The RFX protein, RfxA, is an essential regulator of growth ... · 1/29/2010 · 2 Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. 3 The

The RFX protein, RfxA, is an essential regulator of growth and 1

morphogenesis in Penicillium marneffei. 2

3

Hayley E. Bugeja, Michael J. Hynes and Alex Andrianopoulos*. 4

5

Department of Genetics, 6

University of Melbourne. 7

Victoria, Australia. 3010. 8

9

* corresponding author 10

Department of Genetics, 11

University of Melbourne. 12

Victoria, 3010. Australia. 13

Telephone: 61 3 8344 5164 14

Facsimile: 61 3 8344 5139 15

Email: [email protected] 16

17

Running Title: Regulation of growth and morphogenesis by RfxA 18

Key words: regulatory factor X (RFX), cell division, transcriptional regulation, 19

morphogenesis, fungal pathogen 20

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00226-09 EC Accepts, published online ahead of print on 29 January 2010

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Abstract 1

Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. 2

The opportunistic human pathogen Penicillium marneffei is a dimorphic fungus, displaying 3

vegetative (proliferative) multicellular hyphal growth at 25°C and unicellular yeast growth at 4

37°C. P. marneffei also undergoes asexual development to differentiated multicellular 5

conidiophores bearing uninucleate spores. These morphogenetic processes require regulated 6

changes in cell polarity establishment, cell cycle dynamics and nuclear migration. The RFX 7

(regulatory factor X) proteins are a family of transcriptional regulators in eukaryotes. We 8

sought to determine how the sole P. marneffei RFX protein, RfxA, contributes to the 9

regulation of morphogenesis. Attempts to generate a haploid rfxA deletion strain were 10

unsuccessful but we did isolate a rfxA+/rfxA∆ heterozygous diploid strain. The role of RfxA 11

was assessed using conditional overexpression, RNA-interference (RNAi) and the production 12

of dominant interfering alleles. Reduced RfxA function resulted in defective mitoses during 13

growth at 25°C and 37°C. This was also observed for the heterozygous diploid strain during 14

growth at 37°C. In contrast, overexpression of rfxA caused growth arrest during conidial 15

germination. The data shows that rfxA must be precisely regulated for appropriate nuclear 16

division and to maintain genome integrity. Perturbations in rfxA expression also caused 17

defects in cellular proliferation and differentiation. The data suggests a role for RfxA in 18

linking cellular division with morphogenesis particularly during conidiation and yeast growth, 19

where the uninucleate state of these cell types necessitates a tighter coupling of nuclear and 20

cellular division than that observed during multinucleate hyphal growth. 21

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Introduction 1

We are interested in examining the regulatory networks controlling cell-type specification and 2

development in the opportunistic fungal pathogen Penicilium marneffei, and the role these 3

processes play in pathogenicity. P. marneffei is a thermally dimorphic fungus capable of 4

causing disseminated infection in immunocompromised individuals. Dimorphism is a 5

common morphological process for many fungal pathogens and has been clearly linked to 6

pathogenicity. At room temperature (25°C), P. marneffei exhibits mycelial growth where 7

multinucleate cells are connected in long hyphal filaments. This growth form is also capable 8

of asexual development (conidiation) where single-celled uninucleate spores (conidia) are 9

produced on specialised aerial hyphae (conidiophores). The transition from a multicellular 10

hyphal growth form to a unicellular growth form occurs upon transfer to 37°C. During this 11

process, known as arthroconidiation, cellular and nuclear division become coupled and double 12

septa are deposited between cells. The subsequent fragmentation of these filaments leads to 13

the production of uninucleate yeast cells that divide by fission. It is this growth form of P. 14

marneffei that presents as an intracellular pathogen in phagocytic cells during infection (10, 15

25). 16

17

Transition between multicellular and unicellular morphological states is common to most 18

fungi and serve as an important process within developmental programs such as conidiation 19

and mating. The process of arthroconidiation in P. marneffei is analogous to the transition 20

from a hyphal growth form to a unicellular spore form in Acremonium chrysogenum and 21

Coccidiodes immitis, suggesting there may be common mechanisms underlying these events. 22

The cpcR1 gene of A. chrysogenum, initially identified as a regulator of cephalosporin C 23

biosynthesis genes, was subsequently shown to regulate arthrosporulation, whereby the 24

filamentous mycelium undergoes fragmentation into unicellular arthrospores (31, 56, 58). 25

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CPCR1 is a member of the Regulatory Factor X (RFX) family of transcriptional regulators 2

that have been implicated in the regulation of both developmental and cell cycle events. To 3

date seventeen members of this protein family have been isolated, which are highly conserved 4

from yeast to humans (21). The defining feature of these proteins is the novel RFX DNA-5

binding domain, a member of the winged-helix subfamily of helix-turn-helix DNA binding 6

domains (24). In addition, most of the RFX proteins contain a highly conserved dimerisation 7

domain mediating the formation of homo- and/or heterodimers (21, 52). While specific roles 8

have been assigned to individual RFX proteins, a unified understanding of the processes 9

regulated by these proteins across species has not been forthcoming. 10

11

Variation in tissue and cell-type specific expression of the five RFX genes (RFX1-5) 12

identified in mammals has been observed (33, 53). The prototypical RFX protein, RFX1, is 13

ubiquitously expressed and can act as both an activator and a repressor (34). Potential targets 14

of RFX1 include cell proliferation and DNA damage genes such as PCNA (proliferating cell 15

nuclear antigen), c-myc, MAP1A (microtubule-associated protein), IL-5Rα (interleukin-5 16

receptor α) and the RNR (ribonucleotide reductase) genes (33, 36, 42, 59, 72). Additionally, 17

the role of RFX5 in the regulation of MHC Class II gene expression is well established (60). 18

19

The Caenorhabditis elegans and Drosophila melanogaster RFX proteins, DAF-19 and dRFX, 20

respectively, have recently been assigned roles in the development of ciliated sensory neurons 21

(17, 61). D. melanogaster dRFX2 appears to be involved in the regulation of cell cycle 22

progression, a theme also evident to varying extents in its fungal counterparts (47). In 23

Saccharomyces cerevisiae the RFX homologue Crt1 prevents the expression of the DNA 24

damage-inducible RNR genes in the absence of DNA damage, through recruitment of the 25

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Tup1-Ssn6 co-repressor complex (32). The sak1+ protein of Schizosaccharomyces pombe has 1

been shown to function downstream of protein kinase A (PKA), where it promotes mitotic 2

exit and thereby allows the onset of sexual development or entry into stationary phase (67). 3

Deletion of sak1+ in S. pombe results in lethality, with transient phenotypes indicative of 4

severe mitotic defects. More recently, Rfx2 of the dimorphic pathogen Candida albicans was 5

found to not only regulate elements of the DNA damage response, presumably by a similar 6

mechanism to Crt1 in S. cerevisiae, but also morphogenesis and virulence (27). 7

8

Here, the role of the RFX protein, RfxA, has been investigated during growth and 9

morphogenesis of P. marneffei. The rfxA gene appears to be essential for the viability of P. 10

marneffei and studies involving overexpression, RNA-interference and the production of 11

dominant interfering alleles show that the levels of functional RfxA must be precisely 12

maintained for growth and morphogenesis, suggesting that RfxA participates in the regulation 13

of cell division events. As such, RfxA may be required for linking cell cycle regulation with 14

cellular differentiation during morphogenesis in P. marneffei. 15

16

Materials and Methods 17

Molecular techniques 18

Plasmid DNA was isolated using the Wizard Plus SV DNA Purification System (Promega). 19

Genomic DNA was prepared from frozen mycelia of P. marneffei as previously described (7). 20

For the extraction of RNA, fungal cultures were grown as previously decribed (6). RNA was 21

extracted from 0.1-0.2 g of biomass using the FastRNA Pro Red kit (Bio101). Southern blots 22

were prepared with Hybond N+ membrane (Amersham) using standard procedures (55). For 23

screening of the P. marneffei genomic DNA lambda library, plaque lifts and the isolation of 24

positive clones was performed according to the instructions of the λBlueSTAR vector system 25

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kit (Novagen). Hybridisations were performed with [α-32

P]-dATP-labelled DNA probes using 1

standard methods (55). Oligonucleotides used for PCR are listed in Table 1. RT-PCR was 2

performed using the rfxA specific primers L13 and Q50, benA specific primers F58 and F59, 3

and mobA specific primers HH21 and HH22 on 100 ng of total RNA using the Superscript 4

one-step RT-PCR kit with Platinum Taq (Invitrogen), according to the manufacturers 5

directions. The number of amplification cycles was optimised for each primer pair to ensure 6

that product synthesis was in the exponential phase of amplification. For the Product yields 7

were estimated from ethidium bromide stained gel images using MacBASver2.1 (Fuji 8

PhotoFilm Co. and Kohshin Graphic Systems Inc.). PCR screening of putative rfxA deletion 9

transformants was performed using three primers: L31, specific to the wild-type rfxA locus; 10

L29, a pyrG+ specific primer and L30 an rfxA genomic locus specific primer. A 1.5 kb 11

product generated using the primers L31 and L30 was expected for strains containing the 12

wild-type rfxA locus, while the presence of the rfxA∆::pyrG+ deletion locus would give rise to 13

a 1.9 kb product using the primers L29 and L30. For quantitative real-time RT-PCR, 2 µg of 14

total RNA was subjected to DNase treatment using the RQ1 RNase-free DNase (Promega) 15

prior to cDNA synthesis, performed with the Reverse Transcription System (Promega), 16

according to the manufacturer’s specifications. Real-time PCR was performed in a Rotor-gene 17

RG-3000 instrument (Corbett Research) on ~20 ng of cDNA using the SensiMix Plus SYBR 18

detection kit (Quantace) with an initial denaturation for 10 min at 95°C, followed by 45 19

cycles using the following parameters: 95°C for 20 seconds, 60°C for 20 seconds and 72°C 20

for 15 seconds. The relative fold expression of the sldA (primers FF20 and FF21) and separin 21

(primers FF18 and FF19) transcripts was determined using the comparative Ct method (37), 22

where samples were normalised to the actin (primers GG2 and GG3) transcript abundance to 23

adjust for variations in sample loading amounts. 24

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Cloning and plasmid construction 1

A 650 bp fragment of rfxA was isolated from degenerate PCR performed on genomic DNA of 2

the wild-type strain 2161 using the primers J72 and J73, designed to amplify the region 3

encoding the putative DNA-binding domain which is highly conserved in RFX proteins from 4

the filamentous fungi A. chrysogenum, N. crassa, A. fumigatus and P. chrysogenum. This 5

PCR fragment was used to probe a P. marneffei genomic DNA λ library at high stringency 6

(50% formamide, 0.1X SSC, 65°C) to clone the full-length rfxA gene, contained within the 7

plasmid pHS5584. A 5.9 kb KpnI/SacII sub-clone of P. marneffei rfxA (pHS6520) was 8

generated by combining a 1.4 kb KpnI/SpeI fragment containing the 5’ non-coding region of 9

rfxA with a 4.5 kb SpeI/SacII fragment containing the entire rfxA coding region and additional 10

3’ non-coding region, both derived from pHS5584. 11

A two-step cloning strategy was used to generate the rfxA gene deletion construct pHS5595. 12

First, a 1.1 kb XhoI/EcoICRI fragment of pHS5584, containing the 5’ region of rfxA including 13

the first 149 bp of coding sequence was inserted between the XhoI/EcoRV sites upstream of 14

the A. nidulans pyrG blaster cassette in pAB4626. Subsequently, a 1.4 kb SmaI/SacII 15

fragment of pHS5584, containing the region 3’ of rfxA including the last 132 bp of coding 16

sequence, was inserted downstream of the A. nidulans pyrG blaster cassette. 17

To generate the xylP(p)::rfxA-RNAi construct pHS6521 the 5’ coding region of rfxA (1- 1256 18

bp) was amplified by PCR using the primers Q34 and L6 and blunt cloned into the SmaI site 19

of pBluescript II SK+ (pHS6098). Subsequently, a 1.3 kb ClaI/SmaI and 1.3k b 20

EcoRV/BamHI fragment of pHS6098 were inserted either side of GFP in the vector 21

pAA4111, in opposite orientations. A 3.4 kb NcoI fragment containing GFP flanked by the 22

inverted repeats of the rfxA 5’coding sequence was then inserted downstream of the xylP 23

promoter in the plasmid pHS6103, containing a 2.6 kb EcoRI fragment of areA for targeting 24

to the areA locus (H. Bugeja, M.J. Hynes and A. Andrianopoulos, unpublished). 25

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To create a xylP(p)::rfxA construct a 2.7 kb PCR product containing rfxA was generated using 1

the primers L32 and L33 and blunt cloned into the SmaI site of pBluescript II SK+. An NcoI 2

fragment was isolated by partial digestion and cloned downstream of the xylP promoter in 3

pXylNOM (70)(pHS5705). Since this construct lacked the correct ATG for rfxA, plasmid 4

pHS5705 was modified using partial digestion with NcoI to remove 0.7 kb of 5’ rfxA coding 5

sequence. A second partial NcoI digestion and end-fill reaction was performed to destroy the 6

3’ NcoI site (pHS6097). A 0.8 kb NcoI fragment from pHS6098 was inserted into the 5’ NcoI 7

site of pHS6097 to generate a construct where the entire rfxA coding sequence is downstream 8

of the xylP promoter (pHS6099). In order to target this construct to the areA locus, a 4.5 kb 9

XhoI/SphI fragment of pHS6099, containing part of xylP(p) along with the rfxA coding 10

sequence and trpC(t), was used to replace an equivalent 1.7 kb fragment of pHS6103 giving 11

rise to pHS6529. 12

Inverse PCR using the xylP(P)::rfxA construct (pHS6099) as a template and the primers Q36 13

and Q37, was used to delete the region encoding the conserved DNA-binding domain (721-14

1099). The rfxA-DIM∆ construct pHS6530 was generated by inverse PCR using the 15

xylP(P)::rfxA construct (pHS6099) template and the primers L31 and L13, thereby removing 16

encoding the putative dimerisation domain (1673-2566). To facilitate targeted integration of 17

these constructs at the areA locus in the areADBD∆

strain 41.2.14-3 the constructs contain a 18

2.6kb EcoRI fragment of areA. 19

20

Fungal strains and media 21

The P. marneffei strains used in this study are listed in Table 2. The isolation and 22

transformation of P. marneffei protoplasts was performed as previously described (7). For 23

selection of pyrG+ transformants of strain SPM4, protoplasts were regenerated on osmotically 24

stabilised protoplast medium (PM) containing 1.2 M sucrose and 10mM ammonium tartrate 25

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(NH4)2T. For niaD+ selection, 10 mM sodium nitrate (NaNO3) was used as a sole nitrogen 1

source, whereas 10 mM sodium nitrite (NaNO2) was used as a nitrogen source for selection of 2

areA+ transformants from the areA

DBD∆ strain 41.2.14-3. Strains were grown on 1% glucose 3

minimal medium (ANM) (14), yeast synthetic dextrose medium (SD) (5), or brain heart 4

infusion (BHI) medium (Oxoid). When required medium was supplemented with 10 mM 5

uracil to allow the growth of pyrG- strains. For induction of the xylP promoter, 0.5% xylose 6

and 0.5% sucrose was used in place of 1% glucose (non-induced). The DNA replication 7

inhibitor hydroxyurea (HU) was used at a final concentration of either 2 mM, 5 mM or 8

10mM. To assess the nuclear division arrest, spores were germinated on slides coated with 9

solid medium under inducing conditions for 18 hours in the presence or absence of 2mM HU 10

before being processed for microscopic analysis (see below). Nuclear counts (approximately 11

150 germlings) were determined (n=2). 12

13

Microscopy 14

Strains were grown on slides coated with a thin-layer of solid medium with one end 15

submerged in liquid medium as described previously (6). Slides were fixed by soaking them 16

in a solution of 4% para-n-formaldehyde in PME (50 mM PIPES pH 6.7, 1 mM MgSO4, 20 17

mM EGTA) for 30 minutes, followed by two 5 minute PME washes. Samples were stained 18

using fluorescent brightener 28 (calcofluor white, CAL), Hoechst 33258 or 4’6’-diamino-2-19

phenylindole (DAPI) and visualised using a Reichart Jung Polyvar II microscope with either 20

differential interference contrast (DIC) or epifluorescence optics. Images were captured using 21

a SPOT CCD camera (Diagnostic Instruments) and processed using Adobe Photoshop 22

software. 23

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Sequencing and Bioinformatics 1

DNA sequencing was performed at the Australian Genome Research Facility (AGRF) on 2

purified plasmid DNA. DNA sequence was analysed using Sequencher 3.1.1 (Gene Codes). 3

All sequence analyses, including database searches, were done using the Australian National 4

Genomic Information Service (ANGIS). Pairwise sequence comparisons were performed 5

using the GAP program available through ANGIS and multiple sequence alignments were 6

generated using ClustalW (62) and MacBoxShade. In some instances, sequence data was 7

obtained from the following fungal genome databases: A. nidulans 8

(www.broad.mit.edu/annotation/fungi/aspergillus/); Neurospora crassa 9

(www.broad.mit.edu/annotation/fungi/neurospora/); Aspergillus fumigatus 10

(www.tigr.org/tdb/e2k1/afu1/); S. cerevisiae (www.yeastgenome.org/). 11

Upstream sequences (1000 bp) of annotated genes from A. nidulans, A. fumigatus, Aspergillus 12

terreus and Aspergillus oryzae were downloaded 13

(www.broad.mit.edu/annotation/genome/aspergillus_group/Downloads.htm) and searched for 14

the putative RfxA recognition sequence RTHNYYN{0,3}RGNAAC using the DNA pattern 15

search available at RSAT (http://rsat.ulb.ac.be/rsat/) (65). Common gene sets from Aspergilli 16

containing putative RfxA binding sites were then identified from protein clusters available 17

from the Aspergillus comparative database (www.tigr.org/sybil/asp/index.html) based on 18

preliminary sequence data obtained from The Institute for Genomic Research website at 19

http://www.tigr.org. Putative RfxA target genes were functionally assigned based on the Gene 20

Ontology Consortium classification and description available at http://www.geneontology.org 21

(4). 22

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Results 1

Isolation of P. marneffei rfxA containing the highly conserved RFX DNA-binding 2

domain 3

A degenerate PCR approach was used to isolate a region of P. marneffei rfxA encoding the 4

conserved DNA-binding domain, and this was subsequently used to clone the full-length rfxA 5

gene from a P. marneffei genomic DNA λ library. A 5.8 kb KpnI/SacII sub-clone of P. 6

marneffei rfxA (pHS6520) was sequenced (Genbank accession number DQ666366) and the 7

predicted rfxA gene encodes a protein of 861 amino acids. Orthologues of rfxA were detected 8

in the genome sequences of many filamentous fungi and the P. marneffei RfxA protein 9

contains many sequence features previously characterised in other eukaryotic RFX factors 10

(21)(Fig. 1A). Most importantly, P. marneffei RfxA contains the RFX DNA-binding domain, 11

which is highly conserved among all family members (Fig. 1B), in addition to a putative 12

dimerisation domain (Fig. 1C). It also contains regions common to many transcriptional 13

activators such as a glutamine (Q) rich region (RfxA residues 70-117; 21% Q), and a highly 14

acidic aspartate (D) and glutamate (E) rich region (RfxA residues 782-856; 24.3% D/E). 15

Homology regions B and C, which have not been functionally characterised in RFX proteins, 16

are also present within P. marneffei RfxA. 17

18

The expression of rfxA is up-regulated during conidiation and yeast growth 19

To examine rfxA expression during growth and morphogenesis of P. marneffei, semi-20

quantitative reverse transcriptase PCR (RT-PCR) was performed using RNA isolated from 21

vegetative hyphal cells, cells undergoing asexual development (conidiation) at 25°C, and 22

yeast cells cultured at 37°C. The abundance of rfxA transcript was found to be relatively low 23

during vegetative hyphal growth at 25°C and was approximately 2-fold and 5-fold higher 24

during yeast growth at 37°C and conidiation at 25°C, respectively (Fig. 2). Consistent with 25

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the increased expression of rfxA during conidiation, potential binding sites were identified for 1

the BrlA (5), StuA (1) and AbaA (1) transcriptional regulators within the rfxA promoter 2

region (data not shown; (3, 11, 18). These proteins are involved in mediating the correct 3

temporal and spatial expression of genes involved in conidiation in P. marneffei (A. R. 4

Borneman, K. Tan, M. J. Hynes and A. Andrianopoulos, unpublished, (6, 8). 5

6

Potential RfxA target genes include cell cycle regulators 7

To understand the cellular process(es) which RfxA may regulate, we used a bioinformatic 8

approach to identify putative RfxA target genes in the sequenced and annotated genomes of 9

the P. marneffei-related fungi A. fumigatus, A. nidulans, A. oryzae and A. terreus. These fungi 10

are sufficiently diverged to reveal conserved functional regulatory elements, such as RFX 11

binding sites. This unbiased approach took advantage of the highly conserved DNA binding 12

domain across all the RFX proteins and used the consensus RFX binding sequence 13

RTHNYYN0-3RGNAAC to search the 5’-UTRs of all genes in these fungi. Approximately 14

2500-3300 genes were identified in each species that contained at a least one potential RFX 15

binding site. The presence of a single site has been found to be sufficient to confer regulation 16

in other systems (19, 71). Given that a large proportion of these may represent chance 17

occurrences of the consensus target sequence, the data set was restricted to include only those 18

sites that were present in the promoter sequences of orthologous genes in all four species. 19

This resulted in the identification of 75 orthologous genes containing at least one putative 20

RFX binding site. In six instances the identified binding sites were located in overlapping 21

divergent promoters present in all four species. In these cases it is possible that only one 22

member of the gene pair may represent an RfxA target gene. 23

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Of these 75 genes, no predicted cellular function could be found for 29 (39%) based on either 1

GO annotation or conservation with previously characterised genes. Of the remaining 46 2

genes, 19 (41%) represent genes involved in cell cycle regulation, in particular mitotic spindle 3

dynamics and exit from mitosis, and the rest (27 genes, or 59%) are divided amongst a variety 4

of cellular processes including cellular metabolism, protein synthesis and degradation, 5

chromosome metabolism and cytoskeleton dynamics. In many of the genes examined, the 6

positions of the putative RFX binding sites relative to the initiator codon were also found to 7

be conserved across all four species and in the P. marneffei orthologues (see Table 3). 8

9

In an attempt to understand the regulatory role of RfxA, preliminary expression analysis of 10

nine putative rfxA target genes representing a range of cellular processes related to cell 11

division (see Table 3) was performed using either semi-quantitative or real time RT-PCR. 12

Expression of these genes was examined in hyphal (25°C) and/or yeast (37°C) cells with 13

either reduced or increased rfxA expression using rfxA-RNAi and rfxA OE strains, 14

respectively (Fig. 3A; see below). Of these, six genes orthologous to S. pombe cdc4, cdc15 15

and src1 and A. nidulans kipB, nimA, mpsA and sldA showed no consistent change in 16

expression level when RfxA was either over- or under-expressed, indicating that they are not 17

targets of RfxA under the conditions examined. The remaining genes are known to function in 18

spindle dynamics, chromosome cohesion and mitotic exit. P. marneffei mobA, an orthologue 19

of the S. cerevisiae MOB1 gene encoding a protein kinase required for maintenance of ploidy, 20

showed increased expression in the rfxA-RNAi at 25°C (Fig. 3A). P. marneffei bimB, a 21

homologue of genes encoding separin/separase involved in cleavage of cohesin and sister-22

chromatid separation, showed increased expression in both the rfxA-RNAi and rfxA OE 23

strains under induced conditions during filamentous growth at 25°C, but not during yeast 24

growth at 37°C (Fig. 3B). While these results indicate that P. marneffei RfxA may play a role 25

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in spindle dynamics and mitotic exit, the small fold changes observed may also be a 1

consequence of the experimental conditions required for induction of the dominant alleles in 2

these strains and the inherent asynchrony produced or an indirect consequence of perturbed 3

cell division (see below). Isolation of a conditional allele of RfxA would be important for 4

further investigation. In addition direct DNA-binding studies would be required to distinguish 5

primary RfxA targets from changes in gene regulation due to secondary effects resulting from 6

the perturbed growth observed in strains with reduced or enhanced RfxA function. 7

8

rfxA is essential for viability in P. marneffei 9

To characterise the role of rfxA during growth and development in P. marneffei we attempted 10

to isolate a strain containing a deletion of the single rfxA gene. Strain SPM4 (pyrG-, niaD

-) 11

was transformed with a linear 4.7 kb XhoI/SacII fragment of the rfxA gene deletion construct 12

pHS5595 (Fig. 4A) to replace the majority of the rfxA coding region with the Aspergillus 13

nidulans pyrG+ blaster cassette (8). A total of 246 pyrG

+ transformants were isolated and 14

subsequently screened for integration of the rfxA deletion construct at the rfxA locus using 15

either PCR or Southern blot analysis. Despite the large number of transformants screened a 16

strain lacking the rfxA coding sequences was not identified (data not shown). 17

18

One transformant (44.1.2) was identified which was found by PCR screening to contain both 19

rfxA+ and rfxA∆::pyrG

+ alleles (data not shown) and this was confirmed by Southern blot 20

analysis (Fig. 4B). As P. marneffei is a haploid organism, this result suggested that this 21

represented either a heterokaryotic strain containing nuclei of different genotypes or a 22

heterozygous diploid. The isolation of such strains is not uncommon in many fungi and is 23

often used to study uncharacterised, potentially essential genes (45). While actively growing 24

vegetative hyphal cells are predominantly multinucleate, during conidiation single nuclei are 25

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partitioned into the conidial-producing sterigmata cells, and subsequently into conidia. 1

Genotyping of colonies isolated from purified uninucleate conidia using growth tests and 2

PCR screening showed that the uninucleate conidia of strain 44.1.2 were genetically identical 3

and contained both rfxA+ and rfxA∆::pyrG

+ alleles, showing that these isolates represented 4

heterozygous diploids (data not shown). Microscopic examination of DAPI-stained conidia of 5

strain 44.1.2 verified that each conidium contained only a single nucleus, and that these 6

conidia were larger than the control haploid strain (Fig. 4C). The average volume of 44.1.2 7

conidia was 159.2 ± 23 µm3 (±S.D, n=30), approximately 2-3 times that of the haploid rfxA

+ 8

control strain (33.4.2), 63.6 ± 10.6 µm3. It is well established that conidial size increases with 9

ploidy in fungi (12) and therefore this data is fully consistent with the hypothesis that 44.1.2 10

represents a heterozygous rfxA+/rfxA∆::pyrG

+ diploid strain. The isolation of a diploid strain 11

heterozygous for deletion of rfxA, and the inability to obtain a haploid rfxA deletion strain, 12

strongly suggests that rfxA is an essential gene of P. marneffei. 13

14

Haploinsufficiency of rfxA leads to reduced growth, nuclear division defects and 15

genomic instability at 37°C 16

It was anticipated that deletion of a single copy of rfxA in the rfxA+/rfxA∆::pyrG

+ 17

heterozygous diploid strain (44.1.2) would have no effects on growth since this strain also 18

contains a wild-type allele of rfxA and is therefore able to produce a functional RfxA protein. 19

However, while the ability of the rfxA+/rfxA∆::pyrG

+ strain (44.1.2) to undergo filamentous 20

growth at 25°C was indistinguishable from the wild-type control (2161), growth of this strain 21

was significantly reduced under conditions of yeast growth at 37°C (Fig. 5A). Microscopic 22

examination of the rfxA+/rfxA∆::pyrG

+ strain during growth at 37°C revealed multiple cellular 23

defects including the presence of swollen and irregularly shaped arthroconidial hyphae and 24

cell lysis. Using Hoechst staining of DNA very few intact nuclei were observed and instead 25

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enlarged nuclear masses, characteristic of endoreplication (DNA re-replication in the absence 1

of nuclear division), and small nuclear fragments of irregular shape were prevalent (Fig. 5B). 2

3

The genetic stability of the rfxA+/rfxA∆::pyrG

+ strain was also compromised during growth at 4

37°C. This was initially apparent upon isolation of single cells (protoplasts) from the 5

multicellular arthroconidial hyphae of strain 44.1.2 grown at 37°C, as approximately 90% of 6

viable protoplasts had reverted to uracil auxotrophy (Fig. S2). Genotyping of these putative 7

haploid isolates by PCR confirmed loss of the rfxA∆::pyrG+ allele associated with the onset of 8

uracil auxotrophy, therefore these strains represent wild-type rfxA+ haploids derived from the 9

rfxA+/rfxA∆::pyrG

+ strain (data not shown). The remaining protoplasts displayed uracil 10

prototrophy and contained both rfxA+ and rfxA∆::pyrG

+ alleles as verified by PCR, however 11

many of these isolates displayed an abnormal morphology (Fig. S2, A and B). These strains 12

were highly unstable and frequently reverted to a wild-type growth phenotype, which was 13

subsequently stable (Fig. S2C). No haploids containing only the rfxA∆::pyrG+ allele were 14

isolated. In fungi, the generation of haploid isolates from a diploid progenitor (haploidisation) 15

occurs via random chromosome loss due to nondisjunction of chromosomes, either as a result 16

of genetic mutation or the presence of genotoxic compounds (30, 64). The uracil prototrophic 17

colonies displaying the abnormal growth morphology phenotype are likely to represent 18

aneuploid strains that have not undergone complete haploidisation, since both wild-type and 19

rfxA deleted chromosomes are being maintained,. Reversion to the wild-type growth 20

phenotype may result from imbalanced chromosome segregation in the aneuploid strains 21

leading to restoration of the diploid state. Both the observed nuclear fragmentation and 22

haploidisation of the heterozygous diploid during growth at 37°C, but not at 25°C, is 23

suggestive of nuclear division defects, possibly due to haploinsufficiency of rfxA. In addition, 24

the observation that only wild-type haploids containing the rfxA+ allele, and not the 25

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rfxA∆::pyrG+ allele, were isolated provides additional evidence that a functional rfxA gene is 1

required for the viability of P. marneffei. 2

3

Reduced RfxA levels result in cell division defects 4

To study the consequences of reduced rfxA expression an RNAi strategy was developed in 5

order to silence the endogenous rfxA transcript by driving high-level expression of a rfxA 6

hairpin transcript. For this purpose the construct pHS6521 was generated in which a spacer 7

fragment (GFP) was flanked by inverted repeats of the first 1256 bp of rfxA 5’ coding 8

sequence and placed downstream of the xylP promoter of Penicillium chrysogenum, which is 9

effective in inducing high-level gene expression in P. marneffei in the presence of xylose (51, 10

70). This construct was targeted to the areA locus giving rise to strain 55.2.1. Using semi-11

quantitative RT-PCR, the abundance of the endogenous rfxA transcript was found to be 12

significantly reduced upon induced expression of the rfxA-RNAi hairpin transcript in strain 13

55.2.1 (xylP(p)::rfxA-RNAi) at both 25°C and 37°C (Fig. 3A; data not shown). 14

15

At 25°C the induced expression of the rfxA-RNAi hairpin transcript in strain 55.2.1 resulted 16

in severe growth inhibition. Microscopic examination of colonies revealed multiple growth 17

defects under inducing conditions including aseptate and anucleate hyphae, and deformed 18

apical hyphal cells and lateral branches, often with multiple short swollen hyphal tips 19

exhibiting cell lysis (Fig. 6A). In the older hyphal cells at the centre of the colony the few 20

nuclei that were observed displayed an abnormal highly elongated appearance. 21

22

Due to the severe growth inhibition resulting from over-expression of the rfxA-RNAi hairpin 23

transcript, the effects on conidiation could only be examined under conditions of delayed 24

induction. Abnormal conidiophore morphogenesis was apparent upon induced expression of 25

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the rfxA-RNAi hairpin transcript, with a range of phenotypes observed (Fig. 6B). This 1

included sterigmata cells and conidia that appeared engorged and contained large numbers of 2

nuclei, demonstrating defects in nuclear partitioning. At the most severe end of the spectrum, 3

conidiophores lacked appropriate differentiation of the sterigmata cell types, and consisted of 4

conidiophore stalks with aberrant swollen and misshapen sterigmata cells. These cells lacked 5

intact nuclei and instead contained fragmented nuclear structures. This suggests that reduced 6

rfxA function leads to defects in nuclear division and segregation during differentiation of the 7

uninucleate cell types of the conidiophore. 8

9

The xylP(p)::rfxA-RNAi strain also displayed reduced growth under conditions required for 10

yeast morphogenesis at 37°C. Microscopic examination revealed growth phenotypes similar 11

to the heterozygous rfxA+/rfxA∆::pyrG

+ diploid strain. This included severe growth 12

phenotypes such as cell lysis and the presence of swollen hyphae, which lacked intact nuclear 13

structures and instead contained small nuclear fragments (Fig. 6C). Although reminiscent of 14

arthroconidial hyphae, these cells were aseptate and no hyphal fragments or yeast cells, 15

indicative of arthroconidiation, were observed. 16

17

Overexpression of rfxA results in cell division defects and growth arrest 18

To examine the role of rfxA during growth and morphogenesis further, the rfxA coding region 19

was inserted downstream of the xylP promoter in the plasmid pHS6259 in order to drive high 20

levels of rfxA expression in a xylose-inducible manner. This construct was targeted to the 21

areA locus in single copy. Growth of the xylP(p)::rfxA strain 49.3.1 was almost completely 22

inhibited under inducing conditions at 25°C. Growth arrest was observed at an early stage of 23

germination coinciding with germ-tube elongation and the presence of either one (no nuclear 24

division) or two (one nuclear division) nuclei (data not shown). A small proportion of the 25

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inoculum was able to commence growth and microscopic examination of these hyphal cells 1

during overexpression of rfxA revealed a drastic reduction in cell size and an increase in the 2

number of nuclei partitioned into each sub-apical cell compartment (Table 4). Additional 3

phenotypes identified include an increased occurrence of lateral branches, some resembling 4

short undifferentiated conidiophore stalks (Fig. 7A). These phenotypes were also observed 5

under conditions of delayed induction (see below). 6

7

To assess conidiophore morphogenesis upon overexpression of rfxA, strain 49.3.1 was 8

examined microscopically under conditions of delayed induction. The induced overexpression 9

of rfxA did not lead to any major conidiation defects, however an increase in the frequency of 10

conidiophores, particularly towards the periphery of the colony, was observed. Furthermore, 11

many of these conidiophores appeared rudimentary with shorter stalks and reduced abundance 12

of sterigmata cell types (Fig. 7B). 13

14

Under inducing conditions at 37°C a complete lack of growth was observed for the 15

xylP(p)::rfxA strain (data not shown). Microscopic examination revealed that while many 16

conidia appeared to have initiated germination, as shown by the presence of enlarged cells 17

with some protruding short germ-tubes, the growth of these cells appeared to be arrested with 18

only a single nucleus observed (Fig. 7C). 19

20

Both the DNA-binding and dimerisation domains of RfxA are required to modulate its 21

function 22

The predicted P. marneffei RfxA protein contains the highly conserved RFX DNA-binding 23

domain, as well as a putative dimerisation domain. In some cases these domains have been 24

shown to function independently, where the formation of either homo- or heterodimers is not 25

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a prerequisite for DNA-binding in vitro and protein dimers can be formed when the 1

dimerisation domain is fused to a heterologous DNA-binding domain (21, 35, 52). To assess 2

the co-dependence of these domains for the normal functioning of RfxA, mutant alleles of 3

rfxA encoding RfxA proteins lacking either the DNA-binding domain (DBD∆) or 4

dimerisation domain (DIM∆) were inserted downstream of the xylP promoter in the constructs 5

pHS6522 and pHS6530, respectively. These constructs were targeted to the areA locus. 6

7

During filamentous hyphal growth at 25°C, overexpression of either the rfxA-DBD∆ (56.4.4) 8

or rfxA-DIM∆ (49.4.1) alleles had no effect on growth (data not shown). During yeast 9

morphogenesis at 37°C, overexpression of the rfxA-DIM∆ resulted in slightly reduced growth, 10

however no morphological defects were apparent upon microscopic examination. In 11

comparison, overexpression of the rfxA-DBD∆ allele caused a severe reduction in growth at 12

37°C with cell lysis apparent upon microscopic examination, as well as nuclear division 13

defects characterised by enlarged nuclear masses, possibly due to endoreplication, and nuclear 14

fragmentation (data not shown). These phenotypes recapitulated those previously observed 15

for the rfxA+/rfxA∆ strain 44.1.2 and the induced xylP::rfxA-RNAi strain, during yeast 16

morphogenesis at 37°C. 17

18

Reduced rfxA function leads to defective checkpoint regulation 19

In light of the cellular division defects observed in response to a reduced level of rfxA 20

expression in the xylP(p)::rfxA-RNAi strain we assessed the response of this strain to known 21

inhibitors of the cell cycle. Hydroxyurea (HU), a direct inhibitor of ribonucleoside 22

diphosphate reductase (RNR) function, results in activation of the S-phase checkpoint by 23

preventing completion of DNA replication (16). In addition, the microtubule destabilising 24

compound, Benomyl, causes a block in late mitosis due to activation of the spindle checkpoint 25

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pathway in response to the formation of a defective mitotic spindle (49). Mutants defective in 1

the activation of checkpoints in response to a transient exposure to HU or Benomyl, display 2

reduced viability due to the completion of defective mitotic divisions (20, 23). Both the wild-3

type rfxA+ and xylP(p)::rfxA-RNAi strains were germinated for 24 hours in the presence of HU 4

(2 or 5 mM) or Benomyl (0.01, 0.05, 0.2 or 0.5 µg mL-1

), under both inducing or non-5

inducing conditions prior to being assessed for viability. The wild-type (rfxA+) and 6

xylP(p)::rfxA-RNAi strains displayed a similar viability after transient exposure to Benomyl 7

and under non-inducing conditions in the presence of HU (data not shown). The reduced 8

expression of rfxA in the xylP(p)::rfxA-RNAi strain under inducing conditions caused a 9

significant reduction in viability after treatment with HU (Fig. 8). A similar reduction in 10

viability was observed for the xylP(p)::rfxA-RNAi strain when germlings were exposed to HU 11

for only 6 hours. However, the ability to arrest nuclear division in response to HU was not 12

affected in the xylP(p)::rfxA-RNAi. Approximately 15% of germlings of the xylP(p)::rfxA-13

RNAi (17.9% ±8.6, S.E.M) and wild-type control (15% ±5.5) strains underwent nuclear 14

division in the presence of HU, in contrast to 50% of germlings in the absence of HU (49.2% 15

±7.9 and 47.4% ±6.8 for the xylP(p)::rfxA-RNAi and wild-type control strains, respectively). 16

Thus, although mitosis is inhibited, presumably via activation of the S-phase checkpoint, 17

reduced RfxA function prevents the appropriate response to DNA replication inhibition to 18

ensure survival. Given that the exposure to HU was transient in nature, this may include the 19

ability to slow S-phase progression, stabilise and subsequently re-activate stalled replication 20

forks, thus preventing double stranded DNA breaks, and facilitate DNA repair. 21

22

Discussion 23

Alterations in the expression of rfxA lead to cellular division and growth defects 24

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The rfxA gene of P. marneffei is a member of the RFX transcription factor family, involved in 1

the regulation of cellular differentiation events in eukaryotes. The data presented here shows 2

that rfxA is essential for viability and alterations in the levels of its expression lead to cell 3

division defects with dramatic consequences for growth and morphogenesis. Despite 4

numerous attempts, a haploid rfxA deletion strain could not be isolated. Instead the isolation 5

of a heterozygous diploid strain from which only wild-type haploids could be recovered, 6

suggests that rfxA is essential for survival. Examination of strains with reduced or enhanced 7

rfxA expression demonstrated that rfxA might be involved in cell cycle regulation, consistent 8

with the essential nature of this gene product. In A. nidulans numerous genes involved in cell 9

cycle regulation are essential for viability and were initially identified as temperature sensitive 10

lethal mutants (46). 11

12

The overexpression of rfxA resulted in growth arrest during conidial germination. Although 13

the nature of this growth arrest is unclear, this suggests a role for RfxA in the reactivation of 14

growth in dormant conidia. The observation that hyphal cells display increased nuclear 15

division kinetics (increased numbers of nuclei per cell and shorter cell length) when rfxA is 16

over-expressed implicates RfxA in the temporal regulation of genes affecting cell cycle 17

events. 18

19

In constrast, reduced levels of rfxA caused by expression of the RNAi transcript, resulted in 20

terminal degenerative phenotypes at 25°C characterised by hyphal cells that were aseptate and 21

either produced aberrant elongated nuclei, indicative of a block in mitosis, or were anucleate. 22

Similar phenotypes are observed in cell cycle mutants of A. nidulans affected in chromosome 23

metabolism (sepB, sepJ) and cohesion (bimB, bimD), spindle dynamics (bimC), telomere 24

maintenance (nimU/pot1) and mitotic exit (bimA) (15, 22, 28, 29, 40, 50, 66). In these 25

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mutants, the nuclear phenotypes result from premature entry into mitosis prior to completion 1

of DNA synthesis and/or failure to completely separate chromosomes during division, 2

preventing cells from exiting mitosis. The lack of septation observed for many of these 3

mutants is a secondary consequence of errors in DNA metabolism causing inhibition of 4

cytokinesis via activation of the DNA damage checkpoint pathway (29, 66). The aseptate 5

phenotype of the RNAi strain is also consistent with this hypothesis. 6

7

During growth at 37°C, reduced rfxA expression in the RNAi strain resulted in an inability to 8

undergo yeast morphogenesis. Septation, fundamental to the process of arthroconidiation and 9

the liberation of yeast cells, was not observed either as a direct consequence of reduced RfxA 10

function, or a secondary effect of the terminal degenerative phenotypes observed (cell lysis 11

and nuclear division defects). Mitotic catastrophe as evidenced by enlarged nuclear masses, 12

suggestive of endoreplication, and nuclear fragmentation, was readily apparent. These 13

phenotypes were recapitulated in the heterozygous diploid strain specifically during growth at 14

37°C, presumably as a consequence of gene dosage effects (haploinsufficiency). In support of 15

this hypothesis, haploinsufficiency has also been observed for CRT1/crt1∆ and RFX2/rfx2∆ 16

heterozygous diploids in S. cerevisiae and C. albicans, respectively (27, 32). Such nuclear 17

defects may also account for the apparent loss of genomic stability resulting in the 18

spontaneous haploidisation of the heterozygous diploid strain. Mutations affecting 19

components of the cell cycle apparatus, such as in genes sepB, sepJ, bimA, nimU (pot1) and 20

hfaB of A. nidulans, are known to result in a loss of genomic stability (28, 29, 50, 64, 66). 21

22

Given the role of the related RFX proteins in mediating the DNA damage response (DDR) in 23

closely related organisms, it is interesting to speculate whether RfxA is involved in a similar 24

mechanism in P. marneffei, particularly in light of the increased sensitivity to HU observed 25

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under conditions of rfxA RNAi. Importantly, RFX acts a negative regulator in its role as an 1

effector of the DDR, and thus leads to derepression of the target genes, including the 2

ribonucleotide reductase (RNR) encoding genes in the presence of HU (27, 32, 38). Thus, 3

reduced RFX function would be expected to promote survival in the presence of HU, whereas 4

the results presented here show that in P marneffei the converse is true. Putative RFX binding 5

sites have recently been identified in the promoters of both RNR encoding genes in P. 6

marneffei (data not shown) and the results suggesting that RfxA might positively regulates 7

these genes during DNA replication arrest. 8

9

If RfxA is involved in the DNA damage response in P. marneffei, this is unlikely to be its sole 10

function, as components exclusive to the DDR in closely related fungi are rarely essential for 11

survival under non-DNA damage conditions (26). Almost half of the potential RfxA target 12

genes to which cellular roles could be assigned represent gene products involved in cell cycle 13

events, with particular emphasis on chromosomal mechanics, spindle organisation and mitotic 14

exit. Two of theses putative targets (mobA and bimB) displayed increased expression in 15

strains with altered RfxA activity. The maintenance of ploidy kinase MobA, upregulated 16

during rfxA RNAi, causes defects in mitotic progression and septation when over-expressed in 17

fission yeast (54). The altered expression of the separase BimB, suggests that RfxA may 18

influence chromosome dynamics, by perturbing sister-chromatid cohesion (40, 63). 19

20

RfxA is important for linking cellular division with morphogenesis. 21

The data suggests that the function of RfxA in the regulation of cell cycle progression is 22

strongly influenced by the cellular context. While the overexpression of rfxA increases the 23

rate of cell division in hyphal cells, overexpression of rfxA in conidia caused growth arrest at 24

an early stage of germination. In addition, nuclear division defects were only observed for the 25

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heterozygous diploid and xylP::rfxA-DBD∆ strains during yeast growth at 37°C, and not 1

during filamentous growth at 25°C. During both conidiation and yeast morphogenesis in P. 2

marneffei, nuclear and cellular division are tightly coupled to ensure the maintenance of the 3

uninucleate state of these cell types. In contrast, hyphae are coenocytic/multinucleate and in 4

A. nidulans, aside from the initial nuclear divisions during germination, nuclear division 5

occurs in a parasynchronous wave (13). Thus, hyphal growth may be less sensitive to 6

perturbation in RfxA activity than the tightly regulated nuclear division events occurring 7

during development, yeast morphogenesis and possibly even during germination. 8

9

Regulated changes in cell cycle dynamics are often associated with cellular differentiation 10

events in fungi, including an increased rate of cell cycling during conidiation in A. nidulans 11

(43, 68). Effects on conidiophore morphogenesis were observed in P. marneffei when the 12

levels of rfxA expression were perturbed. Reduced rfxA expression in the rfxA-RNAi strain 13

led to the production of defective conidiophore structures, reflecting disturbances in the 14

coupling of mitosis with cytokinesis. Similar defects resulting in inappropriate conidiophore 15

morphogenesis result from aberrant checkpoint regulation in A. nidulans due to the NimXcdc2-

16

AF mutation, preventing negative regulation of the cyclin-dependent kinase by inhibitory tyr-17

15 phosphorylation (68). Interestingly, while over-expression of rfxA caused an increase in 18

mitotic cycling in basal hyphal cell compartments, the conidiophores produced were 19

morphologically normal, although increased in frequency. This may reflect the normal up-20

regulation of rfxA occurring during conidiation in wild-type cells. It is possible that RfxA 21

directly influences the onset of development through regulation of the transcriptional 22

regulator BrlA, a key component of the central regulatory pathway involved in conidiophore 23

development, since conserved RFX consensus binding sites were identified in the promoters 24

of brlA genes from the Aspergilli and P. marneffei, and induction of brlA expression has 25

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previously been shown to be sufficient for initiation of conidiation (1, 41)(A.R. Borneman, 1

K. Tan, M.. J. Hynes and A. Andrianopoulos, unpublished data). 2

3

Regulation of RfxA activity 4

The underlying question remains as to how RfxA integrates the regulation of cellular division 5

with differentiation events in the different cell types produced by P. marneffei. The possibility 6

that RfxA mediates its cell-type specific roles through interaction with additional regulatory 7

proteins in a cell-type dependent manner is supported by the analysis of strains over-8

expressing rfxA alleles lacking the regions encoding the DNA-binding or dimerisation 9

domains. Overexpression of the rfxA-DBD∆ allele, containing a functional dimerisation 10

domain, resulted in a dominant negative effect suggesting that regulation of RfxA function 11

occurs via protein interactions. This may involve one or more binding partners required for 12

appropriate regulation of target genes, which are sequestered by the RfxA-DBD∆ protein. A 13

similar mode of action has been proposed to account for the dominant interfering effects 14

resulting from a DNA-binding domain deleted version of human RFX1 on the regulation of 15

RFX1 target genes (38). In contrast, the putative dimerisation domain appears to be required 16

for protein function, since overexpression of a mutant allele lacking this domain has no 17

obvious phenotypic effects, contrary to overexpression of the wild-type allele. While the 18

stability of the RfxA-DIM∆ protein has not been assessed in this strain and could account for 19

the lack of phenotype, this finding is consistent with observations in A. chrysogenum where 20

the formation of CPCR1 dimers is essential for both DNA-binding and the interaction with 21

AcFKH1 (57, 58). These data suggest that the formation of RfxA homodimers and the 22

interaction with accessory proteins, possibly involving a forkhead protein, is necessary for 23

appropriate regulation of RfxA target genes. 24

25

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Recent evidence in fission yeast also supports a role for the interaction of RFX and Forkhead 1

proteins in cell cycle regulation. In S. pombe the Forkhead proteins Fkh2 and Sep1 are 2

required for the G2/M specific regulation of genes required for late mitotic events and 3

cytokinesis (9, 44). Interestingly, many of these forkhead-regulated genes have orthologues 4

identified as putative RfxA targets in this study. Both Fkh2 and Sep1 are associated with the 5

PCB (Pombe cell-cycle box) site in the promoters of these genes as components of a multi-6

protein transcription factor PBF (PCB-binding factor) (2, 48). Not only does the PCB 7

sequence reflect the 3’ half site of the RFX consensus sequence, and not a prototypical 8

forkhead consensus sequence, but additional consensus RFX and FKH binding sequences are 9

also present in the promoters of PCB regulated genes (H. Bugeja and A. Andrianopoulos, 10

unpublished). PBF has negative (mediated by Fkh2) and positive (mediated by Sep1) 11

regulatory roles at the promoters of PCB containing genes, (48) consistent with the context-12

dependent regulation of RFX target genes in other organisms. The S. pombe RFX protein 13

Sak1 has an essential, yet biochemically undefined role in the regulation of cell cycle events. 14

Recently, synthetic interactions between Sak1 and anaphase promoting complex/cyclosome 15

(APC/C) components, including separase, have been described (39, 69). Thus, its involvement 16

as a possible component of PBF is worthy of further investigation. 17

18

RFX proteins as regulators of cell division and differentiation in eukaryotes 19

In P. marneffei, the RFX protein RfxA is required for integrating the mitotic cell cycle with 20

cellular differentiation events in a cell context dependent manner. In other organisms where 21

RFX proteins have been found to be involved in cell cycle regulation, such as humans 22

(RFX1), Drosophila (dRFX2) and S. pombe yeast (Sak1), reduced RFX protein function also 23

has dramatic consequences for growth. This is not observed in S. cerevisiae, where crt1 24

mutants are viable, reflecting the underlying differences in checkpoint control mechanisms in 25

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these organisms. Aside from the role of RFX proteins in regulating cell cycle events or the 1

DNA damage response, these factors are also involved in the temporal or spatial regulation of 2

cellular differentiation events including ciliated sensory neuron development in D. 3

melanogaster (dRFX) and C. elegans (DAF-19), and arthrosporulation in A. chrysogenum 4

(CPCR1). Thus not only do RFX proteins have a fundamental role in cell cycle regulation, but 5

this is also manifested in the altered patterns of cell division required for morphogenesis in 6

developmentally complex organisms. 7

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Acknowledgements 1

This work was supported by grants to A. Andrianopoulos from the Australian Research 2

Council, the National Health and Medical Research Council and the Howard Hughes Medical 3

Institute. A. Andrianopoulos is a Howard Hughes Medical Institute International Scholar. H. 4

E. Bugeja was supported by an Australian Postgraduate Award. We thank H. Robertson and 5

K. J. Boyce for critical comments on the manuscript. 6

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FIG. 1. P. marneffei RfxA contains protein motifs characteristic of RFX proteins. (A) Protein 1

structure map of RfxA. Regions represented include the glutamine rich region (Q, residues 2

70-117), the RFX DNA-binding domain (DBD, residues 243-317), the highly conserved B 3

(residues 449-484) and C regions (residues 511-550), the putative dimerisation domain (DIM, 4

residues 567-741) and the acidic aspartate and glutamate rich region (DE, 782-856). (B) 5

Alignment of the region containing the DNA-binding domain from all characterised 6

eukaryotic RFX proteins. Protein sequences included in the alignment are: Homo sapiens 7

RFX1 (HsRFX1; P22670; 429-515); Mus musculus RFX1 (MmRFX1; P48377; 414-500); C. 8

elegans DAF-19 (CeDAF19; Q09555; 250-335); D. melanogaster RFX (DmRFX; Q9U1K2; 9

362-448); D. melanogaster RFX2 (DmRFX2; Q65YQ9; 379-465): A. chrysogenum CPCR1 10

(AcCPCR1; Q9P8F6; 214-303); S. pombe Sak1 (SpSak1; P48383; 91-178); S. cerevisiae Crt1 11

(ScCrt1; P48743; 275-362) and P. marneffei RfxA (PmRfxA; This study; ABG56532; 233-12

319). (C) Alignment of the dimerisation domain from RFX proteins. Protein sequences 13

included in the alignment are: H. sapiens RFX1 (HsRFX1; P22670; 743-909); M. musculus 14

RFX1 (MmRFX1; P48377; 727-893); C. elegans DAF-19 (CeDAF19; Q09555; 605-769); D. 15

melanogaster RFX (DmRFX; Q9U1K2; 686-851); A. chrysogenum CPCR1 (AcCPCR1; 16

Q9P8F6; 547-704); S. pombe Sak1 (SpSak1; P48383; 507-674) and P. marneffei RfxA 17

(PmRfxA; This study; ABG56532; 567-741). 18

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FIG. 2. Expression of rfxA is up-regulated during asexual development. Semi-quantitative 1

RT-PCR was performed on total RNA extracted during vegetative hyphal growth at 25°C 2

(25°C-V), conidiation at 25°C (25°C-C) and yeast growth at 37°C (37°C), using primers 3

specific to rfxA (26 cycles) or benA (22 cycles), encoding β-tubulin, as an RNA abundance 4

standard. The standardised amount of rfxA product (±S.E.M) relative to benA product for the 5

three RNA samples was 0.35 (0.05), 1.7 (0.33) and 0.59 (0.17), respectively. 6

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FIG. 3. Relative expression of putative rfxA target genes in strains with reduced and increased 1

rfxA expression. Total RNA was extracted from the P. marneffei strains rfxA+ (SPM4), 2

xylP(p)::rfxA-RNAi (55.2.1) and xylP(p)::rfxA (49.3.1) during vegetative hyphal growth at 25°C 3

under conditions of delayed induction. Cultures were grown for 3 days in liquid ANM media 4

containing 1% glucose and supplemented with 10 mM GABA, before being harvested and 5

transferred into ANM media containing 0.5% xylose and 0.5% sucrose for 6 hours (A) or 1 6

day (B) of growth. (A) Reverse transcription PCR (RT-PCR) was performed on the genes 7

rfxA (27 cycles), mobA (28 cycles) and benA (25 cycles). The rfxA specific primers bind 8

outside the coding region included in the xylP(p)::rfxA-RNAi construct. (B) The expression of 9

the bimB transcript was determined using quantitative real-time RT-PCR . N-I; non-induced 10

(1% glucose). I; induced (0.5% xylose and 0.5% sucrose). Values were normalised to actin as 11

a loading control and are shown relative to the rfxA+ strain under non-inducing conditions. 12

Error bars represent standard error of the mean (n=3). 13

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FIG. 4. Strain 44.1.2 is a heterozygous diploid containing rfxA+ and rfxA∆::pyrG

+ alleles. (A) 1

Partial restriction map for the wild-type rfxA genomic locus (upper panel) and the expected 2

rfxA deletion construct pHS5595 (lower panel) after integration. The grey box represents the 3

rfxA coding region consisting of four exons, which has been replaced by the A. nidulans pyrG 4

blaster cassette (pyrG+; white box) in the deletion construct. The hatched box indicates the 1 5

kb KpnI/XhoI fragment of pHS6313 used as a probe for the Southern blot hybridisation 6

experiment in B. (B) Southern blot hybridisation of gDNA isolated from the wild-type strain 7

2161 (WT) and strain 44.1.2 (+/∆), containing both rfxA+ and rfxA∆::pyrG

+ alleles. The 8

restriction enzymes used for digestion are indicated above the appropriate panels. The probe 9

used for hybridisation is shown in A (hatched box). (C) The rfxA+ (33.4.2) and rfxA

+/rfxA∆ 10

(44.1.2) strains were point inoculated onto slides containing a thin-layer of 0.1% glucose 11

ANM plus GABA and incubated for 4 days at 25°C. Microscopic images of conidiophores 12

were taken using differential interference contrast (DIC) and epifluorescence optics to 13

observe nuclei stained with DAPI. The scale bar represents 20 µm. 14

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FIG. 5. The heterozygous rfxA+/rfxA∆ strain displays cellular division defects during yeast 1

morphogenesis at 37°C. (A) The rfxA+ pyrG

+ (33.4.2), rfxA

+ pyrG

- (SPM4) and 2

rfxA+/rfxA∆::pyrG

+ (44.1.2) strains were grown on either ANM containing 10 mM GABA at 3

25°C or on SD containing 10 mM (NH4)3SO4 at 37°C, with (+U) and without (-U) the 4

addition of 10 mM uracil. Plates were photographed after 6 days incubation. (B) Microscopic 5

examination of wild-type rfxA+ (2161) and rfxA

+/rfxA∆::pyrG

+ (44.1.2) strains after 4 days 6

growth at 37°C on slides coated with a thin-layer of SD media containing 10 mM (NH4)2SO4. 7

Images were captured using both differential interference contrast (DIC) and epifluorescence 8

optics to observe nuclei stained with Hoechst (H). Two panels of the rfxA+/rfxA∆::pyrG

+ 9

strain are shown. Scale bar represents 20 µm. 10

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FIG. 6. Silencing of the rfxA transcript by RNAi leads to cell division defects and poor 1

growth. Microscopic examination of wild-type rfxA+ (SPM4) and xylP(P)::rfxA-RNAi (55.2.1) 2

strains: (A) After 4 days growth at 25°C on slides coated with a thin-layer of ANM plus 10 3

mM GABA solid medium in the presence of either 1% glucose (non-inducing) or 0.5% xylose 4

and 0.5% sucrose (inducing). Nuclei (single arrowheads) and septae (double arrowheads) are 5

shown. (B) After 2 days growth at 25°C on slides coated with a thin-layer of 0.1% glucose 6

containing ANM plus 10 mM GABA solid medium. Followed by an additional 2 days growth 7

at 25°C after the addition of 5 mL of liquid medium containing either 1% glucose (non-8

induced) or 0.5% xylose and 0.5% sucrose (induced), to the bottom of the slide. The white 9

and black arrowheads represent misshapen sterigmata cells with fragmented nuclei and 10

enlarged sterigmata cells and conidia containing multiple nuclei, respectively. (C) After 4 11

days growth 37°C on slides coated with a thin-layer of SD plus 10 mM (NH4)2SO4 solid 12

medium under non-inducing or inducing conditions. Images were taken using both differential 13

interference contrast (DIC) and epifluorescence optics to observe Calcofluor and Hoechst 14

(C/H) or Hoechst (H) staining. Scale bar represents 20 µm. The inset (C) represents a further 15

2x enlargement. 16

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FIG. 7. Overexpression of rfxA leads to cell division defects and growth arrest. (A) 1

Microscopic examination of wild-type rfxA+ (SPM4) and xylP(P)::rfxA (49.3.1) strains after 4 2

days growth at 25°C on slides coated with a thin-layer of ANM plus 10 mM solid medium in 3

the presence of either 1% glucose (non-induced) or 0.5% xylose and 0.5% sucrose (induced). 4

Images were taken using both differential interference contrast (DIC) and epifluorescence 5

optics to observe Calcofluor and Hoechst (C/H) staining. Scale bar represents 20 µm. (B) 6

Microscopic examination of wild-type rfxA+ (SPM4) and xylP(P)::rfxA (49.3.1) strains after 2 7

days growth at 25°C on slides coated with a thin-layer of 0.1% glucose containing ANM plus 8

10 mM GABA solid medium. Followed by an additional 2 days growth at 25°C after the 9

addition of 5 mL of liquid medium containing either 1% glucose (non-induced) or 0.5% 10

xylose and 0.5% sucrose (induced), to the bottom of the slide. Images were taken using 11

differential interference contrast (DIC) optics. The scale bar represents 50 µm and 12

conidiophores are indicated using arrowheads. (C) Microscopic examination of wild-type 13

rfxA+ (SPM4) and xylP(P)::rfxA (49.3.1) strains after 4 days growth at 37°C on slides coated 14

with a thin-layer of SD plus 10 mM (NH4)2SO4 solid medium in the presence of either 1% 15

glucose (non-induced) or 0.5% xylose and 0.5% sucrose (induced). Images were taken using 16

both differential interference contrast (DIC) and epifluorescence optics to observe Hoechst 17

staining. Scale bar represents 20 µm. 18

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FIG. 8. Silencing of the rfxA transcript by RNAi leads to reduced viability after DNA 1

replication inhibition imposed by Hydroxyurea (HU). Approximately 2x105 conidia of the 2

wild-type rfxA+ (SPM4) and xylP(P)::rfxA-RNAi (55.2.1) strains were inoculated into 1 mL of 3

liquid ANM plus 10 mM GABA medium containing 0.5% xylose and 0.5% sucrose (inducing 4

conditions) and incubated at 25°C for either 24 hours in the presence HU (2, 5 or 10mM) or 5

pre-germinated for 18 hours in the absence of HU followed by a transient 6 hour exposure to 6

HU (2 or 10mM). The conidial suspensions were diluted twice (1/10 dilution) to remove the 7

HU and 100µL spread onto plates containing 1%glucose ANM plus 10 mM GABA medium 8

(non-inducing). Colony forming units were counted after 2 days incubation at 25° C and 9

survival is expressed as the percentage of viability in the absence of HU. 10

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Q DBD B C DIM DE

861 aa

A

AcCPCR1 P NAE RKR QLY AM LWLN SAC VE SKN S.I PRG RVY HTY AS KCT DDRV VV LNP ASF GKL VRV VFP SI KTR RLG VRG ESK YHY CN FQLR DPP 87 PmRfxA G KSE KVK QVF AM VWLK ENC RK SSG S.V RRD RVY CCY AE RCG SEHV SV LNP ASF GKL VRI IFP NV QTR RLG VRG ESK YHY VD LTVI EEK 87 ScCrt1 Q NRE RER QVF AL LWLM KNC KS QHD SYV PRG KIF AQY AS SCS QNNL KP LSQ ASL GKL IRT VFP DL TTR RLG MRG QSK YHY CG LKLT VNE 88 SpSak1 A NSE KFR QVF GI CWLK RAC EE QQD AAV QRN QIY AHY VE ICN SLHI KP LNS ASF GKL VRL LFP SI KTR RLG MRG HSK YHY CG IKLR GQD 88 HsRFX1 S HTT RAS PAT .V QWLL DNY ET AEG VSL PRS TLY CHY LL HCQ EQKL EP VNA ASF GKL IRS VFM GL RTR RLG TRG NSK YHY YG LRIK ASS 87

MmRFX1 S HTT RAS PAT .V QWLL DNY ET AEG VSL PRS TLY CHY LL HCQ EQKL EP VNA ASF GKL IRS VFM GL RTR RLG TRG NSK YHY YG LRIK ASS 87 DmRFX S SSN KIA SAT .I KWLS RNY ET ADG VSL PRS TLY NHY MH HCS EHKL EP VNA ASF GKL IRS VFS GL RTR RLG TRG NSK YHY YG IRIK PGS 87 CeDAF19 P NNQ RAS PAT .V NWLF ENY EI GEG .SL PRC ELY DHY KK HCA EHRM DP VNA ASF GKL IRS VFH NL KTR RLG TRG NSK YHY YG IRLK DSS 86 DmRFX2 . QKK RQH QLF AL VTLI KTF KI SAN AVC PRN IVY LKY VE NCK EHQI SP ICN AAF GKL VKI FHP DI KTR RLG VRG SSR YNY CG LELI KNQ 87

HsRFX1 AF AQT LRRY TSL NHL AQAA RAV LQNT AQI NQM LSD LNR .... ... ... ... .VD FANV QEQ ASWV CR. ..C EDRV VQR LEQ DFK VTL QQQ 73 MmRFX1 AF AQT LRRY TSL NHL AQAA RAV LQNT AQI NQM LSD LNR .... ... ... ... .VD FANV QEQ ASWV CR. ..C EDRV VQR LEQ DFK VTL QQQ 73 DmRFX AF CQT LRRY TSL NHL AQAA RAV LQNG AQI SQM LSD LNR .... ... ... ... .VD FHNV QEQ AAWV SQ. ..C APAV VQR LES DFK AAL QQQ 73 CeDAF19 YL QQG LKRY TSL NHL AHAS RGV LMKP EQV QQM YQD YIR .... ... ... ... .VD INTV HQQ AGWI CG. ..C DSVM VHH VNN AFK HNL QRM 73 SpSak1 IF SNL LSRL LRV NDT AHAA ARF LANP ADR HLI CND WER FVST RFI VHR ELM CND KEAV AAL DEWY SIL STC SNPS ELL DPL KDK HEA SDT 90 AcCPCR1 IF VGI LERM TRV NKT AHAA ARP VALD ANR DQM YAD WLE LVN. ... ... ... ARK IAEC VPT RGMD DVA ELL VREM RYL VDP KNY IPD DET 80 PmRfxA IF CNL LKHM LDV NQA ANAA AAW LCHP DNR NQM WID FAT FVDP KEM LIK AHI PPC SEKA TEQ ILKH DVR .AL LTPL EHP TSP TLL SFY QQT 89

HsRFX1 .. ... ...N SLE QWA AWLD GVV SQVL KPY QGS AG. .FP KAAK LFL LKW SFY SSM VIRD LTL RSAA SFG SFH LIRL LYD EYM YYL IEH RVA 153 MmRFX1 .. ... ...N SLE QWA AWLD GVV SQVL KPY QGS SG. .FP KAAK LFL LKW SFY SSM VIRD LTL RSAA SFG SFH LIRL LYD EYM YYL IEH RVA 153 DmRFX .. ... ...S SLE QWA SWLQ LVV ESAM EEY NGK PT. .YA RAAR QFL LKW SFY SSM IIRD LTL RSAS SFG SFH LIRL LFD EYM FYL VEH KIA 153 CeDAF19 .. ... ...S AME VWA EWLE SIV DQVL AKY HDK PAN VIA NVGK QFL LNW SFY TSM IIRD LTL RSAM SFG SFT LIRL LAD DYM YYL IES KIA 155 SpSak1 .. ... ...S MNR VEL RQID GVL DRMA DFF LEL PSR FPS CSPR MFL LCL GAL QTS VLRE ITV SGGE AFG ALW VIRC WVD EYM TWV AEI GGY 172 AcCPCR1 .. ... ...S NAD AGG ARSP FAV NRWR DFL MSL PGK FPY ASHE DIV WCV ERV GTA IVRE LTL QGGT SFT TWW SIKT FLD EEI MYL AEV GGL 162

PmRfxA GS NTQ DQKS TVE VST GEEY NFP DKWI SFI LGL PSL FPN HPAQ CIV DKV DRL WDC VLHR LTL AGAP SFS AWW MTKV FFH EML LWQ VEK GGF 179

B

C

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rfxA benA

V

C

V

C100 bp

ladder

460 bp

200 bp

Relative

abundance 1 4.9 1.7

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Growth condition

rfxA+ xylP(p)

::rfxA-RNAi xylP(p)

::rfxA

NI NINI I II

Rel

ati

ve

fold

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

2.5

B

benA

mobA

rfxA

rfxA

+

xylP (p

)::r

fxA-R

NA

i

xylP (p

)::r

fxA

A

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DIC

C/H

non-inducing

rfxA+ xylP(p)::rfxA-RNAi

inducing


A

CrfxA+ xylP(p)::rfxA-RNAi

inducingnon-inducing

DIC

H


DIC

C/H

non-inducing


inducing


B

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DIC

C/H

non-inducing

xylP(p)::rfxArfxA+inducing

xylP(p)::rfxArfxA+A

non-inducing

xylP(p)::rfxArfxA+

DIC

H

inducing

xylP(p)::rfxArfxA+C

DIC

non-inducing

xylP(p)::rfxArfxA+inducing

xylP(p)::rfxArfxA+B

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- 2mM 2mM 10mM5mM - HU

100

80

60

40

20

0

Per

cen

tag

e via

bil

ity (

%)

Growth conditions

rfxA+

xylP(p)

::rfxA-RNAi

10mM

24 hrs with HU 18 hrs without HU

+ 6 hrs with HU

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Table 1. Oligonucleotides used in this study.

Name Sequence

F58 5’-GCTCCGGTGTCTACAATGGC-3’

F59 5’-AGTTGTTACCAGCACCGGAC-3’

J72 5’-GGMGARTCMAARTAYCAYTA-3’

J73 5’-RRTCRCAYTCCTYKATCCA-3’

L13 5’-AAGGTTTGGGTGAATAAGCAG-3’

L29 5’-GGACTTTGAGTGTGAGTGGAA-3’

L30 5’-ATTCTGTCCCGTAGATGAAGA-3’

L31 5’-GCAAACCCAGGAAATGACAC-3’

L31 5’-GCAAACCCAGGAAATGACAC-3’

L32 5’-ATGGAGCGCCTGCTAATCCG-3’

L33 5’-ATGGCTAATACCACCCAAGA-3’

L6 5’-CGACTGGTGGTTGAGATATGC-3’

Q34 5’-ATCGATCCATGGCTCCTGAAG-3’

Q36 5’-CATCTGCTTGAGCGTGTAAC-3’

Q37 5’-CAGGATCAAAATACGGCGAAC-3’

Q50 5’-GTCTTTGCGACCTCGCAGTAT-3’

FF18 5’-GGTAAATGCCAATGAACTCG-3’

FF19 5’-CAAGAGACTCACGAGTAGCC-3’

FF20 5’-ATGGACTCAAGCAGAGGAGG-3’

FF21 5’-GCACACTTGATTGTGACTCA-3’

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GG2 5’-CTTCCAGCCTTCCGTCATC-3’

GG3 5’-GCATACGGTCGGAGATACCA-3’

HH21 5’-TGGTTGGCTGTTAATGTGGTC-3’

HH22 5’-AACTGGTGTTGAGGTGTGGCT-3’

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Table 2. P. marneffei strains used in this study.

Strain Genotype Source or Reference

2161 Wild-type J. Pitt (CSIRO Food industries),

ATCCa strain 18224

SPM4 niaD1; pyrG1 Borneman et al., 2001

33.4.2 niaD1; pyrG1; [AnpyrG] S. Zuber and A. Andrianopoulos,

unpublished

41.2.14-3 niaD1; pyrG1; ∆areA H.E. Bugeja, M.J. Hynes and A.

Andrianopoulos, unpublished

44.1.2 niaD1; pyrG1;rfxA/∆rfxA::AnpyrG This study

49.3.1 niaD1; pyrG1; ∆areA;

areA+::xylP(p:: rfxA:: trpC(t)

This study


areA+::xylP(p):: rfxA

∆1673-2566bp::

trpC(t)

This study



1-1256bp:: GFP::

rfxA1256-1bp

:: trpC(t)

This study



∆721-1099bp::

trpC(t)

This study

a ATCC, American type culture collection

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Table 3. Average cell length and nuclear abundance during overexpression of rfxA

Strain Growth condition Average cell length

(µm± SEM, 50 cells,

n=3)

Average number of

nuclei/cell

(±SEM, 50 cells, n=3)

rfxA+ non-inducing

a 66.8 (0.61) 1.17 (0.04)

inducingb

66.7 (1.3) 1.17 (0.05)

xylP(P)::rfxA+ non-inducing 63.3 (3.2) 1.2 (0.02)

inducing 34.7 (2.1) 2.07 (0.08)

a non-inducing, 1% glucose

b inducing, 0.5% xylose and 0.5% sucrose

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Table 4: Conserved genes from filamentous fungi containing RFX consensus binding sites.

Organism Gene Position of binding sitea Position in P. marneffei

homologa

Putative function

A. fumigatus AFUA_7G05950 -609(-)

A. nidulans AN6732.3 -388(-), -534(-)

A. oryzae AO090005000459 -577(-)

A. terreus ATEG_063406 -483(-)

-542(-) Cytokinesis EF-hand protein (Cdc4)

A. fumigatus AFUA_3G10250 -348(-), -967(-)

A. nidulans AN4963.3 -291(+/-)

A. oryzae AO090003000564 -374(+/-)

A. terreus ATEG_04556 -338(+/-)

-318(-) Similarity to protein kinase (Cdc15)

regulating the mitotic exit network

A. fumigatus AFUA_2G03150 -326(+/-)

A. nidulans AN4513.3 (kipB) -272(+/-), -570(-)

A. oryzae AO090120000272 -327(+)

A. terreus ATEG_09578 -262(+), -316(+/-)

-305(-) Related to kinesin protein (KipB)

involved in mitotic spindle dynamics

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A. fumigatus AFUA_6G08120 -246(+)

A. nidulans AN3946.3 -253(+), -311(+)

A. oryzae AO090003000118 -230(-)

A. terreus ATEG_05847 -207(+)

-250(+/-) Similarity to protein kinase

(SldA/Bub1) regulating the spindle

assembly checkpoint


A. nidulans AN8783.3 (BimB) -131(-)

A. oryzae AO090005000276 -156(-)


-150(+) Related to separin protein (BimB)

involved in chromatid segregation

and DNA repair

A. fumigatus AFUA_2G12390 -619(+)

A. nidulans AN6288.3 -195(+), -491(+)

A. oryzae AO090026000371 -497(+)


-352(-), -367(-), -481(+) Maintenance of ploidy protein kinase

(Mob1)

A. fumigatus AFUA_3G08100 -234(+), -668(-)

A. nidulans AN2927.3 -247(-)

A. oryzae AO090005001468 -145(-), 206(+)

-222(-) Spindle checkpoint kinase (Mph1)

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A. fumigatus AFUA_6G02670 -262(+/-)

A. nidulans AN9504.3 -242(+/-), -328 (-)

A. oryzae AO090120000152 -255(+/-)

A. terreus ATEG_07188 -268(+/-)

-766(-) G2-specific protein kinase (NimA)


A. nidulans AN3910.3 -208(-)

A. oryzae AO090001000513 -167(-)


-267(-), -639(+) Sister chromatid separation protein

(Src1)

a Relative to ATG, (+/-) strand

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The RFX protein, RfxA, is an essential regulator of growth ... · 1/29/2010 · 2 Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. 3 The

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