Hyphal Biomechanics in Pathogenic Oomycetes

Miami University, Oxford OH

Investigators

Abstract

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.