Combating Root-Knot (Meloidogyne spp.) and Fusarium oxysporum with Stabilized Allicin (Allium sativum) and siRNA Constructs

Overview

Meloidogyne spp. , or the root -knot nematode, is the most destructive nematode disease worldwide and persists in threatening the sustainability of the vegetable industry in numerous regions. Currently, no environmentally green pesticide is registered to cure this disease and preserve plant health. Fusarium Wilt (Fusarium oxysporum) is a fungal vascular root disease with no registered cure, causing a 95% yield reduction in Cavendish bananas. In this experiment, allicin (Allium sativum) was used to treat root -knot-infected soil, with Velum (Fluopyram) as the standard control. Allicin has a fast degradation rate, chitosan encapsulation was performed to enhance efficacy. Oligonucleotides were designed to silence the virulence factor Secre ted in Xylem 1 (SIX1) in Fusarium Wilt. Root gall index through visual measurements showed a 54.55% decrease, confirmed with plant vigor physical measurement where the plant height increased by 15%. Soil porosity was used to measure the water retention of soil. The macropore content was higher in treated soil by 15%, respectively. Among the different concentrations (100 µM, 500 µM, 2 mM, 5 mM) of allicin, 2 mM was found to be optimal. Oligonucleotide sequence 2 demonstrated a TM of 56.2°Cand 55.6% GC content. The siRNA conserved linear secondary structure, providing strong functionality. Based on the fin dings, allicin can be an effective and sustainable solution for root -knot management, supporting the global agriculture industry, while oligonucleotides have the potential for effective knockout of Fusarium Wilt virulence, with implications on the economy and food security. Optimization of Electroporation Based CRISPR Protocol in Immortalized B-cells: For future studies on the role of a PTPN2 SNP in dysregulation of immune function Amy Xia Cherry Creek High School, Greenwood Village, CO Mentor Dr. Soojin Kim, Barbara Davis Center for Childhood Diabetes - CU Anschutz In this study, I optimized a cost -efficient protocol for editing immortalized human B -cells using CRISPR delivery via nucleofection, GFP, and flow cytometry. In vitro human cell disease models are essential for studying and developing treatments for geneti c diseases. The point mutation rs1893217 in the PTPN2 gene has been associated with multiple genetic autoimmune diseases such as type 1 diabetes through SNP linkage studies. It is unknown how the mutation alters immune response, as it is located within an intron. There is no existing model for this mutation in mice or human cell culture. To study this mutation, I optimized the CRISPR/Cas9 gene editing system for Burkitt lymphoma B -cell lines. B -cells are essential for understanding disease pathogenesis but are difficult to transfect due to their small size. The aim of this study was to use a cost-efficient nucleofection CRISPR delivery system and the GFP reporter gene to determine: optimal nucleofection program, whether to use DNA or RNA, and which B -cell li ne — BJAB, Daudi, Ramos, or Raji — showed the greatest potential to be genetically modified. Using the Lonza nucleofection program DS -104 and sgRNA, I achieved GFP expression of 83.15% in the BJAB cell line. These findings will be used to introduce the rs1893217 point mutation into B-cells to understand its role in B -cell regulation, specifically B -cell signaling. This model will help us understand how the SNP contributes to the dysregulation of the immune system and development of autoimmunity.