Journal: International Journal of Molecular Sciences
Article Title: Aphelenchoides besseyi Ab-FAR-1 Interacts with Arabidopsis thaliana AtADF3 to Interfere with Actin Cytoskeleton, and Promotes Nematode Parasitism and Pathogenicity
doi: 10.3390/ijms232012280
Figure Lengend Snippet: Ab-FAR-1 Δsp interacts with AtADF3. ( a ) The yeast two-hybrid interaction between Ab-FAR-1 Δsp and AtADF3. Left column, cotransformants grown on SD-TL (-trp -leu) agar medium demonstrate that both bait and prey plasmids are present in yeast ( Saccharomyces cerevisiae ); right column, only yeast cells containing the Ab-FAR-1 Δsp bait plus the AtADF3 prey or the positive control interaction of LargeT plus p53 grew and turned blue on the selective medium SD-TLHA (-trp –leu –his -ade) + x-α-gal agar medium. Ab-FAR-1 Δsp , p53 and LaminC were cloned into the pGBKT7 vector, while AtADF3 and LargeT were cloned into the pGADT7 vector, respectively. LargeT was cotransformed with p53 as a positive control. LargeT/LaminC, AtADF3/LaminC and LargeT/Ab-FAR-1 Δsp cotransformations were negative controls. ( b ) Co-immunoprecipitation of AtADF3 with Ab-FAR-1 Δsp . Ab-FAR-1 Δsp :eGFP or eGFP was transiently co-expressed with AtADF3:Flag in tobacco leaf cells. The isolated protein was analysed by immunoblotting with anti-Flag antibodies to detect AtADF3 and anti-GFP antibodies to detect Ab-FAR-1 Δsp . ( c ) Bimolecular fluorescent complementation (BiFC) visualization of the Ab-FAR-1 Δsp with AtADF3 interaction. Ab-FAR-1 Δsp : nYFP and AtADF: cYFP were co-expressed in tobacco leaves. Controls were performed by co-expressing nYFP and AtADF3:cYFP, Ab-FAR-1 Δsp :nYFP and cYFP. Bars = 100 μm as shown. Three independent experiments were performed with similar results.
Article Snippet: After 48 h, total leaf proteins were extracted, and the Co-IP experiment was carried out according to the instructions of the Capturem TM IP & Co-IP Kit (Takara).
Techniques: Positive Control, Clone Assay, Plasmid Preparation, Immunoprecipitation, Isolation, Western Blot, Expressing
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