Patent Application
20190136260
The present invention relates to a process for transient transfection of plants by spraying the plants with an aqueous suspension containing Agrobacterium cells. The invention also provides a process of generating or altering a trait in a plant growing on a field. The invention also relates to a process of producing a protein of interest in a plurality of plants on a field. The invention also relates to a process of protecting crop plants on a field from a pest. Moreover, the invention relates to an aqueous suspension containing cells of an Agrobacterium strain, suitable for large scale transient transfection of plants grown on a farm field for the processes of the invention. The invention also relates to the use of particulate inorganic material for transient transfection of plants by spraying with suspensions containing Agrobacterium cells and the particulate inorganic material.
Current genetic engineering processes for agriculture are all based on stable genetic modification of crop species, demonstrated first in 1983 (Fraley et al 1983; Barton et al 1983) and commercialized since 1996. Although the agriculture process based on plant stable genetic transformation is a reality today and is a basis of a very successful new practices, it has multiple limitations, the main ones being very long time and high cost required for development of transgenic crops. General consensus among the companies involved in plant biotechnology is that the R&D process requires, depending on the crop species, between 8 and 16 years, and the total average cost is estimated to be between $100 and $150 million. Because of these limitations, after more than 25 years since the discovery of a plant genetic transformation process, only a handful traits and few GM crop species have been commercialized thus far.
It is known that plant cells and whole plants can also be re-programmed transiently (i.e. without stable integration of new genetic material on a plant chromosome), and the transient processes, such as viral infections, are fast. Such transient processes could in principle allow a very fast modification of plant metabolism in favor of certain traits or products that are of interest to the user. Clearly, such processes require a DNA or RNA vector (a virus or a bacterium), that has been engineered to effectively and safely transfect the plant, with the resultant effect being devoid of undesired side effects. Earlier attempts to use vectors based on plant viruses have been partially successful in that they allow transfection of plants for manufacturing of high-value recombinant proteins such as certain biopharmaceuticals (Gleba et al 2007, 2008; Lico et al 2008). Use of viruses for manipulation of other traits, such as input traits (for example, herbicide resistance, Shiboleth et al 2001; Zhang and Ghabiral 2006) have been described in the literature, but virus transfection introduces so many undesired changes in the infected host that this kind of transient process is not pursued anymore for input traits. Transient processes can also be built around the ability of Agrobacterium species to transfer part of its Ti plasmid to eukaryotic, in particular, plant cell. Use of Agrobacterium-based transfection is a basis for genetic manipulations such as genetic transformation protocols and of laboratory transient transfection assays. Industrial applications of Agrobacterium-based transfection have also been limited to recombinant protein manufacturing, because the optimal application conditions such as vacuum infiltration of plants with bacterial suspensions cannot be used on a large scale in the field, whereas spraying aerial parts or watering plants with bacterial solutions results in a supposedly very small proportion of plant cells to be transfected, and previous studies simply did not address that specific question. The combination of Agrobacterium delivery and use of virus as a secondary messenger in one process has been successful in manufacturing high-value recombinant proteins including complex biopharmaceuticals such as full IgG antibodies. However, when it comes to traits such as input traits or traits requiring subtle targeted reprogramming of plant cell metabolism, this magnifection process has the same limitations as viral vectors have.
There is considerable knowledge in the area of the use of microorganisms for controlling certain processes that require interaction of microbes with plants, including use of microorganisms such as Lactobacillus and Saccharomyces yeasts for biomass fermentation (preparation of fermented food, drinks), for biocontrol (Agrobacterium, Myrotecium, strains), and use of strains of Rhizobium for improved nitrogen fixation. In research papers and patents in which microorganisms have been explored as biocontrol agents, there is a considerable body of knowledge of how the living cells should be applied to plant surfaces; in particular, studies have been performed that identified spraying conditions and adjuvants (wetters, stickers, etc.) to be used in the spray mixtures. Examples of such research are numerous. The following papers exemplify the state of the art in this area: Arguelles-Arias et al 2009; Nam et al 2009; reviewed by Johnson 2009.
There are registered Agrobacterium rhizogenes/radiobacter strains that have been used for decades for control of crown gall disease in vineyards and orchards. There are two commercially used strains, one being a natural strain carrying plasmid (K84), and the other, a genetically modified derivative that has been modified through deletion of the gene necessary for the conjugative plasmid transfer (K1026). (Kerr and Tate 1984; Vicedo et al 1993; Reader et al 2005; Kim et al 2006; reviewed in Moore 1988).
Agrobacterium tumefaciens and A rhizogenes are broadly used in research laboratories worldwide for transient transfection and stable genetic transformation of plants. These applications are based on the ability of Agrobacterium to transfer genetic information to eukaryotic cells. Many of the genetically modified plants cultivated today, such as soybeans, canola and cotton, have been generated through Agrobacterium-mediated genetic transformation. The essential difference between the transient and stable transformation is that in the process of stable transformation, Agrobacterium-delivered DNA is eventually integrated into a plant chromosome, and is afterwards inherited by the plant progeny. Such integration events are rare even in laboratory experiments specifically designed to provide massive contacts between plant cells and bacteria; thus for the selection of stable transformants, specific selective screening methods have to be utilized. Subsequently, the knowledge accumulated in this science domain is of limited value to those interested in transient processes that have to be designed so as to have a massive character and affect multiple cells of the plant body.
Transient transfection, on the other hand, takes into account only earlier steps of Agrobacterium-driven DNA delivery into a nucleus of a plant cell, along with the fact that such delivered DNA molecules, if property designed to constitute a transcription unit carrying plant-specific promoter and terminator and a coding part, will be transcribed in a nucleus even in the absence of said DNA integration into a plant chromosome, such expression resulting in a transient reprogramming of a plant cell. Such reprogramming has been first achieved early on and has been developed into a standard laboratory tool for rapid evaluation of different genetic experiments. Whereas there is considerable body of knowledge about Agrobacterium-mediated DNA transfer to plant cells, with exception of few cases, that information is limited to laboratory scale experiments, and thus far, there were very few attempts to develop industrial scale applications involving Agrobacterium as a DNA vector. One of the limitations of laboratory applications is the fact that Agrobacterium-based DNA delivery requires certain treatments that are difficult or impossible to apply in open field or on a large scale. In typical transient experiments, cultured plant cells or parts of plants are treated with an excess of bacteria to provide for maximum delivery. In typical research experiments, one is also interested in expression levels that are not economically viable if done on an industrial scale. In general, the research done in this domain has led the inventors to the conclusion that the parameters seriously affecting transient expression are those allowing for the best interaction access of agrobacteria to plant cells within a plant body. Most such studies utilize vacuum infiltration, injection into plant leaf or surfactant treatment, wounding of plant surface e.g. with razor blades, or combination thereof. In fact, the only group that is developing an Agrobacterium-based transfection process for commercial production of recombinant proteins that does not involve further (virus-based) amplification of the original DNA, is the group of Medicago (D'Aoust et al 2008, 2009; Vezina et al, 2009) that is entirely relying on vacuum infiltration as a delivery method. However, because of being based on great excess of bacteria to plant cell ratio, current laboratory protocols used for transient transfection of plants do not have serious translational value, i.e. they cannot be directly replicated on an industrial level. Except in few cases (e.g. Vaquero et al, 1999, D′Aoust et al, 2008, 2009) they also have not addressed quantitatively the issue of efficiency of the transient transfection process. (Examples of such research are multiple, we provide a citation for just a few representative ones: Li et al, 1992; Liu et al, 1992; Clough and Bent, 1998; De Buck et al, 1998, 2000; Chung et al, 2000; Yang et al, 2000; Zambre et al, 2003; Wroblewski et al, 2005; Lee and Yang, 2006; Zhao et al, 2006; Shang et al, 2007; Jones et al., 2009; Li et al, 2009; De Felippes and Weigel, 2010). Except in two cases described below, there were no attempts in the literature to quantify the efficiency of the transient process or to provide sufficient understanding that would lead to potential commercial large-scale exploitation of the phenomenon.
One of the industrial processes being under development today is magnifection, a process that is based on vacuum-infiltration of agrobacteria into leaves of plants. The magnifection process (trademarked by Icon Genetics GmbH as magnICON® and covered by several patents/patent applications) is a simple and indefinitely scalable protocol for heterologous protein expression in plants, which is devoid of stable genetic transformation of a plant, but instead relies on transient amplification of viral vectors delivered to multiple areas of a plant body (systemic delivery) by Agrobacterium as DNA precursors. Such a process is in essence an infiltration of whole mature plants with a diluted suspension of agrobacteria carrying T-DNAs encoding viral RNA replicons. In this process, the bacteria assume the (formerly viral) functions of primary infection and systemic movement, whereas the viral vector provides for cell-to-cell (short distance) spread, amplification and high-level protein expression. Initial demonstration that viral infection can be initiated by agrobacteria delivering a viral genome copy into a plant cell comes from the pioneering work of Grimsley et al, 1986, in which a DNA virus has been delivered, and a first, although very inefficient, infection with tmv, a cytoplasmic RNA virus delivered as a DNA copy, came from the work of Turpen et al 1993. Current technology, however, is extremely efficient and a few adult tobacco plants are sufficient for early construct optimization and fast production of milligram to gram quantities of recombinant protein for pre-clinical or clinical evaluation, or, in case of individualized vaccines, for manufacturing. The scale-up (industrial) version is essentially the same, but is built around fully assembled viral vectors (rather than pro-vectors requiring in planta assembly) and requires apparatuses for high-throughput Agrobacterium delivery to whole plants by vacuum infiltration. The process can be scaled up but it requires submersion of aerial parts of plants into bacterial suspension under vacuum (the process involves inverting plants grown in pots or in trays), a procedure that imposes imitations on the volumes of biomass that can be treated in this way, on the throughput of the process, on the ways the plants can be cultivated prior to treatment, and it also carries certain costs that limit the use of the process to high-cost products, such as recombinant biopharmaceuticals only. The magnifection process is efficient as it allows transfection of almost all leaf cells in treated plants, or approximately 50% of the total aerial plant biomass (the rest being stems and petioles). The process has been optimized in many ways, in particular through improvement of viral replicon release through optimization of the posttranslational modification of the primary DNA transcripts (Marillonnet et al, 2005). However, the current process has been built entirely around bacterial delivery methods such as injection into plant leaf or vacuum-infiltration (e.g. Simmons et al, 2009), wounding of leaves (Andrews and Curtis, 2005), or pouring agrobacteria into soil (‘agrodrenching’, Ryu et al, 2004; Yang et al, 2008), whereas said methods can not be applied for the mass treatment of the plants in a field (reviewed in Gleba et al, 2004, 2007, 2008; Lico et al, 2008; original articles include Giritch et al. 2006; Marillonnet et al., 2004, 2005; Santi et al, 2006; and ideologically similar papers from other research groups—Voinnet et al, 2003; Sudarshana et al, 2006; Gao et al, 2006; Mett et al, 2007; Lindbo, 2007a,b; Plesha et al, 2007, 2009; Huang et al, 2006; Regnard et al 2009; Green et al, 2009; Shoji et al, 2009).
It should be mentioned that although Agrobacterium tumefaciens and A. thizogenes are the DNA vectors that are used in the majority of cases, there are other species of bacteria that can perform similar DNA transfer to plant cells (Broothaerts et al, 2005).
The attempts to quantify Agrobacterium treatment on whole plants after vacuum-infiltration have been performed by a few research groups only. In the papers of Joh et al, 2005, 2006, it has been concluded that the highest used bacteria density of 109 cfu/ml was the best (as opposed to 108 cfu/ml or 107 cfu/ml), as measured by the total recombinant protein expression. In experiments of Lindbo, 2007a,b, essentially similar results have been obtained as in our work, however, no counting of transfected cells has been performed and the conclusions were derived from recombinant protein expression levels.
The attempts to use Agrobacterium treatment on whole plants without vacuum-infiltration result in a very low number of initially transfected cells, thus greatly limiting the practical application of the process. One way of circumventing this initial limitation disclosed in literature is the use of an efficient secondary messenger such as a plant virus that would allow amplifying the initially inefficient process by complementing it with a virus-based cell-to-cell and systemic movement (Azhakanandam et al, 2007). The Agrobacterium-based delivery of DNA copies of plant viruses or plant viral vectors has been described a long time ago (Grimsley et al, 1986, and for TMV—Turpen et al, 1993), and it allows to spread initial replicon delivered to a few cells in plant to the rest of the body by using viral infection process such as cell-to-cell movement and systemic movement of virus. Such a process has limited practical utility for our purposes, because viral infection dramatically changes plant performance; all currently entertained applications are in the area of recombinant protein manufacturing in plants (reviewed by Gleba et al, 2007, 2008). It should also be noted that the cited paper of Azhakanandam et al, 2007 does not even attempt to quantify the efficiency of the initial transfection and it is based on very high amount of agrobacteria in the transfection media.
Departing from the prior art, it is an object of the present invention to provide an efficient process of transiently transfecting plants so as to be applicable to many plants growing on a farm field. It is also an object of the invention to provide a process of altering a trait in plants growing on a farm field. Notably, it is an object of the invention to provide an efficient process allowing transient plant transfection using Agrobacterium on a large scale without the need for the application of pressure differences to introduce Agrobacterium into the intercellular space of plants. It is also an object to provide an Agrobacterium formulation suitable for this purpose.
Accordingly, the present invention provides the following:
The inventors of the present invention have found a way of strongly increasing the probability of achieving plant transfection by Agrobacerium. The inventors have found that addition of a particulate material insoluble in aqueous Agrobacterium suspensions strongly increases the transfection efficiency achievable by spraying of aerial parts of the plant with the suspension. The high efficiency achieved allows for the first time transfection of plants with Agrobacterium suspensions on a large scale such as on agricultural fields, whereby the cumbersome infiltration methods making use of pressure differences can be avoided. The invention also allows transfection of plants that have so far not been amenable to spray transformation with Agrobacterium suspensions.
Spraying: TMV(MP)-GFP (pNMD560).
Nicotiana benthamiana plant was used as a positive control.
Vector: PVX(CP)-GFP (pNMD630). Agrobacterial cells were incubated with 200 μM acetosyringone for 2 hours before spraying. For abrasive treatment, 0.3% carborundum (silicon carbide mixture of F800, F1000 and F1200 particles, Minerallengrosshandel Hausen GmbH, Telfs, Austria) was added to agrobacterial suspension. Dilution factor of agrobacteria of OD600=1.4: 10-2. GFP-expressing spots were leaves are indicated on the right.
Plant material was extracted with 6 volumes of 1× Laemmli buffer containing beta-mercaptoethanol. 10 μl aliquots were separated in 15% polyacrylamide gel under reducing conditions.
For crude extracts, 50 mg plant material (pooled samples of 3 independent leaves) harvested 10 dpi was ground in liquid nitrogen, extracted with 5 vol. 2× Laemmli buffer and denatured at 95° c. for 5 min. 7.5 μl of each sample were analyzed by 10% SDS-PAGE and Coomassie staining.
In the invention, agrobacteria are used for transfecting plants with a sequence or construct of interest by spraying with aqueous suspensions containing cells of an Agrobacterium strain. The Agrobacterium strain may belong to the species Agrobacterium tumefaciens or Agrobacterium rhizogenes that are commonly used for plant transformation and transfection and which is known to the skilled person from general knowledge. The Agrobacterium strain comprises a DNA molecule comprising a nucleic constructs containing a DNA sequence of interest. The DNA sequence of interest encodes a protein or an RNA to be expressed in plants. The nucleic construct is typically present in T-DNA of Ti-plasmids for introduction of the nucleic construct into plant cells by the secretory system of the Agrobacterium strain. On at least one side or on both sides, the nucleic acid construct is flanked by a T-DNA border sequence for allowing transfection of said plant(s) and introduction of cells of said plant with said DNA sequence of interest. In the nucleic acid construct, the DNA sequence of interest is present such as to be expressible in plant cells. For this purpose, the DNA sequence of interest is, in said nucleic acid construct, typically under the control of a promoter active in plant cells. Examples of the DNA sequence of interest are a DNA sequence encoding a DNA viral replicon or an RNA viral replicon or a gene to be expressed. The gene may encode an RNA of interest or a protein of interest to be expressed in cells of the plant(s). Also the viral replicons typically encode an RNA or a protein of interest to be expressed in plants. The DNA construct may comprise, in addition to the DNA sequence of interest, other sequences such as regulatory sequences for expression of the DNA sequence of interest. Agrobacterium-mediated gene transfer and vectors therefor are known to the skilled person, e.g. from the references cited in the introduction or from text books on plant biotechnology such as Slater, Scott and Fowler, Plant Biotechnology, second edition, Oxford University Press, 2008.
In embodiments wherein strong expression of a protein or RNA is desired or wherein accumulation of viral nucleic acids to high amounts in plant cells and possible negative effects on plant health is not a concern, the nucleic acid construct may encode a replicating viral vector that can replicate in plant cells. In order to be replicating, the viral vector contains an origin of replication that can be recognized by a nucleic acid polymerase present in plant cells, such as by the viral polymerase expressed from the replicon. In case of RNA viral vectors, the viral replicons may be formed by transcription, under the control of a plant promoter, from the DNA construct after the latter has been introduced into plant cell nucleic. In case of DNA viral replicons, the viral replicons may be formed by recombination between two recombination sites flanking the sequence encoding the viral relicon in the DNA construct, e.g. as described in WO00/17365 and WO 99/22003. If viral replicons are encoded by the DNA construct, RNA viral replicons are preferred. Use of DNA and RNA viral replicons has been extensively described in the literature at least over the last 15 years. Some examples are the following patent publications by Icon Genetics: WO2008028661, WO2007137788, WO 2006003018, WO2005071090, WO2005049839, WO002097080, WO002088369, WO02068664. An example of DNA viral vectors are those based on geminiviruses. For the present invention, viral vectors or replicons based on plant RNA viruses, notably based on plus-sense single-stranded RNA viruses are preferred. Examples of such viral vectors are tobacco mosaic virus (TMV) and potex virus X (PVX) used in the examples. Potexvirus-based viral vectors and expression systems are described in EP2061890. Many other plant viral replicons are described in the patent publications mentioned above.
The aqueous suspension used for spraying in the processes of the invention may have a concentration of Agrobacterium cells of at most 1.1·10 cfu/ml, which corresponds approximately to an Agrobacterium culture in LB-medium of an optical density at 600 nm of 1. Due to the high transfection efficiency achieved in the invention, much lower concentrations may, however, be used, which allows treatment of many plants such as entire farm fields without the need for huge fermenters for Agrobacterium production. Thus, the concentration is preferably at most 2.2·107 cfu/ml, more preferably at most 1.1·107 cfu/ml, more preferably at most 4.4·106 cfu/ml. In one embodiment, the concentration is at most 1.1·108 cfu/ml of the suspension. For avoiding determination of cell concentrations in terms of cfu/ml, concentrations of agrobacterial suspensions are frequently assessed by measuring the apparent optical density at 600 nm using a spectrophotometer. Herein, the concentration of 1.1·107 cfu/ml corresponds to a calculated optical density at 600 nm of 0.01, whereby the calculated optical density is defined by a 100-fold dilution with water or buffer of a suspension having an optical density of 1.0 at 600 nm. Similarly, the concentrations of 4.4·108 cfu/ml and 1.1·108 cfu/ml correspond to a calculated optical density at 600 nm of 0.004 and 0.001, respectively, whereby the calculated optical densities are defined by a 250-fold or 1000-fold, respectively, dilution with water or buffer of a suspension having an optical density of 1.0 at 600 nm.
The abrasive that may be used in the invention is a particulate material that is essentially insoluble in the aqueous suspension of Agrobacterium cells. The abrasive is believed to weaken, notably if used together with a wetting agent, the surface of plant tissue such as leaves, and thereby facilitates penetration of Agrobacterium cells into the intercellular space of plant tissue. As a result, the transfection efficiency increases.
The particulate material to be used as the abrasive of the invention may be carrier material as commonly used as carriers in wettable powder (WP) of pesticide formulations. In the context of wettable powders, these carriers are also referred to in the field of pesticide formulations as “fillers” or “inert fillers”. Wettable powder formulations are part of the general knowledge in the field of plant protection. Reference is made to the handbook PESTICIDE SPECIFICATIONS, “Manual for Development and Use of FAO and WHO Specifications for Pesticides”, edited by the World Health Organisation (WHO) and the FOOD and Agriculture Organization of the United States, Rome, 2002, ISBN 92-5-104857-6. Wettable powder formulations for plant protection are for example described in EP 1810569, EP1488697, EP1908348 and EP0789510. The abrasive may be a mineral material, typically an inorganic material. Examples of such carrier materials are diatomaceous earth, talc, clay, calcium carbonate, bentonite, acid clay, attapulgite, zeolite, sericite, sepiolite or calcium silicate. It is also possible to use quartz powder such as the highly pure quartz powder described in WO02/087324. Preferred examples are silica, such as precipitated and fumed hydrophilic silica, and carborundum. The abrasive properties of diluents or fillers such as silica used in wettable powders are known (see “Pesticide Application Methods” by G. A. Matthews, third edition, Blackwell Science, 2000, on page 52 thereof).
As commercial products of particulate inorganic materials for use as abrasives in the invention, the hydrophilic silica Sipemat™ 22S and Sipemat™ 50 S, manufactured by Evonic Degussa may be mentioned. Other products are “Hi-Sil™ 257”, a synthetic, amorphous, hydrated silica produced by PPG Industries Taiwan Ltd. or “Hubersorb 600M”, a synthetic calcium silicate, manufactured by Huber Corporation. A commercial sub-micron sized silica is Hi-Sil™ 233 (PPG Industries) having an average particle size of around 0.02 μm.
The abrasive may have a median particle size between 0.01 and 40, preferably between 0.015 and 30, more preferably between 0.05 and 30, even more preferably between 0.1 and 30, even more preferably between 0.1 and 20, even more preferably between 0.5 and 20, and most preferably between 1.0 and 16 μm. In one embodiment, the median particle size is between 0.015 and 1 or between 0.02 and 0.5 μm. The median particle size is the volume median particle size that can be measured by laser diffraction using a Mastersizer™ from Malvern Instruments, Ltd. In order to avoid clogging of spraying nozzles, the maximum particle size of the largest particles contained in the abrasive should be at most 45 μm, preferably at most 40 μm, which may be determined by sieving. This condition is considered fulfilled, if the sieve residue is below 1.5% by weight (following ISO 3262-19). The abrasive may have a D90 value of at most 40 μm, preferably of at most 30 μm, measured by laser diffraction as described above. Typically, the particle sizes above relate to primary particle sizes.
The content of the abrasive in the aqueous suspension of the invention may be between 0.01 and 3, preferably between 0.02 and 2, more preferably between 0.05 and 1 and even more preferably between 0.1 and 0.5% by weight of said suspension.
The aqueous suspension of the invention preferably contains an agricultural spray adjuvant. The spray adjuvant may be a surfactant or wetting agent. The surfactant and wetting agent has multiple advantages in the present invention. It reduces the surface tension of the water of the aqueous suspension and makes the waxy surface of plant leaves more permeable for agrobacteia. It further improves the stability of the suspension and reduces settling of the abrasive in the suspension. Surfactants used in the present invention are not particularly limited, and examples of the surfactants include the following (A), (B), and (C). These may be used singly or in combination.
(A) Nonionic surfactants: A measurement frequently used to describe surfactants is the HLB (hydrophilic/lipophilic balance). The HLB describes the ability of the surfactant to associate with hydrophilic and lipophilic compounds. Surfactants with a high HLB balance associate better with water soluble compounds than with oil soluble compounds. Herein, the HLB value should be 12 or greater, preferably at least 13. As noninionic surfactants, organo-silicone surfactants such as polyalkyleneoxide-modified heptamethyltrisioxane are most preferred in the present invention. A commercial product is Silwet L77M spray adjuvant from GE Advanced Materials.
(A-I) Polyethylene glycol type surfactants: examples of polyethylene glycol type surfactants include polyoxyethylene alkyl (C12-18) ether, ethylene oxide adduct of alkylnaphthol, polyoxyethylene (mono or di) alkyl (C8-12) phenyl ether, formaldehyde condensation product of polyoxyethylene (mono or di) alkyl (C8-12) phenyl ether, polyoxyethylene (mono, di, or tri) phenyl phenyl ether, polyoxyethylene (mono, di, or tri) benzyl phenyl ether, polyoxypropylene (mono, di, or tn) benzyl phenyl ether, polyoxyethylene (mono, di, or td) styryl phenyl ether, polyoxypropylene (mono, di or tnri) styryl phenyl ether, a polymer of polyoxyethylene (mono, di, or td) styryl phenyl ether, a polyoxyethylene polyoxypropylene block polymer, an alkyl (C12-18) polyoxyethylene polyoxypropylene block polymer ether, an alkyl (C8-12) phenyl polyoxyethylene polyoxypropylene block polymer ether, polyoxyethylene bisphenyl ether, polyoxyethylene resin acid ester, polyoxyethylene fatty acid (C12-18) monoester, polyoxyethylene fatty acid (C12-18) diester, polyoxyethylene sorbitan fatty acid (C12-18) ester, ethylene oxide adduct of glycerol fatty acid ester, ethylene oxide adduct of castor oil, ethylene oxide adduct of hardened caster oil, ethylene oxide adduct of alkyl (C12-8) amine and ethylene oxide adduct of fatty acid (C12-18) amide;
(A-2) Polyvalent alcohol type surfactants: examples of polyvalent alcohol type surfactants include glycerol fatty acid ester, polyglycerin fatty acid ester, pentaerythritol fatty acid ester, sorbitol fatty acid (C12-18) ester, sorbitan fatty acid (C12-8) ester, sucrose fatty acid ester, polyvalent alcohol alkyl ether, and fatty acid alkanol amide;
(A-3) Acetylene-type surfactants: examples of acetylene type surfactants include acetylene glycol, acetylene alcohol, ethylene oxide adduct of acetylene glycol and ethylene oxide adduct of acetylene alcohol.
(B) Anionic surfactants:
(B-1) Carboxylic acid type surfactants: examples of carboxylic acid type surfactants include polyacrylic acid, polymethacrylic acid, polymaleic acid, a copolymer of maleic acid and olefin (for example, isobutylene and diisobutylene), a copolymer of acrylic acid and itaconic acid, a copolymer of methacrylic acid and itaconic acid, a copolymer of maleic acid and styrene, a copolymer of acrylic acid and methacrylic acid, a copolymer of acrylic acid and methyl acrylate, a copolymer of acrylic acid and vinyl acetate, a copolymer of acrylic acid and maleic acid, N-methyl-fatty acid (C12-18) sarcosinate, carboxylic acids such as resin acid and fatty acid (C12-18) and the like, and salts of these carboxylic acids.
(B-2) Sulfate ester type surfactants: examples sulfate ester type surfactants include alkyl (C12-18) sulfate ester, polyoxyethylene alkyl (C12-18) ether sulfate ester, polyoxyethylene (mono or di) alkyl (C8-12) phenyl ether sulfate ester, sulfate ester of a polyoxyethylene (mono or di) alkyl (C8-12) phenyl ether polymer, polyoxyethylene (mono, di, or tri) phenyl phenyl ether sulfate ester, polyoxyethylene (mono, di, or tn) benzyl phenyl ether sulfate ester, polyoxyethylene (mono, di, or tn) styryl phenyl ether sulfate ester, sulfate ester of a polyoxyethylene (mono, di, or tn) styryl phenyl ether polymer, sulfate ester of a polyoxyethylene polyoxypropylene block polymer, sulfated oil, sulfated fatty acid ester, sulfated fatty acid, sulfate ester of sulfated olefin and the like, and salts of these sulfate esters.
(B-3) Sulfonic acid type surfactants: examples of sulfonic acid type surfactants include paraffin (C12-22) sulfonic acid, alkyl (C8-12) benzene sulfonic acid, formaldehyde condensation product of alkyl (C8-12) benzene sulfonic acid, formaldehyde condensation product of cresol sulfonic acid, -olefin (C14-16) sulfonic acid, dialkyl (C8-12) sulfosuccinic acid, lignin sulfonic acid, polyoxyethylene (mono or di) alkyl (C8-12) phenyl ether sulfonic acid, polyoxyethylene alkyl (C12-18) ether sulfosuccinate half ester, naphthalene sulfonic acid, (mono, or di) alkyl (CI-6) naphthalene sulfonic acid, formaldehyde condensation product of naphthalene sulfonic acid, formaldehyde condensation product of (mono, or di) alkyl (CI-6) naphthalene sulfonic acid, formaldehyde condensation product of creosote oil sulfonic acid, alkyl (C8-12) diphenyl ether disulfonic acid, Igepon T (tradename), polystyrene sulfonic acid, sulfonic acids of a styrene sulfonic acid—methacrylic acid copolymer and the like, and salts of these sulfonic acids.
(B-4) Phosphate ester type surfactants: examples of phosphate ester type surfactants include alkyl (C8-12) phosphate ester, polyoxyethylene alkyl (C12-18) ether phosphate ester, polyoxyethylene (mono or di) alkyl (C8-12) phenyl ether phosphate ester, phosphate ester of a polyoxyethylene (mono, di, or tni) alkyl (C8-12) phenyl ether polymer, polyoxyethylene (mono, di, or tri) phenyl phenyl ether phosphate ester, polyoxyethylene (mono, di, or tri) benzyl phenyl ether phosphate ester, polyoxyethylene (mono, di, or tri) styryl phenyl ether phosphate ester, phosphate ester of a polyoxyethylene (mono, di, or tri) styryl phenyl ether polymer, phosphate ester of a polyoxyethylene polyoxypropylene block polymer, phosphatidyl choline, phosphate ester of phosphatidyl ethanolimine and condensed phosphoric acid (for example, such as tripolyphosphoric acid) and the like, and salts of these phosphate esters.
Salts of above-mentioned (B-1) to (B-4) include alkaline metals (such as lithium, sodium and potassium), alkaline earth metals (such as calcium and magnesium), ammonium and various types of amines (such as alkyl amines, cycloalkyl amines and alkanol amines).
(C) Amphoteric surfactants: Examples of amphoteric surfactants include betaine type surfactants and amino acid type surfactants.
The above surfactants may be used singly or in combination of two or more surfactants. Notably, the preferred organo-silicone surfactants may be combined with other surfactants. The total concentration of surfactants in the aqueous suspension of the invention may be easily tested by conducting comparative spraying experiments, similarly as done in the examples. However, in general, the total concentration of surfactants may be between 0.005 and 2 volume-%, preferably between 0.01 and 0.5 volume-%, more preferably between 0.025 and 0.2 volume-% of said suspension. Since the density of surfactants is generally close to 1.0 g/ml, the total concentration of surfactants may be defined as being between 0.05 and 20 g per liter of said suspension, preferably between 0.1 and 5.0 g, more preferably between 0.25 and 2.0 g per liter of said suspension (including abrasive). If the above organo-silicone surfactants such as polyalkyleneoxide-modified heptamethyttrisiloxane are used, the concentration of the organo-silicone surfactant in the agrobacterial suspension used for spraying may be between 0.01 and 0.5 volume-%, preferably between 0.05 and 0.2 volume-%. Alternatively, the concentration of the organo-silicone surfactant in the agrobacterial suspension used for spraying may be defined as being between 0.1 and 5.0 g, preferably between 0.5 and 2.0 g per liter of said suspension.
In order to improve the physical properties of the aqueous suspension, it is possible to add highly dispersed sub-micron size silicic acid (silica) or porous polymers such as urea/formaldehyde condensate (Pergopak™). Notably, where the median particle size of the abrasive is between 0.1 and 30 μm, or in one of the preferred sub-ranges of this range given above, it is possible to add a highly dispersed sub-micron size silica to the suspension. Herein, sub-micron size silica is silica having a median particle size between 0.01 and 0.5 μm, preferably between 0.02 and 0.5 μm, more preferably between 0.02 and 0.1 μm. Highly dispersed silicic acid such as Hi-Sil™ 233 (PPG Industries) can contribute to the abrasive properties of the aqueous suspension (see Jensen et al., Bull. Org. mond. Sante, Bull. Wld Hlth Org. 41 (1969) 937-940). These agents may be incorporated in an amount of from 1 to 10 g per liter of the suspension of the invention.
Further possible additives to the agrobacterial suspension are buffer substances to keep maintain the pH of the suspension used for spraying at a desired pH, typically between 7.0 and 7.5. Further, inorganic soluble salts such as sodium chloride by be added to adjust the ionic strength of the suspension. Nutrient broth such as LB medium may also be contained in the suspension.
The aqueous suspension may be produced as follows. In one method, the Agrobacterium stain to be used in the process of the invention is inoculated into culture medium and grown to a high cell concentration. Larger cultures may be inoculated with small volumes of a highly concentrated culture medium for obtaining large amounts of the culture medium. Agrobacteria are generally grown up to a cell concentration corresponding to an OD at 600 nm of at least 1, typically of about 1.5. Such highly concentrated agrobacterial suspensions are then diluted to achieve the desired cell concentration. For diluting the highly concentrated agrobacterial suspensions, water is used. The water may contain a buffer. The water may further contain the surfactant of the invention. Alternatively, the concentrated agrobacterial suspensions may be diluted with water, and any additives such as the surfactant and the optional buffer substances are added after or during the dilution process. The abrasive may be added before, during or after dilution. It is however preferred to agitate the suspension during addition of the abrasive to uniformly disperse the abrasive in the agrobacterial suspension. The step of diluting the concentrated agrobacterial suspension may be carried out in the spray tank of the sprayer used for spraying the diluted suspensions.
The sprayer to be used in the process of the invention mainly depends on the number of plants or the area to be sprayed. For one or a small number of plants to be sprayed, pump sprayers as widely used in household and gardening can be used. These may have volumes of the spray tank of between 0.5 and 2 liters. For applications on a medium scale, manually operated hydraulic sprayers such as lever-operated knapsack sprayers or manually operated compression sprayers may be used. However, the high transfection efficiency achieved in the invention has its full potential in the transfection of many plants such as plants growing on a farm field or in a greenhouse. For this purpose, power-operated hydraulic sprayers such as tractor-mounted hydraulic sprayers equipped with spray booms can be used. Aerial application techniques using helicopters or airplanes are also possible for large fields. All these types of sprayers are known in the art and are described for example in the book “Pesticide Application Methods” by G. A. Matthews, third edition, Blackwell Science, 2000. In order to ensure a homogeneous suspension in the spray tanks of the sprayers, small or medium size sprayers may be shaken at regular intervals or continuously during spraying. Large sprayers such as the tractor-mounted sprayers should be equipped with an agitator in the spray tank.
Considering the presence of agrobacterial cells and abrasive in the suspensions to be sprayed, sprayers used in the invention should produce spray of a droplet size at least of fine spray. Also, medium spray or coarse spray in the classification of sprays used in the above-mentioned book by G. A. Matthews, page 74, may be used. The main purpose of the spraying in the invention is wetting of plant tissue with the suspension. Thus, the exact droplet size is not critical. However, the transfection efficiency may be further improved by providing the spray to plant surfaces with increased pressure.
In the process of the invention, at least parts of plants are sprayed. In an important embodiment, plants growing in soil on a field are sprayed, i.e. plants not growing in movable pots or containers. Such plants cannot be turned upside down and dipped into agrobacterial suspension for vacuum infiltration. At least parts of plants are sprayed such as leaves. Preferably, most leaves are sprayed or entire plants.
The present invention is mainly used for transient transfection of plants with a DNA sequence of interest. The term “transient” means that the no selection methods are used for selecting cells or plants transfected with the DNA sequence of interest in the background of non-transfected cells or plants using, e.g. selectable agents and selectable marker genes capable of detoxifying the selectable agents. As a result, the transfected DNA is generally not stably introduced into plant chromosomal DNA. Instead, transient methods make use of the effect of transfection in the very plants transfected.
The invention is generally used for transfecting multi-cellular plants, notably, higher plants. Both monocot and dicot plants can be transfected, whereby dicot plants are preferred. Plants for the use in this invention include any plant species with preference given to agronomically and horticulturally important crop species. Common crop plants for the use in present invention include alfalfa, barley, beans, canola, cowpeas, cotton, corn, clover, lotus, lentils, lupine, millet, oats, peas, peanuts, rice, rye, sweet clover, sunflower, sweetpea, soybean, sorghum triticale, yam beans, velvet beans, vetch, wheat, wisteria, and nut plants. The plant species preferred for practicing this invention include, but not restricted to, representatives of Gramineae, Compositeae, Solanaceae and Rosaceae.
Further preferred species for the use in this invention are plants from the following genera: Arabidopsis, Agrostis, Allium, Antirrhinum, Apium, Arachis, Asparagus, Atropa, Avena, Bambusa, Brassica, Bromus, Browaalia, Camellia, Cannabis, Capsicum, Cicer, Chenopodium, Chichorium, Citrus, Coffea, Coix, Cucumis, Curcubita, Cynodon, Dactylis, Datura, Daucus, Digitalis, Dioscorea, Elaeis, Eleusine, Festuca, Fragaria, Geranium, Glycine, Helianthus, Heterocallis, Hevea, Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lens, Lilium, Linum, Lolium, Lotus, Lycopersicon, Majorana, Malus, Mangifera, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis, Oryza, Panicum, Pelargonium, Pennisetum, Petunia, Pisum, Phaseolus, Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus, Saccharum, Salpiglossis, Secale, Senecio, Setaria, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum, Vicia, Vigna, Vitis, Zea, and the Olyreae, the Pharoideae and others.
In one embodiment, the process of the invention can be used for producing a protein of interest in a plant or in many plants growing on a field. For this purpose, the plants may be sprayed with the agrobacterial suspension at a desired growth state of the plants. If the main aim is to achieve the highest possible expression levels followed by harvesting plants for obtaining plant material containing high amounts of the protein, viral vectors may be used, since they generally give the highest expression levels.
In another embodiment, the process of the invention is used for generating or altering a trait in a plant such as an input trait. In this embodiment, excessive expression of a protein or RNA of interest may not be desired for avoiding deleterious effects on plant health. For such embodiments, non-replicating vectors (also referred to herein as “transcriptional vectors”), i.e. vectors lacking a functional origin of replication recognised by a nucleic acid polymerase present in the plant cells are preferred. An example of such embodiment is the expression of hormonal molecules as secondary messengers in plant cells. In the example of
Further, the process of the invention allows altering at a desired point in time traits relating to the regulation of flowering time or fruit formation such as tuborisation in potato (Martinez-Garcia et al., Proc. Natl. Acad. Sci. USA 99 (2002) 15211-15216) or the regulation of the flavonoid pathway using a transcription factor (Deluc et al., Plant Physiol. 147 (2008) 2041-2053). Flowering may be induced by transiently expressing the movable florigen protein FT (Zeevaart, Current Opinion in Plant Biology 11 (2008) 541-547; Corbesier et al., Science 316 (2007) 1030-1033). Parthenocarpic fruits in tomatoes may by produced on a large scale using the invention and the method described by Pandolfini et al., BMC Biotechnology 2 (2002). Further applications of the invention are in the context of altering cotton fiber development by way of MYB transcription factors as described by Lee et al., Annals of Botany 100 (2007) 1391-1401 or activation of plant defensive genes (Bergey et al., Proc. Natl. Acad. Sci. USA 93 (1996) 12053-12058. We have demonstrated that transient expression of defensin MsrA2 in Nicotiana benthamiana leaves significantly decreases the Pseudomonas infection symptoms (
The invention also provides a process of protecting crop plants on a field from a pest. In such process, infestation of at least one of the plants from a plurality of plants growing in a lot or farm field may be determined. Due to the rapidness of the process of the invention expression of a protein or RNA detrimental to the pest needs to be caused only if infestation by the pest is determined. Thus, strong and constitutive expression of pest toxins or dsRNA for RNAi even in the absence of a risk of infestation is not necessary. Transient expression of Bacillus thuringiensis endotoxins after the spraying with diluted agrobacterial cultures harbouring corresponding PVX-based expression vectors protected Nicotiana benthamiana plants from feeding damage by larvae of the tobacco homworm Manduca sexta (
The invention is further described in the following by way of examples. The invention is however not limited to these examples.
The concentration of Agrobacterium cells in liquid suspension in terms of colony forming units per ml (cfu/ml) of liquid suspensions can be determined using the following protocol. Cells of Agrobacterium tumefaciens strain ICF 320 transformed with construct pNMD620 were grown in 7.5 ml of liquid LBS medium containing 25 mg/L kanamycin (AppliChem, A1493) and 50 mg/L rifampicin (Carl Roth, 4163.2). The bacterial culture was incubated at 28° C. with continuous shaking. Absorbance or optical density of bacterial culture expressed in absorbance units (AU) was monitored in 1-ml aliquots of the culture using a spectrophotometer at 600 nm wavelength (OD600). The cell concentration estimated as a number of colony-forming units per milliliter of liquid culture (cfu/ml) can be analyzed at OD600 values 1; 1.3; 1.5; 1.7 and 1.8. For this purpose 250-μl aliquots of liquid culture were diluted with LBS-medium to achieve a final volume of 25 ml (dilution 1:100). 2.5 ml of such 1:100 dilution were mixed with 22.5 ml of LBS to achieve the dilution 1:1000. Liquid culture dilutions 1:100; 1:1,000; 1:10,000; 1:100,000; 1:1,000,000; 1:10,000,000 and 1:100,000,000 were prepared similarly. Aliquots of last three dilutions were spread on agar-solidified LBS medium supplemented with 25 mg/L kanamycin and 50 mg/L rifampicin (250 μl of bacterial culture per plate of 90 mm diameter). Plating of aliquots for each dilution was performed in triplicate. After 2 days incubation at 28° C., bacterial colonies were counted for each plate. Plating of 1:1,000,000 and 1:10,000,000 dilutions resulted in few hundred and few dozen colonies per plate, respectively. So far as dilution 1:100,000,000 provided just few colonies per plate, this dilution was not used for calculation of cell concentration. The cell concentration was estimated according to the formula: cfu/ml=4× number of colonies per plate×dilution factor.
For transforming cell concentrations as measured by absorbance measurements at 600 nm (in LB medium) and in terms of cell-forming units, the following relation is used herein: an OD600 of 1.0 corresponds to 1.1×109 cfu/ml.
1% soya peptone (papaic hydrolysate of soybean meal; Duchefa, S1330)
0.5% yeast extract (Duchefa, Y1333)
1% sodium chloride (Carl Roth, 9265.2)
dissolved in water, and the is adjusted to pH 7.5 with 1M NaOH (Carl Roth, 6771.2)
To prepare the solid LBS medium, liquid LBS medium was supplemented with 1.5% agar (Carl Roth, 2266.2). Media were autoclaved at 121° C. for 20 min.
In this study we used standard transcriptional vectors based on 35S CaMV promoter as well as TMV- and PVX-based viral replicons with or without cell-to-cell movement ability.
All transcriptional vectors were created on the basis of pICBV10, a pBIN19-derived binary vector (Marillonnet et al., 2004, 2006). They contained two expression cassettes inserted within right and left borders of same T-DNA region (
TMV-based vectors with cell-to cell movement ability (
TMV-based vectors lacking cell-to cell movement ability were identical to corresponding TMV-based vectors capable of cell-to-cell movement with an exception of point mutation in MP-coding sequence leading to the open reading frame shift that distorted the MP translation (
For cloning of most of PVX-based vectors with cell- to-cell and systemic movement ability, pNMD670 cloning vector was used. Resulting constructs contained, in sequential order, 35S CaMV promoter, coding sequences of RNA-dependent RNA polymerase, coat protein, triple gene block modules comprising 25 kDa, 12 kDa and 8 kDa proteins, gene of interest and 3′untranslated region. The entire fragment was cloned between the T-DNA left and right borders of binary vector (
PVX-based vectors with deletion of coat protein coding sequence were disabled for both systemic and cell-to cell movement. Cloning of these constructs was performed using pNMD694 as a cloning vector. This type of vectors contained, in sequential order, 35S CaMV promoter, coding sequences of RNA-dependent RNA polymerase, triple gene block module, gene of interest and 3′untranslated region inserted between the T-DNA left and right borders of binary vector (
We have shown that Nicotiana benthamiana plants can be transfected by spraying of plants with diluted agrobacterial cultures containing surfactant (
To find the optimal surfactant concentration, we tested 0.1% and 0.05% Silwet L-77 in dipping experiments. For all three types of vectors, the transfection efficiency provided by using of 0.1% Silwet was significantly higher if compared to 0.05% concentration (
10 sec dipping of Nicotiana benthamiana leaves in diluted agrobacterial suspension supplemented with 0.1% Silwet L-77 provided transfection rates close to the efficiency of spraying with same suspension (
The Silwet L-77 used in all examples herein was purchased from Kurt Obermeier GmbH & Co. KG (Bad Berleburg, Germany). The supplier is GE Silicones, Inc., USA. The Silwet L-77 used is an organosilicone product composed of 84.0% of polyalkyleneoxide modified heptamethyttrisioxane (CAS-No. 27306-78-1) and 16% of allyloxypolyethylene-glycol methyl ether (CAS-No. 27252-80-8). All concentrations of Silwet L-77 content given in the examples or figures relate to this commercial product.
We tested the number of plant species using agobacterial transfection with spraying and surfactant. First, we screened each species for optimal expression vector. For this purpose plant leaves were infiltrated using needleless syringe with 1:100 dilutions of OD=1.5 of five agrobacterial cultures harboring GFP expression transcriptional vectors: 1) transcriptional vector 35S-GFP+P19 (pNMD293), 2) TMV-based viral vector capable of cell-to-cell movement TMV(MP)-GFP (pNMD560), 3) TMV-based viral vector disabled for cell-to-cell movement TMV(fsMP)-GFP (pNMD570), 4) PXV-based viral vector capable of both systemic and cell-to-cell movement PVX(CP)-GFP (pNMD630) and 5) PVX-based viral vector disabled for both systemic and cell-to-cell movement PVX(ACP)-GFP (pNMD620). In some cases vacuum infiltration was performed.
We demonstrated the efficient Agrobacterium-mediated transfection for several Solanaceae species including Nicotiana benthamiana (all five vectors), tobacco Nicotiana tabacum (all five vectors), tomato Lycopersicon esculentum (PVX-based and transcriptional vectors), pepper Capsicum annuum, Inca berry Physalis peruviana, eggplant Solanum melongena, potato Solanum tuberosum (all with PVX-based vectors) (
Agrobacterium-mediated transfection was denonstrated for the lettuce Lactuca sativa from Asteraceae family (transcriptional vector), beet Beta vulgaris from Chenopodiaceae family (all five vectors), zucchini Cucurbita pepo from Cucurbitaceae family (transcriptional vector), and cotton Gossypium hirsutum from Malvaceae family (all five vectors (
Treatment of agrobacterial cells with acetosyringone (200 μM, 2 hours) significantly increased the transfection efficiency for several plant species including tomato, eggplant and zucchini (
Based on infiltration data, spraying with diluted agrobacterial suspensions was tested for the number of plant species. The efficient delivery of diluted agrobacteria (10−3) by spraying with suspensions containing 0.1% Silwet was demonstrated for several Nicotiana species (Nicotiana benthamiana, Nicotiana debne, Nicotiana excelsior, Nicotiana exigua, Nicotiana maritima and Nicotiana simulans) as it is shown using PVX with cell-to cell and systemic movement ability in
Delivery of agrobacteia to other species including Solanaceae, Chenopodiaceae, Amarantaceae and Aizoaceae families was demonstrated by both dipping in agrobacterial suspension and spraying with and without abrasive using transcriptional vectors as well as TMV and PVX vectors with and without cell-to-cell movement ability (
Using the GUS gene as a reporter, the successful transfection by spraying with agrobacterial suspension was achieved for rapeseed Brassica napus from Brassicaceae family (
The efficient transfection of plants using the spraying with diluted agrobacterial suspension was demonstrated also for monocot species.
In all examples described herein, spraying was performed either with a pump spray flasks with nominal volume or 500 or 1000 ml (Carl Roth, #0499.1 and #0500.1) based on direct manual pumping or with a pressure sprayer with 1.25 L volume (Gardena, #00864-20) exploiting the increased pressure for pumping. Plants were sprayed so as to wet completely leaves. Sprayers were shaken from time to time to ensure homogeneity of the suspensions to be sprayed, notably if the suspensions contained an abrasive.
The carborundum used in these experiments was a mixture of carborundum (silicon carbide) F800, F1000 and F1200 particles from Mineraliengrosshandel Hausen GmbH, Telfs, Austria. According to the provider, F800, F1000 and F1200 have surface median diameters of 6.5, 4.5 and 3 μm, respectively. 97 mass-% of the particles of F800, F1000 and F1200 have a surface diameter smaller than 14, 10 and 7 μm, respectively. 94 mass-% of the particles have a surface diameter larger than 2, 1, and 1 μm, respectively. F800, F1000 and F1200 were mixed in equal amounts by weight. 0.3% (w/v) of the mixed carborundum was added into the agrobacterial suspensions supplemented with 0.1% Silwet L-77 and used for the spraying of plants using the sprayers described in example 3.
The results shown in
We performed multiple subsequent treatments of Nicotiana benthamiana plants with diluted agrobacteria. For this purpose leaves were dipped in diluted suspensions of Agrobacterium carrying next constructs: pNMD570 (TMV(fsMP)-GFP without cell-to-cell movement ability), pNMD560 (TMV(MP)-GFP with cell-to-cell movement ability), pNMD580 (TMV(MP)-DsRED with cell-to-cell movement ability), pNMD620 (PVX(ACP)-GFP without cell-to-cell movement ability, pNMD600 (PVX(CP)-GFP with cell-to-cell and systemic movement ability) and pNMD610 (PVX(CP)-dsRED with cell-to-cell and systemic movement ability). After the transfection, these vectors form fluorescing spots differing in colour and size (
We demonstrated that spraying of Nicotiana benthamiana plants with 1:100 and 1:1000 dilutions of agrobacterial suspension provides efficient delivery of viral replicons capable of cell to cell-movement, which results in high expression of genes of interest, comparable with expression achieved using infiltration of agrobacteria. This was demonstrated for GFP (
We have demonstrated the induction of anthocyanin biosynthesis in Nicotiana tabacum leaves infiltrated with agrobacterial suspension harbouring the PVX-based viral vector providing the expression of MYB transcription factor anthocyanin 1 (ANT1) from Lycopersicon esculentum (
We have demonstrated the photobleaching of Nicotiana benthamiana leaves caused by silencing of phytoene desaturase (PDS) gene after the spraying of leaves with agrobacterial suspension bearing the PVX-based viral vector containing the fragment of PDS coding sequence in an anti-sense orientation (
We demonstrated the reduction in number of necrotic lesions caused by Pseudomonas syringae pv. syringae infection in Nicotiana benthamiana leaves after the preliminary spraying of plants with agrobacterial suspension bearing the PVX-based vector providing the expression of flagellin gene from Pseudomonas (pNMD1953) (
The sequence listing below contains the following nucleotide sequences:
SEQ ID NO: 1: TNA region of T-DNA region of pNMD280
SEQ ID NO: 2: TNA region of T-DNA region of pNMD033
SEQ ID NO: 3: TNA region of T-DNA region of pNMD035
SEQ ID NO: 4: TNA region of T-DNA region of pNMD661
SEQ ID NO: 5: TNA region of T-DNA region of pNMD670
SEQ ID NO: 6: TNA region of T-DNA region of pNMD694
SEQ ID NO: 7: TNA region of T-DNA region of pNMD1971
SEQ ID NO: 8: TNA region of T-DNA region of pNMD2210
SEQ ID NO: 9: TNA region of T-DNA region of pNMD050
SEQ ID NO: 10: TNA region of T-DNA region of pNMD1953
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 008 267.6 | Aug 2010 | EP | regional |
| 10 008 393.0 | Aug 2010 | EP | regional |
This application is a continuation of U.S. application Ser. No. 13/814,544, which was filed on Feb. 6, 2013, has a § 371(c) date of Apr. 30, 2013, and is the U.S. National Stage of International Application PCT/EP2011/002279, filed May 6, 2011, which designates the U.S and was published by the International Bureau in English on Feb. 16, 2012, and which claims the benefit of European Patent Application No. 10 008 267.6, filed Aug. 7, 2010, and European Patent Application No. 10 008 393.0, filed Aug. 11, 2010, all of which are hereby incorporated herein in their entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13814544 | Apr 2013 | US |
| Child | 16195049 | US |
