Whole-genome sequencing has revealed large quantities of genetic information for many yeast species. These data include gene maps, gene content and gene functions, providing an opportunity for investigating fungal species other than the model organism of baker's yeast, Saccharomyces cerevisiae. One such alternative fungal species is Schizosaccharomyces japonicus. This organism has interesting physiological features that are not present in other fission yeasts: larger cell size, chromosomal behaviour during cell division and hyphal growth. Moreover, the recent development of genetic resources for Sz. japonicus has provided genetic tools comparable to those available for Sz. pombe. As a result, the novel characteristics of Sz. japonicus have begun to yield to genetic analysis. Thus, Sz. japonicus has the potential to serve as a key model organism for understanding the yeast–hyphal cell transition.
History and strains
Two Japanese researchers, Yukawa and Maki (1931), isolated a fission yeast in fermented juices derived from strawberries harvested at a farm of the University of Kyushu, Fukuoka, in May 1928. After characterization, these researchers reported that the isolate represented a new species of fission yeast, which was designated Sz. japonicus (Yukawa and Maki, 1931). This was the first time that a fission yeast had been isolated from a temperate zone, as opposed to tropical or semi-tropical environments. Some 14 years later, an American microbiologist isolated a fission yeast from a batch of home-canned grape juice that was under gas pressure by fermentation. This isolate was reported as a new species of fission yeast designated Sz. versatilis (Wickerham and Duprat, 1945). Subsequently, Sz. versatilis was shown to be a variety of the japonicus-type strain isolated by Yukawa and Maki. In fact, Sz. versatilis and Sz. japonicus cross with each other easily, with a high spore yield (Klar, 2013). Although Sz. japonicus possesses many outstanding characteristics not seen in other Schizosaccharomycetes, only small of group of researchers in East Europe and Japan have further studied the species in the 1970s and 1980s.
One of the unique characteristics of Sz. japonicus is the behaviour of the chromosomes in dividing nuclei in vegetative cells. A series of studies (Robinow and Hyams, 1989) demonstrated, in stained cells under an ordinary microscope, that three chromosomes are separated in mitosis. Surprisingly, the spindle in mitotic nuclei are clearly visible under a phase-contrast microscope. Comparison of Sz. pombe and Sz. japonicus by molecular and structural analyses revealed that tubulin, actin and spindle pole bodies from the two species have distinct properties (Alfa and Hyams, 1990; Horio et al., 2002). Cytological studies, including the use of fluorescently tagged proteins, further demonstrate fundamental differences between the two species in terms of chromosomal and nuclear dynamics (Aoki et al., 2011; Yam et al., 2011).
Hyphal growth is another remarkable property of Sz. japonicus. From the time of its first discovery, Sz. japonicus was noted for its ability, unique among the yeasts, to form hyphae (Wickerham and Duprat, 1945). Many species of fungi are pathogenic to animals and plants, and the pathogenic activity is strongly correlated with hyphal growth, generating a cell type that can penetrate into the host cell. In contrast, a non-pathogenic yeast is an appealing model organism for studying the biology of hyphal growth. Sipiczki et al. (1998a, 1908b) and Enczi et al. (2007) carried out pioneering work analysing hyphal growth by Sz. japonicus, including the isolation of a mutant defective in the formation of hyphae. However, molecular genetic studies had lagged behind because of the paucity of genetic methodologies available for Sz. japonicus. During the past decade, bioresources and genetic tools for studying Sz. japonicus have been developed, as described in the final section of this paper. Notably, whole-genome sequencing has been completed for Sz. japonicus, providing us with the structure of all Sz. japonicus genes (Rhind et al., 2011). We note that research on the fission yeast Sz. pombe has been facilitated by the use of a single clone of the yeast for genetic research. We expect that the exclusive use of the two known strains of Sz. japonicus, the type strain and the variant, which have been distributed to researchers worldwide, will similarly facilitate research on Sz. japonicus.
Fission yeasts in the phylogenetic tree of fungi
The fission yeast clade belongs to the Taphrinomycotina, which is one of three monophyletic subphyla within the Ascomycota and is positioned on a basal branch of the Ascomycota (Rhind et al., 2011) (Figure 1). The fission yeast clade, Schizosaccharomyces, includes four species, Sz. pombe, Sz. octosporus, Sz. cryophilus and Sz. japonicus. All four species exhibit unicellular growth with a binary fission mode of cell division. Among the four species, Sz. japonicus has the largest cell size (Figure 2). In addition to growing as a unicellular yeast, Sz. japonicus generates hyphal cells, forming a true mycelium, a cell type that is common within the larger fungal kingdom. From an evolution/development perspective, a unicellular, yeast-like growth form constitutes one of the earliest lineages during the divergence of the Ascomycota from the Basidiomycota. It appears that a progenitor of the fission yeast acquired a yeast-like growth mode that provided the group with a survival advantage in its early history. Presumably, only Sz. japonicus still harbours this ancestral growth mode derived from the filamentous fungi.
Gene content among the fission yeasts varies widely, consistent with the distinct cellular properties of each species. In fact, each fission yeast species retains hundreds of unique genes, although about 3900 genes are shared among the four species (Rhind et al., 2011) (Figure 3). All four species of the fission yeast have three chromosomes. Despite some subchromosomal synteny, the overall structure of the Sz. japonicus chromosomes differs remarkably from those of Sz. pombe and Sz. octosporus. Comparative analyses of genome structure and gene regulation among the fission yeasts promises to provide tools for investigation across the Schizosaccharomyces clade, thereby potentially elucidating mechanisms of cell division, metabolic activity, and evolutionary history.
Sz. japonicus is able to grow at a high temperature, 40°C, in contrast to Sz. pombe. In spite of larger cell size, the generation time for Sz. japonicus is also faster than that of other yeasts. Sz. japonicus has a doubling time of 100 min at 30°C and 75 min at 37°C in yeast extract–dextran medium, rates that are faster than those of Sz. pombe. Microscopic inspection of vegetatively growing cells reveals dynamic intracellular activities. One of the striking feature of Sz. japonicus is the dynamic behaviour of visibly condensed mitotic chromosomes, so that Sz. japonicus permits better visualization of the process than with other species of fission yeast. Robinow and Hyams (1989) have extensively investigated the behaviour of the nucleus and chromosomes, using phase-constrast microscopy. Even in the absence of a DNA-specific fluorescent stain, chromosomal behaviour can be precisely observed, including the presence of three chromosomes, one of which is associated with the nucleolus. Surprisingly, a single extending band of spindle microtubules can be seen in the nucleus of live Sz. japonicus during anaphase. Indeed, Robinow and Hyams (1989) concluded that: 'the behaviour of the chromosomes in dividing nuclei of vegetative cells is best studied in the larger species of fission yeasts, namely, Sz. japonicus'. More modern approaches are expected to reveal unexpected dynamic behaviour in the nuclei and living cells of Sz. japonicus.
Cells of higher eukaryotes employ open mitosis, meaning that the nuclear envelope breaks down during the process, leaving mitotic chromosomes exposed to the cytoplasm. In contrast, lower eukaryotes, including fungi, employ closed mitosis, whereby segregation of daughter chromosomes occurs within the nucleus while the organelle is still enclosed in an intact nuclear envelope (Sazer, 2005). For instance, an intact nuclear envelope is maintained throughout mitosis in Sz. pombe. However, Sz. japonicus employs an intermediate mode of mitosis (Aoki et al., 2011; Yam et al., 2011). During interphase, the nucleus of Sz. japonicus has a round or ovoid shape. During mitosis, spindle microtubules rapidly extend to separate the daughter chromosomes within the nucleus. By anaphase, extension of spindle microtubules causes the mitotic nucleus to assume a distinctive fusiform shape, with subsequent tearing of the nuclear envelope, exposing the mitotic chromosomes to the cytoplasm. This version of mitosis, intermediate between open and closed, is also referred to as ‘semi-open mitosis’.
Flexibility of the nuclear envelope may control extension of the nucleus and its breakage. Abnormal activity of an enzyme in fatty acid synthesis causes impaired semi-open mitosis because of flexible extension of the nuclear envelope in proportion to the extension of spindle microtubules (Aoki et al., 2013). Anaphase-promoting complex/cyclosome (APC/C) is involved in the regulation of enzyme activity during the metaphase–anaphase transition. Divergent strategies of eukaryotic mitosis could be attributed to control of flexibility of the nuclear envelope via fatty acid composition.
Meiosis and mating type
During vegetative growth, Sz. japonicus cells enter into meiosis following the deterioration of nutrient conditions. Spore formation is also rapidly induced by nitrogen deficiency in both Sz. japonicus and Sz. pombe. The wild-type (type strain) of Sz. japonicus is homothallic (Sipiczki, 1989) and has two cell types for mating; P (plus) and M (minus) are found in cultures of this strain. Switching of the mating types occurs in a manner similar to that observed in Sz. pombe, despite the large sequence divergence and different chromosomal location of mating type cassettes in these organisms (Yu et al., 2013). Following conjugation between the two mating cell types, the nuclei from each cell fuse together in the conjugated cell (karyogamy). As seen in Sz. pombe, dynamic chromosome movement, known as horsetail movement, occurs during the pairing of homologous chromosomes, which is an essential feature of meiosis and begins immediately after nuclear fusion (Chikashige et al., 1994). The horsetail movement of homologous chromosomes in Sz. japonicus was clearly observed under high-resolution microscopy (Figure 4A). As a result of meiosis, an ascus containing eight mature ascospores is formed (Figure 4B). Autolysis of the cell wall releases ascospores from the asci. Interestingly, the crossing of Sz. japonicus and its variety, Sz. versatilis, generates ascospores with high viability for germination (Klar, 2013).
Yeasts constitute a subgroup of fungi, in which cells grow in a unicellular fashion by budding or by binary fission. However, many types of yeast can switch between growth modes, changing from unicellular growth to filamentous branching (multicellular hyphae), as seen in other fungi, namely dimorphism. Sz. japonicus is a dimorphic yeast, one in which yeast cells transform to hyphal cells in response to external stress (Sipiczki et al., 1998a, 1998b). In practice, the hyphal transition is induced by a wide variety of environmental changes, ranging from pH to the nature of the carbon source. Thus, hyphal transition is a simple mode of cellular differentiation that is turned on in response to environmental conditions.
When grown on yeast extract–dextran agar plates (YE medium), Sz. japonicus does not transition to hyphal growth, even after long periods of incubation. On the other hand, Sz. japonicus yeast colonies growing on other types of solid medium are surrounded by extending hyphal cells after long incubation (e.g. at least a week). Inclusion of high concentrations of glucose in such media prevents the hyphal transition, as does the inclusion of caffeine or cAMP (Sipiczki et al., 1998a). In addition, hyphal extension from yeast colonies is induced by a gradient of the nitrogen source across the agar plate (Sipiczki et al., 1998b). Thus, nutrient stress induces the hyphal transition, and the transition also can be induced in the opposite direction upon nutrient provision. Recently, the use of a low flow-rate microchamber was described for induction and observation of hyphal growth in liquid medium (Furuya and Niki, 2010).
In addition to nutrient stress, genotoxic stresses that generate DNA lesions also induce transition from yeast to hyphal growth (Shi et al., 2007; Furuya and Niki, 2010). In Sz. japonicus, a low dose of the topoisomerase inhibitor camptothecin (CPT) induces hyphal differentiation, even under rich nutrient conditions or in liquid medium (Furuya and Niki, 2010). The dose of CPT is lower than that required for the induction of checkpoint arrest by DNA damage. Upon removal of the drug, hyphal cells transition back to yeast cells. Thus, CPT is an inducer for reversible hyphal differentiation, and is expected to facilitate investigation of the transition.
The role of each component of the DNA damage and DNA replication checkpoints is conserved between Sz. japonicus and Sz. pombe. The switching from yeast growth mode to hyphal growth mode by CPT is regulated via a checkpoint kinase, Chk1, which is involved in the DNA damage checkpoint but not in the DNA replication checkpoint (Furuya and Niki, 2010). Moreover, the induction of hyphal growth depends on the components of the Chk1-dependent DNA checkpoint pathway (Furuya and Niki, 2012). Indeed, the hyphal differentiation pathway depends on checkpoint genes that function in the DNA damage checkpoint pathway, but not on genes that function in the DNA replication checkpoint pathway. Thus, the DNA damage checkpoint is the only pathway that governs DNA damage-dependent hyphal growth.
Other studies have demonstrated that the regulation of hyphal transition by nutritional stress occurs downstream of the DNA damage checkpoint. Notably, cAMP levels are altered by by nutritional status, and exogenous cAMP reverses nutrient-dependent hyphal growth as well as CPT-induced hyphal growth (Sipiczki et al., 1998a). Thus, inhibitory regulation by cAMP appears to be located downstream of both DNA damage- and nutrient-stress signals. Similar effects are seen with exogenously added caffeine (Sipiczki et al., 1998a). These findings suggest that hyphal induction and growth are inhibited by some metabolites that would be secreted into solid medium. Alternatively, yeast cells gradually switch to hyphal form after extended incubation on solid substrates, because consumption of the nutrients in the solid substrate causes the subcellular level of cAMP to decline.
Many filamentous fungi sense light and change their metabolic activities and growth mode in response to light (Corrochano and Avalos, 2010). A few yeasts also demonstrate photoresponsivity. One of them is the heterothallic basidiomycetous yeast Cryptococcus neoformans, in which the mating process is influenced by blue light (Idnurm and Heitman, 2005; Lu et al., 2005). Specifically, when vegetatively grown Cryptococcus yeast cells are combined under conditions of nitrogen limitation, individuals of opposing mating type conjugate and form dikaryotic filamentous cells. This process is inhibited by exposure to blue light. Thus, light signals function as negative regulators of spore formation in C. neoformans. In contrast, light is a positive regulator of mating by Sz. japonicus: sexual flocculation, the process of aggregation between cells of opposite mating types, is inhibited in the absence of light (Itoh et al., 1976). In both C. neoformans and Sz. japonicus, light sensing depends on orthologues of the Neurospora crassa white collar complex (WCC) proteins. In N. crassa, the WCC is activated by blue light and controls the induction of carotenoid pigment production, the formation of sexual fruiting bodies and circadian rhythm (Corrochano and Avalos, 2010). Structural studies have shown that the WCC consists of hetero/homo-dimeric transcription factors that include a blue light-responsive flavin adenine dinucleotide (FAD) chromophore as well as several protein domains [light, oxygen or voltage (LOV) and PER–ARNT–SIM (PAS)] implicated in FAD recognition and protein–protein binding.
Orthologues of WCC proteins are widely conserved among the fungi, including more than 80 genomes to date (Idnurm et al., 2010; Okamoto et al., 2013). So far, only three yeasts have been shown to include these proteins: one basidiomycete (C. neoformans) and two ascomycetes (Sz. japonicus and Yarrowia lipolytica). We could not detect photoresponsivity in Y. lipolytica (our unpublished data) and are not aware of any such evidence in the literature. Intriguingly, all three of these species are dimorphic yeasts, with the Sz. japonicus and Y. lipolytica orthologues showing best homology to the basidiomycetous proteins (Figure 1). This pattern suggests that this type of photoresponse is ancient within the fungal kingdom.
In Sz. japonicus, not only do yeasts show light sensitivity of sexual flocculation, but hyphal cells also show a periodic photoresponse (Okamoto et al., 2013). Notably, during incubation at 30°C, a colony of yeasts is formed, and hyphal cells extend from the periphery of the colony. When light cycles (12 h light/ 12 h dark) are applied during hyphal growth, dark- and light-coloured stripes are observed in the hyphal growth zone (Figure 5); septate hyphae are enriched within the dark-coloured stripes. Thus, cytokinesis in growing hyphal cells appears to be synchronously activated by light. Analyses implicate WCC homologues (Wcs1 and Wcs2) in this stripe formation and in blue light sensing.
In addition, alteration of temperature induces a response in hyphal cells similar to that caused by light (Okamoto et al., 2013). Specifically, temperature cycling (12:12 h) between 30°C and 35°C during incubation in the dark results in the formation of alternating dark- and light-coloured stripes. Such stripes in response to temperature cycling persist in wcs gene mutants, demonstrating that the temperature-cycling response is independent of photoperiodicity. Thus, temperature sensing is mediated by an unknown mechanism in Sz. japonicus.
Both light and temperature (daily external cues) lead the growing hyphal cells to synchronously activate the cell division cycle of Sz. japonicus. The coordination of cell growth with a diurnal circadian rhythm is expected to provide the organism with selective advantages, as seen in a range of other organisms.
Bioresources and tools for genetic studies
The wild-type of Sz. japonicus is homothallic (Sipiczki, 1989), making it difficult to isolate mutants in primary screens or to analyse mutations using genetic crosses. This challenge has been one of the primary reasons why Sz. japonicus had not been the subject of further studies. Recent attempts to construct genetic tools for use with Sz. japonicus have yielded several new genetic resources, including the isolation of auxotrophic (nutrient-requiring) mutants and the identification of a shuttle vector (plasmid). One of the largest advances has been the determination of the Sz. japonicus type strain (IFO1609; ATCC10660) genome sequence by the Broad Institute; this information is now publicly available via the Broad Institute website (http://www.broad.mit.edu/) (Rhind et al., 2011). Based on this genome information, mutants harbouring gene knockouts and fluorescently tagged genes have been, and continue to be, constructed. Mutant strain construction can be performed using gene targeting/replacement, as in Sz. pombe.
As a result, many helpful strains of Sz. japonicus are available. All of these strains, which are isogenic with the sequenced type strain, can be obtained via the bioresource centr for Sz. japonicus, the Japonet. Examples of these strains/genetic tools are provided below:
Mating type. Heterothallic strains have been isolated from IFO1609. The true– heterothallic strains (h–-like, h+) and derivatives harbouring selective marker genes are available (Furuya and Niki, 2009).
Diploid construction. The construction of diploid cells is critical for characterization of essential genes. Sz. japonicus diploids can be constructed by using ade6 point mutants that suppress each other via interallelic complementation (Furuya and Niki, 2011). With the ade6 system, strains carrying ade6-domK or ade6-domE mutations exhibit adenine auxotrophy and grow as red colonies on low adenine concentration agar plates. Mating provides interallelic complementation, permitting the isolation of diploid zygotes by selection for adenine independence.
Genetic mapping. To permit the determination of relative genetic distances by mating, a series of insertion-bearing strains were systematically constructed (Furuya et al., 2012). Each carries a positive–negative selection marker inserted at different loci, located every 500 kb throughout the genome. Each marker becomes a ‘scale mark’ on the chromosome, behaving like a yardstick to indicate position on the chromosomes.
Plasmids. An autonomous replicating sequence (ARS) for Sz. japonicus has not yet been formally defined. Notably, Sz. pombe-derived plasmids (with an Sz. pombe ARS) cannot be maintained in Sz. japonicus, consistent with the distinct mitotic behaviours and spindle pole bodies noted above. A DNA fragment including the Sz. japonicus cut1 gene was identified as a potential Sz. japonicus ARS (Bozsik et al., 2002), but this putative ARS exhibited a low efficiency of transformation and was not sufficient for standard cloning. Subsequent efforts identified a sequence that conferred high transformation activity in Sz. japonicus cells (Aoki et al., 2010). This sequence was used to construct pSJU11 (a pUC19-based vector including Sz. pombe ura4+ as a selectable marker) and pSJK11 (which includes a kanamycin resistance-encoding gene). Sz. japonicus can be transformed with these plasmids by electroporation of early log-phase cells. Interestingly, the ARS derived from Sz. japonicus is functional in Sz. pombe cells, permitting construction of interspecies shuttle vectors.
Mating. Sz. japonicus has mating types switching and meiosis that are similar to those of Sz. pombe. When mixtures of h– and h+ heterothallic strains are cultured in liquid synthetic medium lacking nitrogen, conjugation (mating) occurs after several cell division cycles (Furuya and Niki, 2009). The resulting ascus consists of eight ascospores. Notably, the ascus cell wall of the Sz. japonicus type strain lacks dextrose, and so sporulation can be detected as an absence of staining by iodine. In contrast, asci of the versatilis variant are positive for iodine staining (Klar, 2013). Thus, this variant provides a marker that can be used to identify strains defective for meiosis and spore formation.
Fungi differentiate to adapt to environmental challenges. Filamentous (hyphal) growth is one such adaptation, permitting invasion of growth substrates, including plant and animal tissues, by fungal pathogens. Dimorphic yeasts, able to transition between unicellular and hyphal growth, offer an appropriate model for studying differentiation and the requirements for induction of hyphal growth. However, hyphal growth by pathogenic dimorphic yeasts is not always readily achievable in the laboratory, and genetic analysis of the hyphal growth phase and transition is often limited by the lack of appropriate tools. Thus, a genetically tractable non-pathogenic dimorphic yeast constitutes an attractive model for investigating invasive hyphal growth. The non-pathogenic fission yeast Sz. japonicus is evolutionarily similar to the well-characterized fission yeast Sz. pombe, and many of the genetic approaches used with Sz. pombe can be applied to the study of hyphal growth in Sz. japonicus. The confluence of appealing characteristics and genetic tools in Sz. japonicus promises to facilitate research of value to both basic and applied biology.
I would like to thank K. Aoki and S. Okamoto for providing figures. I also thank Amar Klar for critical reading and comments. This work was supported by MEXT KAKENHI (Grant No. 24114516).