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WO2025027165A1 - Résistance accrue par expression d'une protéine ics - Google Patents

Résistance accrue par expression d'une protéine ics Download PDF

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Publication number
WO2025027165A1
WO2025027165A1 PCT/EP2024/071902 EP2024071902W WO2025027165A1 WO 2025027165 A1 WO2025027165 A1 WO 2025027165A1 EP 2024071902 W EP2024071902 W EP 2024071902W WO 2025027165 A1 WO2025027165 A1 WO 2025027165A1
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Prior art keywords
plant
brassica
nucleic acid
identity
plant material
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PCT/EP2024/071902
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English (en)
Inventor
Ruth CAMPE
Brody John DEYOUNG
Flachmann RALF
Anne-Christina HERWIG
Holger Schultheiss
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Basf Plant Science Company Gmbh
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Publication of WO2025027165A1 publication Critical patent/WO2025027165A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/04—Intramolecular transferases (5.4) transferring hydroxy groups (5.4.4)
    • C12Y504/04002—Isochorismate synthase (5.4.4.2)

Definitions

  • the present invention relates to genes, materials, and methods for improving plant health, preferably against infection by phythopathogenic microorganisms. Furthermore, the invention pertains to methods and uses of such genes and materials for creating correspondingly beneficial plant as well as plant material.
  • the present invention also relates to products obtained from such plant or plant material. To this end the invention focuses on facilitating or increasing the production and/or biological activity of an isochorismate synthase (ICS), fragment or homolog thereof in a plant or plant material compared to corresponding wild type plant or plant material.
  • ICS isochorismate synthase
  • the invention also relates to plants, plant parts, and/or plant cells having an increased resistance against fungal pathogens and to material and methods to create or use such plant and plant material or to produce products therefrom.
  • the cultivation of agricultural crop plants serves mainly for production of foodstuffs for humans and animals.
  • Plant pathogenic organisms and particularly fungi have resulted in severe reductions in crop yield in the past, in worst cases leading to famine.
  • Monocultures in particular, which are routine nowadays, are highly susceptible to an epidemic-like spread of diseases. The result is markedly reduced yields.
  • the pathogenic organisms have been controlled mainly by using pesticides.
  • the possibility of directly modifying the genetic disposition of a plant or pathogen is also open to man.
  • natural occurring fungicides produced by the plants after fungal infection can be synthesized and applied to the plants.
  • Resistance generally describes the ability of a plant to prevent, or at least curtail the infestation and colonization by a harmful pathogen. Different mechanisms can be discerned in the naturally occurring resistance, with which the plants fend off colonization by phytopathogenic organisms (Schopfer and Brennicke (1999) convinced to the naturally occurring resistance, with which the plants fend off colonization by phytopathogenic organism
  • race specific resistance also called host resistance
  • a differentiation is made between compatible and incompatible interactions.
  • an interaction occurs between a virulent pathogen and a susceptible plant.
  • the pathogen survives, and may build up reproduction structures, while the host is seriously hampered in development or dies off.
  • An incompatible interaction occurs on the other hand when the pathogen infects the plant but is inhibited in its growth before or after weak development of symptoms (mostly by the presence of R genes of the NBS-LRR family, see below). In the latter case, the plant is resistant to the respective pathogen (Schopfer and Brennicke, vide supra).
  • this type of resistance is mostly specific for a certain strain or pathogen.
  • pathogens are plant-species specific. This means that a pathogen can induce a disease in a certain plant species, but not in other plant species (Heath (2002) Can. J. Plant Pathol. 24: 259-264).
  • the resistance against a pathogen in certain plant species is called non-host resistance.
  • the non-host resistance offers strong, broad, and permanent protection from phytopathogens.
  • Genes providing non-host resistance provide the opportunity of a strong, broad and permanent protection against certain diseases in non- host plants. In particular, such a resistance works for different strains of the pathogen.
  • Fungi are distributed worldwide. Approximately 100000 different fungal species are known to date. Thereof rusts are of great importance. They can have a complicated development cycle with up to five different spore stages (spermatium, aecidiospore, uredospore, teleutospore and basidiospore).
  • the first phases of the interaction between phytopathogenic fungi and their potential host plants are decisive for the colonization of the plant by the fungus.
  • the spores become attached to the surface of the plants, germinate, and the fungus penetrates the plant.
  • Fungi may penetrate the plant via existing ports such as stomata, lenticels, hydatodes and wounds, or else they penetrate the plant epidermis directly as the result of the mechanical force and with the aid of cell-wall-digesting enzymes.
  • Specific infection structures are developed for penetration of the plant.
  • To counteract plants have developed physical barriers, such as wax layers, and chemical compounds having antifungal effects to inhibit spore germination, hyphal growth or penetration.
  • RLKs transmembrane receptor like kinases
  • RLPs receptor-like proteins
  • conserved pathogen associated molecules e.g. flagellin or chitin.
  • the molecular mechanism of defense activation by RLKs and RLPs is quite complex and requires several co-receptors (for review see Yu TY, Sun MK, Liang LK. Receptors in the Induction of the Plant Innate Immunity. Mol Plant Microbe Interact. 2021 Jun;34(6):587-601)
  • SA plays a role in abiotic stresses, like heat stress and drought, but also influences other physiological processes such as photosynthesis, seed germination, induction of flowering and senescence (Rivas-San Vicente, M.; Plasencia, J. Salicylic acid beyond defense: Its role in plant growth and development. J. Exp. Bot. 2011 , 62, 3321-3338.)
  • the soybean rust fungus Phakopsora pachyrhizi directly penetrates through the cuticle and the plant epidermis. After crossing the epidermal cell, the fungus reaches the intercellular space of the mesophyll, where the fungus starts to spread through the leaves. To acquire nutrients the fungus penetrates mesophyll cells and develops haustoria inside the mesophyll cell. During the penetration process the plasma membrane of the penetrated mesophyll cell stays intact.
  • the initial step of pathogenesis of Asian soybean rust disease is the initial penetration of the fungus through the plant cuticle into the epidermal cell.
  • the plant cuticle is an extracellular hydrophobic layer that covers the aerial epidermis and that consists of two major components, the polymer cutin and cuticular waxes (for review about 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, it has been shown that lower cutin amounts in tomato “cd” mutants are associated with increased susceptibility to Botrytis cinerea (Isaacson et al., 2009).
  • To facilitate penetration through the cuticle many fungal pathogens secrete enzymes to degrade or weaken the cuticle, such as e.g. cutinases, a class of small, nonspecific esterases that hydrolyze the cut
  • the epicuticular waxes play an important role in pathogen development and defense.
  • the “inhibitor of rust tube germinationT’ (irg 1 ) mutant of M. truncatula showed less epicuticular wax crystals on the abaxial leaf surface and a strong decrease in wax primary alcohol groups. This surface alteration led to an increased resistance against the fungal pathogens Phakopsora pachyrhizi, Puccinia emaculata and the anthracnose fungus C. trifolii (Uppalapati et al., 2012).
  • Biotrophic phytopathogenic fungi depend for their nutrition on the metabolism of living cells of the plants. This type of fungi belongs to the group of biotrophic fungi, like many rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronospora. Necrotrophic phytopathogenic fungi depend for their nutrition on dead cells of the plants, e.g. species from the genus Fusarium, Rhizoctonia or Mycospaerella. Soybean rust has occupied an intermediate position, since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic. After the penetration, the fungus switches to an obligatory biotrophic lifestyle. The subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy are heminecrotrohic.
  • Soybean rust has become increasingly important in recent times.
  • the disease is caused by the biotrophic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They both belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a wide spectrum of leguminosic host plants.
  • P. pachyrhizi is the more aggressive pathogen on soybean (Glycine max), and is therefore, at least currently, of great importance for agriculture.
  • P. pachyrhizi can be found in nearly all tropical and subtropical soybean growing regions of the world.
  • P. pachyrhizi is capable of infecting 31 species from 17 families of the Leguminosae in nature and is capable of growing on further 60 species in controlled conditions (Sinclair et al. (eds.), Proceedings of the rust workshop (1995), National Soybeana Research Laboratory, Publication No. 1 (1996); Rytter J.L. et al., Plant Dis. 87, 818 (1984)).
  • P. meibomiae has been found in the Caribbean Basin and in Puerto Rico, and has not caused substantial damage as yet.
  • P. pachyrhizi can currently be controlled in the field only by means of fungicides. Soybean plants with resistance to the entire spectrum of the isolates are not available. When searching for resistant soybean accessions, six dominant R genes of the NBS-LRR family, which mediate resistance of soybean to P. pachyrhizi, were discovered. The resistance they conferred was lost rapidly, as P. pachyrhizi develops new virulent races.
  • Phakopsora pachyrhizi infecting its non-host Arabidopsis thaliana induces genes involved in JA/ET mediated defense.
  • JA/ET mediated defense pathways are directed against an infection of necrotrophic pathogens. This is a surprising finding, because Phakopsora pachyrhizi is a rust fungus and should theoretically behave more like a biotrophic pathogen (see R. Campe Molecular aspects of Arabidopsis nonhost resistance to Phakopsora pachyrhizi, Dissertation RWTH Aachen 2014).
  • the object of the present invention is inter alia to provide a method of increasing resistance against fungal pathogens.
  • the fungal resistance is envisaged to comprise resistance against a biotrophic, hemibiotrophic or heminecrotrophic fungus, preferably a rust fungus, downy mildew, powdery mildew, leaf spot, late blight, fusarium and/or septoria.
  • a biotrophic, hemibiotrophic or heminecrotrophic fungus preferably a rust fungus, downy mildew, powdery mildew, leaf spot, late blight, fusarium and/or septoria.
  • a biotrophic, hemibiotrophic or heminecrotrophic fungus preferably a rust fungus, downy mildew, powdery mildew, leaf spot, late blight, fusarium and/or septoria.
  • fungal pathogens of the family Phakopsoraceae more preferably against
  • the present invention accordingly provides a method for increasing or conferring fungal resistance in a plant or plant material, wherein the method comprises a step of increasing the expression and/or biological activity of an isochorismate synthase protein in the plant or plant material in comparison to a respective wild-type plant or plant material.
  • One aspect of the invention provides the method for increasing or conferring fungal resistance in a plant or plant material, wherein the method comprises increasing the expression and/or biological activity of an isochorismate synthase protein in the plant or plant material, wherein the said isochorismate synthase is encoded by i) an exogenous nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity with SEQ ID NO. 1 , or a functional fragment thereof or a splice variant thereof; or ii)an exogenous nucleic acid encoding a protein having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity with SEQ ID NO.
  • an exogenous nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to i) or ii); or iv) an exogenous nucleic acid encoding the same isochorismate synthase protein as the nucleic acids of i) to iii) above, but differing from the nucleic acids of i) to iii) above due to the degeneracy of the genetic code, or v) an exogenous ICS gene of, preferably of Escherichia alba, Escherichia albertii, Escherichia coli, Escherichia faecalis, Escherichia fergusonii, Escherichia hominis, Escherichia marmotae, Escherichia ruysiae, Escherichia senegalensis, Escherichia whittamii
  • Another aspect of the invention provides a method for increasing or conferring fungal resistance in a plant or plant material, comprising the steps of
  • the invention provides a method for increasing or conferring fungal resistance in a plant or plant material, wherein the expression cassettes encoded by a nucleic acid having identity with SEQ ID NO. 3 or SEQ ID NO. 4.
  • the invention provides a method for increasing or conferring fungal resistance in a plant or plant material, wherein the increased production and/or biological activity of the isochorismate synthase protein results in an increase production of salicylic acid.
  • the invention provides a method for production of a genetically modified plant or plant material having increased fungal resistance compared to a respective wild-type plant or wild-type plant material, comprising the steps of
  • the isochorismate synthase protein is encoded by i) an exogenous nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ ID NO. 1 , or a functional fragment thereof, ii) an exogenous nucleic acid encoding a protein having at least 70%, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ ID NO.
  • an exogenous nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to i) or ii); or iv) an exogenous nucleic acid encoding the same isochorismate synthase protein as the nucleic acids of i) to iii) above, but differing from the nucleic acids of i) to iii) above due to the degeneracy of the genetic code, or v) an exogenous ICS gene of, preferably of Escherichia alba, Escherichia albertii, Escherichia coli, Escherichia faecalis, Escherichia fergusonii, Escherichia hominis, Escherichia marmotae, Escherichia ruysiae, Escherichia senegalensis, Escherichia whittamii, most preferably of Escherichia alba,
  • the invention provides the method for production of a genetically modified plant or plant material having increased fungal resistance, further comprising the steps of harvesting the seeds of the transgenic plant and planting the seeds and growing the seeds to plants, wherein the grown plants comprise anyone of the heterologous nucleic acid selected from the group i) to iv) according to the aspect above.
  • the present invention provides a plant or plant material having increased pathogen resistance, wherein the plant or plant material comprises increased production and/or biological activity of an isochorismate synthase protein in comparison to a respective wild-type plant or plant material, wherein the said plant or plant material is selected from the group consisting of members of the taxonomic family Fabaceae, Brassicaceae and Poaceae.
  • the present invention further provides plant or plant material having increased pathogen resistance according to the aspect above, wherein the plant or plant material comprising increased production and/or biological activity of the isochorismate synthase protein encoded by i) an exogenous nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ ID NO. 1 , or a functional fragment thereof, or ii) an exogenous nucleic acid encoding a protein having at least 70%, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ ID NO.
  • an exogenous nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to i) or ii); or iv) an exogenous nucleic acid encoding the same isochorismate synthase protein as the nucleic acids of i) to iii) above, but differing from the nucleic acids of i) to iii) above due to the degeneracy of the genetic code, v) an exogenous ICS gene of, preferably of Escherichia alba, Escherichia albertii, Escherichia coli, Escherichia faecalis, Escherichia fergusonii, Escherichia hominis, Escherichia marmotae, Escherichia ruysiae, Escherichia senegalensis, Escherichia whittamii, most preferably of Escherich
  • the plant or plant material having increased pathogen resistance wherein the plant or plant material is a) the plant or plant material is a genetically modified crop plant, genetically modified plant material or the plant comprising increased production results form an artificially induced heritable mutation of the wild-type genome; and/or b) wherein the gene encoding isochorismate synthase protein is integral in the genome of the plant or the plant material; and/or c) wherein the plant or plant material is homozygous for the gene encoding isochorismate synthase protein or heterozygous for the gene encoding isochorismate synthase protein, and/or d) wherein the plant or plant material, when in meiosis, is non-segregating or segregating for the gene encoding isochorismate synthase protein, and/or e) wherein the gene encoding isochorismate synthase protein is operably linked to a heterologous promoter, and/or f) wherein the gene
  • the plant or the plant material having increased pathogen resistance wherein the plant or the plant material is selected from the group consisting of members of the taxonomic family Fabaceae, Brassicaceae and Poaceae preferably of
  • Phaseoleae Phaseoleae, more preferably of genus Cajanus, Canavalia, Glycine, Phaseolus, Psophocarpus, Pueraria or Vigna, even more preferably of species Cajanus cajan, Canavalia brasiliensis, Canavalia ensiformis, Canavalia gladiata, Glycine gracilis, Glycine max, Glycine soja, Phaseolus acutifolius, Phaseolus lunatus, Phaseolus maculatus, Psophocarpus tetragonolobus, Pueraria montana, Vigna angularis, Vigna mungo, Vigna radiata or Vigna unguiculata, even more preferably of species Glycine gracilis, Glycine max or Glycine soja, even more preferably of species Glycine max,
  • tribus Fabeae more preferably of genus Lathyrus, Lens, Pisum or Vicia, even more preferably of species Lathyrus aphaca, Lathyrus cicera, Lathyrus hirsutus, Lathyrus ochrus, Lathyrus odoratus, Lathyrus sphaericus, Lathyrus tingitanus, Lens culinaris, Pisum sativum, Vicia cracca, Vicia faba or Vicia vellosa,
  • Triticeae more preferably of genus Saccharum, Zea, Oryza, Avena, Hordeum, Secale, Triticum, even more preferably of species Zea mays, Oryza sativa, Avena sativa, Avena strigosa, Hordeum marinum, Hordeum vulgare, Secale cereale or Triticum aestivum, wherein most preferably the plant is soy.
  • the plant or the plant material having increased pathogen resistance wherein the increased pathogen resistance is against pathogen caused by a fungus, preferably a rust fungus, more preferably a fungus of phylum Basidiomycota, even more preferably of subphylum Pucciniomycotina, even more preferably of class Pucciniomycetes, even more preferably of order Pucciniales, even more preferably of family Chaconiaceae, Coleosporiaceae, Cronartiaceae, Melampsoraceae, Mikronegeriaceae, Phakopsoraceae, Phragmidiaceae, Pileolariaceae, Pucciniaceae, Pucci niastraceae, Pucciniosiraceae, Raveneliaceae, Sphaerophragmiaceae or Uropyxidaceae, even more preferably of genus Maravalia, Ochropsora, Olivea, Chrysomyxa
  • the present invention provides a recombinant vector construct comprising a nucleic acid encoding an isochorismate synthase protein selected from the group consisting of: i) a nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ ID NO. 1 , or a functional fragment thereof, ii) a nucleic acid encoding a protein having at least 70%, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ ID NO.
  • an exogenous nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to i) or ii); or iv) an exogenous nucleic acid encoding the same isochorismate synthase protein as the nucleic acids of i) to iii) above, but differing from the nucleic acids of i) to iii) above due to the degeneracy of the genetic code.
  • the recombinant expression vector is a constitutive, pathogen-inducible promoter, a mesophyll-specific promoter or an epidermis specific-promoter.
  • a genetically modified plant or genetically modified plant material transformed with at least one of the recombinant vector constructs according to the aspects above.
  • the present invention provides use of an isochorismate synthase protein or a nucleic acid encoding the isochorismate synthase protein to increase fungal resistance in a plant, preferably wherein the increase of fungal resistance comprises the delay or reduced infection of a plant by a fungus.
  • the present invention also provides a method of controlling a fungus in a field, preferably by reducing or delaying infection of plant in a field and/or reducing or delaying emission of fungal spores from the field, comprising the step of
  • the present invention further provides a method for evaluating the pathogen resistance of the plant according to the aspects above comprising the step of at least one step for detecting presence of a heterologous isochorismate synthase gene or protein expression level in a plant material.
  • the invention also provides molecular markers for selection of the plant or plant material according to aspect above, comprising nucleic acid for detecting isochorismate synthase in a plant material, wherein the nucleic acid is selected from the group consisting of
  • the invention further provides use of the molecular maker for selecting a plant or a plant material having isochorismate synthase induced pathogen resistance.
  • harvestable parts of a plant are provided, wherein the harvestable part of the plant comprises an exogenous nucleic acid encoding the isochorismate synthase protein, wherein the harvestable part is a seed of the plant, preferably genetically modified seed of the genetically modified plant.
  • dead plant material or/and no-producing plant material derived from a plant or from the harvestable part of the plant wherein the product comprises the exogenous nucleic acid encoding the isochorismate synthase protein.
  • product derived from a plant or from the harvestable part of the plant wherein the product comprises the exogenous nucleic acid encoding the isochorismate synthase protein, wherein the product is preferably a soy product, more preferably soybeans, soy oil or soy meal.
  • the present invention also provides use of the plant for modifying genetic variation in a plant population.
  • method for breeding a fungal resistant crop plant which comprises
  • each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.
  • any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
  • Reference throughout this specification to “one embodiment/aspect” or “an embodiment/aspect” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.
  • appearances of the phrases “in one embodiment/aspect” or “in an embodiment/aspect” in various places throughout this specification are not necessarily all referring to the same embodiment, but may do so.
  • the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments.
  • Figure 2 shows the schematic illustration of the plant transformation vector harboring the ICS protein.
  • Figure 3 shows the result of the disease scoring of transgenic soy plants expressing ICS under (left) a constitutive parsley ubiquitin promoter and (right) under control of the Phakopsora induced promoter in T1 generation in greenhouse.
  • Field trials were performed at 2 locations of Campinas (SP) and Uberlandia (MG). In Campinas the field trial were run without any fungicide treatment (no treatment) and with one fungicide treatment at the onset of ASR disease ( ⁇ 35 - 40 days after planting) to increase variability of disease pressure to prove robustness and stability of the resistance increasing effect.
  • SP Campinas
  • MG Uberlandia
  • ICS The expression of ICS was checked by RT-PCR. The evaluation of the diseased leaf area on all leaves was performed 14 days after inoculation by imaging. The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves was considered as diseased leaf area. The average diseased leaf area of all transgenic events (ark grey bar) is shown in comparison to control (diagonally striped bar, labeled wild type control)
  • Field trials were performed at Campinas (SP) location. The field trial was run without any fungicide treatment (no treatment) and with one fungicide treatment at the onset of ASR disease ( ⁇ 35 - 40 days after planting) to increase variability of disease pressure to prove robustness and stability of the resistance increasing effect.
  • the asterisk indicates a significant difference between transgenic and wild type control in the various disease ratings over the season (p ⁇ 0,05; paired one sided T-test)
  • the current invention is focused on the application of isochorismate synthase (ICS) for the protection of plants against fungal infection and progress of such infective diseases.
  • ICS isochorismate synthase
  • composition when used in reference to a measurable value, for example an amount of mass, dose, time, temperature, sequence identity and the like, refers to a variation of ⁇ 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or even 20% of the specified value as well as the specified value.
  • a given composition is described as comprising "about 50% X,” it is to be understood that, in some embodiments, the composition comprises 50% X whilst in other embodiments it may comprise anywhere from 40% to 60% X (i.e., 50% ⁇ 10%).
  • the term "gene” refers to a biochemical information which, when materialized in a nucleic acid, can be transcribed into a gene product, i.e. a further nucleic acid, preferably an RNA, and preferably also can be translated into a peptide or polypeptide.
  • the term is thus also used to indicate the section of a nucleic acid resembling said information and to the sequence of such nucleic acid (herein also termed "gene sequence").
  • alleles or nucleotide sequence variants of the invention have at least, in increasing order of preference, 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide "sequence identity" to the nucleotide sequence of the wild type gene.
  • an "allele” refers to the biochemical information for expressing a peptide or polypeptide
  • the respective nucleic acid sequence of the allele has at least, in increasing order of preference, 30%, 40%, 50%, 60%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid "sequence identity" to the respective wild type peptide or polypeptide.
  • the present invention accordingly provides a method for increasing or conferring fungal resistance in a plant or plant material, wherein the method comprises a step of increasing the expression and/or biological activity of an isochorismate synthase protein in the plant or plant material in comparison to a respective wild-type plant or plant material.
  • resistance refers to an absence or reduction of one or more disease symptoms in a plant caused by a plant pathogen. Resistance generally describes the ability of a plant to prevent, or at least curtail the infestation and colonization by a harmful pathogen. Different mechanisms can be discerned in the naturally occurring resistance, with which the plants fend off colonization by phytopathogenic organisms (Schopfer and Brennicke (1999) convinced physiologicallogie, Springer Verlag, Berlin-Heidelberg, Germany).
  • the invention correspondingly provides a method for conferring or increasing fungal resistance in a plant, a plant part, or a plant cell wherein the method comprises the step of increasing the production and/or biological activity of ICS protein in the plant, plant part, or plant cell in comparison to a respective wild type plant, wild type plant part, or wild type plant cell.
  • plant is used herein in its broadest sense as it pertains to organic material and is intended to encompass eukaryotic organisms that are members of the taxonomic kingdom plantae, examples of which include but are not limited to monocotyledon and dicotyledon plants, vascular plants, vegetables, grains, flowers, trees, herbs, bushes, grasses, vines, ferns, mosses, fungi and algae, etc, as well as clones, offsets, and parts of plants used for asexual propagation (e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.).
  • asexual propagation e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.
  • plant refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same.
  • a plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant.
  • the term "plant” includes whole plants, including descendants or progeny thereof. As used herein unless clearly indicated otherwise, the term “plant” intends to mean a plant at any developmental stage.
  • the plant according to the present invention are (predominantly) self-pollinating, i.e. a significant portion of the seeds produced result from self- pollination and not cross-pollination.
  • Cross-pollination also called allogamy, occurs when pollen is delivered from the stamen of one flower to the stigma of a flower on another plant of the same species.
  • Self-pollination, as opposed to cross-pollination refers to fertilization of ovules/female gametes in a plant by pollen from the same plant.
  • Self- pollination occurs when pollen from one flower pollinates the same flower or other flowers of the same individual.
  • Self- pollination may include autogamy, where pollen is transferred to the female part of the same flower; or geitonogamy, when pollen is transferred to another flower on the same plant.
  • self-pollination involves cleistogamy.
  • at least 25% of the seeds produced result from self-pollination, more preferably at least 50%, even more preferably as at least 75%, most preferably at least 90%.
  • plant part includes any part or derivative of the plant, including particular plant tissues or structures, plant cells, plant protoplast, plant cell or tissue culture from which plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants, such as seeds, kernels, cobs, flowers, cotyledons, leaves, stems, buds, roots, root tips, stover, and the like.
  • Plant parts may include processed plant parts or derivatives, including flower, oils, extracts etc.
  • Parts of a plant are e.g. shoot vegetative organs/structures, e.g., leaves, stems and tubers; roots, flowers and floral organs/structures, e.g.
  • plant tissue e.g. vascular tissue, ground tissue, and the like
  • cells e.g. guard cells, egg cells, pollen, trichomes and the like
  • progeny of the same Parts of plants may be attached to or separate from a whole intact plant. Such parts of a plant include, but are not limited to, organs, tissues, and cells of a plant, and preferably seeds.
  • a "plant cell” is a structural and physiological unit of a plant, comprising a protoplast and a cell wall.
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
  • Plant cell culture means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
  • Plant material refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant. This also includes callus or callus tissue as well as extracts (such as extracts from taproots) or samples.
  • a "plant organ” is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
  • Plant tissue as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
  • wildtype or “corresponding wildtype plant” means the typical form of an organism or its genetic material, as it normally occurs, as distinguished from e.g. mutagenized and/or recombinant forms.
  • control cell or “similar, wildtype, plant, plant tissue, plant cell or host cell” is intended a plant, plant tissue, plant cell, or host cell, respectively, that lacks the particular polynucleotide of the invention that are disclosed herein.
  • wildtype is not, therefore, intended to imply that a plant, plant tissue, plant cell, or other host cell lacks recombinant DNA in its genome, and/or does not possess fungal resistance characteristics that are different from those disclosed herein.
  • One aspect of the invention provides the method for increasing or conferring fungal resistance in a plant or plant material, wherein the method comprises increasing the expression and/or biological activity of an isochorismate synthase protein in the plant or plant material, wherein the said isochorismate synthase is encoded by i) an exogenous nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity with SEQ ID NO. 1 , or a functional fragment thereof or a splice variant thereof; or ii)an exogenous nucleic acid encoding a protein having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity with SEQ ID NO.
  • nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to i) or ii); or iv) an exogenous nucleic acid encoding the same isochorismate synthase protein as the nucleic acids of i) to iii) above, but differing from the nucleic acids of i) to iii) above due to the degeneracy of the genetic code, wherein optionally the nucleic acid according to any of i) - iv) is operably linked with a promoter and a transcription termination sequence.
  • the ICS protein of the present invention is any of the aforementioned homologs or differs, when aligned to SEQ ID NO. 2, from the respective homolog’s sequence only by the amino acids, gaps or deletions.
  • Exogenous nucleic acid refers to the ‘DNA that originates from outside of an organism’ and is typically introduced artificially into cells. This can be done for a variety of reasons such as genetic engineering, gene therapy, or to study gene function. Exogenous nucleic acid can be introduced into cells using various techniques such as transformation, transfection, electroporation, microinjection, and viral vectors.
  • the term “functional” means that the respective plant, plant part or plant cell is more likely to withstand an attempted infection by the pathogenic fungus, preferably Phakopsora pachyrhizi.
  • biological activities refers to a result of certain effects from exposure to a molecule; these affect a metabolic or physiological response.
  • Biological activity is defined as being applied to the simplest and most complex reaction and molecular systems. There are many sorts of biological activities, and these activities can be studied in vivo and in vitro. Biological activity always depends on the dose given to the living organism, so it is logical to show either beneficial or adverse effects that range from low to high. Absorption, distribution, metabolism, and excretion are the main action used to measure biological activity.
  • Protein or nucleic acid variants may be defined by their sequence identity when compared to a parent protein or nucleic acid. Sequence identity usually is provided as “% sequence identity” or “% identity”. To determine the percent-identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e. , a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p.
  • the preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined.
  • Seq B GATCTGA length: 7 bases
  • sequence B is sequence B.
  • the symbol in the alignment indicates gaps.
  • the number of gaps introduced by alignment within the sequence B is 1.
  • the number of gaps introduced by alignment at borders of sequence B is 2, and at borders of sequence A is 1 .
  • the alignment length showing the shorter sequence over its complete length is 8 (one gap is present which is factored in the alignment length of the shorter sequence).
  • the alignment length showing sequence A over its complete length would be 9 (meaning sequence A is the sequence of the invention), the alignment length showing sequence B over its complete length would be 8 (meaning sequence B is the sequence of the invention).
  • %-identity (identical residues I length of the alignment region which is showing the respective sequence of this invention over its complete length) *100.
  • sequence identity in relation to comparison of two amino acid sequences according to the invention is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give “%-identity”.
  • hybridisation is a process wherein substantially complementary nucleotide sequences anneal to each other.
  • the hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution.
  • the hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin.
  • the hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips).
  • the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
  • stringency refers to the conditions under which a hybridisation takes place.
  • the stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20°C below Tm, and high stringency conditions are when the temperature is 10°C below Tm. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore, medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid molecules.
  • the “Tm” is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe.
  • the Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures.
  • the maximum rate of hybridisation is obtained from about 16°C up to 32°C below Tm.
  • the presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher concentrations, this effect may be ignored).
  • Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7°C for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45°C, though the rate of hybridisation will be lowered.
  • Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes.
  • the Tm decreases about 1°C per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:
  • Tm 81.5°C + 16.6xlog([Na+] ⁇ a ⁇ ) + 0.41x%[G/C ⁇ b ⁇ ] - 500x[L ⁇ c ⁇ ]-1 - 0.61x% formamide
  • ⁇ In ⁇ effective length of primer 2* (no. of G/C)+(no. of A/T)
  • Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase.
  • a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68°C to 42°C) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%).
  • annealing temperature for example from 68°C to 42°C
  • formamide concentration for example from 50% to 0%
  • hybridisation typically also depends on the function of post-hybridisation washes.
  • samples are washed with dilute salt solutions.
  • Critical factors of such washes 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.
  • Wash conditions are typically performed at or below hybridisation stringency. A positive hybridisation gives a signal that is at least twice of that of the background.
  • suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions.
  • typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65°C in 1x SSC or at 42°C in 1x SSC and 50% formamide, followed by washing at 65°C in 0.3x SSC.
  • Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50°C in 4x SSC or at 40°C in 6x SSC and 50% formamide, followed by washing at 50°C in 2x SSC.
  • the length of the hybrid is the anticipated length for the hybridising nucleic acid. When nucleic acids of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein.
  • 1 xSSC is 0.15M NaCI and 15mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5x Denhardt's reagent, 0.5-1 .0% SDS, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
  • 5x Denhardt's reagent 0.5-1 .0% SDS
  • 100 pg/ml denatured, fragmented salmon sperm DNA 0.5% sodium pyrophosphate.
  • Another example of high stringency conditions is hybridisation at 65°C in 0.1x SSC comprising 0.1 SDS and optionally 5x Denhardt's reagent, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, followed by the washing at 65°C in 0.3x SSC.
  • isolated DNA molecule refers to a DNA molecule at least partially separated from other molecules normally associated with it in its native or natural state.
  • isolated preferably refers to a DNA molecule that is at least partially separated from some of the nucleic acids which normally flank the DNA molecule in its native or natural state.
  • DNA molecules fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques are considered isolated herein.
  • Such molecules are considered isolated when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules, in that they are not in their native state.
  • PCR polymerase chain reaction
  • Polynucleotide molecules, or fragment thereof can also be obtained by other techniques, such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer.
  • a polynucleotide can be single-stranded (ss) or double- stranded (ds).
  • Double-stranded refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure, generally under physiologically relevant conditions.
  • the polynucleotide is at least one selected from the group consisting of sense single- stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), a double-stranded DNA/RNA hybrid, antisense ssDNA, or anti-sense ssRNA; a mixture of polynucleotides of any of these types can be used.
  • Another aspect of the invention provides a method for increasing or conferring fungal resistance in a plant or plant material, comprising the steps of 1) stably transforming a plant cell with at least one expression cassette comprising an exogenous nucleic acid encoding an isochorismate synthase protein, 2) regenerating a plant from the plant cell; and 3) expressing the said isochorismate synthase protein.
  • the invention provides a method for increasing or conferring fungal resistance in a plant or plant material, wherein the expression cassettes encoded by a nucleic acid having identity with SEQ ID NO. 3 or SEQ ID NO. 4.
  • the present invention provides a recombinant vector construct comprising a nucleic acid encoding an isochorismate synthase protein selected from the group consisting of: i) a nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ ID NO. 1 , or a functional fragment thereof, ii) a nucleic acid encoding a protein having at least 70%, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ ID NO.
  • an exogenous nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to i) or ii); or iv) an exogenous nucleic acid encoding the same isochorismate synthase protein as the nucleic acids of i) to iii) above, but differing from the nucleic acids of i) to iii) above due to the degeneracy of the genetic code.
  • the recombinant expression vector is a constitutive, pathogen-inducible promoter, a mesophyll-specific promoter or an epidermis specific-promoter.
  • the promoter preferably is a constitutive, pathogen-inducible promoter, a mesophyll-specific promoter or an epidermis specific-promoter.
  • any of these promoters allows to produce, in a plant cell, a respectively constitutively increased level of ICS protein, or an increased level in response to a pathogen infection, preferably a fungal pathogen, or to increase the level of ICS specifically in mesophyll or plant epidermis cells.
  • recombinant when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation.
  • a gene sequence open reading frame is recombinant if (a) that nucleotide sequence is present in a context other than its natural one, for example by virtue of being (i) cloned into any type of artificial nucleic acid vector or (ii) moved or copied to another location of the original genome, or if (b) the nucleotide sequence is mutagenized such that it differs from the wild type sequence.
  • the term recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid is a recombinant plant.
  • the terms “cassette”, “plasmid”, and “vector” refer to an extra-chromosomal element often carrying genes that are not part of the native genome of the cell, and usually in the form of double-stranded DNA. Such elements may be autonomously replicating sequences, genome integrating sequences, phage, or nucleotide sequences, in linear or circular form, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a polynucleotide of interest into a cell.
  • expression refers to the production of a functional end-product (e.g., DNA, gene, mRNA, guide R A, or a protein) in either precursor or mature form.
  • the invention provides the method for production of a genetically modified plant or plant material having increased fungal resistance, further comprising the steps of harvesting the seeds of the transgenic plant and planting the seeds and growing the seeds to plants, wherein the grown plants comprise anyone of the heterologous nucleic acid selected from the group i) to iv) according to the aspect above.
  • seed comprises seeds of all types, such as, for example, true seeds, caryopses, achenes, fruits, tubers, seedlings and similar forms.
  • seed refers to true seed(s) unless otherwise specified.
  • the seed can be seed of transgenic plants or plants obtained by site specific mutagenesis, by mutagenesis with a site preference or by traditional breeding methods. Examples of traditional breeding methods are cross-breeding, selfing, back-crossing, embryo rescue, in-crossing, out-crossing, inbreeding, selection, asexual propagation, and other traditional techniques as are known in the art.
  • the invention provides A method for production of a genetically modified plant or plant material having increased fungal resistance compared to a respective wild-type plant or wild-type plant material, comprising the steps of 1) introducing an exogenous nucleic acid encoding an isochorismate synthase protein into a plant or plant material; 2) generating a genetically modified plant or genetically modified plant material; and 3) expressing the isochorismate synthase protein in the genetically modified plant or genetically modified plant material; wherein the said isochorismate synthase protein is encoded by i) an exogenous nucleic acid having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ I D NO.
  • nucleic acid encoding a protein having at least 70%, at least 80% identity, at least 90% identity, at least 95% identity with the SEQ ID NO. 2, or iii) an exogenous nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to i) or ii); or iv) an exogenous nucleic acid encoding the same isochorismate synthase protein as the nucleic acids of i) to iii) above, but differing from the nucleic acids of i) to iii) above due to the degeneracy of the genetic code, wherein the nucleic acid according to any of i) - iv) is operably linked with a promoter and a transcription termination sequence.
  • a "genetically modified organism/plant” is an organism whose genetic characteristics contain alteration(s) that were produced by human effort causing transfection that results in transformation of a target organism with genetic material from another or “source” organism, or with synthetic or modified-native genetic material, or an organism that is a descendant thereof that retains the inserted genetic material.
  • the source organism can be of a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or from the same type of organism (e.g., a GMO plant can contain genetic material from another plant).
  • the term "endogenous”, “native”, or “original” refers to a naturally- occurring nucleic acid or polypeptide/protein.
  • the native nucleic acid or protein may have been physically derived from a particular organism in which it is naturally occurring or may be a synthetically constructed nucleic acid or protein that is identical to the naturally-occurring nucleic acid or protein.
  • transgenic refers to an organism, preferably a plant or part thereof, or a nucleic acid that comprises a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • Transgenic is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell.
  • a "recombinant” organism preferably is a “transgenic” organism.
  • transgenic as used herein is not intended to encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as, e.g., self-fertilization, random cross-fertilization, nonrecombinant viral infection, non-recombinant bacterial transformation, non- recombinant transposition, or spontaneous mutation.
  • mutant refers to an organism or nucleic acid thereof having alteration(s) in the biomolecular sequence of its native genetic material as compared to the sequence of the genetic material of a corresponding wildtype organism or nucleic acid, wherein the alteration(s) in genetic material were induced and/or selected by human action.
  • human action that can be used to produce a mutagenized organism or DNA include, but are not limited to treatment with a chemical mutagen such as EMS and subsequent selection with herbicide(s); or by treatment of plant cells with x-rays and subsequent selection with herbicide(s). Any method known in the art can be used to induce mutations.
  • Methods of inducing mutations can induce mutations in random positions in the genetic material or can induce mutations in specific locations in the genetic material (i.e. , can be directed mutagenesis techniques), such as by use of a genoplasty technique.
  • a nucleic acid can also be mutagenized by using mutagenesis means with a preference or even specificity for a particular site, thereby creating an artificially induced heritable allele according to the present invention.
  • Such means for example site specific nucleases, including for example zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucelases (TALENS) (Mal leopard et al., Cell Biosci, 2017, 7:21) and clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) with an engineered crRNA/tracr RNA (for example as a single-guide RNA, or as modified crRNA and tracrRNA molecules which form a dual molecule guide), and methods of using this nucleases to target known genomic locations, are well-known in the art (see reviews by Bortesi and Fischer, 2015, Biotechnology Advances 33: 41-52; and by Chen and Gao, 2014, Plant Cell Rep 33: 575-583, and references within).
  • ZFNs zinc finger nucleases
  • TALENS transcription activator-like effector nucelases
  • CRISPR/Cas clustered regularly inter
  • the present invention provides a plant or plant material having increased pathogen resistance, wherein the plant or plant material comprises increased production and/or biological activity of an isochorismate synthase protein in comparison to a respective wild-type plant or plant material, wherein the said plant or plant material is selected from the group consisting of members of the taxonomic family Fabaceae, Brassicaceae and Poaceae.
  • the invention in particular pertains to plants in general and preferably to crop plants, that is plants selected from the group consisting of members of the taxonomic family Fabaceae, Brassicaceae and Poaceae, most preferably soy. More preferably, the crop plant is selected from the group consisting preferably of - taxonomic tribus Phaseoleae, more preferably of genus Cajanus, Canavalia, Glycine, Phaseolus, Psophocarpus, Pueraria or Vigna, even more preferably of species Cajanus cajan, Canavalia brasiliensis, Canavalia ensiformis, Canavalia gladiata, Glycine gracilis, Glycine max, Glycine soja, Phaseolus acutifolius, Phaseolus lunatus, Phaseolus maculatus, Psophocarpus tetragonolobus, Pueraria montana, Vigna angularis, Vigna mungo, Vigna radiata or Vigna ungui
  • tribus Fabeae more preferably of genus Lathyrus, Lens, Pisum or Vicia, even more preferably of species Lathyrus aphaca, Lathyrus cicera, Lathyrus hirsutus, Lathyrus ochrus, Lathyrus odoratus, Lathyrus sphaericus, Lathyrus tingitanus, Lens culinaris, Pisum sativum, Vicia cracca, Vicia faba or Vicia vellosa,
  • the present invention in particular provides materials, preferably plants, plant parts or plant cells, or methods to increase fungal resistance.
  • increase of fungal resistance is achieved preferably by reducing, compared to a corresponding wild type, the speed of infection or the extent of infection or delaying the day of earliest infection by the fungus.
  • the ICS protein and gene of the present invention is suitable for conferring, intensifying or stabilizing resistance of plants, plant parts or plant cells against fungal pathogen infections, particularly against biotrophic, hemibiotrophic or heminecrotrophic fungi, and preferably against fungi as described herein.
  • the present invention is particularly useful for fighting against a plant pathogenic fungus.
  • the fungus to fight against is preferably a biotrophic, hemibiotrophic or heminecrotrophic fungus, more preferably a rust fungus, downy mildew, powdery mildew, leaf spot, late blight, fusarium and/or Septoha, and is even more preferably selected from the taxonomic phylum Basidiomycota, more preferably the taxonomic class Pucciniomycetes, more preferably the taxonomic class Pucciniales, more preferably the taxonomic class family Pucciniaceae, more preferably the taxonomic class genus Puccinia, more preferably the taxonomic class species Puccinia graminis', or phylum Basidiomycota, more preferably the taxonomic class class Pucciniomycetes, more preferably the taxonomic class order Pucciniales, more preferably the taxonomic
  • the present invention also provides a method of controlling a fungus in a field, preferably by reducing or delaying infection of plant in a field and/or reducing or delaying emission of fungal spores from the field, comprising the step of 1) planting seed from any of the plants according to the above aspect, 2) optionally, one or more active agents including fungicide can be applied to the plants, such as spraying or splashing.
  • the active agents may be any agent, preferably a chemical agent, that produces a desired effect on the plant or plant material.
  • chemical agents include pesticides (such as fungicides, acaricides, miticides, insecticides, insect repellents, rodenticides, molluscicides, nematicides, bactericides, and fumigants), herbicides, chemical hybridizing agents, auxins, antibiotics and other drugs, biological attractants, growth regulators, pheromones and dyes.
  • chemical agents useful as active ingredients include triticonazole, imidacloprid, tefluthrin, and silthiophenamide (N-allyl-4,5-dimethyl-2-trimethylsilylthiophene-3-caboxamide).
  • Further active agents suitable for use in the present invention are, for example, phytochemicals and antimicrobial agents suitable to protect soybean.
  • Preferred examples thereof are bactericides, antiparasitics and fungicides.
  • the fungicides are for instance selected from the group of classes consisting of strobilurins, triazoles, carboxamides, penflufen, isopyrazam, bixafen, sedaxane, and fluxapyroxad, and mixtures thereof.
  • dead plant material or/and no-producing plant material derived from a plant or from the harvestable part of the plant wherein the product comprises the exogenous nucleic acid encoding the isochorismate synthase protein.
  • the present invention also provides use of the plant for modifying genetic variation in a plant population.
  • genetic variation is used as known in the art.
  • genetic variability or genetic variation may refer to the presence or generation of genetic differences (between individuals or within a population) or the formation of individuals differing in genotype, or the presence of genotypically different individuals (in a population).
  • method for breeding a fungal resistant crop plant which comprises 1) crossing the plant or the plant obtainable by the method above with a second plant; 2) obtaining seed from the cross of step 1); 3) planting said seeds and growing the seeds to plants; and 4) selecting from said plants expressing the isochorismate synthase protein.
  • a seed of descendant may be used.
  • descendant refers to any generation plant.
  • a progeny or decendant plant can be from any filial generation, e.g., F1 , F2, F3, F4, F5, F6, F7, etc.
  • a descendant or progeny plant is a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth generation plant.
  • the invention additionally provides a method for producing a plant or plant material having increased pathogen resistance comprising introducing the ICS protein as defined above using genome editing, preferably using CRISPR system.
  • gene editing refers to strategies and techniques for the targeted, specific modification of any genetic information or genome of a living organism (e.g., soybean) at least one position.
  • the terms comprise gene editing, but also the editing of regions other than gene encoding regions of a genome.
  • Genome editing may comprise targeted or non-targeted (random) mutagenesis.
  • Targeted mutagenesis may be accomplished for instance with designer nucleases, such as for instance with meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system.
  • ZFNs zinc finger nucleases
  • TALEN transcription activator-like effector-based nucleases
  • CRISPR/Cas9 clustered regularly interspaced short palindromic repeats
  • designer nucleases are particularly suitable for generating gene knockouts or knockdowns.
  • designer nucleases are developed which specifically induce a mutation in the ICS gene, as described herein elsewhere, such as to generate a mutated ICS protein or a knockout of the ICS gene.
  • designer nucleases in particular RNA-specific CRISPR/Cas systems are developed which specifically target the ICS mRNA, such as to cleave the ICS mRNA and generate a knockdown of the ICS gene/mRNA/protein. Delivery and expression systems of designer nuclease systems are well known in the art.
  • the CRISPR (clustered regularly interspaced short palindromic repeats) technology may be used to modify the genome of a target organism, for example to introduce any given DNA fragment into nearly any site of the genome, to replace parts of the genome with desired sequences or to precisely delete a given region in the genome of a target organism. This allows for unprecedented precision of genome manipulation.
  • the CRISPR system was initially identified as an adaptive defense mechanisms of bacteria belonging to the genus of Streptococcus (W02007/025097). Those bacterial CRISPR systems rely on guide RNA (gRNA) in complex with cleaving proteins to direct degradation of complementary sequences present within invading viral DNA. The application of CRISPR systems for genetic manipulation in various eukaryotic organisms have been shown (W02013/141680; WO2013/176772; WO2014/093595).
  • gRNA guide RNA
  • Cas9 the first identified protein of the CRISPR/Cas system, is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRSIPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Also a synthetic RNA chimera (single guide RNA or sgRNA) created by fusing crRNA with tracrRNA was shown to be equally functional (WO2013/176772).
  • CRSIPR RNA CRSIPR RNA
  • tracrRNA trans-activating crRNA
  • CRISPR systems from other sources comprising DNA nucleases distinct from Cas9 such as Cpf 1 , C2c1p or C2c3p have been described having the same functionality (WO2016/0205711 , WO2016/205749).
  • Other authors describe systems in which the nuclease is guided by a DNA molecule instead of an RNA molecule.
  • Such system is for example the AGO system as disclosed in LIS2016/0046963.
  • the template for repair allows for editing the genome with nearly any desired sequence at nearly any site, transforming CRISPR into a powerful gene editing tool (WO2014/150624, WO2014/204728).
  • the template for repair is addressed as donor nucleic acid comprising at the 3’ and 5’ end sequences complementary to the target region allowing for homologous recombination in the respective template after introduction of double strand breaks in the target nucleic acid by the respective nuclease.
  • the main limitation in choosing the target region in a given genome is the necessity of the presence of a PAM sequence motif close to the region where the CRISPR related nuclease introduces double strand breaks.
  • various CRISPR systems recognize different PAM sequence motifs. This allows choosing the most suitable CRISPR system for a respective target region.
  • the AGO system does not require a PAM sequence motif at all.
  • the technology may for example be applied for alteration of gene expression in any organism, for example by exchanging the promoter upstream of a target gene with a promoter of different strength or specificity.
  • Other methods disclosed in the prior art describe the fusion of activating or repressing transcription factors to a nuclease minus CRISPR nuclease protein.
  • Such fusion proteins may be expressed in a target organism together with one or more guide nucleic acids guiding the transcription factor moiety of the fusion protein to any desired promoter in the target organism (WO2014/099744; WO2014/099750). Knockouts of genes may easily be achieved by introducing point mutations or deletions into the respective target gene, for example by inducing non-homologous-end-joining (NHEJ) which usually leads to gene disruption (WO2013/176772).
  • NHEJ non-homologous-end-joining
  • the invention also provides an ensemble of at least 50 crop plants according to the present invention, more preferably at least 100 plants, even more preferably at least 1000 plants, even more preferably at least 100000 plants.
  • preferably at least 100000 plants are grown per hectar, more preferably 200000 to 800000 plants per hectar, even more preferably at least 250000 to 650000 plants per hectar.
  • Such plant numbers preferably are observed within one hectar; thus, the invention particularly facilitates ecologically considerate intensive farming with reduced use of fungicides per growing season.
  • the plants according to the invention are preferably growing in a field or greenhouse.
  • the crop plants are soy plants.
  • the invention it is not required that all crop plants of one species growing in the same field or greenhouse are plants of the present invention. Instead, it is sufficient in monoculture plantation if at least about 25% of the plants of one species belong to the present invention, more preferably at least 50%, even more preferably 25%-75% and most preferably 45%-70%, especially when mixed or combined with plants harboring other resistance genes or mechanisms.
  • the combination with plants with other resistance gene can be done by interplanting (mixing), row-wise or blockwise. For example, on a soybean field it is possible to reduce the number of fungicide treatments if approximately every second plant is a plant according to the present invention.
  • At least 25%, more preferably 50%-100% and even more preferably 75%-100% of those plants on the same field that are not plants according to the present invention comprise at least one other biological means for enhancing fungal resistance, most preferably the other means is selected from the list of pathogen resistance polypeptides as described above.
  • oligonucleotides can be affected, for example, in the known fashion using the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896- 897).
  • the cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, phage multiplication and sequence analysis of recombinant DNA, are carried out as described by Sambrook et al. Cold Spring Harbor Laboratory Press (1989), ISBN 0-87969-309-6.
  • the cDNA sequence of ICS gene mentioned in this application was generated by DNA synthesis (Geneart, Regensburg, Germany).
  • the constitutively expressed ICS construct was generated as follows:
  • the ICS gene (as shown in SEQ ID NO. 1) was synthesized in a way that a Asci restriction site is located in front of the start-ATG and a Sbfl restriction site downstream of the stop-codon.
  • the synthesized DNA was digested using the restriction enzymes Sbfl and Asci (NEB Biolabs) and ligated in a Sbfl/Ascl digested Gateway pENTRY-B vector (Invitrogen, Life Technologies, Carlsbad, California, USA) in a way that the full-length fragment is located in sense direction between the parsley ubiquitin promoter and the Agrobacterium tumefaciens derived nopaline synthase terminator (t-nons).
  • the PcUbi promoter regulates constitutive expression of the ubi4-2 gene (accession number X64345) of Petroselinum crispum (Kawalleck et al. 1993 Plant Molecular Biology 21 (4): 673 - 684).
  • the disease induced ICS construct was generated as follows:
  • the ICS gene (as shown in SEQ ID NO. 1) was synthesized in a way that a Asci restriction site is located in front of the start-ATG and a Sbfl restriction site downstream of the stop-codon.
  • the synthesized DNA was digested using the restriction enzymes Sbfl and Asci (NEB Biolabs) and ligated in a Sbfl/Ascl digested Gateway pENTRY-B vector (Invitrogen, Life Technologies, Carlsbad, California, USA) in a way that the full-length fragment is located in sense direction between the Arabidopsis thaliana pdf 1.2 promoter (Genbank ID LR782546.1 18254339- 18252846) and the Agrobacterium tumefaciens derived nopaline synthase terminator (t-nons).
  • the pdf1.2 promoter is systemically activated by fungal pathogens and responds to methyl jasmonate but not to salicylic acid (Manners, J.M., Penninckx, I. A., Vermaere, K. et al. Plant Mol Biol 38, 1071-1080 (1998)
  • a triple LR reaction was performed according to manufacturer’s protocol by using an empty pENTRY-A vector, the Atpdf1.2 promoter::ICS :nos1 terminator in the above described pENTRY-B vector and an empty pENTRY-C.
  • a binary pDEST vector which is composed of: (1) a Kanamycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection under control of its endogenous AtAHAS promoter and t-AtAHAS terminator.
  • the recombination reaction was transformed into E. coli (DH5alpha), mini-prepped and screened by specific restriction digestions. A positive clone from each vector construct was sequenced and submitted soy transformation.
  • the expression vector constructs (see example 2) is transformed into soybean.
  • any seed of any soybean variety can be employed in the method of the invention.
  • a variety of soybean cultivar (including Jack, Williams 82, Jake, Stoddard, CD215 and Resnik) is appropriate for soybean transformation.
  • Soybean seeds are sterilized in a chamber with a chlorine gas produced by adding 3.5 ml 12N HCI drop wise into 100 ml bleach (5.25% sodium hypochlorite) in a desiccator with a tightly fitting lid. After 24 to 48 hours in the chamber, seeds are removed and approximately 18 to 20 seeds are plated on solid GM medium with or without 5 pM 6-benzyl-aminopurine (BAP) in 100 mm Petri dishes.
  • BAP 6-benzyl-aminopurine
  • Seedlings without BAP are more elongated and roots develop especially secondary and lateral root formation.
  • BAP strengthens the seedling by forming a shorter and stockier seedling.
  • Seven-day-old seedlings grown in the light (>100 pEinstein/m2s) at 25 degree C are used for explant material for the three-explant types.
  • the seed coat was split, and the epicotyl with the unifoliate leaves are grown to, at minimum, the length of the cotyledons.
  • the epicotyl should be at least 0.5 cm to avoid the cotyledonary-node tissue (since soybean cultivars and seed lots may vary in the developmental time a description of the germination stage is more accurate than a specific germination time).
  • Method A for inoculation of entire seedlings, see Method A (example 3.3. and 3.3.2) or leaf explants see Method B (example 3.3.3).
  • Method C see example 3.3.4
  • the hypocotyl and one and a half or part of both cotyledons are removed from each seedling.
  • the seedlings are then placed on propagation media for 2 to 4 weeks.
  • the seedlings produce several branched shoots to obtain explants from.
  • the majority of the explants originated from the plantlet growing from the apical bud. These explants are preferably used as target tissue.
  • Agrobacterium cultures are prepared by streaking Agrobacterium (e.g., A. tumefaciens or A. rhizogenes) carrying the desired binary vector (e.g. H. Klee. R. Horsch and S. Rogers 1987 Agrobacterium-Mediated Plant Transformation and its further Applications to Plant Biology; Annual Review of Plant Physiology Vol. 38: 467-486) onto solid YEP growth medium YEP media: 10 g yeast extract. 10 g Bacto Peptone. 5 g NaCI. Adjust pH to 7.0, and bring final volume to 1 liter with H2O, for YEP agar plates add 20g Agar, autoclave) and incubating at 25.
  • Agrobacterium e.g., A. tumefaciens or A. rhizogenes
  • 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
  • Working glycerol stocks (15%) for transformation are prepared and one- mi of Agrobacterium stock aliquoted into 1 .5 ml Eppendorf tubes then stored at -80 °C.
  • 200 ml of YEP are inoculated with 5 pl to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask.
  • the flask is shaken overnight at 25 °C. until the OD600 is between 0.8 and 1.0.
  • the Agrobacteria ARE pelleted by centrifugation for 10 min at 5,500 x g at 20 °C.
  • the pellet is suspended in liquid CCM to the desired density (OD600 0.5-0.8) and placed at room temperature at least 30 min before use.
  • the explants After wounding and preparation, the explants are set aside in a Petri dish and subsequently co-cultivated with the liquid CCM/Agrobacterium mixture for 30 minutes. The explants are then removed from the liquid medium and plated on top of a sterile filter paper on 15x100 mm Petri plates with solid co-cultivation medium. The wounded target tissues are placed such that they are in direct contact with the medium.
  • Agrobacterium AGL1 harboring a plasmid with the gene of interest (GOI) and the AHAS, bar or dsdA selectable marker gene is cultured in LB medium with appropriate antibiotics overnight, harvested and suspended in a inoculation medium with acetosyringone. Freshly prepared epicotyl segments are soaked in the Agrobacterium suspension for 30 to 60 min and then the explants were blotted dry on sterile filter papers. The inoculated explants are then cultured on a co-culture medium with L-cysteine and TTD and other chemicals such as acetosyringone for increasing T-DNA delivery for 2 to 4 d.
  • the infected epicotyl explants are then placed on a shoot induction medium with selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene), or D-serine (for dsdA gene).
  • the regenerated shoots are subcultured on elongation medium with the selective agent.
  • the segments are then cultured on a medium with cytokinins such as BAP, TDZ and/or Kinetin for shoot induction. After 4 to 8 weeks, the cultured tissues are transferred to a medium with lower concentration of cytokinin for shoot elongation. Elongated shoots are transferred to a medium with auxin for rooting and plant development. Multiple shoots are regenerated. Many stable transformed sectors showing strong cDNA expression are recovered. Soybean plants are regenerated from epicotyl explants. Efficient T- DNA delivery and stable transformed sectors are demonstrated.
  • the cotyledon is removed from the hypocotyl.
  • the cotyledons are separated from one another and the epicotyl is removed.
  • the primary leaves, which consist of the lamina, the petiole, and the stipules, are removed from the epicotyl by carefully cutting at the base of the stipules such that the axillary meristems are included on the explant.
  • any pre-formed shoots are removed and the area between the stipules was cut with a sharp scalpel 3 to 5 times.
  • the explants are either completely immersed or the wounded petiole end dipped into the Agrobacterium suspension immediately after explant preparation.
  • the explants are blotted onto sterile filter paper to remove excess Agrobacterium culture and place explants with the wounded side in contact with a round 7 cm Whatman paper overlaying the solid CCM medium (see above).
  • This filter paper prevents A. tumefaciens overgrowth on the soybean-explants. Wrap five plates with Parafilm. TM. "M” (American National Can, Chicago, III., USA) and incubate for three to five days in the dark or light at 25 °C.
  • Method C Propagated Axillary Meristem
  • Axillary meristem explants can be pre-pared from the first to the fourth node. An average of three to four explants could be obtained from each seedling.
  • the explants are prepared from plantlets by cutting 0.5 to 1 .0 cm below the axillary node on the internode and removing the petiole and leaf from the explant. The tip where the axillary meristems lie is cut with a scalpel to induce de novo shoot growth and allow access of target cells to the Agrobacterium.
  • a 0.5 cm explant included the stem and a bud. Once cut, the explants are immediately placed in the Agrobacterium suspension for 20 to 30 minutes. After inoculation, the explants are blotted onto sterile filter paper to remove excess Agrobacterium culture then placed almost completely immersed in solid CCM or on top of a round 7 cm filter paper overlaying the solid CCM, depending on the Agrobacterium strain. This filter paper prevents Agrobacterium overgrowth on the soybean explants. Plates are wrapped with ParafilmTM. "M” (American National Can, Chicago, III., USA) and incubated for two to three days in the dark at 25 °C.
  • explants are transferred onto SIM without selection for one week.
  • the explant should be placed into the medium such that it is perpendicular to the surface of the medium with the petiole imbedded into the medium and the lamina out of the medium.
  • the explant is placed into the medium such that it is parallel to the surface of the medium (basipetal) with the explant partially embedded into the medium.
  • Wrap plates with Scotch 394 venting tape (3M, St. Paul, Minn., USA) are placed in a growth chamber for two weeks with a temperature averaging 25. degree. C.
  • all shoots formed before transformation are removed up to 2 weeks after co-cultivation to stimulate new growth from the meristems. This helped to reduce chimerism in the primary transformant and increase amplification of transgenic meristematic cells.
  • the explant may or may not be cut into smaller pieces (i.e. detaching the node from the explant by cutting the epicotyl).
  • the explants are transferred to SEM medium (shoot elongation medium, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soybean using primary-node explants from seedlings. In Vitro Cell. Dev. Biol. — Plant (2007) 43:536- 549) that stimulates shoot elongation of the shoot primordia.
  • This medium may or may not contain a selection compound.
  • the explants are transferred to fresh SEM medium (preferably containing selection) after carefully removing dead tissue. The explants should hold together and not fragment into pieces and retain somewhat healthy.
  • the explants are continued to be transferred until the explant dies or shoots elongate. Elongated shoots >3 cm are removed and placed into RM medium for about 1 week (Methods A and B), or about 2 to 4 weeks depending on the cultivar (Method C) at which time roots began to form. In the case of explants with roots, they are transferred directly into soil. Rooted shoots are transferred to soil and hardened in a growth chamber for 2 to 3 weeks before transferring to the greenhouse. Regenerated plants obtained using this method are fertile and produced on average 500 seeds per plant.
  • a general rating of plant health is performed before (and partially after) the infection experiment. Only those plants are selected for inoculation that show, in general, a healthy phenotype. Healthy phenotype means normal growth habit, green, fully expanded green leaves, having no or only minor lesions, no obvious yellowing, leaf drop or other stress- associated phenotypes.
  • the plants are inoculated with spores of P. pachyrhizi.
  • soybean leaves which are infected with rust 15-20 days ago, are taken 2-3 days before the inoculation and transferred to agar plates (1 % agar in H2O).
  • the leaves are placed with their upper side onto the agar, which allowed the fungus to grow through the tissue and to produce very young spores.
  • the spores are knocked off the leaves and are added to a Tween-H2O solution.
  • the counting of spores is performed under a light microscope by means of a Thoma counting chamber.
  • the spore suspension is added into a compressed-air operated spray flask and applied uniformly onto the plants or the leaves until the leaf surface is well moisturized.
  • a spore density of 1-5x105 spores/ml is used.
  • a density of >5 x 105 spores I ml is used.
  • the inoculated plants are placed for 24 hours in a greenhouse chamber with an average of 22°C and >90% of air humidity. The following cultivation is performed in a chamber with an average of 25°C and 70% of air humidity.
  • the inoculated leaves of plants are stained with aniline blue 48 hours after infection.
  • the aniline blue staining serves for the detection of fluorescent substances.
  • substances such as phenols, callose or lignin accumulate or are produced and are incorporated at the cell wall either locally in papillae or in the whole cell (hypersensitive reaction, HR).
  • Complexes are formed in association with aniline blue, which lead e.g. in the case of callose to yellow fluorescence.
  • the leaf material is transferred to falcon tubes or dishes containing destaining solution II (ethanol I acetic acid 6/1) and is incubated in a water bath at 90°C for 10-15 minutes.
  • the destaining solution II is removed immediately thereafter, and the leaves are washed 2x with water.
  • the different interaction types are evaluated (counted) by microscopy.
  • An Olympus UV microscope BX61 (incident light) and a UV Longpath filter (excitation: 375/15, Beam splitter: 405 LP) are used. After aniline blue staining, the spores appeared blue under UV light.
  • the papillae can be recognized beneath the fungal appressorium by a green/yellow staining.
  • the hypersensitive reaction is characterized by a whole cell fluorescence. By rating and counting 50-200 individual fungal-plant interaction sites on each transgenic and control leaves it can be observed that resistance inducing genes, show an increased number of defense associated fungal-plant interactions, such as papillae and hypersensitive reactions, whereas more susceptibility associated phenotypes, such as mycelial growth in the mesophyll, can by observed in control leaves.
  • Example 6 Evaluating the susceptibility to soybean rust in greenhouse
  • the progression of the soybean rust disease is scored in percent by the estimation of the diseased area (area which was covered by sporulating uredinia)) of a soybean leaf 14 days after inoculation (see above). Additionally, the yellowing of the leaf is taken into account.
  • a scheme illustrating the disease rating can be found in WO2016124515 and W02020120753. Two independent greenhouse experiments were performed.
  • Non-transgenic control plants are having the same genetic background compared to transgenics and the plants were grown in parallel to the transgenic plants in a fully randomized design.
  • the constitutive expression of ICS by the construct described in example 2.1 leads to enhanced resistance of soybean against Phakopsora pachyrhizi in comparison to wild type control.
  • Non-transgenic control plants are having the same genetic background compared to transgenics and the plants were grown in parallel to the transgenic plants in a fully randomized design.
  • the pathogen induced expression of ICS protein by the construct described in example 2.2 leads to enhanced resistance of soybean against Phakopsora pachyrhizi in comparison to wild type control.
  • Homozygous T2 or T3 seeds were used for field trials. To obtain homozygous seeds, segregating T1 seeds of 5 events per construct were planted. Individual plants that were homozygous for the transgene were selected by using TaqMan® PCR assay as described by the manufacturer of the assay (Thermo Fisher Scientific, Waltham, MA USA 02451).
  • Field trials were planted depending on weather conditions in November or early December to ensure inoculum of Asian soybean rust. Events were tested in split plot trials (2 m long, 4 rows per plot) and 3 replications per event, treatment and trial site. Soybean plants were cultivated using standard cultural practice, e.g. in terms of weed and insect control and fertilization. Concerning fungal disease control two treatment varieties were performed at Campinas location: 1. No fungicide treatment at all (“no treatment”) and 2. only one fungicide treatment at the onset of ASR disease ( ⁇ 35 - 40 days after planting), instead of 3-4 treatments over the season in standard agricultural practice. The 2 different treatment varieties were performed to increase variability of disease pressure and to prove robustness and stability of the resistance increasing effect.
  • Example 8 ASR resistance in field trials
  • the three canopy levels (lower, middle and upper canopy) were rated independently and the average of the infection of all three canopy levels is counted as infection. At all 4-7 ratings were performed, starting at the early onset of disease and repeated mainly every 6-10 days. If weather was not suitable for disease progression the time in between 2 ratings was elongated.
  • DeltaAUDPC AUDPC(event) - AUDPC(control)
  • transgenic plots are more resistant to ASR, the AUDPC of the event plots will be smaller than the AUDPC of the control plots. Therefore, a negative deltaAUDPC indicates a higher resistance of the transgenic events.
  • the deltaAUDPC was calculated on event level (see figure 4 and 5, grey bars)and then averaged over all events harbouring the same construct to come up with the average disease resistance of the construct (see figure 4 and 5, black bars).
  • the statistical significance of the result was calculated based on the relative resistance of every event at every timepoint of rating (paired one-sided t-test).

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Abstract

La présente invention concerne un procédé permettant de conférer ou d'accroître la résistance aux agents pathogènes fongiques dans les plantes et/ou la matière végétale. À cette fin, l'invention vise à faciliter ou à augmenter la production et/ou l'accumulation d'une isochorismate synthase (ICS), d'un fragment ou d'un homologue de celle-ci dans une plante ou une matière végétale par comparaison avec une plante ou une matière végétale de type sauvage correspondante. L'invention concerne également des plantes et des matières végétales présentant une résistance accrue aux agents pathogènes fongiques, ainsi que des matières et des procédés permettant de créer ou d'utiliser de telles plantes, parties de plantes ou de fabriquer des produits à partir de celles-ci.
PCT/EP2024/071902 2023-08-01 2024-08-01 Résistance accrue par expression d'une protéine ics WO2025027165A1 (fr)

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Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999050423A2 (fr) * 1998-03-31 1999-10-07 Mogen International N.V. Genes de la voie de l'acide salicylique et leur utilisation pour induire la resistance chez les vegetaux
WO2002006447A2 (fr) * 2000-07-18 2002-01-24 The General Hospital Corporation Genes de biosynthese d'acide salicylique et utilisations associees
WO2005121345A1 (fr) 2004-06-07 2005-12-22 Basf Plant Science Gmbh Transformation amelioree de soja
WO2007025097A2 (fr) 2005-08-26 2007-03-01 Danisco A/S Utilisation
WO2013141680A1 (fr) 2012-03-20 2013-09-26 Vilnius University Clivage d'adn dirigé par arn par le complexe cas9-arncr
WO2013176772A1 (fr) 2012-05-25 2013-11-28 The Regents Of The University Of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
WO2014093595A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Systèmes de composants de crispr-cas, procédés et compositions pour la manipulation de séquences
WO2014099750A2 (fr) 2012-12-17 2014-06-26 President And Fellows Of Harvard College Modification du génome humain par guidage arn
WO2014150624A1 (fr) 2013-03-14 2014-09-25 Caribou Biosciences, Inc. Compositions et procédés pour des acides nucléiques à ciblage d'acide nucléique
WO2014204728A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Délivrance, modification et optimisation de systèmes, procédés et compositions pour cibler et modéliser des maladies et des troubles liés aux cellules post-mitotiques
WO2016124515A1 (fr) 2015-02-04 2016-08-11 Basf Plant Science Company Gmbh Procédé d'augmentation de la résistance de plantes transgéniques contre la rouille du soja par augmentation de leur teneur en scopolétine
WO2016205749A1 (fr) 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes associés
WO2016205711A1 (fr) 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes
CN111269912A (zh) * 2020-02-05 2020-06-12 中国热带农业科学院热带生物技术研究所 一种驱动水杨酸合成的香蕉异分支酸合酶MaICS启动子及其应用
WO2020120753A1 (fr) 2018-12-14 2020-06-18 Basf Plant Science Company Gmbh Procédés pour augmenter la résistance à la rouiile de soja de végétaux transgéniques par l'augmentation de la teneur en scoparone

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999050423A2 (fr) * 1998-03-31 1999-10-07 Mogen International N.V. Genes de la voie de l'acide salicylique et leur utilisation pour induire la resistance chez les vegetaux
WO2002006447A2 (fr) * 2000-07-18 2002-01-24 The General Hospital Corporation Genes de biosynthese d'acide salicylique et utilisations associees
WO2005121345A1 (fr) 2004-06-07 2005-12-22 Basf Plant Science Gmbh Transformation amelioree de soja
WO2007025097A2 (fr) 2005-08-26 2007-03-01 Danisco A/S Utilisation
WO2013141680A1 (fr) 2012-03-20 2013-09-26 Vilnius University Clivage d'adn dirigé par arn par le complexe cas9-arncr
WO2013176772A1 (fr) 2012-05-25 2013-11-28 The Regents Of The University Of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
WO2014093595A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Systèmes de composants de crispr-cas, procédés et compositions pour la manipulation de séquences
WO2014099744A1 (fr) 2012-12-17 2014-06-26 President And Fellows Of Harvard College Manipulation du génome humain guidée par l'arn
WO2014099750A2 (fr) 2012-12-17 2014-06-26 President And Fellows Of Harvard College Modification du génome humain par guidage arn
WO2014150624A1 (fr) 2013-03-14 2014-09-25 Caribou Biosciences, Inc. Compositions et procédés pour des acides nucléiques à ciblage d'acide nucléique
US20160046963A1 (en) 2013-03-14 2016-02-18 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
WO2014204728A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Délivrance, modification et optimisation de systèmes, procédés et compositions pour cibler et modéliser des maladies et des troubles liés aux cellules post-mitotiques
WO2016124515A1 (fr) 2015-02-04 2016-08-11 Basf Plant Science Company Gmbh Procédé d'augmentation de la résistance de plantes transgéniques contre la rouille du soja par augmentation de leur teneur en scopolétine
WO2016205749A1 (fr) 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes associés
WO2016205711A1 (fr) 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes
WO2020120753A1 (fr) 2018-12-14 2020-06-18 Basf Plant Science Company Gmbh Procédés pour augmenter la résistance à la rouiile de soja de végétaux transgéniques par l'augmentation de la teneur en scoparone
CN111269912A (zh) * 2020-02-05 2020-06-12 中国热带农业科学院热带生物技术研究所 一种驱动水杨酸合成的香蕉异分支酸合酶MaICS启动子及其应用

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
"Proceedings of the rust workshop", 1995, NATIONAL SOYBEANA RESEARCH LABORATORY
BORTESIFISCHER, BIOTECHNOLOGY ADVANCES, vol. 33, 2015, pages 41 - 52
CHENGAO, PLANT CELL REP, vol. 33, 2014, pages 575 - 583
GODOY, C.KOGA, L.CANTERI, M., DIAGRAMMATIC SCALE FOR ASSESSMENT OF SOYBEAN RUST SEVERITY, FITOPATOLOGIA BRASILEIRA, vol. 31, no. 1, 2006
GODOY, C.V.KOGA, L.J.CANTERI, M.G.: "Diagrammatic scale for assessment of soybean rust severity", FITOPATOLOGIA BRASILEIRA, vol. 31, 2006, pages 063 - 068
H. KLEER. HORSCHS. ROGERS: "Agrobacterium-Mediated Plant Transformation and its further Applications to Plant Biology", ANNUAL REVIEW OF PLANT PHYSIOLOGY, vol. 38, 1987, pages 467 - 486
HAO QUNQUN ET AL: "Isochorismate-based salicylic acid biosynthesis confers basal resistance to Fusarium graminearum in barley", MOLECULAR PLANT PATHOLOGY, vol. 19, no. 8, 25 April 2018 (2018-04-25), GB, pages 1995 - 2010, XP093216751, ISSN: 1464-6722, Retrieved from the Internet <URL:https://onlinelibrary.wiley.com/doi/full-xml/10.1111/mpp.12675> DOI: 10.1111/mpp.12675 *
HEATH, CAN. J. PLANT PATHOL., vol. 24, 2002, pages 259 - 264
ICS, MISHRA A.K.BAEK K.-H., SALICYLIC ACID BIOSYNTHESIS AND METABOLISM: A DIVERGENT PATHWAY FOR PLANTS AND BACTERIA, BIOMOLECULES, vol. 11, no. 705, 2021
KAWALLECK ET AL., PLANT MOLECULAR BIOLOGY, vol. 21, no. 4, 1993, pages 673 - 684
M.J. JEGERS.L.H. VILJANEN-ROLLINSON, THE USE OF THE AREA UNDER THE DISEASE-PROGRESS CURVE (AUDPC) TO ASSESS QUANTITATIVE DISEASE RESISTANCE IN CROP CULTIVARS THEOR APPL GENET, vol. 102, 2001, pages 32 - 40
MALZAHN ET AL., CELL BIOSCI, vol. 7, 2017, pages 21
MANNERS, J.M.PENNINCKX, I.A.VERMAERE, K. ET AL., PLANT MOL BIOL, vol. 38, 1998, pages 1071 - 1080
MAUCH F ET AL: "MANIPULATION OF SALICYLATE CONTENT IN ARABIDOPSIS THALIANA BY THE EXPRESSION OF AN ENGINEERED BACTERIAL SALICYLATE SYNTHASE", THE PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 25, no. 1, 1 January 2001 (2001-01-01), pages 67 - 77, XP002907650, ISSN: 0960-7412, DOI: 10.1046/J.1365-313X.2001.00940.X *
MEINKOTHWAHL, ANAL. BIOCHEM., vol. 138, 1984, pages 267 - 284
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1979, pages 443 - 453
NEU ET AL., AMERICAN CYTOPATHOL. SOCIETY, MPMI, vol. 16, no. 7, 2003, pages 626 - 633
OLHOFT ET AL.: "A novel Agrobacterium rhizogenes-mediated transformation method of soybean using primary-node explants from seedlings In Vitro Cell", DEV. BIOL.-PLANT, vol. 43, 2007, pages 536 - 549
OLHOFT ET AL.: "A novel Agrobacterium rhizogenes-mediated transformation method of soybean using primary-node explants from seedlings", IN VITRO CELL. DEV. BIOL.-PLANT, vol. 43, 2007, pages 536 - 549
RIVAS-SAN VICENTE, M.PLASENCIA, J.: "Salicylic acid beyond defense: Its role in plant growth and development", J. EXP. BOT., vol. 62, 2011, pages 3321 - 3338, XP055067867, DOI: 10.1093/jxb/err031
RYTTER J.L. ET AL., PLANT DIS., vol. 87, 1984, pages 818
SANGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 74, 1977, pages 5463 - 897
SIMOH S ET AL: "Metabolic changes of Brassica rapa transformed with a bacterial isochorismate synthase gene", JOURNAL OF PLANT PHYSIOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 167, no. 18, 15 December 2010 (2010-12-15), pages 1525 - 1532, XP027475598, ISSN: 0176-1617, [retrieved on 20100811], DOI: 10.1016/J.JPLPH.2010.06.022 *
VERBERNE M C ET AL: "OVERPRODUCTION OF SALICYLIC ACID IN PLANTS BY BACTERIAL TRANSGENES ENHANCES PATHOGEN RESISTANCE", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 18, 1 July 2000 (2000-07-01), pages 779 - 783, XP002907651, ISSN: 1087-0156, DOI: 10.1038/77347 *
WILDERMUTH MARY C ET AL: "Isochorismate synthase is required to synthesize salicylic acid for plant defence", NATURE,, vol. 414, no. 6863, 29 November 2001 (2001-11-29), pages 562 - 565, XP037675263, DOI: 10.1038/35107108 *
YEATS THROSE JK: "The formation and function of plant cuticles", PLANT PHYSIOL., vol. 163, no. 1, 2013, pages 5 - 20, XP055949029, DOI: 10.1104/pp.113.222737
YU TYSUN MKLIANG LK: "Receptors in the Induction of the Plant Innate Immunity", MOL PLANT MICROBE INTERACT., vol. 34, no. 6, June 2021 (2021-06-01), pages 587 - 601
ZHANG, Y.LI, X.: "Salicylic acid: Biosynthesis, perception, and contributions to plant immunity", CURR. OPIN. PLANT BIOL., vol. 50, 2019, pages 29 - 36, XP085786663, DOI: 10.1016/j.pbi.2019.02.004

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