Poster: Plant-pathogen interactions

Abs # 526: Non-homologous end joining pathway plays a major role in T-DNA integration in Arabidopsis thaliana

Presenter:	Li, Jianxiong , jianxiongli01@hotmail.com
Authors	Li, Jianxiong  (A)   Vaidya, Manjusha  (A)   Citovsky, Vitaly  (A)   Tzfira, Tzvi  (A)
Affiliations:	(A): Dept of Biochemistry and Cell Biology, State University of New York at Stony Brook

Non-homologous end joining (NHEJ) is the predominant pathway for DNA double strand (ds) break repair in higher eukaryotes cells. Several proteins have been shown to function in the NHEJ pathway in both in mammalian and plant cells. During NHEJ, the Ku70K/Ku80 heterodimer plays an essential role in binding the ds DNA molecule and in recruiting the cellular DNA-repair enzymes. T-DNA integration may resemble a NHEJ in plants and was proposed to occur through ds intermediates. Yet, the host proteins that are involved in the integration process, as well as the integration mechanism, are still largely unknown. To study the role that the plant NHEJ pathway plays in T-DNA integration, we isolated Arabidopsis T-DNA insertion mutants knocked-out in each of these key proteins of the NHEJ pathway. The AtKu80 mutant was resistant to stable, but not transient, Agrobacterium-mediated genetic transformation, as indicated by root tumorigenesis and GUS expression assays, respectively. The AtKu70 mutant, on the other hand, remained susceptible to stable and transient Agrobacterium infection. Interestingly, both AtKu80 and AtKu70, which are localized in the plant cell nucleus, remained cytoplasmic in yeast and mammalian cells, indicating their potentially different nuclear import pathways in plant and non-plant systems. Furthermore, over-expression in yeast of AtKu80 and AtKu70 fused to a conventional, basic NLS increased the efficiency of Agrobacterium-mediated stable transformation of these cells nearly 3 fold. We propose that T-DNA integration occurs via the plant NHEJ pathway. In this pathway, binding of Ku70K/Ku80 heterodimer to ds T-DNA integration intermediates may represent the first molecular-mechanical step towards the integration event.