Regulation of fumonisin biosynthesis in Fusarium verticillioides by a zinc binuclear cluster-type gene, ZFR1

Abstract

Fusarium verticillioides, a pathogen of maize, produces a class of mycotoxins called fumonisins in infected kernels. In this study, a candidate regulatory gene, ZFR1, was identified in an expressed sequence tag library enriched for transcripts expressed by F. verticillioides during fumonisin B(1) (FB(1)) biosynthesis. ZFR1 deletion mutants exhibited normal growth and development on maize kernels, but fumonisin production was reduced to less than 10% of that of the wild-type strain. ZFR1 encodes a putative protein of 705 amino acids with sequence similarity to the Zn(II)2Cys6 binuclear cluster family that are regulators of both primary and secondary metabolism in fungi. Expression of ZFR1 in colonized germ and degermed kernel tissues correlated with FB(1) levels. Overexpression of ZFR1 in zfr1 mutants restored FB(1) production to wild-type levels; however, FB(1) was not restored in an fcc1 (Fusarium C-type cyclin) mutant by overexpression of ZFR1. The results of this study indicate that ZFR1 is a positive regulator of FB(1) biosynthesis in F. verticillioides and suggest that FCC1 is required for ZFR1 function.

FIG. 1.

Northern blot analysis of wild type and fcc1 mutant cells cultured on cracked maize kernels. Total RNAs (10 μg) isolated from the wild-type strain (lane 1) and the fcc1Δ mutant strain (lane 2) cultured for 7 days were separated by electrophoresis in a 1.2% agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized with a ³²P-labeled ZFR1-specific probe (A) and a ³²P-labeled β-tubulin-specific probe (B).

FIG. 2.

Northern blot analysis and FB₁ production of the wild type cultured on separated maize kernel germ and degermed tissues. Total RNA (10 μg) isolated from the wild-type strain was cultured for 7 days on maize kernel germ (lane 1) and degermed (lane 2) fractions. The blot was probed with ZFR1 (A) and TUB2 (B) as a loading control. (C) Quantification of FB₁ as determined by HPLC extracted from germ (G) and degermed (DG) kernel fractions. Averages of three repetitions are shown with error bars showing standard errors.

FIG. 3.

Analysis of the ZFR1 deletion and rescued strains. Shown is a Southern analysis of genomic DNAs (2 μg) from the wild-type (strain M-3125, lane 1), zn27ss (zfr1 deletion strain 1, lane 2), zfr1Δ (zfr1 deletion strain 2, lane 3), fcc1Δ (fcc1 deletion strain, lane 4), fcc1Δ GPD::ZFR1 (fcc1Δ mutant transformed with GPD::ZFR1, lane 5), zfr1Δ GPD::ZFR1 (zfr1 deletion strain 2 transformed with GPD::ZFR1, lane 6), and zfr1Δ ZFR1 (zfr1Δ strain transformed with ZFR1, lane 7) strains digested with EcoRI, separated by electrophoresis in a 0.7% agarose gel, transferred to a nylon membrane, and probed with a ³²P-labeled DNA fragment of ZFR1. Molecular size standards are indicated on the left.

FIG. 4.

FB₁ production by the wild type (wt) and the zfr1 deletion mutant on whole cracked maize kernels and separated maize kernel germ and degermed tissues. (A) Wild-type and zfr1Δ mutant (zfr1 deletion) strains cultured on whole cracked maize kernels for 28 days and analyzed for FB₁ every 7 days. (B) Wild-type and zfr1Δ mutant strains cultured for 7 days on separated germ and degermed maize kernel tissue fractions and analyzed for FB₁ production. All values are averages of three repetitions, and error bars indicate standard errors greater than 5% of the mean value.

FIG. 5.

Northern blot analysis of wild-type and ZFR1 overexpression strains. Total RNAs (10 μg) isolated from the zfr1Δ mutant (zfr1 deletion strain, lane 1), the wild type (lane 2), the fcc1Δ/GPD::ZFR1 mutant (fcc1Δ mutant strain transformed with a ZFR1 overexpression construct [GPD::ZFR1], lane 3), the zfr1Δ GPD::ZFR1 mutant (zfr1 deletion strain transformed with GPD::ZFR1, lane 4), and the fcc1Δ mutant (fcc1 deletion strain) extracted from cultures grown for 7 days on whole cracked maize kernels were separated by electrophoresis in a 1.2% agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized with ³²P-labeled FUM1- and FUM8-specific probes (top), a ³²P-labeled ZFR1-specific probe (middle), and a ³²P-labeled β-tubulin-specific probe (bottom).

FIG. 6.

Effect of ZFR1 overexpression on FB₁ production. The wild-type, zfr1Δ mutant (zfr1 deletion mutant), zfr1Δ-R mutant (zfr1Δ mutant transformed with ZFR1), zfr1Δ GPD::ZFR1 mutant (zfr1Δ mutant strain transformed with a GPD::ZFR1 overexpression construct), fcc1Δ mutant, and fcc1Δ GPD::ZFR1 mutant (fcc1Δ mutant transformed with a GPD::ZFR1 overexpression construct) strains were cultured for 7 days on whole cracked maize kernels and analyzed for FB₁ production. All values are averages of three repetitions, and error bars indicate standard errors. n.d. = none detected.