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 (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.

FIG. 1.

Diagram of sequence alignment of TC16321 and TC16322 from F. verticillioides Gene Index Release 4.0 to a portion of the fumonisin cluster sequence and relative position of flanking gene KER1. KER1 encodes a putative carbonyl reductase and is not likely involved in fumonisin biosynthesis since it is not expressed in a manner similar to cluster genes. FUM1 and FUM6 both encode proteins that are required for fumonisin biosynthesis.

FIG. 2.

Diagram of multiple sequence alignment of Zn(II)2Cys6 gene EST/cDNA sequences. The first bar under the base pair scale represents genomic sequence, and the second bar represents the full-length predicted Zn(II)2Cys6 transcript, where each numbered gap represents an intron. Translation of this full-length transcript would yield a 672-amino-acid protein. The next series of bars represent sequences of cDNAs from EST libraries FvF (11F), FvG (1G to 7G, 12G, and 13G) and FvN (8N to 10N). ASFa through ASFd represent ASFs of the Zn(II)2Cys6 transcript. ASFa transcripts, represented by four sequences, had nine fewer nucleotides in the first intron than other ASFs. ASFb transcripts, represented by three sequences, retained the second intron. ASFc, represented by one sequence, retained the fifth intron, and ASFd, represented by one sequence, retained the third, fourth, and seventh introns. Processed transcripts, represented by four sequences, had all of possible introns fully excised, based on available sequence data. Within a cDNA or ASF, dashed lines between two bars represent a portion not sequenced. The three ASFb ESTs were part of TC30496, while all remaining ASF ESTs were from TC30495.

FIG. 3.

Partial sequence alignment of a representative of ASFa and ASFb and genomic sequence, showing the positions of the first and second introns and the corresponding predicted amino acid sequence. The intron splice site sequences are indicated in boldface in the genomic sequence. T1, T2, and T3 refer to the putative translated protein. The number below each cysteine refers to the position of that cysteine in the Zn(II)2Cys6 motif. Excision of the shorter first intron in ASFa introduces a stop codon indicated by the asterisk. The failure to excise the second intron in ASFb results in a frameshift and termination of translation indicated by the asterisk. The putative 72-amino-acid protein would contain a tryptophan in place of the sixth cysteine. The excision of both introns in the “processed” transcript would allow for the translation of a complete Zn(II)2Cys6 motif.

FIG. 4.

Reverse position specific BLAST of the predicted Zn(II)2Cys6 protein (Fum21p) against the NCBI CDD. (A) The bar represents the Fum21p in 100s of amino acids. GAL4 refers to GAL4-like Zn(II)2Cys6 binuclear cluster DNA-binding domain (smart00066) and Fungal_trans refers to a fungal specific transcription factor domain (pfam04082). (B) Alignment of portions of the putative Fum21p with the GAL4 (E = 3 × 10⁻⁷) and Fungal_trans domains (E = 2 × 10⁻⁴). I1, I2, I5, and I6 indicates positions of introns 1, 2, 5, and 6, respectively. The asterisks mark the position of the six, highly conserved cysteines that form the Zn(II)2Cys6 DNA-binding motif.

FIG. 5.

RT-PCR analysis of genes in fum21 mutants. Total RNA from three Δfum21 mutants (strains GMTcp-172, GMTcp-173, and GMTcp-174) and wild-type M-3125 was used as a template for RT-PCR. The 2, 4, and 6 refer to the time point in days. Control reactions without RT are indicated as “−RT”; reactions that included RT are indicated as “+RT.” The primer pairs used for amplification of each gene are described in Materials and Methods.

FIG. 6.

Microarray analysis of FUM21 expression. (A) The upper bars represent the predicted full-length FUM21 transcript, where each numbered gap corresponds to an intron. Each small bar below the full-length transcript represents a single 24-mer oligonucleotide probe. For the exon probes, FUM21 expression was measured by using three different probe sets of between 10 and 12 oligonucleotides each. Exon probes of each probe set were scattered over the length of the FUM21 exon sequence as indicated. For the intron probes, the probe sets for the second, third, fourth, and seventh introns are indicated by the stack of small bars under introns 2, 3, 4, and 7, respectively. (B) Ratio of expression at 96 versus 12 h in liquid GYAM medium for FUM21; FUM21 ASFs retaining introns 2, 3, 4, or 7; FUM1; and FUM8. Relative expression change is indicated on the vertical axis in the logarithmic base 2 ratio. A positive value indicates an increase in expression over time, while a negative value indicates a decrease in expression over time. (C) Fumonisin levels in μg of FB₁ per ml of GYAM medium (•) on the left vertical axis and ratio change in expression in logarithmic base 2 scale over time of FUM1 (⧫) and FUM8 (▪) on the right vertical axis.