FUM and BIK gene expression contribute to describe fumonisin and bikaverin synthesis in Fusarium verticillioides.

Fusarium verticillioides is a maize pathogen that produces toxic secondary metabolites, including fumonisins and bikaverin. The regulation of biosynthetic gene expression and the production of these metabolites are not fully understood and in this study we investigated the influence of water activity (0.955 and 0.990) on the expression of 5 genes (FUM3-FUM8-FUM13-FUM14 and BIK1) in F. verticillioides strains after 14 and 21days incubation. Fumonisin production and biosynthetic gene expression were greatest at a(w)=0.990, and the same trend was observed for bikaverin production, and BIK1 expression. FUM3 and FUM14 were the most highly expressed genes and were positively correlated with the production of FB(1), FB(2) and FB(3). When FUM14 is more highly expressed than FUM3 the amount of FB(3) quantified is higher with respect to FB(1); this could be explained by the role of FUM3 in the hydroxylation of FB(3) to FB(1).

LC-MS/MS method for the determination of the fungal pigment bikaverin in maize kernels as an indicator of ear rot.

Bikaverin is a polyketide-derived pigment produced by multiple species of the fungus Fusarium, some of which can cause ear and kernel rot of maize. A method was developed for the analysis of bikaverin by high-performance liquid chromatography (LC) coupled to electrospray ionisation tandem mass spectrometry (MS/MS). The quantitative nature of the LC-MS/MS method was demonstrated over a range of concentrations of bikaverin in maize. For spike-recovery experiments utilising maize spiked with bikaverin to a level 5 µg g⁻¹ of maize, the measured recovery (%) was 70.6 ± 10.4. Based on the utilised method, the limit of detection (based on a signal-to-noise ratio (S/N) > 3) was better than 0.5 µg g⁻¹ from bikaverin spiked into uncontaminated ground maize. Further, the limit of quantitation (LOQ) was 3 µg g⁻¹ (based on S/N > 10) from bikaverin spiked into ground maize. The method was applied to assess contamination of maize with bikaverin following inoculation of developing maize ears with Fusarium verticillioides under agricultural field conditions.

Involvement of ZFR1 of Fusarium verticillioides in kernel colonization and the regulation of FST1, a putative sugar transporter gene required for fumonisin biosynthesis on maize kernels.

Fumonisins comprise a class of carcinogenic mycotoxins produced by Fusarium verticillioides during colonization of maize kernels. In previous work, we identified ZFR1, which is predicted to encode a Zn(II)2Cys6 zinc finger transcription factor required for fumonisin B(1) (FB(1)) production during growth on kernels. In this study, we characterized the role of ZFR1 in colonizing maize kernels and inducing FB(1) biosynthesis. The ZFR1 deletion strain (Deltazfr1) grew approximately 2.5-fold less than the wild-type on endosperm tissue and a variety of other carbon sources, including glucose and amylopectin. However, the Deltazfr1 strain displayed higher alpha-amylase activity and expression of genes involved in starch saccharification than the wild-type, thus indicating that the reduced growth of the Deltazfr1 strain was not due to inhibition of amylolytic enzymes. In the wild-type strain, expression of six genes encoding putative sugar transporters was significantly greater on endosperm tissue than on germ tissue, and expression of at least three of the six genes was negatively affected by disruption of ZFR1. Intriguingly, disruption of FST1 had no effect on growth, kernel colonization or kernel pH but decreased FB(1) production by approximately 82% on maize kernels. Based on these findings, we hypothesize that ZFR1 controls FB(1) biosynthesis by regulating genes involved in the perception or uptake of carbohydrates.

Genomic analysis of Fusarium verticillioides.

Fusarium verticillioides (teleomorph Gibberella moniliformis) can be either an endophyte of maize, causing no visible disease, or a pathogen-causing disease of ears, stalks, roots and seedlings. At any stage, this fungus can synthesize fumonisins, a family of mycotoxins structurally similar to the sphingolipid sphinganine. Ingestion of fumonisin-contaminated maize has been associated with a number of animal diseases, including cancer in rodents, and exposure has been correlated with human oesophageal cancer in some regions of the world, and some evidence suggests that fumonisins are a risk factor for neural tube defects. A primary goal of the authors' laboratory is to eliminate fumonisin contamination of maize and maize products. Understanding how and why these toxins are made and the F. verticillioides-maize disease process will allow one to develop novel strategies to limit tissue destruction (rot) and fumonisin production. To meet this goal, genomic sequence data, expressed sequence tags (ESTs) and microarrays are being used to identify F. verticillioides genes involved in the biosynthesis of toxins and plant pathogenesis. This paper describes the current status of F. verticillioides genomic resources and three approaches being used to mine microarray data from a wild-type strain cultured in liquid fumonisin production medium for 12, 24, 48, 72, 96 and 120h. Taken together, these approaches demonstrate the power of microarray technology to provide information on different biological processes.

Functional characterization, sequence comparisons and distribution of a polyketide synthase gene required for perithecial pigmentation in some Fusarium species.

Polyketides are a structurally diverse class of secondary metabolites produced by bacteria, fungi, plants and animals. The fungal genus Fusarium includes agronomically important plant pathogenic and mycotoxin-producing species and produces numerous polyketides. The study further characterized a polyketide synthase-encoding gene (PKS3 = PGL1) that was previously identified in F. graminearum and F. verticillioides. Disruption of the F. verticillioides PGL1 indicated that it is required for the production of the dark pigment in perithecial walls, as previously shown in F. graminearum. A third PGL1 orthologue was identified in the genomic sequence of N. haematococca (anamorph F. solani f. sp. pisi). Analysis of the carboxy-terminal end of the deduced PGL1 protein indicated that it had a functional domain related to dehydrogenases/reductases that is sometimes present in non-ribosomal peptide synthetases. Comparison of the genomic regions flanking PGL1 in F. graminearum, F. verticillioides and N. haematococca revealed that the extent of gene synteny in this region was greater between F. graminearum and F. verticillioides than between either of these species and N. haematococca. Southern blot analysis indicated that PGL1 occurs widely within the genus Fusarium including species with no known sexual stage.

Biosynthesis ofFusarium mycotoxins and genomics ofFusarium verticillioides.

Analyses of mycotoxin biosynthetic genes inFusarium indicate that interspecies variation in trichothecene structure can result from differences in gene function and interspecies variation in fumonisin production/non-production can result from differences in the presence/absence of genes. Such variation is not always correlated with phylogenetic relationships of species as determined by sequencing primary metabolic genes; distantly related species can share the same mycotoxin biosynthetic genotype and resulting phenotype, while more closely related species can differ. These findings provide further evidence that the evolution of mycotoxin biosynthesis inFusarium has not always been congruent with the evolution of species.

Aspergillus nidulans mutants defective in stc gene cluster regulation.

The genes involved in the biosynthesis of sterigmatocystin (ST), a toxic secondary metabolite produced by Aspergillus nidulans and an aflatoxin (AF) precursor in other Aspergillus spp., are clustered on chromosome IV of A. nidulans. The sterigmatocystin gene cluster (stc gene cluster) is regulated by the pathway-specific transcription factor aflR. The function of aflR appears to be conserved between ST- and AF-producing aspergilli, as are most of the other genes in the cluster. We describe a novel screen for detecting mutants defective in stc gene cluster activity by use of a genetic block early in the ST biosynthetic pathway that results in the accumulation of the first stable intermediate, norsolorinic acid (NOR), an orange-colored compound visible with the unaided eye. We have mutagenized this NOR-accumulating strain and have isolated 176 Nor(-) mutants, 83 of which appear to be wild type in growth and development. Sixty of these 83 mutations are linked to the stc gene cluster and are likely defects in aflR or known stc biosynthetic genes. Of the 23 mutations not linked to the stc gene cluster, 3 prevent accumulation of NOR due to the loss of aflR expression.

Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus.

Under limiting growth conditions, Aspergillus nidulans produces a carcinogenic secondary metabolite related to aflatoxin and called sterigmatocystin (ST). The genes for ST biosynthesis are co-ordinately regulated and are all found within an approximately 60-kilobase segment of DNA. One of the genes within this region is predicted to encode a CX2CX6CX6CX2CX6CX2 zinc binuclear cluster DNA-binding protein that is related to the Aspergillus flavus and Aspergillus parasiticus aflatoxin regulatory gene aflR. Deletion of the A. nidulans aflR homolog resulted in an inability to induce expression of genes within the ST gene cluster and a loss of ST production. Because A. nidulans aflR mRNA accumulates specifically under conditions that favor ST production we expect that activation of ST biosynthetic genes is determined by A. nidulans aflR. In support of this hypothesis, we demonstrated that induced expression of the A. flavus aflR gene in A. nidulans, under conditions that normally suppress ST gene expression, resulted in activation of genes in the ST biosynthetic pathway. This result demonstrates that AflR function is conserved between Aspergillus spp. and that aflR expression is sufficient to activate genes in the ST pathway.