Transient expression systems are a powerful tool for rapid protein production in plants. Transient expression differs from stable expression on a genetic level. Unlike stable expression where the introduced gene fully integrates into the plant genome and becomes an inheritable trait, transient expression only lasts a few days since the gene of interest exists as an entity extraneous to the plant genome. In transient expression, the gene of interest can be introduced into the plant cell either through the use of bacteria (Agrobacterium tumefaciens) and/or viruses. In agroinfiltration, multiple fragments of transfer DNA containing the gene of interest are introduced to plant cells using agrobacteria. Alternatively, the gene of interest may be delivered in the form of a virus which can take over the cell machinery to replicate the gene and drive the expression of the protein of interest. In both the cases, the gene itself does not integrate into the genome.
Transient systems offer several advantages over stable systems, including increased initial expression speed, very high likelihood of higher yield, reduced overall cost and greater flexibility. Unlike stable transformation, transient expression systems do not require an extensive screening and selection process. Furthermore, they can be used for automated protein expression, making them ideal for large-scale studies.
One of the most common types of transient expression is the use of viral vectors. These are engineered viruses that have been modified to carry genes of interest. When the virus is introduced to a host plant, the vector will replicate such that the foreign gene is expressed and the protein of interest is produced. This method is being used more commonly in molecular biology to deliver genetic material into cells. Viral vectors have many applications in medicine and agriculture and are promising alternative systems for production of pharmaceuticals. Most progress has been achieved using RNA viral vectors from species like Tobacco mosaic virus (TMV), Potato virus X (PVX), Alfalfa mosaic virus (AMV) and Cowpea mosaic virus (CPMV).
Optimization of transient expression systems is an important area of research, as it can greatly increase the yield of the desired protein. Some approaches that have been used to optimize these systems include modifying the promoter sequences used to drive gene expression, using different viral vectors, and manipulating environmental factors such as temperature and light intensity. Vector performance can be optimized by manipulating virus structure, incorporating nonviral and viral enhancer sequences, silencing suppressors, and reducing recombination sites. Several viruses from different families are used for vector design with different advantages and limitations.
Three broad categories are considered; first, second, and third generation viral vectors.
- First-generation viral vectors are typically derived from wild-type viruses and contain the gene of interest under the control of a strong promoter. These vectors are based on full virus genomes, with foreign genes inserted in the same open reading frame as the coat protein. They have limited cloning capacity and can only accommodate relatively small genes. Additionally, they often have safety concerns due to the potential for viral replication and pathogenicity.
- Second-generation viral vectors are safer and more efficient than first-generation vectors. These systems integrate elements of viral machinery with non-viral processes. They are often modified by deleting viral genes essential for replication, thereby rendering them replication-incompetent. These vectors allow for larger foreign gene sizes, higher production levels and broader host plant species options. The gene of interest is usually driven by a strong promoter, and the vector can accommodate larger genes than first-generation vectors. Examples of second-generation viral vectors include lentiviral vectors, adenoviral vectors, and retroviral vectors.
- Third-generation viral vectors have improved safety and efficiency compared to second-generation vectors. They are usually designed with additional modifications, such as removing immunogenic epitopes, further reducing the risk of the host immune response. They also have improved packaging efficiency and can accommodate larger genes than second-generation vectors. Examples of third-generation viral vectors include adeno-associated virus (AAV) vectors and lentiviral vectors with additional modifications.
Once the appropriate vector is identified and prepared, it can be deployed using a number of delivery methods such as mechanical inoculation and/or agroinfiltration. A variety of viral vector types, including RNA viruses, DNA viruses, and adeno-associated viruses, can be delivered with either of these two methods. Alternatively, when the gene of interest is incorporated into transfer DNA within agrobacteria instead of as part of a virus, agroinfiltration is used. The two methods are described below.
Mechanical inoculation involves introducing a virus-containing solution or suspension directly onto plant parts such as leaves or stems. Typically, a rough material saturated in the prepared viral vector is rubbed onto the leaves of a mature plant, creating small tears. The viral vectors enter the plant through these tears and infect plant cells. Another form of mechanical inoculation involves using a gene gun loaded with the viral vector that has been coated in microscopic particles that will carry the genetic material when the gun is fired. When fired, the particles are launched with high enough speed to penetrate cell walls and directly enter the plant cells, where the gene can be expressed to produce the protein of interest.
Agroinfiltration involves introducing a gene of interest into Agrobacterium tumefaciens, a bacterium that naturally infects plants. The gene of interest may be inserted directly into the transfer DNA of agrobacteria during the process of transformation, such that when the bacteria infect the plant cell, multiple copies of the gene of interest are introduced. Viral vectors may also be introduced to agrobacteria as a delivery method. In this case, there is an increased production of gene copies and protein expression when you combine the natural ability of agrobacteria to infect a high percentage of cells with the nature of viruses to produce multiple copies of its viral genome, incorporating the gene of interest within each infected cell. It combines the benefits of a virus (speed and high expression levels), Agrobacterium (transfection efficiency and systemic delivery) and plants (diverse synthetic capabilities and low production cost).
In conclusion, transient expression systems can be a powerful tool for rapid protein production in plants. While first-generation viral vectors may have limited cloning capacity and safety concerns, second and third-generation vectors have improved efficiency, safety, and larger cloning capacity. Additionally, researchers are working to optimize these systems for even greater efficiency and yield. For example, transient expression systems have been tailored to achieve human-like glycosylation modifications on plant expressed proteins. Further, the relatively short production time and scale-up capability of transient expression makes it a suitable system for vaccine production, such as the treatment for Ebola. As further research into this field is conducted, it is likely that transient expression systems will become increasingly beneficial in the area of plant protein production.
