Gene targeting in polymerase theta‐deficient Arabidopsis thaliana

Abstract

SUMMARY Agrobacterium tumefaciens‐mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T‐DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)‐mediated end joining (TMEJ). Targeting of the T‐DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T‐DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ‐proficient plants we predominantly found GT events accompanied by random T‐DNA integrations, GT events obtained in the teb mutant background lacked additional T‐DNA copies, corroborating the essential role of Polθ in T‐DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high‐quality GT events in crop plants.

Engineered Agrobacterium improves transformation by mitigating plant immunity detection

“…3f). As gamete cells are transformed during floral dipping while somatic cells were transformed in Agrobacterium ‐mediated transient expression, the EF‐Tu elicitor may not be perceived well as in somatic cells, consistent with that the expression of EFR gene was not detectable in Arabidopsis egg cells (Wuest et al ., 2010; Schmidt et al ., 2011) and that no increased stable transformation was observed in efr plants compared with Col‐0 (van Tol et al ., 2022). Next, we tested whether the engineered Agrobacterium strains improve transformation in Camelina sativa , an important oil crop and a different member of the Brassicaceae family, and that also contains an EFR homolog.…”

“…3f) as observed. This is consistent with a recent finding that efr mutant exhibited similar stable transformation efficiency as compared to Col‐0 after Agrobacterium ‐mediated floral dip transformations (van Tol et al ., 2022). Previous studies in Arabidopsis have shown that plant factors important for somatic cell transformation may not affect germ cell transformation (Mysore et al ., 2000; Nishizawa‐Yokoi et al ., 2021), indicating germ cell transformation is largely distinct from somatic cell transformation.…”

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana

“…Also the search for infrequent GT events amongst successfully transformed plants would be greatly facilitated by having lost all random integrations. Indeed, we previously found GT events in teb mutant plants to be devoid of any background integration (van Tol et al., 2022). Unfortunately, however, the frequency of GT events in teb mutant plants was found to be 10‐fold reduced as compared to GT frequencies measured in wild‐type plants (van Tol et al., 2022).…”

“…Desired genetic changes can be accomplished by the process of gene targeting (GT), which is based on homologous recombination (HR) between an introduced donor DNA molecule and a target locus, but this procedure is inefficient in plants and thus requires strong selection and screening of large populations of transformants. The frequency of such events is not only low, but positive events are in most cases (>90%) accompanied by the co‐occurrence of random integrations of the donor molecules elsewhere in the genome (Puchta, 1998; van Tol et al., 2022). Additionally, many positive events turn out to be no true GT events upon closer inspection (Huang & Puchta, 2019; van Tol et al., 2022).…”

“…The frequency of such events is not only low, but positive events are in most cases (>90%) accompanied by the co‐occurrence of random integrations of the donor molecules elsewhere in the genome (Puchta, 1998; van Tol et al., 2022). Additionally, many positive events turn out to be no true GT events upon closer inspection (Huang & Puchta, 2019; van Tol et al., 2022). These include “one‐sided” events, in which one side of the donor molecule recombines with the target locus, but the other side attaches to the genome via end‐joining, as well as ectopic GT (EGT) events, in which the donor molecule acquires sequences from the target locus and integrates elsewhere in the genome.…”