Charles P. Woloshuk

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Clustered Pathway Genes in Aflatoxin Biosynthesis

Aflatoxins, a group of polyketide-derived furanocoumarins (Fig. [1][1]), are the most toxic and carcinogenic compounds among the known mycotoxins. Among the at least 16 structurally related aflatoxins characterized, however, there are only four major aflatoxins, B1, B2, G1, and G2 (AFB1, AFG1, AFB2

Efficacy and fumigation characteristics of ozone in stored maize.

This study evaluated the efficacy of ozone as a fumigant to disinfest stored maize. Treatment of 8.9tonnes (350bu) of maize with 50ppm ozone for 3d resulted in 92-100% mortality of adult red flour beetle, Tribolium castaneum (Herbst), adult maize weevil, Sitophilus zeamais (Motsch.), and larval Indian meal moth, Plodia interpunctella (Hübner) and reduced by 63% the contamination level of the fungus Aspergillus parasiticus Speare on the kernel surface. Ozone fumigation of maize had two distinct phases. Phase 1 was characterized by rapid degradation of the ozone and slow movement through the grain. In Phase 2, the ozone flowed freely through the grain with little degradation and occurred once the molecular sites responsible for ozone degradation became saturated. The rate of saturation depended on the velocity of the ozone/air stream. The optimum apparent velocity for deep penetration of ozone into the grain mass was 0.03m/s, a velocity that is achievable in typical storage structures with current fans and motors. At this velocity 85% of the ozone penetrated 2.7m into the column of grain in 0.8d during Phase 1 and within 5d a stable degradation rate of 1ppm/0.3m was achieved. Optimum velocity for Phase 2 was 0.02m/s. At this velocity, 90% of the ozone dose penetrated 2.7m in less than 0.5d. These data demonstrate the potential usefulness of using ozone in managing stored maize and possibly other grains.

PAC1, a pH-regulatory gene from Fusarium verticillioides.

ABSTRACT Fumonisins are a group of mycotoxins that contaminate maize and cause leukoencephalomalacia in equine, pulmonary edema in swine, and promote cancer in mice. Fumonisin biosynthesis in Fusarium verticillioides is repressed by nitrogen and alkaline pH. We cloned a PACC-like gene (PAC1) from F. verticillioides. PACC genes encode the major transcriptional regulators of several pH-responsive pathways in other filamentous fungi. In Northern blot analyses, a PAC1 probe hybridized to a 2.2-kb transcript present in F. verticillioides grown at alkaline pH. A mutant of F. verticillioides with a disrupted PAC1 gene had severely impaired growth at alkaline pH. The mutant produced more fumonisin than the wild type when grown on maize kernels and in a synthetic medium buffered at an acidic pH, 4.5. The mutant, but not the wild type, also produced fumonisin B1 when mycelia were resuspended in medium buffered at an alkaline pH, 8.4. Transcription of FUM1, a gene involved in fumonisin biosynthesis, was correlated with fumonisin production. We conclude that PAC1 is required for growth at alkaline pH and that Pac1 may have a role as a repressor of fumonisin biosynthesis under alkaline conditions.

Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal.

The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions (ITS1 and ITS2) of rDNA. Primers for group-specific detection were designed from the TRI6 gene involved in trichothecene biosynthesis and the FUM5 gene involved in fumonisin biosynthesis. Primer specificity was determined by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillus spp., 9 Fusarium spp., and 10 Penicillium spp. With purified genomic DNA as a template, genus-specific recognition was observed at 10 pg per reaction; group-specific recognition occurred at 100 pg of template per reaction for the trichothecene producer Fusarium graminearum and at 1 ng of template per reaction for the fumonisin producer Fusarium verticillioides. For the application of the PCR assay, a protocol was developed to isolate fungal DNA from cornmeal. The detection of F. graminearum and its differentiation from F. verticillioides were accomplished prior to visible fungal growth at <10(5) CFU/g of cornmeal. This level of detection is comparable to those of other methods such as enzyme-linked immunosorbent assay, and the assay described here can be used in the food industrys effort to monitor quality and safety.

Regulation of Fumonisin B1 Biosynthesis and Conidiation in Fusarium verticillioides by a Cyclin-Like (C-Type) Gene, FCC1

ABSTRACT Fumonisins are a group of mycotoxins produced in corn kernels by the plant-pathogenic fungus Fusarium verticillioides. A mutant of the fungus, FT536, carrying a disrupted gene namedFCC1 (for Fusarium cyclin C1) resulting in altered fumonisin B1 biosynthesis was generated. FCC1 contains an open reading frame of 1,018 bp, with one intron, and encodes a putative 319-amino-acid polypeptide. This protein is similar to UME3 (also called SRB11 or SSN8), a cyclin C of Saccharomyces cerevisiae, and contains three conserved motifs: a cyclin box, a PEST-rich region, and a destruction box. Also similar to the case for C-type cyclins, FCC1was constitutively expressed during growth. When strain FT536 was grown on corn kernels or on defined minimal medium at pH 6, conidiation was reduced and FUM5, the polyketide synthase gene involved in fumonisin B1 biosynthesis, was not expressed. However, when the mutant was grown on a defined minimal medium at pH 3, conidiation was restored, and the blocks in expression ofFUM5 and fumonisin B1 production were suppressed. Our data suggest that FCC1 plays an important role in signal transduction regulating secondary metabolism (fumonisin biosynthesis) and fungal development (conidiation) inF. verticillioides.

Beyond aflatoxin: four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters

Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis has predicted that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in Aspergillus flavus; however, only three metabolic pathways-aflatoxin, cyclopiazonic acid (CPA) and aflatrem-have been assigned to these clusters. To gain an insight into the regulation of and to infer the ecological significance of the 55 secondary metabolite gene clusters predicted in A. flavus, we examined their expression over 28 diverse conditions. Variables included culture medium and temperature, fungal development, colonization of developing maize seeds and misexpression of laeA, a global regulator of secondary metabolism. Hierarchical clustering analysis of expression profiles allowed us to categorize the gene clusters into four distinct clades. Gene clusters for the production of aflatoxins, CPA and seven other unknown compound(s) were identified as belonging to one clade. To further explore the relationships found by gene expression analysis, aflatoxin and CPA production were quantified under five different cell culture environments known to be conducive or nonconducive for aflatoxin biosynthesis and during the colonization of developing maize seeds. Results from these studies showed that secondary metabolism gene clusters have distinctive gene expression profiles. Aflatoxin and CPA were found to have unique regulation, but are sufficiently similar that they would be expected to co-occur in substrates colonized with A. flavus.

Role of AREA, a regulator of nitrogen metabolism, during colonization of maize kernels and fumonisin biosynthesis in Fusarium verticillioides.

Fumonisin B1 (FB(1)) biosynthesis is repressed in cultures containing ammonium as the nitrogen source and when grown on blister kernels, the earliest stages of kernel development. In this study AREA, a regulator of nitrogen metabolism, was disrupted in Fusarium verticilliodes. The mutant (DeltaareA) grew poorly on mature maize kernels, but grew similar to wild type (WT) with the addition of ammonium phosphate. FB(1) was not produced by DeltaareA under any condition or by the WT with added ammonium phosphate. Constitutive expression of AREA (strain AREA-CE) rescued the growth and FB(1) defects in DeltaareA. Growth of WT, DeltaareA, and AREA-CE on blister-stage kernels was similar. After 7 days of growth, none of the strains produced FB(1) and the pH of the kernel tissues was 8.0. Addition of amylopectin to the blister kernels resulted in a pH near 6.6 and FB(1) production by WT and AREA-CE. The results support the hypothesis that FB(1) biosynthesis is regulated by AREA. Also the failure to produce FB(1) in blister kernels is due to high pH conditions generated because of an unfavorable carbon/nitrogen environment.

Regulation of Fumonisin Biosynthesis in Fusarium verticillioides by a Zinc Binuclear Cluster-Type Gene, ZFR1

ABSTRACT Fusarium verticillioides, a pathogen of maize, produces a class of mycotoxins called fumonisins in infected kernels. In this study, a candidate regulatory gene, ZFR1, was identified in an expressed sequence tag library enriched for transcripts expressed by F. verticillioides during fumonisin B1 (FB1) biosynthesis. ZFR1 deletion mutants exhibited normal growth and development on maize kernels, but fumonisin production was reduced to less than 10% of that of the wild-type strain. ZFR1 encodes a putative protein of 705 amino acids with sequence similarity to the Zn(II)2Cys6 binuclear cluster family that are regulators of both primary and secondary metabolism in fungi. Expression of ZFR1 in colonized germ and degermed kernel tissues correlated with FB1 levels. Overexpression of ZFR1 in zfr1 mutants restored FB1 production to wild-type levels; however, FB1 was not restored in an fcc1 (Fusarium C-type cyclin) mutant by overexpression of ZFR1. The results of this study indicate that ZFR1 is a positive regulator of FB1 biosynthesis in F. verticillioides and suggest that FCC1 is required for ZFR1 function.

Cloning and characterization of avfA and omtB genes involved in aflatoxin biosynthesis in three Aspergillus species.

The biosynthesis of aflatoxins (B(1), G(1), B(2), and G(2)) is a multi-enzyme process controlled genetically by over 20 genes. In this study, we report the identification and characterization of the avfA gene, which was found to be involved in the conversion of averufin (AVF) to versiconal hemiacetal acetate (VHA), in Aspergillus parasiticus and A. flavus; a copy of avfA gene was also cloned from a non-aflatoxin producing strain A. sojae. Complementation of an averufin-accumulating, non-aflatoxigenic mutant strain of A. parasiticus, SRRC 165, with the avfA gene cloned from A. flavus, restored the ability of the mutant to convert AVF to VHA and to produce aflatoxins B(1), G(1), B(2), and G(2). Sequence analysis revealed that a single amino acid replacement from aspartic acid to asparagine disabled the function of the enzyme in the mutant strain SRRC 165. The A. parasiticus avfA was identified to be a homolog of previously sequenced, but functionally unassigned transcript, stcO, in A. nidulans based on sequence homology at both nucleotide (57%) and amino acid (55%) levels. In addition to avfA, another aflatoxin pathway gene, omtB, encoding for an O-methyltransferase involved in the conversion of demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and dihydrodemethylsterigmatocystin (DHDMST) to dihydrosterigmatocystin (DHST), was cloned from A. parasiticus, A. flavus, and A. sojae. The omtB gene was found to be highly homologous to stcP from A. nidulans, which has been reported earlier to be involved in a similar enzymatic step for the sterigmatocystin formation in that species. RT-PCR data demonstrated that both the avfA and avfA1 as well as omtB genes in A. parasiticus were expressed only in the aflatoxin-conducive medium. An analysis of the degrees of homology for the two reported genes between the Aspergillus species A. parasiticus, A. flavus, A. nidulans and A. sojae was conducted.