Fig. 4.

Protein-protein assays to detect interaction of Hpa2 with HrpF of X. oryzae pv. oryzicola. (A) Interactions of Hpa2 and HrpF were evaluated using full-length proteins by Y2H and β-gal assays. The reactions of pGADT7-T with pGBKT7-53 and of pGADT7-T with pGBKT-Lam were regarded as the positive (+) and negative (−) controls, respectively. Y2H and β-gal assays were performed according to standard procedures (Clontech). For the image shown, Hpa2 was used as the bait. Similar results were seen when HrpF was used as the bait. (B) Hpa2 interacts with HrpF by a pulldown assay. GST and GST-Hpa2 were immobilized on glutathione Sepharose and were incubated with HrpF-c-Myc. Total-cell lysates (TE) and eluted proteins (Eluate) were analyzed by immunoblotting using antibodies directed against the c-Myc epitope and GST, respectively. Bands corresponding to GST and GST chimeras are marked by asterisks. Arrows indicate c-Myc epitope-tagged proteins.