First Place
Genomes to Gene-editing: Discovery and Validation of Candidate Genes to Enhance Disease Resistance to Spring Black Stem and Leaf Spot Disease
Fungal pathogens cause the majority of crop diseases leading to $200 billion in losses annually. Spring black stem and leaf spot (SBS) is a devastating foliar disease of Medicago sativa (alfalfa) affecting stand survival, yield, and forage quality. The objective of this research is to identify and validate candidate genes involved in host resistance to SBS disease. Robust resistance has been previously reported in the M. truncatula accession SA27063 (HM078). Specifically, two quantitative trait loci (QTL) rnpm1 and rnpm2 have been identified. The recessive nature of these QTL suggests a loss-of-function event in this accession, which fits the inverse gene-for-gene model. We carried out a genome assembly of HM078 to assess structural variants within the QTL regions. We identified a retrotransposon insertion in a ubiquitin conjugating E2, as well as frameshift mutations in several disease resistance related genes including NBS-LRRs, an F-box, and glycosidase. Next, the transcriptomes of HM078 and susceptible accession A17 were profiled after SBS inoculation. Several genes were upregulated in HM078 across multiple time points including lipoxygenase, 3-ketoacyl-CoA synthase, and RLK protein kinase. Isoflavone reductase and protein phosphatase 2C were upregulated in A17, while constitutively expressed in HM078. CRISPR/Cas knock-out and overexpression mutant plants for promising candidate genes were generated in the susceptible M. truncatula accession R108, and reagent efficacy was evaluated. Future research will include disease screening of these mutant lines to validate genes underlying host resistance to this destructive pathogen.
Jacob Botkin, Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108. Abstract Presentation P-1008
Second Place
Establishing Plantago as a Model Species for Plant Vascular Biology
The study of plant vascular tissue is of interest to researchers due to its important role in plant growth and development. Our laboratory has been developing species in the genus Plantago as models for vascular biology studies, due to their unique attribute of having easily extractable vascular tissue. We developed a transformation method for narrowleaf plantain (P. lanceolata), using Agrobacterium tumefaciens to target three-week-old root tissue to achieve a transformation efficiency of ~20%. This method has been published in the Journal of Visualized Experiments (J. Vis. Exp. (193), e64777, doi:10.3791/64777 (2023)). A second, more rapid transformation method was also developed that uses developmental regulators to rapidly generate transgenic plants in vivo. Newly germinated seedlings are incubated in Agrobacterium containing the morphogenic genes, resulting the emergence of transgenic shoots on root surface. This was developed into a paper. Plantago species are unique in the aspect of transporting two types of carbohydrates rather than one: sucrose and sorbitol. However, the phloem-loading mechanisms for these carbohydrates have not been experimentally studied. We have generated transgenic narrowleaf plantains in which the phloem-expressed sucrose transporter gene was knocked out via CRISPR/Cas9. The retarded growth of the knockout plant suggests that sucrose is loaded to the phloem via an apoplastic pathway. Similar CRISPR/Cas9 knockouts are being developed for the sorbitol transporter. We will conduct a series of experimental comparisons between the two types of knockout plants. This study will dissect the phloem loading pathway for sucrose and sorbitol and reveal the importance of the two different phloem mobile carbohydrates to plant growth.
Hannah Levengood, Department of Agronomy, Purdue University, West Lafayette, IN 479061. Abstract Presentation: P-1011
Third Place
Recruiting Novel Cytidine Deaminases for Base Editing in Plants
Cytosine base editors (CBEs) are prominent tools that perform precise C-to-T conversions by catalyzing site-specific deamination. The most employed deaminase in plant base editing consists of an engineered human APOBEC3A (hA3A-Y130F). Nonetheless, the diversity of deaminases across the animal kingdom warrants exploration for base editors with unique editing windows and profiles. Here, we identified novel cytidine deaminases from the tree of life, which were benchmarked for base editing in plants. In total, 29 novel cytidine deaminases and four CBE controls were tested by targeting the OsCGRS55 gene in rice protoplasts. The top-performing deaminases were assessed in tomato protoplasts at two target sites in the SolyA7 gene. Moreover, the best-performing deaminases were evaluated for multiplex base editing in the OsALS2, OsGN1a, OsGS3, and OsGW2 genes in rice protoplasts and stable lines. Base editing efficiencies were quantified using amplicon deep sequencing. In rice protoplasts, we identified 11 novel deaminases that achieved either similar or superior editing efficiency of the established CBE controls. Four of these exhibited higher editing efficiencies than the best CBE control in tomato protoplasts. Notably, low C-to-A/G conversions were carried out by the novel deaminases, ensuring high base editing purities. Several novel deaminases showed distinct base editing windows, offering potential for tailored modifications of specific bases, and directed evolution of target genes. Finally, we unveiled two novel deaminases with equivalent base-editing efficiencies to the hA3A-Y130F control in transgenic rice lines, among which, the OoA3GX2 deaminase enabled biallelic editing with a less stringent motif preference and wider base editing window. Remarkably, unlike hA3A-Y130F, only OoA3GX2 and DlA3G were able to generate multiplex base-edited plants for four target sites. This study expands the repertoire of highly efficient CBEs for several agricultural applications.
Danyel Fernandes Contiliani, Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD and Graduate Program of Genetics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, BRAZIL Abstract Presentation: P-1006
