A Novel Regulator Couples Sporogenesis and Trehalose Biogenesis in Aspergillus nidulans

Trehalose is a compatible osmolyte produced by bacteria, fungi, insects and plants to protect the integrity of cells against various environmental stresses. Spores, the reproductive, survival and infection bodies of fungi require high amounts of trehalose for long-term survival. Here, via a gain-of-function genetic screen, we identify the novel regulator VosA that couples the formation of spores and focal trehalose biogenesis in the model fungus Aspergillus nidulans. The vosA gene is expressed specifically during the formation of both sexual and asexual spores (conidia). Levels of vosA mRNA and protein are high in both types of spore. The deletion of vosA results in the lack of trehalose in spores, a rapid loss of the cytoplasm, organelles and viability of spores, and a dramatic reduction in tolerance of conidia to heat and oxidative stress. Moreover, the absence of vosA causes uncontrolled activation of asexual development, whereas the enhanced expression of vosA blocks sporulation, suggesting that VosA also functions in negative-feedback regulation of sporogenesis. VosA localizes in the nucleus of mature conidia and its C-terminal region contains a potential transcription activation domain, indicating that it may function as a transcription factor primarily controlling the late process of sporulation including trehalose biogenesis. VosA is conserved in most fungi and may define a new fungus-specific transcription factor family.


INTRODUCTION
Organisms have evolved various adaptive mechanisms to survive unfavorable environmental conditions. Trehalose, found in a wide variety of organisms, is a well-suited osmolyte and superior stabilizer of proteins and membranes. This specialized sugar protects cellular integrity and functions from various stresses including dehydration, heat, cold, and oxidation [1,2].
Fungi produce spores as the main means of propagation, survival and infection. Fungal spores are found in every environment inhabited by humankind and have a significant impact on everyday life [3,4]. These spores contain large quantities of trehalose [1], which is necessary for long-term viability. However, the molecular mechanism coupling trehalose biogenesis and the formation of spores is unknown.
The genus Aspergillus includes the most common fungi having both beneficial and detrimental impacts on human activities. All species reproduce asexually by forming long chains of asexual spores (conidia) radiating from a specialized structure called a conidiophore. Among them, Aspergillus nidulans has served as an excellent model for studying the mechanisms of asexual development (conidiation) [5,6].
A key step for conidiation is the activation of brlA, which encodes a C 2 H 2 zinc finger transcription factor (TF) [7]. Further studies identified and characterized abaA and wetA as also being important in this process. The abaA gene encodes a potential TF that is activated by brlA during the middle stages of conidiation [8]. WetA functions in the late phase of development and activates genes involved in the synthesis of spore wall components [9,10]. These three genes have been proposed to define a central regulatory pathway that controls ordered expression of conidiation-specific genes during conidiophore development and spore maturation [5,6,11]. Notably, later studies have revealed that BrlA is required for conidiation in two other aspergilli, the opportunistic human pathogen A. fumigatus [12] and the industrial fungus A. oryzae [13], indicating that its function is conserved in the genus Aspergillus.
However, a simple model depicting the linear activation of brlARabaARwetA is incomplete and unable to explain tight regulation of these genes. For instance, transcripts of brlA and abaA accumulate at the early/middle phases, diminish during the late phase of conidiation and are undetectable in mature conidia [5,14]. On the contrary, wetA mRNA level reaches the maximum during the late phase of conidiation and remains high in mature conidia [5,9]. These suggest that another regulator may coordinate the expression of conidiation-specific genes.
In this study, through a gain-of-function genetic screen, we identify the novel regulator VosA that couples the completion of sporogenesis and focal accumulation of trehalose in spores. VosA is highly conserved in most (if not all) filamentous and dimorphic fungi and is similar to three other proteins including VeA, a wellknown regulator of fungal development and secondary metabolism [15][16][17][18]. VosA is required for the biogenesis of trehalose during spore formation, thereby for the long-term survival of both conidia and sexual spores (ascospores). As it functions as a key negativefeedback regulator of conidiation, vosA is necessary and sufficient to direct repression of brlA and development. We further demonstrate that VosA is a potential TF and localizes in the nucleus of mature conidia, and propose that the conserved VosA/ VeA (or velvet) class proteins may define a new fungal TF family.

Identification of vosA
We hypothesized that a gain-of-function (multi-copy) genetic screen would identify novel negative regulators of development that cannot be defined via chemical mutagenesis. Briefly, a wild type (WT) strain was transformed with the pRG3-AMA1 based WT library [19] and out of more than 50,000 transformants seven exhibiting non-sporulating phenotypes were isolated. By direct sequencing of the insert ends of plasmids recovered from these transformants followed by a genome search [20], three multi-copy repressors of development have been identified: AN6437 and AN1959 from one transformant each, and AN6578 from four transformants. The plasmid from one transformant remains to be identified. We then found that reintroduction of the plasmid containing the AN1959 locus alone resulted in a complete loss of development ( Fig 1A). The AN1959 gene is further studied and found to be essential for the viability of spores, thus named as vosA (see below).
The sequence analyses of the RT-PCR products further led to the identification of the vosA ORF composed of 1950 bp with 10 exons predicted to encode a 430 aa-length protein (DQ856465). VosA is similar to three other proteins including VeA [15], VelB (EF540815) and VelC (EF540816) in A. nidulans. These velvet family proteins are highly conserved in both ascomycetes and basidiomycetes ( Fig 1B).
Levels of the VosA transcripts and protein(s) are high in conidia (C), ascospores (Ac) and during the late phase of conidiation when conidia differentiate and become mature (Fig 1C&D). During vegetative growth, levels of the vosA transcripts and protein(s) quickly drop and continue to be undetectable (or low) until 24 h post induction of conidiation. The vosA gene appears to consist of two overlapping transcripts (1.8 kb and 2.4 kb), where only the 2.4 kb transcript is present in the early phase (6 h) of vegetative growth. Two protein bands (90 KDa and 120 KDa) are also detectable at this time. The A. fumigatus VosA homolog (AfVosA; EF544392) shows 79% aa identity and exhibits the almost identical mRNA accumulation pattern (Fig 1E), implying that VosA function might be conserved in aspergilli.

VosA is required for the viability of spores
The vosA deletion (DvosA) mutant produces light green conidia that differ from WT (Fig 2A). Noticeably, DvosA causes a radical decrease in the viability of spores. When conidia of 2, 5, 10 and 20 d grown colonies of WT and DvosA strains are compared, the mutant conidia quickly lose viability starting from 5 d ( Fig 2B) and become translucent ( Fig 2C). Furthermore, the DvosA mutant produces defective sexual fruiting bodies (cleistothecia) containing few (,1%) viable semi-transparent ascospores ( Fig 2C).
To understand the cellular nature of the defects, conidia of WT and DvosA strains were examined by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). While no clear differences were observed in SEM (not shown), TEM has revealed that an electron-light layer found in the WT spore wall is absent in the DvosA mutant spores. Moreover, both DvosA conidia and ascospores appear to lack cytoplasm and organelles including the nucleus (Fig 2D). In addition, no genomic DNA or RNA was detectable in 8 d or older mutant conidia (not shown). These results indicate that VosA is essential for the integrity of both asexual and sexual spores.

VosA is essential for trehalose accumulation in conidia
The deletion of the A. nidulans tpsA gene encoding trehalose-6phosphate (T-6-P) synthase causes a rapid loss of spore viability [21]. Because no apparent differences in the spore wall composition between the WT and DvosA conidia were detectable, we asked whether VosA is needed for the proper accumulation of trehalose in spores. Measurement of trehalose amount in 2 d old conidia of WT and DvosA strains reveal that trehalose is undetectable in the DvosA conidia ( Fig 3A). This lack of trehalose was observed regardless of the presence (veA + ) or absence (veA1) of the fully functional VeA protein, indicating that VosA's function in trehalose biogenesis is independent of VeA.
To investigate the molecular basis of the defects, we examined the mRNA levels of selected genes associated with synthesis (tpsA, tpsC and orlA) or breakdown (treA and treB) of trehalose [21-24, Christophe d'Enfert, personal communication]. The deletion of vosA results in reduced tpsA, tpsC and orlA mRNA levels during asexual development and in mature conidia ( Fig 3B). On the contrary, mRNA levels of treA (but not treB) increase during the formation and maturation of conidia. These suggest that VosA is required for proper regulation of genes necessary for the accumulation of trehalose in spores. Supporting the positive regulatory role of VosA in trehalose biogenesis, overexpression of vosA is sufficient to direct upregulation of tpsA, tpsC and orlA even in vegetative cells ( Fig S1). We also tested whether the DvosA mutant conidia show altered stress responses and found that the mutant conidia exhibit a drastic reduction in tolerance to heat ( Fig 3C) and H 2 O 2 (Fig 3D), corroborating the idea that trehalose functions as a key protectant. The viability of the DvosA mutant was not affected by increased osmolarity in the medium or storage solution (data not shown).

VosA regulates conidiation-specific gene expression
As VosA plays a key role in modulating conidiation, DvosA results in the formation of conidiophores in liquid submerged culture (arrows in Fig 4A) where WT strains do not develop conidia. Moreover, MvosA blocks conidiation completely even under the conditions favoring development (Fig 1A). To further verify the repressive role of vosA in conidiation, we generated the vosA overexpression mutant by fusing the vosA ORF with the inducible alcA promoter [25]. As elevated expression of vosA is sufficient to block development, overexpression of vosA inhibits conidiation ( Fig 4C). We also found that the VosA-mediated inhibition of development is proportional to the dosage of vosA, and that three copies of vosA are sufficient to block development (not shown).
To further investigate the regulatory role of vosA in conidiation, we examined the mRNA levels of various development-specific genes. As shown in Figure 4B, DvosA results in nearly constitutive accumulation of high levels of brlA (and rodA [26]) mRNA even at 48 h post developmental induction and in conidia. Conversely, levels of wetA (and wA [27]) mRNA decrease considerably in the DvosA mutant (note the differences in conidia). Moreover, consistent with the phenotype, overexpression of vosA causes the blockage in accumulation of brlA mRNA (Fig 4D). Collectively, these results indicate that VosA inhibits brlA expression, but may activate wetA. In addition, DvosA causes altered expression of yA encoding a conidial laccase [28] and wA encoding a polyketide synthase [27], likely leading to lighter conidial pigmentation.
To further dissect genetic interactions among the four regulators, levels of vosA mRNA were examined in various mutants including brlA42, abaA14, wetA6, alcA(p)::brlA, alcA(p)::brlA abaA14 and alcA(p)::abaA (Fig S2 and S3). Expression analyses reveal that both abaA and wetA are required for vosA mRNA accumulation, and vosA and wetA activate each other. In addition, wetA is necessary for the repression of both brlA and abaA (see Discussion).
VosA is a potential TF and localizes in the nucleus of mature conidia Although no known DNA-binding domain is identified in its sequence, VosA contains a nuclear localization signal (NLS)-pat7 (241PVKRQRT247) and has a 65.2% likelihood of being localized in the nucleus as predicted by PSORT II (http://psort. nibb.ac.jp). We carried out the yeast-one hybrid assay by fusing the Gal4 DNA binding domain with full-length (VosA), Nterminal half (VosA-N) or C-terminal half (VosA-C) of VosA as well as AflR, a Gal4 type TF in A. nidulans [29,30]. The chimeric proteins were expressed in yeast under the control of the ADH1 promoter [31]. The transformants expressing VosA and VosA-C grow on the medium lacking histidine in the presence of 3AT as high as 1 mM and 5 mM, respectively (Fig 5A). Moreover, the strain expressing VosA-C shows six times higher b-galactosidase activity than those expressing VosA and VosA-N, implying that the C-terminal region of VosA contains a potential transcriptional activation domain.
We then examined the expression and localization of VosA during conidiophore development (see Fig 5B for structure). At 24 h, when conidia are actively differentiating, VosA-RFP accumulates in the cytoplasm of metulae, phialides and newly generated conidia (yellow arrow), and it begins to localize in the nucleus of mature conidia (white arrow in Fig 5C). A strain expressing RFP under the control of the gpdA promoter shows even distribution of RFP in the cytoplasm of all cell types. At 48 h, when all conidia become mature, VosA-RFP localizes mainly in the nucleus of mature conidia (Fig 5C). These imply that the VosA protein is actively expressed in metulae and phialides, and localizes primarily in the nucleus of mature conidia.

DISCUSSION
In this study, we present the data showing that the multifunctional regulator VosA plays an essential role in long-term survival of both asexual and sexual spores likely via coupling sporogenesis and trehalose biogenesis. We have focused on dissecting the roles of VosA in A. nidulans asexual lifecycle.
A conidiophore is composed of a thick-walled foot cell, a stalk, a multinucleate vesicle, two layers of uninucleate cells (metulae and phialides) and a large number of conidia (Fig 5B; [6,32]). After being generated from phialides through repeated asymmetric mitotic cell divisions, conidia need to undergo a maturation process to enable the long-term viability [9]. Two key events in this process are the formation of a remarkably rigid four-layered conidial wall [9] and accumulation of trehalose [23,33]. We demonstrate that maturation of spores requires VosA, which triggers an active buildup of trehalose and assists in the proper formation of the conidial wall. Importantly, we also found that the deletion of the vosA homolog in A. fumigatus results in reduced trehalose amount (,50% of WT) in conidia and decreased viability (data not shown), suggesting that VosA function may be conserved in other aspergilli.
During the development of conidiophores, the expression and localization of central regulatory components brlA, abaA and wetA is tightly controlled [7][8][9]14,34]. We have presented a series of data supporting the crucial role that VosA plays in the balanced expression of these and other genes. VosA potentially functions as both an activator (for wetA, tpsA and orlA) to complete sporogenesis and a repressor (for brlA and treA) to confer negative-feedback regulation. Notably, vosA itself is subject to strict spatiotemporal regulation: 1) high levels of the VosA protein are first found in the cytoplasm of metulae, phialides and newly generated conidia, and then in the nucleus of mature conidia, and 2) the levels of the vosA mRNA and protein decrease rapidly during conidial germination and vegetative growth, which presumably allows another round of development to occur. However, it is important to note that the VosA protein (90 kDa) is present at low levels during the vegetative growth phases of WT (Fig 1D), and that DvosA results in extremely high levels of brlA mRNA accumulating in liquid culture conditions (Fig 4B). These suggest that VosA may maintain its repressive role on brlA even in vegetative cells to inhibit precocious development. Taken together, we propose that VosA and SfgA, an upstream repressor of conidiation with a Zn(II) 2 Cys 6 domain [35,36], confer differential modulation of conidiation and both are needed for proper control of asexual lifecycle.
A new genetic model depicting the regulation of conidiation is presented (Fig 6). In this model, during the early phases of growth, SfgA (and VosA) stay active thereby repressing the expression of the Flb and brlA genes [36]. The main function of the early developmental activator FluG is to remove the repressive effects imposed by SfgA [36]. This de-repression initiates the subsequent activation of Flbs (encoding TFs) and the central regulatory pathway. In conjunction with the differentiation of conidia, vosA is activated, which in turn represses brlA expression and confers maturation of conidia. As the simplest model, the repressive roles of abaA and wetA on the expression of brlA may be primarily attributed to the VosA functioning in a negative-feedback loop. The observations that both brlA and wetA are subject to autoactivation [5,6] and that wetA functions in negative regulation of abaA (Fig S3) are indicated. While our studies have revealed the important functions of VosA in development, much remains to be learned. The first task is to further investigate the molecular mechanisms for VosAmediated regulation of sporogenesis and trehalose biosynthesis through identification of its potential targets by genome-wide screening and/or targeted approaches. For instance, examining the ability of VosA to bind the promoters of brlA, wetA, tpsA and orlA should help to test the postulated regulatory role of VosA (Fig 6). The second challenge is to characterize the functions of VosA homologs in other fungi. For instance, trehalose is required for plant infection by the rice pathogen M. grisea [37], in which a VosA homolog is found. It is of interest to see if the VosA homolog functions in various pathogenic fungi similarly. Thirdly, identification of VosA interacting proteins (if any) would be crucial to understand the mechanistic nature of VosA-mediated developmental regulation.
Our preliminary data indicate that VeA and VelB may also contain a transcriptional activation domain (not shown). Together with a set of evidence for the likelihood of VosA being a TF, it can be proposed that these velvet family proteins may define a new fungus-specific TF class. Moreover, given the pleiotropic effects caused by the mutational inactivation of VosA and VeA [15][16][17][18], these regulators may act globally at a higher hierarchic level influencing multiple biological processes. Additional studies of the velvet family proteins will further illuminate the molecular mechanisms interconnecting fungal morphogenesis and metabolism.

Fungal strains and growth
Aspergillus strains are listed in supporting Table S1. Standard culture and genetic techniques were used [38]. Strains were grown on minimal solid or liquid medium with appropriate supplements (simplified as MM; [39]). Induction of asexual or sexual development was done as described [35].

Cloning of vosA
The recipient strain RNIW5 (pyrG89; pyroA4) was transformed with the pRG3-AMA1-NotI WT library [19] and four transformants showing a non-sporulating phenotype were isolated. The plasmids were recovered as described [36]. Direct sequencing of the insert ends identified the vosA gene (locus AN1959.2). The vosA ORF was determined by RT-PCR followed by sequence analyses.

Plasmids
The alcA(p)::vosA construct was created as described [40] and cloned into the BamHI site of pJW53 (JW Bok and NP Keller, unpublished) resulting in pNI16. The vosA(p)::VosA::RFP (mRFP1; [41]) construct was cloned into the BamHI site of pSH96 [42] to generate pNI18. The RFP control construct was generated by fusing RFP with the gpdA promoter [43] and cloned into the EcoRI site of pJW53 giving rise to pNI21. For the yeast-one hybrid assay, cDNA of the coding regions of aflR, vosA, vosA-N (N-terminal 1,216 th aa) or vosA-C (C-terminal 217,430 th aa) was amplified via RT-PCR and cloned between the EcoRI and BamHI (for aflR) or BamHI and ClaI (for vosA, vosA-N and vosA-C) sites in pGBDUC1 [31] resulting in pNI32, 33, 34 and 35, respectively.

Nucleic acid isolation and manipulation
Genomic DNA and total RNA isolation, and Northern blot analyses were carried out as described [35]. The DNA probes were prepared by PCR-amplification of the coding regions of individual genes with appropriate oligonucleotide pairs using FGSC4 genomic DNA as template (Table S2).

Western blot
Sample preparation was done as described [44]. Protein concentrations were determined by a BCA protein assay kit (PIERCE). Proteins (20 mg/lane) were separated by electrophoresis in 7.5% SDS-polyacrylamide gel and electroblotted to the PVDF membrane (Bio-Rad). The VosA-FLAG protein was detected using mouse anti-FLAG M2 monoclonal antibody (Sigma), at a 1:1,000 dilution and WesternBreezeH Chromogenic Kit-Anti-Mouse (Invitrogen). MagicMark TM XP Western Protein Size Standard (Invitrogen) was used.

Spore viability test
Two-day old conidia of WT (FGSC26) and the mutant (RNI10.2) were spread on solid MM (10 5 /plate), incubated at 37uC, and the conidia from 2, 5, 10 and 20 day old cultures were collected. Approximately 250 conidia were inoculated on solid MM and incubated for 2,3 days at 37uC until colonies appeared. Survival rate (triplicate/sample) was calculated as a ratio of the number of growing colonies to the number of spores inoculated.

TEM
Conidia and ascospores were collected from the cultures on solid MM. Samples were fixed in Karnovsky's fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M Sorenson's sodium phosphate buffer [PB], pH 7.2) overnight at 4uC and post-fixed in 2% osmium tetroxide in PB for 1 h at RT. The samples were then dehydrated in a graded ethanol series from 35% to 100% and embedded in PolyBed812 resin (Polysciences). Polymerized samples were sectioned on a Leica UC6 ultramicrotome (80 nm) and stained with uranyl acetate and lead citrate. The stained sections were viewed on a JEOL 100CX transmission electron microscope, and documented with a SIS (Soft Imaging Systems, Lakewood, CO) MegaView III digital side mount camera.

Trehalose assay and stress tolerance test
The amount of glucose liberated by the activity of trehalase was measured using a glucose assay kit (Sigma) and converted into the trehalose amount per conidium (23). Each sample not treated with trehalase served as a negative control. The experiments were performed in triplicate. To examine thermal tolerance, WT (FGSC26) and mutant (RNI10.2) conidia were incubated at 50uC for 0, 5, 20, 30, 45 or 60 min. To examine oxidative tolerance, WT or mutant conidia were treated with varying concentrations (0, 0.25, 0.5, 0.75 or 1 M) of H 2 O 2 and incubated for 30 min at room temperature [45]. In both cases, the spores were inoculated on solid MM and incubated at 37uC for 48 h. Colony numbers were counted and calculated as a percentage of the untreated control.

Microscopy
The colony photographs were taken using a Sony DSC-F828 digital camera. Photomicrographs were taken using an Olympus BH2 microscope equipped with the DP-70 digital imaging system. Fluorescence samples were prepared as described [48] and visualized by a Zeiss Axioplan 2 microscope with AxioVision digital imaging software (Zeiss).  Figure S1 Overexpression of vosA is sufficient to direct the expression of trehalose biosynthetic genes in non-developing cells. Northern blot analyses for the levels of tpsA, tpsC and orlA mRNA in control (TJA53.1) and alcA(p)::vosA (TNI9.1) strains are shown.

SUPPORTING INFORMATION
Two strains were grown in liquid glucose medium at 37uC, 250 rpm for 18 h and then transferred onto solid threonine medium (MMT; inducing) for the concomitant induction of conidiation and overexpression of vosA. It needs to be emphasized that such a synchronized induction of both conidiation and overexpression of vosA results in the absence of spore formation due to the prevailing inhibitory role of VosA, whereas a control strain produces a large number of conidia (for reference see Figure 4C). Thus, the mRNA levels of tpsA, tpsC and orlA in an alcA(p)::vosA strain represent those accumulate in undifferentiated hyphae, strongly supporting the role of VosA in activating the genes for trehalose biosynthesis.