Activity of plant non-homologous end-joining DNA repair proteins is not required for Agrobacterium T-DNA integration
K. MYSORE (1), Z. Vaghchipawala (2), B. Vasudevan (2), S. Park (3), Y. Shen (3), E. Kim (3), S. Lee (2), L. Lee (3), W. Waterworth (4), Z. Zhang (5), C. West (6), S. Gelvin (3) (1) The Samuel Roberts Noble Foundation, Ardmore, OK, U.S.A.; (2) The Samuel Roberts Noble Foundation, , U.S.A.; (3) Purdue University, , U.S.A.; (4) University of Leeds, , United Kingdom; (5) University of Missouri, , U.S.A.; (6) University of Leeds, U.S.A.

Agrobacterium tumefaciens causes crown gall disease in many dicotyledonous plants by transferring T-DNA into the plant genome. T-DNA integration is poorly understood and has been proposed to occur via non-homologous end-joining (NHEJ)-mediated double-strand DNA break (DSB) repair. Here we report a negative role for KU70, KU80, and XRCC4, encoding some of the key proteins required for NHEJ, and DNA LIGASE VI (LIG6) in T-DNA integration. Mutation or silencing of these genes in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Mutation of XRCC1 and PARP1, that play a role in the alternative NHEJ pathway, did not alter stable transformation. Higher order mutants that simultaneously inactivate multiple DNA repair and recombination pathways, such as xrcc1/parp1, ku80/xrcc1, ku80/xrcc1/xpf, and ku80/xrcc1/xpf/xrcc2, also did not alter T-DNA integration, although they inhibited plant growth in high light. Over-expression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. XRCC4 directly interacted with Agrobacterium protein VirE2. Overexpression of KU70 or KU80 in Arabidopsis did not increase stable transformation, suggesting that these proteins are already at saturating levels in wild-type plants. We conclude that genes involved in DSB repair are not required for T-DNA integration, and that delay of DSB repair in some of these mutants provides greater opportunity for T-DNA to integrate.

Abstract Number: S5-5
Session Type: Special Session