Construction Of Infectious Clone Of Pod Pepper Vein Yellows Virus And Study On The Transmission Of Efficiency

Pepper vein yellows virus(Pe VYV)is a single-stranded RNA virus in the genus Polerovirus,family Luteoviridae.It can be transmitted by aphids,grafting,etc.The typical viral symptoms of interveinal yellowing and upward leaf curling,which are important factors affecting the quality and yield of pepper.In this study,263 pepper samples were collected from main pepper producing areas in Yunnan,Zhejiang and Anhui provinces.The samples were detected by transcriptome Highthroughput sequencing(HTS)and confirmed by RT-PCR.In Yunnan province,the detection rates of Pe VYVs(Polerovirus)were 60.7 %;the detection rates of Chi RSV(Potyvirus)was44.0 %;and the detection rates of Chi VMV(Potyvirus)was 23.6 %.The prevalence of TZSV(Orthotospovirus)was 15.7 %.In Anhui province,the detection rates of Chi RSV was75.4 %;the detection rates of TMGMV(Tobamovirus)was 100 %.In Zhejiang province,the detetion rates of Chi VMV was 60.0 %;the detection rates of Pe VYV(es)were 60.0 %.In Yunnan province,the incidence of poleroviruses was high and Pe VYVs were obtained in 58 of 89 symptomatic samples in Wenshan city.According to HTS and RACE,a novel full-length of 6015 nt of Pe VYV was obtained in Wenshan city.We designated this virus in pod pepper as Pod pepper vein yellows virus(Po Pe VYV).In the 5’ half of its genome(the 5’ NCR to P3),Po Pe VYV is most similar(93.1% nt identity)to Pe VYV-3(Pepper vein yellows virus 3)(KP326573)but diverges greatly in the 3’-part encoding RTD,where it is most similar(91.7% nt identity)to tobacco vein distorting virus(TVDV,EF529624)suggesting a recombinant origin.Recombination analysis predicted a single recombination event affecting nucleotide positions 4126 to 5192 nt,with Pe VYV-3 as the major parent but with the region 4126-5192 nt derived from TVDV as the minor parent.An infectious clone of the virus was constructed for further investigation.The fulllength Po Pe VYV c DNA was inserted between an upstream 35 S promoter and a downstream hepatitis delta virus(HDV)ribozyme and NOS terminator in the binary vector to construct p CB-Po Pe VYV.This clone was transformed into Agrobacterium tumefaciens which were then delivered to C.frutescens and Nicotiana benthamiana plantlets by infiltration.RT-PCR using primers to detect the coat protein gene in the newly-emerged non-inoculated leaves showed that viral RNA was present and had spread systemically in the inoculated plants.Virions were purified from infected leaves and isometric particles about 25 nm in diameter were observed in the purified preparation from the inoculated plants but not from the controls.Aphid transmission was used to examine the transmission rates of Po Pe VYV in Capsicum frutescens.RT-PCR indicated that all the aphids fed on virus(10/10),but the virus transmission rate by the aphids was low(17-50%).HTS and RT-PCR confirmed two tla RNAs(Tombusvirus-like associated RNAs).The tla RNAs were similar with TBTDa RNA(Tobacco bushy top disease-associated RNA,EF529625).The efficiency of transmission rate was increasing by coinfection with Po Pe VYV and Po Pe VYVassociated RNA.