The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides

Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity.     

Regulation of fumonisin B(1) biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like (C-type) gene, FCC1

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 named FCC1 (for Fusarium cyclin C1) resulting in altered fumonisin B(1) 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, FCC1 was 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 B(1) 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 of FUM5 and fumonisin B(1) production were suppressed. Our data suggest that FCC1 plays an important role in signal transduction regulating secondary metabolism (fumonisin biosynthesis) and fungal development (conidiation) in F. verticillioides.     

Reducing production of fumonisin mycotoxins in <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> by RNA interference

The fungus Fusarium verticillioides is a maize pathogen that can produce fumonisin mycotoxins in ears under certain environmental conditions. Because fumonisins pose health risks to humans and livestock, control strategies with minimal risk to the environment are needed to reduce fumonisin contamination. Host-induced gene silencing is a promising technique in which double-stranded RNA expressed in the plant host is absorbed by an invading fungus and down-regulates genes critical for pathogenicity or mycotoxin production in the fungus. A key preliminary step of this technique is identification of DNA segments within the targeted fungal gene that can effectively silence the gene. Here, we used segments of the fumonisin biosynthetic gene FUM1 to generate double-stranded RNA in F. verticillioides. Several of the resulting transformants exhibited reduced FUM1 gene expression and fumonisin production (24- to 3675-fold reduction in fumonisin FB₁). Similar reductions in fumonisin production resulted from double-stranded RNA constructs with segments of FUM8, another fumonisin biosynthetic gene (3.5- to 2240-fold reduction in fumonisin FB₁). FUM1 or FUM8 silencing constructs were transformed into three isolates of F. verticillioides. Whole genome sequence analysis of seven transformants revealed that reductions in fumonisin production were not due to mutation of the fumonisin biosynthetic gene cluster and revealed a complex pattern of plasmid integration. These results suggest the cloned FUM1 or FUM8 gene segments could be expressed in maize for host-induced gene silencing of fumonisin production.     

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.     

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

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 named FCC1 (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, FCC1 was 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 of FUM5 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) in F. verticillioides.     

Characterization of Four Clustered and Coregulated Genes Associated with Fumonisin Biosynthesis in Fusarium verticillioides

Fumonisins are mycotoxins that cause several fatal animal diseases, including cancer in rats and mice. These toxins are produced by several Fusarium species, including the maize pathogen Fusarium verticillioides, and can accumulate in maize infected with the fungus. We have identified four F. verticillioides genes (FUM6, FUM7, FUM8, and FUM9) adjacent to FUM5, a previously identified polyketide synthase gene that is required for fumonisin biosynthesis. Gene disruption analysis revealed that FUM6 and FUM8 are required for fumonisin production and Northern blot analysis revealed that expression of all four recently identified genes is correlated with fumonisin production. Nucleotide sequence analysis indicated that the predicted FUM6 translation product is most similar to cytochrome P450 monooxygenase-P450 reductase fusion proteins and the predicted products of FUM7, FUM8, and FUM9 are most similar to type III alcohol dehydrogenases, class-II alpha-aminotransferases, and dioxygenases, respectively. Together, these data are consistent with FUM5 through FUM9 being part of a fumonisin biosynthetic gene cluster in F. verticillioides.     

Expression profile analysis of wild-type and fcc1 mutant strains of Fusarium verticillioides during fumonisin biosynthesis

Fusarium verticillioides produces a group of mycotoxins known as fumonisins that are associated with a variety of mycotoxicoses in humans and animals. In this study, DNA microarrays were constructed with expressed sequence tags (ESTs) from F. verticillioides. To identify genes with patterns of expression similar to the fumonisin biosynthetic (FUM) genes, the microarray was probed with labeled cDNAs originating from a wild-type strain and a fcc1 mutant grown on maize and in a defined medium adjusted to either pH 3 or pH 8. The comparative analyses revealed differential expression of genes corresponding to 116 ESTs when the fungal strains were grown on maize. Under different pH conditions, 166 ESTs were differentially expressed, and 19 ESTs were identified that displayed expression patterns similar to the FUM ESTs. These results provide candidate genes with potential roles in fumonisin biosynthesis.     

Transformation-mediated complementation of a FUM gene cluster deletion in Fusarium verticillioides restores both fumonisin production and pathogenicity on maize seedlings

The filamentous ascomycete Fusarium verticillioides is a pathogen of maize and produces the fumonisin mycotoxins. However, a distinct population of F. verticillioides is pathogenic on banana and does not produce fumonisins. Fumonisin-producing strains from maize cause leaf lesions, developmental abnormalities, stunting, and sometimes death of maize seedlings, whereas fumonisin-nonproducing banana strains do not. A Southern analysis of banana strains did not detect genes in the fumonisin biosynthetic gene (FUM) cluster but did detect genes flanking the cluster. Nucleotide sequence analysis of the genomic region carrying the flanking genes revealed that the FUM cluster was absent in banana strains except for portions of FUM21 and FUM19, which are the terminal genes at each end of the cluster. Polymerase chain reaction analysis confirmed the absence of the cluster in all banana strains examined. Cotransformation of a banana strain with two overlapping cosmids, which together contain the entire FUM cluster, yielded fumonisin-producing transformants that were pathogenic on maize seedlings. Conversely, maize strains that possess the FUM cluster but do not produce fumonisins because of mutations in FUM1, a polyketide synthase gene, were not pathogenic on maize seedlings. Together, the data indicate that fumonisin production may have been lost by deletion of the FUM cluster in the banana population of F. verticillioides but that fumonisin production could be restored by molecular genetic complementation. The results also indicate that fumonisin production by F. verticillioides is required for development of foliar disease symptoms on maize seedlings.     

Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis

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.     

Fumonisin B1 production by Fusarium proliferatum strains isolated from Allium fistulosum plants and seeds in Japan

Aims:  To test the fumonisin B₁ - producing ability of Fusarium proliferatum strains isolated from Welsh onion ( Allium fistulosum ) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B₁-producing and nonproducing isolates.
Methods and Results:  Fumonisin B₁ levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B₁. PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates.
Conclusions:  Fusarium proliferatum isolates that can produce fumonisin B₁ were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B₁-producing and nonproducing isolates.
Significance and Impact of the Study:  This study revealed that F. proliferatum producing fumonisin B₁ is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B₁-producing F. proliferatum isolates.     

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C₁₈ chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.     

Genetic and phenotypic variation of <Emphasis Type="Italic">Fusarium proliferatum</Emphasis> isolates from different host species

Fusarium proliferatum (Matsushima) Nirenberg is a common pathogen infecting numerous crop plants and occurring in various climatic zones. It produces large amounts of fumonisins, a group of polyketide-derived mycotoxins. Fumonisin biosynthesis is determined by the presence and activity of the FUM cluster, several co-regulated genes with a common expression pattern. In the present work, we analyzed 38 F. proliferatum isolates from different host plant species, demonstrating host-specific polymorphisms in partial sequences of the key FUM1 gene (encoding polyketide synthase). We also studied growth rates across different temperatures and sample origin and tried to establish the relationships between DNA sequence polymorphism and toxigenic potential. Phylogenetic analysis was conducted based on FUM1 and tef-1α sequences for all isolates. The results indicated the greatest variations of both toxigenic potential and growth patterns found across the wide selection of isolates derived from maize. Fumonisin production for maize isolates ranged from 3.74 to 4,500 μg/g of fumonisin B₁. The most efficient producer isolates obtained from other host plants were only able to synthesize 1,820–2,419 μg/g of this metabolite. A weak negative rank correlation between fumonisin content and isolate growth rates was observed. All garlic-derived isolates formed a distinct group on a FUM1-based dendrogram. A second clade consisted of tropical and sub-tropical strains (isolated from pineapple and date palm). Interestingly, isolates with the fastest growth patterns were also grouped together and included both isolates originating from rice. The sequence of the FUM1 gene was found to be useful in revealing the intraspecific polymorphism, which is, to some extent, specifically correlated with the host plant.     

FUM1--a gene required for fumonisin biosynthesis but not for maize ear rot and ear infection by Gibberella moniliformis in field tests

We have analyzed the role of fumonisins in infection of maize (Zea mays) by Gibberella moniliformis (anamorph Fusarium verticillioides) in field tests in Illinois and Iowa, United States. Fumonisin-nonproducing mutants were obtained by disrupting FUM1 (previously FUM5), the gene encoding a polyketide synthase required for fumonisin biosynthesis. Maize ear rot, ear infection, and fumonisin contamination were assessed by silk-channel injection in 1999 and 2000 and also by spray application onto maize silks, injection into maize stalks, and application with maize seeds at planting in 1999. Ear rot was evaluated by visual assessment of whole ears and by calculating percentage of symptomatic kernels by weight. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of applied strains in kernels was determined by analysis of recovered isolates for genetic markers and fumonisin production. Two independent fumonisin-nonproducing (fum1-3 and fum1-4) mutants were similar to their respective fumonisin-producing (FUM1-1) progenitor strains in ability to cause ear rot following silk-channel injection and also were similar in ability to infect maize ears following application by all four methods tested. This evidence confirms that fumonisins are not required for G. moniliformis to cause maize ear rot and ear infection.     

Growth of Fusarium moniliforme and its biosynthesis of fumonisin B1 on maize grain as a function of different water activities

The production of fumonisin by Fusarium moniliforme during its growth on maize depends on extrinsic factors. In particular, experiments on maize grain at different water activities ( a _w)(1, 0.95, 0.90, 0.85) have demonstrated the influence of a _w on fumonisin biosynthesis, and on fungal growth defined by measurement of ergosterol levels. Fumonisin levels dropped threefold when a _w was lowered by 5%, but growth rate was unchanged. A 10% reduction in a _w from 1 to 0.90 resulted in a 20-fold drop in fungal growth, and fumonisin production was reduced 300-fold. At a threshold a _w of 0.85–0.86, F. moniliforme exhibited virtually no measurable metabolic activity, and hence no fumonisin production.     

A fumonisin biosynthetic gene cluster in Fusarium oxysporum strain O-1890 and the genetic basis for B versus C fumonisin production

Most species of Fusarium that produce fumonisin mycotoxins produce predominantly B fumonisins (FBs). However, Fusarium oxysporum strain O-1890 produces predominantly C fumonisins (FCs). In this study, the nucleotide sequence of the fumonisin biosynthetic gene (FUM) cluster in strain O-1890 was determined. The order and orientation of FUM genes were the same as in the previously described clusters in Fusarium verticillioides and Fusarium proliferatum. Coding regions of F. oxysporum and F. verticillioides FUM genes were 88-92% identical, but regions flanking the clusters did not share significant identity. The FUM cluster gene FUM8 encodes an alpha-oxoamine synthase, and fum8 mutants of F. verticillioides do not produce fumonisins. Complementation of a fum8 mutant with the F. verticillioidesFUM8 restored FB production. Complementation with F. oxysporumFUM8 also restored production, but the fumonisins produced were predominantly FCs. These data indicate that different orthologues of FUM8 determine whether Fusarium produces predominantly FBs or FCs.