A mitogen-activated protein kinase pathway essential for mating and contributing to vegetative growth in Neurospora crassa

Abstract

MAP kinases homologous to Saccharomyces cerevisiae Fus3p/Kss1p have been identified in plant pathogenic fungi and are required for pathogenicity and sexual reproduction. To better understand the role of MAP kinase signaling in Neurospora crassa, and to identify downstream target genes of the pathway, we isolated, cloned, and disrupted the FUS3 homolog mak-2. Ste12p is a transcription factor target of Fus3p that activates genes of the mating pathway in yeast, and we also characterized the N. crassa STE12 homolog pp-1. The mak-2 and pp-1 mutants have reduced growth rate, produce short aerial hyphae, and fail to develop protoperithecia. In addition, ascospores carrying null mutations of either gene are inviable. Subtractive cloning was used to isolate genes having reduced expression in the mak-2 mutant. Expression of some of these genes is protoperithecia specific and three of them are part of a gene cluster potentially involved in the production of a polyketide secondary metabolite. Microarray analysis was used to extend the analysis of gene expression in mak-2 and pp-1 mutants. The role of the MAP kinase pathway in both sexual and asexual development as well as secondary metabolism is consistent with the dual regulation of the mating process and pathogencity observed in fungal pathogens.

Figure 1.—

Construction of mak-2 and pp-1 gene replacement mutants. (A) Physical map of the mak-2 genomic region and gene replacement vector pBP-KOMAK2. The XhoI site introduced from primer MAK1 is indicated with an asterisk. PDL37 and PDL38 are primers used to screen Δmak-2 knockout mutants. The indicated 1.5-kb KpnI fragment was used as a hybridization probe for Southern blot analysis. The restriction sites are X, XhoI; K, KpnI; H, HindIII; B, BamHI. (B) Physical map of pp-1 genomic region and gene replacement vector pBP-KOSTE12. STE11, STE14, STEK4, and CTRP2 are primers used to screen Δpp-1 knockout mutants. (C) Southern analysis of Δmak-2 knockout mutant strains PBM5, PBM7, and PBM49. Genomic DNAs were digested with BamHI (B) or KpnI (K) yielding fragments of the indicated sizes (kb). (D) Southern analysis of the Δpp-1 knockout mutant strain DL14. Genomic DNAs were digested with XhoI (X) or KpnI (K) to yield fragments of the indicated sizes.

Figure 2.—

Phenotypes of wild-type (WT), Δpp-1, and Δmak-2 isolates. (A) Colony growth on VM plates after 4 days at 34°. (B) Protoperithecia (arrow) formation on SC plates after 7 days at 25°. No protoperithecia were observed in the mutants. (C) Perithecia (arrow) development on SC plates after 7 days growth that were fertilized with WT (74-ORS6a) conidia overnight. No perithecia were observed in the mutants. Conidiation phenotype of WT (74-OR23-1VA), Δpp-1, and Δmak-2 strains in SC liquid medium. The cultures were grown at 34° with constant agitation at 250 rpm. At 16 hr of incubation, conidiophores were observed in the mak-2 mutant (arrow) but not in the pp-1 mutant or wild-type cultures. By 24 hr, conidiophore formation was observed in all strains (arrows), but was predominant for the mak-2 mutant.

Figure 3.—

Aerial growth of strains with and without 2 mm cAMP for wild-type, Δpp-1, Δmak-2, and cr-1 mutants. Aerial development of the cr-1, but not pp-1 or mak-2 mutants, was rescued by cAMP.

Figure 4.—

Expression patterns of the pks/mkr gene cluster in the WT, Δpp1 mutant, and Δmak-2 mutant. Strains were grown on SC solid medium for 7 days at 25° (SC solid), and then fertilized with 74-ORS6a conidia for 24 hr at 25° (24 hr fertilized), on VM solid medium for 7 days at 34° (VM solid), in SC liquid medium for 24 hr with 250 rpm shaking at 34° (SC liquid), in VM liquid medium for 24 hr with 250 rpm shaking at 34°. RNA was extracted from harvested cultures and from conidia. (A) Northern hybridization analysis. RNA blots were hybridized in succession with probes for mkr-3, mkr-5, mkr-6, and mkr-2. The blots were then hybridized with a rDNA probe to check the relative amount of RNA in each sample. (B) RT-PCR analysis of pks gene expression. PCR was performed with genomic DNA as a positive control (P) and no template as a negative control (N). The ΛDNA-BstEII digest ladder (New England Biolabs) was used to estimate the size of the PCR products (M). A 661-bp pks fragment (indicated by the arrow) was amplified with the gene-specific primers PDL21 and PDL22 using the first-strand cDNA as a template (top). The same cDNA samples were also used for RT-PCT with a primer pair specific for the actin gene, which was used as a control (bottom). The arrows indicate the PCR products amplified from gDNA (980 bp) and cDNA (917 bp).

Figure 5.—

Relative positions of genes in the pks/mkr gene cluster in N. crassa and an orthologous cluster in M. grisea. Arrows indicate the orientations of mkr genes. The distances between the ORFs are indicated.

Figure 6.—

Model for MAP kinase pathway regulation of gene expression and development. (A) The cAMP-signaling pathway may regulate the mak-2-related MAP kinase pathway to control aerial development or the two pathways may act independently. (B) Conidiophore development is repressed by the cAMP pathway directly or by its effect on the MAK-2 pathway. On the basis of microarray and enhanced conidiation of the mutants, MAK-2 exerts a stronger effect than PP-1 and MAK-2 may act through PP-1 and another (?) factor to repress conidiation. (C) Protoperithecial development and ascospore lethality depend on the MAK-2 pathway and is independent of cAMP signaling.