The Lotus japonicus Temperature-Sensitive Root Development and Nodulation Mutant brush Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Kristina Haage am 16. Mai 2012 Dissertation eingereicht am: 16. Mai 2012 Tag der mündlichen Prüfung: 20. Juli 2012 1. Gutachter: Prof. Dr. Martin Parniske 2. Gutachter: Prof. Dr. Arthur Schüßler Eidesstattliche Versicherung Ich versichere hiermit an Eides statt, dass die vorliegende Dissertation von mir selbständig und ohne unerlaubte Hilfe angefertigt ist. Erklärung Hiermit erkläre ich, dass die Dissertation nicht ganz oder in wesentlichen Teilen einer anderen Prüfungskommission vorgelegt worden ist. Ich habe nicht versucht, anderweitig eine Dissertation einzureichen oder mich einer Doktorprüfung zu unterziehen. Für meine Eltern TABLE OF CONTENTS TABLE OF CONTENTS 1! Introduction .................................................................................................... 1! 1.1! The Arbuscular Mycorrhiza ................................................................................ 1! 1.2! The Root Nodule Symbiosis ............................................................................... 5! 1.3! The Common Symbiosis (SYM) Pathway .......................................................... 6! 1.4! Calcium in Arbuscular Mycorrhiza and Root Nodule Symbiosis Signalling . 8! 2! Results .......................................................................................................... 10! 2.1! A Forward Genetic Screen for Mutants Impaired in Arbuscular Mycorrhiza Development ............................................................................................................... 10! 2.1.1! The AM Screen ............................................................................................. 11! 2.1.2! Putative AM Mutants Impaired in Different Steps during AM ........................ 12! 2.1.3! SL2042-N ...................................................................................................... 16! 2.1.4! SL1856-N ...................................................................................................... 16! 2.1.5! SL1755-N ...................................................................................................... 17! 2.2! The Calcium-Dependent Protein Kinase (CPK) 29 ......................................... 18! 2.2.1! CPK29 of Lotus japonicus ............................................................................. 19! 2.2.1.1! Gene Structure of Lj CPK29 ................................................................................ 19! 2.2.1.2! Expression Patterns of Lj CPK29a and Lj CPK29b in Roots and Nodules ......... 22! 2.2.1.3! Subcellular Localization of Lj CPK29a and Lj CPK29b ....................................... 23! 2.2.2! CPK29 of Medicago truncatula ..................................................................... 24! 2.2.2.1! M. truncatula CPK29 Gene Structure .................................................................. 24! 2.2.2.2! Tnt1 Insertion Lines of Mt CPK29 ....................................................................... 25! 2.2.3! Phylogeny of CPK29 Homologs .................................................................... 25! 2.3! The Temperature-Sensitive Nodulation and Root Development Mutant brush ........................................................................................................................... 31! 2.3.1! Phenotypic Characterization of brush Mutants ............................................. 33! 2.3.1.1! Temperature Shifts at Early Developmental Stages ........................................... 33! 2.3.1.2! Temperature Shift Two Weeks after Germination ............................................... 36! 2.3.1.3! Temperature Shift Six Weeks after Germination ................................................. 38! 2.3.2! Map-Based Cloning of brush ......................................................................... 40! 2.3.2.1! Screening for Recombinants ............................................................................... 40! 2.3.2.2! Phenotypes and Genotypes of F4 and F5 Progenies ......................................... 43! 2.3.3! brush Backcross Mutants Resemble brush Mutants ..................................... 47! 2.3.4! Cyclic Nucleotide-Gated Channel 1 of Lotus japonicus ................................ 51! 2.3.4.1! EMS-Induced Mutation in CNGC1 ...................................................................... 51! 2.3.4.2! Complementation of brush with CNGC1 ............................................................. 51! 2.3.4.3! TILLING Mutants of CNGC1 ............................................................................... 54! 2.3.4.4! ProCNGC1-GUS Analysis ................................................................................... 56! TABLE OF CONTENTS 2.3.4.5! The CNGC Gene Cluster .................................................................................... 56! 2.3.4.6! Phylogenetic Relationships of CNGCs of L. japonicus, M. truncatula and A. thaliana ........................................................................................................................... 58! 3! Discussion .................................................................................................... 60! 3.1.1! A Forward Genetic Screen for Mutants Impaired in Arbuscular Mycorrhiza Development ............................................................................................................ 60! 3.1.2! The Calcium-Dependent Protein Kinase 29 of Lotus japonicus .................... 62! 3.1.3! The Lotus japonicus Mutant brush ................................................................ 67! 4! Summary ...................................................................................................... 72! 5! Materials and Methods ................................................................................ 73! 5.1! Materials ............................................................................................................. 73! 5.1.1! Plant Material, Fungal and Rhizobial Strains ................................................ 73! 5.1.2! Oligonucleotides ............................................................................................ 73! 5.1.3! Media and Solutions ...................................................................................... 73! 5.1.3.1! Plant Cultivation Media ....................................................................................... 73! 5.1.3.1.1! FP-Medium ................................................................................................... 73! 5.1.3.1.2! B5-Medium ................................................................................................... 74! 5.1.3.2! Media for Bacteria Cultivation ............................................................................. 74! 5.1.3.2.1! LB-Medium ................................................................................................... 74! 5.1.3.2.2! TY-Medium ................................................................................................... 74! 5.1.3.3! GUS Staining Buffer ............................................................................................ 75! 5.1.3.4! RNA Extraction Buffer ......................................................................................... 75! 5.1.3.5! Buffers for DNA Extraction .................................................................................. 75! 5.1.3.5.1! DNA Extraction Buffer .................................................................................. 75! 5.1.3.5.2! HB Buffer ...................................................................................................... 76! 5.1.3.5.3! Wash Buffer .................................................................................................. 76! 5.1.3.5.4! CTAB Buffer ................................................................................................. 76! 5.1.3.6! DNA Storage Buffer ............................................................................................ 76! 5.2! Methods .............................................................................................................. 77! 5.2.1! Plant Cultivation and Crossings .................................................................... 77! 5.2.2! AM Inoculation and Staining ......................................................................... 77! 5.2.3! Inoculation with Rhizobia .............................................................................. 77! 5.2.4! GUS-Staining ................................................................................................ 77! 5.2.5! Temperature-Shift Experiments .................................................................... 78! 5.2.5.1! Temperature-Shift Two and Four Days post Germination .................................. 78! 5.2.5.2! Temperature-shift Two Weeks post Germination ................................................ 78! 5.2.5.3! Temperature-Shift Six Weeks post Germination ................................................. 78! 5.2.6! Lotus japonicus Hairy-Root Transformation .................................................. 78! TABLE OF CONTENTS 5.2.7! Transient Transformation of N. benthamiana Leaves ................................... 79! 5.2.8! DNA Extraction from Plant Leaves ................................................................ 79! 5.2.8.1! Small-Scale DNA Extraction ..............................................................................