Abstract

It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (−) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3′ UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection.

Highlights

  • Viroids are non-coding, highly structured, circular, single-stranded RNA molecules of 246–401 nucleotides in length[1]

  • Profiling of viroid derived small RNA (vd-small RNAs (sRNA)) revealed that certain regions of the potato spindle tuber viroid (PSTVd) genome produced more sRNAs than others, that is to say, they are vd-sRNA producing hotspots while less vd-sRNA producing regions on viroids are called as non-hotspots; for details see refs 16, 20 and 21

  • We have shown that sRNA derived from virulence modulating region (VMR) of PSTVd induces can down-regulate callose synthase genes CalS11-like and CalS12-like by RNA silencing

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Summary

Introduction

Viroids are non-coding, highly structured, circular, single-stranded RNA (ssRNA) molecules of 246–401 nucleotides (nts) in length[1]. Either the double-stranded RNA (dsRNA) or the self-complementary RNA triggers RNA silencing through the activity of the DICER-like (DCL) RNase III-type ribonucleases, and results in the production of small interfering RNAs (siRNA). These small RNAs (sRNA) comprise the sequence-specific effectors that regulate gene expression[6]. Due to their high internal base pairing and RNA-RNA mode of replication, viroids are the elicitors of the host defense system via RNA silencing. The least expressed (+) and (−) PSTVd-sRNA were selected as a starting point in order to analyze the fate of their target mRNA during PSTVd infection in tomato plants using both computational and molecular biology approaches

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