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which were then used as an inoculum (Ahlquist
et al.
, 1984; Ahlquist &
Janda, 1984; Allison
,
1986; Xiong & Lommel, 1991). Current methods involve inoculating plants
with cDNA expression constructs. The DNA plasmids can be applied directly
by mechanical inoculation or by agroinfiltration (reviewed in Annamalai
& Rao, 2006). Transcription and expression of the viral RNA lead to the
production of chimeric capsid proteins, which assemble into chimeric VNPs
and spread systemically. Many mutant VNPs have been generated and many
accumulate to titers as high as obtained for wild-type particles, as long as
the mutation does not hinder capsid assembly and/or RNA packaging.
et al.
, 1988; Dessens & Lomonossoff, 1993; Meshi
et al.
3.1.1.1 Agroiniltraion 
This method exploits the plant bacterium
Agrobacterium tumefaciens
,
which
causes crown-gall disease in plants.
can invade
wounded plant cells and transform the cells resulting in tumor growth.
The bacteria contain a tumor-inducing plasmid, the Ti-plasmid. During
infection, a segment of the Ti-plasmid is transferred into the plant cell; this
segment is referred to as transfer or T-DNA. The T-DNA is incorporated into
the plant genome by recombination, resulting in transient expression of the
T-DNA genes. The T-DNA is flanked by 25-bp direct repeats, termed the left
and right borders, that mediate the recombination event. Transfer of the T-
T-DNA is induced by activation of the so-called virulence (vir) genes on the Ti-
Ti-plasmid. Phenolic compounds, mainly acetosyringone, that are produced
and released from wounded plant cells initiate expression of the vir genes.
The T-DNA encodes for growth hormones, which stimulate tumor formation,
as well as for unnatural amino acids such as nopaline, mannopine, and
octopine, which serve as an energy source for the bacterium. The reader is
referred to the following textbooks for more information on the biology of
Agrobacterium
Agrobacterium tumefaciens
and its use as an expression system:
Molecular Biology of the
Cell
(Alberts
et al.
, 2008) and
Agrobacterium: From Biology to Biotechnology
(Tzvi & Vitaly, 2008).
In biotechnology, agrobacteria are exploited to transfer genes of interest
into plant genomes for transient expression (Annamalai & Rao, 2006; D'Aoust
et al.
, 2008; Sheludko, 2008). The T-
T-DNA genes are replaced with the genes of interest, such as the viral genome.
Vectors used are typically binary shuttle vectors that allow replication in
Escherichia coli
, 2009; Fischer
et al.
, 1999a,b; Lico
et al.
as well as in agrobacteria. This facilitates convenient and fast
cloning in
prior to transforming the plasmid into agrobacteria. For
inoculation of Agrobacterium (termed agroinfiltration), a suspension of the
bacteria is injected into the leaves using a syringe (Fig. 3.1). To facilitate
E. coli
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