title: Identification of residues or motif(s) of the rice stripe virus NS3 protein required for self-interaction and for silencing suppressor activity.
abstract: Rice stripe virus (RSV) is an important pathogen of rice. The RSV genome consists of four single-stranded RNA segments that encode seven viral proteins. A previous report found that NS3 is a viral suppressor of RNA silencing and self interacts. Using a model that predicts protein structure, we identified amino acid residues or motifs, including four α-helix motifs, required for NS3 self-interaction. We then used yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays to study the interactions between full-length NS3 and its truncated and alanine substitution mutants. Y2H and BiFC results showed that the N-terminal region of NS3 is essential for self-interaction. All α-helix deletion mutants and substitution mutants lost the ability to self-interact. To identify the relationship between NS3 self-interaction and silencing suppressor activity, we used a GFP silencing system in Nicotiana benthamiana with Agrobacterium-mediated transient overexpression of each mutated NS3 protein. All of the deletion and the α-helix substitution mutants that had lost the ability to self-interact also lost their silencing suppressor ability. The substitution of amino acids with alanine at positions 70-75, 76-83, and 173-177, however, resulted in mutants that were able to self-interact but were unable to function as silencing suppressors. These results suggest that RSV requires NS3 self-interaction to suppress RNA silencing and to thereby counter host defenses.
authors.affiliation: Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea.
authors.forename: Hangil
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea.
authors.forename: Won Kyong
authors.initials: WK
authors.lastname: Cho
authors.affiliation: College of Crop Protection and Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109, China.
authors.forename: Sen
authors.initials: S
authors.lastname: Lian
authors.affiliation: Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea. Electronic address: kookkim@snu.ac.kr.
authors.forename: Kook-Hyung
authors.initials: KH
authors.lastname: Kim
mesh_terms: D004252:DNA Mutational Analysis; D005470:Fluorometry; D054884:Host-Pathogen Interactions; D008958:Models, Molecular; D011487:Protein Conformation; D060066:Protein Interaction Maps; D029364:Tenuivirus / Q000235:genetics / Q000502:physiology*; D014026:Nicotiana / Q000276:immunology* / Q000821:virology*; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017361:Viral Nonstructural Proteins; D037521:Virulence Factors
keywords: GFP silencing; Protein 3D structure prediction; Protein-protein interaction; RNA silencing; Rice stripe virus; Viral suppressor of RNA silencing