EUKARYOTIC CELL, Dec. 2010, p. 1856–1866 Vol. 9, No. 121535-9778/10/$12.00 doi:10.1128/EC.00186-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

A Mutant Defective in Sexual Development ProducesAseptate Ascogonia�†

Sandra Bloemendal,1 Kathryn M. Lord,2 Christine Rech,1 Birgit Hoff,1 Ines Engh,1Nick D. Read,2 and Ulrich Kuck1*

Lehrstuhl fur Allgemeine und Molekulare Botanik, Ruhr-Universitat Bochum, Universitatsstrasse 150, 44780 Bochum, Germany,1

and Institute of Cell Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, United Kingdom2

Received 28 July 2010/Accepted 6 October 2010

The transition from the vegetative to the sexual cycle in filamentous ascomycetes is initiated with theformation of ascogonia. Here, we describe a novel type of sterile mutant from Sordaria macrospora with a defectin ascogonial septum formation. This mutant, named pro22, produces only small, defective protoperithecia andcarries a point mutation in a gene encoding a protein that is highly conserved throughout eukaryotes. Sequenceanalyses revealed three putative transmembrane domains and a C-terminal domain of unknown function.Live-cell imaging showed that PRO22 is predominantly localized in the dynamic tubular and vesicular vacuolarnetwork of the peripheral colony region close to growing hyphal tips and in ascogonia; it is absent from thelarge spherical vacuoles in the vegetative hyphae of the subperipheral region of the colony. This points to aspecific role of PRO22 in the tubular and vesicular vacuolar network, and the loss of intercalary septation inascogonia suggests that PRO22 functions during the initiation of sexual development.

The formation of fruiting bodies in filamentous ascomycetesis a process of multicellular differentiation controlled by manydevelopmentally regulated genes. The self-fertile ascomyceteSordaria macrospora represents an excellent model for study-ing cell differentiation during fungal fruiting body development(reviewed in references 9 and 24).

During the life cycle of S. macrospora, a mature haploidascospore germinates and produces a mycelium composed ofmultinucleate hyphal compartments. After 2 to 3 days, coiledfemale reproductive hyphae, called ascogonia, are formed.Each ascogonium develops further into a more-or-less spher-ical protoperithecium composed of pseudoparenchymatous tis-sue surrounding the original ascogonium (24, 47). Ascogenoushyphae emerge from the ascogonium inside the protoperithe-cium. Within the ascogenous hyphae, the nuclei pair up toform the dikaryotic state, even though S. macrospora is self-fertile and does not require fertilization with an opposite mat-ing type (11, 49). The transition from the spherical protoperi-thecial to the flask-shaped perithecial stage, is believed to bestimulated by the formation of the dikaryon, although this hasnot been experimentally verified (24). The dikaryotic state inindividual hyphal compartments of the growing ascogenoushyphae is maintained by precisely orchestrated crozier forma-tion from the tip of an ascogenous hypha, fusion of the croziertip with the penultimate cell of the ascogenous hypha, conju-gate mitotic divisions, and highly regulated septation (50, 69).Karyogamy of two nuclei in the penultimate cell results in theformation of a diploid ascus mother cell. The diploid state isvery short-lived because the diploid nuclei which are formed

immediately undergo meiosis, followed by an extra mitoticdivision resulting in an ascus containing eight haploid asco-spores (11, 49). An identical process has been observed in theheterothallic fungus Neurospora crassa, with the exception thatcell fusion between opposite mating types is required prior todikaryon formation, and each dikaryotic cell compartmentwithin the ascogenous hyphae contains two nuclei of differentmating types (43, 50).

Although the cytology of ascogenous hyphae with subse-quent crozier formation and karyogamy is well described (3,43), little is known about the nuclear behavior and septation inthe ascogonium or the transition from multinucleate ascogoniato ascogenous hyphae. Recently, we generated a large set of S.macrospora developmental mutants with defects in fruitingbody (perithecium) morphogenesis (21). Mutants of the “pro”type were characterized by forming only protoperithecia andtherefore have sterile phenotypes. Here, we have characterizedthe pro22 mutant and show that, in contrast to all other char-acterized female sterile mutants of Sordaria or Neurospora, it isdefective in septum formation in its ascogonia. To our knowl-edge, this is a novel phenotype for a sterile fungal mutant thatwill allow the analysis of the role of septation during the ear-liest stages of sexual morphogenesis in Sordaria and relatedascomycetes.

MATERIALS AND METHODS

Strains and culture conditions. The S. macrospora mutant pro22 was gener-ated by ethyl methanesulfonate mutagenesis of the wild-type strain S48977, asdescribed previously (39). All S. macrospora strains used in the present study arelisted in Table 1.

Standard growth conditions and DNA-mediated transformation experimentswith recombinant plasmids were performed with the mutant strain pro22 asdescribed previously (10, 26, 31, 64). Culture methods used to compare pro22transcript levels under conditions allowing sexual versus vegetative developmentand RNA isolation for quantitative real-time PCR analysis have been describedby Nowrousian and Cebula (29).

Propagation of recombinant plasmids was performed by using standard labo-

* Corresponding author. Mailing address: Lehrstuhl fur Allgemeineund Molekulare Botanik, Ruhr-Universitat Bochum, ND7/131, Uni-versitatsstrasse 150, 44780 Bochum, Germany. Phone: 49-234-3226212.Fax: 49-234-3214184. E-mail: ulrich.kueck@rub.de.

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 15 October 2010.

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ratory techniques (54). Escherichia coli strain XL1-Blue MRF� served as the hostfor general plasmid construction and maintenance (18).

Construction of plasmids. All plasmids used in the present study are listed inTable 2. Cloning of S. macrospora DNA fragments into various vectors wasperformed by using standard techniques (54). Plasmid pCR6.1-10 containing theentire pro22 open reading frame (ORF), including the 5� flanking region, wasgenerated by ligation of a 4.5-kb amplicon of S. macrospora genomic DNA(oligonucleotides 165.1 and 160.1, Table 3) into pDrive (Qiagen, Hilden, Ger-many).

For complementation analysis of the pro22 mutant strain, a plasmid with amutated pro22 ORF was derived from plasmid pCR6.1-10 as follows. pCR6.1-10was digested with Eco47III and EcoRV, and a 6.8-kb subfragment was self-ligated, generating plasmid p22D153-631, which carries a 1.5-kb in-frame dele-tion (bp positions 459 to 1952) in ORF pro22.

For localization studies, a NcoI fragment containing the entire pro22 ORF wasligated into NcoI-digested pEH3 (32, 40), generating pGFP-22-1, encoding aPRO22-GFP fusion protein. For colocalization experiments, cotransformation ofpGFP-22-1 and pRFP-vam-3 (5) was performed. Cotransformation with pGFP-22-1 carrying the hph gene was required since pRFP-vam-3, coding for a vam-3-tdimer fusion gene under the control of the ccg-1 promoter, lacked any suitableselection marker (7).

Real-time PCR. Reverse transcription of total RNA and quantitative real-timePCR analysis were both performed as described by Nowrousian et al. (33) usinga pPCR Master Mix Plus for SybrGreen (Eurogentec, Seraing, Belgium). pro22amplicons were generated with the oligonucleotides RT-22-FP and RT-22-RP(Table 3). Each reaction was carried out in triplicate with biologically indepen-dent samples. Mean CT values (threshold cycles) were calculated from the trip-licates and used for calculation of expression ratios according to the method ofPfaffl (37). The CT values for an amplicon derived from SSU rRNA (oligonu-cleotides SSU-for and SSU-rev, Table 3) were used as a reference for normal-

ization. The significance of differential expression was verified with REST (i.e.,the pairwise fixed reallocation randomization test [38]).

Live-cell imaging. For all microscopic investigations, strains were grown onsolid BMM fructification medium (11). Samples for the hyphal fusion observa-tions were prepared by using the “inverted-agar-block” method (17) and imagedwith differential interference contrast (DIC) microscopy on a Nikon EclipseTE2000-E inverted microscope, with a 20�/0.50 NA Plan Fluor objective lens, aDXM1200F camera, and ACT-1 image capture software (Nikon, Kingston-upon-Thames, United Kingdom).

For microscopy with the AxioImager microscope (Zeiss, Jena, Germany), S.macrospora strains were grown on glass slides in glass petri dishes at 27°C withcontinuous light for 2 to 6 days, as described previously (10). Samples forconfocal laser scanning microscopy were prepared by using the inverted-agar-block method (17). Fungal cell walls were stained with 0.1 mg of CalcofluorWhite/ml (0.1 mg/ml in 0.01% 0.4 M NaOH).

Fluorescence and light microscopic investigations with the AxioImager micro-scope were carried out using an HBO 100 Hg or XBO 75 xenon lamp forfluorescence excitation. For detection of enhanced green fluorescent protein(EGFP) and tdimer2 fluorescence, the Chroma filter sets 41017 (excitation filterHQ470/40, emission filter HQ525/50, and beamsplitter Q495Ip) and 41035 (ex-citation filter HQ546/11, emission filter HQ605/75, and beamsplitter Q560Ip)(Chroma Technology Corp.) were used. For the detection of Calcofluor, theChroma filter set 31000v2 (excitation filter D350/50, emission filter D460/50, andbeamsplitter 400dclp) (Chroma Technology Corp.) was used. Images were cap-tured with a Photometrix Cool SnapHQ camera (Roper Scientific) and Meta-Morph (version 6.3.1; Universal Imaging). Recorded images were processed withMetaMorph and Adobe Photoshop CS4.

For confocal laser scanning microscopy, the Bio-Rad Radiance 2100 systemequipped with a blue diode and argon ion lasers mounted on a Nikon TE2000UEclipse inverted microscope was used (Bio-Rad Microscience, Hemel Hemp-stead, United Kingdom). EGFP was imaged by excitation at 488 nm and fluo-rescence detection at 500/30 nm. A 100�/1.40 NA oil Plan Apochromat objectivelens was used for imaging (Nikon). Images were captured with LaserSharp 2000software v5.1 (Bio-Rad Microscience), and image analysis was performed byusing ImageJ.

TABLE 2. Plasmids used in this study

Plasmid Characteristics Source orreference

pANsCos1 Cosmid vector 36D2 40.5-kb S. macrospora genomic

DNA in pANsCos141

pDrive Cloning vector (cloning of PCRfragments)

Qiagen, Hilden,Germany

pCR6.1-10 4.5-kb pro22 ORF, including the5� flanking region in pDrive

This study

pEH3 gpd(p)::egfp 32, 40pGFP-22-1 pro22 tagged with egfp in the NcoI

site in pEH3This study

pRFP-vam-3 N. crassa vam-3 tagged withtdimer2 in pMF334

5

TABLE 1. Strains used in this study

Strain Relevant genotype and phenotypea Reference or source

S48977 Wild type Culture collectionb

S55011 pro22; sterile This studyM5571 pro1; sterile 26S63625 pro11; sterile 39S38717 pro40; sterile 10S46357 pro41; sterile 30T548 Primary transformant of S55011 with pGFP-22-1; gpd(p)::pro22::egfp::hphr; fertile This studyT549 Primary transformant of S55011 with pGFP-22-1; gpd(p)::pro22::egfp::hphr; fertile This studyT556 Primary transformant of S55011 with pGFP-22-1; gpd(p)::pro22::egfp::hphr and pRFP-vam-3;

ccg-1(p)::vam-3::tdimer2::his; fertileThis study

T548A Single-spore isolate of T548 This studyT548B Single-spore isolate of T548 This studyT548C Single-spore isolate of T548 This studyT548D Single-spore isolate of T548 This study

a hphr, hygromycin resistant.b That is, the culture collection of the Lehrstuhl fur Allgemeine und Molekulare Botanik, Ruhr-Universitat, Bochum, Germany.

TABLE 3. Oligonucleotides used in this study

Oligonucleotide Sequence (5�–3�)

3305.......................ATCCATGGTACTCCACCCCATCGAGCTCTC3306.......................ATCCATGGCCATCATGAACGCGCTC160.1......................AAAAGCTTCTAACTCCACCCCATCGAGC165.1......................AAAAGCTTATGCTATGTCTGCGGTTGGRT-22-FP..............ACCTTCTGCTCGTGCACTACAART-22-RP .............GTGATGACGCGCATGTTAGACTSSU-for.................ATCCAAGGAAGGCAGCAGGCSSU-rev.................TGGAGCTGGAATTACCGCG

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Two photon microscopy was performed by using a LSM 510 Meta systemmounted on an Axioskop 2 microscope (Zeiss). Calcofluor White was excitedwith a 780-nm line. Fluorescence emission was detected by using a KP 685short-pass filter.

LTSEM. Samples for low-temperature scanning electron microscopy(LTSEM) were cultured on cellophane (uncoated Rayophane) over agar (52). Thecellophane was adhered to the sample stub with Tissue-Tek (Sakura FinetekEurope BV) 4583 (polyvinyl alcohol � 11%, carbowax � 5%) (Agar Scientific,Stanstead, United Kingdom), and specimen preparation was as described previ-ously (51, 52) by using the Hitachi S-4700 cold field emission scanning electronmicroscope fitted with a Gatan Alto 2500 cryospecimen system. The specimenwas partially freeze-dried in the microscope by bringing the temperature of themicroscope cold stage up to approximately �95°C and then sputter coated with60%:40% gold-palladium alloy in the cryopreparation unit.

Sequence analysis. Protein sequence alignments were performed by using theCLUSTAL W program (60; http://clustalw.genome.jp/). Prediction of transmem-brane domains was done by using TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) and TopPred (http://mobyle.pasteur.fr/cgi-bin/portal.py).

Nucleotide sequence accession number. Sequence data for the S. macrosporapro22 gene have been submitted to EMBL database under accession numberAJ627567.1.

RESULTS

The sterile mutant pro22 exhibits a pleiotropic morphoge-netic phenotype. Recently, we generated a large set of S. ma-crospora mutants showing defects in fruiting body maturation(21). We describe here a mutant (pro22) that forms defectiveprotoperithecia that never mature into perithecia. Light mi-croscopy of vegetative hyphae of the wild type and mutant

pro22 (Fig. 1) indicated that the mutant was defective in hy-phal fusion as previously reported (53). Although adjacenthyphae of the pro22 mutant often made contact in the maturecolony, they were never observed to fuse (Fig. 1B) under theexperimental conditions used.

Like the wild type, pro22 produced ascogonia after 2 days ofgrowth, which developed further into young protoperithecia(Fig. 2A to D). After reaching a diameter of �40 �m (Fig. 2Cfor the wild type and Fig. 2D for mutant pro22), protoperithe-cia of the mutant strain did not develop any further and thusled to a sterile phenotype. Externally, the protoperithecia ofthe wild type and pro22 looked very similar. However, afterstaining their cell walls with Calcofluor White, two-photonlaser scanning microscopy revealed that the protoperithecia ofthe two strains clearly had different internal structures (Fig. 3).Figure 3A depicts a schematic overview of the five opticalplanes used to scan through each protoperithecium. The fruit-ing body primordium of pro22 consists of a loose aggregationof thin-walled hyphae without any obvious differentiationwithin the aggregate (Fig. 3B, lower row). In contrast, theaggregated hyphae in the wild-type protoperithecia were moredensely packed, and many fluoresced stronger with Calcofluor,indicating that they had thicker cell walls (Fig. 3B, upper row).

Although pro22 forms ascogonia like the wild type, on closerinspection we observed a clear difference between the strains.In excess of 200 ascogonial coils of both mutant and wild typewere assayed. With the exception of an occasional septum atthe base of the ascogonium adjacent to the parent hypha, theascogonia of pro22 consistently lacked intercalary septa (Fig.4A), whereas wild-type ascogonia always formed one or moreintercalary septa (Fig. 4B). By capturing “z-stacks” of images,we were able to rigorously analyze the three-dimensional struc-ture of the ascogonia, which further confirmed the completeabsence of intercalary septa in pro22 ascogonia (see Movies S2

FIG. 1. (A and B) DIC microscopy of the subperipheral region ofthe young colony in S. macrospora, 5 to 10 mm in from the colony edgeof a 24-h-old culture, inoculated with a mycelial plug. Self-fusion isindicated (circles) and in each case was verified by live-cell observa-tions of cytoplasmic flow through the fusion points. Contacts betweenhyphae, with no evidence of fusion, are also indicated (by stars).(A) Wild-type strain showing vegetative hyphal fusion (circles), butalso a few places of contact (stars), without fusion. (B) In the mutantpro22, there are many points of contact, but no evidence of fusion. Bar,50 �m. Strains were grown on solid BMM fructification medium.

FIG. 2. (A) Ascogonium (ac) of the wild type (wt), which is juststarting to be enwrapped by an enveloping hypha (ev). (B) Ascogo-nium (ac) of the mutant pro22. (C) Protoperithecium of the wild typewith a diameter of �40 �m. At this point, enveloping hyphae havewrapped around the structure. (D) Protoperithecium of the mutantpro22 with a similar diameter of �40 �m. Further development ofpro22 protoperithecia is blocked at this stage. Bar, 10 �m. Strains weregrown on solid BMM fructification medium.

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and S3 in the supplemental material). This contrasted withseptation in the pro22 vegetative mycelium, which was remark-ably similar to that of the wild type (see Fig. S1 in the supple-mental material). Thus, the lack of any septa inside the coiledascogonium is a novel phenotype that is very distinctive forpro22. Interestingly, a homologue of PRO22 from Ashbya gos-sypii, AgFar11, is directly involved in septum formation duringthe development of sporangium-like structures (23), indicatingthat a function in septation during a distinct phase of the lifecycle might be conserved.

To determine whether this nonseptate phenotype is a gen-eral feature of pro mutants, we investigated ascogonia fromfour other well-characterized pro mutants: pro1, pro11, pro40,and pro41 (10, 26, 30, 39). As shown in Fig. 4C to F, all of thesepro mutants produced septate ascogonia that closely resem-bled those of the wild type (Fig. 4B). As shown later, thisdistinctive feature of aseptate ascogonia in pro22 was furtherconfirmed by complementation studies in which the pro22 genewas used to restore the wild-type septate, ascogonial pheno-type in the pro22 mutant (Fig. 4G).

Characterization of the complementing pro22 ORF. Thepro22 gene was identified by a previously described genomiccomplementation strategy (26, 39) with an indexed cosmidlibrary representing the S. macrospora genome (41). A 40.5-kbcosmid clone, designated D2, was isolated due to its ability torestore perithecium and ascospore formation in pro22. In sub-sequent complementation experiments, we succeeded in iso-lating a 3.3-kb subfragment of D2 that was sufficient to restorefertility in the mutant. Oligonucleotides 165.1 and 160.1 (Table3) were used for PCR amplification of the entire S. macrosporagene, including the 5� flanking region (pCR6.1-10, Table 2).We designated the complementing gene as pro22, which ishighly homologous to the ham-2 gene from N. crassa, showing88% sequence identity at the nucleotide level for the codingregion. Similar to ham-2, the pro22 ORF comprises 3,613 bp,interrupted by four introns of 58, 65, 60, and 67 bp, which was

confirmed by sequencing of the corresponding cDNA frag-ments.

For a detailed analysis of the mutant locus, genomic DNAfrom pro22 and the wild type was isolated and subjected toSouthern hybridization analysis, using the insert of pCR6.1-10as a probe. Comparison of the hybridization patterns of genomicDNA digested with EcoRI or HindIII revealed no differencesbetween pro22 and the wild-type strain (data not shown). Wetherefore concluded that no major rearrangements or dele-tions occurred in pro22 during chemical mutagenesis.

To investigate whether pro22 does indeed contain a muta-tion within the pro22 ORF, overlapping genomic DNA frag-ments from the mutant were amplified by PCR, sequenced andcompared to the wild-type pro22 sequence. The mutant pro22gene differs from that of the wild type by a C-to-T transitionwithin the coding region, changing a glutamine codon to a stopcodon. It can therefore be predicted that only a truncatedPRO22 polypeptide of 436 amino acid residues is synthesizedby pro22 (Q437STOP).

Characterization of the PRO22 protein. The complementingpro22 gene encodes a putative protein of 1120 amino acids witha calculated molecular mass of 124 kDa. BLAST searches (1)in public databases identified several predicted proteins asputative PRO22 orthologs in various eukaryotes ranging frombudding yeast to human. The N. crassa homologue of PRO22,HAM-2, is involved in hyphal fusion and sexual development(68). In Saccharomyces cerevisiae, the homologous proteinFar11 belongs to the Far complex and plays a role in phero-mone-induced cell cycle arrest (20). However, no homologueswere detected in genomic sequences from plants and bacteria.The most conserved sequence within the PRO22 homologuesis a domain of unknown function found in the C-terminalregion of the protein shown in Fig. 5A. A sequence alignmentof this domain with homologues of other eukaryotes is de-picted in Fig. 5B. By using in silico analysis, we detected three

FIG. 3. Two-photon microscopy of protoperithecia from S. macrospora wild type and pro22. (A) Schematic overview of the five planes used toscan through each protoperithecium shown in panel B. (B) Protoperithecia of the wild type (wt) and pro22, each with a diameter of �45 �m. Thecell walls were stained with Calcofluor White. Bar, 20 �m. Strains were grown on solid BMM fructification medium.

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putative transmembrane domains in PRO22 at amino acidpositions 364 to 384, 655 to 673, and 1086 to 1104 (Fig. 5A).

For complementation analyses (Fig. 5A), pro22 was trans-formed with pGFP-22-1 (Table 2) coding for a PRO22-GFPfusion protein. We obtained two transformants, T548 and

T549, which showed restoration of the wild-type phenotypewith mature fruiting bodies that released viable ascospores.Microscopy of the ascogonia of these transformants revealedthat both developed normal-looking ascogonia with intercalarysepta (Fig. 4G). These observations clearly demonstrated thataseptate ascogonia are a specific phenotype of pro22 and thatthis phenotype is strictly correlated with the pro22 mutation.

Next, we generated p22D153-631 with a 1.5-kb in-framedeletion in the N terminus of pro22 but still encoding part ofthe PRO22 C terminus. Transformants were still sterile, sug-gesting that essential domains associated with perithecium de-velopment are located within the region deleted in p22D153-631 (Fig. 5A).

Expression analysis of pro22. In common with many otherdevelopmental genes, pro22 is only weakly expressed and is notdetectable in Northern hybridizations with poly(A)-RNA (datanot shown). Using reverse transcription-PCR analysis, we wereable to amplify cDNA fragments lacking intron sequences,thus demonstrating transcriptional expression of pro22 (datanot shown). Subsequently, we performed a comparative anal-ysis of pro22 transcript levels between wild-type mycelia grownunder conditions allowing sexual development versus condi-tions that only result in vegetative growth. pro22 expressionwas found to be increased up to 4-fold after 4 days duringsexual development (Fig. 6). We further analyzed pro22 ex-pression in mutant pro22, which was more than 4-fold reduced,indicating a positive autoregulation (data not shown).

Subcellular localization of PRO22 in living hyphae. Re-cently, we have successfully used the EGFP reporter for thesubcellular localization of developmental proteins in S. mac-rospora (10, 30, 40). Attempts to express fluorescent protein-tagged PRO proteins under the control of their native promot-ers gave very weak signals when imaged by fluorescencemicroscopy (10). Furthermore, pro22 is weakly expressed tran-scriptionally (see above) and therefore, as in previous ap-proaches, the Aspergillus nidulans gpd promoter (42) was usedto express pro22 fused at its 3� end with egfp. This resulted inthe construction of the plasmid pGFP-22-1 (Table 2), whichwas used to transform the pro22 strain. We obtained severaltransformants, with a fertile phenotype, thus indicating that thefusion construct is functional. For confocal laser scanning mi-croscopy, two different transformants, T548 and T549, and foursingle spore isolates of T548 (T548A to T548D, Table 1) wereused. In these strains, fluorescence was restricted to dynamictubular and vesicular structures in the peripheral region of thecolony close to growing hyphal tips although fluorescence wasabsent from a 30 to 40 �m zone immediately behind these tips(Fig. 7B and D). This tubular network with associated vesicularstructures is similar in morphology and dynamics to the vacu-olar system typically found in the peripheral region of filamen-tous fungal colonies (8, 63). However, other organelles (e.g.,mitochondria and endoplasmic reticulum [ER]) can exhibit atubular and/or net-like organization in apical hyphal compart-ments but, significantly, these organelles are not excluded fromthe extreme apical regions of hyphae (12, 16, 17, 25). Local-ization experiments with mitochondrial and ER markers con-firmed that PRO22 is neither localized to the ER nor mito-chondria (data not shown).

To verify localization of PRO22 to vacuoles in S. macros-pora, we used VAM-3, a vacuolar membrane protein, which

FIG. 4. (A to G) Septum formation in ascogonia. The right and leftcolumns show DIC and fluorescence images, respectively, after stain-ing with Calcofluor White. Ascogonia of pro22 lack septa (A), whereasascogonia of the wild type (B) and other pro mutants (pro1, pro11,pro40, and pro41; C to F) are septate (arrowheads). Transformation ofa plasmid coding for PRO22-GFP into mutant pro22 restores septumformation in ascogonia (G). Bar, 10 �m. Strains were grown on solidBMM fructification medium.

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FIG. 5. (A) Physical map of PRO22 (top) encoded by the pro22 ORF (bottom). Three putative transmembrane domains are marked in lightgray, and a conserved domain of unknown function is shown in dark gray. The pro22 ORF is indicated by a gray arrow; the introns are highlightedin white. Recombinant plasmids are shown below the physical map and were used for complementation analysis with the mutant pro22 as recipient.Numbers show the positions of amino acids (aa) and nucleotides (bp). Fertility is indicated by “�”, while “–” marks sterility. (B) Alignment of theconserved C-terminal domain, positions 714 to 1008 of the S. macrospora peptide, containing highly conserved residues. The first two characterson the left indicate the name of an organism: Sm (S. macrospora, accession no. AJ627567 [NCBI]), Nc (N. crassa, accession no. CAC28842 [NCBI]),Mg (Magnaporthe grisea, accession no. MG00731 [Broad Institute]), An (A. nidulans, accession no. XM_659123 [NCBI]), Fg (Fusarium graminea-rum, accession no. XP_387335 [NCBI]), Um (Ustilago maydis, accession no. XP_758764 [NCBI]), Ce (Caenorhabditis elegans, accession no.AAA82421 [NCBI]), Dm (Drosophila melanogaster, accession no. NP_647806 [NCBI]), Hs (Homo sapiens, accession no. AAH19064 [NCBI]), Sp(Schizosaccharomyces pombe, accession no. CAA16899 [NCBI]), and Sc (Saccharomyces cerevisiae, accession no. NP_014272 [NCBI]). Amino acidsthat are invariant are highlighted in black. Identical residues conserved in more than 50% of the aligned sequence are highlighted in dark gray,while conserved similar residues are highlighted in light gray. Sequence analysis was performed as described in Materials and Methods.

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has already been used as a vacuolar marker in different fila-mentous fungi (5, 57). Since N. crassa and S. macrosporaVAM-3 show a high level of sequence similarity (34, 35), wewere able to use the N. crassa VAM-3 (5) for colocalizationexperiments with PRO22. In order to generate strains for co-localization, we performed cotransformation of mutant pro22with pGFP-22-1 and pRFP-vam-3 (Table 2). The latter con-tained a 3� terminal fusion of the N. crassa vam-3 with tdimer(5). Transformants obtained in this experiment regained fer-tility and, for microscopic investigations, transformant T556,expressing both PRO22-GFP and the N. crassa RFP-VAM-3,was chosen. Both fusion proteins colocalized in the same tu-bular and vesicular structures close to hyphal tips (Fig. 8),confirming that PRO22 is localized in the vacuolar system ofthe peripheral colony region. As described previously (5),

FIG. 6. Quantitative real-time PCR analysis of pro22 transcript lev-els. The wild type was grown under vegetative and sexual growthconditions. The data are shown as base 2 logarithmic ratios for themean values (calculated using REST) of three independent experi-ments.

FIG. 7. PRO22 localization in living vegetative hyphae of S. macrospora. (A) Scheme of vacuolar distribution in hyphal compartments. Dynamictubular and vesicular vacuoles are restricted to the peripheral region and are absent from the very tip. These vacuoles disappear in thesubperipheral region and are replaced by more static large spherical vacuoles often stuck at septal pores. (B) Fluorescence microscopy of a growinghyphal tip showing that the PRO22-GFP only starts to label tubular and vesicular components of the vacuolar system 30 to 40 �m back from thehyphal tip. (C and D) Fluorescence microscopy showing localization of PRO22-GFP in dynamic tubular and vesicular components of the vacuolarsystem in the peripheral region of the colony (D) but its absence from the large spherical vacuoles in the subperipheral colony region (C). Bar,10 �m. Strains were grown on solid BMM fructification medium.

FIG. 8. DIC and fluorescence microscopy showing the localizationof PRO22-GFP and the N. crassa vacuolar marker RFP-VAM-3 intubular and vesicular components of the vacuolar system in vegetativehyphae from peripheral regions of the colony (A) and in ascogonia (B).The merged images shows colocalization of the fusion proteins con-firming that PRO22-GFP is a vacuolar protein. Bar, 10 �m. Strainswere grown on solid BMM fructification medium.

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RFP-VAM-3 not only localizes to the vacuolar membrane butalso to the vacuolar lumen, as was observed for PRO22-GFP.

Because of our observed involvement of PRO22 in septumformation in ascogonia and the pro22 mutant being defectivein sexual development, we analyzed these sexual structures ofS. macrospora to determine the localization of PRO22-GFPin them. The DIC micrographs clearly demonstrated theexistence of vacuoles in ascogonia (Fig. 4). Fluorescence ofPRO22-GFP was observed in large and small spherical struc-tures, strongly resembling vacuoles (Fig. 8B). Furthermore,RFP-VAM-3 clearly colocalized within these structures, indi-cating that the stained structures are indeed vacuoles (Fig. 8B).In summary, PRO22 is not only localized to the dynamic tu-bular and spherical vacuoles at the periphery of the colony butalso in ascogonia in the subperipheral region.

Besides tubular and vesicular vacuoles, filamentous fungialso possess large spherical vacuoles that are mostly static andcommonly positioned adjacent to, and on the distal side of,septa (5, 8, 56). PRO22-GFP was found to be absent fromthese spherical vacuolar compartments (Fig. 7C). Figure 7Asummarizes the structure and distribution of vacuoles in dia-grammatic form as it is for S. macrospora and other filamen-tous fungi. In contrast to PRO22-GFP, N. crassa VAM-3 islocalized in both the large spherical vacuoles and the tubular/vesicular vacuoles (data not shown).

DISCUSSION

Septum formation in ascogonia is a prerequisite for fruitingbody formation. In the present study, we used a forward ge-netic approach (21, 26, 39) to characterize the sterile S. mac-rospora mutant pro22, which forms protoperithecia but notmature perithecia. Scanning electron microscopy and extensivelive-cell imaging confirmed our previous observation thatpro22 is defective in hyphal fusion (53) and that this defect iscorrelated with sexual development. A link between defects insexual development and hyphal fusion has been previouslyreported in mutants of S. macrospora and N. crassa (10, 50, 53),suggesting a functional relationship between hyphal anastomo-sis and fertile fruiting body formation. In the wild type, mul-tiple hyphal fusion events result in an interconnected hyphalnetwork that permits translocation of cellular components,such as water, organelles, metabolites, nutrients, or signalingmolecules throughout the colony, which presumably facilitatesgrowth and reproduction (6, 13, 46, 50). Transport of cellularcomponents may be one function of hyphal fusion, but hyphalfusion may play other roles during sexual development. It may,for example, be necessary for the formation of compact, highlydifferentiated, interconnected, multicellular fruiting bodies.The lack of hyphal fusion may be one of the reasons why pro22forms loosely aggregated protoperithecia, and its reduced in-terconnected state may play a role in causing early develop-mental arrest.

Our observation that vegetative hyphae of pro22 are septatewhile ascogonia lack septa seems contradictory. However, thisindicates that PRO22 may play a specific role in septationduring sexual development. Each septum in vegetative hyphaecontains a septal pore that can allow cytoplasmic flow betweenadjacent hyphal compartments and distribution of organellesthroughout the whole colony. Occlusion of septa during sexual

development may serve other important functions. In ascog-enous hyphae of Sordaria humana it has been shown that theseptal pores have highly complex and sometimes very elabo-rate membranous structures associated with them. These poreocclusions are of a type not found in vegetative hyphae and arehighly variable structures from being relatively simple porecaps to having complex swollen rims with associated mem-brane cisternae (2, 49). These membranous structures providecontinuity between the endomembrane systems of adjacentcells in ascogenous hyphae and particularly between the nu-clear envelopes of the two nuclei in the same cell and betweennuclei in adjacent cells. This suggests that they play a special-ized role in communication in ascogenous hyphae that may, insome way, relate to the regulation of the dikaryotic state (2,49). An important aspect of the plugging of septal pores is thatthey may also physically isolate adjacent hyphal compartmentsand thus allow them to undergo different modes of differenti-ation (15). In this way septation and septal pore plugging mustplay important roles in the differentiation of fungal multicel-lular structures such as perithecia (48). However, at present wedo not know whether the septa of ascogonia possess septalpores, and if they do, whether they are plugged and by whattype of septal pore occlusions. We speculate that plugged septain ascogonia might allow for the accumulation of signalingmolecules which trigger subsequent sexual development. Asep-tate ascogonia in pro22 may therefore prevent such an accu-mulation, and this could explain the sterile phenotype.

The homologue of PRO22 in S. cerevisiae, Far11, plays a rolein pheromone-induced cell cycle arrest during the life cycle ofyeast and forms a complex with other Far proteins regulatingG1 arrest (20). Kemp and Sprague (20) further suggested thatFar11 and the other members of the Far complex may berequired for cell fusion during mating. They have proposedthat the Far complex could serve as a checkpoint that monitorsfusion and prevents premature re-entry into mitosis to ensurethat mating cells have sufficient time to fuse. Whether or notPRO22, like its yeast homologue, is directly involved in theregulation of mitosis remains to be determined.

It is very interesting that in S. cerevisiae, the PRO22 homo-logue Far11 directly interacts with the small GTPase RHO4(62). RHO4 is involved in septum formation in many ascomy-cetes; it was first observed in the fission yeast Schizosaccharo-myces pombe, where �rho4 mutants have multiple, abnormalsepta (28, 55). In contrast, a rho4 mutation in N. crassa leads toa complete loss of septation in vegetative hyphae (44). Ras-mussen and Glass further showed that RHO-4 localizes tosepta and identified a difference in the regulation of septationduring conidiation versus vegetative septation in N. crassa (44,45). Recent studies in N. crassa and A. nidulans indicate thatRHO-4 localizes to septa and to the plasma membrane and isinvolved in signaling pathways that regulate septum formation(19, 58).

Another homologue of PRO22 is AgFar11 from A. gossypii,which is directly involved in septum formation during the de-velopment of sporangium-like structures. Normally, in A. gos-sypii, formation of these “sporangia” involves hyphal abscissionby a constriction at the septum between the old mycelium andthe sporangium (67). Interestingly, deletion of the Agfar11gene led to a defect during septum formation with completeabscission of hyphal compartments (23). Thus, in �Agfar11,

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this process already occurs before sporangium formation. Thisphenotype indicates a role for AgFar11 in the regulation ofseptum formation that is dependent on the developmentalstage of the fungus. Interestingly, the pro22 mutant lacks septain ascogonia but forms them in vegetative hyphae. Therefore,the loss of septation in ascogonia suggests that PRO22 in S.macrospora has a different function during the sexual phase ofthe life cycle compared to its role in the vegetative mycelium.

PRO22 is associated with the dynamic vacuolar networknear growing hyphal tips and with vacuoles in ascogonia. Inour attempt to localize PRO22, we analyzed different S. mac-rospora transformants expressing a functional EGFP-taggedversion of the protein. Our finding that PRO22 does not lo-calize to the entire vacuolar system of S. macrospora, but spe-cifically to the dynamic tubular and vesicular vacuolar compo-nents near growing hyphal tips in the peripheral region of thecolony and to vacuoles in ascogonia strongly suggests that theprotein has a unique function in these organelles. It hasbeen previously proposed that the different types of vacu-oles may play different roles in different regions of themycelium (56, 57).

For colocalization experiments, we used the well-establishedvacuolar membrane protein VAM-3 from N. crassa (5). SinceVAM-3 localizes exclusively to the vacuolar membrane in bud-ding yeast (65, 66), it has been used as a vacuolar marker inplants and fungi (5, 22, 57, 61). In N. crassa, VAM-3 localizesto the entire vacuolar system, including tubular and vesicularvacuolar compartments (5). The N. crassa VAM-3 not onlylocalizes to the membrane of spherical vacuoles but also to thelumen, where most probably an internalization of the vacuolarmembrane components occurs. Bowman et al. (5) proposedthat this might be due to a protein turnover process.

A VAM-3 homologue was also used as a vacuolar marker inA. oryzae by labeling AoVam3 with GFP (57). Similar to N.crassa, fluorescence was observed in the entire vacuolar sys-tem, including the lumen of large spherical vacuoles in thesubperiphery of the colony, although the GFP tag was locatedon the cytosolic side of the vacuolar membrane. These authorspostulated that an autophagic degradation of cytosolic mate-rials might have occurred, followed by a subsequent internal-ization of the vacuolar membrane into the lumen. Tubularvacuoles might then transport the degraded material to moreactive regions of the colony.

Significantly, PRO22 could not be identified in the sphericalvacuoles of vegetative hyphae in the subperipheral region ofthe colony and was only found in vacuoles in ascogonia and invery dynamic components of the tubular and vesicular vacuolarnetwork in the colony periphery. Here, PRO22 was not re-stricted to the vacuolar membrane but also occurred in thelumen. It is possible that PRO22 plays a role in nutrient trans-port which has been previously shown to be carried out by thetubular vacuolar system (13), whereas the protein is probablynot involved in vacuolar processes occurring in the sphericalvacuoles in the more distal regions of the colony. Our findingsthat PRO22 is localized to vacuoles in ascogonia seems rea-sonable because the phenotypic characterization of the mutantpoints to a role in this part of the S. macrospora life cycle.

A recent finding supports our observation that PRO22 islocalized to vacuoles. Simonin et al. (59) showed that the N.crassa ham-2 mutant, which corresponds to the pro22 mutant,

exhibits an aberrant vacuolar morphology. In ham-2 and otherham mutants deficient in hyphal fusion, large vacuoles wereobserved in conidial germlings, suggesting a defect in vacuolarprotein sorting. Interestingly, the S. cerevisiae mutant far11 andtwo other far mutants, far9 and far10, were identified in alarge-scale screen for vacuolar protein sorting mutants (4),indicating that Far11, like its homologues PRO22 and HAM-2,may play a role during this process.

Is there a link between septation and vacuolar localization?Our finding that PRO22 localizes to vacuoles and plays a roleduring septum formation in sexual structures raises the ques-tion as to how its localization and function are linked. A can-didate which links septum formation and vacuolar localizationmight be btn1 from the fission yeast S. pombe, the homologueof the human batten disease gene CLN3. Btn1 deletion strainsdisplayed enlarged and more alkaline vacuoles, indicating arole for Btn1 in the vacuolar system, and this correspondedwith the localization of the protein in vacuoles and prevacuolarcompartments (14). Furthermore, btn1 deletion cells had asignificantly higher septation index and an increased number ofbinucleate cells, suggesting a perturbation of cell cycle progres-sion. Although septation in fission yeast and filamentous fungiclearly serves different functions, the molecular mechanismsmay have similarities.

In another study, the entire set of S. pombe ORFs wascloned in a global approach to analyze protein localization infission yeast (27). Interestingly, the PRO22 homologueSpFar11 was shown to localize to vacuoles. This large-scale ex-periment requires verification by a more detailed examinationof SpFar11. However, a connection between this protein andthe vacuolar system seems possible and would corroborate ourfindings.

Taken together, our data point to a specific role for PRO22in the vacuolar network and an involvement in septation dur-ing the initiation of the sexual life cycle. These findings mayprovide new insights into these very early sexual processes, andfuture work will be focused on studies to investigate the inter-action of PRO proteins controlling multicellular development.

ACKNOWLEDGMENTS

We thank Susanne Schlewinski, Regina Ricke, Swenja Ellssel, andIngeborg Godehardt for excellent technical assistance and MinouNowrousian for advice with real-time analysis. We also want to thankHanns Hatt (Bochum, Germany) and Chris Jeffree (Edinburgh,United Kingdom) for assistance with two-photon laser scanning mi-croscopy facilities and scanning electron microscopy facilities, respec-tively. The plasmid pRFP-vam-3 was kindly provided by Barry Bow-man (Santa Cruz, CA).

This study was supported by the Collaborative Research CenterSFB480 (project A1) of the Deutsche Forschungsgemeinschaft (Bonn,Germany) to U.K., a grant from the German Academic ExchangeService DAAD (Bonn, Germany, project D/08/08871) to U.K., anARC grant (project 71/5555/09) to N.D.R., and a Biotechnological andBiological Sciences Research Council grant (project BB/E010741/1) toN.D.R.

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