FIG 8.

Identification of an amino acid critical for PI3P binding present in a subset of effectors containing LED006. (A) Multiple sequence alignment of ten effectors or predicted effectors harboring LED006. The conserved adjacent phenylalanine-lysine and the two adjacent phenylalanine residues, as well as the single lysine or lysine/arginine that were mutated, are marked with red arrows if the mutation affected PI3P binding, with a blue arrow if the mutation had a weak effect on 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 lpg2327 or the three mutants with mutations in the adjacent phenylalanine-lysine, the single lysine, or the two adjacent phenylalanine residues (FK220-221AA, K237A, and FF268-269AA) 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.