New Enzymatic Virulence Factors In Phytophthora Infestans

卵菌是一种真菌类真核生物，在进化上与硅藻和褐藻有关，并在农业和水产养殖中引起一些最具破坏性的植物和动物疾病。疫霉属包含超过140个物种，包括晚疫病病原体致病疫霉（Phytophthora infestans），其在世纪引起爱尔兰马铃薯饥荒，导致超过一百万人死于饥饿。致病疫霉感染马铃薯和番茄作物，并且是农业中最具破坏性的卵菌病原体，每年造成超过60亿美元的经济损失。植物病原体需要穿透植物细胞壁以感染其宿主，并且宿主和病原体之间的分子斗争的主要方面发生在细胞壁中。病原体，如致病疫霉，分泌一系列细胞壁降解酶以促进宿主渗透。这些病原体酶（如多聚半乳糖醛酸酶和果胶甲基酯酶）中的许多酶靶向植物细胞壁的果胶组分。果胶提供了将植物细胞彼此结合的胶水，并且降解果胶允许病原体通过植物细胞之间的中间层生长。作为回应，植物已经进化出一系列对抗措施，包括病原体酶的抑制剂。这些抗病原体反应是在检测到病原体酶分解宿主细胞壁（如果胶寡聚半乳糖醛酸）所产生的小分子后在植物中诱导的。我们最近在卵菌中发现了一类新的溶解性多糖单加氧酶（LPMO），这类酶在植物病原体物种如致病疫霉中特别丰富。致病疫霉中的三个LPMO基因在植物感染过程中非常早期就高度表达，并且阻断这些基因中表达最丰富的基因的作用会阻碍病原体感染植物组织的能力，这表明它对宿主渗透很重要。先前表征的LPMO类是能够消化最难消化的多糖形式如结晶纤维素或几丁质的氧化酶。我们已经表明，表达最多的致病疫霉LPMO氧化攻击聚半乳糖醛酸聚糖，植物中的主要果胶聚合物。这是第一次LPMO已经被证明攻击果胶，表明这种酶已经进化到专门促进宿主渗透，可能有助于病原体逃避宿主产生的多聚半乳糖醛酸酶和果胶甲酯酶抑制蛋白。以前发表的工作表明，氧化的寡聚半乳糖醛酸不会诱导植物中的病原体反应，这表明LPMO攻击可能有助于病原体在宿主的雷达下溜走。基因表达研究表明，一系列其他的，未表征的，分泌的酶在感染早期产生。我们建议研究这些蛋白质在感染过程中的作用，并评估它们在病原体感染plants.To实现我们的目标的能力的重要性，我们将产生一个库存的蛋白质发现在宿主细胞壁在病原体感染早期使用高度敏感的蛋白质组学方法。这将提供病原体和宿主蛋白在感染过程中表达的全面视图。我们将选择这些蛋白质的一个子集进行进一步研究，包括LPMO家族。研究靶标的选择将基于其新奇及其在基于基因沉默研究的发病机制中的重要性。我们将研究重组版本的靶蛋白的生化活性，分析其三维结构，并研究它们在感染过程中在细胞壁中的特异性定位。我们的研究结果将为病原体在感染过程中产生的酶和蛋白质以及宿主植物对此的反应提供新的认识。我们发现的对感染很重要的病原体基因（如LPMO）将为有针对性的作物保护策略提供新的分子靶标。

Oomycetes are fungal-like eukaryotic organisms evolutionarily related to diatoms and brown algae, and cause some of the most devastating plant and animal diseases in agriculture and aquaculture. The Phytophthora genus contains over 140 species, including the late blight pathogen Phytophthora infestans, which caused the Irish potato famine that led to death-by-starvation of over one million people in the 19th century. P. infestans infects both potato and tomato crops and is the most damaging oomycete pathogen in agriculture, causing economic losses in excess of $6 billion annually. Plant pathogens need to penetrate the plant cell wall in order to infect their host, and major aspects of the molecular struggle between the host and pathogen occur in the cell wall. Pathogens, such as P. infestans, secrete a range of cell wall degrading enzymes to facilitate host penetration. Many of these pathogen enzymes (such as polygalacturonase and pectin methylesterase) target the pectin components of plant cell walls. Pectins provide the glue that binds plant cells to one another, and degrading pectin allows the pathogen to grow through the middle lamella between plant cells. In response, plants have evolved a range of counter measures, including inhibitors of pathogen enzymes. These anti-pathogen responses are induced in plants following the detection of small molecules derived from breakdown of the host cell wall (such as pectic oligogalacturonides) by pathogen enzymes.We recently discovered a new class of lytic polysaccharide monooxygenase (LPMO) enzymes in oomycetes, which are notably abundant in plant pathogenic species, such as P. infestans. Three LPMO genes in P. infestans are highly expressed very early during plant infection, ands blocking the action of the most abundantly expressed of these impedes the pathogen's ability to infect plant tissues, suggesting it is important for host penetration. Previously characterised classes of LPMOs are oxidative enzymes able to digest the most recalcitrant forms of polysaccharides such as crystalline cellulose or chitin. We have shown that the most expressed P. infestans LPMO oxidatively attacks polygalacturonan, the main pectic polymer in plants. This is the first time LPMOs have been shown to attack pectins, indicating this enzyme has evolved specifically to facilitate host penetration, perhaps helping the pathogen to evade the polygalacturonase- and pectin methyl esterase-inhibiting proteins produced by the host. Previously published work has shown that oxidised oligogalacturonides do not induce pathogen responses in plants, suggesting that LPMO attack may help the pathogen to slip under the host's radar. Gene expression studies indicate a range of other, uncharacterised, secreted enzymes are produced early in infection. We propose to study the roles of these proteins during infection and assess their importance in the ability of the pathogen to infect plants.To achieve our aims we will generate an inventory of proteins found in the host cell wall during early pathogen infection using highly sensitive proteomic approaches. This will provide a comprehensive view of pathogen and host proteins expressed during infection. We will select a subset of these proteins for further study including the LPMO family. The selection of targets for study will be based on their novelty and their importance in pathogenesis based on gene silencing studies. We will study the biochemical activity of recombinant versions of target proteins, characterise their 3-dimensional structure, and study their specific localisation in the cell wall during infection. The results of our studies will provide new understanding of enzymes and proteins produced by pathogens during infection and by host plants in response to this. The pathogen genes that we show to be important for infection (such as the LPMOs) will provide new molecular targets for targeted crop protection strategies.