Global analysis of serine-threonine protein kinase genes in Neurospora crassa

Abstract

Serine/threonine (S/T) protein kinases are crucial components of diverse signaling pathways in eukaryotes, including the model filamentous fungus Neurospora crassa. In order to assess the importance of S/T kinases to Neurospora biology, we embarked on a global analysis of 86 S/T kinase genes in Neurospora. We were able to isolate viable mutants for 77 of the 86 kinase genes. Of these, 57% exhibited at least one growth or developmental phenotype, with a relatively large fraction (40%) possessing a defect in more than one trait. S/T kinase knockouts were subjected to chemical screening using a panel of eight chemical treatments, with 25 mutants exhibiting sensitivity or resistance to at least one chemical. This brought the total percentage of S/T mutants with phenotypes in our study to 71%. Mutants lacking apg-1, an S/T kinase required for autophagy in other organisms, possessed the greatest number of phenotypes, with defects in asexual and sexual growth and development and in altered sensitivity to five chemical treatments. We showed that NCU02245/stk-19 is required for chemotropic interactions between female and male cells during mating. Finally, we demonstrated allelism between the S/T kinase gene NCU00406 and velvet (vel), encoding a p21-activated protein kinase (PAK) gene important for asexual and sexual growth and development in Neurospora.

Fig. 1.

Venn diagram showing distribution of S/T protein kinase mutants with phenotypes. The 44 viable mutants exhibiting defects in at least one of the three major growth/developmental pathways are indicated as gene names for the deleted genes.

Fig. 2.

Trichogyne chemotropism in the mutant lacking NCU02245/stk-19. The wild type and the ΔNCU02245 (Δstk-19) strain were cultured to produce female reproductive structures (protoperithecia). Sterile agar blocks were then placed on top of protoperithecia. Subsequently, a conidial suspension (males) from a wild-type strain of opposite mating type was applied to the top of the agar block. Trichogyne (TR) growth and migration through the agar blocks toward the males was monitored microscopically 16 to 24 h after application of the conidia.

Fig. 3.

Allelism between the S/T kinase gene NCU00406 and velvet (vel). (A) Colony morphology and vegetative growth of ΔNCU00406 and vel mutants complemented with a construct containing the wild-type NCU00406 gene. C00406-7-1 and Cvel-1-1 are ΔNCU00406 and vel mutants, respectively, transformed with the complementation construct for NCU00406. Strains were grown in VM agar plates (top) and flasks (bottom) for 24 h and 14 days, respectively. (B) Sequence analysis of the NCU00406 ORF region in the vel mutant. After amplification and cloning of the NCU00406 ORF region from the vel mutant, the 2,502-bp sequence was compared with the corresponding sequence from the wild type. The synonymous mutation at position 765 that does not alter the amino acid sequence is shown, along with the premature stop codon at 1144.