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Eukaryot Cell. 2010 April; 9(4): 578–591.
PMCID: PMC2863407

The RFX Protein RfxA Is an Essential Regulator of Growth and Morphogenesis in Penicillium marneffei [down-pointing small open triangle]

Abstract

Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. The opportunistic human pathogen Penicillium marneffei is a dimorphic fungus, displaying vegetative (proliferative) multicellular hyphal growth at 25°C and unicellular yeast growth at 37°C. P. marneffei also undergoes asexual development into differentiated multicellular conidiophores bearing uninucleate spores. These morphogenetic processes require regulated changes in cell polarity establishment, cell cycle dynamics, and nuclear migration. The RFX (regulatory factor X) proteins are a family of transcriptional regulators in eukaryotes. We sought to determine how the sole P. marneffei RFX protein, RfxA, contributes to the regulation of morphogenesis. Attempts to generate a haploid rfxA deletion strain were unsuccessful, but we did isolate an rfxA+/rfxAΔ heterozygous diploid strain. The role of RfxA was assessed using conditional overexpression, RNA interference (RNAi), and the production of dominant interfering alleles. Reduced RfxA function resulted in defective mitoses during growth at 25°C and 37°C. This was also observed for the heterozygous diploid strain during growth at 37°C. In contrast, overexpression of rfxA caused growth arrest during conidial germination. The data show that rfxA must be precisely regulated for appropriate nuclear division and to maintain genome integrity. Perturbations in rfxA expression also caused defects in cellular proliferation and differentiation. The data suggest a role for RfxA in linking cellular division with morphogenesis, particularly during conidiation and yeast growth, where the uninucleate state of these cell types necessitates coupling of nuclear and cellular division tighter than that observed during multinucleate hyphal growth.

We are interested in examining the regulatory networks controlling cell-type specification and development in the opportunistic fungal pathogen Penicillium marneffei and the role these processes play in pathogenicity. P. marneffei is a thermally dimorphic fungus capable of causing disseminated infection in immunocompromised individuals. Dimorphism is a common morphological process for many fungal pathogens and has been clearly linked to pathogenicity. At room temperature (25°C), P. marneffei exhibits mycelial growth in which multinucleate cells are connected in long hyphal filaments. This growth form is also capable of asexual development (conidiation), in which single-celled uninucleate spores (conidia) are produced on specialized aerial hyphae (conidiophores). The transition from a multicellular hyphal growth form to a unicellular growth form occurs upon transfer to 37°C. During this process, known as arthroconidiation, cellular and nuclear division become coupled and double septa are deposited between cells. The subsequent fragmentation of these filaments leads to the production of uninucleate yeast cells that divide by fission. It is this growth form of P. marneffei that presents as an intracellular pathogen in phagocytic cells during infection (10, 25).

Transition between multicellular and unicellular morphological states is common to most fungi and serve as an important process within developmental programs such as conidiation and mating. The process of arthroconidiation in P. marneffei is analogous to the transition from a hyphal growth form to a unicellular spore form in Acremonium chrysogenum and Coccidioides immitis, suggesting there may be common mechanisms underlying these events. The cpcR1 gene of A. chrysogenum, initially identified as a regulator of cephalosporin C biosynthesis genes, was subsequently shown to regulate arthrosporulation, whereby the filamentous mycelium undergoes fragmentation into unicellular arthrospores (31, 56, 58).

CPCR1 is a member of the regulatory factor X (RFX) family of transcriptional regulators that have been implicated in the regulation of both developmental and cell cycle events. To date, 17 members of this protein family that are highly conserved from yeast to humans have been isolated (21). The defining feature of these proteins is the novel RFX DNA-binding domain, a member of the winged-helix subfamily of helix-turn-helix DNA-binding domains (24). In addition, most of the RFX proteins contain a highly conserved dimerization domain mediating the formation of homo- and/or heterodimers (21, 52). While specific roles have been assigned to individual RFX proteins, a unified understanding of the processes regulated by these proteins across species has not been forthcoming.

Variation in tissue- and cell-type-specific expression of the five (RFX1 to -5) RFX genes identified in mammals has been observed (33, 53). The prototypical RFX protein, RFX1, is ubiquitously expressed and can act as both an activator and a repressor (34). Potential targets of RFX1 include cell proliferation and DNA damage genes, such as c-myc and the PCNA (proliferating cell nuclear antigen), MAP1A (microtubule-associated protein), IL-5Rα (interleukin-5 receptor α), and RNR (ribonucleotide reductase) genes (33, 36, 42, 59, 72). Additionally, the role of RFX5 in the regulation of major histocompatibility complex (MHC) class II gene expression is well established (60).

The Caenorhabditis elegans and Drosophila melanogaster RFX proteins, DAF-19 and dRFX, respectively, have recently been assigned roles in the development of ciliated sensory neurons (17, 61). D. melanogaster dRFX2 appears to be involved in the regulation of cell cycle progression, a theme also evident to various extents in its fungal counterparts (47). In Saccharomyces cerevisiae, the RFX homologue Crt1 prevents the expression of the DNA damage-inducible RNR genes in the absence of DNA damage through recruitment of the Tup1-Ssn6 corepressor complex (32). The sak1+ protein of Schizosaccharomyces pombe has been shown to function downstream of protein kinase A (PKA), where it promotes mitotic exit and thereby allows the onset of sexual development or entry into stationary phase (67). Deletion of sak1+ in S. pombe results in lethality, with transient phenotypes indicative of severe mitotic defects. More recently, Rfx2 of the dimorphic pathogen Candida albicans was found to regulate not only elements of the DNA damage response (DDR), presumably by a mechanism similar to that of Crt1 in S. cerevisiae, but also morphogenesis and virulence (27).

Here, the role of the RFX protein RfxA was investigated during the growth and morphogenesis of P. marneffei. The rfxA gene appears to be essential for the viability of P. marneffei, and studies involving overexpression, RNA interference (RNAi), and the production of dominant interfering alleles have shown that the levels of functional RfxA must be precisely maintained for growth and morphogenesis, suggesting that RfxA participates in the regulation of cell division events. As such, RfxA may be required for linking cell cycle regulation with cellular differentiation during morphogenesis in P. marneffei.

MATERIALS AND METHODS

Molecular techniques.

Plasmid DNA was isolated using the Wizard Plus SV DNA Purification System (Promega). Genomic DNA was prepared from frozen mycelia of P. marneffei as previously described (7). For the extraction of RNA, fungal cultures were grown as previously described (6). RNA was extracted from 0.1 to 0.2 g of biomass using the FastRNA Pro Red kit (Bio101). Southern blots were prepared with Hybond N+ membranes (Amersham) using standard procedures (55). For screening of the P. marneffei genomic DNA lambda library, plaque lifts and the isolation of positive clones were performed according to the instructions for the λBlueSTAR vector system kit (Novagen). Hybridizations were performed with [α-32P]dATP-labeled DNA probes using standard methods (55). The oligonucleotides used for PCR are listed in Table 1. Reverse transcriptase (RT)-PCR was performed using the rfxA-specific primers L13 and Q50, the benA-specific primers F58 and F59, and the mobA-specific primers HH21 and HH22 on 100 ng of total RNA using the Superscript one-step RT-PCR kit with Platinum Taq (Invitrogen) according to the manufacturer's directions. The number of amplification cycles was optimized for each primer pair to ensure that product synthesis was in the exponential phase of amplification. Product yields were estimated from ethidium bromide-stained gel images using MacBAS ver2.1 (Fuji PhotoFilm Co. and Kohshin Graphic Systems Inc.). PCR screening of putative rfxA deletion transformants was performed using three primers: L31, specific for the wild-type rfxA locus; L29, a pyrG+-specific primer; and L30, an rfxA genomic-locus-specific primer. A 1.5-kb product generated using the primers L31 and L30 was expected for strains containing the wild-type rfxA locus, while the presence of the rfxAΔ::pyrG+ deletion locus would give rise to a 1.9-kb product using the primers L29 and L30. For quantitative real-time RT-PCR, 2 μg of total RNA was subjected to DNase treatment using RQ1 RNase-free DNase (Promega) prior to cDNA synthesis, performed with the Reverse Transcription System (Promega) according to the manufacturer's specifications. Real-time PCR was performed in a Rotor-Gene RG-3000 instrument (Corbett Research) on ~20 ng of cDNA using the SensiMix Plus SYBR detection kit (Quantace) with an initial denaturation for 10 min at 95°C, followed by 45 cycles using the following parameters: 95°C for 20 s, 60°C for 20 s, and 72°C for 15 s. The relative fold expression of the sldA (primers FF20 and FF21) and bimB (primers FF18 and FF19) transcripts was determined using the comparative threshold cycle (CT) method (37), where samples were normalized to the actin (primers GG2 and GG3) transcript abundance to adjust for variations in sample-loading amounts.

Table 1.
Oligonucleotides used in this study

Cloning and plasmid construction.

A 650-bp fragment of rfxA was isolated from degenerate PCR performed on genomic DNA of the wild-type strain 2161 using the primers J72 and J73, designed to amplify the region encoding the putative DNA-binding domain, which is highly conserved in RFX proteins from the filamentous fungi A. chrysogenum, Neurospora crassa, Aspergillus fumigatus, and Penicillium chrysogenum. This PCR fragment was used to probe a P. marneffei genomic DNA λ library at high stringency (50% formamide, 0.1× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 65°C) to clone the full-length rfxA gene, contained within the plasmid pHS5584. A 5.9-kb KpnI/SacII subclone of P. marneffei rfxA (pHS6520) was generated by combining a 1.4-kb KpnI/SpeI fragment containing the 5′ noncoding region of rfxA with a 4.5-kb SpeI/SacII fragment containing the entire rfxA coding region and an additional 3′ noncoding region, both derived from pHS5584.

A two-step cloning strategy was used to generate the rfxA gene deletion construct pHS5595. First, a 1.1-kb XhoI/EcoICRI fragment of pHS5584, containing the 5′ region of rfxA, including the first 149 bp of coding sequence, was inserted between the XhoI/EcoRV sites upstream of the Aspergillus nidulans pyrG blaster cassette in pAB4626. Subsequently, a 1.4-kb SmaI/SacII fragment of pHS5584, containing the region 3′ of rfxA, including the last 132 bp of coding sequence, was inserted downstream of the A. nidulans pyrG blaster cassette.

To generate the xylPp::rfxA-RNAi construct pHS6521, the 5′ coding region of rfxA (bp 1 to 1256) was amplified by PCR using the primers Q34 and L6 and blunt cloned into the SmaI site of pBluescript II SK(+) (pHS6098). Subsequently, a 1.3-kb ClaI/SmaI and a 1.3-kb EcoRV/BamHI fragment of pHS6098 were inserted on either side of green fluorescent protein (GFP) in the vector pAA4111 in opposite orientations. A 3.4-kb NcoI fragment containing GFP flanked by the inverted repeats of the rfxA 5′ coding sequence was then inserted downstream of the xylP promoter in the plasmid pHS6103, containing a 2.6-kb EcoRI fragment of areA for targeting to the areA locus (H. Bugeja, M. J. Hynes, and A. Andrianopoulos, unpublished data).

To create a xylPp::rfxA construct, a 2.7-kb PCR product containing rfxA was generated using the primers L32 and L33 and blunt cloned into the SmaI site of pBluescript II SK(+). An NcoI fragment was isolated by partial digestion and cloned downstream of the xylP promoter in pXylNOM (70) (pHS5705). Since this construct lacked the correct ATG for rfxA, plasmid pHS5705 was modified using partial digestion with NcoI to remove 0.7 kb of 5′ rfxA coding sequence. A second partial NcoI digestion and end fill reaction was performed to destroy the 3′ NcoI site (pHS6097). A 0.8-kb NcoI fragment from pHS6098 was inserted into the 5′ NcoI site of pHS6097 to generate a construct in which the entire rfxA coding sequence was downstream of the xylP promoter (pHS6099). In order to target this construct to the areA locus, a 4.5-kb XhoI/SphI fragment of pHS6099, containing part of xylPp along with the rfxA coding sequence and trpCt, was used to replace an equivalent 1.7-kb fragment of pHS6103, giving rise to pHS6529.

Inverse PCR using the xylPp::rfxA construct (pHS6099) as a template and the primers Q36 and Q37 was used to delete the region encoding the conserved DNA-binding domain (721 to 1099). The rfxA-DIMΔ construct pHS6530 was generated by inverse PCR using the xylPp::rfxA construct (pHS6099) template and the primers L31 and L13, thereby removing the region encoding the putative dimerization domain (1673 to 2566). To facilitate targeted integration of these constructs at the areA locus in the areA strain 41.2.14-3, the constructs contained a 2.6-kb EcoRI fragment of areA.

Fungal strains and media.

The P. marneffei strains used in this study are listed in Table 2. The isolation and transformation of P. marneffei protoplasts were performed as previously described (7). For selection of pyrG+ transformants of strain SPM4, protoplasts were regenerated on osmotically stabilized protoplast medium (PM) containing 1.2 M sucrose and 10 mM ammonium tartrate [(NH4)2T]. For niaD+ selection, 10 mM sodium nitrate (NaNO3) was used as a sole nitrogen source, whereas 10 mM sodium nitrite (NaNO2) was used as a nitrogen source for selection of areA+ transformants from the areA strain 41.2.14-3. The strains were grown on 1% glucose minimal medium (ANM) with 10 mM γ-aminobutyric acid (GABA) (14), yeast synthetic dextrose medium (SD) with 10 mM ammonium sulfate [(NH4)2SO4] (5), or brain heart infusion (BHI) medium (Oxoid). When required, the medium was supplemented with 10 mM uracil to allow the growth of pyrG strains. For induction of the xylP promoter, 0.5% xylose and 0.5% sucrose were used in place of 1% glucose (noninduced). The DNA replication inhibitor hydroxyurea (HU) was used at a final concentration of either 2 mM, 5 mM, or 10 mM. To assess the nuclear division arrest, spores were germinated on slides coated with solid medium under inducing conditions for 18 h in the presence or absence of 2 mM HU before being processed for microscopic analysis (see below). Nuclear counts (approximately 150 germlings) were determined (n = 2).

Table 2.
P. marneffei strains used in this study

Microscopy.

Strains were grown on slides coated with a thin layer of solid medium with one end submerged in liquid medium, as described previously (6). The slides were fixed by soaking them in a solution of 4% para-n-formaldehyde in PME {PIPES [50 mM piperazine-N,N′-bis(2-ethanesulfonic acid)], pH 6.7, 1 mM MgSO4, 20 mM EGTA} for 30 min, followed by two 5-min PME washes. Samples were stained using fluorescent brightener 28 (calcofluor white [CAL]), Hoechst 33258, or 4′6′-diamino-2-phenylindole (DAPI) and visualized using a Reichart Jung Polyvar II microscope with either differential interference contrast (DIC) or epifluorescence optics. Images were captured using a SPOT charge-coupled device (CCD) camera (Diagnostic Instruments) and processed using Adobe Photoshop software.

Sequencing and bioinformatics.

DNA sequencing was performed at the Australian Genome Research Facility (AGRF) on purified plasmid DNA. DNA sequence was analyzed using Sequencher 3.1.1 (Gene Codes). All sequence analyses, including database searches, were done using the Australian National Genomic Information Service (ANGIS). Pairwise sequence comparisons were performed using the GAP program available through ANGIS, and multiple-sequence alignments were generated using ClustalW (62) and MacBoxShade. In some instances, sequence data were obtained from the following fungal genome databases: A. nidulans (http://www.broad.mit.edu/annotation/fungi/aspergillus/), Neurospora crassa (http://www.broad.mit.edu/annotation/fungi/neurospora/), Aspergillus fumigatus (http://www.tigr.org/tdb/e2k1/afu1/), and S. cerevisiae (http://www.yeastgenome.org/).

Upstream sequences (1,000 bp) of annotated genes from A. nidulans, A. fumigatus, Aspergillus terreus, and Aspergillus oryzae were downloaded (http://www.broad.mit.edu/annotation/genome/aspergillus_group/Downloads.htm) and searched for the putative RfxA recognition sequence RTHNYYN0-3RGNAAC using the DNA pattern search available at RSAT (http://rsat.ulb.ac.be/rsat/) (65). Common gene sets from Aspergillus containing putative RfxA binding sites were then identified from protein clusters available from the Aspergillus comparative database (http://www.tigr.org/sybil/asp/index.html) based on preliminary sequence data obtained from The Institute for Genomic Research website (http://www.tigr.org). Putative RfxA target genes were functionally assigned based on the Gene Ontology Consortium (GO) classification and description available at http://www.geneontology.org (4).

Nucleotide sequence accession number.

The sequence of the 5.8-kb KpnI/SacII fragment of P. marneffei rfxA (pHS6520) sequence was deposited in GenBank under accession number DQ666366.

RESULTS

Isolation of P. marneffei rfxA containing the highly conserved RFX DNA-binding domain.

A degenerate PCR approach was used to isolate a region of P. marneffei rfxA encoding the conserved DNA-binding domain, and this was subsequently used to clone the full-length rfxA gene from a P. marneffei genomic DNA λ library. A 5.8-kb KpnI/SacII subclone of P. marneffei rfxA (pHS6520) was sequenced, and the predicted rfxA gene encoded a protein of 861 amino acids. Orthologues of rfxA were detected in the genome sequences of many filamentous fungi, and the P. marneffei RfxA protein contains many sequence features previously characterized in other eukaryotic RFX factors (21) (Fig. 1A). Most importantly, P. marneffei RfxA contains the RFX DNA-binding domain, which is highly conserved among all family members (Fig. 1B), in addition to a putative dimerization domain (Fig. 1C). It also contains regions common to many transcriptional activators, such as a glutamine (Q)-rich region (RfxA residues 70 to 117; 21% Q) and a highly acidic aspartate (D)- and glutamate (E)-rich region (RfxA residues 782 to 856; 24.3% D/E). Homology regions B and C, which have not been functionally characterized in RFX proteins, are also present within P. marneffei RfxA.

Fig. 1.
P. marneffei RfxA contains protein motifs characteristic of RFX proteins. (A) Protein structure map of RfxA. The regions represented include the glutamine-rich region (Q; residues 70 to 117), the RFX DNA-binding domain (DBD; residues 243 to 317), the ...

The expression of rfxA is upregulated during conidiation and yeast growth.

To examine rfxA expression during the growth and morphogenesis of P. marneffei, semiquantitative RT-PCR was performed using RNA isolated from vegetative hyphal cells, cells undergoing asexual development (conidiation) at 25°C, and yeast cells cultured at 37°C. The abundance of rfxA transcript was found to be relatively low during vegetative hyphal growth at 25°C and was approximately 2-fold and 5-fold higher during yeast growth at 37°C and conidiation at 25°C, respectively (Fig. 2). Consistent with the increased expression of rfxA during conidiation, potential binding sites were identified for the BrlA (5), StuA (1), and AbaA (1) transcriptional regulators within the rfxA promoter region (references 3, 11, and 18 and data not shown). These proteins are involved in mediating the correct temporal and spatial expression of genes involved in conidiation in P. marneffei (6, 8; A. R. Borneman, K. Tan, M. J. Hynes, and A. Andrianopoulos, unpublished data).

Fig. 2.
Expression of rfxA is upregulated during asexual development. Semiquantitative RT-PCR was performed on total RNA extracted during vegetative hyphal growth at 25°C (25°C V), conidiation at 25°C (25°C C), and yeast growth ...

Potential RfxA target genes include cell cycle regulators.

To understand the cellular process(es) that RfxA may regulate, we used a bioinformatics approach to identify putative RfxA target genes in the sequenced and annotated genomes of the P. marneffei-related fungi A. fumigatus, A. nidulans, A. oryzae, and A. terreus. These fungi are sufficiently diverged to reveal conserved functional regulatory elements, such as RFX binding sites. This unbiased approach took advantage of the highly conserved DNA-binding domain across all the RFX proteins and used the consensus RFX binding sequence RTHNYYN0-3RGNAAC to search the 5′ untranslated regions (UTRs) of all genes in these fungi. Approximately 2,500 to 3,300 genes that contained at a least one potential RFX binding site were identified in each species. The presence of a single site has been found to be sufficient to confer regulation in other systems (19, 71). Given that a large proportion of these may represent chance occurrences of the consensus target sequence, the data set was restricted to include only those sites that were present in the promoter sequences of orthologous genes in all four species. This resulted in the identification of 75 orthologous genes containing at least one putative RFX binding site. In six instances, the identified binding sites were located in overlapping divergent promoters present in all four species. In these cases, it is possible that only one member of the gene pair may represent an RfxA target gene.

Of these 75 genes, no predicted cellular function could be found for 29 (39%) based on either GO annotation or conservation with previously characterized genes. Of the remaining 46 genes, 19 (41%) represent genes involved in cell cycle regulation, in particular, mitotic-spindle dynamics and exit from mitosis, and the rest (27 genes, or 59%) are divided among a variety of cellular processes, including cellular metabolism, protein synthesis and degradation, chromosome metabolism, and cytoskeleton dynamics. In many of the genes examined, the positions of the putative RFX binding sites relative to the initiator codon were also found to be conserved across all four species and in the P. marneffei orthologues (Table 3).

Table 3.
Conserved genes from filamentous fungi containing RFX consensus binding sites

In an attempt to understand the regulatory role of RfxA, preliminary expression analysis of nine putative rfxA target genes representing a range of cellular processes related to cell division (Table 3) was performed using either semiquantitative or real-time RT-PCR. The expression of these genes in hyphal (25°C) and/or yeast (37°C) cells with either reduced or increased rfxA expression was examined, using rfxA-RNAi and rfxA OE strains, respectively (Fig. 3) (see below). Of these, six genes orthologous to S. pombe cdc4, cdc15, and src1 and A. nidulans kipB, nimA, mpsA, and sldA showed no consistent change in expression level when RfxA was either over- or underexpressed, indicating that they are not targets of RfxA under the conditions examined. The remaining genes are known to function in spindle dynamics, chromosome cohesion, and mitotic exit. P. marneffei mobA, an orthologue of the S. cerevisiae MOB1 gene encoding a protein kinase required for maintenance of ploidy, showed increased expression in the rfxA-RNAi strain at 25°C (Fig. 3A). P. marneffei bimB, a homologue of genes encoding separin/separase involved in cleavage of cohesin and sister chromatid separation, showed increased expression in both the rfxA-RNAi and rfxA OE strains under induced conditions during filamentous growth at 25°C, but not during yeast growth at 37°C (Fig. 3B and data not shown). While these results indicate that P. marneffei RfxA may play a role in spindle dynamics and mitotic exit, the small fold changes observed may also be a consequence of the experimental conditions required for induction of the dominant alleles in these strains and the inherent asynchrony produced or an indirect consequence of perturbed cell division (see below). Isolation of a conditional allele of RfxA would be important for further investigation. In addition, direct DNA-binding studies would be required to distinguish primary RfxA targets from changes in gene regulation due to secondary effects resulting from the perturbed growth observed in strains with reduced or enhanced RfxA function.

Fig. 3.
Relative expression of putative rfxA target genes in strains with reduced and increased rfxA expression. Total RNA was extracted from the P. marneffei rfxA+ (SPM4), xylPp::rfxA-RNAi (55.2.1), and xylPp::rfxA (49.3.1) strains during vegetative hyphal growth ...

rfxA is essential for viability in P. marneffei.

To characterize the role of rfxA during growth and development in P. marneffei, we attempted to isolate a strain containing a deletion of the single rfxA gene. Strain SPM4 (pyrG niaD) was transformed with a linear 4.7-kb XhoI/SacII fragment of the rfxA gene deletion construct pHS5595 (Fig. 4A) to replace the majority of the rfxA coding region with the Aspergillus nidulans pyrG+ blaster cassette (8). A total of 246 pyrG+ transformants were isolated and subsequently screened for integration of the rfxA deletion construct at the rfxA locus using either PCR or Southern blot analysis. Despite the large number of transformants screened, a strain lacking the rfxA coding sequences was not identified (data not shown).

Fig. 4.
Strain 44.1.2 is a heterozygous diploid containing rfxA+ and rfxAΔ::pyrG+ alleles. (A) Partial restriction map for the wild-type rfxA genomic locus (top) and the expected rfxA deletion construct pHS5595 (bottom) after integration. The gray box ...

One transformant (44.1.2) was identified which was found by PCR screening to contain both rfxA+ and rfxAΔ::pyrG+ alleles (data not shown), and this was confirmed by Southern blot analysis (Fig. 4B). As P. marneffei is a haploid organism, this result suggested that this represented either a heterokaryotic strain containing nuclei of different genotypes or a heterozygous diploid. The isolation of such strains is not uncommon in many fungi and is often used to study uncharacterized, potentially essential genes (45). While actively growing vegetative hyphal cells are predominantly multinucleate, during conidiation, single nuclei are partitioned into the conidium-producing sterigmata cells and subsequently into conidia. Genotyping of colonies isolated from purified uninucleate conidia using growth tests and PCR screening showed that the uninucleate conidia of strain 44.1.2 were genetically identical and contained both rfxA+ and rfxAΔ::pyrG+ alleles, showing that these isolates represented heterozygous diploids (data not shown). Microscopic examination of DAPI-stained conidia of strain 44.1.2 verified that each conidium contained only a single nucleus and that these conidia were larger than those of the control haploid strain (Fig. 4C). The average volume of 44.1.2 conidia was 159.2 ± 23 μm3 (± standard deviation; n = 30), approximately 2 to 3 times that of the haploid rfxA+ control strain (33.4.2), 63.6 ± 10.6 μm3. It is well established that conidial size increases with ploidy in fungi (12), and therefore, these data are fully consistent with the hypothesis that 44.1.2 represents a heterozygous rfxA+/rfxAΔ::pyrG+ diploid strain. The isolation of a diploid strain heterozygous for deletion of rfxA, and the inability to obtain a haploid rfxA deletion strain, strongly suggests that rfxA is an essential gene of P. marneffei.

Haploinsufficiency of rfxA leads to reduced growth, nuclear division defects, and genomic instability at 37°C.

It was anticipated that deletion of a single copy of rfxA in the rfxA+/rfxAΔ::pyrG+ heterozygous diploid strain (44.1.2) would have no effects on growth, since this strain also contains a wild-type allele of rfxA and is therefore able to produce a functional RfxA protein. However, while the ability of the rfxA+/rfxAΔ::pyrG+ strain (44.1.2) to undergo filamentous growth at 25°C was indistinguishable from that of the wild-type control (2161), growth of this strain was significantly reduced under conditions of yeast growth at 37°C (Fig. 5A). Microscopic examination of the rfxA+/rfxAΔ::pyrG+ strain during growth at 37°C revealed multiple cellular defects, including the presence of swollen and irregularly shaped arthroconidial hyphae and cell lysis. Using Hoechst staining of DNA, very few intact nuclei were observed, and instead, enlarged nuclear masses, characteristic of endoreplication (DNA rereplication in the absence of nuclear division), and small nuclear fragments of irregular shape were prevalent (Fig. 5B).

Fig. 5.
The heterozygous rfxA+/rfxAΔ strain displays cellular division defects during yeast morphogenesis at 37°C. (A) The rfxA+ pyrG+ (33.4.2), rfxA+ pyrG (SPM4), and rfxA+/rfxAΔ::pyrG+ (44.1.2) strains were grown on either ANM ...

The genetic stability of the rfxA+/rfxAΔ::pyrG+ strain was also compromised during growth at 37°C. This was initially apparent upon isolation of single cells (protoplasts) from the multicellular arthroconidial hyphae of strain 44.1.2 grown at 37°C, as approximately 90% of viable protoplasts had reverted to uracil auxotrophy (data not shown). Genotyping of these putative haploid isolates by PCR confirmed loss of the rfxAΔ::pyrG+ allele associated with the onset of uracil auxotrophy, and therefore, these strains represented wild-type rfxA+ haploids derived from the rfxA+/rfxAΔ::pyrG+ strain (data not shown). The remaining protoplasts displayed uracil prototrophy and contained both rfxA+ and rfxAΔ::pyrG+ alleles, as verified by PCR; however, many of these isolates displayed an abnormal morphology (data not shown). These strains were highly unstable and frequently reverted to a wild-type growth phenotype, which was subsequently stable (data not shown). No haploids containing only the rfxAΔ::pyrG+ allele were isolated. In fungi, the generation of haploid isolates from a diploid progenitor (haploidization) occurs via random chromosome loss due to nondisjunction of chromosomes, as a result of either genetic mutation or the presence of genotoxic compounds (30, 64). The uracil prototrophic colonies displaying the abnormal growth morphology phenotype likely represented aneuploid strains that had not undergone complete haploidization, since both wild-type chromosomes and chromosomes with rfxA deleted were being maintained. Reversion to the wild-type growth phenotype may result from imbalanced chromosome segregation in the aneuploid strains, leading to restoration of the diploid state. Both the observed nuclear fragmentation and haploidization of the heterozygous diploid during growth at 37°C, but not at 25°C, are suggestive of nuclear division defects, possibly due to haploinsufficiency of rfxA. In addition, the observation that only wild-type haploids containing the rfxA+ allele, and not the rfxAΔ::pyrG+ allele, were isolated provides additional evidence that a functional rfxA gene is required for the viability of P. marneffei.

Reduced RfxA levels result in cell division defects.

To study the consequences of reduced rfxA expression, an RNAi strategy was developed in order to silence the endogenous rfxA transcript by driving high-level expression of an rfxA hairpin transcript. For this purpose, the construct pHS6521 was generated, in which a spacer fragment (GFP) was flanked by inverted repeats of the first 1,256 bp of rfxA 5′ coding sequence and placed downstream of the xylP promoter of Penicillium chrysogenum, which is effective in inducing high-level gene expression in P. marneffei in the presence of xylose (51, 70). This construct was targeted to the areA locus, giving rise to strain 55.2.1. Using semiquantitative RT-PCR, the abundance of the endogenous rfxA transcript was found to be significantly reduced upon induced expression of the rfxA-RNAi hairpin transcript in strain 55.2.1 (xylPp::rfxA-RNAi) at both 25°C and 37°C (Fig. 3A and data not shown).

At 25°C, the induced expression of the rfxA-RNAi hairpin transcript in strain 55.2.1 resulted in severe growth inhibition. Microscopic examination of colonies revealed multiple growth defects under inducing conditions, including aseptate and anucleate hyphae and deformed apical hyphal cells and lateral branches, often with multiple short swollen hyphal tips exhibiting cell lysis (Fig. 6A). In the older hyphal cells at the center of the colony, the few nuclei that were observed displayed an abnormal highly elongated appearance.

Fig. 6.
Silencing of the rfxA transcript by RNAi leads to cell division defects and poor growth. Microscopic examination of wild-type rfxA+ (SPM4) and xylPp::rfxA-RNAi (55.2.1) strains are shown. (A) Strains after 4 days of growth at 25°C on slides coated ...

Due to the severe growth inhibition resulting from overexpression of the rfxA-RNAi hairpin transcript, the effects on conidiation could be examined only under conditions of delayed induction. Abnormal conidiophore morphogenesis was apparent upon induced expression of the rfxA-RNAi hairpin transcript, with a range of phenotypes observed (Fig. 6B). They included sterigmata cells and conidia that appeared engorged and contained large numbers of nuclei, demonstrating defects in nuclear partitioning. At the most severe end of the spectrum, conidiophores lacked appropriate differentiation of the sterigmata cell types and consisted of conidiophore stalks with aberrant swollen and misshapen sterigmata cells. These cells lacked intact nuclei and instead contained fragmented nuclear structures. This suggests that reduced rfxA function leads to defects in nuclear division and segregation during differentiation of the uninucleate cell types of the conidiophore.

The xylPp::rfxA-RNAi strain also displayed reduced growth under conditions required for yeast morphogenesis at 37°C. Microscopic examination revealed growth phenotypes similar to those of the heterozygous rfxA+/rfxAΔ::pyrG+ diploid strain. They included severe growth phenotypes, such as cell lysis and the presence of swollen hyphae, which lacked intact nuclear structures and instead contained small nuclear fragments (Fig. 6C). Although reminiscent of arthroconidial hyphae, these cells were aseptate, and no hyphal fragments or yeast cells, indicative of arthroconidiation, were observed.

Overexpression of rfxA results in cell division defects and growth arrest.

To further examine the role of rfxA during growth and morphogenesis, the rfxA coding region was inserted downstream of the xylP promoter in the plasmid pHS6259 in order to drive high levels of rfxA expression in a xylose-inducible manner. This construct was targeted to the areA locus in single copy. Growth of the xylPp::rfxA strain 49.3.1 was almost completely inhibited under inducing conditions at 25°C. Growth arrest was observed at an early stage of germination, coinciding with germ tube elongation and the presence of either one (no nuclear division) or two (one nuclear division) nuclei (data not shown). A small proportion of the inoculum was able to commence growth, and microscopic examination of these hyphal cells during overexpression of rfxA revealed a drastic reduction in cell size and an increase in the number of nuclei partitioned into each subapical cell compartment (Table 4). Additional phenotypes identified included an increased occurrence of lateral branches, some resembling short undifferentiated conidiophore stalks (Fig. 7A). These phenotypes were also observed under conditions of delayed induction (see below).

Table 4.
Average cell lengths and nuclear abundances during overexpression of rfxA
Fig. 7.
Overexpression of rfxA leads to cell division defects and growth arrest. (A) Microscopic examination of wild-type rfxA+ (SPM4) and xylPp::rfxA (49.3.1) strains after 4 days of growth at 25°C on slides coated with a thin layer of solid ANM plus ...

To assess conidiophore morphogenesis upon overexpression of rfxA, strain 49.3.1 was examined microscopically under conditions of delayed induction. The induced overexpression of rfxA did not lead to any major conidiation defects; however, an increase in the frequency of conidiophores, particularly toward the periphery of the colony, was observed. Furthermore, many of these conidiophores appeared rudimentary, with shorter stalks and reduced abundance of sterigmata cell types (Fig. 7B).

Under inducing conditions at 37°C, a complete lack of growth was observed for the xylPp::rfxA strain (data not shown). Microscopic examination revealed that while many conidia appeared to have initiated germination, as shown by the presence of enlarged cells with some protruding short germ tubes, the growth of these cells appeared to be arrested, with only a single nucleus observed (Fig. 7C).

Both the DNA-binding and dimerization domains of RfxA are required to modulate its function.

The predicted P. marneffei RfxA protein contains the highly conserved RFX DNA-binding domain, as well as a putative dimerization domain. In some cases, these domains have been shown to function independently, where the formation of either homo- or heterodimers is not a prerequisite for DNA binding in vitro and protein dimers can be formed when the dimerization domain is fused to a heterologous DNA-binding domain (21, 35, 52). To assess the codependence of these domains for the normal functioning of RfxA, mutant alleles of rfxA encoding RfxA proteins lacking either the DNA-binding domain (DBDΔ) or the dimerization domain (DIMΔ) were inserted downstream of the xylP promoter in the constructs pHS6522 and pHS6530, respectively. These constructs were targeted to the areA locus.

During filamentous hyphal growth at 25°C, overexpression of either the rfxA-DBDΔ (56.4.4) or rfxA-DIMΔ (49.4.1) allele had no effect on growth (data not shown). During yeast morphogenesis at 37°C, overexpression of rfxA-DIMΔ resulted in slightly reduced growth; however, no morphological defects were apparent upon microscopic examination. In comparison, overexpression of the rfxA-DBDΔ allele caused a severe reduction in growth at 37°C, with cell lysis apparent upon microscopic examination, as well as nuclear division defects characterized by enlarged nuclear masses, possibly due to endoreplication, and nuclear fragmentation (data not shown). These phenotypes recapitulated those previously observed for the rfxA+/rfxAΔ strain 44.1.2 and the induced xylP::rfxA-RNAi strain during yeast morphogenesis at 37°C.

Reduced rfxA function leads to defective checkpoint regulation.

In light of the cellular division defects observed in response to a reduced level of rfxA expression in the xylPp::rfxA-RNAi strain, we assessed the response of this strain to known inhibitors of the cell cycle. HU, a direct inhibitor of RNR function, results in activation of the S-phase checkpoint by preventing completion of DNA replication (16). In addition, the microtubule-destabilizing compound Benomyl causes a block in late mitosis due to activation of the spindle checkpoint pathway in response to the formation of a defective mitotic spindle (49). Mutants defective in the activation of checkpoints in response to a transient exposure to HU or Benomyl display reduced viability due to the completion of defective mitotic divisions (20, 23). Both the wild-type rfxA+ and xylPp::rfxA-RNAi strains were germinated for 24 h in the presence of HU (2.5 or 10 mM) or Benomyl (0.01, 0.05, 0.2, or 0.5 μg ml−1) under both inducing and noninducing conditions prior to being assessed for viability. The wild-type (rfxA+) and xylPp::rfxA-RNAi strains displayed similar viability after transient exposure to Benomyl and under noninducing conditions in the presence of HU (data not shown). The reduced expression of rfxA in the xylPp::rfxA-RNAi strain under inducing conditions caused a significant reduction in viability after treatment with HU (Fig. 8). A similar reduction in viability was observed for the xylPp::rfxA-RNAi strain when germlings were exposed to HU for only 6 h. However, the ability to arrest nuclear division in response to HU was not affected in the xylPp::rfxA-RNAi strain. Approximately 15% of germlings of the xylPp::rfxA-RNAi (17.9% ± 8.6% [standard error of the mean {SEM}]) and wild-type control (15% ± 5.5%) strains underwent nuclear division in the presence of HU, in contrast to 50% of germlings in the absence of HU (49.2% ± 7.9% and 47.4% ± 6.8% for the xylPp::rfxA-RNAi and wild-type control strains, respectively). Thus, although mitosis is inhibited, presumably via activation of the S-phase checkpoint, reduced RfxA function prevents the appropriate response to DNA replication inhibition to ensure survival. Given that the exposure to HU was transient in nature, this may include the ability to slow S-phase progression and to stabilize and subsequently reactivate stalled replication forks, thus preventing double-stranded DNA breaks and facilitating DNA repair.

Fig. 8.
Silencing of the rfxA transcript by RNAi leads to reduced viability after DNA replication inhibition imposed by HU. Approximately 2 × 105 conidia of the wild-type rfxA+ (SPM4) and xylPp::rfxA-RNAi (55.2.1) strains were inoculated into 1 ml of ...

DISCUSSION

Alterations in the expression of rfxA lead to cellular division and growth defects.

The rfxA gene of P. marneffei encodes a member of the RFX transcription factor family involved in the regulation of cellular differentiation events in eukaryotes. The data presented here show that rfxA is essential for viability and that alterations in the levels of its expression lead to cell division defects with dramatic consequences for growth and morphogenesis. Despite numerous attempts, a haploid rfxA deletion strain could not be isolated. Instead the isolation of a heterozygous diploid strain from which only wild-type haploids could be recovered suggests that rfxA is essential for survival. Examination of strains with reduced or enhanced rfxA expression demonstrated that rfxA might be involved in cell cycle regulation, consistent with the essential nature of the gene product. In A. nidulans, numerous genes involved in cell cycle regulation are essential for viability and were initially identified as temperature-sensitive lethal mutants (46).

The overexpression of rfxA resulted in growth arrest during conidial germination. Although the nature of this growth arrest is unclear, it suggests a role for RfxA in the reactivation of growth in dormant conidia. The observation that hyphal cells display increased nuclear division kinetics (increased numbers of nuclei per cell and shorter cell length) when rfxA is overexpressed implicates RfxA in the temporal regulation of genes affecting cell cycle events.

In contrast, reduced levels of rfxA caused by expression of the RNAi transcript, resulted in terminal degenerative phenotypes at 25°C characterized by hyphal cells that were aseptate and either produced aberrant elongated nuclei, indicative of a block in mitosis, or were anucleate. Similar phenotypes are observed in cell cycle mutants of A. nidulans affected in chromosome metabolism (sepB and sepJ) and cohesion (bimB and bimD), spindle dynamics (bimC), telomere maintenance (nimU/pot1), and mitotic exit (bimA) (15, 22, 28, 29, 40, 50, 66). In these mutants, the nuclear phenotypes result from premature entry into mitosis prior to completion of DNA synthesis and/or failure to completely separate chromosomes during division, preventing cells from exiting mitosis. The lack of septation observed for many of these mutants is a secondary consequence of errors in DNA metabolism causing inhibition of cytokinesis via activation of the DNA damage checkpoint pathway (29, 66). The aseptate phenotype of the RNAi strain is also consistent with this hypothesis.

During growth at 37°C, reduced rfxA expression in the RNAi strain resulted in an inability to undergo yeast morphogenesis. Septation, fundamental to the process of arthroconidiation and the liberation of yeast cells, was not observed as either a direct consequence of reduced RfxA function or a secondary effect of the terminal degenerative phenotypes observed (cell lysis and nuclear division defects). Mitotic catastrophe, as evidenced by enlarged nuclear masses, suggestive of endoreplication, and nuclear fragmentation was readily apparent. These phenotypes were recapitulated in the heterozygous diploid strain specifically during growth at 37°C, presumably as a consequence of gene dosage effects (haploinsufficiency). In support of this hypothesis, haploinsufficiency has also been observed for CRT1/crt1Δ and RFX2/rfx2Δ heterozygous diploids in S. cerevisiae and C. albicans, respectively (27, 32). Such nuclear defects may also account for the apparent loss of genomic stability resulting in the spontaneous haploidization of the heterozygous diploid strain. Mutations affecting components of the cell cycle apparatus, such as in the genes sepB, sepJ, bimA, nimU (pot1), and hfaB of A. nidulans, are known to result in loss of genomic stability (28, 29, 50, 64, 66).

Given the role of the related RFX proteins in mediating the DDR in closely related organisms, it is interesting to speculate whether RfxA is involved in a similar mechanism in P. marneffei, particularly in light of the increased sensitivity to HU observed under conditions of rfxA RNAi. Importantly, RFX acts a negative regulator in its role as an effector of the DDR and thus leads to derepression of the target genes, including the RNR-encoding genes, in the presence of HU (27, 32, 38). Thus, reduced RFX function would be expected to promote survival in the presence of HU, whereas the results presented here show that in P. marneffei the converse is true. Putative RFX binding sites have recently been identified in the promoters of both RNR-encoding genes in P. marneffei (data not shown), and the results suggest that RfxA might positively regulate these genes during DNA replication arrest.

If RfxA is involved in the DNA damage response in P. marneffei, this is unlikely to be its sole function, as components exclusive to the DDR in closely related fungi are rarely essential for survival under non-DNA damage conditions (26). Almost half of the potential RfxA target genes to which cellular roles could be assigned represent gene products involved in cell cycle events, with particular emphasis on chromosomal mechanics, spindle organization, and mitotic exit. Two of these putative targets (mobA and bimB) displayed increased expression in strains with altered RfxA activity. The maintenance of the ploidy kinase MobA, upregulated during rfxA RNAi, causes defects in mitotic progression and septation when overexpressed in fission yeast (54). The altered expression of the separase BimB suggests that RfxA may influence chromosome dynamics by perturbing sister chromatid cohesion (40, 63).

RfxA is important for linking cellular division with morphogenesis.

The data suggest that the function of RfxA in the regulation of cell cycle progression is strongly influenced by the cellular context. While the overexpression of rfxA increases the rate of cell division in hyphal cells, overexpression of rfxA in conidia caused growth arrest at an early stage of germination. In addition, nuclear division defects were observed for the heterozygous diploid and xylP::rfxA-DBDΔ strains only during yeast growth at 37°C, and not during filamentous growth at 25°C. During both conidiation and yeast morphogenesis in P. marneffei, nuclear and cellular division are tightly coupled to ensure the maintenance of the uninucleate state of these cell types. In contrast, hyphae are coenocytic/multinucleate, and in A. nidulans, aside from the initial nuclear divisions during germination, nuclear division occurs in a parasynchronous wave (13). Thus, hyphal growth may be less sensitive to perturbation in RfxA activity than the tightly regulated nuclear division events occurring during development and yeast morphogenesis, and possibly even during germination.

Regulated changes in cell cycle dynamics are often associated with cellular differentiation events in fungi, including an increased rate of cell cycling during conidiation in A. nidulans (43, 68). Effects on conidiophore morphogenesis were observed in P. marneffei when the levels of rfxA expression were perturbed. Reduced rfxA expression in the rfxA-RNAi strain led to the production of defective conidiophore structures, reflecting disturbances in the coupling of mitosis with cytokinesis. Similar defects resulting in inappropriate conidiophore morphogenesis result from aberrant checkpoint regulation in A. nidulans due to the NimXcdc2-AF mutation, preventing negative regulation of the cyclin-dependent kinase by inhibitory Tyr-15 phosphorylation (68). Interestingly, while overexpression of rfxA caused an increase in mitotic cycling in basal hyphal cell compartments, the conidiophores produced were morphologically normal, although increased in frequency. This may reflect the normal upregulation of rfxA occurring during conidiation in wild-type cells. It is possible that RfxA directly influences the onset of development through regulation of the transcriptional regulator BrlA, a key component of the central regulatory pathway involved in conidiophore development, since conserved RFX consensus binding sites were identified in the promoters of brlA genes from the aspergilli and P. marneffei, and induction of brlA expression has previously been shown to be sufficient for initiation of conidiation (1, 41; Borneman et al., unpublished).

Regulation of RfxA activity.

The underlying question of how RfxA integrates the regulation of cellular division with differentiation events in the different cell types produced by P. marneffei remains. The possibility that RfxA mediates its cell-type-specific roles through interaction with additional regulatory proteins in a cell-type-dependent manner is supported by the analysis of strains overexpressing rfxA alleles lacking the regions encoding the DNA-binding or dimerization domains. Overexpression of the rfxA-DBDΔ allele, containing a functional dimerization domain, resulted in a dominant-negative effect, suggesting that regulation of RfxA function occurs via protein interactions. This may involve one or more binding partners required for appropriate regulation of target genes, which are sequestered by the RfxA-DBDΔ protein. A similar mode of action has been proposed to account for the dominant interfering effects resulting from a version of human RFX1 with the DNA-binding domain deleted on the regulation of RFX1 target genes (38). In contrast, the putative dimerization domain appears to be required for protein function, since overexpression of a mutant allele lacking this domain has no obvious phenotypic effects, contrary to overexpression of the wild-type allele. While the stability of the RfxA-DIMΔ protein has not been assessed in this strain and could account for the lack of phenotype, this finding is consistent with observations in A. chrysogenum, where the formation of CPCR1 dimers is essential for both DNA binding and the interaction with AcFKH1 (57, 58). These data suggest that the formation of RfxA homodimers and the interaction with accessory proteins, possibly involving a forkhead protein, are necessary for appropriate regulation of RfxA target genes.

Recent evidence in fission yeast also supports a role for the interaction of RFX and forkhead proteins in cell cycle regulation. In S. pombe, the forkhead proteins Fkh2 and Sep1 are required for the G2/M-specific regulation of genes required for late mitotic events and cytokinesis (9, 44). Interestingly, many of these forkhead-regulated genes have orthologues identified as putative RfxA targets in this study. Both Fkh2 and Sep1 are associated with the PCB (pombe cell cycle box) site in the promoters of these genes as components of a multiprotein transcription factor, PBF (PCB-binding factor) (2, 48). Not only does the PCB sequence reflect the 3′ half site of the RFX consensus sequence, and not a prototypical forkhead consensus sequence, but additional consensus RFX and FKH binding sequences are also present in the promoters of PCB-regulated genes (H. Bugeja and A. Andrianopoulos, unpublished data). PBF has negative (mediated by Fkh2) and positive (mediated by Sep1) regulatory roles at the promoters of PCB-containing genes (48), consistent with the context-dependent regulation of RFX target genes in other organisms. The S. pombe RFX protein Sak1 has an essential yet biochemically undefined role in the regulation of cell cycle events. Recently, synthetic interactions between Sak1 and anaphase-promoting complex/cyclosome (APC/C) components, including separase, have been described (39, 69). Thus, its involvement as a possible component of PBF is worthy of further investigation.

RFX proteins as regulators of cell division and differentiation in eukaryotes.

In P. marneffei, the RFX protein RfxA is required for integrating the mitotic cell cycle with cellular differentiation events in a cell context-dependent manner. In other organisms where RFX proteins have been found to be involved in cell cycle regulation, such as humans (RFX1), Drosophila (dRFX2), and S. pombe yeast (Sak1), reduced RFX protein function also has dramatic consequences for growth. This is not observed in S. cerevisiae, where crt1 mutants are viable, reflecting the underlying differences in checkpoint control mechanisms in these organisms. Aside from the role of RFX proteins in regulating cell cycle events or the DNA damage response, these factors are also involved in the temporal or spatial regulation of cellular differentiation events, including ciliated sensory neuron development in D. melanogaster (dRFX) and C. elegans (DAF-19) and arthrosporulation in A. chrysogenum (CPCR1). Thus, not only do RFX proteins have a fundamental role in cell cycle regulation, this is also manifested in the altered patterns of cell division required for morphogenesis in developmentally complex organisms.

ACKNOWLEDGMENTS

This work was supported by grants to A.A. from the Australian Research Council, the National Health and Medical Research Council, and the Howard Hughes Medical Institute. A.A. is a Howard Hughes Medical Institute International Scholar. H.E.B. was supported by an Australian Postgraduate Award.

We thank H. Robertson and K. J. Boyce for critical comments on the manuscript.

Footnotes

[down-pointing small open triangle]Published ahead of print on 29 January 2010.

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