Plant colonization by GFP-labeled Bacillus amyloliquefaciens FZB42 and transcriptomic profiling of its response to plant root exudates [Elektronische Ressource] / Ben Fan. Gutachter: Rainer Borriss ; Thomas Börner ; Rudolf Ehwald

Ben Fan

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 93
• Poids de l'ouvrage: 8 Mo

Extrait

Humboldt-Universität zu Berlin
Dissertation
Plant colonization by GFP-labeled Bacillus
amyloliquefaciens FZB42 and
transcriptomic profiling of its response to
plant root exudates

zur Erlangung des akademischen Grades
doctor rerum naturalium

Mathematisch-Naturwissenschaftlichen Fakultät I
M. Sc. Ben Fan
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Jan-Hendrik Olbertz
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Andreas Herrmann


Gutachter: 1. Prof. Dr. Rainer Borriss
2. Prof. Dr. Thomas Börner
3. Prof. Dr. Rudolf Ehwald

eingereicht: 07.10.2010
Datum der Promotion: 02.02.2011
List of Contents
Humboldt Universität zu Berlin.............................................................................................1
1 Introduction....................................................................................................................1
1.1 Plant growth-promoting rhizobacteria (PGPR) .........................................1
1.2 Bacillus amyloliquefaciens FZB42............................................................2
1.3 Plant root colonization by PGPR...............................................................2
1.4 Roles of plant root exudates in plant-microbe interaction.........................3
1.5 Using DNA microarray to study gene expression .....................................4
1.6 Sigma factors of Bacillus...........................................................................6
A1.6.1 Sigma factor ..........................................................................................7
B1.6.2 Sigma factor 8
D1.6.3 Sigma factor 8
1.6.4 Extracytoplasmic function (ECF) sigma factors........................................9
X1.6.4.1 Sigma factor ..........................................................................................9
W1.6.4.2 Sigma factor .......................................................................................10
M1.6.4.3 Sigma factor 10
Y1.6.4.4 Sigma factor ........................................................................................11
1.7 AbrB and DegU, two important global transcriptional regulators...........11
1.7.1 AbrB.........................................................................................................11
1.7.2 DegU........................................................................................................12
1.8 Small regulatory non-coding RNA in bacteria ........................................13
1.9 Research objectives..................................................................................15
2 Materials and methods.................................................................................................17
2.1 Chemicals and materials ..........................................................................17
2.2 Plasmids, bacterial strains and primers....................................................18
2.3 Media, buffers and solutions....................................................................23
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2.4 Investigation of plant colonization by FZB42 .........................................26
2.4.1 Growth conditions of bacterial strains and plant materials......................26
2.4.2 Construction of fluorescent protein-labeled FZB42 ................................27
2.4.2.1 GFP-labeling of FZB42 ...........................................................................27
2.4.2.2 Red fluorescent protein-labeling of FZB42 .............................................27
2.4.2.3 Comparison of fluorescence intensity......................................................28
2.4.2.4 Test of fluorescence stability of gfp-labeled FZB42 ...............................28
2.4.3 Colonization of plants by FZB42.............................................................28
2.4.3.1 Colonization of maize seedling roots.......................................................28
2.4.3.2 Colonization of Arabidopsis roots ...........................................................29
2.4.3.3 Colonization of Lemna minor..................................................................29
2.4.4 Specimen preparation for microscopy .....................................................29
2.4.5 Microscopy ..............................................................................................30
2.4.5.1 Fluorescent microscopy ...........................................................................30
2.4.5.2 Confocal laser scanning microscopy .......................................................30
2.4.5.3 Transmission electron microscopy ..........................................................30
2.4.5.4 Scanning electron microscopy.................................................................31
2.5 Transcriptomic investigation of FZB42 to root exudates ........................31
2.5.1 Root exudates...........................................................................................31
2.5.2 Standard molecular biology methods ......................................................32
2.5.3 Transformation in Bacillus amyloliquefaciens ........................................33
2.5.4 Design of B. amyloliquefaciens microarray.............................................33
2.5.5 Total RNA preparation ............................................................................34
2.5.6 Synthesis of labeled cDNA, hybridization and image acquisition ..........34
2.5.7 Transcriptome data analysis.....................................................................35
2.5.8 Real-Time PCR........................................................................................35
II

2.6 Northern blot for small RNA identification.............................................36
2.6.1 Radioactive labeling of oligonucleotides.................................................36
2.6.2 RNA separation and northern blotting.....................................................36
2.6.3 Hybridization and detection.....................................................................36
3 Results..........................................................................................................................37
3.1 Plant colonization by B. amyloliquefaciens FZB42.................................37
3.1.1 Fluorescent Protein-labeling of FZB42 ...................................................37
3.1.2 FB01mut, a brighter spontaneous mutant................................................38
3.1.3 The Stability of GFP and its effect on the growth of FZB42 ..................39
3.1.4 Colonization of maize seedlings by FZB42.............................................40
3.1.5 Colonization of Arabidopsis by FZB42...................................................43
3.1.6Lemna minor by FZB42.................................................45
3.2 Transcriptomic analysis of B. amyloliquefaciens FZB42 in
response to maize root exudates..............................................................49
3.2.1 Assay of the compositions of maize root exudates..................................49
3.2.2 Experimental designs and transcriptomic data preprocessing .................50
3.2.3 Determination of the microarray experimental conditions......................51
3.2.4 A general profiling of the genes which were altered in expression by
root exudates…… ...................................................................................52
3.2.5 Validation of the microarray data by real time PCR ...............................53
3.2.6 The regulated genes with known function...............................................55
3.2.6.1 The genes involved in nutrition utilization..............................................56
3.2.6.2 The genes involved in chemotaxis, motility and biofilm formation........59
3.2.6.3 The genes involved in antibiotic production............................................60
3.2.7 The regulated genes with putative function.............................................62
3.2.8 The effect of soil extract on FZB42 transcriptome..................................63
3.2.9 Clustering analysis...................................................................................64
III

3.3 Alternative sigma factors, global transcriptional regulators and the
response of FZB42 to root Exudates.......................................................66
3.3.1 Involvement of SigB in the response of FZB42 to root exudates............67
3.3.2 Involvement of SigD in the response of FZB42 to root exudates ...........67
3.3.3 Involvement of ECF sigma factors in the response of FZB42 to root
exudates……...........................................................................................69
3.3.4 Involvement of AbrB in the response of FZB42 to root exudates...........71
3.3.5 Involvement of DegU in the response of FZB42 to root exudates ..........74
3.4 sRNAs involved in the response of FZB42 to root exudates...................76
3.4.1 Responses of sRNAs to the root exudates ...............................................78
3.4.2 Effects of the alternative factors, AbrB and DegU on the sRNAs .......80
3.4.3 Characterization of the six sRNAs identified ..........................................81
4 Discussion...........................................................................................................