Zellbiologische und biochemische Charakterisierung des Ustilago maydis Virulenzfaktors Mcs1(Myosin-Chitinsynthase 1)

Invasion of the host by phytopathogenic fungi occurs by polar expansion of the fungal cell, a process also called hyphal tip growth. Tip growth requires continuous synthesis of fungal cell wall components at the hyphal apex and is therefore based on polar delivery of enzymes, like chitin synthases (CHS), along cytoskeleton tracks. In particular, class V CHS are fungal virulence factors required for plant infection. These putative motor proteins consist of a myosin-motor domain (MMD) fused to a CHS domain which participates in fungal cell-wall formation. The function of the MMD is unknown, but it might deliver secretory vesicles (chitosomes) to the growth region. In this work, the importance of both domains in Mcs1, the class V CHS of the corn smut fungus Ustilago maydis, was investigated. By quantitative analysis of disease symptoms, tissue colonization and single-cell morphogenic parameters it was demonstrated that both domains play essential though unequal roles during plant infection. While infections with G3Mcs1Chsdead-mutants display phenotypes similar to the deletion mutants, MMD mutants exhibit partial colonization of the plant tissue. mcs1-deletion mutants and strains defective in CHS activity are rapidly recognized and killed by the plant, whereas mutants lacking the MMD can invade the host tissue and cause moderate plant responses. The absence of the class V CHS in mcs1-deletion mutants most likely causes major defects in the fungal cell wall, thereby affecting polarized growth and decelerating the infection process. This results in strong plant defense responses characterized by the formation H2O2 and local cell death which might be elicited by defective cell wall composition of mcs1-deletion and G3Mcs1Chsdead-mutants. Microscopic analysis revealed that apical focussing of Mcs1 depends on a functional MMD, although some chitosomes were rarely able to reach the apex. As a consequence, hyphal growth is partially rescued and U. maydis can partially colonize the host tissue until moderate plant responses stops the infection. Structural analysis revealed that despite low sequence similarities the Mcs1 MMD adapts folding of a myosin head. Furthermore the MMD binds to F-actin and forms dimers and in vivo analysis revealed that Mcs1-bound chitosomes exhibit bi-directional long-distant movements at high velocities. Prior to exocytosis, chitosomes often pause in sub apical regions of the plasma membrane and insertion into the membrane occurs only rarely and randomly. It was determined that residence times at the apex are significantly shorter in mutants lacking the MMD. Whereas apical localisation of Mcs1 depends on F-actin and the MMD, chitosome motility requires both actin and microtubules and persisted when the Mcs1 MMD was deleted. The results suggest that fungal specific exocytosis, but not delivery, of the essential class V CHS is promoted by its own MMD, explaining the importance of both parts for fungal virulence.