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Innovation in life sciences
High Efficiency
Transient Expression
System for Plants
Evaluation and Licensing
Opportunities
For further information on this
technology and evaluation /
licensing opportunities please
contact:
Dr Lars von Borcke
[email protected]
Tel: +44 (0)1603 456500
Fax: +44 (0)1603 456552
Methods to highly express proteins
in plants in days not months
Tech ID: 99.194
Patent Literature
European Patent EP1232274.
US Patent 7,217,854.
International Patent Publication
No. WO 01/038512.
Efficient methods for expressing genes of interest in plants are required for many
applications. Existing methods require the use of either stable transgenic plants
(which are time and resource consuming) or inefficient transient expression
systems.
By combining Suppressors of gene silencing with Agrobacterium infiltration-mediated transient expression, Professor David
Baulcombe and colleagues at the Sainsbury Laboratory, Norwich, have developed a rapid and highly efficient method for
protein expression in plants.
The effectiveness of this Transient Expression system has been demonstrated using both plant- and animal-derived genes.
The system is expected to function in any plant or cell culture where Agrobacterium infiltration-mediated transient expression
can be utilised.
The Transient Expression system can be used to over-express single or multiple genes in the infiltrated leaf, opening up
opportunities for rapid pathway engineering in plants.
The Transient Expression technology is assigned to and patented by Plant Bioscience Limited.
Applications

Rapid and efficient testing of gene expression constructs in non-transgenic plants.

Rapid and high level protein expression for molecular farming in plants. >50-fold enhanced expression.

Rapid analysis of gene function via over-expression.

Rapid pathway engineering via expression of single or multiple genes in non-transgenic plants.
Background
Agrobacterium-mediated transient expression is a useful tool for assessing gene expression constructs in plants. It is rapid;
typically giving results 2 to 3 days after inoculation, and has been demonstrated to work in whole leaf tissue from a range of
plants including tobacco, potato and Arabidopsis1, as well as in a range of different cell culture systems. However, the
method is limited in its applications because expression levels are low and transient in nature (typically expression
disappears after less than 5 days from inoculation).
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Updated September 2012
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The PBL Transient Expression System
Work at the Sainsbury Laboratory has demonstrated that post-transcriptional gene silencing (PTGS) is the limiting factor in
Agrobacterium-mediated transient expression in tobacco.
By mixing Agrobacterium cultures prior to agro-infiltration, one carrying a standard binary expression vector for the gene of
interest and another carrying a standard binary expression vector for a Suppressor of gene silencing, extremely high level
expression of the gene of interest can be achieved. Expression also persists for much longer, up to 12 days post
inoculation and longer. Indeed the time period of expression may only be limited by senescence of the infiltrated plant tissue.
As well as applications where rapid analysis of gene expression constructs in plants is required, the system could easily be
scaled up to be a rapid and efficient protein production system.
Examples
(1)
Green Fluorescent Protein (GFP)
Expression of GFP under the control of the 35S promoter was compared in parallel experiments in Nicotiana benthamiana
with and without co-infiltration with a second Agrobacterium strain carrying the p19 Suppressor from tomato bushy stunt virus
(TBSV). In both Agrobacterium strains the genes were carried on pBIN19 binary vectors. In addition, two independent, stable
transgenic lines carrying a highly expressed single copy GFP transgene (driven by the 35S promoter) were used as a
reference. Expression levels were quantified using Western blots.
Experiment
Relative Expression Levels
(measured at 5 dpi)
Days post inoculation (dpi) when
expression was detectable
Stable 35S-GFP transgenic lines
1
n/a
Transient expression:
35S-GFP
>2
2 - <7
Transient expression: 35S-GFP plus
p19 Suppressor
25 – 50
( ~300 μg/g fresh weight)
2 - >12
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Updated September 2012
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GFP expression measured at 5 dpi by fluorescence and
Western blot, showing high-level expression in the presence
of p19 and another Suppressor Hc-Pro compared to transient
expression in the absence of GFP (left hand side panels) and
to a stable transgenic GFP line (right hand panel).
(2)
Plant-derived Protein Kinase
Similar experiments to those described above were carried out with a truncated form of a tobacco protein kinase gene (driven
by the 35S promoter). Nicotiana benthamiana leaves were infiltrated with the recombinant Agrobacterium suspension and
expression levels compared with co-infiltration with a second Agrobacterium suspension carrying 35S driven p19 Suppressor.
Expression of the protein kinase was detected using Western blots.
Experiment
Relative Maximum Expression
Levels
Days post inoculation (dpi) when
expression was detectable
Transient expression:
35S-Kinase
1
1.5 - <3
Transient expression:
35S-Kinase plus p19
10 – 50*
1.5 - >5
* precise quantification was impaired by the very high amounts of protein made in the presence of p19.
The high level expression of the plant kinase protein achieved with the p19 co-infiltration is particularly surprising given that
this is an unstable protein which is normally rapidly turned over in plants.
Western blot showing high levels of the plant kinase
protein accumulating over time in leaves co-infiltrated with
p19.
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Updated September 2012
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(3)
Secondary metabolite pathway engineering
An example of the Transient Expression Technology being used to engineer a secondary metabolite pathway through the
over-expression of one enzyme in the pathway. The high level increase in the end product, chlorogenic acid, was particularly
unexpected since the enzyme was not previously thought to be a rate-limiting step in the pathway.
a
c
pGFP
p19
relative absorbance
pGFP
pBIN19
200
compound X
1.6fold
p19
100
b
nmol/mg fresh weight
200
pBIN19
800
700
600
500
400
300
200
100
0
100
elution time
pGF P
pBIN19
pTom
pTob
p19
Panel a, N. benthamiana leaves were infiltrated simultaneously with Agrobacterium tumefaciens C58C1 transformed with the
plasmid pGFP, which contains the cDNA encoding GFP under the control of the 35S promoter, and bacteria transformed with
either the plasmid p19, resulting in the expression of a gene-silencing protein, or the control plasmid pBIN19. The expression
of GFP was monitored under UV light.
Panel b, Similar infiltration experiments were
containing the full-length cDNA of the gene from
4 days the infiltrated leaf material was collected
profile of plants expressing the tobacco enzyme
shown (Panel c).
carried out with Agrobacterium tumefaciens transformed with plasmids
tomato (pTom) and tobacco (pTob) in the presence of p19 or pBIN19. After
and the amount of chlorogenic acid measured by HPLC analysis. A HPLC
in the presence of the gene silencing inhibitor (p19) or without (pBIN19) is
References:
Voinnet et al (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the
p19 protein of tomato bushy stunt virus. Plant Journal 33, 949-956.
Voinnet et al (1999). Suppression of gene silencing: a general strategy used in diverse DNA and RNA viruses of plants.
PNAS, 96 (24), 14147-14152.
Plant Science 122 (1997) pp. 101-108; MPMI 13 (2000) pp. 439-446; PNAS 96 (1999) pp.11128-11133
TEC Release: October 2002
Updated September 2012
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