The concerted action of bZip and cMyb transcription factorsFlbB and FlbD induces brlA expression and asexualdevelopment in Aspergillus nidulansmmi_7063 1314..1324

Aitor Garzia,1 Oier Etxebeste,2 Erika Herrero-García,2

Unai Ugalde1 and Eduardo A. Espeso2*1Department of Applied Chemistry, Faculty of Chemistry,University of The Basque Country, San Sebastian20018, Spain.2Department of Cellular and Molecular Medicine, Centrode Investigaciones Biológicas (CSIC), Ramiro deMaeztu, 9, 28040 Madrid, Spain.

Summary

Fungi are capable of generating diverse cell typesthrough developmental processes that stem fromhyphae, acting as pluripotent cells. The formation ofmitospores on emergence of hyphae to the airinvolves the participation of transcription factors,which co-ordinate the genesis of new cell types, even-tually leading to spore formation.

In this investigation, we show that bZip transcrip-tion factor FlbB, which has been attributed to partici-pate in transducing the aerial stimulus signal,activates the expression of c-Myb transcription factorFlbD. Both factors then jointly activate brlA, a C2H2

zinc finger transcription factor, which plays a centralrole in spore formation. This sequence of regulatoryevents resembles developmental control mecha-nisms involving c-Myb and bZip counterparts in meta-zoans and plants.

Introduction

Fungal colonies grow and colonize different environmentsand substrates through the apical extension of tubularcells called hyphae. These pluripotent cells are capable ofundergoing differentiation and give rise to new cell typeswhich fulfil specialized biological functions. In the modelorganism Aspergillus nidulans, hyphae exposed to theatmosphere and light start a developmental processwhich leads to the production of asexual reproductivestructures known as conidiophores. These are composed

of different cell modules: a foot cell, stalk, vesicle, subse-quent layers of primary (metulae) and secondary (phi-alides) sterigmata, and finally, long chains of unicellularmitospores called conidia (Fischer and Kües, 2006).

The morphogenetic transition between pluripotenthyphae and conidiophore formation is regulated by acomplex network of interdependent transcription factors(TF). A number of those act as negative effectors of devel-opmental change under non-inducing environmental con-ditions, and are called sfg genes: SfgA, SfgB, SfgC andSfgD (Seo et al., 2003; Seo et al., 2006). A second groupis composed of the so-called upstream developmentalactivators (UDAs), with a demonstrated positive role onconidiophore production (Seo et al., 2006). UDA factorspurportedly respond to environmental cues by overcom-ing the repression of SFG factors and co-ordinating theexpression of the first conidiation-specific factor: the C2H2-zinc finger TF BrlA (Lee and Adams, 1994; Wieser et al.,1994; Wieser and Adams, 1995).

Mutations in UDAs were identified as they caused fluffy(aconidial, with negligible conidiospore production) colonymorphology and low brlA expression. Consequently, theywere designated as flb (fluffy low bristle mutants; Wieseret al., 1994). Among these, FlbB is a bZip type TF found inthe most apical nucleus, and is also localized at theSpitzenkörper, a fungal organelle involved in apical exten-sion (Harris et al., 2005), together with FlbE, another UDAfactor (Etxebeste et al., 2008; 2009a; Garzia et al., 2009).FlbC and FlbD are putative C2H2–zinc-finger and c-MybTFs respectively (Wieser and Adams, 1995; Yu et al.,2006). Previous epistasis and selective overexpressionanalyses (Wieser and Adams, 1995) had established aputative order of function FlbD > FlbE > FlbB, with FlbCfollowing a parallel route (Yu et al., 2006).

Despite prolonged and continued research on theconidiation regulatory pathway (Fischer and Kües, 2006;Yu et al., 2006), details on the nature of the molecularinteractions underlying the collective control of UDAfactors on brlA activation are still lacking. In this investi-gation, we show that FlbB activates flbD expression andthat both factors are then jointly activating brlAtranscription. These results support the proposal of a non-linear transcriptional regulatory cascade model for conid-iophore development, reminiscent of developmental

Accepted 17 January, 2010. *For correspondence. E-mail eespeso@cib.csic.es; Tel. (+34) 91 837 31 12 ext: 4227; Fax (+34) 91 536 04 32.

Molecular Microbiology (2010) 75(5), 1314–1324 � doi:10.1111/j.1365-2958.2010.07063.xFirst published online 5 February 2010

© 2010 Blackwell Publishing Ltd

control mechanisms involving c-Myb and bZip counter-parts in metazoans and plants.

Results

Evidence of a non-linear signalling cascade ofasexual development

A phenotypic characterization of a DflbD mutant wasundertaken in parallel with the DflbB and double null

mutants (Etxebeste et al., 2008; Garzia et al., 2009) as areference (Fig. 1). In synthetic minimal medium (MMA),DflbB and DflbD mutants yielded fluffy colonies (Fig. 1,control column) with three orders of magnitude lower sporeproduction than the parental wild type strain (3.5 ¥ 106 thewild type and 7 ¥ 103 or 8 ¥ 103 by DflbB and DflbDmutants, respectively, per cm2; Fig. 1). This aconidial phe-notype is a characteristic of flb mutants (Adams et al.,1998). Under stressing growth conditions such as reduc-

Fig. 1. flbD mutant characterization on solid media and comparison with flbB mutant.A. Colony morphologies of wild-type (WT; TN02A3), DflbB (BD177), DflbD (BD197) and double null DflbB DflbD (BD274) strains grown on solidMMA, MMA with 0.4% glucose, MMA with 14 mM sodium nitrate, MMA with 0.6 M KCl and MMA with 0.5 M phosphate (NaH2PO4; seeExperimental procedures) for 72 h are shown. Bar = 1 cm.B. Amount in millions of spores per cm2 produced by each strain under the described growth conditions. The spore counts of the WT strainwere between 25 (control) and 100 (salt stress) millions, and are not displayed due to large differences in the range of the values. Error barsindicate the standard deviations of at least three biological replicates.

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tion in carbon source availability (80% less glucose) lowlevels of conidiation were observed in all three strains(Fig. 1), being the spore amount one order of magnitudelower than the wild type strain. However, this conidiationresponse was not observed with an equivalent reduction innitrogen source (Fig. 1). Finally, under osmotic (0.6 M KCl)or salt (0.5 M NaH2PO4) stress conditions, the DflbD strainshowed sparse conidiation over a mainly aconidial colony,as previously described for DflbB and DflbE mutants(Etxebeste et al., 2008; Garzia et al., 2009). Notably, thedouble null strain showed an additive aconidial phenotypein these conditions (Fig. 1). The latter feature in particularwas interpreted as an indication that the mode of action ofthese factors was interdependent (non-linear) rather thansequential (linear) as previously proposed.

FlbD and FlbB present similar expression patterns butdistinct cellular localizations

To assess this possibility, first, we analysed flbD expres-sion at different stages of development and compared itwith that previously described for flbB (Etxebeste et al.,2008). Transcriptional patterns of flbD and flbB at thosedevelopmental stages were similar. The flbD transcriptwas present during hyphal growth and the early phases ofasexual development preceding the transcriptional activa-tion of brlA. Thereafter, a notable reduction in flbD expres-sion was found (Fig. 2A). As noticed before for flbB

(Etxebeste et al., 2008), the flbD expression was unde-tectable during sexual development (Fig. 2A).

The coincidence in flbD and flbB temporal expressionpatterns was not corresponded by their cellularlocalization. FlbD::GFP or FlbD::CherryRed (FlbD::CR)fusion proteins were accumulated in all nuclei after ger-mination, remaining unaltered in mature hyphae (Fig. 2B).Conversely, FlbB was observed at the tip dome of hyphae,since germling state, and mainly in the most apical nucleialthough not in distal nuclei (Etxebeste et al., 2008). Inaddition, during conidiophore development FlbD::GFPfluorescence was not detected in any of the different celltypes produced, as shown for vesicle and incipient phi-alides stages (Fig. 2B), in which an elevated number ofnuclei accumulate in the vesicle compartment at suchdevelopmental phase (Etxebeste et al., 2009b).

A functional FlbB/E apical complex is required forflbD expression

The relationship between FlbD and FlbB/E complex wasanalysed by transcriptional and proteomic approaches.While the expression of neither factor was affected bydeletion of upstream activator fluG (Adams et al., 1998),the flbD expression and the derived protein levelswere greatly reduced in both DflbB and DflbE strains(Fig. 3A–C). Predictably, fluorescently tagged FlbD wasnot detected in strains lacking FlbB/E complex (Fig. 3D).

In contrast, the transcriptional profile of flbB in a DflbDbackground showed that FlbD was not required forexpression of flbB (Fig. 3B), although lower levels of tran-scription were observed during early phases of asexualdevelopment [6 h after conidiophore development induc-tion (Etxebeste et al., 2008)]. Accordingly, FlbB and FlbEprotein levels in hyphae were also comparable to thosefound in the wild type strain (Fig. 3E) and both, FlbB(nuclear and apical) and FlbE (apical) localizations(Fig. 3F) and apical complex formation (Fig. 3G) wereunaffected. Pull-down analyses ratified the FlbB/Ecomplex formation in the absence of FlbD (Fig. 3G, lanes7 and 8), while mutations previously reported to destabi-lize the complex, FlbE102 and FlbE103 (Garzia et al.,2009), disrupted the in vitro interaction (Fig. 3G, lanes3–6).

Transcription factor FlbB binds the flbD promoter in vivo

Given the dependence of flbD expression on FlbB, weconsidered the hypothesis that the latter may activate flbDexpression. Hence, we analysed the promoter regionof flbD and identified two putative FlbB target sites atpositions -499 to -493 (TCAATAA) and -717 to -711(TCAGTAT) respectively (Fig. 4A). These sites were pro-posed being based on our previous findings showing the

Fig. 2. flbD expression and intracellular localization.A. During the A. nidulans life cycle in a wild-type veA geneticbackground (strain WIM126). Numbers indicate the time (h) ofincubation in liquid MMA plus glucose, corresponding to vegetativegrowth (Veg), or under conditions inducing asexual (Asexual) orsexual development (Sexual) respectively (see Experimentalprocedures).B. In static culture in liquid MMA, the FlbD::GFP proteinaccumulates in nuclei. Bar = 10 mm.

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binding of the GST::FlbBbZIP fusion protein (Etxebesteet al., 2009a). Electrophoretic mobility shift assays(EMSAs) demonstrated that this fusion protein (Etxebesteet al., 2009a) was able to specifically bind promoter frag-ments containing these putative FlbB sites (fragments ABand CD; Fig. 4B and C). Moreover, ChIP (ChromatinImmuno Precipitation) analyses confirmed in vivo bindingof FlbB::GFP to the abovementioned fragments (Fig. 4D).No binding was detected to fragments lacking any puta-tive FlbB target site, such as the EF fragment from theflbD promoter and a control fragment from the gabA pro-moter (Fig. 4D). The overall of these data provide strongevidence for a direct positive role of FlbB in flbDregulation.

FlbD and FlbB are jointly required for brlA activation

In light of the above results, we considered a new linearmodel hypothesis in which FlbB regulated flbD expression

and, in turn, a direct FlbD-dependent brlA activation.Hence, the overexpression of flbD should overcome aDflbB aconidial phenotype, thus promoting conidiophoredevelopment. However, neither overexpression of flbD ina DflbB mutant nor flbB in a DflbD background, were ableto suppress the aconidial phenotype caused by each nullmutation (Fig. 5). Consequently, the linear pathway modelhypothesis was rejected and a concerted action of bothTFs in brlA regulation was postulated.

FlbD is a c-Myb type transcription factor

In order to verify a co-ordinated induction of brlA expres-sion by FlbB and FlbD we first verified that the latter is aDNA-binding protein. The high degree of amino acidsequence conservation between the predicted c-Mybdomain of FlbD and other similar domains (Fig. 6A;Wieser and Adams, 1995) prompted us to verify the func-tionality of this putative DNA-binding domain using a stan-

Fig. 3. Transcriptional and proteomic relationship between FlbD and FlbB/E complex.A. and B. mRNA expression levels at different times during vegetative (0) and asexual development (6, 12, 24 and 48 h), in different nullmutant strains compared with the wild-type veA1 (WT) strain.C. and E. Western blot analyses, using anti-GFP or anti-mRFP and anti-actin primary antibodies.D. and F. Intracellular localization of FlbD in a flbB and flbE null mutant background (BD254 and BD257) and FlbB/FlbE proteins in a flbD nullmutant background (BD209 and BD208) respectively. Bar = 10 mm.G. FlbE specifically interacts with FlbB in pull down assays. Purified GST::FlbB protein was incubated with protein extracts from each of thefollowing strains, being input and pull-down assays of each one, respectively: lines 1–2, flbE100 (FlbE::GFP); lines 3–4, flbE102; lines 5–6,flbE103; lines 7–8, BD205 (DflbD FlbE::GFP); lines 9–10, TN02A3 (WT, control). A double amount of A. nidulans protein extract was added inflbE mutants in order to verify if the interactions with FlbB do not occur or they are debilitated.

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dard synthetic probe recognized by these motifs (Fig. 6B).A GST fusion expressing the region between residues M1and P125 of FlbD, GST::FlbDcMyb, was generated andused in EMSAs against the probe containing the c-Mybconsensus sequence, 5′-YAACKG-3′ (Biedenkapp et al.,1988; Ness et al., 1989). The positive results obtainedwith in vitro experiments confirmed the presence of ac-Myb DNA-binding domain in FlbD (Fig. 6C).

FlbB and FlbD jointly bind the brlA promoter in vivo

To corroborate a co-ordinated induction of brlA expressionby FlbB and FlbD, additional EMSAs were performed withlarge amounts of either GST::FlbDcMyb or GST::FlbBbZIP

(Etxebeste et al., 2009a). As shown in Fig. 4B, FlbB iscapable of binding in vitro DNA sequences different to

those initially described in our previous works (Etxebesteet al., 2009a). Thus, although brlA promoter does notcontain those sequences, we found a similar putativeFlbB-binding site in close proximity to two similar FlbD-binding sites (Fig. 7A).

The two fusion proteins were assayed in binding to amixture of fragments covering a 3 kb 5′-UTR region ofbrlA (being position +1 the brlA cap site described byPrade and Timberlake, 1993; Etxebeste et al., 2009a),and strong binding to fragment G was found (Fig. 7B).

To assess functional analyses of these bZIP- andc-Myb-DNA complexes, ChIP experiments were con-ducted by measuring the in vivo binding of a GFP-taggedFlbB and/or FlbD to the specified fragment in the brlApromoter. These assays showed that FlbB and FlbDbound the brlA promoter fragment specifically in vivo at

Fig. 4. Binding of FlbB to a region in thepromoter flbD.A. Searches using consensus sequences forbZIP TFs (4) allowed the identification of twopossible target sites for FlbB (bold andunderlined sequences) at fragments AB andCD from the flbD promoter. The diagramindicates the relative position of FlbB targetsand fragments studied. Co-ordinates relativeto the initiation codon are indicated.B. and C. Gel shifts using, per reaction,250 ng GST::FlbBbZip fusion protein and 0.3 ngof radiolabelled probes AB and CDrespectively. All competitors were added asnon-radioactive probes to a 500-fold molarexcess (~150 ng). f.p. free probe.D. ChIP assay using anti-GFP antibodydirected against GFP-tagged FlbB. Theinmuno-precipitated DNA was amplified withspecific primers from the flbD promoter. Afragment from the gabA promoter was usedas a negative control. Relative amounts ofprecipitated DNA compared with mock control(without antibody). Error bars indicate thestandard deviations of at least three biologicalreplicates. PCR products are represented onagarose gels.

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Fig. 5. flbB and flbD overexpression experiments in different genetic backgrounds. Control and overexpression strains were grown undernon-inducing conditions for the alcA promoter: MMA plus glucose (MMA), and under alcA promoter-inducing conditions, MMA plus yeastextract (MMT + 5Y). The latter contained 100 mM L-threonine as inducer and 5 g l-1 yeast extract as growth enhancer. Overexpression wasverified by Northern blot analysis (not shown). Photographs were taken after 72 h incubation at 37°C. Bar = 1 cm.

Fig. 6. DNA-binding capacity of the FlbD cMyb domain.A. Schematic representation and alignment of FlbD with various Myb-like proteins according to predicted (Wieser and Adams, 1995; Tahirovet al., 2002). Genedoc software was used (version 2.6.003; http://www.nrbsc.org/downloads/). Abbreviations: A. nid., Aspergillus nidulans;H. sap., Homo sapiens; D. mel., Drosophila melanogaster; A. thal., Arabidopsis thaliana. Database entries for the proteins used in the multiplealignment are ANID_00279.3, AAA52032, NP_511170 and AAL01236 respectively. Arrowheads indicate those residues whose side-chainshave been described to be implicated in direct contacts with the bases at the DNA (see also panel B).B. A model for FlbD binding to the c-Myb consensus site, schematic representation of predicted base-specific contacts (Tahirov et al., 2002).C. EMSA showing the specific recognition of a consensus cMyb DNA probe (annealing of oligonucleotides GST-FlbD c-myb sense andantisense, Supplementary Table SM2, also shown in panel B) by the predicted DNA-binding domain of FlbD. The different amounts of purifiedGST::FlbDcMyb used are indicated in ng. In competition experiments, binding of 100 ng of GST::FlbDcMyb to 0.3 ng of cMyb probe was competedby adding 150 ng of cMyb, AP1 or PLD fragments. For the competitor poly(dIdC)-(dI-dC) 2 mg were used in the assay.

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24 h of vegetative growth (Fig. 7C and D), time at whichbrlA expression was detected (Etxebeste et al., 2008;Garzia et al., 2009). In addition, FlbB binding to the brlApromoter was lost in a DflbD strain (Fig. 7C and D). Thisresult evidenced a combined transcriptional role of bothTFs upon brlA expression and possible molecularinteraction. The reverse experiment, in which FlbDbinding to brlA promoter in a DflbB strain, was not per-formed, because FlbD correct synthesis requires theexpression of flbB (Fig. 3A, C and D).

flbB and flbD transcriptions are not repressed onbrlA overexpression

Once established the concerted activation of brlA by FlbBand FlbD and considering that flbB and flbD transcriptsdisappear shortly after brlA activation (Wieser andAdams, 1995; Etxebeste et al., 2008) we analysedwhether this was a result of BrlA-mediated repression.This hypothesis was plausible as the promoter regions ofboth factors contained potential BRE sites [BrlA responseelements; (C/A)(G/A)AGGG(G/A); (Chang and Timber-lake, 1993), (flbB at -1455 bp, flbD at -259 and -440 bp)].Hence, a strain-expressing brlA independently of UDAfactors was generated, being driven by the inducible pro-moter of the alcohol dehydrogenase I (alcA::brlAab).Northern analyses of mycelium grown for 18 h and sub-sequently incubated in inducing conditions (100 mMthreonine) showed similar transcript levels of either flbB orflbD compared with endogenous levels of brlA (Fig. 8).This result therefore indicated that flbB and flbD down-regulation was not solely BrlA-dependent, although amore complex mechanism also involving this TF may notbe ruled out at this stage.

Discussion

This investigation presents two novel conceptual ele-ments which are relevant in our understanding of asexualdevelopment in filamentous fungi. First, new evidence ispresented to show that flbD activation is FlbB-dependent;and second, that both FlbD and FlbB are jointly requiredfor the activation of brlA (Fig. 9). Considering the firstelement, it appears that flbD expression is positively regu-

Fig. 7. FlbD/FlbB corregulation of the brlA promoter.A. Putative binding sites within the G fragment for FlbD, upper row,and FlbB, lower row. Co-ordinates from the brlA ATG are indicated.B. Gel shift using a mixture of radiolabelled fragments covering the5′-UTR region between co-ordinates -837 and -3030 from the BrlAstart codon. A diagram on the left side of the EMSA indicates theposition and relative size of each fragment. Increased amounts ofGST::FlbBbZip and GST::FlbDcMyb fusion proteins were used (250,500 and 1000 ng). The arrowhead indicates the G fragment boundby both proteins. f.p. free probe.C. As in the experiment in Fig. 4D a ChIP assay was conductedinmunoprecipitating FlbB or FlbD GFP-tagged proteins.Inmunoprecipitation of promoter G fragment was analysed togetherwith a fragment from the gabA promoter taken as control. PCRproducts are represented on agarose gels.D. Relative amounts of precipitated DNA compared with mockcontrol (without antibody). Error bars indicate the standarddeviations of at least three biological replicates.

Fig. 8. flbB and flbD expression levels in a brlAab-overexpressingstrain. Northern analyses of the mRNA expression levels of flbB,flbD and brlA at different times of alcA promoter induction. TotalRNA was extracted from mycelia grown under the conditionsindicated on the top of the figure. First lane, labelled with 18 h veg.,indicates RNA from mycelium grown in MMA for 18 h, which wastransferred either to non-inducing, MMA, or to inducing, MMT,conditions for the alcA promoter expression. The numbers of hoursafter transfer are indicated. rRNA indicates major ribosomalsubunits used as loading controls.

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lated by FlbB. Because FlbB displays nuclear and apicallocalizations it is expected that the nuclear form wouldplay such role. However, our results show that FlbD isfound in all nuclei of different vegetative hyphae, whileFlbB is restricted to most apical nuclei. We cannot excludethat in addition to FlbB, other TFs might also participate inthe regulation of flbD expression, some of which might bealso under FlbB control. In this respect, FlbE plays anon-direct role in flbD transcriptional regulation. The inter-action between FlbE and FlbB in the apical complex isalso a requisite for flbD expression. Thus, the precise formof FlbB taking part in the activation of FlbD remains to beclarified.

The second element, that FlbB and FlbD jointly promotebrlA expression, would most likely involve a post-translationally modified, possibly activated, form of FlbB.This is supported by earlier evidence that native flbBoverexpression did not activate conidiation in liquid media(Etxebeste et al., 2008), while flbD overexpression didindeed result in miss-scheduled conidiation (Wieser andAdams, 1995). It would appear that nuclear FlbD dosagelevels and a specifically activated (but yet unidentified)form of FlbB, presumably translocated from the Spitzen-körper, would be required for the formation of thetranscriptional complex that mediates brlA activation.Possible molecular mechanisms of intracellular FlbB acti-vation are currently under scrutiny in our laboratories.

It has been previously reported that the region betweennucleotides -967 and -414 within the brlA promoter mightcontain target sites for FlbD (Han and Adams, 2001).However, we did not collect any experimental evidence tosupport such conclusion. Interestingly, the same authorspredicted putative binding sequences for FlbC withinregions covering co-ordinates -2901/-2293 and -967/-414 of brlA promoter. Our data suggest an overlapbetween FlbC and FlbB/FlbD regulatory promoterregions. This opens the possibility of a combined effect ofthese three TFs at the same promoter region modulatingthe expression of the master gene for conidiation.Whether FlbC and FlbD/FlbB regulate brlA expressionindependently or not requires further verification. Never-

theless, a complete understanding of brlA regulation willbe laborious. We hypothesize that other specific A. nidu-lans conidiation factors must be present and expected toactivate brlA expression independently of Flb factors, asthe flb- fluffy phenotype is partially suppressed under spe-cific stress conditions. This could occur, for example,through RgsA, an RGS protein that controls conidiationunder altered carbon conditions or nutritional stress in apathway parallel to that one regulated by FlbA (Han et al.,2004), or through the regulatory proteins that might betransported in or out of the nuclei by the nuclear carrierKapI (Etxebeste et al., 2009b).

The concerted activation of brlA by FlbD/FlbB is thefirst example of a developmentally relevant interactionbetween bZip and c-Myb TFs in fungi. Similar interactionshave been described in higher eukaryotes, includinghuman cells (Tahirov et al., 2002). TFs reportedly playvarious roles in development, based on their interactionswith other TFs or proteins (Foster et al., 2009). Onenotable example is the dual role played by the bZip-typefactor PERIANTHIA in flower organ specification. This TFis activated by WUSCHEL, and both factors concertedlyactivate AGAMOUS (a MADS domain TF that plays acentral role in flower patterning). AGAMOUS accumula-tion purportedly activates the downregulation of its activa-tors (Maier et al., 2009).

In this study, BrlA-mediated downregulation of flbB andflbD was plausible on account of the potential BrlA-binding sequences contained in the promoter regions ofboth UDAs. However, miss-scheduled overexpression ofbrlA unsupported the existence of a feedback loop. Earlierreports separately monitoring the pattern of expression ofall three factors during asexual development have indi-cated that FlbB is present up to the transit from the metulato the phialide (Etxebeste et al., 2009a) while onset ofBrlA expression coincides with vesicle formation (Adamset al., 1998). Both FlbB and BrlA expressions thereforeoverlap during development, and it is more likely thatother factors expressed downstream of brlA, and depen-dent on FlbB, may also be involved in UDA downregula-tion (Fig. 9).

Fig. 9. Updated model of the cascadeleading to the transcriptional activation ofmaster regulatory gene brlA by upstreamdevelopmental activators FlbB and FlbD.

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Experimental procedures

A. nidulans techniques

The strains and oligonucleotides employed in this study arelisted in Supplementary Tables SM1 and SM2. Standardminimal (MMA) and complete media (MCA; MMA + 5 g l-1

yeast extract; Pontecorvo et al., 1953; Kafer, 1965) wereused in solid or liquid form with the appropriate supplements.Nutrient depletion experiments were performed as describedby Etxebeste et al. (2008). Spore production was calculatedfrom suspensions obtained by submerging 72-h-old colonieswith 10 ml of a 0.02% Tween 20 solution with gentle scraping.Spore concentrations were determined by microscopicobservation with a Thoma cell counter. Total spore countswere divided by the area of each colony.

Induction of sexual and asexual development (Law andTimberlake, 1980; Aguirre, 1993) and intracellular localizationanalysis (Penalva, 2005; Bernreiter et al., 2007; Stinnettet al., 2007) were carried out as previously described byGarzia et al. (2009) and Etxebeste et al. (2009a).

Aspergillus nidulans strains (null and tagged) were con-structed by using our standardized techniques (Garzia et al.2009 and Yang et al., 2004), transforming TN02A3 (Nayaket al., 2006) with a DNA fragment containing correspondingregions bound to the auxotrophic marker pyrG or riboA ofAspergillus fumigatus.

Strain overexpressing brlAab transcripts were obtained bytransformation of strain TN02A3 (Nayak et al., 2006) withplasmid pAlcA(p)::brlAa/b::pyroA*, which was generated asdescribed by Etxebeste et al. (2009a). Overexpression wasverified by Northern blot analysis.

Isolation and manipulation of nucleic acids and proteins

The isolation of genomic DNA and total RNA, preparation ofDNA probes for Southern and Northern blot analysis andprotocols of these assays were carried out as describedpreviously (Garzia et al., 2009).

For A. nidulans protein extraction, spores were inoculatedand cultivated for 18 h in fermentation medium (Orejas et al.,1995), and filtered through two layers of Miracloth (Calbio-chem), squeezed to eliminate excess liquid, frozen in dry iceand freeze dried overnight. Extraction and Western blotassays were realized as described previously (Garzia et al.,2009), adding the use of anti-RFP rabbit polyclonal anti-body (USBiological; 1/3000) and ECL peroxidase labelledanti-rabbit antibody (Amersham Bioscience; 1/4000) forCherryRed detection. GST::FlbB expression, induction,extraction and pull down assays were performed as inAraújo-Bazán et al. (2008).

EMSAs and in vivo ChIP analysis

pGEX-2T::FlbDcMyb plasmid was constructed inserting thecDNA fragment of flbD encoding amino acids 1–125 thatcontain the c-Myb domain into plasmid pGEX-2T(Pharmacia). Expression and purification of fusion proteinsand EMSA studies were carried out as previously described(Etxebeste et al., 2009a). Synthetic double-stranded oligo-

nucleotides carrying target sequences for bZIP FlbB (AP-1and PLD) (Etxebeste et al., 2009a), the cMyb FlbD (cMyb)(Biedenkapp et al., 1988; Ness et al., 1989), the zinc-fingerTFs PacC (ipnA2) (Tilburn et al., 1995) or CrzA (CDRE-5)(Spielvogel et al., 2008) were prepared as described inTilburn et al. (1995) to be used as specific DNA-bindingcompetitors. For flbD promoter analysis three overlappingfragments (AB, CD and EF; see Fig. 4) were amplified byPCR reactions and products were introduced into pGEM-T-easy vector (Promega). DNA fragments were generated byNotI digestion, radiolabelled and assayed by EMSA.

For Chromatin ImmunoPrecipitation, ChIP, established pro-tocol was followed (Orlando et al., 1997; Bernreiter et al.,2007), using Roche anti-GFP mouse monoclonal antibodycocktail and protein G agarose. Inmunoprecipitated DNA waspurified using a PCR purification column (Macherey-Nagel)and eluted in 50 ml of water and a total of 1 ml of this DNAsolution was used for PCR reaction using the adequateoligonucleotides.

Microscopy

Observation of fluorescently tagged proteins in vegetativegrowth and during conidiophore development from in vivocultures at 37°C were carried out by previously describedmethods (Etxebeste et al., 2009a; Garzia et al., 2009) using aDMI 6000B Leica microscope, with a 63¥ Plan Apo 1.4 N.A.oil immersion lens (Leica), illuminated with a 100 W mercurylamp and fitted with GFP (excitation 470 nm; emission525 nm) and Txred (excitation 562 nm; emission 624 nm)filters, for GFP and Cherry-Red respectively. Images wererecorded with an ORCA-ER digital camera (Hamamatsu Pho-tonics) and processed with Metamorph (Universal Image) orWasabi (Hamamatsu Photonics) software.

Acknowledgements

This work was supported by the Spanish Ministerio deEducación y Ciencia through grants BFU2006-04185 andBFU2004-03499/BMC to E. A. E. and U. O. U., respectively,and by the University of The Basque Country (UPV/EHU) forgrant GIU08/32 to U. O. U. We also thank Diputación Foral deGipuzkoa for project 24/08. A. G. and E. H.-G. held predoc-toral fellowships from the Basque Government. O. E. cur-rently holds a research contract associated with grantBFU2006-04185 at the CIB.

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