Cloning and characterization of a gene (arpA) from ... - Springer Link

Mol Gen Genet (1999) 262: 758±767

Ó Springer-Verlag 1999

ORIGINAL PAPER

K. Hirozumi á H. Nakajima á M. Machida M. Yamaguchi á K. Takeo á K. Kitamoto

Cloning and characterization of a gene (arpA ) from Aspergillus oryzae encoding an actin-related protein required for normal nuclear distribution and morphology of conidiophores Received: 26 January 1999 / Accepted: 22 July 1999

Abstract We have isolated the arpA gene from Aspergillus oryzae as a homologue of the Neurospora crassa ro-4 gene. In N. crassa, mutations in the ro-4 gene, which encodes a major component of the dynactin complex Arp1, causes curling of hyphae and abnormalities in nuclear distribution. The arpA gene contains two introns and encodes a polypeptide of 381 amino acids, with a 78% sequence identity to the N. crassa Arp1. Overexpression of the arpA gene causes a defect in nuclear migration into elongating hyphae of germlings in A. oryzae. We constructed arpA disruptant strains of A. oryzae. The arpA null mutants showed poor growth and hyper-branched mycelia, as well as a nuclear distribution defect. Scanning electron microscopy revealed that the arpA null mutant has an aberrant conidiophore morphology with irregular phialides. Key words Actin-related protein á Dynactin á Nuclear migration á Aspergillus oryzae á Hyper-branching hyphae

Introduction Cytoplasmic dynein is a minus-end-directed, microtubule-dependent motor protein complex (reviewed by Porter and Johnson 1989; Walker and Sheetz 1993). This

Communicated by C. A. M. J. J. van den Hondel K. Hirozumi á H. Nakajima á K. Kitamoto Department of Biotechnology, The University of Tokyo 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan M. Machida Department of Molecular Biology, National Institute of Bioscience and Human Technology Higashi 1-1, Tsukuba, Ibaraki 305-0046, Japan M. Yamaguchi á K. Takeo Research Center for Pathogenic Fungi and Microbial Toxicoses Chiba University, 1-8-1, Inohana, Chuo-ku Chiba, 260-8673, Japan

complex has been proposed to function in a number of intracellular transport processes, including retrograde transport of vesicles in axons, the endocytosis pathway, the organization of the Golgi apparatus, and nuclear migration in fungus cells (Plamann et al. 1994; Xiang et al. 1994). Recently, genes encoding cytoplasmic dynein heavy chain (DHC) have been isolated from a number of organisms including rat, Saccharomyces cerevisiae and Aspergillus nidulans (Zhang et al. 1993; Eshel et al. 1993; Xiang et al. 1994). Disruption of the S. cerevisiae DHC gene results in viable cells, but subsequent delivery of a daughter nucleus into newly formed buds is perturbed (Eshel et al. 1993). In the ®lamentous fungus A. nidulans, mutations in the DHC gene (nudA) a€ect nuclear migration into the elongating hyphae (Xiang et al. 1994). Cytoplasmic dynein is composed of two heavy chains of 530 kDa, three intermediate chains of 74 kDa, and four light intermediate chains of 55±60 kDa (Holzbaur and Vallee 1994). This complex is sucient to support the movement of microtubules in vitro; however, it does not support microtubule-dependent movement of vesicles (Lye et al. 1987). A second protein complex, dynactin, is also required for ecient dynein-powered movement of membrane vesicles in vitro (Schroer and Sheetz 1991). Dynactin, a 1.2-MDa macromolecular complex, is composed of ten subunits, p150Glued, p135Glued, p62, p50, Arp1, actin, actin-capping protein a-subunit, actin-capping protein b-subunit, p27, and p24, in an approximate stoichiometry of 1:1:1:4:9:1: 1:1:1:1 (Schafer et al. 1994). Arp1 is the most abundant component in the dynactin complex and was originally referred to an actin-related protein (actin-RPV) in vertebrates (Lees-Miller et al. 1992b), or as centractin in humans (Clark and Meyer 1992). At the base of the dynactin complex, one actin and nine Arp1 molecules participate in the formation of a 37-nm actin-like ®lament (Schroer et al. 1996). In Neurospora crassa, the cot-1 gene encodes a serine/ threonine protein kinase required for hyphal elongation

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(Yarden et al. 1992). In an e€ort to identify the substrates of Cot1 kinase, several unlinked mutants that show suppression of the hyphal growth defecet of a cot-1 mutant were isolated. These mutant strains have curled hyphae with abnormal nuclear distribution and are de®ned as ropy. Genetic analysis revealed that the genes ro-1, ro-3, and ro-4 encode DHC, pl50Glued and Arp1, respectively (Plamann et al. 1994). The phenotypes of ro-1, ro-3 and ro-4 mutants suggest that the cytoplasmic dynein and dynactin complex plays an important role in maintaining uniform nuclear distribution in N. crassa hyphae. The gene encoding Arp1 protein has been isolated from several eukaryotic organisms, including S. cerevisiae (ACT5), S. pombe (ACT2), Drosophila, and human (centractin) (Clark and Meyer 1994; Lees-Miller et al. 1992a; Fyrberg and Fyrberg 1993; Schroer and Sheetz 1991). The N. crassa Ro4 is 65% identical to centractin and approximately 40% identical to other members of the Arp1 family. Aspergillus oryzae is an important ®lamentous fungus in the fermentation industry, and is used in sake, soy sauce and miso manufacture, as well as for production of commercial enzymes (Hara et al. 1992). A. oryzae is also considered to be a favorable host for heterologous protein production, in view of its ability to secrete large amounts of proteins. We have studied the molecular genetics of A. oryzae, including heterologous protein production (Tada et al. 1991; Tsuchiya et al. 1994), promoter analysis (Hata et al. 1992) and its electrophoretic karyotype (Kitamoto et al. 1994). One of the characteristics by which it di€ers from A. nidulans is that it has multinuclear conidia containing three to ®ve nuclei, but little is known about the mechanism of nuclear migration or intracellular protein transport in A. oryzae. In this paper, we describe the isolation of the ro-4 homolog from A. oryzae, designated arpA, and characterization of both arpA overexpression and null mutants.

Materials and methods Strains, plasmids, and media A. oryzae RIB40 is a wild-type strain and was used as a DNA donor. A. oryzae niaD300 (niaD)) (Minetoki et al. 1996) and NS4 (niaD), sC)) (Yamada et al. 1997) were used as recipient strains. Escherichia coli MC1061 (hsdR mcrB araD D(araABC-leu) DlacX galU galK rpsL thi) (Casadaban and Cohen 1980) and LE392 [e14) (McrA)) hsdR supE supF lacY galK galT metB trpR] (Borck et al. 1976) were used for construction of the A. oryzae gene library and for DNA manipulations (Maniatis et al. 1982). S. cerevisiae BY103-8B (MATa leu2 ura3 his3 trp1 act5::TRP1) was used for complementation tests of the A. oryzae arpA cDNA. The phage vector kDASHII and the plasmid vector pBluescript II were purchased from Toyobo Biochemicals (Osaka, Japan). The plasmid pUNA (Tada et al. 1991), containing the amyB promoter and the niaD gene as a selectable marker, was used for overexpression of the arpA gene. The sC gene on pUSC (Yamada et al. 1997) was used for disruption of the arpA gene. Czapek-Dox (CD) medium, consisting of 0.3% NaNO3, 0.2% KCl, 0.1%

KH2PO4, 0.05% MgSO4á7H2O, 0.002% FeSO4á5H2O and 2% dextrin, and DPY medium, consisting of 2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH2PO4, 0.05% MgSO4á7H2O, were used as minimal and complete media, respectively. The medium for amyB promoter induction (CD + M) contained 2% maltose, instead of glucose, in CD medium (Tada et al. 1991). Cloning and sequence analysis of the arpA gene from A. oryzae A. oryzae RIB40 chromosomal DNA was partially digested with Sau3AI. The resulting 15- to 20-kb fragments were cloned into the BamHI site of the kDASHII phage vector to prepare a genomic library. Two oligonucleotide primers [5¢-CA(T/C)GGIATIGTIACIGA(T/C)TGGGA(T/C)GA(T/C)ATG-3¢ and 5¢-ATI(C/G)(T/A)ICCICC(T/A/G)ATCCAIGTI(G/C)(A/T)(A/G)TA-3¢; I, inosine] were used to isolate an A. oryzae gene homologous to the N. crassa ro-4 gene by PCR, as described previously (Hata et al. 1992). The two primers were designed based on amino acid sequences that are highly conserved among actin-related proteins, which correspond to amino acid residues 76±85 and 343±351 of the N. crassa Ro4 (Fig. 2). Deletion clones for sequencing were constructed by using a Kilo-Sequence deletion kit (Takara Shuzo Kyoto, Japan). Nucleotide sequences were determined with a Model DSQ-1000 DNA sequencer (Shimazu, Tokyo, Japan). mRNA was prepared from A. oryzae RIB40 grown in CD medium, and converted into cDNA using the Marathon cDNA ampli®cation kit (Clontech Laboratories). Two oligonucleotide primers, ARCZ1 (5¢-ACCCCggGCTCTCGCAATGGCCGAGG CCAC-3¢) and ARCZ2 (5¢-GAcccggGAATTAGGCGAATTTCC GATGGATG-3¢) were used to amplify arpA cDNA and introduce a SmaI site (underlined lower case letters) (Fig. 1). The two primers were based on N- and C-terminal regions of the predicted ArpA ORF in the genomic arpA locus. The ampli®ed 1.1-kb fragment was cleaved with SmaI and cloned into pBluescript II. Transformation of A. oryzae was performed according to Rasmussen et al. (1990). Construction of arpA overexpression and arpA-disrupted strains The 1.1-kb SmaI fragment containing the arpA cDNA was inserted into the SmaI site of pUNA, downstream of the amyB promoter (Tada et al. 1991). The resulting plasmid, pAC1, was introduced into A. oryzae niaD300 for overexpression of arpA. To disrupt the arpA gene, a 3.2-kb XbaI-BamHI fragment carrying the sC marker gene, encoding ATP sulfurylase (Buxton et al. 1989), was inserted into the XbaI and BglII sites in the arpA region on pVAD (Fig. 3a). The 6.4-kb HindIII-EcoRV fragment carrying the DarpA::sC gene was ampli®ed by PCR using the M13 primer and M3 primer RV (Takara Shuzo) and introduced into the A. oryzae NS4 strain. Sulfate-assimilating transformants were streaked on fresh medium ®ve times to obtain homokaryotic strains. Chromosomal DNAs of the candidate arpA disruptants were prepared and analyzed by PCR using ARCZ1 and ARCZ2 (Fig. 1) as primers. Analysis of nuclear distribution Nuclear distribution was examined by staining cells with Hoechst 33258 (Harris et al. 1994). Approx. 102 conidia were suspended in DPY liquid media and placed on coverslips in culture plates. The plates were incubated at 30° C for 10 h. Hyphal material on coverslips was treated with ®xing solution (3.7% formaldehyde, 50 mM phosphate bu€er pH 7.0, 0.2% Triton X-100) for 45 min and soaked in 100 ng/ml Hoechst 33258 (Sigma, St. Louis, Mo.) solution at room temperature for 5 min. The coverslips were mounted in mounting solution (10% phosphate bu€er pH 7.0, 50% glycerol, 0.1% n-propyl-gallate) and placed on a slide. Fluorescence was observed with an Olympus BH2-FL microscope with

760 Fig. 1 Nucleotide and deduced amino acid sequences of the Aspergillus oryzae arpA gene. Putative intron sequences are in lower case letters. Numbers at the margin refer to the amino acid sequence. The PCR primers ARCZ1 and ARCZ2 were synthesized based on the underlined sequences, as indicated. A putative TATA box and a StuAp response element (A/TCGCGT/ANA/C) in the 5¢-noncoding region are heavily underlined. The sequence data reported in this paper have been submitted to the DDBJ, EMBL, and NCBS nucleotide sequence databases under the Accession No. AB019144

761 Fig. 2 Comparison of the predicted amino acid sequence of A. oryzae ArpA with those of N. crassa Ro4, S. cerevisiae Act5, vertebrate actin-RPV and human cytoplasmic c-actin. Amino acid residues which are identical in at least four of the ®ve proteins are boxed. The residues in actin that interact with ATP (+) and Ca2+ (#) are indicated below the sequence. The residues involved in actinactin polymerization are indicated by asterisks below the sequence (Holmes et al. 1990). Degenerate PCR primers were synthesized based on the underlined conserved amino acid sequences

a BH-DMU ultraviolet excitation cube (Olympus, Tokyo, Japan) and a Dplan Apo 40´ objective lens.

Results

Analysis of conidiophore morphology

Cloning of an Arp1-encoding gene from A. oryzae

Conidia were placed on the surface of thin agar strips on a coverslip. The coverslips were incubated at 30° C for 3 to 5 days. Hyphae and conidiophores grown on the coverslips were treated with 1% glutalaldehyde for ®xation. After washing with water, the coverslips were transferred to 50%, 70%, 90% and 99.5% ethanol in that order. They were then transferred to isoamyl acetate and critical-point dried. The samples were sputter-coated with platinum-palladium and observed with a Hitachi S-800 scanning electron microscope (Hitachi, Tokyo, Japan).

The 0.9-kb DNA fragment ampli®ed by PCR as described in Materials and methods was used as a hybridization probe. Among approximately 5 ´ 104 plaques of the kDASHII genomic library, six positive clones were identi®ed by plaque hybridization as described (Maniatis et al. 1982). Subcloning and restriction enzyme analysis showed that a 4.0-kb HindIII-EcoRV fragment contained the

762 Fig. 3a, b Disruption of the arpA gene. a Construction of the plasmid pDSCA to disrupt the arpA gene. The 0.8-kb XbaIBglII fragment in the coding region of the arpA gene on pVAD was replaced by the 3.2kb XbaI-BamHI fragment carrying the sC marker gene. The pVAD was constructed by insertion of the 4.0-kb HindIIIEcoRV fragment containing the arpA gene into pUC19. b Southern analysis of the arpA disruptants. Genomic DNA of the wild type RIB40 (lane 1), NS4 (lane 2), and arpA disruptants DPCR7 (lane 3), DPCR6 (lane 4), DPCR5 (lane 5) were digested with BamHI and subjected to Southern analysis with the 1.2-kb HindIII-BamHI fragment of the arpA gene as a probe

entire ro-4 homolog. Southern blots of genomic DNA digested with HindIII or EcoRI were probed with the 0.9kb PCR fragment. In each digest, a single hybridizing band was detected. This suggests that the ro-4 homologue exists as a single copy in A. oryzae RIB40 and we designated the gene as arpA. The sequence of the 2.0-kb arpA region of the genomic clone was determined. In order to isolate a cDNA for the arpA gene we prepared total mRNA from A. oryzae RIB40 and converted it into cDNA. A 1.1-kb fragment was ampli®ed by PCR using the cDNA library as a template, and was cloned into pBluescript II for nucleotide sequence analysis. Figure 1 shows the nucleotide and deduced amino acid sequences of the arpA gene of A. oryzae and its product. Comparison of the cDNA and genomic sequences revealed two introns. Both the 5¢ and 3¢ splice junctions of the introns matched the consensus splice site sequence found in genes from ®lamentous fungi (Gurr et al. 1987). The two introns in the arpA gene were found at the same positions as the second and third of the three introns in the N. crassa ro-4 gene (Plamann et al. 1994). The 5¢-noncoding region contains a TATA-like element (TATAAA) at 392 nucleotides, and a StuAp response element (A/TCGCGT/ANA/C) 204 nucleotides, upstream

of the start codon, respectively. The A. nidulans StuAp regulates multicellular complexity during asexual reproduction by moderating the core developmental program that directs di€erentiation of uninucleate, terminally di€erentiated spores from multinucleate, vegetative hyphae (Dutton et al. 1997). No canonical AATAAA polyadenylation signal exists in the 3¢-noncoding region of the arpA gene. Based on this structural analysis, we concluded that the arpA gene encodes a protein of 381 amino acids. Alignment of the deduced amino acid sequence of A. oryzae ArpA shows a high degree of identity with other members of the Arp1 family and conventional actin (Fig. 2). ArpA was found to be 78%, 62%, 52%, and 55% identical in sequence to N. crassa Ro4 (Plamann et al. 1994), human centractin (Clark and Meyer 1992), S. cerevisiae Act5 (Clark and Meyer 1994; Muhua et al. 1994), and human cytoplasmic c-actin (Erba et al. 1986), respectively. No additional signals were detected on the Southern blots of genomic DNA probed with arpA at low stringency (data not shown), suggesting that A. oryzae has only a single Arp1 gene. In S. cerevisiae, mutants in cytoplasmic dynein are defective in orienting the mitotic spindle. Of S. cerevisiae act5 null mutants, which lack a functional Arp1

763

homologue, are viable but partially defective in nuclear migration into the newly formed daughter cells. More than 10% of budded cells have a binucleate mother cell and an anucleate bud during growth at 14° C. It was reported that the vertebrate actin-RPV cDNA under control of the GAL1 promoter failed to complement the act5 mutation (Muhua et al. 1994). In order to examine whether the A. oryzae arpA cDNA complements the act5 mutation, we constructed a GAL1-arpA cDNA fusion gene and introduced it into the act5 null mutant strain. After 48 h induction on galactose medium at 14° C, the ratio of the cells showing abnormal nuclear distribution was 20% and 22% for the act5 cells with and without arpA expression, respectively. This indicates that the A. oryzae arpA gene is not functionally equivalent to the ACT5 gene in S. cerevisiae. Overexpression of the arpA gene Arp1 is part of the dynactin complex and functions by binding several other polypeptides. In S. cerevisiae, overexpression of Act5p leads to abnormal nuclear migration and defects in spindle orientation (Muhua et al. 1994). To investigate the morphological and physiological e€ects of overexpression of ArpA protein in A. oryzae, we constructed a strain containing the arpA gene placed under the control of the A. oryzae amyB promoter. The amyB promoter is strongly induced by maltose and repressed by glycerol. The activity of the amyB promoter is elevated 31.4-fold with maltose relative to glucose and repressed eightfold with glycerol (Tada et al. 1991). We constructed a plasmid carrying an amyB-arpA fusion gene, pACl, and introduced it into A. oryzae niaD300. Nitrate-utilizing transformants were streaked on fresh medium ®ve times to obtain homokaryotic strains. Integration of the amyB-arpA fusion gene into the chromosome in transformants AC1-3, AC1-6, and AC1-11 was con®rmed by Southern blotting and PCR analyses (data not shown). The transformants showed slow growth on maltose medium but grew normally on glucose or glycerol. Nuclear distribution in germling cells carrying wild-type arpA and amyB-arpA was visualized by staining with Hoechst 33258. In the wild-type germlings, most of the nuclei migrated into the germ tube and were evenly distributed. On the other hand, germlings of the transformants on maltose medium (CD + M) showed poor migration of nuclei into the germ tube, and most of the nuclei remained clustered in the conidia. Figure 4a shows the staining pattern of one of the transformants, AC1-6. These observations indicate that overexpression of ArpA a€ects nuclear distribution. Scanning electron microscopic observations of conidial heads of wild type and the arpA overexpressing strain AC1-6 are shown in Figure 5. Although the wildtype strain formed symmetrical conidial heads, strain AC1-6 grown on maltose medium for 5 days formed distorted conidiophores.

Disruption of the arpA gene To investigate the e€ect of depletion of ArpA protein, we constructed a null mutant for the arpA gene. Chromosomal DNAs of the candidate arpA disruptants were prepared and analyzed by PCR using ARCZ1 and ARCZ2 as primers. Although putative 1.2-kb DNA fragments were ampli®ed from the wild-type strain RIB40 and the recipient strain NS4, the expected 3.7-kb fragments were ampli®ed from the disruption candidates DPCR5, DPCR6, and DPCR7 (data not shown). Southern analysis of BamHI-digested genomic DNA demonstrated that the arpA gene was replaced by the DarpA::sC gene in the candidate strains (Fig. 3b). It has been reported that A. nidulans nudA (which encodes the cytoplasmic dynein heavy chain) mutant shows a temperature-sensitive growth phenotype (Xiang et al. 1994), but the N. crassa ro4 (encoding Arp1) disruptant does not (Plamann et al. 1994). Therefore, we examined the growth phenotype of the arpA disruptant strain. Both the wild type NS4 and the arpA null mutants could grow at 25° C, 37° C, and 40° C but not at 43° C. The arpA null mutants showed much slower growth and poorer conidium formation than wild-type strains at every temperature. This indicates that the arpA null mutant is not temperature sensitive. One of the disruptants, DPCR5, was used for further investigations. Nuclear distribution in the arpA null mutant germlings was observed by staining with Hoechst 33258 (Fig. 4a). Hyper-branched hyphae extended from the conidium and most of the nuclei remained around the conidium region. This clearly indicated that the arpA disruptant strain has a defect in nuclear distribution. Cultures on coverslips revealed that the wild-type strain formed long unbranched hyphae over 100 lm long at the growing tips, but the hyphae of the arpA null strain were hyper-branched (Fig. 4b). Scanning electron microscopic observations con®rmed the morphological changes in the arpA null mutant. The number of conidia was markedly reduced. The number of phialides was reduced by half and the orientation of the phialides on the conidiophores of DPCR5 strain was disordered (Fig. 5). The diameter of the hyphae and stipe in wild-type strains was 8 to 10 lm but was increased to 10 to 15 lm in the arpA null mutant strain. Phialides of the wild-type strain were uniform in length and formed round conidiophores, but the DPCR5 had distorted conidial heads with phialides of irregular length. These observations suggest that the ArpA protein plays an important role in formation of the A. oryzae hyphae, conidiophore, and conidia. It has been reported that the temperature-sensitive growth defect of A. nidulans nud mutants is partially suppressed by low concentrations of the microtubuledestabilizing drug benomyl (Willins et al. 1995). The e€ect of benomyl on the growth of an arpA null mutant strain is shown in Fig. 6. The strain DPCR5 grew much more slowly than the wild-type strain, RIB40, on DPY medium without benomyl. On the other hand, 1.0 lg/ml

764 Fig. 4a, b E€ect of overexpression or disruption of the arpA gene on distribution of nuclei. a Conidia of the wild type RIB40 and the AC1-6 strain carrying the amyB-arpA gene were grown and germinated on maltose (CD + M medium) at 30° C for 15 h. The DPCR5 strain carrying the DarpA::sC gene was grown and germinated on CD medium at 30° C for 12 h. Nuclei were visualized by staining with Hoechst 33258. Bar 20 lm. b Phase-contrast images of growing mycelia of the wild type RIB40 and DPCR5 (DarpA) cultured on coverslips. Bar 20 lm

of benomyl severely limited the growth of RIB40 but not of DPCR5. In the presence of 1.5 lg/ml of benomyl, wild-type RIB40 did not germinate but DPCR5 showed slow growth. This indicates that the growth defect of arpA null mutant is suppressed by low concentrations of benomyl.

Discussion We have cloned and characterized the arpA gene of A. oryzae, encoding a protein which has sequence homology to members of the Arp1 family. We have

shown ArpA is involved in control of nuclear migration. The amino acid sequence of A. oryzae ArpA is 78% identical to N. crassa Ro4. Compared with the Ro4 sequence, Ca2+ binding sites are completely conserved and only 1 of the 15 amino acids involved in ATP interactions has been substituted. ArpA is also homologous to conventional actin (55% sequence identity to human cytoplasmic c-actin), but compared with the c-actin, 20 of the 44 residues involved in actin polymerization are substituted in the ArpA sequence. This suggests that ArpA is not functionally interchangeable with conventional actin. Gene inactivation experiments demonstrated that ArpA is not essential

765 Fig. 5 Scanning electron microscopy of conidial heads. Conidia of the RIB40 (wild type) and DPCR5 (DarpA) strains were grown on DPY medium and the AC1-6 (amyBarpA) conidia were grown on CD + M at 30° C for 5 days, respectively. Bar 20 lm

for viability, although hyphal growth and nuclear migration are severely a€ected by insertion of the sC gene at the arpA locus. These observations are consistent with those reported for the N. crassa ro-4 mutation (Robb et al. 1995). Since it is most similar to Ro4, ArpA is probably part of an analogous dynactin complex, which activates cytoplasmic dynein-mediated movement of nuclei along cytoplasmic microtubules. Overexpression of the arpA gene caused defects in nuclear migration in germlings, indicating that excess ArpA protein a€ects the process of nuclear migration and the formation of hyphae. Biochemical studies have demonstrated that the Arp1 family protein is one of the major components of the dynactin complex which stimulates cytoplasmic dynein to translocate membrane vesicles along microtubules (Schroer and Sheetz 1991;

Lees-Miller et al. 1992b). It has also been reported that no monomeric Arp1 can be detected in cells and all cytosolic Arp1 is assembled into higher structures (Paschal et al. 1993). Ultrastructural analysis of the dynactin complex showed that Arp1 self-associates to form short actin-like ®laments (Schafer et al. 1994). The Arp1 ®laments are proposed not to be dynamic and all Arp1 molecules should be ®xed in the dynactin complex in the ®lamentous form. In A. oryzae cells, overexpression of ArpA may lead to a loss of function of the dynactin complex, owing to formation of many partial but few complete assemblies. In A. nidulans, conidia contain one nucleus each and nuclear division occurs prior to germ tube projection from the conidia. At the eight-nuclei stage of the germling, two or three nuclei are found in the conidium

766

Fig. 6 The arpA disruptant tolerates a higher level of benomyl than the wild type. Conidia of RIB40 (wild type) and DPCR5 (DarpA) were spotted on DPY media containing 0 lg/ml, 1.0 lg/ml or 1.5 lg/ml benomyl and incubated at 30° C for 4 days, 7 days and 9 days, respectively

and the others are evenly distributed in the hypha (Suelmann et al. 1997). On the other hand, in general A. oryzae forms multinuclear conidia containing three to ®ve nuclei. But only one nucleus was found in the conidium and the others were evenly distributed in the hypha of the germlings in wild-type A. oryzae (Fig. 4a). The basis of this di€erence in nuclear distribution pattern between A. nidulans and A. oryzae is unknown. We constructed a null allele of the arpA gene to examine the role of ArpA in A. oryzae. In N. crassa, mutations in ro-4 lead to an unusual hyphal growth phenotype consisting of curled and distorted hyphae and a highly asymmetric nuclear distribution (Plamann et al. 1994). The arpA null strain showed hyper-branched hyphae and defects in nuclear distribution in the germlings of A. oryzae. This observation is similar to what is seen in the N. crassa ro-4 disruptant strain. It indicates that the actin-related protein is not essential for growth but has an important role in determining hyphal morpology. Hoechst staining of conidia did not show any obvious di€erence in numbers of nuclei between wildtype and arpA null mutants. Both low concentrations of benomyl and the tubA22 mutation suppress the growth defect of A. nidulans nuclear distribution mutants (nudA, nudC, nudF, and

nudG). It was proposed that this e€ect is due to suppression of the nuclear migration defect (Willins et al. 1995). We observed that the growth defect of the arpA null mutant is also suppressed by low concentrations of benomyl. This suggested that destabilization of microtubules alternates the defect in the dynactin complex. The eciency of nuclear migration is very low but not zero in arpA null mutant cells. Indeed, the number of nuclei increased to ®ll the conidial shell and some nuclei eventually migrated into the germ tube in the germlings of the arpA null strain. It is possible that inactivation of the dynactin complex could allow an alternative motor protein to substitute for cytoplasmic dynein in the absence of fully active microtubules. Alternative motor proteins and microtubule depolymerization may be related, because several motor proteins, such as yeast minus-end microtubule motor Kar3, can themselves stimulate microtubule depolymerization and couple microtubule depolymerization to chromosome movement (Endow et al. 1994). It has been reported that the A. nidulans apsA ( anucleate primary sterigmata) mutant has hyphae-like metulae (Fischer et al. 1995). The apsA mutant shows a defect in nuclear distribution in hyphae and conidiophores, but the function of microtubule and actin ®laments is normal. We observed that the A. oryzae arpA null mutant has morphologically abnormal conidia and abnormal phialides similar to hyphae-like metulae. A decrease in nuclear migration eciency in the conidiophores in the arpA null mutant probably causes failure of the metulae cells to become nucleated, as in the A. nidulans apsA mutant. Anucleate metulae are likely to become hyphae-like metulae instead of normal sporogenous phialide cells. We observed that the arpA null mutant strain produced almost wild-type levels of amylase on starch plates, even though only small colonies formed (data not shown). This suggests that the arpA null mutant produced more amylase per unit cell volume. Filamentous fungi secrete enzymes mainly from the growing hyphal tips (Punt et al. 1994). The hyper-branching arpA null mutant has a larger number of hyphal tips than the wildtype strain. It is possible that the large number of hyphal tips permits secretion of higher amounts of enzymes in the arpA mutant. Further investigation of the arpA mutant and analysis of additional dynactin complexrelated genes may allow a better understanding of the relationship between hyperbranched mycelia and hypersecretion. Acknowledgements We thank Dr. B. R. Lee for technical assistance and helpful discussions and also thank Ms. Kyoko Yarita for instruction in scanning electron microscopy. We are grateful to Dr. D. I. Meyer and Dr. S. Clark (Princeton University, USA) for the provision of S. cerevisiae BY103-8B containing the ACT5D mutation and to Dr D. B. Archer for critical reading of the manuscript. This study was supported by a Grant-in-Aid for Scienti®c Research (B) (No. 08456045) to K. Kitamoto from the Ministry of Education of Japan and Cooperative Research Program of Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University (`98-15).

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