Progress 07/15/24 to 07/14/25

Outputs
Target Audience:The target audiences reached by this project includeundergraduate researchers, graduate students, and other plant scientists. PI Wong mentored agraduate studentrotating in the Topp lab who was interested in cover crops and gene-editing technologies for manipulating root system architecture. The graduate student shadowed and workedwithPI Wong on parts of cloning out native OGDH genes from pennycress to generate overexpression lines for this project. PI Wong served as as panelist during a career panel at the Danforth Center being held for students from Virginia Tech's Translational Plant Science Center program. Students and postdocs from all levels were in attendance as well as other plant scientist at the Danforth Center and companies such as Bayer and New Leaf Symbiotic. During the panel, PI Wong talked about his work on cover crops, computational protein engineering methods, and manipulating root system architecture via gene editing. In Febraury, PD Wong was invited to and attended a NSF-funded workshop on Plant Synthetic Biology. Issues surrounding plant synthetic biology were discussed from perspectives in industry and academia. At the workshop, PD Wong talked about this project with other attendees allowing him to grow his network and forge new collaborations for future proposals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?PDWong mentored agraduate studentrotating in the Topp lab who was interested in cover crops and gene-editing technologies for manipulating root system architecture. The graduate student shadowed and workedwithPI Wong on parts of cloning out native OGDH genes from pennycress to generate overexpression lines for this project. The graduate student learned about pennycress and workflow for generating overexpression of the E1-OGDH protein,and he gained experience in the floral dip method for transforming pennycress plants. PD Wong has gained more training in the gene editing technologies through collaboration with the Nusinow lab at the Danforth Center. He also gained experienced working on preparing metabolomics samples and analyzing metabolomics data on a separate project. These skills will be employed in the later goals within this project. During this period, PD Wong attended the Rhizosphere 6 conference which allowed him to learn about the state of the art in root biology and plant-microbe interactions. In February, PD Wong was invited to and attended a NSF-funded workshop on Plant Synthetic Biology. Issues surrounding plant synthetic biology were discussed from perspectives in industry and academia. At the workshop, PD Wong talked about this project with other attendees allowing him to grow his network and forge new collaborations for future proposals. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Transformed plants will continue to be self-crossed to generate homozygous knockout mutants of the E1-OGDH. Plants overexpressing the E1-OGDH protein will be generated once plasmid constructs for Agrobacterium-mediated T-DNA insertion are completed. A working protocol for prime editing in pennycress will be developed. Protein simulations to test single amino acid substitutions to the E1-OGDH protein will commence.

Impacts
What was accomplished under these goals? PD Wong has developed extensive hands-on experience with molecular biology techniques by working in the Topp lab. During this period, PD Wong designed guide RNAs to target both copies of the OGDH gene in pennycress accession MN106. Using the guide RNAs along with constructs including the CRISPR/Cas9 gene-editing technology, pennycress plants were successfully transformedto generate OGDH knockout mutants. Currently, plants are being screened over multiple self-crossed generations to obtain successful homozygous mutants with a large deletion of theE1-OGDH gene to knockout gene function for further study. PD Wong has begun to generate pennycress plants overexpressing the E1-OGDH gene as a positive control. Currently, the E1-OGDH gene has been cloned/copied out of wild-type pennycress plants. Plasmids containing the gene and a natural and synthetic promoter sequence are currently being generated. PD Wong has looked into prime editing papers in the closely related model plant Arabidopsis and ordered materials to adapt prime editing constructs for his work. Simulations of the native E1-OGDH protein have been started to develop a protocol for subsequent protein simulations of mutated proteins.

Publications