Institute of Biology, Leiden University, Leiden, The Netherlands.
Lett Appl Microbiol. 2022 Feb;74(2):228-237. doi: 10.1111/lam.13605. Epub 2021 Nov 27.
Agrobacterium tumefaciens is the causative agent of crown gall disease and is widely used as a vector to create transgenic plants. Under laboratory conditions, the yeast Saccharomyces cerevisiae and other yeasts and fungi can also be transformed, and Agrobacterium-mediated transformation (AMT) is now considered the method of choice for genetic transformation of many fungi. Unlike plants, in S. cerevisiae, T-DNA is integrated preferentially by homologous recombination and integration by non-homologous recombination is very inefficient. Here we report that upon deletion of ADA2, encoding a component of the ADA and SAGA transcriptional adaptor/histone acetyltransferase complexes, the efficiency of AMT significantly increased regardless of whether integration of T-DNA was mediated by homologous or non-homologous recombination. This correlates with an increase in double-strand DNA breaks, the putative entry sites for T-DNA, in the genome of the ada2Δ deletion mutant, as visualized by the number of Rad52-GFP foci. Our observations may be useful to enhance the transformation of species that are difficult to transform.
根癌农杆菌是冠瘿病的病原体，被广泛用作创建转基因植物的载体。在实验室条件下，酵母酿酒酵母和其他酵母和真菌也可以被转化，并且根癌农杆菌介导的转化（AMT）现在被认为是许多真菌遗传转化的首选方法。与植物不同，在酿酒酵母中，T-DNA 优先通过同源重组整合，而非同源重组的整合效率非常低。在这里，我们报告说，在删除编码 ADA 和 SAGA 转录适配器/组蛋白乙酰转移酶复合物的组件的 ADA2 后，无论 T-DNA 的整合是否通过同源重组还是非同源重组介导，AMT 的效率都显著增加。这与双链 DNA 断裂的增加相关，双链 DNA 断裂是 T-DNA 的潜在进入位点，在 ada2Δ 缺失突变体的基因组中，通过 Rad52-GFP 焦点的数量来可视化。我们的观察结果可能有助于增强转化那些难以转化的物种。
