PhD candidate Federal University of Paraná Brasilia, Distrito Federal, Brazil
Body of Abstract: The root-knot nematode (RKN) Meloidogyne incognita has caused severe losses in soybean production worldwide. The use of soybean-resistant cultivars is currently the most effective method for its control. The soybean genes Expansin A (GmExA), Glutathione S-Transferase (GmGST), and Pathogenesis-Related Class 10 (GmPR10) are known to be involved in soybean tolerance to nematodes based on omic studies of contrasting genotypes infested with M. incognita. Similarly, the Expansin Like-B gene (GmExpLB) is an ortholog of the AdEXPLB8 gene known for its potential against Meloidogyne species. The overexpression of GmGST and GmPR10 in tobacco plants and soybean hairy roots caused a significant reduction in nematode colonization and reproduction (40.0-58.0%). Likewise, the positive modulation of GmExpA and GmExpLB in soybean hairy roots using CRISPR/dCas9 resulted in a 58.7% and 67.4% decrease in Meloidogyne-induced galls. Histological examination of gall sections revealed a delay in gall development with apparently smaller giant cells containing less cytoplasm area. Confirming these genes’ role in nematode tolerance, we designed a strategy for increasing mRNA translation using CRISPR/Cas9. For that, we evaluated the 5’ leader sequences of these genes for the presence of upstream open reading frames (uORFs) and identified three uORFs for GmExpLB, one for GmGST, and two for GmPR10. The entire 5’ leader region was cloned into a dual-luciferase reporter system harboring the firefly luciferase (LUC) and renilla luciferase (REN) genes. We mutated all uORFs at their start codon (-ATG). The effect of mutated uORFs on luciferase translation was evaluated by transfecting Nicotiana benthamiana protoplasts. RT-qPCR assays indicated that these constructs’ LUC/REN mRNA levels were not significantly different. However, LUC/REN activity presented a 2.0-3.5-fold increase after mutating uORF2 of GmExpLB and uORF1+2 of GmPR10. Our findings suggest the positive modulation of these genes and the depletion of effective uORFs as a transgene-free approach to promote soybean tolerance to phytonematodes.