Genome Sequencing Provides Insight Into the Reproductive Biology, Nutritional Mode and Ploidy of the Fern Pathogen Mixia Osmundae
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Genome sequencing provides insight into the reproductive biology, nutritional mode and ploidy of the fern pathogen Mixia osmundae Toome, M., Ohm, R. A., Riley, R. W., James, T. Y., Lazarus, K. L., Henrissat, B., Albu, S., Boyd, A., Chow, J., Clum, A., Heller, G., Lipzen, A., Nolan, M., Sandor, L., Zvenigorodsky, N., Grigoriev, I. V., Spatafora, J. W. and Aime, M. C. (2014). Genome sequencing provides insight into the reproductive biology, nutritional mode and ploidy of the fern pathogen Mixia osmundae. New Phytologist, 202(2), 554–564. doi:10.1111/nph.12653 10.1111/nph.12653 John Wiley & Sons Ltd. Version of Record http://cdss.library.oregonstate.edu/sa-termsofuse Research Genome sequencing provides insight into the reproductive biology, nutritional mode and ploidy of the fern pathogen Mixia osmundae Merje Toome1, Robin A. Ohm2, Robert W. Riley2, Timothy Y. James3, Katherine L. Lazarus3, Bernard Henrissat4, Sebastian Albu5, Alexander Boyd6, Julianna Chow2, Alicia Clum2, Gregory Heller5, Anna Lipzen2, Matt Nolan2, Laura Sandor2, Natasha Zvenigorodsky2, Igor V. Grigoriev2, Joseph W. Spatafora6 and M. Catherine Aime1 1Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA; 2US Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA; 3Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109, USA; 4Architecture et Fonction des Macromolecules Biologiques, Aix-Marseille University, CNRS UMR 7257, 13288 Marseille, France; 5Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA; 6Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA Summary Author for correspondence: Mixia osmundae (Basidiomycota, Pucciniomycotina) represents a monotypic class contain- M. Catherine Aime ing an unusual fern pathogen with incompletely understood biology. We sequenced and ana- Tel: +1 765 496 7853 lyzed the genome of M. osmundae, focusing on genes that may provide some insight into its Email: [email protected] mode of pathogenicity and reproductive biology. Received: 13 June 2013 Mixia osmundae has the smallest plant pathogenic basidiomycete genome sequenced to Accepted: 19 November 2013 date, at 13.6 Mb, with very few repeats, high gene density, and relatively few significant gene family gains. New Phytologist (2014) 202: 554–564 The genome shows that the yeast state of M. osmundae is haploid and the lack of segrega- doi: 10.1111/nph.12653 tion of mating genes suggests that the spores produced on Osmunda spp. fronds are probably asexual. However, our finding of a complete complement of mating and meiosis genes sug- Key words: biotrophic fungi, CAZy, fungal gests the capacity to undergo sexual reproduction. Analyses of carbohydrate active enzymes genomics, Mixiomycetes, Osmunda spp., suggest that this fungus is a biotroph with the ability to break down several plant cell wall sporogenesis. components. Analyses of publicly available sequence data show that other Mixia members may exist on other plant hosts and with a broader distribution than previously known. Introduction containing numerous small nuclei (Mix, 1947; Kramer, 1958). Spores are exogenously and simultaneously produced on the sur- Mixia osmundae (Nishida) C.L. Kramer (Pucciniomycotina, face of a large sac-like cell that forms from an enlarged swelling Mixiomycetes) is a rarely encountered basidiomycete fungus that within the host epidermal cell wall (Nishida et al., 1995). This has only been isolated from the fronds of ferns belonging to the kind of sporogenous structure and the manner of spore formation genus Osmunda L. (Pteridophyta, Polypodiopsida). The only is unique among fungi. Owing to its sac-like appearance, the spo- records of M. osmundae are from Japan and Taiwan on Osmunda rogenous cell was originally interpreted as an ascus and therefore japonica Thunb. (Nishida et al., 1995; Sugiyama & Katumoto, M. osmundae was first described in the Ascomycota as Taphrina 2008) and from the United States (Georgia and Michigan) on osmundae Nishida (Nishida, 1911). More than 80 yr later, Osmunda cinnamomea L. (Mix, 1947; Kramer, 1958). Mixiomy- detailed morphological and molecular studies showed that cetes is one of only three monotypic classes described in Dikarya M. osmundae holds a rather isolated position within the Basidi- (i.e. Basidiomycota and Ascomycota) and the only one in Pucci- omycota, with the simple-septate species now placed together niomycotina (Aime et al., 2006; Bauer et al., 2006). within Pucciniomycotina (Nishida et al., 1995; Sugiyama, 1998; Most studies of M. osmundae have been confined to the symp- Sjamsuridzal et al., 2002; Aime et al., 2006). tomatic phase on Osmunda spp. Infection is recognizable by the The remaining aspects of the biology and life cycle of small, yellow or brown lesions produced on the fern fronds that M. osmundae are not well known, except that the observed spores develop a powdery white layer of spores (Mix, 1947). Mixia are monokaryotic (Nishida et al., 1995). It is unknown whether osmundae is reported to grow within the plant cell walls, initially these spores are haploid or diploid and, if haploid, whether they forming sparsely septate hyphae that later become coenocytic, are produced as mitospores or meiospores. Since M. osmundae is 554 New Phytologist (2014) 202: 554–564 No claim to original US Government works www.newphytologist.com New Phytologist Ó 2013 New Phytologist Trust New Phytologist Research 555 very rarely collected and the sporogenous cells have only been Prefecture, Japan). The cells were grown on minimal media for observed when growing on the host, spore germination and plant 2 wk, harvested and ground in liquid nitrogen. DNA was infection have never been observed in vivo. When grown on arti- extracted following a modified CTAB protocol described in Pad- ficial media the fungus reproduces by budding, forming creamy amsee et al. (2012). For RNA extraction, a 5-d-old culture grown yeast-like colonies (Nishida et al., 1995, 2011b). As there are sev- on DifcoTM potato dextrose agar (PDA; Becton, Dickinson and eral groups of fungi in Pucciniomycotina that produce basidiosp- Co., Sparks, MD, USA) was used. RNA was isolated using a Ri- ores that can multiply by budding in culture (Swann et al., 2001; boPureTM Yeast Kit (Ambion), followed by DNase treatment with Morrow & Fraser, 2009), it has been speculated that if the spores TURBO DNase (Ambion), all following the manufacturer’s of M. osmundae are meiospores, then the sporogenous cell must recommendations. represent a novel basidium type (Sugiyama, 1998). Although the number of available fungal genomes is increasing Genome sequencing and annotation rapidly, the number of publicly available sequenced genomes within Pucciniomycotina is still small (Grigoriev et al., 2011). For production of the M. osmundae genome, one unamplified These include five species of rust fungi (Pucciniales, Pucciniomy- whole-genome shotgun (WGS) and one 4 kb Long Mate Pair cetes) (Puccinia graminis f. sp. tritici Erikss. & Henning, Puccinia (LMP) library were generated. Quantified sample libraries were striiformis f. tritici Erikss., Puccinia triticina Erikss., Melampsora clustered to flow cells and sequenced on an Illumina HiSeq 2000 larici-populina Kleb. and Cronartium quercuum f. sp. fusiforme platform (Illumina, San Diego, CA, USA). For transcriptome Burds. & G.A. Snow) and five species of Microbotryomycetes sequencing, poly-A RNA was isolated from total RNA and frag- (Sporobolomyces roseus Kluyver & C.B. Niel, Rhodotorula graminis mented for first-strand cDNA synthesis. Digested cDNA was Di Menna, Rhodotorula glutinis (Frensen.) F.C. Harrison, amplified by PCR with Illumina Truseq primers and sequenced Rhodosporidium toruloides I. Banno (Sporidiobolales) and on the Illumina HiSeq 2000 platform, generating 100 bp paired- Microbotryum violaceum (Pers.) G. Deml & Oberw. (Microbot- end reads. ryales)) (Cantu et al., 2011; Duplessis et al., 2011; Kumar et al., The genome was annotated using the JGI annotation Pipeline 2012; www.jgi.doe.gov/fungi; www.broadinstitute.com; www. (Grigoriev et al., 2006), which combines several gene prediction ncbi.nlm.nih.gov). Thus, of the nine classes and 20 orders and annotation methods, and integrates the annotated genome described to date within Pucciniomycotina (Schell et al., 2011; into the web-based fungal resource MycoCosm (Grigoriev et al., Toome et al., 2013; Aime et al., 2014), complete annotated 2012) for comparative genomics. Genome assembly and annota- genomes are only available for two classes and three orders; some tions can be interactively accessed through the JGI fungal incomplete and unannotated genomic data are available for seven genome portal MycoCosm (Grigoriev et al., 2012) at http://jgi. additional Pucciniomycotina species – six additional rust fungi doe.gov/Mosmundae and have also been deposited to DDBJ/ and a draft genome for M. osmundae (Nishida et al., 2011a; EMBL/GenBank under accession no. AYOQ00000000. The ver- www.ncbi.nlm.nih.gov). sion described in this paper is version AYOQ01000000. Some early researchers considered M. osmundae to represent Translated protein sequences were assigned to carbohydrate- the closest extant taxon descended from the common ancestor of active enzyme (CAZy) families using the CAZy pipeline (Canta- Dikarya (Savile, 1955; Kramer, 1987). While modern DNA rel et al., 2009) as described in Floudas et al. (2012). Multigene sequencing studies