Transcriptional regulation during appressorium formation and function in Glomerella cingulata : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology at Massey University, Palmerston North, New Zealand

Abstract

Glomerella cingulata, anamorph Colletotrichum gloeosporioides, causes bitter rot in apples and fruit rot in other subtropical fruits. In response to environmental cues such as contact with the host, Glomerella cingulata forms a special structure called an appressorium, which accumulates glycerol and thereby generates a sufficiently high turgor pressure to push an infection peg into the host tissue. It is known that the cAMP and MAPK signalling transduction pathways control appressorium formation and function in Colletotrichum species and other appressorium-forming fungi. This process is accompanied by a global change in gene expression. Little is known of transcriptional regulation during this process. The aim of this project was to study the transcriptional regulation of appressorium formation and function in G. cingulata. The G cingulata SAP gene had previously been shown to be expressed as a longer transcript during the early stage of appressorium differentiation. It was considered possible that the transcription factors that regulated expression of the longer transcript may be also involved in the regulation of appressorium differentiation. Identification of the transcription factor involved may help to understand the mechanisms that regulate appressorium differentiation. The plan was to use the yeast one-hybrid system to isolate the transcription factor. This required identification of the promoter regions responsible for expression of the longer SAP transcript. Therefore, the G. cingulata SAP promoter was characterized by mapping the transcription start point. Three transcription start points were determined by RLM-RACE. To further characterise the promoters, SAP-GFP reporter plasmids were constructed and transformed into G. cingulata. Even though a reasonable level of GFP expression was observed in RT-PCR experiments, however, no differences in fluorescence intensity were seen between the wild type and GFP reporter transformants. Therefore, no further attempts to study the sap promoter were made. The candidate gene approach was chosen as an alternative way to study the transcriptional regulation of appressorium formation and function in G. cingulata. The G. cingulata StuA gene was cloned using degenerate PCR, single specific primer PCR, subgenomic library screening and plasmid rescue from a disruption mutant. Targeted gene deletion of the G. cingulata StuA gene was successful. Deletion mutants display many phenotypic changes. Complementation mutants were constructed to confirm the function of this gene. A full length copy of this gene together with a second selection marker was reintroduced into the deletion mutant and the wild type phenotype was restored. Deletion mutants form appressoria at the normal rate and with unaltered morphology. In comparison with the wild type, these appressoria did not generate high turgor pressure as shown by a cytorrhysis assay. This resulted in a defect in appressorium penetration of onion epidermal cells. Nor were these mutants able to invade unwounded apples. Therefore, the G. cingulata StuA gene is required for appressorium function. In addition, deletion mutants displayed stunted aerial hyphae, "wettable" mycelium, reduced conidia production, and a defect in conidiophore and perithecium formation. These results suggested that the G. cingulata StuA gene has multiple roles in fungal development.