Impact of glyphosate application to transgenic Roundup Ready® soybean on horizontal gene transfer of the EPSPS gene to Bradyrhizobium japonicum and on the root-associated bacterial community Laura María Arango Isaza Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt Institut für Biochemische Pflanzenpathologie und Abteilung Mikroben-Pflanzen Interaktionen Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie der Ludwig-Maximilians-Universität München Mai 2009 1. Gutachter: Prof. Dr. Anton Hartmann 2. Gutachter: Prof. Dr. Heinrich Jung Eingereicht am: 05.05.2009 Tag der mündlichen Prüfung: 14.07.2009 To my father, my mother and my Felix Contents Abbreviations........................................................................................................1 Abstract.................................................................................................................3 Zusammenfassung................................................................................................5 1 Introduction ...................................................................................................8 1.1 Worldwide situation of genetically modified crops ....................................................8 1.2 Glyphosate mode of action and transgenic resistance mechanism..............................9 1.3 Horizontal gene transfer in the rhizosphere...............................................................11 1.4 Bradyrhizobium as candidate for horizontal gene transfer of the CP4-EPSPS from RR soybean................................................................................................................14 1.5 Rhizosphere and rhizodeposition of translocated glyphosate....................................16 1.6 Effects of glyphosate on non target organisms..........................................................18 1.7 Methodological approach for analysing bacterial diversity ......................................20 1.8 Problem statement and objectives of this work.........................................................22 1.8.1 Horizontal gene transfer of CP4-EPSPS to Bradyrhizobium japonicum ..........22 1.8.2 Effect of glyphosate application on the microbial rhizosphere community......23 2 Materials and Methods ...............................................................................24 2.1 Buffers and solutions.................................................................................................24 2.2 Media.........................................................................................................................25 2.3 Plant material and glyphosate source ........................................................................26 2.4 Reference strains........................................................................................................27 2.5 Growth of bacterial cultures ......................................................................................27 2.5.1 Cultivation of E. coli .........................................................................................27 2.5.2 Cultivation of B. japonicum...............................................................................28 2.6 Growth measurements: Optical density.....................................................................29 2.7 Oligonucleotides........................................................................................................29 2.8 Vectors.......................................................................................................................31 2.9 DNA extraction from RR-Soybean ...........................................................................34 2.10 DNA quantification...................................................................................................34 2.11 PCR amplification of the EPSPS construct from RR-Soybean.................................34 2.12 DNA gel electrophoresis ...........................................................................................35 2.13 DNA size markers.....................................................................................................36 2.14 Plasmid DNA extraction from E. coli cells ...............................................................36 2.15 Sequencing of cloned DNA constructs......................................................................36 2.16 Restriction enzyme cleavage.....................................................................................37 2.17 Extraction and purification of DNA fragments from agarose gels............................37 2.18 Enzyme removal from DNA samples........................................................................37 2.19 Plasmid dephosphorylation........................................................................................38 2.20 Ligation......................................................................................................................38 2.21 Colony PCR for cloned insert screening ...................................................................38 2.22 Cloning EPSPS constructs into TA Cloning® system vectors in E. coli..................38 2.23 Cloning the EPSPS construct under the control of the nptII promoter .....................39 2.24 Generation of electrocompetent cells ........................................................................40 2.24.1 E. coli S17-1 electrocompetent cells .................................................................40 2.24.2 B. japonicum electrocompetent cells.................................................................40 2.25 Transformation experiments......................................................................................41 2.25.1 Electroporation of E. coli...................................................................................41 2.25.2 Electroporation of B. japonicum........................................................................41 2.26 Conjugation – biparental mating ...............................................................................42 2.27 B. japonicum natural transformation on agar plates and in liquid cultures ...............43 2.28 Spontaneous mutations in B. japonicum under glyphosate selection pressure..........43 2.29 Greenhouse experiment .............................................................................................44 2.29.1 Seed sterilization................................................................................................44 2.29.2 Seed germination ...............................................................................................44 2.29.3 RR soybean cultivation conditions on the greenhouse......................................44 2.29.4 Growth conditions in the greenhouse ................................................................45 2.29.5 Experimental design..........................................................................................46 2.30 Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis of RR soybean root-associated bacteria.........................................................................48 2.30.1 T-RFLP analysis................................................................................................48 2.30.2 Total DNA extraction from the microbial community of washed RR soybean roots .....................................................................................................49 2.30.3 PCR for T-RFLP analysis..................................................................................49 2.30.4 Restriction enzyme digestion of labelled PCR products ...................................50 2.30.5 DyeEx Spin purification ....................................................................................51 2.30.6 Detection of end-labelled T-RFs .......................................................................51 2.30.7 16S rRNA gene clone library ............................................................................52 2.30.8 Phylogenetic analysis of the 16S rRNA gene clone library ..............................52 2.30.9 Evaluation of the T-RFLP data..........................................................................53 3 Results...........................................................................................................55 3.1 Amplification of the full length transgene EPSPS ....................................................55 3.2 Cloning into the B. japonicum integration vectors pRJ1035 and pRJ1042...............56 3.3 Cloning the 35S-EPSPS construct into a pCR2.1-TOPO® vector in E. coli .............57 3.4 Cloning the 35S-EPSPS construct into the B. japonicum integration vector pRJ1042.....................................................................................................................57 3.5 Mobilization of the pRJ-35S-EPSPS vector into B. japonicum by biparental mating ........................................................................................................................59 3.6 Monitoring expression of the 35S-EPSPS construct in B. japonicum.......................60 3.7 Cloning the EPSPS gene under control of the nptII promoter ..................................60 3.7.1 nptII promoter joined to the EPSPS gene inside a pCR®-XL-TOPO® vector...60 3.7.2 nptII- EPSPS construct cloned into the suicide vector pRJ1035.......................63 3.8 B. japonicum transformation by electroporation .......................................................64