Analysis The Function Of The Sirnas From TbCSV And Its ? Satellite TbCSB

Tobacco curly shoot virus(TbCSV)is a monopartite begomovirus in family Geminiviridae,which is transmitted by Bemisia tabaci.TbCSV can cause severe curl leaf disease in tomato and tobacco,causing serious economic losses.RNA silencing mediated by vsiRNAs(virus-derived small interfering RNAs)is mainly aimed at targeting virus genome and the post-transcriptional regulation of host mRNA.In this study,Nicotiana benthamiana was used as experimental material to study the role of vsiRNAs derived from TbCSV and its ? satellite(TbCSB)complex in the process of virus infection.This study will help to understand the mechanism of TbCSV/TbCSB inducing host to produce typical symptoms.In order to verify whether vsiRNAs were produced after TbCSV/TbCSB combined infection of Nicotiana benthamiana,this study used small RNA sequencing technology to analyze vsiRNAs on Nicotiana benthamiana infected by TbCSV/TbCSB and infected by TbCSV alone.The results showed that 1165882 vsiRNAs were detected in TbCSV/TbCSB treatment group,87315 were specific;217272 vsiRNAs were detected in TbCSV treatment group,32192 were specific.Most of the vsiRNAs derived from TbCSV and TbCSB was 21 nt and 22 nt in two treatment groups and mainly located in the open reading frames(ORFs)of TbCSV and TbCSB,and the first base of them was Uracil(U)mostly.Hotspot analysis of vsiRNAs showed that there were 55 hotspots in TbCSV genome infected by TbCSV/TbCSB,17 hotspots in TbCSB genome,and 49 hotspots in TbCSV genome infected by TbCSV alone.Finally,55 vsiRNAs with more than 2000 reads from TbCSV/TbCSB co-infection were selected and compared with the hairpin structure sequences of each gene mRNA to obtain 19 small RNAs from hairpin structure of mRNA of TbCSV.Furthermore,14 vsiRNAs were obtained by RT-PCR cloning.In order to verify whether the vsiRNAs produced by the virus play a role in the process of virus infection,14 vsiRNAs were constructed into Cabbage leaf curl virus(CaLCuV)expression vectors(pCVA),which were mediated by Agrobacterium EHA105.The effects of vsiRNAs expression on Nicotiana benthamiana were analyzed.The results showed that three vsiRNAs which caused abnormal growth of Nicotiana benthamiana,named Y35A8,Y35A14 and Y35A18 respectively.The leaves curled downward and had yellow vein symptoms on Y35A8 expressed Nicotiana benthamiana;the heart leaves curled downward seriously and had yellow vein symptoms and the lower leaves had mild yellow vein symptoms on Y35A14 expressed Nicotiana benthamiana;the whole top leaf was curled and had yellow vein symptoms on Y35A14 expressed Nicotiana benthamiana.To study the effect of the expression of vsiRNA Y35A8,Y35A14 and Y35A18 on viral infection,TbCSV/TbCSB infectious clones were inoculated in 15 days after the expression of these three vsiRNAs,respectively.The phenotypic changes of tobacco were observed 8 days and 45 days after inoculation,and the viral accumulation was analyzed by qPCR.The results showed that the viral symptoms of Nicotiana benthamiana expressing the three vsiRNA were more serious than those non-expressing,mainly showing severe leaf curl and stem twist.The virus accumulation was detected by qPCR.In Nicotiana benthamiana expressing Y35A8,the accumulation of virus was higher than control group,while the accumulation of ? satellite was lower than control group.In Nicotiana benthamiana expressing Y35A14,the accumulation of virus and satellite was higher than control group.In Nicotiana benthamiana expressing Y35A18,the accumulation of virus was higher in early stage than control group and lower in later stage than control group,and the accumulation of ? satellite was higher in early stage than control group and basically be the same level in later stage comparing with control group.The above results indicate that the viral vsiRNAs have an impact on both host and virus in the process of viral infection.In order to study which host target genes of Y35A8,Y35A14 and Y35A18 to perform their functions,we combined the psRNA Target target gene prediction website and RT-qPCR method to verify that four target genes conform to the negative regulation of small RNA and down-regulate their expression.They are named Y35A8-6,Y35A14-2,Y35A14-3 and Y35A18-8,respectively.The target gene Y35A8-6 ID is Niben101Scf04410g04006.1 which is a protease inhibitor family gene,the target gene Y35A14-2 ID is Niben101Scf23557g00007.1 which is a leucine repeat sequence receptor protein kinase gene family gene,the target gene Y35A14-3 ID is Niben101Scf03194g01005.1 which is a transmembrane protein family gene,the target gene Y35A18-8 is Niben101101f03009g05001.1 which is a leucine-rich repeat family gene.We studied the effects of down-regulation of these four genes mediated by tobacco fragile virus(TRV)on host and virus.The results showed that there was no difference in phenotype between the treated and control plants after silenced this 4 genes.And TbCSV/TbCSB was inoculated 10 days later after 10 days of gene silencing,viral infection in Y35A8-6 and Y35A18-8 treatment groups resulted in curling of the top leaves but the phenotype of Y35A14-2 and Y35A14-3 treatments was not different from that of the control group.The q-PCR results showed that the accumulation of TbCSV and TbCSB in the treatment group was lower than that of the control group.To summarize,this study indicated that vsiRNAs were produced after TbCSV/TbCSB infected tobacco and some vsiRNAs could affect the normal growth of the host and enhance the symptoms of the virus infection and affect the accumulation of the virus,suggesting that it might be involved in the interaction between the virus and the host.In addition,the accumulation of viruses and satellites was reduced by qPCR analysis in the down-regulation of the expression of 4 target genes,suggesting that they also affect the infection and replication of viruses.