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Agrobacterium T−DNA Integration
Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature.
2. Are Agrobacterium Proteins Involved in T-DNA Integration into the Plant Genome?
3. Where in the Plant Genome Does T-DNA Integrate?
4. T-DNA integrates into plant DNA break sites
T-DNA preferentially integrates into double-strand DNA breaks . This observation was followed by two other reports also showing preferential T-DNA integration into double-strand break sites . In each of these studies, a rare cutting meganuclease (either I-SceI or I-CeuI) was used to cut tobacco DNA during transformation. T-DNA was preferentially “trapped” in these cut sites at frequencies up to several percent of the examined integration events. More recently, scientists used CRISPR technology to generate double-strand breaks in DNA, either to generate site-directed mutations or to attempt homology-dependent repair using recombination with correction templates. In several instances, T-DNA was trapped at these break sites following Cas nuclease cutting (e.g., ). It is thus clear that double-strand DNA breaks can act as a “T-DNA magnet”. However, does Agrobacterium take advantage of naturally occurring host DNA breaks (or nicks), or can Agrobacterium infection perhaps induce host DNA disruptions?
That Agrobacterium can incite DNA breaks would not be unusual, because inoculation by other plant pathogens (bacteria, oomycetes, and fungi) can cause double-strand DNA breaks in host plant genomes . DNA disruptions occur in Arabidopsis cells near the site of Agrobacterium infection, as detected by COMET assays. However, because alkaline pH conditions were used in this study, it is not clear whether these disruptions resulted from single-strand nicks or double-strand breaks in the plant DNA . Recent results indicate that Arabidopsis cells, exposed to Agrobacterium but not stably transformed, contain a higher number of in/dels than would be expected from the natural frequency of such mutations . These results suggest that incubation of cells with Agrobacterium is inherently mutagenic, causing double-strand DNA breaks that are mis-repaired.
There are many hints in the literature that Agrobacterium infection can cause mutations independent of T-DNA integration; these mutations may result from induced double-strand DNA breaks that are subsequently mis-repaired. They may also be generated by “abortive integration” of T-DNA, followed by mis-repair of the abortive integration site. For example, N. plumbaginifolia plants, containing one mutant nitrate reductase (NR) gene, could be converted to fully NR null mutants (chlorate resistant) following Agrobacterium−mediated transformation. However, none of these null mutants contained T-DNA in the NR gene . Mutation of the wild-type NR allele must have occurred by some other mechanism.
5. What Is the Mechanism of T-DNA Integration?
6. The Importance of DNA Polymerase Theta for Agrobacterium Transformation and T-DNA Integration
In 2016 van Kregten et al.  published a seminal paper in which they proposed an essential function for DNA polymerase θ in stable transformation of Arabidopsis and T-DNA integration into its genome. These authors examined two DNA polymerase θ (polQ) mutants, tebichi (teb) 2 and teb5. Although they could not detect differences in transient transformation between wild-type and polQ mutant plants, they were not able to obtain any stable transformants of the polQ mutants using either a flower-dip transformation protocol or a root transformation protocol requiring selection of transgenic calli and regeneration of plants from these calli. The authors noted that DNA polymerase θ can “template switch” during DNA replication, and that it can thereby generate “filler” DNA sequences, a common characteristic of T-DNA/plant DNA junctions at the break site, by copying and joining T-strand DNA and microhomologous plant DNA. They also noted that copying T-strand sequences into both ends of a plant DNA double-strand break could result in integration of T-DNA “head-to-head” (RB-to-RB) dimers, also a common characteristic of many T-DNA insertions. T-DNA integration via theta-mediated end-joining thus became the favored model for T-DNA integration into plant genomes.
Nishizawa-Yokoi et al.  re-examined the role of DNA polymerase θ in T-DNA integration. Using the same Arabidopsis teb2 and teb5 mutants used by van Kregten et al. , as well as three independent rice polQ mutants, this group was able to obtain stable transformants of somatic tissue in all tested polQ mutants. Similar to van Kregten et al. , they were not able to transform Arabidopsis by a flower-dip protocol, except when the incoming T-DNA constitutively expressed a wild-type PolQ gene. These authors additionally showed that transient transformation of roots from the Arabidopsis polQ mutants did decrease relative to transformation of wild-type roots. T-DNA/plant DNA junctions isolated from transformed rice and Arabidopsis polQ mutant calli had characteristics similar to those isolated from wild-type tissue. Finally, the authors showed that both Arabidopsis and rice polQ mutants had growth and/or developmental defects; root segments from Arabidopsis polQ mutants did not form callus well and the calli grew slowly. Calli derived from rice polQ mutants did not regenerate plants even under non-transformation and non-selection conditions. The variable penetrance of the tebichi phenotype was recently examined and was shown to increase under stress, including replication stress, conditions . Similar to the situation with Arabidopsis flower-dip transformation, rice polQ mutants could be transformed and regenerated into plants if the incoming T-DNA contained a constitutively expressed PolQ gene. Thus, transformation and developmental deficiencies resulting from mutation of PolQ could be complemented by transient expression of a wild-type PolQ gene in both Arabidopsis and rice.
The entry is from 10.3390/ijms22168458
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