title: Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing.
abstract: Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.
authors.affiliation: Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA.
authors.forename: Ajith
authors.initials: A
authors.lastname: Anand
authors.forename: Zarir
authors.initials: Z
authors.lastname: Vaghchhipawala
authors.forename: Choong-Min
authors.initials: CM
authors.lastname: Ryu
authors.forename: Li
authors.initials: L
authors.lastname: Kang
authors.forename: Keri
authors.initials: K
authors.lastname: Wang
authors.forename: Olga
authors.initials: O
authors.lastname: del-Pozo
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Martin
authors.forename: Kirankumar S
authors.initials: KS
authors.lastname: Mysore
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D017343:Genes, Plant / Q000235:genetics*; D006657:Histones / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics / Q000382:microbiology; D010940:Plant Proteins / Q000235:genetics; D010941:Plant Tumors / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000382:microbiology; D014170:Transformation, Genetic
chemical_list: D006657:Histones; D008565:Membrane Proteins; D010940:Plant Proteins; C095825:expansin protein, plant; C045625:nodulin