Precise genome modification of agrobacterium based on one-step homologous recombination via endogenous transfer DNA

Here, a simple and precise genome modification method for agrobacterium was described and tested in strain GV3101. Taking the induced endogenous Transfer DNA (T-DNA) as the single-strand homologous repair template, this makes it possible to achieve valid and accurate genome modification for agrobacterium even without the strengthening of gene editing tools such as the CRISPR/Cas9 system. In addition, the introduction of tool plasmids into recipient agrobacteria can be achieved by the freeze-thaw method rather than complex triparental mating or expensive electroporation, which makes it more convenient. Here, the modification rate of agrobacterium strain GV3101 at both the upstream and downstream of VirD2 is 4.5% with 100% sequence accuracy. This research provides a valuable potential tool for genetic engineering in agrobacterium, with potential applications in biotechnology and agricultural sciences.