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

非技术段：将DNA引入植物（将植物转化为转基因或转基因植物）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产生转基因植物最常用的方法。农杆菌可以转移能够编码大量基因的大dna分子（T-DNA），足以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。虽然农杆菌的寄主范围很广，但许多重要的农艺品种或特定的栽培品种仍然难以转化。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，开发新技术来提高更广泛的作物物种和重要农艺品种的转化效率，并将T-DNA插入任何植物基因组的特定位置是很重要的。该项目将开发新的技术来扩大农杆菌的植物寄主范围，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，该项目将开发新的技术，以有效地将基因传递给植物，而无需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无需在试剂完成任务后维持这些试剂非常重要。技术段落：本项目为植物研究界提出工具开发，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首选的DNA转移方法，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。这将通过工程农杆菌菌株来实现，这些菌株可以分泌III型效应物并抑制植物的防御反应，以促进转化。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，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。该系统将利用CRISPR/Cas9在特定的番茄基因组序列中产生断裂，从而“捕获”T-DNA。这些整合位点的选择将最大限度地提高转基因在许多植物世代和田间条件下稳定表达的可能性。将评估靶向和随机整合转基因的表达。农杆菌也被用于向植物输送基因组工程试剂。然而，这些试剂的整合是不可取的，它们通常从工程植物中分离出来。这对无性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基因组编辑试剂的T-DNA的农杆菌菌株，或者通过III型分泌系统分泌这些试剂。这将通过改变农杆菌VirD2来实现，这种蛋白质将T-DNA从细菌引入植物，对T-DNA的整合很重要。

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.