Viroids: how a minimal non-protein-coding RNA initiates disease in its natural host

"Background: Viroids are non-protein-coding RNAs of 246-434 nt infectious to certain plants wherein they replicate in the nucleus (family Pospiviroidae) or in plastids (family Avsunviroidae). Viroids appear very suitable systems to address the question of how viral and sub-viral agents initiate disease. Objectives: Plants react to viroids activating RNA interference (RNAi). Such defensive response has been involved in pathogenesis, but whether it is the initial trigger, or just mediates intermediate-late steps, remains unsolved. We have tackled this issue using peach latent mosaic viroid (PLMVd, Avsunviroidae), some variants of which cause an extreme chlorosis (peach calico, PC) or a yellow mosaic (PYM). Methods: Starting from symptomatic and asymptomatic leaf sectors we have: i) cloned, sequenced and bioassayed full-length PLMVd-cDNAs and analyzed their progeny, ii) deep-sequenced the RNAi-derived PLMVd small RNAs (sRNAs), predicted bioinformatically their host mRNA targets, and verified their cleavage at the expected positions by RNA ligase-mediated RACE and their lower accumulation in symptomatic sectors by RT-qPCR. Results: In contrast with PC, associated with a 12-13-nt insertion, PYM determinant maps at one single nucleotide. Moreover, 21-nt PLMVd-sRNAs containing the PC-associated insertion and the sole PYM change guide, respectively, cleavage of the mRNAs encoding the chloroplastic heat-shock protein cHSP90 and a thylakoid translocase subunit, both required in chloroplast biogenesis. PLMVd-sRNAs triggering PC and PYM have 5'-terminal Us, thus involving Argonaute 1 (with a major defensive role in RNAi) in what likely are the primary alterations eliciting different chloroses, which thus appear early and specific symptoms rather than late and non-specific effects."