Berberine bridge enzyme-like proteins as key virulence factors in plant pathogens

小檗碱桥酶样蛋白作为植物病原体的关键毒力因子

• 批准号: BB/Y003489/1
• 负责人: Federico Sabbadin
• 金额: $ 79.06万
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY003489%2F1
• 关键词: Berberine; bridge; enzyme; like; proteins; Berberine; bridge; enzyme; like; proteins

Plant pathogens cause $300 billion worth of damage to global food production annually. The development of sustainable and targeted disease control approaches underpins global food security and positively impacts public health, social stability and environmental biodiversity. Together, fungi and oomycetes are the most destructive pathogens in modern agriculture and represent a persistent threat to global food security. The oomycete Phytophthora infestans and the fungus Botrytis cinerea are major agricultural pathogens and model species to investigate the molecular mechanisms driving plant-pathogen interactions.The plant cell wall is the first protective barrier against pathogens and is composed of a complex network of cellulose microfibrils embedded in hemicellulose and lignin, plus a layer of pectin forming the bulk of the middle lamella that joins cells together. The plant cell wall structure and molecular composition have driven the evolution of an impressive range of degradative enzymes in both fungi and oomycetes. During infection, plant pathogens produce cell wall degrading enzymes (CWDEs), like glycoside hydrolases, esterases and lyases, to disrupt the plant cell wall and facilitate tissue penetration. Most of these enzymes are yet to be characterised in any detail. As part of the battle against pathogens, plants produce specific inhibitors of CWDEs and use receptors to sense plant cell wall fragments (oligosaccharides) released by the pathogen`s enzymes, and trigger host immunity. This complex arsenal of offensive and defensive mechanisms revolving around plant cell wall polysaccharides testifies to their important roles in plant-pathogen interactions.We previously discovered a family of lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and showed that they are key virulence factors involved in the degradation of pectin, the most abundant charged polysaccharide in the plant cell wall. More recently, we have identified a large number of secreted, uncharacterised FAD-dependent oxidases called berberine bridge enzyme-like proteins (BBEs), that have expanded in fungal and oomycete plant pathogens and are strongly induced during infection. We have produced one P. infestans BBE in yeast, carried out in vitro activity assays and detected specific oxidative activity on negatively charged pectin fragments (oligogalacturonides). Through activity assays, we observed that purified P. infestans LPMO and BBE work synergistically to degrade homogalacturonan (the backbone of pectin). Silencing of the most expressed BBE-coding gene in P. infestans caused complete loss of pathogenicity on potato leaves, confirming that this enzyme has a central role in pathogenesis. Our transcriptomic data also indicate that, like oomycetes, phytopathogenic fungi have co-opted BBEs as part of their offensive arsenal and the coding genes are co-expressed with numerous GHs involved in the degradation of abundant plant cell wall polysaccharides, again supporting an active role during tissue penetration.We hypothesise that oomycetes and fungi secrete BBEs to (i) drive plant cell wall degradation by LPMOs and (ii) oxidatively modify oligosaccharide elicitors released during infection, thus preventing their recognition by plant receptors and dampening the activation of the plant defence responses. Elucidating the molecular roles of BBEs during plant infection and their interplay with other virulence factors will help unlock new strategies to combat plant diseases. In this project, we will use gene silencing to reveal the importance of induced BBE genes during host invasion by fungi and oomycetes and assess their feasibility as targets for crop protection. We will produce recombinant forms of BBE proteins, characterise their biochemical activities and structures, and reveal their synergy with co-secreted enzymes. Finally, we will unveil if and how pathogens use BBEs and their products to manipulate the host immune response.

植物病原体每年对全球粮食生产造成3000亿美元的损害。可持续和有针对性的疾病控制方法的发展是全球粮食安全的基础，并对公共卫生，社会稳定和环境生物多样性产生了积极影响。真菌和卵菌一起是现代农业中最具破坏性的病原体，代表了对全球粮食安全的持续威胁。卵菌菌心血酸菌和真菌灰果酸是主要的农业病原体和模型物种，用于研究驱动植物病原体相互作用的分子机制。植物细胞壁是针对病原体的第一个保护性壁炉。中间片状层将细胞融合在一起。植物细胞壁结构和分子组成驱动了真菌和卵菌中令人印象深刻的降解酶的进化。在感染过程中，植物病原体会产生细胞壁降解酶（CWDES），例如糖苷水解酶，酯酶和裂解酶，以破坏植物细胞壁并促进组织渗透。这些酶中的大多数尚未详细介绍。作为与病原体的战斗的一部分，植物产生了特定的CWDE抑制剂，并使用受体来感知病原体酶释放的植物细胞壁碎片（寡糖），并触发宿主的免疫力。围绕植物细胞壁多糖围绕植物细胞壁多糖的这种进攻和防御机制的复杂武器库证明了它们在植物病原体相互作用中的重要作用。我们先前在植物病原体中发现了一个裂解多糖单一含糖单基因酶（LPMO）家族，并在植物的病原体中表明，它们在植物中涉及的植物均具有供应量的关键因素，以供应量为供应量，并在供应中进行了精致的培养。最近，我们确定了大量分泌的，未表征的FAD依赖性氧化酶，称为Berberine桥式酶样蛋白（BBE），这些蛋白质（BBE）在真菌和卵菌病原体中膨胀，并在感染过程中强烈诱导。我们已经在酵母中产生了一个Infestans bbe，进行了体外活性测定，并在带负电荷的果胶片段（寡乳糖醛酸苷）上检测到了特异性氧化活性。通过活动测定，我们观察到纯化的p. infestans lpmo和bbe协同起作用可降解同型乳糖素（果胶的骨架）。在侵注疟原虫中对最表达的BBE编码基因的沉默导致马铃薯叶片完全丧失致病性，证实该酶在发病机理中具有核心作用。 Our transcriptomic data also indicate that, like oomycetes, phytopathogenic fungi have co-opted BBEs as part of their offensive arsenal and the coding genes are co-expressed with numerous GHs involved in the degradation of abundant plant cell wall polysaccharides, again supporting an active role during tissue penetration.We hypothesise that oomycetes and fungi secrete BBEs to (i) drive plant LPMO和（II）在感染过程中释放出的寡糖引起的细胞壁降解，从而阻止了植物受体的识别并抑制植物防御反应的激活。阐明BBE在植物感染期间的分子作用及其与其他毒力因子的相互作用将有助于解锁对抗植物疾病的新策略。在这个项目中，我们将使用基因沉默来揭示真菌和卵菌在宿主入侵期间诱导的BBE基因的重要性，并评估它们作为作物保护的靶标的可行性。我们将产生重组形式的BBE蛋白质，表征其生化活性和结构，并用共归化的酶揭示其协同作用。最后，我们将揭露病原体使用BBE及其产品来操纵宿主免疫反应。