CaWRKY22 Acts As A Positive Regulator In Pepper's Response To Ralstonia Solanacearum By Modulating Defense Genes Including CaWRKY40

Pepper(Capsicum annuum)is an important vegetable and industrial raw material crop in China and around the world with production efficiency.However,pepper is also a solanaceae with many soil-borne pathogens,and the occurrence of disease often affects its yield and quality and ultimately leads to a decline in its benefits.Cultivating and popularizing disease-resistant pepper cultivars is the most economical and effective solution to the problems caused by diseases during its production,and elucidating the molecular mechanism of disease resistance is an important basis for the effective genetic improvement of pepper disease resistance.In view of the fact that plant disease resistance is greatly regulated by the transcription level,and WRKY transcription factor plays an important role in plant disease resistance,the research on the role and mechanism of WRKY in the resistance of pepper and other solanaceaes is feasible approach to elucidate the mechanism of disease resistance.In view of this,this study carried out to investigate the role of a member of the ?e WRKY transcription factor,CaWRKY22,in pepper resistance to bacterial wilt and analyzed its mechanism of action.The main findings are as follows:1.To identify the WRKY TFs in pepper genome that are probably involved in pepper immunity,a scanning for pathogen responsive cis-elements containing promoters of WRKY TF encoding genes were performed,and putative WRKY genes with pathogen responsive cis-elements including TCA,TGACG,and W-box was identified,its deduced amino acid sequence contains a highly conserved WRKY domain with zinc finger like motif in its C-terminal,indicating that its belong to the group IIe.As the deduced amino acid sequence exhibits the highest sequence identities to WRKY22 among all members in WRKY TF families of Solanum pennellii,Solanum tuberosum,Nicotiana sylvestris,Gossypium raimondii with 91%,91%,88%and 55%sequence identities to SpWRKY22,StWRKY22,NsWRKY22 and GrWRKY22,respectively.2.To examine that modulation of CaWRKY22 expression by Ralstonia solanacearum inoculation,qRT-PCR analysis was carried out and CaWRKY22 transcription levels were up-regulated in leaves inoculated with R.solanacearum but not in the mock treatment,implying that CaWRKY22 is involved in the regulation of response of pepper to R.solanacearum infection.As the phytohormones such as salicylic acid(SA),jasmonic acid(JA),ethylene(ET)and abscisic acid(ABA)are crucial signaling molecules in plant responses to biotic and abiotic stresses,to test their possible involvement in the regulation of CaWRKY22 expression,the expression of CaWRKY22 was assayed by qRT-PCR upon the their exogenous application.The result showed that CaWRKY22 was upregulated by exogenously applied SA,MeJA,ETH,but was downregulated by ABA.The results indicate that CaWRKY22 might be involved in pepper immunity against RSI,probably regulated positively by SA,JA and ET while negatively by ABA.3.To check the subcellular localization of CaWRKY22,a fused CaWRKY22-GFP protein was transiently expressed in the leaves of N.benthamiana by infiltration of GV3101 cells containing 35S::CaWRKY22-GFP(using 35S:GFP as control).A laser scanning confocal microscope(LSCM)was used to observe the GFP signals in the infiltrated leaves.The GFP signal in the control leaves were found all over in the cell including cytosol,plasma membrane and nuclei,whereas GFP signal in CaWRKY22-GFP infiltrated leaves of N.benthamiana were found exclusively in the nuclei.4.To test whether CaWRKY22 play a role in pepper immunity against R.solanacearum infection,the loss of function experiment was conducted by virus-induced gene silencing(VIGS).The efficacy of gene silencing was determined by real time RT-PCR,and the results showed that the transcription level of CaWRKY22 in TRV2::CaWRKY22 pepper plants was less than 10%of that in TRV2::00 plants(control)pepper plants.An obvious wilting phenotype was observed in majority of R.solanacearum FJC100301 inoculated TRV2::CaWRKY22-silenced pepper plants,while no significant wilting phenotype was found in TRV2::00 control pepper plants at 7 dpi.In addition,colony-forming units(cfu)of the pathogen was also checked in TRV2::CaWRKY22-silenced and TRV2::00 control pepper plants at 72 hpi,and TRV2::CaWRKY22-silenced pepper plants showed significantly increase in growth of R.solanacearum as compared to that in the TRV2::00 control pepper plants.Histochemical staining was also employed to assess necrosis and H2O2 production in R.solanacearum-infected CaWRKY22-silenced and control pepper leaves.A strong DAB(dark brown)staining and hypersensitive response(HR)mimic cell death displayed by darker trypan blue staining were detected in the control leaves whereas,the intensities of DAB and trypan blue staining were distinctly reduced in CaWRKY22-silenced leaves.The severity of cell necrosis was also analyzed by ion leakage displayed by electrical conductivity caused by plasma membrane damage after inoculation by a highly virulent strain of R.solanacearum FJC100301,the result showed that TRV::00 pepper plants exhibited high ion leakage as compared to TRV::CaWRKY22 pepper plants leaves.In addition,transcript levels of the known defense-related pepper genes including CaP02,CaPR4,CaACC,CaBPRl,CaDEFl and CaHIRl were found to be down-regulated in CaWRKY22-silenced pepper plants as compared to that in the control plants.All these results suggest that CaWRKY22-silencing in pepper resulted in impaired resistance to RSI.5.CaWRKY22,was transiently over-expressed in pepper plants by infiltration with GV3101 containing 35S::CaWRKY22 and using the empty vector as a control to further confirm the results of loss of function experiment.Histochemical staining was done to assess cell necrosis and H2O2 production.Very obvious cell death and darker staining of trypan blue and DAB were found at the infiltrated sites of GV3101 cells containing 35S::CaWRKY22.Conversely,no noticeable or clear cell death and darker staining of trypan blue or DAB was found in the mock treated pepper leaves.The higher ion leakage was also observed to be triggered by transient overexpression of CaWRKY22 as compared to mock treated pepper leaves.In addition,the defense-related pepper genes including CaP02,CaPR4,CaACC,CaBPR1,CaDEF1 and CaHIR1 were found to be upregulated by transient overexpression of CaWRKY22 compared to that in the control treatment.6.By chromatin Immunoprecipitation(ChIP)assay,CaWRKY22 was found to directly bind to the W-boxes-containing fragment of the promoters of CaPR1,CaDEF1,CaWRKY40 and CaWRKY22,while did not bind to the promoter fragment without W-box,indicating that CaWRKY22 may bind the promoters dependent on the W-box.7.To assay the possible inter-relationship between CaWRKY22 and CaWRKY40,transient overexpression and VIGS based silencing in combination with qRT-PCR were performed,and the results showed that the transcription of CaWRKY22 was increased in CaWRKY40 transiently expressing leaves compared to the control,on the other hand,the transcription of CaWRKY40 was increased in CaWRKY22 transiently expressing leaves.By contrast,CaWRKY40 was significantly higher in TRV::00 than in TRV::CaWRKY22-silenced pepper plants.In addition,when CaWRKY40-HA was transiently overexpressed in CaWRKY22-silenced pepper plants,the transcript levels of the defense-related pepper genes CaPO2,CaPR4,CaACC,CaBPR1,CaDEF1 and CaHIR1 were significantly lower than that in the control plants.9.Transient overexpression and qRT-PCR were also performed to assay the inter-relationship between CaWRKY22 and CaWRKY6,CaWRKY27,CaWRKY40 and CaWRKY58.The results showed that the transcription of CaWRKY22 was increased by transient overexpression of CaWRKY6,CaWRKY27 and CaWRKY40,but was decreased by transient overexpression of CaWRKY58.On the other hand,the transcription of CaWRKY6,CaWRKY27 and CaWRKY40 was increased,but that of CaWRKY58 was decreased,by CaWRKY22 transient overexpression in pepper plants.The above results collectively suggest that CaWRKY22 is induced by challenge of R.solanacearum in pepper plants,thereby enhancing the disease resistance of pepper plants through transcriptional regulation of a series of disease-resistance-related genes including CaWRKY40.CaWRKY22 forms a positive feedback loop with CaWRKY6,CaWRKY27,CaWRKY40,and a negative feedback loop with CaWRKY58,all these WRKYs form a WRKY network,which may play an important role in resistance to bacterial wilt in peppers through finely modulating a large number of disease-resistance-related genes at transcription level.However,the involvement of other WRKY TFs,the hierarchical distribution among the WRKY members,their association in terms of expression and function,etc.remains to be further studied.