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

鉴定半乳糖苷 - 一种新型水解免疫信号通路的抑制剂

• 批准号: BB/T015128/1
• 负责人: Renier Van Der Hoorn
• 金额: $ 82.36万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT015128%2F1
• 关键词: Identifying; Galactosyrin; inhibitor; novel; hydrolytic

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.

植物对病原体的识别对它们的生存和我们的粮食安全至关重要。大多数植物通过鞭毛片段识别细菌病原体。我们最近发现，释放这些免疫原性鞭毛蛋白片段的水解途径始于分泌的β-半乳糖苷酶BGAL 1，其作用于覆盖鞭毛蛋白聚合物的末端聚糖（Science，2019年4月）。细菌可以通过产生BGAL 1不敏感聚糖或产生BGAL 1抑制剂来逃避识别。我们使用感染本氏烟草的假单胞菌进行了我们的发现，但这些发现具有超出该模型系统的意义，因为鞭毛蛋白在植物界中被普遍认可，而BGAL 1在植物中是保守的。此外，聚糖多态性是常见的病原细菌的鞭毛，与植物病原体军备竞赛。BGAL 1抑制剂（“半乳糖苷酶”）的阐明一直受到高度关注，因为它干扰这种新的、保守的免疫途径。该建议旨在阐明半乳糖苷的结构，其生物合成和发现更多的水解酶，在感染过程中受到抑制。我们有令人兴奋的初步数据。首先，我们已经确定了调控基因和操纵子含有两个生物合成基因负责半乳糖苷的生产。将生物合成基因转入E.大肠杆菌促使半乳糖苷酶大量产生。其次，我们已经建立了一个强大的富集方案，半乳糖苷，这是一个稳定的，基本的，亲水性分子产生的细菌生长时，在基本medium. First和主要目标的建议是确定半乳糖苷的结构，通过两条路线：第一，通过经典的分馏使用制备型HPLC和结构解析使用化学方法与詹姆斯McCullagh（化学，牛津大学）。同时，我们将与Peijun Zhang（eBIC，Harwell）合作进行冷冻电子显微镜研究，以阐明半乳糖苷酶在被LacZ编码的β-半乳糖苷酶的活性位点捕获时的结构，该活性位点被用作冷冻EM研究的标准。第二个目标是表征生物合成基因和相应的突变体，以确定其底物和产品使用代谢组学，喂养实验，底物合成，异源表达。第三个目标是使用基于活性的蛋白质组学，发现更多在感染期间或被细菌代谢物抑制的水解酶，如BGAL 1，目前显示>该提案与BBSRC优先计划“农业和粮食安全”相关，并通过增加我们对细菌植物的基础抗性的理解来解决战略优先事项“可持续地提高农业生产”。病原体该项目是可行的，因为我们可以获得大量的耐热半乳糖苷，并且已经建立了浓缩程序。我们还确定了生物合成簇及其调节因子。该项目也很重要，不仅是作为BBSRC支持基础研究的使命的资产，而且还因为该项目将导致新的作物保护策略，例如通过工程抗半乳糖苷BGAL 1。此外，新型BGAL抑制剂及其生物合成将具有重要的医学影响，因为BGAL抑制剂用于治疗代谢紊乱、癌症以及病毒和细菌疾病。

项目成果

The proteome of agroinfiltrated Nicotiana benthamiana is shaped by extensive protein processing

• DOI: 10.1101/2023.11.02.565301
• 发表时间: 2024-01
• 期刊: bioRxiv
• 作者: Kaijie Zheng;Joy C. Lyu;Emma L. Thomas;Mariana Schuster;N. Sanguankiattichai;Sabrina Ninck;F. Kaschani;Markus Kaiser;R. V. D. van der Hoorn

Releasing hidden MAMPs from precursor proteins in plants

• DOI: 10.1016/j.tplants.2023.09.013
• 发表时间: 2024-04-03
• 期刊: TRENDS IN PLANT SCIENCE
• 影响因子: 20.5
• 作者: Chen，Changlong;van der Hoorn，Renier A. L.;Buscaill，Pierre
• 通讯作者: Buscaill，Pierre

How bacteria overcome flagellin pattern recognition in plants.

• DOI: 10.1016/j.pbi.2022.102224
• 发表时间: 2022-05
• 期刊: Current opinion in plant biology
• 影响因子: 9.5
• 作者: N. Sanguankiattichai;Pierre Buscaill;G. Preston

An oomycete effector subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface.

• DOI: 10.7554/elife.65285
• 发表时间: 2021-08-23
• 期刊: eLife
• 影响因子: 7.7
• 作者: Pandey P;Leary AY;Tumtas Y;Savage Z;Dagvadorj B;Duggan C;Yuen EL;Sanguankiattichai N;Tan E;Khandare V;Connerton AJ;Yunusov T;Madalinski M;Mirkin FG;Schornack S;Dagdas Y;Kamoun S;Bozkurt TO
• 通讯作者: Bozkurt TO

Cautionary note on ribonuclease activity of recombinant PR-10 proteins

• DOI: 10.1101/2023.02.27.529914
• 发表时间: 2023-02
• 期刊: bioRxiv
• 作者: Rawit Longsaward;N. Sanguankiattichai;Unchera Viboonjun;R. V. D. van der Hoorn