Protoplast-release-inducing Protein Research Articles

CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex

Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt+) and mating-type minus (mt−). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt+ and mt− cells, respectively. PR-IP mediates the release of protoplasts from mt− cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt− cells of the C. psl. complex. When the knockout mt− cells were mixed with wild-type mt+ cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.

REPRODUCTIVE ISOLATION BY SEX PHEROMONES IN THE CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE COMPLEX (ZYGNEMATALES, CHAROPHYCEAE)(1).

The Closterium peracerosum-strigosum-littorale (C. psl.) complex consists of unicellular algae and is known to be composed of several reproductively isolated mating groups of heterothallic strains. Group I-E is completely isolated from mating groups II-A and II-B, groups II-A and II-B are partially isolated from each other, and only mating-type plus (mt(+) ) cells of group II-A and mating-type minus (mt(-) ) cells of group II-B form zygotes. Based on the alignment of 1506 group I introns, significant phylogenetic relationships were observed among mating groups II-A and II-B, while mating group I-E was distant from groups II-A and II-B. Sexual cell division in both mating-type cells of group II-A was stimulated in conditioned media in which cells of group II-B had been cultured. When mt(-) cells of group II-B were stimulated in conditioned medium derived from group II-A, mt(+) cells of group II-B did not respond to the conditioned medium. Conditioned media derived from group I-E did not exhibit sexual cell division (SCD)-inducing activity against any strain except those within its own group. From the alignment of deduced amino acid sequences from orthologous protoplast-release-inducing protein (PR-IP) Inducer genes, we detected a significant similarity among groups II-A and II-B, and mating group I-E had low similarity to other mating groups. The existing degree of reproductive isolation can be partially explained by differences in molecular structures and physiological activities of sex pheromones of these heterothallic mating groups.

Induction of sex pheromone secretion and observations of the secretory pathway in the Closterium peracerosum-strigosum-littorale complex

Several sex pheromones function in the process of sexual reproduction in the Closterium peracerosum-strigosum-littorale complex. The secretory pathways of two sex pheromones, protoplast release-inducing protein (PR-IP) and PR-IP Inducer, were studied by means of Western blot analysis and immunoelectron microscopy. PR-IP Inducer is secreted from mating-type minus (mt −) cells and induces the release of PR-IP from mating-type plus (mt +) cells. PR-IP causes release of the protoplast from mt − cells. Our studies were carried out using an experimental system that induces the secretion of sex pheromones in mt + or mt − cells cultured separately, without mixing. Active secretion of PR-IP Inducer occurs 6–12 h after inoculation of mt − cells in a nitrogen-depleted medium, and the secretion of PR-IP is substantial by 8 h after inoculation of mt + cells into medium containing PR-IP Inducer. In mt + and mt − cells that actively secrete the pheromones, immunogold particles that label PR-IP Inducer and PR-IP were detected inside and along the surfaces of cell walls, vesicles 80–100 nm in diameter, Golgi cisternae, and in the region of the trans-Golgi networks. As gold particles were distributed entire the cell wall, exocytosis of pheromones may occur throughout the cell surface. This study is the first report to detect algal sex pheromones by means of electron microscopy.

Physiological characterization of the sex pheromone protoplast‐release‐inducing protein from the Closterium peracerosum‐strigosum‐littorale complex (Charophyta)

SUMMARYIn the unicellular charophycean alga Closterium peracerosum‐strigosum‐littorale complex, the protoplast‐release‐inducing protein (PR‐IP), a sex pheromone responsible for gametic protoplast release from mating‐type minus (mt–) cells, was found to stimulate secretion of mucilage from the cells. Induction of sexual cell division by PR‐IP was also confirmed. Bioassays were used to determine the minimum doses required to induce these functions, revealing that 5 · 10−16 M of PR‐IP stimulated mucilage secretion, and that 5 · 10−10 M of PR‐IP were required for protoplast release. Exposure of the cells to 5 · 10−11 M of PR‐IP resulted in the induction of sexual cell division as well as mucilage secretion. These results strongly suggest that PR‐IP is a multifunctional pheromone that independently promotes multiple steps in conjugation at the appropriate times through different induction mechanisms.

A Sex Pheromone, Protoplast Release-inducing Protein (PR-IP) Inducer, Induces Sexual Cell Division and Production of PR-IP in Closterium

The sex pheromone protoplast release-inducing protein (PR-IP) inducer and a sexual cell division-inducing pheromone-minus (SCD-IP-minus) that mediates the sexual reproduction of the heterothallic Closterium peracerosum-strigosum-littorale (C. psl) complex were investigated in this study. Recombinant PR-IP inducer produced by yeast cells was prepared and assayed for production of PR-IP and induction of SCD. Both biological activities were observed after treating mating-type plus (mt+) cells with the recombinant pheromone. SCD was induced by exposure to a lower concentration of the same pheromone and by a shorter treatment period with the pheromone than was production of PR-IP. This indicates that the previously characterized PR-IP inducer has both PR-IP-inducing and SCD-inducing activities with mt+ cells, although the inducing mechanisms of the two pheromones differ.

Mucilage secretion regulated by sex pheromones in Closterium peracerosum-strigosum-littorale complex.

When mating-type plus (mt+) and minus (mt-) cells of the Closterium peracerosum-strigosum-littorale complex were mixed in nitrogen-depleted mating medium, secretion of mucilage containing uronic acid from cells was markedly activated and the mucilage accumulated around the cells. Substances with the ability to stimulate mucilage secretion from mt+ and mt- cells were detected in media in which mt- and mt+ cells had been separately cultured, respectively. We designated the active substances secreted from mt+ and mt- cells mucilage secretion-stimulating pheromone (MS-SP)-plus and MS-SP-minus, respectively. Activity of MS-SP-plus and MS-SP-minus decreased to 20% level by incubation at 80 degrees C for 10 min. Light was indispensable for the secretion of mucilage. The secretion of MS-SP-plus and MS-SP-minus decreased with dark treatment. MS-SP-plus eluted at around 95 k from a gel filtration column, and reacted with antibodies against two subunits of protoplast-release-inducing protein (PR-IP), which induces protoplast release from mt- cells. MS-SP-minus eluted at around 20 k from a gel filtration column, and reacted with an antibody against the PR-IP inducer, which induces the secretion of PR-IP from mt+ cells. In addition, purified PR-IP and PR-IP inducer stimulated mucilage secretion from mt- and mt+ cells, respectively. These results strongly suggested that MS-SP-plus and MS-SP-minus were the same molecules as the PR-IP and the PR-IP inducer, respectively.

CLONING AND CHARACTERIZATION OF A cDNA ENCODING A SEXUAL CELL DIVISION‐INDUCING PHEROMONE FROM A UNICELLULAR GREEN ALGA CLOSTERIUM EHRENBERGII (CHLOROPHYTA)

A full‐length cDNA encoding a sexual cell division‐ inducing pheromone (SCD‐IP) was isolated from a heterothallic green alga, Closterium ehrenbergii Menegh. ex Ralfs, using the rapid amplification of cDNA ends‐PCR method. The cDNA was 907 bp in length and coded for a protein of 209 amino acids with a deduced molecular mass of Mr 23,186. The predicted protein had a possible glycosylation site and a hydrophobic signal peptide for extracellular secretion. Transcripts of the SCD‐IP gene were detected only in mating type minus (mt−) cells and remarkably increased when mt− cells were cocultured with mating type plus (mt+) cells. By contrast, the gene for SCD‐IP was detected in both mating type cells as multicopy genes by Southern blot analysis. Continuous dark conditions and supplementation of a nitrogen source to the culture medium suppressed SCD‐IP expression. The deduced amino acid sequences encoded by genes for SCD‐IP and protoplast‐release‐inducing protein (PR‐IP) Inducer, a sex pheromone isolated from another Closterium species, appeared to share significant homology, suggesting that these sex pheromones were diversified from a single ancestral Closterium pheromone.

Purification and characterization of a pheromone that induces sexual cell division in the unicellular green alga Closterium ehrenbergii

Closterium ehrenbergii is a unicellular charophycean alga consisting of two sexes: mating type plus (mt +) and minus (mt –). The sexual reproductive process consists of five steps: formation of sexual pairs, cell division of each member of a pair, formation of conjugation papillae, release of protoplasts from gametangial cells, and fusion of protoplasts to form a zygote. The second step, called sexual cell division (SCD), produces two gametangial cells from one vegetative mother cell. The SCD of mt + cell is mediated by a diffusible sex pheromone, named SCD-inducing pheromone (SCD-IP). This pheromone is released from mt – cells in the light, and the presence of mt + cells stimulates its secretion from mt – cells. SCD-IP was purified by sequential column-chromatographic fractionation from culture medium in which both mating type cells had been co-cultured. Purified SCD-IP is a glycoprotein with an apparent molecular mass of 20 kDa. The molecular mass of the SCD-IP was estimated to be 18 kDa by mass spectrometry. Amino-terminal and two internal amino acid sequences of the pheromone revealed significant similarity to another Closterium pheromone, protoplast release-inducing protein (PR-IP) inducer of Closterium peracerosum-strigosum-littorale complex ( C. pslc). These two pheromones induced different morphological reactions in each Closterium species. Based on these results, the diversity of sex pheromones is discussed.

Molecular cloning of a novel sex pheromone responsible for the release of a different sex pheromone in Closterium peracerosum-strigosum-littorale complex.

A sex pheromone, protoplast-release-inducing protein (PR-IP) inducer, of the Closterium peracerosum-strigosum-littorale complex is known to induce the release of PR-IP, from mating-type plus (mt+) cells during sexual reproduction. The purified PR-IP inducer was treated with trypsin to obtain internal peptides for determination of partial amino acid sequences. Using these sequences, oligonucleotides were synthesized and used as primers for the combined reverse transcription-PCR. A 296 bp cDNA fragment was amplified, permitting the cloning of corresponding full length cDNA (CpPI; Closterium peracerosum-strigosum-littorale complex PR-IP inducer). The deduced amino acid sequence of CpPI encodes a protein of 212 amino acid residues of M(r) 23,071 whereas portion of the peptide secreted is predicted to have 142 amino acid residues of M(r) 15,717 and shows no significant similarity with known proteins. The predicted protein has three possible consensus sequences for asparagine-linked glycosylation site. The CpPI gene was expressed when mating-type minus (mt-) cells were incubated at a low cell density in the light. Nitrogen deprivation from the medium enhances expression of the CpPI gene. An analysis by genomic Southern hybridization revealed that the cDNA probe hybridized to several DNA fragments obtained from both the genome of mt- and mt+ cells. However, in mt- cells, transcripts for the PR-IP inducer could not be detected by Northern hybridization.

Analysis of genomic sequences encoding a sex pheromone from theClosterium peracerosum-strigosum-littorale complex

Genes for 19-kDa and 42-kDa subunits of protoplast-release-inducing protein (PR-IP) were cloned from theClosterium peracerosum-strigosum-littorale complex mt+ cells. Genomic sequences and transcriptional initiation sites for both genes were determined. Sequence data revealed that typical TATA boxes are located immediately upstream of both transcriptional initiation sites. Several similar sequences were observed at nearly the same location in the promoters of both genes. Based on these data, we propose some possible hypotheses regulating the expression of the PR-IP subunit genes.

BIOCHEMICAL, PHYSIOLOGICAL, AND MOLECULAR ANALYSIS OF SEXUAL ISOLATION IN THE SPECIES COMPLEX CLOSTERIUM PERACEROSUM‐STRIGOSUM‐LITTORALE (CHLOROPHYTA)1

ABSTRACTClosterium strains obtained from Japan (NIES‐64 and ‐65)and Nepal (NIES‐67 and ‐68)have been classified as the same taxonomic species; however, they are sexually isolated from each other. When NIES‐64 and ‐65 cells were separately incubated in a medium in which both strains had previously been cultured together, release of protoplasts from both strains was observed. We suggest that factors responsible for the release of protoplasts from cells of both NIES‐64 and ‐65 are produced in a mixed culture of these cells and function during conjugation. These factors, however, had no effect on the release of protoplasts from cells of strains NIFS‐67 or ‐68. Alternatively, a protein that is responsible for the release of protoplasts from cells of NIES‐68, called the protoplast‐release‐inducing protein (PR‐IP),had no effect on the release of protoplasts from cells of strains NIES‐64 or ‐65. When the media obtained from the culture of NIES‐64 and ‐65 cells at various mixing ratios were analyzed by western blotting with antiserum to a 42‐kDa subunit of PR‐IP, no cross reaction was detected. In Southern hybridization analysis, no hybridizing band was observed when genomic DNAs of NIES‐64 and ‐65 cells were probed with cDNAs encoding the two subunits of PR‐IP. We suggest from these results that the factors responsible for the release of protoplasts from NIES‐64 and ‐65 cells are not structurally similar to PR‐IP. It is known that the release of PR‐IP from NIES‐67 cells can be induced by the action of another sex pheromone (PR‐IP inducer)which is released by NIES‐68 cells. In contrast, no protoplast‐release‐inducing activity was observed from either NIES‐64 or ‐65 in a culture medium conditioned by opposite strains. We suggest that the conjugation systems employed by strains NIES‐64/ NIES‐65 and strains NIES‐67 /NIES‐68 differ, and we propose a possible mechanism of sexual isolation between these biological species.

Regulation of expression of the genes for a sex pheromone by an inducer of the sex pheromone in the Closterium peracerosum-strigosum-littorale complex.

A sex pheromone, protoplast-release-inducing protein (PR-IP), of the Closterium peracerosum-strigosum-littorale complex is known to induce the release of protoplasts from mating-type minus (Mt-) cells during sexual reproduction and to have two subunit polypeptides of 19 and 42 kDa. Here, we describe the regulation mechanism for the release of the PR-IP. The sex pheromone was fractionated to yield subunits of 19 and 42 kDa, respectively, and each subunit was treated with V8 protease and with CNBr. By reference to the partial amino-acid sequences of the digested polypeptides, oligo nucleotides were synthesized and used as primers for the combined reverse transcription-polymerase chain reaction. Amplified fragments of DNA, of 130 bp in the case of the 19-kDa subunit and of 330 bp in the case of the 42-kDa subunit, were obtained, sequenced, and used as probes to identify the respective transcripts. From the results of Northern hybridization, the sizes of transcripts were estimated to be 1.2 kb for the 19-kDa subunit and 1.4 kb for the 42-kDa subunit. These transcripts appeared transiently only when mating-type plus (mt+) cells were treated with another sex pheromone (PR-IP Inducer) for more than 4 h in the light. By immunoblotting with anti-42-kDa-subunit antiserum, it was shown that PR-IP accumulated gradually in the medium but not in the mt+ cells after treatment with PR-IP inducer in the light.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of binding of biotinylated protoplast-release-inducing protein that induces release of gametic protoplasts in the Closterium peracerosum-strigosum-littorale complex

A protoplast-release-inducing protein (PR-IP) which is released from mating-type plus (mt(+)) cells and induces the release of gametic protoplasts from matingtype minus (mt(-)) cells of Closterium was biotinylated and then used to examine the interaction of this protein with mt(-) cells. The protoplast-release-inducing activity of PR-IP was not altered after the biotinylation. When mt(-) cells that had been pre-cultured for 24 h were incubated with biotinylated PR-IP for 6 h in nitrogen-deficient medium that contained 1% (w/v) bovine serum albumin, and then washed with the same medium, only a 19-kDa polypeptide, the smaller subunit of PR-IP, was detected in cells by the avidin and biotinylated horseradish-peroxidase macromolecular complex system. The amount of bound 19-kDa polypeptide increased with increasing doses of PR-IP and reached a maximum at around 10 nM, reflecting the protoplast-release-inducing activity. From a Scatchard plot, the dissociation constant of the polypeptide was calculated to be 2.7 · 10(-8) M. The binding of the polypeptide proceeded only after an appropriate period of pre-culture in the light, and the polypeptide was competitively displaced by non-biotinylated PR-IP. From these results, it appears that the PR-IP induces the release of protoplasts from mt(-) cells by binding of a polypeptide of relative molecular mass 19000 to the receptor on the cell surface in a manner analogous to the binding of peptide hormones in animals.

Biochemical and physiological properties of a protein inducing protoplast release during conjugation in theClosterium peracerosum-strigosum-littorale complex

When mating-type minus (mt(-)) and plus (mt(+)) cells of theClosterium peracerosum-strigosum-littorale complex were mixed together in a nitrogen-deficient mating medium, cells of both types released protoplasts, this release being the first step in the process of conjugation. Release of protoplasts by mt(-) cells also proceeded without pairing in a medium in which mt(-) and mt(+) cells had previously been cultured together. A protein with the ability to induce the release of protoplasts was purified from this medium by sequential column-chromatographic steps, and named PR-IP (protoplast-release-inducing protein). The PR-IP had an apparent molecular mass (Mr) of 95000 on gel filtration and could be separated into several isoforms by anion-exchange chromatography. Each isoform consisted of two glycopolypeptides of Mrs 42000 and 19000, while the deglycosylated polypeptides had Mrs of 34000 and 18000, respectively. From an analysis of dose-response curves, the numbers of PR-IP molecules required for the release of a protoplast by a single cell was calculated as 1.5·10(9) and the concentration required for 50% of the maximum response (ED50) as 4.1·10(-9)M. We suggest that the PR-IP is a biologically active glycoprotein which induces the release of gametic protoplasts from mt(-) cells of thisClosterium complex.