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.     

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.     

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.     

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.     

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.     

FUM cluster divergence in fumonisins-producing Fusarium species

Fumonisins are polyketide-derived mycotoxins, produced by several Fusarium species, and its biosynthetic pathway is controlled by the FUM cluster--a group of genes exhibiting a common expression pattern during fumonisin biosynthesis. The most common are the B analogues with fumonisin B(1) (FB(1)) being the most prevalent. At least a part of the inter- and intraspecific variation in FBs synthesis level can be explained by the sequence differences inside FUM cluster. The aim of our study was to evaluate the toxin production and sequence variability in FUM genes and intergenic regions among thirty isolates of seven species reported as potential fumonisins producers: Fusarium anthophilum, Fusarium fujikuroi, Fusarium nygamai, Fusarium oxysporum, Fusarium proliferatum, Fusarium subglutinans and Fusarium verticillioides, particularly with respect to FBs synthesis. Fumonisins were produced in high amounts (over 1mg g(-1)) by one isolate of F. subglutinans, three of F. verticillioides and all F. proliferatum isolates except one, regardless of the host organism. The remaining isolates produced low amounts of FBs and two F. verticillioides isolates didn't produce it at all. The lowest variation in amount of toxin produced was found among F. proliferatum isolates. Based on the translation elongation factor 1α (tef-1α) sequence of F. fujikuroi, a species-specific marker was developed. The intergenic region presents similar opportunity for F. nygamai identification. The phylogenetic reconstruction based on FUM1 gene generally reflects the scenario presented by tef-1α sequences. Although the sequence similarities for intergenic regions were lower than in coding regions, there are clearly conserved patterns enabling separation of different subsets of species, including the non-producer species.     

Sequence variability in the FUM1 gene of Fusarium verticillioides strains

Fumonisins are mycotoxins, produced mainly by Fusarium verticillioides, that are potentially carcinogenic to humans and toxic to animals. Synthesis of these toxins is directed by a cluster of 15 genes, among which FUM1 is the largest; it encodes a polyketide synthase. This enzyme probably catalyzes the synthesis of a polyketide that forms a large portion of the fumonisin structure. In this study, 27 strains possessing the FUM1 gene, as determined by polymerase chain reaction, were analyzed. A portion of the FUM1 gene was amplified and sequenced from 6 of 27 Brazilian strains isolated from corn and sorghum. The sequence similarity for the six F. verticillioides strains was almost 100%.     

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.     

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.     

Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels

Fusarium verticillioides, a fungal pathogen of maize, produces fumonisin mycotoxins that adversely affect human and animal health. Basic questions remain unanswered regarding the interactions between the host plant and the fungus that lead to the accumulation of fumonisins in maize kernels. In this study, we evaluated the role of kernel endosperm composition in regulating fumonisin B1 (FB1) biosynthesis. We found that kernels lacking starch due to physiological immaturity did not accumulate FB1. Quantitative polymerase chain reaction analysis indicated that kernel development also affected the expression of fungal genes involved in FB1 biosynthesis, starch metabolism, and nitrogen regulation. A mutant strain of F. verticillioides with a disrupted a-amylase gene was impaired in its ability to produce FB1 on starchy kernels, and both the wild-type and mutant strains produced significantly less FB1 on a high-amylose kernel mutant of maize. When grown on a defined medium with amylose as the sole carbon source, the wild-type strain produced only trace amounts of FB1, but it produced large amounts of FB1 when grown on amylopectin or dextrin, a product of amylopectin hydrolysis. We conclude that amylopectin induces FB1 production in F. verticillioides. This study provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.     

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.