WO2011069953A1 - Methods for increasing the resistance of plants to fungi by silencing the fungal smt1-gene - Google Patents

Methods for increasing the resistance of plants to fungi by silencing the fungal smt1-gene Download PDF

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WO2011069953A1
WO2011069953A1 PCT/EP2010/068951 EP2010068951W WO2011069953A1 WO 2011069953 A1 WO2011069953 A1 WO 2011069953A1 EP 2010068951 W EP2010068951 W EP 2010068951W WO 2011069953 A1 WO2011069953 A1 WO 2011069953A1
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nucleic acid
gene
target
smt1
plant
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PCT/EP2010/068951
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French (fr)
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Holger Schultheiss
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Basf Plant Science Company Gmbh
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Priority to US13/514,693 priority Critical patent/US8927812B2/en
Priority to AU2010330050A priority patent/AU2010330050A1/en
Priority to EP10787121A priority patent/EP2510090A1/en
Priority to CA2782545A priority patent/CA2782545A1/en
Priority to BR112012013713-1A priority patent/BR112012013713B1/en
Publication of WO2011069953A1 publication Critical patent/WO2011069953A1/en

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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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/107—1,4-Alpha-glucan branching enzyme (2.4.1.18)

Definitions

  • the present invention relates to methods for generating or increasing resistance to at least one fungi, in particular soy bean rust, in a plant or a part of a plant by the expression of RNA which is at least partial complementary and/ or partial identical to the STM1 -gene, wherein the RNA is capable to provide ds-RNA and/or siRNA and /or miRNA.
  • the invention relates to respective plants, parts thereof and vector constructs capable to provide such RNA and the use of such vector constructs to provide fungal resistant plants.
  • Fungi are distributed worldwide. Approximately 100 000 different fungal species are known to date. The rusts are of great importance. They can have a complicated development cycle with up to five different spore stages (spermatium, aecidiospore, uredospore, teleutospore and basidiospore).
  • 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.
  • the soya rust Phakopsora pachyrhizi directly penetrates the plant epidermis.
  • biotrophic phytopathogenic fungi such as many rusts
  • the necrotrophic phytopathogenic fungi depend for their nutrition on dead cells of the plants. Soybeanrust has occupied an intermediate position since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic. After the penetration, the fungus changes over to an obligatory-biotrophic lifestyle.
  • the subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy will, for the purposes of the present invention, be referred to as being "hemibiotrophic".
  • Soybean rust has become increasingly important in recent times.
  • the disease may be caused by the pathogenic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a wide spectrum of leguminosic host plants.
  • P. pachyrhizi also referred to as Asian soybean rust, is the more aggressive pathogen on soybeans (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.
  • pachyrhizi is capable of infecting 31 species from 17 families of the Leguminosae under natural conditions and is capable of growing on further 60 species under controlled conditions (Sinclair et al. (eds.), Proceedings of the soybean rust workshop (1995), National Soybean 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 plants, four dominant genes Rpp1 -4, which mediate resistance of soya to
  • Ergosterol is a component of fungal cell membranes, serving the same function that cholesterol serves in animal cells.
  • Sterol methyltransferase (SMT1 ) is a key enzyme of the ergosterol biosynthesis pathway. It has been surprisingly found that fungal resistance can be provided by introducing recombinant nucleic acids into plant cells capable of silencing the fungal SMT1 - gene in fungi, in particular by RNAi, mi-RNA, sense and/or antisense techniques. A person skilled in the art would not have assumed that indirectly silencing the fungal SMT-1 gene by transformation of the host plant would provide fungal resistance to the plant.
  • the present invention provides a method for producing a plant and/or a part thereof resistant to a phythopathogenic fungus comprising providing a recombinant nucleic acid comprising a target nucleic acid that is substantial identical and/or substantial complementary to at least contiguous 19 nucleotides of the target SMT1 -sequence,
  • the present invention further provides a vector construct comprising a recombinant nucleic acid comprising a promoter that is functional in the plant cell, operably linked to a target nucleic acid which is substantial identical and/or substantial complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and a terminator regulatory sequence as well as the use of the vector construct for the transformation of plants or parts thereof to provide fungal resistant plants.
  • the present invention also provides a transgenic plant cell, plants or parts thereof comprising a recombinant nucleic acid comprising a target nucleic acid that is substantial identical and/or substantial complementary at least contiguous 19 nucleotides of the target SMT1 -gene.
  • Parts of plants may be plant cells, roots, stems, leaves, flowers and/or seeds.
  • siRNAs small interfering RNAs from the SMT1 construct by using the pathways known in the literature (Andrew Eamens, Ming-Bo Wang, Neil A. Smith, and Peter M. Waterhouse “RNA Silencing in Plants: Yesterday, Today, and Tomorrow"Plant Physiology, June 2008, Vol. 147, pp. 456-468). Due to the close connection between the fungus and its host plant (especially at the haustoria), the siRNAs are able to move or being transported (in complexes with proteins or naked) into the fugus. In the fungus the siRNAs lead to a sequence specific siRNA mediated knock-down of the target gene (in this case SMT1 ). This process is most likely mediated and maintained by protein complexes like RISC (RNA-induced silencing complex) and RdRP (RNA dependent RNA polymerases).
  • RISC RNA-induced silencing complex
  • RdRP RNA dependent RNA polymerases
  • the terms "fungal-resistance, resistant to a fungus” and/or “fungal-resistant” mean reducing or preventing an infection by a fungus. Resistance does not imply that the plant necessarily has 100% resistance to infection. In preferred embodiments, the resistance to infection by a fungus in a resistant plant is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in comparison to a wild type plant that is not resistant to fungus.
  • the wild type plant is a plant of a similar, more preferably identical, genotype as the plant having increased resistance to the fungus, but does not comprise a recombinant nucleic acid comprising the target nucleic acid that is substantial identical and/or complementary to at least 19 nucleotides of the target SMT1 -gene.
  • the terms terms "fungal-resistance, "resistant to a fungus” and/or “fungal-resistant” as used herein refers to the ability of a plant, as compared to a wild type plant, to avoid infection by fungus, to kill fungus, to hamper, reduce, to stop the development, growth and/or multiplication of fungus.
  • the level of fungal resistance of a plant can be determined in various ways, e.g. by scoring/measuring the infected leaf area in relation to the overall leaf area. Another possibility to determine the level of resistance is to count the number of fungal colonies on the plant or to measure the amount of spores produced by these colonies. Another way to resolve the degree of fungal infestation is to specifically measure the amount of fungal DNA by quantitative (q) PCR. Specific probes and primer sequences for most fungal pathogens are available in the literature (Frederick RD, Snyder CL, Peterson GL, et al. 2002 Polymerase chain reaction assays for the detection and discrimination of the soybean rust pathogens Phakopsora pachyrhizi and P-meibomiae PHYTOPATHOLOGY 92(2) 217-227).
  • the term "recombinant nucleic acid” refers to a DNA-molecule comprising a nucleic acid that is substantial identical and/or substantial complementary to at least 19 contiguous nucleotides of the fungal SMT1 -gene, optionally operably linked to a promoter functional in a plant cell and/or other regulatory sequences.
  • the recombinant nucleic acid comprises a sequence which does not naturally occur in the wildtype plant. More preferably, the recombinant nucleic acid comprises a sequence which does occur in fungi but not in plants.
  • target nucleic acid preferably refers to a DNA-molecule capable to prevent the expression, reduce the amount and/or function of the fungal SMT1 -protein in the plant, parts of the plant, fungus and/or parts of the fungus.
  • the term "substantially identical” preferably refers to DNA and/or RNA which is at least 80% identical to 19 or more contiguous nucleotides of a specific DNA or RNA sequence of the SMT1 -gen, more preferably, at least 90% identical to 19 or more contiguous nucleotides, and most preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical or absolutely identical to 19 or more contiguous nucleotides of a specific DNA or RNA- sequence of the SMT1 -gen.
  • the RNA corresponds to the coding DNA-strand of the SMT1 -gen.
  • the term "substantially identical" as applied to DNA of the recombinant nucleic acid, the target nucleic acid and/or the target SMT1 -gene means that the nucleotide sequence is at least 80% identical to 19 or more contiguous nucleotides of the target SMT1 -gene, more preferably, at least 90% identical to 19 or more contiguous nucleotides of the target SMT1 - gene, and most preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical or absolutely identical to 19 or more contiguous nucleotides of the target SMT1 - gene.
  • 19 or more contiguous nucleotides of the target SMT1 -gene corresponds to the target SMT1 -gene, being at least about 19, 20, 21 , 22, 23, 24, 25, 50, 100, 200, 300, 400, 500, 1000, 1500, consecutive bases or up to the full length of the target SMT1 -gene.
  • complementary polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.
  • the term "substantially complementary” means that two nucleic acid sequences are complementary over at least at 80% of their nucleotides. Preferably, the two nucleic acid sequences are complementary over at least at 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more or all of their nucleotides. Preferably, the two nucleic acid sequences are
  • substantially complementary means that two nucleic acid sequences can hybridize under stringency conditions.
  • Hybridization includes "any process by which a strand of nucleic acid molecule joins with a complementary strand through base pairing.” (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid molecules) is impacted by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acid molecules.
  • Tm is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which a population of double- stranded nucleic acid molecules becomes half dissociated into single strands.
  • the equation for calculating the Tm of nucleic acid molecules is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation:
  • Tm 81 .5+0.41 (% G+C), when a nucleic acid molecule is in aqueous solution at 1 M NaCI [see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)].
  • Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of Tm. Stringent conditions, are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6.
  • stringency conditions refers to conditions, wherein 100 contigous nucleotides or more, 150 contigous nucleotides or more, 200 contigous nucleotides or more or 250 contigous nucleotides or more which are a fragment or identical to the complementary nucleic acid molecule (DNA, RNA, ssDNA orssRNA) hybridizes under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 2 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C with a specific nucleic acid molecule (DNA; RNA, ssDNA or ss RNA).
  • SDS sodium dodecyl sulfate
  • the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C, more preferably the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0,1 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C.
  • the complementary nucleotides hybridize with a fragment or the whole SMT1 - target gene.
  • the complementary polynucleotide hybridizes with parts of the SMT1 - target gene capable to provide fungal resistance.
  • the complementary nucleotides hybridize with a fragment or the whole SMT1 - target gene.
  • the complementary polynucleotide hybridizes with parts
  • polynucleotide hybridizes with the coding strand of the SMT1 -gene or a part thereof.
  • RNA complementary to a DNA means that the sequence of the RNA is complementary to the coding strand of the DNA.
  • RNAs are reverse complementary to each other, i.e. form dsRNA.
  • target SMT1 -gene means fungal steroyl-methyl-transferase-genes including any homolog of the sterol methyl transferase.
  • SMT1 -gene refers to a gene having at least 60 % identity with SEQ-ID-No. 1 , SEQ-ID-No. 2 or SEQ-ID-No. 3 or with a sequence coding for a protein having SEQ-ID-No. 5.
  • SMT1 -gene refers to a gene having at least 60 % identity with SEQ-ID-No. 1 , SEQ-ID-No. 2 or SEQ-ID-No. 3 or with a sequence coding for a protein having SEQ-ID-No. 5.
  • homologues of the SMT1 -gene have, at the DNA level or protein level, at least 70%, preferably of at least 80%, especially preferably of at least 90%, quite especially preferably of at least 95%, quite especially preferably of at least 98% or 100% identity over the entire DNA region or protein region given in a sequence specifically disclosed herein.
  • nucleic acid refers preferably in each case over the entire length of the nucleic acid.
  • the identity may be calculated by means of the Vector NTI Suite 7.1 program of the company Informax (USA) employing the Clustal Method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr; 5(2): 151 -1 ) with the following settings:
  • the identity may be determined according to Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment with the Clustal series of programs. (2003) Nucleic Acids Res 31 (13):3497-500, the web page: http://www.ebi.ac.Uk/Tools/clustalw/index.html# and the following settings
  • the complementarity may be calculated as the identity.
  • complementary means that purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
  • G:C guanine paired with cytosine
  • A:T thymine
  • A:U adenine paired with uracil
  • All the nucleic acid sequences mentioned herein can be produced in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • oligonucleotides can, for example, be performed in a known way, by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897).
  • the accumulation of synthetic oligonucleotides and filling of gaps by means of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989), see below.
  • siRNA 19-24 nucleotide fragments
  • RNA-interference RNA-interference
  • mi-RNA micro-RNAi
  • sense RNA sense RNA
  • antisense RNA antisense RNA
  • RNAi refers to the process of sequence-specific post- transcriptional gene silencing, mediated by double-stranded RNA (dsRNA).
  • dsRNA double-stranded RNA
  • dsRNA comprising a first strand that is substantially complementary to at least 19 comtiguous nucleotides of the target SMT1 -gene and a second strand that is complementary to the first strand at least partially has to be provided.
  • a recombinant nucleic acid is introduced into the plant, which is capble to produce such dsRNA.
  • the target SMT1 -gene- specific dsRNA is produced and processed into relatively small fragments (siRNAs) and can subsequently become distributed from the plant to the fungus.
  • miRNA refers to a similar process, except that the produced dsRNA only partially comprises regions substantially identical to the SMT1 -gene (at least 19 contiguous nucleotides).
  • antisense interference refers to the process of sequence-specific post- transcriptional gene silencing, probably also mediated by double-stranded RNA (dsRNA).
  • dsRNA double-stranded RNA
  • ssRNA comprising a first strand that is substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene has to be provided.
  • recombinant nucleic acid is introduced into the plant, which is capable to produce such ssRNA.
  • this RNA moves from the plant to the fungus and subsequently pairs with complementary ssRNA transcribed from the original SMT1 - gene produced by the original SMT1 -gene.
  • the resulting dsRNA is processed into relatively small fragments (siRNAs) and can subsequently become distributed from the plant to the fungus.
  • the target nucleic acid comprises a 19-nucleotide portion which is substantially identical and/or substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene.
  • a target nucleic acid comprising a nucleotide sequence identical and/or identical to a portion of the fungal target SMT1 -gene and/or complementary to the whole sequence and/or a portion of the fungal target SMT1 -gene is preferred for inhibition, the invention can tolerate sequence variations that might be expected due to gene manipulation or synthesis, genetic mutation, strain polymorphism, or evolutionary divergence.
  • the target nucleic acid may also encompass a mismatch with the target SMT1 -gene of at least 1 , 2, or more nucleotides.
  • the target nucleic acid may contain an addition, deletion or substitution of 1 , 2, or more nucleotides, so long as the resulting RNA sequence still interferes with the fungal target SMT1 -gene function.
  • Sequence identity between the recombinant nucleic acid useful according to the present invention and the fungal SMT1 -target gene may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991 , and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 80 % sequence identity, 90% sequence identity, or even 100% sequence identity, between the target nucleic acid and at least 19 contiguous nucleotides of the target gene is preferred. The same preferably applies for the sequence complementarity.
  • the corresponding ds RNA provided therefrom will be cleaved randomly to dsRNAs of about 21 nucleotides within the plant or fungal cell: the siRNAs.
  • Multiple specialized Dicers in plants may generate siRNAs typically ranging in size from 19nt to 24nt (See Henderson et al., 2006. Nature Genetics 38:721 -725.).
  • the cleavage of a longer dsRNA of the invention may yield a pool of 21 mer dsRNAs, derived from the longer dsRNA.
  • the siRNAs may have sequences corresponding to fragments of 19-24 contiguous nucleotides across the entire sequence of the fungal target SMT1 -gene.
  • the siRNA can have a mismatch with the target gene of at least 1 , 2, or more nucleotides. Further, these mismatches are intended to be included in the present invention.
  • the target nucleic acid is substantial identical and/or substantial
  • the target nucleic acid may comprise 19 to 500, preferably 50 to 500, more preferably 250 to 350 nucleotides, wherein preferably at least about 19, 20, 21 , 22, 23, 24, 25, 50, 100, 200, 300, 400, consecutive bases or up to the full length of target nucleic acid are identical and/or complementary and/or identical to the target SMT1 -gene.
  • the recombinant nucleic acid is able to provide dsRNA and/or siRNA and/or miRNA in the plant, a part thereof and/or the fungus infecting the plant or a part thereof once the recombinant nucleic acid is expressed in the plant, wherein preferably at least 19 contiguous nucleotides of the dsRNA and/or si RNA and/or miRNA are substantially complementary to the target SMT1 -gene.
  • One embodiment according to the present invention provides a method for producing a plant and/or a part thereof resistant to a fungus, wherein the recombinant nucleic acid comprises a promoter that is functional in the plant cell, operably linked to a
  • target nucleic acid which is substantial identical and/or substantial complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and a second strand having a sequence at substantially complementary to the first strand and/or parts thereof, and
  • the first strand and the second strand may at least partially form dsRNA.
  • the target nucleic acid comprises 19 to 24 contiguous nucleotides of the target sequence which are substantially identical and/or substantially complementary to the target SMT1 -gene and the remaining nucleotides of the target nucleic acid are not identical and/or not complementary to the target SMT1 -gene.
  • Not-identical means an identity which is lower than 95%, lower that 90%, lower than 80%, lower than 70 %, lower than 60% over the whole sequence of the target nucleic acid.
  • Not-complementary means an complementarity which is lower than 95%, lower that 90%, lower than 80%, lower than 70 %, lower than 60% over the whole sequence of the target nucleic acid.
  • miRNA miRNA
  • One embodiment according to the present invention provides a method for producing a plant and/or a part thereof resistant to a fungus, wherein the recombinant nucleic acid comprises a promoter that is functional in the plant cell, operably linked to a
  • target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary to at least contiguous 19 nucleotides of the target SMT1 -gene, and
  • the first strand generated in the plant forms dsRNA together with a second RNA- strand generated in the fungus which is complementary to the first strand.
  • This technique is also refered to as antisense RNA.
  • One embodiment according to the present invention provides a method for producing a plant and/or a part thereof resistant to a fungus, wherein the recombinant nucleic acid comprises a promoter that is functional in the plant cell, operably linked to a
  • target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially identical to at least contiguous 19 nucleotides of the target SMT1 - gene, and
  • the first strand generated in the plant forms dsRNA together with a second RNA- strand generated in the fungus which is complementary to the first strand.
  • This technique is also refered to as sense RNA.
  • the dsRNA of the invention may optionally comprise a single stranded overhang at either or both ends.
  • the single stranded overhang comprises at least two nucleotides at the 3 ' end of each strand of the dsRNA molecule.
  • the double-stranded structure may be formed by a single self-complementary RNA strand (i.e. forming a hairpin loop) or two complementary RNA strands. RNA duplex formation may be initiated either inside the plant or inside the fungus.
  • the dsRNA of the invention may optionally comprise an intron, as set forth in US 2003/0180945A1 or a nucleotide spacer, which is a stretch of sequence between the complementary RNA strands to stabilize the hairpin transgene in cells.
  • intron as set forth in US 2003/0180945A1
  • nucleotide spacer which is a stretch of sequence between the complementary RNA strands to stabilize the hairpin transgene in cells.
  • plant is intended to encompass plants at any stage of maturity or development, as well as any tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context.
  • Plant parts include, but are not limited to, plant cells, stems, roots, flowers, ovules, stamens, seeds, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, hairy root cultures, and/or the like.
  • the present invention also includes seeds produced by the plants of the present invention. In one embodiment, the seeds are true breeding for an increased resistance to fungal infection as compared to a wild-type variety of the plant seed.
  • a "plant cell” includes, but is not limited to, a protoplast, gamete producing cell, and a cell that regenerates into a whole plant. Tissue culture of various tissues of plants and regeneration of plants therefrom is well known in the art and is widely published.
  • transgenic refers to any plant, plant cell, callus, plant tissue, or plant part that contains all or part of at least one recombinant polynucleotide comprising at least 19 contiguous nucleotides which are substantial identical and/or substantial complementary to the SMT1 -gene.
  • all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations.
  • the plant is selected from the group consisting of beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, and/or groundnut.
  • the plant is a legume, comprising plants of the genus Phaseolus (comprising French bean, dwarf bean, climbing bean (Phaseolus vulgaris), Lima bean (Phaseolus lunatus L), Tepary bean (Phaseolus acutifolius A. Gray), runner bean (Phaseolus coccineus)); the genus Glycine (comprising Glycine soja, soybeans (Glycine max (L.) Merill)); pea (Pisum) (comprising shelling peas (Pisum sativum L. convar. sativum), also called smooth or round-seeded peas; marrowfat pea (Pisum sativum L. convar.
  • Phaseolus comprising plants of the genus Phaseolus (comprising French bean, dwarf bean, climbing bean (Phaseolus vulgaris), Lima bean (Phaseolus lunatus L), Tepary bean (Phaseolus acut
  • sativa L. chickpea (Cicer), lentils (Lens) (Lens culinaris Medik.), lupins (Lupinus); vetches (Vicia), field bean, broad bean (Vicia faba), vetchling (Lathyrus) (comprising chickling pea (Lathyrus sativus), heath pea (Lathyrus tuberosus)); genus Vigna (comprising moth bean (Vigna aconitifolia (Jacq.) Marechal), adzuki bean (Vigna angularis (Willd.) Ohwi & H.
  • Canavalia comprising jack bean (Canavalia ensiformis (L.) DC), sword bean (Canavalia gladiata (Jacq.) DC.)).
  • the fungal resistance is a resistance against a biotrophic fungus, preferably a hemibiotrophic fungus.
  • the biotrophic fungus is selected from the group Basidiomycota, preferably the Uredinales (rusts), especially preferably the Melompsoraceae, and in particular the genus Phakopsora.
  • the pathogen is Phakopsora pachyrhizi and/or P. meibomiae (together also referred to as "soybean rust" or "soya rust”). Preference is being given to the former.
  • the pathogen is selected from the group of the biotrophic pathogens or fungi, it is preferred in some embodiments that the pathogen is other than powdery mildew or downy mildew.
  • the present invention provides a vector construct comprising a promoter that is functional in the plant cell, operably linked to a target nucleic acid which is substantially identical and/or substantially complementary to at least 19 contiguous nucleotides of the target SMT1 - gene and a terminator regulatory sequence.
  • the expression vector may be isolated.
  • the vector construct comprises a promoter that is functional in the plant cell, operably linked to a
  • target nucleic acid which is substantial identical and/or substantial complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and a second strand having a sequence at substantially complementary to the first strand or parts thereof, and a terminator regulatory sequence.
  • first strand and the second strand are capable of hybridizing to form dsRNA at least partially.
  • the vector construct comprises a promoter that is functional in the plant cell, operably linked to a target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary or identical to at least 19 contiguous nucleotides of the target SMT1 -gene, and
  • transcript of the first strand and at least a part of the transcript of the fungal SMT1 -gene are capable of hybridizing to form dsRNA at least partially.
  • the vector construct comprises a target nucleic acid comprising 19 to 500 nucleotides. Further variants of the target nucleic acid are defined in the section referring to the method for producing a plant.
  • the term "operatively linked" is intended to mean that the target nucleic acid is linked to the regulatory sequence, including promotors, terminator regulatory sequences, enhancers and/or other expression control elements (e.g., polyadenylation signals), in a manner which allows for expression of the target nucleic acid (e.g., in a host plant cell when the vector is introduced into the host plant cell).
  • regulatory sequences are described, for example, in Goeddel, Gene Expression
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of dsRNA desired, and the like.
  • the vector constructs of the invention can be introduced into plant host cells to thereby produce ssRNA, dsRNA, siRNA and/or mi RNA in order to prevent and/or reduce fungal infections.
  • the vector construct comprises a promotor operatively linked to a target nucleotide that is a template for one or both strands of the ssRNA- or dsRNA molecules at least substantial complementary to 19 contiguous nucleotides of the target SMT1 -gen.
  • the nucleic acid molecule further comprises two promoters flanking either end of the nucleic acid molecule, wherein the promoters drive expression of each individual DNA strand, thereby generating two complementary RNAs that hybridize and form the dsRNA.
  • the nucleotide sequence is transcribed into both strands of the dsRNA on one transcription unit, wherein the sense strand is transcribed from the 5' end of the transcription unit and the antisense strand is transcribed from the 3' end, wherein the two strands are separated by about 3 to about 500 base pairs, and wherein after transcription, the RNA transcript folds on itself to form a hairpin.
  • the vector contains a bidirectional promoter, driving expression of two nucleic acid molecules, whereby one nucleic acid molecule codes for the sequence
  • a bidirectional promoter is a promoter capable of mediating expression in two directions.
  • the vector contains two promoters, one mediating transcription of the sequence substantially identical to a portion of a SMT1 gene and another promoter mediating transcription of a second sequence being substantially complementary to the first strand and capable of forming a dsRNA, when both sequences are transcribed.
  • the second promoter might be a different promoter.
  • a different promoter means a promoter having a different activity in regard to cell or tissue specificity, or showing expression on different inducers for example, pathogens, abiotic stress or chemicals.
  • Promoters according to the present invention may be constitutive, inducible, in particular pathogen-induceable, developmental stage-preferred, cell type-preferred, tissue-preferred or organ-preferred. Constitutive promoters are active under most conditions.
  • constitutive promoters include the CaMV 19S and 35S promoters (Odell et al., 1985, Nature 313:810-812), the sX CaMV 35S promoter (Kay et al., 1987, Science 236:1299- 1302), the Sep1 promoter, the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171 ), the Arabidopsis actin promoter, the ubiquitin promoter (Christensen et al., 1989, Plant Molec.
  • promoters from the T-DNA of Agrobacterium such as mannopine synthase, nopaline synthase, and octopine synthase, the small subunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter, and/or the like.
  • Promoters that express the dsRNA in a cell that is contacted by fungus are preferred.
  • the promoter may drive expression of the dsRNA in a plant tissue remote from the site of contact with the fungus, and the dsRNA may then be transported by the plant to a cell that is contacted by the fungus, in particular cells of, or close by fungal infected sites.
  • the expression vector of the invention comprises a constitutive promoter, root- specific promoter, a pathogen inducible promoter, or a fungal-inducible promoter.
  • a promoter is inducible, if its activity, measured on the amount of RNA produced under control of the promoter, is at least 30%, 40%, 50% preferably at least 60%, 70%, 80%, 90% more preferred at least 100%, 200%, 300% higher in its induced state, than in its un-induced state.
  • a promoter is cell-, tissue- or organ-specific, if its activity , measured on the amount of RNA produced under control of the promoter, is at least 30%, 40%, 50% preferably at least 60%, 70%, 80%, 90% more preferred at least 100%, 200%, 300% higher in a particular cell-type, tissue or organ, then in other cell-types or tissues of the same plant, preferably the other cell-types or tissues are cell types or tissues of the same plant organ, e.g. a root.
  • the promoter activity has to be compared to the promoter activity in other plant organs, e.g. leaves, stems, flowers or seeds.
  • tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds, or xylem.
  • tissue preferred and organ preferred promoters include, but are not limited to fruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred, integument-preferred, tuber-preferred, stalk-preferred, pericarp-preferred, leaf-preferred, stigma-preferred, pollen-preferred, anther- preferred, a petal-preferred, sepal-preferred, pedicel-preferred, silique-preferred, stem- preferred, root-preferred promoters and/or the like.
  • Seed preferred promoters are preferentially expressed during seed development and/or germination.
  • seed preferred promoters can be embryo-preferred, endosperm preferred and seed coat-preferred. See Thompson et al., 1989, BioEssays 10:108.
  • seed preferred promoters include, but are not limited to cellulose synthase (celA), Cim1 , gamma-zein, globulin-1 , maize 19 kD zein (CZ19B1 ) and/or the like.
  • tissue-preferred or organ-preferred promoters include, but are not limited to, the napin-gene promoter from rapeseed (U.S. Patent No. 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al., 1991 , Mol Gen Genet. 225(3):459-67), the oleosin-promoter from Arabidopsis (PCT Application No. WO 98/45461 ), the phaseolin-promoter from Phaseolus vulgaris (U.S. Patent No. 5,504,200), the Bce4-promoter from Brassica (PCT Application No.
  • WO 91/13980 or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2(2):233-9), as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice, etc.
  • Suitable promoters to note are the Ipt2 or Ipt1 -gene promoter from barley (PCT Application No. WO 95/15389 and PCT Application No. WO
  • Promoters useful according to the invention include, but are not limited to, are the major chlorophyll a/b binding protein promoter, histone promoters, the Ap3 promoter, the ⁇ -conglycin promoter, the napin promoter, the soybean lectin promoter, the maize 15kD zein promoter, the 22kD zein promoter, the 27kD zein promoter, the g-zein promoter, the waxy, shrunken 1 , shrunken 2, bronze promoters, the Zm13 promoter (U.S. Patent No. 5,086,169), the maize polygalacturonase promoters (PG) (U.S. Patent Nos. 5,412,085 and 5,545,546), the SGB6 promoter (U.S. Patent No. 5,470,359), as well as synthetic or other natural promoters.
  • the major chlorophyll a/b binding protein promoter include, but are not limited to, are the major chlorophyll a/b binding protein
  • Epidermisspezific promotors may be seleted from the group consisting of:
  • Prx7 acc. AJ003141 , Kristensen B.K., Ammitzboll H., Rasmussen S.K. and Nielsen K.A., Molecular Plant Pathology, 2(6), 31 1 (2001 );
  • HvPRI a acc. X74939; Bryngelsson et al., Mol. Plant Microbe Interact!. 7 (2), 267 (1994);
  • HvPRI b acc. X74940; Bryngelsson et al., Mol. Plant Microbe Interact. 7(2), 267 (1994);
  • HvB1 ,3gluc acc. AF479647;
  • Constitutve promotors may be selected from the group consisting of - PcUbi promoter from parsley (WO 03/102198)
  • CaMV 35S promoter Cauliflower Mosaic Virus 35S promoter (Benfey et al. 1989 EMBO J. 8(8): 2195-2202),
  • STPT promoter Arabidopsis thaliana Short Triose phosphat translocator promoter
  • - Act1 promoter - Oryza sativa actin 1 gene promoter (McElroy et al. 1990 PLANT CELL 2(2) 163-171 a) and/or
  • vector construct refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector
  • Certain vector constructs are capable of autonomous replication in a host plant cell into which they are introduced. Other vector constructs are integrated into the genome of a host plant cell upon introduction into the host cell, and thereby are replicated along with the host genome. In particular the vector construct is capable of directing the expression of gene to which the vectors is operatively linked.
  • the invention is intended to include such other forms of expression vector constructs, such as viral vectors (e.g., potato virus X, tobacco rattle virus, and/or Gemini virus), which serve equivalent functions.
  • a preferred vector construct comprises sequences selected from the group consisting of SEQ- ID-1 , 2, 3, 4, 6, 7, 8, 9 and/or 10.
  • the present invention further provides a transgenic plant cell comprising a recombinant nucleic acid comprising a target nucleic acid that is substantial identical and/or substantially complementary to at least contiguous 19 nucleotides of the target SMT1 -gene.
  • the present invention further provides a transgenic plant or parts thereof comprising the above transgenic plant cells according or consisting thereof.
  • the present invention also provides transgenic seeds derived from the plant comprising the target nucleic acid. Parts of the plant may be root, leaves and/or flowers.
  • the transgenic plant cells may be transformed with one of the above described vector constructs. Suitable methods for transforming or transfecting host cells including plant cells are well known in the art of plant biotechnology. Any method may be used to transform the recombinant expression vector into plant cells to yield the transgenic plants of the invention.
  • General methods for transforming dicotyledenous plants are disclosed, for example, in U.S. Pat. Nos. 4,940,838; 5,464,763, and the like.
  • Methods for transforming specific dicotyledenous plants, for example, cotton are set forth in U.S. Pat. Nos. 5,004,863; 5,159,135; and 5,846,797. Soybean transformation methods are set forth in U.S. Pat. Nos. 4,992,375; 5,416,01 1 ;
  • Transformation methods may include direct and indirect methods of transformation. Suitable direct methods include polyethylene glycol induced DNA uptake, liposome-mediated transformation (US 4,536,475), biolistic methods using the gene gun (Fromm ME et al., Bio/Technology. 8(9):833-9, 1990;
  • the plasmids used need not meet any particular requirements. Simple plasmids, such as those of the pUC series, pBR322, M13mp series, pACYC184 and the like can be used. If intact plants are to be regenerated from the transformed cells, an additional selectable marker gene is preferably located on the plasmid.
  • the direct transformation techniques are equally suitable for dicotyledonous and monocotyledonous plants.
  • Transformation can also be carried out by bacterial infection by means of Agrobacterium (for example EP 0 1 16 718), viral infection by means of viral vectors (EP 0 067 553; US 4,407,956; WO 95/34668; WO 93/03161 ) or by means of pollen (EP 0 270 356; WO 85/01856; US
  • Agrobacterium based transformation techniques (especially for dicotyledonous plants) are well known in the art.
  • the Agrobacterium strain e.g., Agrobacterium tumefaciens or Agrobacterium rhizogenes
  • the Agrobacterium strain comprises a plasmid (Ti or Ri plasmid) and a T-DNA element which is transferred to the plant following infection with Agrobacterium.
  • the T-DNA (transferred DNA) is integrated into the genome of the plant cell.
  • the T-DNA may be localized on the Ri- or Ti- plasmid or is separately comprised in a so-called binary vector.
  • the Agrobacterium-mediated transformation is best suited to dicotyledonous plants but has also been adapted to monocotyledonous plants.
  • Agrobacteria is described in, for example, White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, Vol. 1 , Engineering and Utilization, edited by S.D. Kung and R. Wu,
  • Transformation may result in transient or stable transformation and expression.
  • a nucleotide sequence of the present invention can be inserted into any plant and plant cell falling within these broad classes, it is particularly useful in crop plant cells.
  • the transgenic plants of the invention may be crossed with similar transgenic plants or with transgenic plants lacking the nucleic acids of the invention or with non-transgenic plants, using known methods of plant breeding, to prepare seeds.
  • the transgenic plant cells or plants of the present invention may comprise, and/or be crossed to another transgenic plant that comprises one or more nucleic acids, thus creating a "stack" of transgenes in the plant and/or its progeny.
  • the seed is then planted to obtain a crossed fertile transgenic plant comprising the nucleic acid of the invention.
  • the crossed fertile transgenic plant may have the particular expression cassette inherited through a female parent or through a male parent.
  • the second plant may be an inbred plant.
  • the crossed fertile transgenic may be a hybrid.
  • seeds of any of these crossed fertile transgenic plants are also included within the present invention.
  • the seeds of this invention can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plant lines comprising the recombinant nucleic acid comprising at least 19 contiguous nucleotides of the target SMT1 -gene.
  • the transgenic is a legume, preferably selected from the group consisting of beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, and/or groundnut.
  • the introduced recombinant nucleic acid may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes.
  • the introduced recombinant nucleic acid may be present on an extra-chromosomal non-replicating vector construct and be transiently expressed or transiently active.
  • the recombinant nucleic acid preferably resides in a plant expression cassette.
  • a plant expression cassette preferably contains regulatory sequences capable of driving gene expression in plant cells that are operatively linked so that each sequence can fulfill its function, for example, termination of transcription by polyadenylation signals.
  • Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al., 1984, EMBO J.
  • a plant expression cassette preferably contains other operatively linked sequences like translational enhancers such as the overdrive-sequence containing the 5'-untranslated leader sequence from tobacco mosaic virus enhancing the polypeptide per RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-871 1 ).
  • Examples of plant expression vectors include those detailed in: Becker, D. et al., 1992, New plant binary vectors with selectable markers located proximal to the left border, Plant Mol. Biol.
  • the target nucleic acid is capable to reduce the protein quantity or function of the SMT1 -protein in plants cell and/or the fungus.
  • the decrease in the protein quantity or function of the SMT1 -protein takes place in a constitutive or tissue-specific manner.
  • an essentially pathogen-induced decrease in the protein quantity or protein function takes place, for example by recombinant expression of the target nucleic acid under the control of a fungal-induceable promoter.
  • the expression of the target nucleic acid takes place on fungal infected sites, where, however, preferably the expression of the target nucleic acid sequence remains essentially unchanged in tissues not infected by fungus.
  • the protein amount of the SMT1 protein in the plant and/or the fungus is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the target nucleic acid.
  • the wild type plant is a plant of a similar, more preferably identical genotype as the plant transformed with the target nucleic acid.
  • SMT1 -protein means fungal steroyl-methyl-transferase-proteins
  • SMT1 -protein refers to a protein having at least 60 % identity with SEQ-ID-No. 5.
  • homologues of the SMT1 -protein have at least 70%, preferably of at least 80%, especially preferably of at least 90%, quite especially preferably of at least 95%, quite especially preferably of at least 98% or 100% identity to SEQ-ID-5 preferably over the entire protein region.
  • the present invention further provides the use of the vector construct according to the present invention for the transformation of plants to provide fungal resistant plants.
  • the fungal pathogens or fungus-like pathogens (such as, for example, Chromista) preferably belong to the group comprising Plasmodiophoramycota, Oomycota, Ascomycota,
  • Chytridiomycetes Chytridiomycetes, Zygomycetes, Basidiomycota and/or Deuteromycetes (Fungi imperfecti).
  • Pathogens which may be mentioned by way of example, but not by limitation, are those detailed in Tables 1 to 4, and the diseases which are associated with them.
  • Rhizoctonia solani Kuhn Rhizoctonia
  • Brown spot black spot, stalk rot
  • Cephalosporium kernel rot Acremonium strictum Cephalosporium
  • Curvularia leaf spot Curvularia clavata, C. eragrostidis, C.
  • Diplodia ear and stalk rot Diplodia frumenti (teleomorph: Botryosphaeria festucae)
  • Green ear downy mildew (graminicola Sclerospora graminicola
  • Dry ear rot (cob, Nigrospora oryzae
  • kernel and stalk rot (teleomorph: Khuskia oryzae)
  • Botrytis cinerea (teleomorph:
  • Botryotinia fuckeliana Cunninghamella sp.
  • Eyespot Aureobasidium zeae Kabatiella zeae
  • Gray ear rot Botryosphaeria zeae Physalospora zeae
  • Helminthosporium root rot Helminthosporium root rot
  • Exserohilum pedicellatum Helminthosporium pedicellatum (teleomorph: Setosphaeria pedicellata)
  • Hormodendrum ear rot Cladosporium cladosporioides
  • sorokinianum H. sativum
  • Epicoccum nigrum
  • Leptosphaeria maydis, Leptothyrium zeae,
  • Penicillium ear rot blue eye, blue Penicillium spp., P. chrysogenum
  • Phaeocytostroma stalk and root rot Phaeocytostroma ambiguum,
  • Phaeosphaeria leaf spot Phaeosphaeria maydis Sphaerulina maydis
  • Red kernel disease ear mold, leaf Epicoccum nigrum
  • Rhizoctonia ear rot (sclerotial rot) Rhizoctonia zeae (teleomorph: Waitea
  • Root rots (minor) Alternaria alternata, Cercospora sorghi,
  • Dictochaeta fertilis Fusarium acuminatum (teleomorph: Gibberella acuminata), F. equiseti (teleomorph: G. intricans), F. oxysporum, F. pallidoroseum, F. poae, F. roseum, G.
  • cyanogena (anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp., Periconia circinata, Phytophthora cactorum, P. drechsleri, P. nicotianae var. parasitica, Rhizopus arrhizus
  • Helminthosporium carbonum, Diplodia maydis, Exserohilum pedicillatum, Exserohilum turcicum Helminthosporium turcicum, Fusarium avenaceum, F. culmorum, F. moniliforme, Gibberella zeae (anamorph: F. graminearum), Macrophomina phaseolina, Penicillium spp., Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicaria sp.
  • Bipolaris maydis Helminthosporium maydis
  • Trichoderma ear rot and root rot Trichoderma viride T. lignorum teleomorph:
  • Plasmodiophoromycota such as Plasmodiophora brassicae (clubroot of crucifers), Spongospora subterranea, Polymyxa graminis,
  • Oomycota such as Bremia lactucae (downy mildew of lettuce), Peronospora (downy mildew) in snapdragon (P. antirrhini), onion (P. destructor), spinach (P. effusa), soybean (P. manchurica), tobacco ("blue mold”; P. tabacina) alfalfa and clover (P. trifolium), Pseudoperonospora humuli (downy mildew of hops), Plasmopara (downy mildew in grapevines) (P. viticola) and sunflower (P.
  • Ascomycota such as Microdochium nivale (snow mold of rye and wheat), Fusarium graminearum, Fusarium culmorum (partial ear sterility mainly in wheat), Fusarium oxysporum (Fusarium wilt of tomato), Blumeria graminis (powdery mildew of barley (f.sp. hordei) and wheat (f.sp. tritici)), Erysiphe pisi (powdery mildew of pea), Nectria galligena (Nectria canker of fruit trees), Uncinula necator (powdery mildew of grapevine),
  • Pseudopeziza tracheiphila red fire disease of grapevine
  • Claviceps purpurea ergot on, for example, rye and grasses
  • Gaeumannomyces graminis take-all on wheat, rye and other grasses
  • Magnaporthe grisea Pyrenophora graminea (leaf stripe of barley), Pyrenophora teres (net blotch of barley), Pyrenophora tritici-repentis (leaf blight of wheat), Venturia inaequalis (apple scab), Sclerotinia sclerotium (stalk break, stem rot),
  • Pseudopeziza medicaginis (leaf spot of alfalfa, white and red clover).
  • Basidiomycetes such as Typhula incarnata (typhula blight on barley, rye, wheat), Ustilago maydis (blister smut on maize), Ustilago nuda (loose smut on barley), Ustilago tritici (loose smut on wheat, spelt), Ustilago avenae (loose smut on oats), Rhizoctonia solani
  • Deuteromycetes such as Septoria (Stagonospora) nodorum (glume blotch) of wheat (Septoria tritici), Pseudocercosporella herpotrichoides (eyespot of wheat, barley, rye), Rynchosporium secalis (leaf spot on rye and barley), Alternaria solani (early blight of potato, tomato), Phoma betae (blackleg on Beta beet), Cercospora beticola (leaf spot on Beta beet), Alternaria brassicae (black spot on oilseed rape, cabbage and other crucifers), Verticillium dahliae (verticillium wilt), Colletotrichum lindemuthianum (bean anthracnose), Phoma lingam (blackleg of cabbage and oilseed rape), Botrytis cinerea (grey mold of grapevine, strawberry, tomato, hops and the like).
  • biotrophic pathogens among which in particular hemibiotrophic pathogens, i.e. Phakopsora pachyrhizi and/or those pathogens which have essentially a similar infection mechanism as Phakopsora pachyrhizi, as described herein.
  • Phakopsora pachyrhizi and/or Phakopsora meibomiae are especially preferred.
  • Harvestable parts of the transgenic plant according to the present invention are part of the invention.
  • the harvestable parts may be seeds, roots, leaves and/or flowers comprising the SMT1 -gene, the complementary SMT1 -gene and/or a part thereof.
  • Preferred parts of soy plants are soy beans comprising the transgenic SMT1 -gene.
  • a preferred product is soybean meal, soybean oil, wheat meal, corn starch, corn oil, corn meal, rice meal, canola oil and/or potato starch.
  • the present invention also includes methods for the production of a product comprising a) growing the plants of the invention and b) producing said product from or by the plants of the invention and/or parts thereof, e.g. seeds, of these plants.
  • the method comprises the steps a) growing the plants of the invention, b) removing the harvestable parts as defined above from the plants and c) producing said product from or by the harvestable parts of the invention.
  • the method for the production of a product comprises
  • the product may be produced at the site where the plant has been grown, the plants and/or parts thereof may be removed from the site where the plants have been grown to produce the product.
  • the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant.
  • the step of growing the plant may be performed only once each time the methods of the invention is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the invention and if necessary further processing of these parts to arrive at the product.
  • step of growing the plants of the invention is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts.
  • steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally the plants are grown for some time before the product is produced.
  • the products produced by said methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic and/or pharmaceutical.
  • Foodstuffs are regarded as compositions used for nutrition and/or for supplementing nutrition.
  • Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs.
  • inventive methods for the production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
  • a plant product consists of one ore more agricultural products to a large extent.
  • Figure 1 shows the full-length-sequence of the SMT1 -gene from Phakopsora pachyrhizi having SEQ-ID-No.1 .
  • Figure 2A shows the sequence of the SMT1 -gene from Phakopsora pachyrhizi used for primer design and construct generation having SEQ-ID-No.2.
  • Figure 2B shows a partial sequence of Figure 1 (SEQ-ID-3).
  • Figure 3 shows the sequence of one vector construct useful according to the present invention (parsley ubiquitin promoter: SMT1 fragment antisense orientation: spacer sequence: SMT1 fragment sense orientation: nopaline synthase promoter)(SEQ-ID-No.4).
  • Figure 4 shows the sequence of the SMT1 -protein (SEQ-ID-5).
  • Figure 5 shows single sequences which are part of the vector construct of Figure 3.
  • Figure 6 shows a schema of one vector construct useful according to the present invention
  • Figure 7 shows the scoring system used to determine the level of resistance of wildtype and transgenic (SMT1 RNAi) soybean plants against the soybean rust fungus P. pachyrhizi.
  • Figure 8 shows the result of the scoring of 17 transgenic soybean events expressing the SMT1 RNAi construct.
  • the average scoring of the wildtype controls was 4.1 (which can be translated to approximately 50% diseased leaf area. The average scoring of the controls was set to zero.
  • For each transgenic soybean event (expressing the SMT1 RNAi construct) 1 -3 clones were scored. The bars represent the deviation of transgenic soybean events (expressing the SMT1 RNAi construct) the from the average of wild type controls.
  • oligonucleotides can be affected, for example, in the known fashion using the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896- 897).
  • the cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, phage multiplication and sequence analysis of recombinant DNA, are carried out as described by Sambrook et al. Cold Spring Harbor Laboratory Press (1989), ISBN 0-87969-309-6.
  • the sequencing of recombinant DNA molecules is carried out with an MWG-Licor laser fluorescence DNA sequencer following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)).
  • RNAi-construct ( Figures 3 and 6) was prepared as follows: By PCR based amplification AscI and Aatll restriction sites were introduced on the first stem (antisense; from position 148 to 454 relative to SEQ ID2) and Sbfl and Xhol sites were introduced on the second stem (sense, Position 199-454) both in the 5' - 3' strand "sense" orientation (relative zu SEQ ID2).
  • SEQ-ID-No.7 was amplified using using primers having SEQ-ID No. 1 1 and 12.
  • nucleic acid of the second stem having SEQ-ID-No.9 was amplified using using primers having SEQ-ID No 13 and 14. Both fragments were cloned using the TOPO cloning system (TOPO® TA Cloning® Kit Invitrogen). Both fragments were sequenced. Each stem was cut with the respective enzymes and gel purified.
  • binary base vector a binary vector was used which is composed of: (1 ) a Kanamycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agorbacteria (3) a pBR322 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection marker under control of a pcUbi-promoter and an Ubi- promoter:MCS::t-Nos cassette for cloning of the target gene (cf. Figures 3 and 6).
  • the base vector was cut within the MCS with Ascl and Sbfl and the backbone purified by gel purification.
  • Example 3 Sterilization and Germination of Soybean Seeds
  • a variety of soybean cultivar (including Jack, Williams 82, and Resnik) is appropriate for soybean transformation. Soybean seeds were sterilized in a chamber with a chlorine gas produced by adding 3.5 ml 12N HCI drop wise into 100 ml bleach (5.25% sodium hypochlorite) in a desiccator with a tightly fitting lid. After 24 to 48 hours in the chamber, seeds were removed and approximately 18 to 20 seeds were plated on solid GM medium with or without 5 ⁇ 6-benzyl- aminopurine (BAP) in 100 mm Petri dishes.
  • BAP 6-benzyl- aminopurine
  • Seedlings without BAP were more elongated and roots developed, especially secondary and lateral root formation.
  • BAP strengthened the seedling by forming a shorter and stockier seedling.
  • Seven-day-old seedlings grown in the light (>100 ⁇ Einstein/m 2 s) at 25°C. were used for explant material for the three-explant types. At this time, the seed coat was split, and the epicotyl with the unifoliate leaves had 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 (Method A, see example 5.1 ) or leaf explants (Method B, see example 5.3), the seedlings were then ready for transformation.
  • the hypocotyl and one and a half or part of both cotyledons were removed from each seedling.
  • the seedlings were then placed on propagation media for 2 to 4 weeks.
  • the seedlings produced several branched shoots to obtain explants from.
  • the majority of the explants originated from the plantlet growing from the apical bud. These explants were preferably used as target tissue.
  • Agrobacterium cultures were prepared by streaking Agrobacterium (e.g. A. tumefaciens or A. rhizogenes) carrying the desired binary vector (e.g. H Klee, R Horsch, and S Rogers 1987 Agrobacterium-Mediated Plant Transformation and its Further Applications to Plant Biology; Annual Review of Plant Physiology Vol. 38: 467-486) onto solid YEP growth medium (YEP media: 10 g yeast extract. 10 g Bacto Peptone. 5 g NaCI. Adjust pH to 7.0, and bring final volume to 1 liter with H20, for YEP agar plates add 20g Agar, autoclave) and incubating at 25°C. until colonies appeared (about 2 days).
  • 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;
  • transformation were prepared and one-ml of Agrobacterium stock aliquoted into 1 .5 ml Eppendorf tubes then stored at -80°C.
  • 200 ml of YEPmedia were inoculated with 5 ⁇ to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask. The flask was shaked overnight at 25 °C until the ⁇ was between 0.8 and 1 .0.
  • the Agrobacteria were pelleted by centrifugation for 10 min at 5, 500. times. g at 20°C. The pellet was resuspended in liquid CCM to the desired density ( ⁇ 0.5-0.8) and placed at room temperature at least 30 min before use.
  • Soybean epicotyl segments prepared from 4 to 8 d old seedlings were used as explants for regeneration and transformation. Seeds of soybean cv L00106CN, 93-41 131 and Jack were germinated in 1/10 MS salts or a similar composition medium with or without cytokinins for 4. about.8 d. Epicotyl explants were prepared by removing the cotyledonary node and stem node from the stem section. The epicotyl was cut into 2 to 5 segments. Especially preferred were segments attached to the primary or higher node comprising axillary meristematic tissue.
  • Agrobacterium AGL1 Lazo GR, Stein PA, Ludwig RA A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology (N Y) 1991 Oct;9(10):963-967) harboring a plasmid with the GUS marker gene and the AHAS, bar or dsdA selectable marker gene was cultured in LB medium with appropriate antibiotics overnight, harvested and resuspended in a inoculation medium with acetosyringone . Freshly prepared epicotyl segments were soaked in the Agrobacterium suspension for 30 to 60 min and then the explants were blotted dry on sterile filter papers.
  • the inoculated explants were then cultured on a co-culture medium with L-cysteine and DTT (Dithiothreitol) and other chemicals such as acetosyringone for enhancing T-DNA delivery for 2 to 4 d.
  • the infected epicotyl explants were then placed on a shoot induction medium with selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene), or D-serine (for dsdA gene).
  • selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene), or D-serine (for dsdA gene).
  • the regenerated shoots were subcultured on elongation medium with the selective agent.
  • cytokinins such as BAP (6-benzylaminopurine), TDZ (thidiazuron) and/or Kinetin for shoot induction. After 4 to 8 weeks, the cultured tissues were transferred to a medium with lower concentration of cytokinin for shoot elongation. Elongated shoots were transferred to a medium with auxin for rooting and plant development. Multiple shoots were regenerated.
  • Method B Leaf Explants
  • the cotyledon was removed from the hypocotyl.
  • the cotyledons were separated from one another and the epicotyl is removed.
  • the primary leaves, which consist of the lamina, the petiole, and the stipules, were removed from the epicotyl by carefully cutting at the base of the stipules such that the axillary meristems were included on the explant.
  • Agrobacterium suspension immediately after explant preparation. After inoculation, the explants are blotted onto sterile filter paper to remove excess Agrobacterium culture and place explants with the wounded side in contact with a round 7 cm Whatman paper overlaying the solid CCM medium (see above). This filter paper prevents A. tumefaciens overgrowth on the soybean explants. Wrap five plates with Parafilm.TM. "M” (American National Can, Chicago, III., USA) and incubate for three to five days in the dark or light at 25°C.
  • Method C Propagated Axillary Meristem
  • Axillary meristem explants could be pre-pared from the first to the fourth node. An average of three to four explants could be obtained from each seedling.
  • the explants were 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 was cut with a scalpel to induce de novo shoot growth and allow access of target cells to the Agrobacterium. Therefore, a 0.5 cm explant included the stem and a bud.
  • the explants were immediately placed in the Agrobacterium suspension for 20 to 30 minutes. After inoculation, the explants were blotted onto sterile filter paper to remove excess Agrobacterium culture then placed almost completely immersed in solid coculture media CCM (see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soybean using primary-node explants from seedlingsln Vitro Cell. Dev. Biol.— Plant (2007) 43:536-549) or on top of a round 7 cm filter paper overlaying the solid CCM, depending on the Agrobacterium strain. This filter paper prevented Agrobacterium overgrowth on the soybean explants. Plates were wrapped with Parafilm.TM. "M” (American National Can, Chicago, III., USA) and incubated for two to three days in the dark at 25°C.
  • the explant For leaf explants (Method B), the explant should be placed into the medium such that it is perpendicular to the surface of the medium with the petiole imbedded into the medium and the lamina out of the medium.
  • Method C For propagated axillary meristem (Method C), the explant was placed into the medium such that it iwas parallel to the surface of the medium (basipetal) with the explant partially embedded into the medium. Wrap plates with Scotch 394 venting tape (3M, St. Paul, Minn., USA) were placed in a growth chamber for two weeks with a temperature averaging 25°C. under 18 h light/6 h dark cycle at 70-100 ⁇ / ⁇ 2 8. The explants remained on the SIM medium with or without selection until de novo shoot growth occured at the target area (e.g., axillary meristems at the first node above the epicotyl). Transfers to fresh medium can occur during this time.
  • the target area e.g., axillary meristems at the first node above the epicotyl.
  • Explants were transferred from the SIM with or without selection to SIM with selection after about one week. At this time, there was considerable de novo shoot development at the base of the petiole of the leaf explants in a variety of SIM (Method B), at the primary node for seedling explants (Method A), and at the axillary nodes of propagated explants (Method C). Preferably, all shoots formed before transformation were removed up to 2 weeks after co- cultivation to stimulate new growth from the meristems. This helped to reduce chimerism in the primary transformant and increase amplification of transgenic meristematic cells. During this time the explant may or may not be cut into smaller pieces (i.e. detaching the node from the explant by cutting the epicotyl).
  • Regenerated plants obtained using this method were fertile and produced on average 500 seeds per plant.
  • Transient GUS expression after 5 days of co-cultivation with Agrobacterium tumefaciens was widespread on the seedling axillary meristem explants especially in the regions wounding during explant preparation (Method A). Explants were placed into shoot induction medium without selection to see how the primary-node responds to shoot induction and regeneration. Thus far, greater than 70% of the explants were formed new shoots at this region . Expression of the GUS gene was stable after 14 days on SIM, implying integration of the T-DNA into the soybean genome. In addition, preliminary experiments resulted in the formation of GUS positive shoots forming after 3 weeks on SIM .
  • soybean rust fungus was a wild isolate from Brazil.
  • the plants were inoculated with
  • soybean leaves which had been infected with soybean rust 15-20 days ago were taken 2-3 days before the inoculation and transferred to agar plates (1 % agar in H20). The leaves were placed with their upper side onto the agar, which allowed the fungus to grow through the tissue and to produce very young spores.
  • the spores were knocked off the leaves and were added to a Tween-H20 solution. The counting of spores was performed under a light microscope by means of a Thoma counting chamber.
  • the spore suspension was added into a compressed-air operated spray flask and applied uniformly onto the plants or the leaves until the leaf surface is well moisturized.
  • a density of 10x105 spores / ml is used.
  • the inoculated plants were placed for 24 hours in a greenhouse chamber with an average of 22°C and >90% of air humidity.
  • the inoculated leaves were incubated under the same conditions in a closed Petri dish on 0,5% plant agar. The following cultivation was performed in a chamber with an average of 25°C and 70% of air humidity.
  • the inoculated leaves of plants were stained with aniline blue.
  • the aniline blue staining served for the detection of fluorescent substances.
  • substances such as phenols, callose or lignin accumulated or were produced and were incorporated at the cell wall either locally in papillae or in the whole cell (hypersensitive reaction, HR).
  • Complexes were formed in association with aniline blue, which lead e.g. in the case of callose to yellow fluorescence.
  • the leaf material was transferred to falcon tubes or dishes containing destaining solution II (ethanol / acetic acid 6/1 ) and was incubated in a water bath at 90°C for 10-15 minutes. The destaining solution II was removed immediately thereafter, and the leaves were washed 2x with water.
  • the hypersensitive reaction (HR) was characterized by a whole cell fluorescence.
  • Plants are screened macroscopically 14 days after inoculation.
  • the progression of the soybean rust disease was scored by the estimation of the diseased area (area which was covered by sporulating uredinia) on the backside (abaxial side) of the leaf. Additionally the yellowing of the leaf was taken into account, (for complete scheme see Figure 7).
  • Negative values show enhanced resistance, whereas positive values indicate enhanced susceptibility (Figure 8). It was shown that the in planta expression of a RNAi construct targeting a steroyl-methyl transferase of P. pachyrhizi leads to a disease scoring of transgenic plants compared to non-transgenic controls. So, the expression of an RNAi construct targeting the SMT1 enzyme of Phakopsora pachyrhizi enhances the resistance of soybean against soybean rust.

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Abstract

The present invention relates to methods for generating or increasing resistance to at least one fungi, in particular soy bean rust, in a plant or a part of a plant by the expression of RNA which is at least partial complementary and/or identical to the STM1-gene, wherein the RNA is capable to provide ds-RNA and/or siRNA and /or miRNA. Moreover, the invention relates to respective plants, parts thereof and vector constructs capable to provide such RNA and the use of such vector constructs to provide fungal resistant plants.

Description

Methods for increasing the resistance of plants to fungi by silencing the fungal SMT1 -gene
The present invention relates to methods for generating or increasing resistance to at least one fungi, in particular soy bean rust, in a plant or a part of a plant by the expression of RNA which is at least partial complementary and/ or partial identical to the STM1 -gene, wherein the RNA is capable to provide ds-RNA and/or siRNA and /or miRNA. Moreover, the invention relates to respective plants, parts thereof and vector constructs capable to provide such RNA and the use of such vector constructs to provide fungal resistant plants.
The cultivation of agricultural crop plants serves mainly for the production of foodstuffs for humans and animals. Monocultures in particular, which are the rule nowadays, are highly susceptible to an epidemic-like spreading of diseases. The result is markedly reduced yields. To date, the pathogenic organisms have been controlled mainly by using pesticides. Nowadays, the possibility of directly modifying the genetic disposition of a plant or pathogen is also open to man.
Fungi are distributed worldwide. Approximately 100 000 different fungal species are known to date. The rusts are of great importance. They can have a complicated development cycle with up to five different spore stages (spermatium, aecidiospore, uredospore, teleutospore and basidiospore).
During the infection of plants by pathogenic fungi, different phases are usually observed. The first phases of the interaction between phytopathogenic fungi and their potential host plants are decisive for the colonization of the plant by the fungus. During the first stage of the infection, the spores become attached to the surface of the plants, germinate, and the fungus penetrates the plant. Fungi may penetrate the plant via existing ports such as stomata, lenticels, hydatodes and wounds, or else they penetrate the plant epidermis directly as the result of the mechanical force and with the aid of cell-wall-digesting enzymes. Specific infection structures are developed for penetration of the plant. The soya rust Phakopsora pachyrhizi directly penetrates the plant epidermis.
The biotrophic phytopathogenic fungi, such as many rusts, depend for their nutrition on the metabolism of live cells of the plants. The necrotrophic phytopathogenic fungi depend for their nutrition on dead cells of the plants. Soybeanrust has occupied an intermediate position since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic. After the penetration, the fungus changes over to an obligatory-biotrophic lifestyle. The subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy will, for the purposes of the present invention, be referred to as being "hemibiotrophic".
Soybean rust has become increasingly important in recent times. The disease may be caused by the pathogenic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a wide spectrum of leguminosic host plants. P. pachyrhizi, also referred to as Asian soybean rust, is the more aggressive pathogen on soybeans (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 under natural conditions and is capable of growing on further 60 species under controlled conditions (Sinclair et al. (eds.), Proceedings of the soybean rust workshop (1995), National Soybean 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 plants, four dominant genes Rpp1 -4, which mediate resistance of soya to
P. pachyrhizi, were discovered. The resistance was lost rapidly. Further, all crosses have only led to sterile progeny.
In recent years, fungal diseases, e.g. soybean rust, has gained in importance as pest in agricultural production. There was therefore a demand in the prior art for developing methods to control fungi and to provide fungal resistant plants.
Ergosterol is a component of fungal cell membranes, serving the same function that cholesterol serves in animal cells. Sterol methyltransferase (SMT1 ) is a key enzyme of the ergosterol biosynthesis pathway. It has been surprisingly found that fungal resistance can be provided by introducing recombinant nucleic acids into plant cells capable of silencing the fungal SMT1 - gene in fungi, in particular by RNAi, mi-RNA, sense and/or antisense techniques. A person skilled in the art would not have assumed that indirectly silencing the fungal SMT-1 gene by transformation of the host plant would provide fungal resistance to the plant.
The present invention provides a method for producing a plant and/or a part thereof resistant to a phythopathogenic fungus comprising providing a recombinant nucleic acid comprising a target nucleic acid that is substantial identical and/or substantial complementary to at least contiguous 19 nucleotides of the target SMT1 -sequence,
introducing said recombinant nucleic acid into in the plant and/or parts thereof.
The present invention further provides a vector construct comprising a recombinant nucleic acid comprising a promoter that is functional in the plant cell, operably linked to a target nucleic acid which is substantial identical and/or substantial complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and a terminator regulatory sequence as well as the use of the vector construct for the transformation of plants or parts thereof to provide fungal resistant plants. The present invention also provides a transgenic plant cell, plants or parts thereof comprising a recombinant nucleic acid comprising a target nucleic acid that is substantial identical and/or substantial complementary at least contiguous 19 nucleotides of the target SMT1 -gene. Parts of plants may be plant cells, roots, stems, leaves, flowers and/or seeds.
Without to be bound by this theory it is assumed that the plant is prodcing small interfering (si)RNAs from the SMT1 construct by using the pathways known in the literature (Andrew Eamens, Ming-Bo Wang, Neil A. Smith, and Peter M. Waterhouse "RNA Silencing in Plants: Yesterday, Today, and Tomorrow"Plant Physiology, June 2008, Vol. 147, pp. 456-468). Due to the close connection between the fungus and its host plant (especially at the haustoria), the siRNAs are able to move or being transported (in complexes with proteins or naked) into the fugus. In the fungus the siRNAs lead to a sequence specific siRNA mediated knock-down of the target gene (in this case SMT1 ). This process is most likely mediated and maintained by protein complexes like RISC (RNA-induced silencing complex) and RdRP (RNA dependent RNA polymerases).
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the examples included herein. Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. In addition to the definitions of terms provided herein, definitions of common terms in molecular biology may also be found in Rieger et al., 1991 Glossary of genetics: classical and molecular, 5th Ed., Berlin: Springer-Verlag; and in Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1998 Supplement). It is to be understood that as used in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.
Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in
Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979 Meth Enzymol. 68; Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101 ; Grossman and Moldave (Eds.) 1980 Meth. Enzymol. 65; Miller (Ed.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose, 1981 Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender 1979 Genetic Engineering: Principles and Methods, Vols. 1 -4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.
As used herein the terms "fungal-resistance, resistant to a fungus" and/or "fungal-resistant" mean reducing or preventing an infection by a fungus. Resistance does not imply that the plant necessarily has 100% resistance to infection. In preferred embodiments, the resistance to infection by a fungus in a resistant plant is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in comparison to a wild type plant that is not resistant to fungus. Preferably the wild type plant is a plant of a similar, more preferably identical, genotype as the plant having increased resistance to the fungus, but does not comprise a recombinant nucleic acid comprising the target nucleic acid that is substantial identical and/or complementary to at least 19 nucleotides of the target SMT1 -gene. The terms terms "fungal-resistance, "resistant to a fungus" and/or "fungal-resistant" as used herein refers to the ability of a plant, as compared to a wild type plant, to avoid infection by fungus, to kill fungus, to hamper, reduce, to stop the development, growth and/or multiplication of fungus. The level of fungal resistance of a plant can be determined in various ways, e.g. by scoring/measuring the infected leaf area in relation to the overall leaf area. Another possibility to determine the level of resistance is to count the number of fungal colonies on the plant or to measure the amount of spores produced by these colonies. Another way to resolve the degree of fungal infestation is to specifically measure the amount of fungal DNA by quantitative (q) PCR. Specific probes and primer sequences for most fungal pathogens are available in the literature (Frederick RD, Snyder CL, Peterson GL, et al. 2002 Polymerase chain reaction assays for the detection and discrimination of the soybean rust pathogens Phakopsora pachyrhizi and P-meibomiae PHYTOPATHOLOGY 92(2) 217-227).
As used herein the term "recombinant nucleic acid" refers to a DNA-molecule comprising a nucleic acid that is substantial identical and/or substantial complementary to at least 19 contiguous nucleotides of the fungal SMT1 -gene, optionally operably linked to a promoter functional in a plant cell and/or other regulatory sequences. Preferably, the recombinant nucleic acid comprises a sequence which does not naturally occur in the wildtype plant. More preferably, the recombinant nucleic acid comprises a sequence which does occur in fungi but not in plants.
As used herein the term "target nucleic acid" preferably refers to a DNA-molecule capable to prevent the expression, reduce the amount and/or function of the fungal SMT1 -protein in the plant, parts of the plant, fungus and/or parts of the fungus.
Generally, the term "substantially identical" preferably refers to DNA and/or RNA which is at least 80% identical to 19 or more contiguous nucleotides of a specific DNA or RNA sequence of the SMT1 -gen, more preferably, at least 90% identical to 19 or more contiguous nucleotides, and most preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical or absolutely identical to 19 or more contiguous nucleotides of a specific DNA or RNA- sequence of the SMT1 -gen. In particular the RNA corresponds to the coding DNA-strand of the SMT1 -gen.
As used herein, the term "substantially identical" as applied to DNA of the recombinant nucleic acid, the target nucleic acid and/or the target SMT1 -gene means that the nucleotide sequence is at least 80% identical to 19 or more contiguous nucleotides of the target SMT1 -gene, more preferably, at least 90% identical to 19 or more contiguous nucleotides of the target SMT1 - gene, and most preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical or absolutely identical to 19 or more contiguous nucleotides of the target SMT1 - gene. The term "19 or more contiguous nucleotides of the target SMT1 -gene" corresponds to the target SMT1 -gene, being at least about 19, 20, 21 , 22, 23, 24, 25, 50, 100, 200, 300, 400, 500, 1000, 1500, consecutive bases or up to the full length of the target SMT1 -gene.
As used herein, "complementary" polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other. As used herein, the term "substantially complementary" means that two nucleic acid sequences are complementary over at least at 80% of their nucleotides. Preferably, the two nucleic acid sequences are complementary over at least at 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more or all of their nucleotides. Preferably, the two nucleic acid sequences are
complementary at least about 19, 20, 21 , 22, 23, 24, 25, 50, 100, 200, 300, 400, 500, 1000, 1500, consecutive bases or up to the full length of the target SMT1 -gene. Alternatively,
"substantially complementary" means that two nucleic acid sequences can hybridize under stringency conditions.
Hybridization: The term "hybridization" as used herein includes "any process by which a strand of nucleic acid molecule joins with a complementary strand through base pairing." (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid molecules) is impacted by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acid molecules. As used herein, the term "Tm" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double- stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acid molecules is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation:
Tm=81 .5+0.41 (% G+C), when a nucleic acid molecule is in aqueous solution at 1 M NaCI [see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)]. Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of Tm. Stringent conditions, are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6.
In particular, the term stringency conditions refers to conditions, wherein 100 contigous nucleotides or more, 150 contigous nucleotides or more, 200 contigous nucleotides or more or 250 contigous nucleotides or more which are a fragment or identical to the complementary nucleic acid molecule (DNA, RNA, ssDNA orssRNA) hybridizes under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 2 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C with a specific nucleic acid molecule (DNA; RNA, ssDNA or ss RNA). Preferably, the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C, more preferably the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0,1 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C. Preferably, the complementary nucleotides hybridize with a fragment or the whole SMT1 - target gene. Preferably, the complementary polynucleotide hybridizes with parts of the SMT1 - target gene capable to provide fungal resistance. In particular, the complementary
polynucleotide hybridizes with the coding strand of the SMT1 -gene or a part thereof.
As used herein a RNA complementary to a DNA means that the sequence of the RNA is complementary to the coding strand of the DNA.
Preferably two complementary RNAs are reverse complementary to each other, i.e. form dsRNA.
As used herein, the term "target SMT1 -gene" means fungal steroyl-methyl-transferase-genes including any homolog of the sterol methyl transferase. In particular, the term SMT1 -gene refers to a gene having at least 60 % identity with SEQ-ID-No. 1 , SEQ-ID-No. 2 or SEQ-ID-No. 3 or with a sequence coding for a protein having SEQ-ID-No. 5. In one embodiment
homologues of the SMT1 -gene have, at the DNA level or protein level, at least 70%, preferably of at least 80%, especially preferably of at least 90%, quite especially preferably of at least 95%, quite especially preferably of at least 98% or 100% identity over the entire DNA region or protein region given in a sequence specifically disclosed herein.
"Identity" or "complementarity" between two nucleic acids refers preferably in each case over the entire length of the nucleic acid.
For example the identity may be calculated by means of the Vector NTI Suite 7.1 program of the company Informax (USA) employing the Clustal Method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr; 5(2): 151 -1 ) with the following settings:
Multiple alignment parameter:
Gap opening penalty 10
Gap extension penalty 10
Gap separation penalty range 8
Gap separation penalty off
% identity for alignment delay 40
Residue specific gaps off
Hydrophilic residue gap off
Transition weighing 0
Pairwise alignment parameter:
FAST algorithm on
K-tuple size 1
Gap penalty 3
Window size 5
Number of best diagonals 5
Alternatively the identity may be determined according to Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment with the Clustal series of programs. (2003) Nucleic Acids Res 31 (13):3497-500, the web page: http://www.ebi.ac.Uk/Tools/clustalw/index.html# and the following settings
DNA Gap Open Penalty 15.0
DNA Gap Extension Penalty 6.66
DNA Matrix Identity
Protein Gap Open Penalty 10.0
Protein Gap Extension Penalty 0.2
Protein matrix Gonnet
Protein/DNA ENDGAP -1
Protein/DNA GAPDIST 4
The complementarity may be calculated as the identity. However, complementary means that purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. All the nucleic acid sequences mentioned herein (single-stranded and double-stranded DNA and RNA sequences, for example cDNA and mRNA) can be produced in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix. Chemical synthesis of oligonucleotides can, for example, be performed in a known way, by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and filling of gaps by means of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989), see below.
There is general agreement that in many organisms, including fungi and plants, large pieces of dsRNA complementary to a specific genes are cleaved into 19-24 nucleotide fragments (siRNA) within cells, and that these siRNAs are the actual mediators for silencing the specific target gene. As used herein siRNA refers to 19-24 nucleotide fragments complementary to the target SMT1 -gen.
There are several possibilities to provide the si-RNA: RNA-interference (RNAi), micro-RNAi (mi- RNA), sense RNA and/or antisense RNA.
As used herein, "RNAi" or "RNA interference" refers to the process of sequence-specific post- transcriptional gene silencing, mediated by double-stranded RNA (dsRNA). In the RNAi process, dsRNA comprising a first strand that is substantially complementary to at least 19 comtiguous nucleotides of the target SMT1 -gene and a second strand that is complementary to the first strand at least partially has to be provided. For this purpose a recombinant nucleic acid is introduced into the plant, which is capble to produce such dsRNA. The target SMT1 -gene- specific dsRNA is produced and processed into relatively small fragments (siRNAs) and can subsequently become distributed from the plant to the fungus. miRNA refers to a similar process, except that the produced dsRNA only partially comprises regions substantially identical to the SMT1 -gene (at least 19 contiguous nucleotides).
As used herein, "antisense interference" refers to the process of sequence-specific post- transcriptional gene silencing, probably also mediated by double-stranded RNA (dsRNA). In the antisenseRNA-process, ssRNA comprising a first strand that is substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene has to be provided. For this purpose recombinant nucleic acid is introduced into the plant, which is capable to produce such ssRNA. Without to be bound by the theory, it is assumed that this RNA moves from the plant to the fungus and subsequently pairs with complementary ssRNA transcribed from the original SMT1 - gene produced by the original SMT1 -gene. The resulting dsRNA is processed into relatively small fragments (siRNAs) and can subsequently become distributed from the plant to the fungus.
As disclosed herein, 100% sequence identity between the target nucleic acid and the target gene is not required to practice the present invention. Preferably, the target nucleic acid comprises a 19-nucleotide portion which is substantially identical and/or substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene. While a target nucleic acid comprising a nucleotide sequence identical and/or identical to a portion of the fungal target SMT1 -gene and/or complementary to the whole sequence and/or a portion of the fungal target SMT1 -gene is preferred for inhibition, the invention can tolerate sequence variations that might be expected due to gene manipulation or synthesis, genetic mutation, strain polymorphism, or evolutionary divergence. Thus the target nucleic acid may also encompass a mismatch with the target SMT1 -gene of at least 1 , 2, or more nucleotides. For example, it is contemplated in the present invention that within 21 contiguous nucleotides the target nucleic acid may contain an addition, deletion or substitution of 1 , 2, or more nucleotides, so long as the resulting RNA sequence still interferes with the fungal target SMT1 -gene function.
Sequence identity between the recombinant nucleic acid useful according to the present invention and the fungal SMT1 -target gene may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991 , and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 80 % sequence identity, 90% sequence identity, or even 100% sequence identity, between the target nucleic acid and at least 19 contiguous nucleotides of the target gene is preferred. The same preferably applies for the sequence complementarity.
When the target nucleic acid of the invention has a length longer than about 19 nucleotides, for example from about 50 nucleotides to about 500 nucleotides, the corresponding ds RNA provided therefrom will be cleaved randomly to dsRNAs of about 21 nucleotides within the plant or fungal cell: the siRNAs. Multiple specialized Dicers in plants may generate siRNAs typically ranging in size from 19nt to 24nt (See Henderson et al., 2006. Nature Genetics 38:721 -725.). The cleavage of a longer dsRNA of the invention may yield a pool of 21 mer dsRNAs, derived from the longer dsRNA. The siRNAs may have sequences corresponding to fragments of 19-24 contiguous nucleotides across the entire sequence of the fungal target SMT1 -gene. One of skill in the art would recognize that the siRNA can have a mismatch with the target gene of at least 1 , 2, or more nucleotides. Further, these mismatches are intended to be included in the present invention.
In one embodiment the target nucleic acid is substantial identical and/or substantial
complementary over a length of at least 19, at least 50, at least 100, at least 200, at least 300, at least 400 or at least 500 nucleotides to the target SMT1 -gene. In particular, the target nucleic acid may comprise 19 to 500, preferably 50 to 500, more preferably 250 to 350 nucleotides, wherein preferably at least about 19, 20, 21 , 22, 23, 24, 25, 50, 100, 200, 300, 400, consecutive bases or up to the full length of target nucleic acid are identical and/or complementary and/or identical to the target SMT1 -gene.
Preferably, the recombinant nucleic acid is able to provide dsRNA and/or siRNA and/or miRNA in the plant, a part thereof and/or the fungus infecting the plant or a part thereof once the recombinant nucleic acid is expressed in the plant, wherein preferably at least 19 contiguous nucleotides of the dsRNA and/or si RNA and/or miRNA are substantially complementary to the target SMT1 -gene.
One embodiment according to the present invention, provides a method for producing a plant and/or a part thereof resistant to a fungus, wherein the recombinant nucleic acid comprises a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which is substantial identical and/or substantial complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and a second strand having a sequence at substantially complementary to the first strand and/or parts thereof, and
a terminator regulatory sequence.
The first strand and the second strand may at least partially form dsRNA.
This technique is also refered to as RNAi. In another embodiment the target nucleic acid comprises 19 to 24 contiguous nucleotides of the target sequence which are substantially identical and/or substantially complementary to the target SMT1 -gene and the remaining nucleotides of the target nucleic acid are not identical and/or not complementary to the target SMT1 -gene. Not-identical means an identity which is lower than 95%, lower that 90%, lower than 80%, lower than 70 %, lower than 60% over the whole sequence of the target nucleic acid. Not-complementary means an complementarity which is lower than 95%, lower that 90%, lower than 80%, lower than 70 %, lower than 60% over the whole sequence of the target nucleic acid. This technique is also refered to as miRNA.
One embodiment according to the present invention, provides a method for producing a plant and/or a part thereof resistant to a fungus, wherein the recombinant nucleic acid comprises a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary to at least contiguous 19 nucleotides of the target SMT1 -gene, and
a terminator regulatory sequence. Preferably, the first strand generated in the plant forms dsRNA together with a second RNA- strand generated in the fungus which is complementary to the first strand. This technique is also refered to as antisense RNA. One embodiment according to the present invention, provides a method for producing a plant and/or a part thereof resistant to a fungus, wherein the recombinant nucleic acid comprises a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially identical to at least contiguous 19 nucleotides of the target SMT1 - gene, and
a terminator regulatory sequence.
Preferably, the first strand generated in the plant forms dsRNA together with a second RNA- strand generated in the fungus which is complementary to the first strand. This technique is also refered to as sense RNA.
The dsRNA of the invention may optionally comprise a single stranded overhang at either or both ends. Preferably, the single stranded overhang comprises at least two nucleotides at the 3' end of each strand of the dsRNA molecule. The double-stranded structure may be formed by a single self-complementary RNA strand (i.e. forming a hairpin loop) or two complementary RNA strands. RNA duplex formation may be initiated either inside the plant or inside the fungus. When the dsRNA of the invention forms a hairpin loop, it may optionally comprise an intron, as set forth in US 2003/0180945A1 or a nucleotide spacer, which is a stretch of sequence between the complementary RNA strands to stabilize the hairpin transgene in cells. Methods for making various dsRNA molecules are set forth, for example, in WO 99/53050 and in U.S. Pat. No.
6,506,559.
The term "plant" is intended to encompass plants at any stage of maturity or development, as well as any tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context. Plant parts include, but are not limited to, plant cells, stems, roots, flowers, ovules, stamens, seeds, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, hairy root cultures, and/or the like. The present invention also includes seeds produced by the plants of the present invention. In one embodiment, the seeds are true breeding for an increased resistance to fungal infection as compared to a wild-type variety of the plant seed. As used herein, a "plant cell" includes, but is not limited to, a protoplast, gamete producing cell, and a cell that regenerates into a whole plant. Tissue culture of various tissues of plants and regeneration of plants therefrom is well known in the art and is widely published.
As used herein, the term "transgenic" refers to any plant, plant cell, callus, plant tissue, or plant part that contains all or part of at least one recombinant polynucleotide comprising at least 19 contiguous nucleotides which are substantial identical and/or substantial complementary to the SMT1 -gene. Preferably, all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations. In one embodiment of the present invention the plant is selected from the group consisting of beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, and/or groundnut. Preferably the plant is a legume, comprising plants of the genus Phaseolus (comprising French bean, dwarf bean, climbing bean (Phaseolus vulgaris), Lima bean (Phaseolus lunatus L), Tepary bean (Phaseolus acutifolius A. Gray), runner bean (Phaseolus coccineus)); the genus Glycine (comprising Glycine soja, soybeans (Glycine max (L.) Merill)); pea (Pisum) (comprising shelling peas (Pisum sativum L. convar. sativum), also called smooth or round-seeded peas; marrowfat pea (Pisum sativum L. convar. medullare Alef. emend. CO. Lehm), sugar pea (Pisum sativum L. convar. axiphium Alef emend. CO. Lehm), also called snow pea, edible-podded pea or mangetout, (Pisum granda sneida L. convar. sneidulo p. shneiderium)); peanut (Arachis hypogaea), clover (Trifolium spec), medick (Medicago), kudzu vine (Pueraria lobata), common lucerne, alfalfa (M. sativa L.), chickpea (Cicer), lentils (Lens) (Lens culinaris Medik.), lupins (Lupinus); vetches (Vicia), field bean, broad bean (Vicia faba), vetchling (Lathyrus) (comprising chickling pea (Lathyrus sativus), heath pea (Lathyrus tuberosus)); genus Vigna (comprising moth bean (Vigna aconitifolia (Jacq.) Marechal), adzuki bean (Vigna angularis (Willd.) Ohwi & H. Ohashi), urd bean (Vigna mungo (L.) Hepper), mung bean (Vigna radiata (L.) R. Wilczek), bambara groundnut (Vigna subterrane (L.) Verde), rice bean (Vigna umbellata (Thunb.) Ohwi & H. Ohashi), Vigna vexillata (L.) A. Rich., Vigna unguiculata (L.) Walp., in the three subspecies asparagus bean, cowpea, catjang bean)); pigeonpea (Cajanus cajan (L.) Millsp.), the genus Macrotyloma (comprising geocarpa groundnut (Macrotyloma geocarpum (Harms) Marechal & Baudet), horse bean (Macrotyloma uniflorum (Lam.) Verde.)); goa bean (Psophocarpus tetragonolobus (L.) DC), African yam bean (Sphenostylis stenocarpa (Hochst. ex A. Rich.) Harms), Egyptian black bean, dolichos bean, lablab bean (Lablab purpureus (L.) Sweet), yam bean (Pachyrhizus), guar bean (Cyamopsis tetragonolobus (L.) Taub.); and/or the genus Canavalia (comprising jack bean (Canavalia ensiformis (L.) DC), sword bean (Canavalia gladiata (Jacq.) DC.)).
In one embodiment according to the present invention the fungal resistance is a resistance against a biotrophic fungus, preferably a hemibiotrophic fungus. In preferred embodiments of the present invention, the biotrophic fungus is selected from the group Basidiomycota, preferably the Uredinales (rusts), especially preferably the Melompsoraceae, and in particular the genus Phakopsora. In especially preferred embodiments, the pathogen is Phakopsora pachyrhizi and/or P. meibomiae (together also referred to as "soybean rust" or "soya rust"). Preference is being given to the former. When the pathogen is selected from the group of the biotrophic pathogens or fungi, it is preferred in some embodiments that the pathogen is other than powdery mildew or downy mildew.
Further, the present invention provides a vector construct comprising a promoter that is functional in the plant cell, operably linked to a target nucleic acid which is substantially identical and/or substantially complementary to at least 19 contiguous nucleotides of the target SMT1 - gene and a terminator regulatory sequence. The expression vector may be isolated.
In one embodiment the vector construct comprises a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which is substantial identical and/or substantial complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and a second strand having a sequence at substantially complementary to the first strand or parts thereof, and a terminator regulatory sequence.
It is preferred that first strand and the second strand are capable of hybridizing to form dsRNA at least partially.
In another embodiment the vector construct comprises a promoter that is functional in the plant cell, operably linked to a target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary or identical to at least 19 contiguous nucleotides of the target SMT1 -gene, and
a terminator regulatory sequence.
It is preferred that the transcript of the first strand and at least a part of the transcript of the fungal SMT1 -gene are capable of hybridizing to form dsRNA at least partially.
In one embodiment the vector construct comprises a target nucleic acid comprising 19 to 500 nucleotides. Further variants of the target nucleic acid are defined in the section referring to the method for producing a plant. With respect to a vector construct and/or the recombinant nucleic acid, the term "operatively linked" is intended to mean that the target nucleic acid is linked to the regulatory sequence, including promotors, terminator regulatory sequences, enhancers and/or other expression control elements (e.g., polyadenylation signals), in a manner which allows for expression of the target nucleic acid (e.g., in a host plant cell when the vector is introduced into the host plant cell). Such regulatory sequences are described, for example, in Goeddel, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, Eds. Glick and
Thompson, Chapter 7, 89-108, CRC Press: Boca Raton, Florida, including the references therein. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of dsRNA desired, and the like. The vector constructs of the invention can be introduced into plant host cells to thereby produce ssRNA, dsRNA, siRNA and/or mi RNA in order to prevent and/or reduce fungal infections.
In one embodiment, the vector construct comprises a promotor operatively linked to a target nucleotide that is a template for one or both strands of the ssRNA- or dsRNA molecules at least substantial complementary to 19 contiguous nucleotides of the target SMT1 -gen.
In one embodiment, the nucleic acid molecule further comprises two promoters flanking either end of the nucleic acid molecule, wherein the promoters drive expression of each individual DNA strand, thereby generating two complementary RNAs that hybridize and form the dsRNA. In alternative embodiments, the nucleotide sequence is transcribed into both strands of the dsRNA on one transcription unit, wherein the sense strand is transcribed from the 5' end of the transcription unit and the antisense strand is transcribed from the 3' end, wherein the two strands are separated by about 3 to about 500 base pairs, and wherein after transcription, the RNA transcript folds on itself to form a hairpin.
In another embodiment, the vector contains a bidirectional promoter, driving expression of two nucleic acid molecules, whereby one nucleic acid molecule codes for the sequence
substantially identical to a portion of SMT1 -gene gene and the other nucleic acid molecule codes for a second sequence being substantially complementary to the first strand and capable of forming a dsRNA, when both sequences are transcribed. A bidirectional promoter is a promoter capable of mediating expression in two directions.
In another embodiment, the vector contains two promoters, one mediating transcription of the sequence substantially identical to a portion of a SMT1 gene and another promoter mediating transcription of a second sequence being substantially complementary to the first strand and capable of forming a dsRNA, when both sequences are transcribed. The second promoter might be a different promoter. A different promoter means a promoter having a different activity in regard to cell or tissue specificity, or showing expression on different inducers for example, pathogens, abiotic stress or chemicals.
Promoters according to the present invention may be constitutive, inducible, in particular pathogen-induceable, developmental stage-preferred, cell type-preferred, tissue-preferred or organ-preferred. Constitutive promoters are active under most conditions. Non-limiting examples of constitutive promoters include the CaMV 19S and 35S promoters (Odell et al., 1985, Nature 313:810-812), the sX CaMV 35S promoter (Kay et al., 1987, Science 236:1299- 1302), the Sep1 promoter, the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171 ), the Arabidopsis actin promoter, the ubiquitin promoter (Christensen et al., 1989, Plant Molec. Biol. 18:675-689); pEmu (Last et al., 1991 , Theor. Appl. Genet. 81 :581 -588), the figwort mosaic virus 35S promoter, the Smas promoter (Velten et al., 1984, EMBO J. 3:2723-2730), the GRP1 - 8 promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Patent No. 5,683,439), promoters from the T-DNA of Agrobacterium, such as mannopine synthase, nopaline synthase, and octopine synthase, the small subunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter, and/or the like. Promoters that express the dsRNA in a cell that is contacted by fungus are preferred. Alternatively, the promoter may drive expression of the dsRNA in a plant tissue remote from the site of contact with the fungus, and the dsRNA may then be transported by the plant to a cell that is contacted by the fungus, in particular cells of, or close by fungal infected sites.
Preferably, the expression vector of the invention comprises a constitutive promoter, root- specific promoter, a pathogen inducible promoter, or a fungal-inducible promoter. A promoter is inducible, if its activity, measured on the amount of RNA produced under control of the promoter, is at least 30%, 40%, 50% preferably at least 60%, 70%, 80%, 90% more preferred at least 100%, 200%, 300% higher in its induced state, than in its un-induced state. A promoter is cell-, tissue- or organ-specific, if its activity , measured on the amount of RNA produced under control of the promoter, is at least 30%, 40%, 50% preferably at least 60%, 70%, 80%, 90% more preferred at least 100%, 200%, 300% higher in a particular cell-type, tissue or organ, then in other cell-types or tissues of the same plant, preferably the other cell-types or tissues are cell types or tissues of the same plant organ, e.g. a root. In the case of organ specific promoters, the promoter activity has to be compared to the promoter activity in other plant organs, e.g. leaves, stems, flowers or seeds.
Developmental stage-preferred promoters are preferentially expressed at certain stages of development. Tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds, or xylem. Examples of tissue preferred and organ preferred promoters include, but are not limited to fruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred, integument-preferred, tuber-preferred, stalk-preferred, pericarp-preferred, leaf-preferred, stigma-preferred, pollen-preferred, anther- preferred, a petal-preferred, sepal-preferred, pedicel-preferred, silique-preferred, stem- preferred, root-preferred promoters and/or the like. Seed preferred promoters are preferentially expressed during seed development and/or germination. For example, seed preferred promoters can be embryo-preferred, endosperm preferred and seed coat-preferred. See Thompson et al., 1989, BioEssays 10:108. Examples of seed preferred promoters include, but are not limited to cellulose synthase (celA), Cim1 , gamma-zein, globulin-1 , maize 19 kD zein (CZ19B1 ) and/or the like.
Other suitable tissue-preferred or organ-preferred promoters include, but are not limited to, the napin-gene promoter from rapeseed (U.S. Patent No. 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al., 1991 , Mol Gen Genet. 225(3):459-67), the oleosin-promoter from Arabidopsis (PCT Application No. WO 98/45461 ), the phaseolin-promoter from Phaseolus vulgaris (U.S. Patent No. 5,504,200), the Bce4-promoter from Brassica (PCT Application No. WO 91/13980), or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2(2):233-9), as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice, etc. Suitable promoters to note are the Ipt2 or Ipt1 -gene promoter from barley (PCT Application No. WO 95/15389 and PCT Application No. WO
95/23230) or those described in PCT Application No. WO 99/16890 (promoters from the barley hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oat glutelin gene, Sorghum kasirin-gene, and/or rye secalin gene)
Promoters useful according to the invention include, but are not limited to, are the major chlorophyll a/b binding protein promoter, histone promoters, the Ap3 promoter, the β-conglycin promoter, the napin promoter, the soybean lectin promoter, the maize 15kD zein promoter, the 22kD zein promoter, the 27kD zein promoter, the g-zein promoter, the waxy, shrunken 1 , shrunken 2, bronze promoters, the Zm13 promoter (U.S. Patent No. 5,086,169), the maize polygalacturonase promoters (PG) (U.S. Patent Nos. 5,412,085 and 5,545,546), the SGB6 promoter (U.S. Patent No. 5,470,359), as well as synthetic or other natural promoters.
Epidermisspezific promotors may be seleted from the group consisting of:
- WIR5 (=GstA1 ); acc. X56012; Dudler & Schweizer,
- GLP4, acc. AJ310534; Wei Y., Zhang Z., Andersen C.H., Schmelzer E., Gregersen P.L., Collinge D.B., Smedegaard-Petersen V. and Thordal-Christensen H., Plant Molecular Biology 36, 101 (1998),
- GLP2a, acc. AJ237942, Schweizer P., Christoffel A. and Dudler R., Plant J. 20, 541 (1999);
Prx7, acc. AJ003141 , Kristensen B.K., Ammitzboll H., Rasmussen S.K. and Nielsen K.A., Molecular Plant Pathology, 2(6), 31 1 (2001 );
- GerA, acc. AF250933; Wu S., Druka A., Horvath H., Kleinhofs A., Kannangara G. and von Wettstein D., Plant Phys Biochem 38, 685 (2000);
- OsROCI , acc. AP004656
- RTBV, acc. AAV62708, AAV62707; Kloti A., Henrich C, Bieri S., He X., Chen G., Burkhardt P.K., Wunn J., Lucca P., Hohn T., Potrykus I. and Futterer J., PMB 40, 249 (1999);
- Chitinase ChtC2-Promotor from potato (Ancillo et al., Planta. 217(4), 566, (2003));
- AtProT3 Promotor (Grallath et al., Plant Physiology. 137(1 ), 1 17 (2005));
SHN-Promotors from Arabidopsis (AP2/EREBP transcription factors involved in cutin and wax production) (Aaron et al., Plant Cell. 16(9), 2463 (2004)); and/or
- GSTA1 from wheat (Dudler et al., WP2005306368 and Altpeter et al., Plant Molecular Biology. 57(2), 271 (2005)).
Mesophyllspezific promotors may be seleted from the group consisting of: - PPCZml (=PEPC); Kausch A.P., Owen T.P., Zachwieja S.J., Flynn A.R. and Sheen J., Plant Mol. Biol. 45, 1 (2001 );
- OsrbcS, Kyozuka et al., PlaNT Phys 102, 991 (1993); Kyozuka J., McElroy D., Hayakawa T., Xie Y., Wu R. and Shimamoto K., Plant Phys. 102, 991 (1993);
- OsPPDK, acc. AC099041 ;
- TaGF-2.8, acc. M63223; Schweizer P., Christoffel A. and Dudler R., Plant J. 20, 541
(1999);
- TaFBPase, acc. X53957;
- TaWISI , acc. AF467542; US 2002201 15849; HvBISI , acc. AF467539; US 2002201 15849;
ZmMISI , acc. AF467514; US 2002201 15849;
HvPRI a, acc. X74939; Bryngelsson et al., Mol. Plant Microbe Interact!. 7 (2), 267 (1994); HvPRI b, acc. X74940; Bryngelsson et al., Mol. Plant Microbe Interact. 7(2), 267 (1994); HvB1 ,3gluc; acc. AF479647;
HvPrx8, acc. AJ276227; Kristensen et al., Molecular Plant Pathology, 2(6), 31 1 (2001 ); and/or
HvPAL, acc. X97313; Wei Y., Zhang Z., Andersen C.H., Schmelzer E., Gregersen P.L., Collinge D.B., Smedegaard-Petersen V. and Thordal-Christensen H. Plant Molecular Biology 36, 101 (1998).
Constitutve promotors may be selected from the group consisting of - PcUbi promoter from parsley (WO 03/102198)
- CaMV 35S promoter: Cauliflower Mosaic Virus 35S promoter (Benfey et al. 1989 EMBO J. 8(8): 2195-2202),
STPT promoter: Arabidopsis thaliana Short Triose phosphat translocator promoter
(Accession NM_123979)
- Act1 promoter: - Oryza sativa actin 1 gene promoter (McElroy et al. 1990 PLANT CELL 2(2) 163-171 a) and/or
- EF1A2 promoter: Glycine max translation elongation factor EF1 alpha (US 20090133159).
One type of vector construct is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vector constructs are capable of autonomous replication in a host plant cell into which they are introduced. Other vector constructs are integrated into the genome of a host plant cell upon introduction into the host cell, and thereby are replicated along with the host genome. In particular the vector construct is capable of directing the expression of gene to which the vectors is operatively linked. However, the invention is intended to include such other forms of expression vector constructs, such as viral vectors (e.g., potato virus X, tobacco rattle virus, and/or Gemini virus), which serve equivalent functions. A preferred vector construct comprises sequences selected from the group consisting of SEQ- ID-1 , 2, 3, 4, 6, 7, 8, 9 and/or 10.
The present invention further provides a transgenic plant cell comprising a recombinant nucleic acid comprising a target nucleic acid that is substantial identical and/or substantially complementary to at least contiguous 19 nucleotides of the target SMT1 -gene. The present invention further provides a transgenic plant or parts thereof comprising the above transgenic plant cells according or consisting thereof. The present invention also provides transgenic seeds derived from the plant comprising the target nucleic acid. Parts of the plant may be root, leaves and/or flowers.
The transgenic plant cells may be transformed with one of the above described vector constructs. Suitable methods for transforming or transfecting host cells including plant cells are well known in the art of plant biotechnology. Any method may be used to transform the recombinant expression vector into plant cells to yield the transgenic plants of the invention. General methods for transforming dicotyledenous plants are disclosed, for example, in U.S. Pat. Nos. 4,940,838; 5,464,763, and the like. Methods for transforming specific dicotyledenous plants, for example, cotton, are set forth in U.S. Pat. Nos. 5,004,863; 5,159,135; and 5,846,797. Soybean transformation methods are set forth in U.S. Pat. Nos. 4,992,375; 5,416,01 1 ;
5,569,834; 5,824,877; 6,384,301 and in EP 0301749B1 may be used. Transformation methods may include direct and indirect methods of transformation. Suitable direct methods include polyethylene glycol induced DNA uptake, liposome-mediated transformation (US 4,536,475), biolistic methods using the gene gun (Fromm ME et al., Bio/Technology. 8(9):833-9, 1990;
Gordon-Kamm et al. Plant Cell 2:603, 1990), electroporation, incubation of dry embryos in DNA- comprising solution, and microinjection. In the case of these direct transformation methods, the plasmids used need not meet any particular requirements. Simple plasmids, such as those of the pUC series, pBR322, M13mp series, pACYC184 and the like can be used. If intact plants are to be regenerated from the transformed cells, an additional selectable marker gene is preferably located on the plasmid. The direct transformation techniques are equally suitable for dicotyledonous and monocotyledonous plants.
Transformation can also be carried out by bacterial infection by means of Agrobacterium (for example EP 0 1 16 718), viral infection by means of viral vectors (EP 0 067 553; US 4,407,956; WO 95/34668; WO 93/03161 ) or by means of pollen (EP 0 270 356; WO 85/01856; US
4,684,61 1 ). Agrobacterium based transformation techniques (especially for dicotyledonous plants) are well known in the art. The Agrobacterium strain (e.g., Agrobacterium tumefaciens or Agrobacterium rhizogenes) comprises a plasmid (Ti or Ri plasmid) and a T-DNA element which is transferred to the plant following infection with Agrobacterium. The T-DNA (transferred DNA) is integrated into the genome of the plant cell. The T-DNA may be localized on the Ri- or Ti- plasmid or is separately comprised in a so-called binary vector. Methods for the Agrobacterium- mediated transformation are described, for example, in Horsch RB et al. (1985) Science
225:1229. The Agrobacterium-mediated transformation is best suited to dicotyledonous plants but has also been adapted to monocotyledonous plants. The transformation of plants by
Agrobacteria is described in, for example, White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, Vol. 1 , Engineering and Utilization, edited by S.D. Kung and R. Wu,
Academic Press, 1993, pp. 15 - 38; Jenes B et al. Techniques for Gene Transfer, Transgenic Plants, Vol. 1 , Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 128-143; Potrykus (1991 ) Annu Rev Plant Physiol Plant Molec Biol 42:205- 225.
Transformation may result in transient or stable transformation and expression. Although a nucleotide sequence of the present invention can be inserted into any plant and plant cell falling within these broad classes, it is particularly useful in crop plant cells. The transgenic plants of the invention may be crossed with similar transgenic plants or with transgenic plants lacking the nucleic acids of the invention or with non-transgenic plants, using known methods of plant breeding, to prepare seeds. Further, the transgenic plant cells or plants of the present invention may comprise, and/or be crossed to another transgenic plant that comprises one or more nucleic acids, thus creating a "stack" of transgenes in the plant and/or its progeny. The seed is then planted to obtain a crossed fertile transgenic plant comprising the nucleic acid of the invention. The crossed fertile transgenic plant may have the particular expression cassette inherited through a female parent or through a male parent. The second plant may be an inbred plant. The crossed fertile transgenic may be a hybrid. Also included within the present invention are seeds of any of these crossed fertile transgenic plants. The seeds of this invention can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plant lines comprising the recombinant nucleic acid comprising at least 19 contiguous nucleotides of the target SMT1 -gene.
In one embodiment the transgenic is a legume, preferably selected from the group consisting of beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, and/or groundnut. According to the present invention, the introduced recombinant nucleic acid may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes. Alternatively, the introduced recombinant nucleic acid may be present on an extra-chromosomal non-replicating vector construct and be transiently expressed or transiently active. Whether present in an extra-chromosomal non-replicating vector construct or a vector construct that is integrated into a chromosome, the recombinant nucleic acid preferably resides in a plant expression cassette. A plant expression cassette preferably contains regulatory sequences capable of driving gene expression in plant cells that are operatively linked so that each sequence can fulfill its function, for example, termination of transcription by polyadenylation signals. Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al., 1984, EMBO J. 3:835) or functional equivalents thereof, but also all other terminators functionally active in plants are suitable. As plant gene expression is very often not limited on transcriptional levels, a plant expression cassette preferably contains other operatively linked sequences like translational enhancers such as the overdrive-sequence containing the 5'-untranslated leader sequence from tobacco mosaic virus enhancing the polypeptide per RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-871 1 ). Examples of plant expression vectors include those detailed in: Becker, D. et al., 1992, New plant binary vectors with selectable markers located proximal to the left border, Plant Mol. Biol. 20:1 195- 1 197; Bevan, M.W., 1984, Binary Agrobacterium vectors for plant transformation, Nucl. Acid. Res. 12:871 1 -8721 ; and Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds.: Kung and R. Wu, Academic Press, 1993, S. 15-38.
According to the present invention the target nucleic acid is capable to reduce the protein quantity or function of the SMT1 -protein in plants cell and/or the fungus. In preferred embodiments, the decrease in the protein quantity or function of the SMT1 -protein takes place in a constitutive or tissue-specific manner. In especially preferred embodiments, an essentially pathogen-induced decrease in the protein quantity or protein function takes place, for example by recombinant expression of the target nucleic acid under the control of a fungal-induceable promoter. In particular, the the expression of the target nucleic acid takes place on fungal infected sites, where, however, preferably the expression of the target nucleic acid sequence remains essentially unchanged in tissues not infected by fungus. In preferred embodiments, the protein amount of the SMT1 protein in the plant and/or the fungus is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the target nucleic acid. Preferably the wild type plant is a plant of a similar, more preferably identical genotype as the plant transformed with the target nucleic acid. Preferably term "SMT1 -protein" means fungal steroyl-methyl-transferase-proteins
including any homolog of the sterol methyl transferase. In particular, the term SMT1 -protein refers to a protein having at least 60 % identity with SEQ-ID-No. 5. In one embodiment homologues of the SMT1 -protein have at least 70%, preferably of at least 80%, especially preferably of at least 90%, quite especially preferably of at least 95%, quite especially preferably of at least 98% or 100% identity to SEQ-ID-5 preferably over the entire protein region.
The present invention further provides the use of the vector construct according to the present invention for the transformation of plants to provide fungal resistant plants. The fungal pathogens or fungus-like pathogens (such as, for example, Chromista) preferably belong to the group comprising Plasmodiophoramycota, Oomycota, Ascomycota,
Chytridiomycetes, Zygomycetes, Basidiomycota and/or Deuteromycetes (Fungi imperfecti). Pathogens which may be mentioned by way of example, but not by limitation, are those detailed in Tables 1 to 4, and the diseases which are associated with them.
Table 1 : Diseases caused by biotrophic phytopathogenic fungi
Disease Pathogen
Leaf rust Puccinia recondita
Yellow rust P. striiformis
Powdery mildew Erysiphe graminis / Blumeria graminis
Rust (common corn) Puccinia sorghi
Rust (Southern corn) Puccinia polysora
Tobacco leaf spot Cercospora nicotianae Rust (soybean) Phakopsora pachyrhizi, P. meibomiae
Rust (tropical corn) Physopella pallescens, P. zeae =
Angiopsora zeae
Table 2: Diseases caused by necrotrophic and/or hemibiotrophic fungi and Oomycetes
Disease Pathogen
Plume blotch Septoria (Stagonospora) nodorum
Leaf blotch Septoria tritici
Ear fusarioses Fusarium spp.
Eyespot Pseudocercosporella herpotrichoides
Smut Ustilago spp.
Late blight Phytophthora infestans
Bunt Tilletia caries
Take-all Gaeumannomyces graminis
Anthrocnose leaf blight Colletotrichum graminicola (teleomorph:
Glomerella graminicola Politis); Glomerella
Anthracnose stalk rot tucumanensis (anamorph: Glomerella falcatum
Went)
Aspergillus ear and Aspergillus flavus
kernel rot
Banded leaf and sheath spot Rhizoctonia solani Kuhn = Rhizoctonia
("Wurzeltoter") microsclerotia J. Matz (telomorph:
Thanatephorus cucumeris)
Black bundle disease Acremonium strictum W. Gams =
alosporium acremonium Auct. non Corda
Black kernel rot Lasiodiplodia theobromae =
Botryodiplodia theobromae
Borde bianco Marasmiellus sp.
Brown spot (black spot, stalk rot) Physoderma maydis
Cephalosporium kernel rot Acremonium strictum = Cephalosporium
acremonium
Charcoal rot Macrophomina phaseolina
Corticium ear rot Thanatephorus cucumeris =
Corticium sasakii Disease Pathogen
Curvularia leaf spot Curvularia clavata, C. eragrostidis, = C.
maculans (teleomorph: Cochliobolus eragrostidis), Curvularia inaequalis, C.
intermedia (teleomorph: Cochliobolus intermedius), Curvularia lunata (teleomorph: Cochliobolus lunatus), Curvularia pallescens (teleomorph: Cochliobolus pallescens), Curvularia senegalensis, C. tuberculata (teleomorph: Cochliobolus tuberculatus)
Didymella leaf spot Didymella exitalis
Diplodia ear and stalk rot Diplodia frumenti (teleomorph: Botryosphaeria festucae)
Diplodia ear and stalk rot, seed rot Diplodia maydis =
and seedling blight Stenocarpella maydis
Diplodia leaf spot or streak Stenocarpella macrospora =
Diplodialeaf macrospora
Brown stripe downy Sclerophthora rayssiae var. zeae
mildew
Crazy top downy mildew Sclerophthora macrospora =
Sclerospora macrospora
Green ear downy mildew (graminicola Sclerospora graminicola
downy mildew)
Dry ear rot (cob, Nigrospora oryzae
kernel and stalk rot) (teleomorph: Khuskia oryzae)
Ear rots (minor) Alternaria alternata = A. tenuis,
Aspergillus glaucus, A. niger,
Aspergillus spp., Botrytis cinerea (teleomorph:
Botryotinia fuckeliana), Cunninghamella sp.,
Curvularia pallescens,
Doratomyces stemonitis =
Cephalotrichum stemonitis,
Fusarium culmorum,
Gonatobotrys simplex,
Pithomyces maydicus,
Rhizopus microsporus Tiegh.,
R. stolonifer = R. nigricans,
Scopulariopsis brumptii
Ergot (horse's tooth) Claviceps gigantea
(anamorph: Sphacelia sp.) Disease Pathogen
Eyespot Aureobasidium zeae = Kabatiella zeae
Fusarium ear and stalk rot Fusarium subglutinans =
F. moniliforme var.subglutinans
Fusarium kernel, root and stalk rot, Fusarium moniliforme
seed rot and seedling blight (teleomorph: Gibberella fujikuroi)
Fusarium stalk rot, Fusarium avenaceum
seedling root rot (teleomorph: Gibberella avenacea)
Gibberella ear and stalk rot Gibberella zeae
(anamorph: Fusarium graminearum)
Gray ear rot Botryosphaeria zeae = Physalospora zeae
(anamorph: Macrophoma zeae)
Gray leaf spot Cercospora sorghi = C. sorghi var. maydis, C. (Cercospora leaf spot) zeae-maydis
Helminthosporium root rot Exserohilum pedicellatum = Helminthosporium pedicellatum (teleomorph: Setosphaeria pedicellata)
Hormodendrum ear rot Cladosporium cladosporioides =
(Cladosporium rot) Hormodendrum cladosporioides, C. herbarum
(teleomorph: Mycosphaerella tassiana)
Leaf spots, minor Alternaria alternata,
Ascochyta maydis, A. tritici,
A. zeicola, Bipolaris victoriae =
Helminthosporium victoriae
(teleomorph: Cochliobolus victoriae), C. sativus
(anamorph: Bipolaris sorokiniana = H.
sorokinianum = H. sativum), Epicoccum nigrum,
Exserohilum prolatum = Drechslera prolata
(teleomorph: Setosphaeria prolata)
Graphium penicillioides,
Leptosphaeria maydis, Leptothyrium zeae,
Ophiosphaerella herpotricha, (anamorph:
Scolecosporiella sp.),
Paraphaeosphaeria michotii, Phoma sp.,
Septoria zeae, S. zeicola,
S. zeina
Northern corn leaf blight (white blast, Setosphaeria turcica (anamorph: Exserohilum crown stalk rot, stripe) turcicum = Helminthosporium turcicum) Disease Pathogen
Northern corn leaf spot Cochliobolus carbonum (anamorph: Bipolaris Helminthosporium ear rot (race 1 ) zeicola = Helminthosporium carbonum)
Penicillium ear rot (blue eye, blue Penicillium spp., P. chrysogenum,
mold) P. expansum, P. oxalicum
Phaeocytostroma stalk and root rot Phaeocytostroma ambiguum, =
Phaeocytosporella zeae
Phaeosphaeria leaf spot Phaeosphaeria maydis = Sphaerulina maydis
Physalospora ear rot (Botryosphaeria Botryosphaeria festucae = Physalospora ear rot) zeicola (anamorph: Diplodia frumenti)
Purple leaf sheath Hemiparasitic bacteria and fungi
Pyrenochaeta stalk and root rot Phoma terrestris =
Pyrenochaeta terrestris
Pythium root rot Pythium spp., P. arrhenomanes,
P. graminicola
Pythium stalk rot Pythium aphanidermatum =
P. butleri L.
Red kernel disease (ear mold, leaf Epicoccum nigrum
and seed rot)
Rhizoctonia ear rot (sclerotial rot) Rhizoctonia zeae (teleomorph: Waitea
circinata)
Rhizoctonia root and stalk rot Rhizoctonia solani, Rhizoctonia zeae
Root rots (minor) Alternaria alternata, Cercospora sorghi,
Dictochaeta fertilis, Fusarium acuminatum (teleomorph: Gibberella acuminata), F. equiseti (teleomorph: G. intricans), F. oxysporum, F. pallidoroseum, F. poae, F. roseum, G.
cyanogena, (anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp., Periconia circinata, Phytophthora cactorum, P. drechsleri, P. nicotianae var. parasitica, Rhizopus arrhizus
Rostratum leaf spot Setosphaeria rostrata, (anamorph:
(Helminthosporium leaf disease, ear xserohilum rostratum = Helminthosporium and stalk rot) rostratum)
Java downy mildew Peronosclerospora maydis =
Sclerospora maydis
Philippine downy mildew Peronosclerospora philippinensis = Sclerospora philippinensis Disease Pathogen
Sorghum downy mildew Peronosclerospora sorghi =
Sclerospora sorghi
Spontaneum downy mildew Peronosclerospora spontanea =
Sclerospora spontanea
Sugarcane downy mildew Peronosclerospora sacchari =
Sclerospora sacchari
Sclerotium ear rot (southern blight) Sclerotium rolfsii Sacc. (teleomorph: Athelia rolfsii)
Seed rot-seedling blight Bipolaris sorokiniana, B. zeicola =
Helminthosporium carbonum, Diplodia maydis, Exserohilum pedicillatum, Exserohilum turcicum = Helminthosporium turcicum, Fusarium avenaceum, F. culmorum, F. moniliforme, Gibberella zeae (anamorph: F. graminearum), Macrophomina phaseolina, Penicillium spp., Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicaria sp.
Selenophoma leaf spot Selenophoma sp.
Sheath rot Gaeumannomyces graminis
Shuck rot Myrothecium gramineum
Silage mold Monascus purpureus, M ruber
Smut, common Ustilago zeae = U. maydis
Smut, false Ustilaginoidea virens
Smut, head Sphacelotheca reiliana = Sporisorium
holcisorghi
Southern corn leaf blight and stalk rot Cochliobolus heterostrophus (anamorph:
Bipolaris maydis = Helminthosporium maydis)
Southern leaf spot Stenocarpella macrospora = Diplodia
macrospora
Stalk rots (minor) Cercospora sorghi, Fusarium episphaeria, F.
merismoides, F. oxysporum Schlechtend, F. poae, F. roseum, F. solani (teleomorph: Nectria haematococca), F. tricinctum, Mariannaea elegans, Mucor sp., Rhopographus zeae, Spicaria sp.
Storage rots Aspergillus spp., Penicillium spp. und weitere
Pilze Disease Pathogen
Tar spot Phyllachora maydis
Trichoderma ear rot and root rot Trichoderma viride = T. lignorum teleomorph:
Hypocrea sp.
White ear rot, root and stalk rot Stenocarpella maydis = Diplodia zeae
Yellow leaf blight Ascochyta ischaemi, Phyllosticta maydis
(teleomorph: Mycosphaerella zeae-maydis)
Zonate leaf spot Gloeocercospora sorghi
Diseases caused by fungi and Oomycetes with unclear classification regarding biotrophic, hemibiotrophic or necrotrophic behavior
Figure imgf000027_0001
The following are especially preferred:
Plasmodiophoromycota such as Plasmodiophora brassicae (clubroot of crucifers), Spongospora subterranea, Polymyxa graminis,
Oomycota such as Bremia lactucae (downy mildew of lettuce), Peronospora (downy mildew) in snapdragon (P. antirrhini), onion (P. destructor), spinach (P. effusa), soybean (P. manchurica), tobacco ("blue mold"; P. tabacina) alfalfa and clover (P. trifolium), Pseudoperonospora humuli (downy mildew of hops), Plasmopara (downy mildew in grapevines) (P. viticola) and sunflower (P. halstedii), Sclerophthora macrospora (downy mildew in cereals and grasses), Pythium (for example damping-off of Beta beet caused by P. debaryanum), Phytophthora infestans (late blight in potato and in tomato and the like), Albugo spec.
Ascomycota such as Microdochium nivale (snow mold of rye and wheat), Fusarium graminearum, Fusarium culmorum (partial ear sterility mainly in wheat), Fusarium oxysporum (Fusarium wilt of tomato), Blumeria graminis (powdery mildew of barley (f.sp. hordei) and wheat (f.sp. tritici)), Erysiphe pisi (powdery mildew of pea), Nectria galligena (Nectria canker of fruit trees), Uncinula necator (powdery mildew of grapevine),
Pseudopeziza tracheiphila (red fire disease of grapevine), Claviceps purpurea (ergot on, for example, rye and grasses), Gaeumannomyces graminis (take-all on wheat, rye and other grasses), Magnaporthe grisea, Pyrenophora graminea (leaf stripe of barley), Pyrenophora teres (net blotch of barley), Pyrenophora tritici-repentis (leaf blight of wheat), Venturia inaequalis (apple scab), Sclerotinia sclerotium (stalk break, stem rot),
Pseudopeziza medicaginis (leaf spot of alfalfa, white and red clover).
Basidiomycetes such as Typhula incarnata (typhula blight on barley, rye, wheat), Ustilago maydis (blister smut on maize), Ustilago nuda (loose smut on barley), Ustilago tritici (loose smut on wheat, spelt), Ustilago avenae (loose smut on oats), Rhizoctonia solani
(rhizoctonia root rot of potato), Sphacelotheca spp. (head smut of sorghum), Melampsora lini (rust of flax), Puccinia graminis (stem rust of wheat, barley, rye, oats), Puccinia recondita (leaf rust on wheat), Puccinia dispersa (brown rust on rye), Puccinia hordei (leaf rust of barley), Puccinia coronata (crown rust of oats), Puccinia striiformis (yellow rust of wheat, barley, rye and a large number of grasses), Uromyces appendiculatus (brown rust of bean), Sclerotium rolfsii (root and stem rots of many plants).
Deuteromycetes (Fungi imperfecti) such as Septoria (Stagonospora) nodorum (glume blotch) of wheat (Septoria tritici), Pseudocercosporella herpotrichoides (eyespot of wheat, barley, rye), Rynchosporium secalis (leaf spot on rye and barley), Alternaria solani (early blight of potato, tomato), Phoma betae (blackleg on Beta beet), Cercospora beticola (leaf spot on Beta beet), Alternaria brassicae (black spot on oilseed rape, cabbage and other crucifers), Verticillium dahliae (verticillium wilt), Colletotrichum lindemuthianum (bean anthracnose), Phoma lingam (blackleg of cabbage and oilseed rape), Botrytis cinerea (grey mold of grapevine, strawberry, tomato, hops and the like).
Especially preferred are biotrophic pathogens, among which in particular hemibiotrophic pathogens, i.e. Phakopsora pachyrhizi and/or those pathogens which have essentially a similar infection mechanism as Phakopsora pachyrhizi, as described herein. Particularly preferred are pathogens from the group Uredinales (rusts), among which in particular the Melompsoraceae. Especially preferred are Phakopsora pachyrhizi and/or Phakopsora meibomiae.
Harvestable parts of the transgenic plant according to the present invention are part of the invention. The harvestable parts may be seeds, roots, leaves and/or flowers comprising the SMT1 -gene, the complementary SMT1 -gene and/or a part thereof. Preferred parts of soy plants are soy beans comprising the transgenic SMT1 -gene.
Products derived from transgenic plant according to the present invention, parts thereof or harvestable parts thereof are part of the invention. A preferred product is soybean meal, soybean oil, wheat meal, corn starch, corn oil, corn meal, rice meal, canola oil and/or potato starch.
The present invention also includes methods for the production of a product comprising a) growing the plants of the invention and b) producing said product from or by the plants of the invention and/or parts thereof, e.g. seeds, of these plants. In a further embodiment the method comprises the steps a) growing the plants of the invention, b) removing the harvestable parts as defined above from the plants and c) producing said product from or by the harvestable parts of the invention. In one embodiment the method for the production of a product comprises
a) growing the plants of the invention or obtainable by the methods of invention and
b) producing said product from or by the plants of the invention and/or parts, e.g. seeds, of these plants. The product may be produced at the site where the plant has been grown, the plants and/or parts thereof may be removed from the site where the plants have been grown to produce the product. Typically, the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant. The step of growing the plant may be performed only once each time the methods of the invention is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the invention and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants of the invention is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts. Also, the steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally the plants are grown for some time before the product is produced.
In one embodiment the products produced by said methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic and/or pharmaceutical. Foodstuffs are regarded as compositions used for nutrition and/or for supplementing nutrition. Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs.
In another embodiment the inventive methods for the production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
It is possible that a plant product consists of one ore more agricultural products to a large extent.
All definitions given to terms used in specific type of category (method for producing a plant and/or part thereof resistant to fungus, transgenic plant cell, vector construct, use of the vector construct etc.) may be also applicable for the other categories.
Figures:
Figure 1 shows the full-length-sequence of the SMT1 -gene from Phakopsora pachyrhizi having SEQ-ID-No.1 .
Figure 2A shows the sequence of the SMT1 -gene from Phakopsora pachyrhizi used for primer design and construct generation having SEQ-ID-No.2. Figure 2B shows a partial sequence of Figure 1 (SEQ-ID-3).
Figure 3 shows the sequence of one vector construct useful according to the present invention (parsley ubiquitin promoter: SMT1 fragment antisense orientation: spacer sequence: SMT1 fragment sense orientation: nopaline synthase promoter)(SEQ-ID-No.4).
Figure 4 shows the sequence of the SMT1 -protein (SEQ-ID-5).
Figure 5 shows single sequences which are part of the vector construct of Figure 3.
Figure 6 shows a schema of one vector construct useful according to the present invention Figure 7 shows the scoring system used to determine the level of resistance of wildtype and transgenic (SMT1 RNAi) soybean plants against the soybean rust fungus P. pachyrhizi.
Figure 8 shows the result of the scoring of 17 transgenic soybean events expressing the SMT1 RNAi construct. The average scoring of the wildtype controls was 4.1 (which can be translated to approximately 50% diseased leaf area. The average scoring of the controls was set to zero. For each transgenic soybean event (expressing the SMT1 RNAi construct) 1 -3 clones were scored. The bars represent the deviation of transgenic soybean events (expressing the SMT1 RNAi construct) the from the average of wild type controls.
Examples
The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the present invention.
Example 1 : General methods
The chemical synthesis of oligonucleotides can be affected, for example, in the known fashion using the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896- 897). The cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, phage multiplication and sequence analysis of recombinant DNA, are carried out as described by Sambrook et al. Cold Spring Harbor Laboratory Press (1989), ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules is carried out with an MWG-Licor laser fluorescence DNA sequencer following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)).
Example 2 : Cloning of RNAi construct RNAi hairpin structure
The RNAi-construct (Figures 3 and 6) was prepared as follows: By PCR based amplification AscI and Aatll restriction sites were introduced on the first stem (antisense; from position 148 to 454 relative to SEQ ID2) and Sbfl and Xhol sites were introduced on the second stem (sense, Position 199-454) both in the 5' - 3' strand "sense" orientation (relative zu SEQ ID2). For the introduction of the resctriction sites the nucleic acid of the first stem having SEQ-ID-No.7 was amplified using using primers having SEQ-ID No. 1 1 and 12. For the introduction of the resctriction sites the nucleic acid of the second stem having SEQ-ID-No.9 was amplified using using primers having SEQ-ID No 13 and 14. Both fragments were cloned using the TOPO cloning system (TOPO® TA Cloning® Kit Invitrogen). Both fragments were sequenced. Each stem was cut with the respective enzymes and gel purified.
Likewise, the spacer having SEQ-ID-No. 8 (Aatll::Stops block:EST166::Stops Block::Xhol) was cloned Fig. 3). It was cut with Aatll and Xhol and gel purified.
As binary base vector a binary vector was used which is composed of: (1 ) a Kanamycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agorbacteria (3) a pBR322 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection marker under control of a pcUbi-promoter and an Ubi- promoter:MCS::t-Nos cassette for cloning of the target gene (cf. Figures 3 and 6). The base vector was cut within the MCS with Ascl and Sbfl and the backbone purified by gel purification.
All four fragments were ligated using T4 ligase under standard conditions and transformed into E. coli, mini-prepped and screened by Ascl & Sbfl digestion. A positive clone (RTP1367) was submitted to soybean transformation.
Soybean transformation
The RTP1376 construct was transformed into soybean. Example 3 - Sterilization and Germination of Soybean Seeds Virtually any seed of any soybean variety can be employed in the method of the invention. A variety of soybean cultivar (including Jack, Williams 82, and Resnik) is appropriate for soybean transformation. Soybean seeds were sterilized in a chamber with a chlorine gas produced by adding 3.5 ml 12N HCI drop wise into 100 ml bleach (5.25% sodium hypochlorite) in a desiccator with a tightly fitting lid. After 24 to 48 hours in the chamber, seeds were removed and approximately 18 to 20 seeds were plated on solid GM medium with or without 5 μΜ 6-benzyl- aminopurine (BAP) in 100 mm Petri dishes. Seedlings without BAP were more elongated and roots developed, especially secondary and lateral root formation. BAP strengthened the seedling by forming a shorter and stockier seedling. Seven-day-old seedlings grown in the light (>100 ^Einstein/m2s) at 25°C. were used for explant material for the three-explant types. At this time, the seed coat was split, and the epicotyl with the unifoliate leaves had 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).
For inoculation of entire seedlings (Method A, see example 5.1 ) or leaf explants (Method B, see example 5.3), the seedlings were then ready for transformation.
For method C (see example 5.4), the hypocotyl and one and a half or part of both cotyledons were removed from each seedling. The seedlings were then placed on propagation media for 2 to 4 weeks. The seedlings produced several branched shoots to obtain explants from. The majority of the explants originated from the plantlet growing from the apical bud. These explants were preferably used as target tissue.
Example 4
Growth and Preparation of Agrobacterium Culture
Agrobacterium cultures were prepared by streaking Agrobacterium (e.g. A. tumefaciens or A. rhizogenes) carrying the desired binary vector (e.g. H Klee, R Horsch, and S Rogers 1987 Agrobacterium-Mediated Plant Transformation and its Further Applications to Plant Biology; Annual Review of Plant Physiology Vol. 38: 467-486) onto solid YEP growth medium (YEP media: 10 g yeast extract. 10 g Bacto Peptone. 5 g NaCI. Adjust pH to 7.0, and bring final volume to 1 liter with H20, for YEP agar plates add 20g Agar, autoclave) and incubating at 25°C. until colonies appeared (about 2 days). Depending on the selectable marker genes present on the Ti or Ri plasmid, the binary vector, and the bacterial chromosomes, different selection compounds were used for A. tumefaciens and rhizogenes selection in the YEP solid and liquid media. Various Agrobacterium strains can be used for the transformation method.
After approximately two days, a single colony (with a sterile toothpick) was picked and 50 ml of liquid YEP was inoculated with antibiotics and shaken at 175 rpm (25°C.) until an OD. sub.600 between 0.8-1 .0 is reached (approximately 2 d). Working glycerol stocks (15%) for
transformation were prepared and one-ml of Agrobacterium stock aliquoted into 1 .5 ml Eppendorf tubes then stored at -80°C. The day before explant inoculation, 200 ml of YEPmedia were inoculated with 5 μΙ to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask. The flask was shaked overnight at 25 °C until the Οϋβοο was between 0.8 and 1 .0. Before preparing the soybean explants, the Agrobacteria were pelleted by centrifugation for 10 min at 5, 500. times. g at 20°C. The pellet was resuspended in liquid CCM to the desired density (Οϋβοο 0.5-0.8) and placed at room temperature at least 30 min before use. Example 5
Explant Preparation and Co-Cultivation (Inoculation) 5.1 Method A: Explant Preparation on the Day of Transformation.
Seedlings at this time had elongated epicotyls from at least 0.5 cm but generally between 0.5 and 2 cm. Elongated epicotyls up to 4 cm in length had been successfully employed. Explants were then prepared with: i) with or without some roots, ii) with a partial, one or both cotyledons, all preformed leaves were removed including apical meristem, and the node located at the first set of leaves was injured with several cuts using a sharp scalpel.
This cutting at the node not only induced Agrobacterium infection but also distributed the axillary meristem cells and damaged pre-formed shoots. After wounding and preparation, the explants were set aside in a Petri dish and subsequently co-cultivated with the liquid CCM/Agrobacterium mixture for 30 minutes. The explants were then removed from the liquid medium and plated on top of a sterile filter paper on 15.times.100 mm Petri plates with solid co-cultivation medium. The wounded target tissues were placed such that they are in direct contact with the medium. 5.2 Modified Method A: Epicotyl Explant Preparation
Soybean epicotyl segments prepared from 4 to 8 d old seedlings were used as explants for regeneration and transformation. Seeds of soybean cv L00106CN, 93-41 131 and Jack were germinated in 1/10 MS salts or a similar composition medium with or without cytokinins for 4. about.8 d. Epicotyl explants were prepared by removing the cotyledonary node and stem node from the stem section. The epicotyl was cut into 2 to 5 segments. Especially preferred were segments attached to the primary or higher node comprising axillary meristematic tissue.
The explants were used for Agrobacterium infection. Agrobacterium AGL1 (Lazo GR, Stein PA, Ludwig RA A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology (N Y) 1991 Oct;9(10):963-967) harboring a plasmid with the GUS marker gene and the AHAS, bar or dsdA selectable marker gene was cultured in LB medium with appropriate antibiotics overnight, harvested and resuspended in a inoculation medium with acetosyringone . Freshly prepared epicotyl segments were soaked in the Agrobacterium suspension for 30 to 60 min and then the explants were blotted dry on sterile filter papers. The inoculated explants were then cultured on a co-culture medium with L-cysteine and DTT (Dithiothreitol) and other chemicals such as acetosyringone for enhancing T-DNA delivery for 2 to 4 d. The infected epicotyl explants were then placed on a shoot induction medium with selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene), or D-serine (for dsdA gene). The regenerated shoots were subcultured on elongation medium with the selective agent.
For regeneration of transgenic plants the segments were then cultured on a medium with cytokinins such as BAP (6-benzylaminopurine), TDZ (thidiazuron) and/or Kinetin for shoot induction. After 4 to 8 weeks, the cultured tissues were transferred to a medium with lower concentration of cytokinin for shoot elongation. Elongated shoots were transferred to a medium with auxin for rooting and plant development. Multiple shoots were regenerated. 5.3 Method B: Leaf Explants
For the preparation of the leaf explant the cotyledon was removed from the hypocotyl. The cotyledons were separated from one another and the epicotyl is removed. The primary leaves, which consist of the lamina, the petiole, and the stipules, were removed from the epicotyl by carefully cutting at the base of the stipules such that the axillary meristems were included on the explant. To wound the explant as well as to stimulate de novo shoot formation, any pre-formed shoots were removed and the area between the stipules was cut with a sharp scalpel 3 to 5 times. The explants are either completely immersed or the wounded petiole end dipped into the
Agrobacterium suspension immediately after explant preparation. After inoculation, the explants are blotted onto sterile filter paper to remove excess Agrobacterium culture and place explants with the wounded side in contact with a round 7 cm Whatman paper overlaying the solid CCM medium (see above). This filter paper prevents A. tumefaciens overgrowth on the 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.
5.4 Method C: Propagated Axillary Meristem For the preparation of the propagated axillary meristem explant propagated 3-4 week-old plantlets were used. Axillary meristem explants could be pre-pared from the first to the fourth node. An average of three to four explants could be obtained from each seedling. The explants were 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 was cut with a scalpel to induce de novo shoot growth and allow access of target cells to the Agrobacterium. Therefore, a 0.5 cm explant included the stem and a bud.
Once cut, the explants were immediately placed in the Agrobacterium suspension for 20 to 30 minutes. After inoculation, the explants were blotted onto sterile filter paper to remove excess Agrobacterium culture then placed almost completely immersed in solid coculture media CCM (see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soybean using primary-node explants from seedlingsln Vitro Cell. Dev. Biol.— Plant (2007) 43:536-549) or on top of a round 7 cm filter paper overlaying the solid CCM, depending on the Agrobacterium strain. This filter paper prevented Agrobacterium overgrowth on the soybean explants. Plates were wrapped with Parafilm.TM. "M" (American National Can, Chicago, III., USA) and incubated for two to three days in the dark at 25°C. Example 6
Shoot Induction After 3 to 5 days co-cultivation in the dark at 25°C, the explants were rinsed in liquid shoot induction medium (SIM, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soybean using primary-node explants from seedlings In Vitro Cell. Dev. Biol.— Plant (2007) 43:536-549; to remove excess Agrobacterium) or Modwash medium ("I X B5 major salts, 1X B5 minor salts, 1X MSIII iron, 3% Sucrose, 1X B5 vitamins, 30 mM MES, 350 mg/L Timentin™ pH 5.6, WO 2005/121345) (Method C) and blotted dry on sterile filter paper (to prevent damage especially on the lamina) before placing on the solid SIM medium. The approximately 5 explants (Method A) or 10 to 20 (Methods B and C) explants were placed such that the target tissue was in direct contact with the medium. During the first 2 weeks, the explants could be cultured with or without selective medium. Preferably, explants were transferred onto SIM without selection for one week.
For leaf explants (Method B), the explant should be placed into the medium such that it is perpendicular to the surface of the medium with the petiole imbedded into the medium and the lamina out of the medium.
For propagated axillary meristem (Method C), the explant was placed into the medium such that it iwas parallel to the surface of the medium (basipetal) with the explant partially embedded into the medium. Wrap plates with Scotch 394 venting tape (3M, St. Paul, Minn., USA) were placed in a growth chamber for two weeks with a temperature averaging 25°C. under 18 h light/6 h dark cycle at 70-100 μΕ/Γη28. The explants remained on the SIM medium with or without selection until de novo shoot growth occured at the target area (e.g., axillary meristems at the first node above the epicotyl). Transfers to fresh medium can occur during this time. Explants were transferred from the SIM with or without selection to SIM with selection after about one week. At this time, there was considerable de novo shoot development at the base of the petiole of the leaf explants in a variety of SIM (Method B), at the primary node for seedling explants (Method A), and at the axillary nodes of propagated explants (Method C). Preferably, all shoots formed before transformation were removed up to 2 weeks after co- cultivation to stimulate new growth from the meristems. This helped to reduce chimerism in the primary transformant and increase amplification of transgenic meristematic cells. During this time the explant may or may not be cut into smaller pieces (i.e. detaching the node from the explant by cutting the epicotyl). Example 7
Shoot Elongation After 2 to 4 weeks (or until a mass of shoots was formed) on SIM medium (preferably with selection), the explants were transferred to SEM medium (shoot elongation medium, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soybean using primary-node explants from seedlingsln Vitro Cell. Dev. Biol.— Plant (2007) 43:536-549) that stimulates shoot elongation of the shoot primordia. This medium may or may not contain a selection compound.
After every 2 to 3 weeks, the explants were transfered to fresh SEM medium (preferably containing selection) after carefully removing dead tissue. The explants should hold together and not fragment into pieces and retain somewhat healthy. The explants were continued to be transferred until the explant dies or shoots elongate. Elongated shoots >3 cm were removed and placed into RM medium (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) for about 1 week (Method A and B), or about 2 to 4 weeks depending on the cultivar (Method C) at which time roots began to form. In the case of explants with roots, they were transferred directly into soil. Rooted shoots were transferred to soil and hardened in a growth chamber for 2 to 3 weeks before transferring to the greenhouse.
Regenerated plants obtained using this method were fertile and produced on average 500 seeds per plant. Transient GUS expression after 5 days of co-cultivation with Agrobacterium tumefaciens was widespread on the seedling axillary meristem explants especially in the regions wounding during explant preparation (Method A). Explants were placed into shoot induction medium without selection to see how the primary-node responds to shoot induction and regeneration. Thus far, greater than 70% of the explants were formed new shoots at this region . Expression of the GUS gene was stable after 14 days on SIM, implying integration of the T-DNA into the soybean genome. In addition, preliminary experiments resulted in the formation of GUS positive shoots forming after 3 weeks on SIM .
[For Method C, the average regeneration time of a soybean plantlet using the propagated axillary meristem protocol was 14 weeks from explant inoculation. Therefore, this method has a quick regeneration time that leads to fertile, healthy soybean plants. 8.1 . Recovery of clones
2-3 clones per TO event were potted into small 6cm pots. For recovery the clones were kept for 12-18 days in the Phytochamber (16 h-day- und 8 h-night-Rhythm at a temeperature of 16° bis 22° C und a humidty of 75 % were grown).
8.2 Inoculation
The soybean rust fungus was a wild isolate from Brazil. The plants were inoculated with
P.pachyrhizi .
In order to obtain appropriate spore material for the inoculation, soybean leaves which had been infected with soybean rust 15-20 days ago, were taken 2-3 days before the inoculation and transferred to agar plates (1 % agar in H20). The leaves were placed with their upper side onto the agar, which allowed the fungus to grow through the tissue and to produce very young spores. For the inoculation solution, the spores were knocked off the leaves and were added to a Tween-H20 solution. The counting of spores was performed under a light microscope by means of a Thoma counting chamber. For the inoculation of the plants, the spore suspension was added into a compressed-air operated spray flask and applied uniformly onto the plants or the leaves until the leaf surface is well moisturized. For the microscopy, a density of 10x105 spores / ml is used. The inoculated plants were placed for 24 hours in a greenhouse chamber with an average of 22°C and >90% of air humidity. The inoculated leaves were incubated under the same conditions in a closed Petri dish on 0,5% plant agar. The following cultivation was performed in a chamber with an average of 25°C and 70% of air humidity.
8.3 Microscopical screening:
For the evaluation of the pathogen development, the inoculated leaves of plants were stained with aniline blue.
The aniline blue staining served for the detection of fluorescent substances. During the defense reactions in host interactions and non-host interactions, substances such as phenols, callose or lignin accumulated or were produced and were incorporated at the cell wall either locally in papillae or in the whole cell (hypersensitive reaction, HR). Complexes were formed in association with aniline blue, which lead e.g. in the case of callose to yellow fluorescence. The leaf material was transferred to falcon tubes or dishes containing destaining solution II (ethanol / acetic acid 6/1 ) and was incubated in a water bath at 90°C for 10-15 minutes. The destaining solution II was removed immediately thereafter, and the leaves were washed 2x with water. For the staining, the leaves were incubated for 1 ,5-2 hours in staining solution II (0.05 % aniline blue = methyl blue, 0.067 M di-potassium hydrogen phosphate) and analyzed by microscopy immediately thereafter. LP) are used. After aniline blue staining, the spores appeared blue under UV light. The papillae could be recognized beneath the fungal appressorium by a green/yellow staining. The hypersensitive reaction (HR) was characterized by a whole cell fluorescence.
8.4 Evaluating the susceptibility to soybean rust
Plants are screened macroscopically 14 days after inoculation.
Screening method:
The progression of the soybean rust disease was scored by the estimation of the diseased area (area which was covered by sporulating uredinia) on the backside (abaxial side) of the leaf. Additionally the yellowing of the leaf was taken into account, (for complete scheme see Figure 7).
Results of screening
The macroscopic disease symptoms of soybean against P. pachyrhizi of 17 independent events (To plants, 2-3 clones per event) were scored 14 days after inoculation. Clones from non- transgenic soybean plants were used as control. The average scoring of non-transgenic control plants was 4.07. After screening all plants, the average of the clones was calculated, which were derived from one event. For analysis of the effect the average of the controls was set to zero, and the average result of all clones from one event was subtracted from the control.
Negative values show enhanced resistance, whereas positive values indicate enhanced susceptibility (Figure 8). It was shown that the in planta expression of a RNAi construct targeting a steroyl-methyl transferase of P. pachyrhizi leads to a disease scoring of transgenic plants compared to non-transgenic controls. So, the expression of an RNAi construct targeting the SMT1 enzyme of Phakopsora pachyrhizi enhances the resistance of soybean against soybean rust.

Claims

36
ims
A method for producing a plant and/or a part thereof resistant to a fungus comprising a) providing a recombinant nucleic acid comprising a target nucleic acid that is
substantial identical and/or substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene,
b) introducing said recombinant nucleic acid into in the plant and/or part thereof.
A method according to claim 1 , wherein the recombinant nucleic acid is able to provide dsRNA and/or si-RNA and/or miRNA in the plant, a part thereof and/or in the fungus infecting the plant or a part thereof, once the recombinant nucleic acid is expressed, wherein at least 19 contiguous nucleotides of the dsRNA and/or siRNA and/or miRNA are substantially complementary to the target SMT1-gene.
A method of claims 1 or 2, wherein the target SMT1-gene having at least 60%, at least 70%, at least 80%, at least 90 %, at least 95 %, at least 98 % or 100% identity to SEQ-ID-No.1 , SEQ-ID-No. 2 or SEQ-ID-No.3 or to a sequence coding for a protein having SEQ-ID- No. 5.
A method of anyone of claims 1 to 3, wherein said recombinant nucleic acid comprises a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which is substantial identical and/or substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary to at least 19 contiguous nucleotides of the target SMT1-gene and a second strand having a sequence substantially complementary to the first strand or parts thereof, and
a terminator regulatory sequence.
A method of anyone of claims 1 to 3, wherein said recombinant nucleic acid comprises a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially identical or substantially complementary to at least contiguous 19 nucleotides of the target SMT1 -gene, and
a terminator regulatory sequence.
The method of anyone of claims 1 to 5, wherein the target nucleic acid comprises 19 to 500 nucleotides. 37
7. The method of claim 6, wherein the target nucleic acid comprises 250 to 350 nucleotides.
8. The method of anyone of claims 1 to 7, wherein the target nucleic acid is substantially
identical and/or substantially complementary to contiguous nucleotides of the SMT1-gene.
9. The method of anyone of claims 1 to 7, wherein 19 to 24 contiguous nucleotides are
substantially identical and/or substantially complementary to the target SMT1 -gene and the remaining nucleotides of the target nucleic acid are not identical and/or complementary to the target SMT1 -gene.
10. The method of anyone of claims 1 to 9, wherein the fungal resistance is a resistance against a hemibiotrophic fungus.
1 1 The method of anyone of the claims 1 to 10, wherein the fungal resistance is a resistance against soybean rust.
12. The method of claim 1 1 , where the soybeanrust is Phakopsora meibomiae and/or
Phakopsora pachyrhizi.
13. Vector construct comprising a recombinant nucleic acid comprising a promoter that is
functional in the plant cell, operably linked to a target nucleic acid which is substantially identical and/or substantially complementary to at least 19 contiguous nucleotides of the target SMT1-gene and
a terminator regulatory sequence.
14. The vector construct of claim 13 comprising, a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which is substantial identical and/or substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary to at least 19 contiguous nucleotides of the target SMT1 -gene and a second strand having a sequence at substantially complementary to the first strand or parts thereof, and
a terminator regulatory sequence.
15. The vector construct of claim 13 comprising,
a promoter that is functional in the plant cell, operably linked to a target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence 38
substantially complementary or substantially identical to at least 19 contiguous nucleotides of the target SMT1-gene, and
a terminator regulatory sequence.
The vector construct of claims 13 to 15, wherein the target nucleic acid comprises 19 to 500 nucleotides.
The vector construct of anyone of claims 13 to 16, wherein the promoter is a constitutive, inducible, in particular pathogen-induceable, developmental stage-preferred, cell type- preferred, tissue-preferred and/or organ-preferred promoter.
Transgenic plant cell comprising a recombinant nucleic acid comprising a target nucleic acid which is substantial identical and/or complementary to at least contiguous19 nucleotides of the target SMT1-gene.
Transgenic plant cell of claim 18, wherein the target SMT1 -gene having at least 60%, at least 70%, at least 80%, at least 90 %, at least 95 %, at least 98 % or 100% identity to SEQ- ID-No.1 , SEQ-ID-No. 2 or SEQ-ID-No.3 or to a sequence coding for a protein having SEQ-ID-No. 5.
Transgenic plant cell of claims 18 or 19, wherein the recombinant nucleic acid is able to provide dsRNA and/or si-RNA and/or miRNA in the plant, parts thereof and/or in the fungus infecting the plant or a part thereof, once the recombinant nucleic acid is expressed.
The transgenic plant cell of anyone of claims 18 to 20 comprising a recombinant nucleic acid comprising
a promoter that is functional in the plant cell, operably linked to a
target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary to at least 19 contiguous nucleotides of the target SMT1-gene and a second strand having a sequence substantially complementary to the first strand or parts thereof, and
a terminator regulatory sequence.
The transgenic plant cell of anyone of claims 18 to 20 comprising a recombinant nucleic acid comprising
a promoter that is functional in the plant cell, operably linked to a target nucleic acid which, when it is transcribed, generates RNA comprising a first strand having a sequence substantially complementary or substantially identical to at least contiguous 19 nucleotides of the target SMT1-gene, and
a terminator regulatory sequence. 39
23. The transgenic plant cell of anyone of claims 18 to 22, wherein the promoter is a constitutive, inducible, in particular pathogen-induceable, developmental stage-preferred, cell type- preferred, tissue-preferred and/or organ-preferred promoter.
24. The transgenic plant cell of anyone of claims 18 to 23, wherein the plant is a legume.
25. The transgenic plant cell of claim 24, wherein the plant is selected from the group consisting of beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, and/or groundnut.
26. Transgenic plant or parts thereof comprising transgenic plant cells according to claims 18 to 25 or consisting thereof.
27. Transgenic seeds derived from the plant of claim 26.
28. Use of the vector construct according to anyone of claims 13 to 17 for the transformation of plant cells to provide fungal resistant plants.
29. The use of claim 28, wherein the fungal resistance is a resistance against soybean rust.
30. The use of claim 29, wherein the soybeanrust is Phakopsora meibomiae and/or Phakopsora pachyrhizi.
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WO2017016963A1 (en) * 2015-07-30 2017-02-02 Bayer Cropscience Aktiengesellschaft Methods and compositions for the control of rust fungi by inhibiting expression of the hxt1 gene
EP3431601A3 (en) * 2013-07-10 2019-04-03 Basf Se Rnai for the control of phytopathogenic fungi and oomycetes by inhibiting the expression of cyp51 genes

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