Location: Genetic Improvement for Fruits & Vegetables Laboratory
Project Number: 8042-21000-283-014-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2018
End Date: Jul 31, 2021
Objective:
Targeting cell wall development should prevent Phytophthora (P.) species from infecting plant tissue and causing disease.
1. Design small double stranded RNAs that target the cell wall production of P. infestans and P. erythroseptica.
2. Test dsRNAs for suppression of Phytophthora species in culture and on tubers.
3. Test dsRNA constructs through expression in transgenic plants, targeting Russet Norkotah, Red Norland, and Kennebec.
Approach:
Initial targets will involve genes that are responsible for cell wall synthesis and composition (cellulose synthases and cellulose binding domain encoding genes). Specific regions within the genes will be optimized through testing of individual dsRNAs obtained commercially from IDT DNA (Iowa, USA). Treatments will be made by application of 50 micromoles of dsRNAi to 1 milliliter potato dextrose broth in 24-well plates, and inoculated with sporangial suspensions. Cut tubers will receive the same treatment of dsRNA followed by inoculation with sporangia. Growth in liquid culture will be measured by dry weight, and tubers will be scored by discoloration symptoms and tuber tissue decay.
Testing of dsRNAs in plants will utilize a high throughput silencing plasmid RNAi-GG (2012 PLoS One 7:e38186). Transgenic potatoes are routinely generated in our lab, using Kennebec and Bintje. We expect to be able to transform and regenerate Russet Norkotah and Red Norland, however, the proof of concept can be demonstrated in Kennebec first. Transgenic plants will be grown to maturity in a greenhouse and tubers harvested for use in resistance screening. Foliar resistance to P. infestans will also be screened during greenhouse cultivation, using detached leaves.
