PROCESSES FOR GENERATING OR ALTERING A TRAIT OF A PLANT, PRODUCTION OF A PROTEIN OF INTEREST ON A PL

专利摘要:
plant transfection process. a process of transfecting a plant comprising spraying parts of said plant with an aqueous suspension containing cells of an agrobacterium strain and at least one abrasive suspended in said suspension, said agrobacterium strain comprising a DNA molecule comprising a DNA construct. nucleic acid containing a DNA sequence of interest to be transfected into the plant.
公开号:BR112013002981B1
申请号:R112013002981-1
申请日:2011-05-06
公开日:2019-07-09
发明作者:Anatoli Giritch；Yuri Symonenko；Simone Hahn；Doreen Tiede；Anton Shvarts；Patrick Roemer；Yuri Gleba
申请人:Nomad Bioscience Gmbh；
IPC主号:

专利说明:

Patent Description for PROCESSES
OF GENERATING OR CHANGING A TRACE IN A PLANT, PRODUCTION OF A PROTEIN OF INTEREST IN A PLANT AND PROTECTION OF CULTURE PLANTS IN A FIELD AGAINST A PRAGUE.
FIELD OF THE INVENTION
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 for generating or altering a trace in a plant that is growing in a field. The invention also relates to a process of producing a protein of interest in a plurality of plants in a field. The invention also relates to a process of protecting crop plants in a field against a pest. In addition, invention 15 relates to an aqueous suspension containing cells of an Agrobacterium strain suitable for transient large-scale transfection of plants grown in a production field for the processes of the present invention. The invention also relates to the use of particulate inorganic material for transient transfection of plants by means of spraying 20 with suspensions containing Agrobacterium cells and inorganic particulate material.
BACKGROUND OF THE INVENTION
Current genetic engineering processes for agriculture are all based on stable genetic modification of crop species, 25 first demonstrated in 1983 (Fraley et al 1983; Barton et al 1983) and marketed since 1996. Although the agricultural process based on genetic transformation plant stability is a reality today and the basis of a very successful new practice, it has several limitations, the main ones being the very long time and high cost necessary for the development of transgenic crops. General consensus among companies involved in plant biotechnology is that the R&D process requires, depending on the crop species, between 8 and 16 years old and the total average cost is estimated on page 1a / 60
Petition 870190011049, of 02/01/2019, p. 11/65
1a / 60 is said to be between $ 100 and $ 150 million. Because of these limitations, after more than 25 years since the discovery of a process of
Petition 870190011049, of 02/01/2019, p. 12/65
2/60 species of GM crops have been commercialized so far.
It is known that plant cells and whole plants can be reprogrammed transiently (for example, without a stable integration of new genetic material into a plant chromosome) and transient processes, such as viral infections, are rapid. Such transient processes could, in principle, allow a very rapid modification of the plant's metabolism in favor of certain traits or products that are of interest to the user. Evidently, such processes require a DNA or RNA vector (a virus or a bacterium), which has been manipulated to efficiently and safely transfect the plant, with the resulting effect being devoid of undesirable side effects. Previous attempts to use vectors based on plant viruses have been partially successful in that they allow transfection of plants to produce high-value recombinant proteins, such as certain biopharmaceuticals (Gleba et al. 2007, 2008; Lico et al. 2008). Use of viruses to manipulate other traits, such as entry traits (eg herbicide resistance, Shiboleth et al. 2001; Zhang and Ghabiral 2006) has been described in the literature, but transfection of the virus introduces so many undesirable changes in the infected host that this type of transitional process is no longer done for input traces. Transient processes can also be developed around the ability of Agrobacterium species to transfer part of their Ti plasmid to a eukaryotic cell, in particular plants. Use of Agrobacterium-based transfection is a basis for genetic manipulations, such as genetic transformation protocols and transient laboratory transfection assays. Industrial applications of transfection based on Agrobacterium have also been limited to the production of recombinant protein due to the optimal application conditions, such as vacuum infiltration of plants with bacterial suspensions, cannot be used on a large scale in the field, while spraying of the aerial parts or irrigation of plants with bacterial solutions results in a supposedly very small proportion of plant cells to be transfected and simple previous studies
3/60 did not address this specific issue. The combination of Agrobacterial distribution and use of viruses as a secondary messenger in one process has been successful in the production of high-value recombinant proteins, including complex biopharmaceuticals, such as complete IgG antibodies. However, when it comes to traits, such as entry traits or traits that require subtle objectively reprogramming of plant cell metabolism, this transfection process has the same limitations as with viral vectors.
There is considerable knowledge in the area of the use of microorganisms to control certain processes that require interaction of microorganisms with plants, including the use of microorganisms, such as Lactobacillus and Saccharomyces yeasts for fermentation of biomass (preparation of fermented foods, drinks), for biocontrol (Agrobacteríum, Myrotecium, strains) and use of Rhizobium strains for improved nitrogen fixation. In research and patent work in which microorganisms have been exploited as biocontrol agents, there is a considerable body of knowledge about how living cells should be applied to plant surfaces; in particular, studies have been carried out that have identified spray conditions and adjuvants (wetting agents, adhesives, etc.) to be used in spray mixes. Examples of such surveys are numerous. The following articles 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 strains of Agrobacteríum rhizogenes / radiobacter that have been used for decades to control crown gall in vineyards and orchards. There are two strains used commercially, one being a natural strain that carries plasmid (K84) and the other, a genetically modified derivative that was modified by deleting the gene necessary for conjugative plasmid transfer (K1026). (Kerr and Tate 1984; Vicedo et al. 1993; Reader et al. 2005; Kim et al. 2006; reviewed in Moore, 1988).
Agrobacteríum tumefaciens and A. rhizogenes are widely
4/60 used in research laboratories worldwide for transfection and stable genetic transformation of plants. These applications are based on the ability of Agrobacteríum to transfer genetic information to eukaryotic cells. Many of the genetically modified plants grown today, such as soybeans, canola and cotton, were generated through Agrobacterium-mediated genetic transformation. The essential difference between transient and stable transformation is that, in the process of stable transformation, DNA distributed by Agrobacteríum is eventually integrated into a plant chromosome and, after that, is inherited by the plant's offspring. 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 used. Subsequently, the knowledge accumulated in this field of science is of limited value for those interested in transient processes that have to be designed to have a massive character and affect multiple cells in the plant's body.
Transient transfection, on the other hand, takes into account only the previous stages of DNA distribution mediated by Agrobacteríum in a nucleus of a plant cell, together with the fact that such distributed DNA molecules, if properly designed to constitute a unit of transcription bringing promoter and specific terminator for plant and a coding part, will be transcribed in a nucleus even in the absence of the said integration of DNA in a plant chromosome, such expression resulting in a transient reprogramming of a plant cell. Such reprogramming was obtained first in the beginning and was developed in a standard laboratory tool for rapid evaluation of different genetic experiments. Considering that there is a considerable body of knowledge on Agrobacterium-mediated DNA transfer to plant cells, with the exception of some cases, this information is limited to laboratory-scale experiments and, until now, there have been few attempts to develop industrial-scale applications involving Agro
5/60 bacterium as a DNA vector. One of the limitations of laboratory applications is that the distribution of DNA based on Agrobacteríum requires certain treatments that are difficult or impossible to apply in the open field or on a large scale. In typical transient experiments, cells from cultured plants or parts of plants are treated with an excess of bacteria to provide maximum distribution. In typical research experiments, there is also an interest in levels of expression that are not economically viable if done on an industrial scale. In general, research in this field has led the inventors to the conclusion that the parameters that severely affect transient expression are those that allow the best access to the interaction of Agrobacteria to plant cells within a plant body. Most of these studies use vacuum infiltration, injection into the leaf of the plant or treatment with surfactant, surface wound of the plant, for example, with razor blades or a 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 additional amplification (based on viruses) of the original DNA is the Medicago group (D'Aoust et al. , 2008, 2009; Vezina et al., 2009) that relies entirely on vacuum infiltration as a distribution method. However, due to the fact that it is based on a greatly excessive proportion of bacteria for plant cells, current laboratory protocols used for transient plant transfection do not have great translational value, that is, they cannot be directly reproduced at a level industrial. Except in some cases (for example, Vaquero et al., 1999, DAoust et al., 2008, 2009), they also did not address the issue of the efficiency of the transient transfection process quantitatively. (Examples of such research are multiple; a quote from only a few of those representative was provided: 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
6/60 two cases described below, there have been no attempts in the literature to quantify the efficiency of the transitional process or provide sufficient understanding that could lead to the potential large-scale commercial exploitation of the phenomenon.
One of the industrial processes that is under development today is Magnifection, a process that is based on the vacuum infiltration of Agrobacterium in plant leaves. The Magnifection process (registered trademark of Icon Genetics GmbH as magnICON® and covered by several patents / patent applications) is a simple and indefinitely scalable protocol for expression of heterologous protein in plants, which lacks the stable genetic transformation of a plant but instead, it has transient amplification of viral vectors distributed to multiple areas of a plant body (systemic distribution) by Agrobacterium as precursors of DNA. Such a process is, in essence, an infiltration of whole mature plants with a diluted suspension of Agrobacteria that bring T-DNAs that encode viral RNA replicons. In this process, the bacteria assume the (formally viral) functions of primary infection and systemic circulation, while the viral vector provides cell-to-cell dissemination (short distance), amplification and high-level protein expression. Initial demonstration that viral infection can be initiated by the distribution of Agrobacteria to a copy of the viral genome in a plant cell comes from the pioneering work of Grimsley et al., 1986, in which a DNA virus was distributed, and a first infection , although very inefficient, with TMV, a cytoplasmic RNA virus distributed as a copy of DNA, derived from the work of Turpen etal., 1993. Current technology, however, is extremely effective and a few adult tobacco plants are sufficient to early construct optimization of the construct and rapid production of milligram quantities to grams of recombinant protein for preclinical or clinical evaluation or, in the case of individualized vaccines, for manufacturing. The scaled (industrial) version is essentially the same, but it is built around fully assembled viral vectors (instead of pro-vectors that require in-plant assembly) and requires trim
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Children for the distribution of Agrobacteríum in high yield to whole plants by means of vacuum infiltration. The process can be staggered, but requires submersion of aerial parts of plants in bacterial suspension under vacuum (the process involves the inversion of plants grown in pots or in trays), a procedure that imposes limitations on the volume of biomass that can be treated in this way. way, on the yield of the process, on the ways in which the plants can be grown before treatment and also brings certain costs that limit the use of the process to high cost products, such as recombinant biopharmaceutical products only. The Magnifection process is efficient since it allows transfection of almost all leaf cells of the treated plants or approximately 50% of the total biomass of the aerial part of the plant (the rest being stems and petioles). The process has been optimized in many ways, in particular by improving the viral replicon release by optimizing the post-translational modification of primary DNA transcripts (Marillonnet et al., 2005). However, the current process was built entirely around bacterial distribution methods, such as injection into plant leaves or vacuum infiltration (eg, Simmons et al., 2009), leaf injury (Andrews and Curtis, 2005) or spillage of Agrobacteríum in the soil (Agrodrenching, Ryu etal., 2004; Yang etal., 2008), while these methods cannot be applied for the mass treatment of 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 documents from other research groups - Voinnet et al ., 2003; Sudarshana et al., 2006; Gao et al., 2006; Mett et al., 2007; Lindbo, 2007a, b; Plesha etal., 2007, 2009; Huang etal., 2006; Regnard etal., 2009 ; Green etal., 2009; Shoji etal., 2009).
It should be mentioned that, although Agrobacteríum tumefaciens and A. rhizogenes are the DNA vectors that are used in most cases, there are other species of bacteria that can carry out similar DNA transfer to plant cells (Broothaerts et al., 2005).
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Attempts to quantify the treatment with Agrobacterium in whole plants after vacuum infiltration were carried out by a few research groups only. In the documents by Joh et al., 2005, 2006, it was concluded that the highest density of bacteria used, of 10 ⁹ cfu / ml, was the best (as opposed to 10 ⁸ cfu / ml or 10 ⁷ cfu / ml), as measured by total recombinant protein expression. In experiments by Lindbo, 2007a, b, essentially similar results were obtained as in our work, however, no count of transfected cells was performed and the conclusions were obtained from the levels of expression of recombinant proteins.
Attempts to use Agrobacterium treatment on whole plants without vacuum infiltration resulted in a very small number of cells initially transfected, thus greatly limiting the practical application of the process. One way to get around this initial limitation disclosed in the literature is the use of an effective secondary messenger, such as a plant virus, which allows amplification of the initially inefficient process by complementing it with a systemic movement and cell to cell based on viruses ( Azhakanandam et al., 2007). Agrobacterium-based distribution of DNA copies of plant viruses or viral plant vectors has been described for a long time (Grimsley et al., 1986 and, for TMV - Turpen et al., 1993) and allows to disperse the initial distributed replicon to a few plant cells for the rest of the body using viral infection process, such cell-to-cell movement and systemic movement of the virus. Such a process has limited practical utility for our purposes because viral infection dramatically alters the plant's performance; all applications currently considered are in the area of production of recombinant proteins in plants (reviewed by Gleba et al., 2007, 2008). It should also be noted that the document cited by Azhakanandam et al., 2007 does not even attempt to quantify the efficiency of the initial transfection and is based on a very high amount of Agrobacterium in the transfection media.
Starting from the prior art, it is an objective of the present invention
9/60 provide an efficient transient plant transfection process to be applicable to many plants growing in an agricultural field. It is also an objective of the present invention to provide a process of altering a trace in plants growing in an agricultural field. Namely, it is an objective of the present invention to provide an efficient process that allows transient transfection of plants using Agrobacteríum on a large scale without the need to apply pressure differences to introduce Agrobacteríum into the intercellular space of plants. It is also an objective to provide an Agrobacterium formulation suitable for this purpose.
SUMMARY OF THE INVENTION
Consequently, the present invention provides the following:
(1) A process of transfecting a plant with a nucleic acid construct or a DNA sequence of interest that comprises spraying said plant, such as aerial parts of said plant, with an aqueous suspension containing cells from an Agrobacterium strain and preferably, at least one abrasive suspended in said suspension, said strain of Agrobacterium comprising a DNA molecule comprising a nucleic acid construct containing a DNA sequence of interest. Said DNA sequence of interest can encode a protein or RNA to be expressed in said plant.
(2) A process of generating or changing a trace in a plant, comprising:
supply of said plant;
expression, in said plant, of a protein or an RNA capable of generating or altering said trace which comprises spraying the aerial parts of said plants with an aqueous suspension containing cells from an Agrobacteríum strain and at least one abrasive suspended in said suspension, said strain of Agrobacteríum comprising a DNA molecule comprising a nucleic acid construct containing a se
10/60 DNA sequence of interest, said DNA sequence of interest encoding said protein or said RNA.
(3) A process of generating or altering a trace in a plant, comprising:
(I) growing said plant to a desired growth state;
(li) expression, in said plant, of a protein or an RNA capable of generating or altering said trace which comprises spraying the aerial parts of said plants with an aqueous suspension containing cells from an Agrobacterium strain and at least one abrasive suspended in the said suspension, said Agrobacterium strain comprising a DNA molecule comprising a nucleic acid construct containing a DNA sequence of interest, said DNA sequence of interest encoding said protein or said RNA.
(4) A process for producing a protein of interest in a plant comprising:
(i) growing said plant to a desired growth state;
(ii) expressing, in said plant, said protein of interest comprising spraying aerial parts of said plants with an aqueous suspension containing cells from an Agrobacterium strain and at least one abrasive suspended in said suspension;
said Agrobacterium strain comprising a DNA molecule comprising a nucleic acid construct containing a DNA sequence of interest, said DNA sequence of interest encoding said protein of interest.
(5) A process for protecting crop plants in a field from a pest comprising:
(i) growth of said plants on the soil of said area;
(ii) determining, in a desired state of growth of said plants, infestation, by a pest, of at least one of the plant wounds;
(iii) expression, in said plant, of a protein or an RNA that is harmful to the pest determined in the previous step comprising spraying aerial parts of said plants with an aqueous suspension containing cells from an Agrobacterium strain and at least one suspended abrasive in said suspension, said Agrobacterium strain comprising a DNA molecule comprising a nucleic acid construct containing a DNA sequence of interest operably linked to a promoter, said DNA sequence of interest encoding said protein or said RNA.
(6) The process according to any one of (1) to (5), wherein said aqueous suspension contains said cells of said Agrobacterium strain in a concentration of maximum 2.2 · 10 ⁷ , preferably in the maximum 1.1 · 10 ⁷ , more preferably at most 4.4 10 ⁶ , even more preferably at most 1.1 · 10 ⁶ cfu / ml of said suspension.
(7) The process according to any one of (1) to (6), wherein said abrasive is an inorganic particulate carrier for wetting powders, such as silica or carborundum.
(8) The process according to (7), wherein said aqueous suspension contains said abrasive in an amount comprised between 0.02 and 2, preferably between 0.05 and 1 and more preferably between 0.1 and 0.5% by weight of said suspension.
(9) The process according to (7) or (8), wherein the average particle size of the abrasive added to the suspension is between 0.1 and 30, preferably between 0.1 and 10, more preferably between 0, 5 and 10 and, even more preferably, between 0.5 and 5 pm.
(10) The process according to any one of (7) to (9), in which the abrasive has a Dgo value of at most 40 pm, preferably at most 30 pm and in which the abrasive does not contain particles of a size above 45 pm, preferably not above 40 pm.
(11) The process under any of (1) to (10), in
12/60 that said suspension further comprises an agricultural spray adjuvant, preferably a non-ionic surfactant or a wetting agent.
(12) The process according to (11), wherein the spray adjuvant is an organo-silicone wetting agent, such as Silwet L-77.
(13) The process according to any one of (1) to (12), wherein said nucleic acid construct is flanked by a T-DNA edge sequence on at least one side, which allows the transfer of the said nucleic acid construct for cells of said plant.
(14) The process according to any one of (1) to (13), wherein said nucleic acid construct encodes a viral replication vector that encodes said protein of interest, said viral vector being incapable of systemic movement in that plant.
(15) The process according to any one of (1) to (13), wherein said DNA sequence of interest is operably linked to a promoter active in plant cells.
(16) Aqueous suspension containing cells from an Agrobacterium strain and at least one abrasive suspended in said suspension, wherein said aqueous suspension contains said cells from said Agrobacterium strain in a concentration of at most 4.4 · 10 ⁷ , preferably at most 1.1 10 ⁷ , more preferably at most 4.4 · 10 ⁶ , even more preferably at most 1.1 · 10 ⁶ cfu / ml of said suspension; said Agrobacterium strain comprising a heterologous DNA molecule comprising a nucleic acid construct containing a heterologous DNA sequence of interest that can be operably linked to a promoter; said suspension further optionally comprising a wetting agent preferably non-ionic, such as an organo-silicone surfactant.
(17) The process according to any one of (1) to (15), wherein said plant is a dicot plant.
(18) The process according to (17), in which said plant is
13/60 tobacco or other species of the genus Nicotiana, sugar beets or other species of the genus Beta, tomato, potato, pepper, soy, alfalfa, peas, beans, rapeseed or other species of the genus Brassica, cotton.
(19) The process according to any one of (1) to (15), wherein said plant is a monocot plant.
(20) The process according to (19), in which said plant is rice, corn, wheat, barley, oats, millet, sorghum.
(21) The process according to (4), wherein said protein is a cellulase used in saccharification of cellulose or hemicellulose polymers.
(22) Use of particulate inorganic material for transient plant transfection. In said use, the plants can be sprayed with an aqueous suspension containing Agrobacterium cells and particulate inorganic material.
(23) A plant transfection process comprising spraying aerial parts of said plant with an aqueous suspension containing at least one abrasive in said suspension, followed by spraying said aerial parts of said plant with an aqueous suspension containing cells from a Agrobacteríum strain comprising a DNA molecule comprising a nucleic acid construct containing a DNA sequence of interest. Said DNA sequence of interest can encode a protein or RNA to be expressed in said plant.
(24) A process of transfecting a plant with a nucleic acid construct or a DNA sequence of interest that comprises spraying said plant, such as aerial parts of said plant, with an aqueous suspension containing cells from an Agrobacterium strain, said strain of Agrobacteríum comprising a DNA molecule comprising a nucleic acid construct containing a DNA sequence of interest; wherein said aqueous suspension contains said cells of said strain of Agrobacteríum in a concentration of a maximum of 2.2 · 10 ⁷ , preferably a maximum of 1.1 10 ⁷ , more preferably
14/60 Reasonably at most 4.4 · 10 ⁶ , even more preferably at most 1.1 10 ⁶ cfu / ml of said suspension and wherein said suspension further comprises an agricultural spray adjuvant, preferably a non-ionic surfactant or a wetting agent.
The inventors of the present invention have found a way to strongly increase the likelihood of achieving plant transfection by Agrobacterium. The inventors found that the addition of an insoluble particulate material in aqueous suspensions of Agrobacterium significantly increases the transfection efficiency that can be achieved by spraying 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 in agricultural fields, so that problematic infiltration methods that make use of pressure differences can be avoided. The invention also allows the transfection of plants that, until now, were not amenable to transformation by spraying with suspensions of Agrobacterium.
DESCRIPTION OF THE FIGURES
Fig. 1 schematically shows vectors used in the Examples. RB and LB represent the right and left edges of T-DNA. P35S: 35S promoter of the cauliflower mosaic virus, O: omega translation enhancer; TNOs: nopaline synthase terminator; TOCs: ocs terminator.
Figs. 2A and B represent TMV-based viral vectors capable of moving from cell to cell. Pact2: promoter of the Arabidopsis actin 2 gene; o: 5 'end from TVCV (turnip obstruction virus); RdRp: Open Reading Frame (ORF) RNA polymerase dependent on cr-TMV RNA (tobamovirus that infects crucifers); MP: cr-TMV motion protein ORF, N: 3 'untranslated cr-TMV region; TNOs or nos: nopaline synthase terminator; SP: signal peptide; white segments interrupting gray segments in the ORFs of RdRp and MMP indicate introns inserted in these ORFs to increase the likelihood of RNA replicon formation in the plant cell cytoplasm, which is described in detail in document W02005049839.
Fig. 3 represents TMV-based vectors without the ability to move cell to cell. A point mutation in ORF MP leads to a frame shift (fs) preventing correct translation of MP.
Figs. 4A and B represent vectors based on PVX (potato X virus) capable of cell to cell movement. PVX-pol: PVX RNA-dependent RNA polymerase; CP: ORF of the capsid protein; 25K, 12K and 8 together indicate the 25 kDa, 12 kDa and 8 kDa triple gene block modules of PVX, N: 3 'untranslated PVX region.
Figs. 5A and B show PVX-based vectors with deletion of the coding sequence of the capsid protein with systemic movement and cell to cell deactivated.
Fig. 6. Photographs showing GFP fluorescence at 4 dpi (days post inoculation), under UV light due to GFP expression after immersing Nicotiana benthamiana plant leaves for 1 minute in diluted agrobacterial cultures containing Silwet surfactant 0.1% by weight L-77, as described in Example 2. Numerals 10 ² and 10 ' ³ show the dilution factor of nocturnal agrobacterial cultures of OD = 1.5 to 600 nm and thus indicate a dilution of 100 times and 1000 times, respectively. The vectors used are indicated and can be associated with the appropriate vector shown in Figs. 1 to 5. 35S-GFP + P19 transcription vector expressing GFP under the control of the 35S promoter and coexpressed with P19 silencing suppressor (pNMD293); TMV (fsMP) -GFP and PVX (ACP) -GFP - viral vectors with no ability to move cell to cell (pNMD570 and pNMD620, respectively). The percentage of cells expressing GFP (indicated) was counted after isolation of protoplasts from the left half of the leaf blade.
Fig. 7 shows photographs of isolated protoplasts for counting cells expressing GFP. Protoplasts that express GFP isolated after immersing Nicotiana benthamiana leaves in agrobacterial suspension (Οϋβοο = 1.5, dilution factor of 10-3). Viral vector based on TMV, TMV (fsMP) -GFP with no cell-to-cell movement capability
16/60 (pNMD570). 0.1% Silwet, immersion of 1 min; protoplasts were isolated at 4 dpi.
Fig. 8. Influence of Silwet L-77 concentration and density of agrobacterial culture on transfection efficacy after immersion of Nicotiana benthamiana leaves in agrobacterial suspension (OD ₆ oo = 1.5, 10 ' ² dilution factor and 10 ' ³ ). 35S-GFP + P19 transcription vector expressing GFP under the control of the 35S promoter and co-expressed with P19 silencing suppressor (pNMD293). Silwet concentration of 0.1% and 0.05%, immersion of 10 sec, 8 dpi.
Fig. 9. Influence of Silwet L-77 concentration and density of agrobacterial culture on transfection efficacy after immersion of Nicotiana benthamiana leaves in agrobacterial suspension (OD ₆ oo = 1.5, 10 ' ² dilution factor and 10 ' ³ ). Viral vector based on TMV, TMV (fsMP) GFP without cell to cell movement capability (pNMD570). Silwet concentration of 0.1% and 0.05%, immersion of 10 sec, 8 dpi. Percentage of cells expressing GFP was counted after protoplast isolation from the left half of the leaf blade.
Fig. 10. Influence of Silwet L-77 concentration and density of agrobacterial culture on transfection efficiency after immersion of Nicotiana benthamiana leaves in agrobacterial suspension (Οϋβοο =
1.5, 10 ' ² and 10 ³ dilution factor). Viral vector based on PVC, PVX (ACP) -GFP without cell to cell movement capacity (pNMD620). Silwet concentration of 0.1% and 0.05%, immersion of 10 sec, 8 dpi. Percentage of cells expressing GFP was counted after protoplast isolation from the left half of the leaf blade.
Fig. 11. Comparison of transfection rates obtained by immersion and spraying of Nicotiana benthamiana in / with agrobacterial suspensions. Dilution factors and transfection rates are indicated. The concentration of Silwet L-77 was 0.1% by weight. Agrobacterial culture (pNMD570, TMV-based viral vector with no cell-to-cell movement capability) was grown to ODeoo = 1.5 and diluted 100 times (10 ' ² ) and 1000 times (10' ³ ) in infiltration buffer supplemented with Silwet L-77 a
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0.1%. Immersion duration of 10 sec. Photographs are taken at 8 dpi. Percentage of cells expressing GFP was counted after protoplast isolation from the left half of the leaf blade.
Fig. 12 shows GFP expression photographs after distribution of diluted Agrobacteria to Nicotiana benthamiana by spraying with surfactant: TMV-based viral vector capable of moving from cell to cell (TMV-GFP, pNMD560); agrobacterial suspensions of ODeoo = 1.5 were diluted in dilution factors of 10 ' ² or 10' ³ , as indicated; 0.1% Silwet L-77, photo taken at 8 dpi.
Fig. 13A shows the results of transfection experiments by means of infiltration (using a needle-free syringe) of different plants with 100-fold dilutions (10 ' ² dilution factor) of agrobacterial cultures of OD ₆ oo = 1.5 . The suspensions used for spraying contained 0.1% by weight Silwet L-77, as described in Example 3. For each case, the same sheet is shown under normal light and under UV light showing GFP expression. Dashed circles indicate the treated leaf area. Numerals close to the treated leaf area indicate the strain / vector used as follows:
A) Infiltration with syringe. Vectors: 1 - TMV (fsMP) -GFP (pNMD570), 2 - TMV (MP) -GFP pNMD560), 3 - PVX (ACP) -GFP (pNMD620), 4 - PVX (CP) -GFP (pNMD630), 5 - 35S-GFP + P19 (pNMD293). Agrobacterial cultures were grown to Οϋβοο = 1.5 and diluted 100 times; photographs taken at 8 dpi.
B) Similarly as in A, however, transfection was performed by means of vacuum infiltration. Vector: PVX (CP) -GFP (pNMD630). Agrobacterial cultures were grown up to OD ₆₀ o = 1.5 and diluted 100 times; photographs taken at 43 dpi.
Fig. 14. Optimal expression vector screening using syringe infiltration of members of the Asteraceae, Chenopodiaceae, Cucurbitaceae and Malvaceae family. Numerals indicate the strains / vectors used as follows: 1 - TMV (fsMP) -GFP (pNMD570), 2 - TMV (MP) -GFP (pNMD560), 3 - PVX (ACP) -GFP pNMD620), 4 - PVX (CP ) -GFP (pNMD630);
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- 35S-GFP + Ρ19 (pNMD293). Dilution factor of agrobacterial cultures (Οϋβοο = 1.5-1.7): 10 '²; photographs taken at 8 dpi.
Fig. 15 shows factors that intensify agrobacterial transfection: aceto-syringone (AS). Vectors: 1 - TMV (fsMP) -GFP (pNMD570), 2 TMV (MP) -GFP (pNMD560) 3 - PVX (ACP) -GFP (pNMD620), 4 - PVX (CP) GFP (pNMD630); 5 - 35S-GFP + P19 (pNMD293). Dilution of agrobacterial cultures (OD ₆ oo = 1.5-1.7): 10 ' ² . For treatment with AS, acetosyringone (AS) at 200 μΜ was added to the agrobacterial suspension 2 hours before transfection. For comparison purposes, leaves not transfected with suspensions without AS are also shown (without AS).
Fig. 16. Photographs showing GFP expression under ultraviolet light in several Nicotiana species after spraying whole plants with agrobacterial suspensions diluted 1000 times of Οϋβοο = 1.0Viral vectors based on PVX with systemic movement capability and cell to cell were used (PVX (+ CP) -GFP, pNMD600). Sprayed suspensions contained 0.1% by weight Silwet L-77. Photographs taken at 12 dpi.
Fig. 17 shows GFP expression after distribution of diluted Agrobacteria to spinach and beetroot plants by immersion with a surfactant. A transcription vector, as well as viral vectors based on TMV and PVX without the ability to move cell to cell, were used. Agrobacterial cultures were grown to an OD ₆ oo = 1.5 and diluted 1: 100, 0.1% Silwet L-77, immersion for 10 seconds, photographs taken at 12 dpi.
Fig. 18 shows the expression of GFP after distribution of diluted Agrobacteria to tomato plants Lycopersicon esculentum by spraying in the presence of surfactant. The vector used was PVX (CP) -GFP (pNMD630). Dilution factor of the agrobacterial culture (OD ₆ oo = 1.5): 10 ² , and Silwet L-77 at 0.1%, aceto-syringone at 200 μΜ; photographs taken at 14 dpi.
Fig. 19 shows the expression of GFP after distribution of diluted Agrobacteria to plants of Inca berry Physalis peruviana by spraying with surfactant. PVX (CP) -GFP vector (pNMD630). Dilution of
19/60 agrobacterial culture (OD ₆ oo = 1.5): 10 ² , 0.1% Silwet L-77, acetosyringone at 200 μΜ; photographs taken at 14 dpi.
Fig. 20 shows a comparison between transfection by infiltration using a syringe and spray. Numerals indicate the Agrobacterium strain used. Distribution of diluted Agrobacteria to cotton sheets Gossypium hirsutum L. using infiltration with a syringe and spraying with agrobacterial suspensions. Nocturnal agrobacterial cultures (strain ICF320) were grown to an OD ₆ oo = 1.7-2.0, diluted in a 10 ' ² factor with an infiltration buffer and incubated with 200 μΜ acetosyringone for 2 hours before use. For spraying, the agrobacterial suspensions were additionally supplemented with 0.1% Silwet L-77.
Infiltration: 1 - TMV (fsMP) -GFP (pNMD570), 2 - TMV (MP) -GFP (pNMD560), 3 - PVX (ACP) -GFP (pNMD620), 4 - PVX (CP) -GFP (pNMD630); 5 - 35S-GFP + P19 (pNMD293).
Spraying: TMV (MP) -GFP (pNMD560).
Nicotiana benthamiana plant was used as a positive control.
Fig. 21 shows GFP expression in cotton leaves Gossipium hirsutum L. infiltrated with Agrobacteria suspension bringing viral and transcription vectors.
A) SDS-PAGE with Coomassie staining,
B) Western blot by hybridization with anti-GFP antibody (1: 3000), second antibody: anti-mouse HRP (1: 5000). The rows are as follows:
- N. benthamiana leaf not infected;
- Uninfected cotton sheet;
- Uninfected red cabbage leaf;
- Ladderde Proteina (Fermentas, # SM0671);
- TMV (fsMP) -GFP (pNMD570) in Nicotiana benthamiana;
- TMV (fsMP) -GFP (pNMD570) in cotton;
- TMV (MP) -GFP (pNMD560) in cotton;
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- PVX (ACP) -GFP (pNMD620) in cotton;
- PVX (CP) -GFP (pNMD630) in cotton;
- 35S9-GFP + P19 (pNMD293) in cotton.
100 mg of leaf material was boiled in 600 µl of 1 x Laemmli buffer containing beta-mercaptoethanol; 2.5 μΙ aliquots were loaded onto the gel.
Fig. 22 shows GFP expression after distribution of Agrobacteria to leaves of Beta vulgaris vulgaris L. using surfactant spray; influence of aceto-syringes and abrasives. Vector: PVX (CP) -GFP (pNMD630). Agrobacterial cells were incubated with 200 μΜ aceto-syringone for 2 hours before spraying. For abrasive treatment, 0.3% carborundum (mixture of silicon carbide particles F800, F1000 and F1200, Mineraliengrosshandel Hausen GmbH, Telfs, Austria) was added to the agrobacterial suspension. Agrobactería dilution factor of OD ₆ oo = 1.4: 10 ' ² . Points expressing GFP on the leaves are indicated on the right.
Fig. 23 shows GFP expression after distribution of diluted Agrobacteria to plants of different species by spraying with a surfactant and abrasives. Vectors: TMV (MP) -GFP (pNM600) and PVX (CP) -GFP (pNMD630), dilution factor of the agrobacterial culture (OD ₆ oo = 1.5): 10 “ ² , Silwet L-77 to 0.1 %, 0.3% silicon carbide.
Fig. 24 shows expression after subsequent triple treatments of Nicotiana benthamina plants with Agrobacteria bringing viral vectors. Leaf immersion was performed with an interval of 7 days in the order:
A) PVX (ACP) -GFP, PVX (CP) -DsRed, TMV (MP) -GFP;
B) TMV (fsMP) -GFP, PVX (CP) -DsRed, PVX (CP) -GFP;
C) PVX (ACP) -GFP, TMV (MP) -DsRed, PVX (CP) -GFP;
D) TMV (fsMP) -GFP, PVX (CP) -DsRed, TMV (MP) -GFP;
E) TMV (fsMP) -GFP, TMV (MP), DsRed, TMV (MP) -GFP;
F) PVX (ACP) -GFP, TMV (MP), DsRed, TMV (MP) -GFP;
G) TMV (fsMP) -GFP, PVX (CP) -DsRed, TMV (MP) -GFP;
H) PVX (ACP) -GFP, PVX (CP) -DsRed, TMV-GFP.
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Simple treatments:
I) TMV (fsMP) -GFP;
J) PVX (ACP) -GFP;
K) TMV (MP) -GFP;
L) PVX (CP) -GFP;
M) TMV (MP); and
N-DsRed) PVX (CP) -DsRed.
Fig. 25. Analysis of GFP expression in Nicotiana benthamiana plants sprayed with agrobacterial suspensions (dilution factor 10-2) bringing TMV (MP) -GFP (pNM600) and PVX (CP) -GFP (pNMD630) vectors.
A) Photograph of Nicotiana benthamiana plants transfected by spray, taken at 15 dpi.
B) SDS-PAGE with Coomassie staining; 12% gel, reduction conditions. Leaves of N. Benthamiana plants transfected by spray expressing GFP) were collected at 15 dpi. The plant material was extracted with 6 volumes of 1 x Laemmli buffer containing betamercaptoethanol. After heating to 95 ° C, 10 μΙ aliquots were loaded onto the gel.
L - Ladderde Proteina (Fermentas, # SM0671);
- TMV (MP) -GFP, plant 1;
- TMV (MP) -GFP, plant 2;
- TMV (MP) -GFP, plant 3;
- PVX (CP) -GFP, plant 1;
- PVX (CP) -GFP, plant 2;
- PVX (CP) -GFP, plant 3;
U - Uninfected leaf tissue of N. benthamiana;
- TMV (MP) -GFP, vacuum infiltrated plant;
- PVX (CP) -GFP, vacuum infiltrated plant.
RbcL - large subunit of RUBISCO.
Fig. 26. SDS-PAGE with Coomassie staining for analysis of human alpha-interferon (Hu-IFN-αΑ) and Klip7-Mini-insulin expressed in
22/60 Nicotiana benthamiana plants sprayed with agrobacterial suspensions of viral vectors based on TMV capable of cell to cell movement.
A) Hu-IFN-αΑ: vectors pNMD38 and pNMD45, 10 ' ² dilution factor of the agrobacterial culture, collected at 12 dpi.
L - Ladderde Proteina (Fermentas, # SM0671);
- pNMD38, syringe infiltration;
- pNMD38, spraying, Folhai;
- pNMD38, spraying, Leaf2;
- pNMD38, spraying, Leaf3;
- pNMD45, spraying, Folhai;
- pNMD45, spraying, Leaf2;
- pNMD45, spraying, Leaf3;
U - Infected N. Benthamiana leaf tissue.
B) Klip27-Mini-insulin: vector pNMD331, dilution factors of 10 ' ² and 10' ³ of the agrobacterial culture, collected at 12 dpi.
L - Ladderde Proteina (Fermentas, # SM0671);
- pNMD331, infiltration with syringe, dilution of 10 '³;
- pNMD331, spraying, 10 ' ³ dilution;
- pNMD331, infiltration with syringe, dilution of 10 ' ³ .
The plant material was extracted with 6 volumes of 1 x Laemmli buffer containing beta-mercaptoethanol. 10 μΙ aliquots were separated on a 15% polyacrylamide gel under reduction conditions.
Fig. 27. Expression of cellulases in N. benthamiana plants obtained by spraying diluted agrobacterial cultures bringing TMV vectors capable of cell-to-cell movement (7 and 10 dpi). N. benthamiana plants were inoculated with 10 ' ² (top panel) and 10' ³ (bottom panel) dilutions of Agrobacterium cultures (OD ₆ oo = 1.3) using a needle-free syringe or spraying with Silwet L-77 0.1%. The levels of cellulase fusion protein were analyzed in crude extracts using SDS-PAGE with Coomassie staining. For crude extracts, 50 mg of plant material (combined samples of 3 independent leaves), paste
23/60 at 10 dpi, were ground in liquid nitrogen, extracted with 5 vol. 2 x Laemmli buffer and denatured at 95 ° C for 5 min. 7.5 μΙ of each sample were analyzed using 10% SDS-PAGE and Coomassie staining.
L - Ladderde protein (Fermentas, # SM0671);
- N. benthamiana leaf tissue not infected;
- Exocellulase E3 of Thermobifida fusca directed to the apoplast,
- Exoglucanase 1 (CBH 1) from Trichoderma reesei directed to the apoplast;
- β-glycosidase BGL4 from Humicola grísea directed to chloroplasts;
- β-glucosidase BGL4 from Humicola grisea expressed in the cytosol;
- β-glucosidase BGL4 from Humicola grisea His-tagged;
- Exocellulase E3 of Thermobifida fusca directed to chloroplasts;
- Endoglucanase E1 from Acidothermus cellulolyticus directed to the apoplast.
Fig. 28. Induction of anthocyanin biosynthesis in leaves of Nicotiana tabacum via transient expression, through infiltration, of anthocyanin MYB transcription factor 1 (ANT1) of Lycopersicon esculentum (AAQ55181). 7 dpi, from Agrobacteríum culture (OD ₆ oo = 1.4), To · ² dilution factor.
Fig. 29. Morphological changes in Nicotiana benthamiana plants caused by the transient expression of the isopentenyl transferase (ipt) gene distributed by spraying with diluted Agrobacteria bringing the transcription vector containing the ipt coding sequence under the control of the 35S promoter. Agrobacteríum culture (OD ₆ oo = 1> 4) was diluted by a factor of 10 ' ² and supplemented with 0.1% Silwet L-77. Photograph taken at 45 dpi.
A) Habitat of transfected plants. IPT - Plants sprayed with diluted agrobacterial culture (construct PNMD460), control - plant
24/60 sprayed with transfection buffer not containing Agrobacteria.
B) Leaves of transfected plants. Upper part: plant leaves sprayed with buffer for transfection not containing Agrobacteria. Bottom: plant leaves sprayed with diluted agrobacterial culture (construct pNMD460).
Fig. 30. Transient expression of Bacillus thuringiensis endotoxins after spraying with diluted agrobacterial cultures bringing corresponding PVX-based expression vectors protects Nicotiana benthamiana plants from damage by feeding by manduca sexta tobacco larvae. The plants were sprayed with Agrobacterium cultures (Οϋβοο = 1.4-1.7) diluted by a factor of 10 ' ² and supplemented with 0.1% Silwet L-77. Two weeks later, three stage 3 larvae were allowed to feed on each plant. Photographs were taken 2 weeks after starting feeding.
Fig. 31. Phenotypes of N. benthamiana transiently expressing defensin MsrA2 and GFP via TMV-based vectors capable of cell-to-cell movement (pNMD1071 and pNMD560, respectively). Inoculation with Pseudomonas was performed 3 days after inoculation with Agrobacterium. Photographs were taken 4 days after inoculation with Pseudomonas. Inoculation of leaves with Agrobacterium and Pseudomonas was performed using a syringe without a needle.
Fig. 32 shows GFP expression after Agrobacteria distribution to eggplant leaves Solanum melongena L. using surfactant spray (0.1% Silwet L-77); abrasive influence. Vector: PVX (CP) GFP (pNMD630). Agrobacterial cells (strain ICF320) were incubated with aceto-syringone at 200 μΜ for 2 hours before spraying. For abrasive treatment, 0.3% carborundum (mixture of silicon carbide particles F800, F1000 and F1200, Mineraliengrosshandel Hausen GmbH, Telfs, Austria) was added to the agrobacterial suspension. Agrobactería dilution factor of OD ₆ oo = 1.3: 10 ' ² . Photographs were taken at 19 dpi. The number of points that express GFP is provided on the right.
Fig. 33 shows GFP expression after A distribution
25/60 grobacteria to pepper leaves Capsicum annuum L. cv Feher Gelb using spray with surfactant (0.1% Silwet L-77), the synergistic action of aceto-syringes and abrasives. Vector: PVX (CP) -GFP (pNMD630). Agrobacterial cells (ICF320 strain) were incubated with aceto-syringone at 200 μΜ for 2 hours before spraying. For abrasive treatment, 0.3% carborundum (mixture of silicon carbide particles F800, F1000 and F1200, Mineraliengrosshandel Hausen GmbH, Telfs, Austria) was added to the agrobacterial suspension. Agrobactería dilution factor of OD ₆ oo = 1.4: 10 ' ² . Photographs were taken at 18 dpi. The number of points that express GFP is provided on the right.
Fig. 34 represents the expression of GFP after distribution of Agrobacteria to potato leaves Solanum tuberosum L. cv Mirage using vacuum infiltration and spray with surfactant (0.1% Silwet L-77); comparison of vacuum infiltration and spraying. Vector: PVX (CP) -GFP (pNMD630). Agrobacterial cells (ICF320 strain) were incubated with aceto-syringone at 200 μΜ for 2 hours before spraying. Agrobacteria dilution factor of Οϋθοο = 1.5: 10 ' ² . Photographs were taken at 14 dpi.
Fig. 35 shows GUS expression after distribution of Agrobacteria to rapeseed leaves Brassica napus L. using syringe infiltration and spraying with surfactant (0.1% Silwet L-77). Vector: 35SGUS + 35S-P19 (pNMD1971). Agrobacterial cells (strain EHA105) were incubated with 200 μΜ aceto-syringone for 2 hours before spraying. Agrobactería dilution factor of OD ₆ oo = 1.3: 10 ' ¹ and 10' ² . Infiltration with syringe: 1: dilution of agrobacterial culture to 10 ' ¹ , 2: dilution of agrobacterial culture to 10' ² . For spraying, dilution of the 10 ' ¹ agrobacterial culture was used. Photographs for infiltrated and pulverized leaves were taken at 5 and 13 dpi, respectively.
Fig. 36 represents the GUS expression after distribution of Agrobacteria to Allium cepa cv Stuttgarter onion leaves sprayed with Agrobacteria using surfactant (0.1% Silwet L-77). Agrobacterial cells (strains EHA105 and GV3101) were incubated with aceto-syringone at 200
26/60 μΜ for 2 hours before spraying. Vectors: 35S-GUS + 35S-P19 (pNMD1971) and rice actin promoter-GUS + 35S-P19 (pNMD2210). ODgoo Agrobacteria dilution factor = 1.3: 10 ' ¹ . Photographs were taken at 11 dpi.
Fig. 37 shows the photo-bleaching, by means of gene silencing, of phytoene desaturase (PDS) in Nicotiana benthamina leaves after distribution, mediated by Agrobacteríum, of PVX constructs bringing the fragment of the PDS coding sequence in orientation antisense; comparison of infiltration with a syringe and spraying with surfactant (0.1% Silwet L-77). Vector: PVX (CP) -antiPDS (pNMD050); strain GV3101 from Agrobacteríum tumefaciens. Agrobacteria dilution factor of OD ₆ oo = 1.5: 10 ' ² . Photographs were taken at 21 dpi and 140 dpi.
Fig. 38 shows the effect of transient flagellin expression on the infection of Nicotiana benthamiana by Pseudomonas. A) Leaves of Nicotiana benthamiana plants infected with Pseudomonas syringae pv. syringe B728a. B) Symptoms of the disease counted as the number of necrotic lesions (see black dots) per leaf. 1 - preliminary plant sprayed with agrobacterial suspension (strain ICF320) bringing vector PVX (CP) that allows the expression of full-length flagellin fusion (YP236536) of Pseudomonas syríngae pv. syringae B728a with barley α-amylase apoplast signaling peptide (pNMD1953), 2 - preliminary plant sprayed with agrobacterial cells (ICF320 strain) without any vector containing T-DNA.
DETAILED DESCRIPTION OF THE INVENTION
In the invention, Agrobacteria are used for transfecting plants with a sequence or construct of interest by spraying with aqueous suspensions containing cells from an Agrobacterium strain. The Agrobacteríum strain may belong to the species of Agrobacteríum tumefaciens or Agrobacteríum rhizogenes that are commonly used for plant transformation and transfection and which are known to those skilled in the field. The Agrobacteríum strain comprises a DNA molecule that comprises a nucleic acid construct containing a
27/60 DNA sequence of interest. The DNA sequence of interest encodes a protein or an RNA to be expressed in a plant. The nucleic acid construct is typically present in the T-DNA of plasmids Ti for the introduction of the nucleic acid construct into plant cells through the secretion system of the Agrobacteríum strain. On at least one side or on both sides, the nucleic acid construct is flanked by a T-DNA border sequence to allow transfection of said plant (s) and introduction of cells from that plant with said DNA sequence of interest. In the nucleic acid construct, the DNA sequence of interest is present in order to be expressed in plant cells. For this purpose, the DNA sequence of interest is, in said nucleic acid construct, normally under the control of an active promoter in plant cells. Examples of the DNA sequence of interest are a DNA sequence that encodes a viral DNA replicon or a viral RNA replicon or a gene to be expressed. The gene can encode an RNA of interest or a protein of interest to be expressed in cells of the plant (s). Also, viral replicons normally encode an RNA or 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. Transference of genes and respective vectors mediated by Agrobacteríum is known to those versed in the field, for example, from the references cited in the introduction or textbooks on plant biotechnology, such as Slater, Scott and Fowler, Plant Biotechnology, second edition, Oxford University Press, 2008.
In modalities where strong expression of a protein or RNA is desired or where accumulation of viral nucleic acids in large amounts in plant cells and possible negative effects on plant health are not an issue, the nucleic acid construct can encode a viral replication vector that can reproduce in plant cells. To be reproducible, the viral vector contains an origin of replication that can be recognized by a nucleic acid polymerase present
28/60 in plant cells, such as viral polymerase expressed from the replicon. In the case of viral RNA vectors, viral replicons can be formed by transcription under the control of a plant promoter from the DNA construct after it has been introduced into the plant cell. In the case of viral DNA replicons, viral replicons can be formed by recombination between two recombination sites that flank the coding sequence of the viral replicon in the DNA construct, for example, as described in documents WOOO / 17365 and WO 99 / 22003. If viral replicons are encoded by the DNA construct, viral RNA replicons are preferred. Use of viral DNA and RNA replicons has been widely described in the literature, at least over the past 15 years. Some examples are the following Icon Genetics Patent Publications: W02008028661, WO2007137788, WO 2006003018, W02005071090, W02005049839, W002097080, WO02088369, WO02068664. An example of viral DNA vectors are those that are based on geminivirus. For the present invention, viral vectors or replicons that are based on plant RNA viruses, namely based on single-stranded + sense RNA viruses, are preferred. Examples of such viral vectors are tobacco mosaic virus (TMV) and Potex X virus (PVX) used in the examples. Viral vectors and expression systems that are based on the Potex virus 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 a maximum of 1.1 10 ⁹ cfu / ml, which corresponds approximately to a culture of Agrobacterium in LB medium of an optical density at 600 nm of 1. Due to the high transfection efficiency obtained in the invention, much lower concentrations can, however, be used, which allows treatment of many plants, such as whole agricultural fields, without the need for large fermenters for the production of Agrobacterium. Thus, the concentration is preferably at most 2.2 10 ⁷ cfu / ml, more preferably at most 1.1 10 ⁷ cfu / ml, more preferably at most 4.4 · 10 ⁶
29/60 cfu / ml. In one embodiment, the concentration is a maximum of 1.1 · 10 ⁶ cfu / ml of suspension. To avoid determining cell concentrations in terms of CFU / ml, concentrations of agrobacterial suspensions are often assessed by measuring the apparent optical density at 600 nm using a spectrophotometer. Here, the concentration of 1.1 10 ⁷ cfu / ml corresponds to a calculated optical density at 600 nm of 0.01, where the calculated optical density is defined by a 100-fold dilution with water or a suspension buffer that has an optical density of 1.0 to 600 nm. Likewise, the concentrations of 4.4 · 10 ⁶ cfu / ml and 1.1 - 10 ⁶ 10 cfu / ml correspond to an calculated optical density at 600 nm of 0.004 and 0.001, respectively, where the calculated optical densities are defined by a dilution of 250 times or 1000 times, respectively, with water or a suspension buffer that has an optical density of 1.0 to 600 nm.
The abrasive that can 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, particularly if used in conjunction with a wetting agent, the surface of the plant tissue, such as leaves, and thus facilitates the penetration of Agrobacterium cells into the intercellular space of the plant tissue. . As a result, transfection efficiency increases.
The particulate material to be used as the abrasive of the present invention can be a support material, as commonly used as wettable powder vehicles (WP) of pesticidal formulations. In the context of wetting powders, these vehicles are also referred to in the field of pesticide formulations as fillers or inert fillers. Wetting powder formulations are part of the general knowledge in the field of plant protection. Reference is made to the book PESTICIDE SPECIFICATION, Manual For Deve30 lopment and Use of FAO and WHO Specifications for Pesticides, edited by the World Health Organization (WHO) and Agriculture Organization of the United States, Rome, 2002, ISBN 92 -5-104857- 6. Wet powder formulations
30/60 civil protection measures are, for example, described in EP 1810569, EP1488697, EP1908348 and EP0789510. The abrasive can be a mineral material, typically an inorganic material. Examples of such carrier materials are diatomaceous earth, talc, clay, calcium carbonate, bentonite, acid clay, atapulgite, zeolite, sericite, sepiolite or calcium silicate. It is also possible to use quartz powder, such as the high purity quartz powder described in WO02 / 087324. Preferred examples are silica, such as fumigated and precipitated hydrophilic silica and carborundum. The abrasive properties of diluents or fillers, such as silica, used in wetting powders are known (see Pesticide Application Methods by G.A. Matthews, third edition, Science Blackwell, 2000, on page 52 thereof).
As commercial products of inorganic particulate materials for use as abrasives in the invention, the hydrophilic silica Sipernat ™ 22S and Sipernat ™ 50S manufactured by Degussa Evonic can be mentioned. Other products are Hi-Sil ™ 257, a synthetic, amorphous, hydrated silica produced by PPG Industries Ltda. Taiwan or Hubersorb ™ 600, a synthetic calcium silicate manufactured by Huber Corporation. A commercial silica of submicron size is Hi-Sil ™ 233 (PPG Industries) having an average particle size of about 0.02 pm.
The abrasive can 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 30. 20, even more preferably between 0.5 and 20 and, even more preferably, between 1.0 and 16 pm. In one embodiment, the median particle size is between 0.015 and 1 or between 0.02 and 0.5 microns. The median particle size is the median particle size by volume that can be measured by laser diffraction using a Mastersizer ™ from Malvern Instruments, Ltd. In order to avoid clogging of the spray nozzles, the maximum particle size of the largest particles contained in the abrasive should be a maximum of 45 pm, preferably a maximum of 40 pm, which can be determined by sieving. This condition is considered
31/60 satisfied if the residue in the sieve is below 1.5% by weight (according to ISO 3262-19). The abrasive can have a D ₉₀ value of at most 40 pm, preferably at most 30 pm, measured by means of laser diffraction, as described above. Typically, the above particle sizes refer to primary particle sizes.
The abrasive content in the aqueous suspension of the invention can 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 aid can be a surfactant or wetting agent. The surfactant and wetting agent have multiple advantages of the present invention. It reduces the surface tension of the water in the aqueous suspension and makes the waxy surface of plant leaves more permeable to Agrobactería. In addition, it improves the stability of the suspension and reduces the settling of the abrasive in the suspension. Surfactants used in the present invention are not particularly limited and examples of surfactants include (A), (B) and (C) below. These can be used alone or in combination.
(A) Non-ionic surfactants: a measure often used to describe surfactants is HLB (hydrophilic / lipophilic balance). HLB describes the ability of the surfactant to associate with hydrophilic and lipophilic compounds. Surfactants with a high HLB balance are better associated with water-soluble compounds than with oil-soluble compounds. Here, the HLB value should be 12 or more, preferably at least 13. As non-ionic surfactants, organo-silicone surfactants, such as heptamethyl tri-siloxane modified with polyalkylene oxide, are more preferred in the present invention. A commercial product is GE Advanced Materials' Silwet L-77 ™ spray adjuvant.
(A-1) Polyethylene glycol type surfactants: Examples of polyethylene glycol type surfactants include alkyl polyoxyethylene (C12-18), ether, ethylene oxide ethylene oxide addition product, polyoxyethylene (mono or di) al
32/60 kil-ether (C8-12) phenyl, condensation product of polyoxyethylene (mono or di) alkyl (C8-12) phenyl polyoxyethylene (mono, di, or tri) phenyl phenyl polyoxyethylene (mono) , di, or tri) -benzyl phenyl ether, polyoxyethylene (mono, di, or tri) benzyl phenyl ether of polyoxyethylene (mono, di, or tri) phenyl ether, polyoxypropylene (mono, di or tri) phenyl ether of styrene, a polyoxyethylene polymer (mono, di, or tri) phenyl styrene ether, a polyoxyethylene polyoxypropylene polymer block, a (C12-18) polyoxyethylene polyoxypropylene block polymer ether, a (C8-12) -phenyl polyoxyethylene polyoxypropylene block ether polymer, bisphenyl polyoxyethylene ether, resin acid polyoxyethylene ester, polyoxyethylene- (C12-18) fatty acid monoester, diester polyoxyethylene fatty (C12-18), polyoxyethylene sorbitan fatty acid (C12-18) ), ethylene oxide adduct ester of glycerin ester of fatty acid, castor oil ethylene oxide adducts, hardened castor oil ethylene adducts, alkyl ethylene oxide (C12 -8 amine) adduct and ethylene oxide adduct of fatty acids (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, (C12-18) fatty acid ester sorbitol, sorbitan fatty acid ester (C12-18), sucrose fatty acid ester, polyvalent alkyl alcohol ether and fatty acid alkanol amide.
(A-3) Acetylene-type surfactants: Examples of acetylene-type surfactants include acetylene glycol, acetylenic alcohol, ethylene oxide adduct of acetylene glycol and ethylene oxide adduct of acetylenic alcohol.
(B) Anionic surfactants:
(B-1) Carboxylic acid type surfactants: Examples of carboxylic acid type surfactants include polyacrylic acid, polymethacrylic acid, polymalleic acid, maleic acid and olefin copolymers (e.g., isobutylene and diisobutylene), an acrylic acid copolymer 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
33/60 co, 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 (C12-18) fatty acids and the like and salts of these carboxylic acids.
(B-2) Sulfate ester type surfactants: examples of sulphate ester type surfactants include (C12-18) alkyl sulfate ester, (C12-18) alkyl ether polyoxyethylene sulfate, (mono or di) (C8-12) alkyl phenoxy polyoxyethylene (mono or di) alkyl (C8-12) alkyl phenyl sulfate ester, sulfate ester of a polyoxyethylene (mono or di) alkyl (C8-12) phenyl ether polymer, polyoxyethylene ether (mono, di , or tri) -phenyl phenyl ester of polyoxyethylene ether sulfate (mono, di, or tri) phenyl benzyl ether sulfate, polyoxyethylene (mono, di, or tri) -ester styryl sulfate phenyl ether, sulfate ester of a polyoxyethylene (mono, di, or tri) phenyl ether styrene polymer, sulfate ester of a polyoxyethylene polyoxypropylene polymer block, sulfated oil, sulfated fatty acid ester, sulfated fatty acids, sulfated olefin sulfate ester and the like and salts of these sulfate esters.
(B-3) Sulfonic acid type surfactants: Examples of sulfonic acid type surfactants include (C12-22) paraffin sulfonic acid, (C8-12) alkyl benzene sulfonic acid, the product of an acid's formaldehyde condensation ( C8-12) alkyl benzene sulfonic, condensation product of formaldehyde and cresol sulfonic acid, sulfonic acid of (C14-16) olefins, (C8-12) dialkyl sulfo-succinic acid, sulfonic acid of lignin, sulfonic acid of (mono or di) (C8-12) polyoxyethylene alkyl phenyl ether, (C12-18) sulfo succinate, polyoxyethylene half-ether alkyl, naphthalene sulfonic acid, (mono- or di) (C1-6) alkyl naphthalene sulfonic acid , the product of condensation of formaldehyde and naphthalene sulfonic acid, the product of condensation of formaldehyde and acid (mono or di) (C1-6) alkyl naphthalene sulfonic acid, the product of condensation of formaldehyde and sulfonic acid of creosote oil, alkyl ( C8-12) diphenyl ether of di-s acid ulfonic, Igepon T (trademark), polystyrene sulfonic acid, sulfonic acid of a copolymer of styrene sulfonic acid - methacrylic acid and the like and salts of these sulphonic acids34 / 60 phonic
(B-4) Phosphate ester-type surfactants: Examples of phosphate ester-type surfactants include (C8-12) alkyl phosphate ester, (C12-18) phosphate ester, polyoxyethylene alkyl, (Phosphate ester) mono or di) (C8-12) alkyl phenyl ether of polyoxyethylene, phosphate ester of a polymer of (mono, di or tri) (08-12) alkyl phenyl ether of polyoxyethylene, phosphate ester of (mono, di, or tri) phenyl phenyl ether of polyoxyethylene, phosphate ester of (mono, di or tri) benzyl phenyl ether of polyoxyethylene, ester phosphate of (mono, di or tri) styrene phenyl ether of polyoxyethylene, phosphate ester of a polymer of (mono , di, or tri) polyoxyethylene phenyl ether, polyoxyethylene - polyoxypropylene block phosphate ester, phosphatidyl choline, phosphatidyl ethanolimine phosphate ester and condensed phosphoric acid (for example, such as tripolyphosphoric acid) and the like and salts of these phosphate esters.
Salts of (B-1) to (B-4) mentioned above include alkali metals (such as sodium, lithium and potassium), alkaline earth metals (such as calcium and magnesium), ammonium and various types of amines (such as alkylamines, cycloalkylamines and alkanol amines).
(C) Amphoteric surfactants: examples of amphoteric surfactants include betaine surfactants and amino acid surfactants.
The above surfactants can be used alone or in combination of two or more surfactants. Notably, the preferred organo-silicone surfactants can be combined with other surfactants. The total concentration of surfactants in the aqueous suspension of the invention can be easily tested by performing comparative spray experiments, similar to those made in the examples. However, in general, the total concentration of surfactants can be between 0.005 and 2% by volume, preferably between 0.01 and 0.5% by volume, more preferably between 0.025 and 0.2% by volume of said suspension. Since the density of surfactants is, in general, close to 1.0 g / ml, the total concentration of surfactants can 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 surfactants
35/60 of organo-silicone above, such as polyalkylene oxide modified hetapmethyl tri-siloxane, are used, the concentration of organosilicone surfactant in the agrobacterial suspension used for spraying can be between 0.01 and 0.5% by volume, preferably between 0.05 and 0.2% by volume. Alternatively, the concentration of organo-silicone surfactant in the agrobacterial suspension used for spraying can 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 submicronic silicic acid (silica) or porous polymers, such as urea / formaldehyde (Pergopak ™). Notably, where the average particle size of the abrasive is between 0.1 and 30 pm or in one of the sub-bands in this range provided above, it is possible to add a highly dispersed silica of submicron size to the suspension. Here, silica with submicron size is silica having an average particle size between 0.01 and 0.5 pm, preferably between 0.02 and 0.5 pm, 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 can be incorporated in an amount of 1 to 10 g per liter of the suspension of the invention.
Other possible additives to the agrobacterial suspension are buffer substances to maintain the pH of the suspension used for spraying at a desired pH, typically between 7.0 and 7.5. In addition, soluble inorganic salts, such as sodium chloride, can be added to adjust the ionic resistance of the suspension. Nutrient broth, like LB medium, can also be contained in the suspension.
The aqueous suspension can be produced as follows. In one method, the Agrobacterium suspension to be used in the process of the invention is inoculated into culture medium and grown in a high cell concentration. Larger cultures can be inoculated with small volumes of
36/60 a highly concentrated culture medium for obtaining large amounts of culture medium. Agrobacteria are, in general, grown to a concentration of cells that corresponds to an OD at 600 nm of at least 1, typically about 1.5. Such highly concentrated agrobacterial suspensions are then diluted to obtain the desired cell concentration. For dilution of highly concentrated agrobacterial suspensions, water is used. The water may contain a buffer. The water may additionally contain the surfactant of the invention. Alternatively, concentrated agrobacterial suspensions can be diluted with water and any additives, such as the surfactant and optional buffer substances, are added during or after the dilution process. The abrasive can be added before, during or after dilution. However, it is preferable to stir the suspension during addition of the abrasive to uniformly disperse the abrasive in the agrobacterial suspension. The dilution step of the concentrated agrobacterial suspension can be carried out in the spray tank of the sprayer used to spray the diluted suspensions.
The sprayer to be used in the process of the invention depends mainly 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 the home and garden, can be used. These can have spray tank volumes between 0.5 and 2 liters. For medium scale applications, manually operated hydraulic sprayers, such as lever operated back sprayers or manually operated compression sprayers, can be used. However, the high transfection efficiency obtained in the invention has its maximum potential in the transfection of various plants, such as plants that grow in a field or in a greenhouse. For this purpose, electric 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 of these types of sprayers are known in the art and are described, for example, in the book Pesticide
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Application Methods by GA. 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 can be agitated at regular intervals or continuously during spraying. Large sprayers, such as tractor mounted sprayers, must be equipped with an agitator in the spray tank.
Considering the presence of agrobacterial and abrasive cells in the suspensions to be sprayed, sprayers used in the invention should produce spray of a droplet size, at least in fine spray. In addition, medium spray or coarse spray in the spray classification used in the book mentioned above by G.A. Matthews, page 74, can be used. The main purpose of spraying in the invention is to moisten the plant tissue with the suspension. Thus, the exact droplet size is not critical. However, the efficiency of transfection can be further enhanced by providing spraying to plant surfaces with increased pressure.
In the process of the invention, at least parts of plants are sprayed. In an important embodiment, plants that grow on the ground in a field are sprayed, that is, plants that are not growing in mobile pots or containers. Such plants cannot be turned upside down and dipped in agrobacterial suspension for vacuum infiltration. At least parts of plants are sprayed, such as leaves. Preferably, most leaves or whole plants are sprayed.
The present invention is mainly used for transient transfection of plants with a DNA sequence of interest. The term transient means that no selection method is used to select cells or plants transfected with the DNA sequence of interest based on non-transfected cells or plants using, for example, selectable agents and selectable marker genes capable of detoxifying selectable agents . As a result, the transfected DNA, in general, is not stably introduced into the plant's chromosomal DNA.
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Instead, transient methods make use of the transfection effect on the same transfected plants.
The present invention is, in general, used for transfection of multicellular plants, namely higher plants. Monocotyledonous and dicotyledonous plants can be transfected, so dicotyledonous plants are preferred. Plants for use in the present invention include any species of plant, with preference given to species of agronomic and agriculturally important crops. Common crop plants for use in the invention include barley, alfalfa, beans, canola, black-eyed beans, cotton, corn, clover, lotus, lentils, lupins, corn, oats, peas, peanuts, rice, rye, sweet clover, sunflower, peas sweet, soy, triticale, sorghum, yam, velvet beans, vetch, wheat, wisteria and nut plants. Preferred plant species for the practice of the present invention include, but are not limited to, representatives of Gramineae, Compositeae, Solanaceae and Rosaceae.
Other preferred species for use in the invention are plants of 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, Ip, Hyoscyam 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, Ranunculus, Ranunculus, Ranunculus Raphanus, Ribes, Ricinus, Rubus, Saccharum, Salpiglossis, Secale, Senecio, Setaria, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum, Vicia, Vigna, Vitis, Zea, Olyreae, Pharoideae and others.
In one embodiment, the process of the invention can be used to produce a protein of interest in one plant or in many plants that grow in a field. For this purpose, plants can
39/60 be sprayed with the agrobacterial suspension at a desired plant growth stage. If the main goal is to achieve the highest possible levels of expression, followed by harvesting plants to obtain plant material containing high amounts of protein, viral vectors can be used since, in general, they allow the highest levels of expression .
In another embodiment, the process of the invention is used to generate or change a trace in a plant, such as an input trace. In this modality, excessive expression of a protein or RNA of interest may not be desired to avoid harmful effects on the health of the plant. For such modalities, non-replication vectors (also referred to here as transcription vectors), that is, vectors without a functional origin of replication recognized by a nucleic acid polymerase present in plant cells are preferred. An example of such a modality is the expression of hormonal molecules as secondary messengers in plant cells. In the example in Fig. 29, the distribution of the key enzyme of cytokinin biosynthesis, isopentenyl transferase, was demonstrated in Nicotiana benthamiana cells by spraying with diluted Agrobacteria, bringing a transcription vector containing the ipt coding sequence under control of a 35S promoter. Morphological changes in transfected plants caused by excessive cytokinin production were observed (Fig. 29). Another application of the invention is RNA expression, for example, for RNA interference, in which the interference signal can be dispersed in the plant from cells that express the signal to other cells. An example is the objectification of unwanted viral DNA in plants, as described by Pooggin in Nat. Biotech. 21 (2003) 131. An example of oncogene silencing that can be adapted to a transient system is described by Escobar et al., Proc. Natl. Acad. Know. USA 98 (2001) 13437-13442. Fig. 37 shows photo-bleaching by means of phytene desaturase (PDS) gene silencing in leaves of Nicotiana benthamina. Another example is the control of coleopteran pest insects through RNA interference similar to that described by Baum et al., Nat.
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Biotech. 25 (2007) 1322-1326, which can be adapted to the transitional process of the invention by means of transient transfection of plants infested with pests with DNA of interest that encodes and expresses the dsRNA. Other methods applicable to the transitional process of the invention are those described by Huang et al., Proc. Natl. Acad. Know. USA 103 (2006) 14302-14306; Chuang et al., Proc .. Natl. Acad. Know. USA 97 (2000) 4985-4990. In the experiment, the results of which are shown in Fig. 38, flagellin expression protects a plant against symptoms of diseases caused by Pseudomonas syringae.
Furthermore, the process of the present invention allows changes, at a desired point in time, to traits related to the regulation of the flowering season or fruit formation, such as potato tuber formation (Martinez-Garcia et al, Proc. Natl. Acad USA 99 (2002) 15211-15216) or regulation of the flavonoid pathway using a transcription factor (Deluc et al., Plant Physiol. 147 (2008) 2041-2053). Flowering can be induced by transiently expressing the mobile FT flowering protein (Zeevaart, Current Opinion in Plant Biology: 11 (2008) 541-547; Corbesier et al., Science 316 (2007) 1030-1033). Parthenocarpic fruits in tomatoes can be produced on a large scale using the present invention and the method described by Pandolfini et al., BMC Biotechnology 2 (2002). Other applications of the present invention are in the context of the development of cotton fibers through 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. It has been shown that transient expression of the MsrA2 defensin in Nicotiana benthamiana leaves significantly decreases the symptoms of Pseudomonas infection (Fig. 31).
The invention also provides a process for protecting crop plants in a field from a pest. In such a process, infestation of at least one of the plants of a plurality of plants growing in a field or agricultural plot can be determined. Due to the speed of the process of the invention, expression of a protein or RNA harmful to
41/60 pest need to be caused only if infestation by the pest is determined. Thus, constitutive and strong expression of toxins for the pest or dsRNA for RNAi, even in the absence of an infestation risk, is not necessary. Transient expression of Bacillus thuringiensis endotoxins after spraying with diluted agrobacterial cultures bringing corresponding PVX-based expression vectors protected Nicotiana benthamiana plants from damage by feeding by manduca sexta tobacco larvae (Fig. 30).
EXAMPLES
The invention is further described below by way of examples. The invention is not, however, limited to these examples.
Reference Example 1: Determination of the concentration of Agrobacteríum cells in liquid culture in terms of colony forming units (cfu)
The concentration of Agrobacteríum cells in liquid suspension in terms of colony forming units per ml (Colony Forming Units - cfu / ml) of liquid suspensions can be determined using the following protocol. Cells from the ICF 320 strain of Agrobacteríum tumefaciens transformed with the pNMD620 construct were grown in 7.5 ml of liquid LBS medium containing 25 mg / l kanamycin (AppIiChem, A1493) and 50 mg / L of rifampicin (Carl Roth, 4163, 2). The bacterial culture was incubated at 28 ° C with continuous shaking. Absorbance or optical density of a bacterial culture, expressed in absorbance units (Absorbance Units - UA), was monitored in 1 ml culture aliquots using a spectrophotometer at a wavelength of 600 nm (OD ₆ oo) · The concentration of cells estimated as a number of colony forming units per milliliter of liquid culture (cfu / ml) can be analyzed at Οϋδοο values of 1, 1.3, 1.5, 1.7 and 1.8. For this purpose, 250 μΙ aliquots of liquid culture were diluted with LBS medium to reach a final volume of 25 ml (dilution at 1: 100). 2.5 ml of such a 1: 100 dilution was mixed with 22.5 ml of LBS to achieve a 1: 1000 dilution. Dilutions of liquid culture of 1: 100; 1: 1000; 1: 10,000; 1: 100,000; 1: 1,000,000; 1: 10,000,000 and
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1: 100,000,000 have been prepared in a similar manner. Aliquots from the last three dilutions were dispersed in LBS medium solidified with agar supplemented with 25 mg / L kanamycin and 50 mg / L rifampicin (250 μΙ of bacterial culture per 90 mm diameter plate). Aliquot culture for each plate dilution was performed in triplicate. After 2 days of incubation at 28 ° C, bacterial colonies were counted for each plate. Plating dilutions at 1: 1,000,000 and 1: 10,000,000 resulted in a few tens and a few dozen colonies per plate, respectively. So far, since the 1: 100,000,000 dilution has provided only a few colonies per plate, this dilution has not been used for calculating cell concentration. The cell concentration was estimated according to the formula: cfu / ml = 4 x number of colonies per plate x dilution factor.
For transforming cell concentrations, as measured by absorbance measurements at 600 nm (in LB medium) and in terms of colony forming units, the following relationship is used here: an OD ₆ oo of 1.0 corresponds at 1.1 χ 10 ⁹ cfu / ml.
LBS medium (liquid)
1% soy peptone (soy bran papaya hydrolyzate;
Shower, S1330)
0.5% yeast extract (Shower, Y1333)
1% sodium chloride (Carl Roth, 9265.2) dissolved in water and pH adjusted to 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). The media were autoclaved at 121 ° C for 20 min.
Example 1: Vectors used in the following examples
In the present study, transcription vectors based on the 35S CaMV promoter were used, as well as viral replicons based on TMV and PVX with or without the ability to move cell to cell.
All transcription vectors were created from pICBVIO, a binary vector derived from pBIN19 (Marillonnet et al., 2004,
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2006). They contained two expression cassettes inserted inside the right and left edges of the same T-DNA region (Fig. 1). For cloning the expression vector pNMD293, two intermediate constructs (pNMD280 and pNMD033) were created. pNMD280 contained an expression cassette comprising, in sequential order, the cauliflower mosaic virus (CaMV) 35S promoter, omega translational enhancer of the Tobacco Mosaic Virus, coding sequence for the P19 Silencing Suppressor of the Ring Spot Virus Tomato (TBSV) (GenBank Accession No. CAB56483 0.1) and terminator of the Agrobacterium tumefaciens octopine synthase gene inserted between the right and left edges of T-DNA. To allow for the next cloning step, two restriction sites, EcoRI and Sphl, were introduced between the right edge of T-DNA and the 35S promoter sequence. The pNMD033 construct contained, between the left and right edges of T-DNA, the expression cassette flanked by restriction sites EcoRI and Sphl and comprised the promoter 35S, omega translational intensifier, coding sequence for the green and jellyfish fluorescent protein terminator of the Agrobacterium tumefaciens octopine synthase gene, listed in sequential order.
For cloning the pNMD293 construct, the GFP expression cassette was excised from the pNMD033 construct using EcoRI and Sphl restriction enzymes and transferred to the linearized pNMD280 vector with the same restriction enzymes. The resulting pNMD293 construct contained two expression cassettes inserted between the right and left edges of TDNA. An expression cassette adjacent to the right edge comprised 35S of CAMV, omega translational enhancer, green fluorescent protein coding sequences and the terminator Nos (listed in sequential order). The expression cassette adjacent to the left edge contained the 35S promoter, followed by the omega translational intensifier, P19 muting suppressor coding sequence and Ocs terminator. All other constructs were created based on the vector pNMD293, replacing the GFP coding sequence with PCR amplified coding sequences of other genes of interest using, for cloning, res sites
44/60 trition Ncol and BamHI. Genes of interest introduced in transcription vector constructs encoded sGFP, modified green fluorescent protein (GFP) from jellyfish Aequorea victoría (GeneBank Accession No. EF030489) (pNMD293); DsRed, red fluorescent protein from a reef coral Discosoma sp. (GeneBank Accession No. AF168419.2) (pNMD1380); flowering factor SP3D of tomato (Accession No. AY186735) (pNMD421); Arabidopsis flowering (FT) locus (GeneBank Accession No. BAA77839) (pNMD655); brassino-steroid regulator DWARF4 from Arabidopsis (NM_114926) (pNMD440); isopentenyl transferase enzyme (IPT), a key cytokinin biosynthesis enzyme from the C58 / ATCC33970 strain of Agrobacterium tumefaciens (GeneBank Accession No. AE007871.2) (pNMD460).
TMV-based vectors with the ability to move cell to cell (Fig. 2) were created based on vectors described in Marillonnet et al. (2006). The construct pNMD035 was used as a cloning vector for the consequent insertion of coding sequences of genes of interest using Bsal cloning sites. The resulting constructs contained, in sequential order, a fragment of the Arabidopsis actin 2 (ACT2) promoter (GenBank Accession No. AB026654); the 5 'end of TVCV (GenBank Accession No. BRU03387, base pairs, 1-5455); and a cr-TMV fragment [GenBank Accession No. Z29370, base pairs 5457-5677, both together containing 16 intron inserts]; a gene of interest; 3 'untranslated cr-TMV region (3' NTR, GenBank Accession No. Z29370) and nopaline synthase terminator (NOS). The entire fragment was cloned between the left and right T-DNA edges of the binary vector. Genes of interest used in these constructs encoded GFP (pNMD560), DsRed (pNMD580), human α-interferon with a signal of rice alpha-amylase apoplast (pNMD38), klip27-mini-insulin with signal of targeting of a plasmid. alpha-amylase of rice (pNMD330), thaumatin 2 of Taumatococcus danielii (pNMD700), β-glucosidase BGL4 of Humicola grisea (pNMD1200), exocellulase E3 of Thermobifida fusca (pNMD1160), exoglucanase 1 , Rafanus sativus Rs-AFP2 defender (pNMD1061), MsrA2 defender (a
45/60 synthetic dermaseptin B1 derivative of Phyllomedusa the frog / οή (pNMD1071), defensina MB39 (modified cecropine of the Moth Cepropia Hyalophora cecropia) (pNMD1280), plectasin defense of the fungus Pseudoplectania nigrella (pNMDxxxx).
TMV-based vectors with no cell-to-cell motion capability were identical to the corresponding TMV-based vectors capable of cell-to-cell motion, except for a point mutation in the MP coding sequence, leading to a deviation from the reading frame that distorted the MP translation (Fig. 3). Cloning of these constructs was performed using pNMD661 as the cloning vector.
For cloning most PVX-based vectors with systemic movement capability and cell to cell, the cloning vector pNMD670 was used. The resulting constructs contained, in sequential order, 35M CaMV promoter, RNA-dependent RNA polymerase coding sequences, coat protein, triple gene block modules comprising 25 kDa, 12 kDa and 8 kDa proteins, gene of interest and region 3 'not translated. The entire fragment was cloned between the left and right T-DNA edges of the binary vector (Fig. 4). Another group of constructs based on PVX had a similar structure with a difference in CP position, which was inserted between PVX polymerase and the triple gene block (for example, pNMD600).
PVX-based vectors with deletion of the coating protein coding sequence had systemic movement and cell to cell disabled. Cloning of these constructs was performed using pNMD694 as a cloning vector. This type of vectors contained, in sequential order, 35M CaMV promoter, RNA-dependent RNA polymerase coding sequences, triple gene block module, gene of interest and 3 'untranslated region inserted between the left and right T borders -DNA of the binary vector (Fig.5).
Example 2: Diluted agrobacteria can be distributed to Nicotiana benthamina using surfactant by spraying
It has been shown that Nicotiana benthamiana plants can be
46/60 transfected by spraying the plants with diluted agrobacterial cultures containing surfactant (Fig. 6). To evaluate the parameters that influence the transfection and to optimize the efficiency of the transfection, immersion of Nicotiana benthamiana leaves in agrobacterial suspension was used. This approach allows for accurate measurements and easy testing of multiple experimental versions. Nocturnal agrobacterial cultures (ODeoo = 1.5) were diluted 1: 100 and 1: 1000 (dilution factors 10 ' ² and 10 ² , respectively) in 10 mM MONTH buffer (pH 5.5) containing magnesium sulfate at 10 mM and supplemented with Silwet L-77 surfactant. Three types of constructs that confer GFP expression were tested: 1) transcription vectors, 2) viral replicons based on TMV and 3) viral replicons based on PVX (Fig. 6). The viral vectors used in these experiments had systemic movement and cell to cell disabled. They allowed expression of the reporter gene only in cells transfected with T-DNA. The percentage of cells expressing GFP was counted after isolation of leaf protoplasts (Fig. 7). Depending on the concentration of agrobacterial suspension and regardless of the type of vector, 2-8% of the total leaf cells were transfected as a result of Agrobacterial-mediated T-DNA transfer when 0.1% by volume Silwet L-77 and a immersion time of 1 min was used.
To find the optimal surfactant concentration, 0.1% and 0.05% Silwet L-77 were tested in immersion experiments. For all three types of vectors, the transfection efficiency provided by the use of 0.1% Silwet was significantly higher compared to the 0.05% concentration (Figs. 8-10).
Immersion of 10 sec of Nicotiana benthamiana leaves in diluted agrobacterial suspension supplemented with 0.1% Silwet L-77 allowed transfection rates close to spray efficiency with the same suspension (Fig. 11). In both cases, the transfection efficiency was higher for the older developed leaves. The transfection rate varied from 1.4 to 3.7% for immersion and from 1.1 to 1.7% for the spraying of agrobacterial culture in a dilution of 1: 100. At 1: 1000 dilution, the
The variation was 0.2% -1.1 for immersion and 0.1-0.6% for spraying.
The Silwet L-77 used in all the examples described here 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 organo-silicon product composed of 84.0% heptamethyl tri-siloxane modified with polyalkylene oxide (CAS-No. 27306-78-1) and 16% allyoxy methyl polyethylene ether glycol (CAS-No. 27252-80-8). All concentrations of Silwet L-77 content provided in the examples or figures refer to this commercial product.
Example 3: Diluted agrobacteria can be distributed to other species by spraying using surfactant and abrasive
The number of plant species was tested using agrobacterial transfection with spray and surfactant. First, each species was analyzed for the optimal expression vector. For this purpose, plant leaves were infiltrated with a needle-less syringe, with 1: 100 dilutions of OD = 1.5 from five agrobacterial cultures bringing the following transcription vectors that express GFP: 1) 35S-GFP + P19 (pNMD293), 2) TMV-based viral vector capable of cell-to-cell movement TMV (MP) -GFP (pNMD560), 3) TMV-based viral vector with cell-to-cell movement disabled TMV (fsMP) -GFP ( pNMD570), 4) PXV-based viral vector capable of systemic and cell-to-PVX (ACP) -GFP cell movement (pNMD630) and 5) PVX-based viral vector with cell-to-cell movement disabled PVX (ACP) -GFP ( pNMD620). In some cases, vacuum infiltration was performed.
Effective Agrobacterium-mediated transfection has been demonstrated for several Solanaceae species, including Nicotiana benthamiana (all five vectors), Nicotiana tabacum tobacco (all five vectors), Lycopersicon esculentum tomato (vectors based on PVX and transcription vectors), Capsicum annuum pepper, Inca Berry Physalis peruviana, be30 eggplant Solanum melongena, potato Solanum tuberosum (all with vectors based on PVX) (Fig. 13).
Agrobacterial-mediated transfection was demonstrated for the
48/60 Lactuca sativa lettuce from the Asteraceae family (transcription vector), Beta vulgaris beet from the Chenopodiaceae family (all five vectors), Cucurbita pepo zucchini from the Cucurbitaceae family (transcription vector) and Gossypium hirsutum cotton from the Malvaceae family (all five vectors) (Fig. 14).
Treatment of agrobacterial cells with aceto-syringone (200 μΜ, 2 hours) significantly increased transfection efficiency in several plant species, including eggplant, tomatoes and zucchini (Fig. 15).
Based on infiltration data, spraying with diluted agrobacterial suspensions was tested for the series of plant species. The efficient distribution of diluted Agrobacteria (10-3) by spraying with suspensions containing 0.1% Silwet has been demonstrated for several Nicotiana species (Nicotiana benthamiana, Nicotiana debne, Nicotiana excelsior, Nicotiana exigua, Nicotiana maritime and Nicotiana simulans), as is shown using PVX with cell-to-cell systemic movement capability in Fig. 16.
The distribution of Agrobacteria to other species, including the families Solanaceae, Chenopodiaceae, Amarantaceae and Aizoaceae was demonstrated through immersion treatment in agrobacterial suspension and spraying with and without abrasive using transcription vectors, as well as TMV and PVX vectors, with and without the ability to cell to cell movement (Figs. 17-21). The list of successfully transfected species includes spinach Spinacea oleracea from the Amaranthaceae family (transcription vectors and based on PVX), Beta vulgaris beet varieties from the Chenopodiaceae family (viral vectors based on TMV and PVX) (Fig. 17), tomatoes Lycopersicon esculentum (vector based on PVX) (Fig. 18), Inca berry Physalis peruviana and potato Solanum tuberosum (Fig. 34) (vector based on PVX) (Fig. 19) from the Solanaceae family, cotton Gossypium hirsutum from the Malvaceae family (vector based on TMV) (Fig. 20). GFP expression in cotton tissues after agrobacterial transfection was confirmed using Western blot by hybridization with specific antibodies to GFP (Fig. 21).
Using the GUS gene as a reporter, successful transfection
49/60 by spraying with the agrobacterial suspension was obtained for the rapeseed Brassica napus of the Brassicaceae family (Fig. 35). The construct pNMD1971 was created based on plasmid pNMD293 by replacing the GFP coding sequence with the beta-glucuronidase (GUS) sequence of Escherichia coli (P05804) containing intron 7 of the PSK7 gene from Petunia hybrrada (AJ224165).
Efficient transfection of plants using spraying with a diluted agrobacterial suspension has also been demonstrated for monocot species. Fig. 36 shows the transfection of Allium cepa onion plants after spraying with agrobacterial suspension supplemented with 0.1% Silwet L-77. The construct pNMD2210 was created based on plasmid pNMD1971 by replacing the 35S promoter in the GUS expression cassette with the Oryza sativa rice actin 2 (Act2) promoter (EU 155408).
In all the examples described here, spraying was carried out with pump spray bottles with a nominal volume of 500 or 1000 ml (Carl Roth, # 0.499.1 and # 0.500.1) based on direct manual pumping or with a pressure with a volume of 1.25 L (Gardena, # 00864-20) that exploits the increased pressure for pumping. The plants were sprayed in order to completely moisten the leaves. Sprayers were periodically agitated to ensure homogeneity of the suspensions to be sprayed, particularly if the suspensions contained abrasive.
Example 4: Transfection of plants using Agrobacterium suspensions containing abrasive
The carborundum used in these experiments was a mixture of carborundum (silicon carbide) particles F800, F1000 and F1200 from Mineraliengrosshandel Hausen GmbH, Telfs, Austria. According to the supplier, F800, F1000 and F1200 have median surface diameters of
6.5, 4.5 and 3 mm, respectively. 97% by mass of the F800, F1000 and F1200 particles have a surface diameter less than 14, 10 and 7 μπι, respectively. 94% by mass of the particles have a surface diameter greater than 2, 1 and 1 μιτι, respectively. F800, F1000 and F1200 were
50/60 mixed in equal amounts by weight. 0.3% (weight / v) of the mixed carborundum was added to the agrobacterial suspensions supplemented with 0.1% Silwet L-77 and used for spraying plants using the sprayers described in Example 3.
The results shown in Figs. 32, 22 and 33 demonstrated that the use of abrasives significantly increases the efficiency of transfection. Spraying of Solanum melongena eggplant plants with agrobacterial suspension containing 0.3% carborundum (silicon carbide, SiC) provided a 2-fold increase in transfection efficiency (Fig. 32). In the case of red beets, the same abrasive treatment resulted in a 15-fold increase in transfection efficiency (Fig. 22). Surprisingly, the use of an abrasive was a decisive factor that allowed the transfection of pepper plants by spraying with the agrobacterial suspension; Combining an abrasive treatment with activation of agrobacterial cells with aceto-syringone further increased transfection efficiency (Fig. 32). List of species transfected using surfactant and abrasive spray also includes yellow root beet, another variety of Beta vulgaris, New Zealand spinach Tetragonia expansa from the Aizoaceae family, Capsicum annuum pepper and Solanum melongena aubergine (Fig. 23).
Example 5: Treatment with Agrobacterium can be repeated: multiple subsequent treatments
Multiple subsequent treatments of diluted Nicotiana benthamiana plants with Agrobacteria were carried out. For this purpose, leaves were dipped in diluted Agrobacterium suspensions that contained the following constructs: pNMD570 (TMV (fsMP) -GFP without cell to cell movement capacity), pNMD560 (TMV (MP) -GFP with cell to cell movement capacity) ), pNMD580 (TMV (MP) -DsRed with cell to cell movement capability), pNMD620 (PVX (áCP) -GFP without cell to cell movement capability), pNMD600 (PVX (áCP) -GFP with and cell systemic movement capability for cell) and pNMD610 (PVX (ÁCP) -DsRed with capacity for systemic movement cell to cell).
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After transfection, these vectors form fluorescent dots that differ in color and size (Fig. 24). The transfection was performed by immersing each leaf tested with 3 different cultures and with 7-day intervals between transfections. For each of the eight vector combinations tested, all transfections with Agrobacteríum were successful, no particular silencing effect was observed (Fig. 24).
Example 6: Spraying with Agrobacteríum can distribute viral replicons capable of cell-to-cell movement
It has been demonstrated that spraying Nicotiana benthamiana plants with 1: 100 and 1: 1000 dilutions of agrobacterial suspension allows efficient distribution of viral replicons capable of cell-to-cell movement, which results in high expression of genes of interest, comparable to the expression obtained using Agrobacteríum infiltration. This has been demonstrated for GFP (Fig. 25), human alpha-interferon and klip2-mini-insulin (Fig. 26) and several cellulases, including E3 exocellulase from Thermobifida fusca, exoglucanase 1 (CBH 1) from Tríchoderma reesei, β-glucosidase BGL4 from Humicola grisea and E3 exocellulase from Thermobifida fusca (Fig. 27).
Example 7: Agrobacteria can be used to distribute transcription factors as secondary messengers
The induction of anthocyanin biosynthesis in Nicotiana tabacum leaves infiltrated with agrobacterial suspension was demonstrated, bringing the PVX-based viral vector, which allows the expression of the anthocyanin transcription factor 1 MYB (ANT1) from Lycopersicon esculentum (Fig. 28).
Example 8: Agrobacteria can be used to distribute RNAi as secondary messengers
The photo-bleaching of Nicotiana benthamiana leaves caused by silencing of the phytoene desaturase (PDS) gene after spraying leaves with an agrobacterial suspension has been shown, bringing the PVX-based viral vector containing the fragment of the PDS coding sequence in an anti-sense orientation (Fig. 37). To generate this construct (pNMD050), a 298-624 bp cDNA fragment of challenge
52/60 phytene turase (PDS) from Nicotiana benthamiana (EU 165355) was inserted into the cloning vector pNMD640 in an antisense orientation using Bsal sites.
Example 9: Agrobacteria can be used to distribute MAMPs (Microbe-Associated Molecular Patterns) as secondary messengers
It has been shown that a reduction in the number of necrotic lesions caused by infection with Pseudomonas syringae pv. syringae in leaves of Nicotiana benthamiana after preliminary spraying of the plants with the agrobacterial suspension bringing the PVX-based vector allows the expression of the Pseudomonas flagellin gene (pNMD1953) (Fig. 38). To create the plasmid pNMD1953, the GFP coding sequence was replaced, in the construct pNMD630, by the sequence comprising the fragment encoding the peptide signaling peptide of the barley alpha-amylase (AMY3) gene (Hordeum vulgare) (FN179391) with the sequence encoding the Pseudomonas syringae pv flagellin. syringae (YP236536). Four Nicotiana benthamiana plants were inoculated with 1: 1000 dilutions of Agrobacterium cultures (ODeoo = 1.3) by spraying. 6 dpi with Agrobacterium cultures, all plants were inoculated with Pseudomonas syringae pv. syringae B728 at 1 x 10 ⁵ cfu / ml by spraying. 7 dpi with Pseudomonas, the symptoms of the disease were classified by counting necrotic spots on two leaves of each plant. The number of necrotic spots caused by infection with Pseudomonas per leaf is given as the average of each 2 leaves of 4 plants.
The sequence listing below contains the following nucleotide sequences:
SEQ ID NO: 1: TNA region of TNNA region of pNMD280 SEQ ID NO: 2: TNA region of TNA region of pNMD033 SEQ ID NO: 3: TNA region of TNA region of pNMD035 SEQ ID NO: 4: TNA region of TNNA region of pNMD661 SEQ ID NO: 5: TNA region of TNA region of pNMD670 SEQ ID NO: 6: TNA region of TNA region of pNMD694
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SEQ ID NO: 7: TNA region of TNNA region of pNMD1971 SEQ ID NO: 8: TNA region of TNA region of pNMD2210 SEQ ID NO: 9: TNA region of TNA region of pNMD050 SEQ ID NO: 10: TNA region of pNMD1953 T-DNA region References
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权利要求:
Claims (11)
[1]
claims
1. Process of generating or altering a trace of a plant, characterized by the fact that it comprises:
(i) growing said plant to a desired growth state;
(ii) expression, in said plant, of a protein or an RNA capable of generating or altering said trace, comprising spraying aerial parts of said plant with an aqueous suspension containing cells from an Agrobacterium strain, at least one abrasive suspended in the said suspension, and, as an agricultural spray adjuvant, a nonionic organo-silicone wetting agent at a concentration between 0.25 and 5.0 g per liter of said suspension;
said strain of Agrobacterium comprising a DNA molecule comprising a nucleic acid construct containing a DNA sequence of interest, said DNA sequence of interest encoding said protein or said RNA wherein the at least one abrasive is carborundum.
[2]
2. Process of producing a protein of interest in a plant, characterized by the fact that it comprises:
(i) growing said plant to a desired growth state;
(ii) expression, in said plant, of said protein of interest comprising spraying the aerial parts of said plant with an aqueous suspension containing cells from an Agrobacteríum strain, at least one abrasive suspended in said suspension, and, as a spray adjuvant agricultural, a non-ionic organo-silicone wetting agent at a concentration between 0.25 and 5.0 g per liter of said suspension, said Agrobacterium strain comprising a DNA molecule comprising a nucleic acid construct containing a sequence of DNA of interest, said DNA sequence of interest encoding said protein of interest,
Petition 870190011049, of 02/01/2019, p. 13/65
2/3 where the at least one abrasive is carborundum.
[3]
3. Process of protection of crop plants in a field against a pest, characterized by the fact that it comprises:
(i) growth of said plants on the soil of said field;
(ii) determining, in a desired growth state of said plants, infestation of at least one of said plants by a pest;
(iii) expression, in said plant, of a protein or an RNA that is harmful to the pest determined in the previous step comprising spraying the aerial parts of said plants with an aqueous suspension containing cells from an Agrobacteríum strain, at least one abrasive suspended in the said suspension, and, as an agricultural spray adjuvant, a nonionic organo-silicone wetting agent at a concentration between 0.25 and 5.0 g per liter of said suspension, said Agrobacteríum strain comprising a DNA molecule which comprises a nucleic acid construct containing a DNA sequence of interest operably linked to a promoter, said DNA sequence of interest encoding said protein or said RNA, wherein the at least one abrasive is carborundum.
[4]
Process according to any one of claims 1 to 3, characterized in that said aqueous suspension contains said cells of said strain of Agrobacteríum in a concentration of maximum 2.2 x 10 ⁷ , preferably maximum 1.1 x 10 ⁷ , more preferably at most 4.4 x 10 ⁶ , even more preferably at most 1.1 x 10 ⁶ cfu / ml of said suspension.
[5]
Process according to claim 1, characterized in that said aqueous suspension contains said abrasive in an amount comprised between 0.02 and 2, preferably between 0.05 and 1 and, more preferably, between 0, 1 and 0.5% by weight of said suspension.
[6]
6. Process according to claim 1 or 5, characterized by the fact that the average particle size of the abrasive added to the
Petition 870190011049, of 02/01/2019, p. 14/65
3/3 suspension is between 0.1 and 30, preferably between 0.1 and 10, more preferably between 0.5 and 10 and, even more preferably, between 0.5 and 5 μm.
[7]
Process according to any one of claims 1 to 6, characterized by the fact that the abrasive has a D90 value of a maximum of 40 μm, preferably of a maximum of 30 μm and in which the abrasive does not contain particles having a size above 45 μm, preferably not above 40 μm.
[8]
8. Process according to claim 1, characterized by the fact that the non-ionic organo-silicone wetting agent is heptamethyl tri-siloxane modified with polyalkylene oxide.
[9]
Process according to any one of claims 1 to 8, characterized in that said nucleic acid construct is flanked by a T-DNA edge sequence on at least one side, which allows the transfer of said construct nucleic acid for cells of said plant.
[10]
Process according to any one of claims 1 to 9, characterized in that said nucleic acid construct encodes a viral replication vector that encodes said protein of interest, said viral replication vector being unable to move the system in that plant.
[11]
Process according to any one of claims 1 to 10, characterized in that said DNA sequence of interest is operably linked to a promoter active in plant cells.

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同族专利:

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公开号 | 公开日

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US20190136260A1|2019-05-09|

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JP2013532992A|2013-08-22|

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EP2601295A1|2013-06-12|

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CA2807544A1|2012-02-16|

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AU2011288653B2|2014-10-09|

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CN103282499A|2013-09-04|

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AU2011288653A1|2013-02-28|

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MX346926B|2017-04-05|

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US20130212739A1|2013-08-15|

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WO2012019660A1|2012-02-16|

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CA2807544C|2020-03-24|

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ES2654583T3|2018-02-14|

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JP5898681B2|2016-04-06|

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EP2601295B1|2017-12-13|

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RU2013110019A|2014-09-20|

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BR112013002981A2|2016-06-07|

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EP2418283A1|2012-02-15|

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MX2013001459A|2013-07-05|

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法律状态:
2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2018-11-06| B07A| Technical examination (opinion): publication of technical examination (opinion)|

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2019-05-21| B09A| Decision: intention to grant|

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2019-07-09| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/05/2011, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/05/2011, OBSERVADAS AS CONDICOES LEGAIS |

优先权:

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申请号 | 申请日 | 专利标题

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EP10008267|2010-08-07|

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EP10008267.6|2010-08-07|

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EP10008393A|EP2418283A1|2010-08-07|2010-08-11|Process of transfecting plants|

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EP10008393.0|2010-08-11|

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PCT/EP2011/002279|WO2012019660A1|2010-08-07|2011-05-06|Process of transfecting plants|

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