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

Abstract

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.

Genome-wide identification of turnip mosaic virus-responsive microRNAs in non-heading Chinese cabbage by high-throughput sequencing

“…Then, plants have evolved sophisticated mechanisms to defend such stresses (Galvez et al, 2014). Recently, miRNAs have turned out to be regulons in plant tolerance to abiotic and biotic stresses like cold, drought, heat, fungi and virus (Taylor et al, 2014), and much effort has been devoted to understanding their role in the responses of viral disease in various plants including: wheat (Fahim et al, 2010), tomato (Feng et al, 2014), cucumber (Du et al, 2014), grape (Miozzi et al, 2013) and rice (Guo et al, 2012). In the present study, deep sequencing technology was used for quantitative determination of genome-wide miRNA expression patterns of non-heading Chinese cabbage in response to TuMV infection.…”

The advance of tomato disease-related microRNAs

“…Transgenic Arabidopsis overexpressing a miR159-resistant MYB33 that contained mutated miRNA-target sequence developed curled-up and round leaves (Millar and Gubler 2005). Recent research demonstrated that miR159 exacerbated viral symptoms through the depression of MYB33 and MYB65 in Arabidopsis (Du et al 2014). It appears that miR159 may be closely associated with disease symptoms, such as reduction of leaflet blades and delayed flowering time in CMV-infected tomato.…”

Virus-induced gene silencing (VIGS) in <i>Lilium leichtlinii</i> using the <i>Cucumber mosaic virus</i> vector

“…Moreover, the new CMV-HL vector was also preserved in dormant bulbs and spread into sprouting shoots in the following year (Figures 2 and 3). In CMV-derived viral vectors, the cloning sites of target genes are usually integrated at the downstream of 2b gene (Hong et al 2012;Kanazawa et al 2011;Otagaki et al 2006) and in only one instance the cloning site has been introduced downstream of the 3b gene (Du et al 2014). Therefore, the present study is the first to demonstrate that host plant gene fragments could be introduced just upstream of the 3b start codon without preventing systemic viral infection.…”