Peach latent mosaic viroid (PLMVd) (Herna?ndez and Flores, PNAS USA 1992) induces distinct phenotypes in its natural host. While most isolates are symptomless in leaves, hence the term latent in the name, some incite peach mosaics (PM) of different severity and a few an extreme albinism (peach calico, PC) (Flores et al., Mol. Plant Pathol. 2006). PC- inducing variants have a characteristic insertion of 12-13 nt in one apical loop and accumulate predominantly in symptomatic but not in adjacent green leaf sectors. We have documented that two 21-nt small RNAs (PLMVd-sRNAs) -containing the insertion associated with PC- guide cleavage of the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90) as predicted by RNA silencing mediated by Argonaute 1 (AGO1), which binds specifically 21-nt sRNAs with a 5'-terminal U, like the two PLMVd-sRNAs holding the PC-associated insertion (Navarro et al., Plant J. 2012). Cleavage of the mRNA coding for cHSP90 most likely is the primary molecular lesion causing eventually PC, since this protein mediates biogenesis of chloroplasts from proplastids. To understand how general such mechanism is, we have extended these studies to PM, a question far more difficult because it implies identification of the molecular determinant, which is not associated with any specific insertion, and because in contrast with the well-defined PC phenotype, PM differs in type and severity of symptoms. Results. After some preliminary studies, we focused on a severe peach yellow mosaic (PYM) expressed in leaf sectors flanked by others green. Full-length PLMVd-cDNAs from both sectors were cloned and sequenced. From two variants, differing in one nucleotide change, we generated dimeric head-to-tail inserts in plasmids, with the corresponding in vitro transcripts being then bioassayed in GF305 peach seedlings. While one of the transcripts incited an intense PYM resembling the original field symptom, the other caused only mild alterations, thus supporting the involvement of one specific PLMVd nucleotide in this syndrome. In addition, the differential nucleotide change: i) was preserved in the progeny of the symptomatic but not in that of adjacent green sectors, ii) is absent in a set of PLMVd natural variants that replicate latently, and iii) turned a severe variant into latent when removed, and a latent variant into severe when inserted by site-directed mutagenesis. Analysis of the PLMVd-sRNAs and their potential mRNAs targets in the known peach genome, combined with RNA ligase mediated-rapid amplification of cDNA ends, have revealed that one 21-nt PLMVd-sRNA, with the PYM-associated change and two 5'-terminal Us, guide cleavage of the mRNA coding for a thylakoid protein as predicted by RNA silencing mediated by AGO1. Accumulation of this mRNA is lower in symptomatic than in asymptomatic sectors as shown by RT-qPCR, and some mutants in the gene coding for this protein, involved in photosynthesis, show yellow phenotypes akin to PYM.
A yellow mosaic incited by peach latent mosaic viroid: strict association with a single-nucleotide change and involvement of RNA silencing-mediated cleavage of the mRNA coding for a thylakoid protein.
Navarro B;Chiumenti M;Di Serio F;
2018
Abstract
Peach latent mosaic viroid (PLMVd) (Herna?ndez and Flores, PNAS USA 1992) induces distinct phenotypes in its natural host. While most isolates are symptomless in leaves, hence the term latent in the name, some incite peach mosaics (PM) of different severity and a few an extreme albinism (peach calico, PC) (Flores et al., Mol. Plant Pathol. 2006). PC- inducing variants have a characteristic insertion of 12-13 nt in one apical loop and accumulate predominantly in symptomatic but not in adjacent green leaf sectors. We have documented that two 21-nt small RNAs (PLMVd-sRNAs) -containing the insertion associated with PC- guide cleavage of the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90) as predicted by RNA silencing mediated by Argonaute 1 (AGO1), which binds specifically 21-nt sRNAs with a 5'-terminal U, like the two PLMVd-sRNAs holding the PC-associated insertion (Navarro et al., Plant J. 2012). Cleavage of the mRNA coding for cHSP90 most likely is the primary molecular lesion causing eventually PC, since this protein mediates biogenesis of chloroplasts from proplastids. To understand how general such mechanism is, we have extended these studies to PM, a question far more difficult because it implies identification of the molecular determinant, which is not associated with any specific insertion, and because in contrast with the well-defined PC phenotype, PM differs in type and severity of symptoms. Results. After some preliminary studies, we focused on a severe peach yellow mosaic (PYM) expressed in leaf sectors flanked by others green. Full-length PLMVd-cDNAs from both sectors were cloned and sequenced. From two variants, differing in one nucleotide change, we generated dimeric head-to-tail inserts in plasmids, with the corresponding in vitro transcripts being then bioassayed in GF305 peach seedlings. While one of the transcripts incited an intense PYM resembling the original field symptom, the other caused only mild alterations, thus supporting the involvement of one specific PLMVd nucleotide in this syndrome. In addition, the differential nucleotide change: i) was preserved in the progeny of the symptomatic but not in that of adjacent green sectors, ii) is absent in a set of PLMVd natural variants that replicate latently, and iii) turned a severe variant into latent when removed, and a latent variant into severe when inserted by site-directed mutagenesis. Analysis of the PLMVd-sRNAs and their potential mRNAs targets in the known peach genome, combined with RNA ligase mediated-rapid amplification of cDNA ends, have revealed that one 21-nt PLMVd-sRNA, with the PYM-associated change and two 5'-terminal Us, guide cleavage of the mRNA coding for a thylakoid protein as predicted by RNA silencing mediated by AGO1. Accumulation of this mRNA is lower in symptomatic than in asymptomatic sectors as shown by RT-qPCR, and some mutants in the gene coding for this protein, involved in photosynthesis, show yellow phenotypes akin to PYM.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
