WRKY TRANSCRIPTION FACTORS: TOOLS FOR IMPROVING DROUGHT RESPONSES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1014820
• Grant No.: 2018-67013-27421
• Cumulative Award Amt.: $475,000.00
• Proposal No.: 2017-06259
• Project Start Date: Jan 1, 2018
• Project End Date: Dec 31, 2023
• Grant Year: 2018
• Program Code: [A1152]- Physiology of Agricultural Plants

Recipient Organization
UNIVERSITY OF NEVADA
4505 S Maryland Pkwy
LAS VEGAS,NV 89154

Performing Department
School of Life Sciences

Non Technical Summary
Environmental stresses depress the yields of major US crops by as much as 70%. Drought is one of the most severe climate-related risks for rice production and a further increase in rice production must come from highly vulnerable, less productive drought-prone rainfed lowland and upland rice areas. Even though rice is a paddy crop, it is susceptible to drought stress and many irrigated paddy lands are now frequented by terminal drought. Likewise, soybean production is threatened by drought, which severity is ever mounting due to global warming.Crops sense and respond to environmental stresses such as drought using their delicate circuits that are more complicated than those used in cell phones, airplanes and satellites. A circuit in plant cells consists molecular switches that are turned on or off by a set of proteins called transcription factors, which are analogous to fingers operating switches. Another set of proteins, analogous to a chain of commanders, instruct whether a "finger" should turn on or off a "switch" under specific physiological/developmental conditions. In the previous project funded by NIFA USDA, we demonstrated that a family of the molecular fingers, called WRKY transcription factors are major players in crops' responses to drought. In 2008, we suggested that such molecular fingers "may present some of the best opportunities for increasing drought tolerance." Our previous project and others around the world have now established that WRKY TFs are indeed major players in drought responses. There is now a pressing need to progress with these genes towards using them to improve drought tolerance in US crop plants. That is the goal of this project.To do this, we have chosen four WRKY transcription factors (molecular fingers) from rice and soybean which our data suggest play important roles in regulating drought responses. We will use state-of-the-art techniques including ChIP-seq, genome editing, protein-protein interaction techniques, promoter analyses, and overexpression. These techniques will allow us to dissect the circuits and pinpoint the key nodes in the circuits that involve the molecular switches and fingers in crops' responses to drought. Our research questions/objectives are: Can we identify interacting protein partners of the finger proteins? Can we determine targets of the finger proteins? Can we use the finger proteins to obtain improved drought phenotypes? Can we use the finger proteins to obtain an increase in soybean and rice?US agriculture has a need to identify key molecular switches and fingers that might be used for improving environmental stress responses in crop plants. This project will further characterize such targets and at the same time will facilitate development of new strategies for improving environmental stress responses based on a better understanding of the molecular mechanisms of the finger proteins outlined in this proposal. This project addresses the Physiology of Agricultural Plants program area priority, "Mechanisms of plant response to abiotic stresses."

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1530	1040	57%
203	1820	1040	43%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1820 - Soybean; 1530 - Rice;

Field Of Science
1040 - Molecular biology;

Keywords

wrky transcription factor

drought

high throughput phenotyping

improved crops

signaling

Goals / Objectives
Our main goal is to increase drought tolerance in US crop plants by the manipulation of WRKY genes that are key regulators of drought responses.In our previous project, we performed extensive transcriptome studies, combined phylogenetic analyses, promoter analyses, overexpression studies, and protein-protein interaction studies. This exciting research proved the validity of the hypothesis from our previous project - WRKY transcription factors are regulators of drought responses. In addition to publishing over a dozen papers and graduating three graduate students, we worked with over half a dozen different plant species including soybean, tobacco, switchgrass, Brachypodium distachyon, rice and barley. We found orthologous WRKY genes that appear to play important roles in regulating abiotic stress responses. We also found apparent species/family-specific regulators. While we produced data on target genes and interacting partners there is now a need to define the roles, targets, and interacting partners of these key WRKY transcription factors, in order to make further progress at the mechanistic and network levels. We will also address our ultimate goal - answering the question of whether we can use WRKY genes to improve drought responses.To do this, we have chosen four WRKY transcription factors from two species which our data suggest play important roles in regulating abiotic stress responses (GmWRKY53, GmWRKY56, OsWRKY24, and OsWRKY70). We will use state-of-the-art techniques including ChIP-seq, genome editing, protein-protein interaction techniques, promoter analyses, and overexpression to place key WRKY transcription factors into signaling networks. Dr. Imre Somssich has joined the project and this ensures that we have hands on experience of all the proposed techniques.Our Research Questions/Objectives are: Can we identify interacting protein partners? Can we determine target genes? Can we use WRKY TFs to obtain improved drought phenotypes? Can we use WRKY TFs to obtain an increase in yields? Can we position WRKY TFs in signaling networks?Drought is one of the most severe climate-related risks for rice production and a further increase in rice production must come from highly vulnerable, less productive drought-prone rainfed lowland and upland rice areas. Even though rice is a paddy crop, it is susceptible to drought stress and many irrigated paddy lands are now frequented by terminal drought.US agriculture has a need to identify key regulatory genes that might be used for improving abiotic stress responses in crop plants. This project will further characterize such genes and at the same time will facilitate development of new strategies for improving abiotic stress responses based on a better understanding of the molecular mechanisms of WRKY transcription factors. This project addresses the Physiology of Agricultural Plants program area priority, "Mechanisms of plant response to abiotic stresses."

Project Methods
We will use state-of-the-art techniques including yeast-two-hybrid screenings, ChIP-seq, genome editing, protein-protein interaction techniques, promoter analyses, and overexpression to place key WRKY transcription factors into signaling networks.For identifications of interacting partners, the Hybrigenics pipeline or in-house screening methods will be used. BiFC interactions will be imaged using a LSM 510 laser scanning microscope (Carl Zeiss). Co-IP followed by identification of protein partners will determine the composition of complexes. The experiments will identify WRKY-interacting proteins, validate these interactions in planta, and establish their subcellular locations.For Chip-seq, high quality reads will be mapped to the relevant genome using Bowtie. To identify genomic DNA regions enriched in sequencing reads in the ChIP sample compared to input control ('peak regions'), the peak calling algorithm of the QuEST program is applied using the transcription factor mode (option "2"), with permissive parameter settings for the peak calling (option "3"). To search for conserved binding motifs in the binding regions, 500 bp of sequence surrounding the peak maximum is extracted and submitted to the online version of MEME-ChIP. FIMO is also run with the peak sequences and the motif of interest (MEME/DREME output) as input and a p-value threshold of 0.001.The Chip-seq results will be used to determine chromatin occupancy over time by a specific WRKY transcription factor. This will establish both direct target genes and changes in target promoter occupancy during drought. These results will be of major interest and will help position specific WRKY proteins in drought signaling networks, facilitating novel strategies to improve drought responses in crops.To define the direct and indirect targets of WRKY transcription factors, the overexpression and knockout lines from rice and soybean will be subjected to RNA-seq analyses to determine which genes show altered expression levels compared to wild type plants during drought. RNA-seq is a standard procedure in our laboratories. The promoters of genes that show differential expression compared to controls will be subjected to MEME and word search analyses to determine whether potential W boxes are present in their promoter regions.Genes with W box-like sequences in their promoters are potentially direct targets of the WRKY transcription factor (this can be validated by the ChIP-seq data and promoters without W box-like sequences are most likely indirect targets of the WRKY protein. These data will help us build signaling networks.To address whether altering WRKY Transcription factor expression result in a drought phenotype, we will use the PlantScreen™ Robotic XYZ System, which controls the severity and duration of the drought stress. The system can be used to both induce defined stresses and identify the degree to which plants are resistant or susceptible. Water loss from plants will be measured by providing controlled watering/fertilization of plants in pots, with the mass of each pot measured on its individual load cell. At the same time a robotic arm carrying imaging devices will go from pot to pot to conduct the following measurements: (a) Laser scattering to monitor plant architecture and make 3D reconstructions of the plant. This would show changes in plant structure and leaf area index during growth and development. (b) RGB analysis for additional morphometric data, as well as for color analysis of the plants related to pigment composition. (c) Chlorophyll fluorescence kinetics to monitor photosynthetic efficiency. This will provide a rapid early indication of water stress, as well as monitor water stress progression and recovery. (d) Thermal imaging to identify variations in temperature. This will allow correlations between stomatal movements, water loss and drought tolerance. (e) Hyperspectral analyses using VNIR (350 - 1000 nm) and SWIR (1000 - 220 nm) cameras to measure plant reflective indices related to leaf biochemical status. Wavebands in the 1400 nm and 1900 nm region will be used to measure relative water content of the plants. This would be vital for any study of water loss and drought tolerance.This represents characterization of drought phenotypes at a level well beyond most published transgenic lines and is, we believe, a major strength of this proposal.Dr. Steve Hunt will analyze the drought response and yield data. The effect on crop yields judged by increased biomass and, in the case of soybean, bean yield will be correlated with drought responses. If a transgenic line reproducibly produces a higher yield after drought than the parental lines, then we have not only validated our hypothesis but we have also produced an improved plant line that could directly or indirectly help US agriculture.To determine positions of the WRKYs in the drought induced signaling network, we will reanalyze the gene expression to infer the co-expression network of the WRKY genes by computing the partial correlation of every gene pair. The final co-expression network will be obtained by keeping the edges that connect genes with high partial correlation that is statistically significant. The obtained co-expression network will be further analyzed by a network-clustering algorithm that subdivides the network into smaller network modules that consist of interacting genes that are functionally coherent. These detected modules may correspond to pathways in the network containing WRKY proteins.In addition to the proposed co-expression network analysis, we will perform a complementary network analysis based on the outcomes of the yeast 2-hybrid analyses to identify potential functional modules. The identified protein-protein interaction (PPI) network can be viewed as a skeletal network that embeds the signaling network induced by drought stress. To unveil the signaling network induced by drought and identify signaling pathways that may be responsible for the drought response of the plants, we will search through the obtained PPI network to detect functional modules. This will be achieved by overlaying the PPI network with the available gene expression data and identifying subnetwork regions that consist of co-expressed genes whose protein products are interconnected on the PPI network.The functional modules detected by these complementary schemes will be compared to identify potential overlap. Significant overlap between modules independently detected by different methods indicates strong potential to be associated with drought induced signaling pathways.The timeline, milestones and deliverables are shown in the following tables:Activity\Month61218243036Yeast 2H ScreensScreeningScreeningPartner identificationBiFC/Co-IPPartner confirmationPartner confirmationPartners confirmedChIP TransgenicsPrepare contructsTransformationSeed productionObtain ChIP LinesChIPTrial experimentsTrial experimentsChip-seqBinding sitesOE LinesOE linesRNA-SeqRNA-seqRNA-seqTarget genesCRISPRPrepare contructsTransformationSeed productionObtain KO LinesDrought PhenotypesDefined phenotypesNetwork AnalysesNetworksThe PD and co-PD will meet once every six months to evaluate the progress of the project, and adjust the strategies and management measures to ensure the successful, on-time completion of the project.

Progress 01/01/18 to 12/31/23

Outputs
Target Audience: Nothing Reported Changes/Problems:The pandemic significantly delayed our research progress. Other unforeseen circumstances also led to major changes in the project. Major Change 1: The soybean part of the project was largely truncated due to the departure of the Co-PD from the company and subsequent termination of project by the company. The subaward was pulled back to the PD's institution to support a postdoc. He made the constructs for the project. However, because soybean and rice require different growing conditions and we didn't have enough greenhouse space on the UNLV campus to work on soybean. Therefore, we decided to focus on the rice project with the following changes: 1) Instead of studying just the two WRKY genes we proposed (OsWRKY24 and -70), we extended the study to include all three close homologs, OsWRKY24, -53 and -70. The fund transferred from the soybean project allowed us to produce 7 different mutants - three single mutants (oswrky24, -53, and -70), three double mutants (oswrky24/53, 24/70, and 53/70), and one triple mutant. We also produced overexpression lines for these three genes using two different promoters, the strong, constitutive Ubiquitin promoter and the drought inducible OsNAC6 promoter. The represents a major increase in the workload for the rice project because the initial proposal only includes two single mutants (oswrky24 and -70) and one double mutant (oswrky24/70). 2) Instead of just doing yeast two-hybrid screening, we adopted a new technology, TurboID-mediated proximity labeling. This new approach will allow us to directly identify proteins interacting with the WRKYs in rice plants. 3) Instead of just studying the WRKY mutants, we extended the project to include two identified interacting proteins, OsNHL61 and OsR3H1. 4) We also performed a comprehensive study of WRKY gene evolution in both domesticated and wild rice species because wild rice species contain elite genes that we can explore to enhance drought tolerance. 5) Our work also extended to include OsWRKY71, whose disruption leads to drought response and germination phenotypes under normal and cold temperatures. Major Change 2: We have tried hard to work with Qubit Systems Inc. for phenotyping, as proposed in our proposal. We chose the company because of its impressive capacity of automated screening (RGB, 3D, Hyperspectral, Multispectral, Thermal, Chlorophyll Fluorescence). However, it was extremely difficult to move rice seeds from the US to Czechia, where the Qubit Systems had full phenotyping operations. We sought alternatives and got support from the Danforth Center in St. Louis, MO. We were planning to do the phenotyping experiments in the first quarter of 2023. However, several reasons prevented us from proceeding with the planned, large scale phenotyping: 1) low budget at the fifth year; 2) lacking of Cas9-free homozygous lines for OsWRKY70 and one more knockout line for OsWRKY24/70 and insufficient time for small scale tests to confirm the drought phenotype of some mutants at the time when the Danforth facility was available to us; and 3) Danforth Center didn't have the hyperspectral imaging capacity. The thermal imaging and hyperspectral imaging systems available on this campus made it possible for us to image the mutants on our campus. Major Change 3: The Co-PD assumed an adjunct professorship at the PD's institution and remained actively involved in various project aspects throughout its duration, including overseeing the postdoc, participating in graduate students' dissertation committees, revising manuscripts and grant reports, and contributing to experiment design. What opportunities for training and professional development has the project provided?This grant facilitated the training of a diverse cohort, including 1 postdoctoral fellow, 4 Ph.D. students (with two graduated), 1 M.S. student, 3 Post-Bacc fellows, and 31 undergraduate students. How have the results been disseminated to communities of interest?Our lab is the only major rice research lab in the state of Nevada. In addition to the publications and presentations listed in the "Products" section, participants in this project delivered over 25 presentations to faculty and students on campus. Notably, one Ph.D. student excelled, winning the competition in "Power in Diversity: Unlocking Creativity and Research Together," with their oral presentation available on YouTube (https://www.youtube.com/watch?v=FOF6H1kIFuY). Furthermore, our work contributing to food security was featured in campus news (https://www.unlv.edu/news/article/food-all) and highlighted on YouTube (https://www.youtube.com/watch?v=CdmdHxsc7ow). Rice plants produced during the course of this project served as a focal point for outreach efforts, engaging students, staff, faculty, and the general community in educational initiatives aimed at deepening understanding of rice biology and its global significance. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Major New Findings OsWRKY24, -53, and -70, all function as negative regulators of drought stress. Knocking out OsWRKY70 confers tolerance to both drought and salinity. OsNHL61 is a membrane bound transcription factor that is regulated by OsWRKY71 and interacts with OsWRKY53, -70, and -71. OsR3H1 is an R3H domain protein that is an important component of drought responses. Interaction of OsR3H1 and OsWRKY71 leads to translocation of OsR3H1 from the cytoplasm to the nucleus. Objective 1: Can we identify interacting protein partners? Major activities and data collected: We screened yeast two hybrid libraries using OsWRKY70 and -71 as baits and identified WRKY-interacting proteins. Transient expression studies tested their functions. Genome-wide RNA-seq data sets were produced to find drought-inducible genes. Confocal microscopy studied the subcellular location of the proteins. Summary and discussion of results: We expanded our list of target genes to six (OsWRKY24, -53, -70, -71, OsNHL61, and OsR3H1). Two proteins from the yeast two hybrid screenings stand out: an NDR1/HIN1-like protein (OsNHL61), which is a membrane bound transcription factor and an R3H-domain containing protein, OsR3H1. Transient expression studies show that both OsNHL61 and OsR3H1 regulate ABA and GA signaling. Excitingly, OsNHL61 is regulated by OsWRKY71, and also OsNHL61 interacts with OsWRKY53, -70, and -71. Transient overexpression of OsNHL61 represses ABA signaling showing it is a negative regulator. OsR3H1 is a positive regulator of drought responses and BiFC assays showed that interaction of OsR3H1 and OsWRKY71 led to translocation of OsR3H1 from the cytoplasm to the nucleus. Key outcomes or impacts: The impact of our results with OsNHL61 and OsR3H1 can hardly be overstated. These data are the first link between NHLs and WRKY transcription factors. We have also obtained important data for objectives 1, 2, and 3. We now have two important interacting partners, parts of two novel signaling pathways, and target genes that play roles in stress responses. Objective 2: Can we determine target genes? Major activities and data collected: RNA-seq was performed on flag leaves using WT and oswrky24/53double mutants under well-watered and 3-day no watering conditions. We also analyzed the transcriptomes of dry and germinating embryos. We identified DEGs and subnetworks underlying the biological processes. Summary and discussion of results: For drought, the numbers of DEGs were: 153 in drought-treated vs well-watered WT, 5,868 in drought-treated vs well-watered oswrky24/53 plants, 7,429 in wrky24/53 drought treated vs WT drought treated plants, and 1,641 in well-watered wrky24/53 vs well-watered WT. Disruption of the two WRKY transcription factor genes in oswrky24/53 plants greatly increases the number of DEGS (5,868 versus 153), presumably due to a major disruption of drought stress signaling. Additionally, OsWRKY71 was determined to be a master regulator, controlling the expression of 9-17% of genes in dry and germinating embryos. Key outcomes or impacts: The RNA-Seq data under water stress with the oswrky24/53 plants provide us with over 5,000 DEGS due to a major disruption of drought stress signaling. These new direct or indirect targets of the OsWRKY24 and -53 transcription factors are useful for improving drought responses in rice. Objective 3: Can we use WRKY TFs to improve drought phenotypes? Major activities and data collected: WE used state-of-the-art non-invasive imaging techniques such as thermal imaging and hyperspectral imaging to obtain high quality data to determine the phenotype of new lines. For OsWRKY24, -53 and -70, we made overexpression lines. Single, double and triple WRKY mutants and OsR3H1 mutants were made. Knockdown and knockout mutants of OsWRKY71 were obtained. Plant responses to drought were non-invasively investigated using thermal and hyperspectral imaging. Photosystem II efficiency and chlorophyll content were also measured. Summary and discussion of results:The plants mutated in OsR3H1 that encodes a WRKY-interacting protein have more dried leaves and higher temperatures than WT. On day 7 after recovery, the PSII efficiency of osr3h1 is much lower than that of WT. The mutant's stomatal conductance initially was significantly higher prior to drought and decreased over the 3-day drought treatment while WT stayed constant. The transpiration rate follows the same trend. The data suggest that the osr3h1 mutants are drought sensitive. Key outcomes or impacts: OsR3H1 is a positive regulator of drought resistance. Knockout mutants have a drought sensitive phenotype. Our data suggest the greater stomatal conductance and transpiration rate in the osr3h1 mutant leads to greater water loss and increased drought sensitivity. We have elucidated part of the molecular mechanism because complementation assays show that interaction of OsR3H1 and OsWRKY71 leads to translocation of OsR3H1 from the cytoplasm to the nucleus. Our novel data provide new targets for increasing drought tolerance in rice. With Dr. Mingon Kang (UNLV) we are developing artificial intelligence models for drought stress in rice based on our thermal and hyperspectral imaging data. Objective 4: Can we use WRKY TFs to obtain an increase in yields? Major activities data collected:In addition to reducing losses due to drought, one way to increase rice yields is to increase seed production. We looked at booting, heading and seed production in single and double knockout lines of oswrky24/53 plus knockout lines of osOsR3H1. Summary and discussion of results: Single and double mutants of OsWRKY24/53 and osOsR3H1 have an accelerated booting and heading compared to WT. Wildtype had a longer panicle length. Mutant lines of OsWRKY24, OsWRKY53, and OsR3H1 all display an early seed production time. Key outcomes or impacts:Single and double knockouts of OsWRKY24 and OsWRKY53 as well as a knockout of OsR3H1 showed early seed production. OsWRKY24, OsWRY53, and OsR3H1 may be parts of the same pathway during development and seed production. Our findings hold promise for the development of novel rice lines with increased grain yields. Crucially, overexpression lines of these genes under field conditions may show later seed production and potentially an increase in grain yields due to an extended growing season. Objective 5: Can we position WRKY TFs in signaling networks?? Major activities and data collected: We have had success in starting to understand the signaling pathways that our four WRKY transcription factors are part of during drought. RNA-seq analyses identified many potential target genes, both direct and indirect of the WRKY proteins and we identified two interacting proteins (OsNHL61 and OsR3H1) that change our understanding of the signaling pathways. We expanded our RNA-seq analyses to the early booting/seed filling phenotype in flag leaf of oswrky24/53 in 3-month-old plants. We also analyzed the transcriptome network of OsWRKY71. Summary and discussion of results:Plant hormone signaling pathways involving cell enlargement and plant growth were upregulated in oswrky24/53. Transcriptional alterations in oswrky24/53 compared to wildtype included upregulation of starch, sucrose, galactose, and riboflavin metabolism, indicating a role in stress mitigation. With OsWRKY71, WGCNA and SC-ION analysis revealed clusters of genes involved in ABA signaling, consistent with the ABA hyposensitive phenotype described above. Key outcomes or impacts: There are links between WRKY transcription factors that regulate drought responses and those that regulate seed germination. Several WRKY proteins play roles in regulating both processes and a common component appears to be the plant hormone ABA. Our new data suggest that WRKY transcription factors are key components in ABA responses including processes such as drought responses and germination and that they hold great promise for the development of improved rice varieties.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Villacastin, A.J., Adams, K.S., Boonjue, R., Rushton, P.J., Han, M., Shen, Q.J.: Dynamic differential evolution schemes of WRKY transcription factors in domesticated and wild rice, Scientific Reports, 2021, 11:14887, doi: 10.1038/s41598-021-94109-4
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Bataller, S., Villacastin, A.J., Shen, Q.J., Bergman, C., Comparative nutritional assessment and metabolomics of a WRKY rice mutant with enhanced germination rates, Agronomy, 2023, 13, 1149
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Victoria Amato, Liyuan Zhang, Linkun Gu and Qingxi J. Shen, Identification and transcription profiling of the NHL (NDR1/HIN1-like) gene family in rice, Plant Biology 2019, August 3-7, San Jose, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Santi Bataller, Linkun Gu, Liyuan Zhang, Victoria Amato, and Qingxi J. Shen: WRKY71 transcription factor mediates the crosstalk of abscisic acid and gibberellin signaling in rice, Plant Biology 2019, August 3-7, San Jose, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Anne Jinky Villacastin, Keeley Adams, Rin Boonjue and Mira Han and Qingxi J. Shen: Evolution of the WRKY transcription factors across Oryza species, Plant Biology 2019, August 3-7, San Jose, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Bataller, S., Shen, Q.J., and Bergman, C.: Comparative nutritional assessment of a WRKY mutant with enhanced germination rates, 2021, Cereals and Grains Association, Online
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Villacastin, A.J., Adams, K.S., Boonjue, R., Rushton, P.J., Han, M., Shen, Q.J.*: Dynamic differential evolution schemes of WRKY transcription factors in domesticated and wild rice, Plant Biology 2021, Online
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Bataller, S., Davis, J., Gu, L., Baca, S., Chen, G., Majid, A, Villacastin, A.J., Barth, D., Han, M. Rushton, P., and Shen, J., The OsWRKY71 transcription factor negatively regulates rice seed germination speed under normal and stress conditions, Plant Biology 2023, August 5-9, Savannah, Georgia
• Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Bataller, S., Davis, J., Gu, L., Baca, S., Chen, G., Majid, A, Villacastin, A.J., Barth, D., Han, M. Rushton, P., and Shen, J., The OsWRKY71 transcription factor negatively regulates rice seed germination speed under normal and stress conditions
• Type: Journal Articles Status: Other Year Published: 2024 Citation: Amato, V., Mahalath, S., Villacastin, A.J., Gu, L., Zhang, L, Rushton, P.J., Shen, Q.J., OsWRKYs interact with OsR3H1 to regulate drought response of rice (Oryza sativa subsp. japonica), In Preparation
• Type: Journal Articles Status: Other Year Published: 2024 Citation: Amato, V., Zhang, L, Gu, L., Rushton, P.J., Shen, Q.J., The roles of the NHL (NDR1/HIN1-like) gene family in plant response to abiotic stresses. In Preparation

Progress 01/01/22 to 12/31/22

Outputs
Target Audience: Nothing Reported Changes/Problems:We have extended the rice project to include three OsWRKY genes, resulting in an increase of the number of mutants to study from 3 to 7. This dramatically increased the workload and time of producing mutants, greenhouse space demands for bulking up seeds, and time for performing pilot experiments. We also need to establish the best treatments and times to see the best differences in phenotypes of the mutants. We have tried hard to work with Qubit Systems Inc. for phenotyping, as proposed in our proposal. We chose the company because of its impressive capacity of automated screening (RGB, 3D, Hyperspectral, Multispectral, Thermal, Chlorophyll Fluorescence). However, it has been very difficult to get a slot from the company in the past years due to the impact of the pandemic. Besides, it is very challenging to move rice seeds from the US to either Canada or Czechia, where the Qubit Systems has full phenotyping operations. We sought alternatives and got support from the Danforth Center in St. Louis, MO. We were planning to do the phenotyping experiments in the first quarter of 2023. However, we still need to have Cas9-free homozygous lines for OsWRKY70 and one more knockout line for OsWRKY24/70 (See Objective 3.1). Also, Danforth Center doesn't have the hyperspectral imaging capacity. The thermal imaging and hyperspectral imaging systems available on this campus made it possible for us to image the mutants on our campus. What opportunities for training and professional development has the project provided?This project helped train 1 postdoc, 4 graduate students and 15 undergraduate students in plant biology research during this interim. How have the results been disseminated to communities of interest?We have one paper published, submitted one and prepared two more to report our discoveries. Five posters were also presented in national/international conferences. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Can we identify interacting protein partners? TurboID-mediated proximity labeling allows us to identify interaction proteins in organisms of interest (rice in our case), rather than yeast. We generated a construct harboring OsWRKY71 driven by its native promoter and tagged with 3X Hemagglutinin (HA) tag and TurboID (W71p-OsWRKY71-HA-TurboID. For the control, we made W71p-GFP-TuboID-3HA. We then generated stable W71p-OsWRKY71-TurboID-3HA and W71p-GFP-TuboID-3HA transgenic lines in the oswrky71-1 background via Agrobacterium-mediated transformation. GFP expression was confirmed in oswrky71-1 transformed with OsWRKY71p-GFP-TurboID-3HA. Western blot analysis is undergoing to identify lines expressing the transgene at high levels. Those lines of the transgenic plants will be studied for OsWRKY71 interaction proteins. If the result is promising, we will produce lines for other WRKY genes of interest. Objective 2: Can we determine target genes? We have RNA-sequencing data for the WT and the oswrky24/53 double mutant showing drought response phenotype in young and flag leaves. Analyzing the data will allow us to determine the direct and indirect targets of these two transcription factors. Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? We have identified all but one of the planned transgenic and mutant lines for this project. The phenotyping results are very promising. All graduate students will help accomplish experiments proposed for this objective. Objective 4: Can we use WRKY TFs to obtain an increase in yields? We have set up non-invasive thermal imaging and hyperspectral imaging to assess the differences of WT and WRKY mutants in drought responses, in addition the setups of equipment/systems for stomata imaging and measuring chlorophyll fluorescence kinetics (Fv/Fm), Quantum yield of fluorescence (PhiPS2), water potentials, iron leakage, and H2O2 contents. In addition, we will continue to study the agronomic traits to assess the trade-offs of the altering drought responses and predict the potential for yield increases.

Impacts
What was accomplished under these goals? Objective 1: Can we identify interacting protein partners? Major activities/experiments conducted: OsDIP1 encodes an R3H domain protein interacting with WRKYs. 4-week-old WT and osdip1-1 and -2 plants were not watered for 13 days before photosystem II efficiency (PhiPS2) was measured using a LI-600 porometer/fluorometer. Data collected: At day 11, PSII efficiency of osdip1-1 and -2 was significantly reduced with a 1.3-fold decrease compared to wild type. This data suggests that osdip1-1 and -2 are drought sensitive. Summary and discussion of results: Knockout mutants osdip1-1 and -2 are drought sensitive. Based on this data, we suggest that the interaction of DIP1 with stress responsive WRKYs mediates drought responses. Key outcomes or impacts: OsDIP1 can be a potential candidate in producing drought-tolerant rice varieties. Objective 2: Can we determine target genes? Major activities/experiments conducted: RNA-seq were performed for WT and oswrky24/53-2 mutant flag leaves under well-watered and 3-day no watering conditions. Data collected: The numbers of differentially expressed genes (DEGs) are: 153 in drought-treated vs well-watered WT, 5,868 in drought-treated vs well-watered oswrky24/53 plants, 7,429 in wrky24/53 drought treated vs WT drought treated plants, and 1,641 in well-watered wrky24/53 vs well-watered WT. Summary and discussion of results: Photosynthesis in the flag leaf provides the majority of the carbohydrates needed for grain filling--so it is the most important leaf for yield potential. RNA-seq data of the well-watered vs drought-treated WT flag leaf revealed key genes crucial for the function of this important leaf in controlling rice crop yields. Comparisons of DEGs in WT and oswrky24/53 identified key DEGs that are either directly or indirectly regulated by OsWRKY24/53. Key outcomes or impacts: This experiment helped us identify target genes of OsWRKY24/53 and hence understand further how WRKYs regulate drought response. Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? Objective 3.1. Production of knockout lines for OsWRKY24, -53 and -70. Major activities/experiments conducted: Following-up genotyping indicated the need of producing two Cas9-negative, homozygous OsWRKY70 and one more OsWRKY24/70 line. Data collected: PCR screening revealed the presence of Cas9 in all oswrky70 lines. Sequencing of one OsWRKY24/70 double mutant line revealed a mutation in OsWRKY53 too. However, the mutation doesn't interrupt the WRKY motif and hence may not affect the function of OsWRKY53. As a backup, we are designing a new OsWRKY70 CRISPR construct to create more OsWRKY24/70 double knockout mutants. Summary and discussion of results: With the additional work, we will have at least two lines of Cas9-negative, homozygous single, double, and triple mutant lines for OsWRKY24, 53 and 70. Key outcomes or impacts: We have generated key genetic reagents essential for the success of this project. Objective 3.2: To investigate the role of the three regulators in rice response to drought. Major activities/ experiments conducted: Two lines each of homozygous single and double mutants of OsWRKY24 and -53 were assayed for responses of 2-week-old plants to a PEG treatment and 1 month-old plants to no-watering in the greenhouse. Also 3-month-old wrky24/53 double-mutant plants were subjected to 3-day no-watering, followed by assays for drought responses. Data collected: 1) Data of quantum yield of fluorescence (PhiPS2), an indicator of PSII efficiency, indicated that after PEG treatment for 2 hours, PhiPS2 was significantly lower in oswrky53 than WT. At day 6 the WT recovered to the optimal PhiPS2 baseline (0.7), while oswrky53 mutants were still below it. 2) Under greenhouse conditions, drought affected the WT more compared to the PEG treatment. After water withholding and rewatering, oswrky24/53 mutants exhibited significantly higher survival rates than the WT or any of the single knockout mutants. 3) For the flag leaves of 3-month plants, PhiPS2 started to decline after 2 days of drought in the oswrky24/53. At day 3, PhiPS2 significantly decreased more in oswrky24/53 than WT. Also, WT had a better recovery than oswrky24/53. Similar results were shown for chlorophyll fluorescence (Fv/Fm). Survival rate was measured 7-days after recovery and showed WT having a 100% recovery rate while oswrky24/53 had a 27.5% recovery rate. Summary and discussion of results: Knocking out both OsWRKY24 and -53, but not individual genes, improved drought (water-withholding) tolerance of greenhouse grown, 1 month-old rice plants. In 3-month-old plants, the effect was totally opposite; the mature plants of oswrky24/53 were more sensitive to drought. We will determine if the individual knockout mutants have the same or similar drought phenotypes at the latter stages of the life cycle. Key outcomes or impacts:Data in this and next section clearly suggest that OsWRKY24 and 53 play significant roles in drought responses. This transgenic double knockout plant can be used to further the field in the understanding of drought responses within the WRKY superfamily. Objective 4: Can we use WRKY TFs to obtain an increase in yields? Objective 4.1. Using non-invasive thermal imaging and hyperspectral imaging to assess the differences of WT and WRKY mutants in drought responses. Major activities/ experiments conducted: Experiments were conducted to study the temperature differences of WT and wrky24/53 double mutants (two lines) using the FLIR T640 camera. A UV-VIS-NIR spectrometer (StellarNet, Inc) was set up in my lab to measure the reflectance of rice leaves by following the method reported 1. Data collected: Thermal images were taken on day 0 and 3 of no-watering and after re-watering for 7-day. The temperature of the whole plant was cooler in the WT at day 3 of drought compared to wrky24/53. After recovery, WT was cooler in temperature compared to wrky24/53. Spectral signatures reported for the leaf reflectance within the visible and infrared wavelengths were reproduced for our plants. Summary and discussion of results:Setting up the imaging system in the lab put us in a great position to phenotype the rice plants with several non-invasive methods. Key outcomes or impacts: We are collaborating with Dr. Mingon Kang in the College of Engineering to develop artificial intelligence models for drought stress responses in rice based on thermal and hyperspectral imaging data. The model can be used to study drought stress in other plants/crops. Objective 4.2. Studying the agronomic traits to reveal trade-offs of the altering drought responses and predict the potential for yield increases. Major activities/ experiments conducted: Single and double oswrky24 and -53 mutants were assessed for changes in plant development (height and seed morphology), leaf senescence, and seed germination. Data collected: Plant height of all mutants were slightly decreased in comparison to WT during tillering stage and during seedling development stage. Grains produced were significantly shorter for mutants, however, seed widths were not reduced. Of the three different mutants, oswrky53 had the greatest reduction in seed length, followed by the double knockout and oswrky24 had just the slightest reduction. This suggests that OsWRKY24 and OsWRKY53 positively regulate height and seed length development. The oswrky24/53 grown in the greenhouse showed early senescence and those mutant seeds decreased germination rates under exogenous ABA treatment (2.5 microM). Summary and discussion of results: OsWRKY24 and 53 play key roles in plant development, seed germination, senescence, and drought response. All these plant processes have been linked to ABA signaling. Key outcomes or impacts: Data obtained here will help us develop better strategies to improve crop yields. Objective 5: Can we position WRKY TFs in signaling networks? No update Reference: 1. Jiang, J. et al. Plant Methods 14, 23, 2018?

Publications

• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Santiago Bataller, Anne J. Villacastin, Qingxi J. Shen and Christine Bergman, Comparative nutritional assessment and metabolomics of a WRKY rice mutant with enhanced germination rates, Agronomy

Progress 01/01/21 to 12/31/21

Outputs
Target Audience:Unfortunately, most students nowadays don't understand the importance of agriculture research and hence don't value it much. Consequently, we don't have many students interested in agriculture research, to say nothing of having agriculture as their career choice. Three of my graduate students, fully or partially supported by this grant, gave two poster presentations in international conferences, and three oral and three posters presentations to the faculty and students in School of Life Sciences UNLV (approximately 60 people each time) about the goals and importance of this research project. Changes/Problems:The subaward has been pulled back to UNLV due to the reason described in the annual report last year. For the soybean project, the postdoc made the constructs for the project. However, because soybean and rice require different growing conditions and we don't have enough greenhouse space on the UNLV campus to work on soybean. Therefore, we decided to focus on the rice project with the following changes: Instead of studying just the two WRKY genes we proposed (OsWRKY24 and -70), we extended the study to include all three close homologs, OsWRKY24, -53 and -70. The fund transferred from the soybean project allowed us to produce 7 different mutants - three single mutants (oswrky24, -53, and -70), three double mutants (oswrky24/53, 24/70, and 53/70), and one triple mutant. The represents a major increase in the workload for the rice project because the initial proposal only includes two single mutants (oswrky24 and -70) and one double mutant (oswrky24/70). Instead of just doing yeast two-hybrid screening, we adopted a new technology, TurboID-mediated proximity labeling. This new approach will allow us to directly identify proteins interacting with the WRKYs in rice plants. Instead of just studying the WRKY mutants, we extended the project to include two identified interacting proteins, OsNHL61 and OsR3H1. We also performed a comprehensive study of WRKY gene evolution in both domesticated and wild rice species because wild rice species contain elite genes that we can explore to enhance drought tolerance. What opportunities for training and professional development has the project provided?This project helped train 1 postdoc, 3 graduate students and 5 undergraduate students in plant biology research during this interim. How have the results been disseminated to communities of interest?We have one paper published and are writing two more to report our discoveries. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Can we identify interacting protein partners? We were planning to perform more rounds of screenings to reach the saturation point of identifying interaction proteins. We have identified two very promising interaction proteins and did follow up studies. Also, a new technology, namely TurboID-mediated proximity labeling, has been recently developed to allow ones identify interaction proteins in organisms of interests, rather than yeast. Drs Zhiyong Wang and Shou-Ling Xu labs at the Carnegie Institution for Science have applied this technology to identify interaction proteins in Arabidopsis 13. Both PIs have agreed to collaborate with us to test the system in rice. In this interim, we generated a construct harboring OsWRKY71 driven by its native promoter and tagged with 3X Hemagglutinin (HA) tag and TurboID (W71p-OsWRKY71-HA-TurboID. For the control, we made W71p-GFP-TuboID-3HA. We then generated stable W71p-OsWRKY71-TurboID-3HA and W71p-GFP-TuboID-3HA transgenic lines in the oswrky71-1 background via Agrobacterium-mediated transformation. So far, we have produced 17 transgenic for W71p-OsWRKY71-TurboID-3HA and 5 plantlets for W71p-GFP-TuboID-3HA. Selected lines of the transgenic plants will be studied for OsWRKY71 interaction proteins. If the result is promising, we will produce lines for other WRKY genes of interest. Objective 2: Can we determine target genes? Now that we have produced knockout mutants and prepared HA-tagged constructs, we are in a better position to produce transgenic plants for this project. We will also perform RNA-sequencing for the wildtype and the wrky mutants showing drought response phenotype. Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? We have produced most of the planned transgenic and mutant lines for this project. The initial phenotyping results are very promising. The lab has just recruited two more new graduate students and they will help us accomplish experiments proposed for this objective. Objective 4: Can we use WRKY TFs to obtain an increase in yields? We have demonstrated that overexpression of OsWRKY53 or -70 resulted in enhanced sensitivity to drought while the oswrky24/53 double mutant is more tolerant to drought tolerance. However, we only did the drought study for a short period of time. We plan to study the performance of the plants under longer drought condition and measure different indexes for yields, such as 1000-seed weights and panicle numbers. Field trials are needed to provide a "real world" answer once new fundings are secured. Objective 5: Can we position WRKY TFs in signaling networks? Once we have more data from Objective 2, we will be at a good position to address this objective using a bioinformatics approach. We plan to focus on WRKY24 and -53 single and double knockout mutants for phenotyping. Once we know the major gene involved in drought resistance, we will focus on the subnetwork centered on that WRKY transcription factor.

Impacts
What was accomplished under these goals? Objective 1: Can we identify interacting protein partners? Major activities/experiments conducted: OsNHL61 interacts with OsWRKY53, -70 and 71. Therefore, studying their interactions will provide insights into the molecular basis of OsNHL61 in different aspects of rice biology including stress responses. Data collected: We created BiFC constructs for this project and made the OsNHL61 reporter construct (OsNHL61pro-GUS). We also produced rice protoplasts in the lab for transient expression studies. OsWRKY71 did not show any effect on OsNHL61 pro-GUS. We also made transgenic plants expressing HA-tagged OsNHL61 (UBIpro-OsNHL61::3XHA) and prepared a binary construct containing GFP::OsNHL61 gene driven by the OsNHL61 native promoter and a construct containing RFP::OsWRKY71 driven by the OsWRKY71 native promoter. These constructs have been introduced into Kitaake rice. Summary and discussion of results: The OsNHL61 protein, being an interacting protein of OsWRKY71, also suppresses GA response. We also have optimized transient assays using protoplasts from rice sheath tissue. This system will allow us to determine if these two proteins interact in rice cells. Key outcomes or impacts: OsNHL61 and OsWRKY53, -70 and -71 as well as their orthologs in other species are involved in both abiotic and biotic stress responses, and/or seed germination. Therefore, these genes could serve as target genes to improve seed germination uniformity and crops' tolerance to both abiotic and biotic stresses. Objective 2: Can we determine target genes? Major activities/experiments conducted: For the CHIP-seq experiment, the HA epitope was used to tag OsWRKY53 and -70. Data collected: The constructs were confirmed by digestion/electrophoresis and DNA sequencing. These constructs will be transformed into rice oswrky mutant plants. Summary and discussion of results: Studies have shown that constitutive activation of transcription factor genes using a strong promoter can result in growth retardation and compromised productivity 1. Native promoters of the WRKY genes of interest were therefore used. Key outcomes or impacts: This study will provide better insight into pathways involved these regulators under drought. We will also explore other possible pathways and therefore novel functions that these proteins may perform apart from stress response. Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? Objective 3.1. Production of knockout lines for OsWRKY24, -53 and -70. Major activities/experiments conducted: Single, double and triple knockout mutants for OsWRKY24, -53 and -70 have been produced using the CRISPR/Cas9 gene editing system2. Transformed rice (cv. Kitaake) were thoroughly genotyped. Data collected: T0 rice seedlings (CASP9 positive) created using Agrobacterium-mediated transformation were screened for mutations in genes OsWRKY24, -53 and -70. Because of the extremely low efficiency of obtaining large deletion mutants, we did Sanger Sequencing of PCR products to screen for mutants. To verify edits, superimposed sequencing chromatograms were decoded using the Degenerate Sequence Decode (DSDecode) program3,4 and CRISP-ID5. Mutation rates for OsWRKY24, -53 and -70 were 72%, 68% and 32%, respectively. Where preferred edit for OsWRKY24 was deletion of 1-5 nucleotides, OsWRKY53 and -70 was a single nucleotide insertion, 86% and 40%, respectively. For the T1 generation lines, a total of 102 samples were assessed. Plants with mutations were 90%, 81% and 69% for OsWRKY24, -53 and -70, respectively. Summary and discussion of results: Sequencing data for 234 T0, 401 T1 and 43 T2 generation samples were performed, for a total of 678 sequencing runs. Key outcomes or impacts: We have generated key genetic reagents essential for the success of this project and have screened up to T2 generation lines for some knockout combinations. To prevent further CRISPR-CAS9 mediated off-target mutations, we have identified transgene-free edited plants in the T1 generation. Objective 3.2: To investigate the role of the three regulators in rice response to drought Major activities/ experiments conducted: Homozygous, transgene-free double knockout mutant lines for OsWRKY24 and -53 were assayed for its response to drought stress, The experiment tested 1-month old plants by withholding watering for 14 days before rewatering for 7 days. Photosynthetic efficiencies and survival rates were determined. Data collected: Survival rate of WT was at least a third less than the double knockouts for OsWRKY24/53 (Student's t-test, p=0.009), which means that simultaneous knockout of the highly homologous genes OsWRKY24 and -53 conferred improved drought tolerance to rice plants. Close examination of the response of photosynthetic efficiency during drought stress shows that upon two weeks of water withholding, WT has significantly dropped its efficiency to almost half that of the double knockouts. Recovery of the WT after rewatering was seen after one week but were still about 30% lower than that of the double knockouts. Summary and discussion of results: Mutants appear to be more tolerant to drought, consistent with our hypothesis that these transcription factors function as negative regulators of drought response. This observation is supported by our prior study: Knocking out OsWRKY70 confers rice tolerance to drought and salinity stress, whereas the overexpression of the reputed repressor made it more susceptible to both stressors 6. Key outcomes or impacts: Our study tests the hypothesis that close homologs OsWRKY24, -53, and -70 function as negative regulators of rice's response to drought stress. Increased tolerance to drought of OsWRKY24/-53 supports our hypothesis. If confirmed, we will proceed with the elucidation of the molecular mechanisms by which these WRKY genes regulate stress response. Future directions involve drought stress assays for homozygous lines of all single, double and triple knockout lines. Objective 4: Can we use WRKY TFs to obtain an increase in yields? Major activities/ experiments conducted: Dwarfed phenotype we observed in previously created overexpression lines of OsWRKY53 driven by the Ubi promoter, which is consistent with studies in other rice cultivars 7,8. To circumvent this issue, we used a 1.8 kb drought-inducible OsNAC6 promoter to overexpress genes of interest in transgenic Kitaake rice. Data collected: To date, 21 of 27 T0 transgenic lines were verified for OsNAC6p-w53cds overexpression mutants and 15 of 20 for OsNAC6p-w70cds. Phenotype of the T0 and T1 generation mutants of the OsNAC6p-w53cds overexpression mutants did not lead to any growth defects, height was not significantly different from WT (Student's t-test, p=0.940) and neither were the seed length, width nor the length to width (LxW) ratio (Student's t-test, p=0.121, p=0.672, and p=0.384). Summary and discussion of results: Production of stress-inducible overexpression lines of our WRKY proteins could allow us to grow these plants under stressful conditions without the growth tradeoffs. Key outcomes or impacts: Producing non-dwarf OsWRKY53 and -70 lines will not only help advance our understanding of the roles of these two genes in plant growth and responses to drought. Objective 5: Can we position WRKY TFs in signaling networks? More results from Objective #2 are needed prior to initiating studies under this objective. References: 1 Wu, X et al. Plant Cell Rep. 28, 21-30 (2009). 2 Xie, K et. al. PNAS USA 112, 3570-3575 (2015). 3 Xie, X. et al. Mol Plant 10, 1246-1249 (2017). 4 Liu, W. et al. Mol Plant 8, 1431-1433 (2015). 5 Dehairs, J. et al. Sci Rep 6, 28973 (2016). 6 Zhang, L. PhD thesis, University of Nevada Las Vegas (2015). 7 Tian, X. et al. Plant Physiol 175, 1337-1349 (2017). 8 Hu, L. et al. Plant Physiol 169, 2907-2921 (2015). 9 Bi, Y. et al. Nat Comm 12, 945 (2021).

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Villacastin, A.J., Adams, K.S., Boonjue, R., Rushton, P.J., Han, M., Shen, Q.J.: Dynamic differential evolution schemes of WRKY transcription factors in domesticated and wild rice, Scientific Reports, 2021, 11:14887

Progress 01/01/20 to 12/31/20

Outputs
Target Audience:Unfortunately, most students nowadays don't understand the importance of agriculture research and hence don't value it much. Consequently, we don't have many students interested in agriculture research, to say nothing of having agriculture as their career choice. Three of my graduate students, fully or partially supported by this grant, gave three presentations to the faculty and students in School of Life Sciences UNLV (approximately 60 people each time) about the goals and importance of this research project. One undergraduate student also gave a presentation in the UNLV Undergraduate Research Forum. Changes/Problems:The subaward has been pulled back to UNLV due to the reason described in the annual report last year. As planned, we hired a postdoc, Dr. James Davis. He has helped accelerate the research progress for both the rice and soybean project. In addition, we worked with UNLV Office of Sponsored Program and USDA NIFA to have our grant extended for one year at no cost. What opportunities for training and professional development has the project provided?This project helped train 1 postdoc, 3 graduate students and 9 undergraduate students in plant biology research during this interim. How have the results been disseminated to communities of interest?We have submitted one manuscript and are writing two more to report our discoveries. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Can we identify interacting protein partners? We were planning to perform more rounds of screenings to reach the saturation point of identifying interaction proteins. However, the graduate students have been tied to work on transforming, screening and analyze rice plants. So, we are planning to have a company to perform this task. We hired a new postdoc James Davis, who will work with graduate student Victoria Amato to perform BiFC to confirm the interactions of OsHNL with OsWRKY70 and OsWRKY24, respectively. They will also do co-immunoprecipitation experiments. Objective 2: Can we determine target genes? Produce OsWRKY70-HA transgenic plants. RNA-sequencing for the wildtype, oswrky24 and -70 mutants, respectively. With the triple and double mutants and the hiring of the postdoc to help speed up the project, we are not in a much better position to produce genetic reagents needed for this objective and perform required experiments. Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? As stated above, we have produced HA tagged-OsWRKY70 and OsWRKY24 overexpression transgenic plants. The postdoc will help us accomplish experiments proposed for this objective. Objective 4: Can we use WRKY TFs to obtain an increase in yields? We have demonstrated that overexpression of OsWRKY53 or -70 resulted in enhanced sensitivity to drought. We predicted that knockout of one or both WRKY genes will make rice plants more resistant to drought. Objective 5: Can we position WRKY TFs in signaling networks? Once we have data from Objective 2, we will be at a good position to address this objective using a bioinformatics approach. We plan to focus on WRKY24 and -70 single and double mutants for phenotyping. Once we know the major gene involved in drought resistance, we will focus on the subnetwork centered on that WRKY transcription factor.

Impacts
What was accomplished under these goals? Objective 1: Can we identify interacting protein partners? Major activities/experiments conducted: During this interim, OsNHL61 was found to be regulated by OsWRKY71, a gene reported to be involved in seed germination1, cold responses2 and disease resistance3. A large-scale RNA-seq analysis was performed with wildtype and a dSpm transposon knockout, oswrky71-1. Data collected: OsNHL61 is among many genes that are found to be directly or indirectly regulated by OsWRKY71. Knock-down of OsWRKY71 decreased the expression of OsNHL61 by more than 2-fold (p = 5.21E-05). This suggests that OsWRKY71 is a positive regulator of OsNHL61, possibly by directly binding to the OsNHL61 promoter. Summary and discussion of results: Our RNA seq data suggests that OsWRKY71 regulates the OsNHL61 gene. We predict that the OsNHL61 protein is membrane-bound transcription factor (MTF). These specific transcription factors can be released from the membrane via the regulated intramembrane proteolysis (RIP) mechanism4, which includes proteolytic cleavage from the membrane via intracellular proteases, or the regulated ubiquitin-proteasome-dependent processing (RUP) mechanism5, which includes ubiquitination and subsequent proteolysis of a specific region on the MTF6. Key outcomes or impacts: The discovery that OsWRKY71 is likely a positively regulator of OsNHL61 reveals a key link between seed germinator and pathogen defense. These genes could serve as target genes to improve seed germination uniformity and crops' tolerance to both abiotic and biotic stresses. Objective 2: Can we determine target genes? Major activities/experiments conducted: Data collected: No reports for the interim Summary and discussion of results: No reports for the interim Key outcomes or impacts: No reports for the interim Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? Objective 3.1. Production of knockout lines for OsWRKY24, -53 and -70. Major activities/experiments conducted: We used the polycistronic tRNA-gRNA gene (PTG) CRISPR/Cas9 system to produce knockout mutants7. In this interim, we produced 95 CAS9-positive T0 rice seedlings using Agrobacterium-mediated transformation with four PTG constructs. The first PTG cassette contains two guide RNAs for each of three targeted WRKY genes, OsWRKY24, -53 and -70. Two double-gene PTG cassettes contain two guide RNAs for each of the two WRKY genes, OsWRKY24/53 and 53/70. Data collected: Of the expected triple KO lines, 21 of 22 had mutations in at least one target sequence of one gene. For double KO lines, 38 of 41 had mutations in gene OsWRKY24 and/ or OsWRKY53, while 27 of 32 had mutations in gene OsWRKY53 and/or OsWRKY70. Of the 21 mutant lines successfully transformed using the Triple KO CRISPR construct, 12 have at least one mutation in each of the three target genes while three of these have mutations in the first gRNA region. Summary and discussion of results: We have produced transgenic plants for single, double and triple knockout mutants of OsWRKY24, 53 and 70. Key outcomes or impacts: We have generated key genetic reagents essential for the success of this project: T0 progeny Kitaake rice plants with triple, double and even single knockout of drought inducible OsWRKY24, -53 and -70. To prevent further CRISPR-CAS9 mediated off-target mutations, we must screen for edited plants that are transgene-free, and then proceed with phenotypic and functional experiments to test our hypothesis that the three WRKY regulators perform necessary functions in the response of rice to drought stress. Objective 3.2: To investigate the role of the three regulators in rice response to drought Major activities/ experiments conducted: We phenotyped the UBI promoter-OsWRKY53 and UBI promoter-OsWRKY70 overexpression transgenic lines in the Nipponbare background, with the rice d1 mutant as a positive control. Polyethylene glycol (PEG) treatment was used to simulate drought stress in 5-week-old rice. Plant stress indexes such as chlorophyll fluorescence (Fv/Fm and Y(II) measurements), chlorosis and survival/recovery were determined. Data collected: Three independent lines each for OsWRKY53-OE and OsWRKY70-OE were drought stressed using 30% PEG 8000 and hourly Fv/Fm values were recorded by FMS2 for eight hours. Higher expression of OsWRKY53 in rice led to more sensitivity to drought. Eight hours of drought treatment showed significantly lower Fv/Fm values for all three OsWRKY53-OE lines and d1 mutant relative to WT. For OsWRKY70-OE plants on the other hand, results from different lines were consistent. Summary and discussion of results: The results for chlorophyll fluorescence analysis suggest higher expression of OsWRKY53 rendered rice plants more sensitive to drought, suggesting that this WRKY transcription factor may function as a negative regulator during the stress response and affect PSII efficiency. Key outcomes or impacts: Our study tests the hypothesis that close homologs OsWRKY24, -53, and -70 function as negative regulators which fine tune expression of defense genes during rice's response to drought stress. Higher susceptibility to the environmental stress upon overexpression of OsWRKY53 and -70 supports our hypothesis that the gene products are involved in the response as negative regulators. Objective 4: Can we use WRKY TFs to obtain an increase in yields? Major activities/ experiments conducted: Overexpression of OsWRKY53 resulted in dwarfed plants that are hypersensitive to drought, suggesting that OsWRKY53 negatively regulates plant growth. We have made constructs containing the promoter of OsNAC6, a gene of the NAC transcription factor family in rice. This gene is induced by abiotic stresses and disease8 but has low expression under optimal growth conditions. To date, we have produced constructs that have the OsNAC6 promoter sequence driving the expression of OsWKRY53 or -24. These binary constructs were used to create transgenic lines of rice cultivar Kitaake using Agrobacteria-mediated transformation. Our hypothesis is that KO of one or both genes should enhance rice plant resistance to drought and hence improve yields. Data collected: Transgenic plants for both constructs have obtained. Summary and discussion of results: The dwarf phenotype we observed is consistent with other studies in rice Longjing 11 cultivar9, and Xiushui 11 cultivar10. Interestingly, another study using CaMV35S to drive overexpression of OsWRKY53 in rice cultivar Nipponbare reported no growth defects11. The OsWRKY70-OE lines we created did not result to any dwarf phenotype, although a previous study have reported reduced growth for rice Xiushui 11 cultivars transformed with CaMV35S pro-OsWRKY7012. These differences in growth may be due to differential expression levels of the gene due to different insertion sites of T-DNA on rice chromosomes. It might also result from differences in rice cultivars. Key outcomes or impacts: Producing non-dwarf OsWRKY53 and -70 lines will not only help advance our understanding of the roles of these two genes in plant growth and responses to drought. Objective 5: Can we position WRKY TFs in signaling networks? Results from Objective #2 are needed prior to initiating studies under this objective. References: 1 Zhang, Z. L. et al. Plant Physiol 134, 1500-1513, (2004). 2 Kim, C.-Y. et al. Plant Biotechnology Reports 10, 13-23, (2016). 3 Liu, X. et al. J Biol Chem 295, 10271-10280, (2020). 5 Hoppe, T. et al. Curr Opin Cell Biol 13, 344-348, (2001). 6 Seo, P.J. et al. Trends Plant Sci 13, 550-556, (2008). 7 Xie, K. et al. PNAS USA 112, 3570-3575, (2015). 8 Nakashima, K. et al. Plant J 51, 617-630, (2007). 9 Tian, X. et al. Plant Physiology 175, 1337-1349, (2017). 10 Hu, L. et al. Plant physiology 169, 2907-2921 (2015). 11 Chujo, T. et al. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1769, 497-505, (2007). 12 Li, R. et al.. Elife 4, e04805 (2015).

Publications

Progress 01/01/19 to 12/31/19

Outputs
Target Audience:Unfortunately, most students nowadays don't understand the importance of agriculture research and hence don't value it either. Consequently, we don't have many students interested in agriculture research, to say nothing of having agriculture as their career choice. three of my graduate students, fully or partially supported by this grant, gave three presentations to the faculty and students in School of Life Sciences UNLV (approximately 60 people each time) about the goals and importance of this research project. Changes/Problems:Research in soybean is delayed because the Co-PD, who was a vice president of 22nd Century Group, Inc, didn't submit required documents for the subaward to the Office of the Sponsored Programs (OSP) at the PD's university in the first year. In the second year, our university was able to reach an agreement with the company and established an account for the subaward. However, the co-PD left the company, which subsequently indicated to the PD that the company liked to terminate the project. Accordingly, we filed a document with Dr. Ed Kaleikau, National Program Leader - Plant Breeding, Genetics and Genomics to justify for the change in project site and provide revised timeline and deliverables. Dr. Kaleikau approved the request on January 29, 2020, with the NIFA Awards Management cc'd on the email. Our OSP has instructed me to act based on the new plan. We are in the process to hire a postdoc and a part time worker to work on the soybean project. Dr. Paul Rushton will continue to supervise the project with me. We plan to ask for a one-year no-cost extension in 2020 so that we will have enough time to finish the proposed experiments for soybean, as detailed in the justification document. What opportunities for training and professional development has the project provided?This project helped train 3 graduate students and 9 undergraduate students in plant biology research. How have the results been disseminated to communities of interest?Three graduate students gave poster presentations in Plant Biology 2019. Anne Jinky Villacastin, Keeley Adams, Rin Boonjue and Mira Han and Qingxi J. Shen: Evolution of the WRKY transcription factors across Oryza species, Plant Biology 2019, August 3-7, San Jose, CA. Santi Bataller, Linkun Gu, Liyuan Zhang, Victoria Amato, and Qingxi J. Shen: WRKY71 transcription factor mediates the crosstalk of abscisic acid and gibberellin signaling in rice, Plant Biology 2019, August 3-7, San Jose, CA. Victoria Amato, Liyaun Zhang, Linkun Gu and Qingxi J. Shen, Identification and transcription profiling of the NHL (NDR1/HIN1-like) gene family in rice, Plant Biology 2019, August 3-7, San Jose, CA. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Can we identify interacting protein partners? We were planning to perform more rounds of screenings to reach the saturation point of identifying interaction proteins. However, the graduate students have been tied to work on transforming, screening and analyze rice plants. So, we are planning to have a company to perform this task. New graduate student Victoria Amato has been training to do BiFC, which will be used to confirm the interactions of OsHNL with OsWRKY70 and OsWRKY24, respectively. She will also be trained to do co-immunoprecipitation experiments. Objective 2: Can we determine target genes? Produce OsWRKY70-HA transgenic plants. RNA-sequencing for the wildtype, oswrky24 and -70 mutants, respectively. This goal was delayed due to lack of double mutants. We are hiring a postdoc to help speed up the project. We also need to request one year no cost extension so that homozygous plants can be obtained in time for this part of the project. Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? Produce OsWRKY70-HA and OsWRKY24 overexpression transgenic plants. We are hiring a postdoc to help speed up the project. We also need to request one year no cost extension so that homozygous mutants can be obtained in time for this part of the project. Objective 4: Can we use WRKY TFs to obtain an increase in yields? Will address this in the fourth year. Objective 5: Can we position WRKY TFs in signaling networks? Will address this in the fourth year.

Impacts
What was accomplished under these goals? Objective 1: Can we identify interacting protein partners? Major activities/experiments conducted: Identifying interaction proteins will allow us to position WRKY transcription factors into drought responsive signaling pathways. In the past interim, we identified an OsWRKY70 interaction protein, NDR1/HIN1-like protein (OsNHL), which modulates the signaling of hormones gibberellins (GA) and abscisic acid (ABA). In the interim, we found that the OsNHL protein also interacts with OsWRKY53, another target gene of the funded project. we focused on two lines of research: the evolution of the NHL gene family and the subcellular location of the OsNHL protein. CRISPR constructs have been designed for the knockout of the gene. Data collected: We performed a genome-wide identification and analysis of the OsNHL families in rice. We found twelve OsNHL genes expressed in all samples analyzed, which suggests that these OsNHLs play regulatory roles at multiple developmental stages. Fifteen genes were only expressed in one tissue, suggesting their roles in specific tissues or conditions. Five OsNHLs were up- or down-regulated in the aleurone layers treated with GA or ABA. Our data suggest this gene family might play roles in regulating seed germination under stress conditions. Localization of proteins helps determine their functions. Prediction via TMHMM program analysis indicated existence of 0-6 transmembrane domains in the OsNHL family. The majority of OsNHL protein contain one transmembrane domain, 13 two transmembrane domains and one 6 transmembrane domains. The OsNHL protein interacting with OsWRKY53 and -70 is a member of one transmembrane domain group. Confocal microscopic studies of the GFP-tagged OsNHL protein indicate that this protein is exclusively located on the plasma membrane. Summary and discussion of results: Studying the OsNHL gene family allows us to have a genomic view of the gene family in term of their gene structures, gene expression patterns, subcellular locations and evolution. To date, there has been no implication of a physical interaction of an NHL protein with a transcription factor. In addition, WRKY transcription factors in pathways parallel to the NDR1-mediated resistance pathway, but no direct link has been made between NHLs and WRKY transcription factors (1, 2). Key outcomes or impacts: Our recent discovery represents a major milestone because WRKY genes were once believed to play a role only in biotic stress responses, but research results from our lab and others have clearly demonstrated that WRKY genes also function in plant responses to abiotic stresses as well as seed germination. Biotic stress pathways intertwined with both the ABA and GA signaling pathways. In plants, biotic stress is largely regulated by three main hormones, salicylic acid (SA), jasmonic acid (JA) and ethylene. It is possible that OsWRKY53 and -70 work in concert with the OsHIL protein to mediate the crosstalk between the biotic and abiotic stress pathways during stress responses and seed germination. Objective 2: Can we determine target genes? Major activities/experiments conducted: Data collected: No reports for the interim Summary and discussion of results: No reports for the interim Key outcomes or impacts: No reports for the interim Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? Major activities/experiments conducted: We used the polycistronic tRNA-gRNA gene (PTG) CRISPR/Cas9 system to produce knockout mutants (3). In the last interim, we prepared double and triple mutant constructs and obtained 11 hygromycin resistant transgenic plants. However, sequencing data indicated that all plants are single gene knockout mutants for OsWRKY70. This kind of "dominance" has been observed with other genes. Therefore, we focused more on three double-gene mutants, with a complementary plan to produce single gene mutants and then make double mutants by crossing. This shift will take more time to produce double/triple mutants. Therefore, shortening transformation procedures became one of our top goals for this interim. In addition, we decided to take an additional approach to address Objective 3: Study the evolution of the WRKY gene family in all 11 Oryza genomes (9 species). Species such as O. nivara shows abundant genetic diversity and elite drought and disease resistance features. By studying selection pressures on different amino acid residues of different WRKY genes in different rice species might help us test the hypothesis that OsWRKY24, -53 and -70 modulate drought stress responses. More important, it will guide us to mutate amino acid residues in these proteins to produce rice cultivars with enhanced drought resistance. Data collected: We have improved the rice transformation protocol that was kindly produced by Dr. Bing Yang in University of Missouri. The improved protocol allows us to produce transgenic Kitaak rice plants within 84 days. We are in the process to produce homozygous mutant lines for OsWRKY24, -53 and -70. For the evolution study, we identified 1,018 WRKY genes across 11 Oryza genomes (9 species) with a total of 107 unique orthologs. Purifying selection indicates genes are possibly responsible for conserved functions; our analysis identified conservative constraints acting on OsWRKY24 and other genes. Forty-nine percent of single copy WRKY orthologs tested positive for selection, closer inspection of protein sequences for all genes evolving under positive selection shows that amino acid selection diversity is extremely low or insignificant in the DNA-binding domain, indicating selective pressure at the WRKY domain is different from pressure acting on the rest of the protein sites. Summary and discussion of results: We have produced 7 CRISPR knockout mutants for OsWRKY70; screening for OsWRKY24 and -53 as well as double and triple mutants are ongoing. We also have initiated collaboration with Dr. Bing Yang's lab to produce rice mutants simultaneously. The parallel effort will increase the probability of success to produce triple mutants within the defined time frame. Our evolution study identified conservative constraints acting on OsWRKY24. The result will guide us to engineer OsWRKY24 for improved drought resistance. Key outcomes or impacts: We have successfully adopted the PTG technology to make CRISPR/Cas9 constructs and improved the rice transformation protocol. These achievements will greatly advance our proposed studies. The study of the WRKY gene evolution in wild and domestic rice species provide invaluable insight into the diversification of an important gene family under strong selective pressure as well as useful information for the biotechnological improvement of the developing world's most valued food crop. Objective 4: Can we use WRKY TFs to obtain an increase in yields? Results from Objective #3 are needed prior to initiating studies under this objective. Objective 5: Can we position WRKY TFs in signaling networks? Results from Objective #2 are needed prior to initiating studies under this objective. References: 1. C. Y. Kim et al, Plant J. 38, 142-151 (2004). 2. M. Schon et al., MPMI 26, 758-767 (2013). 3. K. Xie et al., PNAS 112, 3570-3575 (2015).

Publications

Progress 01/01/18 to 12/31/18

Outputs
Target Audience:Unfortunately, most students nowadays don't understand the importance of agriculture research and hence don't value it either. Consequently, we don't have many students interested in agriculture research, to say nothing of having agriculture as their career choice. One of my graduate students, supported by this grant, gave two presentations to the faculty and students in School of Life Sciences UNLV (approximately 70 people each time) about the goals and importance of this research project. The presentations must have changed the view of some students on agriculture because as many as nine students volunteered to work in my lab (http://shenlab.sols.unlv.edu/shenlab/current_members.html) and one of them applied and was just accepted to be a Ph.D. student in my lab. Changes/Problems:Research in soybean is delayed because the Co-PD hasn't submitted required documents for the subaward to the Office of the Sponsored Programs (OSP) at the PD's university: - Subrecipient Commitment form from 22nd Century - 22nd Century Scope of work - Reporting requirements for 22nd Century (Annual financial and technical reports from the 22nd Century should be prepared based on the USDA guidelines and sent to the PD at least two weeks before the USDA deadlines for compiling and submission. For the current grant, the financial reports are due on Feb. 1, 2019, 2020 and 2021; the technical reports are due on March 31, 2019, 2020 and 2021.) It is my understanding that the company has strict policies on releasing data to public or even the collaborators in this case. The company is working with the OSP and the Technology Transfer & Economic Development Office at the PD's university to ensure the subaward can be released to the company in April 2019. We plan to ask for a one-year no-cost extension in 2020 so that the co-PD will have enough time to finish proposed experiments for soybean. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?What do you plan to do during the next reporting period to accomplish the goals? Objective 1: Can we identify interacting protein partners? Perform 3 more rounds of screenings to reach the saturation point of identifying interaction proteins. Co-immunoprecipitation and BiFC to confirm the interactions of OsHNL with OsWRKY70 and OsWRKY24, respectively. Objective 2: Can we determine target genes? Produce OsWRKY71-HA transgenic plants. RNA-sequencing for the wildtype, oswrky24 and -70 mutants, respectively. Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? Produce OsWRKY71-HA and OsWRKY24 overexpression transgenic plants. Objective 4: Can we use WRKY TFs to obtain an increase in yields? Will address this in the third year. Objective 5: Can we position WRKY TFs in signaling networks? Will address this in the third year.

Impacts
What was accomplished under these goals? Objective 1: Can we identify interacting protein partners? Major activities/experiments conducted: Identifying interaction proteins will allow us to position WRKY transcription factors into drought responsive signaling pathways. In the interim, we made a library from stressed rice root treated with PEG and screened the library using the yeast two hybrid technique with OsWRKY70 as the bait. To further study a key interaction partner, a fusion protein construct, which contains the GFP: OsNHL fusion gene driven by the maize ubiquitin promoter (Ubi) was introduced to aleurone cells using particle-bombardment, followed by fluorescence confocal microscopy for subcellular localization of the fusion protein. We also performed transient expression assays to address the function of the interaction partner in the signaling of hormones gibberellins (GA) and abscisic acid (ABA). Data collected: A total of 18 positive clones were identified from the screening. Among the interaction proteins, the NHL protein is most noticeable. Confocal microscopy data indicate that the NHL-GFP fusion protein was mainly localized to the cell membrane, with detectable signals in endoplasmic reticulum (ER) and other organelles. RNA-seq data(1) indicate that this NHL gene is ABA inducible, but GA repressible. Transient expression studies suggest that NHL is likely an ABA-inducible repressor of both ABA and GA signaling, as OsWRKY70. The effect of the NHL with OsWRKY70 is additive in suppressing ABA induction, but antagonistic in suppressing GA induction. Summary and discussion of results: The first NHL gene was found in tobacco where it was specifically induced by harpin, an elicitor produced by the gram-negative bacterium, Erwinia amylovora. This pathogen causes fire blight in pears, apples, and other rosaceous plants (2, 3). Arabidopsis NDR1 (nonrace-specific disease resistance) shares some sequence similarities with the tobacco NHL protein (4). NHL3 overexpression lines showed disease resistance against pathogenic bacteria (5). In rice, OsHin1 was induced by rice blast, a disease caused by the fungus Magnaporthe grisea (6). The OsNHL protein was found to localize to both the cell membrane and the cytoplasm, function as a negative regulator of both GA and ABA signaling. Key outcomes or impacts: Our recent discovery represents a major milestone because WRKY genes were once believed to play roles only in biotic stress responses, but research results from our lab and others have clearly demonstrated that WRKY genes also function in plant responses to abiotic stresses. Likewise, our recent data suggest that NHL genes might also have functions in plant responses to abiotic stresses even though current literatures only report their roles in biotic stress responses. Consistent with this hypothesis, we have demonstrated that transient overexpression of this OsWRKY70 interacting NHL protein represses ABA signaling in rice. Hence our discovery could open new avenues for exploring the NHL genes to improve the tolerance of crops to abiotic stresses such as drought. Objective 2: Can we determine target genes? Major activities/experiments conducted: We proposed to address this question in rice by targeting OsWRKY70 using ChIP-seq. The POsWRKY70:OsWRKY70-HA construct has been made in a Ti-plasmid vector. Use of the native promoters is preferable to avoid altered expression of the gene as much as possible. We will use the oswrky70 knockout line we have studied so that the level of OsWRKY70 protein is near to wild type. We understand that even a native promoter construct in a knockout background could lead to a range of expression levels. However, there have been many successful reports for this approach. Data collected: No reports for the interim Summary and discussion of results: No reports for the interim Key outcomes or impacts: No reports for the interim Objective 3: Can we use WRKY TFs to obtain improved drought phenotypes? Major activities/experiments conducted: To obtain single and double mutant lines for OsWRKY24 and -70, we used the polycistronic tRNA-gRNA gene (PTG) CRISPR/Cas9 system (7). Briefly, a single synthetic polycistronic gene was designed with four guide RNA (gRNA) sequences specific for OsWRKY24 and -70. Two specific 20 nucleotide sequences within each WRKY gene was selected to create the spacer sequences for the gRNA. The following criteria was followed for the selection of the sequences: (1) both gRNA pairs should flank both WRKY domains for the target locus (8). CRISPT-P 2.0: an improved CRISPR/Cas9 tool for genome editing in plants (9, 10) was used to design the spacer sequences, followed by scans the potential CRISPR guide sequences for possible off-targets (11). The gRNA cassette was assembled using the Golden Gate (GG) Assembly system (12, 13) and ligated into the pRGEB32 vector (7). Data collected: Diagnostic digestion revealed production of correct constructs and no unexpected mutations are present. Agrobacterium-mediated rice transformation was done with pRGEB32-PTG. Callus induction was achieved with 90-100% efficiency from rice embryos and selection was observed 33 days post transfer into selection plates. However, the efficiency in regenerating transgenic plants is lower than what we achieved in the past. Summary and discussion of results: We have produced 11 hygromycin resistant plants and will perform genotypying and sequencing once the plants are old enough for providing materials for DNA extraction. Key outcomes or impacts: We have successfully adopted the PTG technology to make CRISPR/Cas9 constructs and re-established the rice transformation platform in our lab. These achievements will advance our proposed studies tremendously. Objective 4: Can we use WRKY TFs to obtain an increase in yields? Results from Objective #3 are needed prior to initiating studies under this objective. Objective 5: Can we position WRKY TFs in signaling networks? Results from Objective #1 and 2 are needed prior to initiating studies under this objective. References: 1. L. Zhang et al., Plant Sci 236, 214-222 (2015). 2. S. Gopalan, W. Wei, S. He, Plant J. 10, 591-600 (1996). 3. Z.-M. Wei et al., Science 257, 85-88 (1992). 4. P. Dörmann, S. Gopalan, S. Yang He, C. Benning, Plant Physiol. and Biochem. 38, 789-796 (2000). 5. A. Varet, B. Hause, G. Hause, D. Scheel, J. Lee, Plant Physiol 132, 2023-2033 (2003). 6. C. Y. Kim et al., Molecular Plant-Microbe Interact. 13, 470-474 (2000). 7. K. Xie, B. Minkenberg, Y. Yang, PNAS USA 112, 3570-3575 (2015). 8. C. A. Ross, Y. Liu, Q. J. Shen, J. Integ. Plant Biol. 49, 827-842 (2007). 9. H. Liu et al., Mol Plant 10, 530-532 (2017). 10. Y. Ding, H. Li, L.-L. Chen, K. Xie, Frontiers in Plant Science 7, 703 (2016). 11. C. Tennakoon, R. W. Purbojati, W.-K. Sung, Bioinformatics 28, 2122-2128 (2012). 12. C. Engler, R. Kandzia, S. Marillonnet, PloS One 3, e3647 (2008). 13. C. Engler, R. Gruetzner, R. Kandzia, S. Marillonnet, PloS One 4, e5553 (2009).

Publications