Transient gene expression for visualization of protein localization in plant cell

Berestovoy M.A1,2, Tyurin A.A1, Sidorchuk Yu.V.3, FomenkovA.A1, Nosov A.V1, Goldenkova-
Pavlova I.V1

1. Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia 127276,

Botanicheskaya st. 35, tel. +7 (495) 977-94-00;
2. Russian State Agrarian University–Moscow Timiryazev Agricultural Academy, Moscow, Russia
127550, Timiryazevskaya st. 49, tel. +7 (499) 977-14-55;
3. Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk,
Russia 630090, Academician Lavrentiev av. 10, tel. +7 (383) 363-49-80
e-mail: m.berestovoy1181@gmail.com

Transient expression of genes has been shown to be useful in evaluating the intracellular
localiazation of protein products of target genes, studying signaling pathways within the cell and
protein-protein interactions, and also in exploring the functional properties of regulatory sequences
in plants. Two main strategies of transient expression are most popular among researchers:
protoplast transfection and agroinfiltration. Each of them, along with the advantages has its
limitations. This entire demonstrate the need for the development and testing of new approaches for
the transient expression of genes in the plant cell. In this paper, we studied protein localization in
the plant cell using for an example Δ9-acyl-lipid desaturase encoded by the desC gene of
cyanobacteria. For visualization of protein products of the target gene, we used a hybrid desC-egfp
gene, which includes the sequence of the egfp gene, the protein product of which is the green
fluorescent protein. In the expression vectors, we introduced sequences that ensure the localization
of the protein products of the target gene to various cell compartments (into chloroplasts and
endoplasmic reticulum (ER)). Investigation of localization of the target proteins was carried out in
leaves of Nicotiana benthamiana, transformed by agroinfiltration using Agrobacterium tumefaciens,
strain GV3101. Regions with the maximum level of fluorescence of eGFP were taken from
agroinfiltrated leaves, with the subsequent estimation of localization of the hybrid protein by the
method of laser confocal microscopy. However, this method to assess the localization of the fusion
protein, both in chloroplasts and ER has proved difficult, probably due to the intricate outlines of
cells and the spatial structure of the plant compartments themselves. To overcome these difficulties
were isolated protoplasts from the areas of agroinfiltrated leaves with maximum fluorescence and
investigated by fluorescence microscopy. As a result, we were able to determine the localization of
the DesC-EGFP fusion protein in the appropriate cell compartments. We demonstrated that the
extraction of protoplasts from infiltrated leaf tissue after the transient expression simplifies
visualization of the target protein localization in the plant cell.
This study was supported by the Russian Foundation for Basic Research (grant no. №14-04-
01616_a)