Location: Sugarbeet and Potato Research
Project Number: 3060-21000-044-06-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2017
End Date: Jun 30, 2021
Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the most important foliar disease of sugarbeet worldwide. Our objectives are to 1) identify suitable targets from the C. beticola genome for gene silencing using double stranded (DS)RNA; 2) produce dsRNA targets using in vitro transcription; 3) confirm gene silencing using in vitro assays; and 4) test dsRNA in foliar applications to decreases losses to CLS.
The cooperator will carry out the following: 1) Identify dsRNA targets. The genome sequence of C. beticola is available and can be used to extract suitable gene targets for silencing. Initially, targets such as Cyp51 (involved with ergosterol biosynthesis) and Ctb1 (key gene involved in the production of the toxin cercosporin) will be used to test the efficacy of dsRNA-mediated gene silencing. dsRNA of the green fluroescent protein gene, which is not found in the C. beticola genome, will be produced for a negative control. 2) dsRNA synthesis. The production of dsRNA has been thoroughly investigated and is a common laboratory practice particularly in virology pathology. Commercial kits will be utilized to produce dsRNAs. 3) Confirm gene silencing. C. beticola spores will be added to microtiter wells. Treatments (dsRNAs) will be added to the wells in three 10-fold dilutions to identify the optimal concentration for gene silencing. Fungal growth will be quantified using a plater reader. Routine RT-PCR will be carried out to confirm gene silencing in treated samples. 4) Test dsRNA for CLS disease management. Cyp51 enzyme is a target of commercially-available fungicides. The efficacy of dsRNA targeting Cyp51 transcript will be compared to fungicides targeting Cyp51 enzyme for CLS management. Recent advances in application of dsRNA using clay nanosheets will be employed as described by Mitter et al. (Nature Plants 2017).