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Regular Paper A Receptor-Like Kinase, Related to Cell Wall Sensor of Higher Plants, is Required for Sexual Reproduction in the Unicellular Charophycean Alga, Closterium peracerosum–strigosum– littorale Complex Naoko Hirano1, Yuka Marukawa2, Jun Abe2, Sayuri Hashiba2, Machiko Ichikawa1, Yoichi Tanabe3, Motomi Ito3, Ichiro Nishii4, Yuki Tsuchikane2 and Hiroyuki Sekimoto1,2,* 1 Division of Material and Biological Sciences, Graduate School of Science, Japan Women’s University, Bunkyo-ku, Tokyo, 112-8681 Japan Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, Bunkyo-ku, Tokyo, 112-8681 Japan 3 Department of General Systems Studies, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo, 153-8902, Japan 4 Department of Biological Sciences, Faculty of Science, Nara Women’s University, Nara, 630-8506 Japan 2 *Corresponding author: E-mail, [email protected]; Fax: +81-3-5981-3674. (Received February 2, 2015; Accepted April 23, 2015) Here, we cloned the CpRLK1 gene, which encodes a receptorlike protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum–strigosum– littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt+) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt– cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release. Keywords: Cell wall Charophycean alga Closterium peracerosum–strigosum–littorale complex CrRLK1L-1 Receptor-like kinase Sexual reproduction. Abbreviations: CpRLK1, C. psl. complex RLK1; ECD, extracellular domain; FER, FERONIA; PBS, phosphate-buffered saline; PR-IP, protoplast release-inducing protein; RLCK, receptorlike cytoplasmic kinase; RLK, receptor-like protein kinase. Introduction Successful sexual reproduction in flowering plants involves a complex series of interactions between male and female cells (Higashiyama 2010). In recent years, considerable insights have been gained into the molecular mechanisms that control these interactions in higher plants. In vitro analysis of Torenia fournieri identified two small cysteine-rich polypeptides (CRPs), named LURE1 and LURE2, that are secreted from the synergid cells to act as pollen tube attractants (Okuda et al. 2009). When the pollen tube reaches the synergid cells, it arrests growth, bursts and releases the sperm cell. These latter processes are controlled by the female gametophyte via the receptor-like protein kinase (RLK), FERONIA (FER) (Escobar-Restrepo et al. 2007). In contrast, in Arabidopsis, two close homologs of FERRLK, ANXUR1 and ANXUR2, are expressed in the pollen tube and enable it to rupture at the appropriate time to deliver its sperm cell (Boisson-Dernier et al. 2009, Miyazaki et al. 2009). The GENERATIVE CELL SPECIFIC 1 (GCS1)/HAPLESS 2 (HAP2) protein is specifically expressed in sperm cells; in mutants with loss of this protein, the sperm cells fail to fuse with both egg and central cells (Mori et al. 2006, von Besser et al. 2006). Sprunck et al. (2012) showed that EGG CELL 1 (EC1) proteins accumulate in storage vesicles of the egg cell and play an essential role in prevention of multiple sperm cell delivery during double fertilization. In contrast to our increasing understanding of molecular mechanisms associated with sexual reproduction in higher plants, the evolution of these processes is still uncertain. Land plants are thought to have evolved from ancestral charophycean algae (Karol et al. 2001, Graham et al. 2009). Charophyceans comprise five lineages (orders) of freshwater green algae: Charales, Coleochaetales, Zygnematales, Klebsormidiales and Chlorokybales. The desmid Closterium, which belongs to the order Zygnematales, is the most widely studied unicellular charophycean plant in terms of the maintenance of strains and sexual reproduction (Sekimoto et al. 2012). Based on recent phylogenetic analyses, Zygnematales are the closest green algae to land plants (Timme et al. 2012). Their cellular features and metabolism are more similar to those of land plants than those of other algae such as the ‘green yeast’ Chlamydomonas (Graham et al. 2009). Heterothallic strains of the Closterium peracerosum– strigosum–littorale complex (C. psl. complex) have two morphologically indistinguishable sexes: mating-type plus (mt+) and mating-type minus (mt–). Sexual reproduction is easily induced when cells of the two sexes are cultured together in nitrogen-depleted medium under light. Reproduction is regulated by two sex pheromones, protoplast release-inducing protein (PR-IP) and PR-IP Inducer, which are produced from mt+ and mt– cells, respectively. The possible mechanisms involved in sexual reproduction in the C. psl. complex have recently been described (Sekimoto et al. 2012). In a previous study into the Plant Cell Physiol. 56(7): 1456–1462 (2015) doi:10.1093/pcp/pcv065, Advance Access publication on 4 May 2015, available online at www.pcp.oxfordjournals.org ! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] Plant Cell Physiol. 56(7): 1456–1462 (2015) doi:10.1093/pcp/pcv065 molecular mechanisms of intercellular communication during sexual reproduction in the C. psl. complex, a cDNA microarray was constructed. Expression profiles were obtained using mRNAs isolated from cells at various stages of the life cycle. This analysis identified 88 pheromone-inducible, conjugationrelated and/or sex-specific genes (Sekimoto et al. 2006). One of the genes identified in the above exercise encodes a receptor-like protein kinase (RLK) and is named CpRLK1. The gene is expressed specifically in mt+ cells. The expression is elevated during sexual reproduction, and treatment of mt+ cells with the PR-IP Inducer also promotes its expression, indicating that the CpRLK1 protein probably functions during sexual reproduction (Sekimoto et al. 2006). RLKs comprise a large family with several hundred members in land plants (Lehti-Shiu et al. 2009). They have two main configurations: receptor-like kinases (RLKs) with an extracellular domain (ECD), transmembrane domain and intracellular kinase domain; and receptor-like cytoplasmic kinases (RLCKs) that lack an ECD. They are thought to be involved in many different processes, such as the regulation of meristems (Clark et al. 1997), sexual reproduction (Escobar-Restrepo et al. 2007, Boisson-Dernier et al. 2009, Miyazaki et al. 2009, Liu et al. 2013) and hormone perception (Wang et al. 2001, Hirakawa et al. 2008); however, the exact role of most RLKs is unknown. RLKs with an ECD have been found in the charophycean algae Nitella axillaris, Closterium ehrenbergii (Sasaki et al. 2007) and Klebsormidium flaccidum (Hori et al. 2014). In contrast, no ECDencoding RLK genes have been identified in the genomes of early diversified green algae, such as Chlamydomonas reinhardtii and Ostreococcus tauri (Lehti-Shiu et al. 2009), although two RLCK genes have been found in Chlamydomonas reinhardtii. This suggests that the RLK genes evolved in the charophycean–land plant lineage after its divergence from the green algal lineage. Characterization of the C. psl. complex RLK1 (CpRLK1) will provide insight not only into sexual reproduction in Closterium but also into important processes regarding the mechanism and evolution of intercellular communication between the egg and sperm cells of land plants. In the present study, the functions of the CpRLK1 protein during sexual reproduction were evaluated using reverse genetics (Abe et al. 2011). Phylogenetic analysis and investigation of the intracellular localization of the protein support the idea that CpRLK1 belongs to the CrRLK1L-1 subfamily, and that it functions in sensing cell wall integrity and in regulating osmotic pressure in the cell for cytoplasm condensation and gamete release during successful conjugation. Results First, the 3,973 bp full-length cDNA encoding CpRLK1 was cloned using sequential rapid amplification of cDNA ends (RACE)-PCR (accession No. AB920609; see Supplementary Methods S1). The deduced amino acid sequence of CpRLK1 encoded a protein of 1,159 amino acid residues of Mr 121,908 (Supplementary Fig. S1). The CpRLK1 protein had a signal peptide for passing through to the endoplasmic reticulum (amino acids 1–34) and one transmembrane domain (amino acids 631–653). There were 12 possible asparagine-linked glycosylation sites and a malectin-like carbohydrate-binding domain (amino acids 277–583) in the extracellular domain. In the putative cytoplasmic region, a protein kinase domain (amino acids 751–1,061) with ATP-binding (amino acids 757– 790) and active sites (amino acids 884–896) was conserved. Preliminary phylogenetic analysis using 53 RLK subfamily members of land plants (Sasaki et al. 2007, Lehti-Shiu et al. 2009) showed that nine RLK subfamilies had a relatively close relationship with CpRLK1 (Supplementary Table S1). We aligned the kinase domains of the CpRLK1 gene and representative RLK subfamilies (Supplementary Appendix S1) and generated a maximum likelihood tree. Although the phylogenetic relationships were not resolved with high bootstrap values, CpRLK1 showed a relatively close relationship to the CrRLK1L-1 subfamily (Fig. 1). The extracellular domain of CrRLK1L-1 subfamily members contained malectin-like carbohydrate-binding domains as in CpRLK1. Next, we prepared antibodies against synthetic peptides for the extracellular domain of CpRLK1. The production of the protein during conjugation was analyzed by Western blotting. A 150 kDa band, possibly the glycosylated form of CpRLK1, was present at 6 h after mixing both mating types (Fig. 2a). The band became more abundant from 18 to 24 h and then gradually declined. The same band was detected in mt+ cells that had been incubated with a PR-IP Inducer (Fig. 2b). During incubation, the protein content reduced gradually. To elucidate the physiological function of CpRLK1, a cDNA encoding the ECD of the CpRLK1 gene was inserted into the vector pSA0104 in an antisense direction (pSA0104_antiCpRLK1, Supplementary Fig. S2). This antisense construct was introduced into mt+ cells by particle bombardment, and seven transformant strains showing hygromycin resistance were successfully isolated. When the transformants were mixed and incubated with wild-type mt– cells, most showed reduced mating reactions (Fig. 3a) and a reduced level of the CpRLK1 protein (Fig. 3b) compared with controls, although the correlation between CpRLK1 levels and the ratio of cells in mating was not good. Two of the transformants (anti-H13 and H30), which showed severe reduction in CpRLK1 protein, were selected for further investigation of mating. In both strains, sexual pair formation with the wild-type mt– strain appeared to be essentially normal and similar to that for wild-type mt+. However, the subsequent protoplast release step was inhibited in these strains and no zygote formation was observed (Fig. 4). Analysis of time-lapse movies showed that the transformant and wild-type mt– cells could pair and form conjugation papillae; however, they could not release their protoplasts (gametes) (Supplementary Movies S1, S2). Moreover, one of the paired cells often formed an abnormally enlarged papilla (Fig. 5c). Release of gametes was occasionally observed from one of the paired cells (Fig. 5e). Vital staining of the transformant cells, before mixing with wild-type mt– cells, revealed that they were responsible for the formation of the abnormally enlarged papilla and that they did not ever release 1457 N. Hirano et al. | Algal receptor-like kinase for sexual reproduction CpRLK1 MpRLK7 93 XP_001757873 100 80 XP_001757874 At5g54380 69 64 AT3G04690 100 AT5G28680 95 94 94 CrRLK1L-1 AT3G51550 53 Os01g0769700 100 Os05g0318700 AT1G30570 96 At5g38990 At1g67720 56 At4g29990 100 82 LRR-I Os05g0525550 MpRLK21 96 At3g26700 100 100 OsI_19707 RLCK-IXa MpRLK20 80 LRR-I At5g48740 99 MpRLK24 AT2G11520 62 100 100 MpRLK23 65 RLCK-IV At4g00330 LRR-Mp-I MpRLK18 95 At1g49730 87 100 At3g19300 96 URK-1 Os04g0689400 MpRLK15 100 100 LRR-VIII-1 At1g79620 Os05g0486100 At3g59420 At3g55950 100 59 OsJ_16980 CR4L At5g47850 Os08g0109800 0.1 substitutions/site Fig. 1 Phylogenetic analysis of CpRLK1 and representative RLK subfamilies from land plants. The phylogenetic tree is based on alignment of the amino acid sequences of CpRLK1 and 35 RLKs from four species of land plants and was constructed using maximum likelihood methods (Supplementary Table S1; Supplementary Appendix S1). Numbers at branches indicate support values from 100 bootstrap replicates. The scale bar denotes the number of substitutions per site. gametes, i.e. wild-type mt– cells had the ability to release their gametes but the transformants did not (Fig. 5d, f). Indirect immunocytological detection of CpRLK1 protein using confocal laser scanning microscopy showed that the CpRLK1 signal was localized mainly at the inflated region of the conjugation papilla (Fig. 6, arrow; Supplementary Movie S3) in conjugating wild-type paired cells. We also noticed intracellular signal foci in the same cell, suggesting the presence of 1458 the protein on the secreted vesicles (Fig. 6, arrowheads; Supplementary Movie S3). Discussion We found here that the CpRLK1 protein appeared after the mixing of the cells (Fig. 2) and reached a maximum just Plant Cell Physiol. 56(7): 1456–1462 (2015) doi:10.1093/pcp/pcv065 Fig. 2 Immunological detection of CpRLK1 protein in Closterium cells. (a) Mt+ and mt– cells were mixed and incubated in nitrogen-depleted medium to induce sexual reproduction. (b) Mt+ cells were incubated in nitrogen-depleted medium containing PR-IP Inducer. Cells were collected and subjected to SDS–PAGE, followed by immunoblotting with an anti-CpRLK1 antibody. Fig. 3 Phenotypes of transformants expressing an antisense RNA that corresponds to the region for the extracellular domain of CpRLK1. (a) Proportions of mated cells at 48 h after co-incubation of mt– cells (wild type) and mt+ cells (transformants or wild type). Pair-forming, protoplast-releasing and zygote-forming cells are treated as cells undergoing mating. Wild-type mt+ cells were used as a control. Two independent experiments were performed. Vertical bars indicate SEs. H11-13, H21-23 and H30, transformants harboring pSA0104_antiCpRLK1; C1 and C2, control transformants harboring pSA0104. (b) Expression profiles of endogenous CpRLK1 protein in transformants (18 h after the mixing), detected by Western blotting with an antiCpRLK1 antibody. before pairing, followed by a rapid decline after the initiation of zygote formation (Fig. 4; WT). This protein expression pattern is in agreement with the results of a previous real-time PCR analysis (Sekimoto et al. 2006), and suggests that CpRLK1 is required for the maintenance of pairing or for the transition to the gamete-releasing stage. The knockdown transformant strains showed reduced mating reactions (Fig. 3), and inhibition of the gamete-releasing step (Fig. 4). The transformed cells also produced an abnormally enlarged conjugation papilla and did not release protoplasts (Fig. 5). These results suggest that CpRLK1 is involved in the regulation of normal elongation of the conjugation papilla and/or release of gametes after pairing. Indeed, the localization of CpRLK1 to the conjugation papilla of one of a pair of wild-type cells (Fig. 6) strongly implies that the protein plays a role in the gamete-releasing step through the papilla. Although the phylogenetic relationships of CpRLK1 and representative land plant RLKs were not solved here with high bootstrap values (Fig. 1), nevertheless CpRLK1 showed a relatively high relationship to the CrRLK1L-1 subfamily. The presence of a conserved malectin-like carbohydrate domain in the ECD also supported the idea that CpRLK1 is a member of the CrRLK1L-1 subfamily. Members of the LRR-I subfamily also have a malectin-like carbohydrate domain in the ECD; however, they also have LRR_4 and LRR_8 domains, which are not present in CpRLK1. In Arabidopsis thaliana, several proteins belonging to the CrRLK1L-1 subfamily are involved in the control of cell wall integrity and growth regulation (Kanaoka and Torii 2010, Boisson-Dernier et al. 2011, Cheung and Wu 2011, Nibau and Cheung 2011, Lindner et al. 2012). The current view is that members of the CrRK1L-1 subfamily can bind carbohydrate ligands derived from cell wall components, glycoproteins at the plasma membrane or secreted signaling molecules derived from neighboring cells, via their malectin-like ECDs (BoissonDernier et al. 2011). As a consequence of binding, signals are sent to the cytoplasm where they are processed and relayed to the apoplast, for the adjustment of cell wall properties. Based on the current consensus, what might be the role of CpRLK1 in sexual reproduction? Cells of opposite mating types form a pair, and partial degradation of their cell walls is induced in the papillar area (Pickett-Heaps and Fowke 1971). As water constantly enters the cells under osmotic pressure, the cytoplasm of each cell extrudes from the thinned wall to form a papilla. During this process, CpRLK1 proteins localize at the conjugation papilla and sense these cell wall changes (or a specific signaling molecule derived from the mt– cell) and transmit a signal internally to regulate osmotic pressure appropriately for successful gamete release and formation of the zygote. In CpRLK1-knockdown cells, partial loss of cell walls and formation of papillae appeared to be normal. However, we suggest that information regarding cell wall changes or a specific ligand was not received appropriately by CpRLK1. As a result, osmotic pressure in the cell and the condensation of the cytoplasm could not be regulated, and the appropriate protoplast release followed by formation of a zygote could not be accomplished. In addition, an abnormally large papilla is formed as a result of the constant influx of water. In the CrRLK1L-1 subfamily, FER in synergid cells and Anxur1 and Anxur2 in pollen tubes are responsible for the regulation of pollen tube elongation for successful fertilization (EscobarRestrepo et al. 2007, Boisson-Dernier et al. 2009, Miyazaki et al. 2009, Boisson-Dernier et al. 2013). Since CpRLK1 is posited as being responsible for the progress of gamete release during sexual reproduction in Closterium cells, we suggest that the ancestral role of CrRLK1L-1 was control of cell wall integrity during conjugation. Recently, the specific binding of a ‘rapid alkalinization factor’ to FER was identified, although the 1459 Relative number of cells in mating (%) N. Hirano et al. | Algal receptor-like kinase for sexual reproduction WT H13 H30 50 50 50 40 40 40 30 30 30 20 20 20 10 10 10 0 0 0 24 48 72 Paired cells Protoplast-releasing cells Zygotes 0 0 24 48 72 Time after the mixing (h) 0 24 48 72 Fig. 4 Time course of conjugation between mt– and representative transformants. Cells undergoing mating (pair-forming, protoplast-releasing and zygote-forming cells) were counted individually. Wild-type mt+ cells were used as a control. Three independent experiments were performed. Vertical bars indicate SEs. Fig. 5 Phenotype of anti-H30 cells during mating. Cells at 48 h after co-incubation of mt– cells (wild type) and mt+ cells (anti-H30 or wild type) were fixed and visualized. Pair formation (a) and protoplast release (b) between wild-type mt+ and mt– cells. Pair formation (c, d) and protoplast release between vitally stained anti-H30 cells and wild-type mt– cells. Arrows indicate anti-H30. Bright field images (c, e); fluorescence images derived from prior staining by Fluorescent Brightener 28 (d, f). involvement of the malectin-like domain of FER was not apparent (Haruta et al. 2014). We have obtained transcriptome data for sexual reproduction and a draft genome of Closterium using next-generation sequencing. Using this information as a reference source, we are now trying to obtain comparative transcriptome data for conjugation using the wild-type and transformant strains. Materials and Methods Plant materials and induction of sexual reproduction We obtained the heterothallic C. psl. complex strains NIES-67 (mt+) and NIES68 (mt–) from the National Institute for Environmental Studies, Ibaraki, Japan. Vegetative cells were cultured in nitrogen-supplemented medium (C medium; 1460 http://www.nies.go.jp/biology/mcc/home.htm), as previously described (Sekimoto et al. 1990). Sexual reproduction in the C. psl. complex was induced in vegetatively growing cells of the two mating types at the mid-logarithmic phase. The cells were harvested, washed three times with nitrogen-depleted medium (MI medium; Ichimura 1971), and incubated separately in MI medium (3.0 105 cells ml–1) under continuous light for 24 h (high-density pre-culture). Then, cells of both mating types (3.6 105 each) were mixed in 72 ml of fresh MI medium in 300 ml Erlenmeyer flasks, and incubated under continuous light for various time intervals. At each interval, cells were harvested and used in Western blot analyses. Aliquots of the cell cultures were collected before harvest and fixed using 0.6% glutaraldehyde. In these aliquots, the cells in the process of sexual reproduction were counted under a light microscope using a hemacytometer. The experiments were performed twice separately to ensure accuracy in the results. We also incubated mt+ cells (7.2 103 cells ml–1) in MI medium containing PR-IP Inducer (Sekimoto et al. 1993), and harvested these at various time intervals for use in Western blot analyses. Plant Cell Physiol. 56(7): 1456–1462 (2015) doi:10.1093/pcp/pcv065 Fractionated proteins were subjected to SDS–PAGE using an 8% separation gel (Sekimoto et al. 1990). After electrophoresis, the proteins in the gel were transferred to a nitrocellulose membrane (Optitran BA-S 85, Whatman, www.gelifesciences.com) and probed with the affinity-purified anti-CpRLK1 specific polyclonal antibody. Binding of the primary antibody was detected using a horseradish peroxidase-conjugated AffiniPure goat anti-rabbit IgG antibody (Jackson ImmunoResearch, www.jacksonimmuno. com). The CpRLK1 protein was detected by chemiluminescence using a Versadoc (Bio-rad, www.bio-rad.com) or Odyssey Fc Imaging System (LI-COR, www.licor.com). Indirect immunofluorescent detection of CpRLK1 protein Fig. 6 Localization of CpRLK1 proteins on paired cells. Paired cells were fixed and the distribution of CpRLK1 proteins was visualized by indirect immunofluorescence microscopy using an anti-CpRLK1 antibody. Arrow, conjugation papilla; arrowheads, secreted vesicles. Cloning of full-length cDNA encoding CpRLK1 and preparation of CpRLK1-knockdown transformants A full-length cDNA encoding CpRLK1 was cloned as described in the Supplementary Methods S1. A construct (pSA0104_anti-CpRLK1) was prepared and used for transformation to isolate CpRLK1-knockdown clones; preparation of the construct and transformation of cells is detailed in the Supplementary Methods S1. Phylogenetic analysis Sequence alignments were performed with MAFFT E-INS-i (Katoh et al. 2005) (Supplementary Appendix S1). Gaps were removed from the aligned sequences for the phylogenetic analysis. Phylogenetic analysis of CpRLK1 and RLK subfamilies was performed using Molphy, version 2.3b3 (Adachi and Hasegawa 1996). Maximum likelihood distances were calculated using the program PROTML with the conditions of the JTT model (Jones et al. 1992) and an initial Neighbor–Joining (NJ) tree was generated using the program NJDIST. The local bootstrap probability of each branch was estimated by the Nearest-Neighbor Interchanging method from 100 bootstrap replicates. Preparation and affinity purification of antiCpRLK1 antibody Two synthesized peptides (A, Cys-87RALQDQPGSGPDPSA101; and B, Cys-161TKPGATPDDTGTDVN175) that include part of the CpRLK1 ECD were used as antigens. Two rabbits were immunized with both peptides conjugated to keyhole limpet hemocyanin. Antibodies specific to peptide-A and -B were separately purified using affinity columns (NHS-activated Sepharose 4 Fast Flow, GE Healthcare) coupled with peptide-A and -B, respectively. We confirmed the specificity of these antibodies by immunoblotting, and found that the anti-peptide-A antibody gave a better signal; this antibody was used as the affinity-purified anti-CpRLK1-specific polyclonal antibody in this study. The purified antibody was divided into aliquots and stored at –80 C until needed. Immunoblot analysis Cells undergoing sexual reproduction were harvested from cell cultures by centrifugation (1,600 g for 5 min at 4 C) and disrupted in 50 mM Tris–HCl buffer (pH 8.0) containing a protein inhibitor cocktail (Roche, www. roche.com) by ultrasonication (BIORUPTOR, Cosmo Bio, www.cosmobio.co.jp). The cell lysates were fractionated by sequential centrifugation (1,000 g, 5 min and then 13,000 g, 15 min at 4 C) and the final supernatants were collected for analyses of CpRLK1 levels. Protein contents were measured by the standard method (Bradford 1976) using bovine serum albumin as the standard. Cells in the process of mating (16 or 20 h after mixing) were allowed to adhere to 0.01% polyethylenimine-coated coverslips for 5 min. The coverslips were then quickly transferred to a Coplin jar filled with a solution of 75% methanol : 25% acetate, pre-chilled at –80 C and fixed for 5 min. This fixation step was repeated twice by rapid transfer of the coverslips. After fixation, the coverslips were incubated in a Coplin jar containing phosphate-buffered saline (PBS) with 0.05% macerozyme (Yakult, www.yakult.co.jp) for 30 min at room temperature. The coverslips were then washed with PBS for 10 min and then PBS containing 0.1% Tween-20 (PBSt) for 10 min. The coverslips were placed in primary blocking solution (5% bovine serum albumin, 1% fish gelatin, 0.05% NaN3 in PBS) for 30 min, followed by primary blocking solution containing 10% normal goat serum. The coverslips were then incubated in secondary blocking solution (20% primary blocking solution and 0.04% NaN3 in PBSt) containing affinitypurified anti-CpRLK1-specific polyclonal antibody at 1 : 100 dilution overnight at 4 C. The coverslips were washed five times using PBSt for 10 min, blocked again in secondary blocking solution for 30 min and then incubated in secondary blocking solution containing the Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, www.lifetechnologies.com) at 1 : 500 dilution for 4 h at room temperature. The coverslips were washed five times in a Coplin jar containing PBSt for 10 min and mounted in ProLong Gold Antifade Reagents (Life Technologies). Confocal images were obtained using an Olympus FV1200 confocal laser scanning microscope system (model IX-83, Olympus, www.olympusims.com). 3D images were constructed using Volocity software (PerkinElmer, www.perkinelmer.com). Vital staining of cell walls Anti-H30 cells were incubated in MI medium containing 0.01% Fluorescent Brightener 28 (Sigma-Aldrich, www.sigmaaldrich.com) for 30 min and then briefly washed with MI medium. The mutant and wild-type mt– cells were mixed and incubated for 48 h. The samples were observed using a fluorescence microscope (IX-83) under UV light, to identify the mutant cells. Supplementary data Supplementary data are available at PCP online. Funding This work was partly supported by the Japan Society for the Promotion of Science, Japan [Grants-in-Aid for Scientific Research (Nos. 23657161, 24370038, 24247042, 25304012 and 26650147 to H.S., No. 23770277 to J.A., Nos. 23770093 and 26440223 to Y. Tsuchikane]; the Ministry of Education, Culture, Sports, Science and Technology, Japan [Grants-in-Aid for Scientific Research on Innovative Areas ‘Elucidating common mechanisms of allogenic authentication’ (Nos. 22112521 and 24112713 to H.S.)]; the New Technology Development Foundation [to H.S. and Y. Tsuchikane]. 1461 N. Hirano et al. | Algal receptor-like kinase for sexual reproduction Acknowledgments The authors wish to thank Dr. Wolfgang Mages (University of Regensburg) for providing the pHYG4 vector. 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