Using a Viral Vector to Reveal the Role of MicroRNA159 in Disease Symptom Induction by a Severe Strain of Cucumber mosaic virus

In transgenic Arabidopsis (Arabidopsis thaliana), expression of the Cucumber mosaic virus (CMV) 2b silencing suppressor protein from the severe subgroup IA strain Fny disrupted microRNA (miRNA)-regulated development but orthologs from mild subgroup II strains (Q and LS) did not, explaining strain-specific differences in symptom severity. However, it is unknown which miRNAs affected by Fny2b critically affect viral symptoms. Observations that Fny2b-transgenic plants phenocopy microRNA159ab (mir159ab) mutant plants and that Fny2b altered miR159ab-regulated transcript levels suggested a role for miR159ab in elicitation of severe symptoms by Fny-CMV. Using restoration of the normal phenotype in transgenic plants expressing an artificial miRNA as a proof of concept, we developed a LS-CMV-based vector to express sequences mimicking miRNA targets. Expressing a miR159 target mimic sequence using LS-CMV depleted miR159 and induced symptoms resembling those of Fny-CMV. Suppression of Fny-CMV-induced symptoms in plants harboring mutant alleles for the miR159ab targets MYB domain protein33 (MYB33) and MYB65 confirmed the importance of this miRNA in pathogenesis. This study demonstrates the utility of a viral vector to express miRNA target mimics to facilitate functional studies of miRNAs in plants.