Syngenta Biotechnology (China) Co., Ltd, No.25, Life Science Park Road, Beijing, 102206, China.
BMC Plant Biol. 2021 Apr 24;21(1):197. doi: 10.1186/s12870-021-02979-7.
Rice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria. Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants.
We introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants.
We demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.
稻叶枯病是一种世界性的毁灭性疾病，由稻黄单胞菌 pv. 稻种引起（Xoo）。水稻 Xa13 基因启动子区上调转录激活子样 1（UPT）效应盒在 Xoo 致病性中起关键作用。突变 Xa13 的 UPT 盒中关键细菌蛋白结合位点，以消除 PXO99 诱导的 Xa13 表达，是提高水稻对细菌抗性的一种方法。高效产生和选择无转基因编辑植物有助于缩短和简化基于基因编辑的育种过程。T0 植物转基因花粉的选择性消除可以丰富 T1 无转基因后代的比例，种子中颜色标记基因的表达使 T2 植物的选择非常方便和高效。在这项研究中，使用基因组编辑和多重选择系统生成了抗细菌性叶枯病和无转基因的水稻植物。
我们使用 CRISPR/Cas12a 技术在 UPT 盒中引入定点突变，以干扰转录激活因子样效应物（TAL）蛋白结合和基因激活，并生成了具有改良细菌性叶枯病抗性的基因组编辑水稻。T0 植物的转基因花粉通过 Zmaa1 基因的花粉特异性表达被消除，在 T1 代中，单个 T-DNA 插入事件中无转基因植物的比例从 25%增加到 50%。转基因种子通过 DsRed 的特异性 aleuronic 表达被视觉识别和丢弃，这降低了 50%的成本，并使无转基因编辑植物的选择准确率高达 98.64%。
我们证明了 Xa13 启动子 UPT 盒中的核心核苷酸缺失赋予了水稻对叶枯病的抗性，并且通过引入多重选择提高了无转基因植物的选择效率。基因组编辑和无转基因选择的结合是加速功能基因组研究和植物育种的有效策略。
