Characterization of plant virus-encoded gene silencing suppressors.
Agroinfiltration assay using green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c is a powerful method for screening of putative plant virus-encoded gene silencing suppressors. This method allows the investigator to know whether the putative viral suppressor inhibits silencing in a cell (local silencing) and/or spreading of silencing throughout a plant (systemic silencing). Additionally, grafting experiments using transgenic plants expressing the suppressor and the GFP will indicate whether the suppressor blocks systemic silencing steps, which include the production of a silencing signal in a silenced cell, and the cell-to-cell and long-distance movement of a silencing signal throughout a plant. Here, we describe methods and techniques of an agroinfiltration assay and grafting experiments, which were used for the characterization of Apple chlorotic leaf spot virus 50 kDa movement protein as a gene silencing suppressor. This protocol should allow the investigator to characterize putative plant virus-encoded gene silencing suppressors.
Faculty of Agriculture, Plant Pathology Laboratory, Iwate University, Morioka, Japan.
Hajime
H
Yaegashi
Masamichi
M
Isogai
Nobuyuki
N
Yoshikawa
D052677:Flexiviridae / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D053830:Plant Viral Movement Proteins / Q000235:genetics* / Q000378:metabolism*; D034622:RNA Interference; D011994:Recombinant Proteins; D014026:Nicotiana / Q000235:genetics* / Q000821:virology
D053830:Plant Viral Movement Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins