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

NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication.