Table 1.

Strains and culture conditions.

Vogel's minimal medium (VM) was used for vegetative growth and synthetic crossing medium (SCM) for development of female sexual structures (17). Sorbose-containing FGS medium was used for isolation of colonies on plates (17). Hygromycin (Calbiochem, San Diego, CA) was used at 200 μg/ml in media where indicated. Wild-type strains ORS-SL6a (FGSC 4200;mat a) and 74-OR23-IVA (FGSC 2489;mat A) were obtained from the Fungal Genetics Stock Center (FGSC; Kansas City, MO). Gene replacement mutants for S/T kinase genes were generated using a previously described high-throughput method (see Table S1 in the supplemental material) (13).

Mutant constructions.

Deletion cassettes for kinase genes were constructed using yeast recombinational cloning in a 96-well plate system as described previously (12,13). Cassettes were transformed intoNeurospora mutants deficient in nonhomologous end-joining DNA repair (mus-51 ormus-52) by electroporation in 96-well plates, with selection on hygromycin-containing FGS medium. Transformants were picked onto VM agar slants containing hygromycin and then crossed to wild-type 74-OR23-IVA (mat A) on SCM. Ejected ascospores were harvested from each cross and plated on FGS agar plates containing hygromycin. Spot testing was used to identify progeny that lacked themus-51 ormus-52 mutation (both marked withbar, conferring resistance to phosphinothricin [2]) and to determine mating type. Homokaryotic mutant strains were confirmed by Southern blot analysis as described previously (13,67).

In cases where ascospores of viable knockout mutants could not be isolated, attempts were made to isolate homokaryotic mutants in the vegetative phase by isolation of uninucleate microconidia (21) or by serial plating of macroconidia. In both of these cases, the resulting homokaryotic mutant strains retained themus-51 ormus-52 mutant background. For microconidia isolation, transformants were inoculated on 0.1× SCM agar slants containing 0.5% sucrose and 1.0 mM sodium iodoacetate and incubated at 25°C under constant light for at least 10 days (21). Microconidia were harvested using 2.5 ml sterile water and then filtered through a Millex Durapore filter unit (5 μm; Millipore, Bedford, MA; microconidia pass through, while larger macroconidia are retained on the filter). Microconidia were collected by centrifugation, resuspended in sterile water, and plated on FGS solid medium containing hygromycin. For single spore isolation, macroconidia were harvested and plated onto FGS plates containing hygromycin. After incubation of plates at 30°C in the dark, a colony was picked and transferred to a VM-hygromycin slant. After growth for 5 days, macroconidia were isolated and plated onto a new FGS hygromycin plate, and a resistant colony was picked onto a fresh VM-hygromycin slant. This process was repeated 2 to 3 times. The presence of the gene deletion mutation for each kinase was confirmed by Southern blot analysis as described above (13).

We were unable to isolate homokaryotic mutants for nine genes because ascospores, microconidia, or serially transferred single spores carrying the gene replacement were not viable. Knockout cassettes for NCU11235, NCU06419, and NCU11410 were designed based on incorrect gene models later found to include a second gene (Table 1). As a result, two genes were disrupted in these strains, so the corresponding knockout mutants were excluded from the phenotypic analysis. The NCU09071 mutant was generated in ahis-3 background and was supplemented with histidine for phenotypic analysis.

Analysis of growth, morphological, and developmental phenotypes.

Phenotypic analysis of each knockout mutant was performed by students in theNeurospora Genetics and Genomics Summer Research Institute (UCLA) (13,86). Analysis of colony growth and morphology was performed using two different media at two different temperatures: VM and VM containing 2% yeast extract (VM+YE) at 25°C and 37°C. After inoculation, plates were incubated under ambient light/dark conditions in the laboratory. Hyphae at the colony edge and the entire colony were then photographed. Growth rates of basal hyphae were measured in glass race tubes containing VM agar medium at 25°C under ambient light/dark conditions over a 72-h period (86).

Slant tubes containing VM medium were inoculated with strains and grown at room temperature for 6 to 8 days. Production of conidia and aerial hyphae and overall pigmentation were then scored. Aerial hyphal extension was measured in VM or VM+YE standing liquid cultures. Test tubes containing 2 ml of liquid medium were inoculated and incubated statically at 25°C for 24 h. The top edge of the mycelial mat was marked on the tubes, and then the cultures were incubated for an additional 72 h. Total height (in mm) was recorded.

For analysis of female sexual fertility, mutants were inoculated on SCM plates containing 1.5% sucrose and incubated under ambient light/dark conditions at room temperature for 7 to 8 days. Cultures were scored for the formation of protoperithecia and then fertilized using wild-type conidia of the opposite mating type. Perithecial formation and ascospore development were scored 2 weeks after fertilization.

Trichogyne assays.

Formation of trichogynes and chemotropic interactions between trichogynes and conidia of opposite mating type were analyzed as previously described (46).Neurospora wild-type (control) and mutant (ΔNCU02245) strains were grown on SCM agar for 6 days under constant light. A block of mycelia from the SCM plate was inoculated onto a 2% water-agar plate and incubated in a humid atmosphere at 25°C for 4 to 6 days under constant light. The plates were transferred to ambient humidity conditions and incubated 3 to 5 more days. A small square block (0.5 by 0.5 cm) of thin water agar was placed on the water agar plate in a region covering a few protoperithecia. A volume containing 1 μl of a wild-type microconidial suspension was placed on the water agar block. Migration of trichogynes into the block was examined after 16 h and then at 24-h intervals using an Olympus BX41 compound microscope (Olympus America, Lake Success, NY) with UM Plan fluorite objective lenses.

Chemical sensitivity screening.

All viable S/T kinase knockout mutants were screened for responses to the reactive oxygen species (ROS)-generating agent menadione. Race tubes containing 1× VM salts, 1.5% sucrose, 50 ng/ml biotin, and 1.5% agar with or without 100 μM menadione (M5750; Sigma) were inoculated with each mutant. Tubes were incubated in constant light at 25°C for 24 h before transfer to constant darkness at 25°C, after which time the growth front was marked every 24 h. Growth rates were measured and assigned to a growth rate range (±2.5 mm/day) estimated over 4 days. The growth rate in the presence of menadione was normalized to that on medium lacking menadione.

Cytochalasin A (40 ng/ml; Sigma, St. Louis, MO), benomyl (92 ng/ml; Fluka, St. Louis, MO), FK-506 (50 ng/ml; LC Laboratories, Woburn, MA),tert-butyl hydroperoxide (0.13 mM; Sigma, St. Louis, MO), sorbitol (0.8 M; Sigma), sodium chloride (0.35 M; EMD Chemicals, Gibbstown, NJ), and fludioxonil (2.75 ng/ml) were used for chemical screens of viable S/T kinase mutants with growth rates at least 50% of wild type on VM. The concentration of each chemical used was determined as the amount that inhibited wild-type growth by ∼70%. VM agar plates with or without chemical were inoculated with wild-type and knockout strains in quadruplicate. Plates were incubated in constant darkness at 30°C for 22 to 24 h. Colony radii were measured, and the percent growth in the presence versus absence of the chemical was calculated for each of the four measurements. Data from three independent experiments (four measurements/experiment) were subjected to a Q test to remove outliers. Data were then analyzed using at test. Mutants were considered sensitive or resistant if they displayed results within at least 95% confidence in two out of three trials and at least 80% confidence in all three trials.

Complementation of ΔNCU00406 andvelvet mutants.

To test for a possible allelic relationship between NCU00406 and thevelvet (vel) mutation, a construct containing the NCU00406 open reading frame (ORF) and promoter region was generated and transformed intovel and ΔNCU00406 mutants. A fragment containing the NCU00406 ORF, as well as 3 kb upstream and 0.65 kb downstream (total size, ∼6 kb), was amplified by PCR using primers cla45F (5′-TTGAGAGCTCGCAGTTGGTAGGAACACA-3′) and cla43R (5′-CTTCTCTAGACTCCTCCTGATTCGTTGA-3′) and digested with SacI and XbaI. The fragment was then ligated into pTJK1 digested with SacI and XbaI to yield pGP7. pTJK1 contains thebar dominant selectable marker, conferring resistance to phosphinothricin (42). pGP7 was then transformed into conidia from the ΔNCU00406 andvel mutants by using electroporation as described previously (40). Transformants were selected on FGS solid medium containing phosphinothricin (66). Homokaryotic strains were isolated by serial plating of macroconidia as described above and then confirmed by Southern blot analysis (67).

Serine/threonine protein kinase genes in theNeurospora genome.

Currently, 107 serine/threonine protein kinase genes are predicted inN. crassa. At the inception of this study, 89 S/T kinases were annotated and selected for analysis. To classify each of the S/T kinases to a specific group (36,58), protein sequences were submitted as a query for a BLAST algorithm at the Salk Institute's Protein Kinases, Kinomes and Evolution website, using a database of protein kinase genes. Among the 89 genes in our study, 6 appeared to be unique to filamentous fungi, while the remaining 83 could be categorized into the following groups: AGC, CAMK, CK1, CMGC, Other, and STE (Table 1).

To compare S/T kinases across diverse eukaryotic phyla, kinases from human, plant (Selaginella moellendorffii), yeast (S. cerevisiae), andNeurospora crassa were analyzed (Table 2). The distributions of kinases among the AGC, Atypical, CAMK, CK1, CMGC, Other, and STE groups were compared. While additional kinases belonging to other groups (e.g., tyrosine kinases and histidine kinases) are known for each of these organisms, the 83Neurospora kinases with homology to proteins outside the filamentous fungi can be classified within six of the seven groups above. The Atypical kinases were annotated after the inception of this study. While specific kinases within the Atypical group are known to be S/T kinases (87), it is unclear whether all kinases in this group phosphorylate serine and threonine residues. We therefore focused our analyses on the AGC, CAMK, CK1, CMGC, Other, and STE groups.Neurospora has the fewest kinases among the organisms analyzed inTable 2. This is likely caused by the phenomenon of RIP (repeat-induced point mutation), where one copy of a duplicate sequence is detected and mutated (80). This has resulted in a streamlined genome containing fewer paralogs than most eukaryotic organisms (25,26). This lack of redundancy makesNeurospora an ideal organism in which to study the function of kinases. Correcting for differences in the total number of kinases, relative kinase group sizes are essentially uniform across all of the organisms in our comparison. As expected, the relative group sizes of human, yeast, andNeurospora are slightly more similar to one another than they are to those in plants.Neurospora also contains members of protein kinase families and subfamilies that are known to be fungal specific (57).Neurospora S/T kinase gene names were taken from the literature, the e-compendium at Leeds University (http://bmbpcu36.leeds.ac.uk/∼gen6ar/newgenelist/genes/gene_list.htm), or assigned during this study (Table 1).S. cerevisiae orthologs were taken from the literature or assigned during this study based upon analyses of phylogenetic trees available atwww.phylomedb.org (Table 1) (33,34).

We attempted gene replacement of the 89 S/T protein kinase genes as part of theNeurospora Genome Project. In three cases (NCU06419, NCU11235, and NCU11410), the annotation suggests that adjacent genes were also mutated. Therefore, these three genes were excluded from further study (Table 1) (see Materials and Methods). We were able to generate homokaryotic knockout mutants for 49 genes as ascospore progeny from sexual crosses. We obtained homokaryotic mutants for another 28 kinase genes through isolation of uninucleate microconidia or after serial transfer of macroconidia. We were unable to isolate viable knockout mutants for the remaining nine kinase genes (Table 1). This left us with a total of 77 mutants for phenotypic analysis.

A majority of the viable S/T protein kinase mutants exhibit growth or developmental phenotypes.

We analyzed vegetative hyphal growth, asexual development, and sexual development in the 77 viable knockout mutants (13,67).Neurospora grows vegetatively by apical extension, branching, and fusion of basal hyphae. Under the condition of nutrient deprivation or an air-water interface,Neurospora enters the developmental program for production of multinucleate asexual spores, macroconidia. Initially, aerial hyphae are differentiated from basal (vegetative) hyphae. These aerial hyphae then begin a budding routine at their tips, to form conidiophores which ultimately give rise to macroconidia (82). Sexual development is induced by nitrogen starvation inNeurospora, with differentiation of female reproductive structures (protoperithecia) (76). Fertilization is accomplished by chemotropic growth of a specialized female hypha (trichogyne) toward a male cell of opposite mating type, transport of the male nucleus into the protoperithecium, mitosis, and cell proliferation, followed by nuclear fusion. Meiosis ensues, and the protoperithecium develops into the mature fruiting body (perithecium) containing the meiotic progeny (ascospores) (76).

Our analysis revealed that 32 and 31 kinase mutants had abnormalities in basal hyphal growth and asexual development, respectively (Fig. 1 andTable 1; see also Table S1 in the supplemental material for detailed phenotypic data). Among these, 23 mutants were defective in both hyphal growth and asexual development (Fig. 1 andTable 1). Mutants lacking NDR family kinasecot-1, PDK1/PKA family memberstk-23, HAL (halotolerance) familyptk-2, PEK (pancreatic eIF-2α kinase) family membercpc-3, and STE20 family memberstk-4 were distinctive in that their only morphological phenotype was a defect in hyphal growth. Interestingly, thestk-4 orthologste20 is not only required for normal growth inS. cerevisiae but is also essential for mating and pseudohyphal differentiation (99). In contrast to theNeurospora genes specific for hyphal growth, NDR family mutant Δstk-12, PKA family mutant Δpkac-2, CAMKL (Ca²⁺/calmodulin-dependent protein kinase-like) family mutant Δmus-58, and CK2 (casein kinase 2) family mutant Δcka only possessed defects in asexual sporulation.S. cerevisiae does not possess an asexual sporulation pathway; however, loss of thestk-12 ortholog inS. cerevisiae leads to defects in sexual sporulation, along with other phenotypes (83).

A large proportion of S/T kinase mutants displayed defects in sexual development. The genes replaced in STE20 family Δmst-1, STE11 family Δcdc15, CAMKL family Δstk-19, and NEK family Δnim-1 appear to be specific for sexual development, while the majority of genes implicated in sexual development also play roles during asexual growth and development (Fig. 1). Most of the sexual developmental mutants were not able to produce protoperithecia (Table 1; see also Table S1 in the supplemental material). The CAMKL family mutants Δstk-16 and Δstk-31 and the CDK family mutant Δdiv-4 formed protoperithecia but had defects in the timing or number of female structures produced. Three mutants formed normal perithecia but ejected no (Δnim-1), few (Δmst-1), or white ascospores (Δvel; 20% of ascospores were white.). One of theS. cerevisiae vel homologs,cla4, is required for normal pheromone sensitivity (10,41). This suggests that Cla4p acts at an earlier point in sexual development in yeast than VEL inNeurospora.

Most of the 32 mutants that exhibited abnormalities in sexual development also possessed defects in basal hyphae growth and asexual differentiation (Table 1). Among these mutants are those lacking components of the MIK-1/MEK-1/MAK-1 and NRC-1/MEK-2/MAK-2 MAPK pathways. The MAK-1 pathway is required for normal growth, hyphal fusion, conidiation, differentiation of protoperithecia, and cell wall integrity (56,68), while the MAK-2 cascade controls growth, hyphal fusion, conidiation, and protoperithecial development (49,53,56). Strains with mutations in the proteins of the osmotic stress resistance MAPK pathway (OS-4/OS-5/OS-2) have near-normal growth rates but exhibit defects in conidiation and sexual development while displaying sensitivity to osmotic stress and resistance to phenylpyrrole and dicarboximide fungicides (24,42,92). The roles for these MAPK pathways inNeurospora share many parallels with the corresponding pathways inS. cerevisiae and other fungi. For example, inS. cerevisiae, the MAK-2 homologous pathway is required for the pheromone response and filamentous growth, the MAK-1-related pathway is necessary for cell wall integrity and mitotic progression, and the OS-2 homologous MAPK pathway is required for resistance to osmotic stress (11).

The Δstk-19 mutant was of particular interest, since it formed protoperithecia normally but produced very few perithecia after fertilization. We have previously observed such a phenotype in mutants with defects in chemotropic growth of female-specific hyphae, or trichogynes, toward male cells (conidia) (46). In order to determine whether Δstk-19 possessed such a chemotropism defect, we further investigated this mutant in a trichogyne chemotropism assay (46) (Fig. 2). The results showed that although many Δstk-19 protoperithecia form trichogynes, they are largely unable to recognize or encircle the male conidia (Fig. 2). This result suggests that the missing kinase is required for chemotropism of females toward males during fertilization. InS. cerevisiae, thestk-19 ortholog YPL150W is required for a normal growth rate and glycogen accumulation but has no known roles in mating or sexual development (91,99).

Overall, our results showed that 44/77 mutants analyzed (57%) exhibit a defect in at least one of the three major phenotypes analyzed (Fig. 1;Table 1). Among these, 11 and 20 knockout mutants possess defects in two or three traits, respectively (Fig. 1;Table 1). The number of genes implicated in multiple traits (31) in combination with those for which no viable mutant could be isolated (9) suggest that a large proportion ofNeurospora S/T kinases play crucial roles in important fungal life processes (Fig. 1).

Chemical sensitivity assays increase the number of S/T protein kinase mutants with phenotypes.

InS. cerevisiae, most genes that do not exhibit obvious functions during growth and development are essential for normal resistance to at least one chemical treatment (31). In order to better understand the functions of S/T kinases inNeurospora, we compared mutants to the wild type with regard to their sensitivity to a panel of chemical treatments (seeTable 1 and the detailed chemical phenotypes inTable 3). The chemicals included those that induce oxidative (menadione andtert-butyl hydroperoxide) or osmotic (sorbitol and sodium chloride) stress, perturb the cytoskeleton (benomyl and cytochalasin A), act as a fungicide (fludioxonil), or inhibit the Ca²⁺-dependent phosphatase calcineurin (FK-506). In order to avoid issues with slow-growing strains that may have skewed our results, we included in our analysis only those viable mutants (56) that had growth rates of at least 50% of the wild type on normal medium.

Menadione generates a hyperoxidative condition by producing ROS, whiletert-butyl hydrogen peroxide is a source of the oxidant peroxide (6,44). Three mutants, ULK (Unc-51-like kinase) family Δapg-1, STE20 family Δstk-4, and NAK (NF-κB activating kinase) family Δstk-9, were hypersensitive to both menadione andtert-butyl hydrogen peroxide (Table 3), suggesting the missing kinases play general roles in resistance to oxidative stress. Another three and four mutants were more sensitive than the wild type to menadione ortert-butyl peroxide, respectively (Table 3). With a total of seven sensitive mutants,tert-butyl peroxide yielded the greatest number of phenotypes in our study. Reactive oxygen species have previously been implicated in control of conidiation inNeurospora (1). Therefore, it is of interest that four mutants (Δstk-38, Δstk-9, Δstk-17/fi, and Δapg-1) all possess defects in some aspect of conidiation, in addition to altered sensitivity totert-butyl peroxide and/or menadione.

We assayed the response of kinase mutants to two agents that induce osmotic stress in fungi, sorbitol and sodium chloride. One mutant (Δapg-1) that was sensitive to menadione and peroxide also displayed decreased growth in the presence of sorbitol and sodium chloride, consistent with a general sensitivity to environmental stress. InS. cerevisiae, theapg-1 homologous gene,atg1, is required for autophagy, regulation of cellular ROS levels, and sensitivity to the complex chemical propolis but has no reported roles in resistance to osmotic stress (101). In total, sevenNeurospora mutants exhibited significant sensitivity to sodium chloride, while one displayed increased resistance (Table 3). Of the sensitive mutants, two (Δstk-5 and Δstk-30) lack genes of the HAL (halotolerance) family. This result is a significant proof of principle, as the HAL family is known to function in salt tolerance inS. cerevisiae (63).

To explore the role of S/T kinases in cytoskeletal maintenance, the mutants were screened using two chemicals known to alter two major cytoskeletal components. Cytochalasin A interacts with actin filaments and inhibits their polymerization and elongation (14). Benomyl binds to microtubules, resulting in the inhibition of crucial cellular processes such as mitosis, meiosis, and cellular transport (90). The growth of two kinase mutants, Δstk-38 (NAK family) and Δstk-47 (CDK family), was decreased more than the wild type in the presence of cytochalasin A. Two mutants displayed increased resistance relative to the wild type, Δstk-22 (CAMKK [Ca²⁺/calmodulin-dependent protein kinase kinase] family) and Δstk-54 (CLK/SRPK [serine-rich protein kinase]/DYRK [dual-specificity tyrosine-regulated kinase] family) (Table 3). Three kinase mutants (Δnim-1 and Δgsk-3 [GSK family] and Δcamk-4 [CAMK1 family]) were more resistant to benomyl, while three mutants (Δstk-16 [CAMKL family], Δstk-29 [WEE family], and Δstk-38 [NAK family]) exhibited greater sensitivity (Table 3). Notably, mutant Δstk-38 exhibited significant sensitivity to both cytochalasin A and benomyl (Table 3). The observation of altered sensitivity of a mutant to cytochalasin A and/or benomyl is consistent with a defect in the actin and/or β-tubulin cytoskeleton, respectively. Four mutants, Δstk-16, Δstk-22, Δstk-38, and Δstk-47, exhibited defects during vegetative or sexual development in the absence of chemicals, possibly resulting from disturbance of the cytoskeleton. Interestingly, one of theS. cerevisiae stk-16 orthologs,KIN4, inhibits the mitotic exit network and is localized to the mother cell cortex, spindle pole bodies, and bud neck (16,71). Additionally, theS. cerevisiae orthologs ofstk-38,ARK1 andPRK1, are important for regulation of the cortical actin cytoskeleton (15). Deletion ofARK-1 results in formation of actin clumps and a growth defect inS. cerevisiae. Of interest, theNeurosporaΔstk-38 mutant exhibits defects in hyphal growth and asexual and sexual development. Finally, while the sensitivity of theNeurosporaΔstk-47 mutant to cytochalasin A suggests an influence on actin organization, theS. cerevisiae orthologCTK1 may play a role in microtubule maintenance, given the sensitivity of the yeastctk1 mutant to benomyl (69).

FK-506 is an immunosuppressant drug that inhibits calcineurin, an S/T protein phosphatase in the calcium signaling pathway (74). Three S/T kinase mutants, Δapg-1, Δptk-2, and Δstk-9 (NAK family), were more sensitive to FK-506 than the wild type, while two mutants, Δime-2 (RCK [ros cross-hybridizing kinase] family) and Δstk-22 (CAMKK family) were more resistant to FK-506 (Table 3). Altered sensitivity to FK-506 implicates these kinases in calcineurin function inNeurospora. The finding that the Δime-2 mutant has altered sensitivity to FK-506 is a novel finding in any fungal species. Work in the Glass laboratory has shown thatime-2 is not required for meiosis, as inS. cerevisiae, but instead negatively regulates formation of female reproductive structures in response to adequate nitrogen inNeurospora (38). This function is similar to that described for the filamentous fungusCryptococcus neoformans (39,55). In our study, we did not screen for protoperithecial formation under nitrogen-rich conditions. However, we did note thatime-2 mutants had defects in hyphal growth and aerial hyphae formation, in addition to altered sensitivity to FK-506.

Fludioxonil is a member of the phenylpyrrole class of fungicides (64). Fludioxonil inappropriately stimulates theosmotic sensitive-2 (OS-2) MAPK pathway, leading to excessive glycerol production and subsequent cell death in filamentous fungi (100). AllNeurospora os mutants, including those with defects in the three genes of theos MAPK pathway, are resistant to fludioxonil (24,100). In accordance with previous results, we observed that the threeos MAPK pathway mutants were resistant to fludioxonil but sensitive to sorbitol and sodium chloride (data not shown). In addition, we noted another previously uncharacterized S/T kinase mutant (Δstk-16; CAMKL family) that was sensitive to both fludioxonil and sodium chloride, as well as benomyl (Table 3). This mutant is of interest, as it appears to have uncoupled the previously observed association between fungicide resistance and osmotic sensitivity and may participate in a novel pathway that modulates fungicide resistance inNeurospora. Δstk-16 also possesses defects in hyphal growth and protoperithecial development (Table 1). One of the homologous genes inS. cerevisiae,KIN4, modulates mitotic exit but has not been implicated in osmotolerance (16).

Taken together, the results revealed 25 mutants with chemical sensitivity phenotypes. Accounting for mutants that exhibited a significant sensitivity or resistance to more than one chemical, a total of 38 chemical phenotypes were observed. Importantly, these screens uncovered unique phenotypes for eight mutants that did not exhibit any morphological or developmental defects. This corresponds to more than 10% of the viable S/T kinase mutants and brings the total number of viable mutants with phenotypes to 52.

NCU00406 is allelic withVELVET, encoding a PAK with homology to Cla4p and Skm1p fromS. cerevisiae.

While performing phenotypic analysis, we observed that the ΔNCU00406 mutant possessed severe defects in extension of basal and aerial hyphae and a distinctive colony morphology. The mutant was yellow, with aggregated hyphae and little production of conidia. We noted that the phenotype of ΔNCU00406 resembled that of the morphological mutantvelvet (vel), isolated more than 50 years ago (72). Furthermore, thevel mutation maps near cloned genetic markers in the vicinity of NCU00406.

Based on the above information, we investigated whethervel is allelic with NCU00406. We transformedvel and the ΔNCU00406 mutants with a construct containing the NCU00406 open reading frame and promoter region. In both cases, introduction of a wild-type allele of NCU00406 complemented growth and morphological phenotypes (Fig. 3 A). In order to determine the position of the mutation(s) invel, we amplified regions of the NCU00406 gene from thevel and wild-type strains and submitted them for sequencing. The results revealed two single base pair substitutions, a C-to-T change at nucleotide position 765 and another C-to-T change at position 1144 in the 2,502-bp ORF of thevel allele. The substitution at position 765 is synonymous, retaining the codon for phenylalanine. However, the second substitution produces a nonsense mutation at nucleotide position 1144, predicted to result in a truncated protein lacking the protein kinase domain (Fig. 3B). Taken together, these results demonstrate thatvel is allelic with NCU00406, and they highlight the importance of this PAK-encoding gene toNeurospora biology. InS. cerevisiae, the VEL-related protein Cla4p is equally important to yeast biology, being required for normal bud morphology, pheromone responsiveness, filamentation, and invasive growth (10,41,89). In contrast, althoughS. cerevisiae Skm1p is necessary for normal resistance to hyperosmotic and other stresses (45,98), this protein has not been implicated in the wide array of functions ascribed to Cla4p.

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