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

Reference	BB/S003193/1
Principal Investigator / Supervisor	Professor Renier van der Hoorn
Co-Investigators / Co-Supervisors
Institution	University of Oxford
Department	Biology
Funding type	Research
Value (£)	532,138
Status	Completed
Type	Research Grant
Start date	01/03/2019
End date	28/02/2023
Duration	48 months

Abstract

We discovered that the secreted immune protease Pip1 of tomato suppresses infection against unrelated extracellular pathogens, including the devastating oomycete late blight pathogen Phytophthora infestans. All these pathogens secrete Pip1 inhibitors, highlighting the relevance of Pip1. The OVERALL AIM of this project is to identify the substrate(s) of Pip1 by which Pip1 confers its phenotype and to engineer inhibitor-insensitive Pip1 to increase plant protection. This project is focused on the interaction between tomato and P. infestans because the role of Pip1 is strong, P. infestans is an important pathogen, and transient disease assays are established in N. benthamiana. FIRST, we will identify extracellular Pip1 substrates of both host and pathogen using three complementary proteomics approaches: i) in-solution-digest (ISD), to monitor overall protein levels; ii) PROTOMAP, to monitor shifts in molecular weight; and iii) TAILS, to sequence N-termini. All three methods will be applied on the same five biological replicates, on Pip1 cleavage, both in vivo and in vitro. SECOND, we will confirm cleavage by Pip1 and generate un-cleavable substrates by mutagenesis by agroinfiltration of tagged substrates with and without Pip1. THIRD, we will test the role of confirmed Pip1 substrates in transient disease assays by infection of agroinfiltrated leaves by P. infestans. This assay will test the role of Pip1-dependent cleavage of effectors, peptide hormones and proenzymes in defense. The FOURTH objective is to generate Pip1 mutants that are insensitive for inhibition by cystatin-like EpiC inhibitors of P. infestans. This objective is guided by a structural EpiC-Pip1 model and Pip1-like proteases from other plants that are EpiC insensitive. The project is based on feasible, established proteomics methods and transient assays and addresses a BBSRC-relevant question on natural defense in solanaceous plants against a devastating plant pathogen.

Summary

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 Pip1is 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.

Impact Summary

The primary and immediate impact of this project will be in PLANT SCIENCE because this project aims at elucidating the molecular mechanism underling the role of immune protease Pip1. This will not only have an impact on the interaction between solanaeous plants and Phytophthora infestans, but may also explain how Pip1 protects against fungal and bacterial diseases. Beyond this, Pip1 is a representative of extracellular immune proteases that have been described for maize and citrus, which are also targeted by pathogen-derived inhibitors. This project will also have an impact on PROTEASE RESEARCH because it includes a first-time combination of three robust proteomics technologies used for substrate identification. This project will also increase our understanding of how immune proteases select their substrates and how we can prevent their manipulation by pathogen-derived inhibitors. This project will have a significant impact on AGRICULTURE because we study natural resistance mechanisms occurring in a crop (tomato, a very close relative or potato) to the devastating potato blight pathogen, P. infestans. This pathogen is still a major concern in the potato industry and is mostly suppressed by heavy use of agrochemicals. Understanding natural resistance existing in tomato, and engineering improved Pip1 proteases to make them insensitive to inhibitors produced by P. infestans could lead to novel, robust and durable resistance in potato against late blight disease. For instance, engineering an array of inhibitor-sensitive Pip1 paralogs in potato by genome editing could increase sustainable natural resistance. This project will make a substantial impact on the GENERAL PUBLIC by working on a well-known plant pathogen, that caused the notorious Irish potato famine in the 1850s. With 1,5 million deaths and 1 million people emigrating, this disease still speaks to the mind of most UK citizens. Importantly, this oomycete is still a serious threat to the potato industry and ismostly controlled by environment-unfriendly agrochemical use. The general public will have an interest in this problem and realize that research is needed to create a more durable future for crop protection. This topic provides excellent material to engage with the general public and explain the importance on crop protection research. The impact of this project on UK RESEARCH POTENTIAL resides in publications of high impact and the training of highly skilled personnel. These activities will strengthen the position of the UK to sustain its 'Knowledge Based Economy'. It is worth noting that the PI has been a global leader in the study of immune proteases and their pathogen inhibitors over the last ~15 years (see Track Record).

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Crop Science, Microbiology, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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