# Patent

## Title
Phacosporacea resistant soybean plants

## Abstract

The present invention relates to a method of increasing resistance against fungal pathogens of the family Phacosporaceae in transgenic plants and/or plant cells. In these plants, the ethylene signaling pathway and/or activity of the ethylene signaling compounds is changed. This is achieved by priming the ethylene signaling pathway in these plants in comparison to wild type plants and/or wild type plant cells. Depending on the activating or inhibitory function of a particular signaling compound overexpression or knock-down of the cognate gene might be used.

## Background
This application is a National Stage application of International Application No. PCT/IB2012/053193, filed Jun. 25, 2012, which claims the benefit of U.S. Provisional Application No. 61/501,274, filed Jun. 27, 2011. This application also claims priority under 35 U.S.C. §119 to European Patent Application No. 11171484.6, filed Jun. 27, 2011.


The present invention relates to a method of increasing resistance against fungal pathogens of the family Phacosporaceae in transgenic plants and/or plant cells. In these plants, the ethylene signaling pathway and/or activity of the ethylene signaling compounds is changed. This is achieved by priming the ethylene signaling pathway in these plants in comparison to wild type plants and/or wild type plant cells. Depending on the activating or inhibitory function of a particular signaling compound overexpression or knock-down of the cognate gene might be used.
Furthermore, the invention relates to transgenic plants and/or plant cells having an increased resistance against fungal pathogens of the family Phacosporaceae, for example soybean rust and to recombinant expression vectors comprising a sequence that is identical or homologous to a sequence encoding a functional ethylene signaling compound or fragments thereof.
The cultivation of agricultural crop plants serves mainly for the production of foodstuffs for humans and animals. Monocultures in particular, which are the rule nowadays, are highly susceptible to an epidemic-like spreading of diseases. The result is markedly reduced yields. To date, the pathogenic organisms have been controlled mainly by using pesticides. Nowadays, the possibility of directly modifying the genetic disposition of a plant or pathogen is also open to man.
Resistance generally means the ability of a plant to prevent, or at least curtail the infestation and colonization by a harmful pathogen. Different mechanisms can be discerned in the naturally occurring resistance, with which the plants fend off 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).
With regard to the race 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 may build up reproduction structures, while the host mostly dies off. An incompatible interaction occurs on the other hand when the pathogen infects the plant but is inhibited in its growth before or after weak development of symptoms. In the latter case, the plant is resistant to the respective pathogen (Schopfer and Brennicke, vide supra). However, this type of resistance is specific for a certain strain or pathogen.
In both compatible and incompatible interactions a defensive and specific reaction of the host to the pathogen occurs. In nature, however, this resistance is often overcome because of the rapid evolutionary development of new virulent races of the pathogens (Neu et al. (2003) American Cytopathol. Society, MPMI 16 No. 7: 626-633).
Most pathogens are plant-species specific. This means that a pathogen can induce a disease in a certain plant species, but not in other plant species (Heath (2002) Can. J. Plant Pathol. 24: 259-264). The resistance against a pathogen in certain plant species is called non-host resistance. The non-host resistance offers strong, broad, and permanent protection from phytopathogens. Genes providing non-host resistance provide the opportunity of a strong, broad and permanent protection against certain diseases in non-host plants. In particular such a resistance works for different strains of the pathogen.
Immediately after recognition of a potential pathogen the plant starts to elicit defense reactions. Mostly the presence of the pathogen is sensed via so called PAMP receptors, a class of transmembrane receptor like kinases recognizing conserved pathogen associated molecules (e.g. flagellin or chitin). Downstream of the PAMP receptors, the phytohormones salicylic acid (SA), jasmonate (JA) and ethylene (ET) play a critical role in the regulation of the different defense reactions. Depending on the ratio of the different phytohormones, different defense reactions are elicited by the host cell. Generally SA dependent defense is linked with resistance against biotrophic pathogens, whereas JA/ET dependent defense reactions are active against necrotrophic pathogens (and insects). In most plant pathogen interactions ET has been shown to act synergistic to JA and antagonistic to the “biotrophic” defense of SA. For example the well-known JA marker protein PDF1.2 needs the activation of both ET and JA to be up-regulated during defense against necrotrophic pathogens. The crucial involvement of the JA/ET pathway in resistance against necrotrophic pathogens is corroborated by the fact that the overexpression of ERF1, a central protein involved in ET signaling (see FIG. 1) leads to an enhanced resistance against the 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). On the other hand priming of the ET signaling pathway by overexpression of ERF1 increases the susceptibility of Arabidopsis against the biotrophic pathogen Pseudomonas syringae (Berrocal-Lobo et al. 2002, Plant Journal 29:23-32) proving the proposed model that the JA/ET interacts negatively with the SA pathway to balance the nature of the defense reactions according to the attacking pathogen allowing the plant to tailor its defense response. Hence it was generally believed that priming of the ET signaling pathway leads to increased resistance to necrotrophic fungi but at the same time to an increased susceptibility to biotrophic pathogens.
Fungi are distributed worldwide. Approximately 100 000 different fungal species are known to date. The 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).
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 colonization of the plant by the fungus. During the first stage of the infection, the spores become attached to the surface of the plants, germinate, and the fungus penetrates the plant. Fungi may penetrate the plant via existing ports such as stomata, lenticels, hydatodes and wounds, or else they penetrate the plant epidermis directly as the result of the mechanical force and with the aid of cell-wall-digesting enzymes. Specific infection structures are developed for penetration of the plant. The soybean rust Phakopsora pachyrhizi directly penetrates the plant epidermis. After crossing the epidermal cell, the fungus reaches the intercellular space of the mesophyll, where the fungus starts to spread through the leaves. To acquire nutrients the fungus penetrates mesophyll cells and develops haustoria inside the mesophyl cell. During the penetration process the plasmamembrane of the penetrated mesophyll cell stays intact. Therefore the soybean rust fungus establishes a biotrophic interaction with soybean.
Soybean rust has become increasingly important in recent times. The disease may be caused by the biotrophic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a wide spectrum of leguminosic host plants. P. pachyrhizi, also referred to as Asian rust, is the more aggressive pathogen on soy (Glycine max), and is therefore, at least currently, of great importance for agriculture. P. pachyrhizi can be found in nearly all tropical and subtropical soy growing regions of the world. P. pachyrhizi is capable of infecting 31 species from 17 families of the Leguminosae under natural conditions and is capable of growing on further 60 species under controlled conditions (Sinclair et al. (eds.), Proceedings of the rust workshop (1995), National SoyaResearch Laboratory, Publication No. 1 (1996); Rytter J. L. et al., Plant Dis. 87, 818 (1984)). P. meibomiae has been found in the Caribbean Basin and in Puerto Rico, and has not caused substantial damage as yet.
P. pachyrhizi can currently be controlled in the field only by means of fungicides. Soy plants with resistance to the entire spectrum of the isolates are not available. When searching for resistant plants, four dominant genes Rpp1-4, which mediate resistance of soy to P. pachyrhizi, were discovered. The resistance was lost rapidly, as P. pychyrhizi develops new virulent races.
In recent years, fungal diseases, e.g. soybean rust, has gained in importance as pest in agricultural production. There was therefore a demand in the prior art for developing methods to control fungi and to provide fungal resistant plants.
Much research has been performed on the field of powdery and downy mildew infecting the epidermal layer of plants. However, the problem to cope with soybean rust which infects the mesophyll remains unsolved.
Surprisingly we found that the biotrophic fungal pathogens of the family Phacosporaceae, for example soybean rust fungus can be controlled by using the ethylene mediated defense, although prior art teaches, that priming the ethylene mediated defense leads to increased susceptibility to biotrophic fungi (Berrocal-Lobo et al. 2002, Plant Journal 29:23-32). We primed the ET pathway either by over-expression of several proteins involved in ethylene signaling or by downregulation of several proteins involved in suppression of the ET signaling pathway. Generally one should expect that the priming of the ET signaling pathway should lead to enhanced susceptibility against Asian Soybean Rust (ASR), as the ET signaling pathway negatively interacts with the biotrophic defense associated SA pathway. On the other hand one should expect enhanced resistance to ASR by inhibiting the ET signaling pathway, and therefore debottlenecking the SA pathway. Surprisingly we found the ET signaling pathway itself enhances the resistance against soybean rust. Overexpression of several proteins involved in ET signaling pathway (ERF1, ERF2, Pti4, Pti5) increases the resistance of soybean against fungal pathogens of the family Phacosporaceae, for example soybean rust. Downregulation of ET signaling pathway antagonisitc proteins like CTR1 and EBF1 also increases the resistance of soybean to fungal pathogens of the family Phacosporaceae for example soybean rust. Vice versa the overexpression of ET signaling pathwayantagonisitc proteins like CTR1 and EBF1 increases the susceptibility of soybean to fungal pathogens of the family Phacosporaceae for example soybean rust. This clearly demonstrates the positive influence of the ET mediated defense pathways to the resistance of soybean against fungal pathogens of the family Phacosporaceae for example soybean rust.
The object of the present invention is to provide a method of increasing resistance against fungal pathogens of the family Phacosporaceae, preferably against fungal pathogens of the genus Phacospora, most preferably against Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as soy bean rust in transgenic plants and/or transgenic plant cells by using the ethylene signaling pathway, especially by priming the ethylene signaling pathway. This may be achieved by overexpressing one or more nucleic acid of the invention in order to prime the ethylene signaling pathway or downregulating of one or more nucleic acids of the invention that would also lead to the priming of the ethylene signaling pathway or a combination of both, which in turn would lead to increased resistance to fungal pathogens of the family Phacosporaceae for example soybean rust.
The nucleic acids of the invention to be overexpressed in order to prime the ethylene signaling pathway and to achieve increased resistance to fungal pathogens of the family Phacosporaceae for example soybean rust are Pti4, Pti5, ERF1 and/or ERF2 as for example defined by any of SEQ ID NO: 1, 3, 5 or 7 or any homolog, derivative or orthologue or paralogue thereof. The priming of the ethylene signaling pathway may also be achieved by the downregulation of repressors of any of Pti4, Pti5, ERF1 and/or ERF2 such as microRNAs or ta-siRNAs targeting these genes.
The nucleic acids of the invention to be downregulated in order to prime the ethylene signaling pathway and to achieve increased resistance to fungal pathogens of the family Phacosporaceae for example soybean rust are CTR1, EBF1 and/or EBF2 as for example defined by any of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21 or 23 or any fragment, homolog, derivative or orthologue or paralogue thereof. The priming of the ethylene signaling pathway may also be achieved by the overexpression of repressors of any of CTR1, EBF1 and/or EBF2 such as microRNAs or ta-siRNAs targeting these genes.
A further object is to provide transgenic plants resistant against fungal pathogens of the family Phacosporaceae, preferably against fungal pathogens of the genus Phacospora, most preferably against Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as soybean rust, a method for producing such plants as well as a vector construct useful 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 features of the sub-claims.
Definitions
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the examples included herein. Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. In addition to the definitions of terms provided herein, definitions of common terms in molecular biology may 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, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1998 Supplement). It is to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a cell” can mean that at least one cell can be utilized. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.
Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of 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, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.
The term “priming” is to be understood as sensitization of a plant or part thereof to future attack by pests or pathogens in order to induce a resistance against such 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 tissue of the plant that results in a faster and stronger expression of defense mechanisms compared to an unprimed plant once the plant is exposed to pathogen attack. “Priming” refers herein to the sensitization of a plant or part of a plant so that is able to activate defense mechanisms faster and/or stronger when exposed to one or more biotic stresses compared to a non-primed control plant or part thereof which must rely on a direct defense response. Without limiting the scope of the invention, it is believed that priming results in an increased level of signaling factors such as transcription factor (TF) proteins or MAP Kinases and the like in the primed plant or plant tissue compared to non-primed plants or plant tissues. Upon subsequent exposure of the plant or plant tissue to stress such as pest or pathogen attack, these inactive TF proteins become active and regulate gene expression of defense genes, such that a faster and/or stronger defense response is mounted by primed plants or tissues compared to unprimed plants or tissues. Priming may for the application at hand additionally be understood as a constitutive activation of the respective defense mechanism.
The term “priming of the ethylene signaling pathway” means that the effect of priming is achieved by sensitization of the ethylene signaling pathway as shown in FIG. 1 which leads to a faster and stronger defense response of the ethylene dependent defense mechanisms of the plant or plant tissue. The sensitization of the ethylene signaling pathway may be achieved by enhancing the expression of Pti4, Pti5, ERF1 and/or ERF2 protein and/or by suppression of expression of CTR1, EBF1 and/or EBF2 protein.
“Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and/or enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar functional activity as the unmodified protein from which they are derived.
“Homologues” of a nucleic acid encompass nucleotides and/or polynucleotides having nucleic acid substitutions, deletions and/or insertions relative to the unmodified nucleic acid in question, wherein the protein coded by such nucleic acids has similar or higher functional activity as the unmodified protein coded by the unmodified nucleic acid from which they are derived. In particular homologues of a nucleic acid encompass substitutions on the basis of the degenerative amino acid code.
A “deletion” refers to removal of one or more amino acids from a protein or to the removal of one or more nucleic acids from DNA, ssRNA and/or dsRNA.
An “insertion” refers to one or more amino acid residues or nucleic acid residues being introduced into a predetermined site in a protein or the nucleic acid.
A “substitution” refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or beta-sheet structures).
On the nucleic acid level a substitution refers a replacement of nucleic acid with other nucleic acids, wherein the protein coded by the modified nucleic acid has a similar function. In particular homologues of a nucleic acid encompass substitutions on the basis of the degenerative amino acid code.
Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the protein and may range from 1 to 10 amino acids; insertions or deletion will usually be of the order of about 1 to 10 amino acid residues. The amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds) and Table 1 below).







TABLE 1







Examples of conserved amino acid substitutions











Conservative



Residue
Substitutions







Ala
Ser



Arg
Lys



Asn
Gln; His



Asp
Glu



Gln
Asn



Cys
Ser



Glu
Asp



Gly
Pro



His
Asn; Gln



Ile
Leu, Val



Leu
Ile; Val



Lys
Arg; Gln



Met
Leu; Ile



Phe
Met; Leu; Tyr



Ser
Thr; Gly



Thr
Ser; Val



Trp
Tyr



Tyr
Trp; Phe



Val
Ile; Leu










Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation.
Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making 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, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
Orthologues and paralogues encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through specification, and are also derived from a common ancestral gene.
The term “domain” refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein.
Specialist databases exist 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., pp 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): 276-280 (2002)). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics 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 may also be identified using routine techniques, such as by sequence alignment.
Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that 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 percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).
As used herein the terms “soybean rust-resistance”, “resistant to a soybean rust”, “soybean rust-resistant”, “rust-resistance”, “resistant to a rust”, “rust-resistant”, “fungal-resistance”, “resistant to a fungus” and/or “fungal-resistant” mean reducing or preventing an infection by Phacosporacea, in particular Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur) also known as soybean rust or Asian Soybean Rust (ASR). The term “resistance” refers to soybean resistance. Resistance does not imply that the plant necessarily has 100% resistance to infection. In preferred embodiments, the resistance to infection by soy bean rust 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 a similar, more preferably identical, genotype as the plant having increased resistance to the 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.
The terms “soybean rust-resistance”, “resistant to a soybean rust”, “soybean rust-resistant”, “rust-resistance”, “resistant to a rust”, “rust-resistant”, fungal-resistance, resistant to a fungus” and/or “fungal-resistant” as used herein refers to the ability of a plant, as compared to a wild type plant, to avoid infection by fungal pathogens of the family Phacosporaceae, for example of the genus Phacospora, such as Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as soybean rust, to kill rust, to hamper, to reduce, to delay, to stop the development, growth and/or multiplication of soybean rust. The level of fungal resistance of a plant can be determined in various ways, e.g. by scoring/measuring the infected leaf area in relation to the overall 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 (q) PCR. Specific probes and primer sequences for most fungal pathogens are available in the literature (Frederick R D, Snyder C L, Peterson G L, 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, the soybean rust resistance is nonhost-resistance. Nonhost-resistance 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 strains of the soybean rust pathogen, preferably the strains of Phakopsora pachyrhizi. 
The term “hybridization” as used herein includes “any process by which a strand of nucleic acid molecule joins with a complementary strand through base pairing.” (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid molecules) is impacted by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acid molecules. As used herein, the term “Tm” is used in reference to the “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 may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid molecule is in aqueous solution at 1 M NaCl [see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)]. Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of Tm. Stringent 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.
In particular, the term stringency conditions refers to conditions, wherein 100 contiguous nucleotides or more, 150 contiguous nucleotides or more, 200 contiguous nucleotides or more or 250 contiguous nucleotides or more which are a fragment or identical to the complementary nucleic acid molecule (DNA, RNA, ssDNA orssRNA) hybridizes under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. or 65° C., preferably at 65° C., with a specific nucleic acid molecule (DNA; RNA, ssDNA or ss RNA). Preferably, the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C. or 65° C., preferably 65° C., more preferably the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C. or 65° C., preferably 65° C. Preferably, the complementary nucleotides hybridize with a fragment or the whole nucleic acids of the invention. Preferably, the complementary polynucleotide hybridizes with parts of the nucleic acids of the invention capable to provide soybean rust resistance by overexpression or downregulation, respectively.
As used herein, the term “nucleic acid of the invention” or “amino acid of the invention” refers to a gene having 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 coding for a protein having 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 homologues of the nucleic acids of the invention have, at the DNA level and/or protein level, at least 70%, preferably of at least 80%, especially preferably of at least 90%, quite especially preferably of at least 95%, quite especially preferably of at least 98%, 99% or 100% identity over the entire DNA region or protein region given in a sequence specifically disclosed herein and/or a functional fragment thereof.
As used herein, the term “amino acid of the invention” refers to a protein having at least 60% identity to a sequence coding for a protein having 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 have at least 70%, preferably of at least 80%, especially preferably of at least 90%, quite especially preferably of at least 95%, quite especially preferably of at least 98%, 99% or 100% identity over the entire protein region given in a sequence specifically disclosed herein and/or a functional fragment thereof.
“Identity” or “homology” between two nucleic acids and/or refers in each case over the entire length of the nucleic acid of the invention.
For example the identity may be calculated by means of the Vector NTI Suite 7.1 program of the company Informax (USA) employing the Clustal Method (Higgins D G, Sharp P M. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 April; 5(2):151-1) with the following settings:
Multiple Alignment Parameter:
















Gap opening penalty
10



Gap extension penalty
10



Gap separation penalty range
8



Gap separation penalty
off



% identity for alignment delay
40



Residue specific gaps 
off



Hydrophilic residue gap
off



Transition weighing 
0










Pairwise Alignment Parameter:

















FAST algorithm
on



K-tuple size
1



Gap penalty
3



Window size
5



Number of best diagonals
5










Alternatively the identity may 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 Res 31 (13):3497-500, the web page: http://www.ebi.ac.uk/Tools/clustalw/index.html# and the following settings
















DNA Gap Open Penalty
15.0



DNA Gap Extension Penalty
6.66



DNA Matrix
Identity



Protein Gap Open Penalty 
10.0



Protein Gap Extension Penalty
0.2



Protein matrix
Gonnet



Protein/DNA ENDGAP
−1



Protein/DNA GAPDIST
4










All the nucleic acid sequences mentioned herein (single-stranded and double-stranded DNA and RNA sequences, for example cDNA and mRNA) can be produced in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix. 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 filling of gaps by means of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989), see below.
Sequence identity between the nucleic acid useful according to the present invention and the nucleic acids of the invention may 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 percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., 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 even 100% sequence identity, with the nucleic acids having any of SEQ-ID-No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 is preferred.
The term “plant” is intended to encompass plants at any stage of maturity or development, as well as any tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context. Plant parts include, but are not limited to, plant cells, stems, roots, flowers, ovules, 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 true breeding for an increased resistance to fungal infection as compared to a wild-type variety of the plant seed. As used herein, a “plant cell” includes, but is not limited to, a protoplast, gamete producing cell, and a cell that regenerates into a whole plant. Tissue culture of various tissues of plants and regeneration of plants therefrom is well known in the art and is widely published.
In one embodiment of the present invention the plant is selected from the group consisting of beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, and/or groundnut. Preferably, the plant is a legume, comprising plants of the genus Phaseolus (comprising French bean, dwarf bean, climbing bean (Phaseolus vulgaris), Lima bean (Phaseolus lunatus L.), Tepary bean (Phaseolus acutifolius A. Gray), runner bean (Phaseolus coccineus)); the genus Glycine (comprising Glycine soja, soybeans (Glycine max (L.) Merill)); pea (Pisum) (comprising shelling peas (Pisum sativum L. convar. sativum), also called smooth or round-seeded peas; marrowfat pea (Pisum sativum L. convar. medullare Alef. emend. C. O. Lehm), sugar pea (Pisum sativum L. convar. axiphium Alef emend. C. O. Lehm), also called snow pea, edible-podded 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.), chickpea (Cicer), lentils (Lens) (Lens culinaris Medik.), lupins (Lupinus); vetches (Vicia), field bean, broad bean (Vicia faba), vetchling (Lathyrus) (comprising chickling pea (Lathyrus sativus), heath pea (Lathyrus tuberosus)); genus Vigna (comprising moth bean (Vigna aconitifolia (Jacq.) Maréchal), adzuki bean (Vigna angularis (Willd.) Ohwi & H. Ohashi), urd bean (Vigna mungo (L.) Hepper), mung bean (Vigna radiata (L.) R. Wilczek), bambara groundnut (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 asparagus bean, cowpea, catjang bean)); pigeonpea (Cajanus cajan (L.) Millsp.), the genus Macrotyloma (comprising geocarpa groundnut (Macrotyloma geocarpum (Harms) Maréchal & Baudet), horse bean (Macrotyloma uniflorum (Lam.) Verdc.)); goa bean (Psophocarpus tetragonolobus (L.) DC.), African yam bean (Sphenostylis stenocarpa (Hochst. ex A. Rich.) Harms), Egyptian black bean, dolichos bean, lablab bean (Lablab purpureus (L.) Sweet), yam bean (Pachyrhizus), guar bean (Cyamopsis tetragonolobus (L.) Taub.); and/or the genus Canavalia (comprising jack bean (Canavalia ensiformis (L.) DC.), sword bean (Canavalia gladiata (Jacq.) DC.)).
Reference herein to an “endogenous” nucleic acid of the invention” refers to the gene in question as found in a plant in its natural form (i.e., without there being any human intervention). 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 containing such a transgene may, when compared to the expression of the endogenous gene, encounter a substantial increase of the transgene expression or downregulation of the corresponding endogene respectively. The isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis. A transgenic plant according to the present invention includes a recombinant nucleic acid of the invention integrated at any genetic loci and optionally the plant may also include the endogenous gene within the natural genetic background.
For the purposes of the invention, “recombinant” means with regard to, for example, a nucleic acid sequence, a nucleic acid molecule, an expression cassette or a vector construct comprising any one or more nucleic acids of the invention, all those constructions brought about by man by gentechnological methods in which either

    (a) the sequences of the nucleic acids of the invention or a part thereof, or
    (b) genetic control sequence(s) which is operably linked with 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 or have been modified by man by gentechnological methods. The modification may 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 as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library or the combination with the natural promoter.



In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp.
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—becomes a recombinant expression cassette when this expression cassette is modified by man by non-natural, synthetic (“artificial”) methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350, WO 00/15815 or US200405323. Furthermore, 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—becomes a recombinant expression cassette when this expression cassette is not integrated in the natural genetic environment but in a different genetic environment.
It shall further be noted that in the context of the present invention, the term “isolated nucleic acid” or “isolated protein” may in some instances 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 gentechnical methods.
As used herein, the term “transgenic” preferably refers to any plant, plant cell, callus, plant tissue, or plant part that contains the recombinant construct or vector or expression cassette of the invention or a part thereof which is preferably introduced by non-essentially biological processes, preferably Agrobacteria transformation. The recombinant construct or a part thereof is stably integrated into a chromosome, so that it is passed on to successive generations by clonal propagation, vegetative propagation or sexual propagation. Said successive generations are also transgenic. Essentially biological processes may be crossing of plants and/or natural recombination.
A transgenic plant, plants cell or tissue for the purposes of the invention is thus understood as meaning that the recombinant construct or vector or expression cassette of the invention is integrated into the genome.
Preferably, constructs or 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 not at their natural locus of the genome of the original plant.
Natural locus means the location on a specific chromosome, preferably the location between certain genes, more preferably the same sequence background as in the original plant which is transformed.
Preferably, the transgenic plant, plant cell or tissue thereof expresses the constructs or expression cassettes of the invention.
The term “expression” or “gene expression” means the transcription of a specific gene or specifis genes or specific genetic vector construct. The term “expression” or “gene expression” in particular means the transcription of a gene or genes or genetic vector construct into structural RNA (rRNA, tRNA), a regulatory RNA (e.g. microRNA, siRNA, ta-siRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting RNA product.
The term “increased expression” or “enhanced expression” or “overexpression” or “increase of content” as used herein 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 might also be zero (absence of expression).
Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a nonheterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the protein of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., WO9322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
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 other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region (UTR) and/or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-5 intron 1, 2, and 6, the Bronze-1 intron are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The term “functional fragment” refers to any nucleic acid and/or protein which comprises merely a part of the full length nucleic acid and/or full length protein but still provides the same function, i.e. soybean rust resistance 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.
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 90% of the nucleotides of the respective nucleic acid of the invention to the respective nucleic acid of the invention.
In cases where overexpression of nucleic acid of the invention is desired, the term “similar functional activity” or “similar function” means that any homologue and/or fragment provide soybean rust resistance 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 higher of the soybean rust resistance compared with functional activity provided by the 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.
The term “increased activity” as used herein means any protein having increased activity provides an increased soybean rust resistance compared with the wildtype plant merely expressing the respective endogenous nucleic acid of the invention. As far as overexpression is concerned, for the purposes of this invention, the original wild-type expression level might also be zero (absence of expression).
“Repress” or “downregulate” or “suppress” the expression of a nucleic acid molecule in a plant cell are used equivalently herein and mean that the level of expression of the nucleic acid molecule or the level of protein activity of the protein encoded by the nucleic acid molecule in a plant, part of a plant or plant cell after applying a method of the present invention is lower than its expression in the plant, part of the plant or plant cell before applying the method, or compared to a reference plant lacking a recombinant nucleic acid molecule of the invention. The term “repressed” or “downregulated” or “suppressed” as used herein are synonymous and means herein lower, preferably significantly lower expression of the nucleic acid molecule to be expressed or activity of the protein to be expressed. As used herein, a “repression” or “downregulation” or “suppression” of the level of an agent such as a protein, mRNA or RNA means that the level is reduced relative to a substantially identical plant, part of a plant or plant cell grown under substantially identical conditions, lacking a recombinant nucleic acid molecule of the invention, for example lacking the region complementary to at least a part of the precursor molecule of the srRNA, the recombinant construct or recombinant vector of the invention. As used herein, “repression” or “downregulation” or “suppression” of the level of an agent, such as for example a preRNA, mRNA, rRNA, tRNA, snoRNA, snRNA expressed by the target gene and/or of the protein product encoded by it, means that the amount is reduced 10% or more, for example 20% or more, preferably 30% or more, more preferably 50% or more, even more preferably 70% or more, most preferably 80% or more for example 90% relative to a cell or organism lacking a recombinant nucleic acid molecule of the invention. The repression or downregulation can be determined by methods with which the skilled worker is familiar. Thus, the downregulation, repression or suppression of the nucleic acid or protein or protein activity quantity can be determined for example by an immunological detection of the protein. Moreover, 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 immunoassays and fluorescence-activated cell analysis (FACS) can be employed 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 the effect on the phenotype of the organism or the cell may also be determined. Methods for determining the protein quantity are known to the skilled worker. Examples, which may be mentioned, are: the micro-Biuret method (Goa J (1953) Scand J Clin Lab Invest 5:218-222), the Folin-Ciocalteau method (Lowry O H et al. (1951) J Biol Chem 193:265-275) or measuring the absorption of CBB G-250 (Bradford M M (1976) Analyt Biochem 72:248-254).
A method for increasing resistance to Phacosporacea, for example soy bean rust wherein the ethylene signaling pathway is primed in comparison to wild-type plants or wild-type plant cells by enhancing the expression of a Pti4, Pti5, ERF1 and/or ERF2 protein or a functional fragment, orthologue, paralogue or homologue thereof is one embodiment of the invention.
A method for increasing resistance to Phacosporacea, for example soy bean rust wherein the priming of the ethylene signaling pathway may be achieved by enhancing the expression of a Pti4, Pti5, ERF1 and/or ERF2 protein or a functional fragment, orthologue, paralogue or homologue thereof wherein the Pti4, Pti5, ERF1 and/or ERF2 protein is encoded by

    
    
        (i) a recombinant nucleic acid having 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, a functional fragment thereof and/or a recombinant nucleic acid capable of hybridizing under stringent conditions with such nucleic acids thereof and/or by
        (ii) a recombinant nucleic acid encoding a protein having at least 60% 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 orthologue and/or a paralogue thereof is a further embodiment of the invention.
    
    


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 having 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 with such nucleic acids thereof and/or
        (ii) a recombinant nucleic acid coding for a protein having 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 orthologue and/or a paralogue thereof

in functional linkage with a promoter;

    
    
    (b) regenerating the plant from the plant cell; and
    (c) expressing said recombinant nucleic acid which codes for a Pti4, Pti5, ERF1 and/or ERF2 protein in an amount and for a period sufficient to generate or to increase soybean rust resistance in said plant.


A recombinant vector construct comprising:

    (a) (i) recombinant nucleic acid having 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, a functional fragment thereof and/or a nucleic acid capable of hybridizing under stringent conditions with such a nucleic acid and/or
    
        (ii) a recombinant nucleic acid coding for a protein having 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 orthologue and/or a paralogue thereof

operably linked with

    
    
    (b) a promoter and
    (c) a transcription termination sequence is a further embodiment of the invention.


As used herein the term “target nucleic acid” preferably refers to a DNA-molecule capable to prevent the expression, reduce the amount and/or function of the plant CTR1, EBF1 and/or EBF2 gene as for example defined by SEQ ID NO: 9, 11, 13, 15, 17, 19, 21 or 23 in the plant or parts of the plant.
The term “target gene” as used herein refers to a gene the expression of which is to be downregulated or suppressed. In the frame of this application, target genes are preferably plant CTR1, EBF1 and/or EBF2 gene as for example defined by SEQ ID NO: 9, 11, 13, 15, 17, 19, 21 or 23 or homologues, paralogues or functional equivalents thereof.
The present invention provides a method for increasing resistance to fungal pathogens of the family Phacosporaceae, preferably against fungal pathogens of the genus Phacospora, most preferably against Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as soy bean rust in plants and/or plant cells, wherein the ethylene signaling pathway is primed in comparison to wild type plants and/or wild type plant cells by downregulation or suppression of expression of a CTR1, EBF1 and/or an EBF2 protein.
In one embodiment of the invention, the CTR1, EBF1 and/or EBF2 protein is encoded by

    (i) a recombinant nucleic acid having 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% most preferably 99% identity with SEQ ID No. 9, 11, 13, 15, 17, 19, 21 or 23, a functional fragment thereof and/or a recombinant nucleic acid capable of hybridizing under stringent conditions with such nucleic acids thereof and/or by
    (ii) a recombinant nucleic acid encoding a protein having 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% or at least 98% most preferably 99% homology with SEQ ID No. 10, 12, 14, 16, 18, 20, 22 or 24, a functional fragment thereof, an orthologue and/or a paralogue thereof.


A method for increasing resistance to fungal pathogens of the family Phacosporaceae, preferably against fungal pathogens of the genus Phacospora, most preferably against Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur), also known as soy bean rust in plants and/or plant cells, wherein the ethylene signaling pathway is primed in comparison to wild type plants and/or wild type plant cells by downregulation or suppression of expression of a CTR1, EBF1 and/or an EBF2 protein is comprising the steps of

    a) providing a recombinant nucleic acid comprising a target nucleic acid that is substantial identical and/or substantially complementary to at least 19 contiguous nucleotides of the target CTR1, EBF1 and/or EBF2 gene or a homolog, paralogue or ortholog thereof as defined above,
    b) introducing said recombinant nucleic acid into in the plant and/or part thereof is a further embodiment of the invention


It is a further embodiment of the invention, that in the method as defined above, the recombinant nucleic acid is able to provide dsRNA and/or si-RNA and/or miRNA in the plant, a part thereof, once 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 miRNA are substantially complementary to the target CTR1, EBF1 and/or EBF2 gene.
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, operably linked to a target nucleic acid which is substantial 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 CTR1, EBF1 and/or EBF2 gene and which, when it is transcribed, generates RNA comprising a first strand having 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 CTR1, EBF1 and/or EBF2 gene and a second strand having a sequence substantially complementary to the first strand or parts thereof, and a terminator regulatory sequence.
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, operably linked to a target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially identical or substantially complementary to at least contiguous 19 preferably at least 20, more preferably at least 21, for example 22 or 23 nucleotides of the target CTR1, EBF1 and/or EBF2 gene, and a terminator regulatory sequence.
Further embodiments of the invention are recombinant vector constructs comprising a recombinant nucleic acid comprising a promoter that is functional in the plant cell, operably linked to a target nucleic acid which 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 CTR1, EBF1 and/or EBF2 gene and a terminator regulatory sequence.
The recombinant vector constructs of the invention as defined above may further comprise a promoter that is functional in the plant cell, operably linked to a target nucleic acid which is substantial 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 CTR1, EBF1 and/or EBF2 gene and which, when it is transcribed, generates RNA comprising a first strand having 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 CTR1, EBF1 and/or EBF2 gene and optionally a second strand having a sequence at substantially complementary to the first strand or parts thereof, and a terminator regulatory sequence.
The present invention provides a method for producing a plant and/or a part thereof resistant to fungal pathogens of the family Phacosporaceae for example soybean rust comprising

    a) providing a recombinant nucleic acid comprising a target nucleic acid that is substantial identical and/or substantial complementary to at least contiguous 19 preferably at least 20, more preferably at least 21, for example 22 or 23 nucleotides of the target sequence of the invention,
    b) introducing said recombinant nucleic acid into in the plant and/or parts thereof,

wherein the introduction of said recombinant nucleic acid results in downregulation or repression of the expression of the respective target gene. Such target genes are preferably CTR1, EBF1 and EBF2 and homologues, paralogues or functional equivalents thereof as for example defined by SEQ ID NO: 9, 11, 13, 15, 17, 19, 21 or 23.



The present invention further provides a vector construct comprising a recombinant nucleic acid comprising a promoter that is functional in the plant cell, operably linked to a target nucleic acid which is substantial identical and/or substantial 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 and a terminator regulatory sequence as well as the use of the vector construct for the transformation of plants or parts thereof to provide plants resistant to fungal pathogens of the family Phacosporaceae for example soybean rust.
The present invention also provides a transgenic plant cell, plants or parts thereof comprising a recombinant nucleic acid comprising a target nucleic acid that is substantial identical and/or substantial complementary, preferably identical or complementary to at least contiguous 19 preferably at least 20, more preferably at least 21, for example 22 or 23 nucleotides of the target gene of the invention. Parts of plants may be plant cells, roots, stems, leaves, flowers and/or seeds.
There is general agreement that in many organisms, including fungi and plants, large pieces of dsRNA complementary to a specific gene are cleaved into 19-24 nucleotide fragments (siRNA) within cells, and that these siRNAs are the actual mediators for silencing the specific target gene. As used herein siRNA refers to 19-24 nucleotide fragments complementary to the respective target gene.
There are several possibilities to provide the siRNA: RNA-interference (RNAi), micro-RNAi (miRNA), sense RNA and/or antisense RNA for downregulation or suppression of the expression of a target gene of the invention.
As used herein, “RNAi” or “RNA interference” refers to the process of sequence-specific post-transcriptional gene silencing, 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 and a second strand that is complementary to the first strand at least partially has to be provided. For this purpose a recombinant nucleic acid is introduced into the plant, which is capable to produce such dsRNA. The target gene-specific dsRNA is produced and processed into relatively small fragments (siRNAs). miRNA refers to a similar process, except that the produced dsRNA only partially comprises regions substantially identical to the target-gene (at least 19 contiguous nucleotides).
As used herein, “antisense interference” refers to the process of sequence-specific post-transcriptional gene silencing, probably also mediated by double-stranded RNA (dsRNA). In the antisenseRNA-process, ssRNA comprising a first strand that is substantially complementary to at least 19 contiguous nucleotides of the target gene has to be provided. For this purpose recombinant nucleic acid is introduced into the plant, which is capable to produce such ssRNA. Without to be bound by the theory, it is assumed that this RNA pairs with complementary ssRNA transcribed from the original target gene.
As disclosed herein, 100% sequence identity between the target nucleic acid and the target gene is not required to practice the present invention. Preferably, the target nucleic acid comprises a 19-nucleotide portion which is substantially identical and/or substantially complementary to at least 19 contiguous nucleotides of the target gene. While a target nucleic acid comprising a nucleotide sequence identical and/or identical to a portion of the target gene and/or complementary to the whole sequence and/or a portion of the target gene is preferred for inhibition, the invention can tolerate sequence variations that might be expected due to gene manipulation or synthesis, genetic mutation, strain polymorphism, or evolutionary divergence. Thus the target nucleic acid may also encompass a mismatch 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, so long as the resulting RNA sequence still interferes with the respective target gene function.
Sequence identity between the recombinant nucleic acid useful according to the present invention and the target gene may 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 percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 80% sequence identity, 90% sequence identity, or even 100% sequence identity, between the target nucleic acid and at least 19 contiguous nucleotides of the target gene is preferred. The same preferably applies for the sequence complementarity.
When the target nucleic acid of the invention has a length longer than about 19 nucleotides, for example from about 50 nucleotides to about 500 nucleotides, the corresponding dsRNA provided therefrom will be cleaved randomly to dsRNAs of about 21 nucleotides within the plant cell: the siRNAs. Multiple specialized Dicers in plants may generate siRNAs typically ranging in size from 19nt to 24nt (See Henderson et al., 2006. Nature Genetics 38:721-725.). The cleavage of a longer dsRNA of the invention may yield a pool of 21 mer dsRNAs, derived from the longer dsRNA. The siRNAs may have sequences corresponding to fragments of 19-24 contiguous nucleotides across the entire sequence of the target gene. One of skill in the art would recognize that the siRNA can have a mismatch with the target gene of at least 1, 2, or more nucleotides. Further, these mismatches are intended to be included in the present invention.
In one embodiment the target nucleic acid is substantial identical and/or substantial 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 400 or at least 500 nucleotides to the respective target gene. In particular, the target 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 up to the full length of target nucleic acid are identical and/or complementary and/or identical to the target gene.
Preferably, the recombinant nucleic acid is able to provide dsRNA and/or siRNA and/or miRNA in the plant, a part thereof once the recombinant nucleic acid is expressed in the plant, wherein preferably at least 19 contiguous nucleotides of the dsRNA and/or si RNA and/or miRNA are substantially complementary to the respective target gene.
Generally, the term “substantially identical” or “substantially complementary” preferably refers to DNA and/or RNA which is at least 80% identical or complementary to 19 or more contiguous nucleotides of a specific DNA or RNA sequence of the respective target gene, more preferably, at least 90% identical to 19 or more contiguous nucleotides, and most 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 coding DNA-strand of the respective target gene.
As used herein, the term “substantially identical” or “substantially complementary” as applied to DNA of the recombinant nucleic acid, 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%, 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, being at least about 19, 20, 21, 22, 23, 24, 25, 50, 100, 200, 300, 400, 500, 1000, 1500, consecutive bases or up to the full length of the target gene.
One embodiment according to the present invention, provides a method for producing a plant and/or a part thereof resistant to a fungal pathogen of the family Phacosporaceae, for example soybean rust, wherein the recombinant nucleic acid comprises
a promoter that is functional in the plant cell, operably linked to
a target nucleic acid which is substantial identical and/or substantial complementary, or 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 respective target gene and which, when it is transcribed, generates RNA comprising a first strand having 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 having a sequence substantially complementary to the first strand and/or parts thereof, and

a terminator regulatory sequence.

The first strand and the second strand may at least partially form dsRNA. This technique is also referred to as RNAi.
In another embodiment the target nucleic acid comprises 19 to 24 contiguous nucleotides of the target sequence which 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 not complementary to the target gene. Not-identical means an identity which is lower than 95%, lower that 90%, lower than 80%, lower than 70%, lower than 60% over the whole sequence of the target nucleic acid. Not-complementary means a complementarity which is lower than 95%, lower that 90%, lower than 80%, lower than 70%, lower than 60% over the whole sequence of the target nucleic acid. This technique is also referred to as miRNA.
One embodiment according to the present invention, provides a method for producing a plant and/or a part thereof resistant to a fungal pathogens of the family Phacosporaceae, for example soybean rust, wherein the recombinant nucleic acid comprises
a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary, preferably complementary to at least contiguous 19 preferably at least 20, more preferably at least 21, for example 22 or 23 nucleotides of the target gene, and a terminator regulatory sequence.
Preferably, the first strand generated in the plant forms dsRNA together with a second RNA-strand generated in the plant which is complementary to the first strand. This technique is also referred to as antisense RNA.
The dsRNA of the invention may optionally comprise a single stranded overhang at either or both ends. Preferably, the single stranded overhang comprises at least two nucleotides at the 3′ end of each strand of the dsRNA molecule. The double-stranded structure may be formed by a single self-complementary RNA strand (i.e. forming a hairpin loop) or two complementary RNA strands. When the dsRNA of the invention forms a hairpin loop, it may optionally comprise an intron, as set forth in US 2003/0180945A1 or a nucleotide spacer, which is a stretch of sequence between the complementary RNA strands to stabilize the hairpin transgene in cells. Methods for making various dsRNA molecules are set forth, for example, in WO 99/53050 and in U.S. Pat. No. 6,506,559.
In one embodiment the vector construct comprises
a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which is substantial identical and/or substantial 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 it is transcribed, generates RNA comprising a first strand having 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 having a sequence at substantially complementary to the first strand or parts thereof, and a terminator regulatory sequence.
It is preferred that first strand and the second strand are capable of hybridizing to form dsRNA at least partially.
In another embodiment the vector construct comprises a promoter that is functional in the plant cell, operably linked to a target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary 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 terminator regulatory sequence.
It is preferred that the transcript of the first strand and at least a part of the transcript of the target gene are capable of hybridizing to form dsRNA at least partially.
In one embodiment the vector construct comprises a target nucleic acid comprising 19 to 500 nucleotides. Further variants of the target nucleic acid are defined in the section referring to the method for producing a plant.
With respect to a vector construct and/or the recombinant nucleic acid, the term “operatively linked” is intended to mean that the target nucleic acid is linked to the regulatory sequence, including promoters, terminators, enhancers and/or other expression control elements (e.g., polyadenylation signals), in a manner which allows for expression of the target nucleic acid (e.g., 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, Calif. (1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, Eds. Glick and Thompson, Chapter 7, 89-108, CRC Press: Boca Raton, Fla., including the references therein. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of dsRNA desired, and the like. The vector constructs of the invention can be introduced into plant host cells to thereby produce ssRNA, dsRNA, siRNA and/or mi RNA in order to prevent and/or reduce expression of the respective target gene and thereby increase resistance to fungal pathogens of the family Phacosporaceae, for example soybean rust.
In one embodiment, the vector construct comprises a promoter operatively linked to a target nucleotide that is a template for one or both strands of the ssRNA- or dsRNA molecules at least substantial complementary to 19 contiguous nucleotides of the target gene.
In one embodiment, the nucleic acid molecule further comprises two promoters flanking either end of the nucleic acid molecule, wherein the promoters drive expression of each individual DNA strand, thereby generating two complementary RNAs that hybridize and form the dsRNA. In alternative embodiments, the nucleotide sequence is transcribed into both strands of the dsRNA on one transcription unit, wherein the sense strand is transcribed from the 5′ end of the transcription unit and the antisense strand is transcribed from the 3′ end, wherein the two strands are separated by about 3 to about 500 base pairs, and wherein after transcription, the RNA transcript folds on itself to form a hairpin.
In another embodiment, the vector contains a bidirectional promoter, driving expression of two nucleic acid molecules, whereby one nucleic acid molecule codes for the sequence substantially identical to a portion of a target gene of the invention and the other nucleic acid molecule codes for a second sequence being 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.
In another embodiment, the vector contains two promoters, one mediating transcription of the sequence substantially identical to a portion of a target gene of the invention and another promoter mediating transcription of a second sequence being substantially complementary to the first strand and capable of forming a dsRNA, when both sequences are transcribed. The second promoter might be a different promoter.
A different promoter means a promoter having a different activity in regard to cell or tissue specificity, or showing expression on different inducers for example, pathogens, abiotic stress or chemicals.
Promoters according to the present invention may be constitutive, inducible, in particular pathogen-inducible, developmental stage-preferred, cell type-preferred, tissue-preferred or organ-preferred. Constitutive promoters are active under most conditions. Non-limiting examples of constitutive promoters include the CaMV 19S and 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 figwort mosaic virus 35S promoter, the Smas promoter (Velten et al., 1984, EMBO J. 3:2723-2730), the GRP1-8 promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), promoters from the T-DNA of Agrobacterium, such as mannopine synthase, nopaline synthase, and octopine synthase, the small subunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter, and/or the like. Promoters that express the dsRNA in a cell that is contacted by fungus are preferred. Alternatively, the promoter may drive expression of the dsRNA in a plant tissue remote from the site of contact with the fungus, and the dsRNA may then be transported by the plant to a cell that is contacted by the fungus, in particular cells of, or close by fungal infected sites.
Preferably, the expression vector of the invention comprises a constitutive promoter, root-specific promoter, a pathogen inducible promoter, or a fungal-inducible promoter. A promoter is inducible, if its activity, measured on the amount of RNA produced under control of the promoter, is at least 30%, 40%, 50% preferably at least 60%, 70%, 80%, 90% more preferred at least 100%, 200%, 300% higher in its induced state, than in its un-induced state. A promoter is cell-, tissue- or organ-specific, if its activity, measured on the amount of RNA produced under control of the promoter, is at least 30%, 40%, 50% preferably at least 60%, 70%, 80%, 90% more preferred at least 100%, 200%, 300% higher in a particular cell-type, tissue or organ, then in other cell-types or tissues of the same plant, preferably the other cell-types or tissues are cell types or tissues of the same plant organ, e.g. a root. In the case of organ specific promoters, the promoter activity has to be compared to the promoter activity in other plant organs, e.g. leaves, stems, flowers or seeds.
Developmental stage-preferred promoters are preferentially expressed at certain stages of development. Tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds, or xylem. Examples of tissue preferred and organ preferred promoters include, but are not limited to fruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred, integument-preferred, tuber-preferred, stalk-preferred, pericarp-preferred, leaf-preferred, stigma-preferred, pollen-preferred, anther-preferred, a petal-preferred, sepal-preferred, pedicel-preferred, silique-preferred, stem-preferred, root-preferred promoters and/or the like. Seed preferred promoters are preferentially expressed during seed development and/or germination. For example, seed preferred promoters can be embryo-preferred, endosperm preferred and seed coat-preferred. See Thompson et al., 1989, BioEssays 10:108. Examples of seed preferred promoters include, but are not limited to cellulose synthase (celA), Cim1, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1) and/or the like.
Other suitable tissue-preferred or organ-preferred promoters include, but are not limited to, the napin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al., 1991, Mol Gen Genet. 225(3):459-67), the oleosin-promoter from Arabidopsis (PCT Application No. WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoter from Brassica (PCT Application No. WO 91/13980), or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2(2):233-9), as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice, etc. Suitable promoters to note are the Ipt2 or Ipt1-gene promoter from barley (PCT Application No. WO 95/15389 and PCT Application No. WO 95/23230) or those described in PCT Application No. WO 99/16890 (promoters from the barley hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oat glutelin gene, Sorghum kasirin-gene, and/or rye secalin gene)
Promoters useful according to the invention include, but are not limited to, are the major chlorophyll a/b binding protein promoter, histone promoters, the Ap3 promoter, the β-conglycin promoter, the napin promoter, the soybean lectin promoter, the maize 15 kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, the g-zein promoter, the waxy, shrunken 1, shrunken 2, bronze promoters, the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonase promoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546), the SGB6 promoter (U.S. Pat. No. 5,470,359), as well as synthetic or other natural promoters.
Epidermisspezific promotors may 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., Ammitzböll 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; Klöti A., Henrich C., Bieri S., He X., Chen G., Burkhardt P. K., Wünn J., Lucca P., Hohn T., Potrykus I. and Fütterer J., PMB 40, 249 (1999);
Chitinase ChtC2-Promotor from potato (Ancillo et al., Planta. 217(4), 566, (2003));
AtProT3 Promotor (Grallath et al., Plant Physiology. 137(1), 117 (2005));
SHN-Promotors from Arabidopsis (AP2/EREBP transcription factors involved in cutin and wax production) (Aarón et al., Plant Cell. 16(9), 2463 (2004)); and/or
GSTA1 from wheat (Dudler et al., WP2005306368 and Altpeter et al., Plant Molecular Biology. 57(2), 271 (2005)).
Mesophyllspezific promotors may 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., Plant J. 20, 541 (1999);
TaFBPase, acc. X53957;
TaWIS1, acc. AF467542; US 200220115849;
HvBIS1, acc. AF467539; US 200220115849;
ZmMIS1, acc. AF467514; US 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).
Constitutive promotors may be selected from the group consisting of

    
    
        PcUbi promoter from parsley (WO 03/102198)
        CaMV 35S promoter: Cauliflower Mosaic Virus 35S promoter (Benfey et al. 1989 EMBO J. 8(8): 2195-2202),
        STPT promoter: Arabidopsis thaliana Short Triose phosphat translocator promoter (Accession 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: Glycine max translation elongation factor EF1 alpha (US 20090133159).
    
    


The skilled person is aware, that the methods of the invention for upregulation of Pti4, Pti5, ERF1 and/or ERF2 as defined above and downregulation of CTR1, EBF1 and/or an EBF2 as defined above to increase Phacosporacea, for example soybean rust resistance in a plant by priming the ethylene signaling pathway may be combined and 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 downregulation of at least one of CTR1, EBF1 and/or an EBF2.
One type of vector construct is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vector constructs are capable of autonomous replication in a host plant cell into which they are introduced. Other vector constructs are integrated into the genome of a host plant cell upon introduction into the host cell, and thereby are replicated along with the host genome. In particular the vector construct is capable of directing the expression of gene to which the vectors is operatively linked. However, the invention is intended to include such other forms of expression vector constructs, such as viral vectors (e.g., potato virus X, tobacco rattle virus, and/or Gemini virus), which serve equivalent functions.
According to the present invention the target nucleic acid is capable to reduce the protein quantity or function of any of the proteins of the invention in plant cells. In preferred embodiments, the decrease in the protein quantity or function of the target protein takes place in a constitutive or tissue-specific manner. In especially preferred embodiments, an essentially pathogen-induced decrease in the protein quantity or protein function takes place, for example by recombinant expression of the target nucleic acid under the control of a fungal-inducible promoter. In particular, the expression of the target nucleic acid takes place on fungal infected sites, where, however, preferably the expression of the target nucleic acid sequence remains essentially unchanged in tissues not infected by fungus. In preferred embodiments, the protein amount of 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 in comparison to a wild type plant that is not transformed with the target nucleic acid. Preferably the wild type plant is a plant of a similar, more preferably identical genotype as the plant transformed with the target nucleic acid.
The term “introduction” or “transformation” as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
The term “terminator” encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3′ processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
The transgenic plant cells may be transformed with one of the above described vector constructs. Suitable methods for transforming or transfecting host cells including plant cells are well known in the art of plant biotechnology. Any method may be used to transform the recombinant expression vector into plant cells to yield the transgenic plants of the invention. General methods for transforming dicotyledonous plants are disclosed, for example, in U.S. Pat. Nos. 4,940,838; 5,464,763, and the like. Methods for transforming specific dicotyledonous plants, for example, cotton, are set forth in U.S. Pat. Nos. 5,004,863; 5,159,135; and 5,846,797. Soy transformation methods are set forth in U.S. Pat. Nos. 4,992,375; 5,416,011; 5,569,834; 5,824,877; 6,384,301 and in EP 0301749B1 may be used. Transformation methods may include direct and indirect methods of transformation. Suitable direct methods include polyethylene glycol induced DNA uptake, liposome-mediated transformation (U.S. Pat. No. 4,536,475), biolistic methods using the gene gun (Fromm M E et al., Bio/Technology. 8(9):833-9, 1990; Gordon-Kamm et al. Plant Cell 2:603, 1990), electroporation, incubation of dry embryos in DNA-comprising solution, and microinjection. In the case of these direct transformation methods, the plasmids used need not meet any particular requirements. Simple plasmids, such as those of the pUC series, pBR322, M13mp series, pACYC184 and the like can be used. If intact plants are to be regenerated from the transformed cells, an additional selectable marker gene is preferably located on the plasmid. The direct transformation techniques are equally suitable for dicotyledonous and monocotyledonous plants.
Transformation can also be carried out by bacterial infection by means of Agrobacterium (for example EP 0 116 718), viral infection by means of viral vectors (EP 0 067 553; U.S. Pat. No. 4,407,956; WO 95/34668; WO 93/03161) or by means of pollen (EP 0 270 356; WO 85/01856; U.S. Pat. No. 4,684,611). Agrobacterium based transformation techniques (especially for dicotyledonous plants) are well known in the art. The Agrobacterium strain (e.g., Agrobacterium tumefaciens or Agrobacterium rhizogenes) comprises a plasmid (Ti or Ri plasmid) and a T-DNA element which is transferred to the plant following infection with Agrobacterium. The T-DNA (transferred DNA) is integrated into the genome of the plant cell. The T-DNA may be localized on the Ri- or Ti-plasmid or is separately comprised in a so-called binary vector. Methods for the Agrobacterium-mediated transformation are described, for example, in Horsch R B et al. (1985) Science 225:1229. The Agrobacterium-mediated transformation is best suited to dicotyledonous plants but has also been adapted to monocotyledonous plants. The transformation of plants by Agrobacteria is described in, for example, White F F, 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 may result in transient or stable transformation and expression. Although a nucleotide sequence of the present invention can be inserted into any plant and plant cell falling within these broad classes, it is particularly useful in crop plant cells.
The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.
Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
Harvestable parts of the transgenic plant according to the present invention are part of the invention. The harvestable parts may be seeds, roots, leaves and/or flowers comprising the SMT1-gene, the complementary SMT1-gene and/or a part thereof. Preferred parts of soy plants are soy beans comprising the transgenic SMT1-gene.
Products derived from transgenic plant according to the present invention, parts thereof or harvestable parts thereof are part of the invention. A preferred product is soybean meal or soybean oil.
The present invention also includes methods for the production of 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 thereof, e.g. seeds, of these plants. In a further embodiment the method comprises the steps a) growing the plants of the invention, b) removing the harvestable parts as defined above from the plants and c) producing said product from or by the harvestable parts of the invention.
In one embodiment the method for the production of a product comprises

    a) growing the plants of the invention or obtainable by the methods of invention and
    b) producing said product from or by the plants of the invention and/or parts, e.g. seeds, of these plants.


The product may be produced at the site where the plant has been grown, the plants and/or parts thereof may be removed from the site where the plants have been grown to produce the product. Typically, the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant. The step of growing the plant may be performed only once each time the methods of the invention is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the invention and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants of the invention is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts. Also, the steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally the plants are grown for some time before the product is produced.
In one embodiment the products produced by said methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic and/or pharmaceutical. Foodstuffs are regarded as compositions used for nutrition and/or for supplementing nutrition. Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs.
In another embodiment the inventive methods for the production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
It is possible that a plant product consists of one ore more agricultural products to a large extent.



FIGURES
FIG. 1 shows the 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). Binding of ET leads to inactivation of its receptor and in turn to the deactivation of the Raf-like kinase CTR1. 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 up-regulates the expression of ERF1 (and his homologous/orthologous genes). ERF1 (and other ERF-like transcription factors) activate the expression of ethylene regulated defense genes (e.g. PR proteins etc.).
FIG. 2 shows the scoring system used to determine the level of diseased leaf area of wildtype and transgenic soy plants against the rust fungus P. pachyrhizi. 
FIG. 3 shows the full-length-sequence of the ERF-1-gene from Arabidopsis thaliana having SEQ-ID-No. 1.
FIG. 4 shows the sequence of the ERF-1-protein (SEQ-ID-2).
FIG. 5 shows the result of the scoring of 31 transgenic soy plants expressing the ERF-1 over-expression 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 performed 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 31 soybean T0 plants expressing ERF-1 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. The median of the diseased leaf area is shown in FIG. 5. Overexpression of ERF-1 significantly (p<0.001) reduces the diseased leaf area in comparison to non-transgenic control plants.
FIG. 6 shows the full-length-sequence of the Pti-4-gene from Solanum lycopersicum having SEQ-ID-No. 3.
FIG. 7 shows the sequence of the Pti-4-protein (SEQ-ID-4).
FIG. 8 shows the result of the scoring of 33 transgenic soy plants expressing 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 performed 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 33 soybean T0 plants expressing Pti-4 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. The median of the diseased leaf area is shown in FIG. 8. Overexpression of Pti-4 reduces the diseased leaf area in comparison to non-transgenic control plants.
FIG. 9 shows the full-length-sequence of the Pti-5-gene from Solanum lycopersicum having SEQ-ID-No. 5.
FIG. 10 shows the sequence of the Pti-5-protein (SEQ-ID-6).
FIG. 11 shows the result of the scoring of 34 transgenic soy plants expressing the Pti-5 over-expression 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 performed 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 34 soybean T0 plants expressing Pti-5 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. The median of the diseased leaf area is shown in FIG. 11. Overexpression of Pti-5 significantly reduces (p<0.05) the diseased leaf area in comparison to non-transgenic control plants.
FIG. 12 shows the full-length-sequence of the ERF-2-gene from Arabidopsis thaliana having SEQ-ID-No. 7.
FIG. 13 shows the sequence of the ERF-2-protein (SEQ-ID-8).
FIG. 14 shows the result of the scoring of 29 transgenic soy plants expressing 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 performed 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 29 soybean T0 plants expressing ERF-2 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. The median of the diseased leaf area is shown in FIG. 14. Overexpression of ERF-2 significantly (p<0.01) reduces the diseased leaf area in comparison to non-transgenic control plants.
The full-length-sequence of the CTR-1-gene from Arabidopsis thaliana is defined by SEQ-ID-No. 9.
The sequence of the CTR-1-protein is defined by SEQ-ID-10.
The full-length-sequence of the EBF-1-gene from Arabidopsis thaliana is defined by SEQ-ID-No. 11.
The sequence of the EBF-1-protein is defined by SEQ-ID-12.



EXAMPLES
The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the present invention.
Example 1: General Methods
The chemical synthesis of oligonucleotides can be affected, for example, in the known fashion 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, linking DNA fragments, transformation of E. coli cells, bacterial cultures, phage multiplication and sequence analysis of recombinant DNA, are carried out 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 an MWG-Licor laser fluorescence DNA sequencer following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)).
Example 2: Cloning of Overexpression Vector Constructs
The cDNAs of all genes mentioned in this application were generated by DNA synthesis (Geneart, Regensburg, Germany).
The ERF1 cDNA was synthesized in a way that a EcoRV restriction site is located in front of the start-ATG and a SpeI restriction site downstream of the stop-codon. The synthesized cDNA were digested using the restriction enzymes EcoRV and SpeI (NEB Biolabs) and ligated in a EcoRV/SpeI digested Gateway pENTRY vector (Invitrogen, Life Technologies, Carlsbad, Calif., USA) in a way that the full-length fragment is located in sense direction between the parsley ubiquitine promoter (PcUbi) and a Agrobacterium tOCS terminator.
To obtain the binary plant transformation vector, a triple LR reaction (Gateway system, (Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturers protocol by using a pENTRY-A vector containing a parsley ubiquitine promoter, the above described pENTRY-B vector containing the cDNA and a pENTRY-C vector containing a t-StCatpA terminator. As target a binary pDEST vector was used which is composed 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 border an AHAS selection under control of a PcUbi-promoter (FIG. 4). The recombination reaction was transformed into E. coli (DHSalpha), mini-prepped and screened by specific restriction digestions. A positive clone from the vector construct was sequenced and submitted soy transformation.
The Pti4, Pti5, CTR1 cDNA were synthesized in a way that an attB1-recombination site (Gateway system, (Invitrogen, Life Technologies, Carlsbad, Calif., USA) is located in front of the start-ATG and a attB2 recombination site is located directly downstream of the stop-codon. The synthesized cDNAs were transferred to a pENTRY-B vector by using the BP reaction (Gateway system, (Invitrogen, Life Technologies, Carlsbad, Calif., USA) according to the protocol provided by the supplier. To obtain the binary plant transformation vector, a triple LR reaction (Gateway system, (Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturers protocol by using a pENTRY-A vector containing a parsley ubiquitine promoter, the cDNAs in a pENTRY-B vector and a pENTRY-C vector containing a t-Nos terminator. As target a binary pDEST vector was used which is composed 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 border an AHAS selection under control of a pcUbi-promoter (FIG. 4). The recombination reaction was transformed into E. coli (DHSalpha), mini-prepped and screened by specific restriction digestions. A positive clone from each vector construct was sequenced and submitted soy transformation.
The EFB1 and ERF2 cDNA were synthesized in a way that an EcoRV restriction site is located in front of the start-ATG and a SpeI restriction site downstream of the stop-codon. The synthesized cDNAs were digested using the restriction enzymes EcoRV and SpeI (NEB Biolabs) and ligated in a EcoRV/SpeI digested Gateway pENTRY vector (Invitrogen, Life Technologies, Carlsbad, Calif., USA) in a way that the full-length fragment is located in sense direction between the parsley ubiquitine promoter (PcUbi) and a Agrobacterium tOCS terminator. To obtain the binary plant transformation vector, a triple LR reaction (Gateway system, (Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturers protocol by using an empty pENTRY-A vector containing no sequence between the recombination sites, the above described pENTRY-B vector containing the cDNAs, and an empty pENTRY-C vector. As target a binary pDEST vector was used which is composed 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 border an AHAS selection under control of a pcUbi-promoter (FIG. 4). The recombination reaction was transformed into E. coli (DHSalpha), mini-prepped and screened by specific restriction digestions. A positive clone from each vector construct was sequenced and submitted soy transformation.
Example 3 Soy Transformation
The expression vector constructs (see example 2) were transformed into soy.
3.1 Sterilization and Germination of Soy Seeds
Virtually any seed of any soy variety can be employed in the method of the invention. A variety of soycultivar (including Jack, Williams 82, and Resnik) is appropriate for soy transformation. Soy seeds were sterilized in a chamber with a chlorine gas produced by adding 3.5 ml 12N HCl drop wise into 100 ml bleach (5.25% sodium hypochlorite) in a desiccator with a tightly fitting lid. After 24 to 48 hours in the chamber, seeds were removed and approximately 18 to 20 seeds were plated on 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 develop, especially secondary and lateral root formation. BAP strengthens the seedling by forming a shorter and stockier seedling.
Seven-day-old seedlings grown in the light (>100 μEinstein/m2s) at 25 degree C. were used for explant material for the three-explant types. At this time, the seed coat was split, and the epicotyl with the unifoliate leaves have grown to, at minimum, the length of the cotyledons. The epicotyl should be at least 0.5 cm to avoid the cotyledonary-node tissue (since soycultivars and seed lots may vary in the developmental time a description of the germination stage is more accurate than a specific germination time).
For inoculation of entire 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.
For method C (see example 3.3.4), the hypocotyl and one and a half or part of both cotyledons were removed from each seedling. The seedlings were then placed on propagation media for 2 to 4 weeks. The seedlings produce several branched shoots to obtain explants from. The majority of the explants originated from the plantlet growing from the apical bud. These explants were preferably used as target tissue.
3.2—Growth and Preparation of Agrobacterium Culture
Agrobacterium cultures were prepared by streaking Agrobacterium (e.g., A. tumefaciens or A. rhizogenes) carrying the desired binary vector (e.g. 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) onto solid YEP growth medium YEP media: 10 g yeast extract. 10 g Bacto Peptone. 5 g NaCl. Adjust pH to 7.0, and bring final volume to 1 liter with H2O, for YEP agar plates add 20 g Agar, autoclave) and incubating at 25.degree C. until colonies appeared (about 2 days). Depending on the selectable marker genes present on the Ti or Ri plasmid, the binary vector, and the bacterial chromosomes, different selection compounds were be used for A. tumefaciens and rhizogenes selection in the YEP solid and liquid media. Various Agrobacterium strains can be used for the transformation method.
After approximately two days, a single colony (with a sterile toothpick) was picked and 50 ml of liquid YEP was inoculated with antibiotics and shaken at 175 rpm (25.degree. C.) until an OD.sub.600 between 0.8-1.0 is reached (approximately 2 d). Working glycerol stocks (15%) for transformation are prepared and one-ml of Agrobacterium stock aliquoted into 1.5 ml Eppendorf tubes then stored at −80.degree C.
The day before 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 shaked overnight at 25.degree. C. until the OD.sub.600 was between 0.8 and 1.0. Before preparing the soyexplants, the Agrobacteria were pelleted by centrifugation for 10 min at 5,500.times.g at 20.degree. C. The pellet was resuspended in liquid CCM to the desired density (OD.sub.600 0.5-0.8) and placed at room temperature at least 30 min before use.
3.3—Explant Preparation and Co-Cultivation (Inoculation)
3.3.1 Method A: Explant Preparation on the Day of Transformation
Seedlings at this time had elongated epicotyls from at least 0.5 cm but generally between 0.5 and 2 cm. Elongated epicotyls up to 4 cm in length had been successfully employed. 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 apical meristem, and the node located at the first set of leaves was injured with several cuts using a sharp scalpel.
This cutting at the node not only induced Agrobacterium infection but also distributed the axillary meristem cells and damaged pre-formed shoots. After wounding and preparation, the explants were set aside in a Petri dish and subsequently co-cultivated 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 on 15.times.100 mm Petri plates with solid co-cultivation medium. The wounded target tissues were placed such that they are in direct contact with the medium.
3.3.2 Modified Method A: Epicotyl Explant Preparation
Soyepicotyl segments prepared from 4 to 8 d old seedlings were used as explants for regeneration and transformation. Seeds of soyacv L00106CN, 93-41131 and Jack were germinated in 1/10 MS salts or a similar composition medium with or without cytokinins for 4.about.8 d. Epicotyl explants were prepared by removing the cotyledonary node and stem node from the stem section. The epicotyl was cut into 2 to 5 segments. Especially preferred are segments attached to the primary or higher node comprising axillary meristematic tissue.
The explants were used for Agrobacterium infection. Agrobacterium AGL1 harboring a plasmid with the construct of the invention and the AHAS, bar or dsdA selectable marker gene was cultured in LB medium with appropriate antibiotics overnight, harvested and resuspended in a inoculation medium with acetosyringone. Freshly prepared epicotyl segments 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 on a co-culture medium with L-cysteine and TTD and other chemicals such as acetosyringone for enhancing T-DNA delivery for 2 to 4 d. The infected epicotyl explants were then placed on a shoot induction medium with selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene), or D-serine (for dsdA gene). The regenerated shoots were subcultured on elongation medium with the selective agent.
For regeneration of transgenic plants the segments were then cultured on a medium with cytokinins such as BAP, TDZ and/or Kinetin for shoot induction. After 4 to 8 weeks, the cultured tissues were transferred to a medium with lower concentration of cytokinin for shoot elongation. Elongated shoots were transferred to a medium with auxin for rooting and plant development. Multiple shoots were regenerated.
Many stable transformed sectors showing strong expression of the construct of the invention were recovered. Soyplants were regenerated from epicotyl explants. Efficient T-DNA delivery and stable transformed sectors were demonstrated.
3.3.3 Method B: Leaf Explants
For the preparation of the leaf explant the cotyledon was removed from the hypocotyl. The cotyledons were separated from one another and the epicotyl is 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 such that the axillary meristems were included on the explant. To wound the explant as well as to stimulate de novo shoot formation, any pre-formed shoots were removed and the area between the stipules was cut with a sharp scalpel 3 to 5 times.
The explants are either completely immersed or the wounded petiole end dipped into the Agrobacterium suspension immediately after explant preparation. After inoculation, the explants are blotted onto sterile filter paper to remove excess Agrobacterium culture and place explants with the wounded side in contact with a round 7 cm Whatman paper overlaying the solid CCM medium (see above). This filter paper prevents A. tumefaciens overgrowth on the soyexplants. Wrap five plates with ParafilmTM “M” (American National Can, Chicago, Ill., USA) and incubate for three to five days in the dark or light at 25.degree. C.
3.3.4 Method C: Propagated Axillary Meristem
For the preparation of the propagated axillary meristem explant propagated 3-4 week-old plantlets were used. Axillary meristem explants can be pre-pared from the first to the fourth node. An average of three to four explants could be obtained from each seedling. The explants were pre-pared from plantlets by cutting 0.5 to 1.0 cm below the axillary node on the internode and removing the petiole and leaf from the explant. The tip where the axillary meristems lie was cut with a scalpel to induce de novo shoot growth and allow access of target cells to the Agrobacterium. Therefore, a 0.5 cm explant included the stem and a bud.
Once cut, the explants were immediately placed in the Agrobacterium suspension for 20 to 30 minutes. After inoculation, the explants were blotted onto sterile filter paper to remove excess Agrobacterium culture then placed almost completely immersed in solid CCM or on top of a round 7 cm filter paper overlaying the solid CCM, depending on the Agrobacterium strain. This filter paper prevents Agrobacterium overgrowth on the soyexplants. Plates were wrapped with ParafilmTM “M” (American National Can, Chicago, Ill., USA) and incubated for two to three days in the dark at 25.degree. C.
3.4—Shoot Induction
After 3 to 5 days co-cultivation in the dark at 25.degree. C., the explants were rinsed in liquid SIM medium (to remove excess Agrobacterium) (SIM, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soyusing primary-node explants from seedlings In Vitro Cell. Dev. Biol.—Plant (2007) 43:536-549; to remove excess Agrobacterium) or Modwash medium (1×B5 major salts, 1×B5 minor salts, 1×MSIII iron, 3% Sucrose, 1×B5 vitamins, 30 mM MES, 350 mg/L TimentinTM pH 5.6, WO 2005/121345) and blotted dry on sterile filter paper (to prevent damage especially on the lamina) before placing on the solid SIM medium. The approximately 5 explants (Method A) or 10 to 20 (Methods B and C) explants were placed such that the target tissue was in direct contact with the medium. During the first 2 weeks, the explants could be cultured with or without selective medium. Preferably, explants were transferred onto SIM without selection for one week.
For leaf explants (Method B), the explant should be placed into the medium such that it is perpendicular to the surface of the medium with the petiole imbedded into the medium and the lamina out of the medium.
For propagated axillary meristem (Method C), the explant was placed into the medium such that it was parallel to the surface of the medium (basipetal) with the explant partially embedded into the medium.
Wrap plates with Scotch 394 venting tape (3M, St. Paul, Minn., USA) were placed in a growth chamber for two weeks with a temperature averaging 25.degree. C. under 18 h light/6 h dark cycle at 70-100 .mu.E/m.sup.2s. The explants remained on the SIM medium with or without selection until de novo shoot growth occurred at the target area (e.g., axillary meristems at the first node above the epicotyl). Transfers to fresh medium can occur during this time. Explants were transferred from the SIM with or without selection to SIM with selection after about one week. At this time, there was considerable de novo shoot development at the base of the petiole of the leaf explants in a variety of SIM (Method B), at the primary node for seedling explants (Method A), and at the axillary nodes of propagated explants (Method C).
Preferably, all shoots formed before transformation were removed up to 2 weeks after co-cultivation to stimulate new growth from the meristems. This helped to reduce chimerism in the primary transformant and increase amplification of transgenic meristematic cells. During this time the explant may or may not be cut into smaller pieces (i.e. detaching the node from the explant by cutting the epicotyl).
3.5—Shoot Elongation
After 2 to 4 weeks (or until a mass of shoots was formed) on SIM medium (preferably with selection), the explants were transferred to SEM medium (shoot elongation medium, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soy using primary-node explants from seedlings. In Vitro Cell. Dev. Biol.—Plant (2007) 43:536-549) that stimulates shoot elongation of the shoot primordia. This medium may or may not contain a selection compound.
After every 2 to 3 weeks, the explants were transfer to fresh SEM medium (preferably containing selection) after carefully removing dead tissue. The explants should hold together and not fragment into pieces and retain somewhat healthy. The explants were continued to be transferred until the explant dies or shoots elongate. Elongated shoots >3 cm were removed and placed into RM medium for about 1 week (Method A and B), or about 2 to 4 weeks depending on the cultivar (Method C) at which time roots began to form. In the case of explants with roots, they were transferred directly into soil. Rooted shoots were transferred to soil and hardened in a growth chamber for 2 to 3 weeks before transferring to the greenhouse. Regenerated plants obtained using this method were fertile and produced on average 500 seeds per plant.
Transient expression of the construct of the invention after 5 days of co-cultivation with Agrobacterium tumefaciens was widespread on the seedling axillary meristem explants especially in the regions wounding during explant preparation (Method A). Explants were placed into shoot induction medium without selection to see how the primary-node responds to shoot induction and regeneration. Thus far, greater than 70% of the explants were formed new shoots at this region. Expression of the construct of the invention was stable after 14 days on SIM, implying integration of the T-DNA into the soy genome. In addition, preliminary experiments resulted in the formation of positive shoots expressing the construct of the invention forming after 3 weeks on SIM.
For Method C, the average regeneration time of a soyplantlet using the propagated axillary meristem protocol was 14 weeks from explant inoculation. Therefore, this method has a quick regeneration time that leads to fertile, healthy soyplants.
Example 4: Pathogen Assay
4.1. Recovery of Clones
2-3 clones per T0 event were potted into small 6 cm pots. For recovery the clones were kept for 12-18 days in the Phytochamber (16 h-day-und 8 h-night-Rhythm at a temperature of 16° bis 22° C. und a humidity of 75% were grown).
4.2 Inoculation
The rust fungus is a wild isolate from Brazil. The plants were inoculated with P. pachyrhizi. 
In order to obtain appropriate spore material for the inoculation, soyleaves which had been infected with rust 15-20 days ago, were taken 2-3 days before the inoculation and transferred to agar plates (1% agar in H2O). The leaves were placed with their upper side onto the agar, which allowed the fungus to grow through the tissue and to produce very young spores. For the inoculation solution, the spores were knocked off the leaves and were added to a Tween-H2O solution. The counting of spores was performed under a light microscope by means of a Thoma counting chamber. For the inoculation of the plants, the spore suspension was added into a compressed-air operated spray flask and applied uniformly onto the plants or the leaves until the leaf surface is well moisturized. For macroscopic assays we used a spore density of 1-5×105 spores/ml. For the microscopy, a density of >5×105 spores/ml is used. The inoculated plants were placed for 24 hours in a greenhouse chamber with an average of 22° C. and >90% of air humidity. The following cultivation was performed in a chamber with an average of 25° C. and 70% of air humidity.
Example 5 Microscopical Screening
For the evaluation of the pathogen development, the inoculated leaves of plants were stained with aniline blue 48 hours after infection.
The aniline blue staining serves for the detection of fluorescent substances. During the defense reactions in host interactions and non-host interactions, substances such as phenols, callose or lignin accumulated or were produced and were incorporated at the cell wall either locally in papillae or in the whole cell (hypersensitive reaction, HR). Complexes were formed in association with aniline blue, which lead e.g. in the case of callose to yellow fluorescence. The leaf material was transferred to falcon tubes or dishes containing destaining solution II (ethanol/acetic acid 6/1) and was incubated in a water bath at 90° C. for 10-15 minutes. The destaining solution II was removed immediately thereafter, and the leaves were washed 2× with water. For the staining, the leaves were incubated for 1.5-2 hours in staining solution II (0.05% aniline blue=methyl blue, 0.067 M di-potassium hydrogen phosphate) and analyzed by microscopy immediately thereafter.
The different interaction types were evaluated (counted) by microscopy. An Olympus UV microscope BX61 (incident light) and a UV Longpath filter (excitation: 375/15, Beam splitter: 405 LP) are used. After aniline blue staining, the spores appeared blue under UV light. The papillae coul be recognized beneath the fungal appressorium by a green/yellow staining. The hypersensitive reaction (HR) was characterized by a whole cell fluorescence.
Example 6 Evaluating the Susceptibility to Soybean Rust
The progression of the soybean rust disease was scored by the estimation of the diseased area (area which was covered by sporulating uredinia) on the backside (abaxial side) of the leaf. Additionally the yellowing of the leaf was taken into account. (for scheme see FIG. 2)
6.1 Overexpression of ERF-1
T0 soybean plants expressing ERF-1 protein were inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of 31 T0 soybean plants were scored 14 days after inoculation.
The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 31 soybean T0 plants expressing ERF-1 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants were used as control. The median of the diseased leaf area is shown in FIG. 5 for plants expressing recombinant ERF-1 compared with wildtype plants. Overexpression of ERF-1 reduces the diseased leaf area in comparison to non-transgenic control plants by 40%. This data clearly indicate that the in planta expression of the ERF-1 expression vector construct lead to a lower disease scoring of transgenic plants compared to non-transgenic controls. So, the expression of ERF-1 and therefore the priming of the ethylene signaling pathway in soy enhances the resistance of soy against soybean rust.
6.2 Overexpression of Pti-4
T0 soybean plants expressing Pti-4 protein were inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of 33 T0 soybean plants were scored 14 days after inoculation.
The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 31 soybean T0 plants expressing Pti-4 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants were used as control. The median of the diseased leaf area is shown in FIG. 8 for plants expressing recombinant Pti-4 compared with wildtype plants. Overexpression of Pti-4 reduces the diseased leaf area in comparison to nontransgenic control plants by 28%. This data clearly indicate that the in planta expression of the Pti-4 expression vector construct lead to a lower disease scoring of transgenic plants compared to non-transgenic controls. So, the expression of Pti-4 and therefore the priming of the ethylene signaling pathway in soy enhances the resistance of soy against soybean rust.
6.3 Overexpression of Pti-5
T0 soybean plants expressing Pti-5 protein were inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of 34 T0 soybean plants were scored 14 days after inoculation.
The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 34 soybean T0 plants expressing Pti-5 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants were used as control. The median of the diseased leaf area is shown in FIG. 11 for plants expressing recombinant Pti-5 compared with wildtype plants. Overexpression of Pti-5 reduces the diseased leaf area in comparison to nontransgenic control plants by 43%. This data clearly indicate that the in planta expression of the Pti-4 expression vector construct lead to a lower disease scoring of transgenic plants compared to non-transgenic controls. So, the expression of Pti-4 and therefore the priming of the ethylene signaling pathway in soy enhance the resistance of soy against soybean rust.
6.4 Overexpression of ERF-2
T0 soybean plants expressing ERF-2 protein were inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of 29 T0 soybean plants were scored 14 days after inoculation.
The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 29 soybean T0 plants expressing ERF-2 (expression checked by RT-PCR) were evaluated in parallel to nontransgenic control plants. Clones from non-transgenic soy plants were used as control. The median of the diseased leaf area is shown in FIG. 14 for plants expressing recombinant ERF-2 compared with wildtype plants. Overexpression of ERF-2 reduces the diseased leaf area in comparison to non-transgenic control plants by 45%. This data clearly indicate that the in planta expression of the ERF-2 expression vector construct lead to a lower disease scoring of transgenic plants compared to non-transgenic controls. So, the expression of ERF-2 and therefore the priming of the ethylene signaling pathway in soy enhance the resistance of soy against soybean rust.
6.5 Overexpression of ET-Pathway Inhibiting Enzymes CTR-1 and EBF-1
T0 soybean plants expressing the ET-pathway inhibiting proteins CTR-1 and EBF-1 were inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of 27 respectively 28 T0 soybean plants were scored 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 27 CTR-1 overexpressing soybean T0 plants and 28 EBF-1 overexpressing soybean T0 were evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants were used as control. Overexpression of the ethylene signaling pathway inhibiting proteins CTR-1 and EBF-1 enhances the diseased leaf area in comparison to non-transgenic control plants. This data clearly indicate that the in planta inhibition of the ET pathway lead to a higher disease scoring of transgenic plants compared to non-transgenic controls. So, the inhibition of the ethylene signaling pathway in soy reduces the resistance of soy against soybean rust.
6.6 Priming of the Ethylene Signaling Pathway by Inhibiting Enzymes CTR-1 by RNAi
Four sets of transgenic T0 soybean plants are produced expressing RNAi constructs targeting GmCTR1a (SEQ ID 13), GmCTR1 b (SEQ ID 15) or GmCTR1c (SEQ ID 17) individually or all three homologous genes respectively. The RNAi constructs are synthesized and subsequently cloned into transformation vectors under the control of constitutive, pathogen inducible, leaf specific and/or epidermis specific promoters. The RNAi constructs are SEQ ID 25, targeting GmCTR1a, SEQ ID 26 targeting GmCTR1b, SEQ ID 27 targeting GmCTR1c and SEQ ID 28 targeting GmCTR1a, b and c.
The transgenic plants are analysed by RTqPCR for downregulation of the respective gene/s. Plants showing strong repression of CTR1 expression are inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of up to 30 T0 soybean plants per construct are scored 14 days after inoculation.
The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves is considered as diseased leaf area. All soybean T0 plants exhibiting repression of CTR1a, b or c or CTR1a and b and c are evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants are used as control. Repression of CTR1 expression reduces the diseased leaf area in comparison to non-transgenic control plants significantly. The repression of CTR1 expression and therefore the priming of the ethylene signaling pathway in soy enhance the resistance of soy against soybean rust.
6.7 Priming of the Ethylene Signaling Pathway by Inhibiting Enzymes CTR-1 by microRNA Expression
Transgenic T0 soybean plants are produced expressing a recombinant microRNA precursor comprising a microRNA targeting all three homologous GmCTR1a (SEQ ID 13), GmCTR1b (SEQ ID 15) and GmCTR1c (SEQ ID 17) genes. The microRNA precursor is synthesized and subsequently cloned into transformation vectors under the control of constitutive, pathogen inducible, leaf specific and/or epidermis specific promoters. The microRNA precursor is shown in SEQ ID 33.
The transgenic plants are analyzed by RTqPCR for downregulation of the CTR1 homologues. Plants showing strong repression of CTR1 expression are inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of up to 30 T0 soybean plants are scored 14 days after inoculation.
The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves is considered as diseased leaf area. All soybean T0 plants exhibiting repression of CTR1 are evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants are used as control. Repression of CTR1 expression reduces the diseased leaf area in comparison to non-transgenic control plants significantly. The repression of CTR1 expression and therefore the priming of the ethylene signaling pathway in soy enhance the resistance of soy against soybean rust.
6.8 Priming of the Ethylene Signaling Pathway by Inhibiting Enzymes EBF-1 by RNAi
Three sets of transgenic T0 soybean plants are produced expressing RNAi constructs targeting GmEBF1a (SEQ ID 19), GmEBF1b (SEQ ID 21) or GmEBF1c (SEQ ID 23) individually. The RNAi constructs are synthesized and subsequently cloned into transformation vectors under the control of constitutive, pathogen inducible, leaf specific and/or epidermis specific promoters. The RNAi constructs are SEQ ID 29, targeting GmEBF1a, SEQ ID 30 targeting GmEBF1b, SEQ ID 31 targeting.
The transgenic plants are analyzed by RTqPCR for downregulation of the respective gene. Plants showing strong repression of expression of the respective GmEBF1 gene are inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of up to 30 T0 soybean plants per construct are scored 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves is considered as diseased leaf area. All soybean T0 plants exhibiting repression of EBF1a, b or c respectively are evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants are used as control. Repression of EBF1 expression reduces the diseased leaf area in comparison to non-transgenic control plants significantly. The repression of EBF1 expression and therefore the priming of the ethylene signaling pathway in soy enhance the resistance of soy against soybean rust.
6.9 Priming of the Ethylene Signaling Pathway by Inhibiting Enzymes EBF-1 by microRNA Expression
Transgenic T0 soybean plants are produced expressing a recombinant microRNA precursor comprising a microRNA targeting all three homologous GmEBF1a (SEQ ID 19), GmEBF1b (SEQ ID 21) and GmEBF1c (SEQ ID 23) genes. The microRNA precursor is synthesized and subsequently cloned into transformation vectors under the control of constitutive, pathogen inducible, leaf specific and/or epidermis specific promoters. The microRNA precursor is shown in SEQ ID 32.
The transgenic plants are analyzed by RTqPCR for downregulation of the EBF1 homologues. Plants showing strong repression of EBF1 expression are inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of up to 30 T0 soybean plants are scored 14 days after inoculation.
The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves is considered as diseased leaf area. All soybean T0 plants exhibiting repression of EBF1 are evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants are used as control. Repression of EBF1 expression reduces the diseased leaf area in comparison to non-transgenic control plants significantly. The repression of EBF1 expression and therefore the priming of the ethylene signaling pathway in soy enhance the resistance of soy against soybean rust.

## Claims

1. A method for increasing soybean rust resistance in a soybean plant comprising:
(a) stably transforming a soybean plant cell with an expression cassette comprising a recombinant nucleic acid coding for a protein having an amino acid sequence having at least 95% identity with SEQ ID NO: 6, wherein the recombinant nucleic acid is in functional linkage with a promoter;
(b) regenerating a soybean plant from the soybean plant cell;
(c) expressing said recombinant nucleic acid; and
(d) contacting the soybean plant with Phakopsora meibomiae and/or Phakopsora pachyrhizi; 
wherein expression of the recombinant nucleic acid leads to increased soybean rust resistance in said plant as compared to a wild type soybean plant.

2. The method of claim 1, wherein the promoter is a constitutive, pathogen-inducible promoter, a mesophyll-specific promoter and/or an epidermis-specific promoter.