An Investigation into the Underlying Mechanisms of Hyphal Branching in Filamentous Microorganisms

Understanding how hyphal organisms grow and develop is essential in order to manipulate mycelial colonies for purposes such as disease prevention and food production. One aspect of hyphal development that is not well understood is hyphal branching. Hyphal organisms branch as a way of creating new hyphal tips required for the search for nutrients, the acquisition of these nutrients and for hyphal fusion events that facilitate communication of signals within a mycelial colony. This investigation focused on the branching process occurring in the fungus N. crassa and in the oomycete A. bisexualis. An induction technique was developed to study branching in N. crassa involving local application of amino acids towards hyphae. This induced a branch to form along the hypha within the field of view. The use of this technique will enable the study of underlying events occurring internally prior to the visible branching stages. The role of Ca²⁺ hyphal branching was investigated in N. crassa suggesting Ca²⁺ is involved in apical dominance of the hyphal tip. This is based on a dose dependent response of increased branch frequency, decreased colony radius and decreased distance between the hyphal tip and the first branch, to the Ca²⁺ channel inhibitor verapamil. The stretch-activated Ca²⁺ channel inhibitors also had an effect on mycelial morphology. Gd³⁺ resulted in an increased branch frequency and a decreased colony radius and La³⁺ resulted in a decreased colony radius. The local application of verapamil towards N. crassa showed an increase in the number of multiple branches forming. Cytoplasmic Ca²⁺ was imaged in hyphae of A. bisexualis and N. crassa showing a tip-high Ca²⁺ gradient in A. bisexualis and Ca²⁺ sequestered into organelles in N. crassa. The role of F-actin in the process of hyphal branching was investigated using Lifeact N. crassa where F-actin could dynamically be seen at the site of both growing and non-growing hyphal branches. The involvement of F-actin at sites of septa development and associated with suspected vesicles was also observed