Virulence Mechanisms Of Cucumber Green Mottle Mosaic Virus And Functional Analysis Of Its Coat Protein-interacting Protein

CGMMV(Cucumber green mottle mosaic virus)belongs to the genus Tobamovirus,family Virgaviridae and it could be transmitted by seeds.In recent years,the frequent incidence of CGMMV in the word had caused serious economic losses in cucurbits production.In this study,CGMMV capsid protein antiserum was prepared firstly for the detection of virus in subsequent experiments.After that we constructed an infectious cDNA clone of CGMMV and obtained two attenuated mutants of it.Then we analysed the cross protection effects of these attenuated mutants by molecular biological techniques.Finally,we obtained a CP-interacting protein of CGMMV in N.benthamiana plants(NbPI)and we analysed its function by virus-induced gene silencing(VIGS).The experiment results were as follows:(1)Antiserum to CGMMV with high titer and specificity was prepared with CP expressed in E.coli.The resultant antiserum we prepared showed strong positive reaction with CP of CGMMV,but not with that of Tobacco mosaic virus in same genus.The titter of antiserum was 1:16 in agarose immuno diffusion and 1:1024 in ELISA.(2)We constructed an infectious cDNA clones of CGMMV based on Wei Fang isolate,which could infect watermelon.We got the whole genome of the virus by two fragments and inserted an intron of watermelon in the RdRp coding region of CGMMV.And then we put it under 35 S promoter in pCambia0390 vector to obtain a recombinant plasmid named pCGMMV-WF.The plants of watermelon inoculated with pCGMMV-WF through agroinfiltration showed similar symptoms and virus accumulation with the plants inoculated with wild type CGMMV,the inoculation efficiency was 100%.(3)The infectious cDNA clones of CGMMV Ji Nan isolate which could infect N.benthamiana plants had been already constructed in our laboratory.Five amino acid residues were highly conserved in its CP and RdRp coding region by amino acid sequence alignment.They are the 89 th and 114 th amino acids of CP and the 68 th,869th and 1069 th amino acids of RdRp,respectively.We changed these amino acid residues to alanine and the mutants named as CGMMV-CP-D89 A,CGMMV-CP-R114 A,CGMMV-RdRp-E68 A,CGMMV-RdRpK869 A,CGMMV-RdRp-E1069 A.We found the pathogenicity of mutants CGMMV-CP-D89 A and CGMMV-RdRp-E1069 A were significantly reduced after inoculaing with N.benthamiana plants.So we identified that the aspartic at position 89 th of CGMMV CP and glutamic at position 1069 th of RdRp were key amino acid sites which could seriously influence the pathogenicity of CGMMV.These attenuated mutants were then subjected to cross protection experiment on N.benthamiana plants.The results showed that the protective efficiencies of CGMMV-CP-D89 A and CGMMV-RdRp-E1069 A were 91.7% and 100% respectively,which mutants could effectively prevent the severe infection.According to the results on N.benthamiana plants,we remoulded the CGMMV-WF which could infect watermelon to obtain mutants CGMMV-WF-CP-D89 A and CGMMV-WF-RdRp-E1069 A.The results of cross protection assay on watermelon showed that the mutant CGMMV-WF-CP-D89 A could not protect watermelon effectively,the protection efficiency was only 11.1%;however,the mutant CGMMV-WF-RdRp-E1069 A could protect watermelon perfectly and its protection efficiency was up to 100%.(4)We found that the coat protein of CGMMV could interact with a N.benthamiana protease inhibitor(NbPI)through Pull down experiment.In addition,we also found that the CP of TVBMV could interact with the same N.benthamiana protease inhibitor.After verifying the interaction relationship by YTHS and BiFC,we silenced the NbPI gene through VIGS.We found the symptoms of NbPI silenced plants were much more serious and the viral replication level was significantly elevated after challenging CGMMV(or TVBMV).These results suggested that Nb PI affect viral replication through binding CPs of virus.