MXPA05001216A - Protein transport enhancer for transgenic plants. - Google Patents

Abstract

Expression and stability of desirable proteins in transgenic plants are promoted and maintained by treatment with a protein transport enhancer. Preferably, the transgenic plant is a commodity crop that has been modified to express pesticidally effective protein proteins.

Description

PROTEIN TRANSPORTATION ENHANCER FOR TRANSGENIC PLANTS FIELD OF THE INVENTION [0001] The invention relates to a method for the treatment of transgenic plants, especially crop plants that are designed to express pesticidally effective proteins. BACKGROUND OF THE INVENTION [0002] Transgenic plants have had a significant impact on commercial agriculture, with promising benefits but which give rise to new aspects of pest management or administration. Notably, plant-based agricultural crops that have been modified with pesticide-effective crystal protein genes to express pesticide-effective proteins hold the promise of selectively targeting pests by making their food source toxic (plant tissues). , thus reducing or eliminating the amount of chemical insecticides that are used to control pest populations. [0003] Certain members of the gram positive bacteria that belong to the genus Bacillus produce proteins that are insecticidal. Those that are more well characterized are those of Bacillus thuringiensis (BT), where insecticidal proteins are found as crystalline bodies with sporulating bacteria. Insecticidal crystal proteins are characterized by their potency and specificity towards specific insect pests, many of which are agronomically important and their relative safety to non-target insect and vertebrate species, particularly for humans. They have enjoyed a long history of use in horticultural industries where the mixture of crystals and spores is sprayed as well as a chemical pesticide, but they have not been used with great success in field crops with a width of hectares. [0004] Insecticidal crystals are composed of a large protein that is essentially inactive. When a caterpillar ingests some of the insecticidal crystals, the alkaline reducing conditions of the midgut or small intestine of the insects cause the crystals to dissociate and release the crystal protein. At this stage, the protein toxin is inactive, but specific proteases within the insect's gastric juices slice the protein to its protease-resistant core that is now fully active. This activated insecticidal protein then binds to a specific receptor on the brush border membranes of cells that line or line the midgut and insert into cell membranes. When approximately eight of these are aggregated together, they form a pore or channel through the membrane and allow the contents of the cell to escape, causing the death of the cells essential for nutrient absorption. The insect quickly stops eating and eventually dies of hunger or dies of secondary bacterial infections in about 24 hours. The processes of crystal solubilization, proteolytic processing to an active insecticidal protein and the binding to a specific receptor all make BT proteins highly specific and highly desirable from an environmental perspective. [0005] Many thousands of different isolates of B. thuringiensis have been collected and their insecticidal protein content and activity spectra are determined. A large number of BT insecticidal protein genes have also now been cloned and sequenced. To avoid any confusion, the researchers have proposed a uniform nomenclature system for the crystal protein genes (Cry genes) based on their protein sequence and the types of insects for which they were toxic.

Table 1

[0006] The proteins encoded by the Cry I genes are all similar in sequence to proteins and are toxic only to caterpillars, ie larvae of moths and butterflies (Lepidoptera); Cry II genes encode proteins toxic to Lepidoptera and / or Diptera (flies and mosquitoes) while Cry IV proteins are only active against Diptera. The Cry III genes produce active proteins against beetle larvae (Coleoptera). Within these main groupings, smaller divisions have been made when considering the similarities or differences between the different protein sequences. The Cry I group, for example, it was originally divided into Cry IA, IB and IC (although now it reaches Cry IG) where the different subgroups can only be 50% identical at the protein sequence level. Finer sub-divisions have also been made and Cry IA now consists of Cry IA (a), IA (b) and IA (c). [0007] A number of methods have been developed to introduce desired genes into cells of crop plants and to grow fertile plants therefrom. See the patent of the U.S.A. No. 6,329, 574 and htt: // www. cotton.pi. csiro au / publicat / pest / transgen. htm, the contents of which are incorporated herein by reference. The method of selection depends in part on the target species, but modification of cotton is often based on a natural gene transfer agent that has evolved in its own method of genetic plant engineering. The disease called neck gall or crown (Crown Gall) is a disease of plant tumor caused by the bacterial pathogen that contains the soil, Agrobacterium tumefaciens. In the early 1970s, it was recognized that the bacterium causes the disease by transferring some of its own genetic material to the DNA of the cells of plants it infects. These parasitic genes upset the normal biochemical machinery of infected cells and cause them to produce novel compounds that only bacteria can use. This process of genetic colonization by bacteria was just what the genetic engineers were looking for, as long as they could stop the bacteria from causing the symptoms of the disease. After further study, scientists were able to identify which genes caused the disease and because bacteria are very simple organisms to manipulate genetically, they were able to replace the genes that cause the disease with the novel genes they built from parts of potentially useful genes. The bacteria could then be used to load genes from the test tube into the cells of plants. However, not all plant cells exposed to bacteria eventually receive the novel genes. Removal of cells from unmodified plants relative to modified cells utilizes antibiotic purification. [0008] Plant cells are sensitive to many of the antibiotics that are used to control bacterial infections in animals and humans. If a gene could be isolated that would give plant cells tolerance to one of these toxic antibiotics, then if it is physically linked to some desirable gene and inserted into the Agrobacterium, it will provide a useful screening system to kill those cells that do not receive the genes during the "infection" process. Genes have been known in bacteria for many years to give the bacteria resistance to antibiotics by producing enzymes that break or chemically modify the antibiotic, so that it is no longer toxic. By using the techniques of breakdown or separation of genes described above, the researchers have been able to modify the bacterial gene that encodes an enzyme that detoxifies the antibiotic kanamycin and have produced a new hybrid gene that causes the production of this enzyme in plant cells and avoids his death in the presence of potentially lethal doses of kanamycin. It has been possible to combine this antibiotic selection system with plant tissue culture procedures, by using Agrobacterium to supply genes in a wide variety of plants from petunias to cottons. [0009] Cotton is a crop of particular interest. Commercially available forms of transgenic cotton use CryIAc (BOLLARD ™ 1 by Monsanto) or a combination of CryIAc genes with Cry2Ab (BOLLARD ™ 1 II by Monsanto) to express the endotoxin protein of B. thuringierasis. Field efficacy reports indicate a 50-70% reduction in the amount of applied pesticide required to control Helicoverpa armígera and H. punctigera. Also of interest are the modified crop plants with the B. thuringiensis crystal toxin genes designated cryET33 and cryET34 that encode toxic glass proteins to colepterans, the crystal protein CryET33 (29-kDa) and the cryET34 gene encoding the crystal protein CryET34 from 14-kDa. The crystal proteins CryET33 and CryET34 are toxic to the red beetle larvae of flour and Japanese beetle larvae. (See, U.S. Patent 6,399,330.) [0010] The use of transgenic crop plants gives rise to new aspects in the ongoing struggle toward the management of integrated plants. Some of these aspects refer to a reduction in the amount of endotoxin expressed as the plants mature, leading to a loss of efficiency in the later stages of the development season (the last 1/3 of the cotton development season). ) and the increased likelihood of surviving pests that may develop immunity to endotoxin. These disadvantages have led to the development of pest control strategies that dictate a plant "window" relative to the development cycle of local pests and minimum designated threshold values of plague population for pesticide application. [0011] Physiological strain and physical damage to the transgenic plants, can also result in a reduction of the expressed endotoxin protein with a corresponding fall in the efficacy of pest control. In this way, a prolonged drought and / or high temperatures can reduce the rate of endotoxin expression in the transgenic crop and provide a significant drop in pest protection that may dictate the need for pesticide sprays. [0012] The specific reasons for the drop in endotoxin protein expression are not well understood. In BT cotton, it is assumed that the expression of the CryIAc gene falls because the CMV35S promoter concentration declines, the gene is "silenced" or other post-transcription events. It is also considered that the CryIAc protein is reduced due to increased turnover, sequestration within the plant or dilution due to growth and aging. It is understood that CryIAc transcription levels are unstable in both immature and mature BT cotton plants. [0013] It would be convenient to have a system for treating transgenic plants designed to express pesticidally effective proteins that promote the expression of these proteins despite increased plant maturity, physiological stress and physical damage. SUMMARY OF THE INVENTION [0014] It is an object of the invention to provide a method for treating transgenic plants, preferably transgenic crops expressing pesticidal proteins, and especially for transgenic crops expressing insecticidal proteins. [0015] Another objective of the invention is to provide a method for extending the period in which the expressed proteins are presented in sufficient quantity to control populations of insect pests that are fed with the treated plants. [0016] In accordance with these and other objects that will be apparent from the present disclosure, a method for treating transgenic crop plants according to the invention comprises applying to the foliage of transgenic plants that is designed to express pesticidally effective proteins, an enhancer of protein transport that promotes the expression and / or stability of pesticidally effective proteins within the treated plants. [0017] Although it should not be bound by any particular theory of operation, it is considered that the protein transport enhancer acts in one or more of several ways: (a) as a form of protective water substitute for cell membranes during times of tension by deprivation of water, (b) as a protein stabilizer for the desired pesticidal protein, and / or (c) as a binder for proteins that facilitates movement by intraplant transport mechanisms. The result is that the transgenic harvest plants treated according to the invention, express and move pesticide-effective proteins within fruit tissues despite physiological stress due to water shortage damage to the plant. It is considered that the treatment according to the invention will also continue to express effective levels of pesticidal protein through plant growth and maturity. DETAILED DESCRIPTION OF THE INVENTION [0018] Transgenic plants are treated, according to the invention, with a protein transport enhancer that stimulates and / or protects the cell expression and intraplant transport mechanisms, enough that desired protein levels. pesticides are kept in plant tissues, fruits and seeds, despite water deprivation, physical damage to plant tissues, growth and maturity of the plant. The maintenance of expression levels and protein concentrations within the tissue of transgenic plants help to maintain efficacy levels for better pest control, additional reductions in applied pesticide quantities now require counter-attacking reductions in efficacy, and should help in avoiding the survival of exposed pests and the development of resistant pest populations.

[0019] It will be understood that all the percentages identified herein are given by weight with respect to the total weight of the product, unless noted otherwise. [0020] Suitable protein transporters for use in the present invention include one or more compounds and agrochemically acceptable salts of compounds according to the structure of Formula 1: Formula 1 wherein X is N02, Y is H, QL-CS alkyl, CA-C6 alkyloxy, C2-C6 alkenyl and Z is C or N. [0021] In particular, the Y portion can be methyl, ethyl, propyl, butyl, iso-butyl, pentyl, hexyl, methoxy, ethoxy, propoxy, butoxy, iso-butoxy, pentoxy, and hexoxy. [0022] Preferably, the protein transport improvers employed in the present invention include one or more compounds and salts of compounds according to the above structure, wherein X is a nitro group in the ortho or para positions relative to the hydroxy group ,? is hydrogen or Cx-C3 alkyloxy and Z is a carbon atom. Suitable salts include alkali metal salts soluble in water (especially sodium and potassium salts), ammonium salts, and other water soluble salts that are not phytotoxic or environmentally sound. [0023] The most preferred protein transport mej- erator includes a combination of sodium salts of p-nitrophenolate (A), o-nitrophenolate (B), and 2-methoxy-5-nitrophenolate (C). It is particularly preferred that the protein transporting mixture contains a mixture of these salts in a range of proportions within the range of A: B: C of (0.1-10): (0.1-10): 1. A commercial source of these salts are available under the name ATONIK ™ 1 from Asahi Chemical Mfg. Co., Ltd., in a ratio of A: B: C of 2: 3: 1. [0024] Protein transporting agents according to the invention are applied in a proportion generally less than 20 grams of each active ingredient per .4047 ha (1 acre) of the treated field (g Al / ac). Preferably, these improvements are applied in a proportion within the range of 1-20 g of Al / ac and more preferably in a proportion in the range of 3-18 g of Al / ac. Especially preferred when salts of protein transporters are used within the range of 0.01-5% by weight based on total weight and applied in a proportion (combined) in the range of 36.53 to 1461.33 1 / ha ( 0.5-20 fluid ounces per acre (oz / ac)). [0025] Protein transporting agents according to the invention can be applied in combination with one or more active ingredients, spraying aids, dispersing agents or additional agents for agricultural use in the target plant. Exemplary active ingredients that can be applied with the protein transporting enhancer of the invention include herbicides, improved plant growth agents, agents that stunt the growth of plants, foliar fertilizers, fungicides (external and systemic) and insecticides (external and systemic). [0026] Herbicides which may be employed include triazines (for example, atrazine), ureas, glyphosate, sulfosate, glyphosinate and sethoxydim. [0027] Suitable plant growth agents for the present invention include plant growth hormones, such as at least one of the 84 gibberilins identified with preferred GA3, GA, GA5, GA7 and GAg, -cytokinins (e.g. zeatin, kinetin, benzyl adenine, dihydrozeatin, and isopentenyl adenine), auxins (for example, indoleacetic acid (IAA), indole butyric acid (IBA), and naphthaleneacetic acid (NAA)); and polyhydroxycarboxylic acids of 2, 4, 5, and 6 carbon structures; ethephon; and fertilizers.

[0028] Suitable atrophic agents of the growth of suitable plants useful in the invention, include chlormequat chloride, mepiquat chloride, as well as maleic hydrazide and its esters. These plant growth regulators affect and alter plant metabolic processes to improve or retard the growth of plants. All of these agents can be used in accordance with the speeds of application and synchronization specified by the manufacturer on the product label. [0029] Systemic fungicides that will benefit by the invention include tridemorph, metalaxyl, iprodione, fosetil-aluminio, thiophanate, benomyl, triadimefon, carbon, oxycarboxin, carbendazim, thiabendazole, thiophanate, etirimol, bupirimate and dimethirimol. [0030] Suitable systemic insecticides include aldicarb, acephate, carbofuran, dimethoate, phorate and terbufos. [0031] The specific mechanisms by which the mechanisms of expression and transport are effected are not well known. For example, tests on unmodified wheat with radiolabeled nitrophenolate salts have followed the treatment agent with proteins transported to the nucleus of the seed in wheat plants. Another study suggests that phenolic agents act as moisturizing agents for cell membranes.

[0032] In cotton, it is recognized that cottonseed requires high amounts of protein, and the seeds produce carbohydrates within the plant system. Tests with ATONE in normal (that is, not genetically modified or otherwise altered to express pesticidal proteins as a normal part of the plant metabolism function) show an increased yield of seeds and frays or lint. This effect is consistent with the action as a regulator of plant growth, but does not necessarily suggest anything about the effect of treating plants that have been genetically modified to produce a protein designated as part of the normal function of the plant. [0033] Transgenic plants that can be treated according to the invention, are generally those that have been modified from the wild type to contain a gene expressing a desired pesticidally effective protein sequence. Preferably, the transgenic plant includes genes that express pesticidally effective proteins, which are effective to provide resistance against attacks or infections by insects, bacteria, fungi, mildew, mold, mites and the like. [0034] Two genes of particular effectiveness are the CryIAc gene (displaced by the CMV35S promoter) and the Cry2Ab gene. These genes can be inserted individually or in combination in cotton, corn, wheat, sorghum, soybeans and crops of agricultural products to provide pesticide-effective protection against a variety of insect pests. Of special interest is the cotton material that has been modified to express pesticidally effective proteins (e.g., BT cotton). The BT cotton seed material that can be treated according to the invention is commercially available from various sources under the trademark BOLLGARD ™ or BOLLGARD ™ II (Monsanto Co.). [0035] Protein transporters according to the invention are applied to transgenic plants at rates and times in the season or growth cycle of the plant to maintain pesticidal protein expression and stabilization within plant tissues. The following table identifies preferred transgenic plants to be treated and the amount of protein transporter to be applied within generally preferred, preferred and most preferred application rates. Table 1 PLANT VELOCITY SPEED OF TRANSGENIC SPEED APPLICATION APPLICATION APPLICATION M'S 1 / ha (OZ / AC) PREFERRED PREFERRED (Oz / Ac) (Oz / Ac) Cotton Bt 2.47-7306.6 2.47-3653.3 365.3-2192 (1-100) (1 -50) (5-30) Cotton Bt 2.47-7306.6 2.47-3653.3 365.3-2192 (1-100) (1-50) (5-30) EXAMPLES [0036] Transgenic Bt Cotton (Deltapine Nucotn B) and unmodified cotton plants in two liter pots in a controlled environment, were sprayed with ATONI .1 ^, a commercially available product containing a mixture of agents that are considered agents. Regulators of growth but that seem to act well as mej oradores of transport of protein for plants of crops of consumption modified genetically. ATONIK ™ 1 contains the sodium salts of p-nitrophenolate (0.3%), o-nitrophenolate (0.2%), and -nitroguayacolato (0.1%). The cotton plants were treated to the 7th true leaf (TL) and sampled 10 days later for the superior expanded peduncle leaf. The temperature was maintained in the range of 24.4 to 30 degrees C (76 to 86 degrees F) with adequate hydration. After sampling (7th TL + 10 days), the plants were again sprayed with AT0NIKMR and subjected to tension from a high temperature 30-38.9 degrees C (86-102 degrees F) and inadequate water supply (drought conditions) until which were sampled 5 days later (7th TL + 15 days) for expanded main peduncle leaf. Five days later, (7th TL +20 days) under the same conditions, the plants were sampled by leaves and squares. [0037] After each sampled, tissue samples were placed in sealed bags and immediately taken to a worm and cotton worm feeding facility. Maize and cotton worm mortality was measured at 24, 48, 72 and 96 hrs from the start of feeding for the first samples of spray application and at 72 and 96 hrs for the 2nd samples of spray application. [0038] Once the mortality study is completed, a leaf profile of the representative cotton plants is taken for protein analysis. Leaves were collected at nodes 2, 6, 8 and 10 (counting from the top) and stored at -72.2 degrees C (-80 degrees F). [0039] The mortality of the corn worm and the cotton for leaves collected in the first collected sample (7th TL + 10 days) is illustrated in Table 2.

Mortality of the corn worm and the 1st Cotton treatment (% dead at 7a TL + X days) 24 hrs 48 hrs 72 hrs 96 hrs Control 0 28.3 56.7 58.3 (Bt cotton) Mortality of the corn worm and the 1st Cotton treatment (% dead at 7a TL + X days) 24 hrs 48 hrs 72 hrs 96 hrs ?? 0 ??? 365.3 0 35.0 60.0 68.3 1 / ha (5 oz / ac) ATONIK 730.7 1.7 43.3 61.7 71.7 1 / h (10 oz / ac) ATONIK 1461.3 3.3 60.0 78.3 81.7 1 / ha (20 oz / ac) Control 0 1.7 1.7 3.3 ( non-Bt cotton)

[0040] Mortality of corn worm and cotton in leaves collected five and 10 days after the second spray is reported in Table 3. Table 3 Mortality of the corn worm and cotton (% dead at 7a TL + X 2 ° Treatment days) 15 Days 20 Days 72 hrs 96 hrs 72 hrs 96 hrs Control (cotton 68.3 71.1 90.0 95.0 Bt) ATONIK 365.3 63.3 71.1 81.7 91.7 Mortality of the corn worm and cotton (% dead at 7a TL + X 2o Treatment days) 15 Days 20 Days 72 · hrs 96 hrs 72 hrs 96 hrs 1 / ha (5 oz / ac) ATONIK 730.7 1 / h 85.0 90.0 85.0 96.7 (10 oz / ac) ATONIK 1461.3 83.3 90.0 90.0 98.3 1 / ha (20 oz / ac) Control (cotton 0 0 5.0 6.7 no-Bt)

[0041] Mortality of corn worm and cotton in frames collected in 7th TL + 20 days (2nd treatment) sprayed is reported in Table 4. Table 4 Mortality of the corn worm and cotton (% dead to 7th TL 2nd Treatment + 20 days) 72 hrs 96 hrs Control (Bt cotton) 75.0 81.7 ATONIK 365.3 1 / ha (5 95.0 98.3 oz / ac) Corn worm mortality and cotton (% dead at 7a TL 2 ° Treatment + 20 days) 72 hrs 96 hrs ATONIK 730.7 1 / h (10 88.3 93.3 oz / ac) ATONIK 1461.3 1 / ha (20 86.7 95.0 oz / ac) Control (cotton no- Bt) 0 3.3

[0042] Table 5 · shows the effect of treatments with ATONIK on the height and number of nodes in Bt cotton. Table 5 Treatment Height of Plant No. Node (era) (average) Control (Bt cotton) 63.5 16.7 ATONIK 365.3 1 / ha (5 61.7 15.8 oz / c) ATONIK 730.7 1 / h (10 62.0 16.0 oz / ac) ATONIK 1461.3 1 / ha 62.2 17.0 (20 oz / ac) Control (cotton 61.0 13.8 non-Bt) [0043] The results show that the treatment with the protein transport enhancer according to the invention, results in higher mortality of corn worm and cotton both under conditions of optimal growth (Table 2) as under tension conditions both for temperature and lack of water (Tables 3 and 4). Treatment with ATOMIK, however, does not result in increased vegetative growth (Table 5).

Claims (8)

1. CLAIMS: 1. A method for maintaining the effectiveness of pesticides of transgenic plants that includes a gene that expresses a pesticidally effective protein, the method is characterized in that it comprises: treating the transgenic plants with a protein transporting enhancer that stabilizes the transport of transgenic plants. pesticidally effective proteins inside the plant.
2. 2. Method according to claim 1, characterized in that the transgenic plant is cotton or corn.
3. Method according to claim 1, characterized in that the transgenic plant is cotton that has been modified to express protein B. thuringiensis.
4. 4. Method according to claim 1, characterized in that the protein transporting moiety comprises a mixture of phenolate and nitroguayacolate salts.
5. 5. Method according to claim 4, characterized in that the protein transport mej- erator is applied to the plants in a ratio in the range of 2.47-7306. S 1 / ha (1-100 oz / acre).
6. 6. Method according to claim 5, characterized in that the protein transporting enhancer is applied to the plants in a proportion within the range of 2.47-3653.3 1 / ha (1-50 oz / acre).
7. 7. Method according to claim 6, characterized in that the protein transporting enhancer is applied to the plants in a proportion within the range of 365.3-2192 1 / ha (5-30 oz / acre).
8. 8. Method according to claim 1, characterized in that the protein transporting agent comprises polyols that are obtained from reduction of aldo and keto groups in a carbohydrate.