Molecular Mechanisms Of Nbnrp1 Mediated Verticillium Dahliae Elicitor PevD1-induced Disease Resistance In Nicotiana Benthamiana

PevD1 was a protein elicitor isolated from metabolites of Verticillium dahliae XII8 strain.Our previous research has explored the interacting protein of PevD1 in Nicotiana benthamiana,which was designated as Nbnrp1,and the Nbnrp1-RNAi transgenic plants was obtained.The preliminary study also found that Nbnrp1 could regulate PevD1-induced cell death and disease resistance in N.benthamiana.Based on previous results,the function of Nbnrp1 in PevD1-induced disease resistance in N.benthamiana was further clarified and this paper will explain the the mechanism of Nbnrp1modulated PevD1-induced disease resistance in N.benthamiana by RNA-Seq.The main results are as follows:1.The eukaryotic expression system of PevD1 was established and the purified PevD1 protein wasobtained.The eukaryotic expression vector pPICZ?A-PevD1 was constructed,then the expression vector was transfered into Pichia pastoris by electroporation;The positive transformants were selected to express the PevD1 protein;The purified protein was verified by SDS-PAGE and Western blot,and it could also induce HR in N.benthamiana leaves.2.Nbnrp1 positively regulated PevD1-induced defense responses and disease resistance in N. benthamianaThe wild-type and Nbnrp1-RNAi lines were infiltrated with 10?M PevD1 solution,and the early defense responses such as H₂O₂ accumulation,callose sedimentation and MAPK phosphorylation activation were detected at the corresponding time points.At the same time,the systemic resistance of the two lines induced by PevD1 were detected.The results showed that the early defense responses and systemic resistance of Nbnrp1-RNAi lines induced by PevD1 were decreased in comparison with wild-type lines.3.Differentially expressed genes analysis and verificationThe wild-type and Nbnrp1 silencing strains were infiltrated with 10?M PevD1 solution,and local leaf samples were collected at 0 h,6 h,12 h,24 h and 36 h,respectively.After sequencing,differentially expressed genes?DEGs?between WT and Nbnrp1-RNAi lines induced by PevD1 were identified,and then were analyzed by functional annotation and KEGG pathway enrichment.The results showed that DEGs were mainly enriched in the pathway of terpenoids biosynthesis.And then 10 resistance-related genes were selected for verification by qPCR,the results were consistent with the transcript data,which indicated the accuracy of the sequencing data.4.Sesquiterpenoid biosynthesis pathway involved in PevD1-induced disease resistance mediated by Nbnrp1Sesquiterpenes were important components of phytoalexin in N.benthamiana.The DEGs enriched in sesquiterpenes biosynthesis pathway were analysed and six key genes were selected for verification.The results showed that the expression levels of these genes in Nbnrp1-RNAi lines were significantly lower or lagged in response than those in wild type after PevD1 treatment.Which indicated that the silence of Nbnrp1 inhibited the expression of key genes in the synthesis of sesquiterpene,and then affected the synthesis of phytoalexin.Sesquiterpene biosynthesis pathway was involved in PevD1-induced resistance mediated by Nbnrp1 in N.benthamiana.