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An Agrobacterium tumefaciens-mediated gene silencing system for functional analysis in grapevine

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Abstract

An efficient agroinfiltration-based gene silencing assay was established to evaluate candidate genes likely to be involved in resistance to powdery mildew (Uncinula necator) in grapevine (Vitis vinifera L.). Functional assays were performed using two grapevine genotypes, cv. Superior, mildew-susceptible, and cv. Run1 Mtp3294, mildew-resistant. Constructs encoding a self-complementary “hairpin” RNA for phytoene desaturase (PDS) along with the green fluorescent protein gene were introduced into Agrobacterium tumefaciens. These constructs were used for agroinfiltration of leaf tissues, yielding both efficient transformation and gene silencing events. The agroinfiltration procedure did not interfere with the powdery mildew infection steps. Confocal laser-scanning microscopy analysis revealed co-localization of the GFP signal with cells lacking chlorophyll autofluorescence, further supporting PDS gene silencing. The extent of PDS mRNA degradation was evaluated by qRT-PCR in the agroinfiltrated areas and results supported a significant reduction of the gene transcripts in the silenced regions. Findings of the present work suggest that the developed silencing procedures represent a useful tool for functional characterization of grapevine genes involved in powdery mildew resistance.

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Acknowledgments

This work was supported by the Italian national project ‘VItis GeNome Analysis’ (‘VIGNA’), Progetto di Ateneo and Progetto from Agriculture Ministry and funded by MiPAF (Ministero delle Politiche Agricole e Forestali). We thank Dr. Anne-Françoise Adam-Blondon and Mrs. Marion Guillou, INRA (France) for providing the Run1 Mtp3294 grapevine genotype and Dr Mark R. Thomas supplying the PDS EST clone (GeneBank ID: CN007512). We thank Donata Pagani (CRA-GPG) for excellent technical assistance, Dr Alessandro Tondelli for statistical analysis and Dr Elisabetta Barizza for hydroponic plants management.

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Correspondence to Michela Zottini or Giampiero Valè.

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Urso, S., Zottini, M., Ruberti, C. et al. An Agrobacterium tumefaciens-mediated gene silencing system for functional analysis in grapevine. Plant Cell Tiss Organ Cult 114, 49–60 (2013). https://doi.org/10.1007/s11240-013-0305-3

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Keywords

  • Grapevine
  • RNA interference (gene silencing)
  • Transient transformation
  • Agrobacterium tumefaciens
  • Phytoene desaturase
  • Functional genomics