rbcL P SG Т P TG Т accD





Homologous recombination


Chloroplast DNA








Fig. 3. A schematic diagram of plastid transformation. rbcL, gene of the large subunit of ribulose-bisphosphate-carboxylase/oxygenase;

accD, carboxytransferase β-subunit gene; P, promoter; T, terminator; SG, selective marker gene; TG, target gene.

ble. Nevertheless, this method has several serious

drawbacks: plastids do not maintain many posttranslational

modifications of proteins, such as glycosylation;

a number of heterologous proteins in plastids form

inclusion bodies that require repeated folding during

the purification process. Long-term investigations and

optimization of the technology for an individual

plant/construct is needed.

Transient Expression

Transient expression of foreign genes in plants is a

method based on temporary expression of a target

gene in specific plant organs [18]. At present, the following

main approaches are employed for transient

expression of genes in plants: Agrobacterium-mediated

transformation, expression with the use of plant viral

vectors [19], and magnifection.

Agrobacterium-mediated transient expression. As

mentioned above, the process of agrobacterial transformation

can be divided into four steps: attachment

of the bacterium to the plant cell wall, penetration of

T-DNA into the plant cell, integration of T-DNA into

the plant genome, and expression of genes from the

T-DNA region (Fig. 1). In contrast to stable transformation,

no integration of the transgene into the plant

genome occurs in this case, and its expression takes

place directly from the intact DNA of the agrobacterium

through the system of plant cell replication.

The proposed molecular mechanism of transient

expression of genes in plants is described in detail in

review [19]. It was shown, in particular, that after excision,

the T-DNA region exists in the bacterial cell as a

complex of single-stranded DNA (ssT-DNA) with the

bacterial proteins (T complex). The bacterial proteins

not only stabilize the structure of the T complex, they

are also involved in its transport to the host plant cell.

For double-stranded T-DNA to be formed, ssT-DNA

is released from the proteins of the mature T complex.

This process is assumed to involve both the agrobacterial

and plant proteins.

Transient expression with the use of viruses is based

on the knowledge of viral genomes. Viruses, including

plant viruses, have similar genome structures and carry

three main sequences: a sequence encoding the replicase

protein (involved in replication of viruses in the

host organism); a sequence encoding the transport

protein (involved in the spread of the virus in the host

organism), and a sequence encoding the viral envelope

protein (involved in the formation of viral particles).

Figure 4 presents a diagram showing the application of

viruses for transient expression of target genes and

sequences. In most cases, researchers use the following

approaches: substitution of a target sequence for

either the gene encoding the transport protein or the

gene encoding the viral envelope protein; insertion of

a target sequence between the sequences of the viral

genome; insertion of an epitope (an antigen used to

produce vaccines) as a part of the viral envelope protein

(Fig. 4). In the last case, the epitope will be delivered

on the surface of a viral particle.

Magnifection (Agrobacterium-mediated transfection)

is based on a combination of two methods of

transient expression (Icon Genetics). The process

includes vacuum infiltration of whole adult organisms

with dilute suspensions of agrobacteria carrying

T-DNAs encoding RNA replicons. In this case, the

agrobacteria mediate primary infection and movement

in cells, while the viral system mediates the

short-distance cell-to-cell movement, amplification,

and a high-level expression [20].

In general, transient expression of genes is characterized

by a number of advantages (table): a high efficiency

of gene expression and a high level of a target

protein; only several days are required after infiltration

with agrobacteria or viral particles to obtain a maximum

target protein yield (3–4 days for agrobacterial

transformation and 10 days for viral infection). Disadvantages

include the complexity of the agroinfiltration