Robert A. E. Butchko

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.

The Fusarium verticillioides FUM Gene Cluster Encodes a Zn(II)2Cys6 Protein That Affects FUM Gene Expression and Fumonisin Production

ABSTRACT Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Δfum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Δfum21 mutant with a wild-type copy of the gene restored fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis.

FvVE1 regulates biosynthesis of the mycotoxins fumonisins and fusarins in Fusarium verticillioides.

The veA gene positively regulates sterigmatocystin production in Aspergillus nidulans and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus . Whether veA homologues have a role in regulating secondary metabolism in other fungal genera is unknown. In this study, we examined the role of the veA homologue, FvVE1, on the production of two mycotoxin families, fumonisins and fusarins, in the important corn pathogen Fusarium verticillioides . We found that FvVE1 deletion completely suppressed fumonisin production on two natural substrates, corn and rice. Furthermore, our results revealed that FvVE1 is necessary for the expression of the pathway-specific regulatory gene FUM21 and structural genes in the fumonisin biosynthetic gene (FUM) cluster. FvVE1 deletion also blocked production of fusarins. The effects of FvVE1 deletion on the production of these toxins were found to be the same in two separate mating types. Our results strongly suggest that FvVE1 plays an important role in regulating mycotoxin production in F. verticillioides .

The putative monomeric G‐protein GBP1 is negatively associated with fumonisin B1 production in Fusarium verticillioides

SUMMARY Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticillioides that contaminates maize. FB(1) has been linked to a number of human and animal mycotoxicoses worldwide. Despite its significance, our understanding of the FB(1) biosynthesis regulatory mechanisms is limited. Here, we describe F. verticillioides GBP1, encoding a monomeric G-protein, and its role in FB(1) biosynthesis. GBP1 was discovered as an expressed sequence tag (EST) up-regulated in the F. verticillioides fcc1 mutant that showed reduced conidiation and no FB(1) biosynthesis when grown on maize kernels. Sequence analysis showed that GBP1 encodes a putative 368-amino-acid protein with similarity to DRG and Obg subclasses of G-proteins that are involved in development and stress responses. A GBP1 knockout mutant (Deltagbp1) exhibited normal growth, but increased FB(1) production (> 58%) compared with the wild-type when grown on corn kernels. Complementation of Deltagbp1 with the wild-type GBP1 gene restored FB(1) production levels to that of the wild-type. Our data indicate that GBP1 is negatively associated with FB(1) biosynthesis but not with conidiation in F. verticillioides. The deletion of GBP1 led to up-regulation of key FB(1) biosynthetic genes, FUM1 and FUM8, suggesting that the increased FB(1) production in Deltagbp1 is due to over-expression of FUM genes.

FUM9 Is Required for C-5 Hydroxylation of Fumonisins and Complements the Meitotically Defined Fum3 Locus in Gibberella moniliformis

ABSTRACT Deletion of the Gibberella moniliformis FUM9 gene resulted in mutants that produce only fumonisins that lack a C-5 hydroxyl group. This phenotype is identical to that of previously described mutants with defective alleles at the meiotically defined Fum3 locus. Transformation with a wild-type FUM9 gene into a Fum3-defective mutant restored wild-type fumonisin production. These results indicate that the FUM9 protein catalyzes the C-5 hydroxylation of fumonisins and that FUM9 and the Fum3 locus are the same gene.

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 120 h. Taken together, these approaches demonstrate the power of microarray technology to provide information on different biological processes.

Tricarballylic ester formation during biosynthesis of fumonisin mycotoxins in Fusarium verticillioides

Fumonisins are agriculturally important mycotoxins produced by the maize pathogen Fusarium verticillioides. The chemical structure of fumonisins contains two tricarballylic esters, which are rare structural moieties and important for toxicity. The mechanism for the tricarballylic ester formation is not well understood. FUM7 gene of F. verticillioides was predicted to encode a dehydrogenase/reductase, and when it was deleted, the mutant produced tetradehydro fumonisins (DH4–FB). MS and NMR analysis of DH4–FB1 indicated that the esters consist of aconitate with a 3′-alkene function, rather than a 2′-alkene function. Interestingly, the purified DH4–FB1 eventually yielded three chromatographic peaks in HPLC. However, MS revealed that the metabolites of the three peaks all had the same mass as the initial single-peak DH4–FB1. The results suggest that DH4–FB1 can undergo spontaneous isomerization, probably including both cis–trans stereoisomerization and 3′- to 2′-ene regioisomerization. In addition, when FUM7 was expressed in Escherichia coli and the resulting enzyme, Fum7p, was incubated with DH4–FB, no fumonisin with typical tricarballylic esters was formed. Instead, new fumonisin analogs that probably contained isocitrate and/or oxalosuccinate esters were formed, which reveals new insight into fumonisin biosynthesis. Together, the data provided both genetic and biochemical evidence for the mechanism of tricarballylic ester formation in fumonisin biosynthesis.

Identification of genes and gene clusters involved in mycotoxin synthesis.

Abstract: Research methods to identify and characterize genes involved in mycotoxin biosynthetic pathways have evolved considerably. Before whole genome sequences were available (e.g. pre-genomics), work focused primarily on chemistry, biosynthetic mutant strains and molecular analysis of single or relatively small numbers of genes. In recent years, reductions in the cost of DNA sequencing technologies have made genomic methods more widely available. The availability of whole genome sequence for multiple mycotoxin-producing fungi has led to important discoveries both within single genomes and between genomes via comparative genomics (post-genomic). The ability to analyze simultaneously the expression of a large number of genes through expressed sequenced tags (ESTs) and microarrays (transcriptomics) has also had a significant impact on gene cluster identification. This chapter discusses the pre-genomics strategies used to identify toxin biosynthetic genes/gene clusters in fungi as well as more recently developed genomic strategies that greatly enhance the efficiency of the identification process. Pre-genomic researchers focused on analysis of a small number of genes, while post-genomic researchers can compare genomes and examine transcription patterns of thousands of genes at a time to help define biosynthetically related genes involved in mycotoxin synthesis. An examination of two Fusarium secondary metabolites (SMs), the mycotoxins fumonisins and a perithecial pigment, provide models for similar studies of other fungi. The identification of additional mycotoxin gene clusters in other fungi will proceed significantly faster in the future using genomic technologies.