mutant TIR polypeptides to confer disease resistance to the plant when co-expressed with each other. In a particular embodiment, the plant contains a polynucleotide encoding mutant TIRA and TIRB polypeptides, each of the TIRA and TIRB polypeptides having a loss-of-function mutation that reduces the NAD enzymatic activity of these polypeptides, wherein the polynucleotide contains a mutation in the TIRB1 domain of the TIRB polypeptide, such as a glutamate (E) to alanine (A) mutation (or its conservative variant) at the position corresponding to position 87 of SEQ ID NO:2, and no mutation in the TIRB2 domain of the TIRB polypeptide; and a mutation in the TIRA1 domain of the TIRA polypeptide, such as a glutamate (E) to alanine (A) mutation (or its conservative variant) at the position corresponding to position 85 of SEQ ID NO:1. In a particular embodiment, the plant additionally or optionally contains a gain-of-function mutation that increases the NAD enzymatic activity of the TIRA polypeptide, wherein the polynucleotide contains a gain-of-function mutation in the TIRA2 domain of the TIRA polypeptide, such as a valine (V) to glutamate (E) mutation (or its conservative variant) at the position corresponding to position 251 of SEQ ID NO:1.

[0344] In a particular embodiment, a plant containing a fusion protein contains the polynucleotide of SEQ ID NO:21, the fusion protein containing TIRA and TIRB polypeptides having modified NAD enzymatic activity.

[0345] 5. Methods for producing plants with increased disease resistance

[0346] The present invention provides methods for producing plants, plant parts, or seeds having increased disease resistance by introducing a nucleic acid sequence into a plant, a plant part, or a plant cell, the nucleic acid sequence comprising polynucleotides encoding a TIRA polypeptide as shown in SEQ ID NO:1 or an active variant or fragment thereof, and a TIRB polypeptide as shown in SEQ ID NO:2 or an active variant or fragment thereof, wherein co-expression of the TIRA polypeptide and the TIRB polypeptide increases the disease resistance of the plant. In other embodiments, the method comprises introducing a nucleic acid sequence into a plant, a plant cell, or a plant part, the nucleic acid sequence comprising a first polynucleotide as shown in any one of SEQ ID NO:3-5 and a second polynucleotide as shown in any one of SEQ ID NO:6-8 or a variant and fragment thereof, wherein co-expression of the first and second polypeptides increases the disease resistance of the plant.

[0347] The present invention also provides methods for producing plants, plant parts, or seeds having increased disease resistance by introducing a nucleic acid sequence into a plant, a plant part, or a plant cell, the nucleic acid sequence comprising polynucleotides encoding a TIRATIRB fusion protein as shown in any one of SEQ ID NO:9-12 or an active variant or fragment thereof, wherein expression of the TIRATIRB fusion protein increases the disease resistance of the plant. In other embodiments, the method comprises introducing a nucleic acid sequence into a plant, a plant cell, or a plant part, the nucleic acid sequence comprising a polynucleotide as shown in any one of SEQ ID NO:13-19 and 21, wherein expression of the fusion polypeptide encoded by the polynucleotide increases the disease resistance of the plant. In a particular embodiment, the fusion polypeptide comprises a cleavable linker sequence such that after expression of the fusion protein in a plant, a plant part, or a cell, the fusion protein is cleaved into constitutive TIRA and TIRB polypeptides, thereby expressing the TIR polypeptides at relative levels that result in increased disease resistance.

[0348] The nucleic acid sequence can be introduced into a plant cell in a variety of ways, such as by transformation, by genomic modification techniques (e.g., by genome editing or targeted integration), or by breeding. In one aspect, a plant can be produced by transforming a receptor plant with a nucleic acid sequence encoding the TIRA and TIRB polypeptides or the TIR fusion protein disclosed above. In one aspect, the method can comprise editing the genome of a receptor plant such that the resulting plant comprises polynucleotides encoding the TIRA and TIRB polypeptides or the fusion protein disclosed herein or an active variant or fragment thereof. In another aspect, the method can comprise breeding a donor plant comprising polynucleotides encoding the TIRA and TIRB polypeptides or an active variant or fragment thereof provided herein with a receptor plant and selecting for incorporation of the polynucleotide into the genome of the receptor plant.

[0349] a. Methods and compositions for increasing the expression and / or activity of a polypeptide of interest

[0350] The present invention provides methods and compositions for increasing plant disease resistance by increasing the expression and / or activity of TIRA polypeptide or its active variant or fragment, and TIRB polypeptide or its active variant or fragment.

[0351] In particular embodiments, the methods and compositions provided herein increase the co-expression of TIRA and TIRB polypeptides or their fragments and variants. As used herein, "increasing the expression of TIRA and TIRB polypeptides or active variants or fragments of TIRA and / or TIRB polypeptides", "increasing their co-expression", "the increased expression thereof" or "the increased co-expression thereof" means that the level of TIRA and TIRB polypeptides or active variants or fragments of either polypeptide produced by a given plant, plant cell, plant part or seed (including the absolute level of each polypeptide and the relative level of TIRA polypeptide relative to TIRB polypeptide) is statistically higher than the expression level compared to an appropriate control plant, plant part, plant cell or seed. In certain embodiments, the increase in expression or co-expression can include any statistically significant increase in polypeptide concentration (e.g., an increase in TIRA polypeptide concentration, an increase in TIRB polypeptide concentration, or an increase in the concentration of both polypeptides), which is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to an appropriate control. In other embodiments, the increase in expression can include an increase in the concentration level of the target protein of at least 1-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 175-fold, 200-fold or more compared to an appropriate control plant. Methods for determining an increase in the level or expression of the target polypeptide are known and include, for example, detecting the protein via an antibody or detecting an increase in the mRNA expression level encoding the protein.

[0352] In some embodiments, the increase in the expression of TIRA polypeptide or its active variant or fragment in a plant, plant part, plant cell or seed is comparable to the increase in the expression of TIRB polypeptide or its active variant or fragment, for example, where the increase in the expression of both polypeptides is at least 1%, between 1% and 10%, between 10% and 50%, between 25% and 75%, between 50% and 100%, at least 1-fold, between 1-fold and 10-fold, between 10-fold and 50-fold, between 25-fold and 75-fold, between 50-fold and 100-fold or higher compared to an appropriate control.

[0353] In other embodiments, an increase in the expression of the TIRA polypeptide or an active variant or fragment thereof in a plant, plant part, plant cell or seed is different from an increase in the expression of the TIRB polypeptide or an active variant or fragment thereof. In particular embodiments, the increase in the expression of the TIRA polypeptide (or an active variant or fragment thereof) is greater than the increase in the expression of the TIRB polypeptide (or an active variant or fragment thereof), such as in embodiments where the expression of the TIRA polypeptide or an active variant or fragment thereof is driven by a promoter different from the expression of the TIRB polypeptide or an active variant or fragment thereof. In exemplary embodiments, in embodiments where the expression of the TIRA and TIRB polypeptides is driven by different native promoters (e.g., the expression of the TIRA polypeptide is driven by prGCaRG3a (SEQ ID NO:26) and the expression of the TIRB polypeptide is driven by prGCaRG3b (SEQ ID NO:27)), or in embodiments where the expression of the TIRA polypeptide is driven by a constitutive promoter and the expression of the TIRB polypeptide is driven by an inducible promoter (e.g., prLuFIS (SEQ ID NO:25)), the increase in the expression level of the TIRA polypeptide relative to the expression level of the TIRB polypeptide is higher. In other embodiments, the increase in the expression of the TIRB polypeptide (or an active variant or fragment thereof) is less than the increase in the expression of the TIRA polypeptide (or an active variant or fragment thereof). In exemplary embodiments, in a plant, plant part, plant cell or seed, the increase in the expression of the TIRA polypeptide is 1 to 5 times the increase in the expression of the TIRB polypeptide. In other exemplary embodiments, the increase in the expression of the TIRB polypeptide is 0.5 times the increase in the expression of the TIRA polypeptide. In certain embodiments, the higher increase in the expression of the TIRA polypeptide relative to the increase in the expression level of the TIRB polypeptide results in a rebalancing of the expression of the TIRA polypeptide relative to the TIRB polypeptide compared to an appropriate control plant.

[0354] In other particular embodiments, in a plant, plant cell or plant part, the increase in the expression of the TIRA polypeptide or an active variant or fragment thereof and the increase in the expression of the TIRB polypeptide or an active variant or fragment thereof are driven by the expression of both polypeptides through a common bidirectional promoter (e.g., the bidirectional promoter of SEQ ID NO:49), wherein the nucleotide sequence encoding the TIRA polypeptide is operably coupled upstream of the bidirectional promoter and the nucleotide sequence encoding the TIRB polypeptide is operably coupled downstream of the promoter, or the nucleotide sequence encoding the TIRA polypeptide is operably coupled downstream of the bidirectional promoter and the nucleotide sequence encoding the TIRB polypeptide is operably coupled upstream of the promoter.

[0355] In other embodiments, methods and compositions are provided for increasing the activity of TIRA polypeptide or an active variant or fragment thereof and the activity of TIRB polypeptide or an active variant or fragment thereof in a plant, a plant cell, or a plant part. As used herein, "increasing the activity of TIRA and / or TIRB polypeptide or an active variant or fragment thereof" or "its increased activity" means that the level of enzymatic activity or protein functionality of the TIRA and / or TIRB polypeptide is statistically higher compared to an appropriate control. In particular embodiments, an increase in the activity of the TIRA polypeptide or an active variant or fragment thereof means one or more of the following: (i) an increase in the ability of the TIRA polypeptide (or an active variant or fragment thereof) to initiate an immune response in a plant cell, (ii) an increase in the ability of the TIRA polypeptide (or an active variant or fragment thereof) to confer plant disease resistance when co-expressed with the TIRB polypeptide (or an active variant or fragment thereof), and / or (iii) an increase in the NAD enzymatic activity of the polypeptide. In other particular embodiments, an increase in the activity of the TIRB polypeptide or an active variant or fragment thereof means one or more of the following: (i) an increase in the ability of the TIRB polypeptide (or an active variant or fragment thereof) to initiate an immune response in a plant cell, (ii) an increase in the ability of the TIRB polypeptide (or an active variant or fragment thereof) to confer plant disease resistance when co-expressed with the TIRA polypeptide (or an active variant or fragment thereof), and / or (iii) an increase in the NAD enzymatic activity of the polypeptide. In some embodiments, the increased activity means an increase in the ability to confer plant disease resistance when the TIRA and TIRB polypeptides (or active variants or fragments thereof) are co-expressed, while the NAD enzymatic activity of the TIRA and / or TIRB polypeptide (or active variant or fragment thereof) is decreased.

[0356] In certain embodiments, the increase in activity can include an increase in the level of enzymatic activity or protein functionality of the TIRA and / or TIRB polypeptide or an active variant or fragment thereof by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to an appropriate control plant, plant part, plant cell, or seed. In other embodiments, the increase in the activity of the TIRA and / or TIRB polypeptide or an active variant or fragment thereof when compared to an appropriate control plant, plant part, plant cell, or seed can include an increase in the level of enzymatic activity or protein functionality by at least 1-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 175-fold, 200-fold, or more. Methods for determining an increase in enzymatic activity or protein function include directly measuring the activity of a specific protein as well as indirectly measuring.

[0357] In still other embodiments, the increase in the activity and / or expression level of TIRA and TIRB polypeptides includes an increase in the expression level of a fusion protein comprising a TIRA polypeptide and a TIRB polypeptide or an active variant or fragment thereof (e.g., a fusion protein of any one of SEQ ID NOs: 9-12 or an active variant or fragment thereof). In particular embodiments, the fusion protein comprises a cleavable linker sequence such that after the fusion protein is expressed in a plant, plant part, or cell, the fusion protein is cleaved into the constitutive TIRA and TIRB polypeptides, thereby increasing the activity and / or relative expression level of the TIRA and TIRB polypeptides.

[0358] In some embodiments, increasing the activity and / or expression level of TIRA and TIRB polypeptides includes introducing a nucleic acid construct into a plant, resulting in an increase in the expression and / or activity of the TIRA and TIRB polypeptides or an active variant or fragment thereof. In other embodiments, increasing the activity and / or expression level of TIRA and TIRB polypeptides includes introducing a nucleic acid construct into a plant, resulting in an increase in the expression and / or activity of a fusion protein comprising a TIRA and a TIRB polypeptide or an active variant or fragment thereof. The nucleic acid construct can be stably integrated into the genome or provided transiently. For example, the nucleic acid construct can comprise a nucleic acid sequence encoding the TIRA polypeptide of SEQ ID NO: 1 or a variant or fragment thereof and the TIRB polypeptide of SEQ ID NO: 2 or an active variant or fragment thereof. In another example, the nucleic acid construct can comprise a nucleic acid sequence encoding a fusion protein of any one of SEQ ID NOs: 9-12 or an active variant or fragment thereof.

[0359] Accordingly, plants, plant parts, seeds and plant cells are provided that stably incorporate into their genome a polynucleotide operably linked to a promoter active in the plant, wherein the polynucleotide encodes (i) a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:1 or an active variant or active fragment thereof, and (ii) a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:2 or an active variant or active fragment thereof, wherein an increase in the expression or activity of the polypeptide increases the disease resistance and / or pathogen resistance of the plant. In some embodiments, plants, plant parts, seeds and plant cells are provided that stably incorporate into their genome a polynucleotide operably linked to a promoter active in the plant, wherein the polynucleotide encodes a fusion protein having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:9-12 or an active variant or active fragment thereof, wherein an increase in the expression or activity of the fusion protein increases the disease resistance and / or pathogen resistance of the plant.

[0360] After transformation, the target polypeptide is integrated into the genome and expressed in a manner that increases the activity or expression level of the TIRA and TIRB polypeptides in the plant.

[0361] Accordingly, there is provided a method for increasing the pathogen resistance of a plant by introducing a nucleic acid construct into the genome of the plant, the nucleic acid construct resulting in increased expression and / or activity of the following polypeptides: (i) a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NO:1, or an active variant or active fragment thereof, and (ii) a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NO:2, or an active variant or active fragment thereof, wherein the modification increases the pathogen resistance and / or disease resistance of the plant. In one embodiment, the method comprises introducing into the plant genome a nucleic acid sequence encoding the following polypeptides: (i) a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NO:1, or an active variant or active fragment thereof, and (ii) a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NO:2, or an active variant or active fragment thereof, wherein the increased activity or expression of the polypeptide increases the pathogen resistance and / or disease resistance of the plant. There is also provided a method for increasing the pathogen resistance of a plant by introducing a nucleic acid construct into the genome of the plant, the nucleic acid construct resulting in increased expression and / or activity of a fusion protein having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NO:9-12, or an active variant or active fragment thereof, wherein the modification increases the pathogen resistance and / or disease resistance of the plant. In some embodiments, an expression cassette comprising a promoter active in the plant is introduced into the genome of the plant, the promoter being operably linked to a polynucleotide of interest encoding a TIRA and TIRB polypeptide or an active variant or fragment thereof (e.g., an expression cassette comprising a promoter active in the plant is operably linked to a polynucleotide encoding a fusion protein comprising a TIRA and TIRB polypeptide, or a first expression cassette comprising a first promoter active in the plant is operably linked to a first polynucleotide encoding a TIRA polypeptide, and a second expression cassette comprising a second promoter active in the plant is operably linked to a second polynucleotide encoding a TIRB polypeptide).In other embodiments, the polynucleotide of interest can be introduced into the genome of a plant and integrated into a genomic location that permits expression of the polypeptide (e.g., by targeted integration).

[0362] In other embodiments, the method comprises introducing a nucleic acid construct that creates a modification in the genome of the plant, resulting in increased expression or increased activity of the following polypeptides: (i) a TIRA polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of SEQ ID NO:1, or an active variant or fragment thereof, and (ii) a TIRB polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of SEQ ID NO:2, or an active variant or fragment thereof, wherein the modification increases pathogen resistance of the plant.

[0363] In embodiments, methods are also provided for altering the relative expression levels of TIRA polypeptide and TIRB polypeptide or active fragments or variants thereof in a plant, thereby rebalancing their relative expression levels. In embodiments, the method rebalances the expression levels from an unbalanced expression level (where the expression of TIRB polypeptide exceeds the expression of TIRA polypeptide (e.g., the ratio is in the range of 5:1 to 20:1) or where the expression of TIRA polypeptide exceeds the expression of TIRB polypeptide (e.g., the ratio is in the range of 5:1 to 20:1)) to a balanced expression level of 0.5:1 to 2:1. In embodiments, the rebalancing method comprises introducing into the genome of a plant (e.g., a leguminous plant) a first heterologous nucleotide sequence encoding a TIRA polypeptide or an active variant thereof, the polypeptide or its active variant having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1; a second heterologous nucleotide sequence encoding a TIRB polypeptide or an active variant thereof, the polypeptide or its active variant having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2; and a heterologous promoter operably coupled to the first and / or second heterologous nucleotide sequence, wherein the expression level of the TIRA polypeptide or its active variant relative to the expression level of the TIRB polypeptide or its active variant is rebalanced in the plant, plant part or seed as compared to the expression level in a control plant, plant part or seed, wherein the rebalanced expression level of the TIRA polypeptide or its active variant relative to the TIRB polypeptide or its active variant comprises a ratio of about 0.5:1 to about 2:1, and wherein the TIRA polypeptide or its active variant confers disease resistance to the leguminous plant, plant or seed when co-expressed with the TIRB polypeptide or its active variant, and wherein the TIRA polypeptide or its active variant confers disease resistance to the leguminous plant, plant or seed when co-expressed with the TIRA polypeptide or its active variant.

[0364] b. Screening methods

[0365] In some embodiments, methods are provided for screening plants comprising polynucleotides encoding TIRA and TIRB polypeptides, wherein the TIR polypeptides are expressed at balanced levels, thereby conferring increased disease resistance while also improving agronomic performance.

[0366] In particular embodiments, a method of screening for plants having increased disease resistance (e.g., increased ASR resistance or increased powdery mildew resistance or bacterial pathogen resistance or nematode resistance or sucking pest resistance) and increased agronomic performance includes introducing into the plant genome a nucleic acid molecule comprising a heterologous promoter operably coupled to (i) a first polynucleotide encoding a TIRA polypeptide or an active variant thereof, the polypeptide or its active variant comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1; and (ii) a second polynucleotide encoding a TIRB polypeptide or an active variant thereof, the polypeptide or its active variant comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2. In embodiments, the first and second polynucleotides can be introduced via different expression cassettes or via a single expression cassette. In embodiments, the first and second polynucleotides can be linked so as to express the TIRA and TIRB polypeptides or fragments or variants thereof as a fusion protein, as discussed previously herein. In embodiments, the fusion protein comprises a self-cleaving linker that is cleaved after expression of the fusion protein, thereby releasing the constitutive TIR polypeptides into distinct polypeptides.

[0367] The screening method further includes determining the expression level of the TIRA polypeptide or an active variant thereof in the plant, as well as the expression level of the TIRB polypeptide or an active variant thereof in the plant. Any known method for determining expression levels can be used, including but not limited to estimating the transcriptional level (e.g., via qRT-PCR, RNA-seq, RNA blot, etc.). The screening method further includes, after estimating the expression level of the TIR polypeptides, comparing the expression level of the TIRA polypeptide or an active variant thereof in the plant relative to the expression level of the TIRB polypeptide or an active variant thereof in the plant to determine whether the relative expression levels are balanced. In particular embodiments, it is determined whether the relative expression level of the TIRA polypeptide or an active variant thereof in the plant relative to the expression level of the TIRB polypeptide or an active variant thereof is between 0.5:1 and 2:1, thereby determining the balanced expression level of the TIRA polypeptide or an active variant thereof relative to the expression level of the TIRB polypeptide in the plant. The screening method further includes selecting plants having a balanced expression level of the TIRA polypeptide or an active variant thereof relative to the TIRB polypeptide or an active variant thereof. Such plants having a balanced expression level of the TIR polypeptides have increased disease resistance and enhanced agronomic performance (e.g., reduced growth defects and higher yields).

[0368] In particular embodiments, a method of generating a plant having increased disease resistance and increased agronomic performance includes: (a) introducing into the genome of a plant a nucleic acid molecule comprising a heterologous promoter operably coupled to (i) a first polynucleotide encoding a TIRA polypeptide or an active variant thereof, the polypeptide or active variant thereof comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1 and further comprising one or more mutations at positions corresponding to positions 85 and / or 271 of SEQ ID NO:1, wherein the one or more mutations alter the NAD enzymatic activity of the TIRA polypeptide or an active variant thereof; and (ii) a second polynucleotide encoding a TIRB polypeptide or an active variant thereof, the polypeptide or active variant thereof comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2 and further comprising a mutation at a position corresponding to position 87 of SEQ ID NO:2, wherein the mutation reduces the NAD enzymatic activity of the TIRB polypeptide or an active variant thereof. The method further includes determining the expression level of the TIRA polypeptide or an active variant thereof in the plant, as well as the expression level of the TIRB polypeptide or an active variant thereof in the plant; and comparing the expression level of the TIRA polypeptide or an active variant thereof in the plant relative to the expression level of the TIRB polypeptide or an active variant thereof to determine if the relative expression level is between 0.5:1 and 2:1, thereby determining the balanced expression level of the TIRA polypeptide or an active variant thereof relative to the TIRB polypeptide in the plant. A plant having a balanced expression level of the TIRA polypeptide or an active variant thereof relative to the TIRB polypeptide or an active variant thereof is thereby selected.

[0369] In embodiments, methods are also provided for screening and selecting polynucleotides, nucleic acid molecules, and compositions that confer increased disease resistance and also confer increased agronomic performance when expressed in a plant.

[0370] In a particular embodiment, a method of generating a TirA-TirB composition is provided that, when introduced into a plant, is capable of conferring increased disease resistance and enhanced agronomic performance. The method includes generating a first polynucleotide encoding a TIRA polypeptide or an active variant thereof and a second polynucleotide encoding a TIRB polypeptide or an active variant thereof, where the TIRA polypeptide or an active variant thereof comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1, and the TIRB polypeptide or an active variant thereof comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2.

[0371] The screening method further includes mutating the first polynucleotide to introduce one or more mutations at positions corresponding to position 85 and / or position 271 of SEQ ID NO:1, where the one or more mutations alter the NAD enzymatic activity of the TIRA polypeptide or an active variant thereof. In a particular embodiment, the first polynucleotide is mutated to introduce loss-of-function mutations at positions corresponding to position 85 and / or 271 of SEQ ID NO:1. In a specific embodiment, when the TIRA polypeptide or variant thereof comprises a Glu residue at the position corresponding to position 85 of SEQ ID NO:1, where the Glu residue is predicted to have NAD enzymatic cleavage activity, a loss-of-function E85A mutation that reduces the NAD enzymatic activity of the TIRA polypeptide is introduced. In another exemplary embodiment, when the TIRA polypeptide or variant thereof comprises a Glu residue at the position corresponding to position 271 of SEQ ID NO:1, where the Glu residue is predicted to have NAD enzymatic cleavage activity, a loss-of-function E271A mutation that reduces the NAD enzymatic activity of the TIRA polypeptide can be introduced. In yet another exemplary embodiment, when the TIRA polypeptide or variant thereof comprises a non-Glu residue (e.g., a Val residue) at the position corresponding to position 271 of SEQ ID NO:1, where the Val residue is predicted not to have NAD enzymatic cleavage activity, a gain-of-function V271E mutation that increases the NAD enzymatic activity of the TIRA polypeptide is introduced.

[0372] The screening method further comprises mutating the second polynucleotide to introduce a mutation at the position corresponding to position 87 of SEQ ID NO:2, wherein the mutation reduces the NAD enzymatic activity of the TIRB polypeptide or an active variant thereof. In a particular embodiment, when the TIRB polypeptide or a variant thereof comprises a Glu residue at the position corresponding to position 87 of SEQ ID NO:2, wherein the Glu residue is predicted to have NAD enzymatic cleavage activity, a loss-of-function E87A mutation that reduces the NAD enzymatic activity of the TIRB polypeptide is introduced.

[0373] The screening method further comprises expressing in a plant, via an operably linked heterologous promoter, a first polynucleotide comprising one or more mutations and a second polynucleotide comprising one mutation. In an embodiment, the first and second polynucleotides are expressed as separate expression cassettes or via a single expression cassette. In other embodiments, the first and second polynucleotides are ligated to express the TIR polypeptide as a fusion protein. In an embodiment, the fusion protein comprises a self-cleaving linker that is cleaved after expression of the fusion protein, thereby releasing the constitutive TIR polypeptide into distinct polypeptides.

[0374] In an embodiment, the screening method further comprises screening plants for plants having a balanced expression level of the TIRA polypeptide or an active variant thereof relative to the TIRB polypeptide or an active variant thereof, wherein the balanced expression level comprises a ratio of 0.5:1 to 2:1. By selecting a composition comprising a polynucleotide encoding a TIR polypeptide, which when expressed in a plant results in the TIR polypeptide being expressed at a balanced relative expression level, plants into whose genome the composition has been introduced can be generated, thereby conferring increased pathogen resistance and enhanced agronomic performance to the plants.

[0375] c. Transformation method

[0376] In some embodiments, a method of introducing a polynucleotide into a plant comprises transforming a polynucleotide disclosed herein or an active variant or fragment thereof into a recipient plant to obtain a transgenic plant, and the transgenic plant has increased pathogen resistance and / or disease resistance. An expression cassette comprising a polynucleotide encoding a polypeptide as described above can be used for transforming a target plant.

[0377] Plants generated by transformation that incorporate heterologous nucleic acids, including whole plants as well as plant organs (such as leaves, stems, roots, etc.), seeds, plant cells, propagules, and their embryos and progeny. The plant cells can be differentiated or undifferentiated (such as callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). Transformation can result in the stable or transient incorporation of nucleic acids into the cells. "Stable transformation" is intended to mean that the nucleotide construct introduced into the host cell integrates into the genome of the host cell and can be inherited by its progeny. In some embodiments, stable transformation occurs via random integration events. In other embodiments, stable transformation is achieved by targeted integration of the target sequence into the genome using genome modification mechanisms (such as CRISPR or TALEN). "Transient transformation" is intended to mean that the polynucleotide is introduced into the host cell without integrating into the genome of the host cell.

[0378] Methods for transformation typically involve introducing a nucleotide construct into a plant. In some embodiments, the transformation method is Agrobacterium-mediated transformation. In some embodiments, the transformation method is gene gun-mediated transformation. Transformation can also be carried out by: infection, transfection, microinjection, electroporation, microprojection, gene gun or particle bombardment, electroporation, silica / carbon fiber, ultrasound-mediated, PEG-mediated, calcium phosphate co-precipitation, polycation DMSO technology, DEAE dextran procedure, Agrobacterium and virus-mediated (such as, cauliflower virus, geminivirus, RNA plant virus), liposome-mediated, etc.

[0379] Transformation protocols, as well as protocols for introducing polypeptide or polynucleotide sequences into plants, can vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocotyledonous or dicotyledonous plants). Methods for transformation are known in the art and include those shown in the following: U.S. Patent Nos. 8,575,425; 7,692,068; 8,802,934; and 7,541,517; each of which is incorporated herein by reference. See also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. [Cellular and Molecular Biology Letters] 7:849-858; Jones et al. (2005) Plant Methods [Plant Methods], Vol. 1, Article 5; Rivera et al. (2012) Physics of Life Reviews [Reviews on the Physics of Life] 9:308-345; Bartlett et al. (2008) Plant Methods [Plant Methods] 4:1-12; Bates, G.W. (1999) Methods in Molecular Biology [Methods in Molecular Biology] 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology [Annual Review of Microbiology] 42:57Sup' / Sup5-606; Christou, P. (1992) The Plant Journal [The Plant Journal] 2:275-281; Christou, P. (1995) Euphytica [Euphytica] 85:13-27; Tzfira et al. (2004) TRENDS in Genetics [Trends in Genetics] 20:375-383; Yao et al. (2006) Journal of Experimental Botany [Journal of Experimental Botany] 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology [Plant Physiology] 107:1041-1047.

[0380] Methods for the transformation of plant cells or tissues include, but are not limited to, Agrobacterium-mediated transformation methods and gene gun or particle gun-mediated transformation methods. Suitable plant transformation vectors for the purpose of Agrobacterium-mediated transformation include those elements derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens, such as the right border (RB) region and the left border (LB) region, as well as other elements disclosed by Herrera-Estrella et al., Nature 303:209 (1983); Bevan, Nucleic Acids Res. 12:8711-8721 (1984); Klee et al., Bio-Technology 3(7):637-642 (1985). In addition to plant transformation vectors derived from Agrobacterium Ti or root-inducing (Ri) plasmids, alternative methods can be used to insert the DNA constructs of the present invention into plant cells. These methods can involve, but are not limited to, for example, the use of liposomes, electroporation, chemicals that increase the uptake of free DNA, delivery of free DNA by microprojectile bombardment, and transformation using viruses or pollen.

[0381] Methods for the transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87(21):8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90(3):913-917; Staub and Maliga (1993) EMBO J. 12(2):601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome by homologous recombination. In addition, transactivation of plastid-borne transgenes can be achieved by tissue-preferred expression of a nuclear-encoded, plastid-localized RNA polymerase, and plastid transformation can be completed. Such a system has been reported by McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91(15):7301-7305.

[0382] The transformed cells can be cultured into plants in a conventional manner. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown, pollinated with the same transformed line or a different line, and the resulting hybrids with the desired phenotypic characteristics identified. They can be grown for two or more generations to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and then the seeds are harvested to ensure that the expression of the desired phenotypic characteristics has been achieved. In this way, the present invention provides transformed seeds (also referred to as "transgenic seeds") that have the nucleotide constructs of the present invention, such as the expression cassettes of the present invention, stably incorporated into their genomes.

[0383] "Regeneration" refers to the process of growing a plant from a plant cell (e.g., a plant protoplast or an explant). Such regeneration techniques rely on the manipulation of certain plant hormones in the tissue culture growth medium and typically rely on biocide and / or herbicide markers that have been introduced together with the desired nucleotide sequence. The method selection for the regeneration step is not critical. See, for example, Ammirato et al., Handbook of Plant Cell Culture—Crop Species. Macmillan Publ. Co. (1984); Shimamoto et al., Nature 338:274-276 (1989); Fromm, UCLA Symposium on Molecular Strategies for Crop Improvement, April 16-22, 1990. Keystone, Colo. (1990); Vasil et al., Bio / Technology 8:429-434 (1990); Vasil et al., Bio / Technology 10:667-674 (1992); Hayashimoto, Plant Physiol. 93:857-863 (1990); and Datta et al., Bio-technology 8:736-740 (1990). Such regeneration techniques are generally described in Klee et al., Ann. Rev. Plant Phys. 38:467-486 (1987).

[0384] d. Hybridization

[0385] In some embodiments, the method includes hybridizing a donor plant comprising a polynucleotide encoding the following: (i) a TIRA polypeptide or an active variant or fragment thereof and a TIRB polypeptide or an active variant or fragment thereof or (ii) a TIRATIRB fusion protein, and the polypeptide or fusion protein is capable of conferring increased pathogen resistance to the recipient plant. As used herein, the terms “hybridize” and “breed” refer to the fusion of gametes to produce progeny (e.g., by fertilization, such as the production of seeds by pollination in plants). In some embodiments, “hybridize,” “breed,” or “cross-fertilize” is the fertilization of one individual by another (e.g., cross-pollination in plants). The plants disclosed herein can be whole plants, or can be plant cells, seeds or tissues, or plant parts, such as leaves, stems, pollen, or cells that can be grown into whole plants.

[0386] In some embodiments, the progeny plants produced by the hybridization or breeding method are repeatedly backcrossed to one of their parents by a method herein referred to as “backcrossing.” In a backcrossing scheme, the “donor” parent refers to the parent plant having the desired gene or locus to be introgressed. The “recipient” parent (used once or more) or “recurrent” parent (used twice or more) refers to the parent plant into which the gene or locus is introgressed. See, for example, Ragot, M. et al., Marker-assisted Backcrossing: A Practical Example, Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backcross Breeding, Proceedings of the Symposium “Analysis of Molecular Marker Data,” pp. 41-43 (1994). The initial hybridization produces the F1 generation. The term “BC1” refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on.

[0387] In some embodiments, the donor soybean plant is a soybean plant. In some embodiments, the donor soybean plant is a wild soybean plant. In some embodiments, the recipient soybean plant is a elite soybean plant or a elite wild soybean plant.

[0388] e. Gene editing

[0389] Further provided are plants, plant cells, and seeds having genome modifications created by gene editing. Such methods include, but are not limited to, meganucleases, TALENs, and other techniques for precise genome editing designed for a target plant genomic sequence by CRISPR-Cas9 (Feng et al., Cell Research 23:1229-1232, 2013, WO2013 / 026740); Cre-lox site-specific recombination; FLP-FRT recombination (Li et al. (2009) Plant Physiol 151:1087-1095); Bxbl-mediated integration (Yau et al., Plant J (2011) 701:147-166); zinc finger-mediated integration (Wright et al. (2005) Plant J 44:693-705; Cai et al. (2009) Plant Mol Biol 69:699-709); and homologous recombination (Lieberman-Lazarovich and Levy (2011) Methods Mol Biol:51-65).

[0390] Various embodiments of the methods described herein use gene editing. In some embodiments, gene editing is used to mutagenize the genome of a plant to produce a plant having one or more of the polypeptides capable of increasing the disease resistance of the plant. In other cases, gene editing is used to allow the targeted insertion of a nucleotide sequence encoding TIRA and TIRB polypeptides, or a TIRATIRB fusion protein or an active variant or fragment thereof, into the genome.

[0391] "Target site", "target sequence", "target DNA", "target locus", "genomic target site", "genomic target sequence", and "genomic target locus" are used interchangeably herein, and refer to, for example, a polynucleotide sequence in the genome of a cell (including chloroplast and mitochondrial DNA), where an endonuclease is recruited and optionally nicks or cuts the DNA of the target site. The target site can be an endogenous site in the plant genome, or alternatively, the target site can be heterologous to the plant and thus not naturally occurring in the genome, or the target site can be located at a heterologous genomic location compared to its location in nature.

[0392] In some embodiments, provided herein are plants transformed with the gene editing machinery described above and expressing the same, which, when hybridized with a target plant, cause gene editing to occur in the target plant.

[0393] The term "polynucleotide modification template" includes a polynucleotide that contains at least one nucleotide modification compared to the nucleotide sequence to be edited. The nucleotide modification can be at least one nucleotide substitution, addition, or deletion. The polynucleotide modification template can further include homologous nucleotide sequences flanked by at least one nucleotide modification, where the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

[0394] Gene editing generally refers to the use of site-specific nucleases (including but not limited to CRISPR / Cas, zinc fingers, mega nucleases, etc.) to cleave a nucleotide sequence at a desired location. This can result in insertion / deletion ("indel") mutations (i.e., "SDN1"), base editing (i.e., "SDN2"), or allele insertion or replacement (i.e., "SDN3"). SDN2 or SDN3 gene editing can include providing one or more recombinant templates (e.g., in a vector) that contain a target gene sequence (i.e., to be introduced into the plant genome) that can be used for homologous directed repair (HDR) within a plant. In some embodiments, the target gene or allele is a gene or allele capable of conferring an improved trait to the plant (e.g., increased disease resistance). The recombinant template can be introduced into the plant by transformation or breeding of a donor plant containing the recombinant template. Double-strand DNA breaks can be introduced into the interior, upstream, and / or downstream of the target sequence in the plant genome. In some embodiments, double-strand DNA breaks are generated within or near the target sequence locus. In some embodiments, breaks are generated upstream and downstream of the target sequence locus, which can result in its excision from the genome. In some embodiments, one or more single-strand DNA breaks (nicks) are generated within, upstream, and / or downstream of the target sequence (e.g., using a nickase Cas9 variant). Any of these DNA breaks, as well as those introduced by other methods known to those skilled in the art, can induce HDR. Through HDR, the target sequence is replaced by the sequence of the provided recombinant template, which contains the target polynucleotide, e.g., any one of SEQ ID NOs: 8-13 and 15-22 or variants or fragments thereof can be provided on / as the template. By designing the system such that one or more single-strand or double-strand breaks are introduced into the interior, upstream, and / or downstream of the corresponding region in the plant genome that does not contain the target gene sequence, this region can be replaced with the template.

[0395] In some embodiments, mutations in the target genes described herein can be generated by targeted introduction of double-strand DNA breaks without the use of a recombinant template. Such breaks can be repaired by the non-homologous end joining (NHEJ) process, which may result in small insertions or deletions (indels) at the repair site. Such indels may result in frameshift mutations, leading to premature stop codons or other types of loss-of-function mutations in the targeted gene.

[0396] In some embodiments, gene editing may involve transient, inducible, or constitutive expression of gene editing components or systems in a target plant. Gene editing may also involve genomic integration or episomal presence of gene editing components or systems in a target plant.

[0397] In certain embodiments, nucleic acid modification or mutagenesis is achieved by a (modified) zinc finger nuclease (ZFN) system. The ZFN system uses an artificial restriction endonuclease that is generated by fusing a zinc finger DNA-binding domain to a DNA cleavage domain, which can be engineered to target a desired DNA sequence. Exemplary methods of using ZFNs for genome editing can be found, for example, in U.S. Patent Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; and 6,979,539.

[0398] In certain embodiments, nucleic acid modification is achieved by a (modified) meganuclease, which is a deoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of 12 to 40 base pairs). Exemplary methods of using meganucleases can be found in U.S. Patent Nos.: 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134, which are hereby incorporated by reference in their entirety.

[0399] In certain embodiments, nucleic acid modification is achieved by a (modified) CRISPR / Cas complex or system. In certain embodiments, the CRISPR / Cas system or complex is a type II CRISPR / Cas system. In certain embodiments, the CRISPR / Cas system or complex is a type II, V, or VI CRISPR / Cas system or complex. The CRISPR / Cas system does not require the generation of custom proteins to target specific sequences, but rather can program a single Cas protein via an RNA-guided sequence (gRNA) to recognize a specific nucleic acid target. In other words, the short RNA-guided sequence can be used to recruit the Cas enzyme protein to a specific nucleic acid target locus of interest (which may comprise RNA and / or DNA or consist thereof).

[0400] Generally, CRISPR / Cas or a CRISPR system as used herein collectively refers to transcripts and other elements involved in the expression of a CRISPR-associated ("Cas") gene or that direct its activity, including the sequence encoding the Cas gene and one or more of the following: a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or the active portion of tracrRNA), a tracr-pairing sequence (which includes "direct repeats" and the tracrRNA-processed portion of direct repeats in the context of an endogenous CRISPR system), a guide sequence (also referred to as "spacer" in the context of an endogenous CRISPR system), or one or more RNAs as used herein (e.g., one or more RNAs for guiding a Cas such as Cas9, e.g., CRISPR RNA and, where applicable, trans-activating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus. Generally, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the case of forming a CRISPR complex, a "target sequence" refers to the sequence to which the guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. The target sequence can comprise any polynucleotide, such as DNA or RNA polynucleotide.

[0401] In certain embodiments, the gRNA is a chimeric guide RNA or a single guide RNA (sgRNA). In certain embodiments, the gRNA comprises a guide sequence and a tracr-pairing sequence (or direct repeats). In certain embodiments, the gRNA comprises a guide sequence, a tracr-pairing sequence (or direct repeats), and a tracr sequence. In certain embodiments, a CRISPR / Cas system or complex as described herein does not comprise and / or is not dependent on the presence of a tracr sequence (e.g., if the Cas protein is Cas12a).

[0402] Cas proteins as referred to herein, such as but not limited to Cas9, Cas12a (formerly known as Cpf1), Cas12b (formerly known as C2c1), Cas13a (formerly known as C2c2), C2c3, Cas13b proteins, can be derived from any suitable source and thus can include different orthologs from a variety of (prokaryotic) organisms, as is well documented in the art. In certain embodiments, the Cas protein is (modified) Cas9, preferably (modified) Staphylococcus aureus Cas9 (SaCas9) or (modified) Streptococcus pyogenes Cas9 (SpCas9). In certain embodiments, the Cas protein is Cas12a, optionally from the species Acidaminococcus, such as Acidaminococcus species BV3L6 Cpf1 (AsCas12a), or Lachnospiraceae bacterium Cas12a, such as Lachnospiraceae bacterium MA2020 or Lachnospiraceae bacterium MD2006 (LBCas12a). See U.S. Patent No. 10,669,540, which is incorporated herein by reference in its entirety. Alternatively, the Cas12a protein can be from Moraxella bovoculi AAX08_00205 [Mb2Cas12a] or Moraxella bovoculi AAX11_00205 [Mb3Cas12a]. See, WO2017 / 189308, which is incorporated herein by reference in its entirety. In certain embodiments, the Cas protein is (modified) C2c2, preferably Leptotrichia wadei C2c2 (LwC2c2) or Listeria newyorkensis FSL M6-0635 C2c2 (LbFSLC2c2). In certain embodiments, the (modified) Cas protein is C2c1. In certain embodiments, the (modified) Cas protein is C2c3. In certain embodiments, the (modified) Cas protein is Cas13b. Other Cas enzymes can be obtained by those skilled in the art.

[0403] Gene editing methods and compositions are also disclosed in U.S. Patent Nos. 10,519,456 and 10,285,348, the entire contents of which are incorporated herein by reference.

[0404] The gene editing machinery introduced into plants (e.g., DNA modifying enzymes) can be controlled by any promoter capable of driving the expression of a recombinant gene in plants. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter, such as a pollen-specific promoter or a sperm cell-specific promoter, a zygote-specific promoter, or a promoter highly expressed in sperm, egg, and zygote (e.g., prOsActin1). Suitable promoters are disclosed in U.S. Patent No. 10,519,456, the entire content of which is incorporated herein by reference.

[0405] In another aspect, provided herein are methods of editing plant genomic DNA. In some embodiments, the method comprises pollinating a target plant comprising genomic DNA to be edited with a first soybean plant expressing a DNA modifying enzyme and at least one optional guide nucleic acid as described above.

[0406] 6. Stacking

[0407] Polynucleotides encoding the TIRA polypeptide, TIRB polypeptide, and / or TIRATIRB fusion protein provided herein, as well as variants and fragments of the TIRA polypeptide, TIRB polypeptide, and / or TIRATIRB fusion protein, can be stacked with one or more polynucleotides encoding desired traits, such as polynucleotides conferring, for example, pest, disease, or herbicide resistance or other desired agronomic traits, which agronomic traits include, but are not limited to, the following: traits associated with high oil content; traits associated with increased protein content, increased digestibility; balanced amino acid content; improved drought resistance, altered maturity and / or flowering time, and high energy content. Such traits can refer to characteristics of seed and non-seed plant tissues, or characteristics of food or feed prepared from plants or seeds having such traits.

[0408] As used herein, "stacking" of genes or traits involves combining desired genes or traits into a transgenic plant line. Additional polynucleotides can be introduced by a variety of methods, including by transgenic means, by breeding, or by genome editing. As one method, plant breeders stack transgenic traits (so-called "breeding stack") by crossing between parents (each parent having a desired trait) and then identifying the offspring having both desired traits. Another way to stack genes is to transfer two or more genes into the nucleus of a plant simultaneously during transformation. In embodiments, two or more genes can be transferred via different expression cassettes or via a common expression cassette. Another way to stack genes is to re-transform a transgenic plant containing a desired trait with another transgene conferring another desired trait, thereby providing progeny transgenic plants containing a combination of traits. Such methods can include, for example, random integration techniques or targeted integration via a gene editing system (such as Crispr or meganuclease). For ex...

Claims

1. A nucleic acid molecule comprising a nucleotide sequence operably linked to a heterologous regulatory element, wherein the nucleotide sequence comprises: (a) a first polynucleotide encoding a TIRA polypeptide or an active variant thereof, the polypeptide or its active variant comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1; and (b) a second polynucleotide encoding a TIRB polypeptide or an active variant thereof, the polypeptide or its active variant comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2, Among them, introducing the nucleic acid molecule into a plant results in rebalancing in the plant of the expression level of the TIRA polypeptide or its active variant relative to the expression level of the TIRB polypeptide or its active variant compared to the expression level in a control plant, wherein the rebalanced expression level of the TIRA polypeptide or its active variant comprises a ratio of about 0.5:1 to about 2:1 relative to the TIRB polypeptide or its active variant, and wherein the nucleic acid molecule confers disease resistance when expressed in the plant.

2. The nucleic acid molecule according to claim 1, wherein the first polynucleotide has a nucleotide sequence comprising: (i) a nucleotide sequence encoding a TIRA polypeptide having at least 80% sequence identity to SEQ ID NO: 1; (ii) a nucleotide sequence encoding the TIRA polypeptide of SEQ ID NO: 1; or (iii) the nucleotide sequence of (i) or (ii), and further comprising a mutation that alters the NAD enzymatic activity of the TIRA polypeptide; and wherein the second polynucleotide has a nucleotide sequence comprising: (iv) a nucleotide sequence encoding a TIRB polypeptide having at least 80% sequence identity to SEQ ID NO: 2; (v) a nucleotide sequence encoding the TIRB polypeptide of SEQ ID NO: 2; or (vi) the nucleotide sequence of (iv) or (v), and further comprising a loss-of-function mutation that reduces the NAD enzymatic activity of the TIRB polypeptide.

3. The nucleic acid molecule according to claim 2, wherein the TIRA polypeptide or its active variant confers disease resistance to the plant when co-expressed with the TIRB polypeptide or its active variant, and wherein the TIRB polypeptide or its active variant confers disease resistance to the plant when co-expressed with the TIRA polypeptide or its active variant.

4. The nucleic acid molecule according to any one of claims 2-3, wherein the TIRA polypeptide or its active variant and the TIRB polypeptide or its active variant are expressed as a fusion protein.

5. The nucleic acid molecule according to claim 4, wherein the TIRA polypeptide or its active variant is at the N-terminus of the fusion protein or wherein the TIRB polypeptide or its active variant is at the N-terminus of the fusion protein.

6. The nucleic acid molecule according to any one of claims 1-5, wherein the heterologous regulatory element is a heterologous promoter that is active in the plant, and wherein the heterologous promoter is a constitutive promoter, an inducible promoter, or an endogenous promoter, and wherein the inducible promoter is optionally a rust-inducible promoter.

7. The nucleic acid molecule according to claim 6, wherein the heterologous promoter is selected from the group consisting of SEQ ID NOs: 22-27.

8. The nucleic acid molecule according to any one of claims 2-3, wherein the heterologous regulatory element comprises a first heterologous promoter that is active in the plant, which is operably linked to a first polynucleotide in a first expression cassette; and a second heterologous promoter that is active in the plant, which is operably coupled to a second polynucleotide in a second expression cassette.

9. The nucleic acid molecule according to claim 8, wherein the second expression cassette is located transcriptionally upstream of the first expression cassette.

10. The nucleic acid molecule according to any one of claims 8-9, wherein the first heterologous promoter is a constitutive promoter, an inducible promoter, or an endogenous promoter; wherein the second heterologous promoter is a constitutive promoter, an inducible promoter, or an endogenous promoter; and wherein the inducible promoter is optionally a rust-inducible promoter.

11. The nucleic acid molecule according to claim 10, wherein the first heterologous promoter is selected from the group consisting of SEQ ID NOs: 22-24 and 26-27, and wherein the second heterologous promoter is SEQ ID NO: 25 or 27.

12. The nucleic acid molecule according to any one of claims 2-11, wherein the mutation that alters the NADase activity of the TIRA polypeptide or its active variant comprises one or more of the following: (i) a loss-of-function mutation at the NADase site in the TIRA1 region and / or the TIRA2 region of the TIRA polypeptide or its active variant, and (ii) a gain-of-function mutation at the NADase site in the TIRA2 region of the TIRA polypeptide or its active variant; wherein the loss-of-function mutation that reduces the NADase activity of the TIRB polypeptide or its active variant comprises a loss-of-function mutation at the NADase site in the TIRB1 region of the TIRB polypeptide or its active variant, and wherein the TIRB polypeptide or its active variant does not comprise a loss-of-function mutation at the NADase site in the TIRB2 region of the TIRB polypeptide or its active variant.

13. The nucleic acid molecule according to any one of claims 1-12, wherein one or more of the TIRA polypeptide, the TIRB polypeptide, or their active variants are labeled with a detectable label.

14. A vector comprising the nucleic acid molecule according to any one of claims 1-13.

15. A transgenic cell comprising the nucleic acid according to any one of claims 1-13 or the vector according to claim 14, wherein the transgenic cell is a transgenic plant cell, and wherein the transgenic plant cell is a transgenic plant cell of a leguminous plant, optionally wherein the leguminous plant is selected from the group comprising: alfalfa, clover, pea, lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

16. A plant, or a plant part, derived from the transgenic cell according to claim 15, wherein the plant part is a transgenic seed, and wherein the transgenic seed has stably integrated the first polynucleotide and the second polynucleotide into its genome.

17. A harvested product derived from the transgenic seed according to claim 16, wherein the harvested product comprises the first and the second polynucleotides.

18. A processed product derived from the harvested product according to claim 17, wherein the processed product is flour, meal, oil, starch, or a product derived from any of the foregoing, and wherein the processed product comprises the first polynucleotide and the second polynucleotide.

19. A fusion protein comprising: (a) a TIRA polypeptide or an active variant thereof, the polypeptide or its active variant comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1; and (b) a TIRB polypeptide or an active variant thereof, the polypeptide or its active variant comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2, wherein expression of the fusion protein in a plant confers upon the plant increased disease resistance relative to a control plant that does not express the fusion protein.

20. The fusion protein according to claim 19, wherein the TIRA polypeptide or its active variant is at the N-terminus of the fusion protein.

21. The fusion protein according to claim 20, wherein the fusion protein comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:

12.

22. The fusion protein according to claim 19, wherein the TIRB polypeptide or its active variant is at the N-terminus of the fusion protein.

23. The fusion protein according to claim 22, wherein the fusion protein comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 9-11.

24. The fusion protein according to any one of claims 19 - 23, wherein in the plant, compared to the expression level in a control plant, the expression of the fusion protein in the plant re - balances the expression level of the TIRA polypeptide or its active variant relative to the TIRB polypeptide or its active variant, and wherein the re - balanced expression level of the TIRA polypeptide or its active variant relative to the TIRB polypeptide or its active variant comprises a ratio of about 0.5:1 to about 2:

1.

25. The fusion protein according to any one of claims 19 - 24, further comprising a cleavable linker that couples the TIRA polypeptide or its active variant of (a) to the TIRB polypeptide or its active variant of (b), wherein the fusion protein is cleaved at the cleavable linker, and then in the plant, compared to the expression level in a control plant, the expression of the fusion protein in the plant re - balances the expression level of the TIRA polypeptide or its active variant relative to the TIRB polypeptide or its active variant, and wherein the re - balanced expression level of the TIRA polypeptide or its active variant relative to the TIRB polypeptide or its active variant comprises a ratio of about 0.5:1 to about 2:

1.

26. The fusion protein according to any one of claims 19 - 24, wherein the TIRA polypeptide or its active variant has an amino acid sequence comprising: i. an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1; ii. the amino acid sequence of SEQ ID NO:1; or iii. the amino acid sequence of (i) or (ii) and further comprising a mutation that alters the NAD - enzyme activity of the TIRA polypeptide, wherein the mutation comprises a loss - of - function mutation that reduces the NAD - enzyme activity of the TIRA polypeptide and / or a gain - of - function mutation that increases the NAD - enzyme activity of the TIRA polypeptide; and wherein the TIRB polypeptide or its active variant has an amino acid sequence comprising: iv. an amino acid sequence having at least 80% sequence identity with SEQ ID NO:2; v. the amino acid sequence of SEQ ID NO:2; or vi. the amino acid sequence of (iv) or (v), and further comprising a loss - of - function mutation that reduces the NAD - enzyme activity of the TIRB polypeptide, wherein the TIRA polypeptide or its active variant confers disease resistance to the plant when co - expressed with the TIRB polypeptide or its active variant, and wherein the TIRB polypeptide or its active variant confers disease resistance to the plant when co - expressed with the TIRA polypeptide or its active variant.

27. The fusion protein according to claim 26, wherein the mutation that alters the NAD enzyme activity of the modified TIRA polypeptide or its active variant comprises a loss-of-function mutation at the NAD enzyme site in the TIRA1 region and / or TIRA2 region of the TIRA polypeptide or its active variant, and / or a gain-of-function mutation at the NAD enzyme site in the TIRA2 region of the TIRA polypeptide or its active variant; wherein the loss-of-function mutation that reduces the NAD enzyme activity of the TIRB polypeptide or its active variant comprises a loss-of-function mutation at the NAD enzyme site in the TIRB1 region of the TIRB polypeptide or its active variant, and wherein the TIRB polypeptide or its active variant does not comprise a loss-of-function mutation at the NAD enzyme site in the TIRB2 region of the TIRB polypeptide or its active variant.

28. The fusion protein according to any one of claims 19-27, wherein one or more of the TIRA polypeptide, TIRB polypeptide or their active variants are labeled with a detectable label.

29. A nucleic acid molecule encoding the fusion protein according to any one of claims 19-28.

30. A plant, or a plant part, comprising the nucleic acid molecule according to claim 29 or the fusion protein according to any one of claims 19-28, wherein the plant part is a transgenic seed comprising the fusion protein.

31. The plant according to claim 30, wherein the plant is an ASR-resistant soybean plant.

32. The plant according to claim 30, wherein the plant is a powdery mildew-resistant soybean plant.

33. A method for controlling disease resistance in a cultivation area, the method comprising the step of planting a leguminous plant, a plant part or a seed in the cultivation area, wherein the genome of the leguminous plant, the plant part or the seed is stably integrated with: (a) a first heterologous nucleotide sequence encoding a TIRA polypeptide or its active variant, the polypeptide or its active variant having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1; (b) a second heterologous nucleotide sequence encoding a TIRB polypeptide or its active variant, the polypeptide or its active variant having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2; and (c) a heterologous promoter operably coupled to the first and / or second heterologous nucleotide sequence Wherein the expression level of the TIRA polypeptide or its active variant is rebalanced in the plant, plant part or seed compared to the expression level in the control plant, plant part or seed, and wherein the rebalanced expression level of the TIRA polypeptide or its active variant relative to the TIRB polypeptide or its active variant comprises a ratio of about 0.5:1 to about 2:1, and wherein the TIRA polypeptide or its active variant confers disease resistance to the leguminous plant, plant or seed when co-expressed with the TIRB polypeptide or its active variant, and wherein the TIRA polypeptide or its active variant confers disease resistance to the leguminous plant, plant or seed when co-expressed with the TIRA polypeptide or its active variant.

34. The method according to claim 33, wherein one or more of the TIRA polypeptide, TIRB polypeptide or their active variants are labeled with a detectable label.

35. The method according to claim 33 or 34, wherein the TIRA polypeptide or its active variant, and the TIRB polypeptide or its active variant are expressed as a fusion protein in the leguminous plant, plant part or seed.

36. The method according to any one of claims 33-35, wherein the first heterologous nucleotide sequence encoding the TIRA polypeptide or its active variant, and the second heterologous nucleotide sequence encoding the TIRB polypeptide or its active variant are both operably coupled to and transcribed by the heterologous promoter, and wherein the heterologous promoter is an endogenous promoter, a rust-inducible promoter, or a constitutive promoter that is active in the plant.

37. The method according to any one of claims 33-36, wherein the heterologous promoter comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 23-28 or wherein the promoter comprises the sequence of any one of SEQ ID NOs: 22-27.

38. The method according to any one of claims 35-37, wherein the fusion protein comprises one or more loss-of-function mutations that reduce the NAD enzyme activity of the TIRA polypeptide or its active variant and gain-of-function mutations that increase the NAD enzyme activity of the TIRA polypeptide or its active variant, and wherein the TIRA polypeptide or its active variant having the loss-of-function mutation that reduces NAD enzyme activity or the gain-of-function mutation that increases NAD enzyme activity can confer disease resistance to the leguminous plant, plant or seed when co-expressed with the TIRB polypeptide or its active variant.

39. The method according to any one of embodiments 35-38, wherein the fusion protein comprises a loss-of-function mutation that reduces the NAD enzyme activity of the TIRB polypeptide or its active variant, and wherein the TIRB polypeptide or its active variant having the loss-of-function mutation that reduces NAD enzyme activity can confer disease resistance to the leguminous plant, plant or seed when co-expressed with the TIRA polypeptide or its active variant.

40. The method according to any one of claims 38 and 39, wherein the TIRA polypeptide or its active variant has a dumbbell-shaped structure comprising a TIRA1 region and a TIRA2 region, each of the TIRA1 and TIRA2 regions comprising a NAD enzyme site, wherein the TIRB polypeptide or its active variant has a dumbbell-shaped structure comprising a TIRB1 region and a TIRB2 region, each of the TIRB1 and TIRB2 regions comprising a NAD enzyme site, and wherein the loss-of-function mutation that reduces the NAD enzyme activity of the TIRA polypeptide or its active variant comprises a loss-of-function mutation at the NAD enzyme site in the TIRA1 region or the TIRA2 region; wherein the gain-of-function mutation that increases the NAD enzyme activity of the TIRA polypeptide or its active variant comprises a gain-of-function mutation at the NAD enzyme site in the TIRA2 region, wherein the loss-of-function mutation that reduces the NAD enzyme activity of the TIRB polypeptide or its active variant comprises a loss-of-function mutation at the NAD enzyme site in the TIRB1 region; and wherein the fusion protein does not comprise a loss-of-function mutation at the NAD enzyme site in the TIRB2 region of the TIRB polypeptide or its active variant.

41. The method according to any one of claims 38-40, wherein: the loss-of-function mutation that reduces the NAD enzyme activity of the TIRA polypeptide or its active variant comprises at least one mutation at the position corresponding to position 85 of SEQ ID NO:1; the gain-of-function mutation that increases the NAD enzyme activity of the TIRA polypeptide or its active variant comprises at least one mutation at the position corresponding to position 251 of SEQ ID NO:1; and the loss-of-function mutation that reduces the NAD enzyme activity of the TIRB polypeptide or its active variant comprises at least one mutation at the position corresponding to position 87 of SEQ ID NO:

2.

42. A leguminous plant produced by the method according to any one of claims 33-41, wherein the leguminous plant is a leguminous crop plant, and optionally wherein the leguminous crop plant is alfalfa, clover, pea, kidney bean, lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut or tamarind, optionally wherein the leguminous crop plant is an elite soybean plant.

43. The leguminous plant according to claim 42, wherein the plant is resistant to Asian soybean rust and / or powdery mildew and / or Pseudomonas syringae pv. syringae and / or soybean cyst nematode.

44. A cell comprising a heterologous polynucleotide encoding a polypeptide comprising: (a) an amino acid sequence having at least 90% identity to SEQ ID NO:1 or 2, wherein increased expression of the polypeptide in a plant enhances the disease resistance of the plant; (b) an amino acid sequence having at least 95% identity to SEQ ID NO:1 or 2, wherein increased expression of the polypeptide in a plant enhances the disease resistance of the plant; or, (c) Comprising the amino acid sequence of SEQ ID NO:1 or 2.

45. The cell according to claim 44, wherein the encoded polypeptide comprises: (d) An amino acid sequence having at least 90% identity with SEQ ID NO:1 and an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2, wherein increased expression of the polypeptide in a plant enhances the disease resistance of the plant; (e) An amino acid sequence having at least 95% identity with SEQ ID NO:1 and an amino acid sequence having at least 95% sequence identity with SEQ ID NO:2, wherein increased expression of the polypeptide in a plant enhances the disease resistance of the plant; or, (f) Comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:

2.

46. The cell according to claim 44, wherein the polynucleotide comprises: (a) A nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO:4 - 9; (b) A nucleotide sequence having at least 95% identity with any one of SEQ ID NO:4 - 9; or (c) Comprising a nucleotide sequence of any one of SEQ ID NO:4 - 9.

47. The cell according to claim 46, wherein the polynucleotide comprises: (a) A nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO:4 - 6 and a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO:7 - 9; (b) A nucleotide sequence having at least 95% identity with any one of SEQ ID NO:4 - 6 and a nucleotide sequence having at least 95% sequence identity with any one of SEQ ID NO:7 - 9; or (c) Comprising a nucleotide sequence of any one of SEQ ID NO:4 - 6 and comprising a nucleotide sequence of any one of SEQ ID NO:7 - 9.

48. The cell according to any one of claims 43 - 47, wherein the cell is a plant cell, optionally wherein the plant cell is (a) a monocotyledonous plant cell, (b) a dicotyledonous plant cell, (c) a leguminous plant cell, (d) a soybean cell, (e) a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugarcane cell or a wheat cell; or (f) a sunflower cell, a tomato cell, a cotton cell, a beet cell or a tobacco cell.

49. The plant cell according to claim 48, wherein the polynucleotide is stably integrated into the genome of the cell, and wherein the plant cell has an increased polypeptide expression level compared to a control plant cell and the plant cell has increased disease resistance.

50. A plant comprising the plant cell according to any one of claims 48 - 49.

51. The plant according to claim 50, wherein the transgenic plant has increased resistance to Asian soybean rust, wherein the transgenic plant is a leguminous plant, optionally wherein the leguminous plant is a soybean plant.

52. A method for screening plants having increased disease resistance and enhanced agronomic performance, the method comprising: (a) introducing into the genome of the plant a nucleic acid molecule comprising a heterologous promoter operably coupled to (i) a first polynucleotide encoding a TIRA polypeptide or an active variant thereof, the polypeptide or its active variant comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1; and (ii) a second polynucleotide encoding a TIRB polypeptide or an active variant thereof, the polypeptide or its active variant comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2; (b) determining the expression level of the TIRA polypeptide or its active variant, and the expression level of the TIRB polypeptide or its active variant, in the plant; (c) comparing the expression level of the TIRA polypeptide or its active variant relative to the expression level of the TIRB polypeptide or its active variant in the plant to determine whether the relative expression level is between 0.5:1 and 2:1, thereby determining the balanced expression level of the TIRA polypeptide or its active variant relative to the TIRB polypeptide in the plant.

53. The method of claim 52, further comprising selecting a plant having a balanced expression level of the TIRA polypeptide or its active variant relative to the TIRB polypeptide or its active variant.

54. A method for producing a TirA-TirB composition that, when introduced into a plant, is capable of conferring increased disease resistance and enhanced agronomic performance, the method comprising: (a) generating a first polynucleotide encoding a TIRA polypeptide or an active variant thereof and a second polynucleotide encoding a TIRB polypeptide or an active variant thereof, the TIRA polypeptide or its active variant comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1, and the TIRB polypeptide or its active variant comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2; (b) mutating the first polynucleotide to introduce one or more mutations at positions corresponding to position 85 and / or position 271 of SEQ ID NO:1, wherein the one or more mutations alter the NAD enzyme activity of the TIRA polypeptide or its active variant; (c) Mutating the second polynucleotide to introduce a mutation at the position corresponding to position 87 of SEQ ID NO:2, wherein the mutation reduces the NAD enzymatic activity of the TIRB polypeptide or an active variant thereof; (d) Expressing in a plant the first polynucleotide comprising the one or more mutations and the second polynucleotide comprising the mutation via an operably linked heterologous promoter; and (e) Screening the plants for plants having a balanced expression level of the TIRA polypeptide or an active variant thereof relative to the TIRB polypeptide or an active variant thereof, wherein the balanced expression level comprises a ratio of 0.5:1 to 2:

1.

55. A method for identifying a novel disease resistance polypeptide, the method comprising: (a) Providing a set of plant pathogen effector proteins that interact with the TIRA polypeptide of SEQ ID NO:1 or the TIRB polypeptide of SEQ ID NO:2; (b) Determining the interaction of the identified set of plant pathogen effector proteins with a putative TIRA polypeptide having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:1 or a putative TIRB polypeptide having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:2; and (c) Identifying the putative TIRA polypeptide or putative TIRB polypeptide from step (b), thereby identifying a novel disease resistance polypeptide.

56. The method of claim 55, wherein step (b) comprises determining a hypersensitive response or local cell death in a plant or plant cell or plant tissue, thereby indicating that the putative TIRA polypeptide has the same mode of action as the TIRA polypeptide or indicating that the putative TIRB polypeptide has the same mode of action as the TIRB polypeptide.

57. A plant comprising in its genome a stably integrated nucleic acid molecule comprising a heterologous promoter operably coupled to: (a) A first polynucleotide encoding a TIRA polypeptide or an active variant thereof, the polypeptide or active variant thereof comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1; and (b) A second polynucleotide encoding a TIRB polypeptide or an active variant thereof, the polypeptide or active variant thereof comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2, wherein the presence of the nucleic acid molecule in the plant confers increased disease resistance to the plant relative to a control plant that does not comprise the nucleic acid molecule.

58. The plant according to claim 57, wherein the first polynucleotide comprises one or more mutations that alter the NAD enzymatic activity of the TIRA polypeptide or an active variant thereof, and wherein the second polynucleotide comprises one or more mutations that alter the NAD enzymatic activity of the TIRA polypeptide or an active variant thereof, and wherein the presence of the one or more mutations that alter the NAD enzymatic activity of the TIRA polypeptide, TIRB polypeptide, or an active variant thereof in the plant confers enhanced agronomic performance to the plant relative to a control plant that does not comprise the one or more mutations.

59. The plant according to claim 58, wherein the one or more mutations that alter the NAD enzymatic activity of the TIRA polypeptide or an active variant thereof comprise one or more mutations at positions corresponding to positions 85 and / or 271 of SEQ ID NO:1, and wherein the one or more mutations that alter the NAD enzymatic activity of the TIRB polypeptide or an active variant thereof comprise a mutation at a position corresponding to position 87 of SEQ ID NO:

2.

60. The plant according to any one of claims 57-59, wherein the plant is resistant to Asian soybean rust.

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