title: Transgenic Brassica rapa plants over-expressing eIF(iso)4E variants show broad-spectrum Turnip mosaic virus (TuMV) resistance.
abstract: The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage.
authors.affiliation: Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, South Korea.
authors.forename: Jinhee
authors.initials: J
authors.lastname: Kim
authors.forename: Won-Hee
authors.initials: WH
authors.lastname: Kang
authors.forename: Jeena
authors.initials: J
authors.lastname: Hwang
authors.forename: Hee-Bum
authors.initials: HB
authors.lastname: Yang
authors.forename: Kim
authors.initials: K
authors.lastname: Dosun
authors.forename: Chang-Sik
authors.initials: CS
authors.lastname: Oh
authors.forename: Byoung-Cheorl
authors.initials: BC
authors.lastname: Kang
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D029741:Brassica rapa / Q000235:genetics* / Q000821:virology*; D060467:Disease Resistance / Q000235:genetics; D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry / Q000235:genetics; D039642:Eukaryotic Initiation Factors / Q000737:chemistry / Q000235:genetics; D017343:Genes, Plant; D014644:Genetic Variation; D054884:Host-Pathogen Interactions / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010935:Plant Diseases / Q000235:genetics / Q000821:virology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D030821:Plants, Genetically Modified; D017800:Potyvirus / Q000235:genetics / Q000472:pathogenicity*; D011487:Protein Conformation; D039381:RNA Cap-Binding Proteins / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D010940:Plant Proteins; D039381:RNA Cap-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C511734:eIF(iso)4E protein, Arabidopsis
keywords: Chinese cabbage; TuMV; broad-spectrum resistance; eIF(iso)4E; plant transformation; potyvirus; resistance breeding