Early expression of aflatoxin-like dothistromin genes in the forest pathogen Dothistroma septosporum

The forest pathogen Dothistroma septosporum produces the polyketide dothistromin, a mycotoxin very similar in structure to versicolorin B, a precursor of aflatoxin (AF). Dothistromin is a broad-range toxin and possibly involved in red-band needle blight disease. As the role of dothistromin in the disease is unknown the expression of dothistromin genes was studied to reveal clues to its function. Although the genes of AF and dothistromin biosynthesis are very similar, this study revealed remarkable differences in the timing of their expression. Secondary metabolites, like AF, are usually produced during late exponential phase. Previously identified dothistromin genes, as well as a newly reported versicolorin B synthase gene, vbsA, showed high levels of expression during the onset of exponential growth. This unusual early expression was also seen in transformants containing a green fluorescent protein (GFP) gene regulated by a dothistromin gene promoter, where the highest GFP expression occurred in young mycelium. Two hypotheses for the biological role of dothistromin are proposed based on these results. The study of dothistromin genes will improve current knowledge about secondary metabolite genes, their putative biological roles, and their regulation.     

A fragmented aflatoxin-like gene cluster in the forest pathogen Dothistroma septosporum

The polyketide toxin dothistromin is very similar in structure to the aflatoxin precursor, versicolorin B. Dothistromin is made by a pine needle pathogen, Dothistroma septosporum, both in culture and in planta. Orthologs of aflatoxin biosynthetic genes have been identified that are required for dothistromin biosynthesis in D. septosporum. In contrast to the situation in aflatoxin-producing fungi where 25 aflatoxin biosynthetic and regulatory genes are tightly clustered in one region of the genome, the dothistromin gene cluster is fragmented. Three mini-clusters of dothistromin genes have been identified, each located on a 1.3-Mb chromosome and each grouped with non-dothistromin genes. There are no obvious patterns of repeated sequences or transposon relics to suggest recent recombination events. Most dothistromin genes within the mini-clusters are co-regulated, suggesting that coordinate control of gene expression is achieved despite this unusual arrangement of secondary metabolite biosynthetic genes.     

Dothistroma pini, a Forest Pathogen, Contains Homologs of Aflatoxin Biosynthetic Pathway Genes

Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (dotA), oxidase (dotB), major facilitator superfamily transporter (dotC), and thioesterase (dotD). A D. pini dotA mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that dotA is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the dotA mutant confirms that the dotA gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four dot genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene, ddhA, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control Dothistroma needle blight.     

A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of ~1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.     

Dothistroma pini,a forest pathogen,contains homologs of aflatoxin biosynthetic pathway genes

Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (dotA), oxidase (dotB), major facilitator superfamily transporter (dotC), and thioesterase (dotD). A D. pini dotA mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that dotA is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the dotA mutant confirms that the dotA gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four dot genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene, ddhA, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control Dothistroma needle blight.     

The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism

Fungi possess genetic systems to regulate the expression of genes involved in complex processes such as development and secondary metabolite biosynthesis. The product of the velvet gene veA, first identified and characterized in Aspergillus nidulans, is a key player in the regulation of both of these processes. Since its discovery and characterization in many Aspergillus species, VeA has been found to have similar functions in other fungi, including the Dothideomycete Mycosphaerella graminicola. Another Dothideomycete, Dothistroma septosporum, is a pine needle pathogen that produces dothistromin, a polyketide toxin very closely related to aflatoxin (AF) and sterigmatocystin (ST) synthesized by Aspergillus spp. Dothistromin is unusual in that, unlike most other secondary metabolites, it is produced mainly during the early exponential growth phase in culture. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of AF/ST in Aspergillus spp. To begin to address this question, a veA ortholog was identified and its function analyzed in D. septosporum. Inactivation of the veA gene resulted in reduced dothistromin production and a corresponding decrease in expression of dothistromin biosynthetic genes. Expression of other putative secondary metabolite genes in D. septosporum such as polyketide synthases and non-ribosomal peptide synthases showed a range of different responses to loss of Ds-veA. Asexual sporulation was also significantly reduced in the mutants, accompanied by a reduction in the expression of a putative stuA regulatory gene. The mutants were, however, able to infect Pinus radiata seedlings and complete their life cycle under laboratory conditions. Overall this work suggests that D. septosporum has a veA ortholog that is involved in the control of both developmental and secondary metabolite biosynthetic pathways.     

Enzyme reactions and genes in aflatoxin biosynthesis

Aflatoxins are highly toxic and carcinogenic substances mainly produced by Aspergillus flavus and Aspergillus parasiticus. Sterigmatocystin is a penultimate precursor of aflatoxins and also a toxic and carcinogenic substance produced by many species, including Aspergillus nidulans. Recently, the majority of the enzyme reactions involved in aflatoxin/sterigmatocystin biosynthesis have been clarified, and the genes encoding the enzymes have been isolated. Most of the genes constitute a large gene cluster in the fungal genome, and their expression is mostly regulated by a product of the regulatory gene aflR. This review will summarize the enzymatic steps and the genes in aflatoxin/sterigmatocystin biosynthesis.     

Evolutionary relics dominate the small number of secondary metabolism genes in the hemibiotrophic fungus Dothistroma septosporum

Fungal secondary metabolites have important functions for the fungi that produce them, such as roles in virulence and competition. The hemibiotrophic pine needle pathogen Dothistroma septosporum has one of the lowest complements of secondary metabolite (SM) backbone genes of plant pathogenic fungi, indicating that this fungus produces a limited range of SMs. Amongst these SMs is dothistromin, a well-characterised polyketide toxin and virulence factor that is required for expansion of disease lesions in Dothistroma needle blight disease. Dothistromin genes are dispersed across six loci on one chromosome, rather than being clustered as for most SM genes. We explored other D. septosporum SM genes to determine if they are associated with gene clusters, and to predict what their likely products and functions might be. Of nine functional SM backbone genes in the D. septosporum genome, only four were expressed under a range of in planta and in culture conditions, one of which was the dothistromin PKS backbone gene. Of the other three expressed genes, gene knockout studies suggested that DsPks1 and DsPks2 are not required for virulence and attempts to determine a functional squalestatin-like SM product for DsPks2 were not successful. However preliminary evidence suggested that DsNps3, the only SM backbone gene to be most highly expressed in the early stage of disease, appears to be a virulence factor. Thus, despite the small number of SM backbone genes in D. septosporum, most of them appear to be poorly expressed or dispensable for virulence in planta. This work contributes to a growing body of evidence that many fungal secondary metabolite gene clusters might be non-functional and may be evolutionary relics.     

Chromatin‐level regulation of the fragmented dothistromin gene cluster in the forest pathogen Dothistroma septosporum

Genes required for fungal secondary metabolite production are usually clustered, co‐regulated and expressed in stationary growth phase. Chromatin modification has an important role in co‐regulation of secondary metabolite genes. The virulence factor dothistromin, a relative of aflatoxin, provided a unique opportunity to study chromatin level regulation in a highly fragmented gene cluster that is switched on during early exponential growth phase. We analysed three histone modification marks by ChIP‐qPCR and gene deletion in the pine pathogen Dothistroma septosporum to determine their effects on dothistromin gene expression across a time course and at different loci of the dispersed gene cluster. Changes in gene expression and dothistromin production were associated with changes in histone marks, with higher acetylation (H3K9ac) and lower methylation (H3K9me3, H3K27me3) during early exponential phase at the onset of dothistromin production. But while H3K27me3 directly influenced dothistromin genes dispersed across chromosome 12, effects of H3K9 acetylation and methylation were orchestrated mainly through a centrally located pathway regulator gene DsAflR. These results revealed that secondary metabolite production can be controlled at the chromatin‐level despite the genes being dispersed. They also suggest that patterns of chromatin modification are important in adaptation of a virulence factor for a specific role in planta.     

Molecular and physiological aspects of aflatoxin and sterigmatocystin biosynthesis by Aspergillus tamarii and A. ochraceoroseus

Until recently, only three species (Aspergillus flavus, A. parasiticus and A. nomius) have been widely recognized as producers of aflatoxin. In this study we examine aflatoxin production by two other species, A. tamarii and A. ochraceoroseus, the latter of which also produces sterigmatocystin. Toxin-producing strains of A. tamarii and A. ochraceoroseus were examined morphologically, and toxin production was assayed on different media at different pH levels using thin layer chromatography and a densitometer. Genomic DNA of these two species was probed with known aflatoxin and sterigmatocystin biosynthesis genes from A. flavus, A. parasiticus and A. nidulans. Under the high stringency conditions, A. tamarii DNA hybridized to all four of the A. flavus and A. parasiticus gene probes, indicating strong similarities in the biosynthetic pathway genes of these three species. The A. ochraceoroseus DNA hybridized weakly to the A. flavus and A. parasiticus verB gene probe, and to two of the three A. nidulans probes. These data indicate that, at the DNA level, the aflatoxin and sterigmatocystin biosynthetic pathway genes for A. ochraceoroseus are somewhat different from known pathway genes. Received: 21 May 1999 / Received revision: 17 November 1999 / Accepted: 3 December 1999     

Characterization of aflatoxin-producing fungi outside of Aspergillus section Flavi

Most aspergilli that produce aflatoxin are members of Aspergillus section Flavi, however isolates of several Aspergillus species not closely related to section Flavi also have been found to produce aflatoxin. Two of the species, Aspergillus ochraceoroseus and an undescribed Aspergillus species SRRC 1468, are morphologically similar to members of Aspergillus section Circumdati. The other species have Emericella teleomorphs (Em. astellata and an undescribed Emericella species SRRC 2520) and are morphologically distinctive in having ascospores with large flanges. All these aflatoxin-producing isolates were from tropical zones near oceans, and none of them grew on artificial media at 37 C. Aflatoxins and sterigmatocystin production were quantified by high-pressure liquid chromatography (HPLC) and confirmed by HPLC-mass spectrometry (LC-MS) detection. Phylogenetic analyses were conducted on these four species using A. parasiticus and Em. nidulans, (which produce aflatoxin and the aflatoxin precursor sterigmatocystin, respectively) for comparison. Two aflatoxin/sterigmatocystin biosynthesis genes and the beta tubulin gene were used in the analyses. Results showed that of the new aflatoxin-producers, Aspergillus SRRC 1468 forms a strongly supported clade with A. ochraceoroseus as does Emericella SRRC 2520 with Em. astellata SRRC 503 and 512.     

Molecular biology of mycotoxin biosynthesis

Mycotoxins are secondary metabolites produced by many important phytopathogenic and food spoilage fungi including Aspergillus, Fusarium and Penicillium species. The toxicity of four of the most agriculturally important mycotoxins (the trichothecenes, and the polyketide-derived mycotoxins; aflatoxins, fumonisins and sterigmatocystin) are discussed and their chemical structure described. The steps involved in the biosynthesis of aflatoxin and sterigmatocystin and the experimental techniques used in the cloning and molecular characterisation of the genes involved in the pathway are described in detail. The biosynthetic genes involved in the fumonisin and trichothecene biosynthetic pathways are also outlined. The potential benefits gained from an increased knowledge of the molecular organisation of these pathways together with the mechanisms involved in their regulation are also discussed.     

Aflatoxin biosynthetic pathway: Elucidation by using blocked mutants of Aspergillus parasiticus

Two mutant strains of Aspergillus parasiticus, both deficient in aflatoxin production, were used to elucidate the biosynthetic pathway of this mycotoxin. One of the mutants, A. parasiticus ATCC 24551, was capable of accumulating large amounts of averufin, and the other, A. parasiticus 1-11-105 wh-1, accumulated versicolorin A. The averufin producing mutant efficiently converted ¹⁴C-labeled versiconal acetate, versicolorin A, and sterigmatocystin into aflatoxin B₁ and G₁, indicating that averufin preceded these compounds in the aflatoxin biosynthetic pathway. In the presence of dichlorvos (dimethyl 2,2-dichlorovinyl phosphate), a known inhibitor of aflatoxin biosynthesis, the conversion of versicolorin A and sterigmatocystin was unaffected, but the conversion of versiconal acetate was markedly inhibited. The mutant accumulating versicolorin A incorporated ¹⁴C-labeled acetate, averufin, and versiconal acetate into versicolorin A. In the presence of dichlorvos, however, the major conversion product was versiconal acetate. This strongly suggested that dichlorvos inhibited the conversion step of versiconal acetate into versicolorin A. This mutant resumed production of aflatoxin B₁ if sterigmatocystin was added to the resting cell cultures, indicating that the mutant was blocked at the enzymatic step catalyzing the conversion of versicolorin A into sterigmatocystin, and as a result was incapable of aflatoxin production. The experimental evidence is thus provided for the involvement and interrelationship of three anthraquinones (averufin, versiconal acetate, and versicolorin A) and a xanthone (sterigmatocystin) in aflatoxin biosynthesis. A pathway for the biosynthesis of aflatoxin B₁ is proposed to be: acetate →→→ averufin → versiconal acetate → versicolorin A → sterigmatocystin → aflatoxin B₁.     

Increased frequency of sister-chromatid exchange induced by dothistromin in CHO cells and human lymphocytes

Dothistromin is a metabolite produced by Dothistromin pini and Cercospora arachidicola. The latter fungus is a pathogen of the peanuts and thus the mycotoxin may be a contaminant of foodstuffs. Dothistromin induces a dose-dependent increase in sister-chromatid exchange frequency in Chinese hamster ovary cells and stimulated human lymphocytes. The increased frequency in human lymphocytes seen with dothistromin is significantly higher among lymphocytes from smokers compared with those from non-smokers.     

Genetic regulation of aflatoxin biosynthesis: From gene to genome

Aflatoxins are notorious toxic secondary metabolites known for their impacts on human and animal health, and their effects on the marketability of key grain and nut crops. Understanding aflatoxin biosynthesis is the focus of a large and diverse research community. Concerted efforts by this community have led not only to a well-characterized biosynthetic pathway, but also to the discovery of novel regulatory mechanisms. Common to secondary metabolism is the clustering of biosynthetic genes and their regulation by pathway specific as well as global regulators. Recent data show that arrangement of secondary metabolite genes in clusters may allow for an important global regulation of secondary metabolism based on physical location along the chromosome. Available genomic and proteomic tools are now allowing us to examine aflatoxin biosynthesis more broadly and to put its regulation in context with fungal development and fungal ecology. This review covers our current understanding of the biosynthesis and regulation of aflatoxin and highlights new and emerging information garnered from structural and functional genomics. The focus of this review will be on studies in Aspergillus flavus and Aspergillus parasiticus, the two agronomically important species that produce aflatoxin. Also covered will be the important contributions gained by studies on production of the aflatoxin precursor sterigmatocystin in Aspergillus nidulans.     

Further toxic properties of the fungal metabolite dothistromin

The toxicity of the fungal phytotoxin dothistromin (l) to microorganisms, its lysis of human red blood corpuscles and beetroot tissue, and its unexpectedly selective inhibition of radicle elongation for Trigonella foenum-graecum were strongly light-dependent. Dothistromin was also toxic to Artemia salina but without requiring light activation. It was not active as a wilt or necrosis toxin, possible because of its ready adsorption onto external plant tissue.