The Function Of Sly-microRNA482a In The Interaction Between Tobacco Curly Shoot Virus And Nicotiana Benthamiana

Tobacco curly shoot virus(TbCSV)is a monopartite begomovirus of Geminiviridae family with specific betasatellite DNA(TbCSB),and it is an important pathogen that causes tomato and tobacco leaf curl disease,which is widespread and has caused great losses to crops in Yunnan and Sichuan province.Many studies have shown that microRNA is involved in the regulation of plant growth and development,and biotic or abiotic stress response.The current research work on the pathogenic mechanism of TbCSV and TbCSB is mainly focused on the interaction between proteins encoded by the virus and the host factors,while the research on the endogenous non coding RNA of the host is little,so it will be helpful to understand the pathogenic mechanism of the virus.Based on bioinformatics,this study used microRNA sequencing method to analyze the high-expression sly-miR482a in the process of TbCSV/TbCSB infection by using vector construction,RT-qPCR and qPCR technology,revealed the interaction between TbCSV/TbCSB and Nicotiana benthamiana from the perspective of miRNA,and laid a foundation for further analysis of virus pathogenic mechanism and host resistance.To further study the chronological expression of sly-miR482a in N.benthamiana infected with TbCSV/TbCSB,the accumulation of virus and the relative expression of sly-miR482a at 1-25 dpi were detected by qPCR and RT-qPCR,respectively.The results showed that after TbCSV and TbCSB were inoculated,the virus accumulation of TbCSV and TbCSB increased gradually from 1 to 20 dpi,and the accumulation of TbCSV and TbCSB reached the peak at 20 dpi,and tended to be stable at the later stage.sly-miR482a was down-regulated at 3 dpi and significantly down regulated at 5 dpi;at 10 dpi,sly-miR482a was significantly up regulated;at 15 dpi,sly-miR482a was significantly down regulated;at 20 dpi and 25 dpi,sly-miR482a was up regulated.Correlation analysis showed that the accumulation of TbCSB has significant linear correlation with the relative expression of sly-miR482a,and R~2 reached 0.80,but there was no significant linear correlation between the accumulation of TbCSB and the relative expression of sly-miR482a.To clarify the effect of silencing and overexpression of sly-miR482a on plant growth and development of N.benthamiana and the infection of TbCSV/TbCSB,the transient silencing and overexpression vector of sly-miR482a was constructed with PGD vector,and the systematic silencing vector of sly-miR482a was constructed with PVX vector.The accumulation of TbCSV was significantly higher than that of the control group,but there was no significant change in the accumulation of TbCSB when sly-miR482a was transiently down regulated;the accumulation of TbCSV was significantly lower than that of the control group,and the accumulation of TbCSB was also significantly reduced when sly-miR482a was transiently over-expressed.PVX-mediated systematic silenced plants showed the symptoms of uneven and mottled leaves,and the accumulation of TbCSV and TbCSB was promoted when sly-miR482a was down-regulated.To identify the target genes of sly-miR482a,seven predicted target genes were obtained by small RNA sequencing,website prediction and PCR amplification;two potential targets of sly-miR482a named Niben101Scf02248g00001.1 and Niben101Scf10767g03011.1 were initially identified by RT-qPCR and fluorescent vector construction experiments.TRV-mediated silencing of these two target genes showed that down regulation of these two target genes reduced the accumulation of virus.The two target genes showed the opposite regulation mode with sly-miR482a,indicating that sly-miR482a regulated these two target genes to participate in the regulation of virus accumulation.In this study,small RNA sequencing analysis showed that TbCSV and TbCSB infection can induce changes in the relative expression of sly-miR482a,and up-regulation of sly-miR482a and down-regulation of target genes inhibit virus accumulation,while down-regulating the expression of sly-miR482a and up-regulation of target genes promote the virus accumulation.These research results can provide scientific basis for the analysis of the molecular mechanism of interaction between TbCSV with TbCSB and host,as well as the host disease resistance mechanism.