Abstract

Viroid RNAs are targeted by dicer-like (DCLs) enzymes, the activity of which generates viroid-derived small RNAs (vd-sRNAs) of 21-24 nucleotides (nt) in the infected tissues, thus supplying a convincing evidence of a first host anti-viroid barrier based on RNA silencing. Whether a second RNA silencing barrier, based on the RNA-induced silencing complex (RISC), may also target for degradation viroid RNAs remains controversial. The RISC core is always formed by an Argonaute (AGO) protein, which after loading endogenous or viral sRNAs, guides the complex to target for inactivation complementary RNAs (or DNAs). Using RNA immunoprecipitation with a specific antibody against the endogenous AGO1 of Nicotiana benthamiana we showed that, when infected by potato spindle tuber viroid (PSTVd), this protein is also loaded with vd-sRNAs. Moreover, agroexpression in N. benthamiana infected by PSTVd of epitope-tagged versions of nine of the ten AGOs from Arabidopsis thaliana, followed by immunoprecipitation analyses of the agroinfiltrated halos, revealed that AGO1, AGO2 and AGO3 preferentially associated with vd-sRNAs of 21 and 22 nt, while AGO4, AGO5 and AGO9 additionally bound those of 24 nt. In contrast, AGO6, AGO7 and AGO10 did not load vd-sRNAs. Deep-sequencing analysis revealed that the endogenous AGO1 of N. benthamiana and the agroexpressed AGO1, AGO2, AGO4 and AGO5 bound vd-sRNAs according to their size and 5'-terminal nucleotide, with AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Interestingly, agroexpression of AGO1, AGO2, AGO4 and AGO5 in PSTVd-infected tissue attenuated the PSTVd level, suggesting RISC-targeting of the viroid genomic RNAs or their precursors and thus supporting a role of RISC in anti-viroid defense. Moreover, PSTVd RNAs with 5'-termini compatible with degradation mediated by AGO-loaded vd-sRNA have been identified and characterized by 5'-RML-RACE in infected N. benthamiana. Further degradome analysis should provide a description of how the viroid genomic and host RNAs are targeted by AGO-loaded vd-sRNAs, thus supplying a broader view of the biological functions of vd-sRNAs.

Autore Pugliese

• Navarro Ramirez Beatriz , Di Serio Francesco

Tutti gli autori

• Minoia S.; Carbonell A.; Di Serio F.; Gisel A.; Carrington J.C.; Flores R.; Navarro B.

Titolo volume/Rivista

Non Disponibile

Anno di pubblicazione

2015

ISSN

Non Disponibile

ISBN

978-3-95758-006-1

Numero di citazioni Wos

Nessuna citazione

Ultimo Aggiornamento Citazioni

Non Disponibile

Numero di citazioni Scopus

Non Disponibile

Ultimo Aggiornamento Citazioni

Non Disponibile

Settori ERC

Non Disponibile

Codici ASJC

Non Disponibile