The Functional Analysis Of CaPti1/CaERF4 Interaction In Response To Ralstonia Solanacearum Infection In Pepper Plant

Plants are inevitably exposed to pathogen attacks in their natural inhabitants,it is crucial for plant to perceive and transfer the signals downstream to the nuclei,where the signals are translated into appropriate defense reaction through massive transcriptional reprogramming with the action of various transcription factors.Although the involvement of TFs have been intensively investigated in the past several decades,however,how TFs are activated by upstream signaling components and fulfill their functions remains to be elucidated even in the model plants such as rice and Arabidopsis,let alone the non model plants such as pepper.Pto,a resistance protein that recognize avrPto in pathogen and activate resistance to in phytopathogenic bacterium Pseudomonas syringae pv tomato.In the present study,a interacting partner of putative Pto(CaPti1),which was previously isolated from a cDNA library,was functionally characterized in pepper immunity against Ralstonia solanacearum,in addition,its interacting protein,CaERF4,was isolated and confirmed,and the function of CaPtil/CaERF4 interaction in pepper response to Ralstonia solanacearum was also characterized.The results are as followings.1.CaPtil contains a open reading frame of 1200 bps in length,and its deduced amino acid sequence harbors a tyrosine-specific kinase conserved domain,and shares 94%、87%、87%.86%and 78%identities to Ptils in Nicotiana gossei,Lycopersicon esculentum,Solanum tuberosum,Ziziphus jujuba and Arabidopsis thaliana,respectively.2.By qRT-PCR,it was found that CaPtil exhibited constitutive expression in roots,stems,leaves,flowers and fruits of pepper plants,the highest transcript abundance was found in the roots,and lowest was detected in the leaves.Upon the inoculation with Ralstonia solanacearum,the transcript level of CaPtil was induced at 1 hpi and since then it continued to rise.CaPtil upregulated transcriptionally by exogenous application of SA,MeJA or ETH,Upregulation of CaPtil by exogenous applied ETH occur in the early period from 6 to 24 hpt,the highest induction of CaPtil by exogenous application of SA occur in the middle period from 12 to 24 hpt,while that by MeJA occur 48 hpt.These results imply the possible involvement of CaPtil in the pepper immunity against Ralstonia solanacearum as well as other biological processes.3.By employing virus induced gene silencing and transient overexpression in pepper plants,the function of CaPtil in pepper response to Ralstonia solanacearum was assayed.The results showed that the silencing of CaPtil significantly decreased the resistance to Ralstonia solanacearum inoculation,accompanied with upregulations of immunity associated marker genes including CaPR1,CaHIR1,CaPO2 and CaDEF1.By contrast,the transient overexpression of CaPtil triggered the HR cell death and accumulation of H2O2,manifested with darker trypan and DAB staining,enhanced ion leakage and upregulation of immunity associated genes including CaPR1,CaHIR1,CaPO2 and CaDEF1.In addition,CaPtil overexpressing T2 transgenic tobacco lines were acquired,and we found that the overexpression of CaPtil enhanced the resistance of tobacco plants to RSI,accompanied with enhanced the tested immunity associated marker genes.All these results indicate that CaPtil act as a positive regulator in pepper response to RSI.4.As a possible interacting partner of CaPtil,CaERF4,was isolated by yeast hybrid two system from pepper cDNA library,we confirm CaPtil/CaERF4 interaction with co-IP and BiFC,the result confirm their interaction and found that this combination occur in exclusively in the nuclei,and their subcellular localization individually were also performed,that result showed that CaPti1 locates in the whole cell including the nuclei,while CaERF4 locates exclusively in the nuclei.In addition,the transcript abundance of CaERF4 was detected in different organs of pepper plants,the result revealed that CaERF4 exhibit constitutive in all of the tested organs,and the highest level was found in the stems while the lowest was found in the roots.It was also found that transcript levels of CaERF4 were also upregulated by exogenous application of SA,MeJA and ETH,similar to the case in CaPtil,the upregulation of CaERF4 by SA and ETH were found in the early or middle stages,while the regulation of CaERF4 by MeJA was found in the later stage.All these results imply that CaERF4 is involved in that response of pepper to RSI.5.By virus induced gene silencing and transient overexpression in pepper plants,the function of CaERF4 and its interaction with CaPti1 were assayed.The results showed that silencing of CaERF4 decreased the pepper resistance to RSI,enhanced the growth of Ralstonia solanacearum and expression of the tested immunity associated marker genes including CaDEF1,CaPR1,CaPO2,CaHIR1.By contrast,the transient overexpression of CaERF4 triggered significant HR mimic cell death and accumulation of H2O2,manifested with darker trypan blue and DAB staining,as well as enhanced expression of the tested immunity associated marker genes.All these results showed that CaERF4 act as a positive regulator in pepper immunity against Ralstonia solanacearum.6.To assay the possible function of CaPti1/CaERF4 interaction in pepper immunity toward Ralstonia solanacearum,transient overexpression of CaPtil and CaERF4 were performed in combination.The result showed that the transient co-overexpression of CaPtil and CaERF4 significantly enhanced the HR mimic cell death and the upergulation of CaDEF1,CaPR1,CaPO2,CaHIR1 compared to that by their transient overexpression individually.7.To reveal the possible mechanism underlying the enhanced of transcriptional regulation of CaERF4 by CaPtil,we performed chromatin immunoprecipitation analysis of CaERF4 binding to CaPR1 promoter by CaPtil/CaERF4 transient co-expression.The results showed that CaERF4 can bind to the GCC box-containing fragment of the CaPR1 promoter in pepper,which is enhanced by transient overexpression of CaPtil.In summary,we propose that both CaPtil and CaERF4 are induced by R.solanacearum in pepper plants,CaPtil interacts with CaERF4 in the nucleus to increase transcription of PR genes by CaERF4,thereby improving the resistance of pepper to R.solanacearum infection.