Progress 09/01/24 to 08/31/25
Outputs
Target Audience:In the first year of our project we have focused mostly on lab studies and as such have not done any outreach or extension efforts related to this work. Changes/Problems:We had challenges staffing in the first year of the proposal due to a lack of appropriate graduate candidates. This slowed both research progress and spending and will likely necessitate a no cost extension at the end of the grant.We have since recruited one graduate student and recently identified a research technician to round out our team and project healthy progress in the coming year. What opportunities for training and professional development has the project provided?This project has provided training for one graduate student, Max Combest, and three undegraduate trainees assisting him. Max has given a talkon this work aninterdeparmental seminar series. How have the results been disseminated to communities of interest?As we are still in the early stages of the project there have not been many major results to communicate as yet. The graduate student working on the project has given an interdepartmental seminar at Colorado State University on this progress. What do you plan to do during the next reporting period to accomplish the goals?In the coming year we aim to fully staff our research team by bringing on a research technician to enable more rapid progress on research goals. We also plan to use our validated viral vector architecture to trialthe efficacy of targeted infection in palmer using the ribozymes targeted to RDR6.In tandem we plan to compare the impact of targeting different genes in the RNAi pathway, first independently and then in tandem,on the restoration of infectivity. If these are successful, we plan on performing common garden experiments to validate the targeted nature of this infection. We also plan to begin serial passaging studies of PVX in Palmer to identify natural mutations that improve PVX infection and systemic transport in Palmer. Finally, we aim to expand our bioinformatic pipelines to enable both unique target selection and identification of proper folds to enhancereliabilityof Ribozyme-based gene knockdown by the viral vectors.
Impacts
What was accomplished under these goals?
In our first year of the grant, we have focused on prototyping the selective infection of PVX in a model plant system and on setting up to twotargeted infection assaysin Palmer amaranth. We have performed experiments that demonstrate thatour targeted infection assay is reproducibly functional in tomatoes. In these experiments we have demonstrated that the deactivated virus is unable to establish an infection in Nicotiana benthamiana, a model plant, as well as tomato. However, upon addition of a ribozyme that targets tomato's RDR6, a key gene in the RNAi pathway, we see a restoration of infectivity in tomato. Crucially, as this ribozyme only targets the RDR6 gene in tomato but not in benthe we observe that infection is only restored in tomato. A key insight we gained was that the placement of the ribozyme in the viral genome made a big difference to viral competence, with different insertion sites havingsignificantly different effects on viral fitness. Through these experiments we have a viral vector design that we can deploy in palmer amaranth to enable targeted infection. In tandem we have leverage previously developedpangenomic resources for palmer amaranth to identify a set of ribozyme targets for RDR6 in palmer that are cross reactive across palmer genotypes but do not target any major Colorado crops, including maize, soybean, sugar beet and potato. We have also developed a infection protocol for PVX in palmer amaranth.
Publications
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