FIGURE 5.

3MB-PP1 and 1NM-PP1 rescue the interactions of Pto^as(T199A) with bacterial effectors, but not the interaction of Pto^as with AvrPto(I96T). A and B, interactions of Pto forms with AvrPto (A) or AvrPtoB_1-387 (B) measured in a yeast two-hybrid system in the presence of 3MB-PP1 (10 μm), 1NM-PP1 (10 μm), or DMSO using the β-galactosidase assay. β-Galactosidase activity is reported as percentage of that observed for the interaction of Pto with AvrPto or AvrPtoB_1-387 in the presence of DMSO. Data are the means of three independent yeast transformants ± S.E. The assay was repeated three times with similar results. C, kinase activity assay of Pto forms. Autophosphorylation and phosphorylation of GST-Pti1(K96N) by the indicated GST-Pto forms was tested in vitro, and proteins were fractionated by 10% SDS-PAGE. The gel was stained by Coomassie Blue (bottom panel) and exposed to autoradiography (top panel). D, expression of bait proteins in yeast in the presence or absence of 10 μm 3MB-PP1. Wild-type and mutant forms of Pto were expressed in yeast as LexA fusions and visualized in immunoblots using anti-LexA antibodies. E, interactions of Pto and Pto^as with AvrPto or AvrPto(I96T) in the presence of 3MB-PP1 (10 μm), 1NM-PP1 (10 μm), or DMSO. Data are the means of three independent yeast transformants ± S.E. The assay was repeated three times with similar results.