Genetic Diversity And Pathogenicity Analysis Of Zucchini Yellow Mosaic Virus

Zucchini yellow mosaic virus?ZYMV,genus Potyvirus?is one of most important plant viruses infecting cucurbit crops.It can induce mosaic,crinkle,fruit malformation symptoms,resulted in serious losses in cucurbit crops production.Having an accurate system detecting ZYMV and planting resistant cucurbit crops were the most efficient ways to control the virus disease.Here,we prepared ZYMV coat protein?CP?antiserum,analyzed the taxonomic status of ZYMV isolates infecting Shandong province cucurbit crops,obtained a full-length infectious clone of ZYMV,characterized the role of HCpro WxxxG motif during ZYMV infection,screened the resistant cucurbits cultivars.The main results are shown as follows:?1?Preparation of ZYMV CP specific polyclonal antibody.ZYMV CP gene was amplified with specific primers and subsequently cloned into prokaryotic expression vector pEHISTEV.The resultant recombinant plasmid was expressed into Escherichia coli.Screening of different E.coli,IPTG induction time and IPTG concentration showed that expressing ZYMV CP in E.coli Rosetta with 0.2mmol·L^-1 IPTG for four hours was the most suitable condition.The expressed fusion protein with a molecular weight of about 37 kDa was obtained and immunized to healthy New Zealand long-eared rabbits.ZYMV CP antiserum with the titer of1:32768 was obtained.Comparison between ELISA and RT-PCR showed that the antiserum has high accuracy.ELISA detection of Cucurbita pepo?Lvyuandongbao?and Cucurbita moschata?Huoyingua?seeds revealed the incidence of ZYMV was 39.8%and 15.1%,respectively.In addition,the incidence of ZYMV of these two cultivars seedings was 2.56%and 5.13%,respectively.Heating treatment was efficient strategy to get rid of ZYMV in seedings.?2?Cloning and analysis of the complete genome sequence of two ZYMV Shandong isolates?CN:Cm:17,CN:Lc:17?.ZYMV was detected in leaf samples of Luffa cylindrical and Cucurbita moschata collected from Tai?an,Shandong Province.Complete genome sequences of these two isolates were determined using RT-PCR and 5?RACE,and named CN:Cm:17 and CN:Lc:17,respectively.The complete genome sequences of CN:Cm:17 and CN:Lc:17excluding poly?A?tail were 9,593 nt and 9,591 nt,respectively.Both of them encoded a 3 080amino acids polyprotein.CN:Cm:17 and CN:Lc:17 showed 96.7%sequence similarity at polyprotein amino acid level.Sequences comparison with 48 ZYMV isolates obtained from NCBI database showed that CN:Cm:17 and CN:Lc:17,respectively,shared 90.3%⁹7.8%,90.5%⁹8.4%sequence identities at polyprotein amino acid level.Recombination analysis revealed that CN:Lc:17 was recombinant of isolates of KR:KR-PA:05 and CN:Cm:17.Phylogentic analysis based on polyprotein sequences suggested a high correlation between phylo-groups and their geographical origins.Chinese isolates were grouped in A-V and A-VI,respectively.?3?Construction of a ZYMV full-length infectious cDNA clone.Complete genome sequence of ZYMV isolate CN:Cm:17 was cloned into the Agrobacterium binary vector pCam35S under the 35S promoter by homologous recombination,the resultant clone was named pCamZYMV.All Cucurbita pepo and Cucurbita moschata plants infiltrated with agrobacterium carrying pCamZYMV showed similar symptoms to that of wild type ZYMV at14 days post agroinoculation?dpai?,the infection efficiency was 100%.pCamZYMV expressing GFP was obtained by inserting the green fluorescent gfp gene sequence between NIb and CP,the resultant construct was named pCamZYMV-GFP.Green fluorescence was observed on infiltrated leaves and systemic leaves of Cucurbita pepo and Cucurbita moschata plants infiltrated with pCamZYMV-GFP at 5 dpai and 14 dpai,respectively.?4?HCpro WxxxG motif was involved in determining ZYMV pathogenicity and suppressing of RNA silencing.ZYMV mutants W207A,G211A and WG were obtained by site-directed mutagenesis.Results showed that mutation at W₂₀₇ and WG attenuate the viral symptom and reduce virus accumulation,however,mutation on G₂₁₁ residue did not significant effect on viral symptom and virus accumulation.RNA silencing assay revealed that mutation on W₂₀₇ site had no significant effect on HCpro RNA silencing suppression ability,in contrast,mutation on G₂₁₁ site significantly reduced HCpro RNA silencing suppression ability.?5?Resistance screening of 40 cucurbits cultivars to ZYMV.Results showed that cultivars Tangshanqiugua and Qingfengchanghuzigua were highly resistance to ZYMV.Cultivars Xuefengxiaoyu No.5 and Jinfu were resistant to ZYMV.Jinyan No.4,Zhongnong No.28,Nairewangzhongwang,Taiguolvsigua,Xishuaipairouduoduo,Gaokangjulong,YingkeNo.2,Zhepu No.6,Jialipugua were mid-resistant to ZYMV.