Extragenic Suppressors of the nimX2^cdc2 Mutation of Aspergillus nidulans Affect Nuclear Division, Septation and Conidiation

Corresponding author: Sarah Lea McGuire, Millsaps College, P.O. Box 150305, 1701 N. State St., Jackson, MS 39210., mcguisl{at}millsaps.edu (E-mail)

Communicating editor: M. D. ROSE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Aspergillus nidulans NIMX^CDC2 protein kinase has been shown to be required for both the G₂/M and G₁/S transitions, and recent evidence has implicated a role for NIMX^CDC2 in septation and conidiation. While much is understood of its G₂/M function, little is known about the functions of NIMX^CDC2 during G₁/S, septation, and conidiophore development. In an attempt to better understand how NIMX^CDC2 is involved in these processes, we have isolated four extragenic suppressors of the A. nidulans nimX2^cdc2 temperature-sensitive mutation. Mutation of these suppressor genes, designated snxA-snxD for suppressor of nimX, affects nuclear division, septation, and conidiation. The cold-sensitive snxA1 mutation leads to arrest of nuclear division during G₁ or early S. snxB1 causes hyperseptation in the hyphae and sensitivity to hydroxyurea, while snxC1 causes septation in the conidiophore stalk and aberrant conidiophore structure. snxD1 leads to slight septation defects and hydroxyurea sensitivity. The additional phenotypes that result from the suppressor mutations provide genetic evidence that NIMX^CDC2 affects septation and conidiation in addition to nuclear division, and cloning and biochemical analysis of these will allow a better understanding of the role of NIMX^CDC2 in these processes.

THE filamentous fungus Aspergillus nidulans has proven to be a useful genetic system both for the study of cell cycle control and for the study of signals that establish patterns of cell growth and differentiation. It has long been recognized that nuclear division in A. nidulans is linked to septation and asexual development (FIDDY and TRINCI 1976 ; MIRABITO and OSMANI 1994 ); however, the molecular and genetic nature of these relationships has only recently begun to be elucidated. Recent studies have afforded a significant understanding of the molecular regulation of the G₂/M transition of A. nidulans (for a review, see YE and OSMANI 1997 ). Two protein kinases, the NIMX^CDC2 protein kinase and the NIMA protein kinase, are coordinately required to initiate mitosis in A. nidulans (OSMANI and YE 1996 ; YE et al. 1996 ), and their rapid inactivations are essential for progression through mitosis (GLOTZER et al. 1991 ; PU and OSMANI 1995 ). In addition, both protein kinases appear to be the targets of checkpoint regulation (YE et al. 1997A , YE et al. 1998 ). Evidence exists indicating that NIMA may be required for the proper localization of NIMX^CDC2/cyclinB to initiate mitosis (WU et al. 1998 ).

In addition to its mitosis-promoting function, NIMX^CDC2 is required for the G₁/S transition; however, little is known about its G₁/S function and no G₁-specific genes that regulate NIMX^CDC2 have been identified in A. nidulans. S-M checkpoint control in response to incomplete DNA replication and DNA damage functions via regulation of phosphorylation of the Tyr-15 residue of NIMX^CDC2 (YE et al. 1996 ; YE and OSMANI 1997 ). Loss of such checkpoint control regulation over mitosis can also cause defects in DNA rereplication after mitosis (DESOUZA et al. 1999 ). Triggered by the NIMQ^MCM2 DNA licensing factor (YE et al. 1997B ), checkpoint control over mitotic function at the G₁/S transition is transferred from the anaphase promoting complex/cyclosome (APC/C; LIES et al. 1998 ; YE et al. 1998 ; ZACHARIEA and NASMYTH 1999 ) to include Tyr-15 phosphorylation of NIMX^CDC2. One function of the APC/C is to ubiquitinate cyclinB in late mitosis and G₁, thus ensuring that NIMX^CDC2 is inactive during these times. Thus, the activity of NIMX^CDC2/cyclinB is highly regulated at all phases of the nuclear division cycle, but many of the details of this regulation are still not understood in A. nidulans. Identification of proteins that interact with NIMX^CDC2 will allow a better understanding of its regulation.

Vegetative hyphae of A. nidulans grow by apical extension of a germ tube from a single conidium. As the hypha extends, the nuclei undergo repeated mitotic divisions. Beginning with the third nuclear division, crosswalls called septa are typically laid down at uniform intervals along the vegetative hyphae (HARRIS et al. 1994 ; KAMINSKYJ and HAMER 1998 ), producing a multinucleate syncytium. The formation of septa is the equivalent of cytokinesis and is dependent upon cell size, mitosis, and nuclear positioning (WOLKOW et al. 1996 ). Cytokinesis is tightly coupled with mitosis in many organisms (SATTERWHITE and POLLARD 1992 ), and cyclin-dependent kinase activity is believed to coordinate cytokinesis with mitosis (SATTERWHITE et al. 1992 ). In A. nidulans, high levels of NIMX^CDC2 activity are required for septum formation. In the nimX^cdc2AF strain, NIMX^CDC2 is unable to be phosphorylated at positions 14 and 15, leading to increased NIMX^CDC2 activity and premature septation (HARRIS and KRAUS 1998 ). Septation in this strain also occurs inappropriately in the conidiophore stalk, resulting in defects in conidiophore development (YE et al. 1999 ). Recent evidence has shown that mutations in bimA^APC3, which encodes part of the APC/C, indirectly affect septation by leading to errors in DNA metabolism (WOLKOW et al. 2000 ). Thus, both septation and nuclear division are affected by the APC/C and the activity of NIMX^CDC2, but little is understood of the molecular and genetic relationships between the APC/C, NIMX^CDC2, and septation.

The molecular mechanisms controlling conidiophore development have been extensively studied (for a review, see ADAMS et al. 1998 ). Recent work has shown that NIMX^CDC2 is regulated by the BrlA transcriptional regulator, which cues the switch from hyphal growth to conidiophore development, and that the nimX^cdc2AF strain has defects in conidiophore development (YE et al. 1999 ). Proper function of NIMX^CDC2 is therefore essential for nuclear division, septation, and conidiophore development. Elucidation of the regulation of NIMX^CDC2 in A. nidulans will allow a better understanding of how these processes are integrated at the molecular level.

Three temperature-sensitive nimX alleles, nimX1, nimX2, and nimX3, arrest nuclei in interphase (OSMANI et al. 1994 ). nimX1 arrests in G₂, while nimX3 arrests in both G₁ and G₂. The arrest point of nimX2 is less clear, with at least some cells arresting in G₂; however, the homologous mutation in Schizzosaccharomyces pombe, cdc2-45, leads to G₁ arrest (MACNEILL et al. 1991 ). To identify genes that interact with NIMX^CDC2 in A. nidulans, we generated a series of extragenic suppressors of the nimX2 temperature-sensitive allele. Here we describe the genetic and phenotypic characterization of mutants representing four genes that interact with NIMX^CDC2. We call these genes snxA-snxD, for suppressor of nimX. snxA1 affects nuclear division and results in a block during G₁ or early S at the restrictive temperature, snxB1 causes aberrant hyphal septation and increased sensitivity to hydroxyurea, snxC1 leads to aberrant conidiophore septation and development, and snxD1 leads to hydroxyurea sensitivity and slight septation defects. Characterization of these mutations and cloning of the genes will allow a better understanding of the molecular control of NIMX^CDC2 and how its control affects nuclear division, septation, and asexual development.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and growth conditions:
Strains used in this study are listed in Table 1. Media used were the following: rich media, MG (2% malt extract, 2% glucose, 0.2% peptone, trace elements, and vitamins) or YG (1% glucose, 0.5% yeast extract, trace elements, and vitamins) and minimal media (MM; 1% glucose, potassium chloride, nitrate salts, and trace elements). Trace elements, potassium chloride, nitrate salts, and vitamins are described in the appendix to KAFER 1977 . Agar (1.8%) was added for solid medium. Genetic techniques were as described in HARRIS et al. 1994 , except that heterokaryon formation was accomplished by plating strains containing complementary auxotrophic markers 1 cm apart on rich media, incubating at 32° for 2 days, and transferring the zone of mixed mycelia to an MM plate at 32°. When heterokaryotic growth was evident, heterokaryons were subcultured onto MM plates. Spontaneous diploids were isolated as described in ENGLE 1997 .

Mutagenesis and identification of extragenic suppressors with independent phenotypes:
Mutagenesis of strain SO64 (nimX2) was accomplished as described in HOLT and MAY 1996 . Revertant colonies (2500) were isolated to single colony three times and retested for growth at 42° as well as for growth at 20° for 5–7 days (to test for cold sensitivity) or for growth at 32° on solid rich media containing 4% dimethylsulfoxide (DMSO; 4 days), 15 mM hydroxyurea (HU; 4 days), 0.01% methyl methanesulfonate (5 days), or 4 µg/ml benomyl (BEN; 5 days). Sexual crosses between strain A612 and all revertants that had an additional independent phenotype were performed as described (HARRIS et al. 1994 ) to determine if the reversion mutation was intragenic or extragenic and if the independent phenotype cosegregated with reversion. Strains containing extragenic suppressor mutations that cosegregated with the independent phenotype were assigned to linkage group by parasexual genetic analysis (PONTECORVO et al. 1953 ; KAFER 1977 ) using diploids made between mitotic mapping strain A154 and strains carrying the suppressor mutation but not the nimX2 mutation (BC16, BC70, BC52, and BC76). Haploidization was accomplished as described in HOLT and MAY 1996 . Haploid colonies were replica plated onto solid rich media under the appropriate selective conditions to test for specific chromosome markers and the suppressor mutation. Mutations were assigned to linkage group by their linkage with known chromosomal markers (snxA1, snxB1, snxC⁺, and snxD1) as described in ENGLE 1997 . Because snxC1 is dominant, it could not be mapped by following linkage of the snxC1 mutation with chromosomal markers. We thus mapped snxC by following cosegregation of the snxC⁺ allele with chromosomal markers in haploid segregants. The diploids generated with strain A154 were also used to determine dominance or recessiveness of the suppressor mutations by analyzing diploid growth under selective conditions.

Generation and screening of double mutants:
To generate double mutants containing snxB1 or snxC1 and either ankA^wee1 or nimT23^cdc25, the following crosses were made: SWJ108 x BC72, SWJ108 x BC50, ANKA x BC72, and ANKA x BC52. Progeny from each cross were tested for growth under restrictive conditions for each mutation. The nimT23 mutation confers temperature sensitivity at 43°, while the ankA deletion causes sensitivity to 5 mM hydroxyurea. SnxB1 mutants can form a small colony on 5 mM HU but are sensitive to 15 mM HU, and snxC1 confers an inability to conidiate in the presence of 4% DMSO. For each cross, four classes of progeny were clearly identifiable: two parental single-mutant classes and two recombinant classes (one wild type and another distinctly different from either parent and wild type). To determine if progeny in the fourth class were double mutants, five progeny from each cross were streaked to single colony twice, retested for growth under restrictive conditions, and crossed to a wild-type strain (A612). The appearance of both single-mutant phenotypes among the progeny indicated that these were double mutants.

Staining, microscopy, and measurements:
To visualize nuclei and septa, conidia were incubated as described in HARRIS et al. 1994 and fixed and stained with the DNA-specific dye 2,4-diamidino-2-phenylindole (DAPI) as described (OSMANI et al. 1990 ). Septa were simultaneously stained with nuclei by including 4 µg/ml fluorescent brightener 28 (calcofluor; Sigma Chemical Co., St. Louis) in the staining mix. Fluorescence microscopy was performed using a Nikon Alphaphot microscope and a Leica DM-LB microscope. Subapical cell length, defined as the distance between adjacent septa measured at the junction between the septa and the lateral hyphal wall, was measured using SPOT Advanced software (Diagnostic Instruments) calibrated with an ocular micrometer. At least 50 subapical cells were analyzed for each strain. The nonparametric Mann-Whitney test was used to determine the statistical significance of the differences in subapical cell length. The percentage of cells that septate early was determined by counting the number of germlings with four nuclei that contained septa; 100 randomly selected germlings containing four nuclei were counted for each strain.

Reciprocal shift experiments:
Reciprocal shift experiments to determine at which stage of interphase snxA1 cells arrest were performed as described (BERGEN et al. 1984 ; OSMANI et al. 1994 ). Conidia from strains BC7 and MDS250 were first arrested in S phase by inoculation onto coverslips in rich media containing 25 mM HU and incubated 6.5 hr at 32°. The coverslips were either fixed (as a control) or transferred to 32° rich media in the absence of HU or to precooled 20° rich media in the absence of HU, incubated for 25 hr at 20°, and fixed. For the reciprocal experiment, conidia were inoculated into precooled 20° rich media in the absence of HU, incubated for 25 hr at 20°, either fixed (as a control) or transferred to 32° in the presence or absence of 25 mM HU, incubated at 32° for 3 hr, and fixed. These experiments were also performed using 19-hr incubations at 20°. Coverslips were stained with DAPI and the number of nuclei per germling determined.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of nimX2 revertants:
The nimX2 mutation was chosen for these experiments because it arrests nuclei in G₁ or G₂ at the restrictive temperature of 42° (OSMANI et al. 1994 ), resulting in an inability to form colonies at this temperature. Suppressor mutants were isolated after treatment of conidia with NQO by plating survivors onto rich media followed by incubation at 42°. Under these conditions, control treatment with no NQO (solvent only) resulted in no colony formation, whereas conidia treated with 4–8 µg/ml NQO produced viable colonies. These heat-insensitive colonies were streaked to single colony three times and tested for growth at 32° and 42°. To facilitate future genetic analysis and cloning, we tested these revertants following each streak to single colony for the presence of an easily scorable selectable phenotype. A total of 2500 revertants were isolated and scored for growth inhibition at 20° (cold sensitivity, Cs^-) and for growth inhibition in the presence of 4% DMSO, 15 mM HU, 0.01% methyl methanesulfonate (MMS), and 4 µg/ml BEN. A total of 22 revertants were Cs^-, 2 were sensitive to DMSO, 3 were sensitive to HU, 1 was sensitive to MMS, and 12 were sensitive to BEN.

Each of the revertants that possessed an additional phenotype was crossed to strain A612, which has a wild-type nimX allele, to determine if the mutation leading to reversion was intragenic or extragenic. The presence of heat-sensitive colonies in the progeny indicated that suppression of the nimX2 mutation was extragenic (Table 2). Of the 40 revertant colonies with independent phenotypes tested, 15 contained extragenic suppressor mutations. Of these 15, 7 were Cs^-, 2 were HU^-, and 2 were DMSO^-. The crosses also allowed a determination of whether the extragenic suppressor mutations cosegregated with the additional phenotypes possessed by the suppressor strains. Four BEN^- extragenic suppressor strains were also identified, but each of these contained extragenic suppressor mutations that did not cosegregate with the independent phenotype (data not shown). Of the six strains that contained cosegregating extragenic suppressors, MDS250 (Cs^-), CY17 (HU^-), CY1228 (DMSO^-), and S220 (HU^-) had clear suppressor and additional phenotypes and were used for further study. MDS261 was an extremely weak suppressor and MDS545 had only a marginal Cs^- phenotype that could not be scored reliably.

Linkage analysis:
To determine the number of genes represented by the mutations in strains MDS250, CY17, CY1228, and S220, the mutants were crossed in all pairwise combinations. This first required crossing each mutant with strain A612 and with strain SWJ298 to obtain a series of strains with complementing genetic markers, the suppressor mutations, and a wild-type nimX⁺ allele (Table 1). All possible combinations of crosses of suppressor mutations were carried out, and the progeny were analyzed for the presence of recombinant phenotypes among the suppressor mutations (Table 3). Progeny that were either phenotypically wild type or that possessed both suppressor phenotypes were scored as recombinants. For example, for a cross between BC7 and BC52 (which have the suppressor mutations from MDS250 and CY1228, respectively), both wild-type and DMSO^-, Cs^- progeny were scored as recombinants. In a cross between RLC4 and BC72 (which have mutations from MDS250 and CY17, respectively), both parents were HU-sensitive, so the presence of wild-type progeny alone was used to indicate that the genes are not linked. However, double mutants were identified because they were Cs^- and formed extremely small colonies at 32° (not shown). Examination of these strains via fluorescence microscopy confirmed that in addition to being Cs^-, they have the suppressor phenotype originally identified in CY17 (see Fig 3B). In all crosses, progeny with wild-type and/or double-mutant phenotypes were isolated, indicating that four different genes have been identified.

View larger version (102K): In this window In a new window Download PPT slide   Figure 1. Plate growth phenotypes of suppressor mutations in the presence and absence of the nimX2 allele. Solid plates MG (top row), MG + 15 mM HU (bottom left), or MG + 4% DMSO (bottom right) plates were point inoculated with spores as follows: top row, double mutants, left to right, MDS250 (snxA1 nimX2), CY17 (snxB1 nimX2), CY1228 (snxC1 nimX2), or S220 (snxD1 nimX2); middle row, single mutants, BC7 (snxA1), BC70 (snxB1), BC52 (snxC1), or BC93 (snxD1); bottom row, A612 (wild type), SO64 (nimX2). Plate incubations were 42°, 3 days (top left); 32°, 3 days (top middle); 20°, 7 days (top right); or 32°, 5 days (bottom row).

View larger version (40K): In this window In a new window Download PPT slide   Figure 2. Cellular morphologies of snxA+, nimX2, snxA1, and snxA1 nimX2 germlings. Conidia from strains A612 (snxA+ nimX+), SO64 (nimX2), RLC1 (snxA1), and MDS250 (snxA1 nimX2) were inoculated onto coverslips in YG. Coverslips were incubated as indicated below, fixed, and stained with DAPI. Interphase nuclei are identified by diffuse DAPI staining and the presence of prominent nucleoli. (A) A612 incubated at 42° for 8 hr; (B) SO64 incubated at 42° for 16 hr; (C and D) MDS250 incubated at 42° for 16 hr; (E) RLC1 incubated at 42° for 16 hr; (F) RLC1 incubated at 32° for 16 hr; (G) RLC1 incubated at 20° for 25 hr; and (H) RLC1 incubated at 20° for 72 hr. Bars, 5 µm.

View larger version (54K): In this window In a new window Download PPT slide   Figure 3. snxB1 causes increased branching and septation and sensitivity to HU. Conidia from strain A612 (snxB+; A and C) or strain BC70 (snxB1; B and D) were inoculated onto coverslips in YG (A and B) or YG + 15 mM HU (C and D), incubated at 32° for 16 hr, fixed, and stained with DAPI and calcofluor. Arrows indicate septa. Bars, 5 µm.

The four extragenic suppressor mutations identified were designated snxA1–snxD1, for suppressor of nimX. The plate growth phenotypes of strains carrying each of these mutations in the presence and absence of the nimX2 allele are shown in Fig 1. The cold-sensitive snxA1 mutation suppresses nimX2 such that near wild-type growth and normal conidiation are observed in double mutants at 42°, while at 20° colonies do not form. Interestingly, this mutation also confers sensitivity to 15 mM HU. The two mutations originally identified as being HU sensitive, snxB1 and snxD1, both partially suppress nimX2, allowing double mutants to form small aconidial colonies at 42°. These mutations also lead to small but robustly conidiating colonies at 32° and 20°. Both snxB1 and snxD1 strains conidiate normally at 42° and 32° in the absence of the nimX2 mutation; snxB1 is unable to form viable colonies in the presence of 15 mM HU, while snxD1 forms a small colony. The snxC1 mutation allows partial suppression of heat sensitivity due to the nimX2 mutation; it produces sparse conidia at 42° and at 32° and is completely aconidial in the presence of 4% DMSO. Colonies of both double (snxC1 nimX2) and single (snxC1) mutant strains produce long aerial hyphae, which have numerous aberrantly shaped conidiophores extending from them.

Testing for allele specificity of the suppressor mutations:
To determine if the snx mutations are allele-specific, suppressor strains were crossed to SO69 (nimX1) and to SO65 (nimX3) and the progeny analyzed for the presence of double-mutant phenotypes (both temperature-sensitive and sensitive to conditions selective for the particular snx mutation being analyzed). Cold-sensitive, heat-sensitive progeny were isolated from both SO65 (nimX3) x BC16 (snxA1) and SO69 (nimX1) x BC16 (snxA1) crosses, and the resulting double mutants were tested at various temperatures (37°, 41.5°, 43°) to determine if partial suppression occurred. No differences in the heat sensitivity of the progeny were observed compared to the nimX3 or nimX1 strains, indicating that snxA1 is an allele-specific suppressor of nimX2. Double mutants were also isolated for SO65 (nimX3) x MCG1 (snxB1), SO65 (nimX3) x BC76 (snxD1), and SO69 (nimX1) x BC76 (snxD1), also with no partial suppression. Double mutants isolated from SO69 (nimX1) x MCG1 (snxB1) were completely inhibited at 43°, but exhibited some growth at 42°, indicating that snxB1 partially suppresses nimX1. Despite repeated attempts, crosses between SO65 (nimX3) and SO69 (nimX1) with snxC1 strains did not produce viable cleistothecia; thus the allele specificity of snxC1 could not be determined.

Chromosome mapping and dominance testing:
Diploids between mitotic mapping strain A154 and strains containing each of the four suppressor mutations were created to determine the dominant/recessive nature of the mutations and to map each mutation to its specific linkage group. snxA1, snxB1, and snxD1 were shown to be recessive, as the diploids exhibited the wild-type phenotype. snxC1 was classified as partially dominant because the snxC1/snxC⁺ diploid exhibited the aerial hyphae and sparse conidiation observed in the snxC1 haploid in the absence of DMSO, but it was able to conidiate in the presence of 4% DMSO, similar to wild type. Parasexual analysis indicated that snxA is located on chromosome II, snxB is on chromosome VII, snxC is on chromosome I, and snxD is on chromosome VII.

Testing for allelism of snxA and other chromosome II cell cycle genes:
Several known cell cycle genes map to chromosome II, as does nimX^cdc2. These include nimT^cdc25, nimE^cyclinB, and ankA^wee1, which interact with nimX^cdc2, as well as nimG and nimR. To determine if snxA is allelic with any of these, strains carrying the snxA1 mutation were crossed to strains with mutations in each of the chromosome II cell cycle genes. In all cases, recombinant progeny with clear double-mutant phenotypes were obtained (Table 4), indicating that snxA is a novel cell cycle regulatory gene and that the snxA1 mutation does not suppress mutations in other known G₂-specific genes that interact with nimX^cdc2.

Phenotypic characterization of the snx mutants:
Nuclear and hyphal morphologies of each of the snx mutants were examined by DAPI/calcofluor double staining of hyphae. The nimX2 mutation alone leads to arrest of the nuclear division cycle in G₁ or G₂ at 42° (OSMANI et al. 1994 ). The cells arrest with a single interphase nucleus but exhibit some germ tube extension (Fig 2B). Both single mutants (snxA1) and double mutants (snxA1 nimX2) have similar phenotypes at all temperatures tested (42°, 32°, 20°). snxA1 nimX2 double mutants are able to undergo nuclear division and germ tube extension at 42°; however, the majority of the nuclei are smaller than those of wild-type cells (Fig 2C) and the cell wall is swollen in 25% of both single- and double-mutant cells at 42° (n = 100; Fig 2D). The swollen areas may be confined to the conidial head (the part of the germling that corresponds to the original spore) or they may be found as part of the hyphae; in all cases, swollen areas appear to be packed with small interphase nuclei. These nuclei are often obscured in micrographs due to intense DAPI staining. At 32°, both snxA1 nimX2 and snxA1 cells (Fig 2F) appear wild type, with only a small percentage (10%) of cells containing swollen areas. Both snxA1 (Fig 2G) and snxA1 nimX2 cells arrest in interphase of the cell cycle at 20° with a single nucleus and ungerminated conidia. Although an occasional cell (3%) will undergo a single nuclear division and slight germ tube extension, if allowed to incubate for 2–3 days at 20° these arrest with large, aberrantly shaped, nuclei and swollen hyphae (Fig 2H).

snxB1 nimX2 double mutants exhibit nuclear division and germ tube extension at 42° (not shown). Interestingly, at 32° both snxB1 nimX2 and snxB1 cells (Fig 3B) exhibit increased septation compared to wild-type cells (Fig 3A). This same septation pattern is observed in snxB1 cells at 42° (not shown). The average subapical cell (septum to septum) length in wild-type cells was 21.5 ± 8.8 µm, and the average subapical cell length in snxB1 cells was 7.4 ± 2.6 µm, which was determined to be highly statistically significant (P < 0.01).

Both snxB1 and nimX^cdc2AF mutants form hyperseptate hyphae. The nimX^cdc2AF mutation causes cells to form septa earlier than wild-type cells—where wild-type cells do not form septa until eight nuclei are present, nimX^cdc2AF leads to septum formation earlier. To determine if snxB1 causes early septation, BC72 (snxB1), R153 (wild type), and Fry-20-1 (nimX^cdc2AF) conidia were germinated at 32° until the average cell contained four nuclei, and the number of septated cells containing four nuclei was determined. Where 51% of nimX^cdc2AF cells at this stage contained septa, only 4% of snxB1 cells and 0% of wild-type cells contained septa. This indicates that snxB1 does not lead to hyperseptation by allowing septa to form earlier than wild-type cells.

In the presence of 15 mM HU, both snxB1 nimX2 and snxB1 cells germinate, but the nuclei are small and punctate, and very little germ tube extension occurs (Fig 3D). Septation does not normally occur in wild-type cells until after eight nuclei are present and is inhibited in the presence of 15 mM HU (Fig 3C), but septation and the beginnings of branching are observed in snxB1 cells in the presence of 15 mM HU.

The snxC1 mutation suppresses the nimX2 mutation nearly completely at 42°. In both single and double mutants (Fig 4A), hyphae exhibit normal nuclear division and germ tube extension compared to wild type (Fig 3A). Formation of the first septum is often displaced such that rather than occurring at the base of the conidial head, it occurs further into the germ tube, but hyphal septa have spacing and morphology similar to wild type. In both snxC1 nimX2 and snxC1 strains, conidiation is significantly decreased at all temperatures and long aerial hyphae with aberrantly shaped conidiophores are produced (Fig 4, C–E). Conidiation is completely inhibited in the presence of 4% DMSO. Wild-type conidiophores are radially symmetrical and consist of a stalk with a single vesicle from which multiple primary sterigmata (metulae) bud. From each metula two secondary sterigmata (phialides) usually bud, and these bud multiple times to give rise to long chains of conidia. Conidiophores of snxC1 strains exhibit a variety of aberrant morphologies. Some snxC1 conidiophores are normal in appearance, while others have development arrested at various stages or have multiple conidiophore structures extending from the stalk or vesicle (Fig 4E). One frequently observed abnormality is the presence of asymmetric metulae (Fig 4D) that are often unable to form phialides or that begin to form structures resembling hyphae rather than phialides. All aberrantly shaped conidiophores observed contain septa in the conidiophore stalk, and some have septa in the conidiphore head. Wild-type conidiophores do not have septa in the conidiophore stalk and normally stain very calcofluor bright (Fig 4B).

View larger version (66K): In this window In a new window Download PPT slide   Figure 4. snxC1 causes septation in the conidiophore stalk. (A) CY1228 conidia (snxC1 nimX2) were inoculated onto coverslips in YG and incubated at 42°, 16 hr, fixed, and stained with DAPI and calcofluor. (B–E) A612 (B) or BC64 (snxC1; C–E) conidia were inoculated onto MG plates and incubated at 32° for 2 days. To visualize conidiophore structure, conidophores were harvested into fix and stained with DAPI and calcofluor. Arrows indicate septa. Arrowhead indicates a second conidiophore-like structure. Bars, 5 µm.

snxD1 only marginally suppresses the nimX2 mutation, as 36% of snxD1 nimX2 cells undergo mitosis after 20 hr of incubation at 42° (Fig 5A), whereas 100% of wild-type cells undergo division under these conditions (n = 100). snxD1 strains grow well at 32°, with normal nuclear division and germ tube extension, but with slightly increased septation (Fig 5B) as well as an occasional short, anuclear compartment or double septum (Fig 5C). At 32°, subapical cell length is 12.1 ± 3.6 µm compared to 21.5 ± 8.8 µm for wild type, and this difference was determined to be highly statistically significant (P < 0.01). A total of 12% of cells have anuclear compartments or double septa (compared to 1% for wild-type cells; n = 100). Both double and single mutants are also sensitive to 15 mM HU. In the presence of HU, cells undergo nuclear division and germ tube extension much more slowly than wild type, but the nuclei appear normal (Fig 5D).

View larger version (84K): In this window In a new window Download PPT slide   Figure 5. snxD1 partially suppresses the nimX2 mutation and causes aberrant septation, closely spaced nuclei, and HU sensitivity. Conidia from strains S220 (snxD1 nimX2; A) or BC76 (snxD1; B–E) were inoculated onto coverslips in YG, incubated as below, fixed, and stained with DAPI and calcofluor. (A) S220, 42°, 16 hr; (B and C) BC76, 32°, 16 hr; (D) BC76, 32°, 16 hr, + 15 mM HU. Arrows indicate septa; arrowhead indicates a double septum. Bar, 5 µm.

Testing for genetic interactions of snxB and snxC with nimT^cdc25 and ankA^wee1:
The increased septation and HU sensitivity due to the snxB1 mutation are similar to the effects of deletion of ankA^wee1, and snxC1 causes aberrant conidiophores similar to those caused by the nimX^cdc2AF mutation. This suggests that snxB and snxC may be involved in the Tyr-15 phosphorylation of NIMX^CDC2. We therefore wished to determine if either the snxB1 or snxC1 mutations interact with genes known to regulate the tyrosine phosphorylation of nimX^cdc2. Strains carrying the snxB1 or snxC1 mutations were crossed to strains carrying mutations in nimT^cdc25 and ankA^wee1. snxB1 nimT23^cdc25 double mutants grow significantly better than nimT23^cdc25 single mutants under restrictive conditions (42°), indicating that snxB1 suppresses the nimT23^cdc25 mutation (Fig 6). In contrast to the suppression of nimT23^cdc25 by snxB1, snxB1 ankA^wee1 double mutants have a synthetic lethal phenotype. While ankA^wee1 strains are sensitive to 5 mM HU, single mutants can form a colony in the presence of 1 mM HU. The snxB1 ankA^wee1 double mutants are unable to form a colony in the presence of 1 mM HU, similar to nimX^cdc2AF mutants (data not shown).

View larger version (99K): In this window In a new window Download PPT slide   Figure 6. Plate growth phenotypes of snxB1 and snxC1 mutations in the presence and absence of ankAwee1 or nimT23cdc25. Solid plates MG incubated at 42° (top left) or 32° (top right) or MG + 7 mM HU incubated at 32° (bottom left) were point inoculated with spores as follows: top row, double mutants, left to right, MCG3 (ankA snxB1), MCG4 (ankA snxC1), MCG5 (nimT23 snxB1), MCG6 (nimT23 snxC1); middle row, BC72 (snxB1), BC52 (snxC1), ANKA, Fry-20-1 (nimXcdc2AF); bottom row, SWJ108 (nimT23), R153 (wild type). Plate incubations were for 3 days.

Similar analyses indicated that snxC1 does not suppress nimT23^cdc25 but can partially suppress ankA^wee1. The snxC1 nimT23^cdc25 double mutants were more heat sensitive than nimT23^cdc25 alone, as nimT23^cdc25 allows some growth at 42° but the snxC1 nimT23^cdc2 double mutants do not (Fig 6). snxC1 ankA^wee1 double mutants are able to form a small colony in the presence of 5 mM HU and 7 mM HU, concentrations at which ankA^wee1 single mutants were unable to grow. Thus, snxC1 produces a synthetic lethal phenotype in combination with nimT23^cdc25 but partially suppresses ankA^wee1.

snxA1 causes a nuclear division cycle block in G₁ or early S:
Germination of snxA1 strains at 20° for 25 hr followed by DAPI staining (Fig 2G) indicated that snxA1 causes nuclei to arrest in interphase at the restrictive temperature, where wild-type cells normally undergo one to two nuclear divisions under these conditions. That the nuclei were in interphase under these conditions was confirmed by indirect immunofluorescence of microtubules, which exhibited a typical interphase array (data not shown) rather than mitotic spindles. The snxA1 block is reversible until 40 hr of incubation at 20°. If incubation is allowed to proceed for 48 hr at 20°, the block becomes irreversible, as abnormal nuclei begin to accumulate (Fig 2H). To determine at which stage of interphase this nuclear division arrest occurs, reciprocal shift assays were performed on strains BC7 (snxA1; Table 5) and MDS250 (snxA1 nimX2; not shown). Conidia were inoculated into rich media + 25 mM HU and incubated 6.5 hr at 32°, and then shifted to 20° in the absence of HU for 25 hr or shifted to 32° in the absence of HU. After downshift to 20°, 100% of the nuclei underwent mitotic division and then arrested with aberrantly shaped nuclei. This indicates that the cells exit from the S-phase arrest induced by the HU, traverse through G₂ and mitosis, and arrest in G₁ or early S (at a point before the HU arrest) at the restrictive temperature of 20° (see BERGEN et al. 1984 ). Under these same conditions, either a late S-phase arrest (after the HU arrest point) or a G₂ arrest would lead to undivided nuclei. As a control (not shown) the reciprocal experiment was performed, in which conidia were inoculated onto coverslips in rich media and incubated at 20° for 25 hr, and then shifted to 32° in the presence of 25 mM HU. For this experiment, a block in either G₁ or early S would prevent nuclear division. A total of 85% of the cells contained undivided nuclei following the shift from 20° to 32° + 25 mM HU, suggesting a G₁ or early S block. These experiments were repeated four times, either with strain BC7 or strain MDS250, with similar results. As the first experiment rules out G₂ arrest or S arrest after the HU arrest point, and the second supports either early S (before the HU arrest point) or G₁ arrest, these data indicate that the snxA1 nuclear division arrest at 20° occurs either during G₁ or during early S.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In an effort to further understand how the NIMX^CDC2 protein kinase affects nuclear division, septation, and development in A. nidulans, we have generated a set of strains containing extragenic suppressors of the temperature-sensitive nimX2^cdc2 mutation. Extragenic suppressor analysis is designed to allow interacting proteins to be identified; thus the suppressors described here represent genes that interact with NIMX^CDC2. In addition to suppression of the nimX2 mutation, mutations in the four suppressor genes, snxA, snxB, snxC, and snxD, independently affect nuclear division, septation, and conidiation.

snxA is required for progression through G₁ or early S:
Dormant conidia of A. nidulans have a single condensed nucleus that enters the cell cycle at G₁ when dormancy is broken. The spores grow isotropically until a critical cell size is reached and then switch to polar growth and begin to undergo nuclear division. Following the first nuclear division, a switch to polarized cell growth occurs as a germ tube emerges from the conidial head. The NIMX^CDC2 protein kinase has been shown to be required for nuclear division both at G₁ and G₂ in A. nidulans (OSMANI et al. 1994 ) as well as for progression through S phase (YE et al. 1997A ). However, no G₁-specific cyclins or other proteins that might interact with NIMX^CDC2 during G₁ have been identified in this organism. The findings that the cold-sensitive snxA1 suppressor of nimX2 leads to a block in G₁ or early S and that it is allele specific suggest that snxA represents an important gene that interacts with NIMX^CDC2 during G₁ or early S. In addition to its cold sensitivity, the snxA1 mutation also confers slight HU sensitivity at the permissive temperature of 32°. This suggests that snxA may also function during the S-phase checkpoint, possibly as part of the APC/C-dependent checkpoint control mechanism (YE et al. 1997A ). snxA is not allelic with any other previously identified nuclear division regulatory genes located on chromosome II in A. nidulans and therefore represents a novel nuclear division regulator in this organism.

It is interesting that at the permissive temperature snxA1 causes problems in the maintenance of cell polarity, leading to locally swollen regions in the hyphae packed with large numbers of nuclei. MOMANY et al. 1999 found that the establishment of hyphal polarity and the maintenance of polarity are separate events and identified several mutants (swoB, swoE, swoG, and swoH) defective in polarity maintenance. The snxA1 mutation often causes regions similar to those found in the swo mutants, where the cells have switched from polarized growth to isotropic growth. Given that snxA1 mutants are phenotypically similar to swo mutants, it would be interesting to determine if any of the swo mutations affect NIMX^CDC2 activity.

snxB is required for proper septation:
Septation in A. nidulans is the equivalent of cytokinesis and is triggered by mitosis once a critical cell size has been attained (WOLKOW et al. 1996 ). A high level of NIMX^CDC2 activity has been shown to be required for septation, as the presence of a mutation in nimT^cdc25 or nimE^cyclinB causes a delay in septum formation (HARRIS and KRAUS 1998 ). Additionally, the nimX^cdc2AF allele, which causes an increase in NIMX^CDC2 activity due to loss of Tyr-15 regulation, leads to increased septation in both hyphae (HARRIS and KRAUS 1998 ) and conidiophores (YE et al. 1999 ) and to severe HU sensitivity.

snxB1 leads to increased hyphal septation, in addition to its sensitivity to HU, and appears to lower the threshold cell size required to activate septation. A similar phenotype is observed in strains carrying a deletion in ankA^wee1 (data not shown), although the septation defect is more pronounced in snxB1 mutants. It is possible that cytokinesis in snxB1 strains is advanced due to a loss of regulation of Tyr-15 phosphorylation of NIMX^CDC2. This activity is normally controlled by ankA^wee1 and nimT^cdc25; however, both of these genes are located on chromosome II and snxB is not located on this chromosome. The findings that snxB1 is HU sensitive, partially suppresses nimT23, and is synthetic lethal with ankA support the hypothesis that snxB affects Tyr-15 phosphorylation of NIMX^CDC2. snxB could therefore represent a previously uncharacterized gene in the pathway leading to Tyr-15 phosphorylation of NIMX^CDC2, possibly functioning as a "backup" WEE1 kinase.

snxC is required for proper asexual development:
In A. nidulans, asexual development results in the formation of a radially symmetrical conidiophore structure consisting of a stalk, vesicle, metulae, phialides, and conidia. During conidiophore development, changes in septation and nuclear division occur (MIRABITO and OSMANI 1994 ) such that septation is suppressed in the conidiophore stalk but is tightly coupled with nuclear division in the conidiophore head. This allows one daughter nucleus to enter each metula and phialide, followed immediately in each structure by septum formation. It also allows a change from vegetative growth to budding growth. The switch from hyphal growth to conidiophore development is controlled by the brlA transcriptional regulator (ADAMS et al. 1998 ), which has recently been shown to induce the high level of NIMX^CDC2 kinase activity required for conidiophore development (YE et al. 1999 ). Interestingly, snxC1 mutants have phenotypes similar to those of medA (AGUIRRE 1993 ) and brlA42 (MIRABITO et al. 1989 ) developmental mutants in addition to suppressing nimX2, suggesting a role for snxC in the developmental regulation of NIMX^CDC2.

In wild-type A. nidulans, septation and nuclear division are tightly coupled in the hyphae but are uncoupled in the conidiophore stalk, such that multiple nuclei but no septa are present in the stalk. In the nimX^cdc2AF strain, septation and nuclear division are not uncoupled in the conidiophore stalk, leading to multiple septa in conidiophore stalks and aberrantly shaped conidiophore structures. This suggests that regulation of tyrosine phosphorylation of NIMX^CDC2 is involved in uncoupling septation from nuclear division in the conidiophore stalk (YE et al. 1999 ). The snxC1 mutation phenocopies the nimX^cdc2AF mutation in that it affects septation and cell pattern formation during conidiophore development and in that its effects on conidiation are partially dominant. Because snxC1 strains do not exhibit abnormal septation in the hyphae, it is possible that snxC encodes a development-specific regulator that inhibits the septation promoted by NIMX^CDC2 activity and that the snxC1 mutation causes a loss of this regulation, leading to aberrant septation and conidiophore structure. The loss of regulation of Tyr-15 phosphorylation of NIMX^CDC2 observed in the nimX^cdc2AF strain is dominant, and any mutation that leads to such a loss of regulation is also likely to be dominant. Because snxC1 is dominant, phenocopies the conidiophore defects caused by the nimX^cdc2AF mutation, and partially suppresses ankA^wee1, it is likely that it encodes a development-specific protein that regulates Tyr-15 phosphorylation of NIMX^CDC2.

snxD affects hyphal septation:
The snxD1 mutation causes HU sensitivity and leads to defects in septation in the absence of HU. The average subapical cell size is 40% shorter than that of wild-type cells, and 12% of the cells have short anuclear compartments or double septa. snxD1 strains were unable to cross with either nimT23 or ankA strains, making an assessment of possible effects on Tyr-15 phosphorylation difficult. The HU sensitivity of the snxD1 mutation suggests that snxD may be involved in or controlled by the S-phase checkpoint function of NIMX^CDC2.

This work has identified four genes in A. nidulans that interact with the NIMX^CDC2 cell cycle regulatory protein kinase, each of which has distinct effects on the life cycle of the organism. While much is understood regarding the G₂/M function of NIMX^CDC2, little is known about how NIMX^CDC2 participates in G₁/S, septation, or conidiophore development. The identification of the snx genes, which suppress the nimX2 mutation and also affect these processes, genetically implicates these genes in this participation. Each of these genes is likely to be involved in the upstream regulation of NIMX^CDC2 activity. Cloning of the genes and biochemical analysis will allow a better understanding of the regulation of nuclear division in A. nidulans as well as how the NIMX^CDC2 master cell cycle regulator affects septation and conidiophore development.

	ACKNOWLEDGMENTS

We thank Dr. Stephen A. Osmani for gifts of strains, insightful suggestions, and constructive criticisms, Dr. Peter Mirabito for constructive criticisms of the manuscript, Dr. Steve James for gifts of strains and insightful discussions, and Dr. Greg Leno for use of microscopy facilities. This work was supported by grants from the University of Mississippi Medical Center Chapter of Sigma Xi, the National Beta Beta Beta Foundation, the Millsaps College Faculty Development Fund, the George I. Alden Trust, ChemFirst, and the National Institutes of Health grant R15GM55885 to S.L.M.

Manuscript received April 5, 2000; Accepted for publication August 17, 2000.

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