Fig. 2.

Screening of A. lucorum candidate effectors. (A) Agrobacterium tumefaciens infiltration assay for screening candidate effectors inducing cell death. Candidate effectors, the cell death-inducing gene INF1, and empty control (GFP) were expressed in Nicotiana benthamiana leaves via agroinfiltration. GFP is used as the negative control, and INF1 is the positive control that induced cell death. (B and C) A. tumefaciens infiltration assay for screening effectors inhibiting cell death. N. benthamiana leaves were first infiltrated with recombinant strains of A. tumefaciens carrying candidate effector genes or GFP, and INF1 was injected in the same region after 12 h. The numbers in the circle indicate the candidate effector numbers. The leaves were photographed 7 d after agroinfiltration and three biological repeats were performed for each candidate effector. (D) The leaves of N. benthamiana in which INF1-triggered cell death was inhibited by the expression of Al6. GFP is the negative control that cannot induce cell death. The ratios in the circle represent the time of cell death relative to the total time of the experiments. The photographs were taken at 7 d post-infiltration. (E) Western blot detection of the Al6 and INF1 proteins. Anti-GFP and anti-HA antibodies were used to detect the expression of the indicated constructs, and equal loading of each sample is indicated by Ponceau staining of Rubisco protein (PS). (F) Al6 inhibits BAX-induced cell death. (This figure is available in color at JXB online.)