Abstract

Viroid-derived small RNAs of 21-24 nt (vd-sRNAs) accumulate in viroid-infected tissues as a consequence of the activity of Dicer-like (DCL) RNases targeting these infectious RNAs or their double-stranded derivatives. vd-sRNAs are incorporated into Argonaute (AGO) proteins, forming the core of the RNA silencing complex, and guide it to cleave viroid and host mRNAs. To further dissect these molecular pathways and to investigate their involvement in viroid pathogenesis, we applied genome-wide approaches based on high-throughput sequencing of sRNAs and degradome analyses. Two different hosts, tomato and N. benthamiana --which develop similar symptoms of leaf curling/wrinkling and stunting when infected by potato spindle tuber viroid (PSTVd)-- were examined, thus allowing comparative analyses of: i) the mRNAs targeted for cleavage by vd-sRNAs and ii) the effect of PSTVd on the accumulation of miRNAs and their cleaved targets. Results. Although degradome analyses identified about one hundred tomato mRNAs potentially targeted for degradation by vd-sRNA, data from biological replicates and comparisons of healthy and infected samples reduced the number of true targeted mRNAs --i.e. those identified with high confidence in two infected biological replicates and not in healthy controls-- to only eight mostly coding for proteins involved in development. These results were validated by RACE experiments in the three mRNAs examined. Interestingly, RT-qPCR showed that only four of the eight mRNAs actually accumulated at lower levels in the PSTVd-infected tomato, thus showing that cleavage of the mRNAs targeted by the vd-sRNAs is not necessarily associated with the down-regulation of gene expression. When the same degradome analyses were performed with healthy and PSTVd-infected N. benthamiana plants, all the identified mRNAs targeted by vd-sRNAs were different from those observed previously in tomato and, besides, they also differed in the molecular pathways they are involved. Therefore, this comparative analysis failed to pinpoint homologous gene(s), targeted by vd-sRNAs, potentially eliciting the common symptoms induced by PSTVd in tomato and N. benthamiana. Moreover, no significant modifications in the accumulation of miRNAs and their cleaved targeted mRNAs were detected in the PSTVd-infected tomato and N. benthamiana plants with respect to their healthy controls. The implications of these findings on the mechanism of PSTVd pathogenesis will be discussed.

Autore Pugliese

• Navarro Ramirez Beatriz , Di Serio Francesco

Tutti gli autori

• Navarro B.; Gisel A.; Serra P.; Flores R.; Di Serio F.

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Anno di pubblicazione

2018

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