Pathogenic Mechanism Of Papaya Ringspot Virus-Watermelon Strain And Application Of Its Attenuated Mutants

Papaya ringspot virus(PRSV;genus Potyvirus)is grouped into two different strains,P and W.Strain PRSV-P infects both cucurbitaceous crops and papaya,and strain PRSV-W infects cucurbitaceous crops but not papaya.The cucurbitaceous crops(e.g.Cucurbita pepo and Cucumis melo)affected with PRSV-W showed mosaic,curl,distortion,blister on leaves,deformation in fruits and plant stunting,reducing yield and quality and even crop failures.PRSV causes destructive disease of cucurbitaceous crops in many countries including China,Poland,India and Brazil.In this study,we obtained and analyzed the complete genomic sequence of PRSV-SD,constructed an infectious cDNA clone of PRSV-SD,screened the mild mutants and verified the efficiency of cross protection.One zucchini plant showing symptoms of leaf mosaic,distortion and fruit ringspots was collected from Ji’nan city of Shandong province.Primary experiments showed that the sample was infected with PRSV,and resulting virus was designated as PRSV-SD accordingly.The complete genomic fragments of PRSV-SD were obtained with RT-PCR.The results of sequencing showed that the full genomic sequence of PRSV-SD was 10337 nucleotides(nt)excluding the 3′-terminal poly(A)tail.The 5′-and 3′-non-coding region(NCR)were 90 nt and 206 nt respectively.The putative polyprotein was 3346 amino acids in length.Comparison of the PRSV-SD isolate with 18 other PRSV isolates revealed that they shared nucleotide identities of 82.0%~89.3% at the complete genomic levels and amino acid identities of 90.6%~94.7% for the polyprotein.Phylogenetic analysis with complete genomic sequences indicated that the PRSV isolates were clustered into two major groups: Asia and America,and PRSV-SD was clustered to the Asia group.Selection pressure analysis revealed that all of the 11 proteins of PRSV-SD were under negative selection,but positive selection sites were detected in P1,P3,6K1,NIa-pro and NIb.The 35 S promoter and complete genomic fragment of PRSV-SD were inserted into binary vector pCambia0390 to produce the plasmid pCamPRSV-W with DNA homologous recombination in vitro.The pCamPRSV-W was inoculated to cucurbits such as Citrullus lanatus,Cucurbita pepo,Cucumis melo and Cucumis sativus by agroinfiltration.The symptoms and virus accumulation levels of pCamPRSV-W were similar to wild type PRSV-W in all the inoculated plants.The plasmid pCamPRSV-WGFP was obtained via homologous recombination in vitro,by inserting the gfp gene into the genomic region encoding the protease recognition site between NIb and CP from pCamPRSV-W.The pCamPRSV-W-GFP was inoculated to cucurbitaceous crops by agro-infiltration.The virus systemic movement in leaves was detected by GFP expression under UV illumination,and the insertion of GFP didn′t affect infection and movement of PRSV-W.The effects of the conserved amino acid sites in HC-Pro on virulence of PRSV-SD were analyzed based on the infectious clone of p CamPRSV-W-GFP by site-directed mutation in cucurbitaceous crops.The symptoms and virus accumulation of mutations gC26 A,gC57A,gK125 D,gK126D,gN137 A,gG317K,gP328 A,gN346A,gL375 A and gV417 A were significant reduced in C.melo leaves compared with that of the wild type virus at 15 days post agro-infiltration.The mild mutants C57 A,K125D,G317 K and P328 A were obtained from pCamPRSV-W,and inoculated to C.melo by agro-infiltration to the cotyledon.Then,the GFP-tagged wild type PRSV-W was used to mechanically inoculated the upper systemic leaves after different protective interval.The GFP accumulation level was tested by ELISA after 10 days of challenge inoculation.The cross protection results indicated that the mild mutations C57 A and P328 A provided complete cross protection effects when the protective intervals were 10 days.