WO2013113911A2 - Séquences nucléotidiques impliquées dans la résistance aux phytoravageurs - Google Patents

Séquences nucléotidiques impliquées dans la résistance aux phytoravageurs Download PDF

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WO2013113911A2
WO2013113911A2 PCT/EP2013/052101 EP2013052101W WO2013113911A2 WO 2013113911 A2 WO2013113911 A2 WO 2013113911A2 EP 2013052101 W EP2013052101 W EP 2013052101W WO 2013113911 A2 WO2013113911 A2 WO 2013113911A2
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plant
nucleic acid
acid sequence
seq
avrbs4
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WO2013113911A3 (fr
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Tina STRAUß
Thomas Lahaye
Annett STRAUß
Marinus Willem Prins
Remco Martha Prudent VAN POECKE
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Ludwig-Maximilians-Universität München
Keygene N.V.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8281—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 bacterial resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • the present invention relates to isolated nucleotide sequences that are involved in providing plant pest resistance, in particular to isolated nucleotide sequences which encode a plant pest resistance protein and/or which are involved in transcription activation of a plant resistance gene.
  • the invention is further related to the use of such isolated nucleotide sequences in providing pest resistance to a plant and to the use of (parts of) these sequences as a marker for identification of pest resistance genes in plants.
  • Plant pests in the agricultural industry can reduce the yields significantly. It is estimated that plant pests reduce plant yields by about 10% every year in developed countries and that these losses can often exceed 20% in developing countries. Examples of pests that can have a devastating impact are Rice blast, Soybean cyst nematode, Citrus canker, and Potato blight. Hence, providing resistance to pest is of importance for the reliable production of agricultural products.
  • Xanthomonas is a genus of Proteobacteria, many of which cause pests in plants. Xanthomonas can infect a wide variety of plant species including pepper, rice, citrus, cotton, tomato, and soybeans (reviewed by Leyns F. et al. The Botanical Review. 1984, vol.
  • Xanthomonas cause localized leaf spot or leaf streak while others spread systemically and cause black rot or leaf blight pest.
  • the mode of infection of a plant by Xanthomonas includes secreting proteins that have an effect on the plant.
  • proteins include proteins that can act as transcription activators in plants, also termed Transcription Activator Like Effectors (TALE).
  • TALE Transcription Activator Like Effectors
  • endogenous gene expression of the plant may be affected by interaction of one or more TALEs derived from the bacteria.
  • a TALE may affect an endogenous plant gene aiding the infection, e.g. supporting the growth and spread of the bacteria.
  • plants have also evolved countermeasures such as so called R (resistance) genes that can also be activated by the TALE.
  • a TALE comprises in general a central domain of tandem repeats, which functions in DNA binding and gene transcription, and comprises also a nuclear localization signal (NLS) responsible for transport of the TALE to the cell nucleus where it exerts its action.
  • a TALE may comprise an acidic transcriptional activation domain.
  • the central repeat domain comprises substantially identical amino acid motifs, wherein each motif is about 33-35 amino acids in size, which are arranged in tandem to collectively participate in DNA binding.
  • RVDs Repeat Variable Diresidues
  • This code of RVDs also referred to as the TALE code, includes diaminoacids such as Nl, NK, HD and NG, that interact with most affinity with A, G, C, and T, respectively, and NS, that interacts with about equal affinity with A, G, C or T, and NN, that interacts with most affinity with either G or A.
  • the combination and order of RVDs determines the affinity of a TALE to a target sequence in DNA. Recently, the crystal structure of TALE protein PthXol has been solved (Mak et al. Science.
  • Plant pest resistance (R) genes confer monogenic resistance to phytopathogenic microbes and have been employed extensively in many commercial crops. Plant R genes are often transferred from wild to cultivated species by conventional breeding strategies.
  • R gene isolation is generally desirable since it allows gene transfer even if the R gene donor and recipient are sexually incompatible plant species.
  • R gene isolation by positional cloning or other forward genetic approaches is still challenging in most crop species.
  • the tomato Bs4 resistance gene is constitutively expressed, and the TAL effector protein AvrBs4 activates the Bs4 protein thereby providing pest resistance.
  • the presence of a resistance gene termed "Bs4C" (the Bs4 gene was subsequently renamed to Bs4C gene) gene in the pepper species Capsicum pubescens was suspected i.a. by Minsavage et al. (Hypersensitive resistance in Capsicum pubescens PI 235047 to Xanthomonas campestris pv.
  • vesicatoria (Xcv) is elicited by AvrBs3- 2 (now renamed to AvrBs4, Phytopathol. 1999, vol. 89:53).
  • This resistance gene is named after the resistance it provides against bacterial spot pest No.4. However, this suspicion had hitherto not led to detection of any Bs4C gene sequences or any evidence of the expression of such a gene.
  • the present invention provides an isolated nucleic acid sequence comprising a nucleotide sequence of TATAAN1 N2AN3TAN4TCCN5CTN6 (SEQ ID N0.1 ), wherein each N is independently selected from the group consisting of A, C, T and G.
  • N-i and/or N 2 is independently selected from T and A; and/or N 6 is independently selected from T and C.
  • N-i and N 2 are both T, and N 6 is T, or N-i and N 2 is A, and N 6 is
  • nucleic acid sequence is capable of activating gene transcription, preferably plant gene transcription.
  • the isolated nucleic acid sequence may be a binding element for a TALE protein, preferably a functional AvrBs4 protein.
  • the present invention pertains to an isolated nucleotide sequence capable of providing plant pest resistance comprising a nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence of SEQ ID NO.2 or a nucleic acid sequence having at least 60% sequence identity with the nucleic acid sequence of SEQ ID NO.2; b) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO.3 or a polypeptide having at least 60% sequence identity with the polypeptide having an amino acid sequence of SEQ ID NO.3; or c) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of any one of SEQ ID NO.4-10 or a polypeptide having at least 80% sequence identity with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO.4-10.
  • the pest may be a bacterium, preferably wherein the bacterium is from the genus Xanthomonas, preferably wherein the bacterium of the genus Xanthomonas comprises a gene encoding a functional AvrBs4 protein.
  • the nucleotide sequence of the second aspect may be further operably linked to a nucleic acid sequence according to the first aspect.
  • the invention further relates to a chimeric gene comprising a nucleic acid sequence as disclosed herein, a vector comprising a nucleic acid sequence as disclosed herein, or a chimeric gene as disclosed herein, and a host cell comprising such chimeric gene or vector, which host cell may advantageously be selected from a plant cell and a plant protoplast.
  • the present invention is directed to a method for providing a plant having resistance against infection by a pest, preferably wherein the pest is a bacterium, preferably a bacterium of the genus Xanthomonas, preferably a bacterium of the genus Xanthomonas comprising a gene encoding a functional AvrBs4 protein, comprising: a) providing a nucleic acid sequence of the invention operably linked to an endogenous nucleotide sequence encoding a plant pest resistance factor in a plant; or b) transforming a plant with a chimeric gene or vector comprising a nucleic acid sequence according to any one claims 1 -7 operably linked to a nucleotide sequence encoding a plant pest resistance factor; or c) modifying a nucleotide sequence operably linked to an endogenous nucleotide sequence encoding a plant pest resistance factor in a plant such that it comprises the nucleic acid sequence of any one of
  • the present invention relates to a method for providing a plant having resistance against infection by a pest, said method comprising the steps of:
  • nucleotide sequence selected from the group consisting of: i) a nucleic acid sequence of SEQ ID NO.2 or a nucleic acid sequence having at least 60% sequence identity with the nucleic acid sequence of SEQ ID NO.2; ii) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO.3 or a polypeptide having at least 60% sequence identity with the polypeptide having an amino acid sequence of SEQ ID NO.3; or iii) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of any one of SEQ ID NO.4-10 or a polypeptide having at least 80% sequence identity with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO.4-10; operably linked to a promoter active in plant cells, said nucleotide sequence being integrated into the genome of said plant,
  • a plant comprising a chimeric gene as disclosed herein or a vector as disclosed herein, is also encompassed. Additionally, a plant obtainable by the method above is encompassed by the present invention.
  • the invention further relates to an isolated nucleic acid marker for identifying a plant pest resistance gene, wherein the nucleic acid marker has at least 90% sequence identity to a nucleic acid sequence of 20 nucleotides from a nucleic acid sequence selected from the group of SEQ ID NO.21 and SEQ ID NO.22.
  • the nucleic acid sequence of 20 nucleotides is preferably a nucleic acid sequence of 20 contiguous nucleotides.
  • the marker may be a gene expression marker.
  • the invention is concerned with use of such isolated nucleic acid marker for identifying a pest resistance gene, preferably in a plant.
  • the pest may be a bacterium, preferably a bacterium from the genus Xanthomonas, preferably a bacterium of the genus Xanthomonas comprising a gene encoding a TALE protein, preferably a functional AvrBs4 protein.
  • the present invention provides for use of an isolated nucleic acid sequence according to the invention, a chimeric gene according to the invention, a vector according to invention, or a host cell according to the invention for providing pest resistance to a plant.
  • the pest may be a bacterium, preferably a bacterium from the genus
  • Xanthomonas preferably a bacterium of the genus Xanthomonas comprising a gene encoding a TALE protein, preferably a functional AvrBs4 protein.
  • the invention provides a method of identifying a plant pest resistance gene comprising the steps of: - infecting a plant cell with a microorganism encoding a TALE protein, preferably a microorganism from the genus Xanthomonas comprising a gene encoding a functional AvrBs4 protein; - performing an analysis of the transcriptome of the plant cell; and - identifying a plant pest resistance gene.
  • the pest may be a bacterium, preferably a bacterium from the genus Xanthomonas, preferably a bacterium of the genus Xanthomonas comprising a gene encoding a TALE protein, preferably a functional AvrBs4 protein.
  • AvrBs4 triggers Ss4C-mediated hypersensitive response (HR), whereas an AvrBs4 derivative that lacks its transcriptional activation domain (AvrBs4AAD) does not.
  • HR Ss4C-mediated hypersensitive response
  • AvrBs4AAD transcriptional activation domain
  • C. pubescens accession PI 235047 For in planta bacterial growth measurements leaves of C. pubescens accession PI 235047 were blunt-end syringe-infiltrated with Xanthomonas campestris pv. vesicatoria strain 85-10 (Xcv) or an isogenic strain containing AvrBs4
  • a Bs4C candidate is transcriptionally activated by AvrBs4 in the presence of the translation inhibitor cycloheximide.
  • Semiquantitative RT-PCR was carried out on cDNA of Xcv-infected C. pubescens leaves 24 hours after infection with primers matching to the identified Bs4C candidate (Bs4C * ) and another AvrBs4-induced transcript (4766).
  • the TALEs that are present in the given Xcv strains are indicated in superscript font. Inoculations were carried out in the presence (+) or absence (-) of the protein synthesis inhibitor cycloheximide (CHX).
  • CHX protein synthesis inhibitor cycloheximide
  • FIG. 4 The AvrBs3 deletion derivative AvrBs3Arep16 triggers a hypersensitive response in the C. pubescens accessions PI 235047 and PI 585270. Xanthomonas campestris pv.
  • vesicatoria strain 85-10 Xcv
  • XcvAvrBs3Arep16 XcvAvrBs3Arep16
  • AvrBs4 XcvAvrBs4
  • derivatives lacking the transcriptional activation domain XcvAvrBs3Arep16AAD and XcvAvrBs4AAD
  • XcvAvrBs3Arep16AAD and XcvAvrBs4AAD were syringe infiltrated (cultures adjusted to 5x108 colony forming units ml "1 ) into Capsicum pubescens accessions PI 235047 and PI 585270. Dashed lines mark inoculated areas. Leaves were harvested three days post inoculation and cleared in absolute ethanol to visualize the HR (dark areas).
  • C. pubescens orthologs of the C. annuum Bs3 gene mediate recognition of the TALE protein AvrBs3Arep16.
  • Agrobacterium tumefaciens mediated T-DNA transfer was used to deliver the C. annuum R alleles Bs3, Bs3-E or Bs3 orthologs from the C. pubescens accessions PI 235047 (Bs3-P ⁇ 235047) or PI 585270 (Bs3- PI 585270) in combination with the TALE genes avrBs3, avrBs3Arep16 or avrBs4 into N. benthamiana leaves.
  • TALE cauliflower mosaic virus 35S
  • 35S constitutive cauliflower mosaic virus 35S
  • the 35S-promoter driven tomato R gene Bs4 was co-delivered with avrBs4 as a positive control for expression of this TALE. Dashed lines mark inoculated areas.
  • A. tumefaciens strains containing distinct plant R or Xanthomonas TALE genes were adjusted to an OD600 of 0.8 and equal volumes were mixed for infiltration of N. benthamiana leaves. Four days post infiltration, leaves were harvested and cleared with ethanol to visualize the HR (dark areas).
  • C. pubescens orthologs of the C. annuum Bs3 gene are transcriptionally activated by the TALE protein AvrBs3Arep16.
  • Semiquantitative RT-PCR with Ss3-specific primers was carried out on cDNA of Xcv-infected C. pubescens leaves that were harvested 24 hours after inoculation.
  • the TALEs that are present in the given Xcv strains are indicated in superscript font. Inoculations were carried out in the presence (+) or absence (-) of the protein synthesis inhibitor cycloheximide (CHX).
  • CHX protein synthesis inhibitor cycloheximide
  • the constitutively expressed elongation factor 1 alpha (EFIalpha) served as an internal normalization control.
  • a Bs4C candidate gene is genetically linked to the AvrBs4-induced immune response. Ethidium bromide stained 3% agarose gel displaying BglW cleaved PCR fragments. The primers used are located in the 3' region of the Bs4C candidate gene. The PCR fragments are derived from the C. pubescens parental lines PI 235047 (R), PI 585270 (S) and 17 F2 segregants corresponding that were scored as resistant (r) and susceptible (s) upon inoculation with Xanthomonas strains containing AvrBs4. M, GeneRuler 50 bp DNA Ladder (Fermentas, St. Leon-Rot, Germany).
  • Agrobacterium tumefaciens mediated T-DNA transfer was used to deliver the C. pubescens Bs4C candidate genes derived from either PI 235047 ⁇ Bs4C-R) or PI 585270 ⁇ Bs4C-S) into Nicotiana benthamiana leaves.
  • both Bs4C alleles are under transcriptional control of their own promoters.
  • the Bs4C alleles were delivered either solely or in combination with TALE genes avrBs3, avrBs3Arep16 or avrBs4, each driven by the constitutive cauliflower mosaic virus 35S promoter. Dashed lines mark inoculated areas.
  • tumefaciens cultures containing strains with distinct C. pubescens Bs4C alleles or TALE genes were adjusted to an OD600 of 0.8 and equal volumes were mixed prior infiltration of N. benthamiana leaves. Four days post infiltration, leaves were harvested and cleared with ethanol to visualize the HR (dark areas).
  • FIG. 9 Constitutive expression of the coding sequence of the Bs4C-R or Bs4C-S allele triggers HR.
  • the C. pubescens Bs4C-R gene contains a functional L/P7-AvrBs4 box that is required and sufficient to mediate AvrBs4-mediated transcriptional activation.
  • the 35S promoter-driven TALE genes (indicated on the left) and the promoter-i// ' aW reporter constructs (indicated on the top) were delivered via A. tumefaciens into N. benthamiana leaves.
  • GUS assays were carried out at 38 hpi. Leaf discs were stained with 5-bromo-4-chloro-3-indolyl-p- D-glucuronic acid to visualize activity of the GUS reporter.
  • the TALE code suggests that AvrBs4 has a higher affinity for the Bs4C-R as compared to the Bs4C-S promoter.
  • the two uppermost rows show the repeat variable residues (RVDs) of the AvrBs4 DNA binding domain in the single letter code as designated on the far left side.
  • Letters in the third row are base preferences of the AvrBs4 RVDs according to the TALE code.
  • Letter height represents the preferences relative to other nucleotides for that RVD.
  • the two nucleotide sequences on the bottom represent the functional L/P7-AvrBs4 box in the Bs4C-R promoter and the corresponding non-functional i/pf-AvrBs4 box in the Bs4C-S promoter.
  • Grey background highlights the two base pairs that differ between the L/P7-AvrBs4 and the upt-AvrBs4 box.
  • AvrBs4 repeats two (NG type RVD) and three (Nl type RVD) align to TALE-code predicted nucleotides only in the L/P7-AvrBs4 box of the Bs4C-R promoter but not in the i/pfAvrBs4 box of the Bs4C-S promoter.
  • Electrophoretic mobility shift assays show that AvrBs4 has high and low affinity to the C. pubescens Bs4C-R and Bs4C-S promoters, respectively.
  • a 25x, 50x and 100x molar excess of non-labeled Bs4C-R or Bs4C-S fragments were used for competition experiments. Positions of the bound and free probes are indicated.
  • nucleic acid molecule refers to a DNA or RNA molecule in single or double stranded form.
  • isolated nucleic acid sequence refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.
  • protein or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin.
  • isolated protein is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
  • an isolated nucleic acid sequence comprising a nucleotide sequence of TATAAN1 N2AN3TAN4TCCN5CTN6 (SEQ ID N0.1 ), wherein each N is independently selected from the group consisting of A, C, T and G.
  • N-i and/or N 2 is independently selected from T and A; and/or N 6 is independently selected from T and C.
  • isolated nucleic acid sequences are provided comprising a nucleotide sequence according of
  • TATAAN 1 N 2 AN 3 TAN 4 TCCN 5 CTN 6 (SEQ ID NO.1 ), wherein Ni and N 2 are both T, and N 6 is T or wherein N-i and N 2 are both A, and N 6 is C, wherein furthermore N 3 , N 4 and N 5 are independently selected from the group consisting of A, C, T and G.
  • Said isolated nucleic acid sequences are in particular capable of activating gene transcription, preferably in the presence of a TALE protein, more preferably in the presence of a functional AvrBs4 protein. It is understood that according to the invention, activating gene transcription involves increasing transcription levels of a gene. Such an increase in transcription levels is at least 1.5-fold, more preferably at least two-fold, more preferably at least 5-fold. The increase in transcription levels is in comparison to a gene not comprising said isolated nucleic acid sequence of SEQ ID NO.1 , preferably in the presence of a TALE protein, more preferably in the presence of a functional AvrBs4 protein. The skilled person is capable of determining transcription levels of a gene. Preferably, said isolated nucleic acid sequences are capable of activating plant gene transcription.
  • said isolated nucleic acid sequences are binding elements for a TALE protein.
  • Such isolated nucleic acid sequences may also be referred to as UPT, or UPT- box.
  • UPT ⁇ s an abbreviation for UPregulated by Tale.
  • An UPT may indicate an active UPT- box, whereas upt may indicate an inactive UPT-box.
  • a binding element according the invention is a nucleic acid sequence to which a TALE protein can bind.
  • said TALE protein is a functional AvrBs4 protein, preferably the functional AvrBs4 protein is a protein with a sequence as disclosed and described e.g. in Bonas, U., Conrads-Strauch, J., and Balbo, I.
  • AvrBs4 protein advantageously comprises a Repeat Variable Diresidues (RVDs) region having the following putative diaminoacid sequence: NI, NG, NI, NI, NG, NG, NI, NS, NG, NI, NS, NG, HD, HD, NS, HD, NG, NG.
  • RVDs Variable Diresidues
  • Said RVDs of the AvrBs4 protein may have a target sequence of TATAATTAATAATCCACTT (SEQ ID NO.1 1 ) or TATAAAAAATAATC CACTC (SEQ ID NO.12). It may easily be determined whether an isolated nucleic acid sequence according to the invention can serve as a binding element for a TALE protein. This can, for example, be determined under conditions such as described in the examples with an electrophoretic mobility assay (EMSA), the result of which is depicted in figure 12 wherein it is shown for SEQ ID NO.12.
  • ESA electrophoretic mobility assay
  • the isolated nucleic acid sequence is inserted in a gene normally not activated by a functional AvrBs4.
  • transcription can be activated when a functional AvrBs4 protein is present, see i.a. Figure 10 wherein it is shown that by insertion of UPT Av rBs4 (SEQ ID N0.1 ) in the Bs3 gene, the Bs3 gene now becomes activated by the AvrBs4 protein (SEQ ID NO.50).
  • functional AvrBs4 protein or functional TALE protein includes the said AvrBs4 protein (SEQ ID NO.50) or a functional equivalent protein thereof, which is capable of binding to the isolated nucleic acid sequences of the invention, preferably at least the nucleic acid sequence of SEQ ID NO.12, that may serve as a binding element for a TALE protein.
  • Such a functional equivalent protein may be a TALE protein comprising a Repeat Variable Diresidues (RVDs) region having the following putative diaminoacid sequence: Nl, NG, Nl, Nl, NG, NG, Nl, NS, NG, Nl, NS, NG, HD, HD, NS, HD, NG, NG, or variants thereof, which are capable of exerting the same function, i.e. binding to an isolated nucleic acid sequence according to the invention, preferably at least the nucleic acid sequence of SEQ ID NO.12, and capable of activating gene transcription.
  • said functional AvrBs4 protein is derived from a bacterium of the genus Xanthomonas.
  • a plant pest resistance gene comprising an isolated nucleic acid sequence according to the invention that is a target for a TALE protein, e.g. the AvrBs4 protein.
  • Said isolated nucleic acid sequence is a nucleotide sequence according to TATAAAAAATAGTCCTCTC (SEQ ID NO.12).
  • SEQ ID NO.12 When comparing SEQ ID NO.12 with the predicted target sequence SEQ ID N0.1 1 taking into account the TALE rule-set for target nucleic acid sequence recognition as stipulated, only at three positions (underlined nucleotides 6, 7 and 19: TATAAAAAATAGTCCTCTC) there appears to be no perfect match.
  • SEQ ID NO.12 and 13 are derived from genes isolated from the Bs4C-R gene isolated from Capsicum pubescens genotype PI 235047 and Bs4C-S gene isolated from Capsicum pubescens genotype PI 585270, respectively.
  • said isolated nucleic acid sequence according to the invention may comprise a nucleotide sequence having at most four, preferably three, preferably two, preferably one, preferably none of the nucleotides of that sequence binding to the putative diaminoacids as listed above. It can easily be determined whether such a nucleotide sequence complies with this embodiment by taking into account the rules for RVD affinity as stipulated above. As is shown in the examples, nucleic acid sequences that may have more than three nucleotides not binding with the AvrBs4 RVDs may not be activating gene transcription, e.g. such as SEQ ID NO.13 which has five nucleotides not binding to the AvrBs4 RVDs (see i.a. figure 10 and 1 1 ). Nucleotide sequence capable of providing pest resistance
  • the invention provides for an isolated nucleotide sequence capable of providing plant pest resistance comprising a nucleic acid sequence selected from the group consisting of:
  • nucleic acid sequence of SEQ ID NO. 2 or a nucleic acid sequence having at least 60% sequence identity with the nucleic acid sequence of SEQ ID NO.2.
  • nucleic acid sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO.3, wherein at least one amino acid is substituted, deleted, inserted or added, and wherein the polypeptide is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO.3, and
  • nucleic acid sequence that hybridizes under stringent hybridization conditions to the nucleic acid sequences of a), b), c), or d) or the reverse complement thereof and that encodes a plant pest resistance factor.
  • Sequence identity is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity” per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.
  • Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1 ):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference polynucleotide sequence.
  • a polynucleotide having a nucleotide sequence that is at least 95% identical with a reference nucleotide sequence up to 5% of the nucleotides in the reference nucleotide sequence may be deleted and/or substituted with another nucleotide, and/or a number of nucleotides up to 5% of the total nucleotides in the reference nucleotide sequence may be inserted into the reference nucleotide sequence.
  • These mutations of the reference nucleotide sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference nucleotide sequence or in one or more contiguous groups within the reference nucleotide sequence.
  • Stringent hybridisation conditions can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt
  • Stringent conditions for RNA-DNA hybridisations are for example those which include at least one wash in 0.2X SSC at 63°C for 20 min, or equivalent conditions.
  • Stringent conditions for DNA-DNA hybridisation are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001 ).
  • Embraced by the present invention are also functional equivalents of the nucleic acid sequence coding for the plant pest resistance factor of the present invention, i.e., nucleotide sequences that hybridize under stringent conditions to the nucleic acid sequence of SEQ ID NO.2.
  • Functional equivalents of the plant pest resistance factor from other organisms can be found by hybridizing a nucleic acid sequence with SEQ ID NO.2 with genomic DNA isolated from other organisms.
  • the isolated nucleotide sequence capable of providing plant pest resistance comprises a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the nucleic acid sequence of SEQ ID NO.2.
  • a non-limiting example of such nucleotide sequence is the nucleic acid sequence of SEQ ID N0.24.
  • the isolated nucleotide sequence capable of providing plant pest resistance comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO.3, or a polypeptide having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO.3.
  • a non- limiting example of such a polypeptide is a polypeptide comprising the sequence of SEQ ID NO.4-10 and SEQ ID N0.25.
  • the isolated nucleotide sequence capable of providing plant pest resistance comprises a nucleic acid sequence encoding a polypeptide having at least 85%, 90%, 95%, 98% or 99% sequence identity with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO.4-10 and SEQ ID N0.25.
  • nucleic acid sequences having a certain percentage of sequence identity with a reference nucleic acid sequence as listed above may be functionally equivalent to the reference nucleic acid sequence, e.g. to the nucleic acid sequence of SEQ ID NO.2.
  • nucleic acid sequences referred to herein are capable of performing the same function of the reference nucleic acid sequence, for instance, the nucleic acid sequence of SEQ ID NO.2.
  • polypeptide that is encoded by the nucleic acid sequences disclosed herein is a functional polypeptide, i.e. the polypeptide is capable of providing plant pest resistance. Such polypeptides are also referred to as a "plant pest resistance factor".
  • polypeptides that are encoded by the nucleic acid sequences of the present invention advantageously have a function in providing plant pest resistance.
  • the isolated nucleic acid sequences that encode polypeptides having a certain percentage of sequence identity with a reference amino acid sequence as listed above may also be functionally equivalent, i.e. may be capable of providing plant pest resistance.
  • nucleic acid sequences 1 , 2, 3, 4 or more nucleotides may be mutated and in the polypeptide sequences 1 , 2, 3, 4 or more amino acids may be mutated as well, provided that the polypeptide is capable of providing plant pest resistance. Mutations may include deletions, insertions or changes of nucleotides and/or amino acids.
  • the isolated nucleotide sequence according to the invention capable of providing plant pest resistance encodes a polypeptide.
  • the pest is a bacterium, more preferably a bacterium from the genus Xanthomonas, most preferably a bacterium of the genus Xanthomonas encompassing a genome encoding a functional TALE protein, which is preferably a functional AvrBs4 protein.
  • Said isolated nucleotide sequence capable of providing plant pest resistance preferably is operably linked to gene expression elements, such that the polypeptide can be expressed in a plant.
  • gene expression elements may include, without limitation, promoter elements, 5'UTR and 3'UTR encoding sequences, and enhancer elements.
  • an isolated nucleotide sequence capable of providing plant pest resistance may also include intron sequences. This means that the DNA sequence encoding the polypeptide may include intronic sequences. It is to be noted that such intron sequences are not taken into account when determining sequence identity.
  • operably linking said isolated nucleotide sequence capable of providing plant pest resistance to gene expression elements is to be understood that any nucleotide sequence may be included that allows the production of a transcript that can be translated in said polypeptides.
  • Operably linking nucleotide sequences and gene expression elements includes combining them into a contiguous DNA sequence. It is also understood that other elements or sequences may be included as well. Hence, as long as the gene expression elements can have its function such that the polypeptide may be expressed, such operably linking may be contemplated.
  • the present inventors have found that the polypeptide encoded by SEQ ID NO.2, also referred to as BsC4 (SEQ ID NO.3) can induce cell death and by inducing cell death may provide plant pest resistance. Inducing cell death may be advantageous not only upon infection by a bacterium of the genus Xanthomonas comprising a gene encoding a functional AvrBs4 protein, but also in other situations wherein organisms or cells profit from induction of apoptosis, programmed cell death. For example, many plant pests are biotrophic: their growth and spread depends on living tissue.
  • the isolated nucleotide sequences capable of providing plant pest resistance as listed above may be coupled to various other plant pest- inducible gene regulation elements as well.
  • any of the isolated nucleotide sequences as listed above that may be binding elements for a TALE protein such as AvrBs4 may also be operably linked to other nucleotide sequences encoding polypeptides, i.e. not the polypeptides of the invention, e.g., other plant pest resistance factors, to activate its transcription upon binding of a TALE protein, e.g., AvrBs4.
  • plant pest resistance factor may be a factor capable of providing resistance to one or more plant pests.
  • the plant pest resistance factor may perform its function by any means known in the art, for example by inducing cell death and/or other cell death inducing transcripts, e.g.
  • transcripts capable of inducing PTGS or TGS against an essential gene of the cell or any other transcript that may hampers the in planta growth of the pest.
  • the isolated nucleotide sequences as listed above that may be binding elements for a TALE protein such as AvrBs4 show a highly regulated activation of gene expression.
  • a chimeric gene comprising an isolated nucleotide sequence capable of providing plant pest resistance according to the present invention and/or an isolated nucleotide sequence capable of activating gene transcription according to the present invention.
  • Said chimeric gene is preferably capable of providing plant pest resistance such as referred to above.
  • the term "gene” means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA or miRNA) in a cell, operably linked to suitable gene regulatory regions (e.g. a promoter).
  • a gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g.
  • a "chimeric gene” refers to any gene, which is not normally found in nature in a species, in particular a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature.
  • the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region.
  • the Bs4C-R UPT-box (as shown in SEQ ID NO.12) was inserted in a Bs3-E gene promoter. The resulting gene may be regarded s a chimeric gene according to the invention.
  • an isolated nucleotide sequence capable of providing plant pest resistance according to the invention is operably linked to an isolated nucleotide sequence capable of activating gene transcription according to the invention (herein also referred to as "UPT-box").
  • Operably linking these sequences may include inserting (or introducing by modifying nucleotides of an endogenous DNA sequence) an isolated nucleotide sequence capable of activating gene transcription according to the invention 5' of a nucleotide sequence capable of providing plant pest resistance identified herein.
  • the UPT-box is preferably 5' of the transcript encoding sequence.
  • the sequence may be represented as: 5 ' - UPT-box - nucleic acid sequence encoding plant pest resistance factor - 3'.
  • the UPT-box is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides 5' of the transcription start site.
  • the UPT-box is located at most 300, 250, 200, 150. 125, 100
  • the isolated nucleotide sequence according to this embodiment may be at least part of a chimeric gene.
  • the isolated nucleotide sequence according to this embodiment may comprise a sequence according to SEQ ID NO.14, or a functional equivalent thereof, i.e. a nucleic acid sequence having at least 65%, 10 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the nucleic acid
  • SEQ ID NO.14 is the DNA sequence encoding the Bs4C-R gene isolated from Capsicum pubescens PI 235047.
  • a vector comprising
  • an isolated nucleotide sequence capable of providing plant pest resistance according to the present invention operably linked to an isolated nucleotide sequence capable of 20 activating gene transcription of the present invention, and/or
  • a host cell comprising a chimeric gene according to the invention or a vector according to the invention.
  • Said host cell may be an Agrobacterium suitable for transforming a plant cell or plant protoplast, such that the Agrobacterium can 25 confer plant pest resistance to a plant cell or protoplast and thus also to a plant.
  • the host cell according to the invention is a plant cell or a plant protoplast.
  • a "host cell” refers to a new individual cell (or organism) arising as a result of at least one nucleic acid sequence, especially comprising a chimeric gene as defined herein.
  • the host cell is preferably a plant cell or plant protoplast, or a bacterial cell.
  • the host cell may 30 contain the nucleic acid construct as an extra-chromosomally (episomal) replicating molecule, or more preferably, comprises the chimeric gene integrated in the nuclear or plastid genome of the host cell.
  • host may also refer to the host plant species, but this will be clear from the context. Plant species are classified as “host” (including primary and/or secondary hosts) or “non-host” species in relation to, for instance, insect pests.
  • polypeptides of the invention can induce cell death in plant cells and by inducing cell death may provide plant pest resistance. By inducing cell death, the spread of infection by a pest may be prevented. Hence, by expression of a polypeptide according to 5 SEQ ID NO.3 upon infection by a pest, the growth and spread of said pest may be
  • the response invoked by the Bs4C polypeptide may also be referred to as a hypersensitive response (HR).
  • HR hypersensitive response
  • the present invention relates to a polypeptide comprising an amino acid sequence of SEQ ID NO.3 or a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%,
  • polypeptides may be functional polypeptides according to the invention.
  • Functional polypeptides according to the invention indicates that these proteins are capable of providing plant pest resistance, preferably through inducing cell death.
  • functional peptides have the same functionality as the polypeptide referred to as Bs4C (as represented by SEQ ID NO.3). It can easily be determined whether a polypeptide according to the invention is functional, for example, by determining the growth and/or HR score in
  • polypeptides of the invention also include proteins capable of providing plant pest resistance, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO: 1
  • such proteins comprise from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.
  • polypeptides of the invention also include proteins capable of providing plant pest resistance, which have been derived, by way of one or more amino acid substitutions,
  • such proteins comprise from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid
  • sequence of SEQ ID NO. 3 is intended that the amino acid sequence of the polypeptide is identical to the reference amino acid sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid sequence.
  • the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid sequence.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference amino acid sequence may be inserted into the reference amino acid sequence.
  • alterations of the reference amino acid sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference amino acid sequence or in one or more contiguous groups within the reference amino acid sequence.
  • the use is provided of an isolated nucleic acid sequence according to the invention, chimeric gene according to the invention, vector according to the invention or host cell according to the invention for providing pest resistance to a plant.
  • said pest is a bacterium, more preferably a bacterium from the genus Xanthomonas, and most preferably a bacterium of the genus Xanthomonas that comprises (for example, in its genome) a gene encoding a functional AvrBs4 protein.
  • a method for providing a plant having resistance against infection by a pest preferably wherein the pest is a bacterium, preferably a bacterium of the genus Xanthomonas, preferably a bacterium of the genus Xanthomonas comprising a gene encoding a functional AvrBs4 protein, comprising:
  • transforming a plant with a chimeric gene or vector comprising a nucleic acid sequence represented by SEQ ID N0.1 , SEQ ID N0.1 1 or SEQ ID NO.12, operably linked to a nucleotide sequence encoding a plant pest resistance factor; or
  • the plant pest resistance factor may be a plant pest resistance factor according to the invention. Alternatively, it may be any plant pest resistance factor known in the art.
  • the method for providing a plant having resistance against infection by a pest may also include transforming a plant with a nucleic acid sequence encoding a plant pest resistance factor according to the invention, operably linked to a plant pest inducible promoter.
  • Such plant pest inducible promoter may not necessarily comprise the nucleic acid sequence of SEQ ID N0.1 , 1 1 , or 12.
  • chimeric genes and vectors according to the invention may be advantageously used in a method to provide a plant having resistance against infection by a pest.
  • plants may be transformed with these sequences such that a plant not having resistance against a pest may acquire resistance against the pest.
  • a chimeric gene capable of providing pest resistance to a plant may be inserted in a plants genome, e.g. by transformation with an Agrobacterium comprising the chimeric gene and/or vector.
  • a nucleotide sequence operably linked to an endogenous nucleotide sequence encoding a plant pest resistance factor in a plant may be modified such that it comprises the nucleic acid sequence of any one of SEQ ID N0.1 , SEQ ID N0.1 1 or SEQ ID NO.12.
  • the modification is such that the nucleic acid sequence of any one of SEQ ID N0.1 , SEQ ID NO.1 1 or SEQ ID NO.12 is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides 5' of the transcription start site.
  • it is located at most 300, 250, 200, 150, 125, 100 nucleotides 5' of the transcription start site.
  • Capsicum pubescens PI 585270 the nucleotide sequence of SEQ ID NO.13 is operably linked to a nucleic acid sequence encoding a functional plant pest resistance factor.
  • said plant pest resistance factor is not expressed, and said plant is not resistant to Xanthomonas infection.
  • the endogenous gene SEQ ID NO.17
  • the nucleotide sequence that may be expressed may than comprise the sequence according to SEQ ID NO.18), which is a putative transcript sequence. Normally, the endogenous gene is silent and normally does not produce any detectable transcript, at least it is not known how this gene may be activated. By the current invention at least, this gene may be modified such that it may become activated by Xanthomonas infection, i.e. by the AvrBs4 protein, thereby conferring resistance thereto to a plant. Such nucleotide modifications may be introduced into the genome using mutagenesis.
  • Mutagenesis comprises the introduction of mutations into the genome. This can be done randomly, for example by exposing cells to a mutagen, such as for instance UV radiation or a mutagenic agent. Mutagenesis can also be targeted, i.e. agents can be introduced in a cell that are intended to introduce a specific mutation in a specific target endogenous DNA sequence. Such mutagenesis is referred to as targeted mutagenesis. Targeted mutagenesis comprises any technology with which one or more directed mutations can be introduced into the genome of a target organism. Technologies suitable for targeted mutagenesis are for example KeyBase®, Targeted Nucleotide Exchange (TIME), Oligonucleotide Directed
  • ODM Oligonucleotide Directed Targeted Mutagenesis
  • ODTM Oligonucleotide Directed Targeted Mutagenesis
  • Talens Zinc Fingers
  • a plant is provided obtainable or obtained by any of the methods of the present invention.
  • Said plants may comprise a chimeric gene according the invention or a vector according to the invention, or may comprise a one or more mutations in its nucleic acid sequences resulting in the introduction of any one of SEQ ID N0.1 , SEQ ID N0.1 1 or SEQ ID NO.12.
  • Xanthomonas infections occur on hundreds of monocotyledonous and dicotyledonous plants, and as such, the plant may be any monocotyledonous or dicotyledonous plant, particularly such a plant that is sensitive to Xanthomonas infection.
  • the plant is selected from the group consisting of pepper, rice, citrus, cotton, tomato, and soybean plants, monocotyledonous plants or dicotyledonous plants, belonging to the family Brassicaceae or Cruciferae for example, the plant belongs to the genus Brassica (e.g. B. napus, B. juncea, B. oleracea, B. rapa, etc) or any other family, such as to the Solanaceae.
  • Solanum including Lycopersicon
  • Nicotiana Capsicum
  • Petunia and Tobacco Naturaltiana species, e.g. N.
  • benthamiana N. plumbaginifolia, N. tabacum, etc.
  • vegetable species such as tomato (L. esculentum, syn. Solanum lycopersicum) such as e.g. cherry tomato, var. cerasiforme or currant tomato, var. pimpinellifolium) or tree tomato (S. betaceum, syn.
  • Cyphomandra betaceae potato (Solanum tuberosum), eggplant (Solanum melongena), pepino (Solanum muricatum), cocona (Solanum sessiliflorum) and naranjilla (Solanum quitoense), peppers (Capsicum annuum, Capsicum frutescens, Capsicum baccatum), ornamental species (e.g. Petunia hybrida, Petunia axillaries, P. integrifolia). or Gramineae.
  • Suitable host plants include for example maize/corn (Zea species), wheat (Triticum species), barley (e.g. Hordeum vulgare), oat (e.g.
  • glaucum tree species ⁇ Pinus, poplar, fir, plantain, etc), tea, coffee, oil palm, coconut, vegetable species, such as pea, zucchini, beans (e.g. Phaseolus species), cucumber, artichoke, asparagus, broccoli, garlic, leek, lettuce, onion, radish, turnip, Brussels sprouts, carrot, cauliflower, chicory, celery, spinach, endive, fennel, beet, fleshy fruit bearing plants (grapes, peaches, plums, strawberry, mango, apple, plum, cherry, apricot, banana, blackberry, blueberry, citrus, kiwi, figs, lemon, lime, nectarines, raspberry, watermelon, orange, grapefruit, etc.), ornamental species (e.g. Rose, Petunia,
  • Chrysanthemum Lily, Gerbera species
  • herbs mint, parsley, basil, thyme, etc.
  • woody trees e.g. species of Populus, Salix, Quercus, Eucalyptus
  • fibre species e.g. flax (Linum usitatissimum) and hemp (Cannabis sativa).
  • any plant may be suitable in a method of the invention.
  • the plant may be susceptible to infections by a bacterium of the genus
  • an isolated nucleic acid marker for identifying a plant pest resistance gene, wherein the nucleic acid marker has at least 90%, 95%, or 100% sequence identity to a nucleic acid sequence of at least 20 contiguous nucleotides from a nucleic acid sequence SEQ ID NO 14. Said sequence encodes the Bs4C-R gene which has been found to provide for plant pest resistance. Hence, the presence or absence of such nucleic acid markers, e.g. in the genome of a plant, may be indicative of the presence or absence of a plant pest resistance gene.
  • the identified plant pest resistance gene may be active or that the encoded polypeptide is expressed, as is clear from the example section wherein such a marker was used to identify Bs4C-R genes in other plants. Identifying such inactive genes may be advantageous, because, by now having the UPT-box available, e.g. SEQ ID NO.12, pest disease resistance may be introduced in such identified inactive genes.
  • the isolated nucleic acid marker according to the invention is a gene expression marker and the nucleic acid marker has at least 90%, 95%, or 100% sequence identity to a nucleic acid sequence of at least 20 contiguous nucleotides of SEQ ID NO.15 (Bs4C mRNA/cDNA sequence).
  • the presence or absence of said expression marker indicates whether a possible plant pest resistance gene is expressed in the plant, i.e. would provide for the presence of a plant pest resistance factor in the cell.
  • the isolated nucleic acid marker according to the invention is a gene expression marker and the nucleic acid marker has at least 90%, 95%, or 100% sequence identity to a nucleic acid sequence of at least 20 contiguous nucleotides of SEQ ID NO.16.
  • SEQ ID NO.16 was identified as described in the examples.
  • SEQ ID NO.16 comprises the 3' end of a transcript that is expressed, i.e. the 3'-end before the polyA signal.
  • an expressed transcript and/or identified sequence therefrom may also comprise a sequence according to SEQ ID NO.16 (or e.g. SEQ ID NO.15) comprising at the 3' end a polyA sequence.
  • an isolated nucleic acid marker according to the invention for identifying a pest resistance gene, preferably in a plant, and wherein preferably the pest is a bacterium, preferably wherein the bacterium is of the genus Xanthomonas, preferably wherein the bacterium of the genus Xanthomonas comprises (in its genome) a gene encoding a functional AvrBs4 protein.
  • the isolated nucleic acid markers according to the invention are in particular useful in identifying the presence or absence of a plant pest resistance gene.
  • a plant pest resistance gene For example, and as shown in the examples, by analysing genomic sequences of the plant species Capsicum pubescens, Solanum lycopersicon, Vitis vinifera, Nicotiana benthamiana, Solanum pimpinellifolium, Solanum tuberosum homologous genes were identified encoding plant pest resistance factors having substantial identity with SEQ ID NO.2. These identified pest resistance factors are not found in EST (expressed sequence tag) databases. Hence, without wishing to be bound by theory, this may indicate that these genes can also be tightly regulated by a plant pest or may have, when they are expressed, a highly similar function as the amino acid sequence of SEQ ID NO.3.
  • isolated nucleic acid markers according to the invention are useful in identifying plants obtained or obtainable by any of the methods according to the invention for providing a plant having resistance against infection by a pest.
  • a method for identifying a plant pest resistance gene comprising the steps of:
  • a plant cell infecting a plant cell with a microorganism comprising (in its genome) a gene encoding a TALE protein, preferably a microorganism from the genus Xanthomonas comprising (in its genome) a gene encoding a functional AvrBs4 protein;
  • TALE proteins may not access chromatin-embedded DNA
  • putative TALE binding elements such as described above for example for a functional AvrBs4 protein
  • a functional TALE protein such as a functional AvrBs4 protein and analyse the transcriptome to identify genes that are activated, indicating that they are accessible for the TALE protein and not part of inaccessible DNA.
  • the method for identifying a plant pest resistance gene may include the step of comparing the transcriptome of a plant not infected with the microorganism, with the transcriptome infected with the microorganism.
  • the method may also include the step of comparing the transcriptome of a plant infected with the microorganism not encoding a TALE protein, e.g. a functional AvrBs4 protein, with the transcriptome infected with the
  • the analysis of the transcriptome may include determining the relative expression levels of the transcriptome, thereby identifying genes that have an increase in transcription in the presence of a functional AvrBs4 protein.
  • the method for identifying a plant pest resistance gene may comprise in the analysis of the transcriptome the detection of the presence or absence of an isolated genetic marker according to the invention.
  • the expression may be tightly controlled, e.g. when induction of a gene induces a hypersensitive response, in particular genes that are only activated in the presence of a TALE protein, e.g. a functional AvrBs4 protein, may not be detected in the absence of the TALE protein.
  • the method for identifying a plant pest resistance gene may include the application and/or introduction of a functional AvrBs4 protein onto or into a plant or a part of a plant.
  • the method for identifying a plant resistance gene according to the invention includes comparing a transcriptome of a plant resistant to the microorganism with a transcriptome of a plant sensitive to the microorganism, said microorganism encoding a TALE protein, preferably the microorganism from the genus Xanthomonas encoding a functional AvrBs4 protein.
  • a plant pest resistance gene may be more efficiently identified.
  • marker sequences may be used for example in the analysis of sequences, obtained in high throughput sequencing or as available in sequence databases, in alignments therewith.
  • said markers may also be used in microarrays or PCR methods, wherein nucleic acid molecules are used that are (partially) complementary to a genetic marker of the invention.
  • any method with which a marker sequence of the invention may be detected and/or quantified e.g. sequencing, microarray, Southern blot, PCR, qPCR etc, is envisaged to be used in a method of the invention.
  • the transcriptional activation domain of the TALE protein AvrBs4 is necessary in triggering the Bs4C immune response in pepper
  • the TALE protein AvrBs4 triggers HR not only in tomato Bs4 plants but also in certain pepper (C. pubescens) genotypes.
  • C. pubescens C. pubescens
  • PI 235047 and PI 585270 two C. pubescens genotypes, that either show or lack an AvrBs4-triggered HR, respectively.
  • the observed HR was highly specific as it was triggered only by xanthomonads containing AvrBs4 (XcvAvrBs4) but not by an isogenic strain containing AvrBs3 (XcvAvrBs3), a TALE with 96% sequence identity (Fig.1 ).
  • a deletion derivative was tested that lacks its transcriptional activation domain (AvrBs4AAD). Only AvrBs4 but not AvrBs4AAD triggers the Bs4C HR (Fig. 1 ) indicative that recognition of AvrBs4 by pepper Bs4C relies on its function as a transcriptional activator.
  • Transcriptome profiling identifies a transcript that is detectable exclusively upon infection with xanthomonads that contain AvrBs4
  • transcriptome profiling was initiated to identify Bs4C candidates.
  • transcriptome profiling we inoculated the resistant (PI 235047) and susceptible (PI 585270) C. pubescens accessions with XcvAvrBs4 or Xcv. Plant tissue was harvested 0, 6, 12, 18 and 24 hours post inoculation, total RNA was isolated and 18 lllumina 3' end tag-profiling libraries were established. In total, 26,7 M reads of 76 nucleotides (nt) were generated, filtered on quality and clustered based on similarity of the first 31 nt after removal of the sample identification tag. Three mismatches were allowed to accommodate sequencing errors and differences between the two C. pubescens accessions.
  • clusters generally represent transcripts derived from one gene with the normalized number of reads providing a measure for their expression levels. Using these considerations we searched for transcripts that are present only upon inoculation of XcvAvrBs4 but that are nearly absent upon inoculation of the isogenic Xcv strain.
  • XcvAvrBs4-inoculated resistant parent 10-fold increased transcript as compared to Xcv- inoculated plants at any time point as well as non-inoculated control plants. Inspection of the resulting 32 candidates revealed that the gene corresponding to 'cluster 12600' was the most likely Bs4C candidate since corresponding reads were detectable only upon inoculation of XcvAvrBs4 but mostly undetectable upon inoculation of the isogenic Xcv strain.
  • Cluster 12600' defines a Bs4C candidate that is transcriptionally activated by AvrBs4 in the presence of the translational inhibitor
  • 'cluster 12600' contained only the 62 most 3' nucleotides of the Bs4C candidate transcript.
  • the XcvAvrBs3Arep16 induced HR is due to functional copies of the Bs3 gene in both C. pubescens accessions that are transcriptionally activated by AvrBs3Arep16, an AvrBs3 repeat deletion derivative that lacks repeats 1 1 -14, but not by AvrBs3 itself (Fig. 5 and 6).
  • RT- PCR studies showed that transcripts corresponding to the identified Bs4C candidate were not detectable upon inoculation of the HR-inducing strain XcvAvrBs3Arep16, indicating that the Bs4C candidate is activated in the AvrBs4 but not the AvrBs3Arep16 induced defense response (Fig. 3).
  • the Bs4C candidate may not simply activated at the onset of the immune response but may be induced specifically by AvrBs4.
  • the PI 235047- but not the PI 585270-derived Bs4C candidate mediates recognition of AvrBs4
  • To further investigate the Bs4C candidate we established a locus-specific DNA marker that discriminates between PI 235047- and PI 585270-derived amplicons. Genotyping of 17 individuals of a C. pubescens F2 population (PI 235047 X PI 585270) revealed complete linkage of the candidate locus and the Bs4C triggered HR (Fig. 7). Thus genetic mapping supports this candidate as being the Bs4C gene.
  • the Bs4C gene structure was determined by rapid amplification of cDNA ends (RACE) and in silico analysis of the C. annuum sequence 'contig 184552' that contained the 'cluster 12600' A 495-bp Bs4C candidate open reading frame (ORF) (SEQ ID NO.2) including 260 nucleotides of 5' regulatory sequence was PCR amplified from the resistant C. pubescens accession PI 235047 and cloned into a T-DNA vector.
  • RACE rapid amplification of cDNA ends
  • ORF 495-bp Bs4C candidate open reading frame
  • Agrobacterium tumefaciens mediated co-delivery of the PI 235047-derived Bs4C candidate and avrBs4, driven by the constitutive cauliflower mosaic virus 35S (35S) promoter, into Nicotiana benthamiana leaves triggered HR, demonstrating that the identified C. pubescens gene is indeed Bs4C (Fig. 8).
  • the PI 235047-derived Bs4C allele also referred to as 'Bs4C- R' did not mediate recognition of the Xcv TALE proteins AvrBs3 or its derivative AvrBs3-A- rep16.
  • the UPT-AvrBs4 box sequence element was inserted into the pepper Bs3 promoter (SEQ ID NO. 22), which was shown to contain a functional UPT-AvrBs3 but not a functional UPT- AvrBs4 box.
  • GUS assays showed that AvrBs3 and AvrBs4 transcriptionally activate the Bs3 promoter derivative containing the predicted UPT-AvrBs4 box (Fig. 5) whereas the wild-type Bs3 promoter is activated only by AvrBs3 but not AvrBs4.
  • upt-AvrBs4 box SEQ ID NO. 13
  • Two nucleotide polymorphisms were identified that align to repeats two and three of the AvrBs4 repeat domain (Fig. 1 1 ).
  • the UPT-AvrBs4 box of the Bs4C-R promoter may have a higher affinity to AvrBs4 as compared to the upt-AvrBs4 box of the Bs4C-S promoter (Fig. 1 1 ).
  • Electrophoretic mobility shift assays confirmed that the UPT-AvrBs4 box has a significantly enhanced affinity to AvrBs4 as compared with the upt-AvrBs4 box (Fig. 12).
  • the Bs4C-S promoter-derived upt-AvrBs4 box was inserted into the Bs3 promoter (SEQ ID NO 23). GUS assays showed that Bs3 promoter derivatives containing the upt-AvrBs4 box are not AvrBs4 but only AvrBs3 inducible (Fig. 10). This indicates that a two-basepair substitution polymorphism between the Bs4C-R promoter-derived UPT-AvrBs4 box and the Bs4C-S promoter derived uptAvrBs4 box determines resistance or susceptibility to xanthomonads containing the TALE protein AvrBs4.
  • Genomes of other species encode Bs4C-like genes that may be under tight
  • the Bs4C-R gene encodes a putative 164 amino acid protein that shares no significant homology to any other protein of known function.
  • EST databases do not contain corresponding cDNAs.
  • Bs4C homologs may be inactive and/or tightly regulated at the transcriptional level highly similar to the pepper Bs4C gene.
  • Pepper (Capsicum pubescens and C. annuum) and N. benthamiana plants were grown at 60 to 70% humidity (day 22°C [16 h light], night 18°C).
  • leaves of six-week-old pepper plants were syringe-inoculated with Xanthomonas (5x10 8 colony forming units [cfu] /ml in 10 mM MgCI 2 ).
  • HR phenotypes were scored over a period of 2-3 days post inoculation.
  • pepper leaves were infiltrated with 5x10 4 cfu/ml. After 0, 2, 4, 6, 8 and 1 1 days leaf discs (0.28 cm 2 ) were macerated in 1 mM MgCI 2 .
  • Bacterial numbers were determined by plating appropriate dilutions on selective NYG agar plates.
  • Escherichia coli TOP10 and DB3.1 (Life Technologies) were cultivated at 37°C in LB and Agrobacterium tumefaciens GV3101 pMP90 at 30°C in YEB medium.
  • RNA samples were harvested 6,12,18 and 24 hours upon infiltration for RNA isolation. Non Xcv-treated samples from both accessions served as controls. Total RNA was isolated using the RNeasy Plant Midiprep kit (Qiagen). From 18 RNA samples, 18 lllumina 3'end tag-profiling libraries were made with Mse ⁇ as restriction enzyme. Two lllumina GAM lanes were used to sequence the libraries. Samples sequenced in the same lllumina lane were tagged with sample-specific sample identification tags.
  • reads containing more than 46 A's e.g. polyA tails
  • N's or nucleotides with a phred score ⁇ 20 were removed.
  • all reads were trimmed to 36 nt.
  • the remaining 31 nt reads were clustered based on 100% similarity. To enhance clustering-efficiency, only clusters with at least 10 reads in total (i.e.
  • the read counts from these candidates were obtained by using only clusters with at least 10 reads after the first round of clustering, to enhance second round clustering efficiency.
  • Pepper leaves were syringe-infiltrated with the Xcv 85-10 and the isogenic strains and (5 ⁇ ⁇ 8 cfu/ml).
  • leaf tissue was inoculated with bacterial suspensions containing 50 ⁇ cycloheximide.
  • 24 hours upon infiltration 4 leaf discs (6 mm diameter) were harvested and RNA was prepared using the RNeasy Plant Miniprep kit (Qiagen). RNA concentrations were determined with a ND-1000 spectrophotometer
  • Bs4C, Bs3 orthologs and elongation factor (EF-1 a) transcript levels were determined using the oligonucleotides: 12600- fwd2 and 12600-rev1 for Bs4C, Prom+2-fwd and Prom+2-rev for Bs3, and EFrt-F1 and EFrt- R1 for EF-1a. 5 and 3 termini of the Bs4C transcript were isolated with the Clontech SMARTer RACE cDNA kit. Genetic mapping of Bs4C
  • Bs4C genomic fragments from both parental lines (PI 235047 and PI 585270) as well as from 17 F 2 individuals (PI 235047 X PI 585270) were amplified using the primers 12600-fwd2 and 12600-rev1 .
  • BglW cleaved PCR products were analyzed on a 3% agarose gel.
  • the Bs4C-R and Bs4C-S genes (495 bps coding sequence [cds] including the stop codon and 257 bps 5' regulatory sequence) were amplified from genomic DNA of C. pubescens accessions PI 235047 and PI 585270 using the primers 12600box1 -fwd and 12600+stopp- rev. PCR products were cloned into pENTR-D (Life Technologies), sequenced and recombined into the T-DNA vector pGWB3 . Similarly the Bs4C-R and Bs4C-S cds were amplified using the primers 12600 ATG-fwd and 12600+stopp-rev.
  • PCR products were cloned in pENTR-D and recombined into the T-DNA vector pGWB2 (Nakagawa et al., 2007) where the cds is under transcriptional control of the constitutive cauliflower mosaic 35S promoter (35S).
  • 35S constitutive cauliflower mosaic 35S promoter
  • Bs4C-R and Bs4C-S promoters about 1 kb 5' regulatory sequences were amplified using the primers 12600-79prom900-fwd and 12600box1 -rev , cloned in pENTR-D and recombined into pGWB3, which contains an uidA reporter gene to study promoter activity.
  • C. pubescens accessions PI 235047 and PI 585270 were PCR-amplified using the primers A1 -fwd and final-entry-02-rev. PCR fragments were cloned into pENTR-D, sequenced and recombined into the T-DNA vector pGWB1. All pGWB-derivatives were transformed into A. tumefaciens GV3101 pMP90 for transient in planta expression.
  • acetosyringone and adjusted to an OD 6 oonm of 0.8.
  • Equal amounts of A. tumefaciens strains containing a 35S-promoter driven TALE genes (avrBs4, avrBs3 or avrBs3Arep16) and TALE R genes (Bs4C-R, Bs4C-S or Bs3, Bs3-E and Bs3 orthologs; all under transcriptional control of their native promoter) were mixed and infiltrated into N. benthamiana leaves by blunt-end syringe infiltration. Leaves were harvested 4 days post inoculation and cleared in ethanol. beta-Glucuronidase (GUS) assay
  • GUS-staining solution 100 mM sodium phosphate [pH 7], 5 mM EDTA, 0.1 % Triton X-100, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, 1 mM 5-bromo-4-chloro-3-indo
  • Electrophoretic mobility shift assay (EMSA)
  • 6xHis-AvrBs4 was purified from E. coli Rosetta (DE3) pLaql with TALONTM resin (Clontech). Complementary pairs of labeled oligonucleotides of Bs4C-R (5 ' DY682-fwd and 5 ' DY682-rev) or Bs4C-S (5 ' DY782-fwd and 5 ' DY782-rev) or corresponding non-labeled primers were annealed.
  • Binding reactions contained 10 mM Tris-HCI (pH 7.5), 50 mM KCI, 1 mM DTT, 2.5% glycerol, 5 mM MgCI 2 , 25 ng/ ⁇ poly(dl'dC), 0.05% NP-40, 0.2 mM EDTA, 100 fmol 5 ⁇ 682- or 5 ' DY782-labeled DNA and 0, 2.5, 5, or 10 pmol unlabeled DNA and 5 pmol 6xHis-AvrBs4 fusion protein. Binding reactions were incubated at room temperature for 20 min. Reactions were resolved on 6% native polyacrylamide gels and DY682/782 labeled DNA was visualized with the Odyssey infrared imaging system (LI-COR Biosciences).
  • LI-COR Biosciences the Odyssey infrared imaging system
  • Consensus AvrBs4 UPT box (each N is independently selected from A, C, T and G)
  • SLBs4C source: Solanum lycopersicon; tomato
  • homolog on chromosome 8 sequence ID
  • VvBs4C (source: Vitis vinifera, GenBank: AM452480.1 )VvBs4C protein (133 aa)
  • NbBs4C (source: Nicotiana benthamiana) (Sequence ID: Niben.v0.3.Scf25292620); This gene lacks an ATG start codon and has instead an "ATA" codon (displayed in bold green font).
  • NbBs4C protein (133 aa)
  • SpBs4C (source: Solanum pimpinellifolium; contig 6737093).
  • SpBs4C protein (96 aa)
  • StBs4C-01 protein (105 aa) MEFDLRYLILIIINILKSIFLSNEWDPYHISVQLSFMTFMNRLVFLFFFFLHFLHHSYSNSSSNENTHKFKKI LLVFLYLMFSLIFTPTSVILVHFLHYNYGTSF
  • Bs4C-S gene DNA sequence, (Capsicum pubescens; PI 585270; not AvrBs4-inducible)
  • Bs4C-S (the sequence corresponding to the predicted UPTAvrBs4 box of the Bs4C-R promoter is underlined; bold font indicates the predicted translational start)
  • hotbox79in3-fwd Insertion of the UPTAvrBs4 box into the Bs3 promoter (360 bps fragment)) TATAAAAAATAGTCCTCTCCTGGTTAAACAATGAACACGTTTGC SEQ ID N0.42
  • hotbox68in3-fwd Insertion of the upt AvrBs4 box into the Bs3 promoter (360 bps fragment)
  • A1-fwd (Amplification of promotor:cds fragments of Bs3, Bs3-E and Bs3 orthologs)

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Abstract

Cette invention concerne des séquences d'acides nucléiques isolées qui sont impliquées dans la résistance aux phytoravageurs. En particulier, une séquence d'acide nucléique isolée capable d'activer la transcription d'un gène de résistance aux phytoravageurs est décrite, ainsi qu'une séquence d'acide nucléique isolée codant pour un polypeptide destiné à conférer une résistance aux phytoravageurs. Cette invention concerne également une plante à laquelle la résistance à un ravageur a été conférée à l'aide desdites séquences d'acides nucléiques isolées. De plus, des marqueurs d'acides nucléiques isolés destinés à identifier des gènes de résistance aux phytoravageurs et des procédés destinés à identifier un gène de résistance aux phytoravageurs sont également décrits.
PCT/EP2013/052101 2012-02-02 2013-02-01 Séquences nucléotidiques impliquées dans la résistance aux phytoravageurs WO2013113911A2 (fr)

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WO2020014664A1 (fr) * 2018-07-12 2020-01-16 Berkeley Lights, Inc. Le dépistage des protoplastes végétaux pour la recherche de caractéristique de résistance aux maladies

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