An efficient method for stable protein targeting in grasses (Poaceae): a case study in Puccinellia tenuiflora.

Yuanyuan Bu,Mengqing Zhao,Tetsuo Takano,Bo Sun,Xinxin Zhang,Shenkui Liu

doi:10.1186/1472-6750-14-52

Yuanyuan Bu, Mengqing Zhao + Show 4 more

Open Access

https://doi.org/10.1186/1472-6750-14-52

Copy DOI

Abstract

BackgroundAn efficient transformation method is lacking for most non-model plant species to test gene function. Therefore, subcellular localization of proteins of interest from non-model plants is mainly carried out through transient transformation in homologous cells or in heterologous cells from model species such as Arabidopsis. Although analysis of expression patterns in model organisms like yeast and Arabidopsis can provide important clues about protein localization, these heterologous systems may not always faithfully reflect the native subcellular distribution in other species. On the other hand, transient expression in protoplasts from species of interest has limited ability for detailed sub-cellular localization analysis (e.g., those involving subcellular fractionation or sectioning and immunodetection), as it results in heterogeneous populations comprised of both transformed and untransformed cells.ResultsWe have developed a simple and reliable method for stable transformation of plant cell suspensions that are suitable for protein subcellular localization analyses in the non-model monocotyledonous plant Puccinellia tenuiflora. Optimization of protocols for obtaining suspension-cultured cells followed by Agrobacterium-mediated genetic transformation allowed us to establish stably transformed cell lines, which could be maintained indefinitely in axenic culture supplied with the proper antibiotic. As a case study, protoplasts of transgenic cell lines stably transformed with an ammonium transporter-green fluorescent protein (PutAMT1;1-GFP) fusion were successfully used for subcellular localization analyses in P. tenuiflora.ConclusionsWe present a reliable method for the generation of stably transformed P. tenuiflora cell lines, which, being available in virtually unlimited amounts, can be conveniently used for any type of protein subcellular localization analysis required. Given its simplicity, the method can be used as reference for other non-model plant species lacking efficient regeneration protocols.

Highlights

An efficient transformation method is lacking for most non-model plant species to test gene function
Most of the currently available methods for protein subcellular localization can be readily applied to the majority of model plant species such as Arabidopsis and tobacco, which could, in principle, lend themselves as heterologous recipients for localization studies of proteins from nonmodel plant species [18,19]
The relatively high failure rates reported in studies adopting such heterologous approaches indicate that homologous expression systems are preferable whenever possible [10,20]

Summary

Introduction

An efficient transformation method is lacking for most non-model plant species to test gene function. While stable transformation of gametes or regeneration of whole plants from transformed somatic cells is routinely achieved in model plant species (e.g., by floral dipping in Arabidopsis thaliana), for most non-model species, the lack of suitable protocols and the phenomenon of regeneration recalcitrance make the obtainment of whole-plant transformants an arduous task. To circumvent this problem, we here propose a method for protein subcellular localization analyses that does not rely on obtaining whole-plant transformation, but instead focuses on the Agrobacterium-mediated stable transformation of cells in suspension-cultures (Figure 1). We applied this method to express an endoplasmic reticulum-green fluorescent protein (ER-GFP) fusion protein in P. tenuiflora, a grass halophyte with extreme tolerance to alkaline soils (pH ≈ 10)

Methods

Results

Conclusion