Location: Crop Improvement and Genetics Research
Project Number: 2030-21220-003-000-D
Project Type: In-House Appropriated
Start Date: Mar 26, 2023
End Date: Mar 25, 2028
The project will develop new molecular tools that enable the precise and efficient introduction and expression of multiple improvement traits in grapevine, citrus, potato, and potentially other crops. These tools will enable more efficient and precise editing and engineering of plants, while limiting the potential for unintended effects. An integrated set of tools, will be developed for the introduction of desirable traits within these crops including, but not limited to, modifications designed to limit the negative effects of smoke taint in wine, and to confer resistance to citrus greening and potato zebra chip diseases. New technologies targeting transgene insertions to specific genomic loci will improve the quality of genetic engineered events, while limiting the potential for unintended consequences. The research will also augment the existing toolbox by identifying and characterizing additional transcriptional control sequences that confer predictable and precise expression of transgenes and the removal of unwanted DNA sequences within a single target locus. Objective 1: Develop novel methods that enhance the efficiency and/or precision of genome editing and genetic engineering of crops (e.g., grapevine, citrus, potato). Sub-objective 1.A: Design and test genetic engineering technologies that enable genetically edited or engineered plants that are free of unneeded selection markers and other unwanted transgenes. Sub-objective 1.B: Develop genetic engineering technologies that enable recombinase-mediated targeted integration and sequential stacking of traits within the genome of genetically engineered plants. Objective 2: Utilize biotechnology to genetically modify grapes with the goal of reducing the accumulation of smoke taint compounds in fruit tissues. Sub-objective 2.A: Evaluate the available genome and expression data to identify candidate genes that are involved in the metabolism of volatile phenolics in grapevine with special attention on the glycosyltransferase gene family. Sub-objective 2.B: Perform transcriptomic research to better understand the gene expression and metabolic changes that occur during berry development in response to smoke exposure. Sub-objective 2.C: Utilize biotechnology approaches, including CRISPR gene editing and genetic engineering, to validate gene function and modify metabolic pathways involved in the retention of smoke-derived phenolics in wine grapes. Objective 3: Generate molecular tools with multiple defense genes to combat Huanglongbing (citrus greening disease) and potato Zebra Chip disease. Objective 4: Isolate and characterize novel transcriptional control elements (promoters, terminators, and insulators) that confer precise control of transgene expression in genetically engineered plants (e.g., grapevine, citrus, potato).
An inducible site-specific gene excision system utilizing the Cre recombinase will be designed and tested in crops. The system will utilize a chemically inducible promoter and cytoplasmic sequestering system to control recombinase expression, enabling strict control of recombinase activity. In parallel, technology enabling targeted integration combined with precise marker removal will be developed and evaluated in crops. “Exchange” T-DNA vectors will be constructed and transformed into “target” transgenic plants. Selection and molecular screening will be used to identify plants in which the incoming DNA has replaced and/or been added to the original target locus. The efficiencies of different combinations of constructs in performing targeted integration and marker removal will be evaluated. Research into the role that candidate genes have in retaining smoke-derived volatile phenolic compounds following smoke exposure and generating smoke-tainted wines will be investigated in grapevine. Genomic, transcriptomic and gene editing/genetic engineering approaches will be utilized to identify genes that influence the retention of smoke volatiles within developing grape berries. Candidate plant pathogen recognition receptor genes will be introduced into potato and citrus plants using established methods for Agrobacterium–mediated transformation. The defense genes will either be constitutively expressed throughout the plant or expressed specifically in the phloem, the site of infection. Ten or more independent events for each candidate gene will have their susceptibility to zebra chip (in potato) or Huánglóngbìng (in citrus) evaluated. Novel grapevine promoter sequences from constitutively or fruit specifically expressed genes will be isolated. The candidate promoters will be fused to a reporter gene and characterized in transgenic grapevine and/or other plants. The reporter gene expression levels will be measured to identify the sequences that provide the highest levels of expression while preserving the desired tissue/organ expression specificity.