Identifying Galactosyrin - the inhibitor of a novel hydrolytic immune signaling pathway

点击翻译按钮获取中文摘要

The recognition of pathogens by plants is pivotal to their survival and to our food security. Most plants recognize bacterial pathogens through fragments of flagella. We recently discovered that the hydrolytic pathway releasing these immunogenic flagellin fragments starts with the secreted beta-galactosidase BGAL1, which acts on the terminal glycan that covers the flagellin polymer (Science, April 2019). Bacteria can evade recognition by producing BGAL1-insensitive glycans, or by producing a BGAL1 inhibitor. We made our discoveries using Pseudomonas syringae infecting Nicotiana benthamiana, but these findings have implications beyond this model system because flagellin is universally recognized in the plant kingdom and BGAL1 is conserved in plants. Furthermore, glycan polymorphism is common to flagella of pathogenic bacteria, consistent with the plant-pathogen arms race. The elucidation of the BGAL1 inhibitor ('galactosyrin') has been of high interest because it interferes with this novel, conserved immune pathway. This proposal aims to elucidate the structure of galactosyrin, its biosynthesis and discover more hydrolases that are suppressed during infection. We have exciting preliminary data. First, we have identified the regulatory genes and an operon containing two biosynthesis genes responsible for galactosyrin production. Transfer of the biosynthesis genes into E. coli prompts galactosyrin production in large quantities. Second, we have established a robust enrichment protocol for galactosyrin, which is a stable, basic, hydrophilic molecule produced by bacteria when grown in minimal media.The first and main objective of this proposal is to identify the galactosyrin structure through two routes: First, by classical fractionation using preparative HPLC and structure elucidation using chemical methods in collaboration with James McCullagh (Chemistry, Oxford). In parallel we will perform cryo electron microscopy studies in collaboration with Peijun Zhang (eBIC, Harwell) to elucidate the galactosyrin structure when trapped in the active site of the beta-galactosidase encoded by LacZ, which is used as a standard in cryo-EM studies. The second objective is to characterize the biosynthesis genes and the corresponding mutants to identify their substrates and products using metabolomics, feeding experiments, substrate synthesis, and heterologous expression. The third objective is to discover more hydrolases like BGAL1 that are suppressed during infection or by bacterial metabolites, using activity-based proteomics, which currently displays >150 hydrolase activities.This proposal is relevant to the BBSRC priority program 'Agriculture and food security' and addresses the strategic priority 'Sustainably enhancing agricultural production' by increasing our understanding of basal resistance to bacterial plant pathogens. This project is feasible because we have access to large amounts of heat-resistant galactosyrin and enrichment procedures have been established. We also have identified the biosynthesis cluster and its regulators. This project is also important, not only as an asset to the BBSRC mission to support fundamental research, but also because this project will lead to novel crop protection strategies, e.g. by engineering galactosyrin-resistant BGAL1. In addition, a novel BGAL inhibitor and its biosynthesis will have an important medical impact as BGAL inhibitors are used to treat metabolic disorders, cancer, and viral and bacterial diseases.