Innovation in life sciences

Evaluation and Licensing Opportunities For further information on this technology and evaluation / licensing opportunities please contact: Dr Lars von Borcke lars@pbltechnology.com Tel: +44 (0)1603 456500 Fax: +44 (0)1603 456552 Tech ID: 99.194 Patent Literature European Patent EP1232274. US Patent 7,217,854. International Patent Publication No. WO 01/038512.

High Efficiency Transient Expression System for Plants

Methods to highly express proteins in plants in days not months

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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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.
Relative Expression Levels (measured at 5 dpi) 1 >2 25 50 ( ~300 g/g fresh weight) Days post inoculation (dpi) when expression was detectable n/a 2 - <7 2 - >12

Experiment Stable 35S-GFP transgenic lines Transient expression: 35S-GFP Transient expression: 35S-GFP plus p19 Suppressor

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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 Transient expression: 35S-Kinase Transient expression: 35S-Kinase plus p19 Relative Maximum Expression Levels 1 10 50* Days post inoculation (dpi) when expression was detectable 1.5 - <3 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.

pGFP pBIN19

pGFP p19
relative absorbance

200

compound X

1.6fold

p19

100

b
200

nmol/mg fresh weight

pBIN19
800 700 600 500 400 300 200 100 0
100

elution time

pGF P
pBIN19

pTom
p19

pTob

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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