Construction of viral-based expression vectors for high-level production of human interferon alpha 2b in plants.

Human interferon (hINF) alpha 2b is clinically important pharmaceutical product included in combinatory therapy against chronic hepatitis C and B and complex therapy against several cancer diseases. Here, we created the genetic constructions, based on genome elements of potato virus X (PVX), carrying the infα2b gene for transient expression in plant cells. The created plasmid vector constructions were tested through Agrobacterium-mediated transient gene expression method in two plant species-Nicotiana benthamiana and Ocimum basilicum (sweet basil). Production of recombinant hINF alpha 2b was more efficient in N. benthamiana than that in O. basilicum plants. The average yield of hINF alpha 2b produced in N. benthamiana plants was 0.56 mg/g of fresh leaf weight (FW) or 6% of the total soluble cell proteins (TSP). The maximal level reached up to 1.2 mg/g FW or 9% TSP. We estimated that about 0.67 mg of hINF can be obtained from one N. benthamiana plant. The yield of hINF alpha 2b obtained with the PVX-based expression cassette was about 80 times higher than the yield of hINF alpha 2b obtained with a simple expression cassette in which the infα2b gene was controlled by the 35S promoter of cauliflower mosaic virus. KEY POINTS: • PVX-based expression vectors provide efficient transient expression of infα2b gene • N. benthamiana plants can produce human interferon alpha 2b at high levels • The yield of the hINF α2b reached up to 1.2 mg/g of fresh leaf weight.

Accumulation of colicin M protein and its biological activity in transgenic lettuce and mizuna plants.

Food-borne illnesses caused by pathogenic Escherichia coli strains, especially enterohaemorrhagic E. coli (EHEC), are a serious public health problem, as debilitating disease and even death from such food poisonings have been repeatedly reported. Colicin M (ColM), a non-antibiotic antimicrobial protein produced by some strains of E. coli, has shown promising activity in controlling multiple enteropathogenic strains of E. coli and related pathogens. As contaminated green leafy vegetables are a frequent source of pathogenic E. coli infections, we genetically modified (GM) two edible crops, lettuce (Lactuca sativa L.) and mizuna (Brassica rapa subsp. nipposinica var. laciniata), to stably express the ColM gene and assessed the antibacterial activity of tissue extracts from these plants against selected E. coli strains in vitro. Transgenic plants of these species were developed using Agrobacterium-mediated transformation with a vector containing the ColM-coding gene (cma) under the control of the 35S promoter. Western blot analysis of recombinant ColM protein was performed in selected transgenic plants to confirm cma gene expression and quantify ColM accumulation. Extracts of transgenic plants expressing ColM showed significant activity against two major strains of EHEC (O157:H7 and O104:H4) as well as E. coli strains resistant to beta-lactam- and carbapenem-class antibiotics. Importantly, the antibacterial activity persisted in several subsequent generations of transgenic lettuce and mizuna plants that stably expressed the ColM gene. In addition, our results also show that the antibacterial activity of dried (up to 40°C) biomass of transgenic plants remained stable without a decrease for at least three months.

Study of Superoxide Dismutase Activity in Long-Term Cultivated Artemisia and Althaea "hairy" Roots.

The aim of the study was to evaluate the long-term effect of Agrobacterium rhizogenes genes transfer on plant antioxidant system by the study of superoxide dismutase (SOD) activity in "hairy" roots of Artemisia and Althaea spp plants. PCR analyses revealed stability of the transformation and presence of bacterial rol B and rol C genes in the "hairy" roots after 4-6 years from the transformation event. SOD activity in the roots of untransformed in vitro cultivated plants used for the initiation of "hairy" roots growth was in the range of 45.8 ± 8.7 U/µg (Althaea officinalis) and 275 ± 97.1 U/µg (Artemisia ludoviciana). After a long-term in vitro cultivation more than half of tested "hairy" root lines (54%) showed a significant increase in SOD activity values compared to untransformed roots. The highest SOD activity values of "hairy" root lines (24-fold increase) were founded in A. officinalis (1105 ± 174 U/µg) and A. dracunculus (1356 ± 402 U/µg). The increase of the activity was found also in "hairy" roots of A. vulgaris (up to 375 ± 28.2 U/µg, sevenfold increase), A. ludoviciana (1001 ± 191 U/µg, 3.6-fold increase), and A. tilesii (438 ± 104 U/µg, 1.6-fold increase). The results of our study indicate that transformation by wild-type A. rhizogenes not harboring any foreign genes implementing in SOD activity regulation can often stably activate plant antioxidant enzyme system. This effect, observed in the "hairy" roots of five plant species in 4-6 years of the transformation event, obviously, should be taken into account in works aimed at creating transgenic plants by Agrobacterium-mediated transformation.

Robust Agrobacterium-Mediated Transient Expression in Two Duckweed Species (Lemnaceae) Directed by Non-replicating, Replicating, and Cell-to-Cell Spreading Vectors.

Plant-based transient expression systems have recognized potential for use as rapid and cost-effective alternatives to expression systems based on bacteria, yeast, insect, or mammalian cells. The free-floating aquatic plants of the Lemnaceae family (duckweed) have compact architecture and can be vegetatively propagated on low-cost nutrient solutions in aseptic conditions. These features provide an economically feasible opportunity for duckweed-based production of high-value products via transient expression of recombinant products in fully contained, controlled, aseptic and bio-safe conditions in accordance with the requirements for pharmaceutical manufacturing and environmental biosafety. Here, we demonstrated Agrobacterium-mediated high-yield transient expression of a reporter green fluorescent protein using deconstructed vectors based on potato virus X and sweet potato leaf curl virus, as well as conventional binary vectors, in two representatives of the Lemnaceae (Spirodela polyrhiza and Landoltia punctata). Aseptically cultivated duckweed populations yielded reporter protein accumulation of >1 mg/g fresh biomass, when the protein was expressed from a deconstructed potato virus X-based vector, which is capable of replication and cell-to-cell movement of the replicons in duckweed. The expression efficiency demonstrated here places duckweed among the most efficient host organisms for plant-based transient expression systems, with the additional benefits of easy scale-up and full containment.

Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro.

Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), "rejuvenated" through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.

Increased ribonuclease activity in Solanum tuberosum L. transformed with heterologous genes of apoplastic ribonucleases as a putative approach for production of virus resistant plants.

Viral pathogens seriously decrease the yield of potato (Solanum tuberosum L.) -an important agricultural crop. Therefore, there is a demand for potato cultivars resistant to multiple viruses. Ribonucleases (RNases) are supposed to be engaged to antiviral response in plants. Heterologous RNase gene expression provides a tool for production of cultivars with multiple resistance to viruses and viroids. Transgenic potato cultivars Luhivs'ka and Lasynak with heterologous genes bov and ZRNase II of apoplastic RNases from Bos taurus and Zinnia elegans respectively were obtained via Agrobacterium-mediated transformation. The presence of bov and ZRNase II transgenes was confirmed by PCR analysis. RNase activity was examined by modified Oleshko method. Plants with heterologous ribonuclease genes had higher level of RNase activity compared to nontransgenic ones. Transgenic plants inoculated with Potato virus Y, PVY (genus Potyvirus, family Potyviridae) demonstrated delayed and less severe symptoms of viral infection. DAS-ELISA confirmed the presence of viral antigens both in transformed and control plants. Visual manifestations of viral infection in transgenic potatoes were milder than in control plants and their development was delayed, but complete elimination of the virus did not occur.

Rhizoextraction Potential of Convolvulus tricolor Hairy Roots for Cr6+, Ni2+, and Pb2+ Removal from Aqueous Solutions.

This study evaluated the potential of dwarf morning-glory Convolvulus tricolor (Convolvulaceae) plants and their hairy roots induced by Agrobacterium rhizogenes for rhizoextraction of heavy metals ions from the liquid medium under aseptic growth conditions. Both the young C. tricolor plants and the generated hairy root culture efficiently removed Cr6+, Ni2+, and Pb2+ ions from the liquid cultivation medium. As determined by atomic absorption spectroscopy, the hairy roots demonstrated a high level of heavy metal ions accumulation (µg/g dry weight): 3942 ± 210 of chromium, 1529 ± 312 of nickel, and 2613 ± 373 of lead. These data show that the hairy roots of morning glory might be of interest for some phytoremediation strategies due to their high bioaccumulation abilities. The comparison of bioaccumulation potential of established hairy roots and young C. tricolor plants give grounds to suppose that roots of C. tricolor play an active role in the absorption of Cr6+, Ni2+, and Pb2+ from liquid media, whereas the aboveground part rather serves as a storage for the accumulated metal ions.