Molecular coevolution of a sex pheromone and its receptor triggers reproductive isolation in Schizosaccharomyces pombe

The diversification of sex pheromones is regarded as one of the causes of prezygotic isolation that results in speciation. In the fission yeast Schizosaccharomyces pombe, the molecular recognition of a peptide pheromone by its receptor plays an essential role in sexual reproduction. We considered that molecular coevolution of a peptide-mating pheromone, M factor, and its receptor, Map3, might be realized by experimentally diversifying these proteins. Here, we report the successful creation of novel mating-type pairs by searching for map3 suppressor mutations that rescued the sterility of M-factor mutants that were previously isolated. Several strong suppressors were found to also recognize WT M factor. The substituted residues of these Map3 suppressors were mapped to F204, F214, and E249, which are likely to be critical residues for M-factor recognition. These critical residues were systematically substituted with each of the other amino acids by in vitro mutagenesis. Ultimately, we successfully obtained three novel mating-type pairs constituting reproductive groups. These novel mating-type pairs could not conjugate with WT maters. Furthermore, no flow of chromosomally integrated drug-resistance genes occurred between the novel and the WT mating pairs, showing that each experimentally created reproductive group [e.g., M factor(V5H) and Map3(F214H)] was isolated from the WT group. In conclusion, we have succeeded in creating an artificial reproductive group that is isolated from the WT group. In keeping with the biological concept of species, the artificial reproductive group is a new species.Speciation is the most critical step in evolution (1). A new species branches off from an original species when a group of individuals is isolated reproductively (termed “reproductive isolation”) (2). Chemical communication between the two sexes is important in both attracting individuals of the opposite sex and the courtship reaction. Pheromone diversification may be a possible mechanism underlying reproductive isolation.Female-attracting peptide pheromones of newts are providing a promising means to explore this mechanism. A decapeptide called sodefrin was first identified during the analysis of a cDNA library of the abdominal gland of the red-bellied newt Cynops pyrrhogaster (3, 4). A closely related newt, the sword-tailed newt Cynops ensicauda, produces a similar peptide pheromone named Silefrin (5). Interestingly, a sodefrin variant, aonirin, was found in the Nara area of Japan; 1 of 10 amino acids in aonirin differs from those in sodefrin, the prototype peptide (6). This variant peptide was found to not be effective in attracting females in the Niigata and Chiba areas of Japan (7). It was, thus, speculated that altering the primary structure of the female-attracting peptide of the red-bellied newt and coevolution of the corresponding receptor protein may lead to reproductive isolation. To verify this speculation, we are interested in artificially altering a pheromone and its receptor, thereby mimicking coevolution in nature, by using a genetically amenable model organism, the fission yeast Schizosaccharomyces pombe.

Table 1.

Primary structure of peptide pheromones in some red-bellied newts (Cynops sp.) and fission yeasts (Schizosaccharomyces sp.)

Name	Species	Amino acid sequence
Cynops sp.
Sodefrin (prototype)	pyrrhogaster	SIPSKDALLK
Aonirin (Nara area)	pyrrhogaster	SIPSKDAVLK
Silefrin	ensicauda	SILSKDAQLK
Schizosaccharomyces sp.
M factor	pombe	YTPKVPYMC
M factor	octosporus	YQPKPPAMC (presumed)

Open in a separate windowS. pombe has two sexes, which are usually termed mating-type h⁺ [plus (P)] and mating-type h⁻ [minus (M)] (8–10). On nitrogen starvation, two haploid cells of opposite mating type mate to form a diploid zygote (11), which then commences meiosis and finally, culminates in an ascus containing four newly born ascospores. The mating pheromones of S. pombe are small peptides that play essential roles in the courtship reaction. The M-factor pheromone, YTPKVPYMC^Far-OCH₃, is a C-terminally farnesylated nonapeptide secreted by M cells (12–14) that is specifically recognized by a G-protein–coupled receptor, Map3, on the surface of P cells (15). P factor, the mating pheromone secreted by P cells, is a simple peptide composed of 23 amino acids that activates the corresponding receptor, Mam2, on M cells (16, 17). Meiosis also depends on the action of mating pheromone signals (17). Pheromones of S. pombe, thus, play important roles in sexual reproduction, mating, and meiosis.The specificity of mating-type recognition is primarily determined by molecular recognition of the peptide pheromone by its cognate receptor. Mating pheromones play essential roles in sexual maturation, attraction of opposite mating-type cells (cell agglutination), copulation (cell fusion), and mate choice (14, 17–19). For S. pombe, all of the genes encoding the mating pheromones, receptors, and components of the signal transduction cascade emanating from the activated receptors have been identified and thoroughly investigated (11). The primary structures of both mating pheromones and their receptors can be easily altered by in vitro mutagenesis. Because mating competence depends on signaling by both the M- and P-type pheromones, complete impairment of M-factor signaling should prevent the mating reaction (13, 15–17).Activation of the mating pheromone receptor is the initial event during the course of the mating process; thus, structural alteration of the pheromone peptides may affect the downstream signaling pathway. We reasoned that mutational alterations of either the pheromone peptides or the pheromone receptors might result in reproductive isolation from WT cells. If the modified pheromone (a ligand) and the receptor protein are structurally fit and ligand-induced activation of the receptor can be attained, the resulting modified versions of mating-competent cells might constitute a novel reproductive group. Owing to the small size of M factor (9 aa), a full set of single residue-substituted missense mutants of the mfm1 gene (coding for M factor) was previously successfully generated (18). Thorough screening of the 152 mfm1 mutants identified 35 sterile ones that might produce nonfunctional M-factor peptides. These mutant peptides were detected in culture filtrates, indicating that they are likely to be defective in molecular interaction with their specific receptor, Map3 (18).The aim of this study was to identify mutated receptor proteins that could accept any of the mutated M-factor peptides and thereby, create novel mating-type pairs constituting an isolated reproductive group. Here, we report the successful creation of such new reproductive groups isolated from normal mating-type cells. Strict genetic evidence indicates that virtually no gene transfer occurs between the WT and the novel reproductive groups. Our success in prezygotic isolation in the fission yeast population by manipulating mating pheromone recognition systems represents a further advance toward the artificial creation of new species.