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

Simona Urso, Michela Zottini, Cristina Ruberti, Fiorella Lo Schiavo, Antonio Michele Stanca, Luigi Cattivelli, Giampiero Valè

Risultato della ricerca: Contributo su rivistaArticolo in rivistapeer review

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.

Lingua originaleInglese
pagine (da-a)49-60
Numero di pagine12
RivistaPlant Cell, Tissue and Organ Culture
Volume114
Numero di pubblicazione1
DOI
Stato di pubblicazionePubblicato - lug 2013
Pubblicato esternamente

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