Journal: Science Advances
Article Title: OsRNS4-mediated cross-kingdom RNA interference is inhibited by Ustilaginoidea virens effector SCRE2 to promote infection in rice
doi: 10.1126/sciadv.adx7385
Figure Lengend Snippet: ( A ) Yeast two-hybrid assay to show the interaction of SCRE2nsp with OsRNS4. The pGADT7 (AD) and pGBKT7 (BD) constructs were cotransformed into yeast GOLD strain. The transformants were cultured on the dropout medium plates. AD-T and BD-53, a positive control; AD-T and BD-λ, a negative control. OsRNS6, an OsRNS4 homolog in rice RNase T2 family. SCRE1, another U. virens effector. ( B ) Co-IP assay showing a specific interaction between SCRE2 and OsRNS4 in rice protoplasts. SCRE2-FLAG was expressed alone or coexpressed with OsRNS4-HA or OsRNS6-HA in rice protoplasts. Total proteins from transfected protoplasts were incubated with anti-FLAG M2 affinity beads. The inputs and immunoprecipitates were analyzed via immunoblotting with anti-HA (α-HA) and anti-FLAG (α-FLAG) antibodies. IP, immunoprecipitation; WB, Western blot. ( C ) In vitro pull-down assay to detect direct interaction between SCRE2 and OsRNS4. Purified GST- and His-tagged proteins were coincubated with glutathione agarose beads. The input and pull-down proteins were detected with anti-His (α-His) and anti-GST (α-GST) antibodies.
Article Snippet: Yeast two-hybrid assay was performed using the Clontech Matchmaker Gold Yeast Two-Hybrid System following the manufacturer’s instructions (Clontech).
Techniques: Y2H Assay, Construct, Cell Culture, Positive Control, Negative Control, Co-Immunoprecipitation Assay, Transfection, Incubation, Western Blot, Immunoprecipitation, In Vitro, Pull Down Assay, Purification