Location: Vegetable Crops Research
Project Number: 5090-21220-005-022-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2019
End Date: Jun 30, 2021
Objective:
The research will develop a rapid diagnostic assay for identification of Potato Virus Y (PVY)-infected tissue.
Approach:
The research will make use of a synthetic Potato Virus Y (PVY) protease substrate that we have already constructed. The synthetic substrate will be expressed in bacteria and purified using standard procedures. Purified substrate will be mixed with reaction buffer and leaf or tuber sap and development of fluorescence monitored using a fluorescence plate reader. The composition of the buffer, the amount of each component in the reaction mixture and reaction time will be varied to maximize the difference in fluorescence intensity between samples from PVY-infected tissue and uninfected tissue. Sensitivity of the assay relative to standard ELISA and PRC methods for detecting PVY will be quantified. Ability of the protease substrate to identify PVY in vitro will be assessed by expressing the substrate in plant tissues following transient transformation and quantifying fluorescence near the transformation site and in distal tissues. The method will be modified as needed so that a clear fluorescence signal is observed from PVY-infected plants but is lacking from non-infected plants.
