Increased resistance by expression of a defense signal multiplier protein

Abstract

The present invention relates to genes, materials and methods for improving plant health, preferably against infection by phythopathogenic microorganisms. Furthermore, the invention pertains to methods and uses of such genes and materials for creating correspondingly beneficial plant cells, plant parts and whole plants, and relates to products obtained from such plants or plant parts.

Description

[0001] Increased resistance by expression of a defense signal multiplier protein

[0002] The present invention relates to genes, materials and methods for improving plant health, preferably against infection by phythopathogenic microorganisms. Furthermore, the invention pertains to methods and uses of such genes and materials for creating correspondingly beneficial plant cells, plant parts and whole plants, and relates to products obtained from such plants or plant parts.

[0003] BACKGROUND OF THE INVENTION

[0004] Plant pathogenic organisms, in particular fungi, have resulted in severe reductions in crop yield in the past, in worst cases leading to famine. Monocultures, in particular, are highly susceptible to an epidemic-like spreading of diseases. To date, the pathogenic organisms have been controlled mainly by using pesticides. Currently, the possibility of directly modifying the genetic disposition of a plant or pathogen is also open to man. Alternatively, naturally occurring fungicides produced by the plants after fungal infection can be synthesized and applied to the plants.

[0005] The cultivation of agricultural crop plants serves mainly for producing foodstuffs for humans and animals. Plant pathogenic organisms and particularly fungi have resulted in severe reductions in crop yield in the past, in worst cases leading to famine. Monocultures in particular, which are routine nowadays, are highly susceptible to an epidemic-like spread 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. Alternatively, natural occurring fungicides produced by the plants after fungal infection can be synthesized and applied to the plants. Resistance generally describes 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 (Schopfer and Brennicke (1999) Pflanzenphysiologie, Springer Verlag, Berlin-Heidelberg, Germany).

[0006] 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 is seriously hampered in development or 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 (mostly by the presence of R genes of the NBS-LRR family, see below). In the latter case, the plant is resistant to the respective pathogen (Schopfer and Brennicke, vide supra). However, this type of resistance is mostly specific for a certain strain or pathogen.

[0007] 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).

[0008] 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. Fungi are distributed worldwide. Approximately 100 000 different fungal species are known to date. Thereof, rusts are of great importance. For example, in most years Asian soybean rust (Phakopsora pachyrhizi) is the most prominent disease in southern Brazil, whereas target spot (caused by Corynespora cassiicola), is becoming a bigger problem in the north-eastern parts of Brazil. In addition, several so called "late cycle" or "final cycle" diseases, such as Septoria brown spot (Septoria glycines) or Cercospora Leaf Blight (Cercospora kukuchii and other related species) are threatening the soybean crop late in the season, when the maturing pods are filled. In contrast to Brazil, soybean in US is mainly endangered by diseases like Frogeye leaf spot disease (Cercospora sojina) and Sudden death syndrome (SDS, Fusarium virguliforme). A more complete overview of soybean diseases and the respective symptoms and disease management can be found in many publications, e.g. Glen L. Hartman et al. (2015), Compendium of Soybean Diseases and Pests.

[0009] In recent years, fungal diseases, particularly soybean rust, became more important in agricultural production. Soybean rust is a disease caused by the biotrophic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They both belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a wide spectrum of leguminosic host plants.

[0010] P. pachyrhizi is the more aggressive pathogen on soybean (Glycine max), and is therefore, at least currently, of great importance for agriculture. P. pachyrhizi can be found in nearly all tropical and subtropical soybean growing regions of the world. P. pachyrhizi is capable of infecting 31 species from 17 families of the Leguminosae in nature and is capable of growing on further 60 species in controlled conditions (Sinclair et al. (eds.), Proceedings of the rust workshop (1995), National Soybeana Research 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. Soybean plants with resistance to the entire spectrum of the isolates are not available.

[0011] Rusts can have a complicated development cycle with up to five different spore stages (spermatium, aecidiospore, uredospore, teleutospore and basidiospore). Specific infection structures are developed for penetration of the plant. 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, hydathodes and wounds, or else they penetrate the plant epidermis directly as the result of mechanical force with the aid of cell wall digesting enzymes. Specific infection structures are developed for penetration of the plant. To counteract, plants have developed physical barriers, such as wax layers, and chemical compounds having antifungal effects to inhibit spore germination, hyphal growth or penetration.

[0012] The soybean rust Phakopsora pachyrhizi directly penetrates the plant epidermis. After growing through the epidermal cell, the fungus reaches the intercellular space of the mesophyll, where the fungus starts to spread through the leaf. To acquire nutrients, the fungus penetrates mesophyll cells and develops haustoria inside the mesophyll cells. During the penetration process the plasma membrane of the penetrated mesophyll cell stays intact. It is a particularly troubling feature of Phakopsora rusts that these pathogens exhibit an immense variability, thereby overcoming novel plant resistance mechanisms and novel fungicide activities within a few years and sometimes already within one Brazilian growing season.

[0013] Biotrophic phytopathogenic fungi depend for their nutrition on the metabolism of living plant cells. This type of fungi belongs to the group of biotrophic fungi, like many rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronospora. Necrotrophic phytopathogenic fungi depend for their nutrition on dead cells of the plants, e.g. species from the genus Fusarium, Rhizoctonia or Mycospaerella. Soybean rust occupies an intermediate position. It it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic. However, after penetration, the fungus changes over to an obligate-biotrophic lifestyle. The subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy are heminecrotrophic.

[0014] 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). Receptor-like kinases (RLKs) are signaling proteins that feature an extracellular domain connected via a transmembrane domain to a cytoplasmic kinase. This architecture indicates that RLKs perceive external signals, transducing them into the cell. In plants, RLKs were first implicated in the regulation of development, in pathogen responses, and in recognition events. (Santiago A Morillo and Frans E Tax (2006) Functional analysis of receptor-like kinases in monocots and dicots. Current Opinion in Plant Biology9:460-469).

[0015] The presence of several hundreds of RLK coding genes in the genomes of crop plants underlines the importance of RLKs in plant development and pathogen defense but also shows that the specific functions of RLKs are very diverse. Currently it is not possible to predict the specific function and / or the recognized molecule of a RLK from their sequence. Only very few of the RLKs are described to be involved in the recognition of conserved structures of microbes (microbe asociated molecular patterns, PAMPs, for review see Thorsten Nurnberger and Birgit Kemmerling (2006) Receptor protein kinases - pattern recognition receptors in plant immunity. TRENDS in Plant Science 11 (11)519ff).

[0016] 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).

[0017] To further multiply defense responses that are elicited by PAMP recognition, plants have developped amplifications systems. For example Arabidopsis thaliana harbours a "Pamp- Induced-Peptide" (PIP1), that is induced strongly after the recognition of a pathogen a PAMP receptor RLK. PIP1 is a secreted peptide which is generated through the proteolysis processing of its precursor prePIPI . By recognition of apoplastic PIP1 by the PAMP-receptor like kinase RLK7 a secondary PAMP defense signalling is initiated, which amplifies the initital defense signal leading to an even stronger induction of phytohormones and subsequent resistance reactions (Hou S, Wang X, Chen D, Yang X, Wang M, Turra D, Di Pietro A, Zhang W. The secreted peptide PIP1 amplifies immunity through receptor-like kinase 7. PLoS Pathog. 2014, https: / / doi.org / 10.1371 / journal.ppat.1004331)

[0018] It was thus the object of the invention to provide materials and methods to improve plant disease resistance, particularly in crops, and preferably also reducing the negative impact on overall plant health and / or yield which the means of obtaining said improved pathogen resistance may entail. In particular, it was a preferred object of the invention to provide materials and methods which lead to plant material of heritably improved resistance against fungal pathogens with minimised reduction of overall plant health, wherein resistance preferably is directed against a rust fungus and most preferably a fungus in the genus Phakopsora, Fusarium, Sclerotinia, Alternaria, Corynespora, Cercospora, or Septoria. SUMMARY OF THE INVENTION

[0019] Accordingly, the invention provides a method for conferring, modifying or increasing resistance of a plant or plant material against at least one biotrophic or heminecrotrophic fungus, preferably against a rust fungus, wherein the plant or plant material preferably is of taxonomic family Fabaceae, more preferably of genus Glycine, and wherein the method comprises a step of increasing the production and / or accumulation of a PIP protein in the plant or plant material in comparison to a respective wild-type plant or plant material.

[0020] The invention also provides a method for production of a genetically modified plant or plant material, preferably of taxonomic family Fabaceae, more preferably of genus Glycine, having increased resistance, compared to a respective wild-type plant or wild-type plant material, against at least one biotrophic or heminecrotrophic fungus, preferably against a rust fungus, comprising the steps of

[0021] 1) introducing an exogenous nucleic acid encoding a PIP protein of the present invention into a plant or plant material;

[0022] 2) generating a genetically modified plant or genetically modified plant material; and

[0023] 3) expressing the PIP protein in the genetically modified plant or genetically modified plant material.

[0024] Furthermore, the invention provides a vector for creating a genetically modified plant, comprising an expression cassette having a PIP gene operably linked to a promoter, wherein the PIP gene codes for a PIP protein of the present invention.

[0025] The invention also provides a plant or plant material, preferably of taxonomic family Fabaceae, more preferably of genus Glycine, wherein the plant or plant material has increased expression, accumulation or activity of a heterologous PIP protein, wherein the PIP protein is a PIP protein of the present invention.

[0026] Thus, the invention also provides a plant or plant material obtainable or obtained by a method according to the present invention.

[0027] And the invention provides non-propagative plant part or material of a plant or plant part according to the invention, preferably a fermentation product, oil, meal, press cake, pomace, chaff, straw or compost.

[0028] Also provided by the invention is a method for producing a population of plants each having an enhanced resistance to at least one biotrophic or heminecrotrophic fungus, preferably a rust fungus, comprising the steps of i) multiplying seed of a plant according to the present invention, wherein the seed are preferably homozygous for the PIP protein of the present invention, and ii) growing plants of the seed obtained in step i), wherein at least 1000 plants are grown.

[0029] The invention also provides a farming method for controlling or reducing at least one biotrophic or heminecrotrophic fungus in a field, preferably by reducing or delaying infection of plants in a field and / or reducing or delaying emission of fungal spores from the field, comprising the step of growing plants according to the present invention on the field.

[0030] And the invention provides a method of assaying a plant for resistance to a fungus, comprising the screening for a) the overexpression PIP protein of the present invention, b) the expression of a heterologous PIP protein of the present invention, or c) the presence of an exogenous nucleic acid coding for a PIP protein of the present invention.

[0031] The invention also provides an automated plant seed selection method, comprising the steps of i) obtaining, for each seed of a plurality of seeds, a sample comprising genetic material of a tissue body representative for said seed, ii) determining the presence of a PIP protein of the present invention, iii) selecting those seed where the determination in step ii) gave a positive result, wherein the seeds are of one or more plants of taxonomic family Fabaceae, more preferably of genus Glycine.

[0032] And the invention provides a use of a PIP protein for conferring, modifying or increasing resistance of a plant or plant material against at least one biotrophic or heminecrotrophic fungus, preferably against a rust fungus, wherein the PIP protein is a PIP protein of the present invention.

[0033] BRIEF DESCRIPTION OF THE FIGURES

[0034] Figure 1 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 (as described in GODOY, C.V., KOGA, L.J. & CANTERI, M.G. Diagrammatic scale for assessment of soybean rust severity. Fitopatologia Brasileira 31 :063-068. 2006.).

[0035] Figure 2 shows the nucleic acid of PrePIPI I PIP1_ARATH

[0036] Figure 3 shows the amino acid sequence of PrePIPI I PIP1_ARATH

[0037] Figure 4 shows the protein sequence of the mature PIP1 protein

[0038] Figure 5 shows the result of the disease scoring of transgenic soy plants expressing PrePIPI under a constitutive parsley ubiquitin promoter in TO generation in greenhouse. On average the constitutive overexpression of PrePIPI significantly (one sided t-test, p=0.007) reduces the diseased leaf area in comparison to non-transgenic control plants by 38,1 %% relative.

[0039] Figure 6 shows the absolute reduction of the Area Under Disease Progression Curve (AUDPC) of 5 independent events in T2 generation (grey bars) expressing PrePIPI constitutively, in comparison to non-transgenic control (set to 0) in field trials. Negative values indicate less disease severity (= higher resistance) of the transgenic events. The asterisc indicates a statistically significant difference between transgenic events (or construct) and the respective wild type control in the various disease ratings over the season (see example 8 for more information about the statistical approach). It is clearly visible that expression of constitutively expressed PrePIPI leads to a significantly reduced soybean rust disease under both conditions.

[0040] Figure 7 shows a plot of positional amino acid conservation. Per column: The number of stars indicates the degree of conservation (more stars indicate higher degree of conservation); the first letter / "-" below the stars is the respective amino acid (or alignment gap) encountered in PIP1_ARATH (“wt”); all letters / "-" below indicate, in decreasing order of frequency, the amino acids, or, indicated by the alignment gaps encountered in homologous PrePIPI proteins.

[0041] Figure 8 shows a multiple alignment of a PIP sequence of the present invention (SEQ ID NO. 1 , ”SEQ1”) and the corresponding sequences of homologs identified by their Uniprot identifier. The amino acid sequence is given only for the top sequence SEQ ID NO. 1 , for every other sequence per position only the differing amino acids or for a gap are indicated ("." denotes "same amino acid as in top sequence"). The numbering is according to the amino acids of the top sequence. A box surrounds the mature part of PIP1_ARATH and the corresponding amino acids in homolog proteins.

[0042] BRIEF DESCRIPTION OF THE SEQUENCES

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] The technical teaching of the invention is expressed herein using the means of language, in particular by use of scientific and technical terms. However, the skilled person understands that the means of language, detailed and precise as they may be, can only approximate the full content of the technical teaching, if only because there are multiple ways of expressing a teaching, each necessarily failing to completely express all conceptual connections, as each expression necessarily must come to an end. With this in mind the skilled person understands that the subject matter of the invention is the sum of the individual technical concepts signified herein or expressed, necessarily in a pars-pro-toto way, by the innate constrains of a written description. In particular, the skilled person will understand that the signification of individual technical concepts is done herein as an abbreviation of spelling out each possible combination of concepts as far as technically sensible, such that for example the disclosure of three concepts or embodiments A, B and C are a shorthand notation of the concepts A+B, A+C, B+C, A+B+C. In particular, fallback positions for features are described herein in terms of lists of converging alternatives or instantiations. Unless stated otherwise, the invention described herein comprises any combination of such alternatives. The choice of more or less preferred elements from such lists is part of the invention and is due to the skilled person’s preference for a minimum degree of realization of the advantage or advantages conveyed by the respective invention. Such multiple combined instantiations represent the adequately preferred form(s) of the invention.

[0045] In so far as recourse herein is made to entries in public databases, for example Uniprot and PFAM, the contents of these entries are those as of 2023-08-18. Unless stated to the contrary, where the entry comprises a nucleic acid or amino acid sequence information, such sequence information is incorporated herein.

[0046] As used herein, terms in the singular and the singular forms like "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, use of the term "a nucleic acid" optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term "probe" optionally (and typically) encompasses many similar or identical probe molecules. Also as used herein, the word "comprising" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0047] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). The term "comprising" also encompasses the term "consisting of'. The term "about", when used in reference to a measurable value, for example an amount of mass, dose, time, temperature, sequence identity and the like, refers to a variation of ± 0.1%, 0.25%, 0.5%, 0.75%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or even 20% of the specified value as well as the specified value. Thus, if a given composition is described as comprising "about 50% X," it is to be understood that, in some embodiments, the composition comprises 50% X whilst in other embodiments it may comprise anywhere from 40% to 60% X (i.e., 50% ± 10%).

[0048] As used herein, the term "gene" refers to a biochemical information which, when materialised in a nucleic acid, can be transcribed into a gene product, i.e. a further nucleic acid, preferably an RNA, and preferably also can be translated into a peptide or polypeptide. The term is thus also used to indicate the section of a nucleic acid resembling said information and to the sequence of such nucleic acid (herein also termed "gene sequence").

[0049] Also as used herein, the term "allele" refers to a variation of a gene characterized by one or more specific differences in the gene sequence compared to the wild type gene sequence, regardless of the presence of other sequence differences. Alleles or nucleotide sequence variants of the invention have at least, in increasing order of preference, 30%, 40%, 50%, 60%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide "sequence identity" to the nucleotide sequence of the wild type gene. Correspondingly, where an "allele" refers to the biochemical information for expressing a peptide or polypeptide, the respective nucleic acid sequence of the allele has at least, in increasing order of preference, 30%, 40%, 50%, 60%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid "sequence identity" to the respective wild type peptide or polypeptide.

[0050] Protein or nucleic acid variants may be defined by their sequence identity when compared to a parent protein or nucleic acid. Sequence identity usually is provided as "% sequence identity" or "% identity". To determine the percent-identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e., a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program "NEEDLE" (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EBLOSUM62). The preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined.

[0051] The following example is meant to illustrate two nucleotide sequences, but the same calculations apply to protein sequences:

[0052] Seq A: AAGATACTG length: 9 bases

[0053] Seq B: GATCTGA length: 7 bases

[0054] Hence, the shorter sequence is sequence B.

[0055] Producing a pairwise global alignment which is showing both sequences over their complete lengths results in

[0056] Seq A : AAGATACTG-

[0057] Seq B : The "I" symbol in the alignment indicates identical residues (which means bases for DNA or amino acids for proteins). The number of identical residues is 6.

[0058] The symbol in the alignment indicates gaps. The number of gaps introduced by alignment within the sequence B is 1 . The number of gaps introduced by alignment at borders of sequence B is 2, and at borders of sequence A is 1 .

[0059] The alignment length showing the aligned sequences over their complete length is 10.

[0060] Producing a pairwise alignment which is showing the shorter sequence over its complete length according to the invention consequently results in:

[0061] Seq A :

[0062] Seq B :

[0063] Producing a pairwise alignment which is showing sequence A over its complete length according to the invention consequently results in:

[0064] Seq A :

[0065] Seq B :

[0066] Producing a pairwise alignment which is showing sequence B over its complete length according to the invention consequently results in:

[0067] Seq A :

[0068] Seq B :

[0069] The alignment length showing the shorter sequence over its complete length is 8 (one gap is present which is factored in the alignment length of the shorter sequence).

[0070] Accordingly, the alignment length showing sequence A over its complete length would be 9 (meaning sequence A is the sequence of the invention), the alignment length showing sequence B over its complete length would be 8 (meaning sequence B is the sequence of the invention).

[0071] After aligning the two sequences, in a second step, an identity value shall be determined from the alignment. Therefore, according to the present description the following calculation of percent- identity applies:

[0072] %-identity = (identical residues I length of the alignment region which is showing the respective sequence of this invention over its complete length) *100. Thus, sequence identity in relation to comparison of two amino acid sequences according to the invention is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give "%-identity". According to the example provided above, %-identity is: for sequence A being the sequence of the invention (6 / 9) * 100 = 66.7 %; for sequence B being the sequence of the invention (6 / 8) * 100 = 75%.

[0073] The term "nucleic acid construct" as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or is synthetic.

[0074] The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a polynucleotide. The term "control sequence" or "genetic control element" is defined herein to include all sequences affecting the expression of a polynucleotide, including but not limited thereto, the expression of a polynucleotide encoding a polypeptide. Each control sequence may be native or foreign to the polynucleotide or native or foreign to each other. Such control sequences include, but are not limited to, promoter sequence, 5’-UTR (also called leader sequence), ribosomal binding site (RBS), 3’-UTR, and transcription start and stop sites.

[0075] The term "functional linkage" or "operably linked" with respect to regulatory elements is to be understood as meaning the sequential arrangement of a regulatory element (including but not limited thereto a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (including but not limited thereto a terminator) in such a way that each of the regulatory elements can fulfil its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. For example, a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

[0076] A "promoter" or "promoter sequence" is a nucleotide sequence located upstream of a gene on the same strand as the gene that enables that gene's transcription. A promoter is generally followed by the transcription start site of the gene. A promoter is recognized by RNA polymerase (together with any required transcription factors), which initiates transcription. A functional fragment or functional variant of a promoter is a nucleotide sequence which is recognizable by RNA polymerase, and capable of initiating transcription.

[0077] As used herein, the term "isolated DNA molecule" refers to a DNA molecule at least partially separated from other molecules normally associated with it in its native or natural state. The term "isolated" preferably refers to a DNA molecule that is at least partially separated from some of the nucleic acids which normally flank the DNA molecule in its native or natural state. Thus, DNA molecules fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques, are considered isolated herein. Such molecules are considered isolated when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules, in that they are not in their native state.

[0078] Any number of methods well known to those skilled in the art can be used to isolate and manipulate a polynucleotide, or fragment thereof, as disclosed herein. For example, polymerase chain reaction (PCR) technology can be used to amplify a particular starting polynucleotide molecule and / or to produce variants of the original molecule. Polynucleotide molecules, or fragment thereof, can also be obtained by other techniques, such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer. A polynucleotide can be single-stranded (ss) or double- stranded (ds). "Doublestranded" refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure, generally under physiologically relevant conditions. Embodiments of the method include those wherein the polynucleotide is at least one selected from the group consisting of sense single- stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), a double-stranded DNA / RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA; a mixture of polynucleotides of any of these types can be used.

[0079] As used herein, "recombinant" when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation. A gene sequence open reading frame is recombinant if (a) that nucleotide sequence is present in a context other than its natural one, for example by virtue of being (i) cloned into any type of artificial nucleic acid vector or (ii) moved or copied to another location of the original genome, or if (b) the nucleotide sequence is mutagenized such that it differs from the wild type sequence. The term recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid is a recombinant plant.

[0080] The term "transgenic" refers to an organism, preferably a plant or part thereof, or a nucleic acid that comprises a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell. A "recombinant" organism preferably is a "transgenic" organism. The term "transgenic" as used herein is not intended to encompass the alteration of the genome (chromosomal or extra- chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as, e.g., self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non- recombinant transposition, or spontaneous mutation.

[0081] As used herein, "mutagenized" refers to an organism or nucleic acid thereof having alteration(s) in the biomolecular sequence of its native genetic material as compared to the sequence of the genetic material of a corresponding wildtype organism or nucleic acid, wherein the alteration(s) in genetic material were induced and / or selected by human action. Examples of human action that can be used to produce a mutagenized organism or DNA include, but are not limited to treatment with a chemical mutagen such as EMS and subsequent selection with herbicide(s); or by treatment of plant cells with x-rays and subsequent selection with herbicide(s). Any method known in the art can be used to induce mutations. Methods of inducing mutations can induce mutations in random positions in the genetic material or can induce mutations in specific locations in the genetic material (i.e., can be directed mutagenesis techniques), such as by use of a genoplasty technique. In addition to unspecific mutations, according to the invention a nucleic acid can also be mutagenized by using mutagenesis means with a preference or even specificity for a particular site, thereby creating an artificially induced heritable allele according to the present invention. Such means, for example site specific nucleases, including for example zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENS) (Malzahn et al., Cell Biosci, 2017, 7:21) and clustered regularly interspaced short palindromic repeats / CRISPR-associated nuclease (CRISPR / Cas) with an engineered crRNA / tracr RNA (for example as a single-guide RNA, or as modified crRNA and tracrRNA molecules which form a dual molecule guide), and methods of using this nucleases to target known genomic locations, are well known in the art (see reviews by Bortesi and Fischer, 2015, Biotechnology Advances 33: 41-52; and by Chen and Gao, 2014, Plant Cell Rep 33: 575-583, and references within).

[0082] Mutations or alterations of amino or nucleic acid sequences can be any of substitutions, deletions or insertions; the terms "mutations" or "alterations" also encompass any combination of these. Hereinafter, all three specific ways of mutating are described in more detail by way of reference to amino acid sequence mutations; the corresponding teaching applies to nucleic acid sequences such that "amino acid" is replaced by "nucleotide". Amino acids and nucleotides are represented herein according to their respective one-letter code or three-letter code designations (see e.g. https: / / www.bioinformatics.org / sms / iupac.html)

[0083] "Substitutions" are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the substituted amino acid. For example, the substitution of histidine at position 120 with alanine is designated as "His120Ala" or"H120A". "Deletions" are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by "*" or Accordingly, the deletion of glycine at position 150 is designated as "Gly150*", "G150*", "Gly150-" or"G150-". Alternatively, deletions are indicated by e.g. "deletion of D183 and G184".

[0084] "Insertions" are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the original amino acid and the additional amino acid. For example, an insertion at position 180 of lysine next to glycine would be designated as "Gly180GlyLys" or"G180GK". When more than one amino acid residue is inserted, such as e.g. a Lys and Ala after Gly180 this may be indicated as: Gly180GlyLysAla or G180GKA. In cases where a substitution and an insertion occur at the same position, this may be indicated as S99SD+S99A or in short S99AD. In cases where an amino acid residue identical to the existing amino acid residue is inserted, it is clear that degeneracy in the nomenclature arises. If for example a glycine is inserted after the glycine in the above example this would be indicated by G180GG.

[0085] Variants comprising multiple alterations are separated by "+", e.g. "Arg170Tyr+Gly195Glu" or "R170Y+G195E" representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple alterations may be separated by space or a comma e.g. R170Y G195E or R170Y, G195E respectively.

[0086] Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g. "Arg170Tyr, Glu" represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Alternatively, different alterations or optional substitutions may be indicated in brackets e.g. Arg170[Tyr, Gly] or Arg170{Tyr, Gly} or in short R170[Y,G] or R170{Y,G}.

[0087] A special aspect concerning amino acid substitutions are conservative mutations which often appear to have a minimal effect on protein folding resulting in substantially maintained peptide or polypeptide properties of the respective peptide or polypeptide variant compared to the peptide or polypeptide properties of the parent peptide or polypeptide. Conservative mutations are those where one amino acid is exchanged with a similar amino acid. For determination of %-similarity the following applies, which is also in accordance with the BLOSUM62 matrix, which is one of the most used amino acids similarity matrix for database searching and sequence alignments:

[0088] Amino acid A is similar to amino acids S

[0089] Amino acid D is similar to amino acids E, N

[0090] Amino acid E is similar to amino acids D, K and Q

[0091] Amino acid F is similar to amino acids W, Y Amino acid H is similar to amino acids N, Y Amino acid I is similar to amino acids L, M and V Amino acid K is similar to amino acids E, Q and Rm Amino acid L is similar to amino acids I, M and V Amino acid M is similar to amino acids I, L and V Amino acid N is similar to amino acids D, H and S Amino acid Q is similar to amino acids E, K and R Amino acid R is similar to amino acids K and Q Amino acid S is similar to amino acids A, N and T Amino acid T is similar to amino acids S

[0092] Amino acid V is similar to amino acids I, L and M Amino acid W is similar to amino acids F and Y Amino acid Y is similar to amino acids F, H and W Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as a peptide or polypeptide. Preferably such mutations are not pertaining the functional domains of a peptide or polypeptide.

[0093] As used herein, a "genetically modified organism" (GMO) is an organism whose genetic characteristics contain alteration(s) that were produced by human effort causing transfection that results in transformation of a target organism with genetic material from another or "source" organism, or with synthetic or modified-native genetic material, or an organism that is a descendant thereof that retains the inserted genetic material. The source organism can be of a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or from the same type of organism (e.g., a GMO plant can contain genetic material from another plant).

[0094] As used herein, "wildtype" or "corresponding wildtype plant" means the typical form of an organism or its genetic material, as it normally occurs, as distinguished from e.g. mutagenized and / or recombinant forms. Similarly, by "control cell", "wildtype" "control plant, plant tissue, plant cell or host cell" is intended a plant, plant tissue, plant cell, or host cell, respectively, that lacks the particular polynucleotide of the invention that are disclosed herein. The use of the term "wildtype" is not, therefore, intended to imply that a plant, plant tissue, plant cell, or other host cell lacks recombinant DNA in its genome, and / or does not possess fungal resistance characteristics that are different from those disclosed herein.

[0095] As used herein, "descendant" refers to any generation plant. A progeny or descendant plant can be from any filial generation, e.g., F1 , F2, F3, F4, F5, F6, F7, etc. In some embodiments, a descendant or progeny plant is a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth generation plant.

[0096] The term "plant" is used herein in its broadest sense as it pertains to organic material and is intended to encompass eukaryotic organisms that are members of the taxonomic kingdom plantae, examples of which include but are not limited to monocotyledon and dicotyledon plants, vascular plants, vegetables, grains, flowers, trees, herbs, bushes, grasses, vines, ferns, mosses, fungi and algae, etc, as well as clones, offsets, and parts of plants used for asexual propagation (e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). Unless stated otherwise, the term "plant" refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and / or progeny of the same. A plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant.

[0097] The invention particularly applies to plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugar cane, sunflower, tomato, squash, tea and algae, amongst others. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include inter alia soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco.

[0098] The plant is preferably a crop plant, more preferably a plant of tribus Phaseoleae, even more preferably of genus Amphicarpaea, Cajanus, Canavalia, Dioclea, Erythrina, Glycine, Arachis, Lathyrus, Lens, Pisum, Vicia, Vigna, Phaseolus or Psophocarpus, even more preferably of species Amphicarpaea bracteata, Cajanus cajan, Canavalia brasiliensis, Canavalia ensiformis, Canavalia gladiata, Dioclea grandiflora, Erythrina latissima, Phaseolus acutifolius, Phaseolus lunatus, Phaseolus maculatus, Psophocarpus tetragonolobus, Vigna angularis, Vigna mungo, Vigna unguiculata, Glycine albicans, Glycine aphyonota, Glycine arenaria, Glycine argyrea, Glycine canescens, Glycine clandestina, Glycine curvata, Glycine cyrtoloba, Glycine dolichocarpa, Glycine falcata, Glycine gracei, Glycine hirticaulis, Glycine lactovirens, Glycine latifolia, Glycine latrobeana, Glycine microphylla, Glycine peratosa, Glycine pindanica, Glycine pullenii, Glycine rubiginosa, Glycine stenophita, Glycine syndetika, Glycine tabacina, Glycine tomentella, Glycine gracilis, Glycine max, Glycine max x Glycine soja, Glycine soja, more preferably of species Glycine gracilis, Glycine max, Glycine max x Glycine soja, Glycine soja, most preferably of soybean. It is a particular advantage of the present invention that the invention provides material and methods to improve resistance of such plants against fungal pathogens, particularly rust pathogens like Phakopsora, which in these plants are difficult to manage using fungicides and which cause severe losses of plant yield and harvest material quality.

[0099] The fungal pathogen according to the invention preferably is a fungus or a fungus-like organism from the phyla Ascomycota, Basisiomycota or Oomycota, more preferably of phylum Basidiomycota, even more preferably of subphylum Pucciniomycotina, even more preferably of class Pucciniomycetes, even more preferably of order Pucciniales, even more preferably of family Chaconiaceae, Coleosporiaceae, Cronartiaceae, Melampsoraceae, Mikronegeriaceae, Phakopsoraceae, Phragmidiaceae, Pileolariaceae, Pucciniaceae, Pucciniastraceae, Pucciniosiraceae, Raveneliaceae, Sphaerophragmiaceae or Uropyxidaceae, even more preferably of genus Rhizoctonia, Maravalia, Ochropsora, Olivea, Chrysomyxa, Coleosporium, Diaphanopellis, Cronartium, Endocronartium, Peridermium, Melampsora, Chrysocelis, Mikronegeria, Arthuria, Batistopsora, Cerotelium, Dasturella, Phakopsora, Prospodium, Arthuriomyces, Catenulopsora, Gerwasia, Gymnoconia, Hamaspora, Kuehneola, Phragmidium, Trachyspora, Triphragmium, Atelocauda, Pileolaria, Racospermyces, Uromycladium, Allodus, Ceratocoma, Chrysocyclus, Cumminsiella, Cystopsora, Endophyllum, Gymnosporangium, Miyagia, Puccinia, Puccorchidium, Roestelia, Sphenorchidium, Stereostratum, Uromyces, Hyalopsora, Melampsorella, Melampsoridium, Milesia, Milesina, Naohidemyces, Pucciniastrum, Thekopsora, Uredinopsis, Chardoniella, Dietelia, Pucciniosira, Diorchidium, Endoraecium, Kernkampella, Ravenelia, Sphenospora, Austropuccinia, Nyssopsora, Sphaerophragmium, Dasyspora, Leucotelium, Macruropyxis, Porotenus, Tranzschelia or Uro pyxis, even more preferably of species Rhizoctonia alpina, Rhizoctonia bicornis, Rhizoctonia butinii, Rhizoctonia callae, Rhizoctonia carotae, Rhizoctonia endophytica, Rhizoctonia floccosa, Rhizoctonia fragariae, Rhizoctonia fraxini, Rhizoctonia fusispora, Rhizoctonia globularis, Rhizoctonia gossypii, Rhizoctonia muneratii, Rhizoctonia papayae, Rhizoctonia quercus, Rhizoctonia repens, Rhizoctonia rubi, Rhizoctonia Silvestris, Rhizoctonia solani,

[0100] Phakopsora ampelopsidis, Phakopsora apoda, Phakopsora argentinensis, Phakopsora cherimoliae, Phakopsora cingens, Phakopsora coca, Phakopsora crotonis, Phakopsora euvitis, Phakopsora gossypii, Phakopsora hornotina, Phakopsora jatrophicola, Phakopsora meibomiae, Phakopsora meliosmae, Phakopsora meliosmae-myrianthae, Phakopsora montana, Phakopsora muscadiniae, Phakopsora myrtacearum, Phakopsora nishidana, Phakopsora orientalis, Phakopsora pachyrhizi, Phakopsora phyllanthi, Phakopsora tecta, Phakopsora uva, Phakopsora vitis, Phakopsora ziziphi-vulgaris,

[0101] Puccinia abrupta, Puccinia acetosae, Puccinia achnatheri-sibirici, Puccinia acroptili, Puccinia actaeae-agropyri, Puccinia actaeae-elymi, Puccinia antirrhini, Puccinia argentata, Puccinia arrhenatheri, Puccinia arrhenathericola, Puccinia artemisiae-keiskeanae, Puccinia arthrocnemi, Puccinia asteris, Puccinia atra, Puccinia aucta, Puccinia ballotiflora, Puccinia bartholomaei, Puccinia bistortae, Puccinia cacabata, Puccinia calcitrapae, Puccinia calthae, Puccinia calthicola, Puccinia calystegiae-soldanellae, Puccinia canaliculata, Puccinia caricis-montanae, Puccinia caricis-stipatae, Puccinia carthami, Puccinia cerinthes-agropyrina, Puccinia cesatii, Puccinia chrysanthemi, Puccinia circumdata, Puccinia clavata, Puccinia coleataeniae, Puccinia coronata, Puccinia coronati-agrostidis, Puccinia coronati-brevispora, Puccinia coronati-calamagrostidis, Puccinia coronati-hordei, Puccinia coronati-japonica, Puccinia coronati-longispora, Puccinia crotonopsidis, Puccinia cynodontis, Puccinia dactylidina, Puccinia dietelii, Puccinia digitata, Puccinia distincta, Puccinia duthiae, Puccinia emaculata, Puccinia erianthi, Puccinia eupatorii- columbiani, Puccinia flavenscentis, Puccinia gastrolobii, Puccinia geitonoplesii, Puccinia gigantea, Puccinia glechomatis, Puccinia helianthi, Puccinia heterogenea, Puccinia heterospora, Puccinia hydrocotyles, Puccinia hysterium, Puccinia impatientis, Puccinia impedita, Puccinia imposita, Puccinia infra-aequatorialis, Puccinia insolita, Puccinia justiciae, Puccinia klugkistiana, Puccinia knersvlaktensis, Puccinia lantanae, Puccinia lateritia, Puccinia latimamma, Puccinia liberta, Puccinia littoralis, Puccinia lobata, Puccinia lophatheri, Puccinia loranthicola, Puccinia menthae, Puccinia mesembryanthemi, Puccinia meyeri-albertii, Puccinia miscanthi, Puccinia miscanthidii, Puccinia mixta, Puccinia montanensis, Puccinia morata, Puccinia morthieri, Puccinia nitida, Puccinia oenanthes-stoloniferae, Puccinia operta, Puccinia otzeniani, Puccinia patriniae, Puccinia pentstemonis, Puccinia persistens, Puccinia phyllostachydis, Puccinia pittieriana, Puccinia platyspora, Puccinia pritzeliana, Puccinia prostii, Puccinia pseudodigitata, Puccinia pseudostriiformis, Puccinia psychotriae, Puccinia punctata, Puccinia punctiformis, Puccinia recondita, Puccinia rhei-undulati, Puccinia rupestris, Puccinia senecionis-acutiformis, Puccinia septentrionalis, Puccinia setariae, Puccinia silvatica, Puccinia stipina, Puccinia stobaeae, Puccinia striiformis, Puccinia striiformoides, Puccinia stylidii, Puccinia substriata, Puccinia suzutake, Puccinia taeniatheri, Puccinia tageticola, Puccinia tanaceti, Puccinia tatarinovii, Puccinia tetragoniae, Puccinia thaliae, Puccinia thlaspeos, Puccinia tillandsiae, Puccinia tiritea, Puccinia tokyensis, Puccinia trebouxi, Puccinia triticina, Puccinia tubulosa, Puccinia tulipae, Puccinia tumidipes, Puccinia turgida, Puccinia urticae-acutae, Puccinia urticae-acutiformis, Puccinia urticae-caricis, Puccinia urticae-hirtae, Puccinia urticae-inflatae, Puccinia urticata, Puccinia vaginatae, Puccinia virgata, Puccinia xanthii, Puccinia xanthosiae, Puccinia zoysiae, more preferably of species Phakopsora pachyrhizi, Puccinia graminis, Puccinia striiformis, Puccinia hordei or Puccinia recondita, more preferably of genus Phakopsora and most preferably Phakopsora pachyrhizi. As indicated above, fungi of these taxa are responsible for grave losses of crop yield. This applies in particular to rust fungi of genus Phakopsora. It is thus an advantage of the present invention that the method allows to reduce fungicide treatments against Phrakopsora pachyrhizi as described herein. It is a particularly preferable advantage that the materials and methods of the present invention are useful for fighting against rust fungi of genus Phakopsora, in particular and most preferred against Phakopsora pachyrhizi. These fungal pathogens are responsible for huge losses of soybean when soybean plants are left untreated. The present invention thus allows to reduce the number of fungicide treatments by reducing the fungal pathogen pressure.

[0102] The present invention relates to PIP proteins. The PIP protein (also called "PAMP-induced secreted peptide 1", PIP1 or PrePIPI) is a protein of two dominant domains, a signal peptide domain (also called: export signal sequence) and a non-cytoplasmic domain (also called "mature PIP"). In the sequences according to SEQ ID NO. 1 and SEQ ID NO. 3, the signal peptide spans the region of amino acids 1-30 and the non-cytoplasmic domain spans the region of amino acids 31-72. It is expected that the signal peptide sequence facilitates, possibly by becoming embedded in the cell membrane, export of the PIP protein from the cytosol of a plant cell to the apoplast region; after cleaving off of the signal peptide sequence, the non-cytoplasmic domain remains as the "mature PIP" protein in the apoplast region. As used herein, the term "PIP" denotes both the full-length PIP protein comprising both domains and also denotes the mature PIP protein without the signal peptide sequence. In the PIP protein according to Uniprot entry PIP1_ARATH, amino acids at positions 65 and 67 are reported to be modified to 4- hydroxyproline. For the purposes of calculating sequence identities and homolog scores, modifications are not taken into account and the native amino acids are used instead.

[0103] Surprisingly the inventors found that the heterologous expression of a PIP protein as described herein, e.g. PrePIPI from Arabidopsis thaliana (Uniprot entry PIP1_ARATH), in soybean leads to an increased resistance of soybean against soybean rust fungus Phakopsora pachyrhizi. This was even more surprising because the inventors did not transfer or express the cognate receptor protein RLK7 (Uniprot entry RLK7_ARATH). Thus, the PIP protein as such can already induce / amplify resistance reactions in plants other than Arabidopsis thaliana, particularly in crop plants.

[0104] The invention correspondingly provides a method for conferring, modifying or increasing resistance of a plant or plant material against at least one biotrophic or heminecrotrophic fungus. Preferably, the method provides a method for conferring, modifying or increasing resistance of a plant or plant material against at least a rust fungus. The plant or plant material preferably is of taxonomic family Fabaceae, more preferably of genus Glycine. According to the invention, the method comprises a step of increasing the production and / or accumulation of a PIP protein in the plant or plant material in comparison to a respective wild-type plant or plant material. As described above, the PIP protein preferably is a full length PIP protein comprising an export signal peptide domain and a non-cytoplasmic domain, or a homolog or variant thereof.

[0105] As is shown in the examples, by providing a plant with a heterologous gene coding for a PIP protein, the present invention allows to improve fungal resistance in a crop plant even without also providing the cognate receptor RLK7 as a heterologous gene. As given above, the plant preferably is a plant of order Fabaceae, more preferably of genus Glycine, and most preferably of soybean. By improving fungal resistance in soybean and other crop plants, the invention offers several advantages to farmers and to the environment. In particular, fungal diseases require expensive fungicide treatments to control, which can increase production costs. By improving fungal resistance, farmers can reduce the need for fungicides. Fungicides can be expensive, and their frequent application can contribute to a significant portion of a farmer's input costs. By reducing the dependence on fungicides, the invention advantageously allows farmers to save money and improve their profitability. Furthermore, by reducing the need for fungicide treatments, the invention advantageously also decreases the reliance on potentially harmful chemicals, thereby reducing the risk of human exposure to fungicides and improving the safety of crop production. The invention thus also allows to reduce chemical runoff and contamination of water sources or of soil. Furthermore, by reducing severity of infection and / or delaying the onset of disease, the invention allows to reduce or delay the contamination of neighbouring fields with fungal pathogen spores as described below. And by improving fungal resistance, in particular of soybean against Phakopsora rust pathogens, the quality of soybean seeds is improved, which can command a premium price. As soybean is an important source of protein for both humans and animals, improved fungal resistance helps ensure a stable supply of soybean and reduce the risk of food and feed shortages. Overall, increasing resistance against biotrophic or heminecrotrophic fungi, particularly against soybean rust, not only benefits farmers by reducing costs and improving profitability but also contributes to a healthier environment by minimizing the use of fungicides and their associated environmental and health risks.

[0106] The most preferred PIP protein is that of Uniprot entry PIP1_ARATH, shown as SEQ ID NO. 3 herein. Also preferred is a PIP protein variant which differs from SEQ ID NO. 3 (PIP1_ARATH) only by one or more of the following mutations: V9I, 111 L, V12M, F20S, V24A, A29V, V33G, K37N, S42N, T44TV. Even more preferably, the PIP protein differs from the sequence of SEQ ID NO. 1 and / or from the sequence of positions 31-72 of SEQ ID NO. 1 by any one of the following mtuations: I9V, M14V, L19F, V20F, L38S, R42S, T44TV, R46T. More preferably, the mature PIP protein differs from the sequence of positions 31-72 of SEQ ID NO. 1 only at any one of the following mutations: L38S, R42S, T44TV, R46T. Furthermore, the PIP protein preferably differs in the export signal sequence of positions 1-30 of SEQ ID NO. 1 only by one or more of the following mutations: I9V, M14V, L19F. Even more preferably, the PIP protein differs from the sequence according to SEQ ID NO. 3 by at most 4 of the aforementioned 9 mutations, even more preferably at most 3 of the aforementioned mutations, even more preferably at most 2 of the aforementioned mutations, even more preferably at most 1 of the aforementioned mutations.

[0107] However, the invention also relates to homologs thereof. Examples of preferred homolog PIP proteins are indicated below by reference to their respective Uniprot entries.

[0108] Generally, homologs according to the invention will have a sequence similar to the amino acid sequence according to SEQ ID NO. 1 . A candidate protein is considered a homolog according to the present invention if it has a) a sufficiently high sequence identity to SEQ ID NO. 1 and / or SEQ ID NO. 3; and / or b) a sufficiently high homology score to SEQ ID NO. 1 .

[0109] Furthermore, the signal peptide domain can be changed as long as the non-cytoplasmic domain can still be exported to the apoplastic region. Thus, homologs according to the invention are also those which have a different signal peptide domain (but still functional for the purposes of export to the apoplast), but which have a) a sufficiently high sequence identity to amino acids 31-72 of SEQ ID NO. 1 and / or SEQ ID NO. 3; and / or b) a sufficiently high homology score to amino acids 31-72 of SEQ ID NO. 1 .

[0110] A candidate protein has a sufficiently high sequence identity if the a) sequence identity relative to SEQ ID NO. 1 is 80%-100%, preferably of 81-96%, more preferably 87%-96%; and / or b) sequence identity relative to SEQ ID NO. 3 is 74%-100%, preferably of 84-100%, more preferably 90%-100%; and / or c) sequence identity relative to SEQ ID NO. 4 is 80%-100%, preferably of 90-100%, more preferably 92%-100%.

[0111] Alternative c) is the preferred definition for a mature PIP homolog. Thus, this definition applies to mature PIP homologs as such, but also to PIP homologs which comprise a different export signal peptide. Alternatives a) and b) are preferred definitions for full-length PIP proteins. That is, this definition applies to preferred homologs which do not only comprise a preferred mature PIP domain homolog but also the preferred export signal peptide. Most preferably, a PIP homolog is a protein which conforms to all of the above definitions.

[0112] Instead of or in addition to the definition by sequence identity, homologs can also be defined by their homology score, that is, their degree of homology between to the amino acid sequence SEQ ID NO. 1 or to positions 31-72 of SEQ ID NO. 1. To calculate the homology score, for each amino acid position of SEQ ID NO. 1 , a position-specific score is determined, this position-specific score is the number of stars in Figure 7 for the respective position. Thus, the position-specific score for the first amino acid of SEQ ID NO. 1 is 9, the position-specific score for the second amino acid of SEQ ID NO. 1 is 7 and so on. The homology score of the candidate protein is obtained by the aligning the candidate protein sequence to the sequence of SEQ ID NO. 1. Where an amino acid of the candidate protein corresponds to the respective amino acid of SEQ ID NO. 1 , the homology score is increased by the respective position-specific score; in case of a mismatch, the homology score is not increased at the respective position. Where a candidate protein sequence comprises additional amino acids between two positions of SEQ ID NO. 1 , these amino acids are not taken into account. Where the homology score is calculated only relative to the non-cytoplasmic domain of SEQ ID NO. 1 (amino acids 31-72), the alignment and summation is correspondingly performed only over this domain sequence.

[0113] Thus, a candidate protein has a sufficiently high homology score d) relative to SEQ ID NO. 1 if the homology score is 481 to 628, even more preferably 490 to 628, even more preferably 554 to 628, even more preferably 561 to 628, even more preferably 563 to 628, even more preferably 564 to 628, even more preferably 569 to 628, even more preferably 571 to 628, even more preferably 576 to 628, even more preferably 583 to 628, even more preferably 571-603; and / or e) relative to the sequence of positions 31-72 of SEQ ID NO. 1 if the homology score is 272 to 376, even more preferably 335 to 376, even more preferably 337 to 376, even more preferably 338 to 376, even more preferably 346 to 376, even more preferably 347 to 376, even more preferably 351 to 376, even more preferably 351 to 376, even more preferably 352 to 376, even more preferably 354 to 376, even more preferably 355 to 376, even more preferably 359 to 376, even more preferably 351-359.

[0114] Where the PIP protein is a protein according to c) and / or e), i.e. a homolog defined by the sequence identity and / or homology score relative to the mature part of the PIP protein, it is preferred that the PIP protein also comprises an export signal peptide to facilitate localisation of the PIP protein in the apoplastic region. The signal export peptide is preferably linked to the mature part of the PIP protein such as to allow for the export signal peptide to be cleaved off. This advantageously reduces any interference of the export signal peptide in the fungal resistance improvement function of the mature PIP protein. In particular, a mature PIP protein which is not anchored to a plant cell membrane via an export signal peptide is mobile in the apoplast region and thus has more chances to contact a fungal pathogen cell including haustoria cells.

[0115] Preferably, the PIP protein differs from the sequence of SEQ ID NO. 1 and / or from the sequence of positions 31-72 of SEQ ID NO. 1 only at a position having less than 10 stars in Figure 7, even more preferably having less than 9 stars, even more preferably having less than 8 stars. Where the sequence differs from SEQ ID NO. 1 , preferably the difference is according to the respective alternatives shown beneath the respective position in Figure 7. Thus, for example, at position 2 the sequence either has (in the one-letter-code) an R, or one of G, K or M, or a gap relative to SEQ ID NO. 1 (indicated by in Figure 7).

[0116] The sequence identities and homology scores of preferred homologs according to the invention are given below in decreasing order of preference.

[0117] Thus, preferably the homolog has 90%-100% identity to SEQ ID NO. 4, 84-100% sequence identity to SEQ ID NO. 3, 87-96% sequence identity to SEQ ID NO. 1 , a homology score of 351- 362 to amino acids 31-72 of SEQ ID NO. 1 and a homology score of 490 to 611 to SEQ ID NO. 1 . More preferably, the homolog has 92%-100% identity to SEQ ID NO. 4, 90-100% sequence identity to SEQ ID NO. 3, 87-96% sequence identity to SEQ ID NO. 1 , a homology score of 352- 359 to amino acids 31-72 of SEQ ID NO. 1 and a homology score of 571 to 611 to SEQ ID NO. 1 . Even more preferably, the homolog has 97%-100% identity to SEQ ID NO. 4, 97-100% sequence identity to SEQ ID NO. 3, 87-91 % sequence identity to SEQ ID NO. 1 , a homology score of 352- 359 to amino acids 31-72 of SEQ ID NO. 1 and a homology score of 571 to 583 to SEQ ID NO. 1.

[0118] However, as the export signal peptide is preferably cleaved off the PIP protein to obtain a mature PIP protein, it is not required that the export signal peptide and the respective mature part of the PIP protein have a high sequence identity and / or homology score to a PIP homolog of a single species. Instead, an export signal peptide sequence (or homolog thereof) of a PIP protein can be exchanged for an export signal peptide sequence (or homolog thereof) of a PIP protein of another species. For example, the export signal peptide of the PIP protein according to Uniprot entry A0A397L0T3_BRACM is identical to that of the PIP protein according to Uniprot entry AOAOD3DGL5_BRAOL, but the mature PIP protein according to Uniprot entry A0A397L0T3_BRACM is identical to that of the PIP protein according to Uniprot entry M4E5Z7_BRACM. The sequences according to SEQ ID NO. 5-161 exemplify such PIP proteins wherein the export signal peptide sequence is heterologous to the mature part of the PIP protein.

[0119] Preferably, the method comprises the steps of

[0120] 1) stably transforming plant material with at least one expression cassette comprising an exogenous nucleic acid encoding a PIP protein of the present invention,

[0121] 2) regenerating a plant from the plant cell; and 3) expressing said PIP protein.

[0122] It is a particular advantage of the present invention that the PIP protein of the present invention can be transferred to a plant by vector based transformation. Modifying the nucleotide sequence of a gene coding for a distant homolog of a plant to code for a PIP protein of the present invention using mutagenesis means with a preference or even specificity for a particular site (e.g. CRISPR- like technologies) would require potentially many mutation steps. Thus, many plants need to be modified to incrementally obtain a gene coding for the desired PIP protein. The invention, however, advantageously allows to introduce a gene coding for a desired PIP protein in a single transformation step, e.g. using vector-based transformation methods. Such methods are well established in the art and can be applied with comparatively little effort and expenses by the skilled person. In particular, the transformation method of the present invention advantageously allows to introduce a heterologous gene coding for a PIP protein found in another species. When the PIP protein has already occurred in a wild-type plant, then the protein has an advantageously higher probability of being functional upon heterologous expression compared to a fully in-silico created protein (e.g. the protein of SEQ ID NO. 1). In addition to introducing a novel PIP gene, vector-based transformation can also advantageously enable the transfer of one or more further heterologous genes to the plant. These further heterologous genes can, for example, have additional fungicidal properties or can enhance the plant's natural resistance mechanisms.

[0123] The invention also provides a method for production of a genetically modified plant or plant material. Again the plant or plant material preferably is of taxonomic family Fabaceae, more preferably of genus Glycine. The resulting plant or plant material has increased resistance, compared to a respective wild-type plant or wild-type plant material, against at least one biotrophic or heminecrotrophic fungus, preferably against a rust fungus. The method comprises the steps of

[0124] 1) introducing an exogenous nucleic acid encoding a PIP protein of the present invention into a plant or plant material;

[0125] 2) generating a genetically modified plant or genetically modified plant material; and

[0126] 3) expressing the PIP protein in the genetically modified plant or genetically modified plant material.

[0127] It is understood that most preferably the one or more genes coding for the one or more PIP proteins of the present invention is integral in the genome of the plant or plant part, thereby providing a stable enhancement of resistance. Such plants or plant parts according to the invention can be transgenic, thereby preferably comprising the respective gene(s) operably linked to the heterologous promoter - preferred promoters are described herein -, and / or integrated at a different locus than a corresponding wild type gene coding for a PIP-like protein. It is a particular advantage that such stable enhancement of resistance can be achieved by transformation of an exogenous nucleic acid encoding a PIP protein of the present invention into a plant or plant material as described above.

[0128] The gene encoding the PIP protein preferably is operably linked to a constitutively active promoter, b) a tissue-specific or tissue-preferred promoter, c) a promoter inducible by exposition of the plant to a pest, preferably a fungal pest, and / or d) a promoter predominantly active in plants having or developing true leaves. It is a particular advantage of the present invention that while expression of the PIP protein of the present invention may be in all plant tissues, it is sufficient to secure expression of the PIP protein in specific tissues or after exposition of the plant or plant material to a pest. Preferably, the PIP protein of the present invention is expressed in true leaf tissue (also called foliage leaves), i.e. in leaves developing or having developed after the cotyledon leaves or seed leaves. This is particularly advantageous for improving resistance against fungal rust pathogens, because these, like Phakopsora in soybean, infect predominantly true leaves. It is a particular advantage of the present invention that expression of a PIP protein of the present invention for improving fungal resistance can be combined with any of the aforementioned further genes for conferring hypoallergenicity and / or low content of anti-nutrients. This was all the more surprising since it is known that in soybean gene silencing is a major factor contributing to spontaneous decrease of gene expression when two individually effective genes are combined, for example by crossing or co-transformation, into one plant's genetic material.

[0129] The method for production of a genetically modified plant or plant material having increased fungal resistance according to the present invention preferably further comprises the steps of harvesting the seeds of the transgenic plant and growing the seeds to plants, wherein the grown plants comprise a heterologous nucleic acid sequence coding for a PIP protein of the present invention.

[0130] It is a further advantage that the present invention allows, by stably transforming a plant with a single heterologous gene, to create non-segregating offspring. Thus, the PIP fungal resistance gene of the present invention advantageously allows to combine this fungal resistence with further traits (including potentially other genes providing fungal resistance) by established breeding methods, e.g. crossing and selecting. It is a further particular advantage of the present invention that enhancement in fungal resistance as described herein, in particular a statistically significant stable provision or intensification of resistance against biotrophic or heminecrotrophic fungi and most preferably against Phakopsora pachyrhizi can be achieved by transformation also in in polyploid plants, e.g. in Glycine max, already by merely incorporating a gene coding for a heterologous PIP protein into one set of chromosomes. This facilitates the generation, propagation and breeding of plants comprising the PIP protein of the present invention.

[0131] Preferably, the plant or plant material is homozygous for the PIP protein(s) of the present invention. This is of particular advantage because offspring of the plant are according to the Mendelian laws practically guaranteed to share the enhanced resistance phenotype of the homozygous parent plant. While homozygous cells are particularly suitable for breeding, it is noteworthy that the advantages of the present invention are already conferred by cells that are heterozygous with regards to the gene coding for the heterologous PIP protein of the present invention. Thus, the present invention particularly facilitates the generation of hybrid plant varieties, i.e. first generation results of a crossing of parent plants wherein only one parent plant confers the PIP gene of the present invention, such that the hybrid plants already exhibit enhanced resistance to infections by at least one biotrophic or heminecrotrophic fungus. As preferred herein, such plants preferably are of genus Glycine, most preferably of species Glycine max, and the fungus in question preferably is of genus Phakopsora, most preferably of species Phakopsora pachyrhizi.

[0132] Fungal resistance is most useful where it is observed in a whole plant or plant part. This way, the improved resistance allows for a reduction of fungicide use in commercial farming, thereby reducing farming costs and land exposure to pesticides without compromising yield of the finally harvested material. To this end, the plant or plant part according to the present invention comprises a cell according to the present invention or is transformed with an expression construct providing expression of a heterologous PIP protein in the transformed plant cell. Preferably, the plant of the present invention consists of such transformed cells. This way, the plant can make use of the gene coding for the PIP protein of the present invention in all tissues or at least in those tissues where the respective promoter is active. However, it is an advantage of the present invention that the plant can also be a chimeric plant, for example a periclinal or plastidal chimera, or can comprise chimeric plant parts, for example chimeric leaves and / or stems. The skilled person understands that even though a chimeric plant offers enhanced resistance against fungal infections, the degree of protection increases corresponding to the content of cells, particularly leaf tissue cells, capable of expressing the PIP protein according to the invention. Correspondingly, the invention also provides a vector for creating a genetically modified plant, comprising an expression cassette having a PIP gene operably linked to a promoter, wherein the PIP gene codes for a PIP protein as defined herein according to the invention. As described before, transformation vectors advantageously allow to transform a plant in a single transformation step and thereby provides advantageously one or more heterologous genes to the recipient plant.

[0133] The invention also provides a plant or plant material. The plant or plant material has increased expression, accumulation or activity of a heterologous PIP protein, wherein the PIP protein is a PIP protein as defined herein. Accordingly, the preferably recombinant or transgenic plant or plant part of the present invention has reduced or abolished susceptibility, relative to a wild type plant, to infections by a biotrophic or heminecrotrophic fungus, preferably a rust fungus. As described above, this is particularly advantageous where the plant or plant material is of taxonomic family Fabaceae, more preferably of genus Glycine.

[0134] As described herein, such plant or plant material is obtainable or obtained by a method according to the present invention.

[0135] In the plant or plant material according to the present invention, a) the PIP protein preferably comprises an export signal sequence to facilitate export of the PIP protein from the cytosol of a plant cell, and / or b) the plant does not comprise, in the cells capable of expressing the PIP protein, the cognate receptor RLK7.

[0136] As described above, the PIP protein comprises a part which, when cleaved off an export signal peptide, functions as the active, mature PIP protein in the apoplastic region. While the mature part of the PIP protein is required for its fungal pathogen inhibiting activity, the export signal sequence as such seems to be only required to facilitate transport of the PIP protein out of the cytosol. Thus, an export signal sequence of the PIP protein can be essentially any sequence suitable for such transport. It may even be missing, for example, when the PIP protein is provided extraneously (e.g. in irrigation) for circulation through xylem and / or phloem. Preferably, however, the PIP protein comprises an export signal sequence. This way, the PIP protein may be produced within plant material cells, close to the required site of action, thereby reducing the need for wide distribution of the mature PIP protein throughout the plant.

[0137] It is a further advantage of the present invention that the PIP protein as described herein exerts its protective activity as a heterologous protein even in the absence of its cognate receptor. Thus, where a gene coding for a PIP protein is taken from a source plant, it is not necessary to transform the target plant also with a gene coding for the source plant's RLK7 protein. This advantageously reduces the amount of genetic material required in a transformation vectors of the present invention, thereby advantageously leaving room in such vector for additional genetic material. The transformation vector of the present invention thus can comprise one or more further genes for transformation, e.g. further fungal infection reducing or inhibiting genes.

[0138] For the purposes of the present invention, RLK7 means a protein also called "receptor-like protein kinase 7". A paragon of an RLK7 protein is given under Uniprot name RLK7_ARATH, which comprises a protein kinase domain (PFAM accession number PF00069, InterPro identifier IPR000719) at amino acid positions 666-959 and several leucine-rich repeats (positions 71-95, 96-119, 121-145, 168-194, 195-218, 219-242, 244-265, 267-289, 290-312, 313-337, 339-361 , 362-385, 386-409, 411-433, 434-457, 458-481 , 482-505, 507-529, 530-553, 555-578).

[0139] It is a further advantage of the present invention that the enhancement of resistance does not depend on the inactivation or removal of the corresponding wild type PIP gene. However, in order to reduce the likelihood of unwanted homologous recombination events it is preferred that in the plant, plant part or cell of the present invention at least one, preferably all wild-type PIP genes is / are (a) replaced by a gene coding for a PIP protein of the present invention or (b) deleted. Such plants, plant parts or cells can be transgenic or can be the result of an artificially induced heritable genomic alteration as described herein.

[0140] The invention also provides non-propagative plant part or material of a plant or plant part of the present invention, preferably a fermentation product, oil, meal, press cake, pomace, chaff, straw or compost. The material is surprisingly advantageous, because - compared to material obtained from a conventionally treated plant - it required less fungicides for production under pathogen pressure and thus contains fewer fungicide residues. Furthermore, the plant material contains more micronutrients, a lower amount of residual fungicides or metabolic products of fungi such as toxins. Thus, the plant part or plant material has a higher quality compared to the corresponding conventional plant part or material, respectively.

[0141] References to harvestable or collected plant material, preferably of fruit and seed, refer to at least 1000kg (drained weight) of such material, preferably a fill of 1000-10000000kg.

[0142] The invention thus also provides a roduct of a plant or plant part according to the present invention, wherein the product is obtainable or obtained by i) collecting a material of said plant or plant part, preferably a harvestable plant part and most preferably a plant seed, and ii) disrupting the collected material, preferably to obtain a fermentation product, oil, meal, press cake, pomace, chaff, straw or compost.

[0143] By disrupting the collected material, nurients contained in the material become more readily accessible and anti-nutrients are reduced.

[0144] According to the invention, there is also a method provided for producing a population of plants each having an enhanced resistance to at least one biotrophic or heminecrotrophic fungus, preferably a rust fungus, comprising the steps of i) multiplying seed of a plant according to the invention, wherein the seed are preferably homozygous for the PIP protein, and ii) growing plants of the seed obtained in step i), wherein at least 1000 plants are grown.

[0145] Most preferably the plants are grown on a monoculture field of at least 1 ha of the plant and a monoculture field of at least 1 ha of the control plant is determined, respectively. Correspondingly, treatments are preferably performed on such ensemble of plants. Thus, the invention also provides an ensemble of at least 1000 crop plants according to the present invention, more preferably at least 100000 plants. According to the invention, preferably at least 100000 plants are grown per hectar, more preferably 200000 to 800000 plants per hectar, even more preferably at least 250000 to 650000 plants per hectar. Such plant numbers preferably are observed within one hectar; thus, the invention particularly facilitates ecologically considerate intensive farming with reduced use of fungicides per growing season. The plants according to the invention are preferably growing in a field or greenhouse. As indicated above, preferably the plants are soybean crop plants.

[0146] According to the invention it is not required that all crop plants of one species growing in the same field or greenhouse are plants of the present invention. Instead, it is sufficient in monoculture plantation if at least about 25% of the plants of one species belong to the present invention, more preferably at least 50%, even more preferably 25%-75% and most preferably 45%-70%, especially when mixed or combined with plants harboring other resistance genes or mechanisms. The combination with plants with other resistance gene can be done by interplanting (mixing), row-wise or blockwise. For example, on a soybean field it is possible to reduce the number of fungicide treatments if approximately every second plant is a plant according to the present invention. It is particularly preferred that at least 25%, more preferably 50%-100% and even more preferably 75%-100% of those plants on the same field that are not plants according to the present invention comprise at least one other biological means for enhancing fungal resistance, most preferably the other means is selected from the list of pathogen resistance polypeptides as described above.

[0147] As described herein, the invention allows, by way of a heterologous PIP gene provided in plant cells or plant material, to prevent, reduce or delay infections of said plant, plant material or cell by phythopathogenic microorganisms, preferably of rust fungus infections in leguminous plants, most preferably in soybean. Correspondingly, the invention provides a farming method for controlling or reducing at least one biotrophic or heminecrotrophic fungus in a field, preferably by reducing or delaying infection of plants in a field and / or reducing or delaying emission of fungal spores from the field, comprising the step of growing plants according to the invention on the field. According to the invention, increase of fungal resistance is achieved preferably by reducing, compared to a corresponding wild type, the speed of infection or the extent of infection or delaying the day of earliest infection by the fungus. Thus, the PIP protein and gene of the present invention is suitable for conferring, intensifying or stabilising resistance of plants, plant parts or plant cells against fungal pathogen infections, particularly against biotrophic, hemibiotrophic or heminecrotrophic fungi, and preferably against fungi as described herein. Furthermore, by increasing fungal resistance as described in this paragraph the PIP protein and gene of the present invention are suitable to prevent, reduce or delay the spread of fungal spores to other fields, thereby also reducing pathogen pressure in the wider area where the plants expressing the PIP protein of the present invention are grown.

[0148] By increasing fungal resistance as described herein, the PIP protein and gene of the present invention are suitable to reduce the number of fungicide treatments required to protect growing plants. The invention thus also provides a farming method, comprising the step of applying at least one less fungicide treatment to plants than would be required for wild-type control plants grown under the same conditions. For example, in Brazil it may be customary to apply a first fungicide treatment to soybean plants on day 8 after seeding and a second spray on day 18 after seeding. In other regions a scheme may be practiced not depending on mere time of growth but, for example, taking into account first notice of a pest occurrence or passing of a pest incidence threshold. It is a particular and unforeseen advantage of the present invention that the number of pesticide treatments per growth seasons can be reduced compared to a control plant. It was in particular surprising that such treatment reduction is possible not only without reducing yield; instead the farming method according to the invention advantageously allows to maintain or even increase yield despite the reduction in treatments. This greatly improves cost efficiency of farming the plants as provided by the present invention. Of course, the pesticide is preferably applied in pesticidally effective amounts.

[0149] According to the invention the pest is or comprises at least a a biotrophic or heminecrotrophic fungus, more preferably a rust fungus. If during cultivation the plant is also under threat of stress by other pathogens, e.g. nematodes and insects, such other pests are preferably taken care of by respective pesticide treatments. Thus, according to the invention preferably the number of fungicide treatments is reduced as described above, irrespective of other pesticide treatments. The fungicide can be mixed with other pesticides and ingredients preferably selected from insecticides, nematicides, and acaricides, herbicides, plant growth regulators, fertilizers. Preferred mixing partners are insecticides, nematicides and fungicides. It is particularly preferred to reduce, during cultivation of the plant, the number of fungicide treatments per growth season by at least one relative to the control plant, preferably by at least two. Fungicides may include 2- (thiocyanatomethylthio)-benzothiazole, 2-phenylphenol, 8-hydroxyquinoline sulfate, ametoctradin, amisulbrom, antimycin, Ampelomyces quisqualis, azaconazole, azoxystrobin, Bacillus subtilis, Bacillus subtilis strain QST713, benalaxyl, benomyl, benthiavalicarb-isopropyl, benzylaminobenzene- sulfonate (BABS) salt, bicarbonates, biphenyl, bismerthiazol, bitertanol, bixafen, blasticidin-S, borax, Bordeaux mixture, boscalid, bromuconazole, bupirimate, calcium polysulfide, captafol, captan, carbendazim, carboxin, carpropamid, carvone, chlazafenone, chloroneb, chlorothalonil, chlozolinate, Coniothyrium minitans, copper hydroxide, copper octanoate, copper oxychloride, copper sulfate, copper sulfate (tribasic), cuprous oxide, cyazofamid, cyflufenamid, cymoxanil, cyproconazole, cyprodinil, dazomet, debacarb, diammonium ethylenebis-(dithiocarbamate), dichlofluanid, dichlorophen, diclocymet, diclomezine, dichloran, diethofencarb, difenoconazole, difenzoquat ion, diflumetorim, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinobuton, dinocap, diphenylamine, dithianon, dodemorph, dodemorph acetate, dodine, dodine free base, edifenphos, enestrobin, enestroburin, epoxiconazole, ethaboxam, ethoxyquin, etridiazole, famoxadone, fenamidone, fenarimol, fenbuconazole, fenfuram, fenhexamid, fenoxanil, fenpiclonil, fenpropidin, fenpropimorph, fenpyrazamine, fentin, fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, fludioxonil, fluindapyr, flumorph, fluopicolide, fluopyram, fluoroimide, fluoxastrobin, fluquinconazole, flusilazole, flusulfamide, flutianil, flutolanil, flutriafol, fluxapyroxad, folpet, formaldehyde, fosetyl, fosetyl-aluminium, fuberidazole, furalaxyl, furametpyr, guazatine, guazatine acetates, GY-81 , hexachlorobenzene, hexaconazole, hymexazol, imazalil, imazalil sulfate, imibenconazole, iminoctadine, iminoctadine triacetate, iminoctadine tris(albesilate), iodocarb, ipconazole, ipfenpyrazolone, iprobenfos, iprodione, iprovalicarb, isoprothiolane, isofetamide, isopyrazam, isotianil, kasugamycin, kasugamycin hydrochloride hydrate, kresoxim-methyl, laminarin, mancopper, mancozeb, mandipropamid, maneb, mefenoxam, mepanipyrim, mepronil, meptyl- dinocap, mercuric chloride, mercuric oxide, mercurous chloride, metalaxyl, metalaxyl-M, metam, metam- ammonium, metam-potassium, metam-sodium, metconazole, methasulfocarb, methyl iodide, methyl isothiocyanate, metiram, metominostrobin, metrafenone, mildiomycin, myclobutanil, nabam, nitrothal-isopropyl, nuarimol, octhilinone, ofurace, oleic acid (fatty acids), orysastrobin, oxadixyl, oxathiapiprolin, oxine-copper, oxpoconazole fumarate, oxycarboxin, pefurazoate, penconazole, pencycuron, penflufen, pentachlorophenol, pentachlorophenyl laurate, penthiopyrad, phenylmercury acetate, phosphonic acid, phthalide, picoxystrobin, polyoxin B, polyoxins, polyoxorim, potassium bicarbonate, potassium hydroxyquinoline sulfate, probenazole, prochloraz, procymidone, propamocarb, propamocarb hydrochloride, propiconazole, propineb, proquinazid, pydiflumetofen, prothioconazole, pyraclostrobin, pyrametostrobin, pyraoxystrobin, pyraziflumid, pyrazophos, pyribencarb, pyributicarb, pyrifenox, pyrimethanil, pyriofenone, pyroquilon, quinoclamine, quinoxyfen, quintozene, Reynoutria sachalinensis extract, sedaxane, silthiofam, simeconazole, sodium 2-phenylphenoxide, sodium bicarbonate, sodium pentachlorophenoxide, spiroxamine, sulfur, SYP-Z048, tar oils, tebuconazole, tebufloquin, tecnazene, tetraconazole, thiabendazole, thifluzamide, thiophanate-methyl, thiram, tiadinil, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazoxide, tricyclazole, tridemorph, trifloxystrobin, triflumizole, triforine, triticonazole, validamycin, valifenalate, valiphenal, vinclozolin, zineb, ziram, zoxamide, Candida oleophila, Fusarium oxysporum, Gliocladium spp., Phlebiopsis gigantea, Streptomyces griseoviridis, Trichoderma spp., (RS)-N-(3,5-dichlorophenyl)-2- (methoxymethyl)-succinimide, 1 ,2-dichloropropane, l,3-dichloro-l,l,3,3-tetrafluoroacetone hydrate,

[0150] 1-chloro-2,4-dinitronaphthalene, 1-chloro-2-nitropropane, 2-(2-heptadecyl-2-imidazolin-1- yl)ethanol, 2,3-dihydro-5-phenyl-l,4-dithi-ine 1 ,1 ,4,4-tetraoxide, 2-methoxyethylmercury acetate,

[0151] 2-methoxyethylmercury chloride, 2-methoxyethylmercury silicate, 3-(4-chlorophenyl)-5- methylrhodanine, 4-(2-nitroprop- l-enyl)phenyl thiocyanateme, aminopyrifen, ampropylfos, anilazine, azithiram, barium polysulfide, Bayer 32394, benodanil, benquinox, bentaluron, benzamacril; benzamacril-isobutyl, benzamorf, benzovindiflupyr, binapacryl, bis(methylmercury) sulfate, bis(tributyltin) oxide, buthiobate, cadmium calcium copper zinc chromate sulfate, carbamorph, CECA, chlobenthiazone, chloraniformethan, chlorfenazole, chlorquinox, climbazole, copper bis(3-phenylsalicylate), copper zinc chromate, coumoxystrobin, cufraneb, cupric hydrazinium sulfate, cuprobam, cyclafuramid, cypendazole, cyprofuram, decafentin, dichlobentiazox, dichlone, dichlozoline, diclobutrazol, dimethirimol, dinocton, dinosulfon, dinoterbon, dipymetitrone, dipyrithione, ditalimfos, dodicin, drazoxolon, EBP, enoxastrobin, ESBP, etaconazole, etem, ethirim, fenaminosulf, fenaminstrobin, fenapanil, fenitropan, fenpicoxamid, fluindapyr, fluopimomide, fluotrimazole, flufenoxystrobin, furcarbanil, furconazole, furconazole-cis, furmecyclox, furophanate, glyodine, griseofulvin, halacrinate, Hercules 3944, hexylthiofos, ICIA0858, inpyrfluxam, ipfentrifluconazole, ipflufenoquin, isofetamid, isoflucypram, isopamphos, isovaledione, mandestrobin, mebenil, mecarbinzid, mefentrifluconazole, metazoxolon, methfuroxam, methylmercury dicyandiamide, metsulfovax, metyltetra prole, milneb, mucochloric anhydride, myclozolin, N-3,5-dichlorophenyl-succinimide, N-3- nitrophenylitaconimide, natamycin, N-ethylmercurio-4-toluenesulfonanilide, nickel bis(dimethyldithiocarbamate), OCH, oxathiapiprolin, phenylmercury dimethyldithiocarbamate, phenylmercury nitrate, phosdiphen, picarbutrazox, prothiocarb; prothiocarb hydrochloride, pydiflumetofen, pyracarbolid, pyrapropoyne, pyraziflumid, pyridachlometyl, pyridinitril, pyrisoxazole, pyroxychlor, pyroxyfur, quinacetol, quinacetol sulfate, quinazamid, quinconazole, quinofumelin, rabenzazole, salicylanilide, SSF-109, sultropen, tecoram, thiadifluor, thicyofen, thiochlorfenphim, thiophanate, thioquinox, tioxymid, triamiphos, triarimol, triazbutil, trichlamide, triclopyricarb, triflumezopyrim, urbacid, zarilamid, and any combinations thereof.

[0152] The plant, plant material or plant cell may comprise, in addition to the heterologous PIP gene, one or more further heterologous elements. For example, transgenic soybean events comprising herbicide tolerance genes are for example, but not excluding others, GTS 40-3-2, MON87705, MON87708, MON87712, MON87769, MON89788, A2704-12, A2704-21 , A5547-127, A5547-35, DP356043, DAS44406-6, DAS68416-4, DAS-81419-2, GU262, SYHT0H2, W62, W98, FG72 and CV127; transgenic soybean events comprising genes for insecticidal proteins are for example, but not excluding others, MON87701 , MON87751 and DAS-81419. Cultivated plants comprising a modified oil content have been created by using the transgenes: gm-fad2-1 , Pj.D6D, Nc.Fad3, fad2-1A and fatb1-A. Examples of soybean events comprising at least one of these genes are: 260-05, MON87705 and MON87769. Plants comprising such singular or stacked traits as well as the genes and events providing these traits are well known in the art. For example, detailed information as to the mutagenized or integrated genes and the respective events are available from websites of the organizations International Service for the Acquisition of Agri, biotech Applications (ISAAA) (http: / / www.isaaa.org / gmapprovaldatabase) and the Center for Environmental Risk Assessment (CERA) (http: / / cera-qmc.org / GMCropDatabase). Further information on specific events and methods to detect them can be found for soybean events H7- 1 , MON89788, A2704-12, A5547-127, DP305423, DP356043, MON87701 , MON87769, CV127, MON87705, DAS68416-4, MON87708, MON87712, SYHT0H2, DAS81419, DAS81419 x DAS44406-6, MON87751 in WO04 / 074492, W006 / 130436, WC06 / 108674, WC06 / 108675, WO08 / 054747, W008 / 002872, WO09 / 064652, WO09 / 102873, W010 / 080829, W010 / 037016, W011 / 066384, W011 / 034704, WO12 / 051199, WO12 / 082548, W013 / 016527, WO13 / 016516, WO14 / 201235.

[0153] In addition to fungicides, the plant, plant material or plant cell may also be treated with one ore more biopesticides. It is an advantage of the present invention that the PIP gene does not interfere with the beneficial effects of biopesticides. Many biopesticides have been deposited under deposition numbers mentioned herein (the prefices such as ATCC or DSM refer to the acronym of the respective culture collection, for details see e. g. here: http: / / www. wfcc.info / ccinfo / collection / by_acronym / ), are referred to in literature, registered and / or are commercially available: mixtures of Aureobasidium pullulans DSM 14940 and DSM 14941 isolated in 1989 in Konstanz, Germany (e. g. blastospores in BlossomProtect® from bio-ferm GmbH, Austria), Azospirillum brasilense Sp245 originally isolated in wheat reagion of South Brazil (Passo Fundo) at least prior to 1980 (BR 11005; e. g. GELFIX® Gramlneas from BASF Agricultural Specialties Ltd., Brazil), A. brasilense strains Ab-V5 and Ab-V6 (e. g. in AzoMax from Novozymes BioAg Produtos papra Agricultura Ltda., Quattro Barras, Brazil or Simbiose-Malz® from Simbiose-Agro, Brazil; Plant Soil 331 , 413-425, 2010), Bacillus amyloliquefaciens strain AP- 188 (NRRL B-50615 and B-50331 ; US 8,445,255); B. amyloliquefaciens spp. plantarum D747 isolated from air in Kikugawashi, Japan (US 20130236522 A1 ; FERM BP 8234; e. g. Double Nickel™ 55 WDG from Certis LLC, USA), B. amyloliquefaciens spp. plantarum FZB24 isolated from soil in Brandenburg, Germany (also called SB3615; DSM 96-2; J. Plant Dis. Prot. 105, 181- 197, 1998; e. g. Taegro® from Novozyme Biologicals, Inc., USA), B. amyloliquefaciens ssp. plantarum FZB42 isolated from soil in Brandenburg, Germany (DSM 23117; J. Plant Dis. Prot. 105, 181-197, 1998; e. g. RhizoVital® 42 from AbiTEP GmbH, Germany), B. amyloliquefaciens ssp. plantarum MBI600 isolated from faba bean in Sutton Bonington, Nottinghamshire, U.K. at least before 1988 (also called 1430; NRRL B 50595; US 2012 / 0149571 A1 ; e. g. Integral® from BASF Corp., USA), B. amyloliquefaciens spp. plantarum QST-713 isolated from peach orchard in 1995 in California, U.S.A. (NRRL B 21661 ; e. g. Serenade® MAX from Bayer Crop Science LP, USA), B. amyloliquefaciens spp. plantarum TJ1000 isolated in 1992 in South Dakoda, U.S.A, (also called 1 BE; ATCC BAA-390; CA 2471555 A1 ; e. g. QuickRoots™ from TJ Technologies, Watertown, SD, USA), B. firmus CNCM 1-1582, a variant of parental strain EIP-N1 (CNCM I- 1556) isolated from soil of central plain area of Israel (WO 2009 / 126473, US 6,406,690; e. g. Votivo® from Bayer CropScience LP, USA), B. pumilus GHA 180 isolated from apple tree rhizosphere in Mexico (IDAC 260707-01 ; e. g. PRO-MIX® BX from Premier Horticulture, Quebec, Canada), B. pumilus INR-7 otherwise referred to as BU F22 and BU-F33 isolated at least before 1993 from cucumber infested by Erwinia tracheiphila (NRRL B-50185, NRRL B-50153; US 8,445,255), (NRRL B-50754; WO 2014 / 029697; B. pumilus QST 2808 was isolated from soil collected in Pohnpei, Federated States of Micronesia, in 1998 (NRRL B 30087; e. g. Sonata® or Ballad® Plus from Bayer Crop Science LP, USA), B. simplex ABU 288 (NRRL B-50304; US 8,445,255), B. subtilis FB17 also called UD 1022 or UD10-22 isolated from red beet roots in North America (ATCC PTA-11857; System. Appl. Microbiol. 27, 372-379, 2004; US 2010 / 0260735; WO 2011 / 109395); B. thuringiensis ssp. aizawai ABTS-1857 isolated from soil taken from a lawn in Ephraim, Wisconsin, U.S.A., in 1987 (also called ABG 6346; ATCC SD-1372; e. g. XenTari® from BioFa AG, Munsingen, Germany), B. t. ssp. kurstaki ABTS-351 identical to HD-1 isolated in 1967 from diseased Pink Bollworm black larvae in Brownsville, Texas, U.S.A. (ATCC SD-1275; e. g. Dipel® DF from Valent BioSciences, IL, USA), B. t. ssp. kurstaki SB4 isolated from E. saccharina larval cadavers (NRRL B-50753; B. t. ssp. tenebrionis NB-176-1 , a mutant of strain NB-125, a wild type strain isolated in 1982 from a dead pupa of the beetle Tenebrio molitor (DSM 5480; EP 585 215 B1 ; e. g. Novodor® from Valent BioSciences, Switzerland), Beauveria bassiana GHA (ATCC 74250; e. g. BotaniGard® 22WGP from Laverlam Int. Corp., USA), B. bassiana JW-1 (ATCC 74040; e. g. Naturalis® from CBC (Europe) S.r.l., Italy), B. bassiana PPRI 5339 isolated from the larva of the tortoise beetle Conchyloctenia punctata (NRRL 50757), Bradyrhizobium elkanii strains SEMIA 5019 (also called 29W) isolated in Rio de Janeiro, Brazil and SEMIA 587 isolated in 1967 in the State of Rio Grande do Sul, from an area previously inoculated with a North American isolate, and used in commercial inoculants since 1968 (Appl. Environ. Microbiol. 73(8), 2635, 2007; e. g. GELFIX 5 from BASF Agricultural Specialties Ltd., Brazil), B. japonicum 532c isolated from Wisconsin field in U.S.A. (Nitragin 61A152; Can. J. Plant. Sci. 70, 661-666, 1990; e. g. in Rhizoflo®, Histick®, Hicoat® Super from BASF Agricultural Specialties Ltd., Canada), B. japonicum E-109 variant of strain USDA 138 (INTA E109, SEMIA 5085; Eur. J. Soil Biol. 45, 28-35, 2009; Biol. Fertil. Soils 47, 81-89, 2011); B. japonicum strains deposited at SEMIA known from Appl. Environ. Microbiol. 73(8), 2635, 2007: SEMIA 5079 isolated from soil in Cerrados region, Brazil by Embrapa-Cerrados used in commercial inoculants since 1992 (CPAC 15; e. g. GELFIX 5 or ADHERE 60 from BASF Agricultural Specialties Ltd., Brazil), B. japonicum SEMIA 5080 obtained under lab condtions by Embrapa-Cerrados in Brazil and used in commercial inoculants since 1992, being a natural variant of SEMIA 586 (CB1809) originally isolated in U.S.A. (CPAC 7; e. g. GELFIX 5 or AD-HERE 60 from BASF Agricultural Specialties Ltd., Brazil); Burkholderia sp. A396 isolated from soil in Nikko, Japan, in 2008 (NRRL B-50319; WO 2013 / 032693; Marrone Bio Innovations, Inc., USA), Coniothyrium minitans CON / M / 91-08 isolated from oilseed rape (WO 1996 / 021358; DSM 9660; e. g. Contans® WG, Intercept® WG from Bayer CropScience AG, Germany), harpin (alpha-beta) protein (Science 257, 85-88, 1992; e. g. Messenger™ or HARP-N Tek from Plant Health Care pic, U.K.), Helicoverpa armigera nucleopolyhedrovirus (HearNPV) (J. Invertebrate Pathol. 107, 112-126, 2011 ; e. g. Helicovex® from Adermatt Biocontrol, Switzerland; Diplomata® from Koppert, Brazil; Vivus® Max from AgBiTech Pty Ltd., Queensland, Australia), Helicoverpa zea single capsid nucleopolyhedrovirus (HzSNPV) (e. g. Gemstar® from Certis LLC, USA), Helicoverpa zea nucleopolyhedrovirus ABA- NPV-U (e. g. Heligen® from AgBiTech Pty Ltd., Queensland, Australia), Heterorhabditis bacteriophora (e. g. Nemasys® G from BASF Agricultural Specialities Limited, UK), Isaria fumosorosea Apopka-97 isolated from mealy bug on gynura in Apopka, Florida, U.S.A. (ATCC 20874; Biocontrol Science Technol. 22(7), 747-761 , 2012; e. g. PFR-97™ or PreFeRal® from Certis LLC, USA), Metarhizium anisopliae var. anisopliae F52 also called 275 or V275 isolated from codling moth in Austria (DSM 3884, ATCC 90448; e. g. Met52® Novozymes Biologicals Bio- Ag Group, Canada), Metschnikowia fructicola 277 isolated from grapes in the central part of Israel (US 6,994,849; NRRL Y-30752; e. g. formerly Shemer® from Agrogreen, Israel), Paecilomyces ilacinus 251 isolated from infected nematode eggs in the Philippines (AGAL 89 / 030550; W01991 / 02051 ; Crop Protection 27, 352-361 , 2008; e. g. BioAct®from Bayer CropScience AG, Germany and MeloCon® from Certis, USA), Pasteuria nishizawae Pn1 isolated from a soybean field in the mid-2000s in Illinois, U.S.A. (ATCC SD 5833; Federal Register 76(22), 5808, February 2, 2011 ; e.g. Clariva™ PN from Syngenta Crop Protection, LLC, USA), Penicillium bilaiae (also called P. bilaii) strains ATCC 18309 (= ATCC 74319), ATCC 20851 and / or ATCC 22348 (= ATCC 74318) originally isolated from soil in Alberta, Canada (Fertilizer Res. 39, 97-103, 1994; Can. J. Plant Sci. 78(1), 91-102, 1998; US 5,026,417, WO 1995 / 017806; e. g. Jump Start®, Provide® from Novozymes Biologicals BioAg Group, Canada), Reynoutria sachalinensis extract (EP 0307510 B1 ; e. g. Regalia® SC from Marrone BioInnovations, Davis, CA, USA or Milsana® from BioFa AG, Germany), Steinernema carpocapsae (e. g. Millenium® from BASF Agricultural Specialities Limited, UK), S. feltiae (e. g. Nemashield® from BioWorks, Inc., USA; Nemasys® from BASF Agricultural Specialities Limited, UK), Streptomyces microflavus NRRL B-50550 (WO 2014 / 124369; Bayer CropScience, Germany), T. harzianum T- 22 also called KRL-AG2 (ATCC 20847; Bio-Control 57, 687-696, 2012; e. g. Plantshield® from BioWorks Inc., USA or SabrEx™ from Advanced Biological Marketing Inc., Van Wert, OH, USA).

[0154] The invention also provides a method of assaying a plant for resistance to a fungus, comprising the screening for a) the overexpression of a PIP protein of the present invention, b) the expression of a heterologous PIP protein as defined herein, or c) the presence of an exogenous nucleic acid coding for a PIP protein as defined herein.

[0155] It is a particular advantage of the present invention that a single gene, coding for the PIP protein, is all that is required to achieve or improve fungal resistance as described herein. In particular, the invention is not dependent on the co-transformation or presence of further heterologous genes, in particular the cognate receptor RLK7 of the PIP protein. Thus, the invention allows to advantageously screen for the presence of the fungal resistance trait provided by the PIP protein simply by assaying if the PIP protein is overexpressed or if the corresponding gene is present in a plant material. Thus, the invention facilitates breeding methods by allowing a high throughput assay and selection of plant material, in particular seed, for the presence of the fungal resistance trait provided by the present invention.

[0156] In view of these advantages, the invention also provides an automated plant seed selection method, comprising the steps of i) obtaining, for each seed of a plurality of seeds, a sample comprising genetic material of a tissue body representative for said seed, ii) determining the presence of a PIP protein as defined herein, iii) selecting those seed where the determination in step ii) gave a positive result, wherein the seeds are of one or more plants of taxonomic family Fabaceae, more preferably of genus Glycine.

[0157] Ans the invention provides a use of a PIP protein for conferring, modifying or increasing resistance of a plant or plant material against at least one biotrophic or heminecrotrophic fungus, preferably against a rust fungus, wherein the PIP protein is a PIP protein as defined herein. Further advantages and beneficial effects of the present invention are described in the nonlimiting examples and figures appended hereto.

[0158] EXAMPLES

[0159] Example 1 : General methods

[0160] The chemical synthesis of oligonucleotides can be performed, 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).

[0161] Example 2: Cloning of PrePIPI for plant transforamtion

[0162] The cDNA sequence of PrePIPI gene mentioned in this application was generated by DNA synthesis (Geneart, Regensburg, Germany).

[0163] The PrePIPI coding sequence (as shown in SEQ ID NO. 2) was synthesized in a way that a Asci restriction site is located in front of the start-ATG and a Sbfl restriction site downstream of the stop-codon. The synthesized DNA was digested using the restriction enzymes Sbfl and Asci (NEB Biolabs) and ligated in a Sbfl / Ascl digested Gateway pENTRY-B vector (Invitrogen, Life Technologies, Carlsbad, California, USA) in a way that the full-length fragment is located in sense direction between the parsley ubiquitin promoter and the Agrobacterium tumefaciens derived nopaline synthase terminator (t-nos). The PcUbi promoter regulates constitutive expression of the ubi4-2 gene (accession number X64345) of Petroselinum crispum (Kawalleck et al. 1993 Plant Molecular Biology 21 (4): 673 - 684).

[0164] To obtain the binary plant transformation vector, a triple LR reaction (Gateway system, Invitrogen, Life Technologies, Carlsbad, California, USA) was performed according to manufacturer’s protocol by using an empty pENTRY-A vector, the PcUbi promoter::PrePIP1 :nos1 terminator in the above described pENTRY-B vector and an empty pENTRY-C. 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 ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection under control of its endogenous AtAHAS promoter and t-AtAHAS terminator. The recombination reaction was transformed into E. coli (DH5alpha), mini-prepped and screened by specific restriction digestions. A positive clone from each vector construct was sequenced, transformed into Agrobacterium and submitted soy transformation.

[0165] Example 3: Soybean transformation

[0166] The expression vector constructs (see example 2) is transformed into soybean.

[0167] 3.1 Sterilization and Germination of Soybean Seeds

[0168] Virtually any seed of any soybean variety can be employed in the method of the invention. A variety of soybean cultivar (including Jack, Williams 82, Jake, Stoddard, CD215 and Resnik) is appropriate for soybean transformation. Soybean seeds are sterilized in a chamber with a chlorine gas produced by adding 3.5 ml 12N HCI 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 are removed and approximately 18 to 20 seeds are plated on solid GM medium with or without 5 pM 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.

[0169] Seven-day-old seedlings grown in the light (>100 pEinstein / m2s) at 25 degree C are used for explant material for the three-explant types. At this time, the seed coat was split, and the epicotyl with the unifoliate leaves are 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 soybean cultivars and seed lots may vary in the developmental time a description of the germination stage is more accurate than a specific germination time).

[0170] For inoculation of entire seedlings, see Method A (example 3.3.1 and 3.3.2) or leaf explants see Method B (example 3.3.3).

[0171] For method C (see example 3.3.4), the hypocotyl and one and a half or part of both cotyledons are removed from each seedling. The seedlings are 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 are preferably used as target tissue.

[0172] 3.2 - Growth and Preparation of Agrobacterium Culture

[0173] Agrobacterium cultures are 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 NaCI. Adjust pH to 7.0, and bring final volume to 1 liter with H2O, for YEP agar plates add 20g 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 are to be used for A. tumefaciens and A. rhizogenes selection in the YEP solid and liquid media. Various Agrobacterium strains can be used for the transformation method.

[0174] After approximately two days, a single colony (with a sterile toothpick) is picked and 50 ml of liquid YEP is inoculated with antibiotics and shaken at 175 rpm (25 °C.) until an GD600 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 °C.

[0175] The day before explant inoculation, 200 ml of YEP are inoculated with 5 pl to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask. The flask is shaken overnight at 25 °C. until the GD600 is between 0.8 and 1 .0. Before preparing the soybean explants, the Agrobacteria ARE pelleted by centrifugation for 10 min at 5,500 x g at 20 °C. The pellet is suspended in liquid CCM to the desired density (GD600 0.5-0.8) and placed at room temperature at least 30 min before use.

[0176] 3.3 - Explant Preparation and Co-Cultivation(lnoculation)

[0177] 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 are successfully employed. Explants are then prepared with: i) with or without some roots, ii) with a partial, one or both cotyledons, all preformed leaves are removed including apical meristem, and the node located at the first set of leaves is injured with several cuts using a sharp scalpel.

[0178] This cutting at the node not only induces Agrobacterium infection but also distributes the axillary meristem cells and damaged pre-formed shoots. After wounding and preparation, the explants are set aside in a Petri dish and subsequently co-cultivated with the liquid CCM / Agrobacterium mixture for 30 minutes. The explants are then removed from the liquid medium and plated on top of a sterile filter paper on 15x100 mm Petri plates with solid co-cultivation medium. The wounded target tissues are placed such that they are in direct contact with the medium.

[0179] 3.3.2 Modified Method A: Epicotyl Explant Preparation

[0180] Soybean epicotyl segments prepared from 4 to 8 d old seedlings are used as explants for regeneration and transformation. Seeds of soybean are germinated in 1 / 10 MS salts or a similar composition medium with or without cytokinins for 4 to 8 d. Epicotyl explants are prepared by removing the cotyledonary node and stem node from the stem section. The epicotyl is cut into 2 to 5 segments. Especially preferred are segments attached to the primary or higher node comprising axillary meristematic tissue.

[0181] The explants are used for Agrobacterium infection. Agrobacterium AGL1 harboring a plasmid with the gene of interest (GOI) and the AHAS, bar or dsdA selectable marker gene is cultured in LB medium with appropriate antibiotics overnight, harvested and suspended in a inoculation medium with acetosyringone. Freshly prepared epicotyl segments are soaked in the Agrobacterium suspension for 30 to 60 min and then the explants were blotted dry on sterile filter papers. The inoculated explants are then cultured on a co-culture medium with L-cysteine and TTD and other chemicals such as acetosyringone for increasing T-DNA delivery for 2 to 4 d. The infected epicotyl explants are 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 are subcultured on elongation medium with the selective agent.

[0182] For regeneration of transgenic plants, the segments are 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 are transferred to a medium with lower concentration of cytokinin for shoot elongation. Elongated shoots are transferred to a medium with auxin for rooting and plant development. Multiple shoots are regenerated. Many stable transformed sectors showing strong cDNA expression are recovered. Soybean plants are regenerated from epicotyl explants. Efficient T- DNA delivery and stable transformed sectors are demonstrated.

[0183] 3.3.3 Method B: Leaf Explants

[0184] For the preparation of the leaf explant the cotyledon is removed from the hypocotyl. The cotyledons are separated from one another and the epicotyl is removed. The primary leaves, which consist of the lamina, the petiole, and the stipules, are removed from the epicotyl by carefully cutting at the base of the stipules such that the axillary meristems are included on the explant. To wound the explant as well as to stimulate de novo shoot formation, any pre-formed shoots are 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 soybean- explants. Wrap five plates with Parafilm. TM. "M" (American National Can, Chicago, III., USA) and incubate for three to five days in the dark or light at 25 °C.

[0185] 3.3.4 Method C: Propagated Axillary Meristem

[0186] For the preparation of the propagated axillary meristem explant propagated 3-4 week-old plantlets are 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 are prepared 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 is 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 are immediately placed in the Agrobacterium suspension for 20 to 30 minutes. After inoculation, the explants are blotted onto sterile filter paper to remove excess Agrobacterium culture then placed almost completely immersed in solid COM or on top of a round 7 cm filter paper overlaying the solid COM, depending on the Agrobacterium strain. This filter paper prevents Agrobacterium overgrowth on the soybean explants. Plates are wrapped with Parafilm. TM. "M" (American National Can, Chicago, III., USA) and incubated for two to three days in the dark at 25 °C.

[0187] 3.4 - Shoot Induction

[0188] After 3 to 5 days co-cultivation in the dark at 25 °C., the explants are rinsed in liquid SIM medium (to remove excess Agrobacterium) (SIM, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soybean using primary-node explants from seedlings In Vitro Cell. Dev. Biol. — Plant (2007) 43:536-549; to remove excess Agro bacterium) or Modwash medium (1X B5 major salts, 1X B5 minor salts, 1X MSIII iron, 3% Sucrose, 1X B5 vitamins, 30 mM MES, 350 mg / L Timentin 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 are 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 are transferred onto SIM without selection for one week.

[0189] 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.

[0190] For propagated axillary meristem (Method C), the explant is placed into the medium such that it is parallel to the surface of the medium (basipetal) with the explant partially embedded into the medium.

[0191] Wrap plates with Scotch 394 venting tape (3M, St. Paul, Minn., USA) are 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 pE / m2s. The explants remain 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 are transferred from the SIM with or without selection to SIM with selection after about one week. At this time, there is 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).

[0192] Preferably, all shoots formed before transformation are removed up to 2 weeks after cocultivation 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).

[0193] 3.5 - Shoot Elongation

[0194] After 2 to 4 weeks (or until a mass of shoots is formed) on SIM medium (preferably with selection), the explants are transferred to SEM medium (shoot elongation medium, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soybean 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.

[0195] After every 2 to 3 weeks, the explants are transferred 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 are continued to be transferred until the explant dies or shoots elongate. Elongated shoots >3 cm are removed and placed into RM medium for about 1 week (Methods 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 are transferred directly into soil. Rooted shoots are 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 are fertile and produced on average 500 seeds per plant.

[0196] After 5 days of co-cultivation with Agrobacterium tumefaciens transient expression of the gene of interest (GOI) is widespread on the seedling axillary meristem explants especially in the regions wounding during explant preparation (Method A). Explants are 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 GOI is stable after 14 days on SIM, implying integration of the T-DNA into the soybean genome. In addition, preliminary experiments results in the formation of cDNA expressing shoots forming after 3 weeks on SIM.

[0197] For Method C, the average regeneration time of a soybean plantlet using the propagated axillary meristem protocol is 14 weeks from explant inoculation. Therefore, this method has a quick regeneration time that leads to fertile, healthy soybean plants.

[0198] Example 4: Pathogen assay for soybean

[0199] 4.1. Recovery of clones

[0200] 2-3 clones per TO event were generated by cutting tillers of the primary transformed TO plant before transfer of the TO plant to the greenhouse. Clones were rooted in the phytochamber and then potted into small 6cm 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).

[0201] 4.2 Rating for plant health

[0202] A general rating of plant health is performed before (and partially after) the infection experiment. Only those plants are selected for inoculation that show, in general, a healthy phenotype. Healthy phenotype means normal growth habit, green, fully expanded green leaves, having no or only minor lesions, no obvious yellowing, leaf drop or other stress-associated phentotypes. 4.3 Inoculation

[0203] The plants are inoculated with spores of P.pachyrhizi .In order to obtain appropriate spore material for the inoculation, soybean leaves, which are infected with rust 15-20 days ago, are taken 2-3 days before the inoculation and transferred to agar plates (1 % agar in H2O). The leaves are 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 are knocked off the leaves and are added to a Tween-H2O solution. The counting of spores is performed under a light microscope by means of a Thoma counting chamber. For the inoculation of the plants, the spore suspension is 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 a spore density of 1-5x105 spores / ml is used. For the microscopy, a density of >5 x 105 spores I ml is used. The inoculated plants are placed for 24 hours in a greenhouse chamber with an average of 22°C and >90% of air humidity. The following cultivation is performed in a chamber with an average of 25°C and 70% of air humidity.

[0204] Example 5: Microscopical evaluation

[0205] For the evaluation of the pathogen development, the inoculated leaves of plants are 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 accumulate or are produced and are incorporated at the cell wall either locally in papillae or in the whole cell (hypersensitive reaction, HR). Complexes are formed in association with aniline blue, which lead e.g. in the case of callose to yellow fluorescence. The leaf material is transferred to falcon tubes or dishes containing destaining solution II (ethanol I acetic acid 6 / 1) and is incubated in a water bath at 90°C for 10-15 minutes. The destaining solution II is removed immediately thereafter, and the leaves are washed 2x with water. For the staining, the leaves are 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.

[0206] The different interaction types are 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 can be recognized beneath the fungal appressorium by a green / yellow staining. The hypersensitive reaction (HR) is characterized by a whole cell fluorescence.

[0207] By rating and counting 50-200 individaul fungal-plant interaction sites on each trangenic and control leaves it can be observed that resitance inducing genes, show an incrased number of defense associated fungal-plant interactions, such as papillae and hypersensitive reactions, whereas more susceptibility associated phenotypes, such as mycelial growth in the mesophyll, can by observed in control leaves.

[0208] Example 6: Evaluating the susceptibility to soybean rust in greenhouse

[0209] The progression of the soybean rust disease is scored in percent by the estimation of the diseased area (area which was covered by sporulating uredinia)) of a soybean leaf 14 days after inoculation (see above). Additionally, the yellowing of the leaf is taken into account. A scheme illustrating the disease rating can be found in WO2016124515 and W02020120753.

[0210] In total 18 TO soybean plants dervied from 8 independent transgenic events (2-3 clones per event) expressing PrePIPI protein constitutively plus 25 control plants (same genetic background, same treatment as transgenics, see above) were inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soybean against P. pachyrhizi of the inoculated soybean plants were scored 14 days after inoculation. The the ratio of the leaf area showing fungal colonies or strong yellowing / browning on all leaves was considered as diseased leaf area.

[0211] At all 18 primary transgenic TO plants showing constitutive expression of the PrePIPI protein under control of a parsley ubiquitin promoter (Pcllbi) and and 25 non-transgenic wild type control plants were inoculated with spores of Phakopsora pachyrhizi. The 18 transgenic plants were clones derived from 8 independent transgenic events with 2 or 3 clones of each event. The non- transgenic control plants had the same genetic backgrond as transgenic plants. All plants (transgenic and control plants) were grown in parallel in a fully randomized design.

[0212] The expression of PrePIPI was checked by RT-PCR. 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 (for reference see figure 1). The average diseased leaf area of all trangenic events (black bar) is shown in comparison to control (diagonally striped bar, labeled wild type control)

[0213] The constitutive expression of PrePIPI by the construct described in example 2 leads to enhanced resistance of soybean against Phakopsora pachyrhizi in comparison to wild type control.

[0214] As shown in Figure 5 the constitutive expression of PrePIPI protein leads to an average reduction of the diseased leaf area from 29,6 % in wild type (n=25) to 18,3 % in the transgenic plants (construct level; n=18), which represents an average increase of soybean rust resistance by 38,1 %. This result is close to be statistically significant as a one sided Students t-test delivered a p-value of 0,007.

[0215] Example 7: Field trials

[0216] Homozygous T2 seeds were used for field trials. To obtain homozygous seeds, segregating T1 seeds of 5 events per construct were planted. Individual plants that were homozygous for the transgene were selected by using TaqMan® PCR assay as described by the manufacturer of the assay (Thermo Fisher Scientific, Waltham, MA USA 02451).

[0217] Per event 10-30 homozygous plants were grown under standard conditions (12 h daylength, 25°C) and selfed (in-bred). Mature homozygous seeds were harvested approx. 120 days after planting. Harvested seeds of all 10-30 homozygous plants per event were pooled.

[0218] Homozygous seeds of 5 events per construct were tested in the field for "real world" resistance against soybean rust. Field trials were performed in Brazil in a field trial site close to Londina (state of Parana)

[0219] Field trials were planted depending on weather conditions in November or early December to ensure inoculum of Asian soybean rust. Events were tested in split plot trials (2 m long, 4 rows per plot) and 3 replications per event and treatment. Soybean plants were cultivated using standard cultural practice, e.g. in terms of weed and insect control and fertilization. Concerning fungal disease control two treatment varieties were perfomed:

[0220] 1 . No fungicide treatment at all ("no treatment") and

[0221] 2. only one fungicide treatment at the onset of ASR disease (~35 - 40 days after planting), instead of 3-4 treatments over the season in standard agricultural practice.

[0222] The 2 different treatment variaties were performed to increase variability of disease pressure and to prove robustness and stability of the resistance increasing effect. Depending on the trial site and experimental design 8-15 replicates per treatment and location were planted with control seeds. Depending on trial design the untransformed wild-type (WT) mother line or bulk of seeds harvested from null-segregants, grown in parallel to the transgenic mother plants (see above) were used as control.

[0223] Example 8: Soybean rust resistance in field trials

[0224] Field trials as described in example 7 were performed at Londrina (Parana, Brazil). Field trials were run without any fungicide treatment (no treatment) and with one fungicide treatment at the onset of ASR disease (~35 - 40 days after planting) to increase variability of disease pressure to prove robustness and stability of the resistance increasing effect.

[0225] Soybean rust disease severity in field trials was rated by experts using the scheme published by Godoy et al (2006) (citation Godoy, C., Koga, L., Canteri, M. (2006) Diagrammatic scale for assessment of soybean rust severity, Fitopatologia Brasileira 31 (1)).

[0226] In short, the three canopy levels (lower, middle and upper canopy) were rated independently and the average of the infection of all three canopy levels is counted as infection. At all 4 ratings were performed, starting at the early onset of disease and repeated mainly every 6-10 days. If weather was not suitable for disease progression the time in between two ratings was elongated.

[0227] To eliminate transgene insertion effects, which would be only dependent on the integration locus, 5 independent transgenic events were planted per field trial.

[0228] To compare the overall disease severity in different events over the whole growing season we calculated the Area Under Disease Progression Curve (AUDPC) for each event, location and treatment (for reference see: M.J. Jeger and S.L.H. Viljanen-Rollinson (2001) The use of the area under the disease-progress curve (AUDPC) to assess quantitative disease resistance in crop cultivars Theor Appl Genet 102:32-40.).

[0229] To compare the soybean rust resistance across different treatments and sites the deltaAUDPC was calculated using the following formula:

[0230] DeltaAUDPC = AUDPC(event) - AUDPC(control)

[0231] If transgenic plots are more resistant to soybean rust, the AUDPC of the event plots will be smaller than the AUDPC of the control plots. Therefore a negative deltaAUDPC indicates a higher resistance of the transgenic events.

[0232] The deltaAUDPC was calculated on event level.

[0233] Figure 6 shows, the delta AUDPC per event and both treatments. The black bar in Figure 6 ("construct average") indcates the average of all events per treatment (n cmparison to control). The statistical significance of the result of the individual events was calculated based on a 95% confidence level based on the least significant difference (LSD) calculated by a Dunnett's test. The statistical significance of the construct average was calculated using a one sided paired T- test to compare the AUDPC of the ndividual events to the respective average AUDPC of the wt control (construct average no treatment: p=0,01 , construct average one fungiide spray: p=0,009).

[0234] Figure 6 shows that the consitutve expression of PrePIPI leads to an significantly increased disease resitance against soybean rust disease under field conditions, corroborating the results obtained in greenhouse (see example 7).

Claims

CLAIMS1 . A method for conferring, modifying or increasing resistance of a plant or plant material against at least one biotrophic or heminecrotrophic fungus, preferably against a rust fungus, wherein the plant or plant material preferably is of taxonomic family Fabaceae, more preferably of genus Glycine, and wherein the method comprises a step of increasing the production and / or accumulation of a PIP protein in the plant or plant material in comparison to a respective wild-type plant or plant material.

2. The method according to claim 1 , wherein the PIP protein is a) a protein having a sequence identity relative to SEQ ID NO. 1 of 80%-100%, preferably of 81-96%, more preferably 87%-96%; and / or b) a protein having a sequence identity relative to SEQ ID NO. 3 of 74%-100%, preferably of 84-100%, more preferably 90%-100%; and / or c) a protein having a sequence identity relative to SEQ ID NO. 4 of 80%-100%, preferably of 90-100%, more preferably 92%-100%; and / or d) a protein having a homology score relative to SEQ ID NO. 1 of 481 to 628, even more preferably 490 to 628, even more preferably 554 to 628, even more preferably 561 to 628, even more preferably 563 to 628, even more preferably 564 to 628, even more preferably 569 to 628, even more preferably 571 to 628, even more preferably 576 to 628, even more preferably 583 to 628, even more preferably 571-603; and / or e) a protein having a homology score relative to the sequence of positions 31-72 of SEQ ID NO. 1 of 272 to 376, even more preferably 335 to 376, even more preferably 337 to 376, even more preferably 338 to 376, even more preferably 346 to 376, even more preferably 347 to 376, even more preferably 351 to 376, even more preferably 351 to 376, even more preferably 352 to 376, even more preferably 354 to 376, even more preferably 355 to 376, even more preferably 359 to 376, even more preferably 351-359; wherein in case of a protein according to c) or e), the protein preferably further comprises an export signal sequence.

3. The method according to claim 2, comprising the steps of1) stably transforming plant material with at least one expression cassette comprising an exogenous nucleic acid encoding a PIP protein as defined in claim 2,2) regenerating a plant from the plant cell; and3) expressing said PIP protein.

4. A method for production of a genetically modified plant or plant material, preferably of taxonomic family Fabaceae, more preferably of genus Glycine, having increased resistance, compared to a respective wild-type plant or wild-type plant material, against at least one biotrophic or heminecrotrophic fungus, preferably against a rust fungus, comprising the steps of1) introducing an exogenous nucleic acid encoding a PIP protein as defined in claim 2 into a plant or plant material;2) generating a genetically modified plant or genetically modified plant material; and3) expressing the PIP protein in the genetically modified plant or genetically modified plant material.

5. The method for production of a genetically modified plant or plant material having increased fungal resistance according to claim 4, further comprising the steps of harvesting the seeds of the transgenic plant and growing the seeds to plants, wherein the grown plants comprise a heterologous nulceic acid sequence coding for a PIP protein as defined in claim 2.

6. A vector for creating a genetically modified plant, comprising an expression cassette having a PIP gene operably linked to a promoter, wherein the PIP gene codes for a PIP protein as defined in claim 2.

7. A plant or plant material, preferably of taxonomic family Fabaceae, more preferably of genus Glycine, wherein the plant or plant material has increased expression, accumulation or activity of a heterologous PIP protein, wherein the PIP protein is a PIP protein as defined in claim 2.

8. A plant or plant material obtainable or obtained by a method according to any of claims 1 -5.

9. A plant or plant material according to any of claim 8-9, wherein a) the PIP protein comprises an export signal sequence to facilitate export of the PIP protein from the cytosol of a plant cell, and / or b) the plant does not comprise, in the cells capable of expressing the PIP protein, the cognate receptor RLK7.

10. Non-propagative plant part or material of a plant or plant part according to any of claims 7- 9, preferably a fermentation product, oil, meal, press cake, pomace, chaff, straw or compost.11 . Product of a plant or plant part according to any of claims 7-9, wherein the product is obtainable or obtained by i) collecting a material of said plant or plant part, preferably a harvestable plant part and most preferably a plant seed, and ii) disrupting the collected material, preferably to obtain a fermentation product, oil, meal, press cake, pomace, chaff, straw or compost.

12. A method for producing a population of plants each having an enhanced resistance to at least one biotrophic or heminecrotrophic fungus, preferably a rust fungus, comprising the steps of i) multiplying seed of a plant according to any of claims 7-9, wherein the seed are preferably homozygous for the PIP protein, and ii) growing plants of the seed obtained in step i), wherein at least 1000 plants are grown.

13. A farming method for controlling or reducing at least one biotrophic or heminecrotrophic fungus in a field, preferably by reducing or delaying infection of plants in a field and / or reducing or delaying emission of fungal spores from the field, comprising the step of growing plants according to any of claims 7-9 on the field.

14. Method of assaying a plant for resistance to a fungus, comprising the screening for a) the overexpression protein as defined in claim 2, b) the expression of a heterologous PIP protein as defined in claim 2, or c) the presence of an exogenous nucleic acid coding for a PIP protein as defined in claim 2.

15. Automated plant seed selection method, comprising the steps of i) obtaining, for each seed of a plurality of seeds, a sample comprising genetic material of a tissue body representative for said seed, ii) determining the presence of a PIP protein as defined in claim 2, iii) selecting those seed where the determination in step ii) gave a positive result, wherein the seeds are of one or more plants of taxonomic family Fabaceae, more preferably of genus Glycine.

16. Use of a PIP protein for conferring, modifying or increasing resistance of a plant or plant material against at least one biotrophic or heminecrotrophic fungus, preferably against a rust fungus, wherein the PIP protein is a PIP protein as defined in claim 2.

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