Sexual development and sclerotial formation in the black aspergilli

Brett, Paul Michael (2023) Sexual development and sclerotial formation in the black aspergilli. PhD thesis, University of Nottingham.

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The black aspergilli are a group of species from the genus Aspergillus that are typically characterised by the production of dark brown or black conidia. This group is formally referred to as section Nigri and includes the species Aspergillus niger and Aspergillus tubingensis, which are of biotechnological importance due to their production of organic acids such as citric acid, enzymes such as amylases, and their emerging utility in bioremediation. The group was formally believed to be comprised of solely asexual species. However, there have been recent successes in elucidating sexual cycles in A. tubingensis and A. sclerotiicarbonarius. These studies demonstrated that the production of sclerotia is a necessary prerequisite to sexual reproduction in the black aspergilli with cleistothecia (ascomata) developing within the interior of the sclerotia, linked to a sexual Aspergillus sexual morph known as saitoa (superseding the use of the sexual morph petromyces). Sclerotia are hardened, melanised and metabolically active structures that are resistant to adverse environmental conditions.

The reporting of sexual reproduction in the black aspergilli have encouraged efforts to try and induce sex in A. niger, as this would allow strain improvement via sexual breeding and also provide a valuable genetic tool for this economically important species. Prior to the present study, conditions had been described to produce sclerotia in A. niger under laboratory conditions. In addition, analyses of the MAT locus of A. niger had revealed a proto-heterothallic nature, with isolates of two separate mating types found in global collections, which were defined by the presence of either a MAT1-1-1 α-box- or MAT1-1-2 HMG-encoding gene at the MAT locus, HMG and α-box proteins are key transcriptional regulators for sex.

The present study aimed to gain knowledge about processes governing sclerotial formation and mating in the black aspergilli, with the ultimate goal of inducing sexual reproduction in A. niger. Work was first completed to reproduce and optimise the conditions used to produce sclerotia in the black aspergilli with a focus on A. niger. Media and environmental conditions were identified which could be used to reliably induce formation of sclerotia, although the conditions were species specific and there was much variation in numbers of sclerotia depending on the strain used. Secondly, genetic characterisation of two transcription factors, termed SclR and SclB, thought to be involved in sclerotia production in the aspergilli was also completed. SclR was shown to be required, but not essential, for sclerotia formation in A. niger whilst deletion of sclB resulted in enhanced production of sclerotia in the majority but not all strains of A. niger, Investigations into production of sclerotia were complemented by studies examining whether any chemical factor(s) with a role in sclerotia formation could be uncovered. No lipid morphogen was found despite extracting from mated cultures over a wide period of maturation of sclerotia.

Attempts were then made to induce sexual reproduction in A. niger, together with a series of other Aspergillus species as controls. A mating-type diagnostic was applied to allow expedient PCR determination of mating type for isolates of A. niger to assist with crossing efforts by identifying putative compatible MAT1-1 and MAT1-2 strains. The accuracy of the diagnostic was confirmed through later genome sequencing of representative isolates. These studies were complemented with the use of scanning electron microscopy (SEM) to examine sclerotia for the possible presence of ascomata, confocal microscopy to examine changes within sclerotia during ‘mating’, and the development of flow cytometry techniques to apply cell sorting to separate different spore types (i.e. conidia and ascospores). Sexual reproduction was indeed induced in certain of the control Aspergillus species. However, all studies failed to yield direct observation of the expected products of sexual recombination, namely cleistothecia or ascospores, in any crosses involving A. niger despite extensive efforts involving the crossing of multiple MAT1-1 and MAT1-2 isolates under a range of conditions and using a variety of crossing techniques. However, there were observations of formation a distinct red structure within the sclerotia of certain ‘mated’ cultures that were absent from control unmated axenic cultures of A. niger. This was complemented by the identification via SEM of a rare and distinct tissue type at the centre of sclerotia, as well as distinct channels within certain sclerotia. Fluorescent strains of MAT1-1 and MAT1-2 mating type of A. niger were created and confocal microscopy used to reveal that germinating conidia of one mating type could produce hyphae with the potential for ingrowth into sclerotia of the opposite mating type. Finally, studies were completed to analyse whether a suite of meiosis-specific genes were transcriptionally upregulated during this ‘mating’. This confirmed that during the ’mating’ process, certain meiosis genes (dmc1, rec8, hop2 and msh4) as well as sclR (a regulator of sclerotia formation) were indeed upregulated.

Taken as whole, results from the present study add to increasing evidence of an extant but cryptic sexual cycle in Aspergillus niger.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Dyer, Paul S.
Brock, Matthias
Keywords: Aspergillus; Aspergillus niger; Sclerotium (Mycelium); Sexual cycle
Subjects: Q Science > QK Botany
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 74379
Depositing User: Brett, Paul
Date Deposited: 13 Dec 2023 04:40
Last Modified: 13 Dec 2023 04:40

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