title: Two amino acids near the N-terminus of Cucumber mosaic virus 2b play critical roles in the suppression of RNA silencing and viral infectivity.
abstract: Cucumber mosaic virus (CMV) 2b suppresses RNA silencing primarily through the binding of double-stranded RNA (dsRNA) of varying sizes. However, the biologically active form of 2b remains elusive. Here, we demonstrate that the single and double alanine substitution mutants in the N-terminal 15th leucine and 18th methionine of CMV 2b exhibit drastically attenuated virulence in wild-type plants, but are efficiently rescued in mutant plants defective in RNA-dependent RNA polymerase 6 (RDR6) and Dicer-like 4 (DCL4). Moreover, the transgenic plants of 2b, but not 2blm (L15A/M18A), rescue the high infectivity of CMV-Δ2b through the suppression of antiviral silencing. L15A, M18A or both weaken 2b suppressor activity on local and systemic transgene silencing. In contrast with the high affinity of 2b to short and long dsRNAs, 2blm is significantly compromised in 21-bp duplex small interfering RNA (siRNA) binding ability, but maintains a strong affinity for long dsRNAs. In cross-linking assays, 2b can form dimers, tetramers and oligomers after treatment with glutaraldehyde, whereas 2blm only forms dimers, rather than tetramers and oligomers, in vitro. Together, these findings suggest that L15 and M18 of CMV 2b are required for high affinity to ds-siRNAs and oligomerization activity, which are essential for the suppression activity of 2b on antiviral silencing.
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Dong
authors.affiliation: Department of Plant Pathology, China Agricultural University, Beijing, 100193, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
authors.forename: Long-Xiang
authors.initials: LX
authors.lastname: Chai
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Tong
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
authors.forename: Xian-Bing
authors.initials: XB
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D017360:Arabidopsis / Q000821:virology; D029681:Arabidopsis Proteins / Q000378:metabolism; D001483:Base Sequence; D017799:Cucumovirus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D024202:Electrophoretic Mobility Shift Assay; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D017354:Point Mutation / Q000235:genetics; D055503:Protein Multimerization; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D019076:Transgenes; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C404227:2b protein, cucumber mosaic virus; D000596:Amino Acids; D029681:Arabidopsis Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D012367:RNA, Viral; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C514575:RDR6 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase
keywords: 2b; Cucumber mosaic virus; RNA binding; RNA silencing; oligomerization; suppressor