Tobacco rattle virus infection alters Arabidopsis gene expression via virus-derived SiRNAs and host MiRNA network disruption

Virus-induced gene silencing (VIGS) technology based on tobacco rattle virus (TRV) has been widely employed in plant gene function research. However, the regulatory roles of TRV-derived small interfering RNAs (siRNAs) in Arabidopsis gene expression and the impact of TRV infection on the endogenous microRNA (miRNA) regulatory network in Arabidopsis remain undefined. In this study, many TRV-derived sRNAs were identified in infected Arabidopsis leaves. Target validation confirmed that 26 viral siRNAs specifically targeted 17 host mRNAs involved in critical biological processes, including chlorophyll biosynthesis, protein translation, metabolic homeostasis, and stress response pathways. Additionally, TRV infection induced the differential expression of 120 endogenous miRNAs (with 108 showing downregulation), disrupting flowering regulatory modules such as the miR156-SQUAMOSA promoter binding protein-like gene (SPL) and the miR172-APETALA2 (AP2), as well as miRNA regulatory networks associated with plant innate immunity and drought stress responses. Further investigations revealed that TRV infection significantly suppressed the expression of trans-acting small interfering RNAs(tasiRNAs)triggered by miR173, thereby affecting the regulation of genes involved in carbon metabolism and ethylene biosynthesis. This study elucidates the broad-spectrum interference of TRV-siRNAs with host gene expression and the remodeling of plant endogenous miRNA/tasiRNA regulatory networks by TRV infection. It provides critical insights into the unintended impacts of TRV infection on plant systems and suggests that the inherent effects of TRV on gene expression and physiology should be carefully considered when employing TRV as a genetic engineering tool.