Biosynthesis of Nodulisporic Acids: A Multifunctional Monooxygenase Delivers a Complex and Highly Branched Array.

Nodulisporic acids (NAs) are structurally complex potent antiinsectan indole diterpenes. We previously reported the biosynthetic gene cluster for these metabolites in Hypoxylon pulicicidum and functionally characterised the first five steps of the biosynthetic pathway. Here we reveal a highly complex biosynthetic array, furnishing multiple end products through expression of cluster components in Penicillium paxilli. We show that seven additional cluster-encoded gene products comprise the biosynthetic machinery that elaborate precursor NAF in this highly branched pathway. The combined action of these enzymes delivers 37 NA congeners including four major end products, NAA, NAA1 , NAA2 and NAA4 . The plethora of intermediates arises due to modification of the carboxylated prenyl tail by a single promiscuous P450 monooxygenase, NodJ, a pivotal branchpoint enzyme which produces four distinct biosynthetic products giving rise to the complex metabolic grid that characterises NA biosynthesis.

Chitin Deacetylases Are Required for Epichloë festucae Endophytic Cell Wall Remodeling During Establishment of a Mutualistic Symbiotic Interaction with Lolium perenne.

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network and also grows as an epiphyte. However, little is known about the cell wall-remodeling mechanisms required to avoid host defense and maintain intercalary growth within the host. Here, we use a suite of molecular probes to show that the E. festucae cell wall is remodeled by conversion of chitin to chitosan during infection of L. perenne seedlings, as the hyphae switch from free-living to endophytic growth. When hyphae transition from endophytic to epiphytic growth, the cell wall is remodeled from predominantly chitosan to chitin. This conversion from chitin to chitosan is catalyzed by chitin deacetylase. The genome of E. festucae encodes three putative chitin deacetylases, two of which (cdaA and cdaB) are expressed in planta. Deletion of either of these genes results in disruption of fungal intercalary growth in the intercellular spaces of plants infected with these mutants. These results establish that these two genes are required for maintenance of the mutualistic symbiotic interaction between E. festucae and L. perenne.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Regulation of host-infection ability in the grass-symbiotic fungus Epichloë festucae by histone H3K9 and H3K36 methyltransferases.

Recent studies have identified key genes that control the symbiotic interaction between Epichloë festucae and Lolium perenne. Here we report on the identification of specific E. festucae genes that control host infection. Deletion of setB, which encodes a homologue of the H3K36 histone methyltransferase Set2/KMT3, reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae ΔclrD mutant, which lacks the gene encoding the homologue of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect L. perenne. Alleles that complement the culture and plant phenotypes of both mutants also complement the histone methylation defects. Co-inoculation of either ΔsetB or ΔclrD with the wild-type strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild type. Deletion of crbA, which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae.

Regulation of subtelomeric fungal secondary metabolite genes by H3K4me3 regulators CclA and KdmB.

Studies on the regulation of fungal secondary metabolism highlight the importance of histone H3K4 methylation regulators Set1, CclA (Ash2) and KdmB (KDM5), but it remains unclear whether these proteins act by direct modulation of H3K4me3 at the target genes. In filamentous fungi, secondary metabolite genes are frequently located near telomeres, a site where H3K4 methylation is thought to have a repressive role. Here we analyzed the role of CclA, KdmB and H3K4me3 in regulating the subtelomeric EAS and LTM cluster genes in Epichloë festucae. Depletion of H3K4me3 correlated with transcriptional activation of these genes in ΔcclA, similarly enrichment of H3K4me3 correlated with transcriptional repression of the genes in ΔkdmB which was accompanied by significant reduction in the levels of the agriculturally undesirable lolitrems. These transcriptional changes could only be explained by the alterations in H3K4me3 and not in the subtelomerically-important marks H3K9me3/K27me3. However, H3K4me3 changes in both mutants were not confined to these regions but occurred genome-wide, and at other subtelomeric loci there were inconsistent correlations between H3K4me3 enrichment and gene repression. Our study suggests that CclA and KdmB are crucial regulators of secondary metabolite genes, but these proteins likely act via means independent to, or in conjunction with the H3K4me3 mark.

Complex epigenetic regulation of alkaloid biosynthesis and host interaction by heterochromatin protein I in a fungal endophyte-plant symbiosis.

Epichloë festucae forms mutualistic symbiotic interactions with grasses of the Lolium and Festuca genera. Protection from insect and mammalian herbivory are the best-documented host benefits of these associations. The two main classes of anti-mammalian alkaloids synthesized by E. festucae are the ergot alkaloids and indole diterpenes, of which ergovaline and lolitrems are the principal terminal products. Synthesis of both metabolites require multiple gene products encoded by clusters of 11 genes located at the subtelomeric regions of chromosomes I and III respectively. These loci are essentially unexpressed in axenic culture but among the most highly expressed genes in planta. We show here that heterochromatin 1 protein (HepA) is an important component of the regulatory machinery that maintains these loci in a silent state in culture. Deletion of this gene led to derepression of eas and ltm gene expression under non-symbiotic culture conditions. Although there was no obvious culture phenotype, RNAseq analysis revealed that around 1000 genes were differentially expressed in the ΔhepA mutant compared to wild type with just one-third upregulated. Inoculation of the ΔhepA mutants into seedlings of Lolium perenne led to a severe host interaction phenotype characterized by a reduction in tiller length but an increase in tiller number. Hyphae within the leaves of these associations were much more abundant in the intercellular spaces of the leaves and aberrantly colonized the vascular bundles. This physiological change was accompanied by a dramatic change in the transcriptome with around 900 genes differentially expressed, with two thirds of these upregulated. This major physiological change was accompanied by a decrease in ltm gene expression and loss of the ability to synthesize lolitrems. These results show that HepA has an important role in controlling the chromatin state of these sub-telomeric secondary metabolite genes, including their symbiosis-specific regulation.

Molecular and cellular analysis of the pH response transcription factor PacC in the fungal symbiont Epichloë festucae.

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 resulting in activation of the PacC transcription factor at alkaline pH. The role of PacC in regulating fungal virulence and pathogenicity has been described in several pathogenic fungi but to date not in a symbiotic fungus. Epichloë festucae is a biotrophic fungal endophyte that forms a stable mutualistic interaction with Lolium perenne. In this study, pacC deletion (ΔpacC) and dominant active (pacC(C)) mutants were generated in order to study the cellular roles of PacC in E. festucae. Deletion of pacC resulted in increased sensitivity of the mutant to salt-stress but surprisingly did not affect the ability of the mutant to grow under alkaline pH conditions. Alkaline pH was observed to induce conidiation in wild-type E. festucae but not in the ΔpacC mutant. On the other hand the pacC(C) mutant had increased conidiation at neutral pH alone. Null pacC mutants had no effect on the symbiotic interaction with ryegrass plants whereas the pacC(C) mutant increased the tiller number. Examination of the growth of the pacC(C) mutant in the plant revealed the formation of aberrant convoluted hyphal structures and an increase in hyphal breakage, which are possible reasons for the altered host interaction phenotype.

Epichloë hybrida, sp. nov., an emerging model system for investigating fungal allopolyploidy.

Endophytes of the genus Epichloë (Clavicipitaceae, Ascomycota) frequently occur within cool-season grasses and form interactions with their hosts that range from mutualistic to antagonistic. Many Epichloë species have arisen via interspecific hybridization, resulting in species with two or three subgenomes that retain all or nearly all of their original parental genomes, a process termed allopolyploidization. Here, we characterize Epichloë hybrida, sp. nov., a mutualistic species that has increasingly become a model system for investigating allopolyploidy in fungi. The Epichloë species so far identified as the closest known relatives of the two progenitors of E. hybrida are E. festucae var. lolii and E. typhina. We confirm that the nuclear genome of E. hybrida contains two homeologs of most protein-coding genes from E. festucae and E. typhina, with genome-wide gene expression analysis indicating a slight bias in overall gene expression from the E. typhina subgenome. Mitochondrial DNA is detectable only from E. festucae, whereas ribosomal DNA is detectable only from E. typhina. Inheriting ribosomal DNA from just one parent might be expected to preferentially favor interactions with ribosomal proteins from the same parent, but we find that ribosomal protein genes from both parental subgenomes are nearly all expressed equally in E. hybrida. Finally, we provide a comprehensive set of resources for this model system that are intended to facilitate further study of fungal hybridization by other researchers.