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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.
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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
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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
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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
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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;
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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].
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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.
Disclosures
The authors have no conflicts of interest to declare.
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