Engineered nlrs for disease resistance

Abstract

Disclosed herein are engineered nucleotide-biding site leucine-rich repeat (NLR) fusion polypeptides along with polynucleotides encoding the engineered NLR fusion polypeptides and methods for improving or enhancing pathogen resistance in plants, such as legume plants, using the polynucleotides and encoded polypeptides. Methods of using NLR polynucleotides to make transgenic or genome edited pathogen resistant plants are also disclosed.

Description

ENGINEERED NLRs FOR DISEASE RESISTANCEREFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The official copy of the sequence listing is submitted electronically via Patent Center as an XML formatted sequence listing with a file named 108742_SequenceListing created on September 3, 2023, and having a size of 1,355,767 bytes and is filed concurrently with the specification. The sequence listing comprised in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.REFERENCE TO 3D PROTEIN STRUCTURAL COORDINATES SUBMITTED ELECTRONICALLY

[0002] The official copy of the 3D protein structural coordinates in protein data bank (PDB) format for each of the Roql(APLTD) NLR scaffold, the Roql(700:ID) NLR scaffold, the GmTNL_3(719:ID) NLR scaffold, the GmTNL_5(700:ID) NLR scaffold, and the GmTNL_9(718:ID) NLR scaffold are submitted electronically via Patent Center. The 3D protein structural coordinates, filed concurrently with the specification, submitted as Tables A-E, were created on September 7, 2023. The 3D protein structural coordinates comprised in this format are part of the specification and are herein incorporated by reference in their entirety.FIELD

[0003] This disclosure relates to the field of molecular biology, and particularly, relates to compositions and methods useful in enhancing pathogen resistance in plants. The disclosure further relates to fusion polypeptides, and polynucleotides encoding the fusion polypeptides, capable of enhancing pathogen resistance in plants and methods of using these polynucleotide sequences to make transgenic or genome edited plants having enhanced resistance to a pathogen.BACKGROUND

[0004] Plant diseases are responsible for considerable economic losses due to the reduced crop yield and quality that they can cause. Diverse species of plant pathogens encompassing bacteria, fungi, viruses, insects, and nematodes are responsible for these diseases in susceptible crops. Common practices designed to mitigate the damage caused by these pathogens include plantingdisease-resistant varieties and treating crops with chemical pesticides. Planting disease-resistant crops is the ideal strategy due to the high cost of treating crops with chemical pesticides and the negative environmental impact that these treatments can have. However, the conventional breeding methods typically used to develop disease-resistant crop varieties are time-consuming and can fail to develop resistant crop varieties against important plant diseases.

[0005] Advances in biotechnology, and a better understanding of how the plant immune system functions, have enabled scientists to develop disease-resistant plant varieties without conventional breeding methods. For example, thale cress (Arabidopsis thcilianc ) and soybean plants have been engineered to be resistant against viral pathogens. A genetic component of the rice immune system has been modified to provide resistance against a viral pathogen when expressed in a close relative of tobacco (Nicotiana benthamiand). However, the technologies that these approaches rely on are either costly, time-consuming, and / or difficult to adapt to different plant diseases.

[0006] Accordingly, due to a lack of resistant crop varieties against some important plant diseases, and the rapid pace at which pathogens can evolve to overcome resistant plants, new compositions and methods to quickly create disease-resistant plant varieties are needed to avoid substantial economic losses. This disclosure provides such compositions and methods.SUMMARY

[0007] Provided are nucleotide-binding leucine-rich repeat (NLR) fusion polypeptides comprising an NLR scaffold and a heterologous pathogen effector interacting domain, the NLR scaffold comprising a TOLL / interleukin-1 receptor (TIR) domain, a nucleotide binding site (NB) domain, and a leucine rich repeat (LRR) domain, or functional fragments thereof comprising at least one leucine-rich repeat. Further provided are polynucleotides encoding the NLR fusion polypeptides, and host cells, plant cells, plants, and plant parts comprising the polynucleotides.

[0008] Also provided are plants and plant cells comprising a polynucleotide encoding an NLR fusion polypeptide comprising an NLR scaffold and a heterologous pathogen effector interacting domain, the NLR scaffold comprising a TIR domain, an NB domain, and an LRR domain, or functional fragments thereof comprising at least one leucine-rich repeat.

[0009] Further provided are methods for increasing resistance to a pathogen in a plant comprising expressing in a plant cell a polynucleotide encoding an NLR fusion polypeptidecomprising an NLR scaffold and a heterologous pathogen effector interacting domain, the NLR scaffold comprising a TIR domain, an NB domain, and an LRR domain, or functional fragments thereof comprising at least one leucine-rich repeat, and generating a plant from the plant cell wherein the plant expresses the NLR fusion polypeptide and has increased resistance to a pathogen as compared to a control plant. In certain embodiments, the method for increasing resistance comprises introducing into a regenerable plant cell the polynucleotide encoding the NLR fusion polypeptide optionally operably linked to at least one regulatory element, and generating a plant from the plant cell wherein the plant comprises the recombinant polynucleotide and expresses the NLR fusion polypeptide. In certain embodiments, the method for increasing resistance comprises introducing in a regenerable plant cell a genetic modification at an endogenous TIR domain containing NLR (TIR-NLR) gene to generate a modified TIR- NLR gene encoding the NLR fusion polypeptide, and generating a plant from the plant cell expressing the NLR fusion polypeptide.BRIEF DESCRIPTION OF THE DRAWINGS AND THE SEQUENCE LISTING

[0010] The disclosure can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application. The sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§1.831-1.835.

[0011] Fig. 1 A provides the predicted structure for the Roql(APL:ID) NLR scaffold (SEQ ID NO: 1). Fig. IB provides the predicted structure for the Roql(700:ID) NLR scaffold (SEQ ID NO: 2). The structures were predicted using AlphaFold2 and are oriented with the TIR domain on the left (black) followed by the NB-ARC domain in the middle (dark grey) and the LRR domain on the right (light grey).

[0012] Fig. 2A provides the predicted structure for a full-length soybean TIR domain containing NLR protein from soybean referred to as GmTNL_3 (SEQ ID NO: 62). Fig. 2B provides the predicted structure for a truncated version of GmTNL_3 (GmTNL_3(719:ID), SEQ ID NO: 392) for use as a NLR scaffold. The structures were predicted using AlphaFold2.

[0013] Fig. 3A provides the predicted structure for a full-length soybean TIR domain containing NLR protein from soybean referred to as GmTNL 5 (SEQ ID NO: 84). Fig. 3B provides thepredicted structure for a truncated version of GmTNL_5 (GmTNL_5(700:ID), SEQ ID NO: 393) for use as a NLR scaffold. The structures were predicted using AlphaFol d2.

[0014] Fig. 4A provides the predicted structure for a full-length soybean TIR domain containing NLR protein from soybean referred to as GmTNL_9 (SEQ ID NO: 121). Fig. 4B provides the predicted structure for a truncated version of GmTNL_9 (GmTNL_9(718:ID), SEQ ID NO: 396) for use as a NLR scaffold. The structures were predicted using AlphaFold2.DETAILED DESCRIPTION

[0015] The plant immune system relies on nucleotide-binding leucine-rich repeat (NLR) intracellular immune receptors that recognize secreted proteins from invading pathogens called effectors. The NLR genes are comprised of two subclasses. Class 1 NLR genes contain a TIR- Toll / Interleukin-1 like domain at their N-terminus. The second class of NLR genes contain either a coiled-coil domain or an NT domain at their N-terminus. The leucine-rich repeat (LRR) domain of some plant NLRs bind directly to pathogen effectors to activate the NLR protein. For NLRs that contain an N-terminal Toll / interleukin-1 receptor (TIR) domain, the catalytically active TIR domain produces a signaling molecule that initiates a signaling cascade culminating in an immune response against the invading pathogen.

[0016] One aspect of the disclosure provides engineered nucleotide-binding leucine-rich repeat (NLR) fusion polypeptides comprising, consisting essentially of, or consisting of an NLR scaffold and a heterologous pathogen effector interacting domain. In certain embodiments, the engineered NLR fusion polypeptide confers resistance or increased resistance to one or more pathogens, such as, for example, the pathogens causing Asian Soybean Rust (hereinafter “ASR”) when expressed in plants.

[0017] In certain embodiments, the NLR scaffold is fused directly to the pathogen effector interacting domain. The term “directly” defines fusions in which the polypeptides are joined without a peptide linker. In certain embodiments, the NLR scaffold is fused through a linker to the pathogen effector interacting domain. The linker is generally a polypeptide of between about 1 and about 500 amino acids in length. The linkers joining the NLR scaffold and pathogen effector interacting domain are preferably designed to (1) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered secondary structure which could interfere with the functional domains of the two proteins, and (3) haveminimal hydrophobic or charged characteristic which could interact with the functional protein domains. Typically surface amino acids in flexible protein regions include Gly, Asn, and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn, and Ser would be expected to satisfy the above criteria for a linker sequence. Other neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the fusion.

[0018] As used herein, the term "resistance" refers to an absence or reduction of one or more disease symptoms in a plant caused by a plant pathogen. Resistance can mean that disease symptoms, such as, for example, number of lesions, defoliation, and associated yield loss, are reduced, minimized or lessened, when compared to a plant that is susceptible to the disease or a plant that does not contain or express an engineered NLR fusion polypeptide. Further, resistance can include the prevention or delay of proliferation of a pathogen (e.g., fungi).

[0019] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0020] In certain embodiments, the NLR scaffold of the compositions and methods described herein comprises, consists essentially of, or consists of a TOLL / interleukin-1 receptor (TIR) domain or a functional fragment thereof, a nucleotide binding site (NB) domain or a functional fragment thereof, and a leucine rich repeat (LRR) domain, or a functional fragment thereof comprising at least one leucine-rich repeat.

[0021] In certain embodiments, the TIR domain, NB domain, and LRR domain of the NLR scaffold are arranged, from N-terminus to C-terminus, in the order TIR domain, NB domain, and LRR domain, or functional fragment(s) thereof, respectively. In certain embodiments, the TIR domain, NB domain, and LRR domain are isolated from the same NLR polypeptide. In certain embodiments, the TIR domain, NB domain, and LRR domain are isolated from two or more NLR polypeptides, such that at least one of the TIR domain, the NB domain, or the LRR domain are heterologous to one or more of the other domains. In certain embodiments, the TIR domain, NB domain, and LRR domain are each isolated from a different NLR polypeptide.TIR Domain

[0022] Toll / interleukin- 1 receptor domains (TIR domains) are conserved structures which often display the Rossman fold. In plants, TIR domains predominantly comprise six highly conserved motifs referred to as (3 A, aA, PC, aC, PD-aDl, and aD3 (a and refer to helical and beta-strand elements and A-D correspond to structural modules of the TIR domain). The PA motif comprises the amino acid sequence YX1VFX2SFRGEDX3R wherein Xi is aspartic acid (D) or glutamic acid (E), X2 is leucine (L), isoleucine (I), or valine (V), and Xs is threonine (T), isoleucine (I), or valine (V) (SEQ ID NO: 31). The aA motif comprises the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G). The PC motif comprises the amino acid sequence SX1X2X X4X5X6X7 wherein Xi is any amino acid, X2 is leucine (L), isoleucine (I), or valine (V), X3 is any amino acid, X-ris leucine (L), isoleucine (I), or valine (V), X5 is isoleucine (I) or valine (V), Xe is isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L). The aC motif comprises the amino acid sequence X1CLX2EL wherein Xi is tryptophan (W), phenylalanine (F), or tyrosine (Y) and X2is any amino acid (SEQ ID NO: 32). The PD- Dl motif comprises the amino acid sequence X1X2PX3FYX4X5X6PSX7VRX8Q wherein Xi and X2 are leucine (L), isoleucine (I), or valine (V), X3 and X5 are isoleucine (I) or valine (V), Xe is aspartic acid (D) or glutamic acid (E), and X4, X7 and Xs are any amino acid (SEQ ID NO: 33). The aD3 motif comprises the amino acid sequence WX1X2ALX3X4X5X6X7JX8G wherein Xi is lysine (K) or arginine (R), X2, X3 X4 and X7 are any amino acid, X5 and Xs are alanine (A), serine (S), or valine (V), and Xe is alanine (A) or serine (S) (SEQ ID NO: 34).

[0023] In certain embodiments, the TIR domain of the engineered NLR fusion polypeptides described herein comprises at least one amino acid motif selected from the group consisting of a PA motif comprising SEQ ID NO: 31, an aA motif comprising the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), X5 and Xe are isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a PD-aDl motif comprising SEQ ID NO: 33, and a aD3 motif comprising SEQ ID NO: 34 or any combination thereof. In certain embodiments, the TIR domain of the engineered NLR fusion polypeptides comprises a PA motif comprising SEQ ID NO: 31, an aA motif comprising the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I),valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), X5 and X6 are isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a pD-aDl motif comprising SEQ ID NO: 33, and a aD3 motif comprising SEQ ID NO: 34. In certain embodiments, the TIR domain of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 40 and 183-196. In certain embodiments, the TIR domain of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 40 and 183-196 and comprises at least one amino acid motif selected from the group consisting of a A motif comprising SEQ ID NO: 31, an 01A motif comprising the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), X5 and Xe are isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a PD-aDl motif comprising SEQ ID NO: 33, a aD3 motif comprising SEQ ID NO: 34 or any combination thereof. In certain embodiments, the TIR domain of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 40 and 183-196 and comprises a PA motif comprising SEQ ID NO: 31, an aA motif comprising the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), X5 and Xe are isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a PD-aDl motif comprising SEQ ID NO: 33, and a aD3 motif comprising SEQ ID NO: 34. In certain embodiments, the TIR domain of the engineered NLR fusion polypeptide comprises an aminoacid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 40, 185, 187, or 190. In certain embodiments, the TIR domain of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 40, 185, 187, or 190 and comprises a PA motif comprising SEQ ID NO: 31, an aA motif comprising the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), X5 and Xe are isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a PD-aDl motif comprising SEQ ID NO: 33, a aD3 motif comprising SEQ ID NO: 34 or any combination thereof. In certain embodiments, the TIR domain of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 40, 185, 187, or 190 and comprises a PA motif comprising SEQ ID NO: 31, an aA motif comprising the amino acid sequence FX1X2HI wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), X5 and Xe are isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a PD-aDl motif comprising SEQ ID NO: 33, and a aD3 motif comprising SEQ ID NO: 34.

[0024] In certain embodiments, the polynucleotide encoding the TIR domain of the engineered NLR fusion polypeptides described herein comprises a nucleic acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 367-380 and 429. In certain embodiments, the polynucleotide encoding the TIR domain of the engineered NLR fusion polypeptide comprises a nucleic acid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 429.

[0025] In certain embodiments, the TIR domain of the engineered NLR fusion polypeptides described herein comprises an AlphaFol d2 predicted structure having a template modeling score (TM-score) of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 40 and 183-196. In certain embodiments, the TIR domain of the engineered NLR fusion polypeptides described herein comprises an amino acid sequence that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 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% identical to SEQ ID NO: 40 or 183-196 and comprises an AlphaFol d2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 40 and 183-196.

[0026] In certain embodiments, the TIR domain of the engineered NLR fusion polypeptides described herein comprises an AlphaFol d2 predicted structure having a template TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of SEQ ID NO: 40, 185, 187, or 190. In certain embodiments, the TIR domain of the engineered NLR fusion polypeptides described herein comprises an amino acid sequence that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 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% identical to SEQ ID NO: 40, 185, 187, or 190 and comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NO: 40, 185, 187, or 190.NB Domain

[0027] The NB Domain, also referred to as NB-ARC domain orNBS domain, functions as an “on / off switch” in an NLR polypeptide by changing its configuration upon activation by a pathogen effector. NB domains predominantly comprise three subdomains referred to as the nucleotide binding domain, helical domain, also referred to as ARC1, and the winged helix domain, also referred to as ARC2. NB domains often comprise six highly conserved regions, occurring over the span of the three subdomains or motifs, which are referred to as P-loop, Walker B, RNBS-B, RNBS-D, GLPL, and MHD. The P-loop motif comprises the amino acid sequence GX1GGX2GKX3X4 wherein Xi is methionine (M), isoleucine (I), leucine (L) or serine(S), X2 is isoleucine (I), leucine (L) or valine (V), X3 is threonine (T) or serine (S) and X4 is threonine (T) or isoleucine (I) (SEQ ID NO: 35). The Walker B motif comprises the amino acid sequence X1X2X3X4X5LX6X7JDDVX8X9 wherein Xi, X2, X3, Xs, and X9 are any amino acid, X4 is lysine (K) or arginine (R), X5 is valine (V), isoleucine (I), or proline (P), Xe is isoleucine (I), leucine (L) or valine (V), and X7 is isoleucine (I) or valine (V) (SEQ ID NO: 36). The RNBS-B motif comprises the amino acid sequence GX1GSX2X3X4X5TSRDX6X7X8J wherein Xi, Xe, and X7 are any amino acid, X2 is lysine (K) or arginine (R), X3 and X4 are isoleucine (I) or valine (V), X5 and Xs are isoleucine (I), leucine (L) or valine (V), and Xe is threonine (T) or serine (S) (SEQ ID NO: 38). The RNBS-D motif comprises the amino acid sequence ZX1X2X3FLDJAX4X5X6X7X8 wherein Xi is lysine (K), arginine (R), or glutamine (Q), X2 and X7 are any amino acid, X3 is isoleucine (I) or valine (V), X4 and X5 are phenylalanine (F) or cysteine (C), Xe is phenylalanine (F), tyrosine (Y) or leucine (L), and Xs is glycine (G), arginine (R) or threonine (T) (SEQ ID NO: 39). The GLPL motif comprises the amino acid sequence GJPLX wherein X is alanine (A) or valine (V) (SEQ ID NO: 37). The MHD motif comprises the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N).

[0028] In certain embodiments, the NB domain of the engineered NLR fusion polypeptides comprises at least one amino acid motif selected from a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQ ID NO: 37, an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N), or any combination thereof. In certain embodiments, the NB domain of the engineered NLR fusion polypeptide comprises a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQ ID NO: 37, and an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N). In certain embodiments, the NB domain of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 41 and 169-182. In certain embodiments, the NB domain of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 41 and 169-182 and comprises at least one amino acid motif selected from the group consisting of a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQ ID NO: 37, an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N) or any combination thereof. In certain embodiments, the NB domain of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 41 and 169-182 and comprises a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQ ID NO: 37, and an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N). In certain embodiments, the NB domain of the engineered NLR fusion polypeptides comprises an amino acid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 41, 171, 173, or 176. In certain embodiments, the NB domain of the engineered NLR fusion polypeptides comprises an amino acid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 41, 171, 173, or 176 and comprises a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQ ID NO: 37, an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N) or any combination thereof. In certain embodiments, the NB domain of the engineered NLR fusion polypeptides comprises an amino acid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 41, 171, 173, or 176 and comprises a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQID NO: 37, and an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N).

[0029] In certain embodiments, the polynucleotide encoding the NB domain of the engineered NLR fusion polypeptide comprises a nucleic acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 353-366 and 430. In certain embodiments, the polynucleotide encoding the NB domain of the engineered NLR fusion polypeptide comprises a nucleic acid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 430.

[0030] In certain embodiments, the NB domain of the engineered NLR fusion polypeptides described herein comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 41 and 169-182. In certain embodiments, the NB domain of the engineered NLR fusion polypeptides described herein comprises an amino acid sequence that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 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% identical to SEQ ID NO: 41 and 169-182 and comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 41 and 169-182.

[0031] In certain embodiments, the NB domain of the engineered NLR fusion polypeptides described herein comprises an AlphaFold2 predicted structure having a template TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of SEQ ID NO: 41, 171, 173, or 176. In certain embodiments, the NB domain of the engineered NLR fusion polypeptides described herein comprises an amino acid sequence that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 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% identical to SEQ ID NO: 41, 171, 173, or 176 and comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65,0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NO: 41, 171, 173, or 176.LRR Domain

[0032] The leucine rich repeat domain or LRR domain predominantly comprise 2-45 leucine- rich repeats with each repeat being roughly 15-35 amino acids in length. The repeats often start with an LxxLxL motif in which L is a hydrophobic amino acid, usually leucine, and X is any residue. Structurally LRR domains form an arc or horseshoe shape, and the repeats are held together via an H-bond network formed among the LxxLxL stretches on the ventral side of the horseshoe. In certain embodiments, the LRR domain of the engineered NLR fusion polypeptides comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 leucine-rich repeats and less than 70, 65, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47,46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 leucine-repeats. In certain embodiments, the LRR domain of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 42 and 155-168 or a functional fragment thereof. In certain embodiments, the LRR domain of the engineered NLR fusion polypeptides comprises an amino acid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 42, 157, 159, or 162 or a functional fragment thereof.

[0033] In certain embodiments, the polynucleotide encoding the LRR domain of the engineered NLR fusion polypeptide comprises a nucleic acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 339-352 and 431 or a functional fragment thereof. In certain embodiments, the polynucleotide encoding the LRR domain of the engineered NLR fusion polypeptide comprises a nucleic acid sequence that is at least 50%, 55%, 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% identical to SEQ ID NO: 431 or a functional fragment thereof.

[0034] As used herein, "functional fragment," "fragment that is functionally equivalent," "functionally equivalent fragment" and the like refer to a portion or subsequence of a polypeptide sequence of the present disclosure in which its native ability is retained. For example, a functional fragment of an LRR domain polypeptide, such as those described herein, produces an engineered NLR fusion polypeptide that maintains the ability to recognize and bind to a pathogen effector protein. Accordingly, in certain embodiments, a functional fragment of an LRR domain polypeptide (e.g., SEQ ID NOs: 42 and 155-168) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 leucine-rich repeats and less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 leucine-repeats. In certain embodiments, the functional fragment of an LRR domain is produced by removing at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50 or more) leucine-rich repeats from the C-terminus of an LRR domain polypeptide (e.g., SEQ ID NOs: 42 and 155-168). In certain embodiments, the functional fragments of the polypeptides described herein comprises an N-terminal and / or a C-terminal truncation of at least 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or 650 or more amino acids. In certain embodiments, the LRR domain functional fragment of the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 407-417.

[0035] NLR polypeptides may be held in an inactive state by intramolecular interactions between the NB domain and the N-terminal portion of the LRR domain. Typically, the interaction involves hydrogen bonds between residues in the NB domain and the LRR domain. Upon binding to an effector these interactions are disrupted and the NLR transitions into an active complex that initiates an immune response. Accordingly, in certain embodiments, the functional fragment of the LRR domain comprises, consists essentially of, or consists of the leucine-rich repeats predicted to associate with the NB domain (e.g., residues important for maintaining the hydrogen bonds between the domain). In certain embodiments, the structure of an LRR domain is predicted using AlphaFold2 and the prediction is used to identify the last leucine-rich repeat predicted to interact with the NB domain so that the functional fragment comprises, consists essentially of, or consists of the N-terminal portion of an LRR domain up to and including the end of the predicted last leucine-rich repeat predicted to interact with the NB domain. In certainembodiments, the structure of the engineered NLR fusion polypeptide is modeled using AlphaFold2 and the pathogen effector interacting domain is inserted into a position of the NLR scaffold so as not to disrupt the interaction between the LRR domain and the NB domain in the inactive state.

[0036] In certain embodiments, the LRR domain or functional fragment thereof of the engineered NLR fusion polypeptides described herein comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 42, 155-168, and 407-417. In certain embodiments, the LRR domain or functional fragment thereof of the engineered NLR fusion polypeptides described herein comprises an amino acid sequence that is at least 10%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 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% identical to a sequence selected from SEQ ID NOs: 42, 155-168, and 407-417 and comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 42, 155-168, and 407-417.

[0037] In certain embodiments, the LRR domain or functional fragment thereof of the engineered NLR fusion polypeptides described herein comprises an AlphaFold2 predicted structure having a template TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of a sequence selected from SEQ ID NO: 42, 157, 159, or 162, 408, 409, 412 or 417. In certain embodiments, the LRR domain or functional fragment thereof of the engineered NLR fusion polypeptides described herein comprises an amino acid sequence that is at least 10%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 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% identical to a sequence selected from SEQ ID NO: 42, 157, 159, or 162, 408, 409, 412 or 417 and comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NO: 42, 157, 159, or 162, 408, 409, 412 or 417.

[0038] As used herein "percent (%) sequence identity" with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidatesequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence * 100).

[0039] Unless otherwise stated, sequence identity / similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-402).

[0040] In certain embodiments, the NLR scaffold of the engineered NLR fusion polypeptides described herein comprises an amino acid sequence that is at least 50%, 55%, 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% identical to a sequence selected from SEQ ID NO: 1, 2 or 391-400.

[0041] In certain embodiments, the NLR scaffold of engineered NLR fusion polypeptides described herein comprises an AlphaFol d2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the NLR scaffold of any one of SEQ ID NOs: 1, 2 and 391- 400. Representative predicted structures of various NLR scaffolds for use in the compositions and method described herein generated using AlphaFold2 can be found in Figs. 1A, IB, 2A, 2B, 3A, 3B, 4A, and 4B. Additionally, the reference PDB coordinates for the structure of Fig. 1A, IB, 2B, 3B, and 4B can be found in the 3D protein structural coordinate fdes Table A, Table B, Table C, Table D, and Table E, respectively.

[0042] In certain embodiments, the NLR scaffold of engineered NLR fusion polypeptides described herein comprises an amino acid sequence that is at least 10%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 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% identical to a sequence selected from SEQ ID NO: 1, 2 or 391-400 and comprises an AlphaFol d2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the NLR scaffold of any one of SEQ ID NOs: 1, 2 or 391-400.

[0043] In certain embodiments, the NLR scaffold of engineered NLR fusion polypeptides described herein comprises an amino acid sequence that is at least 10%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 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% identical to a sequence selected from SEQ ID NO: 1, 2, 392, 393, or 396 and comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the NLR scaffold of any one of SEQ ID NOs: 1, 2, 392, 393, or 396.

[0044] In certain embodiments, the NLR scaffold of the NLR fusion polypeptides described herein comprise a TIR domain comprising an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the TIR domain of any one of SEQ ID NOs: 40 and 183-196; an NB domain comprising an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the NB domain of any one of SEQ ID NOs: 41 and 169-182; and an LRR domain comprising an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the LRR domain of any one of SEQ ID NOs: 42 and 155-168 or a functional fragment thereof.

[0045] In certain embodiments, the NLR scaffold of the NLR fusion polypeptides described herein comprise a TIR domain comprising an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the TIR domain of any one of SEQ ID NOs: 40 and 183-196; an NB domain comprising an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 41 and169-182 and comprises the amino acid motifs set forth in SEQ ID NOs: 35-39; and an LRR domain comprising an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the LRR domain of any one of SEQ ID NOs: 42 and 155-168 or a functional fragment thereof.

[0046] In certain embodiments, the NLR scaffold of the NLR fusion polypeptides described herein comprise a TIR domain comprising an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 40 and 183-196 and comprises a PA motif comprising SEQ ID NO: 31, an aA motif comprising the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), Xs and Xe are isoleucine (I) or valine (V), and X7 is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a pD-aDl motif comprising SEQ ID NO: 33, and a aD3 motif comprising SEQ ID NO: 34; an NB domain comprising an amino acid sequence that is at least 50%, 55%, 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% identical to any one of SEQ ID NOs: 41 and 169-182 and comprises a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQ ID NO: 37, and an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N); and an LRR domain comprising an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the LRR domain of any one of SEQ ID NOs: 42 and 155-168 or a functional fragment thereof.

[0047] In certain embodiments, the NLR scaffold of the NLR fusion polypeptides described herein comprise a TIR domain comprising an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 55%, 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% identical to any one of SEQ ID NOs: 40 and 183-196 and comprises a 0A motif comprising SEQ ID NO: 31, anaA motif comprising the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), X5 and Xs are isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a D-aDl motif comprising SEQ ID NO: 33, and a aD3 motif comprising SEQ ID NO: 34, and comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the TIR domain of any one of SEQ ID NOs: 40 and 183-196; an NB domain comprising an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 55%, 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% identical to any one of SEQ ID NOs: 41 and 169-182 and comprises a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQ ID NO: 37, and an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N) and comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the NB domain of any one of SEQ ID NOs: 41 and 169-182; and an LRR domain comprising an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 55%, 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% identical to any one of SEQ ID NOs: 42 and 155-168 or a functional fragment thereof and comprises an AlphaFold2 predicted structure having a TM-score of at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the AlphaFold2 predicted structure of the LRR domain of any one of SEQ ID NOs: 42 and 155-168 or a functional fragment thereof.

[0048] AlphaFold is a computational method that can predict protein structures with atomic accuracy even in cases in which no similar structure is known. The AlphaFold network directly predicts the 3D coordinates of all heavy atoms for a given protein using the primary amino acid sequence and aligned sequences of homologues as inputs. The AlphaFold methods are scalable to very long proteins with accurate domains and domain-packing, and the model providesprecise, per-residue estimates of its reliability that enables confident use of its structure predictions. (Jumper, J., Evans, R., Pritzel, A. etal., Nature 596, 583-589 (2021)). The structures provided herein were predicted using AlphaFol d2 version 2.3.1 in the monomer mode with relaxed model prediction and “uniref90” was used as the reference database (accessible using the prefix “www” on the internet at .uniprot.org).

[0049] As used herein, a “template modeling score” “TM-score” “structural similarity score” or the like refers to a measure of structural similarity between two protein tertiary structures. TM- scores range from 0 (no structural identity) to 1 (perfect structural identity) and can be computed using multiple different publicly available approaches, including TM-align (zhanggroup.org / TM- align / ) or Foldseek (search.foldseek.com / search), accessible using the prefix “www” on the internet. Those skilled in the art can determine appropriate parameters for optimally aligning structures.

[0050] Unless otherwise stated, TM-scores provided herein refer to the value obtained using the TM-align program using default parameters to compare protein structures predicted using AlphaFold2 version 2.3.1 in the monomer mode with relaxed model prediction and the “uniref90” reference database.

[0051] As used herein a “pathogen effector interacting domain” (also referred to herein as an integrated domain or ID) refers to a polypeptide that binds to an effector protein. Pathogens, such as Phakopsora pcichyrhizi and Phakopsora meibomiae, which cause ASR, can overcome first- tier immunity of a plant by secreting molecules known as “effector proteins” or “effectors.”

[0052] The effector recognized by the pathogen effector interacting domain may be secreted from any pathogen known in the art to cause a plant (e.g., soybean) disease. Similarly, the sequence of the effector recognized by the pathogen effector interacting domain may be any effector sequence known in the art. In certain embodiments, the effector is secreted from a pathogen selected from the group consisting of Rhizocionia solani, Phakopsora pachyrhizi, Phakopsora meibomiae, Colletotrichum truncatum, Phialophora gregata, Cercospora kukuchii, Macrophomina phaseolina, Diaporthe phaseolorum, Peronospora manshurica, Cercospora sojina, Phytophthora sojae, Microsphaera diffusa, Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne javanica, Heterodera glycines, Fusarium virguliforme, Septoria glycines, Corynespora cassiicola, and Sclerotinia sclerotioru . In certain embodiments, the effector is secreted from Phakopsora pachyrhizi and / or Phakopsora meibomiae, which cause ASR. Incertain embodiments, the pathogen effector interacting domain recognizes an effector having an amino acid sequence having at least that is at least 50%, 55%, 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% identical to a sequence selected from SEQ ID NO: 4, 7, 10, 13, 16, 20, 23, 26, 29, 436, 444, 450, 458, 466, 472, 480, 490, 537, 539, 541, 543, 545, or 547.

[0053] In certain embodiments, effectors that are conserved among multiple pathogen species are identified using methods including, but not limited to, AlphaFold or homology-based searching. In certain embodiments, the pathogen effector interacting domains recognize the identified conserved effectors such that the effector interacting domain recognizes multiple pathogen species.

[0054] The sequence of the pathogen effector interacting domain for use in the engineered NLR fusion polypeptides described herein is not particularly limited and can be any polypeptide sequence that binds to an effector described herein or known in the art. Methods for identifying pathogen effector interacting domains are known in the art and include for example, yeast two- hybrid, co-immunoprecipitation, and proximity labeling. In certain embodiments, the pathogen effector interacting domain is identified using co-immunoprecipitation, yeast two-hybrid, and / or proximity labeling. In certain embodiments, the pathogen effector interacting domain comprises an amino acid sequence having at least 50%, 55%, 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% identical to a sequence selected from SEQ ID NO: 3, 6, 9, 12, 15, 19, 22, 25, 28, 435, 443, 449, 457, 465, 471, 479, 489, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, or 583.

[0055] In certain embodiments, the engineered NLR fusion polypeptides described herein comprise two or more pathogen effector interacting domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 50, 100 or more) fused to a single NLR scaffold. Any pathogen effector interacting domain described herein may be used in the NLR fusion polypeptides comprising two or more pathogen effector interacting domains. In certain embodiments, a single pathogen effector interacting domain targets effectors conserved among multiple pathogen species. In certain embodiments, when two or more pathogen effector interacting domains are fused to a single NLR scaffold each of the pathogen effector interacting domains recognize (a) effectors secreted from the same pathogen, (b) effectors secreted from different pathogens, or (c) a combination thereof.

[0056] In certain embodiments, the engineered NLR fusion polypeptides described herein comprise two or more NLR scaffolds (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 50, 100 or more). In certain embodiments the two or more NLR scaffolds are linked via a linker sequence. In certain embodiments, each of the two or more NLR scaffolds are fused to at least one pathogen effector interacting domain (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 50, 100 or more). In certain embodiments, each of the NLR scaffolds are independently fused to the same effector interacting domain, different effector interacting domains, or a combination thereof. In certain embodiments, the elements are arranged, from N-terminus to C-terminus, in the order effector interacting domain 1, LRR domain or functional fragment thereof 1, NB domain 1, TIR domain 1, linker, TIR domain 2, NB domain 2, LRR domain or functional fragment thereof 2, and effector interacting domain 2. In certain embodiments, one or more domains of the NLR scaffold is shared between the two or more NLR scaffolds, for example, in certain embodiments, the elements are arranged, from N-terminus to C-terminus, in the order effector interacting domain 1, LRR domain or functional fragment thereof 1, NB domain 1, TIR domain, NB domain 2, LRR domain or functional fragment thereof 2, and effector interacting domain 2. Any NLR scaffold described herein may be used in the NLR fusion polypeptides comprising two or more NLR scaffolds. Similarly, any pathogen effector interacting domain described herein may be used in the NLR fusion polypeptides comprising two or more NLR scaffolds.

[0057] As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a fusion polypeptide may comprise an NLR scaffold polypeptide and a heterologous pathogen effector interacting domain that is from a species different from the species from which the NLR scaffold polypeptide was derived, or, if from the same / analogous species, one or both are substantially modified from their original form and / or genomic locus, or the pathogen effector interacting domain is not the native pathogen effector interacting domain for the NLR scaffold polypeptide.

[0058] In certain embodiments, the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 50%, 55%, 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% identical to a sequence selected from SEQ ID NO: 5, 8, 11, 14, 17, 18, 21, 24, 27, 30, 402, 404,406, 437, 439, 445, 451 , 453, 459, 461, 467, 473, 475, 481, 483, 485, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, or 535.

[0059] Also provided herein are polynucleotides encoding the engineered NLR fusion polypeptides described herein. In certain embodiments, the polynucleotide encoding the polypeptide comprises a nucleotide sequence that is at least 50%, 55%, 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% identical to a sequence selected from SEQ ID NO: 313, 316, 319, 322, 325, 326, 329, 332, 335, 338, 401, 403, 405, 434, 438, 442, 448, 452, 456, 460, 464, 470, 474, 478, 482, 484, 488, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534.

[0060] As used herein “encoding,” “encoded,” or the like, with respect to a specified nucleic acid, means comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons.Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as is present in some plant, animal and fungal mitochondria, the bacterium Mycoplasma capricolum (Yamao, et al., (1985) Proc. Natl. Acad. Set. USA 82:2306-9) or the ciliate Macromicleus, may be used when the nucleic acid is expressed using these organisms.

[0061] When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledonous plants or dicotyledonous plants as these preferences have been shown to differ (Murray, et al., (1989) Nucleic Acids Res. 17:477-98 and herein incorporated by reference).

[0062] In certain embodiments, the polynucleotide encoding the engineered NLR fusion polypeptide is operably linked to at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7 or more) regulatory element. In certain embodiments, the regulatory element is a promoter. In certain embodiments, the regulatory element is a heterologous regulatory element (e.g., heterologous promoter). Incertain embodiments, the heterologous regulatory element is heterologous to the polynucleotide sequence encoding the polypeptide. In certain embodiments, in which the polynucleotide operably linked to the heterologous regulatory element is introduced in a cell the regulatory element is heterologous to the cell.

[0063] As used herein “operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide encoding an engineered NLR fusion polypeptide and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, operably linked is intended that the coding regions are in the same reading frame.

[0064] As used herein “regulatory element” generally refers to a transcriptional regulatory element involved in regulating the transcription of a nucleic acid molecule such as a gene or a target gene. The regulatory element is a nucleic acid and may include a promoter, an enhancer, an intron, expression modulating elements (EMEs), a 5 ’-untranslated region (5’-UTR, also known as a leader sequence), or a 3’-UTR, or a combination thereof. A regulatory element may act in "cis" or "trans", and generally it acts in "cis", i.e., it activates expression of genes located on the same nucleic acid molecule, e.g., a chromosome, where the regulatory element is located.

[0065] An “enhancer” element is any nucleic acid molecule that increases transcription of a nucleic acid molecule when functionally linked to a promoter regardless of its relative position. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al., (1989) Molecular Biology ofRNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9: 1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments described herein.

[0066] A “repressor” (also sometimes called herein silencer) is defined as any nucleic acid molecule which inhibits the transcription when functionally linked to a promoter regardless of relative position. The term "cis-element" generally refers to a transcriptional regulatory element that affects or modulates expression of an operably linked transcribable polynucleotide, wherethe transcribable polynucleotide is present in the same DNA sequence. A cis-element may function to bind transcription factors, which are trans-acting polypeptides that regulate transcription.

[0067] An “intron” is an intervening sequence in a gene that is transcribed into RNA but is then excised in the process of generating the mature mRNA. The term is also used for the excised RNA sequences. An “exon” is a portion of the sequence of a gene that is transcribed and is found in the mature mRNA derived from the gene but is not necessarily a part of the sequence that encodes the final gene product. The 5' untranslated region (5’UTR) (also known as a translational leader sequence or leader RNA) is the region of an mRNA that is directly upstream from the initiation codon. This region is involved in the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes. The “3' non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0068] As used herein “promoter” refers to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. In certain embodiments, the polynucleotides described herein are operably linked to a promoter that drives expression in a plant cell. Any promoter known in the art can be used in the methods of the present disclosure including, but not limited to, constitutive promoters, pathogeninducible promoters, wound-inducible promoters, tissue-preferred promoters, and chemical- regulated promoters. The choice of promoter may depend on the desired timing and location of expression in the transformed plant as well as other factors, which are known to those of skill in the art. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter; rice actin; ubiquitin; pEMU; MAS; ALS; and the like. Other constitutive promoters include, for example, those disclosed in U.S. Patent Nos.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 known in the art, and can be contemplated for use in the present disclosure.

[0069] Generally, it can be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen, e.g., PR proteins, SAR proteins, beta-1,3 -glucanase, chitinase, etc.

[0070] Of interest are promoters that are expressed locally at or near the site of pathogen infection. Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter can be used in the constructions of the disclosure. Such woundinducible promoters include potato proteinase inhibitor (pin II) gene, wunl and wun2, winl and win2, systemin, WIP1, MPI gene, and the like.

[0071] Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter can be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemi cal -inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (e.g., the glucocorticoid-inducible promoter, and tetracycline-inducible and tetracycline-repressible promoters).

[0072] Tissue-preferred promoters can be utilized to target enhanced expression of the target genes or proteins within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include 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; Canevascini 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. Such promoters can be modified.

[0073] Leaf-specific promoters are known in the art. See, for example, 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.

[0074] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seedgerminating" promoters (those promoters active during seed germination). Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message), cZ19Bl (maize 19 kDa zein), milps (myo-inositol- 1 -phosphate synthase), and celA (cellulose synthase) (see WO 00 / 11177, herein incorporated by reference). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean P-phaseolin, napin, -conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00 / 12733, where seed-preferred promoters from endl and end2 genes are disclosed; herein incorporated by reference.

[0075] In certain embodiments, the polynucleotides of the present disclosure can involve the use of the intact, native NLR genes, wherein the expression is driven by a cognate 5' upstream promoter sequence(s).

[0076] Specific soybean promoters include but are not limited to soy ubiquitin (subi-1), elongation factor 1A, and S-adenosyl methionine synthase for constitutive expression and Rpp4, RPG1-B, and promoters contained in gene models such as Glyma promoters.

[0077] Also provided is a nucleic acid (e.g., DNA) construct comprising any of the polynucleotides encoding the engineered NLR fusion polypeptides described herein. The use of the term "nucleotide constructs" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in themethods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and- loop structures and the like.

[0078] In certain embodiments, the polynucleotides encoding the engineered NLR fusion polypeptides described herein are provided in expression cassettes (e.g., a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear or circular single-stranded or doublestranded DNA or RNA nucleotide sequence) for expression in a plant of interest or any organism of interest. The cassette can include 5' and 3' regulatory sequences operably linked to a polynucleotide encoding the engineered NLR fusion polypeptide. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for insertion of the polynucleotide encoding the engineered NLR fusion polypeptide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0079] The expression cassette can include in the 5'-3 ' direction of transcription, a transcriptional and translational initiation region (e.g., a promoter), a polynucleotide encoding the engineered NLR fusion polypeptide, and a transcriptional and translational termination region (e.g., termination region) functional in plants. The regulatory regions (e.g., promoters, transcriptional regulatory regions, and translational termination regions) and / or the polynucleotide encoding the engineered NLR fusion polypeptide may be native / analogous to the host cell or to each other. Alternatively, the regulatory regions and / or the polynucleotide encoding the engineered NLR fusion polypeptide may be heterologous to the host cell or to each other.

[0080] The termination region may be native with the transcriptional initiation region, with the plant host, or may be derived from another source (i.e., foreign or heterologous) than the promoter, the polynucleotide encoding the engineered NLR fusion polypeptide, the plant host, or any combination thereof.

[0081] The expression cassette may additionally contain a 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include viral translational leader sequences.

[0082] Generally, the expression cassette can comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present disclosure.

[0083] In preparing the expression cassette, the various DNA fragments may be manipulated, to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0084] In certain embodiments, the nucleic acid construct or expression cassette, described herein, is expressed in a plant or seed. In certain embodiments, the plant or seed is a soybean plant or soybean seed. The nucleic acid constructs or expression cassettes disclosed herein may be used for transformation of any plant species.

[0085] In certain embodiments, the polynucleotide sequences encoding an engineered NLR fusion polypeptide, described herein, can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. This stacking can be accomplished by a combination of genes within a nucleic acid construct, or by crossing one or more plants having transgenes with another plant line that comprises a desired combination. Forexample, the polynucleotides of the present disclosure or variants thereof can be stacked with any other polynucleotides of the disclosure, or with other genes. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the present disclosure can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including and not limited to traits desirable for animal feed such as high oil genes, balanced amino acids, increased digestibility, insect, disease or herbicide resistance, avirulence and disease resistance genes, agronomic traits (e g, male sterility, flowering time) and / or transformation technology traits (e.g., cell cycle regulation or gene targeting).

[0086] These stacked combinations can be created by any method including and not limited to cross breeding plants by any conventional or known methodology, genetic transformation, or genome engineering. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a cotransformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that can suppress the expression of the polynucleotide of interest. This can be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant.

[0087] In one embodiment, the stacked combination includes one or more genes encoding pesticidal proteins including, but not limited to: Pesticidal proteins include but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7: 1-13); from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58: 12343- 12349) and from Pseudomonas pseudoalcaligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89: 159-168); insecticidal proteinsfrom Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal, 3: 101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069); US Patent Number 6,048,838, and US Patent Number 6,379,946; a PIP-1 polypeptide of US 9,688,730; an AflP-lA and / or AflP-lB polypeptide of US9,475,847; a PIP-47 polypeptide of US Publication Number US20160186204; an IPD045 polypeptide, an IPD064 polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077 polypeptide of PCT Publication Number WO 2016 / 114973; an IPD080 polypeptide of International Patent Application Publication Number WO2018 / 075350; an IPD078 polypeptide, an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, an IPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide of International Patent Application Publication Number WO2018 / 084936; PIP-72 polypeptide of US Patent Publication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide of US Publication Number US20170166921; an IPD098 polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109 polypeptide of International Patent Application Publication Number WO2018 / 232072; a PtIP-83 polypeptide of US Publication Number US20160347799; a PtIP-96 polypeptide of US Publication Number US20170233440; an IPD079 polypeptide of PCT Publication Number WO2017 / 23486; an IPD082 polypeptide of PCT Publication Number WO 2017 / 105987, an IPD090 polypeptide of International Patent Application Publication Number WO2017 / 192560, an IPD093 polypeptide of International Patent Application Publication Number WO2018 / 111551; an IPD103 polypeptide of International Patent Application Publication Number WO2018 / 005411; an IPD101 polypeptide of International Patent Application Publication Number WO2018 / 118811; an IPD121 polypeptide of International Patent Application Publication NumberWO20 18 / 208882; and 5-endotoxins including, but not limited to a Cryl, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, CrylO, Cryl l, Cryl2, Cry 13, Cryl4, Cryl5, Cryl6, Cryl7, Cryl8, Cry 19, Cry20, Cry21, Cry 22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, and Cry 72 classes of 5-endotoxin polypeptides and the B. thuringiensis cytolytic cytl and cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins can be found in Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011), atlifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / which can be accessed on the world-wide web using the "www" prefix).

[0088] In another embodiment, the stacked combination includes a polynucleotide encoding resistance to an herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J. 7: 1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449, respectively. See also, US Patent Numbers 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and 5,378,824; US Patent Application Serial Number 11 / 683,737 and International Publication WO 1996 / 33270.

[0089] In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to Glyphosate (resistance imparted by mutant 5-enolpyruvl-3- phosphikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes). See, for example, US Patent Number 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSPS which can confer glyphosate resistance. US Patent Number 5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes. See also, US Patent Numbers 6,566,587; 6,338,961; 6,248,876; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 5,094,945, 4,940,835; 5,866,775; 6,225,114; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;5,510,471; Re. 36,449; RE 37,287 and 5,491,288 and International Publications EP 1173580; WO 2001 / 66704; EP 1173581 and EP 1173582, which are incorporated herein by reference for this purpose. Glyphosate resistance is also imparted to plants that express a gene encoding a glyphosate oxido-reductase enzyme as described more fully in US Patent Numbers 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose. In addition, glyphosate resistance can be imparted to plants by the over expression of genes encoding glyphosate N-acetyltransf erase. See, for example, US Patent Numbers 7,462,481; 7,405,074 and US Patent Application Publication Number US 2008 / 0234130. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC® Accession Number 39256, and the nucleotide sequence of the mutant gene is disclosed in US Patent Number 4,769,061 to Comai. EP Application Number 0 333 033 to Kumada, et al., and US Patent Number 4,975,374 toGoodman, et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in EP Application Numbers 0 242 246 and 0 242 236 to Leemans, et al.; De Greef, et al., (1989) Bio / Technology 7:61, describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. See also, US Patent Numbers 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 and 5,879,903, which are incorporated herein by reference for this purpose. Exemplary genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and hal oxyfop, are the Accl- Sl, Accl-S2 and Accl-S3 genes described by Marshall, et al., (1992) Theor. Appl. Genet. 83:435.

[0090] In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell 3: 169, describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in US Patent Number 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC® Accession Numbers 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes, et al., (1992) Biochem. J. 285: 173.

[0091] In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants (see, e.g., Hattori, et al., (1995) Mol Gen Genet. 246:419). Other genes that confer resistance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994) Plant Physiol 106: 17), genes for glutathione reductase and superoxide dismutase (Aono, et al., (1995) Plant Cell Physiol 36: 1687) and genes for various phosphotransferases (Datta, et al., (1992) Plant Mol Biol 20:619).

[0092] In another embodiment, the stacked combination includes a polynucleotide encoding resistance to an herbicide targeting Protoporphyrinogen oxidase (protox) which is necessary for the production of chlorophyll. The protox enzyme serves as the target for a variety of herbicidalcompounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in US Patent Numbers 6,288,306; 6,282,83 and 5,767,373 and International Publication WO 2001 / 12825.

[0093] In another embodiment, the stacked combination includes an aad-1 gene (originally from Sphingobium herbicidovorans) encoding the aryloxyalkanoate dioxygenase (AAD-1) protein. The trait confers tolerance to 2,4-dichlorophenoxyacetic acid and aryl oxy phenoxy propionate (commonly referred to as “fop” herbicides such as quizalofop) herbicides. The aad-1 gene, itself, for herbicide tolerance in plants was first disclosed in WO 2005 / 107437 (see also, US 2009 / 0093366). The aad-12 gene, derived from Delftia acidovorans, which encodes the aryloxy alkanoate dioxygenase (AAD-12) protein that confers tolerance to 2,4- dichlorophenoxyacetic acid and pyridyloxyacetate herbicides by deactivating several herbicides with an aryloxyalkanoate moiety, including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxy auxins (e.g., fluroxypyr, triclopyr).

[0094] In another embodiment, the stacked combination includes a polynucleotide encoding an herbicide resistant dicamba monooxygenase disclosed in US Patent Application Publication 2003 / 0135879 for imparting dicamba tolerance.

[0095] In another embodiment, the stacked combination includes a polynucleotide encoding bromoxynil nitrilase (Bxn) disclosed in US Patent Number 4,810,648 for imparting bromoxynil tolerance.

[0096] In another embodiment, the stacked combination includes a polynucleotide encoding CcRppl disclosed in U.S. 10,842,097 for imparting ASR resistance and / or a polynucleotide encoding CcRpp2 disclosed in U.S. Patent Application Publication 2024 / 0043861 for imparting ASR resistance.

[0097] In another embodiment, the stacked combination includes a polynucleotide encoding phytoene (crtl) described in Misawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994) Plant J. 6:481-489 for norflurazon tolerance.

[0098] In another embodiment, the stacked combination includes a polynucleotide encoding a protein that confers or contributes to an altered grain characteristic, such as altered fatty acids, for example, by:

[0099] (1) Down-regulation of stearoyl-ACP to increase stearic acid content of the plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA 89:2624 and WO 1999 / 64579 (Genes to Alter Lipid Profiles in Com).

[0100] (2) Elevating oleic acid via FAD-2 gene modification and / or decreasing linolenic acid via FAD-3 gene modification (see, US Patent Numbers 6,063,947; 6,323,392; 6,372,965 and WO 1993 / 11245).

[0101] (3) Altering conjugated linolenic or linoleic acid content, such as in WO 2001 / 12800.

[0102] (4) Altering LEC1, AGP, Dekl, Superall, mil ps, and various Ipa genes such as Ipal, Ipa3, hpt or hggt. For example, see, WO 2002 / 42424, WO 1998 / 22604, WO 2003 / 011015, WO 2002 / 057439, WO 2003 / 011015, US Patent Numbers 6,423,886, 6,197,561, 6,825,397 and US Patent Application Publication Numbers US 2003 / 0079247, US 2003 / 0204870 and Rivera- Madrid, et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624.

[0103] (5) Genes encoding delta-8 desaturase for making long-chain polyunsaturated fatty acids (US Patent Numbers 8,058,571 and 8,338,152), delta-9 desaturase for lowering saturated fats (US Patent Number 8,063,269), Primula A6-desaturase for improving omega-3 fatty acid profiles.

[0104] (6) Isolated nucleic acids and proteins associated with lipid and sugar metabolism regulation, in particular, lipid metabolism protein (LMP) used in methods of producing transgenic plants and modulating levels of seed storage compounds including lipids, fatty acids, starches or seed storage proteins and use in methods of modulating the seed size, seed number, seed weights, root length and leaf size of plants (EP 2404499).

[0105] (7) Altering expression of a High-Level Expression of Sugar-Inducible 2 (HSI2) protein in the plant to increase or decrease expression of HSI2 in the plant. Increasing expression of HSI2 increases oil content while decreasing expression of HSI2 decreases abscisic acid sensitivity and / or increases drought resistance (US Patent Application Publication Number 2012 / 0066794).

[0106] (8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oil content in plant seed, particularly to increase the levels of omega-3 fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (US Patent Application Publication Number 2011 / 0191904).

[0107] (9) Nucleic acid molecules encoding wrinkled 1 -like polypeptides for modulating sugar metabolism (US Patent Number 8,217,223).

[0108] Further provided are plants, plant cells, plant parts, seeds, and grain expressing at least one engineered NLR fusion polypeptide described herein. In certain embodiments, the plants, plant cells, plant parts, seeds, and grain comprise at least one of the engineered NLR fusion polypeptide polynucleotide sequences, nucleic acid constructs, or expression cassettes described herein, so that the plants, plant cells, plant parts, seeds, and / or grain express any of the engineered NLR fusion polypeptides described herein. In certain embodiments, the plants, plant cells, plant parts, seeds, and / or grain have stably incorporated at least one polynucleotide sequence encoding an engineered NLR fusion polypeptide into its genome. In certain embodiments, the plants, plant cells, plant parts, seeds, and grain comprise two or more of the engineered NLR fusion polypeptide polynucleotide sequences, nucleic acid constructs, or expression cassettes described herein, so that the plants, plant cells, plant parts, seeds, and / or grain express two or more of the engineered NLR fusion polypeptides described herein. In certain embodiments, the plants, plant cells, plant parts, seeds, and / or grain have stably incorporated two or more polynucleotide sequence encoding an engineered NLR fusion polypeptide into its genome. In embodiments in which two or more of the engineered NLR fusion polypeptides are expressed they can recognize effectors secreted from the same pathogen, effectors secreted from different pathogens, or a combination thereof.

[0109] For example, in certain embodiments the plant, plant cell, plant part, seed, or grain comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 50%, 55%, 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% identical to a sequence selected from SEQ ID NO: 5, 8, 11, 14, 17, 18, 21, 24, 27, 30, 402, 404, 406, 437, 439, 445, 451, 453, 459, 461, 467, 473, 475, 481, 483, 485, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, or 535. In certain embodiments, the polynucleotide is operably linked to a regulatory element, such as, for example, a heterologous promoter. In certain embodiments, the plant or a plant generated from the plant cell, plant part, or seed, has resistance to a pathogen, such as, for example, Asian soybean rust. In certain embodiments, the plant or a plant generated from the plant cell, plant part, or seed, has increased resistance to Asian soybean rust for example, as compared to a control plant (e.g., a plant of the same genetic background not comprising the polynucleotide).

[0110] In certain embodiments, the polynucleotides are transiently expressed in the plant, plant cell, plant part, seed or grain using a transient transformation technique. In certain embodiments, the polynucleotides are stably expressed in the plant, plant cell, plant part, seed or grain using a stable transformation technique. In certain embodiments, the polynucleotides are introduced into the plant, plant cell, plant part, seed or grain using a nucleic acid construct, or an expression cassette described herein. In certain embodiments, the polynucleotides are introduced into the plant, plant cell, plant part, seed or grain using a genome editing technique.

[0111] In certain embodiments, the plant cells or plant parts are grown into plants. The method for generating the plants from the plant cells or plant parts is not particularly limited. These plants can then be grown, and either pollinated with the same strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations can be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In some aspects of the present disclosure, the transformed seed or transgenic seed having a nucleotide construct, or an expression cassette is stably incorporated into their genome.

[0112] In certain embodiments, the present disclosure encompasses seeds comprising a polynucleotide sequence disclosed herein that can develop into or can be used to develop a plant or plants with increased or enhanced resistance to a pathogen (e.g., ASR) or infection caused by a pathogen as compared to, for example, a wild-type variety of the plant seed. In an aspect, the present disclosure features seeds from transgenic legume crop plants wherein the seed comprises a polynucleotide disclosed herein.

[0113] In certain embodiments, the plants described herein are elite plant lines (e.g., elite soybean line). In certain embodiments, the plant cells, plant parts, seeds, and grain are isolated from or produced by an elite plant line. As used herein, “elite line” refers to any line that has resulted from breeding and selection for superior agronomic performance that allows a producer to harvest a product of commercial significance. Numerous elite lines are available and known to those of skill in the art of plant breeding (e.g., soybean, canola, and sunflower breeding). An “elite population” is an assortment of elite individuals or lines that can be used to represent the state of the art in terms of agronomically superior genotypes of a given crop species, such as soybean.

[0114] As used herein, the term “plant” includes plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the disclosure, provided that these parts comprise the introduced polynucleotides.

[0115] The plant species of the compositions and methods of the present disclosure can be any plant species for which expression of an engineered NLR fusion polypeptide, or polynucleotide encoding the NLR fusion polypeptide, described herein is desired, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica spp. (e g., Brassica napus, Brassica rapa, Brassica j uncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp ), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Per sea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[0116] In certain embodiments, the plant of the compositions and methods described herein is a legume crop species, including, but not limited to, alfalfa (Medicago sativa); clover or trefoil (Trifolium spp.); pea, including (Pisum satinum), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata) and Lathyrus spp.; bean (Fabaceae or Leguminosae); lentil (Lens culinaris); lupin (Lupinus spp.); mesquite (Prosopis spp.); carob (Ceratonia siliqua), soybean (Glycine max), peanut (Arachis hypogaea) or tamarind (Tamarindus indica). The terms "legume species" and"legume crop species" are used herein to refer to plants, and can be for example, a plant of interest. In some aspects, the legume species or legume crop species is a plant, plant part or plant cell.

[0117] Also provided are host cells that are engineered (e.g., transduced, transformed, or transfected) to express one or more of any of the engineered NLR fusion polypeptides described herein and / or express one or more of any of the polynucleotides or nucleic acid constructs described herein. The NLR fusion polypeptides, polynucleotides encoding the NLR fusion polypeptides, or nucleic acid constructs encoding the NLR fusion polypeptides described herein can be expressed in any organism, including in non-animal cells such as yeast, fungi, bacteria and the like. Details regarding non-animal cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture,' Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin, Heidelberg, New York); and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

[0118] Host cells of interest can include, for example, a eukaryotic cell, an animal cell, a protoplast, a tissue culture cell, prokaryotic cell, a bacterial cell, such as E. coli, B. subtilis, Streptomyces, Salmonella typhimurium, a gram positive bacteria, a purple bacteria, a green sulfur bacteria, a green non-sulfur bacteria, a cyanobacteria, a spirochetes, a thermatogale, a flavobacteria, bacteroides; a fungal cell, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa, an insect cell such as Drosophila and Spodoptera frugiperda,' a mammalian cell such as CHO, COS, BHK, HEK 293 or Bowes melanoma, archaebacteria (i.e., Korarchaeota, Thermoproteus, Pyrodictium, Thermococcales, Methanogens, Archaeoglobus, and extreme Halophiles).

[0119] Also provided are methods for providing, conferring, or enhancing resistance to a pathogen in a plant. The pathogen for which resistance is provided, conferred, or enhanced may be any pathogen described herein, or known in the art, for which resistance to is desired, such as, for example, Rhizoctonia solani, Phakopsora pachyrhizi, Phakopsora meibomiae, Colletotrichum truncatum, Phialophora gregata, Cercospora kukuchii, Macrophomina phaseolina, Diaporthe phaseolorum, Peronospora manshurica, Cercospora sojina, Phytophthora sojae, Microsphaera diffusa, Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne javanica, Heterodera glycines, Fusarium virguliforme, Septaria glycines,Corynespora cassiicola, and Sclerotinia sclerotiorum . In certain embodiments, the pathogen is a pathogen causing ASR (e.g., Phakopsora pachyrhizi and Phakopsora meibomiae such that method provides, confers, or enhances resistance of a plant to ASR. As used herein the term "enhance" means to improve, increase, amplify, multiply, elevate and / or raise, thereby reducing one or more disease symptoms. Accordingly, plants (e.g., soybean) exhibit an increased resistance to a disease (e.g., ASR) when compared to plants that are susceptible or tolerant to Phakopsora spp. In an aspect, methods described herein can reduce one or more symptoms (i.e., disease symptoms) of a plant disease (e.g., ASR).

[0120] In certain embodiments, the method for providing, conferring, or enhancing resistance to a pathogen in a plant comprises introducing into a regenerable plant cell at least one of the polynucleotides, nucleic acid constructs, or expression cassettes described herein and generating a plant which expresses an engineered NLR fusion polypeptide described herein and has increased resistance to a pathogen as compared to a control plant. In certain embodiments, the plant is a legume crop species (e.g., soybean). In certain embodiments, the pathogen is a pathogen causing ASR (e.g., Phakopsora pachyrhizi and Phakopsora meibomiae)' . In certain embodiments, the engineered NLR fusion polypeptide comprises an NLR scaffold described herein and a pathogen effector interacting domain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 3, 6, 9, 12, 15, 19, 22, 25, 28, 435, 443, 449, 457, 465, 471, 479, 489, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, or 583. In certain embodiments, the engineered NLR fusion polypeptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 5, 8, 11, 14, 17, 18, 21, 24, 27, 30, 402, 404, 406, 437, 439, 445, 451, 453, 459, 461, 467, 473, 475, 481, 483, 485, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, or 535.

[0121] In certain embodiments of the methods for providing, conferring, or enhancing resistance of a plant to a pathogen (e.g., ASR) described herein, the method comprises introducing a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 5, 8, 11, 14, 17, 18, 21, 24, 27, 30, 402, 404, 406, 437, 439,445, 451, 453, 459, 461 , 467, 473, 475, 481 , 483, 485, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, or 535, optionally operably linked to a regulatory element (e.g., heterologous promoter); and generating a plant from the plant cell wherein the plant comprises the polynucleotide and has enhanced resistance, increased resistance, or resistance to the pathogen (e.g., ASR).

[0122] In certain embodiments, the method for providing, conferring, or enhancing resistance to a pathogen in a plant comprises expressing in a regenerable plant cell a nucleic acid construct or expression cassette comprising a polynucleotide described herein; and generating the plant from the plant cell. In certain embodiments, the polynucleotide is operably linked to at least one regulatory sequence. In certain embodiments, the at least one regulatory sequence is a heterologous promoter. The nucleic acid construct or expression cassette for use in the method may be any nucleic acid construct or expression cassette provided herein. In certain embodiments the nucleic acid construct or expression cassette is expressed by introducing into a plant, plant cell, plant part, seed, and / or grain the nucleic acid construct or expression cassette, whereby the engineered NLR fusion polypeptide is expressed in the plant, plant cell, plant part, seed, and / or grain. In certain embodiments the nucleic acid construct or expression cassette is incorporated into the genome of the plant.

[0123] Various methods can be used to introduce the polynucleotide sequences into a plant, plant part, plant cell, seed, and / or grain. "Introducing" is intended to mean presenting to the plant, plant cell, seed, and / or grain the polynucleotide or resulting polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the disclosure do not depend on a particular method for introducing a sequence into a plant, plant cell, seed, and / or grain, only that the polynucleotide or polypeptide gains access to the interior of at least one cell of the plant.

[0124] "Stable transformation" is intended to mean that the polynucleotide introduced into a plant integrates into the genome of the plant of interest and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant of interest and does not integrate into the genome of the plant or organism, or a polypeptide is introduced into a plant or organism.

[0125] Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e.,monocot or di cot, targeted for transfor ation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Patent No. 5,563,055 and U.S. Patent No. 5,981,840), Ochrobacterium-mediated transformation (U.S. Patent Application Publication 2018 / 0216123 and WO20 / 092494) direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717 -2722), and ballistic particle acceleration (see, for example, U.S. Patent Nos. 4,945,050; U.S. Patent No. 5,879,918; U.S. Patent No. 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00 / 28058). D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens , all of which are herein incorporated by reference.

[0126] In certain embodiments, the polynucleotide sequences can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the engineered NLR fusion polypeptide directly into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44:53-58,' Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference.

[0127] In other embodiments, the polynucleotides encoding the engineered NLR fusion polypeptides disclosed herein may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the disclosure within a DNA or RNA molecule. It is recognized that the polynucleotide sequence may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters disclosed herein also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, forexample, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5 209-221; herein incorporated by reference.

[0128] Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99 / 25821, WO99 / 25854, WO99 / 25840, WO99 / 25855, and WO99 / 25853, all of which are herein incorporated by reference. Briefly, the polynucleotide disclosed herein can be contained in a transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided, and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome. Other methods to target polynucleotides are set forth in WO 2009 / 114321 (herein incorporated by reference), which describes “custom" meganucleases produced to modify plant genomes, in particular the genome of maize. See, also, Gao et al. (2010) Plant Journal 7: 176-187.

[0129] One of skill will recognize that after the expression cassette containing the polynucleotide encoding the engineered NLR fusion polypeptide is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

[0130] Parts obtained from the regenerated plants described herein, such as flowers, seeds, leaves, branches, fruit, and the like are included, provided that these parts comprise cells comprising the polynucleotide encoding the engineered NLR fusion polypeptide. Progeny and variants, and mutants of the regenerated plants are also included, provided that these parts comprise the introduced nucleic acid sequences.

[0131] In one embodiment, a homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced. Back- crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

[0132] In certain embodiments, the method for providing, conferring, or enhancing resistance to a pathogen in a plant comprises introducing a polynucleotide encoding a polypeptide describedherein into the genome of the regenerable plant cell using genome editing technologies. In certain embodiments, the method comprises editing an endogenous TIR domain containing NLR gene or previously introduced TIR domain containing NLR gene to produce a polynucleotide encoding an engineered NLR fusion polypeptide described herein. In certain embodiments, the endogenous TIR domain containing NLR gene comprises a nucleic acid sequence having at least 50%, 55%, 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% identity to any one of SEQ ID NOs: 197-308. In certain embodiments, the method comprises replacing an endogenous post- LRR (PL) domain of the endogenous TIR domain containing NLR gene or a PL domain of a previously introduced TIR domain containing NLR gene with one or more heterologous pathogen effector interacting domains described herein. When two or more heterologous pathogen effector interacting domains are present the domains can interact with effectors secreted from the same pathogen, different pathogens, or a combination thereof. In certain embodiments, the method comprises using genome editing to introduce a polynucleotide encoding the engineered NLR fusion polypeptide into the genome at a pre-determined locus.

[0133] The genome editing technology for use in the methods and compositions described herein is not particularly limited and may be any genome editing technique that allows for the introduction and / or targeted introduction of the desired polynucleotide.

[0134] In certain embodiments the genome editing technique is selected from the group consisting of a polynucleotide-guided endonuclease, CRISPR-Cas endonucleases, base editing deaminases, zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), engineered site-specific meganuclease, or Argonaute.

[0135] In certain embodiments, the genome modification may be facilitated through the induction of a double-stranded break (DSB) or single-strand break, in a defined position in the genome near the desired alteration. DSBs can be induced using any DSB-inducing agent available, including, but not limited to, TALENs, meganucleases, zinc finger nucleases, Cas9- gRNA systems (based on bacterial CRISPR-Cas systems), guided cpfl endonuclease systems, and the like. In some embodiments, the introduction of a DSB can be combined with the introduction of a polynucleotide modification template.

[0136] The process for editing a genomic sequence combining DSB and modification templates generally comprises: providing to a host cell, a DSB-inducing agent, or a nucleic acid encoding aDSB-inducing agent, that recognizes a target sequence in the chromosomal sequence and is able to induce a DSB in the genomic sequence, and at least one polynucleotide modification template comprising at least one nucleotide alteration when compared to the nucleotide sequence to be edited. The polynucleotide modification template can further comprise nucleotide sequences flanking the at least one nucleotide alteration, in which the flanking sequences are substantially homologous to the chromosomal region flanking the DSB.

[0137] The endonuclease can be provided to a cell by any method known in the art, for example, but not limited to, transient introduction methods, transfection, microinjection, and / or topical application or indirectly via recombination constructs. The endonuclease can be provided as a protein or as a guided polynucleotide complex directly to a cell or indirectly via recombination constructs. The endonuclease can be introduced into a cell transiently or can be incorporated into the genome of the host cell using any method known in the art. In the case of a CRISPR-Cas system, uptake of the endonuclease and / or the guided polynucleotide into the cell can be facilitated with a Cell Penetrating Peptide (CPP) as described in WO2016073433 published May 12, 2016.

[0138] TAL effector nucleases (TALEN) are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism (Miller et al. (2011) Nature Biotechnology 29: 143-148).

[0139] Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA at specific sites without damaging the bases, and meganucleases, also known as homing endonucleases (HEases), which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18 bp or more (patent application PCT / US12 / 30061, filed on March 22, 2012). Meganucleases have been classified into four families based on conserved sequence motifs. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganuclease is similar to the convention for other restriction endonuclease. Meganucleases are also characterized by prefix F-, I-, or PI- for enzymes encoded by freestanding ORFs, introns, and inteins, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. The cleaving activity can be used toproduce a double-strand break. For reviews of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7. In some examples the recombinase is from the Integrase or Resolvase families.

[0140] Zinc finger nucleases (ZFNs) are engineered double-strand break inducing agents comprised of a zinc finger DNA binding domain and a double-strand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprising two, three, or four zinc fingers, for example having a C2H2 structure, however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example nuclease domain from a Type Ils endonuclease such as Fokl. Additional functionalities can be fused to the zinc-finger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases. In some examples, dimerization of nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3-finger domain recognized a sequence of 9 contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind an 18-nucleotide recognition sequence.

[0141] Genome editing using DSB-inducing agents, such as Cas9-gRNA complexes, has been described, for example in U.S. Patent Application US 2015-0082478 Al, WO2015 / 026886 Al, WO20 16007347, and WO201625131 all of which are incorporated by reference herein.

[0142] In certain embodiments the genetic modification is introduced without introducing a double strand break using base editing technology, see e.g., Gaudelli et al., (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551(7681):464- 471; Komor et al., (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, Nature 533(7603):420-4.

[0143] In certain embodiments, base editing comprises (i) a catalytically impaired CRISPR- Cas9 mutant that is mutated such that one of their nuclease domains cannot make DSBs; (ii) a single-strand-specific cytidine / adenine deaminase that converts C to U or A to G within an appropriate nucleotide window in the single-stranded DNA bubble created by Cas9; (iii) a uracil glycosylase inhibitor (UGI) that impedes uracil excision and downstream processes that decreasebase editing efficiency and product purity; or (iv) nickase activity to cleave the non-edited DNA strand, followed by cellular DNA repair processes to replace the G-containing DNA strand.

[0144] In certain embodiments of the method for providing, conferring, or enhancing resistance to a pathogen in a plant crossing a first plant comprising a polynucleotide encoding an engineered NLR fusion polypeptide described herein with a second different plant line and harvesting the seed produced thereby and generating a second-generation progeny plant. In certain embodiments, the second-generation progeny plant comprises the polynucleotide and has resistance or increased resistance to the pathogen.

[0145] In certain embodiments, the engineered NLR fusion polypeptide confers resistance to ASR such that the second-generation progeny plant comprises the polynucleotide encoding the engineered NLR fusion polypeptide and has resistance or increased resistance to ASR. In certain embodiments, the second plant line is susceptible to ASR. In certain embodiments, the second plant line comprises a different ASR resistance polypeptide than the polypeptide expressed in the first plant line, such as, for example CcRppl and / or CcRpp2.

[0146] In certain embodiments, the method further comprises backcrossing the second- generation progeny plant to the second plant to produce a backcross progeny plant that comprises the polypeptide and produces backcrossed seed with ASR resistance.

[0147] Introgressing is sometimes called "backcrossing" when the process is repeated two or more times. In introgressing or backcrossing, the "donor" parent refers to the parental plant with the desired gene or locus to be introgressed. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. The initial cross gives rise to the Fl generation; the term "BC1 " then refers to the second use of the recurrent parent, and "BC2" refers to the third use of the recurrent parent, and so on.

[0148] The present disclosure also provides a method for screening or assaying plants comprising an engineered NLR fusion polypeptide, described herein, for resistance, immunity, or susceptibility to a plant disease. General methods for determination of resistance, immunity, or susceptibility of a plant to a particular pathogen are known to one skilled in the art. For example, a method for screening or assaying plants for resistance, immunity or susceptibility to a plant disease may comprise exposing a plant cell, tissue or organ (e.g., leaf) to a pathogen (e.g., Phakopsora pachyrhizi) and then determining and / or measuring in the exposed plant, the degreeof resistance, immunity and / or susceptibility to a plant disease (e ., ASR) caused by the pathogen. The method can further comprise measuring any observable plant disease symptoms on the plant exposed to the plant pathogen and then comparing the plant disease symptoms to a reference standard to determine the degree or extent of disease resistance.

[0149] Methods of exposing a plant cell, tissue or organ to a pathogen are known in the art. Methods of measuring, comparing, and determining the level of resistance, immunity and / or susceptibility (e.g., plant disease symptoms) to a disease, such as, for example, ASR, caused by the pathogen are also known in the art. The exposed plants can be further assessed to isolate polynucleotides, amino acid sequences and / or genetic markers that are associated with, linked to, and / or confer resistance, immunity or susceptibility of a plant to a particular pathogen or disease. Further assessments include, but are not limited to, isolating polynucleotides, nucleic acids, or amino acids sequences from the exposed plant, carrying out an assay of the isolated polynucleotides or nucleic acids, for example, to detect one or more biological or molecular markers associated with one or more agronomic characteristics or traits, including but not limited to, resistance, immunity and / or susceptibility. The information gleaned from such methods can be used, for example, in a breeding program.

[0150] In certain embodiments, the plants expressing the polynucleotides and engineered NLR fusion polypeptides disclosed herein, such as, for example, an engineered NLR fusion polypeptide comprising a pathogen effector interacting domain recognizing an effector from Phakopsora pachyrhizi or Phakopsora meibomiae, may also have one or more fungicides applied to the plants as a method of further preventing ASR associated damage to a legume crop species. These fungicidal compounds may also be applied to supplement the protection of a legume crop species comprising the engineered NLR fusion polypeptide to a wider variety of undesirable diseases. These fungicides may be formulated or tank-mixed with other fungicide(s) disclosed herein or applied sequentially with the other fungicide(s). Such fungicides may include 2-(thiocyanatomethylthio)-benzothiazole, 2-phenylphenol, 8-hydroxyquinoline sulfate, ametoctradin, aminopyrifen, amisulbrom, antimycin, Ampelomyces quisqualis, azaconazole, azoxystrobin, Bacillus subtilis, Bacillus subtilis strain QST713, benalaxyl, benomyl, benthiavalicarb-isopropyl, benzovindiflupyr, benzylaminobenzene-sulfonate (BABS) salt, bicarbonates, biphenyl, bismerthiazol, bitertanol, bixafen, blasticidin-S, borax, Bordeaux mixture, boscalid, bromuconazole, bupirimate, calcium polysulfide, captafol, captan,carbendazim, carboxin, carpropamid, carvone, chlazafenone, chloroinconazide, chloroneb, chlorothalonil, chlozolinate, Coniothyrium minitans, copper hydroxide, copper octanoate, copper oxychloride, copper sulfate, copper sulfate (tribasic), cuprous oxide, cyazofamid, cyflufenamid, cymoxanil, cyproconazole, cyprodinil, dazomet, debacarb, diammonium ethylenebis- (dithiocarbamate), dichlofluanid, dichlorophen, diclocymet, diclomezine, dichloran, diethofencarb, difenoconazole, difenzoquat ion, diflumetorim, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinobuton, dinocap, diphenylamine, dithianon, dodemorph, dodemorph acetate, dodine, dodine free base, edifenphos, enestrobin, enestroburin, epoxiconazole, ethaboxam, ethoxyquin, etridiazole, famoxadone, fenamidone, fenarimol, fenbuconazole, fenfuram, fenhexamid, fenoxanil, fenpiclonil, fenpropidin, fenpropimorph, fenpyrazamine, fentin, fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, fludioxonil, fluindapyr, flumorph, fluopicolide, fluopyram, fluoroimide, fluoxapiprolin, fluoxastrobin, fluquinconazole, flusilazole, flusulfamide, flutianil, flutolanil, flutriafol, fluxapyroxad, folpet, formaldehyde, fosetyl, fosetyl-aluminium, fuberidazole, furalaxyl, furametpyr, guazatine, guazatine acetates, GY-81, hexachlorobenzene, hexaconazole, hymexazol, imazalil, imazalil sulfate, imibenconazole, iminoctadine, iminoctadine triacetate, iminoctadine tris(albesilate), inpyrfluxam, iodocarb, ipconazole, ipfenpyrazolone, iprobenfos, iprodione, iprovalicarb, isofetamide, isoflucypram, isoprothiolane, isopyrazam, isotianil, kasugamycin, kasugamycin hydrochloride hydrate, kresoxim-methyl, laminarin, mancopper, mancozeb, mandipropamid, maneb, mefenoxam, mepanipyrim, mepronil, meptyl-dinocap, mercuric chloride, mercuric oxide, mercurous chloride, metalaxyl, metalaxyl-M, metam, metam- ammonium, metam-potassium, metam-sodium, metconazole, methasulfocarb, methyl iodide, methyl isothiocyanate, metiram, metominostrobin, metrafenone, mildiomycin, myclobutanil, nabam, nitrothal-isopropyl, nuarimol, octhilinone, ofurace, oleic acid (fatty acids), orysastrobin, oxadixyl, oxathiapiprolin, oxine-copper, oxpoconazole fumarate, oxycarboxin, pefurazoate, penconazole, pencycuron, penflufen, pentachlorophenol, pentachlorophenyl laurate, penthiopyrad, phenylmercury acetate, phosphonic acid, phthalide, picoxystrobin, polyoxin B, polyoxins, polyoxorim, potassium bicarbonate, potassium hydroxyquinoline sulfate, probenazole, prochloraz, procymidone, propamocarb, propamocarb hydrochloride, propi conazole, propineb, proquinazid, prothioconazole, pydiflumetofen, pyrametostrobin, pyraoxystrobin, pyraclostrobin, pyraziflumid, pyrazophos, pyribencarb, pyributicarb, pyrifenox,pyrimethanil, pyriofenone, pyroquilon, quinoclamine, quinoxyfen, quintozene, Reynoutria sachalinensis extract, sedaxane, silthiofam, simeconazole, sodium 2-phenylphenoxide, sodium bicarbonate, sodium pentachlorophenoxide, spiroxamine, sulfur, SYP-Z048, tar oils, tebuconazole, tebufloquin, tecnazene, tetraconazole, thiabendazole, thifluzamide, thiophanate- methyl, thiram, tiadinil, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazoxide, tricyclazole, tridemorph, trifloxystrobin, triflumizole, triforine, tri ti conazole, validamycin, valifenalate, valiphenal, vinclozolin, zineb, ziram, zoxamide, Candida oleophila, Fusarium oxysporum, Gliocladium spp., Phlebiopsis gigantea, Streptomyces griseoviridis, Trichoderma spp., (RS)-N-(3,5-dichlorophenyl)-2-(methoxymethyl)-succinimide, 1,2-di chloropropane, 1,3- di chi oro-1, 1,3, 3 -tetrafluoroacetone hydrate, l-chloro-2,4-dinitronaphthalene, l-chloro-2- nitropropane, 2-(2-heptadecyl-2-imidazolin- 1 -yl)ethanol, 2,3 -dihydro-5-phenyl- 1 ,4-dithi-ine 1,1,4,4-tetraoxide, 2-methoxy ethylmercury acetate, 2-methoxy ethylmercury chloride, 2- methoxyethylmercury silicate, 3-(4-chlorophenyl)-5-methylrhodanine, 4-(2-nitroprop-l- enyl)phenyl thiocyanateme, ampropylfos, anilazine, azithiram, barium polysulfide, Bayer 32394, benodanil, benquinox, bentaluron, benzamacril, benzamacril-isobutyl, benzamorf, binapacryl, bis(methylmercury) sulfate, bis(tributyltin) oxide, buthiobate, cadmium calcium copper zinc chromate sulfate, carbamorph, CECA, chlobenthiazone, chloraniformethan, chlorfenazole, chlorquinox, climbazole, copper bis(3-phenylsalicylate), copper zinc chromate, coumoxystrobin, cufraneb, cupric hydrazinium sulfate, cuprobam, cyclafuramid, cypendazole, cyprofuram, decafentin, dichlobentiazox, dichlone, dichlozoline, diclobutrazol, dimethirimol, dinocton, dinosulfon, dinoterbon, dipymetitrone, dipyrithione, ditalimfos, dodicin, drazoxolon, EBP, enoxastrobin, ESBP, etaconazole, etem, ethirim, fenaminstrobin, fenaminosulf, fenapanil, fenitropan, fenpicoxamid, florylpicoxamid, flubeneteram, flufenoxystrobin, fluopimomide, fluotrimazole, furcarbanil, furconazole, furconazole-cis, furmecyclox, furophanate, glyodine, griseofulvin, halacrinate, Hercules 3944, hexylthiofos, ICIA0858, ipfentrifluconazole, ipflufenoquin, isopamphos, isovaledione, mandestrobin, mebenil, mecarbinzid, mefentrifluconazole, metazoxolon, methfuroxam, methylmercury dicyandiamide, metsulfovax, metyltetraprole, milneb, mucochloric anhydride, myclozolin, N-3,5-dichlorophenyl-succinimide, N-3-nitrophenylitaconimide, natamycin, N-ethylmercurio-4-toluenesulfonanilide, nickel bis(dimethyldithiocarbamate), OCH, phenylmercury dimethyldithiocarbamate, phenylmercury nitrate, phosdiphen, prothiocarb; prothiocarb hydrochloride, pyracarbolid, pyrapropoyne,pyridachlometyl, pyridinitril, pyrisoxazole, pyroxychlor, pyroxyfur, quinacetol; quinacetol sulfate, quinazamid, quinconazole, quinofumelin, rabenzazole, salicylanilide, SSF-109, sultropen, tecoram, thiadifluor, thicyofen, thiochlorfenphim, thiophanate, thioquinox, tioxymid, triamiphos, triarimol, triazbutil, trichlamide, triclopyricarb, triflumezopyrim, urbacid, zarilamid, (2S,3S)-3-(o-tolyl)butan-2-yl (4-methoxy-3-(propionyloxy)picolinoyl)-L-alaninate, and any combinations thereof.

[0151] Accordingly, provided is a method for preventing pathogen (e.g., ASR) associated damage to a legume crop species comprising planting a field with seed comprising a polynucleotide encoding an engineered NLR fusion polypeptide comprising a pathogen effector interacting domain recognizing an effector from a pathogen of interest (e.g., Phakopsora pachyrhizi or Phakopsora meibomiae) described herein. In certain embodiments, the method further comprises treating the field with a fungicide.

[0152] Also provided herein is a method of identifying germplasm or plants comprising a polynucleotide encoding an engineered NLR fusion polypeptide of the disclosure. The method comprising obtaining a nucleic acid sample from one or more plants, and contacting said nucleic acid sample with a nucleic acid sequence that specifically binds to the polynucleotide of the disclosure and detecting the specific binding of the nucleic acid to its target sequence. For example, the method can detect the target sequence through the use of a labeled probe or by conducting a PCR reaction with suitable PCR primers that only produce an amplicon in the presence of the target sequence. In one embodiment the method comprises obtaining a nucleic acid sample from one or more plants, and contacting the nucleic acid sample with either:

[0153] i) a polynucleotide that comprises a sequence of at least 8 nucleotides that are identical or have at least 90-95% sequence identity to a contiguous sequence selected from the group consisting of SEQ ID NOs: 313, 316, 319, 322, 325, 326, 329, 332, 335, 338, 401, 403, 405, 434, 438, 442, 448, 452, 456, 460, 464, 470, 474, 478, 482, 484, 488, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534 or complements thereof; wherein said method further comprises subjecting said sample and said polynucleotide to stringent hybridization conditions; and assaying said sample for hybridization of said polynucleotide to said DNA; or

[0154] ii) a pair of PCR primers, wherein a first and second PCR primer each specifically bind to a sequence selected from the group consisting of SEQ ID NOs: 313, 316, 319, 322, 325, 326,329, 332, 335, 338, 401 , 403, 405, 434, 438, 442, 448, 452, 456, 460, 464, 470, 474, 478, 482, 484, 488, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, wherein said first and second PCR primers are capable of producing an amplicon when bound to their target complementary sequences and subjected to standard PCR reaction conditions; subjecting said sample to polymerase chain reaction conditions; and assaying for an amplicon generated between said first and second primers.

[0155] As used herein, “stringent conditions” encompass conditions under which hybridization will only occur if there is less than 20% mismatch between the hybridization molecule and a sequence within the target nucleic acid molecule. “Stringent conditions” include further particular levels of stringency. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 20% sequence mismatch will not hybridize; conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize; and conditions of “very high stringency” are those under which sequences with more than 5% mismatch will not hybridize. The following are representative, non-limiting hybridization conditions.

[0156] High Stringency condition (detects sequences that share at least 90% sequence identity): Hybridization in 5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 16 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 20 minutes each.

[0157] Moderate Stringency condition (detects sequences that share at least 80% sequence identity): Hybridization in 5x-6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65-70 °C for 16-20 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 5-20 minutes each; and wash twice in lx SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 55-70 °C for 30 minutes each.

[0158] Non-stringent control condition (sequences that share at least 50% sequence identity will hybridize): Hybridization in 6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature to 55 °C for 16-20 hours; wash at least twice in 2x-3x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature to 55 °C for 20-30 minutes each.

[0159] As used herein, the term "germplasm" refers to genetic material of or from an individual (e g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell or can be separate from the organism or cell. The germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. Germplasm in the context of the present disclosure includes cells, seed or tissues from which new plants can be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.

[0160] Further provided are methods for identifying TIR domains, NB domains, LRR domains and / or NLR scaffolds for use in the NLR fusion polypeptides described herein. In certain embodiments, the method comprises determining and / or predicting the tertiary structure of one or more test proteins, aligning the structure to the Roql TIR domain, NB domain, LRR domain and / or NLR scaffold, and selecting test proteins as domains or scaffolds for use in the NLR fusion polypeptides described herein when the TM-score is at least 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1 as compared to the structure of the Roql TIR domain, NB domain, LRR domain and / or NLR scaffold. The method of predicting or determining the structure of the test protein can be any method known in the art. In certain embodiments, the test protein is determined or predicted using X-ray crystallography, NMR spectroscopy, and electron microscopy or artificial-intelligence systems such as AlphaFold2, trRosetta, and ESMFold. The structural alignment may be conducted using any method known in the art or described herein. In certain embodiments, structural alignment is determined using Foldseek or TM-align.

[0161] The following are examples of specific embodiments of some aspects of the invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way.EXAMPLE 1

[0162] This example demonstrates the generation of engineered NLR fusion polypeptides using TIR domain containing NLR polypeptides as a scaffold.

[0163] Roql is a TIR-NLR from N. benthamiana that provides disease resistance against Xanthomonas species by recognizing an effector secreted by Xanthomonas called XopQ (Schultink, Qi et al. 2017). Recognition of XopQ by Roql occurs when XopQ binds directly to the LRR domain and a post-LRR (PL) domain of Roql (Martin, Qi et al. 2020).

[0164] To generate the engineered NLR fusion polypeptide, the soybean substrate proteins of P. pachyrhizi effectors were incorporated into the LRR domain of Roql. The P. pachyrhizi effector substrates integrated into the LRR domain of Roql were referred to as pathogen effector interacting domains, also referred to herein as integrated domains (IDs). Briefly, for the construction of the fusion polypeptide AlphaFold was used to model the structure of Roql to ensure that incorporation of the IDs does not disturb important NLR polypeptide intramolecular interactions. The pathogen effector interacting domains were inserted at two locations along the LRR domain of Roq l to create two scaffolds for pathogen effector interacting domain incorporation. The Roql(APL:ID) scaffold (SEQ ID NO: 1) has had the PL domain removed and replaced with the ID while the Roql(700:ID) scaffold (SEQ ID NO: 2) has most of the LRR domain removed and the pathogen effector interacting domains added after amino acid 700 of Roql. The Roql(700:ID) scaffold was created by using AlphaFold2 to identify the last LRR repeat that is predicted to associate with the NB-ARC domain and the ID was incorporated after this LRR repeat to reduce the chances of creating a Roql variant that induces an immune response in the absence of an effector.

[0165] The structure of the Roql(APLTD) (Fig. 1 A) and Roql(700:ID) (Fig. IB) scaffold was predicted using AlphaFold 2 (Jumper, Evans et al. 2021). The TIR domain is the portion of the structure depicted in black, the NB-ARC domain is dark grey, and the LRR domain is light grey.EXAMPLE 2

[0166] This example demonstrates that the engineered NLR fusion polypeptides recognize their cognate effector.

[0167] To determine if the engineered NLR fusion polypeptides Roql(APL:ID) (SEQ ID NO: 1) and Roql(700:ID) (SEQ ID NO: 2) from Example 1 could recognize their cognate effector, therespective engineered NLR fusion polypeptides were co-expressed with their cognate effector in N. benthamiana leaves. Briefly, pathogen effector interacting domains (IDs) were identified in a yeast-two hybrid screen and fused to the Roql NLR scaffolds Roql(APL:ID) and Roql(700:ID). The pathogen effector interacting domains were numbered such that Roql(APL:55) indicates that the NLR scaffold is the Roql(APL) scaffold and the pathogen effector interacting domain is ID 55 (SEQ ID NO: 9). Agrobacterium-mediated transformation was used to co-express the engineered NLR fusion polypeptide and cognate effect pairs in N. benthamiana leaves.Successful recognition of an effector by an NLR typically results in localized visual tissue death that indicates an immune response has been activated. After expression was confirmed, the leaves were analyzed for visual signs of cell death that indicate an immune response has been activated, and the leaves were scored for the presence or absence of tissue death across multiple experiments. XopQ and AvrPikD are effectors recognized by the TIR-NLR Roql and NLR Pikml, respectively, in N. benthamiana and served as a positive control.

[0168] As shown in Table 1, the Roql(APLTD) variant containing ID 55 (SEQ ID NO: 11) induced reproducible cell death in N. benthamiana leaves, whereas four Roql(700:ID) variants containing ID 28a (SEQ ID NO: 24), 29b (SEQ ID NO: 27), 44 (SEQ ID NO: 30), or 56 (SEQ ID NO: 8) induced cell death in N. benthamiana leaves, indicating that the engineered NLR fusion polypeptides recognized the cognate effector. The same IDs were fused to the non-TIR domain containing NLR polypeptide Pikm-1, expressed in N. benthamiana and analyzed for visual signs of cell death. However, as shown in Table 1, none of the IDs that functioned in the Roql(APLTD) or Roql(700:ID) functioned in Pikm-1. Additionally, 20 out of the 33 tested variants were autoactive as compared to only 5 of the 29 Roql(APLTD) variants tested and 5 of the 22 Roql(700:ID) variants tested.Table 1 : Screening in N. benthamiana for Engineered NLR Fusion Polypeptide Activity

[0169] Taken together, these results indicate that the engineered fusion polypeptides using a TIR domain containing scaffold can recognize their cognate effector, and also highlights the limitations of using non-TIR domain containing NLR polypeptides such as Pikm-1 as a scaffold for IDs.EXAMPLE 3

[0170] This example demonstrates that the engineered NLR fusion polypeptides can induce an immune response.

[0171] To determine if the cell death phenotype observed in N. benthamiana leaves was associated with an immune response, the expression of marker genes for immune activation, PRla and HSR201, were assessed in N. benthamiana leaves co-expressing Roql (APL:55) and PpnA3_5_378.

[0172] As shown in Table 2, expression of both marker genes is higher in N. benthamiana leaves co-expressing PpnA3_5_378 and Roql(APL:55) than either gene alone. XopQ is an effector recognized by the TIR-NLR Roq 1 in N. benthamiana and serves as a positive control for HR cell death induction and activation of plant immunity.Table 2: Expression of Marker Genes for Immune Activation

[0173] These results demonstrate that engineered NLR fusion polypeptides can induce immune activation upon expression of the cognate effector.EXAMPLE 4

[0174] This example demonstrates that an engineered NLR fusion polypeptide induces cell death in legume protoplasts upon recognition of a P. pachyrhizi effector.

[0175] To determine if Roql(APL:55) recognizes the P. pachyrhizi effector PpnA3 5 378 (SEQ ID NO: 10), Roql(APL:55) and PpnA3_5_378 were separately cloned into a protoplast expression vector. The vectors were then transfected into legume protoplasts with a luciferase reporter plasmid. Cells dying from HR exhibit reduced luciferase activity resulting in reduced luminescence. As shown in Table 3, co-expression of PpnA3_5_378 and Roql(APL:55) in protoplasts resulted in lower luminescence than expression of either gene alone. These results indicate that Roql(APL:55) recognizes PpnA3_5_378 and activates an immune response in legume protoplasts.Table 3: Immune Response Activation of NLR Fusion Polypeptides in Protoplast Activity Assay

[0176] These results demonstrate that engineered NLR fusion polypeptide induce cell death in legume protoplasts.EXAMPLE 5

[0177] This example demonstrates the expression of an NLR fusion polypeptide in soybean.

[0178] The engineered NLR fusion polypeptides are expressed in legumes, such as soybean, by generating transgenic plants in which a ubiquitous promoter such as GmUbi or ZmH2B are used to drive expression. Alternatively, or in addition, SDN3 gene editing is used to incorporate IDs into the natural genomic location of the TIR-NLR scaffold, such that expression will be driven by the native promoter of the TIR-NLR scaffold. Soybean plants expressing engineered NLR fusion polypeptides having an ASR specific effector interacting domain demonstrate resistance or demonstrate increased resistance against ASR.EXAMPLE 6

[0179] This example demonstrates the generation of engineered NLR fusion polypeptides using TIR domain containing NLR polypeptides from soybean.

[0180] The predicted structures of three full-length TIR-NLR proteins from soybean and the truncated versions of these proteins were used as NLR scaffolds for engineered NLR fusion polypeptides. The structures were predicted using AlphaFold 2. GmTNL_3(719:ID) (SEQ ID NO: 382) was created by truncating GmTNL_3 (SEQ ID NO: 62) after amino acid residue 719 (Figs. 2A-2B). GmTNL_5(700:ID) (SEQ ID NO: 383) was created by truncating GmTNL_5 (SEQ ID NO: 84) after amino acid residue 700 (Figs. 3A-3B). GmTNL_9(718:ID) (SEQ ID NO: 386) was created by truncating GmTNL_9 (SEQ ID NO: 121) after amino acid residue 718 (Figs. 4A-4B). The generated NLR fusion polypeptides were tested in N. benthamiana to determine if they can trigger cell death when they are co-expressed with their cognate effector PpnA3_5_303 (SEQ ID NO: 7).

[0181] As shown in Table 4, GmTNL_3(719:ID), GmTNL_5(700:ID), and GmTNL_9(718:ID) linked to an effector interacting domain trigger cell death when co-expressed with their cognate effector.Table 4: Screening in N. benthamiana for Engineered NLR Fusion Polypeptide Activity

[0182] These results indicate that engineered NLR fusion polypeptides comprising a soybean NLR scaffold and a soybean effector interacting domain could be used as cisgenic disease resistance genes against a pathogen (e.g., ASR) since both the NLR scaffold and effector interacting domain are soybean genes.EXAMPLE 7

[0183] This example demonstrates the generation of engineered NLR fusion polypeptides having stacked IDs.

[0184] Stacking effector interacting domains (e.g., fusion of multiple effector interacting domains to a single NLR scaffold) that recognize multiple and unique effectors (e.g., multiple modes of action) is expected to provide more durable resistance since the pathogen (e.g., ASR) will have to evolve to overcome multiple resistance genes instead of a single resistance gene.

[0185] The engineered NLR fusion polypeptides can be stacked as separate genes in soybean to provide more durable resistance against a pathogen, such as ASR. In addition to stacking individual genes, multiple IDs are integrated (e.g., fuses) onto the same NLR scaffold. This will allow a single gene to provide multiple modes of resistance by incorporating multiple effector interacting domains (IDs) allowing the engineered NLR fusion polypeptide to recognize multiple effectors, for example multiple ASR effectors. The engineered NLR fusion polypeptide comprising multiple effector interaction domains are tested in N. benthamicma for recognition of their cognate effectors. Based upon the results in N. benthamiana the soybean plants expressing the engineered NLR fusion polypeptide comprising multiple effector interaction domains are expected to demonstrate resistance against a pathogen, such as ASR.EXAMPLE 8

[0186] This example demonstrates the generation of engineered NLR fusion polypeptides having broad pathogen effector recognition.

[0187] The identification of pathogen effector interacting domains that recognize effectors secreted from multiple pathogens would provide engineered NLR fusion polypeptides with multiple modes of action. To identify these pathogen effector interacting domains AlphaFold and / or homology-based searches can be used to identify effectors conserved among multiple pathogen species. Pathogen effector interacting domains that target these effectors can be identified using binding assays such as yeast-two hybrid. The pathogen effector interacting domains from these screens can be fused to any of our NLR scaffolds to produce engineered NLR fusion polypeptides that recognize multiple pathogens.

[0188] Additionally, these pathogen effector interacting domains can be stacked, as described in Example 7. NLR scaffolds fused to two or more pathogen effector interacting domainstargeting different effectors conserved among multiple pathogens would allow for the development of a single engineered NLR fusion polypeptide that convers multiple MO As against multiple pathogen species.

[0189] All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.

[0190] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

[0191] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0192] Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

Claims

We claim:

1. A nucleotide-binding leucine-rich repeat (NLR) fusion polypeptide comprising an NLR scaffold and a heterologous pathogen effector interacting domain, the NLR scaffold comprising a TOLL / interleukin-1 receptor (TIR) domain, a nucleotide binding site (NB) domain, and a leucine rich repeat (LRR) domain, or functional fragments thereof comprising at least one leucine-rich repeat.

2. The NLR fusion polypeptide of claim 1, wherein the TIR domain comprises an amino acid motif selected from the group consisting of a PA motif comprising SEQ ID NO: 31, an aA motif comprising the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), X5 and Xe are isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a pD-aDl motif comprising SEQ ID NO: 33, a aD3 motif comprising SEQ ID NO: 34 or any combination thereof.

3. The NLR fusion polypeptide of claim 1 or 2, wherein the TIR domain comprises a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 40 and 183-196.

4. The NLR fusion polypeptide of any one of claims 1-3, wherein the NB domain comprises an amino acid motif selected from the group consisting of a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQ ID NO: 37, an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N), or any combination thereof.

5. The NLR fusion polypeptide of any one of claims 1-4, wherein the NB domain comprises a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 41 and 169-182.

6. The NLR fusion polypeptide of any one of claims 1-5, wherein the LRR domain comprises a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 42 or 155-168 or a functional fragment thereof comprising at least one leucine-rich repeat.

7. The NLR fusion polypeptide of any one of claims 1-6, wherein the LRR domain functional fragment comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2, 376, 378, or 380.

8. The NLR fusion polypeptide of any one of claims 1-7, wherein the LRR domain or LRR domain functional fragment of the NLR scaffold comprises an AlphaFold2 predicted structure having a TM-score of at least 0.50 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 42 or 155-168.

9. The NLR fusion polypeptide of any one of claims 1-8, wherein the TIR domain, NB domain, and LRR domain of the NLR scaffold are arranged, from N-terminus to C-terminus, in the order TIR domain, NB domain, and LRR domain.

10. The NLR fusion polypeptide of any one of claims 1-9, wherein the NLR scaffold comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 2 or 391-400.

11. The NLR fusion polypeptide of any one of claims 1-10, wherein the NLR scaffold comprises an AlphaFold2 predicted structure having a TM-score of at least 0.50 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 1, 2 or 391-400.

12. The NLR fusion polypeptide of any one of claims 1-11, wherein the pathogen effector interacting domain is fused to the C-terminus of the NLR scaffold.

13. The NLR fusion polypeptide of any one of claims 1-12, wherein the pathogen effector interacting domain binds to two or more effectors.

14. The NLR fusion polypeptide of claim 13, wherein the two or more effectors are secreted from different pathogens.

15. The NLR fusion polypeptide of any one of claims 1-14, wherein the pathogen effector interacting domain binds to an Asian soybean rust (ASR) effector polypeptide.

16. The NLR fusion polypeptide of claim 15, wherein the ASR effector polypeptide comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4, 7, 10, 13, 16, 20, 23, 26, 29, 436, 444, 450, 458, 466, 472, 480, 490, 537, 539, 541, 543, 545, or 547.

17. The NLR fusion polypeptide of any one of claims 1-16, wherein the effector interacting domain comprises a polypeptide sequence having at least 90% identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 19, 22, 25, 28, 435, 443, 449, 457, 465, 471, 479, 489, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, or 583 or a functional fragment thereof.

18. The NLR fusion polypeptide of any one of claims 1-17, wherein the NLR fusion polypeptide further comprises at least one additional pathogen effector interacting domain.

19. The NLR fusion polypeptide of claim 18, wherein the at least one additional pathogen effector interacting domain is fused to the C-terminus of the heterologous pathogen effector interacting domain.

20. The NLR fusion polypeptide of any one of claims 18-19, wherein the at least one additional pathogen effector interacting domain and the heterologous pathogen effector interacting domain recognize effectors secreted from different pathogens.

21. A polynucleotide encoding the NLR fusion polypeptide of any one of claims 1-20.

22. The polynucleotide of claim 21, wherein the polynucleotide comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 313, 316, 319,322, 325, 326, 329, 332, 335, 338, 401, 403, 405, 434, 438, 442, 448, 452, 456, 460, 464,470, 474, 478, 482, 484, 488, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514,516, 518, 520, 522, 524, 526, 528, 530, 532, 534.

23. A nucleic acid construct comprising the polynucleotide of claim 21 or 22 operably linked to a regulatory element.

24. The nucleic acid construct of claim 23, wherein the regulatory element is a promoter.

25. A host cell comprising the polynucleotide of claim 21 or 22 or the nucleic acid construct of claim 23 or 24.

26. The host cell of claim 25, wherein the host cell is a plant cell.

27. A plant or plant part comprising the polynucleotide of claim 21 or 22 or the nucleic acid construct of claim 23 or 24.

28. The plant or plant part of claim 27, wherein the plant is a legume crop plant.

29. The plant or plant part of claim 28, wherein the legume crop plant is selected from the group consisting of alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

30. The plant or plant part of claim 29, wherein the legume crop plant is soybean.

31. A plant cell comprising a polynucleotide encoding a nucleotide-binding leucine-rich repeat (NLR) fusion polypeptide comprising an NLR scaffold and a heterologous pathogen effector interacting domain, the NLR scaffold comprising a TOLL / interleukin-1 receptor (TIR) domain, a nucleotide binding site (NB) domain, and a leucine rich repeat (LRR) domain, or functional fragments thereof comprising at least one leucine-rich repeat.

32. The plant cell of claim 31, wherein the polynucleotide is operably linked to a regulatory element.

33. The plant cell of claim 32, wherein the regulatory element is a promoter.

34. The plant cell of any one of claims 31-33, wherein the TIR domain comprises an amino acid motif selected from the group consisting of a A motif comprising SEQ ID NO: 31, an aA motif comprising the amino acid sequence FX1X2HJ wherein Xi is leucine (L), isoleucine (I), valine (V) or threonine (T) and X2 is serine (S) or glycine (G), a PC motif comprising the amino acid sequence SX1X2X3X4X5X6X7 wherein Xi and X3 are any amino acid, X2 and X4 are leucine (L), isoleucine (I), or valine (V), X5 and Xe are isoleucine (I) or valine (V), and X?is phenylalanine (F) or leucine (L), an aC motif comprising SEQ ID NO: 32, a D-aDl motif comprising SEQ ID NO: 33, a aD3 motif comprising SEQ ID NO: 34 or any combination thereof.

35. The plant cell of any one of claims 31-34, wherein the TIR domain comprises a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 40 and 183-196.

36. The plant cell of any one of claims 31-35, wherein the NB domain comprises an amino acid motif selected from the group consisting of a P-loop motif comprising SEQ ID NO: 35, a Walker B motif comprising SEQ ID NO: 36, an RNBS-B motif comprising SEQ ID NO: 38, an RNBS-D motif comprising SEQ ID NO: 39, a GLPL motif comprising SEQ ID NO: 37, an MHD motif comprising the amino acid sequence MHX wherein X is aspartic acid (D) or asparagine (N), or any combination thereof.

37. The plant cell of any one of claims 31-36, wherein the NB domain comprises a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 41 and 169-182.

38. The plant cell of any one of claims 31-37, wherein the LRR domain comprises a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 42 and 155-168 or a functional fragment thereof comprising at least one leucine-rich repeat.

39. The plant cell of any one of claims 31-38, wherein the LRR domain of the NLR scaffold comprises an AlphaFold2 predicted structure having a TM-score of at least 0.50 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 42 or 155-168.

40. The plant cell of any one of claims 31-39, wherein the LRR domain functional fragment comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 407-417.

41. The plant cell of any one of claims 31-40, wherein the NLR scaffold comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 2 or 391-400.

42. The plant cell of any one of claims 31-41, wherein the NLR scaffold comprises an AlphaFold2 predicted structure having a TM-score of at least 0.50 as compared to the AlphaFold2 predicted structure of any one of SEQ ID NOs: 1, 2 or 391-400.

43. The plant cell of any one of claims 31-42, wherein the pathogen effector interacting domain is fused to the C-terminus of the NLR scaffold.

44. The plant cell of any one of claims 31-43, wherein the pathogen effector interacting domain binds to two or more effectors.

45. The plant cell of claim 44, wherein the two or more effectors are secreted from different pathogens.

46. The plant cell of any one of claims 31-45, wherein the pathogen effector interacting domain binds to an Asian soybean rust (ASR) effector polypeptide.

47. The plant cell of any one of claims 31-46, wherein the ASR effector polypeptide comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs 4, 7, 10, 13, 16, 20, 23, 26, 29, 436, 444, 450, 458, 466, 472, 480, 490, 537, 539, 541, 543, 545, or 547.

48. The plant cell of any one of claims 31-47, wherein the effector interacting domain comprises a polypeptide sequence having at least 90% identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 19, 22, 25, 28, 435, 443, 449, 457, 465, 471, 479, 489, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, or 583 or a functional fragment thereof.

49. The plant cell of any one of claims 31-48, wherein the NLR fusion polypeptide further comprises at least one additional pathogen effector interacting domain.

50. The plant cell of claim 49, wherein the at least one additional pathogen effector interacting domain is fused to the C-terminus of the heterologous pathogen effector interacting domain.

51. The plant cell of any one of claims 49-50, wherein the at least one additional pathogen effector interacting domain and the heterologous pathogen effector interacting domain recognize effectors secreted from different pathogens.

52. The plant cell of any one of claims 31-51, wherein the NLR fusion polypeptide does not comprise a post-LRR (PL) domain.

53. A plant or plant part comprising the plant cell of any one of claims 31-52.

54. The plant of claim 53, wherein the plant is a legume crop plant.

55. The plant of claim 54, wherein the legume crop plant is selected from the group consisting of alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

56. The plant of claim 55, wherein the legume crop plant is soybean.

57. A seed comprising the plant cell of any one of claims 36-52.

58. A method for increasing resistance to a pathogen in a plant, the method comprising:(a) expressing in a plant cell a polynucleotide encoding the NLR fusion polypeptide of any one of claims 1-20; and(b) generating a plant from the plant cell wherein the plant expresses the NLR fusion polypeptide and has increased resistance to a pathogen as compared to a control plant.

59. The method of claim 58, wherein the method comprises:(a) introducing into a regenerable plant cell the polynucleotide encoding the NLR fusion polypeptide operably linked to at least one regulatory element; and(b) generating a plant from the plant cell wherein the plant comprises the recombinant polynucleotide and expresses the NLR fusion polypeptide.

60. The method of claim 59, wherein the at least one regulatory element comprises a promoter sequence.

61. The method of claim 59 or 60, wherein the polynucleotide encoding the NLR fusion polypeptide operably linked to at least one regulatory element is introduced into the regenerable plant cell by genome editing.

62. The method of claim 59 or 60, wherein the polynucleotide encoding the NLR fusion polypeptide operably linked to at least one regulatory element is introduced into the regenerable plant cell by backcrossing or transformation.

63. The method of claim 58, wherein the method comprises:(a) introducing in a regenerable plant cell a genetic modification at an endogenous TIR domain containing NLR (TIR-NLR) gene to generate a modified TIR-NLR gene encoding the NLR fusion polypeptide; and(b) generating a plant from the plant cell expressing the NLR fusion polypeptide.

64. The method of claim 63, wherein the targeted genetic modification replaces an endogenous post-LRR (PL) domain with the effector interacting domain.

65. The method of any one of claims 58-64, wherein the pathogen is a bacteria or virus.

66. The method of claim 65, wherein the pathogen is Phakopsora pachyrhizi or Phakopsora meibomiae, or a combination thereof.

67. The method of any one of claims 58-66, wherein the effector interacting domain binds to an Asian soybean rust (ASR) effector polypeptide and the generated plant has increased resistance to ASR.

68. The method of any one of claims 58-67, wherein the plant is a legume crop plant.

69. The method of claim 68, wherein the legume crop plant is selected from the group consisting of alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

70. The method of claim 69, wherein the legume crop plant is soybean.

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