Differential Effects of Mild and Severe Cucumber mosaic virus Strains in the Perturbation of MicroRNA-Regulated Gene Expression in Tomato Map to the 3′ Sequence of RNA 2

Abstract

Viral infections interfere with the microRNA (miRNA)-mediated regulation of gene expression, determining developmental defects. In tomato leaves, the accumulation levels of six miRNA species and their target transcripts corresponding to transcription factors with roles in plant development and leaf morphogenesis and two genes involved in the short RNA processing, DCL1 and AGO1, were significantly enhanced upon infection with the severe strain Cucumber mosaic virus (CMV)-Fny, while that of AGO4 was reduced. In plants harboring the infection of the mild strain CMV-LS, the effects on miRNA pathway were reduced, although AGO1, DCL1, and NAC1 also were shown to overaccumulate during infections exhibiting a mild phenotype. The use of the recombinant strain CMV-Fny(LS2b), in which the 3'-terminal region of CMV-Fny RNA 2, including the 2b coding sequence, was replaced with the corresponding region of CMV-LS RNA 2, provided evidence that the exchanged region was implicated in the perturbation of miRNA metabolism. In tomato plants infected with CMV-Fny supporting the ameliorative satellite (sat)RNA variant Tfn-satRNA, the symptomless phenotype correlated, with the exception of NAC1 upregulation, with the absence of effects on mitochondrial RNA and miRNA expression. Some of the aspects of miRNA pathway perturbation described were peculiar to CMV-tomato interactions and involved in the etiology of the disease phenotype elicited in this host.

RNA binding is more critical to the suppression of silencing function ofCucumber mosaic virus2b protein than nuclear localization

“…It could therefore be possible that transport of siRNAs and miRNAs to the nucleus could be aimed either at their functional removal from the cytoplasm, or with the intent of causing specific effect in the nucleus, such as perhaps interference with DNA methylation (Guo and Ding 2002). In addition, interaction of 2b with AGO4 has been demonstrated in vivo and visualized inside the nucleus (González et al 2010;Hamera et al 2012) and this interaction could also constitute another link between nuclear localization and suppression of silencingmediated interference with gene expression, as reported (Cillo et al 2009;Ye et al 2009). …”

“…It has been proposed that interaction of 2b with AGO1 could inhibit RISCs involved in antiviral RNA silencing (Zhang et al 2006) and that AGO4 activity is negatively affected by its interaction with 2b protein (Hamera et al 2012). Besides these direct interactions with AGO proteins, transcription of AGO4 mRNA has been shown to be negatively affected by the 2b protein (Cillo et al 2009;Ye et al 2009). It is therefore probable that the 2b protein neutralizes at least two different levels of antiviral RNA silencing by binding to siRNAs and host proteins.…”

“…The protein also interferes with the long-range signaling of silencing in the plant (Guo and Ding 2002), with the accumulation of siRNAs generated by the plant DICER-like proteins 2, 3, and 4 (Díaz-Pendó n et al 2007), with the metabolism of some miRNAs involved in development (Lewsey et al 2007;Cillo et al 2009), and also with transgene DNA methylation (Guo and Ding 2002). The ability of the 2b protein to facilitate the transit of sRNAs into the nucleus has also been demonstrated recently and used to facilitate inheritable epigenetic modifications of endogenous plant gene promoters, using dsRNAs originating from a CMV-derived vector (Kanazawa et al 2011).…”

Attenuated plant viruses: preventing virus diseases and understanding the molecular mechanism

“…CMV 2b inhibits AGO1-mediated cleavage, implying that 2b is involved in biogenesis of the host's small RNA and affects symptom expression (Cillo et al 2009;Lewsey et al 2009;Zhang et al 2006). A severe strain (CM95R) of CMV is a spontaneous mutant from an attenuated strain (CM95), which was selected through a series of local lesion isolations (Kosaka and Fukunishi 1997).…”

Symptom induction and RNA silencing suppression by the cucumber mosaic virus 2b protein

“…Known or suspected functional domains include nuclear localization sequences (NLS), 16,17 an RNA binding domain (overlapping the NLSs), 13,14 putative phosphorylation sites, 17 as well as the N and C termini (involved in DNA binding and, of host mRNA accumulation by a class of sRNAs known as microRNAs varies between CMV strains and correlates with symptom induction, the degree to which 2b proteins inhibit silencing directed by the class of sRNAs known as short-interfering (si) RNAs (which includes antiviral silencing) does not. 11,12 This suggests that symptoms may not after all be an incidental effect of suppressing antiviral silencing and conceivably interference with microRNA-mediated silencing may provide some unknown fitness advantage to the virus. The 2b protein can bind double-stranded sRNAs and interact directly with the AGO 1 and 4 proteins.…”