Transgenic plants having increased resistance to fungal diseases and methods of producing same

Abstract

The present disclosure provides compositions and methods for producing transgenic plants that exhibit increased resistance to fungal diseases, the plants so produced, and parts of the plants so produced. The present disclosure provides compositions and methods for the generation and creation of plant varieties that are highly resistant, or even immune, to Asian Soybean Rust and other fungal pathogens, while lacking undesirable off-types. In one embodiment, the present disclosure provides effective transgenic expression of Lr67 in soybean varieties to reduce the need for at least one, if not more, fungicide applications, while simultaneously protecting against fungal pathogens developing resistance to both the chemical control agents as well as other native or engineered resistance genes.

Description

TRANSGENIC PLANTS HAVING INCREASED RESISTANCE TO FUNGALDISEASES AND METHODS OF PRODUCING SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Appl. Ser. No. 63 / 499,305, filed May 1, 2023, the entire disclosure of which is incorporated herein by reference.INCORPORATION OF SEQUENCE LISTING

[0002] A sequence listing containing the file named “MONS539US_ST26.xml” which is 458 kilobytes (measured in MS-Windows®) and created on April 17, 2024, and comprises 230 sequences, is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of genetically engineering plants, and more specifically to methods and compositions for producing plants exhibiting increased resistance to fungal diseases.BACKGROUND OF THE INVENTION

[0004] There are numerous fungal diseases that affect a variety of plant species. Disease control is commonly managed by application of fungicides before infection or immediately after the first symptoms are observed. However, there are examples of fungi that have acquired various levels of resistance to certain fungicides. In addition, there is the cost of pesticide application, and there is increasing pressure to reduce the pesticide load in the environment.

[0005] Therefore, there is a need for plants having increased resistance to fungal diseases.SUMMARY

[0006] The present disclosure solves these and other problems in the art by providing plants exhibiting increased resistance to fungal diseases, and methods for making such plants.

[0007] The present disclosure provides a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 1 15, 117, 119, 121 , 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221 or 223, wherein the amino acid sequence comprises a glycine to arginine, glycine to lysine, or glycine to histidine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO: 4, and wherein the heterologous promoter is a constitutive or above-ground promoter. In certain embodiments, the heterologous promoter is an above-ground promoter.

[0008] In some embodiments, the amino acid sequence comprises a glycine to arginine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO: 4. In other embodiments, the nucleotide coding sequence of the recombinant DNA construct is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1, 3, 5, 6, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 or 222. In yet other embodiments, the nucleotide coding sequence of the recombinant DNA construct is at least 99% identical to SEQ ID NO: 1, 3, 5, 6, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 or 222, wherein the coding sequence encodes an amino acid sequence comprising a glycine to arginine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO: 4. In particular embodiments, the nucleotide coding sequence of the recombinant DNA construct encodes the amino acid sequence of SEQ ID NO: 2 or 7. In additional embodiments, the nucleotide coding sequence of the recombinant DNA construct comprises the nucleotide sequence of SEQ ID NO: 1, 5 or 6.

[0009] In certain embodiments, the protein encoded by the nucleotide coding sequence further comprises a valine to leucine mutation at the amino acid position corresponding to amino acid 387of SEQ ID NO: 4. In other embodiments, the protein encoded by the nucleotide coding sequence further comprises a valine to leucine, valine to isolcucinc, valine to methionine, or valine to phenylalanine mutation at the amino acid position corresponding to amino acid 387 of SEQ ID NO: 4. In particular embodiments, the nucleotide coding sequence of the recombinant DNA construct encodes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleotide coding sequence of the recombinant DNA construct comprises SEQ ID NO: 1. In further embodiments, the nucleotide coding sequence of the recombinant DNA construct comprises SEQ ID NO: 5.

[0010] In some embodiments, the heterologous promoter comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to one or more of SEQ ID NOs: 8-23 and 225-230. In other embodiments, the heterologous promoter comprises a nucleotide sequence that is 100% identical to one or more of SEQ ID NOs: 8-23 and 225-230. In certain embodiments, the above-ground promoter drives expression of the operably linked nucleotide coding sequence in a plant at a higher level in one or more above-ground tissues of the plant relative to the expression level of the operably linked nucleotide coding sequence in below-ground root tissues of the plant. In particular embodiments the above-ground promoter drives expression of the operably linked nucleotide coding sequence at least 10-fold, at least 20- fold, at least 30-fold, at least 40-fold, or at least 50-fold higher in the one or more above-ground tissues relative to the below-ground root tissues. In yet other embodiments, the heterologous promoter comprises a fragment of one of SEQ ID NOs: 8-23 and 225-230, wherein the fragment has promoter activity as a constitutive or above-ground promoter. In additional embodiments, the heterologous promoter fragment has promoter activity as an above-ground promoter.

[0011] The present disclosure also provides a DNA molecule comprising a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 or 223, whereinthe amino acid sequence comprises a glycine to arginine, glycine to lysine, or glycine to histidine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO: 4, and wherein the heterologous promoter is an above-ground promoter.

[0012] The present disclosure additionally provides a DNA plasmid or vector comprising a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221 or 223, wherein the amino acid sequence comprises a glycine to arginine, glycine to lysine, or glycine to histidine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO:4, and wherein the heterologous promoter is a constitutive or above-ground promoter.

[0013] The present disclosure further provides a transgenic plant comprising a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 or 223, wherein the amino acid sequence comprises a glycine to arginine, glycine to lysine, or glycine to histidine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO:4, and wherein the heterologous promoter is a constitutive or above-ground promoter, and wherein the plant has increased resistance to a fungal disease compared to a control plant lacking the recombinant DNA construct.

[0014] In certain embodiments, the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, target spot and / or brown spot. In some embodiments the transgenic plant is a dicotyledonous plant. In other embodiments, the transgenic plant is a leguminous plant. In still other embodiments the transgenic plant is a soybean plant.

[0015] The present disclosure also provides a transgenic plant part of the transgenic plant comprising a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221 or 223, wherein the amino acid sequence comprises a glycine to arginine, glycine to lysine, or glycine to histidine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO: 4, and wherein the heterologous promoter is a constitutive or above-ground promoter, and wherein the plant has increased resistance to a fungal disease compared to a control plant lacking the recombinant DNA construct. In certain embodiments, the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, target spot and / or brown spot. In some embodiments, the transgenic plant part is from a dicotyledonous plant. In other embodiments the transgenic plant part is from a leguminous plant. In still other embodiments the transgenic plant part is from a soybean plant. In particular embodiments, the transgenic plant part is a seed, a root, a stem, a leaf, a flower, an embryo, or a meristem. In some embodiments, the transgenic plant part is a seed.

[0016] Additionally, the present disclosure provides a method of making a transgenic plant resistant to a fungal disease, comprising introducing into at least one cell of an explant a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 1 11 , 113, 115, 117, 119, 121 , 123, 125, 127, 129, 131 , 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221 or 223, wherein the amino acid sequence comprises a glycine to arginine, glycine to lysine, or glycine to histidine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO:4, and wherein the heterologous promoter is a constitutive or above-ground promoter, and wherein the plant has increased resistance to a fungal disease compared to a control plant lacking the recombinant DNA construct, and regenerating or developing the transgenic plant from the explant. In certain embodiments, the recombinant DNA molecule is introduced into the plant by Agrobacterium-mediated transformation, microprojectile bombardment, calcium phosphate precipitation, polyethylene glycol treatment, electroporation, site-directed integration, gene editing, or a combination thereof. In additional embodiments, the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, target spot or brown spot. In some embodiments, the plant is a dicotyledonous plant. In other embodiments the plant is a leguminous plant. In yet other embodiments, the plant is a soybean plant.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0018] FIG. 1. Illustration of efficacy and mRNA expression data for 8 events from plants transformed with construct pM992. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0019] FIG. 2. Illustration of efficacy and mRNA expression data for 8 events from plants transformed with construct pM367. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0020] FIG. 3. Illustration of efficacy and mRNA expression data for 17 events from plants transformed with construct pM369. Top Panel: Percentage disease severity tested in soybean planttransformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgcnc.

[0021] FIG. 4. Illustration of efficacy and mRNA expression data for 7 events from plants transformed with construct pM370. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0022] FIG. 5. Illustration of efficacy and mRNA expression data for 22 events from plants transformed with construct pM372. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0023] FIG. 6. Pictures from controlled environment tested events with construct pM372, having a high leaf tissue, low root expression profile. Pictured on the left (Panel A) is Event 6 which showed extreme resistance and normal growth and development. On the right (Panel B) is an empty vector negative control, and the inoculated leaves are almost completely lost to disease. Note that only the first trifoliate was inoculated in this study.

[0024] FIG. 7. Illustration of efficacy and mRNA expression data for 9 events from plants transformed with construct pM838. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0025] FIG. 8. Illustration of efficacy and mRNA expression data for 8 events from plants transformed with construct pM839. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0026] FIG. 9. Illustration of efficacy and mRNA expression data for 8 events from plants transformed with construct pM840. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0027] FIG. 10. Illustration of efficacy and mRNA expression data for 4 events from plants transformed with construct pM842. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0028] FIG. 11 . Illustration of efficacy and mRNA expression data for 8 events from plants transformed with construct pM843. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0029] FIG. 12. Illustration of efficacy and mRNA expression data for 4 events from plants transformed with construct pM844. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0030] FIG. 13. Illustration of efficacy and mRNA expression data for 8 events from plants transformed with construct pM967. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0031] FIG. 14. Illustration of efficacy and mRNA expression data for 8 events from plants transformed with construct pM678 Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0032] FIG. 15. Illustration of efficacy and mRNA expression data for 4 events from plants transformed with construct pM400. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0033] FIG. 16. Illustration of efficacy and mRNA expression data for 4 events from plants transformed with construct pM494. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene. Bottom Panel: Relative mRNA expression level of Ta.Lr67res transgene.

[0034] FIG. 17. Illustration of efficacy and mRNA expression data for 4 events from plants transformed with construct pM285. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0035] FIG. 18. Illustration of efficacy and mRNA expression data for 5 events from plants transformed with construct pM479. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0036] FIG. 19. Illustration of efficacy data for 3 events from plants transformed with construct pM838 in CE-2. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0037] FIG. 20. Illustration of efficacy data for 2 events from plants transformed with construct pM839 in CE-2. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0038] FIG. 21. Illustration of efficacy data for 1 event from plants transformed with construct pM840 in CE-2. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0039] FIG. 22. Illustration of efficacy data for 6 events from plants transformed with construct pM841 in CE-2. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0040] FIG. 23. Illustration of efficacy data for 3 events from plants transformed with construct pM843 in CE-2. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0041] FIG. 24. Illustration of efficacy data for 10 events from plants transformed with construct pM844 in CE-2. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0042] FIG. 25. Illustration of efficacy data for 10 events from plants transformed with construct pM967 in CE-2. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0043] FIG. 26. Illustration of efficacy data for 9 events from plants transformed with construct pM968 in CE-2. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0044] FIG. 27. Illustration of efficacy data for 10 events from plants transformed with construct pM678 in CE-2. Percentage disease severity tested in soybean plant transformed with Ta.Lr67res transgene.

[0045] FIG. 28. Pictures from controlled environment (CE-2) tested events with construct pM840, having a high above ground and low in vascular tissues and root expression profile. Event- 5 from transformed plant which showed extreme resistance and normal growth and development. WT control, the inoculated leaves are chlorotic and infected with pathogen.

[0046] FIG. 29 Partial sequence alignment of wild-type (or disease susceptible Ta.Lr67) and disease resistant Ta.Lr67 (Ta.Lr67rcs) protein sequences from wheat along with protein sequences of other Lr67 protein homologs identified from various plant species through a NCBI BLAST program.BRIEF DESCRIPTION OF THE SEQUENCES

[0047] SEQ ID NO: 1 Triticum aestivum Lr67 resistant DNA sequence.

[0048] SEQ ID NO: 2 Triticum aestivum Lr67 resistant protein sequence.

[0049] SEQ ID NO: 3 Triticum aestivum Lr67 susceptible DNA sequence.

[0050] SEQ ID NO: 4 Triticum aestivum Lr67 susceptible protein sequence.

[0051] SEQ ID NO: 5 Synthetic Lr67 resistant codon optimized DNA sequence

[0052] SEQ ID NO: 6 Synthetic Lr67 resistant G144R DNA sequence

[0053] SEQ ID NO: 7 Synthetic Lr67 resistant G144R protein sequence.

[0054] SEQ ID NO: 8 Glycine max promoter P-Gm.13G046200:2.

[0055] SEQ ID NO: 9 Glycine max promoter P-Gm.08G282100:2.

[0056] SEQ ID NO: 10 Glycine max promoter P-Gm.l 1G155000:2.

[0057] SEQ ID NO: 11 Glycine max promoter P-Gm.02G215700:2.

[0058] SEQ ID NO: 12 Synthetic promoter P-At.GSP571.nno:l

[0059] SEQ ID NO: 13 Glycine max promoter P-Gm.PSII:l.

[0060] SEQ ID NO: 14 Glycine max promoter P-Gm.15G057600: 1.

[0061] SEQ ID NO: 15 Glycine max promoter P-Gm.l6G089000_trunc:l.

[0062] SEQ ID NO: 16 Glycine max promoter P-Gm.05G007100:1.

[0063] SEQ ID NO: 17 Glycine max promoter P-Gm.l9G007700:l.

[0064] SEQ ID NO: 18 Glycine max promoter P-Gm.l7G020600:l.

[0065] SEQ ID NO: 19 Glycine max promoter P-Gm.01G238800:l.

[0066] SEQ ID NO: 20 Glycine max promoter P-Gm.07G156100_trunc:l.

[0067] SEQ ID NO: 21 Glycine max promoter P-Gm.02G101100:1 .

[0068] SEQ ID NO: 22 Glycine max promoter P-Gm.07G156100: 1.

[0069] SEQ ID NO: 23 Glycine max promoter P-Gm.16G089000: 1.

[0070] SEQ ID NO: 24 Aegilops tauschii subsp. strangulata XM_020291770.2 DNA sequence.

[0071] SEQ ID NO: 25 Aegilops tauschii subsp. strangulata XP_020147359.1 protein sequence.

[0072] SEQ ID NO: 26 Triticum aestivum KR604816.1 DNA sequence.

[0073] SEQ ID NO: 27 Triticum aestivum ALL26330.1 protein sequence.

[0074] SEQ ID NO: 28 Triticum aestivum XM_044514814.1 DNA sequence.

[0075] SEQ ID NO: 29 Triticum aestivum XP_044370749.1 protein sequence.

[0076] SEQ ID NO: 30 Triticum aestivum KR604817.2 DNA sequence.

[0077] SEQ ID NO: 31 Triticum aestivum ALL26331.2 protein sequence.

[0078] SEQ ID NO: 32 Triticum dicoccoicles XM_037569898.1 DNA sequence.

[0079] SEQ ID NO: 33 Triticum dicoccoicles XP_037425795.1 protein sequence.

[0080] SEQ ID NO: 34 Triticum turgidum subsp. durum LT934118.1 DNA sequence.

[0081] SEQ ID NO: 35 Triticum turgidum subsp. durum VAI08283.1 protein sequence.

[0082] SEQ ID NO: 36 Triticum urartu XMJ148716059.1 DNA sequence.

[0083] SEQ ID NO: 37 Triticum urartu XP_048572016.1 protein sequence.

[0084] SEQ ID NO: 38 Triticum dicoccoides XM_037564139.1 DNA sequence.

[0085] SEQ ID NO: 39 Triticum dicoccoides XP_037420036.1 protein sequence.

[0086] SEQ ID NO: 40 Triticum aestivum XM_044506760.1 DNA sequence.

[0087] SEQ ID NO: 41 Triticum aestivum XP_044362695. 1 protein sequence.

[0088] SEQ ID NO: 42 Hordeum vulgare subsp. vulgare XM_045126453.1 DNA sequence.

[0089] SEQ ID NO: 43 Hordeum vulgare subsp. vulgare XP_044982388.1 protein sequence.

[0090] SEQ ID NO: 44 Hordeum vulgare J ACSIPO 10000626.1 DNA sequence.

[0091] SEQ ID NO: 45 Hordeum vulgare KAI4964990.1 protein sequence.

[0092] SEQ ID NO: 46 Hordeum vulgare JACSIPO 10000004.1 DNA sequence.

[0093] SEQ ID NO: 47 Hordeum vulgare KAI4993357.1 protein sequence.

[0094] SEQ ID NO: 48 Brachypodium distachyon XM_003558432.4 DNA sequence.

[0095] SEQ ID NO: 49 Brachypodium distachyon XP_003558480.1 protein sequence.

[0096] SEQ ID NO: 50 Setaria italica CM003536.1 DNA sequence.

[0097] SEQ ID NO: 51 Setaria italica RCV45878.1 protein sequence.

[0098] SEQ ID NO: 52 Setaria italica XM_004985118.4 DNA sequence.

[0099] SEQ ID NO: 53 Setaria italica XP_004985175.1 protein sequence.

[0100] SEQ ID NO: 54 Panicum hallii XM_025939222.1 DNA sequence.

[0101] SEQ ID NO: 55 Panicum hallii XP_025795007.1 protein sequence.

[0102] SEQ ID NO: 56 Zea mays NM_001323285.1 DNA sequence.

[0103] SEQ ID NO: 57 Zea mays NP_001310214.1 protein sequence.

[0104] SEQ ID NO: 58 Sorghum bicolor XM_002465591.2 DNA sequence.

[0105] SEQ ID NO: 59 Sorghum bicolor XP_002465636.1 protein sequence.

[0106] SEQ ID NO: 60 Sorghum bicolor CM027680.1 DNA sequence.

[0107] SEQ ID NO: 61 Sorghum bicolor KAG0552145.1 protein sequence.

[0108] SEQ ID NO: 62 Miscanthus lutarioriparius CAJGY0010000001.1 DNA sequence.

[0109] SEQ ID NO: 63 Miscanthus lutarioriparius CAD6202690.1 protein sequence.

[0110] SEQ ID NO: 64 Panicum hallii var. hallii CM009757.1 DNA sequence.

[0111] SEQ ID NO: 65 Panicum hallii var. hallii PUZ42154.1 protein sequence.

[0112] SEQ ID NO: 66 Eleusine coracana subsp. coracana BQKI01000015.1 DNA sequence.

[0113] SEQ ID NO: 67 Eleusine coracana subsp. coracana GJN08635.1 protein sequence.

[0114] SEQ ID NO: 68 Eleusine coracana subsp. coracana BQKTO 1000082.1 DNA sequence.

[0115] SEQ ID NO: 69 Eleusine coracana subsp. coracana GJN30721.1 protein sequence.

[0116] SEQ ID NO: 70 Digitaria exilis JACEF0010001710.1 DNA sequence.

[0117] SEQ ID NO: 71 Digitaria exilis KAF8718418.1 protein sequence.

[0118] SEQ ID NO: 72 Lolium rigidum XM_047233893.1 DNA sequence.

[0119] SEQ ID NO: 73 Lolium rigidum XP_047089849.1 protein sequence.

[0120] SEQ ID NO: 74 Miscanthus lutarioriparius C A JGYOO 10000002.1 DNA sequence.

[0121] SEQ ID NO: 75 Miscanthus lutarioriparius CAD6210216.1 protein sequence.

[0122] SEQ ID NO: 76 Digitaria exilis JACEFOO 10002202.1 DNA sequence.

[0123] SEQ ID NO: 77 Digitaria exilis KAF8673769.1 protein sequence.

[0124] SEQ ID NO: 78 Panicum hallii var. hallii CM009757.1 DNA sequence.

[0125] SEQ ID NO: 79 Panicum hallii var. hallii PUZ42152.1 protein sequence.

[0126] SEQ ID NO: 80 Oryza sativa Japonica Group XM_015774963.1 DNA sequence.

[0127] SEQ ID NO: 81 Oryza sativa Japonica Group XP_015630449.1 protein sequence.

[0128] SEQ ID NO: 82 Digitaria exilis LR792836.1 DNA sequence.

[0129] SEQ ID NO: 83 Digitaria exilis CAB3496654.1 protein sequence.

[0130] SEQ ID NO: 84 Oryza sativa Japonica Group AY342321.1 DNA sequence.

[0131] SEQ ID NO: 85 Oryza sativa Japonica Group AAQ24871.1 protein sequence.

[0132] SEQ ID NO: 86 Oryza brachyantha XM_006649580.2 DNA sequence.

[0133] SEQ ID NO: 87 Oryza brachyantha XP_006649643.1 protein sequence.

[0134] SEQ ID NO: 88 Panicum virgatum XM_039923333.1 DNA sequence.

[0135] SEQ ID NO: 89 Panicum virgatum XP_039779267.1 protein sequence.

[0136] SEQ ID NO: 90 Oryza meyeriana var. granulata SPHZ02000006.1 DNA sequence.

[0137] SEQ ID NO: 91 Oryza meyeriana var. granulata KAF0912539.1 protein sequence.

[0138] SEQ ID NO: 92 Zizania palustris JAAALKO 10000283.1 DNA sequence.

[0139] SEQ ID NO: 93 Zizania palustris KAG8074070.1 protein sequence.

[0140] SEQ ID NO: 94 Panicum virgatum XM_039973091.1 DNA sequence.

[0141] SEQ ID NO: 95 Panicum virgatum XP_039829025.1 protein sequence.

[0142] SEQ ID NO: 96 Digitaria exilis JACEF0010000735.1 DNA sequence.

[0143] SEQ ID NO: 97 Digitaria exilis KAF8758196.1 protein sequence.

[0144] SEQ ID NO: 98 Zizania palustris JAAALK010000283.1 DNA sequence.

[0145] SEQ ID NO: 99 Zizania palustris KAG8074071.1 protein sequence.

[0146] SEQ ID NO: 100 Oryza sativa Japonica Group CM000140.1 DNA sequence.

[0147] SEQ ID NO: 101 Oryza sativa Japonica Group EEE58602.1 protein sequence.

[0148] SEQ ID NO: 102 Zizania palustris JAAALK010000079.1 DNA sequence.

[0149] SEQ ID NO: 103 Zizania palustris KAG8096992.1 protein sequence.

[0150] SEQ ID NO: 104 Zizania palustris JAAALKO 10002011.1 DNA sequence.

[0151] SEQ ID NO: 105 Zizania palustris KAG8038400.1 protein sequence.

[0152] SEQ ID NO: 106 Oryza sativa Indica Group CM000128.1 DNA sequence.

[0153] SEQ ID NO: 107 Oryza sativa Indica Group EEC74774.1 protein sequence.

[0154] SEQ ID NO: 108 Panicum miliaceum PQ1B 02000001.1 DNA sequence.

[0155] SEQ ID NO: 109 Panicum miliaceum RLN38896.1 protein sequence.

[0156] SEQ ID NO: 110 Musa acuminata subsp. malaccensis XM_009412997.2 DNA sequence.

[0157] SEQ ID NO: 111 Musa acuminata subsp. malaccensis XP_009411272.1 protein sequence.

[0158] SEQ ID NO: 112 Musa troglodytarum CP097510.1 DNA sequence.

[0159] SEQ ID NO: 113 Musa troglodytarum URE40612.1 protein sequence.

[0160] SEQ ID NO: 114 Cocos nucifera CM017876.1 DNA sequence.

[0161] SEQ ID NO: 1 15 Cocos nucifera KAG1342218.1 protein sequence.

[0162] SEQ ID NO: 116 Elaeis guineensis XM_010906918.3 DNA sequence.

[0163] SEQ ID NO: 117 Elaeis guineensis XP_010905220.1 protein sequence.

[0164] SEQ ID NO: 118 Dioscorea alata JAFBIIO 10000019.1 DNA sequence.

[0165] SEQ ID NO: 119 Dioscorea alata KAH7653817.1 protein sequence.

[0166] SEQ ID NO: 120 Musa balbisiana PYDT01000005.1 DNA sequence.

[0167] SEQ ID NO: 121 Musa balbisiana THU59244.1 protein sequence.

[0168] SEQ ID NO: 122 Phoenix dactylifera XM_008803478.4 DNA sequence.

[0169] SEQ ID NO: 123 Phoenix dactylifera XP_008801700.1 protein sequence.

[0170] SEQ ID NO: 124 Dioscorea cayenensis subsp. rotundata XM_039258588.1 DNA sequence.

[0171] SEQ ID NO: 125 Dioscorea cayenensis subsp. rotundata XP_039114522.1 protein sequence.

[0172] SEQ ID NO: 126 Musa troglodytarum CP097506.1 DNA sequence.

[0173] SEQ ID NO: 127 Musa troglodytarum URD97174.1 protein sequence.

[0174] SEQ ID NO: 128 Musa acuminata subsp. malaccensis XM_009396448.2 DNA sequence.

[0175] SEQ ID NO: 129 Musa acuminata subsp. malaccensis XP_009394723.1 protein sequence.

[0176] SEQ ID NO: 130 Ananas comosus XM_020249632.1 DNA sequence.

[0177] SEQ ID NO: 131 Ananas comosus XP_020105221.1 protein sequence.

[0178] SEQ ID NO: 132 Zingiber officinale XM_042564728.1 DNA sequence.

[0179] SEQ ID NO: 133 Zingiber officinale XP_042420662.1 protein sequence.

[0180] SEQ ID NO: 134 Cannabis sativa XM_030651537.1 DNA sequence.

[0181] SEQ ID NO: 135 Cannabis sativa XP_030507397.1 protein sequence.

[0182] SEQ ID NO: 136 Trema orientale JXTCO 1000005.1 DNA sequence.

[0183] SEQ ID NO: 137 Trema orientale POO02217.1 protein sequence.

[0184] SEQ ID NO: 138 Parasponia andersonii JXTB01000002.1 DNA sequence.

[0185] SEQ ID NO: 139 Parasponia andersonii PON80119.1 protein sequence.

[0186] SEQ ID NO: 140 Colocasia esculenta NMUH01012884.1 DNA sequence.

[0187] SEQ ID NO: 141 Colocasia esculenta MQM22426.1 protein sequence.

[0188] SEQ ID NO: 142 Olea europaea subsp. europaea CACTIH010005529.1 DNA sequence.

[0189] SEQ ID NO: 143 Olea europaea subsp. europaea CAA2996553.1 protein sequence.

[0190] SEQ ID NO: 144 Olea europaea var. sylvestris XM_022990986.1 DNA sequence.

[0191] SEQ ID NO: 145 Olea europaea var. sylvestris XP_022846754.1 protein sequence.

[0192] SEQ ID NO: 146 Dendrobium chrysotoxum JAGFB R010000005.1 DNA sequence.

[0193] SEQ ID NO: 147 Dendrobium chrysotoxum KAH0467165.1 protein sequence.

[0194] SEQ ID NO: 148 Phalaenopsis equestris XM_020724504.1 DNA sequence.

[0195] SEQ ID NO: 149 Phalaenopsis equestris XP_020580163.1 protein sequence.

[0196] SEQ ID NO: 150 Olea europaea subsp. europaea CACTIH010009456.1 DNA sequence.

[0197] SEQ ID NO: 151 Olea europaea subsp. europaea CAA3O31477.1 protein sequence.

[0198] SEQ ID NO: 152 Nelumbo nucifera XM_010257690.1 DNA sequence.

[0199] SEQ ID NO: 153 Nelumbo nucifera XP_010255992.1 protein sequence.

[0200] SEQ ID NO: 154 Camelina sativa XM_010456685.2 DNA sequence.

[0201] SEQ ID NO: 155 Camelina sativa XP_010454987.1 protein sequence.

[0202] SEQ ID NO: 156 Raphanus sativus XM_018596920.1 DNA sequence.

[0203] SEQ ID NO: 157 Raphanus sativus XP_018452422.1 protein sequence.

[0204] SEQ ID NO: 158 Acacia mangium AB839219.1 DNA sequence.

[0205] SEQ ID NO: 159 Acacia mangium BAO45901 . 1 protein sequence.

[0206] SEQ ID NO: 160 Capsella rubella XM_006287435.2 DNA sequence.

[0207] SEQ ID NO: 161 Capsella rubella XP_006287497.1 protein sequence.

[0208] SEQ ID NO: 162 Camelina sativa XM_010423201.1 DNA sequence.

[0209] SEQ ID NO: 163 Camelina sativa XP_010421503.1 protein sequence.

[0210] SEQ ID NO: 164 Herrania umbratica XM_021434422.1 DNA sequence.

[0211] SEQ ID NO: 165 Herrania umbratica XP_021290097.1 protein sequence.

[0212] SEQ ID NO: 166 Sesamum indicum XM_011092436.2 DNA sequence.

[0213] SEQ ID NO: 167 Sesamum indicum XP_011090738.1 protein sequence.

[0214] SEQ ID NO: 168 Cinnamomum micranthum f. kanehirae QPKB01000001.1 DNA sequence.

[0215] SEQ ID NO: 169 Cinnamomum micranthum f kanehirae RWR73752.1 protein sequence.

[0216] SEQ ID NO: 170 Mangifera indica XM_044644096.1 DNA sequence.

[0217] SEQ ID NO: 171 Mangifera indica XP_044500031.1 protein sequence.

[0218] SEQ ID NO: 172 Aristolochia fimbriata JAINDJ010000002.1 DNA sequence.

[0219] SEQ ID NO: 173 Aristolochia fimbriata KAG9456452.1 protein sequence.

[0220] SEQ ID NO: 174 Arabidopsis suecica JAEFB JO 10000005.1 DNA sequence.

[0221] SEQ ID NO: 175 Arabidopsis suecica KAG7610463.1 protein sequence.

[0222] SEQ ID NO: 176 Corchorus olitorius AWUE01014431.1 DNA sequence.

[0223] SEQ ID NO: 177 Corchorus olitorius OMP03741.1 protein sequence.

[0224] SEQ ID NO: 178 Dendrobium nobile JAGYWB010000005.1 DNA sequence.

[0225] SEQ ID NO: 179 Dendrobium nobile KAI0523383.1 protein sequence.

[0226] SEQ ID NO: 180 Spirodela intermedia LR746267.1 DNA sequence.

[0227] SEQ ID NO: 181 Spirodela intermedia CAA7395464.1 protein sequence.

[0228] SEQ ID NO: 182 Arabidopsis thaliana NM_122535.4 DNA sequence.

[0229] SEQ ID NO: 183 Arabidopsis thaliana NP_198006.1 protein sequence.

[0230] SEQ ID NO: 184 Smallanthus sonchifolius JAKNSE010000010.1 DNA sequence.

[0231] SEQ ID NO: 185 Smallanthus sonchifolius KAI3803991.1 protein sequence.

[0232] SEQ ID NO: 186 Dendrobium catenation XM_020823020.2 DNA sequence.

[0233] SEQ ID NO: 187 Dendrobium catenatum XP_020678679.1 protein sequence.

[0234] SEQ ID NO: 188 Smallanthus sonchifolius JAKNSE010000016.1 DNA sequence.

[0235] SEQ ID NO: 189 Smallanthus sonchifolius KAI3774343.1 protein sequence.

[0236] SEQ ID NO: 190 Nelumbo nucifera XM_010279359.2 DNA sequence.

[0237] SEQ ID NO: 191 Nelumbo nucifera XP_010277661.1 protein sequence.

[0238] SEQ ID NO: 192 Camelina saliva XM_010495595.2 DNA sequence.

[0239] SEQ ID NO: 193 Camelina sativa XP_010493897.1 protein sequence.

[0240] SEQ ID NO: 194 Shorea leprosula BPVZ01000161.1 DNA sequence.

[0241] SEQ ID NO: 195 Shorea leprosula GKV42763.1 protein sequence.

[0242] SEQ ID NO: 196 Apostasia shenzhenica KZ452037.1 DNA sequence.

[0243] SEQ ID NO: 197 Apostasia shenzhenica PKA49860.1 protein sequence.

[0244] SEQ ID NO: 198 Papaver somniferum XM_026582532.1 DNA sequence.

[0245] SEQ ID NO: 199 Papaver somniferum XP_026438317.1 protein sequence.

[0246] SEQ ID NO: 200 Castanea mollissima JRKL02003281.1 DNA sequence.

[0247] SEQ ID NO: 201 Castanea mollissima KAF3955770.1 protein sequence.

[0248] SEQ ID NO: 202 Durio zibethinus XM_022866338.1 DNA sequence.

[0249] SEQ ID NO: 203 Durio zibethinus XP_022722073.1 protein sequence.

[0250] SEQ ID NO: 204 Brassica napus XM_013882001.3 DNA sequence.

[0251] SEQ ID NO: 205 Brassica napus XP_013737455.2 protein sequence.

[0252] SEQ ID NO: 206 Papaver somniferum XM_026600778. 1 DNA sequence.

[0253] SEQ ID NO: 207 Papaver somniferum XP_026456563.1 protein sequence.

[0254] SEQ ID NO: 208 Durio zibethinus XM_022862426.1 DNA sequence.

[0255] SEQ ID NO: 209 Durio zibethinus XP_022718161.1 protein sequence.

[0256] SEQ ID NO: 210 Telopea speciosissima XM_043836660.1 DNA sequence.

[0257] SEQ ID NO: 211 Telopea speciosissima XP_043692595.1 protein sequence.

[0258] SEQ ID NO: 212 Telopea speciosissima XM_043836659.1 DNA sequence.

[0259] SEQ ID NO: 213 Telopea speciosissima XP_043692594.1 protein sequence.

[0260] SEQ ID NO: 214 Papaver atlanticum JAJJMBO 10014022.1 DNA sequence.

[0261] SEQ ID NO: 215 Papaver atlanticum KAI3864014.1 protein sequence.

[0262] SEQ ID NO: 216 Pistacia vera XM_031416243.1 DNA sequence.

[0263] SEQ ID NO: 217 Pistacia vera XP_031272103.1 protein sequence.

[0264] SEQ ID NO: 218 Papaver bracteatum JAJJWX010001598.1 DNA sequence.

[0265] SEQ ID NO: 219 Papaver bracteatum KAI3880823.1 protein sequence.

[0266] SEQ ID NO: 220 Theobroma cacao XM_007034647.2 DNA sequence.

[0267] SEQ ID NO: 221 Theobroma cacao XP_007034709.1 protein sequence.

[0268] SEQ ID NO: 222 Macadamia integrifolia XM_042656840.1 DNA sequence.

[0269] SEQ ID NO: 223 Macadamia integrifolia XP_042512774.1 protein sequence.

[0270] SEQ ID NO: 224 - Triticum aestivum Lr67 resistant genomic DNA sequence.

[0271] SEQ ID NO: 225 - Arabidopsis thaliana promoter P-At.CeresRep74_Mdhg:l.

[0272] SEQ ID NO: 226 - Medicago truncatula promoter P-Mt.Lhcb2:l.

[0273] SEQ ID NO: 227 - Cucumis melo promoter P-CUCme.CipLhcb:l.

[0274] SEQ ID NO: 228 - Cucumis melo promoter P-CUCme.CipLhcb:3.

[0275] SEQ ID NO: 229 - Medicago truncatula promoter P-Mt.Lhcb2:l.

[0276] SEQ ID NO: 230 - Arahidopsis thaliana P-At.RbcS4:2.DETAILED DESCRIPTION

[0277] Soybean rust, also referred to as Asian soybean rust (ASR) is caused by Phakopsora pachyrhizi and is an aggressive fungal pathogen that has spread from Asia to Africa, South America, and now the U.S. Soybean rust was first detected in the U.S. in the fall of 2004. Control of ASR and other fungal diseases relies heavily on the application of fungicides, requiring multiple sprays each year. Genetic resistance has the potential to be both sustainable and durable when combining multiple modes of action; however, native sources of genetic resistance are few and most have already been overcome by the pathogen.

[0278] Previous studies have shown that a wheat Lr67 protein variant can confer broadspectrum resistance to leaf rust, stripe rust, stem rust, and powdery mildew (Hiebert et al., Theor. Appl. Genet. 121:1083-1091, 2010, Herrera-Foessel et al., Theor. Appl. Genet. 127:781-789, 2014). However, in addition to the observed pathogen resistance, the presence of the resistant Lr67 allele in wheat also results in leaf-tip necrosis, particularly evident in older leaves (Herrera- Foessel et al., supra). Similarly, barley plants transformed with the resistant Lr67 allele conferred multi-pathogen resistance, but improper expression resulted in moderate to severe necrosis and / or lack of pathogen resistance.

[0279] The present disclosure overcomes the limitations of the prior art and demonstrates for the first time that a resistance Lr67 allele, when transgenically expressed in soybean (Glycine max) under optimal levels and localization, can confer exceedingly high levels of resistance to Asian Soybean Rust, caused by the fungal pathogen Phakopsora pachyrhizi', and such resistance is achievable in adult plants as well as seedlings. Moreover, the present disclosure demonstrates for the first time that it is possible to have the resistance phenotype while minimizing or reducing undesirable off-types. As such, the beneficial effects yielded by the present disclosure are extremely useful in the generation and creation of plant varieties that are highly resistant, or even immune, to ASR and other fungal pathogens. For example, the present disclosure provides effective transgenic expression of Lr67 in soybean varieties to reduce the need for at least one, if not more, fungicide applications, while simultaneously protecting against fungal pathogens developing resistance to both the chemical control agents as well as other native or engineeredresistance genes. The present disclosure thus represents a significant advance in the art in that it permits the production of ASR resistant soybean plants lacking undesirable off-types.

[0280] The present disclosure generally describes transgenic plants having increased resistance to various fungal diseases and methods for making such transgenic plants. The following sections provide embodiments that describe the subject matter in greater detail.I. Nucleic Acid and Polypeptide Sequences

[0281] Certain embodiments of the current disclosure concern nucleic acid sequences (polynucleotides) and the corresponding amino acid sequences (proteins or polypeptides) for increasing resistance of plants to fungal diseases. Complements to any nucleic acid or protein sequences described herein arc also provided.

[0282] As used herein, the term “DNA” or “DNA molecule” refers to a double- stranded DNA molecule of genomic or synthetic origin, z.e., a polymer of deoxyribonucleotide bases or a polynucleotide molecule, read from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to that of by Title 37 of the United States Code of Federal Regulations § 1.822, and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

[0283] As used herein, the term “isolated DNA molecule” or an equivalent refers to a DNA molecule at least partially separated from other molecules normally associated with it in its native or natural state or environment and / or alone or in combination with other compositions or elements, but not within its natural state or environment. Nucleic acid elements, such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, that are naturally found within the DNA of the genome of an organism are not considered to be “isolated” so long as the element is within the genome of the organism, and at the location within such genome, where it is naturally found. However, each of these elements, and subparts of these elements, would be “isolated” within the scope of this disclosure so long as the element is not within the genome of the organism and at the location within the genome in which it is naturally found. Similarly, a nucleotide sequence encoding a Lr67 protein or any variant or homolog of that protein would be an isolated nucleotide sequence so long as the nucleotide sequence is not within the DNA of the plant genome from which thenucleotide sequence encoding the protein is naturally found and not present at the native location with the native orientation for such nucleotide sequence in such plant genome. In present embodiments with regard to DNA molecules, segments, fragments or sequences, the term “isolated” refers to a DNA molecule, segment, fragment, element or sequence that is at least partially separated from one or more nucleic acid segments, fragments, sequences or elements which are normally associated with, or adjacent, flanking or operably linked to, the DNA molecule, segment, fragment or sequence in its native or natural state. Such as “isolated” DNA molecule, segment, fragment, element or sequence may be associated with, or adjacent, flanking or operably linked to, a DNA molecule, segment, fragment or sequence with which it is not associated with, or adjacent, flanking or operably linked to, in nature. Thus, for purposes of this disclosure, a DNA molecule, segment, fragment or sequence fused to a regulatory or coding sequence with which they are not normally associated or linked in nature, for example as the result of a recombinant technique, are considered isolated herein. Such molecules are considered isolated when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules, not normally associated with in their native state. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., the nucleotide sequence of the DNA inserted into the genome of the cells of a plant or bacterium, or present in an extrachromosomal vector, would be considered to be an isolated nucleotide sequence whether it is present within the plasmid vector or similar structure used to transform cells, within the genome of the plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commodity products derived from the plant or bacterium. An isolated DNA molecule, segment, fragment, element or sequence is a chemical molecule, segment, fragment, element or sequence, regardless of whether it is referred to as a nucleic acid, a nucleic acid sequence, a polynucleotide sequence, a construct, a cassette, or the like. Such an isolated molecule, etc., is a novel and inventive molecule that exhibits industrial applicability both when present in a plant cell, bacterial cell or in a plant or bacterial genome, and when present outside of a plant cell, and therefore, exhibits and is intended to have or exhibit utility regardless of where the molecule, etc., is located outside its native context or environment. The term “isolated” may also be used to similarly describe a protein or other biomolecule that is at least partially separated from its native or natural state.

[0284] Any number of methods well-known to those skilled in the ail can be used to isolate and manipulate a DNA molecule, or fragment thereof, as presently disclosed. For example, PCR™(polymerase chain reaction) technology can be used to amplify a particular starting DNA molecule and / or to produce variants of the original molecule. DNA molecules, or fragment thereof, can also be obtained by other techniques such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer.

[0285] “Identity,” as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. Methods to determine “identity” are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. “Identity” can be readily calculated by any of the many methods known to those of skill in the art. Computer programs can be used to determine “identity” between two sequences these programs include but are not limited to, GCG; suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, NCBI NLM NIH, Bethesda, Md. 20894). The well-known Smith Waterman algorithm can also be used to determine identity. As used herein, the term “sequence identity” refers to the extent to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. An optimal sequence alignment is created by manually aligning two sequences, e.g.. a reference sequence and another sequence, to maximize the number of nucleotide matches in the sequence alignment with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term “reference sequence” refers to a sequence provided as the polynucleotide sequences of SEQ ID NOs: 1, 3, 5, 6, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 or 222, or a complement thereof; or a sequence provided as the polypeptide sequences of SEQ ID NOs: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131 , 133, 135, 137, 139, 141 , 143, 145, 147, 149, 151 , 153, 155, 157, 159, 161 , 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 or 223.

[0286] In accordance with the present disclosure, a polynucleotide or polypeptide sequence as described herein may exhibit at least from about 34%, 40%, 50%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90% or 95% to about 100% sequence identity to at least one of the sequences set forth herein. For example, in one embodiment, a nucleic acid sequence, such as a nucleotide coding sequence, as described herein may comprise, for example, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1, 3, 5, 6, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220 or 222, or a complement thereof. In other embodiments, an amino acid or polypeptide sequence or protein as described herein may comprise for example, at least 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, or 100% sequence identity to an amino acid or polypeptide sequence selected from SEQ ID NOs: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 or 223.

[0287] Parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch ( / . Mol. Biol. 48:443-453, 1970); Comparison matrix: BLOSUM62 fromHentikoff and Hentikoff, (Proc. Natl. Acad. Sci. USA 89:1091 -10919, 1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program that can be used with these parameters is publicly available as the “gap” program from Genetics Computer Group, Madison WI. The above parameters along with no penalty for end gap may serve as default parameters for peptide comparisons.

[0288] Parameters for nucleic acid sequence comparison include the following: Algorithm: Needleman and Wunsch (supra) Comparison matrix: matches=+10; mismatches=0; Gap Penalty: 50; and Gap Length Penalty: 3. A program that can be used with these parameters is publicly available as the “gap” program from Genetics Computer Group, Madison Wis. The above parameters may serve as the default parameters for nucleic acid comparisons.

[0289] As used herein, “hybridization,” “hybridizes,” or “capable of hybridizing” means the forming of a double- stranded hybridization structure, or partial double- stranded hybridization structure, between two polynucleotide sequences, molecules or segments as understood in the art. Such hybridization may take place under relatively high- stringency conditions, including low salt and / or high temperature conditions, such as provided by a wash in about 0.02 M to about 0.15 M NaCl at temperatures of about 50 °C to about 70 °C for 10 min. In one embodiment of the present disclosure, the conditions are 0.15 M NaCl and 70 °C. Stringent conditions tolerate little mismatch between a nucleic acid and a target strand. Such conditions are well-known to those of ordinary skill in the art and may be preferred for applications requiring high selectivity and defining sequences that hybridize under varying stringency conditions to a reference sequence or its complement.

[0290] “Fragment”, with respect to a nucleic acid sequence or molecule disclosed herein, refers to any part of the nucleic acid sequence or molecule that retains a usable, functional characteristic of the nucleic acid sequence or molecule. Useful fragments include oligonucleotides and polynucleotides that may be used as probes or primers in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A polynucleotide fragment refers to any subsequence of a polynucleotide, typically, of at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 21 consecutive nucleotides, at least about 22 consecutive nucleotides, at least about 23 consecutive nucleotides, at least about 24 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about35 nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides, or at least about 50 nucleotides or more, of any of the nucleic acid sequences provided herein.

[0291] Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide, as disclosed herein. Fragments may have antigenic potential, or may be a subsequence of the polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. Fragments can vary in size from as few as 5 amino acids to the full length of the intact polypeptide, but are preferably at least about 10 amino acids in length, at least about 15 amino acids in length, at least about 20 amino acids in length, at least about 25 amino acids in length, at least about 30 amino acids in length, at least about 35 amino acids in length, at least about 40 amino acids in length, at least about 45 amino acids in length, at least about 50 amino acids in length, at least about 55 amino acids in length, or at least about 60 amino acids in length or more, of any of the amino acid sequences provided herein.

[0292] The nucleic acids provided herein as SEQ ID NOs: 1, 3, 5, 6, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 or 222, and amino acids provided herein as SEQ ID NOs: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 or 223, may be from any source, e.g., identified as naturally occurring in a plant, or synthesized, e.g., identified from a naturally occurring gene in a plant or other species, or modified or synthesized relative to the sequences provided by SEQ ID NOs: 1, 3, 5, 6, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220or 222, for example to create a codon optimized sequence or a coding sequence with a G / C content more like the G / C content of naturally occurring genes from a particular plant.II. DNA Constructs

[0293] As used herein, a “DNA construct” is a recombinant DNA molecule comprising two or more heterologous DNA sequences. DNA constructs are useful for transgene expression and may be comprised in vectors and plasmids. DNA constructs may be used in vectors for transformation (that is, the introduction of heterologous DNA into a host cell) to produce recombinant bacteria or transgenic plants and cells (and as such may also be contained in the plastid DNA or genomic DNA of a transgenic plant, seed, cell, or plant part). As used herein, a “vector” means any recombinant DNA molecule that may be used for bacterial or plant transformation. DNA molecules provided herein can, for example, be inserted into a vector as part of a DNA construct having the DNA molecule operably linked to a heterologous gene expression element that functions in a plant to affect expression of the protein encoded by the DNA molecule. Methods for making and using DNA constructs and vectors are well known in the ail and described in detail in, for example, handbooks and laboratory manuals including Michael R. Green and Joseph Sambrook, “Molecular Cloning: A Laboratory Manual” (Fourth Edition) ISBN:978- 1-936113-42- 2, Cold Spring Harbor Laboratory Press, NY (2012). The components for a DNA construct, or a vector comprising a DNA construct, include one or more gene expression elements operably linked to a transcribable nucleic acid sequence, such as the following: a promoter for the expression of an operably linked DNA, an operably linked protein-coding DNA molecule, and an operably linked 3’ untranslated region (UTR). Gene expression elements that are useful include, but are not limited to, one or more of the following type of elements: promoter, 5’ UTR, enhancer, leader, cis-acting element, intron, transit sequence, 3’ UTR, and one or more selectable marker transgenes.

[0294] The term “transgene” refers to a DNA molecule artificially incorporated into the genome of an organism as a result of human intervention, such as by plant transformation methods. As used herein, the term “transgenic” means comprising a transgene, for example a “transgenic plant” refers to a plant comprising a transgene in its genome and a “transgenic trait” refers to a characteristic or phenotype conveyed or conferred by the presence of a transgene incorporated into the plant genome. As a result of such genomic alteration, the transgenic plant is something distinctly different from the related wild-type plant and the transgenic trait is a trait not naturallyfound in the wild-type plant. Transgenic plants provided herein comprise the recombinant DNA molecules and proteins of the present disclosure.

[0295] As used herein, the term “heterologous” refers to the relationship between two or more elements, proteins, polynucleotides, substances and / or sequences not normally associated with each other in nature. For instance, the two or more elements, etc., may be derived from different sources or not normally found in nature together in the same manner. For example, a DNA molecule, sequence or protein may be heterologous with respect to another DNA molecule, sequence, protein, cell, plant, seed, or organism if not normally found in nature together or in the same context. In certain embodiments, a first DNA molecule or sequence is heterologous to a second DNA molecule or sequence if the two DNA molecules or sequences are not normally found in nature together in the same context. For instance, a protein-coding recombinant DNA molecule (z.e., a nucleotide coding sequence) is heterologous with respect to an operably linked promoter if such a combination is not normally found in nature. Similarly, a protein is heterologous with respect to a second operably linked protein, such as a transit peptide, if such combination is not normally found in nature. In another embodiment, a recombinant DNA molecule encoding a protein is heterologous with respect to an operably linked promoter that is functional in a plant cell if such combination is not normally found in nature. A recombinant DNA molecule also may be heterologous with respect to a cell, seed, or organism into which it is inserted when it would not naturally occur in that cell, seed, or organism.

[0296] A “heterologous protein” is a protein present in a plant, seed, cell, tissue, or organism in which it does not naturally occur or operably linked to a protein with which it is not naturally linked. An example of a heterologous protein is an Lr67 protein or homolog thereof comprising at least a first amino acid substitution described herein that is expressed in any plant, seed, cell, tissue, or organism. Another example is a protein operably linked to a second protein, such as a transit peptide, with which it is not naturally linked, or a protein introduced into a plant cell in which it does not naturally occur using the techniques of genetic engineering.

[0297] As used herein, “operably linked” means two or more DNA molecules or sequences or two or more proteins linked in manner so that one may affect the function of the other. Operably linked DNA molecules or sequences may be part of a single contiguous molecule, chromosome or vector and may or may not be adjacent or continuous. For example, a promoter is operably linkedwith a protein-coding sequence of a gene or transgene in a DNA construct or molecule where the promoter and the protein-coding sequence arc so arranged that the promoter may affect the expression of the protein-coding sequence of the gene or transgene.

[0298] The DNA constructs of the present disclosure may include a promoter operably linked to a protein-coding DNA molecule provided herein, whereby the promoter drives expression of the protein. Promoters useful in practicing the contemplated embodiments include those that function in a plant cell for expression of an operably linked DNA molecule or nucleotide coding sequence of a transgene or expression cassette. Plant promoters may include, for instance, those that are viral, synthetic, constitutive, temporally regulated, spatially regulated, or spatio- temporally regulated.

[0299] As used herein, “transgene expression,” “expressing a transgene,” “protein expression,” and “expressing a protein” mean the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into polypeptide chains, which are ultimately folded into proteins. A protein-coding DNA molecule may be operably linked to a heterologous promoter in a DNA construct for use in expressing the protein in a cell transformed with the recombinant DNA molecule.A. Plant Transformation Constructs

[0300] Vectors used for plant transformation may include, for example, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or any other suitable cloning system, as well as fragments of DNA therefrom. Thus when the term “vector” or “expression vector” is used, all of the foregoing types of vectors, as well as nucleic acid sequences isolated therefrom, are included. It is contemplated that utilization of cloning systems with large insert capacities will allow introduction of large DNA sequences comprising more than one selected gene. In accordance with the present disclosure, this could be used to introduce genes corresponding to an entire biosynthetic pathway into a plant. Introduction of such sequences may be facilitated by use of bacterial or yeast artificial chromosomes (BACs or YACs, respectively), or even plant artificial chromosomes. For example, the use of BACs for graZzacterzw / n-mediated transformation was disclosed by Hamilton etal. Proc. Natl. Acad. Sci. USA 93:9975-9979, 1996).

[0301] Particularly useful for transformation are expression cassettes that have been isolated from such vectors. DNA segments used for transforming plant cells will, of course, generallycomprise the cDNA, gene or genes that one desires to introduce into and have expressed in the host cells. These DNA segments can further include structures such as promoters, enhancers, polylinkers, terminators or even regulatory genes as desired. The DNA segment or gene chosen for cellular introduction will often encode a protein that will be expressed in the resultant recombinant cells resulting in a screenable or selectable trait and / or that will impart an improved phenotype to the resulting transgenic plant. However, this may not always be the case, and the present disclosure also encompasses transgenic plants incorporating non-expressed transgenes. Components that may be included with vectors used in the current disclosure are as follows.B. Promoters and Other Regulatory Elements

[0302] In certain embodiments, the presently disclosed recombinant DNA vectors or expression cassettes further comprise one or more promoters. As used herein, the term “promoter” refers generally to a DNA molecule that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. A promoter may be initially isolated from the upstream region or the 5' untranslated region (5' UTR) of a gene. Alternatively, promoters may be synthetically produced or manipulated DNA molecules. Promoters may also be chimeric, such as a promoter produced through the fusion of two or more heterologous DNA molecules. Promoters useful in practicing the present disclosure include SEQ ID NOs: 8-23 and 225-230, or fragments or variants thereof. In some embodiments of the present disclosure, such molecules or sequences and any variants or derivatives thereof as described herein, are further defined as comprising promoter activity, i.e., are capable of acting as a promoter in a host cell, such as in a transgenic plant, plant pail or plant cell. In still further specific embodiments, a fragment of a promoter may be defined as exhibiting promoter activity possessed by the promoter from which it is derived, or a fragment of a promoter may comprise a “minimal promoter” which provides a basal level of transcription and is comprised of a TATA box or equivalent sequence for recognition and binding of the RNA polymerase II complex for initiation of transcription. According to embodiments, a nucleotide coding sequence of the present disclosure is operably linked to a heterologous promoter, wherein the heterologous promoter is an above-ground promoter, a green tissue promoter, or a constitutive promoter. According to embodiments of the present disclosure, such a heterologous promoter is an above-ground promoter.

[0303] As used herein, the term “above-ground promoter” refers to a promoter that drives expression of a nucleotide coding sequence operably linked to the promoter in a plant, such as a soybean, leguminous or dicot plant, at a much higher level in one or more above-ground tissues of the plant (e.g., leaf, stem and / or branch tissues) relative to the expression level of the operably linked nucleotide coding sequence in below-ground root tissues of the plant. In certain embodiments, the level of expression of the operably linked nucleotide coding sequence is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50-fold higher in the one or more above-ground tissues of the plant relative to the below-ground root tissues. In certain embodiments, an above-ground promoter drives expression of the operably linked nucleotide coding sequence at a medium or high level in one or more above-ground tissues of the plant. In certain embodiments, an above-ground promoter drives expression of the operably linked nucleotide coding sequence at a low or very low level in one or more below-ground root tissues of the plant. In certain embodiments, an above-ground promoter drives expression of the operably linked nucleotide coding sequence at an undetectable level in the one or more below-ground root tissues of the plant. In certain embodiments, an above-ground promoter drives a medium or high level of expression of the operably linked nucleotide coding sequence in one or more green photosynthetic above-ground tissues of a plant. In certain embodiments, the above-ground promoter drives a medium or high level of expression of the operably linked nucleotide coding sequence in one or more leaf tissues of a plant. In certain embodiments, the above-ground promoter drives a medium or high level of expression of the operably linked nucleotide coding sequence in one or more stem and / or branch tissues of a plant. In certain embodiments, the aboveground promoter drives a medium or high level of expression of the operably linked nucleotide coding sequence in trifolate leaf tissues of a plant. In certain embodiments, an above-ground promoter drives expression of the operably linked nucleotide coding sequence at a medium or high level in one or more non-vascular tissues, such as one or more above-ground, non-vascular tissues, of a plant. In certain embodiments, the above-ground promoter drives a low, very low or undetectable level of expression of the operably linked nucleotide coding sequence in one or more vascular tissue(s) of a plant. Vascular tissues of a plant include the tissues of the xylem and / or phloem of the plant, whereas non-vascular tissues of a plant include other tissues of the plant that are not part of the xylem and / or phloem of the plant. In certain embodiments, the above-ground promoter drives a low, very low or undetectable level of expression of the operably linkednucleotide coding sequence in one or more meristem tissue(s) of a plant. In certain embodiments, the above-ground promoter drives expression of the operably linked nucleotide coding sequence at a medium or high level in one or more above-ground leaf tissues of a plant and at a low or very low level in the one or more below-ground root tissues of the plant.

[0304] According to many embodiments of the present disclosure, an above-ground promoter is a green tissue promoter. As used herein, a “green tissue promoter” refers to a promoter that drives expression of a nucleotide coding sequence operably linked to the promoter in a plant, such as a soybean, leguminous or dicot plant, at a higher level in one or more above-ground and green (or photosynthetic) tissues of the plant relative to the expression level of the operably linked nucleotide coding sequence in other above-ground, non-green (or non-photosynthetic) tissues of the plant. According to certain embodiments, the non-green (or non-photosynthetic) tissues of the plant include one or more vascular tissues of the plant. According to certain embodiments, the green tissue promoter is an above-ground promoter. In certain embodiments, a green tissue promoter drives a medium or high level of expression of the operably linked nucleotide coding sequence in one or more green (or photosynthetic) above-ground tissues of a plant. In certain embodiments, a green tissue promoter drives expression of an operably linked nucleotide coding sequence at a low or very low level in the one or more vascular tissues of the plant. In certain embodiments, the above-ground promoter or green tissue promoter drives expression of an operably linked nucleotide coding sequence at a low or very low level in the one or more reproductive tissues of the plant. In certain embodiments, the level of expression of a nucleotide coding sequence operably linked to the green tissue promoter is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50-fold higher in one or more above-ground, green (or photosynthetic) tissues of the plant relative to other above-ground, non-green (or nonphotosynthetic) tissues of the plant, to vascular tissues of the plant, and / or to below-ground root tissues. A green-tissue promoter may include a promoter isolated from one of a number of photosynthetic or other genes that are at least preferentially expressed in green or photosynthetic tissues of a plant as known in the art, such as, for example, a promoter sequence isolated from a Rubisco (large or small subunit) (see, e.g., Fischhoff et al. (1992) Plant Mol. Biol. 20:81-93), Rubisco activase, cytochrome, light harvesting chlorophyll (Lhc), aldolase and pyruvate orthophosphate dikinase (PPDK) (see, e.g., Taniguchi et al. (2000) Plant Cell Physiol. 41(1):42- 48), or photosystem I and II gene, or any promoter sequence having at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any such promoter sequence, or a functional fragment thereof. Sec, c.g., Berry, J.O. ct al., Photosynthetic gene expression in higher plants, Photosynth Res 117:91-120 (2013), the entire contents and disclosure of which are hereby incorporated herein by reference. Examples of green tissue promoters (and above-ground promoters) include Cucumis melo Lhc promoters, P- CUCme.CipLhcbri (SEQ ID NO: 227) and P-CUCme.CipLhcb:3 (SEQ ID NO: 228), Medicago truncatula Lhc promoter, P-Mt.Lhcb2:l (SEQ ID NO: 229), and an Arabidopsis thaliana Rubisco small subunit promoter, P-At.RbcS4:2 (SEQ ID NO: 230).

[0305] In some embodiments, an above-ground promoter or green tissue promoter comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to one or more of SEQ ID NOs: 8-23 and 225-230, or a functional fragment thereof. In some embodiments, the heterologous promoter comprises a nucleotide sequence that is 100% identical to one or more of SEQ ID NOs: 8-23 and 225-230, or a functional fragment thereof. As used herein, a “functional fragment” of an above-ground promoter, green tissue promoter or constitutive promoter is a DNA fragment or portion of the DNA sequence of the above-ground promoter, green tissue promoter or constitutive promoter, respectively, that drives expression of a nucleotide coding sequence operably linked to the fragment in a manner that is similar to the full-length DNA sequence of the above-ground promoter, green tissue promoter or constitutive promoter, respectively, and itself meets the definition for an above-ground promoter, green tissue promoter or constitutive promoter, respectively, based on its pattern of tissue expression.

[0306] According to certain embodiments, the expression level of the nucleotide coding sequence operably linked to the heterologous above-ground promoter, green tissue promoter or constitutive promoter in one or more tissues of a plant may be quantified and defined in terms of the “fragments per kilobase of transcript per million mapped reads” or “FPKM” of mRNA transcripts expressed from the nucleotide coding sequence in such plant tissue(s), respectively, as known in the art. See, e.g., Zhao et al., J. Transl. Med. 19: 269, 2021, the entire content and disclosure of which is hereby incorporated by reference into this disclosure. Briefly, the total reads in a sample are counted and divided by 1 million (as a scaling factor), the read counts for the transcript encoded by the gene (or transgene) of interest are then divided by the “per million” scaling factor (to normalize the sequencing depth) to provide reads per million (RPM) for thetranscript encoded by the gene (or transgene) of interest, and then the RPM value is divided by the length of the coding sequence of the gene (or transgcnc) encoding the transcript (in kilobascs) to generate the FPKM for the expression of the gene (or transgene) in the sample. Thus, a FPKM for a heterologous above-ground promoter or green tissue promoter operably linked to a nucleotide coding sequence in one or more tissues of a plant can be determined by taking a sample from the one or more tissues of the plant and determining the FPKM for transcripts expressed from the nucleotide coding sequence. Although a FPKM for a nucleotide coding sequence operably linked to a heterologous above-ground promoter or green tissue promoter in one or more tissues of a plant may vary among more specific cells or tissues within the one or more tissues of the plant, the FPKM would be the average or collective FPKM for the one or more tissues of the plant. While a FPKM value for a native promoter of an endogenous gene in a plant may also be experimentally determined based on the expression of the endogenous gene in the plant, the FPKM values for such above-ground promoter or green tissue promoter operably linked to a heterologous nucleotide coding sequence as provided herein are defined in terms of the expression of the operably linked nucleotide coding sequence. However, the FPKM value for a promoter of an endogenous gene may provide a strong indication of the FPKM value for such promoter when operably linked to a heterologous nucleotide coding sequence as described herein.

[0307] For the present disclosure, a promoter may be described as driving a “low” level of expression in one or more tissues of a plant if the FPKM value for the operably linked nucleotide coding sequence in the one or more plant tissues is less than about 7 but greater than or equal to about 4, such as a FPKM value of about 4, about 5, or about 6, and a promoter may be described as driving a “very low” level of expression in one or more tissues of a plant if the FPKM value for the operably linked nucleotide coding sequence in the one or more plant tissues is less than about 4, such as a FPKM value of less than or equal to 3, less than or equal to 2, or less than or equal to 1. For the present disclosure, a promoter may be described as driving a “medium” level of expression in one or more tissues of a plant if the FPKM value for the operably linked nucleotide coding sequence in the one or more plant tissues is greater than or equal to about 7 but less than about 10, such as a FPKM value of about 7, about 8, or about 9, and a promoter may be described as driving a “high” level of expression in one or more tissues of a plant if the FPKM value for the operably linked nucleotide coding sequence in the one or more plant tissues is greater than or equalto about 10, such as a FPKM value of greater than or equal to 11 , greater than or equal to 12, greater than or equal to 13, greater than or equal to 14, or greater than or equal to 15.

[0308] In certain embodiments, the heterologous above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence in one or more above-ground tissues of the plant at a FPKM value of greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In certain embodiments, the heterologous above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence in one or more below-ground root tissues of the plant at a FPKM value of less than or equal to 4, less than or equal to 3, less than or equal to 2, or less than or equal to 1. In certain embodiments, the heterologous above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence in green photosynthetic above-ground tissues of a plant at a FPKM value of greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In certain embodiments, the heterologous above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence in one or more leaf tissues of a plant at a FPKM value of greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In certain embodiments, the heterologous above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence in one or more stem and / or branch tissues of a plant at a FPKM value of greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In certain embodiments, the heterologous above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence in trifolate leaf tissues of a plant at a FPKM value of greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In certain embodiments, the heterologous above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence in one or more vascular tissue(s) of a plant at a FPKM value of less than or equal to 4, less than or equal to 3, less than or equal to 2, or less than or equal to 1. In certain embodiments, the heterologous above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence in one or more meristem tissue(s) of a plant at a FPKM value of less than or equal to 4, less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0309] In addition to above-ground promoters disclosed herein, other promoters for expression of a nucleotide coding sequence may include any constitutive plant promoter known in the art that is functional in a dicot or soybean plant cell, such as a cauliflower mosaic virus (CaMV) 35S or 19S promoter (see, e.g., U.S. Pat. Nos. 5,352,605 and 5,858,742; Odell et al., Nature 313:810-812, 1985; and Lawton et al., Plant Mol. Biol. 9:315-324, 1987), an enhanced CaMV 35S promoter, such as a CaMV 35S promoter with Omega region (see, e.g., Holtorf, S. et al., Plant Molecular Biology, 29: 637-646 (1995) or a dual enhanced CaMV promoter (see, e.g., U.S. Pat. No. 5,322,938), a Figwort Mosaic Virus (FMV) 35S promoter (see, e.g., U.S. Pat. No. 6,372,211), a Mirabilis Mosaic Virus (MMV) promoter (see, e.g., U.S. Pat. No. 6,420,547), a Peanut Chlorotic Streak Caulimovirus promoter (see, e.g., U.S. Pat. No. 5,850,019), a nopaline or octopine promoter, a ubiquitin promoter, such as a soybean polyubiquitin promoter (see, e.g., U.S. Pat. No. 7,393,948), a nopaline synthase (nos) promoter or octopine synthase (ocs) promoter (see, e.g., Ebert et al., Proc. Natl. Acad. Sci. USA 84:5745-5749, 1987), an Arabidopsis S- Adenosylmethionine synthetase promoter (see, e.g., U.S. Pat. No. 8,809,628), an Adh promoter (see, e.g., Walker et al., Proc. Natl. Acad. Sci. USA 84:6624-6628, 1987), a sucrose synthase promoter (see, e.g., Yang and Russell, Proc. Natl. Acad. Sci. USA 87:4144-4148, 1990), an a- tubulin or actin promoter (see, e.g., U.S. Pat. No. 5,641,876; and Wang et al., Mol. Cell Biol. 12:3399-3406, 1992), a cab promoter (see, e.g., Sullivan et al., Mol. Gen. Genet. 215:431-440, 1989), a PEPCase promoter (see, e.g., Hudspeth and Grula, Plant Mol. Biol. 12:579-589, 1989); or a promoter associated with a R gene complex (see, e.g., Chandler et al., Plant Cell 1:1175-1183, 1989), the entire contents and disclosures of any of the foregoing references are hereby incorporated herein by reference, or any similar promoter sequence or functional fragment of any of the foregoing.

[0310] The DNA sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence or 5’ untranslated region (5’ UTR), can also influence gene expression. One may wish to employ a particular leader sequence with a transformation construct of the present disclosure. Leader sequences are contemplated to include those that comprise sequences predicted to direct optimum expression of the attached gene, i.e., to include a consensus leader sequence that may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be known to those ofskill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants may be desirable.

[0311] It is envisioned that presently disclosed coding sequences may be introduced under the control of a novel promoter, leader, enhancer, etc., or a homologous or tissue specific promoter and / or expression control element. Vectors for use in tissue-specific targeting of genes in transgenic plants may include tissue- specific promoters and may also include other tissue-specific control elements such as enhancer sequences. Promoters that direct specific or enhanced expression in certain plant tissues will be known to those of skill in the ail in light of the present disclosure.C. Transcription Terminators

[0312] In certain embodiments, the presently disclosed expression cassettes further comprise one or more transcription terminators. Transformation constructs prepared in accordance with the present disclosure will typically include a 3' end DNA sequence that acts as a signal to terminate transcription and allow for the polyadenylation of the mRNA produced by coding sequences operably linked to a promoter. In one embodiment of the present disclosure, the native terminator associated with a nucleic acid coding sequence disclosed herein is used. Alternatively, a heterologous terminator relative to the nucleic acid coding sequence may be used for expression of the transgene. In addition to any terminator sequences disclosed herein, other transcription terminators known in the art may be used, such as those from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3’ end) (Bevan et al., Nucl. Acids Res. 11:369-385, 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato. Regulatory elements such as an Adh intron (Callis et al., Genes Dev. 1:1183-1200, 1987), sucrose synthase intron (Vasil et al., Plant Physiol. 91:1575-1579, 1989) or TMV omega element (Gallic and Kado, Proc. Natl. Acad. Sci. USA 86:129-132, 1989), may further be included where desired.D. Transit or Signal Peptides

[0313] In certain embodiments of the present disclosure, transit or signal sequences may be incorporated into the presently disclosed coding sequences. Sequences that are joined to the coding sequence of an expressed gene, which are removed post-translationally from the initial translation product and that facilitate the transport of the protein into or through intracellular orextracellular membranes, are termed transit (usually into vacuoles, vesicles, plastids and other intracellular organelles) and signal sequences (usually to the endoplasmic reticulum, golgi apparatus and outside of the cellular membrane). In some embodiments, a DNA construct provided herein includes a DNA sequence encoding a transit sequence that is operably linked to a heterologous DNA or nucleotide sequence encoding a Lr67 protein or homolog thereof as provided herein, whereby the transit sequence facilitates localizing the protein molecule within the cell. Transit sequences are known in the art as signal sequences, targeting peptides, targeting sequences, localization sequences, and transit peptides. An example of a transit sequence is a chloroplast transit peptide (CTP), a mitochondrial transit sequence (MTS), or a dual chloroplast and mitochondrial transit peptide. By facilitating protein localization within the cell, the transit sequence may increase the accumulation of recombinant protein, protect the protein from proteolytic degradation, or enhance the level of herbicide tolerance, and thereby reduce levels of injury in the cell, seed, or organism after herbicide application. CTPs and other targeting molecules that may be used in connection with the present disclosure are well known in the ail.

[0314] By facilitating the transport of the protein into compartments inside and outside the cell, these sequences may increase the accumulation of gene product protecting them from proteolytic degradation. These sequences also allow for additional mRNA sequences from highly expressed genes to be attached to the coding sequence of the genes. Since mRNA being translated by ribosomes is more stable than naked mRNA, the presence of translatable mRNA in front of the gene may increase the overall stability of the mRNA transcript from the gene and thereby increase synthesis of the gene product. Since transit and signal sequences are usually post-translationally removed from the initial translation product, the use of these sequences allows for the addition of extra translated sequences that may not appear on the final polypeptide. It further is contemplated that targeting of certain proteins may be desirable in order to enhance the stability of the protein (U.S. Patent No. 5,545,818, incorporated herein by reference in its entirety).

[0315] Additionally, vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment. This generally will be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit, or signal, peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and will then be post-translationally removed.E. Marker Genes

[0316] By employing a selectable or screenable marker protein, one can provide or enhance the ability to identify transformants. “Marker genes” are genes that impart a distinct phenotype to cells expressing the marker protein and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait that one can “select” for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i.e., by “screening” (e.g., the green fluorescent protein). Many examples of suitable marker proteins are known to the art and can be employed in the practice of the present disclosure.

[0317] Included within the terms “selectable” or “screenable” markers also are genes that encode a “secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers that are secretable antigens that can be identified by antibody interaction, or even secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; small active enzymes detectable in extracellular solution (e.g., a-amylase, P-lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR S).

[0318] Many selectable marker coding regions are known and could be used with the present disclosure including, but not limited to, neo (Potrykus etal., Mol. Gen. Genet. 199:169-177, 1985), which provides kanamycin resistance and can be selected for using kanamycin, G418, paromomycin, etc , bar, which confers bialaphos or phosphinothricin resistance; a mutant EPSP synthase protein conferring glyphosate resistance; a nitrilase such as bxn from Klebsiella ozaenae, which confers resistance to bromoxynil (Stalker et al., J. Biol. Chem. 263:6310-6314, 1988); a mutant acetolactate synthase (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS inhibiting chemicals (European Patent Application 154,204, 1985); a methotrexate resistant DHFR (Thillet et al., J. Biol. Chem. 263:12500-12508, 1988), a dalapon dehalogenase that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase that confers resistance to 5-methyl tryptophan.

[0319] An illustrative embodiment of selectable marker capable of being used in systems to select transformants arc those that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viriclochromo genes . The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, causing rapid accumulation of ammonia and cell death.

[0320] Screenable markers that may be employed include a 0 glucuronidase (GUS) or uidA gene, which encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues; a 0 lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA 75:3737-3741, 1978), which encodes an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA 80:1101- 1105, 1983), which encodes a catechol dioxygenase that can convert chromogenic catechols; an a-amylase gene (Ikuta et al., Biotechnology 8:241-242, 1990); a tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714, 1983), which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form the easily -detectable compound melanin; a 0 galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., Science 234:856-859, 1986), which allows for bioluminescence detection; an aequorin gene (Prasher et al., Biochem. Biophys. Res. Commun. 126:1259-1268, 1985), which may be employed in calcium-sensitive bioluminescence detection; or a gene encoding for green fluorescent protein (GFP; Sheen et al., Plant J. 8:777-784, 1995; Haseloff et al., Proc. Natl. Acad. Sci. USA 94:2122-2127, 1997; Reichel et al., Proc. Natl. Acad. Sci. USA 93:5888-5893, 1996; WO 97 / 41228) is also contemplated as a useful reporter gene. Expression of green fluorescent protein may be visualized in a cell or plant as fluorescence following illumination by particular wavelengths of light.III. Gene Editing

[0321] One method for producing the transgenic plants of the present disclosure is through genome modification using site-specific integration or genome editing. Targeted modification of plant genomes through the use of genome editing methods can be used to create improved plant lines through modification of plant genomic DNA and / or incorporation of a transgene at the target site. As used herein “site-directed integration” refers to genome editing methods that enabletargeted insertion of one or more nucleic acids of interest, such as a recombinant DNA molecule, construct, transgcnc or cassette, into a plant genome. Suitable methods for altering a preexisting transgenic sequence or for inserting DNA into a plant genome at a pre-determined chromosomal target site include any method known in the art. Exemplary methods include the use of sequence specific nucleases, such as zinc-finger nucleases, engineered or native meganucleases, TALE- endonucleases, or RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) / Cas9 system, a CRISPR / Cpfl system, a CRISPR / CasX system, a CRISPR / CasY system, or a CRISPR / Cascade system). Several embodiments relate to methods of genome editing by using single- stranded oligonucleotides or guide RNAs to introduce precise base pair modifications in a plant genome. Methods of genome editing to modify, delete, or insert nucleic acid sequences into genomic DNA are known in the art.

[0322] In certain embodiments, the present disclosure provides modification or replacement of an existing coding sequence, such as an existing transgenic insert, within a plant genome with a sequence encoding a different protein, or an expression cassette comprising such a protein. Several embodiments relate to the use of a known genome editing methods, such as zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, or an RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) / Cas9 system, a CRISPR / Cpfl system, a CRISPR / CasX system, a CRISPR / CasY system, or a CRISPR / Cascade system).

[0323] Several embodiments may therefore relate to a recombinant DNA construct comprising an expression cassette(s) encoding a site-specific nuclease and, optionally, any associated protein(s) and / or guide RNAs (gRNAs) to carry out genome modification. These nuclease- expressing cassette(s) may be present in the same molecule or vector as a donor template for templated editing or targeted insertion or integration of a transgene(s) of interest. Several methods for site-directed integration are known in the art involving different sequence- specific nucleases (or complexes of proteins or guide RNA or both) that cut the genomic DNA to produce a double strand break (DSB) or nick at a desired genomic site or locus. As understood in the ait, during the process of repairing the DSB or nick introduced by the nuclease enzyme, the donor template DNA, transgene, or expression cassette may become integrated into the genome at the site of the DSB or nick. The presence of the homology arm(s) in the DNA to be integrated may promote the adoption and targeting of the insertion sequence into the plant genome during the repair process throughhomologous recombination, although an insertion event may occur through non-homologous end joining (NHEJ). As used herein, the term “double- strand break inducing agent” refers to any agent that can induce a double-strand break (DSB) in a DNA molecule. In some embodiments, the double-strand break inducing agent is a site-specific genome modification enzyme.

[0324] As used herein, the term “site- specific genome modification enzyme” refers to any enzyme that can modify a nucleotide sequence in a sequence- specific manner. In some embodiments, a site-specific genome modification enzyme modifies the genome by inducing a single-strand break. In some embodiments, a site-specific genome modification enzyme modifies the genome by inducing a double-strand break. In some embodiments, a site-specific genome modification enzyme comprises a cytidine deaminase. In some embodiments, a site specific genome modification enzyme comprises an adenine deaminase. Site-specific genome modification enzymes include endonucleases, recombinases, transposases, deaminases, helicases and any combination thereof. In some embodiments, the site-specific genome modification enzyme is a sequence-specific nuclease.

[0325] In one aspect, the endonuclease is selected from a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nucleases (TALEN), an Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), and Natronobacterium gregoryi Argonaute (NgAgo)), or an RNA-guided nuclease, such as a CRISPR associated nuclease (non-limiting examples of CRISPR associated nucleases include, but are not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, CasX, CasY, homologs thereof, or modified versions thereof).

[0326] In some embodiments, the site-specific genome modification enzyme is a recombinase. Non-limiting examples of recombinases include a tyrosine recombinase attached to a DNA recognition motif and is selected from the group consisting of a Cre recombinase, a Gin recombinase, a Flp recombinase, and a Tnpl recombinase. In one aspect, a Cre recombinase or a Gin recombinase is tethered to a zinc-finger DNA-binding domain, or a TALE DNA binding domain, or a Cas9 nuclease. In another aspect, a serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901integrase. In another aspect, a DNA transposase attached to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE-Mutator.

[0327] Any DNA of interest as provided herein can be integrated into a target site of a chromosome sequence by introducing the DNA of interest and the disclosed site-specific genome modification enzymes. Any method provided herein can utilize any site-specific genome modification enzyme disclosed herein.IV. Transformation

[0328] In some embodiments, transgenic plants of the present disclosure are created by transforming the selected natural plants with one or more of the expression cassettes or constructs disclosed herein. The natural plants prior to transformation are not naturally resistant to the disclosed fungal diseases. In certain embodiments, the selected plants for transformation include wild-type, or untransformed, or non-transformed plants, which may not be resistant to the disclosed fungal diseases, provided that such plants may comprise one or more mutations, edits and / or transgenes that may or may not provide fungal disease resistance.

[0329] Suitable methods for transformation of plant or other cells for use with the current disclosure are believed to include virtually any method known in the art by which DNA can be introduced into a cell, such as by direct delivery of DNA such as by PEG-mediated transformation of protoplasts (Omirulleh et al., Plant. Mol. Biol. 21:414-428, 1993), by desiccation / inhibition- mediated DNA uptake (Potrykus et al. , Mol. Gen. Genet. 199: 169-177, 1985), by electroporation (U.S. Patent No. 5,384,253, specifically incorporated herein by reference in its entirety), by agitation with silicon carbide fibers (U.S. Patent Nos. 5,302,523 and 5,464,765, specifically incorporated herein by reference in their entirety), by AgroZwcterzum-mediated transformation (U.S. Patent No. 5,591,616 and U.S. Patent No. 5,563,055; both specifically incorporated herein by reference in their entirety) and by acceleration of DNA coated particles (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,877; and U.S. Patent No. 5,538,880; each specifically incorporated herein by reference in their entirety), etc. Through the application of techniques such as these, the cells of virtually any plant species may be transiently transformed, or stably transformed, and these cells developed into transgenic plants.A. Agrobacterium -mediated T ransformation

[0330] Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agro&acten'um-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described by Fraley et al. , (Proc. Natl. Acad. Sci. USA 80:4803-4807, 1985), and U.S. Patent No. 5,563,055, specifically incorporated herein by reference in its entirety.

[0331] Agrobacterium-mediated transformation is most efficient in dicotyledonous plants and is an efficient method for transformation of dicots, including Arabidopsis. soybean, tobacco, tomato, alfalfa and potato. Indeed, while Agrobacterium-mediated transformation has been routinely used with dicotyledonous plants for a number of years, it has only recently become applicable to monocoty ledonous plants. Advances in Agrobacterium-mediated transformation techniques have now made the technique applicable to nearly all monocotyledonous plants. For example, Agrobacterium-mediated transformation techniques have now been applied to rice (Hiei et al., Plant Mol. Biol. 35:205-218, 1997; U.S. Patent No. 5,591,616, specifically incorporated herein by reference in its entirety), wheat and barley (McCormac et al., Mol. Biotechnol. 9:155- 159, 1998), alfalfa and maize (Ishida et al., Nat. Biotechnol. 14:745-750, 1996). Similarly, Agrobacterium-mediated transformation has also proven to be effective in switchgrass.

[0332] Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations. Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.B. Electroporation

[0333] To effect transformation by electroporation, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Examples of some species that have been transformed by electroporation of intact cells include maize (U.S. Patent No. 5,384,253, incorporated herein by reference in its entirety; Rhodes et al., Methods Mol. Biol. 55:121-131, 1995; D’Halluin et al., Plant Cell 4:1495-1505, 1992), wheat (Zhou et al., Plant Cell Rep. 12:612- 616, 1993), tomato (Tsukada et al., Plant Cell Physiol. 30:599-603, 1989), soybean (Christou et al., Proc. Natl. Acad. Sci. USA 84:3962-3966, 1987) and tobacco (Riggs and Bates, Proc. Natl. Acad. Sci. USA 83:5602-5606, 1986).

[0334] One also may employ protoplasts for electroporation transformation of plants (Bates, Mol. Biotechnol. 2:135-145, 1994; Lazerri, Methods Mol. Biol. 49:95-106, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described in WO 9217598 (specifically incorporated herein by reference). Other examples of species for which protoplast transformation has been described include barley (Lazerri, supra), sorghum (Battraw et al., Theor. Appl. Genet. 82:161-168, 1991), maize (Rhodes et al., Science 240:204-207, 1988), wheat (He et al., Plant Cell Rep. 14:192-196, 1994) and tomato (Tsukada, supra).C. Microprojectile Bombardment

[0335] Another method for delivering transforming DNA segments to plant cells in accordance with the present disclosure is microprojectile bombardment (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT Application WO 94 / 09699; each of which is specifically incorporated herein by reference in its entirety). In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and often, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.

[0336] For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.

[0337] An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with monocot plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species. Examples of species that have been transformed by microprojectile bombardment include monocot species such as maize (PCT Application WO 95 / 06128), barley (Ritala et al., Plant Mol. Biol. 24:317-325, 1994; Hensgens et al., Plant Mol. Biol. 22:1101-1127, 1993), wheat (U.S. Patent No. 5,563,055, specifically incorporated herein by reference in its entirety), rice (Hensgens et al., supra), oat (Torbet etal., Crop Science 38:226-231, 1998), rye (Hensgens et al., supra), sugarcane (Bower et al., Plant J. 2:409-416, 1992), and sorghum (Casas et al., Proc. Natl. Acad. Sci. USA 90:11212-11216, 1993; Hagio et al., Plant Cell Rep. 10:260-264, 1991); as well as a number of dicots including tobacco (Tomes et al., Plant Mol. Biol. 14:261-268, 1990), soybean (U.S. Patent No. 5,322,783, specifically incorporated herein by reference in its entirety), sunflower (Knittel et al., Plant Cell Rep. 14:81-86, 1994), peanut (Singsit et al., Transgenic Res. 6:169-176, 1997), cotton (McCabe and Martinell, Nat. Biotechnol. 11:596-598, 1993), tomato (VanEck et al., Plant Cell. Rep. 14:299-304, 1995), switchgrass (Richards et al., Plant Cell Rep. 20:48-54, 2001) and legumes in general (U.S. Patent No. 5,563,055, specifically incorporated herein by reference in its entirety).D. Other Transformation Methods

[0338] Transformation of protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., supra; Omirulleh et al., supra;). Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain fromprotoplasts. Illustrative methods for the regeneration of cereals from protoplasts have been described (Toriyama et al., Nat. Biotechnol. 6:1072-1074, 1988; Abdullah et al., Nat. Biotechnol. 4:1087-1090, 1986; Omirulleh et al., supra, and U.S. Patent No. 5,508,184; each specifically incorporated herein by reference in its entirety). Examples of the use of direct uptake transformation of cereal protoplasts include transformation of rice (Ghosh-Biswas et al., J. Biotechnol. 32:1-10, 1994), sorghum (Battraw et al., supra), barley (Lazzeri, supra), oat, and maize (Omirulleh et al., supra).

[0339] To transform plant strains that cannot be successfully regenerated from protoplasts, other ways to introduce DNA into intact cells or tissues can be utilized. For example, regeneration of cereals from immature embryos or explants can be effected as described (Vasil, supra). Also, silicon carbide fiber-mediated transformation may be used with or without protoplasting (Kaeppler et al., Theor. Appl. Genet. 84:560-566, 1992; U.S. Patent No. 5,563,055, specifically incorporated herein by reference in its entirety). Transformation with this technique is accomplished by agitating silicon carbide fibers together with cells in a DNA solution. DNA passively enters as the cells are punctured. This technique has been used successfully with, for example, the monocot cereals maize (PCT Application WO 95 / 06128, specifically incorporated herein by reference in its entirety) and rice (Nagatani et al., Biotechnol. Tech. 11:471-473, 1997).E. Tissue Culture

[0340] Tissue culturing may be used in certain transformation techniques for the preparation of cells for transformation and for the selection and regeneration of plants therefrom. Maintenance of tissue cultures requires use of media and controlled environments. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. The medium usually is a suspension of various categories of ingredients (salts, amino acids, growth regulators, sugars, buffers) that are required for growth of most cell types. However, each specific cell type requires a specific range of ingredient proportions for growth, and an even more specific range of formulas for optimum growth. Rate of cell growth also will vary among cultures initiated with the array of media that permit growth of that cell type.

[0341] Nutrient media is prepared as a liquid, but this may be solidified by adding the liquid to materials capable of providing a solid support. Agar may be commonly used for this purpose.

[0342] Some cell types will grow and divide either in liquid suspension or on solid media. As disclosed herein, plant cells will grow in suspension or on solid medium, but regeneration of plantsfrom suspension cultures typically requires transfer from liquid to solid media at some point in development. The type and extent of differentiation of cells in culture will be affected not only by the type of media used and by the environment, for example, pH, but also by whether media is solid or liquid.

[0343] Tissue that can be grown in a culture includes meristem cells, Type I, Type II, and Type III callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. Type I, Type II, and Type III callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, root, leaf, microspores and the like. Those cells that are capable of proliferating as callus also are recipient cells for genetic transformation. Tissue culturing and regeneration may also involve organogenic processes without the use of callus culture. See, e.g., U.S. Patent No. 8,362,317.

[0344] Somatic cells are of various types. Embryogenic cells are one example of somatic cells that may be induced to regenerate a plant through embryo formation. Non-embryogenic cells are those that typically will not respond in such a fashion. Certain techniques may be used that enrich recipient cells within a cell population. For example, Type II callus development, followed by manual selection and culture of friable, embryogenic tissue, generally results in an enrichment of cells. Manual selection techniques that can be employed to select target cells may include, e.g., assessing cell morphology and differentiation, or may use various physical or biological means. Cryopreservation also is a possible method of selecting for recipient cells.

[0345] Manual selection of recipient cells, e.g., by selecting embryogenic cells from the surface of a Type II callus, is one means that may be used in an attempt to enrich for particular cells prior to culturing (whether cultured on solid media or in suspension).

[0346] Where employed, cultured cells may be grown either on solid supports or in the form of liquid suspensions. In either instance, nutrients may be provided to the cells in the form of media, and environmental conditions controlled. There are many types of tissue culture media comprised of various amino acids, salts, sugars, growth regulators and vitamins. Most of the media employed in the practice of the present disclosure will have some similar components, but may differ in the composition and proportions of their ingredients depending on the particular application envisioned. For example, various cell types usually grow in more than one type of media, but will exhibit different growth rates and different morphologies, depending on the growth media. In some media, cells survive but do not divide. Various types of media suitable for cultureof plant cells previously have been described. Examples of these media include, but are not limited to, the N6 medium described by Chu ct al., (Sci. Sin. [Peking] 18:659-668, 1975) and MS media (Murashige and Skoog, Physiol. Plant 15:473-479, 1962).V. Production and Characterization of Stably Transformed Plants

[0347] After effecting delivery of exogenous DNA to recipient cells, the next steps generally concern identifying the transformed cells for further culturing and plant regeneration. In order to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene with a transformation vector prepared in accordance with the present disclosure. In this case, one would then generally assay the potentially transformed cell population by exposing the cells to a selective agent or agents, or one would screen the cells for the desired marker gene trait.A. Selection

[0348] It is believed that DNA is introduced into only a small percentage of target cells in any one study. In order to provide an efficient system for identification of those cells receiving DNA and integrating it into their genomes one may employ a means for selecting those cells that are stably transformed. One exemplary embodiment of such a method is to introduce into the host cell a marker gene that confers resistance to some normally inhibitory agent, such as an antibiotic or herbicide. Examples of antibiotics that may be used include the aminoglycoside antibiotics neomycin, kanamycin and paromomycin, or the antibiotic hygromycin. Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphotransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I, whereas resistance to hygromycin is conferred by hygromycin phosphotransferase.

[0349] Potentially transformed cells then are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA.

[0350] One herbicide that constitutes a desirable selection agent is the broad spectrum herbicide bialaphos. Bialaphos is a tripeptide antibiotic produced by Streptomyces hygroscopicus and is composed of phosphinothricin (PPT), an analogue of L-glutamic acid, and two L-alanine residues. Upon removal of the L-alanine residues by intracellular peptidases, the PPT is releasedand is a potent inhibitor of glutamine synthetase (GS), a pivotal enzyme involved in ammonia assimilation and nitrogen metabolism (Ogawa et al., Sci. Rep. Meiji Seika 13:42-48, 1973). Synthetic PPT, the active ingredient in the herbicide Liberty™ also is effective as a selection agent. Inhibition of GS in plants by PPT causes the rapid accumulation of ammonia and death of the plant cells.

[0351] The organism producing bialaphos and other species of the genus Streptomyces also synthesizes an enzyme phosphinothricin acetyl transferase (PAT), which is encoded by the bar gene in Streptomyces hygroscopicus and the pat gene in Streptomyces viridochromogen.es. The use of the herbicide resistance gene encoding phosphinothricin acetyl transferase (PAT) is referred to in DE 3642 829 A, wherein the gene is isolated from Streptomyces viridochromogenes . In the bacterial source organism, this enzyme acetylates the free amino group of PPT preventing autotoxicity (Thompson et al., EMBO J. 6:2519-2523, 1987). The bar gene has been cloned (Thompson et al., supra and expressed in transgenic tobacco, tomato, potato (De Block et al., EMBO J. 6:2513-2518, 1987) Brassica (De Block et al., Plant Physiol. 91:694-701, 1989) and maize (U.S. Patent No. 5,550,318, incorporated herein by reference in its entirety).

[0352] Another example of a herbicide that is useful for selection of transformed cell lines in the practice of the present disclosure is the broad spectrum herbicide glyphosate. Glyphosate inhibits the action of the enzyme EPSPS, which is active in the aromatic amino acid biosynthetic pathway. Inhibition of this enzyme leads to starvation for the amino acids phenylalanine, tyrosine, and tryptophan and secondary metabolites derived thereof. U.S. Patent No. 4,535,060 (incorporated herein by reference in its entirety) describes the isolation of EPSPS mutations that confer glyphosate resistance on the Salmonella typhimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zea mays and mutations similar to those found in a glyphosate resistant aroA gene were introduced in vitro. Mutant genes encoding glyphosate resistant EPSPS enzymes are described in, for example, International Patent WO 97 / 4103.

[0353] To use the bar-bialaphos or the EPSPS-glyphosate selective system, transformed tissue is cultured for 0-28 days on nonselective medium and subsequently transferred to medium containing from 1-3 mg / 1 bialaphos or 1-3 mM glyphosate as appropriate. While ranges of 1-3 mg / 1 bialaphos or 1-3 mM glyphosate may be beneficial, it is proposed that ranges of 0.1-50 mg / 1 bialaphos or 0.1-50 mM glyphosate will find utility.

[0354] An example of a screenable marker trait is the enzyme luciferase. Tn the presence of the substrate luciferin, cells expressing luciferase emit light that can be detected on photographic or x-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. These assays are nondestructive and transformed cells may be cultured further following identification. The photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells that are expressing luciferase and manipulate those in real time. Another screenable marker that may be used in a similar’ fashion is the gene coding for green fluorescent protein.B. Regeneration and Seed Production

[0355] Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. In an exemplary embodiment, MS and N6 media may be modified by including further substances such as growth regulators. One such growth regulator is dicamba or 2,4-D. However, other growth regulators may be employed, including NAA, NAA + 2,4-D or picloram. Media improvement in these and like ways has been found to facilitate the growth of cells at specific developmental stages. Tissue may be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, at least 2 weeks, then transferred to media conducive to maturation of embryoids. Cultures are transferred every 2 weeks on this medium. Shoot development will signal the time to transfer to medium lacking growth regulators.

[0356] The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, will then be allowed to mature into plants. Developing plantlets are transferred to soilless plant growth mix, and hardened, e.g., in an environmentally controlled chamber, for example, at about 85% relative humidity, 600 ppm CO2, and 25-250 microeinsteins m2s"1of light. Plants may be matured in a growth chamber or greenhouse. Plants can be regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Cons. Regenerating plants can be grown at about 19 to 28°C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.

[0357] Seeds on transformed plants may occasionally require embryo rescue due to cessation of seed development and premature senescence of plants. To rescue developing embryos, they arc excised from surface-disinfected seeds 10-20 days post-pollination and cultured. An embodiment of media used for culture at this stage comprises MS salts, 2% sucrose, and 5.5 g / 1 agarose. In embryo rescue, large embryos (defined as greater than 3 mm in length) are germinated directly on an appropriate media. Embryos smaller than that may be cultured for 1 week on media containing the above ingredients along with 10‘5M abscisic acid and then transferred to growth regulator-free medium for germination.C. Characterization

[0358] To confirm the presence of the exogenous DNA or “transgene(s)” in the regenerating plants, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays, such as Southern and northern blotting and PCR; “biochemical” assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also by analyzing the phenotype of the whole regenerated plant.

[0359] The plants, seeds, cells, plant parts, and commodity products of the disclosure may be used for detection of DNA or protein molecules indicative of the presence of the recombinant DNA molecules provided herein. Detection of the presence of such recombinant DNA molecules may be done by using methods known in the art, such as thermal amplification of nucleic acid or nucleic acid hybridization techniques (such as northern blotting and Southern analysis). Methods for designing and using primers and probes are well known in the art; and can readily be designed by one of skill in the art using the sequences provided herein. For example, DNA molecules comprising the full length of, or fragments of, SEQ ID NO: 3, 8-23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 223, 224, 225, or 226 are useful as primers and probes for detecting the presence of a recombinant DNA molecule provided herein.D. DNA Integration, RNA Expression and Inheritance

[0360] Genomic DNA may be isolated from cell lines or any plant parts to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art. Note that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell. The presence of DNA elements introduced through the methods of this disclosure may be determined, for example, by polymerase chain reaction (PCR). Using this technique, discreet fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a gene is present in a stable transformant, but does not prove integration of the introduced gene into the host cell genome. It is typically the case, however, that DNA has been integrated into the genome of all transformants that demonstrate the presence of the gene through PCR analysis. In addition, it is not typically possible using PCR techniques to determine whether transformants have exogenous genes introduced into different sites in the genome, i.e., whether transformants are of independent origin. It is contemplated that using PCR techniques it would be possible to clone fragments of the host genomic DNA adjacent to an introduced gene.

[0361] Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, i.e., confirm that the introduced gene has been integrated into the host cell genome. The technique of Southern hybridization provides information that is obtained using PCR, e.g., the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant.

[0362] It is contemplated that using the techniques of dot or slot blot hybridization, which are modifications of Southern hybridization techniques, one could obtain the same information that is derived from PCR, e.g., the presence of a gene.

[0363] Both PCR and Southern hybridization techniques can be used to demonstrate transmission of a transgene to progeny. In most instances the characteristic Southern hybridizationpattern for a given transformant will segregate in progeny as one or more Mendelian genes (Spencer ct al., 1992) indicating stable inheritance of the transgcnc.

[0364] Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA will only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues. PCR techniques also may be used for detection and quantitation of RNA produced from introduced genes. In this application of PCR it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species also can be determined using dot or slot blot northern hybridizations. These techniques are modifications of northern blotting and will only demonstrate the presence or absence of an RNA species.E. Gene Expression

[0365] While Southern blotting and PCR may be used to detect the gene(s) in question, they do not provide information as to whether the corresponding protein is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression.

[0366] Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physicalchemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.

[0367] Assay procedures also may be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed and may include assays for PAT enzymatic activity by following production of radiolabeled acetylated phosphinothricin from phosphinothricin and14C-acetyl CoA or for anthranilate synthase activity by following loss of fluorescence of anthranilate, to name two.

[0368] Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including, but not limited to, analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered by expression of genes encoding enzymes or storage proteins that change amino acid composition and may be detected by amino acid analysis, or by enzymes that change starch quantity, which may be analyzed by near infrared reflectance spectrometry. Morphological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays.VI. Breeding Plants

[0369] In addition to direct transformation of a particular plant genotype with a construct prepared according to the current disclosure, transgenic plants may be made by crossing a plant having a selected DNA of the present disclosure to a second plant lacking the construct. For example, a selected coding sequence can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the current disclosure not only encompasses a plant directly transformed or regenerated from cells that have been transformed in accordance with the current disclosure, but also the progeny of such plants.

[0370] As used herein the term “progeny” denotes the offspring of any generation of a parent plant prepared in accordance with the instant disclosure, wherein the progeny comprises a selected DNA construct. “Crossing” a plant to provide a plant line having one or more added transgenes relative to a starting plant line, as disclosed herein, is defined as the techniques that result in a transgene of the present disclosure being introduced into a plant line by crossing a starting linewith a donor plant line that comprises a transgene of the present disclosure. To achieve this one could, for example, perform the following steps:(a) plant seeds of the first (starting line) and second (donor plant line that comprises a transgene of the present disclosure) parent plants;(b) grow the seeds of the first and second parent plants into plants that bear flowers;(c) pollinate a flower from the first parent plant with pollen from the second parent plant; and(d) harvest seeds produced on the parent plant bearing the fertilized flower.

[0371] Backcrossing is herein defined as the process including the steps of:(a) crossing a plant of a first genotype containing a desired gene, DNA sequence or element to a plant of a second genotype lacking the desired gene, DNA sequence or element;(b) selecting one or more progeny plant containing the desired gene, DNA sequence or element;(c) crossing the progeny plant to a plant of the second genotype; and(d) repeating steps (b) and (c) for the purpose of transferring a desired DNA sequence from a plant of a first genotype to a plant of a second genotype.

[0372] Introgression of a DNA element into a plant genotype is defined as the result of the process of backcross conversion. A plant genotype into which a DNA sequence has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid. Similarly a plant genotype lacking the desired DNA sequence may be referred to as an unconverted genotype, line, inbred, or hybrid.VII. Commodity Products

[0373] The present disclosure provides a commodity product comprising DNA molecules according to the present disclosure. As used herein, a “commodity product” refers to any composition or product that is comprised of material derived from a plant, seed, plant cell or plant part comprising a DNA molecule of the present disclosure. Commodity products may be sold to consumers and may be viable or nonviable. Nonviable commodity products include, but are not limited to, nonviable seeds and grains; processed seeds, seed parts, and plant parts; dehydrated plant tissue, frozen plant tissue, and processed plant tissue; seeds and plant pails processed for animal feed for terrestrial and / or aquatic animals consumption, oil, meal, flour, flakes, bran, fiber, milk, cheese, paper, cream, wine, and any other food for human consumption; and biomasses andfuel products. Viable commodity products include, but are not limited to, seeds and plant cells. Plants comprising a DNA molecule according to the present disclosure can thus be used to manufacture any commodity product typically acquired from plants or parts thereof.VIII. Fungal Diseases

[0374] The transgenic plants of the present disclosure have increased resistance to a variety of fungal diseases. The following paragraphs describe in greater detail some, but not all, of these fungal diseases.

[0375] Asian Soybean Rust (ASR)

[0376] Asian Soybean Rust (ASR), caused by Phakopsora pcichyrhizi, is the single greatest biotic threat to soybean yields in Brazil. Control of this disease relies heavily on the application of fungicides, requiring multiple sprays each year. Genetic resistance has the potential to be both sustainable and durable when combining multiple modes of action; however, native sources of genetic resistance are few and most have already been overcome by the pathogen. Yield losses can be severe, and losses of 10-80% have been reported.

[0377] Soybean rust symptoms are most commonly observed on the leaves and will start in the lower canopy. Lesions can also develop on petioles, pods and stems. Symptom development occurs rapidly once the plant starts flowering and can result in significant levels of defoliation under favorable environmental conditions. Lesions first appear- as small yellow and irregularly shaped spots. As the disease progresses, lesions enlarge to l / 16th to 1 / 12th of an inch in diameter, and are tan to dark reddish brown in color. Within each lesion are a few to several volcano- shaped uredinia (spore producing structures). As rust severity increases, plants prematurely lose their leaves and commonly mature early.

[0378] Powdery Mildew

[0379] Powdery mildew, also known as white mold, is caused by many different species of ascomycete fungi in the order Erysiphales, and affects a wide variety of plant species. Symptoms of Powdery Mildew during the initial stages of infection may include visible light-green to bright- yellow blotches appearing on upper and lower surfaces of leaves followed by a powdery, white growth caused by the sporulation of the fungus. Under some environmental conditions these areas may later turn necrotic. Infected leaves may also curl upward and exhibit a visible powdery, white growth on the underside of leaves. When lesions are numerous, they often coalesce, resulting in general chlorosis and leaf drop. The disease generally progresses from older to younger leaves.

[0380] Airborne conidia (asexual fungal spores) from previously infected crops or weeds can be carried long distances by wind and act as initial sources of inoculum. The wide host range of these fungi exacerbate disease spread and reduce the ability of agronomic practice to control disease incidence. Disease control is commonly managed by application of fungicides before infection or immediately after the first symptoms are observed. In addition to the cost of pesticide application, there is increasing social pressure to reduce the pesticide load in the environment.

[0381] Target Spot

[0382] Target spot, caused by Corynespora cassiicola, can infect more than 500 species of plants, including many economically important crops such as cotton, soybean, tomato, and cucumber. Target spot symptoms begin as small dark lesions on the leaves that enlarge to form light brown lesions with concentric pattern and a yellow halo around it in the transplants. Lesions can be seen inside the canopy and the micro-climate favors the infection process.

[0383] Target spot has been a concern for farmers and researchers due to its increasing occurrence especially on soybean and cotton, owing to monoculture farming, adoption of conservation tillage systems, susceptibility of current cultivars, lack of crop rotation and optimal weather patterns for disease development. Yield losses up to 1,009 kg / ha of soybean and up to 448 kg / ha of seed + lint of cotton have been reported as a consequence of C. cassiicola infection.

[0384] Brown Spot

[0385] Brown spot, caused by Septoria glycines, is a highly prevalent foliar disease in the United States and other soybean (Glycine max) production areas, such as Argentina, Brazil, and China. The estimated yield losses in the northern United States between 2010-2014 ranged from 173,625 to 688,786 metric tons each year. When the symptoms of the disease reach 30% vertical progress of the plant at the R6 physiological state there is a 10% yield loss, but if the symptoms reach 80% vertical progress then the there is a 27% yield loss. This disease limits the yield by causing premature defoliation and reducing seed weight when severe infection occurs.

[0386] The typical symptoms of brown spot are dark and irregular spots surrounded by chlorosis on the leaves. The pathogen infects the plant through stomata without forming an appressorium. It can also infect pods and seeds, but the pathogen is rarely seed-borne. In the field, symptoms on leaves can be observed as early as V2 to V3 stage, and the disease gradually develops to the upper canopy throughout the growing season. The incubation period (the time betweeninfection to showing visible symptoms) of S. glycines has been reported to vary depending on host maturity.IX. Amino Acid Substitutions

[0387] The present disclosure provides resistant forms of the Lr67 protein, in which the glycine at the position corresponding to amino acid 144 in SEQ ID NO: 4 has been changed to an arginine, lysine or histidine or other positively charged amino acid, and in some embodiments comprising a further change in which the valine at the position corresponding to amino acid 387 in SEQ ID NO: 4 has been changed to a leucine, isoleucine, methionine or phenylalanine or other hydrophobic amino acid. However, variations at other amino acid positions in a Lr67 protein are also encompassed by the present disclosure.

[0388] Variants of the presently disclosed polypeptides also include polypeptides that vary from the sequences set forth in the Sequence Listing by conservative amino acid substitutions, substitution of a residue by another with like characteristics. Those of ordinary skill in the art are aware that modifications in the amino acid sequence of a peptide, polypeptide, or protein can result in equivalent, or possibly improved, second generation peptides, etc., that display equivalent or superior functional characteristics when compared to the original amino acid sequence. The present disclosure accordingly encompasses such modified amino acid sequences. Alterations can include amino acid insertions, deletions, substitutions, truncations, fusions, shuffling of subunit sequences, and the like, provided that the peptide sequences produced by such modifications have substantially the same functional properties as the naturally occurring counterpart sequences disclosed herein.

[0389] One factor that can be considered in making such changes is the hydropathic index of amino acids. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein has been discussed by Kyte and Doolittle ( J. Mol. Biol. 157:105- 132, 1982). It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resultant protein. This, in turn, affects the interaction of the protein with molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0390] Based on its hydrophobicity and charge characteristics, each amino acid has been assigned a hydropathic index as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamatc / glutaminc / aspartatc / asparaginc (-3.5); lysine (-3.9); and arginine (-4.5).

[0391] As is known in the ait, certain amino acids in a peptide or protein can be substituted for other amino acids having a similar hydropathic index or score and produce a resultant peptide or protein having similar biological activity, i.e., which still retains biological functionality. In making such changes, it is preferable that amino acids having hydropathic indices within ±2 are substituted for one another. More preferred substitutions are those wherein the amino acids have hydropathic indices within ±1. Most preferred substitutions are those wherein the amino acids have hydropathic indices within ±0.5.

[0392] Like amino acids can also be substituted on the basis of hydrophilicity. U.S. Patent No. 4,554,101 discloses that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acids: arginine / lysine (+3.0); aspartate / glutamate (+3.0 +1); serine (+0.3); asparagine / glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 +1); alanine / histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine / isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). Thus, one amino acid in a peptide, polypeptide, or protein can be substituted by another amino acid having a similar hydrophilicity score and still produce a resultant protein having similar biological activity, i.e., still retaining correct biological function. In making such changes, amino acids having hydropathic indices within +2 are preferably substituted for one another, those within +1 are more preferred, and those within +0.5 are most preferred.

[0393] As outlined above, amino acid substitutions in the proteins of the present disclosure can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take various of the foregoing characteristics into consideration in order to produce conservative amino acid changes resulting in silent changes within the present peptides, etc., can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids. Representative amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, andlysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral non-polar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. It should be noted that changes which are not expected to be advantageous can also be useful if these result in the production of functional sequences.

[0394] Changes may be made at a specific amino acid position in a protein by substituting an alternate amino acid for the amino acid found in that position in the wild-type protein sequence. As used herein, the term “substitution,” “substituting” or like terms, refers to replacing one amino acid with another amino acid. A substitution is indicated in standard scientific nomenclature by X#Y (where X is the original or wild-type amino acid, # is the amino acid position in the protein’s amino acid sequence, and Y is the amino acid to be substituted for X). DNA sequences encoding Lr67 proteins, and homologs thereof, with an amino acid substitution(s) can be produced by introducing changes into the DNA sequence encoding the Lr67 protein using methods known in the art and the information provided in Table 1.

[0395] Because of the degeneracy of the genetic code, a variety of different DNA sequences can encode proteins, such as the protein sequences disclosed herein. For example, Table 1 provides the universal genetic code chart showing all possible mRNA triplet codons (where T in the DNA molecule is replaced by U in the RNA molecule) and the amino acid encoded by each codon. DNA sequences encoding Lr67 proteins or homologs thereof with the amino acid substitutions described herein can be produced by introducing mutations into the DNA sequence encoding a Lr67 protein or homolog thereof using methods known in the art and the information provided in Table 1. It is well within the capability of one of skill in the art to create alternative DNA sequences encoding the same, or essentially the same, altered or engineered proteins as described herein. These variant or alternative DNA sequences are within the scope of the embodiments described herein. As used herein, references to “essentially the same” sequence refers to sequences that encode amino acid substitutions, deletions, additions, or insertions that do not materially alter the functional activity of the protein encoded by the DNA molecule of the embodiments described herein. Allelic variants of the nucleotide sequences encoding a protein disclosed herein are also encompassed within the scope of the embodiments described herein.Table 1Ss tod Base i Cod nX. Additional Definitions

[0396] The following definitions or interpretations of technical terms will be used throughout the present disclosure. The technical terms used herein are generally to be given the meaning commonly applied to them in the pertinent art of plant biology, molecular biology, bioinformatics, and plant breeding. All of the following term definitions apply to the complete content of this application.

[0397] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives arc mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device,the method being employed to determine the value, or the variation that exists among the study subjects.

[0398] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of.” As used herein, the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property / ies, method / process steps or limitation^ )) only.

[0399] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0400] As used herein, words of approximation such as, without limitation, “about,” “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0401] The terms “peptides,” “oligopeptides,” “polypeptide,” or “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds, unless mentioned herein otherwise. The terms “polynucleotide(s),” “nucleic acid sequence(s),” “nucleotide sequence(s),” “nucleic acid(s),” “nucleic acid molecule” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.

[0402] As used herein, the term “endogenous” or “native” nucleic acid and / or protein refers to a nucleic acid and / or protein as found in a plant or other organism in its natural form (i.e., without there being any human intervention, such as recombinant DNA engineering technology),

[0403] As used herein, the term “exogenous” (in contrast to “endogenous”) means a nucleic acid or protein that has been introduced in a plant or other organism by means of recombinant DNA technology. An “exogenous” nucleic acid or protein can either not occur in a plant in its natural form, be different from the nucleic acid or protein as found in a plant in its natural form, be present at a higher or lower level than the nucleic acid or protein naturally present in a plant, or in the case of a nucleic acid can be identical to a nucleic acid found in a plant in its natural form, but integrated at a location different that its natural genetic environment.

[0404] As used herein, an “expression cassette” refers to a nucleic acid sequence of interest operably linked to one or more control sequences (at least to a promoter) as described herein. An expression cassette can also include additional transcriptional and / or translational enhancers. An expression cassette can also include terminator, silencer and enhancer sequences, intron sequences added to the 5' untranslated region (UTR) or in the coding sequence of the nucleic acid sequence, and / or other control sequences such as protein and / or RNA stabilizing elements. An expression cassette may be integrated into the genome of a host cell and replicated together with the genome of the host cell, or transiently present in a host cell.

[0405] As used herein, “genetic transformation” refers to the process of introducing a DNA sequence or construct (e.g., a vector or expression cassette) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication.

[0406] As used herein, “modulation” refers to when the expression level is changed in comparison to the expression seen in a control plant. Modulation refers to an expression level that is either increased or decreased.

[0407] “Obtaining,” when used in conjunction with a transgenic plant cell or transgenic plant, obtaining means cither transforming a non-transgcnic plant cell or plant to create the transgenic plant cell or plant, or planting transgenic plant seed to produce the transgenic plant cell or plant. Such a transgenic plant seed may be from an RO transgenic plant or may be from a progeny of any generation thereof that inherits a given transgenic sequence from a starting transgenic parent plant.

[0408] As used herein, the term “plant” encompasses whole plants, ancestors and progeny of the plants and plant parts, including fruits, seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene / nucleic acid of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene / nucleic acid of interest. In certain embodiments the plants are dicot, leguminous or soybean plants.

[0409] As used herein “ploidy” or “chromosomal ploidy” refers the number of complete sets of chromosomes occurring in the nucleus of a cell. Somatic cells, tissues, and individual organisms can be described according to the number of sets of chromosomes present (the “ploidy level”): monoploid (1 set), diploid (2 sets), triploid (3 sets), tetrapioid (4 sets), pentapioid (5 sets), hexapioid (6 sets), heptapioid or septapioid (7 sets), etc. The generic term polyploidy is used herein to describe cells with three or more chromosome sets.

[0410] As used herein, the term R0 transgenic plant refers to a plant that has been genetically transformed or has been regenerated from a plant cell or cells that have been genetically transformed.

[0411] As used herein, the term “recombinant” refers to a non-natural DNA, protein, or organism that would not normally be found in nature and was created by human intervention. As used herein, a “recombinant DNA molecule” is a DNA molecule comprising a combination of DNA molecules that would not naturally occur together and is the result of human intervention, for example, a DNA molecule that is comprised of a combination of at least two DNA molecules heterologous to each other, such as a DNA molecule that comprises a transgene and the plant genomic DNA adjacent to the transgene. As used herein, a “recombinant plant” is a plant that would not normally exist in nature, is the result of human intervention, and contains a transgenic DNA molecule. As a result of such genomic alteration, the recombinant plant is something new and distinctly different from the related wild-type plant.

[0412] As used herein, the term “regeneration” refers to the process of growing a plant from a plant cell (e.g., plant protoplast, callus or cxplant).

[0413] As used herein, the term “selected DNA” refers to a DNA segment that one desires to introduce or has introduced into a plant genome by genetic transformation.

[0414] As used herein, “terminator” refers to a DNA control sequence at the end of a transcriptional unit that signals 3' processing and polyadenylation of a primary transcript and termination of transcription.

[0415] As used herein, the term “transformation construct refers to a chimeric DNA molecule that is designed for introduction into a host genome by genetic transformation. Transformation constructs will often comprise all of the genetic elements necessary to direct the expression of one or more exogenous genes. In particular embodiments of the instant disclosure, it may be desirable to introduce a transformation construct into a host cell in the form of an expression cassette.

[0416] As used herein, a “transformed cell” refers to a cell the DNA complement of which has been altered by the introduction of an exogenous DNA molecule into that cell.EXAMPLES

[0417] The following examples are included to demonstrate illustrative embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute one embodiment of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 Transformation constructs for expression of Ta.Lr67res in soybean

[0418] Plant transformation vectors comprising transgene cassettes designed to express wheat Lr67 resistant protein (also referred to as “Ta.Lr67res”) were used to stably transform soybean plants. A codon optimized coding sequence for expression of a Ta.Lr67res protein in soybean was cloned into plant transformation vector constructs identified in Table 2 to allow for testing of theTa.Lr67res protein expressed in soybean with different promoters and expression elements. The transformation vector constructs comprised a first transgcnc cassette for expression of the Ta.Lr67res protein operably linked to plant-expressible promoter (P) and terminator (T) elements, in addition to possibly an intron (I) element; and a second transgene cassette for expression of adenylyltransferase (aadA) for selection of transformed plant cells using spectinomycin as the selection agent.

[0419] Table 2 provides a list of constructs for different expression patterns of the proteincoding wheat Lr67res transgene to provide enhanced resistance against Asian Soy Rust (ASR). For each construct, Table 2 provides the “Construct ID” for each given construct; “Expression element name,” which identifies the plant-expressible promoter present in the construct; “SEQ ID NO,” which provides the DNA sequence of the respective promoter; and “Specific expression pattern,” which describes the expected expression pattern based on the particular promoter element.Table 2. Constructs and associated promoters for expression of Ta.Lr67res in soybean.

[0420] A coding sequence of the Ta.Lr67res gene from wheat is provided as SEQ ID NO: 1, a codon optimized sequence for expressing the Ta.Lr67res protein in soybean is provided as SEQ ID NO: 5, the amino acid sequence for the Ta.Lr67res protein is provided as SEQ ID NO: 2, and the genomic DNA sequence for the Ta.Lr67res gene is provided as SEQ ID NO: 224. The coding sequence of a wild-type Ta.Lr67 gene from wheat is provided as SEQ ID NO: 3, and the amino acid sequence for a wild-type Lr67 protein is provided as SEQ ID NO: 4. The wild-type Ta.Lr67 gene from wheat may also be referred to as a disease-susceptible gene sequence.

[0421] Meristem explants excised from soybean seeds were transformed with each of the transformation constructs in Table 2 via Agro / zacterzwm-mediated transformation. Transgenic R0 plants were regenerated following spectinomycin selection and self-pollinated to produce R1 plants. Typically, eight events were selected per construct for controlled environment efficacy screening, although greater or fewer than eight events may have been created and tested in the controlled environment for some of the constructs. Eight R1 seeds per event were also planted in controlled environment and sampled at the V 1 growth stage to confirm transgene presence and expression level through mRNA analysis. The plant leaf samples were collected from the same events evaluated in the disease efficacy assay below.Example 2Pathogen assay and evaluation of disease resistance efficacy

[0422] This example describes the controlled environment (CE) pathogen assay for evaluating ASR disease susceptibility or resistance of soybean plants stably transformed with the Ta.Lr67res transgenes relative to the resistance of control plants. ASR disease resistance efficacy evaluations were performed in one or two controlled environment settings (CE-1 and / or CE-2). CE-1 refersto controlled environment tests conducted in the United States, and CE-2 refers to controlled environment tests conducted in Brazil.

[0423] Production of inoculum for the pathogen assay. For the production of Phakopsora pachyrhizi (P. pachyrhizi) spores to use as an inoculum for ASR disease resistance efficacy evaluation in controlled environment testing, wild-type soybean plants were seeded into 4 inch pots, and plants grown from these seeds were typically inoculated at V4-V7, placed in a mist tent with an ultrasonic fogger overnight before being removed from the tent in the controlled environment to allow the disease to progress. Spores from these plants were harvested by tapping sporulating leaves over aluminum foil and funneling those spores into microfuge tubes. Spores were then suspended in a 0.05% Tween 20 in sterile filtered water solution, counted using the hemacytometer, and diluted / adjusted to 3 x 104spores / ml.

[0424] Plant inoculation and pathogen assay. R1 transgenic seeds comprising one of the transgenic events from each of the tested constructs in Table 2 were planted into 2.5 inch pots. Seven or eight R1 plants for each Ta.Lr67res transgene event were selected for testing. Where available, soybean plants of the same variety and containing an empty expression vector with only the spectinomycin resistance transgene (Empty Vector), and / or untransformed wild-type soybean plants of the same variety as the transformed plants without either expression cassette (WT), were each used as a negative control. The first trifoliate of both control and Ta.Lr67res transgene positive plants were inoculated at the V2 growth stage with at least two different isolates of P. pachyrhizi, when the first trifoliate was fully developed. An air brush sprayer was used to apply the spore suspension to the first trifoliate until near run-off. The inoculated plants were then placed in a mist tent with ultrasonic foggers to incubate overnight.

[0425] To evaluate the efficacy of transgenic plants expressing the Ta.Lr67res transgene against ASR disease, both control and test plants were scored 12-14 days after incubation for total disease severity, which incorporates both percent leaf infection (e.g., number and size of pustules) and symptom development severity e.g., percent leaf chlorosis and / or necrosis caused by the infection). This rating scale would roughly be 1%, 5%, 10%, 20%, etc., in 10% increments for ratings above 10%. Control plants are evaluated to ensure that the overall test has a high and uniform level of infection. Plants were sub-irrigated twice daily with weekday fertilization. The growth chamber was kept at a temperature of about 25 °C during the day and 20 °C at night, with a relative humidity of about 70% and a 13 hour day length.

[0426] Resistance efficacy trial results: Transgenic soybean plants expressing the Ta.Lr67res transgcnc under the control of different promoters and expression elements were evaluated in two CEs (CE-1 and / or CE-2) against the fungal pathogens after inoculation at V2 stage as described above. Multiple transformation events were generated with each of these constructs (e.g., seven or more events per construct in one or more sets of experiments), and transgenic plants containing these events were evaluated in the CE to determine if they have enhanced efficacy to P. pachyrhizi. The percent disease severity rating as described above for each event per construct tested was calculated as an average across 7-8 replicates for such event (see FIGs. 1-5 and 7-18 and Tables 3-19 for each event, WT and Empty Vector controls for each construct for CE-1). Table 3 and FIG. 1 provides event level data for the pM992 construct; Table 4 and FIG. 2 provides event level data for the pM367 construct; Table 5 and FIG. 3 provides event level data for the pM369 construct; Table 6 and FIG. 4 provides event level data for the pM370 construct; and Table 7 and FIG. 5 provides event level data for the pM372 construct; Table 8 and FIG. 7 provides event level data for the pM838 construct; Table 9 and FIG. 8 provides event level data for the pM839 construct; Table 10 and FIG. 9 provides event level data for the pM840 construct; Table 11 and FIG. 10 provides event level data for the pM842 construct; Table 12 and FIG. 11 provides event level data for the pM843 construct; Table 13 and FIG. 12 provides event level data for the pM844 construct; Table 14 and FIG. 13 provides event level data for the pM967 construct; Table 15 and FIG. 14 provides event level data for the pM678 construct; Table 16 and FIG. 15 provides event level data for the pM400 construct; Table 17 and FIG. 16 provides event level data for the pM494 construct; Table 18 and FIG. 17 provides event level data for the pM285 construct; and Table 19 and FIG. 18 provides event level data for the pM479 construct;

[0427] The average results for each event per construct were further averaged across all events for each construct as summarized in Table 20 for CE-1 testing. Standard errors were calculated for the transgenic soybean and control plants, which are represented as error bars in the figures. The average disease efficacy ratings were statistically different at alpha = 0.05. The alpha (a) measurement is the statistical probability of a Type I error in any hypothesis test (incorrectly rejecting the null hypothesis), whereas the beta (0) measurement is the statistical probability of a Type II error in any hypothesis test (incorrectly failing to reject the null hypothesis).

[0428] In an additional experiment, Event- 14 from expression construct pM372 along with 3 kilobases (kb) of 5’ and 3’ genomic sequences that flank the Event-14 insertion site were excised,added to a transformation vector (defined as pM400) and retransformed into soybean. This pM400 event demonstrated efficacy in CE without any off types (intra-vcinal leaf chlorosis or stunting) that were observed in a few events from this expression construct. Table 16 and FIG. 15 provides event level data for the pM400 construct.Table 3. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM992.Table 4. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM367.Table 5. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM369.Table 6. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM370.Table 7. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM372.Table 8. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM838.Table 9. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM839.Table 10. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM840.Table 11. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM842.Table 12. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM843.Table 13. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM844.Table 14. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM967.Table 15. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM678.Table 16. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM400.Table 17. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM494.Table 18. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM285.Table 19. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM479.

[0429] The expression of a Ta.Lr67 transgene was measured using primers specific for Lr67 transgene using TaqMan using PerfeCTa® qPCR FastMix® II (QuantaBio, Beverly, MA, USA,cat.no 95119) according to manufacturer’s instructions. Soybean plant leaf disk samples were collected from the same events evaluated for the disease efficacy. 7-8 replicates per event were assayed for the gene expression analysis by qRT-PCR. The relative expression level of transgenic Ta.Lr67 mRNA transcripts were calculated by normalizing to the geometric mean of Glycine max poly adenylate-binding protein RBP45 and Glycine max casein kinase II subunit alpha- 1 -like reference genes.

[0430] Depending on the type and level of expression, transgenic plants expressing the Ta.Lr67res transgene showed significant efficacy against at least two different isolates of P. pachyrhizi. The efficacy trial results in this example demonstrated that the level and / or type of transgene expression is an important factor for Ta.Lr67res to achieve efficacy as illustrated, for example, in FIGs. 1-5 and 7-18.

[0431] The data from this example indicate that expression of Ta.Lr67res transgene using a constitutive promoter (pM992) was effective at reducing disease severity, particularly for events having higher levels of expression. Events with higher mRNA expression levels in this experiment were associated with higher levels of efficacy to the pathogen (Events 1-3, 6, and 7), whereas events with lower mRNA expression levels had lower resistance (Events 4, 5, and 8) as shown, for example, in FIG 1. In this experiment, a few events from the pM992 construct also showed chlorosis and stunting off-types.

[0432] As described above, disease efficacy was evaluated at V2 (seedling) growth stage for each of the tested constructs. Adult plants having the same events from construct pM992 were also evaluated for the disease resistance. Disease efficacy results with adult plants having those same events were similar to those from the seedling assay, with the exception that older leaves did appear to show a small amount of increased disease suppression over younger leaves. However, the increase was small enough as to be deemed not biologically relevant.

[0433] This example further indicates that expression of Ta.Lr67res transgene under the control of a promoter having medium to low expression in leaf tissues (pM367) was less effective at providing resistance against tested pathogen as compared to higher constitutive expression. As can be seen in FIG. 2, the percent disease severity with pM367 construct was mostly neutral between transgenic events and the controls. As shown in FIGs. 3 and 4, expression of Ta.Lr67res gene under the control of promoters having medium expression in leaf tissues (pM369 and pM370)resulted in varying levels of efficacy in this experiment. In this experiment, a few events from the pM370 construct also showed chlorotic off-type.

[0434] The data in this example further indicates that expression of Ta.Lr67res transgene under the control of a promoter having high expression in leaf tissues of the plant (pM372) was effective at reducing disease severity. All 22 tested events from the pM372 construct in this experiment showed high levels of efficacy against the fungal pathogen (FIG. 5). See also FIG. 6 with plant images showing the difference in disease progression between an Empty Vector control plant and a Tci.Lr67res transgenic plant containing Event 6 of the pM372 construct. In this experiment, a few events from the pM372 construct also showed chlorosis and stunting off-types.

[0435] As shown in FIG. 7, expression of Ta.Lr67res gene under the control of a promoter having high expression in leaf tissues (pM838) resulted in varying levels of efficacy in this experiment. Several events (Events 6 to 9) in this experiment were associated with higher levels of efficacy to the pathogen relative to the controls. In this experiment, a few events from the pM838 construct also showed some level of stunting and slight chlorosis off-types.

[0436] The data in this example further indicates that expression of Ta.Lr67res transgene under the control of a promoter having high expression in leaf tissues of the plant (pM839) was effective at reducing disease severity. All 8 tested events from the pM839 construct in this experiment showed high levels of efficacy against the fungal pathogen (FIG. 8). In this experiment, a few events from the pM839 construct also showed some level of stunting and slight chlorosis off-types.

[0437] As demonstrated in FIG. 9, expression of Ta.Lr67res transgene under the control of a promoter having high expression in leaf tissues (pM840) was less effective at providing resistance against tested pathogen. As can be seen in FIG. 9, the percent disease severity with pM840 construct was mostly neutral between transgenic events and the controls.

[0438] As shown in FIG. 10, expression of Ta.Lr67res gene under the control of a promoter having high expression in leaf tissues (pM842) resulted in varying levels of efficacy in this experiment. Several events (Event 1 and Event 4) in this experiment were associated with moderate to higher levels of efficacy to the pathogen. A few events from the pM842 construct also showed off-types including some level of stunting and chlorosis in this experiment.

[0439] As can be seen in FIGs. 11 and 12, expression of Ta.Lr67res transgene under the control of promoters having high expression in leaf tissues (pM843 and pM844) was less effectiveat providing resistance against tested pathogen as compared to the controls. The percent disease severity with constructs pM843 and pM844 was mostly neutral between transgenic events and the controls.

[0440] The data in this example further indicates that expression of Ta.Lr67res transgene under the control of a promoter having medium expression in leaf tissues of the plant (pM967) was less effective at reducing disease severity. All tested events from this construct in this experiment showed neutral efficacy against the fungal pathogen as shown in FIG. 13.

[0441] As shown in FIG. 14, expression of Ta.Lr67res gene under the control of a promoter having medium expression in leaf tissues (pM678) resulted in varying levels of efficacy in this experiment. In this experiment, a few events from the pM678 construct also showed some level of stunting and slight chlorosis off-types.

[0442] All 4 tested events from the pM400 construct in this experiment showed high levels of efficacy against the fungal pathogen (FIG. 15). A few events from the pM400 construct also showed off-types including moderate level of stunting and, in some events, chlorosis in this experiment.

[0443] As demonstrated in FIG. 16, expression of Ta.Lr67res transgene under the control of a promoter having preferential expression in green tissues (pM494) was less effective at providing resistance against tested pathogen. As can be seen in FIG. 16, the percent disease severity with pM494 construct was mostly neutral between transgenic events and the WT plants.

[0444] As demonstrated in FIG. 17, expression of Ta.Lr67res transgene under the control of a promoter having low constitutive preferential expression in leaf and flower tissues (pM285) was less effective at providing resistance against tested pathogen. As can be seen in FIG. 19, the percent disease severity with pM285 construct was mostly neutral between transgenic events and the WT plants.

[0445] As shown in FIG. 18, expression of Ta.Lr67res gene under the control of a promoter having preferential expression in above-ground green tissues (pM479) resulted in varying levels of efficacy in this experiment. A few events from the pM479 construct also showed off-types including some level of stunting in this experiment.Table 20. Summary of observations of transgenic plants with various constructs and promoters for expression of the Ta.Lr67res transgene evaluated in CE-1.

[0446] In additional experiments, a subset of constructs from Table 2 were evaluated in CE-2 for ASR disease susceptibility or resistance of soybean plants stably transformed with the Ta.Lr67res transgenes relative to the resistance of WT plants. Expression constructs pM841 and pM968 were evaluated only under CE-2 settings.

[0447] Production of inoculum for the pathogen assay for CE-2 were conducted as described above except spores were diluted to 5 x 104 spores / ml following suspension in 0.05% Tween 20 in sterile filtered water solution and counting using the hemacytometer.

[0448] To evaluate the efficacy of transgenic plants expressing the Ta.Lr67res transgene against ASR disease, both WT and test plants were scored 12-14 days after incubation for total disease severity. Disease severity was estimated based on visual observations of the symptoms using a standard area diagram as described in Godoy et al. 2006. This rating scale used a standard area diagram in roughly 1% increments from 1 to 20%, and in 5% increments for ratings above 20%.

[0449] Transgenic soybean plants expressing the Ta.Lr67res transgene under the control of different promoters and expression elements were evaluated in CE-2 against the fungal pathogens after inoculation at V2 stage as described above. Up to 10 (1 to 10) transformation events were generated with each of these constructs and transgenic plants containing these events were evaluated in the CE-2 to determine if they have enhanced efficacy to P. pachyrhizi. The percent disease severity rating as described above for each event per construct tested was calculated as an average across 6-8 replicates for such event (see FIGs. 19-27 and Tables 21-29 for each event, WT and Empty Vector controls for each construct for CE-2). FIGs. 19-27 and Tables 21-29 provides event level data for constructs compared with WT evaluated in CE-2.

[0450] Table 21 and FIG. 19 provides event level data for the pM838 construct; Table 22 and FIG. 20 provides event level data for the pM839 construct; Table 23 and FIG. 21 provides event level data for the pM840 construct; Table 24 and FIG. 22 provides event level data for the pM841 construct; Table 25 and FIG. 23 provides event level data for the pM843 construct; Table 26 and FIG. 24 provides event level data for the pM844 construct; Table 27 and FIG. 25 provides event level data for the pM967 construct; Table 28 and FIG. 26 provides event level data for the pM968 construct; and Table 29 and FIG. 27 provides event level data for the pM678 construct.Table 21 Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM838.Table 22. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM839.Table 23. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM840.Table 24. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM841.Table 25. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM843.Table 26. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM844.Table 27. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM967.Table 28. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM968.Table 29. Transgenic soybean plants expressing Ta.Lr67res transgene from construct pM678.

[0451] As shown in FIG. 19, expression of Ta.Lr67res gene under the control of a promoter having high expression in leaf tissues (pM838) was highly effective at reducing disease severity in CE-2. All 3 tested events from the pM838 construct in this experiment showed high levels of efficacy against the fungal pathogen. In this experiment, a few events from the pM838 construct also showed moderate stunting and slight chlorosis.

[0452] The data in this example indicates that expression of Ta.Lr67res transgene under the control of a promoter having high expression in leaf tissues of the plant (pM839) was effective at reducing disease severity in CE-2. Both tested events from the pM839 construct in this experiment showed high levels of efficacy against the fungal pathogen (FIG. 20). A few events from thepM839 construct also showed off-types including moderate level of stunting and, in some events, slight chlorosis in this experiment.

[0453] As demonstrated in FIG. 21, expression of Ta.Lr67res transgene under the control of a promoter having high expression in leaf tissues (pM840) was effective at providing resistance against tested pathogen in CE-2. Only one event was tested from the pM840 construct in this experiment and it showed high levels of efficacy against the fungal pathogen. See also FIG. 28 with plant images showing the difference in disease progression between a WT control plant and a transgenic plant containing Event-5 of the pM840 construct.

[0454] The data in this example further indicates that expression of Ta.Lr67res transgene under the control of a promoter having high expression in leaf tissues of the plant (pM841) was highly effective at reducing disease severity in CE-2. All 6 tested events from the pM841 construct in this experiment showed high levels of efficacy against the fungal pathogen (FIG. 22).

[0455] As can be seen in FIG. 23, expression of Ta.Lr67res transgene under the control of a promoter having high expression in leaf tissues (pM843) was less effective at providing resistance against tested pathogen as compared to the controls in CE-2. The percent disease severity with constructs pM843 was mostly neutral between transgenic events and the controls.

[0456] As shown in FIG. 24, expression of Ta.Lr67res gene under the control of a promoter having high expression in leaf tissues (pM844) resulted in varying levels of efficacy in CE-2 experiment.

[0457] The data in this example further indicates that expression of Ta.Lr67res transgene under the control of a promoter having medium expression in leaf tissues of the plant (pM967) was highly effective at reducing disease severity CE-2. All 10 tested events from this construct in this experiment showed high levels of efficacy against the fungal pathogen (FIG. 25).

[0458] As demonstrated in FIG. 26, expression of Ta.Lr67res gene under the control of a promoter having medium expression in leaf tissues (pM968) resulted in varying levels of efficacy in CE-2 experiment.

[0459] As shown in FIG. 27, expression of Ta.Lr67res gene under the control of a promoter having medium expression in leaf tissues (pM678) had high levels of efficacy in this CE-2 experiment. 9 out of 10 tested events from this construct in this experiment showed high levels of efficacy and one event (Event-2) showed moderate level of efficacy against the fungal pathogen.

[0460] Depending on the construct and expression level, transgenic plants expressing Ta.Lr67res gene showed high level of disease resistance with higher expression levels being associated with a greater reduction in disease severity. Some events from these constructs also had moderate to severe levels of stunting and intra-veinal leaf chlorosis. By modifying the expression pattern of Ta.Lr67res gene to restrict or eliminate expression in the roots, the observed stunting off-types were reduced or eliminated. Thus, while higher levels of expression of the Ta.Lr67res transgene in above ground tissues (e.g.. trifoliate leaves) may be effective at reducing disease severity, reducing Ta.Lr67res transgene expression in plant roots may be effective in recuing or eliminating apparent stunting off-types.Example 3Disease efficacy trial results of soybean plants comprising Ta.Lr67res gene

[0461] Field trials of soybean transgenic plants expressing Ta.Lr67res protein (polypeptide SEQ ID NO: 2) under the control of expression elements (a subset of constructs listed in the Table 2) were conducted to assess plant resistance to Phakopsora pachyrhizi utilizing two planting years across two geographies. Soybean field trials were conducted to identify events that can reduce disease severity (efficacy) and improve or maintain broad-acre yield (BAY) under standard agronomic practices.

[0462] The field trials were conducted using a randomized complete block design for each of two planting years. Plots in this evaluation consisted of 2 rows 15’ in length with 30” spacing between them. 3-4 replicates and up to 7 fields locations were averaged for each year of field trials. Efficacy parameters were scored throughout the field trial season and at the end of the season yield were determined. Trait efficacy was measured by scoring disease prevalence 3-4 times after inoculating the plots with the rust spores. Disease Ratings were taken at a scale of 1-10 indicative of the overall plot infection. To determine the progression of the disease from the time of infection (DAI: Days After Infection), area under disease progress curve (AUDPC) was calculated. Results of the disease efficacy trials conducted under standard agronomic practice in the USA (Geography - 1) and Brazil (Geography-2) for Year-1 (2022) and Year-2 (2023) are shown in Tables 30-32, and soybean yield was compared to WT plants. In some instances, a fungicide and / or commercially available traits were used as a positive control. Disease efficacy parameters were scored severaltime points (DAI) throughout the field trial season. Tn this example disease efficacy and AUDPC data at ~35 DAI arc presented.

[0463] As shown in Table 30, in Year-1 field data, several events from soybean plant transformed with Ta.Lr67res transgene demonstrated significant efficacy relative to WT control plants. Table 30 shows disease efficacy and AUDPC data from middle and upper canopy for field trials conducted within Geography- 1. Four expression constructs and 2-10 events per construct were evaluated in this study.Table 30. Year-1 disease efficacy results of transgenic plant comprising Ta.Lr67res gene versus the WT plants under standard agronomic conditions in the field, relative to WT control plants, in Geography-1.

[0464] As shown in Table 31 , the soybean plants expressing Ta.Lr67res gene such as Event- 10 from plants transformed with construct pM369 demonstrated efficacy as compared to WT plants in Geography- 1, Year-2 trial. Four expression constructs and up to 3 events per construct were evaluated in this study at ~35 DAI.Table 31. Year-2 disease efficacy results of transgenic plant comprising Ta.Lr67res gene versus the WT plants under standard agronomic conditions in the field, relative to WT control plants, in Geography-1.

[0465] All tested transgenic events from 5 expression constructs (up to 4 events per construct) did not demonstrate significant efficacy as compared to WT plants in Geography-2, Year-2 trial as shown in Table 32. One event (Event- 14) from plants transformed with construct pM372 demonstrated efficacy comparable to the WT plant at -35 DAI. However, the efficacy of Event- 14 at late infection time-period was not statistically different than WT plants (data not shown). This event also showed off-types including low vigor, chlorosis and stunting. Disease severity values provided in Table 32 are reported as the difference (delta) of the mean value for a given transgenic plants with the Ta.Lr67res transgene versus WT plants (unmodified soybean plants). Disease Ratings were taken at a scale of 1-9 indicative of the overall plot infection for the Geography -2.Table 32. Year-2 disease efficacy results of transgenic plant comprising Ta.Lr67res gene versus the WT plants under standard agronomic conditions in the field, relative to WT control plants, in Geography-2.Example 4Broad Acre Yield (BAY) trial results of soybean plants comprising Ta.Lr67res gene

[0466] In this example, results from broad acre yield (BAY) trials are presented. BAY can be defined as the harvestable plant parts per unit area, for example seeds, or weight of seeds, per acre, pounds per acre, bushels per acre, tones per acre, tons per acre, kilo per hectare. BAY of transgenic soybean plants expressing a Ta.Lr67res transgene are grown on broad acres in the field according to standard agronomic practice (SAP). BAY trials were conducted at 6-7 field locations across a variety of soybean growing geographies within the United States (Geography- 1) and Brazil (Geography-2). The results are shown in Tables 33-35 below. Yield values described in these tables are reported as the difference (delta) of the mean value for transgenic plants comprising a given Ta.Lr67res transgene versus the WT plants (unmodified soybean plants).

[0467] Table 33 shows BAY results of soybean plants comprising a Ta.Lr67res transgene under standard agronomic conditions in the field, relative to WT control plants for Year-1 within Geography- 1. Four expression constructs and 2-10 events per construct were evaluated in this trial. Several events demonstrated significant yield advantage relative to WT plants in the presence of disease. Several events showed a yield penalty in the absence of disease across locations.Table 33. Year-1 BAY results of transgenic plant comprising Ta.Lr67res gene versus the WT plants under standard agronomic conditions in the field, relative to WT control plants, in Geography- 1.

[0468] Table 34 shows BAY results of soybean plants comprising a Ta.Lr67res transgene under standard agronomic conditions in the field, relative to WT control plants for Year-2 within Geography- 1. Four expression constructs (up to 3 events per construct) were evaluated in this trial. A few events (Event-3 and Event-7) from plants transformed with construct pM369 maintained neutral yield compared to WT plants in the presence of disease. Several events demonstrated yieldpenalty in the presence of disease. All tested transgenic events within Geography- 1 and Year-2 trial showed yield penalty in the absence of disease across locations (Table 34).Table 34. Year-2 BAY results of transgenic plant comprising Ta.Lr67res gene versus the WT plants under standard agronomic conditions in the field, relative to WT control plants, in Geography-1.

[0469] Table 35 shows BAY results of soybean plants comprising a Ta.Lr67res transgene under standard agronomic conditions in the field, relative to WT control plants for Year-2 within Geography-2 in the presence and absence of disease. Transgenic soybean plants expressing a Ta.Lr67res transgene maintained neutral yield relative to WT plants across locations in the presence of disease. Event- 14 (pM372) showed a yield penalty compared to WT plants in the presence of disease. All tested transgenic events in this trial showed yield penalty in the absence of disease across locations.Table 35. Year-2 BAY results of transgenic plant comprising Ta.Lr67res gene versus the WT plants under standard agronomic conditions in the field, relative to WT control plants, in Geography-2.Example 5 Identification of the Lr67 homologs

[0470] This example provides the identification of homologs from other plant species of the wild-type wheat (disease susceptible) Ta.Lr67 protein sequence (SEQ ID NO: 4). Relative to the wild-type wheat Ta.Lr67 sequence, a glycine at position 144 is replaced by arginine, and a valine at position 387 is replaced by leucine, in the Ta.Lr67res protein (SEQ ID NO: 2). Thus, the Ta.Lr67 protein homologs identified in this example may be similarly modified to have a glycine to arginine mutation at the amino acid position of the protein homolog corresponding to amino acid 144 of SEQ ID NO: 4, and / or a valine to leucine mutation at the amino acid position of the protein homolog corresponding to amino acid 387 of SEQ ID NO: 4. The amino acid positions for mutations in the protein homolog sequences identified in these examples corresponding to the G144R and / or V387L mutations present in the Ta.Lr67res protein, as well as any other mutations that may be made at these corresponding positions, can be identified by sequence alignment, and transgenes encoding mutant versions of the Ta.Lr67 homologs with a corresponding or related mutation can be inserted into plants, such as soybean, dicot or leguminous plants for improved fungal or ASR disease resistance.

[0471] Lr67 homologs were identified using NCBI Basic Local Alignment Search Tool (BLAST). The NCBI Non-rcdundant protein sequences (nr) database was queried based on the wild-type wheat Ta.Lr67 protein sequence (SEQ ID NO: 4) using National Center for Biotechnology Information (NCBI) “blastp” program with default search parameters. The top 100 sequence hits across plant species were kept, sorted by %Identity, and separated by organism (genus / species) names. The Multiple Sequence Comparison by Log-Expectation (MUSCLE) sequence alignment software tool was used to generate a visual alignment of a subset of the amino acid sequences of the Ta.Lr67 protein homologs along with the wild-type (ALL26327.1) and disease resistant (ALL26331.2) Ta.Lr67 protein sequences from wheat (FIG. 29). In FIG. 31, the wild-type Ta.Lr67 amino acid sequence is provided in the top row as a reference for the Ta.Lr67res and other sequence homologs. Only fourteen (14) additional protein homologs are shown in FIG. 29 as a representative set from various plant species, but similar sequence alignments could be generated for the other protein homologs. For the Ta.Lr67res protein and each of the homologs, dots (.) indicate positions in which the corresponding amino acid at that position is the same residue as the wild-type Ta.Lr67 protein. Amino acid residues of the homolog and Ta.Lr67res protein sequences that differ from the wild-type Lr67res protein are indicated by the single letter amino acid abbreviation for the amino acid present at such position in the respective protein, with the locations of the G144R and V387L mutations in the Ta.Lr67res protein indicated above. As can be seen in FIG. 29, glycine at a position corresponding to amino acid number 144 of the Lr67res protein and a valine at position 387 of the wild-type Lr67res protein are generally conserved among the homologs shown in FIG. 29 (see vertical box for each corresponding residue of the aligned sequences). Aligned residues between amino acid position coordinates 136 to 204 and 304 to 414 are shown in FIG. 29, while amino acids outside of these coordinates are not shown in this alignment.

[0472] Table 36 provides a list of one-hundred (100) Ta.Lr67 homolog genes identified in the BLAST search from different plant species, the elements of which are described as follows: “PRT SEQ ID NO.” identifies the amino acid sequence of the protein homolog encoded by the homolog gene; “Homolog PRT ID” refers to the unique identifier or accession for the protein sequence in the NCBI GenBank protein record; “NUC SEQ ID NO.” identifies the nucleotide coding sequence of the homolog gene; “Homolog NUC ID” refers to the unique identifier or accession for the nucleotide sequence in the NCBI GenBank nucleotide record; and “Organism” refers to thescientific name (e.g., genus / species) of the source organism for the homolog gene; and “Description” provides cither the protein name or a short functional description of the protein encoded by the identified gene homolog. Table 37 provides the percent identity and percent homolog and query coverage for each protein homolog listed in Table 8 relative to the wild-type Ta.Lr67 protein (query) sequence.Table 36. Ta.Lr67 Homolog gene information.Table 37. List of homologs showing the percent alignment identity and coverage for each protein homolog relative to the wild-type Ta.Lr67 protein (query) sequence.

[0473] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the ail that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. For example, all of the disclosed components of the preferred and alternative embodiments arc interchangeable providing disclosure herein of many systems having combinations of all the preferred and alternative embodiment components. This application is therefore intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this disclosure pertains and which fall within the limits of the appended claims. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

CLAIMS1. A recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 7, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221 or 223, wherein the amino acid sequence comprises a glycine to arginine, glycine to lysine, or glycine to histidine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO: 4, and wherein the heterologous promoter is a constitutive promoter, an aboveground promoter or a green tissue promoter.

2. The recombinant DNA construct of claim 1, wherein the amino acid sequence comprises a glycine to arginine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO: 4.

3. The recombinant DNA construct of claim 1 or 2, wherein the nucleotide coding sequence of the recombinant DNA construct is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1, 3, 5, 6, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 or 222.

4. The recombinant DNA construct of any one of claims 1-3, wherein the nucleotide coding sequence of the recombinant DNA construct is at least 99% identical to SEQ ID NO: 1, 3, 5, 6, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 or 222, wherein the coding sequence encodes an amino acid sequence comprising a glycine to arginine mutation at the amino acid position corresponding to amino acid 144 of SEQ ID NO: 4.

5. The recombinant DNA construct of any one of claims 1-4, wherein the nucleotide coding sequence of the recombinant DNA construct encodes the amino acid sequence of SEQ ID NO: 2 or 7.

6. The recombinant DNA construct of any one of claims 1-5, wherein the nucleotide coding sequence of the recombinant DNA construct comprises the nucleotide sequence of SEQ ID NO: 1, 5 or 6.

7. The recombinant DNA construct of any one of claims 1-6, wherein the protein encoded by the nucleotide coding sequence further comprises a valine to leucine, valine to isoleucine, valine to methionine, or valine to phenylalanine mutation at the amino acid position corresponding to amino acid 387 of SEQ ID NO: 4.

8. The recombinant DNA construct of any one of claims 1-7, wherein the protein encoded by the nucleotide coding sequence further comprises a valine to leucine mutation at the amino acid position corresponding to amino acid 387 of SEQ ID NO: 4.

9. The recombinant DNA construct of any one of claims 1-8, wherein the nucleotide coding sequence of the recombinant DNA construct encodes the amino acid sequence of SEQ ID NO: 2.

10. The recombinant DNA construct of any one of claims 1-9, wherein the nucleotide coding sequence of the recombinant DNA construct comprises SEQ ID NO: 1.

11. The recombinant DNA construct of any one of claims 1-10, wherein the nucleotide coding sequence of the recombinant DNA construct comprises SEQ ID NO: 5.

12. The recombinant DNA construct of any one of claims 1-11, wherein the heterologous promoter is an above-ground promoter.

13. The recombinant DNA construct of any one of claims 1-12, wherein the heterologous promoter comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to one or more of SEQ ID NOs: 8-23 and 225-230.Il l14. The recombinant DNA construct of any one of claims 1-13, wherein the heterologous promoter comprises a nucleotide sequence that is 100% identical to one or more of SEQ ID NOs: 8-23 and 225-230.

15. The recombinant DNA construct of any one of claims 1-14, wherein the above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence in a plant at a higher level in one or more above-ground tissues of the plant relative to the expression level of the operably linked nucleotide coding sequence in below-ground root tissues of the plant.

16. The recombinant DNA construct of any one of claims 1-15, wherein the above-ground promoter or green tissue promoter drives expression of the operably linked nucleotide coding sequence at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50-fold higher in the one or more above-ground tissues relative to the below-ground root tissues.

17. The recombinant DNA construct of any one of claims 1-16, wherein the heterologous promoter comprises a fragment of one of SEQ ID NOs: 8-23 and 225-230, wherein the fragment has promoter activity as a constitutive promoter, an above-ground promoter or a green tissue promoter.

18. The recombinant DNA construct of any one of claims 1-17, wherein the heterologous promoter fragment has promoter activity as an above-ground promoter or a green tissue promoter.

19. A DNA molecule comprising the recombinant DNA construct of claim 1.

20. A DNA plasmid or vector comprising the recombinant DNA construct of claim 1.2E A transgenic plant comprising the recombinant DNA construct of claim 1, wherein the plant has increased resistance to a fungal disease compared to a control plant lacking the recombinant DNA construct.

22. The transgenic plant of claim 21, wherein the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, target spot and / or brown spot.

23. The transgenic plant of claim 21 or 22, wherein the transgenic plant is a dicotyledonous plant.

24. The transgenic plant of claim 23, wherein the transgenic plant is a leguminous plant.

25. The transgenic plant of claim 24, wherein the transgenic plant is a soybean plant.

26. A transgenic plant part of the transgenic plant of any one of claims 21-25.

27. The transgenic plant part of claim 26, wherein the transgenic plant part is a seed, a root, a stem, a leaf, a flower, an embryo, or a meristem.

28. The transgenic plant part of claim 27, wherein the transgenic plant pail is a seed.

29. A method of making a transgenic plant resistant to a fungal disease, comprising introducing into at least one cell of an explant a recombinant DNA construct according to claim 1, and regenerating or developing the transgenic plant from the explant.

30. The method of claim 29, wherein the recombinant DNA construct is introduced into the plant by Agro&acte / 'ZMm-mediated transformation, microprojectile bombardment, calcium phosphate precipitation, polyethylene glycol treatment, electroporation, site-directed integration, gene editing, or a combination thereof.

31. The method of claim 29 or 30, wherein the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, target spot or brown spot.

32. The method of any one of claims 29-31, wherein the plant is a dicotyledonous plant.

33. The method of claim 32, wherein the plant is a leguminous plant.

34. The method of claim 33, wherein the plant is a soybean plant.