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Applied and Environmental Microbiology, June 2006, p. 3924-3932, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.00963-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The PKS4 Gene of Fusarium graminearum Is Essential for Zearalenone Production

Bioforsk—Norwegian Institute for Agricultural and Environmental Research, Plant Health and Plant Protection Division, Høgskoleveien 7, N-1432 Ås, Norway,¹ Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway,² The Department of Ecology, The Royal Veterinary and Agricultural University, DK-1871 Fredriksberg C, Copenhagen, Denmark,³ Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads 221, DK-2800 Kgs. Lyngby, Denmark⁴

Received 26 April 2005/ Accepted 22 March 2006

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.

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