Flexible tools for gene expression and silencing in tomato.
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.
Fernandez
Ana I
AI
Department of Plant Systems Biology, Flanders Institute for Biotechnology, 9052 Ghent, Belgium.
Viron
Nicolas
N
Alhagdow
Moftah
M
Karimi
Mansour
M
Jones
Matthew
M
Amsellem
Ziva
Z
Sicard
Adrien
A
Czerednik
Anna
A
Angenent
Gerco
G
Grierson
Donald
D
May
Sean
S
Seymour
Graham
G
Eshed
Yuval
Y
Lemaire-Chamley
Martine
M
Rothan
Christophe
C
Hilson
Pierre
P
D017360:Arabidopsis / Q000235:genetics; D015415:Biomarkers / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002999:Clone Cells; D003001:Cloning, Molecular; D005638:Fruit / Q000166:cytology / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D020868:Gene Silencing; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018551:Solanum lycopersicum / Q000166:cytology / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D013045:Species Specificity; D015533:Transcriptional Activation / Q000235:genetics
D015415:Biomarkers; D035683:MicroRNAs; D049452:Green Fluorescent Proteins; D005966:Glucuronidase