A Ras1-Cdc24 signal transduction pathway mediates

A Ras1-Cdc24signal transduction pathway mediates thermotolerance in the fungal pathogen Cryptococcus neoformansConnie B.Nichols,1Zahra H.Perfect 1and J.Andrew Alspaugh 1,2*Departments of 1Medicine and 2Molecular Genetics and Molecular,Microbiology,Duke University Medical CenterDurham,NC 27710,USA.SummaryPathogenic microorganisms must precisely regulate morphogenesis to survive and proliferate within an infected host.This regulation is often controlled by conserved signal transduction pathways that direct morphological changes in varied species.One such pathway,whose components include Ras proteins and the PAK kinase Ste20,allows the human fungal pathogen Cryptococcus neoformans to grow at high temperature.Previously,we found that Ras1signal-ling is required for differentiation,thermotolerance and pathogenesis in C.neoformans .We show here that the guanine nucleotide exchange factor Cdc24is a Ras1effector in C.neoformans to mediate the ability of this fungus to grow at high temperature and to cause disease.In addition,we provide evidence that the Ras1-Cdc24signalling cascade functions specifically through one of the three Cdc42/Rac1homologues in C.neoformans .In conclusion,our studies illustrate how components of conserved sig-nalling cascades can be specialized for different downstream functions,such as pathogenesis.IntroductionAs microbial pathogens enter an infected host,they encounter many external stresses,including elevated temperatures,nutrient deprivation and the host immune system.In order to respond to such changes in their environment,microorganisms often alter their cell shape,size,or volume.These morphogenic changes in fungi may be as dramatic as the transition from a round,yeast-like cell to an elongated hyphal form.Subtler morphogenic processes include establishing polarity,or subcellularasymmetry,to allow proper and efficient budding in the face of cell stress.A precise regulation of morphogenesis can therefore be an important adaptive response to the host environment.Guanine nucleotide-binding proteins from the Ras superfamily play major roles to regulate morphogenesis in small eukaryotes.For example,the Rho-like GTPase Cdc42establishes and maintains cellular polarity both in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe (Johnson,1999).Similarly,Ras and Ras-like proteins also help to direct cell polarity and differentiation in these yeasts (Fukui et al .,1986;Nadin-Davis et al .,1986;Chang et al .,1994;Mosch et al .,1996;Mosch et al .,1999;Ho and Bretscher,2001).Recent studies in the human patho-genic yeast Candida albicans have also demonstrated that homologues of Ras and Cdc42are required for the yeast-to-hyphal transition,and therefore for pathogenesis (Feng et al .,1999;Leberer et al .,2001;Ushinsky et al .,2002;Bassilana et al .,2003;VandenBerg et al .,2004).In addition to their roles in yeasts,Cdc42and Ras homologues also help to control morphogenesis in fila-mentous fungi.For example,homologues of Ras and Cdc42both control hyphal formation in Neurospora crassa (Kana-uchi et al .,1997;Seiler and Plamann,2003).Additionally,Cdc42proteins regulate various aspects of hyphal growth in Schizophyllum commune ,Ashbya gossypii ,Wangiella dermatitidis and Claviceps purpurea (Ye and Szaniszlo,2000;Wendland and Phil-ippsen,2001;Scheffer et al .,2005;Weber et al .,2005).Ras proteins are required for proper hyphal growth in plant and animal fungal pathogens such as Ustilago maydis and Aspergillus fumigatus (Lee and Kronstad,2002;Fortwendel et al .,2005).Ras and Cdc42proteins have been demonstrated to function together to mediate morphogenesis in two patho-genic fungi.In the filamentous plant pathogen Colletotri-chum trifolii and the dimorphic human pathogen Penicillium marneffei,Cdc42functions downstream of Ras to mediate hyphal growth and spore germination (Boyce et al .,2005;Chen et al .,2006).The P .marneffei Ras and Cdc42proteins also direct polarized growth of the yeast stage of this fungus (Boyce et al .,2001;Boyce et al .,2005).Accepted 7December,2006.*For correspondence.E-mail alspa001@;Tel.(+1)9196840045;Fax(+1)9196848902.Molecular Microbiology (2007)63(4),1118–1130doi:10.1111/j.1365-2958.2006.05566.x First published online 18January 2007©2007The AuthorsJournal compilation ©2007Blackwell Publishing LtdRas and Cdc42proteins are activated by guanine nucleotide exchange factors(GEFs)that facilitate the exchange of GDP for GTP.S.cerevisiae Cdc24,the GEF for Cdc42,is essential for cell viability,helping to establish cell polarity required for budding(Johnson,1999).In con-trast,the Cdc24homologue in S.pombe,Scd1,is not essential,but scd1mutants are not able to maintain normal polarity.Instead,S.pombe scd1mutants grow as round cells,in contrast to the rod-shaped morphology of wild-type cells.The striking morphogenesis phenotype exhibited by S.pombe scd1mutant strains is identical to that of the ras1mutant strains.In fact,S.pombe Scd1has been demonstrated to be a target of Ras1,providing a mechanistic link between Ras and Cdc42proteins(Chang et al.,1994).Guanine nucleotide exchange factors for Ras and Cdc42also regulate cell polarity infilamentous fungi such as A.gossypii,U.maydis and N.crassa(Wendland and Philippsen,2001;Weinzierl et al.,2002;Muller et al., 2003;Seiler and Plamann,2003).Both Cdc25and Cdc24,GEFs for Ras1and Cdc42,respectively,are also required for the transition from yeast cell to hyphae in C.albicans(Enloe et al.,2000;Bassilana et al.,2003). Previously,we demonstrated that the Ras1protein is required for morphogenesis and virulence in the human fungal pathogen C.neoformans(Alspaugh et al.,2000; Waugh et al.,2003). C.neoformans is the causative agent of cryptococcosis,a life-threatening fungal menin-gitis that primarily infects immunocompromised individu-als(Mitchell and Perfect,1995).Ras1regulates two distinct signalling pathways in C.neoformans,a mating pathway and a morphogenesis pathway.The Ras1 protein mediates its effect on mating through transcrip-tional control of the pheromone response pathway (Waugh et al.,2003).Strains with a ras1mutation are therefore unable to undergo fusion andfilamentation during sexual differentiation,resulting in sterility.Through a separate signalling pathway,Ras1controls cell morpho-genesis and thermotolerance(Alspaugh et al.,2000; Waugh et al.,2002). C.neoformans ras1mutant cells exhibit a temperature-dependent defect in actin polariza-tion,arresting as large unbudded cells at37°C.Accord-ingly,ras1mutants are avirulent in animal models of cryptococcal disease.To further define the mechanisms by which Ras1con-trols cell morphology and high temperature growth,we have identified a Cdc24homologue as a downstream effector of C.neoformans Ras1.Strains deleted for the CDC24gene have temperature-sensitive growth and morphology defects similar to ras1mutant strains.The cdc24mutant is avirulent in a murine model of cryptococcosis.Our genetic and two-hybrid data also suggest that C.neoformans Cdc24functions in a con-served signalling cascade comprised of Ras1,Cdc24,Cdc42and the PAK kinase Ste20to mediate thermotol-erance,polarized growth and pathogenicity.ResultsIdentification of Ras1downstream signalling componentsRas,or Ras-like proteins,activate Cdc42in the model yeasts S.pombe and S.cerevisiae through Cdc24 (also known as Scd1in S.pombe).Using comparative genomics we identified a Cdc24homologue in the C.neoformans genome database(C.neoformans var. grubii).BLAST searches revealed a gene predicted to encode a Cdc24homologue of S.pombe Scd1and other fungal Cdc24proteins(Miyamoto et al.,1987;Chang et al.,1994;Wendland and Philippsen,2001;Bassilana et al.,2003).Protein domain analysis using Pfam soft-ware revealed that the putative C.neoformans Cdc24 protein contained conserved Cdc24domains including those for Rho-GEF activity[DH(Dbl homology)and PH (pleckstrin homology)domains,a PB1(Pbox and Bem1) protein binding domain and a CH(calponin homology) domain(Gulli and Peter,2001;Bateman et al.,2004)]. To determine if Cdc24functions downstream of Ras1in C.neoformans,we overexpressed CDC24in the ras1 mutant strain.Previously,we demonstrated that overex-pression of the gene encoding the PAK kinase Ste20 suppressed the temperature-sensitive growth defect of the Cryptococcus neoformans var.neoformans(serotype D)ras1mutant strain(Vallim et al.,2005).Overexpression of STE20also suppresses the high temperature growth defect of a ras1mutant strain(LCC70)generated in the highly pathogenic C.neoformans var.grubii(serotype A) (Fig.1).Similar to our Ste20overexpression studies, CDC24overexpression suppresses the high temperature growth phenotype of the LCC70ras1mutant strain (Fig.1).Together,these results suggest that C.neoformans Cdc24and Ste20function as downstream effectors of Ras1to mediate high temperature growth.Growth of a C.neoformans cdc24mutant is inhibitedby high temperatureTo further define the role of Cdc24in C.neoformans,we generated a cdc24mutant in which the entire CDC24 coding region was replaced with a neomycin-resistance cassette(Fraser et al.,2003).Several viable cdc24 mutants were obtained,indicating that CDC24is not essential in C.neoformans.Southern analysis confirmed the deletion of the CDC24gene,and reverse transcription polymerase chain reaction(RT-PCR)demonstrated that there was no CDC24transcript produced in these cdc24 mutant strains(data not shown).All strains had identicalC.neoformans Cdc241119©2007The AuthorsJournal compilation©2007Blackwell Publishing Ltd,Molecular Microbiology,63,1118–1130morphogenic phenotypes,and one strain,CBN16,was designated for subsequent experiments.The cdc24 mutant strain did not have an apparent growth defect at 30°C but exhibited reduced growth at37°C and no growth at39°C(Fig.2A).This is a similar but less severe temperature-sensitive phenotype compared with the ras1 mutant strain.Reintroduction of the CDC24gene into the cdc24mutant strain restored growth to wild-type level at 37°C and39°C(Fig.2A).Microscopic examination of cdc24mutant cells revealed striking alterations in cell morphology.cdc24mutant cells incubated in YPD medium at30°C were larger than wild-type cells,a phenotype shared by ras1mutant cells (Fig.2B and data not shown).The severity of the cdc24 mutant strain morphogenic defect correlated directly with increased temperature.When cdc24mutant cells were incubated to mid-log phase at30°C in YPD medium and shifted to either37°C or39°C for6h,these mutant cells were markedly larger than either cdc24mutant cells incu-bated at30°C or wild-type cells grown at37°C and39°C (Fig.2B).In addition,the cdc24mutant cells ceased mul-tiplying at39°C and arrested as large unbudded cells, similar to the terminal phenotype of the ras1mutant incu-bated at elevated temperatures(Waugh et al.,2002).To ensure that the large cell size of the cdc24mutant cells did not influence our interpretation of growth,we carefully examined the growth characteristics of the cdc24 mutant strain compared with wild type.Equal numbers (106ml-1)of wild type and cdc24mutant cells were inocu-lated in YPD medium at30°C.At various time points during exponential growth,samples were collected,fixed and counted to determine the doubling time of each strain. Samples were counted using a haemocytometer rather than measuring optical density to account for the size difference between the wild type and the cdc24mutant strain.We found no significant difference in the doubling time for the cdc24mutant strain compared with wild type; the average doubling time for both strains was108min. Thus,the altered cell morphology and cell size of the cdc24mutant strain is not associated with an altered growth rate at30°C.ras1and cdc24mutant cells exhibit an actin polarization defectIn S.cerevisiae,some mutant strains that arrest with a large unbudded phenotype,including cdc24and cdc42 mutants,accumulate two or more nuclei(Hartwell et al., 1973;1974;Adams et al.,1990).To determine if the C.neoformans cdc24mutant strain exhibited a similar defect,we DAPI-stained the nuclei of wild type and cdc24 mutant cells shifted from30°C to39°C(data not shown). We found that wild type and cdc24mutant cells contained a single nucleus at each temperature.In S.cerevisiae,ras2mutant cells are much larger than wild type at35°C due to a temperature-sensitiveFig.1.Overexpression of downstreamsignalling components suppresses thetemperature-sensitive growth defect of theC.neoformans var.grubii(Serotype A)ras1mutant strain.The ras1ura5mutant strainLCC70was transformed with plasmidsexpressing STE20,CDC24,CDC42,DCH2,RAC1and a control vector.Transformantswere incubated on YPD medium at30°C and37°C for48h.Wild type(H99)and the ras1prototrophic strain LCC1were included ascontrols.1120 C.B.Nichols,Z.H.Perfect and J.A.Alspaugh©2007The AuthorsJournal compilation©2007Blackwell Publishing Ltd,Molecular Microbiology,63,1118–1130actin polarization defect(Ho and Bretscher,2001).C.neoformans ras1mutants also exhibit a temperature-sensitive actin polarization defect(Waugh et al.,2002).To determine if the cdc24mutant strain has an actin polar-ization defect similar to the ras1mutant strain,we com-pared actin localization in wild type,ras1and cdc24 mutant cells.Strains were initially incubated at30°C in YPD then shifted to37°C.At various time points,samples werefixed and stained with rhodamine-conjugated phal-loidin to visualize actin localization.For each sample,100 cells containing small to medium sized buds were scored for the ability to polarize actin to the bud.Individual cells were considered polarized if50%or more of the actin localized to the bud(Ho and Bretscher,2001).At30°C,we observed that actin was properly polarized in100%of the wild-type cells but was depolarized in a small proportion of the ras1(10%)and cdc24(3%)mutant cells.When shifted to37°C for2h,actin was depolarized in6%of the wild-type cells.In contrast,depolarization of actin in the ras1and cdc24mutant cells increased to36%and24%, respectively,when incubated under identical conditions (Fig.3).These results indicate the ras1and cdc24mutant cells have a defect maintaining actin polarization in the bud.Ras1and Cdc24function in a common signalling pathwayThe morphology of the ras1and cdc24mutant strains is quite similar,suggesting that Ras1and Cdc24may func-tion in the same pathway.To further define the relationship between the RAS1and CDC24gene products,we deleted the CDC24gene in a ras1mutant strain.The resulting ras1cdc24double mutant strain wasFig.2.The cdc24mutant strain has atemperature-sensitive morphology defect.Wildtype(H99),cdc24and cdc24+CDC24mutantstrains were incubated on YPD medium for48h at30°C,37°C and39°C(A)or grown tomid-log in YPD medium at30°C then shiftedto37°C and39°C for6h(B).Cells wereimaged(¥40)with DIC optics.Bar,10mm.C.neoformans Cdc241121©2007The AuthorsJournal compilation©2007Blackwell Publishing Ltd,Molecular Microbiology,63,1118–1130temperature-sensitive for growth at 37°C (Fig.4A).Phe-notypically,the ras1cdc24mutant strain was similar to both the cdc24and ras1mutant strains;cells from all three mutant strains were larger than wild type at 30°C and arrested as large unbudded cells at the higher restric-tive temperatures (Fig.4B).There was no phenotypic dif-ference among these three mutant strains and therefore no apparent additive effects of the double mutation over the single ras1mutation.This result is consistent with Ras1and Cdc24acting in the same signalling pathway,as opposed to parallel pathways.To more precisely determine where Cdc24functions with respect to Ras1and Ste20,we overexpressed the RAS1and STE20genes in the cdc24mutant strain.Over-expression of RAS1did not suppress the temperature sensitivity of the cdc24mutant strain (Fig.4C and data not shown).However,overexpression of the STE20gene par-tially suppressed the temperature-sensitivity of the cdc24mutant;the cdc24+STE20mutant strain was able to grow at 39°C but was not able to grow as vigorously as the wild type or CDC24reconstituted strains (Fig.4C).We also examined if overexpression of STE20suppressed the morphology defect of the cdc24mutant.cdc24mutant cells overexpressing STE20were of an intermediate size at 30°C and 37°C;larger than wild type but not as large as the cdc24mutant (Fig.4D).Together with the observation that CDC24overexpression suppresses the ras1mutant growth defect at 37°C,these results suggest that Cdc24functions downstream of Ras1but upstream of Ste20in a signal transduction pathway mediating cell morphology and growth.However,overexpression of STE20does not completely restore the morphology of the cdc24mutant strain,suggesting that there may be other downstream effectors of Cdc24in addition to Ste20regulating morphology.The PAK kinase Pak1does not appear to be this effector because overexpression of PAK1does not suppress the growth or morphology defects of the cdc24mutant strain (data not shown).Cdc24and Ras1interactIn S.pombe ,Ras1physically interacts with Scd1,the Cdc24homologue.To determine if Ras1interacts with Cdc24in C.neoformans ,we utilized the yeast two-hybrid interaction system.The cDNA of the C.neoformans RAS1gene was modified to alter the C-terminal CAAX motif to prevent membrane localization (ras1C207A ).This gene was fused in frame to the GAL4DNA binding domain of vector pGBKT7.Similarly the CDC24cDNA was fused in frame to the GAL4DNA activation domain of vector pGADT7.Coexpression of GAL4BD-Ras1and GAL4AD-Cdc24transgenes restored adenine (Fig.5,middle panel)and histidine prototrophy (Fig.5,bottom panel)in the S.cerevisiae strain AH109.This result supports a model in which C.neoformans interacts with the Ras1protein.In addition,Cdc24interacted with a GTP-lockedRas1Fig.3.Actin is depolarized in ras1and cdc24mutant cells.Wild type (H99),cdc24and ras1mutant cells were incubated to mid-log phase in YPD medium at 30°C.Cells were shifted to 37°C for either 0or 120min,fixed,and stained for actin with rhodamine-conjugated phalloidin.One hundred budded cells were observed using fluorescent microscopy (¥100)and scored for actin polarization at each time point.The percentage of polarized cells for each time point is included in each panel.Arrowheads and asterisks refer to examples of cells scored as having either polarized actin or unpolarized actin respectively.Bar,10m m.1122 C.B.Nichols,Z.H.Perfect and J.A.Alspaugh©2007The AuthorsJournal compilation ©2007Blackwell Publishing Ltd,Molecular Microbiology ,63,1118–1130C.neoformans Cdc241123Fig.4.A and B.Epistasis analysis of CDC24.The ras1cdc24double mutant has a temperature-sensitive morphology defect at37°C.Wild type(H99),cdc24,ras1and ras1cdc24mutant strains were(A)incubated on YPD medium at30°C and37°C for48h or(B)grown to mid-log in YPD medium at30°C then shifted to37°C or39°C for6h.C and D.Overexpression analysis of the cdc24mutant.The cdc24ura5mutant strain CBN30was transformed with plasmids expressingCDC24,STE20,CDC42,DCH2,RAC1or a control plasmid.Transformants were(C)incubated on YPD medium at30°C and39°C for48h or (D)grown to mid-log in YPD medium at30°C then shifted to39°C for6h.Wild type(H99)and the cdc24prototrophic strain CBN16were included as controls.Cells were imaged(¥40)with DIC optics.Bar,10m m.©2007The AuthorsJournal compilation©2007Blackwell Publishing Ltd,Molecular Microbiology,63,1118–1130mutant (Ras1G12V,C207A )but did not interact with a dominant negative GDP-locked Ras1mutant (Ras1S23N,C207A ),sug-gesting that Cdc24preferentially interacts with the GTP-bound form of Ras1,further supporting our hypothesis that Cdc24acts downstream of Ras1.Cdc42functions in the Ras-Cdc24signalling pathway As previously reported,the genome of C.neoformans includes two Cdc42homologues;Cdc42and Dch2(Dual Cdc42homologue)(Vallim et al .,2005),differing primarily in the Rho insert region.In addition,C.neoformans also contains a Rac1homologue.Cdc42and Rac1proteins belong to the same family of Rho GTPases.Both Cdc42and an activated form of Rac1are able to bind the CRIB (Cdc42/Rac interaction binding)domain of Ste20in the two-hybrid interaction system (Wang et al .,2002;Vallim et al .,2005).Similarly,we found that Dch2is also able to bind to the CRIB domain of Ste20in the two-hybrid inter-action system (data not shown).Thus,all three of the C.neoformans Cdc42/Rac1homologues have potential Ste20binding ability.In an attempt to determine specificity for Cdc24inter-action among these three related proteins,we overex-pressed Cdc42,Dch2and Rac1in the cdc24mutantstrain.Overexpression of Cdc42,but not Dch2or Rac1,was able to restore the ability of the cdc24mutant strain to grow at 39°C (Fig.4C and D).Similarly,overexpression of Cdc42suppressed the growth defect of the ras1mutant strain.However,overexpression of Dch2weakly sup-pressed the growth defect of the ras1mutant strain (Fig.1).Overexpression of Rac1also suppresses the growth defects of the ras1mutant strain (Fig.1;Vallim et al .,2005).Taken together,these results suggest that Cdc42is the primary target of Ras1-Cdc24signalling mediating high temperature growth in C.neoformans .In addition,these results also indicate that Ras1may also have Cdc24-independent roles mediating high tempera-ture growth.Cdc24is required for C.neoformans pathogenicity The ability to grow at 37°C is important for the pathoge-nicity of C.neoformans,and the ras1mutant strain is attenuated for virulence in animal models of cryptococco-sis (Alspaugh et al .,2000).cdc24mutants are able to grow at 37°C but not at 39°C.However,cdc24mutant cells display significant defects in actin polarization even at 37°C.We therefore tested the role of cdc24in C.neoformans pathogenicity using a murine inhalation model of cryptococcosis (Cox et al .,2000).A/Jcr mice (8per strain)were inoculated by nasal inhalation (5¥106cells/mouse)with the wild type,cdc24mutant,or the reconstituted cdc24+CDC24mutant strain.By 29days after infection,all mice infected with the wild type or the reconstituted strain had succumbed to the infection (Fig.6).However,seven out of eight mice infected with the cdc24mutant strain survived to the completion of the experiment (57days).Interestingly,lung and spleen tissue harvested from the animals inoculated with the cdc24mutant still contained viable cryptococcal cells at the end of the experiment,although the mice were clini-cally well-appearing and gaining weight throughout the experiment.Phenotypic and genetic analysis of the recov-ered cryptococcal cells confirmed that they were cdc24mutant cells.These results confirm the importance of the Cdc24signalling pathway in C.neoformans virulence.DiscussionRas proteins control several distinct cellular processes in C.neoformans .However,one of the main functions of Ras in this pathogenic fungus is to maintain cytoskeletal integrity,allowing this organism to respond to changing environments and to external stress.To better understand the mechanisms by which Ras1controls cellular growth under stressful conditions such as elevated temperatures,we identified a Cdc24homologue in C.neoformans ,hypothesizing that it might act as a downstream effectorofFig.5.Ras1interacts with Cdc24.The cDNA sequences of the CDC24,RAS1,ras1G17V and ras1S23N genes were cloned into the two-hybrid vectors pGADT7and pGBKT7and cotransformed in pairwise combinations into the yeast strain AH109.One isolate transformed with the empty vectors was also tested as a control.Transformants were selected on synthetic medium agar plates lacking tryptophan and leucine (SD-Trp-Leu).These strains were assessed for growth on synthetic medium lacking tryptophan,leucine and adenine (SD-Trp-Leu-Ade)and synthetic medium lacking tryptophan,leucine and histidine and including 5mM 3-aminotriazole (SD-Trp-Leu-His).1124 C.B.Nichols,Z.H.Perfect and J.A.Alspaugh©2007The AuthorsJournal compilation ©2007Blackwell Publishing Ltd,Molecular Microbiology ,63,1118–1130Ras1.We show that Cdc24functions in a Ras1signalling cascade that mediates morphogenesis and the ability to grow at high temperature,and that these processes are important for the pathogenicity of C.neoformans.In addi-tion,our results indicate that this signalling cascade func-tions specifically through Cdc42,one of three highly related proteins in C.neoformans.Ras activation of Cdc24and Cdc42proteins–a conserved signalling motifIn thefission yeast S.pombe,Ras1activates two distinct pathways:a pheromone response pathway and an actin polarization(morphogenesis)pathway.Specificity between the pathways is mediated by the Ras1GEFs Ste6and Efc25and also by spatial compartmentalization of the Ras1protein(Onken et al.,2006).Components of the morphogenesis pathway consist of Efc25,Ras1, Scd1,Cdc42and Shk1(Papadaki et al.,2002).Activated Ras1directly interacts with the Scd1protein,a GEF that controls the activity of the highly conserved GTPase Cdc42.In turn,activated Cdc42binds to and activates the PAK kinase Shk1.Mutation of any of these genes pro-duces cells that are round instead of rod shaped due to the loss of actin polarity(Fukui et al.,1986;Fukui and Yamamoto,1988;Chang et al.,1994;Miller and Johnson, 1994;Marcus et al.,1995;Ottilie et al.,1995;Tratner et al.,1997).Similar to S.pombe,C.neoformans Ras1regulates the mating process as well as mating-independent morpho-logical events.Our two observations that cdc24mutant strains have a ras1-like temperature-sensitive morpho-genesis defect and that these two proteins physically interact suggest that Cdc24is acting as an effector of Ras1signalling.In addition,our epistasis data also suggest that Ras1,Cdc24,Cdc42and Ste20function in a linear pathway that mediates morphogenesis and thermotolerance.Also,similar to S.pombe,overexpres-sion of Cdc24does not suppress the mating defect of the ras1mutant strain(data not shown)suggesting that the Cdc24-dependent branch of Ras1signalling does not mediate mating.Thus,the core components of morpho-genic signalling appear to be conserved between these two organisms.Our data also place C.neoformans Ras1upstream of Cdc42.The relationship between Ras and Cdc42is an emerging theme in fungal biology.In the dimorphic fungal pathogen P.marneffei,the Ras homologue RasA acts upstream of CflA(Cdc42)to regulate polarized growth of hyphal and yeast cells,septation,branch emergence and spore germination(Boyce et al.,2005).In the plant patho-gen C.trifolii,CtCdc42functions downstream of CtRas1 to mediate spore germination and proper hyphal growth (Chen et al.,2006).To date Cdc24homologues have not been characterized for either organism,although the sequence for a P.marneffei Cdc24homologue has been recently deposited into the NCBI database(Woo et al., 2006).Cdc24has therefore only been conclusively demonstrated as a Ras effector in C.neoformans and S.pombe.A different signalling paradigm exists in S.cerevisiae. The yeast cell polarity is regulated by morphogenesis proteins such as Cdc24,Cdc42and the PAK kinases Ste20and Cla4(Johnson,1999).However,Cdc24is recruited to the membrane and activated by the Ras-like GTPase Rsr1p/Bud1p(Gulli and Peter,2001;Park et al., 2002).The S.cerevisiae Ras protein Ras2is involved in Cdc42signalling,but this interaction role appears to beFig.6.Cdc24is required for pathogenesis ina murine model of cryptococcosis.The wildtype(H99),cdc24mutant and cdc24+CDC24reconstituted strains were intranasallyinoculated into A/Jcr mice.Animal survivalwas monitored for58days.C.neoformans Cdc241125©2007The AuthorsJournal compilation©2007Blackwell Publishing Ltd,Molecular Microbiology,63,1118–1130。