Browsing by Author "Lukito, Yonathan"

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    Epigenetic regulation of Epichloë festucae secondary metabolite biosynthesis and symbiotic interaction with Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2017) Lukito, Yonathan
    Histone methylation is one of several epigenetic layers for transcriptional regulation. Most studies on the importance of this histone modification in regulating fungal secondary metabolite gene expression and pathogenicity have focussed on the role of histone methyltransferases, while few studies have focussed on the role of histone demethylases that catalyse the reversal of the modification. Epichloë festucae (Ascomycota) is an endophyte that forms a mutualistic interaction with perennial ryegrass. The fungus contributes to the symbiosis by the production of several classes of secondary metabolites, these have anti-insect and/or anti-mammalian activity. The EAS and LTM clusters in E. festucae are located subtelomerically and contain the biosynthetic genes for two of these important metabolites which are only synthesised in planta. Thus, in the host plant these genes are highly expressed, but they are tightly silenced in culture conditions. Previous study has shown that histone H3K9 and H3K27 methylation and their corresponding histone methyltransferases are important for this process. In this study, the role of histone lysine demethylases (KDMs) in regulating these genes and the symbiotic interaction is described. Eight candidate histone demethylases (Jmj1-Jmj8) were identified in E. festucae, among these proteins are homologues of mammalian KDM4, KDM5, KDM8, JMDJ7, and N. crassa Dmm-1. The genes for the proteins were overexpressed in E. festucae and histone methylation levels were determined in the strains. Overexpression of the genes was not observed to cause any change to the culture and symbiotic phenotypes of the fungus. Western blot analysis subsequently identified one of the proteins, KdmB, as the histone H3K4me3 demethylase. Further analysis by ChIP- and RT-qPCR showed that demethylation of H3K4me3 by KdmB at the eas/ltm genes is crucial for the activation of these genes in planta. The full expression of several other telomeric genes was similarly found to require KdmB. On the other hand, the COMPASS H3K4 methyltransferase complex subunit CclA that is required for H3K4 trimethylation in E. festucae represses the eas/ltm genes in culture conditions by maintaining H3K4me3 levels at the loci. Thus, these findings suggest a repressive role for H3K4me3 at these subtelomeric secondary metabolite loci and are consistent with the role of H3K4me3 in yeast telomeric silencing. Disruption of kdmB did not affect the symbiotic interaction of E. festucae with the host grass but severely reduced the levels of lolitrem B, an animal neurotoxin. At the same time, the levels of ergovaline, another animal toxin, and peramine, an insect feeding deterrent, were not affected. Therefore, disruption or inhibition of KdmB may also serve as a promising approach for future endophyte improvement programmes. The E. festucae homologue of KDM8 (an H3K36me2 demethylase), Jmj4, was further investigated in this study but no H3K6 demethylase activity was found for the protein. Both disruption and overexpression of the gene encoding Jmj4 similarly had no effect on the culture and symbiotic phenotypes of E. festucae. However, deletion of setB, encoding the homologue of yeast Set2 (H3K36 methyltransferase) specifically reduced histone H3K36me3 levels in E. festucae. This contrasts with deletion of Set2 in other fungi which affected H3K36 mono-, di- and trimethylation. The ΔsetB mutant was severely impeded in development, and was unable to establish infection of the host plant. Introduction of the wild-type setB gene reversed these phenotypes. This study shows that H3K4 trimethylation controlled by CclA and KdmB is an important regulator of subtelomeric secondary metabolite genes in E. festucae but not for the symbiotic interaction of the fungus with perennial ryegrass. On the other hand, the histone H3K36 methyltransferase SetB specifically controls H3K36 trimethylation in E. festucae and is required for normal vegetative growth and ability of the fungus to infect the host plant.
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    Investigation into the role of PacC in Epichloë festucae development and symbiosis with perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2014) Lukito, Yonathan
    In order to survive and adapt to the environment, it is imperative for fungi to be able to sense and respond to changes in extracellular pH conditions. In ascomycetes, sensing of extracellular pH is mediated by the Pal pathway which is activated by alkaline pH. The signal is subsequently relayed to changes in gene expression by activation of the transcription factor PacC, which is known to regulate various fungal metabolic pathways; including ion tolerance, cell-wall integrity and secondary metabolism. The role of PacC in regulating fungal virulence and pathogenicity has also been studied in several pathogenic fungi, but to date not in a symbiotic fungus. Epichloë festucae is a biotrophic fungal endophyte that forms a stable symbiosis with the perennial ryegrass Lolium perenne. In this mutualistic interaction, secondary metabolites are produced by the fungus that confer bioprotection for the host, and the host in turn provides nutrients and a means of dissemination for the fungus. In this study, deletion (ΔpacC; acid-mimicking) and constitutively active (pacCCA; alkaline-mimicking) mutants were generated to study the cellular roles of PacC in E. festucae. Deletion of pacC resulted in increased sensitivity to salt-stress and reduction in aerial hyphae formation, but did not affect the ability of the mutant to grow under alkaline pH conditions. The pacCCA mutant on the other hand showed greater sensitivity to cell-wall and temperature stresses, and was able to grow under both acidic and alkaline conditions. Aberrant intrahyphal hyphae formation and abnormal conidiation were additionally observed in the pacCCA mutant in culture, but hyphal fusion was unaffected. The expression of secondary metabolite genes both in culture and in planta was largely unaffected in both ΔpacC and pacCCA mutants, and expression analysis of genes known to be pH- and PacC-dependent in other fungi showed that these genes were generally pH- and PacC-independent in E. festucae. The deletion and constitutive active pacC mutants were still able to sense and modify extracellular pH. Deletion of pacC did not affect the endophyte-host interaction, but ryegrass plants infected with the pacCCA mutant were mildly hypertillered. Further examination of the growth of the pacCCA mutant in the plant revealed formation of aberrant convoluted hyphal structures and an increase in hyphal breakage; possible reasons for the observed host plant phenotype. This study provides novel insights into the role of PacC in the agriculturally important endophyte, E. festucae. In this fungus, PacC regulates salt tolerance and cell-wall integrity, but not secondary metabolism and growth at non-neutral pH conditions. The results also show that PacC is involved in the symbiotic interaction between E. festucae and perennial ryegrass but is not crucial in the establishment and maintenance of the symbiosis.