Molecular analyses on the mechanism of nonhost resistance of barley (Hordeum vulgare L.) to the wheat powdery mildew fungus (Blumeria graminis f.sp. tritici) [Elektronische Ressource] / submitted by Ruth Eichmann

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2006
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 11 Mo

Extrait

Institute of Phytopathology and Applied Zoology
Head: Prof. Dr. Karl-Heinz Kogel



Molecular Analyses on the
Mechanism of Nonhost Resistance of
Barley (Hordeum vulgare L.)
to the Wheat Powdery Mildew Fungus
(Blumeria graminis f.sp. tritici)



Inaugural Dissertation for the Achievement of the Degree
Doktor der Agrarwissenschaften
at the Faculty of Agricultural and Nutritional Sciences, Home Economics and
Environmental Management

Justus-Liebig-Universität Giessen



Submitted by
Dipl.-Ing. agr. Ruth Eichmann
from Usingen

























Board of Examiners

Chairman of the Committee Prof. Dr. Ernst-August Nuppenau
1. Referee Prof. Dr. Karl-Heinz Kogel
2. Referee Prof. Dr. Wolfgang Friedt
3. Referee Prof. Dr. Sylvia Schnell
Examiner Prof. Dr. Bernd Honermeier
Examiner Prof. Dr. Wolfgang Köhler
Examiner PD Dr. Ralph Hückelhoven

Date of oral examination: 22.12.2005
Parts of this work have already been published:

EICHMANN, R., SCHULTHEISS, H., KOGEL, K.-H. AND HÜCKELHOVEN, R. (2004) The barley
apoptosis suppressor homologue Bax Inhibitor-1 compromises nonhost penetration
resistance of barley to the inappropriate pathogen Blumeria graminis f.sp. tritici. Mol.
Plant-Microbe Interact. 17: 484-490.

EICHMANN, R., BIEMELT, S., SCHÄFER, P., SCHOLZ, U., JANSEN, C., FELK, A., SCHÄFER, W.,
LANGEN, G., SONNEWALD, U., KOGEL, K.-H. AND HÜCKELHOVEN, R. (in press) Macroarray
expression analysis of barley susceptibility and nonhost resistance to Blumeria
graminis. J. Plant Physiol. doi:10.1016/j.jplph.2005.06.019.

1 Introduction 1
1.1 Host-pathogen relationship 1
1.2 The interaction of barley with cereal powdery mildew fungi 2
1.3 The compatible interaction 4
1.4 Defense mechanisms 6
1.4.1 Formation of cell wall appositions 6
1.4.2 The plant Hypersensitive Reaction and regulation of programmed cell
death in animals 7
1.4.3 Antimicrobial compounds and pathogenesis related proteins 9
1.4.4 Generation and role of Reactive Oxygen Intermediates in plant defense 10
2+1.4.5 The role of Ca in defense responses 13
1.5 Establishment of compatibility 14
1.6 Genetics and molecular mechanisms of resistance to powdery mildew fungi 16
1.6.1 Quantitative resistance 16
1.6.2 Race-specific resistance 16
1.6.3 mlo-mediated broad-spectrum resistance 19
1.6.4 Nonhost resistance 20
1.7 Objectives 23
2 Materials and methods 24
2.1 Plants, pathogens and inoculation 24
2.2 Macroarray-based identification of differentially expressed genes 24
2.2.1 Macroarray generation 24
332.2.2 Synthesis of P-cDNA and hybridization procedure 25
+2.2.2.1 Isolation of poly(A) -RNA 25
2.2.2.2 Synthesis of first strand cDNA 26
2.2.2.3 Random prime labeling 27
2.2.2.4 Pre-hybridization and hybridization of macroarray membranes 28
2.2.3 Data analysis 28
2.2.4 Confirmation of differential gene expression 29
2.2.4.1 Northern analysis 29
2.2.4.2 Semi-quantitative RT-PCR 30
2.3. Structural and functional characterization of the cell-death suppressor
BAX INHIBITOR-1 (BI-1) 32
2.3.1 Expression analysis of BI-1 32
2.3.2 Construction of pGFP-BI-1 32
2.3.3 Mutagenesis of barley BI-1 33
2.3.4 Transient transformation and evaluation of penetration efficiency 35
2.3.5 Localization of BI-1 fusion constructs 36
2.3.6 H O staining of transiently transformed leaf segments 382 2
2.3.7 Cell death assay in barley 38
2.3.8 DAPI staining of transiently transformed barley leaf segments 39
2.3.9 Assessment of BAX suppression in stably transformed, GFP-BI-1
expressing barley plants 40
2.3.9.1 Construction of sGFPHdel as marker for cytoplasmic movement 40
2.3.9.2 BAX expression and assessment of cell viability 40
2.3.10 Yeast transformation and yeast viability assay 41
2.3.11 Protein extraction from yeast and immunoblot analysis 43

3 Results 45
3.1 Macroarray-based expression analysis of barley host susceptibility and
nonhost resistance to Blumeria graminis 45
3.1.1 Macroarray construction and differential hybridization 45
3.1.2 Differentially expressed genes 47
3.1.3 Reliability of macroarray data 49
3.1.4 Functional classification 50
3.2 Structural and functional characterization of the potential cell death
suppressor BAX INHIBITOR-1 52
3.2.1 Yeast transformation and cell viability assay 53
3.2.2 BAX-induced collapse of single barley epidermal cells 54
3.2.3 Overexpression of barley BI-1 delays BAX-induced collapse of the
cytoplasm 55
3.2.4 Analysis of BAX-dependent cell death in stably transformed barley
plants expressing a GFP-BI-1 fusion protein 57
3.2.5 Expression of barley BI-1 in response to Bgt 60
3.2.6 BI-1 overexpression compromises penetration resistance of barley to Bgt 61
3.2.7 Simultaneous overexpression of BI-1 and MLO 62
3.2.8 Localization of a GFP-BI-1 fusion protein 63
3.2.9 H O staining in BI-1 overexpressing barley epidermal cells during 2 2
the interaction with B. graminis 66
3.2.10 Site-directed mutagenesis of barley BI-1 cDNAs 67
4 Discussion 71
4.1 Macroarray-based identification of differentially regulated genes in the host
and nonhost interaction of barley with powdery mildew fungi 71
4.1.1 Analysis of gene expression during the interaction of barley with Bgh
and Bgt 72
4.1.1.1 Genes up-regulated after inoculation with powdery mildew fungi 73
4.1.1.2 Genes down-regulated after inoculation with powdery mildew fungi 80
4.1.2 General considerations on the macroarray results 82
4.2. Molecular characterization of BAX INHIBITOR-1 and its role in nonhost
resistance of barley to the wheat powdery mildew fungus 85
4.2.1 Barley BI-1 delays BAX-induced death of barley epidermal cells 86
4.2.2 Expression of barley BI-1 in response to inoculation with Bgt 91
4.2.3 BI-1 overexpression compromises penetration resistance of barley to Bgt 92
4.2.4 Subcellular localization of GFP-BI-1 fusion proteins 95
4.2.5 Overexpression of BI-1 modulates local H O accumulation 972 2
4.2.6 The BI-1 motif is important for protein function in powdery mildew
susceptibility 98
4.2.6 General considerations on the BI-1 results 100
5 Summary / Zusammenfassung 103
6 References 105
7 Supplement 127


INTRODUCTION
1 Introduction
Crop plants are confronted with a huge array of potentially phytopathogenic viruses,
bacteria and fungi. Considering the large number of possible combatants, it is quite
astonishing that only very few ‘specialists’ eventually succeed in colonizing a plant.
Under certain conditions however, these pathogens can cause severe damage with
high yield losses and reduction in crop quality and monetary gain. Oerke and Dehne
(1997) have estimated that about 17.5 % of the possible yield worldwide is lost due to
pathogen infections. Taking into account that resources are limited and that more
and more arable land is eroded, it will be even more difficult to supply a growing
world population with adequate amounts of food. This goal demands the cultivation of
highly productive crops in monocultures, which, through enormous selection
pressure, leads to the emergence of fungicide resistant pathogen races or the
breakdown of established genetic resistances. In order to continue to provide
increasing crop quality and quantity it will be important to develop and realize new
sophisticated resistance strategies. It is thus necessary to gain comprehensive
information on both the pathogen’s infection strategy and the processes that underlie
the plant’s defense reactions.

1.1 Host-pathogen relationship
Phytopathogenic agents like bacteria, viruses and fungi pursue various strategies in
order to utilize plants for their own propagation. When a pathogen succeeds in
colonizing a plant and accomplishes its lifecycle, the interaction is considered as
being compatible and the host plant then is susceptible to the virulent microorganism.
In case of successful plant defense prior to pathogen propagation, the interaction
between the resistant host and the avirulent pathogen is referred to as incompatible
(Schlösser 1997).
Fungal pathogens are the most prevalent agents, causing severe diseases of plants.
They show high variability in terms of morphology, infection strategy, and evoked
symptoms. According to their general lifestyle or their infection process, most fungal
pathogens can be classified into two major categories: biotrophs and necrotrophs.
Biotrophs derive their nutrients from the living host cell. They are mostly, though not
always generating specialized feeding structures, called haustoria, and their infection
1INTRODUCTION_________________________________________________________________

is often controlled on the level of race-specific resistance, frequently involving death
of the infected host cell to destroy the pathogen’s means of existence. Examples <