Cloning and characterization of th BPR1 gene (Botrytis cinerea and Pseudomonas syringae pv. phaseolicola susceptibility and Root length 1) from Arabidopsis thaliana [Elektronische Ressource] / vorgelegt von Gholam Reza Sharifi-Sirchi

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• Biologie

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2006
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Cloning and characterization of the BPR1 gene („Botrytis cinerea
and Pseudomonas syringae pv. phaseolicola susceptibility and Root
length 1”) from Arabidopsis thaliana.
Gholam Reza Sharifi-Sirchi„Cloning and characterization of the BPR1 gene („Botrytis cinerea and
Pseudomonas syringae pv. phaseolicola susceptibility and Root length 1”)
from Arabidopsis thaliana“
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades eines Doktors
der Naturwissenschaften genehmigte Dissertation
vorgelegt von
Master of science in Plant breeding
Gholam Reza Sharifi-Sirchi
aus
Kerman, Iran
Berichter: Universitätsprofessor Alan J. Slusarenko, Ph. D.
Privatdozent Dr. Nikolaus L. Schlaich
Tag der mündlichen Prüfung: 01.06.2006
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.Contents
CONTENTS
Page
1 Introduction 1
1-1 Plant-pathogen associations and the genetic basis of plant defence 1
1-1-1 Gene-for-gene hypothesis 2
1-1-2 Guard hypothesis 6
1-1-3 RPM1 (resistance to P. syringae pv. maculicola)6
1-1-4 RPS2 (resistance to Pst)7
1-1-5 NDR1 (Non-race-specific disease resistance1) 8
1-1-6 EDS1 (Enhanced disease susceptibility 1) and PAD4 (Phytoalexin deficient 4) 8
1-1-7 Salicylic acid-dependent resistance 10
1-1-8 EDS5 (enhanced disease susceptibility) and SID2 (SA induction deficient 2) 12
1-1-9 NPR1 (Non-expressor of PR1)13
1-1-10 Jasmonic acid (JA) and ethylene (ET) – dependent signalling 14
1-2 Pathogens 15
1-2-1 Botrytis cinerea and Alternaria brassicicola 15
1-2-2 Prevoius work in our laboratory 16
2 Materials and Methods 18
2-1 Organisms 18
2-1-1 Plants 18
2-1-2 Bacteria 18
2-1-3 Yeast (Saccharomyses cerevisiae)19
2-2 Vectors 20
2-3 Seed Cultivation 20
2-4 Molecular methods 21
2-4–1 Isolation of plasmid DNA 21
2-4-2 Isolation of plant genomic DNA 21
2-4-3 Isolation of DNA fragments from agarose gel 22
2-4-4 Polymerase chain reaction (PCR) 22
2-4-5 Restriction enzyme digestion 23
2-4-6 DNA sequencing 23
2-4-7 Isolation of total RNA from plant leaves 23
2-4-8 First strand cDNA synthesis from RNA 24Contents
2-4-9 Northern blot 25
2-5 Microbiological methods 27
2-5-1 Collection of fungal spores (Botrytis and Alternaria)27
2-5-2 Infection of Arabidopsis with A. brassicicola 28
2-5-3 Infection of with B. cinerea 29
2-5-4 Culture of bacteria 29
2-5-4-1 Culture of Pseudomonas syringae 29
2-5-4-2 Infiltration of P. syringae 30
2-5-4-3 Culture of Escherichia coli 30
2-5-4-4 Culture of Agrobacterium tumefaciens 31
2-5-5 Infiltration and Re-isolation of P. syringae 31
2-5-6 Transformation of bacteria 32
2-5-6-1 Making of and transformation of electrocompetent E. coli 32
2-5-6-2 Making of rmation of chemically competent E.coli 33
2-5-6-3 Making of and transforemical competent Agrobacterium cells 34
2-6 Biochemical methods 35
2-6-1 Recombinant protein expression in bacteria 35
2-6-2 Purification of GST-fusion protein proteins by GST column 36
2-6-3 Extraction of proteins from plant leaves 38
2-6-4 Determination of protein concentrations in leaf extracts 38
2-6-5 SDS-Polyacrylamide Gel electrophoresis 39
2-6-6 Rapid ethanol-based Coomassie Blue staining of SDS-polyacrylamide gels 40
2-6-7 Western blot 41
2-6-7-1 Wet blotting 41
2-6-7-2 Semi dry blotting 42
2-7 Protein interaction using lexA Yeast-two-hybrid system 43
2-7-1 Quick while yeast cell extract procedure 43
2-7-2 Yeast plasmid extraction 44
2-7-3 Yeast transformation 44
2-8 GUS histochemical analyses 45
2-8-1 Quick GUS staining protocol I 45
2-8-2 GUS staining protocol II 46
2-9 Generation and characterization of transgenic plants 46
2-9-1 Transformation of Arabidopsis plants 46Contents
2-9-2 Selection of the transgenic plants 47
2-10 Statistical analyses 47
3 Results 48
3-1 Comparison of genomic DNA and cDNA of PCC1 paralogs 49
3-2 Analysis of expression of the various PCC1 superfamily members 52
3-2-1 PCC1 superfamily members did not indicate rhythmic expression patterns 52
3-2-2 BPR1 shows induction by avirulent P. syringae (Pst)53
3-2-3 BPR1 was marginally induced by high concentrations of virulent Pst 54
3-2-4 RPM1 (resistance to P. s. pv. maculicola) activated BPR1 and PCC1 expression 55
3-2-5 BPR1 induction depended on RPS2 but PCC1 did not 56
3-2-6 BPR1 induction by Pst(avrRpt2) depends to NDR1 57
3-2-7 Induction of BPR1 and PCC1 by Pst(avrRpt2) is independent from EDS1 58
3-2-8 PCC1 induction after Pst(avrRpt2) treatment was dependent on EDS5
but BPR1 induction was not 59
3-2-9 BPR1 and PCC1 induction after Pst(avrRpt2) infiltration is independent
from SID2 60
3-2-10 Pathogen-mediated BPR1-induction does not depend on NPR1 but pathogen
induction and circadian clock expression of PCC1 depends on NPR1 61
3-2-11 In NahG plants, neither pathogen-mediated BPR1- and PCC1-induction
nor rhythmic expression of PCC1 was observed 61
3-2-12 Exogenous application of salicylic acid activates BPR1 and PCC1 expression 62
3-2-13 Exogtion of ethylene does not trigger BPR1 expression 63
3-2-14 Exogenous application of jasmonic acid does not induce BPR1 expression 64
3-2-15 Pathogen-mediated induction of PCC1 and BPR1 is independent
from COI1 (coronatine insensitive 1) 65
3-2-16 Botrytis cinerea infection resulted in increased BPR1 expression and
reduced PCC1 expression 66
3-2-17 A. brassicicola does not induce PCC1 and BPR1 expression 66
3-2-18 BPR1 is expressed in roots, flowers and pathogen infected leaves, but
PCC1 only in non-treated and pathogen-treated leaves 67
3-3 Functional analysis of BPR1 68
3-3-1 BPR1 knockout and over-expression lines show hypersensitive response
to Pst (avrRpt2) like Arabidopsis ecotype Col-0 72
3-3-2 BPR1 appears to have no role in the compatible interaction of ArabidopsisContents
with Pst DC3000 73
3-3-3 Non-host resistance to Psp is reduced in over-expression BPR1 plants 74
3-3-4 BPR1 knockout and over-expression plants do not show any difference
in symptom development as compared to Col-0 after inoculation with A. brassicicola 76
3-3-5 BPR1 over-expression plants were more susceptible than Col-0 and BPR1
knockout plants to B. cinerea infection 77
3-3-6 BPR1 over-expression plants have longer roots in comparison to Col-0
and BPR1 knockout plants 78
3-3-7 BPR1 is expressed in roots, flowers and infected leaves in BPR1
promoter::GUS plants 80
3-4 Natural variation and polymorphisms in the PCC1 superfamily of Arabidopsis 82
3-4-1 Polymorphic PCC1 in St-0 and Oy-0 showed rhythmic expression pattern
as PCC1 in Col-0 85
3-5 Expression and purification of a recombinant BPR1-GST fusion protein 86
3-6 Identification of BPR1 interacting proteins in yeast EGY48 87
3-7 Summary of Results 91
4 Discussion 93
4-1 The PCC1 superfamily 93
4-2 BPR1 and PCC1 expression analysis 94
4-2-1 BPR1 is induced by avirulent and virulent Pseudomonas 94
4-2-2 BPR1 induction by pathogens depended on resistance genes and their
signalling pathway 94
4-2-3 Is BPR1 dependent on SA and SA-dependent signalling pathways? 95
4-2-4 Is pathogen-mediated BPR1 induction under control of JA and ET and their
signalling pathways? 96
4-2-5 B. cinerea induces BPR1 but A. brassicicola does not 97
4-2-6 Organ-specific BPR1 expression 98
4-3 PCC1 pathogen induction is related to SA-signalling pathway 98
4-3-1 PCC1 and R genes 99
4-3-2 Reduction of PCC1 expression in plants infected with B. cinerea and
increase of PCC1 expression in plants inoculated with A. brassicicola 100
4-3-3 Specific expression of PCC1 in leaves 101
4-4 Functional analyses BPR1 101
4-4-1 BPR1 over-expression and knockout plants do not show visible differencesContents
in symptom development after inoculation with Pst, Pst(avrRpt2), H. parasitica
and A. brassicicola as compared to Col-0 plants 101
4-4-2 BPR1 over-expression lines showed susceptibility to P. syringae pv. phaseolicola 102
4-4-3 BPR1 over-expres showed susceptibility to Botrytis cinerea 104
4-4-4 BPR1 over-expression lines had longer roots 105
4-4-5 BPR1 promoter was active in root vascular tissue, flowers, and infected leaves 105
4-5 Natural variation in the PCC1 superfamily 106
4-6 Future prospects 107
5 Abstract 109
6 Literature 111
7 Appendices 129
7-1 Abbreviations 129
7-2 Figures 133
7-3 Tables 136