Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters.

Bingqi Wu,Ronghua Chen,Fei Lin,Xiaohui Xu,Siwei Wang,Hanhong Xu,Zhiting Chen

doi:10.3390/plants10030524

Abstract

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

Highlights

Both the uptake and transport of agrochemicals are important factors influencing whether agrochemicals reach sites of pest damage and subsequently exert their activity.Agrochemicals must pass through the barrier of the plant plasma membrane before they arrive at the vascular systems for long-distance transport [1]
The nicotine content absorbed by the pEAQ-NtNUP1-transformed foliar discs was significantly higher than that absorbed by the control foliar discs, and this trend was maintained at different nicotine concentrations (100–500 μM), which clearly indicated that NtNUP1 functions in the uptake of nicotine into leaf cells (Figure 1b)
These results further indicated that the increase in the nicotine content in leaf discs originated from the uptake of the nicotine solution and not from the transfer of endogenous nicotine from other parts of the plants

Summary

Introduction

Both the uptake and transport of agrochemicals are important factors influencing whether agrochemicals reach sites of pest damage and subsequently exert their activity. Agrochemicals must pass through the barrier of the plant plasma membrane before they arrive at the vascular systems for long-distance transport [1]. Transporter-mediated agrochemical transport provides a new approach to increase agrochemical uptake and directs their site-targeted distribution by genetically modifying plant membrane transporters [2]. Direction of transporter (inword or outword) and localization of transporter in plant is very important [3,4,5]. Most of the information on the physiological roles of these transporters was obtained from studies carried out in model plants species (e.g., Arabidopsis thaliana and Oryza sativa L.) by screening mutants following genetic mapping, which is a time-consuming and laborious processes. Other in vivo heterologous expression systems, such as Xenopus oocytes and yeast, have been effectively used as tools for the functional characterization of these types of transporter proteins

Methods

Results

Conclusion