Fungal Genetics and Biology 45 (2008) S15–S21

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Review

Sex in smut fungi: Structure, function and evolution of mating-type complexes Guus Bakkeren a, Jörg Kämper b, Jan Schirawski c,* a b c

Agriculture & Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, BC, Canada V0H 1Z0 University of Karlsruhe, Institute for Applied Biosciences, Department of Genetics, 76187 Karlsruhe, Germany Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany

a r t i c l e

i n f o

Article history: Received 4 February 2008 Accepted 11 April 2008 Available online 22 May 2008 Keywords: Pheromone receptor Homeodomain Sporisorium reilianum Ustilago hordei Malassezia globosa

a b s t r a c t Smut fungi are basidiomycete plant pathogens that pose a threat to many important cereal crops. In order to be pathogenic on plants, smut fungal cells of compatible mating-type need to fuse. Fusion and pathogenicity are regulated by two loci, a and b, which harbor conserved genes. The functions of the encoded mating-type complexes have been well-studied in the model fungus Ustilago maydis and will be briefly reviewed here. Sequence comparison of the mating-type loci of different smut and related fungi has revealed that these loci differ substantially in structure. These structural differences point to an evolution from tetrapolar to bipolar mating behavior, which might have occurred several independent times during fungal speciation. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Smut fungi are important cereal crop pathogens that depend on sex to cause disease. There are approximately 1200 smut species known that together can infect more than 4000 different plant species. The vast majority of plant species serving as hosts are from the grass family (Graminaceae) and includes the world’s most important crops: corn, barley, wheat, oats, sorghum, sugarcane and forage grasses. Smut symptoms are characterized by the formation of fruiting structures containing black masses of teliospores that give the infected tissue a ‘‘sooty” or ‘‘smutted” appearance. While most smut fungi develop sexual spores exclusively in the inflorescence and symptoms are visible only late in the infection upon heading, Ustilago maydis, the corn smut pathogen, is a notable exception that can induce tumors, in which the fungal spores develop, on all above-ground parts of the plant. Since fungal biomass usually develops in inflorescences of infected plants, considerable yield reductions are suffered. Therefore, it is not surprising that smuts have been the subject of intense study for the last century. However, molecular insight on mating systems and their function has so far only been obtained for three smut species, the corn smuts U. maydis and Sporisorium reilianum and the barley smut Ustilago hordei. All three species show a close phylogenetic relationship (Bakkeren et al., 2000) that is reflected by the ease with which molecular tools developed for one species can be transferred to the other species. The elucidation of the complete genome sequence of U. maydis (Kämper et al., 2006) has enormously ad* Corresponding author. Fax: +49 6421 178609. E-mail address: [email protected] (J. Schirawski). 1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2008.04.005

vanced our understanding of the biology of smut fungi. The U. maydis genome sequence is publicly available through the Broad Institute (http://www.broad.mit.edu/annotation/fungi/ustilago_maydis/) and has been manually annotated by the Munich Institute for Protein Sequences (MIPS; http://MIPS.gsf.de/genre/ proj/ustilago/). Genome sequencing efforts are under way for S. reilianum (R. Kahmann and J. Schirawski, unpublished) and for U. hordei (R. Kahmann, J. Schirawski and G. Bakkeren, unpublished) that will allow whole genome comparison of these related smut species and is expected to lead to insights into the determinants of host selection and symptom formation. Common to all smut species investigated so far is their need to undergo a successful mating reaction to form dikaryotic hyphae before being able to infect a host plant. On the other hand, the infectious dikaryon requires a host for proliferation and for the formation of sexual spores. Thus, sex and pathogenesis are intimately intertwined in these species. For mating to occur, two haploid cells of different mating-type need to recognize each other and fuse to form the infectious dikaryon. Mating is regulated by two loci, a and b, which harbor conserved genes. At the a locus, these genes encode pheromones and pheromone receptors while at the b locus two subunits of a heterodimeric transcription factor are encoded. In S. reilianum and U. maydis that have a tetrapolar mating system, these genetic loci segregate independently, while in bipolar species, such as U. hordei, the a and b loci are linked and MAT segregates as one locus. Despite the similarity in gene function and sequence, in the three smuts that have been analyzed at a molecular level (U. maydis, U. hordei and S. reilianum), the mating-type loci differ substantially in locus structure. These differences will be reviewed to cover

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function, structure and evolution of mating complexes in comparison to a few more distantly related fungal basidiomycete species for which molecular data exist. 2. Structure of mating-type loci The b mating-type genes encode two subunits of a homeodomain (HD) transcription factor, consisting of an HD1 class and an HD2-class protein. In general, the HD1 and HD2 proteins are not related to each other in primary sequence; however, they share a common domain organization. The N-terminal regions of the HD proteins contain the highest degree of variation when different alleles are compared and are thus designated as variable regions, while the C-terminal regions of the proteins, including the homeodomains, are highly conserved (Gillissen et al., 1992; Kronstad and Leong, 1990; Schulz et al., 1990). The tetrapolar species U. maydis and S. reilianum as well as the bipolar species U. hordei possess one divergently transcribed gene pair encoding the homeodomain proteins bE (HD1) and bW (HD2; Fig. 1). For the b mating-type genes of U. maydis, S. reilianum and the U. hordei MAT-1 locus, gene order, orientation, as well as the genomic context are conserved (Fig. 1). The orientation of the divergently transcribed b gene pair seems inverted in the U. hordei MAT-2 locus. In the absence of additional sequence information for the MAT-2 locus of U. hordei, it cannot be assessed, whether this gene pair is part of an inversion covering a large genomic context. While from U. hordei two

different mating-type alleles are known (Bakkeren and Kronstad, 1994), five different b alleles have been described in S. reilianum (Schirawski et al., 2005) and at least 19 exist in U. maydis (J. Kämper and R. Kahmann, unpublished). Interestingly, both bE and bW mating-type genes are present in the genome of the Ustilaginomycete Malassezia globosa (Table 1), a human pathogenic fungus involved in dandruff disease, for which no sexual cycle is known (Xu et al., 2007). Even the distantly related, opportunistic human pathogen, the Tremellomycete Cryptococcus neoformans (Table 1), carries HD1- and HD2-encoding genes within the mating-type region (Loftus et al., 2005). For C. neoformans, two mating types are known but in this species each mating type has retained only one of the b mating-type genes, with the HD1 protein being specific for the MATa mating type and the HD2 protein being present only in the MATa mating-type region (Fig. 1; Fraser et al., 2004; Lengeler et al., 2002). In addition to the b mating-type complexes, smut fungi contain genes necessary for cell–cell recognition located in the a matingtype loci, encoding pheromones and pheromone receptors. The detailed structure of these loci has been determined for both a alleles of U. maydis, for all three a alleles of S. reilianum and for the MAT-1 allele of U. hordei (Fig. 1). For the MAT-2 allele of U. hordei only partial information is available (Fig. 1). Both U. maydis and U. hordei have two alleles of an a mating system with one pheromone receptor (pra) and one functional pheromone gene (mfa) per locus (Anderson et al., 1999; Bakkeren and

Fig. 1. Genetic organization of the mating-type loci of selected basidiomycetes. Genes are indicated by arrows with the arrow denoting direction of transcription. Related genes are denoted by the same color and respective gene functions are explained in the lower part of the Figure.  indicates that the relative order and orientation of these genes has not been determined. In the tetrapolar species U. maydis and S. reilianum the a and b specific sequences reside on different chromosomes while they are linked by spacer regions (which are not drawn to scale and whose length is indicated) in the bipolar species U. hordei and C. neoformans, as well as in M. globosa. The black bars on top of the figure indicate the regions of the b locus, which covers the two homeodomain protein genes bE and bW, and the a locus (that expands to different length in the different loci, indicated by a broken line) from the lba gene to the rba gene. Sequence information was obtained from the following Accession Nos. AF043940, AM118080, AACP01000083, AACP01000013, AJ884588, AJ884583, AJ884590, AJ884585, AJ884589, AJ884584, U37796, M84182, AF184070, AF184069, Z18531, AAYY01000003, AF542530, and AF542531.

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G. Bakkeren et al. / Fungal Genetics and Biology 45 (2008) S15–S21 Table 1 Taxonomic information on the basidiomycete fungi compared Class Agaricomycotina Agaricomycetes

Tremellomycetes Ustilaginomycotina Ustilaginomycetes

Species

Mating system

Reference

Coprinopsis cinerea (syn. Coprinus cinereus) Pholiota nameko Pleurotus djamor Schizophyllum commune Laccaria bicolor Filobasidiella (syn. Cryptococcus) neoformans; Cryptococcus gattii

Tetrapolar Bipolar Tetrapolar Tetrapolar Tetrapolar Bipolar

Casselton and Kües (2007) Aimi et al. (2005), Ratanatragooldacha et al. (2002) James et al. (2004) Fowler and Vailllancourt (2007), Kothe (1996) Martin et al. (2008) Fraser et al. (2004), Lengeler et al. (2002)

Malassezia globosa Sporisorium reilianum Ustilago hordei Ustilago maydis

Bipolar Tetrapolar Bipolar Tetrapolar

Xu et al. (2007) Schirawski et al. (2005) Anderson et al. (1999), Bakkeren et al. (2006) Kämper et al. (2006)

Kronstad, 1996; Bölker et al., 1992). The sequences of the pheromone and receptor genes in the a2 locus differ from those in the a1 locus. Additionally, sequences immediately surrounding the pheromone and receptor genes are unrelated between different a mating types. Thus, the a1 locus of U. maydis encompasses a 4-kb region, while the a2 locus extends over 8 kb and contains two additional a2-specific genes, rga2 and lga2, with a possible function in uniparental mitochondrial inheritance (Bortfeld et al., 2004). The a1 and a2 loci of S. reilianum are syntenic to the a1 and a2 loci of U. maydis, respectively, with conservation of gene content, order, position and genomic context (Fig. 1). However, in S. reilianum, both loci contain an additional pheromone gene that allows recognition of S. reilianum strains of a3 mating type (Schirawski et al., 2005). Correspondingly, the a3 mating-type locus of S. reilianum also contains one pheromone receptor and two pheromone genes, one each for recognition of a1 and a2 mating partners (Schirawski et al., 2005). Interestingly, the genome of the anamorphic species M. globosa also contains one pheromone and one pheromone receptor gene (Xu et al., 2007), raising the interesting possibility that this species is mating competent. Pheromone and pheromone receptor genes are also present in both the MATa and the MATa mating-type regions of C. neoformans. Each region contains one pheromone receptor gene and three pheromone genes (Fraser et al., 2004; Lengeler et al., 2002). However, while the two pheromone genes in the S. reilianum a locus encode different pheromones, the three pheromone genes of the C. neoformans MAT locus code for identical pheromones (McClelland et al., 2002). Comparison of the 527-kb large MAT-1 locus of U. hordei with the genome of U. maydis revealed that more than 90% of the genes encoded in the U. hordei MAT-1 region have orthologs in the vicinity of either the U. maydis a or the b mating-type genes (Bakkeren et al., 2006). Gene order is conserved over several regions of the U. hordei MAT-1 locus, but inversions are apparent and in some areas the U. hordei genes have homologs in unrelated regions of the U. maydis genome. One of the hallmarks of the U. hordei MAT-1 locus is an accumulation of repetitive elements covering more than 50% of this large region. These repetitive elements are not found in the syntenic regions (or any other part of the genome) of U. maydis. Many of the elements show long terminal repeat (LTR) structures reminiscent of (retro-) transposable elements, some of which are of the copia and gypsy type (Bakkeren et al., 2006). Many of the classes of elements are scattered over the U. hordei genome but certain classes seem to have accumulated disproportionately in and near the MAT-1 region. Specifically, the repetitive elements are often found in-between syntenous gene-rich stretches and bordering inversions in the MAT-1 locus, suggesting their involvement in genome rearrangement during evolution (Bakkeren et al., 2006).

Overall, sequence analysis and comparison of the mating-type regions of tetrapolar and bipolar smut fungi revealed that they are not fundamentally different. Both bipolar and tetrapolar smuts and related species contain the genes for the a and b mating-type complexes. In the tetrapolar species U. maydis and S. reilianum, the a and b mating-type genes reside on different chromosomes and therefore segregate independently during meiosis giving rise to progeny with four different mating types. In contrast, the mating-type complexes of the bipolar species U. hordei and C. neoformans are encoded on the same chromosome and in a recombination-suppressed region ensuring genetic linkage (Bakkeren et al., 2006; Bakkeren and Kronstad, 1994; Fraser et al., 2004). The recently published genome of the dandruff-causing fungus M. globosa reveals an average of 50% amino acid similarity with predicted proteins in U. maydis, although gene synteny among these species is only 15% for adjacent genes and