Plant viruses cause significant damage to crops worldwide, and once infected there are no effective treatments. Large numbers of viral-derived small RNAs (vsRNA) are generated in infected plants. We are interested in the function of vsRNA and how they contribute to symptom development. We fully sequenced an Australian strain of Turnip mosaic virus (TuMV), and performed deep small-RNA sequencing on TuMV infected Arabidopsis thaliana. Mapping revealed “hotspots” of highly abundant vsRNA. We hypothesise abundant vsRNA could target host transcripts in the plant, contributing to symptom development. A thaliana transcript sequences were screened to predict targets of abundant vsRNAs. Using an optimised transient assay that combines Agro-infiltration with the quantitative dual luciferase system, we demonstrate that vsRNAs can target plant transcript sequences, reducing expression. Mutating the plant transcript target sequences restored expression levels.
Artificial microRNA (amiRNA) refers to the strategy of replacing the mature miRNA sequence within a native microRNA precursor gene, to target a different expressed sequence of interest. Artificial microRNA predominantly process one mature miRNA that will target only highly complementary expressed gene sequences. This provides an advantage over other strategies such as RNA interference, where larger numbers of short interfering RNAs are generated increasing the likelihood of off-target effects. We successfully applied an amiRNA approach to develop tomato lines resistant to Cucumber mosaic virus (CMV). The amiRNA was designed to directly target the RNA genome of CMV. ELISA testing of CMV inoculated T1 populations from two independent amiRNA lines confirmed a reduction in CMV levels relative to controls. Symptom severity scoring and counts of harvested quality fruits further confirmed enhanced resistance to CMV in the amiRNA lines.