Hyphal Biomechanics in Pathogenic Oomycetes

该项目关注丝状真菌中进化的生物力学策略，使它们能够穿透植物和动物的固体组织并从其中获取能量。虽然外切酶被认为在减少这些组织的机械阻力中起重要作用，但在菌丝顶端施加的力是一个关键的，但在很大程度上被忽视的入侵因素。所选择的实验设计反映了比较生理学的方法，并将检查一对密切相关的卵菌（最近被分配到Straviila王国）从腐霉属：Pythium insidiosum导致人类和其他哺乳动物的侵入性疾病，Pythium graminicola是禾本科植物的病原体。该项目利用了一套由P.I.开发的生物力学技术。这将允许分析微观菌丝的机械行为。新的方法采用超灵敏硅桥应变计（或力传感器）与UN（百万分之一牛顿）的分辨率将允许直接测量微牛顿力产生的个别菌丝，也将测量所需的力推动玻璃微探针到动物和植物组织的样品。通过使用显微操作器将应变计定位在菌丝顶端前几微米处来测量菌丝力。随着细胞生长并推压硅梁的末端，仪器的电输出与施加的力成比例地变化。比较这些组织的菌丝力和物理阻力将建立机械入侵的潜力，而不需要组织降解酶的作用。机械渗透与外源酶在侵入过程中的相对重要性（这仍然是真菌学中一个基本的未解决的问题），然后将通过比较菌丝力和各种真菌酶处理的宿主组织的强度进行测试。将进一步测量菌丝膨压、菌丝壁的抗张强度、细胞骨架活性和细胞大小，以确定控制菌丝顶端力的关键变量。这些实验将探索决定菌丝和宿主组织所呈现的物理微环境之间相互作用的细胞机制。虽然其中一些过程可能是宿主特异性的，但其他过程可能是侵入性生长的普遍特征。总之，计划中的实验将揭示生物力学适应在定义感染动物和植物的卵菌的宿主范围（或生态位）方面的重要性。

This project is concerned with the biomechanical strategies that have evolved among filamentous fungi enabling them to penetrate and harvest energy from the solid tissues of plants and animals. While exoenzymes are presumed to play an important role in reducing the mechanical resistance presented by these tissues, the exertion of force at the hyphal apex represents a critical, yet largely ignored factor in invasion. The chosen experimental design reflects a comparative physiological approach and will examine a pair of closely related species of oomycetes (recently assigned to the Kingdom Stramenopila) from the genus Pythium: Pythium insidiosum causes an invasive disease in humans and other mammals, and Pythium graminicola is a pathogen of grasses. The project draws upon a suite of biomechanical techniques developed by the P.I. that will allow analysis of the mechanical behavior of microscopic hyphae. New methods employing ultra-sensitive silicon bridge strain gauges (or force transducers) with UN (millionths of one Newton) resolution will allow direct measurement of the micronewton forces produced by individual hyphae, and will also measure the force required to push glass microprobes into samples of animal and plant tissues. Hyphal force is measured by positioning the strain gauge a few micrometers in advance of a hyphal apex using a micromanipulator. As the cell grows and pushes against the end of the silicon beam, the electrical output from the instrument changes in proportion to the applied force. Comparisons between hyphal forces and the physical resistance of these tissues will establish the potential for mechanical invasion without the action of tissue-degrading enzymes. The relative significance of mechanical penetration versus exogenous enzymes in the invasive process (which remains a fundamental unresolved question in mycology) will then be tested by comparing hyphal forces and the strength of host tissues treated with a variety of fungal enzymes. Further measurements of hyphal turgor pressure, the tensile strength of the hyphal wall, cytoskeletal activity, and cell size will be made to identify key variables that control force at the hyphal apex. These experiments will explore the cellular mechanisms that determine the interaction between hyphae and the physical microenvironment presented by host tissues. While some of these processes may be host specific, others are likely to be universal features of invasive growth. Together, the planned experiments will reveal the significance of biomechanical adaptations in defining the host range (or ecological niche) of oomycetes that infect animals and plants.