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

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

Content Provider	Springer Nature Link
Author	Jelly, émie S. Schellenbaum, Paul Walter, Bernard Maillot, Pascale
Copyright Year	2012
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: amiR$^{CP}$-1 or amiR$^{CP}$-2. Transient expression of these two pre-amiRNA constructs was tested in grapevine somatic embryos after co-cultivation with Agrobacterium tumefaciens. Expression of amiR$^{CP}$-1 and amiR$^{CP}$-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 (G$^{CP}$-1 and G$^{CP}$-2) were obtained by fusing the target sequence of amiR$^{CP}$-1 or amiR$^{CP}$-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.
Starting Page	1319
Ending Page	1327
Page Count	9
File Format	PDF
ISSN	09628819
Journal	Transgenic Research
Volume Number	21
Issue Number	6
e-ISSN	15739368
Language	English
Publisher	Springer Netherlands
Publisher Date	2012-03-17
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Vitis vinifera Fanleaf degeneration Agrobacterium tumefaciens Transient transformation Animal Genetics and Genomics Molecular Medicine Plant Sciences Human Genetics Plant Genetics & Genomics
Content Type	Text
Resource Type	Article
Subject	Genetics Animal Science and Zoology Biotechnology Agronomy and Crop Science

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