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

Abstract

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.

Dothideomycete–Plant Interactions Illuminated by Genome Sequencing and EST Analysis of the Wheat Pathogen Stagonospora nodorum

“…was identified in the genome of S. nodorum as was found for F. graminearum (Gaffoor et al, 2005) and C. heterostrophus (Lee et al, 2005). C. heterostrophus NPS7 and SNOG_00308.2 do not appear to be closely related, and it is likely that they evolved independently.…”

“…NRPS genes from C. heterostrophus have been analyzed in detail (Lee et al, 2005). Comparison of both protein sequences and domain structure of the C. heterostrophus NRPS genes (NPS1-11) with the eight identified in S. nodorum was undertaken (Table 6).…”

“…Interestingly, a further seven NRPS genes from C. heterostrophus (NPS3,5,8,10,11,and 12) have no obvious ortholog in S. nodorum, suggesting expansion of this subfamily in the maize pathogen (Yoder and Turgeon, 2001). Intensive studies in C. heterostrophus showed that individual gene knockouts produced altered phenotypes only for NPS6 (Lee et al, 2005), indicating redundancy of function. The smaller complement of NRPS genes in M. grisea (eight) and S. nodorum indicate that these organisms may be more fruitful models to study NRPS Each species name is preceded by a unique AFTOL ID number (www.aftol.org).…”

“…M. grisea has nine TECAT/CATE/CAT/ CATE/C/T/C/T a Domain abbreviations: A, adenylation; C, condensation; Cy, Cyclization; E, epimerization; T, thiolation; Te, Thioesterase. Potential orthologs were identified by best BLASTP hits to NRPS genes in C. heterostrophus (Lee et al, 2005) and by best informative hit to the nonredundant database at NCBI of e-value <1 3 10 À10 . b The best hit of the identified gene in the S. nodorum gene set was observed reciprocally.…”

Phylogenomic analysis of polyketide synthase-encoding genes in Trichoderma

“…Non-ribosomal peptides and polyketides comprise a major portion of these products, which are synthesized by large proteins composed of various domains for the individual enzymic steps (Cox, 2007). Both broad and genus-specific phylogenomic and functional analysis of polyketide synthase (PKS)-and non-ribosomal peptide synthetase (NRPS)-encoding genes have been performed, which has accelerated the pace at which genes and pathways are being linked to specific secondary metabolites (Bushley & Turgeon, 2010;Bushley et al, 2008;Chiang et al, 2010;Cramer et al, 2006;Gaffoor et al, 2005;Kroken et al, 2003;Kubicek et al, 2011;Lee et al, 2005).…”