The Role Of CaWRKY71 And Its Interaction With CaWRKY40 In Peppers’ Response To Ralstonia Solanacearum Inoculation

Papper(Capsicum annuum L.),regarded as one of the most important vegetables in China,is a kind of typical Solanaceae crop of soil-borne diseaes.Disease often and seriously occurs in high temperature and high humidity conditions,which affects its yield and quality.So,carrying out the molecular mechanism in pepper responses to disease contributed to the development of pepper genetic improvements and cultivating new varieties resistance to disease resistance.The WRKY proteins,a superfamily of transcription factors,widely involve in the regulation of various physio-logical programs,especially in plant response to diseases infection and the respone regulation to other environmental stresses.The analysis of the role of WRKY family members in plant disease resistance or tolerance is an important part and practical method to clarify the molecular mechanism in plant response to disease resistance.Although previous studies have found pepper WRKY family members CaWRKY6,CaWKRY27,CaWRKY40 and CaWRKY58 pepper play a role in pepper response to their important soil-borne pathogen Ralstonia solanacearum inoculated(RSI),high temperature high humidity(HTHH),the molecular mechanism is still unknown.Here we found a protein CaWKRY71 interacted with CaWRKY40 by yeast two-hybrid.Researchs on the expression of CaWRKY71 as well as the interaction of CaWRKY71 and CaWRKY40 in pepper response to bacterial Ralstonia solanacearum have been done,and the main results are as follows:(1)Sequence analysis showed that CaWRKY71 contained a 993 bp complete ORF,encoding 331 peptide amino acid residues,and the deduced amino acid sequences has 90%,86%,78%,78%,34%sequence homology similarity with potatoes,tomatoes,tobacco,rice homologous genes,respectively.Based on CaWRKY71-GFP transient overexpression in the Nicotiana benthamiana leaves combined with confocal laser scanning microscopy,showed that CaWRKY71 mainly localized in the nuclei in plant cells.(2)The result of real-time RT-PCR analysis showed that Ca WRKY71 can involve the processes of the response to Ralstonia solanacearum infection in pepper plants,and the transcriptional expression of CaWRKY71 was inducted by exogenous plant hormones salicylic acid(SA)and ethephon(ETH).(3)The result of virus-induced CaWRKY71-silencing in pepper plants showed that it resulted in enhancing the sensitivity to Ralstonia solanacearum inoculation,meanwhile down-regulated the expression of immunity or thermotolerance associated marker genes such as CaPRl,CaNPR1,CaDEFl and CaABR1.(4)Overexpression of 35S::CaWRKY71 in pepper leaves,showed CaWRKY71 transient overexpression triggered serious HR-mediated cell death with deep color of trypan blue staining and DAB staining and high ion leakage.And it observably up-regulated the expression of immunity or thermotolerance-associated marker genes CaPRl,CaNPRl,CaDEFl and CaABRl,while 35S::Ca WRKY71-SRDX transient overexpression had no obvious effect.(5)The interaction between CaWRKY71 and CaWRKY40 in the nuclei was verified by co-Immunoprecipitation(coIP)and bimolecular fluorescence interaction(BiFC).The result of chromatin immunoprecipitation(ChIP)analysis suggested that CaWRKY71 directly bound to the W-box in CaPR1,CaNPR1,CaDEF1 and CaABR1 promoters,and the capacity to bind the promoters of above-mentioned genes was enhanced by the Ralstonia solanacearum inoculation though transient overexpression of 35S::CaWRKY71 in the pepper plants.In contrast,CaWRKY40-silencing significantly decreased binding capacity of CaWRKY71 with the W-box in CaPR1,CaNPR1,CaDEFl and CaABR1 promoters.These results illustrated CaWRKY71 can directly regulate the expression of defense-related or thermotolerance-associated marker genes CaPR1,CaNPR1,CaDEFl and CaABR1,and CaWRKY40 has a significantly positive regulation effect on CaWRKY71 when it regulats CaPR1,CaNPR1,CaDEF1 and the CaABR1.(6)The ion leakage rate and the HR-mediated cell death of CaWRKY71 and CaWRKY40 transiently overexpressing pepper leaves were significantly improved.Comparing co-overexpression CaWRKY71 and CaWRKY40 with individual transient expression,the ion leakage rate and the HR-mediated cell death can be significantly improved.Meanwhile,the expression of immunity or thermotolerance-associated marker genes CaPR1,CaNPR1,CaDEF1 and CaABR1 were up-regulated comparing with the transient overexpression Ca WRKY71 and Ca WRKY40,separately.(7)The result of ChIP-PCR showed the DNA precipitated by CaWRKY40 harbored W-box at CaWRKY71 promoter,suggesting that CaWRKY40 can directly bind to CaWRKY71 promoter.The result of Real-time RT-PCR showed that the transient overexpression of CaWRKY40 can significantly improve the expression of CaWRKY71,indicating that CaWRKY40 can directly bind to W-box in the CaWRKY71 promoter and promote the expression of CaWRKY71.While the DNA precipitated by CaWRKY71 without any CaWRKY40 promoter sequence.Real-time RT-PCR detected the transcription levels of CaWRKY40 and found that transient overexpression of CaWRKY71 increases the tanscriptional expression level of Ca WRKY40.These results indicate that CaWRKY71 is able to indirectly promote the expression of Ca WRKY40.In conclusion,there is an inter-relationship on the expression and function between CaWRKY71 and CaWRKY40,constructing a WRKY feedback loop plays a key role in pepper response to Ralstonia solanacearum inoculation.