Construction of TRV-mediated virus induced gene silencing (VIGS) system in sweet cherry fruit

Abstract: 【Objective】Sweet cherry (Prunus avium L.) is an important horticultural crop cultivated in temperate areas for notable economic value. However, to date, our understanding of the molecular mechanisms dealing with the regulation of fruit quality in sweet cherry and other horticultural fruit trees is still quite limited, and few genes related to the molecular mechanisms have been identified for the limitation of the tools for gene function analysis. The genomics sequences of sweet cherry have been published. Therefore, the establishment of transient genetic transformation system of sweet cherry fruit is urgently needed for identification and characterization of genes associated with fruit quality to illuminate the corresponding molecular mechanisms. Virus induced gene silencing (VIGS) is one of RNA-mediated post transcriptional gene silencing (PTGS) in that RNAs are specifically degraded if there is a similar sequence between viruses and plant genes. In recent years, VIGS has been developed as an effective genetics tool used for functional gene characterization in a range of dicot plant species, including Nicotiana benthamiana (tobacco) , Solanum lycopersicum (tomato) , Gossypium spp. (cotton) , Triticum aestivum (wheat) , Hordeum vulgare (barley) , Zea mays (maize) , Oryza sativa (rice) , Malus pumila (apple) , Citrus reticulata (citrus) , and Vitis vinifera (grape) . In this study, a highly efficient TRV-based VIGS system in sweet cherry fruit was established, and the VIGS technique could be applied in sweet cherry fruit, and would be a powerful new tool for corresponding studies of fruit quality.【Methods】InFusion cloning technology was performed to generate the pTRV2-ANS plasmid for constructing TRVVIGS vector. A 370 bp fragment of PaANS was amplified with the primers PaANS-F and PaANS-R, including 17 bp-long overhangs identical to the corresponding pTRV2 sequence digested with Eco RⅠ orKpnⅠ, by PCR and inserted into the pTRV2 vector digested with Eco RⅠ and KpnⅠ by In-Fusion cloning to generate the pTRV2-ANS plasmid. Then plasmid pTRV2-ANS was transformed into the Agrobacterium tumefaciens GV3101 strain via electroporation. TRV-mediated gene silencing was performed with A. tumefaciens-mediated transformation. A. tumefaciens strain GV3101 was resuspended in the Agrobacterium infiltration buffer (10 mmol· L^-1 MgCl2, 10 mmol· L^-1 MES, pH 5.6, 400 μmol· L^-1 acetosyringone) to a final OD600 of 0.8-1.0. Mixed Agrobacterium GV3101 strains containing the pTRV1, pTRV2 or p TRV2-ANS vectors were infiltrated using a needle-less syringe into the basal pedicel of sweet cherry fruit 14 days after full bloom (DAFB) until the whole fruit was permeated. The inoculated fruits were treated with bagging for 3 days. Morphological analysis of sweet cherry fruit, including flesh color and peel color, molecular detection and q RT-PCR technology were used to evaluate TRV mediated VIGS silencing efficiency.【Results】Quantification of the TRV virus biomass in fruit tissue of sweet cherry were performed using RT-PCR on 7 days post inoculation (dpi) with specific primers pTRV1-F/pTRV1-R and pTRV2-F/pTRV2-R for testing whether the TRV-vector could directly infect sweet cherry fruit. The results showed that only a 647 bp fragment was amplified using the primer pair pTRV1-F/pTRV1-R in the cDNA of TRV::ANS-infected fruits and the TRV::00-infected fruits. Meanwhile, the TRV-RNA2-specific PCR product was detected only in the TRV-infiltrated sweet cherry fruit, suggesting that TRV virus could directly infect sweet cherry fruit. In order to assess the effectiveness of TRV-VIGS in sweet cherry fruit, the expression of endogenous PaANS in the landrace sweet cherry cultivar‘Brooks' was detected by a semi-quantitative RT-PCR assay using cDNA samples from infiltrated fruit at 10 dpi. The expression of PaANS was markedly reduced in the TRV::ANS-infected fruit compared with the TRV::00-infected fruit., The fruit skin of TRV::00-infected fruits sector developed a yellow bottom with flush color phenotype at 14 dpi. Meanwhile, the fruit skin of TRV::ANS-infected fruits sector did not turn yellow like the wild type fruit. At 28 dpi, compared with TRV:: 00-infected sweet cherry fruit, the skin of the TRV::ANS-infected sweet cherry fruit sector still remained green or light yellow green. In contrast, the skin of TRV::00-infected sweet cherry fruit sector turned red. Furthermore, the pulp of TRV::00-infected sweet cherry fruit sector developed yellow or pink color, while the pulp of the TRV::ANS-infected sweet cherry fruit sector was still green or white.【Conclusion】The ans gene was effectively silenced in TRV:: ANS-infected sweet cherry fruit, and the fruit peel and pulp of TRV::ANS-infected sweet cherry fruit showed significant defects compared with TRV::00-infected sweet cherry fruit, indicating that TRV mediated VIGS system was successfully established in sweet cherry fruit. These findings revealed the great potential of application of VIGS for studying fruit development and ripening, and could be an alternative to transgenic technology. This system in sweet cherry fruit would likely prove a valuable tool in future research on functional identification of genes contributing to fruit quality formation and controlling the metabolic networks of fruit development in sweet cherry.