Preliminary Study Of OsWRKY5 And OsMYB1R In Regulating Pik-H₄-mediated Resistance To Rice Blast

Rice blast,which is caused by Magnaporthe oryzae,is one of the most serious diseases in rice production.It is an effective measure to improve the resistance of rice blast by pyramiding the main disease resistance genes of rice blast.There is still insufficient understanding of the disease resistance pathways activated by these major genes.Previous studies have shown that disease resistance genes may regulate immune network by regulating a series of transcription factors.In-depth functional study of these transcription factors not only helps to analyze the interaction mechanisms between plants and pathogenic microorganisms,but also provides theoretical basis for the research of rice resistance breeding.In our previous work for identifying Pik-H₄and Avr-Pik H₄interacting proteins by co-immunoprecipitation（Co-IP）,the transcription factors OsWRKY5 and OsMYB1R were found to interact with resistance protein Pik₁-H₄and the rice blast fungus effector protein Avr-Pik H₄,respectively.In this study,H4（carries Pik-H4）,a rice blast-resistant rice cultivar,was used to study the possible molecular functions of OsWRKY5 and OsMYB1R involved in regulating Pik-H4-mediated resistance in rice.The main conclusions are as follows:（1）Gene sequence analysis reveals that OsWRKY5 belongs to subgroup a of the major second subfamily of the WRKY family:C-Xm-C-Xn-HXC/H;OsMYB1R falls into the CCA1 subfamily of the MYB family,and OsMYB1R is closely related to RVE1 and LHY2,so it may be involved in the regulation of circadian rhythm.Both OsWRKY5 and OsMYB1R have transcriptional activation ability,the WRKY domain is only responsible for binding to genomic DNA and has no transcriptional activation abilitiy;while the MYB domain has the transcriptional activation ability.（2）Tissue-specific analysis showed that OsWRKY5 and OsMYB1R are expressed in various tissues.OsWRKY5 is relatively high expressed in leaves and leaf sheaths,while OsMYB1R is expressed more in stems and internodes.（3）The interaction of the two protein pairs（Pik₁-H₄-OsWRKY5,Avr-Pik H₄-OsMYB1R）were confirmed by yeast two-hybrid assays.Further results showed that the interaction between Pik₁-H₄and OsWRKY5 occurs between the coiled-coil domain（CC domain）of Pik₁-H₄and the WRKY domain of OsWRKY5,and the interaction between Avr-Pik H₄and OsMYB1R occurs at the Avr-Pik H₄and C-terminus of OsMYB1R instead of the MYB domain.GST Pull-down assays showed Pik₁-H₄-CC interacts with OsWRKY5-WRKY and Avr-Pik H₄interacts with OsMYB1R-C.Subcellular localization analysis showed that OsWRKY5 and OsMYB1R are localized in the nucleus,which is consistent with the roles of transcriptional factors.（4）The expression patterns of OsWRKY5 and OsMYB1R showed that they both were induced by M.oryzae,but the trend is opposite.Resistance detection analysis showed that loss of function of OsWRKY5 could reduce resistance,while overexpression of OsMYB1R increase resistance,suggesting that OsWRKY5 positively regulates rice blast resistance.Overexpressing of OsMYB1R negatively regulates rice blast resistance.（5）Quantitative analysis of the key genes in ABA hormone systhesis with reported and the TGAC core sequence analysis of W-box cis-acting element for its 2 kb promoters showed that OsWRKY5 may be involved in the regulation of ABA pathway.The interaction protein speculated and the MYBR sequence analysis of 2 kb promoter of key genes suggested that OsMYB1R may be involved in the GA signal pathway through interaction with DELLA-like protein GAI.