GAC1, a gene encoding a putative GTPase-activating protein, regulates bikaverin biosynthesis in Fusarium verticillioides

Fusarium verticillioides (teleomorph Gibberella moniliformis) is an ascomycete known to produce a variety of secondary metabolites, including fumonisins, fusaric acid and bikaverin. These metabolites are synthesized when the fungus is under stress, notably nutrient limitations. To date we have limited understanding of the complex regulatory process associated with fungal secondary metabolism. In this study we generated a collection of F. verticillioides mutants by using REMI (restriction enzyme mediated integration) mutagenesis and in the process identified a strain, R647, that carries a mutation in a gene designated GAC1. Mutation in the GACI locus, which encodes a putative GTPase activating protein, resulted in the increased production of bikaverin, suggesting that GAC1 is negatively associated with bikaverin biosynthesis. Complementation of R647 with the wildtype GAC1 gene restored the bikaverin production level to that of the wild-type progenitor, demonstrating that gac1 mutation was directly responsible for the overproduction of bikaverin. We also demonstrated that AREA, encoding global nitrogen regulator, and PKS4, encoding polyketide synthase, are downstream genes that respectively are regulated positively and negatively by GAC1. Our results suggest that GAC1 plays an important role in signal transduction regulating bikaverin production in F. verticillioides.     

Sequence variability in the FUM1 gene of Fusarium verticillioides strains

Fumonisins are mycotoxins, produced mainly by Fusarium verticillioides, that are potentially carcinogenic to humans and toxic to animals. Synthesis of these toxins is directed by a cluster of 15 genes, among which FUM1 is the largest; it encodes a polyketide synthase. This enzyme probably catalyzes the synthesis of a polyketide that forms a large portion of the fumonisin structure. In this study, 27 strains possessing the FUM1 gene, as determined by polymerase chain reaction, were analyzed. A portion of the FUM1 gene was amplified and sequenced from 6 of 27 Brazilian strains isolated from corn and sorghum. The sequence similarity for the six F. verticillioides strains was almost 100%.     

The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides

Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity.     

Identification of gene clusters associated with fusaric acid, fusarin, and perithecial pigment production in Fusarium verticillioides

The genus Fusarium is of concern to agricultural production and food/feed safety because of its ability to cause crop disease and to produce mycotoxins. Understanding the genetic basis for production of mycotoxins and other secondary metabolites (SMs) has the potential to limit crop disease and mycotoxin contamination. In fungi, SM biosynthetic genes are typically located adjacent to one another in clusters of co-expressed genes. Such clusters typically include a core gene, responsible for synthesis of an initial chemical, and several genes responsible for chemical modifications, transport, and/or regulation. Fusarium verticillioides is one of the most common pathogens of maize and produces a variety of SMs of concern. Here, we employed whole genome expression analysis and utilized existing knowledge of polyketide synthase (PKS) genes, a common cluster core gene, to identify three novel clusters of co-expressed genes in F. verticillioides. Functional analysis of the PKS genes linked the clusters to production of three known Fusarium SMs, a violet pigment in sexual fruiting bodies (perithecia) and the mycotoxins fusarin C and fusaric acid. The results indicate that microarray analysis of RNA derived from culture conditions that induce differential gene expression can be an effective tool for identifying SM biosynthetic gene clusters.     

Heterologous expression of two trichothecene P450 genes in Fusarium verticillioides

Fusarium graminearum Z-3639 and F. sporotrichioides NRRL3299 produce the trichothecene mycotoxins 15-acetyldeoxynivalenol and T-2 toxin, respectively. These toxins differ in oxygenation at C-4, C-7, and C-8. In F. sporotrichioides, Tri1 (FsTri1) controls C-8 hydroxylation. To determine the function of an apparent F. graminearum Tri1 (FgTri1) homolog, both FsTri1 and FgTri1 genes were heterologously expressed in the trichothecene-nonproducing species F. verticillioides by fusing the Tri1 coding regions to the promoter of the fumonisin biosynthetic gene FUM8. FsTri1 and FgTri1 have been partially characterized by disruption analysis, and the results from these analyses suggest that FsTri1 most likely has a single function but that FgTri1 may have two functions. Transgenic F. verticillioides carrying the FsTri1 (FvF8FsTri1) converted exogenous isotrichodermin and calonectrin to 8-hydroxyisotrichodermin and 8-hydroxycalonectrin, respectively. Transgenic F. verticillioides carrying FgTri1 (FvF8FgTri1) converted isotrichodermin to a mixture of 7-hydroxyisotrichodermin and 8-hydroxyisotrichodermin but converted calonectrin to a mixture of 7-hydroxycalonectrin, 8-hydroxycalonectrin, and 3,15-diacetyldeoxynivalenol. A fourth compound, 7,8-dihydroxycalonectrin, was identified in large-scale F. verticillioides FvF8FgTri1 cultures fed isotrichodermin. Our results indicate that FgTri1 controls both C-7 and C-8 hydroxylation but that FsTri1 controls only C-8 hydroxylation. Our studies also demonstrate that F. verticillioides can metabolize some trichothecenes by adding an acetyl group to C-3 or by removing acetyl groups from C-4 or C-15. In addition, wild-type F. verticillioides can convert 7,8-dihydroxycalonectrin to 3,15-diacetyldeoxynivalenol.     

Segregation of secondary metabolite biosynthesis in hybrids of Fusarium fujikuroi and Fusarium proliferatum

Fusarium fujikuroi and Fusarium proliferatum are two phylogenetically closely related species of the Gibberella fujikuroi species complex (GFC). In some cases, strains of these species can cross and produce a few ascospores. In this study, we analyzed 26 single ascospore isolates of an interspecific cross between F. fujikuroi C1995 and F. proliferatum D4854 for their ability to produce four secondary metabolites: gibberellins (GAs), the mycotoxins fusarin C and fumonisin B(1), and a family of red polyketides, the fusarubins. Both parental strains contain the biosynthetic genes for all four metabolites, but differ in their ability to produce these metabolites under certain conditions. F. fujikuroi C1995 produces GAs and fusarins, while F. proliferatum D4854 produces fumonisins and fusarubins. The segregation amongst the progeny of these traits is not the expected 1:1 Mendelian ratio. Only eight, six, three and three progeny, respectively, produce GAs, fusarins, fumonisin B(1) and fusarubins in amounts similar to those synthesized by the producing parental strain. Beside the eight highly GA(3)-producing progeny, some of the progeny produce small amounts of GAs, predominantly GA(1), although these strains contain the GA gene cluster of the non-GA-producing F. proliferatum parental strain. Some progeny had recombinant secondary metabolite profiles under the conditions examined indicating that interspecific crosses can yield secondary metabolite production profiles that are atypical of the parent species.     

Recent advances in genome mining of secondary metabolite biosynthetic gene clusters and the development of heterologous expression systems in Aspergillus nidulans

Fungi are prolific producers of secondary metabolites (SMs) that show a variety of biological activities. Recent advances in genome sequencing have shown that fungal genomes harbor far more SM gene clusters than are expressed under conventional laboratory conditions. Activation of these “silent” gene clusters is a major challenge, and many approaches have been taken to attempt to activate them and, thus, unlock the vast treasure chest of fungal SMs. This review will cover recent advances in genome mining of SMs in Aspergillus nidulans. We will also discuss current updates in gene annotation of A. nidulans and recent developments in A. nidulans as a molecular genetic system, both of which are essential for rapid and efficient experimental verification of SM gene clusters on a genome-wide scale. Finally, we will describe advances in the use of A. nidulans as a heterologous expression system to aid in the analysis of SM gene clusters from other fungal species that do not have an established molecular genetic system.     

Draft genome and secondary metabolite biosynthetic gene clusters of Streptomyces sp. strain 196

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