Investigating the role of chitin deacetylation in Magnaporthe oryzae

The fungal cell wall is a highly complex and dynamic structure. It plays a critical role in maintaining cellular integrity, as well as forming the interfacial barrier with the environment. It comprises a matrix of interconnected polysaccharides and proteins, which can change in response to altered environmental conditions, during interactions with other organisms, or during cellular growth and morphogenesis. One such observed change is the deacetylation of chitin to chitosan, a reaction catalysed by a family of carbohydrate esterase enzymes known as chitin deacetylases (CDAs). In phytopathogenic species, such as the rice blast fungus Magnaporthe oryzae, chitosan has been shown to be a component of the cell wall during pathogenic interactions. Here, it was hypothesized that the deacetylation of chitin acts as a 'stealth mechanism', by both protecting the fungal cell wall from plant chitinase activity, and by preventing the activation of plant defence responses. In addition, differences in the physical and chemical properties of chitin and chitosan are such that chitin deacetylation may also be required for cell wall modification during cellular morphogenesis. In order to test these hypotheses, the role of chitin deacetylation in appressorium development and vegetative growth were investigated. During germling morphogenesis, chitosan staining revealed that chitin deacetylation occurs both prior to, and during, appressorium development. Through the creation of single, double and triple deletion strains of three CDAs (CBP1, CDA2 and CDA3), chitosan was shown to be necessary for appressorium development on artificial surfaces. However, chitin deacetylation is not required for appressorium development on plant surfaces, arguing against its role in morphogenesis. Exogenous application of chitosan and chemical inducers of appressorium development revealed that chitosan is instead required for germling adhesion and surface sensing. Fluorescent tagging of these CDAs and colocalization with chitin synthase VII suggests that the deacetylation of chitin may involve close association/collaboration between these two families of enzymes. Chitin deacetylation also occurs in the septa and lateral walls of vegetative hyphae. Two further CDAs (CDA1 and CDA4) were characterized by gene deletion and uorescent protein tagging, and were found to be required for chitin deacetylation in these locations. Although chitin deacetylation was dramatically reduced in the deletion strains, no defects in growth or morphology were observed. This again argues against a role for chitin deacetylation in morphogenesis, and also suggests that chitosan is not a crucial structural component of the cell wall. However, mCherry fusions of Cda4 provided further evidence of possible collaboration between CDAs and chitin synthases. Lastly, the role of chitosan hydrolysis was investigated. Cell wall hydrolases have long been hypothesized to have roles in cell wall modification during morphogenesis. To test this hypothesis, strains were created in which the gene encoding the sole chitosanase (CSN) in M.oryzae was either deleted or overexpressed. However, these strains proved to be identical to the WT strain under all conditions tested. In addition, a promoter fusion revealed only weak expression of CSN in vegetative hyphae. Chitosanase therefore appears not to have a role in cell wall modification during morphogenesis, but its true purpose remains unclear. In summary, the data in this thesis do not support the original hypotheses regarding the roles of chitin deacetylation or chitosan hydrolysis. Instead, novel roles for chitosan were unmasked, which demonstrate the complex and multifaceted nature of the cell wall. Novel mechanistic insights into chitin deacetylation were also revealed, which suggest that our current knowledge of chitin synthesis and deacetylation is far from complete.