Increase plant resistance to fungal infections

Abstract

The present invention relates to methods for conferring or increasing resistance to fungal pathogens in plants, plant parts and / or plant cells. To this end, the present invention focuses on promoting or increasing the production and / or accumulation of Myb41-type transcription factors (Myb41), fragments thereof or homologs thereof in plants, plant parts and / or plant cells compared to corresponding wild-type plants, wild-type plant parts and / or wild-type plant cells. The present invention also relates to plants, plant parts and / or plant cells having increased resistance to fungal pathogens, as well as materials and methods for producing or using such plants, plant parts or products produced therefrom.

Description

Summary of the Invention

[0001] The present invention relates to methods for conferring or increasing resistance to fungal pathogens in plants, plant parts and / or plant cells. To this end, the present invention focuses on promoting or increasing the production and / or accumulation of Myb41-type transcription factors (Myb41), fragments thereof, or homologs thereof, in plants, plant parts and / or plant cells compared to corresponding wild-type plants, wild-type plant parts and / or wild-type plant cells. The present invention also relates to plants, plant parts and / or plant cells having increased resistance to fungal pathogens, as well as materials and methods for producing or using such plants, plant parts, or producing products therefrom. Background of the Invention

[0003] The cultivation of crop plants plays a major role in producing food for humans and animals. Currently conventional monocultures are particularly susceptible to the epidemic spread of diseases. As a result, yields are significantly reduced. To date, pathogenic organisms have been primarily controlled by the use of pesticides. Today, for humans, there is also the possibility of directly changing the genetic configuration of plants or pathogens. Alternatively, naturally occurring fungicides produced by plants after fungal infection can also be synthesized and applied to plants. Resistance generally describes the ability of plants to prevent or at least reduce infection and colonization by harmful pathogens. Different mechanisms can be identified in naturally occurring resistance, which allow plants to avoid colonization by phytopathogenic organisms (Schopfer and Brennicke (1999) PflanzenPhysiologie, Springer-Verlag, Berlin-Heidelberg, Germany).

[0004] With regard to physiological race-specific resistance, also known as host resistance, a distinction is made between compatible and incompatible interactions. In a compatible interaction, an interaction occurs between a virulent pathogen and a susceptible plant. The pathogen survives and can establish reproductive structures, while the host is severely stunted in development or dies. On the other hand, an incompatible interaction occurs when the pathogen infects the plant but growth is inhibited before or after the slight development of symptoms (mainly due to the presence of R genes of the NBS-LRR family, see below). In the latter case, the plant becomes resistant to the corresponding pathogen (Schopfer and Brennicke, see above). However, this type of resistance is mainly specific to a certain strain or pathogen.

[0005] In compatible and incompatible interactions, host defense and specific responses to pathogens occur. However, in nature, this resistance is often overcome due to the rapid evolutionary development of new virulent races of pathogens (Neu et al. (2003) American Cytopathol. Society, MPMI 16 No. 7: 626-633).

[0006] Most pathogens are plant species specific. This means that pathogens can induce disease in certain plant species, but not in other plant species (Heath (2002) Can. J. Plant Pathol. 24: 259-264). Resistance to pathogens in certain plant species is called non-host resistance. Non-host resistance provides strong, widespread and permanent protection against plant pathogens. Genes that provide non-host resistance provide strong, widespread and permanent protection against specific diseases in non-host plants. In particular, this resistance works on different strains of pathogens.

[0007] Fungi are distributed worldwide. Approximately 100,000 different fungal species are known to date. Rust fungi are particularly important. They can have a complex developmental cycle with up to five distinct spore stages (sexual spore, rust spore, summer spore, winter spore, and basidiospore).

[0008] During the infection of plants by pathogenic fungi, different stages are generally observed. The first stage of the interaction between plant pathogenic fungi and their potential host plants is decisive for the colonization of the fungus in the plant. During the first stage of infection, spores attach to the surface of the plant, germinate, and the fungus penetrates the plant. The fungi can penetrate the plant via existing openings (such as stomata, lenticels, hydatids and wounds), or as a result of mechanical forces and with the help of cell wall digestive enzymes, they directly penetrate the plant epidermis. Specific infection structures are formed for the penetration of plants. In order to resist, plants have formed physical barriers, such as wax layers, as well as chemical compounds with antifungal action to inhibit spore germination, hyphal growth or penetration.

[0009] The soybean rust fungus Phakopsora pachyrhizi penetrates the cuticle and epidermis directly. After penetrating the epidermal cells, the fungus reaches the intercellular spaces of the mesophyll, where it begins its spread through the leaf. To obtain nutrients, the fungus penetrates the mesophyll cells and forms haustoria within them. During this penetration, the plasma membrane of the penetrated mesophyll cells remains intact.

[0010] The initial step in the pathogenesis of Asian soybean rust is the initial penetration of the fungus into epidermal cells through the plant cuticle. The plant cuticle is an extracellular hydrophobic layer covering the aerial epidermis and is composed of two main components, the polymer cutin and cuticular waxes (for a review of the plant cuticle, see Yeats TH, Rose JK. The formation and function of plant cuticles. Plant Physiol. 2013; 163(1): 5-20). The cuticle provides protection against desiccation, external environmental stresses, and pathogens. For example, lower cuticle mass in tomato "cd" mutants has been shown to be associated with increased susceptibility to Botrytis cinerea (Isaacson et al., 2009). To facilitate penetration of the cuticle, many fungal pathogens secrete enzymes to degrade or weaken the cuticle, such as, for example, cutinases, which are a class of small, nonspecific esterases that hydrolyze cutin polymers.

[0011] In addition to cutin, cuticular waxes also play an important role in pathogen development and defense. For example, it has been shown that the "inhibitor of germination 1" (IRG1) mutant of M. trunctula displays fewer cuticular wax crystals on the underside of leaves and a strong reduction in wax primary alcohol groups. This surface modification leads to increased resistance against the fungal pathogens Puccinia pachyrhizi, Puccinia emaculata, and the anthracnose fungus Colletotrichum trifolii (Uppalapati et al., 2012). The authors found that IRG1 encodes a Cys(2)His(2) zinc finger transcription factor, also known as PALM1.

[0012] The nutrition of biotrophic phytopathogenic fungi depends on the metabolism of living plant cells. This type of fungi belongs to the biotrophic fungi group, such as many rust fungi, powdery mildew fungi or oomycete pathogens such as Phytophthora (Phytophthora) or Peronospora (Peronospora). The nutrition of necrotrophic phytopathogenic fungi depends on the dead cells of plants, for example from the species of Fusarium (Fusarium), Rhizoctonia (Rhizoctonia) or Mycospaerella (Mycospaerella). Soybean rust has occupied an intermediate position because it directly penetrates the epidermis and the penetrated cells become necrotic therewith. After penetration, the fungus switches to a dedicated biotrophic life mode. The subgroup of biotrophic fungal pathogens that basically follow this infection strategy is semi-necrotic.

[0013] Soybean rust has recently become increasingly important. The disease is caused by the biotrophic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). Both belong to the phylum Basidiomycota, order Uredinales, family Phakopsoraceae. Both rust fungi infect a broad spectrum of leguminous host plants.

[0014] Layer puccinia pachyrhizi is the pathogen that goes up more invasive on soybean (Glycine max), and is therefore very important to agriculture at least at present.Can find layer puccinia pachyrhizi in nearly all tropical and subtropical soybean growing areas in the world.Ph. puccinia pachyrhizi can infect 31 kinds of 17 leguminous families in nature, and can grow (Sinclair etc. (editor), Proceedings of the Rust Workshop (1995), NationalSoybeanaResearchLaboratory, PublicationNo.1(1996), RytterJL etc., PlantDis.87,818(1984)) on other 60 kinds under controlled condition.Ph. meibomiae (P.meibomiae) has been found in Caribbean basin and Puerto Rico, and has not yet caused substantial damage.

[0015] Psoralea pachyrhizi is currently controlled in the field only with fungicides. Soybean plants resistant to the entire spectrum of isolates are unavailable. The search for resistant soybean germplasm led to the discovery of six dominant R genes from the NBS-LRR family that mediate soybean resistance to Psoralea pachyrhizi. The resistance conferred by these genes is rapidly lost as the fungus develops new virulent races.

[0016] In recent years, fungal diseases (such as soybean rust) have become more important in agricultural production. Therefore, there is a need in the art to develop methods for controlling fungi and providing plants that are resistant to fungal diseases.

[0017] Much research has been done on powdery and downy mildews that infect the epidermis of plants. However, tackling soybean rust, which infects the mesophyll, or Fusarium fungi, which infect inaccessible internal tissues, remains an open question.

[0018] It is an object of the present invention in particular to provide a method for increasing resistance against fungal pathogens, preferably against fungal pathogens of the family Phakopsora, more preferably against fungal pathogens of the genus Phakopsora, most preferably against Phakopsora pachyrhizi (Sydow) and / or Phakopsora argenteus (Arthur), also known as soybean rust.

[0019] Surprisingly, we found that fungal pathogens, particularly those of the family Pseudocordiaceae, such as soybean rust, can be controlled by expression of the Myb41 protein, which was originally identified as a repressor of cuticle biosynthesis (Cominelli et al. (2008) Over expression of the Arabidopsis AtMYB41 gene alters cell expansion and leaf surface permeability. Plant J. 53(1):53-64). Cominelli et al. have found that overexpression of AtMYB41 results in a dwarf phenotype similar to that exhibited by some mutants affecting cuticle biosynthesis.

[0020] The MYB superfamily of transcription factors is a large and functionally diverse family of proteins that can be found in all eukaryotic organisms. In plants, the Myb family has been selectively expanded. For example, in Arabidopsis thaliana, 196 Myb transcription factor homologs can be found, belonging to four different families (for a comprehensive review of Myb transcription factors in Arabidopsis, see Dubos et al. (2010), Trends in Plant Science, Vol. 15, No. 10; 573-581).

[0021] The Myb41 protein described in the present invention belongs to the R2R3-MYB family, which consists of 126 members in Arabidopsis thaliana. R2R3-MYB genes are reported to be involved in many different regulatory networks that control development, metabolism, and responses to biotic and abiotic stresses.

[0022] In Arabidopsis, AtMYB41 is expressed at high levels in response to drought, ABA and salt treatment, indicating a possible role in stress response. Transgenic lines that overexpress this transcription factor in Arabidopsis show similar pleiotropic phenotypes (dwarf appearance) as those presented by some mutants that affect cuticle biosynthesis (Cominelli et al. (2008) Over-expression of the Arabidopsis AtMYB41 gene alters cell expansion and leaf surface permeability. Plant J. 53 (1): 53-64). However, this publication does not analyze the real reason for the phenotype. Further characterization of AtMyb41 overexpression lines by transcriptome and metabolome analysis showed that AtMyb41 is involved in several cellular processes, including the control of primary metabolism and the negative regulation of short-term transcriptional responses to osmotic stress (Lippold et al., AtMyb41 regulates transcriptional and metabolic responses to osmotic stress in Arabidopsis. Plant Physiol. 149: 1761-1772 (2009)). Further evaluation of the role of Myb41 in salt and desiccation tolerance revealed that it is regulated by phosphorylation of Ser251 by the mitogen-activated protein kinase MPK6; phosphorylation of MYB41 by MPK6 is required for the biological function of MYB41 in salt tolerance (Hoang et al., Biochem Biophys Res Commun. 2012 May; 422(1): 181-186).

[0023] Recently, the molecular mechanism of AtMYB41 gene overexpression has been linked to the synthesis of suberin in leaves. (Kosma et al., AtMYB41 activates ectopic suberin synthesis and assembly in multiple plant species and cell types. Plant J. 80: 216-229 (2014)). Suberin is a lipid and phenolic cell wall heteropolymer found in the roots and vascular tissues of all plants. Suberin plays a key role in plant water relations and in protecting plants from biotic and abiotic stresses. Kosma et al. showed that the expression of AtMYB41 can activate all the steps necessary for suberin synthesis and the deposition of cell wall-associated suberin-like lamellae. Thus, overexpression of AtMYB41 will increase the transcripts of suberin, lignin, and phenylpropanoid biosynthesis genes and will increase the amount of monolignols in leaves, leading to the formation of suberin-like lamellae in the epidermal and mesophyll cells of leaves.

[0024] The formation of suberin lamellae in leaves has never been associated with increased resistance to fungal pathogens. Therefore, it is surprising to find that the resistance to Phakopsora pachyrhizi is increased after overexpressing AtMyb41 in soybean. In addition, as described in (Cell Wall Metabolism in Response to Abiotic Stress.Plants 2015,112-166) for the differences in salt response in Rosaceae and Arabidopsis, for different species, there seem to be different responses to stressors. Therefore, it is impossible to extrapolate the stress response found in, for example, Arabidopsis to other plant species. SUMMARY OF THE INVENTION

[0026] Accordingly, the present invention provides a method for conferring or increasing fungal resistance in a plant, plant part or plant cell, wherein the method comprises the step of increasing the production and / or accumulation of Myb41 in the plant, plant part or plant cell compared to a corresponding wild-type plant, wild-type plant part or wild-type plant cell.

[0027] Also provided is a method of conferring or increasing fungal resistance in a plant, plant part or plant cell, wherein the method comprises increasing the expression and / or biological activity of a Myb41 protein in the plant, plant part or plant cell as compared to a corresponding wild-type plant, wild-type plant part or wild-type plant cell, wherein the Myb41 protein is encoded by:

[0028] (i) an exogenous nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO. 1, or a functional fragment or splice variant thereof;

[0029] (ii) an exogenous nucleic acid encoding a protein or a functional fragment thereof that is at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO. 2 or 5;

[0030] (iii) an exogenous nucleic acid capable of hybridizing under stringent conditions to the complement of any nucleic acid according to (i) or (ii); or

[0031] (iv) An exogenous nucleic acid encoding the same Myb41 protein as the nucleic acid of (i) to (iii) above, but different from the nucleic acid of (i) to (iii) above due to the degeneracy of the genetic code.

[0032] The present invention also provides a recombinant vector construct comprising a nucleic acid encoding the Myb41 protein of the present invention.

[0033] Also provided are genetically modified plants, genetically modified plant parts, or genetically modified plant cells transformed with one or more recombinant vector constructs of the invention.

[0034] The present invention also provides crop plants, crop plant parts or crop plant cells that overexpress the Myb41 protein of the present invention.

[0035] Furthermore, the present invention provides a method for producing a genetically modified crop plant, a genetically modified crop plant part or a genetically modified crop plant cell having increased fungal resistance compared to a corresponding wild-type plant, plant part or plant cell, the method comprising

[0036] (a) introducing an exogenous nucleic acid encoding a Myb41 protein into a plant, a plant part or a plant cell,

[0037] (b) producing genetically modified plants, genetically modified plant parts or genetically modified plant cells from plants, plant parts or plant cells; and

[0038] (c) expressing the Myb41 protein in a genetically modified plant, a genetically modified plant part or a genetically modified plant cell derived from said plant.

[0039] According to one aspect of the present invention, Myb41 protein or a nucleic acid encoding a Myb41 protein is used to increase fungal resistance in plants.

[0040] The present invention also provides a method of controlling fungi in a field, preferably by reducing or delaying infection of plants in the field and / or reducing or delaying emission of fungal spores from the field, the method comprising the steps of: (a) planting seeds from any of the plants of the present invention and / or (b) increasing suberin lamellae formation in the plant.

[0041] The present invention also provides harvestable parts of the plant of the present invention, wherein the harvestable parts of the plant comprise an exogenous nucleic acid encoding the Myb41 protein of the present invention.

[0042] The present invention also provides products derived from the plants of the present invention, from plants producible by the methods of the present invention, or from harvestable parts of the plants of the present invention, wherein the product comprises an exogenous nucleic acid encoding a Myb41 protein and / or a Myb41 protein of the present invention. Preferred products according to the present invention are fruits, more preferably seeds, and products derived from such fruits, preferably dried fruits and dried fruit pieces, meals, and oils. Most preferred according to the present invention are soybeans, soybean flour, and soybean oil.

[0043] The present invention also provides a method for producing a product, comprising a) growing a plant of the present invention or a plant obtainable by a method of the present invention and b) producing the product from or by the plant and / or plant part (preferably seeds), wherein the product comprises an exogenous nucleic acid encoding a Myb41 protein of the present invention and / or a Myb41 protein of the present invention.

[0044] The present invention also provides a method for determining resistance of a plant to fungi, which comprises screening the overexpression of the Myb41 gene in cells of the plant.

[0045] In the context of the present invention, fungal resistance is envisaged to include resistance against biotrophic, hemibiotrophic or heminecrotrophic fungi, preferably against rust, downy mildew, powdery mildew, leaf spot, late blight, Fusarium and / or Septoria.

[0046] Also in the context of the present invention, the plant is preferably selected from members of the taxonomic families Fabaceae, Cruciferae and Poaceae, most preferably soybean.

[0047] The present invention also provides a method for breeding fungus-resistant crop plants, the method comprising

[0048] (a) crossing a plant according to the invention or a plant obtainable by a method according to the invention with a second plant;

[0049] (b) obtaining seeds from the hybridization of step (a);

[0050] (c) planting the seeds and growing the seeds into plants; and

[0051] (d) selecting plants expressing the Myb41 protein of the present invention from among the plants.

[0052] As stated above, the objects of the invention are primarily achieved by the subject matter of the independent claims. Preferred embodiments of the invention are defined by the subject matter of the dependent claims or are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Shown is a scoring system used to determine the level of diseased leaf area resistance of wild-type and transgenic soybean plants to the rust fungus Phakopsora pachyrhizi (as described in Godoy, CV, Koga, LJ & Canteri, MG Graphical scale for assessment of soybean rust severity. Fitopatologia brasileira 31:063-068. 2006).

[0055] Figure 2 A schematic diagram of a plant transformation vector used in the Examples of the present invention is shown, containing a Myb41 DNA fragment under the control of a parsley ubiquitin promoter.

[0056] Figure 3 A graph showing positional amino acid conservation. In each column: the number of stars indicates the degree of conservation (more stars indicates higher conservation); the first letter / "-" below the asterisk is the corresponding amino acid encountered in SEQ ID NO. 2 (or alignment gap); all following letters / "-" represent amino acids in decreasing order of frequency, or "-" indicates alignment gaps encountered in the homologous Myb 41 gene.

[0057] Figure 4 A multiple alignment of the Myb41 sequence of the present invention (SEQ ID NO. 2) and the corresponding sequences of homologs identified by their UniProt identifiers is shown. The amino acid sequence is given only for the top sequence, SEQ ID NO. 2, with each other sequence at each position indicating only a different amino acid or a gap (a "." represents "same amino acid as in the top sequence").

[0058] Figure 5Results of scoring of 107 transgenic soybean plants derived from 9 independent transformation events (11-12 plants per event) expressing a Myb41 overexpression vector construct are shown. The experiments were performed using T1 generation plants. The plants were checked for transgenesis by PCR. Non-transgenic plants were discarded. T1 soybean plants carrying the Myb41 expression cassette were inoculated with spores of Phakopsora pachyrhizi. The expression of the Myb41 gene was checked by RT-PCR. The diseased leaf area was assessed on all leaves 14 days after inoculation. The mean of the percentage of leaf area showing fungal colonies or strong yellowing / browning on all leaves was considered the diseased leaf area. All 107 soybean T1 plants expressing Myb41 (expression checked by RT-PCR) were assessed in parallel with non-transgenic control plants. The mean values of the diseased leaf area of plants expressing Myb41 and wild type control plants are shown in Table 1. Figure 5 Overexpression of Myb41 significantly (***: p < 0.001) reduced diseased leaf area by 33.5% compared to non-transgenic control plants.

[0059] Sequence Description

[0060] Detailed Description of the Invention

[0062] The present invention focuses on the use of Myb41 for protecting plants against fungal infections and the progression of such infectious diseases.

[0063] The technical teachings of the present invention use language means in this article, particularly by using scientific and technical terms to express.But, those skilled in the art understand that, if only because there is a variety of ways to express teaching, each way must not fully express all conceptual connections, then language means (as they may be detailed and accurate) can only approximate the full content of technical teaching, because each expression must end.With this in mind, those skilled in the art understand that subject matter of the present invention is the summation of each technical concept represented herein or expressed in a general manner by the inherent constraints of written description.Especially, those skilled in the art will understand that the meaning of each technical concept is carried out in this article as an abbreviation of each possible combination of spelling concepts when technically reasonable, so that the disclosure of, for example, three concepts or embodiments A, B and C is a shorthand annotation of concept A+B, A+C, B+C, A+B+C.

[0064] As used herein, singular terms and singular forms such as "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant," "the plant," or "a plant" also includes a plurality of plants; further, depending on the context, use of the term "plant" may also include genetically similar or identical progeny of that plant or plants derived therefrom by hybridization; indeed, use of the term "nucleic acid" optionally includes many copies of that nucleic acid molecule; similarly, the term "probe" optionally (and typically) encompasses many similar or identical probe molecules. Also as used herein, the word "comprising" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0065] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in the alternative ("or"). The term "comprising" also encompasses the term "consisting of."

[0066] When used with respect to a measurable value (e.g., an amount of mass, dose, time, temperature, sequence identity, etc.), the term "about" refers to a variation of ±0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or even 20% of the specified value, as well as the specified value. Thus, if a given composition is described as comprising "about 50% X," it will be understood that in some embodiments, the composition comprises 50% X, while in other embodiments, it may comprise any value from 40% to 60% X (i.e., 50% ± 10%).

[0067] As used herein, the term "gene" refers to biochemical information that, when embodied in nucleic acid, can be transcribed into a gene product, i.e., another nucleic acid, preferably RNA, and preferably also translated into a peptide or polypeptide. Thus, the term is also used to refer to portions of nucleic acids that are similar to the information and the sequence of such nucleic acids (also referred to herein as "gene sequences").

[0068] Also as used herein, the term "allele" refers to a variation of a gene characterized by one or more specific differences in the gene sequence compared to the wild-type gene sequence, regardless of the presence or absence of other sequence differences. An allele or nucleotide sequence variant of the present invention has, in increasing order of preference, at least 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide "sequence identity" to the nucleotide sequence of the wild-type gene. Accordingly, where "allele" refers to the biochemical information for expressing a peptide or polypeptide, the corresponding nucleic acid sequence of the allele has, in increasing order of preference, at least 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid "sequence identity" to the corresponding wild-type peptide or polypeptide.

[0069] The mutation or change of the amino acid or nucleic acid sequence can be any of substitution, deletion or insertion; the term "mutation" or "alteration" also encompasses any combination of these. Below, all three specific mutation modes are described in more detail by reference to amino acid sequence mutations; corresponding teachings apply to nucleic acid sequences, such that an "amino acid" is replaced by a "nucleotide".

[0070] "Substitutions" are described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the substituted amino acid. For example, a substitution of histidine at position 120 with alanine is designated "His120Ala" or "H120A."

[0071] "Deletions" are described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by "*" or "-". Thus, a deletion of glycine at position 150 is designated as "Gly150*", "G150*", "Gly150-", or "G150-". Alternatively, a deletion is indicated by, for example, "deletion of D183 and G184".

[0072] "Insertion" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the original amino acid and another amino acid. For example, a lysine inserted next to a glycine at position 180 would be designated as "Gly180GlyLys" or "G180GK." When more than one amino acid residue is inserted, such as, for example, inserting Lys and Ala after Gly180, this can be represented as: Gly180GlyLysAla or G180GKA. When substitution and insertion occur at the same position, this can be represented as S99SD+S99A or simply S99AD. When inserting an amino acid residue identical to an existing amino acid residue, degeneracy in the nomenclature is apparent. If, for example, a glycine is inserted after glycine in the above example, this would be represented by G180GG.

[0073] Variants containing multiple alterations are separated by "+", for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" indicate that the arginine and glycine at positions 170 and 195 are substituted with tyrosine and glutamic acid, respectively. Alternatively, multiple alterations can be separated by spaces or commas, for example, R170Y G195E or R170Y, G195E, respectively.

[0074] Where different alterations can be introduced at one position, the different alterations are separated by commas, e.g., "Arg170Tyr, Glu" indicates that the arginine at position 170 is substituted with tyrosine or glutamic acid. Alternatively, the different alterations or optional substitutions can be indicated in brackets, e.g., Arg170[Tyr, Gly] or Arg170{Tyr, Gly} or simply R170[Y, G] or R170{Y, G}.

[0075] A special aspect of amino acid substitutions is conservative mutations, which generally appear to have minimal effects on protein folding, resulting in substantial retention of the peptide or polypeptide properties of the corresponding peptide or polypeptide variant compared to those of the parent peptide or polypeptide. A conservative mutation is one in which an amino acid is replaced with a similar amino acid. For the determination of % similarity, the following applies, which is also based on the BLOSUM62 matrix, one of the most commonly used amino acid similarity matrices for database searching and sequence alignment:

[0076] Amino acid A is similar to amino acid S

[0077] Amino acid D is similar to amino acids E and N

[0078] Amino acid E is similar to amino acids D, K, and Q

[0079] Amino acid F is similar to amino acids W and Y

[0080] Amino acid H is similar to amino acids N and Y

[0081] Amino acid I is similar to amino acids L, M, and V

[0082] Amino acid K is similar to amino acids E, Q, and R

[0083] Amino acid L is similar to amino acids I, M, and V

[0084] Amino acid M is similar to amino acids I, L, and V

[0085] Amino acid N is similar to amino acids D, H, and S

[0086] Amino acid Q is similar to amino acids E, K, and R

[0087] Amino acid R is similar to amino acids K and Q

[0088] Amino acid S is similar to amino acids A, N, and T

[0089] Amino acid T is similar to amino acid S

[0090] Amino acid V is similar to amino acids I, L, and M

[0091] Amino acid W is similar to amino acids F and Y

[0092] Amino acid Y is similar to amino acids F, H, and W

[0093] Conservative amino acid substitutions can occur over the entire length of a functional protein (e.g., a peptide or polypeptide). Preferably, such mutations do not occur within a functional domain of the peptide or polypeptide. According to the present invention, the Myb41 gene is a gene encoding a Myb41 polypeptide.

[0094] In comparison with SEQ ID NO. 2, the Myb41 protein of the present invention preferably differs in the following aspects:

[0095] - differs from the corresponding amino acid of SEQ ID NO. 2 at 3 or fewer, more preferably at 2 or fewer, even more preferably at 1 and even more preferably at 0 positions at any of positions 89, 90, 92, 93, 96, 97, 99, 100, 102, 103, 105, 106, 108, 114 and 170,

[0096] - differs from the corresponding amino acid of SEQ ID NO. 2 at positions 15, 17, 21, 22, 25, 28, 29, 33, 37, 40, 44, 45, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 74, 78, 81, 82, 86, 87, 89, 90, 92, 93, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 114 and 170, more preferably at 3 or fewer, even more preferably at 2 or fewer, even more preferably at 1 and even more preferably at 0 positions,

[0097] - at positions 2, 15, 17, 18, 21, 22, 25, 28, 29, 33, 35, 37, 40, 41, 44, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 67, 68, 74, 75, 78, 81, 82, 86, 87, 89, 90, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 114, 123, 125, 128, and 170 The corresponding amino acids of NO.2 more preferably differ in 3 or fewer, even more preferably in 2 or fewer, even more preferably in 1 and even more preferably in 0 positions,

[0098] - or, at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24, 25, 28, 29, 32, 33, 35, 37, 38, 40, 41, 44, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 73, 74, 75, 78, 79, 80, 81, 82, 83, 86, 87, 89, 90, 91, 92, 93, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 120, 121, 122, 123, 125, 126, 128, 129, 130, 131, 132, 133, 134, 136, 137, 139, 146, 158, 166, 170, 171, 191, 243, 248, 258, 269 and 272 The corresponding amino acids of ID NO. 2 more preferably differ in 5 or fewer, even more preferably in 4 or fewer, even more preferably in 3 or fewer, even more preferably in 2 or fewer, even more preferably in 1 and even more preferably in 0 positions, as long as the amino acids at positions 89, 90, 92, 93, 96, 97, 99, 100, 102, 103, 105, 106, 108, 114 and 170 are conserved.

[0099] Therefore, in the alignment of the Myb41 protein of the present invention with SEQ ID NO. 2, it is preferred to retain at least the following positions of SEQ ID NO. 2: 89, 90, 92, 93, 96, 97, 99, 100, 102, 103, 105, 106, 108, 114 and 170. In addition, the Myb41 protein of the present invention preferably comprises the minimum consensus sequence SEQ ID NO. 4. More preferably, at least the following positions of SEQ ID NO. 2 are retained: 15, 17, 21, 22, 25, 28, 29, 33, 37, 40, 44, 45, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 74, 78, 81, 82, 86, 87, 89, 90, 92, 93, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 114 and 170. Even more preferably, at least the following positions of SEQ ID NO. 2 are retained: 2, 15, 17, 18, 21, 22, 25, 28, 29, 33, 35, 37, 40, 41, 44, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 67, 68, 74, 75, 78, 81, 82, 86, 87, 89, 90, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 114,

[0100] 123, 125, 128 and 170. And most preferably retain at least the following positions of SEQ ID NO. 2: 2, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24, 25, 28, 29, 32, 33, 35, 37, 38, 40, 41, 44, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 73, 74, 75, 78, 79, 80, 81, 82, 83, 86, 87 , 89, 90, 91, 92, 93, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 120, 121, 122, 123, 125, 126, 128, 129, 130, 131, 132, 133, 134, 136, 137, 139, 146, 158, 166, 170, 171, 191, 243, 248, 258, 269, and 272.

[0101] At those positions in the alignment where the Myb41 protein of the invention differs from SEQ ID NO. 2 or 5, ie where the numbering according to SEQ ID NO. 2 is not retained, the differences are preferably according to Figure 3 The amino acids depicted at the corresponding positions or gaps and / or phospho-mimetic substitutions are preferred. Figure 3 The provided amino acids or gaps are selected from any deletion or substitution of amino acids of SEQ ID NO. 2.

[0102] In addition to the amino acid selections or deletions at the above positions, the Myb41 protein may also contain an insertion. Examples of acceptable insertions are Figure 4 Preferably, in the case where the Myb41 protein contains an insertion, the insertion is only numbered according to SEQ ID NO. Figure 4 Insertions are found between those amino acids. Insertions may be of any length, preferably 1 to 70 amino acids, even more preferably 1 to 50 amino acids, and most preferably 1 to 28 amino acids.

[0103] In the particular case where the Myb41 protein of the present invention comprises a phosphomimetic mutation, the mutation is preferably or comprises the substitution S251E or S251D numbered according to SEQ ID NO. 2. The corresponding preferred Myb41 amino acid sequences comprising such phosphomimetic substitutions are represented by SEQ ID NOs. 6, 7, 8, or 9. The phosphomimetic mutation renders the Myb41 protein of the present invention in a state similar to the phosphorylated form of Myb41, thereby obviating the need for a phosphorylation step and increasing the constitutive availability of the activated form of Myb41. This, in turn, reduces the delay between Myb41 production and the onset of fungal resistance.

[0104] The following UniProt entries describe the Myb41 protein and its homologs from Arabidopsis thaliana based on their corresponding sequences on November 5, 2019: Q9M0J5 (MYB41_ARATH), K7LU39 (K7LU39_SOYBN), A0A445HNS7 (A0A445HNS7_GLYSO), A0A445KFX1 (A0A445KFX1_GLYSO), I1KQR0 (I1KQR0_SOYBN), R0GQH1(R0GQH1_9BRAS), D7MDM8(D7MDM8_ARALL), V4LUX5(V4LUX5_EUTSA), A0A0D3A772(A0A0D3A772_ BRAOL), M4EBX2 (M4EBX2_BRARP), A0A398AMS3 (A0A398AMS3_BRACM), A0A078G2Y0 (A0A078G2Y0_BRANA) , A0A0D3DGI8(A0A0D3DGI8_BRAOL), A0A078JP44(A0A078JP44_BRANA), A0A397KYM7(A0A397KYM7_BRAC M), A0A3S3NZN7(A0A3S3NZN7_9MAGN), M4E620(M4E620_BRARP), A0A078GXG4(A0A078GXG4_BRANA), A0A 1P8B402 (A0A1P8B402_ARATH), A0A2U1Q9L5 (A0A2U1Q9L5_ARTAN), A0A2U1LGR0 (A0A2U1LGR0_ARTAN), A0A3Q7JEJ1 (A0A3Q7JEJ1_SOLLC), A0A3N7G9J4 (A0A3N7G9J4_POPTR) and A0A498JL09 (A0A498JL09_MALDO). Preferably, the Myb41 protein of the present invention is any of the above homologues, or when aligned with SEQ ID NO. 2, is identified only by the method according to Figure 3 amino acids, gaps or deletions and / or according to Figure 4Even more preferably, the Myb41 protein of the present invention is any of the above homologues, or when aligned with SEQ ID NO. 2, differs only by the insertion of Figure 3 The sequences of the corresponding homologs differ from those of the corresponding homologs by a few amino acids, gaps or deletions.

[0105] Most preferably, the Myb41 protein of the present invention comprises or consists of the following protein: the protein of SEQ ID NO.2.

[0106] The Myb41 gene of the present invention is preferably

[0107] (i) a nucleic acid sequence that is at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO. 1 or a functional fragment or splice variant thereof;

[0108] (ii) a nucleic acid sequence encoding a protein or a functional fragment thereof that is at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO. 2 or 5;

[0109] (iii) a nucleic acid capable of hybridizing under stringent conditions to the complement of any nucleic acid according to (i) or (ii); or

[0110] (iv) A nucleic acid that encodes the same Myb41 protein as the nucleic acid of (i) to (iii) above, but is different from the nucleic acid of (i) to (iii) above due to the degeneracy of the genetic code.

[0111] When compared with the parent protein or nucleic acid, protein or nucleic acid variants can be defined by their sequence identity. Sequence identity is usually provided as "% sequence identity" or "% identity". In order to determine the percent identity between two amino acid sequences in a first step, a paired sequence alignment is generated between the two sequences, wherein the two sequences are aligned over their entire length (i.e., paired global alignment). Alignment is generated using a program (J. Mol. Biol. (1979) 48, pp. 443-453) implementing the Needleman and Wunsch algorithm, preferably using the program "NEEDLE" (The European Molecular Biology Open Software Suite (EMBOSS)) with program default parameters (gapopen=10.0, gapextend=0.5, and matrix=EBLOSUM62). For purposes of the present invention, a preferred alignment is one that determines the highest sequence identity.

[0112] The following example is intended to illustrate two nucleotide sequences, but the same calculations apply to protein sequences:

[0113] SEQ A: AAGATACTG Length: 9 bases

[0114] SEQ B: GATCTGA Length: 7 bases

[0115] Therefore, the shorter sequence is sequence B.

[0116] Producing a pairwise global alignment showing two sequences over their entire length results in

[0117]

[0118] The "|" symbol in the alignment indicates identical residues (which means bases of DNA or amino acids of proteins). The number of identical residues is 6.

[0119] The symbol "-" in the alignment indicates a gap. The number of gaps introduced by the alignment within sequence B is 1. The number of gaps introduced by the alignment at the boundaries of sequence B is 2, and at the boundaries of sequence A is 1.

[0120] The aligned sequences are shown with an alignment length of 10 over their entire length.

[0121] Thus, according to the invention, pairwise alignments are generated which show shorter sequences over their entire length, resulting in:

[0122]

[0123] Thus, according to the invention, a pairwise alignment showing sequence A over its entire length is generated resulting in:

[0124]

[0125] Thus, according to the invention, a pairwise alignment showing sequence B over its entire length is generated resulting in:

[0126]

[0127] The alignment length of the shorter sequence is shown to be 8 over its entire length (there is one gap which is factored in the alignment length of the shorter sequence).

[0128] Thus, the alignment length showing sequence A over its entire length would be 9 (meaning sequence A is a sequence of the invention), and the alignment length showing sequence B over its entire length would be 8 (meaning sequence B is a sequence of the invention).

[0129] After the two sequences have been aligned, in a second step, the identity value should be determined from the alignment. Therefore, according to this specification, the following calculation of percent identity applies:

[0130] %-identity=(identical residues / length of the alignment region showing the corresponding sequence of the present invention over its full length)*100. Thus, the sequence identity relevant to the comparison of two amino acid sequences according to the present invention is calculated by dividing the number of identical residues by the length of the alignment region, which shows the corresponding sequence of the present invention over its full length. This value is multiplied by 100 to give "%-identity". According to the examples provided above, %-identity is: for sequence A, which is a sequence of the present invention, (6 / 9)*100=66.7%; for sequence B, a sequence of the present invention, (6 / 8)*100=75%.

[0131] As defined herein, term "hybridization" is a process in which substantially complementary nucleotide sequences anneal to each other. Hybridization can occur completely in solution, i.e., two complementary nucleic acids are in solution. Hybridization can also occur when one of complementary nucleic acids is fixed on a matrix (such as magnetic beads, agarose beads or any other resin). Hybridization can also occur when one of complementary nucleic acids is fixed on a solid support (such as nitrocellulose or nylon membrane) or is fixed on, for example, a siliceous glass support (the latter being referred to as nucleic acid array or microarray or nucleic acid chip) by, for example, photolithography. In order to allow hybridization to occur, usually nucleic acid molecules are thermally denatured or chemically denatured to melt the double-stranded chain into two strands and / or remove hairpin or other secondary structures from single-stranded nucleic acid.

[0132] The term "stringency" refers to the conditions under which hybridization occurs. The stringency of hybridization is affected by conditions such as temperature, salt concentration, ionic strength, and hybridization buffer composition. Typically, low stringency conditions are selected to be approximately 30°C lower than the thermal melting point (Tm) of a specific sequence under a defined ionic strength and pH. Moderate stringency conditions are when the temperature is 20°C lower than Tm, and high stringency conditions are when the temperature is 10°C lower than Tm. High stringency hybridization conditions are typically used to separate hybridization sequences with high sequence similarity to the target nucleic acid sequence. However, due to the degeneracy of the genetic code, nucleic acids may deviate from and still encode substantially the same polypeptide in sequence. Therefore, sometimes moderate stringency hybridization conditions may be needed to identify this type of nucleic acid molecules.

[0133] "Tm" is the temperature under defined ionic strength and pH at which 50% of the target sequence hybridizes to a perfectly matched probe. Tm depends on the solution conditions as well as the base composition and length of the probe. For example, longer sequences hybridize specifically at higher temperatures. The maximum hybridization rate is obtained from about 16°C below the Tm to 32°C. The presence of monovalent cations in the hybridization solution reduces the electrostatic repulsion between the two nucleic acid chains, thereby promoting hybrid formation; this effect is significant for sodium concentrations up to 0.4 M (for higher concentrations, this effect is negligible). Formamide lowers the melting temperature of DNA-DNA and DNA-RNA duplexes by 0.6 to 0.7°C per percentage formamide, and the addition of 50% formamide allows hybridization to proceed at 30 to 45°C, although the hybridization rate will be reduced. Base pair mismatches reduce the hybridization rate and thermal stability of the duplex. On average, and for large probes, Tm decreases by about 1°C per % base mismatch. Depending on the type of hybrid, the Tm can be calculated using the following equation:

[0134] DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):

[0135] Tm=81.5℃+16.6xlog([Na+]{a})+0.41x%[G / C{b}]-500×[L{c}]-1-0.61x%formamide

[0136] DNA-RNA or RNA-RNA hybrids:

[0137] Tm=79.8+18.5(log10[Na+]{a})+0.58(%G / C{b})+11.8(%G / C{b})2-820 / L{c}

[0138] Oligo-DNA or oligo-RNA hybrids:

[0139] For <20 nucleotides: Tm = 2 ({In})

[0140] For 20-35 nucleotides: Tm = 22 + 1.46 ({In})

[0141] in:

[0142] {a} or for other monovalent cations, but accurate only in the range 0.01-0.4M

[0143] {b} Accurate for %GC only in the 30% to 75% range

[0144] {c}L = length of the duplex in base pairs

[0145] {g} oligonucleotide

[0146] {In} effective length of primer = 2×(number of G / C) + (number of A / T)

[0147] Nonspecific binding can be controlled using any of a number of known techniques, such as, for example, blocking the membrane with a protein-containing solution, adding heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase.

[0148] For non-related probes, a series of hybridizations can be performed by changing one of (i) gradually lowering the annealing temperature (e.g., from 68° C. to 42° C.) or (ii) gradually lowering the formamide concentration (e.g., from 50% to 0%). Those skilled in the art are aware of the various parameters that can be changed during hybridization and which will maintain or alter stringency.

[0149] In addition to hybridization conditions, the specificity of hybridization is often determined by the performance of post-hybridization washes. To remove background caused by nonspecific hybridization, the sample is washed with a dilute salt solution. Key factors in this wash include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Washing conditions are typically performed at or below hybridization stringency. A positive hybridization produces a signal at least twice the background signal.

[0150] Usually, the suitable stringent conditions for nucleic acid hybridization determination or gene amplification detection program are as described above.Also can select more stringent or less stringent conditions.Those skilled in the art will know that can change and will maintain or change the various parameters of stringent conditions during the washing period.

[0151] For example, typical high stringency hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 1× SSC at 65°C or in 1× SSC and 50% formamide at 42°C, followed by a wash in 0.3× SSC at 65°C. Examples of moderately stringent hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 4× SSC at 50°C or in 6× SSC and 50% formamide at 40°C, followed by a wash in 2× SSC at 50°C. The length of the hybrid is the expected length of the hybridizing nucleic acid. When hybridizing nucleic acids of known sequence, the hybrid length can be determined by aligning the sequences and identifying the conserved regions described herein. 1× SSC is 0.15 M NaCl and 15 mM sodium citrate; hybridization and wash solutions may additionally include 5× Denhardt's reagent, 0.5-10% SDS, 100 μg / ml denatured, fragmented salmon sperm DNA, and 0.5% sodium pyrophosphate. Another example of high stringency conditions is hybridization in 0.1X SSC containing 0.1X SDS and optionally 5X Denhardt's reagent, 100 μg / ml denatured fragmented salmon sperm DNA, 0.5% sodium pyrophosphate at 65°C, followed by a wash in 0.3X SSC at 65°C.

[0152] To determine the level of stringency, reference may be made to Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989 and annual updates).

[0153] As used herein, the term "isolated DNA molecule" refers to a DNA molecule that is at least partially separated from other molecules that are usually combined with it under its natural or native state. The term "isolated" preferably refers to a DNA molecule that is at least partially separated from some nucleic acids that are usually located at the DNA molecule flank under its natural or native state. Therefore, it is considered herein that the DNA molecule (for example, as a result of recombinant technology) that is fused to its usually unrelated regulatory or coding sequence is separated. When integrated into the chromosome of a host cell or present in a nucleic acid solution together with other DNA molecules, such molecules are considered to be separated because they are not in their native state.

[0154] The polynucleotides disclosed herein or their fragments can be separated and manipulated using any number of methods well known to those skilled in the art. For example, polymerase chain reaction (PCR) technology can be used to amplify specific starting polynucleotide molecules and / or generate variants of the original molecule. Polynucleotide molecules or their fragments can also be obtained by other techniques, such as directly synthesizing fragments by chemical methods, such as typically by using an automated oligonucleotide synthesizer to implement. Polynucleotides can be single-stranded (ss) or double-stranded (ds). "Double-stranded" refers to the base pairing that occurs between fully complementary, antiparallel nucleic acid chains to form a double-stranded nucleic acid structure, typically under physiologically relevant conditions. Embodiments of the method include wherein the polynucleotide is selected from at least one of sense single-stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), double-stranded DNA / RNA hybrids, antisense ssDNA or antisense ssRNA; A mixture of polynucleotides of any one of these types can be used.

[0155] As used herein, "recombinant" in reference to a nucleic acid or polypeptide means that the material has been altered as a result of human application of recombinant techniques, such as by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation. A gene sequence open reading frame is recombinant if (a) the nucleotide sequence is present in an environment other than its natural environment, for example, as a result of (i) cloning into any type of artificial nucleic acid vector or (ii) movement or copying to another location in the original genome, or if (b) the nucleotide sequence is mutagenized so that it differs from the wild-type sequence. The term recombinant can also refer to an organism with recombinant material, for example, a plant comprising a recombinant nucleic acid is a recombinant plant.

[0156] The terms "genetically modified" or "transgenic" are used interchangeably herein and refer to organisms, preferably plants or parts thereof, or nucleic acids comprising heterologous polynucleotides. Preferably, heterologous polynucleotides are stably integrated within the genome so that the polynucleotides are passed to successive generations. Heterologous polynucleotides can be integrated into the genome alone or as part of a recombinant expression cassette. Heterologous polynucleotides are preferably cis-genetic or intragenic. "Transgenic" and "genetically modified" are used herein to refer to any cell, cell line, callus, tissue, plant part or plant whose genotype has been so changed by the presence of heterologous nucleic acids, including those transgenic organisms or cells that have initially changed, and those produced from the initial transgenic organisms or cells by hybridization or asexual propagation. A "recombinant" organism is preferably a "transgenic" organism. As used herein, the term "transgenic" is not intended to encompass changes in genomes (chromosomes or extra chromosomes) by conventional plant breeding methods (e.g., hybridization) or by naturally occurring events (e.g., self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation).

[0157] As used herein, "mutagenic" refers to an organism or its nucleic acid having an altered sequence of a biomolecule in its native genetic material compared to the sequence of the genetic material of a corresponding wild-type organism or nucleic acid, wherein the alteration in the genetic material is induced and / or selected by human behavior. Examples of human behavior that can be used to produce mutagenic organisms or DNA include, but are not limited to, treatment with a chemical mutagen (such as EMS) followed by selection with a herbicide; or by treating plant cells with x-rays followed by selection with a herbicide. Any method known in the art can be used to induce mutations. The method of inducing mutations can induce mutations at random positions in the genetic material, or can induce mutations at specific positions in the genetic material (i.e., can be directed mutagenesis techniques), such as by using gene shaping techniques. In addition to non-specific mutations, according to the present invention, nucleic acids can also be induced by using a mutagenic means that has a preference for or even specificity for a specific site, thereby producing artificially induced heritable alleles according to the present invention. Such means, for example, site-specific nucleases, including, for example, zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENS) (Malzahn et al., Cell Biosci, 2017, 7:21), and clustered regularly interspaced short palindromic repeats / CRISPR-associated nucleases (CRISPR / Cas) with engineered crRNA / tracrRNA (e.g., as single guide RNA, or as modified crRNA and tracrRNA molecules that form a bimolecular guide), and methods of using such nucleases to target known genomic locations are well known in the art (see Bortesi and Fischer, 2015, Biotechnology Advances 33:41-52; and reviews by Chen and Gao, 2014, Plant Cell Rep 33:575-583, and references therein).

[0158] As used herein, a "genetically modified organism" (GMO) is an organism whose genetic characteristics contain changes resulting from human effort that cause transfection, which results in the transformation of the target organism with genetic material from another, or "source," organism, or with synthetic or modified natural genetic material, or an organism that retains the inserted genetic material as its progeny. The source organism can be a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or from the same type of organism (e.g., a GMO plant can contain genetic material from another plant).

[0159] As used herein, "wild-type" or "corresponding wild-type plant" means a typical form of an organism or its genetic material, as it normally occurs, as distinguished from, for example, mutagenized and / or recombinant forms. Similarly, a "control cell" or "similar wild-type plant, plant tissue, plant cell, or host cell" means a plant, plant tissue, plant cell, or host cell, respectively, that lacks a particular polynucleotide of the invention disclosed herein. Thus, the use of the term "wild-type" is not intended to imply that a plant, plant tissue, plant cell, or other host cell lacks recombinant DNA in its genome and / or does not possess fungal resistance characteristics different from those disclosed herein.

[0160] As used herein, "progeny" refers to any generation of plants. Progeny or offspring plants can be from any daughter generation, such as F1, F2, F3, F4, F5, F6, F7, etc. In some embodiments, the progeny or offspring plant is a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth generation plant.

[0161] The term "plant" is used herein in its broadest sense as it relates to organic material and is intended to encompass eukaryotic organisms that are members of the plant taxonomic kingdom, examples of which include, but are not limited to, monocotyledons and dicotyledons, vascular plants, vegetables, cereals, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi, and algae, as well as clones, suckers, and plant parts used for asexual propagation (e.g., cuttings, tubes, buds, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). Unless otherwise indicated, the term "plant" refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, including any of the following: a whole plant, a plant component or organ (e.g., a leaf, stem, root, etc.), a plant tissue, a seed, a plant cell, and / or its progeny. A plant cell is a biological cell of a plant that is taken from a plant or derived by culturing a cell taken from a plant.

[0162] The present invention particularly relates to plants in general, and preferably to crop plants, i.e. plants selected from the taxonomic families Fabaceae, Cruciferae and Poaceae, most preferably soybean. More preferably, the crop plants are selected from the following:

[0163] - the taxonomic family Phaseoleae, more preferably the genus Cajanus, Canavalia, Glycine, Phaseolus, Psophocarpus, Pueraria or Vigna, even more preferably the species Cajanus cajan, Canavalia brasiliensis, Canavalia ensiformis, Canavalia gladiata, Glycinegracilis, Glycine max, Glycine soja, Phaseolus acutifolius, Phaseolus lunatus, Phaseolus maculatus, Psophocarpus tetragonolobus, Pueraria montana, Vigna angularis, Vigna mungo), mung bean (Vigna radiata) or cowpea (Vigna unguiculata), even more preferably the species Glycine gracilis, Glycine max or Glycine soja, even more preferably the species Glycine max; or

[0164] - the taxonomic tribe Fabeae, more preferably the genus Lathyrus, Lens, Pisum or Vicia, even more preferably the species Lathyrus aphaca, Lathyrus cicera, Lathyrus hirsutus, Lathyrus ochrus, Lathyrus odoratus, Lathyrus sphaericus, Lathyrustingitanus, Lens culinaris, Pisum sativum, Vicia cracca, Vicia faba or Vicia vellosa; or

[0165] - the taxonomic family Brassiceae, more preferably the genus Brassica, Crambe, Raphanus or Sinapis, even more preferably the species Brassica aucheri, Brassica balearica, Brassica barrelieri, Brassica bourgeaui, Brassica carinata, Brassica cretica, Brassica deflexa, Brassica desnottesii, Brassica drepanensis, Brassica elongata, Brassica fruticulosa, Brassica gravinae, Brassica hilarionis, Brassica incana, Brassica insularis, Brassica juncea, Brassica macrocarpa, Brassica maurorum, Brassica montana, Brassica napus, Brassica nigra, Brassica oleracea, Brassica oxyrrhina, Brassica procumbens, Brassica rapa, Brassica repanda, Brassica rupestris, Brassica souliei, Brassica spinescens, Brassica tournefortii, Brassica villosa, or hybrids of any of these species, and even more preferably Brassica napus, Brassica nigra, Brassica oleracea, Brassica oxyrrhina, Brassica procumbens, Brassica rapa, Brassica repanda, Brassica rupestris, Brassica souliei, Brassica spinescens, Brassica tournefortii, Brassica villosa, or hybrids of any of these species, and even more preferably Brassica napus, Brassica nigra, Brassica oleracea, Brassica oxyrrhina, Brassica procumbens, Brassica rapa, Brassica repanda, Brassica rupestris, Brassica souliei, Brassica spinescens, Brassica tournefortii, Brassica villosa, or hybrids of any of these species. cabbage), Brassica rapa (Brassica rapa), or a hybrid of any of these species, and even more preferably Brassica napus,

[0166] Species Radish (Raphanus sativus),

[0167] species white mustard (Sinapis alba); or

[0168] - the taxonomic tribes Tribus Andropogoneae, Bambuseae, Oryzeae, Poeae, Triticeae, more preferably the genera Saccharum, Zea, Oryza, Avena, Hordeum, Secale, Triticum, even more preferably the species Zea mays, Oryza sativa, Avena sativa, Avenastrigosa, Hordeum marinum, Hordeum vulgare, Secale cereale or Triticum aestivum,

[0169] And preferably the plant is soybean.

[0170] The term "seed" includes all types of seeds, such as, for example, true seeds, caryopsis, achenes, fruits, tubers, seedlings and similar forms. Preferably, "seed" refers to real seeds, unless otherwise indicated. For example, seed can be a transgenic plant or the seed of a plant obtained by site-specific mutagenesis, by site-preferred mutagenesis or by traditional breeding methods. Examples of traditional breeding methods are crossbreeding, selfing, backcrossing, embryo rescue, inbreeding, outcrossing, inbreeding, selection, asexual reproduction and other traditional techniques known in the art.

[0171] The present invention is particularly useful for combating phytopathogenic fungi. According to the present invention, the fungi to be combated are preferably biotrophic, hemibiotrophic or heminectotrophic fungi, more preferably rusts, downy mildews, powdery mildews, leaf spots, late blights, Fusarium and / or Septoria, and even more preferably rusts selected from the group consisting of:

[0172] - Basidiomycota, more preferably the taxonomy class Pucciniomycetes, more preferably the taxonomy order Pucciniales, more preferably the taxonomy family Pucciniaceae, more preferably the taxonomy genus Pucciniaceae, more preferably the taxonomy species Puccinia graminis; or

[0173] - phylum Basidiomycota, more preferably the taxonomy class Pucciniomycetes, more preferably the taxonomy order Pucciniales, more preferably the taxonomy family Phakopsoraceae, more preferably the taxonomy genus Phakopsora, more preferably the taxonomic species Phakopsora pachyrhizi or Phakopsora meibomiae; or

[0174] - phylum Ascomycota, more preferably the taxonomy Sordariomycetes, more preferably the taxonomy Hypocreales, more preferably the taxonomy Nectriaceae, more preferably the taxonomy Fusarium, more preferably the taxonomy species Fusarium graminearum or Fusarium verticillioides.

[0175] A particularly preferred advantage is that the materials and methods of the present invention can be used against rust fungi of the genus Phakopsora, particularly and most preferably Phakopsora pachyrhizi. These fungal pathogens are responsible for significant losses in soybeans when soybean plants are not treated. Thus, the present invention allows for a reduction in the number of fungicide treatments by reducing the fungal pathogen pressure.

[0176] The present invention particularly provides materials, preferably plants, plant parts or plant cells, or methods for increasing fungal resistance. According to the present invention, the increase in fungal resistance is preferably achieved by reducing the infection rate or infection extent or delaying the earliest day of fungal infection compared to the corresponding wild type. Thus, the Myb41 proteins and genes of the present invention are suitable for conferring, enhancing or stabilizing resistance of plants, plant parts or plant cells to infection by fungal pathogens, particularly resistance to biotrophic, hemibiotrophic or heminecrotizing fungi, and preferably resistance to fungi as described herein. In addition, by increasing fungal resistance as described in this paragraph, the Myb41 proteins and genes of the present invention are suitable for preventing, reducing or delaying the spread of fungal spores to other fields, thereby also reducing pathogen pressure in a wider area where plants expressing the Myb41 proteins of the present invention are planted. Furthermore, by increasing fungal resistance as described in this paragraph, the Myb41 proteins and genes of the present invention are suitable for reducing the number of fungicide treatments required to protect growing plants.

[0177] The present invention accordingly provides a method for conferring or increasing fungal resistance in a plant, plant part or plant cell, wherein the method comprises the step of increasing the production and / or accumulation of Myb41 in the plant, plant part or plant cell as compared to a corresponding wild-type plant, wild-type plant part or wild-type plant cell. As mentioned above, it is surprising that genes known to function only in controlling osmotic stress can have the beneficial effect of increasing fungal resistance, particularly in species other than Arabidopsis.

[0178] The present invention also provides a method for conferring or increasing fungal resistance in a plant, plant part or plant cell, wherein the method comprises increasing the expression and / or biological activity of a Myb41 protein in the plant, plant part or plant cell as compared to a corresponding wild-type plant, wild-type plant part or wild-type plant cell, wherein the Myb41 protein is encoded by:

[0179] (i) an exogenous nucleic acid having at least 70%, at least 80%, at least 90%, or at least 95% identity to SEQ ID NO. 1 or a functional fragment thereof or a splice variant thereof;

[0180] (ii) an exogenous nucleic acid encoding a protein or a functional fragment thereof that is at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO. 2 or 5;

[0181] (iii) an exogenous nucleic acid capable of hybridizing under stringent conditions to the complement of any nucleic acid according to (i) or (ii); or

[0182] (iv) an exogenous nucleic acid encoding the same Myb41 protein as the nucleic acid of (i) to (iii) above, but differing from the nucleic acid of (i) to (iii) above due to the degeneracy of the genetic code. Again, this method surprisingly and beneficially increases fungal resistance as described herein.

[0183] Preferably, the method comprises the following steps:

[0184] (a) stably transforming a plant cell with at least one expression cassette comprising an exogenous nucleic acid encoding a Myb41 protein,

[0185] (b) regenerating a plant from the plant cell; and

[0186] (c) expressing the Myb41 protein. Expression of the Myb41 protein of the present invention surprisingly confers or increases fungal resistance in plants as described herein.

[0187] Preferably, the above-mentioned method of the present invention comprises the step of phosphorylating the Myb41 protein in the corresponding plant, plant part or plant cell, preferably at serine 251 of SEQ ID NO. 2. Without being bound by any particular theory, it is expected that phosphorylation increases the availability of functional Myb41 protein to plant cells. Accordingly, the phosphomimetic forms of the Myb41 protein of the present invention are described above, in particular with respect to SEQ ID NO. 6, 7, 8 or 9.

[0188] The term "functional" means that the respective plant, plant part or plant cell is more likely to withstand an attempted infection by a pathogenic fungus, preferably Phakopsora pachyrhizi.

[0189] The present invention also provides a recombinant vector construct comprising a nucleic acid encoding the Myb41 protein, wherein the nucleic acid is selected from:

[0190] (i) a nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO. 1 or a functional fragment thereof or a splice variant thereof;

[0191] (ii) a nucleic acid encoding a protein having at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO. 2 or 5, or a functional fragment thereof;

[0192] (iii) an exogenous nucleic acid capable of hybridizing under stringent conditions to the complement of any nucleic acid according to (i) or (ii); or

[0193] (iv) a nucleic acid encoding the same Myb41 protein as the nucleic acid of (i) to (iii) above, but differing from the nucleic acid of (i) to (iii) above due to the degeneracy of the genetic code;

[0194] The nucleic acid is operably linked to a promoter and a transcription termination sequence, and wherein the nucleic acid does not encode a protein having the amino acid sequence of SEQ ID NO. 2. Such vectors are particularly useful for transforming plants to express the Myb41 gene of the present invention, thereby conferring or increasing fungal resistance in plants, plant parts or plant cells.

[0195] The promoter is preferably a constitutive promoter, a pathogen-inducible promoter, a mesophyll-specific promoter, or an epidermis-specific promoter. The selection of any of these promoters allows for the production of constitutively increased Myb41 protein levels in plant cells, or increased levels in response to infection with a pathogen (preferably a fungal pathogen), or specifically increasing Myb41 levels in mesophyll or plant epidermal cells, respectively.

[0196] Accordingly, the present invention provides transgenic plants, transgenic plant parts or transgenic plant cells transformed with one or more recombinant vector constructs according to the present invention.

[0197] And particularly beneficial for fungal resistance, the present invention provides a crop plant, crop plant part or crop plant cell that overexpresses a Myb41 protein, wherein the Myb41 protein is encoded by:

[0198] (i) a nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO. 1 or a functional fragment thereof or a splice variant thereof;

[0199] (ii) a nucleic acid encoding a protein having at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO. 2 or 5, or a functional fragment thereof;

[0200] (iii) an exogenous nucleic acid capable of hybridizing under stringent conditions to the complement of any nucleic acid according to (i) or (ii); or

[0201] (iv) a nucleic acid encoding the same Myb41 protein as the nucleic acid of (i) to (iii) above, but differing from the nucleic acid of (i) to (iii) above due to the degeneracy of the genetic code;

[0202] wherein the nucleic acid according to any one of (i) to (iv) is operably linked to a promoter and a transcription termination sequence, and preferably wherein

[0203] (a) the crop plant, crop plant part or crop plant cell is a transgenic crop plant, crop plant part or crop plant cell, or overexpresses artificially induced heritable mutations that result in the formation of a wild-type genome; and / or

[0204] (b) wherein the gene encoding Myb41 is integrated into the genome of the plant, plant part or plant cell, and / or

[0205] (c) wherein the plant or plant part is homozygous for a gene encoding Myb41 or heterozygous for a gene encoding Myb41, and / or

[0206] (d) wherein the plant or plant part is non-disjunctive or segregating during meiosis with respect to the gene encoding Myb41, and / or

[0207] (e) wherein the gene encoding Myb41 is operably linked to a heterologous promoter, and / or

[0208] (f) wherein the gene encoding Myb41 is integrated in the genome of the plant or plant part at a locus different from that of the corresponding wild-type Myb41 gene.

[0209] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology can be used to modify the genome of a target organism, such as introducing any given DNA fragment into almost any site in the genome, replacing part of the genome with a desired sequence, or precisely deleting a given region in the genome of a target organism. This allows for unprecedented precision in genome manipulation.

[0210] The CRISPR system was initially identified as an adaptive defense mechanism (WO2007 / 025097) of bacteria belonging to the genus Streptococcus. Those bacterial CRISPR systems rely on a guide RNA (gRNA) complexed with a cleavage protein to guide the degradation of complementary sequences present in invading viral DNA. Applications of CRISPR systems for genetic manipulation in various eukaryotic organisms have been shown (WO2013 / 141680, WO2013 / 176772, WO2014 / 093595). Cas9 (the first identified protein of the CRISPR / Cas system) is a large monomeric DNA nuclease that is guided to a DNA target sequence adjacent to a PAM (protospacer sequence adjacent motif) sequence motif by a complex of two non-coding RNAs: CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA).

[0211] In addition, synthetic RNA chimeras (single guide RNA or sgRNA) produced by fusing crRNA to tracrRNA have shown equivalent functions (WO2013 / 176772). CRISPR systems from other sources have been described, which contain DNA nucleases different from Cas9, such as Cpf1, C2c1p or C2c3p, which have the same function (WO2016 / 0205711, WO2016 / 205749). Other authors have described systems in which nucleases are guided by DNA molecules rather than RNA molecules. Such systems are, for example, the AGO system disclosed in US2016 / 0046963.

[0212] Several research groups have discovered that the CRISPR cutting property can be used to destroy target regions in the genome of almost any organism with unprecedented ease. Recently, it has become clear that providing a template for repair allows the genome to be edited with almost any desired sequence at almost any site, transforming CRISPR into a powerful gene editing tool (WO2014 / 150624, WO2014 / 204728). The template used for repair is called a donor nucleic acid, which contains sequences complementary to the target region at the 3' and 5' ends, allowing homologous recombination in the corresponding template after the introduction of a double-strand break in the target nucleic acid by the corresponding nuclease.

[0213] A major limitation in selecting a target region within a given genome is the necessity of a PAM sequence motif near the region where the CRISPR-associated nuclease introduces the double-strand break. However, various CRISPR systems recognize different PAM sequence motifs. This allows for the selection of the most appropriate CRISPR system for the respective target region. Furthermore, the AGO system does not require a PAM sequence motif at all.

[0214] This technology can be applied to, for example, change the gene expression in any organism, for example, by exchanging the promoter upstream of the target gene with a promoter of different strengths or specificity. Other methods disclosed in the prior art describe activation or inhibition of the fusion of transcription factors and nuclease minus CRISPR nuclease protein. Such fusion protein can be expressed in the target organism together with one or more guide nucleic acids, which guide the transcription factor portion of the fusion protein to any desired promoter in the target organism (WO2014 / 099744; WO2014 / 099750). By introducing point mutations or deletions into the corresponding target gene, for example, by inducing non-homologous end joining (NHEJ) that generally causes gene disruption, it is possible to easily achieve gene knockout (WO2013 / 176772).

[0215] Therefore, the present invention also provides a collection of at least 50 crop plants according to the present invention, more preferably at least 100 plants, even more preferably at least 1,000 plants, even more preferably at least 100,000 plants. According to the present invention, preferably at least 100,000 plants are grown per hectare, more preferably 200,000 to 800,000 plants are grown per hectare, even more preferably at least 250,000 to 650,000 plants are grown per hectare. Such a number of plants is preferably observed within one hectare; therefore, the present invention is particularly conducive to ecologically sound intensive agriculture that reduces the use of fungicides in each growing season. The plants according to the present invention are preferably grown in fields or greenhouses. Preferably, the crop plants are soybean plants.

[0216] According to the present invention, it is not required that all crop plants of a species grown in the same field or greenhouse are plants of the present invention. On the contrary, it is sufficient in a monoculture plantation if at least about 25% of the plants of a species belong to the present invention, more preferably at least 50%, even more preferably 25%-75%, and most preferably 45%-70%, especially when mixed or combined with plants carrying other resistance genes or mechanisms. The combination with plants with other resistance genes can be carried out by intercropping (mixing), row type or block type. For example, in a soybean field, if approximately every other plant is a plant of the present invention, the number of fungicide treatments can be reduced. It is particularly preferred that at least 25%, more preferably 50%-100%, even more preferably 75%-100% of those plants on the same field that are not plants according to the present invention contain at least one other biological means for enhancing fungal resistance, most preferably that the other means are selected from the list of pathogen resistance polypeptides as described above.

[0217] The present invention also provides a method for producing a transgenic crop plant, a transgenic crop plant part or a transgenic crop plant cell having increased fungal resistance compared to a corresponding wild-type plant, plant part or plant cell, comprising

[0218] (a) introducing an exogenous nucleic acid encoding a Myb41 protein into a plant, a plant part or a plant cell,

[0219] (b) generating transgenic plants, transgenic plant parts or transgenic plant cells from plants, plant parts or plant cells; and

[0220] (c) expressing said Myb41 protein in a transgenic plant, a transgenic plant part or a transgenic plant cell derived from a plant,

[0221] The Myb41 protein is encoded by:

[0222] (i) a nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO. 1 or a functional fragment thereof or a splice variant thereof;

[0223] (ii) a nucleic acid encoding a protein having at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO. 2 or 5, or a functional fragment thereof;

[0224] (iii) an exogenous nucleic acid capable of hybridizing under stringent conditions to the complement of any nucleic acid according to (i) or (ii); or

[0225] (iv) a nucleic acid encoding the same Myb41 protein as the nucleic acid of (i) to (iii) above, but differing from the nucleic acid of (i) to (iii) above due to the degeneracy of the genetic code;

[0226] The nucleic acid is operably linked to a promoter and a transcription termination sequence. As described above, this method is particularly suitable for conferring or increasing fungal resistance in plants, plant parts or plant cells.

[0227] The present invention also provides a method for determining antifungal resistance in plants, comprising screening cells of the plant for overexpression of the Myb41 gene. As described herein, overexpression of Myb41 increases resistance to antifungal infection, particularly in crop plants, particularly against infection by Phakopsora pachyrhizi. It is useful to prepare the plants to be screened without osmotic stress to prevent an unintended increase in Myb41 expression levels.

[0228] The present invention also provides a method for breeding fungus-resistant crop plants, comprising:

[0229] (a) crossing a plant according to the invention or a plant obtainable by a method according to the invention with a second plant;

[0230] (b) obtaining seeds from the hybridization of step (a);

[0231] (c) planting the seeds and growing the seeds into plants; and

[0232] (d) selecting plants expressing the Myb41 protein as defined above from said plants. This method advantageously allows conferring the increased fungal resistance trait achieved according to the present invention to plants, in addition to transformation or other direct intervention (e.g., by CRISPR-mediated methods). Thus, this breeding method advantageously allows for high-speed plant propagation.

[0233] Further advantages and beneficial effects of the invention are described in the accompanying examples and drawings. Example

[0234] The following examples are not intended to limit the scope of the claims to the present invention, but are intended to illustrate certain embodiments.Any variations of the exemplary methods that occur to one skilled in the art are intended to fall within the scope of the present invention.

[0235] Example 1: General method

[0236] Chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoramidite method (Voet, Voet, 2nd edition, Wiley Press New York, pp. 896-897). The cloning steps carried out for the purposes of the present invention, such as, for example, restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, ligation of DNA fragments, transformation of E. coli cells, bacterial culture, phage propagation and sequence analysis of recombinant DNA, are carried out as described in Sambrook et al., Cold Spring Harbor Laboratory Press (1989), ISBN 0-87969-309-6. Sequencing of recombinant DNA molecules is carried out using a MWG-Licor laser fluorescence DNA sequencer according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 546 3 (1977)).

[0237] Example 2: Cloning of overexpression vector constructs

[0238] The cDNA sequence of the Myb41 gene mentioned in the present application was generated by DNA synthesis (Geneart, Thermo Fisher Scientific, Waltham, Massachusetts, USA).

[0239] Myb41 cDNA (shown as SEQ ID NO. 1) was synthesized with an AscI restriction site located before the initiator ATG and an SbfI restriction site located downstream of the stop codon. The synthesized DNA was digested with the restriction enzymes SbfI and AscI (NEB Biolabs) and ligated into an SbfI / AscI-digested binary plant transformation vector with the full-length fragment positioned in the sense orientation between the parsley ubiquitin promoter and the Agrobacterium tumefaciens-derived nopaline synthase terminator (t-nos). The PcUbi promoter regulates constitutive expression of the parsley ubi4-2 gene (EMBL accession number X64345) (Kawalleck et al., 1993 Plant Molecular Biology 21(4): 673-684).

[0240] The binary plant transformation vector used in this application consists of the following: (1) a kanamycin resistance cassette for bacterial selection; (2) a pVS1 origin of replication in Agrobacterium; (3) a ColE1 origin of replication for stable maintenance in Escherichia coli; and (4) a double mutant AHAS (acetohydroxyacid synthase large subunit) selection marker between the right and left borders, which is derived from Arabidopsis thaliana (P-AtAHASL5', see Figure 2The ligation reaction was transformed into Escherichia coli (DH5α), miniprepped, and screened by specific restriction digestion. Positive clones were sequenced and transformed into soybean.

[0241] Example 3: Soybean transformation

[0242] The expression vector construct (see Example 2) was transformed into soybean.

[0243] 3.1 Sterilization and germination of soybean seeds

[0244] In fact, any seed of any soybean variety can be used in the method of the present invention. Various soybean cultivars (including Jack, Williams 82, Jake, Stoddard and Resnik) are suitable for soybean transformation. Soybean seeds are sterilized in a sealed chamber with chlorine gas, which is produced by adding 3.5ml 12N HCl dropwise to 100ml bleach (5.25% sodium hypochlorite) in a desiccator with a tightly fitting lid. After 24 to 48 hours in the sealed chamber, the seeds are taken out and approximately 18 to 20 seeds are spread on solid GM medium containing or not containing 5μM 6-benzyl-aminopurine (BAP) in a 100mm culture dish. Seedlings without BAP are longer and have root development, particularly secondary and lateral root formation. BAP enhances seedlings by forming shorter and sturdier seedlings.

[0245] The 25 ° C under light (> 100 μ Einstein / m 2 Seven-day-old seedlings grown under the condition of s) are used as the explant material of three explant types.At this moment, seed coat splits, and epicotyl has grown to the length of at least cotyledon together with single compound leaf.Epicotyl should be at least 0.5cm to avoid cotyledon-stem node tissue (because soybean cultivar and seed batch may be different in development time, so the description of germination stage is more accurate than specific germination time).

[0246] For inoculation of whole seedlings, see Method A (Examples 3.3 and 3.3.2), or for inoculation of leaf explants, see Method B (Example 3.3.3).

[0247] For method C (see embodiment 3.3.4), remove the hypocotyl and a part of one and a half cotyledons or two cotyledons from each seedling. Then the seedling is placed on a propagation medium for 2 to 4 weeks. The seedling produces several branched buds to obtain explants therefrom. Most explants are derived from the plantlets grown from the terminal bud. These explants are preferably used as target tissues.

[0248] 3.2-Growth and preparation of Agrobacterium cultures

[0249] Agrobacterium cultures are prepared by streaking Agrobacterium (e.g., Agrobacterium tumefaciens or Agrobacterium rhizogenes) carrying the desired binary vector (e.g., H. Klee. R. Horsch and S. Rogers 1987 Agrobacterium-Mediated Plant Transformation and its further Applications to Plant Biology; Annual Review of Plant Physiology Vol. 38: 467-486) onto solid YEP growth medium (YEP medium: 10 g yeast extract, 10 g bacto-peptone, 5 g NaCl. Adjust pH to 7.0 and adjust to a final volume of 1 liter with H2O. For YEP agar plates, add 20 g agar and autoclave) and incubate at 25°C until colonies appear (approximately 2 days). Different selection compounds are used for Agrobacterium tumefaciens and Agrobacterium rhizogenes selection in YEP solid and liquid medium, depending on the selection marker gene present on the Ti or Ri plasmid, binary vector, and bacterial chromosome. Various Agrobacterium strains can be used for transformation methods.

[0250] After about two days, a single colony was picked (with a sterile toothpick) and inoculated into 50 mL of liquid YEP with antibiotics and shaken at 175 rpm (25°C) until the OD 600 Reach between 0.8-1.0 (approximately 2 days). Prepare working glycerol stock (15%) for transformation and aliquot 1 ml of Agrobacterium stock into 1.5 ml Eppendorf tubes and store at -80°C.

[0251] One day before explant inoculation, inoculate 200 ml of YEP with 5 μl to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask. Shake the flask overnight at 25 °C until the OD 600 Between 0.8 and 1.0. Before preparing soybean explants, Agrobacterium was pelleted by centrifugation at 5,500 x g for 10 min at 20°C. The pellet was resuspended in liquid CCM to the desired density (OD 600 0.5-0.8) and place at room temperature for at least 30 min before use.

[0252] 3.3-Explant preparation and co-cultivation (inoculation)

[0253] 3.3.1 Method A: Explant preparation on the day of transformation.

[0254] The seedlings at this point have an elongated epicotyl of at least 0.5 cm but typically between 0.5 and 2 cm. Elongated epicotyls up to 4 cm have been successfully used. Explants are then prepared with or without some roots, or with part, one or two cotyledons, removing all preformed leaves, including the apical meristem, and using a sharp scalpel to injure the nodes at the first set of leaves with several cuts.

[0255] This incision at the stem node not only induces Agrobacterium infection but also disperses axillary meristem cells and damages preformed shoots. After wounding and preparation, the explants are placed in a Petri dish and subsequently co-cultivated with the liquid CCM / Agrobacterium mixture for 30 minutes. The explants are then removed from the liquid culture medium and plated on sterile filter paper in a 15 x 100 mm Petri dish with solid co-cultivation medium. The wounded target tissues are positioned so that they are in direct contact with the culture medium.

[0256] 3.3.2 Improved Method A: Epicotyl Explant Preparation

[0257] The soybean epicotyl fragment prepared from 4 to 8 day old seedlings is used as the explant for regeneration and conversion.Seeds of soybean cultivars (cv.) L00106CN, 93-41131 and Jack are germinated 4 to 8 days in the culture medium with or without 1 / 10MS salt of cytokinin or similar composition.Prepare epicotyl explant by removing cotyledonary node and stem node from stem section.Epicotyl is cut into 2 to 5 fragments.Particularly preferably be connected to the segment that comprises the primary or higher section of axillary meristem.

[0258] Explant is used for agrobacterium infection.To carry the Agrobacterium AGL1 of the plasmid with target gene (GOI) and AHAS, bar or dsdA selective marker gene and incubate overnight in the LB culture medium containing suitable antibiotic, gather in the crops and be resuspended in the inoculation culture medium containing acetosyringone.The epicotyl fragment of fresh preparation is soaked 30 to 60min in Agrobacterium suspension, then explant is blotted on sterile filter paper.Then the explant of inoculation is cultivated 2 to 4 days on the co-culture medium with other chemicals (such as acetosyringone) that T-DNA sends for increasing.Then the epicotyl explant of infection is placed on the bud induction culture medium containing selective agent such as imazapyr (for AHAS gene), glufosinate (for bar gene) or D-serine (for dsdA gene).The bud of regeneration is subcultured on the elongation culture medium with selective agent.

[0259] To regenerate transgenic plants, the fragments are then cultured on a medium containing cytokinins (e.g., BAP, TDZ, and / or kinetin) to induce shoots. After 4 to 8 weeks, the cultured tissue is transferred to a medium containing a lower concentration of cytokinins for shoot elongation. The elongated shoots are then transferred to a medium containing auxins for rooting and plant development. Multiple shoots are regenerated.

[0260] Many stably transformed segments showing strong cDNA expression were collected. Soybean plants were regenerated from epicotyl explants, demonstrating efficient T-DNA delivery and stable transformed segments.

[0261] 3.3.3 Method B: Leaf Explants

[0262] To prepare leaf explants, remove the cotyledons from the hypocotyl. Separate the cotyledons from each other and remove the epicotyl. Remove the primary leaves consisting of blade, petiole and stipules from the epicotyl by carefully cutting at the stipule base so that the axillary meristem is included on the explant. In order to wound the explant and stimulate new bud formation, remove any pre-formed buds and cut the area between the stipules 3 to 5 times with a sharp scalpel.

[0263] After explant preparation, immediately explant is immersed in the Agrobacterium suspension fully or the petiole end that is injured is immersed in the Agrobacterium suspension.After inoculation, explant is dipped into dry to remove excessive Agrobacterium culture on sterile filter paper, and explant is placed into and makes the injured side contact with the circular 7cm Whatman paper that covers solid CCM culture medium (referring to above).This filter paper prevents Agrobacterium tumefaciens from overgrowing on soybean explant.Wrap five flat boards with Parafilm.TM. " M" (American National Can, Chicago, III., USA), and under dark or illumination, under 25 ℃, hatch 3 to 5 days.

[0264] 3.3.4 Method C: Propagation of axillary meristems

[0265] In order to prepare the axillary meristem explants of breeding, use the 3-4 week old plantlets of breeding. Axillary meristem explants can be prepared from the first to the fourth node. Each seedling can obtain 3-4 explants on average. By cutting 0.5-1.0cm below the axillary nodes of the internodes and removing the petiole and leaves from the explant, explants are prepared from the plantlets. With a scalpel, the tip where the axillary meristem is located is used to induce new shoot growth and allow target cells to approach Agrobacterium. Therefore, the 0.5cm explant includes stem and bud.

[0266] Once cut, immediately place the explant in an Agrobacterium suspension for 20 to 30 minutes. After inoculation, the explant is dried on a sterile filter paper to remove excess Agrobacterium culture and then almost completely immersed in solid CCM or placed on top of a circular 7 cm filter paper covering solid CCM, depending on the Agrobacterium strain. This filter paper prevents Agrobacterium from overgrowing on the soybean explant. Wrap the flat plate with Parafilm.TM. "M" (American National Can, Chicago, III., USA) and incubate in the dark at 25°C for 2 to 3 days.

[0267] 3.4-Bud induction

[0268] After 3 to 5 days of co-cultivation at 25°C in the dark, the explants were rinsed in liquid SIM medium (to remove excess Agrobacterium) (SIM, see Olhoft et al. 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soy using primary-node explants from serlings invitro cells. Dev. Biol.-Plant (2007) 43:536-549; to remove excess Agrobacterium) or Modwash medium (1×B5 major salts, 1×B5 minor salts, 1×MSI11 iron, 3% sucrose, 1×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 leaves) and then placed on solid SIM medium. Approximately 5 explants (method A) or 10 to 20 explants (methods B and C) are placed so that the target tissue is in direct contact with the culture medium. During the first 2 weeks, the explants can be cultured with or without selective medium. Preferably, the explants are transferred to SIM without a selective agent for 1 week.

[0269] For leaf explants (Method B), the explant should be placed in the medium so that it is perpendicular to the surface of the medium with the petiole embedded in the medium and the leaf blade protruding from the medium.

[0270] For propagating axillary meristems (method C), the explant was placed in the medium so that it was parallel to the medium surface (base) and the explant was partially embedded in the medium.

[0271] The plates were wrapped with Scotch 394 breathable tape (3M, St. Paul, Minn., USA) and placed in a growth chamber for two weeks at an average temperature of 25°C and a flow rate of 70-100 μE / m2 s under 18h light / 6h dark cycle.Explant remains on SIM culture medium with or without selection, until new sprout growth takes place in target area (for example, the axillary meristem at the first node above the epicotyl).Can be transferred to fresh culture medium during this period.After about one week, explant is transferred to the SIM with selection from the SIM with or without selection.At this moment, at the petiole base (method B) of the leaf explant in various SIMs, at the primary node place (method A) of the seedling explant, and at the axillary node place (method C) of the explant of breeding, there is considerable new sprout development.

[0272] Preferably, all shoots formed before transformation are removed at most 2 weeks after co-cultivation to stimulate new growth of meristems. This helps to reduce mosaicism in primary transformants and increase the expansion of transgenic meristem cells. During this period, the explants may or may not be cut into smaller pieces (i.e., the nodes are separated from the explants by cutting the epicotyl).

[0273] 3.5-Bud elongation

[0274] After 2 to 4 weeks (or until a large number of shoots are formed) on SIM medium (preferably containing a selection agent), the explants are transferred to SEM medium (shoot elongation medium, see Olhoft et al., a novel Agrobacterium rhizogenes-mediated transformation method of soy using primary-node explants from sidelings. In Vitro Cell. Dev. Biol.-Plant (2007) 43: 536-549) which stimulates shoot elongation of the shoot primordium. This medium may or may not contain a selection compound.

[0275] After every 2 to 3 weeks, after carefully removing dead tissue, explant is transferred in the fresh SEM culture medium (preferably containing selection agent).Explant should gather together and do not disperse into small pieces and keep a certain degree of health.Continue to transfer explant until explant death or bud elongation.Take out the elongation bud of >3cm and place RM culture medium for about 1 week (method A and B), or about 2 to 4 weeks, this depends on cultivar (method C), and now, root begins to form.Under the situation of the explant with root, they are directly transferred in the soil.The bud that takes root is transferred in the soil and hardened 2 to 3 weeks in the growth chamber, then transferred in the greenhouse.The regenerated plant that uses this method to obtain is fertile, and every strain plant on average produces 500 seeds.

[0276] After 5 days of co-cultivation with Agrobacterium tumefaciens, transient expression of the gene of interest (GOI) was widely distributed in the axillary meristem explants of the seedlings, particularly in the area injured during explant preparation (Method A). The explants were placed in a shoot induction medium without a selection agent to observe how the primary nodes responded to shoot induction and regenerated. So far, more than 70% of the explants have formed new shoots in this area. After 14 days on SIM, expression of the GOI was stable, indicating that the T-DNA had integrated into the soybean genome. In addition, preliminary experiments resulted in the formation of shoots expressing the cDNA after 3 weeks on SIM.

[0277] The average regeneration time for soybean plantlets using the axillary meristem protocol for propagation was 14 weeks from explant inoculation for Method C. Thus, this method has a rapid regeneration time that results in fertile, healthy soybean plants.

[0278] Example 4: Pathogen Assay

[0279] 4.1. Plant growth

[0280] Ten T1 plants per event were potted and grown in a plant room for 3-4 weeks (16-h-day- and 8-h-night-rhythm, temperature of 16° C. and 22° C., humidity of 75%), until the first two trifoliate leaves were fully expanded.

[0281] 4.2 Inoculation

[0282] Plants are inoculated with spores of Phakopsora pachyrhizi. To obtain appropriate spore material for inoculation, soybean leaves that have been infected with rust for 15-20 days previously are removed 2-3 days before inoculation and transferred to agar plates (1% agar in H2O). The leaves are placed on the agar with their upper side, which allows the fungus to grow through the tissue and produce very young spores. For the inoculation solution, spores are knocked off the leaves and added to a Tween-H2O solution. Spore counting is performed under a light microscope using a Thoma counting chamber. To inoculate plants, the spore suspension is added to a compressed air-driven spray flask and applied evenly to the plants or leaves until the leaf surface is thoroughly wetted. For macroscopic determinations, we use 1-5×10 6 Spore density is spores / ml. For microscopy, use >5 × 10 5 The inoculated plants were placed in a greenhouse at an average temperature of 22°C and >90% humidity for 24 hours. Subsequent cultivation was carried out in a room at an average temperature of 25°C and 70% humidity.

[0283] Example 5: Microscopic screening:

[0284] To assess pathogen development, inoculated plant leaves were stained with aniline blue 48 h after infection.

[0285] Aniline blue staining is used to detect fluorescent substances. During the defense reaction in host interaction and non-host interaction, substances such as phenols, callose or lignin are accumulated or produced and are incorporated into the cell wall locally in the papilla or in intact cells (hypersensitive reaction, HR). Combined with aniline blue to form a complex, which, for example, causes yellow fluorescence in the case of callose. The leaf material is transferred to a falcon tube or culture dish containing decolorization solution II (ethanol / acetic acid 6 / 1) and incubated in a 90°C water bath for 10-15 minutes. Immediately thereafter, the decolorization solution II is removed and the leaves are washed twice with water. For staining, the leaves are incubated in staining solution II (0.05% aniline blue = methyl blue, 0.067M dipotassium hydrogen phosphate) for 1.5-2 hours and analyzed by microscopy immediately thereafter.

[0286] The different interaction types were evaluated (counted) microscopically. An Olympus UV microscope BX61 (incident light) and a UV long-pass filter (excitation: 375 / 15, beam splitter: 405LP) were used. After staining with aniline blue, spores appear blue under UV light. The papillae beneath the fungal attachment wall can be identified by green / yellow staining. Hypersensitive reactions (HR) were characterized by whole-cell fluorescence.

[0287] Example 6: Assessment of Susceptibility to Soybean Rust

[0288] 14 days after inoculation (see Example 4), the progression of soybean rust was scored by estimating the diseased area (area covered by sporulated rust fungi) on the adaxial surface of the leaves. In addition, yellowing of the leaves was taken into account (for protocol see Figure 1 ).

[0289] All 107 T1 soybean plants expressing the Myb41 protein, representing nine independent transformation events (11-12 plants per event), were inoculated with spores of P. pachyrhizi (see Example 4). Macroscopic disease symptoms of P. pachyrhizi infection of soybeans were scored 14 days after inoculation. Plants were grown and maintained under the conditions described in Example 4.

[0290] The mean of the percentage of leaf area showing fungal colonies or strong yellowing / browning was considered the diseased leaf area. All 107 transgenic T1 soybean plants expressing Myb41 (expression checked by RT-PCR) were grown and evaluated in parallel with non-transgenic control plants having the same genetic background as used for transformation (see Example 3).

[0291] The mean values of diseased leaf areas of plants expressing recombinant Myb41 protein and corresponding wild-type control plants are shown in Figure 5 Transgenic T1 soybean plants expressing Myb41 and non-transgenic wild-type control plants were based on the same genetic background.

[0292] Overexpression of the Myb41 protein reduced diseased leaf area by an average of 33.5% across all events and resulting plants compared to non-transgenic control plants. This difference was statistically significant at the p < 0.001 level (two-sided Student's t-test). The data clearly demonstrate that in planta expression of the Myb41 expression vector construct (see Figure 2 ) resulted in lower disease in transgenic plants. Thus, expression of the Myb41 protein (as shown in SEQ ID NO. 2) in soybeans increased the resistance of soybeans to soybean rust.

Claims

1. A method for conferring or increasing resistance to Phakopsora pachyrhizi in a soybean plant, soybean plant part, or soybean plant cell, wherein the method comprises the step of overexpressing Myb41 in the soybean plant, soybean plant part, or soybean plant cell as compared to a corresponding wild-type soybean plant, wild-type soybean plant part, or wild-type soybean plant cell, wherein the Myb41 protein is encoded by a nucleic acid encoding SEQ ID NO.

2.

2. The method according to claim 1, comprising the step of phosphorylating the Myb41 protein in the corresponding soybean plant, soybean plant part or soybean plant cell, or wherein the Myb41 protein comprises a phosphomimetic mutation.

3. A method for conferring or increasing resistance to Phakopsora pachyrhizi in a soybean plant, soybean plant part, or soybean plant cell, wherein the method comprises overexpressing a Myb41 protein in the soybean plant, soybean plant part, or soybean plant cell as compared to a corresponding wild-type soybean plant, wild-type soybean plant part, or wild-type soybean plant cell, wherein the Myb41 protein is encoded by a nucleic acid encoding SEQ ID NO.

2.

4. The method according to claim 3, comprising the steps of: (a) stably transforming soybean plant cells with at least one expression cassette comprising an exogenous nucleic acid encoding a Myb41 protein, (b) regenerating a soybean plant from the soybean plant cell; and (c) Overexpression of the Myb41 protein.

5. The method of claim 3, further comprising the step of phosphorylating the Myb41 protein in the corresponding soybean plant, soybean plant part or soybean plant cell, or wherein the Myb41 protein comprises a phosphomimetic mutation.

6. A method for producing a genetically modified crop soybean plant, genetically modified crop soybean plant part, or genetically modified crop soybean plant cell having increased resistance to Phakopsora pachyrhizi compared to a corresponding wild-type soybean plant, soybean plant part, or soybean plant cell, comprising: (a) introducing an exogenous nucleic acid encoding a Myb41 protein into a soybean plant, a soybean plant part or a soybean plant cell, (b) producing a genetically modified soybean plant, genetically modified soybean plant part, or genetically modified soybean plant cell from the soybean plant, soybean plant part, or soybean plant cell; and (c) overexpressing a Myb41 protein in a genetically modified soybean plant cell, a genetically modified soybean plant part or a genetically modified soybean plant from a soybean plant, The Myb41 protein is encoded by the nucleic acid encoding SEQ ID NO. 2, It is operably linked to a promoter and transcription termination sequences.

7. Use of an overexpressed Myb41 protein or a nucleic acid encoding a Myb41 protein for increasing resistance to Phakopsora pachyrhizi in soybean plants, wherein the Myb41 protein is encoded by a nucleic acid encoding SEQ ID NO.

2.

8. The use according to claim 7, wherein the increase in resistance to Phakopsora pachyrhizi comprises delaying or reducing infection of soybean plants by Phakopsora pachyrhizi.

9. A product derived from a soybean plant producible by the method of claim 6, wherein the product is non-reproductive and comprises a Myb41 protein and / or an exogenous nucleic acid encoding a Myb41 protein as defined in claim 6, which is soybean oil or soybean meal.

10. A method for producing a product, comprising: a) growing a soybean plant obtainable by the method according to claim 6, and b) producing the product from or by a soybean plant and / or soybean plant part, wherein the product comprises an exogenous nucleic acid encoding the Myb41 protein and / or the Myb41 protein, wherein the Myb41 protein is encoded by a nucleic acid encoding SEQ ID NO.

2.

11. The method according to claim 10, wherein the soybean plant part is a seed.

12. The method according to claim 10 or 11, wherein the product is a soy product.

13. The method according to claim 10 or 11, wherein the product is soybeans, soybean oil or soybean meal.

14. A method according to claim 10 or 11, comprising a) growing soybean plants obtainable by the method of claim 6 and removing harvestable parts from the soybean plants, and b) producing said product from or by harvestable parts of a soybean plant.

15. A method for determining resistance of a soybean plant to Phakopsora pachyrhizi, comprising screening cells of the soybean plant for overexpression of the Myb41 gene, wherein the Myb41 protein is encoded by a nucleic acid encoding SEQ ID NO.

2.

16. The method according to claim 15, wherein the Myb41 gene comprises a phosphomimetic mutation.

17. A method for breeding soybean plants resistant to Phakopsora pachyrhizi, comprising (a) crossing a soybean plant obtainable by the method of claim 6 with a second soybean plant; (b) obtaining seeds from the hybridization of step (a); (c) planting the seeds and allowing the seeds to grow into soybean plants; and (d) selecting soybean plants overexpressing the Myb41 protein from the soybean plants.

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