Fig. 5.

PIP binding is required for the accumulation of CMPG1 and AVR3a in planta. (A) Immunoblots probed with anti-myc and anti-FLAG antibodies following coexpression of 4×-myc-CMPG1 and FLAG-AVR3a derivatives, respectively, in N. benthamiana. Protein loading is shown by Coomassie blue (CBB) staining. (B) Immunoblots probed with anti-FLAG antibody following expression of FLAG-AVR3a mutants in N. benthamiana (in vivo) and purified His-tagged AVR3a mutant proteins after incubation overnight at room temperature (in vitro). (C) Circular dichroism spectra of the purified His-tagged AVR3a proteins (WT, black; K85E, gray). (D) Interaction of AVR3a derivatives with CMPG1 in the yeast two-hybrid system. The expression of lacZ was monitored to check the interaction. (E) PIP binding of the Lys85 derivatives WT (AVR3a^KI), AVR3a^K85E, AVR3a^K85A, and AVR3a^K85G. The protein lipid overlay assay was performed as in Fig. 2A. (F) Immunoblots probed with anti-FLAG antibody following expression of FLAG-tagged Lys85 derivatives in N. benthamiana. (G) Percentage of sites with INF1-induced cell death upon coexpression of INF1 with Lys85 derivatives. Error bars indicate SD of the means (n = 3).