Molecular mechanisms underlying late blight resistance by Pip1 immune protease of tomato

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Plant pathogens cause starvation and economic ruin and alter natural and managed ecosystems. Agrochemicals are commonly used against plant disease but they are increasingly difficult because of environmental concerns and pathogens becoming resistant. Therefore, exploitation of natural disease resistance is an increasingly attractive alternative. In this proposal we aim to exploit our detailed knowledge of a unique and effective natural defense mechanism acting in plants involving extracellular (apoplastic) papain-like cysteine proteases (PLCPs). These PLCPs accumulate during defense in tomato, maize, citrus and other plants and are targeted by pathogen-derived inhibitors. The Pip1 protease of tomato is a well-studied representative of these immune PLCPs.Pip1 suppresses susceptibility of tomato plants to the devastating oomycete late blight pathogen Phytophthora infestans, but also to the fungal leaf mold pathogen Cladosporium fulvum and the bacterial leaf spot pathogen Pseudomonas syringae (Ilyas et al., Curr. Biol. 2015). All these unrelated tomato pathogens colonize the extracellular space (apoplast), where Pip1 resides. All these three pathogens secrete unrelated inhibitors targeting Pip1, further highlighting the importance of Pip1. And indeed, transgenic antisense Pip1 (asPip1) plants lack Pip1 and are hyper-susceptible for all tested apoplastic pathogens, demonstrating its significance in immunity. However, the molecular mechanism underpinning Pip1-mediated resistance is unknown. Pip1 may act broadly by degrading pathogen-derived proteins such as apoplastic effectors, but Pip1 can also act in releasing peptide elicitors or activate host-derived hydrolases. The AIM of this project is to identify the substrate(s) of Pip1 by which Pip1 confers its immunity phenotype and to engineer inhibitor-insensitive Pip1. We focus this proposal on the role of Pip1 suppressing susceptibility to P. infestans because this is an economically relevant pathogen, defense by Pip1 is very strong, and transient disease assays are well established.The OBJECTIVES are to FIRST identify apoplastic candidate Pip1 substrates from both host and pathogen using three complementary proteomics methods. SECOND, we will clone and express these candidate substrates transiently in Nicotiana benthamiana with and without Pip1 to confirm cleavage. Also, un-cleavable mutant substrates will be selected. THIRD, we will investigate the role of the substrate and its cleavage by infecting agroinfiltrated N. benthamiana expressing (mutant) substrates with and without Pip1 with P. infestans. FOURTH, guided by a structural model of inhibitor-Pip1 complexes, we will engineer Pip1 such that it is insensitive for inhibition and decrease P. infestans susceptibility in transient assays.This project takes advantage of a powerful set of complementary proteomics approaches to identify candidate substrates and well-established assays with P. infestans and N. benthamiana. This project will identify Pip1-dependent modulators of host susceptibility, both from host and pathogen. This work will increase our knowledge on natural resistance of solanaceous plants against late blight disease that caused the Irish potato famine and is still a major concern in agriculture. Given the broad role of Pip1 in immunity against unrelated apoplastic pathogens, this information could explain the role of Pip1 in defense against other pathogens and how immune PLCPs act in other plants.