NGS of sRNA reads in plants. NGS of sRNA reads in plants

Previous works had found correlation between the association of small RNAs (sRNAs) to Potyvirus HCPro variants in infected plants, and their abilities to suppress silencing. Moreover, we had shown a preference by HCPro for sRNAs of 21-22 nt with 5´-end adenines, and of viral sequence. Conformational properties can explain preferences for sRNAs by size and/or 5´-ends, but preference for sRNAs of viral over plant sequence would require their having different accessibilities to the suppressor, or chemical properties. To investigate this, we expressed suppressor-competent and –deficient HCPro variants by agro-infiltration or from potex- and potyviral vectors and characterized the sRNAs in infiltrated tissues and associated to the purified proteins. In input infiltrated or virus-infected tissues, methylation levels in selected sRNAs of either T-DNA or plant sequence were assessed, and the former were found significantly less methylated in the presence of suppression-competent HCPro. In purified preparations of suppression-competent HCPro, sRNAs of non-plant sequence of 21 ntwere selectively enriched in those having 5´-end adenines. Our data indicate that the ratio of sRNAs of plant vs. non-plant sequence associated to HCPro is influenced by the context in its expression, and suggest that also by a concurrent interference by HCPro on the methylation of newly generated sRNAs of non-plant sequence. Overall design: 14 different samples analyzed by NGS

已有研究证实，感染植物中小分子RNA（small RNAs, sRNAs）与马铃薯Y病毒属（Potyvirus）辅助成分蛋白酶（helper component-proteinase, HCPro）变体的结合，与其沉默抑制活性存在相关性。此外，本团队此前的研究表明，HCPro偏好结合带有5'端腺嘌呤的21~22 nt长度的病毒来源sRNAs。构象特性可解释HCPro对sRNAs的长度及/或5'端的偏好性，但HCPro对病毒来源sRNAs相较于植物来源sRNAs的偏好性，则需归因于两类sRNAs与该沉默抑制子的结合可及性差异，或是其化学特性不同。为探究该问题，本研究通过农杆菌浸润法，或是利用马铃薯X病毒属（Potexvirus）及马铃薯Y病毒属（Potyvirus）载体，分别表达具有沉默抑制活性及无沉默抑制活性的HCPro变体，并对浸润组织中以及与纯化蛋白结合的sRNAs进行了表征分析。在初始的浸润组织或病毒感染组织中，本研究对选定的转移DNA（Transfer DNA, T-DNA）来源及植物来源sRNAs的甲基化水平进行了检测，结果发现，在具备沉默抑制活性的HCPro存在时，T-DNA来源sRNAs的甲基化水平显著更低。在具备沉默抑制活性的HCPro的纯化制剂中，带有5'端腺嘌呤的21 nt非植物来源sRNAs呈现出选择性富集现象。本研究数据表明，与HCPro结合的植物来源与非植物来源sRNAs的比例，受其表达环境的影响；同时，数据也提示HCPro可通过同时干扰新生成的非植物来源sRNAs的甲基化过程，实现这一偏好性。整体实验设计：通过下一代测序（Next Generation Sequencing, NGS）技术对14份不同样本进行分析。