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AgrobacteriumT‐DNA integration into the plant genome is essential for the process of transgenesis and is widely used for genome engineering. The importance of the non‐homologous end‐joining (NHEJ) protein DNA polymerase Θ, encoded by thePolQgene, for T‐DNA integration is controversial, with some groups claiming it is essential whereas others claim T‐DNA integration inArabidopsisand ricepolQmutant plant tissue. Because of pleiotropic effects of PolQ loss on plant development, scientists have previously had difficulty regenerating transgenicpolQmutant plants. We describe a protocol for regenerating transgenicpolQmutant rice plants using a sequential transformation method. This protocol may be applicable to other plant species.

 

Agrobacterium transfers T-DNA to plants where it may integrate into the genome. Non-homologous end-joining (NHEJ) has been invoked as the mechanism of T-DNA integration, but the role of various NHEJ proteins remains controversial. Genetic evidence for the role of NHEJ in T-DNA integration has yielded conflicting results. We propose to investigate the formation of T-circles as a proxy for understanding T-DNA integration. T-circles are circular double-strand T-DNA molecules, joined at their left (LB) and right (RB) border regions, formed in plants. We characterized LB-RB junction regions from hundreds of T-circles formed in Nicotiana benthamiana or Arabidopsis thaliana . These junctions resembled T-DNA/plant DNA junctions found in integrated T-DNA: Among complex T-circles composed of multiple T-DNA molecules, RB-RB/LB-LB junctions predominated over RB-LB junctions; deletions at the LB were more frequent and extensive than those at the RB; microhomology was frequently used at junction sites; and filler DNA, from the plant genome or various Agrobacterium replicons, was often present between the borders. Ku80 was not required for efficient T-circle formation, and a VirD2 ω mutation affected T-circle formation and T-DNA integration similarly. We suggest that investigating the formation of T-circles may serve as a surrogate for understanding T-DNA integration. 

Agrobacterium effector protein VirE2 is important for plant transformation. VirE2 likely coats transferred DNA (T-DNA) in the plant cell and protects it from degradation. VirE2 localizes to the plant cytoplasm and interacts with several host proteins. Plant-expressed VirE2 can complement a virE2 mutant Agrobacterium strain to support transformation. We investigated whether VirE2 could facilitate transformation from a nuclear location by affixing to it a strong nuclear localization signal (NLS) sequence. Only cytoplasmic-, but not nuclear-localized, VirE2 could stimulate transformation. To investigate the ways VirE2 supports transformation, we generated transgenic Arabidopsis plants containing a virE2 gene under the control of an inducible promoter and performed RNA-seq and proteomic analyses before and after induction. Some differentially expressed plant genes were previously known to facilitate transformation. Knockout mutant lines of some other VirE2 differentially expressed genes showed altered transformation phenotypes. Levels of some proteins known to be important for transformation increased in response to VirE2 induction, but prior to or without induction of their corresponding mRNAs. Overexpression of some other genes whose proteins increased after VirE2 induction resulted in increased transformation susceptibility. We conclude that cytoplasmically localized VirE2 modulates both plant RNA and protein levels to facilitate transformation. 

Integration ofAgrobacterium tumefacienstransferred DNA (T‐DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T‐DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end‐joining (NHEJ) pathways. Recent evidence suggests that inArabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T‐DNA integration.

We conducted quantitative transformation assays of wild‐type andpolQmutantArabidopsisand rice, analyzed T‐DNA/plant DNA junction sequences, and (forArabidopsis) measured the amount of integrated T‐DNA in mutant and wild‐type tissue.

Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency ofpolQmutants was c.20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice andArabidopsis polQmutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events.polQmutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation.

We suggest that either multiple redundant pathways function in T‐DNA integration, and/or that integration requires some yet unknown pathway.