Download A simple and effective method for protein subcellular

Biologia, Bratislava, 62/5: 529—532, 2007
Section Cellular and Molecular Biology
DOI: 10.2478/s11756-007-0104-6
A simple and effective method for protein subcellular localization
using Agrobacterium-mediated transformation
of onion epidermal cells
Wei Sun, Ziyi Cao, Yan Li, Yanxiu Zhao & Hui Zhang*
Key Laboratory of Plant Stress Research, College of Life Science, Shandong Normal University, Jinan 250014, People’s
Republic of China; e-mail [email protected]
Abstract: A modified Agrobacterium-mediated transformation protocol has been successfully used for transient expression
of the intrinsically fluorescent proteins and their fusion proteins in onion epidermis. The mean of the transformed cells rate
per peel is about 10.5±0.9%, while that of the particle bombardment method is at the range 2.0±0.4%. To compare with
the prevailing method of micro-projectile bombardment, the modified Agrobacterium-mediated transformation may provide
with higher efficiency and even more simplified manipulability on a lower budget.
Key words: Agrobacterium-mediated transformation; intrinsically fluorescent protein localization; onion epidermal cell.
Abbreviations: DsRed, red fluorescent protein from Discosoma sp. reef coral; EGFP, enhanced green fluorescent protein;
GFP, green fluorescent protein; IFPs, intrinsically fluorescent proteins;
Introduction
In order to understand gene functions in cellular physiological events in vivo, a large number of reporter genes
have been very widely used to visualize the subcellular localization and the traffic of target proteins. However, some of these traditional reporter genes such as
β-glucuronidase, chloramphenicol acetyltransferase and
luciferase have many drawbacks and sometimes make
the results unconvincing (Sheen et al. 1995; Scott et
al. 1999). Recently, the intrinsically fluorescent proteins
(IFPs), such as the green, cyan, red and yellow fluorescent proteins, have been used as new reporter genes
or targeted markers to monitor directly the gene functions in cellular events. The IFPs isolated from coelenterates, such as the Pacific jellyfish and corals, have
become a useful tool in contemporary cell biological research because of their auto-fluorescence visibility in
living cells and other desirable traits (Chiu et al. 1996).
The IFPs have been now widely used as a marker for
gene localization and protein interaction in Escherichia
coli, yeast, Caenorhabditis elegans, Drosophila, mammals and plants (Chalfie et al. 1994; Wang & Hazelrigg
1994; Delagrave et al. 1995; Heim et al. 1995). Originally IFPs were mainly used for transient expression in
suspension culture cells and protoplasts transformation
mediated by polyethyleneglycol, electroporation, bombardment method and stable transformation (Allen et
al. 1993; Chiu et al. 1996; Gerenuk et al. 1997). How-
ever, the suspension-cultured cells and protoplasts as
well as the stable transformation are time-consuming,
expensive and need high proficiency of special techniques and great patience (Scott et al. 1999; Sheen
2001).
Scott et al. (1999) reported that particle bombardment is an efficient method for the green fluorescent
protein (GFP) imaging in living onion epidermal cells.
Compared with suspension-cultured cells, protoplasts
and stable transformation, particle bombardment is
simple, inexpensive, rapid and powerful as an experimental tool for studying protein localization (Scott et
al. 1999). Therefore the bombardment of onion cells for
testing the localization and dynamics of GFP fusion
proteins in vivo has been widely used in plants (Köhler
et al. 1997). Unfortunately, in spite of these compelling
advantages, the particle bombardment method has a
few potential drawbacks and the application is limited
by exorbitant expense, special equipment and complex
manipulation. In addition, the bombardment which is a
physical method is easily influenced by the parameters
of biolistic system, such as, vacuum, target distance,
helium pressure and gold microcarriers.
In this paper, a modified protocol for transient transformation of onion epidermis mediated by
Agrobacterium, presenting high efficiency and low cost,
is proven as successful in subcellular localization of target proteins using GFP and other IFPs as reporter
genes.
* Corresponding author
c
2007
Institute of Molecular Biology, Slovak Academy of Sciences
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W. Sun et al.
530
Fig. 1. Diagram of the constructs used in plant transformation experiments. (A) EGFP; (B) DsRed; (C) AtNsLTP1::DsRed fused
gene; (D) p35S promoter fragment of pRT101 vector; (E) pCAMBIA3301 T-DNA fragment.
Material and methods
Plant material
Onion (Allium cepa cv. Tango) bulbs were purchased from
market.
Plasmid constructions
For plant expression, EGFP (enhanced GFP) (Yang et al.
1996) and DsRed (red fluorescent protein from Discosoma
sp. reef coral) (Matz et al. 1999) (Clontech, USA) genes
were fused with the Camv 35S promoter in pRT101 vector (Töpfer et al. 1987) between Xho I and Xba I sites
(Fig. 1A,B,D). The 35S::EGFP and 35S::DsRed were cut
off from the fused pRT101 vector with the Hind III and
Pst I, respectively and inserted into the binary vector
pCAMBIA3301 (CAMBIA, Australia) in Hind III or Pst
I site (Fig. 1E). The Arabidopsis AtLTP1 (At2g38540) encodes a nonspecific lipid transfer protein, whose cDNA was
obtained by RT-PCR and fused to pRT101::DsRed between the Xhol I and NcoI sites at the 5’end of DsRed
(Fig. 1C). The 35S::AtLTP1::DsRed sequence was obtained from pRT101::AtLTP1::DsRed and inserted into the
Pst I site of the pCAMBIA3301 plasmid. The primers
were 5’-TCTCGAGTTGCAAACGAACATAAAAC-3’ and
5’-TCCATGGTCACGGTTTTGCAGTTGG-3’ for the
AtLTP1 (At2g38540).
Onion inner epidermal transformation
All the recombinant vectors were transferred into Agrobacterium GV3101 using the standard cloning techniques (Sambrook et al. 1989).
The protocols for onion inner epidermal peels preparation and Agrobacterium-mediated transformation were similar to those used for tobacco transformation described previously (Horsch et al. 1984), with some modifications. Healthy
and fresh onion scales (1–1.5×1 cm) were placed on a 9
cm plate and their inner surfaces were immersed into 6
mL resuspension Agrobacterium solution (OD600 = 1–1.5)
consisting of 5% (g/v) sucrose, 100 mg acetosyringone/L
and 0.02% (v/v) Silwet-77 for 6–12 hours at 28 ◦C. Then
the onion scales were transferred to a Petri dish containing
about 25 mL 1/2 MS0 (Murashige and Skoog salts, 30 g
sucrose/L and 0.7% (g/v) agar, pH 5.7) and co-cultivated
with Agrobacterium for 1–2 days.
Fluorescence microscopy
The scales were rinsed with water, onion epidermal cell layers were peeled and directly transferred to glass slides. Fluorescence images were screened using a confocal laser microscope LSM 5 PASCAL (Carl Zeiss) equipped with 10× and
20× lenses and analyzed by LSM 5 Image Browser software.
Particle bombardment transformation
Gold particles (1.0 µm; Bio-Rad, USA) coated with DNA
(0.1 µg/µL) were delivered into onion epidermal cells by
Biolistic PDS-1000/He system (Bio-Rad, USA) with 1100
psi rupture discs under a vacuum of 27 inch Hg according
to the manufacturer’s directions as described by Scott et al.
(1999).
Statistical analysis and estimation of transformation rate
In order to calculate the mean of both the transformed scales
number and the transformed scales rate of Agrobacteriummediated and particle bombardment transformation, respectively, we calculated the transformed scales number and
transformed scales rate of the four separate experiments and
then used the Sigmaplot 9.0 software to calculate the mean
and standard error. In addition, we calculated the number
of transformed cells and the transformed cells rate of every
transformed scale and all the data we obtained were analyzed using the Sigmaplot 9.0 software to calculate the mean
and standard error, respectively. The transformed cells rate
of transformed scales was calculated using the number of
transformed cells divided by the number of the whole cells of
transformed scales every time. The number of transformed
cells was calculated using the confocal laser microscope LSM
5 PASCAL. For calculating the number of the whole cells
on transformed scales, we used the area of the transformed
scale divided by the average area of one onion cell analyzed
by LSM 5 Image Browser software.
Results
We utilized the Agrobacterium-mediated transformation and particle bombardment for transient expression
of EGFP and DsRed in onion epidermal peels. It was
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A method for protein subcellular localization
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Fig. 2. High transformation rates are seen in this overview of onion cells 24 h after Agrobacterium-mediated transformation. (A)
35S::EGFP and (C) 35S::DsRed appears in the cytoplasm byAgrobacterium-mediated transformation. (B) Soluble 35S::EGFP and
(D) 35S::DsRed appears in the cytoplasm transformed by particle bombardment. (E) 35S::AtLTP1: DsRed appears in the cell wall
byAgrobacterium-mediated transformation. Onion cells were plasmolyzed in 30% (g/v) sucrose. The arrows indicate cell wall and
protoplast. Scale bars in all panel are equal to 50µm.
Table 1. The efficiency of Agrobacterium-mediated transformation and particle bombardment.a
Total onion
Transformed Transformed
Transformed
Transformed
scales (every time) scales (± SE) cells (± SE) scales rate (± SE) cells rate (± SE)
Agrobacterium-mediated transformation
Particle bombardment transformation
a
40
6
12.0 ± 1.5
5.0 ± 0.70
177.6 ± 10.8
26.2 ± 3.6
30.0 ± 3.7%
83.3 ± 11.8%
10.5 ± 0.9%
2.0 ± 0.4%
SE, standard error.
found that EGFP and DsRed had the same subcellular localization in onion epidermal peel by these two
methods (Fig. 2 A–D). However, the Agrobacteriummediated transformation showed more transformed
cells than the particle bombardment. In most cases
the transformed cells appeared as an adjacent community, which facilitated easy capturing of the fluorescence image. The time of the maximum fluorescence could be observed as early as 12–24 h and
might last even two weeks. All transformed and nontransformed cells could be plasmolyzed in 30% sucrose indicating that Agrobacterium has no deleterious
effects on cells. Using Agrobacterium-mediated transformation, AtLTP1::DsRed fusion protein was transferred into onion inner epidermal peels and the resultant red fluorescence was mainly observed in the cell
wall (Fig. 2E), since AtLTP1 is a secretion protein and
localizes in cell wall, which was proven using the specific
stain method (Thoma et al. 1993).
In order to compare both the stability and the efficiency of our methods with microbombardment, four
separate experiments were evaluated for DsRed fluorescence. Forty and six onion scales were used for
Agrobacterium-mediated transformation and particle
bombardment transformation every time, respectively.
We calculated the transformed scales and the transformed cells on transformed scales every time. Data
was subjected to statistical analysis and presented (Table 1). Our results demonstrated that Agrobacteriummediated transformation was better than bombardment in two aspects: the transformed cells and the
transformation cells rate. The mean ± SE of the
transformed scales rate using Agrobacterium-mediated
method was 30.0 ± 3.7%, while the bombardment resulted in 83.3 ± 11.8%. The mean ± SE number of the
transformed cells and the transformation cells rate were
177.6 ± 10.8 and 10.5 ± 0.9% of the Agrobacteriummediated method, while the bombardment resulted in
26.2 ± 3.6 and 2.0 ± 0.4%, respectively (Table 1).
Discussion
In this paper, using onion epidermal cells as a model
system, we describe a rapid, transient gene expression system mediated by Agrobacterium. Although the
Agrobacterium-mediated transformation is not a novel
method, it has not been reported that this method
could be used in onion epidermis cells for protein subcellular localization so far. In comparison to particle
bombardment and other traditional methods, this system exhibited very high transformation rate and clear
fluorescence images in all experiments we did and the
protocols were more stable, simpler, and more economical. Agrobacterium-mediated transformation has lower
rate of transformed scales, but it transformed more cells
per epidermal peel than particle bombardment. These
evidences indicate that Agrobacterium-mediated transformation is a powerful experimental tool for studying
GFP fusion protein subcellular localization and other
physiological events in vivo.
We obtained higher transformation efficiency with
the onion epidermal peel than reported by Eady et
al. (2000). The maximum transformation frequency
2.7% was obtained by Eady et al. (2000) using the
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onion immature embryos as the explant source after
co-cultivation with theAgrobacterium. The reasons that
are responsible for transformation frequency differences
might be partially due to the differential sensitivity of
the specific somatic cells to Agrobacterium tumefaciens.
It is also possible that the low regenerating frequency
of transformed cells, rather than the transforming efficiency, is the bottle-neck of acquiring lower frequency of
transformed plantlets. Our results may imply that the
frequency of transformants can be much higher, if the
regenerating efficiency can be improved to some extent.
Acknowledgements
This work was supported by the Chinese National Basic Research Project (2006CB100104) and National Science Foundation of China Research Grants (30470158).
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Received January 29, 2007
Accepted July 6, 2007
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