Impaginato 257 Adv. Hort. Sci., 2019 33(2): 257-262 DOI: 10.13128/ahs-23240 TuMV as an efficient transient vector for expressing heterologous proteins in Nicotiana tabacum and N. benthamiana M. Modarresi 1, M. Jalali-Javaran 1 (*), M. Shams-Bakhsh 2, S. Zeinali 3, M. Mirzaee 1 1 D e p a r t m e n t o f P l a n t B r e e d i n g a n d B i o t e c h n o l o g y , F a c u l t y o f Agriculture, Tarbiat Modares University, Tehran, I.R. Iran. 2 Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, I.R. Iran. 3 Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, I.R. Iran. Key words: green fluorescent protein, recombinant protein, tobacco plant, tran- sient expression, viral vector. Abstract: Nowadays the production of recombinant proteins such as drugs and commercial protein compounds in plants is called molecular farming. It has some benefits such as fast and large quantity production of recombinant pro- teins with low cost. In this research, the green fluorescent protein (GFP) was transiently expressed in two tobacco species via turnip mosaic virus (TuMV) derived vector, a virus which can infect a wide range of plant species. Florescence microscopy results indicated that TuMV could infect tobacco plants and accumulate GFP protein in plant leaves. In addition, RT-PCR, Dot-Blot and ELISA assays demonstrated the recombinant gene transcription, translation and stability. This is the first report of using TuMV-based viral vectors for producing recombinant proteins in tobacco. Optimized TuMV-based viral vectors could be used for producing recombinant proteins in tobacco. 1. Introduction Various expression systems, such as bacteria, yeast, plants, insects and mammalian cell cultures can produce recombinant proteins. The benefits of expressing recombinant proteins in plants include economic, agricul- tural scale, safe and authentic production (Sijmons et al., 1990; Ma et al., 2003; Mardanova et al., 2015;). Molecular farming (also known as molec- ular pharming or biopharming) uses genetically engineered plants for the production of biopharmaceutical products, vaccine subunits, industrial enzymes therapy peptides and other compounds of interest (Boothe et al., 1997; Wang and Ma, 2011; Yarbakht et al., 2015). Recombinant proteins in plants may be gained by stable genetic trans- formation (nuclear or plastid) or through transient expression. Transient expression is usually used for fast and flexible expression of genes of (*) Corresponding author: m_jalali@modares@ac.ir Citation: MODARRESI M., JALALI-JAVARAN M., SHAMS- BAKHSH M., ZEINALI S., MIRZAEE M., 2019 - TuMV as an efficient transient vector for expres- sing heterologous proteins in Nicotiana tabacum and N. benthamiana. - Adv. Hort. Sci., 33(2): 257- 262 Copyright: © 2019 Modarresi M., Jalali-Javaran M., Shams- Bakhsh M., Zeinali S., Mirzaee M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 23 March 2018 Accepted for publication 7 August 2018 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(2): 257-262 258 interest (GOI), evaluation of expression system per- formance and components such as promoter and enhancers (Chiera et al., 2008). In plants, a number of virus-based vectors are utilized for the transient expression of foreign genes, such as tobacco yellow dwarf virus (TYDV) for transient expression of chal- cone synthase in Petunia hybrida (Atkinson et al., 1998), tobacco mosaic virus (TMV) for transient expression of GFP (jellyfish, Aequorea victoria green- fluorescent protein) in tobacco (Shivprasad et al., 1999), bean pod mottle virus (BPMV) for expression the GFP in the soybean (Zhang et al., 2010), wheat streak mosaic virus (WSMV) for expression the GFP in cereals (Tatineni et al., 2011) etc. Turnip mosaic virus (TuMV) belongs to Potyviridae family and infects a wide range of plant species espe- cially cruciferous (Brassicaceae family). It is a posi- tive-sense single stranded RNA virus with a linear and monopartite genome and average length of 720 nm (Brunt et al., 1996). Previously Beauchemin et al. (2005) strongly expressed GFP and GUS (bacterial β- glucuronidase) reporters genes in Brassica perviridis plants via TuMV virus. Furthermore, Chen et al. (2007) introduced GFP in some Brassica hosts such as B. campestris and B. juncea and high levels of the recombinant protein expression were observed. Therefore, in this study, to investigate the perfor- mance of recombinant protein production, the GFP reporter gene was introduced into the tobacco (Nicotiana tabacum and N. benthamiana) plants by using TuMV vector. 2. Materials and Methods Plant material and growth conditions Nicotiana tabacum cv. Xanthi and cv. Samsun and N. benthamiana seeds were grown in pots containing autoclaved soil, including 40% farm soil, 30% peat moss and 30% perlite. They were kept at 25°C in a phytotron under a 16-hour photoperiod (16:8 h L: D). Plasmid and viral constructs The TuMV-GFP construct (Fig. 1) was kindly pro- v i d e d b y D r . S h y i - D o n g Y e h , P l a n t P a t h o l o g y D ep a rt men t , Na t i o n a l C h en g H s i n g Un i vers i t y, Taichnug, Taiwan. The plasmid contains a cauliflower mosaic virus 35S promoter (CaMV 35S) and GFP cod- ing sequence between the NIb (nuclear inclusion pro- tein b) and CP (coat protein) positions. Recombinant viral construct, pTuMV-GFP, was transferred into bacterial (Escherichia coli DH5α) competent cells (Sambrook and Russell, 2001). Bacteria were grown in 200 ml Luria-Bertani medium and then, pTuMV- GFP was extracted (Engebrecht et al., 1991). Plant Rub-inoculation with TuMV-derived vector Wild-type TuMV (for control plants) and pTuMV- GFP was mechanically inoculated on upper surface of two top leaves (10 µg in 10 µl per leaf), using a cot- ton stick and carborandum powder according to Hosseini et al. (2013). Systemically infected (non- inoculated leaves) were used for further analysis. Total RNA extraction and reverse transcription-poly- merase chain reaction (RT-PCR) The presence of the GFP gene in inoculated leaves was determined by RT-PCR. Total RNA was extracted from inoculated and control plant leaves (five inde- pendent samples) by Qiagene kit (South Korea) twelve days after incubation according to the manu- facturer’s instruction. RNA was extracted from non- inoculated leaves for confirming replication and movement of the virus. After treating with DNase I (Thermo Fisher Scientific, USA), cDNA was synthe- sized using the RevertAid Reverse Transcriptase (Thermo Fisher Scientific, USA) and GFP reverse primer (5’ –TTG TAC TCC AGC TTG TGC CC-3’) accord- ing to the producer’s instructions. RT-PCR was con- ducted using the cDNA and the following forward and reverse primers under the following cycling con- ditions: forward (5’- ACG ACG GCA ACT ACA AGA CC - 3’) and reverse (5’- TTG TAC TCC AGC TTG TGC CC - 3’). PCR cycling conditions were as follows: 94°C for 3 min for initial denaturation; 35 cycles of 94°C for 30 s, 51°C for 30s, and 72°C for 30 s; and 72°C for 10 min for a final extension. Then PCR products were ana- lyzed by 1% TAE agarose gel. Fluorescence microscopy Leaves from TuMV-based vector inoculated and control plants were observed under an Olympus fluo- rescent microscope 6 (version IX71, Tokyo, Japan). Fig. 1 - Schematic representation of the viral construct contai- ning GFP under the 35S promoter that was used in this expression analysis. The foreign gene insertion site is between NIb (nuclear inclusion protein b) and CP (the virus coat protein gene) provided by NcoI and NheI restriction endonuclease enzymes. Modarresi et al. - Heterologous proteins production via viruses 259 The fluorescence photographs were taken using a mounted high-resolutionm7 Olympus DP70 DP71 digital camera at 12 days post-inoculation (dpi). Protein extraction and GFP analysis Proteins were extracted from 0.5g tissues of con- trol and inoculated tobacco leaves (five independent samples) with extraction buffer, including 0.2M Tris- HCl (pH 8.0), 5mM ethylenediaminetetraacetic acid ( E D T A ) , 1 0 0 m M s u c r o s e , a n d 0 . 1 m M 2 - m e r - capthoethanol (Abdoli-Nasab et al., 2013) and the concentration was assessed by Bradford’s assays (Bradford, 1976). Dot-Blot (Stott, 1989) and indirect enzyme-linked immunosorbent assay (ELISA) (Wang and Gonsalves, 1990) were carried out to quantita- tive detection of the GFP protein in the inoculated tobacco plants. Statistical analysis All experiments were done according to a com- pletely randomized design at five independent sam- ples. Data analyses were performed using Microsoft Excel program software and SPSS version 22. When significant differences were found least significant difference (LSD) test at P<0.05 was applied to sepa- rate means. 3. Results and Discussion The main benefits of the plant made proteins (PMPs) are lower costs and potential to produce a very large scale of recombinant proteins. Viral vec- tors have the ability to express transgenes in hosts and they are suitable and rapid platform for produc- tion high-level of recombinant proteins. In this research, we utilized a TuMV viral vector (Fig. 1) under the control of the CaMV 35S promoter for transient expression of the GFP in tobacco plants. Although systemic symptoms of TuMV were not observed on infected plants, GFP was detected by the fluorescence microscopy (Fig. 2) twelve days post-inoculation. This research has displayed for the first time that recombinant protein (GFP) can accumulate in tobacco plants via TuMV based viral vector with CaMV 35S promoter. TuMV can infect tens different plant species (Chen et al., 2007) (to compare common viral vectors which can affected specific plant species), therefore, TuMV based viral vector can be economical. In this study, two different tobacco species, N. benthamiana and N. tabacum (two different cultivars Xanthi and Samsun) were investigated. Fluorescence microscopy analysis of GFP (Fig. 2), RT-PCR (Fig. 3), Dot-Blot analyses (Fig. 4) and ELISA assay (Fig. 5) indi- cated that recombinant protein expression in tobac- co plants leaves occrued. RT-PCR (Fig. 3) showed that, as expected, 160 bp bands were found in infect- ed plants, while not observed in the negative control (wild type). It shows that the TuMV virus can infect the plant and replicate its genome. Viruses (like TuMV from Potyviridae family) have developed pro- teins such as Helper Component Proteinase (HCPro), Fig. 2 - Fluorescence microscopy analysis of GFP expression in tobacco plant's leaves which infected by pTuMV-GFP. (A) Nicotiana benthamiana, (B) N. tabacum cv. Xanthi, (C) N. tabacum cv. Samsun (D) Negative control (tobac- co plant infected with wild-type TuMV). Green color indicated GFP expression and the red indicated chlo- rophyll autofluorescence. Fig. 3 - RT-PCR amplified a 160 bp fragment from the GFP with specific primers in 1% agarose gel. C- 1= negative con- trol (water template), C- 2= negative control (RNA tem- plate), C- 3= negative control (Wild type (non-inoculated plant)), C+= positive control, Lane 1= Nicotiana bentha- miana transformed plants, Lane 2= N. tabacum cv. Xanthi, Lane 3= N. tabacum cv. Samsun transformed plants, M= molecular weight marker (1 kb standard GeneRuler). Adv. Hort. Sci., 2019 33(2): 257-262 260 which suppress the plants silencing defense (Voinnet, 2001). Furthermore, HCPro has protease activity and it is necessary for virus genome replication and viral movement and transmission (Klein et al., 1994; Chiera et al., 2008). Dot-Blot assay (Fig. 4) indicated that GFP protein was recognized by specific antibody and developed brown color in transformed plants and positive con- trol. ELISA assay indicated that expression levels of GFP was estimated approximately ≤0.5% of total sol- uble protein (TSP) of fresh weight of tobacco leaves. These results show lower accumulation of recombi- nant proteins compared with a number of previous studies which expressed by viral vectors such as Artichoke mottled crinckle virus (Lombardi et al., 2009), Beet curly top virus (Kim et al., 2012) etc. Some strategies, such as codon-optimization (Love et al., 2012) and the use of improved viral vector ele- ments including strong viral promoters (Gleba et al., 2007) can increase the expression of recombinant protein. It seems that, N. benthamiana lacks RNA-depen- dent RNA polymerases (RdRPs) which required for defense against viruses, therefore N. benthamiana infected plant displays more strong symptoms and its products more than do other tobacco species (Yang et al., 2004). In many previous studies (Kumar and Kirti, 2010; Sasaki et al., 2015; Vojta et al., 2015; Park et al., 2 0 1 6 ) , R h i z o b i u m r a d i o b a c t e r ( f o r m e r l y Agrobacterium tumefaciens) delivery systems has been used to express the transient expression of recombinant protein using a viral vector. However, in this study, a direct virus inoculation system via dust- ed with carborundum has been used. Our study indi- cated that this method is useful to accelerate the production of recombinant proteins in tobacco plants. 4. Conclusions In conclusion, our results showed that TuMV, as a virus that could infect a wide range of plant species, could be used to produce recombinant proteins in tobacco. In this investigation, all inoculated plants expressed GFP protein. Results showed that incubat- ed N. benthamiana has more accumulated recombi- nant protein compared to the two N. tabacum culti- vars. Although the level of expression is low and should be optimized for future studies. 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