Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense

Abstract

To successfully infect plants, viruses must counteract small RNA-based host defense responses. During infection of Arabidopsis, Cauliflower mosaic pararetrovirus (CaMV) is transcribed into pregenomic 35S and subgenomic 19S RNAs. The 35S RNA is both reverse transcribed and also used as an mRNA with highly structured 600 nt leader. We found that this leader region is transcribed into long sense- and antisense-RNAs and spawns a massive quantity of 21, 22 and 24 nt viral small RNAs (vsRNAs), comparable to the entire complement of host-encoded small-interfering RNAs and microRNAs. Leader-derived vsRNAs were detected bound to the Argonaute 1 (AGO1) effector protein, unlike vsRNAs from other viral regions. Only negligible amounts of leader-derived vsRNAs were bound to AGO4. Genetic evidence showed that all four Dicer-like (DCL) proteins mediate vsRNA biogenesis, whereas the RNA polymerases Pol IV, Pol V, RDR1, RDR2 and RDR6 are not required for this process. Surprisingly, CaMV titers were not increased in dcl1/2/3/4 quadruple mutants that accumulate only residual amounts of vsRNAs. Ectopic expression of CaMV leader vsRNAs from an attenuated geminivirus led to increased accumulation of this chimeric virus. Thus, massive production of leader-derived vsRNAs does not restrict viral replication but may serve as a decoy diverting the silencing machinery from viral promoter and coding regions.

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

“…These small RNAs mapped to a restricted number of defined genomic loci, and whether they are bona fide Dicer products remains unclear (55). In plants (Arabidopsis), the pararetrovirus Cauliflower mosaic virus (CaMV) also produces long antisense transcripts (56,57). Furthermore, CaMV vsiRNAs mapped to both strands of the genome, indicating that antisense transcripts base-pair with sense transcripts to produce dsRNA substrates for members of the Dicer-like protein family.…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…RDR1 (also known as SDE1/SGS2) in Arabidopsis and its homologs in other plant species contribute to RNA silencing-based resistance to virus infection (Vaistij et al, 2002;Yu et al, 2003;Muangsan et al, 2004;Yang et al, 2004;Diaz-Pendon et al, 2007;Garcia-Ruiz et al, 2010;Blevins et al, 2011;Leibman et al, 2011;Cao et al, 2014). It was also implicated in biogenesis of tasiRNAs during juvenile development (Peragine et al, 2004) …”

“…It participates in biogenesis of endogenous siRNAs (natsiRNAs) (Brosnan et al, 2007;Borges and Martienssen, 2015). RDR2 is not required for production of viral siRNAs from the Cauliflower mosaic pararetrovirus (Blevins et al, 2011), Cabbage leaf curl geminivirus (Aregger et al, 2012). RDR3 converts PolIV transcripts into dsRNA, which is processed by DCL3 into 24 nt siRNAs loaded onto AGO4 (Pontes et al, 2006;Zhang et al, 2007).…”

“…RDR6-dependent antiviral response includes the cucumber mosaic virus in Arabidopsis (Wang et al, 2010) or tobacco mosaic virus in Nicotiana benthamina (Qu et al, 2005) but not the cauliflower mosaic virus in Arabidopsis (Blevins et al, 2011). RDR6 was also implicated in the biogenesis of tasiRNAs and development (Peragine et al, 2004;Li et al, 2005a;Vaucheret, 2005).…”

The role of virus-derived small interfering RNAs in RNA silencing in plants

“…At present, evidence for the role of vsiRNAs in translational repression is limited. Blevins et al [67] found that vsiRNAs derived from the leader sequence of CaMV did not restrict virus replication, but served as a decoy diverting the silencing machinery from the viral promoter and coding regions. More studies are needed to find out whether other plant viruses utilize RNA decoys to evade RNA silencing mechanisms.…”