Objective
1. Double-stranded RNAs (dsRNAs) targeting eight distinct genes in Leptosphaeria maculans (Lm) were designed and tested as a single, combined dsRNA construct aimed at silencing all eight genes simultaneously. Efficacy of each of these eight genes and their orthologues from Plasmodiophora brassicae (Pb), or Verticillium longisporum (Vl) in suppressing infection by Lm, Pb, or Vl will be evaluated and compared to identify the most effective dsRNA target.
2. Effect of dsRNA length and sequence in silencing the target gene will be compared to determine the optimal length and the most effective sequence for the most potent target dsRNA (identified under Objective 1). The most effective dsRNA configuration will be applied once (before) or twice (before and after) inoculation to determine the effect of single vs multiple dsRNA application.
3. Naked dsRNA and dsRNA-coated carbon dots will be sprayed and their efficacy in controlling each of the three pathogens (Lm, Pb and Vl) will be compared.
4. To evaluate application of Spray-Induced Gene Silencing (SIGS) technology in controlling disease of canola in the field, the most effective dsRNA and application method will be tested in disease nurseries in Alberta in year 3 and year 4 of the project.
Project Description
Genetic resistance and chemical fungicides are the two primary tools for protecting crops against many but not all pathogens. Repeated use of disease resistance (R) genes and fungicides leads to the emergence of highly virulent pathogens that overcome R genes and fungicide applications. For example, in canola R genes against Blackleg (BL) and Clubroot (CR) disease, were reported to break down within a couple of years of being launched. Another pathogen of concern is Vl a soil-borne fungus that causes Verticillium stripe (VS) disease. Currently, there are no available fungicides against Vl and genetic resistance is governed by multiple minor effect genes (QTL) making them difficult to manipulate in breeding programs. In recent years, a new technology called Spray Induced Gene Silencing (SIGS) has been promoted as an environmentally safe and effective alternative to chemical fungicides. SIGS is based on the well-studied and widely conserved RNA interference (RNAi) phenomenon in eukaryotes. Among many other biological functions, RNAi in plants serves as a natural defence mechanism against viral pathogens by generating small interfering RNA (siRNA) from dsRNA (produced during virus replication/transcription) to inhibit viral replication. In a greenhouse pilot experiment, we successfully demonstrated control against BL and VS by applying a dsRNA fragment corresponding to eight target genes in Lm and Vl. A single foliar application of the same dsRNA, with homology to orthologous genes in Lm and Vl, protected Westar against both pathogens from the seedling to mature stages. We also explored the effect of SIGS to protect canola against clubroot disease. Using a dsRNA spray targeting clubroot homologues of the genes targeted in Lm and Vl, we observed significant reduction in clubroot disease severity in B. napus seedlings inoculated with the Pb pathotype 3H (Pb3H). It is very encouraging to see such a robust effect of the same target sequences providing effective protection against root and foliar diseases that co-occurs in the field. This is the first successful application of dsRNA against Lm, Pb and Vl and, to our knowledge, the first report of foliar application of dsRNA against root pathogens. To build on this initial success, we propose to conduct additional research to determine the silencing effect of each of the eight target genes individually and in various combinations, optimize the application methods and verify the efficiency of dsRNA spray in the field to control Lm, Pb and Vl.
