Shared and Independent Roles for a G{alpha}i Protein and Adenylyl Cyclase in Regulating Development and Stress Responses in Neurospora crassa

Growth and development are regulated using cyclic AMP (cAMP)-dependentand -independent pathways in Neurospora crassa. The cr-1 adenylylcyclase mutant lacks detectable cAMP and exhibits numerous defects,including colonial growth habit, short aerial hyphae, prematureconidiation on plates, inappropriate conidiation in submergedculture, and increased thermotolerance. Evidence suggests thatthe heterotrimeric G ${alpha}$ protein GNA-1 is a direct positive regulatorof adenylyl cyclase. ${Delta}$ gna-1 strains are female-sterile, and ${Delta}$ gna-1strains have reduced apical extension rates on normal and hyperosmoticmedium, greater resistance to oxidative and heat stress, andstunted aerial hyphae compared to the wild-type strain. In thisstudy, a ${Delta}$ gna-1 cr-1 double mutant was analyzed to differentiatecAMP-dependent and -independent signaling pathways regulatedby GNA-1. ${Delta}$ gna-1 cr-1 mutants have severely restricted colonialgrowth and do not produce aerial hyphae on plates or in standingliquid cultures. Addition of cAMP to plates or standing liquidcultures rescues cr-1, but not ${Delta}$ gna-1 cr-1, defects, which isconsistent with previous results demonstrating that ${Delta}$ gna-1 mutantsdo not respond to exogenous cAMP. The females of all strainscarrying the ${Delta}$ gna-1 mutation are sterile; however, unlike cr-1and ${Delta}$ gna-1 strains, the ${Delta}$ gna-1 cr-1 mutant does not produce protoperithecia.The ${Delta}$ gna-1 and cr-1 mutations were synergistic with respect toinappropriate conidiation during growth in submerged culture.Thermotolerance followed the order wild type < ${Delta}$ gna-1 <cr-1 = ${Delta}$ gna-1 cr-1, consistent with a cAMP-dependent process.Taken together, the results suggest that in general, GNA-1 andCR-1 regulate N. crassa growth and development using parallelpathways, while thermotolerance is largely dependent on cAMP.

INTRODUCTION

Top

Introduction

Development in fungal systems frequently occurs in responseto specific environmental cues and stressors. In the presenceof abundant nutrients, the filamentous fungus Neurospora crassaextends hyphae that elongate and fuse to form the multicellularmycelium (for a review, see reference 51). Desiccation or nutrientdeprivation causes the mycelium to differentiate aerial hyphaethat give rise to conidiophores and multinucleate asexual spores,macroconidia (referred to here as conidia). Elaboration of conidiophoresand production of conidia require an air-water interface; however,submerged cultures can be induced to undergo conidiation bycarbon or nitrogen starvation or exposure to high temperatures(7, 18, 43, 55). Nitrogen limitation initiates the sexual cycleby stimulating production of female reproductive structures,or protoperithecia (reviewed in reference 44). Fertilizationby a conidium or hypha of the opposite mating type results information of the fertilized structure (perithecium), withinwhich sexual spores (ascospores) develop. Mature ascosporesare subsequently ejected from the perithecium in the directionof blue light.

Heterotrimeric GTP-binding proteins, consisting of ${alpha}$ , ß,and ${gamma}$ subunits, transduce various environmental signals to stimulatemorphogenesis and cellular responses in fungi (for reviews,see references 5, 19, and 34). Ligands, such as nutrients andpheromones, are recognized by seven transmembrane helical receptorscoupled to G proteins. Ligand binding results in a conformationalchange within the receptor, leading to the exchange of GDP forGTP on the G ${alpha}$ subunit and subsequent dissociation of the GTP-G ${alpha}$ and Gß ${gamma}$ moiety. Depending on the system, both the freedGTP-G ${alpha}$ and Gß ${gamma}$ moiety may regulate downstream effectors,such as adenylyl cyclase and mitogen-activated protein kinase(MAPK) cascades.

The N. crassa cr-1 mutant lacks adenylyl cyclase activity andcyclic AMP (cAMP) (47, 53, 54), and studies support cr-1 asthe structural gene for the adenylyl cyclase enzyme (28, 29).When the cr-1 mutant is cultured on a solid medium, it growscolonially, lacks appreciable aerial hyphae, and prematurelyproduces macroconidia on the surface of the mycelium, yieldinga "crisp" appearance. Previous work from our laboratory suggeststhat the G ${alpha}$ subunit GNA-1 positively stimulates CR-1 activityand participates in pathways regulating female fertility, apicalextension, osmotic sensitivity, conidiation, and resistanceto heat and oxidative stress (23, 24, 59).

Incongruous development has been observed in signal transductionmutants from N. crassa and the related filamentous fungus Aspergillusnidulans. Deletion of the N. crassa G ${alpha}$ subunit gna-3 gene orthe putative glucose sensor rco-3 gene causes inappropriatedifferentiation of conidiophores in submerged cultures and transcriptionof the conidiation-specific gene con-10 (27, 38). The cr-1 mutantalso conidiates in submerged culture; however, the submergedconidiation phenotype is not as dramatic as that observed in ${Delta}$ gna-3 and rco-3 strains (27, 38, 41). These results suggestthat development in N. crassa utilizes both cAMP-dependent and-independent pathways. In A. nidulans, overexpression of theRGS (named for regulator of G-protein signaling) protein geneflbA, expression of the dominant negative G ${alpha}$ allele fadA^G203R,or deletion of the Gß gene sfaD leads to submergedculture conidiation (32, 33, 46, 60). Deletion of the cAMP-dependentprotein kinase A catalytic subunit gene pkaA does not causeinappropriate conidiation in submerged cultures, suggestingthat the conidiation defects of FlbA, FadA, and SfaD are cAMPindependent (50).

Results from studies of N. crassa and Saccharomyces cerevisiaesupport an inverse correlation between cAMP levels and resistanceto high temperatures. In N. crassa, cr-1 and gna-1 mutants possessincreased thermotolerance (8, 59). Conversely, strains withGTPase-deficient alleles of gna-1 are more sensitive to lethaltemperatures than the wild type (59). Low cAMP levels inducedby loss-of-function mutations in ras2 or by deletion of theG ${alpha}$ subunit GPA2 lead to increased heat stress resistance in S.cerevisiae (6, 22). In contrast, the elevated cAMP levels ofstrains carrying the constitutively activated ras2^Val19 mutationare correlated with hypersensitivity to lethal temperatures(11, 26).

In this study, we investigate cAMP-dependent and -independentpathways regulated by adenylyl cyclase and the stimulatory G ${alpha}$ protein GNA-1 in N. crassa. Wild-type, ${Delta}$ gna-1, cr-1, and ${Delta}$ gna-1cr-1 strains are analyzed for phenotypes and expression of G-proteinsubunits and adenylyl cyclase. Thermotolerance, submerged cultureconidiation, and transcript levels of a conidiation-regulatedgene are also investigated. Our results suggest that thermotoleranceis largely dependent on cAMP and that GNA-1 and CR-1 act synergisticallyto regulate development in N. crassa.

MATERIALS AND METHODS

Top

Materials and Methods

Strains, media, and culture conditions. N. crassa strain 74-OR23-1A (mating type A) (Fungal GeneticsStock Center [FGSC]) (University of Kansas Medical Center, KansasCity, Kans.) was used as the wild type in all experiments exceptthose involving fertilization of mating type A strains; in suchcases, wild-type strain Sta73a (mating type a) was used as themale. A ${Delta}$ gna-1 cr-1 mating type A double mutant (strain 1.2)was constructed through a sexual cross between the parentalcr-1 mating type A strain (FGSC 4008; allele B123) and ${Delta}$ gna-1::hph⁺mating type a strain (3B10a) (24). Ascospores were plated onmedium containing sorbose (9) containing 200 µg of hygromycinB per ml. Southern blot analysis was performed (23) to verifythat hygromycin-resistant progeny contained the ${Delta}$ gna-1::hph⁺gene replacement (data not shown). Since the cr-1 gene and themating type gene are linked, hygromycin B-resistant progenywere tested for mating type to determine the cr-1 allele present(9, 41). Escherichia coli strain DH5 ${alpha}$ was used to maintain plasmids(20).

N. crassa strains were cultured on Vogel's minimal medium containing1.5% sucrose (VM) (56) to facilitate production of vegetativehyphae in liquid cultures or production of vegetative hyphae,aerial hyphae, and conidia on plates. Synthetic crossing medium(SCM) (57) was used to induce production of female reproductivestructures during the sexual cycle. Conidia were used to inoculateshaken or standing liquid cultures and agar plates. Media weresupplemented with peptone (2% wt/vol) or cAMP (2.0 mM) whereindicated.

Overexpression of N. crassa adenylyl cyclase (CR-1) and antiserum generation. Cosmid pSV50-4:7C (4:7C) containing the structural gene forN. crassa adenylyl cyclase (nac/cr-1) has previously been identified(28). This cosmid was used to generate a construct to allowoverexpression of a 56-kDa protein corresponding to the 5' endof the cr-1 open reading frame (ORF). PCR mutagenesis was usedto create restriction sites for cloning into the pET22 vector(Novagen, Madison, Wis.) (allows protein overexpression in E.coli) and to remove the first two introns from the 5' end ofthe ORF in the 4:7C genomic clone. Templates for PCR were generatedby cloning (i) a 9.14-kb NdeI fragment from 4:7C into the NdeIsite of pGEM5Zf (pDIV6), (ii) the PstI-BamHI fragment from pDIV6into pBluescript II KS⁺ digested with the same enzymes (pDIV6.5),and (iii) a 4-kb EcoRI fragment from 4:7C that contains the5' end of the cr-1 ORF and $~$ 1-kb untranslated sequence into pBluescriptII KS⁺ to yield pDIV8.

The first cr-1 intron was deleted through creation of a silentmutation at the intron splice junction that also encodes anNheI restriction site. Two PCR products flanking the intronwere generated using pDIV8 as a template and ligated togetherto create a fragment extending from the ATG translational initiationcodon to the end of the second exon of cr-1. The primers usedfor the two PCRs are (i) NAC1 (creates an NdeI site at the translationalstart site of nac; plus strand) (G GTG CAT ATG ACG AGA AAT GAC),(ii) NAC2 (encodes an NheI site at the 3' end of exon 1; minusstrand) (TCA GCT AGC AAA TTG GGT GCC GTC), (iii) NAC3 (createsan NheI site at the 5' end of exon 2; plus strand) (ATC GCTAGC AGG TCA TCG AGA G), and (iv) NAC4 (recognizes an EcoRI sitein exon 2; minus strand) (GG GAA TTC AAT GAC AAC TGC). The NAC1/NAC2and NAC3/NAC4 PCRs were performed using Pfu polymerase and 625ng (each) of the two indicated primers according to the manufacturer'srecommendations (Stratagene, La Jolla, Calif.). The NAC1/NAC2and NAC3/NAC4 PCR products were then digested with the appropriateenzymes and then ligated into pSP72 (Promega, Madison, Wis.)digested with NdeI and EcoRI to create pDIV13.

The second intron in cr-1 was removed using inverse PCR withpDIV6.5 as a template. The primers were NAC5 (recognizes the5' end of intron 2 junction; minus strand) (GTC GGC TTC TTGGTA CAG AAA CGG) and NAC6 (anneals at 3' end of intron 2 junction;plus strand) (GAC ATC GCT CGC TAT GGC GAA GCA CCC). The NAC5/NAC6reactions were performed using Pfu polymerase and 125 ng (each)of the two indicated primers according to the manufacturer'srecommendations (Stratagene), except for the presence of 5%dimethyl sulfoxide. The NAC5/NAC6 3.7-kb linear PCR productcontaining vector and insert was ligated and transformed intoE. coli DH5 ${alpha}$ cells. Approximately 56% (27 of 48) of the resultingclones contained a 700-bp PstI-BamHI fragment, indicating deletionof the second intron. Sequence analysis revealed that all clonescontained additional small deletions at the intron splice site.Clone pDIV12 contained a n^-3 mutation that resulted in an in-framedeletion of one of two successive aspartate residues (at position441 or 442 in the amino acid-coding sequence). The absence ofone amino acid is not expected to dramatically affect the abilityof the protein to function as an effective antigen in rabbits;therefore, this clone was used to make the final overexpressionconstruct (see below).

The 700-bp PstI-BamHI fragment from pDIV12 and the 673-bp NdeI-PstIfragment from pDIV13 were ligated into pET22b (Qiagen, Inc.,Valencia, Calif.) digested with NdeI and BamHI to create thefinal overexpression construct, pDIV18. E. coli strain HMS174(plysS)(52) was transformed with pDIV18, and the strain was culturedto facilitate overexpression of the CR-1 protein fragment asrecommended by the manufacturer (Qiagen). The 56-kDa proteinwas subsequently purified by virtue of its carboxy-terminalsix-His epitope tag using Ni-nitrilotriacetic acid agarose accordingto the manufacturer's recommendations (Qiagen). The fractionsenriched for the CR-1 protein were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (10% polyacrylamide),and the CR-1 protein band was excised and used to raise a specificantiserum in rabbits (Cocalico Biologicals, Reamstown, Pa.).

Western and Northern blot analyses. The particulate fractions used for the adenylyl cyclase assaysdescribed above were subjected to Western blot analysis usingGNA-1 (23), GNA-2 (3), GNB-1 (24), or CR-1 antisera at finaldilutions of 1:2,500, 1:5,000, 1:5,000, and 1:10,000, respectively.The proteins were detected by using the enhanced chemiluminescencesystem (Amersham Pharmacia Biotech, Piscataway, N.J.) with ahorseradish peroxidase-conjugated goat anti-rabbit antibody(Bio-Rad, Hercules, Calif.) as the secondary antibody.

For isolation of total RNA, 8-day-old conidia were inoculatedinto 50-ml portions of liquid VM and cultured in constant darknessfor 16 h at 30°C with shaking at 200 rpm. Mycelia were collectedby vacuum filtration and ground to a fine powder in liquid nitrogen,and total RNA was isolated as previously described (48). Northernblot analysis was performed as described previously (4), usinga 200-bp BamHI-EcoRI fragment of pBW100 containing the con-10cDNA (45) or the cox-5 gene insert from pSRCOX-5 (48) as probes.

Adenylyl cyclase assays. Eight-day-old conidia were inoculated at a final concentrationof 2.5 x 10⁶ cells/ml in 500 ml of VM and cultured at 30°Cfor 16 h in darkness with shaking at 200 rpm. Mycelia were collectedby filtration and ground to a fine powder in liquid nitrogen.Samples were suspended in 10-ml portions of extraction buffer[25 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (pH6.9), 1 mM MgCl₂, 0.25 mM EDTA, 1 mM phenylmethylsulfonyl fluoride]and stirred gently at 4°C for 20 min. Extracts were centrifugedat 1,000 x g for 15 min at 4°C. The resulting supernatantwas centrifuged at 180,000 x g at 4°C. The pellet (particulatefraction) was resuspended in extraction buffer, and proteinconcentration was determined using the Bradford microassay kit(Bio-Rad) with bovine serum albumin as a standard. Samples wereassayed for Mn²⁺-ATP- and Mg²⁺ -ATP-dependent adenylyl cyclaseactivity as described previously (24).

Phenotypic analysis and thermotolerance measurements. Colony growth and morphology were assessed by inoculating VMplates with conidia followed by incubation at 30°C in thedark for approximately 36 h. Standing liquid cultures were inoculatedand grown as previously described (27) in the presence or absenceof 2.0 mM cAMP. For analysis of conidiation of submerged cultures,liquid VM cultures were inoculated with conidia at a densityof 1 x 10⁶ or 3 x 10⁶ cells/ml, incubated for 16 h with shakingat 200 rpm in the dark, and then photographed. For analysisof female fertility, strains were cultured on SCM plates for10 days in constant light at room temperature to induce productionof protoperithecia. Perithecia were photographed as describedpreviously (23) 6 days after fertilization with conidia of theopposite mating type. Resistance of 3-h-old germlings to a 52°Clethal heat treatment with and without prior exposure to 45°C(induced versus uninduced thermotolerance, respectively) wasmeasured as previously described (59). Percent survival wasobtained by dividing the number of colonies from plates containingheated germlings (induced or uninduced) by the number of colonieson plates from germlings held at 30°C throughout the experiment(controls).

RESULTS

Top

Results

${Delta}$ gna-1 cr-1 strains contain wild-type levels of GNA-2 and GNB-1 but lack full-length CR-1 protein and adenylyl cyclase activity. A regulatory component is required for adenylyl cyclase to utilizeMg²⁺-ATP as a substrate for both basal and GTP-stimulated activities;in contrast, Mn²⁺-ATP adenylyl cyclase activity is independentof auxiliary proteins and thus serves as a measurement of theamount of active enzyme (15). N. crassa cr-1 strains do notcontain Mg²⁺- or Mn²⁺-ATP adenylyl cyclase activity or intracellularcAMP (47). We have previously shown that while Mn²⁺-ATP adenylylcyclase activity is only slightly reduced in ${Delta}$ gna-1 mutants,basal and GTP-stimulated Mg²⁺-ATP activities are severely affectedin these strains (24). These results, in combination with theobservation that an anti-GNA-1 antibody inhibits Mg²⁺-ATP-dependentadenylyl cyclase activity in preparations of the wild-type strain,suggested that GNA-1 is the regulatory component in N. crassaadenylyl cyclase assays (24).

Certain ${Delta}$ gna-1 defects, such as female sterility, are not sharedby cr-1 strains, suggesting that the affected functions areregulated using cAMP-independent pathways. Furthermore, exogenouscAMP does not rescue ${Delta}$ gna-1 phenotypes (23), despite the apparentrequirement for GNA-1 in regulation of adenylyl cyclase activity.To differentiate between cAMP-dependent and -independent functionsof gna-1, a ${Delta}$ gna-1 cr-1 mutant was constructed through a sexualcross. An observation of identical defects in cr-1 and ${Delta}$ gna-1cr-1 strains would be evidence for cAMP-dependent functionsfor gna-1. In contrast, an observation of different phenotypesfor these two strain backgrounds would offer support for cAMPindependence.

The ${Delta}$ gna-1 mutation (strain 3b10a [mating type a]) is markedby an E. coli hph gene insertion, and the cr-1 mutation (FGSC4008 [mating type A]) is followed by linkage to the mating type(41). Therefore, homokaryotic hygromycin-resistant (hyg^R) ascosporeprogeny should carry the gna-1 gene replacement (data not shown)and should not contain the GNA-1 protein (Fig. 1A). Hyg^R progenythat were phenotypically identical to the ${Delta}$ gna-1 parent weretested for mating type, and all were mating type a, consistentwith the presence of the cr-1⁺ allele. Of 10 hyg^R phenotypicallycrisp progeny, 2 were mating type a and 8 were mating type A.Southern blot analysis demonstrated that all 10 strains carriedthe ${Delta}$ gna-1::hph⁺ deletion mutation (data not shown). Thus, themating type a hyg^R crisp progeny represented strains that werecr-1 mating type recombinants.

View larger version (48K):
[in this window]
[in a new window]
FIG. 1. Levels of G ${alpha}$ , Gß, and adenylyl cyclase proteins. (A) Levels of GNA-1, GNA-2, and GNB-1. Samples containing 30 µg of total membrane protein from strains with the indicated genotypes were subjected to Western blot analysis using specific antisera. WT, wild type. (B) Levels of CR-1. Samples containing 30 µg of total membrane protein were analyzed by Western blot analysis using a CR-1 antiserum. The position of full-length CR-1 in the wild-type (WT) and ${Delta}$ gna-1 mutant preparations is indicated to the right of the blot. The migration position of the amino-terminal 56-kDa CR-1 protein fragment (IB) used as an antigen is indicated. The asterisk indicates the 150-kDa species found in the cr-1 strain preparation.

Western blot analysis showed that cr-1 and wild-type strainscontain wild-type levels of GNA-1, GNA-2, and GNB-1, as previouslyobserved (Fig. 1A) (27). The ${Delta}$ gna-1 cr-1 mutants lack GNA-1 butcontain wild-type levels of the G ${alpha}$ and Gß subunitsGNA-2 and GNB-1, respectively (Fig. 1A). A 56-kDa protein correspondingto the extreme amino terminus of CR-1 was overexpressed in E.coli and used to generate a polyclonal antibody in rabbits.Western blot analysis with the preimmune serum showed no hybridizingproteins (data not shown). Analysis with the immune serum indicatedreaction with a 220-kDa species present at similar levels in ${Delta}$ gna-1 and wild-type strains (Fig. 1B). The 220-kDa protein wasnot observed in cr-1 preparations (Fig. 1B), but a prominent $~$ 150-kDa species was present. Since the CR-1 antibody is directedagainst the amino terminus, this 150-kDa protein may be theresult of a nonsense mutation at the carboxy terminus, producinga truncated protein. Mutation of gna-1 in the cr-1 backgroundleads to loss of the $~$ 150-kDa species, implying that GNA-1 influencesthe level of the mutant protein; however, we do not know themechanism underlying this effect. The observation that levelsof the 220-kDa protein are similar in wild-type and ${Delta}$ gna-1 strainsis consistent with the modest effect of the ${Delta}$ gna-1 mutation onMn²⁺-ATP activity noted in our earlier study (24).

As expected, no Mn²⁺-ATP or Mg²⁺-ATP-dependent adenylyl cyclaseactivity could be detected in cr-1 or ${Delta}$ gna-1 cr-1 strains (datanot shown). ${Delta}$ gna-1 mutant preparations contain substantial levelsof Mn²⁺-ATP-dependent activity, consistent with previous results(87% of wild-type activity [24]). Similar to what has been notedpreviously (24), ${Delta}$ gna-1 mutants have basal and GTP-stimulatedMg²⁺-ATP-dependent activities that are only 18 and 9.5% of wild-typeactivity, respectively.

cr-1 and gna-1 contribute to female fertility. Previous work from our laboratory demonstrated that ${Delta}$ gna-1 mutantsare male-fertile but female-sterile. ${Delta}$ gna-1 strains have aberrantprotoperithecia that do not develop into normal perithecia afterfertilization and that only rarely produce ascospores (Fig.2) (23). In contrast, cr-1 mutants are able to function as malesor females during a sexual cross but exhibit delayed perithecialand ascospore development compared to the wild type (Fig. 2)(41). These observations supported the hypothesis that cAMPis not essential for female fertility in N. crassa.

View larger version (132K):
[in this window]
[in a new window]
FIG. 2. Female fertility. Strains with the indicated genotypes were cultured on SCM for 7 days prior to fertilization using wild-type (WT) strain 74-OR23-1A or Sta73a conidia. Perithecia were photographed 6 days after fertilization. Perithecia (in cr-1 and wild-type strains) and aberrant perithecia (in a ${Delta}$ gna-1 strain) are indicated by arrows.

Functions for GNA-1 and cAMP in regulating fertility were furtherexamined by analyzing the sexual cycle in strains lacking bothgna-1 and cr-1. Similar to ${Delta}$ gna-1 mutants, the males of ${Delta}$ gna-1cr-1 strains are fertile but the females are sterile (data notshown and Fig. 2). However, unlike ${Delta}$ gna-1 strains, ${Delta}$ gna-1 cr-1double mutants do not form observable protoperithecia or perithecia(data not shown and Fig. 2). Therefore, the cr-1 mutation blocksprotoperithecial formation in the ${Delta}$ gna-1 background, suggestingthat cAMP may play a role in protoperithecial development. Thiscontrasts with the known effects of cr-1 on perithecial developmentand may reflect a requirement for cAMP during protoperithecialformation that is exacerbated by mutation of gna-1 and whichimpedes later perithecial development.

Loss of gna-1 blocks the response of cr-1 mutants to exogenous cAMP. As mentioned above, cr-1 mutants have a greatly reduced apicalextension rate and produce much shorter aerial hyphae comparedto those of the wild type. The apical extension rate of ${Delta}$ gna-1strains is approximately 50% of the wild-type rate, and ${Delta}$ gna-1mutants produce shorter aerial hyphae (23). Supplementationof cr-1 plate and standing liquid cultures with cAMP restoresthe wild-type morphology and growth rate to cr-1 mutants buthas no effect on ${Delta}$ gna-1 mutants (23, 27; unpublished observationsin reference 53).

The inability to rescue the apical extension rate and aerialhypha height defects of ${Delta}$ gna-1 strains by adding cAMP could stemfrom the fact that these processes are controlled using distinctcAMP-dependent (cr-1) and cAMP-independent (gna-1) mechanismsin N. crassa. Alternatively, our previous observations couldreflect a more general requirement for gna-1 in the responseto exogenous cAMP. To distinguish between these possibilities,we explored the effect of cAMP supplementation on the ${Delta}$ gna-1cr-1 double mutant. Loss of gna-1 in a cr-1 background causesa greater restriction of apical growth than that observed ina cr-1 background alone (57% decrease) (Fig. 3 and data notshown). Similar to previous observations with ${Delta}$ gna-1 mutants,exogenous cAMP has no effect on plate cultures of ${Delta}$ gna-1 cr-1strains (data not shown). In standing liquid cultures, cr-1strains produce short aerial hyphae, while hyphae are completelyabsent in ${Delta}$ gna-1 cr-1 mutants (Fig. 3B). Addition of cAMP restoredwild-type aerial hypha height to cr-1 mutants but had no effecton ${Delta}$ gna-1 cr-1 (Fig. 3B). Thus, not only are ${Delta}$ gna-1 strains refractoryto exogenous cAMP, but the presence of the ${Delta}$ gna-1 mutation alsoblocks the normally robust response of cr-1 mutants. A similareffect is observed upon loss of gna-1 in the ${Delta}$ gna-3 genetic background(A.M. Kays and K. A. Borkovich, unpublished observations). Theseobservations are consistent with a more general requirementfor gna-1 in the response of N. crassa to exogenous cAMP.

View larger version (68K):
[in this window]
[in a new window]
FIG. 3. Vegetative phenotypes in the presence of an air-water interface. (A) Growth of colonies on solid medium. Strains with the indicated genotypes were grown on VM plates at room temperature under constant light and photographed when the ${Delta}$ gna-1 colony had reached the edge of the plate (approximately 36 h). Wild-type colonies are approximately two times larger than ${Delta}$ gna-1 colonies under these conditions (data not shown). (B) Standing liquid cultures. Strains were inoculated into 2-ml portions of liquid VM and incubated in a stationary position first for 3 days at 30°C in the dark and then for 4 days at room temperature in constant light. The presence (+) or absence (-) of 2.0 mM cAMP is indicated below the test tubes.

Relationship between cr-1 and gna-1 in regulating thermotolerance. The heat shock response is a biological phenomenon that occursin all cells as a mechanism to survive the stress of nonphysiologicaltemperatures. In response to heat stress, cells quickly synthesizeheat shock proteins (Hsps) that act to stabilize endogenousproteins, prevent denaturation, and protect cells from subsequentlethal heat exposure. The mechanism of Hsp induction is referredto as induced thermotolerance (for reviews, see references 39and 49). A sudden increase in temperatures is lethal when cellsdo not have the opportunity to adapt and is referred to as alethal or uninduced heat shock. Exposure to temperatures over50°C is lethal to N. crassa; however, previous exposureof cells to temperatures of 40 to 47°C allows the inductionof Hsps (25, 42).

Studies have shown that mutation of cr-1 or gna-1 results inheightened thermotolerance in N. crassa (8, 59). Analysis ofthermotolerance in ${Delta}$ gna-1, cr-1, and ${Delta}$ gna-1 cr-1 strains was performedto determine the relationship between ${Delta}$ gna-1 and cr-1 in regulatingthis phenomenon. If GNA-1 exerts its effect through controlof cAMP levels and cAMP alone controls thermotolerance, thenthe response to high temperatures in the cr-1 and ${Delta}$ gna-1 cr-1strains should be the same. Alternatively, if GNA-1 also controlsa cAMP-independent pathway that negatively regulates the heatshock response, loss of gna-1 in the cr-1 background shouldcause increased thermotolerance.

In agreement with previous work from our laboratory, ${Delta}$ gna-1 mutantspossess approximately sixfold-greater uninduced thermotolerancethan that of the wild type and 4.5-fold-greater induced thermotolerancethan that of the wild type (Fig. 4). In contrast to a previousreport (8), we found that thermotolerance in cr-1 strains isinducible; strains containing the cr-1 mutation have a greaterthan twofold-higher survival if preexposed to a nonlethal heattreatment (Fig. 4). cr-1 and ${Delta}$ gna-1 cr-1 strains were extremelythermotolerant relative to wild-type and ${Delta}$ gna-1 strains, andthermotolerance is inducible in both cr-1 and ${Delta}$ gna-1 cr-1 mutants.Importantly, the thermotolerance of cr-1 and ${Delta}$ gna-1 cr-1 strainsis similar within experimental error (Fig. 4), implying thatGNA-1 regulates thermotolerance through modulation of cAMP levels.The data are consistent with previous reports that the observedincrease and decrease in thermotolerance of strains carryingeither null or constitutively activated gna-1 alleles resultfrom their lower or higher levels of cAMP, respectively (59).

View larger version (26K):
[in this window]
[in a new window]
FIG. 4. Thermotolerance assays. The viability of 3-h-old germlings was determined after exposure to a lethal temperature (52°C) with (induced thermotolerance) and without (uninduced thermotolerance) prior exposure to a sublethal heat shock temperature (45°C). The error bars show standard errors of the means.

${Delta}$ gna-1 mutants conidiate in submerged cultures at higher cell densities. Macroconidiation in N. crassa requires an air-water interface;thus, conidiation typically occurs on solid surfaces. However,wild-type N. crassa strains can be induced to conidiate in submergedcultures when starved for carbon or nitrogen. Conidiation ofsubmerged cultures has also been observed for cr-1, rco-1, andgna-3 mutants (27, 38, 41). On the basis of these observations,we postulated that the ${Delta}$ gna-1 cr-1 mutant would be derepressedfor conidiation in submerged cultures.

The wild-type control strain grew to confluence and did notconidiate in submerged culture under any of the conditions tested(Fig. 5A). The ${Delta}$ gna-1 mutant conidiated in submerged cultureonly when cells were inoculated at a high cell density (3 x10⁶ cell/ml [Fig. 5A]). A threefold decrease (10⁶ cell/ml) incell density resulted in small, tight mycelial aggregates, withno observable conidiophores (Fig. 5A). cr-1 and ${Delta}$ gna-1 cr-1 strainsproduced large numbers of conidiophores at both cell densities,with more conidiophores visible in the ${Delta}$ gna-1 cr-1 mutant (Fig.5A). Therefore, submerged culture conidiation in the ${Delta}$ gna-1 mutantis cell density dependent, and the ${Delta}$ gna-1 mutation intensifiesthe conidiation defect of cr-1 strains.

View larger version (77K):
[in this window]
[in a new window]
FIG. 5. Conidiophore development and conidiation-specific gene expression in submerged cultures. (A) Conidiophore development. Conidia were inoculated into liquid VM at either 1 x 10⁶ or 3 x 10⁶ cells/ml, incubated with shaking in the dark at 30°C for 16 h, and then photographed. Arrows indicate conidiophores. (B) Correlation between conidiophore development and conidiation-specific gene expression. Total RNA (20 µg) isolated from strains with the indicated genotypes was analyzed by Northern blot analysis using a ³²P-labeled con-10 cDNA as a conidiation-specific probe; the cox-5 gene was used as a probe to control for RNA loading and transfer.

It has been previously demonstrated that cr-1 mutants expressthe conidiation-specific gene con-10 in submerged culture (27).To determine if the conidiation observed in ${Delta}$ gna-1 and ${Delta}$ gna-1cr-1 strains correlated with expression of con-10, RNA was isolatedand analyzed for the presence of the con-10 transcript usingNorthern blot analysis. Although no conidiophores were observed,con-10 was weakly transcribed at the higher cell density forthe wild-type strain, suggesting that conidiation may just beinitiating. For the ${Delta}$ gna-1 strain, con-10 was expressed at bothcell densities, with greater transcription occurring at thehigher inoculation density (Fig. 5B). cr-1 and ${Delta}$ gna-1 cr-1 strainstranscribe the con-10 message at both cell densities (Fig. 5B),with the highest level produced by the ${Delta}$ gna-1 cr-1 mutant atthe high cell density. The relative amounts of con-10 in cr-1and ${Delta}$ gna-1 cr-1 cultures inoculated at the lower concentrationwere more variable; however, the amounts of transcript producedby ${Delta}$ gna-1 cr-1 strains were generally the same as or greaterthan that made by cr-1 mutants (data not shown and Fig. 5B).

These results indicate that loss of cr-1 and gna-1 is synergisticwith respect to derepression of con-10 expression, particularlyin cultures inoculated at a high cell density, as the ${Delta}$ gna-1cr-1 strain contains more con-10 mRNA than the cr-1 strain.Furthermore, because adenylyl cyclase activity is absent fromboth cr-1 and ${Delta}$ gna-1 cr-1 strains, the observation that the ${Delta}$ gna-1cr-1 strain contains the greatest amount of con-10 transcriptindicates that con-10 gene expression is also regulated by apathway involving GNA-1, independent of cAMP.

DISCUSSION

Top

Discussion

Previous work has demonstrated that GNA-1 is necessary for normalstress responses and female fertility and that GNA-3 is a globalnegative regulator of conidiation (23, 27, 59). Biochemicalstudies indicate dual regulation of adenylyl cyclase by GNA-1and GNA-3 in N. crassa: GNA-1 positively modulates adenylylcyclase activity, and GNA-3 maintains adenylyl cyclase enzymelevels (24, 27). Here we have further defined the role of GNA-1in cAMP-dependent and -independent pathways by constructingand analyzing a ${Delta}$ gna-1 cr-1 double mutant. In contrast to thecr-1 strain, ${Delta}$ gna-1 cr-1 grows even more slowly and is refractoryto exogenous cAMP. Deletion of gna-1 in the cr-1 backgroundleads to female sterility. Thermotolerance experiments showthat ${Delta}$ gna-1 cr-1 and cr-1 strains are similarly resistant tohigh temperatures. Conidiation in ${Delta}$ gna-1 submerged cultures isdependent on cell density, and the ${Delta}$ gna-1 cr-1 strain conidiatesmore abundantly than the cr-1 strain.

The inability of ${Delta}$ gna-1 cr-1 strains to respond to exogenouscAMP was consistent with our earlier observation that ${Delta}$ gna-1mutants are insensitive to cAMP supplementation (23). As mentionedabove, the same phenomenon is seen in ${Delta}$ gna-1 ${Delta}$ gna-3 strains (Kaysand Borkovich, unpublished). Previous work has shown that bothwild-type and ${Delta}$ gna-1 strains secrete similar amounts of cAMPinto the medium during growth in submerged cultures (24). Thisindicates that cAMP may function as an extracellular signalin N. crassa and that the mechanism that allows movement ofcAMP in and out of the cell is intact in ${Delta}$ gna-1 mutants. Therefore,GNA-1 may function in a pathway that senses extracellular cAMPlevels to inhibit conidiation of submerged cultures or otherdevelopmental processes. Alternatively, GNA-1 may be requiredfor the expression or stability of a component in the cAMP sensingor signaling pathway in N. crassa.

A requirement for a G ${alpha}$ protein in the response to exogenous cAMPhas not been previously reported in N. crassa or other relatedfungi. In fact, there are several instances of fungal adenylylcyclase and G ${alpha}$ mutants that can respond to exogenous cAMP. Asmentioned above, the wild-type morphology is restored to standingliquid cultures of cr-1 and ${Delta}$ gna-3 strains by cAMP supplementation(27, 53). Treatment with cAMP suppresses the developmental defectsof the Magnaporthe grisea ${Delta}$ mac1 and Ustilago maydis ${Delta}$ uac1 adenylylcyclase mutants (1, 16). Phenotypic defects resulting from mutationof the gna-3 homologue in the saprophyte Podospora anserinaand the pathogens U. maydis, Ustilago hordei, and Cryptococcusneoformans are all rescued by exogenous cAMP (2, 31, 35, 37).Addition of cAMP to a mutant of the gna-1 homologue, cpg-1,in the plant pathogen Cryphonectria parasitica suppresses itsvirulence defect (13).

Sexual development in N. crassa is initiated by formation ofprotoperithecia in response to nitrogen limitation. Deletionof gna-1 causes an early stage defect in the sexual cycle, resultingin the formation of aberrant protoperithecia (23, 59). In contrast,although cr-1 strains lack intracellular cAMP, they are stillable to complete the sexual cycle and are only delayed in postfertilizationdevelopment, including perithecial and ascospore production(41). Female sterility in the cr-1 strain background is observedonly upon mutation of gna-1, supporting a cAMP-independent rolefor GNA-1 in regulating female fertility. Furthermore, cAMPsupplementation has no effect on the sexual cycle defects ofeither ${Delta}$ gna-1 or ${Delta}$ gna-3 mutants (27; F. D. Ivey and K. A. Borkovich,unpublished observations). In contrast to N. crassa, the sexualcycle of M. grisea and U. maydis is dependent on cAMP, and the ${Delta}$ mac1 and ${Delta}$ uac1 adenylyl cyclase mutants mate only when exogenouscAMP is supplied (1, 16).

Thermotolerance in N. crassa and S. cerevisiae is inverselycorrelated with cAMP levels (12). Strains with mutations ingenes encoding either adenylyl cyclase or its positive regulatorsare more resistant to heat stress (6, 8, 22, 59), and heat shocktranscription factors are active during periods of low proteinkinase A activity in S. cerevisiae (17). The thermotoleranceof ${Delta}$ gna-1 cr-1 and cr-1 strains is identical, consistent withthis process being largely dependent on cAMP in N. crassa. However,the observation that thermotolerance can be induced in strainscarrying the cr-1 mutation suggests that there may also be acAMP-independent mechanism for the induction of heat shock stressregulators. Studies in S. cerevisiae support cAMP-independentpathways in regulating the heat shock response. Disturbing theintegrity of the cell wall by chemical agents or by mutationof genes important for cell wall synthesis leads to phosphorylationand activation of the MAPK Slt2p and changes in cell wall composition(10). Such disruptions to the cell wall also result in an increasedresistance to high temperatures, supporting the observationthat additional kinases may also function during the heat shockresponse (10, 14).

Conidiation of submerged cultures occurs in ${Delta}$ gna-1 mutants ina cell density-dependent manner. Conidiation of submerged culturesis much more extensive in cr-1 cultures than in ${Delta}$ gna-1 mutantcultures. Deletion of gna-1 and cr-1 is synergistic, with respectto expression of the conidiation-specific gene con-10 at highcell densities. The observations that ${Delta}$ gna-1 strains produceconidiophores only at higher cell densities and that ${Delta}$ gna-1 cr-1strains exhibit more conidiation than cr-1 mutants suggest thatGNA-1 functions independently of cAMP as a negative regulatorof conidiation of submerged cultures. The GNA-1 homologue FadAin A. nidulans also serves as a negative regulator of conidiationof submerged cultures (21, 60). cAMP independence is supportedby the observation that A. nidulans pkaA catalytic subunit mutantsdo not conidiate in submerged culture (50).

The results from comparison of ${Delta}$ gna-1, cr-1, and ${Delta}$ gna-1 cr-1 strainssupport the hypothesis that hyphal development and conidiationinvolve cAMP-dependent and MAPK pathways. A gene encoding aMAPK kinase kinase (MAPKKK), nrc-1, has been identified in N.crassa (30). NRC-1 is most similar to the MAPKKK proteins Ste11pand Byr-2 from S. cerevisiae and Schizosaccharomyces pombe,respectively (30). Mutation of nrc-1 results in formation ofshort aerial hyphae, constitutive conidiation on solid mediumand in submerged culture, thin basal hyphae, semicolonial growthmorphology, defective conidial separation, and female sterility(30); some of these phenotypes are also found in cr-1 and ${Delta}$ gna-1strains. The presence of shared defects for nrc-1 and gna-1mutants suggests that GNA-1 may act upstream of NRC-1 to suppresssubmerged conidiation. Another possibility is that a conidiation-specific,cAMP-dependent pathway influences the NRC-1 MAPK cascade. Crosstalk between cAMP and MAPK pathways during regulation of fungaldevelopment has been observed in M. grisea; the MAPK PMK-1,the GNA-1 homologue MAGB, and cAMP all regulate the invasivegrowth habit of this organism (36, 40).

The results presented here provide further evidence that GNA-1controls multiple signal transduction pathways in N. crassa.The previous demonstration of differential complementation of ${Delta}$ gna-1 phenotypes by mammalian G ${alpha}$ protein genes (58), as wellas the results from functional analysis of N. crassa gna-3 andP. anserina modD mutants, showed that these three G ${alpha}$ subunitsfunction in more than one pathway (27, 37). GNA-1 regulatescAMP-dependent functions, such as thermotolerance, by positivelymodulating adenylyl cyclase activity. Female fertility and submergedconidiation are regulated by GNA-1 through cAMP-independentpathways. Of the three N. crassa G ${alpha}$ subunits, GNA-1 has the greatestrole in resistance to cellular stresses, including hyperosmoticconditions and elevated temperatures. Identification of signalingcomponents that act upstream and downstream of GNA-1 will elucidatethe molecular mechanisms that enable filamentous fungi to senseand respond to environmental stresses and will further revealthe extent of conservation between signaling pathways in yeastand filamentous fungal species.

ACKNOWLEDGMENTS

We thank Jennifer Bieszke, Svetlana Krystofova and Qi Yang formany helpful discussions and Thomas Vida and Dale Hereld forassistance with microscopy.

This work was supported in part by Public Health Service grantGM-48626 from the National Institutes of Health (to K.A.B.).

FOOTNOTES

* Corresponding author. Present address: Department of Plant Pathology, University of California, 2338 Webber Hall, 900 University Ave., Riverside, CA 92521. Phone: (909) 787-2753. Fax: (909) 787-4294. E-mail: Katherine.Borkovich{at}ucr.edu.

Present address: Biology Department, Boston College, ChestnutHill, MA 02467.

Present address: Whitehead Institute, Massachusetts Instituteof Technology, Cambridge, MA 02142-1493.

REFERENCES

Top