METHOD FOR INCREASING THE RESISTANCE TO PHACOSPORACEA SOY RUST IN SOYBEAN PLANTS, METHOD FOR THE PRO

专利摘要:
METHOD FOR INCREASING THE RESISTANCE TO PHACOSPORACEA IN PLANTS AND / OR PLANT CELLS, RECOMBINANT VECTOR CONSTRUCT, METHOD FOR THE PRODUCTION OF A TRANSGENIC PLANT THAT HAS INCREASED RESISTANCE AGAINST PHACOSPORACEA AND THE USE OF THE VETERINARY CONSTRUCTOR. A method is provided to increase resistance against pathogenic fungi of the Phacosporaceae family in transgenic plants and / or plant cells. In these plants, the ethylene signaling pathway and / or activity of the ethylene signaling compounds is altered. This is achieved by priming the ethylene signaling pathway in these plants, as compared to wild type plants and / or wild type plant cells. Depending on the activation or inhibitory function of a specific signaling compound, overexpression or knockdown of the cognate gene may be used.
公开号:BR112013033494B1
申请号:R112013033494-0
申请日:2012-06-25
公开日:2021-04-27
发明作者:Holger Schultheiss
申请人:Basf Plant Science Company Gmbh；
IPC主号:

专利说明:

[001] The present invention relates to a method for increasing resistance against pathogenic fungi of the Phacosporaceae family in transgenic plants and / or plant cells. In these plants, the ethylene signaling pathway and / or activity of the ethylene signaling compounds is altered. This is achieved by priming the ethylene signaling pathway in these plants, as compared to wild type plants and / or wild type plant cells. Depending on the activation or inhibitory function of a specific signaling compound, overexpression or knockdown of the cognate gene may be used.
[002] In addition, the invention relates to transgenic plants and / or plant cells that have an increased resistance against pathogenic fungi of the Phacosporaceae family, for example, soybean rust and recombinant expression vectors that comprise a sequence that is identical or homologous to a sequence encoding a functional ethylene signaling compound or fragments thereof.
[003] The cultivation of agricultural crop plants mainly serves for the production of foodstuffs for humans and animals. Monocultures, in particular, which are the norm today, are highly susceptible to a spread of disease similar to the epidemic. The result is noticeably reduced productions. Until today, pathogenic organisms have been mainly controlled by the use of pesticides. Nowadays, the possibility of directly modifying the genetic disposition of a plant or pathogen is also open to man.
[004] Resistance generally means the ability of a plant to prevent, or at least reduce, infestation and colonization by a harmful pathogen. Different mechanisms can be discerned in the naturally occurring resistance, in which plants defend themselves from colonization by phytopathogenic organisms. These specific interactions between the pathogen and the host determine the course of infection (Schopfer and Brennicke (1999) Pflanzenphysiologie, Springer Verlag, Berlin-Heidelberg, Germany, Germany).
[005] With reference to the race to specific resistance, also called host resistance, a differentiation is made between compatible and incompatible interactions. In the compatible interaction, an interaction occurs between a virulent pathogen and a susceptible plant. The pathogen survives, and can accumulate reproductive structures, while the host most often dies. An incompatible interaction occurs, on the other hand, when the pathogen infects the plant but is inhibited in its growth before or after the weak development of symptoms. In the latter case, the plant is resistant to the respective pathogen (Schopfer and Brennicke, see above). However, this type of resistance is specific to a certain strain or pathogen.
[006] In both compatible and incompatible interactions, a host specific and defensive reaction to the pathogen occurs. In nature, however, this resistance is often overcome because of the rapid evolutionary development of new virulent breeds of pathogens (Neu et al. (2003) American Cytopathol. Society, MPMI 16 no 7: 626-633).
[007] Most pathogens are specific to plant species. This means that the pathogen can induce a disease in certain plant species, but not in other plant species (Heath (2002) Can. J. Plant Pathol. 24: 259-264). Resistance to a pathogen in certain plant species is called non-host resistance. Non-host resistance offers strong, broad and permanent protection from phytopathogens. The genes that provide non-host resistance provide the opportunity for strong, broad and permanent protection against certain diseases in non-host plants. In particular, this resistance works for different strains of the pathogen.
[008] Immediately after the recognition of a potential pathogen, the plant begins to produce defense reactions. Most of the time, the presence of the pathogen is perceived through the so-called PAMP receptors, a class of kinase-like transmembrane receptor that recognize conserved molecules associated with the pathogen (for example, flagellin or chitin). Downstream of PAMP receptors, phytohormones of salicylic acid (SA), jasmonate (JA) and ethylene (ET) play an important role in the regulation of different defense reactions. Depending on the reason for the different phytohormones, different defense reactions are produced by the host cell. Generally, SA-dependent defense is linked to resistance against biotrophic pathogens, while JA / ET-dependent defense reactions are active against necrotrophic pathogens (and insects). In most plant pathogens, ET interactions have been shown to act synergistically with JA and antagonistic to the biotrophic defense of SA. For example, the well-known protein PDF1.2, a JA marker, requires the activation of ET and JA to be overloaded during defense against necrotrophic pathogens. The crucial involvement of the JA / ET pathway in resistance to necrotrophic pathogens is corroborated by the fact that overexpression of ERF1, a central protein involved in ET signaling (see Figure 1) leads to improved resistance against necrotrophic fungi (Botrytis cinerea, Fusarium oxysporum and Plectosphaerella cucumerina (Berrocal-Lobo et al. 2002, Plant Journal 29: 23-32, Berrocal-Lobo and Molina 2004, MPMI 17: 763ff). ERF1 increases the susceptibility of Arabidopsis to the biotrophic pathogen Pseudomonas syringae (Berrocal-Lobo et al. 2002, Plant Journal 29: 23-32), providing the proposed model that JA / ET interacts negatively with the SA pathway to balance the nature of the defense reactions according to the attack pathogen, allowing the plant to adjust its defense response, so priming the ET signaling pathway is generally believed to lead to increased fungal resistance necrotrophic, but at the same time, to an increased susceptibility to biotropic pathogens.
[009] Fungi are distributed worldwide. Approximately 100,000 different species of fungi are known to date. Rusts are of great importance. They can have a complicated development cycle with up to five different spore stages (spermatium, aecidiospore, uredospore, teleutospore and basidiospore).
[010] During the infection of plants by pathogenic fungi, different phases are usually observed. The first phases of the interaction between phytopathogenic fungi and their potential host plants are decisive for the plant's colonization by the fungus. During the first stage of infection, the spores become attached to the plant's surface, germinate, and the fungus penetrates the plant. Fungi can penetrate the plant through existing ports, such as stomata, lenticels, hydatodes and wounds, or else they penetrate the plant's epidermis directly as a result of mechanical strength and with the help of enzymes that digest the cell wall. Specific infection structures are developed to penetrate the plant. The soybean rust, Phakopsora pachyrhizi, penetrates directly into the plant's epidermis. After crossing the cell of the epidermis, the fungus reaches the intercellular space of the mesophile, where the fungus starts dispersing through the leaves. To acquire nutrients, the fungus penetrates the mesophilic cells and develops haustories inside the mesophilic cell. During the penetration process, the plasma membrane of the penetrated mesophilic cell remains intact. Therefore, the soybean rust fungus establishes a biotopic interaction with the soybean.
[011] Soy rust has become increasingly important in recent times. The disease can be caused by biotropic rust Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a broad spectrum of host plants. P. pachyrhizi, also mentioned as an Asian fungus, is the most aggressive pathogen in soy (Glycine max), and is therefore, at least currently, of great importance for agriculture. P. pachyrhizi can be found close to all tropical and subtropical soy growing regions in the world. P. pachyrhizi is able to infect 31 species of 17 Legume families under natural conditions and is able to grow in more than 60 species under controlled conditions (Sinclair et al. (Eds.), Proceedings of the rust workshop (1995), National SoyaResearch Laboratory, Publication No 1 (1996); Rytter JL et al., Plant Dis. 87, 818 (1984)). P. meibomiae was discovered in the Caribbean Basin and Puerto Rico, and has not caused any substantial damage so far.
[012] P. pachyrhizi can currently be controlled in the field only by means of fungicides. Soy plants with resistance to the full spectrum of isolates are not available. When looking for resistant plants, four dominant Rpp1-4 genes were found, which mediate soybean resistance to P. pachyrhizi. Resistance was lost quickly, as P. pychyrhizi develops new virulent breeds.
[013] In recent years, fungal diseases, for example, soybean rust, have gained importance as a plague in agricultural production. There was, therefore, a requirement in the prior art to develop methods for controlling fungi and providing fungal resistant plants.
[014] Much research has been carried out in the field of dusty and feathery mold that infects the epidermis layer of plants. However, the problem of dealing with soybean rust that infects mesophilic remnants has not been solved.
[015] Surprisingly, we found that biotropic pathogenic fungi of the Phacosporaceae family, for example, soybean rust fungus, can be controlled by the use of ethylene-mediated defense, although the prior art teaches, that the priming of ethylene-mediated defense leads to increased susceptibility to biotropic fungi (Berrocal-Lobo et al. 2002, Plant Journal 29: 23-32). We perform the priming of the ET pathway both by overexpression of several proteins involved in ethylene signaling and by the infregulation of several proteins involved in the suppression of the ET signaling pathway. Generally, it is expected that priming the ET signaling pathway should lead to improved susceptibility against Asian Soybean Rust (ASR), since the ET signaling pathway interacts negatively with the biotropic defense associated with the SA pathway. On the other hand, improved resistance to ASR is expected by inhibiting the ET signaling pathway and, therefore, decongesting the SA pathway. Surprisingly, we found that the ET signaling pathway itself increases soybean rust resistance. The overexpression of several proteins involved in the ET signaling pathway (ERF1, ERF2, Pti4, Pti5) increases the resistance of soybeans against pathogenic fungi of the Phacosporaceae family, for example, soybean rust. The lack of regulation of antagonistic proteins in the ET signaling pathway such as CTR1 and EBF1 also increases the resistance of soybeans to pathogenic fungi of the Phacosporaceae family, for example, soybean rust. Vice versa, the overexpression of proteins of the ET signaling antagonist pathway, such as CTR1 and EBF1, increases the susceptibility of soybeans to pathogenic fungi of the Phacosporaceae family, for example, soybean rust. This clearly demonstrates the positive influence of ET-mediated defense pathways for soy resistance against pathogenic fungi of the Phacosporaceae family, for example, soybean rust.
[016] The object of the present invention is to provide a method of increasing resistance against pathogenic fungi of the family Phacosporaceae, preferably against pathogenic fungi of the genus Phacospora, more preferably against Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as rust of soy in transgenic plants and / or transgenic plant cells by using the ethylene signaling pathway, especially by priming the ethylene signaling pathway. This can be achieved by overexpression of one or more nucleic acids of the invention to perform the priming of the ethylene signaling pathway or infrarregulation of one or more nucleic acids of the invention that would also lead to increased resistance to pathogenic fungi of the Phacosporaceae family, for example, soybean rust.
[017] The nucleic acids of the invention for priming the ethylene signaling pathway and for achieving increased resistance to pathogenic fungi of the Phacosporaceae family, for example, soybean rust, are Pti4, Pti5, ERF1 and / or ERF2, for example , as defined by any one of SEQ ID NO: 1, 3, 5 or 7 or any homologue, derivative, ortholog or similar thereto. The priming of the ethylene signaling pathway can also be achieved by infrarregulation of repressors of any one of Pti4, Pti5, ERF1 and / or ERF2 such as microRNAs or ta-siRNAs that direct these genes.
[018] The nucleic acids of the invention to be unregulated to perform the priming of the ethylene signaling pathway and to achieve increased resistance to pathogenic fungi of the Phacosporaceae family, for example, soybean rust, are CTR1, EBF1 and / or EBF2, for example example, as defined by any of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21 or 23 or any fragment, homolog, derivative, ortholog or similar thereof. The priming of the ethylene signaling pathway can also be achieved by overexpression of repressors of any of CTR1, EBF1 and / or EBF2 as microRNAs or ta-siRNAs that direct these genes.
[019] An additional object is to provide transgenic plants resistant to pathogenic fungi of the family Phacosporaceae, preferably against pathogenic fungi of the genus Phacospora, with the utmost preference against Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as soybean rust, a method for producing these plants, as well as a useful vector construct for the above methods. This object is achieved by the subject matter of the main claims. Preferred embodiments of the invention are defined by the characteristics of the subclaims. DEFINITIONS
[020] The present invention can be more easily understood by reference to the following detailed description of the preferred embodiments of the invention and the Examples included in the present application. Unless otherwise noted, the terms used in the present application are to be understood in accordance with conventional usage by those skilled in the art in the relevant technique. In addition to the definitions of terms provided in the present application, definitions of common terms in molecular biology can also be found in Rieger et al., 1991 Glossary of genetics: classical and molecular, 5th Ed., Berlin: Springer-Verlag; and in Current Protocols in Molecular Biology, F.M.Ausubel et al., Eds., Current Protocols, the joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (Supplement 1998). It is understood that as used in the specification and in the claims, "one" or "one" can mean one or more, depending on the context in which it is used. So, for example, reference to "a cell" can mean that at least one cell can be used. It is understood that the terminology used in this application is intended to describe only specific accomplishments described, and is not intended to be limiting.
[021] Throughout this request, several publications are referred to. The divulations of all of these publications and those references cited within those publications in their entirety are incorporated as a reference in this application to more fully describe the state of the art to which this invention forms part. Standard techniques for cloning, DNA isolation, amplification and purification, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and generally employed by those skilled in the art. Several standard techniques are described in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y .; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y .; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979 Meth Enzymol. 68; Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101; Grossman and Moldave (Eds). 1980 Meth. Enzymol. 65; Miller (Ed.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y .; Old and Primrose, 1981 Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender 1979 Genetic Engineering: Principles and Methods, Vols. 1-4, Pleem a Press, New York. Abbreviations and nomenclature, where used, are considered standard in the field and are generally used in professional publications such as those cited in this application.
[022] The term “priming” should be understood as the sensitization of a plant or part of it to the future attack by pests or pathogens, in order to induce resistance against these pests or pathogens. The resistance induced by priming is not based on a direct activation of a defense mechanism, but on a sensitization of the plant or plant tissue that results in a faster and stronger expression of the defense mechanisms, compared to a non-primate plant. , since the plant is exposed to pathogen attack. “Priming” refers in this application to the sensitization of a plant or part of a plant, so that it is able to activate defense mechanisms faster and / or stronger when exposed to one or more biotic stresses, in comparison with a non-primate control plant or part of it that must have a direct defense response. Without being limited to the scope of the invention, priming is believed to result in an increased level of signaling factors such as transcription factor (TF) proteins or MAP kinases and the like in the plant or primate plant tissue, compared to plants or tissues non-primate vegetables. Upon subsequent exposure of the plant or plant tissue to stress such as plague or pathogen attack, these inactive TF proteins become active and regulate the gene expression of defense genes, so that a faster and / or stronger defense response is assembled by plants or primate tissues, compared to plants or non-primate tissues. Priming can, for the present application, additionally be understood as a constitutive activation of the respective defense mechanism.
[023] The term “ethylene signaling pathway priming” means that the priming effect is accomplished by sensitizing the ethylene signaling pathway, as shown in Figure 1, which leads to a faster and stronger defense response from defense mechanisms dependent on plant ethylene or plant tissue. Sensitization of the ethylene signaling pathway can be achieved by improving the expression of Pti4, Pti5, ERF1 and / or ERF2 protein and / or by suppressing the expression of CTR1, EBF1 and / or EBF2 protein.
[024] "Homologues" of a protein include peptides, oligopeptides, polypeptides, proteins and / or enzymes that have substitutions, deletions and / or insertions of amino acids in relation to the unmodified protein in question, and which have similar biological and functional activity to of the unmodified protein from which they are derived.
[025] Nucleic acid "homologues" encompass nucleotides and / or polynucleotides that have nucleic acid substitutions, deletions and / or insertions in relation to the unmodified nucleic acid in question, in which the protein encoded by that nucleic acid has functional activity similar to or greater than the unmodified protein encoded by the unmodified nucleic acid from which they are derived. In particular, homologues of a nucleic acid include substitutions in the degenerative amino acid code.
[026] A "deletion" refers to the removal of one or more amino acids from a protein or the removal of one or more nucleic acids from DNA, ssRNA and / or dsRNA.
[027] An "insert" refers to one or more amino acid residues or nucleic acid residues to be introduced at a predetermined site on a protein or nucleic acid.
[028] A "substitution" refers to the replacement of amino acids in the protein by other amino acids that have similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or beta-leaf structures).
[029] At the nucleic acid level, a substitution refers to a substitution of nucleic acid for other nucleic acids, in which the protein encoded by the modified nucleic acid has a similar function. In particular, nucleic acid homologues include substitutions in the degenerative amino acid code.
[030] Amino acid substitutions are typically from single residues, but can be grouped depending on the functional restrictions placed on the polypeptide and can vary from 1 to 10 amino acids; insertions or deletions will usually be in the order of about 1 to 10 amino acid residues. Amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see, for example, Creighton (1984) Proteins. WH Freeman and Company (Eds) and Table 1 below).

[031] Amino acid substitutions, deletions and / or insertions can easily be done using well-known peptide synthesis techniques, such as solid-phase peptide synthesis and the like, or by manipulating recombinant DNA.
[032] Methods for manipulating DNA sequences to produce substitution, insertion or deletion of variants of a protein are well known in the art. For example, techniques for producing substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gene in vitro mutagenesis (USB, Cleveland, OH), QuickChange site-directed mutagenesis (Stratagene, San Diego, CA), PCR mediated by site-directed mutagenesis or other site-directed mutagenesis protocols.
[033] Orthologists and analogues encompass evolutionary concepts used to describe the ancestral relationships of genes. Parallels are genes of the same species that were originated by duplicating an ancestral gene, orthologists are genes from different organisms that were originated through speciation, and are also derived from a common ancestor gene.
[034] The term "domain" refers to a set of amino acids conserved at specific positions along an alignment of evolutionarily related protein sequences. Although amino acids in other positions may vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely to be essential in the structure, stability or function of a protein.
[035] There are specialized databases for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pages 53- 61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32: D134- D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30 (1): 276280 (2002) ). A set of tools for analyzing protein sequences in silico is available on the ExPASy proteomic server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31: 3784 - 3788 (2003)) Domains or motifs can also be identified with the use of routine techniques, such as by sequence alignment.
[036] Methods for aligning sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) algorithm to find the global alignment (that is, covering the complete strings) of two strings, which maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates the percentage of sequence identity and performs a statistical analysis of the similarity between the two sequences. The computer program for performing the BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI). Homologues can be easily identified using, for example, the ClustalW multi-sequence alignment algorithm (version 1.83), with standard paired alignment parameters, and a percentage scoring method. The global percentages of similarity and identity can also be determined using one of the methods available in the MatGAT computer program package (Campanella et al., BMC 2003 Jul 10; 4: 29. MatGAT: an application that generates matrices of similarity / identity with the use of protein or DNA sequences). Secondary manual editing can be performed to optimize the alignment between preserved motifs, as would be apparent to a technician in the subject. In addition, instead of using the entire strings to identify homologues, specific domains can also be used. Sequence identity values can be determined across the complete nucleic acid, the amino acid sequence or across the selected domains or conserved motif (s), using the programs mentioned above using standard parameters . For local alignments, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147 (1); 195-7).
[037] As used in this application, the terms "soybean rust resistance", "soybean rust resistant", "soybean rust resistant", "rust resistant", "rust resistant", " fungus resistance ”,“ fungus resistant ”and / or“ fungus resistant ”means to reduce or prevent an infection by Phacosporacea, in particular Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur) also known as soybean rust or Asian Rust of Soy (ASR). The term "resistance" refers to the resistance of soybeans. Resistance does not imply that the plant necessarily has 100% resistance to infection. In preferred embodiments, resistance to soybean rust infection in a resistant plant is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in comparison to a wild type plant that is not resistant to soybean rust. Preferably the wild type plant is a plant of similar genotype, more preferably identical to the plant that has increased resistance to soybean rust, but does not comprise a recombinant nucleic acid of the invention, functional fragments thereof and / or a nucleic acid capable of hybridizing with a nucleic acid of the invention.
[038] The terms “soybean rust resistance”, “soybean rust resistant”, “soybean rust resistant”, “rust resistance”, “rust resistant”, “rust resistant”, “ fungal resistant ”,“ fungus resistance ”and / or“ fungal resistant ”, as used in the present application, refer to the ability of a plant, in comparison to a wild type plant, to prevent infection by pathogenic fungi of the family Phacosporaceae , for example, of the genus Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as soybean rust, to destroy, hinder, reduce, delay rust, stop the development, growth and / or multiplication of soybean rust. The level of fungal resistance of a plant can be determined in several ways, for example, by scoring / measuring the infected leaf area in relation to the total leaf area. Another possibility to determine the level of resistance is to count the number of soybean rust colonies on the plant or to measure the amount of spores produced by these colonies. Another way to resolve the degree of fungal infestation is to specifically measure the amount of rust DNA by quantitative PCR (q). Specific probes and primer sequences for most pathogenic fungi are available in the literature (Frederick RD, Snyder CL, Peterson GL, et al. 2002 Polymerase chain reaction assays for the detection and discrimination of the rust pathogens Phakopsora pachyrhizi and P. meibomiae PHYTOPATHOLOGY 92 (2) 217-227). Preferably, soybean rust resistance is non-host resistance. The resistance of non-hosts means that the plants are resistant to at least 80%, at least 90%, at least 95%, at least 98%, at least 99% and preferably 100% of the soybean rust pathogen strains, preferably the strains of Phakopsora pachyrhizi.
[039] The term "hybridization" as used in the present application includes "any process by which a strand of nucleic acid molecule joins a complementary strand through base pairing". (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the hybridization strength (that is, the strength of the association between the nucleic acid molecules) is affected by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the hybrid formed, and the G: C ratio within the nucleic acid molecules. As used in the present application, the term "Tm" is used in reference to "melting temperature". The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acid molecules is well known in the art. As indicated by standard references, a simple estimate of the Tm value can be calculated by the equation: Tm = 81.5 + 0.41 (% G + C), when a nucleic acid molecule is in an aqueous solution in 1 M NaCl [ see, for example, Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)]. Other references include more sophisticated calculations, which take into account structural as well as sequence characteristics for calculating Tm. Strict conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
[040] In particular, the term stringent conditions refers to conditions, in which 100 contiguous nucleotides or more, 150 contiguous nucleotides or more, 200 contiguous nucleotides or more or 250 contiguous nucleotides or more that are a fragment or identical to the molecule of complementary nucleic acid (DNA, RNA, ssDNA or ssRNA) hybridizes under conditions equivalent to hybridization in sodium dodecyl sulfate (SDS) 7%, 0.5 M NaPO4, 1 mM EDTA at 50 ° C with washing in 2X SSC, SDS 0.1% at 50 ° C or 65 ° C, preferably at 65 ° C, with a specific nucleic acid molecule (DNA; RNA, ssDNA or ssRNA). Preferably, the hybridization conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 ° C with washing in 1X SSC, 0.1% SDS at 50 ° C or 65 ° C, preferably 65 ° C, more preferably the hybridization conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 ° C with washing in 0.1X SSC , 0.1% SDS at 50 ° C or 65 ° C, preferably 65 ° C. Preferably, the complementary nucleotides hybridize to a fragment or to the entire nucleic acids of the invention. Preferably, the complementary polynucleotide hybridizes with parts of the nucleic acids of the invention capable of providing resistance to soybean rust by overexpression or infraregulation, respectively.
[041] As used in the present application, the term "nucleic acid of the invention" or "amino acid of the invention" refers to a gene that has at least 60% identity with any of SEQ-ID-No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 or with a sequence encoding for a protein that has at least 60% identity with SEQ-ID-No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 and / or functional fragments thereof. In one embodiment, nucleic acid homologues of the invention have at the level of DNA and / or protein level, at least 70%, preferably at least 80%, especially preferably at least 90%, quite especially preferably at least 95 %, most especially preferably at least 98%, 99% or 100% identity over the entire DNA region or protein region provided in a sequence specifically disclosed in the present application and / or a functional fragment thereof.
[042] As used in the present application, the term "amino acid of the invention" refers to a protein that has at least 60% identity to a sequence encoding for a protein that has SEQ-ID-No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 and / or a fragment thereof. In one embodiment, homologues of the amino acids of the invention are at least 70%, preferably at least 80%, especially preferably at least 90%, very especially preferably at least 95%, very especially preferably at least 98%, 99% or 100% identity over the entire protein region provided in a sequence specifically disclosed in the present application and / or a functional fragment thereof.
[043] The "identity" or "homology" between two nucleic acids and / or in each case refers to the entire length of the nucleic acid of the invention.
[044] For example, identity can be calculated using the Vector NTI Suite 7.1 program from Informax (USA), which employs the Clustal Method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. April 1989; 5 (2): 151-1) with the following configurations:


[045] Alternatively, identity can be determined according to Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment with the Clustal series of programs. (2003) Nucleic Acids

[046] All nucleic acid sequences mentioned in the present application (single-stranded and double-stranded DNA and RNA sequences, for example, cDNA and mRNA) can be produced in a manner known by chemical synthesis from the building blocks of nucleotides, for example, by condensation of an individual overlapping fragment, complementary nucleic acid building blocks of the double helix. The chemical synthesis of oligonucleotides can, for example, be performed in a known way, by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and gap filling through the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989), see below.
[047] The sequence identity between the useful nucleic acid according to the present invention and the nucleic acids of the invention can be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press , 1991, and references cited in that regard) and calculating the percentage difference between the nucleotide sequences, for example, the Smith-Waterman algorithm as implemented in the BESTFIT computer program using standard parameters (for example, University of Wisconsin Genetic Computing Group). At least 60% sequence identity, preferably at least 70% sequence identity, 80% 90%, 95%, 98%, 99% sequence identity, or up to 100% sequence identity, with the nucleic acids that have any SEQ-ID-No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 are preferred.
[048] The term "plant" is intended to include plants at any stage of maturity or development, as well as any tissues or organs (plant parts) taken or derived from any of these plants unless clearly indicated otherwise by context. Plant parts include, but are not limited to, plant cells, stems, roots, flowers, eggs, stamens, seeds, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, hairy root cultures and / or the like. The present invention also includes seeds produced by the plants of the present invention. Preferably, the seeds comprise the recombinant nucleic acids of the invention. In one embodiment, the seeds are of true reproduction for increased resistance to fungal infection compared to a wild type variety of the plant's seed. As used in the present application, a "plant cell" includes, but is not limited to, a protoplast, gamete producing cell and a cell that regenerates in an entire plant. The tissue culture of various plant tissues and plant regeneration from this point on is well known in the art and is widely published.
[049] In one embodiment of the present invention the plant is selected from the group consisting of beans, soy, peas, clover, kudzu, lucerne, lentils, lupins, vetch and / or peanuts. Preferably, the plant is a vegetable, which comprises plants of the genus Phaseolus (which comprises French beans, dwarf beans, string beans (Phaseolus vulgaris), Lima beans (Phaseolus lunatus L.), Tepari beans (Phaseolus acutifolius A. Gray) , runner beans (Phaseolus coccineus)); the Glycine genus (which comprises Glycine soybeans, soybeans (Glycine max (L.) Merill)); peas (Pisum) (comprising shelling peas (Pisum sativum L. convar. sativum), also called smooth peas or round seeds; marrowfat peas (Pisum sativum L. convar. medullare Alef. emend. CO Lehm), sugar peas (Pisum sativum L. convar. axiphium Alef emend. CO Lehm), also called fresh pea, edible pea or mangetout, (Pisum granda sneida L. convar. sneidulo p. shneiderium)); peanut (Arachis hypogaea), clover (Trifolium spec.), medick (Medicago), kudzu vine (Pueraria lobata), common lucerne, alfalfa (M. sativa L.), chickpeas (Cicer), lentils (Lens) (Lens) culinaris Medik.), lupine (Lupinus); vetch (Vicia), field beans, broad bean (Vicia faba), vetchling (Lathyrus) (comprising chickling pea (Lathyrus sativus), bush pea (Lathyrus tuberosus)); genus Vigna (comprising moth beans (Vigna aconitifolia (Jacq.) Maréchal), adzuki beans (Vigna angularis (Willd.) Ohwi & H. Ohashi), urd beans (Vigna mungo (L.) Hepper), mung beans (Vigna radiata (L.) R. Wilczek), bambara peanuts (Vigna subterrane (L.) Verdc.), Rice bean (Vigna umbellata (Thunb.) Ohwi & H. Ohashi), Vigna vexillata (L.) A. Rich., Vigna unguiculata (L.) Walp., in the three subspecies beans asparagus, black-eyed beans, cow beans)); pigeon pea (Cajanus cajan (L.) Millsp.), the genus Macrotyloma (comprising peanut geocarpa (Macrotyloma geocarpum (Harms) Maréchal & Baudet), horse bean (Macrotyloma uniflorum (Lam.) Verdc.)); goa beans (Psophocarpus tetragonolobus (L.) DC.), African yam beans (Sphenostylis stenocarpa (Hochst. ex A. Rich.) Harms), Egyptian black beans, dolichos beans, lablab beans (Lablab purpureus (L.) Sweet), yam beans (Pachyrhizus), guar beans (Cyamopsis tetragonolobus (L.) Taub.); and / or the genus Canavalia (comprising pork beans (Canavalia ensiformis (L.) DC.), sword beans (Canavalia gladiata (Jacq.) DC.)).
[050] The reference in the present application to an "endogenous" nucleic acid of the invention refers to the gene in question as found in a plant in its natural form (that is, without any human intervention). The recombinant nucleic acid of the invention refers to the same gene (or a substantially homologous nucleic acid / gene) in an isolated form subsequently (re) introduced into a plant (a transgene). For example, a transgenic plant that contains this transgene may, when compared to the expression of the endogenous gene, find a substantial increase in the expression of the transgene or infrarregulation of the corresponding endogen, respectively. The isolated gene can be isolated from an organism, or it can be manufactured artificially, for example, by chemical synthesis. A transgenic plant according to the present invention includes a recombinant nucleic acid of the invention integrated into any genetic loci and optionally the plant can also include the endogenous gene within the natural genetic background.
[051] For the purposes of the invention, "recombinant" means with respect to, for example, a nucleic acid sequence, a nucleic acid molecule, an expression cassette or a vector construct comprising any one or more of the nucleic acids of the invention, all of these man-made constructs by genetic technology methods in which either (a) the nucleic acid sequences of the invention or a part thereof, or (b) genetic control sequence (s) that are functionally linked to the nucleic acid sequence of the invention according to the invention, for example, a promoter, or (c) a) and b) are not located in their natural genetic environment nor have they been modified by man by methods of genetic technology. The modification can take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood to mean the natural genomic or chromosomal locus in the original plant, the presence in a genomic library or the combination with the natural promoter.
[052] In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably maintained, at least in part. The environment flanks the nucleic acid sequence on at least one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, with the most preference at least 5000 bp.
[053] A naturally occurring expression cassette, for example, the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a protein useful in the methods of the present invention, as defined above, makes a recombinant expression cassette is used when that expression cassette is modified by humans by unnatural, synthetic (“artificial”) methods, such as, for example, mutagenic treatment. Suitable methods are described, for example, in US patent 5,565,350, WO 00/15815 in US patent 200405323. In addition, a naturally occurring expression cassette, for example, the naturally occurring combination of the naturally occurring promoter of nucleic acid with the corresponding nucleic acid sequence encoding a protein useful in the methods of the present invention, as defined above, becomes a recombinant expression cassette when that expression cassette is not integrated in the natural genetic medium, but in a genetic medium different. It should also be noted that, in the context of the present invention, the term "isolated nucleic acid" or "isolated protein" can, in some cases, be considered as a synonym for a "recombinant nucleic acid" or a "recombinant protein", respectively and refers to a nucleic acid or protein that is not located in its natural genetic environment and / or that has been modified by methods of genetic technology.
[054] As used in the present application, the term "transgenic" preferably refers to any plant, plant cell, callus, plant tissue or part of the plant that contains the recombinant construct, vector or expression cassette of the invention or a part of same that is preferably introduced by non-essentially biological processes, preferably by transformation of Agrobacteria. The recombinant construct or a part of it is stably integrated into a chromosome, so that it is passed on to successive generations by clonal propagation, vegetative propagation or sexual propagation. These successive generations are also transgenic. Essentially biological processes can be plant crossing and / or natural recombination.
[055] A transgenic plant, cell or plant tissue for the purposes of the invention is thus understood to mean that the recombinant construct, vector or expression cassette of the invention is integrated into the genome.
[056] Preferably, constructs, vectors or expression cassettes of the invention are not present in the genome of the original plant or are present in the genome of the transgenic plant without its natural locus from the genome of the original plant.
[057] Natural locus means the location on a specific chromosome, preferably the location between certain genes, more preferably the same antecedent sequence as in the original plant that is transformed.
[058] Preferably, the transgenic plant, cell or plant tissue of the same expresses the constructs or expression cassettes of the invention.
[059] The term "expression" or "gene expression" means the transcription of a specific gene, specific genes or specific genetic vector construct. The term "expression" or "gene expression", in particular, means the transcription of a gene, genes or genetic vector construct into the structural RNA (rRNA, tRNA), a regulatory RNA (for example, microRNA, siRNA, ta- siRNA) or mRNA, with or without subsequent translation of the latter into a protein. The process includes transcribing DNA and processing the resulting RNA product.
[060] The term "augmented expression", "enhanced expression", "overexpression" or "content increase" as used in this application, means any form of expression that is additional to the original wild type expression level. For the purposes of this invention, the original wild-type expression level can also be zero (no expression).
[061] Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression directed by the appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids that serve as promoters or enhancer elements can be introduced into the appropriate position (typically upstream) in a nonheterologous form of a polynucleotide in order to suppress the expression of a nucleic acid encoding the protein of interest. For example, endogenous promoters can be altered in vivo by mutation, deletion and / or substitution (see, Kmiec, US patent 5,565,350, Zarling et al, WO 9322443), or isolated promoters can be introduced into a plant cell in the appropriate orientation and distance from a gene of the present invention, in order to control the expression of the gene.
[062] If protein expression is desired, it is generally desirable to include a polyadenylation region at the 3 'end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of genes from other plants, or from T-DNA. The 3 'end sequence to be added can be derived, for example, from the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryote gene.
[063] An intron sequence can be added to the 5 'untranslated region (RTU) and / or to the coding sequence of the partial coding sequence to increase the amount of mature message that accumulates in the cytosol. The inclusion of a junction intron in the transcription unit in both constructs for expression in plants and animals has been shown to increase gene expression in both mRNA and proteins, up to 1000-fold levels (Buchman and Berg (1988) Mol. Cell Biol 8: 4395-4405; Callis et al. (1987) Genes Dev 1: 1183-1200). This intron increase in gene expression is typically greater when placed near the 5 'end of the transcription unit. The use of Adh1-S corn introns 1, 2 and 6, and Bronze-1 introns are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
[064] The term "functional fragment" refers to any nucleic acid and / or protein that merely comprises part of the entire nucleic acid and / or whole protein, but still provides the same function, that is, resistance to soybean rust. , when expressed or repressed in a plant, respectively. Preferably, the fragment comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%, at least 98%, at least 99% of the original sequence. Preferably, the functional fragment comprises contiguous nucleic acids or amino acids as in the original nucleic acid and / or original protein.
[065] In one embodiment, the fragment of any of the nucleic acids of the invention has an identity as defined above over a length of at least 20%, at least 30%, at least 50%, at least 75%, at least minus 90% of the nucleotides of the respective nucleic acid of the invention to the respective nucleic acid of the invention.
[066] In cases where nucleic acid overexpression of the invention is desired, the term "similar functional activity" or "similar function" means that any homolog and / or fragment provides resistance to soybean rust when expressed in a plant. Preferably similar functional activity means at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% or more resistance to soybean rust compared to functional activity provided by recombinant expression of any of the nucleotide sequences of the invention, as defined by SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 and / or recombinant protein of the invention, as defined by SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24.
[067] The term "increased activity", as used in the present application, means any protein that has increased activity and provides increased resistance to soybean rust compared to the wild type plant, merely expressing the respective endogenous nucleic acid of the invention. Insofar as overexpression is related, for the purposes of this invention, the original wild type expression may also be zero (no expression).
[068] "Suppressing", "infrared" or "suppressing" the expression of a nucleic acid molecule in a plant cell are used equivalently in this application and mean that the level of expression of the nucleic acid molecule or the level of activity of protein of the protein encoded by the nucleic acid molecule in a plant, part of a plant or vegatal cell, after application of a method of the present invention, is lower than its expression in the plant, part of the plant or plant cell before application of the method, or in comparison with a reference plant lacking a recombinant nucleic acid molecule of the invention. The term "suppressed", "unregulated" or "suppressed", as used in this application, are synonyms and mean in this application, lower, preferably significantly lower expression of the nucleic acid molecule to be expressed or activity of the protein to be expressed expressed. As used in the present application, a "repression", "downregulation" or "suppression" of the level of an agent such as a protein, mRNA or RNA means that the level is reduced in relation to a substantially identical plant, part of a plant or cell vegetable grown under substantially identical conditions, being devoid of a recombinant nucleic acid molecule of the invention, for example, devoid of a complementary region to at least a part of the precursor molecule of the srRNA, recombinant construct or recombinant vector of the invention. As used in the present application, "repression", "downregulation" or "suppression" of the level of an agent such as a pre-RNA, mRNA, rRNA, tRNA, snoRNA, snRNA expressed by the target gene and / or the product protein encoded by this, means that the amount is reduced by 10% or more, for example, 20% or more, preferably 30% or more, more preferably 50% or more, even more preferably 70% or more, with the maximum preferably 80% or more, for example, 90% with respect to a cell or organism lacking a recombinant nucleic acid molecule of the invention. The crackdown or lack of regulation can be determined by methods with which the person skilled in the art is familiar. Thus, the lack of regulation, repression or suppression of the nucleic acid or protein or the amount of protein activity can be determined, for example, by an immunological detection of the protein. In addition, techniques such as protein assay, fluorescence, Northern hybridization, nuclease protection assay, reverse transcription (quantitative RT-PCR) ELISA (enzyme linked immunosorbent assay), Western blotting, radioimmunoassay (RIA) or other immunoassay and cell analysis fluorescence activated (FACS) can be used to measure a specific protein or RNA in a plant or plant cell. Depending on the type of the target protein product, its activity or effect on the organism's phenotype or cell can also be determined. Methods for determining the amount of protein are known to those skilled in the art. Examples that can be mentioned are: the micro-Biuret method (Goa J (1953) Scand J Clin Lab Invest 5: 218-222), the Folin-Ciocalteau method (Lowry OH et al. (1951) J Biol Chem 193 : 265-275) or measurement of CBB G-250 absorption (Bradford MM (1976) Analyt Biochem 72: 248-254).
[069] A method to increase resistance to Phacosporacea, for example, soybean rust in which the ethylene signaling pathway is paramount, compared to wild-type plants or wild-type plant cells, by increasing the expression of a Pti4 protein, Pti5, ERF2 and / or ERF1 or a functional, orthologous, analogous or homologous fragment thereof is an embodiment of the invention.
[070] A method to increase resistance to Phacosporacea, for example, soybean rust in which the priming of the ethylene signaling pathway can be achieved by increasing the expression of a Pti4, Pti5, ERF1 and / or ERF2 protein or a functional fragment , orthologist, paragon or homologue thereof in which the Pti4, Pti5, ERF1 and / or ERF2 protein is encoded by: (i) a recombinant nucleic acid that has at least 60% identity, 70% sequence identity, 80% 90%, 95%, 98%, 99% sequence identity, or even 100% sequence identity with SEQ ID No. 1, 3, 5 or 7, a functional fragment thereof and / or a recombinant nucleic acid able to hybridize under stringent conditions with those nucleic acids of the same and / or by (ii) a recombinant nucleic acid encoding a protein that has at least 60%, preferably at least 70% sequence identity, 80% 90%, 95 %, 98%, 99% sequence identity, or even 100% sequence identity with the SEQ ID No. 2, 4, 6 or 8, a functional fragment of the same, an orthologist and / or a catalog thereof is a further embodiment of the invention.
[071] In a further method of the invention, the priming of the ethylene signaling pathway is achieved by a method comprising the steps of: (a) stably transforming a plant cell with an expression cassette, comprising: (i) a recombinant nucleic acid that has at least 60% identity, preferably at least 70% sequence identity, 80% 90%, 95%, 98%, 99% sequence identity, or even 100% sequence identity with SEQ ID No. 1, 3, 5 or 7, and / or a functional fragment thereof and / or a recombinant nucleic acid capable of hybridizing under stringent conditions to those nucleic acids therein and / or (ii) a recombinant nucleic acid which encodes a protein that has at least 60% identity, preferably at least 70% sequence identity, 80% 90%, 95%, 98%, 99% sequence identity, or even 100% sequence identity with SEQ ID No. 2, 4, 6 or 8, a functional fragment thereof, an orthologist and / or a p atogo of the same in functional connection with a promoter; (b) regenerating the plant from the plant cell; and (c) expressing said recombinant nucleic acid encoding a Pti4, Pti5, ERF1 and / or ERF2 protein in an amount and for a period sufficient to generate or to increase resistance to soybean rust in said plant.
[072] CONSTRUCT, with recombinant vector, characterized by the fact that it comprises: (a) (i) a recombinant nucleic acid that has at least 60% identity, 70% sequence identity, 80% 90%, 95%, 98 %, 99% sequence identity, or even 100% sequence identity with SEQ ID No. 1, 3, 5 or 7, a functional fragment thereof and / or a nucleic acid capable of hybridizing under stringent conditions with these nucleic acids and / or (ii) a recombinant nucleic acid encoding a protein that has at least 60% identity, preferably at least 70% sequence identity, 80% 90%, 95%, 98%, 99% identity of sequence, or even 100% sequence identity with SEQ ID No. 2, 4, 6 or 8, a functional fragment thereof, an orthologist and / or a catalog thereof functionally linked with (b) a promoter and (c) a transcription termination sequence is a further embodiment of the invention.
[073] As used in the present application, the term "target nucleic acid" preferably refers to a DNA molecule capable of preventing expression, reducing the amount and / or function of the plant's CTR1, EBF1 and / or EBF2 gene as defined, for example, by SEQ ID NO: 9, 11, 13, 15, 17, 19, 21 or 23 on the plant or part of the plant.
[074] The term "target gene", as used in the present application, refers to a gene the expression of which is unregulated or suppressed. In the structure of this application, target genes are preferably plant CTR1, EBF1 and / or EBF2 genes as, for example, defined by SEQ ID NO: 9, 11, 13, 15, 17, 19, 21 or 23 or homologues, parallels or functional equivalents of the same.
[075] The present invention provides a method to increase resistance to pathogenic fungi of the family Phacosporaceae, preferably against pathogenic fungi of the genus Phacospora, more preferably against Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as soybean rust in plants and / or plant cells, in which the ethylene signaling pathway is preferred over wild-type plants and / or wild-type plant cells by misregulation or suppression of the expression of a CTR1, EBF1 and / or EBF2 protein.
[076] In one embodiment of the invention, the CTR1, EBF1 and / or EBF2 protein is encoded by: (i) a recombinant nucleic acid that is at least 60%, preferably at least 70%, for example at least 75%, more preferably at least 80%, for example at least 85%, even more preferably at least 90%, for example at least 95%, or at least 96%, or at least 97%, or at least 98%, with the maximum preferably 99% of with SEQ ID No. 9, 11, 13, 15, 17, 19, 21 or 23, a functional fragment of the same and / or a recombinant nucleic acid capable of hybridizing under stringent conditions with those nucleic acids therein and / or by (ii) a recombinant nucleic acid encoding a protein that has at least 60% identity, preferably at least 70%, for example, at least 75%, more preferably at least 80%, for example at least 85% , even more preferably at least 90%, for example at least 95%, or at least 96%, or at least 97%, u at least 98%, with maximum preference at least 99% homology with SEQ ID No. 10, 12, 14, 16, 18, 20, 22 or 24, a functional fragment thereof, an orthologist and / or a similar the same.
[077] A method to increase resistance to pathogenic fungi of the family Phacosporaceae, preferably against pathogenic fungi of the genus Phacospora, more preferably against Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as soybean rust on plants and / or plant cells, in which the ethylene signaling pathway is given priority over wild-type plants and / or wild-type plant cells by misregulation or suppression of the expression of a CTR1, EBF1 and / or EBF2 protein comprises the steps of: a) providing a recombinant nucleic acid that comprises a target nucleic acid that is substantially identical and / or substantially complementary to at least 19 contiguous nucleotides from the target gene of CTR1, EBF1 and / or EBF2 or a homolog, parallel or ortholog thereof, as defined above. b) introducing said recombinant nucleic acid into the plant and / or part of it is a further claim of the invention.
[078] It is a further embodiment of the invention, that in the method as defined above, the recombinant nucleic acid is able to supply dsRNA, si-RNA and / or miRNA in the plant, a part of it, since the recombinant nucleic acid is expressed, wherein at least 19, preferably at least 20, more preferably at least 21, for example 22 or 23 contiguous nucleotides of the dsRNA and / or siRNA and / or dsRNA are substantially complementary to the target gene of CTR1, EBF1 and / or EBF2 .
[079] In a specific embodiment of the method of the invention as defined above, said recombinant nucleic acid comprises a promoter that is functional in the plant cell, functionally linked to a target nucleic acid that is substantially identical and / or substantially complementary to hair. at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides from the target gene of CTR1, EBF1 and / or EBF2 and which, when transcribed, generates RNA comprising a first strand that has a sequence substantially complementary to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides from the target gene of CTR1, EBF1 and / or EBF2 and a second strand having a sequence substantially complementary to the first tape or parts thereof, and a terminating regulatory sequence.
[080] In another specific embodiment of the method of the invention, as defined above, said recombinant nucleic acid comprises a promoter that is functional in the plant cell, functionally linked to a target nucleic acid that, when it is transcribed, generates RNA that comprises a first strand having a sequence substantially identical or substantially complementary to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides from the target gene of CTR1, EBF1 and / or EBF2, and a terminating regulatory sequence.
[081] Additional embodiments of the invention are recombinant vector constructs that comprise a recombinant nucleic acid that comprises a promoter that is functional in the plant cell, functionally linked to a target nucleic acid that is substantially identical and / or substantially complementary to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides from the target gene of CTR1, EBF1 and / or EBF2, and a regulatory terminator sequence.
[082] The recombinant vector constructs of the invention, as defined above, can further comprise a promoter that is functional in the plant cell, functionally linked to a target nucleic acid that is substantially identical and / or substantially complementary to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides from the target gene of CTR1, EBF1 and / or EBF2 and which, when transcribed, generates RNA comprising a first strand that has a substantially complementary sequence to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides from the target gene of CTR1, EBF1 and / or EBF2 and optionally a second strand that has a sequence substantially complementary to the first strand or parts of it, and a regulatory terminator sequence.
[083] The present invention provides a method for producing a straw and / or a part of it resistant to pathogenic fungi of the Phacosporaceae family, for example, soybean rust, which comprises: a) providing a recombinant nucleic acid comprising a nucleic acid target that is substantially identical and / or substantially complementary to at least 19, preferably at least 20, more preferably at least 21, for example 22 or 23 contiguous nucleotides of the target sequence of the invention, b) introducing said recombinant nucleic acid into the plant and / or parts thereof, in which the introduction of said recombinant nucleic acid results in the downregulation or repression of expression of the respective target gene. These target genes are preferably CTR1, EBF1 and EBF2 and homologues, analogues or functional equivalents thereof as defined, for example, by SEQ ID NO: 9, 11, 13, 15, 17, 19, 21 or 23.
[084] The present invention further provides a vector construct that comprises a recombinant nucleic acid that comprises a promoter that is functional in the plant cell, functionally linked to a target nucleic acid that is substantially identical and / or substantially complementary, preferably identical or complement at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides of the target gene of the invention and a regulatory terminator sequence, as well as the use of the vector construct, for the transformation of plants or parts thereof to provide plants resistant to pathogenic fungi of the Phacosporaceae family, for example, soybean rust.
[085] The present invention also provides a transgenic plant cell, plants or parts thereof, which comprises a recombinant nucleic acid which comprises a target nucleic acid which is substantially identical and / or substantially complementary, preferably identical or complementary to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides of the target gene of the invention. Plant parts can be plant cells, roots, stems, leaves, flowers and / or seeds.
[086] There is a general agreement that in many organisms, including fungi and plants, large pieces of dsRNA complementary to a specific gene are cleaved into 19 to 24 nucleotide fragments (siRNA) within cells, and that these siRNAs are the real mediators to silence the specific target gene. As used in the present application, siRNA refers to 19 to 24 nucleotide fragments complementary to the respective target gene.
[087] There are several possibilities for providing siRNA: interference RNA (RNAi), micro-RNAi (miRNA), sense RNA and / or antisense RNA for infrared regulation or suppression of the expression of the target gene of the invention.
[088] As used in the present application, "RNAi" or "interference RNA" refers to the process of silencing the sequence-specific post-transcription gene, mediated by double-stranded RNA (dsRNA). In the RNAi process, dsRNA comprising a first strand that is substantially complementary to at least 19 contiguous nucleotides of the target gene of the invention must be provided and a second strand that is at least partially complementary to the first strand. For this purpose, a recombinant nucleic acid is introduced into the plant, which is capable of producing this dsRNA. The target gene's specific dsRNA is produced and processed into relatively small fragments (siRNAs). miRNA refers to a similar process, except that the dsRNA produced only partially comprises regions substantially identical to the target gene (at least 19 contiguous nucleotides).
[089] As used in the present application, "antisense interference" refers to the process of silencing the sequence-specific post-transcription gene, probably also mediated by double-stranded RNA (dsRNA). In the antisense RNA process, ssRNA comprising a first strand that is substantially complementary to at least 19 contiguous nucleotides of the target gene must be provided. For this purpose, recombinant nucleic acid is introduced into the plant, which is capable of producing that ssRNA. Without being limited to theory, these RNA pairs with complementary ssRNA are supposed to be transcribed from the original target gene.
[090] As disclosed in the present application, 100% of the sequence identity between the target nucleic acid and the target gene is not required to practice the present invention. Preferably, the nucleic acid comprises a portion of 19 nucleotides that is substantially identical and / or substantially complementary to at least 19 contiguous nucleotides of the target gene. Although a target nucleic acid comprising an identical nucleotide sequence and / or identical to a portion of the target gene and / or complementary to the entire sequence and / or a portion of the target gene is preferred for inhibition, the invention can tolerate sequence variations that they may be expected due to gene manipulation or synthesis, genetic mutation, lineage polymorphism or evolutionary divergence. Thus, the target nucleic acid may also comprise an incompatibility with the target gene of at least 1, 2 or more nucleotides. For example, it is contemplated in the present invention that within 21 contiguous nucleotides the target nucleic acid may contain an addition, deletion or substitution of 1, 2 or more nucleotides, as long as the resulting RNA sequence still interferes with the respective function of the target gene.
[091] The sequence identity between the recombinant nucleic acid useful according to the present invention and the target gene can be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press , 1991, and references cited therein) and calculating the percentage difference between the nucleotide sequences, for example, by the Smith-Waterman algorithm, as implemented in the software program, using standard parameters (for example, University of Wisconsin Genetic Computing Group). More than 80% sequence identity, 90% sequence identity, or even 100% sequence identity, is preferred between the target nucleic acid and at least 19 contiguous nucleotides of the target gene. The same preferably applies for sequence complementarity.
[092] When the target nucleic acid of the invention has a length greater than approximately 19 nucleotides, for example, from about 50 nucleotides to about 500 nucleotides, the corresponding dsRNA supplied from it will be randomly cleaved to the dsRNAs of about 21 nucleotides inside the plant cell: siRNAs. The specialized multi-plant Dicers can generate siRNAs typically ranging in size from 19nt to 24nt (See Henderson et al., 2006. Nature Genetics 38: 721-725.). Cleavage of a longer dsRNA of the invention can produce a 21mer cluster of dsRNAs, derived from the longer dsRNA. SiRNAs can have sequences corresponding to fragments of 19 to 24 contiguous nucleotides along the entire sequence of the target gene. One skilled in the art would recognize that siRNA may have an incompatibility with the target gene of at least 1, 2 or more nucleotides. In addition, these incompatibilities are intended to be included in the present invention.
[093] In one embodiment, the nucleic acid is substantially identical and / or substantially complementary, preferably identical or complementary over a length of at least 19, at least 50, at least 100, at least 200, at least 300, at least at least 400 or at least 500 nucleotides to the respective target gene. In particular, the nucleic acid may comprise 19 to 500, preferably 50 to 500, more preferably 250 to 350 nucleotides, wherein preferably at least about 19, 20, 21, 22, 23, 24, 25, 50, 100, 200 , 300, 400 consecutive bases or even the entire length of the target nucleic acid are identical and / or complementary and / or identical to the target gene.
[094] Preferably, the recombinant nucleic acid is capable of delivering dsRNA and / or siRNA and / or miRNA to the plant, a part of which since the recombinant nucleic acid is expressed in the plant, where preferably at least 19 contiguous nucleotides from the plant. dsRNA and / or siRNA and / or miRNA are substantially complementary to the respective target gene.
[095] Generally, the term "substantially identical" or "substantially complementary" preferably refers to DNA and / or RNA that is at least 80% identical or complementary to 19 or more contiguous nucleotides from a specific DNA or RNA sequence in the respective target gene, more preferably, at least 90% identical to 19 or more contiguous nucleotides, and more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% Identical or complementary or absolutely identical or absolutely complementary to 19 or more contiguous nucleotides of a specific DNA or RNA sequence of the respective target gene. In particular, the identical RNA corresponds to the DNA encoding strand of the respective target gene.
[096] As used in the present application, the term "substantially identical" or "substantially complementary", as applied to the recombinant nucleic acid DNA, the target nucleic acid and / or the target gene means that the nucleotide sequence is at least 80 % identical or complementary to 19 or more contiguous nucleotides of the target gene, more preferably, at least 90% identical or complementary to 19 or more contiguous nucleotides of the target gene, and most preferably at least 95%, at least 96%, by at least 97%, at least 98% or at least 99% identical or complementary or absolutely identical or absolutely complementary to 19 or more contiguous nucleotides of the target gene. The term “19 or more contiguous nucleotides of the target gene” corresponds to the target gene, having at least about 19, 20, 21, 22, 23, 24, 25, 50, 100, 200, 300, 400, 500, 1000, 1500 consecutive bases or up to the entire length of the target gene.
[097] An embodiment according to the present invention provides a method for the production of a plant and / or part of it resistant to a pathogenic fungus of the family Phacosporaceae, for example, soybean rust, in which the recombinant nucleic acid comprises a promoter that is functional in the plant cell, functionally linked to a target nucleic acid, which is substantially identical and / or substantially complementary, or preferably identical or complementary to at least 19, preferably at least 20, more preferably at least 21, by for example, 22 or 23 contiguous nucleotides of the respective target gene and which, when transcribed, generates RNA comprising a first strand that has a sequence substantially complementary to at least 19, preferably at least 20, more preferably at least 21, for example , 22 or 23 contiguous nucleotides of the target gene and a second strand that has a sequence substantially complementary to the first strand and / or parts of the same, and a terminating regulatory sequence.
[098] The first strand and the second strand can at least partially form the dsRNA. This technique is also referred to as RNAi.
[099] In another embodiment, the target nucleic acid comprises 19 to 24 contiguous nucleotides of the target sequence that are substantially identical and / or substantially complementary to the target gene, and the remaining nucleotides of the target nucleic acid are not identical and / or complementary to the gene target. Non-identical means identity that is less than 95%, less than 90%, less than 80%, less than 70%, less than 60% over the entire target nucleic acid sequence. Non-complementary means a complementarity that is less than 95%, less than 90%, less than 80%, less than 70%, less than 60% over the entire target nucleic acid sequence. This technique is also referred to as miRNA.
[0100] An embodiment according to the present invention provides a method for producing a plant or part of it resistant to a pathogenic fungus of the Phacosporaceae family, for example, soybean rust, in which the recombinant nucleic acid comprises a promoter which is functional in the plant cell, functionally linked to a target nucleic acid which, when transcribed, generates RNA comprising a first strand that has a substantially complementary sequence, preferably complementary to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides of the target gene, and a regulatory terminator sequence.
[0101] Preferably, the first strand generated in the plant forms dsRNA together with a second strand of RNA generated in the plant that is complementary to the first strand. This technique is also referred to as antisense RNA.
[0102] The dsRNA of the invention can optionally comprise a single strand attached at both ends. Preferably, the single strand attached comprises at least two nucleotides at the 3 'ends of each strand of the dsRNA molecule. The double-stranded structure can be formed by a single RNA strand complementary to itself (i.e., forming a clamp loop) or two complementary RNA strands. When the dsRNA of the invention forms a clamp loop, it can optionally comprise an intron, as disclosed by US patent 2003 / 0180945A1 or a nucleotide spacer, which is a sequence extension between complementary strands of RNA to stabilize the clamp transgene in the cells. Methods for producing various dsRNA molecules are presented, for example, in WO 99/53050 and in US patent 6,506,559.
[0103] In one embodiment, the vector construct comprises a promoter that is functional in the plant cell, functionally linked to a target nucleic acid that is substantially identical and / or substantially complementary to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides of the target gene and which, when transcribed, generates RNA comprising a first strand that has a sequence substantially complementary to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides of the target gene, and a second strand that has a sequence substantially complementary to the first strand or parts thereof, and a regulatory terminator sequence.
[0104] It is preferred that the first strand and the second strand are able to hybridize to form dsRNA at least partially.
[0105] In another embodiment, the vector construct comprises a promoter that is functional in the plant cell, functionally linked to a target nucleic acid that, when transcribed, generates RNA that comprises a first strand that has a substantially complementary sequence or identical to at least 19, preferably at least 20, more preferably at least 21, for example, 22 or 23 contiguous nucleotides of the target gene, and a regulatory terminator sequence.
[0106] It is preferred that the transcript of the first strand and at least part of the transcript of the target gene are able to hybridize to form dsRNA at least in part.
[0107] In one embodiment, the vector construct comprises a target nucleic acid comprising 19 to 500 nucleotides. Additional variants of the target nucleic acid are defined in the section referring to the method for producing a plant.
[0108] With respect to a vector construct and / or recombinant nucleic acid, the term "functionally linked" means that the target nucleic acid is linked to the regulatory sequence, including promoters, terminators, enhancers and / or other control elements expression (for example, polyadenylation signals) in a way that allows expression of the target nucleic acid (for example, in a host plant cell when the vector is introduced into the host plant cell). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, Eds. Glick and Thompson, Chapters 7, 89-108, CRC Press: Boca Raton, Florida, including references there. Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct the direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be considered by these technicians on the subject that the design of the vector may depend on such factors as the choice of the host cell to be transformed, the desired level of dsRNA expression, and the like. The vector constructs of the invention can be introduced into plant host cells to thereby produce ssRNA, dsRNA and / or siRNA and / or miRNA to prevent and / or reduce expression of the respective target gene and thus increase resistance to pathogenic fungi of the Phacosporaceae family. , for example, soybean rust.
[0109] In one embodiment, the vector construct comprises a promoter functionally linked to a target nucleotide that is a template for one or both strands of the ssRNA or dsRNA molecules at least substantially complementary to 19 contiguous nucleotides of the target gene.
[0110] In one embodiment, the nucleic acid molecule further comprises two promoters flanking both ends of the nucleic acid molecule, in which the promoters direct the expression of each individual DNA strand, thus generating two complementary RNAs that hybridize and form the dsRNA.
[0111] In alternative embodiments, the nucleotide sequence is transcribed on both strands of the dsRNA in a transcription unit, where the sense strand is transcribed from the 5 'end of the transcription unit and the antisense strand is transcribed from from the 3 'end, where the two strands are separated by about 3 to about 500 base pairs, and where after transcription, the RNA transcript folds to form a clamp.
[0112] In another embodiment, the vector contains a bidirectional promoter, directing the expression of two nucleic acid molecules, through which one nucleic acid molecule encodes a sequence substantially identical to a portion of a target gene of the invention and the other The nucleic acid molecule encodes a second sequence substantially complementary to the first strand and capable of forming a dsRNA, when both sequences are transcribed. A bidirectional promoter is a promoter capable of mediating expression in two directions.
[0113] In another embodiment, the vector contains two promoters, one mediating the transcription of the sequence substantially identical to a portion of a target gene of the invention and the other promoter mediating the transcription of a second sequence being substantially complementary to the first strand and capable of form a dsRNA when both sequences are transcribed. The second prosecutor may be a different prosecutor.
[0114] A different promoter means a promoter that has a different activity in relation to cell or tissue specificity, or that exhibits expression in different inducers, for example, pathogens, abiotic stress or chemicals.
[0115] Promoters according to the present invention can be constitutive, inducible, in particular, inducible by pathogen, preferred for stages of development, preferred for cell type, preferred for tissue, preferred for organ. Constitutive promoters are active under most conditions. Non-limiting examples of constitutive promoters include the CaMV 19S w 35S promoters (Odell et al., 1985, Nature 313: 810-812), the sX CaMV 35S promoter (Kay et al., 1987, Science 236: 1299-1302), the Sep1 promoter, the rice actin promoter (McElroy et al., 1990, Plant Cell 2: 163-171), the Arabidopsis actin promoter, the ubiquitin promoter (Christensen et al., 1989, Plant Molec. Biol. 18: 675-689); pEmu (Last et al., 1991, Theor. Appl. Genet. 81: 581-588), the 35S promoter of the figwort mosaic virus, the Smas promoter (Velten et al., 1984, EMBO J. 3: 27232730) , the GRP1-8 promoter, the cinnamyl alcohol dehydrogenase promoter (US patent 5,683,439), Agrobacterium T-DNA promoters, such as mannopine synthase, nopaline synthase, and octopine synthase, the small subunit of the ribulose bisphosphate carboxylase promoter (ssuRUBISCO) , and / or the like. Promoters that express dsRNA in a cell that is contacted by the fungus are preferred. Alternatively, the promoter can direct the expression of dsRNA in plant tissue remote from the site of contact with the fungus, and the dsRNA can then be transported by the plant to a cell that is contacted by the fungus, in particular, cells from fungal-infected sites. or close to these.
[0116] Preferably, the expression vector of the invention comprises a constitutive promoter, a root-specific promoter, a pathogen-inducible promoter, or a fungus-inducible promoter. A promoter is inducible, if its activity, measured in the amount of RNA produced under the promoter's control, is at least 30%, 40%, 50%, preferably at least 60%, 70%, 80%, 90% more preferably at least 100%, 200%, 300% higher in its induced state than in its non-induced state. A promoter is a cell-, tissue- or organ-specific, if its activity, measured in the amount of RNA produced under the control of the promoter, is at least 30%, 40%, 50%, preferably at least 60%, 70%, 80 %, 90% more preferably at least 100%, 200%, 300% higher in a specific cell type, tissue or organ, than in other cell types or tissues of the same plant, preferably the other cell types or tissues of the same organ of plant, for example, a root. In the case of organ-specific promoters, the activity of the promoter has to be compared to the activity of the promoter in other organs of the plant, for example, leaves, stems, flowers or seeds.
[0117] Preferred promoters for the stage of development are preferably expressed at certain stages of development. Preferred tissue and organ promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds or xylem. Examples of preferential promoters for tissue and preferred for organ include, but are not limited to fruit-preferred, egg-preferred, male-preferred, seed-preferred, integral-preferred, tuber-preferred, stem-preferred, pericarp-preferred, preferred-leaf, preferred-stigma, preferred-pollen, anther-preferred, petal-preferred, sepal-preferred, pedicel-preferred, silica-preferred, stem-preferred, root-preferred and / or similar. Preferred seed promoters are preferentially expressed during seed development and / or germination. For example, preferred seed promoters can be embryo-preferred, endosperm-preferred and seed coating-preferred. See Thompson et al., 1989, BioEssays 10: 108. Examples of preferred seed promoters include, but are not limited to, cellulose synthase (celA), Cim1, gamma-zein, globulin-1, 19 kD corn zein (cZ19B1) and / or the like.
[0118] Other tissue-preferred or organ-preferred promoters include, but are not limited to, rapeseed napkin gene promoter (US patent 5,608,152), the USP promoter of Vicia faba (Baeumlein et al., 1991 , Mol Gen Genet. 225 (3): 459-67), the Arabidopsis oleosin promoter (PCT application WO 98/45461), the phaseolin promoter of Phaseolus vulgaris (US patent 5,504,200), the Bce4 promoter Brassica (PCT application document WO 91/13980), or the B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9), as well as promoters that confer specific expression on seeds in monocotyledonous plants such as corn, barley, wheat, rye, rice, etc. Suitable promoters to be targeted are the barley lpt2 or lpt1 gene promoter (PCT application document WO 95/15389 and PCT application document WO 95/23230) or those described in PCT application document 99/16890 (promoters of the barley hordein gene, glutelin gene from rice, orizine gene from rice, prolamin gene from rice, gliadin gene from wheat, glutelin gene from wheat, glutelin gene from wheat, casirin gene from sorghum, secalin gene from rye)
[0119] Useful promoters according to the invention include, but are not limited to, a major promoter of chlorophyll a / b binding protein, histone promoters, Ap3 promoter, β-conglycine promoter, napine promoter, lecithin promoter soybean, corn zein promoter with 15kD, corn zein promoter with 22kD, corn zein promoter with 27kD, g-zein promoter, waxy, shrunken 1, shrunken 2, bronze promoters, Zm13 promoter (US patent 5,086,169), corn polygalacturonase (PG) promoters (US patent 5,412,085 and 5,545,546), SGB6 promoter (US patent 5,470,359), as well as synthetic or other natural promoters.
[0120] The epidermis-specific promoters can be selected from the group consisting of: WIR5 (= GstA1); acc. X56012; Dudler & Schweizer, GLP4, acc. AJ310534; Wei Y., Zhang Z., Andersen C.H., Schmelzer E., Gregersen P.L., Collinge D.B., Smedegaard-Petersen V. and Thordal-Christensen H., Plant Molecular Biology 36, 101 (1998), GLP2a, acc. AJ237942, Schweizer P., Christoffel A. and Dudler R., Plant J. 20, 541 (1999); Prx7, acc. AJ003141, Kristensen B.K., Ammitzboll H., Rasmussen S.K. and Nielsen K.A., Molecular Plant Pathology, 2 (6), 311 (2001); GerA, acc. AF250933; Wu S., Druka A., Horvath H., Kleinhofs A., Kannangara G. and von Wettstein D., Plant Phys Biochem 38, 685 (2000); OsROC1, acc. AP004656 RTBV, acc. AAV62708, AAV62707; Kloti A., Henrich C., Bieri S., He X., Chen G., Burkhardt PK, Wünn J., Lucca P., Hohn T., Potrykus I. and Fütterer J., PMB 40, 249 (1999) ; Potato chitinase promoter (Ancillo et al., Planta. 217 (4), 566, (2003)); AtProT3 promoter (Grallath et al., Plant Physiology. 137 (1), 117 (2005)); SHN promoters of Arabidopsis (AP2 / EREBP transcription factors involved in the production of cutiona and wax) (Aarón et al., Plant Cell. 16 (9), 2463 (2004)); and / or wheat GSTA1 (Dudler et al., WP2005306368 and Altpeter et al., Plant Molecular Biology. 57 (2), 271 (2005)).
[0121] Mesophilic specific promoters can be selected from the group consisting of: PPCZm1 (= PEPC); Kausch A.P., Owen T.P., Zachwieja S.J., Flynn A.R. and Sheen J., Plant Mol. Biol. 45, 1 (2001); OsrbcS, Kyozuka et al., PlaNT Phys 102, 991 (1993); Kyozuka J., McElroy D., Hayakawa T., Xie Y., Wu R. and Shimamoto K., Plant Phys. 102, 991 (1993); OsPPDK, acc. AC099041; TaGF-2.8, acc. M63223; Schweizer P., Christoffel A. and Dudler R., Plantam J. 20, 541 (1999); TaFBPase, acc. X53957; TaWIS1, acc. AF467542; US patent 200220115849; HvBIS1, acc. AF467539; US patent 200220115849; ZmMIS1, acc. AF467514; US patent 200220115849; HvPR1a, acc. X74939; Bryngelsson et al., Mol. Plant Microbe Interacti. 7 (2), 267 (1994); HvPR1b, acc. X74940; Bryngelsson et al., Mol. Plant Microbe Interact. 7 (2), 267 (1994); HvB1,3gluc; acc. AF479647; HvPrx8, acc. AJ276227; Kristensen et al., Molecular Plant Pathology, 2 (6), 311 (2001); and / or HvPAL, acc. X97313; Wei Y., Zhang Z., Andersen C.H., Schmelzer E., Gregersen P.L., Collinge D.B., Smedegaard-Petersen V. and Thordal-Christensen H. Plant Molecular Biology 36, 101 (1998).
[0122] The constitutive promoters can be selected from the group consisting of: - PcUbi promoter of parsley (document WO 03/102198) - 35S promoter of CaMV: 35S promoter of the cauliflower mosaic virus (Benfey et al. 1989 EMBO J. 8 (8): 2195-2202), - STPT promoter: Arabidopsis thaliana short phosphate translocator promoter (Access NM_123979) - Act1 promoter: - Oryza sativa actin 1 gene promoter (McElroy et al. 1990 PLANT CELL 2 (2) 163-171 a) and / or - EF1A2 promoter: alpha lengthening factor for translation of Glycine max (US patent 20090133159).
[0123] The person skilled in the art is aware that the methods of the invention for supra-regulation of Pti4, Pti5, ERF1 and / or ERF2, as defined above, and infra-regulation of CTR1, EBF1 and / or EBF2, as defined above, to increase resistance to soybean rust, for example, Phacosporacea, in a plant by priming the ethylene signaling pathway, can be combined and can be applied to one plant at a time. This means that the vector or plant or plant part of the invention may comprise one or more constructs for overexpression of at least one of Pti4, Pti5, ERF1 and / or ERF2 and, at the same time, one or more constructs for the downregulation of at least one of CTR1, EBF1 and / or EBF2.
[0124] One type of vector construct is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be linked. Another type of vector is a viral vector, in which additional DNA segments can be linked to the viral genome. Certain vector constructs are capable of autonomous replication in a host plant cell in which they are introduced. Other vector constructs are integrated into the genome of a host plant cell by introducing them into a host cell, and thereby being replicated together with the host genome. In particular, the vector construct is capable of directing the gene expression to which the vector is functionally linked. However, the invention is intended to include these other forms of expression vector constructs, such as viral vectors (for example, potato X virus, tobacco rattle virus and / or twin viruses), which serve equivalent functions .
[0125] In accordance with the present invention the target nucleic acid is capable of reducing the protein amount or function of any of the proteins of the invention in plant cells. In preferred embodiments, the reduction in the amount of protein or function of the target protein occurs in a constitutive or tissue-specific manner. In especially preferred embodiments, a pathogen-induced reduction in the amount of protein or protein function occurs, for example, by recombinant expression of the target nucleic acid under the control of a fungus-inducible promoter. In particular, the expression of the target nucleic acid takes place in fungus-infected sites where, however, preferentially the expression of the target nucleic acid sequence remains essentially unchanged in non-fungus-infected tissues. In preferred embodiments, the amount of protein in a target protein in the plant is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90% or at least 95% or more, compared to a wild type plant that is not transformed with the target nucleic acid. Preferably, the wild type plant is a plant with a similar genotype, more preferably identical to that of the plant transformed with the target nucleic acid.
[0126] The term "introduction" or "transformation", as mentioned in the present application, encompasses the transfer of an exogenous polynucleotide in a host cell, regardless of the method used for the transfer. Plant tissue capable of subsequent clonal propagation, through organogenesis or embryogenesis, can be transformed by a vector construct of the present invention and regenerate an entire plant from it. The specific tissue chosen varies according to the clonal propagation systems available and best suited to the particular species being transformed. Exemplary tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callous tissue, existing merismatic tissue (eg apical meristem, axillary buds and root meristems), and induced meristem system (eg cotyledon meristem) and hypocotyl meristem). The polynucleotide can be introduced transiently or stably into a host cell and can be kept unintegrated, for example, as a plasmid. Alternatively, it can be integrated into the host genome. The resulting transformed plant cell can then be used to regenerate a transformed plant in a manner known to those skilled in the art.
[0127] The term "terminator" encompasses a control sequence that is a DNA sequence at the end of a transcription unit that signals 3 'processing and polyadenylation of a primary transcript and transcription termination. The terminator can be derived from the natural gene, from and a variety of other plant genes, or from T-DNA. The terminator to be added can be derived, for example, from the genes of nopaline synthase or octopine synthase, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
[0128] Transgenic plant cells can be transformed with one of the vector constructs described above. Suitable methods for transformation or transfection host cells including plant cells are well known in the art of plant biotechnology. Any method can be used to transform the recombinant expression vector into plant cells to produce transgenic plants of the invention. General methods for transforming dicot plants are disclosed, for example, in US patents 4,940,838, 5,464,763, and the like. Methods for transforming specific dicot plants, for example, cotton, are disclosed in patents 5,004,863, 5,159,135 and 5,846,797. Soy processing methods that are disclosed in US patents 4,992,375, 5,416,011, 5,569,834, 5,824,877, 6,384,301 and EP 0301749B1 can be used. Transformation methods can include direct and indirect transformation methods. Suitable direct methods include glycol-induced DNA absorption, liposome-mediated transformation (US patent 4,536,475), biological methods using a genetic weapon (Fromm ME et al., Bio / Technology. 8 (9): 833-9 , 1990; Gordon-Kamm et al. Plant Cell 2: 603, 1990), electroporation, incubation of dry embryos in a solution comprising DNA, and microinjection. In the case of these direct transformation methods, the plasmids used do not need to satisfy any specific requirements. Simple plasmids, such as those in the pUC, pBR322, M13mp, pACYC184 and similar series can be used. If intact plants will be regenerated from the transformed cells, a selectable marker gene is preferably located on the plasmid. Direct transformation techniques are equally suitable for dicotyledonous and monocotyledonous plants.
[0129] The transformation can also be carried out by bacterial infection by means of Agrobacterium (for example, patent EP 0 116 718), viral infection by means of viral vectors (patent EP 0 067 553, US patent 4,407,956, document WO 95 / 34668, document WO 93/03161) or by means of pollen (patent EP 0 270 356, document WO 85/01856, US patent 4,684,611). Transformation techniques based on Agrobacterium (especially for dicot plants) are well known in the art. The Agrobacterium strain (for example, Agrobacterium tumefaciens or Agrobacterium rhizogenes) comprises a plasmid (plasmid Ti or Ri) and a T-DNA element that is transferred to the plant after infection with Agrobacterium. T-DNA (transferred DNA) is integrated into the plant cell's genome. T-DNA can be located on plasmid Ri or Ti or is comprised separately in a so-called binary vector. Methods for Agrobacterium-mediated transformation are described, for example, in Horsch RB et al. (1985) Science, 225: 1229. Agrobacterium-mediated transformation is more suitable for dicotyledonous plants, but has been adapted for monocotyledonous plants. The transformation of plants by Agrobacteria is described, for example, in White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 15 - 38; Jenes B et al. Techniques for Gene Transfer, Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42: 205- 225. Transformation can result in temporary or stable transformation and expression. Although a nucleotide sequence of the present invention can be inserted into any plant and plant cell included within these broad classes, it is particularly useful in cultured plant cells.
[0130] Genetically modified plant cells can be regenerated using all the methods with which a person skilled in the art is familiar. Suitable methods can be found in the publications mentioned above by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
[0131] Generally after transformation, cells or clusters of plant cells are selected for the presence of one or more markers that are encoded by the expressible genes in the plant cotransfected with the gene of interest, after which the transformed material is regenerated in a whole plant. In order to select the transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that the transformed plants can be distinguished from unprocessed plants. For example, seeds obtained in the manner described above can be planted and, after a period of initial growth, subjected to an appropriate selection by spray. An additional possibility is the cultivation of seeds, if appropriate after sterilization, on agar plates with the use of an appropriate selection agent, so that only transformed seeds can grow on plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as those described above.
[0132] After DNA transfer and regeneration, putatively transformed plants can also be evaluated, for example, with the use of Southern analysis for the presence of the gene of interest, number of copies and / or genomic organization. Alternatively or additionally, the expression levels of the newly introduced DNA can be monitored using Northern and / or Western analysis, both techniques being well known to those skilled in the art.
[0133] Regenerated transformed plants can be propagated in a variety of ways, such as clonal propagation or classical reproduction techniques. For example, a transformed first generation (or T1) plant can be self-fertilized and homozygous second generation (or T2) transformers selected, and T2 plants can then be further propagated using classical reproduction techniques. The transformed organisms generated can take a variety of forms. For example, they can be chimeras of transformed cells and untransformed cells; clonal transformants (for example, all cells transformed to contain the expression cassette); grafts of transformed and unprocessed tissues (for example, in plants, a transformed rootstock grafted to an untransformed seedling).
[0134] The collectable parts of the transgenic plant according to the present invention are part of the invention. The collectable parts can be seeds, roots, leaves and / or flowers that comprise the SMT1 gene, the complementary SMT1 gene and / or part of it. Preferred parts of soy plants are soy beans that comprise the transgenic SMT1 gene.
[0135] Products derived from transgenic plants according to the present invention, parts thereof or collectable parts thereof are part of the invention. A preferred product is soy meal or soy oil.
[0136] The present invention also includes methods for producing a product comprising a) growing the plants of the invention and b) producing said product from, or by the plants of the invention and / or parts of it, for example , seeds of these plants. In a further embodiment, the method comprises the steps of a) growing the plants of the invention, b) removing the collectable parts, as defined above, from the plants and c) producing said product from, or by the collectable parts according to with the invention.
[0137] In one embodiment, the method for producing a product comprises: a) growing the plants of the invention or which are obtained by the methods of the invention and, b) producing said product from or by the plants of the invention and / or parts, for example, seeds, of these plants.
[0138] The product can be produced in the place where the plant was grown, or the plants and / or parts of it can be removed from the place where the plants were grown to produce the product. Typically, the plant is grown, the desired collectable parts are removed from the plant, if possible, in repeated cycles, and the product is produced from the collectable parts of the plant. The plant growth step can be carried out only once each time the methods of the invention are carried out, while allowing the production steps of the product repeatedly, for example, by repeated removal of collectable parts of the plants of the invention and, if necessary, further processing of these parts to arrive at the product. It is also possible for the plant cultivation step of the invention to be repeated and the collectable plants or parts to be stored until the production of the product is then carried out once for the plants or parts of the accumulated plants. Also, the stages of plant growth and product production can be carried out with an overlap of time, even if simultaneously to a great extent, or sequentially. Usually the plants are grown for some time before the product is produced.
[0139] In one embodiment, the products produced by said methods of the invention are products of plants such as, but not limited to, a foodstuff, animal feed, a food supplement, feed supplement, fiber, cosmetic or pharmaceutical product. Foodstuffs are considered as compositions used for nutrition and / or for supplementary nutrition. Animal feed and animal feed supplements, in particular, are considered to be foodstuffs.
[0140] In another embodiment, inventive methods for production are used to produce agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins and the like.
[0141] It is possible that a plant product consists of one or more agricultural products to a large extent. FIGURES
[0142] Figure 1 shows a schematic illustration of the ET signaling pathway (taken from Adie et al. J Plant Growth Regul 2007 26: 160ff, DOI 10.1007 / s00344-007-0012-6). The binding of ET leads to the inactivation of its receptor and successively to the deactivation of the CTR1 kinase similar to Raf. This allows EIN2 to activate the Ein3 family of transcription factors. On the other hand, Ein3 is regulated by EBF1 and EBF2, leading to the degradation of EIN3. Activated Ein3 suppresses the expression of ERF1 (and its counterpart / ortholog genes). ERF1 (and other ERF-like transcription factors) activate the expression of ethylene-regulated defense genes (eg, PR proteins, etc.).
[0143] Figure 2 shows the scoring system used to determine the level of diseased leaf of wild type and transgenic soybean plants against the rust fungus P. pachyrhizi.
[0144] Figure 3 shows the entire sequence of the Arabidopsis thaliana ERF-1 gene that has SEQ-ID-No.1.
[0145] Figure 4 shows the sequence of the ERF-1 protein (SEQ-ID-2).
[0146] Figure 5 shows the result of the score of 31 transgenic soybean plants that express the ERF-1 overexpression vector construct. T0 soybean plants expressing ERF-1 protein were inoculated with spores of Phakopsora pachyrhizi. The evaluation of the diseased leaf area on all leaves was carried out 14 days after inoculation. The average percentage of leaf area showing colonies of fungi or strong yellowish / brownish in all leaves was considered as diseased leaf area. All 31 T0 soybean plants that express ERF-1 (expression verified by RT-PCR) were evaluated in parallel to non-transgenic control plants. The median of the diseased leaf area is shown in Figure 5. Overexpression of ERF-1 significantly reduces (p <0.001) the diseased leaf area compared to non-transgenic control plants.
[0147] Figure 6 shows the entire sequence of the Pti-4 gene from Sola in a lycopersicum that has SEQ-ID-No.3.
[0148] Figure 7 shows the sequence of the Pti-4 protein (SEQ-ID-4).
[0149] Figure 8 shows the result of the score of 33 transgenic soybean plants that express the Pti-4 overexpression vector construct. T0 soybean plants expressing Pti-4 protein were inoculated with spores of Phakopsora pachyrhizi. The evaluation of the diseased leaf area on all leaves was carried out 14 days after inoculation. The average percentage of leaf area showing colonies of fungi or strong yellowish / brownish in all leaves was considered as diseased leaf area. All 33 T0 soybean plants that express Pti-4 (expression verified by RT-PCR) were evaluated in parallel to non-transgenic control plants. The median of the diseased leaf area is shown in Figure 8. Overexpression of Pti-4 reduces the diseased leaf area compared to non-transgenic control plants.
[0150] Figure 9 shows the entire sequence of the Pti-5 gene from Sola in a lycopersicum that has SEQ-ID-No.5.
[0151] Figure 10 shows the sequence of the Pti-5 protein (SEQ-ID-6).
[0152] Figure 11 shows the result of the score of 34 transgenic soybean plants that express the Pti-5 overexpression vector construct. T0 soybean plants expressing Pti-5 protein were inoculated with spores of Phakopsora pachyrhizi. The evaluation of the diseased leaf area on all leaves was carried out 14 days after inoculation. The average percentage of leaf area showing colonies of fungi or strong yellowish / brownish in all leaves was considered as diseased leaf area. All 34 T0 soybean plants expressing Pti-4 (expression verified by RT-PCR) were evaluated in parallel to non-transgenic control plants. The median of the diseased leaf area is shown in Figure 11. Overexpression of Pti-4 significantly reduces (p <0.05) the diseased leaf area compared to non-transgenic control plants.
[0153] Figure 12 shows the entire sequence of the Arabidopsis thaliana ERF-2 gene that has SEQ-ID-No.7.
[0154] Figure 13 shows the sequence of the ERF-2 protein (SEQ-ID-8).
[0155] Figure 14 shows the result of the score of 29 transgenic soybean plants that express the ERF-2 overexpression vector construct. T0 soybean plants expressing ERF-2 protein were inoculated with spores of Phakopsora pachyrhizi. The evaluation of the diseased leaf area on all leaves was carried out 14 days after inoculation. The average percentage of leaf area showing colonies of fungi or strong yellowish / brownish in all leaves was considered as diseased leaf area. All 29 T0 soybean plants that express ERF-2 (expression verified by RT-PCR) were evaluated in parallel to non-transgenic control plants. The median area of diseased leaf is shown in Figure 14. Overexpression of ERF-2 significantly reduces (p <0.01) the area of diseased leaf compared to non-transgenic control plants.
[0156] Figure 15 shows the entire sequence of the Arabidopsis thaliana CTR-1 gene that has SEQ-ID-No.9.
[0157] Figure 16 shows the sequence of the CTR-1 protein (SEQ-ID-10).
[0158] Figure 17 shows the entire sequence of the EBF-1 gene from Arabidopsis thaliana that has SEQ-ID-No.11.
[0159] Figure 18 shows the sequence of the EBF-1 protein (SEQ-ID-12). EXAMPLES
[0160] The following examples are not intended to limit the scope of the claims of the invention, but are especially intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to be included in the scope of the present invention. EXAMPLE 1 GENERAL METHODS
[0161] The chemical synthesis of oligonucleotides can be affected, for example, in a manner known for using the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896-897). The cloning steps carried out for the purposes of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, DNA binding fragments, transformation of E. coli cells, bacterial cultures, multiplier phages and analysis of recombinant DNA sequences, are performed as described by Sambrook et al. Cold Spring Harbor Laboratory Press (1989), ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules is carried out with a DNA sequencer with laser fluorescence following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)). EXAMPLE 2 CLONING OF OVEREXPRESSION VECTOR CONSTRUCTS
[0162] The cDNAs of all genes mentioned in this application were generated by DNA synthesis (Geneart, Regensburg, Germany).
[0163] The ERF1 cDNA has been synthesized so that an EcoRV restriction site is located in front of the initial ATG and a SpeI restriction site downstream of the stop codon. The synthesized cDNAs were digested with the use of restriction enzymes EcoRV and SpeI (NEB Biolabs) and ligated into a Gateway pENTRY vector digested by EcoRV / SpeI (Invitrogen, Life Technologies, Carlsbad, California, USA) so that the entire fragment was located in the sense direction between the salsa ubiquitin promoter (PcUbi) and an Agrobacterium tOCS terminator.
[0164] To obtain the plant transformation binary vector, a LR triple reaction system (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA) was performed according to the manufacturers protocol by using a vector pENTRY-A containing a salsa ubiquitin promoter, the pENTRY-B vector described above containing the cDNA and a pENTRY-C vector containing a t-StCatpA terminator. As a target, a binary vector pDEST was used which consists of: (1 ) a kanamycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a pBR322 origin of replication for stable maintenance in E.coli and (4) between the right and left limit an AH selection under control from a PcUbi promoter (Figure 4). The recombination reaction was transformed into E. coli (DH5alpha), mini prepared and selected by specific restriction digestions. A positive clone of the vector construct was sequenced and subjected to soybean transformation.
[0165] The Pti4, Pti5, CTR1 cDNAs were synthesized so that an attB1 recombination site (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA) was located in front of the initial ATG and a recombination site attB2 was located directly downstream of the stop codon.The synthesized cDNAs were transferred to a pENTRY-B vector using the reaction system (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA) according to the protocol provided To obtain the plant transformation binary vector, a LR triple reaction system (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA) was performed according to the manufacturers protocol by using a vector pENTRY-A containing a salsa ubiquitin promoter, the cDNAs in a pENTRY-C vector and a pENTRY-C vector containing a t-Nos terminator. res cassette kanamycin resistance to bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a pBR322 origin of replication for stable maintenance in E.coli and (4) between the right and left limit an AH selection under the control of a pcUbi promoter (Figure 4). The recombination reaction was transformed into E. coli (DH5alpha), mini prepared and selected by specific restriction digestions. A positive clone of each vector construct was sequenced and subjected to soybean transformation.
[0166] The EFB1 and ERF2 cDNAs were synthesized so that an EcoRV restriction site was located in front of the initial ATG and a SpeI restriction site downstream of the stop codon. The synthesized cDNAs were digested with the use of restriction enzymes EcoRV and SpeI (NEB Biolabs) and ligated into an Gateway pENTRY vector digested by EcoRV / SpeI (Invitrogen, Life Technologies, Carlsbad, California, USA) so that the entire fragment is located in the sense direction between the salsa ubiquitin promoter (PcUbi) and an Agrobacterium tOCS terminator. To obtain the plant transformation binary vector, a LR triple reaction system (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA) was performed according to the manufacturers protocol by using a pENTRY-A vector empty containing no sequence between the recombination sites, the pENTRY-B vector described above containing the cDNAs and an empty pENTRY-C vector As a target, a binary vector pDEST was used which is composed of: (1) a resistance cassette to kanamycin for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a pBR322 origin of replication for stable maintenance in E.coli and (4) between the right and left limit an AH selection under the control of a pcUbi promoter ( Figure 4) The recombination reaction was transformed into E. coli (DH5alpha), mini prepared and selected by specific restriction digestions A positive clone of each vector construct was sequenced and subjected to soybean transformation. EXAMPLE 3 TR SOY ANSFORMATION
[0167] The constructs containing the expression vector (see example 2) were transformed into soy. 3.1 STERILIZATION AND GERMINATION OF SOYBEAN SEEDS
[0168] Virtually any seed of any variety of soybeans can be used in the method of the invention. A variety of soybean cultivars (including Jack, Williams 82 and Resnik) are suitable for processing soybeans. The soybean seeds were sterilized in a chamber with a chlorine gas by adding 3.5 ml of 12N HCl dropwise in 100 ml of bleach (sodium hypochlorite 5.25%) in a desiccator with a suitable lid. After 24 to 48 hours in the chamber, seeds were removed and approximately 18 to 20 seeds were plated in solid GM medium with or without 5 μM 6-benzyl-aminopurine (BAP) in 100 mm petri dishes. Seedlings without BAP are more elongated and roots developed, especially the formation of lateral and secondary roots. BAP strengthens the seedling by forming a shorter and more stout seedling.
[0169] Plants with seven to ten days grown in light (> 100 μEinstein / m2s) at 25 ° C were used for explant material for the three explant types. This time, the seed lining was broken, and the epicotyl with the unifoliated leaves grew at least to the length of the cotyledons. The epicotyl should be at least 0.5 cm to avoid knots in cotyledon tissue (since soybean cultivars and seed lots can vary in development time, a description of the germination stage is more accurate than a specific germination time).
[0170] For inoculation of whole seedlings (Method A, see example 3.3. And 3.3.2) or leaf explants (Method B, see example 3.3.3), the seedlings were then ready for transformation.
[0171] For method C (see example 3.3.4), the hypocotyl and one, half or part of both cotyledons were removed from each seedling. The seedlings were then placed in propagation media for 2 to 4 weeks. The seedlings produce several branched shoots to obtain explants from them. Most explants originated from the growth of the apical bud seedling. These explants were preferably used as the target tissue. 3.2 - AGROBACTERIUM CULTURE GROWTH AND PREPARATION
[0172] Agrobacterium cultures were prepared by scraping Agrobacterium (for example, A. tumefaciens or A. rhizogenes) carrying the desired binary vector (for example, H. Klee. R. Horsch and S. Rogers 1987 Agrobacterium-Mediated Plant Transformation and its further Applications to Plant Biology; Annual Review of Plant Physiology Vol. 38: 467-486) on YEP media of solid YEP growth medium: 10 g of yeast extract. 10 g of Bacto Peptona. 5 g of NaCl. Adjust pH to 7.0, and bring final volume to 1 liter with H2O, for YEP agar plates add 20g of agar, autoclave) and incubate at 25 ° C until colonies appear (approximately 2 days). Depending on the selectable marker genes in the Ti or Ri plasmid, the binary vector and the bacterial chromosomes, different selection compounds were used for A. tumefaciens and selection of rhizogenes in the solid medium and YEP liquid medium. Several strains of Agrobacterium can be used for the transformation method.
[0173] After approximately two days, a single colony (with a sterile toothpick) was selected and 50 ml of YEP liquid was inoculated with antibiotics and stirred at 175 rpm (25 ° C) until an OD.sub.600 between 0 , 8-1.0 was reached (approximately 2 d). Working glycerol stocks (15%) for transformation are prepared and 1 ml of Agrobacterium stock aliquoted in 1.5 ml Eppendorf tubes then stored at -80 ° C.
[0174] The day before the explant inoculation, 200 ml of YEP were inoculated with 5. mu.l to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask. The flask was shaken overnight at 25 ° C. until OD.sub.600 was between 0.8 and 1.0. Before preparing the soy explants, Agrobacteria were pelleted by centrifugation for 10 min at 5,500.times.ga at 20 ° C. The pellet was resuspended in CCM liquid to the desired density (OD.sub.600 0.5-0.8 ) and placed at room temperature for at least 30 min before use. 3.3 - PREPARATION OF EXPLANTATION AND CO-CULTIVATION (INOCULATION) 3.3.1 METHOD A: PREPARATION OF EXPLANTATION ON THE DAY OF TRANSFORMATION
[0175] The seedlings this time had elongated epicotyls of at least 0.5 cm, but generally between 0.5 and 2 cm. Epicotyls elongated to 4 cm in length have been successfully employed. The explants were then prepared with: i) with or without some roots, ii) with a partial, one or both cotyledons, all preformed leaves were removed including the apical meristem, and the node located in the first set of leaves was removed bruised with several cuts by the use of an acute scalpel.
[0176] This cut in the knot not only induced infection by Agrobacterium, but also distributed the cells of the axillary meristem and damaged preformed shoots. After injury and preparation, the explants were discarded in a Petri dish and subsequently co-cultured with the liquid CCM / Agrobacterium mixture for 30 minutes. The explants were then removed from the liquid medium and plated on top of a sterile filter paper 15 times. 100 mm Petri dishes with solid co-culture medium. The injured target tissues were placed so that they were in direct contact with the medium. 3.3.2 MODIFIED METHOD: EPICOTYLE EXPLANTATION PREPARATION
[0177] The segments of soybean epicotyl prepared from seedlings with 4 to 8 days were used as explants for regeneration and transformation. Soybean seeds L00106CN, 93-41131 and Jack were germinated in 1/10 MS salts or a medium of similar composition with or without cytokines for epicotyl explants with 4 to about 8 days was prepared by removing the cotyledon node from the stem of the stem section. The epicotyl was cut into 2 to 5 segments. Particularly preferred are segments attached to the primary node or the highest node comprising axillary meristematic tissue.
[0178] The explants were used for infection with Agrobacterium. Agrobacterium AGL1 which houses a plasmid with the construct of the invention and the selectable marker gene AHAS, bar or dsdA were grown in LB medium with appropriate antibiotics overnight, harvested and resuspended in an acetoseringone inoculation medium. Freshly prepared segments of epicotyl were soaked in the Agrobacterium suspension for 30 to 60 min and then the explants were blotted dry on sterile filter papers. The inoculated explants were then cultured in a co-culture medium with L-cysteine and TTD and other chemicals such as acetosyringone to improve the distribution of T-DNA for 2 to 4 days. The infected epicotyl explants were then placed in a bud-inducing medium with selection agents such as imazapyrine (for AHAS gene), glufosinate (for bar gene) or D-serine (for dsdA gene). The regenerated shoots were subcultured in elongation medium with the selective agent.
[0179] For the regeneration of transgenic plants, the segments were then grown in a medium with cytokines such as BAP, TDZ and / or kinetin for bud induction. After 4 to 8 weeks, the cultured tissues were transferred to a medium with a lower concentration of cytokinin for elongation of the bud. The elongated shoots were transferred to a medium with auxin for rooting and plant development. Multiple shoots have been regenerated.
[0180] Many sectors transformed in a stable manner that showed strong expression of the construct of the invention have been recovered. Soy plants were regenerated from epicotyl explants. The efficient distribution of T-DNA and stable transformed sectors has been demonstrated. 3.3.3 METHOD B: LEAF EXPLANTS
[0181] To prepare the leaf explant, the cotyledon was removed from the hypocotyl. The cotyledons were separated from each other and the epicotyl was removed. The primary leaves, which consist of the lamina, the petiole and the stipules, were removed from the epicotyl by carefully cutting at the base of the stipules so that the axillary meristems were included in the explant. To injure the explant, as well as to stimulate bud formation again, any preformed shoots were removed and the area between the stipules was cut with an acute scalpel 3 to 5 times.
[0182] The explants are completely immersed or the end of the injured petiole dipped in the Agrobacterium suspension immediately after the explant is prepared. After inoculation, the explants are smeared on sterile filter paper to remove excess culture of Agrobacterium and place the explants with the injured side in contact with a 7 cm round Whatman paper superimposed on the CCM solid medium (see above). This filter paper prevents the excessive growth of A. tumefaciens in soybean explants. Plates were wrapped five times with Parafilm.TM. “M” American National Can, Chicago, Ill., USA) and incubated for three to five days in the dark or light at 25 ° C. 3.3.4 METHOD C: PROPAGATED AXILLARY MERISTEMA
[0183] Seedlings propagated with 3 to 4 weeks were used to prepare the propagated axillary meristem explant. Axillary meristem explants can be pre-prepared from the first to the fourth node. An average of three to four explants could be obtained from each seedling. The explants were prepared from seedlings by cutting 0.5 to 1.0 cm below the axillary node in the internode and removing the petiole and leaf from the explant. The tip where the axillar meristem is located was cut with a scalpel to again induce the growth of the bud and allow access to the target cells for Agrobacterium. Therefore, a 0.5 cm explant included the stem and the yolk.
[0184] Once cut, the explants were immediately placed in the Agrobacterium suspension for 20 to 30 minutes. After inoculation, the explants were smeared on sterile filter paper to remove excess culture of Agrobacterium, then placed almost completely immersed in solid MCC or on top of a 7 cm round filter paper overlapping the solid MCC, depending on the strain Agrobacterium. This filter paper prevents the overgrowth of Agrobacterium in soy explants. The plates were wrapped with Parafilm.TM “M” (American National Can, Chicago, IL., USA) and incubated for two to three days in the dark at 25 ° C. 3.4 - BROTH INDUCTION
[0185] After 3 to 5 days of co-cultivation in the dark at 25 ° C, the explants were rinsed in liquid SIM medium (to remove excess Agrobacterium) (YES, see Olhoft et al 2007 The novel Agrobacterium rhizogenes- mediated transformation method of soyusing primary-node explants from seedlings In Vitro Cell. Dev. Biol.-Plant (2007) 43: 536-549) or Modwash medium (1X B5 main salts, 1X B5 secondary salts, 1X MSIII iron, Sucrose 3%, 1X B5 vitamins, 30 mM MES, 350 mg / L TimentinTM pH 5.6, WO 2005/121345) and blotted to dry on sterile filter paper (prevent damage, especially on the slide) before being placed in the solid SIM medium. Approximately 5 explants (Method A) or 10 to 20 explants (Methods B and C) were placed so that the target tissue was in direct contact with the medium. During the first 2 weeks, the explants could be grown with or without selective medium. Preferably, the explants were transferred over SIM without selection for one week.
[0186] For leaf explants (Method B), the explant should be placed in the middle so that it is perpendicular to the surface of the medium with the petiole soaked in the middle and the blade out of the medium.
[0187] For the propagated axillary meristem (Method C), the explant was placed in the middle so that it was parallel to the surface of the medium (towards the base) with the explant partially embedded in the middle.
[0188] The plates wrapped with adhesive tape 394 (3M, St. Paul, Minn., USA) were placed in a growth chamber for two weeks with a temperature varying on average 25 ° C through an 18 h light / 6 h cycle from dark to 70-100 .mu.E / m.sup.2s. The explants remained in the SIM medium with or without selection until the growth of new shoots occurred again in the target area (for example, meristems in the first node above the epicotyl). Transfers to the fresh medium can occur during this time. The explants were transferred from SIM with or without selection to SIM with selection after about a week. This time, there was considerable new bud development at the base of the leaf explants petiole in a variety of SIM (Method B), in the primary node for seedling explants (Method A), and in the axillary nodes of propagated explants (Method C) .
[0189] Preferably, all shoots formed before transformation were removed up to 2 weeks after co-cultivation to stimulate new growth of meristems. This helped to reduce chimerism in the primary transformant and increased amplification of transgenic meristematic cells. During this time the explant may or may not be cut into smaller pieces (that is, untie the explant knot by cutting the epicotyl). 3.5 - STRETCHING THE BROTH
[0190] After 2 to 4 weeks (or until a mass of shoots has been formed) in the SIM medium (preferably with selection), the explants were transferred to SEM medium (bud elongation medium, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soy using primary-node explants from seedlings. This medium may or may not contain a selection compound.
[0191] After every 2 to 3 weeks, the explants were transferred to fresh SEM-free medium (preferably containing selection) after carefully removing the dead tissue. The explants must remain joined and not fragmented into pieces, and kept healthy in some way. The explants continued to be transferred until the explant died or the sprouts elongated. The shoots that stretched> 3 cm were removed and placed in RM medium for about 1 week (Method A and B), or about 2 to 4 weeks depending on the cultivar (Method C), in that period the roots started to form . In the case of explants with roots, they were transferred directly to the soil. Rooted shoots were transferred to the soil and hardened in a growth chamber for 2 to 3 weeks before being transferred to the greenhouse. Regenerated plants obtained using this method were fertile and produced an average of 500 seeds per plant.
[0192] The temporary expression of the construct of the invention after 5 days of co-cultivation with Agrobacterium tumefaciens was widespread in the axillary meristem explants of the seedlings, especially in the wound regions during the preparation of the explant (Method A). The explants were placed in the sprout induction medium without selection to observe how the main nodes respond to sprout induction and regeneration. So far, more than 70% of explants have formed new shoots in this region. The expression of the construct of the invention was stable after 14 days in SIM, implying integration of T-DNA in the soybean genome. In addition, preliminary experiments resulted in the formation of positive shoots that express the construct of the invention that formed after 3 weeks in the SIM.
[0193] For Method C, the average regeneration time for a soybean plant using the propagated axillary meristem protocol was 14 weeks from the explant inoculation. Therefore, this method has a quick regeneration time which leads to healthy and fertile soy plants. EXAMPLE 4 PATHOGEN TEST 4.1. CLONES RECOVERY
[0194] 2 to 3 clones per T0 event small 6 cm pots were planted. For recovery, the clones were kept for 12 to 18 days in the greenhouse (they were grown at a pace of 16h-day and 8-night at a temperature of 16 ° to 22 ° C and humidity of 75%). 4.2 INOCULATION
[0195] The rust fungus is a wild isolate from Brazil. The plants were inoculated with P.pachyrhizi.
[0196] In order to obtain appropriate spore material for inoculation, soybean leaves that were infected with rust between 15 to 20 days prior, were taken 2 to 3 days before inoculation and transferred to agar plates (1 % agar in H2O). The leaves were placed with their upper side on the agar, which allowed the fungus to grow through the tissue and produce very young spores. For the inoculation solution, the spores were removed from the leaves and added to a solution of Tween-H2O. Spore counting was performed with a light microscope, using a Thoma counting chamber. For the inoculation of the plants, the spore suspension was added to a spray bottle operated with compressed air and applied evenly over the plants or the leaves, until the surface of the leaf was well moist. For macroscopic assays we use a spore density of 1- 5x105 spores / ml. For microscopic assays, a spore density> 5 x 105 spores / ml is used. The inoculated plants were placed for 24 hours in a greenhouse chamber with an average of 22 ° C and> 90% air humidity. The following culture was carried out in a chamber with an average of 25 ° C and 70% humidity in the air. EXAMPLE 5 MICROSCOPIC SELECTION
[0197] To assess the development of the pathogen, the leaves of inoculated plants were stained with blue aniline 48 hours after infection.
[0198] The blue aniline that stains is used for the detection of fluorescent substances. During defense reactions in host and non-host interactions, substances such as phenols, callose or lignin have accumulated or been produced and have been incorporated into the cell wall both locally in the papilla and in the entire cell (hypersensitive reaction, HR). The complexes were formed in association with blue aniline which leads, for example in the case of callose, to yellow fluorescence. The leaf material was transferred to falcon tubes or dishes containing bleaching solution II (ethanol / acetic acid 6/1) and was incubated in a water bath at 90 ° C for 10 to 15 minutes. The bleach solution II was removed immediately afterwards, and the leaves were washed 2x with water. For staining, the leaves were incubated for 1.5 to 2 hours in dye solution II (aniline blue 0.05% = methyl blue, potassium hydrogen diphosphate 0.067 M) and analyzed by microscopy immediately afterwards.
[0199] The different types of interaction were evaluated (counted) by microscopy. A BX61 Olympus UV microscope (incident light) and a Longpath UV filter (excitation: 375/15, beam splitter: 405 LP) are used. After staining with blue aniline, the spores appeared blue under UV light. The papilla can be recognized under the fungal aphorium by a green / yellow color. The hypersensitive reaction (HR) was characterized by an entire cell fluorescence. EXAMPLE 6 EVALUATION OF SUSCEPTIBILITY TO SOY RUST
[0200] The progression of soybean rust disease was assessed by estimating the diseased area (area that was covered by sporulating uredinia) in the rear (abaxial side) of the leaf. Additionally, the yellowing of the leaf was taken into account. (for schematic see Figure 2) 6.1 ERF-1 OVEREXPRESSION
[0201] T0 soybean plants expressing ERF-1 protein were inoculated with spores of Phakopsora pachyrhizi. The symptoms of macroscopic soybean disease against P. pachyrhizi from 31 T0 soybean plants were evaluated 14 days after inoculation.
[0202] The average percentage of leaf area showing colonies of fungi or strong yellowish / brownish on all leaves was considered as diseased leaf area. All 31 T0 soybean plants that express ERF-1 (expression verified by RT-PCR) were evaluated in parallel to non-transgenic control plants. Clones of non-transgenic soybean plants were used as controls. The median of diseased leaf area is shown in Figure 5 for plants that express ERF-1 compared to wild type plants. . Overexpression of ERF-1 reduces the diseased leaf area compared to non-transgenic control plants by 40%. These data clearly indicate that in plant expression the ERF-1 expression vector construct leads to a lower score for transgenic plant disease compared to non-transgenic controls. Therefore, the expression of ERF-1 and, therefore, the priming of the ethylene signaling pathway in soybeans increases the resistance of soybeans against soybean rust. 6.2 ATI-4 OVEREXPRESSION
[0203] T0 soybean plants expressing Pti-4 protein were inoculated with spores of Phakopsora pachyrhizi. Symptoms of macroscopic soybean disease against P. pachyrhizi from 33 T0 soybean plants were evaluated 14 days after inoculation.
[0204] The average percentage of leaf area showing colonies of fungi or strong yellowish / brownish in all leaves was considered as diseased leaf area. All 31 T0 soybean plants expressing Pti-4 (expression verified by RT-PCR) were evaluated in parallel to non-transgenic control plants. Clones of non-transgenic soybean plants were used as controls. The median of diseased leaf area is shown in Figure 8 for plants that express Pti-4 compared to wild type plants. Overexpression of Pti-4 reduces diseased leaf area compared to non-transgenic control plants by 28%. These data clearly indicate that in plant expression the Pti-4 expression vector construct leads to a lower disease score for transgenic plants compared to non-transgenic controls. Therefore, the expression of Pti-4 and, therefore, the priming of the ethylene signaling pathway in soybeans improves the resistance of soybeans against soybean rust. 6.3 PTI-5 OVEREXPRESSION
[0205] T0 soybean plants expressing Pti-5 protein were inoculated with spores of Phakopsora pachyrhizi. Symptoms of macroscopic soybean disease against P. pachyrhizi from 34 T0 soybean plants were evaluated 14 days after inoculation.
[0206] The average percentage of leaf area showing colonies of fungi or strong yellowish / brownish in all leaves was considered as diseased leaf area. All 34 T0 soybean plants expressing Pti-5 (expression verified by RT-PCR) were evaluated in parallel to non-transgenic control plants. Clones of non-transgenic soybean plants were used as controls. The median of diseased leaf area is shown in Figure 11 for plants expressing Pti-5 compared to wild type plants. Overexpression of Pti- 5 reduces the diseased leaf area compared to non-transgenic control plants by 43%. These data clearly indicate that in plant expression the Pti-4 expression vector construct leads to a lower disease score for transgenic plants compared to non-transgenic controls. Therefore, the expression of Pti-4 and, therefore, the priming of the ethylene signaling pathway in soy increases the resistance of soy against soybean rust. 6.4 ERF-2 OVEREXPRESSION
[0207] T0 soybean plants expressing ERF-2 protein were inoculated with spores of Phakopsora pachyrhizi. Symptoms of macroscopic soybean disease against P. pachyrhizi from 29 T0 soybean plants were evaluated 14 days after inoculation.
[0208] The average percentage of leaf area showing colonies of fungi or strong yellowish / brownish in all leaves was considered as diseased leaf area. All 29 T0 soybean plants that express ERF-2 (expression verified by RT-PCR) were evaluated in parallel to non-transgenic control plants. Clones of non-transgenic soybean plants were used as controls. The median of diseased leaf area is shown in Figure 14 for plants that express ERF-2 compared to wild type plants. Overexpression of ERF-2 reduces the diseased leaf area compared to non-transgenic control plants by 45%. These data clearly indicate that in plant expression the ERF-2 expression vector construct leads to a lower disease score for transgenic plants compared to non-transgenic controls. Therefore, the expression of ERF-2 and, therefore, the priming of the ethylene signaling pathway in soybeans increases the resistance of soybeans against soybean rust. 6.5 Overexpression of CTR-1 and EBF-1 EN-INHIBITION ENVIRONMENTS
[0209] T0 soybean plants that express CTR-1 and EBF-1 proteins that inhibit the ET pathway were inoculated with spores of Phakopsora pachyrhizi. The symptoms of macroscopic soybean disease against P. pachyrhizi of 27, respectively 28 soybean T0 plants were evaluated 14 days after inoculation.
[0210] The average percentage of the leaf area showing colonies of fungi or strong yellowish / brownish on all leaves was considered to be a diseased leaf area. All 27 T0 soybean plants that overexpress CTR-1 and 28 T0 soybean plants that overexpress EBF-1 were evaluated in parallel to non-transgenic control plants. Clones of non-transgenic soybean plants were used as controls. Overexpression of CTR-1 and EBF-1 inhibiting the ethylene signaling pathway increases the diseased leaf area compared to non-transgenic control plants. These data clearly indicate that plant inhibition of the ET pathway leads to a lower disease score for transgenic plants compared to non-transgenic controls. So, inhibition of the ethylene signaling pathway in soy reduces the resistance of soy against soybean rust. 6.6 PRIMING OF THE ETHYLENE SIGNALING ROUTE BY RNAI CTR-1 ENZYME INHIBITION
[0211] Four sets of transgenic T0 soybean plants that express RNAi constructs that target GmCTR1a (SEQ ID 13), GmCTR1b (SEQ ID 15) or GmCTR1c (SEQ ID 17) are produced individually or all three homologous genes respectively. The constructs of RNAi are synthesized and subsequently cloned into transformation vectors under the control of constitutive, pathogen-inducible, leaf-specific and / or epidermis promoters. The RNAi constructs are SEQ ID 25, which target GmCTR1a, SEQ ID 26 which target GmCTR1b, SEQ ID 27 which target GmCTR1c and SEQ ID28 which target GmCTR1 a, b and c.
[0212] Transgenic plants are analyzed by RTqPCR for the infregulation of the respective gene (s). Plants that show strong repression of the expression of CTR1 are inoculated with spores of Phakopsora pachyrhizi. Symptoms of macroscopic soybean disease against P. pachyrhizi of up to 30 T0 soybean plants per construct are evaluated 14 days after inoculation.
[0213] The average percentage of leaf area showing fungal colonies or strong yellowing / brown on all leaves is considered to be a diseased leaf area. All T0 soybean plants that exhibit repression of CTR1a, b or c or CTR1 a and b and c are evaluated in parallel to non-transgenic control plants. Clones of non-GM soybean plants are used as controls. The repression of CTR1 expression significantly reduces the diseased leaf area compared to non-transgenic control plants. The repression of CTR1 expression and, therefore, the priming of the ethylene signaling pathway in soybeans increases the resistance of soybeans against soybean rust. 6.7 PRIMING OF THE ETHYLENE SIGNALING ROUTE BY CTR-1 ENZYME INHIBITION BY MICROWAVE EXPRESSION
[0214] Transgenic T0 soybean plants that express a recombinant microRNA precursor that comprises a microRNA that targets all three homologous genes GmCTR1a (SEQ ID 13), GmCTR1b (SEQ ID 15) and GmCTR1c (SEQ ID 17) are produced. The RNAi precursor is synthesized and subsequently cloned into transformation vectors under the control of constitutive, pathogen-inducible, leaf-specific and / or epidermis promoters. The microRNA precursor is shown in SEQ ID 33.
[0215] Transgenic plants are analyzed by RTqPCR to infrarregulate CTR1 homologues. Plants that show strong repression of the expression of CTR1 are inoculated with spores of Phakopsora pachyrhizi. Symptoms of macroscopic soybean disease against P. pachyrhizi of up to 30 T0 soybean plants are evaluated 14 days after inoculation.
[0216] The average percentage of leaf area showing fungal colonies or strong yellowing / brown on all leaves is considered to be a diseased leaf area. All T0 soybean plants that exhibit CTR1 repression are evaluated in parallel to the non-GM control plants. Clones of non-GM soybean plants are used as controls. The repression of CTR1 expression significantly reduces the diseased leaf area compared to non-transgenic control plants. The repression of CTR1 expression and, therefore, the priming of the ethylene signaling pathway in soybeans increases the resistance of soybeans against soybean rust. 6.8 PRIMING OF ETHYLENE SIGNALING ROUTE BY EBF-1 ENZYME INHIBITION BY RNAI
[0217] Three sets of transgenic T0 soybean plants that express RNAi constructs that target GmEBF1a are produced individually, GmEBF1a (SEQ ID 19), GmEBF1b (SEQ ID 21) or GmEBF1c (SEQ ID 23). Constructs of RNAi are synthesized and subsequently cloned into transformation vectors under the control of specific, constitutive, pathogen-inducible, leaf-specific and / or epidermis-specific promoters. The RNAi constructs are SEQ ID 29, which target GmEBF1a, SEQ ID 30 which target GmEBF1b, SEQ ID 31.
[0218] Transgenic plants are analyzed by RTqPCR for the infregulation of the respective gene (s). Plants that show strong repression of the expression of the respective GmEBF1 gene are inoculated with spores of Phakopsora pachyrhizi. Symptoms of macroscopic soybean disease against P. pachyrhizi of up to 30 T0 soybean plants per construct are evaluated 14 days after inoculation.
[0219] The average percentage of leaf area showing fungal colonies or strong yellowing / brown on all leaves is considered to be a diseased leaf area. All T0 soybean plants that exhibit EBF1a, b or c repression, respectively, are evaluated in parallel to non-transgenic control plants. Clones of non-GM soybean plants are used as controls. The repression of EBF1 expression significantly reduces the diseased leaf area compared to non-transgenic control plants. The repression of EBF1 expression and, therefore, the priming of the ethylene signaling pathway in soybeans increases the resistance of soybeans against soybean rust. 6.9 PRIMING OF THE ETHYLENE SIGNALING ROUTE BY EBF-1 ENZYME INHIBITION BY MICROWAVE EXPRESSION
[0220] Transgenic T0 soybean plants that express a recombinant microRNA precursor that comprises a microRNA that targets all three homologous genes GmEBF1a (SEQ ID 19), GmEBF1b (SEQ ID 21) and GmEBF1c (SEQ ID 23) are produced. The RNAi precursor is synthesized and subsequently cloned into transformation vectors under the control of constitutive, pathogen-inducible, leaf-specific and / or epidermis promoters. The microRNA precursor is shown in SEQ ID 32.
[0221] Transgenic plants are analyzed by RTqPCR to infrarregulate EBF1 homologs. Plants that show strong repression of the expression of EBF1 are inoculated with spores of Phakopsora pachyrhizi. Symptoms of macroscopic soybean disease against P. pachyrhizi of up to 30 T0 soybean plants are evaluated 14 days after inoculation.
[0222] The average percentage of leaf area showing fungal colonies or strong yellowing / brown on all leaves is considered to be a diseased leaf area. All T0 soybean plants that exhibit EBF1 repression are evaluated in parallel with the non-GM control plants. Clones of non-GM soybean plants are used as controls. The repression of EBF1 expression significantly reduces the diseased leaf area compared to non-transgenic control plants. The repression of EBF1 expression and, therefore, the priming of the ethylene signaling pathway in soybeans increases the resistance of soybeans against soybean rust. 1/1

权利要求:
Claims (5)
[0001]
1. METHOD TO INCREASE THE RESISTANCE TO PHACOSPORACEA SOY RUST IN SOYBEAN PLANTS, characterized by understanding the stage of expression of a protein of SEQ ID NO: 6 in plant cells.
[0002]
2. METHOD FOR THE PRODUCTION OF A SOYBEAN PLANT THAT HAS AN INCREASED RESISTANCE AGAINST SOY RUST PHACOSPORACEA, characterized by comprising: a) transforming the plant cell with an expression cassette comprising a recombinant nucleic acid, in which the recombinant nucleic acid of SEQ ID NO: 5 encodes a protein of SEQ ID NO: 6 functionally linked with a promoter; b) regenerating a plant from said transformed plant cell.
[0003]
METHOD, according to any one of claims 1 to 2, characterized in that the promoter is a constitutive promoter, inducible by pathogen, specific promoter of mesophile and / or promoter specific to epidermis.
[0004]
METHOD according to any one of claims 1 to 3, characterized in that the soybean rust Phacosporacea is caused by Phakopsora meibomiae and / or Phakopsora pachyrhizi.
[0005]
5. TRANSFORMATION VECTOR, characterized in that it comprises an expression cassette comprising a recombinant nucleic acid, wherein the recombinant nucleic acid of SEQ ID NO: 5 encodes a protein of SEQ ID NO: 6 functionally linked with a promoter.

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法律状态:
2019-07-09| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-08-18| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-02-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-04-27| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 25/06/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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US61/501,274|2011-06-27|

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EP11171484.6|2011-06-27|

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EP11171484|2011-06-27|

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PCT/IB2012/053193|WO2013001435A1|2011-06-27|2012-06-25|Phacosporacea resistant soybean plants|

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