Detection And Molecular Studies On Begomoviruses In Fujian Province Of China

Geminiviruses form an important family of plant viruses with characteristic twinned quasi-icosahedral virions and a small,single-stranded circular DNA genome.The family Geminiviridae consists of nine genera,which are Becurtovirus,Begomovirus,Capulavirus,Curtovirus,Eragrovirus,Grablovirus,Mastrevirus,Topocuvirus,and Turncurtovirus,respectively.Geminiviruses,especially begomoviruses,cause substantial economic losses in tropical and subtropical regions worldwide or even famine in some underdeveloped countries.In recent years,certain begomoviruses have become prevalent infecting many crop and ornamental plants in South China.However,the diversity of begomoviruses in Fujian province is poorly characterized.In the first section of this study,plant species including Hibiscus rosa-sinensis,H.sabdariffa,H.esculentus,Tomato(Lycopersicon esculentum L),Jute(Corchorus olitorius L),Ramie(Boehmeria nivea),Ageratum weed(Ageratum conyzoides)and Pueraria montana(Kudzu weed)with typical begomovirus symptoms were collected from different locations of Fujian province during 2015-2017.Using degenerate PCR followed by full-genome sequencing(when necessary),six distinct begomoviruses,namely Cotton leaf curl Multan virus,Tomato yellow leaf curl virus(TYLCV),Corchorus yellow vein Vietnam virus(CoYVV),Ramie mosaic virus(RamV),Ageratum yellow vein China virus(AYVCnV)and Kudzu mosaic virus(KuMV)were detected from these samples.CLCuMuV was detected in four different cities of Fujian including Fuzhou,Xiamen,Zhangzhou and Sanming.Besides H.rosa-sinensis and H.esculentus,which are known host plants of CLCuMuV,we detected CLCuMuV in H.sabdariffa,thus finding a new host for this virus.TYLCV,a destructing virus for tomato,was previously reported only in Fuzhou,but was found also in Sanming in this study.RamV was first detected in Longyan by our lab several years ago.In this study,it was found to have a high incidence in many cities of Fujian including Fuzhou,Xiamen and Sanming.CoYVV is a begomovirus first reported from Vietnam and has never been reported from China.However,this virus was detected in Fuzhou and Sanming in this study.Overall,these results revealed a high diversity of begomoviruses in Fujian and suggested a high speed of spreading of some begomoviruses in this province.Among the six begomoviruses detected here,three,namely CLCuMuV,CoYVV and RamV,are of great or potential economic importance,but are poorly studied.In the second section of this study,the infectious clones of these three begomoviruses were constructed to facilitate further studies on these begomoviruses.By using traditional molecular techniques,corresponding plasmids were obtained and introduced into the cells of Agrobacterium tumefaciens.By agro-infiltration,the infectious clones of CLCuMuV and RamV were inoculated onto Nicotiana benthamina and that of CoYVV onto jute.For all the three inoculations,typical symptoms were observed on N.benthamina or jute,although the time of appearance of symptoms varied greatly.Southern Blotting was used to detect corresponding viruses after the onset of symptoms.The results showed that all the three viruses replicated and accumulated in the inoculated plants.It is noteworthy that the infectious clone of CoYVV has never been reported before.Thus,this is the first study an infectious clone was obtained to confirm the infectivity and pathogenicity of CoYVV in jute.Geminiviruses use the host transcriptional machinery to synthesize their mRNAs.They are considered as an attractive model to understand the mechanism of transcription of their host plants.With this in mind,experiments were set out to identity transcriptional start sites(TSSs)of the three begomoviruses mentioned above.To do this,we first rub-inoculated Rice stripetenuivirus(RSV),a segmented negative-sense RNA virus that uses cap-snatching to produce capped viral mRNAs,into N.benthamina.Two weeks after the inoculation,RSV-infected N.benthamina were super-infected by CoYVV,CLCuMuV or RamV,respectively.Total RNA was extracted from N.benthamina co-infected by RSV and each of the three begomoviruses.The capped-RNA leaders snatched by RSV were obtained by determining the 5’ ends of a RSV mRNA with a high throughput sequencing strategy.After that,the snatched capped-RNA leaders of RSV were mapped onto the genome of each begomovirus and those matching the begomoviral genome were considered to come from the 5’ ends of assumed begomoviral mRNAs.In this way,the TSSs of the three begomoviruses were obtained.After mapping these TSSs onto the genome of the respective begomoviruses,it was found very common that a begomovirus can use many different TSSs to transcribe the same gene,producing many different mRNA isoforms containing the same open reading frames(ORFs).Overall,surveys were conducted to gain information on the incidence of begomoviruses in Fujian,a mountainous province that is relatively isolated from other parts of China.Six begomoviruses,with differences in their incidence and biogeography,were identified.The infectious clones of three begomoviruses that may pose a serious threat plant cultivation in Fujian were constructed.With these infectious clones in hand,the TSSs of the three begomoviruses were identified,paving a way for further studies on the molecular basis of their infection.