Molecular studies on compatibility in the mutualistic plant root-Piriformospora indica interaction [Elektronische Ressource] / vorgelegt von Behanm Khatabi

Khatabi, Behnam

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 84
• Poids de l'ouvrage: 2 Mo

Extrait

Molecular studies on compatibility in the
mutualistic plant root-Piriformospora indica
interaction



Dissertation zur Erlangung des Doktorgrades
(Dr. rer. nat.)
der Naturwissenschaftlichen Fachbereiche
der Justus-Liebig-Universität Gießen


durchgeführt am
Institut für Phytopathologie und Angewandte Zoologie


vorgelegt von
M.Sc. Behanm Khatabi
aus Iran


Gießen 2009



Dekan: Prof. Dr. Ingrid-Ute Leonhäuser
1. Gutachter: Prof. Dr. Karl-Heinz Kogel
2. Gutachter: Prof. Dr. Annegret Wilde




































Board of Examiners


Chairman of the Committee: Prof. Dr. Annegret Wilde

1. Referee: Prof. Dr. Karl-Heinz Kogel
2. Referee: Prof. Dr. Annegret Wilde
Examiner: Prof. Dr. Katja Becker iner: Prof. Dr. Sylvia Schnell



Date of oral examination: 29.06.2009

IContents

1. Introduction 1
1.1 Plant-microbe interactions 1
1.2 Plant immune system 2
1.2.1 Basal plant defense 3
1.2.2 R-gene mediated resistance 5
1.2.3 Non-host resistance 6
1.2.4 Induced resistance 6
1.3 Compatibility in plant-microbe interactions 7
1.3.1 Microbial effector proteins 8
1.3.2 Plant compatibility factors 9
1.4 Beneficial fungal microorganisms 10
1.4.1 Mycorrhizal fungi 10
1.4.2 Endophytic microorganisms 12
1.4.3 Piriformospora indica 12
1.4.3.1 Phylogeny of Sebacinales ordo nov. 13
1.4.3.2 Bacteria associated with P. indica 14
1.4.3.3 Biological activities of P. indica 14
1.4.3.4 Plant root colonisation by P. indica 15
1.5 Objectives 17
2. Materials and Methods 18
2.1 Plant materials and fungal inoculation 18
2.2 Cyto-histological techniques 18
2.3 RNA isolation, biotin-labeled cDNA synthesis and subsequent hybridisation
for TSH assay 19
2.4 Generation of a subtracted cDNA library, cloning and sequencing 21
2.5 Evaluation of efficiency and accuracy of TSH assay and YSST method 22
2.6 Quantification of ACC in Arabidopsis / barley roots during P. indica association 22
2.7 Colonisation of Arabidopsis mutants by P. indica 23
2.8 Quantification of fungal colonisation by QPCR 23
II2.9 Histochemical analyses of Arabidopsis ethylene- and polyamine reporter lines 24
2.10 Exogenous application of 1-aminocyclopropane 1-carboxylic acid (ACC) and
1-methylcyclopropene (MCP) 24
2.11 cDNA library construction for YSST screening 25
2.12 Ligation of cDNA library into pSMASH, yeast transformation and screening 26
2.13 Prediction strategy for putative proteins and domain delineation 27
2.14 Methods for protein modelling of PIALH43 RING finger domain 28
2.15 Isolation of full-length α- expansin and PIALH43 cDNAs 28
2.16 Gene expression profile of PIALH43 in planta compared with axenic cultures 29
2.17 Construction of PIALH43-His , production of recombinant protein and purification 6
29
2.18 In vitro ubiquitin ligase assay 31
2.19 Statistical analysis 32
3 Results 33
3.1 Establishment of the yeast signal sequence trap assay to identify proteins secreted
during the interaction of barley roots with P. indica 33
3.2 Isolation of secreted proteins using the yeast signal sequence trap approach 35
3.3 In silico-based analysis of isolated genes and homology modeling of PIALH43 37
3.4 In vitro E3 ubiquitin ligase assay showed the involvement of PIALH43 in protein
degradation 42
3.5 Establishment of TSH method for the identification P. indica-responsive genes 43
3.6 Identification of genes involved in ethylene and polyamine biosynthesis by
subtractive hybridisation 47
3.7 Ethylene accumulation in barley roots colonized by P. indica 49
3.7.1 De novo ethylene synthesis during P. indica colonisation 50
3.7.2 Ethylene – a compatibility factor during plant root colonisation by P. indica
52
3.8 Polyamines – compatibility factors for plant root colonisation by P. indica 55
4. Discussion 56
4.1 The protein secretion system in eukaryotic cells 56
III4.2 The yeast signal sequence trap (YSST) assay identifies PIALH43 as a putative P.
indica effector protein 58
4.2.1 The ubiquitin proteasome pathway and its function in plants 61
4.2.2 PIALH43 as a putative P. indica effector protein and symbiotic
reprogramming 64
4.3 Plant factors influencing root colonisation by P. indica 65
4.4 Plant genes identified by Transcript Subtractive Hybridization (TSH) are induced
by P. indica during root colonisation 66
4.5 Ethylene acts as a compatibility factor in plant root colonisation by P. indica 69
4.6 Diamine and polyamine recruitment to support the Arabidopsis root-
symbiosis 74
5. Summary /Zusammenfasssung 76
6. References 81
7. Supplements 97





















List of Abbreviations
IV
ACC 1-aminocyclopropane-1-carboxylic acid
ACO ACC-oxidase
ACS ACC synthase
Avr Avirulence
BAK1 BRI1-associated kinase 1
Bgh Blumeria graminis f.sp. hordei
BI-1 Bax Inhibitor -1
CIP Calf intestine phosphatase
CSPs Cold-shock proteins
CTR1 Constitutive triple reponse 1
DEPC Diethylpyrocarbonate
E1 Ubiquitin activating enzymes
E2 Ubiquitin conjuga