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.