The PKS4 gene of Fusarium graminearum is essential for zearalenone production

Abstract

Zearalenones are produced by several Fusarium species and can cause reproductive problems in animals. Some aurofusarin mutants of Fusarium pseudograminearum produce elevated levels of zearalenone (ZON), one of the estrogenic mycotoxins comprising the zearalenones. An analysis of transcripts from polyketide synthase genes identified in the Fusarium graminearum database was carried out for these mutants. PKS4 was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An Agrobacterium tumefaciens-mediated transformation protocol was used to replace the central part of the PKS4 gene with a hygB resistance gene through double homologous recombination in an F. graminearum strain producing a high level of ZON. PCR and Southern analysis of transformants were used to identify isolates with single insertional replacements of PKS4. High-performance liquid chromatography analysis showed that the PKS4 replacement mutant did not produce ZON. Thus, PKS4 encodes an enzyme required for the production of ZON in F. graminearum. Barley root infection studies revealed no alteration in the pathogenicity of the PKS4 mutant compared to the pathogenicity of the wild type. The expression of PKS13, which is located in the same cluster as PKS4, decreased dramatically in the mutant, while transcription of PKS4 was unchanged. This differential expression may indicate that ZON or its derivatives do not regulate expression of PKS4 and that the PKS4-encoded protein or its product stimulates expression of PKS13. Furthermore, both the lack of aurofusarin and ZON influenced the expression of other polyketide synthases, demonstrating that one polyketide can influence the expression of others.

FIG. 1.

(A) Reverse transcriptase PCR with the F. pseudograminearum wild type (WT) and aurofusarin mutants PK2.1 and KHt62.1 and with primers designed for PKSs with an ER domain. (B) Reverse transcriptase PCR with the F. pseudograminearum wild type and mutants on three PKSs without an ER domain. The F. pseudograminearum strains were cultivated for 2 weeks on a medium described by Bell et al. (2). (C) Reverse transcriptase PCR with PKS4-specific primers for F. graminearum strain 1104-14, which produces high levels of ZON, after 4, 7, and 11 days of growth on polished rice. All PCR products are about 400 bp long. The PKS designations are based on those of Kroken et al. (23).

FIG. 2.

Schematic diagram of the PKS4 (FG12055) gene in F. graminearum, showing putative catalytic domains and, at the bottom, the replacement cassette from pAg1-H3-PKS4 used to disrupt the gene. The small open boxes in PKS4 are putative introns. Primers PKS4-Hyg-A1 and PKS4-Hyg-A2 and primers PKS4-Hyg-B1 and PKS4-Hyg-B2 were used to verify replacement of PKS4 (Fig. 3). These primers are located inside the hygB gene and outside the replacement cassette in vector pAg1-H3-PKS4, but they are within the flanking PKS4 gene sequence and give a product only if homologous recombination occurs. For Southern blot analysis (Fig. 4), the following three probes were used: the 1,684-bp hygB probe from BglII/AsiSI, the 1,511-bp PKS4-1 probe from BglII/XhoI, and the 794-bp delPKS4 probe made with primers PKS4-del-1 and PKS4-del-2 from the deleted sequence of PKS4. KS, β-ketoacyl synthase; AT, acyl transferase; ACP, acyl carrier protein; DH, dehydratase; KR, keto reductase; ER, enoyl reductase.

FIG. 3.

PCR products from genomic DNA of wild-type strain F. graminearum 1104-14 (WT) and ΔPKS4 mutants amplified with primers PKS4-Hyg-A1 and PKS4-Hyg-A2 (lanes A) and with primers PKS4-Hyg-B1 and PKS4-Hyg-B2 (lanes B). The 2.4-kb and 2.6-kb bands resulted from homologous recombination and replacement of the central part of PKS4 with hygB. For the locations of the primers, see Fig. 2. ΔPKS4 mutants T4, T5, T9, and T11 are double homologous recombinants, while T7, T8, and T12 are single homologous recombinants.

FIG. 4.

Southern blot analysis of F. graminearum PH1, wild type (WT), and ΔPKS4 mutants. Genomic DNA was digested with BglII. (A) Analysis with probe made from a BglII/AsiSI digest of the hygB sequence from pAg1-H3-PKS4. (B) Analysis with probe made from a PCR product obtained with primers PKS4-del-1 and PKS4-del-2 from the deleted sequence of PKS4. (C) Analysis with probe made from a BglII/XhoI digest of the PKS4-1 sequence from pAg1-H3-PKS4. For the locations of probes, see Fig. 2.

FIG. 5.

HPLC traces for the wild type (WT) and ZON mutant ΔPKS4-T9 after 11 days of growth on rice.

FIG. 6.

Relative expression of PKS4 and PKS13 in the wild-type strain (WT) and the ΔPKS4-T9 mutant cultured for 4, 7, 11, and 14 days on rice. Expression data were normalized to the BestKeeper Index based on the internal control genes encoding β-tubulin (FG09530), translation elongation factor 1α (FG08811), and ubiquitin conjugating enzyme (FG10805) and were calibrated with the level of expression of PKS4 in ΔPKS4-T9 cultured for 4 days under ZON-producing conditions. The results are the averages for three replicate cultures and a minimum of three independent amplifications.

FIG. 7.

Reverse transcriptase PCR for F. graminearum wild type (WT) and ZON mutant ΔPKS4-T9 with primers designed for all PKS genes except PKS4 and PKS13. The strains were cultivated for 7 days on rice at 25°C in the dark.