Figure 2. Lipid overlay assay of oomycete RXLR effectors, AVR3a, AVR3a4, AVR3a11 and ATR1. (A) Escherichia coli strain BL21-AI was transformed with pDEST24 constructs for AVR3a (Asp23-Tyr147), AVR3a4 (Asn22-Tyr122), AVR3a11 (Asn22-Val132) or ATR1-Emwa1 (Ser22-Glu324). Protein expression and purification were performed as described in Yaeno et al.⁵ The purified C-terminal GST fusion proteins were checked by SDS-PAGE stained with InstantBlue (Expedeon) and equal amounts of proteins were used for the lipid overlay assay. (B) Nitrocellulose membranes spotted with 100 pmol of various lipids (PIP Strips; Echelon Biosciences) were blocked in 1% nonfat milk in PBS for 1 h and then incubated with 1 μg/mL C-terminal GST fusions of P. infestans AVR3a, P. capsici AVR3a4, P. capsici AVR3a11 and H. arabidopsidis ATR1 overnight at 4°C. After washing with PBS-T, the bound proteins were detected using anti–GST-HRP antibodies (GE Healthcare) diluted to 1:2,000. PA, phosphatidic acid; PC, phosphatidyl-choline; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI3P, PI-3-phosphate; PI4P, PI-4-phosphate; PI5P, PI-5-phosphate; PI3,4P2, PI-3,4-biphosphate; PI3,5P2, PI-3,5-biphosphate; PI4,5P2, PI-4,5-biphosphate; PI3,4,5P3, PI-3,4,5-triphosphate; PS, phosphatidylserine; S1P, sphingosine-1-phosphate.