Non Technical Summary
Beneficial microbes are known to associate with plants as endophytes, symbionts or just closely located next to the plant in the rhizosphere. These relationships provide favorable effects on plant growth and health under stressed environment conditions and protection against pathogens. Many of these microbes are genetically intractable and difficult to study. Nitrogen fixation by actinorhizal and leguminous plants is an important part of the nitrogen budget of the planet. Actinorhizal plants provide an excellent mechanism to restore disrupted environmental sites. A major hindrance in the application of these association is the lack of genetic tools for the beneficial microbes associated with plants. The purpose of this study is the development of gene editing tools that will allow the genetic analysis of plant-associated microbes and their interactions with their host plants. The establishment of gene editing technology is a major step in manipulating these important beneficial plant-associated microbes and provide a greater understanding of the plant-microbe interactions including a variety of agricultural crops.
Animal Health Component
Research Effort Categories
Goals / Objectives
A wide variety of microbes are associated with plants as endophytes, symbionts or just closed connected to the plant. Many of these microbes have beneficial effects on plant growth and health but have proved to be genetically intractable and difficult to study. The overall goal of this project is to establish gene editing approaches in bacteria that are genetically intractable. The bacterium, Frankia, is an example of one of these plant beneficial symbionts that is considered genetically intractable. It forms a nitrogen-fixing association with woody plants (termed actinorhizal plants) and ensures successful growth and survival of the plant in stressed environments. Nitrogen fixation by actinorhizal plants is an important part of the nitrogen budget of the planet. The plants are involved in land reclamation, reforestation, soil stabilization, landscaping, fuel, and as a food source for ruminant animals. The symbiotic association greatly accelerates growth of the host plant and indirectly does the same for neighboring plants. The goals of this project are: (1) Establishing the use of CRISPR-Cas9 for gene editing in Frankia and (2) Initiate protocol learned in Objective 1 to other genetically intractable actinobacteria. The project addresses the expansion of gene editing technologies for use in agriculturally-important microorganisms that are associated with plants and/or animals. The establishment of gene editing technology is a major step in manipulating these important beneficial plant-associated microbes that have been considered genetically intractable and understudied. These tools are necessary to provide a greater understanding of the plant-microbe interactions and may be extended to other microbes for a variety of crops. The discovery of gene editing approaches with these genetically difficult microbes could also have broader implications and beneficial effects in biotechnology technologies for use in agriculturally-important microorganisms that are associated with plants.
Building off our recent successfully efforts developing stable gene transfer protocols for a genetically intractable plant-associated actinobacteria, Frankia, we will focus on establishing gene editing or CRISPR-Cas9 in the genetically intractable Frankia as our first plant-associated microbe and this tool will be extended to other genetically intractable plant-associated actinobacteria.Objective 1. Establishing the use of CRISPR-Cas9 for gene editing in Frankia.Several modified CRISPR-Cas9 plasmids having different antibiotic resistance genes have been constructed. A synthesized Frankia 16S promoter was cloned in front of the Cas9 gene to allow expression of Cas9 gene in Frankia. The constructs have been successfully introduced and stability maintained in Frankia. The plasmid inside Frankia expresses Cas9 gene so we are at a good starting point to generate mutants by this gene editing approach. Potential genes involved in plant-microbe interactions or growth under harsh environmental conditions will serve as potential targets for the gene-editing experiments. To target a specific gene and generate a large deletion (starting with a 200-300 bp deletion), two editing templates are required for both ends of the targeted genes (1 Kbp each in length). Once generated, the mutants will be confirmed by molecular approaches and the physiological properties including plant infectivity will be determined.Objective 2. Initiate protocol learned in Objective 1 to other genetically intractable actinobacteriaWe will use lessons learned in Objective 1 to guide our efforts to utilize gene editing technology with other plant-associated actinobacteria. For these studies, we have initially chosen three different actinobacteria, Micromonospora strain L5, Nocardia casuarinae BMG51109, and Nocardia sp. strain BMG111209. Genome databases are available for all three actinobacteria. We have shown that the Nocardia strains are beneficial to plant health. Other plant-associated actinobacteria will also be included in these studies to expand the range of this gene editing tool. Several actinobacteria have been isolated from other plants and their genomes have been sequenced to provide background information for this gene editing tool.