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

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价值3000亿美元的损失。制定可持续和有针对性的疾病控制方法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积极影响。真菌和卵菌是现代农业中最具破坏性的病原体，对全球粮食安全构成持续威胁。卵菌Phytophthora infestans和灰葡萄孢Botrytis cinerea是主要的农业病原菌，也是研究植物-病原菌相互作用的分子机制的模式物种。植物细胞壁是抵御病原菌的第一道保护屏障，由嵌入半纤维素和木质素中的纤维素微纤丝组成的复杂网络，以及形成将细胞连接在一起的中间层大部分的果胶层组成。植物细胞壁结构和分子组成驱动了真菌和卵菌中一系列令人印象深刻的降解酶的进化。在感染过程中，植物病原体产生细胞壁降解酶（CWDE），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。这些酶中的大多数还没有被详细描述。作为对抗病原体的一部分，植物产生特异性的CWDEs抑制剂，并使用受体来感知病原体酶释放的植物细胞壁片段（寡糖），并引发宿主免疫。这种复杂的进攻和防御机制围绕植物细胞壁多糖证明了他们的重要作用，在植物-病原菌interactions.We以前发现了一个家庭的溶解性多糖单加氧酶（LPMO）在植物病原卵菌，并表明他们是关键的毒力因子参与的果胶，最丰富的带电多糖在植物细胞壁中的降解。最近，我们已经确定了大量的分泌，未表征的FAD依赖性氧化酶称为小檗碱桥酶样蛋白（BBE），已扩大在真菌和卵菌植物病原体，并强烈诱导感染过程中。我们已经在酵母中产生了一种致病疫霉BBE，进行了体外活性测定，并检测了对带负电荷的果胶片段（寡聚半乳糖醛酸）的特异性氧化活性。通过活性测定，我们观察到纯化的致病疫霉LPMO和BBE协同作用以降解同型半乳糖醛酸（果胶的主链）。在致病疫霉中表达最多的BBE编码基因的沉默导致马铃薯叶片上的致病性完全丧失，证实了这种酶在发病机制中具有核心作用。我们的转录组学数据还表明，与卵菌纲一样，植物病原性真菌也选择BBE作为其进攻性武器库的一部分，并且编码基因与参与降解丰富的植物细胞壁多糖的许多GH共表达，我们假设卵菌和真菌分泌BBE（i）通过LPMO驱动植物细胞壁降解和（ii）氧化修饰在感染期间释放的寡糖激发子，从而防止它们被植物受体识别并抑制植物防御反应的激活。阐明BBE在植物感染过程中的分子作用及其与其他毒力因子的相互作用将有助于揭示对抗植物疾病的新策略。在本项目中，我们将利用基因沉默来揭示诱导的BBE基因在真菌和卵菌入侵寄主过程中的重要性，并评估其作为作物保护靶标的可行性。我们将生产重组形式的BBE蛋白，研究它们的生化活性和结构，并揭示它们与共分泌酶的协同作用。最后，我们将揭示病原体是否以及如何使用BBE及其产物来操纵宿主免疫反应。