title: The fungal core effector Pep1 is conserved across smuts of dicots and monocots.
abstract: The secreted fungal effector Pep1 is essential for penetration of the host epidermis and establishment of biotrophy in the Ustilago maydis-maize pathosystem. Previously, Pep1 was found to be an inhibitor of apoplastic plant peroxidases, which suppresses the oxidative burst, a primary immune response of the host plant and enables fungal colonization. To investigate the conservation of Pep1 in other pathogens, genomes of related smut species were screened for pep1 orthologues. Pep1 proteins were produced in Escherichia coli for functional assays. The biological function of Pep1 was tested by heterologous expression in U. maydis and Hordeum vulgare. Pep1 orthologues revealed a remarkable degree of sequence conservation, indicating that this effector might play a fundamental role in virulence of biotrophic smut fungi. Pep1 function and its role in virulence are conserved in different pathogenic fungi, even across the monocot-dicot border of host plants. The findings described in this study classify Pep1 as a phylogenetically conserved fungal core effector. Furthermore, we documented the influence of Pep1 on the disease caused by Blumeria graminis f. sp. hordei which is a non-smut-related pathosystem.
authors: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Str. 10, D-35043, Marburg, Germany.|Lehrstuhl für Phytopathologie, Technische Universität München, Emil-Ramann-Str. 2, D-85350, Freising-Weihenstephan, Germany.
authors: Christoph
authors: C
authors: Hemetsberger
authors: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Str. 10, D-35043, Marburg, Germany.
authors: André N
authors: AN
authors: Mueller
authors: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Str. 10, D-35043, Marburg, Germany.
authors: Alexandra
authors: A
authors: Matei
authors: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Str. 10, D-35043, Marburg, Germany.
authors: Christian
authors: C
authors: Herrberger
authors: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, D-06466, Stadt Seeland/OT Gatersleben, Germany.
authors: Götz
authors: G
authors: Hensel
authors: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, D-06466, Stadt Seeland/OT Gatersleben, Germany.
authors: Jochen
authors: J
authors: Kumlehn
authors: Biodiversity and Climate Research Centre (BiK-F), Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, D-60325, Frankfurt am Main, Germany.|Integrative Fungal Research Cluster (IPF), Georg-Voigt-Str. 14-16, D-60325, Frankfurt am Main, Germany.|Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt am Main, Max-von-Laue-Str. 13, D-60438, Frankfurt am Main, Germany.
authors: Bagdevi
authors: B
authors: Mishra
authors: Biodiversity and Climate Research Centre (BiK-F), Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, D-60325, Frankfurt am Main, Germany.|Integrative Fungal Research Cluster (IPF), Georg-Voigt-Str. 14-16, D-60325, Frankfurt am Main, Germany.|Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt am Main, Max-von-Laue-Str. 13, D-60438, Frankfurt am Main, Germany.
authors: Rahul
authors: R
authors: Sharma
authors: Biodiversity and Climate Research Centre (BiK-F), Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, D-60325, Frankfurt am Main, Germany.|Integrative Fungal Research Cluster (IPF), Georg-Voigt-Str. 14-16, D-60325, Frankfurt am Main, Germany.|Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt am Main, Max-von-Laue-Str. 13, D-60438, Frankfurt am Main, Germany.
authors: Marco
authors: M
authors: Thines
authors: Lehrstuhl für Phytopathologie, Technische Universität München, Emil-Ramann-Str. 2, D-85350, Freising-Weihenstephan, Germany.
authors: Ralph
authors: R
authors: Hückelhoven
authors: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Str. 10, D-35043, Marburg, Germany.|Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Biocenter, Zuelpicher Str. 47a, 50674 Cologne, Germany.
authors: Gunther
authors: G
authors: Doehlemann
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D005658:Fungi / Q000235:genetics* / Q000472:pathogenicity; D016681:Genome, Fungal; D001467:Hordeum / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D054884:Host-Pathogen Interactions / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D017382:Reactive Oxygen Species / Q000378:metabolism; D016415:Sequence Alignment; D014588:Ustilago / Q000235:genetics / Q000472:pathogenicity
chemical_list: D003062:Codon; D005656:Fungal Proteins; D017382:Reactive Oxygen Species
keywords: Ustilago maydis; biotrophy; effector; immune suppression; pathogen evolution
references: Aichinger C, Hansson K, Eichhorn H, Lessing F, Mannhaupt G, Mewes W, Kahmann R. 2003. Identification of plant-regulated genes in Ustilago maydis by enhancer-trapping mutagenesis. Molecular Genetics and Genomics 270: 303-314.
references: Anderson RG, Casady MS, Fee RA, Vaughan MM, Deb D, Fedkenheuer K, Huffaker A, Schmelz EA, Tyler BM, McDowell JM. 2012. Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants. Plant Journal 72: 882-893.
references: Babaeizad V, Imani J, Kogel KH, Eichmann R, Hückelhoven R. 2009. Over-expression of the cell death regulator Bax inhibitor-1 in barley confers reduced or enhanced susceptibility to distinct fungal pathogens. TAG. Theoretical and Applied Genetics 118: 455-463.
references: Bolwell GP, Butt VS, Davies DR, Zimmerlin A. 1995. The origin of the oxidative burst in plants. Free Radical Research 23: 517-532.
references: Bozkurt TO, Schornack S, Win J, Shindo T, Ilyas M, Oliva R, Cano LM, Jones AME, Huitema E, van der Hoorn RAL et al. 2011. Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface. Proceedings of the National Academy of Sciences, USA 108: 20832-20837.
references: Brefort T, Doehlemann G, Mendoza-Mendoza A, Reissmann S, Djamei A, Kahmann R. 2009. Ustilago maydis as a pathogen. Annual Review of Phytopathology 47: 423-445.
references: Djamei A, Kahmann R. 2012. Ustilago maydis: dissecting the molecular interface between pathogen and plant. PLoS Pathogens 8: e1002955.
references: 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 Pathogens 5: e1000290.
references: Dong S, Stam R, Cano LM, Song J, Sklenar J, Yoshida K, Bozkurt TO, Oliva R, Liu Z, Tian M et al. 2014. Effector specialization in a lineage of the Irish potato famine pathogen. Science 343: 552-555.
references: Dunford HB. 1999. Heme peroxidases. New York, NY, USA: Wiley VCH.
references: Eichmann R, Bischof M, Weis C, Shaw J, Lacomme C, Schweizer P, Duchkov D, Hensel G, Kumlehn J, Hückelhoven R. 2010. BAX INHIBITOR-1 is required for full susceptibility of barley to powdery mildew. Molecular Plant-Microbe Interactions 23: 1217-1227.
references: van Esse HP, van't Klooster JW, Bolton MD, Yadeta KA, van Baarlen P, Boeren S, Vervoort J, de Wit PJGM, Thomma BPHJ. 2008. The Cladosporium fulvum virulence protein Avr2 inhibits host proteases required for basal defense. Plant Cell 20: 1948-1963.
references: Hemetsberger C, Herrberger C, Zechmann B, Hillmer M, Doehlemann G. 2012. The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity. PLoS Pathogens 8: e1002684.
references: Hensel G, Valkov V, Middlefell-Williams J, Kumlehn J. 2008. Efficient generation of transgenic barley: the way forward to modulate plant-microbe interactions. Journal of Plant Physiology 165: 71-82.
references: Himmelbach A, Liu L, Zierold U, Altschmied L, Maucher H, Beier F, Muller D, Hensel G, Heise A, Schutzendubel A et al. 2010. Promoters of the barley germin-like GER4 gene cluster enable strong transgene expression in response to pathogen attack. Plant Cell 22: 937-952.
references: Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H. 2001. A large family of class III plant peroxidases. Plant and Cell Physiology 42: 462-468.
references: Hof A, Zechmann B, Schwammbach D, Hückelhoven R, Doehlemann G. 2014. Alternative cell death mechanisms determine epidermal resistance in incompatible barley-Ustilago interactions. Molecular Plant-Microbe Interactions 27: 403-414.
references: Hoffman CS, Winston F. 1987. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57: 267-272.
references: Hogenhout SA, Van der Hoorn RA, Terauchi R, Kamoun S. 2009. Emerging concepts in effector biology of plant-associated organisms. Molecular Plant-Microbe Interactions 22: 115-122.
references: Hückelhoven R, Kogel K-H. 1998. Tissue-specific superoxide generation at interaction sites in resistant and susceptible near-isogenic barley lines attacked by the powdery mildew fungus (Erysiphe graminis f. sp. hordei). Molecular Plant-Microbe Interactions 11: 292-300.
references: Johrde A, Schweizer P. 2008. A class III peroxidase specifically expressed in pathogen-attacked barley epidermis contributes to basal resistance. Molecular Plant Pathology 9: 687-696.
references: Jones JD, Dangl JL. 2006. The plant immune system. Nature 444: 323-329.
references: de Jonge R, van Esse HP, Kombrink A, Shinya T, Desaki Y, Bours R, van der Krol S, Shibuya N, Joosten MH, Thomma BP. 2010. Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants. Science 329: 953-955.
references: Kämper J, Kahmann R, Bölker M, Ma LJ, Brefort T, Saville BJ, Banuett F, Kronstad JW, Gold SE, Müller O et al. 2006. Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444: 97-101.
references: Kaschani F, Shabab M, Bozkurt T, Shindo T, Schornack S, Gu C, Ilyas M, Win J, Kamoun S, van der Hoorn RAL. 2010. An effector-targeted protease contributes to defense against Phytophthora infestans and is under diversifying selection in natural hosts. Plant Physiology 154: 1794-1804.
references: Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059-3066.
references: Katoh K, Toh H. 2008. Recent developments in the MAFFT multiple sequence alignment program. Briefings in Bioinformatics 9: 286-298.
references: Koga H, Bushnell WR, Zeyen RJ. 1990. Specificity of cell type and timing of events associated with papilla formation and the hypersensitive reaction in leaves of Hordeum vulgare attacked by Erysiphe graminis f.sp. hordei. Canadian Journal of Botany 68: 2344-2352.
references: Korber B. 2000. HIV signature and sequence variation analysis. In: Learn AGRAGH, ed. Computational analysis of HIV molecular sequences. Dordrecht, the Netherlands: Kluwer Academic, 55-72.
references: Laurie JD, Ali S, Linning R, Mannhaupt G, Wong P, Güldener U, Münsterkötter M, Moore R, Kahmann R, Bakkeren G et al. 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: 1733-1745.
references: Lozano-Torres JL, Wilbers RH, Gawronski P, Boshoven JC, Finkers-Tomczak A, Cordewener JH, America AH, Overmars HA, Van‘t Klooster JW, Baranowski L et al. 2012. Dual disease resistance mediated by the immune receptor Cf-2 in tomato requires a common virulence target of a fungus and a nematode. Proceedings of the National Academy of Sciences, USA 109: 10 119-10 124.
references: Ma W, Dong FF, Stavrinides J, Guttman DS. 2006. Type III effector diversification via both pathoadaptation and horizontal transfer in response to a coevolutionary arms race. PLoS Genetics 2: e209.
references: Ma W, Guttman DS. 2008. Evolution of prokaryotic and eukaryotic virulence effectors. Current Opinion in Plant Biology 11: 412-419.
references: Macho AP, Zipfel C. 2014. Plant PRRs and the activation of innate immune signaling. Molecular Cell 54: 263-272.
references: Marshall R, Kombrink A, Motteram J, Loza-Reyes E, Lucas J, Hammond-Kosack KE, Thomma BP, Rudd JJ. 2011. Analysis of two in planta expressed LysM effector homologs from the fungus Mycosphaerella graminicola reveals novel functional properties and varying contributions to virulence on wheat. Plant Physiology 156: 756-769.
references: Mentlak TA, Kombrink A, Shinya T, Ryder LS, Otomo I, Saitoh H, Terauchi R, Nishizawa Y, Shibuya N, Thomma BP et al. 2012. Effector-mediated suppression of chitin-triggered immunity by Magnaporthe oryzae is necessary for rice blast disease. Plant Cell 24: 322-335.
references: Mueller AN, Ziemann S, Treitschke S, Assmann D, Doehlemann G. 2013. Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2. PLoS Pathogens 9: e1003177.
references: Mukhtar MS, Carvunis AR, Dreze M, Epple P, Steinbrenner J, Moore J, Tasan M, Galli M, Hao T, Nishimura MT et al. 2011. Independently evolved virulence effectors converge onto hubs in a plant immune system network. Science 333: 596-601.
references: O'Brien JA, Daudi A, Butt VS, Bolwell GP. 2012. Reactive oxygen species and their role in plant defence and cell wall metabolism. Planta 236: 765-779.
references: Rice P, Longden I, Bleasby A. 2000. EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics 16: 276-277.
references: Saitoh H, Fujisawa S, Mitsuoka C, Ito A, Hirabuchi A, Ikeda K, Irieda H, Yoshino K, Yoshida K, Matsumura H et al. 2012. Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens. PLoS Pathogens 8: e1002711.
references: Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory.
references: Schirawski J, Mannhaupt G, Munch K, Brefort T, Schipper K, Doehlemann G, Di Stasio M, Rossel N, Mendoza-Mendoza A, Pester D et al. 2010. Pathogenicity determinants in smut fungi revealed by genome comparison. Science 330: 1546-1548.
references: 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 Biology and Evolution 6: 2034-2049.
references: Song J, Win J, Tian M, Schornack S, Kaschani F, Ilyas M, van der Hoorn RA, Kamoun S. 2009. Apoplastic effectors secreted by two unrelated eukaryotic plant pathogens target the tomato defense protease Rcr3. Proceedings of the National Academy of Sciences, USA 106: 1654-1659.
references: Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688-2690.
references: Stergiopoulos I, van den Burg HA, Okmen B, Beenen HG, van Liere S, Kema GH, de Wit PJ. 2010. Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots. Proceedings of the National Academy of Sciences, USA 107: 7610-7615.
references: Stergiopoulos I, De Kock MJ, Lindhout P, De Wit PJ. 2007. Allelic variation in the effectorgenes of the tomato pathogen Cladosporium fulvum reveals different modes of adaptive evolution. Molecular Plant-Microbe Interactions 20: 1271-1283.
references: Veitch N. 2004. Structural determinants of plant peroxidase function. Phytochemistry Reviews 3: 3-18.
references: Yang Z. 2007. PAML 4: phylogenetic analysis by maximum likelihood. Molecular Biology and Evolution 24: 1586-1591.
references: Yang Z, Wong WSW, Nielsen R. 2005. Bayes empirical Bayes inference of amino acid sites under positive selection. Molecular Biology and Evolution 22: 1107-1118.