P-126 - High-throughput in vitro identification of rice blast effector targets

In the previous studies, we reported a rice blast (Magnaporthe oryzae) effector AVR-PikD bound the heavy metal associated domain (HMA)-integrated domain (ID) of rice Pik-1 sensor NLR, which led to the recognition of AVR-PikD. In the rice plant without Pik, AVR-PikD is supposed to bind host small HMA-containing (sHMA) proteins and exert virulence. AVR-PikD is one of the MAX (Magnaporthe AVRs and ToxB-like) fold effectors widely distributed in M. oryzae. In this study, we addressed the interactions between MAX effectors and sHMA proteins by a high-throughput in vitro protein-protein interaction assay. Eighty-five sHMA proteins and 37 MAX effectors were synthesized using a wheat germ cell-free protein expression system, and their pairwise interactions were studied by the AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) method. The studied MAX effectors were categorized into the three types according to their binding patterns to sHMA proteins: the effectors binding to (1) a large number of sHMA proteins, (2) specific sHMA proteins, and (3) no sHMA proteins. A subset of the MAX effectors interacted with a large number of sHMA proteins are highly conserved in M. oryzae isolates. Functional analysis of the interactions between these MAX effectors and the interacting sHMA proteins will provide important information for understanding the pathogen virulence and host resistance.