Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
(N/A)
Non Technical Summary
Plant genetic engineering is a set of technologies that, along with conventional plant breeding practices, has the potential to make major contributions to crop productivity and resilience to mounting disease and climate-related pressures. However, in many economically important crop species and individual lines, genetic engineering is currently either infeasible or only marginally productive, requiring a large amount of time, labor, and resources to achieve. Research directed toward improving engineering processes has focused on approaches such as optimizing the nutrient and hormone composition of tissue culture media and delivering genes that boost the formation of new plantlets after gene transmission. So far, there has been relatively little focus on another potential avenue to improvement--genetically enhancing the organisms most commonly used as a gene delivery vehicle in plant genetic engineering to improve their capacity for transmitting genetic material.Microbes in the Agrobacterium species complex are known to cause disease symptoms in a wide range of plants by delivering a genetic sequence into a host plant's cells, which is then incorporated into its genome. This unique capability makes Agrobacterium a "natural genetic engineer" and thus this naturally occurring process has been coopted by scientists and crop developers to deliver genetic sequences of choice into crop plant genomes for the purpose of affecting a desired trait (i.e., genetic engineering). To employ an Agrobacterium strain for this end, a (typically) disease-causing strain referred to as a "wild-type" strain must first be engineered to remove the genetic sequence that it transfers into host plants, while leaving the cellular machinery necessary for transferring gene sequences intact. This operation, referred to as "disarmament," results in an Agrobacterium strain that can no longer generate disease symptoms in plants, but can instead be used as a vehicle to deliver genetic material supplied by the researcher.Today, only a handful of disarmed Agrobacterium strains, most of which were originally derived from their wild-type progenitor strains in the late 1980s or early 90s, are widely available for plant researchers to use in developing genetic engineering processes for plant lines. Many advances have been made in biotechnology since these few strains were initially disarmed. Newly developed tools for genetic manipulations of bacteria can enable the rapid development of additional disarmed strains, taking advantage of the genetic diversity within wild-type Agrobacterium (some of which seem to be specialized to particular plant hosts) to further improve genetic engineering capabilities in plants which are currently difficult to modify. Additionally, a few tools have been developed which can dramatically improve the efficiency of transferring a gene sequence of interest from Agrobacterium to target plant tissues. However, "installing" these sequences into Agrobacterium cells, especially in combination with each other can be quite difficult or impossible, at least in their current form.The aim of this project is to "engineer the engineer" by generating a toolkit for making a variety of modifications to various Agrobacterium strains, both those commonly used in plant genetic engineering processes as well as novel wild-type ones, all directed toward the goal of improving the efficiency and applicability of plant genetic engineering processes. We will also design a new system for rapidly and efficiently installing existing genetic tools that have been shown to boost the rate of gene delivery from Agrobacterium to plants into new strains. After using these new resources to produce several enhanced strains as a proof-of-concept, we will put them to the test by using them in genetic engineering processes for difficult plant species (e.g., hops, trees), evaluating their gene delivery capacities against those of the more conventional strains regularly used in these protocols. The genetic tools and resources resulting from this project will be made available for other researchers and crop developers to use for following the same strategy, which ideally will help to develop or improve genetic engineering processes in many additional crops.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Goals / Objectives
Goal 1 (Research): Expand the capacity for plant transformation by enhancing transgene delivery from Agrobacterium to target plantsObjectives:Assemble an Agrobacterium strain engineering toolkit with the best available toolsDevelop a new system for stacking plant transformation-enhancing genes in agrobacteriaAs proof-of-concept, generate modified Agrobacterium strains using the developed engineering toolkit and gene-stacking systemTest performance of newly developed strains for plant transformation, compared to standard strainsGoal 2 (Career development): Gain the skills and knowledge necessary to launch a plant biotechnology startupObjectives:Identify trait needs and opportunities in individual specialty crop sectorsChart regulatory pathways for bringing potential biotech plant lines to marketUnderstand technology licensing processes and gain competence in IP protectionContinue acquiring formal and informal business trainingSecure startup funding for a project to demonstrate the feasibility of producing potential biotech plant products
Project Methods
To assess the relative functionality of the published Agrobacterium engineering tools (base-editor, INTEGRATE, and recombineering systems) for accomplishing different genetic alterations (gene knockout, sequence insertion, and sequence replacement) across diverse genetic backgrounds, experiments will be performed in four "test" strains of Agrobacterium consisting of the conventional laboratory strains AGL1 and LBA4404, as well as two wild-type strains. Experiments will consist of parallel delivery of plasmids containing each tool into the relevant strains by room-temperature electroporation, followed by dilution and plating of each culture. Depending on the specific modification being made, efficiency of the different tools will be assessed by taking colony counts as well as colony PCR and/or sequencing on a subset of colonies.For the purpose of identifying antibiotic resistance marker genes to use as components in the design of the new gene stacking system, a series of antibiotic susceptibility screens will be performed using at least 10 diverse wild-type Agrobacterium strains. The cell density in liquid cultures for each strain will be equalized and then serially diluted to an appropriate concentration for scoring numbers of discreet colonies. Then, cultures will be plated on a series of MGYS media plates with at least three different concentrations of each antibiotic being evaluated. Colony formation on these plates will be evaluated against an antibiotic-free control plate. Using the cell density levels described above, strains will be considered susceptible to an antibiotic at a given concentration if no colonies grow on the test plate versus >100 colonies formed on the control plate.Incorporation of vetted engineering tools into a cloning kit as well as assembling components of the gene stacking system will be accomplished by standard molecular cloning techniques including restriction digest/ligation, Golden Gate assembly, and Gibson assembly. When necessary, synthetic gene fragments may be generated using a commercial service, otherwise they will be PCR-amplified or digested from pre-existing plasmids acquired through Addgene. Plasmids generated using the engineering toolkit components will be used to "install" the AgroStack system in Agrobacterium tester strains. The performance of the system in these strains as well as the system's potential cross-reactivity with the GAANTRY system will be evaluated by making at least two gene stacking iterations (in each system) and screening colonies resulting from each modification for susceptibility to the relevant antibiotics. Sequencing of PCR fragments or whole genome sequencing of a subset of bacterial clones will be perform as appropriate to verify that outcomes of the stacking steps were achieved as expected.The potential "enhancement" of engineered Agrobacterium strains resulting from work on this project will be evaluated by incorporating these strains into established transformation protocols used for hop and/or poplar in the primary mentor's laboratory. Strains will be evaluated against equivalent standard laboratory disarmed strains on the basis of their transformation efficiency, which will be quantified using fluorescent marker expression within the target plant tissue as well as the number of transgenic events produced per number of infected explants as responses. For each of these responses, treatments will be compared to each other, and to controls, using ANOVA and Duncan's multiple range test (if needed, after mathematical transformation to improve normality of data).The outputs of this project will be evaluated for their impact on the field of Agrobacterium biology and plant genetic engineering based on tracking the usage of molecular components (plasmids) and Agrobacterium strain resources by the research community. For example, the number of plasmid requests the tools receive on Addgene as well as the number of requests our lab receives for the newly engineered strains can serve as quantitative measures of this project's impact.