Molecular Mechanism Of NbRFP Regulating HR Induced By ODV RepA On Tobacco Plants

Oat dwarf virus(ODV)is a typical member in Geminiviridae Mastrevirus.RepA protein encoded by ODV had been reported to function as a hypersensitive response(HR)elicitor.A RepA-interacting NbRFP protein had been identified from Nicotiana benthamiana(N.benthamiana)cDNA library in the yeast two-hybrid screening assay by using RepA as bait.To better elucidate the molecular mechanism of RepA-induced HR,our research was focused on confirming the in vivo and in vitro RepA-NbRFP interaction and analyzing the biological function of this interaction in regulating RepA-induced HR.Full length NbRFP cDNA sequence was cloned from N.benthamiana library,sequence alignment showed that it shares 97%amino acid sequence similarity with NtRFP isolated from Nicotiana tabacum(N.Tabacum),which had been confirmed to be a functional RING E3 ubiquitin ligase.To determine the expression pattern of NbRFP,quantitative reverse transcript qRT-PCR was performed with total RNA from various tobacco tissues as template.The highest NbRFP mRNA level was detected in the root and leaf before florescence,and in the root and flower during florescence.Besides,NbRFP protein was observed to locate in cell nucleus and cytoplasm in tobacco epidermic cells through laser scanning confocal microscope.The in vitro RepA-NbRFP interaction was confirmed in yeast two hybrid assay.In the meantime,bimolecular fluorescence complementation(BiFC)assay was also introduced to confirm their in vivo interaction,and the fluorescence showed that these two proteins mainly interacted in cell nucleus and cytoplasm in epidermic cells.Next,full-length NbRFP and RepA cDNAs were cloned into pBA-GFP and pBA-Flag vectors separately for Co-Immunoprecipitation(Co-IP)assay in N.benthamiana cells.It also confirmed the in planta interaction between RepA and NbRFP protein.Virus induced gene silencing(VIGS)system was used to systemically knock down the expression of indigenous NbRFP gene.RepA-induced HR was significantly delayed in NbRFP silenced tobacco,while in transgenic tobacco expressing NbRFP gene,RepA-induced HR appeared in advance compared with that in wild type plant,indicating that NbRFP was involved in RepA-induced HR.It was worth noticing that the RepA protein level was affected by neither overexpressing NbRFP nor silencing NbRFP.It suggested that NbRFP regulated RepA-induced HR other than affecting RepA protein stability.Putative functional domains of NbRFP protein were deduced through online software InterProScan.NbRFP contains a von Willebrand factor(vWF)type A domain(VWA)and a RING finger domain(RING).To locate the domains necessary for RepA-NbRFP interaction,different deletion mutants of NbRFP were tested in the BiFC assay and the RepA binding region of NbRFP was narrowed down to its RING domain.Also transient co-expressed NbRFP deletion mutants together with RepA in tobacco cells showed that NbRFP mutants with RING domain deletion was unable to regulate RepA-induced HR any more,indicating that RING domain is the key element of NbRFP in regulating RepA-induced HR.In order to dissect the molecular mechanism of this regulation by NbRFP toward RepA-induced HR,gene expression profiles of RepA-induced HR in transgenic tobacco overexpressing NbRFP and Wild type tobacco were analyzed and compared.Two different time points,18 h and 36 h after transient expression of RepA in tobacco leaves were chosen for further gene expression profiles analysis.In total,the transcriptional levels of 22 genes were significantly up regulated and 15 down regulated in the 18 h sample.While in the 36 h sample,the transcriptional levels of 384 genes were found up regulated and 232 down regulated.Based on KEGG database,pathway enrichment analysis of differential expression genes(DEGs)showed that proteasome degradation pathway,plant-pathogen interaction pathway,ubiquitin mediated proteolysis pathway,autophagy pathway,plant hormone signal transduction pathway and other HR related pathways were altered.JAZ from jasmonica acid(JA)signal transduction pathway was chosen for analysis.The transcriptional level of JAZ was dramatically decreased in NbRFP overexpressed plant,suggesting that overpression of NbRFP might influence the expression of JAZ gene,so as to regulate RepA-induced HR through affecting JA signaling pathway.