WO2011149429A1

WO2011149429A1 - Use of glycosidases and glycosyltransferases for enhanced protein production

Info

Publication number: WO2011149429A1
Application number: PCT/SI2011/000024
Authority: WO
Inventors: David Dobnik; Špela BAEBLER; Jana ŽEL; Kristina Gruden; Dejan ŠTEBIH
Original assignee: Nacionalni Inštitut Za Biologijo
Priority date: 2010-05-24
Filing date: 2011-05-18
Publication date: 2011-12-01
Also published as: SI23374A

Abstract

Method for enhanced production of recombinant proteins in plants or plant cells solves a problem of complex protein production with fast, reliable and economically viable solution. Preferably in N. tabacum plants it is introduced at least one polynucleotide comprising a sequence coding a protein to be produced, and at least one polynucleotide comprising a sequence for at least one further modulating protein or polyribonucleotide which increases the cell-cell permeability of the target cells, preferably from group of glycosidases, or it is introduced at least one polynucleotide which comprises the sequence for protein of interest and a sequence increasing the cell-cell permeability of the target cells, preferably from group of glycosidases. Polynucleotide sequences preferably originate from plant viruses, preferably TMV and/or PVX.

Description

Use of glycosidases and glycosy!transferases for enhanced protein production

The present invention relates to a method for production of recombinant proteins in plants or plant cells.

The method of the invention enables enhanced production of recombinant proteins in plants or plant cells and solves a problem of low efficiencies of so far existing methods for plant protein production. Production system of the invention is a plant, in which it is introduced together with the sequence of protein of interest also the sequence for enzyme of the glycosidase group or the sequence for inhibitor of enzymes of glycosyltransferase group, what will facilitate and speed up the spreading of the sequence for protein of interest throughout the plant. Result of the use of this method is more protein of interest in shorter time.

Proteins, e.g. therapeutic proteins like antibodies, vaccines, enzymes, antibiotics, hormones, cytokines, interferons etc. are needed in industrial amounts. Traditional methods to produce recombinant proteins are microbial fermentation and animal cell cultures. The increasing world-wide demand for recombinant proteins cannot be met by traditional production methods alone. Therefore, it has been searched for further options, one of which is plantal protein production.

Since the late 1970's attempts have been made to efficiently produce protein in plants, but those attempts have been hampered by the long time needed to create plants with a stable transformation with the genetic code of the protein to be produced and the low yield obtained by both stable and transient transformation. Further obstacles were bio-safety concerns when cultivating the transformed plants in the open field and the scale-up to make the methods commercially viable. Meanwhile, a lot of progress has been made. Routinely used is transient expression of heterologous genes delivered to the plant by a bacterium or a virus. Plants transformed by Agrobacterium express the protein after a short time; however, they are usually characterized by low expression. Plants transformed by viral vectors need a longer time to express the protein after transformation, but can spread within the plant by using the virus's abilities for cell-to-cell movement. Deom et al (1992) found that the viral spread from cell to cell was occurring in the plasmodesmata and was mediated by viral movement proteins.

By now, large-scale production methods of recombinant proteins in plantal cells are known in the art but they are not competitive for many proteins compared to traditional methods. In the last decades, it has been attempted to increase the efficiency of plantal protein production.

In 1999, US5939541 disclosed a method for enhancing the expression of genes in plants by supplying a virally encoded booster sequence. In 2000, US6011198 disclosed a method for enhancing protein expression in plastids of photosynthetic organisms by increasing the light harvesting capability. In 2004, US2004/0214318 disclosed a method for enhancing protein expression in plants by using non- native 5' untranslated enhancer sequences. In 2006, US7087811 disclosed a method for enhancing protein production by using fusion products of the protein of interest with ubiquitin. In principle, these methods try to enhance the expression of the protein of interest on the individual cellular level.

To be efficient, however, the expression of the protein should not only be high on individual cellular level, but across the whole plant or plantal tissue. This can be realized when the polynucleotide sequence coding the protein to be produced is present across the whole plant or plantal tissue.

One attempt to provide a distribution of the vector across the whole plant or plantal tissue is to introduce the polynucleotide sequence across the whole plant or plantal tissue.

Such an approach has been described by Icon Genetics and by a method that is called Magnifection (WO2007/137788, WO2006/079546, S. Marillonnet et al., Nature Biotechnol. 2005, Gleba et al., Vaccine. 2005). It requires the simultaneous introduction of Agrobacterium to many leaves of the plant.

If the disadvantages of plantal protein production can be overcome, it would be an alternative to existing methods like microbial fermentation and animal cell cultures, and could result in a more cost-efficient method of protein production as it is using natural, renewable resources and is not limited to special manufacturing facilities. Production in plants can be less expensive because fewer resources like culture media and bioreactors are needed. Plants can be very efficient protein producers and can do so without propagation of human pathogens or other mammalian contaminants which cuts down costs related to security precautions concerning biohazard. Plants have a better ability to assimilate genetic information and produce complex recombinant proteins compared to other existing production methods. Furthermore, plant cells produce glycosylation patterns more closely resembling human proteins. This allows skipping of additional post- translational modification steps as are needed in other methods.

The current approaches are still not satisfying, and further improvements, especially concerning industrial-scale applications are necessary to increase efficiency and profit.

It was the object of the present invention to provide a method with improved expression of protein in plant cells and to provide a fast, reliable and commercially viable way to produce complex proteins.

Furthermore, it was the object of the present invention to efficiently transform parts of a plant or plantal tissue and to provide means for efficient spread within the plant and for efficient expression of the protein of interest throughout the plant.

The present invention provides a method of producing one or more proteins of interest in plants or plantal tissue that leads to high yield of protein and that is easily scalable to large-scale production. The present invention also provides a method for producing transgenic plants which have the polynucleotide sequence coding the protein of interest stably integrated into the plantal genome and can be used for commercially viable production of protein. The present invention also provides a method for producing transgenic plants which have the polynucleotide sequence coding the modulating sequence stably integrated into the plantal genome and can be used for commercially viable production of protein of interest which has been transiently introduced into the plant.

The present invention comprises a method for producing at least one protein in plantal cells comprising the step of

a) introducing into a cell at least one polynucleotide comprising at least one sequence i) coding a protein to be produced, and at least one polynucleotide which comprises coding sequence ii) for at least one further protein or polyribonucleotide which increases the cell-cell permeability of the target cells or

b) introducing into a cell at least one polynucleotide which comprises both polynucleotide i) and polynucleotide ii).

Surprisingly, it was found that by using enzymes which increase the cell-cell-permeability or by inhibiting enzymes which decrease cell-cell-permeability, polynucleotide sequences coding protein(s) to be produced are spread faster throughout the plant, which results in the desired increase in protein production and yield. Thus, by using a known method for producing protein in plantal cells that includes the step of claim 1 of the present invention, the protein production can be increased. In the context of the present invention the following expressions have the following definitions: The expressions "plant" and "plantal tissue" are used interchangeably. The term "plant" shall also comprise plantal tissue or parts of a plant.

"Heterologous" in the context of the present invention refers to any genetic material that is introduced into the plant cell.

"Transgenic plant" is a plant that carries the introduced heterologous polynucleotides stably integrated in its genome.

"To be derived from" regarding the origin of a RNA replicon in the context of the present invention means that parts of the original polynucleotide sequence from the virus are used, other parts can be deleted or modified. This includes "full virus" vector strategy, where only the sequence of the protein is added to the viral replicon, and "deconstructed virus" vector strategy, where the vector genome is custom tailored to only include the parts beneficial or necessary for the application. Persons skilled in the art can select the suitable strategy.

Polynucleotide sequences ii) which can increase the cell-cell permeability will in the context of this invention also be referred to as "modulators" or "modulating sequences". In one embodiment of the present invention, these modulating sequences can be stably integrated into the plant genome.

The step as defined in claim 1 of the present invention can be used in any method of plantal protein production.

An important part of the present invention is the increase of the cell-cell permeability, which facilitates the spread of the heterologous sequences within the plant tissue and results in better expression and yield of the protein of interest.

Cell-cell contact in plant cells occurs in the plasmodesmata. Plants of the Archaeplastida group, which includes the land plants and green algae, have plasmodesmata. Plasmodesmata show a dynamic nature and can be opened or closed.

The plasmodesmatal permeability is increased by enzymatic degradation of polysaccharides present in the plasmodesmatum. The plasmodesmatal permeability is decreased by synthesis of new polysaccharide which is then deposited in the plasmodesmatum.

These polysaccharides act as gate keeper and the amount of the polysaccharide present at the plasmodesmata regulates the size exclusion limit, which is the size of the largest molecule that can move from cell to cell. Increasing the polysaccharide deposit is a natural defence mechanism to fight a viral infection and limit its spread within the plant.

The plasmodesmatal permeability is increased by enzymatic degradation of the polysaccharide. Another mechanism to increase the cell-cell permeability is to inhibit enzymes which synthesize the polysaccharide, which results in enhanced plasmodesmatal trafficking and therefore enhanced spread of the introduced material.

The polysaccharides belong to the class of glucans, and are usually β-glucans, particularly β-1,3- glucans. It has been shown by Levy et al (2007) that the amount of one specific p-l,3-glucan, callose, in the plasmodesmata is correlated with the cell-cell permeability. Callose is a substrate for β-1,3- glucanases and controls plasmodesmatal trafficking. So far enhanced symptoms of viral infection within tobacco have been described by Bucher et al (The Plant Journal, 2001) and have been linked to virus-induced increase of cell-cell permeability by enzymatic degradation of callose.

According to the present invention this mechanism is used to influence the cell-cell permeability, and thus, the spread of polynucleotides within the plant by introducing into the plant a polynucleotide which comprises a coding sequence for a modulator. As either the polysaccharides shall be degraded or the polysaccharide synthesis shall be inhibited the modulator can be either a substance that increases the enzymatic degradation or inhibits the synthesis. Some substances that are particularly useful for this purpose are enzymes that are active in this regard.

The polysaccharides that are influenced by the enzymes are those polysaccharides that are normally present in the plasmodesmata, i.e. glucans, particularly p-l,3-glucans. Enzymes that have an influence on the polysaccharides that increase the enzymatic degradation or inhibit the synthesis are well known in the art. They have been disclosed and their sequences are published. As examples for degrading enzymes p-l,3-glucanases can be cited belonging to the class of E.C.3.2.1.39 of the enzyme nomenclature. This type of enzyme has been found inter alia i n Arabidopsis thaliana. The function of the p-l,3-glucanases is the hydrolysis of the p-l,3-D-glucosidic linkage in p-l,3-D-glucanes. As these enzymes degrade the polysaccharide in the plasmodesmatum, at the same time they increase the cell-cell permeability.

Therefore in one embodiment of the present invention enzymes are used to degrade polysaccharides, like P-l,3-glucans.

In a preferred embodiment of the present method the modulating polynucleotide sequence ii) comprises the coding sequence for p-l,3-glucanase.

The modulating polynucleotide sequence ii) is introduced into the plantal cell. The transformation can be either transient or can result in stable integration. For example, it is possible to create transgenic plants where the polynucleotide sequence coding the modulating sequence is stably integrated into the plant genome. In a preferred embodiment a polynucleotide sequence comprising the coding sequence for -l,3-glucanase is stably integrated into the plant.

Furthermore, for increasing the cell-cell permeability it is possible to inhibit the synthesis of the polysaccharides, particularly by inhibiting enzymes that are active in the synthesis of these polysaccharides. Enzymes having this activity are known in the art, for example p-l,3-D-glucan- synthases (EC.2.4.1.34). Those enzymes have been disclosed in the prior art and their sequences have been published.

Therefore, in one embodiment of the present invention, enzymes which synthesize the polysaccharides are inhibited to increase cell-cell permeability.

Methods or substances used to inhibit these enzymes are known in the art. For example, the enzymes can be inhibited on DNA level by silencing with microRNA, or on mRNA level by siRNA or shRNA, or on protein level by using antibodies.

This inhibition can be done via inhibiting substances such as small interfering RNA (siRNA), small hairpin RNA (shRNA), dsRNA, MicroRNA, Spiegelmere, which are ..-oligonucleotides, DNA-, RNA- or peptide aptamere, ribozymes, abzymes, which are catalytic antibodies or enzymes.

The term "inhibition" when used in connection with the synthesizing enzymes means that the activity of these enzymes is down-regulated by at least 30%, preferably 50%. The activity of the enzyme can be determined by methods well known to the skilled person. By selecting the percentage of inhibition the spreading can be influenced accordingly. The two approaches for influencing the cell-cell permeability can be used separately or in a combined manner. Moreover, more than one type of hydrolyzing enzyme of the group 3.2.1 and/or more than one inhibiting substance for enzymes of the group EC.2.4.1 can be used. Hydrolyzing enzymes and inhibiting substances for the synthesizing enzymes can be used separately, consecutively or simultaneously. In other words, the action of one enzyme can be influenced at the same time as the activity of the other enzyme, the activity of one enzyme can be first influenced and thereafter the activity of the other enzyme can be influenced or the activity of both types of enzymes can be influenced at different times and/or sites.

"Modulating the activity of the enzyme" shall refer to the increase of the hydrolyzing enzyme or the inhibition of the synthesizing enzyme. Increase of the hydrolyzing enzyme comprises also that this enzyme is introduced in a plant where it was not present before.

Plants used for the protein production can be any plants that have plasmodesmata with polysaccharides controlling the cell-cell permeability. Plants that have plasmodesmata with callose deposits e.g. are from the group of Archaeplastida, which comprises all land plants and green algae. Plants can be monocots or eudicots, preferably from the plant families Solanaceae, Poaceae, Fabaceae, Brassicaceae, Musaceae, Cucurbitaceae, Apiaceae, Rosaceae, and Salicaceae.

Most preferably, plants of the genus Solanum and Nicotiana are used, e.g. N. tabacum, N. benthamiana and Solanum tuberosum, because these plants are among the best researched and most used plants for plantal protein production.

Surprisingly, it was found that by weakening the natural defence mechanism of plants to viral infection by targeted and controlled increase of the cell-cell permeability by lowering the amount of callose present at plasmodesmata, the protein production can be increased.

It is the gist of the present invention that polynucleotide sequences i) and ii) are introduced in plant cells to allow spreading of polynucleotide sequence i). The polynucleotide sequences i) and ii) can be present on one nucleotide sequence or can be on separate nucleotide sequences. The introduction of both sequences can be either combined or done separately. Thus, in one embodiment polynucleotide sequence ii) is first introduced into a plant to be stably integrated into plantal genome. Afterwards the polynucleotide sequence i) is introduced, its spread within the plant facilitated by polynucleotide sequence ii) and then expressed to produce the protein of interest. In another embodiment both sequences i) and ii) are introduced into the plant cell at the same time. The heterologous nucleotide sequence or sequences are typically DNA and comprise a nucleotide sequence coding a RNA replicon.

The source of said RNA replicon is not critical to the present invention. It can be any replicon suitable for plantal protein production. A person skilled in the art knows how to select the suitable replicon. For example, the replicon can be derived from a group of viruses able to infect plants as listed in the VIDE (Virus Identification Data Exchange), published in "Viruses of Plants" by A A Brunt, Horticulture Research International, Wellesbourne, UK.

These viruses can be derived from any group of viruses, dsRNA viruses, ssRNA viruses, negative ssRNA viruses, ssDNA, dsDNA. Examples for virus replicon origins are Tobacco Mosaic Virus (TMV), Potato Virus X (PVX), Cowpea Mosaic Virus (CPMV), Plum Pox Virus (PPV), Alstroemeria Mosaic Virus (AIMV), Tomato Bushy Stunt Virus (TBSV), Cucumber Mosaic Virus (CMV), Zucchini Yellow Mosaic Virus (ZYMV), Potato Virus A(PVA) and Turnip Mosaic Virus (TuMV). Acronym standard can be found in "A list of proposed standard acronyms for plant viruses and viroids", Archives of Virology, 1991. In the method of the present invention at least one sequence i) is introduced which codes at least one protein of interest. The protein of interest can be any protein, for example proteins that are useful for therapeutic use, such as therapeutic proteins like antibodies, vaccines, enzymes, antibiotics, hormones, cytokines and interferons.

The present invention provides several options for producing proteins in a plant. It is possible to introduce the sequence of only one protein or sequences for more than one protein to be produced. Moreover, the polynucleotide can comprise more than one copy of sequence i).

One example for a protein to be produced is an antibody where the sequence of the light and the heavy chain can be on the same polynucleotide or can be on separate polynucleotides.

Furthermore, as outlined above, only one modulator can be used or two or more modulators can be used which can have the same activity or different activities. If more than one modulator is used, the sequences coding for the different modulators can be on the same polynucleotide or can be on separate polynucleotides. Using more than one modulator can be useful to optimally adapt the cell- cell permeability. This adaptation can also be obtained by regulating or controlling the expression of the sequences. Methods for induction or control of expression of sequences are well known to the skilled person.

In a preferred embodiment sequence ii) encodes the enzyme p-l,3-glucanase. This sequence can be on the same heterologous nucleotide sequence as the sequence encoding for the protein(s) to be produced. It can also be on a separate heterologous nucleotide sequence.

In addition to p-l,3-glucanase encoding nucleotide sequences one or more further modulating sequences or sequences encoding modulating substances respectively can be used.

Any combination of polynucleotide sequences i) and ii) can be used, i.e. any combination of numbers and types of sequences. Particularly preferred is the combination of a sequence encoding β-1,3- glucanase with a sequence encoding a protein of interest.

In a preferred embodiment the polynucleotide sequence i) comprises the sequence of one protein to be produced. The method of the present invention can be used to introduce sequences that encode one protein of interest or more, or to introduce different sequences encoding different proteins.

Thus, the polynucleotide sequence i) can also comprise sequences of at least two proteins to be produced, for example the light and heavy chain of an antibody.

In one embodiment at least two separate polynucleotide sequences i) that each comprise sequences of at least one or of two or more proteins to be produced, can be introduced.

-l,3-glucanase can be on the same heterologous nucleotide sequence as the at least one polynucleotide sequence i) comprising the sequence(s) for the protein(s) of interest.

-l,3-glucanase can be the only modulating sequence or one of several modulating sequences. The modulating sequences can be on one heterologous nucleotide sequence or on at least two different heterologous nucleotide sequences.

Any combination of polynucleotide sequences i) and ii), including p-l,3-glucanase, concerning the quantity and quality is possible.

Preferably, the combination used is the sequence for the protein of interest in combination with the sequence for p-l,3-glucanase on two different heterologous nucleotide sequences, preferably non- competing, synergistic vectors, preferably controlled by inducible promoters.

The polynucleotide sequences i) and/or ii) can be part of one large heterologous sequence or from different viral replicons.

In one embodiment the polynucleotide sequences i) and ii) are part of one large heterologous sequence which is derived from the same viral replicon.

In one embodiment the polynucleotide sequences i) and ii) are from separate large heterologous sequences, which are derived from different viral replicons which can be synergistic or non- competing replicons, such as TMV and PVX.

Any polynucleotide of the present invention comprises also at least a promoter. The promoter used is not critical. It can be any promoter suitable for the expression in a plant and suitable for the expression of the sequence to be expressed. The selection of the optimal promoter is routine for a person skilled in the art and methods for selecting suitable promoters are also well known. Examples of suitable promoters are p35s and Act2.

Usually upstream of any sequence a promoter is present. In the method of the present invention the same promoter can be used for any polynucleotide sequence or different promoters can be used for different polynucleotide sequences. Thus, it is possible to use different modulating sequences with the same promoter or to use different promoters for each modulating sequence. This allows controlling the level of expression.

For example, the expression of the protein to be produced can be under the control of a stronger promoter than the promoter controlling the expression of the modulating sequences. The promoter can be a constitutive promoter or it can be an inducible promoter. The type of promoter can be the same for all polynucleotides or it can be different. Thus, in one embodiment the promoter for the modulating sequence can be inducible whereas the promoter for the protein to be produced can be constitutive, or both can be inducible. It can be useful if the promoter is different for multi-step induction.

The promoter can be a chemically-regulated promoter, including promoters whose transcriptional activity is regulated by the presence or absence of alcohol, tetracycline, steroids, metal and other compounds or physically-regulated promoters, including promoters whose transcriptional activity is regulated by the presence or by the absence of light and low or high temperatures.

In one embodiment sequences i) and ii) are on different vectors downstream of two different inducible promoters. This set-up allows a controlled expression of the sequences which facilitate viral spread from cell to cell in a distribution step, followed by a switch-off step, which is followed by an induction of the sequence i) in the production step.

The polysaccharide amount can be modified by two different ways, separately, consecutively or simultaneously. One way is to lower the amount of already present callose by using hydrolyzing enzymes of the EC 3.2.1 group, the other way is to inhibit the enzymes (EC 2.4.1.) which synthesize new callose. Thus it is possible to use both ways by using inhibition of synthesizing enzymes and by using degrading enzymes at the same time.

The heterologous polynucleotide sequences i) and ii) of the present invention can be part of a viral expression system and can comprise one or several vectors. The one or several polynucleotide sequences i) can be on the same vector or on separate vectors. The one or several polynucleotide sequences ii) can be on the same vector or on separate vectors. The one or several polynucleotide sequences i) and ii) can be on the same vector.

In one embodiment of the present invention the polynucleotide sequences i) and ii) are on two different vectors, preferably non-competing synergistic viral vectors, such as TMV and PVX.

In one embodiment of the present invention the polynucleotide sequence ii) is prepared by using pMDC32 as a vector backbone. Preferably, polynucleotide sequence ii) comprises pMDC32 as a vector backbone and the sequence coding p-l,3-glucanase. The vector pMDC32 has been published by Curtis in 2003 on Plant Physiol. 2003 Oct;133(2):462-9. One variation of this vector is published on GenBank under Accession number FJ172534.

In the method of the present invention, the polynucleotides are introduced into plant cells.

Methods to introduce large heterologous nucleotide sequences into plant cells are known in the art. A person skilled in the art can select a method adapted for the plant-vector combination in question. Such methods can be chemical methods, such as transfection with calcium phosphate, cyclodextrin, liposomes (Lipofection) and transformation of plastids with polyethylene glycol (PEG), or direct non- chemical methods such as biolistic bombardment with a particle gun, electroporation, magnetofection and sonoporation, or transduction by viral methods.

All of these methods and combinations thereof can be used.

One method is ^grobacfer/um-mediated plant transformation. The bacteria carrying the viral expression system can be from a group comprising Agrobacterium tumefaciens, A. rhizogenes, Sinorhizobium meliloti, Rhizobium sp. and Mesorhizobium loti, without being limited to this group. Moreover, heterologous sequences can be introduced into the piantal cells by high-pressure spray of the bacterium suspension after preparing the piantal tissue for transfection with a mechanical abrasive, such as silicon carbide (carborundum), which enhances the transfection efficiency by wounding the plant tissue.

Moreover, heterologous sequences can be introduced into the piantal cells by high-pressure spray of the bacterium suspension together with mechanical abrasive, such as silicon carbide (carborundum), which enhances the transfection efficiency by wounding the plant tissue.

Furthermore, heterologous sequences can be introduced into the plant by Magnifection.

Although the introduction can be done across the whole plant, it is the advantage of the present method that introduction of the heterologous sequence(s) can occur on one or few entry points and nevertheless the sequences are spread across the whole plant by the expression of polynucleotide sequence(s) ii).

The introduction can be stable and lead to transgenic plants or it can be transient.

In one embodiment of the present invention the modulating sequences is stably integrated in to the piantal genome and the sequence coding the protein of interest is introduced transiently.

The piantal cells can be part of a whole plant or part of piantal cell tissue.

The polynucleotide sequence(s) i) can code any protein of interest, such as therapeutic proteins like antibodies, vaccines, enzymes, antibiotics, hormones, cytokines and interferons.

The proteins can be modified to facilitate downstream processing by being marked with a tag, or localisation signals, for example a His-tag or a localization signal which marks the protein to be excreted via guttation.

The polynucleotide sequence(s) ii) can be any polynucleotide which can enhance the cell-cell permeability after expression or translation.

A modulating sequence which increases cell-cell permeability is defined as a sequence which upon expression increases the viral spread by at least about 5%, preferably 10% to 200%, when compared to the viral spreading without the modulating sequences.

Methods for testing the spread of a sequence within piantal tissue are known in the art. One approach is introducing into a plant a sequence i) coding a reporter gene like Green Fluorescent Protein (GFP) and a candidate modulating sequence ii); as a c ontrol, seque nee i) is introduced without the candidate modulating sequence. The increase of spread can then be measured by comparing the reporter signals, for example fluorescence if GFP is the reporter molecule. Spread of the reporter protein can be measured, in the case of GFP as fluorescence across the plant.

Another method for testing the viral spread is to inoculate the lower leafs of a plant with a sequence i) coding a protein of interest and a candidate modulating sequence ii); as a control, sequence i) is introduced without the candidate modulating sequence. After a time interval the expression of the protein of interest is measured in the un-inoculated upper leaves and the protein amount is compared. Alternatively, to control the spreading of the vector, the quantity of mRNA of a reporter gene can be measured by real time PCR.

Furthermore, the present invention provides a vector system that can be used in the method as described above. The vector system can either comprises at least one vector comprising a promoter, optionally an inducible promoter, and at least one polynucleotide comprising sequences i) and ii) as defined in claim 1; or it can comprise a combination of two vectors, wherein one vector comprises a promoter, optionally an inducible promoter, and at least sequence i) as defined in claim 1, and wherein a second vector comprises a promoter, optionally an inducible promoter, and at least sequence ii) as defined in claim 1.

A bacterium containing the above mentioned vector system and a plant containing the said bacterium are also part of the present invention.

Description of drawings

Fifi.l illustrates the vector of pENTR_glulll.

Fig.2 illustrates the vector of pMDC85_glulll.

Fig.3 illustrates the vector of pMDC32_glulll.

Examples

Example 1:

The gene for p-l,3-glucanase was amplified from Solarium tuberosum cv. Igor cDNA by PCR with primers glucBF (SEQ ID NO: 1) and glucl3R (SEQ ID NO: 2) resulting in glulll (SEQ ID NO: 3) and cloned into pENTR D-TOPO plasmid, using pENTR™ Directional TOPO^® Cloning Kit (Invitrogen, USA), resulting in pENTR_glulll plasmid (Fig.l).

The gene then was cloned into pMDC85 plasmid (Curtis and Grossniklaus, 2003, in Plant Physiol. 2003 Oct;133(2):462-9.) using Gateway^® LR Clonase™ II Enzyme Mix (Invitrogen, USA), resulting in pMDC85_glulll (Fig.2). This plasmid was electroporated into ElectroMAX™ Agrobacterium tumefaciens LBA4404 cells (Invitrogen, USA) using an Eppendorf 2510 electroporator.

Transformation of potato (Solanum tuberosum cv. Desiree) was carried out according to Visser et al. 1989 (Plant Molecular Biology 12, No. 3:329-337) with Agrobacterium tumefaciens LBA4404 cells harbouring pMDC85_glulll. Three transgenic lines (positive for pMDC85_glulll construct), Gl, A6 and A2, with different level of glulll expression were selected for further experiments.

Transgenic lines and control (wild-type) plants were transferred from tissue cultures into the soil and grown in growth chambers at 21 ± 2 °C in the light and 18 ± 1 °C in the dark, at a relative humidity of 75% ± 2%, with 70-90 μη"ΐοΙ/ητι2/52 radiation (L36W/77 lamp, Osram, Germany) and a 16h photoperiod. After four weeks in the soil, the three lower leaves of each transgenic and control plant were inoculated with potato virus Y^NTN (PVY). The lower inoculated and upper non-inoculated leaves were collected after 7 days. Total RNA was isolated from the samples and converted to cDNA. Relative amounts of RNA coding PVY's coat protein (CP) were determined in the leaf samples using quantitative real-time PCR with NTN F primer, NTN R primer and NTN probe (Kogovsek et al. 2008, Journal of Virological Methods, 149:1-11). For normalization cox gene was used with primers COX-F, COX-R and probe COX-P (Weller et al., 2000, Applied and Environmental Microbiology, 66(7):2853- 2858). Since the amount of inoculum could not be completely equalized, the amount of PVY CP in lower leaves was normalized to the value of 1000 units for all samples in the calculations. The amount of CP in control plants in upper non-inoculated leaves was 1 unit, whereas the amount of CP in all transgenic lines was on average 130 units.

Example 2:

The glulll gene (SEQ ID NO: 3) in pENTR_glulll plasmid (Fig.l) was cloned into pMDC32 plasmid (Curtis and Grossniklaus, 2003, in Plant Physiol. 2003 Oct;133(2):462-9.) using Gateway^® LR Clonase™ II Enzyme Mix (Invitrogen, USA), resulting in pMDC32_glulll (Fig.3). This plasmid was electroporated into ElectroMAX™ Agrobacterium tumefaciens LBA4404 cells (Invitrogen, USA) using an Eppendorf 2510 electroporator.

Transformation of potato (Solarium tuberosum cv. Desiree) was carried out according to Visser et al. 1989 (Plant Molecular Biology 12, No. 3:329-337) with Agrobacterium tumefaciens LBA4404 cells harbouring pMDC32_glulll. The transgenic line (positive for pMDC32_glulll construct) J3 was selected for further experiments.

Transgenic and control (wild-type) plants were transferred from tissue cultures into the soil and grown in growth chambers at 21 ± 2 °C in the light and 18 ± 1 °C in the dark, at a relative humidity of 75% ± 2%, with 70-90 ηηοΙ/Γη2/52 radiation (L36W/77 lamp, Osram, Germany) and a 16h photoperiod. After four weeks in the soil, the three lower leaves of each transgenic and control plant were inoculated with potato virus Y^NTN (PVY). The lower inoculated and upper non-inoculated leaves were collected after 1, 4 and 7 days. Total RNA was isolated from the samples and converted to cDNA. Relative amounts of RNA coding PVY's coat protein (CP) were determined in the leaf samples using quantitative real-time PCR with NTN F primer, NTN R primer and NTN probe (Kogovsek et al. 2008, Journal of Virological Methods, 149:1-11). For normalization cox gene was used with primers COX-F, COX-R and probe COX-P (Weller et al., 2000, Applied and Environmental Microbiology, 66(7):2853-2858). Since the amount of inoculum could not be completely equalized, the amount of PVY CP in lower leaves was normalized to the value of 1000 units for all samples in the calculations. The amount of CP after 7 days in control plants in upper non-inoculated leaves was 8 units, whereas the amount of CP in transgenic line was on average 28 units.

Claims

Claims:

1. Method for producing at least one protein in plantal cells comprising the step of

a. introducing into a cell at least one polynucleotide comprising at least one sequence i) coding a protein to be produced, and at least one polynucleotide which comprises the coding sequence ii) for at least one further protein or polyribonucleotide which increases the cell-cell permeability of the target cells or

b. introducing into a cell at least one polynucleotide which comprises both polynucleotide i) and polynucleotide ii).

2. The method of claim 1 wherein the plantal cells are part of a plantal cell tissue or of a whole plant wherein the tissue or the plant is from monocots or eudicots, preferably from the plant families Solanaceae, Poaceae, Fabaceae, Brassicaceae, Musaceae, Cucurbitaceae, Apiaceae, Rosaceae, and Salicaceae, preferably from the genus Nicotiana, preferably N. tabacum or N. benthamiana, or from the genus Solarium, preferably S. tuberosum.

3. The method of claim 1 wherein the method of introducing the polynucleotides is Magnifection and/or high-pressure spraying of a suspension comprising the polynucleotides and/or an abrasive, preferably carborundum.

4. The method of claim 3 wherein the polynucleotides are introduced into one or several separate parts of the plant or plantal tissue or into the whole plant or tissue and/or wherein the polynucleotides are introduced simultaneously and/or consecutively.

5. The method of any of the preceding claims wherein the polynucleotide sequences are introduced via a bacterium from the group comprising Agrobacterium tumefaciens, A. rhizogenes, Sinorhizobium meliloti, Rhizobium sp. and Mesorhizobium loti.

6. The method of any of the preceding claims wherein the polynucleotide sequences are derived from any plantal virus, preferably TMV and/or PVX, preferably the combination of non-competing TMV and PVX vectors.

7. The method of any of the preceding claims wherein the polynucleotide sequence(s) ii) encode enzymes capable of degradation of polysaccharides.

8. The method of any of the preceding claims wherein the polynucleotide sequence(s) ii) encode a substance that down-regulates and/or inhibits the enzymes capable of synthesizing polysaccharides which regulate cell-cell permeability.

9. The method any of the preceding claims wherein the method of claim 7 is combined with the method of claim 8.

10. The method of claim 7 or 9 wherein the enzyme is from group EC 3.2.1.

11. The method of claim 8 or 9 wherein the down-regulated and/or inhibited enzymes are from group EC 2.4.1.

12. The method of any of the preceding claims wherein the proteins capable of facilitating spread are expressed in a first step by induction, and switched off after spreading, and wherein in a further step the proteins to be produced are induced.

13. The method of claim 8 or 9 wherein down-regulation and/or inhibition is done via inhibiting substances such as small interfering RNA (siRNA), small hairpin RNA (shRNA), dsRNA, MicroRNA, Spiegelmere, which are ..-oligonucleotides, DNA-, RNA- or peptide aptamere, ribozymes, abzymes, which are catalytic antibodies or enzymes.

14. The method of claim 13 wherein a transgenic plant with stable introduction is generated.

15. A vector system comprising at least one vector comprising a promoter, optionally an inducible promoter, and at least one polynucleotide comprising sequences i) and ii) as defined in claim 1; or comprising a combination of two vectors, wherein one vector comprises a promoter, optionally an inducible promoter, and at least one sequence i) as defined in claim 1, and wherein a second vector comprises a promoter, optionally an inducible promoter, and at least one sequence ii) as defined in claim 1.

16. A bacterium containing the vector system of claim 15.

17. A plant containing the bacterium of claim 16.