A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies

Abstract

SUMMARYFluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter (P UBQ10 ) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicletrafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K + channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P UBQ10 promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P UBQ10 promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant advantages for the study of membrane dynamics and cellular development, as well as events associated with environmental stimuli in guard cells and nutrient acquisition in roots.

ARABIDOPSIS THALIANA HOMEOBOX25 Uncovers a Role for Gibberellins in Seed Longevity

“…To confirm the relationship between ATHB25 and seed longevity, we generated transgenic plants in which the complementary DNA (cDNA) of the gene was cloned in sense and antisense orientations under the control of two promoters expressed in nearly all tissues: the strong 35S promoter (Benfey and Chua, 1990) and the moderate UBIQUITIN10 promoter (Grefen et al, 2010). Transgenic lines overexpressing ATHB25 from the UBQ10 promoter expressed ATHB25 at levels similar to those of the athb25-1D mutant (5-to 6-fold wildtype levels; Fig.…”

Pseudomonas syringae type III effector HopAF1 suppresses plant immunity by targeting methionine recycling to block ethylene induction

“…S6 B and C) (60). We investigated these potentially different localizations of HopAF1 and Arabidopsis MTNs using bimolecular fluorescence complementation (BiFC) experiments (61). We generated constructs with MTN1 and MTN2 fused at their C termini to the C-terminal half of YFP (MTN1-cYFP and MTN2-cYFP).…”

Specialized Roles of the Conserved Subunit OST3/6 of the Oligosaccharyltransferase Complex in Innate Immunity and Tolerance to Abiotic Stresses

“…Binary expression vector p40 is derived from p20F by replacing the cauliflower mosaic virus 35S promoter with the Arabidopsis ubiquitin10 promoter (Grefen et al, 2010). The vector for expression of the truncated OST3/6-1 fused to GFP was generated by PCR amplification using the primers At1g61790_3F and At1g61790_10R.…”