title: 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.
authors.affiliation: Institute of Bioengineering, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Aixia
authors.initials: A
authors.lastname: Xu
authors.forename: Zhijing
authors.initials: Z
authors.lastname: Zhao
authors.forename: Wenhu
authors.initials: W
authors.lastname: Chen
authors.forename: Hengmu
authors.initials: H
authors.lastname: Zhang
authors.forename: Qiansheng
authors.initials: Q
authors.lastname: Liao
authors.forename: Jishuang
authors.initials: J
authors.lastname: Chen
authors.forename: John P
authors.initials: JP
authors.lastname: Carr
authors.forename: Zhiyou
authors.initials: Z
authors.lastname: Du
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D017360:Arabidopsis / Q000254:growth & development / Q000821:virology; D029681:Arabidopsis Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D017799:Cucumovirus / Q000502:physiology*; D005814:Genes, Viral / Q000235:genetics; D006824:Hybridization, Genetic; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010935:Plant Diseases / Q000821:virology*; D030821:Plants, Genetically Modified; D011485:Protein Binding; D034622:RNA Interference; D043244:Ribonuclease III / Q000378:metabolism; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C404227:2b protein, cucumber mosaic virus; D000596:Amino Acids; D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; D014764:Viral Proteins; C504982:DCL4 protein, Arabidopsis; C511919:DCL2 protein, Arabidopsis; D043244:Ribonuclease III