ETD

• Fol4287
• meiosis-related genes
• NCRAD21
• phenotypic characterization
• REC8
• SGE1
• SPO11

Fusarium oxysporum is a pathogenic fungus that infects hundreds of plant species and is known to reproduce sexually only rarely. The Fol4287 is a reference strain of Fusarium oxysporum f.sp. lycopersici, a soil-borne fungal pathogen that causes tomato wilt disease. The genome of Fol4287 was one of the first Fusarium genomes to be completely sequenced and thoroughly annotated (Ma et al., Nature, 2010). Moreover, this strain is widely available and genetically well-characterized and allows for highly reproducible experiments. These characteristics make Fol4287 a suitable choice for this study.
The meiosis detection toolkit is a set of meiotic genes that serve as reliable markers for the presence of meiosis. The identification of these meiotic genes enables the application of comparative genomics to all eukaryotes. The study aims to investigate the functions (both novel or conserved) of meiosis-related genes in Fusarium oxysporum f. sp lycopersici (Fol4287) by generating null mutants and phenotypically characterising the resulting strains during vegetative growth. The genes under investigation include SPO11, RAD21 paralogs (REC8 and NCRAD21), and SGE1.
This follows up on a study conducted by Pareek et al. (2019), which proposed “Alternative functional RAD21 Paralogs in Fusarium oxysporum.” The main work of the thesis is on phenotypic characteristics of RAD21 paralogs (REC8, NCRAD21). In addition, the study also focuses on the generation of knock-out mutants and phenotypic characterization of the SPO11 and SGE1 genes. All the mutant strains were analysed using various assays, including viability (CFUs), PI staining, growth rate, cellular size and complexity, cell cycle, sensitivity to abiotic stress and antifungals, and cellophane penetration capacity.
Firstly, REC8 (RAD21) is the sister chromatid cohesion is regulated by cohesion, a multisubunit complex comprised of RAD21, which links SMC1 and SMC3 subunits. In meiosis, RAD21 is largely replaced by its meiosis-specific paralog REC8, which remains bound until the onset of anaphase II. In addition, according to the study by Patreek et al. (2019), they demonstrated that NCRAD21 functions as part of a cohesion complex by observing that the non-conversed region retains some classic α-kleisin domains (Smc1, Smc3 binding) (Bergerat et al., 1997; Keeney et al.,1997). The rec8Δ and ncrad21Δ were obtained from Pareek et al. (2019). Based on the results of various assays assessing vegetative growth, we concluded that ncrad21Δ has a significant effect on most aspects of vegetative development. The ncrad21Δ mutant exhibited reduced cell viability, as indicated by a lower number of viable cells. However, it demonstrated a higher growth rate and enhanced metabolic activity. Furthermore, ncrad21Δ induced alterations in the cell cycle, especially leading to an accumulation of cells in the G2 phase. No notable differences in sensitivity to abiotic stress or antifungal agents were observed. Finally, the ncrad21Δ strain displayed a reduced capacity for penetration compared to the WT. On the other hand, the rec8Δ also played a role during the vegetative growth of Fol4287, although its effect was less pronounced than that of the ncrad21Δ. For instance, rec8Δ significantly affected the fungal proliferation, promoting the accumulation in the G2 phase.
SPO11 is a dispensable gene in meiosis whose protein is a homolog of an archaebacterial topoisomerase VI subunit. SPO11 is required for DNA double-strand breaks, which are essential to initiate meiotic recombination (Hearing et al., 2003), which is unexpected in mitotic growth. The finding from this study raises questions about the roles of SPO11 in the introduction of DNA double-strand breaks and potentially its involvement in the activation of the DNA damage checkpoint. The effects were associated with changes in metabolic activity, a slightly higher proportion of cells in the G2 phase, as well as alterations in cell size and cell complexity, as observed from the results.
The final work from this study also outlines the future work on SGE1. We obtained the knock-out mutant strains of SGE1 through the protoplast transformation, and our subsequent efforts will focus on the phenotypic characterisation of sge1Δ mutant strains. SGE1 is not a meiotic-specific gene; it was chosen because SGE1 (Six gene expression 1) was identified as showing homology to the transcriptional regulator WOR1 of Candida albicans (Michiele et al., 2009). In Candida albicans, WOR1 is one of the key genes regulating the phenotypic switch from the white form to the opaque form, enabling cells to become mating-competent (Hull et al., 2000; Magee et al., 2000; Miller et al., 2002).
Overall, this study assumes that the meiosis-related genes REC8 and NCRAD21 in Fusarium oxysporum have alternative functional roles in mitosis. The SPO11 should be investigated further to better understand its potential functions during mitosis. Finally, ongoing work on the phenotypic characterization of SGE1.