sRNA sequencing of TRV-infected Nicotiana benthamiana 16c

To systematically investigate viral sRNA production and sRNA-target interaction, we sequenced sRNAs from Tobacco Rattle Virus (TRV)-infected Nicotiana benthamiana at an early (1 week post infection) and late time point (3 weeks post infection). The N. benthamiana 16c plants were infected with TGS-inducing viruses (TRV-35S and TRV-35-2M) and PTGS-inducing viruses (TRV-GFP and TRV-GFP-2M), respectively. TRV-35S is a recombinant TRV containing a 120 nt segment of the 35S promoter. Its derivative, TRV-35S-2M, carrying single nucleotide substitutions (SNS) at every 10 nt within the 120 nt 35S target segment. Same strategy was used to create recombinant TRV-GFP and TRV-GFP-2M targeting GFP coding sequence. According to SNS content, sRNAs from TRV-35S-2M/TRV-GFP-2M infected plants can be separated to yield primary (containing SNSs) and secondary sRNAs (lacking SNSs). Wild Type TRV was used along as viral infection control. Libraries were indexed during PCR amplification (16 cycles) according to the Illumina protocol. See individual sample information for specific index primers used.

为系统探究病毒小RNA（small RNA, sRNA）的产生机制及其与靶标的互作关系，我们对感染烟草脆裂病毒（Tobacco Rattle Virus, TRV）的本氏烟（Nicotiana benthamiana）分别在侵染早期（接种后1周）与晚期（接种后3周）开展了小RNA测序。本氏烟16c植株分别被诱导转录基因沉默（Transcriptional Gene Silencing, TGS）的病毒（TRV-35S与TRV-35S-2M）以及诱导转录后基因沉默（Post-transcriptional Gene Silencing, PTGS）的病毒（TRV-GFP与TRV-GFP-2M）侵染。TRV-35S为携带一段120 nt 35S启动子片段的重组烟草脆裂病毒；其衍生株TRV-35S-2M在该120 nt的35S靶标片段内每10 nt位置引入1处单核苷酸替换（single nucleotide substitutions, SNS）。针对GFP编码序列构建重组病毒TRV-GFP与TRV-GFP-2M的策略与此一致。根据单核苷酸替换的携带情况，可将感染TRV-35S-2M与TRV-GFP-2M植株的小RNA划分为两类：携带单核苷酸替换的初级小RNA，以及不携带单核苷酸替换的次级小RNA。野生型烟草脆裂病毒作为病毒侵染对照。所有样本文库均参照Illumina建库流程，在PCR扩增（16个循环）阶段完成索引标记，具体所用索引引物信息详见各样本的详细说明。