Analysis Of WRKY62 And WRKY76 Transcription Factors In Rice

Plants have evolved pathogen-associated molecular patterns(PAMPs)-triggered immunity(PTI)and effector-triggered immunity(ETI)to protect themselves from invading pathogens.WRKY transcription factors function as transcriptional activators or repressors in PTI and ETI defense signaling pathway.Here we report that OsWRKY76 and OsWRKY62,two WRKY ?a subfamily genes,showed a similar induction pattern in rice seedlings treated with chitin,flg22,methyl jasmonate and wound.The full-length OsWRKY62.1 and OsWRKY76.1 interacted both physically through N-or C-terminal coiled-coil domain to regulate rice defense responses.Overexpression lines of OsWRKY76.1 or OsWRKY62.1 were more susceptible to the rice pathogens Magnaporthe oryzae SZ and Xanthomonas oryzae pv oryzae(Xoo)J18,while RNAi lines and loss-of-function knockout(KO)mutants using the CRISPR/Cas9 technology exhibited elevated resistance.Using a transient transformation assay system in Nicotiana benthamiana,we showed that OsWRKY76.1 and OsWRKY62.1 could produce visible transcriptional repression activity.The data indicate that OsWRKY76.1 and OsWRKY62.1 are negative regulators of disease resistance against M.oryzae and Xoo in rice.Interestingly,the double RNAi plants(dsOW76/62)harboring lesion-mimic phenotype displayed enhanced resistance to both M.oryzae SZ and J18 compared to the dsOW76 or dsOW62 plants,and accompanied by elevated expresssion of many defense-related genes and accumulation of phytoalexins.These results suggest that the two transcription factors may function collaboratively in pathogen defense.Surprisingly,the dsOW76 and dsOW76/62 plants exhibited significant and simultaneous accumulation of OsWRKY76 and OsWRKY62 transcripts.We observed prominent alternative transcripts for OsWRKY76.2,OsWRKY62.2 by RT-PCR and,most interestingly,the ratio of full-length vs.truncated transcripts changed in dsOW76/62 plants as well as in response to pathogen infection.The short OsWRKY76.2 and OsWRKY62.2 proteins encoded by the alternative transcripts could interact each other and with full-length proteins.OsWRKY76.2 or OsWRKY62.2 significantly decreased the interaction of full-length OsWRKY76.1 with OsWRKY62.1.OsWRKY62.2 showed a reduced repressor activity in planta and two sequence determinants required for the repressor activity were identified in the N-terminus of OsWRKY62.1.The N-termini of OsWRKY62.2 and OsWRKY76.2 also showed reduced binding to the canonical W box motif.These data demonstrate that the function of the alternative transcripts are different in the complicated disease resistance in rice.OsWRKY76 and OsWRKY62 may form a highly complex interacting regulatory network that mediates plant disease resistanse responses.Expression of OsWRKY76 and OsWRKY62 significantly increased in knockout(KO)mutants.We further certified that OsWRKY62.1 could directly bind to W-box clusters of its own promoter and OsWRKY76 promoter through chromatin immunoprecipitation(ChIP)analysis.These results suggest that OsWRKY76 and OsWRKY62 may exist mutual regulation mechanism.Posttranslational modification in OsWRKY62.1 by tandem mass spectrometry analysis was characterized,such as phosphorylation and ubiquitination.