Agrobacterium-mediated transformation remains a cornerstone of plant biology, fueling advancements in molecular genetics, new genomic techniques (NGTs), and the biotech industry. However, recalcitrant crops and technical hurdles persist as bottlenecks. The goal was to develop super-infective ternary vector systems that integrate a novel SA-degrading enzyme, GABA, and ethylene–degrading enzymes, targeting the transformation of crops by neutralizing plant defense system on Agrobacterium. Firstly, both the effect and activity of introducing enzymes were validated in EHA105, an important Agrobacterium strain. Our study demonstrates that all ternary vector (Tv) system variants significantly enhance reporter expression in transient assays with Nicotiana benthamiana and Cannabis sativa. Specifically, incorporating a constitutive virG mutation with novel enzyme combinations increased GFP and RUBY expression in C. sativa by over 5-fold and 13-fold, respectively. The Tv system, combined with a geminivirus replicon, markedly boosted GUS gene expression in tomato, enhancing genome editing efficiency. Notably, compared to controls, Tv-VS demonstrated up to 18-fold and 4.5-fold increases in genome editing efficiency in C. sativa and tomato, respectively. Additionally, stable transformation rates in tomato and Arabidopsis improved significantly, with Tv-VS showing a remarkable 2.5-fold increase in transformation efficiency compared to control strains. The research marks notable progress in Agrobacterium-mediated plant transformation. The innovative ternary vectors overcome plant defense mechanisms, enabling genetic manipulation in previously challenging plant species. This development is anticipated to broaden the applications of plant genetic engineering, contributing to advancements in crop genome editing.

中文翻译：

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开发超感染性三元载体系统以增强农杆菌介导的植物转化和基因组编辑效率


农杆菌介导的转化仍然是植物生物学的基石，推动了分子遗传学、新基因组技术 (NGT) 和生物技术行业的进步。然而，顽固的作物和技术障碍仍然是瓶颈。目标是开发整合新型SA降解酶、GABA和乙烯降解酶的超感染性三元载体系统，通过中和农杆菌上的植物防御系统来靶向作物转化。首先，在重要的农杆菌菌株EHA105中验证了引入酶的效果和活性。我们的研究表明，所有三元载体 (Tv) 系统变体都能显着增强烟草和大麻瞬时测定中的报告基因表达。具体来说，将组成型 virG 突变与新型酶组合相结合，可使 C. sativa 中的 GFP 和 RUBY 表达分别增加超过 5 倍和 13 倍。 Tv系统与双生病毒复制子相结合，显着提高了番茄中GUS基因的表达，提高了基因组编辑效率。值得注意的是，与对照相比，Tv-VS 在苜蓿和番茄中的基因组编辑效率分别提高了 18 倍和 4.5 倍。此外，番茄和拟南芥中的稳定转化率显着提高，与对照菌株相比，Tv-VS 的转化效率显着提高了 2.5 倍。该研究标志着农杆菌介导的植物转化取得了显着进展。创新的三元载体克服了植物防御机制，从而能够对以前具有挑战性的植物物种进行基因操作。 这一进展预计将扩大植物基因工程的应用，促进作物基因组编辑的进步。