Loss of gibberellin production in Fusarium verticillioides (Gibberella fujikuroi MP-A) is due to a deletion in the gibberellic acid gene cluster

Abstract

Fusarium verticillioides (Gibberella fujikuroi mating population A [MP-A]) is a widespread pathogen on maize and is well-known for producing fumonisins, mycotoxins that cause severe disease in animals and humans. The species is a member of the Gibberella fujikuroi species complex, which consists of at least 11 different biological species, termed MP-A to -K. All members of this species complex are known to produce a variety of secondary metabolites. The production of gibberellins (GAs), a group of diterpenoid plant hormones, is mainly restricted to Fusarium fujikuroi (G. fujikuroi MP-C) and Fusarium konzum (MP-I), although most members of the G. fujikuroi species complex contain the GA biosynthesis gene cluster or parts of it. In this work, we show that the inability to produce GAs in F. verticillioides (MP-A) is due to the loss of a majority of the GA gene cluster as found in F. fujikuroi. The remaining part of the cluster consists of the full-length F. verticillioides des gene (Fvdes), encoding the GA(4) desaturase, and the coding region of FvP450-4, encoding the ent-kaurene oxidase. Both genes share a high degree of sequence identity with the corresponding genes of F. fujikuroi. The GA production capacity of F. verticillioides was restored by transforming a cosmid with the entire GA gene cluster from F. fujikuroi, indicating the existence of an active regulation system in F. verticillioides. Furthermore, the GA(4) desaturase gene des from F. verticillioides encodes an active enzyme which was able to restore the GA production in a corresponding des deletion mutant of F. fujikuroi.

FIG. 1.

GA-biosynthetic pathway in F. fujikuroi. The major pathway is indicated by bold arrows.

FIG. 2.

Southern blot analysis of F. fujikuroi IMI58289 and several F. verticillioides isolates. The genomic DNA of all strains was digested with HindIII and hybridized with a 1:2 mixture of the gene probes from F. fujikuroi and F. verticillioides (for des and P450-4) or with the F. fujikuroi gene (P450-1). Ff, F. fujikuroi; Fv, F. verticillioides.

FIG. 3.

Comparison of the GA gene clusters in F. fujikuroi and F. verticillioides. Crosses indicate gene inversions, dashed lines demonstrate putative inversions, and arrows show the orientation of transcription. GA genes are shown in gray, and genes not belonging to the cluster are colored.

FIG. 4.

Northern blot analysis of F. verticillioides isolates and the F. fujikuroi wild-type strain IMI58289. Total RNA was hybridized with the probes as indicated. P450-3 is not present in the genome of F. verticillioides and was used as a negative control. Fv, F. verticillioides; Ff, F. fujikuroi.

FIG. 5.

Analysis of ent-kaurene oxidase P450-4 expression. (A) Northern blot analysis of F. fujikuroi wild-type strain IMI58289, the F. fujikuroi mutant B1-41a carrying a point mutation in the gene P450-4, and transformants of B1-41a transformed (+) with the F. verticillioides ent-kaurene oxidase gene FvP450-4, driven either by its own promoter or the promoter of the F. fujikuroi P450-4 gene (FfP4_prom::FvP4). (B) RT-PCR analysis of the P450-4 gene in different fusaria and mutant strains. Lanes: 1 and 10, markers; 2, F. verticillioides cDNA; 3, F. verticillioides genomic DNA; 4, F. fujikuroi cDNA; 5, B1-41a cDNA; 6, B1-41a FvP450-4 T6 cDNA; 7, B1-41a FfP4_prom::FvP4 T3 cDNA; 8, F. fujikuroi cDNA; 9, F. fujikuroi genomic DNA. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.

FIG. 6.

(A) Northern blot analysis of strains F. fujikuroi IMI58289, the F. fujikuroi FfΔdes mutant, and transformants of the FfΔdes mutant complemented (+) with Fvdes. Total RNA was hybridized with the probes as indicated. (B) GC-MS analysis of culture filtrates of FfΔdes and of transformants of FfΔdes complemented (+) with Fvdes (T2, T3, and T4). Total ion currents are shown for ethyl acetate extracts as methyl esters trimethylsilyl ethers. Components were identified by comparison of their mass spectra and GC retention times with published data (15). Peaks: 1, GA₉; 2, GA₂₅; 3, GA₁₄; 4, GA₂₀; 5, GA₂₄; 6, 7β-hydroxykaurenolide; 7, GA₄; 8, gibberellenic acid; 9, GA₇; 10, GA₄₀; 11, GA₁₃; 12, fujenoic acid; 13, GA₄₇; 14, GA₁₆; 15, iso-GA₃; 16, GA₃₆; 17, GA₁; 18, 7β,18-dihydroxykaurenolide; 19, GA₃. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.

FIG. 7.

Northern blot analysis of F. fujikuroi wild-type strain IMI58289, F. verticillioides wild-type strain A00149, and A00149 transformed (+) with additional copies of the Fvdes gene from strain A00149. Total RNA was hybridized with Fvdes. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.

FIG. 8.

Transformation (+) of F. verticillioides A00149 with the cosmid (pCos1) carrying the entire GA gene cluster from F. fujikuroi. (A) Southern blot analysis of F. fujikuroi IMI58289, F. verticillioides A00149, and transformants of A00149 carrying pCos1. The genomic DNA was restricted with HindIII. The filter was probed with GA-biosynthetic genes from F. fujikuroi as indicated. (B) Northern blot analysis of F. fujikuroi IMI58289, F. verticillioides A00149, and transformants of strain A00149 with pCos1. The filter was probed with GA-biosynthetic genes from F. fujikuroi as indicated. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.

FIG. 9.

GC-MS analysis of culture filtrates of wild-type strains F. fujikuroi IMI58289 and F. verticillioides A00149 and strains of A00149 transformed (+) with pCos1 carrying the entire F. fujikuroi GA gene cluster. Total ion current for ethyl acetate extracts after derivatization to methyl esters trimethylsilyl ethers is shown. Peak identities from comparison with published mass spectra (15) are as listed for Fig. 6B, with, additionally, GA₄₂ (peak 20). Ff, F. fujikuroi; Fv, F. verticillioides.

FIG. 10.

Heterologous expression of Fvdes in F. fujikuroi mutant SG139. The entire GA gene cluster is missing in strain SG139. (A) Southern blot analysis of F. fujikuroi IMI58289 (wild type), F. fujikuroi SG139, and transformants (+) of SG139 with the gene Fvdes. Genomic DNA was digested with HindIII and probed with the genomic fragment of gene Fvdes. (B) Northern blot analysis of strains F. fujikuroi IMI58289 (wild type) and F. fujikuroi SG139 and transformants of SG139 which were transformed (+) with the gene Fvdes. The filter was probed with the genomic fragment of gene Fvdes. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.