Characterization of an Nmr Homolog That Modulates GATA Factor-Mediated Nitrogen Metabolite Repression in Cryptococcus neoformans

Nitrogen source utilization plays a critical role in fungal development, secondary metabolite production and pathogenesis. In both the Ascomycota and Basidiomycota, GATA transcription factors globally activate the expression of catabolic enzyme-encoding genes required to degrade complex nitrogenous compounds. However, in the presence of preferred nitrogen sources such as ammonium, GATA factor activity is inhibited in some species through interaction with co-repressor Nmr proteins. This regulatory phenomenon, nitrogen metabolite repression, enables preferential utilization of readily assimilated nitrogen sources. In the basidiomycete pathogen Cryptococcus neoformans, the GATA factor Gat1/Are1 has been co-opted into regulating multiple key virulence traits in addition to nitrogen catabolism. Here, we further characterize Gat1/Are1 function and investigate the regulatory role of the predicted Nmr homolog Tar1. While GAT1/ARE1 expression is induced during nitrogen limitation, TAR1 transcription is unaffected by nitrogen availability. Deletion of TAR1 leads to inappropriate derepression of non-preferred nitrogen catabolic pathways in the simultaneous presence of favoured sources. In addition to exhibiting its evolutionary conserved role of inhibiting GATA factor activity under repressing conditions, Tar1 also positively regulates GAT1/ARE1 transcription under non-repressing conditions. The molecular mechanism by which Tar1 modulates nitrogen metabolite repression, however, remains open to speculation. Interaction between Tar1 and Gat1/Are1 was undetectable in a yeast two-hybrid assay, consistent with Tar1 and Gat1/Are1 each lacking the conserved C-terminus regions present in ascomycete Nmr proteins and GATA factors that are known to interact with each other. Importantly, both Tar1 and Gat1/Are1 are suppressors of C. neoformans virulence, reiterating and highlighting the paradigm of nitrogen regulation of pathogenesis.


Introduction
With over 1,500,000 species estimated to be present in the Earth's biosphere, the kingdom Fungi consists of a spectacularly large group of eukaryotes [1]. To successfully thrive in their broad range of niches, many fungal species have evolved mechanisms that enable them to utilize a wide variety of nitrogen sources. The expression of permease and catabolic enzyme-encoding genes needed for the scavenging of most nitrogenous compounds requires activation by global transcription factors belonging to the GATA family. These nitrogen regulatory GATA factors are conserved throughout the phyla Ascomycota and Basidiomycota. In the model ascomycetes, two positively acting GATA factors GLN3 and GAT1 are encoded in the genome of the yeast Saccharomyces cerevisiae while single positively acting factors nit-2 and areA are encoded in the genomes of the moulds Neurospora crassa and Aspergillus nidulans, respectively [2,3,4,5,6,7,8]. Loss-of-function mutations in these ascomycete GATA genes result in an inability to utilize a diverse array of nitrogen sources apart from the readily assimilated and hence generally preferred ammonium or glutamine [9]. Thus, both ammonium and glutamine are metabolites that likely trigger nitrogen metabolite/catabolite repression, resulting in the generation of signals that antagonize activation of secondary (nonpreferred) nitrogen gene expression by GATA factors [10,11,12].
Although GATA factors are employed to globally control nitrogen metabolism in S. cerevisiae, N. crassa and A. nidulans, there are compelling differences in key aspects of the molecular circuitry that underlies the regulation of these transcription factors in different fungal species. Studies thus far have shown that GATA factor activity is regulated in response to nitrogen availability in the surroundings and/or the nitrogen status of the cell. In S. cerevisiae, transcriptional activation of GAT1 is cross-regulated by both the positively acting factor Gln3 and negatively acting Dal80 [13]. The functions of Gln3 and Gat1 are also regulated posttranscriptionally via protein subcellular localization, through phosphorylation and interaction with the prion-forming glutathione S-transferase Ure2 [14,15,16,17].
In N. crassa, the function of Nit2 is instead regulated via interaction with a co-repressor protein known as Nmr1 (Nitrogen metabolic regulation 1) [18]. Loss-of-function mutations in nmr-1 result in derepression of a range of secondary nitrogen catabolic genes under normally repressing conditions (nitrogen sufficient conditions, e.g. in the presence of ammonium and/or glutamine) [19]. Unlike S. cerevisiae Ure2, there is no evidence suggesting that the structurally unrelated N. crassa Nmr1 affects Nit2 subcellular localization. Rather, Nmr1 likely exerts its effect by modulating the trans-activation function of Nit2 by interfering with its DNA binding activity [20]. Like N. crassa Nit2, the function of A. nidulans AreA is also regulated by an Nmr ortholog, NmrA [12,21,22,23]. In addition, AreA activity is further controlled by autogenous regulation and control of transcript stability mediated through an element in the 39 untranslated region of the areA mRNA [23,24,25]. These pioneering studies of nitrogen metabolism in S. cerevisiae, N. crassa and A. nidulans have played a crucial role in our current understanding of gene regulation in eukaryotes.
Over the past decade, interest in nitrogen regulation has expanded to encompass a number of important human fungal pathogens, implicating gene regulation by nitrogen availability in virulence. For example, during nitrogen limitation, Candida albicans dimorphic transition from the budding yeast to filamentous growth form that facilitates tissue invasion is dependent on the Mep2 ammonium permease, whose expression is regulated by the GATA factors Gln3 and Gat1 [26,27]. Both gln3D and gat1D mutants exhibit reduced virulence in a murine model of disseminated candidiasis [28,29]. Likewise, the GATA factor AreA enables nutritional versatility that is a key attribute influencing the ability of Aspergillus fumigatus to cause disease [30]. Consistent with this notion, the AreA ortholog in Penicillium marneffei has recently been proposed to contribute to pathogenicity by regulating the production of extracellular proteases that are potential virulence factors [31]. Impact of nitrogen regulation on virulence is not merely limited to members of the Ascomycota but is also observed in the basidiomycete Cryptococcus neoformans, a species that causes lifethreatening meningoencephalitis predominantly in immunocompromised individuals [32]. For instance, apart from nitrogen catabolism, the GATA factor Gat1/Are1 regulates multiple virulence attributes including infectious basidiospore production, capsule biosynthesis, high temperature growth and melanin pigment formation [33,34].
Gaining insights into the molecular mechanism governing the regulation of GATA factor activity is therefore important for the understanding of how these pathogenic fungi establish disease in a mammalian host. In C. albicans, Gln3 is proposed to be regulated by an Ure2-like mechanism, similar to other closely related yeasts [35,36]. In contrast, AreA of both A. fumigatus and P. marneffei are likely to be regulated in an Nmr-like fashion, similar to the related moulds [31,37]. A recent study by Jiang et al. suggests that the yeast C. neoformans may follow this previously mould-specific paradigm [38]. The potential Nmr homolog Tar1 (Temperature associated repressor 1) was first identified in C. neoformans through the overproduction of melanin at 37uC in a clone from a random insertional mutagenesis library [38]. The expression of TAR1 was induced at high temperature (37uC), and Tar1 was found to negatively regulate LAC1-encoded laccase that catalyses the formation of melanin in C. neoformans [38]. Like N. crassa Nmr1 and A. nidulans NmrA, Tar1 harbours a predicted canonical Rossmann fold motif that is also found in other co-repressors such as S. cerevisiae Gal80 and mammalian CtBP [21,38,39,40,41,42,43].
As part of an ongoing effort to elucidate the global nitrogen regulatory circuit in the most clinically prevalent form of C. neoformans (var. grubii strain H99), we have investigated the genetic and physical interactions between Tar1 and the GATA factor Gat1/Are1. In addition, we have examined the role of Tar1 in virulence factor expression and pathogenesis. Overall, our study provides evidence of divergence between different fungal species in the evolution of Nmr-associated proteins, and demonstrates the importance of Tar1 in modulating C. neoformans virulence.

Construction and complementation of C. neoformans mutant strains
All primers and plasmids used in this study are listed in Table  S2 and S3, respectively. Gene deletion mutants were created using overlap PCR and biolistic transformation as described previously [54]. Briefly, to construct the tar1D mutant strain in the H99 background, the 1,067 bp TAR1 (CNAG04934.2) coding sequence was replaced with the neomycin phosphotransferase II-encoding selectable marker NEO using a construct created by overlap PCR combining a ,1 kb fragment upstream the TAR1 start codon, the NEO marker and a ,1 kb fragment downstream the TAR1 stop codon. Strain H99 genomic DNA and plasmid pJAF1 were used as PCR templates [55]. The construct was transformed into C. neoformans cells via particle bombardment and transformants selected on YPD plates supplemented with G418. A similar approach was adopted to delete TAR1 in the H99 gat1/are1::NEO and wild-type KN99a strains using the NAT selectable marker from pCH233, with transformants selected on YPD supplemented with nourseothricin. Deletion of TAR1 was confirmed by diagnostic PCR and Southern blot [56]. To complement the H99 tar1D mutant, the TAR1 gene including ,1 kb promoter and terminator was amplified from genomic DNA using high fidelity PCR, cloned into pCR2.1-TOPO (Invitrogen) to give pIRL25, and sequenced. The TAR1 fragment of pIRL25 was then subcloned into pCH233, creating the complementation construct pIRL26. pIRL26 was subsequently linearised and biolistically transformed into the tar1D mutant. Stable transformants were selected on YPD supplemented with nourseothricin and complemented strains containing a single copy of the wild-type TAR1 gene were identified by Southern blot.
Quantitative real-time PCR C. neoformans strains were grown in YNB or L-DOPA suppflemented with 10 mM of the specified nitrogen source and shaken at 30 or 37uC for 16 hr. For nitrogen starvation cultures, ammonium-grown cells were gently centrifuged at 1,000 rpm for 3 min, the loose pellets washed with YNB, and cells transferred to fresh YNB medium lacking a nitrogen source for an additional 4 hr. Overnight cultures were harvested, cell pellets frozen and lyophilized, total RNA isolated using TRIzol reagent (Invitrogen) and cDNA generated using the SuperscriptIII First-Strand Synthesis System (Invitrogen). Primers for genes PUT1 (CNAG02049.2), GAT1/ARE1 (CNAG00193.2), TAR1 (CNAG04934.2), LAC1 (CNAG03465.2) and LAC2 (CNAG03464.2) were designed to span exon-exon boundaries and tested to verify that they bind specifically to cDNA but not genomic DNA. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green Supermix (Applied Biosystems) and an Applied Biosystems 7900HT Fast Real Time PCR System with the following cycling conditions: denaturation at 95uC for 10 min, followed by amplification and quantification in 45 cycles at 95uC for 15 sec and 60uC for 1 min, with melting curve profiling at 95uC for 2 min, 60uC for 15 sec and 95uC for 15 sec. Dissociation analysis confirmed the amplification of a single PCR product for each primer pair and an absence of primer dimer formation. Relative gene expression was quantified using SDS software 1.3.1 (Applied Biosystems) based on the 2 2DDCT method [57]. The housekeeping actin-encoding gene ACT1 was used as a control for normalization. One-way analysis of variance was performed using the unpaired, two-tailed t test in GraphPad Prism Version 5.0c. P values of ,0.05 were considered statistically significant.
Nitrogen utilization, toxic analog sensitivity, melanization, high temperature growth and capsule assays Starter C. neoformans cultures were prepared by growth in YPD at 30uC overnight with shaking, diluted to OD 595 nm = 0.05 in water, then further diluted tenfold in series. Each diluted cell suspension was then spotted onto YPD or YNB, L-DOPA, norepinephrine and caffeic acid medium supplemented with the specified nitrogen source or toxic analog. Results were imaged after 2-3 days incubation at 30uC (nitrogen utilization and toxic analog sensitivity assays), or both 30 and 37uC (melanization and high temperature growth assays). For capsule assays, cells were scrapped off the YNB plates, stained with India ink (Becton Dickinson), and visualized under a ZEISS Axioplan 2 epifluorescent/light microscope.

C. elegans killing assays
Starter cultures of C. neoformans strains were prepared by growth in YPD at 30uC overnight with shaking. 10 mL overnight cultures were spread onto both brain-heart infusion (BHI) (Becton Dickinson) and pigeon guano agar plates (35 mm), and incubated at 25uC overnight. ,50 young adult C. elegans worms were then transferred from a lawn of E. coli OP50 on NGM to BHI and pigeon guano medium-grown C. neoformans [34,61]. Plates were incubated at 25uC and worms examined for viability at 24 hr intervals using a dissecting microscope, with worms that did not respond to a touch with a platinum wire pick considered dead. Each experimental condition was performed in triplicate. Survival was plotted against time, and P values were calculated by plotting a Kaplan-Meier survival curve and performing a log-rank (Mantel-Cox) test using Graphpad Prism Version 5.0c. P values of ,0.05 were considered statistically significant.

Murine inhalation model of cryptococcosis
For murine virulence assays, C. neoformans were used to infect 6 weeks old female BALB/c mice by nasal inhalation [62]. For every tested strain, ten mice were each inoculated with a 50 mL drop containing 5610 5 cells. Mice were weighed before infection and daily thereafter; animals were sacrificed using CO 2 inhalation once their body weight had decreased to 80% of the pre-infection weight. Survival was plotted against time, and P values were calculated by plotting a Kaplan-Meier survival curve and performing a log-rank (Mantel-Cox) test using Graphpad Prism Version 5.0c. P values of ,0.05 were considered statistically significant.

Ethics statement
This study was carried out in strict accordance with the recommendations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes by the National Health and Medical Research Council. The protocol was approved by the Molecular Biosciences Animal Ethics Committee of The University of Queensland (AEC approval number: SCMB/008/11/UQ/ NHMRC). Infection was performed under methoxyflurane anaesthesia, and all efforts were made to minimize suffering through adherence to the Guidelines to Promote the Wellbeing of Animals Used for Scientific Purposes as put forward by the National Health and Medical Research Council.

Tar1 is not required for nitrogen source utilization
Although the genome of VNI strain H99 of C. neoformans contains multiple open reading frames encoding putative GATA factors, only Gat1/Are1 regulates nitrogen catabolism [34]. An equivalent nitrogen regulatory system that is dependent on a single positively acting GATA factor also occurs in A. nidulans and N. crassa where AreA and Nit2, respectively, have been extensively characterized [2,3,8]. Like the ascomycete moulds, a predicted Nmr homolog Tar1 has been identified in C. neoformans but curiously, Jiang et al. have suggested that Tar1 plays a negative role in nitrogen metabolism as indicated by the tar1D mutants faster growth on potassium nitrate as compared to wild-type [38]. This observation is counterintuitive as the inability of C. neoformans to utilize nitrate is a classic trait that has long been used as a diagnostic tool to identify this fungus; our recent bioinformatic and phenotypic analyses also supported this lack of nitrate utilization [34,63,64].
A set of deletion mutants lacking TAR1, GAT1/ARE1 or both was therefore created via homologous recombination in strain H99 to investigate if Tar1 plays a regulatory role in nitrogen utilization ( Figure 1). All three generated mutants were viable and had a growth rate indistinguishable from wild-type on rich undefined YPD medium. However, on YNB defined medium supplemented with a panel of different sole nitrogen sources including ammonium, amino acids, purines, nitrate or nitrite, both the gat1/are1D and double gat1/are1D tar1D mutants only had the ability to proliferate on proline. This result is consistent with previous studies indicating that the gat1/are1D mutant is unable to utilize ammonium or glutamine but is able to consume proline, a marked difference in phenotype compared to well-characterized ascomycete GATA factor mutants [33,34]. On the other hand, the tar1D mutant displayed wild-type growth on all tested nitrogenous compounds except nitrate and nitrite, which all strains were unable to grow on. Together, our results indicate that unlike Gat1/Are1, Tar1 is not required for nitrogen source utilization. Additionally, the similar nitrogen utilization phenotypes displayed by the gat1/are1D and double gat1/are1D tar1D mutants suggest that if Tar1 does indeed play a regulatory role in these nitrogen catabolic pathways, Gat1/Are1 functions downstream of Tar1.

Tar1 modulation of nitrogen metabolite repression at the phenotypic level
Gat1/Are1 has been shown to play a role in nitrogen metabolite repression, a regulatory mechanism that enables preferential utilization of readily assimilated nitrogen sources such as ammonium and glutamine [34]. We sought to determine if Tar1 is also functionally associated with this metabolic response. We note that Jiang et al. have previously assessed the existence of inappropriate derepression of secondary nitrogen catabolism in the tar1D mutant by growing the strain solely on potassium chlorate (a toxic analog of potassium nitrate) in the absence of a preferred nitrogen source [38]. However, such sensitivity assays used to determine constitutive expression of non-preferred nitrogen structural genes that are subjected to nitrogen metabolite repression could only be interpreted if a repressing nitrogen source is simultaneously present [21,23].
We therefore tested the tar1D mutants sensitivity to thiourea (a toxic analog of urea) in the presence of ammonium or glutamine ( Figure 2A). Like ascomycete nmrD mutants that exhibit partially derepressed phenotypes due to derepression of the urea permease, the tar1D mutant displayed a slight increase in sensitivity relative to wildtype while the negative control gat1/are1D and double gat1/are1D tar1D mutants were unable to proliferate due to their inability to utilize these nitrogen sources [65]. This result suggests that the tar1D mutant is metabolizing thiourea to a greater extent than wild-type, despite the simultaneous presence of preferred nitrogen sources. Complementation of the tar1D mutant subsequently restored the wild-type phenotype ( Figure S1). Together, these results indicate that Tar1 plays a negative role in the regulation of genes involved in utilization of more complex nitrogen sources. We also tested sensitivity to potassium chlorate in the presence of ammonium or glutamine and found that the tar1D mutant grew to the same extent as wild-type, consistent with C. neoformans lacking nitrate reductase needed to catabolize potassium chlorate (Figure 2A) [9].
The S. cerevisiae Dal80 and Gzf3, and A. nidulans AreB proteins are negatively acting GATA factors involved in nitrogen regulation [66,67]. In C. neoformans, six additional GATA factors have been identified: Gat201 and Gat204 work in concert to prevent phagocytosis by macrophages, Bwc2 regulates cell fusion and hyphal development in response to blue light, Cir1 controls iron acquisition, while CNAG04263.2 and CNAG03401.2 have unknown functions [34,68,69,70,71]. To determine if any of these GATA factors play a negative role in nitrogen catabolism, we tested the gat201D, gat204D, bwc2D, cir1D, CNAG04263.2D and CNAG03401.2D mutants sensitivity to thiourea in the presence of ammonium or glutamine (not shown). The six GATA deletion mutants exhibited wild-type sensitivity, suggesting that negatively acting nitrogen regulatory GATA factors are likely to be a unique evolutionary feature of the ascomycetes. Consistent with this notion, all seven C. neoformans GATA factors including Gat1/Are1, lack putative leucine zippers that are present in the negative acting S. cerevisiae Dal80 and Gzf3, or A. nidulans AreB. Overall, our analysis thus far revealed that only Tar1 plays a negative role in secondary nitrogen catabolism.

Tar1 modulation of nitrogen metabolite repression at the transcriptional level
We sought to provide support for our sensitivity assays and prove that Tar1 is implicated for the response to nitrogen metabolite repression in a range of metabolic pathways. We therefore analysed the transcriptional regulation of the proline oxidase-encoding gene PUT1 using qRT-PCR on RNA extracted from the wild-type, tar1D, gat1/are1D and double gat1/are1D tar1D mutant strains grown in YNB supplemented with proline or proline plus ammonium ( Figure 2B). The proline catabolic pathway was chosen to be examined as this traditionally non-preferred nitrogen source supports the most robust growth in the gat1/are1D and double gat1/are1D tar1D mutants, thus enabling the isolation of RNA from a defined medium. Furthermore, PUT1 has previously been shown to be sensitive to nitrogen metabolite repression, providing an ideal condition to test how Tar1 and Gat1/ Are1 are affiliated to this regulatory control mechanism [34].
As expected, high levels of PUT1 expression were observed when the wild-type strain was cultured in proline as the sole nitrogen source, and this upregulation was largely abolished (,30fold difference) with the simultaneous presence of ammonium. In the tar1D mutant, intermediate levels of PUT1 mRNA were observed in the presence of proline, and this upregulation was only partially reduced (,5-fold difference) when ammonium was simultaneously present. While the 2-3-fold more PUT1 transcript produced by the tar1D mutant relative to wild-type when grown in proline plus ammonium was anticipated, the 2-3-fold less PUT1 expression of the tar1D mutant compared to wild-type in the sole presence of proline was surprising (WT nitrogen metabolite repression sensitivity vs tar1D nitrogen metabolite repression sensitivity, P = 0.0195). The former indicates that PUT1 transcription in the tar1D mutant was not completely repressed in the presence of a preferred nitrogen source, and reiterates the fact that Tar1 prevents the expression of secondary nitrogen structural genes under repressing conditions. The latter, intriguingly, suggests that Tar1 also plays a positive role in the activation of secondary nitrogen structural genes under non-repressing conditions, despite the fact that the tar1D mutant exhibited indistinguishable phenotypic growth rate relative to wild-type on various nitrogen sources (Figure 1). Tar1 therefore contributes to the expression of nitrogen catabolism associated genes, however, it does not overcome the requirement for Gat1/Are1.
The expression of GAT1/ARE1, but not TAR1, is regulated in response to nitrogen availability In A. nidulans, NmrA modulation of AreA activity is finely tuned; this well-coordinated control mechanism begins at the transcriptional level as expression of areA and nmrA are inversely regulated in response to the quality of the nitrogen source available [37]. To determine if a similar phenomenon occurs in C. neoformans, we analysed the transcriptional regulation of both GAT1/ARE1 and TAR1 using qRT-PCR on RNA extracted from wild-type H99 grown in YNB supplemented with different nitrogen sources ( Figure 3). During growth in ammonium, a low level of GAT1/ ARE1 mRNA was observed, while an elevated level (,7-fold) of GAT1/ARE1 transcript was present when grown in proline (Pro vs NH 4 , P = 0.0003). A marked increase (.10-fold) in GAT1/ARE1 transcript level was seen when cells were starved for nitrogen (N free vs NH 4 , P = 0.003). In stark contrast to the expression profile of GAT1/ARE1, the level of TAR1 transcript was relatively unaltered when cultured under these different nitrogen conditions. Overall, our qRT-PCR data indicates that GAT1/ARE1 expression is transcriptionally induced in response to nitrogen limitation, whereas TAR1 transcription is unaffected by nitrogen availability.
Tar1 plays a dual function in positive and negative regulation of GAT1/ARE1 expression according to the nitrogen source present Given that Gat1/Are1 is involved in the utilization of most nitrogen sources, we sought to determine if Tar1 regulates GAT1/ ARE1 expression, and vice versa. We first analysed the transcriptional regulation of both GAT1/ARE1 and TAR1 using qRT-PCR on RNA extracted from the wild-type, tar1D and gat1/are1D mutant strains grown in YNB supplemented with proline ( Figure 4A). As expected, no GAT1/ARE1 or TAR1 transcripts were detected in the gat1/are1D and tar1D mutants, respectively, validating our diagnostic PCR and Southern blot analyses for confirmation of gene deletion. Consistent with nitrogen independence of TAR1 expression, the gat1/are1D mutant produced wildtype levels of the TAR1 transcript. On the other hand, the tar1D mutant expressed ,3-fold less GAT1/ARE1 mRNA compared to wild-type (WT vs tar1D, P = 0.0008). This result provides a plausible explanation for why the tar1D mutant transcribed less PUT1 mRNA relative to wild-type in the sole presence of proline ( Figure 2B), and indicates that Tar1 positively regulates GAT1/ ARE1 expression under non-repressing conditions.
To provide support for the model that Tar1 plays a dual function in positive and negative regulation of nitrogen catabolism, we analysed the transcriptional regulation of GAT1/ARE1 using qRT-PCR on RNA extracted from the wild-type and tar1D mutant strains grown in YNB supplemented with ammonium ( Figure 4B). As expected, the wild-type strain produced low levels of GAT1/ ARE1 transcripts under such nitrogen sufficient conditions, while the tar1D mutant transcribed ,3-fold more GAT1/ARE1 mRNA relative to wild-type (WT vs tar1D, P = 0.0481). This result indicates that Tar1 negatively regulates GAT1/ARE1 expression under repressing conditions. Collectively, Tar1 can function to elicit both activation and repression of GAT1/ARE1 autoregulation according to the quality of the nitrogen source present.
Gat1/Are1 lacks the highly conserved extreme C-terminus domain of A. nidulans AreA and N. crassa Nit2 known to be involved in Nmr recognition Our phenotypic and transcriptional data suggest that the molecular mechanism of action of Tar1 may operate in a fashion similar to ascomycete Nmr homologs. In A. nidulans and N. crassa, certain mutations of the GATA DNA-binding domain or extreme Figure 1. Tar1 is not required for nitrogen source utilization. Tenfold spot dilution assays for nitrogen utilization showed that the tar1D mutant exhibited wild-type growth on YNB supplemented with ammonium, glutamine, glutamate, asparagine, proline, glycine, phenylalanine, leucine, uric acid, urea and creatinine (10 mM each nitrogen source). In contrast, both the gat1/are1D and double gat1/are1D tar1D mutants were only able to proliferate on proline as the sole nitrogen source. All tested strains were unable to grow on 7% potassium nitrate or sodium nitrite. doi:10.1371/journal.pone.0032585.g001 Figure 2. Tar1 plays a role in modulating nitrogen metabolite repression. (A) Tenfold spot dilution assays for nitrogen utilization showed that the tar1D mutant exhibited slight sensitivity to 5 mM thiourea when compared to wild-type in the simultaneous presence of 10 mM ammonium or glutamine. (B) cDNA from wild-type H99 and the mutant strains grown in YNB supplemented with proline or proline plus ammonium (10 mM each nitrogen source) were amplified via qRT-PCR using primers against the proline oxidase-encoding gene PUT1 and the control gene ACT1. In the wildtype strain, PUT1 expression was significantly increased in the presence of proline but upregulation was largely abolished when ammonium was also present. In the tar1D mutant, PUT1 transcription was intermediate in the presence of proline and upregulation was only partially reduced when ammonium was simultaneously present. Nitrogen metabolite repression sensitivity of PUT1 in the wild-type strain statistically differs from that in the tar1D mutant (* denotes P,0.05). In the gat1/are1D and double gat1/are1D tar1D mutants, PUT1 expression was low under both tested growth conditions. Error bars represent standard errors across three biological replicates. doi:10.1371/journal.pone.0032585.g002 C-terminus of AreA and Nit2 lead to partially derepressed phenotypes under nitrogen sufficient conditions similar to that observed for nmrD mutants, suggesting that these regions are critical for interaction with Nmr proteins and modulation of GATA factor transcriptional activity [18,23].
However, global alignment of C. neoformans Gat1/Are1 showed little overall sequence conservation to AreA or Nit2 (15 and 16% identity, respectively) ( Figure S2). Importantly, the extreme Cterminus of AreA and Nit2 known to be critical for modulating activities by the co-repressors NmrA and Nmr1, respectively, is absent in Gat1/Are1 ( Figure 5A and E) [18,21,23]. In fact, the last nine and 12 C-terminus residues of A. nidulans AreA are identical to N. crassa Nit2 and Penicillium chrysogenum GATA factor Nre, respectively ( Figure 5E and not shown) [2,23,72]. Nonetheless, three notable blocks of conservation do exist between Gat1/Are1 and AreA or Nit2: two regions of unknown function (in AreA and Nit2, these two regions lie near the N-terminus while in Gat1/ Are1, they lie further towards the middle or C-terminus region of the protein) ( Figure 5A, B and C), and the GATA DNA-binding domain consisting of a zinc finger motif followed by an adjacent basic region ( Figure 5A and D). The zinc finger domain has been shown to play a role in the DNA-binding activity of both AreA and Nit2, and the same scenario is likely occurring in C. neoformans given the high level of conservation seen among the zinc fingers of Gat1/Are1, AreA and Nit2 [2,3]. In summary, one of the two regions of ascomycete GATA factors known to be important for interaction with Nmr proteins is absent in Gat1/Are1: the Cryptococcus GATA DNA-binding domain is conserved but the extreme C-terminus has diverged.

Tar1 lacks the conserved C-terminus domain of A. nidulans NmrA known to be important for interaction with AreA
We next analysed the sequences of the Nmr homologs of A. nidulans, N. crassa and C. neoformans. In A. nidulans, the conserved ahelix at the C-terminus of NmrA is also known to be critical for interaction with AreA [40,41]. Given that the sequence of N. crassa Nmr1 is highly conserved with A. nidulans NmrA, it is likely that both these proteins share similar structural features [21,43]. In contrast, global alignment of C. neoformans Tar1 revealed only moderate overall sequence conservation to both NmrA and Nmr1 (19 and 16% identity, respectively) ( Figure S3). Tar1 harbours the residues GlyXXGlyXXGly that are predicted to form a Rossmann fold in the N-terminus, while NmrA and Nmr1 contain a slightly diverged canonical Rossmann motif AsnXXGlyXXAla [38,41,73]. However, Tar1 lacks the long C-terminus region of NmrA and Nmr1 that is predicted to play a role in interaction with GATA factors. Based on bioinformatics conducted on these GATA factors and Nmr proteins, it was not apparent whether Tar1 is likely to physically interact with and inhibit the function of Gat1/Are1.

Interaction between Tar1 and Gat1/Are1 was not detectable in vivo
The direct interaction of N. crassa Nit2 and Nmr1 was originally shown via yeast two-hybrid experiments. The DNA-binding domain or extreme C-terminus of Nit2 fused to the Gal4 activation domain (AD) interacted with an Nmr1-Gal4 DNAbinding domain (BD) fusion protein, with stronger interaction detected when both the DNA-binding domain and C-terminus regions of Nit2 were present [20]. To investigate if a similar situation occurs in C. neoformans, we also performed a yeast twohybrid assay to test if protein-protein interaction occurs between Gat1/Are1 and Tar1 (Table S4). We fused the first-third (nucleotides 1-1,284), second-third (nucleotides 1,285-2,562; contains the first conserved motif of unknown function) and final-third (nucleotides 2,563-3,834; contains the second conserved motif of unknown function, GATA DNA-binding domain and C-terminus) GAT1/ARE1 fragments, as well as full-length GAT1/ARE1 and TAR1 cDNA, to both the GAL4 AD and BD. Every possible combination of the clones were then expressed in S. cerevisiae AH109 in which the GAL promoter regulates ADE2, HIS3 and lacZ reporter genes. If any protein-protein interaction did exist, it would likely be the final-third Gat1/Are1 fragment and/or full-length Gat1/Are1 (both contains the conserved GATA DNAbinding domain) that may interact with Tar1.
In AH109 strains expressing the first-third fragment or fulllength Gat1/Are1-Gal4 BD fusion protein, auto-activation of the bait occurred indicating that an activation domain is present within the nucleotides 1-1,284 of GAT1/ARE1. However, in AH109 strains expressing any of the three individual fragments or full-length Gat1/Are1-Gal4 AD fusion protein and Tar1-Gal4 BD fusion protein, cells failed to grow in the absence of adenine and histidine, and did not produce b-galactosidase activity. Our bioinformatics coupled with yeast two-hybrid data therefore suggest that no protein-protein interaction is likely occurring between Gat1/Are1 and Tar1 when expressed in the nucleus of the hemiascomycete S. cerevisiae. Additionally, our two-hybrid Figure 3. The expression of GAT1/ARE1, but not TAR1, is regulated in response to nitrogen availability. cDNA from wildtype H99 grown in YNB supplemented with ammonium, proline (10 mM each nitrogen source) or no nitrogen source were amplified via qRT-PCR using primers against GAT1/ARE1, TAR1 and the control gene ACT1. In the presence of a preferred nitrogen source (ammonium), the expression of GAT1/ARE1 was low but in the presence of a traditionally non-preferred nitrogen source (proline), or under nitrogen starvation conditions, GAT1/ARE1 transcription was significantly upregulated (** denotes P,0.01, *** denotes P,0.001). In contrast, TAR1 expression was relatively unaltered under these different nitrogen growth conditions. Error bars represent standard errors across three biological replicates. doi:10.1371/journal.pone.0032585.g003 analysis provides no indication of Gat1/Are1 or Tar1 homodimerization.
Gat1/Are1 negatively regulates laccase-encoding LAC1 expression at 37uC Jiang et al. originally identified Tar1 through its negative role in antioxidant melanin production at 37uC on norepinephrinecontaining asparagine medium, which eventually led to their finding that Tar1 regulates laccase-encoding LAC1 expression [38]. The two paralogs LAC1 and LAC2 encode laccases that catalyse the formation of melanin in C. neoformans, with Lac1 being the main enzyme responsible [45,74,75]. Interestingly, our group has also observed that Gat1/Are1 negatively regulates melanin production at 37uC on L-DOPA-containing proline medium [34]. We therefore investigated the genetic epistasis between TAR1 and GAT1/ARE1regulated production of melanin and expression of laccases.
Given that the gat1/are1D and double gat1/are1D tar1D mutants are unable to utilize asparagine as a nitrogen source, we analysed melanin production of the deletion mutants on L-DOPA (proline) agar grown at both 30 and 37uC ( Figure 6A). All strains melanized robustly when cultivated at 30uC. However, at 37uC, the gat1/ are1D and double gat1/are1D tar1D mutants produced more melanin than the wild-type and tar1D mutant strains, reiterating the observation that the gat1/are1 mutation is dominant over the tar1 mutation but disagreeing with those seen by Jiang et al. [38]. We then analysed the transcriptional regulation of LAC1 and LAC2 using qRT-PCR on RNA extracted from the wild-type and mutant strains grown in L-DOPA (proline) at both 30 and 37uC ( Figure 6B). As previously reported, transcription of LAC1 was severely decreased (50-100-fold) at 37uC compared to 30uC [76]. Consistent with our melanin phenotypic plate assays, both the gat1/are1D and double gat1/are1D tar1D mutants transcribed more (,2-fold) LAC1 mRNA as compared to the wild-type or tar1D mutant strains at 37uC (WT vs gat1/are1D, P = 0.0003; WT vs double gat1/are1D tar1D, P = 0.0131). No significant difference in the expression of LAC1 at 30uC or LAC2 at 30 and 37uC was observed among the tested strains. Altogether, our data indicates that Gat1/Are1, but not Tar1, represses melanin production and LAC1 expression at human body temperature when L-DOPA is used as the laccase substrate.
The different substrate used for melanin production may have resulted in conflicting observations made by Jiang et al. and our group [38]. We therefore tested melanin production of wild-type H99 and three independent H99 tar1D mutants, as well as the isogenic wild-type MATa strain KN99a and six independent KN99a tar1D mutants, at both 30 and 37uC on additional laccase substrates: norepinephrine and caffeic acid, supplemented with asparagine as the nitrogen source ( Figure S4). Under all tested growth conditions, no visual difference in melanin production was observed between the wild-type and tar1D mutant strains. Therefore, in our hands, we are unable to reproduce the increased melanin production and LAC1 expression phenotypes of the tar1D mutant as described by Jiang et al. [38]. We speculate that the passage of H99 during subculture between laboratories may have possibly caused the phenotypic variation. In support of this notion, Figure 4. Tar1 plays both positive and negative roles in regulating GAT1/ARE1 transcription according to the nitrogen source available. (A) cDNA from wild-type H99, gat1/are1D and tar1D mutant strains grown in YNB supplemented with 10 mM proline were amplified via qRT-PCR using primers against GAT1/ARE1, TAR1 and the control gene ACT1. The expression of GAT1/ARE1 was significantly lower in the tar1D mutant compared to wild-type (*** denotes P,0.001). Error bars represent standard errors across three biological replicates. (B) cDNA from wild-type H99 and tar1D mutant strains grown in YNB supplemented with 10 mM ammonium were amplified via qRT-PCR using primers against GAT1/ARE1 and ACT1. The expression of GAT1/ ARE1 was significantly higher in the tar1D mutant compared to wildtype (* denotes P,0.05). Error bars represent standard errors across three biological replicates. doi:10.1371/journal.pone.0032585.g004 Morrow et al. have recently reported that various H99 subcultures have differing melanization abilities [77]. Additionally, the indistinguishable growth rate of the tar1D mutants in comparison to their wild-type H99 or KN99a counterparts on YPD medium at 37uC suggests that Tar1 does not play a role in high temperature growth.
Expanding our analysis on virulence factor expression, we examined the polysaccharide capsule that has an antiphagocytic function [78]. We grew the wild-type and tar1D mutant strains on YNB supplemented with creatinine, uric acid or urea that are known inducers of capsule formation, and found that both the wild-type and tar1D cells possess equally large capsule on each of these individual medium (not shown) [34]. Given that nitrogen metabolite repression plays a role in the regulation of capsule production, we also examined the capsule of both strains when grown on the same capsule-inducing media but with the simultaneous presence of ammonium (not shown) [34]. Under these growth conditions, the wild-type cells should possess completely repressed (small) capsule since ammonium is a poor inducer of capsule formation, while we were predicting that the tar1D cells would possess partially repressed (intermediate) capsule due to inappropriate depression of secondary nitrogen (creatinine, uric acid, urea) catabolism [34]. Unexpectedly, the capsule size was equally small and indistinguishable between the wild-type and tar1D mutant strains under such nitrogen metabolite repression conditions. The subtle role of Tar1 in modulating nitrogen metabolite repression may explain why the tar1D mutants capsule was not detectably derepressed, as can be seen in the toxic thiourea assays where the tar1D mutant only exhibited slight sensitivity relative to wild-type (Figure 2A). Taken together, Tar1 does not appear to affect well-established virulence factors including, but not limited to, melanin production, growth at human body temperature and capsule biosynthesis.
Tar1 is not required for killing of C. elegans but modestly represses virulence in a murine inhalation model of cryptococcosis Our overarching interest in C. neoformans lies in better understanding its pathogenicity in an animal host. We therefore investigated the role of Tar1 in pathogenesis by performing both in vitro and in vivo virulence assays. First, we conducted C. elegans killing assays using two different media: the standard BHI medium for nematode killing experiments, as well as pigeon guano medium to mimic the C. neoformans ecological niche ( Figure 7A). Under both growth conditions, killing of C. elegans by the tar1D [LT 50 (time for half of the worms to die) = 5 and 3 days, for BHI and pigeon guano medium, respectively] and double gat1/are1D tar1D mutants (LT 50 = 5 and 4 days, for BHI and pigeon guano medium, respectively) was not significantly different to that observed for wild-type (LT 50 = 6 and 3 days, for BHI and pigeon guano medium, respectively). The undiminished pathogenicity of the double gat1/are1D tar1D mutant is consistent with our previous work showing that Gat1/Are1 does not affect killing of C. elegans [34].
Since the C. elegans virulence assays were conducted at 25uC, we questioned if Tar1 is required for infection of a host at mammalian body temperature given that the TAR1 promoter contains putative heat shock sequence elements [38]. To test this hypothesis, we performed a murine inhalation model of cryptococcosis ( Figure 7B). Mice infected with the tar1D mutant succumbed to infection slightly faster (between 16 and 19 days post infection, median survival of 18 days) than mice infected with the wild-type strain (between 18 and 24 days post infection, median survival of 21.5 days) (WT vs tar1D, P = 0.0021). It should be noted that mice infected with the complemented tar1D+TAR1 strain did not progress to morbidity at the same rate as wild-type (between 17 and 21 days post infection, median survival of 18 days). However, the difficulty associated with the restoration of wild-type phenotype of the tar1D mutant in certain physiological aspect has previously been reported [38]. Alternatively, the role of Tar1 in virulence may be dependent on its expression levels, which could have been adversely affected by integration of TAR1 into a non-native locus. Mice infected with the double gat1/ are1D tar1D mutant also succumbed to infection slightly faster than mice infected with the wild-type strain, consistent with our previous work demonstrating that Gat1/Are1 modestly represses virulence during murine infection (between 15 and 22 days post infection, median survival of 19 days) (WT vs gat1/are1D tar1D, P = 0.0342) [34]. Together, these results suggest that both the tar1D and double gat1/are1D tar1D mutants are slightly more virulent than wild-type during in vivo infection. Hence, in addition to fine-tuning regulation of nitrogen catabolism, Tar1 also modulates pathogenesis in a mammalian host.

Discussion
Nitrogen starvation stimulates the initiation of the infection cycle of the ubiquitous C. neoformans, whereby sexual reproduction occurs, leading to the production of potentially infectious basidiospores [69,79,80]. This mating process is controlled by multiple signalling cascades including the high affinity ammonium permease Amt2, which is in turn regulated by the GATA factor Gat1/Are1 [34,81]. In addition to regulating nitrogen catabolism, Gat1/Are1 also impacts multiple virulence attributes such as melanin production, high temperature growth and capsule biosynthesis [33,34]. Therefore, the underlying mechanism regulating Gat1/Are1 activity in response to nitrogen availability is important for the understanding of the C. neoformans life cycle in the environment and successful proliferation during infection of a host.
In the model hemiascomycete S. cerevisiae, the function of the GATA factors Gln3 and Gat1 are negatively regulated by interaction with the prion-forming glutathione S-transferase Ure2 [14,15,16,17]. As in the system of the filamentous ascomycete A. nidulans, there is no genetic or molecular evidence for the existence of an Ure2 homolog in C. neoformans [82,83]. Although we have identified a putative protein sequence (CNAG04110.2) in the H99 genome which contains a glutathione S-transferase domain that shows weak overall sequence similarity to Ure2, CNAG04110.2 lacks several features unique to Ure2 including an N-terminus extension and loop region [84,85].
In A. nidulans and N. crassa, the function of the GATA factors AreA and Nit2 are negatively regulated by interaction with Nmr proteins that are unrelated to glutathione S-transferases [12,18,20,21,22,23,82,83]. Recently, the group of Jiang et al. identified the C. neoformans potential Nmr homolog Tar1 during their search for gene disruption mutants that hypermelanize at 37uC [38]. In this study, we further characterized Tar1 function from a different perspective based on our interest in regulation of nitrogen metabolism. Our findings indicate that Tar1 possesses several unique regulatory and functional features in comparison to A. nidulans NmrA and N. crassa Nmr1.
In A. nidulans, nmrA expression is low during nitrogen limitation but increases during nitrogen sufficiency [37]. The bZIP Figure 6. Gat1/Are1 negatively regulates melanin production and LAC1 expression at 376C. (A) Tenfold spot dilution assays for melanization showed that the gat1/are1D and double gat1/are1D tar1D mutants produce more melanin than the wild-type and tar1D mutant strains on L-DOPA medium supplemented with 10 mM proline at 37uC. (B) cDNA from wild-type H99 and the mutant strains grown in L-DOPA (10 mM proline) at 30 and 37uC were amplified via qRT-PCR using primers against the laccase-encoding genes LAC1 and LAC2, and the control gene ACT1. LAC1 expression was significantly higher in the gat1/are1D and double gat1/are1D tar1D mutants compared to wild-type or the tar1D mutant when strains were grown at 37uC (* denotes P,0.05, *** denotes P,0.001). Error bars represent standard errors across three biological replicates. doi:10.1371/journal.pone.0032585.g006 Figure 7. The tar1D mutant kills C. elegans as efficiently as wild-type, but exhibits modestly enhanced virulence in a murine host. (A) C. elegans infection: ,50 nematode worms were transferred to a lawn of wild-type H99, tar1D, tar1D+TAR1 or double gat1/are1D tar1D cells as the sole food source on both BHI and 1% pigeon guano medium, and survival was monitored at 24 hr intervals. There was no observable difference in C. elegans killing by all four strains on both media. (B) Mus musculus infection: 10 mice were each intranasally infected with either 5610 5 cells of wildtype H99, tar1D, tar1D+TAR1 or double gat1/are1D tar1D strains, and survival was monitored daily. Mice infected with the tar1D, tar1D+TAR1 and double gat1/are1D tar1D strains progress to morbidity slightly more rapidly than mice infected with the wild-type strain. doi:10.1371/journal.pone.0032585.g007 transcription factor MeaB activates the expression of nmrA, and MeaB homologs are found in all the available filamentous ascomycetes genome databases [37]. In contrast, TAR1 transcription is unaffected by nitrogen availability and our bioinformatic analyses indicate that the C. neoformans H99 genome does not encode an obvious homolog of MeaB. In addition, the promoter region of TAR1 lacks the conserved element, TTGCACCAT; in vitro studies have shown that A. nidulans MeaB binds to TTGCACCAT and this binding site is also present in the promoters of NmrA homologs in other filamentous ascomycetes [37]. Instead, Jiang et al. have reported that TAR1 expression is upregulated at high temperature (37uC), consistent with the fact that its promoter contains three TTC/GAA repeats that are putative binding sites for the heat shock factor [38].
However, Tar1 does contain the characteristic NAD/NADH dinucleotide binding motif, GlyXXGlyXXGly, found in members of the short-chain dehydrogenase/reductase family such as the negatively acting Gal80 of S. cerevisiae, which acts to block the activation domain of Gal4 required for transcription of galactoseinducible genes [42,86,87,88]. Like the negative transcription regulators A. nidulans NmrA and N. crassa Nmr1, Tar1 retains the evolutionary conserved function of Nmr proteins of preventing activation of secondary nitrogen catabolism when preferred nitrogenous compounds are present. However, the role of Tar1 in this regulatory aspect is quite subtle, indicative that additional factors are likely operating to modulate nitrogen metabolite repression or to repress Gat1/Are1 activity in C. neoformans. For example, in A. nidulans, the activity of AreA is also controlled through autogenous regulation and differential transcript stability [23,24,25].
Intriguingly, we also discovered a novel role of Tar1 in positively regulating the expression of the proline oxidaseencoding gene PUT1 under non-repressing conditions. The dual function of Tar1 in fine-tuning nitrogen catabolism appears to be mediated through modulation of GAT1/ARE1 transcription in response to the quality of the nitrogen source available ( Figure S5). Given that Tar1 is not required for nitrogen source utilization, we speculate that only basal-to-intermediate levels of GAT1/ARE1 transcripts are needed to efficiently activate secondary nitrogen catabolism under non-repressing conditions. The exact mechanism by which Tar1 regulates Gat1/Are1 activity remains unclear as no physical interaction could be detected amongst these two proteins. Since Tar1 does not appear to have a DNA-binding domain, this protein may possibly control the activity of an unknown co-factor(s)/accessory transcription apparatus that in turn influences GAT1/ARE1 autoregulation in response to nitrogen availability. Further work will be required to dissect the nature and origin of intracellular signals that govern the activity of Gat1/Are1.
Notwithstanding, although a plethora of information about nitrogen metabolism is known in A. nidulans and N. crassa, studies into the regulation of GATA factor and Nmr protein activity have had their fair share of controversies. For example, while Lamb et al. have previously suggested that the N-terminus is required for modulating derepression activity of A. nidulans AreA, Caddick and Arst later disagreed with this theory [89,90]. In more recent times, Lamb et al. demonstrated that the extreme nine C-terminus residues of AreA that are highly conserved across a range of filamentous ascomycete homologs does not affect the affinity for NmrA binding, contradicting the findings of Pan et al. and Xiao et al. [18,20,40]. Most recently, Wagner et al. asserted that nmrA expression in A. nidulans is not dependent on MeaB, an observation that contrasts those of Wong et al. [37,91]. Hence, the mechanism of action governing the activity of GATA factors and Nmr proteins in model ascomycetes, too, remain elusive.
Such research into nitrogen regulation is of immense interest as nitrogen source utilization influences key aspects of fungal biology including development, secondary metabolite production and pathogenesis. In the context of virulence, nitrogen regulated pathogenesis has been documented in clinically prevalent human pathogens such as C. albicans and A. fumigatus [28,29,30]. Our analysis of TAR1 and GAT1/ARE1 reiterated this dogma; both genes are suppressors of C. neoformans virulence. A parsimonious explanation as to why no opposing regulatory effects on pathogenesis were observed between the tar1D and gat1/are1D or double gat1/are1D tar1D mutants may relate to the scarce nutrient availability during in vivo infection, a condition in which Tar1 is predicted to positively regulate GAT1/ARE1 activity [34]. Nonetheless, we cannot rule out the possibility that Tar1 is not exclusively involved in regulation of nitrogen catabolism but may also control targets involved in a broader spectrum of metabolic processes. The fact that the TAR1 promoter contains putative heat shock elements supports this notion [38]. Using whole transcriptome sequencing, we are currently attempting to identify novel virulence and stress adaptation associated genes that are regulated by both Tar1 and Gat1/Are1 during infection of a mammalian host. Indeed, Kronstad et al. have recently highlighted transcriptional profiling and genetic studies as invaluable research tools, with the means to provide insights into C. neoformans adaptation to key features within a mammalian host environment including nitrogen availability [92].
In summary, there is a pressing need to combat infection from the killer fungus C. neoformans that is responsible for .625,000 deaths annually in predominantly AIDS patients [93]. Our study has provided deeper insights into the molecular mechanisms that wire the Cryptococcus global nitrogen regulatory circuit. Specifically, we provide an indication of how Tar1 controls Gat1/Are1 activity in response to changes in nitrogen availability, as would occur when C. neoformans leaves its purine rich ecological niche of pigeon guano to infect a human host which in comparison is nutrient limiting [94]. Understanding the intricate details of how Gat1/ Are1 function is pivotal, as candidate genes in several nitrogen catabolic pathways represent potential drug targets for therapeutic intervention in this important pathogen of humans. Figure S1 Complementation of toxic analog sensitivity phenotype to wild-type levels upon the re-introduction of TAR1 into the tar1D mutant. Tenfold spot dilution assays for nitrogen utilization showed that the tar1D+TAR1 strain exhibited wild-type growth on 5 mM thiourea plus 10 mM ammonium. (DOC) Figure S2 ClustalW multiple sequence alignment of A. nidulans AreA (XP_681936.1), N. crassa Nit2 (P19212.2) and C. neoformans Gat1/Are1 (CNAG00193.2). Identical amino acid residues are shaded dark grey while similar residues are shaded light grey. Gat1/Are1 shows little overall sequence conservation to AreA and Nit2 at the entire protein level. However, three notable blocks of conversation do exist between Gat1/Are1 and AreA or Nit2: two regions of unknown function (boxed in red and orange) and the GATA DNA-binding domain (boxed in blue). The second conserved motif of unknown function (boxed in orange) was identified through Pustell dot plot protein matrix. The extreme C-terminus domain of AreA and Nit2 (boxed in green) is highly conserved but that of Gat1/Are1 has diverged. Enlarged versions of all four domains are shown in Figure 5B, C, D and E. (DOC) Figure S3 ClustalW multiple sequence alignment of A. nidulans NmrA (AAC39442.1), N. crassa Nmr1 (P23762.2) and C. neoformans Tar1 (CNAG04934.2). Identical amino acid residues are shaded dark grey while similar residues are shaded light grey. Tar1 shows moderate overall sequence conservation to NmrA and Nmr1. The predicted Rossmann fold motif at the N-terminus is boxed in red. The long C-terminus region of NmrA and Nmr1 is absent in Tar1. (DOC) Figure S4 Multiple independently generated tar1D mutants all produced equal amount of pigment melanin in comparison to their wild-type H99 or KN99a counterparts. The tenfold spot dilution assays for melanization on L-DOPA, norepinephrine and caffeic acid agar (supplemented with 10 mM asparagine as the nitrogen source) were conducted at both 30 and 37uC. (DOC) Figure S5 Scheme representing the dual roles of Tar1 in modulating GAT1/ARE1 transcription that in turn influences PUT1 expression according to the nitrogen source available. In the presence of the traditionally nonpreferred proline, the Nmr homolog Tar1 positively regulates the transcription of the GATA factor-encoding gene GAT1/ARE1 that is required for induction of the proline oxidase-encoding gene PUT1. In the presence of the preferred ammonium, Tar1 negatively regulates GAT1/ARE1 transcription leading to reduce levels of PUT1 expression. (DOC)    Table S4 Interaction between Tar1 and Gat1/Are1 could not be detected in a yeast two-hybrid assay. S. cerevisiae AH109 was co-transformed with both the bait and prey constructs, and selected for growth on double dropout medium (-Leu -Trp). Interaction (denoted by + symbol) was assessed by growth on quadruple dropout medium (-Leu -Trp -His -Ade), and b-galactosidase activity. 2 symbol denotes no protein-protein interaction. A.A. denotes auto-activation. N.T. denotes not tested.

Supporting Information
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