Mycological Progress

, Volume 18, Issue 5, pp 763–768 | Cite as

Saprotrophic yeasts formerly classified as Pseudozyma have retained a large effector arsenal, including functional Pep1 orthologs

  • Rahul Sharma
  • Bilal Ökmen
  • Gunther Doehlemann
  • Marco ThinesEmail author
Original Article


The basidiomycete smut fungi are predominantly plant parasitic, causing severe losses in some crops. Most species feature a saprotrophic haploid yeast stage, and several smut fungi are only known from this stage, with some isolated from habitats without suitable hosts, e.g. from Antarctica. Thus, these species are generally believed to be apathogenic, but recent findings that some of these might have a plant pathogenic teleomorph counterpart cast doubts on the validity of this hypothesis. Here, four genomes of species previously assigned to the polyphyletic genus Pseudozyma were re-annotated and compared with published smut pathogens. It was found that 113 genes coding for putative secreted effector proteins were conserved among smut-causing and Pseudozyma genomes. Among these were several validated effector genes, including Pep1. Orthologs of this well-characterised effector from Pseudozyma yeasts were further analysed and checked for their ability to complement a Pep1-deficient mutants of Ustilago maydis. By genetic complementation, we show that Pep1 homologs from the supposedly apathogenic yeasts restore virulence in Pep1-deficient mutants Ustilago maydis. Thus, it is concluded that Pseudozyma species have likely retained a suite of effectors, which hints at the possibility that Pseudozyma species have kept an unknown plant pathogenic stage for sexual recombination. However, it cannot be excluded that these effectors might also have positive effects also when colonising plant surfaces.


Core effectors Effector complementation Plant pathogens Pseudozyma Ustilago Yeast 



We thank Melanie Kastl and Raphael Wemhöner for assistance in generation of U. maydis strains and plant infection assays. This study was first submitted for publication in the summer of 2017. While the main conclusions have stayed the same—Pseudozyma species known only from the yeast stage have a hidden pathogenic stage—claims have been softened due to the criticism of reviewers. None of the reviewers had questioned the validity of the experimental results, but equally none believed in the conclusions deduced from them.

Authors’ contributions

RS carried out bioinformatics work and participated in data analysis; BO carried out the molecular lab work, participated in data analysis, and edited the manuscript; GD designed the study, participated in data analysis, and drafted the manuscript; MT conceived of the study, designed the study, participated in data analysis, coordinated the study, and drafted the manuscript.

Funding information

This study was funded by the LOEWE initiative of the government of Hessen, in the framework of the cluster for Integrative Fungal Research (IPF) and the LOEWE Centre for Translational Biodiversity Genomics (TBG), as well as the Centre of Excellence on Plant Science (CEPLAS).

Supplementary material

11557_2019_1486_Fig3_ESM.png (422 kb)
Figure S1

Southern blot analysis to confirm single integration events. All complementation events were performed in the ip locus in SG200∆umpep1 background. Restriction enzyme, DNA probe that were used and the expected fragments sizes for each southern blot analysis are indicated below each picture. Red arrows indicate single integration event in the correct genomic locus. (PNG 422 kb)

11557_2019_1486_MOESM1_ESM.tif (1.3 mb)
High Resolution Image (TIF 1288 kb)
11557_2019_1486_MOESM2_ESM.doc (190 kb)
ESM 1 (DOC 190 kb)


  1. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676CrossRefGoogle Scholar
  2. Djamei A, Schipper K, Rabe F, Ghosh A, Vincon V, Kahnt J, Osorio S, Tohge T, Fernie AR, Feussner I, Feussner K, Meinicke P, Stierhof YD, Schwarz H, Macek B, Mann M, Kahmann R (2011) Metabolic priming by a secreted fungal effector. Nature 478:395–398CrossRefGoogle Scholar
  3. Doehlemann G, Van Der Linde K, Aßmann D, Schwammbach D, Hof A, Mohanty A, Jackson D, Kahmann R (2009) Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells. PLoS Path 5:e1000290CrossRefGoogle Scholar
  4. Doehlemann G, Reissmann S, Aßmann D, Fleckenstein M, Kahmann R (2011) Two linked genes encoding a secreted effector and a membrane protein are essential for Ustilago maydis-induced tumour formation. Mol Microbiol 81:751–766CrossRefGoogle Scholar
  5. Franceschetti M, Maqbool A, Jiménez-Dalmaroni MJ, Pennington HG, Kamoun S, Banfield MJ (2017) Effectors of filamentous plant pathogens: commonalities amid diversity. Microbiol Mol Biol Rev 81:e00066–e00016CrossRefGoogle Scholar
  6. Gafni A, Claudia C, Raviv H, Buxdorf K, Avis D, Berger ML (2015) Biological control of the cucurbit powdery mildew pathogen Podosphaera xanthii by means of the epiphytic fungus Pseudozyma aphidis. Frontiers Pl Sci 6:1–11Google Scholar
  7. Hawksworth DL, Crous PW, Redhead SA, Reynolds DR, Samson RA, Seifert KA, Taylor JW, Wingfield MJ, Abaci O, Aime C, Asan A, Bai FY, de Beer ZW, Begerow D, Berikten D, Boekhout T, Buchanan PK, Burgess T, Buzina W, Cai L, Cannon PF, Crane JL, Damm U, Daniel HM, van Diepeningen AD, Druzhinina I, Dyer PS, Eberhardt U, Fell JW, Frisvad JC, Geiser DM, Geml J, Glienke C, Gräfenhan T, Groenewald JZ, Groenewald M, de Gruyter J, Guého-Kellermann E, Guo LD, Hibbett DS, Hong SB, de Hoog GS, Houbraken J, Huhndorf SM, Hyde KD, Ismail A, Johnston PR, Kadaifciler DG, Kirk PM, Kõljalg U, Kurtzman CP, Lagneau PE, Lévesque CA, Liu X, Lombard L, Meyer W, Miller A, Minter DW, Najafzadeh MJ, Norvell L, Ozerskaya SM, Oziç R, Pennycook SR, Peterson SW, Pettersson OV, Quaedvlieg W, Robert VA, Ruibal C, Schnürer J, Schroers HJ, Shivas R, Slippers B, Spierenburg H, Takashima M, Taşkın E, Thines M, Thrane U, Uztan AH, van Raak M, Varga J, Vasco A, Verkley G, Videira SI, de Vries RP, Weir BS, Yilmaz N, Yurkov A, Zhang N (2011) The Amsterdam declaration on fungal nomenclature. IMA Fungus 2:105–112CrossRefGoogle Scholar
  8. Hemetsberger C, Mueller AN, Matei A, Herrberger C, Hensel G, Kumlehn J, Mishra B, Sharma R, Thines M, Hückelhoven R, Doehlemann G (2015) The fungal core effector Pep1 is conserved across smuts of dicots and monocots. New Phytol 206:1116–1126CrossRefGoogle Scholar
  9. Kämper J, Kahmann R, Bölker M, Ma LJ, Brefort T, Saville BJ, Banuett F, Kronstad JW, Gold SE, Müller O, Perlin MH, Wösten HA, de Vries R, Ruiz-Herrera J, Reynaga-Peña CG, Snetselaar K, McCann M, Pérez-Martín J, Feldbrügge M, Basse CW, Steinberg G, Ibeas JI, Holloman W, Guzman P, Farman M, Stajich JE, Sentandreu R, González-Prieto JM, Kennell JC, Molina L, Schirawski J, Mendoza-Mendoza A, Greilinger D, Münch K, Rössel N, Scherer M, Vranes M, Ladendorf O, Vincon V, Fuchs U, Sandrock B, Meng S, Ho EC, Cahill MJ, Boyce KJ, Klose J, Klosterman SJ, Deelstra HJ, Ortiz-Castellanos L, Li W, Sanchez-Alonso P, Schreier PH, Häuser-Hahn I, Vaupel M, Koopmann E, Friedrich G, Voss H, Schlüter T, Margolis J, Platt D, Swimmer C, Gnirke A, Chen F, Vysotskaia V, Mannhaupt G, Güldener U, Münsterkötter M, Haase D, Oesterheld M, Mewes HW, Mauceli EW, DeCaprio D, Wade CM, Butler J, Young S, Jaffe DB, Calvo S, Nusbaum C, Galagan J, Birren BW (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444:97–101CrossRefGoogle Scholar
  10. Kemen E, Gardiner A, Schultz-Larsen T, Kemen AC, Balmuth AL, Robert-Seilaniantz A, Bailey K, Holub E, Studholme DJ, Maclean D, Jones JD (2011) Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana. PLoS Biol 9:e1001094CrossRefGoogle Scholar
  11. Konishi M, Hatada Y, Horiuchi J (2013) Draft genome sequence of the basidiomycetous yeast-like fungus Pseudozyma hubeiensis SY62, which produces an abundant amount of the biosurfactant mannosylerythritol lipids. Genome Announc 1:13–14CrossRefGoogle Scholar
  12. Kruse J, Doehlemann G, Kemen E, Thines M (2017) Asexual and sexual morphs of Moesziomyces revisited. IMA Fungus 8:117–129CrossRefGoogle Scholar
  13. Kruse J, Dietrich W, Zimmermann H, Klenke F, Richter U, Richter H, Thines M (2018) Ustilago species causing leaf-stripe smut revisited. IMA Fungus 9:49–73CrossRefGoogle Scholar
  14. Lanver D, Mendoza-Mendoza A, Brachmann A, Kahmann R (2010) Sho1 and Msb2-related proteins regulate appressorium development in the smut fungus Ustilago maydis. Plant Cell 22:2085–2101CrossRefGoogle Scholar
  15. Lanver D, Tollot M, Schweizer G, Lo Presti L, Reissmann S, Ma LS, Schuster M, Tanaka S, Liang L, Ludwig N, Kahmann R (2017) Ustilago maydis effectors and their impact on virulence. Nat Rev Microbiol 15:409CrossRefGoogle Scholar
  16. Latijnhouwers M, De Wit PJGM, Govers F (2003) Oomycetes and fungi: similar weaponry to attack plants. Trends Microbiol 11:462–469CrossRefGoogle Scholar
  17. Laurie JD, Ali S, Linning R, Mannhaupt G, Wong P, Güldener U, Münsterkötter M, Moore R, Kahmann R, Bakkeren G, Schirawski J (2012) Genome comparison of barley and maize smut fungi reveals targeted loss of RNA silencing components and species-specific presence of transposable elements. Plant Cell 24:1733–1745CrossRefGoogle Scholar
  18. Lefebvre F, Joly DL, Labbé C, Teichmann B, Linning R, Belzile F, Bakkeren G, Bélanger RR (2013) The transition from a phytopathogenic smut ancestor to an anamorphic biocontrol agent deciphered by comparative whole-genome analysis. Plant Cell 25:1946–1959CrossRefGoogle Scholar
  19. Lorenz S, Guenther M, Grumaz C, Rupp S, Zibek S, Sohn K (2014) Genome sequence of the basidiomycetous fungus Pseudozyma aphidis DSM70725, an efficient producer of biosurfactant mannosylerythritol lipids. Genome Announc 2:e00053CrossRefGoogle Scholar
  20. Martínez-Soto D, Robledo-Briones AM, Estrada-Luna A, Ruiz-Herrera J (2013) Transcriptomic analysis of Ustilago maydis infecting Arabidopsis reveals important aspects of the fungus pathogenic mechanisms. Plant Signal Behav 8:1–13CrossRefGoogle Scholar
  21. Morita T, Koike H, Koyama Y, Hagiwara H, Ito E, Fukuoka T, Imura T, Machida M, Kitamoto D (2013) Genome sequence of the basidiomycetous yeast Pseudozyma antarctica T-34, a producer of the lycolipid biosurfactants mannosylerythritol lipids. Genome Announc 1:e00064–e00013CrossRefGoogle Scholar
  22. Morita T, Koike H, Hagiwara H, Ito E, Machida M, Machida M, Sato S, Habe H, Kitamoto D (2014) Genome and transcriptome analysis of the basidiomycetous yeast Pseudozyma antarctica producing extracellular glycolipids, mannosylerythritol lipids. PLoS One 9:e86490CrossRefGoogle Scholar
  23. Müller O, Schreier PH, Uhrig JF (2008) Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis. Mol Gen Genomics 279:27–39CrossRefGoogle Scholar
  24. Ökmen B, Kemerich B, Hilbig D, Wemhöner R, Aschenbroich J, Perrar A, Huesgen P, Schipper K, Doehlemann G (2018) Dual function of a secreted fungalysin metalloprotease in Ustilago maydis. New Phytol 220:249–261CrossRefGoogle Scholar
  25. Oliveira JV d C, Dos Santos RAC, Borges TA, Riaño-Pachón DM, Goldman GH (2013) Draft genome sequence of Pseudozyma brasiliensis sp. nov. Strain GHG001, a high producer of endo-1,4-xylanase isolated from an insect pest of sugarcane. Genome Announc 1:e00920–e00913Google Scholar
  26. Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R (2005) InterProScan: protein domains identifier. Nucleic Acids Res 3:W116–W120CrossRefGoogle Scholar
  27. Quinn L, O’Neill PA, Harrison J, Paskiewicz KH, Mccracken AR, Cooke LR, Grant MR, Studholme DJ (2013) Genome-wide sequencing of Phytophthora lateralis reveals genetic variation among isolates from Lawson cypress (Chamaecyparis lawsoniana) in Northern Ireland. FEMS Microbiol Lett 344:179–185CrossRefGoogle Scholar
  28. Redkar A, Villajuana-Bonequi M, Doehlemann G (2015) Conservation of the Ustilago maydis effector See1 in related smuts. Plant Signal Behav 10:e1086855CrossRefGoogle Scholar
  29. Schirawski J, Mannhaupt G, Munch K, Brefort T, Schipper K, Doehlemann G, Di Stasio M, Rössel N, Mendoza-Mendoza A, Pester D, Müller O, Winterberg B, Meyer E, Ghareeb H, Wollenberg T, Münsterkötter M, Wong P, Walter M, Stukenbrock E, Güldener U, Kahmann R (2010) Pathogenicity determinants in smut fungi revealed by genome comparison. Science 330:1546–1548CrossRefGoogle Scholar
  30. Sharma R, Mishra B, Runge F, Thines M (2014) Gene loss rather than gene gain is associated with a host jump from monocots to dicots in the smut fungus Melanopsichium pennsylvanicum. Genome Biol Evol 6:2034–2049CrossRefGoogle Scholar
  31. Sharma R, Xia X, Riess K, Bauer R, Thines M (2015) Comparative genomics including the early-diverging smut fungus Ceraceosorus bombacis reveals signatures of parallel evolution within plant and animal pathogens of fungi and oomycetes. Genome Biol Evol 7:2781–2798CrossRefGoogle Scholar
  32. Tanaka S, Brefort T, Neidig N, Djamei A, Kahnt J, Vermerris W, Koenig S, Feussner K, Feussner I, Kahmann R (2014) A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize. Elife 3:e01355lCrossRefGoogle Scholar
  33. Tanaka E, Koitabashi M, Kitamoto H (2019) A teleomorph of the ustilaginalean yeast Moesziomyces antarcticus on barnyardgrass in Japan provides bioresources that degrade biodegradable plastics. Antonie Van Leeuwenhoek in press.Google Scholar
  34. Ter-Hovhannisyan V, Lomsadze A, Chernoff YO, Borodovsky M (2008) Gene prediction in novel fungal genomes using an ab initio algorithm with unsupervised training. Genome Res 18:1979–1990CrossRefGoogle Scholar
  35. Wang QM, Begerow D, Groenewald M, Liu XZ, Theelen B, Bai FY, Boekhout T (2015) Multigene phylogeny and taxonomic revision of yeasts and related fungi in the Ustilaginomycotina. Stud Mycol 81:55–83CrossRefGoogle Scholar

Copyright information

© German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Senckenberg Gesellschaft für NaturforschungSenckenberg Biodiversity and Climate Research CentreFrankfurt am MainGermany
  2. 2.Department of Biosciences, Institute of Ecology, Evolution and DiversityGoethe UniversityFrankfurt am MainGermany
  3. 3.Botanical Institute and CEPLASUniversity of Cologne, BioCenterCologneGermany
  4. 4.Integrative Fungal Research Cluster (IPF)Frankfurt am MainGermany

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