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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 Unauthenticated Download Date | 6/11/17 10:08 PM 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 Unauthenticated Download Date | 6/11/17 10:08 PM A method for protein subcellular localization 531 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 Unauthenticated Download Date | 6/11/17 10:08 PM 532 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). References Allen G.C., Hall G.E., Childs L.C., Weissinger A.K., Spiker S. & Thompson W.F. 1993. Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. Plant Cell 5: 603–613. Chalfie M., Tu Y., Euskirchen G., Ward W.W. & Prasher D.C. 1994. 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