Mapping cytosine methylation on BYVMV genome

Geminiviruses are molecular pathogens, contains small circular single stranded DNA genome which is prone for Transcriptional gene silencing (TGS). To counteract this TGS mediated host defence, Geminiviruses encodes silencing suppressor proteins. Bhendi Yellow vein mosaic virus is a monopartite geminivirus contains DNA A genome and assiciated DNA Beta. To find out whether BYVMV interferes in TGS Bisulfite sequencing using Next Generation Sequencing (NGS) was performed on wild type BYVMV viral DNA isolated from infiltrated N. benthamiana plants and the base level cytosine methylation was identified. further to know the involvement of DNA A genes in TGS suppression, C2 and C4 genes of BYVMV was mutated by introducing stop codons in the N-terminal of C2 and C4 ORFs seperately. C2 mutant and C4 mutant BYVMV A DNA was infiltrated alone or along with betasatellite in N. benthamiana. the viral DNA was isolated from infiltrated plants at 16 dpi and Bisulfite sequencing was performed by using NGS. the NGS Data was analysed for the identification of transcriptional silencing suppressor. Through this study the role of BYVMV A DNA genes in TGS suppression can be identified.

双生病毒（Geminiviruses）是一类分子病原体，其基因组为小型环状单链DNA，极易引发转录基因沉默（Transcriptional gene silencing, TGS）。为抵御宿主通过TGS介导的免疫防御，双生病毒会编码沉默抑制蛋白。秋葵黄脉花叶病毒（Bhendi Yellow vein mosaic virus, BYVMV）属于单分体双生病毒，其基因组包含DNA A组分，并伴随有DNA Beta（DNAβ）卫星分子。为探究BYVMV是否能够干扰TGS通路，研究人员对从浸润接种的本氏烟（Nicotiana benthamiana）植株中分离得到的野生型BYVMV病毒DNA，开展了基于二代测序（Next Generation Sequencing, NGS）的亚硫酸氢盐测序（Bisulfite sequencing），成功鉴定了碱基水平的胞嘧啶甲基化修饰情况。为明确DNA A基因组的基因在TGS抑制过程中的作用，研究人员分别在C2和C4开放阅读框（Open Reading Frame, ORF）的N端引入终止密码子，对BYVMV的C2、C4基因进行了定点突变。随后将C2突变体、C4突变体BYVMV A DNA单独或与DNAβ卫星分子共同浸润接种本氏烟植株，并于接种后16天分离侵染植株中的病毒DNA，再次通过NGS开展亚硫酸氢盐测序。研究人员对获取的NGS数据进行分析，以鉴定转录沉默抑制因子。本研究可明确BYVMV DNA A基因组的基因在TGS抑制中的具体作用。