Role of RNA silencing in triggering the initial molecular lesion of viroid pathogenesis.

"How subcellular pathogens incite plant diseases and, specifically, which is the molecular lesion initiating a signaling cascade ultimately resulting in symptom expression is still unclear. Viroids, small (246-434 nt), circular RNAs infecting and frequently causing diseases in certain higher plants, appear appropriate systems to address this question because they do not code for any protein and rely almost completely on host factor for replication and systemic plant invasion. Early ideas on viroid pathogenesis focused on a direct interaction of the genomic RNA with host factors as the initial molecular event eliciting ultimately symptoms. With the advent of RNA silencing and the discovery of its role in plant defense response against invasive nucleic acids, including viroids, it was alternatively proposed that viroid-derived small RNAs (vd-sRNAs, 21-24 nt) might target for cleavage cell mRNAs and initiate a signal cascade, eventually leading to symptoms. We have addressed this issue using the plastid-replicating viroid peach latent mosaic viroid (PLMVd, family Avsunviroidae). While most variants of this viroid are latent, some cause symptoms of albinism (peach calico, PC) or yellow mosaic (PYM). Analysis of the PLMVd sequence variants from the symptomatic and asymptomatic leaf sectors, and bioassays of single variants and mutants, has led to the identification of the pathogenesis determinant associated to PC and PYM. PC is strictly associated with a 12-nt hairpin insertion located in the left arm of the PLMVd secondary structure, whereas PYM determinant maps at one single nucleotide change. Using deep sequencing and 5' RNA ligase-mediated rapid amplification of cDNA ends (RML-RACE), we have established that the vd-sRNAs containing the PC-associated insertion (PC-sRNA8) and the PYM-associated nucleotide change (PYM-sRNA40) target and guide cleavage, as predicted by RNA silencing, of the mRNAs encoding the chloroplastic heat-shock protein 90 (cHSP90) and a thylakoid translocase subunit (cpSecA), respectively, which are two proteins required for chloroplast development. RT-qPCR showed lower accumulation of cHSP90 and cpSecA mRNAs in PC- and PYM-expressing tissue, respectively. Interestingly, the vd-sRNAs inducing PC and PYM have a U at 5' end, which is the preferential 5'-terminal nucleotide for AGO1-loading. Altogether these data are consistent with the targeting by PC-sRNA8 and PYM-sRNA40 for AGO1(RISC)-mediated cutting of their cognate mRNAs. Moreover, since the mRNAs targeted by PC-sRNA8- and PYM-sRNA40 code for proteins involved in chloroplast biogenesis, the initial molecular lesion and the expected phenotype (different chloroses) are connected by short signaling cascades that permit tracing a direct cause-effect relationship."