TRTech-PGR: Engineering Agrobacterium to Express a Type III Secretion System to Improve Plant Transformation and Genome Editing

植物基因工程是生物技术发展最快的领域之一。对耐高温、干旱、病原体、除草剂或产生理想性状的转基因植物的需求很大。分子生物学和植物转化的最新进展使从许多作物物种中培育转基因植物成为可能。农杆菌介导的植物转化(AMT)是获得转基因植物最常用的方法。与其他转化方法相比，它是首选的方法，因为它相对便宜，易于使用，并且导致低拷贝数的基因插入。AMT的主要缺点是目前不能用于许多植物物种/变种，特别是单子叶植物。AMT抗性的两个主要原因是植物对根癌农杆菌的强烈防御反应和植物组织不能再生。该项目试图通过改造农杆菌来弥补这些不足，这种农杆菌可以传递能够抑制植物防御反应和/或促进植物转化和再生的蛋白质。在这样做的过程中，该项目可能会克服基因编辑的一些缺点，方法是通过一种工程农杆菌菌株传递基因编辑试剂。该项目产生的信息和材料将免费提供给科学界。该项目还将为植物基因组学和转化方面的培训和推广活动提供绝佳的机会。转化植物的能力使植物生物技术行业发生了革命性的变化。农杆菌介导的植物转化(AMT)是植物转化的首选形式。然而，目前只有某些植物物种/品种的有效AMT是可用的。本项目致力于克服这一限制，通过将假单胞菌III型分泌系统(T3SS)改造为农杆菌，该系统可以输送细菌效应蛋白来抑制植物防御反应，和/或植物生长调节因子或其他可以促进植物再生和转化的植物蛋白。此外，该项目旨在克服目前基因组编辑的一些限制。基于CRISPR-Cas9的植物基因组编辑/工程作为一种改善作物田间表现的方法正变得流行起来。基于CRISPR-Cas9的基因组编辑的主要挑战之一是需要将Cas9表达为转基因。Cas9的结构性表达可以导致植物中的非靶标突变，并为解除调控创造障碍。这一缺点可以通过表达T3SS的工程农杆菌菌株直接将Cas9蛋白转移到植物中来克服。利用农杆菌工程菌株加强小麦转化和基因组编辑将被用作概念验证。来自美国境内研究机构和大学的各级科学家将通过动手研讨会接受植物转化方面的培训。这项研究中产生的数据和知识将发表在同行评议的期刊上，通过俄克拉荷马州立大学和普渡大学的网站传播，并在科学会议上发表。生物资源将根据要求和通过拟南芥生物资源中心(ABRC)获得。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Plant genetic engineering is one of the most rapidly developing fields of biotechnology. There is a great need for engineered plants that are resistant to heat, drought, pathogens, herbicides, or that produce a desirable trait. Recent developments in molecular biology and plant transformation have made it possible to produce transgenic plants from a number of crop species. Agrobacterium-mediated plant transformation (AMT) is the most commonly used method to produce transgenic plants. It is preferred over other means of transformation because it is relatively inexpensive, easy to use, and results in low copy number gene insertions. The main drawback of AMT is that it cannot currently be used for many plant species/varieties, especially monocots. The two main reasons for recalcitrance to AMT are a strong plant defense response against Agrobacterium tumefaciens and the inability of the plant tissue to regenerate. This project seeks to remedy these deficiencies by engineering Agrobacterium that can deliver proteins that can dampen plant defense responses and/or promote plant transformation and regeneration. In doing so, the project may overcome some of the drawbacks of gene editing by delivering gene editing reagents through an engineered Agrobacterium strain. Information and materials generated from this project will be made freely available to the scientific community. This project will also offer outstanding opportunities for training and outreach activities in plant genomics and transformation.The ability to transform plants has revolutionized the plant biotechnology industry. Agrobacterium-mediated plant transformation (AMT) is the preferred form of plant transformation. However, efficient AMT of only certain plant species/varieties is currently available. This project seeks to overcome this limitation by engineering Agrobacterium with the Pseudomonas type III secretion system (T3SS) that can deliver bacterial effector proteins to dampen plant defense responses, and/or plant growth regulating factors or other plant proteins that can enhance plant regeneration and transformation. In addition, this project aims to overcome some of the current limitations of genome editing. CRISPR-Cas9 based plant genome editing/engineering is becoming popular as a method to improve crop performance in the field. One of the main challenges of CRISPR-Cas9 based genome editing is the need to express Cas9 as a transgene. Constitutive expression of Cas9 can cause off-target mutations in plants and create a hurdle for de-regulation. This drawback may be overcome by directly delivering Cas9 protein into plants through an engineered Agrobacterium strain expressing a T3SS. Enhancement of wheat transformation and genome editing with the engineered Agrobacterium strains will be used as a proof of concept. Scientists of all levels from research institutes and universities within the United States will be trained in plant transformation through a hands-on workshop. Data and knowledge generated in this study will be published in peer reviewed journals, disseminated via Oklahoma State University and Purdue University websites, and presented at scientific meetings. Biological resources will be accessible upon request and through the Arabidopsis Biological Resource Center (ABRC).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.