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

Oklahoma State University, Stillwater OK

Investigators

Abstract

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.