TRANSFORM-PGR: Manipulating Agrobacterium-mediated transformation and T-DNA integration for plant synthetic biology and genome engineering

TRANSFORM-PGR：操纵农杆菌介导的转化和 T-DNA 整合，用于植物合成生物学和基因组工程

• 批准号: 1725122
• 负责人: Stanton Gelvin
• 金额: $ 146.71万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1725122&HistoricalAwards=false
• 关键词: TRANSFORM; PGR; Manipulating; Agrobacterium; mediated

Non-technical paragraph:The introduction of DNA into plants (plant transformation to generate genetically modified or transgenic plants) has become a core technology for basic plant research and the agricultural biotechnology industry. Agrobacterium-mediated plant genetic transformation is the most commonly used method to generate transgenic plants. The bacterium Agrobacterium can transfer large DNA-molecules (T-DNA) capable of encoding numerous genes sufficient to, e.g., encode a novel biosynthetic pathway. Thus, Agrobacterium-mediated transformation is a key tool for synthetic biology. Although the Agrobacterium host range is broad, many agronomic important species or specific cultivars remain recalcitrant to transformation. In addition, T-DNA integrates randomly into the plant genome. Consequently, T-DNA may disrupt genes important for plant development and productivity. Random T-DNA integration often occurs in genomic regions that silence encoded transgenes, leading to unpredictable and unstable transgene expression. It is therefore important to develop novel technologies to increase transformation efficiency of a broader range of crop species and agronomic important varieties, and to insert T-DNA into defined locations of any plant genome. This project will develop novel technologies to broaden the plant host range of Agrobacterium, and to direct the integration of T-DNA to specific plant chromosomal regions pre-selected by the scientist. In addition, this project will develop novel technologies to deliver genes to plants efficiently without subsequent integration into the plant genome. This latter technology is important for delivering plant genome engineering reagents without maintaining these reagents after they have accomplished their tasks. Technical paragraph:This project proposes tool development for the plant research community to help enable functional genomics for a broad spectrum of species. Because Agrobacterium-mediated transformation is the preferred DNA transfer approach, one goal of this project is to increase the efficiency of transformation by debilitating plant defense responses to Agrobacterium. This will be accomplished by engineering Agrobacterium strains that can secrete Type III effectors and suppress plant defense responses to enhance transformation. Because controlled T-DNA integration and predictable transgene expression is important for synthetic biology, a second goal is to build a system to facilitate effective and precise DNA integration into plant genomes. This system will be developed using CRISPR/Cas9 to generate breaks in specific tomato genomic sequences that will "trap" T-DNA. These integration sites will be chosen to maximize the probability of stable transgene expression through numerous plant generations and under field conditions. The expression of targeted and randomly integrated transgenes will be assessed. Agrobacterium is also used to deliver genome engineering reagents to plants. However, integration of these reagents is undesirable, and they usually are segregated out of the engineered plants. This is difficult for vegetative-propagated species. The third goal of this project is to develop Agrobacterium strains that can efficiently deliver but not integrate T-DNA encoding genome editing reagents, or will secrete these reagents through a Type III secretion system. This will be accomplished by altering Agrobacterium VirD2, the protein that leads T-DNA from the bacterium into the plant and which is important for T-DNA integration.

非技术性段落：将DNA引入植物（植物转化以产生基因修饰或转基因植物）已成为基础植物研究和农业生物技术产业的核心技术。农杆菌介导的植物遗传转化是获得转基因植物最常用的方法。农杆菌属细菌可以转移能够编码许多基因的大DNA分子（T-DNA），这些基因足以例如，编码一种新的生物合成途径。因此，农杆菌介导的转化是合成生物学的关键工具。虽然农杆菌的宿主范围很广，但许多重要的农杆菌物种或特定的农杆菌品种仍然难以转化。此外，T-DNA随机整合到植物基因组中。因此，T-DNA可能会破坏对植物发育和生产力重要的基因。随机T-DNA整合通常发生在沉默编码的转基因的基因组区域，导致不可预测和不稳定的转基因表达。因此，重要的是开发新技术以提高更广泛的作物物种和农艺学重要品种的转化效率，并将T-DNA插入任何植物基因组的限定位置。该项目将开发新技术，以扩大农杆菌的植物宿主范围，并指导T-DNA整合到科学家预先选择的特定植物染色体区域。此外，该项目还将开发新技术，将基因有效地传递到植物中，而不会随后整合到植物基因组中。后一种技术对于递送植物基因组工程试剂而在它们完成其任务后不维护这些试剂是重要的。技术段落：该项目提出为植物研究界开发工具，以帮助实现广泛物种的功能基因组学。由于农杆菌介导的转化是优选的DNA转移方法，因此本项目的一个目标是通过削弱植物对农杆菌的防御反应来提高转化效率。这将通过工程改造农杆菌菌株来实现，所述农杆菌菌株可以分泌III型效应物并抑制植物防御反应以增强转化。由于受控的T-DNA整合和可预测的转基因表达对合成生物学很重要，因此第二个目标是建立一个系统，以促进有效和精确的DNA整合到植物基因组中。该系统将使用CRISPR/Cas9开发，以在特定的番茄基因组序列中产生断裂，从而“捕获”T-DNA。将选择这些整合位点以最大化通过许多植物世代和在田间条件下稳定转基因表达的可能性。将评估靶向和随机整合的转基因的表达。农杆菌还用于向植物递送基因组工程试剂。然而，这些试剂的整合是不期望的，并且它们通常从工程化植物中分离出来。这对于植物繁殖的物种来说是困难的。该项目的第三个目标是开发能够有效递送但不整合编码T-DNA的基因组编辑试剂的农杆菌菌株，或者将通过III型分泌系统分泌这些试剂。这将通过改变农杆菌VirD 2来实现，VirD 2是一种将T-DNA从细菌引导到植物中的蛋白质，对于T-DNA整合非常重要。

项目成果

CRISPR/Cas9-mediated targeted T-DNA integration in rice

• DOI: 10.1007/s11103-018-00819-1
• 发表时间: 2019-03-01
• 期刊: PLANT MOLECULAR BIOLOGY
• 影响因子: 5.1
• 作者: Lee, Keunsub;Eggenberger, Alan L.;Wang, Kan
• 通讯作者: Wang, Kan

Agrobacterium-delivered VirE2 interacts with host nucleoporin CG1 to facilitate the nuclear import of VirE2-coated T complex.

农杆菌递送的 VirE2 与宿主核孔蛋白 CG1 相互作用，促进 VirE2 包被的 T 复合物进入核。

• DOI: 10.1073/pnas.200964517
• 发表时间: 2020
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Li, X.;Yang, Q.;Peng, L.;Tu, H.;Lee, L.-Y.;Gelvin, S.B.;Pan, S.Q.
• 通讯作者: Pan, S.Q.

Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco

• DOI: 10.1007/s11103-019-00907-w
• 发表时间: 2019-11-01
• 期刊: PLANT MOLECULAR BIOLOGY
• 影响因子: 5.1
• 作者: Hsu, Chen-Tran;Cheng, Yu-Jung;Lin, Choun-Sea
• 通讯作者: Lin, Choun-Sea