Self‐interaction of the cucumber mosaic virus 2b protein plays a vital role in the suppression of RNA silencing and the induction of viral symptoms

Abstract

The cucumber mosaic virus (CMV) 2b protein is an RNA silencing suppressor protein that can also play direct and indirect roles in symptom induction. Previous work has shown that a hybrid virus, FRad35(2b) -CMV (renamed here as CMV-FRad2b-Pro), generated by replacement of the 2b gene of strain Fny-CMV with that from Rad35-CMV, displays markedly lower pathogenicity than Fny-CMV on Nicotiana species. However, the replacement of proline with leucine at position 55 of the 2b protein of CMV-FRad2b-Pro (protein Rad2b-Pro) created a virus (CMV-FRad2b-Leu) that induced severe symptoms. Infection of Arabidopsis thaliana mutants defective in the expression of DICER-like (DCL) endoribonucleases 2 and 4, which mediate antiviral RNA silencing, as well as of dcl3 and dcl2/3/4 triple-mutant plants, indicated that Rad2b-Pro was a weaker RNA silencing suppressor than the protein Rad2b-Leu. This was confirmed in Nicotiana benthamiana using agroinfiltration assays, showing that, compared with either Rad2b-Leu or the Fny2b protein, Rad2b-Pro was ineffective at inhibiting local or systemic silencing of expression of a green fluorescent protein reporter gene. Transgenic expression of Rad2b-Leu, but not of Rad2b-Pro, in Arabidopsis induced symptom-like phenotypes and rescued the accumulation of the 2b-deletion mutant Fny-CMVΔ2b. Bimolecular fluorescent complementation indicated that, in planta, Rad2b-Leu, but not Rad2b-Pro, self-interacts. Thus, self-interaction is crucial to the ability of the 2b protein to suppress silencing and induce a symptom-like phenotype, and is dependent on the properties of the residue at position 55.

Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing

“…However, such interactions by themselves seem to be insufficient to sustain the VSR activity of p37 because the interaction with AGO4 was maintained by all the assayed p37 variants, and the interaction with AGO1, although notably diminished regarding p37-W 28 , was retained at WT levels in the case of the loss-of-function form p37-3R 67 . The great importance of the sRNA binding capability over other protein properties is in agreement with recent reports on other VSRs, such as cucumber mosaic virus 2b, which, like p37, exhibits nucleolar localization, self-interaction, and interaction with AGO1 and AGO4 proteins (42,62,75,78,82).…”

“…In any case, such subcellular localization seems to be dispensable for suppressor activity, as deduced from results with mutant p37-2R 15 that is able to inhibit RNA silencing despite being excluded from the nucleolus. On the other hand, dimerization has been shown to be a requirement for distinct VSRs, including the well characterized tombusviral p19 (33,39,71,75,81). Although we have shown here that PLPV p37 is able to interact with itself, all of the assayed protein mutants retained this capacity, and, thus, we could not critically assess whether this property is a prerequisite for the suppressor function.…”

“…pressor Activity-Although binding to sRNAs seems fundamental for the activity of many VSRs, other properties, such as dimerization, specific subcellular localization, or interaction with AGO proteins, have been proposed to be important in some instances (34,42,46,47,(73)(74)(75). Thus, experiments were conducted to assess the potential relevance of the latter features in p37 suppressor function.…”

Nuclear-Cytoplasmic Partitioning of Cucumber Mosaic Virus Protein 2b Determines the Balance between Its Roles as a Virulence Determinant and an RNA-Silencing Suppressor

“…tumefaciens cells carrying p35S:GFP were mixed with an equal amount of cells carrying pBI121-EGFP-Fny2b, pBI121-EGFP-Fny2bQ24R, pBI121-EGFP-Fny2bNLS, pBI121-EGFP-Fny2b6His, or control plasmids pBI121-GUS (β-glucuronidase) or pBI121-EGFP as described previously (37). A.…”

Using a Viral Vector to Reveal the Role of MicroRNA159 in Disease Symptom Induction by a Severe Strain of Cucumber mosaic virus

“…RNA gel blotting for analysis of CMV genomic RNAs was performed as previously described (Xu et al, 2013). For analysis of low-M r RNAs, including miRNAs and U6, 15 mg total RNA was used for RNA-blot…”