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. 14 different samples analyzed by NGS