Putative Polyketide Synthase and Laccase Genes for Biosynthesis of Aurofusarin in Gibberella zeae

Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.     

The velvet gene, FgVe1, affects fungal development and positively regulates trichothecene biosynthesis and pathogenicity in Fusarium graminearum

Trichothecenes are a group of toxic secondary metabolites produced mainly by Fusarium graminearum (teleomorph: Gibberella zeae) during the infection of crop plants, including wheat, maize, barley, oats, rye and rice. Some fungal genes involved in trichothecene biosynthesis have been shown to encode regulatory proteins. However, the global regulation of toxin biosynthesis is still enigmatic. In addition to the production of secondary metabolites belonging to the trichothecene family, F. graminearum produces the red pigment aurofusarin. The gene regulation underlying the production of aurofusarin is not well understood. The velvet gene (veA) is conserved in various genera of filamentous fungi. Recently, the veA gene from Aspergillus nidulans has been shown to be the key component of the velvet complex regulating development and secondary metabolism. Using blast analyses, we identified the velvet gene from F. graminearum, FgVe1. Disruption of FgVe1 causes several phenotypic effects. However, the complementation of this mutant with the FgVe1 gene restores the wild-type phenotypes. The in vitro phenotypes include hyperbranching of the mycelium, suppression of aerial hyphae formation, reduced hydrophobicity of the mycelium and highly reduced sporulation. Our data also show that FgVe1 modulates the production of the aurofusarin pigment and is essential for the expression of Tri genes and the production of trichothecenes. Pathogenicity studies performed on flowering wheat plants indicate that FgVe1 is a positive regulator of virulence in F. graminearum.     

Two novel classes of enzymes are required for the biosynthesis of aurofusarin in Fusarium graminearum

Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated γ-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to- aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.     

Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum)

Polyketides are a class of secondary metabolites that exhibit a vast diversity of form and function. In fungi, these compounds are produced by large, multidomain enzymes classified as type I polyketide synthases (PKSs). In this study we identified and functionally disrupted 15 PKS genes from the genome of the filamentous fungus Gibberella zeae. Five of these genes are responsible for producing the mycotoxins zearalenone, aurofusarin, and fusarin C and the black perithecial pigment. A comprehensive expression analysis of the 15 genes revealed diverse expression patterns during grain colonization, plant colonization, sexual development, and mycelial growth. Expression of one of the PKS genes was not detected under any of 18 conditions tested. This is the first study to genetically characterize a complete set of PKS genes from a single organism.     

Characterization of two polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae

Zearalenone, a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. Zearalenone is a polyketide derived from the sequential condensation of multiple acetate units by a polyketide synthase (PKS), but the genetics of its biosynthesis are not understood. We cloned two genes, designated ZEA1 and ZEA2, which encode polyketide synthases that participate in the biosynthesis of zearalenone by Gibberella zeae (anamorph Fusarium graminearum). Disruption of either gene resulted in the loss of zearalenone production under inducing conditions. ZEA1 and ZEA2 are transcribed divergently from a common promoter region. Quantitative PCR analysis of both PKS genes and six flanking genes supports the view that the two polyketide synthases make up the core biosynthetic unit for zearalenone biosynthesis. An appreciation of the genetics of zearalenone biosynthesis is needed to understand how zearalenone is synthesized under field conditions that result in the contamination of grain.     

The regulatory gene nit-2 of Neurospora crassa complements a nnu mutant of Gibberella zeae (Fusarium graminearum).

Summary The nnu mutant of Gibberella zeae (= Fusarium graminearum) is unable to catabolize many of the nitrogen sources utilized by its wild-type parent, and may have suffered a mutation in the major nitrogen regulatory locus. Transformation of this mutant with the major nitrogen regulatory gene from Neurospora crassa, nit-2, restored the wild-type phenotype, thus confirming that the nnu mutation is in the major nitrogen regulatory locus of G. zeae. Our results are consistent with the premise of conservation of the structure of regulatory factors and suggest the possibility that functional DNA homologues of this regulatory element occur across a broad range of ascomycetous fungi.     

A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A.

Fumonisins are toxins associated with several mycotoxicoses and are produced by the maize pathogen Gibberella fujikuroi mating population A (MP-A). Biochemical analyses indicate that fumonisins are a product of either polyketide or fatty acid biosynthesis. To isolate a putative polyketide synthase (PKS) gene involved in fumonisin biosynthesis, we employed PCR with degenerate PKS primers and a cDNA template prepared from a fumonisin-producing culture of G. fujikuroi. Sequence analysis of the single PCR product and its flanking DNA revealed a gene (FUM5) with a 7.8-kb coding region. The predicted FUM5 translation product was highly similar to bacterial and fungal Type I PKSs. Transformation of a cosmid clone carrying FUM5 into G. fujikuroi enhanced production in three strains and restored wild-type production in a fumonisin nonproducing mutant. Disruption of FUM5 reduced fumonisin production by over 99% in G. fujikuroi MP-A. Together, these results indicate that FUM5 is a PKS gene required for fumonisin biosynthesis.     

禾谷镰孢菌β-微管蛋白基因克隆及其与多菌灵抗药性关系的分析

应用3对引物,从禾谷镰孢菌(Gibberella zeae)对多菌灵(MBC)的敏感菌株(MBC^R)和田间及室内诱导抗药性菌株(MBC^R)中扩增β-微管蛋白基因。该基因全长1631bp,包含3个内含子,编码447aa,与其他常见植物病原丝状真菌β-微管蛋白基因的氨基酸同源性达95．12％～99．30％。MBC^R和MBC^R菌株核苷酸序列分析表明,MBCR菌株未发生任何位点的突变,说明G．zeae对MBC的抗药性机制并非像其他丝状真菌一样由β-微管蛋白198位氨基酸突变所致。     

小麦赤霉病镰孢菌毒素的生物合成及其分子调控

禾谷镰刀菌是小麦赤霉病的主要致病菌,其真菌次生代谢产生的单端孢霉烯类B型毒素,如雪腐镰刀菌烯醇(nivalenol,NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和其它乙酰化衍生物等污染小麦籽粒后对人畜健康构成威胁。综述了近年来国内外对小麦赤霉病镰孢菌单端孢霉烯类B型毒素生物合成的主要途径及分子调控研究进展,对毒素合成过程中的重要调控基因如TRI5、TRI7和TRI13在农业中的应用进行了阐述。     

A novel F-box protein involved in sexual development and pathogenesis in Gibberella zeae

Gibberella zeae is an ascomyceteous fungus that causes serious diseases in cereal crops. Severe epidemics require strains that are virulent and that can reproduce sexually. We characterized an insertional mutant (designated ZH436) with a pleiotropic defect in both traits, and identified a novel F-box protein gene encoding FBP1 (F-box protein 1) that is similar to fungal F-box proteins including Saccharomyces cerevisiae Grr1, a well-characterized component of the Skp1-Cullin-F-box protein (SCF(Grr1)) E3 ligase complex required for protein degradation. FBP1 also can bind both S. cerevisiae Skp1 protein, the other component of the SCF(Grr1) complex, and its G. zeae sequence homologue SKP1. Two putative protein interacting domains in FBP1 are essential for in vivo function. FBP1 and ScGRR1 are not so interchangeable between S. cerevisiae and G. zeae, but FBP1 can partially complement several defects of a yeast grr1 deletion mutant. Functional analyses confirmed that FBP1 is required for several phenotypes including both sexual development and virulence in G. zeae; the phenotype of DeltaFBP1 strains is different from those of null mutants for F-box proteins in other filamentous fungi as well as from S. cerevisiae grr1Delta strains. Thus, FBP1 is a versatile F-box protein that presumably participates in the formation of the SCF(FBP1) complex that probably controls the ubiquitin-mediated degradation of proteins involved in sexual reproduction and virulence important for disease development by G. zeae.     

On the trail of a cereal killer: recent advances in Fusarium graminearum pathogenomics and host resistance

The ascomycete fungal pathogen Fusarium graminearum (sexual stage: Gibberella zeae) causes the devastating head blight or scab disease on wheat and barley, and cob or ear rot disease on maize. Fusarium graminearum infection causes significant crop and quality losses. In addition to roles as virulence factors during pathogenesis, trichothecene mycotoxins (e.g. deoxynivalenol) produced by this pathogen constitute a significant threat to human and animal health if consumed in respective food or feed products. In the last few years, significant progress has been made towards a better understanding of the processes involved in F. graminearum pathogenesis, toxin biosynthesis and host resistance mechanisms through the use of high-throughput genomic and phenomic technologies. In this article, we briefly review these new advances and also discuss how future research can contribute to the development of sustainable plant protection strategies against this important plant pathogen.     

Media for identification of Gibberella zeae and production of F-2-(Zearalenone).

Media are described for the isolaton of Fusarium graminearum in the perithecial state, Gibberella zeae, and for the production of F-2 (zearalenone) by Fusarium species. On soil extract-corn meal agar isolated medium, G. Zeae produced perithecia in 9 to 14 days under a 12-h photoperiod. Species of Fusarium were screened for F-2 production on a liquid medium. From strains that produced F-2, the yields, from stationary cultures of G. zeae and F. culmorum after 12 days of incubation, ranged from 22 to 86 mg/liter. Three strains produced no F-2. Glumatic acid, starch, yeast extract,and the proper ratio of medium volume-to-flask volume were necessary for F-2 synthesis.