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2019/03/13 ABRC Seminar

Speaker: Dr. Stanton Bruce Gelvin (H. Edwin Umbarger Distinguished Professor, Department of Biological Sciences, Purdue University, USA)
Topic: Mechanism of Agrobacterium T-DNA integration into the plant genome

The mechanism of T-DNA integration into the plant genome remains controversial. Because T-DNAs containing sequences homologous to the plant genome rarely integrate using homologous recombination, it has been assumed that a non-homologous end-joining (NHEJ) pathway is used for integration. Although some studies have indicated less stable transformation of Arabidopsis or rice NHEJ mutants compared to wild-type plants, our laboratory has shown that single or higher order mutants in both the classical (cNHEJ) pathway and the alternative NHEJ pathway (using microhomology-mediated end-joining; MMEJ) maintain stable transformation frequencies similar to or even greater than that of wild-type plants. These transformation results were confirmed by blotting of high molecular weight DNA from transformed but not selected plant tissues, and by suppression PCR analysis of T-DNA/plant DNA junctions from these tissues. We hypothesize that delayed repair of double-strand DNA breaks in these mutants provides more opportunity for T-DNA integration. Recently, the Hooykaas laboratory proposed that DNA polymerase theta (PolQ) is required for T-DNA integration into the Arabidopsis genome. We have examined both Arabidopsis tebichi (teb/polQ) and rice CRISPR-generated polQ mutants for transient and stable Agrobacterium-mediated transformation. These mutants show decreased transient transformation but significant (~20% wild-type) levels of stable transformation. T-DNA/plant DNA junctions isolated from stably transformed rice and Arabidopsis tissues are similar to those isolated from wild-type plants. Both rice and Arabidopsis polQ mutants show growth/developmental phenotypes that may be responsible for decreased transformation frequency: Arabidopsis polQ mutant root calli grow slowly, and rice polQ mutant calli fail to regenerate plants. Taken together, these results indicate either that these various DNA repair pathways function redundantly, or that some unknown pathway is used for T-DNA integration into the plant genome.

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