Functional characterization of cellulose and chitin synthase genes in Oomycetes Johanna Fugelstad Doctoral thesis in Biotechnology Division of Glycoscience Stockholm, Sweden 2011 © Johanna Fugelstad, 2011 School of Biotechnology Royal Institute of Technology AlbaNova University Center 106 91 Stockholm Sweden Printed at US-AB universitetsservice TRITA-BIO Report 2011:13 ISBN 978-91-7415-971-4 ISSN 1654-2312 Cover: Saprolegnia monoica mycelium stained with Congo Red (left) and human cell expressing the pleckstrin homology domain of S. monoica cellulose synthase 2, fused with green fluorescent protein (right). Micrographs by author. Johanna Fugelstad (2011) Functional characterization of cellulose and chitin synthase genes in Oomycetes. Doctoral thesis in Biotechnology. Royal Institute of Technology (KTH), Division of Glycoscience, Stockholm, Sweden. TRITA‐BIO Report 2011:13 ISBN 978‐91‐7415‐971‐4 ISSN 1654‐2312 ABSTRACT Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of β‐(1Æ3) and β‐(1Æ6)‐glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica. A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N‐terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F‐actin and microtubules in vitro and can co‐localize with F‐actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up‐regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full‐length chitin synthase genes (Chs) were analyzed from S. monoica. Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro. Altogether these results show that at least some of the CesA1‐4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase. Keywords: cellulose biosynthesis; chitin biosynthesis; cellulose synthase genes; chitin synthase genes; Oomycetes; Phytophthora infestans; Saprolegnia monoica; pleckstrin homology domain. © Johanna Fugelstad, 2011 Johanna Fugelstad (2011) Funktionell karaktärisering av cellulosa‐ och kitinsyntasgener i oomyceter. Doktorsavhandling i Bioteknologi. Kungliga Tekniska högskolan, avdelningen för Glykovetenskap, Stockholm, Sverige. TRITA‐BIO Report 2011:13 ISBN 978‐91‐7415‐971‐4 ISSN 1654‐2312 SAMMANFATTNING Många oomyceter är välstuderade patogena arter som orsakar ansenliga ekonomiska förluster inom jordbruks‐ och fiskodlingsindustrin. För närvarande finns inga kemikalier till hands som effektivt bekämpar oomyceter och på samma gång är miljövänliga. Oomyceters cellvägg består av β‐(1Æ3)‐ och β‐(1Æ6)‐glukaner, cellulosa och i vissa arter små mängder kitin. Hur oomyceter syntetiserar cellulosa och kitin är relativt okänt, dock är cellväggssyntesen ett potentiellt mål för nya oomycetinhibitorer. Cellulosa‐ och kitinsyntasgener och dess genprodukter, från växtpatogenen Phytophthora infestans och fiskpatogenen Saprolegnia monoica, har analyserats i den här avhandlingen. En ny familj cellulosasyntasgener från oomyceter har identifierats, innehållande fyra underklasser av gener, utsedda till CesA1 till 4. N‐terminalerna i genprodukterna från CesA1, 2 och 4 innehåller en pleckstrinhomolog(PH)‐domän, vilket är unikt för CesAs från oomyceter. Våra resultat visar att PH‐domänen från SmCesA2 binder till fosfoinositider, F‐aktin och mirotubuli in vitro och kan samlokalisera med F‐aktin in vivo. Tystande av CesA‐generna i P. infestans ledde till en minskad mängd cellulosa i cellväggen. Utöver detta inhiberade cellulosasyntasinhibitorerna DCB och Kongo Rött tillväxten av S. monoica mycel och hade en uppreglerande effekt på uttrycket av SmCesA‐generna. Zoosporer från P. infestans behandlade med DCB var oförmögna att infektera potatisblad. Dessutom analyserades två fullängds kitinsyntasgener (Chs) från S. monoica. Rekombinant uttryck av SmChs2 i jäst resulterade i ett aktivt protein. Den biokemiska analysen av in vitro‐produkten från SmChs2 bekräftade att proteinet syntetiserar kitin. Kitinsyntasinhibitorn nikkomycin Z inhiberade SmChs2 både in vivo och in vitro. Sammataget visar dessa resultat att åtminstone några av generna CesA1‐4 är involverade i cellulosabiosyntes och att syntes av cellulosa är nödvändigt för P. infestans infektionsförmåga. PH‐domänen är involverad i interaktionen mellan CesA och cytoskelettet. Dessutom har vi bevisat att SmChs2‐genen kodar för ett katalytiskt aktivt kitinsyntas. Nyckelord: cellulosabiosyntes; kitinbiosyntes; cellulosasyntasgen; kitinsyntasgen, Oomyceter, Phytophthora infestans; Saprolegnia monoica; pleckstrinhomolog‐domän © Johanna Fugelstad, 2011 LIST OF PUBLICATIONS Publication I Grenville‐Briggs L. J., Anderson V. L., Fugelstad J., Avrova A. O., Bouzenzana J., Williams A., Wawra S., Whisson S. C., Birch P. R., Bulone V. and van West P., Cellulose synthesis in Phytophthora infestans is required for normal appressorium formation and successful infection of potato, 2008, Plant Cell 20(3):720‐38 Publication II Fugelstad J., Bouzenzana J., Djerbi S., Guerriero G., Ezcurra I., Teeri T.T., Arvestad L. and Bulone V. Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity, 2009, Fungal Genet. Biol. 46(10):759‐67 Publication III Fugelstad J.*, Brown C.*, Hukasova E., Sundqvist G., Lindqvist A. and Bulone V., Functional characterization of the pleckstrin homology domain of a cellulose synthase from the Oomycete Saprolegnia monoica, 2011, Manuscript Publication IV Guerriero G., Avino M., Zhou Q., Fugelstad J., Clergeot P.H. and Bulone V. Chitin synthases from Saprolegnia are involved in tip growth and represent a potential target for anti‐oomycete drugs, 2010, PLoS Pathog. 6(8): 1‐12 *Both authors contributed equally to the work AUTHOR’S CONTRIBUTION Publication I: Johanna Fugelstad’s contribution is the biochemical characterization of the cell wall, participation in the co‐localization studies using antibodies and some of the writing. Publication II: Johanna Fugelstad did most of the experimental work and the writing with assistance from Dr. Lars Arvestad for the bioinformatic sections. Publication III: Johanna Fugelstad performed the cloning, expression, purification, bioinformatics and parts of the localization of the experimental section, and wrote the manuscript with assistance from Christian Brown in the Materials and Methods section. Publication IV: Johanna Fugelstad’s contribution is the Southern blot experiment and participation in some of the writing. RELATED PUBLICATIONS NOT INCLUDED IN THE THESIS Haas B.J., Kamoun S., Zody M.C., Jiang R.H., Handsaker R.E., Cano L.M., Grabherr M., Kodira C.D., Raffaele S., Torto‐Alalibo T., Bozkurt T.O., Ah‐Fong A.M., Alvarado L., Anderson V.L., Armstrong M.R., Avrova A., Baxter L., Beynon J., Boevink P.C., Bollmann S.R., Bos J.I., Bulone V., Cai G., Cakir C., Carrington J.C., Chawner M., Conti L., Costanzo S., Ewan R., Fahlgren N., Fischbach M.A., Fugelstad J., Gilroy E.M., Gnerre S., Green P.J., Grenville‐Briggs L.J., Griffith J., Grünwald N.J., Horn K., Horner N.R., Hu C.H., Huitema E., Jeong D.H., Jones A.M., Jones J.D., Jones R.W., Karlsson E.K., Kunjeti S.G., Lamour K., Liu Z., Ma L., Maclean D., Chibucos M.C., McDonald H., McWalters J., Meijer H.J., Morgan W., Morris P.F., Munro C.A., O'Neill K., Ospina‐Giraldo M., Pinzón A., Pritchard L., Ramsahoye B., Ren Q., Restrepo S., Roy S., Sadanandom A., Savidor A., Schornack S., Schwartz D.C., Schumann U.D., Schwessinger B., Seyer L., Sharpe T., Silvar C., Song J., Studholme D.J., Sykes S., Thines M., van de Vondervoort P.J., Phuntumart V., Wawra S., Weide R., Win J., Young C., Zhou S., Fry W., Meyers B.C., van West P., Ristaino J., Govers F., Birch P.R., Whisson S.C., Judelson H.S., Nusbaum C., Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans, 2009, Nature, 461(7262):393‐8. Guerriero G., Fugelstad J. and Bulone V., What do we really know about cellulose biosynthesis in higher plants? 2010, J. Integr. Plant Biol. 52(2):161‐75, review LIST OF CONTENTS 1 INTRODUCTION 1 2 INTRODUCTION TO OOMYCETES 1 2.1 General characteristics of Oomycetes