WO2013092275A2 - Genes to enhance the defense against pathogens in plants - Google Patents

Abstract

The present invention pertains to methods, means and uses of nucleic acids and polypeptides for conferring, modifying or improving plant resistance against fungal infections. Particularly, the invention provides nucleic acids and polypeptides for conferring, modifying or improving plant resistance against fungal infections. The invention also provides vectors, cells and plants. Also, the invention provides methods for creating corresponding plant cells and plants, and for identification of agents for conferring, modifying or improving plant resistance against fungal infections.

Description

Genes to enhance the defense against pathogens in plants

Summary of the invention The present invention relates to a method of increasing resistance against fungal pathogens in transgenic plants and/or plant cells. In these plants, specific genes are overexpressed or silenced, which show differential expression pattern in Arabidopsis and soybean after inoculation with soybean rust. Depending on the activating or inhibitory function of a particular signaling compound overexpression or knock-down of the cognate gene might be used.

Furthermore, the invention relates to transgenic plants and/or plant cells having an increased resistance against fungal pathogens, for example soybean rust and to recombinant expression vectors comprising a sequence that is identical or homologous to a sequence encoding a functional ethylene signaling compound or fragments thereof.

Background of the invention

The cultivation of agricultural crop plants serves mainly for the production of foodstuffs for humans and animals. Monocultures in particular, which are the rule nowadays, are highly suscep- tible to an epidemic-like spreading of diseases. The result is markedly reduced yields. To date, the pathogenic organisms have been controlled mainly by using pesticides. Nowadays, the possibility of directly modifying the genetic disposition of a plant or pathogen is also open to man.

Resistance generally means the ability of a plant to prevent, or at least curtail the infestation and colonization by a harmful pathogen. Different mechanisms can be discerned in the naturally occurring resistance, with which the plants fend off colonization by phytopathogenic organisms. These specific interactions between the pathogen and the host determine the course of infection (Schopfer and Brennicke (1999) Pflanzenphysiologie, Springer Verlag, Berlin- Heidelberg, Germany).

With regard to the race specific resistance, also called host resistance, a differentiation is made between compatible and incompatible interactions. In the compatible interaction, an interaction occurs between a virulent pathogen and a susceptible plant. The pathogen survives, and may build up reproduction structures, while the host mostly dies off. An incompatible interaction oc- curs on the other hand when the pathogen infects the plant but is inhibited in its growth before or after weak development of symptoms. In the latter case, the plant is resistant to the respective pathogen (Schopfer and Brennicke, vide supra). However, this type of resistance is specific for a certain strain or pathogen. In both compatible and incompatible interactions a defensive and specific reaction of the host to the pathogen occurs. In nature, however, this resistance is often overcome because of the rapid evolutionary development of new virulent races of the pathogens (Neu et al. (2003) American Cytopathol. Society, MPMI 16 No. 7: 626-633). Most pathogens are plant-species specific. This means that a pathogen can induce a disease in a certain plant species, but not in other plant species (Heath (2002) Can. J. Plant Pathol. 24: 259-264). The resistance against a pathogen in certain plant species is called non-host resistance. The non-host resistance offers strong, broad, and permanent protection from phyto- pathogens. 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. The rusts are of great importance. They can have a complicated development cycle with up to five different spore stages (spermatium, aecidiospore, uredospore, teleutospore and ba- sidiospore).

During the infection of plants by pathogenic fungi, different phases are usually observed. The first phases of the interaction between phytopathogenic fungi and their potential host plants are decisive for the colonization of the plant by the fungus. During the first stage of the infection, the spores become attached to the surface of the plants, germinate, and the fungus penetrates the plant. Fungi may penetrate the plant via existing ports such as stomata, lenticels, hydatodes and wounds, or else they penetrate the plant epidermis directly as the result of the mechanical force and with the aid of cell-wall-digesting enzymes. Specific infection structures are developed for penetration of the plant. The soybean rust Phakopsora pachyrhizi directly penetrates the plant epidermis. After crossing the epidermal cell, the fungus reaches the intercellular space of the mesophyll, where the fungus starts to spread through the leaves. To acquire nutrients the fungus penetrates mesophyll cells and develops haustoria inside the mesophyl cell. During the penetration process the plasma membrane of the penetrated mesophyll cell stays intact. Therefore the soybean rust fungus establishes a biotrophic interaction with soybean.

Immediately after the attachment of the spore to the plant cell, the plants senses the presence of the pathogen. Mostly the presence of the pathogen is sensed via so called PAMP receptors, a class of trans-membrane receptor like kinases recognizing conserved pathogen associated molecules (e.g. flagellin or chitin). Downstream of the PAMP receptors, a signaling cascade is initiated leading to the elicitation of defense reactions. On the other hand the pathogen is counteracting the defense related signaling pathway by injecting so-called effector proteins into the host cell. The effectors lead to the inhibition, weakening or deregulation of the defense regula- tion, in a way that the pathogen can further colonize the plant. But these effectors might also be recognized by the plant by major R- (resistance) genes of the NBS-LRR class, leading to a strong defense response.

During million years of evolution, every pathogen shaped its effector set in a way to block exact- ly those defense reactions that are most effective against the particular pathogen. So, the most interesting genes, in terms of creating a disease resistant plant, are those genes that are suppressed by the pathogen in their respective host plant.

Immediately after recognition of a potential pathogen the plant starts to elicit defense reactions. Soybean rust has become increasingly important in recent times. The disease may be caused by the biotrophic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a wide spectrum of leguminosic host plants. P. pachyrhizi, also referred to as Asian rust, is an aggressive pathogen on soy (Glycine max, Soja hispida or Soja max), and is therefore, at least currently, of great importance for agriculture. P. pachyrhizi can be found in nearly all tropical and subtropical soy growing regions of the world. P. pachyrhizi is capable of infecting 31 species from 17 families of the Leguminosae under natural conditions and is capable of growing on further 60 species under controlled conditions (Sinclair et al. (eds.), Proceedings of the rust workshop (1995), National SoyaResearch Laboratory, Publication No. 1 (1996); Rytter J.L. et al., Plant Dis. 87, 818 (1984)). P. meibomiae has been found in the Caribbean Basin and in Puerto Rico, and has not caused substantial damage as yet. P. pachyrhizi can currently be controlled in the field only by means of fungicides. Soy plants with resistance to the entire spectrum of the isolates are not available. When searching for resistant plants, four dominant genes Rpp1 -4, which mediate resistance of soy to P. pachyrhizi, were discovered. The resistance was lost rapidly, as P. pychyrhizi develops new virulent races. In recent years, fungal diseases, e.g. soybean rust, has gained in importance as pest in agricultural production. There was therefore a demand in the prior art for developing methods to control fungi and to provide fungal resistant plants.

Much research has been performed on the field of powdery and downy mildew infecting the epidermal layer of plants. However, the problem to cope with soybean rust which infects the mesophyll remains unsolved. This problem is worsened by the fact that prior to the present invention no prediction could be made regarding plant genes involved in resistance against soybean rust. Thus, constructing resistant plants by means of genetic modification and/or breeding was a matter of try and error, severely reducing the chances of obtaining resistant plants. Also, as no prediction could be made regarding plant genes involved in resistance against soybean rust, it was also not possible to screen in any technically meaningful way for chemical agents increasing expression of genes involved in soybean rust resistance.

Brief summary of the invention

The object of the present invention is thus to provide means for conferring, improving or modifying resistance of plant cells and plants against infections by a fungal pathogen, preferably against infections by a pathogen of the class Basidiomycota, preferably of the order Uredinales, more preferably of the family Phakopsoraceae, even more preferably against soybean rust, even more preferably of the genus Phacopsora, most preferably of the species Phakopsora pachyrhizi (Sydow) and/or Phakopsora meibomiae (Arthur). The invention further provides corresponding nucleic acids, proteins, vectors, host cells, plant cells and plants. Also, it was an object of the present invention to provide means and methods for identification of agents for conferring, increasing or modifying resistance against the fungal infections of a plant as com- pared to a corresponding wild type plant.

Surprisingly the inventors found new genes mediating resistance of soybean against soybean rust by comparing gene expression in two different species, one being a host for soybean rust (i.e. soybean) and another being a non-host for soybean rust (i.e. Arabidopsis). In contrast to the usal way of selecting defense associated genes, only those genes were selected that were downregulated after soybean rust inoculation in soybean (DIS - downregulated in susceptible). To further filter the candidate genes the inventors selected only those that were upregulated in Arabidopsis after infection with soybean rust.

Brief description of the Tables and several views of the drawings

Table 1 shows the list of the candidates selected by the method described in Examples 1 -3. Table 2 shows a list of primers.

Table 3 shows a compilation of nucleic acids and amino acids for the respective DIS genes together with the corresponding sequence identifier. 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.

Figure 2 shows the result of the scoring of 16 transgenic soy plants expressing the DIS-1 over- expression vector construct. TO soybean plants expressing DIS1 protein were inoculated with spores of Phakopsora pachyrhizi. The evaluation of the diseased leaf area on all leaves was performed 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 16 soybean TO plants expressing DIS1 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. The average of the diseased leaf area is shown in Fig 2. Overexpression of DIS1 significantly (p<0.001 ) reduces the diseased leaf area in comparison to non-transgenic control plants.

Figure 3 shows the result of the scoring of 24 transgenic soy plants expressing the DIS-13 overexpression vector construct. TO soybean plants expressing DIS13 protein were inoculated with spores of Phakopsora pachyrhizi. The evaluation of the diseased leaf area on all leaves was performed 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 24 soybean TO plants expressing DIS13 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. The average of the diseased leaf area is shown in Fig 3. Overexpression of DIS13 significantly (p<0.001 ) reduces the diseased leaf area in comparison to non-transgenic control plants.

Figure 4 shows the result of the scoring of 24 transgenic soy plants expressing the DIS-4 over- expression vector construct. TO soybean plants expressing DIS4 protein were inoculated with spores of Phakopsora pachyrhizi. The evaluation of the diseased leaf area on all leaves was performed 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 24 soybean TO plants expressing DIS4 (expression checked by RT-PCR) were evalu- ated in parallel to non-transgenic control plants. The average of the diseased leaf area is shown in Fig 4. Overexpression of DIS13 significantly (p<0.01 ) reduces the diseased leaf area in comparison to non-transgenic control plants.

Figure 5 shows the description of the sequence listing.

Detailed description of the invention

As described above and further explained by the examples hereinafter, it has now been found that proteins encoded by the nucleic acids of the invention are able to confer improved re- sistance to plant cells and plants against infections by a fungal pathogen, preferably against infections by a pathogen of the class Basidiomycota, preferably of order Uredinales, more preferably of family Phakopsoraceae, even more preferably against soybean rust, even more preferably of genus Phacopsora, most preferably of species Phakopsora pachyrhizi (Sydow) and/or Phakopsora meibomiae (Arthur).

The nucleic acids of the present invention can confer such resistance to plant cells and plants that are, in their corresponding wild type, prone to or not resistant to such infections. Susceptibility of infection is determined by exposing a plant to the respective fungal pathogen, allowing time for the pathogen to infect the plant, and determine if the plant leaves show signs of infec- tion, e.g. discoloured or wrinkled areas.

The present invention is thus directed to DIS nucleic acids and the use of a DIS nucleic acid for enhancing fungal resistance in a plant. Preferably the DIS nucleic acid is an isolated nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of: (i) a nucleic acid having in increasing order of preference at least 40%, at least 50%, at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, or a functional fragment, derivative, orthologue, or paralogue thereof;

(ii) a nucleic acid encoding a DIS protein having in increasing order of preference at least

40%, at least 50%, at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, or a functional fragment, derivative, orthologue, or paralogue thereof; preferably the DIS protein has essentially the same biological activity as a DIS protein encoded by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28; preferably the DIS protein confers enhanced fungal resistance relative to control plants;

(iii) a nucleic acid encoding the same DIS protein as the DIS nucleic acids of (i) or (ii) above, but differing from the DIS nucleic acids of (i) or (ii) above due to the degeneracy of the genetic code.

Percentages of identity of a nucleic acid are indicated with reference to the entire nucleotide region given in a sequence specifically disclosed herein.

The DIS nucleic acids described herein are useful in the constructs, methods, plants, harvesta- ble parts and products of the invention.

Another embodiment of the present invention are DIS polypeptides. Preferably, the DIS polypeptide is a polypeptide consisting of or comprising an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence having in increasing order of preference at least 40%, at least 50%, at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, or a functional fragment, derivative, orthologue, or paralogue thereof; preferably the DIS protein has essentially the same biological activity as a DIS protein encoded by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28; preferably the DIS protein confers enhanced fungal resistance relative to control plants; or

(ii) an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 40%, at least 50%, at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, or a functional fragment, derivative, orthologue, or paralogue thereof; preferably the DIS protein confers enhanced fungal resistance relative to control plants.

Preferably, the DIS protein consists of or comprises an amino acid sequence represented by SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27 with one or more conservative amino acid substitutions of the corresponding amino acid residues of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27. Preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 1 -10, 10-20, 20-30, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-1 10, 1 10-120, or 120-130 amino acid residues of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27 are conservative amino acid substitutions of the cor- responding amino acid residue of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27.

More preferably, the DIS protein consists of or comprises an amino acid sequence having at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with an amino acid sequence as represented by SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, wherein at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 of the non-identical amino acid residues, or wherein 1 -10, 10-20, 20-30, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 or even all of the non-identical amino acid residues are conservative amino acid substitutions of the corresponding amino acid residue of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27. Percentages of identity of a polypeptide or protein are indicated with reference to the entire amino acid sequence specifically disclosed herein.

The terms "protein" and "polypeptide" are used herein interchangeably. The DIS proteins described herein are useful in the constructs, methods, plants, harvestable parts and products of the invention.

According to the invention there is thus provided a plant cell comprising, as a heterologous nucleic acid, a nucleic acid selected from the group consisting of

a) a nucleic acid comprising any of the nucleic acid sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28,

b) a nucleic acid coding for a polypeptide comprising any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

c) a nucleic acid coding for a polypeptide having an amino acid sequence identity of at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27. According to the invention, the plant cells comprise the above nucleic acids as heterologous nucleic acids. That is to say that any of the following criteria are met:

a) The nucleic acids are not part of the genome of the corresponding wild type plant, or b) the nucleic acids are not expressed in the genome of the corresponding wild type plant, e.g. susceptibility of the wild type plant to fungal infections does not differ from the susceptibility of a corresponding knockout plant, or

c) the copy number of the nucleic acids in functional form, i.e. that can be expressed by the plant cell, is higher in the plant cells according to the present invention compared to the corresponding wild type plant, or

d) the nucleic acid is under the control of a transcription regulating element which allows to increase transcription of the nucleic acid.

Preferably, the criteria a) and/or b) apply; most preferably criterium a) applies. For the purposes of the present invention, the term "wild type plant" refers to the plant community to which the plant transformed according to the present invention originally belonged to.

The nucleic acids of subgroup c) preferably are similar to the nucleic acids of subgroup a) and/or b). That is, they code for a polypeptide the amino acid sequence of which has a degree of identity of at least 40% to one or more of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27.

Such sequences can differ from the sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28. For example, the nucleic acids may code for a protein consisting of any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27 but differ from the corresponding nucleic acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28 due to the degeneration of the genetic code, i.e. by exchanging a codon for another codon coding for the same amino acid. Such exchange may for example allow to optimize the nucleic acid according to the codon preference of the respective plant cell or plant, thereby allowing an improvement of expression of the respective gene.

For Glycine max, the codon preference inherent in the plant cells is as follows (the most preferred codon per amino acid is marked by an asterisk):

Sequences of sub-group c) can also differ from the sequences of sub-groups a) and b) by coding for a protein mutated by one or more acceptable point mutations. For example, the following amino acid substitutions (indicated by an asterisk) are considered acceptable point mutations according to the present invention:

Preferably, the degree of identity is even higher, e.g. 48%, 58%, 68%, 74%, 78%, 84%, 88%, 90%, 94%, 95%, 96%, 97%, 98% or 99%. Good protection against infection by fungal pathogens can be achieved when the nucleic acid codes for a protein the amino acid of which is any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27.

According to the invention, identity of amino acid sequences and nucleic acid sequences, respectively, is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default pa- rameters and preferably with sequences of mature proteins (i.e. without taking into account secretion signals or transit peptides).

The nucleic acids advantageously are expressed in the plant cell of the present invention. The term "expression" or "gene expression" means the transcription of a specific gene or specific genes or specific genetic construct, particularly of any of the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27. The term "expression" or "gene expression" in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.

The term "increased expression" or "overexpression" as used herein means any form of expression that is additional to the original wild-type expression level. For the purposes of this invention, the original wild-type expression level might also be zero, i.e. absence of expression or immeasurable expression. To allow for expression of the nucleic acid of the present invention, the nucleic acid preferably is under the control of a transcription modifying element.

The invention correspondingly provides a plant cell comprising, as a heterologous polypeptide, a polypeptide selected from the group consisting of

a) polypeptides having any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

b) polypeptides having an amino acid sequence identity of at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any of the amino acid sequences SEQ

ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27.

Again, a protein or polypeptide is considered heterologous if any of the following criteria apply: a) the protein is not part of the proteome of the corresponding wild type plant, or

b) the protein is not expressed in the corresponding wild type plant, or

c) the protein expression in the plant cell according to the present invention is higher than compared to the corresponding wild type plant.

Preferably, the criteria a) and/or b) apply; most preferably criterium a) applies.

The polypeptides allow to confer, modify or increase resistance of the plant cell against infection by fungal pathogens, preferably against infections by a pathogen of the class Basidiomycota, preferably of order Uredinales, more preferably of family Phakopsoraceae, even more preferably against soybean rust, even more preferably of genus Phacopsora, most preferably of spe- cies Phakopsora pachyrhizi (Sydow) and/or Phakopsora meibomiae (Arthur).

As described above for nucleic acids, the amino acid sequence of the polypeptide in the plant cell of the present invention may differ from the amino acid given for sub-group a). That is to say, the plant cell of the present invention can according to sub-group b) comprise a polypep- tide having an amino acid sequence identity of at least 40% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27. Also as described above, the amino acid sequence of the polypeptide may differ from any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27 by one or more acceptable point mutations. Preferably, the degree of identity is higher than 40%, e.g. 48%, 58%, 68%, 74%, 78%, 84%, 88%, 90%, 94%, 95%, 96%, 97%, 98% or 99%.

The invention also provides a vector, preferably an expression vector, comprising a nucleic acid selected from the group consisting of

a) nucleic acids comprising any of the nucleic acid sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28,

b) nucleic acids coding for a polypeptide comprising any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and c) nucleic acids coding for a polypeptide having an amino acid sequence identity of at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27.

Such vectors allow to transfer easily the nucleic acid into a plant of interest. Useful methods of plant transformations are known to the skilled person, e.g. transformation by Agrobacterium tumefaciens. The term "introduction" or "transformation" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meri- stems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

The transfer of foreign genes into the genome of a plant is called transformation. Transfor- mation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injec- tion of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1 102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein TM et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacte- ria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the fol- lowing: European patent application EP 1 198985 A1 , Aldemita and Hodges (Planta 199: 612- 617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271 -282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1 ): 13- 22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991 ) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 871 1 ). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant de- velopment, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:1 -9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternative methods are based on the re- peated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551 -558; Katavic (1994). Mol Gen Genet, 245: 363- 370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pres sure are treated with an agrobacterial suspension [Bechthold, N

(1993). C R Acad Sci Paris Life Sci, 316: 1 194-1 199], while in the case of the "floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non- transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001 ) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21 ; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21 , 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229). The genetically modified plant cells can be regen- erated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Will- mitzer.

With respect to soybean transformation, methods are known which are based on somatic em- bryogenesis: Embryos are induced from immature soybean cotyledons by placing the explant on high levels of 2,4-D (40 mg/L) and the embryogenic tissues are subsequently proliferated on induction medium (Finer (1988) Plant Cell Rep 7:238-241 ) or liquid suspension culture (Finer and Nagasawa (1988) Plant Cell Tissue Organ Cult 15:125-136). Hinchee et al. describes the production of transgenic soybean plants via Agrobacterium- mediated transformation. The production of transgenic plants is based on a regeneration protocol in which shoot organogenesis is induced on cotyledons of soybeans (see Hinchee et al. (1988) Nature Biotechnology, 6:915-922). Also known are methods based on Agrobacterium-mediated transformation of zygotic immature cotyledons (Parrott et al. (1989) Plant Cell Rep 7:615-617; Yan et al. (2000) Plant Cell Rep 19:1090-1097; Ko et al. (2003) Theor Appl Genet. 107:439-447). However, in Parrott et al. the three plants produced were chimeric, from a multicellular origin, and did not transmit the transgene to the next generation. Yan et al. (2000) Plant Cell Rep 19:1090-1097 reported a low transformation frequency of 0.03%. The plant produced transmitted the transgene into the next generation, presumably due to the continuous selection of transformed primary embryos for the production of secondary embryos thereby resulting in non-chimeric plants. Recently, Ko et al. (2003) Theor Appl Genet. 107:439-447 has reported the recovery of transgenic plants at 1 .7% transformation frequencies, however, the method relies on using a partially disarmed (oncogen- ic) Agrobacterium strain, pKYRT, with a functional TR-DNA sequence in order to stimulate em- bryogenesis (Ko et al. (2004) Planta 218:536-541 ). These methods use the immature cotyledons as the target tissue with subsequent proliferation and selection on solid medium.

US2009/0049567 discloses Agrobacterium-mediated soybean transformation utilizing meriste- matic cells of primary or higher leaf nodes as target tissues and subsequent regeneration of the transformed cells into a whole plant.

Further beneficial transformation methods are described in European patent application

EP1 1 175038.6 and US patent application 61/510513, both filed on 22.07.201 1 and incorporated herein by reference in their entirety.

Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art. The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1 ) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chi- meras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

Also provided is a host cell comprising the vector, preferably the expression vector, of the pre- sent invention. Such host cell allows to reproduce or multiply the vector of the present invention, and/or to transform a plant or plant cell. Useful host cells according to the present invention preferably are Escherichia coli and Agrobacterium tumefaciens.

The plant cell according to the present invention preferably is a cell taxonomically belonging to a crop plant species. Particularly preferred are cells of dicotyledon plants, preferably of family Fa- baceae, more preferably of tribe Phaseoleae, even more preferably of genus Glycine, e.g. Glycine soja, Soja hispida, Soja max and Glycine max, and most preferably of Glycine max. Such plants are agriculturally important and suffer, as described above, from fungal pathogen infections.

The invention also provides a plant comprising, consisting essentially of or consisting of one or more plant cells of the present invention. This way, resistance of the plant as a whole against infections by fungal pathogens, preferably against infections by a pathogen of the class Basidi- omycota, preferably of order Uredinales, more preferably of family Phakopsoraceae, even more preferably against soybean rust, even more preferably of genus Phacopsora, most preferably of species Phakopsora pachyrhizi (Sydow) and/or Phakopsora meibomiae (Arthur), can be improved compared to the wild type. As indicated above, the plant preferably is a dicotyledon plant, preferably of family Fabaceae, more preferably of tribe Phaseoleae, even more preferably of genus Glycine, e.g. Glycine soja, Soja hispida, Soja max and Glycine max, and most preferably of Glycine max.

The invention thus provides a plant, preferably a crop plant as described above, exhibiting increased or modified resistance as compared to a wild type plant against infections by a fungus of the class Basidiomycota, preferably of order Uredinales, more preferably of family

Phakopsoraceae, even more preferably of genus Phacopsora, most preferably of species Phakopsora pachyrhizi and/or Phakopsora meibomiae, wherein said plant

i) comprises, consists essentially of or consists of a plant cell as described above; and/or ii) comprises a nucleic acid selected from the group consisting of

a) nucleic acids comprising any of the nucleic acid sequences SEQ ID NO: 2, 4, 6, 8,

10, 12, 14, 16, 18, 20, 22, 24, 26 or 28,

b) nucleic acids coding for a polypeptide comprising any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

c) nucleic acids coding for a polypeptide having an amino acid sequence identity of at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27; and/or

iii) comprises a polypeptide selected from the group consisting of

a) polypeptides having any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 ,

13, 15, 17, 19, 21 , 23, 25 or 27, and

b) polypeptides having an amino acid sequence identity of at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any of the amino acid se- quences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and wherein said polypeptide is heterologous to said plant.

As described herein, the nucleic acid(s) can be constitutively or temporarily expressed to produce the polypeptide(s) in the plant or plant cells. Where the nucleic acid(s) is/are constitutively expressed the respective plant or plant cells permanently comprises a level of the corresponding polypeptide and thus exhibits permanently increased or modified resistance against infection by the pathogen. However, expression of the nucleic acid or nucleic acids can also be regulated such that expression is induced or repressed in predefined conditions. For example, the nucleic acid(s) can be inducibly expressed after application of an inductor to the plant or plant cells. Such inductor can be an artificial agent like IPTG, or can be a constituent of the infecting fungus. This way, production of the corresponding polypeptide or polypeptides can be started or increased in the plant or plant cell in the imminent danger of fungal infection, thereby saving resources when there is no imminent danger of infection. Also, expression can be limited to defined growth stages or organs of the plant, further saving resources of the plant. The invention also provides a method for creating a plant, preferably a crop plant, particularly preferably a dicotyledon plant, preferably of the family Fabaceae, more preferably of the tribe Phaseoleae, even more preferably of the genus Glycine, e.g. Glycine soja, Soja hispida, Soja max and Glycine max, and most preferably of Glycine max, exhibiting increased or modified resistance as compared to a wild type plant against infections by a fungus of the class Basidio- mycota, preferably of order Uredinales, more preferably of family Phakopsoraceae, even more preferably of genus Phacopsora, most preferably of species Phakopsora pachyrhizi and/or Phakopsora meibomiae, wherein the method comprises the steps of

i) introducing a nucleic acid selected from the group consisting of

a) nucleic acids comprising any of the nucleic acid sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28,

b) nucleic acids coding for a polypeptide comprising any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

c) nucleic acids coding for a polypeptide having an amino acid sequence identity of at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27

into a plant cell, and

ii) growing a plant from said plant cell.

The method facilitates the production of plants having modified or increased resistance against infection by the above mentioned pathogen.

The invention also provides a method for creating a plant exhibiting increased or modified resistance as compared to a wild type plant against infections by a fungus of the class Basidiomy- cota, preferably of order Uredinales, more preferably of family Phakopsoraceae, even more preferably of genus Phacopsora, most preferably of species Phakopsora pachyrhizi and/or Phakopsora meibomiae, wherein the method comprises the step of reproducing a plant obtainable or obtained by the method as described above. Reproduction can be by means of seed generation or by artificial reproduction, e.g. using callus cultures.

Thus, the invention also provides a seed of a plant of the present invention. Such seeds benefi- cially allow to grow plants e.g. on a field having increased or modified resistance as compared to a wild type plant against infections by a fungus of the class Basidiomycota, preferably of order Uredinales, more preferably of family Phakopsoraceae, even more preferably of genus Phacopsora, most preferably of species Phakopsora pachyrhizi and/or Phakopsora meibomiae. Also corresponding to the above description, the seed preferably is a seed of a preferably a crop plant, particularly preferably a dicotyledon plant, preferably of family Fabaceae, more preferably of tribe Phaseoleae, even more preferably of genus Glycine, e.g. Glycine soja, Soja hispida, Soja max and Glycine max, and most preferably of Glycine max.

The invention also provides a method of conferring, increasing or modifying, in a plant, re- sistance as compared to a wild type plant against infections by a fungus of the class Basidiomy- cota, preferably of the order Uredinales, more preferably of the family Phakopsoraceae, even more preferably of the genus Phacopsora, most preferably of the species Phakopsora pachyrhi- zi and/or Phakopsora meibomiae, wherein the method comprises the step of

i) inducing or increasing expression of a polypeptide selected from the group consisting of a) polypeptides having any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

b) polypeptides having an amino acid sequence identity of at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and c) homologs of the polypeptides having any of the amino acid sequences SEQ ID NO:

1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27. By inducing the expression of the polypeptide, preferably by inducing the expression using a nucleic acid as described above, production of the corresponding polypeptide or polypeptides can be started or increased in the plant or plant cell in the imminent danger of fungal infection, thereby saving resources when there is no imminent danger of infection. Also, expression can be limited to defined growth stages or organs of the plant, further saving resources of the plant.

In view of the present invention, "induction" or "inducing" is understood to comprise both the increase of a previously lower level of expression, and the commencement of previously not detectable expression. In the above method, expression of homologs of the polypeptides having any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27 can be induced. A polypeptide is considered to be a homolog if its pattern of expression resembles that of one or more of the aforementioned polypeptides. Such expression pattern can be determined as described in the examples. Also, homologs can be found in databases, e.g. in the HomoloGene database maintained at the US National Center for Biotechnology Information (NCBI). The term "homolog" therefor includes orthologs and paralogs.

Harvestable parts of the transgenic plant according to the present invention are part of the invention. Preferably, the harvestable parts comprise the DIS nucleic acid or DIS protein. The harvestable parts may be seeds, roots, leaves and/or flowers comprising the DIS nucleic acid or DIS protein or parts thereof. Preferred parts of soy plants are soy beans comprising the DIS nucleic acid or DIS protein.

Products derived from a transgenic plant according to the present invention, parts thereof or harvestable parts thereof are part of the invention. A preferred product is meal or oil, preferably, soybean meal or soybean oil. Preferably, the soybean meal and/or oil comprises the DIS nucleic acid or DIS protein.

One embodiment is a method for the production of a product comprises a) growing the plants of the invention or obtainable by the methods of the invention and b) producing said product from or by the plants of the invention and/or parts, e.g. seeds, of these plants. In a further embodiment the method comprises the steps a) growing the plants of the invention, b) removing the harvestable parts as defined above from the plants and c) producing said product from or by the harvestable parts of the invention.

The product may be produced at the site where the plant has been grown, the plants and/or parts thereof may be removed from the site where the plants have been grown to produce the product. Typically, the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant. The step of growing the plant may be performed only once each time the methods of the invention is performed, while allowing repeated times the steps of product production e.g. by repeated re- moval of harvestable parts of the plants of the invention and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants of the invention is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts. Also, the steps of growing the plants and producing the product may be performed with an overlap in time, even simulta- neously to a large extend or sequentially. Generally the plants are grown for some time before the product is produced.

In one embodiment the products produced by said methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic and/or pharmaceutical. Foodstuffs are regarded as compositions used for nutrition and/or for supplementing nutrition. Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs.

In another embodiment the inventive methods for the production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.

It is possible that a plant product consists of one or more agricultural products to a large extent. The transgenic plants of the invention may be crossed with similar transgenic plants or with transgenic plants lacking the nucleic acids of the invention or with non-transgenic plants, using known methods of plant breeding, to prepare seeds. Further, the transgenic plant cells or plants of the present invention may comprise, and/or be crossed to another transgenic plant that comprises one or more exogenous nucleic acids, thus creating a "stack" of transgenes in the plant and/or its progeny. The seed is then planted to obtain a crossed fertile transgenic plant comprising the DIS nucleic acid. The crossed fertile transgenic plant may have the particular expression cassette inherited through a female parent or through a male parent. The second plant may be an inbred plant. The crossed fertile transgenic may be a hybrid. Also included within the present invention are seeds of any of these crossed fertile transgenic plants. The seeds of this invention can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plant lines comprising the exogenous nucleic acid. Thus, one embodiment of the present invention is a method for breeding a fungal resistant plant comprising the steps of

(a) crossing a transgenic plant described herein or a plant obtainable by a method described herein with a second plant;

(b) obtaining a seed or seeds resulting from the crossing step described in (a);

(c) planting said seed or seeds and growing the seed or seeds to plants; and

(d) selecting from said plants the plants expressing a DIS protein, preferably encoded by

(i) a heterologous nucleic acid having at least 40% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, a functional fragment thereof, an orthologue or a paralogue thereof, or a splice variant thereof;

(ii) a heterologous nucleic acid encoding a protein having at least 40% identity with

SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, or a functional fragment thereof, an orthologue or a paralogue thereof; preferably the encoded protein confers enhanced fungal resistance relative to control plants;

(iii) a heterologous nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); preferably encoding a DIS protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27; preferably the encoded protein confers enhanced fungal resistance relative to control plants; and / or by (iv) a heterologous nucleic acid encoding the same DIS protein as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.

Another preferred embodiment is a method for plant improvement comprising

(a) obtaining a transgenic plant by any of the methods of the present invention;

(b) combining within one plant cell the genetic material of at least one plant cell of the plant of (a) with the genetic material of at least one cell differing in one or more gene from the plant cells of the plants of (a) or crossing the transgenic plant of (a) with a second plant;

(c) obtaining seed from at least one plant generated from the one plant cell of (b) or the plant of the cross of step (b);

(d) planting said seeds and growing the seeds to plants; and

(e) selecting from said plants, plants expressing the nucleic acid encoding the DIS protein; and optionally

(f) producing propagation material from the plants expressing the nucleic acid encoding the DIS protein.

The transgenic plants may be selected by known methods as described above (e.g., by screening for the presence of one or more markers which are encoded by plant-expressible genes co- transferred with the DIS gene or screening for the DIS nucleic acid itself). The invention is hereinafter further described using examples, tables and figures, particularly detailing preferred embodiments of the present invention. However, neither of these is to be construed as limiting the scope of the claims. Examples

The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the present in- vention.

Example 1 : General methods

The chemical synthesis of oligonucleotides can be affected, for example, in the known fashion using the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896- 897). The cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, phage multiplication and sequence analysis of recombinant DNA, are carried out as described by Sambrook et al. Cold Spring Harbor Laboratory Press

(1989), ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules is carried out with an MWG-Licor laser fluorescence DNA sequencer following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)). Example 2: Performing microarray analysis with Arabidopsis thaliana plants inoculated with soybean rust

To obtain the material Arabidopsis thaliana Col-0 plants were grown under short day conditions (8 h photoperiod), a temperature of 22 °C, a relative humidity of 65 % and a light intensity of 120 μηηοΙ m-2 s-1 for five weeks and inoculated with P. pachyrhizi uredospores (1 mg/ml in

0.01 % Tween-20) brushed from infested soybean plants at two to three weeks post inoculation. After foliar spray application plants were covered by transparent moistened domes to ensure high levels of humidity for 24 h and kept under short day conditions. For microarray analysis, three leaf samples were harvested per plant from three plants at 0 and 48 hours post inocula- tion and frozen in liquid nitrogen. Leaf samples were pooled for every point of time, ground in liquid nitrogen and RNA was extracted following a modified guanidinium thiocyanate-phenol protocol (Chomczynski and Sacchi, 1987) in combination with the Rneasy® Mini Kit (Qiagen). Thereto, 1 ml of RNA extraction reagent was added to 100 mg of homogenized plant material and the mixture was vortexed thoroughly. After an incubation of 5 min at RT, 200 μΙ of chloro- form were added and the samples mixed again for at least 15 sec. After incubation for 3 more minutes the samples were centrifuged at -4 °C and 14,000 rpm for 15 min in a tabletop centrifuge. Subsequently, 400 μΙ of the upper aqueous phase was transferred into a new microcenti- fuge tube and RNA was precipitated adding 200 μΙ ethanol (96 %). From here, the Rneasy® Mini Kit (Qiagen) was followed starting from step 6 (Protocol: Purification of Total RNA from Plant Cells and Tissues and Filamentous Fungi). 10 to 15 μg of RNA of each sample was sent for microarray analysis, which was performed by IFG Munster using Affymetrix ATH1 gene chips. Microarray signal data obtained from IFG Munster was processed and normalized by dCHIP Software ((Li and Wong, 2003), normalization method invariant set).

Example 3: Bioinformatic analysis

Expression data for soybean inoculated with P. pachyrhizi were retrieved from a publication (van de Mortel et al., 2007), in which the authors measured abundance changes of soybean mRNAs over a time period of seven days after infection with P. pachyrhizi using the GeneChip Soybean Genome Array. All data obtained in their study are deposited and available in the Plant Expression database (PLEXdb, accession number GM2). Data from infected samples at 24, 36, and 48 hours post inoculation of the susceptible genotype Embrapa-48 were analyzed with respect to their corresponding mock-inoculated samples. Genes with a threshold of four-fold dif- ferential reduction in transcript level in the P. pachyrhizi-treated samples in comparison to mock treatment were selected.

The 150 genes which showed the most prominent reduction in gene expression were compared to the Arabidopsis thaliana genome by BLASTX resulting in the identification of orthologous genes in Arabidopsis. These orthologs identified were further examined by comparison with the result of the above decribed microarray analysis of Arabidopsis accession Columbia-0 (Col-0) plants inoculated with P. pachyrhizi.

At all 14 genes match all criteria (see Table 1 ). All Arabidopsis genes that are orthologs to the downregulated soybean genes and which were upregulated at 48 hpi in Arabidopsis were selected as candidate genes and named DIS (downregulated in susceptible) genes.

Example 4 cloning of Arabidopsis knock-down vectors To analyze the the contribution of the DIS genes to Arabidopsis' nonhost resistance against

P. pachyrhizi by double stranded RNA silencing either parts of the candidate gene sequence or the full coding region was used in a silencing vector to mediate post-transcriptional gene silencing. If available, clones for Arabidopsis candidate genes were obtained from TAIR (www. arabidopsis. org, see

Table 1 ) as either entry clones containing full length coding region (AT1 G19210, AT1 G49000, AT2G24180, AT3G24550, AT5G44020) or parts of it (AT1 G68320, AT3G25500, AT5G21 105) or as expression vectors containing artificial microRNA sequences specifically targeting the gene of interest (AT1 G19210, AT1 G49000, AT1 G64640, AT3G24550, AT4G17670,

AT5G17300, AT5G21 105). Entry clones were used in the LR reaction to clone the gene-specific insert into the destination vector pJawohl8 (B. Lllker, Genbank accession number AF408413.1 ). The destination vectors include two lambda integration sites, one in a sense and one in an anti- sense direction, to create a double-stranded hairpin RNA which will be recognized by the post- transcriptional gene silencing machinery and, therefore, mediate specific degradation of targeted mRNA. Expression clones were used for plant transformation (see below). For several candidates, multiple expression constructs were used (see

Table 1 ). For candidate genes without corresponding clones at TAIR, a fragment for silencing (full length (AT2G30490) or a fragment of the coding regions' 3' ends part (AT1 G66250, AT5G18270)) was amplified by PCR using primers which were designed using the Primer 3 program. The Gateway-specific regions attbl and attb2 were added at the primers' 5' ends for cloning with Gateway® technology (Invitrogen). Amplification was done using proofreading Phusion® High-Fidelity DNA Polymerase (Finnzymes). Primer sequences are listed in Table 2. PCR products were cleaned using the QIAquick® PCR Purification Kit (Qiagen). Gateway® cloning was performed according to manufacturer's instructions using Gateway® BP Clonase® II enzyme mix and Gateway® LR Clonase® II enzyme mix (both Invitrogen). As donor vector, pDONR207 (Invitrogen) was used and as destination vector, the sequences were cloned into the binary silencing vector pJawohl8. Both, Entry and expression vectors were amplified in E. coli DH5a and isolated from bacteria using the ZyppyTM Plasmid Miniprep kit (Zymo Research).

Example 5 Transformation of Arabidopsis

For plant transformation, the expression vectors were transformed into Agrobacterium tumefa- ciens strain GV3101 and Arabidopsis Col-0 and pen2-1 plants (Lipka et al., 2005) were dipped into Agrobacterium suspension following a modified protocol (Bechtold and Pelletier, 1998) of Clough and Bent (1998). Only CSHL expression clones (s.

Table 1 ) were co-transformed with Agrobacteria containing pSoup (Hellens et al., 2000). Dipped plants were allowed to set seed. Seeds produced by dipped plants were sown on soil drenched with 1 ml/l (v/v) Basta® (Bayer CropScience) for selection of plants with transformation events. The pJawohl8-based vectors carry the coding sequence of a phosphinotricin-acetyltransferase which provides resistance against the glufosinate herbicide Basta®. Healthy-looking seedlings were transplanted into fresh soil at approximately two weeks after germination. Example 6 Evaluation of gene silencing by qPCR

The transformed plants were tested for positive silencing using reverse transcription quantitative PCR (RT-qPCR). Thereto, five-week-old plants were inoculated with P. pachyrhizi together with control plants Col-0 and pen2-1 as described above. Leaf material was harvested for each plant after 48 hpi for RNA extraction (one leaf per plant) and microscopy (three leaves per plant). RNA was extracted from Arabidopsis leaves following a modified guanidinium thiocyanate- phenol protocol (Chomczynski and Sacchi, 1987). 1 ml of RNA extraction reagent was added to 100 mg of homogenized plant material and the mixture was vortexed thoroughly. After an incubation of 5 minutes at RT, 200 μΙ of chloroform were added and the samples mixed again for at least 15 sec. After incubation for 3 more minutes the samples were centrifuged at -4 °C and 14,000 rpm for 15 min in a tabletop centrifuge. Subsequently, the upper aqueous phase was transferred into a new microcentifuge tube. To precipitate the RNA 500 μΙ of isopropanol were added, the samples shortly mixed on a Vortex shaker, incubated at RT for 10 min, and centri- fuged at -4 °C and 14,000 rpm for 10 min. The resulting pellet was washed with 70 % (v/v) eth- anol and centrifuged again for 5 minutes. Afterwards the supernatant was removed, the pellet dried for 10 min and then 30 μΙ of DEPC-treated water were added. RNA was dissolved by heating the samples for 10 min at 60 °C and pipetting up and down. RNA was stored at -20 °C. Be- fore cDNA synthesis, a 1 μg of RNA of each sample was subjected to DNAse 1 treatment of the RNA samples was performed according to manufactures instructions (Fermentas). For synthesis of cDNA 1 μΙ random primer (50 μΜ stock solution, Invitrogen) and 1 μΙ DEPC-treated water were added to each sample, followed by an incubation at 70 °C for 5 min, to allow primer annealing. Afterwards, 4 μΙ 5x M-MuLV buffer (Fermentas), 2 μΙ dNTPs (10 mM stock solution), 1 μΙ_ RevertAid™ M-MuLV Reverse Transcriptase (Fermentas) and 1 μΙ DEPC-treated water were added to each sample. Reverse transcription of RNA was initiated by incubation of the samples at 37 °C for 60 min. With a last incubation step at 70 °C for 10 min the enzyme was inactivated. cDNA was stored at -20 °C. To perform RT-qPCR a master mix of 5 μΙ Platinum® SYBR® Green qPCR Super-Mix-UDG with ROX (Invitrogen), 7 μΙ H20, and 0.15 μΙ of each primer (end concentration 150 nM) per reaction was prepared and pipetted in 96-well plates (Sarstedt). Before use cDNA was diluted 1 :10 for Arabidopsis and 1 :2 for soybean, respectively. 2 μΙ cDNA were added to each well and for each reaction three technical replicates were done. The PCR reaction was performed in a iQ™5 Real-Time PCR Detection System (Bio-Rad) under the following conditions: 2 min

(50 °C), 10 min (95 °C), 40 cycles [15 s (95 °C), 1 min (60 °C)]. The iQ5 optical system software was used to analyze the data. According to the mathematic model of Livak and Schmittgen (2001 ) every gene specific transcript level was normalized to the respective housekeeping gene using the 2-AACt method. ACTIN2 was used as the supposedly constitutively expressed refer- ence gene for Arabidopsis while for soybean, the nitrate transporter 1 (NRT1 -5) was chosen according to Choi et al. (2008). Genes were defined to be silenced at a relative expression level of 50 % or below in comparison to the non-transformed control.

Example 7: Resistance evaluation in DIS13 silenced Arabidopsis thaliana

Successfully silenced plants were analyzed microscopically after trypan blue staining to determine if the interaction of the plant with P. pachyrhizi has changed in silenced plants compared to untransformed control plants. Thereto, Arabidopsis leaves were incubated in trypan blue solution (10% (v/v) lactic acid, 10% (v/v) glycerol, 10% (v/v) deionized H20, 70% (v/v) Ethanol, 0,025% (w/v) trypan blue) and incubated for 1 min at 80 °C to stain fungal structures and collapsed plant cells (Keogh et al., 1980). After incubation for 10 min at RT the staining solution was replaced by chloral hydrate (2.5 g/ml (w/v)) destaining solution. Leaves were kept in destaging solution for at least two days, before they were mounted on object slides in 50 % (v/v) glycerol. For microscopic analysis a Nikon Eclipse50i and a Leica DMRBE microscope were used. 100 interaction sites were examined on each leaf. Statistical evaluation of microscopic results was done with help of the software R (version 2.10.1 ).

Successful silencing of the resistance gene DIS13 either by RNAi or by miRNA leads to an increased susceptibility of Arabidopsis to soybean rust. Col-0 plants transformed with pAMIR- DIS13 (miRNA) as well as Col-0 plants transformed with pJawohl8-DIS13 (RNAi), which had significant transcript reduction for DIS13, showed up to 7 and 4.5 % enhanced fungal growth in the plant's mesophyll layer in comparison to untransformed Col-0 plants at 2 dpi with P. pach- yrhizi during microscopical analysis of the interaction.

Example 8 : Cloning of overexpression vector constructs

The cDNAs of all genes mentioned in this application were generated by DNA synthesis (Gene- art, Regensburg, Germany).

The DIS1 cDNA was synthesized in a way that a attB1 -recombination site (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA) is located in front of the start-ATG and a attB2 recombination site is located directly downstream of the stop-codon. The synthesized cDNA were transferred to a pENTRY-B vector by using the BP reaction (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA ) according to the protocol provided by the supplier. To obtain the binary plant transformation vector, a triple LR reaction (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA) was performed according to manufacturers protocol by using a pENTRY-A vector containing a parsley ubiquitine promoter, the cDNA in a pENTRY-B vector and a pENTRY-C vector containing a StCAT-pA terminator. As target a binary pDEST vector was used which is composed of: (1 ) a Spectinomy- cin/Streptomycin cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a pBR322 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection under control of a pcUbi-promoter. The recombination reaction was transformed into E. coli (DH5alpha), mini-prepped and screened by specific restriction di- gestions. A positive clone from each vector construct was sequenced and submitted soy transformation.

The cDNA of all other DIS-genes (e.g. DIS13, DIS4, etc.)was synthesized in a way that a Pad restriction site is located in front of the start-ATG and a Ascl restriction site downstream of the stop-codon. The synthesized cDNA were digested using the restriction enzymes Pad and Ascl (NEB Biolabs) and ligated in a Pacl/Ascl digested Gateway pENTRY vector (Invitrogen, Life Technologies, Carlsbad, California, USA) in a way that the full-length fragment is located in sense direction between the parsley ubiquitine promoter (PcUbi) and a Solanum tuberosum CAT-pA terminator.

To obtain the binary plant transformation vector, a triple LR reaction (Gateway system, (Invitrogen, Life Technologies, Carlsbad, California, USA) was performed according to manufacturers' protocol by using an empty pENTRY-A vector, the above described pENTRY-B vector containing the cDNA and an empty pENTRY-C vector. As target a binary pDEST vector was used which is composed of: (1 ) a Spectinomycin/Streptomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a pBR322 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection under control of a PcUbi-promoter. 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 and submitted soy transformation.

Example 9 Soy transformation

The expression vector constructs (see example 8) were transformed into soy.

9.1 Sterilization and Germination of Soy Seeds Virtually any seed of any soy variety can be employed in the method of the invention. A variety of soy cultivar (including Jack, Williams 82, and Resnik) is appropriate for soy transformation. Soy seeds were sterilized in a chamber with a chlorine gas produced by adding 3.5 ml 12N 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 were removed and approximately 18 to 20 seeds were plated on solid GM medium with or without 5 μΜ 6-benzyl-aminopurine (BAP) in 100 mm Petri dishes. Seedlings without BAP are more elongated and roots develop, especially secondary and lateral root formation. BAP strengthens the seedling by forming a shorter and stockier seedling. Seven-day-old seedlings grown in the light (>100 μΕίη8ί.βίη/Γη28) at 25 °C were used for explant material for the three-explant types. At this time, the seed coat was split, and the epicotyl with the unifoliate leaves have grown to, at minimum, the length of the cotyledons. The epicotyl should be at least 0.5 cm to avoid the cotyledonary-node tissue (since soycultivars and seed lots may vary in the developmental time a description of the germination stage is more accurate than a specific germination time).

For inoculation of entire seedlings (Method A, see example 3.3. and 3.3.2) or leaf explants (Method B, see example 3.3.3), the seedlings were then ready for transformation. For method C (see example 3.3.4), the hypocotyl and one and a half or part of both cotyledons were removed from each seedling. The seedlings were then placed on propagation media for 2 to 4 weeks. The seedlings produce several branched shoots to obtain explants from. The majority of the explants originated from the plantlet growing from the apical bud. These explants were preferably used as target tissue.

9.2 - Growth and Preparation of Agro bacterium Culture

Agrobacterium cultures were prepared by streaking Agrobacterium (e.g., A. tumefaciens or A. rhizogenes) carrying the desired binary vector (e.g. H. Klee. R. Horsch and S. Rogers 1987 Ag- robacterium-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 H20, for YEP agar plates add 20g Agar, autoclave) and incubating at 25° C. until colonies appeared (about 2 days). Depending on the selectable marker genes present on the Ti or Ri plasmid, the binary vector, and the bacterial chromosomes, different selection compounds were be used for A. tumefaciens and rhizogenes selection in the YEP solid and liquid media. Various Agrobacterium strains can be used for the transformation method. After approximately two days, a single colony (with a sterile toothpick) was picked and 50 ml of liquid YEP was inoculated with antibiotics and shaken at 175 rpm (25° C.) until an Οϋβοο 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.

The day before explant inoculation, 200 ml of YEP were inoculated with 5 μΙ to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask. The flask was shaked overnight at 25° C. until the Οϋβοο was between 0.8 and 1 .0. Before preparing the soyexplants, the Agrobacteria were pelleted by centrifugation for 10 min at 5,500xg at 20° C. The pellet was resuspended in liquid CCM to the desired density (Οϋβοο 0.5-0.8) and placed at room temperature at least 30 min before use.

9.3 - Explant Preparation and Co-Cultivation (Inoculation) 9.3.1 Method A: Explant Preparation on the Day of Transformation.

Seedlings at this time had elongated epicotyls from at least 0.5 cm but generally between 0.5 and 2 cm. Elongated epicotyls up to 4 cm in length had been successfully employed. Explants were then prepared with: i) with or without some roots, ii) with a partial, one or both cotyledons, all preformed leaves were removed including apical meristem, and the node located at the first set of leaves was injured with several cuts using a sharp scalpel.

This cutting at the node not only induced Agrobacterium infection but also distributed the axillary meristem cells and damaged pre-formed shoots. After wounding and preparation, the explants were set aside in a Petri dish and subsequently co-cultivated with the liquid CCM/Agrobacterium mixture for 30 minutes. The explants were then removed from the liquid medium and plated on top of a sterile filter paper on 15x100 mm Petri plates with solid co-cultivation medium. The wounded target tissues were placed such that they are in direct contact with the medium. 9.3.2 Modified Method A: Epicotyl Explant Preparation

Soy epicotyl segments prepared from 4 to 8 d old seedlings were used as explants for regeneration and transformation. Seeds of soyacv L00106CN, 93-41 131 and Jack were germinated in 1/10 MS salts or a similar composition medium with or without cytokinins for 4 to 8 d. Epicotyl explants were prepared by removing the cotyledonary node and stem node from the stem section. The epicotyl was cut into 2 to 5 segments. Especially preferred are segments attached to the primary or higher node comprising axillary meristematic tissue.

The explants were used for Agrobacterium infection. Agrobacterium AGL1 harboring a plasmid with the GUS marker gene and the AHAS, bar or dsdA selectable marker gene was cultured in LB medium with appropriate antibiotics overnight, harvested and resuspended in a inoculation medium with acetosyringone . Freshly prepared epicotyl segments were soaked in the Agrobac- terium suspension for 30 to 60 min and then the explants were blotted dry on sterile filter pa- pers. The inoculated explants were then cultured on a co-culture medium with L-cysteine and TTD and other chemicals such as acetosyringone for enhancing T-DNA delivery for 2 to 4 d. The infected epicotyl explants were then placed on a shoot induction medium with selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene), or D-serine (for dsdA gene). The regenerated shoots were subcultured on elongation medium with the selective agent.

For regeneration of transgenic plants the segments were then cultured on a medium with cyto- kinins such as BAP, TDZ and/or Kinetin for shoot induction. After 4 to 8 weeks, the cultured tissues were transferred to a medium with lower concentration of cytokinin for shoot elongation. Elongated shoots were transferred to a medium with auxin for rooting and plant development. Multiple shoots were regenerated.

Many stable transformed sectors showing strong expression were recovered. Soyplants were regenerated from epicotyl explants. Efficient T-DNA delivery and stable transformed sectors were demonstrated.

9.3.3 Method B: Leaf Explants

For the preparation of the leaf explant the cotyledon was removed from the hypocotyl. The coty- ledons were separated from one another and the epicotyl is removed. The primary leaves, which consist of the lamina, the petiole, and the stipules, were removed from the epicotyl by carefully cutting at the base of the stipules such that the axillary meristems were included on the explant. To wound the explant as well as to stimulate de novo shoot formation, any pre-formed shoots were removed and the area between the stipules was cut with a sharp scalpel 3 to 5 times.

The explants are either completely immersed or the wounded petiole end dipped into the Agro- bacterium suspension immediately after explant preparation. After inoculation, the explants are blotted onto sterile filter paper to remove excess Agrobacterium culture and place explants with the wounded side in contact with a round 7 cm Whatman paper overlaying the solid CCM medium (see above). This filter paper prevents A. tumefaciens overgrowth on the soyexplants. Wrap five plates with Parafilm™ "M" (American National Can, Chicago, III., USA) and incubate for three to five days in the dark or light at 25° C. 9.3.4 Method C: Propagated Axillary Meristem

For the preparation of the propagated axillary meristem explant propagated 3-4 week-old plant- lets were used. Axillary meristem explants can be pre-pared from the first to the fourth node. An average of three to four explants could be obtained from each seedling. The explants were pre- pared from plantlets by cutting 0.5 to 1 .0 cm below the axillary node on the internode and removing the petiole and leaf from the explant. The tip where the axillary meristems lie was cut with a scalpel to induce de novo shoot growth and allow access of target cells to the Agrobacte- rium. Therefore, a 0.5 cm explant included the stem and a bud.

Once cut, the explants were immediately placed in the Agrobacterium suspension for 20 to 30 minutes. After inoculation, the explants were blotted onto sterile filter paper to remove excess Agrobacterium culture then placed almost completely immersed in solid CCM or on top of a round 7 cm filter paper overlaying the solid CCM, depending on the Agrobacterium strain. This filter paper prevents Agrobacterium overgrowth on the soyexplants. Plates were wrapped with Parafilm™ "M" (American National Can, Chicago, III., USA) and incubated for two to three days in the dark at 25° C.

9.4 - Shoot Induction

After 3 to 5 days co-cultivation in the dark at 25° C, the explants were rinsed in liquid SIM medium (to remove excess Agrobacterium) (SIM, see Olhoft et al 2007 A novel Agrobacterium rhi- zogenes-mediated transformation method of soyusing primary-node explants from seedlings In Vitro Cell. Dev. Biol.— Plant (2007) 43:536-549; to remove excess Agrobacterium) or Modwash medium (1 X B5 major salts, 1X B5 minor salts, 1X MSI 11 iron, 3% Sucrose, 1 X 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 were placed such that the target tissue was in direct contact with the medium. During the first 2 weeks, the ex- plants could be cultured with or without selective medium. Preferably, explants were transferred onto SIM without selection for one week.

For leaf explants (Method B), the explant should be placed into the medium such that it is perpendicular to the surface of the medium with the petiole imbedded into the medium and the lam- ina out of the medium.

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

Wrap plates with Scotch 394 venting tape (3M, St. Paul, Minn., USA) were placed in a growth chamber for two weeks with a temperature averaging 25° C. under 18 h light/6 h dark cycle at 70-100 μΕ/Γη²8. The explants remained on the SIM medium with or without selection until de novo shoot growth occured at the target area (e.g., axillary meristems at the first node above the epicotyl). Transfers to fresh medium can occur during this time. Explants were transferred from the SIM with or without selection to SIM with selection after about one week. At this time, there was considerable de novo shoot development at the base of the petiole of the leaf ex- plants in a variety of SIM (Method B), at the primary node for seedling explants (Method A), and at the axillary nodes of propagated explants (Method C). Preferably, all shoots formed before transformation were removed up to 2 weeks after co- cultivation to stimulate new growth from the meristems. This helped to reduce chimerism in the primary transformant and increase amplification of transgenic meristematic cells. During this time the explant may or may not be cut into smaller pieces (i.e. detaching the node from the explant by cutting the epicotyl).

9.5 - Shoot Elongation After 2 to 4 weeks (or until a mass of shoots was formed) on SIM medium (preferably with selection), the explants were transferred to SEM medium medium (shoot elongation medium, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soy using primary-node explants from seedlings. In Vitro Cell. Dev. Biol.— Plant (2007) 43:536-549) that stimulates shoot elongation of the shoot primordia. This medium may or may not contain a selection compound.

After every 2 to 3 weeks, the explants were transfer to fresh SEM medium (preferably containing selection) after carefully removing dead tissue. The explants should hold together and not fragment into pieces and retain somewhat healthy. The explants were continued to be trans- ferred until the explant dies or shoots elongate. Elongated shoots >3 cm were removed and placed into RM medium for about 1 week (Method A and B), or about 2 to 4 weeks depending on the cultivar (Method C) at which time roots began to form. In the case of explants with roots, they were transferred directly into soil. Rooted shoots were transferred to soil and hardened in a growth chamber for 2 to 3 weeks before transferring to the greenhouse. Regenerated plants obtained using this method were fertile and produced on average 500 seeds per plant.

Transient GUS expression after 5 days of co-cultivation with Agrobacterium tumefaciens was widespread on the seedling axillary meristem explants especially in the regions wounding during explant preparation (Method A). Explants were placed into shoot induction medium without selection to see how the primary-node responds to shoot induction and regeneration. Thus far, greater than 70% of the explants were formed new shoots at this region . Expression of the gene was stable after 14 days on SIM, implying integration of the T-DNA into the soygenome. In addition, preliminary experiments resulted in the formation of positive shoots forming after 3 weeks on SIM.

For Method C, the average regeneration time of a soy plantlet using the propagated axillary meristem protocol was 14 weeks from explant inoculation. Therefore, this method has a quick regeneration time that leads to fertile, healthy soy plants. Example 10: Pathogen assay

10.1 . Recovery of clones 2-3 clones per TO event were 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). 10.2 Inoculation

The rust fungus is a wild isolate from Brazil. The plants were inoculated with P.pachyrhizi.

In order to obtain appropriate spore material for the inoculation, soy leaves which had been infected with rust 15-20 days ago, were taken 2-3 days before the inoculation and transferred to agar plates (1 % agar in H20). The leaves were placed with their upper side onto the agar, which allowed the fungus to grow through the tissue and to produce very young spores. For the inoculation solution, the spores were knocked off the leaves and were added to a Tween-H20 solution. The counting of spores was performed under a light microscope by means of a Thoma counting chamber. For the inoculation of the plants, the spore suspension was added into a compressed-air operated spray flask and applied uniformly onto the plants or the leaves until the leaf surface is well moisturized. For macroscopic assays the inventors used a spore density of 1 -5x105 spores/ml. For the microscopy, a density of >5 x 105 spores / ml is used. The inoculated plants were placed for 24 hours in a greenhouse chamber with an average of 22°C and >90% of air humidity. The following cultivation was performed in a chamber with an average of 25°C and 70% of air humidity.

Example 1 1 Microscopical screening:

For the evaluation of the pathogen development, the inoculated leaves of plants were stained with aniline blue 48 hours after infection.

The aniline blue staining serves for the detection of fluorescent substances. During the defense reactions in host interactions and non-host interactions, substances such as phenols, callose or lignin accumulated or were produced and were incorporated at the cell wall either locally in papillae or in the whole cell (hypersensitive reaction, HR). Complexes were formed in association with aniline blue, which lead e.g. in the case of callose to yellow fluorescence. The leaf material was transferred to falcon tubes or dishes containing destaining solution II (ethanol / acetic acid 6/1 ) and was incubated in a water bath at 90°C for 10-15 minutes. The destaining solution II was removed immediately thereafter, and the leaves were washed 2x with water. For the staining, the leaves were incubated for 1 .5-2 hours in staining solution II (0.05 % aniline blue = me- thyl blue, 0.067 M di-potassium hydrogen phosphate) and analyzed by microscopy immediately thereafter.

The different interaction types were evaluated (counted) by microscopy. An Olympus UV microscope BX61 (incident light) and a UV Longpath filter (excitation: 375/15, Beam splitter: 405 LP) are used. After aniline blue staining, the spores appeared blue under UV light. The papillae coul be recognized beneath the fungal appressorium by a green/yellow staining. The hypersensitive reaction (HR) was characterized by a whole cell fluorescence.

Example 12 Evaluating the susceptibility to soybean rust The progression of the soybean rust disease was scored by the estimation of the diseased area (area which was covered by sporulating uredinia) on the backside (abaxial side) of the leaf. Additionally the yellowing of the leaf was taken into account (for scheme see Figure 1 ).

12.1 Overexpression of DIS1

TO soybean plants expressing DIS1 protein were inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of 16 TO soybean plants were scored 14 days after inoculation.

The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 16 soybean TO plants expressing DIS1 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants were used as control. The average of the diseased leaf area is shown in Fig 2 for plants expressing recombinant DIS1 compared with wildtype plants. Overexpression of DIS1 reduces the diseased leaf area in comparison to non- transgenic control plants by 46%. This data clearly indicate that the in planta expression of the DIS1 expression vector construct lead to a lower disease scoring of transgenic plants compared to non-transgenic controls. So, the expression of DIS1 in soy enhances the resistance of soy against soybean rust.

12.2 Overexpression of DIS13 TO soybean plants expressing DIS13 protein were inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of 24 TO soybean plants were scored 14 days after inoculation.

The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 24 soybean TO plants expressing DIS13 (expression checked by RT-PCR) were evaluated in parallel to non- transgenic control plants. Clones from non-transgenic soy plants were used as control. The average of the diseased leaf area is shown in Fig 3 for plants expressing recombinant DIS13 compared with wildtype plants. Overexpression of DIS13 reduces the diseased leaf area in comparison to non-transgenic control plants by 40%. This data clearly indicate that the in planta expression of the DIS13 expression vector construct lead to a lower disease scoring of transgenic plants compared to non-transgenic controls. So, the expression of DIS13 in soy enhances the resistance of soy against soybean rust.

12.3 Overexpression of DIS4

TO soybean plants expressing DIS4 protein were inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of 24 TO soybean plants were scored 14 days after inoculation. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. At all 24 soybean TO plants expressing DIS4 (expression checked by RT-PCR) were evaluated in parallel to non-transgenic control plants. Clones from non-transgenic soy plants were used as control. The average of the diseased leaf area is shown in Fig 4 for plants expressing recombinant DIS4 compared with wildtype plants. Overexpression of DIS4 reduces the diseased leaf area in comparison to non- transgenic control plants by 31 % (p<0.01 ). This data clearly indicate that the in planta expression of the DIS13 expression vector construct lead to a lower disease scoring of transgenic plants compared to non-transgenic controls. So, the expression of DIS4 in soy enhances the resistance of soy against soybean rust.

Comparable results can be obtained using the other DIS genes.

Table 1 : List of the candidate genes selected by the method described in Example 3

Table 2: Primers for Gateway®-based cloning

Name 5'-3' sequence Amplicon |bpj

AT1 G66250si_ F GGGGACAAGT T TGTACAAAAAAGCAGGC TTAGCATGC TAACTACGCGT TTG 217

AT1 G66250si_ R GGGGACCACT T TGTACAAGAAAGCTGGGTACAAATCAC TCCGTAT TCCAGA

AT2G30490oe _F GGGGACAAGT T TGTACAAAAAAGCAGGC TTAATGGACC TCC TC TTGC TG 1518

AT2G30490oe _R GGGGACCACT T TGTACAAGAAAGCTGGGTAT TAACAGT TCC TTGGT TTCATAACG

AT5G18270si_ F GGGGACAAGT TTGTACAAAAAAGCAGGCT TAATGGCGGT TGTGGTTGAAG 1008

AT5G18270si_ R GGGGACCACT T TGTACAAGAAAGCTGGGTAT TAGAAGTCCCACAAGTCC

Table 3: Compilation of genes and sequence identifiers

gene protein nucleic acid

DIS SEQ ID NO: SEQ ID NO:

1 1 2 5 6

7 8

9 10

1 1 12

13 14

15 16

17 18

19 20

21 22

23 24

25 26

27 28

Claims

Claims

A method for conferring, increasing or modifying resistance in a plant, a plant part, or a plant cell as compared to a wild type plant, a wild type plant part or a wild type plant cell against infections by a fungus of the class Basidiomycota, preferably of the order Uredi- nales, more preferably of the family Phakopsoraceae, even more preferably of the genus Phacopsora, most preferably of the species Phakopsora pachyrhizi and/or Phakopsora meibomiae, wherein the method comprises the step of increasing the expression and / or activity of a polypeptide comprising an amino acid sequence having at least 40% identity with anyone of the amino acid sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27 in the plant, the plant part, or the plant cell in comparison to the wild type plant, the wild type plant part or the wild type plant cell.

The method according to claim 1 , wherein the polypeptide is encoded by

(i) a heterologous nucleic acid having at least 40% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, or a functional fragment thereof;

(ii) a heterologous nucleic acid encoding a protein having at least 40% identity with SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27 or a functional fragment thereof; and / or by

(iii) a heterologous nucleic acid encoding the same polypeptide as anyone of the nucleic acids of (i) or (ii) above, but differing from the nucleic acids of (i) or (ii) above due to the degeneracy of the genetic code.

The method according to claim 1 or 2, comprising

(a) stably transforming a plant cell with an expression cassette comprising

(i) a heterologous nucleic acid having at least 40% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, or a functional fragment thereof;

(ii) a heterologous nucleic acid encoding a protein having at least 40% identity with SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27 or a functional fragment thereof; and / or by

(iii) a heterologous nucleic acid encoding the same polypeptide as anyone of the nucleic acids of (i) or (ii) above, but differing from the nucleic acids of (i) or (ii) above due to the degeneracy of the genetic code,

in functional linkage with a promoter;

(b) regenerating the plant from the plant cell; and

(c) expressing said exogenous nucleic acid.

A transgenic plant, transgenic plant part or transgenic plant cell

i) comprising a heterologous nucleic acid selected from the group consisting of

a) a heterologous nucleic acid comprising any of the nucleic acid sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, b) a heterologous nucleic acid coding for a polypeptide comprising any of the amino acid sequences SEQ ID NO:1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27,

c) a heterologous nucleic acid coding for a polypeptide having an amino acid sequence identity of at least 40% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

d) a heterologous nucleic acid encoding the same polypeptide as any of the nucleic acids of (b) or (c) above, but differing from the nucleic acids of (b) or (c) above due to the degeneracy of the genetic code; and/or

ii) comprising a heterologous polypeptide selected from the group consisting of

a) polypeptide having any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

b) polypeptide having an amino acid sequence identity of at least 40% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27.

A method for creating a transgenic plant, transgenic plant part or transgenic plant cell exhibiting increased or modified resistance as compared to a wild type plant, a wild type plant part or a wild type plant cell against infections by a fungus of the class Basidiomyco- ta, preferably of the order Uredinales, more preferably of the family Phakopsoraceae, even more preferably of the genus Phacopsora, most preferably of the species Phakopsora pachyrhizi and/or Phakopsora meibomiae, wherein the method comprises the steps of i) introducing a heterologous nucleic acid selected from the group consisting of

a) a heterologous nucleic acid comprising any of the nucleic acid sequences SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28,

b) a heterologous nucleic acid coding for a polypeptide comprising any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27,

c) a heterologous nucleic acid coding for a polypeptide having an amino acid sequence identity of at least 40% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

d) a heterologous nucleic acid encoding the same polypeptide as any of the nucleic acids of (b) or (c) above, but differing from the nucleic acids of (b) or (c) above due to the degeneracy of the genetic code;

into a plant, plant part or plant cell, and

ii) generating a transgenic plant, transgenic plant part or transgenic plant cell from said plant, plant part or plant cell.

The method of claim 5, further comprising the step of harvesting the seeds of the transgenic plant and planting the seeds and growing the seeds to plants, wherein the grown plants comprise anyone of the heterologous nucleic acid selected from the group consisting of a) a heterologous nucleic acid comprising any of the nucleic acid sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28,

b) a heterologous nucleic acid coding for a polypeptide comprising any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, c) a heterologous nucleic acid coding for a polypeptide having an amino acid sequence identity of at least 40% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

d) a heterologous nucleic acid encoding the same polypeptide as any of the nucleic acids of (b) or (c) above, but differing from the nucleic acids of (b) or (c) above due to the degeneracy of the genetic code.

A method of identifying an agent for conferring, increasing or modifying resistance of a plant, plant part or plant cell as compared to a wild type plant, plant part or plant cell against infections by a fungus of the class Basidiomycota, preferably of the order Uredi- nales, more preferably of the family Phakopsoraceae, even more preferably of the genus Phacopsora, most preferably of the species Phakopsora pachyrhizi and/or Phakopsora meibomiae, wherein the method comprises the steps of

i) applying a putative agent to a plant, plant part or plant cell exhibiting a desired degree of resistance against said fungus,

ii) determining expression by the plant, plant part or plant cell of a polypeptide selected from the group consisting of

a) a polypeptide having any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

b) a polypeptide having an amino acid sequence identity of at least 40% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and

iii) selecting the agent of step i) if application of the agent in step i) leads to an increase of expression of a polypeptide as determined in step ii).

Use of

i) a nucleic acid selected from the group consisting of

a) a nucleic acid comprising any of the nucleic acid sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28,

b) a nucleic acid coding for a polypeptide comprising any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and c) a nucleic acid coding for a polypeptide having an amino acid sequence identity of at least 40% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27; and/or

ii) a polypeptide selected from the group consisting of

a) a polypeptide having any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and b) a polypeptide having an amino acid sequence identity of at least 40% to any of the amino acid sequences SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27,

for conferring, increasing or modifying resistance of a plant as compared to a wild type plant against infections by a fungus of the class Basidiomycota, preferably of the order

Uredinales, more preferably of the family Phakopsoraceae, even more preferably of the genus Phacopsora, most preferably of the species Phakopsora pachyrhizi and/or

Phakopsora meibomiae. 9. Harvestable part of a transgenic plant described in claim 4, wherein the harvestable part is preferably a transgenic seed of the transgenic plant.

10. Product derived from a plant described in claim 4, from a plant producible by the method of claim 5 or 6 or from the harvestable part of the plant according to claim 9, wherein the product is preferably soybean meal or soy oil.

1 1 . Method for the production of a product comprising

a) growing a plant of claim 4 or obtainable by the method of claim 5 or 6 and b) producing said product from or by the plant and/or part, preferably seeds, of the plant.

12. Method according to claim 1 1 comprising

a) growing the plant of claim 4 or obtainable by the method of claim 5 or 6 and removing the harvestable parts as defined in claim 9 from the plant; and

b) producing said product from or by the harvestable parts of the plant.

13. Method according to claim 1 1 or 12, wherein the product is meal or oil, preferably, soybean meal or soybean oil. 14. A method for breeding a fungal resistant plant comprising

(a) crossing a plant of claim 4 or a plant obtainable by the method of claim 5 or 6 with a second plant;

(b) obtaining seed from the cross of step (a);

(c) planting said seeds and growing the seeds to plants; and

(d) selecting from said plants plants expressing a polypeptide encoded by

a) the heterologous nucleic acid comprising any of the nucleic acid sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, b) the heterologous nucleic acid coding for a polypeptide comprising any of the amino acid sequences SEQ ID NO:1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, or

c) the heterologous nucleic acid coding for a polypeptide having an amino acid sequence identity of at least 40% to any of the amino acid sequences SEQ ID NO:1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27. The method according to anyone of claims 1 to 3, 5, 6, 7, or 1 1 to 14, or the transgenic plant, transgenic plant part, or transgenic plant cell according to claim 4, or the use according to claim 8, the harvestable part according to claim 9 or the product according to claim 10, wherein the plant is selected from the group consisting of beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, groundnut, rice, wheat, barley, arabidopsis, lentil, banana, canola, cotton, potatoe, corn, sugar cane, alfalfa, and sugar beet, preferably wherein the plant is soy.