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Identification of susceptibility targets of bacterial leaf blight and development of genome edited rice lines with increased resistance

Publikation: Qualifikations-/StudienabschlussarbeitDissertation

Autoren

  • Stefanie Mücke

Organisationseinheiten

Details

OriginalspracheEnglisch
QualifikationDoctor rerum naturalium
Gradverleihende Hochschule
Betreut von
  • Jens Boch, Betreuer*in
Datum der Verleihung des Grades14 Sept. 2020
ErscheinungsortHannover
PublikationsstatusVeröffentlicht - 2020

Abstract

Pflanzenpathogene Xanthomonas oryzae Bakterien infizieren Reis und verursachen hohe Ernteverluste, die eine sichere Lebensmittelversorgung gefährden. Transcription activator-like effectors (TALEs) sind Hauptvirulenzfaktoren dieser Pathogene und manipulieren die Genexpression des Wirts zu Gunsten der Infektion. Mithilfe des Programms AnnoTALE können TALEs Klassen zugeordnet werden, die eine enge Verwandtschaft der TALEs zeigen und auf ein mögliches gemeinsames Zielgen hinweisen. Eine Analyse von 34 asiatischen Xanthomonas oryzae pv. oryzae (Xoo)-Stämmen enthüllte insgesamt 45 TALE-Klassen und einen Basissatz aus 10 TALE Kernklassen, die in mehr als 80% der Stämme vorkommen. Eine Kombination aus computerbasierten TALE-Zielgen-Vorhersagen und Transkriptom-daten von infiziertem Reis resultierte in 61 TALE-Zielgenkandidaten für die drei selektierten Xoo-Stämme. Repräsentative TALEs dieser Stämme wurde nachgebaut und in einen Xoo-Stamm eingebracht, der keine TALE-Gene trägt, um die Korrelation zwischen einzelnen TALEs und spezifischen, induzierten Genen zu prüfen. Für 13 Reisgene konnte eine TALE-spezifische Induktion nach Infektion nachgewiesen werden. Durch den Einsatz von Reporterassays konnten direkte Interaktionen zwischen den TALEs und ihren Zielpromotoren gezeigt werden. Durch diese neu identifizierten Zielgene konnte eine konvergente Evolution zwischen Xoo und den reispathogenen Xanthomonas oryzae pv. oryzicola aufgedeckt werden, welche mehr gemeinsame Zielgene besitzen als zuvor angenommen und diese mit verschiedenen TALEs adressieren. Einzelne TALEs hatten keinen großen Einfluss auf die Virulenz von Xoo in gain-of-function-Assays, aber mehrere TALE-Klassen manipulieren vermutlich den Salicylsäurehaushalt, der Xoo-Resistenz in Reis kontrolliert. Reispflanzen wurden mithilfe von CRISPR/Cas9 verändert, um Zielgene auszuknocken oder mehrere TALE-Bindestellen in Zielpromotoren zu mutieren. Initiale Ergebnisse deuten auf eine reduzierte Virulenz von Wildtyp Xoo-Stämmen auf Reispflanzen hin, in denen 7 TALE-Bindestellen von Zielpromotoren mutiert wurden, die in dieser Arbeit identifiziert wurden. Zusammengefasst konnte ein detailliertes Bild von TALE-manipulierten Pflanzen-prozessen etabliert werden, dass unser Verständnis von Xanthomonas oryzae-Virulenz signifikant erweitert. Zusätzlich wurde der Grundstein für die Entwicklung neuer Resistenzen gelegt, um diese wichtigen Reiskrankheiten zu bezwingen.

Zitieren

Identification of susceptibility targets of bacterial leaf blight and development of genome edited rice lines with increased resistance. / Mücke, Stefanie.
Hannover, 2020. 141 S.

Publikation: Qualifikations-/StudienabschlussarbeitDissertation

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title = "Identification of susceptibility targets of bacterial leaf blight and development of genome edited rice lines with increased resistance",
abstract = "Phytopathogenic Xanthomonas oryzae bacteria infect rice and cause severe harvest loss, which challenges the stable food supply. Transcription activator-like effectors (TALEs) are major virulence factors of these pathogens and manipulate the host gene expression to benefit infection. With the help of the AnnoTALE tool, TALEs can be assigned into classes, which represent closely related TALEs that likely activate the same gene. Analyzing the TALome diversity of 34 Asian Xanthomonas oryzae pv. oryzae (Xoo) strains revealed a total of 45 TALE classes and a common core set of 10 TALE classes present in more than 80% of strains. Combining computational TALE target gene predictions and transcriptomics data of infected rice produced 61 likely TALE target genes for three selected Xoo strains. Representative TALEs of these strains were reconstructed and introduced into a naturally TALE-free Xoo strain to verify the correlation between individual TALEs and specific induced genes. 13 genes were shown to be TALE-dependently induced upon infection in rice. Using reporter assays, direct interaction between TALEs and their corresponding target promoters could be demonstrated. These newly identified target genes unveiled convergent evolution between Xoo and rice-pathogenic Xanthomonas oryzae pv. oryzicola, which share more common target genes addressed by different TALE classes than previously believed. Individual TALEs did not have a strong influence on virulence in gain-of-function assays but several TALE classes might be linked to salicylic acid manipulation, which controls Xoo resistance reactions. Rice plants were edited using CRISPR/Cas9 to knockout target genes or mutate multiple TALE binding sites in target promoters. Initial results indicate reduced virulence of wild type Xoo strains on rice plants with 7 mutated TALE binding sites in TALE target promoters identified in this thesis. Taken together, a detailed picture of TALE-induced plant processes could be established that significantly expands understanding of Xanthomonas oryzae virulence strategies. Additionally, the groundwork was laid to facilitate the development of novel resistances to overcome this important rice disease.",
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note = "Doctoral thesis",
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school = "Leibniz University Hannover",

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Download

TY - BOOK

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AU - Mücke, Stefanie

N1 - Doctoral thesis

PY - 2020

Y1 - 2020

N2 - Phytopathogenic Xanthomonas oryzae bacteria infect rice and cause severe harvest loss, which challenges the stable food supply. Transcription activator-like effectors (TALEs) are major virulence factors of these pathogens and manipulate the host gene expression to benefit infection. With the help of the AnnoTALE tool, TALEs can be assigned into classes, which represent closely related TALEs that likely activate the same gene. Analyzing the TALome diversity of 34 Asian Xanthomonas oryzae pv. oryzae (Xoo) strains revealed a total of 45 TALE classes and a common core set of 10 TALE classes present in more than 80% of strains. Combining computational TALE target gene predictions and transcriptomics data of infected rice produced 61 likely TALE target genes for three selected Xoo strains. Representative TALEs of these strains were reconstructed and introduced into a naturally TALE-free Xoo strain to verify the correlation between individual TALEs and specific induced genes. 13 genes were shown to be TALE-dependently induced upon infection in rice. Using reporter assays, direct interaction between TALEs and their corresponding target promoters could be demonstrated. These newly identified target genes unveiled convergent evolution between Xoo and rice-pathogenic Xanthomonas oryzae pv. oryzicola, which share more common target genes addressed by different TALE classes than previously believed. Individual TALEs did not have a strong influence on virulence in gain-of-function assays but several TALE classes might be linked to salicylic acid manipulation, which controls Xoo resistance reactions. Rice plants were edited using CRISPR/Cas9 to knockout target genes or mutate multiple TALE binding sites in target promoters. Initial results indicate reduced virulence of wild type Xoo strains on rice plants with 7 mutated TALE binding sites in TALE target promoters identified in this thesis. Taken together, a detailed picture of TALE-induced plant processes could be established that significantly expands understanding of Xanthomonas oryzae virulence strategies. Additionally, the groundwork was laid to facilitate the development of novel resistances to overcome this important rice disease.

AB - Phytopathogenic Xanthomonas oryzae bacteria infect rice and cause severe harvest loss, which challenges the stable food supply. Transcription activator-like effectors (TALEs) are major virulence factors of these pathogens and manipulate the host gene expression to benefit infection. With the help of the AnnoTALE tool, TALEs can be assigned into classes, which represent closely related TALEs that likely activate the same gene. Analyzing the TALome diversity of 34 Asian Xanthomonas oryzae pv. oryzae (Xoo) strains revealed a total of 45 TALE classes and a common core set of 10 TALE classes present in more than 80% of strains. Combining computational TALE target gene predictions and transcriptomics data of infected rice produced 61 likely TALE target genes for three selected Xoo strains. Representative TALEs of these strains were reconstructed and introduced into a naturally TALE-free Xoo strain to verify the correlation between individual TALEs and specific induced genes. 13 genes were shown to be TALE-dependently induced upon infection in rice. Using reporter assays, direct interaction between TALEs and their corresponding target promoters could be demonstrated. These newly identified target genes unveiled convergent evolution between Xoo and rice-pathogenic Xanthomonas oryzae pv. oryzicola, which share more common target genes addressed by different TALE classes than previously believed. Individual TALEs did not have a strong influence on virulence in gain-of-function assays but several TALE classes might be linked to salicylic acid manipulation, which controls Xoo resistance reactions. Rice plants were edited using CRISPR/Cas9 to knockout target genes or mutate multiple TALE binding sites in target promoters. Initial results indicate reduced virulence of wild type Xoo strains on rice plants with 7 mutated TALE binding sites in TALE target promoters identified in this thesis. Taken together, a detailed picture of TALE-induced plant processes could be established that significantly expands understanding of Xanthomonas oryzae virulence strategies. Additionally, the groundwork was laid to facilitate the development of novel resistances to overcome this important rice disease.

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