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Eukaryotic Cell, March 2005, p. 545-555, Vol. 4, No. 3
1535-9778/05/$08.00+0     doi:10.1128/EC.4.3.545-555.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Analysis of All Nonribosomal Peptide Synthetases in Cochliobolus heterostrophus Reveals a Factor, NPS6, Involved in Virulence and Resistance to Oxidative Stress{dagger}

Bee-Na Lee,{ddagger} Scott Kroken,§ David Y. T. Chou, Barbara Robbertse,|| O. C. Yoder,# and B. Gillian Turgeon*

Torrey Mesa Research Institute, San Diego, California

Received 1 October 2004/ Accepted 9 December 2004

Nonribosomal peptides, made by nonribosomal peptide synthetases, have diverse biological activities, including roles as fungal virulence effectors. Inspection of the genome of Cochliobolus heterostrophus, a fungal pathogen of maize and a member of a genus noted for secondary metabolite production, revealed eight multimodular nonribosomal peptide synthase (NPS) genes and three monomodular NPS-like genes, one of which encodes a nonribosomal peptide synthetase/polyketide synthase hybrid enzyme presumed to be involved in synthesis of a peptide/polyketide molecule. Deletion of each NPS gene and phenotypic analyses showed that the product of only one of these genes, NPS6, is required for normal virulence on maize. NPS6 is also required for resistance to hydrogen peroxide, suggesting it may protect the fungus from oxidative stress. This and all other nps mutants had normal growth, mating ability, and appressoria. Real-time PCR analysis showed that expression of all NPS genes is low (relative to that of actin), that all (except possibly NPS2) are expressed during vegetative growth, and that expression is induced by nitrogen starvation. Only NPS6 is unfailingly conserved among euascomycete fungi, including plant and human pathogens and saprobes, suggesting the possibility that NPS6 activity provides oxidative stress protection during both saprobic and parasitic growth.

* Corresponding author. Mailing address: Department of Plant Pathology, 334 Plant Science Bldg., Cornell University, Ithaca, NY 14853. Phone: (607) 254 7458. Fax: (607) 255 8835. E-mail: bgt1{at}cornell.edu.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

{ddagger} Present address: School of Biological Science, Nanyang Technology University, Singapore.

§ Present address: Department of Plant Sciences, Division of Plant and Microbial Biology, University of Arizona, Tucson, Ariz.

Present address: Biology Department, San Diego State University, San Diego, Calif.

|| Present address: Department of Plant Pathology, Cornell University, Ithaca, N.Y.

# Present address: Diversa Corp., San Diego, Calif.

Eukaryotic Cell, March 2005, p. 545-555, Vol. 4, No. 3
1535-9778/05/$08.00+0     doi:10.1128/EC.4.3.545-555.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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