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Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos

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Abstract

Grapevines are affected worldwide by viruses that compromise fruit yield and quality. Grapevine fanleaf virus (GFLV) causes fanleaf degeneration disease, a major threat to grapevine production. Transgenic approaches exploiting the RNA silencing machinery have proven suitable for engineering viral resistance in several crop species. However, the artificial microRNA (amiRNA)-based strategy has not yet been reported in grapevine. We developed two amiRNA precursors (pre-amiRNAs) targeting the coat protein (CP) gene of GFLV and characterised their functionality in grapevine somatic embryos. To create these pre-amiRNAs, natural pre-miR319a of Arabidopsis thaliana was modified by overlapping PCR in order to replace miR319a with two amiRNAs targeting different regions of the CP gene: amiRCP-1 or amiRCP-2. Transient expression of these two pre-amiRNA constructs was tested in grapevine somatic embryos after co-cultivation with Agrobacterium tumefaciens. Expression of amiRCP-1 and amiRCP-2 was detected in plant tissues by an endpoint stem-loop RT-PCR as early as 1 day after a 48-h co-cultivation, indicating active processing of pre-amiRNAs by the plant machinery. In parallel, GUS-sensor constructs (GCP-1 and GCP-2) were obtained by fusing the target sequence of amiRCP-1 or amiRCP-2 to the 3′ terminus of the GUS gene. Co-transformation assays with GUS-sensors and the pre-amiRNA constructs provided evidence for in vivo recognition and cleavage of the 21-nt target sequence of GUS-sensors by the corresponding amiRNA. This is the first report of amiRNA ectopic expression in grapevine. The constructs we developed could be useful for engineering GFLV-resistant grapes in the future.

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Abbreviations

amiRNA:

Artificial microRNA

GFLV:

Grapevine fanleaf virus

GUS:

β-Glucuronidase

CP:

Coat protein

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Acknowledgments

This study was funded by the French Ministry for Research. The authors thank Sascha Laubinger (University of Tuebingen, Germany) for his input concerning stem-loop RT-PCR, Marc Lollier (Université de Haute-Alsace, France) for statistical analysis, Leon Otten (Institut de Biologie Moléculaire des Plantes, France) for providing A. tumefaciens strain GV3101 (pMP90), Detlef Weigel (Max Planck Institute for Developmental Biology, Germany) for providing pRS300, and Ignacio Rubio Somoza (Max Planck Institute for Developmental Biology, Tuebingen, Germany) for his helpful support.

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  1. Noémie S. Jelly

    Present address: Institut de Biologie Moléculaire et Cellulaire, UPR9022 CNRS, Université de Strasbourg, 15 rue René Descartes, 67084, Strasbourg, France

Authors and Affiliations

  1. Laboratoire Vigne, Biotechnologies et Environnement, Université de Haute-Alsace, 33 rue de Herrlisheim, 68008, Colmar, France

    Noémie S. Jelly, Paul Schellenbaum, Bernard Walter & Pascale Maillot

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  1. Noémie S. Jelly
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Correspondence to Pascale Maillot.

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Jelly, N.S., Schellenbaum, P., Walter, B. et al. Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos. Transgenic Res 21, 1319–1327 (2012). https://doi.org/10.1007/s11248-012-9611-5

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  • DOI: https://doi.org/10.1007/s11248-012-9611-5

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