WiseR_SOW_EMSA V12 CleanV3.doc

Wise Y1H/EMSA Project V12:

Barley transcription factors that activate disease defense to destructive pathogens

GENERAL INFORMATION

This requirement is critical to the mission of the congressionally appropriated USDA Barley Pest Initiative, which serves to manage 20 major barley diseases and insect pests across 15 states for the benefit of growers and producers, resulting in an affordable food supply for American consumers.

1.0 Scope of Work:

ADDIN EN.CITE

· A predicted barley gene regulatory network (GRN) has provided a list of transcription factor (TF) candidates and their role in disease resistance of barley to the powdery mildew pathogen, Blumeria hordei. Four of these TFs [a NAC domain protein, a MADS box protein, a homeobox (HB) protein, and a WRKY2 protein] were selected for further analysis, and affinity purified antibodies were created for each by PhytoAB. We utilized chromatin immunoprecipitation sequencing (ChIP-Seq) to test the differential binding of these TFs in two barley lines: CI 16151 (Mla6), which is resistant to the powdery mildew fungus, and it’s fast-neutron derived susceptible mutant, mla6-m18982. We now require yeast 1-hybrid (Y1H) and gel electrophoretic mobility shift assays (EMSA) to test specific DNA binding site(s) in the promoter regions of a series of TF in the barley GRN.

Following are the general tests we will need: SEE APPENDIX A for detailed sequences of Mla6/Nac promoters and NAC, MADS, HB, and WRKY2 transcription factors (TF)

· Summary of tests:

1. Test the NAC, MADS, HB, and WRKY2 TF for binding to the 575 bp Mla6 and 500 bp Nac promoters with yeast 1-hybrid (Y1H) (4 x 2 = 8 confirmatory assays)

2. Test promoter-TF interactions with specific overlapping 60-70 bp probes selected from the 575 bp Mla6 and 500 bp Nac promoters in electrophoretic mobility shift assays (EMSA)

3. Use competitive assays combined with mutational analysis to test the positive promoter-TF interactions from Step #2 to validate specificity in electrophoretic mobility shift assays (EMSA)

· To validate initial TF protein-DNA binding, vendor will perform Y1H assays optimized for Eukaryotic (specifically plant) proteins.

1. Vendor will use plant optimized protein expression systems, such as Nicotiana benthamiana, to generate and purify NAC, MADS, HB, and WRKY2 transcription factors (TF) from the DNA/amino acid sequence provided by client (SEE APPENDIX A).

2. Vendor will synthesize Mla6 promoter (575 bp) and Nac promoter (500 bp) into Y1H vector for testing with Y1H assays with NAC, MADS, HB, and WRKY2 TF, SEE APPENDIX A.

3. Vendor will synthesize specific overlapping 60-70 bp probes selected from the 575 bp Mla6 and 500 bp Nac promoters to test promoter : NAC, MADS, HB, and WRKY2 TF interactions in electrophoretic mobility shift assays (EMSA).

4. Vendor will use positive interactions from step #3 to test TF protein-DNA binding in competitive assays with mutant /cold 60-70 bp probe concentration gradients to understand the specificity/stoichiometry of TF protein-DNA binding.

a. Binding probe: Biotin-labeled probe that binds to the target protein.

b. Mutant probe: Biotin-labeled probe with mutations that don't bind to the target protein.

c. and cold competitor probe: Unlabeled probe with the same sequence as the binding probe, which competes with the binding probe for protein binding.

5. Gel Electrophoresis/Autoradiography

6. Based on the above results and consultation with the client, vendor will perform bioinformative analysis and deliver full report.

2.0 Background:

Transcriptional reprogramming is a key component of disease resistance in all organisms. The barley MLA immune receptor interacts with several transcription factors (TFs) and thus, represents a model to explore the regulation of nucleotide binding, leucine rich repeat (NLR) receptors during disease defense. Previous work presented evidence of an interconnection between MLA-signaling and transcriptional reprogramming. Using time-course RNA-Sequencing data, a gene regulatory network (GRN) of barley disease defense was reconstructed. Functional validation of these transcriptional relationships will reveal novel mechanisms of the immune receptor and disease resistance in crops. Experimental approaches to validate these TF-target associations include yeast 1-hybrid (Y1H) and gel Electrophoretic Mobility Shift Assays (EMSA) to detect protein complexes with nucleic acids. These are core technologies underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems.

VENDOR REQUIREMENTS

3.0 Technical Requirements/Tasks:

· Vendor’s personnel must have expertise in yeast 1-hybrid (Y1H) and electrophoretic mobility shift assays (EMSA) and bioinformatic analysis to identify and validate the specific target / binding site(s) of each transcription factor.

· The vendor should offer expertise in expression of protein in Nicotiana benthamiana, or equivalent eukaryotic systems to produce EMSA quality plant protein. The (nuclear) protein isolation, synthesis, DNA binding, and electrophoretic mobility shift assays are key steps in the success of the project and require demonstrated expertise. Required Y1H and EMSA includes a full service starting with expressed transcription factor proteins to be used in Step 1: Y1H assays with NAC, MADS, HB, and WRKY2 transcription factors (TF) for the each of the 575 bp Mla6 and 500 bp Nac promoters to confirm TF-promoter binding. Step 2: Test the promoter-TF interactions with overlapping 60-70 bp probe predictions for EMSA (see Appendix A). Step 3: Competitive assays with mutant/cold probe concentration gradients to validate specificity and understand the stoichiometry of TF protein-DNA binding will be performed on the positive probes from Step 2 (see Appendix A).

· All original Y1H yeast colony and EMSA gel images must be retained for use in publication by client.

· Vendor must be then able to identify the specific identity of the target(s), including bioinformatics analysis.

· Vendor should consult with USDA-ARS client and provide expert analysis of differential TF binding between and within CI 16151 (Mla6), which is resistant to the powdery mildew fungus, and it’s susceptible derived mutant mla6-m18982.

4.0 Government Furnished:

· Government will furnish DNA sequence of 575 bp Mla6- and 500 bp Nac promoters.

· Government will furnish DNA and amino acid sequence corresponding to 4 transcription factors (NAC, MADS, HB, and WRKY2; see APPENDIX A).

5.0 Deliverables / Schedule:

Required parts of the base project include Yeast 1-Hybrid (Y1H) and gel Electrophoretic Mobility Shift Assays (EMSA) to detect protein complexes with nucleic acids, which control gene expression.

Polyacrylamide Gel Electrophoresis visualization of mobility shifts Required to compare the samples and find genomic regions of interest

Needed for documentation and publication 1-2 weeks after Item #4; EMSA tests

We anticipate this to take 6 months, not to exceed 8 months.

6.0 Travel: N/A

7.0 Vendor’s Key Personnel:

· Vendors personnel must have expertise in yeast 1-hybrid (Y1H) and electrophoretic mobility shift assays (EMSA) and bioinformatic analysis to identify and validate the specific target / binding site(s) of each transcription factor, including EMSA quality nuclear protein isolation and purification. The nuclear protein isolation and immunoprecipitation are key steps in the success of the project and require demonstrated expertise.

8.0 Security Requirements: N/A

9.0 Data Rights: All material and date are the sole property of USDA-ARS Project 5030-21220-068-000-D.

10.0 Section 508 – Electronic and Information Technology Standards: N/A

Wise Y1H/EMSA Project V12:

Barley transcription factors that activate disease defense to destructive pathogens

APPENDIX A

Mla6 promoter (575 bp):

GTCATTGGCACCTCACCTTCTGTCTCTCTCGTTAAATTTGTATCGATATATAAGTGCTTTTGAGTACTTGCATATATAAGTGCTTTTGGATCTAAAAAGTTATTAGTTTTCATGCTTAAGTATCTGATCAATTTGCGGTGGTAGTGGCATCTTTCTTGCTATTCTGCTCTAATGAAATCTTTCACGTCCACACGTTCTTGTTATAGATCTGCTGATTTGCTTAGATTATAAGTTCTTCTTATTCTTCCAGATCGATTGGAGCGACCCTCACGCCTCTGGTGCGCCGTCGCTGTGTTCTGCTCCGCCGTGAAGAATCAAGGTGGGCTTGGTCCAGATCTAGCTAAGCTTTAATTTCGCAGCTTGTTCAAGGCTTCACACAATTTGGATTGCGTTACAGCTCCCTTTATTCATCAATTTACAGGCTTCCAGCTGATTGATACGGAGATCTCGTCCCTCCTGCTCTCATGGATATTGTCACCGGTGCCATTTCCAACCTGATTCCCAAGTTGGGGGAGCTGCTCACGGAGGAGTTCAAGCTGCACAAGGGTGTCAAGAAAAATATTGAGGACCTCGGG CAAGGC: predicted NAC motif:

ATG: start codon

Highlighted region identified with Chip-Seq for NAC

Predicted motifs for NAC protein on the Mla6 promoter (4) (Probe name=Promoter + Transcription factor) Mla6-NAC-1: TGCTGATTTGCTTAGATTATAAGTTCTTCTTAT https://jaspar.elixir.no/matrix/MA0938.3/ Mla6-NAC-2: TGGAGCGACCCTCACGCCTCTGGTGCGCCGTC https://jaspar.elixir.no/matrix/MA0935.1/ Mla6-NAC-3: TTCGCAGCTTGTTCAAGGCTTCACACAATTTG https://jaspar.elixir.no/matrix/MA2018.1/ Mla6-NAC-4: CACACAATTTGGATTGCGTTACAGCTCCCTTT https://jaspar.elixir.no/matrix/MA1045.1/ Reference: NAC TFs regulate many target genes by binding to the CATGTG motif in the promoter region https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2013.00248/full

Predicted motifs for MADS box on the Mla6 promoter (2)

Mla6-MADS-1: CGTCCACACGTTCTTGTTATAGATCTGCTGATTT https://jaspar.elixir.no/matrix/MA0497.2/ Mla6-MADS-2: GGTGGGCTTGGTCCAGATCTAGCTAAGCTTTAAT https://jaspar.elixir.no/matrix/MA0555.1/ Predicted motifs for HB on the Mla6 promoter (3) Mla6-HB-1: TAAAAGTTATTAGTTTTCATGCTTAAGTATCTGATCA https://jaspar.elixir.no/search?q=HD-ZIP&collection=all&tax_group=all&tax_id=all&type=all&class=all&family=all&version=all Mla6-HB-2: GCTATTCTGCTCTAATGAAATCTTTCACGT https://jaspar.elixir.no/matrix/MA0008.2/ Mla6-HB-3: TCTAGCTAAGCTTTAATTTCGCAGCTTGTT https://jaspar.elixir.no/matrix/MA1327.3/ No corresponding motif was found on the Mla6 promoter for WRKY ***Extended probes to be synthesized by vendor 5’ -> 3’ (Probe design can be modified as required by the client.)

Probes designed for NAC (and MADS) on the Mla6 promoter (3) Mla6-NAC-V1 & Mla6-MADS-V1:

AATCTTTCACGTCCACACGTTCTTGTTATAGATCTGCTGATTTGCTTAGATTATAAGTTCTTCTTATTCT (position 176-245 / 70bp) Mla6-NAC-V1mut & Mla6-MADS-V1： AATCTTTCACGTCCACACGTTCTTGTTATAGATCTGCTGATTTGaTTAGATTATAAtTTCTTCTTATTCT

Mla6-NAC-V1 & Mla6-MADS-V1 mut： AATCTTTCACGTCCACACGTTaTTGTTATAtATCTGCTGATTTGCTTAGATTATAAGTTCTTCTTATTCT Mla6-NAC-V2 TCTTCCAGATCGATTGGAGCGACCCTCACGCCTCTGGTGCGCCGTCGCTGTGTTCTGCTC (position 243-302 / 60bp) Mla6-NAC-V2mut： TCTTCCAGATCGATTGGAGCGACCCTCAttaaTCTGGTGCGCCGTCGCTGTGTTCTGCTC

Mla6-NAC-V3&V4

CTAAGCTTTAATTTCGCAGCTTGTTCAAGGCTTCACACAATTTGGATTGCGTTACAGCTCCCTTTATTC (position 341-409 / 69bp) Mla6-NAC-V3&V4 mut

CTAAGCTTTAATTTCGCAGCTTGTTCAAttCTTCACACAATTTGGATTaaaTTACAGCTCCCTTTATTC

Probes designed for MADS (and HB) on the Mla6 promoter (1) Mla6-MADS-V2 & Mla6-HB-V3： AATCAAGGTGGGCTTGGTCCAGATCTAGCTAAGCTTTAATTTCGCAGCTTGTTCAAGGCT (position 313-373 / 60bp) Mla6-MADS-V2mut & Mla6-HB-V3： AATCAAGGTGGGCTTGGTtaAGATCTAtCTAAGCTTTAATTTCGCAGCTTGTTCAAGGCT

Mla6-MADS-V2& Mla6-HB-V3mut： AATCAAGGTGGGCTTGGTCCAGATCTAGCTAAGCTTccggcTCGCAGCTTGTTCAAGGCT

Probes designed for HB on the Mla6 promoter (2)

Mla6-HB-V1： TGCTTTTGGATCTAAAAAGTTATTAGTTTTCATGCTTAAGTATCTGATCAATTTGCGGTG (position 81-141 / 60bp) Mla6-HB-V1mut： TGCTTTTGGATCTAAAAAacccggcGTTTTCATGCTccgGgATCTGATCAATTTGCGGTG

Mla6-HB-V2： AGTGGCATCTTTCTTGCTATTCTGCTCTAATGAAATCTTTCACGTCCACACGTTCTTGTT (position 100-160 / 60bp) Mla6-HB-V2mut： AGTGGCATCTTTCTTGCTATTCTGCTCccgGgAAATCTTTCACGTCCACACGTTCTTGTT

Additional potential Mla6 promoter motif for TF binding site Mla6-add1: GTCATTGGCACCTCACCTTCTGTCTCTCTCGTTAAATTTGTATCGATATATAAGTGCTTTTGAGTACTTGCATATATAAGTGCTTT (position1-86 / 86bp)

Mla6-add2:

ATTCATCAATTTACAGGCTTCCAGCTGATTGATACGGAGATCTCGTCCCTCCTGCTCTCATGGATATTGTCACCGGTGCCATTTCC (position 406-491 bp / 86bp)

Mla6-add3:

TTCCAACCTGATTCCCAAGTTGGGGGAGCTGCTCACGGAGGAGTTCAAGCTGCACAAGGGTGTCAAGAAAAATATTGAGGACCTCGGG (position 488-575 bp / 87bp) Nac promoter (500 bp):

Sequence for testing of all transcription factors (test with WRKY2, MADS- priority 2, HB, NAC?)

CACCTAGAGGCAATATATATATATATCCAGCCGAGCCGGGCAAATGAACATGAAGTGGGCAATGACCTCATGGCCCTGGCCATGGCCAAGTCGCCATCACAAAAAGCTTCCATACCCTTTTTTTCTTCTTTTTTTGGCTTCAGGCCAGGTACAATGAATGAACCACCACCGCGACGGACCCCGTTACCATCTCGTCGCCTGCCTGCCGCCTGCTGCTGCTCCTCCTCAGCCGTCGCCACACGTGCTCCATCTCCCGGACGATCCGCCGTCGCCTCTTGACCCACCCTTTGACTCCTTTCCCGCCACCAACACCTCCCCACGAACCTTCTCCTCCTCCTCCTCCTCCCCGCGCGCTATTTATCCCCGCGCACGCCATTTCCCAGCATCCATTCGAACACACTCAACACAGTCCACCACCCAAGTACGTCCACCAGCAGCCAGCGCGCGCCCGAGAACCTCGTTCCCATCGACGGAGAAGCAGGGCCAGGCCGGAGGCGGCA

Predicted motif for NAC protein on its own NAC promoter (1)

NAC-NAC: TTTATCCCCGCGCACGCCATTTCCCAGCATC

https://jaspar.elixir.no/matrix/MA1044.2/ Extended probes to be synthesized by vendor 5’ -> 3’ (Probe design can be modified as required by the client.)

NAC-NAC-V1:

TCCTCCTCCTCCTCCTCCCCGCGCGCTATTTATCCCCGCGCACGCCATTTCCCAGCATCCATTCGAACACACT (329-401 bp) NAC-NAC-V1mut:

TCCTCCTCCTCCTCCTCCCCGCGCGCTATTTATCCCCGCGatttaaATTTCCCAGCATCCATTCGAACACACT

Predicted motif for MADS-1 on the NAC promoter (1)

NAC-MADS: CCTTTTTTTCTTCTTTTTTTGGCTTCAGGCCAGG

https://jaspar.elixir.no/matrix/MA0001.2/ Extended probes for synthesis

NAC-MADS-V1：

AAAGCTTCCATACCCTTTTTTTCTTCTTTTTTTGGCTTCAGGCCAGGTACAATGAATGAAC (position 103-164 / 61bp) NAC-MADS-V1mut： AAAGCTTCCATACCCTTTTTTTCTTaaTTTTTTaaCTTCAGGCCAGGTACAATGAATGAAC

Predicted motif for WRKY on the NAC promoter (1) NAC-WRKY: TCGCCTCttgacCCACCCTttgacTCCTTTC https://jaspar.elixir.no/matrix/MA1077.2/ Extended probes for synthesis

NAC-WRKY-V1:

CCGGACGATCCGCCGTCGCCTCttgacCCACCCTttgacTCCTTTCCCGCCACCAACACCT (position 254-314 / 61bp) NAC-WRKY-V1mut:

CCGGACGATCCGCCGTCGCCTCtttttCCACCCTttaaaTCCTTTCCCGCCACCAACACCT

NO motif on NAC promoter for HB.

Additional potential Nac promoter motif for TF binding site Nac-add1： CACCTAGAGGCAATATATATATATATCCAGCCGAGCCGGGCAAATGAACATGAAGTGGGCAATGACCTCATGGCCCTGGCCATGGCCAAGTCGCCATCACAAAA (position 1-104 / 104bp)

Nac-add2： TGAACCACCACCGCGACGGACCCCGTTACCATCTCGTCGCCTGCCTGCCGCCTGCTGCTGCTCCTCCTCAGCCGTCGCCACACGTGCTCCATCTCCCG (position159-256 / 97bp)

Nac-add3： ACTCAACACAGTCCACCACCCAAGTACGTCCACCAGCAGCCAGCGCGCGCCCGAGAACCTCGTTCCCATCGACGGAGAAGCAGGGCCAGGCCGGAGGCGGCA (position 399-500 / 101 bp)

Wise Y1H/EMSA Project V12:

Barley transcription factors that activate disease defense to destructive pathogens

TRANSCRIPTION FACTOR SEQUENCES:

1) NAC domain-containing protein: HORVU2Hr1G004130, HORVU.MOREX.r2.6HG0455190, HORVU.MOREX.r3.6HG0547700

Nac Nucleotide sequence:

"ATGGCGCGGCGGCGGGACGCGGAGGCGGAGCTGAGCCTGCCGCCGGGGTTCCGGTTCCACCCGACGGACGAGGAGCTGGTGGGGGACTACCTCTGCGCGCGCGCGGCCGGCCGGGCCCCGCCGGCGCACGTCATCGCCGAGGTCGACCTGTACCGGCACGACCCGTGGGAGCTACCGGAGCGGGCGCTCTTCGGGGTGCGGGAGTGGTACTTCTTCACGCCGCGGGACCGCAAGTACCCCAACGGCTCCCGCCCTAGCAGGGCCGCCGGGGGCGGCTACTGGAAGGCCACCGGCGCCGACAAGCCCGTCGCGCGCGCCGGTAGGACTGTCGGGGTCAAGAAGTCGCTCGTCTTCTACCATGGGAGGCCGGGGGCTGCGGTCAAGACCGACTGGATCATGCACGAGTACCGCCTCGCCGGCGCTGAGGGGCGCGCCGCGAAGAAGAGGGACGGCGCCTCGCTCAGGCTCGACGACTGGGTGCTCTGCCGCCTCTACGATAAGAAGAACCAGTGGGAGAAGATGCAGCAGCAGCTGCAGGAGGCGGCGGTCAAGGCTGCGGCGTCACGGTCGGTCTCGTGGGGCGAGATGCAGACGCCGGAGTCCGATGTCGACAACGATGCGTTCCCGGAGTTGGATTCGCTACCGCCGACACTCCAGACGACAAACGCGGCGATGTTGCCGAAGGAGGAGGTGCAGGAGTTGGAGCTGGACACTGACGAGTGGCTCATGGGGATCAGCCTCGATGACCTGCAGGGCCCAGGCTCCCTGATGCTGCCCTGGGATGACTCCTACGCCGCCTCTTTCCTGTCGCCAGTGGCCACGATGAAGATGGAGCAGGACATCAGCCCGTTCCTGTTCTGA"

NAC Amino acid sequence

MARRRDAEAELSLPPGFRFHPTDEELVGDYLCARAAGRAPPAHVIAEVDLYRHDPWELPERALFGVREWYFFTPRDRKYPNGSRPSRAAGGGYWKATGADKPVARAGRTVGVKKSLVFYHGRPGAAVKTDWIMHEYRLAGAEGRAAKKRDGASLRLDDWVLCRLYDKKNQWEKMQQQLQEAAVKAAASRSVSWGEMQTPESDVDNDAFPELDSLPPTLQTTNAAMLPKEEVQELELDTDEWLMGISLDDLQGPGSLMLPWDDSYAASFLSPVATMKMEQDISPFLF

2) MADS box transcription factor: HORVU4Hr1G077850, HORVU.MOREX.r2.4HG0338120, HORVU.MOREX.r3.4HG0406150

MADS box Nucleotide sequence:

"ATGGCGGGGAAGAGGGAGAGGATTGCGATACGGAGGATCGAGAATCTGGCGGCCAGGCAGGTCACCTTCTCCAAGCGCCGGAGGGGCCTCTTCAAGAAGGCCGAGGAGCTCTCCATCCTCTGCGACGCCGAGGTCGGCCTCGCCGTCTTCTCCGCCACCGGCAAGCTCTTCCAGTTCGCCAGCTCCAGCATGAACCAGATCATTGATCGGTATAACTCTCATTCCAAGATACTTAAGAAAGTAGACGAGCCATCTCAGCTGGATTTGCATGAGGACAGTAATTGTGCAAGACTAAGGGACGAGCTTGCAGAAGCAAGCCTCTGGCTCCAGCAGATGAGAGGGGAGGAACTCCAGAGCCTGAACGTCCAGCAGCTTCAGGCTCTAGAGAAGAGCCTCGAGTCCGGGCTCAGCTCCGTTCTGAAAACCAAGAGCCAGAAGATCATGGACCAGATCAGCGAGCTAGAAAAAAAGAGGGTGCAACTGATAGAGGAGAACGCAAGGCTAAAGGAGCAAGCGTCCAAGATGGAGATGCAGGTCGCCGCTGATCCACTGGTGGTGGTGTACGATGAAGGGCAGTCATCTGAGTCTGTCACCAACACGTCGTATCCACGCCCTCCCCTCGACACCGAG

GACAGCTCCGACACATCTCTCAGGCTCGGATTATCACTCTTCAACTCGAAGTGA"

MADS box Amino acid sequence

MAGKRERIAIRRIENLAARQVTFSKRRRGLFKKAEELSILCDAEVGLAVFSATGKLFQFASSSMNQIIDRYNSHSKILKKVDEPSQLDLHEDSNCARLRDELAEASLWLQQMRGEELQSLNVQQLQALEKSLESGLSSVLKTKSQKIMDQISELEKKRVQLIEENARLKEQASKMEMQVAADPLVVVYDEGQSSESVTNTSYPRPPLDTEDSSDTSLRLGLSLFNSK

3) Homeobox protein: HORVU4Hr1G078410, HORVU.MOREX.r2.4HG0338560, HORVU.MOREX.r3.4HG0406710

Homeobox Nucleotide sequence:

"ATGAAGCGGCAGATCAAGAGGCCGGCCGGCAGCAAAGAATCGCCTGAGCCAGCAGGGGAGAAGCTGGCGTTCGCTGAGAAAGAGAACCTGCTGGCGGCCTACATGGATCACGAGGAGCCCGAGCTGGAGGAGGCCGACGAGGAGGAGGAGGAGGAGGAGGAGCGCGCCATGTCGTGCGGGCTGGGCGGGAAGAAGCGGCGGCTGGCGCTGGAGCAGGTGCGCGCGCTGGAGCGCAGCTTCGAGACGGACAACAAGCTGGACCCGGAGCGCAAGGCCCGCATCGCCCGCGACCTCGGCCTGCACCCGCGCCAGGTCGCCGTCTGGTTCCAGAACCGCCGCGCCCGCTGGAAGACAAAGCAGCTCGAGCGCGACTTCAACGCCCTCCGCGCCCGCCACGACGCGCTCCGCGCCGACTGCGACGCGCTCCGCCGCGACAAGGACGCCCTCGCCGCCGAGATACACGAGCTGAGGGAAAAGCTGTCGACCAAGCCGGAGACGGCGGTGAAGGCGGAGGCCACCGGCAATGTCGAGGCCGCGGAGGAGCGCCTGCAGCAGGCGACGATGGTCGGCGCGGCGGTCTGCAAGGACGGATCCTCGGACAGCGACTCGAGCGCGGTGTTCAACGACGAGGCGTCGCCCTACTCCGGCGCGGTCTTCGAGCAGCAGGGATTCATGGGGTTCGGCGCGTCGTTCCTGGACACGGCGTCGGCGGCCGCGGCCACCACGGGCTGCACGTCCCTGCCCATGCTGGAACCCAAATGGCCCAGCGCGTACCCGTACGACGCGAGCAGGTCCAGCGCCTACGGCTTCACGGAGGAATGGCTGTCCGGGTCGGACGCGATTGGCAACGACGGCAGCTCCGCCTTCTTCTCCGAGGAGCACGTCTCCAACCTCAACTTCGGCTGGTGTAGCAGTGGCGCCGAGGGTTTTGACCTCCAGAGTTACTGTAAAAAGTAA"

Homeobox Amino acid sequence:

MKRQIKRPAGSKESPEPAGEKLAFAEKENLLAAYMDHEEPELEEADEEEEEEEERAMSCGLGGKKRRLALEQVRALERSFETDNKLDPERKARIARDLGLHPRQVAVWFQNRRARWKTKQLERDFNALRARHDALRADCDALRRDKDALAAEIHELREKLSTKPETAVKAEATGNVEAAEERLQQATMVGAAVCKDGSSDSDSSAVFNDEASPYSGAVFEQQGFMGFGASFLDTASAAAATTGCTSLPMLEPKWPSAYPYDASRSSAYGFTEEWLSGSDAIGNDGSSAFFSEEHVSNLNFGWCSSGAEGFDLQSYCKK

4) WRKY2 transcription factor: HORVU7Hr1G113830, HORVU.MOREX.r2.7HG0616250, HORVU.MOREX.r3.7HG0743270

WRKY2 Nucleotide sequence:

"ATGGAGGAGCAGTGGATGATCGGGCAGACTTGCCTCAGCCTCGCCCTCGGTGTCGGCCAGCCGACGGCGCCGCGAGGTGCTCCCCCGGTGACCAAGGTGCTCGTGGAGGAGGACTTCATGTCCTCCAAGAAAAATCACGAGGTTGAGGCGCTGGAGGCGGAGCTCCGGCGAGTGGGCGAGGAGAACAGGAGGCTGGTCGACATGCTCCGCGCGCTGGTGGCCAAGTACTCCGACCTGCAGGGAAAGGTCAGCGGCATGATGGCGGCGGCCAACAACCACCACCAGTCGTTGACTACGTCTGAGGGCAGTTCCGCGGCCTCGCCGGCCAGGAAGCGCGCCCGCAGCGACAGCCTGAACACCGCCGACCGCAATCCTTCGCCACCGCTCGCCGCAGCCGGCAGCGTCAACGTAGGCCCAGGCCAGCCGGAGTGCACGTCCGTCCACGAGCCCGGCGACAGCAAGCGCGTCCGCGCCGACGAGGACAAGGCCAGCAGGGTCTCCAAGGTCTACGTCCACGCCGACCCCTCCGACCTCAGCCTCGTGGTGAAGGATGGGTACCAATGGCGGAAGTACGGGCAGAAGGTGACCAAGGACAACCCGTGCCCGAGGGCCTACTTCCGGTGCTCGTCCGCGCCGTCGTGCCAGGTGAAGAAGAAGGTGCAGCGCAGCGCCGAGGACAAGACCGTGCTTGTGGCCACGTACGACGGCGACCACAACCACGCACCGCCGCCCAAGCATCAAGGCTCTGGTGGCAGGAAGAGCGGCGACGCAGCCCCCGTCCGCGTGTCACCACCGGCGCCGGTGCTTGTCCAGCAGCAGCGGGAGCAGGAAGCTTCGACGGCGGATCAGGTGGCCGACAGGAAGAAGCTTGTGGAGCAGATGGCGGCGACGCTGACGAGGGACCCCGGGTTCAAGGCGGCACTCGTCTCCGCGCTTTCTGGTCGGATCCCTGTTGCTTGA"

WRKY2 Amino acid sequence:

MEEQWMIGQTCLSLALGVGQPTAPRGAPPVTKVLVEEDFMSSKKNHEVEALEAELRRVGEENRRLVDMLRALVAKYSDLQGKVSGMMAAANNHHQSLTTSEGSSAASPARKRARSDSLNTADRNPSPPLAAAGSVNVGPGQPECTSVHEPGDSKRVRADEDKASRVSKVYVHADPSDLSLVVKDGYQWRKYGQKVTKDNPCPRAYFRCSSAPSCQVKKKVQRSAEDKTVLVATYDGDHNHAPPPKHQGSGGRKSGDAAPVRVSPPAPVLVQQQREQEASTADQVADRKKLVEQMAATLTRDPGFKAALVSALSGRIPVA