Microtubule Dynamics during Infection-Related Morphogenesis of Colletotrichum lagenarium

Fungal Genetics and Biology 34, 107–121 (2001) doi:10.1006/fgbi.2001.1293, available online at http://www.idealibrary.com on

Microtubule Dynamics during Infection-Related Morphogenesis of Colletotrichum lagenarium

Yoshitaka Takano, 1 Eriko Oshiro, and Tetsuro Okuno Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Accepted for publication July 3, 2001; published online August 22, 2001

Takano, Y., Oshiro, E., and Okuno, T. 2001. Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium. Fungal Genetics and Biology 34, 107–121. Using a green fluorescent protein (GFP)–tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two ␣-tubulin genes of C. lagenarium were isolated, and GFP–␣-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation

which is distinct from their random orientation inside appressoria. © 2001 Academic Press

Index Descriptors: microtubule; ␣-tubulin genes; appressorium formation; green fluorescent protein; plant pathogenic fungus; Colletotrichum lagenarium. Many plant pathogenic fungi, including Colletotrichum lagenarium, the causal agent of anthracnose disease of cucumber, produce highly specialized infection structures called appressoria (Emmett and Parbery, 1975). In C. lagenarium, conidia first attach tightly on the host plant surface. Following germination, germ tubes differentiate into swollen appressoria accompanied by nuclear duplication. Subsequently, appressoria form a dense layer of pentaketide-derived melanin essential for appressorial penetration (Kubo and Furusawa, 1991). Melanized appressoria form a penetration peg that perforates the host surface directly. Melanized appressoria of Colletotrichum and Magnaporthe species have been shown to generate enormous turgor pressure which enables mechanical penetration into plants (Bechinger et al., 1999; Howard et al., 1991; Howard and Valent, 1996). In both Magnaporthe and Colletotrichum species, genes involved in differentiation of this specialized infection structure have been identified and characterized. Several of the genes encode components of conserved signal transduction pathways (Dean, 1997; Hamer et al., 1997; Fang and Dean, 2000; Kim et al., 2000; Thines et al., 2000). In C. lagenarium, a mitogenactivated protein kinase has been demonstrated to play a crucial role in appressorium formation, conidial germination, and invasive growth inside plants (Takano et al., 2000).

1 To whom correspondence should be addressed. Fax: 81-75-7536131. E-mail: [email protected].

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Cytological analyses of appressoria have been performed in several Colletotrichum and Magnaporthe species by use of electron microscopy (EM) and immunofluorescence methods, but there are few reports about the organization of microtubule cytoskeletons during appressorium formation (Bourett and Howard, 1990; Kubo and Furusawa, 1991; O’Connell et al., 1985). Organization of the microtubule cytoskeleton has been studied in the rust fungus Uromyces appendiculatus (Hoch and Staples, 1987; Kwon et al., 1991). However, the nonmelanized appressorium formed by the uredospore of U. appendiculatus produces an infection peg through the plant stomatal aperture without breaking of the host surface, which suggests that the appressorium structure and function are different from those of appressoria formed by Colletotrichum and Magnaporthe species. Microtubules are found in all eukaryotic cells and are composed of two types of tubulin proteins, ␣-tubulin and ␤-tubulin. The microtubule cytoskeleton is responsible for the coordinated movement of many structures in eukaryotic cells, including chromosomes, organelles, and nuclei (Avila, 1992; Desai and Mitchison, 1997). It has also been reported for several organisms that microtubules contribute to the establishment of cell polarity (Goode et al., 2000; Sawin and Nurse, 1998). In the filamentous fungi Aspergillus nidulans and Neurospora crassa, experiments with antimicrotubule agents have demonstrated that nuclear migration is mediated primarily by the microtubule system (Oakley and Morris, 1980; That et al., 1988). Molecular genetic analysis revealed that cytoplasmic dynein is involved in nuclear migration mediated by microtubules in A. nidulans, N. crassa, and Nectria haematococca (Inoue et al., 1998; Plamann et al., 1994; Xiang et al., 1994). Immnofluorescence experiments have been performed to study microtubule organization in filamentous fungi such as A. nidulans, N. crassa, and U. appendiculatus (Kwon et al., 1991; Minke et al., 1999; Osmani et al., 1988), although the application of this method is restricted to fixed cells. A green fluorescent protein (GFP)–tubulin fusion protein allowed for visualization of the microtubule cytoskeleton in living cells of yeast (Carminati and Stearns, 1997; Maddox et al., 1999; Straight et al., 1997). This GFP–tubulin fusion system facilitated detection of more cytoplasmic microtubules in living cells than those detected in fixed cells by immunofluorescence methods. Time-lapse views of the microtubule organization in yeast expressing the GFP fusions gave new insights for both organization and function of microtubules. Here we demonstrate dynamic rearrangements of microtubules during infection structure formation of the

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Takano, Oshiro, and Okuno

fungal pathogen C. lagenarium. Two ␣-tubulin genes were isolated from C. lagenarium, and one of them was fused to the GFP gene. Expression of the GFP–tubulin fusion gene successfully enabled visualization of microtubules in living cells of C. lagenarium. We found dynamic rearrangements during appressorium formation. The effects of microtubule depolymerization on appressorium formation were also investigated. These studies support the concept of multiple roles of microtubules during infection structure formation.

MATERIALS AND METHODS Fungal Strains and Culture Conditions The C. lagenarium (Pass.) Ellis et Halsted (syn. C. orbiculare (Berk. et Mont.) von Arx) strain 104-T (stock culture of the laboratory of Plant Pathology, Kyoto University) was used as the wild-type strain. All C. lagenarium cultures were maintained on potato dextrose agar medium (3.9% (w/v) PDA; Difco) at 24°C in the dark. Conidia were obtained by gentle scraping of cultures incubated for 5 to 7 days.

Isolation and Nucleotide Sequence Analysis of ␣-Tubulin Genes Nested PCR was performed to isolate the ␣-tubulin genes by use of cDNA synthesized from polyadenylated RNA that was prepared from conidia 2 h after the start of incubation for appressorium formation (Inagaki et al., 2000). Three degenerate primers, 5⬘-CCCAGAATTC(C/ G)NTG(C/T)TGGGA(A/G)(C/T)TNTA-3⬘ (TUBS1), 5⬘ACTTGCGGCCGCC(C/T)TC(C/T)TCCATNCC(C/ T)TCNCC-3⬘ (TUBAS1), and 5⬘-CCCAGAATTCTGGTG(T/C)CCNACNGGNTT-3⬘ (TUBS2), were designed based on the conserved amino acids in Saccharomyces cerevisiae, A. nidulans, and human ␣-tubulin genes. TUBAS1 contains a terminal NotI site, whereas TUBS1 and TUBS2 contain a terminal EcoRI site. The first PCR was performed with TUBS2 and TUBAS1 with Ex Taq polymerase (Takara, Ohtsu, Japan). Nested PCR was performed with primers TUBS1 and TUBAS1. PCR conditions were as follows: 30 cycles (94°C, 1 min; 52°C, 2 min; 72°C, 2 min) with a final extension at 72°C for 5 min. Nested PCR products were digested with both NotI and EcoRI, cloned into pBluescript II KS- (Stratagene, La Jolla, CA), and sequenced. The PCR clones pTUB1 and

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pTUB2, having homology with ␣-tubulin genes of other organisms, were used as a probe to screen a C. lagenarium genomic library by colony hybridization. Bacterial colonies of the library were grown on nylon membranes (Biodyne A transfer membrane; PALL). The membranes were treated with 10% SDS for 5 min, denaturing solution (0.5 M NaOH, 1.5 M NaCl) for 5 min, neutralizing solution (0.5 M Tris–HCl, pH 7.5, 1.5 M NaCl) for 5 min, and wash solution [2⫻ standard saline citrate (SSC); 1⫻ SSC is 0.15 M NaCl, 0.015 M sodium citrate] for 5 min. For hybridization, AlkPhos Direct labeling kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) was used. Labeling of probes, hybridization, washing of membranes, and signal detection were performed according to manufacturer’s instructions. Colony hybridization analysis identified cosmid clones containing ␣TUB1 or ␣TUB2. Two fragments (HindIII 3.5-kb and PstI 9.5-kb fragments) containing ␣TUB1 and three fragments (EcoRI 2.5-kb, HindIII 6.5kb, and XhoI 0.6-kb fragments) containing ␣TUB2 were subcloned into pBluescript II KS- (Stratagene) from each cosmid clone. Nucleotide sequences of ␣TUB1 and ␣TUB2 in subcloned fragments were determined with Big Dye Termination Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Warrington, UK) and an automated DNA sequencer (Model 310; Applied Biosystems).

Plasmid Constructs All recombinant DNA manipulations followed standard protocols (Sambrook et al., 1989). SmaI–ClaI fragment containing the SGFP (S65T)–TYG gene in pOTEFSG (Spellig et al., 1996) was introduced into SmaI–ClaI sites in pBluescript (KS-) and the resultant plasmid was designated pBSGFP. The eGFP gene (P64L and S65T) was generated by a site-directed mutagenesis of SGFP-TYG in pBSGFP with primer P64LEGFP (5⬘-CACCCTCGTGACCACCCTCACCTACGGCGTGCAG-3⬘). For the eGFP–␣TUB1 fusion gene, the eGFP gene and the ␣TUB1 gene were linked with a short spacer coding for five glycine residues. The full region of the eGFP open reading frame was amplified by PCR with primers EGFPX (5⬘-GCCCTCTAGACAGACACAATGGTGAGCAAGGGCGAG-3⬘) and EGFPG5B (5⬘-CGGGATCCAGCCACCACCACCACCCTTGTACAGCTCGTCCAT-3⬘). The primer EGFPX contains a terminal XbaI site, whereas EGFPG5B contains a terminal BamHI site. The amplified product was digested with XbaI and BamHI, introduced into pCB1636 carrying hygromycin resistant gene hph (Sweigard et al., 1997), and designated pCB16EGFP. The 221-bp 5⬘ upstream region of the SCD1 gene was ampli-

fied by PCR with SCD1PNS (5⬘-CAGGTTGCGGCCGCGTGTTTTGCGGCAGTCC-3⬘) and SCD1PNA (5⬘AACCTGGCGGCCGCCTGATAGGTGGGATATT-3⬘) from the plasmid pCBSD (Kubo et al., 1996). Amplified product was digested with NotI and introduced into the NotI site of pCB16GFP. The resultant clone was named pCB16EGFPSP. The entire ␣TUB1 gene was amplified by PCR with primers TUB1P (5⬘-GCCGCCTGCAGCGATGAAGGGAGAGGTAAGT-3⬘) and TUB1E (5⬘-CGGCGGAATTCAAGCCAATCCAGTACAAT-3⬘). The amplified product was digested with PstI and EcoRI and introduced into PstI and EcoRI sites of pCB16EGFPSP. The resultant clone was named pHGFPTUB1.

Fungal Transformation To obtain protoplasts, hyphae of C. lagenarium wildtype strain 104-T were incubated for 3 days in 20 ml of potato sucrose broth supplemented with yeast extract (liquid extract from 200 g potato, 20 g sucrose, 2 g yeast extract per liter; PSY). Mycelia were harvested by filtration and treated with enzyme solution containing 5 mg/ml of lysing enzyme from Trichoderma harzianum (Sigma Chemical Co., St. Louis, MO) in 1.2 M MgSO 4 and 10 mM Na 2HPO 4 for 3 to 4 h to release the protoplasts (Rodriguez and Yoder, 1987). Transformations were performed according to the method described previously (Voller and Yanofsky, 1986). Twenty microliters of plasmid DNA solution (0.5 ␮g/␮l) was added to 1 ml of protoplast solution [5 ⫻ 10 7 protoplasts in 1 M sorbitol/50 mM Tris–HCl, pH 8.0/50 mM CaCl 2 (STC solution)]. The protoplast solution mixed with plasmid DNA was incubated for 30 min on ice. Subsequently, 10 ml of 60% polyethylene glycol/50 mM Tris–HCl, pH 8.0/50 mM CaCl 2 (PEG solution) was added gradually to the protoplast solution and then incubated for 15 min at room temperature. After removal of PEG solution by centrifugation, protoplasts were suspended in 1 ml of STC solution and mixed with 9 ml of regeneration agar medium (3.12% PDA/0.6 M glucose). The mixture was poured onto selection plates for hygromycin-resistant transformants [3.9% PDA/0.6 M glucose/100 ␮g/ml hygromycin B (Wako Pure Chemicals, Osaka, Japan)]. Colonies growing on selection plates were picked to PDA medium and screened.

Western Blot Analysis Western blot analysis was performed with an enhanced chemiluminescence (ECL) detection system. Proteins

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were extracted from mycelia of the wild-type stain and the GFP–␣TUB1transformant incubated in PSY media for 3 days. Mycelia were ground in protein extraction buffer (5 mM EDTA, 50 mM Tris, 1 mM phenylmethylsulfonyl fluoride, pH 7.5) under liquid nitrogen. Homogenates were collected into polypropylene centrifuge tubes and 500 ␮l extraction buffer was added, the homogenates were kept on ice for 5 min and subjected to centrifugation at 4°C. Supernatant was collected, and cracking buffer (62.5 mM Tris–HCl (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.01% bromphenol blue) was added before being boiled at 100°C for 2 min. Ten micrograms of proteins were separated on a 12.5% polyacrylamide gel containing 0.1% SDS. Western blot analysis was carried out according to the method described previously (Towbin et al., 1979). Electrophoresed proteins were transferred onto an Immobilon-P membrane (Millipore Corp., Bedford, MA) and probed with a polyclonal anti-GFP antibody (diluted 1:3000) (Molecular Probes, Inc., Eugene, OR) as primary antibody. The protein concentration of the fungal extracts was determined by the dye-binding method of Bradford (1976).

Microscopy For appressorium formation assays, conidia were harvested from 5-day-old cultures on PDA plates with the tips of toothpicks. Twenty microliters of conidial suspension (1 ⫻ 10 5 conidia/ml in 0.1% yeast extract solution) was placed on a glass slide (8-well multitest slide; ICN Biomedicals, Santa Clara, CA) and incubated in a humid environment at 24°C. After 1 h of incubation, 0.1% yeast extract solution was changed to distilled water. Cells were viewed on a fluorescent microscope (Model Axioskop; Carl Zeiss, Feldbach, Switzerland) equipped with epifluorescent and differential-interference contrast (DIC) optics. For staining of nuclei, samples on the glass slide were fixed with 3.75% formaldehyde, 50 mM phosphate buffer (pH 7.0), and 0.2% Triton X-100 and left at room temperature for 30 min. Fixed samples were rinsed with distilled water prior to the staining and were stained with 1 ␮g/ml DAPI (Sigma) for 5 min before another rinsing with distilled water. DAPI-stained samples were observed with the microscope with a Zeiss filter set (excitation 365 nm, dichroic 395 nm, emission 420 nm). To observe microtubules labeled by GFP, cells in water were directly observed without fixation. For observation of GFP fluorescence, a Zeiss filter set (excitation 450 – 490 nm, dichroic 510 nm, emission 515–565 nm) was used, and GFP-fluorescence images

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Takano, Oshiro, and Okuno

were acquired with 400-ms exposures. Both DIC and fluorescent images were captured with a chilled chargecoupled device camera (Model Argus 50; Hamamatsu Photonics, Hamamatsu, Japan).

RESULTS Kinetics of Nuclear Division and Morphogenesis during Infection Structure Formation Conidia of C. lagenarium are elliptical cells which contain a single nucleus as revealed by DAPI staining (Fig. 1A). When conidia land on a host plant, they germinate, first attaching and then producing a germ tube. Subsequently the tip of the germ tube swells to form a swollen melanized appressoria (Fig. 1A). One round of nuclear division occurs during this infection-related morphogenesis, which results in conidia and appressoria each having one nucleus (Fig. 1A). Hydroxyurea, an inhibitor of DNA synthesis, prevents nuclear division during appressorium formation (unpublished results). This indicates that the nucleus of dormant conidia is arrested before the DNA synthesis (S) phase and they enter into the S phase at the start of germination. Conidia proceed through morphological development including nuclear division on both the artificial surface and the plant leaf surface. Incubation in yeast extract solution enables conidia to germinate more synchronously than incubation in water, although a long incubation in yeast extract results in multiple germination (Takano et al., 2000). We have found that conidia are able to germinate synchronously without multiple germination when incubated in yeast extract solution for 1 h followed by incubation in water (Fig. 1, and see Materials and Methods). Germination and appressorium formation were evaluated carefully under these conditions (Fig. 1B). At 2 h after the start of incubation, there was little evidence of germination on glass slides. In contrast, at 3 h, about 60% of conidia had formed visible germ tubes. Most conidia (⬎95%) completed germination by 4 h. Appressorium formation from germ tubes was completed by 6 h. DAPI staining revealed that nuclear division occurred almost synchronously at 4 h, i.e., 1 h after germination (Fig. 1B). After nuclear division, septation was observed between conidia and appressoria (data not shown), and appressoria became darkly pigmented with melanin (Fig. 1A).

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Identification and Characterization of C. lagenarium ␣-Tubulin Genes In yeast, GFP-fused ␣-tubulin proteins were successfully incorporated into microtubules (Carminati and Stearns, 1997). Conversely, overexpression of ␤-tubulin caused lethality in yeast (Weinstein and Solomon, 1990). Based on these results, we fused the ␣-tubulin gene to GFP for visualization of microtubules in C. lagenarium. First, the ␣-tubulin genes of C. lagenarium were isolated with a PCR-based strategy (see Materials and Methods). The screening identified two different ␣-tubulin genes designated ␣TUB1 and ␣TUB2. The ␣TUB1 gene contained a 1758-bp open reading frame with four introns, whereas the ␣TUB2 gene contained a 1885-bp open reading frame with six introns (Fig. 2A, and data not shown). Homology between the deduced amino acid sequences in ␣TUB1 and ␣TUB2 was 68% (Figs. 2A and 2B). ␣Tub1 shared significant homology with the A. nidulans TubB (84%) and the N. crassa Tub␣A (82%). In contrast, ␣Tub2 showed high homology to the A. nidulans TubA (89%) and the N. crassa Tub␣B (90%), whereas it showed relatively low homology to the A. nidulans TubB (68%) and the N. crassa Tub␣A (66%). The ␣TUB1 gene was used for construction of a fusion gene with the GFP gene.

Visualization of Microtubules with GFP–␣Tubulin Expression

FIG. 1. Formation of appressoria and nucleation state in C. lagenarium. (A) Morphological development and nuclear division. Before incubation (at 0 h), conidia have a single nucleus. After incubation for about 3 h, conidia form germ tubes. At 6 h, germ tubes form swollen appressoria accompanied by nuclear division, resulting in each cell (conidium and appressorium) possessing one nucleus. Cells were fixed and stained with DAPI (right) or viewed under differential-interference contrast (left). Ap, appressorium; Co, conidium; Ge, germ tube; N, nucleus. Bar, 20 ␮m. (B) Kinetics of germination and nuclear division. Conidia were incubated at 24°C in 0.1% yeast extract solution for 1 h followed by incubation in water. White circles show germination rate, and black circles show nuclear division rate.

An improved version of GFP, eGFP, was fused to amino terminus of ␣TUB1 and designated GFP–␣TUB1. Expression of GFP under the control of the 221-bp short promoter region of the melanin gene SCD1 resulted in constitutive GFP fluorescence at all fungal stages examined (unpublished results), although the intact SCD1 promoter showed inductive expression during appressorium formation (Takano et al., 1997). The plasmid pHGFPTUB1 containing the 221-bp SCD1 promoter and GFP–␣TUB1 was transformed into the C. lagenarium wild-type strain (see Materials and Methods). The GFP green fluorescence of 50 hygromycin-resistant transformants was examined. Seven transformants (GTB1 to GTB7) clearly exhibited GFP fluorescence. Western blot analysis with antiGFP antibody suggested that the GFP–␣Tub1 fusion protein was expressed in vegetative mycelia of a transformant GTB1 (Fig. 3A). In addition, fluorescent filaments were observed in vegetative mycelia in GTB1 (Fig. 3B). Similar results were obtained in the other six transformants (data not shown). Benomyl (5 ␮g/ml), a microtubule-depolymerizing drug, caused fragmentation of fila-

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FIG. 2. The two ␣-tubulin genes of C. lagenarium. (A) Amino acid sequence alignment of ␣Tub1 and ␣Tub2 of C. lagenarium (C. l.) with Tub␣A and Tub␣B of Neurospora crassa (N. c.) and with TubA and TubB of Aspergillus nidulans (A. n.). Sequences were aligned with the CLUSTAL W program (Thompson et al., 1994). Amino acids identical with those of C. lagenarium ␣Tub1 are indicated as white letters on black background. Similar residues are shown on gray background. Gaps introduced for the alignment are indicated by hyphens. (B) Relatedness of C. lagenarium ␣Tub1 and ␣Tub2 to N. crassa Tub␣A and Tub␣B, A. nidulans TubA and TubB, Schizosaccharomyces pombe (S.p.) tubulin ␣-1 and tubulin ␣-2, Arabidopsis thaliana (A. t.) TubA, and human (H. s.) tubulin ␣-1. The phylogram was prepared with the CLUSTAL W program. Data base accession numbers: AF321052 (GenBank) for ␣Tub1 (C. l.), AF321053 (GenBank) for ␣Tub2 (C. l.), P38668 (Swiss Prot) for Tub␣A (N. c.), P38669 (Swiss Prot) for Tub␣B (N. c.), S13336 (PIR) for TubA (A. n.), S13337 (PIR) for TubB (A. n.), A25072 (PIR) for tubulin ␣-1 (S. p.), B25072 (PIR) for tubulin ␣-2 (S. p.), M17189 (GenBank) for TubA (A. t.), and 177403 (PIR) for ␣-tubulin 1 (H. s.).

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FIG. 3. Visualization of microtubules with the GFP–␣TUB1 fusion gene. (A) Western blot of protein extracts of mycelia probed with antiGFP antibody. Lane 1, the wild type 104-T; lane 2, the transformant GTB1. In strain GTB1, the GFP–␣Tub1 fusion protein was detected. (B) Detection of fluorescent microtubules labeled with the GFP–␣Tub1 protein in vegetative hyphae. Fluorescent microtubules were detected in vegetative mycelia of the strain GTB1 (⫺BEN). Benomyl (5 ␮g/ml) disrupted the fluorescent microtubules (⫹BEN). Arrows indicate interphase microtubules.

ments in GTB1 (Fig. 3B), demonstrating that the GFP fusion proteins were successfully incorporated into endogenous microtubule structures. All aspects of growth and development of strain GTB1, including radial growth rate, conidiation, germination, appressorium formation, and pathogenicity, were identical to the wild type (data not shown). From these results, we concluded that the expression of the GFP–␣Tub1 fusion protein visualized microtubules of C. lagenarium without any other negative effects. Strain GTB1 was chosen for further experiments.

Microtubule Rearrangements during Morphological Development of Appressoria Temporal rearrangement of microtubules during appressorium formation was investigated in detail with fluorescence microscopy. In dormant conidia (before incubation), abundant microtubule structures were observed inside conidia (Figs. 4A– 4C). These microtubules were

considered interphase cytoplasmic microtubules because dormant conidia arrest before the S phase. The microtubules formed aster-like structures connected at a single spot (Figs. 4A and 4C). These microtubule-associated centers were found in various positions in dormant conidia. The distal ends of these microtubules elongated in random directions and appeared to attach to the cell cortex. Examples of microtubules not associated with the center of aster-like structures were also observed. In concurrence with germ tube emergence at 2.5 h, microtubule rearrangement, in that some of the interphase microtubules extended toward the apical site of a germ tube (Figs. 4D and 4E), was observed. At 4 h, most germ tubes differentiated into swollen appressoria (Figs. 4F and 4H). In parallel, vacuoles appeared in conidial cells followed by movement of cytoplasm from the conidia to appressorial cells with a concurrent growth and fusion of vacuoles (Figs. 4F and 4H). At this stage, most of the microtubules extended into the appressoria. Several filaments extending into appressoria showed greater fluorescence than other interphase microtubules (Figs. 4G and 4I). We considered these structures to be bundles of microtubules. We also observed that most of the interphase microtubules apparently associated with a certain spot at the cell cortex of conidia. These microtubule-associated centers were located opposite the germination site in most cases (Figs. 4G and 4I).

Microtubule Rearrangements at Mitosis during Appressorium Formation Interphase microtubules disassembled immediately when conidia entered into mitosis at about 4 h. Instead of interphase microtubules, mitotic spindles appeared, which demonstrates that the GFP–␣TUB1 gene could visualize spindle microtubules as well as interphase cytoplasmic microtubules (Figs. 5 and 6A– 6D). Figure 5 shows timelapse images of microtubules from the G2 phase to early mitosis in appressorium-differentiating conidia. In the image at 0 min in Fig. 5, both interphase and spindle microtubules are observed in conidia. Remarkably, this image shows that a microtubule-associated center in the cell cortex observed before mitosis was different from a nuclear spindle pole body (SPB). Interphase microtubules were found to associate with the microtubule-associated center at the cell cortex, whereas the SPB was visible inside conidia at the same time (Fig. 5, 0 min). This result demonstrates that interphase microtubules connect with a microtubule-associated center that is distinct from the nuclear SPB. A mitotic spindle containing SPBs was

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FIG. 4. Microtubule organization during morphological development of appressoria. (A–C) Before incubation (at 0 h), dormant conidia contain abundant interphase microtubules. Frequently, microtubules were associated with a certain spot (arrows) in conidia (a microtubule-associated center). (D and E) At 2 to 3 h, interphase microtubules were oriented toward a polar germination site. (F–I) At 3 to 4 h, germ tubes differentiate into swollen appressoria accompanying formation of vacuoles in conidia. Interphase microtubules elongated into appressoria. Most of these microtubules contacted a microtubule-associated center (arrows) in the cell cortex of conidia. A, C, E, G, and I are GFP images. B, D, F, and H are DIC images of C, E, G, and I, respectively. Ap, appressorium; Co, conidium; Ge, germ tube; Va, vacuole. Bar, 10 ␮m.

formed in various positions in conidia (Figs. 6A and 6B), whereas a microtubule-associated center was located opposite the germination site in conidia (Figs. 4F– 4I). In Fig. 5, disassembly of interphase microtubules started within less than 2 min after the recognition of visible spindle microtubules. At 4 min, interphase microtubules were almost completely disassembled. Similar results were obtained from repeated experiments (data not shown). Spindles were not arrayed parallel with the axis of germination at first appearance; however, prior to elongation, the spindle direction was modified to align with the axis of germination (Fig. 5, 4 min to 7 min). The fluorescence

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intensity of spindle microtubules was higher than that of interphase microtubules (Fig. 5, 0 min to 2 min), presumably because spindles contain a large number of closely spaced microtubules. After adjustment of direction, spindles elongated into the developing appressoria, and formation of astral microtubules was observed (Figs. 6D and 7). Fig. 7 shows the temporal progress of spindle microtubule elongation, later in mitosis in conidia that formed appressoria. In the image at 0 min in Fig. 7, short spindle microtubules showing high fluorescence intensity were formed between duplicated SPBs in conidia. Within 1 min, mitotic spindles elongated rapidly and reached ap-

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FIG. 5. Formation of mitotic spindles in a conidium during appressorium formation. At 0 min, spindle microtubules emanating from SPBs, appearing as a fluorescent spot, appear in a conidium (upper left). An arrowhead indicates a microtubule-associated center in the cell cortex of the conidium. Note that interphase microtubules are associated with the microtubule-associated center that is distinct from the fluorescent spot of the SPBs. Fluoresence of spindle microtubules increases rapidly, and interphase microtubules are disassembled by 4 min, whereas the other conidium (lower right) still displays interphase microtubules. The orientation of mitotic spindle in the conidium changes. At 7 min, the orientation is adjusted to align with the axis of germination (toward the appressorium). Ap, appressorium; Co, conidium; Im, interphase microtubule; Sp, spindle pole body; Sm, spindle microtubules. Bar, 5 ␮m.

pressorial cells. After segregation of SPBs into each cell, mitotic astral microtubules were clearly seen emanating from each SPB at 2 min. These astral microtubules seemed to attach to the cell cortex (Fig. 7, 2 min to 7 min). Duplicated SPBs emanating astral microtubules moved adjacent to the polar cell cortex in each cell. After almost reaching the cell cortex, SPBs still showed oscillatory movement around the cell cortex (Fig. 7, SPBs are marked by arrowheads). On completion of mitosis, astral and spindle microtubules were not clearly visible in appressoria, implying that they were depolymerized. Instead of astral and spindle microtubules, interphase cytoplasmic microtubules appeared in appressoria with a random pattern of orientation (Figs. 6E and 6F). In contrast, microtubule arrays were little changed inside conidia after mitosis, suggesting that mitotic microtubules remained in conidia (Figs. 6E and 6F).

Roles of Microtubules in Appressorium Formation Appressorium formation of C. lagenarium displays temperature sensitivity. At 32°C, conidia efficiently germinate but produce no appressoria (Takano et al., 1997). To

compare microtubule organization between appressorium formation and non-appressorium formation of conidia, we investigated microtubule organization in conidia in a noninductive condition for appressorium formation. When conidia are incubated at 24°C in either water or yeast extract solution, conidia form appressoria. In contrast, when conidia are incubated at 32°C in yeast extract solution, conidia germinate efficiently and germ tubes form thick vegetative hyphae without appressoria (Fig. 8A). The occurrence of conidial germination under this condition was at 3 h, similar to 2.5 h for conidia in an environment conductive for appressorium formation. In contrast, with similar timing of germination, nuclear division occurred at 8.5 h, i.e., at 5.5 h after germination in noninductive condition, which was 4.5 h later than that during appressorium formation. Germinating conidia at 32°C exhibited microtubule organization similar to that of germinating conidia under appressorium formation process (data not shown). During nuclear division (around 8.5 h), both the appearance of spindle microtubules and the disassembly of interphase microtubules were observed to be similar to those of the appressorium formation process (data not shown). During postnuclear division, organization of interphase microtubules in conidia elaborating vegetative

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FIG. 6. Microtubule organization at mitosis during appressorium formation. (A and B) Appearance of mitotic spindles in conidia. At nuclear division, mitotic spindles showing high-intensity fluorescence are formed, whereas interphase microtubules are depolymerized. (C and D) Elongation of mitotic spindles accompanying formation of astral microtubules. (E and F) Microtubule organization after completion of nuclear division (at 6 h). Appressoria are pigmented with melanin, and vacuoles occupy the whole space of conidia. In appressorial cells, mitotic microtubules disappear, and interphase microtubules reappear. B, D, and F are GFP images. A, C, and E are DIC images of B, D, and F, respectively. Ap, appressorium; Co, conidium; Va, vacuole; Am, astral microtubule; Sp, spindle pole body; Sm, spindle microtubule. Bar, 10 ␮m.

hyphae was quite different from that of appressoriumdifferentiating conidia (Figs. 8A and 8B). Microscopic observation showed that interphase microtubules had no uniform direction inside appressoria (Figs. 6E and 6F). On the other hand, in conidia producing hyphae, microtubules showed polarized elongation toward the apical site of hyphae (Figs. 8A and 8B). They also exhibited synchronous formation of multiple mitotic spindles during disassembly of interphase microtubules (Figs. 8C and 8D). We investigated the effects of benomyl on appressorium formation. At 1 h of incubation in the presence of benomyl, fragmentation of microtubules occurred in conidia, which indicated depolymerization of microtubules. These short-fragmented microtubules showed relatively strong fluorescence compared with normal cytoplamic microtubules (data not shown). After benomyl treatment, conidia germinated similar to controls. When germ tubes were visible, fragmented microtubules were still observed (data not shown). Subsequently, germ tubes differentiated into swollen appressoria accompanying movement of cytoplasm from conidia to appressoria. Unlike mature appres-

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FIG. 7. Elongation of mitotic spindles from a conidium into an appressorium. At 0 min, mitotic spindles are oriented toward the appressorium. At 1 min, mitotic spindles have elongated rapidly and reached to the appressorium. Astral microtubules are emanating from each SPB. It can be seen that some astral microtubules reach to the cortex of each cell. After segregation of SPBs, each SPB is still mobile until it contacts the cell cortex. Arrowheads indicate SPBs. Ap, appressorium; Co, conidium; Sm, spindle microtubule. Bar, 5 ␮m.

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soria of the control, most appressoria showed curling under the benomyl treatment (Fig. 9A). This demonstrates that benomyl interfered with normal appressorial development. In the absence of benomyl, the two daughter nuclei segregated precisely to the conidiium and the appressorium during appressorium formation. In contrast, benomyl blocked the distribution of nuclei. With benomyl treatment, DAPI staining detected two (occasionally several) nuclei (Fig. 9A). This suggested that benomyl permitted duplication of nuclei in conidial cells; however, only 3.3% of conidia treated with benomyl achieved distribution of duplicated nuclei to the appressorium and conidium, whereas 99% of conidia in the control did so (Fig. 9B). These results suggested that microtubules are essential for precise distribution of nuclei during appressorium formation. The disruption of microtubules caused 70% of duplicated nuclei to move together to appressoria, whereas 26% of duplicated nuclei remained together in conidial cells (Fig. 9B). We suggest that microtubules have an important role for proper postmitotic nuclear distribution. FIG. 9. Benomyl treatment of conidia during appressorium formation. (A) Morphological changes and inhibition of nuclear distribution caused by benomyl treatment. Conidia were incubated at 24°C in 0.1% yeast extraction solution for 1 h followed by incubation in water for 8 h. Under the benomy treatment (5 ␮g/ml), appressoria curled toward conidia (DIC image). In these cells, nuclear distribution was inhibited, and both daughter nuclei located in the appressoria (DAPI image). Ap, appressorium; Co, conidium; N, nucleus. (B) Inhibition of nuclear distribution at appressorium formation under benomyl treatment. In the absence of benomyl (⫺BEN), conidia and appressoria mainly possess one nucleus each (I). In contrast, in the presence of benomyl (⫹BEN), 70% of appressorium-differentating conidia have both daughter nuclei in appressoria (II), whereas 26% of them have both daughter nuclei in conidia (III). I, conidia and appressoria contain one nucleus each; II, appressoria contain both nuclei; III, conidia contain both nuclei.

DISCUSSION FIG. 8. Microtubule organization in conidia under noninductive condition for appressorium formation. (A and B) Conidia were incubated in 0.1% yeast extract solution at 32°C for 12 h, which resulted in elongation of hyphal structures from conidia without appressorium formation. In conidia elongating vegetative hyphae, interphase microtubules elongate toward the apical site of the hypha. (C and D) Synchronous formation of mitotic spindles. When conidia are subjected to further incubation (for approximately 18 h), division of three nuclei starts synchronously, causing formation of three mitotic spindles. B and D are GFP images. A and C are DIC images of B and D, respectively. Bar, 10 ␮m.

Dynamic Changes of Microtubule Organization during Infection-Related Morphogenesis We successfully visualized microtubules during appressorium formation by the fungal plant pathogen C. lagenarium. This is the first report of visualization of microtubules in living cells during infection structure formation by a fungal pathogen. PCR-based screening identified two

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␣-tubulin genes, ␣TUB1 and ␣TUB2, in C. lagenarium. The two genes exhibited high divergence (32%) between their derived amino acid sequences. It has been reported that other filamentous fungi such as A. nidulans and N. crassa possess two divergent ␣-tubulins (Doshi et al., 1991; Monnat et al., 1997). We found that ␣-tubulin sequences of C. lagenarium, A. nidulans, and N. crassa formed two distinct phylogenetic classes. This suggests that a progenitor gene evolved two distinct tubulin genes before divergence of these fungi. In A. nidulans, the two ␣-tubulin genes showed a developmental stage-specific expression pattern. Disruption of each gene resulted in different phenotypes, suggesting that the two ␣-tubulins have distinct roles (Doshi et al., 1991). However, it has been reported that either ␣-tubulin product is sufficient for microtubule function of this fungus, indicating that the ␣-tubulins are functionally identical (Kirk and Morris, 1993). The C. lagenarium GFP–␣TUB1 fusion also visualized both interphase and mitotic microtubules in all stages examined. From these results, we considered that two divergent ␣-tubulins of C. lagenarium would be functionally identical despite their high divergence. The GFP– ␣Tub1 fusion proteins were incorporated into intracellular microtubules without an apparent affect on microtubule function. Immunofluorescence analysis of microtubules showed interphase and astral microtubules exhibiting relatively weak fluorescence compared with that of mitotic spindle microtubules, thus often making them undetectable. However, we readily detected these microtubules when they were labeled by GFP fluorescence. These results demonstrate that the GFP–␣TUB1 fusion gene is a powerful tool for monitoring microtubule organization in C. lagenarium and possibly related fungi. Microtubules are thought to originate from nuclearassociated SPBs, whereas the origin of interphase microtubules in fungal hyphae is not well understood. In C. lagenarium, most of the interphase microtubules, arranged in aster-like structures, were apparently associated with a region in the cell cortex of conidia that produced appressoria. Time-lapse views showed that this microtubule-associated center in conidia was not the nuclear SPB. Formation of microtubule-organizing centers (MTOCs), distinct from nuclear SPBs, has been reported in several fungi (Hagan and Hyams, 1988; Thompson-Coffe and Zickler, 1992). There is the possibility that the microtubule-associated center in appressorium-differentiating conidia of C. lagenarium is a MTOC distinct from an SPB. In addition, in dormant conidia, a portion of the microtubules was formed into aster-like structures. It is technically difficult to observe both DAPI-stained nuclei and GFP-

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Takano, Oshiro, and Okuno

labeled interphase microtubules in the same sample due to the cell fixation process for DAPI staining which causes disruption of interphase microtubules and reduction of GFP fluorescence. Thus, it remains to be determined whether the microtubule-associated centers in dormant conidia are distinct from nuclear-associated SPBs. In C. lagenarium, interphase microtubules disassembled obviously at the mitotic stage of appressorium formation. Similar disassembly of microtubules occurred also in conidia elongating hyphal structures. This demonstrates that C. lagenarium disassembles interphase microtubules at mitosis of both infection structure formation and hyphal elongation stages. Breakdown of interphase microtubules at mitosis has also been reported during hyphal extension in A. nidulans (Osmani et al., 1988). In contrast, this phenomenon is not observed in N. crassa (Minke et al., 1999), showing that regulation of interphase microtubules at mitosis is not identical among filamentous fungi. We also found that conidia elongating hyphae of C. lagenarium underwent synchronous division of multiple nuclei. Interestingly, synchronous nuclear division in vegetative hyphae is observed in A. nidulans but not in N. crassa (Minke et al., 1999). These data might suggest that breakdown of interphase microtubules is linked with synchronous nuclear division in filamentous fungi. Time-lapse views of microtubule arrays revealed that formation of mitotic spindles preceded disassembly of interphase microtubules at appressorium formation. This observation suggests either that disassembly of interphase microtubules is triggered before mitotic spindle formation and proceeds slowly or that it is triggered after mitotic spindle formation. Time-lapse observation demonstrated that short mitotic spindles adjusted their direction toward appressoria before their rapid elongation. This could imply the presence of a regulator inside appressoria which modulates the orientation of mitotic spindles formed in conidia. In budding yeast, interaction of mitotic astral microtubules with dynein in the cell cortex was shown to be necessary for establishment of correct spindle orientation (Carminati and Stearns, 1997). It is possible that putative dynein in the cell cortex of appressoria regulates spindle orientation by interacting with astral microtubules in C. lagenarium. At completion of mitosis in many organisms, mitotic microtubules, i.e., spindle and astral microtubules, disappear and interphase microtubules reappear. Consistently, the mitotic microtubules of appressorial cells were broken down after nuclear division and interphase microtubules reappeared. However, mitotic microtubules remained in conidial cells after mitosis. This result suggests that the

Microtubule Dynamics of Colletotrichum lagenarium

regulation system for the disassembly of mitotic microtubules might not work in conidia after mitosis. When conidia form appressoria, vacuoles grow in the conidium to fill the entire cell. On the other hand, in conidia elongating hyphae at 32°C that hardly showed vacuole formation, mitotic microtubules were depolymerized in the conidia after mitosis. Significant displacement of cytoplasm by vacuoles might cause retention of mitotic microtubules in conidia.

Roles of Microtubules in Nuclear Distribution and Cellular Morphogenesis During hyphal growth of filamentous fungi such as A. nidulans, N. crassa, and N. haematococca, nuclear distribution and migration were shown to be dependent on the microtubule-dependent motor, cytoplasmic dynein (Xiang et al., 1994; Plamann et al., 1996; Inoue et al., 1998). Benomyl treatment caused complete inhibition of nuclear distribution during appressorium formation of C. lagenarium. This result indicates that microtubules function in nuclear distribution during infection structure formation of this pathogen. Benomyl treatment caused approximately 70% of daughter nuclei to move together into appressoria, showing that nuclei could move from conidia to appressoria without microtubules. We considered that transfer of nuclei under benomyl treatment was passive and dependent on cytoplasm movement to appressoria, which in turn was based on vacuole formation inside conidia. In conidia forming hyphae, benomyl inhibited nuclear distribution and normally caused retention of divided nuclei in the region of conidia (data not shown). These results suggest that mitotic microtubules during appressorium formation are necessary for the retention of one nucleus in the conidia rather than for the transfer of it to appressoria. It has been shown, in several organisms, that microtubules are necessary for cell polarity establishment. In fission yeast, microtubules were shown to be involved in establishment of cell polarity at certain stages (Sawin and Nurse, 1998). In N. crassa, benomyl treatment resulted in multiple germination, indicating that benomyl impaired polar germination (That et al., 1988). In contrast, it was shown that microtubules are not essential for the establishment of polar budding of the yeast S. cerevisiae (Huffaker et al., 1988; Jacobs et al., 1988). In germinating conidia of C. lagenarium, interphase microtubules were rearranged to elongate toward a polar germination site, whereas they elongated in random directions during the dormant stage of conidia. A microtubule-associated center

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was observed at a site in the conidium opposite to the germination region. These observations suggest the possibility of an interaction of microtubules with the cell polarity determinants in the conidia. However, benomyl treatment had no effect on conidial germination of C. lagenarium, whereas it interfered with normal appressorium development, indicating that microtubules are not essential for the germination of conidia. In benomyl treatment, short fragmented microtubules still remained when germ tubes became visible, which implies incomplete depolymerization of microtubules. At present, it remains to be elucidated whether or not microtubules are involved in germination of C. lagenarium. Benomyl did show effects on later steps of infection-related morphogenesis. Under benomyl treatment, appressoria showed curling toward conidial cells. Similarly, benomyl caused curling of hyphae when conidia germinated at 32°C (data not shown). This indicates that functioning microtubules are necessary for correct extension of fungal hyphae and for normal appressorium formation. It is unclear whether curling caused by benomyl is due to disassembly of interphase microtubules or inhibition of nuclear distribution based on depolymerization of mitotic microtubules. Interphase microtubules showed a random orientation in appressoria at the postmitotic stage, which presented significant contrast to the microtubule array in conidia elongating hyphae. Consistent with our result, at an early stage of appressorium formation in the rust fungus, U. appendiculatus, random orientation of microtubules in appressoria was observed (Kwon et al., 1991). It has also been shown that germlings of the rust fungus treated with an actin-depolymerizing drug shifted from polarized apical growth to spherical expansion, suggesting that the establishment of cell polarity mediated by actin filaments is repressed in appressoria of this fungus (Tucker et al., 1986). An actin-depolymerizing drug caused formation of a swollen structure in the hyphal apex of C. lagenarium (unpublished results). It is possible that random array of interphase microtubules in appressoria is linked to the repression of cell polarity establishment in appressoria in C. lagenarium. The GFP– ␣TUB1 fusion gene enabled us to describe temporal and spatial organization of microtubules during infection structure formation of C. lagenarium. We have isolated several nonpathogenic mutants showing morphological defects during the appressorium formation process (Takano et al., 2000, and unpublished results). Application of the GFP–␣TUB1 fusion gene will contribute to efforts to investigate changes of microtubules in these morphological mutants.

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ACKNOWLEDGMENTS We acknowledge Drs. Ralph A. Dean, Thomas K. Mitchell, and Yasuyuki Kubo for critical reading of the manuscript. We are grateful to Dr. Regine Kahmann for providing the plasmid pOTEFSG. We thank Drs. Iwao Furusawa and Kazuyuki Mise for valuable suggestions and support during the course of this work. This work was supported in part by research grants (No. 10760031) from the Ministry of Education, Science, Sports and Culture of Japan, and a Grant-in-Aid (JSPSRFTF96L00603) from the “Research for the Future” program of the Japan Society for the Promotion of Science.

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