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Phosphatidylinositol phospholipase C mediatescarbon sensing and vegetative nuclear duplicationrates in Aspergillus nidulans

Ana Paula de Figueiredo Conte Vanzela, Suraia Said, and Rolf Alexander Prade

Abstract: In this work, we disrupted one of three putative phosphatidylinositol phospholipase C genes of Aspergillus nidu-lans and studied its effect on carbon source sensing linked to vegetative mitotic nuclear division. We showed that glucosedoes not affect nuclear division rates during early vegetative conidial germination (6–7 h) in either the wild type or theplcA-deficient mutant. Only after 8 h of cultivation on glucose did the mutant strain present some decrease in nuclear dupli-cation. However, decreased nuclear division rates were observed in the wild type when cultivated in media amended withpolypectate, whereas our plcA-deficient mutant did not show slow nuclear duplication rates when grown on this carbonsource, even though it requires induction and secretion of multiple pectinolytic enzymes to be metabolized. Thus, plcA ap-pears to be directly linked to high-molecular-weight carbon source sensing.

Key words: Aspergillus nidulans, phosphatidylinositol phospholipase C, plcA, carbon source sensing, nuclear division.

Résumé : Dans ce travail, nous avons interrompu trois gènes présumés de phosphatidylinositol phospholipase C d’Aspergil-lus nidulans et en avons étudié l’effet sur la détection de sources de carbone liée à la division nucléaire végétative par mi-tose. Nous montrons que le glucose n’affecte pas les taux de division nucléaire lors de la germination végétative précocedes conidies (6–7 h) chez la souche sauvage ou mutante déficiente en plcA. Ce n’était qu’après 8 h de culture sur glucoseque la souche mutante présentait une certaine diminution de la duplication nucléaire. Cependant, chez la souche sauvage,les taux de division nucléaire étaient diminués lorsqu’elle était cultivée sur un milieu amendé avec du polypectate, alors quele mutant déficient en plcA ne montrait pas de ralentissement de duplication nucléaire lorsqu’il était cultivé sur cette sourcede carbone, même si ceci requiert l’induction et la sécrétion de plusieurs enzymes pectinolytiques devant être métabolisées.Ainsi, la plcA semble être directement liée à la détection de sources de carbone de haut poids moléculaire.

Mots‐clés : Aspergillus nidulans, phosphatidylinositol phospholipase C, plcA, détection de sources de carbone, divisionnucléaire.

[Traduit par la Rédaction]

Fungi, such as Aspergillus nidulans, and other organismssense nutrients in the environment. These signals are neededto coordinate vegetative growth and cellular processes, suchas gene expression, protein synthesis, DNA replication, andcell division (Han et al. 2004; Muthuvijayan and Marten2004; Reyes et al. 2006).

The carbon source effect on nuclear division has been re-ported for Aspergillus niger and Aspergillus oryzae, in whichhigh-glucose levels result in apical compartments containingmultiple nuclei and low-glucose levels lead to a reduced ac-cumulation of nuclei (Müller et al. 2000). It was also demon-strated that polypectate and pectin reduce the nuclear division

Received 23 September 2010. Revision received 16 March 2011. Accepted 17 March 2011. Published at www.nrcresearchpress.com/cjmon 19 July 2011.

A.P.F.C. Vanzela.* Laboratório de Enzimologia Industrial, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de SãoPaulo, Avenida do Café S/No., Ribeirão Preto-SP 14040-903, Brazil; Fungal Genetics Laboratory, Department of Microbiology andMolecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA.S. Said. Laboratório de Enzimologia Industrial, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,Avenida do Café S/No., Ribeirão Preto-SP 14040-903, Brazil.R.A. Prade. Fungal Genetics Laboratory, Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK74078, USA.

Corresponding author: Ana Paula de Figueiredo Conte Vanzela (e-mail: apvanzela@yahoo.com.br).

*Present address: Laboratório de Biologia Molecular e Biotecnologia, Departamento Farmácia, Universidade Federal dos Vales doJequitinhonha e Mucuri, Campus Juscelino Kubitschek, Prédio Farmácia Básica, Rodovia MGT367, 5000, Diamantina-MG, 39100-000Brazil.

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rate during the initial stages of A. nidulans vegetative growthcompared with cultures supplemented with glucose (Vanzelaand Said 2002; Yuvamoto and Said 2007). Additionally, nu-clear division is diminished in response to protein kinase C(PKC) activation by a phorbol ester, even on glucose. Thus,at least one PKC isoform may control the nuclear duplicationcycle when the carbon source is polypectate (Vanzela andSaid 2002). Such signaling might be important for fungal ger-mination on organic materials because polypectate is the maincomponent of pectin, the cellular cement of vegetal tissues.In Saccharomyces cerevisiae, signaling for glucose and ni-

trogen appears to be integrated by phosphatidylinositol phos-pholipase C (PI-PLC), which is activated by the glucosereceptor (Flick and Thorner 1993; Lorenz et al. 2000). Phos-pholipase C function was also investigated in Neurosporacrassa, and the null mutants presented aberrant growth and abranching pattern (Gavric et al. 2007).Considering the involvement of PKC in the control of nu-

clear division in A. nidulans, and the well-established actionof PI-PLC on PIP2 (phosphatidylinositol 4,5-bisphosphate),which leads to the activation of PKC isoforms (Suh et al.2008), we reasoned that PI-PLC–PKC signaling could be in-vestigated by constructing a disruption mutant. In the presentwork, A. nidulans FGSC A851 (yA1, pabaA1, DargB::trpC,trpC801) was used for disruption of a gene coding for PI-PLC. The mutant strain, named AP27, and A. nidulansFGSC A26 (control strain) were used to analyze sodiumpolypectate (NaPP) signaling and nuclear division.Initially, a true genomic DNA fragment of the plcA gene

(812 bp) was recovered by PCR from FGSC A26 genomic DNAand oligonucleotides (PLC1F, 5′-TACATCTCCTCCTCCCAC-aayacntayyt-3′, and PLC1R, 5′-GGTCTGCCAGTTCAGG-

gcngccatytg-3′) designed to align inside the region codingfor PLCX and PLCY domains, which were both describedto be specific for and conserved among PI-PLC enzymes(Rebecchi and Pentyala 2000). After cloning and sequenc-ing, the PI-PLC gene insert was confirmed by BLASTXand used as a radioactive probe to search an A. nidulanscosmid library previously ordered and assembled into aphysical map (Prade et al. 1997). Two cosmids were iso-lated, both of which mapped to chromosome VIII. Analysisof the genomic region of the plcA gene suggested that it islocated on chromosome VIII, flanked by aldA and riboB(Fig. 1A). A putative open reading frame from the full-length gene sequence was translated, and the PI-PLCA pro-tein product (1141 amino acid residues) was analyzed em-ploying the ExPASy proteomics tool server. The openreading frame coincided with AN0664 (Broad Institute Se-quencing Project), and its general organization encodes allthe necessary domains to make a phospholipase C: pleck-strin homology domain (PH), catalytic domain (PLCcx andPLCcy), and kinase domain (C2_2) (Fig. 1B). The C2_2domain is a Ca(2+)-dependent membrane-targeting modulefound in many cellular proteins involved in signal transduc-tion and membrane trafficking (Ananthanarayanan et al.2002). The PH domain performs functions like lipid bind-ing, attachment to membranes, and binding to b/g subunitsof heterotrimeric G proteins (Seifert et al. 2004). Taken to-gether, these domains strongly suggest that plcA may play arole in PIP2-, IP3 (inositol(1,4,5)trisphosphate)-, calcium-,and PKC-mediated signaling (Rasmussen et al. 1990; Su etal. 2006). In A. nidulans, there are two additional geneswith putative PLC domains, AN2947 (614 amino acids, lo-cated on chromosome VI) and AN6382 (630 amino acids,

Catalytic Domain

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AN0664 (1141 aa, chromosome VIII)

AN6382 (630 aa, chromosome I)

AN2947 (614 aa, chromosome VI)

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Fig. 1. Structure and physical characterization of the plcA locus. (A) Genomic organization of the plcA locus. The plcA gene maps to chro-mosome VIII and appears to have no introns, with one continuous open reading frame encoding an 1141 amino acid putative phospholipaseC. Primer sets for PCR analysis (APSF–APSR, yielding a 1.6 kb amplicon from intact locus; M13F–PLC1R, yielding a 1 kb amplicon fromtransformant DNA) are shown. (B) PlcA protein domain organization. Two additional Aspergillus nidulans putative plc genes were found bysearching the entire genomic DNA sequence; however, only PlcA (AN0664) included all the necessary and sufficient domains, namely thecatalytic domains PLCcx and PLCcy, as well as the C2_2 and PH domains. Two other putative Plc proteins, AN2947 and AN6382, showedboth or only one of the predicted phospholipase-catalytic domains, respectively, and neither of them showed the C2_2 domain.

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located on chromosome I), whereas only AN0664 hybrid-izes to our 812 bp chromosome VIII probe and contains allthe predicted domains (Fig. 1B). Indeed, other reports haveshown the existence of three genes coding for PLC-A, PLC-B, and PLC-C enzymes in A. nidulans (Tuckwell et al.2006). These authors found some conserved residues acrossPLC-A to PLC-D fungal proteins and no differences at theactive sites. However, according to our results, the proteinsencoded by AN0664, AN2947, and AN6382 present pecu-liar overall structures, which suggests they may perform dif-ferent roles (Fig. 1B). We also found differences among thepredicted catalytic domains of these three putative PLC pro-teins (Fig. 1B).A disruption vector, named pAP1 (Fig. 1) was constructed

by cloning a plcA fragment adjacent to argB, which wasmoved from pDC1 (Stringer et al. 1991) for complementationof arginine auxotrophy of A851. pAP1 was next used totransform A. nidulans A851 protoplasts prepared as describedby Yelton et al. (1984), with the exception that the cell walldigestion cocktail contained 10 mg/mL of Driselase (Sigma),5 mg/mL of b-D-glucanase G (InterSpex), 5 mg/mL of lyso-zyme (Sigma), and 12 mg/mL of bovine serum albumin frac-

tion V (Sigma). The transforming protoplast fusion mixturewas plated onto selective minimal medium (MM)(Pontecorvo et al. 1953; Clutterbuck 1992) containing1.2 mol/L sorbitol and 5 µmol/L PABA. Transformants ap-pearing after 48 h were sequentially replicated three times onselective media to select a recombinant homokaryon, andthey were further analyzed by spore PCR to assure DNAmediated integration, by Southern blotting to verify themode of integration, and by Northern blotting to verify thelack of PI-PLC mRNA accumulation.A PCR was performed with primers PLC1R, 5′-GGTCTG-

CCAGTTCAGGgcngccatytg-3′, and M13For, 5′-GTAAAA-CGACGGCCAGTC-3′, aligning within the vector to ensurethat amplification of a 1 kb fragment reports pAP1 integra-tion. Spore microwaving (Ferreira et al. 1996) and primer setM13F–PLC1R yielded the 1 kb PCR product expected fromtransformants selected as arginine prototrophs (see Fig. S11).Transformants containing an intact plcA locus were excludedemploying primers APSF, 5′-GACGCAGGCAAAGTCTAC-TGGGAT-3′, and APSR, 5′-GTGATTCGGTAACAGGTC-GAACCC-3′, for amplification of a 1.6 kb DNA fragment.This fragment was expected from genomic DNA according

Fig. 2. Effect of plcA disruption on nuclear cell division as a response to the carbon source. Germlings from Aspergillus nidulans strains A26(wild type; A and C) and AP27 (DplcA mutant; B and D) were stained with DAPI after 6, 7, and 8 h of cultivation in minimal medium amendedwith glucose (MMG; A and B) or sodium polypectate (MMN; C and D). One, two, and four nuclei were scored as percentages of total germlingscounted.

1Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/cjm.

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to the distance between primers APSF–APSR in the intactplcA gene (see Fig. S11), whereas integration of the disrup-tion vector would increase this distance to approximately6 kb and the extension time set (2 min) was not enough forlong products. PCR (APSF–APSR) of over a 100 independ-ent transformants previously recognized on selective media(arginine prototrophs) revealed that only 22% of them couldbe plcA disruptants because they did not amplify the 1.6 kbproduct (see Fig. S11). All other recombination events hap-pened at heterologous sites suggesting that homologous inte-gration events at the plcA location on chromosome VIII arerare (1 in 5) (Martinelli et al. 1984; Martinelli 1994). Trans-formants AP27, AP34, and AP41 selected by means of sporePCR were next analyzed by Southern blotting (Sambrook etal. 1989) using EcoRV–ScaI (Invitrogen) digestion and radio-active pDC1 DNA as probe. The resulting profiles of AP27,AP34, and AP41 were similar and contained four bands (2.9,1.7, 1.6, and 0.9 kb) as expected from plcA::argB disruptants(see Fig. S11). However, none of the arginine prototrophs an-alyzed by Southern blotting showed the profile correspondingto a single insertion of the pAP1 vector. AP27, AP34, andAP41 were also analyzed by Northern blotting (Sambrook etal. 1989) using a radioactively labeled plcA gene fragment asprobe. After confirmation of gene disruption, AP27 was se-lected to study plcA gene function in the nuclear duplicationcycle and carbon source sensing.Conidia of FGSC A26 and AP27 were inoculated into

liquid MM supplemented with either glucose (MMG) orNaPP (MMN) to a final concentration of 106/mL. Samplesof each culture were dropped onto glass coverslips after 6, 7,and 8 h of incubation at 30 °C, and the nuclei of adherentgermlings were stained with 4′-6′-diamidino-2-phenylindole(DAPI) as previously described (Osherov and May 2000;Vanzela and Said 2002). Nuclei were counted with a fluores-cence microscope (Zeiss Axioskop) under an 100× objective.

The number of nuclei was counted for at least 100 cells ineach sample, and experiments were done in duplicate.The number of nuclei in A26 (wild type) and AP27

(DplcA mutant) germlings cultivated on glucose was similar(Figs. 2A, 2B, 3A, and 3B). After 6 and 7 h of incubationin MMG, about 30% of germlings contained a single nuclei,while more than 50% contained two nuclei in both A26 andAP27. Germlings with four nuclei were rarely observed after6–7 h (Fig. 2). However, after 8 h about 35% of the wild-type (A26) germlings contained four nuclei, while 20% ofthe AP27 germlings were multinucleated (Figs. 2A and 2B,respectively). At the same time (8 h), the two-nuclei stage de-creased to 45% in wild-type (A26) cultures (Fig. 2A), whilein the DplcA mutant (AP27), two-nuclei-containing germ-lings were kept at 60% (Fig. 2B). Therefore, in media supple-mented with glucose, the overall nuclei content in AP27 wassimilar to that in A26, except after 8 h, when AP27 seemedto have decreased the second round of mitosis (four-nucleistage). NaPP as the sole carbon source increased the fre-quency of A26 mononucleated germlings (Fig. 2C). After7 h of incubation in MMN, 70% of A26 germlings weremononucleated, while germlings with two nuclei decreasedto approximately 25%. On the other hand, the DplcA mutant(AP27) maintained nuclear divisions at similar rates whencultivated on glucose or NaPP (Figs. 2B and 2D).As a general trend, there seems to be a predominance of

germlings with two nuclei in the DplcA mutant (AP27) inde-pendent of the carbon source (glucose or NaPP), which is insharp contrast with the wild type (A26). A26 mononucleatedgermlings predominated in media amended with NaPP(Figs. 2A and 2C). Figure 3 shows microscopic images ofnuclei contained in A26 and AP27 germlings grown for 7 hin minimal medium amended with glucose (Figs. 3A and 3B)or NaPP (Figs. 3C and 3D). These images highlight the clearpredominance of two-nuclei-containing germlings in the mu-

Fig. 3. Representative DAPI-stained nuclear distribution of Aspergillus nidulans A26 wild type (A and C) and DplcA (AP27) mutant (B and D)germlings after 7 h of incubation in minimal medium supplemented with glucose (MMG; A and B) and sodium polypectate (MMN; C and D).

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tant strain AP27 and corroborate the findings depicted inFig. 2. Therefore, our plcA-deficient mutant did not showslow nuclear duplication rates when grown on NaPP, eventhough it requires the induction and secretion of pectinolyticenzymes before the carbon source can be metabolized(Figs. 2B and 2D). Thus, plcA appears to play a role in sens-ing high-molecular-weight carbon sources, such as pecticsubstrates, during the early stages of vegetative growth inA. nidulans. These findings are in agreement with other re-ports that showed that the PLC inhibitors Spm and C48/80delayed the first nuclear division in A. nidulans wild typegrowing on glucose but stimulated it in media supplementedwith pectin (Chellegatti et al. 2010). These authors reportedthat PLC inhibitors presented minor effects on AP27 andthat this mutant slowed the second round of mitosis after 8 hof growth on glucose, which is in accordance with the resultspresented herein. However, we followed nuclear divisionfrom 6 to 8 h and determined that the frequencies of two-nuclei-stage germlings (first nuclear division) of both AP27and A26 were quite similar after 6–7 h of cultivation onglucose (Fig. 2). Thus, the second nuclear division roundmight be activated by plcA when glucose is available, anda polymeric carbon source would lead to an opposite re-sponse via the same signaling protein. Nevertheless, NaPPand pectin triggered different responses in AP27, since thenuclear division was slowed when the mutant was cultivatedon the second carbon source (Chellegatti et al. 2010). Thismay be due to the fact that pectin is a much more complexcarbohydrate that requires induction of different enzyme ac-tivities to be metabolized, or perhaps other signaling path-ways might be involved.Future research into gene expression, enzyme levels, and

the effects of other carbon polymers will be pursued toachieve a clearer comprehension on how nutrient sensing inA. nidulans is coordinated with growth and nuclear duplica-tion. However, disruption of plcA in the present work con-firms the idea that carbon source sensing is involved in earlynuclear duplication decisions in A. nidulans and that thismechanism is likely under the control of a PI-PLC–PKCmediated pathway.

AcknowledgmentsThis work was supported by grants from Fundação de Am-

paro a Pesquisa do Estado de São Paulo (FAPESP) No.2002/11874-7 and is part of a thesis submitted by Ana Paulade Figueiredo Conte Vanzela to the Faculdade de CiênciasFarmacêuticas de Ribeirão Preto, Universidade de São Paulo,in partial fulfillment of the requirements for the Doctor’s de-gree. A.P.F.C.V. received a fellowship from CAPES that sup-ported her work in Brazil and at the Fungal GeneticsLaboratory at Oklahoma State University.

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