EUKARYOTIC CELL, Oct. 2010, p. 1495–1503 Vol. 9, No. 101535-9778/10/$12.00 doi:10.1128/EC.00083-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

A Novel Gene, ROA, Is Required for Normal Morphogenesis andDischarge of Ascospores in Gibberella zeae�†

Kyunghun Min,1 Jungkwan Lee,1 Jin-Cheol Kim,2 Sang Gyu Kim,1 Young Ho Kim,1 Steven Vogel,3Frances Trail,4 and Yin-Won Lee1*

Department of Agricultural Biotechnology and Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921, Republic ofKorea1; Chemical Biotechnology Center, Korea Research Institute of Chemical Technology, Daejon 305-600, Republic of

Korea2; Department of Biology, Duke University, Durham, North Carolina 27708-03383; and Department ofPlant Biology and Department of Plant Pathology, Michigan State University,

East Lansing, Michigan 48824-13124

Received 9 April 2010/Accepted 13 August 2010

Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley,and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly dischargedfrom sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, whichis required for normal sexual development. Deletion of ROA (�roa) resulted in an abnormal size and shape ofasci and ascospores but did not affect vegetative growth. The �roa mutation triggered round ascospores andinsufficient cell division after spore delimitation. The asci of the �roa strain discharged fewer ascospores fromthe perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressurewithin the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROAgene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the �roa mutantasci and the mutant spore shape contributed to the longer distance dispersal. When the �roa mutant wasoutcrossed with a �mat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in placeof the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROAplays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.

Gibberella zeae (anamorph: Fusarium graminearum) causesFusarium head blight in wheat, barley, and rice, as well as earrot and stalk rot in maize (20, 23). The infected grains arefrequently contaminated by mycotoxins, such as trichothecenesand zearalenone, which are harmful to humans and animals(6). The fungus overwinters in crop debris in the form ofstorage hyphae and develops ephemeral fruiting bodies (peri-thecia) at warmer temperatures. Ascospores formed within theperithecia are forcibly discharged into the air and are believedto serve as the primary inoculum of the disease (7, 27, 37,39–42). Therefore, sexual development and ascospore dis-charge are important factors in fungal survival and diseaseinitiation.

In fungi of the phylum Ascomycota, the sexual cycle is initi-ated when two genetically distinct nuclei combine to form abinucleate cell (31). As a homothallic fungus, G. zeae possessesthe two mating type genes MAT1-1 and MAT1-2 in the haploidgenome and therefore does not require a mating partner forsexual development (22, 46). Perithecium initials give rise tosmall, coiled initials that develop into perithecia filled withasci, tubular sacs of ascospores, which are the products of

meiosis. Mature asci extend through the ostiole of peritheciaand discharge their ascospores (40).

Unique features of cell differentiation are involved in ascusand ascospore morphogenesis. Ascospore delimitation withinthe ascus and the development of a cell wall between the ascusand ascospore membranes are unique features of the process(31). Most studies of morphogenesis have described thesechanges in detail; however, much of these data have beenlimited to microscopic observations. Several genes involved inascospore morphogenesis have been identified in Neurosporacrassa (30), but the detailed mechanisms and genes involved inascus and ascospore morphogenesis remain to be elucidated.The Round spore (R) mutant of N. crassa was shown to haveround ascospores (24), and the gene responsible for this phe-notype, rsp, was subsequently cloned (28). However, in G. zeae,no genes have been identified that are involved in ascus andascospore morphogenesis.

Although recent research has shed light on the physiologicalbasis of ascospore discharge, the genetic basis remains largelyunknown (38). The main force responsible for the observedshooting is turgor pressure within the extended asci. In G. zeae,a buildup of K� and Cl� ions drives the influx of water andcauses turgor pressure that stretches the asci (41). Asci canaccumulate polyols as well as ions. In a previous study, it wasshown that the polyols are comprised mainly of mannitol andglucose; however, the concentration of these polyols is too lowto make a significant contribution to turgor pressure (42).When the turgor pressure exceeds the threshold of the asci,apical pores rupture and ascospores are forcibly discharged(38). Trail et al. (41) estimated that the acceleration of asco-

* Corresponding author. Mailing address: Department of Agricul-tural Biotechnology and Center for Fungal Pathogenesis, Seoul Na-tional University, Seoul 151-921, Republic of Korea. Phone: (82)2-880-4671. Fax: (82) 2-873-2317. E-mail: lee2443@snu.ac.kr.

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

� Published ahead of print on 27 August 2010.

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spores in G. zeae is 8,500,000 m s�2 using an iterative model topredict initial velocity. Recently, Yafetto et al. (44) used high-speed video photography to examine several large-spore fungi,including Ascobolus immerses, and to predict acceleration dur-ing dispersal. The asci of A. immerses are more than 12-foldlarger in diameter than the asci of G. zeae (38). The sizedifference between these fungi greatly affects the behavior oftheir projectiles and results in an initial speed for G. zeae thatis too great for application of the video photography method(for further discussion, see the supplemental material).

To date, only one gene from G. zeae, the calcium ion channelgene cch1, has been shown to be involved in ascospore dis-charge (12). Deletion of this gene was shown to arrest asco-spore discharge without affecting spore and ascus morphology.Since the genomic sequence of G. zeae is now available, thefunctional analysis of genes involved in sexual development hasbeen accelerated. Random insertional mutagenesis is onestrategy that has been used to identify novel genes associatedwith sexual development (13, 34). Previously, we produced acollection of more than 20,000 mutants from G. zeae by usingthe restriction enzyme-mediated integration (REMI) transfor-mation procedure (13). In this study, the G. zeae mutantZ43R9901, which was isolated from a screening of REMItransformants, showed an unusual phenotype during sexualdevelopment. Further analysis demonstrated that the novelgene ROA is involved in ascospore morphogenesis and dis-charge in G. zeae. The results of this study increase our under-standing of sexual development in the fungus.

MATERIALS AND METHODS

Strains and culture conditions. The wild-type strain Z03643 of G. zeae (3) andmutants derived from this strain were used for this study (see Table S1 in thesupplemental material). The mutant strain Z43R9901 was generated by REMImutagenesis (13). All strains were stored as mycelia and conidia in a 20%glycerol solution at �80°C. Standard laboratory methods and culture media forthe Fusarium species were used (23). For conidial production, the strains wereinoculated in carboxymethyl cellulose medium (4).

Nucleic acid manipulations, primers, and PCR conditions. Fungal genomicDNA was extracted as previously described (23). Total RNA was isolated frommycelia that were ground in liquid nitrogen using an Easy-Spin total RNAextraction kit (Intron Biotech, Seongnam, Republic of Korea). Standard proce-dures were used for restriction endonuclease digestion, agarose gel electrophore-sis, and Southern and Northern analysis (33). The PCR primers (see Table S2 inthe supplemental material) for this study were synthesized by the Bionics oligo-nucleotide synthesis facility (Seoul, Republic of Korea). General PCRwas performed, following the manufacturer’s instructions (Takara Bio Inc., Otsu,Japan). Plasmid DNA was purified from Escherichia coli grown in 3 ml oflysogeny broth using a plasmid purification kit (Intron Biotech, Seongnam, Re-public of Korea). DNA sequence analysis of the rescued plasmids was initiatedat a location close to the HindIII site on the REMI vector pUCH1 (43) andperformed with specific primers for pUCH1-P1 and pUCH1-P5.

Targeted gene deletion and complementation. Fusion PCR products for tar-geted gene deletion were constructed using a slightly modified double-joint PCR(DJ-PCR) procedure (45). The 5� and 3� regions of the target gene were amplified byPCR with the primer pairs ROA-5F/ROA-5R and ROA-3F/ROA-3R, respec-tively. The Geneticin resistance gene cassette (gen), which carries the Aspergillusnidulans trpC promoter and terminator, was amplified from pII99 (26). The threeamplicons (5� region, 3� region, and gen) were mixed in a 1:1:3 molar ratio andfused by a second round of PCR. The fused constructs were amplified with thenew nested primers ROA-NF and ROA-NR. To complement the �roa mutant,the entire ROA gene, including the promoter and terminator, was amplified fromthe wild-type strain using the ROA-NF/ROA-NR primer pair. The amplicon wascloned into the pGEM-T-Hyg vector, which contained the hygromycin B resis-tance gene, and the plasmid was transformed into the �roa strain to generate a�roa::ROA strain using the polyethylene glycol (PEG)-mediated fungal transfor-mation method as previously described (18). Transformants were first selected

from regeneration medium amended with 50 �g/ml of Geneticin (Sigma-Aldrich,St. Louis, MO) or hygromycin (Sigma-Aldrich), and each transformant wastransferred to fresh complete medium amended with 100 �g/ml of the antibiotic.The numbers of independent transformants are listed in Table S1 in the supple-mental material.

Self-fertilization. One hundred microliters of conidial suspension (105 spores/ml) was spread across a petri dish (9-cm diameter) containing carrot agar andincubated at 25°C for 5 days (12). The cultures were mock fertilized with 1 ml of2.5% Tween 60 solution to induce sexual development (23) and were continu-ously incubated under near-UV light (wavelength, 365 nm; HKiv Co., Ltd.,Xiamen, China) at 25°C.

Ascospore discharge was observed in small acrylic chambers (1 by 2.5 by 5 cm)that were constructed to minimize free convection (1). A semicircular agar block(11 mm in diameter) that was covered with mature perithecia was placed on acoverslip in the chamber for 24 h (41). This placement allowed the ascospores tobe discharged horizontally down the length of the chamber onto the coverslip.Images were captured on a DE/Axio Imager A1 microscope (Carl Zeiss,Oberkochen, Germany) with a charge-coupled-device (CCD) camera, and theshooting distance of the ascospores was measured at intervals of 0.5 mm usingthe AxioVision release 4.7 software program (Carl Zeiss).

Outcrosses. Outcrosses were performed to characterize the inheritance of thetrait of the �roa mutant under the conditions described above. A MAT1-2deleted mutant (�mat2) tagged with cytoplasmic green fluorescent protein(GFP) (22), a �roa �mat2 double mutant, and a MAT1-1-1-deleted mutant(�mat1) tagged with hH1-GFP (14) were fertilized individually with 1 ml of aconidial suspension (106 spores/ml in 2.5% Tween 60 solution) of the �roamutant or the wild-type strain. Following perithecium formation, progeny werecollected and phenotypically characterized.

Epiplasmic fluid analysis. Five days after induction (DAI) of sexual develop-ment, cultures were inverted so that the ascospores could discharge onto petridish lids. On 14 DAI, distilled water was added to the petri dish lids and theascospores were collected. Ascospores were collected by centrifugation for 10min at 4,000 � g, resuspended in distilled water, and counted with a hemocy-tometer. Spore washes were frozen at �20°C and then dried in a Speed Vacinstrument (Savant Instrument, Inc., Farmingdale, NY) as previously described(42). The identification and quantification of simple sugar alcohols in the epi-plasmic fluid were accomplished by high-performance anion-exchange chroma-tography with pulsed amperometric detection (Dionex Corp., Sunnyvale, CA)using an anion-exchange column (CarboPac MA1, 4 by 250 mm; Dionex Corp.).The mobile phase was a 0.48 M NaOH solution at a flow rate of 0.4 ml/min.Confirmation of the sugar alcohols was performed by gas chromatography-massspectrometry (GC-MS). Sugar alcohols in the epiplasmic fluid were derivatizedby the acetylation method as previously described (17). Briefly, each extract wasdissolved in dry pyridine (1.2 ml) followed by acetic anhydride (3 ml), and themixture was stirred at 100°C. After 1 h, the mixture was cooled at room tem-perature, and 20 ml of cold water containing ice was added. The aqueous layerwas extracted twice with 5 ml of diethyl ether. The combined extracts were thendried over anhydrous MgSO4 and evaporated until completely dry. The residuewas dissolved in 500 �l of ethyl acetate, and 1 �l of the solution was subsequentlyinjected into a capillary Shimadzu QP-5050 GC-MS (Shimadzu, Kyoto, Japan).The analytical conditions were as follows: a DB-5 fused silica column was used(30 m by 0.25 mm [inside diameter], 0.25-�m film; J&W Scientific, Folsom, CA);the column temperature was 80°C for 4 min and then was increased to 220°C ata rate of 10°C/min; the injector temperature was 250°C; the interface tempera-ture was 250°C; the ionizing voltage was 70 eV; and the ionizing current was 300�A. The K� and Na� contents were determined on an inductively coupledplasma-atomic emission spectrometer (ICP-AES) at an emission-line wavelengthof 766.490 nm using the Jobin-Yvon Model 170 ICP emission spectrometerUltrace (Jobin-Yvon Ultima, Longjumeau, France). The Cl� content was quan-tified by ion chromatography (IC) using a Metrohm model 761 Compact IC(Metrohm, Herrisau, Switzerland) with a suppressor module and equipped withan ICSep AN2 analytical column (4.6 by 250 mm) and an ICSep AN2 guardcolumn (4.6 by 50 mm). The ions were detected using a suppressed conductivitydetector that had a full scale of 250 �S/cm, which was optimized with respect tothe maximum signal-to-noise ratio for the anions being analyzed.

Mathematical model for predicting initial velocity and turgor pressure. Pre-viously, we used a computer program which predicted initial velocity based onsuccessive approximations, working back from the mean distance traveled (41).Determining the launch speed of the spores emerging from the ascus is anessential step in predicting the turgor pressure. We used two methods for thisprediction here, modifying the iterative program to use Stokes’ law and a “full-model” approach. The program was the same for both except in the way itapproximated drag, varying drag coefficients for the “full model” and using the

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standard 24/Re drag coefficient reflected in Stokes’ law. The effective size wascalculated using predictions of drag at two extremes (spore flying lengthwise andspore flying crosswise with respect to the trajectory). The results were averaged,and then the sphere diameter that gives an equivalent result was used (41). Thediameter for the wild type and revertant was 11.03 �m; for the mutant, it was11.40 �m; density was taken as 1,200 kg m�3. The iterative interval was 20 ms,which produced about 1,000 steps, and the launch pitch angle was 2 degrees.Launch speeds were determined from calculated trajectories that produced theobserved horizontal ranges, as done previously (41).

The following formulas were used to approximate the drag (D) of sporeshapes, cylinders or long ellipsoids, with their long axes normal and parallel toflow, respectively: D � (4��vl)/[ln(l/a) � 0.193] and D � (24��vl)/[ln(l/a) �0.807], where l and a are length and radius, respectively, of the cylinder orellipsoid. An earlier publication with these formulas contained an error in thesecond equation (41).

Staining of asci and ascospores. Nuclei and chromosomes of G. zeae werestained with acriflavin following the procedure described by Raju (29). Dis-charged ascospores and perithecia were hydrolyzed in 4 M HCl at 30°C for 15 to20 min. The samples were washed twice with distilled water and stained inacriflavin solution (100 �g of acriflavin and 5 mg of K2S2O in 1 ml of 0.1 M HCl)for 20 min. The stained samples were washed three times (2 min each) in anHCl–70% ethanol mixture (2:98 [vol/vol]) and then twice in distilled water.

Specimen fixation and preparation for light microscopy. The perithecia werecollected from the carrot agar surface by gently scraping with a scalpel. Thecollected perithecia were fixed, dehydrated, and embedded in Spurr’s resin (36)as previously described (19). The resin blocks were sectioned at 1 �m with a glassknife on an MT-X ultramicrotome (RMC, Tucson, AZ) and stained with 1%toluidine blue for light microscopy (40).

GFP tagging. To check the cellular localization of ROA, the ROA openreading frame (ORF), which included its own terminator region, was amplifiedfrom the wild-type genomic DNA with ROA-AUG(�) and ROA-3R primers.The 5� flanking region of ROA, which included its own promoter, was amplifiedwith the ROA-promoter and ROA-GFP 5R primers. The GFP was amplifiedfrom pIGPAPA (15) with the EGFP-M and EGFP-p1 primers and fused to theN terminus and 5� flanking region of ROA by DJ-PCR. The fused construct wasthen amplified with the nested primers ROA-promoter N and ROA-3N. Thefusion construct was cloned into the pGEM-T easy vector (Promega Corp.,Madison, WI) and subcloned into pUCH1 (43). The clone was transformed into�roa or the wild-type strain, and the phenotypes of the transformants werecompared with those of the wild-type strain.

Microscopy. The perithecia were dissected on glass slides in a drop of 20%glycerol, and the asci were flattened under the cover glass. Both GFP-tagged andacriflavin-stained nuclei were examined with the 488-nm excitation and 515/530-nm emission wavelength filters. Differential interference contrast (DIC) andfluorescence images were captured on a DE/Axio Imager A1 microscope (CarlZeiss) with a CCD camera. The sizes of the asci and ascospores were measuredusing the AxioVision release 4.7 software program (Carl Zeiss).

RESULTS

Phenotype of REMI mutant Z43R9901. Following self-fer-tilization, the wild-type Z03643 strain produced eight normal,spindle-shaped ascospores containing two to four cells each,while the asci produced by the mutant Z43R9901 containedeight ascospores that were round or oval and contained one ortwo cells each. The numbers of perithecia and ascospores perperithecium that were produced by the mutant were similar tothose of the wild-type strain. In addition, we did not observeany significant difference between the wild-type strain and theREMI mutant in mycelial growth, conidiation, spore germina-tion, or virulence (see Table S3 in the supplemental material).

Molecular characterization of the ROA gene. The genomicDNA from strain Z43R9901 was digested with HindIII orBglII for Southern analysis. The digested DNA was hybridizedwith the entire REMI vector pUCH1. The hybridization pat-tern confirmed a single integration site of the vector in theZ43R9901 genome. Genomic DNA of Z43R9901 digested withBglII was self-ligated and transformed in E. coli for sequenc-

ing. The BLAST results from the sequence flanking the in-serted vector showed that the vector integrated at a site 407 bpupstream of an ORF annotated as FGSG_08667.3. We desig-nated this putative ORF round ascospore (ROA) after thephenotype of its REMI mutant. ROA encodes a 770-amino-acid protein and has three putative introns (see Fig. S1 in thesupplemental material). ROA is conserved in filamentous fungi(see Fig. S2), and the predicted protein sequence of ROA hashigh identity (60%) to the hypothetical proteins in Podosporaanserina, Magnaporthe grisea, N. crassa, Chaetomium globosum,and other Fusarium species. The protein sequence containedthe conserved domain ketopantoate reductase, which was lo-cated near the N terminus (1 to 315 amino acid residues) andis known to be involved in the coenzyme A biosynthesis path-way (8). Examination of expression of FGSG_08667 using Af-fymetrix GeneChip data in culture during sexual developmentand wheat colonization (9–11) indicated that it is consistentlyhighly expressed across a range of conditions.

Targeted deletion of the ROA gene and complementation.The ROA ORF from the wild-type genome was replaced withthe gen cassette to generate the ROA deletion mutant (�roa).When probed with the 5� flanking region, the genomic DNAdigested with EcoRV from four independent transformantshad a single 12-kb hybridized DNA fragment instead of the2.2-kb fragment found in the wild-type strain (Fig. 1A), whichconfirmed that the 2.5-kb ROA locus in G. zeae had beenreplaced with the gen cassette. The �roa mutant was comple-mented by introducing ROA into the �roa strain, and thepresence of the inserted ROA gene was confirmed by Southernblot analysis (Fig. 1B). The shapes of asci and ascospores in the�roa::ROA strain were restored to those of the wild-type strain.

Sexual development of the �roa mutant. Similarly to theREMI mutant, the �roa mutants showed no difference in my-celial growth, conidiation, and germination from the wild-typestrain. Both �roa and wild-type perithecia contained rosettesof asci of similar sizes, with each ascus containing eight asco-spores (Fig. 2). The average ascus length in the �roa mutantwas 61 �m, which was shorter than that of the wild-type asci(66 �m) (P 0.01). The shape of �roa ascospores was roundor oval, while the wild-type ascospores were slender and spin-dle shaped. The wild-type ascospores usually contained fourcells; however, the mutant ascospores had one or two cells(Fig. 2). The nuclei in the ascospores were counted by acrifla-vin staining, showing that approximately 40% of the �roa as-cospores had two cells, with each one containing one nucleus.In addition, 26% of the ascospores were composed of one cellwith two nuclei. Some of the �roa ascospores had three nucleiin one or two cells. In contrast, most of the wild-type asco-spores (�85%) had four cells, with one nucleus per cell (Fig.2). A few of the wild-type ascospores had only two cells, butascospores with one cell were not found in the wild-type strain.The average length and width of the �roa ascospores were 12�m and 7.0 �m, respectively, while those of the wild-typeascospores were 23 �m and 4.5 �m, respectively. Therefore,ascospores from the �roa strain were shorter and wider thanwild-type ascospores (P 0.01). The ascus shape is partiallydetermined by the ascospores, and the shorter ascus may bedue to the wider, shorter ascospores. We estimated a cylindri-cal shape for the volume calculation of the wild-type asco-spores and an ellipsoid shape for the �roa ascospores. A cy-

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lindrical shape overestimates volume, since the ascosporestaper at their end, and the ascospore volume was divided by 1.2to adjust the overestimation (41). Based on these measurementcriteria, the average volumes of the wild-type and �roa asco-spores were by chance the same and were estimated to be2.94 � 10�16 m3.

Approximately 300 perithecia from the wild-type and �roastrains were fixed and sectioned for microscopic observation.The number and shape of perithecia from �roa strain weresimilar to those of the wild-type strain (Fig. 3) despite havinga reduced number of discharged ascospores. In contrast to thetightly packed asci in wild-type perithecia, the arrangement of�roa asci was loose and sparse. Intact ascus rosettes wererarely observed in the old perithecia from �roa at the 9-DAItime point, while perithecia from the wild-type strain at thesame time point exhibited intact asci (Fig. 3). When matureperithecia from the �roa strain were dissected with needles

under a microscope, the ascospores were scattered from theperithecia without forming the typical rosettes of asci. How-ever, wild-type perithecia at the 14-DAI time point still con-tained intact asci that were ready for the release of ascospores.

Ascospore discharge. The wild-type strain discharged asco-spores for more than 9 days after 5 DAI (maturation point),while the �roa strain discharged ascospores for only 3 days(from 6 to 8 DAI). Approximately 67% of the ascospores fromthe �roa strain were discharged at 6 DAI. We collected asco-spores from the lid of the culture plate until 14 DAI andestimated the total number of discharged ascospores per peri-thecium. The wild-type strain discharged approximately 3,800ascospores per perithecium on average, while the �roa straindischarged 630 ascospores per perithecium (P 0.01).

The shooting distance of more than 1,000 ascospores fromeach strain was measured in still air. The mean distances ofdischarged ascospores were 3.6 mm, 5.0 mm, and 4.0 mm in the

FIG. 1. Targeted deletion and complementation of ROA. For each panel, the left and right sides show the strategy and Southern analysis,respectively. (A) Targeted deletion of ROA from the genome of the wild-type strain. E, EcoRV; gen, Geneticin resistance gene; lane 1, wild-typestrain; lane 2, �roa mutant. The 5� flanking region of ROA (black bar) was used as a probe. (B) Complementation of ROA in the �roa strain. H,HindIII; hygB, hygromycin resistance gene; lane 1, wild-type strain; lane 2, �roa; lanes 3, �roa::ROA. The partial ROA ORF (black bar) and hygBORF (gray bar) were used as probes for the left and right blots, respectively. The sizes of the standards (kb) are indicated on the left of each blot.

FIG. 2. Morphology of asci rosettes and ascospores. (A and D) Microscopic observation of 8-DAI asci rosettes of the wild-type (WT) (A) or�roa (D) strain. (B and E) Discharged ascospores of the wild-type (B) or �roa (E) strain. (C and F) Nuclei of the discharged ascospores werestained with acriflavin. Scale bar � 20 �m.

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wild type, �roa, and �roa::ROA strains, respectively (Fig. 4 and5). The �roa strain discharged ascospores farther than thewild-type strain and showed a broader range of shooting dis-tance. The discharge pattern of the ascospores from the�roa::ROA strain was similar to that of the wild type.

The launch speed of the ascospores was estimated from thedischarge distance. In addition, the turgor pressure requiredfor discharge was estimated from the launch speed (Table 1).The discharge distance was set to a range where 95% of theascospores dispersed from the mean distance. Ascosporesfrom the �roa strain were launched at a higher speed thanwild-type ascospores, suggesting that they possessed a higherturgor pressure in the asci. The estimated launch speed and

FIG. 3. Light microscopy of developing asci and ascospores. Perithecia of wild-type (A, B, and C) or �roa (D, E, and F) strains were stainedwith toluidine blue. Perithecia were collected from carrot agar 5, 7, and 9 days after sexual induction (DAI). Scale bar � 20 �m.

FIG. 4. Forcible ascospore discharge of the wild-type (WT), �roa,and �roa::ROA strains. Photographs were taken 48 h after the assaywas initiated. A semicircular agar block (arrowhead) covered withperithecia was placed on a coverslip in the chamber. This placementallowed for ascospores (arrow) to be discharged horizontally down thelength of the chamber onto the coverslip.

FIG. 5. Number of ascospores discharged at indicated distances instill air. Wild-type (black bar), �roa (dashed bar), and complement�roa::ROA strains (white bar) are shown. One thousand spores fromeach strain were assessed.

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turgor pressure of the �roa::ROA strain were similar to thoseof the wild type.

Epiplasmic fluid analysis. Based on estimates of epiplasmicfluid volume, the �roa strain contained a higher concentrationof ions (K�, Na�, and Cl�) and sugar alcohols in the fluid perascus than the wild-type strain (Fig. 6). The asci from the �roastrain contained approximately four times more ions than thewild-type asci (Fig. 6A). Glycerol, arabitol, mannitol, and glu-cose were found in the epiplasmic fluids from both the �roaand wild-type strains (see Fig. S3 in the supplemental mate-rial). Among the polyols, glycerol was the major component ofthe epiplasmic fluid of both strains (Fig. 6B), and both thewild-type and �roa strains had similar patterns of polyol pro-duction (Fig. 7). The concentration of polyols in the mutantasci was also markedly higher than that in the wild-type asci.The total concentration of the identified sugar alcohols perascus was approximately 20 times higher in the �roa strain thanin the wild-type strain.

The volume of epiplasmic fluid was calculated as the volumeof extended asci minus the measured volume of spores (41).Since the ascospore volumes were equal in the wild-type, �roa,and �roa::ROA strains, the volume of the epiplasmic fluid wasset as the same (5.77 � 10�15 m3). The volume of epiplasmic

fluid in the unextended mature ascus was calculated previously(2.1 � 10�15 m3) (41). The concentration of osmolytes in theextended asci was calculated from the mass of the osmolytesand the volume of epiplasmic fluid. The turgor pressure in theextended asci was estimated from the concentration ofosmolytes (Table 1). The �roa strain showed a significantlyhigher predicted turgor pressure than the wild-type strain (P 0.01). In contrast, the turgor pressures of the �roa::ROA andwild-type strains were similar.

Effects of ROA in outcrossing. The outcrossing between thefemale �mat1 strain tagged with hH1-GFP and the male �roastrain showed that the GFP and non-GFP ascospores within anindividual ascus had a 1:1 segregation ratio (Fig. 8). However,the shapes of eight ascospores in each individual ascus wereidentical. Approximately 10% of asci contained eight roundascospores (5 to 14 �m in length), similar to case with the �roastrain, while �60% had eight spores with wild-type shapes (20to 30 �m in length). In addition, �30% of ascospores had ashape that resembled an intermediate form (15 to 19 �m inlength). The progeny of the cross between the female �mat2

TABLE 1. Estimation of launch speed and turgor pressure in G. zeae

Parameter

Value in model for genotyped

Full Stokes’ law

WT �roa �roa::ROA WT �roa �roa::ROA

Measured distance (mean, range) (mm) 3.6 (1.4–5.5) 5 (1.1–8.7) 4 (1.7–6.2)Estimated mean launch speed (m s�1)a 12.0 (3.9–20.6) 16.9 (2.7–36.0) 12.5 (4.9–24.2) 8.4 (3.2–12.9) 11.0 (2.4–19.0) 8.8 (3.9–14.5)Estimated pressure (MPa)b 0.57 1.13 0.62 0.27 0.69 0.31Measured turgor pressure (MPa)c 0.41 1.6 0.40

a Launch speed was estimated from measured distance.b Estimated pressure was the pressure that is required for discharging ascospores calculated from the launch speed.c Measured pressure was the osmotic pressure calculated using the concentration of osmolytes in the epiplasmic fluid.d WT, wild type.

FIG. 6. Mass of epiplasmic fluid components per ascus. Wild-type(black bar), �roa (dashed bar), and complement �roa::ROA (whitebar) strains are shown.

FIG. 7. High-performance liquid chromatography (HPLC) chro-matogram of polyol standards (A), polyols in the epiplasmic fluid ofthe wild-type strain (B), or polyols in the epiplasmic fluid of the �roastrain (C). Peaks 1 to 5 are glycerol, erythritol, arabitol, mannitol, andglucose, respectively.

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and male �roa strains and the female �roa �mat2 and malewild-type strains showed the same phenotype (data notshown). When we randomly isolated ascospores from the threeoutcrossing sets, cultured them, and induced sexual develop-ment, they produced their respective wild-type or round asco-spores regardless of the initial shape. When ascospores thatdischarged from the outcrossing between the female �mat2(genotype mat1-2 ROA) strain and the male �roa (genotypeMAT1-2 roa) strain were randomly isolated, the segregationratio of the genotypes was 25:7:10:24 (mat1-2 ROA/mat1-2roa/MAT1-2 ROA/MAT1-2 roa, respectively). The genetic dis-tance of the two linked genes (MAT1-2 and ROA) was esti-mated to be 19 cM based on chromosome maps of G. zeae (21),and therefore, the segregation ratio was consistent with theexpected number (�2 � 2.8).

GFP localization. We tried complementing the �roa strainby introducing a plasmid that contained the ROA gene fused toGFP. We obtained 60 transformants from three independenttransformation trials, and GFP expression was detected in 24transformants, of which only one strain fully restored the wild-type phenotype. However, this complemented strain carriedmultiple copies of the construct, and other strains that carrieda single copy of the construct did not restore the wild-type

phenotype. We amplified the construct from each transfor-mant for sequencing and found each construct carried severalpoint mutations resulting in nonsynonymous substitution. Re-gardless of the patterns of point mutation and integrated copynumber, GFP in the 24 transformants was localized in thecytoplasm in all stages of the life cycle, including ascospores,conidia, and mycelia, but was not detected in young asci beforespore delimitation. We could not determine GFP expression inperithecia of the transformants, because perithecia producedby the wild-type strain had strong autofluorescence. We alsointroduced the construct into the wild-type strains and selected30 transformants. Southern blot analyses showed that five in-dependent mutants carried a single integration of the constructin their genome and that two of them carried an integration atthe 5� flanking region of ROA (see Fig. S4 in the supplementalmaterial). In both strains, GFP was also localized in the cyto-plasm in all fungal stages except for young asci (Fig. 9).

DISCUSSION

In this study, we identified a novel gene, ROA, from thescreening of a REMI mutant collection. The �roa mutantshowed unique characteristics of ascospore morphology and

FIG. 8. Outcrosses between �mat1 female and �roa male. In the heterothallic �mat1 strain, the histone H1 gene was fused with GFP. DIC,differential interference contrast image; GFP, GFP fluorescence image; DIC � GFP, DIC images merged with GFP fluorescence image. Scalebar � 20 �m.

FIG. 9. Localization of ROA in mycelia (A and B) or ascospores (C and D). Young asci before spore delimitation and ascospores within asciare indicated by arrowheads and arrows, respectively. DIC, DIC image; GFP, GFP fluorescence image. Scale bar � 20 �m.

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discharge without any defects in vegetative growth, conidia-tion, and conidia germination. Despite their abnormal shape,germination of �roa ascospores occurred normally. These re-sults suggest that ROA has a specific role in ascospore mor-phology and discharge in G. zeae. The most striking pheno-types of the �roa mutant are the reduction in the number ofascospores discharged from the mature perithecia and the in-creased distance that these spores are fired.

We hypothesized that the mutant had a defect in the shoot-ing of ascospores, since the total number of ascospores perperithecium was the same in the �roa and wild-type strains.The turgor pressure in asci is known to be the predominantdriving force behind the discharge of ascospores from asci (32,38, 42). In addition, components of the ascus epiplasmic fluidhave been identified and quantified in G. zeae (39). Therefore,we analyzed the osmolyte components of the ascus fluid. As-suming the volumes of the epiplasmic fluid are equal in asci ofthe mutant and the wild type, the measured turgor pressure ofthe �roa asci is four times higher than that of the wild-typestrain.

The difference between the estimated and measured pressures(Table 1) for both the mutant and the wild type is relatively small,and the full model did a better job of predicting the measuredpressure for the mutant. If our assumption that the epiplasmicfluid volume is equal in the mutant and wild-type is incorrect,and there is greater fluid in the mutant, then the measuredturgor pressure would be lower and may align more closelywith the estimated pressure (Table 1). Wild-type spores appearto nest against each other in the ascus, whereas the shape ofthe mutants prevents this (Fig. 2A and D), which may increasethe fluid in the discharging mutant ascus. For further discus-sion of the model, see the supplemental material.

The change in spore shape may also contribute to the dis-tance fired. Recent work (32) weighed the evolutionary pres-sures on spore shape from friction at the ascus pore to reduc-tion of drag and suggests the latter as the driving force forspores without appendages or multiple cells. Our data from aprevious publication (41) were used to develop this model,although G. zeae spores have multiple cells. Additionally, oneof the assumptions of the model is that spores exit the ascussingly and without accompanying ascus fluid, an assumption,which was based on our previous results (42). The figure inquestion, however, shows accompanying fluid clinging to 7 of 8discharged spores. However, for the �roa mutants, the aspectratio (length divided by width) plotted against Reynold’s num-ber (Re) fits within 1% of that predicted by the model forminimum drag, whereas the wild-type spores fall outside thisregion. The study also predicts that the rounder shape of themutant would minimize friction during exit of the ascus pore,resulting in a longer shooting distance.

The asci of G. zeae were shown previously to accumulatepredominantly mannitol and glucose (42). In this study, weidentified several other sugar alcohols in the epiplasmic fluid ofG. zeae asci, including glycerol and arabitol (Fig. 6). The polyolprofile of epiplasmic fluid is consistent with several reports ofother fungi (16). Polyol profiles may vary according to condi-tions under which the fungi are grown. Magnaporthe oryzae wasshown to use glycerol to drive turgor pressure in the appres-soria (5). However, the concentration of the glycerol (4.6 mM)in G. zeae asci is too low to reach the required turgor pressure

(0.57 MPa) for the release of ascospores. In contrast, M. griseaaccumulates a large amount of glycerol (3.3 M) in the appres-sorium that creates a sufficient force (5.8 MPa) for penetration(5). Therefore, G. zeae uses ions (predominantly K� and Cl�)instead of polyols to increase the turgor pressure in asci, aspreviously reported (41). An analysis of ion components hasnot been published for the M. oryzae system.

Although the morphologies of the ascopores from the �roaand wild-type strains were quite different, acriflavin stainingshowed that the �roa strain maintained normal meiosis and athird division of the nuclei. Three nuclear divisions (2 meioticand 1 mitotic) that generated eight nuclei occurred normally inthe young �roa asci before spore delimitation; however, fur-ther cell division in these ascospores did not accompany thesedivisions as expected. Ascospores are formed from the com-partmentalization of the ascus cytoplasm during ascospore de-limitation (2), and the shape of the young ascus may govern theshape of ascospores produced within it (30). Therefore, insuf-ficient cell division after spore delimitation in the �roa strainmay be a consequence of abnormal ascus physiology.

Unusual phenotype segregation occurred in the outcrossbetween the �mat1 mutant tagged with hH1-GFP and the �roamutant (Fig. 8). All eight spores from one ascus had the sameshape; however, the GFP/non-GFP segregation ratio was 1:1.Genotypes of F1 progeny ascospores had an expected segre-gation ratio and confirmed that the outcross underwent normalsexual recombination. This result showed that the initial shapeof F1 ascospores from the outcross was not determined by thespore genotype. This unusual phenomenon may be caused by adiffusible factor, such as a protein or RNA that gets capturedby asci at various concentrations, since the asci produced bythe outcross have haploinsufficiency in ROA. This phenome-non also can result independently of haploinsufficiency. Ge-netic studies of N. crassa showed a phenomenon similar to theunusual phenotypic segregation observed in this study, but thisphenomenon in N. crassa is not related to haploinsufficiency.Crosses of the N. crassa wild-type strain with the Round sporemutant (R) strain produced 100% round-spore asci, suggestingthat R is ascus dominant (24). Shiu et al. (35) hypothesized thatthis phenomenon was a result of meiotic silencing by unpairedDNA (MSUD) and involved a molecular mechanism that wassimilar to RNA silencing: when DNA from the parental cells isunpaired in early stages of meiosis, both the unpaired DNAand its homologue were silenced. When SAD-1, an importantgene involved in MSUD, is self-silenced, the ascus dominanceof R becomes suppressed (semidominance). A cross of sad-1UV

with R produced 35% round-spore asci in Neurospora (35). Inthis regard, MSUD in G. zeae may be partially functioning,since this semidominance is similar to the outcross results of�mat1 and �roa in G. zeae in our study and major genes ofMSUD are conserved in G. zeae (25).

In conclusion, the novel gene ROA is required for propersexual development in G. zeae. Interestingly, the round-sporemutant has a possible selective advantage in that it shootsfarther, which would help in distribution, but it does not shootas many spores. The round-spore mutant has not been previ-ously identified in nature, which may reflect the balance ofpropagule number versus firing distance. Deletion of the ROAgene suggests an increase in turgor pressure in the ascusthrough the accumulation of ions. In addition, this mutation

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triggered the formation of round ascospores and caused insuf-ficient cell divisions after spore delimitation. In this study, wehave shown a close relationship between ascospore morphol-ogy, turgor pressure, and ascospore discharge and have iden-tified additional polyol components in asci. Future work willexplore the mechanisms by which ROA affects turgor pressure.These studies will also help explain how the physiology andstructure of asci affects turgor pressure and the morphogenesisof ascospores.

ACKNOWLEDGMENTS

We thank Namboori B. Raju for valuable advice on cytology, HansolChoi for technical assistance, and Wonsang Woo for estimating thelaunch speed of the ascospores.

This work was supported by a grant (CG 1140) from the CropFunctional Genomics Center of the 21st Century Frontier ResearchProgram funded by the Korean Ministry of Education, Science, andTechnology and by the National Research Foundation of Korea(NRF) grant funded by the Korean government (MEST) (2010-0001826). K. Min was supported by graduate fellowships from theKorean Ministry of Education, Science, and Technology through theBrain Korea 21 project. The contributions of F. Trail were supportedin part by the Michigan Agricultural Experiment Station.

REFERENCES

1. Aylor, D. E., and S. L. Anagnostakis. 1991. Active discharge distance ofascospores of Venturia inaequalis. Phytopathology 81:548–551.

2. Beckett, A. 1981. Ascospore formation, p. 107–129. In G. Turian, and H.Hohl (ed.), The fungal spore: morphogenetic controls. Academic Press,London, United Kingdom.

3. Bowden, R. L., I. Fuentes-Bueno, J. F. Leslie, J. Lee, and Y.-W. Lee. 2008.Methods for detecting chromosome rearrangements in Gibberella zeae. Ce-real Res. Commun. 36(Suppl. 6):603–608.

4. Cappellini, R. A., and J. L. Peterson. 1965. Macroconidium formation insubmerged cultures by a non-sporulating strain of Gibberella zeae. Mycologia57:962–966.

5. De Jong, J. C., B. J. McCormack, N. Smirnoff, and N. J. Talbot. 1997.Glycerol generates turgor in rice blast. Nature 389:244–245.

6. Desjardins, A. E. 2006. Fusarium mycotoxins: chemistry, genetics, and biol-ogy. APS Press, St. Paul, MN.

7. Fernando, W. G. D., T. C. Paulitz, W. L. Seaman, P. Dutilleul, and J. D.Miller. 1997. Head blight gradients caused by Gibberella zeae from areasources of inoculum in wheat field plots. Phytopathology 87:414–421.

8. Genschel, U. 2004. Coenzyme A biosynthesis: reconstruction of the pathwayin archaea and an evolutionary scenario based on comparative genomics.Mol. Biol. Evol. 21:1242–1251.

9. Guenther, J. C., H. E. Hallen-Adams, H. Bucking, Y. Shachar-Hill, and F.Trail. 2009. Triacylglyceride metabolism by Fusarium graminearum duringcolonization and sexual development on wheat. Mol. Plant Microbe Interact.22:1492–1503.

10. Guldener, U., K.-Y. Seong, J. Boddu, S. Cho, F. Trail, J.-R. Xu, G. Adam,H.-W. Mewes, G. J. Muehlbauer, and H. C. Kistler. 2006. Development of aFusarium graminearum Affymetrix GeneChip for profiling fungal gene ex-pression in vitro and in planta. Fungal Genet. Biol. 43:316–325.

11. Hallen, H. E., M. Huebner, S.-H. Shiu, U. Guldener, and F. Trail. 2007.Gene expression shifts during perithecium development in Gibberella zeae(anamorph Fusarium graminearum), with particular emphasis on ion trans-port proteins. Fungal Genet. Biol. 44:1146–1156.

12. Hallen, H. E., and F. Trail. 2008. The L-type calcium ion channel cch1 affectsascospore discharge and mycelial growth in the filamentous fungus Gib-berella zeae (anamorph Fusarium graminearum). Eukaryot. Cell 7:415–424.

13. Han, Y.-K., T. Lee, K.-H. Han, S.-H. Yun, and Y.-W. Lee. 2004. Functionalanalysis of the homoserine O-acetyltransferase gene and its identification asa selectable marker in Gibberella zeae. Curr. Genet. 46:205–212.

14. Hong, S.-Y., J. So, J. Lee, K. Min, H. Son, C. Park, S.-H. Yun, and Y.-W. Lee.2010. Functional analyses of two syntaxin-like SNARE genes, GzSYN1 andGzSYN2, in the ascomycete Gibberella zeae. Fungal Genet. Biol. 47:364–372.

15. Horwitz, B. A., A. Sharon, S.-W. Lu, V. Ritter, T. M. Sandrock, O. C. Yoder,and B. G. Turgeon. 1999. A G protein alpha subunit from Cochliobolusheterostrophus involved in mating and appressorium formation. FungalGenet. Biol. 26:19–32.

16. Jennings, D. H., and R. M. Burke. 1990. Compatible solutes—the mycolog-ical dimension and their role as physiological buffering agents. New Phytol.116:277–283.

17. Jerkovic, I., and J. Mastelic. 2004. GC-MS characterization of acetylated -D-glucopyranosides: transglucosylation of volatile alcohols using almond -glucosidase. Croat. Chem. Acta 77:529–535.

18. Kim, J.-E., J. Jin, H. Kim, J.-C. Kim, S.-H. Yun, and Y.-W. Lee. 2006. GIP2,a putative transcription factor that regulates the aurofusarin biosyntheticgene cluster in Gibberella zeae. Appl. Environ. Microbiol. 72:1645–1652.

19. Kim, K. W., and J.-W. Hyun. 2007. Nonhost-associated proliferation ofintrahyphal hyphae of citrus scab fungus Elsinoe fawcettii: refining the per-ception of cell-within-a-cell organization. Micron 38:565–571.

20. Lee, J., I.-Y. Chang, H. Kim, S.-H. Yun, J. F. Leslie, and Y.-W. Lee. 2009.Genetic diversity and fitness of Fusarium graminearum populations from ricein Korea. Appl. Environ. Microbiol. 75:3289–3295.

21. Lee, J., J. E. Jurgenson, J. F. Leslie, and R. L. Bowden. 2008. Alignment ofgenetic and physical maps of Gibberella zeae. Appl. Environ. Microbiol.74:2349–2359.

22. Lee, J., T. Lee, Y.-W. Lee, S.-H. Yun, and B. G. Turgeon. 2003. Shifting fungalreproductive mode by manipulation of mating type genes: obligatory het-erothallism of Gibberella zeae. Mol. Microbiol. 50:145–152.

23. Leslie, J. F., and B. A. Summerell. 2006. The Fusarium laboratory manual.Blackwell Publishing, Ames, IA.

24. Mitchell, M. B. 1966. A round-spore character in Neurospora crassa. Neu-rospora Newslett. 10:6.

25. Nakayashiki, H. 2005. RNA silencing in fungi: mechanisms and applications.FEBS Lett. 579:5950–5957.

26. Namiki, F., M. Matsunaga, M. Okuda, I. Inoue, K. Nishi, Y. Fujita, and T.Tsuge. 2001. Mutation of an arginine biosynthesis gene causes reducedpathogenicity in Fusarium oxysporum f. sp. melonis. Mol. Plant MicrobeInteract. 14:580–584.

27. Parry, D. W., P. Jenkinson, and L. McLeod. 1995. Fusarium ear blight (scab)in small grain cereals—a review. Plant Pathol. 44:207–238.

28. Pratt, R. J., D. W. Lee, and R. Aramayo. 2004. DNA methylation affectsmeiotic trans-sensing, not meiotic silencing, in Neurospora. Genetics 168:1925–1935.

29. Raju, N. B. 1986. A simple fluorescent staining method for meiotic chromo-somes of Neurospora. Mycologia 78:901–906.

30. Raju, N. B. 1992. Genetic control of the sexual cycle in Neurospora. Mycol.Res. 96:241–262.

31. Read, N. D., and A. Beckett. 1996. Ascus and ascospore morphogenesis.Mycol. Res. 100:1281–1314.

32. Roper, M., R. E. Pepper, M. P. Brenner, and A. Pringle. 2008. Explosivelylaunched spores of ascomycete fungi have drag-minimizing shapes. Proc.Natl. Acad. Sci. U. S. A. 105:20583–20588.

33. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratorymanual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

34. Seong, K., Z. Hou, M. Tracy, H. C. Kistler, and J.-R. Xu. 2005. Randominsertional mutagenesis identifies genes associated with virulence in thewheat scab fungus Fusarium graminearum. Phytopathology 95:744–750.

35. Shiu, P. K. T., N. B. Raju, D. Zickler, and R. L. Metzenberg. 2001. Meioticsilencing by unpaired DNA. Cell 107:905–916.

36. Spurr, A. R. 1969. A low-viscosity epoxy resin embedding medium for elec-tron microscopy. J. Ultrastruct. Res. 26:31–43.

37. Sutton, J. C. 1982. Epidemiology of wheat head blight and maize ear rotcaused by Fusarium graminearum. Can. J. Plant Pathol. 4:195–209.

38. Trail, F. 2007. Fungal cannons: explosive spore discharge in the Ascomycota.FEMS Microbiol. Lett. 276:12–18.

39. Trail, F. 2009. For blighted waves of grain: Fusarium graminearum in thepostgenomics era. Plant Physiol. 149:103–110.

40. Trail, F., and R. Common. 2000. Perithecial development by Gibberella zeae:a light microscopy study. Mycologia 92:130–138.

41. Trail, F., I. Gaffoor, and S. Vogel. 2005. Ejection mechanics and trajectory ofthe ascospores of Gibberella zeae (anamorph Fuarium graminearum). FungalGenet. Biol. 42:528–533.

42. Trail, F., H. Xu, R. Loranger, and D. Gadoury. 2002. Physiological andenvironmental aspects of ascospore discharge in Gibberella zeae (anamorphFusarium graminearum). Mycologia 94:181–189.

43. Turgeon, B. G., R. C. Garber, and O. C. Yoder. 1987. Development of afungal transformation system based on selection of sequences with promoteractivity. Mol. Cell. Biol. 7:3297–3305.

44. Yafetto, L., L. Carroll, Y. Cui, D. J. Davis, M. W. F. Fischer, A. C. Henterly,J. D. Kessler, H. A. Kilroy, J. B. Shidler, J. L. Stolze-Rybczynski, Z. Sug-awara, and N. P. Money. 2008. The fastest flights in nature: high-speed sporedischarge mechanisms among fungi. PLoS One 3:e3237.

45. Yu, J.-H., Z. Hamari, K.-H. Han, J.-A. Seo, Y. Reyes-Dominguez, and C.Scazzocchio. 2004. Double-joint PCR: a PCR-based molecular tool for genemanipulations in filamentous fungi. Fungal Genet. Biol. 41:973–981.

46. Yun, S.-H., T. Arie, I. Kaneko, O. C. Yoder, and B. G. Turgeon. 2000.Molecular organization of mating type loci in heterothallic, homothallic, andasexual Gibberella/Fusarium species. Fungal Genet. Biol. 31:7–20.

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