Probing the molecular interactions at the extrahaustorial membrane interface

面对气候变化和人口增长，粮食安全是人类面临的重大挑战;然而，在全球范围内，每年高达15%的作物因病原体而损失。保护作物免受病原体的侵害是一项关键的、持续的全球倡议。许多农业上重要的疾病是由白粉病和锈菌以及真菌样卵菌病原体引起的。这些进化上不同的病原体的一个共同特征是，它们发展出类似的进食结构，称为吸器，以入侵植物并从宿主细胞中提取营养。尽管在了解植物对病原体抗性的分子机制方面取得了巨大进展，但在植物-吸器界面中如何执行宿主防御仍然缺乏特征。该研究的目的是使用模型病理系统和最先进的遗传学和生物化学工具来识别和功能表征宿主-病原体界面（称为吸器外膜）的新型分子。该项目的新发现不仅有助于更好地了解宿主-病原体界面的分子组成，还有助于设计新的策略来对抗吸器形成病原体并减少作物损失，从而促进可持续农业。吸器形成病原体如白粉病真菌在宿主组织中形成称为吸器的进食结构，以分泌能够抑制宿主免疫应答并从宿主细胞中提取营养物的效应蛋白。吸器与宿主细胞之间由一层界面膜隔开，称为吸器外膜（EHM）。EHM被认为是最关键的宿主-病原体战场，其中宿主防御被安装以限制吸器，并且病原体部署效应器以破坏宿主免疫并从宿主中提取营养。然而，尽管EHM的重要性，其分子组成和宿主防御或发病机制的特点是很差的。本项目的长期目标是了解EHM的蛋白质组成和在宿主-病原体界面上植物-吸器相互作用的机制。以拟南芥-白粉病互作为模式致病系统，RPW8.2（一种广谱抗性蛋白）作为第一个和最好的EHM特异性驻留蛋白，一个合作团队将（i）通过基于邻近的标记结合吸器复合物的富集/纯化，然后通过质谱鉴定和表征在EHM处与RPW8.2相互作用的蛋白质和其他EHM驻留蛋白;（ii）解析RPW8.2的蛋白质结构，并了解RPW8.2的EHM定向囊泡运输如何受到跨高尔基体网络上的候选SNARE复合物的调节;和（iii）鉴定宿主易感性因子，如糖转运蛋白，这可能是招募到EHM的白粉病真菌营养收购通过遗传和细胞生物学的方法。这一奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估的支持。

Food security is a grand challenge facing humanity in the face of climate change and population growth; yet, on a global scale up to 15% of crops are lost to pathogens each year. Protecting crops from pathogens is a critical, ongoing global initiative. Many agriculturally important diseases are caused by powdery mildew and rust fungi, and fungus-like oomycete pathogens. One common feature of these evolutionarily distinct pathogens is that they develop similar feeding structures called haustoria to invade plants and extract nutrients from host cells. Despite tremendous progress towards understanding the molecular mechanisms of plant resistance against pathogens, how the host defense is executed in the plant-haustorial interface is still poorly characterized. The research aims to identify and functionally characterize novel molecular players at the host-pathogen interface termed the extrahaustorial membrane using a model pathosystem and state-of-the-art genetics and biochemistry tools. New findings from this project will not only lead to a better understanding of the molecular composition of the host-pathogen interface but also help design novel strategies to fight against haustorium-forming pathogens and reduce crop losses, thereby contributing to sustainable agriculture. Haustorium-forming pathogens such as powdery mildew fungi develop a feeding structure termed the haustorium in host tissues to secrete effector proteins capable of suppressing the host immune response and extracting nutrients from the host cell. The haustorium is physically separated from the host cell by a host-derived interfacial membrane named the extrahaustorial membrane (EHM). The EHM is believed to be the most critical host-pathogen battleground, where the host defense is mounted to constrain the haustorium, and the pathogen deploys effectors to subvert host immunity and extracts nutrients from the host. However, despite the importance of the EHM, its molecular components and the mechanisms associated with host defense or pathogenesis are poorly characterized. The long term goal of this project is to understand the protein composition of the EHM and the mechanisms of plant-haustorium interactions at this host-pathogen interface. Using the Arabidopsis-powdery mildew interaction as a model pathosystem and RPW8.2 (a broad-spectrum resistance protein) as the first and best EHM-specific resident protein, a collaborative team will (i) identify and characterize proteins interacting with RPW8.2 at the EHM and additional EHM-resident proteins via proximity-based labeling coupled with enrichment/purification of haustorial complexes followed by mass spectrometry; (ii) resolve the protein structure of RPW8.2 and understand how EHM-oriented vesicle transport of RPW8.2 is regulated by a candidate SNARE complex at the Trans-Golgi Network; and (iii) identify host susceptibility factors, such as sugar transporters, which may be recruited to the EHM by powdery mildew fungi for nutrient acquisition through both genetic and cell biological approaches.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.