Nicotiana cavicola as a host for production of recombinant proteins by Agrobacterium -mediated transient gene expression.

Aim. To analyze a novel plant species as a host for obtaining recombinant proteins via transient gene expression. Methods. Agrobacterium -mediated transient gene expression, protein analysis, statistical data processing. Results. N. cavicola plants demonstrate good biotechnological characteristics; they are susceptible to agrobacterial infection and plant viruses. Green fluorescent protein (GFP) and human interferon alpha were produced in N. cavicola after transient gene expression using two different vector systems. The level of recombinant proteins de-pended on the gene and the system used. GFP content reached 6.0 % and 12.6 % TSP (0.44 mg/g FW). The interferon antiviral activity of the leaf extracts was 840 IU/g FW and 1710 IU/g FW. Conclusion. Here we propose N. cavicola species as a novel host for obtaining recombinant proteins which can be used as an alternative to the N. benthamiana host.


Introduction
The plant transient expression (TE) of transgenes is a fast, reliable and simple method that extensively used for fundamental physiological research and applied biotechnological purposes [1][2][3]. Potentially TE can be used for rapid production of pharmaceutically valuable proteins, including antibodies [4][5][6][7]. Rapid production of antibodies or vaccine proteins is preferable in the treatment of advanced cancer diseases or in case of season epidemic diseases. In case of TE, the transgenes are not integrated into plant genome thus avoiding the position effects and providing considerably increased levels of recombinant proteins [8].
The protocols for Agrobacterium-mediated TE of transgenes in Nicotiana benthamiana plants are well developed and widely applicable. N. benthamiana demonstrates high sus-ceptibility to agrobacterial and viral infections [9,10], and is considered as a model host for TE. However, common utilization of one species gives some limitations and hides the advantages of other species, e.g. increased recombinant protein levels or better biotechnological characteristics. Earlier, it was proved the important role of host species for successful production of recombinant proteins [11].
Here we describe a new host for TE of transgenes, namely Nicotiana cavicola. This species can be easily infiltrated, it is susceptible to agrobacterial transfection, assures transgene expression from various expression cassettes (different vector systems), and produces recombinant proteins in large amounts. The final levels of green fluorescent protein (GFP) and human interferon alpha 2b obtained in N. cavicola plants were comparable to or surpassing the corresponding levels obtained in N. benthamiana plants.

Materials and Methods
Plant growth conditions. Nicotiana spp. seeds were obtained from "National Germplasm Bank of World Flora of the Institute of Cell Biology and Genetic Engineering" (Kyiv, Ukraine). Seeds were germinated either in unsterile conditions (commercial soil) or in aseptic conditions on MS agar medium [12] after surface sterilization [13]. Plants grew in greenhouse conditions: 16-hour light day at 22-28 o C, 3000-4000 lux. Leaves of 6-8-week old plants were used for the experiments.
Agrobacterial strains and genetic constructions. Vector constructions were harbored into Agrobacterium tumefaciens GV3101 strain. The used plasmids (pICH5290, pICH7410, pICH10570, pICH10881, pICH17311, pICH6692) were obtained for scientific purposes from Icon Genetics GmbH (Halle, Germany) and described in [14,15]. The plasmid pCB125 was described in [16]. The sequence coding for the human interferon alpha 2b was fused with calreticulin signal from N. plumbaginifolia for the protein targeting to intracellular space [15].
Two different vector systems used in the experiments were described in the earlier publication [11]. One of them represents a simple vector construction where a gene of interest was driven by the 35S CaMV promoter (named herein as 35S-system).
Another vector system (named herein as recombinase vector (Rec) system) includes combination of viral genome elements and binary plasmid of A. tumefaciens. This vector system requires simultaneous introduction of three vector modules into the same cell for in planta assembly of the RNA replicon [11,14].
Bacteria growing and infiltration procedure. Growth and preparation of agrobacteria for the infiltration were performed as described [11,15]. Infiltration was carried out according to Schob et al. [17] with minor modifications [15]. Mixed bacterial suspension (100 µl/sample) was injected at the same leaf position to exclude sample variability [18]. There were two control groups of plants: the first group was infiltrated with the buffer only; the second one was infiltrated just with the agrobacterium clone which harbored the vector construction with the p19 gene (a suppressor of post-transcriptional gene silencing). All experiments were done in 7-10 replications.
Extraction of total soluble proteins. Plant material was collected on the 4 th day post infiltration (dpi) if 35S expression system was used or on the 16 th dpi if the recombinase expression system was used.
GFP extraction procedure: 0.1-0.2 g of fresh plant material was ground on ice in 1.5 ml tubes in 500 µl of cold sodium phosphate buffer, and debris was precipitated by centrifugation at the top speed. The supernatant was transferred into clean tube. A new portion of buffer (500 µl) was added to the sediment and procedure was repeated. The total supernatant was used for the next analysis.
Interferon extraction procedure: plant leaf tissue was collected, weighted and ground on ice with a mortar and a pestle in one volume of cold Tris-base buffer. Grinded leaves were transferred in 1.5 ml tubes and the debris was precipitated by two-round top speed centrifugation to clarify a supernatant. The supernatant was used for the next analysis.
Measurement of the concentration of total soluble proteins. The quantity of total soluble proteins (TSP) was measured according to Bradford's method [19] using bovine serum albumin as a standard.
GFP assay. The presence of recombinant GFP in leaf tissues was determined and its quantity was calculated as described earlier [11]. The percentage of GFP per TSP was calculated as well.
Assessment of antiviral activity of interferon. The protective antiviral activity of experimental extracts was demonstrated in titration assay described in [15]. The dilution of sample where 50 % of the cells survived after vesicular stomatitis virus infection was determined as the titer of interferon. Based on titers of standard interferon alpha 2b the activity of extracts was estimated in International Units (IU).
Statistical data processing. For statistical data manipulations, standard deviation (SD) and Student's test were used. Bars on diagrams demonstrate standard deviation [20].

Results and Discussion
Nicotiana cavicola, an Australian species of tobacco is classified together with N. benthamiana in the same subgenus. Several studies revealed a high sensitivity of N. cavicola to a wide range of plant viruses [21,22]. This feature is distinctive also for N. benthamiana, which suggests that N. cavicola along with N. benthamiana may be used for the heterologous protein production via transient expression (TE) if the gene is located in a viral based vector system.
To investigate a biological potential of N. cavicola, two mentioned species were compared for several parameters: growth in greenhouse conditions, plant biomass, easiness of infiltration procedure, possibility of Agrobacteriummediated TE of transgenes and the production of desired proteins.
Growth in greenhouse conditions. The mature seeds of N. cavicola sprouted on the 4 th -6 th days similarly to N. benthamiana ones, and we did not observe any difference in the plant germination rate between the surface sterilized or non-sterilized seeds. Subsequently, the nonsterilized seeds were germinated directly in a commercial soil mixture. We found that N. cavicola can be used for TE experiments at the age of 6-8 weeks but before appearance of flowers [11]. The 6-8 weeks old N. benthamiana plants were often used for TE as well [23,24]. The N. cavicola plants demonstrate efficient growth in greenhouse and their fully expanded leaves are bigger than those of N. benthamiana ones grown under the same conditions (Fig. 1).
Plant biomass and infiltration procedure. Total biomass weights of N. cavicola and N. benthamiana plants available for the protein production were compared. As shown in the Table 1, one fully expanded leaf of N. cavi cola weighs more than that of N. benthamiana. The difference was significant (P<0.001). N. benthamiana demonstrates the best production of recombinant proteins in five upper mature leaves [11], whereas only three upper mature leaves can be used in N. cavicola. Thus, there was no difference between the species in the total leaf biomass useful for protein production.
N. cavicola leaves are nicely infiltrated by bacterial suspension without lateral vein restriction. Simplicity of leaf infiltration is typical for young N. benthamiana plants.

Recombinant protein production: Green Fluorescent Protein (GFP).
Vital reporter protein, GFP, was used for the assessment of recombinant protein production in N. cavicola. Two vector systems carrying the gfp gene were used for the experiments. One vector includes the gfp gene driven by 35S CaMV promoter, whereas another system, a recombinase vector (Rec) system [8,14], includes two pro-vectors and the gfp gene appears under the viral subgenomic promoter after the recombination event.
Green fluorescence was detected under shot-wave UV-light within infiltrated areas that confirmed the presence of heterologous GFP   ( Fig. 2). The viral suppressor of gene silencing p19 was used to increase the heterologous protein production [25,26]. Total soluble proteins (TSP) were extracted from the infiltrated leaf areas and the contents of GFP and TSP were calculated as described.
The GFP content in N. cavicola leaves reached 6.0±1.51 % TSP when 35S system was used. In N. benthamiana leaves the GFP level reached 3.84±1.31 % TSP (Fig. 3). The difference between the means was significant (P<0.05). The data were compared with the data obtained earlier for other Nicotiana spp. [11] and it was noted that the percentage of GFP in N. cavicola was the highest. In general, a higher percentage of heterologous protein will invest in facilitation of the next purification procedure.
The GFP content calculated per a plant biomass unit was 0.440±0.140 mg/g fresh weight (FW) in N. cavicola and 0.312±0.075 mg/g FW in N. benthamiana. The difference was not statistically significant. The average amount of TSP in N. benthamiana tissues was higher than that in N. cavicola: 9.43±3.07 mg/g FW and 7.36±2.01 mg/g FW, respectively.
Then GFP content was measured after applying the Rec system. Usage of this system resulted in an increase of GFP percentage compared to 35S system but the amount of GFP per gram of fresh weight was similar to 35S system: 12.59±6.48 % TSP (or 0.438±0.218 mg/g FW) in N. cavicola and 16.2±5.7 % TSP (or 0.365±0.122 mg/g FW) in N. benthamiana (Fig. 4). Further analysis revealed that the TSP level in experimental leaves reduced: 3.86±1.32 mg/g FW in N. cavicola and 2.26±1.04 mg/g FW in N. benthamiana. The possible explanation is senescence of leaves and diminution of proteins during the experimental period: the maximal level of recombinant protein accumulation after using the Rec system is reached on the 14 th -16 th days post infiltration (dpi), whereas the 35S system al-  lows collection of leaves on the 4 th dpi. An increased GFP percentage after applying the Rec system may ensure advantages for the following protein purification steps. In the previous paper [11] it was shown that not all Nicotiana species were sensitive to the Rec system and some of them did not accumulate GFP in measurable quantities.
The obtained data proved that N. cavicola is susceptible to agrobacterial infection, and T-DNA is transferred efficiently in the plant cells. Additionally, N. cavicola demonstrates visible sensitivity to different expression systems. This confirms that N. cavicola is a perspective host for obtaining heterologous proteins via the Agrobacterium-mediated transient gene expression.
Human interferon (INF) alpha 2b production. To check if the tested host can produce the proteins not as stable as GFP, human interferon α2b was chosen for the next experiments. To reach the aim, the identical expression systems (the 35S system and Rec system) were used but the gfp gene was replaced by the inf gene. The experimental conditions were the same except for the extraction procedure. In the course of experiment, it was confirmed the presence of antiviral activity in the extracts prepared from the infiltrated leaves. This can be regarded as a proof of the bioactive heterologous interferon production in N. cavicola cells.
If the 35S system was used, the average antiviral activity detected in the N. cavicola leaf extracts was 844±302 International Units (IU)/g FW (variation range 770-1570 IU/g FW), and the average antiviral activity detected in the N. benthamiana leaf extracts was 281±105 IU/g FW (variation range 170-370 IU/g FW) (Fig. 5). The INF production in N. cavicola was about 3 times higher than in N. benthamiana. The difference between the means was significant (P<0.01). These data agree with the data shown for the GFP production.
Further, antiviral activity was measured in the leaf extracts obtained after using the Rec system. In this case antiviral activity of extracts increased if compared with the data shown for the 35S system: the average activity was 1714±855 IU/g FW (variation range 800-2400 IU/g FW).
In general, the total amount of INF was less than the total amount of GFP obtained in the same manner. The possible explanation of such results is a different degradation rate of the various proteins in plant cells. GFP conformation provides its high proteolytic stability [27] whereas native human INF is attacked by plant proteases more frequently. Moreover, optimal conditions for accumulation of recombinant proteins can vary significantly.