Asian soybean rust resistance gene

Abstract

Compositions and methods are provided for enhancing the resistance of a soybean plant to Asian soybean rust caused by one or more races of puccinia pachyrhizus. The compositions comprise nucleic acid molecules encoding resistance (R) gene products and / or modulator gene products and variants thereof, as well as plants, seeds and plant cells comprising such nucleic acid molecules. The methods for enhancing the resistance of a plant to Asian soybean rust caused by one or more races of puccinia puccinia comprise introducing a nucleic acid molecule encoding an R gene product into a plant cell, and may further comprise introducing a nucleic acid molecule encoding a modulator gene product. Also provided are methods for using the soybean plants in agriculture to limit plant disease.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 383,775, filed on November 15, 2022, which is hereby incorporated by reference in its entirety.

[0003] Reference to Electronic Sequence Listing

[0004] The contents of the electronic sequence listing (070294 - 0219SEQLST.xml; size: 322,337 bytes; created on November 10, 2023) are hereby incorporated by reference in their entirety. Technical Field

[0005] The present invention relates to compositions and methods for identifying, selecting, and producing enhanced disease - and / or pathogen - resistant soybean plants. Background Art

[0006] Soybean (Glycine max (L.) Merr) is one of the most important economic crops worldwide. The Food and Agriculture Organization of the United Nations (FAO) estimates that the total global value of soybean crops exceeded $120 billion USD in 2020, second only to rice, maize, and wheat (FAOSTAT, available on the World Wide Web at fao.org / faostat / en / #data / QV).

[0007] As is the case with other crops, soybean yields are reduced by pests and pathogens, including, for example, insects, nematodes, viruses, bacteria, and fungi. To limit the damage caused by such pests and pathogens, farmers use agrochemicals, cultural practices, and / or plants containing resistance (R) genes against such pests and pathogens.

[0008] Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is an economically damaging foliar disease of soybeans. Although ASR has been a serious disease in Asia for decades, ASR has spread to other soybean - growing regions of the world (Rupe and Sconyers (2008) Plant Health Instructor DOI: 10.1094 / PHI - I - 2008 - 0401 - 01). ASR was first detected in Africa in 1997, in South America in 2001, and in the United States in 2004. Ibid.

[0009] The Asian soybean rust fungus (Phakopsora pachyrhizi) is not only a pathogen of soybeans, but can also grow on a variety of leguminous plants (subfamily Papilionoideae, also known as Faboideae) of the specific legume family that have keel-shaped flowers (College of Agriculture and Biological Sciences, South Dakota State University, “Asian Soybean Rust” (2005), College of Agriculture and Biological Sciences Publications. Paper 2; available on the World Wide Web at openprairie.sdstate.edu / coabs_pubs / 2). This legume subfamily includes, for example, peas, kidney beans (Phaseolus species), and a variety of forage grasses, including sweet clover and red clover. Ibid.

[0010] Over 90 plant species are known hosts of Phakopsora pachyrhizi, including but not limited to common dry beans (e.g., field bean, kidney bean, navy bean, pinto bean; Phaseolus vulgaris var. vulgaris), common snap beans (e.g., green bean, wax bean, string bean, and yellow wax bean; Phaseolus vulgaris var.), fava or broad bean (Vicia faba), hyacinth bean or lablab (Lablab purpureus), lima bean (Phaseolus lunatus var. lunatus), mung bean (Vigna radiata), scarlet runner bean (Phaseolus coccineus), winged bean or goa bean (Psophocarpus tetragonolobus), yam bean (Pachyrhizus ahipa, P. erosus), black-eyed pea, cowpea or long bean (Vigna unguiculata), calopo (Calopogonium mucunoides), alysicarp or single-leaf clover (Alysicarpus vaginalis), crimson clover (Trifolium incarnatum), yellow clover (Trifolium aureum), spiny clover (Trifolium lappaceum), white clover (Trifolium repens), rattlebox (Crotalaria anagyroides, C. spectabilis), crownvetch (Securigera varia), fenugreek (Trigonella foenum-graicum), Florida beggarweed (Desmodium tortuosum), kudzu (Pueraria montana var. lobata), bush clover (Lespedeza spp., Kummerowia striata, K. stipulaceae), lupine (Lupinus spp.), medic (Medicago spp.), milkvetch (Astragalus cicer, A.Glycyphyllos), field pea (Pisum sativum), giant sesbania or Colorado River hemp (Sesbania exaltata), pigeon pea (Cajanus cajan), purple-winged bean (Macroptilium atropurpureum), soybean (Glycine max), sword bean (Canavalia gladiata), clover (Lotus species), black gram (Vigna mungo), wild soybean (Neonotonia wightii), hairy pod vetch (Vicia villosa subsp. varia), and sweet clover (Melilotus officinalis) (Rupe & Sconyers, 2008, “Soybean Rust,” Plant Health Instr. DOI: 10.1094 / PHI-I-2008-0401-01).

[0011] Sustainable intensification of agriculture will require greater use of genetic solutions rather than chemical solutions (such as pesticides) to protect crops from pathogens and pests (Jones et al. (2014) Philos. T. Roy. Soc. B 369: 20130087). Wild relatives of domesticated crops such as soybean contain a large number of useful R genes, which are valuable resources for sustainable disease control. Summary of the Invention

[0012] The present invention provides nucleic acid molecules capable of conferring resistance to Asian soybean rust (ASR) caused by one or more races of Phakopsora pachyrhizi on leguminous plants, particularly soybean plants. Such nucleic acid molecules contain one or more resistance (R) genes and may optionally contain one or more other genes, such as genes that regulate the expression of R genes (i.e., regulator genes).

[0013] The present invention further provides leguminous plants whose genomes contain one or more nucleic acid molecules of the present invention and which have enhanced resistance to ASR caused by one or more races of Phakopsora pachyrhizi when compared to control plants that do not contain one or more nucleic acid molecules. Also provided are leguminous plant cells, plant parts, and seeds containing one or more nucleic acid molecules.

[0014] The present invention provides methods for enhancing the resistance of leguminous plants to at least one race of Phakopsora pachyrhizi that is known to cause ASR in leguminous plants. Such methods include introducing a heterologous polynucleotide comprising a nucleic acid molecule of the present invention into at least one leguminous plant cell. Preferably, the heterologous polynucleotide or a portion thereof is stably incorporated into the genome of the leguminous plant cell. These methods may also optionally include regenerating the leguminous plant cell into a leguminous plant that contains the heterologous polynucleotide in its genome. Preferably, such leguminous plants contain enhanced resistance to ASR caused by at least one race of Phakopsora pachyrhizi relative to the resistance of control leguminous plants that do not contain the heterologous polynucleotide. More preferably, such leguminous plants contain enhanced resistance to ASR caused by at least two, three, four, five or more races of Phakopsora pachyrhizi relative to the resistance of control leguminous plants that do not contain the heterologous polynucleotide.

[0015] Preferably, such soybean plants contain enhanced resistance to ASR caused by at least one race of Phakopsora pachyrhizi relative to the resistance of control soybean plants that do not contain the heterologous polynucleotide. More preferably, such soybean plants contain enhanced resistance to ASR caused by at least two, three, four, five or more races of Phakopsora pachyrhizi relative to the resistance of control soybean plants that do not contain the heterologous polynucleotide.

[0016] The present invention additionally provides methods for identifying soybean or other plants that contain a nucleic acid molecule capable of conferring enhanced resistance to ASR caused by at least one race of Phakopsora pachyrhizi. Such methods include detecting the presence of at least one nucleotide molecule of the present invention in the plant.

[0017] Also provided are methods for using the soybean plants of the present invention in crop production to limit ASR. Such methods include planting soybean seeds produced by the plants of the present invention in a field or outdoor growing area or indoor greenhouse, wherein the seeds contain at least one nucleic acid molecule of the present invention. Such methods also include growing the soybean plants under conditions favorable for the growth and development of the soybean plants, and particularly in an environment where ASR is known to occur, and optionally harvesting at least one seed or plant part from the soybean plants. Such methods can limit the economic losses caused by ASR. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figures 1A to 1B . Glycine argyrea germplasm at 14 days post inoculation (dpi) with Phakopsora pachyrhizi. Figure 1A . Resistant genotype PI 653478 A: Lesions formed but no sporulation. Figure 1B . Susceptible genotype PI595798: Abundant sporulation at 14 dpi.

[0019] Figure 1A To Figure 2C. Fine mapping of GaRpp1 resistance and overview of the resistance locus containing the candidate genes. The fine mapping steps are illustrated by key loss-of-function recombinants. Figure 2A. QTL mapping on the F2 population led to the identification of a resistance interval (2.8 Mbp) between markers GA_155 and GA_206. Figure 2B. Fine mapping steps on F2 and F3 recombinants narrowed the resistance interval to a region (109.6 Kb) between markers CAPS_CG2 and GA_204. Figure 2C. Overview of the GaRpp1 resistance interval containing four candidate genes, GaRpp1_CG1, GaRpp1_CG4, GaRpp1_CG5, and GaRpp1_CG10. The gene GaRpp1_CG1 (black) is essential for resistance, as verified by VIGS experiments. The region upstream of CAPS_CG2 (outside the resistance interval defined by fine mapping) is shown to display the presence of neighboring genes that are paralogs of GaRpp1_CG1 or GaRpp1_CG4.

[0020] Figure 2A to Figure 3H . Determination results of detached leaves of VIGS plants attacked with PPUFV02. Figures 3A to 3B : Leaves of Gargy_1900 plants silenced with BPMV:cg1 showed sporulation at 13 dpi; Figures 3C to 3D : Leaves of Gargy_1900 plants silenced with BPMV:cg1-2 showed sporulation at 13 dpi; Figures 3E to 3F : Leaves of Gargy_1900 plants silenced with BPMV:cg3-9 remained resistant at 13 dpi; Figure 3G : Leaves of Gargy_1900 inoculated with BPMV:EV (empty vector) remained resistant after rust inoculation; Figure 3H : PI 595798 (susceptible control of the experiment) showed sporulation at 13 dpi.

[0021] Figure 4 . Percent disease severity (top) and mRNA expression (bottom) of soybean transformation events for construct-4 (light gray bars), construct-6 (dark gray bars), and construct-7 (black bars).

[0022] Figure 5 . Images of the abaxial side of leaves taken from three different transgenic soybean events containing construct 7 and one wild-type soybean plant. The images highlight the differences in both disease severity and sporulation observed in the transgenic soybean events containing the construct.

[0023] Figure 6.Genomic intervals containing the sequences of genes GaRpp1_CG1 and GaRpp1_CG4.

[0024] Figure 7 .Agrobacterium-mediated transient expression of GaRpp1 candidate genes in Nicotiana benthamiana at 4 dpi. Circles indicate leaf areas infiltrated with specific constructs. Photos were taken under UV light. Positive control for PsCRN63 = HR.

[0025] Figure 8 .Promoter analysis of the GaRpp1 intergenic region based on the PlantCARE database. Predicted cis-acting elements are annotated in the sequence.

[0026] Figure 9 .GaRpp1-mediated cell death in Nicotiana benthamiana is independent of major NLR and PRR pathway signaling components. Using the Agrobacterium system, the GaRpp1_CG1, GaRpp1_CG4, or GaRpp1_CG1+GaRpp1_CG4 candidate genes were transiently overexpressed in wild-type Nicotiana benthamiana (upper left), BAK1 overexpressing plants (Nb BAK, upper middle), bak1 mutants (Nb bakl, upper right), Nicotiana benthamiana quadruple knockout mutants of EDS1 family genes EDS1a, PAD4, SAG101a, and SAG101b (Nb epss; lower left), NRG1 and ADR1 mutants (Nb nrg1 / adr1 middle lower), and Nicotiana benthamiana with VIGS-silenced SOBIR1 (Nb TRV:SOBIR1, lower right). Positive controls of PsCRN63 and HopQ1 = HR Empty vector were used as negative controls. Four-week-old plants were infiltrated at OD0.8 and imaged at 4 dpi.

[0027] Figure 10 .Schematic diagram of the GaRPP1_CG1 truncated protein. The top line represents the full-length GaRPP1_CG1. GaRPP1_CG1_v1, GaRPP1_CG1_v2, GaRPP1_CG1_v3 are truncated proteins with 1, 2, and 3 transmembrane domains (TMhelix in the figure) removed respectively. GaRPP1_CG1_v4 is a truncated protein in which the first 200 amino acids at the N-terminus are removed. All truncations have an ATG added for translation initiation.

[0028] Figure 11. GaRpp1_CG1-mediated cell death requires all three TM domains, while the GaRpp1_CG1 N-terminus is required for the inhibition of GaRpp1_CG1-mediated cell death by GaRpp1_CG4. Transient overexpression of full-length GaRpp1_CG1, truncated proteins GaRPP1_CG1_v1, GaRPP1_CG1_v2 (left lobe), GaRPP1_CG1_v3, GaRPP1_CG1_v4, and GaRPP1_CG1_v4+GaRpp1_CG4 (right lobe) was performed using an Agrobacterium-mediated expression system. PsCRN63 and HopQ1=HR were used as positive controls. The empty vector was used as a negative control.

[0029] Sequence Listing

[0030] The nucleotide and amino acid sequences listed in the attached sequence listing are shown using the standard letter abbreviations for nucleotide bases and the three-letter codes for amino acids. The nucleotide sequences follow the standard convention of starting at the 5′ end of the sequence and proceeding in the forward direction (i.e., from left to right in each row) to the 3′ end. Although only one strand of each nucleotide sequence is shown, it should be understood that the complementary strand is included whenever the shown strand is referred to. The amino acid sequences follow the standard convention of starting at the amino terminus of the sequence and proceeding in the forward direction (i.e., from left to right in each row) to the carboxyl terminus.

[0031] SEQ ID NO: 1 shows the nucleotide sequence of a genomic interval containing GaRpp1_CG1, a 306 bp intergenic region, and GaRpp1_CG4 from the Glycine soja germplasm PI 653478 A.

[0032] SEQ ID NO: 2 shows the genomic nucleotide sequence of GaRpp1_CG1 contained in SEQ ID NO: 1.

[0033] SEQ ID NO: 3 shows the genomic nucleotide sequence of the intergenic region between GaRpp1_CG1 and GaRpp1_CG4 contained in SEQ ID NO: 1.

[0034] SEQ ID NO: 4 shows the genomic nucleotide sequence of GaRpp1_CG4 contained in SEQ ID NO: 1.

[0035] SEQ ID NO: 5 shows the cDNA sequence of GaRpp1_CG1. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3′ end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 5. The natural stop codon of this cDNA is TGA.

[0036] SEQ ID NO: 6 shows the amino acid sequence of the protein encoded by GaRpp1_CG1 (SEQ ID NO: 5 and SEQ ID NO: 32).

[0037] SEQ ID NO: 7 shows the cDNA sequence of GaRpp1_CG4. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 8. The native stop codon of this cDNA is TAA.

[0038] SEQ ID NO: 8 shows the amino acid sequence of the protein encoded by GaRpp1_CG4 (SEQ ID NO: 7).

[0039] SEQ ID NO: 9 shows the nucleotide sequence of a genomic interval comprising GaRpp1_CG2, a 130 bp intergenic region, GaRpp1_CG5, GaRpp1_CG1, a 306 bp intergenic region, and GaRpp1_CG4 from the Glycine soja germplasm PI 653478 A.

[0040] SEQ ID NO: 10 shows the nucleotide sequence of a genomic interval comprising GaRpp1_CG2 and a 130 bp intergenic region and GaRpp1_CG5 from the Glycine soja germplasm PI 653478 A.

[0041] SEQ ID NO: 11 shows the genomic nucleotide sequence of GaRpp1_CG2 contained in SEQ ID NO: 9.

[0042] SEQ ID NO: 12 shows the genomic nucleotide sequence of the intergenic region between GaRpp1_CG2 and GaRpp1_CG5 contained in SEQ ID NO: 9.

[0043] SEQ ID NO: 13 shows the genomic nucleotide sequence of GaRpp1_CG5 contained in SEQ ID NO: 9.

[0044] SEQ ID NO: 14 shows the cDNA sequence of GaRpp_CG2. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 14. The native stop codon of this cDNA is TGA.

[0045] SEQ ID NO: 15 shows the amino acid sequence of the protein encoded by GaRpp1_CG2 (SEQ ID NO: 14).

[0046] SEQ ID NO: 16 shows the cDNA sequence of GaRpp1_CG5. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3′ end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 16. The native stop codon of this cDNA is TAA.

[0047] SEQ ID NO: 17 shows the amino acid sequence of the protein encoded by GaRpp1_CG5 (SEQ ID NO: 16).

[0048] SEQ ID NO: 18 shows the nucleotide sequence of a genomic interval comprising MSTRG.57, a 433 bp intergenic region, GaRpp1_CG3, GaRpp1_CG2, a 130 bp intergenic region, GaRpp1_CG5, GaRpp1_CG1, a 306 bp intergenic region, and GaRpp1_CG4 from the Glycine soja germplasm PI 653478 A.

[0049] SEQ ID NO: 19 shows the nucleotide sequence of a genomic interval comprising MSTRG.57 and a 433 bp intergenic region and GaRpp1_CG3 from the Glycine soja germplasm PI 653478 A.

[0050] SEQ ID NO: 20 shows the genomic nucleotide sequence of MSTRG.57 contained in SEQ ID NO: 18.

[0051] SEQ ID NO: 21 shows the genomic nucleotide sequence of the intergenic region between MSTRG.57 and GaRpp1_CG3 contained in SEQ ID NO: 18.

[0052] SEQ ID NO: 22 shows the genomic nucleotide sequence of GaRpp1_CG3 contained in SEQ ID NO: 18.

[0053] SEQ ID NO: 23 shows the cDNA sequence of MSTRG.57. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3′ end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 23. The native stop codon of this cDNA is TGA.

[0054] SEQ ID NO: 24 shows the amino acid sequence of the protein encoded by MSTRG.57 (SEQ ID NO: 21) starting from the first start codon in the cDNA sequence.

[0055] SEQ ID NO: 25 shows the cDNA sequence of GaRpp1_CG3. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3′ end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 25. The native stop codon of this cDNA is TAA.

[0056] SEQ ID NO: 26 shows the amino acid sequence of the protein encoded by GaRpp1_CG3 (SEQ ID NO: 25).

[0057] SEQ ID NO: 27 shows the nucleotide sequence of a genomic interval comprising GaRpp1_CG1 and a 306 bp intergenic region from Glycine soja germplasm PI 653478 A.

[0058] SEQ ID NO: 28 shows the nucleotide sequence of a genomic interval comprising a 306 bp intergenic region and GaRpp1_CG4 from Glycine soja germplasm PI 653478 A.

[0059] SEQ ID NO: 29 shows the nucleotide sequence of a fragment of the intergenic region between GaRpp1_CG1 and GaRpp1_CG4 consisting of nucleotides 1 to 106 of SEQ ID NO: 3.

[0060] SEQ ID NO: 30 shows the nucleotide sequence of a fragment of the intergenic region between GaRpp1_CG1 and GaRpp1_CG4 consisting of nucleotides 103 to 306 of SEQ ID NO: 3.

[0061] SEQ ID NO: 31 shows the nucleotide sequence of a fragment of the intergenic region between GaRpp1_CG1 and GaRpp1_CG consisting of nucleotides 103 to 190 of SEQ ID NO: 3.

[0062] SEQ ID NO: 32 shows the coding sequence (CDS) of GaRpp1_CG1. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3′ end of the nucleic acid molecule. The native stop codon of this cDNA is TGA. The native stop codon of this cDNA is TGA. The amino acid sequence encoded by SEQ ID NO: 32 is as shown in SEQ ID NO: 6.

[0063] SEQ ID NO: 33 shows the nucleotide sequence of a fragment of the GaRpp1_CG1 CDS consisting of nucleotides 1 to 1032 of SEQ ID NO: 32. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3′ end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 33. TAG is used as the stop codon for expressing this fragment, as described in Example 10 below.

[0064] SEQ ID NO: 34 shows the amino acid sequence of the protein encoded by SEQ ID NO: 33.

[0065] SEQ ID NO: 35 shows the nucleotide sequence of a fragment of the GaRpp1_CG CDS consisting of nucleotides 1 to 939 of SEQ ID NO: 32. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3′ end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 35. TAG is used as the stop codon for expressing this fragment, as described in Example 10 below.

[0066] SEQ ID NO: 36 shows the amino acid sequence of the protein encoded by SEQ ID NO: 35.

[0067] SEQ ID NO: 37 shows the nucleotide sequence of a fragment of the GaRpp1_CG CDS consisting of nucleotides 1 to 870 of SEQ ID NO: 32. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3′ end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 37. TGA is used as the stop codon for expressing this fragment, as described in Example 10 below.

[0068] SEQ ID NO: 38 shows the amino acid sequence of the protein encoded by SEQ ID NO: 37.

[0069] SEQ ID NO: 39 shows the nucleotide sequence of a fragment of the GaRpp1_CG1 CDS consisting of nucleotides 580 to 1224 of SEQ ID NO: 32. The ATG codon is added at the 5′ end as the start codon. If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3′ end of a nucleic acid molecule comprising or consisting of SEQ ID NO: 39. The natural stop codon of GaRpp1_CG1 (TGA) is used as the stop codon for expressing this fragment, as described in Example 10 below.

[0070] SEQ ID NO: 40 shows the amino acid sequence of the protein encoded by SEQ ID NO: 39. Detailed Description

[0071] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. In fact, the 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 always indicate like elements.

[0072] Benefiting from the foregoing description and the teachings given in the related drawings, those skilled in the art to which the present invention pertains will envision many modifications and other embodiments of the invention set forth herein. Accordingly, it is to be understood that the invention is not 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.

[0073] The present invention relates to the identification of certain genomic intervals in the genome of a germplasm of Glycine soja, the wild relative of domesticated soybean (Glycine max). The Glycine soja germplasm (PI 653478 A) is known to be resistant to ASR caused by multiple races of Phakopsora pachyrhizi. The identified genomic intervals correspond to loci containing the resistance trait to ASR caused by multiple races of Phakopsora pachyrhizi. The present invention also relates to the isolation of nucleic acid molecules corresponding to the genomic intervals. Such nucleic acid molecules may comprise the entire genomic interval or a part or parts thereof. For example, a part may comprise a single gene (e.g., an R gene), two adjacent genes and the intergenic region between them, or even a part of a single gene (e.g., a promoter).

[0074] As disclosed hereinafter, transgenic soybean plants expressing GaRpp1_CG1 ("CG1") under the control of an operably linked heterologous constitutive promoter exhibit enhanced resistance to ASR caused by Phakopsora pachyrhizi. In the genome of the Glycine soja germplasm PI 653478 A, CG1 and GaRpp1_CG4 ("CG4") are arranged in a head-to-head orientation, sharing a short intergenic region (the starting point of the transcription start site from one gene to the other is 306 bp, Figure 6)。This arrangement coupled with the short intergenic region may suggest that the short intergenic region contains a bidirectional promoter that drives the expression of both genes and can be regulated by pathogen infection. Although the present invention is not bound by a particular biological mechanism, CG4 can act as a regulator of CG1, which restricts its own activity in plant tissues, as disclosed in Example 6 below. Thus, in certain embodiments of the present invention, it may be desirable to co-express CG4 and CG1 in transgenic plants.

[0075] The present invention provides nucleic acid molecules comprising the nucleotide sequences of the R gene CG1 and its paralog GaRpp1_CG2 ("CG2") and MSTRG.57, and their naturally occurring (e.g., allelic, orthologous, paralogous) and synthetic or artificial (i.e., non-naturally occurring) variants. The present invention also provides nucleic acid molecules comprising the nucleotide sequences of the regulator gene CG4 and its paralogs GaRpp1_CG5 ("CG5") and GaRpp1_CG3 ("CG3"), and their naturally occurring and synthetic or artificial variants. In addition, the present invention provides nucleic acid molecules comprising the intergenic regions between the gene pairs CG1 and CG4, CG2 and CG5, and MSTRG.57 and CG3, and naturally occurring and synthetic or artificial variants of such intergenic sequences. Examples of such R gene, regulator gene, and intergenic region nucleotide sequences include the nucleotide sequences shown in SEQ ID NO: 2-5, 7, 11-14, 16, 20-23, 25, 27, 32, 33, 35, 37, and 39. In a preferred embodiment of the present invention, the nucleic acid molecule comprises the CG1 nucleotide sequence or a variant thereof. If desired, such nucleic acid molecules may also comprise the CG4 nucleotide sequence or a variant thereof.

[0076] The R gene and regulator nucleotide sequences of the present invention include, but are not limited to, the nucleotide sequences of the wild-type or native CG1, CG4, CG2, CG5, MSTRG.57, and CG3 genes, which comprise the native promoter and the native 3′ flanking region containing the coding region; cDNA sequences; and nucleotide sequences comprising only the coding region. Examples of such R gene and regulator nucleotide sequences include the nucleotide sequences shown in SEQ ID NO: 1, 2, 4, 5, 7, 9-11, 13, 14, 16, 18-20, 22, 23, 25, 27, 28, 32, 33, 35, 37, and 39 and their variants, as well as nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 6, 8, 15, 17, 24, 26, 34, 36, 38, and 40. In embodiments where a native promoter is not used to drive the expression of the nucleotide sequence encoding the R protein or regulator protein, a heterologous promoter may be operably linked to the nucleotide sequence encoding the R protein or regulator protein of the present invention to drive the expression of the nucleotide sequence encoding the protein in plants.

[0077] Preferably, the R protein encoded by the R nucleotide sequence of the present invention is a functional R protein or a part or domain thereof that can confer resistance to one, two, three, four, five or more races of Phakopsora pachyrhizi known to cause ASR in plants, particularly soybean plants or other plants of the genus Glycine.

[0078] Preferably, the regulator protein encoded by the regulator nucleotide sequence of the present invention is a functional regulator protein or a part or domain thereof. Such a regulator protein is encoded by a regulator gene that can regulate the activity of at least one R gene of the present invention in a plant when co-expressed with the R gene of the present invention.

[0079] As used herein, a regulator gene is a gene that can regulate the activity of an R protein encoded by an R gene when the regulator gene and the R gene are co-expressed in a plant. Although such a regulator gene generally cannot confer resistance to plant diseases caused by plant pathogens on its own, when the regulator gene is co-expressed with at least one R gene of the present invention in a plant, it can, for example, enhance or increase the resistance conferred by the R gene alone or improve at least one harmful or other undesirable phenotype produced when the R gene is expressed in the plant. Such a harmful or other undesirable phenotype can be any harmful or other undesirable phenotypic change in the plant caused by or otherwise associated with the expression of the R gene in the plant (particularly in soybean plants, more particularly in soybean varieties). Examples of such harmful or other undesirable phenotypes (compared to the same plant without the R gene) include, but are not limited to, reduced agricultural yield, reduced plant height and / or appearance density, a mosaic pattern on the leaves with patches of wrinkled tissue, a negative transmission bias of the resistance allele, delayed plant growth and development, and an auto-catalytic hypersensitive response (HR).

[0080] The present invention further provides a plant comprising a heterologous polynucleotide comprising the R gene nucleotide sequence and / or the regulator gene nucleotide sequence of the present invention. Preferably, such an R gene nucleotide sequence encodes the full-length R protein of the present invention, or at least a functional part or domain thereof. Preferably, such a regulator gene nucleotide sequence encodes the full-length regulator protein of the present invention, or at least a functional part or domain thereof. In some embodiments, such heterologous polynucleotides of the present invention are 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.

[0081] In other embodiments, plants comprising a heterologous polynucleotide are produced using the methods of the invention that involve genome editing to modify the nucleotide sequence of a native or non-native gene in a plant genome, the heterologous polynucleotide comprising an R gene nucleotide sequence of the invention and / or a regulator gene. The native or non-native gene comprises a nucleotide sequence that is different (i.e., not identical) from the R gene or regulator gene nucleotide sequence of the invention, and after modification by the methods disclosed in further detail below, the modified native or non-native gene comprises the R gene or regulator nucleotide sequence of the invention. Generally, such methods include using a plant that comprises a native or non-native gene in its genome, wherein the native or non-native gene comprises a nucleotide sequence that is homologous to the R gene or regulator nucleotide sequence of the invention, and such methods further include introducing into the plant a nucleic acid molecule comprising at least a portion of the R gene or regulator nucleotide sequence of the invention. Preferably, the nucleotide sequence of the native or non-native gene has about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher nucleotide sequence identity to at least one R gene or regulator nucleotide sequence of the 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 not known to be able to confer resistance to plant diseases in a plant. It should be appreciated that when the modified native or non-native gene is stably incorporated into the genome of a plant, the plant produced by genome engineering as disclosed herein is a stably transformed plant.

[0082] Methods for stable and transient transformation of plants and for genome editing are disclosed elsewhere herein or are otherwise known in the art. In one embodiment of the invention, the plant is a stably transformed soybean plant that comprises the heterologous polynucleotide of the invention stably incorporated into its respective genome and further comprises enhanced resistance to at least one race of Phakopsora pachyrhizi that is known to cause ASR. In a preferred embodiment of the invention, such stably transformed plants comprise resistance to multiple races of Phakopsora pachyrhizi that are known to cause ASR.

[0083] In certain embodiments, the plants of the invention comprise a heterologous polynucleotide that comprises a nucleotide sequence encoding an R protein and / or a regulator protein of the invention and a heterologous promoter operably linked for expression of the nucleotide sequence encoding the R protein or regulator protein. The choice of heterologous promoter can depend on many factors, such as the desired time of expression, localization and pattern, and responsiveness to specific biotic or abiotic stimuli. Promoters of interest include, but are not limited to, pathogen-inducible promoters, constitutive promoters, tissue-preferred promoters, wound-inducible promoters, and chemically regulatable promoters.

[0084] The present invention also provides methods for enhancing the resistance of soybean plants to ASR caused by Phakopsora pachyrhizi. These methods include introducing the heterologous polynucleotides of the present invention into at least one soybean plant cell. In certain embodiments, the heterologous polynucleotides are stably incorporated into the genome of the soybean plant cell. If desired, these methods may further include regenerating the plant cell into a plant that contains the heterologous polynucleotide in its genome. Preferably, such regenerated soybean plants contain enhanced resistance to ASR caused by at least one race of Phakopsora pachyrhizi. More preferably, such regenerated soybean plants contain enhanced resistance to ASR caused by two, three, four, five, six, seven, eight, nine, ten or more races of Phakopsora pachyrhizi.

[0085] In other embodiments, plants containing heterologous polynucleotides are produced using the methods of the present invention that involve genome editing to modify the nucleotide sequence of a native or non-native gene in the plant genome, the heterologous polynucleotide containing the R gene nucleotide sequence and / or regulator gene of the present invention. The native or non-native gene contains a nucleotide sequence that is different (i.e., not identical) from the R gene or regulator gene nucleotide sequence of the present invention, and after modification by the methods disclosed in further detail below, the modified native or non-native gene contains the R gene or regulator nucleotide sequence of the present invention. Generally, such methods include using a plant that contains a native or non-native gene in its genome, where the native or non-native gene contains a nucleotide sequence that is homologous to the R gene or regulator nucleotide sequence of the present invention, and such methods further include introducing a nucleic acid molecule containing at least a portion of the R gene or regulator nucleotide sequence of the present invention into the plant. Preferably, the nucleotide sequence of the native or non-native gene contains about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher nucleotide sequence identity to at least one R gene or regulator 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 capable or not known to be capable of conferring resistance to plant diseases in plants. It should be recognized that when the modified native or non-native gene is stably incorporated into the genome of the plant, the plants produced by genome engineering as disclosed herein are stably transformed plants.

[0086] Methods for stable and transient transformation of plants and genome editing are disclosed elsewhere herein or are otherwise known in the art. In one embodiment of the present invention, the plant is a stably transformed soybean plant that contains the heterologous polynucleotides of the present invention stably incorporated into their respective genomes and also contains enhanced resistance to at least one race of Phakopsora pachyrhizi known to cause ASR. In a preferred embodiment of the present invention, such stably transformed plants contain resistance to multiple races of Phakopsora pachyrhizi known to cause ASR.

[0087] The plants disclosed herein can be used in methods for limiting Asian soybean rust (ASR) caused by at least one race of Phakopsora pachyrhizi in agricultural crop production, particularly in regions where ASR is prevalent and is known or at least likely to have a negative impact on agricultural yields. The methods of the invention include planting the soybean seeds of the invention, wherein the seeds comprise at least one R gene nucleotide sequence of the invention and optionally comprise at least one regulator gene nucleotide sequence of the invention. These methods also include growing the plants derived from the seeds under conditions favorable for plant growth and development and optionally harvesting at least one seed from the plant.

[0088] The invention further provides methods for identifying plants, particularly soybean plants, comprising the R gene or regulator gene nucleotide sequences of the invention. These methods can be used to breed soybean plants resistant to ASR caused by Phakopsora pachyrhizi. Such resistant plants can be used in the agricultural production of soybean seeds for human or livestock consumption or other uses. These methods include detecting the presence of the R gene and / or regulator gene nucleotide sequences of the invention in the plant or at least a part or cell thereof. In some embodiments of the invention, detecting the presence of the R gene and / or regulator gene nucleotide sequences comprises detecting the complete R gene or regulator gene nucleotide sequence in genomic DNA isolated from the soybean plant. However, in preferred embodiments, detecting the presence of the R gene or regulator gene nucleotide sequence comprises detecting the presence of at least one marker within the R gene or regulator gene nucleotide sequence, respectively. In other embodiments of the invention, detecting the presence of the R gene or regulator gene comprises detecting the presence of the R protein encoded by the R gene nucleotide sequence or the regulator protein encoded by the regulator gene nucleotide sequence using, for example, an immunological detection method involving an antibody preparation that specifically binds to the R protein or regulator protein.

[0089] In a method for identifying a plant, particularly a soybean plant, comprising the nucleotide sequence of an R gene or a regulator gene of the present invention, detecting the presence of the nucleotide sequence of the R gene and / or the regulator gene in the soybean plant may involve one or more of the following molecular biology techniques 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 nucleotide sequence of the R gene and / or the regulator gene and / or a marker by PCR amplification, sequencing the nucleic acid molecules comprising the nucleotide sequence of the R gene and / or the regulator gene and / or a marker, identifying the nucleotide sequence of the R gene and / or the regulator gene, a marker or one or more transcripts of the nucleotide sequence of the R gene and / or transcripts of the nucleotide sequence of the regulator gene by nucleic acid hybridization, and performing an immunological assay to detect the R protein encoded by the nucleotide sequence of the R gene and / or the regulator gene. It should be recognized that oligonucleotide probes and PCR primers can be designed to identify the nucleotide sequence of the R gene and / or the regulator gene of the present invention, and such probes and PCR primers can be used in the methods disclosed elsewhere herein or otherwise known in the art to rapidly identify one or more plants in a plant population that contain the nucleotide sequence of the R gene and / or the regulator gene of the present invention.

[0090] Depending on the desired result, the heterologous polynucleotide of the present invention can be stably incorporated into the genome of a plant cell or unstably incorporated into the genome of a plant cell. For example, if the desired result is to produce a stably transformed plant with enhanced resistance to ASR caused by Phakopsora pachyrhizi, the heterologous polynucleotide can be, for example, fused to a plant transformation vector suitable for stably incorporating the heterologous polynucleotide into the genome of a plant cell. Generally, stably transformed plant cells will regenerate into transformed plants that contain the heterologous polynucleotide in their genome. Such stably transformed plants are capable of transmitting the heterologous polynucleotide to progeny plants through sexual reproduction and / or asexual reproduction. For monocotyledonous and dicotyledonous plants, plant transformation vectors, methods for stably transforming plants with the introduced heterologous polynucleotide, and methods for regenerating plants from transformed plant cells and tissues are generally known in the art or described elsewhere herein.

[0091] In other embodiments of the invention where stable incorporation of the heterologous polynucleotide into the genome of the plant is not desired, transient transformation methods can be utilized to introduce the heterologous polynucleotide into one or more plant cells of the plant. Such transient transformation methods include, for example, virus-based methods involving the use of viral particles or at least viral nucleic acids. Generally, such virus-based methods involve constructing a modified viral nucleic acid comprising the heterologous polynucleotide of the invention operably linked to the viral nucleic acid, and then contacting the plant 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 the modified viral nucleic acid can be applied to the plant or parts thereof, for example, according to conventional methods used in agriculture (e.g., by spraying, irrigation, dusting, etc.). The modified virus and / or the modified viral nucleic acid can be applied by spraying, atomizing, dusting, broadcasting, or pouring in the form of a directly sprayable solution, powder, suspension or dispersion, emulsion, oil dispersion, paste, dustable product, material for spreading, or granules. It should be appreciated that preparations comprising the modified virus and / or the modified viral nucleic acid may need to be prepared prior to application to the plant or one or more of its parts. Methods for preparing pesticidal formulations are generally known in the art or described elsewhere herein.

[0092] The present invention provides nucleic acid molecules comprising at least one R gene nucleotide sequence, nucleic acid molecules comprising at least one regulator gene nucleotide sequence, and nucleic acid molecules comprising at least one R gene nucleotide sequence and at least one regulator gene nucleotide sequence. Preferably, the nucleic acid molecule comprising at least one R gene nucleotide sequence is capable of conferring resistance to ASR caused by at least one race of Phakopsora pachyrhizi to a host plant, particularly a soybean plant. Preferably, the nucleic acid molecule comprising at least one regulator gene nucleotide sequence is capable of regulating the activity of the R protein encoded by the R gene when co-expressed with the R gene of the present invention in a plant. Thus, such nucleic acid molecules can be used to limit plant diseases caused by ASR caused by at least one race of Phakopsora pachyrhizi in agricultural production. The nucleic acid molecules of the present invention include, but are not limited to, nucleic acid molecules comprising the R gene and / or regulator gene nucleotide sequences disclosed herein, as well as additional paralogs, orthologs, and other variants of the R gene and regulator gene nucleotide sequences. Preferably, such paralogs, orthologs, and other variants of the R gene of the present invention are capable of conferring resistance to plant diseases caused by ASR caused by at least one race of Phakopsora pachyrhizi to the plant. Preferably, such paralogs, orthologs, and other variants of the regulator gene of the present invention are capable of regulating the activity of the R protein encoded by at least one R gene of the present invention in the plant.

[0093] Methods for determining the resistance of soybean plants to ASR caused by at least one race of Phakopsora pachyrhizi and for determining the ability of a regulator gene to modulate the activity of an R protein encoded by an R gene are known in the art or are disclosed elsewhere herein, including, for example, the assays described below.

[0094] Methods are also provided for introducing one or more nucleic acid molecules of the invention into a plant, particularly a soybean plant. These methods include crossing (i.e., cross-pollinating) a first plant that contains at least one copy of the nucleic acid molecule of the invention in its genome with a second plant that lacks the nucleic acid molecule in its genome. In such methods, the first plant or the second plant can be the pollen donor plant. For example, if the first plant is the pollen donor plant, the second plant is the pollen recipient plant. Similarly, if the second plant is the pollen donor plant, the first plant is the pollen recipient plant. After the cross, the pollen recipient plant is grown under conditions favorable for plant growth and development for a sufficient time for the seeds to mature or reach the desired growth stage for subsequent in vitro germination procedures, such as embryo rescue described below. The seeds can then be harvested and those seeds containing the nucleic acid molecule can be identified by any method known in the art, including, for example, the methods described elsewhere herein for identifying plants containing the nucleotide sequence of an R gene or a regulator gene of the invention.

[0095] Methods are also provided for introducing the nucleic acid molecules of the invention into leguminous plants that lack the nucleic acid molecule in their genomes. Such nucleic acid molecules can include, for example, any one or more of the foregoing genomic sequences or regions that contain an R gene (particularly CG1). Such nucleic acid molecules include, but are not limited to, nucleic acid molecules that contain the nucleotide sequences shown in SEQ ID NO: 1, 2, 3, 4, 9, 10, 11, 12, 13, 18, 19, 20, 21, 22, and / or 27. Preferred nucleic acid molecules are those that contain a nucleotide sequence encoding CG1, which nucleotide sequence includes, but is not limited to, the nucleotide sequences shown in SEQ ID NO: 1, 2, 9, 18, and 27. These methods include crossing (i.e., outcrossing) a first leguminous plant that contains at least one copy of the nucleic acid molecule of the invention in its genome with a second leguminous plant that lacks the nucleic acid molecule in its genome. The first leguminous plant and the second leguminous plant can be the same leguminous species or can be different leguminous species. For example, the first leguminous plant can be the Glycine soja germplasm PI 653478, and the second leguminous plant can be Glycine max. Such crossing of a first species of plant with a second species of plant is called an interspecific cross and can be used to introgress one or more genes of interest (e.g., CG1) from one species into a related species that lacks one or more genes of interest and generally involves backcrossing the progeny to the related species for multiple generations and selecting progeny that contain one or more genes of interest in each generation. Such interspecific crosses, gene introgression, and backcrossing methods are well known in the art and can be used in the methods of the invention and have been reported for Glycine soja and Glycine max (Grant et al., 1986, J. Heredity 77(6): 423-426, doi.org / 10.1093 / oxfordjournals.jhered.a110274). 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.

[0096] In the method of the invention for introducing a nucleic acid molecule of the invention into a leguminous plant lacking the nucleic acid molecule in the genome, the first leguminous plant or the second leguminous plant can be a pollen donor plant. For example, if the first leguminous plant is the pollen donor plant, then the second leguminous plant is the pollen receptor plant. Similarly, if the second leguminous plant is the pollen donor plant, then the first leguminous plant is the pollen receptor plant. After hybridization, the pollen receptor plant is grown under conditions favorable for plant growth and development and for a sufficient time to allow the seeds to mature or reach the desired growth stage for subsequent in vitro germination processes, such as embryo rescue described below. The seeds can then be harvested and those seeds containing the nucleic acid molecule of the invention can be identified by any method known in the art, including, for example, the methods for identifying leguminous plants containing an R gene against plant diseases caused by ASR described elsewhere herein.

[0097] However, it should be recognized that in certain embodiments of the invention involving interspecific hybridization, it may be advantageous to harvest the seeds resulting from such interspecific hybridization at an immature growth stage and then germinate the immature seeds in culture (i.e., in vitro), thereby using the "embryo rescue" method known in the art to germinate the seeds in culture. See Reed (2005) "Embryo Rescue", Plant Development and Biotechnology, edited by Trigiano and Gray, 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 commonly used when few or no mature seeds resulting from interspecific hybridization germinate, thereby rarely or never producing interspecific hybrid plants.

[0098] Plants of interest are plant species susceptible to the Asian soybean rust fungus caused by Phakopsora pachyrhizi. Such plants of interest include, for example, leguminous plants in the subfamily Faboideae (Caesalpiniaceae; also known as Fabaceae). Preferred plants are plants in the subfamily Faboideae that are used as food for humans and / or other animals (such as livestock, fish), including but not limited to common dry beans (such as field beans, kidney beans, navy beans, pinto beans), common fleshy beans, broad beans, hyacinth beans, lima beans, mung beans, winged beans, yam beans, black-eyed peas, cowpeas, red clover, white clover, fenugreek, lupins, garden peas, field peas, pigeon peas, soybeans, jack beans, black beans, sweet clover, and soybeans. In a preferred embodiment of the invention, the plant of interest is soybean.

[0099] The leguminous plants of the present invention include, for example, leguminous plants that are hosts of Phakopsora pachyrhizi. Such leguminous plants include, but are not limited to, common dry beans (e.g., field beans, kidney beans, navy beans, pinto beans; Phaseolus vulgaris var.), common fleshy beans (e.g., green beans, fava beans, snap beans, and yellow podded beans; Phaseolus vulgaris var.), fava or broad bean (Vicia faba), hyacinth bean or lablab (Lablab purpureus), lima bean (Phaseolus lunatus var. lunatus), mung bean (Vigna radiata), runner bean (Phaseolus coccineus), winged bean or goa bean (Psophocarpus tetragonolobus), yam bean (Pachyrhizus ahipa, P. erosus), black-eyed pea, cowpea or long bean (Vigna unguiculata), calopo (Calopogonium mucunoides), alysicarp or single-leaf bean (Alysicarpus vaginalis), crimson clover (Trifolium incarnatum), yellow clover (Trifolium aureum), spiny clover (Trifolium lappaceum), white clover (Trifolium repens), crotalaria (Crotalaria anagyroides, C. spectabilis), crown vetch (Securigera varia), fenugreek (Trigonella foenum-graicum), Florida beggarweed (Desmodium tortuosum), kudzu (Pueraria montana var. lobata), lespedeza (Lespedeza spp., Japanese clover, roundleaf lespedeza), lupine (Lupinus spp.), alfalfa (Medicago spp.), milk vetch (Astragalus cicer, A. glycyphyllos), garden pea (Pisum sativum), giant sesbania or Colorado River hemp (Sesbania exaltata), pigeon pea (Cajanus cajan), purple butterfly pea (Macroptilium atropurpureum), soybean (Glycine max), sword bean (Canavalia gladiata), lotus (Nelumbo spp.), black gram (Vigna mungo), neonotonia (Neonotonia wightii), hairy vetch (Vicia villosa subsp. varia), and sweet clover (Melilotus officinalis).

[0100] The method of the present invention can be used to produce plants that have enhanced resistance to plant diseases caused by ASR caused by at least one race of Phakopsora pachyrhizi. Typically, when compared to the resistance of a control plant to the same race of Phakopsora pachyrhizi, the method 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. Unless otherwise stated or obvious from the context of use, the control plant of the present invention is a plant that does not contain the nucleotide sequence of the R gene and / or regulator gene of the present invention. Preferably, the control plant is substantially the same as the plant containing the nucleotide sequence of the R gene and / or regulator gene (e.g., the same species, subspecies, and variety), except that the control does not contain the nucleotide sequence of the R gene and / or regulator gene. In some embodiments, the control will contain a heterologous control polynucleotide (e.g., a vector control) that does contain one or more R genes and / or regulator gene nucleotide sequences.

[0101] In addition, the present invention provides transformed plants, seeds, and plant cells produced by the method of the present invention and / or containing the heterologous polynucleotide of the present invention. Such a heterologous polynucleotide contains at least one R gene or regulator gene nucleotide sequence of the present invention. Also provided are progeny plants and their seeds containing the heterologous polynucleotide of the present invention. The present invention also provides fruits, seeds, leaves, stems, roots, and other plant parts produced by the transformed plants and / or progeny plants of the present invention, as well as food products and other agricultural products containing the plant or any one or more of its parts (including but not limited to fruits, leaves, stems, roots, and seeds) or produced or derived from the plant or any one or more of its parts. 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).

[0102] The term "plant" is intended to cover plants at any stage of maturity or development, as well as any cell, tissue, or organ (plant part) obtained or derived from any such plant, unless the context clearly indicates otherwise. Plant parts include but are not limited to fruits, stems, tubers, roots, flowers, ovules, stamens, leaves, embryos, meristematic regions, callus, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, etc. The present invention also includes seeds produced by the plants of the present invention.

[0103] The present invention provides nucleic acid molecules capable of conferring resistance to ASR caused by at least one race of Phakopsora pachyrhizi to plants, particularly soybean plants, plants and plant cells containing such nucleic acid molecules, and related methods.

[0104] In a preferred embodiment of the present invention, a nucleic acid molecule comprising at least one R gene nucleotide sequence is capable of conferring resistance to ASR in soybean plants caused by at least two races of Phakopsora pachyrhizi. In a more preferred embodiment, a nucleic acid molecule comprising at least one R gene nucleotide sequence is capable of conferring resistance to plant diseases caused by three, four, five, six, seven, eight, nine, ten or more races of Phakopsora pachyrhizi.

[0105] In one embodiment of the present invention, the nucleotide sequence encoding the R protein has at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the complete nucleotide sequence or a fragment thereof shown in SEQ ID NO: 2, 5, 11, 14, 20, 23 and 32.

[0106] In another embodiment of the present invention, the nucleotide sequence encoding the regulator protein has at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the complete nucleotide sequence or a fragment thereof shown in SEQ ID NO: 4, 7, 13, 16, 22, 25 and 28.

[0107] The present invention encompasses isolated or substantially purified polynucleotide (also referred to herein as "nucleic acid molecule", "nucleic acid", etc.) or protein (also referred to herein as "polypeptide") compositions. An "isolated" or "purified" polynucleotide or protein or a biologically active portion thereof is substantially or essentially free of components that are normally associated with or interact with the polynucleotide or protein as found in its natural 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. Preferably, an "isolated" polynucleotide is free of 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 (preferably protein-coding sequences). For example, in various embodiments, an 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 sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes a protein preparation having less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein. When the proteins of the present invention or biologically active portions thereof are produced recombinantly, preferably the culture medium represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or chemical substances that are not the protein of interest.

[0108] The present invention also encompasses fragments and variants of the disclosed polynucleotides and the proteins encoded thereby. A "fragment" refers to a portion of a polynucleotide or a portion of an amino acid sequence and thus refers to a protein encoded thereby. A polynucleotide fragment that contains a coding sequence can encode a protein fragment that retains the biological activity of the full-length or native protein. A polynucleotide fragment that contains a promoter sequence can retain the biological activity of the full-length or native promoter (i.e., be capable of driving the expression of an operably linked nucleotide sequence). A polynucleotide fragment that contains a bidirectional promoter sequence can retain the biological activity of the full-length or native promoter in one or both directions. Alternatively, a polynucleotide fragment that is used as a hybridization probe generally does not encode a protein that retains biological activity or does not retain promoter activity. Thus, fragments of a nucleotide sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides and up to the full-length polynucleotide of the present invention.

[0109] "Variant" is intended to mean a substantially similar sequence. For polynucleotides, variants include polynucleotides having deletions (i.e., truncations) at the 5' and / or 3' end; deletions and / or additions of one or more nucleotides at one or more internal sites in the native polynucleotide; and / or substitutions of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, "native" polynucleotide or polypeptide encompasses a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that encode the amino acid sequence of one of the R proteins of the invention due to the degeneracy of the genetic code. Naturally occurring allelic variants such as these can be identified with well-known molecular biology techniques (e.g., polymerase chain reaction (PCR) and hybridization techniques as described below). Variant polynucleotides also include synthetically derived polynucleotides, such as those generated by site-directed mutagenesis but still encoding an R protein of the invention. Typically, 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 higher sequence identity to that particular polynucleotide, as determined by sequence alignment programs and parameters as described elsewhere herein. In certain embodiments of the invention, 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 higher sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5, 7, 9-14, 16, 18-23, 25, 27-32 and 33, 35, 37 and 39, and optionally contain a non-naturally occurring nucleotide sequence that differs from the nucleotide sequences shown in SEQ ID NOs: 1-5, 7, 9-14, 16, 18-23, 25, 27-32 and 33, 35, 37 and 39 by at least one nucleotide modification selected from the group consisting of substitution of at least one nucleotide, addition of at least one nucleotide, and deletion of at least one nucleotide. It should be understood that the addition of at least one nucleotide can be the addition of one or more nucleotides within the nucleotide sequence of the invention (e.g., SEQ ID NOs: 1-5, 7, 9-14, 16, 18-23, 25, 27-32 and 33, 35, 37 and 39), the addition of one or more nucleotides to the 5' end of the nucleotide sequence of the invention, and / or the addition of one or more nucleotides to the 3' end of the nucleotide sequence of the invention.

[0110] Variants of a particular polynucleotide of the invention (i.e., a reference polynucleotide) can also be evaluated by comparing the percent sequence identity between a polypeptide encoded by the variant polynucleotide and a polypeptide encoded by the reference polynucleotide. Thus, for example, polynucleotides encoding polypeptides having a given percent sequence identity to at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 15, 17, 24, 26, 34, 36, 38, and 40 are disclosed. The percent sequence identity between any two polypeptides can be calculated using the sequence alignment programs and parameters described elsewhere herein. When evaluating any given pair of polynucleotides of the invention by comparing 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 higher 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 higher sequence identity to at least one amino acid sequence shown in SEQ ID NO: 6, 8, 15, 17, 24, 26, 34, 36, 38, and 40 and optionally contain a non-naturally occurring amino acid sequence that is different from at least one amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 15, 17, 24, 26, 34, 36, 38, and 40, the difference being at least one amino acid modification selected from the group consisting of substitution of at least one amino acid, addition of at least one amino acid, and deletion of at least one amino acid. It should be understood that addition of at least one amino acid can be addition of one or more amino acids within the amino acid sequence of the invention (e.g., SEQ ID NO: 6, 8, 15, 17, 24, 26, 34, 36, 38, and 40), addition of one or more amino acids to the N-terminus of the amino acid sequence of the invention, and / or addition of one or more amino acids to the C-terminus of the amino acid sequence of the invention.

[0111] A "variant" protein is intended to mean a protein derived from a native protein by deletion (so-called truncation) of one or more amino acids at the N-terminus and / or C-terminus 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 can be produced, for example, by genetic polymorphism or by human manipulation. A bioactive variant of an R protein or a regulator protein will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence of the native protein (such as the amino acid sequences shown in SEQ ID NO: 6, 8, 15, 17, 24, 26, 34, 36, 38 or 40), as determined by the sequence alignment programs and parameters described elsewhere herein. A bioactive variant of the protein of the present invention can differ from the 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.

[0112] The proteins of the present invention can be modified in a variety of ways, including amino acid substitution, deletion, truncation and insertion. Methods for such manipulations are well known in the art. Methods for mutagenesis and polynucleotide alteration 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 regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), which is incorporated herein by reference. Conservative substitutions (such as exchanging one amino acid for another with similar properties) are likely to be optimal.

[0113] Accordingly, the genes and polynucleotides of the present invention include naturally occurring sequences as well as mutant and other variant forms. Similarly, the proteins of the present invention encompass naturally occurring proteins and their variants and modified forms. More preferably, variants of the R protein confer resistance to ASR in a plant or a part thereof containing the variant, caused by at least one race of Phakopsora pachyrhizi, and variants of the regulator protein are capable of regulating the activity of the R protein in a plant. In some embodiments, the mutations made in the DNA encoding the variant do not place the sequence out of the reading frame. Desirably, the mutations do not create complementary regions that could give rise to secondary mRNA structures. See EP Patent Application Publication No. 75,444.

[0114] Deletions, insertions, and substitutions of the protein sequences covered herein are not expected to result in a radical change in the properties of the protein. However, when it is difficult to predict in advance the exact effect of a substitution, deletion, or insertion, one of ordinary skill in the art will understand that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by the assays disclosed below.

[0115] Variant polynucleotides and proteins also include sequences and proteins derived from mutagenesis and recombination processes 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 et 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.

[0116] The polynucleotides of the present invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this way, methods such as PCR, hybridization, etc. can be used to identify such sequences based on their sequence homology to the sequences described herein. The present invention encompasses sequences isolated based on their sequence identity to the complete sequences described herein or their variants and fragments. Such sequences include sequences that are orthologs and paralogs of the disclosed sequences. "Orthologs" are intended to mean genes derived from a common ancestral gene and found in different species due to speciation. When the nucleotide sequences of genes found in different species and / or the amino acid sequences encoded by them share at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, these genes are considered orthologs. The functions of orthologs are generally highly conserved between species. "Paralogs" are homologous genes produced by gene duplication events in a species. Paralogs usually have similar structures and functions in related pathways and protein complexes. When the nucleotide sequences of genes found in the same species and / or the amino acid sequences encoded by them share at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, these genes are considered paralogs.

[0117] In one embodiment, the orthologs of the present invention have a coding sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher nucleotide sequence identity to at least one nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 1, 2, 4, 5, 7, 9-11, 13, 14, 16, 18-20, 22, 23, 25, 27, 28, 32, 33, 35, 37 and 40 and / or a protein having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid sequence identity to at least one amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 6, 8, 15, 17, 24, 26, 34, 36, 38 and 40.

[0118] In the PCR method, oligonucleotide primers can be designed for use in a PCR reaction to amplify the corresponding DNA sequence from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and for PCR cloning are well known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd 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 PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specificity primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.

[0119] In hybridization techniques, all or part of a known polynucleotide is used as a probe which 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 selected organism. The hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and can be labeled with a detectable group such as 32 p or any other detectable marker. Thus, for example, a probe for hybridization can be prepared by labeling a synthetic oligonucleotide based on the polynucleotides of the present invention. Methods for preparing probes for hybridization and for constructing cDNA and genomic libraries are well known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0120] For example, the complete polynucleotides or one or more portions thereof disclosed herein can be used as probes capable of specifically hybridizing to the corresponding polynucleotides and messenger RNAs. To achieve specific hybridization under various conditions, such probes include sequences unique in the gene sequence or cDNA sequence of interest and are optimally at least about 10 nucleotides in length, most desirably at least about 20 nucleotides in length. Such probes can be used to amplify by PCR the corresponding polynucleotides of a specific gene of interest from a selected plant. This technique can be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of a coding sequence in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York)). Detailed guidelines for nucleic acid hybridization are 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 (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0121] The term "substantially identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of amino acid residues or nucleotides that are the same as or equivalent to (e.g., have similar side chains) a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common domain and / or common functional activity. For example, amino acid or nucleotide sequences that contain a common 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 substantially identical.

[0122] To determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between 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) × 100). In one embodiment, the two sequences have the same length. The percent identity between two sequences can be determined using techniques similar to the following techniques, with or without allowing gaps. When calculating percent identity, exact matches are typically counted.

[0123] Determining the percent identity between two sequences can be accomplished using a mathematical algorithm. Preferred non-limiting examples of mathematical algorithms for comparing two sequences are the algorithms of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, which is modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. This 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 the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterative search for detecting distant relationships between molecules. See Altschul et al. (1997) supra. When using the 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 for sequence comparison is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Alignment can also be performed manually by inspection.

[0124] Unless otherwise indicated, the sequence identity / similarity values provided herein refer to values obtained by multiple alignment using the full-length sequences of the present invention and the program AlignX included in the software package Vector NTI Suite version 7 (InforMax, Inc., Bethesda, MD, USA) or any equivalent program using the algorithm Clustal W (Nucleic Acid Research, 22(22):4673-4680, 1994) with default parameters. An "equivalent program" means any sequence comparison program that, for any two sequences under discussion, generates an alignment having the same nucleotide or amino acid residue matches and the same percent sequence identity when compared to the corresponding alignment generated by CLUSTALW (version 1.83) with default parameters (available at the European Bioinformatics Institute website ebi.ac.uk / Tools / clustalw / index on the World Wide Web).

[0125] The use of the terms "nucleic acid molecule" and the equivalent terms "polynucleotide molecule" and "polynucleotide" is not intended to limit the invention to nucleic acid molecules, polynucleotide molecules, and polynucleotides containing DNA. Those of ordinary skill in the art will recognize that nucleic acid molecules, polynucleotide molecules, and polynucleotides can include ribonucleotides as well as combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs. The nucleic acid molecules, polynucleotide molecules, and polynucleotides of the present invention also encompass all forms of sequences, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like.

[0126] A heterologous polynucleotide or polynucleotide construct comprising a coding region for an R protein or a regulator protein can be provided in an expression cassette for expression in a plant or other organism or non-human host cell of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the coding region for the R protein or regulator protein. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide or gene of interest and a regulatory sequence (i.e., a promoter) is a functional linkage that permits expression of the polynucleotide of interest. The elements operably linked may be contiguous or non-contiguous. When used to refer to the linkage of two protein coding regions, operably linked means that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene may be provided on multiple expression cassettes. Such expression cassettes have multiple restriction sites and / or recombination sites for insertion of the coding region to be under the transcriptional regulation of the regulatory region. The expression cassette may additionally contain a selectable marker gene.

[0127] The expression cassette will include a transcription and translation initiation region (i.e., a promoter) in the 5'-3' transcription direction, the R protein or regulator protein coding region of the present invention, and a transcription and translation termination region (i.e., a terminator) that functions in a plant or other organism or non-human host cell. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translation termination regions) and / or coding regions of the present invention can be native / similar to the host cell or similar to each other. Alternatively, the regulatory regions and / or protein coding regions of the present invention can be heterologous to the host cell or heterologous to each other.

[0128] As used herein, "heterologous" with respect to a nucleic acid molecule, polynucleotide, nucleotide sequence, or polynucleotide construct is a nucleic acid molecule, polynucleotide, nucleotide sequence, or polynucleotide construct that is derived from a foreign species, or, if from the same species, has been modified in composition and / or genomic locus from its native form 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 derived polynucleotide is obtained, or, if from the same / similar species, one or both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter of the operably linked polynucleotide. As used herein, a chimeric gene contains a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0129] As used herein, a "native gene" is intended to mean a gene that is naturally present in its native or natural position in the plant genome. Such a native gene has not been genetically engineered or otherwise modified in nucleotide sequence and / or in its position in the plant genome by human intervention, nor has such a native gene been introduced into the plant genome by artificial means such as plant transformation.

[0130] 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 contains a nucleotide sequence that is not naturally present in the plant. Non-native genes include, for example, genes introduced into a plant by plant transformation methods (e.g., R genes). Additionally, when a native gene in a plant genome is modified, for example, by a genome editing method, to contain a nucleotide sequence that is different (i.e., not identical) from the nucleotide sequence of the native gene, the modified gene is a non-native gene.

[0131] The present invention provides a host cell comprising at least one of the nucleic acid molecule, expression cassette, and vector of the present invention. In a preferred embodiment of the present invention, the host cell is a plant cell. In other embodiments, the host cell is selected from the group consisting of bacteria, fungal cells, and animal cells. In certain embodiments, the host cell is a non-human animal cell. However, in some other embodiments, the host cell is a human cell cultured in vitro.

[0132] Although expression of an R protein or regulator protein using a heterologous promoter may be optimal, the native promoter of the corresponding R gene or regulator gene can be used.

[0133] The terminator region can be native to the transcriptional initiation region, native to the protein-coding region of interest to which it is operably linked, native to the plant host, or can be derived from another source (i.e., foreign or heterologous to the promoter, protein of interest, and / or plant host), or any combination thereof. Convenient terminator regions can be obtained from the Ti plasmid of Agrobacterium tumefaciens, such as the octopine synthase (OCS) and nopaline synthase terminator regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0134] Where appropriate, the polynucleotide can be optimized for increased expression in the transformed plant. That is, the polynucleotide can be synthesized using plant-preferred codons to improve expression. See, e.g., Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods for synthesizing plant-preferred genes are available in the art. See, e.g., U.S. Patent Nos. 5,380,831 and 5,436,391 and Murray et al. (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference.

[0135] Additional sequence modifications are known to enhance gene expression in cellular hosts. These include elimination of sequences encoding false polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be detrimental to gene expression. The G-C content of the sequence can be adjusted to the average level of a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0136] Expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can function to enhance translation. Translational leader sequences are known in the art and include: picornavirus leader sequences, such as the EMCV leader sequence (encephalomyocarditis 5′ non-coding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leader sequences, such as the TEV leader sequence (tobacco etch virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader sequence (maize dwarf mosaic virus) (Virology 154:9-20), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); the untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader sequence (TMV) (Gallie et al. (1989), Molecular Biology of RNA, edited by Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader sequence (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

[0137] In preparing expression cassettes, various DNA fragments may be manipulated so as to provide the DNA sequences in the correct orientation and, where appropriate, in the correct reading frame. To this end, linkers or adaptors may be employed to join the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove superfluous DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, substitution, such as transitions and transversions, may be involved.

[0138] Many promoters can be used in the practice of the present invention. The promoter can be selected based on the desired result. The nucleic acid can be combined with a constitutive, tissue-preferred, or other promoter for expression in plants. Such constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163_171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); ALS promoter (U.S. Patent No. 5,659,026), etc. 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.

[0139] Tissue-preferred promoters can be used to target enhanced expression of R protein-encoding sequences within specific plant tissues. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include those described in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if desired, for weak expression.

[0140] In general, it will be beneficial to express genes from inducible promoters, particularly pathogen-inducible promoters. Such promoters include those from pathogenesis-related proteins (PR proteins) that are induced after pathogen infection; e.g., PR proteins, SAR proteins, β-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, which is incorporated herein by reference.

[0141] Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9: 335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2: 325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83: 2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2: 93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93: 14972-14977. See also Chen et al. (1996) Plant J. 10: 955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91: 2507-2511; Warner et al. (1993) Plant J. 3: 191-201; Siebertz et al. (1989) Plant Cell 1: 961-968; U.S. Patent No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter of the maize PRms gene, the expression of which is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41: 189-200).

[0142] In addition, when a pathogen enters a plant through a wound or insect damage, wound-inducible promoters can be used in the heterologous polynucleotides of the present invention. Such wound-inducible promoters include the potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopathh. 28: 425-449; Duan et al. (1996) Nature Biotechnology 14: 494-498); wun1 and wun2, U.S. Patent No. 5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215: 200-208); systemin (McGurl et al. (1992) Science 225: 1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2): 141-150); etc., which are incorporated herein by reference.

[0143] Chemical-regulated promoters can be used to regulate the expression of genes in plants by applying exogenous chemical regulators. Depending on the purpose, the promoter can be a chemically inducible promoter, in which the application of a chemical substance induces gene expression, or a chemically repressible promoter, in which the application of a chemical substance represses gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter activated by the benzenesulfonamide herbicide safener, the maize GST promoter activated by a hydrophobic electrophilic compound used as a pre-emergence herbicide, and the tobacco PR-1a promoter activated by salicylic acid. Other chemically regulated promoters of interest include glucocorticoid-inducible promoters in steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoters in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237 and U.S. Patent Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[0144] The expression cassette may also contain a selectable marker gene for selecting transformed cells. The selectable marker gene is used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicide compounds such as glufosinate, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85: 610-9 and Fetter et al. (2004) Plant Cell 16: 215-28), cyan fluorescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117: 943-54 and Kato et al. (2002) Plant Physiol 1129: 913-42), and yellow fluorescent protein (PhiYFP from Evrogen TM, see Bolte et al. (2004) J. Cell Science 117: 943-54). For additional alternative markers, generally see 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), 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. Sci. 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 et al. (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653; Hillenand-Wissman (1989) Topics Mol. Struct. Biol. 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc.Natl. Acad. Sci. USA 89: 5547 - 5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36: 913 - 919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334: 721 - 724. Such disclosures are incorporated herein by reference.

[0145] The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene can be used in the present invention.

[0146] Many 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′alluin et al. (1992) Bio / Technol. 10: 309-314; Dhir et al. (1992) Plant Physiol. 99: 81-88; Casas et al. (1993) Proc. Nat. Acad Sci. USA 90: 11212-11216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P: 119-124; Davies et al. (1993) Plant Cell Rep. 12: 180-183; Dong, J. A. and Mchughen, A. (1993) Plant Sci. 91: 139-148; Franklin, C. I. and Trieu, T. N. (1993) Plant. Physiol. 102: 167; Golovkin et al. (1993) Plant Sci. 90: 41-52; Guo Chin Sci. Bull. 38: 2072-2078; Asano et al. (1994) Plant CellRep. 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. HiTech. 5: 17-27; Eapen et al. (1994) Plant Cell Rep.13:582 - 586; Hartman et al. (1994) Bio - Technology 12:919 - 923; Ritala et al. (1994) Plant. Mol. Biol. 24:317 - 325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol. 104:37 - 48.

[0147] The methods of the present invention relate to introducing a heterologous polynucleotide or polynucleotide construct into a plant. "Introducing" means presenting the heterologous polynucleotide or polynucleotide construct to the plant in such a way that the construct can enter the interior of a plant cell. The methods of the present invention do not depend on a particular method of introducing the heterologous polynucleotide or polynucleotide construct into the plant, only on the ability of the heterologous polynucleotide or polynucleotide construct to enter the interior of at least one cell of the plant. Methods of introducing a heterologous polynucleotide or polynucleotide construct into a plant are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus - mediated methods.

[0148] "Stable transformation" means that the heterologous polynucleotide or polynucleotide construct introduced into the plant integrates into the genome of the plant and can be inherited by its progeny. "Transient transformation" means that the heterologous polynucleotide or polynucleotide construct introduced into the plant does not integrate into the genome of the plant. It is recognized that stable transformation and transient transformation methods involve 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, a plant cell, or other host cell or organism.

[0149] To transform plants and plant cells, the nucleotide sequences of the present invention are inserted into any vector known in the art suitable for expressing nucleotide sequences in plants or plant cells using standard techniques. The choice of vector depends on the preferred transformation technique and the target plant species to be transformed.

[0150] Methods 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, tumor-inducing (Ti) plasmid vectors can be used to introduce foreign DNA into plants. Other methods for foreign DNA delivery include using PEG-mediated protoplast transformation, electroporation, microinjection, whiskers, and gene guns or particle bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Patent No. 5,405,765 to Vasil et al.; Bilang et al. (1991) Gene 100:247-250; Scheid et al., (1991) Mol. Gen. Genet., 228:104-112; Guerche et al., (1987) Plant Science 52:111-116; Neuhause et al., (1987) Theor. Appl Genet. 75:30-36; Klein et al., (1987) Nature 327:70-73; Howell et al., (1980) Science 208:1265; Horsch et al., (1985) Science 227:1229-1231; DeBlock et al., (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (edited by Weissbach and Weissbach) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (edited by Schuler and Zielinski) Academic Press, Inc. (1989). The transformation method depends on the plant cells to be transformed, the stability of the vector used, the expression level of the gene product, and other parameters.

[0151] Other suitable methods for introducing a nucleotide sequence into a plant cell and subsequently inserting it into the plant genome include: microinjection, as described in Crossway et al. (1986) Biotechniques 4: 320-334; electroporation, as described in Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606; Agrobacterium-mediated transformation, as described in Townsend et al., U.S. Patent No. 5,563,055, Zhao et al., U.S. Patent No. 5,981,840; direct gene transfer, as described in Paszkowski et al. (1984) EMBO J. 3: 2717-2722; and ballistic particle acceleration, as described, for example, in 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”, Plant Cell, Tissue, and Organ Culture: Fundamental Methods, edited by Gamborg and Phillips (Springer-Verlag, Berlin), McCabe et al. (1988) Biotechnology 6: 923-926); and Lec1 transformation (WO 00 / 28058). See also 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", Plant Cell, Tissue, and Organ Culture: Fundamental Methods, edited by 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) The Experimental Manipulation of Ovule Tissues, edited by 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 these documents are incorporated herein by reference.

[0152] The polynucleotides of the present invention can be introduced into plants by contacting the plants with a virus or viral nucleic acid. Generally, such methods involve incorporating the heterologous polynucleotide or polynucleotide construct of the present invention into a viral DNA or RNA molecule. In addition, it should be appreciated that the promoters of the present invention also encompass promoters for transcription by viral RNA polymerases. Methods for introducing a polynucleotide construct into a plant and expressing the 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; which are incorporated herein by reference.

[0153] If desired, the modified virus or modified viral nucleic acid can be formulated into a preparation. Such preparations are prepared in a known manner (see, for example, the reviews US 3,060,084, EP-A 707 445 (for liquid concentrates), Browning, “Agglomeration”, Chemical Engineering, December 4, 1967, 147 - 48, Perry's Chemical Engineer's Handbook, 4th Edition, McGraw-Hill, New York, 1963, page 8 - 57 and the following: WO91 / 13546, US 4,172,714, US 4,144,050, US 3,920,442, US 5,180,587, US 5,232,701, US 5,208,030, GB 2,095,558, US 3,299,566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al. Weed Control Handbook, 8th Edition, 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 formulating agrochemicals, such as solvents and / or carriers, if desired, emulsifiers, surfactants and dispersants, preservatives, defoamers, antifreeze agents, and for seed treatment preparations also optionally colorants and / or binders and / or gelling agents.

[0154] In specific embodiments, the polynucleotides, polynucleotide constructs, and expression cassettes of the invention can be provided to plants using a variety of transient transformation methods known in the art. Such methods include, for example, microinjection or particle bombardment. See, e.g., 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 incorporated herein by reference. Alternatively, polynucleotides can be transiently transformed into plants using techniques known in the art. Such techniques include viral vector systems and Agrobacterium tumefaciens-mediated transient expression, as described elsewhere herein.

[0155] The transformed cells can be grown into plants by conventional methods. See, e.g., McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown, pollinated with the same or a different strain of the transformation, and the resulting hybrids with constitutive expression of the desired phenotypic characteristics identified. The plants can be grown for two or more generations to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and then the seeds are harvested to ensure that the expression of the desired phenotypic characteristics has been achieved. In this way, the invention provides transformed seeds (also referred to as "transgenic seeds") having the heterologous polynucleotides or polynucleotide constructs of the invention (e.g., the expression cassettes of the invention) stably incorporated into their genomes.

[0156] Any method known in the art for modifying DNA in a plant genome can be used to modify a genomic nucleotide sequence in a plant, for example, to generate or insert a resistance gene or even replace or modify an endogenous resistance gene or its alleles. Such methods include, but are not limited to, genome editing (or gene editing) techniques, such as 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 these documents are incorporated herein by reference in their entirety. Methods for gene modification or gene replacement including homologous recombination can involve the use of zinc finger nucleases (ZFNs), TAL (transcription activator-like) effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats / CRISPR-associated nucleases (CRISPR / Cas nucleases), or homing endonucleases to induce double-strand breaks in DNA, which homing endonucleases have been engineered to create double-strand breaks at specific recognition sequences in the genomes of plants, other organisms, or host cells. See, e.g., Durai et al., (2005) Nucleic Acids Res 33:5978-90; Mani et al. (2005) Biochem. Biophys. Res. Comm. 335:447-57; U.S. Patent Nos. 7,163,824, 7,001,768, and 6,453,242; Arnould et al. (2006) J. Mol. Biol. 355:443-58; Ashworth et al., (2006) Nature 441:656-9; Doyon et al. (2006) J. Am. Chem. Soc. 128:2477-84; Rosen et al., (2006) Nucleic Acids Res. 34:4791-800; and Smith et al., (2006) Nucleic Acids Res. 34:e149; U.S. Patent Application Publication No. 2009 / 0133152; and U.S. Patent Application Publication No. 2007 / 0117128; all of these documents are incorporated herein by reference in their entirety.

[0157] Unless otherwise specified or obvious from the context of use, the term "gene replacement" is intended to mean the replacement of any part of a first polynucleotide molecule or nucleic acid molecule (e.g., a chromosome) involved in homologous recombination with a second polynucleotide molecule or nucleic acid molecule using a genome editing technique as disclosed elsewhere herein, whereby at least a portion of the nucleotide sequence of the first polynucleotide molecule or nucleic acid molecule is replaced by the second polynucleotide molecule or nucleic acid molecule. It should be 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 replacement of an entire gene or multiple genes, replacement of any part or multiple parts of a single gene, or replacement of non-gene sequences in the first polynucleotide molecule or nucleic acid molecule.

[0158] TAL effector nucleases (TALENs) can be used to generate double-strand breaks at specific recognition sequences in the plant genome for gene modification or gene replacement by homologous recombination. TAL effector nucleases are a class of sequence-specific nucleases that can be used to generate double-strand breaks at specific target sequences in the genome of plants or other organisms. TAL effector nucleases are generated by fusing a native or engineered transcription activator-like (TAL) effector or a functional portion thereof to the catalytic domain of an endonuclease (e.g., FokI). The unique modular TAL effector DNA-binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA-binding domain of a TAL effector nuclease can be engineered to recognize a specific DNA target site and thus be used to generate a double-strand break at the desired target sequence. See WO 2010 / 079430; Morbitzer et al. (2010) PNAS 10.1073 / pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi:10.1093 / nar / gkq704; and Miller et al. (2011) Nat. Biotechnol. 29:143-148; all of which are incorporated herein by reference.

[0159] The CRISPR / Cas nuclease system can also be used to generate double-strand breaks at specific recognition sequences in the plant genome for gene modification or gene replacement by homologous recombination. The CRISPR / Cas nuclease is an RNA-guided (single guide RNA, sgRNA for short) DNA endonuclease system that makes sequence-specific double-strand breaks in DNA segments homologous to the designed RNA. The sequence specificity can be designed (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).

[0160] In addition, ZFNs can be used to generate double-strand breaks at specific recognition sequences in the plant genome for gene modification or gene replacement by homologous recombination. Zinc finger nucleases (ZFNs) are fusion proteins that contain a portion of the FokI restriction endonuclease protein responsible for DNA cleavage and zinc finger proteins that recognize specific, designed genomic sequences and cleave double-stranded DNA at those sequences, thereby generating free DNA ends (Urnov et al. (2010) Nat. Rev. Genet. 11: 636-46; Carroll (2011) Genetics. 188: 773-82).

[0161] Breaking DNA using site-specific nucleases (such as those described above) can increase the rate of homologous recombination in the broken region. Thus, the coupling of such effectors with nucleases as described above enables the generation of targeted changes in the genome, including additions, deletions, and other modifications.

[0162] The nucleic acid molecules, expression cassettes, vectors, and heterologous polynucleotides of the present invention can be used for the transformation and / or genome editing of any plant species, including but not limited to monocotyledonous and dicotyledonous plants.

[0163] 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 intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, tubers, propagules, leaves, flowers, shoots, fruits, roots, root tips, anthers, and the like. Offspring, variants, and mutants of regenerated plants are also included within the scope of the present invention, provided that these parts contain the introduced polynucleotide. As used herein, "offspring" and "offspring plant" include any subsequent generation of a plant, whether produced by sexual reproduction and / or asexual propagation, unless otherwise expressly stated or obvious from the context of use.

[0164] As used herein, the terms "transgenic plant" and "transformed plant" are equivalent terms to "plant" as described above, wherein the plant contains a heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct introduced into the plant by any stable and transient transformation method, such as those disclosed elsewhere herein or otherwise known in the art. Such transgenic plants and transformed plants also refer to, for example, the plant in which the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct was first introduced, and any of its offspring plants that contain the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct.

[0165] In certain embodiments of the present invention, these methods involve growing seedlings, then growing such seedlings to produce plants derived therefrom, and optionally harvesting one or more plant parts from the plants. As used herein, a "seedling" refers to an immature plant that is typically grown under greenhouse or other controlled or semi-controlled (e.g., cold frame) environmental conditions and then planted or replanted outdoors or in a greenhouse to produce harvestable plant parts (e.g., seeds).

[0166] In some embodiments of the present invention, plant cells are transformed with a heterologous polynucleotide encoding the R protein and / or regulator protein of the present invention. As used herein, the term "expression" refers to the biosynthesis of a gene product, including the transcription and / or translation of the gene product. "Expressing" or "producing" 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 "expressing" or "producing" 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 encoding the R protein and regulator protein are described elsewhere herein.

[0167] The use of the terms "DNA" or "RNA" herein is not intended to limit the invention to polynucleotide molecules that contain DNA or RNA. Those of ordinary skill in the art will recognize that the methods and compositions of the invention encompass polynucleotide molecules that consist of deoxyribonucleotides (i.e., DNA), ribonucleotides (i.e., RNA), or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs, including but not limited to synthetic, naturally occurring, and non-naturally occurring nucleotide analogs or modified backbone residues or linkages that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include but are not limited to phosphorothioates, phosphoroamidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). The polynucleotide molecules of the invention also encompass all forms of polynucleotide molecules, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc. In addition, those of ordinary skill in the art will understand that the nucleotide sequences disclosed herein also encompass the complementary sequences of such exemplary nucleotide sequences.

[0168] The present invention relates to compositions and methods for enhancing plant resistance to plant diseases, particularly compositions and methods for enhancing plant resistance to ASR caused by at least one race of Phakopsora pachyrhizi. "Disease resistance" refers to a plant's avoidance of disease symptoms as a result of a plant-pathogen interaction. That is, preventing a pathogen from causing a plant disease and associated disease symptoms, or minimizing or alleviating the disease symptoms caused by the pathogen.

[0169] The present invention also provides promoters that contain nucleotide sequences of the intergenic regions between gene pairs CG1 and CG4, CG2 and CG5, and MSTRG.57 and CG3, as well as naturally occurring and synthetic or artificial variants of such intergenic sequences. It is believed that the intergenic regions are promoters, particularly bidirectional promoters that can drive the expression of two paired R genes, which are arranged in a head-to-head orientation in the genome of the Glycine soja germplasm PI 653478A (Figure 2C). Such promoters include, for example, promoters that contain nucleotide sequences selected from the group consisting of SEQ ID NO: 3, 12, and 21, as well as naturally occurring and synthetic or artificial variants of such intergenic sequences or fragments of such promoters that are capable of driving the expression of an operably linked nucleotide sequence in a plant cell. Examples of such fragments include but are not limited to fragments that contain the nucleotide sequences shown in SEQ ID NO: 29, 30, and 31.

[0170] Preferably, the promoter of the present invention is a bidirectional promoter or a pathogen-inducible promoter. More preferably, the promoter of the present invention is a bidirectional promoter and a pathogen-inducible promoter. Methods for determining promoter activity are known in the art or disclosed elsewhere herein, including, for example, bidirectional promoter activity and pathogen-inducible promoter activity.

[0171] The following examples are provided by way of illustration and not limitation.

[0172] Examples

[0173] Example 1: Identification of candidate ASR resistance genes

[0174] The F2 population was generated by crossing two Glycine argyrea germplasms: PI 653478 A (a germplasm known to be resistant to Phakopsora pachyrhizi) and PI 595798 (a germplasm known to be susceptible to Phakopsora pachyrhizi). PI 653478 A has been reported to be resistant to nine Australian races of Phakopsora pachyrhizi (Jarosz and Burdon (1990) Heredity 64: 347-353, doi.org / 10.1038 / hdy.1990.43). In addition, the resistance of PI 653478 A and PI 595798 to Phakopsora pachyrhizi isolates PPUFV02, Aus-1, K1-2, and T1-4 has been tested. PI 653478 A was resistant to each isolate, and PI 595798, PI 653478 A was resistant to each isolate (data not shown).

[0175] Bulked segregant analysis and QTL mapping were performed on the resulting F2 segregating population to identify the major resistance QTL, designated GaRpp1. The resistance region was mapped to linkage group 3 of Glycine argyrea, which is syntenic with soybean Chr03.

[0176] Using PacBio single molecule real-time (SMRT) sequencing (available on the World Wide Web at pacb.com) and the Dovetail Genomics (Santa Cruz, CA, US) Hi-C scaffolding method (Genome Res. March 2016; 26(3): 342-350. doi: 10.1101 / gr.193474.115), a high-quality genome was assembled for the resistant genotype PI 653478 A, generating a 1.02 Gb genome in 690 large scaffolds. All markers associated with the resistance locus were mapped to contig 17, providing a complete overview of the physical map.

[0177] The first round of fine mapping of additional F2 recombinants using the Phakopsora pachyrhizi isolate PPUFV02 delimited the resistance interval to the region between markers GA_155 and GA_206 (Figure 2A, represented by two loss-of-function recombinants Gargy_0292 and Gargy_0059). Screening of the progeny of F2 and F3 recombinants for this genomic interval ultimately narrowed the GaRpp1 resistance locus to a 109.6 Kb region between flanking markers CAPS_CG2 and GA_204 (Figure 2B). The presence of a single copy of the resistance allele in this interval was sufficient to confer resistance to plants 14 days after inoculation with the PPUFV02 isolate. Recombinants that did not carry any copies of the resistance allele within this interval were susceptible.

[0178] Four candidate genes were present in this interval, named: GaRpp1_CG1, GaRpp1_CG4, GaRpp1_CG5, and GaRpp1_CG10. Marker CAPS_CG2 was developed on the sequence of candidate gene GaRpp1_CG2, thus excluding this gene as a resistance candidate (Figure 2C).

[0179] Example 2: Candidate gene validation by virus-induced gene silencing

[0180] The virus-induced gene silencing (VIGS) protocol using the Bean pod mottle virus (BPMV) described by Whitham et al. ((2016) Curr. Protoc. Plant Biol. 1:263-283, doi:10.1002 / cppb.20012) was optimized for Glycine soja. To silence candidate genes for functional validation, fragments of the GaRpp1 candidate genes were cloned into the BPMV vector to develop four constructs: BPMV:cg1 (targeting GaRpp1_CG1), BPMV:cg2 (targeting GaRpp1_CG2), BPMV:cg1-2 (targeting CG1 and CG2), and BPMV:cg3-9 (simultaneously targeting CG 3, 4, and 5).

[0181] The VIGS assay was performed using the resistant genotype Gargy_1900, which was derived from a cross between PI 653478 A and PI 595798 and is known to carry the resistance allele GaRpp1. The Gargy_1900 genotype was used instead of the resistant germplasm PI 653478 A for the VIGS experiment to avoid the possibility that PI 653478 A has other genetic resistance genes to Phakopsora pachyrhizi that could interfere with the results of silencing GaRpp1.

[0182] Initial VIGS assays were performed using constructs BPMV:cg2 and BPMV:cg3-9 against the resistant genotype Gargy_1900. After inoculation with Phakopsora pachyrhizi, the silenced leaves remained resistant. However, silencing experiments targeting GaRpp1_CG1 with two different constructs, BPMV:cg1 and BPMV:cg1-2, resulted in clear sporulation at 13 dpi in the silenced leaves (Figure 3).

[0183] At 13 dpi with PPUFV02, the leaves of Gargy_1900 plants silenced with BPMV:cg1 and BPMV:cg1-2 showed a susceptible phenotype( Figures 3A to 3D ), similar to the susceptible control PI 595798( Figure 3H ). The leaves of Gargy_1900 plants infected with BPMV:cg3-9 and with the BPMV:EV construct remained resistant to soybean rust( Figures 3E to 3G ). These results indicate that GaRpp1_CG1 is the resistance gene GaRpp1.

[0184] Example 3: Validation by stable transformation of GaRpp1_CG1 in soybean

[0185] Transgenic soybean plants were generated by transforming the susceptible genotype Williams 82 with the coding sequence (CDS) of the resistance gene GaRpp1_CG1 driven by a high-expression promoter. Transgenic plants transformed with three constructs, "Construct-4", "Construct-6", and "Construct-7", showed a significant reduction in disease severity( Figure 4 ).

[0186] All events transformed with Construct-4 exhibited a "slow rusting" phenotype, characterized by a delayed latency period, smaller lesion size, and reduced sporulation at 14 dpi. Transformants with Construct-6 showed a 36%-50% reduction in disease severity in three events( Figure 4 , medium gray bars). Events transformed with Construct-7 differed in the severity of the disease phenotype (ranging from high to low) and the level of sporulation caused by the disease (ranging from normal sporulation to no sporulation, Figure 5 ).

[0187] Transformation with Construct-4 resulted in moderate expression of the transgene( Figure 4 , light gray bars), Construct 6 resulted in high expression( Figure 4 , medium gray bars), and Construct-7 resulted in moderate targeted expression( Figure 4 , black bars).

[0188] Constitutive expression of GaRpp1_CG1 at moderate to high levels results in stunted plant growth, strange branching, leaf malformation, and strong transmission bias, imposing negative selection on transgene transmission.

[0189] The deleterious effects of GaRpp1_CG1 overexpression suggest that it requires strict transcriptional regulation, which may involve GaRpp1_CG4 and the intergenic region between CG1 and CG4 (bidirectional promoter). Additional constructs containing genomic sequences spanning GaRpp1_CG1 and CG4, including the native intergenic region, are generated. Transgenic soybean plants are produced by transforming the susceptible genotype Williams 82 with constructs containing genomic sequences of GaRpp1_CG1, the intergenic region, and GaRpp1_CG4. When testing the ASR resistance of the transgenic plants, the transgenic plants exhibit enhanced resistance to ASR relative to the susceptible control plants.

[0190] Example 4: Genomic orientation of GaRpp1_CG1 and GaRpp1_CG4 indicates that these genes are expressed by a shared bidirectional promoter

[0191] The genes GaRpp1_CG1 and GaRpp1_CG4 are arranged in a head-to-head orientation in the genome, sharing a short intergenic region (306 bp from the start of the transcription initiation site of one gene to the other), Figure 6 )). This arrangement coupled with the short intergenic region may suggest that the short intergenic region contains a bidirectional promoter that drives the expression of both genes and can be regulated by pathogen infection.

[0192] Bidirectional promoters include the intergenic region of two adjacent genes located on complementary DNA strands, driving their transcription in opposite directions (Arnaiz et al., 2019). Bidirectional promoters are prevalent in eukaryotes including plants. Genome-wide analysis of Arabidopsis bidirectional promoters identified 2,471 bidirectional gene pairs and found that they are generally co-expressed and tend to be involved in the same biological functions (Wang et al., 2009).

[0193] Example 5: GaRpp1 locus in other Glycine soja germplasms

[0194] Five additional Glycine soja accessions (PI509452, PI509452, PI599400, PI595794, and PI595795) that exhibited contrasting phenotypes after inoculation with Phakopsora pachyrhizi were sequenced, and reads were mapped to the GaRpp1 locus of the reference assembly of PI 653478 A. We observed that the upstream region of GaRpp1_CG1, which contains intergenic sequence and the GaRpp1_CG4 gene, was conserved in two resistant Glycine soja accessions but not in three susceptible accessions tested (data not shown). The fact that the GaRpp1_CG4 sequence, as well as the intergenic sequence, is conserved in resistant accessions but not in susceptible accessions may indicate their importance for the proper activity of GaRpp1_CG1.

[0195] Example 6: Transient expression of GaRpp1 gene in Nicotiana benthamiana

[0196] Agrobacterium-mediated transient expression of GaRpp1_CG1 in Nicotiana benthamiana elicited a hypersensitive response (HR) at 2 to 3 dpi. HR is a common and effective mechanism for plants to defend against pathogens, but the fact that CG1 expression triggers HR in the absence of pathogens (autoactivity) suggests that CG1 may require regulators to limit its own activity.

[0197] Given that GaRpp1_CG1 and GaRpp1_CG4 are arranged head-to-head in the genome and may share a bidirectional promoter as described in Example 2 above (see also Figure 6 ), GaRpp1_CG4 was co-infiltrated with GaRpp1_CG1. The results can be visualized in Figure 7 . CG1 triggered an HR response when infiltrated alone, while GaRpp1_CG2 and CG4 did not (no autoactivity was observed). After co-expression with CG4, CG1 did not elicit an HR response, indicating that CG4 can regulate the activity of CG1 in plants.

[0198] For infiltration, the Phytophthora sojae effector PsCRN63 was used as a positive control for the HR response, and the empty vector was used as a negative control. All genes were under the regulation of the Lotus japonicus LjUBI promoter.

[0199] Although the present invention does not depend on a specific biological mechanism, the transient expression results are consistent with a biological mechanism in which GaRpp1_CG1 and GaRpp1_CG4 act together to confer ASR resistance to plants. The VIGS results of the above BPMV:cg3-9 construct are also consistent with this biological mechanism, since silencing a positive regulator of the resistance gene does not result in susceptibility.

[0200] Example 7: Paralogues of GaRpp1_CG1 and GaRpp1_CG4

[0201] The region upstream of the GaRpp1 interval contains two additional gene pairs that are paralogous to the CG1+CG4 pair (Figure 2C): GaRpp1_CG2 and MSTRG.57 are paralogous to CG1, while GaRpp1_CG5 and GaRpp1_CG3 are paralogous to CG4. These gene pairs are also arranged in a head-to-head layout in the genome and share up to 91.9% identity at the protein level in the case of MSTRG.57 compared to GaRpp1_CG1 (Table 1). GaRpp1_CG3 has 89.7% identity with GaRpp1_CG4 at the protein level (Table 2).

[0202] The orthologue of GaRpp1_CG1 in soybean is Glyma.03G108000.1, which has 79.6% identity at the nucleotide sequence level (comparison of CDS sequences) and 64.6% identity at the amino acid sequence level (Table 1). The orthologue of GaRpp1_CG4 is Glyma.03G107900.1, which encodes a longer protein (705 bp, compared to 456 bp for CG4), has 60.4% identity at the nucleotide sequence level (based on comparison of CDS sequences), and 86.8% identity at the amino acid sequence level (Table 2).

[0203] Table 1. Distance matrix protein alignment of GaRpp1_CG1 paralogues (% amino acid sequence identity)

[0204]

[0205] *MSTRG.57 is a pseudogene (i.e., lacks a start codon). The predicted protein sequence of MSTRG.57 starting from the first Met in the sequence encodes a peptide that is 100 bp shorter than GaRpp1_CG1.

[0206] Table 2. Distance matrix protein alignment of GaRpp1_CG4 paralogues (% amino acid sequence identity)

[0207]

[0208] Example 8: Promoter analysis of the intergenic region between GaRpp1_CG1 and GaRpp1_CG4

[0209] The 306 bp intergenic region between GaRpp1_CG1 and GaRpp1_CG4 was used as a query to predict cis - acting elements using the PlantCARE online database (accessible at bioinformatics.psb.ugent.be / webtools / plantcare / html / ) and to detect transcription factor binding sites (TFBS) using the Plant Promoter Analysis Navigator (PlantPAN 2.0; accessible at PlantPAN2.itps.ncku.edu.tw).

[0210] The core promoter element, the TATA box, is present in the region around position 101 in both forward and reverse orientations (Table 3). The GaRpp1 intergenic region also has multiple putative cis - acting elements present in both orientations, such as the CAAT box, the CGTCA / TGACG motif (methyl jasmonate response element), and the ABRE (abscisic acid response element). A putative TGA box element (auxin response element) is present in the reverse strand at positions 82 - 89 (Table 3; Figure 8 ).

[0211] Table 3. Promoter analysis of the GaRpp1 intergenic region using the PlantCARE online database.

[0212]

[0213] Automated promoter analysis using the PlantPAN 2.0 platform returned a long list of transcription factor binding sites in both DNA strands (Table 4). Two regions in the reverse strand represent putative WRKY DNA - binding domains (positions 153 - 157 and 225 - 229). The WRKY transcription factors are known to play important roles in plant defense responses (Cheng et al., 2012, Plant Physiol. 159(2):810 - 825, doi.org / 10.1104 / pp.112.196816).

[0214] A putative CG - 1 DNA - binding domain is also present in both DNA strands and is associated with the CAMTA (for CAlModulin - binding Transcription Activator) transcription factor. CAMTA belongs to a family of transcription factors that play key roles in plant responses to biotic and abiotic stresses (Bouche et al., 2022, J. Biol. Chem. 277(24):21851 - 2186, doi.org / 10.1074 / jbc.M200268200).

[0215]

[0216]

[0217] Example 9: GaRpp1-mediated cell death in Nicotiana benthamiana is independent of major NLR and PRR pathway signaling components

[0218] NLR signaling components including EDS1, PAD4, SAG101, NRG, and ADR1 form an important network in plant immunity. EDS1 acts as a central integrator, coordinating the regulation of defense gene expression and immune responses in concert with PAD4 and SAG101. PAD4 plays a key role in controlling the expression of defense-related genes, while SAG101 contributes to signal transduction during NLR-mediated immune responses. NRG enhances resistance to a variety of pathogens by working together with EDS1 and PAD4. ADR1, as a transcriptional co-activator, further strengthens the plant's defense response. In parallel, PRR signaling components such as BAK1 and SOBIR1 are crucial in initiating immune responses upon detection of pathogen-associated molecular patterns (PAMPs). BAK1 acts as a co-receptor, amplifying PRR activation, while SOBIR1 interacts with PRRs and co-receptors, transmitting signals that lead to the activation of robust defense mechanisms (reviewed in Ramirez-Zavaleta et al., 2022, doi.org / 10.3390 / ijms2321129 74 ). These components together form a complex signaling network essential for plant immunity, enabling plants to effectively defend against a variety of pathogens. To test whether any of these components are involved in GaRpp1 signaling, we utilized GaRpp1_CG1-mediated cell death in Nicotiana benthamiana. We have tested various mutants in different PRR and NLR signaling components( Figure 8 ), and shown that GaRpp1_CG1 triggers cell death independently of all mutants. This suggests that GaRpp1 functions through different, potentially novel mechanisms.

[0219] Example 10: GaRpp1-CG1-mediated cell death requires all three transmembrane domains

[0220] To further investigate the possible mechanisms underlying GaRpp1_CG1-induced cell death, we generated truncated versions of the protein: GaRpp1_CG1_v1, GaRpp1_CG1_v2, GaRpp1_CG1_v3, in which one, two, and three transmembrane domains were truncated, respectively, and GaRpp1_CG1_v4, in which the N-terminus was truncated to three transmembrane domains (amino acids 1-200). The truncated fragments were cloned under the LjUBI promoter. All constructs (including two positive controls (PsCRN63 and HopQ1) and one negative control (empty vector)) were agro-infiltrated into four-week-old wild-type Nicotiana benthamiana plants at 0.8 OD and imaged at 4 dpi. Cell death imaging showed that truncation of even one transmembrane domain led to the loss of cell death compared to transient expression of full-length GaRpp1_CG1. However, transient expression of GaRpp1_v4 exhibited strong cell death. This experiment demonstrated that the transmembrane domains are sufficient and necessary for the cell death phenotype in N. benthamiana.

[0221] Since GaRpp1_CG1_v4 can induce cell death, it was co-infiltrated with GaRpp1_CG4 to investigate whether N-terminal truncation would result in inhibition of cell death. The strong cell death phenotype indicated that GaRpp1_CG4 requires the N-terminal region of GaRpp1_CG1 to regulate or inhibit the cell death phenotype and possible pathogen resistance.

[0222] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" means one or more elements.

[0223] Throughout the specification, the word "comprising" or variations such as "comprises" or "having" will be understood to imply the inclusion of the 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.

[0224] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which the present invention pertains. All publications and patent applications are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent application had been specifically and individually indicated to be incorporated by reference.

[0225] Although the foregoing invention has been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A nucleic acid molecule, the nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence shown in SEQ ID NO: 1, 2, 4, 5, 7, 9, 10, 11, 13, 14, 16, 18, 19, 20, 22, 23, 25, 27, 28 or 32; (b) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6, 8, 15, 17, 24 or 26; (c) a nucleotide sequence having at least 80% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NO: 2, 5, 11, 14, 20, 23, 27 and 32, wherein relative to a control plant not comprising the nucleic acid molecule, the nucleic acid molecule is capable of conferring resistance to Asian soybean rust (ASR) caused by at least one race of Phakopsora pachyrhizi; (d) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity with at least one of the amino acid sequences shown in SEQ ID NO: 6, 15 and 24, wherein relative to a control plant not comprising the nucleic acid molecule, the nucleic acid molecule is capable of conferring resistance to ASR caused by at least one race of Phakopsora pachyrhizi; (e) a nucleotide sequence having at least 80% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NO: 4, 7, 13, 16, 22, 25 and 28, wherein relative to a control plant not comprising the nucleic acid molecule, the nucleic acid molecule is capable of regulating the activity of at least one R gene in the plant; and (f) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity with at least one of the amino acid sequences shown in SEQ ID NO: 8, 17 and 26, wherein relative to a control plant not comprising the nucleic acid molecule, the nucleic acid molecule is capable of regulating the activity of at least one R gene in the plant.

2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is an isolated nucleic acid molecule.

3. An expression cassette or vector, the expression cassette or vector comprising the nucleic acid molecule according to claim 1 or 2.

4. A host cell, the host cell being transformed with the nucleic acid molecule according to claim 1 or 2 or the expression cassette or vector according to claim 3.

5. A plant or plant cell, the plant or plant cell comprising the nucleic acid molecule according to claim 1 or 2 or the expression cassette or vector according to claim 3.

6. The plant or plant cell according to claim 5, wherein the plant is a soybean plant and the plant cell is a soybean plant cell.

7. The plant according to claim 6, wherein the soybean plant comprises enhanced resistance to ASR caused by at least one race of Phakopsora pachyrhizi relative to the resistance of a control soybean plant.

8. A soybean plant, plant cell, or seed, wherein the soybean plant, plant cell, or seed contains a heterologous polynucleotide stably incorporated into its genome, and the heterologous polynucleotide contains a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence shown in SEQ ID NO: 1, 2, 4, 5, 7, 9, 10, 11, 13, 14, 16, 18, 19, 20, 22, 23, 25, 27, 28, or 32; (b) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6, 8, 15, 17, 24, or 26; (c) a nucleotide sequence having at least 80% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NO: 2, 5, 11, 14, 20, 23, 27, and 32, wherein the nucleic acid molecule can confer resistance to Asian soybean rust (ASR) caused by at least one race of Phakopsora pachyrhizi relative to a control plant that does not contain the nucleic acid molecule; (d) a nucleic acid molecule containing a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity with at least one of the amino acid sequences shown in SEQ ID NO: 6, 15, and 24, wherein the nucleic acid molecule can confer resistance to ASR caused by at least one race of Phakopsora pachyrhizi relative to a control plant that does not contain the nucleic acid molecule; (e) a nucleotide sequence having at least 80% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NO: 4, 7, 13, 16, 22, 25, and 28, wherein the nucleic acid molecule can regulate the activity of at least one R gene in the plant relative to a control plant that does not contain the nucleic acid molecule; and (f) a nucleic acid molecule containing a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity with at least one of the amino acid sequences shown in SEQ ID NO: 8, 17, and 26, wherein the nucleic acid molecule can regulate the activity of at least one R gene in the plant relative to a control plant that does not contain the nucleic acid molecule.

9. The soybean plant, plant cell, or seed according to claim 8, wherein the heterologous polynucleotide further contains a promoter operably linked for expressing the nucleotide sequence in the plant.

10. The soybean plant, plant cell, or seed according to claim 8, wherein the heterologous polynucleotide contains the nucleotide sequence shown in SEQ ID NO:

1.

11. The soybean plant, plant cell, or seed according to claim 8 or 9, wherein the heterologous polynucleotide contains the nucleotide sequence shown in SEQ ID NO: 2 or 5 or a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

6.

12. The soybean plant, plant cell, or seed according to claim 11, wherein the heterologous polynucleotide further comprises the nucleotide sequence set forth in SEQ ID NO: 4 or 7, or wherein the soybean plant, plant cell, or seed further comprises an additional heterologous polynucleotide, the additional heterologous polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 4 or 7.

13. The soybean plant according to any one of claims 8 - 12, wherein the soybean plant comprises enhanced resistance to Asian soybean rust (ASR) caused by at least one race of Phakopsora pachyrhizi, relative to the resistance of a control soybean plant.

14. A method for enhancing the resistance of a soybean plant to ASR caused by at least one race of Phakopsora pachyrhizi, the method comprising introducing a heterologous polynucleotide into at least one soybean plant cell and regenerating a soybean plant comprising the heterologous polynucleotide in its genome, wherein the heterologous polynucleotide comprises a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1, 2, 5, 9, 10, 11, 14, 18, 19, 20, 23, 27, or 32; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 6, 15, and 24; (c) a nucleotide sequence having at least 80% sequence identity with at least one of the nucleotide sequences set forth in SEQ ID NO: 2, 5, 11, 14, 20, 23, 27, and 32, wherein the nucleic acid molecule is capable of conferring resistance to Asian soybean rust (ASR) caused by at least one race of Phakopsora pachyrhizi, relative to a control plant that does not comprise the nucleic acid molecule; and (d) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity with at least one of the amino acid sequences set forth in SEQ ID NO: 6, 15, and 24, wherein the nucleic acid molecule is capable of conferring resistance to ASR caused by at least one race of Phakopsora pachyrhizi, relative to a control plant that does not comprise the nucleic acid molecule.

15. The method according to claim 14, wherein the heterologous polynucleotide further comprises an additional nucleotide sequence selected from the group consisting of: (e) the nucleotide sequence set forth in SEQ ID NO: 4, 7, 13, 16, 22, 25, or 28; (f) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 8, 17, or 26; (g) a nucleotide sequence having at least 80% sequence identity with at least one of the nucleotide sequences set forth in SEQ ID NO: 4, 7, 13, 16, 22, 25, and 28, wherein the nucleic acid molecule is capable of regulating the activity of at least one R gene in a plant, relative to a control plant that does not comprise the nucleic acid molecule; and (h) A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity to at least one of the amino acid sequences shown in SEQ ID NO: 8, 17, and 26, wherein the nucleic acid molecule is capable of regulating the activity of at least one R gene in a plant relative to a control plant that does not contain the nucleic acid molecule.

16. The method according to claim 14, wherein the regenerated soybean plant comprises an additional nucleotide sequence selected from the group consisting of: (e) The nucleotide sequence shown in SEQ ID NO: 4, 7, 13, 16, 22, 25, or 28; (f) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 8, 17, or 26; (g) A nucleotide sequence having at least 80% sequence identity to at least one of the nucleotide sequences shown in SEQ ID NO: 4, 7, 13, 16, 22, 25, and 28, wherein the nucleic acid molecule is capable of regulating the activity of at least one R gene in a plant relative to a control plant that does not contain the nucleic acid molecule; and (h) A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity to at least one of the amino acid sequences shown in SEQ ID NO: 8, 17, and 26, wherein the nucleic acid molecule is capable of regulating the activity of at least one R gene in a plant relative to a control plant that does not contain the nucleic acid molecule.

17. The method according to any one of claims 14-16, wherein the heterologous polynucleotide is stably incorporated into the genome of the soybean plant cell.

18. The method according to any one of claims 14-17, wherein the heterologous polynucleotide further comprises a promoter operably linked to the nucleotide sequence for expression of the nucleotide sequence in a plant.

19. The method according to any one of claims 14-18, wherein the soybean plant comprising the heterologous polynucleotide comprises enhanced resistance to ASR caused by at least one race of Phakopsora pachyrhizi relative to the resistance of a control soybean plant.

20. A soybean plant capable of being produced or produced by the method according to any one of claims 14-19.

21. Seeds of the plant according to any one of claims 5-13 and 20, wherein the seeds comprise the heterologous polynucleotide.

22. A method for limiting ASR caused by at least one race of Phakopsora pachyrhizi in crop production, the method comprising planting seeds of the plant according to any one of claims 5-13 and 20 and growing the seeds under conditions favorable for the growth and development of the resulting plant, wherein the seeds comprise the nucleic acid molecule, expression cassette, vector, or heterologous polynucleotide.

23. A method for identifying a plant comprising an R gene and / or a regulator gene, the method comprising detecting the presence of a nucleotide sequence selected from the group consisting of: (a) The nucleotide sequence shown in SEQ ID NO: 1, 2, 4, 5, 7, 9, 10, 11, 13, 14, 16, 18, 19, 20, 22, 23, 25, 27, 28 or 32; (b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6, 8, 15, 17, 24 or 26; (c) A nucleotide sequence having at least 80% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NO: 2, 5, 11, 14, 20, 23, 27 and 32, wherein the nucleic acid molecule can confer resistance to Asian soybean rust (ASR) caused by at least one race of Phakopsora pachyrhizi relative to a control plant that does not contain the nucleic acid molecule; (d) A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity with at least one of the amino acid sequences shown in SEQ ID NO: 6, 15 and 24, wherein the nucleic acid molecule can confer resistance to ASR caused by at least one race of Phakopsora pachyrhizi relative to a control plant that does not contain the nucleic acid molecule; (e) A nucleotide sequence having at least 80% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NO: 4, 7, 13, 16, 22, 25 and 28, wherein the nucleic acid molecule can regulate the activity of at least one R gene in a plant relative to a control plant that does not contain the nucleic acid molecule; and (f) A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity with at least one of the amino acid sequences shown in SEQ ID NO: 8, 17 and 26, wherein the nucleic acid molecule can regulate the activity of at least one R gene in a plant relative to a control plant that does not contain the nucleic acid molecule.

24. The method according to claim 23, wherein the presence of the R gene or the regulator gene is detected by detecting at least one marker nucleotide sequence.

25. The method according to claim 23 or 24, wherein the nucleotide sequence comprises or consists of the nucleotide sequence of (a).

26. The method according to any one of claims 23-25, wherein detecting the presence of the nucleotide sequence comprises a member selected from the group consisting of PCR amplification, nucleic acid sequencing, nucleic acid hybridization and immunological assays for detecting the R protein encoded by the R gene or the regulator protein encoded by the regulator gene.

27. Use of the plant or seed according to any one of claims 5-13, 20 and 21 in agriculture.

28. A human or animal food product, the human or animal food product comprising the plant and / or seed according to any one of claims 5-13, 20 and 21 or one or more parts of the plant and / or seed, or produced using them.

29. A polypeptide, the polypeptide comprising an amino acid sequence selected from the group consisting of: (a) The amino acid sequences shown in SEQ ID NO: 6, 15, and 24; (b) The amino acid sequences shown in SEQ ID NO: 8, 17, and 26; (c) An amino acid sequence having at least 80% sequence identity to at least one of the amino acid sequences shown in SEQ ID NO: 6, 15, and 24, wherein the polypeptide comprising the amino acid sequence is capable of conferring resistance to ASR caused by at least one race of Phakopsora pachyrhizi relative to a control plant that does not contain the polypeptide; and (d) An amino acid sequence having at least 80% sequence identity to at least one of the amino acid sequences shown in SEQ ID NO: 8, 17, and 26, wherein the polypeptide comprising the amino acid sequence is capable of regulating the activity of at least one R gene in a plant relative to a control plant that does not contain the polypeptide.

30. A promoter, the promoter comprising a nucleotide sequence selected from the group consisting of: (a) The nucleotide sequences shown in SEQ ID NO: 3, 12, 21, 29, 30, and 31; and (b) A nucleotide sequence having at least 90% sequence identity to at least one of the nucleotide sequences shown in SEQ ID NO: 3, 12, 21, 29, 30, and 31, wherein the promoter is capable of driving expression on a polynucleotide operably linked in a plant cell.

31. The promoter according to claim 30, wherein the promoter is a bidirectional promoter capable of driving the expression of two operably linked polynucleotide sequences.

32. The promoter according to claim 30, wherein the promoter is a pathogen-inducible promoter capable of driving the expression of the operably linked polynucleotide sequence in response to infection by a pathogen.

33. The promoter according to claim 31, wherein the promoter is a pathogen-inducible promoter capable of driving the expression of the two operably linked polynucleotide sequences in response to infection by a pathogen.

34. The promoter according to claim 30 or 31, wherein the promoter is capable of being induced by at least one race of Phakopsora pachyrhizi.

35. The promoter according to claim 30 or 31, wherein the promoter is capable of being induced by two, three, four, five, or more races of Phakopsora pachyrhizi.

36. A method for introducing CG1 into a leguminous plant, the method comprising: (a) Crossing a first leguminous plant that contains at least one copy of CG1 in its genome with a second leguminous plant that lacks CG1 in its genome, thereby producing at least one offspring plant; and (b) Selecting at least one offspring plant that contains CG1 in its genome by detecting the presence of CG1 in the offspring plant.

37. The method according to claim 36, wherein the first leguminous plant is a Glycine soja plant, and the second leguminous plant is a soybean plant that lacks CG1 in its genome.

38. The method according to claim 36 or 37, wherein the first leguminous plant further contains at least one copy of CG4 in its genome.

39. The method according to claim 38, wherein CG4 is connected to CG1.

40. A progeny plant obtainable by a method according to any one of claims 36 - 39.

41. A seed of a progeny plant according to claim 40.

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