Identification and functional characterization of powdery mildew effectors [Elektronische Ressource] / vorgelegt von Sarah Maria Schmidt
132 Pages
English

Identification and functional characterization of powdery mildew effectors [Elektronische Ressource] / vorgelegt von Sarah Maria Schmidt

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Published 01 January 2009
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Language English
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IDENTIFICATION AND FUNCTIONAL
CHARACTERIZATION OF POWDERY MILDEW
EFFECTORS





Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln
vorgelegt von
Sarah Maria Schmidt
aus Marburg an der Lahn



Köln, September 2009



Die vorliegende Arbeit wurde am Max-Planck-Institut für Züchtungsforschung in Köln
in der Abteilung für Molekulare Phytopathologie (Direktor: Prof. Dr. P. Schulze-
Lefert) angefertigt.











Berichterstatter: Prof. Dr. Paul Schulze-Lefert
Prof. Dr. Martin Hülskamp
Ass.-Prof. Dr. Frank Takken

Prüfungsvorsitzender: Prof. Dr. Ulf-Ingo Flügge

Tag der Disputation: 30. November 2009
I
II
Publications
Schmidt, S.M., and Panstruga, R. (2007). Cytoskeleton functions in plant-microbe
interactions. Physiol. Mol. Plant Pathol. 71, 135-148.
III
IV
Table of Contents
PUBLICATIONS III
TABLE OF CONTENTS V
ABBREVIATIONS IX
SUMMARY XIII
ZUSAMMMENFASSUNG XV
1 INTRODUCTION 1
1.1 Plants defend themselves with an evolutionary ancient innate immune system 1
1.1.1 First layer- recognition of conserved microbial patterns 1
1.1.2 Second layer- recognition of microbial activity 2
1.1.3 Limitations 3
1.2 Prokaryotic and eukaryotic pathogens have adapted to cause disease in plants 4
1.3 Co-evolution of plant resistance and pathogen effector proteins 5
1.3.1 Direct effector recognition 5
1.3.2 Indirect effector recognition 6
1.3.3 Resistance genes against haustoria-forming, obligate biotrophic pathogens 7
1.3.4 Avirulence genes of haustoria-forming, obligate biotrophic pathogens 8
1.4 Effectors – executive pathogenicity agents 9
1.4.1 Effector translocation into the host 10
1.4.2 Virulence function of bacterial effectors 11
1.4.3 Virulence function of oomycete effectors 12
1.4.4 Virulence function of fungal effectors 13
1.5 Aim of thesis 14
2 MATERIALS AND METHODS 15
2.1 Plant and Fungal Material 15
2.2 EC and AC cDNA Isolation and Sequence Analysis 15
V
2.3 Plasmid constructs 16
2.4 Transient gene expression in single barley epidermal cells 16
2.5 RNA isolation and quantitative RT-PCR 17
2.6 Transformation of Arabidopsis thaliana 17
2.7 Transformation of yeast 17
2.8 Yeast signal sequence trap 17
2.9 Yeast BAX Assay 18
2.10 Yeast Growth Inhibition Assay 18
2.11 Fluorescent Microscopy 18
2.12 FRET analysis 19
2.13 Yeast two-hybrid screen 20
2.14 Yeast Crude Protein Extracts 21
2.15 Phylogenetic Analyses 21
3 RESULTS 23
3.1 Identification and functional characterization of effector candidates 23
3.1.1 Candidate effectors were identified bioinformatically 23
3.1.2 ECs enhance Bgh haustorium formation 26
3.1.3 EC proteins have a signal peptide that is functional in yeast 29
3.1.4 EC proteins do not alter nonhost resistance to Bgt 30
3.1.5 EC proteins are encoded by single copy genes and are in part conserved among powdery mildew
species 31
3.1.6 EC proteins are highly sequence conserved among six Bgh isolates 32
3.1.7 EC genes are differentially expressed during the infection process 33
3.1.8 EC proteins localize to the plant cytoplasm 36
3.1.9 EC proteins do not suppress BAX-induced cell death 38
3.1.10 EC proteins do not cause growth inhibition in yeast 38
3.1.11 EC4 interacts with a HvUBC-E2 and a HvARF-GAP; EC6 interacts with a HvTPMT in yeast 39
3.1.12 EC4 interacts with a HvUBC-E2; EC6 interacts with a HvTPMT in planta 43
3.1.13 FRET analysis 45
3.1.14 EC4 activates HvUBC-E2; EC6 activates HvTPMT 47
VI