FIG 7.

Identification of amino acids critical for PI3P binding by Ceg22. (A) Multiple sequence alignment of ten effectors or predicted effectors harboring LED006. The conserved adjacent phenylalanine-lysine, histidine-arginine, and the two phenylalanine residues that were mutated are marked with red arrows if the mutation affected PI3P binding or with black arrows if the mutation did not affect PI3P binding (see panel B). (B) Binding of affinity-purified GST fusion proteins of the wild-type effector Ceg22 or the three mutants with mutations in the adjacent phenylalanine-lysine, the adjacent histidine-arginine, and the adjacent phenylalanine residues (FK189-190AA, HR207-208AA, and FF245-246AA) of the same effector (300 pmol) to 2-fold serial dilutions of PIs (100 to 1.56 pmol) immobilized on nitrocellulose membranes was analyzed by a protein-lipid overlay assay using an anti-GST antibody.