Functional characterization of two cytochrome P450 monooxygenase genes, P450-1 and P450-4, of the gibberellic acid gene cluster in Fusarium proliferatum (Gibberella fujikuroi MP-D)

Abstract

Gibberella fujikuroi is a species complex with at least nine different biological species, termed mating populations (MPs) A to I (MP-A to MP-I), known to produce many different secondary metabolites. So far, gibberellin (GA) production is restricted to Fusarium fujikuroi (G. fujikuroi MP-C), although at least five other MPs contain all biosynthetic genes. Here, we analyze the GA gene cluster and GA pathway in the closest related species, Fusarium proliferatum (MP-D), and demonstrate that the GA genes share a high degree of sequence homology with the corresponding genes of MP-C. The GA production capacity was restored after integration of the entire GA gene cluster from MP-C, indicating the existence of an active regulation system in F. proliferatum. The results further indicate that one reason for the loss of GA production is the accumulation of several mutations in the coding and 5' noncoding regions of the ent-kaurene oxidase gene, P450-4.

FIG. 1.

Gibberellin biosynthesis pathway and gene cluster. (A) Pathway showing genes, enzymes, and final products. The major pathway is denoted by enlarged arrows and letters. GGPP, geranylgeranyl-pyrophosphate; CPP, copalylpyrophosphate. (B) Gene cluster of the GA biosynthesis pathway (about 20 kb). Numbers 1 to 6 indicate the different primer combinations for amplification of the overlapping regions between two cluster genes (1, orf3-Fus1/P450-4-Fus2; 2, P4-Fus1/P1-Fus2; 3, P450-1-Fus3/P450-2-Fus4; 4, P450-2-Fus5/ggs2-Fus6; 5, ggs2-Fus3/cps-Fus5; 6, cps-Fus7/P450-3-Fus8).

FIG. 2.

Comparison of des and P450-1 gene expression for wild-type IMI58289, MP-D02945, and transformant cTcos1D-2. rRNA (rDNA) was used as a loading control. For hybridization, a 1-kb des (C) and a 1.5-kb P450-1 (C) cDNA clone were used.

FIG. 3.

Northern blot analysis of strains SG139, IMI58289 (MP-C), and D02945 and transformants containing P450-4 (D), P450-4 (C), and P450-1 (C). Strains were grown for 3 days in 20% ICI medium. (A) P450-4 (D) was transformed into strain SG139. A 1.5-kb HindIII fragment of plasmid pP450-4GKD was used for hybridization. (B) P450-4 (D) was transformed into strain D02945. A 1.5-kb HindIII fragment of plasmid pP450-4GKD was used for hybridization. (C) P450-4 (C) was transformed into strain D02945. A 1.5-kb cDNA clone of P450-4 (C) was used for hybridization. (D) P450-1 (C) was transformed into strain D02945. A 1.5-kb cDNA clone of P450-1 (C) was used for hybridization.

FIG. 4.

Northern blot analysis of strains SG139 and IMI58289 and transformants of strain SG139 containing P450-1 (D). Strains were grown for 3 days in 20% ICI medium. A 1.6-kb SphI fragment of plasmid pP450-1GKD was used for hybridization.

FIG. 5.

Enzyme activity test of P450-1 genes from MP-D and MP-C in both genetic backgrounds in a time course experiment after incubation with [¹⁴C]GA₁₂-aldehyde. Strains were precultivated as described in Materials and Methods and harvested. Mycelia were then cultivated in 10 ml of 0% ICI medium, and at each time course, a 2-ml aliquot was taken from the culture. Values shown are the results of four independent experiments. Error bars indicate standard deviations. (A) Comparison of P450-1 activity in transformants cTP1-C SG139 [carrying P450-1 (C)] and dTP1-C SG139 [carrying P450-1 (D)]. (B) Comparison of P450-1 activity in F. proliferatum D00502 and transformant cTP1-D2, carrying P450-1 (C).

FIG. 6.

Sequence alignment of the bidirectional promoter region between genes P450-1 and P450-4 of the GA gene cluster in MP-C and MP-D. Gene directions are marked with arrows, and GATA-binding motifs are indicated as boxes. Mutations in GATA-binding motifs are marked with asterisks above (in the case of MP-C) and below (in the case of MP-D) the GATA boxes. The bidirectional promoter region sequence has been deposited in the GenBank database (accession number AJ628021).

FIG. 7.

GUS reporter assays. (A) Plate assay with dP1GUS-C transformants of SG139, all carrying more than two gene copies of the promoter construct pDP1::GUS. Plates were incubated for 5 days on X-Gluc agar. (B) Quantitative measurement of GUS activity. The different promoter constructs were fused to the E. coli uidA gene. Relative GUS activity is given on the right of the corresponding promoter construct. Positions of GATA motifs are indicated as ovals, and grey ovals show GATA elements differing from the MP-C promoter (cP4GUS-C).