RU2285045C2 - System for eradication of vegetable cells - Google Patents

Abstract

FIELD: biotechnology, agriculture.

SUBSTANCE: invention relates to a system used in eradication of vegetable cells. Method involves transformation of plant with chimeric gene wherein the encoding gene sequence encodes PAP-S α-protein or PAP-S β-protein or its fragment and this gene comprises promoter that shows activity with response to specific irritating effect on plant. Also, the plant can be transformed with either a single construction comprising two chimeric genes wherein one of them encodes inactivated antiviral protein from phytolacca Americana and another gene encodes activator of inactivated antiviral protein from phytolacca Americana or with the construction set wherein each abovementioned chimeric gene is in the separated construction. Invention provides the effective removing required cells from the plant body.

EFFECT: valuable biological properties of system.

37 cl, 15 dwg

Description

The present invention relates to a system for killing plant cells, and in particular to transgenic plants that carry a chimeric gene in their genome, upon expression of which a cytotoxic protein is formed.

One of the known ways for plant breeders to obtain new varieties is the creation of hybrids of existing varieties that carry the required characteristics. Hybrids, as a rule, differ from any of the parents in improved various characteristics, this phenomenon is called hybrid strength (heterosis). Such hybrid crosses can be carried out using manual cross-pollination, which is a tedious and time-consuming procedure.

In the process of implementing such hybrid crosses, the prevention of self-pollination is crucial. To prevent self-pollination, the parent plant can be emulsified manually, for example, when producing hybrid corn by removing the panicle. However, large-scale emasculation of species with hermaphroditic flowers is impossible from an economic point of view. It is also possible to obtain maternal lines (with male sterility) using genetic male sterility; this trait, well-known in many plants, is usually recessive and monogenic. A problem associated with the use of this approach is the difficulty in obtaining, for each cross, pure parental lines with male sterility. The system for obtaining male sterility most widely used to create hybrids is the cytoplasmic male sterility system (cms). In this case, the underdevelopment of pollen is due to cytoplasmic factors. In those cultivated plants in which cms was detected in the germplasm, for example, in corn and sunflower, this trait is widely used to create hybrids. Several disadvantages of this system are known: a cytoplasm with male sterility may carry other undesirable characteristics, for example, maize T-cytoplasm and sensitivity to Helminthosporium maydis; when using it, it is necessary to maintain lines with isogenic male fertility; and the use of this system is limited to species in which a cytoplasmic source of sterility is available.

Another advantage of the system based on the use of male sterility is that pollen plants can be obtained with it. This is required for many varieties of ornamental flowering plants and can also be used to combat undesirable genetic traits by preventing outcrossing.

Another valuable feature based on the use of sterility of the system is the ability to obtain plants with female sterility, in which the development of the fruit does not occur seed formation. Seedless fruit varieties have advantages when processing, for example, tomatoes and are also preferred for the consumer, for example, melons. Seedless varieties are currently known and appropriate selection programs exist, but the creation of seedless fruits is limited by the lack of suitable germplasm in many plant species.

In cases where the genetic source of sterility is absent or unavailable for other reasons, you can apply the method of genetic modification, which provides sterility using a system to destroy cells, while necrosis occurs in specific cells of reproductive tissues.

WO 89/10396 describes a system for killing plant cells by using which a chimeric gene containing an anther-specific promoter associated with an RNase protein or polypeptide is introduced into the plant, the expression of which violates cellular metabolism. Thus, the expression of a chimeric gene leads to necrosis of anther cells and, as a consequence, to male sterility of plants.

To create female sterility in plants, a similar system can be used to destroy cells whose target is the ovule.

WO 93/18170 and WO 92/04453 describe systems for killing plant cells specific for controlling infection by nematodes. According to the systems described in WO 93/18170 and WO 92/04453, a gene containing a coding sequence is introduced into certain types of host plants, the coding sequence encoding a product that reduces the harmful effect of nematodes. The gene also contains a promoter region, which controls the expression of the coding sequence in such a way that expression occurs upon attack of the nematodes and occurs almost specifically in the cells of the regions that the nematodes feed on, or in neighboring cells. To reduce the harmful effect of nematodes, the product can either have an adverse effect on plant cells, which, as a result of differentiation, turn into cells of the regions that nematodes feed on, or on neighboring cells, or can have an adverse effect directly on nematodes.

Plant parasitic nematodes of economic importance include cyst-forming nematodes such as potato nematodes (Globodera rostochiensis and G. pallida), soybean nematodes (Heterodera glycines), beet nematodes (Heterodera schachtii), and grain nematodes (Heterodera and aaeae aaeae aaeae like Meloidogyne spp. Such nematode parasitic plants are the main pathogens of many cultivated plants around the world, such as vegetables, fodder legumes, tomatoes, watermelons, grapes, peanuts, tobacco and cotton.

The most widespread modern means of controlling nematodes are based on the use of chemical plant protection products, agrotechnical methods and resistant varieties of plants, and they are often used in an integrated system for controlling parasitic nematodes on plants. There is a need to improve the system for combating nematodes, since the indicated current approaches do not provide reliable protection of crops. The safety of nematicides for the environment is problematic, and they do not always have a sufficiently high efficiency. However, the application of a crop protection system may be unprofitable for farmers for various reasons. A wide range of gall nematode hosts limits the possibility of using economically significant crops that are not targeted by nematodes. Effective resistant varieties are often not available, and those that the farmer can use, sometimes with a low number of nematodes, are worse than sensitive varieties. In addition, stability can decrease at high soil temperatures, which is typical for tropical and subtropical zones.

Other ways of using systems to destroy plant cells can be considered. For example, the target may be certain parts of the flower, which causes a change in the morphology of the flower. In another embodiment, the target may be lateral roots, spikes, or burning hairs. It is possible to achieve separation (abscision) of leaves or fruits when the target is the release layer of the leaf or fruit. You can also achieve easier dropping of seeds in the case when the target is the seed shaft. When using other organs as targets, such as trichomes, which, as a rule, are glandular (glandular), the production of chemical substances in these organs can be stopped or prevented. Another application may be the inducible separation of roots, leaves, flowers or fruits at the end of the growing season.

NZ 260511 proposes a system for killing plant cells, characterized by increased tissue specificity. This system allows the expression of a cytotoxic molecule (under the control of the first promoter, where the first promoter provides expression in specific target cells and in one or more other areas of the plant) in combination with a protective molecule (under the control of the second promoter, where the second promoter provides expression in all areas in which the first promoter is active, except for specific target cells). Examples of suitable cytotoxic and protective molecules are proteases and protease inhibitors, respectively, or nucleases and nuclease inhibitors, respectively. WO 93/10251 describes the use of cytotoxic barnase ribonuclease in combination with a barstar protective inhibitor molecule.

WO 98/44138 describes a method for increasing the specificity of gene expression by targeted transfer of a specific expression site of a target gene. In this way, a chimeric gene is obtained, wherein the chimeric gene contains a promoter that provides expression in more than one region of the body to be exposed. The promoter is associated with an agent that affects the function of the endogenous gene in the plant, which is also expressed in more than one region of the plant. The promoter and agent are selected so that their expression sites overlap in one or more of the desired regions. The presence of this (their) overlapping (their) site (s) provides an increase in the specificity and direction of gene expression.

Ribosome Inactivating Proteins (RIPs) are a group of toxic plant proteins that catalytically inactivate ribosomes of eukaryotic organisms (Stirpe and Barbieri, 1986). RIPs function as N-glycosidases, removing certain adenine in the conservative loop of a large rRNA molecule and thereby prevents the binding of lengthening factor 2, blocking the synthesis of cellular protein as a result.

Three forms of RIP are described. RIP type 1, such as the American phytolacca antiviral protein (PAP) and barley translation inhibitor, each of which has one polypeptide chain, the relative molecular weight (M _r ) of which is approximately 30,000. All RIP type 2, such as ricin, abrin and Modeccin, have two polypeptide chains. One RIP-active polypeptide (A chain) is linked via a disulfide bridge to a galactose-binding lectin (B chain; Stirpe et al., 1978). The M _{r of} each RIP type 2 is approximately 60,000. RIP type 3, such as maize RIP, have one polypeptide chain, which then undergoes proteolytic cleavage to release two active peptide domains.

Phytolacca americana (Phytolacca americana) produces three different antiviral proteins, namely PAP ', PAPII and PAP-S, which are characteristic of spring leaves, summer leaves and seeds, respectively. The similarity of amino acid sequences of these proteins has been established. As used herein, “PAP” refers to all three of these antiviral proteins. PAP 'or PAPII are found in the cell membrane matrix of leaf mesophilic cells and can be isolated by aqueous extraction of macerated leaf tissue (Ready et al., 1986). It has been found that by exogenously applying them to the surface of plant leaves, several different host plants can be protected from infection by a wide range of viruses (Chen et al., 1991; Lodge et al., 1993).

US Pat. No. 6015940 describes the creation of a PAP cDNA clone obtained from the spring leaves of American phytolacca and its use under the control of a constitutive promoter (either the 35S promoter of the cauliflower mosaic virus or the 35S promoter of the pineal mosaic virus) to produce transgenic tobacco and potato plants, virus resistant polyoma PVX and PVY. As a negative characteristic of the stable phenotype, it should be noted that in plants whose PAP 'expression levels exceeded 10 ng / mg protein, leaf mottling and growth retardation were detected. Plants accumulating higher PAP 'levels were sterile.

Transgenic plants containing PAP forms typical of summer leaves, i.e. PAP-II are described in WO 99/60843. A large number of full-sized and shortened PAP-II gene sequences were screened for PAP-II protein variants that retained antiviral activity but were not phytotoxic. Transgenic plants had both antiviral and fungicidal activity.

The PAP gene is first expressed in leaves in vivo to produce the inactive Pro-PAP protein. It is known that after translation there is a directed transfer of the Pro-PAP 'protein molecule into the cell membrane. At a certain stage in this process, the N- and C-terminal extension segments of the Pro-PAP 'molecule are cleaved to form the activated PAP' molecule (mature PAP '). Cellular localization is known for PAP-S (which is expressed in seeds). However, the N-terminal processed region of the PAP-S probably has properties similar to those of signal sequences providing directional transfer.

The structure of the mature PAP-S protein, i.e. a protein in which the N- and C-terminal extension segments are deleted can be characterized by the presence of two separate domains corresponding to RIP type 3 or two RIP type 2 polypeptides, i.e. the binding domain of the ribosome and the catalytic domain. The molecules described herein are recombinant PAP-S molecules containing either the PAP-S α sequence or the PAP-S β sequence. Based on structural features, it can be assumed that PAP-S α contains regions of an RNA recognizing motif and ribosome binding domain, and PAP-S β contains a site critical for catalysis of the residue. When creating the present invention, it was unexpectedly found that the expression of only the protein PAP-S α or protein PAP-S β leads to a significant inhibition of the activity of ribosomes.

The present invention relates to a method for inducing necrotic action in specific plant cells, according to which the plant is transformed with a chimeric gene, where the coding sequence of the gene encodes a mature American phytolacca antiviral protein or fragment thereof, this gene contains a promoter that is active in response to a specific irritating effect on it plant, as a result of which the mature American phytolacca antiviral protein or its fragment is expressed in specific cells plants.

The present invention also relates to a plant transformed with a chimeric gene, wherein the coding sequence of the gene encodes a mature American phytolacca antiviral protein or fragment thereof, this gene contains a promoter that is active in response to a specific irritating effect on this plant, resulting in this mature phytoluca antiviral protein American or its fragment are expressed in specific plant cells.

The present invention also relates to a recombinant plant cell transformed with a chimeric gene, where the coding sequence of the gene encodes a mature American phytolacca antiviral protein or fragment thereof, this gene contains a promoter that is active in response to a specific irritating effect on this plant, resulting in this mature antiviral American phytolacca protein or its fragment is expressed in specific plant cells.

In addition, the present invention provides an isolated DNA molecule containing a chimeric gene, where the coding sequence of the gene encodes a mature American phytolacca antiviral protein or fragment thereof, this gene contains a promoter that is active in response to a specific irritating effect on this plant, resulting in this mature American phytolacca antiviral protein or a fragment thereof is expressed in specific plant cells.

The present invention also relates to a biologically functional expression vector containing a chimeric gene, wherein the coding sequence of the gene encodes a mature American phytolacca antiviral protein or fragment thereof, this gene contains a promoter that is active in response to a specific irritating effect on this plant, resulting in this mature American phytolacca antiviral protein or a fragment thereof is expressed in specific plant cells.

In the context of the present description, the term "fragment" refers to a fragment of a gene encoding the American phytolacca antiviral protein, and this fragment has activity against protein synthesis inhibition.

The chimeric gene preferably encodes a mature PAP-S protein, the nucleotide sequence of which is presented in SEQ. ID No. 3, and the amino acid sequence is presented in SEQ. ID No. 4, or PAP-S α, the nucleotide sequence of which is presented in SEQ. ID No. 5, and the amino acid sequence is presented in SEQ. ID No. 6, or PAP-S β, the nucleotide sequence of which is presented in SEQ. ID No. 7, and the amino acid sequence is presented in SEQ. ID No. 8, or the mature PAP ′ protein or a variant thereof described in US Pat. No. 6,015,940 (i.e., nucleotides 290-1076 SEQ. ID. No. 30 and 31) or the mature PAPII protein described in WO 99 / 60843 (i.e., nucleotides 75-903 of SEQ. ID. No. 32).

Depending on the required homology of the nucleotide sequences, various hybridization stringency conditions can be applied to screen for similar sequences. As an example (but not limited to), the following stringent conditions are used for the hybridization process: hybridization with filter-bound DNA in 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C and washing in 0.1 × SSC / 0.1% SDS at 68 ° C (Ausubel FM et al., (Eds.), 1989, Current Protocols in Molecular Biology, Volume I, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., New York, c. 2.10.3). Other stringent conditions that can be used are well known in the art. Hybridization processes can be applied under the following conditions of moderate severity: hybridization with filter-bound DNA in 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C and washing in 0.2 × SSC / 0 , l% SDS at 42 ° С (Ausubel et al., 1989, supra). Other conditions of moderate severity that can be used are well known in the art. Other solutions can be used for hybridization processes, such as a standard solution of sodium chloride and sodium citrate (SSC) or sodium chloride and EDTA phosphate (SSPE).

Acceptable homologous sequences are sequences that are at least 70%, preferably 80%, and even more preferably 90 or 95% homologous to each of the sequences described herein, which homologous sequences retain the desired enzymatic activity.

Acceptable sequences may also represent variants thereof. According to the present invention, the variant may be any substitution, variation, modification, substitution or deletion or addition of one or more nucleotides / amino acids, allowing to obtain a sequence that expresses or exhibits the desired enzymatic activity. A derivative or mutation may also be used according to the invention. The derivative has certain modifications, usually chemical, in comparison with the naturally occurring polypeptide expressed by nucleic acid.

Another object of the present invention is a method for inducing necrotic action in specific plant cells, where the plant is transformed with two chimeric genes, the coding sequence of one of the indicated genes encoding the inactivated American phytolacca antiviral protein, and the coding sequence of the other of the indicated genes encoding the second molecule, which is an activator American inactivated phytolacca antiviral protein, each of the two genes contains a promo op, these promoters operate together in response to a specific irritant effect on the plant as a result of the inactivated pokeweed antiviral protein is activated in specific cells of the plant.

The present invention also relates to a plant transformed with two chimeric genes, wherein the coding sequence of one of these genes encodes an American inactivated phytolacca antiviral protein, and the coding sequence of the other of these genes encodes a second molecule, which is an activator of an inactivated American phytolacca antiviral protein, each of two gene contains a promoter, and these promoters act together in response to a specific irritant e impact on the plant, as a result of the inactivated virus protein pokeweed activated in specific cells of the plant.

The present invention also relates to a recombinant plant cell, an isolated DNA molecule of two chimeric genes, and a biologically functional expression vector containing two chimeric genes, where each chimeric gene falls within the second aspect of the present invention.

As used herein, the term “inactivated American phytolacca antiviral protein” refers to a mature American phytolacca antiviral protein, such as, for example, PAP-S, operably linked to a heterologous or homologous N- or C-terminal blocking sequence.

The chimeric gene preferably encodes a mature PAP-S protein, the nucleotide sequence of which is presented in SEQ. ID No. 3, and the amino acid sequence is presented in SEQ. ID No. 4, or PAP-S α, the nucleotide sequence of which is presented in SEQ. ID No. 5, and the amino acid sequence of which is presented in SEQ. ID No. 6, or PAP-S β, the nucleotide sequence of which is presented in SEQ. ID No. 7, and the amino acid sequence of which is presented in SEQ. ID No. 8, or mature PAP 'or a variant thereof described in US Pat. No. 6,015,940 (i.e., nucleotides 290-1076 SEQ. ID. No. 30 and 31), or mature PAPII described in international application published under No. WO 99/60843 (i.e., nucleotides 75-903 of SEQ. ID. No. 32), each associated with a heterologous or homologous N- or C-terminal blocking sequence, as described above to obtain an inactivated American phytolucentus antiviral protein.

The terminal blocking sequence is preferably a sequence that may have an additional necrotic effect on the cell or pathogen.

Preferably, the terminal blocking sequence is an oryzacystatin sequence (Δ86 cystatin sequence), presented herein as SEQ. ID No. 17. The Δ86 sequence of cystatin blocks the action of naturally occurring proteases, thereby preventing the naturally occurring expression of the PAP coding sequence. Cystatins are small (~ 100 amino acids) cysteine protease protein inhibitors found in many plant species [Ryan, 1990; Richardson, 1991], and a large number of genes (> 60), the sequence of which is established. Phytocystatins are a class different from type I and II cystatins [Kondo et al., 1991]. Orizizistatin I (OS-I) of rice seeds [Abe et al., 1987] is an effective inhibitor, but significantly less effective than animal cystatins, such as egg protein cystatin (CEWC). Recombinant variants of OS-1 obtained by protein construction (Uwrin et al., 1995) and a deletion mutant Δ-Asp86 (OS-1Δ86), which has a higher inhibitory activity, are described.

In another embodiment, the terminal blocking sequence is only a molecule that inactivates the mature PAP molecule. Preferably, the terminal blocking sequence is a naturally occurring end sequence of Pro-PAP derived from any Pro-PAP molecule.

The blocking sequence may be located at the N-terminus or at the C-terminus of the mature PAP sequence.

Preferably, the blocking sequence is a C-terminal blocking sequence.

To ensure the reactivation of the blocked mature PAP sequence in the inactivated American phytolacca antiviral protein, a specific cleavage site located between the mature American phytolacca antiviral protein sequence and the blocking sequence must also be present. The cleavage site is preferably located between the mature American phytolacca antiviral protein and the blocking sequence.

Preferably, the specific cleavage site is a cleavage site specific for tobacco engraving virus (TEV) NIa protease (Carrington and Dougherty, 1998), presented herein as SEQ. ID NO 28 and SEQ. ID No. 29. The site recognized by said proteinase is a Glu-Xaa-Xaa-Tyr-Xaa-Gln-Gly heptapeptide or a Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser heptapeptide, wherein cleavage occurs between the Gin and Gly residues (or Ser) .

The coding sequence of the second molecule is preferably a protease that has the ability to cleave a specific cleavage site. When the specific cleavage site is a TEV protease (PCS) cleavage site, the protease may be a TEV NIa protease. Specialists in this field can use other specific protease cleavage sites. For example, a site-specific protease enterokinase can be used to cleave the corresponding cleavage site associated with the mature American phytolacca antiviral protein.

According to one aspect of the invention, said irritating effect on a plant may be caused by an attack of a pathogen. In another embodiment, the irritating effect may be chemical induction or induction as a result of the natural development of the plant.

As should be obvious to experts in this field, the first and second objects of the invention can find wide application for all representatives of the plant kingdom in order to protect them, for example, from fungi, nematodes, bacteria and viruses, as well as for other purposes.

It should be understood that in the context of the present description, the term "necrotic effect" means a significant metabolic disturbance, so that when applying the invention to achieve the desired effect, for example, to protect against diseases.

Preferably, according to a second aspect of the present invention, promoters (a two-component system) have overlapping expression region (s). The characteristics of the overlapping expression of the two promoters and their corresponding reactions to these effects are such that they affect the direction of expression of the inactivated antiviral protein of American phytolacca and its activator molecule, leading to the fact that a lethal or harmful effect affects specific cells and only them.

Alternatively, promoters may have the same expression sites, or expressed proteins may otherwise accumulate in the target region.

A third object of the present invention is a method for inducing necrotic action in specific plant cells, according to which the plant is transformed with a chimeric gene, wherein the coding sequence of the gene encodes a precursor of the PAP molecule or its C-terminal deletion variant, the gene containing a promoter that acts in response to a specific irritating effect on the plant, as a result, the protein expressed by the coding sequence is expressed in specific cells plants, and the specified promoter should be selected in such a way as to provide one of the following effects: reduction of the harmful effect of nematodes, sterility, changes in the morphology of the flower, separation (of leaves or fruits), dropping of seeds or the development of trichomes.

The present invention also relates to a plant transformed according to the method, which is the third object of the invention. The present invention also relates to a recombinant plant cell isolated by the DNA of a chimeric gene and a biologically functional expression vector, where they all contain the chimeric gene described in the third aspect of the invention.

According to a third aspect of the invention, the coding sequence preferably encodes one of the following sequences: the nucleotide sequence of Pro-PAP-S shown in SEQ. ID No. 1, and the amino acid sequence shown in SEQ. ID No. 2, or PAP 'or a variant thereof described in US Pat. No. 6,015,940, which has the sequence set forth in SEQ. ID No. 30 and 31, or PAPII, the sequence of which is described in the international patent application published under No. WO 99/60843 and presented herein as SEQ. ID No.32.

The selection of acceptable promoters should depend on the choice of specific cells. If the goal is to protect against the attack of nematodes, then specific cells can be cells of the regions that nematodes feed on. In this case, according to a second aspect of the invention, suitable promoters may be the KNT1 promoter, acting in conjunction with the TobRB7 promoter. According to the first and third objects of the invention, these promoters can be used separately. The method for isolating the KNT1 promoter is described in New Zealand Patent 260511, and is also further described below, and the method for isolating the TobRB7 promoter is described in international patent application WO 94/17194. An object of the invention of this application regarding the allocation of a promoter is incorporated into this description by reference. Lemmi promoters are also suitable promoters, the disclosure of which is described in the international patent application published under No. WO 92/21757.

According to the second object, for example, the coding sequence of the American phytolacca antiviral protein may be controlled by the TobRB7 promoter, while the coding sequence of the activator molecule is controlled by the KNT1 promoter. The KNT1 promoter provides expression in the cells of the regions that nematodes feed on, the root tips and to a lesser extent in other types of meristem, while the TobRB7 promoter provides expression in roots and giant cells (Conkling et al., 1990). In these studies, it was demonstrated that the TobRB7 promoter can express in the root mass, but not in the root tips. Thus, the zone of overlapping expression of the KNT1 and TobRB7 promoters are giant plant cells (cells of the regions that nematodes feed on).

In another embodiment, when it is necessary to ensure male sterility, specific cells may be anther cells of plants. Examples of suitable promoters are described by Twell et al. (1991) and Mariani et al. (1990).

According to a first embodiment of the present invention, the target region may be the region that the nematodes feed on. In the case where the target region is the region that the nematodes feed on, the promoter that is induced in the region that the nematodes feed on and / or in the neighboring region is selected as a promoter. Induction of such a promoter preferably occurs when the plant is affected by nematodes. An example of an acceptable promoter is the KNT1 promoter. Other suitable promoters include the TobRB7 promoter and Lemmi promoters.

The area that the nematodes feed on may include, for example, plant cells located in the local lesion area, which subsequently undergo differentiation, forming syncytium (in the case of cyst-forming nematodes), or giant cells and / or concomitant hypertrophic cells (in the case of gall nematodes), and / or one or more syncytium cells, giant cells, and concomitant hypertrophic cells.

When using the target area of the nematodes, nematode-resistant plants can be obtained. The term "nematode-resistant plant" refers to a plant that, when infected with parasitic nematodes on a plant, has the ability to prevent, slow down or otherwise adversely affect the growth and development of nematodes that affect the plant, thereby preventing the number of nematodes parasitizing on plants from reaching an economically significant level during the growing season of the culture. It is understood that nematodes can, for example, die or the nematode life cycle can slow down, which leads to an increase in the time required to achieve puberty and egg laying, or female nematodes may have a reduced size and therefore reduced ability to lay eggs, which occurs only after the female nematodes reach the minimum critical size.

The present invention is applicable to the following types of nematodes (but not limited to): Globodera spp., Heterodera spp. and Meloidogyne spp.

According to a second embodiment of the present invention, the method may not be aimed at achieving resistance to nematodes, but at obtaining male sterility in plants. For example, the target area may be one or more of plant organs such as pollen, anther, or tapetum. If the target region is, for example, tapetum, then the selected promoter is a promoter that is induced in tapetum and / or in regions adjacent to the tapetum. An example of a tapetum-acceptable promoter is the TA29 tobacco promoter described by Mariani et al. (1990). Anther specific promoters are described by Twell et al. (1991).

According to a third embodiment of the present invention, the method is used to achieve female sterility of plants. For example, the target region may be the ovule of a plant. This provides that the selected promoter is a promoter that is induced in the ovule and / or adjacent region. An example of an acceptable promoter is the AGL15 promoter described by Perry et al. (1996).

According to a fourth embodiment of the present invention, the morphological structure of the flower is affected. For example, the target area is certain parts of the flower, with the goal being that these specific parts of the flower do not develop and the morphological structure of the flower changes. In this case, the selected promoter is a promoter that is induced in the sepals, carpels, petals and / or stamens, or in adjacent areas. Examples of acceptable promoters are promoters of the genes that cause asexuality (lack of petals (apetala3), sphericality (globosa), pestle (pistillata) and various other deficiencies (Sieburth and Meyerowitz, 1997; Samach et al., 1997 and those cited in these publications links).

According to a fifth embodiment of the present invention, the method of the invention is used to promote or enhance the abscission of leaves and / or fruits in plants. For example, the target region may be a release layer of the sheet and / or fetus. The promoter selected in this case is a promoter that is induced in the release layer and / or in the neighboring region.

According to a sixth embodiment of the present invention, the target is trichomes, the trichomes being generally glandular. The selected promoter is a promoter that is induced in trichomes and / or neighboring regions. Causing necrosis of plant trichomes, it is possible to stop or prevent the production of chemical substances by trichome.

According to a seventh embodiment of the present invention, the target is lateral roots, spikes, or stinging hairs.

According to an eighth embodiment of the present invention, the method is for controlling a viral infection. In viral infections, to a large extent affected by the virus, cells specifically or practically specifically induce numerous genes. The selected promoter is a promoter that is induced in virus-infected cells and / or in an adjacent region.

According to a ninth embodiment of the present invention, the method is intended to facilitate dropping seeds from a plant when the target is seeds. A large number of genes are known which, in the process of seed development, are specifically or practically specifically induced in certain cells / parts of the seed.

According to a tenth embodiment of the present invention, a promoter is selected which is induced by an external stimulus and which is induced, for example, in the roots of a plant. Such a promoter can be used to separate the root at the end of the growing season. To separate leaves, flowers or fruits at the end of the growing season, promoters that are induced, for example, in leaf petioles, pedicels or stalks, can be used.

Each chimeric gene may further comprise a 3 ′ untranslated terminator sequence. The terminator sequence can be obtained from plant genes, bacteria, or viruses. Suitable terminator sequences are, for example, the rbcS terminator sequence of the pea E9 gene, the Nos terminator sequence derived from the Agrobacterium tumefaciens nopaline synthase gene, and the cauliflower mosaic virus 35S terminator sequence. Other sequences of terminators are known to those skilled in the art. The chimeric gene may also optionally contain transcription or translation enhancer sequences, such as those described in international patent application published under No. WO 97/20056, for example, intracellular, providing directed transfer of the sequence, and introns, as well as nucleotide sequences, the function of which is to facilitate the process of transformation and stable expression of the chimeric gene, such as border regions of T-DNA, regions that provide accession to the hyaloplasm, and sequences excision / recombination.

Plant transformation methods are well known in the art and include, for example, Agrobacterium mediated transformation. Typically, in an Agrobacterium-mediated transformation, a binary vector carrying a foreign DNA of interest, i.e. a chimeric gene, is transferred from the corresponding Agrobacterium strain to the target plant by co-culturing Agrobacterium with explants of the target plant. The transformed plant tissue is then regenerated on selection media, the selection media containing a selectable marker and plant growth hormones.

Suitable transformation methods include, for example, direct gene transfer into protoplasts using polyethylene glycol or electroporation, particle bombardment, microinjection and the use of silicon carbide fibers.

Acceptable plant species that can be transformed according to the present invention include, but are not limited to, rice, wheat, corn, canola, potatoes, tobacco, sugar beets, soybeans, tomatoes, peanuts, cotton, grapes, watermelon, papaya, vegetables and fodder bean plants.

According to a second aspect of the invention, both chimeric genes are introduced into a plant in one expression cassette. In another embodiment, each chimeric gene is introduced into a plant in different expression cassettes by sequential or simultaneous transformation using two transgenic constructs. Such a two-component system can also be obtained by crossing two plants, each of which contains one individual component.

To facilitate understanding and practical implementation of the invention is described in more detail below with reference to the accompanying drawings, which show:

figure 1 is a schematic representation of promoter constructs for the expression of PAP in protoplasts;

figure 2 - vector pDVM;

figure 3 - plant transforming vector pATC;

Fig. 4 is a schematic representation of nematode-induced promoter constructs for the expression of PAP in transgenic plants;

figure 5 - effect of the protein PAP-S on tobacco ribosomes, evaluated by the synthesis of the GUS protein;

Fig.6 - the effect of modified PAP-S proteins on tobacco ribosomes, evaluated by the synthesis of GUS protein;

Fig.7 - the effect of PAP-S α and PAP-S β on protoplasts;

Fig. 8 is a schematic illustration of a PAP-S fusion construct / protease cleavage site (PCS) / cystatin ΔD86 and constructs in pBluescript vectors used in experiments using TnT;

figure 9 - effect of PAP proteins on the translation of luciferase protein in rabbit reticulocyte lysates;

figure 10 - the effect of the modified protein PAP-S / cystatin on tobacco ribosomes, evaluated by the synthesis of GUS protein;

11 is a schematic representation of the construction of an attack-induced nematode promoter designed to regulate the expression of PAP-S-PCS-cystatin ΔD86 and TEV NIa protease in transgenic plants;

Fig. 12 is a screening of the resistance of potato plants transformed with Pro-PAP-S and infected with a 2/3 potato (cyst-forming) nematode, in comparison with the sensitive control (Hermes) and the resistant variety (Sante) that is commercially available;

on Fig - experimental data on the resistance to the Javanese gall nematode of tobacco plants containing the construction of Pro-PAP-S (pATC05502) compared with control plants grown using tissue culture (ncc). The sizes of parasitic nematodes are expressed in units of the ocular scale;

on Fig is a schematic diagram of obtaining variants of PAP-S using PCR;

on Fig - comparison of the average number of cysts on potato plants transformed with genes containing Pro PAP-S, antisense promoter KNT1 or trypsin inhibitor of cow pea (CpTI).

Examples

Cloning and sequencing of American phytolacca antiviral protein (PAP-S), derived from genomic DNA of American phytolacca

The Pro-PAP-S sequence was isolated from DNA of American phytolacca leaves by polymerase chain reaction (PCR) using Pfu polymerase. The sequence was isolated as two segments, which were then combined to remove the XbaI site located in the central region of the molecule. The 5'-region of the molecule was amplified using PPS1BF and PSXDR primers (SEQ. ID. No. 9 and 16, respectively), and the 3'-region was amplified using primers PSXDF and PPS2SR (SEQ. ID. No. 15 and 10, respectively). Two PCR products were combined and overlapping DNA fragments were extended using Pfu polymerase and re-amplified using PPS1BF and PPS2SR primers (SEQ. ID. No. 9 and 10, respectively). The PCR product was digested with restriction enzymes XbaI and SalI and cloned into pBluescript to give the clone pBS / Pro-PAP-S (FIG. 14).

Using the clone pBS / Pro-PAP-S as a template, the PAP-S sequence was amplified by PCR using primers PS1BF and PS2SR (SEQ. ID. No. 11 and 12, respectively) to remove the N- and C-terminal regions. Using the PCR primers, the start codon of methionine and the stop codon of TAA were added. The sequence of start and stop codons is presented in SEQ. ID No. 3, but in SEQ. ID No. 3 does not show enzyme cloning sites. The added methionine residue is presented in SEQ. ID No. 4. The PCR product was digested with restriction enzymes XbaI and SalI and cloned into pBluescript to give the clone pBS / PAP-S. Sequencing of the PAP-S clone revealed only one nucleotide substitution relative to the sequence obtained from the database. Suitable primer combinations were used to remove either the N- or C-terminal regions independently of one another.

After comparing PAP-S and maize RIP proteins, the nominal polypeptide domains PAP-S α and PAP-S β were determined relative to the corresponding regions of RIP α, β and the central domain of maize (these data are presented in the pending application of the same authors, filed simultaneously with this application). The PAP-S α and PAP-S β polypeptide domains were amplified from the plasmid DNA of the PAP-S clone using a pair of primers PS1BF plus PS1SR (SEQ. ID. No. 11 and 14, respectively) and PS2BF plus PS2SR, respectively (SEQ. ID. No. 13 and 12, respectively). Starting codons of methionine and stop codons of TAA were added using PCR primers. The start and stop codon sequences are presented in SEQ. ID No. 5 and 7, but enzyme cloning sites are not represented in SEQ. ID No. 5 and 7. The added methionine residue is presented in SEQ. ID No. 6 and 8. The sequences of the amplified recombinant PAP-S α and PAP-S β were cloned in different vectors.

Isolation of a promoter that is induced in the target region and / or neighboring regions

The method for isolating a promoter described herein is given as an example only. Alternative methods for isolating an acceptable promoter for use in accordance with the present invention are well known to those skilled in the art, some of which are referenced above.

The method for isolating the KNT1 promoter is as follows.

Growing and infecting tobacco plants

C319 tobacco seeds were germinated in Fisons F1 compost under the following conditions: light intensity 4500-5000 lux; 16 hours of light / 8 hours of darkness; temperature 20-25 ° C. After about 3 weeks, the seedlings were carefully washed with tap water to remove soil and transferred into bags (Northrup-King), 2 plants per bag, and grown for another 1 week in a Conviron chamber at 25 ° C and under the above light regime. The roots at the back of the bag were released and their tips supported using Whatman GF / A fiberglass paper. Then, the tips of the roots were infected with three-day nematodes (M. javanica), adding aliquots of 10 μl (50 nematodes), and a second piece of GF / A paper was placed on top in order to completely close the root tip from all sides. 24 hours after infection, GF / A paper was removed in order to ensure synchronization of infection. 3 days after infection, the root nodes were cut off (leaving a healthy part of the root and tissue of the root tip behind) and immediately frozen in liquid nitrogen. Approximately 0.5-1.0 g of infected root tissue was obtained from 80 inoculated plants.

Staining to visualize nematodes in infected roots

The infection level was evaluated by the number of nematodes that infected one root tip. The roots were separated 3 days after infection of the plants and immersed for 90 s at 95 ° C in lactophenol containing 0.1% Cotton Blue dye. After washing for 5 s in water, the roots were kept in lactophenol at room temperature (CT) for 3-4 days until bleaching. Stained nematodes were visualized using a light microscope.

Isolation of RNA from healthy and infected root tissue

The root tissue was crushed to obtain finely ground powder using a mortar and pestle while cooling in liquid nitrogen. Then, aliquots of approximately 100 mg were transferred into similarly cooled microcentrifuge tubes and 300 μl of hot phenolic extraction buffer (50% phenol, 50% extraction buffer: 0.1 M lithium chloride, 0.1 M Tris-HCl pH 8.0 was added (CT), 10 mM EDTA, 1% SDS) and incubated at 80 ° C for 5 min. Then an equal volume of chloroform was added and the homogenate was centrifuged using a microcentrifuge for 15 min at 4 ° C. After that, the aqueous phase was extracted with 600 μl phenol / chloroform and again centrifuged using a microcentrifuge as described above. The aqueous phase was again separated and RNA was precipitated using an equal volume of lithium chloride at 4 ° C. overnight. The pellet was pelletized using a microcentrifuge for 15 minutes at RT and washed in 70% ethanol. Debris was lyophilized, resuspended in DEPC-treated water and analyzed spectrophotometrically. RNA levels were evaluated using denaturing gel electrophoresis (adapted for research purposes, the method described by Shirzadegan et al., 1991).

Subtractive cloning of specific infectious cDNA

Poly (A) ⁺ RNA (mRNA) was isolated from 200 mg of total RNA samples obtained from healthy and infected root tissue of C319 tobacco using Dynabead magnetic loaded oligo-dT granules according to the manufacturer's instructions. The first cDNA strand was synthesized in situ using a Dynabead-bound poly (A) ⁺ fraction isolated from healthy tissue to obtain driver DNA. The first and second strands were synthesized in situ using the Dynabead-bound poly (A) ⁺ fraction isolated from the infected tissue to obtain the target DNA. All cDNA reactions were performed using the cDNA synthesis kit according to the manufacturer's instructions (Pharmacia). The three oligonucleotides used, i.e. SUB21 (5 'CTCTTGCTTGAATTCGGACTA 3'), designated as SEQ. ID No. 30, SUB25 (5 'TAGTCCGAATTCAAGCAAGAGCACA 3') (sequences described by Duguid and Dinauer, 1990 and designated as SEQ. ID. No. 31) and LDT15 (5 'GACAGAAGCGGATCCd (T) ₁₅ 3') (O'Reilly, 1991 ), denoted as SEQ. ID No. 32, were treated with T4 phage polynucleotide kinase according to the method described by Maniatis et al., (1982). Then SUB21 and SUB25 were “annealed” to obtain a linker, which was then ligated to the target DNA using T4 phage DNA ligase according to the method described by King and Blakesley (1986). Then, the pellets carrying the target DNA were washed extensively with TE and the second cDNA strand was eluted at 95 ° C in a five-fold (5x) SSC.

RNA associated with Dynabead granules that are associated with driver DNA was removed by heating and the eluted target DNA was hybridized with driver DNA at 55 ° C in 5 × SSC for 5 hours. Unreacted hybridization of the target DNA was separated from the bound with driver-DNA granules at room temperature (CT) according to the manufacturer's instructions, after which the target DNA that has entered the hybridization reaction was also separated from the driver-DNA bound to the granules at 95 ° C. The target DNA obtained by elution at room temperature was again added to the driver DNA and the hybridization was repeated. This process was repeated until the amount of the target DNA that entered the hybridization reaction with the driver DNA no longer exceeded the amount that did not enter the hybridization reaction. DNA concentrations were evaluated using a DNA Dipstick kit (Invitrogen) according to the manufacturer's instructions.

Aliquots of the final fraction, obtained by elution at room temperature, were used for PCR amplification (Eckert and Kunkel, 1990) to obtain double-stranded cDNA for cloning in a plasmid vector. The amplification of the target DNA was performed using primers SUB21 and LDT15 under the conditions described by Frohman et al., 1988. Then, PCR products were inserted by ligation into the Smal digested pBluescript vector according to the method described by King and Blakesley (1986).

Subtractive library screening using reverse Northern blotting

Recombinants were identified by colonial PCR (Gussow and Clackson, 1989). Amplified inserts were analyzed by Southern blotting in triplicate on Pall Biodyne type membranes according to the manufacturer's recommendations. Both pre-hybridization and primary hybridization were carried out at 42 ° C in 5 × SSPE, 0.05% BLOTTO, 50% formamide. The membranes were hybridized separately with cDNA probes (see below) obtained from healthy and infected tissue, and with a probe containing an amplified target DNA from the latter obtained by separation (subtraction) of the fraction. Clones that showed a hybridization signal only with a cDNA probe from infected tissue or showed a hybridization signal only with a probe from the fraction obtained by separation (subtraction) were selected for further analysis.

Obtaining cDNA probe

Samples containing 10 μg of total RNA from healthy and infected tissue were treated with 2.5 U of DNase1 at 37 ° C for 15 min. Then, DNase1 was denatured at 95 ° C for 10 min before cDNA synthesis, which was carried out using the manufacturer's protocol (Pharmacia). Then the RNA was removed in the presence of 0.4 M sodium hydroxide for 10 min at RT and the cDNA was purified using a Sephacryl 400HR rotary column. The yield and concentration were determined using a DNA Dipsticks type kit (Invitrogen). cDNA was labeled with oligonucleotides at a rate of about 35 ng / probe using the standard Pharmacia oligonucleotide labeling protocol.

Northern blotting

To evaluate the profile, expression of clones selected using reverse Northern blot analysis was used as probes for Northern blot analysis of either total RNA or poly (A) ⁺ RNA obtained from healthy and infected roots, stems , leaves and flowers. Blots of total RNA contained 25 μg RNA per lane, while blots of poly (A) ⁺ RNA contained 0.5-1.0 μg RNA per lane. RNA was subjected to electrophoresis on formaldehyde gels and blotted on a Pall Biodyne B type membrane according to the method described by Fourney et al. (1988). The probes were labeled and hybridized according to the methods described above.

Southern blotting

To determine whether the selected cDNA is from a plant or a nematode, C319 tobacco DNA and M. javanica nematodes were obtained according to the method described by Gawel and Jarret (1991). Southern blots were prepared for analysis by blots containing 10 μg of DNA digested with EcoRI and HindII per lane. Blots were hybridized with oligonucleotide-labeled probes as described above.

In situ hybridization

In situ hybridization was performed to determine the localization of the expression of the cDNA of interest in the region that the nematodes feed on. The tissue of the infected and healthy roots was immersed in wax, cut and hybridized with probes according to the method described by Jackson (1991).

Isolation of 5'-ends of mRNA

The structure of the 5'-ends of the RNAs of interest was determined using 5'-RACE according to the method described by Frohman et al. (1988).

Isolation of promoter regions

The promoter regions of the genes of interest were isolated by PCR using a ligated vector. 100 ng of restriction enzyme digested genomic DNA samples of tobacco cultivar C319 were ligated for 4 hours at CT (King and Blakesley, 1986) with 100 ng of pBluescript vector samples (digested with restriction enzymes to give compatible ends). Typically, enzymes such as EcoRI, BamHI, HindIII, BglII, XhoI, ClaI, SalI, KpnI, PstI and SstI were used. Then, PCR was carried out using ligation products as a template using a vector primer such as a sequencing primer-40 and a primer complementary to the 5 ′ end of the mRNA. Then the PCR products were cloned and sequenced. If necessary, the process was repeated using a new primer complementary to the 5'-end of the promoter fragment to ensure that the control sequences of the promoters were isolated.

Using the procedures described above, the KNT1 gene was identified and isolated from tobacco plants. The KNT1 promoter fragment, approximately 0.8 kb in length, extending from the transcription initiation site, was isolated and inserted into the GUS reporter carrying the pBI101 vector (Jefferson et al., 1987). The resulting construct pBIN05101 was used to transform tobacco plants. When M. javanica was infected, a high level of expression of the GUS gene was detected in the areas that nematodes feed on.

The KNT1 gene has been found to be homologous to the gene present in plants other than tobacco. These plants include, but are not limited to, Solanum tuberosum (potatoes), Lycopersicon esculentum (tomatoes), and Beta vulgaris (beets). The KNT1 gene was induced by both gall and cyst-forming nematode species.

In accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, pBIN05101 was deposited by Advanced Technologies (Cambridge) Limited, 210 Cambridge Science Park, Cambridge CB4 OWA, United Kingdom at the National Collections of Industrial , Food and Marine Bacteria, NCIMB), 23 St. Machar Street, Aberdeen, Scotland, March 20, 1997 under registration number NCIMB 40870. The vector contains the left and right T-DNA border sequences of Agrobacterium tumefaciens C58 strain. Between the border sequences there is a multiple cloning site and a gene that determines resistance to kanamycin, under the control of a plant promoter (Nos). Outside of the border sequences, a bacterial gene is located in the vector that determines resistance to kanamycin. The insertion into the vector includes the KNT1 promoter, the coding sequence of glucuronidase, the Nos terminator.

Obtaining PAP-S constructs

PAP-S transgene constructs were created in vectors suitable for use in a protoplast short-term expression system (FIG. 1). Constructs were created in the pDE4 vector with a large number of copies (Denecke et al., 1990) using the constitutive 35S CaMV promoter. The following designs were created:

1.pDE4 / Pro-PAP-S

2. pDE4 / PAP-S ΔN

3. pDE4 / PAP-S ΔС

4.pDE4 / PAP-S

5. pDE4 / PAP-S α

6. pDE4 / PAP-S β

For experiments on plant transformation, PAP-S constructs containing effector components in binary vectors using various promoters were also created. PAP-S sequences were first cloned into pDVM donor vectors (as shown in FIG. 2) containing the nematode-induced KNT1 promoter and the Nos terminator. Then the promoter-gene-terminator cassettes were cut out and cloned in the pATC binary vector (Fig. 3) and introduced by electroporation into the Agrobacterium tumefaciens LBA4404 strain.

Similarly, constructs suitable for use in accordance with other embodiments of the invention were obtained using techniques similar to those described above and selecting promoters corresponding to the target.

Rapid analysis of PAP-S constructs in tobacco protoplasts

In this assay, PAP-S-mediated ribosome inactivation was determined by evaluating GUS protein synthesis. Protoplasts were obtained from the leaves of in vitro tobacco plants and electroporated using PAP-S constructs. The effectiveness of ribosomes with respect to protein translation in tobacco protoplasts was evaluated using the GUS reporter gene in pDE4 construct under the control of the 35S CaMV promoter. The GUS gene bearing pDE4 construct was electroporated with each PAP-S construct. To obtain data on the optimal levels of GUS protein synthesis in protoplasts under conditions of competition with the second protein, a GUS-positive control was used, which was created by electroporation of tobacco protoplasts using the non-toxic BiP chaperone protein construction pDE800 (Leborgna-Castel et al., 1999) and GUS carrier pDE4. A GUS-negative control was also used, to create which co-electroporation of tobacco protoplasts was carried out using the unloaded pDE4 vector and the non-toxic chaperone protein BiP bearing the construct.

(I) Mature PAP-S.

Tobacco protoplasts were transformed by co-electroporation with PAP-S constructs and a GUS-bearing construct.

The effect of PAP-S activity on ribosomes was evaluated by GUS activity levels after expression for 24 hours (Fig. 5). The results indicated that the PAP-S protein effectively inactivates tobacco ribosomes. Therefore, when compared with the GUS-positive control, only background GUS levels were detected.

(II) Constructs containing modified PAP-S

Using tobacco protoplasts, various versions of the PAP-S proteins were analyzed (FIG. 6).

It was found that intact Pro-PAP-S was as effective as the mature PAP-S protein in inhibiting protein translation, which supports the hypothesis of its activation by endogenous proteases. However, modifications to the Pro-PAP-S sequences inhibit this activation. The role of the N- and C-terminal extensions of the PAP-S protein is probably different, since a preliminary deletion of the C-terminal region of Pro-PAP-S had little effect on subsequent inactivation of the ribosomes, and an equivalent deletion of the N-terminal region of Pro-PAP-S led to the synthesis of a molecule that could not be processed to form an active form.

This allows you to create a two-component activation system based on the use of Δ-N Pro-PAP-S with a specific cleavage site, built next to the C-terminal region, or alternatively or in addition to it, based on the use of Δ-C Pro- PAP-S with a sequence that blocks endogenous proteases, but acts as a specific cleavage site, integrated in the vicinity of the N-terminal region.

(III) Various PAP-S domains

The polypeptide regions of PAP-S α and PAP-S β were also expressed either individually or in combination with each other in tobacco protoplasts (Fig. 7).

Unexpectedly, it was found that the expression of any one protein PAP-S α or PAP-S β led to a significant decrease in GUS activity. Based on assumptions based on structural features, PAP-S α contains the RNA recognition motif and the region of the ribosome binding domain, while PAP-S β contains the crucial residue of the catalytic site. Protein PAP-S α can prevent protein translation by binding to ribosomes and terminating protein translation. The results suggest that PAP-S α is adapted to the correct folded conformation and has the ability to specifically recognize molecules. PAP-S β contains the remainder of the active site necessary for the depurinization of ribosomes, but it is unlikely that it has the ability to recognize and interact with the ribosome. However, these results unexpectedly demonstrated that PAP-S β actually has the ability to inhibit protein synthesis. This suggests that, when used individually, PAP-S β is adapted to the correct folding conformation and has the ability to specifically recognize molecules and catalytic cleavage. The inactivation of ribosomes that occurs with PAP-S β alone is higher than with PAP-S α.

Simultaneous expression of both PAP-S α and PAP-S β in protoplasts did not lead to an additional decrease in GUS activity compared to the expression of only PAP-S β protein. This differs from the situation typical for the use of maize RIP α and RIP β domains, during the joint expression of which there was an additional decrease in activity, which is described in a pending application of the same authors, filed simultaneously with this application.

Design containing structural inactivated PAP-S form:

Inhibition of PAP activity by preventing the binding of rRNA can be mediated by the formation of a fusion protein that extends into the active site near the PAP.

A suitable component selected to create the fusion protein was rice cystatin protein (oryzacystatin), and a synthetic version of the gene containing the most common codon was synthesized for optimal expression in potatoes (SEQ. ID. No. 17). To ensure that cystatin will be positioned in the region of the active site of PAP, 7 amino acids were deleted by deletion from the C-terminus of PAP. A cystatin fusion was created using a linker sequence encoding the protease cleavage site NIa of the tobacco engraving virus (TEV).

The primers described below were used to construct a fusion between PAP-S and rice cystatin ΔD86 with the TEV protease cleavage site (PCS) between them as a linker (Fig. 8). The fusion protein encoding sequence was amplified from cloned DNA using two PCR fragments; (I) PAP-S plus PCS were amplified using PS1BF and PCSPAPSR (SEQ. ID. No. 11 and 20), and (II) cystatin Δ86 with PCS was amplified using primers PCSDelta86F and SYNPOTDelta86SR (SEQ. ID. No. 21 and 22). Then, two PCR fragments were used to amplify a full-size fusion protein (PAPS-PCS-cystatin Delta86) by providing overlapping PCR extension using primers PS1BF and SYNPOTDelta86SR (SEQ. ID. No. 11 and 22).

According to this procedure, 8 amino acids from the C-terminus of the PAP-S were removed and the NIa TEV protease cleavage site (7 amino acids) was inserted using the PCSDelta86F primer (SEQ. ID. No. 21) encoding the heptapeptide: Glu Pro Val Tyr Phe Gln Gly. Cystatin was then added as a protein fused to the C-terminus of the PAPS-PCS sequence.

The fused PCR product was cloned into pBluescript vector and its sequence was confirmed by sequence analysis.

Functional analysis of inactivated PAP-S protein in an in vitro transcription / translation system

The activity of the constructs pBS / Pro PAP-S, pBS / PAP-S and pBS / PAP-S / PCS / cystatin ΔD86 was evaluated using a rabbit reticulocyte lysate system. A fast fusion transcription and translation system based on TnT phage T7 phage (Promega Corporation) was used.

TnT-containing reaction mixtures were incubated at 30 ° C. for a total of 60 minutes with various PAP constructs using luciferase-containing constructs as a positive control. After an initial 30-minute incubation of PAP constructs with TnT-containing reaction mixtures, an equal amount of luciferase matrix was added to all reaction mixtures and incubation was continued for another 30 minutes. Aliquots of the various reaction mixtures were mixed with the luciferase assay reagent (Luciferase Assay Reagent) and the luciferase activity was evaluated using a luminometer for 10 s (Fig. 9).

The translation evaluation results of the Pro PAP-S and PAP-S Proteins indicated that, as expected, the proteins inactivated translation using rabbit ribosomes. The modified PAP fusion protein was found to be relatively inactive compared to PAP. The translation of various PAP proteins was confirmed by the inclusion of biotinylated lysyl tRNA, which was used as a label in the translation process and which was detected using streptavidin conjugated with alkaline phosphatase. It was not possible to detect translated Pro PAP-S and PAP-S proteins due to the rapid inactivation of the ribosomes, while relatively large amounts of the modified PAP fusion protein were detected, indicating a relatively weak activity of this PAP fusion protein in relation to rabbit ribosomes.

Cloning of NEV Protease TEV

The TEV NIa protease gene was amplified from a TEV cDNA clone. For cloning purposes, the SpeI restriction site was removed using overlapping deletion primers. The NIa protease was first amplified as two PCR fragments using NIAPROBF primers (SEQ. ID. No. 23) plus NIAPPROSR (SEQ. ID. No. 26) and NIAPROSDF (SEQ. ID. NO. 25) plus NIAPROSR (SEQ. (ID. NO. 24) and then the full-length protease was amplified using overlapping PCR extension using NIAPROBF primers (SEQ. ID. No. 23) plus NIAPROSR (SEQ. ID. No. 24).

Rapid analysis in the protoplast fused protein PAP-S

The effect of modified PAP-S / cystatin on tobacco ribosomes was analyzed in protoplasts, and it was found that the protein had significantly lower activity compared to unmodified protein (figure 10). However, with co-expression of the TEV NIa protease in combination with the protein-modified PAP-S / cystatin, an increase in ribosome inactivation was found. Separate expression of the TEV NIa protease did not affect GUS protein synthesis.

Two-component system for nematode-induced expression of modified PAP-S

To evaluate the effectiveness of the modified PAP-S protein and its activation using the NIa protease TEV, a double construct was created using two nematode-induced promoters KNT1 (described above) and KNT2 (SEQ. ID. No. 27). KNT2 provided expression in the mass of the root (but not at the tips of the roots) and in the giant cells of the nematode-affected plant.

The TEV NIa protease gene was amplified using Pfu DNA polymerase, and the PCR product was cloned as an XbaI and SalI fragment in pBluescript vectors. The recombinant clone was sequenced to confirm its identity. The fusion genes PAP-S-PCS-cystatin ΔD86 NIa protease TEV were cloned individually in pDVM donor vectors as separate constructs using KNT2 and KNT1 promoters, respectively. The double construct (Fig. 11) was created in the pDVM intermediate donor vector and re-cloned in the pATC binary plant transforming vector to obtain transgenic plants and a sign of resistance to nematodes.

Getting transgenic plants

Two constructs, namely, pATC05501, containing a fully processed mature version of PAP-S, and pATC05502, containing the precursor Pro-PAP-S (Fig. 4), were introduced using the Agrobacterium tumefaciens LBA4404 strain into tobacco and potato plants using a leaf cultivation method discs described by Horsch et al. (1985).

Attempts to transform tobacco plants using pATC05501 (mature PAP-S) failed to produce callus or seedlings, despite repeated experiments. When using control constructs, as expected, transgenic seedlings were obtained, which indicated the dependence of this phenomenon on the mature PAP-S sequence.

In contrast, transgenic tobacco plants were obtained using pATC05502 (Pro-PAP-S).

During the transformation of potato plants, it was not difficult to obtain transgenic plants using either constructs containing mature PAP-S or using constructs containing the precursor Pro-PAP-S.

Screening for resistance to nematodes of tobacco plants transformed with PAP-S constructs

Transgenic Pro-PAP-S-bearing tobacco plants were used in standard resistance tests for gall nematodes Meloidogyne javanica. Transgenic and control tobacco seedlings were randomly planted blindly in devices to moisten the roots [(rootrainer]) filled with fertilizer-free humus compost of high quality and the largest leaves were cut in half. The plants were covered with polyethylene to maintain high humidity until until engraftment takes place. Before completion of the engraftment, holes were made in polyethylene to reduce moisture. A week after the engraftment, small plants were infected 200 hatched nematodes of the second age (J2) Meloidogyne javanica. Watering was carried out with liquid top dressing only after the soil became dry enough to start leaf withering. Rutrainers were placed in boxes on a heated insulating coating to maintain the soil temperature at 25-30 ° С. cut once a week to stop their growth and prevent the growth points from covering larger leaves due to the density of planting. Plants were collected for their analysis approximately 4 weeks after infection and the roots were washed from the soil and bleached for 4 minutes in a 1% sodium hypochlorite solution. The bleach was removed by washing with water and then the roots were soaked in a large volume of water for 15 minutes with periodic stirring. Then the roots were placed in 10-15 ml diluted in a ratio of 1: 500 of a mother liquor of acidic fuchsin in 5% acetic acid (a mother liquor of acidic fuchsin was obtained according to the method described in Introduction to Plant Nematology by V. N. Dropkin, ISBN 0- 471-85268-6. To prepare it, 0.35 g of acidic fuchsin was dissolved in 100 ml of a 1: 3 mixture of glacial acetic acid and distilled water). For staining, the samples were placed in a boiling water bath for 4 min and then transferred to a 37 ° C bath for 4 hours. The dye was decanted and the samples were clarified by adding acidified glycerin and incubated at 37 ° C overnight.

Then the clarified roots with stained nematodes were placed in Petri dishes (the sample was placed on the inside of the lid of the Petri dish and the base of the Petri dish was used to distribute and press the sample in order to facilitate analysis using a microscope). Samples were analyzed at an increase of 20-100 times and the nematode population was analyzed in several ways. Gall nematodes were divided into 3 groups: a) parasitiform nematodes, b) encapsulated nematodes in which no eggs are formed, and c) encapsulated nematodes in which eggs are formed. The diameters of almost completely encapsulated nematodes were evaluated visually using an ocular scale.

The size of the nematode parasitic on the plant was significantly reduced on the transgenic tobacco plants of the Pro-PAP-S line compared to the size of the nematode on the control infected plants (Fig. 13). This suggests that plants expressing Pro-PAP-S have an inhibitory effect on the development of nematodes.

Screening for resistance to nematodes of tobacco plants transformed with PAP-S constructs

For the experiment on resistance assessment against potato (cyst-forming) nematodes Globodera pallida, pathogenic variant 2/3 (MCC), 21 transgenic lines carrying the Pro-PAP-S construct were used. For the experiment, the control sensitive (Hermes) and partially resistant (Sante) varieties were used.

The initial assessment of the resistance of transgenic plants was carried out using random blind experiments using groups comprising 20-25 transgenic lines, and using at least 10 replicates for two or more control lines. Routiners or containers were filled with a 50:50 mixture of loam and sand. 12 l of a mixture of loam and sand moistened 1250 ml of water, obtaining a mixture with a water concentration of 40%. Three cysts were placed on the roots of each seedling, which was then introduced into the hole in the compost and the roots were carefully covered with compost. The plants took root and after that they were watered only once a week with liquid top dressing or only when the soil became sufficiently dry. After the plants reached a height of about 10 cm, their tops were cut off, preventing further growth. Plants were grown for approximately 3 months, allowing cysts to mature. Then the plants were allowed to dry for 1 month. Cysts were isolated from plants, intensively washing the soil and roots in a beaker with 250 ml of water. The soil was allowed to settle for several minutes and the supernatant was poured into a large filter funnel with a Whatman No.1 filter paper disc 32 cm in diameter placed in it. The supernatant was allowed to stand in the funnel for 1 min and then a drop of detergent was introduced into the central region of the solution surface Hederol for shifting the surface material of the meniscus of the solution to the side of the filter. Then the filter base was punctured to remove the remainder of the solution. The filter paper disc was removed and the number of cysts adhered was counted.

Plants were considered as showing signs of resistance if they were infected with fewer cysts than sensitive control lines (Fig. 12). The population of transgenic plants as a whole was significantly less affected by MCC. At least two transgenic lines (namely, pATC05502 - 6, 8 16) showed the same level of resistance as the commercially available resistant variety Sante.

Comparison of potato plants transformed with Pro PAP-S, antisense KNT1, or cow pea trypsin inhibitor (CpTI) under the control of the KNT1 promoter showed that Pro PAP-S gives a higher level of resistance compared to the resistance level of transgenic lines containing either antisense KNT1 or CpTI (Fig. 15).

KNT1 is a gene that is almost specifically induced in giant cells when infected with nematodes. KNT1 probably plays an important role in the process of infection and development of giant cells. The introduction of the antisense sequence KNT1 probably inhibits the nematode infection process.

Cow Pea Trypsin Inhibitor (CpTI) is a protein that inhibits the enzymes involved in cleavage in nematodes, and its expression should impede the nutrition and development of nematodes. This is not a system that causes cell death per se, but has a direct effect on nematodes.

Comparative data suggest that the transformation of plants with a construct containing Pro PAP-S has a more noticeable effect on reducing nematode infection than the action of lines containing either antisense KNT1 or CpTI.

Claims (37)

1. A method of inducing necrotic action in specific cells of a plant where the plant is transformed with a chimeric gene, wherein the coding sequence of the gene encodes a PAP-S α protein or PAP-S β protein, or a fragment thereof, this gene contains a promoter that is active in response to a specific irritating effect on this plant, as a result of which the specified protein or its fragment is expressed in specific plant cells. 2. The method according to claim 1, characterized in that the chimeric gene encodes a mature protein PAP-S α, wherein said coding sequence is the sequence shown in SEQ. ID No: 5, or a sequence at least 70% identical to this sequence and retaining the desired enzymatic activity, and the amino acid sequence is given in SEQ. ID No: 6 or is its variant that retains the required enzymatic activity. 3. The method according to claim 1, characterized in that the chimeric gene encodes a protein PAP-S β, and the specified coding sequence is a sequence shown in SEQ. ID No: 7, or a sequence at least 70% identical to this sequence and retaining the desired enzymatic activity, and the amino acid sequence is given in SEQ. ID No: 8 or represents its variant, which retains the required enzymatic activity. 4. The method according to any one of claim 2 or 3, characterized in that said coding sequence is at least 80% identical to the sequences given in these SEQs. ID 5. The method according to claim 4, characterized in that said coding sequence is at least 90% identical to the sequences given in these SEQs. ID 6. A method of inducing necrotic action in specific cells of a plant, where the plant is transformed with two chimeric genes, the coding sequence of the first of these genes encoding the inactivated American phytolacca antiviral protein and the coding sequence of the second gene encoding the second molecule, which is the activator of the inactivated American phytolacca antiviral protein , each of these two genes contains a promoter, and the promoters act together in response to a specific skoe irritant effect on the plant, resulting in a virus-inactivated protein pokeweed activated in specific cells of the plant. 7. The method according to claim 6, characterized in that the terminal blocking sequence of the inactivated antiviral protein of American phytolacca has an additional necrotic effect on the cell or pathogen. 8. The method according to claim 7, characterized in that the terminal blocking sequence is the sequence of oryzacystatin shown in SEQ. ID No: 17. 9. The method according to claim 7, characterized in that the terminal blocking sequence of the indicated American phytolacca antiviral protein is an inactivating mature PAP molecule. 10. The method according to claim 6, characterized in that the end blocking sequence is a native end sequence of Pro-PAP from any Pro-PAP molecule. 11. The method according to any one of claims 7, 9 or 10, characterized in that the terminal blocking sequence is a C-terminal blocking sequence. 12. The method according to any one of claims 7, 9 or 10, characterized in that the terminal blocking sequence is an N-terminal blocking sequence. 13. A method according to any one of claims 6-12, characterized in that said coding sequence of an inactivated American phytolacca antiviral protein contains a specific cleavage site between a mature American phytolacca antiviral protein and a blocking sequence. 14. The method according to item 13, wherein the cleavage site is a protease cleavage site NIa of tobacco engraving virus (TEV) (SEQ. ID. Nos: 28 and 29). 15. The method according to any one of claims 6-14, wherein the coding sequence of the second molecule is a protease sequence that cleaves a specific cleavage site. 16. The method according to clause 15, when it is dependent on clause 14, wherein the protease is a TEV NIa protease. 17. The method according to any one of claims 6-16, characterized in that the promoters have an overlapping expression site. 18. The method according to any one of claims 1 to 17, characterized in that the irritating effect is associated with the attack of pathogens, chemical inducers or the natural development of the plant. 19. The method according to any one of claims 1 to 18, characterized in that the promoter is chosen to provide one of the following effects: reducing the harmful effects of nematodes, sterility, changes in flower morphology, abscision, shedding of seeds or the development of trichome. 20. A chimeric gene whose function is to induce necrotic activity in specific plant cells, where the coding sequence of the gene encodes a PAP-S α protein, PAPS β protein or fragment thereof, this gene contains a promoter that is active in response to a specific irritating effect on it plant, resulting in the specified protein or its fragment is expressed in specific plant cells. 21. The chimeric gene according to claim 20, where the specified coding sequence is shown in any of claims 2-5. 22. The chimeric gene according to claim 20 or 21, characterized in that the promoter is selected to provide one of the following effects: reduce the harmful effects of nematodes, sterility, changes in flower morphology, abscision, shedding of seeds or the development of trichome. 23. The chimeric gene according to claims 20, 21 or 22, characterized in that the promoter is active in response to an irritating effect that is associated with the attack of pathogens, chemical inducers or the natural development of the plant. 24. A design that includes two chimeric genes whose function is to induce necrotic action in specific plant cells, where the coding sequence of one of these genes encodes the inactivated American phytolacca antiviral protein, and the coding sequence of the second gene encodes the second molecule, which is an activator of the inactivated antiviral protein American phytolacchi, with each of these two genes containing a promoter, the promoters act together in response to a digital irritant effect on this plant, as a result of which the inactivated antiviral protein of American phytolacca is activated in certain plant cells. 25. The design according to paragraph 24, where the specified coding sequence of the first gene includes the sequence that is shown in any of claims 2-5. 26. The construction according to paragraph 24 or 25, characterized in that the end blocking sequence of the inactivated antiviral protein of American phytolacca is given in any of paragraphs.7-12. 27. The construction according to paragraphs 24, 25 or 26, characterized in that the coding sequence of the inactivated antiviral protein of American phytolacca is given in paragraph 13 or 14. 28. The construction according to paragraphs 24-27, characterized in that the coding sequence of the second molecule is given in paragraph 15 or 16. 29. The construct according to any one of paragraphs.24-28, where the promoters have a region of overlapping expression. 30. The construction according to any one of paragraphs.24-29, where the protomotor is selected to provide one of the following effects: reduce the harmful effects of nematodes, sterility, changes in flower morphology, abscision, dropping seeds or the development of trichome. 31. A set of constructs whose function is to induce a necrotic effect in specific plant cells, including two constructs, where each construct includes a chimeric gene, the coding sequence of one of these genes encodes the inactivated antiviral protein of the American phytolacca, and the coding sequence of the second gene encodes the second molecule which is an activator of the inactivated antiviral protein of American phytolacca, and each of these two genes contains romotor promoters act together in response to a specific irritant effect on the plant, resulting in the inactivated pokeweed antiviral protein is activated in specific cells of the plant. 32. The set of constructs according to p, where the specified coding sequence of the first gene includes the sequence that is shown in any of claims 2-5. 33. A set of designs according to p. 31 or 32, characterized in that the terminal blocking sequence of the inactivated antiviral protein of American phytolacca is given in any one of claims 7-12. 34. A set of constructs according to claims 31, 32 or 33, characterized in that the coding sequence of the inactivated antiviral protein of American phytolacca is given in claims 13 or 14. 35. A set of constructs according to claims 31-34, characterized in that the coding sequence of the second molecule is given in clause 15 or 16. 36. A set of constructs according to any one of paragraphs.31-35, where the promoters have a region of overlapping expression. 37. A set of constructs from paragraphs 31-36, where the promoter is chosen to provide one of the following effects: reduce the harmful effects of nematodes, sterility, changes in flower morphology, abscision, dropping seeds or the development of trichome.

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