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LaeA regulates mycelium growth by mediating H3K9 methylation in Myceliophthora thermophila [WGBS]

ABSTRACT: The low efficiency of fungal growth and substrate utilization is a barrier to high yield and productivity of target products during fungal fermentation. However, elaborated and intricate regulatory mechanisms of fungal growth remains inconclusive. In this study, we found deletion of putative methyltransferase LaeA enhanced sugar consumption and fungal growth rate of M. thermophila. The exploration of the mechanism of LaeA regulation revealed that transcription factor (Cre-1, Grf-1, Grf-2 and Grf-3) acted as negative repressor of intracellular metabolism, of which expression was indirectly regulated by LaeA. Moreover, higher gluconeogenesis activity is crucial for elevated fugal growth rate and PCK might be the core notes of metabolism network relative to fungal vegetative growth. Further in vivo global histone methylation assay and in vitro biochemical analysis declared LaeA was a methyltransferase with histone H3 lysine 9 (H3K9) as the substrate and regulated growth properties by directly modulating H3K9 methylation levels. ChIP-seq analysis displayed significantly reduced H3K9me3 in region of pck locus in strain ΔlaeA, implying methylation modification of pck might be the way of LaeA regulating growth phenotypes. Additionally, whole-genome bisulfite sequencing (WGBS) analysis demonstrated methylation of cytosines (5mC) in genomic DNA was carried out and correlated to the methylation of H3K9 modified by LaeA. Taken together, our research declared LaeA possessed methyltransferase activity, targeting H3K9 and participated in regulation of fugal properties, including on sugar consumption, mycelium growth, cellulase production, secondary metabolism, and oxidative stress tolerance, providing new insights into the epigenetics of filamentous fungi.

ORGANISM(S): Thermothelomyces thermophilus ATCC 42464

PROVIDER: GSE213575 | GEO | 2023/04/12

REPOSITORIES: GEO

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Project description:Genetic and molecular evidence to support the hypothesis that fungal secondary metabolites play a significant role in protecting the fungi against fungivory is scarce. We investigated the impact of fungal secondary metabolites on transcript regulation of stress related expressed sequence tags (ESTs) of the Collembola Folsomia candida feeding on mixed vs. single diets. Aspergillus nidulans wildtype (WT; Ascomycota) able to produce secondary metabolites including sterigmatocystin (ST) and a knockout mutant with reduced secondary metabolism (A. nidulans ?LaeA) were combined with the high quality fungus Cladosporium cladosporioides as mixed diets or offered as single diets. We hypothesized that (i) A. nidulans WT triggers more genes associated with stress responses compared to the A. nidulans ?laeA strain with suppressed secondary metabolism, (ii) C. cladosporioides causes significantly different transcript regulation than the A. nidulans strains ?laeA and WT, and (iii) mixed diets will cause significantly different transcript expression levels than single diets. All three hypotheses are generally supported despite the fact that many functions of the affected ESTs are unknown. The results bring molecular evidence for the existence of a link between fungal secondary metabolites and responses in springtails supporting the hypothesis that fungal secondary metabolites act as a shield against fungivory. Twenty-three day old Folsomia candida were fed ad libitum for five days to fungal cuts respectively Cladosporium cladosporoides, Aspergillus nidulans WT, Aspergillus nidulans ?LaeA and two mixed diets of C.cladosporoides/A. nidulans WT (mix 1) and C. cladosporoides/A. nudlans ?LaeA (mix2) respectively. Four biological replicates were used for every treatment and a dye swap was used with the Cy3/Cy5 labels. This resulted in 20 samples which were analysed in 10 hybridisations executed in an interwoven loop design. The C. cladosporoides diet was used as the reference in the data analysis.

Project description:Aspergillus flavus is a common saprophyte and opportunistic pathogen producing aflatoxin (AF) and many other secondary metabolites. 5-Azacytidine (5-AC), a derivative of nucleoside cytidine, is widely used for studies in epigenetics and cancer biology as an inactivator of DNA methyltransferase and is also used for studying secondary metabolism in fungi. Our previous studies showed that 5-AC affects development and inhibits AF production in A. flavus, and that A. flavus lacks DNA methylation. How this common DNA methyltransferase inhibitor affects development and AF production is not clear. In this study, we applied an RNA-Seq approach to elucidate the mechanism of 5-ACM-bM-^@M-^Ys effect on A. flavus. In our current study, we identified 240 significantly differently expressed (Q-value<0.05) genes after 5-AC treatment, including two backbone genes in secondary metabolite clusters #27 and #35, which are involved in development or survival of sclerotia. With 5-AC treatment, about three quarters of the genes in the AF biosynthetic gene cluster in A. flavus were down-regulated to a certain degree. Strikingly, at least two genes aflI and aflLa, were completely inhibited. Interestingly, several genes involved in fungal development were down-regulated, especially veA, which is a gene that encodes protein bridges VelB and LaeA. This result supports the hypothesis that 5-AC affects development and AF production through weakening or even interrupting the connection between VelB and LaeA and then causing dysregulation of the expression pattern of genes involved in development and secondary metabolism. Our results improved the A. flavus genome annotation, provided a comprehensive view of the transcriptome of A. flavus responding to 5-AC and confirmed that fungal development and secondary metabolism are co-regulated. In additon, the RNA-Seq data of another sample treated with gallic acid was used to improve A. flavus genome annotation. mRNA of Aspergillus flavus cultured in three different culture media PDB, PDB+5-AC(5-Azacytidine),and PDB+GA(gallic acid) was subjected to sequence independently.

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LaeA regulates mycelium growth by mediating H3K9 methylation in Myceliophthora thermophila [WGBS]

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