Gene Silencing

..................................................................................................................................64 INTRODUCTION............................................................................................................................65 MATERIAL AND METHODS ..........................................................................................................68 Microorganisms and plants....................................................................................................68 Cloning procedures ................................................................................................................68 VIGS constructs for agroinoculation .....................................................................................69 Rolling circle amplification Restriction fragment length polymorphism (RCA-RFLP) ......69 Inoculation of plants...............................................................................................................69 Analysis of viral DNAs ...........................................................................................................69 Microscopy .............................................................................................................................70 Reverse Transcription – PCR (RT-PCR) and small interfering RNA (siRNA) isolation and detection .................................................................................................................................71 RESULTS......................................................................................................................................73 Plant inoculation with pBIN-TR227 (Hsc70 ) .....................................................................73 DISCUSSION.................................................................................................................................79 REFERENCES ...............................................................................................................................82 THE COMPLEX ROLE OF ABUTILON MOSAIC VIRUS (ABMV) AC2 IN REMODELING OF VIRAL AND TRANSREPLICON MINICHROMOSOMES ......................................................................................................85 ABSTRACT...................................................................................................................................85 INTRODUCTION............................................................................................................................87 MATERIAL AND METHODS ..........................................................................................................91 Microorganisms, plants, and general methods ......................................................................91 Cloning of viral constructs.....................................................................................................91 Transreplicon cloning ............................................................................................................92 Plant transformation and inoculation ....................................................................................92 Agroinfiltration assay.............................................................................................................92 Rolling circle amplification Restriction fragment length polymorphism (RCA-RFLP) ......93 Analysis of viral DNA conformations.....................................................................................93 Small interfering RNA (siRNA) isolation and detection.........................................................94 Western blot analysis .............................................................................................................94 Microscopy and GFP imaging ...............................................................................................95 RESULTS......................................................................................................................................96 Trans-replication of GFPexpress replicon during AbMV infection.......................................96 Rep is necessary and sufficient to release GFPexpress replicon...........................................98 Co-infiltration assays ...........................................................................................................100 Analysis of topoisomer distribution to recognize changes in chromatin condensation.......101 DISCUSSION...............................................................................................................................104 REFERENCES .............................................................................................................................109 GESAMTLITERATURVERZEICHNIS .....................................................................113 DANKSAGUNG ....................................................................................................................125 ERKLÄRUNG........................................................................................................................126 CURRICULUM VITAE.....................................................................................................127