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.

Transient expression assays in grapevine: a step towards genetic improvement

“…This also provides a straightforward method for transient expression assays. Up to now, these assays, based on short‐term high‐level expression of transgenes after cocultivation, mainly involved somatic embryos selected at the mid‐cotyledonary stage of development of Thompson seedless (Li et al ., , , , ) and Chardonnay (Jelly et al ., ). Naturally colourless embryos are particularly convenient for monitoring the expression of fluorescence‐ (Li et al ., & Li et al ., ) and colour‐based reporter genes (Jelly et al ., ; Li et al ., ).…”

“…New synthetic promoters were tested in Thompson seedless somatic embryos using gus and gfp (Li et al ., and Li et al ., ). To validate amiRNA constructs designed to control virus infection, a GUS‐based RNAi reporter was used in somatic embryos of Chardonnay (Jelly et al ., ).…”

“…Moreover, a large set of Gateway ® ‐compatible destination vectors are available for many applications (Karimi et al ., ; Murai, ). Binary vectors most often used for transient expression assays in grapevine are pBIN19 and derivative vectors (Le Henanff et al ., ; Li et al ., and Li et al ., ; Santos‐Rosa et al ., ; Visser et al ., ), pCambia (He et al ., ; Xu et al ., ) and varied Gateway ® destination vectors (Jelly et al ., ; Li et al ., ; Urso et al ., ). Xu et al .…”

Top 10 plant‐parasitic nematodes in molecular plant pathology

“…12B). However, the most significant impact of X. index is a result of its activity as the vector of Grapevine fanleaf virus (GFLV) (Hewitt et al, 1958), one of the most important viruses of grapevine (Jelly et al, 2012). Virus particles are attached to the cuticular lining and lumen of the odontophore and the pharynx.…”

Small Non-Coding RNAs in Plant Immunity

“…Indeed several studies demonstrated that artificial miRNAs (amiRNAs) targeting key components of the viral replication machinery can efficiently impair viral growth upon infection [59][60][61][62][63][64][65]. These efforts have revealed that such resistance is cell-autonomous, inheritable, more efficient than siRNA-mediated strategies and successful in blocking viral replication and movement [59,66].…”