Progress 08/15/09 to 08/14/10
Outputs
OUTPUTS: Activities: 1) Which WRKY genes regulate responses to water stress We performed water stress experiments using either soybean or tobacco grown under well-defined hydroponic conditions. Oligo array analysis was performed in the two plant species in parallel. In tobacco leaves, thirteen WRKY genes were induced at least 8-fold by water stress and an additional ten were induced at least 5-fold. In the parallel soybean experiments, 21 WRKY genes were induced at least 8-fold by water stress. This represents over 10% of the soybean WRKY gene family. 2) Do phylogenetically similar WRKY genes regulate water stress responses We performed a phylogenetic analysis of the WRKY gene family in the green lineage and compared the water stress-regulated WRKY genes from tobacco and soybean. WRKY genes from all subfamilies are up-regulated by water stress, but we found clear "hotspots" in the phylogeny in Groups I, IIa and III. This forms the first part of our predictive phylogenetic tree. 3) Do drought responsive WRKY genes share similar drought responsive promoter elements Five promoters from tobacco WRKY genes that are strongly up-regulated by water stress are currently being evaluated for the direction of water-stress inducible gene expression in several systems including transgenic plants. Twelve soybean WRKY promoters are being analyzed in transgenic soybean. 4) How do WRKY genes regulate drought responses and can we use them to increase drought tolerance We have identified at least 800 potential gene targets for the improvement of drought responses. These may be regulated by WRKY transcription factors and we are focusing on the identification of downstream target genes. In the rice portion of the project, OsWRKYb and OsWRKYf are likely to be the key regulators of drought responses. Genotyping and phenotyping of OsWRKYf knockout mutants is being performed. Overexpression, RNAi, and promoter-GUS constructs are also being tested, together with three transposon insertional knockout mutants for OsWRKYf. Phenotyping of these OsWRKYf mutants under drought and ABA treatments are ongoing. We are also pursuing a characterization of the OsWRKYb and OsWRKYf proteins and results indicate that OsWRKYb, but not OsWRKYf, can bind to W boxes. It is likely that both proteins are positive regulators of drought response in rice, but that they regulate different subsets of genes. Events: Paul Rushton established his laboratory at South Dakota State University and was an invited speaker at the Plant and Animal Genome Conference in San Diego in January 2010. Services: In 2010, Paul Rushton served as an ad hoc reviewer for the NSF. Together with Dr Imre Somssich (Cologne, Germany) the PI and Co-PI, wrote a review on WRKY transcription factors for Trends in Plant Science. Dissemination of results: Some project data were presented at the Plant and Animal Genome Conference in San Diego in January 2010. PARTICIPANTS: PARTICIPANTS: During the last year, the personnel on the project has grown to include one postdoc (Jun Lin), two graduate students (Prateek Tripathi and Roel Rabara) and four undergraduate student (Deena Rinerson, Ashley Boken, Tanner Langum and Lucas Smidt). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: This year's work has provided evidence that 10-20% of WRKY genes appear to be involved in drought responses in soybean, rice and tobacco. In addition, there appear to be "hotspots" in the phylogenetic tree of the WRKY gene family (Groups I, IIa and III) where orthologous WRKY genes may regulate water stress responses in multiple species and these genes may represent a commonality in water stress responses in plants. We now have many promising candidate WRKY genes and their promoters to form the focus of continuing work. The transcriptome work has also identified at least 800 potential gene targets for the improvement of drought responses in crop plants.
Publications
- Rushton, P.J., Somssich, I.E., Ringler, P., and Shen, J. (2010) WRKY transcription factors. Trends in Plant Science Volume: 15 Issue: 5 Pages: 247-258
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: Activities: Progress has been made towards answering all four of our research questions: 1) Which WRKY genes regulate responses to water stress We have performed three separate water stress experiments using either soybean or tobacco. qRT-PCR was used to assess the drought inducibility of ten WRKY genes that were chosen based on phylogenetic similarity to previously reported drought inducible WRKY genes. More than half of the tested WRKY genes from both soybean and tobacco showed significant induction. To further investigate drought responses in tobacco, a custom oligo array containing over 40,000 tobacco gene space sequences, the NTGI ESTs from tobacco and the complete TOBFAC transcription factor collection was used to monitor global gene expression changes during water stress in leaves. This has enabled us identify over 800 genes that appear to be involved in drought responses and provides the framework for detailed analysis of regulatory genes and the formulation of new hypotheses. 2) Do phylogenetically similar WRKY genes regulate water stress responses A major activity for the first year of the project was the characterization of the complete WRKY gene family from soybean. This has allowed the construction of a combined WRKY phylogenetic tree from ten complete plant genomes. This phylogeny is being used as a framework for the comparison of results in different plant species. 3) Do drought responsive WRKY genes share similar drought responsive promoter elements Twelve soybean WRKY promoters, including several from drought inducible genes, have been sent to Prof John Finer at The Ohio State University. Prof Finer's group have performed initial promoter analyses in lima bean cotyledons and the promoters are now being introduced into soybean plants to test for drought inducibility. 4) How do WRKYs regulate drought responses and can we use them to increase drought tolerance During the first year of the project, we have identified several candidate genes that will form the focus of this activity. Events Paul Rushton accepted a position at South Dakota State University (SDSU) and was promoted to Associate Professor. The project was transferred to SDSU from the University of Virginia. Since the start of the project, Paul Rushton was a plenary speaker at the Solanaceae Conference in Cologne Germany and also chaired one of the sessions. He was also an invited speaker at the Plant and Animal Genome Conference in San Diego. Services In summer 2009, Paul Rushton served on a USDA panel as a result of his work and funding on WRKY transcription factors. Since the transfer of the award to SDSU, one graduate student (Prateek Tripathi) and one undergraduate student (Ashley Boken) have joined the project and after initial tutoring are now members of the project team. Products Both the PI and Co-PI have been invited to write a review on WRKY transcription factors. This work is ongoing and should be published during the second year of the project PARTICIPANTS: Since the transfer of the award to SDSU, one graduate student (Prateek Tripathi) and one undergraduate student (Ashley Boken) have joined the project and after initial tutoring are now members of the project team. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge The first year of the project has provided evidence that 10-15% of WRKY genes appear to be involved in drought responses as shown by transcriptome analysis in tobacco and qRT-PCR in soybean. We have identified eleven WRKY genes in tobacco that are at least 8-fold upregulated by water stress. Strikingly, after several of the well characterized DREB transcription factors, the next most up-regulated transcription factor gene was a WRKY gene. This is the first evidence to support our hypothesis that WRKYs are key regulators of water stress responses and that similar genes regulate these responses in different plant species. The highest up-regulated WRKY gene is induced over 50-fold. We now have promising candidate WRKY genes to form the focus of continuing work. The transcriptome work has also identified a number of new aspects of water stress responses and suggests an interplay with plant hormones that have not previous been known to play major roles in these responses. Change in actions The results from year one have identified specific WRKY genes that will be the focus of future work. Change in conditions The project, along with the principle investigator, has moved to South Dakota State University. Two additional members have been recruited to the project team.
Publications
- No publications reported this period
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