Source: TEXAS A&M UNIVERSITY submitted to NRP
PROTEIN PHOSPHORYLATION AND POLY(ADP-RIBOSYL)ATION IN COTTON DROUGHT STRESS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022409
Grant No.
2020-67013-31615
Cumulative Award Amt.
$475,000.00
Proposal No.
2019-05630
Multistate No.
(N/A)
Project Start Date
Jun 1, 2020
Project End Date
May 31, 2024
Grant Year
2020
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Biochemistry and Biophysics
Non Technical Summary
Cotton sustains one of the world’s largest industries (textiles) and serves as a significant source of fiber, feed and oil products. With increasingly dwindling water supplies, water deficit is the major abiotic stress limiting cotton productivity. However, the genetic and molecular basis of cotton drought stress responses remains largely unknown. Recent advances in cotton genome sequencing and functional genomics make it possible to understand cotton drought stress at the genome and proteome level and to leverage genetic resources for improving cotton drought tolerance by molecular breeding and genetic engineering. The proposed research is based on our recent findings that reveal a novel phosphorelay cascade and protein poly(ADP-ribosyl)ation (PARylation) in cotton drought stress. We have identified a key transcription factor that is phosphorylated by an evolutionarily conserved mitogen-activated protein kinase (MAPK) cascade and PARylated by poly(ADP-ribose) polymerases (PARPs) in regulating cotton drought responses in a phytohormone abscisic acid (ABA)-independent manner. The goal of this application is to understand the mechanisms and the intertwined crosstalk of these two key post-translational modifications, protein phosphorylation and PARylation, in orchestrating cotton drought stress responses, and genetically improve cotton drought tolerance by rewiring this unique transcriptional regulon. The proposed objectives represent a systems approach to decipher genetic and biochemical mechanisms controlling an important trait in the complex genome of an essential crop and establish platforms to generate drought tolerant cotton cultivars.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20317101040100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1710 - Upland cotton;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
Cotton (Gossypium spp.) serves as a significant source of fiber, feed and oil products. Cotton production is limited by various abiotic stresses, in particular drought stress, which cause substantial losses of cotton yield worldwide. Moreover, 57% of global cotton is grown in regions with high water stress. Thus, improving water use efficiency and developing drought tolerant cotton cultivars are essential for the sustainable cotton industry and reducing the tension between crop production and water supply. The recent advances in cotton genome sequencing and functional genomics make it possible to understand how cotton responds to drought stress at the genome and proteome level and to leverage genetic resources for improving cotton drought tolerance. With the previous USDA-NIFA support, we have identified a novel phosphorelay cascade, consisting of a mitogen-activated protein kinase (MAPK) module with three tiers of protein kinases and a key transcriptional factor, and revealed the previously unrecognized protein poly(ADP-ribosyl)ation (PARylation) in cotton drought stress. In this application, we aim to understand the underlying molecular and biochemical mechanisms and the crosstalk of these two key posttranslational modifications (phosphorylation and PARylation) in controlling cotton drought stress, and apply these genetic resources to improve cotton adaption to the changing environment . We propose two specific objectives:Decipher the MAPK-WRKY-DREB2 phosphorylation and transcriptional regulatory cascade and its agricultural application in cotton drought stress. We aim to elucidate the mechanism of MAPK phosphorylation on GhWRKY59 function in drought stress by chromatin immunoprecipitation, proteomics, molecular and phenotypic analyses, identify WRKY59-DREB2 regulon by RNA-Seq and feedback regulation by transactivation and EMSA assays, and improving cotton drought tolerance by rewiring the GhWRKY59 regulon.Elucidate protein PARylation, its targets and crosstalk with protein phosphorylation in cotton drought stress. We aim to systematically characterize cotton PARylation enzymes PARPs and PARG with the comprehensive biochemical, molecular and functional genomic approaches, elucidate the interplay between phosphorylation and PARylation on GhWRKY59 function, and identify PARylation targets in cotton drought stress with quantitative proteomics.
Project Methods
Protein phosphorylation assay, protein ADP-Ribosylation assay, transcriptional activity assay, RNA-Seq assay, fusion protein isolation, molecular cloning, plant transformation, drought physiology assays.

Progress 06/01/20 to 05/31/24

Outputs
Target Audience:Our target audiences are researchers and scientists with an aim to advance knowledge in cotton biology and cotton stress tolerance; farmers and produces with an aim to improve the production and value of cotton; students and graduates with an aim to train the next generation of scientists. agricultural professionals and leaders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students supported: Graduate student Ms. Catherine Danmaigona Clementcontinues to give multiple virtual workshops that have impacted thousands of early-career researchers in Africa. Catherine chaired Plant Breeding Circle Symposia at Texas A&M, and invited speakers with different backgrounds. She obtained Ph.D in Spring 2023. Graduate student Mr. Brendan Mormile, has organized five consecutive Texas A&M Genome Editing Symposia (2018-2021) (https://genome-editing-symposium-tamu.com/) with a huge success and impact every year. He was the Chair of the symposium in 2021.He obtained Ph.D in Spring 2023. Graduate student Barbara Rodrigues has organized Texas A&M Genome Editing Symposium in 2022. She obtained Ph.D in Spring 2023. Will Dodge, Ph. D., Texas A&M University. "UAS-based phenotyping tools to enable cotton germplasm selection for limited groundwater resources and changing climate." Supervisors: J. K. Dever and S. Hague. Committee members: Murilo Maeda, Mauricio Ulloa, and Dana Porter. Status: Completed Spring 2023. How have the results been disseminated to communities of interest?The PIs gave multiple lectures about cotton genomics and drought resistance todifferent audiences in 2022, including: Ping He Department of Cell Biology and Molecular Genetics, University of Maryland, October 2022 Molecular Plant Virtual Seminar, September 2022 Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, August 2022 The University of Missouri, Columbia, MO, March 2022 Libo Shan 1. 12th Japan-US Seminar, Mobilization of molecular defenses by cell surface receptor signaling, Ithaca, NY, August 2022 2. 7th Xanthomonas Genomics Conference, Clearwater Beach, FL, June 2022 3. Department of Biochemistry & Molecular Biology,Michigan State University, April 2022 4.Danforth Plant Science Center, St. Louis, MO, March 2022 5. Interdisciplinary Plant Group, The University of Missouri, Columbia, MO, March 2022 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In this cycle, we mainly focused on Protein ADP-ribosylation in cotton drought stress. The process is mediated by ADP-ribosyltransferases (ARTs), which transfer single ADP-ribose (MAR, mono-ADP-ribose) or multiple ADP-ribose (PAR, poly-ADP-ribose) from nicotinamide adenine dinucleotide (NAD+) to acceptor proteins, termed mono(ADP-ribosyl)ation (MARylation) or poly(ADP-ribosyl)ation (PARylation), respectively. The PARylated or/and MARylated proteins could be immunoprecipitated by macrodomain affinity resin (MD-resin), whereas MARylated proteins are only immunoprecipitated by PARP14m3 resin. Therefore, we can identify ADP-ribosylated proteins in cotton response to drought stress using MD-resin and PARP14m3 resin coupled with label-free quantitative proteomics. We collected 14-day-old cotton seedlings grown in hydroponic medium with or without 5% PEG (a reagent mimicking drought stress) treatment for 12 hrs. Total protein extracts were incubated with MD-resin and PARP14m3 resin at 4 ? for 3-4 hrs. Mock and PEG treatment samples includes three independent repeats, respectively. The immunoprecipitated cotton PARylated and MARylated proteins are identified using mass spectrometry-based label-free quantitative proteomics. As a results, we identified total 1330 candidates (include three PARPs, Table 1) for PARylated or MARylated proteins, and we also identified total 2231 candidates only for MARylated proteins. For PARylated or/and MARylated proteins, 22 candidates show increased ADP-ribosylation, whereas 325 candidates show decreased ADP-ribosylation upon PEG treatment. For only MARylated proteins, 416 candidates show increased MARylation, whereas 14 candidates show decreased MARylation upon PEG treatment . In addition, there are 942 overlapped candidates identified by MD-resin and PARP14m3 resin, which means that 388 among 1330 candidates are likely PARylated proteins. Among the PARylated and/or MARylated candidates, a subset of candidates belonging to RNA-binding proteins (RBP) were identified response to drought stress. Importantly, their homologs in mammalian involved in forming stress granules response to different biotic and abiotic stresses including H2O2, heat, temperature, and pathogens. Also, mammalian system research show that RNA-proteins function as ADP-ribosylation substrates to regulate various cellular processes. These information give us more rationale to focus on the function of RBPs in cotton response to the drought stress. We have made the VIGS constructs of these RBPs to knockdown them in cotton using our previously established VIGS-based gene silence system. We are in the process of screening the growth and drought phenotypes to focus on certain genes that involved in regulating drought stress. We will also study the molecular mechanisms of these RBPs in regulating drought stress through PARylation and/or MARylation. Table 1: List of RNA-binding proteins (RNPs) identified in the ADP-ribosylation proteomics Accession ID Function description Unique Peptides PAR-IP MAR-IP Gh_D11G137700.1 PARP1, Poly [ADP-ribose] polymerase 1 61 0 Gh_A11G121000.1 PARP2, Poly [ADP-ribose] polymerase 2 23 0 Gh_D11G122900.1 PARP2, Poly [ADP-ribose] polymerase 2 7 0 Gh_D13G091600.1 Putative G3BP-like protein (RNA-binding protein) 0 9 Gh_D08G186800.1 RGG repeats nuclear RNA binding protein B 9 3 Gh_A08G160800.1 RGG repeats nuclear RNA binding protein B 4 0 Gh_A09G215700.1 RGG repeats nuclear RNA binding protein B 3 3 Gh_D08G160300.1 RGG repeats nuclear RNA binding protein B 3 4 Gh_A08G160900.1 RGG repeats nuclear RNA binding protein B 2 1 Gh_D08G160200.1 RGG repeats nuclear RNA binding protein B 0 5 Gh_A03G223900.1 Glycine-rich RNA-binding protein GRP1A 5 4 Gh_D13G034800.1 Glycine-rich RNA-binding protein 2 2 Gh_A12G029400.1 Glycine-rich RNA-binding protein GRP1A 2 2 Gh_D07G073400.1 Glycine-rich RNA-binding protein 3, mitochondrial 2 2

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ayele, A. G., J. K. Dever, C. M. Kelly, M. Sheehan, V. Morgan, and P. Payton. 2020. Responses of upland cotton (Gossypium hirsutum L.) lines to irrigated and rainfed conditions of Texas High Plains." Plants 9(11): 1598. https://doi.org/10.3390/plants9111598.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Dever, J. K., C. M. Kelly, A. Ayele, J. Zwonitzer, P. Payton, and D. Jones. 2020. Registration of CA 4007 cotton germplasm line for water-limited production. Journal of Plant Registrations. 14(1):49-56. https://doi.org/10.1002/plr2.20034.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Maeda, A. B., L. W. Wells, M. A. Sheehan, and J. K. Dever. 2021. Stories from the greenhouse  A brief on cotton seed germination. Plants. https://doi.org/10.3390/plants10122807.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Zhang, Z., G. Gia, F. S. Bao, A. Ghosh, K. Attri, J. K. Dever, and Z. Xie. 2022. Evidence for accelerated evolution of a polyphenol oxidase-targeting miRNA family facilitated by allotetraploid formation in Gossypium. TTU Institute of Genomics for Crop Abiotic Stress Tolerance (IGCAST) Symposium. December 7, 2022. Lubbock, TX, USA.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 1. de Moura, S.M., Babilonia, K., de Macedo, L.L.P., Grossi-de-S�, M.F., Shan, L., He, P., and Alves-Ferreira, M. (2022). The oral secretion from Cotton Boll Weevil (Anthonomus grandis) induces defense responses in cotton (Gossypium spp) and Arabidopsis thaliana. Current Plant Biology 31: 100250.
  • Type: Book Chapters Status: Published Year Published: 2022 Citation: Liu, Z., No., E.G., Danmaigona Clement, C., He, P., and Shan., L. (2022) Isolation of high-molecular-weight (HMW) DNA from Fusarium oxysporum for MinIONlong-read sequencing. Methods Mol Biol. 2391: 21-30.


Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Our target audiences are researchers and scientists with an aim to advance knowledge in cotton biology and cotton stress tolerance; farmers and produces with an aim to improve the production and value of cotton; students and graduates with an aim to train the next generation of scientists. agricultural professionals and leaders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students supported: Graduate student Ms. Catherine Danmaigona Clementcontinues to give multiple virtual workshops that have impacted thousands of early-career researchers in Africa. Catherine chaired Plant Breeding Circle Symposia at Texas A&M, and invited speakers with different backgrounds. She obtained Ph.D in Spring 2023. Graduate student Mr. Brendan Mormile, has organized five consecutive Texas A&M Genome Editing Symposia (2018-2021) (https://genome-editing-symposium-tamu.com/) with a huge success and impact every year. He was the Chair of the symposium in 2021.He obtained Ph.D in Spring 2023. Graduate student Barbara Rodrigues has organized Texas A&M Genome Editing Symposium in 2022. She obtained Ph.D in Spring 2023. Will Dodge, Ph. D., Texas A&M University. "UAS-based phenotyping tools to enable cotton germplasm selection for limited groundwater resources and changing climate." Supervisors: J. K. Dever and S. Hague. Committee members: Murilo Maeda, Mauricio Ulloa, and Dana Porter. Status: Completed Spring 2023. How have the results been disseminated to communities of interest?The PIs gave multiple lectures about cotton genomics and drought resistance todifferent audiences in 2022, including: Ping He Department of Cell Biology and Molecular Genetics, University of Maryland, October 2022 Molecular Plant Virtual Seminar, September 2022 Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, August 2022 The University of Missouri, Columbia, MO, March 2022 Libo Shan 1. 12thJapan-US Seminar, Mobilization of molecular defenses by cell surface receptor signaling, Ithaca, NY, August 2022 2. 7thXanthomonas Genomics Conference, Clearwater Beach, FL, June 2022 3. Department of Biochemistry & Molecular Biology,Michigan State University, April 2022 4.Danforth Plant Science Center, St. Louis, MO, March 2022 5. Interdisciplinary Plant Group, The University of Missouri, Columbia, MO, March 2022 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In this cycle, we mainly focused on Protein ADP-ribosylation in cotton drought stress. The process is mediated by ADP-ribosyltransferases (ARTs), which transfer single ADP-ribose (MAR, mono-ADP-ribose) or multiple ADP-ribose (PAR, poly-ADP-ribose) from nicotinamide adenine dinucleotide (NAD+) to acceptor proteins, termed mono(ADP-ribosyl)ation (MARylation) or poly(ADP-ribosyl)ation (PARylation), respectively. The PARylated or/and MARylated proteins could be immunoprecipitated by macrodomain affinity resin (MD-resin), whereas MARylated proteins are only immunoprecipitated by PARP14m3 resin. Therefore, we can identify ADP-ribosylated proteins in cotton response to drought stress using MD-resin and PARP14m3 resin coupled with label-free quantitative proteomics. We collected 14-day-old cotton seedlings grown in hydroponic medium with or without 5% PEG (a reagent mimicking drought stress) treatment for 12 hrs. Total protein extracts were incubated with MD-resin and PARP14m3 resin at 4 ? for 3-4 hrs. Mock and PEG treatment samples includes three independent repeats, respectively. The immunoprecipitated cotton PARylated and MARylated proteins are identified using mass spectrometry-based label-free quantitative proteomics. As a results, we identified total 1330 candidates (include three PARPs, Table 1) for PARylated or MARylated proteins, and we also identified total 2231 candidates only for MARylated proteins. For PARylated or/and MARylated proteins, 22 candidates show increased ADP-ribosylation, whereas 325 candidates show decreased ADP-ribosylation upon PEG treatment. For only MARylated proteins, 416 candidates show increased MARylation, whereas 14 candidates show decreased MARylation upon PEG treatment . In addition, there are 942 overlapped candidates identified by MD-resin and PARP14m3 resin, which means that 388 among 1330 candidates are likely PARylated proteins. Among the PARylated and/or MARylated candidates, a subset of candidates belonging to RNA-binding proteins (RBP) were identified response to drought stress. Importantly, their homologs in mammalian involved in forming stress granules response to different biotic and abiotic stresses including H2O2, heat, temperature, and pathogens. Also, mammalian system research show that RNA-proteins function as ADP-ribosylation substrates to regulate various cellular processes. These information give us more rationale to focus on the function of RBPs in cotton response to the drought stress. We have made the VIGS constructs of these RBPs to knockdown them in cotton using our previously established VIGS-based gene silence system. We are in the process of screening the growth and drought phenotypes to focus on certain genes that involved in regulating drought stress. We will also study the molecular mechanisms of these RBPs in regulating drought stress through PARylation and/or MARylation. Table 1: List of RNA-binding proteins (RNPs) identified in the ADP-ribosylation proteomics Accession ID Function description Unique Peptides PAR-IP MAR-IP Gh_D11G137700.1 PARP1, Poly [ADP-ribose] polymerase 1 61 0 Gh_A11G121000.1 PARP2, Poly [ADP-ribose] polymerase 2 23 0 Gh_D11G122900.1 PARP2, Poly [ADP-ribose] polymerase 2 7 0 Gh_D13G091600.1 Putative G3BP-like protein (RNA-binding protein) 0 9 Gh_D08G186800.1 RGG repeats nuclear RNA binding protein B 9 3 Gh_A08G160800.1 RGG repeats nuclear RNA binding protein B 4 0 Gh_A09G215700.1 RGG repeats nuclear RNA binding protein B 3 3 Gh_D08G160300.1 RGG repeats nuclear RNA binding protein B 3 4 Gh_A08G160900.1 RGG repeats nuclear RNA binding protein B 2 1 Gh_D08G160200.1 RGG repeats nuclear RNA binding protein B 0 5 Gh_A03G223900.1 Glycine-rich RNA-binding protein GRP1A 5 4 Gh_D13G034800.1 Glycine-rich RNA-binding protein 2 2 Gh_A12G029400.1 Glycine-rich RNA-binding protein GRP1A 2 2 Gh_D07G073400.1 Glycine-rich RNA-binding protein 3, mitochondrial 2 2

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ayele, A. G., J. K. Dever, C. M. Kelly, M. Sheehan, V. Morgan, and P. Payton. 2020. Responses of upland cotton (Gossypium hirsutum L.) lines to irrigated and rainfed conditions of Texas High Plains." Plants 9(11): 1598. https://doi.org/10.3390/plants9111598.
  • Type: Book Chapters Status: Published Year Published: 2022 Citation: Liu, Z., No., E.G., Danmaigona Clement, C., He, P., and Shan., L. (2022) Isolation of high-molecular-weight (HMW) DNA from Fusarium oxysporum for MinIONlong-read sequencing. Methods Mol Biol. 2391: 21-30.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Dever, J. K., C. M. Kelly, A. Ayele, J. Zwonitzer, P. Payton, and D. Jones. 2020. Registration of CA 4007 cotton germplasm line for water-limited production. Journal of Plant Registrations. 14(1):49-56.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Maeda, A. B., L. W. Wells, M. A. Sheehan, and J. K. Dever. 2021. Stories from the greenhouse  A brief on cotton seed germination. Plants. https://doi.org/10.3390/plants10122807.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: de Moura, S.M., Babilonia, K., de Macedo, L.L.P., Grossi-de-S�, M.F., Shan, L., He, P., and Alves-Ferreira, M. (2022). The oral secretion from Cotton Boll Weevil (Anthonomus grandis) induces defense responses in cotton (Gossypium spp) and Arabidopsis thaliana. Current Plant Biology 31: 100250.


Progress 06/01/21 to 05/31/22

Outputs
Target Audience:The PIs gave multiple lectures about cotton genomics and drought resistance todifferent audiences in 2021, including: Signal Transduction Symposia, Mexican Society of Biochemistry (SMB), November 2021 (Online, Invited speaker) 6th International Conference on Biotic Plant Interactions, Xi'an, China, October 2021 (Online, Invited speaker) Department of Plant Pathology, University of California, Davis, October 2021 (Online, Invited speaker) Department of Biological Sciences, University of North Texas, September 2021 School of Life Sciences, Technical University of Munich, Germany, July 2021 (online) School of Biological Sciences, The University of Hong Kong, April 2021 (online) Department of Plant Science and Landscape Architecture, University of Maryland, December 2021 (Invited speaker) Beltwide Cotton Conference, New Orleans, LA, January 2021 (Online, Invited speaker) Co-PI Sixue Chen edits an ebook on plant proteomics that covers post-translational modifications. In addition, he has a commissioned book chapter on phosphoprotein analysis to be published this year. Chen, S., Komatsu, S. (2021) Plant Proteomic Research 4.0: Frontiers in Stress Resilience. International Journal of Molecular Sciences 22, 13362. https://doi.org/10.3390/ijms222413362 Perron, N., Tan, B., Dufresne, C.P., Chen, S. (2022) Proteomics and phosphoproteomics of C3 to CAM transition in the common ice plant. Methods in Enzymology (invited) Graduate student Ms. Catherine Danmaigona Clementcontinues to give multiple virtual and face-face workshops that have impacted thousands of early-career researchers not only in Africa but also in Asia, South America, Europe, the US, and Australia. Catherine chairs Plant Breeding Circle Symposia at Texas A&M, and invited speakers with different backgrounds. She has been featured in seed world magazine (https://seedworld.com/napb-puts-a-focus-on-breaking-down-barriers). The greatest impact is that African students are gaining confidence in analyzing and interpreting their data, and most importantly, they teach others in their various schools and communities in their countries. Graduate student Mr. Brendan Mormile, has organized four consecutive Texas A&M Genome Editing Symposia (2018-2021) (https://genome-editing-symposium-tamu.com/) with a huge success and impact every year. He was the Chair of the symposium in 2021. Dr. Sixue Chen has co-organized a National Science Foundation sponsored national posttranscriptional and posttranslational modifications workshop on October 18-20th, 2021 (https://biotech.ufl.edu/event/nsf-sponsored-workshop-cross-disciplinary-study-of-post-transcriptional-and-post-translational -modifications/). Changes/Problems:Due to the Covid19, the progress wasdelayed. One hired postdoc cannot come last year. One new postdoc just came early this year to work on this project. What opportunities for training and professional development has the project provided?We are making every effort to mentor undergraduate and graduate students and postdoc fellows, and outreach activities in the community. We provided a combination of molecular, genetics, biochemical, and plant physiology training to the students. The undergraduate students in 2021: Lauren Kearns (Biochemistry, 2 credit for Fall 2021, 2 credit for Spring 2022) Tengyang Wu (Biochemistry, 1 credit for Fall 2021, 2 credit for Spring 2022) Jesph Gallucci (Biochemistry, 1 credit for Fall 2021) Sarah Mattison Edgar (491 student from Genetics, 2 credit hr for Fall 2019, 2 credit for Fall 2020,1 credit for Spring 2021), The graduate students as the major advisor: Ms. Lahong Xu, a Ph. D student in Molecular & Environmental Plant Sciences (MEPS) program, joined lab in September 2018. Ms. Catherine Danmaigona Clement (a joint Ph.D student with Soil and Crop Sciences at TAMU), an African-American female graduate student. Ms. Suji Ye, a Ph. D student in Biochemistry & Biophysics (MEPS) program. Ms. Lahong Xu, a Ph. D student in Molecular & Environmental Plant Sciences (MEPS) program. Mentoring of postdoctoral researcherrelated to this project: Dr. Mingli Yong is a new postdoctoral researcher working on PARylation proteomics in cotton drought stress. How have the results been disseminated to communities of interest?The PIs gave multiple lectures about cotton genomics and drought resistance todifferent audiences in 2021, including: Signal Transduction Symposia, Mexican Society of Biochemistry (SMB), November 2021 (Online, Invited speaker) 6th International Conference on Biotic Plant Interactions, Xi'an, China, October 2021 (Online, Invited speaker) Department of Plant Pathology, University of California, Davis, October 2021 (Online, Invited speaker) Department of Biological Sciences, University of North Texas, September 2021 School of Life Sciences, Technical University of Munich, Germany, July 2021 (online) School of Biological Sciences, The University of Hong Kong, April 2021 (online) Department of Plant Science and Landscape Architecture, University of Maryland, December 2021 (Invited speaker) Beltwide Cotton Conference, New Orleans, LA, January 2021 (Online, Invited speaker) Co-PI Sixue Chen edits an ebook on plant proteomics that covers post-translational modifications. In addition, he has a commissioned book chapter on phosphoprotein analysis to be published this year. Dr. Sixue Chen has co-organized a National Science Foundation sponsored national posttranscriptional and posttranslational modifications workshop on October 18-20th, 2021 (https://biotech.ufl.edu/event/nsf-sponsored-workshop-cross-disciplinary-study-of-post-transcriptional-and-post-translational -modifications/). What do you plan to do during the next reporting period to accomplish the goals?We will focus on how ADP-ribosylation regulates gene expression and protein modification under cotton drought stress. The prepared RNA samples will be sent to Texas A&M University Molecular Genomics Workplace for sequencing with NovaSeq s4-XP workflow 2x150 (2-2.5 billion clusters). The graduate student Ms. Catherine Danmaigona Clement will assist with bioinformatic analysis to identify PARylation-regulated genes. To identify ADP-ribosylation targets, we will use label-free quantitative proteomics in Co-PI Dr. Chen's facility. Proteins from cotton plants treated with and without drought stress will be isolated for label-free quantitative proteomics. We will continue to investigate phosphorylation and dephosphorylation in regulating cotton drought stresses. In particular, we are interested in GhTOPP targets. Do GhTOPPs dephosphorylate GhWRKYs that are phosphorylated by GhMAP3K15-GhMKK4-GhMPK6 cascade? Or, do GhTOPPs directly dephosphorylate GhMAP3K15-GhMKK4-GhMPK6 cascade? What are the additional targets of GhTOPPs and GhMAP3K15-GhMKK4-GhMPK6 cascade?

Impacts
What was accomplished under these goals? Objective 1: Drought is a key limiting factor for cotton production since more than half of global cotton is grown in regions with a high level of water shortage. However, the underlying mechanism of cotton response to drought stress remains elusive. We combined genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing and identified GhWRKY59 as an important transcription factor that regulates drought stress response in cotton. We performed biochemical and genetic analyses and elucidated a drought stress-activated MAP kinase cascade consisting of GhMAP3K15-GhMKK4-GhMPK6 that directly phosphorylates GhWRKY59. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of GhDREB2, which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2 in controlling cotton drought response. We further show that GhMAP3K15-GhMKK4-GhMPK6 cascade phosphorylates GhWRKY59 at residue serine 221. Importantly, the Arabidopsis transgenic plants expressing GhWRKY59S221D, a phospho-mimetic mutant of GhWRKY59 with a substitution of aspartic acid (D), were more tolerant to drought stress than the plants expressing WT GhWRKY59. The data support our hypothesis that phosphorylation of GhWRKY59 by GhMAP3K15-GhMKK4-GhMPK6 cascade is important for its function in cotton drought tolerance. Phosphorylation and dephosphorylation are two reversible events. We are looking for potential phosphatases that are involved in cotton drought tolerance. By using a genome-wide virus-induced gene silencing (VIGS) screen, we identified a type one protein phosphatase, GhTOPP6, that may positively regulate cotton drought response. VIGS-GhTOPP6 plants showed more sensitive to drought stress than control plants. The expression of GhTOPP6 was up-regulated by ABA and PEG treatment. Overexpression of GhTOPP6 in Arabidopsis improved plant drought tolerance. We further confirmed that GhTOPP6 is involved in drought tolerance with cotton transgenic plants. Overexpression of GhTOPP6 in G. hirsutum enhanced plant drought tolerance with a higher fresh weight under PEG mimics osmotic stress treatment. Interestingly, the silencing of a homolog of GhTOPP6, GhTOPP4, showed increased tolerance to drought stress. The data suggest that different members of type one protein phosphatases differentially regulate cotton drought stress response. Significantly, GhWRKY1 is a target of GhTOPP6. We are investigating the relationship of GhMAP3K15-GhMKK4-GhMPK6 cascade and GhTOPPs in the phosphorylation and dephosphorylation of different WRKYs in regulating cotton drought stress. We also collaborated with bioinformaticians for modeling drought signaling pathways with the RNA-seq data we generated and available in the literature and identified two novel components, MYC2 and ATAF1 in the drought stress tolerance. One manuscript about this study has been published. Objective 2: Poly(ADP-ribosyl)ation (PARylation) regulated by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs) plays a vital role in diverse biological processes. We have previously shown a potential role of PARylation in cotton drought stress. Cotton has three PARPs and one PARG. We have generated VIGS constructs to silence individual cotton PARPs (GhPARP1, GhPARP2, and GhPARP3) and PARG (GhPARG1), and silence three GhPARPs simultaneously (GhPARP1,2,3). Interestingly VIGS-GhPARP1, and GhPARP1/2/3 plants showed increased resistance to PEG (a reagent mimicking drought stress) treatment when growing in the hydroponic condition and to drought stress when growing in soil. However, VIGS-GhPARP2 plants showed increased sensitivity to PEG treatment when growing in hydroponic conditions and to drought stress when growing in soil. The data suggest the coordination of different PARP members in regulating cotton drought stress. We have treated VIGS-GhPARG and VIGS-GhPARP2 plants with 3% PEG-6000 as a dehydration treatment three weeks after VIGS, and collected samples at 0, 6, 12 hours after PEG treatment for RNA-Seq analysis. Meanwhile, we have tested different resins for immunoprecipitating PARylated proteins for quantitative proteomics to identify differentially PARylated proteins in cotton under drought stress. We have shown that the macrodomain affinity resin (MD-resin) could bind both PARylated and mono(ADP-ribosy)lated (MARylated) proteins, and PARP14m3 resin only binds MARylated proteins. Compared to PARylation, the chemistry and physiological functions of mono(ADP-ribosy)lation (MARylation) remain elusive. We have treated VIGS-GhPARG and VIGS-GhPARP2 plants with 5% PEG-6000 as a dehydration treatment three weeks after VIGS, and collected samples at 0, 6, 12 hours after PEG treatment for MD-resin (AF1521) and PARP14m3 resin to identify PARylated and MARylated proteins with quantitative proteomics. Samples of three repeats (3 plants for each repeat) for three time points (0, 6, 12 h) have been prepared for immunoprecipitation using AF1521 and PARP14m3 resins.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 2. Wang, P., Mormile, B., and He, P. (2021) A GLoRyBattle for Cotton against Fusarium. Trends in Plant Science 26: P671-673
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 3. Babilonia, K., Wang, P., Liu, L., Jamieson, P., Mormile B., Rodrigues, O., Lin, W., Danmaigona Clement, C., Menezes de Moura, S., Alves-Ferreira, M., Finlayson, S.A., Nichols, R.L., Wheeler, T.A., Dever, J.K., Shan, L., and He, P. (2021) A non-proteinaceous Fusarium cell wall extract triggers receptor-like protein-dependent immune responses in Arabidopsis and cotton. New Phytologist 230: 275-289.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Chen, S., Komatsu, S. (2021) Plant Proteomic Research 4.0: Frontiers in Stress Resilience. International Journal of Molecular Sciences 22, 13362. https://doi.org/10.3390/ijms222413362
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 1. Lahiri, A., Zhou, L., He, P., and A Datta, A. (2021) Detecting drought regulators using stochastic inference in Bayesian networks. PloS one 16 (8): e0255486.


Progress 06/01/20 to 05/31/21

Outputs
Target Audience: The PIs gave multiple lectures about cotton genomics and drought resistance to different audiences in 2020, including: School of Integrative Plant Sciences (SIPS) Plenary Seminar, Cornell University, September 2020 Faculty of Genetics, Texas A&M University, September 2020 Beltwide Cotton Conference, New Orleans, LA, January 2021 Molecular Biosciences Symposium, Emerging Approaches in Plant Biology, Austin, Texas, August 2020 Graduate student Ms. Catherine Danmaigona Clementhas given multiple workshops for students and scientists on Africa in collaboration with the JRBiotek Foundation (2019 onsite on Molecular Biology, 2020 virtual workshop on cotton SNP and GWAS data analysis with R with 1400+ Africans registered). Giving the high impact and reach of the workshops she gave, theJRBiotekfoundationhas asked Ms. Catherine Danmaigona Clement to coordinateand organize more workshops, lectures, and training for African participants. In 2020,graduate student Mr. Brendan Mormile has given a workshop about cotton CRISPR-CAS and Barabara Rodrigues has given a workshop about the application of virus-induced gene silencing or RNAi, which was first developed by PI's lab in cotton to African students and scientists. Changes/Problems:Due to the Covid19, the progress was slightly delayed. One hired postdoc cannot come last year. We are in the process in hiring another postdoc. What opportunities for training and professional development has the project provided?We are making every effort to mentoring undergraduate and graduate students and postdoc fellows, and outreach activities in the community. We provided a combination of molecular, genetics, biochemical, and plant physiology training to the students. The undergraduate students in 2020-2021: Taylon Prevost (491 student from Genetics, 2 credit hr for Fall 2019, 1 credit for Spring 2020, 1 credit for Fall 2020,), an African-American female undergraduate student. Michael Lynn, (491 student from Genetics, 2 credit hr for Fall 2019, 1 credit for Spring 2020, 3 credit Fall 2020), Amir Melek (491 student from Genetics, 2 credit hr for Fall 2019, 1 credit for Spring 2020, 3 credit Fall 2020), Sarah Mattison Edgar (491 student from Genetics, 2 credit hr for Fall 2019, 2 credit for Fall 2020,1 credit for Spring 2021), The graduate students as the major advisor: Ms. Catherine Danmaigona Clement (a joint Ph.D student among Dr. Dever, Shan and He), an African-American female graduate student Mr. Chao Zhang, a Ph.D student in Plant Pathology and Microbiology Ms. Suji Ye, a Ph. D student in Biochemistry & Biophysics (MEPS) program Ms. Lahong Xu, a Ph. D student in Molecular & Environmental Plant Sciences (MEPS) program Mentoring of postdoctoral researchers related to this project: Dr. Liang Kong works on PARG1 target, and presented a talk at The Cold Spring Harbor Laboratory "The PARP Family and ADP-ribosylation" meeting, December 2020. Dr. JunHyeok Kim works on PARP regulation and presented a poster at The Cold Spring Harbor Laboratory's "The PARP Family and ADP-ribosylation" meeting, December 2020. How have the results been disseminated to communities of interest?The PIs gave multiple lectures about cotton genomics and drought resistance todifferent audiences in 2020, including: School of Integrative Plant Sciences (SIPS) Plenary Seminar, Cornell University, September 2020 Faculty of Genetics, Texas A&M University, September 2020 Beltwide Cotton Conference, New Orleans, LA, January 2021 Molecular Biosciences Symposium, Emerging Approaches in Plant Biology, Austin, Texas, August 2020 Graduate student Ms. Catherine Danmaigona Clementhas given multiple workshops for students and scientists on Africa in collaboration with the JRBiotek Foundation (2019 onsite on Molecular Biology, 2020 virtual workshop on cotton SNP and GWAS data analysis with R with 1400+ Africans registered). Giving the high impact and reach of the workshops she gave, theJRBiotekfoundationhas asked Ms. Catherine Danmaigona Clement to coordinateand organize more workshops, lectures, and training for African participants. In 2020,graduate student Mr. Brendan Mormile has given a workshop about cotton CRISPR-CAS and Barabara Rodrigues has given a workshop aboutthe application of virus-induced gene silencing orRNAi, which was first developed by PI's lab in cotton to African students and scientists. What do you plan to do during the next reporting period to accomplish the goals?We will continue to investigate phosphorylation and dephosphorylation in regulating cotton drought stresses. In particular, we will study the relationship of GhMAP3K15-GhMKK4-GhMPK6 cascade and GhTOPPs in the phosphorylation and dephosphorylation of different WRKYs in regulating cotton drought stress by molecular, biochemical, proteomic and physiology studies. Do GhTOPPs dephosphorylate GhWRKYs that are phosphorylated by GhMAP3K15-GhMKK4-GhMPK6 cascade? Or, do GhTOPPs directly dephosphorylate GhMAP3K15-GhMKK4-GhMPK6 cascade? What are the additional targets of GhTOPPs and GhMAP3K15-GhMKK4-GhMPK6 cascade? We will further characterize the functions of different PARPs in cotton drought stress by identifying the targets and characterizing the activities as outlined in the proposal. To identify ADP-ribosylation targets, we plan to use a two-step purification system coupled with label-free quantitative proteomics. We will usea-PAR-coupled beads orWWE domain resin coupled with MD beads to immunoprecipitate PARylated proteins. We will usePARP14m3 resin coupled with MD beads to immunoprecipitate MARylated proteins. Proteins from cotton plants treated with and without drought stress will be isolated for label-free quantitative proteomics.

Impacts
What was accomplished under these goals? Objective 1: Drought is a key limiting factor for cotton production since more than half of global cotton is grown in regions with high level of water shortage. However, the underlying mechanism of cotton response to drought stress remains elusive. We combined genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, and identified GhWRKY59 as an important transcription factor that regulates drought stress response in cotton. We performed biochemical and genetic analyses and elucidated a drought stress-activated MAP kinase cascade consisting of GhMAP3K15-GhMKK4-GhMPK6 that directly phosphorylates GhWRKY59. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regualted MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of GhDREB2, which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2 in controlling cotton drought response. We further show that GhMAP3K15-GhMKK4-GhMPK6 cascade phosphorylates GhWRKY59 at residue serine 221. Importantly, the Arabidopsis transgenic plants expressing GhWRKY59S221D, a phospho-mimetic mutant of GhWRKY59 with a substitution of aspartic acid (D), were more tolerant to drought stress than the plants expressing WT GhWRKY59. The data support our hypothesis that phosphorylation of GhWRKY59 by GhMAP3K15-GhMKK4-GhMPK6 cascade is important for its function in cotton drought tolerance. Phosphorylation and dephosphorylation are two reversible events. We are looking for potential phosphatases that are involved in cotton drought tolerance. By using a genome-wide virus-induced gene silencing (VIGS) screen, we identified a type one protein phosphatase, GhTOPP6, that may positively regulate cotton drought response. VIGS-GhTOPP6 plants showed more sensitive to drought stress than control plants. The expression of GhTOPP6 was up-regulated by ABA and PEG treatment. Overexpression of GhTOPP6 in Arabidopsis improved plant drought tolerance. Interestingly, silencing of a homolog of GhTOPP6, GhTOPP4, showed increased resistance to drought stress. The data suggest that different members of type one protein phosphatases differentially regulate cotton drought stress response. Significantly, GhWRKY1 is a target of GhTOPP6. We are investigating the relationship of GhMAP3K15-GhMKK4-GhMPK6 cascade and GhTOPPs in the phosphorylation and dephosphorylation of different WRKYs in regulating cotton drought stress. We also collaborated with bioinformaticians for modeling drought signaling pathways with the RNA-seq data we generated and available in the literature and identified two novel components, MYC2 and ATAF1 in the drought stress tolerance. One manuscript about this study has been submitted. Lahiri, A., Zhou, L., He, P., Datta, A. Detecting Drought Regulators using Stochastic Inference in Bayesian Networks. (Under review at BMC Plant Biology). Background: Drought is a natural hazard that affects crops by inducing water stress. Water stress, induced by drought, accounts for more loss in crop yield than all the other causes combined. With the increasing frequency and intensity of droughts worldwide, it is essential to develop drought-resistant crops to ensure food security. In this paper, we model multiple drought signaling pathways in plants using Bayesian networks to identify potential regulators of drought-responsive reporter genes. Genetically intervening at these regulators can help develop drought-resistant crops. Result: We create the Bayesian network model from biological literature and determine its parameters from publicly available data. We conduct inference on this model using a stochastic simulation technique known as likelihood weighting to determine the best regulators of drought-responsive reporter genes. Our analysis reveals that activating MYC2 or inhibiting ATAF1 is the best single node intervention strategies to regulate the drought-responsive reporter genes. Additionally, we observe simultaneously activating MYC2 and inhibiting ATAF1 is a better strategy. Conclusion: The Bayesian network model indicated that MYC2 and ATAF1 are possible regulators of drought response. Validation experiments showed that ATAF1 negatively regulated drought response. Thus intervening at ATAF1 has the potential to create drought-resilient crops. Objective 2: Poly(ADP-ribosyl)ation (PARylation) regulated by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs) plays a vital role in diverse biological processes. We have previously shown a potential role of PARylation in cotton drought stress. Cotton has three PARPs and one PARG. We have generated VIGS constructs to silence individual cotton PARPs (GhPARP1, GhPARP2, and GhPARP3) and PARG (GhPARG1), and silence three GhPARPs simultaneously (GhPARP1,2,3). Interestingly VIGS-GhPARP1, and GhPARP1/2/3 plants showed increased resistance to PEG (a reagent mimicking drought stress) treatment when growing in the hydroponic condition and to drought stress when growing in soil. However, VIGS-GhPARP2 plants showed increased sensitivity to PEG treatment when growing in hydroponic condition and to drought stress when growing in soil. The data suggest the coordination of different PARP members in regulating cotton drought stress. We are investigating the underlying mechanisms by identifying the PARP targets and characterizing the activities. Meanwhile, we have tested different resins for immunoprecipitating PARylated proteins for quantitative proteomics to identify differentially PARylated proteins in cotton under drought stress. We have shown that the macrodomain affinity resin (MD-resin) could bind both PARylated and mono(ADP-ribosy)lated (MARylated) proteins, and PARP14m3 resin only binds MARylated proteins. Comparing to PARylation, the chemistry and physiological functions of mono(ADP-ribosy)lation (MARylation) remain elusive. We will use MD-resin and PARP14m3 resin to identify ADP-ribosylated proteins (both PARylated and MARylated) and MARylated proteins during cotton response to drought stress coupled with quantitative proteomics (see research plan for details).

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang, P., Zhou, L., Jamieson, P., Zhang, L., Zhao, Z., Babilonia, K., Shao, W., Wu, L., Mustafa, R., Amin, I., Diomaiuti, A., Pontiggia, D., Ferrari, S., Hou, Y., He, P., and Shan, L. (2020) The cotton wall-associated kinase GhWAK7A mediates responses to fungal wilt pathogens by complexing with the chitin sensory receptors. Plant Cell 32: 39784001.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Babilonia, K., Wang, P., Liu, L., Jamieson, P., Mormile B., Rodrigues, O., Lin, W., Danmaigona Clement, C., Menezes de Moura, S., Alves-Ferreira, M., Finlayson, S.A., Nichols, R.L., Wheeler, T.A., Dever, J.K., Shan, L., and He, P. (2020) A non-proteinaceous Fusarium cell wall extract triggers receptor-like protein-dependent immune responses in Arabidopsis and cotton. New Phytologist. 10.1111/nph.17146.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wang, P., Mormile, B., and He, P. (2021) A GLoRy battle for cotton against Fusarium. Trends in Plant Science https://doi.org/10.1016/j.tplants.2021.04.007.
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Lahiri, A., Zhou, L., He, P., Datta, A. Detecting Drought Regulators using Stochastic Inference in Bayesian Networks. (Under review at BMC Plant Biology).