WO2025019221A1

WO2025019221A1 - Broad-spectrum polerovirus resistance gene

Info

Publication number: WO2025019221A1
Application number: PCT/US2024/037280
Authority: WO
Inventors: Robert Peter James HEAL; Jonathan D.G. Jones; Kamil WITEK
Original assignee: Two Blades Foundation
Current assignee: Two Blades Foundation
Priority date: 2023-07-15
Filing date: 2024-07-10
Publication date: 2025-01-23
Anticipated expiration: 2026-01-15

Abstract

Compositions and methods for enhancing the resistance of plants to plant diseases caused by poleroviruses are provided. The compositions comprise nucleic acid molecules encoding Rl_adg resistance (R) gene products and variants thereof and plants, seeds, and plant cells comprising such nucleic acid molecules. The methods for enhancing the resistance of a plant to plant disease caused by a polerovirus comprise introducing a nucleic acid molecule encoding an R gene product into a plant cell. Additionally provided are methods for using the plants in agriculture to limit plant disease.

Description

BROAD-SPECTRUM POLEROVIRUS RESISTANCE GENE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/513,869 filed July 15, 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 (070294-0228SEQLST.xml;

Size: 24,254 bytes; and Date of Creation: July 9, 2024) is herein incorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to the fields of gene isolation and plant improvement, particularly to enhancing the resistance of plants to plant diseases through the use of disease resistance genes.

BACKGROUND OF THE INVENTION

[0004] The Polerovirus genus of plant viruses infect many important crop species. Among these viruses are, Turnip yellows virus (TuYV), beet mild yellowing virus (BMYV) and Pepper vein yellows virus (PeVYV) which infect oilseed rape, sugar beet, and pepper respectively. The polerovirus type species is Potato leafroll virus (PLRV) which infects potato (Solanum tuberosum). PLRV is among the most economically important viruses of potato. Infection affects tuber quality and can cause yield loss between 50% and 80% (Kondrak et al., 2020, PloS one 15, e0224534-e0224534; Cameiro et al., 2017, Crop Breeding and Applied Biotechnology 17:242-249). Coinfection with other viruses can increase the severity of infection, with greater viral titre and more severe symptoms being observed (Hameed et al., 2014, Plant Pathol J. 30; 407-15; Jingwei et al., 2013, Plant Cell. Tissue and Organ Culture 114:313-324). The vegetative propagation of tubers means viral resistance in potato is more important than other crop species, viral titres in seed potatoes are tightly regulated (Cameiro et al., 2017, Crop Breeding and Applied Biotechnology 17:242- 249; Mondal et al.. 2017, Plant Dis. 101 : 1812-1818).

[0005] Currently, most poleroviruses are managed with broad-spectrum insecticides targeting the aphid vector, Myzus persicae. The effectiveness of this control is limited by the development of insecticide resistance. The impact of M. persicae vectored viruses is likely to be exacerbated in a warming climate. Warmer winters have been linked to early flight and may result in the build-up of aphid populations on alternative hosts before transmission to crops (Hemming et al., 2022, J. Econ. Entomol. 1 15: 1342-1349). Prevalence of aphid borne viruses will be affected by greater regulation of insecticides. Neonicotinoids, one of the most effective insecticide classes, have been restricted due to their adverse effects on pollinating insects (Bass and Field, 2018, Curr. Biol. 28:R772-R773). Elevating plant immunity could provide an alternative, chemical free method to reducing the impact of plant viruses.

[0006] Intracellular recognition of pathogens by plants is mediated by the nucleotide binding leucine rich repeat (NLR) class of proteins, one product of resistance (R) genes. Plant NLRs can be divided into three groups based on their N-terminal domain, 'Toll, interleukin-1 receptor’ (TIR-NLR), ‘Coiled-coil’ (CC-NLR) or 'Resistance to powdery mildew 8’ (RPW8- NLR). All three groups also include a central NB-ARC domain and a C-terminal LRR domain. Several R genes which confer resistance to plant viruses have been cloned, many encode NLR proteins. R genes conferring resistance to Potato virus X (Rx-1, Rx-2) and Potato virus Y (Rysto, Ny-1) have been positionally mapped and cloned. No R genes conferring resistance to PLRV have been reported as cloned. Most instances of reported PLRV resistance in potato have drawbacks, being linked to unwanted traits, or having complex genetic basis and multiple loci responsible for resistance (Marczewski et al., 2004, Theor. Appl. Genet. 109: 1604-9; Barker etal., 1994, Theor. Appl. Genet. 88:754-8). Resistance to both TuYV and PLRV was observed in Nicotiana glutinosa and elicited by the P0 suppressor of RNA silencing. This gene, named RPO1 (Resistance to Poleroviruses 1) w as found to be simply inherited in a 3: 1 ratio. However, the gene underlying this resistance has not yet been published (Wang et al., 2015, Mol. Plant Pathol. 16:435-448; Wang et al., 2023, Virology 578:24-34).

[0007] Mihovilovich et al. (2007, Crop Sci. 47: 1091-1103) discovered that three S. tuberosum ssp. andigena landraces have high levels of heritable PLRV resistance; LOP-868, HUA-332 and OCH-7643. This resistance is strong and even upon graft inoculation plants had low viral titre relative to other accessions. LOP-868 was further characterised by Velasquez et al. (2007, Theor. Appl. Genet. 114: 1051-8), who found that a single major J? gene is responsible. This gene, designated Rladg. maps to the long arm of chromosome 5. Annotation of the S. tuberosum genome shows the Rladg locus contains many NLR encoding genes (Jupe et al., 2012, BMC Genomics 13:75; Kuang et al., 2005, Plant J. 44:37-51). Whilst Rladg is present in S. tuberosum germplasm, its use is largely restricted by complex tetrapioid genetics. Cameiro et al. (2017, Crop Breeding and Applied Biotechnology 17:242- 249) crossed LOP-868 with several different potato clones, resulting progeny had large variation in yield tuber size and poor appearance. Further genetics could be performed to transfer Rl_ad_g and eliminate drag of unwanted traits. This would be simplified by the identification of Rladg, and the development of a perfect marker.

SUMMARY OF THE INVENTION

[0008] The present invention provides isolated nucleic acid molecules of resistance (R) genes that are capable of conferring to a plant resistance to at least one polerovirus that is known to cause a plant disease in the plant. In certain embodiments of the invention, the nucleic acid molecules of R genes are capable of conferring to a plant, resistance to at least one polerovirus that is known to cause a plant disease in the plant. Preferably, such nucleic acid molecules comprise R genes that provide broad-spectrum resistance against two, three, four, five or more species of poleroviruses. Such poleroviruses can cause plant disease in the same or different plant species. In one embodiment, the present invention provides nucleic acid molecules of Rladg, an R gene against potato leafroller virus (PLRV) that was isolated from potato (Solanum tuberosum), and its variants including, for example, orthologs, and other naturally and non-naturally occurring variants of Rladg.

[0009] The present invention further provides plants, plant cells, and seeds comprising in their genomes one or more heterologous polynucleotides of the invention. The heterologous polynucleotides comprise a nucleotide sequence encoding a resistance (R) protein of the present invention. Such R proteins are encoded by the R genes of the present invention, particularly Rladg, its orthologs and other naturally and non-naturally occurring variants. In a preferred embodiment, the plants and seeds are transgenic plants and seeds that have been transformed with one or more heterologous polynucleotides of the invention. Preferably, such plants comprise enhanced resistance to at least one polerovirus that is known to cause a plant disease in a plant, when compared to the resistance of a control plant that does not comprise the heterologous polynucleotide. [0010] The present invention provides methods for enhancing the resistance of a plant to at least one polerovirus that is known to cause a plant disease in the plant. In certain embodiments of the invention, the methods involve enhancing the resistance of a plant, to a plant disease caused by at least one polerovirus. Such methods comprise introducing into at least one plant cell a heterologous polynucleotide comprising a nucleotide sequence of an R gene of the present invention. Preferably, the heterologous polynucleotide or part thereof is stably incorporated into the genome of the plant cell. The methods can optionally further comprise regenerating the plant cell into a plant that comprises in its genome the heterologous polynucleotide. Preferably, such a plant comprises enhanced resistance to a plant disease caused by at least one polerovirus, relative to the resistance of a control plant not comprising the heterologous polynucleotide.

[0011] The present invention additionally provides methods for identifying a plant that comprises an R gene for a plant disease caused by at least one polerovirus. The methods comprise detecting in the plant the presence of an Rladg nucleotide sequence of the present invention.

[0012] Methods of using the plants of the present invention in agricultural crop production to limit plant disease caused by at least one polerovirus are also provided. The methods comprise planting a plant (e.g. a seedling) or a seed of the present invention, wherein the plant, tuber, or seed comprises at least one R gene nucleotide sequence of the present invention. The methods further comprise growing a plant under conditions favorable for the growth and development of the plant, and optionally harvesting at least one fruit, tuber, leaf, or seed from the plant.

[0013] Additionally provided are plants, plant parts, seeds, plant cells, other host cells, expression cassettes, and vectors comprising one or more of the nucleic acid molecules of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1. Expression of the PLRV P1-P2 region activates a hypersensitive response (HR) in LOP-868, which carries Rladg. To identify the protein recognized by the PLRV resistance gene Rladg., PLRV open reading frames (ORFs) were cloned into binary vector pICSLUS00040D (35S promoter, Ocs terminator) and agroinfiltrated into LOP-868 (carries Rladg) and the PLRV susceptible cultivar Maris piper. No HR was observed when the movement protein (MP). RNA silencing suppressor (P0) or coat protein (CP) were expressed in either accession. LOP-868 specific HR was observed when the P1-P2 PLRV genomic region was expressed. Codelivery of 35S:Rpi-amr3 and 35S:Avramr3 was used as a control for agroinfiltration and cell death. Constructs were transformed into agrobacterium strain Agll. Agrobacterium was infiltrated at 0.1 OD. Images were taken at 3 days post infiltration. [0015] FIG. 2. Rladg is encoded by a TIR-NLR with homology' to Bs4. Rladg was previously mapped to the R1 and Bs4 gene clusters, NLRs from these clusters were cloned into the pICSLUS00040D binary vector under control of a 35S promoter and Ocs terminator. Candidates were tested by agroinfiltration into N. tabacum. Upon co-delivery with the P1-P2 region of PLRV, a single NLR (Rladg) was found to confer cell death. No Rladg induced cell death was observed upon coexpression with 35S:Avramr3. Another cloned Bs4 homologue (NLR 153 1) does not confer recognition of PLRV P1-P2 indicating cell death is specific to Rladg. No tested R1 homologues caused cell death (not shown). Constructs were transformed into agrobacterium strain Agll. Agrobacterium was infiltrated at 0.5 OD. Images were taken at 3 days post infiltration.

[0016] FIG. 3. Rladg functions through recognition of PLRV Pl, specifically the protease domain. Initial screening of Rladg candidates was performed using the P1-P2 genomic region. This region encodes three proteins; Rapl, Pl and a P1-P2 protein produced by a -1 frameshift from the Pl ORF to the P2 ORF. Truncation to remove most of the P2 ORF and mutate the ATG codon ( Rapl demonstrates that Pl is recognized by Rladg. Pl self-cleaves by serine protease action to produce three proteins, an N-terminal domain, a serine protease and a C- terminal domain. The protease domain of Pl alone is sufficient to activate Rladg and cause a HR. Constructs were transformed into agrobacterium strain Agll. Agrobacterium was infiltrated at 0.5 OD. Images were taken at 3 days post infiltration.

[0017] FIG. 4. Rladg confers recognition of PLRV -protease in transgenic S. tuberosum. To test whether Rladg is sufficient to confer recognition of PLRV Pl protease, stable transgenic S. tuberosum lines expressing Rladg under either 35 S promoter and Ocs terminator or its native regulatory elements were generated. Native regulated Rladg was defined as from 1.6 Kb prior to the start codon to 648 bp after the stop codon. For each construct two independent transgenic events are show n. A lack of HR indicates that wild-type (WT) Maris piper has no recognition of PLRV Pl protease. Transgenic lines expressing Rladg show strong cell death in response to Pl -protease expression. Constructs were transformed into agrobacterium strain Agll. Agrobacterium was infiltrated at 0. 1 OD. Images were taken at 2 days post infiltration. [0018] FIG. 5. All poleroviruses contain a Pl -protease orthologue. Neighbor joining tree produced using amino acid sequences of Polerovirus protease domains, sequences were extracted from published genomes and the protease domain identified using the boundaries defined within the PLRV genome. Pl protease from three Enamoviruses; Gravevine enamovirus-1, Citrus vein enation virus and Pea enation mosaic virus-1 are included as an outgroup.

[0019] FIG. 6. In transient assays Rladg recognizes the serine proteases from multiple poleroviruses. The proteases from 9 additional poleroviruses were synthesized and tested for recognition by Rladg. Upon coexpression with the NLR, cell death was observed for all these proteases. Coexpression of the proteases with the non-functional Rladg paralogue NLR_153_1 resulted in no visible cell death. Constructs were transformed into agrobacterium strain Agll. Agrobacterium was infiltrated at 0.5 OD. Images were taken at 4 days post infiltration.

[0020] FIG. 7. Rladg confers recognition of PLRV -protease in transgenic N. tabacum. To test for resistance to different poleroviruses, N. tabacum were stably transformed with Rladg under the control of an operably linked CaMV35S promoter and an operably linked Ocs terminator. To confirm Rladg function, these lines were initially tested for recognition of PLRV protease. Progeny from two independent transgenic events are shown. WT N. tabacum has no recognition of PLRV Pl protease. Transgenic lines expressing Rladg show strong cell death in response to Pl -protease expression. Constructs were transformed into agrobacterium strain Agll. Agrobacterium was infiltrated at 0.5 OD. Images were taken at 2 days post infiltration.

[0021] FIG. 8. Transgenic lines carrying Rladg activate HR upon infection with PLRV and TuYV. To test for recognition and resistance to poleroviruses we used infectious clones of PLRV and TuYV delivered by Agrobacterium. Upon whole leaf infiltration of WT N tabacum, no response w as observed for either virus. Progeny from two independent transgenic events expressing Rladg under 35 S promoter and Ocs terminator activate HR against both PLRV and TuYV. Constructs were transformed into agrobacterium strain Agll. Agrobacterium was infiltrated at 0.5 OD. Pictures w ere taken 10 days after agroinfiltration.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0022] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and one 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 (z.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.

[0023] SEQ ID NO: 1 sets forth the nucleotide sequence of the R gene. Rladg from Solanum tuberosum.

[0024] SEQ ID NO: 2 sets forth the amino acid sequence Rladg, the R protein encoded by Rl_ad_g (SEQ ID NO: 1).

[0025] SEQ ID NO: 3 sets forth the nucleotide sequence of the coding region of the cDNA of Rladg (SEQ ID NO: 1). SEQ ID NO: 3 encodes the amino acid sequence set forth in SEQ ID NO: 2. If desired, a stop codon (e.g. TAA, TAG, TGA) can be operably linked to the 3' end of nucleic acid molecule comprising SEQ ID NO: 3. The native stop codon of this cDNA is TAA.

[0026] SEQ ID NO: 4 sets forth the nucleotide sequence of the Rladg polynucleotide construct used for the stable transformation of plants as described below in the Examples. This polynucleotide construct is a modified version of Rladg (SEQ ID NO: 1) in which the third intron was truncated from 2.23 kilobase pairs (kbp) to 349 base pairs (bp). SEQ ID NO: 5 encodes the amino acid sequence set forth in SEQ ID NO: 2.

DETAILED DESCRIPTION OF THE INVENTION

[0027] 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.

[0028] 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.

[0029] The present invention relates to the isolation of plant resistance (R) genes, particularly R genes that confer upon a plant resistance to plant disease(s) caused by one or more poleroviruses. As disclosed hereinbelow, an R gene, Rladg, an R gene that is known to confer to potato plants resistance to potato leafroller virus (PLRV), was isolated from LOP- 86 (CIP-Number 702853). a cultivar of Solatium tuberosum subsp. andigena that is known to be highly resistant to PLRV (Mihovilovich et al. (2007) Crop Set. 47: 1091-1103). S. tuberosum subsp. andigena is a native cultivated species that is genetically similar to commercial potato ( . tuberosum L. subsp. tuberosum). Id.

[0030] Rladg encodes an TIR-NB-LRR protein which functions through recognition of the PLRV Pl protease which is required for Pl maturation and whose function was previously shown to be required for PLRV replication. PLRV Pl is a polyprotein which cleaves itself through the action of a central serine protease domain. Cleavage products include an N- terminal protein, the protease and the C-terminal Vpg. As disclosed hereinbelow in the Examples, the dissection of the elicitor showed that the serine protease domain of Pl is sufficient for recognition by Rladg, co-expression with either of the tw o other products of processed Pl does not activate HR.

[0031] Moreover, the Pl -protease structure is largely conserved within the poleroviridae leading to the hypothesis that Rladg may be able to recognize the Pl protease of other poleroviruses including poleroviruses that are pathogens of species other than potato. Indeed, the present inventors disclose hereinbelow in the Examples that RLd_g can recognize not only the PLRV P 1 protease but also many other poleroviruses that are known to be pathogens of dicotyledonous and monocotyledonous plant species, indicating that Rladg is capable of conferring broad-spectrum resistance against multiple poleroviruses species that are pathogens of dicotyledonous and monocotyledonous plant species. Accordingly, the present invention provides methods and compositions that find in use in the engineering of plants, particularly crop plants, for the enhanced resistance to poleroviruses.

[0032] The present invention provides nucleic acid molecules comprising the nucleotide sequences of Rladg, particularly the nucleotide sequences of Rladg and other naturally occurring (e.g. orthologs and allelic variants) and synthetic or artificial (i.e. non-naturally occurring) variants thereof. As used herein, such nucleic acid molecules are referred to herein as “Rladg nucleic acid molecules”. Likewise, the nucleotide sequences of Rladg and other naturally occurring (e.g. orthologs and allelic variants) and synthetic or artificial (i.e. non-naturally occurring) variants thereof are referred to herein as “Rladg nucleotide sequences”. It is recognized that as used herein, the term “R genes of the present invention” encompasses the Rladg nucleic acid molecules described above.

[0033] The Rladg nucleotide sequences of the present invention are nucleotide sequences of R genes, which are also referred to herein as R gene nucleotide sequences. Preferably, such nucleotide sequences of R genes encode R proteins. Rl dg nucleotide sequences include, but not limited to: the nucleotide sequences of the wild-type or native Rladg gene comprising a native promoter and the native 3' adjacent region comprising the coding region; cDNA sequences; and nucleotide sequences comprising only the coding region. Examples of such Rladg nucleotide sequences include the nucleotide sequences set forth in SEQ ID NOS: 1. 3, and 4, and variants thereof. In embodiments in which the native Rladg gene promoter is not used to drive the expression of the nucleotide sequence encoding the R protein, a heterologous promoter can be operably linked a nucleotide sequence encoding an R protein of the invention to drive the expression of nucleotide sequence encoding an R protein in a plant.

[0034] Preferably, the Rladg nucleic acid molecules of the present invention are capable of conferring to a plant enhanced resistance to a plant disease caused by at least one polerovirus and include, for example, the Rladg nucleic acid molecules comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, and 4. More preferably, such the Rladg nucleic acid molecules are capable of conferring to a plant enhanced resistance to plant disease caused by multiple strains of a polerovirus. As used herein, “multiple strains of a polerovirus” is intended to mean at least two strains, but preferrable at least three, four, five, six, or more stains, of a particular polerovirus species.

[0035] In certain preferred embodiments, the Rladg nucleic acid molecules of the present invention comprise broad-spectrum resistance to multiple poleroviruses and include, for example, the Rladg nucleic acid molecules comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, and 4. In certain other preferred embodiments, such Rladg nucleic acid molecules of the present invention comprise broad-spectrum resistance to multiple strains of multiple poleroviruses.

[0036] Preferably, the R proteins encoded by the Rladg of the present invention are functional R proteins, or part(s), or domain(s) thereof, which are capable of conferring to a plant. comprising the R protein, enhanced resistance to a plant disease caused by at least one polerovirus.

[0037] In certain preferred embodiments, the R proteins of the present invention comprise broad-spectrum resistance to multiple poleroviruses and include, for example, RLdg (SEQ ID NO: 2). Such an R protein is encoded by the Rladg nucleotide sequences set forth in SEQ ID NOS: 1, 3, and 4 and variants thereof. In certain other preferred embodiments, such R proteins of the present invention comprise broad-spectrum resistance to multiple strains of multiple poleroviruses.

[0038] The present invention further provides plants comprising a heterologous polynucleotide which comprises an R gene nucleotide sequence of the present invention. Preferably, such an R gene nucleotide sequence encodes a full-length R protein of the present invention, or at least a functional part(s) or domain(s) thereof. In some embodiments, such a heterologous polynucleotide of the present invention is stably incorporated into the genome of the plant, and in other embodiments, the plant is transformed by a transient transformation method and the heterologous polynucleotide is not stably incorporated into the genome of the plant.

[0039] In other embodiments, a plant comprising a heterologous polynucleotide which comprises an R gene nucleotide sequence of the present invention is produced using a method of the present invention that involves genome editing to modify the nucleotide sequence of a native or non-native gene in the genome of the plant. The native or non-native gene comprises a nucleotide sequence that is different from (i.e. not identical to) an R gene nucleotide sequence of the present invention, and after modification by methods disclosed in further detail hereinbelow, the modified native or non-native gene comprises an R gene nucleotide sequence of the present invention. Generally, such methods comprise the use of a plant comprising in its genome a native or non-native gene wherein the native or non-native gene comprises a nucleotide sequence that is homologous to an R gene nucleotide sequence of the present invention and further comprises introducing into the plant a nucleic acid molecule comprising at least part of an R gene nucleotide sequence of the present invention. Preferably, a nucleotide sequence of native or non-native gene comprises about 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater nucleotide sequence identity to at least one R gene nucleotide sequence of the present invention. Such a native or non-native gene can be, for example an R gene, or a non-functional homolog of such an R gene that is not, or is not known to be, capable of conferring to a plant, resistance to a plant disease. It is recognized that a plant produced by genome engineering as disclosed herein is a stably transformed plant when the native or non-native gene that is modified is stably incorporated in the genome of the plant.

[0040] Methods for both the stable and transient transformation of plants and genome editing are disclosed elsewhere herein or otherwise known in the art. In one embodiment of the invention, the plants are stably transformed plants comprising a heterologous polynucleotide of the present invention stably incorporated into their respective genomes and further comprising enhanced resistance to plant disease caused by at least one polerovirus. In another embodiment of the invention, the plants are stably transformed plants comprising a heterologous polynucleotide of the present invention stably incorporated into their respective genomes and further comprising enhanced resistance to plant disease caused by at least two, three, or more poleroviruses.

[0041] In certain embodiments, a plant of the invention comprises a heterologous polynucleotide which comprises a nucleotide sequence encoding an R protein of the present invention and a heterologous promoter that is operably linked for expression of the nucleotide sequence encoding an R protein. The choice of heterologous promoter can depend on a number of factors such as, for example, the desired timing, localization, and pattern of expression as w ell as responsiveness to particular biotic or abiotic stimulus. Promoters of interest include, but are not limited to, pathogen-inducible, constitutive, tissue-preferred, wound-inducible, and chemical-regulated promoters.

[0042] The present invention further provides methods for enhancing the resistance of a plant to a plant disease caused by at least one polerovirus, particularly at least one polerovirus. The methods comprise modifying at least one plant cell to comprise a heterologous polynucleotide, and optionally regenerating a plant from the modified plant comprising the heterologous polynucleotide. In a first aspect, the methods for enhancing the resistance of a plant to a plant disease caused by at least one polerovirus comprise introducing a heterologous polynucleotide of the invention into at least one plant cell, particularly a plant cell from a plant. In certain embodiments, the heterologous polynucleotide is stably incorporated into the genome of the plant cell.

[0043] In a second aspect, the methods for enhancing the resistance of a plant to a plant disease caused by at least one polerovirus involve the use of a genome-editing method to modify the nucleotide sequences of a native or non-native gene in the genome of the plant cell to comprise a heterologous polynucleotide of the present invention. The methods comprise introducing a nucleic acid molecule into the plant cell, wherein the nucleic acid molecule comprises a nucleotide sequence comprising at least a part of the R gene nucleotide sequence of the present invention and wherein at least a part of the nucleotide sequence of the native or non-native gene is replaced with at least a part of the nucleotide sequence of the nucleic acid molecule. Thus, the methods of the invention involve gene replacement to produce a heterologous polynucleotide of the present invention in the genome of a plant cell. [0044] If desired, the methods of the first and/or second aspect(s) can further comprise regenerating the plant cell into a plant comprising in its genome the heterologous polynucleotide. Preferably, such a regenerated plant comprises enhanced resistance to a plant disease caused by at least one polerovirus. at least one polerovirus, relative to the resistance of a control plant to the plant disease or diseases caused by the same polerovirus(es).

[0045] The plants disclosed herein find use in methods for limiting plant disease caused by at least one polerovirus in agricultural crop production, particularly in regions where such a plant disease is prevalent and is known to negatively impact, or at least has the potential to negatively impact, agricultural yield. The methods of the invention comprise planting a plant (e.g. a seedling), tuber, or seed of the present invention, wherein the plant, tuber, or seed comprises at least one R gene nucleotide sequence of the present invention. The methods further comprise growing the plant that is derived from the seedling, tuber, or seed under conditions favorable for the growth and development of the plant, and optionally harvesting at least one fruit, tuber, leaf, or seed from the plant.

[0046] The present invention additionally provides methods for identifying a plant that comprises an R gene for a plant disease caused by at least one polerovirus. The methods find use in breeding plants for resistance to plant diseases caused by poleroviruses. Such resistant plants find use in the agricultural production of fruits, tubers, leaves, and/or seeds for human or livestock consumption or other use. The methods comprise detecting in a plant, or in at least one part or cell thereof, the presence of a Rladg nucleotide sequence of the present invention. In some embodiments of the invention, detecting the presence of the Rladg nucleotide sequence comprises detecting the entire Rladg nucleotide sequence in genomic DNA isolated from a plant. In preferred embodiments, however, detecting the presence of a Rladg nucleotide sequence comprises detecting the presence of at least one marker within the Rl dg nucleotide sequence, respectively. In other embodiments of the invention, detecting the presence of a Rladg nucleotide sequence comprises detecting the presence of either one. or both, of the R proteins encoded by the Rladg nucleotide sequence using, for example, immunological detection methods involving an antibody preparation specific to.

[0047] In the methods for identifying a plant that comprises an R gene for a plant disease caused by at least one polerovirus, detecting the presence of the Rladg nucleotide sequence in the plant can involve one or more of the following molecular biology techniques that are disclosed elsewhere herein or otherwise known in the art including, but not limited to, isolating genomic DNA and/or RNA from the plant, amplifying nucleic acid molecules comprising the Rl dg nucleotide sequence and/or marker(s) therein by PCR amplification, sequencing nucleic acid molecules comprising the Rladg nucleotide sequence and/or marker(s), identifying the Rladg nucleotide sequence, the marker(s), or a transcript or transcripts of the Rladg nucleotide sequence by nucleic acid hybridization, and conducting an immunological assay for the detection of the R protein(s) encoded by the Rladg nucleotide sequence. It is recognized that oligonucleotide probes and PCR primers can be designed to identity the Rladg nucleotide sequence of the present invention and that such probes and PCR primers can be utilized in methods disclosed elsewhere herein or otherwise known in the art to rapidly identify in a population of plants one or more plants comprising the presence of an Rladg nucleotide sequence of the present invention.

[0048] Depending on the desired outcome, the heterologous polynucleotides of the invention can be stably incorporated into the genome of the plant cell or not stably incorporated into genome of the plant cell. If. for example, the desired outcome is to produce a stably transformed plant with enhanced resistance to a plant disease caused by at least one polerovirus, then the heterologous polynucleotide can be, for example, fused into a plant transformation vector suitable for the stable incorporation of the heterologous polynucleotide into the genome of the plant cell. Typically, the stably transformed plant cell will be regenerated into a transformed plant that comprises in its genome the heterologous polynucleotide. Such a stably transformed plant is capable of transmitting the heterologous polynucleotide to progeny plants in subsequent generations via sexual and/or asexual reproduction. Plant transformation vectors, methods for stably transforming plants with an introduced heterologous polynucleotide and methods for plant regeneration from transformed plant cells and tissues are generally known in the art for both monocotyledonous and dicotyledonous plants or described elsewhere herein.

[0049] In other embodiments of the invention in which it is not desired to stably incorporate the heterologous polynucleotide in the genome of the plant, transient transformation methods can be utilized to introduce the heterologous polynucleotide into one or more plant cells of a plant. Such transient transformation methods include, for example, viral-based methods which involve the use of viral particles or at least viral nucleic acids. Generally, such viral-based methods involve constructing a modified viral nucleic acid comprising a heterologous polynucleotide of the invention operably linked to the viral nucleic acid and then contacting the plant either with a modified virus comprising the modified viral nucleic acid or with the viral nucleic acid or with the modified viral nucleic acid itself. The modified virus and/or modified viral nucleic acids can be applied to the plant or part thereof, for example, in accordance with conventional methods used in agriculture, for example, byspraying, irrigation, dusting, or the like. The modified virus and/or modified viral nucleic acids can be applied in the form of directly sprayable solutions, powders, suspensions or dispersions, emulsions, oil dispersions, pastes, dustable products, materials for spreading, or granules, by means of spraying, atomizing, dusting, spreading or pouring. It is recognized that it may be desirable to prepare formulations comprising the modified virus and/or modified viral nucleic acids before applying to the plant or part or parts thereof. Methods for making pesticidal formulations are generally known in the art or described elsewhere herein. [0050] The present invention provides nucleic acid molecules comprising Rladg nucleotide sequences. Preferably, such nucleic acid molecules are capable of conferring upon a host plant enhanced resistance to a plant disease caused by at least one polerovirus. Thus, such nucleic acid molecules find use in limiting a plant disease caused by a polerovirus in agricultural production. The nucleic acid molecules of the present invention include, but are not limited to, nucleic acid molecules comprising at least one of the Rladg nucleotide sequence disclosed herein but also additional orthologs and other variants of the Rladg nucleotide sequences that are capable of conferring to a plant resistance to a plant disease caused by at least one polerovirus. Methods are known in the art or otherwise disclosed herein for determining resistance of a plant to a plant disease caused by at least one polerovirus including, for example, the assays described hereinbelow.

[0051] Additionally provided are methods for introducing Rladg into a plant lacking in its genome Rladg (SEQ ID NO: 1, 3. or 4). The methods comprise crossing (i.e. crosspollinating) a first plant comprising in its genome at least one copy of Rladg with a second plant lacking in its genome Rladg. The first and second plants can be the same species or can be different species. Such a crossing of a first species of a plant to a second species of a plant is known as an interspecific hybridization and can be used to introgress a gene or genes of interest (e.g. Rladg) from one species into a related species lacking the gene or genes of interest and typically involves multiple generations of backcrossing of the progeny with the related species and selection at each generation of progeny comprising the gene or genes of interest. Such interspecific hybridization, introgression, and backcrossing methods are well known in the art and can be used in the methods of the present invention. See “Principals of Cultivar Development.” Fehr, 1993, Macmillan Publishing Company, New York; and “Fundamentals of Plant Genetics and Breeding.” Welsh, 1981, John Wiley & Sons, Inc.. New York.

[0052] In methods of the present invention for introducing Rladg into a plant lacking in its genome Rladg, either the first plant or the second plant can be the pollen donor plant. For example, if the first plant is the pollen donor plant, then the second plant is the pollenrecipient plant. Likewise, if the second plant is the pollen donor plant, then the first plant is the pollen-recipient plant. Following the crossing, the pollen-recipient plant is grow n under conditions favorable for the grow th and development of the plant and for a sufficient period of time for seed to mature or to achieve an otherwise desirable growth stage for use in a subsequent in vitro germination procedure such as, for example, embryo rescue that is described below. The seed can then be harvested and those seed comprising Rladg identified by any method known in the art including, for example, the methods for identifying a plant that comprises an R gene for a plant disease caused by at least one polerovirus that are described elsewhere herein.

[0053] It is recognized, however, that in certain embodiments of the invention involving interspecific hybridizations, it may be advantageous to harvest the seed resulting from such interspecific hybridizations at an immature growth stage and then to germinate the immature seeds in culture (i.e. in vitro), whereby the seeds are allowed germinate in culture using methods known in art as “embryo rescue” methods. See Reed (2005) “Embryo Rescue,” in Plant Development and Biotechnology, Trigiano and Gray, eds. CRC Press, Boca Raton, pp. 235-239; and Sharma et al. (1996) Euphytica 89: 325-337. It is further recognized that “embryo rescue methods are typically used when mature seeds produced by an interspecific cross display little or no germination, whereby few or no interspecific hybrid plants are produced.

[0054] The methods of the present invention find use in producing plants with enhanced resistance to a plant disease caused by at least one polerovirus. Typically , the methods of the present invention will enhance or increase the resistance of the subject plant to the plant disease by at least 25%, 50%, 75%, 100%, 150%, 200%, 250%, 500% or more when compared to the resistance of a control plant to the same polerovirus(es). Unless stated otherwise or apparent from the context of a use, a control plant for the present invention is a plant that does not comprise the heterologous polynucleotide, particularly a Rladg nucleotide sequence of the present invention. Preferably, the control plant is essentially identical (e.g. same species, subspecies, and variety) to the plant comprising the heterologous polynucleotide of the present invention except the control does not comprise the heterologous polynucleotide. In some embodiments, the control will comprise a heterologous, control polynucleotide (e.g. vector control) that does comprise the Rladg nucleotide sequence that is in a heterologous polynucleotide of the present invention.

[0055] Additionally, the present invention provides transformed plants, seeds, and plant cells produced by the methods of present invention and/or comprising a heterologous polynucleotide of the present invention. Also provided are progeny plants and seeds thereof comprising a heterologous polynucleotide of the present invention. The present invention also provides fruits, seeds, tubers, leaves, stems, roots, 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 comprising, or produced or derived from, the plants or any part or parts thereof including, but not limited to, fruits, tubers, leaves, stems, roots, and seed. Other agricultural products include, for example, smoking products produced from tobacco leaves (e.g.. cigarettes, cigars, and pipe and chewing tobacco) and food and industrial starch products produced from potato tubers. It is recognized that such food products can be consumed or used by humans and other animals including, but not limited to, pets (e.g., dogs and cats), livestock (e.g., pigs, cows, chickens, turkeys, and ducks), and animals produced in freshwater and marine aquaculture systems (e.g. fish, shrimp, prawns, crayfish, and lobsters). [0056] Non-limiting examples of the compositions and methods of the present invention are as follows:

[0057] 1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;

(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, and optionally, wherein the nucleotide sequence is not naturally occurring; (c) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, and 4. wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule and optionally, wherein the nucleotide sequence is not naturally occurring; and

(d) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 2, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule and optionally, wherein the nucleotide sequence is not naturally occurring.

[0058] 2. An expression cassette or vector comprising the nucleic acid molecule of embodiment 1.

[0059] 3. A host cell transformed with the nucleic acid molecule of embodiment 1 or the expression cassette or vector of embodiment 2.

[0060] 4. A plant or plant cell comprising the nucleic acid molecule of embodiment 1 or the expression cassette or vector of embodiment 2.

[0061] 5. The host cell of embodiment 3. or the plant or plant cell of embodiment 4, wherein the host cell is not a Solatium tuberosum cell, the plant is not a Solarium tuberosum plant and the plant cell is not a Solatium tuberosum plant cell.

[0062] 6. A plant or plant cell comprising stably incorporated in its genome a heterologous polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;

(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2;

(c) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, and 4, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule; and (d) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 2, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule.

[0063] 7. The plant or plant cell of embodiment 6. wherein the plant is not Solanum tuberosum and the plant cell is not a Solanum tuberosum plant cell.

[0064] 8. The plant or plant cell of embodiment 6 or 7, wherein the heterologous polynucleotide comprises the nucleotide sequence of any one of (a)-(d) and further comprises a promoter operably linked for the expression of the nucleotide sequence in a plant.

[0065] 9. The plant or plant cell of any one of embodiments 6-8, wherein the polerovirus is selected from the group consisting of potato leafroller virus (PLRV), chickpea chlorotic stunt virus (CchSV), wheat yellow dwarf virus (WYDV), tobacco virus 2 (TV2), cotton leafroller dwarf virus (CLRDV). pepper vein yellows virus (PeVYV), beet mild yellowing virus (BMYV), cucurbit aphid-bome yellows virus (CaBYV), maize yellow dwarf virus (MYDV), and turnip yellows virus (TuYV).

[0066] 10. The plant or plant cell of any one of embodiments 6-9, wherein the plant or plant cell comprises enhanced resistance to a plant disease caused by at least one polerovirus, relative to the resistance of a control plant.

[0067] 11 . A method for enhancing the resistance of a plant to a plant disease caused by at least one polerovirus, the method comprising introducing at least one plant cell a heterologous polynucleotide, the heterologous polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;

(c) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: I, 3, and 4. wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule; and (d) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 2, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule.

[0068] 12. The method of embodiment 11, wherein the plant is not Solarium tuberosum and the plant cell is not a Solanum tuberosum plant cell.

[0069] 13. The method of embodiment 11 or 12, wherein the heterologous polynucleotide is stably incorporated into the genome of the plant cell.

[0070] 14. The method of any one of embodiments 11-13, wherein the plant cell is regenerated into a plant comprising in its genome the heterologous polynucleotide.

[0071] 15. The method of any one of embodiments 11-14, wherein the heterologous polynucleotide comprises the nucleotide sequence of any one of (a)-(d) and further comprises a promoter operably linked for the expression of the nucleotide sequence in a plant.

[0072] 16. The method of any one of embodiments 11-15, wherein the plant comprising the heterologous polynucleotide comprises enhanced resistance to a plant disease caused by at least one polerovirus, relative to the resistance of a control plant.

[0073] 17. The method of any one of embodiments 11-16, wherein the polerovirus is selected from the group consisting of potato leafroller virus (PLRV), chickpea chlorotic stunt virus (CchSV), wheat yellow dwarf virus (WYDV), tobacco virus 2 (TV2), cotton leafroller dwarf virus (CLRDV), pepper vein yellows virus (PeVYV), beet mild yellowing virus (BMYV), cucurbit aphid-bome yellows virus (CaBYV), maize yellow dwarf virus (MYDV), and turnip yellows virus (TuYV).

[0074] 18. A plant producible by the method of any one of embodiments 11-17.

[0075] 19. A fruit, leaf, root, or seed of the plant of any one of embodiments 4-10 and 18, wherein the fruit, leaf, root, or seed comprises the heterologous polynucleotide.

[0076] 20. A method of limiting a plant disease caused by at least one polerovirus in agricultural crop production, the method comprising planting a seedling or seed of the plant of any one of embodiments 4-10 and 18 and growing the seedling or seed under conditions favorable for the growth and development of a plant resulting therefrom, wherein the seedling or seed comprises the nucleic acid molecule, expression cassette, vector, or heterologous polynucleotide. [0077] 21. The method of embodiment 20, further comprising harvesting at least one seed, fruit, leaf, root, or other plant part from the plant, and optionally processing the harvested seed, fruit, leaf, root, or other plant part into a food product.

[0078] 22. A seed, fruit, leaf, root, or other plant part, or a food product, obtainable using the method of embodiment 21.

[0079] 23. A method for identifying a plant that comprises an R gene for a plant disease caused by at least one polerovirus. the method comprising detecting in the plant, or in at least one part or cell thereof, the presence of an Rladg nucleotide sequence, wherein the Rladg nucleotide sequence is selected from the group consisting of:

(a) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;

(c) a nucleotide sequence having at least 90% sequence identify to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, and 4, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus. relative to a control plant not comprising the nucleic acid molecule; and

(d) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 2, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule.

[0080] 24. The method of embodiment 23, wherein the plant is not Solanum tuberosum.

[0081] 25. The method of embodiment 23 or 24, wherein the presence of the Rladg nucleotide sequence is detected by detecting at least one marker within the Rladg nucleotide sequence.

[0082] 26. The method of any one of embodiments 23-25, wherein detecting the presence of the Rladg nucleotide sequence comprises a member selected from the group consisting of PCR amplification, nucleic acid sequencing, nucleic acid hybridization, and an immunological assay for the detection of the R protein encoded by the Rladg nucleotide sequence. [0083] 27. A method for introducing Rladg into a plant, the method comprising:

(a) crossing a first plant comprising in its genome at least one copy of an Rladg polynucleotide with a second plant lacking in its genome an Rladg polynucleotide, whereby at least one progeny plant is produced; and

(b) selecting at least one progeny plant comprising in its genome the Rladg polynucleotide by detecting in the progeny plant the presence of an Rladg polynucleotide; wherein the Rladg polynucleotide comprises a nucleotide sequence is selected from the group consisting of:

(i) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;

(ii) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2;

(ii) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, and 4. wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus. relative to a control plant not comprising the nucleic acid molecule; and (iv) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 2, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule.

[0084] The method of embodiment 27, wherein the plant is not Solanum tuberosum. [0085] A progeny plant obtainable using the method of embodiment 27 or 28.

[0086] A seed, fruit, leaf, root, or other plant part obtainable from the plant of embodiment 29.

[0087] Use of the plant, seed, fruit, leaf, root, or other plant part of any one of embodiments 4-10, 18, 22. 29. and 30 in agriculture or in production of a food product.

[0088] A human or animal food product comprising, or produced using, the plant, seed, fruit, leaf, root, or other plant part of any one of embodiments 4-10, 18, 22, 29, and 30.

[0089] An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 2;

(b) the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4; and

(c) an amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 2, wherein a polypeptide comprising the amino acid sequence is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the polypeptide.

[0090] Additional embodiments of the methods and compositions of the present invention are described elsewhere herein.

[0091] Plants of interest for the methods and compositions of the present invention include, for example, any plant that is a host to a Polerovirus. Examples of other plant species of interest for the methods and compositions of the present invention include, but are not limited to, com (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 (x Triticosecale or Triticum Secale) sorghum (Sorghum bicolor, Sorghum vulgar e), teff (Er agrost is 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 x 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 occidental), 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), spinach {Spinacia oleracea), sugarcane {Saccharum spp.), quinoa {Chenopodium quinoa), oat {Avena saliva), barley {Hordeum vulgare), cannabis {Cannabis saliva, C. indica, C. ruderalis), poplar {Populus spp ). eucalyptus {Eucalyptus spp.), Arabidopsis thaliana, Arabidopsis rhizogenes, Nicoliana 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.).

[0092] Vegetables include tomatoes {Lycopersicon esculenlum), eggplant (also know n as “aubergine” or “brinjal”) {Solanum melongena), pepper {Capsicum annuum). lettuce (e.g., Lactuca saliva), green beans {Phaseohis vulgaris), lima beans {Phaseolus limensis), peas {Lalhyrus spp.), chickpeas {Cicer arielinum), and members of the genus Cucumis such as cucumber (C salivus), 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.

[0093] The present invention provides resistance nucleic acid molecules that are capable of conferring to a plant resistance to a plant disease caused by at least one plant pathogen, plants and plants cells comprising such nucleic acid molecules and related methods. Plant pathogens include, for example, viruses, bacteria, fungi, oomycetes, nematodes, and the like. Preferred plant pathogens of the present invention are viruses, particularly viruses in the family Solemoviridae , more particularly viruses in the genus Polerovirus. Virus species of interest in the in the genus Polerovirus include, but are not limited to, beet chlorosis virus, beet mild yellowing virus, beet western yellows virus, carrot red leaf virus, cereal yellow dwarf virus RPS. cereal yellow dwarf virus RPV, chickpea chlorotic stunt virus, cotton leafroll dwarf virus, cucurbit aphid-borne yellows virus, faba bean polerovirus 1, maize yellow dwarf virus RMV, maize yellow mosaic virus, melon aphid-bome yellows virus, pepo aphid-bome mosaic vims, pepper vein yellows virus 1, pepper vein yellows virus 2, vein yellows virus 3, pepper vein yellows virus 4, pepper vein yellows vims 5, pepper vein yellows virus 6, potato leafroll virus, pumpkin polerovirus, suakwa aphid-bome yellows virus, sugarcane yellow leaf virus, tobacco vein distorting virus, and turnip yellows virus. Preferred poleroviruses include, for example, potato leafroller virus (PLRV), chickpea chlorotic stunt virus (CchSV), wheat yellow dwarf virus (WYDV), tobacco virus 2 (TV2), cotton leafroller dwarf virus (CLRDV), pepper vein yellows virus (PeVYV), beet mild yellowing virus (BMYV), cucurbit aphid-bome yellows vims (CaBYV), maize yellow dwarf vims (MYDV), and turnip yellows virus (TuYV).

[0094] In one embodiment of the invention, the nucleotide sequences encoding R proteins have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the entire nucleotide sequence set forth in SEQ ID NO: 2, or to a fragment thereof. Such fragments include, for example, those comprising or consisting of the entire nucleotide sequence set forth in SEQ ID NO: 2.

[0095] 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. 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. [0096] Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of polynucleotides comprising coding sequences may encode protein fragments that retain biological activity⁷ of the full-length or native protein. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the invention.

[0097] In certain embodiments of the invention, the fragments and variants of the disclosed polynucleotides and proteins encoded thereby are those that are capable of conferring to a plant resistance to a plant disease caused by at least one polerovirus. Preferably, a polynucleotide comprising a fragment of a native R polynucleotide of the present invention is capable of conferring resistance to a plant disease caused by at least one polerovirus to a plant comprising the polynucleotide. Likewise, a protein or polypeptide comprising a native R protein of the present invention is preferably capable of conferring resistance to a plant disease caused by at least one polerovirus.

[0098] Polynucleotides that are fragments of a native R polynucleotide comprise at least 16, 20, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500, 1000. 2000, 3000, 4000, 5000. 6000, 7000, or 8000 contiguous nucleotides, or up to the number of nucleotides present in a full-length R polynucleotide disclosed herein (for example, 8662, 3369, and 6748 nucleotides for of SEQ ID NOS: 1, 3, and 4 respectively).

[0099] “Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having deletions (i. e. , truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the R proteins of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology⁷ techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode an R protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein. In certain embodiments of the invention, vanants of a particular polynucleotide of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, and 4, and optionally comprise a non-naturally occurring nucleotide sequence that differs from the nucleotide sequence set forth in SEQ ID NO: 1, 3, and/or 4 by at least one nucleotide modification selected from the group consisting of the substitution of at least one nucleotide, the addition of at least one nucleotide, and the deletion of at least one nucleotide. It is understood that the addition of at least one nucleotide can be the addition of one or more nucleotides within a nucleotide sequence of the present invention (e.g. SEQ ID NO: 1, 3. and 4), the addition of one or more nucleotides to the 5’ end of a nucleotide sequence of the present invention, and/or the addition of one or more nucleotides to the 3’ end of a nucleotide sequence of the present invention.

[0100] Variants of a particular polynucleotide of the invention (i.e.. the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, a polynucleotide that encodes a polypeptide with a given percent sequence identity to at least one polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 is disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 60%, 65%, 70%. 75%. 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99% or more sequence identity’. In certain embodiments of the invention, variants of a particular polypeptide of the invention will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, and optionally comprises a non-naturally occurring amino acid sequence that differs from at least one amino acid sequence set forth in SEQ ID NO: 2 by at least one amino acid modification selected from the group consisting of the substitution of at least one amino acid, the addition of at least one amino acid, and the deletion of at least one amino acid. It is understood that the addition of at least one amino acid can be the addition of one or more amino acids within an amino acid sequence of the present invention (e.g. SEQ ID NO: 2), the addition of one or more amino acids to the N- terminal end of an amino acid sequence of the present invention, and/or the addition of one or more amino acids to the C-terminal end of an amino acid sequence of the present invention. [0101] “Variant’’ protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C- terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of an R protein will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein (e.g. the amino acid sequence set forth in SEQ ID NO: 2) as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10. as few as 5, as few as 4, 3. 2, or even 1 amino acid residue.

[0102] The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D C ), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal. [0103] Thus, the genes and polynucleotides of the invention include both the naturally occurring sequences as well as mutant and other variant forms. Likewise, the proteins of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. More preferably, such variants confer to a plant or part thereof comprising the variant enhanced resistance a plant disease caused by at least one polerovirus. In some embodiments, the mutations that will be made in the DNA encoding the variant will not place the sequence out of reading frame. Optimally, the mutations will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

[0104] The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity⁷ can be evaluated by assays that are disclosed herein below.

[0105] Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri el al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391 :288-291 ; and U.S. Patent Nos. 5,605,793 and 5,837,458.

[0106] The polynucleotides of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identify to the entire sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. "Orthologs” is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or greater sequence identify. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode R proteins and which hybridize under stringent conditions to at least one of the R proteins disclosed herein or otherwise known in the art, or to variants or fragments thereof, are encompassed by the present invention.

[0107] In one embodiment, the orthologs of the present invention have coding sequences comprising at least 80%, 85%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or greater nucleotide sequence identity to at least one nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, and 4 and/or encode proteins comprising least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0108] In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning 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 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.

[0109] In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as ²P. or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the polynucleotides of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries 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). [0110] For example, an entire polynucleotide disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding polynucleotide and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among the sequence of the gene or cDNA of interest sequences and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length. Such probes may be used to amplify corresponding polynucleotides for the particular gene of interest from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.. Cold Spring Harbor Laboratory Press, Plainview, New York). An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology’ — Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory' Press, Plainview, New York).

[0111] It is recognized that the R protein coding sequences of the present invention encompass polynucleotide molecules comprising a nucleotide sequence that is sufficiently identical to the nucleotide sequence of any one or more of SEQ ID NOS: 1 , 3, and 4. The term “sufficiently identical” is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g.. with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 45%, 55%, or 65% identity, preferably 75% identity, more preferably 85%. 90%. 95%. 96%. 97%. 98% or 99% identity are defined herein as sufficiently identical.

[01 12] To determine the percent identify of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identify between the two sequences is a function of the number of identical positions shared by the sequences (i.e.. percent identity = number of identical positions/total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

[0113] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Set. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the polynucleotide molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSLBlast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e g., XBLAST and NBLAST) can be used. BLAST, Gapped BLAST, and PSI-Blast, XBLAST and NBLAST are available on the World Wide Web at ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Alignment may also be performed manually by inspection.

[0114] Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the full-length sequences of the invention and using multiple alignment by mean of the algorithm Clustal W (Nucleic Acid Research, 22(22):4673-4680, 1994) using the program AlignX included in the software package Vector NTI Suite Version 7 (InforMax, Inc., Bethesda, MD, USA) using the default parameters; or any equivalent program thereof. By ‘"equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by CLUSTALW (Version 1.83) using default parameters (available at the European Bioinformatics Institute website on the World Wide Web at ebi . ac. uk/T ools/ clustal w/index) .

[0115] 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.

[0116] The heterologous polynucleotides or polynucleotide constructs comprising R protein coding regions can be provided in expression cassettes for expression in the plant or other organism or non-human host cell of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the R 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 regulatory⁷ 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 frame. 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 R protein coding region to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0117] The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e.. a promoter), a R 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 regulatory⁷ regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the R protein coding region or of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the R protein coding region of the invention may be heterologous to the host cell or to each other.

[01 18] As used herein, “heterologous’’ in reference to a nucleic acid molecule, polynucleotide, nucleotide sequence, or polynucleotide construct is a nucleic acid molecule, polynucleotide, nucleotide sequence, or polynucleotide construct that originates from a foreign species, or. if from the same species, 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 comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0119] As used herein, a “native gene’’ is intended to mean a gene that is a naturally- occurring gene in its natural or native position in the genome of a plant. Such a native gene has not been genetically engineered or otherwise modified in nucleotide sequence and/or positon in the genome the plant through human intervention, nor has such a native gene been introduced into the genome of the plant via artificial methods such as, for example, plant transformation.

[0120] As used herein, a “non-native gene” is intended to mean a gene that has been introduced into a plant by artificial means and/or comprises a nucleotide sequence that is not naturally occurring in the plant. Non-native genes include, for example, a gene (e.g. an R gene) that is introduced into the plant by a plant transformation method. Additionally, when a native gene in the genome of a plant is modified, for example by a genome-editing method, to comprise a nucleotide sequence that is different (i.e. non-identical) from the nucleotide sequence of native gene, the modified gene is a non-native gene.

[0121] 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 cells is 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. [0122] While it may be optimal to express the R protein using heterologous promoters, the native promoter of the corresponding R gene may be used.

[0123] The termination region may be native with the transcriptional initiation region, may be native with the operably linked R protein coding region of interest, may be native with the plant host, or may be derived from another source (/. e. , foreign or heterologous to the promoter, the R 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 (OCS) and nopaline synthase termination regions. See also Guerineau et al. (1991)A/o/. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen el a/. (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.

[0124] 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 ATI -498, herein incorporated by reference.

[0125] 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.

[0126] 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: picomavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); polerovirus 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.

[0127] 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 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.

[0128] 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); nee 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.

[0129] 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, seedpreferred promoters, and stem-preferred promoters. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol.

38(7): 792-803; Hansen et al. (1997) Mo/. Gen Genet. 254(3):337-343; Russell e? 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.

[0130] Generally, it will be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g.. PR proteins, SAR proteins, beta-1.3-glucanase, chitinase, etc. 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.

[0131] 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 :2507-251 1; 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).

[0132] Additionally, as pathogens find entry' into plants through wounds or insect damage, a wound-inducible promoter may be used in the heterologous polynucleotides 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); wunl and wun2, U.S. Patent No. 5,428,148; winl and win2 (Stanford et al.

(1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225:1570- 1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2): 141-150); and the like, herein incorporated by reference.

[0133] 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- la 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 tetracyclineinducible 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.

[0134] 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 markers include phenotypic markers such as P-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 55:610-9 and Fetter et al. (2004) Plant Cell 76:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) ./. Cell Science 777:943-54 and Kato et al. (2002) Plant Physiol 729:913-42), and yellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al. (2004) J. Cell Science 777:943-54). 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;

Fuerst 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 etal. (1993) roc. Natl. Acad. Sci. USA 90: 1917-1921; Labo eta/. (1990)A o/. Cell. Biol. 10:3343-3356;

Zambretti etal. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956: Bairn 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 etal. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavkac 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.

[0135] The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene can be used in the present invention.

[0136] 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.-Planf, 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 Tneu, 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 ai. (1994) Plant. Mol. Biol. 24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol. 104:3748.

[0137] The methods of the invention involve introducing a heterologous polynucleotide or polynucleotide construct into a plant. By “introducing'’ is intended presenting to the plant the heterologous polynucleotide or 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 heterologous polynucleotide or polynucleotide construct to a plant, only that the heterologous polynucleotide or polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing heterologous polynucleotides or polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0138] By “stable transformation” is intended that the heterologous polynucleotide or 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 heterologous polynucleotide or polynucleotide construct introduced into a plant does not integrate into the genome of the plant. It is recognized that stable and transient transformation methods comprise introducing one or more nucleic acid molecules (e.g.

DNA), particularly one or more recombinant nucleic acid molecules (e.g. recombinant DNA) into a plant, plant cell, or other host cell or organism.

[0139] 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.

[0140] 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)Afo/. Gen. Genet. , 22 '. 104-112; Guerche etal., (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.

[0141] Other suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection as Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described by Riggs et al. (1986) roc. Natl. Acad. Set. USA 83:5602-5606. Agrobacterium-mediated transformation as described by Townsend etal., U.S. Patent No. 5,563,055, Zhao et al., U.S. Patent No. 5,981,840, direct gene transfer as described by Paszkowski etal. (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 Led 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.

[0142] 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 heterologous polynucleotide or 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 transcription 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. [0143] 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 707 445 (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., New⁷ 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.

[0144] In specific embodiments, the polynucleotides, 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-me^taXsA transient expression as described elsewhere herein. [0145] 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 then 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 heterologous polynucleotide or polynucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

[0146] Any methods known in the art for modifying DNA in the genome of a plant can be used to modify genomic nucleotide sequences in planta, for example, to create or insert a resistance gene or even to replace or modify an endogenous resistance gene or allele thereof. Such methods include, but are not limited to, genome-editing (or gene-editing) techniques, such as, for example, methods involving targeted mutagenesis, homologous recombination, and mutation breeding. 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 double 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 example, Durai et al., (2005) Nucleic Acids Re.s 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; Amould et a/. (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:el49; 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.

[0147] Unless stated otherwise or apparent from the context of a use. the term “gene replacement” is intended to mean the replacement of any portion of a first polynucleotide molecule or nucleic acid molecule (e.g. a chromosome) that involves homologous recombination with a second polynucleotide molecule or nucleic acid molecule using a genome-editing technique as disclosed elsewhere herein, whereby at least a part of the nucleotide sequence of the first polynucleotide molecule or nucleic acid molecule is replaced with the second polynucleotide molecule or nucleic acid molecule. It is recognized that such gene replacement can result in additions, deletions, and/or modifications in the nucleotide sequence of the first polynucleotide molecule or nucleic acid molecule and can involve the replacement of an entire gene or genes, the replacement of any part or parts of one gene, or the replacement of non-gene sequences in the first polynucleotide molecule or nucleic acid molecule.

[0148] 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, Fokl. 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) P TS 10.1073/pnas. 1013133107; Scholze & Boch (2010) Virulence 1 :428-432;

Christian et al. Genetics (2010) 186:757-761; Li et al. (2Q G) Nuc. Acids Res. (2010) doi: 10. 1093/nar/gkq704; and Miller et al. (201 1) Ato. Biotechnol. 29:143-148; all of which are herein incorporated by reference.

[0149] The CRISPR/Cas nuclease system can also 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 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).

[0150] 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 cleavage 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 (Umov et al. (2010) Nat. Rev. Genet. 11:636-46; Carroll (2011) Genetics. 188:773-82).

[0151] 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.

[0152] The nucleic acid molecules, expression cassettes, vectors, and heterologous polynucleotides of the present invention may be used for transformation and/or genome editing of any plant species, including, but not limited to, monocots and di cots.

[0153] As used herein, the term "plant" includes seeds, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, tubers, propagules, leaves, flowers, branches, fruits, roots, root tips, anthers, and the like. 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” and “progeny 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.

[0154] As used herein, the terms “transgenic plant” and “transformed plant” are equivalent terms that refer to a “plant” as described above, wherein the plant comprises a heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct that is introduced into a plant by, for example, any of the stable and transient transformation methods disclosed elsewhere herein or otherwise known in the art. Such transgenic plants and transformed plants also refer, for example, the plant into which the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct was first introduced and also any of its progeny plants that comprise the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct.

[0155] In certain embodiments of the invention, the methods involve the planting of seedlings and/or tubers and then growing such seedlings and tubers so as to produce plants derived therefrom and optionally harvesting from the plants a plant part or parts. As used herein, a "seedling" refers to a less than fully mature plant that is typically grown in greenhouse or other controlled- or semi-controlled (e.g. a cold frame) environmental conditions before planting or replanting outdoors or in a greenhouse for the production a harvestable plant part, such as, for example, a tomato fruit, a potato tuber or a tobacco leaf. As used herein, a "tuber" refers to an entire tuber or part or parts thereof, unless stated otherwise or apparent from the context of use.

[0156] In the methods of the invention involving planting a tuber, a part of tuber preferably comprises a sufficient portion of the tuber whereby the part is capable of growing into a plant under favorable conditions for the growth and development of a plant derived from the tuber. It is recognized that such favorable conditions for the growth and development of crop plants are generally known in the art.

[0157] In some embodiments of the present invention, a plant cell is transformed with a heterologous polynucleotide encoding an R protein of the present invention. 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. Examples of heterologous polynucleotides and nucleic acid molecules that encode R proteins are described elsewhere herein.

[0158] 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 polynucleotide molecules 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, 2-0-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 ordinary⁷ skill in the art that the nucleotide sequences disclosed herein also encompasses the complement of that exemplified nucleotide sequence.

[0159] The invention is drawn to compositions and methods for enhancing the resistance of plants to plant disease, particularly to compositions and methods for enhancing the resistance of a plant to a plant disease caused by at least one polerovirus. By “disease resistance’' is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen is minimized or lessened.

[0160] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

EXAMPLE 1: Two Classes of NLR-Encoding Genes Co-Segregate with Rl_a<i_g in a Dihaploid Population

[0161] In previous studies, Rladg was mapped using a dihaploid population derived from crossing the tetrapioid LOP-868 to a diploid Solarium phureja dihaploid inducer line (Velasquez et al., 2007, Theor. Appl. Genet. 114: 1051-8). Genetic map construction was used to position Rladg on the long arm of Chromosome 5. More recently, annotation of the DM potato reference genome revealed that this region contains three groups of NLRs, the Rl, Bs4, and Prf clusters (Jupe et al., 2012, BMC Genomics 13:75). We predicted that Rladg is likely to belong to one of these families and used RenSeq to identify orthologues from the resistant parent.

[0162] SMRT RenSeq was used to assemble NLRs of all LOP-868 haplotypes, an assembly of high confidence contigs was produced, each assembled from at least 20 reads and a minimum depth of 5 reads. Using the population described by (Velasquez et al., 2007, Theor. Appl. Genet. 114: 1051-8), we generated bulked susceptible and resistant groups of 30 plants. Mapping of short read RenSeq from LOP-868, bulked susceptible and bulked resistant plants and subsequent analysis of polymorphisms resulted in several genes being identified as absent from susceptible haplotypes. LOP-868 cDNA RenSeq reads were used to predict gene models and confirm expression of the candidates. Phylogenetic analysis of NB-ARC domains against a library of cloned NLRs showed TIR-NLRs have highest homology to Bs4 and CC- NLRs have highest homology to Rl. The NLRs identified as linked to resistance are found on the same region of Chromosome 5 previously reported as containing.

EXAMPLE 2: Rl_td_s Confers HR in Potato After Transient Expression of the PLRV Pl- Pl Region

[0163] To facilitate rapid testing of Rladg candidates, we identified the PLRV protein detected by Rladg. The ssRNA genome of PLRV encodes several major proteins; P0, a suppressor of RNA silencing (P0 - ORFO), a viral proteinase (Pl - ORF1), a P1-P2 fusion protein (ORFs 1/2), the Rapl protein and movement protein (ORF4). A two-part coat protein is produced from ORF3 and a readthrough translation (ORF5). We PCR-amplified ORFO, ORFsl/2, ORF4 and ORFs3/5 from a cDNA clone (Nurkiyanova et al.. 2000, J. Gen. Virol. 81:617-26) and cloned fragments into the 35S: expression vector pICSLUS00040D following the method previously described (Witek et al., 2016, Nature Biotechnology 34:656- 660). Transient expression of the P1-P2 region in young leaves of LOP-868 resulted in HR after 3 days, no response was observed after expression of other PLRV ORFs (FIG. 1). Response to P1-P2 appeared to be Rladg dependent and was not observed in the susceptible cultivar Maris piper (FIG. 1). We used the P1 -P2 region as a diagnostic test to identify the functional Rl dg.

EXAMPLE 3: A Single Bs4 Homologue Confers PLRV Recognition in Tobacco

[0164] To identify Rladg, candidate ORFs (as determined using cDNA RenSeq data) were cloned into pICSLUS00040D. When transiently codelivered into N. tabacum with 35S:P1- P2, no HR was observed for any of the six Rl homologues (Figure . . . ). Of the three TIR- NLRs identified as Rladg candidates, two were successfully cloned; NLR.97.1 and NLR. 153.1. Upon co-expression with 35S:P1-P2. strong HR was observed for NLR.97.1 indicating this candidate could be Rladg. Whilst we were unable to isolate a clone of the third TIR-NLR (NLR.106.1), this candidate shows lower amino acid identify to NLR.97.1 than to the non-functional NLR.153.1. This NLR.97.1 dependent HR was also observed when expressed under native promoter (FIG. 2). Hereafter, NLR.97.1 is referred to as Rladg. EXAMPLE 4: Rlad Recognises the PLRV Pl Protease

[0165] Rladg elicits a response when expressed with the P1-P2 region of the PLRV genome. This region encodes three proteins: Pl, a P1-P2 fusion and the short Rapl protein. By truncating our original construct and mutating the Rapl start codon, we showed that Pl alone is sufficient to induce HR. No response was observed upon expression with the Rapl or P2 ORFs (FIG 3). P l is a polyprotein which cleaves itself through the action of a central serine protease domain. Cleavage products include an N-terminal protein, the protease and the C-terminal Vpg. Further dissection of the elicitor showed that the protease domain is sufficient for recognition by Rladg, co-expression with the other two products does not activate HR (FIG. 3).

[0166] Next, we investigated whether recognition of the PLRV Pl protease is dependent on its function. This serine protease contains a HDS catalytic triad, mutation of either of these residues has been shown to compromise its function (Sadowy et al., 2001, J. Gen. Virol. 82: 1517-1527). We reconstructed a HDS protease mutant and tested for recognition by Rladg. HR appears to be unaffected by mutation of these residues suggesting recognition is independent of protease function. This indicates that Rladg does not recognise a cleaved host protein or guarded protein.

EXAMPLE 5: Rladg Confers PLRV Resistance in Transgenic Potato

[0167] Next, we next tested stably transformed Nicotiana tabacum and Solanum tuberosum (Maris piper). Consistent with transient assays, Plprotease expression resulted in HR in transformants expressing Rladg under both 35S and native promoters (FIGS. 4 and 7). Transgenic N. tabacum lines were screened for resistance to PLRV by agroinfiltration of an infectious clone (Cowan et al., 2023, J. Virol. Methods. 315:114691).

EXAMPLE 6: Rladg Confers Recognition of the Protease Domain of a Diverse Set of Poleroviruses

[0168] Rysto, the Stolanum stoloniferum R gene which confers resistance to PVY, has been shown to recognise many different potyviruses (Grech-Baran et al., 2019, MPMI 32:68- 68; Grech-Baran et al., 2022, New Phytol. 235: 1179-1195). We hypothesised that Rladg could confer recognition of other poleroviruses. Proteases of poleroviruses have high variation in amino acid sequence, ranging between 38 and 83 % identity relative to PLRV. Despite this sequence variation, Alphafold (www.deepmind.com) structure predictions appear to be highly similar (FIG. 5). Nine additional polerovirus proteases were tested by co-expression with Rladg in N. tabacum. Remarkably, the polerovirus proteases of chickpea chlorotic stunt virus (CchSV), wheat yellow dwarf virus (WYDV), tobacco virus 2 (TV2), cotton leafroller dwarf virus (CLRDV), pepper vein yellows virus (PeVYV), beet mild yellowing virus (BMYV), cucurbit aphid-bome yellows virus (CaBYV), maize yellow dwarf virus (MYDV), and turnip yellows virus (TuYV), in addition to P l protease PLRV (FIG. 6).

[0169] Next, we tested whether the neighbouring enamovirus family contain members that are recognised by Rladg. This family is known both to share a similar genome structure to poleroviruses and to encode a self-cleaving Pl protein. We synthesised the proteases from Citrus vein enation virus, Grapevine enamovirus- 1, and Pea enation mosaic virus- 1 and tested for recognition by Rladg through transient expression in N. tabacum (data not shown). No HR was observed upon expression with these proteases indicating their divergence is too great to be recognised by Rladg.

[0170] Rl dg confers extraordinarily wide recognition of distantly related viruses, we defined the breadth of this recognition across the polerovirus and enamovirus families. From these observations, it seems that the recognition of proteases by Rladg is based upon high structural conservation of the serine protease.

EXAMPLE 7: Rladg Confers Resistance to TuYV in N tabacum Transgenic Lines [0171] Rladg confers recognition of diverse polerovirus Pl proteases, we aimed to investigate whether this can be utilised to engineer resistant crops. Using N. tabacum lines stably expressing Rladg, we assayed resistance to two economically important viruses, Turnip yellows virus (TuYV) infects oilseed rape. Agroinfiltration of infectious cDNA clones resulted in systemic infection of TuYV in WT plants (FIG. 8). Transgenic lines carrying Rladg show ed HR upon infection with PLRV (FIG. 8).

[0172] TuYV is an economically important virus of Brassicaceae species. To determine whether Rladg can function in a Brassicaceae species, we performed transient expression and cell death assays in the Turnip cultivar ‘Just right’, which, unlike Arabidopsis thaliana, species is amenable to agroinfiltration and transient expression assays (Sohn et al., 2009, Plant J. 57: 1079-1091). We found that co-expression of Rladg with the PLRV serine protease activates HR indicating Rladg gene is functional in some Brassicaceae species (data not shown). Discussion

[0173] Genetic resistance to PLRV is limited, the rare instance of a single major gene against PLRV is reported in accession LOP-868 and is due to Rladg. Previous publication positioned Rladg at an NLR rich region of Chr5, we hypothesised that Rladg belongs to one of these gene clusters and encodes an NLR protein. We identified Rladg using resistance gene enrichment sequencing coupled with bulk segregant analysis and pacbio sequencing. Rladg encodes a TNL with homology to Bs4 and functions through recognition of the PLRV Pl protease.

[0174] We screened the recognition capacity' of Rladg using a panel of proteases from other poleroviruses, surprisingly Rladg recognised all tested poleroviruses despite their highly diverse amino acid sequences. By testing a catalytic mutant of the PLRV protease, we found that recognition was independent of protease function. Instead, we found that tested proteases share a highly similar predicted structure. It is likely that Rladg directly recognises viral proteases regardless of their activity. By observing this broad-spectrum resistance, we hypothesise that Rladg will be durable when deployed against PLRV and display resistance to multiple strains of PLRV. Because this protease is strictly required for polerovirus replication and virus maturation, the protease cannot be simply lost to evade resistance.

[0175] The article “a” and “an” are used herein to refer to one or more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element.

[0176] 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.

[0177] 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.

[0178] 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.

Claims

1. A plant or plant cell comprising stably incorporated in its genome a heterologous polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;

(c) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, and 4, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule; and

(d) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity’ to the amino acid sequences set forth in SEQ ID NO: 2, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule; wherein the plant is not Solanum tuberosum and the plant cell is not a Solanum tuberosum plant cell.

2. The plant or plant cell of claim 1, wherein the heterologous polynucleotide comprises the nucleotide sequence of any one of (a)-(d) and further comprises a promoter operably linked for the expression of the nucleotide sequence in a plant.

3. The plant or plant cell of claim 1 or 2, wherein the polerovirus is selected from the group consisting of potato leafroller virus (PLRV), chickpea chlorotic stunt virus (CchSV). wheat yellow dwarf virus (WYDV). tobacco virus 2 (TV 2), cotton leafroller dwarf virus (CLRDV), pepper vein yellows virus (PeVYV), beet mild yellowing virus (BMYV), cucurbit aphid-bome yellows virus (CaBYV), maize yellow dwarf virus (MYDV), and turnip yellows virus (TuYV).

4. The plant or plant cell of any one of claims 1-3, wherein the plant or plant cell comprises enhanced resistance to a plant disease caused by at least one polerovirus, relative to the resistance of a control plant.

5. A method for enhancing the resistance of a plant to a plant disease caused by at least one polerovirus, the method comprising introducing at least one plant cell a heterologous polynucleotide, the heterologous polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;

(d) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 2, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule; wherein the plant is not Solarium tuberosum and the plant cell is not a Solarium tuberosum plant cell.

6. The method of claim 5, wherein the heterologous polynucleotide is stably incorporated into the genome of the plant cell.

7. The method of claim 5 or 6, wherein the plant cell is regenerated into a plant comprising in its genome the heterologous polynucleotide.

8. The method of any one of claims 5-7, wherein the heterologous polynucleotide comprises the nucleotide sequence of any one of (a)-(d) and further comprises a promoter operably linked for the expression of the nucleotide sequence in a plant.

9. The method of any one of claims 5-8, wherein the plant comprising the heterologous polynucleotide comprises enhanced resistance to a plant disease caused by at least one polerovirus, relative to the resistance of a control plant.

10. The method of any one of claims 5-9, wherein the polerovirus is selected from the group consisting of potato leafroller virus (PLRV), chickpea chlorotic stunt virus (CchSV), wheat yellow dwarf virus (WYDV). tobacco virus 2 (TV2), cotton leafroller dwarf virus (CLRDV), pepper vein yellows virus (PeVYV), beet mild yellowing virus (BMYV), cucurbit aphid-bome yellows virus (CaBYV), maize yellow dwarf virus (MYDV), and turnip yellows virus (TuYV).

11. A plant producible by the method of any one of claims 5-10.

12. A fruit, leaf, root, or seed of the plant of any one of claims 1-4 and 11, wherein the fruit, leaf, root, or seed comprises the heterologous polynucleotide.

13. A method of limiting a plant disease caused by at least one polerovirus in agricultural crop production, the method comprising planting a seedling or seed of the plant of any one of claims 1-4 and 11 and growing the seedling or seed under conditions favorable for the growth and development of a plant resulting therefrom, wherein the seedling or seed comprises the nucleic acid molecule, expression cassette, vector, or heterologous polynucleotide.

14. The method of claim 13, further comprising harvesting at least one seed, fruit, leaf, root, or other plant part from the plant, and optionally processing the harvested seed, fruit, leaf, root, or other plant part into a food product.

15. A seed, fruit, leaf, root, or other plant part, or a food product, obtainable using the method of claim 14.

16. A method for identifying a plant that comprises an R gene for a plant disease caused by at least one polerovirus, the method comprising detecting in the plant, or in at least one part or cell thereof, the presence of an Rl_ad_g nucleotide sequence, wherein the Rladg nucleotide sequence is selected from the group consisting of:

(a) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;

(c) a nucleotide sequence having at least 90% sequence identify to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, and 4, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule; and

(d) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identify to the ammo acid sequences set forth in SEQ ID NO: 2, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule; wherein the plant is not Solanum tuberosum.

17. The method of claim 1 , wherein the presence of the Rl_ad_g nucleotide sequence is detected by detecting at least one marker within the Rladg nucleotide sequence.

18. The method of claim 16 or 17, wherein detecting the presence of the Rladg nucleotide sequence comprises a member selected from the group consisting of PCR amplification, nucleic acid sequencing, nucleic acid hybridization, and an immunological assay for the detection of the R protein encoded by the Rladg nucleotide sequence.

19. A method for introducing Rl_ad_g into a plant, the method comprising:

(a) crossing a first plant comprising in its genome at least one copy of an Rladg polynucleotide with a second plant lacking in its genome an Rladg polynucleotide, whereby at least one progeny plant is produced; and (b) selecting at least one progeny plant comprising in its genome the Rladg polynucleotide by detecting in the progeny plant the presence of an Rladg polynucleotide; wherein the Rladg polynucleotide comprises a nucleotide sequence is selected from the group consisting of:

(z) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 4;

(zz) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2;

(zz) a nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, and 4, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule; and

(zv) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the ammo acid sequences set forth in SEQ ID NO: 2, wherein the nucleic acid molecule is capable of conferring to a plant resistance to a plant disease caused by a polerovirus, relative to a control plant not comprising the nucleic acid molecule; wherein the plant is not Solanum tuberosum.

20. A progeny plant obtainable using the method of claim 19.

21. A seed, fruit, leaf, root, or other plant part obtainable from the plant of claim 20.

22. Use of the plant, seed, fruit, leaf, root, or other plant part of any one of claims 1-4, 11, 15, 20. and 21 in agriculture or in production of a food product.

23. A human or animal food product comprising, or produced using, the plant, seed, fruit, leaf, root, or other plant part of any one of claims 1-4, 11, 15, 20, and 21.

24. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 2;