Progress 08/01/02 to 07/31/05
Outputs
The primary objectives of the project are to identify the members of the rice WRKY gene family, characterize their expression and analyze their biological functions. We have collaborated in this project with two research groups in China (Dr. Zejia Guo at Chinese Agricultural University and Dr. Deqiu Yu at Chinese Academy of Sciences). We have identified 85 genes in rice that encode WRKY proteins that can be divided into three major groups based on the number and structures of the zinc-finger WRKY domains. Phylogenetic analyses based on protein structures indicate that many rice WRKY proteins are structurally closely related and may be functionally redundant. We have performed comprehensive expression analyses of these identified rice WRKY genes and found that a large number of rice WRKY genes are differentially regulated during infection by the rice blast fungal pathogen or treatment of various abiotic stresses (cold and drought). We have generated dsRNAi mutants for
identified rice WRKY genes but no substantially altered phenotype was observed in these loss-of-function mutants. In addition, we have generated transgenic rice and Arabidopsis plants that overexpress rice WRKY genes. A number of the transgenic rice lines overexpressing rice WRKY genes exhibited alterred disease resistance and/or abiotic stress tolerance in our collaborating laboratories in China. Because of the relative ease for transformation and short life cycle, we have also generated transgenic Arabidopsis plants overexpressing more than 30 pathogen-regulated rice WRKY genes. We have tested these transgenic Arabidopsis plants for their response to the bacterial pathogen Pseudomonas syringae. These experiments have shown that a number of transgenic lines became more resistant to the bacterial pathogen and expressed constitutively defense-related genes. These results suggest that some of the pathogen-regulated genes function as positive regulators of plant defense responses and
disease resistance. Surprisingly, a substantial percentage (>30%) of the tested WRKY genes rendered transgenic plants more susceptible to the bacterial pathogen when they were overexpressed, suggesting that they function as negative regulators of plant defense responses. We have also found that three rice WRKY proteins can enhance drought tolerance when overexpressed in transgenic Arabidopsis plants. These results indicate that WRKY proteins are involved in plant responses to both biotic and abiotic stress. During this funding period, we have also continued our studies on WRKY proteins in both Arabidopsis and tobacco for two main reasons. First, we failed to observe strong mutant phenotypes in the rice WRKY RNA silencing mutants because of the functional redundancy of structurally related WRKY genes. In Arabidopsis, we can address this issue through generating of double or even triple WRKY gene knockout mutants. Second, WRKY proteins are found in all plants and comparative analysis of
the gene family in both the monocot rice and dicots tobacco and Arabidopsis will provide a better understanding about the evolution of the family of transcription.
Impacts
Plant WRKY genes encode novel DNA-binding transcription factors with possible roles in a variety of biological processes in plants, particularly in plant defense responses and disease resistance. Functional analysis of this family of genes in several different plant species have revealed important information with respect to the organization, structures and biological functions of the important family of plant transcription factors. Parallel analyses of the gene families in tobacco, Arabidopsis and rice have also revealed that many important regulatory factors and mechanisms of plant stress response and disease resistance are conserved in different plant species. A number of WRKY genes from rice, Arabidopsis and tobacco have been shown to enhance plant stress tolerance and disease resistance and therefore can be further explored for introduction into crop plants for genetic engineering.
Publications
- Yu, D. Chen, C. and Chen, Z. (2001) Evidence for an important role of WRKY DNA-binding proteins in the regulation of the NPR1 gene expression. Plant Cell 13:1527-1539.
- Yang, P. Fan, B. and Chen, Z. (2001). A family of dispersed repetitive DNA sequences in tobacco contain clusters of W-box elements recognized by pathogen-induced WRKY DNA-binding proteins. Plant Sciences 161:655-664.
- Chen, C. and Chen, Z. (2002) Potentiation of developmentally regulated defense response by AtWRKY18, a pathogen-induced Arabidopsis transcription factor. Plant Physiol. 129:706-716.
- Dong, J., Chen, C. and Chen, Z. (2003) Expression profiles of the WRKY gene superfamily from Arabidopsis during plant defense responses. Plant Mol. Biol. 51:21-37.
- Chen, K., Du, L., and Chen, Z. (2003) Sensitization of defense response and activation of programmed cell death by a pathogen-induced receptor-like protein kinase from Arabidopsis. Plant Mol. Biol. 53:61-74.
- Chen, K., Fan, B., and Chen, Z. (2004) Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from Arabidopsis. Plant Mol. Biol. 56:271-283.
- Menke, F. L. H., Kang, H.-G., Chen, Z., Park, J. M., Kumar, D., and Klessig, D. F. (2005) Transcription factor NtWRKY1 is phosphorylated by MAP kinase SIPK and mediates HR-like cell death in tobacco. Mol. Plant-Microbe Interact. 18:1027-1034.
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Progress 10/01/03 to 09/29/04
Outputs
This project seeks to determine the biological function of the rice WRKY gene family through identifying the members of the gene family, analyzing their expression patterns and assessing possible phenotypes of gain- or loss-of function mutants for the rice WRKY genes. We have been collaborating on this project with two research groups in China (Dr. Zejia Guo at Chinese Agricultural University and Dr. Deqiu Yu at Chinese Academy of Sciences). We have previously identified approximately 85 WRKY genes and obtained a comprehensive expression pattern of these identified WRKY genes. We have also generated dsRNAi mutants for most of the identified rice WRKY genes but no substantially altered phenotype was observed in these loss-of-function mutants. This observation is consistent with our work with the Arabidopsis WRKY gene family where we have generated T-DNA knockout mutants for more than 40 WRKY genes but observed no significantly altered phenotype. The failure to observe
altered phenotypes in this single WRKY knockout mutant plants is mostly likely resulted from functional redundancy among many of these WRKY genes. In Arabidopsis, we are addressing this issue through generating of double or even triple WRKY gene knockout mutants. A similar approach will be necessary to address the biological functions of many of these rice WRKY genes. In addition, we have generated transgenic rice and Arabidopsis plants that overexpress pathogen-induced rice WRKY genes. A number of the transgenic rice lines overexpressing rice WRKY genes are being analyzed for possible alteration in defense responses and disease resistance in our collaborating laboratories in China. Because of the relative ease for transformation and short life cycle, we have also generated transgenic Arabidopsis plants overexpressing more than 30 pathogen-regulated rice WRKY genes. We are currently testing these transgenic Arabidopsis plants for their response to the bacterial pathogen Pseudomonas
syringae. These experiments have shown that a number of transgenic lines became more resistant to the bacterial pathogen and expressed constitutively defense-related genes. These results suggest that some of the pathogen-regulated genes function as positive regulators of plant defense responses and disease resistance. Surprisingly, a substantial percentage (>30%) of the tested WRKY genes rendered transgenic plants more susceptible to the bacterial pathogen when they were overexpressed, suggesting that they may function as negative regulators of plant defense responses. These results obtained in Arabidopsis will be very valuable to further analyze the biological functions of these pathogen-induced rice WRKY genes in rice.
Impacts
Plant WRKY genes are novel regulatory genes with important roles in plant growth, development and response to both abiotic and biotic stress. The proposed studies will lead to determination of the biological functions of specific WRKY genes in these important biological processes.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
This project seeks to identify, analyze expression patterns and generate mutants lines for the WRKY genes from rice. We are collaborating with Dr. Zejia Guo's group at Chinese Agricultural University from China on various aspects of the project. We have previously identified approximately 85 WRKY genes through analysis of published rice genome sequence data. For analysis of their expression, we have previously used northern blotting analysis and determined a number of rice WRKY genes. For expression analysis of the wholw WRKY gene family, we are in the process of making a WRKY gene oligo chip that will enable detection of expression of all identified WRKY genes. To generate overexpression and dsRNA suppression transgenic plants, we have generated a rice cDNA library and isolated a number of rice WRKY gene cDNA clones. Some of these cDNA clones have been subcloned into overexpression vector and are in the process of being transformed into rice. The dsRNA silencing
constructs for some rice WRKY genes are also being constructed.
Impacts
Plant WRKY genes are novel regulatory genes with important roles in plant growth, development and response to both abiotic and biotic stress. The proposed studies will lead to determination of the biological functions of specific WRKY genes in these important biological processes.
Publications
- No publications reported this period
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