Abstract

Introduction and Objective. While most isolates of peach latent mosaic viroid (PLMVd) (Hernández and Flores, PNAS USA 1992) are latent in leaves, hence its name, some incite peach mosaic (PM) or albinism (peach calico, PC). PC-inducing variants have a characteristic insertion of 12-13 nucleotides and accumulate preferentially in the symptomatic but not in the surrounding green foliar sectors. We have shown previously that two 21-nt small RNAs (PLMVd-sRNAs) containing the PC-associated insertion, guide cleavage of the mRNA encoding the chloroplastic heat-shock protein as predicted by RNA silencing (Navarro et al., Plant J. 2012), with this event likely representing the primary alteration eventually resulting in symptoms. Here we have extended these studies to PM, an issue more complex because entails identification of the corresponding molecular determinant, which is not associated with a specific insertion, and because in contrast with the well-defined PC phenotype, PM differs in type and intensity of symptoms.Material and Methods. Deep sequencing was applied to host- and PLMVd-sRNAs, RT-PCR to synthesize complete PLMVd-cDNAs, semi-quantitative RT-PCR to evaluate host mRNA levels, and RNA ligase-mediated rapid amplification of cDNA ends (RACE) to map cleavage sites in mRNAs.Results. We focused on one severe yellow mosaic (peach yellow mosaic, PYM) expressed in leaf sectors interspersed with others green. Full-length PLMVd-cDNAs from both sectors were cloned and sequenced. Two clones, differing in one nucleotide substitution, served to generate dimeric head-to-tail inserts in plasmids from which in vitro transcripts were produced and bioassayed in GF-305 peach seedlings. Whereas one of the transcripts incited an intense yellow mosaic, the other caused only mild lesions, supporting the involvement of one specific PLMVd nucleotide in PYM. Additionally, the differential nucleotide substitution: i) was preserved in the progeny of symptomatic but not in that of the adjacent green sectors, and ii) is absent in a set of PLMVd variants that replicate latently. Examination in silico of the PLMVd-sRNAs and their potential mRNAs targets of peach, whose genome is completely sequenced, has shown that one PLMVd-sRNA containing the PYM-associated substitution could guide cleavage of the mRNA coding for a thylakoid protein as predicted by RNA silencing. RACE experiments indicate that that is indeed the case and that accumulation of this mRNA is lower in symptomatic than in asymptomatic sectors. Moreover, mutants in the gene coding for this thylakoid protein exhibit yellow phenotypes.Conclusions. PLMVd variants with a single nucleotide substitution elicit a yellow mosaic by cleaving, via RNA silencing, the mRNA encoding a thylakoid protein.

Autore Pugliese

• Navarro Ramirez Beatriz , Di Serio Francesco

Tutti gli autori

• Delgado S.;Navarro B.; Serra P.; Di Serio F.; Flores F.

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Anno di pubblicazione

2017

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