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Fusarium verticillioides causes multiple diseases of Zea mays (maize) including ear and seedling rots, contaminates seeds and seed products worldwide with toxic chemicals called fumonisins. The role of fumonisins in disease is unclear because, although they are not required for ear rot, they are required for seedling diseases. Disease symptoms may be due to the ability of fumonisins to inhibit ceramide synthase activity, the expected cause of lipids (fatty acids, oxylipins, and sphingolipids) alteration in infected plants. In this study, we explored the impact of fumonisins on fatty acid, oxylipin, and sphingolipid levels in planta and how these changes affect F. verticillioides growth in maize. The identity and levels of principal fatty acids, oxylipins, and over 50 sphingolipids were evaluated by chromatography followed by mass spectrometry in maize infected with an F. verticillioides fumonisin-producing wild-type strain and a fumonisin-deficient mutant, after different periods of growth. Plant hormones associated with defense responses, i.e., salicylic and jasmonic acid, were also evaluated. We suggest that fumonisins produced by F. verticillioides alter maize lipid metabolism, which help switch fungal growth from a relatively harmless endophyte to a destructive necrotroph.

Background: Sphingolipids are structural components and signaling molecules in eukaryotic membranes, and many organisms produce compounds that inhibit sphingolipid metabolism. Some of the inhibitors are structurally similar to the sphingolipid biosynthetic intermediate sphinganine and are referred to as sphinganine-analog metabolites (SAMs). The mycotoxins fumonisins, which are frequent contaminants in maize, are one family of SAMs. Due to food and feed safety concerns, fumonisin biosynthesis has been investigated extensively, including characterization of the fumonisin biosynthetic gene cluster in the agriculturally important fungi Aspergillus and Fusarium. Production of several other SAMs has also been reported in fungi, but there is almost no information on their biosynthesis. There is also little information on how widely SAM production occurs in fungi or on the extent of structural variation of fungal SAMs., Results: Using fumonisin biosynthesis as a model, we predicted that SAM biosynthetic gene clusters in fungi should include a polyketide synthase (PKS), an aminotransferase and a dehydrogenase gene. Surveys of genome sequences identified five putative clusters with this three-gene combination in 92 of 186 Fusarium species examined. Collectively, the putative SAM clusters were distributed widely but discontinuously among the species. We propose that the SAM5 cluster confers production of a previously reported Fusarium SAM, 2-amino-14,16-dimethyloctadecan-3-ol (AOD), based on the occurrence of AOD production only in species with the cluster and on deletion analysis of the SAM5 cluster PKS gene. We also identified SAM clusters in 24 species of other fungal genera, and propose that one of the clusters confers production of sphingofungin, a previously reported Aspergillus SAM., Conclusion: Our results provide a genomics approach to identify novel SAM biosynthetic gene clusters in fungi, which should in turn contribute to identification of novel SAMs with applications in medicine and other fields. Information about novel SAMs could also provide insights into the role of SAMs in the ecology of fungi. Such insights have potential to contribute to strategies to reduce fumonisin contamination in crops and to control crop diseases caused by SAM-producing fungi.

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 (Deltafum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Deltafum21 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.

It has been almost 10 years since Joan Bennett suggested that fungal biologists create a "wish list" for fungal genome sequences (Bennett JW. White paper: Genomics for filamentous fungi. Fungal Genet Biol 1997; 21: 3-7). The availability of over 200 review papers concerning fungal genomics is a reflection of significant progress with a diversity of fungal species. Although much progress has been made, the use of genomic data to study mycotoxin synthesis and function, pathogenesis and other aspects of fungal biology is in its infancy. Here, we briefly present the status of publicly available genomic resources for Fusarium, a genus of important plant pathogenic and mycotoxin-producing fungi of worldwide concern. Preliminary examination of microarray data collected from F. verticillioides liquid cultures provides evidence of widespread differential gene expression over time.

Fusarium verticillioides (teleomorph Gibberella moniliformis) is a pathogen of maize worldwide and produces fumonisins, a family of mycotoxins that have been associated with several animal diseases as well as cancer in humans. In this study, we sought to identify fungal genes that affect fumonisin production and/or the plant-fungal interaction. We generated over 87,000 expressed sequence tags from nine different cDNA libraries that correspond to 11,119 unique sequences and are estimated to represent 80% of the genomic complement of genes. A comparative analysis of the libraries showed that all 15 genes in the fumonisin gene cluster were differentially expressed. In addition, nine candidate fumonisin regulatory genes and a number of genes that may play a role in plant-fungal interaction were identified. Analysis of over 700 FUM gene transcripts from five different libraries provided evidence for transcripts with unspliced introns and spliced introns with alternative 3' splice sites. The abundance of the alternative splice forms and the frequency with which they were found for genes involved in the biosynthesis of a single family of metabolites as well as their differential expression suggest they may have a biological function. Finally, analysis of an EST that aligns to genomic sequence between FUM12 and FUM13 provided evidence for a previously unidentified gene (FUM20) in the FUM gene cluster.

Production of the carcinogenic mycotoxins fumonisins has been reported in several Fusarium species, most of which are members of the Gibberella fujikuroi (Gf) complex. In this study, we examined 15 Fusarium species in the Gf complex and 12 other species for fumonisin production and the presence of fumonisin biosynthetic genes (FUM). Among the species within the Gf complex, fumonisin production was detected only in F. fujikuroi, F. globosum, F. proliferatum, F. nygamai, F. oxysporum and F. verticillioides. These five species include members of two of the three major clades delineated in the Gf complex. The FUM genes were detected in these same five species and in F. anthophilum, a member of the third clade. Among the species outside the Gf complex, fumonisin production and FUM genes were detected only in F. oxysporum. Phylogenetic analyses of nucleotide sequences from two FUM gene fragments inferred relationships similar but not identical to those inferred from previous analyses of other genes. The results indicate the FUM genes are discontinuously distributed in the Gf complex and that this distribution gives rise to the differences in the abilities of closely related Fusarium species to produce fumonisins.

Fumonisins are mycotoxins produced by the maize pathogen Gibberella moniliformis and are associated with cancer in rodents. In this study, we determined the nucleotide sequence of a 75-kb region of G. moniliformis DNA and identified 18 heretofore undescribed genes flanking a cluster of five previously identified fumonisin biosynthetic (FUM) genes. Ten of the newly identified genes downstream of the cluster were coregulated with FUM genes and exhibited patterns of expression that were correlated with fumonisin production. BLASTX analyses indicated that the predicted functions of proteins encoded by the 10 genes were consistent with activities expected for fumonisin biosynthesis or self-protection. These data indicate that the 10 newly identified genes and the previously identified FUM genes constitute a fumonisin biosynthetic gene cluster. Disruption of two of the new genes, encoding longevity assurance factors, had no apparent effect on fumonisin production, but disruption of a third, encoding an ABC transporter, had a subtle effect on ratios of fumonisins produced.

Fungal genetic systems ideally combine molecular tools for genome manipulation and a sexual reproduction system to create an informative assortment of combinations of genomic modifications. When employing the sexual cycle to generate multi-mutants, the background genotype variations in the parents may result in progeny phenotypic variation obscuring the effects of combined mutations. Here, to mitigate this variation in Fusarium verticillioides, we generated a MAT1-2 strain that was near isogenic to the sequenced wild-type MAT1-1 strain, FGSC7600. This was accomplished by crossing FGSC7600 with the divergent wild-type MAT1-2 strain FGSC7603 followed by six sequential backcrosses (e.g., six generations) of MAT1-2 progeny to FGSC7600. We sequenced each generation and mapped recombination events. The parental cross involved twenty-six crossovers on nine of the eleven chromosomes. The dispensable chromosome 12, found in FGSC7603 but lacking in FGSC7600, was not present in the progeny post generation five. Inheritance of complete chromosomes without crossover was frequently observed. A deletion of approximately 140 kilobases, containing 54 predicted genes on chromosome 4, occurred in generation 4 and was retained in generation 5 indicating that these genes are dispensable for growth and both asexual and sexual reproduction. The final MAT1-2 strain TMRU10/35 is about 93% identical to FGSC7600. TMRU10/35 is available from the Fungal Genetics Stock Center as FGSC27326 and from the ARS Culture Collection as NRRL64809. [ABSTRACT FROM AUTHOR]

Abstract: The filamentous fungus Fusarium verticillioides can cause disease of maize and is capable of producing fumonisins, a family of toxic secondary metabolites linked to esophageal cancer and neural tube defects in humans and lung edema in swine and leukoencephalomalacia in equines. The expression of fumonisin biosynthetic genes is influenced by broad-domain transcription factors (global regulators) and Fum21, a pathway-specific transcription factor. LaeA is a global regulator that in Aspergillus nidulans, affects the expression of multiple secondary metabolite gene clusters by modifying heterochromatin structure. Here, we employed gene deletion analysis to assess the effect of loss of a F. verticillioides laeA orthologue, LAE1, on genome-wide gene expression and secondary metabolite production. Loss of Lae1 resulted in reduced expression of gene clusters responsible for synthesis of the secondary metabolites bikaverin, fumonisins, fusaric acid and fusarins as well as two clusters for which the corresponding secondary metabolite is unknown. Analysis of secondary metabolites revealed that, in contrast to a previously described Fusarium fujikuroi lae1 mutant, bikaverin production is reduced. Fumonisin production is unchanged in the F. verticillioides lae1 mutant. Complementation of the F. verticillioides mutant resulted in increased fumonisin production. [Copyright &y& Elsevier]

The disaccharide trehalose has long been recognized for its role as a stress solute, but in recent years some of the protective effects previously ascribed to trehalose have been suggested to arise from a function of the trehalose biosynthesis enzyme trehalose-6-phosphate (T6P) synthase that is distinct from its catalytic activity. In this study, we use the maize pathogenic fungus Fusarium verticillioides as a model to explore the relative contributions of trehalose itself and a putative secondary function of T6P synthase in protection against stress as well as to understand why, as shown in a previous study, deletion of the TPS1 gene coding for T6P synthase reduces pathogenicity against maize. We report that a TPS1 -deletion mutant of F. verticillioides is compromised in its ability to withstand exposure to oxidative stress meant to simulate the oxidative burst phase of maize defense and experiences more ROS-induced lipid damage than the wild-type strain. Eliminating T6P synthase expression also reduces resistance to desiccation, but not resistance to phenolic acids. Expression of catalytically-inactive T6P synthase in the TPS1 -deletion mutant leads to a partial rescue of the oxidative and desiccation stress-sensitive phenotypes, suggesting the importance of a T6P synthase function that is independent of its role in trehalose synthesis. • T6P synthase expression protects against oxidative stress and lipid damage. • T6P synthase expression protects against desiccation stress. • Protective effect of T6P synthase due to role distinct from transferase activity. [ABSTRACT FROM AUTHOR]

Spore killers are meiotic drive elements that can block the development of sexual spores in fungi. In the maize ear rot and mycotoxinproducing fungus Fusarium verticillioides, a spore killer called SkK has been mapped to a 102-kb interval of chromosome V. Here, we show that a gene within this interval, SKC1, is required for SkK-mediated spore killing and meiotic drive. We also demonstrate that SKC1 is associated with at least 4 transcripts, 2 sense (sense-SKC1a and sense-SKC1b) and 2 antisense (antisense-SKC1a and antisense-SKC1b). Both antisense SKC1 transcripts lack obvious protein-coding sequences and thus appear to be noncoding RNAs. In contrast, sense-SKC1a is a protein-coding transcript that undergoes A-to-I editing to sense-SKC1b in sexual tissue. Translation of sense-SKC1a produces a 70-aminoacid protein (Skc1a), whereas the translation of sense-SKC1b produces an 84-amino-acid protein (Skc1b). Heterologous expression analysis of SKC1 transcripts shows that sense-SKC1a also undergoes A-to-I editing to sense-SKC1b during the Neurospora crassa sexual cycle. Sitedirected mutagenesis studies indicate that Skc1b is responsible for spore killing in Fusarium verticillioides and that it induces most meiotic cells to die in Neurospora crassa. Finally, we report that SKC1 homologs are present in over 20 Fusarium species. Overall, our results demonstrate that fungal meiotic drive elements like SKC1 can influence the outcome of meiosis by hijacking a cell's A-to-I editing machinery and that the involvement of A-to-I editing in a fungal meiotic drive system does not preclude its horizontal transfer to a distantly related species. [ABSTRACT FROM AUTHOR]

Highlights: [•] Temperature stress alters trehalose and polyol levels in Fusarium verticilliodes. [•] The trehalose analog validamycin A reduces mycelial trehalose and polyols. [•] Deleting the TPS1 gene abolishes trehalose synthesis and increases glycogen content. [•] ΔTPS1 mutants synthesize less fumonisins and cause less disease than WT. [ABSTRACT FROM AUTHOR]

Abstract: Fusarium (Hypocreales, Nectriaceae) is one of the most economically important and systematically challenging groups of mycotoxigenic phytopathogens and emergent human pathogens. We conducted maximum likelihood (ML), maximum parsimony (MP) and Bayesian (B) analyses on partial DNA-directed RNA polymerase II largest (RPB1) and second largest subunit (RPB2) nucleotide sequences of 93 fusaria to infer the first comprehensive and well-supported phylogenetic hypothesis of evolutionary relationships within the genus and 20 of its near relatives. Our analyses revealed that Cylindrocarpon formed a basal monophyletic sister to a ‘terminal Fusarium clade’ (TFC) comprising 20 strongly supported species complexes and nine monotypic lineages, which we provisionally recognize as Fusarium (hypothesis F1). The basal-most divergences within the TFC were only significantly supported by Bayesian posterior probabilities (B-PP 0.99–1). An internode of the remaining TFC, however, was strongly supported by MP and ML bootstrapping and B-PP (hypothesis F2). Analysis of seven Fusarium genome sequences and Southern analysis of fusaria elucidated the distribution of genes required for synthesis of 26 families of secondary metabolites within the phylogenetic framework. Diversification time estimates date the origin of the TFC to the middle Cretaceous 91.3million years ago. We also dated the origin of several agriculturally important secondary metabolites as well as the lineage responsible for Fusarium head blight of cereals. Dating of several plant-associated species complexes suggests their evolution may have been driven by angiosperm diversification during the Miocene. Our results support two competing hypotheses for the circumscription of Fusarium and provide a framework for future comparative phylogenetic and genomic analyses of this agronomically and medically important genus. [Copyright &y& Elsevier]