Abstract

Introduction. Chrysantemum chlorotic mottle viroid (CChMVd) (Navarro and Flores, PNAS USA 1997), a member of the genus Pelamoviroid (type member peach latent mosaic viroid, PLMVd), displays the higest mutation rate reported for any biological entity (Gago et al., Science 2009). To understand how CChMVd colonizes, evolves and incites disease in its natural host, two natural variants, one with a UUUC tetraloop tightly associated with symptoms and the other with an alternative GAAA tetraloop and asymptomatic (De la Pen?a et al., PNAS USA 1999), were assayed.Results. As anticipated, the second one induced no phenotype, and RT-PCR, cloning and sequencing of its progeny showed variants with only the GAAA tetraloop. In contrast, the first variant induced early symptoms consisting in adjacent chlorotic and green leaf sectors, with progeny analysis disclosing that variants with the UUUC tetraloop predominate in chlorotic sectors, while in green sectors they coexisted with others having one or two mutations in the pathogenic determinant. Bioassay of the latter mutant variants led to asymptomatic infections (without the UUUC tetraloop in the progeny), showing that one single substitution in this tetraloop abrogates pathogenicity. Therefore, symtomatic and non- symptomatic CChMVd variants display distinct evolutionary trajectories, with the high mutation rate of CChMVd and the ability of some variants to colonize preferentially some leaf sectors and exclude other variants, accounting most likely for the observed results. Thus, CChMVd shows a territorial behaviour, with clearly segregated populations in a single leaf. The finding that a single substitution in the UUUC tetraloop disrupts pathogenicity is consistent with the involvement of RNA silencing. To gain support for this notion, we first examined by deep sequencing the viroid-derived small RNAs (CChMV-sRNAs) generated by RNA silencing: a significant prevalence of those of 21 nt was observed, in line with previous results for PLMVd. On the other hand, the chrysanthemum transcriptome of a cultivar in which CChMVd incites clear symptoms was determined. Combining both set of data we found a 21-nt CChMV-sRNA (containing the pathogenic determinant and with two 5'- terminal Us) potentially targetting for cleavage, as predicted by RNA silencing mediated by AGO1, the mRNA of an enzyme of the Calvin's cycle, the light-independent part of photosyntesis occurring in the chloroplast stroma. RNA ligase mediated-rapid amplification of cDNA ends confirmed that this mRNA was cleaved in the expected site (between positions 10 and 11 of the complementary 21-nt CChMV-sRNA) only in symptomatic sectors. Three additional lines of evidence support that such RNA silencing mechanism mediates the CChMVd-induced primary lesion. First, RT-qPCR showed a reduced level of this mRNA in symptomatic sectors, where the complementary 21-nt CChMV-sRNA accumulates preferentially. Second, a full-length CChMVd mutated so that the mismatch a

Autore Pugliese

• Navarro Ramirez Beatriz , Di Serio Francesco

Tutti gli autori

• Serra P.; Navarro B.; Gisel A.; Di Serio F.; Flores R.

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Anno di pubblicazione

2018

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