Establishment of an efficient virus-induced gene silencing (VIGS) assay in Arabidopsis by Agrobacterium-mediated rubbing infection.
Several VIGS protocols have been established for high-throughput functional genomic screens as it bypasses the time-consuming and laborious process of generation of transgenic plants. The silencing efficiency in this approach is largely hindered by a technically demanding step in which the first pair of newly emerged true leaves at the 2-week-old stage are infiltrated with a needleless syringe. To further optimize VIGS efficiency and achieve rapid inoculation for a large-scale functional genomic study, here we describe a protocol of an efficient VIGS assay in Arabidopsis using Agrobacterium-mediated rubbing infection. The Agrobacterium inoculation is performed by simply rubbing the leaves with Filter Agent Celite(®) 545. The highly efficient and uniform silencing effect was indicated by the development of a visibly albino phenotype due to silencing of the Cloroplastos alterados 1 (CLA1) gene in the newly emerged leaves. In addition, the albino phenotype could be observed in stems and flowers, indicating its potential application for gene functional studies in the late vegetative development and flowering stages.
Center of Biosciences & Biotechnology, North Rio de Janeiro State University, Campos dos Goytacazes, RJ, 28013-602, Brazil.
Ana Marcia E de A
AM
Manhães
Marcos V V
MV
de Oliveira
Libo
L
Shan
D060054:Agrobacterium / Q000235:genetics / Q000502:physiology / Q000821:virology*; D017360:Arabidopsis / Q000235:genetics / Q000382:microbiology / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D010641:Phenotype; D018515:Plant Leaves / Q000235:genetics / Q000382:microbiology / Q000502:physiology*; D010942:Plant Viruses / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics
D029681:Arabidopsis Proteins; C099778:CLA1 protein, Arabidopsis