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And Xanthomonas Translucens Pv Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2011 Genetic characterization of the interaction between Italian ryegrass (Lolium multiflorum Lam.) and Xanthomonas translucens pv. graminis Wichmann, Fabienne Abstract: Italian ryegrass (Lolium multiflorum Lam.) is a widely used for hay and silage production. Xanthomonas translucens pv. graminis (Xtg) is a plant pathogen and causes bacterial wilt on a number of forage grass species including L. multiflorum. The pathogen spreads through wounds caused when mowing with contaminated equipment. Resistance mechanisms of L. multiflorum and virulence mecha- nisms of Xtg are largely unknown. Therefore, the main objective of this thesis was to understand the interaction of Xtg with L. multiflorum. The first aim was to elucidate the existence of race-specificity in the L. multiflorum-Xtg interaction and to identify genomic regions that contribute to Xtg resistance. No race-specific interactions were found in 62 L. multiflorum genotypes infected with six Xtg isolates. Molecular marker-resistance associations revealed one simple sequence repeat marker on linkage group 5 to be significantly associated with bacterial wilt resistance. The second aim was to identify candidate genes for bacterial wilt resistance using a cDNA microarray of perennial ryegrass (L. perenne). Transcrip- tome analyses revealed candidate genes for bacterial wilt resistance such as the low silicon transporter and the germin-like protein 6. Virulence factors of Xtg were investigated by screening for genes encoding the type III secretion system. This revealed a sequence similar to the hrpG gene. A mutant deficient of the hrpG gene was obtained and infection with the ΔhrpG mutant lead to significantly reduced symp- toms. In planta analyses revealed that this mutant was still able to multiply inside its host. The data presented here provides comprehensive knowledge of this plant-pathogen interaction and represents a valuable basis for the development of tools for marker-assisted selection. Zusammenfassung Italienisches Raigras (Lolium multiflorum) wird häufig als Futtergras angebaut. Bakterienwelke ist eine Krankheit, die durch Xanthomonas translucens pv. graminis (Xtg), hervorgerufen wird. Die Züchtung von resistenten Sorten ist die effizienteste Kontrollmassnahme gegen Bakterienwelke. Die Quelle der Krankheitsresistenz und Virulenzmechanismen der Bakterien sind noch immer unbekannt. Deshalb war das Hauptziel dieser Arbeit, die Interaktion von L. multiflorum und Xtg besser zu charakterisieren. Das erste Ziel stellte die Aufklärung der Existenz von Rassenspezifität in der L. multiflorum-Xtg Interaktion dar. Zudem wur- den molekulare Marker untersucht, die mit Bakterienwelke Resistenz assoziiert sind. Es konnte keine Rassenspezifität gefunden werden in den 62 L. multiflorum Genotypen, die mit sechs verschiedenen Xtg Isolaten infiziert wurden. Die Analyse mit molekularen Markern zeigte, dass ein Mikrosatelliten Marker auf der Kopplungsgruppe 5 signifikant mit Bakterienwelke- Resistenz assoziiert war. Das zweite Ziel war, Kandidatengene für Xtg Resistenz in L. multiflorum zu identifizieren mittels einer Genexpressions- analyse. Dazu wurde ein cDNA Microrarray verwendet, der mit Sequenzen von Englischem Raigras (L. perenne) entwickelt worden war. Die Genexpressionsstudie führte zur Identifizierung von interessanten Kandidatengenen, die mit Bakterienwelke Resistenz assoziiert sein könnten. Um auch Gene von Xtg zu kennen, die für die Interaktion mit L. multiflorum von grosser Wichtigkeit sein könnten, wurde das Xtg Genom nach Virulenzgenen des Typ III Sekretionssystems untersucht. So wurde das hrpG Gen gefunden und eine Mutante generiert, die das hrpG Gen nicht mehr hat. Diese Mutante war signifikant weniger virulent bei der Infektion von L. multiflorum Pflanzen. In planta Analysen zeigten jedoch, dass die ΔhrpG Mutanten sich noch immer vermehren konnten. Die Erkenntnisse aus dieser Studie stellen einen elementaren Baustein dar, diese Interaktion in grossem Umfang zu verstehen und das erarbeitete Wissen stellt eine essentielle Grundlage dar für die Entwicklung von Marker gestützter Züchtung. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-59375 Dissertation Published Version Originally published at: Wichmann, Fabienne. Genetic characterization of the interaction between Italian ryegrass (Lolium mul- tiflorum Lam.) and Xanthomonas translucens pv. graminis. 2011, University of Zurich, Facultyof Science. 2 Genetic Characterization of the Interaction between Italian Ryegrass (Lolium multiflorum Lam.) and Xanthomonas translucens pv. graminis Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Fabienne Wichmann von Münchenstein BL Promotionskomitee: Prof. Dr. Beat Keller (Vorsitz) Prof. Dr. Robert Dudler Dr. Roland Kölliker (Leitung der Dissertation) Zürich, 2011 Table of contents Table of contents ....................................................................................................................... 1 Summary ................................................................................................................................... 6 Zusammenfassung .................................................................................................................... 8 1 General introduction ........................................................................................................... 10 1.1 Grasslands – important ecosystems ................................................................................ 11 1.1.1 The importance of ryegrasses in grassland production ........................................... 11 1.1.2 Systematics and genetics of the genus Lolium ........................................................ 12 1.2 Xanthomonas spp. – destructive plant pathogens ........................................................... 14 1.2.1 Diseases caused by Xanthomonas spp. ................................................................... 14 1.2.2 Systematics of Xanthomonas .................................................................................. 15 1.2.2 Xanthomonas translucens pv. graminis causing bacterial wilt of forage grass ...... 17 1.3 Virulence factors from Xanthomonas spp. ..................................................................... 19 1.3.1 The type III secretion system (T3SS) ...................................................................... 20 1.3.2 Other secretion systems ........................................................................................... 22 1.3.3 Extracellular polysaccharides (EPS) ....................................................................... 23 1.3.4 Lipopolysaccharides (LPS) ..................................................................................... 24 1.4 Concepts of disease resistance in plants ......................................................................... 25 1.4.1 Qualitative (race-specific) resistance ...................................................................... 26 1.4.2 Quantitative disease resistance ................................................................................ 27 1.5 Improving resistance to bacterial wilt ............................................................................ 27 1.5.1 Breeding for resistant forage grasses with recurrent selection ................................ 27 1.5.2 Genetic linkage maps and quantitative trait loci (QTL) .......................................... 28 1.5.3 Transcriptome analyses of host–bacterial pathogen interactions ............................ 30 1.5.4 Marker-assisted selection for disease resistance ..................................................... 32 1.6 Recent advances in the characterization of bacterial wilt resistance in Italian ryegrass 33 1.7 Objectives ....................................................................................................................... 35 1 Table of contents 2 Phenotypic and molecular genetic characterization indicate no major race-specific interactions between Xanthomonas translucens pv. graminis and Lolium multiflorum ... 36 2.1 Abstract .......................................................................................................................... 37 2.2 Introduction .................................................................................................................... 37 2.3 Materials and Methods ................................................................................................... 40 2.3.1 Bacterial isolates and plant material ........................................................................ 40 2.3.2 Experimental design and disease scoring ................................................................ 40 2.3.3 Data analyses and statistics ..................................................................................... 41 2.3.4 Molecular marker and association analysis ............................................................. 42 2.4 Results ............................................................................................................................ 44 2.4.1 Data structure and distribution ................................................................................ 44 2.4.2 Regression modelling and race-specificity ............................................................
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