The Role Of CaWRKY17 And Its Interaction With CaWRKY40 In Peppers' Response To High-temperature-high-humidity Or Ralstonia Solanacearum Inoculation

Pepper is a kind of vegetable which had the largest plant acreage and economic value in China.It is also a typical plant in Solanaceae which likely suffer soil-borne disease that reduces its yield and quality.So,it is important to elucidate the underlying mechanism of pepper resistance.The plant that responses pathogenic inoculation is largely regulated by transcriptional regulation.Transcription factors and its network play an important role in this process.WRKYs are one of the largest transcription factor families in plant.It is a significant way to elucidate the molecular mechanism of pepper disease resistance by analysis the roles and mechanisms of transcription factors and its transcriptional activated network.CaWRKY40 was known as a positive regulator in the response of high-temperature-high-humidity(HTHH)and Ralstonia solanacearum inoculation(RSI)in pepper.However,the underlying mechanism remains unclear.In this study,CaWRKY17 was found to interact with CaWRKY40 by yeast-two-hybrid system,and we analyzed the expression of CaWRKY17 and its interaction with CaWRKY40 in pepper response to HTHH or RIS.The main results are as follows:(1)Real-time RT-PCR analysis showed that CaWRKY17 is able to response to HTHH and RSI and had a high expression in pepper root.We found that CaWRKY17-GFP was localized in the whole cell by transient overexpression 35::CaWRKY17-GFP in N.benthamiana leaves,but forms a multimer in the nuclei with under the treatment of RSI or HTHH.(2)The result of virus-induced gene silencing(VIGS)experiment showed that silenced Ca WRKY17 in pepper plant significantly attenuated their resistance to RSI and tolerance to HTHH,accompanied by down-regulation of immunity and thermotolerance-associated genes CaPR1,CaNPR1,CaDEF1,CaHSP24 compared with the control.(3)Transient overexpression 35S::CaWRKY17 in pepper leaves showed that hypersensitive response(HR)mediates cell death would be activated,and enhance thermotolerance of pepper leaves,accompanied by up-regulation immunity associated marker genes.On the contrary,transient overexpression 35S::CaWRKY17-SRDX could not activate cell death,and down-regulate the transcriptional expression of immunity and thermotolerance-associated marker genes.(4)The interaction between CaWRKY17 and CaWRKY40 in the nuclei was demonstrated by co-immunprecipitation(co-IP)and bimolecular fluorescence complementation(BiFC).HR and the ability of resistance to RSI and tolerance to HTHH were significantly improved by co-overexpression of CaWRKY17 and CaWRKY40.In addition,the data showed that the interaction between CaWRKY17 and CaWRKY40 would enhance the binding activity of CaWRKY17 and CaWRKY40 to the promoters of their target genes,and improve the ability of transcription regulation.(5)The transcriptional expression level of CaWRKY40 was up-regulated by transient overexpression of CaWRKY17 and vice versa.The data suggested that there is a relationship of mutual expression between Ca WRKY17 and Ca WRKY40,and Ca WRKY17 and Ca WRKY40 can form a positive feedback loop to amplify the stress signal.The results will help to clarify the positive regulation mechanism of Ca WRKY40 under the treatment of HTHH and RSI,and reveal the role of interaction of WRKY transcription factors and complex regulatory network composed of protein interaction and expression regulation in pepper against to HTHH and RSI.