Flexible Tools for Gene Expression and Silencing in Tomato    

Abstract

As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.

Alteration of the Interconversion of Pyruvate and Malate in the Plastid or Cytosol of Ripening Tomato Fruit Invokes Diverse Consequences on Sugar But Similar Effects on Cellular Organic Acid, Metabolism, and Transitory Starch Accumulation

“…However, certain speciesspecific differences exist in the dynamics of other metabolite pools across ripening, with, for example, grape (Vitis vinifera), strawberry (Fragaria 3 ananassa), prune (Prunus domestica), and pepper (Capsicum annuum) displaying slightly different metabolic programs from tomato (Solanum lycopersicum; Zamboni et al, 2010;Lombardo et al, 2011;Osorio et al, 2011Osorio et al, , 2012Zhang et al, 2011). This fact notwithstanding, tomato has become the primary experimental model in which to study the development and ripening of fleshy fruits (Giovannoni, 2004;Fernandez et al, 2009). Furthermore, it is arguably also the best characterized fruit at the biochemical level (Rose and Bennett, 1999;Fraser and Bickmore, 2007;Moco et al, 2007).…”

Genetic Transformation in Tomato: Novel Tools to Improve Fruit Quality and Pharmaceutical Production

“…In addition, novel resources for temporal and tissue-specific manipulation of gene expression in tomato plants are now available for the scientific community. In this regard, it is noteworthy the work from Fernandez et al (2009) andEstornell et al (2009) that created new Solanaceae genetic toolkit for targeted gene expression and silencing in tomato fruits. Recently, as the information provided by the tomato genome sequencing become available, the demand for efficient functional genomics tools are increasing.…”

Micro-Tom mutants for functional analysis of target genes and discovery of new alleles in tomato

“…Because the generation of large enough transfer DNA (T-DNA) or transposon-tagged lines remains out of reach in tomato (Emmanuel and Levy 2002), functional analysis in planta of the target genes is usually done using stable genetic transformation (over-expression, RNA interference and chimeric repressor silencing technologies) with constitutive or tissue-specific promoters (Fernandez et al 2009) or transient expression by agro-infiltration or VIGS (Virus Induced Gene Silencing) (Orzaez et al 2009;Quadrana et al 2011). Recently, association mapping has also been shown to be an effective tool for assessing the molecular basis of fruit quality traits in tomato (Xu et al 2013).…”