|Institution:||Iowa State University|
|Keywords:||genome editing; genome modification; TAL effector; TAL effector nuclease; Genetics|
|Full text PDF:||http://lib.dr.iastate.edu/etd/13875
The transcription activator-like effectors (TALE) are the largest family of the type III effector proteins from Xanthomonas spp. Each TALE contains an N-terminal signal for its secretion and translocation into host cells, a central modular DNA-binding repeat domain, and three nuclear localization signals (NLS) and an acidic activation domain (AD) at the C-terminus. Once internalized, TALEs facilitate the bacterial parasitism by transcriptionally activating the host target genes. TALE repeats recognize and bind to the target DNA sequence in a simple and unique "one to one" fashion, i.e., one repeat corresponds to one nucleotide and one type of repeat preferentially binds one of four necleotides, a code that makes TALE an ideal material to achieve the recognition of any preselected DNA sequence. In my thesis I describe the development and functional characterization of TALENs, fusion proteins of native or artificial TALEs and the cleavage domain of the restriction enzyme FokI, in vitro and in yeast, and demonstrate the feasibility of customizing the novel TALEN to target and inactivate the yeast endogenous genes. I also investigated the efficiency of the gene editing through the repair of TALEN-caused double-stranded DNA breaks by non-homologous end-joining (NHEJ) and homologous recombination (HR). Furthermore, I utilized the designer TALENs to edit the TAL effector binding elements (EBEs) in the promoters of the disease susceptibility (S) genes targeted by the rice blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). The gene editing disrupted the induction of the S genes by the naturally occurring TALEs (PthXo1 and AvrXa7) and thus generated the disease resistant rice lines. Finally, using Xoo-delivered designer TALEs to induce the expression of rice SWEET genes that encode sugar transporters in rice, I identified two disease susceptibility genes potentially targeted by the as yet unidentified TALEs in Xanthomonas oryzae pv. oryzae.