title: Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos.
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.
authors.affiliation: Laboratoire Vigne, Biotechnologies et Environnement, Université de Haute-Alsace, 33 rue de Herrlisheim, 68008 Colmar, France.
authors.forename: Noémie S
authors.initials: NS
authors.lastname: Jelly
authors.forename: Paul
authors.initials: P
authors.lastname: Schellenbaum
authors.forename: Bernard
authors.initials: B
authors.lastname: Walter
authors.forename: Pascale
authors.initials: P
authors.lastname: Maillot
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D035683:MicroRNAs / Q000502:physiology*; D017860:Nepovirus / Q000235:genetics*; D034622:RNA Interference; D018749:RNA, Plant / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D027843:Vitis / Q000196:embryology / Q000235:genetics* / Q000254:growth & development
chemical_list: D035683:MicroRNAs; D018749:RNA, Plant; D012367:RNA, Viral; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005966:Glucuronidase