Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing.
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.
Anand
Ajith
A
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA.
Vaghchhipawala
Zarir
Z
Ryu
Choong-Min
CM
Kang
Li
L
Wang
Keri
K
del-Pozo
Olga
O
Martin
Gregory B
GB
Mysore
Kirankumar S
KS
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
D006657:Histones; D008565:Membrane Proteins; D010940:Plant Proteins; C095825:expansin protein, plant; C045625:nodulin