An STE12 Homolog From the Asexual, Dimorphic Fungus Penicillium marneffei Complements the Defect in Sexual Development of an Aspergillus nidulans steA Mutant

Corresponding author: Alex Andrianopoulos, Department of Genetics, University of Melbourne, Victoria, 3010 Australia., alex{at}genetics.unimelb.edu.au (E-mail)

Communicating editor: R. H. DAVIS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species identified in its genus. At 25° P. marneffei exhibits true filamentous growth, while at 37° P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. Members of the STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number of fungi. We have cloned a homolog of the S. cerevisiae STE12 gene from P. marneffei, stlA, which is highly conserved. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12 genes, form a distinct subclass of STE12 homologs that have a C₂H₂ zinc-finger motif in addition to the homeobox domain that defines STE12 genes. To examine the function of stlA in P. marneffei, we isolated a number of mutants in the P. marneffei-type strain and, in combination with selectable markers, developed a highly efficient DNA-mediated transformation procedure and gene deletion strategy. Deletion of the stlA gene had no detectable effect on vegetative growth, asexual development, or dimorphic switching in P. marneffei. Despite the lack of a detectable function, the P. marneffei stlA gene complemented the sexual defect of an A. nidulans steA mutant. In addition, substitution rate estimates indicate that there is a significant bias against nonsynonymous substitutions. These data suggest that P. marneffei may have a previously unidentified cryptic sexual cycle.

SIGNAL transduction pathways play a crucial role in linking genetically programmed cellular events to external stimuli in eukaryotic organisms. This is achieved through protein-mediated transduction of signals from the cell membrane to the nucleus, which ultimately leads to changes in gene expression. The most common and intensively studied signaling pathway is the mitogen-activated protein kinase (MAPK) pathway, which has been shown to be conserved throughout eukaryotes including fungi, flies, worms, and humans. In each of these organisms the MAPK module consists of a phosphorylation cascade of three protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAPK. The yeast Saccharomyces cerevisiae contains some of the best-characterized MAPK pathways and these have been shown to regulate a variety of processes including growth in high osmolarity environments, cell integrity, spore formation, mating, and pseudohyphal/filamentous growth (for a review see GUSTIN et al. 1998 ). It has also been shown that the activation of the MAPK modules of each of these pathways leads to the phosphorylation-mediated regulation of specific downstream transcription factors to alter gene expression (COOPER 1994 ; MARSHALL 1994 ; COBB and GOLDSMITH 1995 ).

Ste12p is the direct target of the MAPK for both the mating and pseudohyphal growth pathways. These two pathways share their MAPKKK (Ste7p) and MAPKK (Ste11p) proteins but the MAPK of each cascade is unique with Fus3p regulating Ste12p function in response to mating pheromone and Kss1p in response to pseudohyphal signals (COOK et al. 1997 ; MADHANI et al. 1997 ; MADHANI and FINK 1998 ). Specificity of Ste12p activation is achieved despite receiving signals from two different pathways through combinatorial activation of target genes. In response to pheromone, Ste12p homodimers or Ste12p-Mcm1p heterodimers activate transcription from PREs (pheromone response elements) to induce the mating response (KRONSTAD et al. 1987 ; HAGEN et al. 1991 ; SONG et al. 1991 ). During pseudohyphal growth, Ste12p requires the presence of a second, filamentation-specific transcription factor, Tec1p, for activation from FREs (filamentation response elements) found upstream of genes responsible for pseudohyphal development (GAVRIAS et al. 1996 ; MADHANI and FINK 1997 ).

The study of STE12 homologs in other fungi has shown that this regulator has been conserved at both the sequence and functional levels. STE12 homologs identified in the dimorphic yeast Candida lusitaniae (CLS12) and the filamentous fungus Aspergillus nidulans (steA) have been shown to regulate the mating response without affecting any other cellular processes (VALLIM et al. 2000 ; YOUNG et al. 2000 ). This phenotype is specific to sexual development such that the A. nidulans steA mutant fails to complete sexual reproduction, but is unaffected in asexual reproduction (conidiation; VALLIM et al. 2000 ). STE12 homologs have also been identified from two pathogenic species of fungi, Cryptococcus neoformans and Candida albicans, where Ste12p regulates processes other than mating. In C. neoformans, an STE12 homolog is found only in the MAT mating type of the fungus and, while largely dispensable for mating, Ste12 is required for haploid fruiting and regulates virulence depending on the serotype under investigation (WICKES et al. 1997 ; YUE et al. 1999 ; CHANG et al. 2000 ). C. albicans has been proposed to be an asexual diploid yeast that would not require genes involved in mating. However, mating-type loci have recently been described in C. albicans and mating between strains of different mating types demonstrated (HULL et al. 2000 ; MAGEE and MAGEE 2000 ). In C. albicans, the STE12 homolog CPH1 is required for hyphal growth on solid media and, in combination with a second developmental regulator, Efg1p is required for virulence of C. albicans in an animal host (LIU et al. 1994 ; LO et al. 1997 ). The function of CPH1 in the cryptic sexual cycle has yet to be determined, however.

Penicillium marneffei is an asexual ascomycete that displays a temperature-dependent dimorphic growth switch (GARRISON and BOYD 1973 ; CHAN and CHOW 1990 ). Unlike dimorphic fungi such as C. albicans and C. neoformans, which are primarily yeast-like with a relatively minor filamentous form, P. marneffei is predominantly a filamentous species that has a significant unicellular growth form. As discussed above, STE12 homologs have been shown to regulate filamentous growth associated with yeast-hyphal dimorphism in S. cerevisiae, C. albicans, and C. neoformans. We therefore wished to analyze the function of an STE12 homolog in P. marneffei to determine if, like the STE12 homologs in these species, it plays a role during dimorphic development in a species that is primarily filamentous.

We have cloned the P. marneffei STE12 homolog stlA and it is predicted to encode a protein that has two potential DNA-binding domains: a homeobox domain and a C₂H₂ Zn²⁺ finger motif. A DNA-mediated transformation system was developed for P. marneffei to allow disruption of the stlA locus. The stlA deletion strain displayed no detectable mutant phenotype despite the high degree of stlA sequence conservation observed. The stlA gene was shown to be functional in A. nidulans, however, because it was able to complement the mating defect of an A. nidulans steA deletion strain.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fungal strains and media:
P. marneffei and A. nidulans strains were grown on either Aspergillus nitrogen-free medium (ANM; COVE 1966 ) supplemented with either 10 mM -amino butyric acid (GABA), sodium nitrate (NaNO₃), or ammonium tartrate (NH₄T) as a sole nitrogen source, S. cerevisiae synthetic dextrose (SD) medium (AUSUBEL et al. 1994 ), or brain heart infusion (BHI) broth (Oxoid). Media for protoplast regeneration contained 1.2 M sucrose as an osmotic stabilizer. The P. marneffei-type strain FRR2161 was obtained from Dr. J. Pitt (CSIRO Food Industries, Sydney). Strain SPM3 was isolated as a FRR2161 sector resistant to chlorate, unable to grow on nitrate as a sole nitrogen source, and presumed to be a nitrate reductase mutant on the basis of previously defined growth tests (COVE 1972 ). Strain SPM4 was isolated as a SPM3 sector resistant to 1 mg/ml 5-fluoroorotic acid (5-FOA) that was unable to grow in the absence of uridine (5 mM) and uracil (5 mM). A. nidulans strain UI139 (biA1; argB; steA::argB) was obtained from Dr. B. Miller (University of Idaho; VALLIM et al. 2000 ).

Protoplast transformation:
The transformation procedure for P. marneffei germlings is based on previously described methods for A. nidulans transformation (TILBURN et al. 1983 ; YELTON et al. 1984 ). Briefly, 1 x 10⁸ P. marneffei spores were harvested from ANM + GABA solid medium, inoculated into 400 ml of appropriately supplemented SD broth, and incubated at 37° for 40 hr. Highly branched germlings were isolated by filtration through Miracloth (Calbiochem, La Jolla, CA) and washed with 0.6 M MgSO₄. Approximately 5 g wet weight tissue was resuspended in 10 ml of chilled osmotic buffer (1.2 M MgSO₄, 10 mM NaOP, pH 5.8) and placed on ice. Lytic enzyme (Sigma, St. Louis) and bovine serum albumin were added to final concentrations of 5 mg/ml and 1.2 mg/ml, respectively, and the mixture incubated for 1 hr at 30° with gentle agitation. Protoplasts were harvested and processed according to the method previously described for the transformation of A. nidulans (ANDRIANOPOULOS and HYNES 1988 ). Osmotically stabilized selection plates for SPM4 contained either 10 mM NH₄T as the sole nitrogen source (selecting for pyrG complementation), 10 mM NaNO₃, and 5 mM uridine and uracil (selecting for niaD complementation), or 1.5 µg/ml bleomycin, 10 mM NH₄T, and 5 mM uridine and uracil selecting for bleomycin resistance. A. nidulans protoplast transformations were performed as described previously using 1 µg/ml bleomycin (final concentration) in the selection plates (ANDRIANOPOULOS and HYNES 1988 ).

Molecular techniques:
The plasmids used in this study are listed in Table 1. DNA for transformation was isolated using either the Qiafilter 100 kit (QIAGEN, Chatsworth, CA) or the High Purity Plasmid kit (Roche). To isolate genomic DNA, 100 ml of SD medium was inoculated with 1 x 10⁶ P. marneffei spores and incubated at 37° for 40 hr. Germlings were isolated by filtration through Miracloth (Calbiochem), washed with water, blotted to remove excess fluid, and stored at -20°. Genomic DNA was prepared as described previously (LEE and TAYLOR 1990 ). RNA was isolated from P. marneffei cultures using the RNA Red fast prep kit (BIO 101, Vista, CA). For the 25° vegetative samples, FRR2161 conidia were inoculated into SD medium and incubated at 25° for 2 days with shaking and mycelia harvested by filtration through Miracloth (Calbiochem). Conidiating cultures were prepared by filtering the 25° vegetative cultures onto Whatman paper circles after 2 days of growth and placing the filters onto 0.1% glucose, ANM + GABA agar plates for 4 days to allow for the production of conidiophores. Yeast cultures were grown by inoculating BHI (Oxoid) liquid medium with FRR2161 conidia and allowing growth to proceed at 37° with shaking for 4 days. At this time, there was a mixture of both yeast cells and hyphal filaments. A sample of the supernatant was taken after the hyphal material was allowed to settle and transferred to new medium for an additional 2 days of growth. Cells were harvested by filtration onto Whatman paper. After harvesting, all samples were immediately frozen at -70°. RNA was subjected to gel electrophoresis on 1.2% agarose, formaldehyde denaturing gels. Southern and Northern blotting were performed using H bond N+ membrane (Amersham) according to manufacturer's instructions. Filters were hybridized with [-³²P]dATP-labeled probes (random primer) and processed using standard procedures (SAMBROOK et al. 1989 ).

Cloning and disruption of the stlA locus:
The stlA gene was isolated by degenerate PCR using the primers STE1 (GAARAARTTYGARGARGGNRT) and STE2 (AAIACYTTYTGYTTYTTYTGNGT), designed using the conserved homeodomain regions of S. cerevisiae (X16112), C. albicans (L16451), Kluyveromyces lactis (L21156), and C. neoformans STE12 (AF012924) homologs. PCR conditions consisted of an initial denaturation step of 94° for 2 min after which Taq DNA polymerase was added and 30 cycles of 94° for 30 sec, 45° for 30 sec, and 72° for 30 sec were performed. One broad band of 125 bp in size was observed. Sequence analysis of individual clones revealed that this band was composed of two major species, one of which had significant homology to STE12 homologs. This PCR product was used to probe a 3- to 4-kb EcoRI-SalI size-selected FRR2161 genomic library in pBluescript II SK⁺ (Stratagene, La Jolla, CA). One positive clone (pAB4458) that contained the entire region shown to hybridize to the PCR probe, but which lacked the 5' portion of the gene, was isolated. A 4.1-kb XbaI-BglII fragment that overlapped the 5' end of pAB4458 was obtained from a -GEM11 FRR2161 genomic library and cloned into pLitmus 29 (New England Biolabs, Beverly, MA) to give pAB4623. The XbaI-BglII fragment of pAB4623 was cloned into the XbaI-BglII sites of pAB4458 to give the full-length clone pAB4624. To disrupt stlA, a 1.4-kb EcoRV-BamHI fragment containing the pyrG cassette of pAB4342 was cloned into the SmaI-BglII sites of pAB4624 to give pAB4625. pAB4625 was digested at the unique ApaI site in the pBluescript II SK⁺ polylinker and subjected to gel electrophoresis, and the single digested band gel purified using the Bresaclean Gel purification kit (Geneworks). A total of 500 ng of digested vector was transformed into SPM4 and transformants selected for complementation of the uridine/uracil auxotrophy.

Microscopy and cellular staining:
P. marneffei was grown on microscope slides coated with thin layers of either ANM + GABA or SD solid medium and incubated at 25° or 37° as indicated (BORNEMAN et al. 2000 ). Slides were viewed on a Reichart Jung Polyvar II microscope using either differential interference contrast (DIC) or epifluorescence optics to detect green fluorescent protein (GFP) fluorescence (band pass 450–495 nm, dichroic mirror 510 nm, barrier filter 520 nm). Microscope images were captured with a SPOT CCD camera (Diagnostic Instruments, Sterling Heights, MI) and processed using Adobe Photoshop 4.0.

Complementation of the A. nidulans steA mutation:
Strain UI139 (steA; VALLIM et al. 2000 ) was cotransformed with 1.5 µg of pAB4624 (stlA) and 1 µg of pAmPh520 (ble^R; AUSTIN et al. 1990 ). Colonies resistant to 1 µg/ml bleomycin were isolated and screened for their ability to self-fertilize. Spores from single transformant colonies were stab inoculated onto ANM + NO₃ media and allowed to grow for 2 days at 37°. Plates were sealed to promote the sexual cycle and examined for the presence of sexual structures (cleistothecia) using a dissection microscope after 7 days growth at 37°.

Analysis of stlA conservation:
Pairwise protein and DNA alignments were performed using GAP from the Wisconsin Package (DEVERAUX et al. 1984 ) on the Australian National Genomics Information Service. For the synonymous/nonsynonymous rate analysis, minor manual modifications were made to the DNA alignments to optimize the alignments and ensure codons were not split by gaps. Estimates of synonymous and nonsynonymous substitution rates were made using the YN00 program from the PAML package (YANG and NEILSEN 2000 ). The YN00 algorithm takes into account both transition/transversion rate bias and base/codon frequency bias.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

P. marneffei has an STE12 homolog:
Degenerate PCR primers were designed to the conserved homeodomain encoding region of the S. cerevisiae STE12, C. albicans CPH1, and C. neoformans STE12 genes. This PCR produced a fragment of 125 bp, which was shown by sequence analysis to be homologous to STE12. This product was used to probe P. marneffei genomic libraries (see MATERIALS AND METHODS), resulting in the isolation of a full-length genomic clone, pAB4624. Sequencing of this clone revealed an open reading frame (ORF), interrupted by four introns with extensive similarity to STE12, and was designated stlA (sterile twelve like; accession no. AF284062). GenBank database searches (BLAST) also identified a second ORF in this clone that had significant identity to the SUC1 sucrose transporter of Arabidopsis thaliana (S38197) and a putative sucrose transporter from Schizosaccharomyces pombe (CAB16264) (Fig 1A). On the basis of the gene structure of the A. nidulans steA gene, stlA is predicted to encode a 689-amino-acid protein with significant similarity to all identified STE12 homologs. This similarity is primarily restricted to the region predicted to encode the homeobox DNA-binding motif common to all the Ste12 proteins (Fig 1B). StlA shows the highest degree of similarity to SteA of A. nidulans (71% identity and 84% similarity) and Ste12 of C. neoformans (58% similarity and 39% identity). In addition to the homeodomain motif, these three proteins contain two C₂H₂ Zn²⁺ finger domains (Fig 1C). This second putative DNA-binding motif is absent from the S. cerevisiae, K. lactis, and C. albicans Ste12p proteins. The P. marneffei, A. nidulans, and C. neoformans proteins also lack homology to the regions of Ste12p that have previously been shown to be important for regulation by Dig1p and Dig2p (Ste12p residues 305–311) as well as the tyrosine residues (residues 307 and 314) required for repression of transcription by Ste12p in the absence of MAPK activation (PI et al. 1997 ).

View larger version (54K): In this window In a new window Download PPT slide   Figure 1. The stlA gene of P. marneffei encodes a homeodomain and C2H2 Zn2+ finger domain protein. (A) The restriction map of the stlA genomic locus and diagrammatic representation of the predicted position of the coding region (black boxes) separated by four introns. The position of the homeodomain (hatched box) and the two C2H2 Zn2+ fingers (open boxes) is indicated. The region of DNA that is predicted to encode a sucrose transporter homolog is also indicated (shaded box). (B) Alignment of the predicted homeodomain of StlA with several other Ste12 homologs: A. nidulans SteA (AnSteA), C. neoformans Ste12 (CnSte12), S. cerevisiae Ste12p (ScSte12), K. lactis Ste12p (KlSte12), and C. albicans Cph1p (CaCph1). The regions predicted to form the three -helices of the homeodomain are indicated by thick black lines. (C) Alignment of the C2H2 Zn2+ fingers identified in the A. nidulans, C. neoformans, and P. marneffei STE12 homologs. The cysteine and histidine residues predicted to comprise the Zn2+ coordinating residues of the C2H2 fingers are indicated by an asterisk.

The promoter of the stlA gene was examined for recognition sequences of known fungal developmental regulators to attempt to infer the pattern of regulation expected for the stlA transcript. There are no sequences present in the stlA promoter region that match the consensus of a S. cerevisiae PRE (YUAN and FIELDS 1991 ) or FRE (MADHANI and FINK 1997 ). A number of putative binding sites were identified for regulators of asexual development in A. nidulans, however, with six BrlA consensus binding sites (CHANG and TIMBERLAKE 1993 ), four AbaA sites (ANDRIANOPOULOS and TIMBERLAKE 1994 ), and one StuA site (DUTTON et al. 1997 ) identified, the majority of which are within 1 kb of the translational start of stlA.

stlA is expressed primarily in vegetative tissues:
The data from the promoter analysis suggested that stlA may be developmentally regulated, most likely being upregulated during conidiation due to the action of BrlA, AbaA, and StuA. This pattern is observed in A. nidulans, with expression of steA being almost undetectable in vegetative tissues, but expressed at high levels upon the commencement of asexual development despite SteA not being required for conidiation (VALLIM et al. 2000 ). In S. cerevisiae, STE12 expression is 5- to 10-fold higher in a or haploids than in a/ diploids (FIELDS and HERSKOWITZ 1987 ). Therefore, upregulation of STE12 expression appears to be correlated with induction of the pathways that it regulates. Northern analysis was performed to determine if any correlations between stlA expression and developmental stages in P. marneffei could be found. In P. marneffei the stlA gene produces a single transcript of 2.8 kb that is present during both filamentous and yeast growth states (Fig 2). The stlA transcript is abundant in the vegetative filamentous form, with expression decreasing to 40% of the vegetative level during conidiation. The level of stlA transcript in the yeast form of P. marneffei was approximately equivalent to that observed in the vegetative filamentous form. Therefore the expression of stlA is opposite to that of the A. nidulans steA gene and suggests that, instead of functioning after asexual development, stlA may function during vegetative growth. The decrease in stlA expression levels during conidiation may reflect a lack of stlA expression in the developing conidiophore, because asexual developmental cultures contain a mixture of vegetative and developmental tissue types.

View larger version (62K): In this window In a new window Download PPT slide   Figure 2. stlA is expressed in vegetative tissue. RNA was isolated from P. marneffei vegetative hyphal cells grown at 25°, filamentous hyphal cells induced to undergo asexual development (25°C dev), and yeast cells grown at 37° (see MATERIALS AND METHODS). Northern blots were hybridized with probes specific for stlA to examine expression levels or A. nidulans histone H3 (EHINGER et al. 1990 ) as a loading control.

Isolation of pyrG and niaD mutant strains and transformation of P. marneffei:
To examine the function of the stlA gene in P. marneffei, it was necessary to develop a DNA-mediated transformation procedure and to establish targeted gene deletion strategies. One of the key elements of such procedures is suitable selectable markers to allow identification of transformed strains. P. marneffei orotidine 5'-monophosphate decarboxylase (pyrG) and nitrate reductase (niaD) mutants were isolated by selecting for 5-FOA and chlorate resistance, respectively, with the precise genetic defect determined using defined growth tests (see MATERIALS AND METHODS). The pyrG and niaD genes were chosen due to the availability of positive selection regimes that allow spontaneous mutants to be isolated, the nonleaky phenotypes of the mutants under selection conditions, and the availability of heterologous pyrG and niaD genes for use as transformation markers. The mutations that gave rise to the 5-FOA and chlorate resistance in SPM4 were confirmed by complementation using the cloned A. nidulans pyrG (AnpyrG) and A. oryzae niaD (AoniaD) genes (OAKLEY et al. 1987 ; UNKLES et al. 1989 ).

A transformation procedure was developed based on polyethylene glycol-mediated cell fusion using P. marneffei protoplasts. Protoplasting trials showed that germlings grown for 40 hr at 37° produced cells with the greatest protoplasting efficiency (data not shown). These growth conditions were used for subsequent transformation experiments and were optimized for digestion time. DNA-mediated transformation of the protoplasts used a technique developed for A. nidulans protoplasts that makes use of polyethylene glycol-induced protoplast fusion to introduce the transforming DNA into the cytosol (TILBURN et al. 1983 ; YELTON et al. 1984 ). This technique was also shown to be effective in P. marneffei using the same polyethylene glycol concentrations and times used for A. nidulans transformations (ANDRIANOPOULOS and HYNES 1988 ).

The pAB4342 (AnpyrG) and pSTA14 (AoniaD) plasmids were shown to complement the pyrG and niaD mutant phenotypes of SPM4 at frequencies up to 1400 transformants per microgram of plasmid DNA. This efficiency was dependent on both the concentration of DNA used and whether the DNA was circular or linear (Table 2). Southern blot analysis of the pyrG⁺ transformants showed that pAB4342 DNA was present in the genome of the transformants and was associated with high molecular weight genomic DNA rather than being present as independent episomes (Fig 3). Estimates of plasmid copy number in the transformants ranged from a single copy to multiple copies that were often integrated in tandem. Analysis of transformants generated with linearized plasmid DNA showed that most integration events were single copy, accounting for the reduced transformation frequency observed when this type of DNA was used.

View larger version (65K): In this window In a new window Download PPT slide   Figure 3. Analysis of the fate of plasmid DNA in P. marneffei transformants. (A) Southern hybridization of genomic DNA from 18 independent transformants using differing amounts and forms of DNA (as indicated) digested with PstI, transferred to membrane, and probed with a 1.4-kb, 32P-labeled PstI-BamHI fragment of pAB4342 containing the A. nidulans pyrG gene. PstI does not cut within pAB4342, therefore producing a single hybridizing band per integrated plasmid copy. (B) Southern hybridization of undigested genomic DNA from the identical transformants used in A and probed using the same fragment of pAB4342. Hybridizing sequences are associated with high-molecular-weight DNA, suggesting a chromosomally integrated location.

The bleomycin resistance plasmid pAmPh520 (AUSTIN et al. 1990 ) was also shown to be an effective dominant selectable marker. Transformants obtained using this plasmid were isolated at a frequency lower than that for the pSTA14 and pAB4342 plasmids with 150 transformants obtained per microgram of pAmPh520 (data not shown). Selection schemes based on other dominant selectable markers such as hygromycin resistance were shown to be unsuitable for use in P. marneffei due to high-level natural resistance.

Cotransformation has previously been shown to be extremely useful for transforming fungi with plasmids that do not possess a selectable marker (FINCHAM 1989 ; HYNES 1996 ). Cotransformation was tested in P. marneffei by transforming SPM4 with a combination of pAB4342 and the plasmid pRS31, which encodes GFP fused to the region encoding the N terminus of the A. nidulans StuA protein expressed constitutively from the gpdA promoter. This protein fusion has previously been shown to localize GFP to the nucleus of A. nidulans transformants (SUELMANN et al. 1997 ). A total of 50 pyrG⁺ transformants were chosen at random and screened for GFP expression and localization. Of the 50 transformants tested, 46% displayed GFP fluorescence that was localized to the nucleus in both the filamentous and yeast forms (Fig 4). Southern analysis confirmed the presence of both plasmids in the genomes of the GFP-expressing transformants (data not shown).

View larger version (62K): In this window In a new window Download PPT slide   Figure 4. Microscopic analysis of pAB4342-pRS31 cotransformants expressing GFP. A representative cotransformant for the StuA-GFP-expressing pRS31 plasmid is shown. Colonies were grown on microscope slides covered with a thin layer of SD solid medium and incubated at either 25° for 2 days to induce filamentous growth (A) or 37° for 7 days to induce yeast growth (B). Slides were examined using both differential interference (DIC) and fluorescence optics (GFP) to detect the cellular localization of the StuA-GFP protein. Both the filamentous and yeast-like growth forms show the StuA-GFP protein localized in the nucleus, as noted by the concentrated fluorescence in these organelles.

Targeted gene deletion of the stlA locus:
To examine the role of stlA, a targeted deletion of the stlA locus was obtained. Plasmid pAB4625 was created by inserting the AnpyrG cassette of pAB4342 into pAB4624, thereby deleting the majority of the stlA coding region including the predicted translational start codon, homeodomain, and both C₂H₂ fingers. Strain SPM4 was transformed with pAB4625, previously linearized by restriction enzyme digestion at a unique ApaI site to increase the frequency of homologous integration at the stlA locus (Fig 5A). A total of eight pyrG⁺ transformants were obtained from 500 ng of digested plasmid. None of the transformants displayed any detectable phenotype when grown at 25°. All eight transformants were screened by Southern blot analysis and one of these transformants, TAB19008, displayed a genomic restriction pattern consistent with deletion of the stlA locus and integration of a single copy of the pyrG selectable marker (Fig 5B).

View larger version (58K): In this window In a new window Download PPT slide   Figure 5. Deletion of the stlA by targeted gene replacement. (A) Restriction maps of the wild-type (top) and predicted gene-replaced stlA (bottom) loci showing the position of the stlA coding region (black boxes) and the region replaced by the A. nidulans pyrG selectable marker (shaded box). The region of stlA DNA used as a probe for the Southern blotting analysis is represented by an open rectangle. (B) Southern blot analysis of the wild type and eight transformants obtained from the ApaI digested pAB4625 deletion construct indicated in A. Genomic DNA was isolated from each transformant (labeled 1–8) and the recipient strain (W), restriction digested (SalI or BglII), gel fractionated, and Southern blotted. The Southern blot was probed using the P32-labeled stlA fragment indicated in A. The stlA deletion transformant, TAB19008, which has the AnpyrG marker integrated at stlA, is indicated by an asterisk.

stlA is not required for growth or development:
The stlA strain (TAB19008) was examined for any phenotypes that may be associated with loss of stlA function. There were no detectable differences in growth rate, colony morphology, or conidiation density observed at 25° during filamentous growth. Microscopic examination showed that the stlA strain had normal hyphal and conidiophore morphology (Fig 6A). At 37°, colony morphology and growth rate were shown to be normal and both the wild-type and stlA strains were capable of switching from the yeast to filamentous growth forms following transfer from 37° to 25° (Fig 6B). In addition, the stlA strain showed normal yeast cell morphology at the microscopic level, including normal positioning of nuclei (4'6-diamidino-2-phenylindole staining) and septa (calcofluor staining; data not shown).

View larger version (100K): In this window In a new window Download PPT slide   Figure 6. Loss of stlA does not affect vegetative growth or conidiation in P. marneffei. (A) Wild-type (FRR2161) and stlA (TAB19008) strains were grown on ANM + GABA medium at 25° for 10 days to examine hyphal growth and conidiation. The edges of single colonies show the fluffy appearance indicative of conidiophores (left). Individual conidiophores were also examined by growing both wild type and stlA on slides coated in ANM + GABA for 5 days. Colonies were fixed and viewed using DIC microscopy to examine the structure of individual conidiophores (right). Bar, 20 µM. (B) Wild-type (FRR2161) and stlA (TAB19008) strains were grown on SD media for 4 days at 37° to examine yeast growth. Single colonies were viewed using brightfield microscopy (left). The ability to undertake the yeast-hyphal transition was then investigated by shifting the plates to 25° for 3 days to induce the dimorphic switch and result in hyphal growth (right).

stlA can complement the sexual defect of an A. nidulans steA mutant:
To assess whether the stlA gene actually encoded a functional homolog of STE12, we tested the ability of stlA to complement the sexual cycle defect of the A. nidulans steA deletion strain UI139, which is self-sterile and is therefore unable to form cleistothecia unless crossed to an steA⁺ strain (VALLIM et al. 2000 ).

UI139 was cotransformed with pAmPh520 (ble^R; AUSTIN et al. 1990 ) and pAB4624 (stlA). Bleomycin-resistant transformants were picked at random and tested for their ability to produce cleistothecia without outcrossing. From 50 colonies screened, 34 were shown to produce morphologically normal cleistothecia, with two representative colonies shown in Fig 7. Cleistothecia from several of these transformants were analyzed and shown to contain viable ascospores. Southern blot analysis of these transformants showed that all the complemented strains contain copies of the stlA gene while retaining the steA deletion locus (data not shown).

View larger version (63K): In this window In a new window Download PPT slide   Figure 7. stlA can complement the steA mutation in A. nidulans. The sexual cycle was induced in wild type (steA+), UI139 (steA), and UI139 transformed with stlA by incubation at 37° for 14 days. Wild-type strains of A. nidulans form numerous cleistothecia (white arrowheads). UI139 does not produce cleistothecia unless mated to an steA+ strain. UI139 was cotransformed with pAB4624 (stlA) and pAmPh520 (bleR). Transformants that had regained the ability to form cleistothecia without outcrossing (white arrowheads) were isolated, indicating complementation of the steA phenotype. Cleistothecia formed by two representative transformants (TAB770002 and TAB770017) are shown.

StlA is conserved at the DNA level:
P. marneffei stlA encodes a functional gene product capable of activating the sexual developmental pathway in an A. nidulans steA mutant. If stlA plays an important role in P. marneffei, then it should still be under selective pressure. To assess this, a comparison of the synonymous and nonsynonymous base substitution rates (d_N/d_S) was performed on the entire coding region of the P. marneffei and A. nidulans genes, as well as the two putative DNA-binding motifs, which show the highest level of conservation. Synonymous and nonsynonymous nucleotide substitutions were assigned, counted, and corrected for multiple substitutions on a codon-for-codon basis of DNA alignments. The d_N/d_S rates were 0.069, 0.011, and 0.010 for the entire coding region, the homeodomain DNA-binding motif, and the C₂H₂ DNA-binding motif, respectively. The P. marneffei and A. nidulans abaA genes, which have been shown to be required for asexual development in both of these organisms (BORNEMAN et al. 2000 ), show similar values of 0.091 for the entire coding region and 0.004 for the ATTS DNA-binding motif. Similarly, comparison of the conserved region from homologous chitin synthase genes in these two organisms, P. marneffei chsA (PmCHS1, accession no. U60515) and A. nidulans chsC (accession no. AB023911), yielded a d_N/d_S rate of 0.0261.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

P. marneffei as a model for fungal dimorphism:
P. marneffei is a pathogen that is rapidly emerging as a public health problem, not only in Asia where P. marneffei has been classed as an "AIDS defining pathogen," but also throughout other parts of the world with cases of P. marneffei infection having been recorded in Australia, Europe, and North America (COOPER 1998 ; COOPER and HAYCOCKS 2000 ). Like a number of other fungal pathogens, P. marneffei displays a temperature-dependent dimorphic switch; however, unlike most other dimorphic fungi that are classified as yeasts, P. marneffei is primarily a filamentous species that adopts a yeast morphology in its host. The control of this developmental process is of interest because, with other dimorphic fungi, it is thought that the dimorphic switch is required for pathogenicity and may therefore provide a target for controlling infection. To investigate these processes in P. marneffei at the molecular level, we have isolated mutant strains and developed a protocol to transform them. We have also demonstrated that it is possible to create precise mutations in genes by targeted gene replacement. These techniques provide the basic tools required for in-depth study of P. marneffei pathogenicity and development at the genetic level through the use of gene knockout strains and gene reporter and protein localization constructs.

P. marneffei has a conserved STE12 homolog that is distinct from S. cerevisiae STE12:
P. marneffei stlA was shown to be highly conserved when compared to the recently cloned A. nidulans homolog, steA (VALLIM et al. 2000 ). The predicted StlA protein, like A. nidulans SteA and C. neoformans Ste12, possesses two potential DNA-binding domains, a homeodomain found in all Ste12p homologs, in addition to two C₂H₂ Zn²⁺ motifs. These three proteins clearly define a second class of Ste12 protein, present in higher ascomycetes and basidiomycetes, which is distinct from the yeast proteins that lack the C₂H₂ Zn²⁺ motif. The P. marneffei, A. nidulans, and C. neoformans proteins also lack homology to the regions that are important for the regulation of Ste12p by Dig1p and Dig2p (PI et al. 1997 ). These proteins may therefore be regulated directly by a MAPK without the requirement for Dig1p or Dig2p homologs or, alternatively, the C₂H₂ Zn²⁺ finger domains may be responsible for regulating the activity of the proteins through protein-protein interactions (MACKAY and CROSSLEY 1998 ).

StlA is a conserved gene:
STE12 homologs in other fungi are required for sexual development, yeast-hyphal switching, or both. Although deletion of the stlA locus from P. marneffei has no discernible effect on growth, development, or dimorphic switching, there are a number of lines of evidence that suggest stlA plays a role in P. marneffei. First, the P. marneffei gene shows a highly regulated pattern of expression with relatively high levels in vegetative hyphal cells and yeast cells. Second, the stlA gene is able to complement the sexual development defect of an A. nidulans steA mutant, showing that the P. marneffei stlA gene is correctly regulated at the transcriptional level and that the protein is fully functional in A. nidulans. Third, measurements of the rate of synonymous and nonsynonymous base substitutions in the stlA and steA genes show that there is a strong bias against nonsynonymous changes over the entire coding region but especially for the two putative DNA-binding motifs. In comparison to the stlA/steA genes, the abaA developmental regulatory genes, which are required for asexual development and yeast cell morphogenesis in P. marneffei and for asexual development in A. nidulans (BOYLAN et al. 1987 ; ANDRIANOPOULOS and TIMBERLAKE 1994 ; BORNEMAN et al. 2000 ), show a slightly weaker bias against nonsynonymous changes over the entire coding region but a stronger bias over the DNA-binding domain. This suggests that very similar selective pressures are operating for both pairs of genes. Similar results were obtained for a pair of chitin synthase homologs.

P. marneffei may have a sexual cycle:
Although the coding regions of stlA and steA are highly conserved, the promoter regions share very little similarity and searches for binding sites of known transcription factors failed to identify any motifs conserved between species. This result, coupled with the different transcriptional profiles of the P. marneffei and A. nidulans genes, demonstrates the divergence between these two fungi. Therefore, the high degree of conservation of stlA in P. marneffei may be due to an involvement in controlling sexual development and to the fact that P. marneffei has a previously undefined teleomorphic (sexual) state. This is supported by the identification of an A. nidulans stuA homolog (which is also involved in controlling mating) in P. marneffei, which, like stlA, is highly conserved between the two species (MILLER et al. 1992 ; A. R. BORNEMAN, M. J. HYNES and A. ANDRIANOPOULOS, unpublished results). This may be a similar situation to C. albicans, which was also thought to be strictly asexual, but from which numerous, highly conserved homologs of S. cerevisiae mating genes had been identified (SADHU et al. 1992 ; LEBERER et al. 1996 ; RAYMOND et al. 1998 ). Recently, it has been shown that C. albicans diploids possess homologs of both the a and mating loci of S. cerevisiae. Deletion of one of each of these mating loci from reciprocal strains allows C. albicans to be induced to mate, albeit in an artificial manner, to form tetraploid cells (HULL et al. 2000 ; MAGEE and MAGEE 2000 ).

Alternatively, loss of sexual reproduction in P. marneffei may be a relatively recent event. Phylogenetic studies of teleomorphic Talaromyces species with Penicillium asexual states and anamorphic (asexual) Penicillium species of the Biverticilliate group have clearly demonstrated that sexual and asexual species are closely related. The asexual species also appear to be relatively recently derived, probably due to loss of the sexual cycle (LOBUGLIO et al. 1993 ). P. marneffei is a member of the biverticilliate group and has a number of closely related teleomorphic species (LOBUGLIO and TAYLOR 1995 ). Studies of members of the Aspergilli also show close phylogenetic pairing between a teleomorphic and an anamorphic species (GEISER et al. 1996 ).

If P. marneffei is capable of sexual reproduction, there are a number of reasons why the sexual state may not have been observed. P. marneffei may be heterothallic, requiring two strains of different mating type to be brought together for mating to be successful. If one mating type of P. marneffei is rare, avirulent, or even monomorphic, it may not have been isolated or may have failed to be identified as P. marneffei. While this seems unlikely, a similar situation exists in C. neoformans Serotype D strains where the MAT strain is 30-fold more prevalent in the environment and 40-fold more prevalent in infections than the MATa strain (KWON-CHUNG and BENNETT 1978 ). The MAT strain has also been shown to be more virulent than the MATa strain (KWON-CHUNG et al. 1992 ). This dichotomy is more pronounced in Serotype A isolates of C. neoformans as a MATa strain of this serotype has never been isolated. If Serotype A were considered in isolation from Serotype D, it would be classed as asexual due to the lack of an opposite mating type. This situation may also be complicated further by environmental constraints on P. marneffei mating. There may be any number of stimuli that may be required for mating between strains and may include infection of multiple strains in one host, as was the case observed for the artificially induced mating of C. albicans (HULL et al. 2000 ). Further work is therefore required in this area to discriminate among the many possibilities that are presented with respect to the sexuality of P. marneffei and the function of stlA. The presence of an STE12 homolog in an "asexual" species, however, makes this gene an excellent marker for the study of the evolution of sexual/asexual life cycles in fungi.

	ACKNOWLEDGMENTS

We thank Dave Geiser and Jon Martin for their helpful discussions and Rhonni Croft for her expert technical assistance. This work was supported by an Australian Research Council grant. A.R.B. was supported by an Australian Postgraduate Award (APA) scholarship.

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