The Function Of CaWRKY17 That Interact With CaCDPK17/29 In Response To High-temperature-high-Humidity And Ralstonia Solanacearum Inoculation

WRKY transcription factors play an important role in plant disease resistance by integrating the signal from the cytoplasm immune and transcriptional regulating resistance-related gene expression in the nuclei,but the mechanism about how to transmit the signal from cytoplasm to the WRKY protein is still not very unclear.In this study,we obtain two Ser/Thr protein kianase genes by yeast two hybrid separation,which were named CaCDPK17 and CaCDPK29,and interacted with CaWRKY17,respectively.We analyzed the expression of two protein-coding genes and the role of this two genes response to high-temperature-high-humidity and Ralstonia solanacearum infection in pepper plants.In addition,we verified the interaction,and the fuction of response to high-temperature-high-humidity and Ralstonia solanacearum infection in pepper plants.The main results were as follows:(1)The result of real-time RT-PCR showed that the transcriptional expression of CaCDPK17 can be induced by high-temperature-high-humidity,Ralstonia solanacearum infection and exogenous phytohorm-ones ABA.CaCDPK29 can be activated by high-temperature-high-humidity,Ralstoniasolanacearum,MeJA and SA.But its expression levels were restrained by exogenous phytohormones ABA.The subcellular localization experiment result showed that CaCDPK17-GFP and CaCDPK29-GFP located in the plasma membrane by transient overexpression 35s::CaCDPK17-GFP and 35s::CaCDPK29-GFP in N.benthamiana.We performed the VIGS assay and the result showed that silenced CaCDPK17 can enhance the pepper plants capacity of resistant to Ralstonia solanacearum infection,but slack the capacity of resistant high-temperature-high-humidity.The Ca CDPK29-silensed pepper plants exhibited significiently decreased resistance to Ralstonia solanacearum infection or tolerance to high-temperature-high-humidity compared with the wild-type control plants.(2)We have performed yeast two-hybrid,BiFC and co-IP to prove the result that CaWRKY17 interact with CaCDPK17 and CaCDPK29 in plasma membrane,respectively.HR-mediated cell death induced by CaWRKY17 and expression of defense-related genes were markedly reduced by co-transient overexpression of CaCDPK17 and CaWRKY17,but the ability that increased the transcript expression of thermotolerance-associated gene by transient overexpression of CWRKY17 was enhanced.The resistance of pepper plants to Ralstonia solanacearum infection or high-temperature-high-humidity by transient overexpression of CaWRKY17 was ulteriorly improved though co-transient overexpression of CaCDPK29 and CaWRKY17 in pepper leaves.In view of our another topic for study that CaWRKY40 interact with CaWRKY17 under the condition of high-temperature-high-humidity or Ralstonia solanacearum inoculation in nuclei,which means that CaWRKY17 could enter into nuclei.The influence that CaCDPK17 or CaWRKY29 affect CaWRKY17 to enter into nucleus also needs further research.(3)We further analyzed the result that CaWRKY40 and CaWRKY17 may directly regulate transcriptional expression of CaCDPK17 and CaCDPK29 which contain typical W-box in promoters by ChIP-qPCR.The result showed that CaWRKY 17 can not bind to the promoters of CaCDPK17 or CaCDPK29,but CaWRKY40 can bind to the promoters of CaCDPK17 or CaCDPK29.In addition,the ability that CaWRKY40 bind to CaCDPK29 promoter was strengthened by co-transient overexpression of CaWRKY40 and CaWRKY17 in pepper leaves.Accordingly,real-time RT-PCR indicated that transcriptional expression of CaCDPK17 and CaCDPK29 was not affected by transient overexpression of CaWRKY17.But transient overexpression of CaWRKY40 could significantly improve transcriptional expression of CaCDPK29.In conclution that CaCDPK17 and CaCDPK29 may play an important role in the process of transmitting the pathogen signal into nucleus and the function regulation of CaWRKY17 though interacting with CaWRKY17.Our data will help to further elucidate the underlying defense reaction molecular mechanisms of pepper’s response to Ralstonia solanacearum.