A p21-Activated Kinase Is Required for Conidial Germination in Penicillium marneffei

Asexual spores (conidia) are the infectious propagules of many pathogenic fungi, and the capacity to sense the host environment and trigger conidial germination is a key pathogenicity determinant. Germination of conidia requires the de novo establishment of a polarised growth axis and consequent germ tube extension. The molecular mechanisms that control polarisation during germination are poorly understood. In the dimorphic human pathogenic fungus Penicillium marneffei, conidia germinate to produce one of two cell types that have very different fates in response to an environmental cue. At 25 °C, conidia germinate to produce the saprophytic cell type, septate, multinucleate hyphae that have the capacity to undergo asexual development. At 37 °C, conidia germinate to produce the pathogenic cell type, arthroconidiating hyphae that liberate uninucleate yeast cells. This study shows that the p21-activated kinase pakA is an essential component of the polarity establishment machinery during conidial germination and polarised growth of yeast cells at 37 °C but is not required for germination or polarised growth at 25 °C. Analysis shows that the heterotrimeric G protein α subunit GasC and the CDC42 orthologue CflA lie upstream of PakA for germination at both temperatures, while the Ras orthologue RasA only functions at 25 °C. These findings suggest that although some proteins that regulate the establishment of polarised growth in budding yeast are conserved in filamentous fungi, the circuitry and downstream effectors are differentially regulated to give rise to distinct cell types.


Introduction
The generation of an axis of cell polarity is central to the activity of many cells and the establishment of a wide variety of cell morphologies. It relies on the ability to mark different cellular regions by specific protein localisation. The establishment of polarised growth requires selection of a site to which proteins and components of the cytoskeleton are recruited. Growth is then directed specifically to this site via targeted cellular trafficking and concomitant cell wall deposition. Fungi are small eukaryotes that exhibit highly polarised growth patterns and provide excellent models for the study of the molecular mechanisms underlying cell polarity. Saccharomyces cerevisiae establishes a polarised axis of growth during the processes of budding cell division, schmoo formation during mating, and pseudohyphal growth. Under conditions of nitrogen starvation, S. cerevisiae diploid cells undergo pseudohyphal growth, a morphological switch that involves changes in cell shape and division [1]. Pseudohyphal growth requires the initiation of polarised growth for cellular elongation and is under the control of two signaling pathways: a cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and a mitogen-activated protein kinase (MAPK) cascade. The cAMP/PKA pathway is activated by a glucose/sucrose sensitive receptor Gpr1p, which activates the G protein Gpa2p (a subunit), which in turn is inhibited by the novel kelch-Gb subunits Gpb1/2p, a third subunit Gpg1p, and a negative regulator Rgs2p [2][3][4][5][6][7][8]. The Gpa2-Gpb1/2 complex regulates the cAMP/PKA pathway directly in association with the Ras2p GTPase and the RasGAP neurofibromin homologues Ira1/2p [6][7]9]. Ras2p can also activate the MAPK cascade by activating the guanine nucleotide exchange factor Cdc24p, which catalyzes the guanosine diphosphate (GDP) to guanosine triphosphate (GTP) exchange of the Rho GTPase Cdc42p [1,10,11]. GTP-bound Cdc42p is required to initiate actin polarisation and recruits and activates additional proteins required for polarised growth such as septins, myosins, and the p21-activated kinase (PAK) Ste20p (reviewed in [12]). In turn, Ste20p activates the MAPK cascade by phosphorylating Ste11p (MAPKKK); Ste11p phosphorylates Ste7p (MAPKK), which then activates the Kss1p MAPK [13][14][15].
Filamentous fungi exhibit a highly polarised axis during vegetative hyphal growth, asexual (conidiation) and sexual (mating) development, and initiation of polarised filamentous growth during the germination of asexual (conidia) and sexual (ascospores) spores. Spherical conidia germinate under favorable environmental conditions by initially grow-ing isotropically. This growth is followed by the establishment of a de novo axis of polarised growth to allow a germ tube to emerge [16][17][18]. Conidial germination is a central aspect of fungal cell propagation, initiating the formation of the extensive radiating hyphal network necessary for colonization of substrates from a dormant conidium. Conidia are also the infectious propagules of many pathogenic fungi, and germination of conidia in the lung or leaves of potential hosts is likely to be a key pathogenicity determinant. Studies in the filamentous fungus Aspergillus nidulans have shown that the cAMP-PKA and Ras pathways play a role [18][19][20]. However, the molecular mechanisms governing germination of fungal conidia are not well understood in any system.
Penicillium marneffei is an opportunistic human pathogen with a thermally regulated dimorphic switch. At 25 8C, in the saprophytic growth phase, conidia germinate to produce highly polarised, septate, branched, multinucleate hyphae. Conidia can also germinate at 37 8C to produce polarised arthroconidiating hyphae, in which nuclear division and septation are coupled, double septa are laid down, and fragmentation occurs along this plane to liberate uninucleate yeast cells that consequently divide by fission [21]. The yeast cells are the pathogenic growth form and multiple yeast cells are observed in the pulmonary alveolar macrophages of infected individuals. P. marneffei infection is thought to occur through inhalation of conidia, which bind to the laminin in the broncholalvelolar epithelia. Conidia are then ingested by pulmonary alveolar macrophages and germinate, generating the uninucleate yeast cells [21]. Therefore in P. marneffei, conidial germination can lead to two very different morphological programs in response to different temperatures.
Some of the core components regulating polarised growth establishment in S. cerevisiae are conserved during polarity establishment in germinating conidia of filamentous fungi. The P. marneffei Gpa2p homologue, encoded by gasC, is required during conidial germination to produce hyphae. Deletion of gasC results in delayed germination, whereas expression of a dominant activating allele shows a significantly accelerated germination rate [22]. Likewise, the P. marneffei Ras homologue, RasA, is required for conidial germination where expression of either a dominant negative or activated allele results in a germination delay and conidia with abnormal isotropic growth [23]. Expression of either a dominant negative or constitutively active allele of the P. marneffei CDC42 homologue (cflA) results in a decrease or increase in the rate of germination at 25 8C, respectively [24]. The role of GasC, RasA, and CflA during conidial germination in P. marneffei suggests that the core components regulating polarised growth establishment in S. cerevisiae may be conserved during polarity establishment in germinating conidia of filamentous fungi. To investigate if any of the downstream effectors of this core pathway are also conserved in function, a PAK STE20 homologue (designated pakA) was identified and deleted in P. marneffei. Characterization of pakA in P. marneffei has shown that this gene is essential for conidial germination at 37 8C and for polarised growth of yeast cells and is downstream of both a heterotrimeric G protein and Cdc42 pathway. In contrast, PakA plays only a minor role during germination of conidia at 25 8C and is not required for polarised growth of hyphae. Germination in this case is controlled by a heterotrimeric G protein-Ras-Cdc42 pathway. These data suggest that although some proteins that regulate the establishment of polarised growth in budding yeast and filamentous fungi may be conserved, the downstream effectors are likely to be different or regulated differently to give rise to the distinct cell types in these two modes of growth.

Results
Cloning the STE20 p21-Activated Kinase Orthologue from P. marneffei An A. nidulans sequence was identified from the genome sequence (http://www.broad.mit.edu/annotation/genome/ aspergillus_group/MultiHome.html) with strong homology to Candida albicans Cst20p (67% identity, 81% similarity) and other PAKs, including S. cerevisiae Ste20p (71% identity, 81% similarity, accession number AAA35039). Primers were designed to amplify the sequence encoding the conserved kinase domain and a PCR product was generated using A. nidulans genomic DNA. The A. nidulans STE20 homologous sequence was used to screen a P. marneffei genomic library at low stringency. Five positive clones were identified, which fell into two classes based on restriction enzyme digestion patterns. A 6.4 kb NotI/BglII hybridizing fragment from one of these classes was subcloned into NotI/BamHI digested pBluescript II SK þ (pKB5751). Sequencing revealed strong sequence homology to STE20-like PAKs from Magnaporthe grisea (78% identity, 89% similarity, accession number AAP93639), Ustilago maydis (74% identity, 83% similarity, accession number AAM97788), and S. cerevisiae (72% identity, 83% similarity

Author Summary
Many fungal infections are initiated by the entry of dormant fungal spores into their host. Once inside the host these dormant spores must reactivate (germinate) for the infection to proceed. Productive infections necessitate that the fungus grow and divide within the host, which makes understanding the mechanisms that control germination crucial to developing preventative or prophylactic treatments for fungal infections. The molecular mechanisms that control spore germination are poorly understood and studies of the opportunistic fungal pathogen Penicillium marneffei have shown that a group of highly conserved signalling and cell polarity factors, known as small GTPases, play important roles in germination and other aspects of morphogenesis. In this study we have shown that a downstream target of these small GTPases, a p21-activated kinase plays a crucial role in germination at the host temperature of 37 8C but not at 25 8C. This is the first component of germination, which shows temperature-dependent effects and provides insights into the different mechanisms used by fungal pathogens to infect their host or to grow saprophytically in non-host environments.
cells grown for 8 d in liquid medium at 37 8C. A pakA transcript was detected under all conditions. The amount of pakA transcript was approximately equivalent under all conditions when compared with the histone H3 control (unpublished data).

Deletion of pakA in P. marneffei
A pakA construct, which deleted from À425 to þ2030 of pakA, was linearised and transformed into P. marneffei strain SPM4 (niaD1 pyrG1) and pyrG þ transformants selected. PyrG þ transformants were screened by genomic Southern blotting and one strain was isolated that possessed a restriction pattern consistent with replacement of pakA by pyrG at the genomic locus. The deletion strain was plated on medium containing 5-fluoroorotic acid (5-FOA) to generate a DpakA pyrG strain (Materials and Methods). This strain was cotransformed with plasmids containing pakA þ and pyrG þ genes and co-transformants confirmed by Southern blot analysis. The transformants contained from 1-7 copies of pakA.
In S. cerevisiae, the H345G mutation in the conserved CRIB domain of Ste20p results in a mutant protein that shows reduced interaction with the upstream activator Cdc42p (GTPase) in a two-hybrid assay and loss of correct localisation to sites of polarised growth [25]. The phenotype of the STE20 H345G strain is almost equivalent to the null, indicating that the interaction of Cdc42p and Ste20p is essential for Ste20p function [25]. The equivalent mutation was generated in P. marneffei pakA (pakA H108G ) by inverse PCR (Materials and Methods). The pakA H108G construct was co-transformed with pyrG þ into the DpakA pyrG À strain and transformants selected for PyrG þ . Co-transformation was confirmed by Southern blot analysis of genomic DNA and four representative transformants with copy numbers ranging from 3 to 12 were selected for further analysis.

PakA Is Localised to Sites of Polarised Growth
The gfp::pakA and gfp::pakA H108G fusion constructs were generated and co-transformed with the pyrG þ gene into the DpakA pyrG strain to investigate the localisation of PakA and to assess whether the pakA H108G mutation affects PakA localisation. Transformants were selected for PyrG þ and confirmed by Southern blot analysis of genomic DNA. Four transformants of each genotype were selected for further analysis and had copy numbers ranging from 4 to 9 for gfp::pakA and from 2 to 20 for gfp::pakA H108G . The gfp::pakA and gfp::pakA H108G strains were grown on agar-coated slides for 2 and 4 d at 25 8C and 37 8C, respectively. At 25 8C, the GFP::PakA fusion protein was concentrated at the hyphal apex ( Figure 1A) and localised as spots along the subapical cells of the hyphae ( Figure 1B). In contrast, the GFP::Pa-kA H108G fusion protein was visible as diffuse fluorescence in the cytoplasm but not concentrated at the hyphal apex or as spots along the hyphae ( Figure 1A and 1B). At 37 8C, the GFP::PakA fusion protein was concentrated at the apex of arthroconidiating hyphae, although this fluorescence was less intense than that visualized in the same transformants at 25 8C. The GFP::PakA H108G fusion protein was not observed at the apex of arthroconidiating hyphae (unpublished data).
To investigate whether the GFP::PakA fusion protein colocalises with actin at the hyphal apex and at nascent septation sites, immunostaining using mouse anti-actin and rabbit anti-GFP antibodies was performed on two of the gfp::pakA and two of the gfp::pakA H108G strains at both 25 8C and 37 8C. At 25 8C, actin is localised as cortical actin spots along the hyphae and is concentrated at nascent septation sites and the hyphal apex ( Figure 1C-1F). The GFP::PakA fusion protein co-localised with actin at all of these locations ( Figure 1C-1F). The GFP::PakA H108G fusion protein also colocalised with actin at cortical actin patches, nascent septation sites, and the hyphal apex, in addition to showing diffuse staining throughout the cytoplasm ( Figure 1C-1F).
At 37 8C, actin is also localised as cortical patches along the arthroconidiating hyphae, concentrated at nascent septation sites and the apex of arthroconidiating hyphae ( Figure S1). The GFP::PakA and GFP::PakA H108G fusion proteins colocalised with actin at all of these sites ( Figure S1).
The Role of pakA during Asexual Development and Hyphal Growth at 25 8C At 25 8C, P. marneffei colonies are comprised of highly polarised vegetative hyphae growing on the agar surface and bearing asexual structures (conidiophores) that appear green due to the presence of pigmented asexual spores (conidia) on the conidiophores. After 5 d growth at 25 8C, surface hyphae are visible in the wild-type strain ( Figure 2A). The DpakA pakA þ strains appeared wild-type after 5 d at 258 C, whereas both the DpakA and DpakA pakA H108G strains showed a reduction in growth ( Figure 2A). Despite the initial reduction in growth, after 10 d all strains were producing conidia. P. marneffei colonies are yeast-like at 37 8C and DpakA pakA þ strains were indistinguishable from the wild type when grown for 4 d at 37 8C ( Figure 2B). In contrast, the DpakA and DpakA pakA H108G strains displayed reduced growth at 37 8C ( Figure  2B).
Both the DpakA and DpakA pakA H108G strains exhibit a delay in growth after 5 d at 25 8C (Figure 2A). One explanation for this difference could be a delay in the germination of conidia. The kinetics of germination were measured at 25 8C in the wild-type (pakA þ ), DpakA, DpakA pakA þ , and two DpakA pakA H108G strains by counting the number of ungerminated versus germinated conidia (conidia with a visible germ tube) in a population of 100 in three independent experiments after 8, 12, or 15 h incubation in liquid media (Table S1). The complemented DpakA strain (DpakA pakA þ ) is indistinguishable from wild type ( Figure 3A and 3B). The DpakA and DpakA pakA H108G strains show a minor delay in germination at 25 8C ( Figure 3A and 3B).
To investigate if the deletion of pakA or the pakA H108G mutation results in aberrant hyphal morphology or asexual development at 25 8C the wild-type, DpakA, DpakA pakA þ , and DpakA pakA H108G strains were grown on agar-coated slides for 2 or 4 d at 25 8C, stained with calcofluor (to observe cell walls) or 496-diamidino-2-phenylindole (DAPI; to observe nuclei), and examined microscopically. After 2 d at 25 8C, wild-type P. marneffei grows as septate, branched hyphae of which subapical cells are predominately uninucleate and apical cells are multinucleate. The DpakA, DpakA pakA þ , and DpakA pakA H108G strains were indistinguishable from wild type after 2 d, with normal morphology, septation, branching, and nuclear index. After 4 d at 25 8C, wild-type P. marneffei begins to undergo asexual development, with the production of a specialized stalk from which differentiated cells are produced sequentially in a budding fashion: metulae bud from the stalk, phialides bud from metulae, and uninucleate conidia bud from phialides. The DpakA, DpakA pakA þ , and DpakA pakA H108G strains produced conidiophores with wild-type morphology (unpublished data).
pakA Is Essential for Conidial Germination and Correct Yeast Cell Morphogenesis at 37 8C In contrast to the wild-type and DpakA pakA þ strains, the DpakA and DpakA pakA H108G strains displayed reduced growth rates at 37 8C ( Figure 2B). To assess if the basis of this difference is because pakA is required during the germination of conidia or during yeast morphogenesis and growth at 37 8C, the wild-type (pakA þ ), DpakA, DpakA pakA þ , and DpakA pakA H108G strains were inoculated on agar-coated slides and incubated for 4 d at 37 8C. It was immediately apparent that the DpakA and DpakA pakA H108G strains possessed a severe germination defect and almost all of the conidia remained ungerminated. The germination kinetics were measured by counting the number of ungerminated versus germinated conidia (conidia with a visible germ tube) in a population of 100 in three independent experiments after 8, 12, 15, or 20 h in liquid medium (Table S2). Germination is slower and germlings appear fatter at 37 8C compared with 25 8C ( Figure  3A and 3C). In contrast to wild-type and the DpakA pakA þ strains, both the DpakA strain and the DpakA pakA H108G strains showed a severe defect in germination ( Figure 3C and 3D). Despite the majority of conidia remaining ungerminated in the DpakA and DpakA pakA H108G strains, a small proportion do germinate, and it is presumably these cells that go on to establish the colony.
Growth of the pakA strains in liquid medium showed that the small proportion of conidia that germinate go on to form arthroconidiating hyphae that fragment at septation sites to liberate uninucleate yeast cells, just like the wild type. The wild-type (pakA þ ), DpakA, DpakA pakA þ , and DpakA pakA H108G strains were grown in liquid brain heart infusion (BHI) for 6 d at 37 8C and cells were stained with calcofluor or DAPI and observed microscopically ( Figure 4). In wild type, the culture consists of a mixture of fragmented arthroconidiating hyphae and uninucleate yeast cells. The DpakA strain produced swollen arthroconidiating hyphae and yeast cells with increased septation ( Figure 4) but normal nuclear index. These defects were complemented when the strain was transformed with pakA þ . The DpakA pakA H108G strains also produced swollen arthroconidiating hyphae and yeast cells, but the phenotype was more severe than the DpakA strain, with very few yeast cells produced and-in contrast to the DpakA strain and wild type-a decrease in the septation index ( Figure 4). Therefore, the pakA H108G allele has an inhibitory activity on septation.

PakA Is Required for Conidial Germination In Vivo
P. marneffei infection occurs through the inhalation of conidia. The conidia are ingested by host pulmonary alveolar macrophages where they germinate into unicellular yeast cells that divide by fission. Multiple yeast cells are seen in the pulmonary alveolar macrophages and peripheral blood mononuclear cells of infected individuals [21]. To investigate if perturbing conidial germination will influence pathogenicity, the ability of the mutant pakA strains to germinate into pathogenic yeast cells was investigated. LPS activated J774 murine macrophages were infected with no conidia or with conidia of the wild-type (pakA þ ), DpakA, DpakA pakA H108G , or DpakA pakA þ strains (Materials and Methods). After 24 h, numerous yeast cells dividing by fission were observed in macrophages infected with wild-type (pakA þ ) or DpakA pakA þ conidia ( Figure 5). Only 16.3 6 2.50% or 21.5 6 2.07% of wild-type (pakA þ ) or DpakA pakA þ conidia, respectively, remained ungerminated in infected macrophages. In contrast, conidia of the DpakA or DpakA pakA H108G strains remained predominately ungerminated in infected macrophages ( Figure 5). 60.0 6 3.80% of DpakA conidia and 66.1 6 7.16% of DpakA pakA H108G conidia remained ungerminated in macrophages 24 h post infection. This suggests that PakA is required for conidial germination during infection of host macrophages.
Differential Regulation of Conidial Germination at 25 8C and 37 8C The temperature-dependent regulation of conidial germination could be the result of the presence of a temperaturespecific factor on which PakA depends or the result of a change in the thermostability of a complex in which PakA operates. To distinguish between these two possibilities, wildtype and DpakA conidia were incubated in liquid medium at different temperatures ranging from 25 8C to 37 8C, in 2 8C increments, and germination rates were measured (Materials and Methods). In contrast to the wild type, the DpakA strain showed a gradual decrease in the percentage of germination as the temperature increased, indicating that there is no critical temperature during the switch ( Figure 3E). This result supports the latter hypothesis and suggests PakA-dependent thermosensitivity. To investigate if DpakA conidia at 37 8C are waiting for the signal to germinate or have aborted, DpakA conidia were incubated at 37 8C for 20 h, then at 25 8C for 20 h. The majority of conidia germinated upon switching to 25 8C, indicating that after 20 h incubation at 37 8C, the DpakA conidia remained viable (unpublished data).
To identify other factors involved in the pakA-dependent differences in germination at 25 8C and 37 8C, conidial germination was analyzed in detail at both temperatures in strains carrying mutations in cflA (CDC42 orthologue), gasC (GPA2 orthologue), and rasA (RAS2 orthologue), which have been shown previously to affect germination at 25 8C but which had not been characterized at 37 8C [22][23][24]. The role of CflA, GasC, and RasA in germination at 37 8C was characterized by assessing the percentage of germinated conidia after 8, 12, 15, and 20 h at both 25 8C and 37 8C in two strains of each genotype (Tables S1 and S2). Two-level nested ANOVA was performed on the data for each time point at both 25 8C and 37 8C to test if germination rates differed significantly between genotypes and also between transformants of the same genotype (Materials and Methods). ANOVA showed there was a significant difference between genotypes in all time points except 0 h. In a few instances, there was variation within transformants of the same genotype (Tables S1 and S2). Strains expressing the dominant negative cflA D120A allele showed delayed germination at both 25 8C and 37 8C ( Figure 6A and 6C). Dominant activated cflA G14V strains displayed accelerated germination at both 25 8C and 37 8C (Figure 6B and 6D). These results indicate that active CflA promotes conidial germination at both 25 8C and 37 8C. Likewise, the dominant interfering gasC G207R strains showed delayed germination at both 25 8C and 37 8C ( Figure  7A and 7C). The dominant activated gasC G45R strains showed accelerated germination at 25 8C and 37 8C, suggesting that, like CflA, GasC is required for conidial germination at both 25 8C and 37 8C (Figure 7B and 7D). Both the dominant activated (rasA G19V ) and dominant negative (rasA D125A ) rasA strains showed a decrease in germination at 25 8C (Table S1). In contrast, both the dominant negative and dominant activated strains showed wild type germination patterns at 37 8C, suggesting that RasA is required for conidial germination at 25 8C but not at 37 8C (Table S2).
Genetic Interactions Between cflA, pakA, and rasA during Conidial Germination To investigate any genetic interaction between pakA and cflA, double mutants were generated (DpakA cflA þ , DpakA cflA D120A , DpakA cflA G14V , DpakA pakA H108G cflA þ , DpakA pakA H108G cflA D120A , and DpakA pakA H108G cflA G14V ) and germination was characterized by assessing the percentage of germinated conidia after 8, 12, 15, and 20 h at both 25 8C and 37 8C (Tables S1 and S2). It should be noted that multiple copy integrants may result in significant overexpression. Two strains of each genotype were assessed and a single representative strain is shown in Figure 6. ANOVA was performed on the data for each time point at both 25 8C and 37 8C to test if germination rates differed significantly between genotypes and also between transformants of the same genotype (Materials and Methods). ANOVA showed there was a significant difference between genotypes at all time points except 0 h. In a few instances, there was variation within transformants of the same genotype (Tables S1 and  S2). The control DpakA cflA þ and DpakA pakA H108G cflA þ strains showed germination patterns at 25 8C and 37 8C that were indistinguishable from the parental DpakA and DpakA pakA H108G strains. At 25 8C, the DpakA cflA D120A and DpakA pakA H108G cflA D120A strains displayed delayed conidial germination at rates similar to the single cflA D120A mutant strains ( Figure 6A). At 37 8C, the DpakA cflA D120A and DpakA pakA H108G cflA D120A strains displayed dramatically reduced germination like the single DpakA and DpakA pakA H108G strains ( Figure 6C).
In contrast to the accelerated germination observed in cflA G14V mutants at 25 8C, which is much faster than wild-type ( Figure 6B), the DpakA cflA G14V and DpakA pakA H108G cflA G14V double mutants display slower than wild-type germination at 25 8C, similar to the DpakA and DpakA pakA H108G single mutant strains ( Figure 6B). This suggests that the accelerated germination observed in cflA G14V strains at 25 8C requires active PakA and an interaction of CflA and PakA via the PakA CRIB domain.
Likewise at 37 8C, in contrast to the accelerated germination observed in cflA G14V mutants, the DpakA cflA G14V and DpakA pakA H108G cflA G14V double mutants display dramatically reduced germination at rates equivalent to the DpakA and DpakA pakA H108G single mutant strains ( Figure 6D). The inability of the cflA G14V dominant activated allele to suppress the reduced germination phenotype of the DpakA and DpakA pakA H108G mutants suggests that at 37 8C, CflA acts upstream of PakA during germination and that an interaction between CflA and the CRIB domain of PakA is required for germination to proceed.

GasC Activates Two Pathways Regulating Conidial Germination at 25 8C and 37 8C
To investigate the genetic interaction between pakA and gasC, double mutants were generated (DpakA gasC þ , DpakA gasC G207R , and DpakA gasC G45R ). Germination was characterized in five strains of each double mutant genotype by assessing the percentage of germinated conidia after 8, 12, 15, and 20 h at both 25 8C and 37 8C (Tables S1 and S2). ANOVA was performed on the data for each time point at both 25 8C and 37 8C (Materials and Methods) and revealed there was a significant difference between genotypes at all time points except 0 h. In a few instances, there was variation within transformants of the same genotype (Tables S1 and S2). DpakA gasC þ strains were indistinguishable from DpakA. At 25  The wild-type (pakA þ ), DpakA, DpakA pakA þ , and DpakA pakA H108G strains were grown in liquid BHI medium at 37 8C for 6 d. Wild type (pakA þ ) produces arthroconidiating hyphae and a mixture of fragmented and separated yeast cells. The DpakA pakA þ strains are indistinguishable from wild type. The DpakA mutant produces swollen arthroconidiating hyphae with increased septation. Fewer yeast cells, which are also swollen and highly septate, are produced in the DpakA mutant. The DpakA pakA H108G strains produce swollen arthroconidiating hyphae that have reduced septation, and there are reduced numbers of yeast cells. Images were captured using DIC or with epifluorescence to observe calcofluor stained cell walls (CAL 8C, DpakA gasC G207R strains have delayed germination, which is slower than both the gasC G207R single mutant strains and the DpakA mutant ( Figure 7A). At 37 8C DpakA gasC G207R strains have a severe reduction in germination similar to DpakA ( Figure 7C). In contrast to the accelerated germination of the gasC G45R single mutant strains and the delayed germination of the DpakA mutant, the DpakA gasC G45R double mutant strains show wild-type germination at 25 8C ( Figure  7B). In addition, DpakA gasC G45R double mutant strains show germination rates at 37 8C, which are lower than those of wild-type and the gasC G45R mutants but higher than that of the DpakA mutant ( Figure 7D). This indicates that expression of the gasC G45R mutant allele partially suppresses the germination defects of DpakA and suggests that GasC regulates two pathways during germination, one of which is independent of PakA.

Conserved Proteins Initiate Filamentous Growth but the Downstream Effectors Are Differentially Regulated
The establishment of an axis of polarised growth is orchestrated by the asymmetric distribution of cellular components through the localisation and activation of proteins required for growth. Some of the core components regulating polarised growth establishment in S. cerevisiae are conserved both in the genome and functionally during polarity establishment in more complex organisms. However, the question remains as to how multi-cellular organisms generate the greater diversity of distinct cell types with the same set of core components. Unlike small eukaryotes like fungi, larger eukaryotes such as flies and mammals often have an increased number of factors involved in polarity establish- ment with significant redundancy [26][27][28]. One possible mechanism is to alter the activity of the key establishment proteins in different cell types, while another is to differentially regulate the effector proteins. In the dimorphic pathogen P. marneffei, the germination of conidia gives rise to two different developmental pathways and cell types. The regulation of conidial germination by CflA (CDC42 orthologue) and GasC (GPA2 orthologue) at both 25 8C and 37 8C suggests that the core components regulating polarised growth establishment in S. cerevisiae may be conserved during polarity establishment in germinating conidia of filamentous fungi. However, mutations in pakA, a potential downstream effector of cflA, result in a dramatic reduction in the rates of germination at 37 8C but not 25 8C. This suggests that in P. marneffei conserved polarity establishment proteins regulate germination, but the downstream effectors are differentially regulated to give rise to distinct cell types. The results suggest a model in which, at 25 8C, GasC activates two pathways regulating conidial germination (Figure 8). In one pathway, RasA activates CflA, which activates PakA-by association via the CRIB domain-and a proposed additional effector to establish polarised hyphal growth (Figure 8). At 37 8C, GasC also activates two pathways regulating conidial germination. In one pathway, similar to 25 8C, CflA activates PakA via the CRIB domain, and this interaction is required for PakA function. Unlike 25 8C, RasA does not activate CflA, PakA plays a crucial role during the establishment of polarised arthroconidiating hyphal growth, and no additional effector is required (Figure 8).
The G-protein GasC Regulates Two Signaling Pathways Required for the Initiation of Filamentous Growth in P. marneffei gasC encodes a heterotrimeric guanine nucleotide-binding (G-protein) a-subunit with homology to S. cerevisiae GPA2. Gpa2p is required for the initiation of filamentous growth (pseudohyphal growth) in response to nitrogen starvation via the activation of the cAMP-PKA pathway [2,3]. In P. marneffei, expression of dominant activated gasC G45R and dominant interfering gasC G207R alleles at both 25 8C and 37 8C results in accelerated or delayed conidial germination, respectively, and suggests that GasC acts as a general upstream activator initiating filamentous growth. This study suggests that GasC activates two pathways regulating germination at both 25 8C and 37 8C, one of which is dependent on PakA (Figure 8). In contrast to the accelerated germination of gasC G45R strains and the slightly delayed germination of DpakA, DpakA gasC G45R double mutant strains display wild-type germination at 25 8C. A reduction in the germination rates suggests that PakA is acting downstream of GasC at 25 8C. However, as germination of the DpakA gasC G45R strains is higher than DpakA, GasC must also regulate an additional PakA-independent pathway activating germination at 25 8C. This hypothesis is supported by the germination rates observed in the DpakA gasC G207R strains, which are lower than both the single mutant strains, suggesting these mutations have an additive effect. Likewise, at 37 8C, partial suppression of the DpakA germination defect by expression of the gasC G45R allele indicates that GasC acts as an upstream regulator of two pathways activating conidial germination, one of which is dependent on PakA. The activation of two pathways regulating the initiation of filamentous growth in P. marneffei differs from S. cerevisiae. In S. cerevisiae, the Gpa2p regulation of pseudohyphal growth occurs via the activation of the cAMP-PKA pathway, which is independent of the Ste20p/MAPK cascade [3]. The pseudohyphal growth defect of a gpa2 mutant can be suppressed by addition of cAMP or overexpression of the dominant activated RAS2 G19V allele. However, the pseudohyphal defect cannot be suppressed by the overexpression of the dominant active STE11-4 allele and has no effect on expression of a reporter gene (FRE-lacZ), which is known to be regulated by the MAP kinase cascade [3]. These results together suggest that, unlike P. marneffei GasC, S. cerevisiae, Gpa2p regulates the cAMP pathway but not the Ste20p-MAPK cascade, thus regulating the initiation of filamentous growth.
The Cdc42 Orthologue in P. marneffei Activates PakA during the Initiation of Filamentous Growth In S. cerevisiae Cdc42p localises and activates Ste20p (reviewed in [12]). Therefore P. marneffei PakA is a potential downstream effector of the CflA (Cdc42p orthologue) and may be involved in similar processes. Like cflA D120A strains, Figure 8. Models for the Regulation of the Initiation of Filamentous Growth in S. cerevisiae and P. marneffei (A) In S. cerevisiae, nitrogen starvation triggers a switch from the unicellular budding growth form to a filamentous growth form (pseudohyphal growth). The activated GTPase Ras2p, activates the GEF Cdc24p, which catalyses the activation of the Rho GTPase Cdc42p. Cdc42p activates the PAK kinase Ste20p, which leads to the activation of a MAPK kinase cascade, which culminates in the activation of the transcription factor Ste12p. The activation of a heterotrimeric G protein is also involved in the initiation of filamentous growth in S. cerevisiae, via activation of cAMP signaling. (B) In P. marneffei, GasC (Gpa2p homologue) activates two pathways regulating conidial germination at 25 8C. In one pathway, GasC activates RasA (Ras2p homologue), which activates CflA (Cdc42p homologue). CflA activates PakA (Ste20p homologue) via the CRIB domain and this interaction is required for PakA function. CflA also activates an additional unknown effector at 25 8C, which plays a more important role than PakA in germination. It is possible that GasC directly acts on PakA and does not act upstream of RasA and CflA. B. At 37 8C, GasC also activates two pathways regulating conidial germination. In one pathway, GasC activates CflA, which activates PakA. Similar to 25 8C, at 37 8C CflA activates PakA via the CRIB domain and this interaction is required for PakA function. Unlike 25 8C, at 37 8C RasA is not required for germination, PakA plays a crucial role during the establishment of polarised arthroconidiating hyphal growth, and no additional effector is required. doi:10.1371/journal.ppat.0030162.g008 which show delayed germination at 37 8C, the DpakA mutant exhibits a dramatic reduction in conidial germination at 37 8C, suggesting that PakA is crucial during the establishment of polarised growth to give rise to arthroconidiating hyphae. However, unlike CflA, PakA plays only a minor role during germination at 25 8C, as strains expressing the dominant negative cflA D120A allele show a severe delay in germination compared with only a slight delay in conidial germination for the DpakA and DpakA pakA H108G strains. These results suggest that PakA acts downstream of CflA at both 25 8C and 37 8C in P. marneffei. This hypothesis is also supported by the observation that the accelerated germination seen in cflA G14V strains is abrogated by the DpakA at both 25 8C and 37 8C in DpakA cflA G14V strains.
PAKs contain a conserved N-terminal CRIB domain and a C-terminal kinase domain (reviewed in [27]). The CRIB domain of S. cerevisiae Ste20p negatively inhibits the kinase domain preventing signaling [25]. This autoinhibition is relieved by interaction of the CRIB domain with Cdc42p, and this interaction is also necessary for localisation of Ste20p to sites of polarised growth [25]. The H345G mutation in the Ste20p CRIB domain shows reduced interaction with Cdc42p in a two-hybrid assay and the loss of correct localisation to sites of polarised growth. The phenotype of the STE20 H345G strain is almost equivalent to the null, indicating that the interaction of Cdc42p and Ste20p is essential for Ste20p function (and for relief of autoinhibition of the kinase domain) [25]. The H108G CRIB domain mutation in P. marneffei is equivalent to the H345G of S. cerevisiae and was found to have a similar effect. The H108G mutation resulted in reduced localisation of PakA to sites of polarised growth and a phenotype equivalent to the deletion mutant. Compared with the accelerated germination of cflA G14V single mutant strains at both 25 8C and 37 8C, the DpakA pakA H108G cflA G14V mutant strains exhibited reduced germination, suggesting that the interaction of CflA and PakA via the CRIB domain is required during conidial germination at both 25 8C and 37 8C. The interaction of CflA and PakA is also required during polarised growth of yeast cells, as the DpakA pakA H108G strains showed a swollen, abnormal yeast morphology at 37 8C, similar to the deletion strain. In S. cerevisiae, the CRIB domain is essential for pseudohyphal growth but dispensable for G protein-mediated pheromone signaling [11]. In P. marneffei the CRIB domain of PakA is also required for the initiation of filamentous growth, but unlike S. cerevisiae the initiation of filamentous growth in P. marneffei by PakA is partially dependent on G-protein signaling.
The minor role played by PakA during germination and hyphal growth at 25 8C suggests that another CflA effector, possibly a Cla4p orthologue, is required for these processes. The genomes of A. nidulans, M. grisea, U. maydis, Coprinopsis cinerea, Neurospora crassa, and C. albicans encode two PAKs, one with homology to Ste20p and the other to Cla4p (http://www. broad.mit.edu/annotation/fgi/). In addition to the CRIB and kinase domains of Ste20p orthologues, Cla4p homologues have a pleckstrin homology domain. Cla4p in S. cerevisiae is required for septation but does not play a role in pseudohyphal growth. However, deletion of CLA4 in the yeasts Yarrowia lipolytica and C. albicans blocks filament formation, and the cla4 deletion mutant of the plant pathogen U. maydis is unable to form filaments during infection [29][30][31][32].
PAKs in P. marneffei May Have Overlapping Roles during Polarised Growth of Hyphae at 25 8C cflA has been previously shown to play a pivotal role during hyphal morphogenesis with mutations resulting in grossly aberrant hyphae [24]. It was therefore expected that the DpakA strain may have a similar phenotype, albeit less severe, as CflA is proposed to interact and activate numerous effector proteins. The lack of a DpakA hyphal phenotype suggests that PakA is not required for hyphal growth. Like P. marneffei, deletion of the Ste20p homologues in C. albicans and U. maydis does not result in defects in hyphal morphology [33,34]. However, the co-localisation of the GFP::PakA fusion protein with actin and the localisation to the same cellular locations as CflA at nascent septation sites and to the hyphal apex suggests a role during polarised growth of hyphae [23]. In S. cerevisiae, Ste20p directly phosphorylates Bni1p, a component of the polarisome [35]. The polarisome is a protein complex, which contains Bni1p, Spa2p, Pea2p, and Bud6p, that promotes polarised morphogenesis during filamentous growth [35,36]. The Bni1p homologue, SepA, plays a conserved role in polarised growth in A. nidulans [37]. However, A. nidulans lacks a Pea2p homologue and the Spa2 homologue, SpaA, is only partially conserved in sequence and function, indicating that the polarisome in filamentous fungi likely consists of a modified set of components with different contributions to polarisome function [36]. The implication is that in P. marneffei, in addition to specific developmental roles, pakA and pakB play complementary, and possibly overlapping, roles in the establishment of polarised growth during conidial germination and in the maintenance of an axis of polarisation during hyphal growth. How the two PAKs coordinately regulate different aspects of development of multi-cellular fungi still remains unclear. The analysis of a CLA4 orthologue from P. marneffei may resolve many of these issues.

Materials and Methods
Molecular techniques. P. marneffei genomic DNA and RNA was isolated as previously described [38,39]. Southern and northern blotting was performed with Amersham Hybond Nþ membrane according to the manufacturer's instructions. Filters were hybridized using [aÀ 32 P]dATP-labeled probes by standard methods [40].
Cloning and plasmid construction. Primers L18 (59-TGATCCCA-CAAAACTTTACT-39) and L19 (59-GCTCGTTTCTCAGGGTCCAC-39) were used to amplify the A. nidulans genomic sequence encoding the conserved kinase domain of the STE20 homologue. The PCR product was sequenced and used to screen a P. marneffei genomic library (constructed in kGEM-11) at low stringency (50% formamide, 2 x SSC, 37 8C). A 6.4 kb NotI/BglII hybridizing fragment from a positively hybridizing clone was subcloned into NotI/BamHI digested pBluescript II SK þ (pKB5751). Sequencing was performed by the Australian Genome Research Facility and analyzed using Sequencher 3.1.1 (Gene Codes Corporation). The Genbank accession number of the P. marneffei pakA gene is AY621630.
A pakA deletion construct (pKB5792) was generated by replacing the 2.5 kb EcoRV/ClaI fragment of pKB5751 with the 2.5 kb SmaI/ClaI fragment containing the pyrG þ selectable marker. This resulted in pyrG þ flanked by 2.6 kb of 59 and 1.1 kb of 39 pakA sequence, and deleted from À425 to þ2030. Inverse PCR using the mutagenic primers N30 (59-ACATGAGTAACACCGACAGGG-39) and N32 (59-TGGATACGACAATCAGACTGG-39) was used to introduce the H108G mutation into pakA generating pKB5908. The integrity of the construct was confirmed by sequencing.
Fungal strains and media. Strains used in this study are shown in Table 1. The DpakA strain (DpakA::pyrG þ ) was generated by transforming the strain SPM4 with linearised pKB5792 and selecting for pyrG þ . Transformation was performed using the previously described protoplast method [38]. The DpakA pyrG À strain was isolated by plating the DpakA strain (DpakA::pyrG þ ) on medium containing 1 mg/ mL À1 5-FOA supplemented with 10 mM c-amino butyric acid (GABA) and 5 mM uracil to select for the loss of the pyrG marker. A 5-FOA resistant sector was isolated that had a restriction pattern consistent with loss of pyrG at the pakA locus. The strain is unable to grow in the absence of 5 mM uracil.
In vivo macrophage assay. J774 murine macrophages (1 3 10 5 ) were seeded into each well of a 6-well microtitre tray containing one sterile coverslip and 2 mL of complete Dulbecco's Modified Eagle Medium (complete DMEM: DMEM, 10% fetal bovine serum, 2 mM Lglutamine and penicillin-streptomycin). Macrophages were incubated at 37 8C for 24 h before activation with 0.1lg/mL À1 lipopolysaccharide (LPS) from E. coli (Sigma). Macrophages were incubated a further 24 h at 37 8C and washed 3 times in phosphate buffered saline, and 2 mL of complete DMEM medium containing 1 3 10 6 conidia was added. A control lacking conidia was also performed. Macrophages were incubated for 2 h at 37 8C (to allow conidia to be engulfed), washed once in phosphate buffered saline (to remove free conidia), and incubated a further day at 37 8C. Macrophages were fixed in 4% paraformaldehyde and stained with 1 mg/mL À1 fluorescent brightener 28 (calcofluor, CAL) to observe fungal cell walls. The numbers of germinated conidia was measured microscopically by counting the numbers of germinated conidia (conidia with a visible germ tube or yeast cells) in a population of approximately 100. Three independent experiments were performed. Mean and standard error of the mean values were calculated using GraphPad Prism3.
Microscopy. P. marneffei strains were grown on slides covered with a thin layer of solid medium, with one end resting in liquid medium [38]. Wild-type (pakA þ ), DpakA, DpakA pakA þ , and DpakA pakA H108G strains were grown on ANM medium supplemented with GABA at 25 8C for 2 or 4 d. At 37 8C, strains were grown on BHI medium for 4 d or in liquid BHI medium for 6 d.
Immunofluorescence microscopy for examination of the actin cytoskeleton was performed using standard protocols [43]. Double staining was performed with the mouse C4 monoclonal anti-actin (Chemicon International) and rabbit anti-GFP polyclonal primary antibodies, as well as ALEXA 488 rabbit anti-mouse (Molecular Probes) and ALEXA 594 anti-rabbit (Molecular Probes) secondary antibodies. Single immunostaining controls and a minus primary antibody control were also performed. The gfp::pakA and gfp::pakA H108G strains were grown on agar-coated slides containing ANM plus GABA for 2 d or SD with 5 mM ammonium tartrate (NH 4 T) for 4 d at 37 8C.
Slides were examined using differential interference contrast (DIC) and epifluorescence optics for GFP, antibody fluorescence, cell wall staining with fluorescent brightener 28 (calcofluor, CAL), or nuclear staining with DAPI and viewed on a Reichart Jung Polyvar II microscope. Images were captured using a SPOT CCD camera (Diagnostic Instruments) and processed in Adobe Photoshop.
Germination experiments. Approximately 10 6 spores were inoculated into 300 lL of SD plus 10 mM (NH 4 ) 2 SO 4 and incubated for 8, 12, or 15 h at 25 8C or for 8, 12, 15, or 20 h at 37 8C. The rates of germination were measured microscopically by counting the numbers of germinating conidia (conidia with a visible germ tube) in a population of 100. Three independent experiments were performed. Mean and standard error of the mean values were calculated using GraphPad Prism3. Two-level nested ANOVA was performed on the data for each time point at both 25 8C and 37 8C to test if germination rates differed significantly between genotypes and between transformants of the same genotype. ANOVA simultaneously tests two null hypotheses; there is no difference between the means of the data sets from all genotypes and there is no difference between the means of the data sets between transformants of the same genotype. The generation of two F-statistics and probability values allow rejection or acceptance of these null hypothesis at a 99% confidence. Values with an asterisk in Tables S1 and S2 showed significant differences between transformants of the same genotype.
To investigate the differential regulation of conidial germination at 25 8C and 37 8C, wild-type and DpakA conidia were incubated in liquid medium at different temperatures ranging from 25 8C to 37 8C. The 20-h incubation was performed in a gradient thermocycler using 2 8C temperature increments from 25 8C to 37 8C. The media was then transferred to a microtitre tray and the rates of germination were measured microscopically by counting the numbers of germinating conidia (conidia with a visible germ tube) in a population of 100. Three independent experiments were performed. Mean and standard error of the mean values were calculated using GraphPad Prism3.