Identification of novel genes controlling sexual development in Aspergillus species
Publication Date
December 15, 2022
Creator
Abstract
The overall aim of this PhD project was to gain a better understanding of the genetic controls of sexual development in Aspergillus species, a topic of both of fundamental and practical importance. Work covered three areas. First, over 70 genes have already been identified associated with sexual reproduction in A. nidulans. In order to maintain A. nidulans as a comprehensive model for sexual development in the Ascomycotina, genetic studies were undertaken to determine any role that genes involved in sexual reproduction identified from other ascomycete species, but not yet studied in A. nidulans, might have in A. nidulans i.e. to investigate whether such genes were universal ascomycete sex genes. To achieve this goal, putative gene homologs were identified in A. nidulans by bioinformatic analysis and then gene function characterised by screening for sexual fertility of control versus gene deletion strains. Gene AN2701.2, encoding a putative MAPK (mitogen activated protein kinase) scaffold protein, was found to be pivotal for sexual development with gene deletion causing a significant reduction in size of cleistothecia, number of ascospores and ascospore viability. The STRIPAK (striatin-interacting phosphatase and kinases) complex has an essential role in signal transduction. The STRIPAK subunits AN0164.2 (PP2Ac regulatory unit) and AN6611.2 (STRIP1/2 unit) were also found to be required for normal sexual development and growth. However, deletion of AN4632.2 (SLAMP unit) did not show any significant impact on sexual development. The putative transcription factors AN4813.2, AN1217.2, AN7170.2 and AN7736.2 were investigated. The mybA homolog AN4813.2 was found to be required for normal sexual development, with gene deletion leading to a significant reduction in numbers of cleistothecia and ascospores, and lowered ascospore viability. AN4813.2 deletion in addition blocked conidiophore formation, indicating a dual role in sexual and asexual development. Deletion of AN1217.2 also caused abnormal development of cleistothecia whereas deletion of AN7170.2 and AN7736.2 had no impact on cleistothecial formation. Deletion of AN3147.2, encoding a glycolipid protein, reduced the number of cleistothecia and ascospores produced. Deletion of AN10631.2 had no impact on fruit body formation whilst AN4891.2, predicted to encode a histone chaperone, appeared essential for general growth.
Second, the role of high mobility group (HMG) genes in controlling sexual development in A. fumigatus was studied. HMG proteins have previously been shown to regulate a wide range of developmental processes in eukaryotic organisms but in the case of fungi, almost all studies have focused only on HMG genes involved in determination of sexual identity. However, recent studies with A. nidulans and Podospora anserina had revealed the presence of a network of additional HMG genes in the genome, many of which were shown to regulate sexual reproduction in these homothallic/pseudohomothallic species. Studies were therefore undertaken to investigate the possible role of such additional HMG genes in the heterothallic species A. fumigatus. Bioinformatic analysis identified seven putative HMG gene homologs in the genome of A. fumigatus. Subsequent gene deletion and fertility screening via sexual crossing revealed that five of the HMG homologs were required for normal sexual development. As part of this work, gene deletion of the known sexual development activator gene nsdC was conducted as pilot work, and it was found interestingly that loss of fertility was only observed when both mating partners were of the ΔnsdC background. This necessitated the construction of highly fertile ΔkuB strains of both MAT1-1 and MAT1-2 genotype for later characterisation of HMG gene function.
Third, any possible dual role of HMG genes in the regulation of secondary metabolism of A. fumigatus was investigated. Levels of the mycotoxins gliotoxin, fumagillin, pseurotin A and helvolic acid were investigated via HPLC in HMG deletant strains of A. fumigatus. Both fumagillin and pseurotin A production was reduced in five of the gene deletants, indicating that these HMG genes positively upregulated these secondary metabolites. By contrast, gene deletion had no impact on gliotoxin production.
Overall, studies led to the identification of novel genes involved with sexual reproduction of A. nidulans, identified a network of HMG genes regulating sexual development in A. fumigtaus, and provided evidence of the dual role of certain HMG genes in the regulation of both fruit body formation and secondary metabolism.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
71226
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