Detection of Arthropod Transmitted Viruses of Cereal Crops and RNAi Induced Resistance to Wheat Streak Mosaic Virus

Abstract

Bread wheat (Triticum aestivum) is one of the most important crops cultivated worldwide both in terms of nutrition and economic value. Some of the greatest challenges wheat growers face around the world are plant viruses, which may induce symptoms such as stunting or discoloration and can lead to yield losses, or in extreme cases, total crop failure. To identify potential solutions to the threat posed by plant viruses in wheat, one of the most important steps is to accurately and quickly detect and discriminate between viruses so the appropriate management strategy may be applied. Two of the most powerful technologies currently available for the detection and discrimination of plant viruses are PCR and massive parallel sequencing (MPS). The objectives of this study are to utilize multiplex PCR in combination with high-resolution melting (HRM), as well as the MPS based Electronic-Probe Diagnostic Nucleic Acid Analysis (EDNA), to develop new tools for the detection and discrimination of viruses of wheat such as Wheat streak mosaic virus (WSMV). Next, to test the efficacy of RNA interference (RNAi) as a potential treatment to induce resistance to WSMV in wheat. Primers were designed to simultaneously detect WSMV, Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV) in multiplex, and to discriminate between the three species of BYDV using HRM. E-probes capable of detecting 21 different viruses of cereal in metagenomic data were designed and the theoretical limit of detection was assessed for WSMV using in silico predictive models. These models were validated by sequencing known ratios of WSMV to wheat nucleic acids in a series of dilutions, then analyzed using EDNA on the subsequent metagenomic data. Gallagher, a wheat variety susceptible to WSMV, was injected with an RNAi construct specific to WMSV, then inoculated with WSMV. Viral titer of WSMV was then monitored at 7, 14 and 21 days post inoculation with qPCR and results were compared to WSMV inoculated and uninoculated, susceptible and resistant wheat varieties. It was determined that susceptible wheat treated with RNAi specific to WSMV prior to inoculation with WSMV had significantly reduced viral expression.