Methods of improving the thermostability of plant immune receptors

Abstract

Methods are provided of improving the thermostability of plant immune receptors, particularly nucleotide-binding leucine-rich repeat proteins. The methods comprise making one or more amino substitution at one or more predetermined positions in an α-helix in the nucleotide-binding domain or the β-sheet in the leucine-rich repeat (LRR) domain of the NLR protein to produce a thermostable NLR protein. Methods are further provided for making plants comprising the thermostable NLR proteins. Additionally provided are the thermostable NLR proteins as well as nucleic acid molecules encoding the thermostable NLR proteins, and plants and plant cells comprising the thermostable NLR proteins.

Description

METHODS OF IMPROVING THE THERMOSTABILITY OF PLANT IMMUNE RECEPTORS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 513,618 filed July 14, 2023, which is hereby incorporated herein in its entirety by reference. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (070294-0229SEQLST.xml; Size: 56,306 bytes; and Date of Creation: July 11, 2024) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION

[0003] The present invention relates to protein engineering, particularly to engineering plant immune receptors for improved thermostability. BACKGROUND OF THE INVENTION

[0004] World population growth is driving up the demand for food. To feed the estimated 10 billion people by 2050, food production will need to increase by 60% (www.fao.org). However, climate change imposes a severe threat to agricultural systems, food safety and security, making the goal of a 60% increase in food production by 2050 even more challenging. Because global average temperatures are expected to continue to rise, agricultural scientists need to find new solutions to improve agricultural crop production at elevated temperatures.

[0005] Elevated temperatures are known to disturb immune responses of plants to pathogen attacks by inactivating plant immune receptors and / or blocking systemic acquired resistance (Kim et al., 2022, Nature 607:339–344, doi.org / 10.1038 / s41586-022-04902-y). However, the underlying molecular mechanisms remain largely unknown, leaving a 105735300.1 - 1 - 070294.0229significant gap in our understanding of how frequent and extreme heat waves influence the effectiveness of the plant immune system (Hua and Dong, 2022, Cell Res.32:1038–1039, doi.org / 10.1038 / s41422-022-00710-1).

[0006] Plant temperature-sensing was already observed as a complete loss of resistance to tobacco mosaic virus (TMV) when the temperature was raised from the permissive temperature 22°C to restrictive 30°C (Whitham et al., 1994, Cell 78(6):1101-15, doi: 10.1016 / 0092-8674(94)90283-6, erratum in: Cell 81(3):466). TMV resistance is conferred by N gene encoding a resistance (R) immune receptor, N, that belongs to the largest family of R proteins. The family comprises nucleotide binding-leucine rich repeat (NLR) proteins, with either a coiled-coil (CC) or a toll-interleukin-1 receptor (TIR) domain at the N terminus, followed by a central nucleotide-binding (NB) domain and a C-terminal leucine-rich repeat (LRR) domain (van Ooijrn et al, 2008, J. Exp. Biol.59(6): 1383–1397, doi.org / 10.1093 / jxb / ern04). The NB domain of plant NLR proteins belongs to a class of NB domains known as NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domains (Pan et al., 2022, Front. Genet., vol.13, article 887217, doi.org / 10.3389 / fgene.2022.887217).

[0007] Plant NLR receptors initiate immunity by perceiving specific pathogens compounds – avirulence factors (Avrs) (Ngou et al., 2022, Plant Cell 34:1447–1478, doi.org / 10.1093 / plcell / koac041). Many other NLR receptors, like Mi-1, SNC-1, RPS-4, Ny- 1, Sr35, Rpi-amr1, and Roq1, show the same temperature- sensitive phenotype as N (Cohen and Leach, 2020, Curr. Opin. Plant Biol.56:235–241, doi.org / 10.1016 / j.pbi.2020.02.008). On the other hand, at least two of the R receptors whose encoding sequences were cloned, namely Rx and Ry, are temperature-insensitive and can confer resistance over 30°C (Richard et al., 2020, Front. Genet., vol.11, article 417, doi.org / 10.3389 / fgene.2020.00417; Grech- Baran et al., 2020, Plant Biotechnol J.18:655–667, doi.org / 10.1111 / pbi.13230). Historically, NLRs were mostly studied as sensors of pathogen Avrs (“sensor” NLRs). Recent studies, however, showed that some also work downstream of pathogen perception by helping to activate the immune response (“helper” NLRs; Wu et al., 2017, New Phytol.222:938–953, doi.org / 10.1111 / nph.15665).

[0008] Understanding why some NLR receptors are temperature sensitive, and others are not temperature sensitive may lead to new strategies for designing or engineering NLR receptors with improved thermostability. 105735300.1 - 2 - 070294.0229SUMMARY OF THE INVENTION

[0009] The present invention provides methods for improving the thermostability of plant NLR proteins that are capable of conferring to a plant resistance to a plant disease. Such methods comprise PDNLQJ^DW^OHDVW^RQH^DPLQR^VXEVWLWXWLRQ^DW^D^SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į- helix in the NB-ARC domain or the ȕ-sheet in the LRR domain of the NLR protein so as to produce a thermostable NLR protein by generating a interdomain interaction between a first DPLQR^DFLG^LQ^WKH^Į-KHOL[^DQG^D^VHFRQG^DPLQR^DFLG^LQ^WKH^ȕ-sheet. Interdomain interactions include, for example, electrostatic interactions (i.e., anionic-cationic interactions, hydrophobic interactions, and hydrogen bonding. The amino substitution comprises replacing an amino acid in the NLR protein at the amino acid position of the first amino acid or the second amino acid with a different amino acid. In some embodiments of the invention, two, three, four, five, or more amino acid substitutions are made at two, three, four, five, or more predetermined positions LQ^DQ^Į-helix in the NB-ARC domain and / or the ȕ-sheet in the LRR domain of the NLR protein to generate a thermostable NLR protein with two, three, four, five, or more interdomain interactions.

[0010] The present invention further provides methods for improving the thermostability of an NLR protein in a plant. Such methods modifying the nucleotide sequence of an NLR gene encoding the NLR protein in at least one plant cell to produce at least one modified plant cell comprising the modified nucleotide sequence, whereby the modified nucleotide sequence encodes a thermostable NLR protein with at least one amino substitution at a SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or the ȕ-sheet in the LRR domain, wherein the least one amino acid substitution generates a interdomain interaction EHWZHHQ^D^ILUVW^DPLQR^DFLG^LQ^WKH^Į-KHOL[^DQG^D^VHFRQG^DPLQR^DFLG^LQ^WKH^ȕ-sheet.

[0011] Further provided are thermostable NLR proteins, nucleic acid molecules encoding the thermostable NLR proteins, and plants, plant cells, and other host cells comprising the thermostable NLR proteins and / or the nucleic acid molecules encoding the thermostable NLR proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG.1 is a structural model of a representative NLR immune protein. Regions involved in protein stabilization are marked in orange (alpha-helix) and blue (beta-sheets). 105735300.1 - 3 - 070294.0229

[0013] FIG.2 is a zoom-in on the region of the temperature-stability of a representative NLR protein. The alpha-helix (orange) and beta-sheet (blue) are stabilised with three interdomain electrostatic interactions (bright green arrows).

[0014] FIGS.3A-3B show the regions of the alpha-helix and beta-sheet in SNC 1 (FIG. 3A) and N (FIG.3B). The interdomain electrostatic interactions between alpha-helix (orange) and beta-sheet (blue) stabilising the structure are marked with sticks and arrows.

[0015] FIG.4 shows the region of the alpha-helix and beta-sheet in Rystoprotein. The interdomain electrostatic interactions between alpha-helix (orange) and beta-sheet (blue) stabilising the structure are marked with sticks and arrows.

[0016] FIGS.5A-5C show structural changes in the NB-ARC region of the Rystoprotein remove its ability to initiate HR at elevated temperatures. Cell death phenotypes (FIG.5A) and scoring box plots (FIG.5B) of Rystovariants challenged by PVY CP under permissive and elevated temperatures. (FIG.5C) Accumulation of Rystoprotein variants in Nicotiana benthamiana under tested temperatures. The cell death was quantified and documented at 5 dpi; protein extraction, antibody detection and Ponceau S staining were performed at 3 dpi.

[0017] FIG.6 shows that K624E change in the Rystoprotein remove its ability to respond to PVY in planta at elevated temperatures. Cell death response of Rysto_WT and Rysto_K624E variant to PVY under permissive (FIG.6A) and elevated temperatures (FIG.6B). The photographs were taken at 3 dpi.

[0018] FIG.7 shows the region of the alpha-helix and beta-sheet in Roq1 protein. The interdomain electrostatic interactions between alpha-helix (orange) and beta-sheet (blue) stabilizing the structure are marked with sticks and arrows.

[0019] FIGS.8A-8C show the structural changes in the NB-ARC region of the Roq1 protein enable it to initiate HR at high temperatures. Cell death phenotypes (FIG.8A) and scoring box plots (FIG.8B) of Roq1 variants challenged by XopQ under permissive and elevated temperatures. (FIG.8C) Accumulation of Roq1 protein variants in roq1 N. benthamiana under tested temperatures. The cell death was quantified and documented at 5 dpi; protein extraction, antibody detection and Ponceau S staining were performed at 3 dp.

[0020] FIGS 9A-9B. Cell death phenotypes (FIG.9A) and scoring plots (FIG.9B) of NRG1 variants co-delivered with Rysto(Ry) and PVY CP (CP) into N. benthamiana / nrg1 mutant under permissive (22°C) and elevated (30°C) temperatures. The cell death was quantified and documented at 5 dpi. Each dot represents biological replicate, n=10. 105735300.1 - 4 - 070294.0229BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0021] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and one-letter code or three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5' end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.

[0022] SEQ ID NO: 1 is the cDNA sequence of wild-type Rystofrom Solanum stoloniferum. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising this nucleotide sequence. The native stop codon of this cDNA is TGA.

[0023] SEQ ID NO: 2 is the amino acid sequence of wild-type Rysto from Solanum stoloniferum.

[0024] SEQ ID NO: 3 is a synthetic or artificial nucleotide sequence encoding an engineered, temperature-sensitive variant of Rystocomprising the amino acid sequence set forth in SEQ ID NO: 4. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising this nucleotide sequence.

[0025] SEQ ID NO: 4 is the amino acid sequence of an engineered, temperature-sensitive variant of Rystocomprising one amino acid substitution (K624E) relative to the amino acid sequence of wild-type Rystoset forth in SEQ ID NO: 2.

[0026] SEQ ID NO: 5 is the cDNA sequence of wild-type Roq1 from Nicotiana benthamiana. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising this nucleotide sequence. The native stop codon of this cDNA is TAG.

[0027] SEQ ID NO: 6 is the amino acid sequence of wild-type Roq1 from Nicotiana benthamiana.

[0028] SEQ ID NO: 7 is a synthetic or artificial nucleotide sequence encoding an engineered, thermostable variant of Roq1 comprising the amino acid sequence set forth in SEQ ID NO: 8. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising this nucleotide sequence. 105735300.1 - 5 - 070294.0229

[0029] SEQ ID NO: 8 is the amino acid sequence of an engineered, thermostable variant of Roq1 comprising one amino acid substitution (R451P) relative to the amino acid sequence of wild-type Roq1 set forth in SEQ ID NO: 6.

[0030] SEQ ID NO: 9 is a synthetic or artificial nucleotide sequence encoding an engineered, thermostable variant of Roq1 comprising the amino acid sequence set forth in SEQ ID NO: 10. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising this nucleotide sequence.

[0031] SEQ ID NO: 10 is the amino acid sequence of an engineered, thermostable variant of Roq1 comprising two amino acid substitutions (A455L, A459L) relative to the amino acid sequence of wild-type Roq1 set forth in SEQ ID NO: 6.

[0032] SEQ ID NO: 11 is a synthetic or artificial nucleotide sequence encoding an engineered, thermostable variant of Roq1 comprising the amino acid sequence set forth in SEQ ID NO: 12. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising this nucleotide sequence.

[0033] SEQ ID NO: 12 is the amino acid sequence of an engineered, thermostable variant of Roq1 comprising three amino acid substitutions (R451P, A455L, A459L) relative to the amino acid sequence of wild-type Roq1 set forth in SEQ ID NO: 6.

[0034] SEQ ID NO: 13 is the cDNA sequence of wild-type NRG1 from Nicotiana benthamiana. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising this nucleotide sequence. The native stop codon of this cDNA is TAA.

[0035] SEQ ID NO: 14 is the amino acid sequence of wild-type NRG1 from Nicotiana benthamiana.

[0036] SEQ ID NO: 15 is a synthetic or artificial nucleotide sequence encoding an engineered, thermostable variant of NRG1 comprising the amino acid sequence set forth in SEQ ID NO: 16. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising this nucleotide sequence.

[0037] SEQ ID NO: 16 is the amino acid sequence of an engineered, thermostable variant of NRG1 comprising one amino acid substitution (R638Q) relative to the amino acid sequence of wild-type NRG1 set forth in SEQ ID NO: 14.

[0038] SEQ ID NO: 17 is a synthetic or artificial nucleotide sequence encoding an engineered, thermostable variant of NRG1 comprising the amino acid sequence set forth in SEQ ID NO: 18. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising this nucleotide sequence. 105735300.1 - 6 - 070294.0229

[0039] SEQ ID NO: 18 is the amino acid sequence of an engineered, thermostable variant of NRG1 comprising one amino acid substitution (R638N) relative to the amino acid sequence of wild-type NRG1 set forth in SEQ ID NO: 14. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

[0041] 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.

[0042] The present invention is based in part on the discovery by the present inventors that the thermostability of certain plant immune receptors, particularly nucleotide-binding leucine-rich repeat (NLR) proteins, can be improved by making one or more amino acid substitutions at predetermined positions in the amino acid sequences of such NLR proteins. Using protein modeling, the present inventors compared the structures of temperature- sensitive NLR proteins with the structures of thermostable NLR proteins. Such temperature- sensitive NLR proteins of the present invention are capable of conferring to a plant a certain level of resistance to a plant pathogen at a permissive temperature, but the resistance level is at a lower or is lost at an elevated temperature (also referred to herein as a “restrictive temperature”). Thus, such temperature-sensitive NLR proteins display instability of resistance function at an elevated temperature. In contrast, thermostable or temperature- insensitive NLR proteins do not, or are not known to, display instability of resistance function at an elevated temperature.

[0043] As disclosed hereinbelow in the Examples, the present inventors determined that interdomain interactions between a first amino acid in an Į-helix in the NB-ARC domain and 105735300.1 - 7 - 070294.0229a second amino acid in the ȕ-sheet in the LRR domain of the NLR protein are important for stabilizing the resistance function of an NLR protein at elevated temperatures. Such interdomain interactions of present invention relate to the chemical properties of the side chains of the amino acids and include, for example, electrostatic or anionic-cationic interactions, hydrophobic interactions, and hydrogen bonding. As further disclosed hereinbelow, the present inventors have demonstrated that the thermostability of an NLR protein that is known to display functional instability at an elevated temperature can be improved by engineering or designing the NLR protein to increase the number of interdomain interactions between an Į-helix in the NB-ARC domain the ȕ-sheet in the LRR domain. Moreover, the present inventors have demonstrated the importance of such interdomain interactions for the thermostability of NLR proteins by creating a temperature-sensitive NLR protein from a thermostable (i.e temperature-insensitive) NLR protein by decreasing the number of such interdomain interactions.

[0044] In one aspect, the present invention relates to methods for improving the thermostability of plant NLR proteins that are capable of conferring to a plant resistance to a plant disease. Such methods comprise making at least one amino substitution at a SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or the ȕ-sheet in the LRR domain of the NLR protein so as to produce a thermostable NLR protein by generating a interdomain LQWHUDFWLRQ^EHWZHHQ^D^ILUVW^DPLQR^DFLG^LQ^WKH^Į-helix and a second amino acid in WKH^ȕ-sheet. The at least one amino substitution comprises replacing an (original) amino acid in the NLR protein with a substitute amino acid. The predetermined position can be either the position of the first amino acid or the second amino acid. In some embodiments, an amino substitution is made at the position of both the first and second amino acid to generate the desired interdomain interaction.

[0045] Interdomain interactions of the present invention include, for example, electrostatic interactions (i.e., anionic-cationic interactions), hydrophobic interactions, and hydrogen bonding. Amino acids comprise side chains that are charged and capable of electrostatic interactions, particularly anionic and cationic amino acids. Anionic amino acids include, for example, aspartic acid and glutamic acid. Cationic amino acids include, for example, lysine, arginine, and histidine.

[0046] In certain embodiments, the methods comprise making at least one amino VXEVWLWXWLRQ^DW^D^SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or the ȕ-sheet in the LRR domain of the NLR to create or generate an electrostatic interaction between a ILUVW^DPLQR^DFLG^LQ^WKH^Į-KHOL[^DQG^D^VHFRQG^DPLQR^DFLG^LQ^WKH^ȕ-sheet, wherein the substitute 105735300.1 - 8 - 070294.0229amino acid is a charged amino acid and the first amino acid is aspartic acid or glutamic acid and the second amino acid is lysine, arginine, or histidine, or the first amino acid is lysine, arginine, or histidine and the second amino acid is aspartic acid or glutamic acid.

[0047] In other embodiments, the methods comprise making at least one amino VXEVWLWXWLRQ^DW^D^SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or the ȕ-sheet in the LRR domain of the NLR to create or generate a hydrophobic interaction between a first DPLQR^DFLG^LQ^WKH^Į-KHOL[^DQG^D^VHFRQG^DPLQR^DFLG^LQ^WKH^ȕ-sheet, wherein the substitute amino acid is hydrophobic amino acid and both the first and second amino acids are hydrophobic amino acids. Such hydrophobic acids include, for example, leucine, isoleucine, methionine, phenylalanine, proline, tyrosine, tryptophan, and valine. Additionally, the -CH2-, groups of the side chains of lysine and glutamic acid can make hydrophobic contacts which additionally support their cation-anion interaction.

[0048] In still other embodiments of the invention, the methods comprise making at least RQH^DPLQR^VXEVWLWXWLRQ^DW^D^SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or the ȕ-sheet in the LRR domain of the NLR to create or generate a hydrogen bond between a ILUVW^DPLQR^DFLG^LQ^WKH^Į-KHOL[^DQG^D^VHFRQG^DPLQR^DFLG^LQ^WKH^ȕ-sheet, wherein the substitute amino acid is an uncharged, polar amino acid and both the first and second amino acids are uncharged, polar amino acids. Such uncharged, polar amino acids include, for example, serine, threonine, asparagine, glutamine, tyrosine, and cysteine.

[0049] :KHQ^VHOHFWLQJ^DQ^DPLQR^DFLG^IRU^VXEVWLWXWLRQ^RI^WKH^RULJLQDO^DPLQR^DFLG^LQ^WKH^Į- helix in the NB-$5&^GRPDLQ^RU^WKH^ȕ-sheet in the LRR domain, an importation consideration is not to distort the existing local structure. Therefore, it is desirable to not change the size of an amino acid residue. For example, the replacements of aspartic acid with asparagine (and vice versa) and glutamic acid with glutamine (and vice versa) are preferred. Similarly, for short distances, the replacement serine with threonine (and vice versa) is preferred.

[0050] The methods of present invention comprise making at least one amino substitution at a predetermined position. In some embodiments of the invention, two, three, four, five, or more amino acid substitutions are made at two, three, four, five, or more predetermined SRVLWLRQV^LQ^WKH^LQ^DQ^Į-helix in the NB-ARC domain and / or the ȕ-sheet in the LRR domain of the NLR protein to generate a thermostable NLR protein with two, three, four, five, or more interdomain interactions.

[0051] For example, in embodiments of the methods of the present invention comprising two amino acid substitutions, the methods comprising making: (1) a first amino acid substitution at a first predetermined position in an Į-helix in the NB-ARC domain or the ȕ- 105735300.1 - 9 - 070294.0229sheet in the LRR domain, wherein the first amino acid substitution generates a first interdomain interaction EHWZHHQ^D^ILUVW^DPLQR^DFLG^LQ^WKH^Į-helix and a second amino acid in WKH^ȕ-sheet, and wherein the first amino substitution comprises replacing a first amino acid in the NLR protein with a first substitute amino acid; and (2) a second amino acid substitution at a second SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or the ȕ-sheet in the LRR domain, wherein the second amino acid substitution generates a second interdomain interaction between a third DPLQR^DFLG^LQ^WKH^Į-helix and a fourth DPLQR^DFLG^LQ^WKH^ȕ-sheet, and wherein the second amino substitution comprises replacing a second amino acid in the NLR protein with a second substitute amino acid.

[0052] For example, in embodiments of the methods of the present invention comprising three amino acid substitutions, the methods comprising making: (1) a first amino acid substitution at a first predetermined position in an Į-helix in the NB-ARC domain or the ȕ- sheet in the LRR domain, wherein the first amino acid substitution generates a first interdomain interaction EHWZHHQ^D^ILUVW^DPLQR^DFLG^LQ^WKH^Į-helix and a second amino acid in WKH^ȕ-sheet, and wherein the first amino substitution comprises replacing a first amino acid in the NLR protein with a first substitute amino acid; (2) a second amino acid substitution at a second SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or WKH^ȕ-sheet in the LRR domain, wherein the second amino acid substitution generates a second interdomain interaction between a third DPLQR^DFLG^LQ^WKH^Į-helix and a fourth DPLQR^DFLG^LQ^WKH^ȕ-sheet, and wherein the second amino substitution comprises replacing a second amino acid in the NLR protein with a second substitute amino acid; and (3) a third amino acid substitution at a third SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or WKH^ȕ-sheet in the LRR domain, wherein the third amino acid substitution generates a third interdomain interaction between a fifth DPLQR^DFLG^LQ^WKH^Į-helix and a sixth DPLQR^DFLG^LQ^WKH^ȕ-sheet, and wherein the third amino substitution comprises replacing a third amino acid in the NLR protein with a third substitute amino acid.

[0053] For example, in embodiments of the methods of the present invention comprising four amino acid substitutions, the methods comprising making: (1) a first amino acid substitution at a first predetermined position in an Į-helix in the NB-ARC domain or WKH^ȕ- sheet in the LRR domain, wherein the first amino acid substitution generates a first interdomain interaction EHWZHHQ^D^ILUVW^DPLQR^DFLG^LQ^WKH^Į-helix and a second amino acid in WKH^ȕ-sheet, and wherein the first amino substitution comprises replacing a first amino acid in the NLR protein with a first substitute amino acid; (2) a second amino acid substitution at a second SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or WKH^ȕ-sheet in the 105735300.1 - 10 - 070294.0229LRR domain, wherein the second amino acid substitution generates a second interdomain interaction between a third DPLQR^DFLG^LQ^WKH^Į-helix and a fourth DPLQR^DFLG^LQ^WKH^ȕ-sheet, and wherein the second amino substitution comprises replacing a second amino acid in the NLR protein with a second substitute amino acid; (3) a third amino acid substitution at a third SUHGHWHUPLQHG^SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or WKH^ȕ-sheet in the LRR domain, wherein the third amino acid substitution generates a third interdomain interaction between a fifth DPLQR^DFLG^LQ^WKH^Į-helix and a sixth DPLQR^DFLG^LQ^WKH^ȕ-sheet, and wherein the third amino substitution comprises replacing a third amino acid in the NLR protein with a third substitute amino acid; and (4) a fourth amino acid substitution at a fourth predetermined SRVLWLRQ^LQ^DQ^Į-helix in the NB-ARC domain or WKH^ȕ-sheet in the LRR domain, wherein the fourth amino acid substitution generates a fourth interdomain interaction between a fifth DPLQR^DFLG^LQ^WKH^Į-helix and a sixth DPLQR^DFLG^LQ^WKH^ȕ-sheet, and wherein the fourth amino substitution comprises replacing a fourth amino acid in the NLR protein with a fourth substitute amino acid.

[0054] The predetermined positions for making amino acid substitutions can be selected or identified by using a protein modeling program to identify for a pair of amino acids—a first amino acid in an Į-helix in the NB-ARC domain and a second amino acid in WKH^ȕ-sheet in the LRR domain that are located sufficiently close to each and otherwise in favorable orientations for the generation of a interdomain interaction following an amino acid substitution at one or both of the first and second amino acids with one or two substitute amino acids. The methods do not depend on a particular protein modeling program. As disclosed hereinbelow, the present inventors used the Yasara protein structure package (www.yasara.org). Other suitable protein modeling programs for use in the methods of the present invention include, but are not limited to, AlphaFold (www.deepmind.com).

[0055] As disclosed hereinbelow, a thermostable NLR protein typically comprises at least three of such interdomain interactions between a pair of amino acids—a first amino acid in an Į-helix in the NB-ARC domain and a second amino acid in WKH^ȕ-sheet in the LRR domain. Thus, if a particular temperature-sensitive NLR protein comprises 0, 1, or 2 of interdomain interactions, then methods of the present invention can be used to generate at least 3, 2, or 1 additional interdomain interactions, respectively.

[0056] It is recognized that preferred substitute amino acids are those that do not, or are not expected to, alter or disrupt the secondary structure of the NLR protein at or in the vicinity of the predetermined position(s). It is further recognized that one amino acid in a first domain of an NLR protein may interact with amino acids in a second domain. 105735300.1 - 11 - 070294.0229

[0057] Th methods for improving the thermostability of plant NLR proteins can further comprise HOLPLQDWHV^D^GHVWDELOL]LQJ^LQWHUDFWLRQ^EHWZHHQ^WKH^ILUVW^DPLQR^DFLG^LQ^WKH^Į-helix and WKH^VHFRQG^DPLQR^DFLG^LQ^WKH^ȕ-sheet of an NLR protein. Such destabilizing interactions include, but are not limited to, an anionic-anionic interaction or a cationic-cationic interaction. For example, a destabilizing glutamic acid-glutamic acid interaction can be replaced with a neutral or minutely favorable (i.e. stabilizing) glutamine-glutamic acid or glutamic acid-glutamine interaction with minimal effect to the local structure of the Į-helix and / or WKH^ȕ-sheet.

[0058] So, it is possible to cancel unfavorable ones, making them neutral or moderately favorable (e.g. unfavorable E-E could be replaced by neutral or minutely favorable Q-E or E- Q ) with minimal effect to the local structure.

[0059] A thermostable NLR protein produced by the methods of the present invention is capable conferring to a plant comprising the thermostable NLR protein increased resistance resistant to a plant pathogen at an elevated temperature, when compared to the NLR protein prior to modification by the methods of the present invention. Any know NLR protein can be used in the methods of the present invention including, but not limited to, ZAR1, Roq1, Bs2, Bs4, Mi 1.1, Mi-1.2, Gpa2, Y-1, Ry, Tm2, Rx2, Rx, Rpi-amr1, Rpi-amr3, N, RPP1, Roq1, Ry, Hero, I2, Pvr4, R1, R2, Sr35, Rp1-D, Rpi- blb1 and Rpi-blb3. Preferred NLR proteins are those that are known to be temperature sensitive with respect to resistance function at an elevated temperature including, but not limited to, Mi1.1 (resistance to root-knot nematodes), Mi 1.2 (resistance to root-knot nematodes), Sr35 (resistant to wheat stem rust), Rp1-D21 (resistance to maize common rust), Rpi-amr1 (resistance to potato late blight), Rpi-amr3 (resistant to potato late blight), SNC-1 (resistance to Pseudomonas syringae), RPS-4 (resistance to Pseudomonas syringae pv.tomato), and Ny-1 (resistance to potato virus Y).

[0060] While the methods of the present invention do not depend on particular methods for making the one or more amino acid substitutions to produce the thermostable NLR proteins, it is recognized that such amino acid substitutions can be made by modifying the nucleotide sequence of a nucleic acid molecule encoding the NLR protein to encode one or more substitute amino acids at the one or more predetermined positions. It is recognized that modifying the nucleotide sequence comprises substituting 1-3 nucleotides in each of the codons encoding each of the amino acids at the predetermined positions. It is further recognized that any methods for modifying the nucleotide sequence of a nucleic acid molecule can be used in the methods of the present invention including, for example, in vitro 105735300.1 - 12 - 070294.0229methods and in vivo methods (e.g. gene editing), which are described elsewhere herein or are otherwise known in the art.

[0061] In another aspect, the present invention further relates to methods for improving the thermostability of an NLR protein in a plant that comprises a polynucleotide encoding the NLR protein stably incorporated in its genome. Such methods modifying the nucleotide sequence of the polynucleotide encoding the NLR protein (e.g. an NLR gene) in at least one plant cell to produce at least one modified plant cell comprising the modified nucleotide sequence, whereby the modified nucleotide sequence encodes a thermostable NLR protein with at least one amino substitution at a predetermined position LQ^DQ^Į-helix in the NB-ARC domain or WKH^ȕ-sheet in the LRR domain, wherein the least one amino acid substitution generates a interdomain LQWHUDFWLRQ^EHWZHHQ^D^ILUVW^DPLQR^DFLG^LQ^WKH^Į-helix and a second amino DFLG^LQ^WKH^ȕ-sheet. If desired, the methods can further comprise regenerating the modified plant cell comprising the modified nucleotide sequence encoding the thermostable NLR.

[0062] Preferably, a thermostable NLR protein made by the method of the present invention comprises at least three long-range interactions between three amino acids in the IRXUWK^Į-helix in the NB-$5&^GRPDLQ^DQG^WKUHH^DPLQR^DFLGV^LQ^WKH^ȕ-sheet in the LRR domain. More preferably, a thermostable NLR made by the methods of the present invention comprises at least three long-UDQJH^LQWHUDFWLRQV^EHWZHHQ^WKUHH^DPLQR^DFLGV^LQ^WKH^IRXUWK^Į- helix in the NB-$5&^GRPDLQ^DQG^WKUHH^DPLQR^DFLGV^LQ^WKH^ȕ-sheet in the LRR domain, ZKHUHLQ^WKUHH^DPLQR^DFLGV^LQ^WKH^ȕ-VKHHW^LV^LQ^D^GLIIHUHQW^VWUDQG^RI^WKH^ȕ-sheet (i.e., the first of WKH^WKUHH^DPLQR^DFLGV^LQ^WKH^ȕ-sheet is in a first strand, the second acid is a second strand, and the third amino acid is in a third strand).

[0063] Because the methods for improving the thermostability of an NLR protein in a plant are essentially identical to the embodiments of the methods for improving the thermostability of a plant NLR proteins except that the one or more amino acid substitutions are made by gene editing at least one plant cell, the various embodiments of methods for improving the thermostability of a plant NLR proteins described above are applicable to the methods for improving the thermostability of an NLR protein in a plant.

[0064] In yet another aspect, the present invention relates to compositions comprising the thermostable NLR proteins, nucleic acid molecules encoding such thermostable NLR proteins and plants, plant cells, and other host cells comprising such thermostable NLR proteins and / or nucleic acid molecules encoding such thermostable NLR proteins. 105735300.1 - 13 - 070294.0229

[0065] The present invention encompasses thermostable NLR proteins comprising an amino acid sequence disclosed herein or in the accompanying sequence listing and / or drawings.

[0066] Such thermostable NLR proteins include, but are not limited, to a thermostable NLR protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 10, and 12 or a protein encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOS: 7, 9, and 11.

[0067] The present invention also encompasses nucleic acid molecules comprising one or more of the nucleotide sequences encoding NLR proteins disclosed herein or in the accompanying sequence listing and / or drawings. Such nucleic acid molecules include, but are not limited to, a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 7, 9, and 11 or a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 10, and 12.

[0068] The present invention further encompasses plants, plant cells, host cells, expression cassettes, polynucleotide constructs and vectors comprising at least one of such nucleic acid molecules, as well as food products produced from such plants. Additionally encompassed by the present invention are uses of plants comprising at least one of such nucleic acid molecules in the methods disclosed elsewhere herein such as, for example, methods of limiting plant diseases in agricultural crop production.

[0069] In certain embodiments of present invention, the plants and plant cells of the present invention comprise at least one heterologous polynucleotide construct comprising a nucleic acid molecule encoding a thermostable NLR protein of the present invention. Such a heterologous polynucleotide can be introduced into a plant or a cell thereof by a stable or transient plant transformation method disclosed elsewhere herein or otherwise known in the art.

[0070] Additionally, the present invention provides plants, seeds, and plant cells produced by the methods of present invention and / or comprising a polynucleotide construct of the present invention. Also provided are progeny plants and seeds thereof comprising a polynucleotide construct of the present invention. The present invention also provides seeds, vegetative parts, and other plant parts produced by the transformed plants and / or progeny plants of the invention as well as food products and other agricultural products produced from such plant parts that are intended to be consumed or used by humans and other animals including, but not limited to pets (e.g., dogs and cats) and livestock (e.g., pigs, cows, chickens, turkeys, and ducks). 105735300.1 - 14 - 070294.0229

[0071] The present invention encompasses isolated or substantially purified polynucleotide (also referred to herein as “nucleic acid molecule”, “nucleic acid” and the like) or protein (also referred to herein as “polypeptide”) compositions including, for example, polynucleotides and proteins comprising the sequences set forth in the accompanying Sequence Listing as well as variants and fragments of such polynucleotides and proteins. An “isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0072] Methods are known in the art for modifying DNA in the genome of a plant including, for example, and genome editing techniques, such as, for example, methods involving targeted mutagenesis, site-directed integration (SDI), and homologous recombination. Targeted mutagenesis or similar techniques are disclosed in U.S. Patent Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972, 5,871,984, and 8,106,259; all of which are herein incorporated in their entirety by reference. Methods for gene modification or gene replacement comprising homologous recombination can involve inducing single- strand or double-strand breaks in DNA using zinc-finger nucleases (ZFN), TAL (transcription activator-like) effector nucleases (TALEN), Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated nuclease (CRISPR / Cas nuclease), or homing endonucleases that have been engineered endonucleases to make double-strand breaks at specific recognition sequences in the genome of a plant, other organism, or host cell. See, for 105735300.1 - 15 - 070294.0229example, Durai et al., (2005) Nucleic Acids Res.33:5978-90; Mani et al. (2005) Biochem. Biophys. Res. Comm 335:447-57; U.S. Pat. Nos.7,163,824, 7,001,768, and 6,453,242; Arnould et al. (2006) J Mol. Biol.355:443-58; Ashworth et al., (2006) Nature 441:656-9; Doyon et al. (2006) J Am Chem Soc 128:2477-84; Rosen et al., (2006) Nucleic Acids Res. 34:4791-800; and Smith et al., (2006) Nucleic Acids Res.34:e149; U.S. Pat. App. Pub. No. 2009 / 0133152; and U.S. Pat. App. Pub. No.2007 / 0117128; all of which are herein incorporated in their entirety by reference.

[0073] TAL effector nucleases (TALENs) can be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. TAL effector nucleases 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. TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, FokI. The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See, WO 2010 / 079430; Morbitzer et al. (2010) PNAS 10.1073 / pnas.1013133107; Scholze and Boch (2010) Virulence 1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi:10.1093 / nar / gkq704; and Miller et al. (2011) Nature Biotechnology 29:143–148; all of which are herein incorporated by reference.

[0074] The CRISPR / Cas nuclease system can also be used to make single-strand or double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. The CRISPR / Cas nuclease is an RNA-guided (simple guide RNA, sgRNA in short) DNA endonuclease system performing sequence-specific double-stranded breaks in a DNA segment homologous to the designed RNA. It is possible to design the specificity of the sequence (Cho S.W. et al., Nat. Biotechnol.31:230-232, 2013; Cong L. et al., Science 339:819-823, 2013; Mali P. et al., Science 339:823-826, 2013; Feng Z. et al., Cell Research: 1-4, 2013).

[0075] In addition, a ZFN can be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. The Zinc Finger Nuclease (ZFN) is a fusion protein comprising the part of the FokI restriction endonuclease protein responsible for DNA 105735300.1 - 16 - 070294.0229cleavage and a zinc finger protein which recognizes specific, designed genomic sequences and cleaves the double-stranded DNA at those sequences, thereby producing free DNA ends (Urnov F.D. et al., Nat Rev Genet.11:636-46, 2010; Carroll D., Genetics.188:773-82, 2011).

[0076] Breaking DNA using site specific nucleases, such as, for example, those described herein above, can increase the rate of homologous recombination in the region of the breakage. Thus, coupling of such effectors as described above with nucleases enables the generation of targeted changes in genomes which include additions, deletions and other modifications.

[0077] PCR amplification can be used in certain embodiments of the methods of the present invention. Methods for designing PCR primers and PCR amplification are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR amplification include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.

[0078] The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides, can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0079] The polynucleotide constructs comprising thermostable NLR protein coding regions can be provided in expression cassettes for expression in the plant or other organism or in a host cell of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the protein coding region. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide or gene of interest and a regulatory sequence (i.e., a promoter) is functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading 105735300.1 - 17 - 070294.0229frame. 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 protein coding region to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0080] The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a thermostable NLR protein coding region of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants or other organism or non-human host cell. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and / or the thermostable NLR protein coding region or of the invention may be native / analogous to the host cell or to each other. Alternatively, the NLR gene, the regulatory regions and / or NLR protein coding region of the invention may be heterologous to the host cell or to each other.

[0081] As used herein, “heterologous” in reference to a nucleic acid molecule or nucleotide sequence that is present in a species of interest is a nucleic acid molecule or nucleotide sequence that originates from a different species than the species of interest and that is not introduced by introgression or other method that involves sexual reproduction, or, if from the same species, the nucleic acid molecule or nucleotide sequence that is present in a species of interest is modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide 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 promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene or chimeric polynucleotide construct comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0082] The present invention provides host cells comprising at least of the nucleic acid molecules, expression cassettes, and vectors of the present invention. In preferred embodiments of the invention, a host cell is a plant cell. In other embodiments, a host cell is selected from the group consisting of a bacterium, a fungal cell, and an animal cell. In certain embodiments, a host cell is non-human animal cell. However, in some other embodiments, the host cell is an in-vitro cultured human cell. 105735300.1 - 18 - 070294.0229

[0083] The termination region may be native with the transcriptional initiation region, may be native with the operably linked NLR protein coding region of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the protein of interest, and / or the plant host), or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res.15:9627-9639.

[0084] Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol.92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

[0085] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well- characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0086] Additionally, the polynucleotides can be modified to alter the amino acid sequences of the NLR proteins, for example, to improve translational efficiency, protein stability and / or any other desired property or properties, and / or to reduce any one or more undesirable properties, while improving or at least not reducing significantly the biological activity of the NLR proteins. For example, the polynucleotides can be modified to remove potential allergenic regions in the proteins encoded thereby. See, the AllergenOnline database for a comprehensive list of known and putative allergens (Goodman et al. (2016) Mol. Nutr. Food Res.60(5):1183-1198; available on the World Wide Web at: allergenonline.org). 105735300.1 - 19 - 070294.0229

[0087] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol.84:965-968.

[0088] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters (also referred to as “adaptors) 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.

[0089] A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants. Such constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J.3:2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, 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.

[0090] Tissue-preferred promoters can be utilized to target enhanced expression of the R protein coding sequences 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 105735300.1 - 20 - 070294.0229Yamamoto 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, if necessary, for weak expression.

[0091] The transgene can be expressed using an inducible promoter, such as, for example, 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. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol.89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol.4:111-116. See also WO 99 / 43819, herein incorporated by reference.

[0092] Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol.9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet.2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J.3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Patent No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

[0093] Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter may be used in the constructions of the invention. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath.28:425-449; Duan et al. (1996) Nature Biotechnology 14:494- 498); wun1 and wun2, U.S. Patent No.5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet.215:200-208); systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol.22:783-792; Eckelkamp et al. (1993) FEBS Letters 105735300.1 - 21 - 070294.0229323:73-76); MPI gene (Corderok et al. (1994) Plant J.6(2):141-150); and the like, herein incorporated by reference.

[0094] 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 may 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. Chemical-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-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J.14(2):247-257) and tetracycline- inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet.227:229-237, and U.S. Patent Nos.5,814,618 and 5,789,156), herein incorporated by reference.

[0095] The expression cassette can also 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 glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable PDUNHUV^LQFOXGH^SKHQRW\SLF^PDUNHUV^VXFK^DV^ȕ-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFP¥ from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech.3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; 105735300.1 - 22 - 070294.0229Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094- 1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol.78 ( Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.

[0096] The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene can be used in the present invention.

[0097] Numerous plant transformation vectors and methods for transforming plants are available. See, for example, An, G. et al. (1986) Plant Pysiol., 81:301-305; Fry, J., et al. (1987) Plant Cell Rep.6:321-325; Block, M. (1988) Theor. Appl Genet.76:767-774; Hinchee, et al. (1990) Stadler. Genet. Symp.203212.203-212; Cousins, et al. (1991) Aust. J. Plant Physiol.18:481-494; Chee, P. P. and Slightom, J. L. (1992) Gene.118:255-260; Christou, et al. (1992) Trends. Biotechnol.10:239-246; D’Halluin, et al. (1992) Bio / Technol.10:309-314; Dhir, et al. (1992) Plant Physiol.99:81-88; Casas et al. (1993) Proc. Nat. Acad Sci. USA 90:11212-11216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P:119-124; Davies, et al. (1993) Plant Cell Rep.12:180-183; Dong, J. A. and Mchughen, A. (1993) Plant Sci. 91:139-148; Franklin, C. I. and Trieu, T. N. (1993) Plant. Physiol.102:167; Golovkin, et al. (1993) Plant Sci.90:41-52; Guo Chin Sci. Bull.38:2072-2078; Asano, et al. (1994) Plant Cell Rep.13; Ayeres N. M. and Park, W. D. (1994) Crit. Rev. Plant. Sci.13:219-239; Barcelo, et al. (1994) Plant. J.5:583-592; Becker, et al. (1994) Plant. J.5:299-307; Borkowska et al. (1994) Acta. Physiol Plant.16:225-230; Christou, P. (1994) Agro. Food. Ind. Hi Tech.5: 17-27; Eapen et al. (1994) Plant Cell Rep.13:582-586; Hartman, et al. (1994) Bio-Technology 12: 919923; Ritala, et al. (1994) Plant. Mol. Biol.24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol.104:3748.

[0098] Plant transformation vectors that find use in the present invention include, for example, T-DNA vectors or plasmids, which are suitable for use in Agrobacterium-mediated transformation methods that are disclosed elsewhere herein or otherwise known in the art. 105735300.1 - 23 - 070294.0229

[0099] The methods of the invention involve introducing a polynucleotide construct into a plant. By “introducing” is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a polynucleotide construct to a plant, only that the polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0100] By “stable transformation” is intended that the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof. By “transient transformation” is intended that a polynucleotide construct introduced into a plant does not integrate into the genome of the plant.

[0101] For the transformation of plants and plant cells, the nucleotide sequences of the invention are inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell. The selection of the vector depends on the preferred transformation technique and the target plant species to be transformed.

[0102] Methodologies for constructing plant expression cassettes and introducing foreign nucleic acids into plants are generally known in the art and have been previously described. For example, foreign DNA can be introduced into plants, using tumor-inducing (Ti) plasmid vectors. Other methods utilized for foreign DNA delivery involve the use of PEG mediated protoplast transformation, electroporation, microinjection whiskers, and biolistics or microprojectile bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Pat. No.5,405,765 to Vasil et al.; Bilang et al. (1991) Gene 100: 247-250; Scheid et al., (1991) Mol. Gen. Genet., 228: 104-112; Guerche et al., (1987) Plant Science 52: 111-116; Neuhause et al., (1987) Theor. Appl Genet.75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208:1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlock et al., (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). The method of transformation depends upon the plant cell to be transformed, stability of vectors used, expression level of gene products and other parameters.

[0103] Other suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection as Crossway et al. (1986) 105735300.1 - 24 - 070294.0229Biotechniques 4:320-334, electroporation as described by Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation as described by Townsend et al., U.S. Patent No.5,563,055, Zhao et al., U.S. Patent No.5,981,840, direct gene transfer as described by Paszkowski et al. (1984) EMBO J.3:2717-2722, and ballistic particle acceleration as described in, for example, Sanford et al., U.S. Patent No.4,945,050; Tomes et al., U.S. Patent No.5,879,918; Tomes et al., U.S. Patent No.5,886,244; Bidney et al., U.S. Patent No.5,932,782; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” 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 Lec1 transformation (WO 00 / 28058). Also see, Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Patent No.5,240,855; Buising et al., U.S. Patent Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker- mediated transformation); 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.

[0104] The polynucleotides of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide construct of the invention within a viral DNA or RNA molecule. Further, it is recognized that promoters of the invention also encompass promoters utilized for 105735300.1 - 25 - 070294.0229transcription by viral RNA polymerases. Methods for introducing polynucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.

[0105] If desired, the modified viruses or modified viral nucleic acids can be prepared in formulations. Such formulations are prepared in a known manner (see e.g. for review US 3,060,084, EP-A 707445 (for liquid concentrates), Browning, “Agglomeration”, Chemical Engineering, Dec.4, 1967, 147-48, Perry’s Chemical Engineer’s Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages 8-57 and et seq. WO 91 / 13546, US 4,172,714, US 4,144,050, US 3,920,442, US 5,180,587, US 5,232,701, US 5,208,030, GB 2,095,558, US 3,299,566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al. Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989 and Mollet, H., Grubemann, A., Formulation technology, Wiley VCH Verlag GmbH, Weinheim (Germany), 2001, 2. D. A. Knowles, Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, Dordrecht, 1998 (ISBN 0-7514- 0443-8), for example by extending the active compound with auxiliaries suitable for the formulation of agrochemicals, such as solvents and / or carriers, if desired emulsifiers, surfactants and dispersants, preservatives, antifoaming agents, anti-freezing agents, for seed treatment formulation also optionally colorants and / or binders and / or gelling agents.

[0106] In specific embodiments, the polynucleotide constructs and expression cassettes of the invention can be provided to a plant using a variety of transient transformation methods known in the art. 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) PNAS Sci.91: 2176-2180 and Hush et al. (1994) J. Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and Agrobacterium tumefaciens-mediated transient expression as described elsewhere herein.

[0107] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and 105735300.1 - 26 - 070294.0229then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a polynucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

[0108] Unless expressly stated or apparent from the context of usage, the methods and compositions of the present invention can be used with any plant species including, for example, monocotyledonous plants, dicotyledonous plants, and conifers. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), triticale (× Triticosecale or Triticum × Secale) sorghum (Sorghum bicolor, Sorghum vulgare), teff (Eragrostis tef), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), switchgrass (Panicum virgatum), 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), strawberry (e.g. Fragaria × ananassa, Fragaria vesca, Fragaria moschata, Fragaria virginiana, Fragaria chiloensis), sweet potato (Ipomoea batatus), yam (Dioscorea spp., D. rotundata, D. cayenensis, D. alata, D. polystachya, D. bulbifera, D. esculenta, D. dumetorum, D. trifida), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), oil palm (e.g. Elaeis guineensis, Elaeis oleifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea 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), date (Phoenix dactylifera), cultivated forms of Beta vulgaris (sugar beets, garden beets, chard or spinach beet, mangelwurzel or fodder beet), sugarcane (Saccharum spp.), oat (Avena sativa), barley (Hordeum vulgare), cannabis (Cannabis sativa, C. indica, C. ruderalis), poplar (Populus spp.), eucalyptus (Eucalyptus spp.), Arabidopsis thaliana, Arabidopsis rhizogenes, Nicotiana benthamiana, Brachypodium distachyon vegetables, ornamentals, and conifers and other trees. In specific embodiments, plants of the present invention are crop plants (e.g. maize, sorghum, wheat, millet, rice, barley, oats, sugarcane, alfalfa, soybean, peanut, sunflower, cotton, safflower, Brassica spp., lettuce, strawberry, apple, citrus, etc.).

[0109] Vegetables include tomatoes (Lycopersicon esculentum), eggplant (also known as “aubergine” or “brinjal”) (Solanum melongena), pepper (Capsicum annuum), lettuce (e.g., 105735300.1 - 27 - 070294.0229Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), chickpeas (Cicer arietinum), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Fruit trees and related plants include, for example, apples, pears, peaches, plums, oranges, grapefruits, limes, pomelos, palms, and bananas. Nut trees and related plants include, for example, almonds, cashews, walnuts, pistachios, macadamia nuts, filberts, hazelnuts, and pecans.

[0110] In certain embodiments, the plants of the present invention are crop plants such as, for example, maize (corn), soybean, wheat, rice, cotton, alfalfa, sunflower, canola (Brassica spp., particularly Brassica napus, Brassica rapa, Brassica juncea), rapeseed (Brassica napus), sorghum, millet, barley, triticale, safflower, peanut, sugarcane, tobacco, potato, tomato, and pepper.

[0111] The term “plant” is intended to encompass plants at any stage of maturity or development, as well as any cells, tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context. Plant parts include, but are not limited to, fruits, stems, tubers, roots, flowers, ovules, stamens, petals, leaves, hypocotyls, epicotyls, cotyledons, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, seeds, and the like. It is recognized that the plant protoplasts of the present invention can be prepared from any one or more of the aforementioned plant parts and at any stage of development and / or maturity.

[0112] Likewise, the term “plant cell” is intended to encompass plant cells obtained from or in plants at any stage of maturity or development unless otherwise clearly indicated by context. Plant cells can be from or in plant parts including, but are not limited to, fruits, stems, tubers, roots, flowers, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, in vitro-cultured tissues, organs or cells and the like. It is recognized that the plant protoplasts of the present invention can be prepared from any one or more of the aforementioned plant cells and at any stage of development and / or maturity. As used herein, unless expressly stated otherwise or apparent from the context of usage, the term “plant cell” is intended to encompass a plant protoplast. 105735300.1 - 28 - 070294.0229

[0113] Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides. As used herein, “progeny”, “progeny plant” and “progeny of a plant” comprise any subsequent generation of a plant whether resulting from sexual reproduction and / or asexual propagation, unless it is expressly stated otherwise or is apparent from the context of usage.

[0114] The term “expression” as used herein refers to the biosynthesis of a gene product, including the transcription and / or translation of said gene product. The “expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while the “expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide.

[0115] The use of the terms “DNA” or “RNA” herein is not intended to limit the present invention to polynucleotide molecules comprising DNA or RNA. Those of ordinary skill in the art will recognize that the methods and compositions of the invention encompass nucleic acid molecules, polynucleotides, polynucleotide constructs, expression cassettes, and vectors comprised of deoxyribonucleotides (i.e., DNA), ribonucleotides (i.e., RNA) or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues including, but not limited to, nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). The polynucleotide molecules of the invention also encompass all forms of polynucleotide molecules including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. Furthermore, it is understood by those of ordinary skill in the art that the nucleotide sequences disclosed herein also encompasses the complement of that exemplified nucleotide sequence.

[0116] The invention relates to methods for improving the thermostability of plant NLR proteins. Such thermostable NLR proteins are capable of conferring to a plant enhanced or increased resistance to a plant pathogen at an elevated temperature, when compared to the resistance conferred by the original or unimproved NLR protein. By “resistance to a plant disease” or “disease resistance” is intended that the plants avoid the disease symptoms that 105735300.1 - 29 - 070294.0229are the outcome of plant-pathogen interactions. That is, one or more pathogens are prevented from causing a plant disease or plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the one or more pathogens is minimized or lessened.

[0117] The methods of the present invention are broadly applicable to NLR proteins that are R proteins against any plant pest including, but not limited to, plant pathogens (e.g. fungi, oomycetes, bacteria, viruses, and nematodes) and insects and acarids that cause damage to plants. Thus, the term “plant pathogen” as used herein encompasses any plant pest unless expressly stated or apparent from the context of usage. Similarly, term “plant disease” or “disease” as used herein encompasses any damage caused to a plant by a plant pest unless expressly stated or apparent from the context of usage.

[0118] Plant pathogens include, for example, bacteria, fungi, oomycetes, viruses, nematodes, and the like. Specific pathogens for the major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticillium albo-atrum, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas 105735300.1 - 30 - 070294.0229syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia graminis f.sp. hordei, Puccinia graminis f.sp. avenae, Puccinia graminis f.sp. secalis, Puccinia recondita f.sp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, High Plains Virus, European wheat striate virus; Sunflower: Plasmopora halstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Corn: Colletotrichum graminicola, Fusarium moniliforme var. subglutinans, Erwinia stewartii, Gibberella zeae (Fusarium graminearum), Fusarium verticilloides, Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Maize Chlorotic Mottle Virus, 105735300.1 - 31 - 070294.0229High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum, C. sublineolum, Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium verticillioides, Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola, etc.; Tomato: Corynebacterium michiganense pv. michiganense, Pseudomonas syringae pv. tomato, Ralstonia solanacearum, Xanthomonas vesicatoria, Xanthomonas perforans, Alternaria solani, Alternaria porri, Collectotrichum spp., Fulvia fulva Syn. Cladosporium fulvum, Fusarium oxysporum f. lycopersici, Leveillula taurica / Oidiopsis taurica, Phytophthora infestans, other Phytophthora spp., Pseudocercospora fuligena Syn. Cercospora fuligena, Sclerotium rolfsii, Septoria lycopersici, Meloidogyne spp.; Potato: Ralstonia solanacearum, Pseudomonas solanacearum, Erwinia carotovora subsp. Atroseptica Erwinia carotovora subsp. Carotovora, Pectobacterium carotovorum subsp. Atrosepticum, Pseudomonas fluorescens, Clavibacter michiganensis subsp. Sepedonicus, Corynebacterium sepedonicum, Streptomyces scabiei, Colletotrichum coccodes, Alternaria alternate, Mycovellosiella concors, Cercospora solani, Macrophomina phaseolina, Sclerotium bataticola, Choanephora cucurbitarum, Puccinia pittieriana, Aecidium cantensis, Alternaria solani, Fusarium spp., Phoma solanicola f. foveata, Botrytis cinerea, Botryotinia fuckeliana, Phytophthora infestans, Pythium spp., Phoma andigena var. andina, Pleospora herbarum, Stemphylium herbarum, Erysiphe cichoracearum, Spongospora subterranean Rhizoctonia solani, Thanatephorus cucumeris, Rosellinia sp. Dematophora sp., Septoria lycopersici, Helminthosporium solani, Polyscytalum pustulans, Sclerotium rolfsii, Athelia rolfsii, Angiosorus solani, Ulocladium atrum, Verticillium albo-atrum, V. dahlia, Synchytrium endobioticum, Sclerotinia sclerotiorum, Candidatus Liberibacter solanacearum; Banana: Fusarium oxysporum f. sp. cubense, Colletotrichum musae, Armillaria mellea, Armillaria tabescens, Pseudomonas solanacearum, Phyllachora musicola, Mycosphaerella fijiensis, 105735300.1 - 32 - 070294.0229Rosellinia bunodes, Pseudomas spp., Pestalotiopsis leprogena, Cercospora hayi, Pseudomonas solanacearum, Ceratocystis paradoxa, Verticillium theobromae, Trachysphaera fructigena, Cladosporium musae, Junghuhnia vincta, Cordana johnstonii, Cordana musae, Fusarium pallidoroseum, Colletotrichum musae, Verticillium theobromae, Fusarium spp., Acremonium spp., Cylindrocladium spp., Deightoniella torulosa, Nattrassia mangiferae, Dreschslera gigantean, Guignardia musae, Botryosphaeria ribis, Fusarium solani, Nectria haematococca, Fusarium oxysporum, Rhizoctonia spp., Colletotrichum musae, Uredo musae, Uromyces musae, Acrodontium simplex, Curvularia eragrostidis, Drechslera musae-sapientum, Leptosphaeria musarum, Pestalotiopsis disseminate, Ceratocystis paradoxa, Haplobasidion musae, Marasmiellus inoderma, Pseudomonas solanacearum, Radopholus similis, Lasiodiplodia theobromae, Fusarium pallidoroseum, Verticillium theobromae, Pestalotiopsis palmarum, Phaeoseptoria musae, Pyricularia grisea, Fusarium moniliforme, Gibberella fujikuroi, Erwinia carotovora, Erwinia chrysanthemi, Cylindrocarpon musae, Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica, Pratylenchus coffeae, Pratylenchus goodeyi, Pratylenchus brachyurus, Pratylenchus reniformia, Sclerotinia sclerotiorum, Nectria foliicola, Mycosphaerella musicola, Pseudocercospora musae, Limacinula tenuis, Mycosphaerella musae, Helicotylenchus multicinctus, Helicotylenchus dihystera, Nigrospora sphaerica, Trachysphaera frutigena, Ramichloridium musae, Verticillium theobromae, Phytophthora infestans, Phytophthora parasitica, Phytophthora ramorum, Phytophthora ipomoeae, Phytophthora mirabilis, Phytophthora capsici, Phytophthora porri, Phytophthora sojae, Phytophthora palmivora, and Phytophthora phaseoli.

[0119] Bacterial pathogens include, but are not limited to, Agrobacterium tumefaciens, Candidatus Liberibacter asiaticus, Candidatus Liberibacter solanacearum, Clavibacter michiganensis, Clavibacter sepedonicus, Dickeya dadantii, Dickeya solani, Erwinia amylovora, Pectobacterium atrosepticum, Pectobacterium carotovorum, Pseudomonas andropogonis, Pseudomonas avenae, Pseudomonas alboprecipitans, Pseudomonas fluorescens, Pseudomonas savastanoi, Pseudomonas solanacearum, Pseudomonas syringae, Ralstonia solanacearum, Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas citri, Xanthomonas perforans, Xanthomonas vesicatoria, Xanthomonas oryzae, and Xylella fastidiosa.

[0120] Oomycete pathogens include, but are not limited to, Phytophthora infestans, Phytophthora ipomoeae, Phytophthora mirabilis, Phytophthora phaseoli, Phytophthora 105735300.1 - 33 - 070294.0229megasperma fsp. glycinea, Phytophthora megasperma, Phytophthora cryptogea, Peronospora spp. and Pythium spp.

[0121] Nematode pathogens include, but are not limited to, Anguina tritici, Aphelenchoides besseyi, Bursaphelenchus xylophilus, Ditylenchus dipsaci, Globodera spp., *ORERGHUD^SDOOLGD^^*ORERGHUD^URVWRFKLHQVLV^^+HWHURGHUD^VSS^^^+HWHURGHUD^DYHQDH^^ +HWHURGHUD^ILOLSMHYL^^+HWHURGHUD^JO\FLQHV^^0HORLGRJ\QH spp., Meloidogyne graminicola, Meloidogyne hapla, Meloidogyne incógnita, Meloidogyne enterolobii, Merlinius spp., Nacobbus aberrans, Paratylenchus VSS^^^3UDW\OHQFKXV^FRIIHDH^^3UDW\OHQFKXV^QHJOHFWXV^^ Pratylenchus penetraQV^^3UDW\OHQFKXV^SHQHWUDQV^^3UDW\OHQFKXV^WKRUQHL^^3UDW\OHQFKXV^YXOQXV^^ 3UDW\OHQFKXV^]HDH^^5DGRSKROXV^VLPLOLV^^5RW\OHQFKXOXV^UHQLIRUPLV^^7\OHQFKRUK\QFKXV spp., and Xiphinema index.

[0122] Insect pests include, but are not limited to, insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Thysanoptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.

[0123] Insects of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the family Noctuidae Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. segetum Denis & Schiffermüller (turnip moth); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hübner (cotton leaf worm); Anticarsia gemmatalis Hübner (velvetbean caterpillar); Athetis mindara Barnes and McDunnough (rough skinned cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Egira (Xylomyges) curialis Grote (citrus cutworm); Euxoa messoria Harris (darksided cutworm); Helicoverpa armigera Hübner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Heliothis virescens Fabricius (tobacco budworm); Hypena scabra Fabricius (green cloverworm); Hyponeuma taltula Schaus; (Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Melanchra picta Harris (zebra caterpillar); Mocis latipes Guenée (small mocis moth); Pseudaletia unipuncta Haworth (armyworm); Pseudoplusia includens Walker (soybean looper); Richia albicosta Smith (Western bean cutworm);Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Trichoplusia ni Hübner (cabbage looper); borers, casebearers, webworms, coneworms, and skeletonizers from the families Pyralidae and Crambidae such as Achroia grisella Fabricius (lesser wax moth); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella 105735300.1 - 34 - 070294.0229Walker (almond moth); Chilo partellus Swinhoe (spotted stalk borer); C. suppressalis Walker (striped stem / rice borer); C. terrenellus Pagenstecher (sugarcane stemp borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leaf roller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea flavipennella Box; D. grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Papaipema nebris (stalk borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Hedylepta accepta Butler (sugarcane leafroller); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Loxostege sticticalis Linnaeus (beet webworm); Maruca testulalis Geyer (bean pod borer); Orthaga thyrisalis Walker (tea tree web moth); Ostrinia nubilalis Hübner (European corn borer); Ostrinia furnacalis (Asian corn borer); Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); and leafrollers, budworms, seed worms, and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Hellula phidilealis (cabbage budworm moth); Adoxophyes orana Fischer von Rösslerstamm (summer fruit tortrix moth); Archips spp. including A. argyrospila Walker (fruit tree leaf roller) and A. rosana Linnaeus (European leaf roller); Argyrotaenia spp.; Bonagota salubricola Meyrick (Brazilian apple leafroller); Choristoneura spp.; Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (codling moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hübner (vine moth); Grapholita molesta Busck (oriental fruit moth); Lobesia botrana Denis & Schiffermüller (European grape vine moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth); and Suleima helianthana Riley (sunflower bud moth).

[0124] Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J.E. Smith (orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese Oak Silkmoth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus 105735300.1 - 35 - 070294.0229Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hübner (elm spanworm); Erannis tiliaria Harris (linden looper); Erechthias flavistriata Walsingham (sugarcane bud moth); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guérin-Méneville (grapeleaf skeletonizer); Heliothis subflexa Guenée; Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Malacosoma spp.; Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Orgyia spp.; Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail, orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée (omnivorous looper); Schizura concinna J.E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Telchin licus Drury (giant sugarcane borer); Thaumetopoea pityocampa Schiffermüller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer) and Yponomeuta padella Linnaeus (ermine moth).

[0125] Of interest are larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Chrysomelidae, and Curculionidae including, but not limited to: Bruchus pisorum (pea weevil), Callosobruchus maculatus (cowpea weevil), Anthonomus grandis Boheman (boll weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepes abbreviatus Linnaeus (Diaprepes root weevil); Hypera punctata Fabricius (clover leaf weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Metamasius hemipterus hemipterus Linnaeus (West Indian cane weevil); M. hemipterus sericeus Olivier (silky cane weevil); Sitophilus zeamais (maize weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug); S. livis Vaurie (sugarcane weevil); Rhabdoscelus obscurus 105735300.1 - 36 - 070294.0229Boisduval (New Guinea sugarcane weevil); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leafminers in the family Chrysomelidae including, but not limited to: Cerotoma trifurcata (bean leaf beetle), Chaetocnema ectypa Horn (desert corn flea beetle); C. pulicaria Melsheimer (corn flea beetle); Colaspis brunnea Fabricius (grape colaspis); Diabrotica barberi Smith & Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber (southern corn rootworm); D. virgifera virgifera LeConte (western corn rootworm); Leptinotarsa decemlineata Say (Colorado potato beetle); Oulema melanopus Linnaeus (cereal leaf beetle); Phyllotreta cruciferae Goeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflower beetle); beetles from the family Coccinellidae including, but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle); chafers and other beetles from the family Scarabaeidae including, but not limited to: Antitrogus parvulus Britton (Childers cane grub); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Dermolepida albohirtum Waterhouse (Greyback cane beetle); Euetheola humilis rugiceps LeConte (sugarcane beetle); Lepidiota frenchi Blackburn (French’s cane grub); Tomarus gibbosus De Geer (carrot beetle); T. subtropicus Blatchley (sugarcane grub); Phyllophaga crinita Burmeister (white grub); P. latifrons LeConte (June beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus majalis Razoumowsky (European chafer); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp. including M. communis Gyllenhal (wireworm); Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae; beetles from the family Tenebrionidae; beetles from the family Cerambycidae such as, but not limited to, Migdolus fryanus Westwood (longhorn beetle); and beetles from the Buprestidae family including, but not limited to, Aphanisticus cochinchinae seminulum Obenberger (leaf-mining buprestid beetle).

[0126] Adults and immatures of the order Diptera are of interest, including leafminers Agromyza parvicornis Loew (corn blotch leafminer); midges including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Neolasioptera murtfeldtiana Felt, (sunflower seed midge); Sitodiplosis mosellana Géhin (wheat midge); fruit flies (Tephritidae), Bactrocera oleae (olive fruit fly), Ceratitis capitata (Mediterranean fruit fly), Oscinella frit Linnaeus (frit flies); maggots including, but not limited to: Delia spp. including Delia platura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly); Fannia canicularis Linnaeus, F. femoralis Stein (lesser house flies); Meromyza americana Fitch (wheat stem maggot); Musca domestica Linnaeus (house flies); 105735300.1 - 37 - 070294.0229Stomoxys calcitrans Linnaeus (stable flies)); face flies, horn flies, blow flies, Chrysomya spp.; Phormia spp.; and other muscoid fly pests, horse flies Tabanus spp.; bot flies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deer flies Chrysops spp.; Melophagus ovinus Linnaeus (keds); and other Brachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.; biting midges, sand flies, sciarids, and other Nematocera.

[0127] Agronomically important members from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Acyrthisiphon pisum Harris (pea aphid); Adelges spp. (adelgids); Adelphocoris rapidus Say (rapid plant bug); Anasa tristis De Geer (squash bug); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacaspis tegalensis Zehntner (sugarcane scale); Aulacorthum solani Kaltenbach (foxglove aphid); Bemisia argentifolii (silverleaf whitefly); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Blissus leucopterus leucopterus Say (chinch bug); Blostomatidae spp.; Brevicoryne brassicae Linnaeus (cabbage aphid); Cacopsylla pyricola Foerster (pear psylla); Calocoris norvegicus Gmelin (potato capsid bug); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Cimicidae spp.; Coreidae spp.; Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); C. notatus Distant (suckfly); Deois flavopicta Stål (spittlebug); Dialeurodes citri Ashmead (citrus whitefly); Diaphnocoris chlorionis Say (honeylocust plant bug); Diuraphis noxia Kurdjumov / Mordvilko (Russian wheat aphid); Duplachionaspis divergens Green (armored scale); Dysaphis plantaginea Paaserini (rosy apple aphid); Dysdercus suturellus Herrich-Schäffer (cotton stainer); Dysmicoccus boninsis Kuwana (gray sugarcane mealybug); Empoasca fabae Harris (potato leafhopper); Eriosoma lanigerum Hausmann (woolly apple aphid); Erythroneoura spp. (grape leafhoppers); Eumetopina flavipes Muir (Island sugarcane planthopper); Eurygaster spp.; Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (complex of seed bugs); and Hyalopterus pruni Geoffroy (mealy plum aphid); Icerya purchasi Maskell (cottony cushion scale); Labopidicola allii Knight (onion plant bug); Laodelphax striatellus Fallen (smaller brown planthopper); Leptoglossus corculus Say (leaf- footed pine seed bug); Leptodictya tabida Herrich-Schaeffer (sugarcane lace bug); Lipaphis erysimi Kaltenbach (turnip aphid); Lygocoris pabulinus Linnaeus (common green capsid); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western 105735300.1 - 38 - 070294.0229tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Macrosiphum euphorbiae Thomas (potato aphid); Macrosteles quadrilineatus Forbes (aster leafhopper); Magicicada septendecim Linnaeus (periodical cicada); Mahanarva fimbriolata Stål (sugarcane spittlebug); M. posticata Stål (little cicada of sugarcane); Melanaphis sacchari Zehntner (sugarcane aphid); Melanaspis glomerata Green (black scale); Metopolophium dirhodum Walker (rose grain aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stål (rice leafhopper); Nezara viridula Linnaeus (southern green stink bug); Nilaparvata lugens Stål (brown planthopper); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Orthops campestris Linnaeus; Pemphigus spp. (root aphids and gall aphids); Peregrinus maidis Ashmead (corn planthopper); Perkinsiella saccharicida Kirkaldy (sugarcane delphacid); Phylloxera devastatrix Pergande (pecan phylloxera); Planococcus citri Risso (citrus mealybug); Plesiocoris rugicollis Fallen (apple capsid); Poecilocapsus lineatus Fabricius (four-lined plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Pseudococcus spp. (other mealybug complex); Pulvinaria elongata Newstead (cottony grass scale); Pyrilla perpusilla Walker (sugarcane leafhopper); Pyrrhocoridae spp.; Quadraspidiotus perniciosus Comstock (San Jose scale); Reduviidae spp.; Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Saccharicoccus sacchari Cockerell (pink sugarcane mealybug); Scaptacoris castanea Perty (brown root stink bug); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Sogatella furcifera Horvath (white- backed planthopper); Sogatodes oryzicola Muir (rice delphacid); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Therioaphis maculata Buckton (spotted alfalfa aphid); Tinidae spp.; Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid); and T. citricida Kirkaldy (brown citrus aphid); Trialeurodes vaporariorum (greenhouse whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood (greenhouse whitefly); Trioza diospyri Ashmead (persimmon psylla); Typhlocyba pomaria McAtee (white apple leafhopper);Homalodisca vitripennis (glassy winged sharpshooter); Cicadulina mbila (maize leafhopper); Circulifer tenellus (beet leafhopper); Daktulosphaira vitifoliae (grape phylloxera); Coccus pseudomagnoliarum (citricola scale); Coccus hesperidum (soft brown scale); Pulvinaria regalis (horse chestnut scale); Pulvinaria psidii (green shield scale); Aonidiella aurantii (California citrus scale); Aonidiella taxus (Asiatic 105735300.1 - 39 - 070294.0229red scale); Aspidiotus excisus (Cyanotis scale); Aspidiotus nerii (oleander scale); Aulacaspis rosarum (Asiatic rose scale); Aulacaspis tubercularis (white mango scale); Chionaspis lepineyi (oak scurfy scale); Hemiberlesia lataniae (latania scale); Kuwanaspis pseudoleucaspis (bamboo diaspidid scale; Lepidosaphes pini (pine oystershell scale); Lopholeucaspis japonica (Japanese maple scale); Oceanaspidiotus spinosus (spined scale insect); Parlatoria ziziphi (black parlatoria scale); Pseudaonidia duplex (camphor scale); Unaspis yanonensis (arrowhead scale); Phenacoccus solani (Solanum mealybug); Planococcus citri (citrus mealybug); Planococcus (ficus vine mealybug); Pseudococcus longispinus (long-tailed mealybug); Pseudococcus affinis (glasshouse mealybug); Diaphorina citri (Asian citrus psyllid); and Bactericera cockerelli (potato psyllid).

[0128] Insects of the order Thysanoptera include, but are not limited to, Thrips tabaci (potato thrips) and Frankliniella occidentalis (western flower thrips).

[0129] Other insects of interest include, but are not limited to, grasshopper species (e.g. Schistocerca americana and crickets (e,g, Teleogryllus taiwanemma, Teleogryllus emma).

[0130] Acarids are arachnids (Class Arachnida) that are members of the subclass Arci which comprise mites and ticks. While acarids are not true insects, acarids are often grouped together with insect pests of plants because both acarids and insects are members of the phylum Arthropoda. As used herein, the term “insects” encompasses both true insects and acarids unless stated otherwise or apparent from the context of usage. Acarids of interest include, but are not limited to: Aceria tosichella Keifer (wheat curl mite); Panonychus ulmi Koch (European red mite); Petrobia latens Müller (brown wheat mite); Steneotarsonemus bancrofti Michael (sugarcane stalk mite) spider mites and red mites in the family Tetranychidae, Oligonychus grypus Baker & Pritchard, O. indicus Hirst (sugarcane leaf mite), O. pratensis Banks (Banks grass mite), O. stickneyi McGregor (sugarcane spider mite); Tetranychus urticae Koch (two spotted spider mite); T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite), flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and bud mites in the family Eriophyidae. 105735300.1 - 40 - 070294.0229EXAMPLES EXAMPLE 1: Identification and characterisation of the Interactions in the Region of NLR Immune Receptors Critical for Maintaining Functionality at Elevated Temperatures

[0131] To reveal the molecular mechanism underlying temperature-dependent defence response, we performed structural modelling by homology (using Yasara) on a set of CC- and TIR-NLR proteins that are temperature-sensitive (e.g. N, SNC1, Roq1) or maintain functionality above 28 degrees Celsius (e.g. Ry, Rx, Sr21). This led us to formulate a model that the thermal stability of R proteins depends on the proper spatial stabilization of the LRR domain by the distal region of the NB-ARC domain. There have to be at least three more pairs of specific interdomain interactions than any other interactions between alpha-helix of the NB-ARC and three / four beta-sheet strands of LRR (FIG.1). Depending on the specific sequence of the investigated protein, these specific interactions include anion-cation keyrequirement for the spatial stabilization is the presence of anion-cation interactions between both ends of the alpha-helix strand and spatially compatible (or aligned) beta-sheet strands. Based on our model, a thermolabile protein can be modified to gain thermostability while105735300.1 - 41 - 070294.0229suitable amino acid replacements; these replacements are made based on the rules of amino acid biochemistry. The presented model is universal for all the R proteins possessing theEXAMPLE 2: A Model for the Mechanism of SNC1 and N Thermostability Gained by Random Mutagenesis

[0133] SNC1 and N are temperature-sensitive proteins (Zhu et al., 2010, Proteins. PLOS Pathogens 6, e1000844, doi.org / 10.1371 / journal.ppat.1000844). E640K mutation in SNC1 acquired by random mutagenesis restored resistance at 28°C and the same was shown for the equivalent mutations in N (Y646K and N648D; Zhu et al., 2010, Proteins. PLOS Pathogens 6, e1000844, doi.org / 10.1371 / journal.ppat.1000844); however, the authors did not propose a mechanism of this phenomenon. We speculated that these mutations stabilised the interaction between NB-ARC and LRR in accordance with our model. To test it, we modelled the structures of these two proteins and shown that, for SNC1, the introduction of lysine instead of glutamate at position 640 of LRR beta-sheet introduced anion-cation interaction with glutamate 441 in the NB-ARC alpha-helix. Together with already existing interdomain interactions between lysine at position 616 and aspartic acid at position 447, and tyrosine at position 444 and asparagine at position 594, it resulted in the proper spatial stabilization of SNC1 (FIG.3A).

[0134] Similarly, for N protein, the substitution of tyrosine 452 to lysine and asparagine 648 to aspartate adds the anion-cation interaction needed to stabilise the protein, together with preexisting interactions between tyrosine 458 and arginine 622, glutamine 455 and threonine 646, and a cluster of hydrophobic folds that also contribute to stability (FIG.3B). Summarising, our analysis revealed that the previously unexplained mechanism of thermostability of SNC1 and N protein variants fully aligns with our model. EXAMPLE 3: The Model is Validated by Creating a Thermolabile Variant of Rysto

[0135] Rysto(SEQ ID NO: 2) is a thermostable TIR-NB-LRR (TNL) protein conferring resistance against potato virus Y (PVY) by recognition of the viral coat protein CP (Grech- Baran et al., 2020, Plant Biotechnol. J.18:655–667, doi.org / 10.1111 / pbi.13230; Grech-Baran et al., 2022, New Phytol.235:1179–1195, doi.org / 10.1111 / nph.18183).

[0136] Based on our model, we predicted three interactions crucial for retaining Rystofunction at elevated temperatures; glutamic acid 456 in the alpha-helix with lysine 624 in the 105735300.1 - 42 - 070294.0229beta-sheet, aspartic acid 450 with histidine 625 as well as isoleucine 453 with lysine 624 (FIG.4).

[0137] To create a temperature-sensitive variant of Rysto, we generated a GFP-tagged Rysto_K624E mutant (SEQ ID NO: 4), predicted to lose stability at the elevated temperature, and a GFP-Rysto_H625E variant predicted to disrupt the interaction with D450. The GFP- tagged mutated proteins and wild-type Rysto-GFP control were then transiently co-expressed with PVY CP under permissive and elevated temperatures. As predicted, all variants were similarly functional at 22°C (FIGS.5A, 5B). At elevated temperature, the Rysto_K624E variant fully abolished cell death. As expected, hypersensitive response (HR) was still observed for wild-type (WT) protein, but also for Rysto_H625E mutant, which suggests that not all interdomain interactions contribute equally to stabilisation and in vivo verification is crucial (FIGS.5A, 5B). No differences in protein expression between Rystovariants at each temperature were observed (FIG.5C).

[0138] To further investigate the Rysto_K624E variant, we grew N. benthamiana plants at 22°C or 30°C, infected them with PVY and three weeks later infiltrated leaves with either Rysto_K624E or Rysto_WT. After three days, we observed resistance response to PVY manifested by cell death for Rysto_WT at both temperatures, while for Rysto_K624E only at 22°C, as expected (FIGS.6A-6B). This result further confirms the importance of interdomain interactions between NB-ARC and LRR domains for NLR thermostability at elevated temperatures. EXAMPLE 4: The Replacement of Amino Acids Indicated by the Model Restores Thermostability of Roq1

[0139] Next, we engineered Roq1 (SEQ ID NO: 6), a temperature-sensitive TNL protein that confers resistance to economically important bacteria Xanthomonas spp. and Ralstonia solanacearum. Our structural modelling singled out three residues, arginine 451, alanine 455 and alanine 459, as targets for replacement for the electrostatic stabilisation by establishing hydrophobic interactions with threonine 626 (FIG.7). Another preexisting interaction occurs between aspartic acid 452 and arginine 650.

[0140] Based on the modelling outcome, we generated single (Roq1_R451P) (SEQ ID NO: 8), double (Roq1_A455L_A459L) (SEQ ID NO: 10), and triple (Roq1_R451P_A455L_A459L) (SEQ ID NO: 12) mutant Roq1 variants. We tested the engineered proteins by transiently co-expressing them with Roq1 avirulence factor – XopQ – in N. benthamiana roq1 knockout background (as Roq1 is endogenously present in N. 105735300.1 - 43 - 070294.0229benthamiana genome; FIG.8A). Unlike WT Roq1, all three engineered Roq1 variants were functional at permissive temperature as well as at 29°C (FIGS.8A, 8B). The strongest cell- death phenotype was observed for the triple mutant of Roq1 (SEQ ID NO: 12), predicted in silico as the most stable at elevated temperature (FIGS.8B, 8C). EXAMPLE 5: The Model Predicts Thermostability of Helper NLRs

[0141] Wu et al. (2017, New Phytol.222:938–953, doi.org / 10.1111 / nph.15665) shown that NLRs signal via network and two main classes of NLRs can be distinguished, namely sensor NLRs which perceive pathogen-derived signals, and helper NLRs that function downstream in signal transduction. A large diversity of sensor NLRs relies on a small set of helper NLRs, with CC-NLRs (CNLs) generally relying on NRC class of helpers, while TNLs tend to signal via NRG1 protein. As both thermostable and thermolabile sensor NLRs use the same small set of helpers, and we showed that modifying sensor NLRs only is sufficient to restore thermostability, it implies that the helper NLRs are thermostable.

[0142] To validate this, we applied our model to both NRC and NRG classes of helper NLRs. In NRCs, we detected a significant number of strong electrostatic interactions on the interface between the beta-sheets of the LRR and the alpha-helix of the NB-ARC. For NRC2a, NRC2b and NRC3, the stabilisation is warranted by two hydrogen bonds created between alpha-helix arginine at position 430 (NRC2a and b) or 427 (NRC3) that interacts with two histidine residues of the beta-sheet, at positions 640 and 642 (NRC2a and b) or 639 and 641 (NRC3). Additional anion-cation stabilising interactions were detected between glutamic acid at position 435 and arginine at positions 541 (NRC2a), 569 (NRC2b) or 592 (NRC3). In case of NRC4, the stabilisation arises from the interaction of glutamic acid 434 and arginine 540, as well as creation of sulphide bridges, most likely via cystines 429 and 641.

[0143] For NRG1, stabilisation comes from the three anion-cation interactions of arginine at position 638 with glutamic acid at positions 470 and 642, as well as aspartic acid at position 466. The region is further stabilised by the hydrophobic interaction of histidine 457 and 460 with glutamic acid 594.

[0144] To create temperature-sensitive variants of NRG1(SEQ ID NO: 13), we substituted arginine at position 638 with either glutamine, to create variant NRG1_R638Q (SEQ ID NO: 15), or with asparagine, to create variant NRG1_R638N (SEQ ID NO: 17). Since N. benthamiana has native NRG1 gene, we used a knockout N. benthamiana / nrg1 105735300.1 - 44 - 070294.0229mutant to test NRG1 variants under permissive and elevated temperatures. Co-infiltration of Rystoand PVY CP into WT N. benthamiana causes cell death (FIGS.5A, 5B), but not in N. benthamiana / nrg1 mutant, confirming dependence of Rystosignalling on NRG class of helpers (FIGS.9A, 9B). In contrast to NRG1_WT, which restores Rysto+CP-dependent cell death under permissive and elevated temperatures when delivered to N. benthamiana / nrg1 mutant, NRG1_R638N variant restored cell death only at permissive, but not elevated temperatures (FIG.9A, 9B). The NRG1_R638Q variant showed full functionality at permissive temperature and only partial functionality at elevated temperature (FIG.9A, 9B). These results confirm that helper class of NLRs is also stabilised by long range interactions between NB-ARC and LRR domains, which is consistent with our model.

[0145] The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element.

[0146] Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0147] All publications and patent applications mentioned in the specification are indicative of the level of those skilled 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 to be incorporated by reference.

[0148] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. 105735300.1 - 45 - 070294.0229

Claims

Claims 1. A method for improving the thermostability of a plant nucleotide-binding leucine-rich repeat (NLR) protein that is capable of conferring to a plant resistance to a plant disease, the method comprising making at least one amino substitution at a predetermined position in an Į-helix in the nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC) domain or WKH^ȕ-sheet in the leucine-rich repeat (LRR) domain of the NLR protein so as to produce a thermostable NLR protein, wherein the least one amino acid substitution: (a) generates a interdomain LQWHUDFWLRQ^EHWZHHQ^D^ILUVW^DPLQR^DFLG^LQ^WKH^Į-helix and a VHFRQG^DPLQR^DFLG^LQ^WKH^ȕ-sheet and / or (b) eliminates a destabilizing interaction EHWZHHQ^WKH^ILUVW^DPLQR^DFLG^LQ^WKH^Į-helix and the second DPLQR^DFLG^LQ^WKH^ȕ-sheet; wherein the at least one amino substitution comprises replacing an amino acid in the NLR protein with a substitute amino acid.

2. The method of claim 1, wherein the at least one amino acid substitution does QRW^^RU^LV^QRW^SUHGLFWHG^WR^^DIIHFW^WKH^VHFRQGDU\^VWUXFWXUH^RI^WKH^Į-KHOL[^RU^WKH^ȕ-sheet.

3. The method of claim 1 or 2, wherein the substitute amino acid is a charged amino acid.

4. The method of claim 3, wherein the charged amino acid is selected from the group consisting of aspartic acid, glutamic acid, lysine, arginine, and histidine.

5. The method of claim 3 or 4, wherein the interdomain interaction is an anionic- cationic interaction.

6. The method of any one of claims 3-5, wherein: the first amino acid is aspartic acid or glutamic acid and the second amino acid is lysine, arginine, or histidine; or the first amino acid is lysine, arginine, or histidine and the second amino acid is aspartic acid or glutamic acid.

7. The method of claim 1 or 2, wherein the substitute amino acid is a hydrophobic amino acid. 105735300.1 - 46 - 070294.02298. The method of claim 7, wherein the hydrophobic amino acid is selected from the group consisting of leucine, isoleucine, methionine, phenylalanine, proline, tyrosine, tryptophan, and valine.

9. The method of claim 7 or 8, wherein the interdomain interaction is a hydrophobic interaction.

10. The method of claim 1 or 2, wherein the substituted amino acid is an uncharged, polar amino acid.

11. The method of claim 10, wherein the uncharged, polar amino acid amino acid is selected from the group consisting of serine, threonine, asparagine, glutamine, tyrosine, and cysteine.

12. The method of claim 10 or 11, wherein the interdomain interaction is hydrogen bonding.

13. The method of any one of claims 1-12, wherein the predetermined position is identified using a protein structure modeling program.

14. The method of any one of claims 1-13, wherein the thermostable NLR protein is capable of conferring to a plant enhanced resistance to a plant pathogen at an elevated temperature, when compared to the NLR protein at the elevated temperature.

15. The method of any one of claims 1-14, wherein making the at least one amino acid substitution comprises making one or more nucleotide substitutions in the nucleotide sequence of a polynucleotide encoding the NLR protein so as to produce a modified polynucleotide encoding the thermostable NLR protein.

16. The method of claim 15, wherein the one or more nucleotide substitutions are made in a plant or plant cell using a genome editing technique. a 105735300.1 - 47 - 070294.022918. A plant or plant cell comprising the thermostable NLR protein and / or the nucleic acid molecule of claim 17.

19. A method for improving the thermostability of an NLR protein in a plant, the method comprising modifying the nucleotide sequence of an NLR gene encoding the NLR protein in at least one plant cell to produce at least one modified plant cell comprising the modified nucleotide sequence, whereby the modified nucleotide sequence encodes a thermostable NLR protein with at least one amino substitution at a predetermined position in DQ^Į-helix in the NB-ARC domain or WKH^ȕ-sheet in the LRR domain of the NLR protein, wherein the least one amino acid substitution: (a) generates a interdomain interaction between D^ILUVW^DPLQR^DFLG^LQ^WKH^Į-KHOL[^DQG^D^VHFRQG^DPLQR^DFLG^LQ^WKH^ȕ-sheet and / or (b) eliminates a destabilizing interaction between the first amino acid in the Į-helix and the second amino DFLG^LQ^WKH^ȕ-sheet; wherein the at least one amino substitution comprises replacing an amino acid in the NLR protein with a substitute amino acid.

20. The method of claim 19, wherein the at least one amino acid substitution does QRW^^RU^LV^QRW^SUHGLFWHG^WR^^DIIHFW^WKH^VHFRQGDU\^VWUXFWXUH^RI^WKH^Į-KHOL[^RU^WKH^ȕ-sheet.

21. The method of claim 19 or 20, wherein the substitute amino acid is a charged amino acid.

22. The method of claim 21, wherein the charged amino acid is selected from the group of aspartic acid, glutamic acid, lysine, arginine, and histidine.

23. The method of claim 21 or 22 wherein the interdomain interaction is an anionic-cationic interaction.

24. The method of any one of claims 21-23, wherein: the first amino acid is aspartic acid or glutamic acid and the second amino acid is lysine, arginine, or histidine; or the first amino acid is lysine, arginine, or histidine and the second amino acid is aspartic acid or glutamic acid. 105735300.1 - 48 - 070294.022925. The method of claim 19 or 20, wherein the substitute amino acid is a hydrophobic amino acid.

26. The method of claim 25, wherein the hydrophobic amino acid is selected from the group consisting of leucine, isoleucine, methionine, phenylalanine, proline, tyrosine, tryptophan, and valine.

27. The method of claim 25 or 26, wherein the interdomain interaction is a hydrophobic interaction.

28. The method of claim 19 or 20, wherein the substituted amino acid is an uncharged, polar amino acid.

29. The method of claim 28, wherein the uncharged, polar amino acid amino acid is selected from the group consisting of serine, threonine, asparagine, glutamine, tyrosine, and cysteine.

30. The method of claim 28 or 29, wherein the interdomain interaction is hydrogen bonding.

31. The method of any one of claims 19-30, wherein the predetermined position is identified using a protein structure modeling program.

32. The method of any one of claims 19-31, wherein the thermostable NLR protein is capable of conferring to a plant enhanced resistance to a plant pathogen at an elevated temperature, when compared to the NLR protein at the elevated temperature.

33. The method of any one of claims 19-31, further comprising regenerating the at least one modified plant cell into a plant comprising the modified nucleotide sequence encoding thermostable NLR protein.

34. A plant or plant cell produced by the method of any one of claims 19-33, or a progeny plant or cell thereof comprising the modified nucleotide sequence encoding thermostable NLR protein. 105735300.1 - 49 - 070294.022935. A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 7, 9, or 11; and (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 8, 18, or 12.

36. The nucleic acid molecule of claim 35, wherein the nucleic acid molecule is an isolated nucleic acid molecule.

37. The nucleic acid molecule of claim 35, wherein the nucleic acid molecule is contained in a plant cell or other host cell.

38. An expression cassette comprising a promoter operably linked to the nucleic acid molecule of claim 35 or a vector comprising the nucleic acid molecule.

39. A plant or plant cell comprising the nucleic acid molecule of claim 35 or the expression cassette or vector of claim 38.

40. A polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence set forth in SEQ ID NO: 8, 10, or 12; and (b) the amino acid sequenced encoded the nucleotide sequence set forth in SEQ ID NO: 7, 9, or 11.

41. A plant or plant cell comprising the polypeptide of claim 40. 105735300.1 - 50 - 070294.0229

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