Physiological and molecular basis of Azospirillum-Arabidopsis Interaction Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades angefertigt am Institut für Molekulare Pflanzenphysiologie und Biophysik an der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Nazeer Ahmed aus Quetta (Pakistan) Würzburg 2010 Eingereicht am: Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Thomas Dandekar 1. Gutachter: PD Dr. Dirk Becker 2. Gutachter: PD Dr. Susanne Berger Tag des Promotionskolloquiums: …………………………… Doktorurkunde ausgehändigt am ……………………… Dedicated to My father Table of contents Table of contents 1 Introduction ........................................................................................... 14 1.1 Plants and the rhizospheric microbes ..................................................... 14 1.2 Mycorrhizal interactions .......................................................................... 15 1.3 Diazotrophs ............................................................................................. 18 1.3.1 Rhizobia-legume mutualism .................................................................... 19 1.3.2 Plant growth promoting Rhizobacteria / associative symbionts ............... 21 1.3.3 Azospirillum ............................................................................................. 22 1.3.3.1 Interaction with plants .......................................................................... 25 1.3.3.2 Azospirillum and plant growth promotion ............................................. 28 1.3.3.3 Azospirillum and plant root morphology ............................................... 33 1.3.3.4 Azospirillum and biocontrol .................................................................. 35 1.3.3.5 Azospirillum and plant tolerance to a biotic stress ............................... 35 1.3.3.6 Azospirillum and plant improved mineral and water uptake ................. 36 1.4 Plant pathogenesis and mutualism ......................................................... 38 1.5 Aims of the project .................................................................................. 41 2 Material and methods ........................................................................... 42 2.1 Plant material and growth conditions ...................................................... 42 2.1.1 For microarray ......................................................................................... 42 2.1.2 For Reactive species (ROS) measurements ........................................... 42 2.1.3 For Arabidopsis and tomato cell cultures ................................................ 43 2.2 Bacterial cultures and elicitor preparations ............................................. 44 2.2.1 Bacterial cultures .................................................................................... 44 2.2.2 Elicitor preparations ................................................................................ 45 2.2.2.1 Flagella isolation .................................................................................. 45 2.2.2.2 Bacterial lysates ................................................................................... 46 2.2.2.3 Live bacteria ......................................................................................... 46 2.3 Bacterial DNA isolation and PCR conditions ........................................... 46 2.4 Inoculation of plants ................................................................................ 47 2.5 Bioassays ................................................................................................ 47 2.5.1 Medium alkalinization responses ............................................................ 47 2.5.2 Reactive Oxygen species ........................................................................ 48 2.6 RNA isolation .......................................................................................... 48 2.6.1 Total RNA concentration ......................................................................... 49 2.6.2 RNA gel ................................................................................................... 49 2.7 Microarray Hybridization and stastical analysis ....................................... 49 Table of Contents 2.8 DNA digestion ......................................................................................... 50 2.9 Reverse transcription .............................................................................. 50 2.9.1 Gel electrophoresis ................................................................................. 51 2.10 Real time qRT-PCR ................................................................................ 51 2.10.1 Primer designing ..................................................................................... 51 2.10.2 Determination of annealing temperatures of primers .............................. 52 2.10.3 Gel documentation .................................................................................. 54 2.10.4 PCR Product Purification ........................................................................ 54 2.10.5 Spectrophotometery ................................................................................ 54 2.10.6 Making of Standards1-10 for qRT-PCR .................................................. 54 2.10.7 Buffers ..................................................................................................... 55 3 Results ................................................................................................... 57 3.1 Early signalling events ............................................................................ 57 3.1.1 Extracellular alkalinization and PAMP perception ................................... 57 3.1.1.1 Validation of the system ....................................................................... 57 3.1.1.2 Azospirillum flagella do not induce extracellular alkalinization ............. 58 3.1.1.3 Azospirillum lysates and medium alkalinization. .................................. 59 3.1.1.4 Nature of elicitor ................................................................................... 61 3.1.1.5 Activity of Azospirillum lysates in tomato cells ..................................... 61 3.1.2 Reactive oxygen species (ROS) production ............................................ 62 3.1.2.1 Validation of the experimental system ................................................. 62 3.1.2.2 Azospirillum Sp7 induces an oxidative burst in Col-0 plants ................ 63 3.1.2.3 The Arabidopsis efr mutant are insensitive to Sp7 lysates .................. 65 3.2 Transcriptome profiling ............................................................................ 67 3.2.1 Genetic responses-6 hours post inoculation (6hpi) ................................. 67 3.2.1.1 Signaling .............................................................................................. 69 3.2.1.2 Defence ................................................................................................ 69 3.2.1.3 Others .................................................................................................. 70 3.2.2 Genetic responses-24 hours post inoculation (24hpi) ............................. 71 3.2.2.1 Lipid transfer proteins (LTPs) ............................................................... 73 3.2.2.2 Cell wall ................................................................................................ 73 3.2.2.3 Hormone metabolism ........................................................................... 74 3.2.2.4 Stress / defense ................................................................................... 75 3.2.2.5 Others .................................................................................................. 76 3.2.3 Genetic responses-96 hours post inoculation (96hpi) ............................. 77 3.2.3.1 Cell wall ................................................................................................ 82 ~ 5 ~ Table of Contents 3.2.3.2 Secondary metabolism ........................................................................ 85 3.2.3.3 Hormone metabolism ........................................................................... 87 3.2.3.4 Stress ................................................................................................... 90 3.2.3.5 RNA ..................................................................................................... 93 3.2.3.6 Miscellaneous ...................................................................................... 97 3.2.3.7 Signalling ........................................................................................... 101 3.2.3.8 Not assigned ...................................................................................... 103 3.2.4 Common Genetic responses in all three time points ............................. 105 3.2.5 Comparative analysis ............................................................................ 107 3.2.6 Validation of microarray results by qRT-PCR ........................................ 109 4 Discussion ........................................................................................... 111 4.1 Early signalling events .......................................................................... 111 4.2 Genome wide studies - a comparative approach .................................. 116 4.2.1 Genes with alike regulation trend .........................................................