Broadening of miRNA and secondary siRNA targets in virus-infected plants by integrated genome- wide approaches

Plant viruses modify gene expression in infected tissues by altering the micro (mi)RNA-mediated regulation of genes. Among conserved miRNA targets there are transcripts codifying for transcription factors, RNA silencing core and disease resistance proteins. Paralogs in these gene families are widely present in plant genomes and are known to respond differently to miRNA-mediated regulation during plant virus infections. Using genomic-wide approaches applied to Solanum lycopersicum infected by a nuclear-replicating virus we highlighted miRNA-mediated cleavage events that could not be revealed in virus-free systems: among them, we confirmed targeting of one paralogous Argonaute1, seven transcriptional factors from five different families cleaved by miR156, miR160, miR166, miR169 and miR172 and one RX-Coiled-coil (RX-CC), nucleotide binding (NBS) mRNA cleaved by miR6024. Interestingly, in most cases short indels close to the target sites discriminated cleavage of duplicates, indicating a functional significance of indels in fine tuning gene expression in plant-virus interaction. The same analysis was extended to Cicer arietinum plants and we could confirm that indels in close vicinity to the miRNA target sites of duplicated genes could discriminate miRNA-mediated post-transcriptional control of genes. Furthermore, we show that the miR6024 target site overlap to some extent to that of miR482 in the NBS-LRR. miR6024-mediated cleavage, uniquely in virus-infected tissues, triggers the production of several unique 21nt secondary siRNAs. These secondary siRNAs explained cleavages of several mRNAs annotated as involved in plant-pathogen interaction and appearance of viral symptoms.