Pathogenic Analysis Of The Coat Protein And Replicase Gene Mutants Of Cucumber Green Mottle Mosaic Virus

Cucumber green mottle mosaic virus(CGMMV)is one of the most devastating viruses infecting members of the family Cucurbitaceae.CGMMV causes huge threat to cucurbit production.In the last 10 years,it has been spread to larger areas in the word.To find the critical cites of CGMMV infectivity and investigate the inside molecular mechanism of the virus' s pathogenicity,different mutants were used as material based on the CGMMV infectious cDNA clone,and we had identified the pathogenicity of these mutants.Coat protein is not only involved in virion assembly but also a key factor determining the long-distance movement of the virus.The pathogenicity of the mutants of CGMMV CP alanine scanning library were identified by agrobacterium-mediated inoculation assay in Nicotiana benthamiana and watermelon.We also had a further detection of virus infection by DAS-ELISA and one step RT-PCR.We find the symptom of 23 alanine mutants had changed which showed yellow or chlorisis in Nicotiana benthamiana in 134 mutants.Most of these amino acid of mutants which showed yellow or chlorisis were nonpolar amino acid before the mutation.We also find 49 mutation sites showed no symptom after inoculation in Nicotiana benthamiana which showed negative by DAS-ELISA.Futhermore,these mutants have a certain rule which were located in the inside hollow structure area of four alpha helix and on the outer surface side of CP subunit.Assembly initiation site is located in the viral genome RNA,which will start virion assembly when CP subunit aggregate binding with this special region.After inoculation in N.benthamiana,V94 A,V97 and T104 A showed delayed systemic symptoms We found a spontaneous and substitution mutation in the coat protein when we identified the mutants V94 A,V97 and T104 A about assembly initiation site and the adjacent region.All of these three mutants induced mutation from glutamic acid to lysine at residue 96 of CP gene.Next,we constructed two single point mutants E96 A and E96 K and three double mutants V94A-E96 K,V97-E96 K and T104A-E96 K based on V94 A,V97 and T104 A.After inoculation in N.benthamiana,E96 A showed delayed systemic symptoms,but the E96 K and three double-sites mutants exhibited typical symptoms of mosaic when wild type also showed mosaic at seven days post-inoculation.Then,sap from CGMMV-infected N.benthamiana leaves was mechanically inoculated on watermelon plants.The results showed that the 96 th amino acid of CP affected the infectivity of CGMMV.and the assembly initiation sites and the adjacent region affected the infectivity of CGMMV.This is the first study about the relationship between virus assembly initiation site and pathogenicity.In order to obtain the attenuated strain of CGMMV,we constructed mutants E480 G,A1124V,N1157 D,P1397S,K546 R,V557T,V651 A,S762L,G86 S,S534F,P1362 L,480-1124,546-557,86-534,480-1124-1157,546-557-651,86-534-1362,GVDS and RTAL,refered to the attenuated related sites in the CGMMV genome RNA.Compared these mutation sites,we found the intervening region residue 480 in replicase induced a obvious delay in the systemic symptom.The infectivity will be lost when V557-T and P1397-S exist in the genome RNA.A closer look at the results indicates that these mutation sites were essential for the normal infection of the virus,and further clarify the direction of our screening of the CGMMV attenuated strain.