Invasive aspergillosis (IA) is associated with high mortality rates and can be difficult and expensive to treat with current drugs. The drugs used to treat IA are also associated with undesirable, and often severe, side-effects of the patient. The main causative agent of this disease is the opportunistic pathogen Aspergillus fumigatus. This study identifies genes which play a role in a fungal-specific type of programmed cell death (PCD) in A. fumigatus, known as heterokaryon incompatibility. The development of drugs specifically targeting the products of these genes could lead to fewer side-effects than those arising from currently available anti-fungal drugs. The drug amphotericin B is currently used to treat IA and has been shown to induce an apoptotic-like phenotype in A. fumigatus; however, the sterols targeted are present in both fungal and mammalian cell membranes. HI is a fungal-specific self/non-self recognition system that results in rapid compartmentalisation and cell death of hyphal fusion sites if the two fusing fungi are not genetically compatible. The HI system could be exploited as a novel drug target against invasive fungal pathogens through targeting a component of the molecular pathway to induce cell death. In contrast to current drugs, novel drugs could target HI components to induce PCD without affecting non-desirable targets that cause side-effects. The non-self recognition systems used by Neurospora crassa, Aspergillus Nidulans and Podospora anserina are the well characterised, and they each differ significantly in their modes of action. BLAST searches found 30 homologues of HI genes from other the systems of characterised species in A. fumigatus, with 8 containing the fungal-specific het domain. The first assay to determine whether disruption of het genes could affect HI was to observe the barrage phenotype between incompatible A. fumigatus individuals. However, there was no barrage visible as the leading edge of colonies stopped growing when in close proximity to another colony. Instead, nitrate non-utilising (Nit) A. fumigatus mutant strains were generated using chlorate and pair-wise crosses of 46 environmentally and clinically isolates on nitrate-containing media resulted in the formation of 16 viable heterokaryons. All of the heterokaryons fell into exclusive compatibility groups where no intergroup crossing was possible. Homologous recombination was used to disrupt five of the identified het domain genes with gene replacement cassettes, generated through fusion-PCR, in an akuB(KU80Delta) A. fumigatus strain. The mutant strains displayed both detrimental growth on standard agar growth media and reduced ability to recognise non-self strains. Full and partial heterokaryons were formed during intergroup pair-wise compatibility crosses using the mutants and strains that the akuB(KU80Delta) parent strain was previously incompatible with. This was followed with a non-bias approach of gene disruption using the Fusarium oxysporum impala160 transposable element in a Nit A. fumigatus mutant. Inducing transposon mutagenesis through exposure to low temperature generated a mutant library of spores in which the transposon had disrupted different open reading frames at different locations across the A. fumigatus genome. The mutant spore library was also screened for the ability to form viable intergroup heterokaryons with strains belonging to different compatibility groups. PCR recovery and DNA sequencing was able to identify the locus of impala160 in three isolates able to form viable heterokaryons. The sequences revealed the transposable element had disrupted the same gene, AFUA_2G05070, in each of the three isolates. This gene encodes an uncharacterised conserved hypothetical protein which may be a critical component for non-self recognition in A. fumigatus HI, and a potential target for novel anti-fungal drugs to induce PCD.

The sexual functions of the mating type gene (mt) of Neurospora crassa include specification of mating identity (Shear and Dodge, 1927) and perithecial maturation (Griffiths and DeLange, 1978; Staben and Yanofsky, 1990). The gene also acts as a vegetative incompatibility locus, so that A + a heterokaryons (Beadle and Coonradt, 1944) or A/a duplication strains (Newmeyer and Taylor, 1967) grow poorly or not at all. An intriguing question regarding the mating type gene is this: How does it control both the switch between somatic and meiotic events and heterokaryon incompatibility? Several research groups (Glass, et al., 1990; Staben and Yanofsky, 1990) are presently studying the sexual functions of the mating type genes. I present a study of the incompatibility function. Two experiments were performed. The first was a search for new suppressors of mating type-associated incompatibility, which resulted in the identification of seven new suppressors, none of which was allelic with the one known suppressor, tol. The second was the comparison of growth rates of a mating type mutant (fertile, heterokaryon compatible) in a mixed mating type heterokaryon and in a mixed mating type duplication to determine whether or not cytoplasmic incompatibility is separable from nuclear incompatibility. The results obtained suggest that the mating type mutant, am33, eliminates heterokaryon incompatibility without eliminating nuclear incompatibility. The search for suppressors was attempted in order to define more of the genes involved in A/a incompatibility. The analysis of heterokaryon versus nuclear incompatibility was done to investigate the cellular interactions involved in A/a incompatibility. / Science, Faculty of / Botany, Department of / Graduate

Toxigenic Aspergillus species produce mycotoxins that are carcinogenic, hepatotoxic and teratogenic immunosuppressing agents in both human and animals. Kenya frequently experiences outbreaks of aflatoxicosis with the worst occurring in 2004, which resulted in 125 deaths. This study sought to find possible reasons for frequent aflatoxicosis outbreaks in Kenya by isolating Aspergillus flavus strains from maize kernels sampled from different climatic regions of Kenya. Using diagonal transect random sampling, maize kernels were collected from Makueni, Homa Bay, Nandi, and Kisumu regions. The genetic diversity and variation among the isolates was examined by characterising the strains according to morphology, phenotype, vegetative compatible groups and molecular systematics. Selected atoxigenic and aflatoxigenic A. flavus isolates were also further analysed for aflatoxin production potential using quantitative real-time PCR and various bioanalytical techniques. The influence of the maize lines grown in Kisumu, Homa Bay, Nandi and Makueni region on A. flavus infection and aflatoxin production was also examined and served as the basis for an in vitro biocontrol assay. Out of 37 isolates identified, nitrate non-utilizing auxotroph’s complementation test revealed 20 vegetative compatibility groups. These groups were further designated using the prefix ʻʻKVCGʼʼ, where ʻʻKʼʼ represented Kenya and consequently assigned numbers 1 to 20 based on our findings. KVCG14 and KVCG15 had highest distribution frequency (n = 13; 10.8 %). The distribution of the L, S and S/L- morphotypes across the regions were 57 % (n = 21); 7 % (n = 3) and 36 % (n = 13) respectively. The phylogenetic analysis exhibited high diversity of A. flavus isolates from Makueni. ITS1 and ITS2 markers did not reveal significant information within intraspecies speciation of A. flavus. Furthermore, a unique isolate (KSM015) was identified that had characteristics of S-morphotype, but produced both aflatoxins B and G. Coconut agar medium (CAM) assay, TLC, HPLC and LCMS/MS analyses confirmed the presence or absence of aflatoxins in selected toxigenic and atoxigenic isolates. qPCR analysis revealed aflP, aflS, aflR and aflO transcripts as the most upregulated genes across the tested isolates whereas false detection of aflD gene transcript was observed in both induced and uninduced A. flavus isolates. Diversity Index (H) analyses ranged from 0.11 (Nandi samples) to 0.32 (Kisumu samples). Heterokaryon compatibility ranged from 33 % (for the Makueni samples, n = 3) to 67 % (Nandi samples, n = 6). The KDV1 maize line was more sensitive to A. flavus infection in comparison to GAF4. We also tested the biocontrol of atoxigenic isolates to inhibit toxin production by aflatoxigenic strains on infected maize kernels. It was shown that the atoxigenic strain (KSMO12) could inhibit the aflatoxigenic strain (KSM014) depending on the atoxigenic concentration during infection. To our knowledge, this is the first reported study for A. flavus genetic diversity, variation and distribution in Nandi, Homa Bay and Kisumu regions in comparison to and could assist researchers in the selection of biocontrol strategies to mitigate aflatoxin contamination, especially in Makueni and neighbouring regions.