CaWRKY40b Act As A Negative Regulator In Pepper’s Response To Ralstonia Solanacearum By Modulating Defense Genes Including CaWRKY40

Despite the agricultural significance of pepper and crucial role of WRKY transcriptional factors（TFs）in plant responses to biotic stress,the roles of the majority of pepper WRKY s in immunity remain uninvestigated.Herein,CaWRKY40b of pepper,a homologue of AtWRKY40 in Arabidopsis,was functionally characterized in pepper immunity,and the main findings are as followings:1.A highly conserved WRKY domain containing（C-X5-C-X23-H-X1-H）zinc-finger-like motif is found in the CaWRKY40b,which belong to Group Ⅱa WRKY family.CaWRKY40b share 87%,82%,71%,58%and 45%amino acid sequence identity to its homologues in Solanum lycopersicum（XP₀06341684.1）,S.tuberosum（XP₀06356251.1）,N.sylvestris（XP₀09802478.1）,Arabidopsis thaliana（NP₁78199.1）and Oryza sativa（Q6IEK5.1）.In addition,CaWRKY40b shares 59.46%sequence identity to CaWRKY40 which is a positive regulator in pepper’s response to R.solaanacearum and it shares the highest sequence identity to At WRKY40 among all members of WRKY family in Arabidopsis.2.For subcellular localization of Ca WRKY40b,a fused Ca WRKY40b-GFP protein was expressed in N.benthamiana leaves by infiltration of GV3101 cells containing 35S::Ca WRKY40b-GFP with 35S:GFP construct as control,the GFP signal was observed under a laser scanning confocal microscopy（LSCM）in the infiltrated leaves,the GFP signal in 35S:GFP transient overexpressed leaves was found throughout the cell including plasma membrane,cytosol and the nuclei,while GFP signal in the majority of cells（58/60）in Ca WRKY40b-GFP overexpressing N.benthamiana leaves was found in the nuclei.Interestingly,two cells out of 60 exhibited GFP signal in the whole cells including the cytosol.The presence of HSE and W-box in the promoter of CaWRY40b indicating its possible involvement in pepper immunity as well as in its response to heat stress.To test this possibility,the transcription of CaWRKY40b was assayed in pepper plant challenged with to Ralstonia solanacearum inoculation（RSI）and high-temperature-high-humidity（HTHH）treatment by real time RT-PCR,and the result showed that CaWRKY40b was significantly down-regulated by RSI,implying that CaWKRY40b is involved in pepper’s response to RSI.3.To further determine the role of CaWRKY40b in pepper immunity,the effect of gene silencing of CaWKRY40b in pepper plants on pepper immunity was assayed by virus-induced gene silencing（VIGS）using two independent VIGS vectors of CaWRKY40b.The gene silencing efficacy was determined by real time RT-PCR,and the result showed that the transcript level of CaWRKY40b in TRV2::CaWRKY40b-1 and TRV2::CaWRKKY40b-2 pepper plants was less than 10%of that in TRV2::00 plants.A clear wilting phenotype was observed in majority of FJC100301 inoculated TRV2::00 pepper plants,while no obvious or only a slight wilting phenotype was found in both TRV2::CaWRKY40b-1 and TRV2::CaWRKY40b-2 pepper plants at 7 dpi.In addition,colony-forming units（cfu）of the pathogen at 24 or 48 hpi were also measured,and TRV2::Ca WRKY40b-1 TRV2::Ca WRKY40b-2 pepper plants showed significantly decreased in Ralstonia solanacearum growth compared to that in the wild-type plants.All these results suggest that CaWRKY40b-silencing in pepper resulted in plants resulted in enhancing the resistance to RSI.4.To further confirm the result of loss of function analysis,transient overexpression of CaWRKY40b and CaWRKY40b-SRDX（chimeric repressor version of CaWRKY40b）in pepper leaves by filtration with GV3101 containing 35S::CaWRKY40b,35S::Ca WRKY40b-SRDX,using the empty vector as a control,and the HR cell death and H2O2 accumulation was detected by trypan blue staining and DAB staining,respectively.Clear cell death and darker staining of trypan blue and DAB were consistently found around infiltrated sites of GV3101 cells containing 35S::CaWRKY40b-SRDX.However,no obvious cell death and darker staining of trypan blue or DAB was observed in CaWRKY40b transiently overexpressing or in the mock treated pepper leaves.The higher ion leakage was also found to be triggered by 35S::CaWRKY40b-SRDX than that of ion leakage by 35S::CaWRKY40b.5.The genes CA00g87690（CaWRKY40）,CA05g11520（jasmonic acid-amido synthetase JAR1-like isoform X2,JAR1）,CA06g24540（heat shock cognate 70 kDa protein 1,HSC701）,CA01g13570（ETHYLENE INSENSITIVE 3-like,EIN3）,CA02g12020（LRR receptor-like serine/threonine-protein kinase FLS2,FLS2）,CA10g01730（LRR receptor-like serine/threonine-protein kinase At3g47570 isoform X1,RLK1）,CA05g11620（putative cyclic nucleotide-gated ion channel 8,CNGIC8）and CA09g10430（calcium-dependent protein kinase 13,CDPK13）were previously found to be the potential target genes of CaWRKY40b by Chromatin immune-precipitation ChIP-seq.To confirm this possibility,ChIP-real time RT-PCR was employed and the result showed that the promoters of all these genes were found to be bound by CaWRKY40b.However,the immunity associated genes including CaPR1,CaNPR1 and CaDEF1 which were not the target genes of CaWRKY40b by ChIP-seq that exhibited no binding of their promoters to CaWRKY40b.6.To test if CaWRKY40b can transcriptionally regulated the its potential target genes,their transcription levels were assayed in RSI challenged CaWRKY40b-VIGS pepper plants,and the results showed that the transcriptional level of CaWRKY40,JAR1,BPR1,EIN3,FLS2,RLK1,CNGIC8 or CDPK13 was enhanced significantly by RSI,but was significantly higher in CaWRKY40b silenced pepper plants than that in the control plants with or without RSI.By contrast,the transcription of CaHSC70 was down-regulated by RSI,and its expression in CaWRKY40b silenced pepper plants was significantly lower than that in the control plants.In addition,the transcriptional levels of these target genes were also assayed in CaWRKY40b-SRDX transiently expressing pepper leaves,and the result showed that the transient overexpression of CaWRKY40b-SRDX enhanced the expression of CaWRKY40,JAR1,BPR1,EIN3,FLS2,RLK1,CNGIC8 and CDPK13 compared to the control,while the transient overexpression of CaWRKY40b significantly down-regulated the transcription of these genes compared to the control.7.By ChIP analysis,CaWKRY40b was found to bind the promoter of CaWRKY40b,and the self-regulation of CaWRKY40b was also tested comparatively by effect of transient overexpression of CaWRKY40b on expression of GUS driven pCaWRKYb and on the transcription of CaWRKY40b.the result showed that the transient overexpression of CaWRKY40b enhanced significantly expression of GUS and activity of GUS driven by promoter of CaWRKY40b,and transcript level of CaWRKY40b was also enhanced by real time RT-PCR using a pair of specific primers based the sequence of 3-UTR’ of Ca WRKY40b.Overall,CaWRKY40b was also found to bind promoter of CaWRKY40b,the expression of CaWRKY40b was activated by transient expression of CaWRKY40b itself.The data validate the role of CaWRKY40b as a negative regulator by transcriptional modulation of a subset of immunity-associated genes including CaWRKY40,repressing immunity in the absence of pathogens and de-repressing immunity upon the challenge of pathogens.