Exploitation of fungal sex for biotechnology and disease control

Ashton, George David (2019) Exploitation of fungal sex for biotechnology and disease control. PhD thesis, University of Nottingham.

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Abstract

The overall theme of work in the present thesis was to gain knowledge about aspects of the fungal sexual cycle that would allow the practical exploitation of sexual reproduction for use in biotechnology and disease control.

Investigations were first made into the potential sexual cycle in Aspergillus niger and Aspergillus sclerotiicarbonarius, members of the black aspergilli. Both species are proto-heterothallic, showing distinct MAT1-1 and MAT1-2 genotypes. Furthermore, both could be induced to produce sclerotia, a prerequisite for sex in the black aspergilli. In A. niger crosses involving one isolate, 8-160, sclerotia were found to contain red-pigmented structures following incubation for 6 months. Putative ascospores were then visualized from these structures through the use of high-resolution imaging techniques. This is the first report of ascospore formation by A. niger, indicating a potential sexual cycle in this species. However, it was not possible to induce germination of the putative ascospore progeny or demonstrate that recombination had occurred. In parallel, studies were undertaken to examine possible recombination within progeny from a sexual cross of A. sclerotiicarbonarius. SNP analysis provided evidence of recombination, adding new insights to the ‘sexual revolution’ within supposed asexual fungi.

Secondly, it was assessed whether the technique of bulk segregant analysis (BSA) combined with next-generation sequencing could determine the genetic basis of unknown forms of azole resistance in A. fumigatus. This species is the primary agent of aspergillosis, with a mortality rate of >50% and shows increasing resistance to current treatment therapies. Here, the sexual cycle of A. fumigatus was exploited for the first time in BSA studies to identify a linked region of the genome containing an itraconazole resistance mutation. The efficiency of BSA was then increased by manipulation of the number of backcrosses in tandem with an increased number of progeny in each DNA pool. This identified smaller linked regions of the genome containing fewer candidate genes. Therefore, BSA may provide a valuable technique to identify novel antifungal resistance genes in A. fumigatus.

Thirdly, investigations were made to assess the presence and bioactivity of fungal sex hormones as a potential basis for novel disease control. The homothallic species Neosartorya (Aspergillus) fischeri was used as a model organism. Lipid extractions were made from mating and asexual cultures and bioassayed for activity. Extracts from mating cultures were found to increase production of cleistothecia in a dose-dependent manner. However, the sexual extract had no clear impact on asexual conidiation in N. fischeri, and no clear developmental impacts on closely related species. Therefore, it was uncertain whether the extract contained a true sex morphogen.

Finally, the role of oxygenase family ppo and lox genes in sexual reproduction of A. fumigatus was explored. Using deletion and overexpression mutants, it was found that ΔppoB and OE: loxAB strains showed increased cleistothecia development in mating cultures, whereas any alteration of expression of ppoA, ppoC, loxA or loxB genes led to absence of cleistothecia production. This demonstrates for the first time the important role of oxygenase family genes in sexual reproduction of A. fumigatus, indicating a possible wider role in regulation of sexual reproduction in heterothallic pezizomycete species.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Dyer, Paul S.
Avery, Simon
Keywords: Fungal sexual cycle; Antifungal resistance genes; Fungal sex hormones
Subjects: Q Science > QK Botany
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 56806
Depositing User: Ashton, George
Date Deposited: 26 Apr 2022 08:49
Last Modified: 26 Apr 2022 08:50
URI: http://eprints.nottingham.ac.uk/id/eprint/56806

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