P-350 - An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function

NLR family proteins act as intracellular receptors to detect pathogen effectors, thereby inducing effector-triggered immunity. Gene duplication events amplify the number of NLR family genes, and subsequent random mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLR genes remain largely unclear. Here, we report that the rice NLR protein Pit1 and its paralogue Pit2 were generated by gene duplication, and Pit1 forms a hetero-immune complex with Pit2. The two are required for the resistance to rice blast fungus but have different functions: the helper NLR Pit1 induces cell death, while the sensor NLR Pit2 competitively attenuates Pit1 homocomplexes by forming heteromers with Pit1 to suppress Pit1-mediated cell death. We identified the fate-determining residues, which account for functional differences between Pit1 and Pit2. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two critical residues in the NB-ARC domain of Pit2 that are involved in plasma membrane localization. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary history of tandemly duplicated helper and sensor NLRs after gene duplication and how they orchestrate plant immunity.