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Role of the Defensin-Like Protein Gene Family in Plant Reproductive Isolation and Disease Resistance in Arabidopsis Thaliana

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Role of the Defensin-Like Protein Gene Family in Plant Reproductive Isolation and Disease Resistance in Arabidopsis Thaliana Role of the defensin-like protein gene family in plant reproductive isolation and disease resistance in Arabidopsis thaliana DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG Vorgelegt von Ajay John Arputharaj aus Dubai, U.A.E im Januar 2017 Das Promotionsgesuch wurde eingereicht am: 7.1.2017 Die Arbeit wurde angeleitet von: Dr. Mariana Mondragón-Palomino Unterschrift: (Ajay John Arputharaj) To the Glory of Jesus Christ Table of Contents Table of Contents List of Figures ........................................................................................................................................ vi List of Tables ........................................................................................................................................... x Abbreviations ......................................................................................................................................... xi 1. Summary ............................................................................................................................................ 1 2. Zusammenfassung ............................................................................................................................... 3 3. Introduction ......................................................................................................................................... 5 3.1 Double fertilization ........................................................................................................................ 5 3.2 The role of cysteine-rich peptides in reproduction ......................................................................... 7 3.3 Role of DEFLs in species -preferential manner during reproduction ............................................ 8 3.3.1 Arabidopsis species are an ideal experimental model for studying reproductive isolation .... 9 3.4 Role of defensins in immunity ..................................................................................................... 10 3.5 Other functional roles of defensins .............................................................................................. 12 3.6 Structure of Defensins .................................................................................................................. 13 3.6.1 Defensins under selection pressure....................................................................................... 14 3.7 Fusarium graminearum ................................................................................................................ 15 3.8 Aims of the study ......................................................................................................................... 19 4. Material and Methods ........................................................................................................................ 20 4.1 Plant materials and growth conditions ......................................................................................... 20 4.1.1 Surface sterilization of Arabidopsis seeds ............................................................................ 20 4.1.2 Growth conditions of Arabidopsis species ........................................................................... 20 4.1.3 Arabidopsis thaliana root germination in MS plates ............................................................ 20 4.1.4 Pollen grain germination and tube growth ........................................................................... 21 4.2 Pollination related work ............................................................................................................... 21 4.2.1 Emasculation of Arabidopsis species flowers ...................................................................... 21 4.2.2 Pollination experiments ........................................................................................................ 21 4.2.3 Aniline blue staining of the pistils ........................................................................................ 21 4.3 Fusarium graminearum work ....................................................................................................... 22 4.3.1 Fusarium graminearum strain .............................................................................................. 22 4.3.2 F. graminearum culturing in potato dextrose agar (PDA) plates ......................................... 22 4.3.3 Preparation of F. graminearum culture for infection ........................................................... 22 4.3.4 Inoculation of Arabidopsis species for RNAseq .................................................................. 22 4.3.5 Chloral hydrate method for clearing infected tissue ............................................................. 23 4.3.6 Wheat germ agglutinin-tetramethylrhodamine (WGA-TMR) staining of the pistil ............. 23 4.4 RNAseq related work ................................................................................................................... 23 i Table of Contents 4.4.1 Total RNA extraction of tissue samples ............................................................................... 23 4.4.2 Preparation of cDNA libraries for RNAseq and sequencing ................................................ 24 4.4.3 RNAseq analysis and transcriptomic analysis of differential gene expression .................... 24 4.4 Bacterial related work .................................................................................................................. 25 4.4.1 Preparation of chemically competent Escherichia coli cells ................................................ 25 4.4.2 Escherichia coli transformation of ligation reaction ............................................................ 25 4.4.3. Preparation of competent Agrobacterium cells ................................................................... 25 4.4.4 Agrobacterium tumefaciens transformation ......................................................................... 26 4.4.5 Agrobacterium mediated transformation of Arabidopsis thaliana ....................................... 26 4.5 Molecular biology work ............................................................................................................... 27 4.5.1 Isolation of Genomic DNA from plants using CTAB method ............................................. 27 4.5.2 Primer design ........................................................................................................................ 28 4.5.3 Polymerase chain reaction (PCR) ......................................................................................... 28 4.5.3.1 Amplification of PCR products for cloning .................................................................. 28 4.5.3.2 Colony screening from LB plates .................................................................................. 28 4.5.3.3 Genotyping of transgenic plants .................................................................................... 29 4.5.4 Digestion of the plasmid ....................................................................................................... 29 4.5.5 DNA ligation of digested fragments ..................................................................................... 30 4.5.6 Agarose gel electrophoresis .................................................................................................. 30 4.5.7 Gel Elution............................................................................................................................ 30 4.5.8 Miniculture of bacterial colony ............................................................................................ 31 4.5.9 Isolation of plasmid DNA .................................................................................................... 31 4.5.10 Cloning strategies ............................................................................................................... 31 4.5.10.1 pENTR/D-TOPO cloning reaction .............................................................................. 31 4.5.10.2 LR reaction .................................................................................................................. 32 4.5.10.3 Cloning for subcellular localization ............................................................................ 32 4.5.10.4 Cloning for promoter analysis. .................................................................................... 33 4.5.10.5 Cloning of transgenic RNAi lines ............................................................................... 33 4.6 qPCR related work ....................................................................................................................... 34 4.6.1 Pistil collection for qPCR analysis ....................................................................................... 34 4.6.1.1 Pollination study ................................................................................................................ 34 4.6.1.2 Pollination - infection study .............................................................................................. 34 4.6.1.3 Infection (aging) study ...................................................................................................... 34 4.6.2 cDNA synthesis .................................................................................................................... 35 4.6.3
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