Ki-Beom Moon

The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B

Disease prevention through vaccination is considered to be the greatest contribution to public health over the past century. Every year more than 100 million children are vaccinated with the standard World Health Organization (WHO)-recommended vaccines including hepatitis B (HepB). HepB is the most serious type of liver infection caused by the hepatitis B virus (HBV), however, it can be prevented by currently available recombinant vaccine, which has an excellent record of safety and effectiveness. To date, recombinant vaccines are produced in many systems of bacteria, yeast, insect, and mammalian and plant cells. Among these platforms, the use of plant cells has received considerable attention in terms of intrinsic safety, scalability, and appropriate modification of target proteins. Research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over 30 diseases, most frequently HepB and influenza. More inspiring, approximately 12 plant-made antigens have already been tested in clinical trials, with successful outcomes. In this study, the latest information from the last 10 years on plant-derived antigens, especially hepatitis B surface antigen, approaches are reviewed and breakthroughs regarding the weak points are also discussed.

Transient erythropoietin overexpression with cucumber mosaic virus suppressor 2b in Nicotiana benthamiana

Erythropoietin (EPO) is a glycoprotein hormone that regulates red blood cell formation in mammals. It also plays an important role in wound healing and the brain’s response to neuronal injury. Recombinant EPO has been produced in various hosts including Escherichia coli, insect cells, and yeast. Plant systems are also useful for the production of recombinant proteins. In this study, we used Nicotiana benthamiana for the production of recombinant human EPO (rhEPO) using an Agrobacterium-mediated transient expression system. We evaluated the effect of repeated agroinfiltration and Agrobacterium density on the rhEPO production. In addition, the rhEPO expression vector was coinfiltrated with a vector expressing cucumber mosaic virus suppressor 2b (CMV2b), which suppresses posttranscriptional gene silencing. We found that rhEPO expression was increased approximately 5.5-fold in N. benthamiana leaves coinfiltrated with CMV2b. In contrast, neither Agrobacterium density nor the number of infiltrations influenced rhEPO expression.

Nicotiana benthamiana Matrix Metalloprotease 1 (NMMP1) gene confers disease resistance to Phytophthora infestans in tobacco and potato plants

We previously isolated Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) from tobacco leaves. The NMMP1 gene encodes a highly conserved, Zn-containing catalytic protease domain that functions as a factor in the plants defense against bacterial pathogens. Expression of NMMP1 was strongly induced during interactions between tobacco and one of its pathogens, Phytophthora infestans. To elucidate the role of the NMMP1 in defense of N. benthamiana against fungal pathogens, we performed gain-of-function and loss-of-function studies. NMMP1-overexpressing plants had stronger resistance responses against P. infestans infections than control plants, while silencing of NMMP1 resulted in greater susceptibility of the plants to the pathogen. This greater susceptibility correlated with fewer NMMP1 transcripts than the non-silenced control. We also examined cell death as a measure of disease. The amount of cell death induced by the necrosis-inducing P. infestans protein 1, PiNPP1, was dependent on NMMP1 in N. benthamiana. Potato plants overexpressing NMMP1 also had enhanced disease resistance against P. infestans. RT-PCR analysis of these transgenic potato plants revealed constitutive up-regulation of the potato defense gene NbPR5. NMMP1-overexpressing potato plants were taller and produced heavier tubers than control plants. We suggest a role for NMMP1in pathogen defense and development.

The laccase promoter of potato confers strong tuber-specific expression in transgenic plants

Tissue-specific promoters for efficient expression of transgenes at specific times or in specific plant tissues can be applied to develop new transgenic plants. To exploit a promoter capable of driving strong expression in potato tubers, we isolated the promoter region of the laccase gene from potato (Solanum tuberosum L. cv. Desiree) and characterized its activity in transgenic Solanaceae plants, such as potato, tobacco and tomato. The ability of the laccase promoter to induce the β-glucuronidase (GUS) reporter was evaluated in independent transgenic potato lines and compared with that of the constitutive CaMV35S promoter. To determine the tissue specificity of expression in transgenic potato, GUS levels in shoot tips, leaves, stems, roots and tubers were measured by histochemical analysis. The laccase promoter conferred tuber-specific expression in transgenic potato regardless of the developmental stage, and there was no GUS reporter expression in leaves, stems or roots. Serial 5′ deletion analysis of the laccase promoter revealed that the tuber-specific regulatory elements might be scattered throughout the promoter. The laccase promoter responded weakly to salt stress, mannitol stress, and mechanical wounding but not to cold stress in the leaves and stems of transgenic potato. In transgenic tobacco, weak GUS expression driven by the laccase promoter was detected throughout the entire plant, whereas in transgenic tomato, GUS expression was detected only in the roots and seeds. Our data show that the laccase promoter represents a feasible candidate to drive high and preferential expression of genes in potato tubers.

Development of rice(Oryza sativa L.) transformation system to improve callus utilization

Plant molecular farming has attracted a lot of attention lately in the field of mass production of industrially valuable materials by extending application of the plant as a kind of factory concept. Among them, protein expression system using rice(Oryza sativa L.) callus is a technology capable of mass culture and industrialization because of a high expression rate of a target protein. This study was carried out to develop an Agrobacterium-mediated transformation system to increase the utilization of rice callus. The transformation efficiency was improved by using the hand when seeds were de-husked for callus induction. Furthermore, we were possible induction of callus from 6 years old seed smoothly. Selection of the callus contained the target gene was required a cultivation period of at least 3 weeks, and the most efficient selection period was after 6 weeks of culture including one passage. This selection was confirmed that the gene was stably inserted into the genomic DNA of the plant cell by the southern blot analysis and progeny test. Such an efficient selection system of rice callus that can be cultured in the long term will be contribute to the industrialization of useful recombinant proteins using rice.