BackgroundGenes belonging to the pathogenesis related 10 (PR10) group have been studied in several plant species, where they form multigene families. Until now, such an analysis has not been performed in Vitis vinifera, although three different PR10 genes were found to be expressed under pathogen attack or abiotic stress, and during somatic embryogenesis induction. We used the complete genome sequence for characterising the whole V. vinifera PR10 gene family. The expression of candidate genes was studied in various non-treated tissues and following somatic embryogenesis induction by the auxin 2,4-D.ResultsIn addition to the three V. vinifera PR10 genes already described, namely VvPR10.1, VvPR10.2 and VvPR10.3, fourteen different PR10 related sequences were identified. Showing high similarity, they form a single cluster on the chromosome 5 comprising three pseudogenes. The expression of nine different genes was detected in various tissues. Although differentially expressed in non-treated plant organs, several genes were up-regulated in tissues treated with 2,4-D, as expected for PR genes.ConclusionsPR10 genes form a multigene family in V. vinifera, as found in birch, apple or peach. Seventeen closely related PR10 sequences are arranged in a tandem array on the chromosome 5, probably reflecting small-scale duplications during evolution. Various expression patterns were found for nine studied genes, highlighting functional diversification. A phylogenetic comparison of deduced proteins with PR10 proteins of other plants showed a characteristic low intraspecific variability. Particularly, a group of seven close tandem duplicates including VvPR10.1, VvPR10.2 and VvPR10.3 showed a very high similarity, suggesting concerted evolution or/and recent duplications.

Little is known about the genes expressed during grapevine somatic embryogenesis. Both groups of Somatic Embryogenesis Receptor Kinase (SERK) and Leafy Cotyledon (LEC and L1L) genes seem to play key roles during somatic embryogenesis in various plant species. Therefore, we identified and analysed the sequences of VvSERK and VvL1L (Leafy cotyledon1-Like) genes. The deduced amino acid sequences of VvSERK1, VvSERK2 and VvSERK3 are very similar to that of registered SERK proteins, with highest homologies for the kinase domain in the C-terminal region. The amino acid sequence of VvL1L presents all the domains that are characteristic for LEC1 and L1L proteins, particularly, the 16 amino acid residues that serve as signature of the B-domain. Phylogenetic analysis distinguishes members of subclass LEC1 and subclass L1L, and VvL1L is closely related to L1L proteins. Using semi-quantitative RT-PCR, we studied gene expression of VvSERK1, VvSERK2, VvSERK3 and VvL1L in calli and somatic embryos obtained from anther culture of Vitis vinifera L. cv Chardonnay. Expression of VvSERK2 is relatively stable during in vitro culture. In contrast, VvSERK1, VvSERK3 and VvL1L are expressed more 4 to 6 weeks after transfer of the calli onto embryo induction medium, before the visible appearance of embryos on the calli as seen by environmental scanning electron microscopy. Later on (8 weeks after transfer) VvSERK1 expression is maintained in the embryogenic calli and VvSERK3 in the embryos, whereas VvL1L expression is very low. All together, these data suggest the involvement of VvSERK and VvL1L genes in grapevine somatic embryogenesis.

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A culture model was developed in Vitis vinifera L., cultivar 'Chardonnay' for studying SE (Somatic Embryogenesis). The auxin 2,4-D (2,4-Dichlorophenoxyacetic acid) was used to induce indirect secondary embryogenesis at a high rate, starting from embryos derived from embryogenic cultures previously obtained. Cotyledonary embryos were shown to be more responsive to SE induction than embryos at the torpedo-stage and were used for molecular analyses. The expression of SERK (Somatic Embryogenesis Receptor Kinase), L1L (Leafy Cotyledon1 Like) and a set of PR (Pathogenesis-Related) genes was monitored during the whole SE process. VvSERK1, VvSERK2 and VvL1L were down-regulated by the 2,4-D treatment but expressed in embryonic tissues. On the contrary, VvPR1, VvPR8, VvPR10.1 and VvPR10.3 were strongly up-regulated by the 2,4-D treatment, and their transcripts were not or only weakly detected in clusters of secondary embryos. VvSERK3, VvPR3 and VvPR10.2 were more stably expressed in all tissues examined. The discussion deals with the putative role of the different genes in grapevine SE.

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.