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.

FIG. 1.

Structural organization of C. heterostrophus predicted NPS proteins. Abbreviations: A, adenylation; dA, degenerate adenylation; T, thiolation; C, condensation; E, epimerization; M, N-methyltransferase; D, dehydrogenase; KS, beta-ketoacyl synthase; AT, acyl transferase; DH, dehydratase; KR, ketoreductase. Shaded regions indicate portions of genes that were deleted to make nps mutations. Each box represents an open reading frame, the size of which is indicated on the right in kilobases. The dotted vertical line in NPS9 indicates incomplete sequence. No two predicted proteins are alike in their domain structure.

FIG. 2.

Conserved A, C, and E motifs. A comparison of ten A, five C, and two E conserved motifs is shown (41). For each motif, the fungal consensus, based on amino acid sequences encoded by all fungal NPS genes examined in this study (Fig. 7; see Table S2 for complete list) is compared with the bacterial consensus reported (41). The fungal C domains have only C2 to C5 and lack the C1, C6, and C7 domains found in bacterial nonribosomal peptide synthetases. Similarly, the fungal E domains have only E1 and E2 and lack the E3 to E7 domains found in bacterial nonribosomal peptide synthetases.

FIG. 3.

Expression of 11 NPS genes in wild-type strain C4 grown under various conditions. Blue/purple: 6 h of germination in liquid complete medium. Orange: 20 h of vegetative growth in liquid complete medium. Yellow: 72 h of asexual development on solid complete medium with 2% xylose. Light blue: 120 h in liquid Fries medium. Maroon: 24 h in liquid minimal medium minus nitrogen. Total RNA from the wild type was tested. Relative abundance was determined by normalizing changes to actin expression (23) (see Table S1).

FIG. 4.

nps6 mutant has reduced virulence on maize. Leaves were inoculated with progeny of a cross between an nps6 mutant and the wild type. All hygromycin B-resistant progeny are nps6 mutants (from left, leaves 3 to 7). Hygromycin B-sensitive progeny are the wild type (from left, leaves 8 to 13). The photo was taken 4 days postinoculation.

FIG. 5.

Evaluation of growth of nps6 mutant strains versus wild-type strains. nps6 mutants grow normally. Symbols: diamond = nps6 (N26-R-9); square = nps6 (N26-R-10); triangle = NPS6 (N26-R-11); and circle = NPS6 (C4). Measurements were taken at 12-h intervals.

FIG. 6.

Colony diameter increase in 24 h of colonies measured on 7 mM H₂O₂ amended medium, at 2 and 3 days after setting up the assay. Parents and progeny segregate 1:1 for three phenotypes: sensitivity to hydrogen peroxide, resistance to hygromycin B, and reduced virulence. For strain genotypes, see Table 1 and Materials and Methods. Bar, standard deviation.

FIG. 7.

Homology of NPS6. Genealogy of 125 NPS AMP-binding adenylation (A) domains of 48 NPS and NPS-like proteins, inferred by maximum parsimony analysis. Vertical bars mark major clades that include functionally similar proteins, and subclades that are comprised of apparently homologous proteins that are predicted to be identical or nearly identical in their basic function. Branch length indicates number of inferred amino acid changes. Numbers below branches indicate percent bootstrap support (when >70%) for each clade (performed with 100 repetitions). Accession numbers for all sequences obtained from GenBank are given in Table S2. Shown is one of four most-parsimonious trees (only the branching order of the NPS6 A domains was ambiguous) (arrow).

FIG. 8.

Cartoon of the organization of proteins encoded by nine NPS6 genes and the amino acid specificity code (42) of A module 1 for each of nine homologous NPS6 proteins. Note conservation of specificity residues; numbers correspond to specificity residue positions, (42). The homolog from M. grisea was inferred to have the same modular arrangement; the contig containing this gene terminates prematurely after the first module, and the second module was not found elsewhere in the genomic sequence. Ch, C. heterostrophus; Fg, F. graminearum; Bc, B. cinerea; Nc, N. crassa; Mg, M. grisea; Fv, F. verticillioides; Af, A. fumigatus; An, A. nidulans; Ao, A. oryzae; A, T, C, and dA, see the legend to Fig. 1.