| 研究実績の概要 |
Our initial strategy is to express the fusion protein, Venus-HAC701, into rice protoplast of mutant HAC701 lines to be able to (1) complement the loss of HAC701; (2) isolate a stable Venus-HAC701 expressing transgenic lines; (3) identify interacting partners of HAC701 by immunoprecipitation (IP) using Venus antibody; (4) identify the DNA binding site of HAC701 by IP using Venus antibody. However, the expression of the vector in rice protoplast is unstable and yielded poor results. Instead of completely relying on the fusion vector to answer the above questions, we utilized molecular genetic analysis and NGS approach. Our RNA-seq analysis found that HAC701 mutants possibly targets and potentiates WRKY45 locus before and after pathogen treatment. As WRKY45 is upregulated in HAC701 mutants, we then compared the target genes of our mutant and the overexpression WRKY45 transgenic lines (OE-WRKY45). Our results showed that HAC701 single mutants phenocopy the OE-WRKY45 lines. These results indicate that there is genetic interaction of HAC701 and WRKY45. In addition, these results also showed the range of possible targets of HAC701 during pathogen infection. We also reported previously that we have performed ChIP-seq analysis of HAC701 mutant, 9-12b-/-. Our ChIP-seq experiment and analysis is a success. In brief, we have confirmed that H3K9ac is possibly catalyzed by HAC701 acetyltransferase. Also, we found enrichment of WRKY45 sequences in H3K9ac fraction adding more evidence of the possible physical interaction of HAC701 with WRKY45 locus.
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| 今後の研究の推進方策 |
(A)Continue with the expression of HAC701 fusion protein in rice protoplast and callus in preparation for Agrobacterium transformation. Aside from a range of possible interacting factors that might be found in doing this experiment, we are also very interested in finding a CREB-like protein. This protein serves as the link between HAC701 and WRKY45 interaction through CRE-binding motif.
(B)Express in vitro the HAC701 full-length and domains and perform binding assays with rice total histones and histone peptides. Although, we have evidences that HAC701 targets H3K9ac, we are still keen in generating evidences of direct binding of HAC701 full or domains to H3K9ac site.
(C)Cross HAC701 9-5-/- and 9-12b-/- single mutants with WRKY45 knockdown transgenic lines. The genetic interaction of HAC701 and WRKY45 is supported by evidences from the analysis of HAC701 single mutants and OE-WRKY45 lines differentially expressed genes and also their morpho-phenotypes. Crossing HAC701 single mutants and WRKY45 knockdown lines and showing the opposite disease phenotype (i.e. from resistant HAC701-/-WRK45+/+ to susceptible HAC701-/-WRKY45-/-) will be the strongest evidence for this interaction.
(D)Finalize the manuscript about the story of negative regulation function of HAC701 on rice immunity through WRKY45 modulation.
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