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The eln3 gene involved in fruiting body morphogenesis of Coprinus cinereus encodes a putative membrane protein with a general glycosyltransferase domain
Fungal Genetics and Biology 41 (2004) 805–812 www.elsevier.com/locate/yfgbi
The eln3 gene involved in fruiting body morphogenesis of Coprinus cinereus encodes a putative membrane protein with a general glycosyltransferase domain Toshihide Arima,a,b,1 Maki Yamamoto,c Aiko Hirata,c Shigeyuki Kawano,c and Takashi Kamadab,* a Research Fellow of the Japanese Society for the Promotion of Science (JSPS), Japan Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, Japan Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan b
c
Received 10 February 2004; accepted 19 April 2004 Available online 18 May 2004
Abstract We identified and characterized elongationless3 (eln3-1), a restriction enzyme-mediated integration (REMI) mutation affecting fruiting body morphogenesis in Coprinus cinereus. The mutant produces an aberrant fruiting body in which the stipe hardly elongates during fruiting body maturation. In the wild type, cylindrical stipe cells, elongation growth of which is responsible for stipe elongation, make side-by-side contact with one another and run parallel to the stipe axis, whereas in the mutant, the organization of the stipe tissue is disturbed and much space is produced between stipe cells. This disorganization of the stipe tissue, together with reduced elongation of the stipe cells, causes the mutant stipe short and bulgy. After a plasmid rescue, the eln3 gene was identified as a DNA fragment that complements the eln3-1 mutation. The eln3 ORF is predicted to encode a protein of 927 amino acids with a general glycosyltransferase domain and to be located in the plasma membrane. Transcription of the eln3 gene is specifically activated in rapidly elongating stipes. Possible involvement of the putative Eln3 enzyme in cell-to-cell connection is discussed. 2004 Elsevier Inc. All rights reserved. Index Descriptors: Homobasidiomycete; Coprinus cinereus; Fruiting body morphogenesis; REMI mutant; Stipe elongation; Cell-tocell connection; Membrane protein; Glycosyltransferase
1. Introduction The homobasidiomycete Coprinus cinereus develops a multicellular structure, the fruiting body, under proper environmental conditions. Fruiting body development of C. cinereus is rapid and predictable in the laboratory, providing an excellent opportunity for studies of fungal multicellular morphogenesis (Kamada, 2002; K€ ues, 2000; Moore, 1981, 1996; Muraguchi and Kamada, 1998). The development starts with the formation of hyphal knots, aggregations of hyphae of less than * Corresponding author. Fax: +81-86-251-7876. E-mail address: [email protected] (T. Kamada). 1 Present address: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan.
1087-1845/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2004.04.003
0.2 mm in diameter that lack recognizable tissue differentiation. Some of hyphal knots develop into fruiting body primordia with differentiation of the rudimentary cap and stipe. In the final phase of development (fruiting body maturation), basidia on the undersurface of the cap undergo meiosis, which is immediately followed by basidiospore formation, and stipe elongation and cap expansion occur for efficient spore dispersal to form a mature fruiting body (Figs. 1A and B). C. cinereus fruiting body is ephemeral and autolyzes soon after the completion of the development. Thus, fruiting body development consists of a number of processes, including switching from mycelial growth to fruiting body development, cell differentiation and organization into tissues to form the fruiting body primordium, and meiosis, basidiospore formation and cellular morphogenesis
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Fig. 1. Photographs showing young and mature fruiting bodies of Coprinus cinereus. (A and B) Wild-type; (C and D) elongationless3; and (E) elongationless3 rescued by the wild-type eln3 gene. (A and C) Young fruiting bodies at 11th hour after the start of illumination on the day when fruiting body maturation occurs, a developmental phase just before the stipe elongating rapidly in the wild type; (B, D, and E) mature fruiting bodies. The brackets indicate the stipe portion that elongates during fruiting body maturation. Note that the stipe portion is enclosed by the cap before its rapid elongation. The bar represents 1 cm.
to form the mature fruiting body. A promising approach to molecular mechanisms for these developmental processes is isolation and characterization of mutations affecting the respective developmental processes. To date, a number of mutants defective in various processes of fruiting body development have been isolated (Chiu and Moore, 1990; Cummings et al., 1999; Gibbins and Lu, 1982; Kamada et al., 1984; Kanda and Ishikawa, 1986; Kanda et al., 1989; Kimura and Fujio, 1961; Muraguchi et al., 1999; Takemaru and Kamada, 1972; Zolan et al., 1988). Apart from genes involved in meiosis (see Kamada, 2002; K€ ues, 2000), however, only two genes, ich1 and eln2, have been characterized at the molecular level by analyses of such developmental mutations: ich1 was identified by analysis of ichijiku1 mutation that prevents differentiation of the cap during the development of the fruiting body primordium and its product is suggested to function in the nucleus (Muraguchi and Kamada, 1998). eln2 was identified by analysis of elongationless2 mutation that affects pattern formation during the development of the fruiting body primordium, which in turn interferes with differentiation of the stipe tissue, resulting in a much smaller stipe tissue as compared with other parts of the fruiting body (Muraguchi and Kamada, 2000). The eln2 gene is predicted to encode a microsomal cytochrome P450 enzyme. Thus, information about molecular mechanisms for fruiting body morphogenesis in C. cinereus is only fragmentary at present. In the present study, we identify and characterize elongationless3, a restriction enzyme-mediated integration (REMI) mutation, which affects stipe elongation during fruiting body maturation in C. cinereus (Fig. 1). In the mutant, the organization of the stipe tissue is disturbed and much space is produced between stipe cells, in contrast to the wild type, in which the stipe cells run parallel to the stipe axis and make side-by-side contact with one another. The eln3 gene responsible for the mutation is predicted to encode a protein with a
general glycosyltransferase and to be located in the plasma membrane.
2. Materials and methods 2.1. Strains, culture conditions, and genetic techniques Strains of C. cinereus used in the present study are listed in Table 1. Malt extract–yeast extract–glucose (MY) medium (Rao and Niederpruem, 1969) solidified with 2% (w/v) agar in 9-cm petri dishes was used for routine mycelial cultures and for fruiting. Slants of MY agar medium in test tubes were also used for fruiting. MY medium without agar in 9-cm petri dishes was used for mycelial cultures for extraction of DNA and RNA. The minimal medium was that of Shahriari and Casselton (1974) modified by Binninger et al. (1987). Cultures were maintained at 28 C under a 12-h light/12-h dark regime throughout this study. Crosses and isolation of basidiospore germlings were performed as described previously (Inada et al., 2001). 2.2. Microscopy The lengths of cylindrical stipe cells were measured using a micrometer under a light microscope equipped
Table 1 Coprinus cinereus strains used in this study Strain
Genotype/description
Source
5302 5401 326 292 198 198 F1 #36
A2B2 A1B1 AmutBmut pab1-1 A3B1 trp1-1, 1-6 AmutBmut pab1-1 eln3-1 AmutBmut pab1-1 eln3-1/ a progeny of 198 5401
This laboratory This laboratory P.J. Pukkila P.J. Pukkila This study This study
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with phase-contrast optics after the stipe tissue was squashed between a glass slide and a coverslip. For electron microscopy, tissues excised from the middle part of stipes were prefixed in 2.5% glutaraldehyde dissolved in 0.05 M sodium/potassium phosphate buffer (pH 6.95) at room temperature for 2 h. At the beginning of prefixation, samples were degassed for several minutes until they sank in the solution. After being washed in water, they were postfixed in a 2% KMnO4 aqueous solution at room temperature for 2 h, washed in water, and then soaked in 0.5% uranyl acetate for 2 h. Specimens were dehydrated in a graded ethanol series, and then acetone was substituted for ethanol before being embedded in Spurr resin. Ultrathin sections were obtained on a Leica ULTRACUT UCT. The sections were stained with a 3% uranyl acetate aqueous solution for 2 h and then with lead citrate (Reynolds, 1963) for 10 min before being examined in a Hitachi H7600 transmission electron microscope with an accelerating voltage at 100 kV. 2.3. Southern and Northern analysis DNA or RNA was transferred to Hybond-Nþ (Amersham, Arlington Heights, IL) according to Sambrook et al. (1989). The enhanced chemiluminescence (ECL) direct system (Amersham) and the Gene Images system (Amersham) were used for probe-labeling and detection for Southern and Northern analysis, respectively. For Northern analysis, about 15 lg of total RNA was fractionated by electrophoresis in 1.3% agarose formaldehyde gel, and the 50 RACE product 3#2, which covers 83% of the eln3 transcript, was used as the probe. 2.4. Plasmid rescue The genomic DNA from the mutant strain, 198, was digested with EcoRI, self-ligated, and then introduced into Escherichia coli DH10B cells (Invitrogen) by electroporation, as described by Makino and Kamada (2004). Plasmids were then isolated from 13 of the resulting ampicillin-resistant transformants. The 13 plasmids all displayed the same band pattern when double-digested with EcoRI and HindIII: one 4-kb HindIII–EcoRI band, which is considered to be derived from pPHT1, plus two bands (0.61-kb EcoRI–HindIII band and 0.58-kb HindIII–HindIII band), both of which are considered to be derived from the genomic DNA (see Fig. 4). One of the 13 plasmids, named pSL1, was used in later experiments. 2.5. Cosmid library construction and screening, transformation of C. cinereus, and sequencing A cosmid library of chromosome I from the wild-type strain, 5302, was constructed using vector LLC5200,
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which contains the C. cinereus trp1 gene as a selectable marker (Pukkila and Casselton, 1991), as described by Zolan et al. (1992). The library was composed of 960 clones (96 clones · 10 plates). To screen the library for the eln3 gene, groups of 12 clones were cultured on plates of LB/ampicillin solid medium, and subjected to miniprep. The pooled DNAs were digested with EcoRI, electrophoresed in 1% agarose gel, and subjected to Southern hybridization using the mixture of the 0.61-kb EcoRI–HindIII and 0.58-kb HindIII–HindIII genomic fragments (see above) as the probe. Sib selection was then performed to identify a positive, single clone. To examine whether a cosmid or plasmid carries a DNA fragment containing the eln3 gene, the cosmid or plasmid was mixed with pPAB2 carrying the pab1 gene at a 2:1 ratio (w/w), and introduced into the eln3-1 mutant strain, 198#36, as described by Binninger et al. (1987) with minor modifications. Pabþ transformants were isolated, cultured on minimal medium for 3 days to purify the transformed mycelium, and then transferred to MY slants to test for the fruiting body phenotype. Plasmid pPAB2 is the generous gift of U. K€ ues. To sequence the 8.2-kb XbaI fragment containing the whole length of the eln3 gene, we deleted the fragment from each end with the Nested Deletion Kit (Pharmacia Biotech) and sequenced the nested deletion products with a Model 373S DNA sequencer (Perkin–Elmer) using the PRISM Dye Primer Cycle Sequencing Kit (Perkin–Elmer). 2.6. 50 and 30 RACE experiments eln3 cDNA was amplified from an existing cDNA library (Muraguchi and Kamada, 1998) using a genespecific primer for 50 RACE, pSL3-5 (AACGGTACGA GGGCAGGGTTTTCATAG), and two gene-specific primers for 30 RACE, pSL5-3 (GATTTATGGGATGC TTGGCTACAC) and 3RACE-1 (GTTTCTTGGACA TTGGATTCTTGG). PCR was performed with the Advantage cDNA PCR Kit (Clontech) and PCR products were cloned into pGEM-T Easy vector (Promega) according to the manufacturer’s instruction. The genomic DNA sequence of eln3 and the deduced amino acid sequence data reported in this paper will appear in the DDJB/EMBL/GenBank databases with the Accession No. AB111462. 2.7. DNA and RNA isolations Cosmid and plasmid DNAs were isolated with the FlexiPrep Kit (Pharmacia Biotech). Genomic DNAs from C. cinereus were prepared as described by Zolan and Pukkila (1986). Total RNAs were prepared as described previously (Inada et al., 2001).
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2.8. Predictions of protein structure and location, and motif scan Protein structure of Eln3 and its location were predicted by using TMpred at ch.EMBnet.org (http:// www.ch.embnet.org/), PSORT II at PSORT (http:// psort.nibb.ac.jp/), TMHMM at CBS (http://www.cbs. dtu.dk/), and HMMTOP at Institute of Enzymology. Motifs in Eln3 were scanned by using PFSCAN at ch.EMBnet.org.
3. Results 3.1. Identification and phenotypic analysis of elongationless3 mutation We have previously isolated 3225 hygromycin-B-resistant transformants of the homokaryotic fruiting strain, 326 (AmutBmut), after REMI mutagenesis using plasmid pPHT1 (Inada et al., 2001). Plasmid pPHT1 carries the hygromycin-B-resistance gene as the selectable marker (Cummings et al., 1999). In this study we screened the 3225 transformants for strains defective in fruiting body morphogenesis and identified strain 198 producing an aberrant fruiting body with a short and bulgy stipe (Fig. 1). In the wild type, the stipe elongates remarkably during the final phase of fruiting body development (fruiting body maturation) (Figs. 1A and B), while in the mutant it hardly elongates (Figs. 1C and D). The development of the cap appeared normal in the mutant: the cap bore abundant basidiospores, expanded, and finally autolyzed (Fig. 1D). Vegetative mycelial growth was a little slower in the mutant than in the wild type (data not shown). The stipe of C. cinereus is composed of large, cylindrical cells, which are arranged parallel to the stipe axis and interspersed by thin vegetative-like hyphae (Hammad et al., 1993), and its elongation during fruiting body maturation is the result of longitudinal growth of the cylindrical stipe cells (Kamada and Takemaru, 1977). Therefore it was possible that the defect in stipe elongation was due to a defect in the elongation growth of the stipe cells. We tested this possibility by comparing the lengths of cylindrical, stipe cells between the wild type and the mutant. In the wild type, the lengths of the stipe cells before and after rapid stipe elongation were 137 41.2 lm (n ¼ 50) and 628 171 lm (n ¼ 50), respectively, whereas in the mutant they were 152 39.3 lm (n ¼ 60) and 312 95.5 lm (n ¼ 30). Thus, the increment in cell length was reduced by 50% in the mutant. However, this reduction in longitudinal growth only partially accounts for the reduction of stipe elongation in the mutant (see Fig. 1). This suggested another defect in the mutant, that is, the disorganization of stipe cells. To examine this hypothesis, stipes from the mutant as well
as from the wild type were fixed and embedded in resin, and their thin sections were observed under the electron microscope at a low magnification (Fig. 2). We found that in the wild type, cylindrical stipe cells run parallel to the stipe axis and make side-by-side contact with one another, whereas in the mutant, much space was produced between the cells and the parallel arrangement of the cells was disturbed. Based on these observations, we conclude that a combination of defects in cellular morphogenesis and in tissue organization in the stipe causes a serious defect in stipe elongation. In addition, vegetativelike hyphae with electron-dense cytoplasm occurred more frequently in the mutant than in the wild type, suggesting the possibility that the mutation also affects the differentiation of vegetative cells into cylindrical cells. There appears not to be much reduction in the overall cell number in the mutant stipe, as deduced from the size and shape of young fruiting bodies before rapid stipe elongation (see Figs. 1A and C). Also, no clear difference in cell-wall construction was recognizable by electron microscopy, although the mutant walls appeared to be less even at the edges than the wild-type walls (Fig. 3). 3.2. Genetic analysis of elongationless3 mutation To examine whether the eln3-1 mutation is the direct result of insertion of plasmid pPHT1, we crossed the mutant strain, 198 (AmutBmut pab1-1 eln3-1), to the wild-type homokaryon, 5401 (A1B1 + +), and examined its F1 progeny for segregation of the fruiting body phenotype and the hygromycin B resistance. Because the A and B mating-type loci are located on different chromosomes and segregate independently from each other, one fourth of the progeny were expected to carry AmutBmut and hence be capable to produce fruiting bodies. Of 60 progeny examined, 13 strains produced mature fruiting bodies. Of the 13 strains, 12 were resistant to hygromycin B and produced mutant fruiting bodies with short stipes, and the remaining strain was hygromycin B-sensitive and developed wild-type fruiting bodies. This result suggested that the eln3-1 mutation is the direct result of insertion of the plasmid, and also that the eln3 gene is linked to either the A or B locus. 3.3. Cloning of the eln3 gene On the basis of the above result suggesting that the gene responsible for the eln3-1 mutation is tagged by the plasmid, we performed a plasmid rescue and cloned a genomic DNA fragment [1.19-kb EcoRI–HindIII (0.61kb EcoRI–HindIII + 0.58-kb HindIII–HindIII)] adjacent to pPHT1 in the mutant genome (see Fig. 4). On the assumption that the eln3 gene may be linked to the A locus, we then screened a cosmid library from chromosome I, on which the A locus is located (Pukkila, 1993), by Southern hybridization using the mixture of the
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Fig. 2. Electron micrographs showing longitudinal sections of the stipe tissue in the wild type (A) and elongationless3 (B). The samples were taken at 15th hour after the start of illumination on the day of fruiting body maturation, the developmental phase when the stipe elongates most rapidly in the wild type. The bar represents 30 lm.
Fig. 4. Physical map of the eln3 gene inserted by plasmid pPHT1, and the eln3 ORF. Exons are shown as black boxes and introns as white boxes. H, HindIII; E, EcoRI; X, XbaI; and Hyg, hygromycin-B-resistance gene.
Fig. 3. Electron micrographs showing the walls of stipe cells in the wild type (A) and elongationless3 (B). The samples were taken at 15th hour after the start of illumination on the day of fruiting body maturation. *, intercellular space. The bar represents 1 lm.
0.61-kb and 0.58-kb fragments as the probe, and identified a positive cosmid clone, 5F2. To examine whether clone 5F2 has the eln3 activity or not, we co-transformed 198F1 #36 (AmutBmut pab1-1
eln3-1) by cosmid 5F2 and plasmid pPAB2 carrying the wild-type pab1 gene. The eln3-1 mutation was rescued in 22% (10/45) of pabþ transformants (Fig. 1E). When strain 198 F1 #36 was transformed only with pPAB2, as a control experiment, the mutation was not rescued in any of 38 pabþ transformants examined. These results indicate that clone 5F2 carries the whole eln3 gene. To define the active region in cosmid clone 5F2, we digested the clone with various restriction enzymes and examined each digest for the rescuing activity by co-transformation with plasmid pPAB2. We found that XbaI did not destroy the activity. Southern analysis of the XbaI digest using the mixture of the 0.61- and 0.58-kb genomic fragments as the probe indicated that a 8.2-kb XbaI fragment contains the eln3 gene.
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To confirm that the 8.2-kb XbaI fragment contains the whole eln3 gene, we inserted the fragment into pGEM-7zf+ (Promega), mixed the resulting plasmid (pXba9#5) with pPAB2, linearized with KpnI, and then introduced the mixture into strain 198F1 #36. The mutation was rescued in 26% (26/99) of pabþ transformants. With circular plasmids, the rate of rescue was as low as 3% (4/123). These results demonstrate that the 8.2-kb XbaI fragment contains the whole eln3 gene. 3.4. eln3 encodes a putative membrane protein with a general glycosyltransferase domain Sequencing of the 8.2-kb XbaI genomic fragment, together with 30 - and 50 -RACE experiments, identified an ORF interrupted by 16 introns, which is predicted to encode a protein of 927 amino acids (Fig. 5). The 50 and 30 splice sites agree with the consensus sequences GTRNGT and YAG found for filamentous fungi, respectively (Gurr et al., 1987), except that the ninth and 15th 50 splice sites are GTAAGA and GTGAGG, respectively, and the seventh 30 splice site is AAG. The size of the introns ranges between 54 and 582 bp. The eln3 mRNA is predicted to have a 260-nt 50 - and an 846-nt 30 -untranslated region. The promoter region of eln3 contains a CAAT-box-like sequence (CAAT) and a TATA-box-like sequence (TATAAA), which are located at 123–120 and 118–113 bp upstream of the predicted transcriptional start site, respectively. Hydropathy analyses with the programs, TMpred, TMHMM, HMMTOP, and PSORT II, all predict that Eln3 is a membrane protein with seven transmembrane helices (Fig. 5). TMpred and HMMTOP predict that the N-terminus is outside, while PSORT II and TMHMM predict that it is inside. Also, PSORT II predicts that Eln3 is likely located in the plasma membrane (47.8%, plasma membrane; 26.1%, endoplasmic reticulum;
13.0%, mitochondrial; 4.3%, Golgi; 4.3%, vacuolar; and 4.3%, vesicles of secretory system). Motif Scan analysis using the program, PFSCAN, revealed that Eln3 has a general glycosyltransferase domain at amino acid residues 386–563 (rawscore ¼ 379, N score ¼ 10.188, E value ¼ 0.0014). Database searches using the BLAST procedure (Altschul et al., 1997) revealed that Eln3 has high similarities to Neurospora crassa hypothetical protein, CAC28725.2 (Expect ¼ e-145, Identities ¼ 35%), N. crassa hypothetical protein, CAD36996.1 (Expect ¼ e-137, Identities ¼ 43%), and Cryptococcus neoformans var. neoformans NCP1 protein (Expect ¼ 6e-76, Identities ¼ 32%). NCP1 is present within the mating-type locus of the serotype D MAT a strain JEC20 of C. neoformans var. neoformans (Lengeler et al., 2002). 3.5. eln3-1 mutant allele Sequencing of plasmid pSL1 indicated that plasmid pPHT1 linearized with HindIII is inserted in the HindIII site in the fourth exon, which is located 1089–1094 bp downstream of the predicted translational start site. This insertion creates a stop codon at 21–23 bp downstream of the HindIII site, which in turn truncates most of the Eln3 peptide, including the seven transmembrane domains and the general glycosyltransferase domain. 3.6. Developmental regulation of eln3 expression Using the 30 -RACE product, 30 #2, which covers 83% of the eln3 transcript, as a probe, we examined total RNAs from the vegetative mycelium of the wild-type homokaryon, 5401, and the vegetative mycelium of the wild-type dikaryon, 5401 5302, and fruiting body primordia and various tissues of fruiting bodies at various developmental phases from the dikaryon (Fig. 6).
Fig. 5. Predicted amino acid sequence of Eln3 (A) and its hydropathy plot (B). Putative transmembrane domains are underlined and a putative general glycosyltransferase domain is double-underlined.
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Fig. 6. Expression of the eln3 gene. For each lane, 15 lg of total RNAs from various mycelia and fruiting body tissues were electrophoresed: lane 1, wild-type homokaryon 5401; lane 2, wild-type dikaryon 5401 5302; lane 3, fruiting body primordia from the dikaryon on one day before fruiting body maturation; lane 4, whole fruiting bodies from the dikaryon at 11 h after the start of illumination on the day of fruiting body maturation; lane 5, stipes at the 11-h stage; and lane 6, caps at the 11-h stage. Lanes 7–10 are stipes at 6, 11, 18, and 22 h after the start of the illumination. The rate of stipe elongation changes during fruiting body maturation: most rapid elongation occurs between 11 and 18 h and ends by 22-h stage after the start of the illumination. After the ribosomal RNA was visualized under UV, the gels were blotted and hybridized.
As expected from the eln3 cDNA analysis, we identified a transcript at 3.9 kb. We also found that the transcription of eln3 was specifically activated in the stipe, especially in rapidly elongating ones.
4. Discussion In this study we identified and characterized elongationless3, a REMI mutation that affects stipe elongation during fruiting body maturation, the final phase of fruiting body development, in C. cinereus. The mutant strain was apparently normal in vegetative mycelial growth as well as in fruiting body phenotypes other than stipe elongation, including the development of the cap and the formation of basidiospores. These observations indicate that elongationless3 is defective in a function that is specific of stipe development. This interpretation is consistent with the fact that the transcription of the eln3 gene responsible for the mutation is specifically activated in rapidly elongating stipes and is very low in fruiting body tissues other than the stipe as well as in vegetatively growing, homokaryotic and dikaryotic mycelia. Stipe elongation in the C. cinereus fruiting body is the result of elongation growth of cylindrical stipe cells, which run parallel to the stipe axis (Kamada, 1994; Kamada and Takemaru, 1977). Light microscopy showed that elongation growth of the stipe cells is
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reduced by 50% in the mutant, as compared with that in the wild type. However, this reduction in cell elongation only partially accounts for the fact that the mutant stipe hardly elongates during fruiting body maturation. This prompted us to examine the organization of the stipe tissue. In the wild-type stipe, cylindrical stipe cells make side-by-side contact with one another and run parallel to the stipe axis, and hence the elongation of the stipe cells directly results in the elongation of the stipe. In the mutant, however, much space is produced between the stipe cells and the parallel arrangement of the cells is disturbed, and hence the elongation growth of the stipe cells does not lead to stipe elongation but makes the stipe bulgy. The disorganization of the stipe tissue forming much space between stipe cells suggests that the mutant has a defect in the mechanism for cell-to-cell connection in the stipe. This hypothesis is supported by the fact that the mutant stipe was softer and more fragile than the wildtype stipe when taken between our fingers. It has been reported in C. cinereus that two galectins (Cgl1 and Cgl2), b-galactoside binding lectins, are produced during mushroom development (Boulianne et al., 2000). Although galectins are present throughout the fruiting body, the highest level of their expression is within a group of cells that form the outer portion of the stipe. On the basis of this fact, together with the reasonable hypothesis that a strong tension would be applied on the outer portion of the stipe and hence the stipe tissue would require an increased connectivity of the cells forming them, Boulianne et al. (2000) speculate that galectins are involved in cell-to-cell interaction, mediated by their ability to bind specific carbohydrates of the cell wall. Because Eln3 is predicted to be a protein with a general glycosyltransferase domain and to be located in the plasma membrane, it is a tempting hypothesis that Eln3 is involved in cell-to-cell connection by producing a cell-wall carbohydrate that binds to galectins. Because various extracellular matrices (ECMs) exist in fruiting bodies of homobasidiomycetes (see Walser et al., 2003 for review), it is also possible that Eln3 adds a carbohydrate moiety to one of ECMs and modulates its function. A future challenge would be identification of the presumptive carbohydrate produced by Eln3 through analysis of cell walls and/or ECMs in the stipe from elongationless3 mutant as well as from the wild type. Database searches revealed that Eln3 shows high similarities to two N. crassa hypothetical proteins and the C. neoformans var. neoformans NCP1 protein. This suggests that Eln3 belongs to a protein family conserved among basidiomyceteous and ascomyceteous fungi. Examination of developmental regulation of the expression of the orthologs in N. crassa and C. neoformans would be instrumental in identifying the Eln3 function.
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Acknowledgments We thank Professor Ursula K€ ues for the gift of plasmid pPAB2. This work was supported in part by a Grant-in-Aid for JSPS fellows (to T.A.). References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Boulianne, R.P., Liu, Y., Aebi, M., Lu, B.C., K€ ues, U., 2000. Fruiting body development in Coprinus cinereus: regulated expression of two galectins secreted by a non-classical pathway. Microbiology 146, 1841–1853. Binninger, D.M., Skrzynia, C., Pukkila, P.J., Casselton, L.A., 1987. DNA-mediated transformation of the basidiomycete Coprinus cinereus. EMBO J. 6, 835–840. Chiu, S.W., Moore, D., 1990. A mechanism for gill pattern formation in Coprinus cinereus. Mycol. Res. 94, 320–326. Cummings, W.J., Celerin, M., Crodian, J., Brunick, L.K., Zolan, M.E., 1999. Insertional mutagenesis in Coprinus cinereus: use of a dominant selectable marker to generate tagged, sporulation-defective mutants. Curr. Genet. 36, 371–382. Gibbins, A.M.V., Lu, B.C., 1982. An ameiotic mutant of Coprinus cinereus halted prior to pre-meiotic S-phase. Curr. Genet. 5, 119– 126. Gurr, S.J., Unkles, S.E., Kinghorn, J.R., 1987. The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn, J.R. (Ed.), Gene Structure in Eukaryotic Microbes. IRL Press, London, pp. 93–139. Hammad, F., Watling, R., Moore, D., 1993. Cell population dynamics in Coprinus cinereus: narrow and inflated hyphae in the basidiome stipe. Mycol. Res. 97, 275–282. Inada, K., Morimoto, Y., Arima, T., Murata, Y., Kamada, T., 2001. The clp1 gene of the mushroom Coprinus cinereus is essential for Aregulated sexual development. Genetics 157, 133–140. Kamada, T., 1994. Stipe elongation in fruit bodies. In: Wessels, J.G.H., Meinhardt, F. (Eds.), The Mycota, Vol. I. Growth, Differentiation and Sexuality. Springer-Verlag, Berlin, Heidelberg, pp. 367–379. Kamada, T., 2002. Molecular genetics of sexual development in the mushroom Coprinus cinereus. BioEssays 24, 449–459. Kamada, T., Takemaru, T., 1977. Stipe elongation during basidiocarp maturation in Coprinus macrorhizus: mechanical properties of stipe cell wall. Plant Cell Physiol. 18, 831–840. Kamada, T., Katsuda, H., Takemaru, T., 1984. Temperature sensitive mutants of Coprinus cinereus defective in hyphal growth and stipe elongation. Curr. Microbiol. 11, 309–312. Kanda, T., Ishikawa, T., 1986. Isolation of recessive developmental mutants in Coprinus cinereus. J. Gen. Appl. Microbiol. 32, 541– 543. Kanda, T., Goto, A., Sawa, K., Arakawa, H., Yasuda, Y., Takemaru, T., 1989. Isolation and characterization of recessive sporeless mutants in the basidiomycete Coprinus cinereus. Mol. Gen. Genet. 216, 526–529. Kimura, K., Fujio, M., 1961. Studies on abnormal fruit-bodies of the hymenomycetous fungi. I. Undeveloped fruit-bodies of Coprinus macrorhizus f. microsporus. Rep. Tottori Mycol. Inst. 1, 19–28.
K€ ues, U., 2000. Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiol. Mol. Biol. Rev. 64, 316–353. Lengeler, K.B., Fox, D.S., Fraser, J.A., Allen, A., Forrester, K., Dietrich, F.S., Heitman, J., 2002. Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes. Eukaryot. Cell 1, 704–718. Makino, R., Kamada, T., 2004. Isolation and characterization of mutations that affect nuclear migration for dikaryosis in Coprinus cinereus. Curr. Genet. 45, 149–156. Moore, D., 1981. Developmental genetics of Coprinus cinereus: genetic evidence that carpophores and sclerotia share a common pathway of initiation. Curr. Genet. 3, 145–150. Moore, D., 1996. Inside the developing mushroom—cells, tissues and tissue patterns. In: Chiu, S.W., Moore, D. (Eds.), Patterns in Fungal Development. Cambridge University Press, Cambridge, UK, pp. 1–36. Muraguchi, H., Kamada, T., 1998. The ich1 gene of the mushroom Coprinus cinereus is essential for pileus formation in fruiting. Development 125, 3133–3141. Muraguchi, H., Kamada, T., 2000. A mutation in the eln2 gene encoding a cytochrome P450 of Coprinus cinereus affects mushroom morphogenesis. Fungal Genet. Biol. 29, 49–59. Muraguchi, H., Takemaru, T., Kamada, T., 1999. Isolation and characterization of developmental variants in fruiting using a homokaryotic fruiting strain of Coprinus cinereus. Mycoscience 40, 227–233. Pukkila, P.J., 1993. Methods of genetic manipulation in Coprinus cinereus. In: Chang, S.T., Buswell, J.A., Miles, P.G. (Eds.), Culture Collection and Breeding of Edible Mushrooms. Gordon & Breach, Philadelphia, pp. 249–264. Pukkila, P.J., Casselton, L.A., 1991. Molecular genetics of the agaric Coprinus cinereus. In: Bennet, J.W., Lasure, L.L. (Eds.), More Gene Manipulations in Fungi. Academic Press, San Diego, pp. 126–150. Rao, P.S., Niederpruem, D.J., 1969. Carbohydrate metabolism during morphogenesis of Coprinus lagopus (sensu Buller). J. Bacteriol. 100, 1222–1228. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 19, 208– 212. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Shahriari, H., Casselton, L.A., 1974. Suppression of methionine mutants in Coprinus. I. Complementation and allele specificity as criteria of suppressor gene action. Mol. Gen. Genet. 134, 85–92. Takemaru, T., Kamada, T., 1972. Basidiocarp development in Coprinus macrorhizus. I. Induction of developmental variations. Bot. Mag. Tokyo 85, 51–57. Walser, P.J., Velagapudi, R., Aebi, M., K€ ues, U., 2003. Extracellular matrix proteins in mushroom development. Recent Res. Dev. Microbiol. 7, 381–415. Zolan, M.E., Pukkila, P.J., 1986. Inheritance of DNA methylation in Coprinus cinereus. Mol. Cell. Biol. 6, 195–200. Zolan, M.E., Crittenden, J.R., Heyler, N.K., Seitz, L.C., 1992. Efficient isolation and mapping of rad genes of the fungus Coprinus cinereus using chromosome-specific libraries. Nucleic Acids Res. 20, 3993–3999. Zolan, M.E., Tremel, C.J., Pukkila, P.J., 1988. Production and characterization of radiation-sensitive meiotic mutants of Coprinus cinereus. Genetics 120, 379–387.
The eln3 gene involved in fruiting body morphogenesis of Coprinus cinereus encodes a putative membrane protein with a general glycosyltransferase domain Toshihide Arima,a,b,1 Maki Yamamoto,c Aiko Hirata,c Shigeyuki Kawano,c and Takashi Kamadab,* a Research Fellow of the Japanese Society for the Promotion of Science (JSPS), Japan Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, Japan Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan b
c
Received 10 February 2004; accepted 19 April 2004 Available online 18 May 2004
Abstract We identified and characterized elongationless3 (eln3-1), a restriction enzyme-mediated integration (REMI) mutation affecting fruiting body morphogenesis in Coprinus cinereus. The mutant produces an aberrant fruiting body in which the stipe hardly elongates during fruiting body maturation. In the wild type, cylindrical stipe cells, elongation growth of which is responsible for stipe elongation, make side-by-side contact with one another and run parallel to the stipe axis, whereas in the mutant, the organization of the stipe tissue is disturbed and much space is produced between stipe cells. This disorganization of the stipe tissue, together with reduced elongation of the stipe cells, causes the mutant stipe short and bulgy. After a plasmid rescue, the eln3 gene was identified as a DNA fragment that complements the eln3-1 mutation. The eln3 ORF is predicted to encode a protein of 927 amino acids with a general glycosyltransferase domain and to be located in the plasma membrane. Transcription of the eln3 gene is specifically activated in rapidly elongating stipes. Possible involvement of the putative Eln3 enzyme in cell-to-cell connection is discussed. 2004 Elsevier Inc. All rights reserved. Index Descriptors: Homobasidiomycete; Coprinus cinereus; Fruiting body morphogenesis; REMI mutant; Stipe elongation; Cell-tocell connection; Membrane protein; Glycosyltransferase
1. Introduction The homobasidiomycete Coprinus cinereus develops a multicellular structure, the fruiting body, under proper environmental conditions. Fruiting body development of C. cinereus is rapid and predictable in the laboratory, providing an excellent opportunity for studies of fungal multicellular morphogenesis (Kamada, 2002; K€ ues, 2000; Moore, 1981, 1996; Muraguchi and Kamada, 1998). The development starts with the formation of hyphal knots, aggregations of hyphae of less than * Corresponding author. Fax: +81-86-251-7876. E-mail address: [email protected] (T. Kamada). 1 Present address: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan.
1087-1845/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2004.04.003
0.2 mm in diameter that lack recognizable tissue differentiation. Some of hyphal knots develop into fruiting body primordia with differentiation of the rudimentary cap and stipe. In the final phase of development (fruiting body maturation), basidia on the undersurface of the cap undergo meiosis, which is immediately followed by basidiospore formation, and stipe elongation and cap expansion occur for efficient spore dispersal to form a mature fruiting body (Figs. 1A and B). C. cinereus fruiting body is ephemeral and autolyzes soon after the completion of the development. Thus, fruiting body development consists of a number of processes, including switching from mycelial growth to fruiting body development, cell differentiation and organization into tissues to form the fruiting body primordium, and meiosis, basidiospore formation and cellular morphogenesis
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Fig. 1. Photographs showing young and mature fruiting bodies of Coprinus cinereus. (A and B) Wild-type; (C and D) elongationless3; and (E) elongationless3 rescued by the wild-type eln3 gene. (A and C) Young fruiting bodies at 11th hour after the start of illumination on the day when fruiting body maturation occurs, a developmental phase just before the stipe elongating rapidly in the wild type; (B, D, and E) mature fruiting bodies. The brackets indicate the stipe portion that elongates during fruiting body maturation. Note that the stipe portion is enclosed by the cap before its rapid elongation. The bar represents 1 cm.
to form the mature fruiting body. A promising approach to molecular mechanisms for these developmental processes is isolation and characterization of mutations affecting the respective developmental processes. To date, a number of mutants defective in various processes of fruiting body development have been isolated (Chiu and Moore, 1990; Cummings et al., 1999; Gibbins and Lu, 1982; Kamada et al., 1984; Kanda and Ishikawa, 1986; Kanda et al., 1989; Kimura and Fujio, 1961; Muraguchi et al., 1999; Takemaru and Kamada, 1972; Zolan et al., 1988). Apart from genes involved in meiosis (see Kamada, 2002; K€ ues, 2000), however, only two genes, ich1 and eln2, have been characterized at the molecular level by analyses of such developmental mutations: ich1 was identified by analysis of ichijiku1 mutation that prevents differentiation of the cap during the development of the fruiting body primordium and its product is suggested to function in the nucleus (Muraguchi and Kamada, 1998). eln2 was identified by analysis of elongationless2 mutation that affects pattern formation during the development of the fruiting body primordium, which in turn interferes with differentiation of the stipe tissue, resulting in a much smaller stipe tissue as compared with other parts of the fruiting body (Muraguchi and Kamada, 2000). The eln2 gene is predicted to encode a microsomal cytochrome P450 enzyme. Thus, information about molecular mechanisms for fruiting body morphogenesis in C. cinereus is only fragmentary at present. In the present study, we identify and characterize elongationless3, a restriction enzyme-mediated integration (REMI) mutation, which affects stipe elongation during fruiting body maturation in C. cinereus (Fig. 1). In the mutant, the organization of the stipe tissue is disturbed and much space is produced between stipe cells, in contrast to the wild type, in which the stipe cells run parallel to the stipe axis and make side-by-side contact with one another. The eln3 gene responsible for the mutation is predicted to encode a protein with a
general glycosyltransferase and to be located in the plasma membrane.
2. Materials and methods 2.1. Strains, culture conditions, and genetic techniques Strains of C. cinereus used in the present study are listed in Table 1. Malt extract–yeast extract–glucose (MY) medium (Rao and Niederpruem, 1969) solidified with 2% (w/v) agar in 9-cm petri dishes was used for routine mycelial cultures and for fruiting. Slants of MY agar medium in test tubes were also used for fruiting. MY medium without agar in 9-cm petri dishes was used for mycelial cultures for extraction of DNA and RNA. The minimal medium was that of Shahriari and Casselton (1974) modified by Binninger et al. (1987). Cultures were maintained at 28 C under a 12-h light/12-h dark regime throughout this study. Crosses and isolation of basidiospore germlings were performed as described previously (Inada et al., 2001). 2.2. Microscopy The lengths of cylindrical stipe cells were measured using a micrometer under a light microscope equipped
Table 1 Coprinus cinereus strains used in this study Strain
Genotype/description
Source
5302 5401 326 292 198 198 F1 #36
A2B2 A1B1 AmutBmut pab1-1 A3B1 trp1-1, 1-6 AmutBmut pab1-1 eln3-1 AmutBmut pab1-1 eln3-1/ a progeny of 198 5401
This laboratory This laboratory P.J. Pukkila P.J. Pukkila This study This study
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with phase-contrast optics after the stipe tissue was squashed between a glass slide and a coverslip. For electron microscopy, tissues excised from the middle part of stipes were prefixed in 2.5% glutaraldehyde dissolved in 0.05 M sodium/potassium phosphate buffer (pH 6.95) at room temperature for 2 h. At the beginning of prefixation, samples were degassed for several minutes until they sank in the solution. After being washed in water, they were postfixed in a 2% KMnO4 aqueous solution at room temperature for 2 h, washed in water, and then soaked in 0.5% uranyl acetate for 2 h. Specimens were dehydrated in a graded ethanol series, and then acetone was substituted for ethanol before being embedded in Spurr resin. Ultrathin sections were obtained on a Leica ULTRACUT UCT. The sections were stained with a 3% uranyl acetate aqueous solution for 2 h and then with lead citrate (Reynolds, 1963) for 10 min before being examined in a Hitachi H7600 transmission electron microscope with an accelerating voltage at 100 kV. 2.3. Southern and Northern analysis DNA or RNA was transferred to Hybond-Nþ (Amersham, Arlington Heights, IL) according to Sambrook et al. (1989). The enhanced chemiluminescence (ECL) direct system (Amersham) and the Gene Images system (Amersham) were used for probe-labeling and detection for Southern and Northern analysis, respectively. For Northern analysis, about 15 lg of total RNA was fractionated by electrophoresis in 1.3% agarose formaldehyde gel, and the 50 RACE product 3#2, which covers 83% of the eln3 transcript, was used as the probe. 2.4. Plasmid rescue The genomic DNA from the mutant strain, 198, was digested with EcoRI, self-ligated, and then introduced into Escherichia coli DH10B cells (Invitrogen) by electroporation, as described by Makino and Kamada (2004). Plasmids were then isolated from 13 of the resulting ampicillin-resistant transformants. The 13 plasmids all displayed the same band pattern when double-digested with EcoRI and HindIII: one 4-kb HindIII–EcoRI band, which is considered to be derived from pPHT1, plus two bands (0.61-kb EcoRI–HindIII band and 0.58-kb HindIII–HindIII band), both of which are considered to be derived from the genomic DNA (see Fig. 4). One of the 13 plasmids, named pSL1, was used in later experiments. 2.5. Cosmid library construction and screening, transformation of C. cinereus, and sequencing A cosmid library of chromosome I from the wild-type strain, 5302, was constructed using vector LLC5200,
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which contains the C. cinereus trp1 gene as a selectable marker (Pukkila and Casselton, 1991), as described by Zolan et al. (1992). The library was composed of 960 clones (96 clones · 10 plates). To screen the library for the eln3 gene, groups of 12 clones were cultured on plates of LB/ampicillin solid medium, and subjected to miniprep. The pooled DNAs were digested with EcoRI, electrophoresed in 1% agarose gel, and subjected to Southern hybridization using the mixture of the 0.61-kb EcoRI–HindIII and 0.58-kb HindIII–HindIII genomic fragments (see above) as the probe. Sib selection was then performed to identify a positive, single clone. To examine whether a cosmid or plasmid carries a DNA fragment containing the eln3 gene, the cosmid or plasmid was mixed with pPAB2 carrying the pab1 gene at a 2:1 ratio (w/w), and introduced into the eln3-1 mutant strain, 198#36, as described by Binninger et al. (1987) with minor modifications. Pabþ transformants were isolated, cultured on minimal medium for 3 days to purify the transformed mycelium, and then transferred to MY slants to test for the fruiting body phenotype. Plasmid pPAB2 is the generous gift of U. K€ ues. To sequence the 8.2-kb XbaI fragment containing the whole length of the eln3 gene, we deleted the fragment from each end with the Nested Deletion Kit (Pharmacia Biotech) and sequenced the nested deletion products with a Model 373S DNA sequencer (Perkin–Elmer) using the PRISM Dye Primer Cycle Sequencing Kit (Perkin–Elmer). 2.6. 50 and 30 RACE experiments eln3 cDNA was amplified from an existing cDNA library (Muraguchi and Kamada, 1998) using a genespecific primer for 50 RACE, pSL3-5 (AACGGTACGA GGGCAGGGTTTTCATAG), and two gene-specific primers for 30 RACE, pSL5-3 (GATTTATGGGATGC TTGGCTACAC) and 3RACE-1 (GTTTCTTGGACA TTGGATTCTTGG). PCR was performed with the Advantage cDNA PCR Kit (Clontech) and PCR products were cloned into pGEM-T Easy vector (Promega) according to the manufacturer’s instruction. The genomic DNA sequence of eln3 and the deduced amino acid sequence data reported in this paper will appear in the DDJB/EMBL/GenBank databases with the Accession No. AB111462. 2.7. DNA and RNA isolations Cosmid and plasmid DNAs were isolated with the FlexiPrep Kit (Pharmacia Biotech). Genomic DNAs from C. cinereus were prepared as described by Zolan and Pukkila (1986). Total RNAs were prepared as described previously (Inada et al., 2001).
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2.8. Predictions of protein structure and location, and motif scan Protein structure of Eln3 and its location were predicted by using TMpred at ch.EMBnet.org (http:// www.ch.embnet.org/), PSORT II at PSORT (http:// psort.nibb.ac.jp/), TMHMM at CBS (http://www.cbs. dtu.dk/), and HMMTOP at Institute of Enzymology. Motifs in Eln3 were scanned by using PFSCAN at ch.EMBnet.org.
3. Results 3.1. Identification and phenotypic analysis of elongationless3 mutation We have previously isolated 3225 hygromycin-B-resistant transformants of the homokaryotic fruiting strain, 326 (AmutBmut), after REMI mutagenesis using plasmid pPHT1 (Inada et al., 2001). Plasmid pPHT1 carries the hygromycin-B-resistance gene as the selectable marker (Cummings et al., 1999). In this study we screened the 3225 transformants for strains defective in fruiting body morphogenesis and identified strain 198 producing an aberrant fruiting body with a short and bulgy stipe (Fig. 1). In the wild type, the stipe elongates remarkably during the final phase of fruiting body development (fruiting body maturation) (Figs. 1A and B), while in the mutant it hardly elongates (Figs. 1C and D). The development of the cap appeared normal in the mutant: the cap bore abundant basidiospores, expanded, and finally autolyzed (Fig. 1D). Vegetative mycelial growth was a little slower in the mutant than in the wild type (data not shown). The stipe of C. cinereus is composed of large, cylindrical cells, which are arranged parallel to the stipe axis and interspersed by thin vegetative-like hyphae (Hammad et al., 1993), and its elongation during fruiting body maturation is the result of longitudinal growth of the cylindrical stipe cells (Kamada and Takemaru, 1977). Therefore it was possible that the defect in stipe elongation was due to a defect in the elongation growth of the stipe cells. We tested this possibility by comparing the lengths of cylindrical, stipe cells between the wild type and the mutant. In the wild type, the lengths of the stipe cells before and after rapid stipe elongation were 137 41.2 lm (n ¼ 50) and 628 171 lm (n ¼ 50), respectively, whereas in the mutant they were 152 39.3 lm (n ¼ 60) and 312 95.5 lm (n ¼ 30). Thus, the increment in cell length was reduced by 50% in the mutant. However, this reduction in longitudinal growth only partially accounts for the reduction of stipe elongation in the mutant (see Fig. 1). This suggested another defect in the mutant, that is, the disorganization of stipe cells. To examine this hypothesis, stipes from the mutant as well
as from the wild type were fixed and embedded in resin, and their thin sections were observed under the electron microscope at a low magnification (Fig. 2). We found that in the wild type, cylindrical stipe cells run parallel to the stipe axis and make side-by-side contact with one another, whereas in the mutant, much space was produced between the cells and the parallel arrangement of the cells was disturbed. Based on these observations, we conclude that a combination of defects in cellular morphogenesis and in tissue organization in the stipe causes a serious defect in stipe elongation. In addition, vegetativelike hyphae with electron-dense cytoplasm occurred more frequently in the mutant than in the wild type, suggesting the possibility that the mutation also affects the differentiation of vegetative cells into cylindrical cells. There appears not to be much reduction in the overall cell number in the mutant stipe, as deduced from the size and shape of young fruiting bodies before rapid stipe elongation (see Figs. 1A and C). Also, no clear difference in cell-wall construction was recognizable by electron microscopy, although the mutant walls appeared to be less even at the edges than the wild-type walls (Fig. 3). 3.2. Genetic analysis of elongationless3 mutation To examine whether the eln3-1 mutation is the direct result of insertion of plasmid pPHT1, we crossed the mutant strain, 198 (AmutBmut pab1-1 eln3-1), to the wild-type homokaryon, 5401 (A1B1 + +), and examined its F1 progeny for segregation of the fruiting body phenotype and the hygromycin B resistance. Because the A and B mating-type loci are located on different chromosomes and segregate independently from each other, one fourth of the progeny were expected to carry AmutBmut and hence be capable to produce fruiting bodies. Of 60 progeny examined, 13 strains produced mature fruiting bodies. Of the 13 strains, 12 were resistant to hygromycin B and produced mutant fruiting bodies with short stipes, and the remaining strain was hygromycin B-sensitive and developed wild-type fruiting bodies. This result suggested that the eln3-1 mutation is the direct result of insertion of the plasmid, and also that the eln3 gene is linked to either the A or B locus. 3.3. Cloning of the eln3 gene On the basis of the above result suggesting that the gene responsible for the eln3-1 mutation is tagged by the plasmid, we performed a plasmid rescue and cloned a genomic DNA fragment [1.19-kb EcoRI–HindIII (0.61kb EcoRI–HindIII + 0.58-kb HindIII–HindIII)] adjacent to pPHT1 in the mutant genome (see Fig. 4). On the assumption that the eln3 gene may be linked to the A locus, we then screened a cosmid library from chromosome I, on which the A locus is located (Pukkila, 1993), by Southern hybridization using the mixture of the
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Fig. 2. Electron micrographs showing longitudinal sections of the stipe tissue in the wild type (A) and elongationless3 (B). The samples were taken at 15th hour after the start of illumination on the day of fruiting body maturation, the developmental phase when the stipe elongates most rapidly in the wild type. The bar represents 30 lm.
Fig. 4. Physical map of the eln3 gene inserted by plasmid pPHT1, and the eln3 ORF. Exons are shown as black boxes and introns as white boxes. H, HindIII; E, EcoRI; X, XbaI; and Hyg, hygromycin-B-resistance gene.
Fig. 3. Electron micrographs showing the walls of stipe cells in the wild type (A) and elongationless3 (B). The samples were taken at 15th hour after the start of illumination on the day of fruiting body maturation. *, intercellular space. The bar represents 1 lm.
0.61-kb and 0.58-kb fragments as the probe, and identified a positive cosmid clone, 5F2. To examine whether clone 5F2 has the eln3 activity or not, we co-transformed 198F1 #36 (AmutBmut pab1-1
eln3-1) by cosmid 5F2 and plasmid pPAB2 carrying the wild-type pab1 gene. The eln3-1 mutation was rescued in 22% (10/45) of pabþ transformants (Fig. 1E). When strain 198 F1 #36 was transformed only with pPAB2, as a control experiment, the mutation was not rescued in any of 38 pabþ transformants examined. These results indicate that clone 5F2 carries the whole eln3 gene. To define the active region in cosmid clone 5F2, we digested the clone with various restriction enzymes and examined each digest for the rescuing activity by co-transformation with plasmid pPAB2. We found that XbaI did not destroy the activity. Southern analysis of the XbaI digest using the mixture of the 0.61- and 0.58-kb genomic fragments as the probe indicated that a 8.2-kb XbaI fragment contains the eln3 gene.
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To confirm that the 8.2-kb XbaI fragment contains the whole eln3 gene, we inserted the fragment into pGEM-7zf+ (Promega), mixed the resulting plasmid (pXba9#5) with pPAB2, linearized with KpnI, and then introduced the mixture into strain 198F1 #36. The mutation was rescued in 26% (26/99) of pabþ transformants. With circular plasmids, the rate of rescue was as low as 3% (4/123). These results demonstrate that the 8.2-kb XbaI fragment contains the whole eln3 gene. 3.4. eln3 encodes a putative membrane protein with a general glycosyltransferase domain Sequencing of the 8.2-kb XbaI genomic fragment, together with 30 - and 50 -RACE experiments, identified an ORF interrupted by 16 introns, which is predicted to encode a protein of 927 amino acids (Fig. 5). The 50 and 30 splice sites agree with the consensus sequences GTRNGT and YAG found for filamentous fungi, respectively (Gurr et al., 1987), except that the ninth and 15th 50 splice sites are GTAAGA and GTGAGG, respectively, and the seventh 30 splice site is AAG. The size of the introns ranges between 54 and 582 bp. The eln3 mRNA is predicted to have a 260-nt 50 - and an 846-nt 30 -untranslated region. The promoter region of eln3 contains a CAAT-box-like sequence (CAAT) and a TATA-box-like sequence (TATAAA), which are located at 123–120 and 118–113 bp upstream of the predicted transcriptional start site, respectively. Hydropathy analyses with the programs, TMpred, TMHMM, HMMTOP, and PSORT II, all predict that Eln3 is a membrane protein with seven transmembrane helices (Fig. 5). TMpred and HMMTOP predict that the N-terminus is outside, while PSORT II and TMHMM predict that it is inside. Also, PSORT II predicts that Eln3 is likely located in the plasma membrane (47.8%, plasma membrane; 26.1%, endoplasmic reticulum;
13.0%, mitochondrial; 4.3%, Golgi; 4.3%, vacuolar; and 4.3%, vesicles of secretory system). Motif Scan analysis using the program, PFSCAN, revealed that Eln3 has a general glycosyltransferase domain at amino acid residues 386–563 (rawscore ¼ 379, N score ¼ 10.188, E value ¼ 0.0014). Database searches using the BLAST procedure (Altschul et al., 1997) revealed that Eln3 has high similarities to Neurospora crassa hypothetical protein, CAC28725.2 (Expect ¼ e-145, Identities ¼ 35%), N. crassa hypothetical protein, CAD36996.1 (Expect ¼ e-137, Identities ¼ 43%), and Cryptococcus neoformans var. neoformans NCP1 protein (Expect ¼ 6e-76, Identities ¼ 32%). NCP1 is present within the mating-type locus of the serotype D MAT a strain JEC20 of C. neoformans var. neoformans (Lengeler et al., 2002). 3.5. eln3-1 mutant allele Sequencing of plasmid pSL1 indicated that plasmid pPHT1 linearized with HindIII is inserted in the HindIII site in the fourth exon, which is located 1089–1094 bp downstream of the predicted translational start site. This insertion creates a stop codon at 21–23 bp downstream of the HindIII site, which in turn truncates most of the Eln3 peptide, including the seven transmembrane domains and the general glycosyltransferase domain. 3.6. Developmental regulation of eln3 expression Using the 30 -RACE product, 30 #2, which covers 83% of the eln3 transcript, as a probe, we examined total RNAs from the vegetative mycelium of the wild-type homokaryon, 5401, and the vegetative mycelium of the wild-type dikaryon, 5401 5302, and fruiting body primordia and various tissues of fruiting bodies at various developmental phases from the dikaryon (Fig. 6).
Fig. 5. Predicted amino acid sequence of Eln3 (A) and its hydropathy plot (B). Putative transmembrane domains are underlined and a putative general glycosyltransferase domain is double-underlined.
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Fig. 6. Expression of the eln3 gene. For each lane, 15 lg of total RNAs from various mycelia and fruiting body tissues were electrophoresed: lane 1, wild-type homokaryon 5401; lane 2, wild-type dikaryon 5401 5302; lane 3, fruiting body primordia from the dikaryon on one day before fruiting body maturation; lane 4, whole fruiting bodies from the dikaryon at 11 h after the start of illumination on the day of fruiting body maturation; lane 5, stipes at the 11-h stage; and lane 6, caps at the 11-h stage. Lanes 7–10 are stipes at 6, 11, 18, and 22 h after the start of the illumination. The rate of stipe elongation changes during fruiting body maturation: most rapid elongation occurs between 11 and 18 h and ends by 22-h stage after the start of the illumination. After the ribosomal RNA was visualized under UV, the gels were blotted and hybridized.
As expected from the eln3 cDNA analysis, we identified a transcript at 3.9 kb. We also found that the transcription of eln3 was specifically activated in the stipe, especially in rapidly elongating ones.
4. Discussion In this study we identified and characterized elongationless3, a REMI mutation that affects stipe elongation during fruiting body maturation, the final phase of fruiting body development, in C. cinereus. The mutant strain was apparently normal in vegetative mycelial growth as well as in fruiting body phenotypes other than stipe elongation, including the development of the cap and the formation of basidiospores. These observations indicate that elongationless3 is defective in a function that is specific of stipe development. This interpretation is consistent with the fact that the transcription of the eln3 gene responsible for the mutation is specifically activated in rapidly elongating stipes and is very low in fruiting body tissues other than the stipe as well as in vegetatively growing, homokaryotic and dikaryotic mycelia. Stipe elongation in the C. cinereus fruiting body is the result of elongation growth of cylindrical stipe cells, which run parallel to the stipe axis (Kamada, 1994; Kamada and Takemaru, 1977). Light microscopy showed that elongation growth of the stipe cells is
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reduced by 50% in the mutant, as compared with that in the wild type. However, this reduction in cell elongation only partially accounts for the fact that the mutant stipe hardly elongates during fruiting body maturation. This prompted us to examine the organization of the stipe tissue. In the wild-type stipe, cylindrical stipe cells make side-by-side contact with one another and run parallel to the stipe axis, and hence the elongation of the stipe cells directly results in the elongation of the stipe. In the mutant, however, much space is produced between the stipe cells and the parallel arrangement of the cells is disturbed, and hence the elongation growth of the stipe cells does not lead to stipe elongation but makes the stipe bulgy. The disorganization of the stipe tissue forming much space between stipe cells suggests that the mutant has a defect in the mechanism for cell-to-cell connection in the stipe. This hypothesis is supported by the fact that the mutant stipe was softer and more fragile than the wildtype stipe when taken between our fingers. It has been reported in C. cinereus that two galectins (Cgl1 and Cgl2), b-galactoside binding lectins, are produced during mushroom development (Boulianne et al., 2000). Although galectins are present throughout the fruiting body, the highest level of their expression is within a group of cells that form the outer portion of the stipe. On the basis of this fact, together with the reasonable hypothesis that a strong tension would be applied on the outer portion of the stipe and hence the stipe tissue would require an increased connectivity of the cells forming them, Boulianne et al. (2000) speculate that galectins are involved in cell-to-cell interaction, mediated by their ability to bind specific carbohydrates of the cell wall. Because Eln3 is predicted to be a protein with a general glycosyltransferase domain and to be located in the plasma membrane, it is a tempting hypothesis that Eln3 is involved in cell-to-cell connection by producing a cell-wall carbohydrate that binds to galectins. Because various extracellular matrices (ECMs) exist in fruiting bodies of homobasidiomycetes (see Walser et al., 2003 for review), it is also possible that Eln3 adds a carbohydrate moiety to one of ECMs and modulates its function. A future challenge would be identification of the presumptive carbohydrate produced by Eln3 through analysis of cell walls and/or ECMs in the stipe from elongationless3 mutant as well as from the wild type. Database searches revealed that Eln3 shows high similarities to two N. crassa hypothetical proteins and the C. neoformans var. neoformans NCP1 protein. This suggests that Eln3 belongs to a protein family conserved among basidiomyceteous and ascomyceteous fungi. Examination of developmental regulation of the expression of the orthologs in N. crassa and C. neoformans would be instrumental in identifying the Eln3 function.
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