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

Abstract

The present disclosure provides compositions and methods for making transgenic plants expressing a resistant Lr34 protein that exhibit increased resistance to fungal diseases, and transgenic plants produced by these methods, and plant parts thereof. The present disclosure provides transgenic plants, and plant parts thereof, that are resistant or immune, or have reduced susceptibility, to Asian Soybean Rust (ASR) and other fungal pathogens, while demonstrating minimized or reduced undesirable off-types. The present disclosure provides effective transgenic expression of Lr34 in soybean varieties to reduce the need for one or more fungicide applications, and / or protecting against fungal pathogens developing resistance to both the chemical control agents and other native or engineered disease resistance genes.

Description

TRANSGENIC PLANTS HAVING INCREASED RESISTANCE TO FUNGAL DISEASES AND METHODS OF PRODUCING SAME REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States provisional application No.63 / 711,949, filed October 25, 2024, herein incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A sequence listing containing the file named “MONS599WO_ST26.xml” which is 806 kilobytes (measured in MS-Windows®) and created on September 24, 2025, and comprises 226 sequences, is incorporated herein by reference in its entirety.FIELD

[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

[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 numerous 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 social 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 codingsequence encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, or wherein the nucleotide coding sequence encodes a protein having 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: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115, and the protein comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7 and / or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7, and wherein the heterologous promoter is a constitutive or aboveground promoter. In certain embodiments the heterologous promoter is an above-ground promoter. According to some embodiments, the protein comprises a deletion of a non-polar amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a histidine.

[0008] In some embodiments, the nucleotide coding sequence of the recombinant DNA construct may have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 8, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, or 215. In some embodiments, the nucleotide coding sequence of the recombinant DNA construct may have at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 8, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, or 215, and wherein the coding sequence encodes an amino acid sequence comprising a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid 546 of SEQ ID NO: 7 and / or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7. In some embodiments, the nucleotide coding sequence of the recombinant DNA construct encodes the amino acid sequence of SEQ ID NO: 6. In some embodiments, the nucleotide coding sequence of the recombinant DNA construct comprises the nucleotide sequence of SEQ ID NO: 1, 2, 3 or 4.

[0009] In some embodiments, the heterologous promoter comprises a nucleotide sequence that may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to one or more of SEQ ID NOs: 9-15 and 216-226. In other embodiments the heterologous promoter comprises a nucleotide sequence that is 100% identical to one or more of SEQ ID NOs: 9-15 and 216-226. In certain embodiments the aboveground 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: 9-15 and 216-226, wherein the fragment has promoter activity as a constitutive orabove-ground promoter. In additional embodiments the heterologous promoter fragment has promoter activity as an above-ground promoter.

[0010] 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 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, or wherein the nucleotide coding sequence encodes a protein with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115, and the protein comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid 546 of SEQ ID NO: 7 and / or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid 634 of SEQ ID NO: 7, and wherein the heterologous promoter is an above-ground promoter. According to some embodiments, the protein comprises a deletion of a non-polar amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a histidine.

[0011] The present disclosure additionally provides a DNA plasmid or vector comprising a recombinant DNA construct comprising a nucleotide coding sequence operably linked to aheterologous promoter, wherein the nucleotide coding sequence encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, or 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: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115, and the protein comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid 546 of SEQ ID NO: 7 and / or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid 634 of SEQ ID NO: 7, and wherein the heterologous promoter is a constitutive or above-ground promoter.

[0012] The present disclosure further provides a transgenic plant or plant part thereof comprising a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, or 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: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115, and the protein comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid 546 of SEQ ID NO: 7 and / or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid 634 of SEQ ID NO: 7, 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. According to some embodiments, the protein comprises a deletion of a non-polar amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ IDNO: 7. According to some embodiments, the protein comprises a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a histidine.

[0013] In certain embodiments the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, target spot, frogeye leaf spot and / or brown spot. In some embodiments, the transgenic plant is a dicotyledonous plant. In some embodiments, the transgenic plant is a leguminous plant. In some embodiments, the transgenic plant is a soybean plant.

[0014] The present disclosure also provides a transgenic plant part comprising a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, or wherein the nucleotide coding sequence encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115, and the protein comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid 546 of SEQ ID NO: 7 and / or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid 634 of SEQ ID NO: 7, and wherein the heterologous promoter is a constitutive or above-ground promoter. According to some embodiments, the protein comprises a deletion of a non-polar amino acid at an amino acid position of the proteincorresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a histidine. In some embodiments, the transgenic plant part is from a dicotyledonous plant. In some embodiments, the transgenic plant part is from a leguminous plant. In some embodiments, the transgenic plant part is from a soybean plant. In some embodiments, the transgenic plant part is a seed, a root, a stem, a leaf, a flower, an embryo, an explant or a meristem. In some embodiments, the transgenic plant part is a seed.

[0015] In addition, 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 of a plant a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequence encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, or wherein the nucleotide coding sequence encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115, and the protein comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid 546 of SEQ ID NO: 7 and / or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid 634 of SEQ ID NO: 7, and wherein the heterologouspromoter 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. According to some embodiments, the protein comprises a deletion of a non-polar amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a histidine. In some 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 some embodiments, the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, target spot, frogeye leaf spot or brown spot. In some embodiments, the plant is a dicotyledonous plant. In some embodiments, the plant is a leguminous plant. In some embodiments, the plant is a soybean plant.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] FIG. 1. Graphs showing disease efficacy and mRNA expression data for 8 events from plants transformed with construct pM383 (comprising the coding sequence of wheat Lr34 resistant gene (SEQ ID NO:1) and promoter P-At. GSP571.nno:5 (SEQ ID NO:9)) relative to wild-type(WT) and empty vector controls. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene. Bottom Panel: Relative mRNA expression level of Ta. Lr34res transgene.

[0018] FIG. 2. Graphs showing disease efficacy and mRNA expression data for 7 events from plants transformed with construct pM384 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-At. GSP571.nno: 5 (SEQ ID NO:9)) relative to WT and empty vector controls. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene. Bottom Panel: Relative mRNA expression level of Ta. Lr34res transgene.

[0019] FIG. 3. Graphs showing disease efficacy and mRNA expression data for 8 events from plants transformed with construct pM386 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:4) and promoter P-At. GSP571.nno: 5 (SEQ ID NO:9)) relative to WT and empty vector controls. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene. Bottom Panel: Relative mRNA expression level of Ta. Lr34res transgene.

[0020] FIG. 4. Graphs showing disease efficacy and mRNA expression data for 4 events from plants transformed with construct pM499 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l3G046200:2 (SEQ ID NO: 10)) relative to WT and empty vector controls. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene. Bottom Panel: Relative mRNA expression level of Ta. Lr34res transgene.

[0021] FIG. 5. Graphs showing disease efficacy and mRNA expression for two sets of data including a total of 17 events from plants transformed with construct pM524 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_l:l (SEQ ID NO: 12)) relative to WT and empty vector controls. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene. Bottom Panel: Relative mRNA expression level of Ta. Lr34res transgene.

[0022] FIG. 6. Graphs showing disease efficacy and mRNA expression data for 4 events from plants transformed with construct pM525 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_2: 1 (SEQ ID NO: 13)) relativeto WT and empty vector controls. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene. Bottom Panel: Relative mRNA expression level of Ta. Lr34res transgene.

[0023] FIG. 7. Graphs showing disease efficacy and mRNA expression data for 4 events from plants transformed with construct pM526 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l7G020600:l (SEQ ID NO: 14)) relative to WT and empty vector controls. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene. Bottom Panel: Relative mRNA expression level of Ta. Lr34res transgene.

[0024] FIG. 8. Graphs showing disease efficacy and mRNA expression data for 3 events from plants transformed with construct pM527 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.02G101100:1 (SEQ ID NO: 15)) relative to WT and empty vector controls. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene. Bottom Panel: Relative mRNA expression level of Ta. Lr34res transgene.

[0025] FIG. 9. Graphs showing disease efficacy and mRNA expression data for 8 events from plants transformed with construct pM679 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. PSII:l (SEQ ID NO: 11)) relative to WT and empty vector controls. Top Panel: Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene. Bottom Panel: Relative mRNA expression level of Ta. Lr34res transgene.

[0026] FIG. 10. Graph showing disease efficacy data for 8 events from plants transformed with construct pM499 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l3G046200:2 (SEQ ID NO: 10)) in CE-2 relative to a WT control. Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene.

[0027] FIG. 11. Illustration of disease efficacy data for 10 events from plants transformed with construct pM524 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_l:l (SEQ ID NO: 12)) in CE-2 relative to a WTcontrol. Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene.

[0028] FIG. 12. Illustration of disease efficacy data for 8 events from plants transformed with construct pM525 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_2: 1 (SEQ ID NO: 13)) in CE-2 relative to a WT control. Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene.

[0029] FIG. 13. Illustration of disease efficacy data for 6 events from plants transformed with construct pM526 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l7G020600:l (SEQ ID NO: 14)) in CE-2 relative to a WT control. Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene.

[0030] FIG. 14. Illustration of disease efficacy data for 6 events from plants transformed with construct pM527 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.02G101100:1 (SEQ ID NO:15)) in CE-2 relative to a WT control. Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene.

[0031] FIG. 15. Illustration of disease efficacy data for 10 events from plants transformed with construct pM679 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. PSII:l (SEQ ID NO: 11)) in CE-2 relative to a WT control. Percentage disease severity tested in soybean plant transformed with Ta. Lr34res transgene.BRIEF DESCRIPTION OF THE SEQUENCES

[0032] SEQ ID NO:1 Triticum aestivum Lr34 resistant (Ta. Lr34res) DNA sequence (coding sequence of the Ta. Lr34res gene).

[0033] SEQ ID NO:2 Triticum aestivum Lr34 resistant codon optimized DNA sequence.

[0034] SEQ ID NO:3 Triticum aestivum Lr34 resistant codon optimized DNA sequence.

[0035] SEQ ID NO:4 Triticum aestivum Lr34 resistant codon optimized DNA sequence.

[0036] SEQ ID NO:5 Triticum aestivum Ta. Lr34 susceptible DNA sequence (coding sequence of a wild-type, disease-susceptible Ta. Lr34 gene sequence).

[0037] SEQ ID NO:6 Triticum aestivum Ta. Lr34 resistant Protein (i.e., amino acid) sequence.

[0038] SEQ ID NO:7 Triticum aestivum Ta. Lr34 susceptible Protein (i.e., amino acid) sequence (wild-type Ta. Lr34 protein).

[0039] SEQ ID NO:8 Triticum aestivum Ta. Lr34 resistant (Ta. Lr34res) Genomic DNA sequence.

[0040] SEQ ID NO:9 Synthetic Promoter P-At. GSP571.nno:5 sequence.

[0041] SEQ ID NO: 10 Glycine max Promoter P-Gm.13G046200:2 sequence.

[0042] SEQ ID NO: 11 Glycine max Promoter P-Gm. PSII: 1 sequence.

[0043] SEQ ID NO: 12 Glycine max Promoter P-Gm. Cba_l: 1 sequence.

[0044] SEQ ID NO: 13 Glycine max Promoter P-Gm. Cba_2: 1 sequence.

[0045] SEQ ID NO: 14 Glycine max Promoter P-Gm.17G020600: 1 sequence.

[0046] SEQ ID NO: 15 Glycine max Promoter P-Gm.02G101100: 1 sequence.

[0047] SEQ ID NO: 16 Aegilops tauschii subsp. strangulata XP_020162164.1 Protein sequence.

[0048] SEQ ID NO: 17 Aegilops tauschii ACL36480.1 Protein sequence.

[0049] SEQ ID NO: 18 Triticum aestivum XP_044442862.1 Protein sequence.

[0050] SEQ ID NO: 19 Triticum aestivum XP_044442863.1 Protein sequence.

[0051] SEQ ID NO:20 Triticum aestivum XP_044442860.1 Protein sequence.

[0052] SEQ ID NO:21 Triticum dicoccoides XP_037423359.1 Protein sequence.

[0053] SEQ ID NO:22 Triticum aestivum XP_044366008.1 Protein sequence.

[0054] SEQ ID NO:23 Triticum turgidum subsp. durum VAH98361.1 Protein sequence.

[0055] SEQ ID NO:24 Triticum aestivum KAF7045545.1 Protein sequence.

[0056] SEQ ID NO:25 Oryza sativa Japonica Group XP_015618071.1 Protein sequence.

[0057] SEQ ID NO:26 Panicum hallii PVH66410.1 Protein sequence.

[0058] SEQ ID NO:27 Oryza glaberrima XP_052139756.1 Protein sequence.

[0059] SEQ ID NO:28 Panicum hallii var. hallii PUZ75757.1 Protein sequence.

[0060] SEQ ID NO:29 Panicum hallii var. hallii PUZ75758.1 Protein sequence.

[0061] SEQ ID NO:30 Oryza sativa Japonica Group KAF2908023.1 Protein sequence.

[0062] SEQ ID NO:31 Oryza sativa Japonica Group NP_001410444.1 Protein sequence.

[0063] SEQ ID NO:32 Oryza sativa Japonica Group XP_025877988.1 Protein sequence.

[0064] SEQ ID NO:33 Panicum hallii var. hallii PUZ75754.1 Protein sequence.

[0065] SEQ ID NO:34 Panicum hallii var. hallii PUZ75753.1 Protein sequence.

[0066] SEQ ID NO:35 Panicum hallii XP_025812506.1 Protein sequence.

[0067] SEQ ID NO:36 Panicum hallii var. hallii PUZ75755.1 Protein sequence.

[0068] SEQ ID NO:37 Panicum hallii var. hallii PUZ75756.1 Protein sequence.

[0069] SEQ ID NO:38 Oryza brachyantha XP_015698314.1 Protein sequence.

[0070] SEQ ID NO:39 Oryza sativa Indica Group EEC69371.1 Protein sequence.

[0071] SEQ ID NO:40 Lolium rigidum XP_047091328.1 Protein sequence.

[0072] SEQ ID NO:41 Lolium rigidum XP_047091359.1 Protein sequence.

[0073] SEQ ID NO:42 Lolium perenne XP_051180455.1 Protein sequence.

[0074] SEQ ID NO:43 Lolium perenne XP_051180457.1 Protein sequence.

[0075] SEQ ID NO:44 Lolium perenne XP_051180458.1 Protein sequence.

[0076] SEQ ID NO:45 Lolium rigidum XP_047091327.1 Protein sequence.

[0077] SEQ ID NO:46 Lolium rigidum XP_047091358.1 Protein sequence.

[0078] SEQ ID NO:47 Lolium perenne XP_051180453.1 Protein sequence.

[0079] SEQ ID NO:48 Panicum hallii var. hallii PUZ38223.1 Protein sequence.

[0080] SEQ ID NO:49 Setaria italica XP_022678987.1 Protein sequence.

[0081] SEQ ID NO:50 Setaria italica XP_004982570.1 Protein sequence.

[0082] SEQ ID NO:51 Panicum virgatum KAG2536935.1 Protein sequence.

[0083] SEQ ID NO:52 Setaria viridis XP_034576553.1 Protein sequence.

[0084] SEQ ID NO:53 Setaria viridis TKV92838.1 Protein sequence.

[0085] SEQ ID NO:54 Setaria italica XP_012698408.1 Protein sequence.

[0086] SEQ ID NO:55 Digitaria exilis KAF8682044.1 Protein sequence.

[0087] SEQ ID NO:56 Panicum hallii XP_025792512.1 Protein sequence.

[0088] SEQ ID NO:57 Sorghum bicolor XP_021304767.1 Protein sequence.

[0089] SEQ ID NO:58 Sorghum bicolor KAG0548825.1 Protein sequence.

[0090] SEQ ID NO:59 Panicum virgatum XP_039783626.1 Protein sequence.

[0091] SEQ ID NO:60 Digitaria exilis KAF8722245.1 Protein sequence.

[0092] SEQ ID NO:61 Setaria italica XP_004982568.1 Protein sequence.

[0093] SEQ ID NO:62 Panicum virgatum XP_039783624.1 Protein sequence.

[0094] SEQ ID NO:63 Sorghum bicolor KAG0548823.1 Protein sequence.

[0095] SEQ ID NO:64 Sorghum bicolor OQU91495.1 Protein sequence.

[0096] SEQ ID NO:65 Setaria italica XP_012698407.1 Protein sequence.

[0097] SEQ ID NO:66 Setaria viridis TKV92833.1 Protein sequence.

[0098] SEQ ID NO:67 Setaria viridis TKV92832.1 Protein sequence.

[0099] SEQ ID NO:68 Panicum virgatum XP_039783623.1 Protein sequence.

[0100] SEQ ID NO:69 Setaria viridis XP_034575835.1 Protein sequence.

[0101] SEQ ID NO:70 Setaria viridis XP_034575838.1 Protein sequence.

[0102] SEQ ID NO:71 Sorghum bicolor XP_021306845.1 Protein sequence.

[0103] SEQ ID NO:72 Phoenix dactylifera XP_038979886.1 Protein sequence.

[0104] SEQ ID NO:73 Elaeis guineensis XP_029118044.1 Protein sequence.

[0105] SEQ ID NO:74 Elaeis guineensis XP_010914761.1 Protein sequence.

[0106] SEQ ID NO:75 Rhynchospora tenuis KAJ3687424.1 Protein sequence.

[0107] SEQ ID NO:76 Ananas comosus XP_020101536.1 Protein sequence.

[0108] SEQ ID NO:77 Dioscorea zingiberensis KAI0966922.1 Protein sequence.

[0109] SEQ ID NO:78 Asparagus officinalis XP_020241545.1 Protein sequence.

[0110] SEQ ID NO:79 Asparagus officinalis XP_020277113.1 Protein sequence.

[0111] SEQ ID NO:80 Dioscorea alata KAH7669104.1 Protein sequence.

[0112] SEQ ID NO:81 Dioscorea cayenensis subsp. rotundata XP_039114166.1 Protein sequence.

[0113] SEQ ID NO:82 Rhynchospora breviuscula KAJ1696028.1 Protein sequence.

[0114] SEQ ID NO:83 Rhynchospora pubera KAJ4807677.1 Protein sequence.

[0115] SEQ ID NO:84 Dioscorea alata KAH7655209.1 Protein sequence.

[0116] SEQ ID NO:85 Dioscorea alata KAH7655208.1 Protein sequence.

[0117] SEQ ID NO:86 Nelumbo nucifera XP_019054935.1 Protein sequence.

[0118] SEQ ID NO:87 Macleaya cordata OVA16669.1 Protein sequence.

[0119] SEQ ID NO:88 Rhynchospora tenuis KAJ3687421.1 Protein sequence.

[0120] SEQ ID NO:89 Dioscorea alata KAH7655222.1 Protein sequence.

[0121] SEQ ID NO:90 Dioscorea cayenensis subsp. rotundata XP_039114165.1 Protein sequence.

[0122] SEQ ID NO:91 Musa acuminata subsp. malaccensis CAG1859253.1 Protein sequence.

[0123] SEQ ID NO:92 Spirodela intermedia CAA2614131.1 Protein sequence.

[0124] SEQ ID NO:93 Dioscorea alata KAH7655223.1 Protein sequence.

[0125] SEQ ID NO:94 Musa acuminata subsp. malaccensis XP_009390975.1 Protein sequence.

[0126] SEQ ID NO:95 Dioscorea alata KAH7655211.1 Protein sequence.

[0127] SEQ ID NO:96 Spirodela intermedia CAA7388374.1 Protein sequence.

[0128] SEQ ID NO:97 Musa acuminata subsp. malaccensis CAG1848804.1 Protein sequence.

[0129] SEQ ID NO:98 Parasponia andersonii PON68433.1 Protein sequence.

[0130] SEQ ID NO:99 Trema orientate PON46936.1 Protein sequence.

[0131] SEQ ID NO: 100 Vitis vinifera CBI26001.3 Protein sequence.

[0132] SEQ ID NO: 101 Prunus dulcis KAI5349097.1 Protein sequence.

[0133] SEQ ID NO: 102 Quillaja saponaria KAJ7973220.1 Protein sequence.

[0134] SEQ ID NO:103 Dioscorea cayenensis subsp. rotundata XP_039118207.1 Protein sequence.

[0135] SEQ ID NO: 104 Camellia sinensis XP_028110576.1 Protein sequence.

[0136] SEQ ID NO: 105 Nelumbo nucifera XP_019054946.1 Protein sequence.

[0137] SEQ ID NO: 106 Musa troglodytarum URD74552.1 Protein sequence.

[0138] SEQ ID NO: 107 Rosa rugosa XP_061994445.1 Protein sequence.

[0139] SEQ ID NO: 108 Zingiber officinale XP_042388306.1 Protein sequence.

[0140] SEQ ID NO: 109 Quercus suber XP_023885614.1 Protein sequence.

[0141] SEQ ID NO: 110 Melia azedarach KAJ4721905.1 Protein sequence.

[0142] SEQ ID NO: 111 Nyssa sinensis KAA8527979.1 Protein sequence.

[0143] SEQ ID NO: 112 Rosa rugosa XP_061994444.1 Protein sequence.

[0144] SEQ ID NO:113 Dipteronia sinensis KAK3189944.1 Protein sequence.

[0145] SEQ ID NO: 114 Corylus avellana XP_059437806.1 Protein sequence.

[0146] SEQ ID NO: 115 Comusflorida XP_059632028.1 Protein sequence.

[0147] SEQ ID NO: 116 Aegilops tauschii subsp. strangulata XM_020306575.3 DNA sequence.

[0148] SEQ ID NO: 117 Aegilops tauschii FJ436986.1 DNA sequence.

[0149] SEQ ID NO: 118 Triticum aestivum XM_044586927.1 DNA sequence.

[0150] SEQ ID NO: 119 Triticum aestivum XM_044586928.1 DNA sequence.

[0151] SEQ ID NO: 120 Triticum aestivum XM_044586925.1 DNA sequence.

[0152] SEQ ID NO: 121 Triticum dicoccoides XM_037567462.1 DNA sequence.

[0153] SEQ ID NO: 122 Triticum aestivum XM_044510073.1 DNA sequence.

[0154] SEQ ID NO: 123 Triticum turgidum subsp. durum LT934117.1 DNA sequence.

[0155] SEQ ID NO: 124 Triticum aestivum CM022220.1 DNA sequence.

[0156] SEQ ID NO: 125 Oryza sativa Japonica Group XM_015762585.2 DNA sequence.

[0157] SEQ ID NO: 126 Panicum hallii CM008046.2 DNA sequence.

[0158] SEQ ID NO: 127 Oryza glaberrima XM_052283796.1 DNA sequence.

[0159] SEQ ID NO: 128 Panicum hallii var. hallii CM009749.1 DNA sequence.

[0160] SEQ ID NO: 129 Panicum hallii var. hallii CM009749.1 DNA sequence.

[0161] SEQ ID NO: 130 Oryza sativa Japonica Group CM020532.1 DNA sequence.

[0162] SEQ ID NO: 131 Oryza sativa Japonica Group NM_001423515.1 DNA sequence.

[0163] SEQ ID NO: 132 Oryza sativa Japonica Group XM_026022203.1 DNA sequence.

[0164] SEQ ID NO: 133 Panicum hallii var. hallii CM009749.1 DNA sequence.

[0165] SEQ ID NO: 134 Panicum hallii var. hallii CM009749.1 DNA sequence.

[0166] SEQ ID NO:135 Panicum hallii XM_025956721.1 DNA sequence.

[0167] SEQ ID NO: 136 Panicum hallii var. hallii CM009749.1 DNA sequence.

[0168] SEQ ID NO: 137 Panicum hallii var. hallii CM009749.1 DNA sequence.

[0169] SEQ ID NO: 138 Oryza brachyantha XM_015842828.1 DNA sequence.

[0170] SEQ ID NO: 139 Oryza sativa Indica Group CM000137.1 DNA sequence.

[0171] SEQ ID NO: 140 Lolium rigidum XM_047235372.1 DNA sequence.

[0172] SEQ ID NO: 141 Lolium rigidum XM_047235403.1 DNA sequence.

[0173] SEQ ID NO: 142 Lolium perenne XM_051324495.1 DNA sequence.

[0174] SEQ ID NO: 143 Lolium perenne XM_051324497.1 DNA sequence.

[0175] SEQ ID NO: 144 Lolium erenne XM_051324498.1 DNA sequence.

[0176] SEQ ID NO: 145 Lolium rigidum XM_047235371.1 DNA sequence.

[0177] SEQ ID NO: 146 Lolium rigidum XM_047235402.1 DNA sequence.

[0178] SEQ ID NO: 147 Lolium perenne XM_051324493.1 DNA sequence.

[0179] SEQ ID NO: 148 Panicum hallii var. hallii CM009757.1 DNA sequence.

[0180] SEQ ID NO: 149 Setaria italica XM_022823252.1 DNA sequence.

[0181] SEQ ID NO: 150 Setaria italica XM_004982513.3 DNA sequence.

[0182] SEQ ID NO:151 Panicum virgatum CM029054.1 DNA sequence.

[0183] SEQ ID NO: 152 Setaria viridis XM_034720662.1 DNA sequence.

[0184] SEQ ID NO: 153 Setaria viridis CM016560.1 DNA sequence.

[0185] SEQ ID NO: 154 Setaria italica XM_012842954.2 DNA sequence.

[0186] SEQ ID NO:155 Digitaria exilis JACEF0010002109.1 DNA sequence.

[0187] SEQ ID NO: 156 Panicum hallii XM_025936727.1 DNA sequence.

[0188] SEQ ID NO: 157 Sorghum bicolor XM_021449092.1 DNA sequence.

[0189] SEQ ID NO: 158 Sorghum bicolor CM027680.1 DNA sequence.

[0190] SEQ ID NO: 159 Panicum virgatum XM_039927692.1 DNA sequence.

[0191] SEQ ID NO:160 Digitaria exilis JACEF0010001675.1 DNA sequence.

[0192] SEQ ID NO: 161 Setaria italica XM_004982511.4 DNA sequence.

[0193] SEQ ID NO: 162 Panicum virgatum XM_039927690.1 DNA sequence.

[0194] SEQ ID NO: 163 Sorghum bicolor CM027680.1 DNA sequence.

[0195] SEQ ID NO: 164 Sorghum bicolor CM000760.3 DNA sequence.

[0196] SEQ ID NO: 165 Setaria italica XM_012842953.2 DNA sequence.

[0197] SEQ ID NO: 166 Setaria viridis CM016560.2 DNA sequence.

[0198] SEQ ID NO: 167 Setaria viridis CM016560.2 DNA sequence.

[0199] SEQ ID NO: 168 Panicum virgatum XM_039927689.1 DNA sequence.

[0200] SEQ ID NO: 169 Setaria viridis XM_034719944.1 DNA sequence.

[0201] SEQ ID NO: 170 Setaria viridis XM_034719947.1 DNA sequence.

[0202] SEQ ID NO:171 Sorghum bicolor XM_021451170.1 DNA sequence.

[0203] SEQ ID NO: 172 Phoenix dactylifera XM_039123958.1 DNA sequence.

[0204] SEQ ID NO: 173 Elaeis guineensis XM_029262211.1 DNA sequence.

[0205] SEQ ID NO: 174 Elaeis guineensis XM_010916459.3 DNA sequence.

[0206] SEQ ID NO:175 Rhynchospora tenuis J AMRDG010000002.1 DNA sequence.

[0207] SEQ ID NO: 176 Ananas comosus XM_020245947.1 DNA sequence.

[0208] SEQ ID NO: 177 Dioscorea zingiberensis IAGGNH010000007.1 DNA sequence.

[0209] SEQ ID NO: 178 Asparagus officinalis XM_020385956.1 DNA sequence.

[0210] SEQ ID NO: 179 Asparagus officinalis XM_020421524.1 DNA sequence.

[0211] SEQ ID NO:180 Dioscorea alata IAFB11010000011.1 DNA sequence.

[0212] SEQ ID NO:181 Dioscorea cayenensis subsp. rotundata XM_039258232.1 DNA sequence.

[0213] SEQ ID NO: 182 Rhynchospora breviuscula JAMQYH010000003.1 DNA sequence.

[0214] SEQ ID NO:183 Rhynchospora pubera JAMFTS010000001.1 DNA sequence.

[0215] SEQ ID NO:184 Dioscorea alata JAFBII010000019.1 DNA sequence.

[0216] SEQ ID NO:185 Dioscorea alata JAFBII010000019.1 DNA sequence.

[0217] SEQ ID NO: 186 Nelumbo nucifera XM_019199390.1 DNA sequence.

[0218] SEQ ID NO:187 Macleaya cordata MVGT01000481.1 DNA sequence.

[0219] SEQ ID NO: 188 Rhynchospora tenuis JAMRDG010000002.1 DNA sequence.

[0220] SEQ ID NO:189 Dioscorea alata JAFBII010000019.1 DNA sequence.

[0221] SEQ ID NO:190 Dioscorea cayenensis subsp. rotundata XM_039258231.1 DNA sequence.

[0222] SEQ ID NO: 191 Musa acuminata subsp. malaccensis HG996466.1 DNA sequence.

[0223] SEQ ID NO: 192 Spirodela intermedia LR743588.1 DNA sequence.

[0224] SEQ ID NO:193 Dioscorea alata JAFBII010000019.1 DNA sequence.

[0225] SEQ ID NO: 194 Musa acuminata subsp. malaccensis XM_009392700.2 DNA sequence.

[0226] SEQ ID NO:195 Dioscorea alata JAFBII010000019.1 DNA sequence.

[0227] SEQ ID NO: 196 Spirodela intermedia LR746264.1 DNA sequence.

[0228] SEQ ID NO: 197 Musa acuminata subsp. malaccensis HG996468.1 DNA sequence.

[0229] SEQ ID NO: 198 Parasponia andersonii IXTB01000063.1 DNA sequence.

[0230] SEQ ID NO: 199 Trema orientale JXTC01000544.1 DNA sequence.

[0231] SEQ ID NO:200 Vitis vinifera FN595749.1 DNA sequence.

[0232] SEQ ID NO:201 Prunus dulcis IAJFAZ020000001.1 DNA sequence.

[0233] SEQ ID NO:202 Quillaja saponaria JARAO00010000004.1 DNA sequence.

[0234] SEQ ID NO:203 Dioscorea cayenensis subsp. rotundata XM_039262273.1 DNA sequence.

[0235] SEQ ID NO:204 Camellia sinensis XM_028254775.1 DNA sequence.

[0236] SEQ ID NO:205 Nelumbo nucifera XM_019199401.1 DNA sequence.

[0237] SEQ ID NO:206 Musa troglodytarum CP097502.1 DNA sequence.

[0238] SEQ ID NO:207 Rosa rugosa XM_062138461.1 DNA sequence.

[0239] SEQ ID NO:208 Zingiber officinale XM_042532372.1 DNA sequence.

[0240] SEQ ID NO:209 Quercus suber XM_024029846.1 DNA sequence.

[0241] SEQ ID NO:210 Melia azedarach J AQMWS010000003.1 DNA sequence.

[0242] SEQ ID NO:211 Nyssa sinensis CM018045.1 DNA sequence.

[0243] SEQ ID NO:212 Rosa rugosa XM_062138460.1 DNA sequence.

[0244] SEQ ID NO:213 Dipteronia sinensis JANJYJ010000009.1 DNA sequence.

[0245] SEQ ID NO:214 Corylus avellana XM_059581823.1 DNA sequence.

[0246] SEQ ID NO:215 Comusflorida XM_059776045.1 DNA sequence.

[0247] SEQ ID NO:216 Glycine max Promoter P-Gm.08G282100:2 sequence.

[0248] SEQ ID NO:217 Glycine max Promoter P-Gm.02G215700:2 sequence

[0249] SEQ ID NO:218 Glycine max Promoter P-Gm.11G155OOO:2 sequence

[0250] SEQ ID NO:219 Glycine max Promoter P-Gm.01G238800:1 sequence

[0251] SEQ ID NO:220 Glycine max Promoter P-Gm.16G089000: 1 sequence

[0252] SEQ ID NO:221 Glycine max Promoter P-Gm.16G089000_trunc: 1 sequence

[0253] SEQ ID NO:222 Glycine max Promoter P-Gm.15G057600: 1 sequence

[0254] SEQ ID NO:223 Glycine max Promoter P-Gm.07G156100:1 sequence

[0255] SEQ ID NO:224 Glycine max Promoter P-Gm.07G156100_trunc: 1 sequence

[0256] SEQ ID NO:225 Arabidopsis thaliana Promoter P-At. CeresRep74_Mdhg: 1 sequence

[0257] SEQ ID NO:226 Medicago truncatula Promoter P-Mt. Lhcb2: 1 sequenceDETAILED DESCRIPTION

[0258] Soybean, Glycine max, is one of the major economic crops grown worldwide. Asian Soybean Rust (herein referred to as ASR) is an aggressive fungal pathogen (Phakopsora pachyrhizi) that threatens soybean crops worldwide having been reported in the Eastern and Western Hemispheres. In the Eastern Hemisphere, ASR has been reported in Australia, China, India, Japan, Taiwan and Thailand. In the Western Hemisphere, ASR has been observed in Brazil, Columbia, Costa Rica and Puerto Rico. ASR can be a devastating disease, causing potentially severe yield losses as reported in some field trials. Plants that are heavily infected have fewer pods and smaller seeds of poor quality (see, e.g., Frederick et al., Mycology 92: 217-227 (2002)). Control of ASR and other fungal diseases relies heavily on the application of fungicides, requiringmultiple sprays each year. Thus, genetic traits and alleles for disease resistance in soybean have the potential to be both sustainable and durable, especially when combining multiple modes of action. However, native sources of genetic resistance are few and most have already been overcome by the pathogen. Thus, there is a need for novel genetic or transgenic approaches for improving resistance and / or increasing durability of resistance of soybean plants to ASR, alone or in combination with other modes of action.

[0259] The present disclosure provides recombinant constructs and transgenic soybean plants or plant parts that express a resistant Lr34 protein to confer resistance to infection by a fungal pathogen, Phakopsora pachyrhizi, which is the causal agent of Asian Soybean Rust (ASR) in soybean. The Lr34 protein encoded by this gene functions as an ATB-binding cassette (ABC) transporter, and a resistant Ta. Lr34 allele has been found to impart increased fungal pathogen resistance (and decreased fungal pathogen susceptibility) in wheat plants. A resistant Ta. Lr34 allele has also been transgenically expressed and shown to confer rust resistance in both durum wheat and barley species. See, e.g., Published PCT Application No. W02010 / 022443; Plant Biotechnol J., 11(7):847-54 (2013); and Science, 323(5919): 1360-1363 (2009).

[0260] The present disclosure overcomes the limitations of the prior art and demonstrates that a resistance Lr34 transgene, when transgenically expressed in soybean (Glycine max) under optimal levels and localization, can confer higher levels of resistance to Asian Soybean Rust, caused by the fungal pathogen Phakopsora pachyrhizi, and such resistance is observed in seedlings and mature plants. Moreover, the present disclosure demonstrates that it is possible to have a resistance phenotype with a resistance Lr34 transgene while minimizing or reducing undesirable off-types. As such, the beneficial effects provided by the present disclosure are useful in the generation and creation of transgenic plants that are resistant to ASR and other fungal pathogens. In some embodiments, the present disclosure provides effective transgenic expression of resistant Lr34 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 chemical control agents and possibly other native or engineered resistance genes. The present disclosure thus provides a significant advance in the art for the production of ASR resistant soybean plants demonstrating minimized or reduced undesirable off-types.

[0261] 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

[0262] 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 are also provided.

[0263] As used herein, the term “DNA” or “DNA molecule” refers to a double- stranded DNA molecule of genomic or synthetic origin, i.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 for nucleotide and amino acids as set forth at 37 CFR §§ 1.831 through 1.835 is used and as set forth in WIPO Standard ST.26 (2021), Annex I, Tables 1 and 3.

[0264] As used herein, "synthetic nucleotide sequence”, “synthetic polynucleotide sequence", or “synthetic promoter” refers to a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. The polynucleotides of the present disclosure comprise synthetic polynucleotide sequences. The recombinant DNA constructs of the present disclosure comprise synthetic polynucleotide sequences. Preferably, synthetic polynucleotide sequences share little or no extended homology to natural sequences. Extended homology in this context generally refers to 100% sequence identity extending beyond about 25 nucleotides of contiguous sequence.

[0265] As used herein, the term “isolated DNA molecule” refers to a DNA molecule at least partially separated from other molecules normally associated with it in its native or natural state. In one embodiment, the term “isolated” refers to a DNA molecule that is at least partially separated from some of the nucleic acids which normally flank the DNA molecule in its native or natural state. Thus, DNA molecules fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques, 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, in that they are not in their native state.

[0266] Any number of methods well-known to those skilled in the art 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.

[0267] “Identity”, as is 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 known Smith Waterman algorithm and any other known sequence alignment algorithm or program 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 aligning two sequences, e.g., a reference sequence and another sequence, to maximize the number of nucleotide matches in the sequence alignment with appropriate or minimal internal nucleotide insertions, deletions, or gaps. As used herein with regard to polynucleotide sequences, the term “reference sequence” may refer to any one of the polynucleotide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 8, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, or 215, or a complement thereof; or any one of the polypeptide sequences of SEQ ID NOs: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115.

[0268] According to some embodiments, a polynucleotide or polypeptide sequence as described herein may have at least 40%, 50%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity or 100% sequence identity to at least one of the sequences set forth herein. In some embodiments, a nucleic acid or polynucleotide sequence, such as a nucleotide coding sequence, as described herein may have or comprise, for example, about or at least 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%, or 99% sequence identity, or 100% sequence identity, to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 8, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, and / or 215, or a complement thereof. In some embodiments, an amino acid or polypeptide sequence or protein as provided herein may have or comprise, for example, about or at least 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%, or 99% sequence identity, or 100% sequence identity, to one or more of SEQ ID NOs: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, and / or 115.

[0269] Parameters for polypeptide sequence comparison may include, for example, the following: Algorithm: Needleman and Wunsch ( / . Mol. Biol. 48:443-453, 1970); Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, (Proc. Natl. Acad. Sci. USA 89:10915-10919, 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.

[0270] Parameters for nucleic acid sequence comparison may include, for example, 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.

[0271] 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.

[0272] “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 nucleic acid fragments include oligonucleotides and polynucleotides that may be used as probes or primers in hybridization oramplification technologies or in the regulation of replication, transcription or translation (for example, promoter fragments). A polynucleotide fragment may refer to any subsequence, part or portion of a polynucleotide, which may be 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 about 35 consecutive 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. In some embodiments, fragments of a promoter sequence disclosed herein are provided. Promoter fragments may comprise promoter activity or gene regulatory activity, as described above, and may be useful alone or in combination with other promoters and / or promoter fragments, such as in constructing chimeric promoters, or in combination with other expression or regulatory elements and expression or regulatory element fragments. In specific embodiments, fragments of a promoter are provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, at least about 1000, or at least about 1500 consecutive nucleotides, or longer, of the reference sequence from which they are derived that is, of any of SEQ ID NOs: 9-15 and 216-226. Methods for producing such fragments from a starting promoter are well known in the art. Fragments of any of SEQ ID NOs: 9-15 and 216-226 may have the activity of the base sequence, for example the promoter activity of the base sequence.

[0273] Fragments may also include a subsequences, portions or parts of polypeptides and protein molecules, or a subsequence, portion or part of a polypeptide disclosed herein. Protein fragments may have antigenic potential or may perform at least one biological function of the intact full-length polypeptide in substantially the same manner, or to a similar extent, as does the intact full-length polypeptide. Protein fragments may vary in size from as few as 5 amino acids to almost the full length of the intact polypeptide, but may preferably be 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 leastabout 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 a subsequence, portion or part of any of the amino acid sequences provided herein. The nucleic acids provided herein, such as SEQ ID NO: 1, 2, 3, 4, 5, 8, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, or 215, and amino acids provided herein as SEQ ID NOs: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115, may be from any source, e.g., identified from a naturally occurring plant species, or synthesized, e.g., identified from a naturally occurring gene in a plant or other species, or modified or synthesized relative to any one of the sequences provided herein. For example, any one of SEQ ID NOs: 1, 2, 3, 4, 5, 8, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, and / or 215, may be modified to create a codon optimized sequence for expression in a plant, such as a legume or soybean plant species, or a coding sequence with a G / C content more like or similar to the G / C content of naturally occurring genes in a particular plant, such as a legume or soybean plant species.II. DNA Constructs

[0274] 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 art 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 molecule, an operably linked protein-coding DNA molecule, and an operably linked 3’ untranslated region (UTR) for example, for the termination of transcription. 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.

[0275] 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 naturally found in the wild-type plant. Transgenic plants provided herein comprise the recombinant DNA molecules and proteins of the present disclosure.

[0276] 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 DNAmolecule, 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 (i.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.

[0277] 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 Lr34 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.

[0278] 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 linked with a protein-coding sequence of a gene or transgene in a DNA construct or molecule where the promoter and the protein-coding sequence are so arranged that the promoter may affect the expression of the protein-coding sequence of the gene or transgene.

[0279] 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 thatfunction 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. An example of a synthetic promoter useful in practicing the present disclosure is presented as SEQ ID NO:9.

[0280] 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

[0281] 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 Agrobacterium-mediated transformation was disclosed by Hamilton et al. (Proc. Natl. Acad. Sci. USA 93:9975-9979, 1996).

[0282] Particularly useful for transformation are expression cassettes that have been isolated from such vectors. DNA segments used for transforming plant cells will, of course, generally comprise 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, poly linkers, 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 improvedphenotype 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

[0283] 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 5' untranslated region (5' UTR) of a genomic copy of a gene. Alternately, promoters may be synthetically produced or manipulated DNA molecules. Promoters may also be chimeric, that is a promoter produced through the fusion of two or more heterologous DNA molecules. Promoters useful in practicing the present disclosure include SEQ ID NOs: 9-15 and 216-226, or fragments or variants thereof. In specific embodiments of the present disclosure, such molecules 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. In still further specific embodiments, a fragment may be defined as exhibiting promoter activity possessed by the starting promoter molecule from which it is derived, or a fragment 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.

[0284] As used herein, the term “variant” refers to a second DNA molecule, such as a promoter, that is in composition similar, but not identical to, a first DNA molecule, and wherein the second DNA molecule still maintains the general functionality, i.e. the same or similar expression pattern, for instance through more or less equivalent transcriptional activity, of the first DNA molecule. A variant may be a shorter or truncated version of the first DNA molecule or an altered version of the sequence of the first DNA molecule, such as one with different restriction enzyme sites and / or internal deletions, substitutions, or insertions. A “variant” can also encompass a promoter having a nucleotide sequence comprising a substitution, deletion, or insertion of one or more nucleotides of a reference sequence, wherein the derivative regulatory element has more or less or equivalent transcriptional or translational activity than the corresponding parent regulatorymolecule. Promoter “variants” will also encompass variants arising from mutations that naturally occur in bacterial and plant cell transformation. In the present disclosure, any polynucleotide sequence provided as SEQ ID NOs: 9-15 and 216-226 may be used to create variants that are similar in composition, but not identical to, the DNA sequence of the original regulatory element, while still maintaining the general functionality, i.e., the same or similar expression pattern, of the original promoter. Production of such variants of the present disclosure is well within the ordinary skill of the art in light of the disclosure and is and is contemplated herein.

[0285] 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 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 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 belowground 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 aboveground 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, 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. 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 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. Above-ground promoters useful in practicing the present disclosure include SEQ ID NOs: 10-15, 216-226 or fragments or variants thereof.

[0286] In some embodiments, a 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: 9-15 and 216-226, or a functional fragment or variant thereof. In some embodiments the heterologous promoter comprises a nucleotide sequence that is 100% identical to one or more of SEQ ID NOs: 9-15 and 216-226, or a functional fragment or variant thereof. In some embodiments, the heterologous promoter comprises a functional fragment of one of SEQ ID NOs: 9-15 and 216-226. As used herein, a “functional fragment” of a constitutive or an above-ground promoter is a DNA fragment or portion of the DNA sequence of the constitutive or above-ground promoter that drives expression of a nucleotide coding sequence operably linked to the fragment in a manner that is similar to the longer DNA sequence of the constitutive or aboveground promoter and itself meets the definition for a constitutive or an above-ground promoter.

[0287] According to certain embodiments, the expression level of the nucleotide coding sequence operably linked to the heterologous above-ground 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), as known in the art. See, e.g., Zhao et al., I. 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 the transcript 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 transgene) encoding the transcript (in kilobases) to generate the FPKM for the expression of the gene (or transgene) in the sample. Thus, a FPKM for a heterologous above-ground promoter operably linked to anucleotide 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 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 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.

[0288] 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 equal to 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.

[0289] In certain embodiments, the heterologous above-ground 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 to9, or greater than or equal to 10. In certain embodiments, the heterologous above-ground 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 aboveground 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 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 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 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 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 aboveground 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.

[0290] In addition to the promoter sequences disclosed herein, other exemplary promoters for expression of a nucleic acid sequence may include a constitutive plant promoter such as the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985), or others such as CaMV 19S (Lawton et al., Plant Mol. Biol. 9:315-324, 1987), nos (Ebert et al., Proc. Natl. Acad. Sci. USA 84:5745-5749, 1987), Adh (Walker et al., Proc. Natl. Acad. Sci. USA 84:6624-6628, 1987), sucrose synthase (Yang and Russell, Proc. Natl. Acad. Sci. USA 87:4144-4148, 1990), a-tubulin, actin (Wang et al., Mol. Cell Biol. 12:3399-3406, 1992), cab (Sullivan et al., Mol. Gen. Genet. 215:431-440, 1989), PEPCase (Hudspeth and Grula, Plant Mol. Biol. 12:579-589, 1989) or those associated with the Rgene complex (Chandler et al., Plant Cell 1:1175-1183, 1989). Tissue specific promoters such as root cell promoters (Conkling et al., Plant Physiol. 93:1203-1211, 1990) and tissue specific enhancers are also contemplated to be useful, as are inducible promoters such as ABA- and turgorinducible promoters. The PAL2 promoter may also be useful with the disclosure (U. S. Patent Application Publication No. 2004 / 0049802, the entire disclosure of which is specifically incorporated herein by reference). In one embodiment of the present disclosure, the native promoter associated with one or more of the nucleic acid sequences disclosed herein is used. In some embodiments, the promoter is a strong promoter or a weak promoter.

[0291] The DNA sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can also influence gene expression. One may thus 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 of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants may be desirable.

[0292] It is envisioned that presently disclosed coding sequences may be introduced under the control of novel promoters or enhancers, etc., or homologous or tissue specific promoters or control elements. 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 art in light of the present disclosure.C. Transcription Terminators

[0293] 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 expressionof the transgene. 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 transcription terminator T-Zm. GST59.nno:l known from International Patent Application WO2018 / 136594 or fragments or variants thereof, the transcription terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, or 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)orTMV 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

[0294] 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 or extracellular 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 Lr34 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 art.

[0295] 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 fromproteolytic 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).

[0296] 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

[0297] 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.

[0298] 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, diffusibleproteins detectable, e.g., by ELISA; small active enzymes detectable in extracellular solution (e.g., a-amylase, 0-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).

[0299] Many selectable marker coding regions are known and could be used with the present disclosure including, but not limited to, neo (Potrykus et al., 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.

[0300] An illustrative embodiment of selectable marker capable of being used in systems to select transformants are those that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes. 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.

[0301] Screenable markers that may be employed include a β 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 β 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 tyrosineto DOPA and dopaquinone, which in turn condenses to form the easily -detectable compound melanin; a p 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

[0302] 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 enable targeted insertion of one or more nucleic acids of interest, such as a recombinant DNA molecule, construct, transgene 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.

[0303] 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).

[0304] 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 art, 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 through homologous 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.

[0305] 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.

[0306] 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 Thermits 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, Cast, 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 (also known as Casl2a), CasX, CasY, homologs thereof, or modified versions thereof).

[0307] 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-901 integrase. 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.

[0308] 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

[0309] 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 thedisclosed 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.

[0310] Suitable methods for transformation of plant or other cells for use with the cunent 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 Agrobacterium-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 Transformation

[0311] 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 Agrobacterium-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.

[0312] 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 Agrobacteri / / / H- mediated transformation techniques have now made the technique applicable to nearly all monocoty ledonous plants. For example, Agrobacterium-mediated transformation techniques have now been applied to rice (Hieiet 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.

[0313] 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

[0314] 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 etal., Plant Cell 4:1495-1505, 1992), wheat (Zhou etal., Plant Cell Rep. 12:612-616, 1993), tomato (Tsukada et al., Plant Cell Physiol. 30:599-603, 1989), soybean (Christou etal., Proc. Natl. Acad. Sci. USA 84:3962-3966, 1987) and tobacco (Riggs and Bates, Proc. Natl. Acad. Sci. USA 83:5602-5606, 1986).

[0315] 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 ofspecies 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

[0316] 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-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.

[0317] 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.

[0318] 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 etal., 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 etal., 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

[0319] 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 from protoplasts. 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).

[0320] 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, forexample, 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

[0321] 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.

[0322] 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.

[0323] 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 plants from 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.

[0324] 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.

[0325] Somatic cells are of various types. Embry ogenic cells are one example of somatic cells that may be induced to regenerate a plant through embryo formation. Non-embryogenic cells arethose 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.

[0326] 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).

[0327] 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 culture of plant cells previously have been described. Examples of these media include, but are not limited to, the N6 medium described by Chu et al., (Sei. 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

[0328] 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

[0329] 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.

[0330] 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.

[0331] 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 released and 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.

[0332] 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 viridochromogenes. The use of the herbicide resistance gene encoding phosphinothricin acetyl transferase (PAT) is referred to in DE 3642829 A, wherein the gene is isolated from Streptomyces viridochromogenes. In the bacterial source organism, this enzyme acetylates the free amino group of PPT preventing auto-toxicity (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).

[0333] 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 Application WO 97 / 4103.

[0334] 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.

[0335] An example of a screenable marker trait is the enzyme luciferase. In 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

[0336] 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 anexemplary 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.

[0337] 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.

[0338] 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 are 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

[0339] 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 (ELIS As 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.

[0340] 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 sequence of, or a fragment of, SEQ ID NO: 1, 2, 3, 4, 5, 8, 9-15, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, or 216-226 may be useful as primers and probes for detecting the presence of a recombinant DNA molecule provided herein.D. DNA Integration, RNA Expression and Inheritance

[0341] 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 butdoes 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.

[0342] 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.

[0343] 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.

[0344] Both PCR and Southern hybridization techniques can be used to demonstrate transmission of a transgene to progeny. In most instances the characteristic Southern hybridization pattern for a given transformant will segregate in progeny as one or more Mendelian genes (Spencer et al., 1992) indicating stable inheritance of the transgene.

[0345] 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 mostinstances 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

[0346] 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.

[0347] 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.

[0348] 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 fromphosphinothricin and14C-acetyl CoA or for anthranilate synthase activity by following loss of fluorescence of anthranilate, to name two.

[0349] 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

[0350] 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.

[0351] As used herein the term “progeny” refers to 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 line with 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.

[0352] Backcrossing is herein defined as the process including the steps of: (a) crossing a plant of a first genotype containing a desired gene, trait, DNA sequence or element to a plant of a second genotype lacking the desired gene, trait, DNA sequence or element; (b) selecting one or more progeny plants containing the desired gene, trait, 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 gene, trait, DNA sequence or element from a plant of a first genotype to a plant of a second genotype.

[0353] Introgression of a DNA element into a plant genotype may be 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

[0354] 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 parts 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 and fuel 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

[0355] 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.

[0356] Asian Soybean Rust (ASR)

[0357] Asian Soybean Rust (ASR), caused by Phakopsora pachyrhizi, 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.

[0358] 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 1 / 16th to l / 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.

[0359] Powdery Mildew

[0360] 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.

[0361] 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.

[0362] Target Spot

[0363] 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.

[0364] 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.

[0365] Frogeye Leaf Spot

[0366] Frogeye leaf spot (FLS), caused by the fungal plant pathogen Cercospora sojina Hara, is a common foliar disease of soybean in the United States and other soybean production areas in the world. The disease may cause severe defoliation during warm, humid weather. Frogeye leaf spot can be distinguished from other soybean foliar diseases by the reddish-brown or purple ring surrounding the round leaf spots. For many decades, FLS was prevalent in the southern U. S. but has more recently become endemic throughout the Midwest and upper Midwest. Significant yield losses of soybean (10-60%) from this disease have occurred and severity has increased in the last five years due to continued cropping of susceptible soybean varieties over large acreage.

[0367] Symptoms of FLS are most visible and typically seen on leaves, but may also occur on stems, pods, and seeds late in the growing season with prolonged conditions that favor disease development (warm and humid conditions). Infection can occur at any stage of soybean development, but most often occurs during reproductive growth stages (from bloom to maturity) and may develop sooner in continuous soybean fields and / or under optimal environmental conditions. The pathogen readily infects young, developing foliage as leaflets are expanding.

[0368] Brown Spot

[0369] 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, andChina. 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.

[0370] 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 between infection to showing visible symptoms) of S. glycines has been reported to vary depending on host maturity.IX. Amino Acid Substitutions

[0371] The present disclosure provides resistant forms of a Lr34 protein, which may include of an amino acid at an amino acid position of the protein corresponding to amino acid 546 of SEQ ID NO: 7 and / or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid 634 of SEQ ID NO: 7, and wherein the heterologous promoter is an above-ground promoter. According to some embodiments, the protein comprises a deletion of a non-polar amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, the protein comprises an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine. According to some embodiments, the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a histidine. However, variations at other amino acid positions in a Lr34protein are also encompassed by the present disclosure. A resistant Lr34 protein may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, or at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115. According to some embodiments, a resistant Lr34 protein may comprise a deletion of a non-polar amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, a resistant Lr34 protein may comprise a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, a resistant Lr34 protein may comprise a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, a resistant Lr34 protein may comprise an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7. According to some embodiments, the tyrosine at an amino acid position of a resistant Lr34 protein corresponding to amino acid position 634 of SEQ ID NO: 7 may be replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine. According to some embodiments, the tyrosine at an amino acid position of a resistant Lr34 protein corresponding to amino acid position 634 of SEQ ID NO: 7 may be replaced with a histidine.

[0372] 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 havesubstantially the same functional properties as the naturally occurring counterpart sequences disclosed herein.

[0373] 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.

[0374] 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); glutamate / glutamine / aspartate / asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0375] As is known in the art, 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.

[0376] Likewise, 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.

[0377] 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, and lysine; (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.

[0378] 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 Lr34 proteins, and homologs thereof, with an amino acid substitution(s) can be produced by introducing changes into the DNA sequence encoding the Lr34 protein using methods known in the art and the information provided in Table 1.

[0379] 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. DNAsequences encoding Lr34 proteins or homologs thereof with the amino acid substitutions described herein can be produced by introducing mutations into the DNA sequence encoding a Lr34 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. As used herein, the term "codon-optimized" refers to a polynucleotide sequence that has been modified to exploit the codon usage bias of a particular plant. The modified polynucleotide sequence still encodes the same, or substantially similar polypeptide as the original sequence but uses codon nucleotide triplets that are found in greater frequency in a particular plant. Allelic variants of the nucleotide sequences encoding a protein disclosed herein are also encompassed within the scope of the embodiments described herein.Table 1: The universal genetic code chart shows all possible mRNA triplet codons (where Tin the DNA molecule is replaced by U in the RNA molecule) and the amino acid encoded by each codon (shown in 3-letter notation).X. Additional Definitions

[0380] 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.

[0381] 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 are 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.

[0382] 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.

[0383] 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%.

[0384] 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),

[0385] 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.

[0386] 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.

[0387] 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.

[0388] 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.

[0389] “Obtaining,” when used in conjunction with a transgenic plant cell or transgenic plant, obtaining means either transforming a non-transgenic 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 R0 transgenic plant or may be from a progeny of any generation thereof that inherits a given transgenic sequence from a starting transgenic parent plant.

[0390] 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 may be dicotyledonous, leguminous or soybean plants.

[0391] 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.

[0392] As used herein, the terms “RO transgenic plant” and “RO plant” refer to an initial regenerated transformant that is, a plant that has been genetically transformed or has been regenerated from a plant cell or cells that have been genetically transformed. As used herein, the terms “R1 transgenic seed” and “R1 seed” refer to seed produced from selfing RO transgenic plants or RO plants. As used herein, the terms “R1 transgenic plant” and “R1 plant” refers to a plant grown from R1 transgenic seed or R1 seed. As used herein, the terms “R2 transgenic seed” and “R2 seed” refer to seed produced from selfing R1 transgenic plants or R1 plants. As used herein, the terms “R2 transgenic plant” and “R2 plant” refer to a plant grown from R2 transgenic seed or R2 seed.

[0393] As used herein, the “vegetative phase” of plant development is the period of growth between germination and flowering. The stages in the vegetative phase of soybean are as follows: VE (emergence), VC (cotyledon stage), V 1 (first trifoliolate leaf), V2 (second trifoliolate leaf), V3 (third trifoliolate leaf), V(n) (nth trifoliolate leaf), and V6 (flowering will soon start). As used herein, the “reproductive phase” of plant development is the period between flowering and the end of harvest. The stages in the reproductive phase of soybean are as follows R1 (beginning bloom, first flower); R2 (full bloom, flower in top 2 nodes); R3 (beginning pod, 3 / 16" pod in top 4 nodes); R4 (full pod, 3 / 4" pod in top 4 nodes); R5 (1 / 8" seed in top 4 nodes); R6 (full size seed in top 4nodes); R7 (beginning maturity, one mature pod); and, R8 (full maturity, 95% of pods on the plant have reached mature color). Soybean vegetative and reproductive stages are well known to those of skill in the art and numerous publications describing these stages can be found on the world wide web and elsewhere, such as North Dakota State University publication A- 1174, June 1999, Reviewed and Reprinted August 2004.

[0394] 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.

[0395] As used herein, the term “regeneration” refers to the process of growing a plant from a plant cell (e.g., plant protoplast, callus or explant).

[0396] 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.

[0397] As used herein, the terms “terminator”, “transcription terminator” and “3’UTR” refer 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.

[0398] 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.

[0399] 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.

[0400] As used herein, “off-types” may refer to any undesirable phenotypes in plants, specifically in crop plants, for example, stunting (characterized by, for example, reduced growth) or chlorosis (characterized by, for example, yellowing of leaves due to insufficient chlorophyll production), as occasionally observed in some of the transgenic plants that express a transgene compared to the wild-type (WT) control plants that do not express the transgene. Additionally, undesirable phenotypes may include yield penalties (characterized by, for example, decrease in crop yield), reduced germination, negative transmission bias (characterized by, for example, a decreased likelihood that a transgene is passed on to the next generation) etc.EXAMPLES

[0401] 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 1Transformation constructs for expression of Ta. Lr34res transgene in soybean

[0402] Plant transformation vectors comprising transgene cassettes designed to express a wheat Lr34 resistant protein (also referred to as “Ta. Lr34res”) were used to stably transform soybean plants. A wheat coding sequence or codon optimized coding sequence for expression of a Ta. Lr34res protein in soybean was cloned into plant transformation vector constructs identified in Table 2 to allow for testing of the Ta. Lr34res protein expressed in soybean with different promoters and expression elements. The transformation vector constructs comprised a first transgene cassette for expression of the Ta. Lr34res 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.

[0403] Table 2 provides a list of constructs with different promoters for different expression patterns of the protein-coding wheat Lr34res 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. Lr34res in soybean.

[0404] A coding sequence of the Ta. Lr34res gene from wheat is provided as SEQ ID NO:1, three codon optimized sequences for expressing the Ta. Lr34res protein in soybean are provided as SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, the amino acid sequence for the Ta. Lr34res protein used on these experiments is provided as SEQ ID NO: 6, and the genomic DNA sequence for the Ta. Lr34res gene in wheat is provided as SEQ ID NO: 8. The coding sequence of a wildtype susceptible Ta. Lr34 gene from wheat is provided as SEQ ID NO: 5, and the amino acid sequence for the wild-type susceptible Lr34 protein is provided as SEQ ID NO: 7. The wild-type Ta. Lr34 gene from wheat may also be referred to as a disease- susceptible gene sequence.

[0405] Meristem explants excised from soybean seeds were transformed with each of the transformation constructs in Table 2 via AgroZ ctenum-mediated transformation. Transgenic RO plants were regenerated following spectinomycin selection and self-pollinated to produce R1 plants. On average, eight events were selected per construct for controlled environment efficacy screening. Eight R1 seeds per event were also planted in controlled environment and sampled at the VI 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

[0406] This example describes the controlled environment (CE) pathogen assay used in these experiments for evaluating ASR disease susceptibility or resistance of soybean plants stablytransformed with the Ta. Lr34res transgenes relative to the resistance of control plants. However, efficacy testing data is not provided for the pM385 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:3) and promoter P-At. GSP571.nno:5 (SEQ ID NO:9)) construct. ASR disease resistance efficacy evaluations were performed in one or two controlled environment settings (CE-1 and / or CE-2). CE-1 refers to controlled environment tests conducted in the United States, and CE-2 refers to controlled environment tests conducted in Brazil.

[0407] 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.

[0408] Plant inoculation and pathogen assay. For transgenic events from each of the tested constructs in Table 2, R1 transgenic seeds were planted into 2.5 inch pots. Seven or eight R1 plants for each Ta. Lr34res 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. Lr34res 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.

[0409] To evaluate the efficacy of transgenic plants expressing the Ta. Lr34res 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%. A lower percentage rating corresponds to a lower disease severity, which consequently corresponds to a higher efficacy of the examined plants against ASR disease. 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 a fertilization from Monday to Friday (i.e., on five consecutive days) each week during the entire duration of the pathogen assay. 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.

[0410] Resistance efficacy trial results: Transgenic soybean plants expressing the Ta. Lr34res transgene under the control of different promoters and expression elements were evaluated in CE against the fungal pathogens after inoculation at V2 stage as described above. Multiple transformation events were generated with each of these constructs (e.g., three 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. 1-9 and Tables 3-11 for each event, WT and Empty Vector controls for each construct). 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).

[0411] Table 3 and FIG. 1 present the disease efficacy and / or gene expression for each event transformed with construct pM383 (comprising the coding sequence of wheat Lr34 resistant gene (SEQ ID NO:1) and promoter P-At. GSP571.nno:5 (SEQ ID NO:9)); Table 4 and FIG. 2 present the disease efficacy and / or gene expression for each event transformed with construct pM384 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-At. GSP571,nno:5 (SEQ ID NO:9)); Table 5 and FIG. 3 present the disease efficacy and / or gene expression for each event transformed with construct pM386 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:4) and promoter P-At. GSP571.nno:5 (SEQ ID N0:9)); Table 6 and FIG. 4 present the disease efficacy and / or gene expression for each event transformed with construct pM499 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l3G046200:2 (SEQ ID NO: 10)); Table 7 and FIG. 5 present the disease efficacy and / or gene expression for each event transformed with construct pM524 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_l:l (SEQ ID NO: 12)); Table 8 and FIG. 6 present the disease efficacy and / or gene expression for each event transformed with construct pM525 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_2:l (SEQ ID NO: 13)); Table 9 and FIG. 7 present the disease efficacy and / or gene expression for each event transformed with construct pM526 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l7G020600:l (SEQ ID NO: 14)); Table 10 and FIG. 8 present the disease efficacy and / or gene expression for each event transformed with construct pM527 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.02G101100:1 (SEQ ID NO: 15)); and Table 11 and FIG. 9 present the disease efficacy and / or gene expression for each event transformed with construct pM679 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. PSIEl (SEQ ID NO: 11)).

[0412] The average results for each event per construct were further averaged across all events for each construct as summarized in Table 12 for CE-1. 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 (P) measurement is the statistical probability of a Type II error in any hypothesis test (incorrectly failing to reject the null hypothesis).Table 3. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM383 (comprising the coding sequence of wheat Lr34 resistant gene (SEQ ID NO:1) and promoter P-At. GSP571.nno:5 (SEQ ID NO:9)).Table 4. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM384 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-At. GSP571.nno:5 (SEQ ID NO:9)).Table 5. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM386 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:4) and promoter P-At. GSP571.nno:5 (SEQ ID NO:9)).Table 6. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM499 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l3G046200:2 (SEQ ID NO: 10)).Table 7. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM524 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_l:l (SEQ ID NO: 12)).Table 8. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM525 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_2:l (SEQ ID NO: 13)).Table 9. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM526 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l7G020600:l (SEQ ID NO: 14)).Table 10. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM527 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.02G101100:1 (SEQ ID NO: 15)).Table 11. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM679 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. PSII: 1 (SEQ ID NO: 11)).

[0413] The expression of a Ta. Lr34 transgene was measured using primers specific for Lr34 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. Lr34 mRNA transcripts were calculated by normalizing to the geometric mean of Glycine max polyadenylate-binding protein RBP45 and Glycine max casein kinase II subunit alpha- 1 -like reference genes.

[0414] Depending on the type and level of expression, transgenic plants expressing the Ta. Lr34res 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. Lr34res to achieve efficacy as illustrated, for example, in FIGs. 1-9.

[0415] The data from this example indicate that expression of Ta. Lr34res transgene using constitutive promoters (pM383, pM384, pM386) were effective at reducing disease severity with varying levels of efficacy as shown in FIG. 1-3 and Tables 3-5 respectively. Plants transformed with the construct pM383 (comprising the coding sequence of wheat Lr34 resistant gene (SEQ ID NO:1) and promoter P-At. GSP571.nno:5 (SEQ ID NO:9)) expressing the wheat Lr34res protein in this experiment showed at least some activity across all 8 events (FIG. 1; Table 3). Plants transformed with the construct pM384 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-At. GSP571.nno:5 (SEQ ID NO:9)) were effective at reducing disease severity with varying levels of efficacy across all 7 events (FIG. 2; Table 4). The Event-4 originating from plants transformed with construct pM384, demonstrated high levels of disease efficacy compared to the controls. Similarly, plants transformed with the construct pM386 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:4) and promoter P-At. GSP571.nno:5 (SEQ ID NO:9)) were effective at reducing disease severity with varying levels of efficacy across all 8 events (FIG. 3; Table 5). The Event-4 originating from plants transformed with construct pM386, demonstrated very high levels of efficacy compared to the controls.

[0416] This example further indicates that expression of Ta. Lr34res transgene under the control of a promoter having high above-ground expression in leaf tissues (pM499) was highly effective at providing resistance against the pathogen. As can be seen in FIG. 4 and Table 6, all 4 tested events from the construct pM499 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l3G046200:2 (SEQ ID NO: 10)) showed high levels of efficacy in soybean plants against the tested fungal pathogen.

[0417] As shown in FIG. 5 and Table 7, expression of Ta. Lr34res gene under the control of a promoter having high above-ground expression in leaf tissues (pM524) resulted in high levels of efficacy in this experiment. Almost all 17 tested events from the construct pM524 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_l: 1 (SEQ ID NO: 12)) in two sets of experiments showed high levels of efficacy against the tested fungal pathogen.

[0418] As shown in FIG. 6 and Table 8, expression of Ta. Lr34res transgene under the control of a promoter having high above-ground expression in leaf tissues of the plant (pM525) was effective at reducing disease severity. All 4 tested events from the pM525 construct (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_2:l (SEQ ID NO: 13)) in this experiment showed high levels of efficacy against the tested fungal pathogen.

[0419] As shown in FIG. 7 and Table 9, expression of Ta. Lr34res transgene under the control of a promoter having medium above-ground expression in leaf tissues (pM526) was less effective at providing resistance against tested pathogen. The percent disease severity with pM526 construct (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l7G020600:l (SEQ ID NO: 14)) was mostly neutral between transgenic events and the controls.

[0420] As shown in FIG. 8 and Table 10, expression of Ta. Lr34res transgene under the control of a promoter providing medium above-ground expression in leaf tissues of the plant was neutral or less effective at reducing disease severity across the three events of the pM527 construct (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.02G101100:1 (SEQ ID NO: 15)).

[0421] As shown in FIG. 9 and Table 11, expression of Ta. Lr34res gene under the control of a promoter driving medium above-ground expression in leaf tissues (pM679) resulted in high levels of efficacy in this experiment. All 8 tested events from the construct pM679 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. PSII:l (SEQ ID NO: 11)) in this experiment showed medium to high levels of efficacy against the tested fungal pathogen.Table 12: Summary of observations of transgenic plants with various constructs and promoters for expression of the Ta. Lr34res transgene.

[0422] 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. Lr34res transgenes relative to the resistance of WT plants.

[0423] 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.

[0424] To evaluate the efficacy of transgenic plants expressing the Ta. Lr34res 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%.

[0425] Transgenic soybean plants expressing the Ta. Lr34res 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 (6 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. 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).

[0426] Table 13 and FIG. 10 present the disease efficacy data for each event transformed with construct pM499 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l3G046200:2 (SEQ ID NO: 10)); Table 14 and FIG. 11 present the disease efficacy data for each event transformed with construct pM524 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_l:l (SEQ ID NO: 12)); Table 15 and FIG. 12 present the disease efficacy data for each event transformed with construct pM525 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_2:l (SEQ ID NO: 13)); Table 16 and FIG. 13 present the disease efficacy data for each event transformed with construct pM526(comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l7G020600:l (SEQ ID NO: 14)); Table 17 and FIG. 14 present the disease efficacy data for each event transformed with construct pM527 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.02G101100: 1 (SEQ ID NO: 15)); and Table 18 and FIG. 15 present the disease efficacy data for each event transformed with construct pM679 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. PSII:l (SEQ ID NO: 11)).

[0427] The average results for each event per construct were further averaged across all events for each construct as summarized in Table 19 for CE-2. 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).Table 13. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM499 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l3G046200:2 (SEQ ID NO: 10)).Table 14. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM524 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_l:l (SEQ ID NO: 12)).Table 15. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM525 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_2:l (SEQ ID NO: 13)).Table 16. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM526 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l7G020600:l (SEQ ID NO: 14)).Table 17. Transgenic soybean plants expressing Ta. Lr34res transgene from construct pM527 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.02G101100:1 (SEQ ID NO: 15)).Table 18. Transgenic soybean plants expressing Ta.34res transgene from construct pM679 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. PSII: 1 (SEQ ID NO: 11)).

[0428] As shown in FIG. 10 and Table 13, plants transformed with Ta. Lr34res gene under the control of a promoter providing high above-ground expression in leaf tissues (pM499) was highly effective at reducing disease severity in CE-2. All 8 tested events from the construct pM499 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l3G046200:2 (SEQ ID NO: 10)) in this experiment showed high levels of efficacy against the tested fungal pathogen.

[0429] As shown in FIG. 11 and Table 14, plants transformed with Ta. Lr34res gene under the control of a promoter providing high above-ground expression in leaf tissues (pM524) resulted in high levels of efficacy in this experiment in CE-2. All 10 tested events from the construct pM524 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_l:l (SEQ ID NO: 12)) in this experiment showed high levels of efficacy against the tested fungal pathogen.

[0430] As shown in FIG. 12 and Table 15, plants transformed with Ta. Lr34res transgene under the control of a promoter providing high above-ground expression in leaf tissues of the plant (pM525) was effective at reducing disease severity in CE-2. All 8 tested events from the construct pM525 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. Cba_2:l (SEQ ID NO: 13)) in this experiment showed high levels of efficacy against the tested fungal pathogen.

[0431] As shown in FIG. 13 and Table 16, plants transformed with Ta. Lr34res transgene under the control of a promoter providing medium above-ground expression in leaf tissues (pM526) showed medium or low level of resistance against tested pathogen in CE-2. The Event-3 originating from plants transformed with the construct pM526 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.l7G020600:l (SEQ ID NO: 14)), demonstrated medium levels of disease efficacy compared to the controls.

[0432] As shown in FIG. 14 and Table 17, plants transformed with Ta. Lr34res transgene under the control of a promoter providing medium above-ground expression in leaf tissues of the plant (pM527) resulted in high levels of efficacy in this experiment in CE-2. All 6 tested events transformed with construct pM527 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm.02G 101100:1 (SEQ ID NO: 15)) in this experiment showed high levels of efficacy against the fungal pathogen.

[0433] As shown in FIG. 15 and Table 18, plants transformed with Ta. Lr34res gene under the control of a promoter providing medium above-ground expression in leaf tissues (pM679) resulted in high levels of efficacy in this experiment in CE-2. All ten tested events from the construct pM679 (comprising the codon optimized coding sequence of wheat Lr34 resistant gene (SEQ ID NO:2) and promoter P-Gm. PSII:l (SEQ ID NO: 11)) in this experiment showed very high levels of efficacy against the tested fungal pathogen.Table 19. Summary of observations of transgenic plants with various constructs and promoters for expression of the Ta. Lr34res transgene evaluated in CE-2.

[0434] Depending on the construct and expression pattern, transgenic plants expressing Ta. Lr34res gene showed high level of disease resistance. These results indicate that the transgenic soybean plants comprising a wheat Ta. Lr34res transgene (SEQ ID NO:2) demonstrated significant disease efficacy against at least two different isolates of P. pachyrhizi.Example 3Identification of the Lr34 homologs

[0435] This example provides the identification of homologs from other plant species of the wild-type wheat (disease susceptible) Ta. Lr34 amino acid sequence (SEQ ID NO: 7). Relative to said wild-type wheat Ta. Lr34 amino acid sequence as described herein, the Ta. Lr34res (SEQ ID NO: 6) amino acid sequence lacks a phenylalanine residue at a position corresponding to amino acid number or position 546 of SEQ ID NO: 7 (also referred to as “F546”) and has a histidine (H) in place of a tyrosine (Y) residue at a position corresponding to amino acid number or position 634 of SEQ ID NO: 7 (also referred to as “Y634H”). However, other amino acid differences may exist between various Lr34 protein homologs and between ASR disease resistant and / or susceptible versions of Lr34 proteins, and resistance Lr34 proteins may comprise a deletion of a different amino acid at an amino acid position of the protein corresponding to amino acid 546 of SEQ IDNO: 7 and / or may comprise a substitution of a different amino acid at an amino acid position of the protein corresponding to amino acid 634 of SEQ ID NO: 7. Thus, the Ta. Lr34 protein homologs identified in this example may be similarly modified to have a deletion at the amino acid position of the protein homolog corresponding to amino acid 546 of SEQ ID NO: 7, and / or a substitution of an amino acid at an amino acid position of the protein homolog corresponding to amino acid 634 of SEQ ID NO: 7. According to some embodiments, a modified Lr34 protein homolog comprises a deletion of a non-polar amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, a modified Lr34 protein homolog comprises a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, a modified Lr34 protein homolog comprises a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. According to some embodiments, a modified Lr34 protein homolog comprises an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7. According to some embodiments, the tyrosine at an amino acid position of a modified Lr34 protein homolog corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine. According to some embodiments, the tyrosine at an amino acid position of a modified Lr34 protein homolog corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a histidine. The amino acid positions for mutations in the protein homolog sequences identified in these examples corresponding to the F546 and / or Y634H mutations, for example, which are present in the Ta. Lr34res protein, as well as any other mutations that may be made at these and other corresponding positions, can be identified by sequence alignment, and transgenes encoding mutant versions of the Ta. Lr34 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 as described herein.

[0436] Lr34 homologs were identified using NCBI Basic Local Alignment Search Tool (BLAST). The NCBI Non-redundant protein sequences (nr) database was queried based on the wild-type wheat Ta. Lr34 protein sequence (SEQ ID NO: 7) 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 %Sequence Identity, and separated byorganism (genus / species) names. The Multiple Sequence Comparison by Log-Expectation (MUSCLE) sequence alignment software tool was used to generate an alignment of a subset of the amino acid sequences of the Ta. Lr34 protein homologs from various other species along with the wild-type Ta. Lr34 protein sequence (SEQ ID NO: 7) and inter alia disease resistant Ta. Lr34 protein sequences (ACL36480.1, XP_044442862.1, XP.044442863.1, XP_044442860.1; see also SEQ ID NO: 6) from wheat (alignment not shown). A phenylalanine or other non-polar residue, such as leucine, at a position corresponding to amino acid number 546 of the Lr34res protein and a tyrosine at position 634 of the wild-type Lr34res protein are generally conserved among the homologs, but are deleted or substituted in the Ta. Lr34res proteins. For example, the protein sequences ACL36480.1 (SEQ ID NO:17), XP_044442862.1 (SEQ ID NO: 18), XP_044442863.1 (SEQ ID NO: 19), and XP_044442860.1 (SEQ ID NO: 20) are homologous to the Ta. Lr34 protein and comprise a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7. In addition, XP_044442862.1 (SEQ ID NO: 18), XP_044442863.1 (SEQ ID NO: 19), and XP_044442860.1 (SEQ ID NO: 20) have a substitution of a tyrosine for a histidine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7.

[0437] Table 20 provides a list of one-hundred (100) Ta. Lr34 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 the scientific name (e.g., genus / species) of the source organism for the homolog protein / gene; and “Description” provides either the protein name or a short functional description of the protein encoded by the identified gene homolog. Table 21 provides the percent sequence identity and percent homolog and query coverage for each protein homolog listed in Table 20 relative to the wild-type Ta. Lr34 protein (query) sequence.Table 20. Ta. Lr34 Homolog protein and gene informationTable 21. List of homologs showing the percent alignment identity and coverage for each protein homolog relative to the wild-type Ta. Lr34 protein (query) sequence (SEQ ID NO: 7).

[0438] 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 art 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 are 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.EMBODIMENTS

[0439] For further illustration, additional non-limiting embodiments of the present disclosure are set forth below.

[0440] Embodiment 1 is a recombinant DNA construct comprising a nucleotide coding sequence operably linked to a heterologous promoter, wherein the nucleotide coding sequenceencodes a protein with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115, wherein the protein comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7 or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7, and wherein the heterologous promoter is a constitutive or above-ground promoter.

[0441] Embodiment 2 is the recombinant DNA construct of embodiment 1, wherein the protein comprises a deletion of a non-polar amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7.

[0442] Embodiment 3 is the recombinant DNA construct of embodiment 1 or 2, wherein the protein comprises a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7.

[0443] Embodiment 4 is the recombinant DNA construct of embodiment 1 or 2, wherein the protein comprises a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7.

[0444] Embodiment 5 is the recombinant DNA construct of any one of embodiments 1-4, the protein comprises an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7.

[0445] Embodiment 6 is the recombinant DNA construct of embodiment 5, wherein the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine.

[0446] Embodiment 7 is the recombinant DNA construct of embodiment 5 or 6, wherein the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a histidine.

[0447] Embodiment 8 is the recombinant DNA construct of any one of embodiments 1-7, wherein the nucleotide coding sequence of the recombinant DNA construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 8, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, or 215.

[0448] Embodiment 9 is the recombinant DNA construct of any one of embodiments 1-8, wherein the nucleotide coding sequence of the recombinant DNA construct encodes a protein having the amino acid sequence of SEQ ID NO: 6.

[0449] Embodiment 10 is the recombinant DNA construct of any one of embodiments 1-9, wherein the nucleotide coding sequence of the recombinant DNA construct comprises the nucleotide sequence of SEQ ID NO: 1, 2, 3, or 4.

[0450] Embodiment 11 is the recombinant DNA construct of any one of embodiments 1-10, wherein the protein encoded by the nucleotide coding sequence comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7 and a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7.

[0451] Embodiment 12 is the recombinant DNA construct of any one of embodiments 1-11, wherein the heterologous promoter is an above-ground promoter.

[0452] Embodiment 13 is the recombinant DNA construct of any one of embodiments 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: 9-15 and 216-226.

[0453] Embodiment 14 is the recombinant DNA construct of any one of embodiments 1-13, wherein the heterologous promoter comprises a nucleotide sequence that is 100% identical to one or more of SEQ ID NOs: 9-15 and 216-226.

[0454] Embodiment 15 is the recombinant DNA construct of any one of embodiments 1-14, wherein 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.

[0455] Embodiment 16 is the recombinant DNA construct of any one of embodiments 1-15, wherein 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.

[0456] Embodiment 17 is the recombinant DNA construct of any one of embodiments 1-16, wherein the heterologous promoter comprises a fragment of one of SEQ ID NOs: 9-15 and 216-226, wherein the fragment has promoter activity as a constitutive or above-ground promoter.

[0457] Embodiment 18 is the recombinant DNA construct of any one of embodiments 1-17, wherein the heterologous promoter fragment has promoter activity as an above-ground promoter.

[0458] Embodiment 19 is a DNA molecule comprising the recombinant DNA construct of any one of embodiments 1-18.

[0459] Embodiment 20 is a DNA plasmid or vector comprising the recombinant DNA construct of any one of embodiments 1-18.

[0460] Embodiment 21 is a transgenic plant comprising the recombinant DNA construct of any one of embodiments 1-18, wherein the plant has increased resistance to a fungal disease compared to a control plant lacking the recombinant DNA construct.

[0461] Embodiment 22 is the transgenic plant of embodiment 21, wherein the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, frogeye leaf spot, target spot and / or brown spot.

[0462] Embodiment 23 is the transgenic plant of embodiment 21 or 22, wherein the transgenic plant is a dicotyledonous plant.

[0463] Embodiment 24 is the transgenic plant of embodiment 23, wherein the transgenic plant is a leguminous plant.

[0464] Embodiment 25 is the transgenic plant of embodiment 24, wherein the transgenic plant is a soybean plant.

[0465] Embodiment 26 is a transgenic plant part of the transgenic plant of any one of embodiments 21-25.

[0466] Embodiment 27 is the transgenic plant part of embodiment 26, wherein the transgenic plant part is a seed, a root, a stem, a leaf, a flower, an embryo, or a meristem.

[0467] Embodiment 28 is the transgenic plant part of embodiment 27, wherein the transgenic plant part is a seed.

[0468] Embodiment 29 is 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 any one of embodiments 1-18, and regenerating or developing the transgenic plant from the explant.

[0469] Embodiment 30 is the method of embodiment 29, wherein the recombinant DNA construct 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.

[0470] Embodiment 31 is the method of embodiment 29 or 30, wherein the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, frogeye leaf spot, target spot or brown spot.

[0471] Embodiment 32 is the method of any one of embodiments 29-31, wherein the plant is a dicotyledonous plant.

[0472] Embodiment 33 is the method of embodiment 32, wherein the plant is a leguminous plant.

[0473] Embodiment 34 is the method of embodiment 33, wherein the plant is a soybean plant.

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%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, 7, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115, wherein the protein comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7 and / or a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7, and wherein the heterologous promoter is a constitutive or above-ground promoter.

2. The recombinant DNA construct of claim 1, wherein the protein comprises a deletion of a non-polar amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7.

3. The recombinant DNA construct of claim 1, wherein the protein comprises(i) a deletion of a phenylalanine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7, or(ii) a deletion of a leucine at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7.

4. The recombinant DNA construct of claim 1, wherein the protein comprises an amino acid substitution of a tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7.

5. The recombinant DNA construct of claim 4, wherein the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a basic or positively charged amino acid, such as histidine, lysine or arginine.

6. The recombinant DNA construct of claim 5, wherein the tyrosine at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7 is replaced with a histidine.

7. The recombinant DNA construct of claim 1, wherein the nucleotide coding sequence of the recombinant DNA construct has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 8, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, or 215.

8. The recombinant DNA construct of claim 1, wherein the nucleotide coding sequence of the recombinant DNA construct encodes a protein having the amino acid sequence of SEQ ID NO: 6.

9. The recombinant DNA construct of claim 7, wherein the nucleotide coding sequence of the recombinant DNA construct comprises the nucleotide sequence of SEQ ID NO: 1, 2, 3, or 4.

10. The recombinant DNA construct of claim 1, wherein the protein encoded by the nucleotide coding sequence comprises a deletion of an amino acid at an amino acid position of the protein corresponding to amino acid position 546 of SEQ ID NO: 7 and a substitution of an amino acid at an amino acid position of the protein corresponding to amino acid position 634 of SEQ ID NO: 7.

11. The recombinant DNA construct of claim 1, wherein(i) the heterologous promoter is an above-ground promoter, or(ii) 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: 9-15 and 216-226, or(iii) the heterologous promoter comprises a nucleotide sequence that is 100% identical to one or more of SEQ ID NOs: 9-15 and 216-226.

12. The recombinant DNA construct of claim 1, wherein the promoter is an above-ground promoter andIll(i) wherein said 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, or(ii) wherein said 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.

13. The recombinant DNA construct of claim 1, wherein the heterologous promoter comprises a fragment of any one of SEQ ID NOs: 9-15 and 216-226, wherein the fragment has promoter activity as a constitutive or above-ground promoter.

14. A DNA plasmid or vector comprising the recombinant DNA construct of claim 1.

15. A transgenic plant, plant part, or seed 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.

16. The transgenic plant of claim 15, wherein the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, frogeye leaf spot, target spot and / or brown spot.

17. The transgenic plant of claiml5, wherein the transgenic plant is a dicotyledonous plant, or a leguminous plant, or a soybean plant.

18. 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.

19. The method of claim 18, wherein the fungal disease is Asian Soybean Rust (ASR), powdery mildew, white mold, frogeye leaf spot, target spot or brown spot.

20. The method of claim 19, wherein the plant is a dicotyledonous plant, or a leguminous plant, or a soybean plant.

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