WO2010131960A2 - New fungal elicitor protein and its use as resistance marker - Google Patents

New fungal elicitor protein and its use as resistance marker Download PDF

Info

Publication number
WO2010131960A2
WO2010131960A2 PCT/NL2010/050275 NL2010050275W WO2010131960A2 WO 2010131960 A2 WO2010131960 A2 WO 2010131960A2 NL 2010050275 W NL2010050275 W NL 2010050275W WO 2010131960 A2 WO2010131960 A2 WO 2010131960A2
Authority
WO
WIPO (PCT)
Prior art keywords
ecp6
plant
fgi
broad
protein
Prior art date
Application number
PCT/NL2010/050275
Other languages
French (fr)
Other versions
WO2010131960A3 (en
Inventor
Bart Pierre Hélène Joseph THOMMA
Ronnie De Jonge
Hendrikus Pieter Van Esse
Original Assignee
Wageningen Universiteit
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wageningen Universiteit filed Critical Wageningen Universiteit
Publication of WO2010131960A2 publication Critical patent/WO2010131960A2/en
Publication of WO2010131960A3 publication Critical patent/WO2010131960A3/en

Links

Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi

Definitions

  • the present invention is in the field of plant biotechnology. More in particular, the present invention relates to fungal resistance in plants, especially the interaction between the fungus and the plant. The invention further relates to fungal elicitor molecules, and their use as a marker to determine fungal resistance in plants.
  • Cladosporium ful ⁇ um is a biotrophic pathogen that causes leaf mold of tomato (Lycopersicon esculentum). After germination of conidia, the fungus produces runner hyphae that penetrate stomata predominantly on the lower side of the leaf. Once inside the apoplast, C. ful ⁇ um does not penetrate host cells or develop haustoria but remains confined to the intercellular space between plant mesophyll cells. Despite much research on the C. ful ⁇ um-tom&to interaction, the molecular components that C. ful ⁇ um utilizes for infection and colonization are largely unknown (Thomma, B. P. H.J. et al., 2005, MoI. Plant Pathol. 6:379-393).
  • HR acquired resistance
  • elicitor also called effector
  • the pathogen resistance is elicited by response to elicitor (also called effector) compounds, which are frequently found to be of proteinaceous nature (Arlat, M., et al., EMBO J., 13, 543-553, 1994; Baker, CJ. et al., Plant Physiol. 102, 1341-1344, 1993; Staskawicz, B.J. et al., Proc. Natl. Acad. Sci. USA 81, 6024-6028, 1984; Vivian, A. et al., Physiol. MoI. Plant Pathol. 35, 335-344, 1989; Keen, N.T., Ann. Rev. Gen. 24, 447-463, 1990; Ronald, P.C.
  • the elicitor proteins are characterized by that they are race-specific and only are able to elicit the response with a cognate (also called corresponding or specific) resistance protein.
  • a cognate also called corresponding or specific
  • the concept of avirulence-gene based resistance is also known under the name of the gene-for-gene response.
  • Avirulence genes have been cloned from bacterial and viral pathogens (such as TMV, Pseudomonas and Xanthomonas) and from fungal pathogens (such as Cladosporium ful ⁇ um, Rhynchosporium secalis and Phytophthora parasitica).
  • plant genes coding for some of the corresponding resistance genes have been cloned (such as the tomato gene Cf9 corresponding to the avirulence gene avr9 from Cladosporium fulvum, RPMl from Arabidopsis corresponding to the avirulence gene avrRPMl from Pseudomonas syringae pv. Maculicola, Pi-ta from Oryza sativa corresponding to AvrPita from Magnaporthe grisea and the N-gene from Nicotiana tabacum which corresponds with TMV-helica from Tobacco Mosaic Virus).
  • the invention now comprises a fungal effector protein comprising at least one LysM domain according to the sequence of SEQ ID NO: 4 or a sequence which is 95% or more identical thereto.
  • a fungal effector comprises the amino acid sequence as depicted in SEQ ID NO:3 or a protein that is more than 95% identical thereto.
  • said effector is an ortholog of Ecp6, selected from the orthologs of Fig. 11 or a protein that is more than 95% identical thereto.
  • the invention further comprises a method for detecting the presence of a resistance gene cognate for an effector protein according to the invention, also identified as CfEcp ⁇ orthologs, in a plant comprising introducing a fungal effector protein according to the invention to a plant or a plant part; and detecting whether a resistance reaction in said plant or plant part occurs.
  • Said presence of CfEcp ⁇ orthologs preferably confers fungal resistance to said plant.
  • the introduction of said fungal effector protein is established by infecting the plant or plant part with a fungus capable of expressing said protein, preferably wherein introduction of said fungal effector protein is established by application of said protein to the plant or a plant, more preferably wherein said application is application into the apoplastic space.
  • the introduction of said fungal effector protein is by transformation of a plant with a construct encoding said protein, more preferably wherein said effector protein is transiently expressed.
  • a method wherein introduction of said fungal effector protein is achieved by transient Agrobacterium transformation (ATTA) or through a viral construct, preferably a potatoviral construct.
  • an effector protein according to the invention for assaying pathogen resistance in plants.
  • a method for providing pathogen resistant plants comprising: a. Selecting a plant having pathogen resistance and containing a resistance gene cognate for an effector protein according to the invention; b. Crossing said plant with a plant that needs to be provided with pathogen resistance; c. Assaying offspring of said crossing by testing for the presence of the resistance gene cognate for an effector protein according to the invention; d. Selecting those plants that contain said resistance gene.
  • step (c) is performed with a method according to the invention.
  • Fig. 1 Disease progression of Cladosporium ful ⁇ um on tomato.
  • the fungus is not visible at early stages of infection (3 dpi) but develops white patches of conidiophores (6 dpi) that expand and cover almost the whole leaf (9 dpi). Subsequently, the conidiophores start to produce conidia (13 dpi) which give the leaf a green-brownish velvet-like appearance (16 dpi).
  • B Quantitative real-time reverse transcription PCR to measure C. fulvum growth on resistant MM-Cf-4 tomato plants (white) and on susceptible MM-Cf- 0 tomato plants (grey) at 3, 6, 9, 13 and 16 dpi.
  • the extent of colonization is determined by the relative quantification (RQ) of transcript levels of the constitutively expressed C. fulvum actin gene (measure for fungal biomass) to the constitutively expressed tomato glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (measure for plant biomass) shown on a logarithmic scale. Bars represent mean values and standard errors of three leaflets taken from two plants at each time point analysed. The experiment was repeated twice with similar results.
  • Fig. 2 The apoplast proteome of Cladosporium ful ⁇ um-infected tomato analysed with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Coomassie brilliant blue-stained 2D-PAGE gels obtained after electrophoresis of soluble proteins present in apoplastic fluid collected from a compatible (A; race 5 C. ful ⁇ um strain inoculated onto MM-Cf-O plants) and an incompatible (B; race 5 C. fulvum strain inoculated onto MM-Cf-4 plants) interaction at 14 days post inoculation. The proteins were focused over a non-linear gradient of pH 4—7. Molecular weight markers for the second dimension are indicated on the left. The part of the gel containing the C. fulvum-derived differentially accumulated proteins is shown. Protein spots for which identification was pursued are numbered.
  • Fig. 3 Expression analysis of the newly identified Cladosporium fulvum extracellular proteins.
  • the expression of CfPhiA, Ecp6, Ecp7 and A ⁇ r9 genes was monitored during the interaction of C. ful ⁇ um with MM-Cf-4 tomato (incompatible; white bars) and MM-Cf-O tomato (compatible; grey bars) at 3, 6, 9, 13 and 16 days post inoculation.
  • Real-time reverse transcription PCR was used for the relative quantification (RQ) of transcript levels of the C. ful ⁇ um CfPhiA, Ecp6 and Ecp7 genes relative to the constitutively expressed C. ful ⁇ um actin gene as an endogenous control.
  • the RQ of the A ⁇ r9 gene is shown as an example of the expression profile of a typical C. ful ⁇ um effector gene.
  • Fig. 4 Symptoms caused by wild-type Fusarium oxysporum f. sp. lycopersici and heterologous Ecp6 overexpression transformants on susceptible tomato.
  • a and B Side view (A) and top view (B) of the disease phenotype caused by F. oxysporum f. sp. lycopersici wild-type (WT) and four independent heterologous Ecp6 overexpression transformants (Ecp6-1 to Ecp6-4) on susceptible tomato MoneyMaker plants when compared with mock-inoculated tomato (mock) at 14 days post inoculation.
  • Ecp6 is monitored during a compatible interaction between C. fulvum and MM-Cf-O tomato involving the wild-type (WT) C. ful ⁇ um and RNAi transformants at 10 days post inoculation.
  • Real-time PCR was used to measure the relative quantification (RQ) of transcript levels of the Ecp6 genes, as compared with the constitutively expressed C. ful ⁇ um actin gene as an endogenous control. Bars represent mean values and standard error of the results obtained from three leaflets taken from two infected plants.
  • Fig. 6 Typical symptoms caused by C. ful ⁇ um wild-type (WT) and RNAi transformants silenced for Ecp6 at 10 days post inoculation onto susceptible tomato plants (MM-Cf-O).
  • Fig. 7 Allelic variation of the Cladosporium ful ⁇ um Ecp6 gene. Open reading frames are shown as light grey boxes and introns as black boxes. The predicted signal peptide is indicated as dark grey box. The white flag indicates a single- nucleotide polymorphism (SNP) that leads to an amino acid substitution in the Ecp6 protein. Silent mutations are indicated by a T. The figure is drawn to scale.
  • SNP single- nucleotide polymorphism
  • Fig. 8 Homologues of Ecp6 in other fungal species. Neighbour- joining tree of 17 Ecp6-like sequences from different fungal species. The evolutionary history of Ecp6-like protein sequences was inferred by neighbour- joining analysis and bootstrap values (%) are indicated at the nodes. The tree is drawn to scale, with branch lengths representing evolutionary distances. The positions containing alignment gaps were eliminated in pair- wise sequence comparisons. A total of 220 positions were calculated in the final data set.
  • Fig. 9 Homology models for the LysM domains of Cladosporium ful ⁇ um Ecp6.
  • PrChi-A Identical amino acid residues are shaded in black and similar residues (75% threshold according to Blosum62 score) are shaded in grey.
  • Panels 1, 2 and 3 display the three-dimensional ribbon structures of the Ecp6 LysM domains 1, 2 and 3 respectively.
  • Panel 4 shows the computed molecular surface of Ecp6 LysM domain 1.
  • the arrow indicated in panel 1 indicates the direction of looking to obtain the view in panel 4.
  • the arrow in panel 4 indicates the shallow groove described as the site of interaction of PrChi-A with chitin oligomers.
  • Fig. 10 Recognition of Cladosporium ful ⁇ um Ecp6 in various wild tomato varieties.
  • a strong response in the tomato varieties L. cheesmanii v.typ. PI266375 Gl.1615 (A) and L. pimpinellifolium Gl.1914 (B) results in a hypersensitive response including tissue collapse.
  • a less strong response in L. pimpinellifolium Gl.1310 (C) and L. pimpinellifolium PI344102 Gl.1594 (D) results in clear chlorosis.
  • No phenotypic responses are observed upon injection of Ecp6 in MoneyMaker (E) and Motelle (F) plants.
  • Fig. 11 Nucleotide and amino acid sequences of Ecp6 orthologs.
  • Fig. 12 Nucleotide and amino acid sequences of CfPhiA and Ecp7.
  • genes include coding sequences and/or the regulatory sequences required for their expression.
  • gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • the term “native” or wild type” gene refers to a gene that is present in the genome of an untransformed cell, i. e., a cell not having a known mutation. The term “native” or wild type” is intended to encompass allelic variants of the gene.
  • a "marker gene” encodes a selectable or screenable trait. The term
  • selectable marker refers to a polynucleotide sequence encoding a metabolic trait which allows for the separation of transgenic and non-transgenic organisms and mostly refers to the provision of antibiotic resistance.
  • a selectable marker is for example the aphLl encoded kanamycin resistance marker, the nptll gene, the gene coding for hygromycin resistance.
  • Other selection markers are for instance reporter genes such as chloramphenicol acetyl transferase, ⁇ -galactosidase, luciferase and green fluorescence protein. Identification methods for the products of reporter genes include, but are not limited to, enzymatic assays and fluorimetric assays.
  • Reporter genes and assays to detect their products are well known in the art and are described, for example in Current Protocols in Molecular Biology, eds. Ausubel et al., Greene Publishing and Wiley- Inter science: New York (1987) and periodic updates.
  • chimeric gene refers to any gene that contains 1) DNA sequences, including regulatory and coding sequences that are not found together in nature, or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.
  • transgene refers to a gene that has been introduced into the genome by transformation. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed.
  • endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • foreign gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.
  • oligonucleotide e. g., for use in probing or amplification reactions, may be about 30 or fewer nucleotides in length (e. g., 9, 12, 15, 18, 20, 21 or 24, or any number between 9 and 30).
  • primers are upwards of 14 nucleotides in length.
  • primers 16 to 24 nucleotides in length may be preferred.
  • probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or even 1000's of nucleotides in length.
  • Coding sequence refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", i. e., lacking an intron, such as in a cDNA or it may include one or more introns bound by appropriate splice junctions.
  • An "intron” is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.
  • regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. As is noted above, the term “suitable regulatory sequences” is not limited to promoters.
  • Promoter refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • Promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.
  • Promoter also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an "enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
  • Constant expression refers to expression using a constitutive promoter.
  • Transient expression is expression as a result of a transient transformation event. Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus.
  • Constutive promoter refers to a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant.
  • open reading frame and “ORF” refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • initiation codon and “termination codon” refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
  • “Operably-linked” refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other.
  • a regulatory DNA sequence is said to be "operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i. e., that the coding sequence or functional RNA is under the transcriptional control of the promoter).
  • Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
  • “Expression” refers to the transcription and/or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants. Expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein.
  • heterologous DNA sequence each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
  • a "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
  • “Homologous to” in the context of nucleotide or amino acid sequence identity refers to the similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (as described in Haines and Higgins (eds.), Nucleic Acid Hybridization, IRL Press, Oxford, U. K.), or by the comparison of sequence similarity between two nucleic acids or proteins. Two nucleotide or amino acid sequences are homologous when their sequences have a sequence similarity of more than 60%, preferably more than 70%, 80%, 85%, 90%, 95%, or even 98%.
  • substantially similar refers to nucleotide and amino acid sequences that represent functional and/or structural equivalents of sequences disclosed herein. For example, altered nucleotide sequences which simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to a particular amino acid sequence are substantially similar to the particular sequences. In addition, amino acid sequences that are substantially similar to a particular sequence are those wherein overall amino acid identity is at least 65% or greater to the instant sequences. Modifications that result in equivalent nucleotide or amino acid sequences are well within the routine skill in the art.
  • nucleotide sequences encompassed by this invention can also be defined by their ability to hybridize, under low, moderate and/or stringent conditions (e. g., 0. IX SSC, 0.1% SDS, 65°C), with the nucleotide sequences that are within the literal scope of the instant claims.
  • transformation refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.
  • Host cells containing the transformed nucleic acid fragments are referred to as "transgenic” cells, and organisms comprising transgenic cells are referred to as "transgenic organisms”.
  • Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation (De Blaere et al., 1987) particle bombardment technology (Klein et al. 1987; U.S. Patent No. 4,945,050), microinjection, CaPCu precipitation, lipofection (liposome fusion), use of a gene gun and DNA vector transporter (Wu et al., 1992).
  • Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan (see, for example, Fromm et al., 1990).
  • Transformed refers to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the heterologous nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook et al., 1989. See also Innis et al., 1995 and Gelfand, 1995; and Innis and Gelfand, 1999.
  • “transformed”, “transformant”, and “transgenic” plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome.
  • the term “untransformed” refers to normal plants that have not been through the transformation process.
  • Transiently transformed refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance.
  • “Stably transformed” refers to cells that have been selected and regenerated on a selection media following transformation.
  • Genetically stable and “heritable” refer to chromosomally- integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations. "Chromosomally-integrated” refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not “chromosomally integrated” they may be “transiently expressed”.
  • Gene refers to the complete genetic material of an organism.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine.
  • the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985; Rossolini et al. 1994).
  • a "nucleic acid fragment” is a fraction of a given nucleic acid molecule.
  • deoxyribonucleic acid DNA
  • RNA ribonucleic acid
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single-or double-stranded, optionally containing synthetic, non- natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • nucleic acid or “nucleic acid sequence” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.
  • nucleotide sequences used in aspects of the invention include both the naturally occurring sequences as well as mutant (variant) forms. Such variants will continue to possess the desired activity, i. e., either promoter activity or the activity of the product encoded by the open reading frame of the non-variant nucleotide sequence.
  • variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
  • variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein.
  • nucleotide sequence variants of the invention will have at least 40,50,60, to 70%, e. g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e. g., 81%-84%, at least 85%, e. g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide sequence identity to the native (wild type or endogenous) nucleotide sequence.
  • nucleotide sequence identity or "nucleotide sequence homology” as used herein denotes the level of similarity, respectively the level of homology, between two polynucleotides.
  • Polynucleotides have “identical” sequences if the sequence of nucleotides in the two sequences is the same.
  • Polynucleotides have “homologous” sequences if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence.
  • Sequence comparison between two or more polynucleotides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity.
  • the comparison window is generally from about 20 to 200 contiguous nucleotides.
  • the "percentage of sequence identity " or "percentage of sequence homology" for polynucleotides, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence identity or homology may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and (c) multiplying the result by 100 to yield the percentage of sequence homology.
  • Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms, or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990; Altschul et al., 1997) and ClustalW programs, both available on the internet.
  • BLAST Basic Local Alignment Search Tool
  • nucleic acid sequences of the invention can be "optimized" for enhanced expression in plants of interest. See, for example, EP 0359472 or WO 91/16432. In this manner, the open reading frames in genes or gene fragments can be synthesized utilizing plant-preferred codons. Thus, the nucleotide sequences can be optimized for expression in any plant.
  • variant polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
  • polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred. Individual substitutions deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are "conservatively modified variations", where the alterations result in the substitution of an amino acid with a chemically similar amino acid.
  • variants have a degree of homology (or identity) of preferably more than 90%, more preferably more than 95%, more preferably more than 97% and most preferably more than 98% or 99%.
  • a "homologous" gene is a gene related to a second gene by descent from a common ancestral DNA sequence. The term, homolog, may apply to the relationship between genes separated by the event of speciation (ortholog) or to the relationship between genes separated by the event of genetic duplication (paralog).
  • orthologs are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes.
  • "Paralogs” are genes related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.
  • “Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue or organ or stage of development.
  • vector refers to a construction comprised of genetic material designed to direct transformation of a targeted cell.
  • a vector contains multiple genetic elements positionally and sequentially oriented, i.e., operatively linked with other necessary elements such that the nucleic acid in a nucleic acid cassette can be transcribed and when necessary, translated in the transformed cells.
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e. g.
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e. g. higher plant, mammalian, yeast or fungal cells).
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e. g. bacterial, or plant cell.
  • the vector may be a bi- functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • Codoning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • a “transgenic plant” is a plant having one or more plant cells that contain an expression vector.
  • “Significant increase” is an increase that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 10%-50%, or even 2-fold or greater.
  • “Significantly less” means that the decrease is larger than the margin of error inherent in the measurement technique, preferably a decrease by about 2-fold or greater.
  • Virtually any DNA composition may be used for delivery to recipient plant cells.
  • DNA segments in the form of vectors and plasmids, or linear DNA fragments, in some instances containing only the DNA element to be expressed in the plant, and the like may be employed.
  • the construction of vectors which may be employed in conjunction with the present invention will be known to those of skill of the art in light of the present disclosure (see, e. g., Sambrook et al., 1989; Gelvin et al., 1990).
  • Vectors including plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) and DNA segments for use in transforming cells, according to the present invention will, of course, comprise the cDNA, gene or genes necessary for production of the desired protein in the transformant.
  • the vector of the invention can be introduced into any plant.
  • the genes and sequences to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest.
  • the transcriptional cassette will include in the 5'-to-3' direction of transcription, transcriptional and translational initiation regions, a DNA sequence of interest, and transcriptional and translational termination regions functional in plants.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) MoI. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671- 674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.
  • Suitable methods of transforming plant cells include, but are not limited to, microinjection (Crossway et al., 1986), electroporation (Riggs et al., 1986), Agrobacterium -mediated transformation (Hinchee et al., 1988), direct gene transfer (Paszkowski et al., 1984), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wis. And BioRad, Hercules, Calif, (see, for example, Sanford et al., U. S. Pat. No. 4,945,050; and McCabe et al., 1988).
  • Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti et al., 1985 : Byrne et al., 1987 ; Sukhapinda et al., 1987; Park et al., 1985: Hiei et al., 1994).
  • the use of T-DNA to transform plant cells has received extensive study and is amply described (EP 120516; Hoekema, 1985 ; Knauf, et al., 1983; and An et al.,
  • chimeric genes of the invention can be inserted into binary vectors as described in the examples.
  • transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see EP 0295959), techniques of electroporation (Fromm et al., 1986) or high velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (Kline et al., 1987, and U. S. Patent No. 4,945,050). Once transformed, the cells can be regenerated by those skilled in the art.
  • rapeseed (De Block et al., 1989), sunflower (Everett et al., 1987), soybean (McCabe et al., 1988; Hinchee et al., 1988 ; Chee et al., 1989; Christou et al., 1989 ; EP 301749), rice (Hiei et al., 1994), and corn (Gordon Kamm et al., 1990; Fromm et al., 1990).
  • Agrobacterium tumefaciens cells containing a vector comprising an expression cassette of the present invention, wherein the vector comprises a Ti plasmid are useful in methods of making transformed plants. Plant cells are infected with an Agrobacterium tumefaciens as described above to produce a transformed plant cell, and then a plant is regenerated from the transformed plant cell. Numerous Agrobacterium vector systems useful in carrying out the present invention are known. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, 1984).
  • Methods using either a form of direct gene transfer or Agrobacterium-mediated transfer usually, but not necessarily, are undertaken with a selectable marker which may provide resistance to an antibiotic (e. g., kanamycin, hygromycin or methotrexate) or a herbicide (e. g., phosphinothricin) .
  • a selectable marker which may provide resistance to an antibiotic (e. g., kanamycin, hygromycin or methotrexate) or a herbicide (e. g., phosphinothricin) .
  • the nucleotide sequence coding for an effector protein is placed under control of the CaMV 35S promoter and introduced into an Agrobacterium strain which is also used in protocols for stable transformation. After incubation of the bacteria with acetosyringon or any other phenolic compound which is known to enhance Agrobacterium T-DNA transfer, 1 ml of the Agrobacterium culture is infiltrated into an in situ plant by injection after which the plants are placed in a greenhouse. After 2-5 days the leaves can be scored for occurrence of HR symptoms.
  • the invention now comprises a new effector protein that has been identified in the interaction between tomato plants and Cladosporium fulvum.
  • Said effector molecule, Ecp-6 of which both the amino acid sequence and the nucleotide sequence encoding said polypeptide are given below, is clearly different from known effector molecules, like Avr's and other Ecp effector proteins.
  • Ecp6_Genomic DNA start and stop codons bold underlined and intron sequences in italics and underlined
  • the mature Ecp6 protein contains 199 amino acids and has an estimated molecular mass of 21 kDa, making it the largest of the abundantly secreted effector proteins of C. fulvum identified so far.
  • Previous studies on the genes encoding secreted C. fulvum effectors have shown that A ⁇ r genes accumulated considerably more polymorphisms than Ecp genes (Stergiopoulos et al., 2007). This was suggested to be due to the lack of selection pressure imposed on the pathogen to overcome resistance mediated by resistance genes that recognize Ecps, as these have not been deployed yet in commercial tomato lines. In line with these findings, polymorphisms in Ecp6 were only rarely observed. Of the 50 C.
  • Allelic variant 1 (G > A at 128 bp downstream of the putative start codon in intron, shown in gray shading)
  • Allelic variant 2 (G > A at 494 bp downstream of the putative start codon in intron, C >T at 335 bp downstream of the putative start codon, silent mutation, G > A at 662 bp downstream of the putative start codon, silent mutation, and C > A at 142 bp downstream of the putative start codon, amino acid substitution Thr25 > Asn, all shown in gray shading).
  • ATGCAGTCGATGATTCTTTTCGCTGCCGCTCTTATGGGCGCCGCCGTAAACGGCTTCGTTCTCCCA CGCGAGAACAAGGCCACAGACTGCGGTTCGACCAGCAACATCAAATACACTGTCGTCAAGGGTGAC
  • Ecp6 protein contains three lysin motifs (LysM domains) that were originally found in a variety of enzymes that bind to and hydrolyse peptidoglycans present in bacterial cell walls, of which lysozyme is the best- known example.
  • cysteine residues are found at positions 32 and 34 in the fungal LysM consensus sequence. It has been shown for several C. ful ⁇ um effectors that the formation of disulphide bridges between cysteine residues is required for stability upon secretion in the host, which might be true also for fungal LysM effectors.
  • the consensus sequence that can be defined for the common LysM domain(s) in Ecp6 orthologs has the following sequence: (SEQ ID NO: 4)
  • part of the invention are effector proteins that have at least a domain comprising the amino acid sequence of SEQ ID NO:4 or a domain that is at least 95% or more identical to the sequence as shown in SEQ ID NO:4. It will be clear to the skilled person that - in general - the effector proteins of the present invention lack a further functional domain. This is in contrast with other Lysm containing proteins (not being effector proteins) that are found in fungi, wherein the LysM domain(s) is/are coupled with other functional domains, such as domains having an enzymatic function.
  • the LysM domains are thought to be involved in chitin binding, and thus protecting the chitin structure of the fungus against any chitinase activity produced by the plant.
  • the hypothesis is that, in its turn, the plant has used this protection mechanism of the fungus as a signal that a fungal infection is taken place. Accordingly, the plant has developed a recognition system, which puts in motion a signaling cascade that eventually results in a hypersensitive response.
  • the hypothetical receptor or resistance gene product that recognizes Ecp6 is denominated c/Ecp6 (i.e. the cognate counterpart of C. ful ⁇ um Ecp6).
  • c/Ecp6 is capable to recognize not only Ecp6 itself, but also orthologs of this effector protein, i.e. orthologs derived from many, different fungal species. These species include tomato pathogens, but also pathogens of other crop plants.
  • the invention also comprises a method for the testing of the presence of the c/Ecp6 ortholog in a plant by using an effector protein according to the invention.
  • a plant is tested for the presence of c/Ecp6 (or ortholog of c/Ecp6) by providing a plant with an effector protein of the invention and checking for a hypersensitive response.
  • Providing the effector protein to the plant(s) in such an assay can be achieved in many ways, which will be clear to the person of skill in the art.
  • a first method to provide a plant with an effector protein is by injecting the protein into a part of the plant, e.g. a leaf or a stem segment.
  • a part of the plant e.g. a leaf or a stem segment.
  • said c/Ecp6 or ortholog will recognize the effector protein and the cascade leading to the hypersensitive reaction will be started. As discussed before, and as is shown in Fig. 10, this will lead to a local necrosis of the plant tissue, or, in a less strong reaction, to chlorosis of the injected site, which effect can be visually observed.
  • nucleotide construct encoding said protein.
  • ATTA Agrobacterium Transient Transformation Assay
  • nucleotide sequences encoding the effector proteins are disclosed in this description or can be derived from the amino acid sequences that are also provided in the present description, any person of skill will be able to clone a coding sequence into an expression vector that is suitable for Agrobacterium transformation.
  • a viral vector can be used.
  • a sequence encoding an effector protein of the invention can be cloned into a viral vector (such as potatovirus X) after which the viral particles are used to infect the plant or plant part and to express the protein.
  • a viral vector harbouring the sequence encoding the effector protein is constructed after which this viral vector is introduced into A. tumefaciens.
  • Using a viral vector in which the virus stills maintains its infectious properties has a further advantage.
  • the absence of the resistance gene will also be visible, because in such plants the disease that is caused by the virus will develop.
  • Such signal sequences are available for a person skilled in the art and one example is the signal sequence of the tobacco PR- Ia gene.
  • Alternative sequences can be obtained from the Avr4 peptide, the carrot extension gene or from studies on N-terminal signal sequences (Small, I. et al., 2004, Proteomics 4:1581-1590). It is envisaged that the assay system as described above will be of use when breeding or constructing plants with a fungal resistance based on the presence of the Ecp6-c/Ecp6 resistance mechanism. In case of a breeding program for the introduction of c/Ecp6 based resistance, the breeder normally will depart from a plant that contains c/Ecp6 or an ortholog thereof.
  • This plant line will then be used as a parent line and crossed with another plant to generate offspring.
  • This offspring can be tested for the presence of the resistance mechanism in an assay as described above. This process then will be repeated until the desired end product is obtained.
  • Podo ⁇ pora an ⁇ e ⁇ na Pa_3_3275 7 49 86 133 Podo ⁇ pora an ⁇ e ⁇ na Pa_3_780 131 176 Podo ⁇ pora an ⁇ e ⁇ na Pa_4_1460 35 80 160 205 Podo ⁇ pora an ⁇ e ⁇ na Pa_4_5520 51 97 172 217 250 295 332 377 410 455 Podo ⁇ pora an ⁇ e ⁇ na Pa_5_1 130 12 57 93 149 170 204 Podo ⁇ pora an ⁇ e ⁇ na Pa_5_1560 51 95 151 196 229 274 31 1 356 389 434 Podo ⁇ pora an ⁇ e ⁇ na Pa_5_2020 397 444 Podo ⁇ pora an ⁇ e ⁇ na Pa_5_3800 96 141 230 275 344 386 Podo ⁇ pora an ⁇ e ⁇ na Pa 6 3590 223 266
  • the wild-type race 5 strain of C. ful ⁇ um was stored in 50% glycerol at -80 0 C until revitalized on potato dextrose agar (PDA; Oxoid, Hampshire, England) and was grown at room temperature in the dark.
  • PDA potato dextrose agar
  • Two-week-old C. fulvum PDA plate cultures were used to harvest conidia by adding sterile water to the plates and rubbing the surface with a sterile glass rod to release the conidia.
  • Conidial suspensions were filtered through Miracloth (Calbiochem-Behring, La Jolla, CA), centrifuged at 4000 r.p.m. and washed twice with sterile water after which the conidial concentration was determined.
  • the conidia were used for plant inoculations or Agrobacterium tumefaciens- mediated transformation.
  • All tomato plants were grown under standard greenhouse conditions: 21°C during the 16 h day period, 19°C at night, 70% relative humidity (RH) and 100 Watt m-2 supplemental light when the sunlight influx intensity was below 150 Watt m-2.
  • the tomato (S. esculentum) cultivar MoneyMaker, containing no resistance genes against C. fulvum (MM-Cf-O), and a MoneyMaker near isogenic line containing the Cf-4 locus (MM-Cf-4) were used for all inoculations.
  • fulvum was inoculated as described previously (de Wit, P.J.G.M.,1977, Neth. J. Plant Pathol. 44:337-366).
  • 5 ml of conidial suspension (1 x 10 6 conidia per ml) was used for spray inoculation on the lower surface of the leaves until drop-off. Plants were kept at 100% RH under a plastic cover for 48 h after inoculation. All experiments, starting from plant inoculations, were repeated at least twice.
  • Leaves were harvested from C. fulvum-infected MM-Cf-O and MM-Cf-4 lines at 14 dpi and apoplastic fluid (AF) was isolated by vacuum infiltration (van Esse, H.P. et al., 2006, MoI. Plant Microbe Interact. 20:1092-1101) using demineralized water followed by centrifugation for 5 min and stored at -20 0 C until further analysis. AF from both interactions was freeze dried and the residue was resuspended in 3.5 ml of water. After centrifugation (10 min at 4000 g) samples were desalted using a PD-10 desalting column (GE Healthcare, UK), freeze-dried again and stored at -20 0 C.
  • a PD-10 desalting column GE Healthcare, UK
  • Freeze-dried protein samples were dissolved in 340 ml of Rehydration Buffer [7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT, 0.002% (w/v) bromophenol blue] along with 3.4 ml of IPG buffer pH 4-7 (GE Healthcare). The samples were vortexed briefly and centrifuged (10 min at 4000 g). The protein samples were applied to Immobiline DryStrips of 18 cm with a non-linear pH 4-7 gradient (GE Healthcare), covered with paraffin oil and allowed to rehydrate overnight at room temperature. Isoelectric focusing was performed using the Ettan IPGphor electrophoresis apparatus (GE Healthcare) at 20 0 C maintaining 50 mA per strip. A total focusing of 70 k Vh was achieved by following a running protocol using a step-n-hold gradient (1.5 h 0-3500 V, 6 h 3500 V).
  • the strips were stored at -20 0 C. Subsequently, strips were placed in equilibration buffer [EB; 50 mM Tris, pH 8.8, 6 M urea, 30% (v/v) glycerol and 2% (w/v) SDS] supplemented with 65 mM DTT. After 15 min, the buffer was replaced by EB supplemented with 135 mM iodoacetamide, and the strips were incubated for another 15 min. The proteins were subsequently separated on 12.5% polyacrylamide gels; the gels were run at 70 V for the first 30 min and subsequently at 200 V until the bromophenol blue reached the bottom of the gels. Gels were stained with Coomassie brilliant blue overnight and de-stained with 10% ethanol and 7.5% HAc in water.
  • EB equilibration buffer
  • Protein spots were excised from the gel and digested with trypsin with an in- gel method (Shevchenko A. et al., 1996, Anal. Chem. 69:850-858).
  • the collected extracts of the resulting tryptic peptides were freeze dried and stored at -20 0 C.
  • the peptides were redissolved in 8 ml of 50% acetonitrile, 5% formic acid.
  • MS and MS/MS information was acquired with a Q-Tofl (Waters, Manchester, UK) coupled with a nano-LC Ultimate system (LC Packings Dionex, Sunnyvale, CA).
  • peptides were separated on a nano-analytical column (75 mm inside diameter x l ⁇ cm Cl ⁇ PepMap, LC Packings, Dionex) using a gradient of 2-50% acetonitrile, 0.1% formic acid in 20 min.
  • the forward degenerate primer Deg-PhiA along with an oligo-(dT) primer (Table S2) was used to isolate the CfPhiA coding sequence.
  • degenerate forward primers (Table S2) were designed matching the ETKATDCG and QITTQDFG sequences from the N-terminal sequences of Ecp6 and Ecp7 respectively.
  • Using the degenerate primers and a poly T primer PCR products were amplified from a cDNA library derived from a compatible interaction between C. ful ⁇ um and tomato using the high fidelity polymerase ExTaq (Takara, Shiga, Japan). Products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and sequenced.
  • RNAi constructs for overexpression of inverted-repeat constructs for RNAi based on two different parts of the Ecp6 coding sequence were generated.
  • 218 bp oi Ecp ⁇ was PCR-amplified from cDNA using a forward primer that added an Ncol restriction site to the 5' end (Ecp6i-F) and a reverse primer that added EcoRI and Notl restriction sites to the 3' end (Ecp6i-R; Table S2).
  • PCR reactions were carried out under the following conditions: an initial denaturation step for 2 min followed by denaturation for 15 s at 94°C, annealing for 30 s at 55°C and extension for 1 min at 72°C for 30 cycles, followed by a final elongation step at 72°C for 5 min.
  • PCR products were separated on 1% agarose gels and were purified using the DNeasy kit (Qiagen, Valencia, CA). Subsequently, PCR products were cloned into the pGemT-Easy vector. Vectors were digested with Ncol and Notl or with Ncol and EcoRI.
  • the plasmid pFBB302 is constructed in the backbone of the pGreen II binary vector (Hellens R. P. et al., 2000, Plant MoI. Biol. 42:819-832) and contains a nourseothricin resistance cassette (Malonek S. et al., 2004, J. Biol. Chem.
  • RNAid pFBT004 is a modified version of pFBB302, in which the nourseothricin resistance cassette is replaced by a hygromycin resistance cassette (Punt et al., supra).
  • RNAi plasmids were transformed into A. tumefaciens strain LBAlIOO [containing the binary vector pSoup (Hellens et al., supra] by electroporation.
  • a 3 ml culture of A. tumefaciens was grown overnight in Ix YT (Sambrook and Russell, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press, 2001) supplemented with kanamycin (25 mg ml-1).
  • MM fresh minimal medium
  • the culture was centrifuged and resuspended in 10 ml of fresh MM.
  • One millilitre of resuspended bacteria was used to inoculate 50 ml of induction medium [IM; MM salts plus 40 mM 2-(Nmorpholino) methanesulphonic acid (MES), pH 5.3, 10 mM glucose and 0.5% (w/v) glycerol] supplemented with 200 mM acetosyringone (AS) and was grown for an additional 4—5 h until the culture reached an optical density (OD600) of 0.25. At that point, the A. tumefaciens culture was centrifuged and resuspended in 10 ml of sterile water.
  • IM induction medium
  • AS mM acetosyringone
  • the filter was transferred to PDA supplemented with 50 mg ml 1 nourseothricin (Werner BioAgents, Jena, Germany) or with 100 mg ml-1 hygromycin B (Duchefa Biochemie BV, Haarlem, the Netherlands) as a selection agent for transformants and 200 mg ml 1 cefotaxime (Duchefa Biochemie BV, Haarlem, the Netherlands) to kill A. tumefaciens cells. Individual transformants were transferred to new selection plates and incubated until conidiogenesis under normal growth conditions. Conidia from these plates were stored in 50% glycerol at -80 0 C until further analysis.
  • Leaf samples were composed of three leaflets from the second, third and fourth tomato leaves of two tomato plants taken at each time point, immediately frozen in liquid nitrogen and stored at -80 0 C until used for RNA analysis. A similar procedure was used for RNAi transformant analysis.
  • Ecp ⁇ RNAi transformants Ecp6i-1 and Ecp6i-4 along with Ecp7 RNAi transformants Ecp7i-1, Ecp7i-3 and Ecp7i-7 were randomly chosen for inoculation and analysis with the progenitor race 5 wild-type strain inoculated on MM-Cf-O plants.
  • Leaf samples were taken at 10 dpi, immediately frozen in liquid nitrogen and stored at -80 0 C until used for RNA analysis.
  • Quantitative real-time PCR was conducted using an ABI7300 PCR machine (Applied Biosystems, Foster City, CA) with the GoldStar SYBR green PCR kit (Eurogentec, Seraing, Belgium). All primer sequences are shown in Table S2.
  • RNAi constructs were designed so that the reverse primer was not included in the RNAi construct to prevent detection of the constitutively expressed RNAi construct.
  • primer pair Ecp6-RNAi-RQ-F and Ecp6-RNAi-RQ-R was used, and for the second RNAi construct primer pair Ecp6-RNAi2-RQ-F and Ecp6-RNAi2-RQ-R.
  • Real-time PCR conditions were as follows: an initial 95°C denaturation step for 10 min followed by denaturation for 15 s at 95°C, annealing for 30 s at 60 0 C and extension for 30 s at 72°C for 40 cycles, and analysed on the 7300 System SDS software (Applied Biosystems, Foster City, CA). To ensure no genomic DNA contaminated RNA samples, real-time PCR was also carried out on RNA without the addition of reverse transcriptase. All experiments, including leaf inoculations, were repeated twice.
  • C. fulvum Ecp6 the cDNA corresponding to the mature protein was amplified using primer Ecp6OE-F that also contained the sequence encoding the C. fulvum Avr4 signal peptide for extracellular targeting (Table S2).
  • C. fulvum Ecp7 the cDNA corresponding to the mature protein was amplified in two steps. As the 5' coding sequence was lacking from our cDNA clone, a primer was designed to add a 5' codon-optimized sequence stretch based on the N-terminal protein sequence (Ecp7NtermF) and used in combination with the reverse primer Ecp7OE-R (Table S2).
  • the resulting PCR product was used as template for a second PCR with primer Ecp7OE-F that also contained the sequence encoding the C. fulvum Avr4 signal peptide for extracellular targeting and a HindIII restriction site in combination with the reverse primer Ecp7OE-R that contained a Xmal restriction site (Table S2). All PCR reactions were carried out under the following conditions: an initial denaturation step for 2 min followed by denaturation for 15 s at 94°C, annealing for 30 s at 56°C and extension for 1 min at 72°C for 30 cycles, followed by a final elongation step at 72°C for 5 min.
  • PCR products were separated on 1% agarose gels and purified using the DNeasy kit (Qiagen, Valencia, CA). Subsequently, PCR products were cloned into the pGemT-Easy vector and sequenced. A correct clone was digested with EcoRI (for Ecp6) or HindIII and Xmal (for Ecp7), cleaned from gel, and ligated into the EcoRI- (for Ecp6) or HindIII- and Xmal- (for Ecp7) digested plasmid pFBT004. The constructs were transformed into A.
  • TSPl Three primers designed on the region encoding the mature Ecp6 protein (TSPl, TSP2 and TSP3; Table S2) were used to amplify the genomic DNA sequence upstream of the region that encodes the mature Ecp6 protein using the DNAWalking SpeedUpTM Premix Kit (Seegene, Rockville, MD) according to the manufacturer's instructions. Amplified products were cloned in the pGEM-T Easy vector (Promega, Madison, WI) and sequenced. Putative open reading frames (ORFs) were predicted using the FGENESH program (Salamov, A.A. and Solovyev, V.V., 2000, Genome Res.
  • the generated cDNA was used as template for the primers Ecp6_ChrWal_Fl and Ecp6_R (Table S2) to amplify the predicted Ecp6 ORF.
  • the primers Ecp6_F3, Ecp6_F2, Ecp6_R3, Ecp6_R2 (Table S2) that hybridized outside the predicted Ecp6 ORF were used as negative controls.
  • the 50 ml PCR reaction mixes contained 5.0 ml of 1Ox SuperTaq PCR reaction buffer, 10 mM of each dNTP (Promega Benelux bv, Leiden, the Netherlands), 20 mM of each primer, 1 unit of SuperTaq DNA polymerase (HT Biotechnology, Cambridge, UK) and approximately 100 ng of cDNA as template.
  • the PCR programme consisted of an initial 5 min denaturation step at 94°C, followed by 35 cycles of denaturation at 94°C (30 s), annealing at 55°C (30 s) and extension at 72°C (60 s). A final extension step at 72°C (7 min) concluded the reaction. Amplified products were cloned in the pGEM-T Easy vector (Promega, Madison, WI) and sequenced.
  • the 50 ml PCR reaction mixes contained 5.0 ml of 10 ⁇ SuperTaq PCR reaction buffer, 10 mM of each dNTP (Promega Benelux bv, Leiden, the Netherlands), 20 mM of each primer, 1 unit of SuperTaq DNA polymerase (HT).
  • the PCR programme consisted of an initial 5 min denaturation step at 94°C, followed by 35 cycles of denaturation at 94°C (30 s), annealing at 55°C (30 s) and extension at 72°C (60 s). A final extension step at 72°C (7 min) concluded the reaction.
  • Amplified PCR products were excised from 0.8% agarose gels, purified using the GFXTM PCR DNA and Gel Band Purification Kit (Amersham Biosciences UK limited, Buckinghamshire, England), and sequenced using the forward primers Ecp6_F2 and Ecp6_F in combination with the reverse primer Ecp6_R3 (Table S2).
  • Hmmpfam analysis of each identified candidate was performed by running a customized Perl script for Pfam HMM detection, available at ftp://ftp.sanger.ac.uk/pub/databases/Pfam, using Bioperl version 1.4 (http://bioperl.org) and HMMER version 2.3.2 (http://hmmer.janelia.org), which was loaded with the current Pfam Is and fs models (02.10.2007), for whole domain and fragment models respectively. An E- value of 0.001 was used as cut-off. The retained sequences were analysed in BioEdit version 7.0.5.3 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).
  • Fig. 1 Proteins present in 2 ml of apoplastic fluid isolated from the two different interactions were analysed with 2D-PAGE. Separation of the proteins in the first dimension was carried out on Immobiline DryStrips (pH 4—7) and for the second dimension 12.5% polyacrylamide gels were used.
  • CfPhiA This is a protein that typically occurs on phialides, which are sporogenous cells that release conidia from their apex by budding (Melin, P. et al., 2003, Fungal Genet. Biol. 40:234-241).
  • N- terminal sequencing of spot 12 resulted in a 26-amino-acid sequence harbouring the identified MS/MS sequence tags and the corresponding protein was designated Ecp6.
  • the 25-amino-acid sequence that was obtained matched the corresponding MS/MS sequence tags and the protein was designated Ecp7.
  • sequence information based on MS/MS was available for protein spot 15, this protein was not considered for further study because N-terminal sequence failed repeatedly.
  • CfPhiA a 720 bp fragment encoding the mature protein and part of the 3'UTR was cloned (Fig. Sl).
  • the predicted mature CfPhiA protein contains 175 amino acids and has a predicted molecular mass of about 19 kDa and an isoelectric point (pi) of 5.0.
  • pi isoelectric point
  • BLASTP analysis of the amino acid sequence showed that this protein shares similarity to putative proteins of several fungal species including A. nidulans, A. fumigatus and Neurospora crassa. Of these orthologues, the PhiA protein from A.
  • nidulans has been functionally characterized (Melin et al., supra), and was found to be essential for growth and sporulation of the fungus as phiA mutants were found to be impaired in phialide development. Therefore, it is likely that the C. fulvum putative orthologue CfPhiA has a similar function.
  • a 742 bp fragment with the coding region for the mature Ecp6 protein and the 3'UTR was cloned.
  • Ecp6 encodes a mature protein of 199 amino acids, including eight cysteines, and has a predicted molecular mass of 21 kDa and a pi of 4.6.
  • Ecp6 contains five predicted N-glycosylation sites, explaining the location of the Ecp6 protein spots on the 2D-gel. Based on BLASTP analysis, Ecp6 was found to share significant homology to the glycoprotein CIHl identified in the plant pathogenic fungus Colletotrichum lindemuthianum (Perfect, S. E. et ⁇ /.,1998, Plant J. 15:273-279). Although the contribution of CIHl to pathogenicity is unknown, it has been shown in this reference to accumulate during infection on bean in the walls of intracellular hyphae and the interfacial matrix which separates the hyphae from the invaginated host plasma membrane.
  • Ecp7 For Ecp7, a 464 bp cDNA fragment was cloned containing the coding region for 84 amino acids of the mature Ecp7 protein. N-terminal sequencing of Ecp7 revealed that a stretch of 16 amino acids precedes the peptide that was identified as an MS tag, and based on which the degenerate primer for cloning the cDNA was designed. Therefore it should be concluded that Ecp 7 encodes a mature protein of 100 amino acids which includes six cysteines and has a predicted molecular mass of 11 kDa and a pi of 6.0. BLASTP analysis of the amino acid sequence revealed no significant homology of Ecp7 to other protein sequences deposited in public databases.
  • CfPhiA expression is induced already early in the compatible interaction, at 6 dpi, and maintains this level of expression for all time points analysed.
  • CfPhiA is also induced, although its expression level is approximately half of that found in the compatible interaction (Fig. 3).
  • Ecp6 and Ecp7 show a low but steady level of expression in the incompatible interaction when compared with that of the C. fulvum actin gene, while the genes are clearly induced in the compatible interaction. While Ecp7 peaks at 9 dpi (Fig. 3), Ecp6 is maximally expressed at 13 dpi (Fig. 3).
  • the patterns of Ecp6 and Ecp 7 typically resemble those of other genes encoding secreted C. fulvum effectors.
  • C. fulvum Avr9 is highly expressed throughout the compatible interaction, with maximum expression at 9 dpi, whereas its expression in the incompatible interaction remains low (Fig. 3). Nevertheless, the expression level of the Avr9 gene is much higher than those of Ecp6 and Ecp7 (Fig. 3).
  • RNAi-mediated silencing of Ecp6 compromises C. fulvum virulence on tomato.
  • RNAi has been successfully employed for gene functional analysis in filamentous fungi (Nakayashiki, H. et al., 2005, Fungal Genet. Biol. 42:275- 283). This is particularly relevant for fungi like C. fulvum for which homologous recombination is not straightforward.
  • Recent evidence has shown that PEG-mediated transformation may generate somaclonal variation that may be circumvented by Agrobacterium-mediated transformation which is, however, significantly less efficient (van Esse et ⁇ l., supra). Therefore, RNAi was recently successfully implemented to silence the expression of C. fulvum effector genes (van Esse et al., supra). Based on the results obtained with heterologous expression of C. fulvum Ecp6 in F.
  • RNAi-mediated silencing for functional analysis of the C. fulvum Ecp 6 gene using Agrobacterium-mediated transformation with constructs aimed at generating double- stranded RNA that targets these genes (RNAi).
  • T-DNA transfer DNA
  • ToxA promoter To target the expression of the Ecp6 gene, two RNAi constructs were generated based on different sections of the Ecp6 coding region.
  • RNAi constructs generated several antibiotic-resistant transformants for each construct. Analysis of the transformants indicated that their growth in vitro was indistinguishable from that of the progenitor race 5 isolate (data not shown).
  • C. fulvum effector genes show variable expression when cultured in vitro (Thomma et ⁇ l., 2006), 4-week-old MM-Cf-O tomato plants were inoculated with three transgenic C. fulvum strains to determine whether the introduc- tion of the inverted-repeat construct resulted in Ecp6 silencing.
  • RNAi transformants To measure the extent of fungal growth of RNAi transformants compared with the parental wild-type strain, the constitutively expressed C. fulvum actin gene was used as a marker in real-time PCR analyses (Fig. 5B).
  • the constitutively expressed tomato chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a reference for the ratio of fungal biomass to plant biomass to determine the degree of colonization.
  • GPDH tomato chloroplast glyceraldehyde-3-phosphate dehydrogenase
  • Ecp6 is a virulence factor of C. fulvum
  • sequence variation of Ecp6 in a worldwide collection of strains We first obtained 691 bp of genomic sequence upstream of the region that encodes the mature Ecp6 protein by gene walking. Sequence analysis using the gene prediction algorithm FGENESH identified a putative start codon and predicted intron/exon boundaries using the genetic codes of several fungi present as models in the database. These were confirmed by cloning the Ecp6 cDNA from infected plant material, showing that the Ecp6 ORF is 669 bp, interrupted by two introns of 68 and 111 bp, respectively, and encodes a protein of 222 amino acids (Fig. 7).
  • the full-length sequence of Ecp6 was obtained from a collection of 50 C. fulvum strains (Table Sl). Analysis of the sequence 62 bp upstream of the start codon to 91 bp downstream of the stop codon revealed that variation within Ecp6 was very limited, resulting in a total of five single-nucleotide polymorphisms (SNPs) within these strains (Fig. 7).
  • SNPs single-nucleotide polymorphisms
  • One SNP (G > A at 494 bp downstream of the putative start codon) occurred inside the second intron oiEcp ⁇ , and was only detected in one Canadian strain (#34; Table Sl). The other four SNPs all occurred in seven strains originating from North America (#31, #34, #40, #41; Table Sl), and Japan (#67, #71, #74; Table Sl).
  • Ecp6 protein contains three lysin motif (LysM) domains. These domains are widespread protein modules of approximately 40 amino acids, originally identified in a bacterial autolysin that degrades bacterial cell walls (Joris, B. et ah, 1992, FEMS Microbiol. Lett. 70:257-264).
  • LysM domains are also found in eukaryotic proteins, and presently LysM domains are implicated in binding of diverse carbohydrates that occur in bacterial peptidoglycan, fungal chitin and Nod factor signals that are produced by Rhizobium bacteria during the initiation of root nodules on legumes.
  • Fig. S2 a multiple sequence alignment analysis was performed (Fig. S2). In addition to the LysM domains, the positions of the cysteine residues that flank the LysM domains, and the high abundance of proline, serine and threonine residues in the LysM linker regions appear to be conserved (Fig. S2). Subsequently a neighbour-joining tree) was constructed to reveal evolutionary relationships (Fig. 8).
  • the 16 Ecp6-like proteins can be divided into three groups.
  • the second group of Ecp6-like proteins encompasses the two M. grisea Ecp6-like proteins and CIHl from C. lindemuthianum that are shorter than other Ecp6-like proteins and have only two LysM domains (Group 2, Fig. 8).
  • LysM domains have been identified in over 1500 proteins, the three- dimensional (3D) structure of only three LysM domains has been reported. Two of these are of bacterial origin, the 3D structure of a LysM domain of the Escherichia coli membrane-bound lytic murein transglycosylase D (MItD; PDB code: IEOG; Bateman, A. and Bycroft M., 2000, J. MoI. Biol. 299:1113-1119) and the LysM domain of the Bacillus subtilis spore protein ykuD of unknown function (PDB code: 1Y7M; Bielnicki, J. et al., 2006, Proteins 62:144-151).
  • MItD Escherichia coli membrane-bound lytic murein transglycosylase D
  • PDB code IEOG
  • Bateman A. and Bycroft M.
  • LysM domain of the Bacillus subtilis spore protein ykuD of unknown function PB code: 1Y7M
  • the 3D structure of the LysM domain of the human hypothetical protein SB145 was determined using nuclear magnetic reso- nance (NMR) imaging (PDB code: 2DJP).
  • NMR nuclear magnetic reso- nance
  • the structural organization of the three LysM domains from these different proteins is highly similar, and characterized by a ⁇ fold, with the two helices stacking on one side of the plate generated by a double-stranded antiparallel ⁇ -sheet.
  • the first characterization of an interaction of a LysM domain with its ligand was reported (Ohnuma, T. et al., 2008, J. Biol. Chem. 283:5178-5187).
  • ligand binding can be modelled according to the interaction between chitin oligomers and PrChi-A LysM domains.
  • the molecular surface of the first LysM domain of Ecp6 (Fig. 9B, panel 1) was computed and is shown in panel 4 of Fig. 9B.
  • a cavity is observed that fulfils the requirements to act as binding site of chitin oligomers, based on the structural homology with PrChi- A.
  • Ecp6 was fused to the Avr4 signal peptide and a His6-FLAG-tag by overlap extension PCR, and transformed into Pichia pastoris. Subsequently, Ecp6 was produced and purified using a Ni-NTA column. Finally the fractions containing Ecp6 were isolated and dialyzed against ddH20. The protein concentration was measured using BCA, and determined to be approximately 5.5 mg/ml.
  • the binary PVX vector pGrlO6 (Jones, L. et al., 1999, Plant Cell 11:2291-2301) was used as a backbone for all PVX expression constructs.
  • the coding sequences of the Ecp6 orthologs were fused to the tobacco PR- Ia signal sequence, for extracellular targeting, using overlap extension PCR and directionally cloned into the Clal-Notl restriction sites of pGrlO ⁇ .
  • the resulting plasmids were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. The A.
  • tumefaciens strains were cultured on plates containing modified LB medium (10 g 1/1 bacto-peptone; 5 g 1/1 yeast extract; 2.5 g 1/1 NaCl; 10 g 1/1 mannitol) for 48 h at 28 degrees Celcius. Subsequently, colonies were selected and inoculated on 2-week-old tomato plants by toothpick inoculation.
  • modified LB medium 10 g 1/1 bacto-peptone; 5 g 1/1 yeast extract; 2.5 g 1/1 NaCl; 10 g 1/1 mannitol
  • Ecp6 was purified from the culture medium and used to screen a collection of 28 tomato lines for the occurrence of a hypersensitive response upon injection with Ecp6 (Table S3). Ecp6 injection triggered the development of strong chlorotic and necrotic lesions in the center of the injected area within 5 days in leaves of 7 lines ( Figure 10; Table S3). In another 6 lines a less strong response was observed. Inoculation of these lines with a similar concentration of Avr4 caused no symptoms. The remaining 15 lines and the control cultivars MoneyMaker and Motelle, that both lack functional C. fulvum resistance genes, exhibited no symptoms upon injection of Ecp6.
  • Cf-Ecp6 a single dominant gene, designated Cf-Ecp6
  • Ecp6-responsive plants were crossed with MoneyMaker Cf-O plants that lack functional Cf resistance genes.
  • the progeny of the cross (Fl) was tested for Ecp6 responsiveness by injection of P. pastoris produced Ecp6 and tested for C. ful ⁇ um resistance by challenge inoculation with various strains of C. ful ⁇ um that all possess Ecp6. All Fl plants obtained in the diverse crosses showed Ecp6-responsiveness as well as resistance against all C. ful ⁇ um strains that were tested.
  • F2 progenies were generated to study the heritability of HR upon exposure to Ecp6.
  • the three F2 populations exhibited a 3:1 ratio for Ecp6- responsiveness as well as for C. ful ⁇ um resistance, showing that both traits are conferred by a single dominant gene, designated Cf-Ecp6, in the three wild tomato species.
  • Cf-Ecp6-responsiveness a single dominant gene, designated Cf-Ecp6, in the three wild tomato species.
  • pairwise crosses were made and F2 progeny was obtained. Since all individuals of the diverse F2 populations exhibited Ecp6-responsiveness, it is concluded that Ecp6 responsiveness in the three wild tomato species is conferred by different alleles of the same gene.
  • Cf-Ecp6 recognizes Ecp6 orthologs of multiple fungal species
  • responsiveness towards various Ecp6 orthologs was tested. Screenings were carried out by using Potato Virus X (PVX) for systemic production of the Ecp6 orthologs that were targeted to the apoplast of virus- infected plants.
  • PVX Potato Virus X
  • the Ecp6 orthologs were cloned from Mycosphaerella fijiensis, M. graminicola, Cercospora beticola and Septoria lycopersici that, similar to C.
  • Ecp6 orthologs were cloned from the distantly related tomato pathogens Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Verticillium dahliae.
  • Cf-Ecp6 plants are resistant to multiple, Ecp6 expressing, 2 fungal pathogens
  • Ecp6 recognition also occurs in Arabidopsis
  • 280 Arabidopsis accessions were screened for responsiveness to C. fulvum Ecp6 in the greenhouse. Even though C. fulvum is not a tomato pathogen, two accessions that clearly responded with an HR to Ecp6 injection were identified.
  • the screen of 280 Arabidopsis accessions was performed in the climate chamber under controlled conditions. This time, three different accessions were retained. So, in total five Arabidopsis accessions were retained that respond with an HR upon injection with C. fulvum Ecp6. These same five accessions respond with an HR upon injection with Ecp6 orthologs from other fungal pathogens. This demonstrates that Ecp6 recognition is conserved across plant families.
  • Ecp6i-F CCATGGAGATCGAGAACCCAGATGCC
  • Ecp6OE-R TTATGCCACAGCAGTAGTGA
  • Ecp ⁇ over-expression Ecp7NtermF CACTACTTGACCATCTACAGCAACATCGG
  • Ecp7 over-expression CTGCCGCAAGGGCAGCCAGATTACGACGC primer to obtain coding AGGATTTTGGTCACGAG sequence for mature protein (bold)
  • Ecp7OE-F AAGCTTATGGGATTTGTTCTCTTTTCACA Ecp7 over-expression with ATTGCCTTCTTTCTTCTTGTCTCTACACT coding sequence for C.
  • Avr4 signal peptide GCCGTGCCCAAAATCACTACTTGACCATC (bold) and Hmdlll TAC

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to a new fungal effector protein Ecp6 and its orthologs and the use of these effector proteins in an assay for detecting fungal resistance in plants.

Description

Title: New fungal elicitor protein and its use as resistance marker
FIELD OF THE INVENTION
The present invention is in the field of plant biotechnology. More in particular, the present invention relates to fungal resistance in plants, especially the interaction between the fungus and the plant. The invention further relates to fungal elicitor molecules, and their use as a marker to determine fungal resistance in plants.
BACKGROUND OF THE INVENTION
Cladosporium fulυum is a biotrophic pathogen that causes leaf mold of tomato (Lycopersicon esculentum). After germination of conidia, the fungus produces runner hyphae that penetrate stomata predominantly on the lower side of the leaf. Once inside the apoplast, C. fulυum does not penetrate host cells or develop haustoria but remains confined to the intercellular space between plant mesophyll cells. Despite much research on the C. fulυum-tom&to interaction, the molecular components that C. fulυum utilizes for infection and colonization are largely unknown (Thomma, B. P. H.J. et al., 2005, MoI. Plant Pathol. 6:379-393).
Yet some tomato plants are resistant to specific pathovarieties of C. fulυum. This resistance is effected through a hypersensitive response (HR) resulting in rapid cell death of the infected plant cells. This rapid cell death or necrosis inhibits the pathogen from further growth and thus stops the infection. This mechanism is known already for a long time (Klement, Z., In: Phytopathogenic Prokaryotes, Vol. 2, eds.: Mount, M.S. and Lacy, G. H., New York, Academic Press, 1982, pp. 149-177). The HR is often confused with other lesion-like phenomena, but a typical HR gives local cell death and is associated with secondary responses such as callus deposition, generation of active oxygen species, induction of phytoalexins, changes in ion fluxes across membranes and induction of acquired resistance (AR) (Hammond-Kosack, K.E., et al., Plant Physiol. 110, 1381-1394, 1996).
The pathogen resistance is elicited by response to elicitor (also called effector) compounds, which are frequently found to be of proteinaceous nature (Arlat, M., et al., EMBO J., 13, 543-553, 1994; Baker, CJ. et al., Plant Physiol. 102, 1341-1344, 1993; Staskawicz, B.J. et al., Proc. Natl. Acad. Sci. USA 81, 6024-6028, 1984; Vivian, A. et al., Physiol. MoI. Plant Pathol. 35, 335-344, 1989; Keen, N.T., Ann. Rev. Gen. 24, 447-463, 1990; Ronald, P.C. et al., J. Bacteriol. 174, 1604-1611, 1992; Whitham ,S. et al., Cell 78, 1-20, 1994;Kobe, B. and Deisenhofer, J., Trends Biochem. Sci. 19, 415, 1994; and Honee G. et al., Plant MoI. Biol. 29, 909-920, 1995). These elicitor proteins (encoded by avirulence genes) are produced by the pathogen and are thought to signal through a resistance protein available in the plant, therewith starting a cascade of events resulting in the HR-response. The elicitor proteins are characterized by that they are race-specific and only are able to elicit the response with a cognate (also called corresponding or specific) resistance protein. The concept of avirulence-gene based resistance is also known under the name of the gene-for-gene response. Avirulence genes have been cloned from bacterial and viral pathogens (such as TMV, Pseudomonas and Xanthomonas) and from fungal pathogens (such as Cladosporium fulυum, Rhynchosporium secalis and Phytophthora parasitica). Also plant genes coding for some of the corresponding resistance genes have been cloned (such as the tomato gene Cf9 corresponding to the avirulence gene avr9 from Cladosporium fulvum, RPMl from Arabidopsis corresponding to the avirulence gene avrRPMl from Pseudomonas syringae pv. Maculicola, Pi-ta from Oryza sativa corresponding to AvrPita from Magnaporthe grisea and the N-gene from Nicotiana tabacum which corresponds with TMV-helica from Tobacco Mosaic Virus).
As the complete set of effectors of a potential pathogen determines the outcome of the interaction with a possible host, it is important to make an inventory of this effector catalogue. Many plant pathogenic bacteria inject effector proteins into the cytoplasm of host cells by means of the type III secretion system (TTSS) to subvert host cellular physiology to the bacterium's advantage (e.g. Tang, X. et al., 2006, MoI. Plant Microbe Interact. 19:1159- 1166). This process is orchestrated by specific cis-elements in the promoters of genes encoding type III effector proteins, a feature that has widely been exploited to identify such effectors in genome-wide functional screens (Chang, J.H. et al., 2005, Proc. Natl. Acad. Sci USA 102:2549-2554). In a similar way, the discovery that several oomycete effector molecules enter the host cytoplasm through a specific host targeting motif (Whisson, S. C. et al., 2007, Nature 450: 115-118) has been exploited to identify oomycete effector catalogues. It is currently predicted that the genomes of oomycete plant pathogens contain hundreds of such effectors.
The effectors of extracellularly growing plant pathogenic fungi are usually very rich in cysteine residues involved in disulphide bridges, thereby protecting them against proteinases that occur frequently in apoplastic spaces of their host plants (Tomma et al., supra, Kamoun, S., 2006, Ann. Rev. Phytopathol. 44:41-60). At present, relatively few whole-genome sequences of plant pathogenic fungi are available when compared with bacteria. As most effector proteins from extracellular pathogenic fungi are secreted, apoplastic extract from colonized plants is an important resource for the discovery of molecular factors important in several plant diseases.
As C. fulvum is restricted to the tomato apoplast during colonization, all communication and exchange of molecular components between C. fulvum and its host occurs in the apoplastic space. So far, analysis of the protein composition of the apoplastic space of C. fulvum infected tomato leaves has mainly resulted in the identification of race-specific avirulence proteins (Avrs) that are secreted by the fungus during infection and which invoke a resistance response in tomato genotypes carrying cognate C. fulvum resistance (Cf) genes (van Kan, J.A.L. et al., 1991,, MoI. Plant Microbe Interact 4:52-59; Joosten, M.H.A.J. et al., 1994, Nature 367:384-386; Westerink, N. et al., 2004, MoI. Microbiol. 54:533-545). As indicated above, these avirulence proteins and their corresponding Cf proteins have been proposed in providing fungal resistance in (transgenic) plants by provoking a hypersensitive response. Engineering this hypersensitive response in plants has been the subject of much research (e.g. WO 96/39802; WO 98/24297; US 7,045,123; US 7,029,667; US 6,998,515; US2005246799; US2005076406; WO 02/02787; US2004016029; US2003182683; WO 03/054211; WO 03/000288; WO 02/12293; WO 01/55347; US 6,774,281, WO 99/45125 and numerous more). In adition, a number of extracellular proteins (Ecps) secreted during infection by all strains of C. fulvum have been identified (Wubben, J. P. et al., 1994, MoI. Plant Microbe Interact. 7:516-524; Lauge, R. et al., 1997, MoI. Plant Microbe Interact. 10:725-734; Haanstra, J.P.W. et al., 2000, Theor. Appl. Genet. 101:661-668). Like Avrs, Ecps induce a resistance response in tomato accessions carrying as yet unindentified Cf- Ecp resistance genes. Collectively, the Avrs and Ecps are the secreted effector proteins. In total, eight C. fulvum secreted effector proteins have been characterized in detail and their corresponding genes have been cloned (Westerink et al., supra; Thomma et al., supra). All these secreted effector proteins are relatively small (ranging from 3 to 15 kDa) and contain a high and even number of cysteine residues that appear to be involved in disulphide bridge formation. These bridges provide a compact tertiary structure that contributes to stability and activity of the secreted effector proteins in the protease-rich tomato apoplast. All of these effector proteins elicit a defence response in plants carrying the cognate Cf genes in a 'gene-for-gene' manner (Kruijt, M. et al., 2005, MoI. Plant Pathol. 6:85-97).
Although already several of these effector proteins are known, it is still desirable to find novel effector molecules. SUMMARY OF THE INVENTION
The invention now comprises a fungal effector protein comprising at least one LysM domain according to the sequence of SEQ ID NO: 4 or a sequence which is 95% or more identical thereto. Preferably such a fungal effector comprises the amino acid sequence as depicted in SEQ ID NO:3 or a protein that is more than 95% identical thereto. Alternatively, said effector is an ortholog of Ecp6, selected from the orthologs of Fig. 11 or a protein that is more than 95% identical thereto.
The invention further comprises a method for detecting the presence of a resistance gene cognate for an effector protein according to the invention, also identified as CfEcpδ orthologs, in a plant comprising introducing a fungal effector protein according to the invention to a plant or a plant part; and detecting whether a resistance reaction in said plant or plant part occurs. Said presence of CfEcpδ orthologs preferably confers fungal resistance to said plant. In a preferred embodiment the introduction of said fungal effector protein is established by infecting the plant or plant part with a fungus capable of expressing said protein, preferably wherein introduction of said fungal effector protein is established by application of said protein to the plant or a plant, more preferably wherein said application is application into the apoplastic space. In another preferred embodiment the introduction of said fungal effector protein is by transformation of a plant with a construct encoding said protein, more preferably wherein said effector protein is transiently expressed. Further preferred herein is a method wherein introduction of said fungal effector protein is achieved by transient Agrobacterium transformation (ATTA) or through a viral construct, preferably a potatoviral construct.
Also part of the invention is the use of an effector protein according to the invention for assaying pathogen resistance in plants. Further part of the invention is a method for providing pathogen resistant plants, comprising: a. Selecting a plant having pathogen resistance and containing a resistance gene cognate for an effector protein according to the invention; b. Crossing said plant with a plant that needs to be provided with pathogen resistance; c. Assaying offspring of said crossing by testing for the presence of the resistance gene cognate for an effector protein according to the invention; d. Selecting those plants that contain said resistance gene.
Preferably in said method said assaying of step (c) is performed with a method according to the invention.
LEGENDS TO THE FIGURES
Fig. 1. Disease progression of Cladosporium fulυum on tomato.
A. Typical symptoms caused by C. fulvum on susceptible MM-Cf-O tomato plants at 3, 6, 9, 13 and 16 days post inoculation (dpi). The fungus is not visible at early stages of infection (3 dpi) but develops white patches of conidiophores (6 dpi) that expand and cover almost the whole leaf (9 dpi). Subsequently, the conidiophores start to produce conidia (13 dpi) which give the leaf a green-brownish velvet-like appearance (16 dpi).
B. Quantitative real-time reverse transcription PCR to measure C. fulvum growth on resistant MM-Cf-4 tomato plants (white) and on susceptible MM-Cf- 0 tomato plants (grey) at 3, 6, 9, 13 and 16 dpi. The extent of colonization is determined by the relative quantification (RQ) of transcript levels of the constitutively expressed C. fulvum actin gene (measure for fungal biomass) to the constitutively expressed tomato glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (measure for plant biomass) shown on a logarithmic scale. Bars represent mean values and standard errors of three leaflets taken from two plants at each time point analysed. The experiment was repeated twice with similar results.
Fig. 2. The apoplast proteome of Cladosporium fulυum-infected tomato analysed with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Coomassie brilliant blue-stained 2D-PAGE gels obtained after electrophoresis of soluble proteins present in apoplastic fluid collected from a compatible (A; race 5 C. fulυum strain inoculated onto MM-Cf-O plants) and an incompatible (B; race 5 C. fulvum strain inoculated onto MM-Cf-4 plants) interaction at 14 days post inoculation. The proteins were focused over a non-linear gradient of pH 4—7. Molecular weight markers for the second dimension are indicated on the left. The part of the gel containing the C. fulvum-derived differentially accumulated proteins is shown. Protein spots for which identification was pursued are numbered.
Fig. 3. Expression analysis of the newly identified Cladosporium fulvum extracellular proteins. The expression of CfPhiA, Ecp6, Ecp7 and Aυr9 genes was monitored during the interaction of C. fulυum with MM-Cf-4 tomato (incompatible; white bars) and MM-Cf-O tomato (compatible; grey bars) at 3, 6, 9, 13 and 16 days post inoculation. Real-time reverse transcription PCR was used for the relative quantification (RQ) of transcript levels of the C. fulυum CfPhiA, Ecp6 and Ecp7 genes relative to the constitutively expressed C. fulυum actin gene as an endogenous control. The RQ of the Aυr9 gene is shown as an example of the expression profile of a typical C. fulυum effector gene.
The mean and standard error of the results obtained from three leaflets taken from two plants at each time point assayed are shown. The experiment was repeated twice with similar results. Fig. 4. Symptoms caused by wild-type Fusarium oxysporum f. sp. lycopersici and heterologous Ecp6 overexpression transformants on susceptible tomato. A and B. Side view (A) and top view (B) of the disease phenotype caused by F. oxysporum f. sp. lycopersici wild-type (WT) and four independent heterologous Ecp6 overexpression transformants (Ecp6-1 to Ecp6-4) on susceptible tomato MoneyMaker plants when compared with mock-inoculated tomato (mock) at 14 days post inoculation.
C. Reverse transcription PCR to detect in planta transcription of heterologously expressed C. fulυum Ecp6 in F. oxysporum f. sp. lycopersici wild-type and four independent heterologous Ecp6 overexpression transformants (Ecp6-1 to Ecp6-4) on susceptible tomato MoneyMaker plants when compared with mock-inoculated tomato (mock) at 14 days post inoculation.
Fig. 5. Expression analysis and quantification of growth of Cladosporium fulvum RNAi transformants silenced for Ecp6.
A. The expression of Ecp6 is monitored during a compatible interaction between C. fulvum and MM-Cf-O tomato involving the wild-type (WT) C. fulυum and RNAi transformants at 10 days post inoculation. Real-time PCR was used to measure the relative quantification (RQ) of transcript levels of the Ecp6 genes, as compared with the constitutively expressed C. fulυum actin gene as an endogenous control. Bars represent mean values and standard error of the results obtained from three leaflets taken from two infected plants.
B. Growth of WT C. fulυum and RNAi transformants was quantified on MM- Cf-O tomato plants. The transcript levels of the constitutively expressed C. fulυum actin gene (measure for fungal biomass) relative to the levels of the constitutively expressed tomato glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (measure for plant biomass) are shown to determine the degree of fungal colonization of the MM-Cf-O tomato leaves. Bars represent mean values and standard error of the results obtained from three infected leaflets taken from two plants.
Fig. 6. Typical symptoms caused by C. fulυum wild-type (WT) and RNAi transformants silenced for Ecp6 at 10 days post inoculation onto susceptible tomato plants (MM-Cf-O).
Fig. 7. Allelic variation of the Cladosporium fulυum Ecp6 gene. Open reading frames are shown as light grey boxes and introns as black boxes. The predicted signal peptide is indicated as dark grey box. The white flag indicates a single- nucleotide polymorphism (SNP) that leads to an amino acid substitution in the Ecp6 protein. Silent mutations are indicated by a T. The figure is drawn to scale.
Fig. 8. Homologues of Ecp6 in other fungal species. Neighbour- joining tree of 17 Ecp6-like sequences from different fungal species. The evolutionary history of Ecp6-like protein sequences was inferred by neighbour- joining analysis and bootstrap values (%) are indicated at the nodes. The tree is drawn to scale, with branch lengths representing evolutionary distances. The positions containing alignment gaps were eliminated in pair- wise sequence comparisons. A total of 220 positions were calculated in the final data set.
Fig. 9. Homology models for the LysM domains of Cladosporium fulυum Ecp6.
A. Alignment of the individual LysM domains of C. fulυum Ecp6 (this study), Escherichia coli MItD (Bateman and Bycroft, 2000) and Pteris ryukyuensis
PrChi-A. Identical amino acid residues are shaded in black and similar residues (75% threshold according to Blosum62 score) are shaded in grey.
B. LysM domains modelled based on the MItD LysM solution structure. Panels 1, 2 and 3 display the three-dimensional ribbon structures of the Ecp6 LysM domains 1, 2 and 3 respectively. Panel 4 shows the computed molecular surface of Ecp6 LysM domain 1. The arrow indicated in panel 1 indicates the direction of looking to obtain the view in panel 4. The arrow in panel 4 indicates the shallow groove described as the site of interaction of PrChi-A with chitin oligomers.
Fig. 10. Recognition of Cladosporium fulυum Ecp6 in various wild tomato varieties. A strong response in the tomato varieties L. cheesmanii v.typ. PI266375 Gl.1615 (A) and L. pimpinellifolium Gl.1914 (B) results in a hypersensitive response including tissue collapse. A less strong response in L. pimpinellifolium Gl.1310 (C) and L. pimpinellifolium PI344102 Gl.1594 (D) results in clear chlorosis. No phenotypic responses are observed upon injection of Ecp6 in MoneyMaker (E) and Motelle (F) plants.
Fig. 11. Nucleotide and amino acid sequences of Ecp6 orthologs.
Fig. 12. Nucleotide and amino acid sequences of CfPhiA and Ecp7.
DETAILED DESCRIPTION OF THE INVENTION
Definitions The term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. For example, gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. The term "native" or "wild type" gene refers to a gene that is present in the genome of an untransformed cell, i. e., a cell not having a known mutation. The term "native" or "wild type" is intended to encompass allelic variants of the gene. A "marker gene" encodes a selectable or screenable trait. The term
"selectable marker" refers to a polynucleotide sequence encoding a metabolic trait which allows for the separation of transgenic and non-transgenic organisms and mostly refers to the provision of antibiotic resistance. A selectable marker is for example the aphLl encoded kanamycin resistance marker, the nptll gene, the gene coding for hygromycin resistance. Other selection markers are for instance reporter genes such as chloramphenicol acetyl transferase, β-galactosidase, luciferase and green fluorescence protein. Identification methods for the products of reporter genes include, but are not limited to, enzymatic assays and fluorimetric assays. Reporter genes and assays to detect their products are well known in the art and are described, for example in Current Protocols in Molecular Biology, eds. Ausubel et al., Greene Publishing and Wiley- Inter science: New York (1987) and periodic updates.
The term "chimeric gene" refers to any gene that contains 1) DNA sequences, including regulatory and coding sequences that are not found together in nature, or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.
A "transgene" refers to a gene that has been introduced into the genome by transformation. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.
An "oligonucleotide", e. g., for use in probing or amplification reactions, may be about 30 or fewer nucleotides in length (e. g., 9, 12, 15, 18, 20, 21 or 24, or any number between 9 and 30). Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16 to 24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use in processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or even 1000's of nucleotides in length.
The terms "protein", "peptide" and "polypeptide" are used interchangeably herein.
"Coding sequence" refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", i. e., lacking an intron, such as in a cDNA or it may include one or more introns bound by appropriate splice junctions. An "intron" is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.
"Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. As is noted above, the term "suitable regulatory sequences" is not limited to promoters. "Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
"Constitutive expression" refers to expression using a constitutive promoter. "Transient expression" is expression as a result of a transient transformation event. Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus. "Constitutive promoter" refers to a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant.
The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. The terms "initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation). "Operably-linked" refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i. e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
"Expression" refers to the transcription and/or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants. Expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein.
The terms "heterologous DNA sequence", "exogenous DNA segment" or "heterologous nucleic acid", as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
"Homologous to" in the context of nucleotide or amino acid sequence identity refers to the similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (as described in Haines and Higgins (eds.), Nucleic Acid Hybridization, IRL Press, Oxford, U. K.), or by the comparison of sequence similarity between two nucleic acids or proteins. Two nucleotide or amino acid sequences are homologous when their sequences have a sequence similarity of more than 60%, preferably more than 70%, 80%, 85%, 90%, 95%, or even 98%.
The term "substantially similar" refers to nucleotide and amino acid sequences that represent functional and/or structural equivalents of sequences disclosed herein. For example, altered nucleotide sequences which simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to a particular amino acid sequence are substantially similar to the particular sequences. In addition, amino acid sequences that are substantially similar to a particular sequence are those wherein overall amino acid identity is at least 65% or greater to the instant sequences. Modifications that result in equivalent nucleotide or amino acid sequences are well within the routine skill in the art. Moreover, the skilled artisan recognizes that equivalent nucleotide sequences encompassed by this invention can also be defined by their ability to hybridize, under low, moderate and/or stringent conditions (e. g., 0. IX SSC, 0.1% SDS, 65°C), with the nucleotide sequences that are within the literal scope of the instant claims. The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms". Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation (De Blaere et al., 1987) particle bombardment technology (Klein et al. 1987; U.S. Patent No. 4,945,050), microinjection, CaPCu precipitation, lipofection (liposome fusion), use of a gene gun and DNA vector transporter (Wu et al., 1992). Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan (see, for example, Fromm et al., 1990).
"Transformed", "transgenic" and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The heterologous nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook et al., 1989. See also Innis et al., 1995 and Gelfand, 1995; and Innis and Gelfand, 1999. For example, "transformed", "transformant", and "transgenic" plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal plants that have not been through the transformation process.
"Transiently transformed" refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance.
"Stably transformed" refers to cells that have been selected and regenerated on a selection media following transformation.
"Genetically stable" and "heritable" refer to chromosomally- integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations. "Chromosomally-integrated" refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not "chromosomally integrated" they may be "transiently expressed".
"Genome" refers to the complete genetic material of an organism. The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e. g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985; Rossolini et al. 1994). A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid (DNA) is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA which can be single-or double-stranded, optionally containing synthetic, non- natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid" or "nucleic acid sequence" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.
The nucleotide sequences used in aspects of the invention include both the naturally occurring sequences as well as mutant (variant) forms. Such variants will continue to possess the desired activity, i. e., either promoter activity or the activity of the product encoded by the open reading frame of the non-variant nucleotide sequence.
Thus, by "variant" is intended a substantially similar sequence. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein. Generally, nucleotide sequence variants of the invention will have at least 40,50,60, to 70%, e. g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e. g., 81%-84%, at least 85%, e. g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide sequence identity to the native (wild type or endogenous) nucleotide sequence.
The term "nucleotide sequence identity" or "nucleotide sequence homology" as used herein denotes the level of similarity, respectively the level of homology, between two polynucleotides. Polynucleotides have "identical" sequences if the sequence of nucleotides in the two sequences is the same. Polynucleotides have "homologous" sequences if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence. Sequence comparison between two or more polynucleotides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is generally from about 20 to 200 contiguous nucleotides. The "percentage of sequence identity " or "percentage of sequence homology" for polynucleotides, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence identity or homology may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and (c) multiplying the result by 100 to yield the percentage of sequence homology. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms, or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990; Altschul et al., 1997) and ClustalW programs, both available on the internet. Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity, and FASTA in the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), Madison, WI, USA) (Devereux et al., 1984). The nucleic acid sequences of the invention can be "optimized" for enhanced expression in plants of interest. See, for example, EP 0359472 or WO 91/16432. In this manner, the open reading frames in genes or gene fragments can be synthesized utilizing plant-preferred codons. Thus, the nucleotide sequences can be optimized for expression in any plant. By "variant" polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
Thus, the polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred. Individual substitutions deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are "conservatively modified variations", where the alterations result in the substitution of an amino acid with a chemically similar amino acid. This would mean that variants have a degree of homology (or identity) of preferably more than 90%, more preferably more than 95%, more preferably more than 97% and most preferably more than 98% or 99%. A "homologous" gene is a gene related to a second gene by descent from a common ancestral DNA sequence. The term, homolog, may apply to the relationship between genes separated by the event of speciation (ortholog) or to the relationship between genes separated by the event of genetic duplication (paralog). "Orthologs" are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes. "Paralogs" are genes related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.
"Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.
The term "vector" as used herein refers to a construction comprised of genetic material designed to direct transformation of a targeted cell. A vector contains multiple genetic elements positionally and sequentially oriented, i.e., operatively linked with other necessary elements such that the nucleic acid in a nucleic acid cassette can be transcribed and when necessary, translated in the transformed cells. "Vector" is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e. g. autonomous replicating plasmid with an origin of replication). Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e. g. higher plant, mammalian, yeast or fungal cells). Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e. g. bacterial, or plant cell. The vector may be a bi- functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
"Cloning vectors" typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
A "transgenic plant" is a plant having one or more plant cells that contain an expression vector.
"Significant increase" is an increase that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 10%-50%, or even 2-fold or greater.
"Significantly less" means that the decrease is larger than the margin of error inherent in the measurement technique, preferably a decrease by about 2-fold or greater.
Virtually any DNA composition may be used for delivery to recipient plant cells. For example, DNA segments in the form of vectors and plasmids, or linear DNA fragments, in some instances containing only the DNA element to be expressed in the plant, and the like, may be employed. The construction of vectors which may be employed in conjunction with the present invention will be known to those of skill of the art in light of the present disclosure (see, e. g., Sambrook et al., 1989; Gelvin et al., 1990). Vectors, including plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) and DNA segments for use in transforming cells, according to the present invention will, of course, comprise the cDNA, gene or genes necessary for production of the desired protein in the transformant. The vector of the invention can be introduced into any plant. The genes and sequences to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest. The transcriptional cassette will include in the 5'-to-3' direction of transcription, transcriptional and translational initiation regions, a DNA sequence of interest, and transcriptional and translational termination regions functional in plants.
The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) MoI. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671- 674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.
Methodologies for the construction of plant transformation constructs are described in the art. Obtaining sufficient levels of transgene expression in the appropriate plant tissues is an important aspect in the production of genetically engineered crops. Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Transformation of plants can be undertaken with a single DNA molecule or multiple DNA molecules (i. e., co-transformation), and both these techniques are suitable for use with the expression cassettes of the present invention. Numerous transformation vectors are available for plant transformation, and the expression cassettes of this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation.
Suitable methods of transforming plant cells include, but are not limited to, microinjection (Crossway et al., 1986), electroporation (Riggs et al., 1986), Agrobacterium -mediated transformation (Hinchee et al., 1988), direct gene transfer (Paszkowski et al., 1984), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wis. And BioRad, Hercules, Calif, (see, for example, Sanford et al., U. S. Pat. No. 4,945,050; and McCabe et al., 1988). Also see, Weissinger et al., 1988; Sanford et al., 1987 (onion); Christou et al., 1988 (soybean); McCabe et al., 1988 (soybean); Datta et al., 1990 (rice); Klein et al., 1988 (maize); Klein et al., 1988 (maize); Klein et al., 1988 (maize); Fromm et al., 1990 (maize); and Gordon- Kamm et al., 1990 (maize); Svab et al., 1990 (tobacco chloroplast); Koziel et al., 1993 (maize); Shimamoto et al., 1989 (rice); Christou et al., 1991 (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al., 1993 (wheat); Weeks et al., 1993 (wheat). In one embodiment, the protoplast transformation method for maize is employed (European Patent Application EP 0 292 435, U. S. Pat. No. 5,350,689).
It is particularly preferred to use the binary type vectors of Ti and Ri plasmids of Agrobacterium spp. Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti et al., 1985 : Byrne et al., 1987 ; Sukhapinda et al., 1987; Park et al., 1985: Hiei et al., 1994). The use of T-DNA to transform plant cells has received extensive study and is amply described (EP 120516; Hoekema, 1985 ; Knauf, et al., 1983; and An et al.,
1985). For introduction into plants, the chimeric genes of the invention can be inserted into binary vectors as described in the examples.
Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see EP 0295959), techniques of electroporation (Fromm et al., 1986) or high velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (Kline et al., 1987, and U. S. Patent No. 4,945,050). Once transformed, the cells can be regenerated by those skilled in the art. Of particular relevance are the methods to transform foreign genes into commercially important crops, such as rapeseed (De Block et al., 1989), sunflower (Everett et al., 1987), soybean (McCabe et al., 1988; Hinchee et al., 1988 ; Chee et al., 1989; Christou et al., 1989 ; EP 301749), rice (Hiei et al., 1994), and corn (Gordon Kamm et al., 1990; Fromm et al., 1990).
Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i. e., monocotyledonous or dicotyledonous. Agrobacterium tumefaciens cells containing a vector comprising an expression cassette of the present invention, wherein the vector comprises a Ti plasmid, are useful in methods of making transformed plants. Plant cells are infected with an Agrobacterium tumefaciens as described above to produce a transformed plant cell, and then a plant is regenerated from the transformed plant cell. Numerous Agrobacterium vector systems useful in carrying out the present invention are known. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, 1984).
Methods using either a form of direct gene transfer or Agrobacterium-mediated transfer usually, but not necessarily, are undertaken with a selectable marker which may provide resistance to an antibiotic (e. g., kanamycin, hygromycin or methotrexate) or a herbicide (e. g., phosphinothricin) .
In order to provide a quick and simple test if a new plant species indeed can yield a hypersensitive response upon presentation of the effector proteins of the invention, the person skilled in the art can perform one of two tests. One of the most reliable is the infiltration of the elicitor protein in the leaves, and scoring for the HR. Another one is a rapid transient expression test known under the name of ATTA (Agrobacterium tumefaciens Transient expression Assay). In this assay (of which a detailed description can be found in Van den Ackerveken, G., et al., Cell 87, 1307-1316, 1996) the nucleotide sequence coding for an effector protein is placed under control of the CaMV 35S promoter and introduced into an Agrobacterium strain which is also used in protocols for stable transformation. After incubation of the bacteria with acetosyringon or any other phenolic compound which is known to enhance Agrobacterium T-DNA transfer, 1 ml of the Agrobacterium culture is infiltrated into an in situ plant by injection after which the plants are placed in a greenhouse. After 2-5 days the leaves can be scored for occurrence of HR symptoms.
The invention now comprises a new effector protein that has been identified in the interaction between tomato plants and Cladosporium fulvum. Said effector molecule, Ecp-6, of which both the amino acid sequence and the nucleotide sequence encoding said polypeptide are given below, is clearly different from known effector molecules, like Avr's and other Ecp effector proteins.
Wild-type C. fulvum Ecp6. SEQ ID NO:1 Ecp6_Genomic DNA (start and stop codons bold underlined and intron sequences in italics and underlined)
Figure imgf000027_0001
ATCGAATTC
SEQ ID NO:2 Ecp6_CDNA
Figure imgf000028_0001
GCTGTGGCATAA SEQ ID NO:3 Ecp6_Protein (signal sequence underlined)
MQSMILFAAALMGAAVNGFVLPRETKATDCGSTSNIKYTWKGDTLTSIAKKFKSGICNIV SVNKLANPNLIELGATLIIPENCSNPDNKSCVSTPAEPTETCVPGLPGSYTIVSGDTLTNI SQDFNITLDSLIAANTQIENPDAIDVGQIITVPVCPSSQCEAVGTYNIVAGDLFVDLAATY HTTIGQIKALNNNVNPSKLKVGQQIILPQDCKNVTTAVA
The mature Ecp6 protein contains 199 amino acids and has an estimated molecular mass of 21 kDa, making it the largest of the abundantly secreted effector proteins of C. fulvum identified so far. Previous studies on the genes encoding secreted C. fulvum effectors have shown that Aυr genes accumulated considerably more polymorphisms than Ecp genes (Stergiopoulos et al., 2007). This was suggested to be due to the lack of selection pressure imposed on the pathogen to overcome resistance mediated by resistance genes that recognize Ecps, as these have not been deployed yet in commercial tomato lines. In line with these findings, polymorphisms in Ecp6 were only rarely observed. Of the 50 C. fulvum strains tested, only seven strains contained allelic variants of Ecpβ. All seven of these strains contained the same four SNPs, while one strain contained an additional fifth SNP. Of these five SNPs, only one resulted in an amino acid change, while the four others concerned silent or intron mutations. The occurrence of mostly synonymous modifications in Ecp genes was hypothesized to imply selective constraints for maintaining Ecp protein sequences or, alternatively, a recent common ancestor gene (Stergiopoulos et al., 2007). However, our finding that Ecp6 markedly contributes to C. fulvum virulence, and that Ecp6 has orthologues in other fungal species, favours the second hypothesis.
The nucleotide sequences of the two other versions of Ecp6 are given below:
Allelic variant 1 (G > A at 128 bp downstream of the putative start codon in intron, shown in gray shading)
ATGCAGTCGATGATTCTTTTCGCTGCCGCTCTTATGGGCGCCGCCGTAAACGGCTTCGTTCTCCCA CGTACTGATGACCCTGATTGTATAGTCGATGGCTATTAGAGCTGCAGAGACCGGCTGACACMiTTTC
TAGGCGAGACCAAGGCCACAGACTGCGGTTCGACCAGCAACATCAAATACACTGTCGTCAAGGGTG
TCGCCAACCCCAACCTCATCGAGCTCGGCGCAACCCTCATCATCCCAGAGAACTGTTCTAACCCCG
Figure imgf000029_0001
AGTGCGAGGCTGTCGGTACTTACAACATTGTGGCCGGTGACCTTTTCGTCGATTTGGCCGCTACCT
GTCAGCAGATCATTCTGCCACAGGACTGCAAGAACGTCACTACTGCTGTGGCATiUV
Allelic variant 2 (G > A at 494 bp downstream of the putative start codon in intron, C >T at 335 bp downstream of the putative start codon, silent mutation, G > A at 662 bp downstream of the putative start codon, silent mutation, and C > A at 142 bp downstream of the putative start codon, amino acid substitution Thr25 > Asn, all shown in gray shading). ATGCAGTCGATGATTCTTTTCGCTGCCGCTCTTATGGGCGCCGCCGTAAACGGCTTCGTTCTCCCA CGCGAGAACAAGGCCACAGACTGCGGTTCGACCAGCAACATCAAATACACTGTCGTCAAGGGTGAC
GCCAACCCCAACCTCATCGAGCTCGGCGCAACCCTCATCATCCCAGAGAACTGTTCTAACCCCGAC TACACCATCGTCAGCGGCGACACTCTCACCAACATCTCCCAGGACTTCAACATCACGCTCGACTCC
CCAGTCTGCCCATCGTCCCAGTGCGAGGCTGTCGGTACTTACAACATTGTGGCCGGTGACCTTTTC GTCGATTTGGCCGCTACCTACCACACCACTATCGGTCAGATTAAGGCTCTCAACAACAACGTTAAC GTGGCATAA
Ecp6_Protθin (amino acid substitution Thr25 > Asn)
MQSMILFAAALMGAAVNGFVLPRENKATDCGSTSNIKYTWKGDTLTSIAKKFKSGI
QDFNITLDSLIAANTQIENPDAIDVGQI ITVPVCPSSQCEAVGTYNIVAGDLFVDLAATYHTTIGQ IKALNNNVNPSKLKVGQQIILPQDCKNVTTAVA The Ecp6 protein contains three lysin motifs (LysM domains) that were originally found in a variety of enzymes that bind to and hydrolyse peptidoglycans present in bacterial cell walls, of which lysozyme is the best- known example.
A further difference with many of the known effector proteins is that it appears that many orthologs are present in pathogenic fungi. 70 fungal species (see Table 1) were tested for the occurrence of Ecp6 orthologs (see Table 1). Of the 70 species tested, LysM-encoding genes are not found in 14 species. It was found that most of the orthologs have more than one LysM domains, and some have up to 7 repeats. Table 2 shows the fungal species and the identification code of the orthologs found therein, and also an indication of the start and stop position of the LysM domains in these orthologs is given. To study the conservation between LysMs of these Ecp6 orthologs, a multiple sequence alignment of the 669 LysMs found in the 302 LysM ortholog proteins was generated and used to build a consensus sequence. When comparing this consensus to the published LysM consensus that is mainly based on prokaryotic LysMs (Pfam ID: PF01476) it seemed that, like in bacteria, the first 16 amino acid residues are most conserved, whereas the final 10 residues seem less conserved for fungal LysMs. The most remarkable difference, however, is the presence of two highly conserved cysteine residues at positions 9 and 44 in the fungal LysMs. Moreover, two additional, albeit less-conserved, cysteine residues are found at positions 32 and 34 in the fungal LysM consensus sequence. It has been shown for several C. fulυum effectors that the formation of disulphide bridges between cysteine residues is required for stability upon secretion in the host, which might be true also for fungal LysM effectors.
The consensus sequence that can be defined for the common LysM domain(s) in Ecp6 orthologs has the following sequence: (SEQ ID NO: 4)
[YIH]X[VIAT]XX[GN] [DEQ] [TSY] [CYIVL]XX[LIV] [AS]XXX[GNCS] [LIVR] [ST]XXX
[LIVF]XXXNXXXXXXXXX[LIV]XX[GDN]XX[LIV] [CK] [LIVT]X in which X can be any amino acid, and the amino acids indicated between square brackets indicate alternatives at that position.
Thus, part of the invention are effector proteins that have at least a domain comprising the amino acid sequence of SEQ ID NO:4 or a domain that is at least 95% or more identical to the sequence as shown in SEQ ID NO:4. It will be clear to the skilled person that - in general - the effector proteins of the present invention lack a further functional domain. This is in contrast with other Lysm containing proteins (not being effector proteins) that are found in fungi, wherein the LysM domain(s) is/are coupled with other functional domains, such as domains having an enzymatic function.
The LysM domains are thought to be involved in chitin binding, and thus protecting the chitin structure of the fungus against any chitinase activity produced by the plant. The hypothesis is that, in its turn, the plant has used this protection mechanism of the fungus as a signal that a fungal infection is taken place. Accordingly, the plant has developed a recognition system, which puts in motion a signaling cascade that eventually results in a hypersensitive response. The hypothetical receptor or resistance gene product that recognizes Ecp6 (or one of its orthologs) is denominated c/Ecp6 (i.e. the cognate counterpart of C. fulυum Ecp6).
It now has appeared (see the experimental section) that c/Ecp6 is capable to recognize not only Ecp6 itself, but also orthologs of this effector protein, i.e. orthologs derived from many, different fungal species. These species include tomato pathogens, but also pathogens of other crop plants.
It has also appeared that it is possible to induce a hypersensitive response in other plants than tomato (e.g. Arabidopsis, see Example 3) when introducing Ecp6. These two findings indicate that a similar mechanism for pathogen resistance as is found in tomato with this specific fungal effector protein and its cognate hypothetical receptor also exists in other plants. This opens up the possibility of using the effector proteins of the invention (Ecp6 and its orthologs) to search for the cognate receptor/resistance protein in any plant. If plants are found that show a hypersensitive response upon introduction of Ecp6, such a plant will possess a c/Ecp6 protein or an ortholog thereof. Such a plant then can be used as a parental line in the breeding of (fungal) resistance in plants.
Thus, the invention also comprises a method for the testing of the presence of the c/Ecp6 ortholog in a plant by using an effector protein according to the invention. In such a method a plant is tested for the presence of c/Ecp6 (or ortholog of c/Ecp6) by providing a plant with an effector protein of the invention and checking for a hypersensitive response. Providing the effector protein to the plant(s) in such an assay can be achieved in many ways, which will be clear to the person of skill in the art.
A first method to provide a plant with an effector protein is by injecting the protein into a part of the plant, e.g. a leaf or a stem segment. In a plant that does not have the functional cognate resistance gene the injection of the protein will have no clear visible result. However, in a plant that harbours a functional resistance gene, said c/Ecp6 or ortholog will recognize the effector protein and the cascade leading to the hypersensitive reaction will be started. As discussed before, and as is shown in Fig. 10, this will lead to a local necrosis of the plant tissue, or, in a less strong reaction, to chlorosis of the injected site, which effect can be visually observed.
In stead of direct injection of the protein into a plant (part) it is also possible to provide the protein to the plant through a nucleotide construct encoding said protein. One of the most suitable ways to enable expression of the protein in a plant or plant part is through the so-called ATTA (Agrobacterium Transient Transformation Assay), in which an Agrobacterium tumefaciens bacterium is provided with a construct encoding an effector protein according to the invention. Since the nucleotide sequences encoding the effector proteins are disclosed in this description or can be derived from the amino acid sequences that are also provided in the present description, any person of skill will be able to clone a coding sequence into an expression vector that is suitable for Agrobacterium transformation.
Alternatively, a viral vector can be used. A sequence encoding an effector protein of the invention can be cloned into a viral vector (such as potatovirus X) after which the viral particles are used to infect the plant or plant part and to express the protein. As is shown in the experimental section a combination of both techniques can be used in which a viral vector harbouring the sequence encoding the effector protein is constructed after which this viral vector is introduced into A. tumefaciens. Using a viral vector in which the virus stills maintains its infectious properties has a further advantage. Next to the observation of local necrosis or chlorosis, which indicates the presence of the resistance gene, the absence of the resistance gene will also be visible, because in such plants the disease that is caused by the virus will develop. Thus, even if no clear signs of a hypersensitive response will be detected, a lack of disease progress will indicate the presence of a resistance mechanism. Of course, such a system will only be feasible with plants that are normally susceptible for the virus that is used as a viral vector. Ideally, it should be ensured that the effector protein is excreted to the apoplastic space, because this is the location where the effector protein in nature is found. When the assay is based on injecting the protein into the plant, the protein automatically will be present in the apoplastic space. If an assay is used where the protein is introduced by a construct having the nucleotide sequence encoding the effector protein, expression into the apoplastic space can be achieved by providing the nucleotide sequence with a signal sequence for extracellular targeting. Such signal sequences are available for a person skilled in the art and one example is the signal sequence of the tobacco PR- Ia gene. Alternative sequences can be obtained from the Avr4 peptide, the carrot extension gene or from studies on N-terminal signal sequences (Small, I. et al., 2004, Proteomics 4:1581-1590). It is envisaged that the assay system as described above will be of use when breeding or constructing plants with a fungal resistance based on the presence of the Ecp6-c/Ecp6 resistance mechanism. In case of a breeding program for the introduction of c/Ecp6 based resistance, the breeder normally will depart from a plant that contains c/Ecp6 or an ortholog thereof. This plant line will then be used as a parent line and crossed with another plant to generate offspring. This offspring can be tested for the presence of the resistance mechanism in an assay as described above. This process then will be repeated until the desired end product is obtained. In stead of breeding, it will also be possible to engineer the resistance using recombinant techniques. In that case the gene encoding the resistance gene will be cloned into an appropriate vector and a target plant will be transformed with this vector. Functional expression of the resistance gene can then be assessed by an assay wherein the plant is provided with the effector protein of the invention.
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Table 2
SPECIES RELEA INSTITUTE PROTEIN ID LYSM LYS LYS LYS LYS LYS LYS LYS LYS LYS LYS LYS LYS LYS LYS LYS LYS
SE V. EFFECTOR M1 - Mi- M2- M2- M3- M3- M4- M4-E M5- M5- M6- M6- M7- M7- M8- M8-
Start End Start End Start End Start Start End Start End Start End Start End
Aspergillus clavatuε 1 Broad - FGI ACLA_004670 108 154 160 208 256 301 365 41 1 448 500 537 582 609 654
Aspergillus clavatuε 1 Broad - FGI ACLA_019900 47 93 123 168
Aspergillus clavatuε 1 Broad - FGI ACLA_022990 252 297 342 387 427 472 524 561
Aεpergilluε clavatuε 1 Broad - FGI ACLA_054430 258 301
Aεpergilluε clavatuε 1 Broad - FGI ACLA_067420 236 280 286 331 376 421 456 501
Aεpergilluε clavatuε 1 Broad - FGI ACLA_071270 48 93 143 189 231 278 296 341
Aεpergilluε flavuε 2 Broad - FGI AFL2T_00905 172 217 250 295
Aεpergilluε flavuε 2 Broad - FGI AFL2T_0171 1 1 16 161 167 214 367 407
Aεpergilluε flavuε 2 Broad - FGI AFL2T_02043 46 92 180 227 284 329 359 405 435 483
Aεpergilluε flavuε 2 Broad - FGI AFL2T_05105 254 297
Aεpergilluε flavuε 2 Broad - FGI AFL2T_06185 1 10 155
Aεpergilluε flavuε 2 Broad - FGI AFL2T_0801 1 33 76 109 158 172 214
Aεpergilluε flavuε 2 Broad - FGI AFL2T_10474 144 188 194 235 246 291 354 41 1 418 453 505 550
Aspergillus flavus 2 Broad - FGI AFL2T_1 1586 49 87 123 168 Aspergillus fumigatus 1 Broad - FGI Afu1 g15420 48 81 126 174 Aspergillus fumigatus 1 Broad - FGI Afu4g04000 257 300 Aspergillus fumigatus 1 Broad - FGI Afu5g03980 40 108 154 199 247 292 332 377 Aspergillus fumigatus 1 Broad - FGI Afu6g00720 86 131 Aspergillus fumigatus 1 Broad - FGI Afu6g09320 305 350 403 448 Aspergillus fumigatus 1 Broad - FGI Afu6g09790 220 261 292 347 375 420 429 477 Aspergillus nidulans 1 Broad - FGI AN1 T_00850 35 78 151 196 214 259 Aspergillus nidulans 1 Broad - FGI AN1 T_00857 60 104 1 10 149 329 374 412 457 497 542 Aspergillus nidulans 1 Broad - FGI AN1 T_00885 75 1 17 125 174 344 389 425 470 Aspergillus nidulans 1 Broad - FGI AN1 T_00886 52 100 136 182 221 266 305 350 388 433 468 513 Aspergillus nidulans 1 Broad - FGI AN1 T_00896 1 167 1212 1219 1269 1318 1365 Aspergillus nidulans 1 Broad - FGI AN1 T_01384 32 79 Aspergillus nidulans 1 Broad - FGI AN1 T_02157 246 290 296 342 387 432 464 509 Aspergillus nidulans 1 Broad - FGI AN1 T_03077 39 97 142 187 223 268 Aspergillus nidulans 1 Broad - FGI AN1 T_03091 1 166 121 1 1218 1268 1316 1363 Aspergillus nidulans 1 Broad - FGI AN1 T_05043 47 93 140 186 227 272 309 354 402 448 Aspergillus nidulans 1 Broad - FGI AN1 T_05473 75 122 154 206 243 288 331 377 Aspergillus nidulans 1 Broad - FGI AN1 T_07232 91 138 Aspergillus nidulans 1 Broad - FGI AN1 T_09096 23 70 1 1 1 156 322 367 422 467 502 547 585 630 Aspergillus nidulans 1 Broad - FGI AN1 T_09687 30 73 130 175 212 257 298 346 Aspergillus nidulans 1 Broad - FGI AN1 T_10078 32 77 135 180 219 264 Aspergillus oryzae 1 Broad - FGI AO090003000985 46 92 180 227 Aspergillus oryzae 1 Broad - FGI AO090003001360 203 248 254 301 Aspergillus oryzae 1 Broad - FGI AO090005000926 1 19 164 197 242 Aspergillus oryzae 1 Broad - FGI AO090009000182 213 257 263 306 329 355 418 475 505 550 Aspergillus oryzae 1 Broad - FGI AO090010000417 49 94 130 175 Aspergillus oryzae 1 Broad - FGI AO090103000216 42 86 134 180 228 273 Aspergillus oryzae 1 Broad - FGI AO090124000032 33 76 109 158 172 214 Aspergillus niger 1 Broad - FGI ASN IG_C_19000036.1 1 16 160 Aspergillus niger 1 Broad - FGI ASNIG_C_22000043.1 1 10 151 Aspergillus niger 1 Broad - FGI ASN IG_C_24000019.1 1 137 1 182 1 189 1239 1286 1333 Aspergillus niger 1 Broad - FGI ASNIG_C_4000282.1 49 92 123 176 Aspergillus niger 1 Broad - FGI ASNIG_C_6000613.1 180 224 230 277 443 488 525 570 608 653 Aspergillus niger 1 Broad - FGI ASNIG_C_8000051.1 257 300 Aspergillus niger 1 Broad - FGI
Figure imgf000040_0001
ASNIGe_gw1_4.239.1 144 185 217 263 306 351 360 412
Aspergillus niger 1 Broad - FGI ASNIGe_gw1_6.238.1 12 58 82 128 204 251 303 349 381 427 463 511
Aspergillus niger 1 Broad - FGI ASNIGg_C_5000650.1 34 78 1 10 156
Aspergillus niger 1 Broad - FGI ASNIGgw1_1.2101.1 9 53 83 135
Aspergillus niger 1 Broad - FGI ASNIGgw1_1 1.857.1 17 62 83 128
Aspergillus terreuε 1 Broad - FGI ATET_02209 266 311 318 368 419 466
Aspergillus terreuε 1 Broad - FGI ATET_02239 262 305
Aspergillus terreuε 1 Broad - FGI ATET_04459 42 88 126 173 208 254 283 329 365 411
Aεpergilluε terreuε 1 Broad - FGI ATET_06271 23 76 151 196
Aεpergilluε terreuε 1 Broad - FGI ATET_06462 240 285 292 350 401 448
Aεpergilluε terreuε 1 Broad - FGI ATET_07073 212 258 264 312 369 414 487 526 528 581 621 666 693 738
Aεpergilluε terreuε 1 Broad - FGI ATET_07293 121 167 267 314 365 411 443 489
Aεpergilluε terreuε 1 Broad - FGI ATET_07445 1181 1225 1232 1282 1332 1379
Aεpergilluε terreuε 1 Broad - FGI ATET_08589 49 94 139 171 215 260 304 350 368 413
Aεpergilluε terreuε 1 Broad - FGI ATET_09999 49 94 106 170
Aεpergilluε terreuε 1 Broad - FGI ATET_10189 46 89 138 183 220 265 301 346
Botrytiε cinerea 1 Broad - FGI BC1T_03016 250 293
Botrytiε cinerea 1 Broad - FGI BC1T_08058 193 238
Botrytiε cinerea 1 Broad - FGI BC1T_13415 63 106
Botrytiε cinerea 1 Broad - FGI BC1T_13975 36 79 109 157
Batrachochytrium 1 Broad - FGI BDET_02830 108 151 dendrobatidiε
Batrachochytrium 1 Broad - FGI BDET 07591 57 106 143 188 dendrobatidiε
Copπnopεiε cmereuε 1 Broad - FGI CC1 T_ 00641 145 188
Copπnopεiε cmereuε 1 Broad - FGI CC1 T_ 03239 152 195
Copπnopεiε cmereuε 1 Broad - FGI CC1 T_ 05860 28 74 84 129
Copπnopεiε cmereuε 1 Broad - FGI CC1 T 06682 17 44 45 86
Copπnopεiε cmereuε 1 Broad - FGI CC1 T_.06684 29 73 83 128
Chaetomium globoεum 1 Broad - FGI CHGT._03555 49 95 114 160 234 281 336 382 487 535 604 647
Chaetomium globoεum 1 Broad - FGI CHGT._04103 29 72
Chaetomium globoεum 1 Broad - FGI CHGT._04702 232 275
Chaetomium globoεum 1 Broad - FGI CHGT._05423 48 94 132 178 215 259
Chaetomium globoεum 1 Broad - FGI CHGT._05464 223 266 315 361 444 490 532 567
Chaetomium globoεum 1 Broad - FGI CHGT._06158 1313 1358 1365 1422 1433 1479
Chaetomium globoεum 1 Broad - FGI CHGT._08588 42 87 93 140 185 230 392 438 476 522 559 605 643 689
Chaetomium globoεum 1 Broad - FGI CHGT._09456 213 261 267 313 359 404 442 487 533 577 586 630
Chaetomium globoεum 1 Broad - FGI
Figure imgf000041_0001
CHGT 09900 47 93 185 231 287 332 362 408 443 488 558 585
CIHT_04241 42 86 92 139 180 225 394 440 480 529 CIHT_09818 275 318
CIMT_04999 222 266 272 319 360 405 574 620 660 709 CIMT_08979 275 318 CIRT_04614 222 266 272 319 360 405 574 620 660 709
CIRT_06615 275 318
CIST_04150 275 318
CIST_06992 42 86 92 139 180 225 394 440 480 529
CNAT_02514 184 233
CNAT_04233 159 202
CNAT_05522 26 72 82 127
CocheC5_1 19747 144 184 236 284 346 383
CocheC5_123134 40 84
CocheC5_124328 228 272 278 325 366 411 490 531 569 615 655 701 745 791 831 877
CocheC5_127948 702 745
CocheC5_130417 304 349 450 488 537 563
CocheC5_132914 80 123 159 204 216 259
CocheC5_141005 1 16 160
CocheC5_166157 36 81 133 178 221 266
CocheC5_168348 280 330 389 438 490 539 584 633
CocheC5_171410 43 89 173 219 275 320 350 396 447 492
Figure imgf000042_0001
CocheCδ 172945 207 251 257 307 330 365 428 484 517 561 heteroεtrophuε
Cochliobolus 1 JGI - DOE CocheC5_18916 161 206 heterostrophus
Coccidioides posadasii 1 Broad - FGI CPAT_04969 295 338
RMSCC 3488
Coccidioides posadasii 1 Broad - FGI CPAT_06616 222 266 272 319 360 405 574 620 660 709
RMSCC 3488
Coccidioides posadasii 1 Broad - FGI CPST_06416 257 300
Silveira
Coccidioides posadasii 1 Broad - FGI CPST_08933 239 283 289 336 377 422 584 630 670 719
Silveira
Candida tropicalis 3 Broad - FGI CTRT_04177 39 86 1 1 1 161
Candida tropicalis 3 Broad - FGI CTRT_05676 35 87 1 12 163 240 281 315 367 443 494 518 570
Debaryomyceε hanεenn 1 Broad - FGI DEHA0B13563g.1 78 127
Fuεaπum graminearum 3 Broad - FGI FGST. 02218 247 290 Fusaπum graminearum 3 Broad - FGI FGST. 02560 128 171 Fusarium graminearum 3 Broad - FGI FGST. 02586 58 103 Fusarium graminearum 3 Broad - FGI FGST. 03241 82 126 146 193 Fusarium graminearum 3 Broad - FGI FGST. 07342 49 93 145 190 230 275 315 360 393 438 472 517 Fusarium graminearum 3 Broad - FGI FGST. 08300 121 164 Fusarium graminearum 3 Broad - FGI FGST..10052 166 213 321 370 Fusarium graminearum 3 Broad - FGI FGST..10471 217 257 264 314 356 404 467 512 Fusarium graminearum 3 Broad - FGI FGST..12127 32 76 151 196 239 284 319 364 Fusarium oxyεporum 2 Broad - FGI FOXT. 03822 128 171 Fusarium oxyεporum 2 Broad - FGI FOXT. 04195 247 290 Fuεanum oxyεporum 2 Broad - FGI FOXT. 05730 31 76 153 198 241 286 Fuεanum oxyεporum 2 Broad - FGI FOXT. 05750 46 91 123 168 204 249 Fuεanum oxyεporum 2 Broad - FGI FOXT..1 1839 41 87 200 246 276 322 358 403 Fuεanum oxyεporum 2 Broad - FGI FOXT..13204 58 103 Fuεanum oxyεporum 2 Broad - FGI FOXT..14328 136 180 187 237 280 327 390 435 Fuεanum oxyεporum 2 Broad - FGI FOXT..15152 136 180 187 237 280 327 390 435 Fuεanum oxyεporum 2 Broad - FGI FOXT..16948 44 89 134 180 220 266 306 351 392 437 480 525 556 601 Fuεanum oxyεporum 2 Broad - FGI FOXT..16949 159 204 210 260 312 360 Fuεanum oxyεporum 2 Broad - FGI FOXT..17276 35 76 94 139 Fuεanum oxyεporum 2 Broad - FGI
Figure imgf000043_0001
FOXT 17687 56 93 139 184 219 264 305 350 357 402 435 480
Fusaπum verticillioides 3 Broad - FGI FVET_03621 31 76 163 208 248 293
Fuεaπum verticillioideε 3 Broad - FGI FVET_03627 42 87 1 15 160 196 241
Fuεaπum verticillioideε 3 Broad - FGI FVET_05343 125 168
Fuεaπum vertiαllioideε 3 Broad - FGI FVET_07320 247 290
Fuεaπum vertiαllioideε 3 Broad - FGI FVET_07599 80 125 162 207 244 289 322 367
Fuεaπum vertiαllioideε 3 Broad - FGI FVET_12054 128 171
Fuεaπum vertiαllioideε 3 Broad - FGI FVET_12343 58 103
Fuεaπum vertiαllioideε 3 Broad - FGI FVET_13785 159 204 210 260 312 360
Fuεaπum vertiαllioideε 3 Broad - FGI FVET_13786 44 89 134 180 220 266 306 351 392 437 480 525 556 601
Laccaria bicolor 1 JGI - DOE LACB1163800 9 54 64 103
Laccaria bicolor 1 JGI - DOE LACBI297101 146 195
Laccaria bicolor 1 JGI - DOE LACBI301644 182 225
Laccaria bicolor 1 JGI - DOE LACBI313870 132 175
Magnaporthe gπεea 5 Broad - FGI MGT_00421 237 280
Magnaporthe gπεea 5 Broad - FGI MGT_01259 46 94 172 219 274 320 358 398 449 493
Magnaporthe gπεea 5 Broad - FGI MGT_01814 48 96
Magnaporthe gπεea 5 Broad - FGI MGT_03949 46 90 145 191 229 275 307 352
Magnaporthe gπεea 5 Broad - FGI MGT_07020 137 180
Magnaporthe gπεea 5 Broad - FGI MGT_09368 53 98
Magnaporthe gπεea 5 Broad - FGI MGT_09848 159 202
Magnaporthe gπεea 5 Broad - FGI MGT_09958 43 86 1 15 158
Magnaporthe gπεea 5 Broad - FGI MGT_12666 2 48
Mycoεphaerella fψenεiε 1 JGI - DOE MYCFI30096 1 13 156
Mycoεphaerella fψenεiε 1 JGI - DOE MYCFI34332 3 46 53 96
Mycoεphaerella fψenεiε 1 JGI - DOE MYCFI42954 1 19 162
Mycoεphaerella 1 JGI - DOE MYCGR39835 133 178 184 232 279 324 graminicola
Mycoεphaerella 1 JGI - DOE MYCGR40100 39 86 graminicola
Mycoεphaerella 1 JGI - DOE MYCGR44256 308 358 446 495 550 599 graminicola
Mycoεphaerella 1 JGI - DOE MYCGR85230 81 127 166 207 graminicola
Mycoεphaerella 1 JGI - DOE MYCGR8922 2 45 75 1 19 graminicola
Neuroεpora craεεa 7 Broad - FGI NCUT_04902 1 14 154
Neuroεpora craεεa 7 Broad - FGI NCUT_05084 34 77 144 189 228 273 312 357 375 420
Neuroεpora craεεa 7 Broad - FGI
Figure imgf000044_0001
NCUT_06293 37 75
Neurospora crassa NCUT_07078 214 258 265 322 516 567 642 687 738 783 Neuroεpora crassa NCUT_07396 35 79 138 183 219 264 Nectria haematococca NECHA106587 1 139 1 183 1 189 1239 1286 1333 Nectria haematococca NECHA42325 37 84 125 170 234 292 323 369 416 461 500 545 Nectria haematococca NECHA55494 214 258 265 316 373 422 Nectria haematococca NECHA55547 36 80 Nectria haematococca NECHA56796 27 64 105 150 187 232 270 315 350 395 Nectria haematococca NECHA66955 167 210 Nectria haematococca NECHA74344 219 263 269 316 Nectria haematococca NECHA84348 48 87 92 137 177 223 282 317 Nectria haematococca NECHA84350 210 254 260 307 Nectria haematococca NECHA84640 41 85 Nectria haematococca NECHA87919 303 348 354 401 443 488 565 605 659 704 Nectria haematococca NECHA87984 325 370 379 427 Nectria haematococca NECHA90567 33 76 124 169 214 259 293 338 Neoεartorya fiεcheπ NFIA_003680 82 1 18 230 273 376 412 Neosartorya fischeri NFIA_004980 1 1 12 1 157 1 164 1214 1263 1310 Neoεartorya fischeri NFIA_007660 84 129 162 207 Neoεartorya fiεcheπ NFIA_009990 48 92 137 183 213 259 Neoεartorya fiεcheπ NFIA_029390 257 300 Neoεartorya fiεcheπ NFIA_038140 40 87 133 178 228 273 312 357 Neoεartorya fiεcheπ NFIA_041570 2 37 77 122 161 206 242 287 Neoεartorya fiεcheπ NFIA_044370 1219 1263 1270 1324 1371 1420 Neoεartorya fiεcheπ NFIA_047880 80 125 157 202 Neoεartorya fiεcheπ NFIA_055510 50 95 Neoεartorya fiεcheπ NFIA_069820 47 92 146 192 234 279 340 387 405 450 Ophioεtoma novo-ulmi COGEME OnCon[0025] 50 94 198 242
Podoεpora anεeπna Pa_3_3275 7 49 86 133 Podoεpora anεeπna Pa_3_780 131 176 Podoεpora anεeπna Pa_4_1460 35 80 160 205 Podoεpora anεeπna Pa_4_5520 51 97 172 217 250 295 332 377 410 455 Podoεpora anεeπna Pa_5_1 130 12 57 93 149 170 204 Podoεpora anεeπna Pa_5_1560 51 95 151 196 229 274 31 1 356 389 434 Podoεpora anεeπna Pa_5_2020 397 444 Podoεpora anεeπna Pa_5_3800 96 141 230 275 344 386 Podoεpora anεeπna
Figure imgf000045_0001
Pa 6 3590 223 266
Podospora anseπna 2 CNRS Pa_7_2050 158 201
Paracoccidioides 1 Broad- FGI PAAG_03769T0 37 82 147 190 bras i liens is Pb01
Paracoccidioides 1 Broad- FGI PAAG_08395T0 267 310 brasiliensis PbO2
Paracoccidioides 1 Broad- FGI PABG_07802T0 263 306 brasiliensis PbO3
Paracoccidioides 1 Broad- FGI PADG_08655T0 263 306 brasiliensis Pb18
Phycomyces 1.1 JGI-DOE PHYBL58843|1 707 131 180 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL68092|21 144 29 77 289 338 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL71631|44_31 29 77 149 213 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL71632|44 32 29 77 142 191 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL71633|44_33 29 77 218 267 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL71635|44_35 29 77 218 267 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL71636|44 36 29 77 142 191 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL79000|210059 30 79 153 202 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL79037|210143 38 86 150 199 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL79038|210145 44 92 135 184 blakesleeanus
Phycomyces 1.1 JGI-DOE PHYBL80444|620032 137 182 blakesleeanus
Phanerochaete 2.1 JGI-DOE PHYCH137048 5 50 60 106 cryεoεponum
Phanerochaete 2.1 JGI-DOE PHYCH137130 18 63 73 118 cryεoεponum
Phanerochaete 2.1 JGI-DOE PHYCH4212 172 221 crysoεpoπum
Phanerochaete 2.1 JGI-DOE PHYCH43875 5 50 60 99 cryεoεponum
Phanerochaete 2.1 JGI-DOE PHYCH44013 5 51 61 100 cryεoεpoπum
Phanerochaete 2.1 JGI-DOE
Figure imgf000046_0001
PHYCH7273 130 173
crysospoπum
Phanerochaete 2.1 JGI - DOE PHYCH7732 31 76 86 131 crysosporium
Postia placenta 1 JGI - DOE POSPL112449 179 222
Postia placenta 1 JGI - DOE POSPL116985 50 95 105 150
Postia placenta 1 JGI - DOE POSPL128517 152 201
Pyrenophora tπtici- 1 Broad - FGI PTRT_00260 129 172 repentiε
Pyrenophora tπtici- 1 Broad - FGI PTRT_01 191 96 141 repentis
Pyrenophora tπtici- 1 Broad - FGI PTRT_04680 283 330 397 441 repentis
Pyrenophora tπtici- 1 Broad - FGI PTRT_05421 42 87 105 150 repentiε
Pyrenophora tπtici- 1 Broad - FGI PTRT_10505 1 18 162 repentis
Pyrenophora tπtici- 1 Broad - FGI PTRT_1 1 100 219 262 repentiε
Rhizopuε oryzae 1 Broad - FGI RO3T_00459 37 82
Rhizopus oryzae 1 Broad - FGI RO3T_02852 27 74 181 231
Rhizopus oryzae 1 Broad - FGI RO3T_05055 31 78
Rhizopus oryzae 1 Broad - FGI RO3T_07020 31 78
Rhizopus oryzae 1 Broad - FGI RO3T_08993 72 1 18
Rhizopus oryzae 1 Broad - FGI RO3T_09412 29 75
Rhizopus oryzae 1 Broad - FGI RO3T_1 1087 31 78
Rhizopus oryzae 1 Broad - FGI RO3T_1 1093 31 78
Rhizopus oryzae 1 Broad - FGI RO3T_1 1427 26 69 169 219
Rhizopus oryzae 1 Broad - FGI RO3T_14383 75 120
Rhizopus oryzae 1 Broad - FGI RO3T_16328 31 78
Stagonospora nodorum 1 Broad - FGI SNOT_02002 1 17 161
Stagonospora nodorum 1 Broad - FGI SNOT_08478 139 184 190 240 290 338
Stagonospora nodorum 1 Broad - FGI SNOT_12762 222 265
Stagonospora nodorum 1 Broad - FGI SNOT_14804 131 176
Sclerotinia εclerotiorum 1 Broad - FGI SS1T_00772 240 284 291 341 381 431 478 523
Sclerotinia εclerotiorum 1 Broad - FGI SS1T_03535 36 79 109 158
Sclerotinia εclerotiorum 1 Broad - FGI SS1T_05453 201 245 251 298 341 395 428 473
Sclerotinia εclerotiorum 1 Broad - FGI SS1T_06971 185 228
Sclerotinia εclerotiorum 1 Broad - FGI
Figure imgf000047_0001
SS1T 07580 249 292
Sclerotinia sclerotiorum 1 Broad - FGI SS1T_12509 211 256 262 312 359 406
Sclerotinia sclerotiorum 1 Broad - FGI SS1T_12513 47 95 146 192 231 277 320 355
Trichoderma atroviπde 1 JGI-DOE TRIAT152087 187 230
Trichoderma atroviπde 1 JGI-DOE TRIAT156453 35 82 123 168 342 388 432 477 524 569 617 662 690 735
Trichoderma atroviπde 1 JGI-DOE TRIAT29053 27 72 103 148
Trichoderma atroviπde 1 JGI-DOE TRIAT31285 1 44 108 153
Trichoderma atroviπde 1 JGI-DOE TRIAT31372 120 163
Trichoderma atroviπde 1 JGI-DOE TRIAT43321 40 87 128 173 231 291 324 369 438 483
Trichoderma atroviπde 1 JGI-DOE TRIAT45936 55 93 137 182 222 267 301 335
Trichoderma atroviπde 1 JGI-DOE TRIAT81465 224 268 275 326 382 430
Trichoderma atroviπde 1 JGI-DOE TRIAT81699 216 259
Trichoderma atroviπde 1 JGI-DOE TRIAT85797 210 255 261 310 340 385 449 496
Trichoderma reesei 2 JGI-DOE TR I RE 105336 41 88 131 176 356 401 427 472
Trichoderma reesei 2 JGI-DOE TRIRE2916 124 167
Trichoderma reesei 2 JGI-DOE TRIRE54723 44 92 142 187 228 273
Trichoderma virens 1 JGI-DOE TRIVE15018 19 62 126 171 210 255 301 345
Trichoderma virens 1 JGI-DOE TRIVE21922 157 200
Trichoderma virens 1 JGI-DOE TRIVE22885 51 89 133 178 218 263 299 344
Trichoderma virens 1 JGI-DOE TRIVE28703 224 268 275 326 382 430
Trichoderma virens 1 JGI-DOE TRIVE40373 124 167
Trichoderma virens 1 JGI-DOE TRIVE42694 38 87 136 181 226 271
Trichoderma virens 1 JGI-DOE TRIVE53098 30 76 132 178 230 275
Trichoderma virens 1 JGI-DOE TRIVE53625 35 82 123 168 346 391 441 486 543 588 616 661
Trichoderma virens 1 JGI-DOE TRIVE66683 58 103 187 232 273 318
Trichoderma virens 1 JGI-DOE TRIVE9027 39 84 121 166
Uεtilago maydiε 1 Broad - FGI UM02090 33 79 89 132
Uεtilago maydiε 1 Broad - FGI UM05087 219 262
Unαnocarpuε reeεn 1 Broad - FGI URET_02673 263 306
Unαnocarpuε reeεn 1 Broad - FGI URET_03624 38 86 144 189 226 271 310 355 394 439
Unαnocarpuε reeεn 1 Broad - FGI URET_04770 1097 1142 1149 1199 1249 1296
Unαnocarpuε reeεn 1 Broad - FGI URET_07182 47 93 176 222 278 323 353 399 433 479
Verticillium dahliae 1 Broad - FGI VDAG_01171T0 123 166
Verticillium dahliae 1 Broad- FGI VDAG_04781T0 205 249 255 302 343 388 463 509 551 597 641 687
Verticillium dahliae 1 Broad - FGI VDAG_05180T0 33 76 98 141
Verticillium dahliae 1 Broad - FGI VDAG_06426T0 128 171
Verticillium dahliae 1 Broad - FGI
Figure imgf000048_0001
VDAG 08171 TO 83 128 161 206 239 284 315 360 391 436 469 514
Verticillium dahliae 1 Broad - FGI VDAG_09032T0 232 274
Verticillium albo-atrum 1 Broad - FGI VDBG_03944T0 122 165
Verticillium albo-atrum 1 Broad - FGI VDBG_04345T0 232 274
Verticillium albo-atrum 1 Broad - FGI VDBG_08506T0 84 129 162 207 240 286 317 362 393 438 471 516
Verticillium albo-atrum 1 Broad - FGI VDBG_08764T0 162 205
Verticillium albo-atrum 1 Broad - FGI VDBG_09550T0 205 249 255 302 343 388 463 509 551 597 645 691
Verticillium albo-atrum 1 Broad - FGI VDBG_09766T0 1 1 1 155 171 216 249 294 325 370 404 449
Cladosporium fulvum COGEM COGEME CfEcp6 44 87 1 17 161 174 217 Ev.1.6
Leptosphaeria COGEM COGEME Lma99039186 12 55 85 127 140 183 maculans Ev.1.6
10
EXAMPLES
Fungal and plant materials, and infection assays
The wild-type race 5 strain of C. fulυum was stored in 50% glycerol at -800C until revitalized on potato dextrose agar (PDA; Oxoid, Hampshire, England) and was grown at room temperature in the dark. Two-week-old C. fulvum PDA plate cultures were used to harvest conidia by adding sterile water to the plates and rubbing the surface with a sterile glass rod to release the conidia. Conidial suspensions were filtered through Miracloth (Calbiochem-Behring, La Jolla, CA), centrifuged at 4000 r.p.m. and washed twice with sterile water after which the conidial concentration was determined. Subsequently, the conidia were used for plant inoculations or Agrobacterium tumefaciens- mediated transformation. All tomato plants were grown under standard greenhouse conditions: 21°C during the 16 h day period, 19°C at night, 70% relative humidity (RH) and 100 Watt m-2 supplemental light when the sunlight influx intensity was below 150 Watt m-2. The tomato (S. esculentum) cultivar MoneyMaker, containing no resistance genes against C. fulvum (MM-Cf-O), and a MoneyMaker near isogenic line containing the Cf-4 locus (MM-Cf-4) were used for all inoculations. C. fulvum was inoculated as described previously (de Wit, P.J.G.M.,1977, Neth. J. Plant Pathol. 44:337-366). Per 5-week-old tomato plant, 5 ml of conidial suspension (1 x 106 conidia per ml) was used for spray inoculation on the lower surface of the leaves until drop-off. Plants were kept at 100% RH under a plastic cover for 48 h after inoculation. All experiments, starting from plant inoculations, were repeated at least twice.
Preparation of protein samples and 2D-PAGE
Leaves were harvested from C. fulvum-infected MM-Cf-O and MM-Cf-4 lines at 14 dpi and apoplastic fluid (AF) was isolated by vacuum infiltration (van Esse, H.P. et al., 2006, MoI. Plant Microbe Interact. 20:1092-1101) using demineralized water followed by centrifugation for 5 min and stored at -200C until further analysis. AF from both interactions was freeze dried and the residue was resuspended in 3.5 ml of water. After centrifugation (10 min at 4000 g) samples were desalted using a PD-10 desalting column (GE Healthcare, UK), freeze-dried again and stored at -200C. Freeze-dried protein samples were dissolved in 340 ml of Rehydration Buffer [7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT, 0.002% (w/v) bromophenol blue] along with 3.4 ml of IPG buffer pH 4-7 (GE Healthcare). The samples were vortexed briefly and centrifuged (10 min at 4000 g). The protein samples were applied to Immobiline DryStrips of 18 cm with a non-linear pH 4-7 gradient (GE Healthcare), covered with paraffin oil and allowed to rehydrate overnight at room temperature. Isoelectric focusing was performed using the Ettan IPGphor electrophoresis apparatus (GE Healthcare) at 200C maintaining 50 mA per strip. A total focusing of 70 k Vh was achieved by following a running protocol using a step-n-hold gradient (1.5 h 0-3500 V, 6 h 3500 V).
After first dimensional isoelectric focusing, the strips were stored at -200C. Subsequently, strips were placed in equilibration buffer [EB; 50 mM Tris, pH 8.8, 6 M urea, 30% (v/v) glycerol and 2% (w/v) SDS] supplemented with 65 mM DTT. After 15 min, the buffer was replaced by EB supplemented with 135 mM iodoacetamide, and the strips were incubated for another 15 min. The proteins were subsequently separated on 12.5% polyacrylamide gels; the gels were run at 70 V for the first 30 min and subsequently at 200 V until the bromophenol blue reached the bottom of the gels. Gels were stained with Coomassie brilliant blue overnight and de-stained with 10% ethanol and 7.5% HAc in water.
Mass spectrometry
Protein spots were excised from the gel and digested with trypsin with an in- gel method (Shevchenko A. et al., 1996, Anal. Chem. 69:850-858). The collected extracts of the resulting tryptic peptides were freeze dried and stored at -200C. The peptides were redissolved in 8 ml of 50% acetonitrile, 5% formic acid. MS and MS/MS information was acquired with a Q-Tofl (Waters, Manchester, UK) coupled with a nano-LC Ultimate system (LC Packings Dionex, Sunnyvale, CA). After the dilution of 1-2 ml of sample 12 times with water, peptides were separated on a nano-analytical column (75 mm inside diameter x lδ cm Clδ PepMap, LC Packings, Dionex) using a gradient of 2-50% acetonitrile, 0.1% formic acid in 20 min.
The flow of 300 nl min-1 was directly infused into the Q-Tofl, operating in data-dependent MS and MS/MS modes. The resulting MS/MS spectra were processed with Masslynx software (Waters, Manchester, UK) and used to search in MASCOT using the MSDB database. As sequence data of both C. fulυum and tomato are far from complete, MS/MS data from un-assigned spectra were analysed by using the Masslynx Pepseq software for de noυo sequence information. Both BLAST (http://www.expasy.org/tools/blast) and MSBLAST were used to search for possible homologous proteins with the generated sequence information. For MALDI-TOF analysis, a 1 ml volume was spotted on a target plate after mixing the samples 1:1 (v/v) with a solution of 10 mg ml-1 a-Cyano-4-hydroxycinnamic acid in 50% ethanol/50% acetonitrile/0.1% TFA. Reflectron MALDI-TOF spectra were acquired on a TofSpec 2E (Waters, Manchester, UK). For peptide mass fingerprinting the resulting peptide mass lists were used to search in MASCOT using the same MSDB database.
Cloning of CfPhiA, Ecp6 and Ecp7
Based on the N-terminal CfPhiA sequence MDPIDWWK, the forward degenerate primer Deg-PhiA along with an oligo-(dT) primer (Table S2) was used to isolate the CfPhiA coding sequence. Likewise, degenerate forward primers (Table S2) were designed matching the ETKATDCG and QITTQDFG sequences from the N-terminal sequences of Ecp6 and Ecp7 respectively. Using the degenerate primers and a poly T primer PCR products were amplified from a cDNA library derived from a compatible interaction between C. fulυum and tomato using the high fidelity polymerase ExTaq (Takara, Shiga, Japan). Products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and sequenced.
Construction of plasmids for RNAi in C. fulvum
Two constructs for overexpression of inverted-repeat constructs for RNAi based on two different parts of the Ecp6 coding sequence were generated. For the first RNAi construct targeting the 3' end oi Ecpβ, 218 bp oi Ecpβ was PCR-amplified from cDNA using a forward primer that added an Ncol restriction site to the 5' end (Ecp6i-F) and a reverse primer that added EcoRI and Notl restriction sites to the 3' end (Ecp6i-R; Table S2). PCR reactions were carried out under the following conditions: an initial denaturation step for 2 min followed by denaturation for 15 s at 94°C, annealing for 30 s at 55°C and extension for 1 min at 72°C for 30 cycles, followed by a final elongation step at 72°C for 5 min. PCR products were separated on 1% agarose gels and were purified using the DNeasy kit (Qiagen, Valencia, CA). Subsequently, PCR products were cloned into the pGemT-Easy vector. Vectors were digested with Ncol and Notl or with Ncol and EcoRI. Both digested inserts were cleaned from gel using the QIAquick gel extraction kit (Qiagen) and subsequently ligated with a Notl- and EcoRI- digested 129 bp spacer segment from the Pichia pastoris Aox-1 gene into the Ncol-digested plasmid pFBB302 (Dr Brandwagt, Wageningen University). The plasmid pFBB302 is constructed in the backbone of the pGreen II binary vector (Hellens R. P. et al., 2000, Plant MoI. Biol. 42:819-832) and contains a nourseothricin resistance cassette (Malonek S. et al., 2004, J. Biol. Chem. 279:25075-25084) to select for fungal transformants, and the UidA reporter gene flanked by the constitutive ToxA fungal promoter (Ciuffetti L.M. et al., 1997, Plant Cell 9:135-144) and trpC terminator (Punt P.J. et al., 1987, Gene 56:117-124). Digestion with Ncol releases the UidA coding sequence and allows ligation of the inverted-repeat RNAi sequence. For the second RNAi construct targeting the 5' end of Ecp6, two Ecp6 PCR products were generated of 250 and 318 bp with the same forward primer that added an EcoRI restriction site to the 5' end (Ecp6i2-F) and two different reverse primers that added a Notl restriction sites to the 3' end (Ecp6i2 k-R and Ecp6i2 1-R respectively; Table S2). PCR reactions and gel cleaning were performed similar as for the first RNAi construct. Subsequently, PCR products were cloned into the pGemT-Easy vector, digested with Notl and EcoRI, cleaned from gel and ligated into the EcoRI- digested plasmid pFBT004. The plasmid pFBT004 is a modified version of pFBB302, in which the nourseothricin resistance cassette is replaced by a hygromycin resistance cassette (Punt et al., supra).
A. tumefaciens-medtαfed transformation of C. fulvum
RNAi plasmids were transformed into A. tumefaciens strain LBAlIOO [containing the binary vector pSoup (Hellens et al., supra] by electroporation. A 3 ml culture of A. tumefaciens was grown overnight in Ix YT (Sambrook and Russell, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press, 2001) supplemented with kanamycin (25 mg ml-1). The following day, the culture was centrifuged and resuspended in 50 ml of fresh minimal medium (MM) (Hooykaas et al., 1979) supplemented with kanamycin (25 mg ml-1) and grown overnight.
The following day, the culture was centrifuged and resuspended in 10 ml of fresh MM. One millilitre of resuspended bacteria was used to inoculate 50 ml of induction medium [IM; MM salts plus 40 mM 2-(Nmorpholino) methanesulphonic acid (MES), pH 5.3, 10 mM glucose and 0.5% (w/v) glycerol] supplemented with 200 mM acetosyringone (AS) and was grown for an additional 4—5 h until the culture reached an optical density (OD600) of 0.25. At that point, the A. tumefaciens culture was centrifuged and resuspended in 10 ml of sterile water. In addition, while A. tumefaciens cultures were growing in IM+AS medium, C. fulvum conidia were harvested and subsequently suspended in 50 ml of B5 medium (Duchefa Biochemie BV, Haarlem, the Netherlands) at a concentration of approximately 1 x 106 conidia ml 1 and placed in a rotary shaker (125 r.p.m.) at room temperature to induce germination of conidia. After 4—5 h, germinated conidia were centrifuged twice at 4000 r.p.m. and re-suspended in sterile water to a final volume of 1 x 107 conidia ml 1.
Five hundred microlitres from the induced A. tumefaciens cell suspension was mixed with 10 ml of germinated conidia and plated (200 ml per plate) on a 0.45-mm-pore, 45-mmdiameter nitrocellulose filter (Whatman, Hillsboro, OR) and placed on cocultivation medium (IM + 200 mM AS and 5 mM glucose and 1.5% technical agar). The co-cultivation mixture was incubated at 22°C for 2 days. Following incubation, the filter was transferred to PDA supplemented with 50 mg ml 1 nourseothricin (Werner BioAgents, Jena, Germany) or with 100 mg ml-1 hygromycin B (Duchefa Biochemie BV, Haarlem, the Netherlands) as a selection agent for transformants and 200 mg ml 1 cefotaxime (Duchefa Biochemie BV, Haarlem, the Netherlands) to kill A. tumefaciens cells. Individual transformants were transferred to new selection plates and incubated until conidiogenesis under normal growth conditions. Conidia from these plates were stored in 50% glycerol at -800C until further analysis.
Real-time PCR analyses
Three leaflets were harvested from inoculated MM-Cf-O and MM-Cf-4 plants at 3, 6, 9, 13 and 16 dpi. Leaf samples were composed of three leaflets from the second, third and fourth tomato leaves of two tomato plants taken at each time point, immediately frozen in liquid nitrogen and stored at -800C until used for RNA analysis. A similar procedure was used for RNAi transformant analysis.
Ecpβ RNAi transformants Ecp6i-1 and Ecp6i-4 along with Ecp7 RNAi transformants Ecp7i-1, Ecp7i-3 and Ecp7i-7 were randomly chosen for inoculation and analysis with the progenitor race 5 wild-type strain inoculated on MM-Cf-O plants. Leaf samples were taken at 10 dpi, immediately frozen in liquid nitrogen and stored at -800C until used for RNA analysis.
Total RNA was isolated from infected leaf material using the RNeasy kit (Qiagen, Valencia, CA), including an in-column DNase treatment (Qiagen) according to manufacturer's instruction. Total RNA was used for cDNA synthesis using an oligo-(dT) primer and the Superscript II reverse transcriptase kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Quantitative real-time PCR was conducted using an ABI7300 PCR machine (Applied Biosystems, Foster City, CA) with the GoldStar SYBR green PCR kit (Eurogentec, Seraing, Belgium). All primer sequences are shown in Table S2. Expression primers were designed so that the reverse primer was not included in the RNAi construct to prevent detection of the constitutively expressed RNAi construct. For the first RNAi construct, primer pair Ecp6-RNAi-RQ-F and Ecp6-RNAi-RQ-R was used, and for the second RNAi construct primer pair Ecp6-RNAi2-RQ-F and Ecp6-RNAi2-RQ-R. Real-time PCR conditions were as follows: an initial 95°C denaturation step for 10 min followed by denaturation for 15 s at 95°C, annealing for 30 s at 600C and extension for 30 s at 72°C for 40 cycles, and analysed on the 7300 System SDS software (Applied Biosystems, Foster City, CA). To ensure no genomic DNA contaminated RNA samples, real-time PCR was also carried out on RNA without the addition of reverse transcriptase. All experiments, including leaf inoculations, were repeated twice.
Heterologous expression of C. fulvum Ecp6 in F. oxysporum f. sp. lycopersici
For C. fulvum Ecp6, the cDNA corresponding to the mature protein was amplified using primer Ecp6OE-F that also contained the sequence encoding the C. fulvum Avr4 signal peptide for extracellular targeting (Table S2). For C. fulvum Ecp7, the cDNA corresponding to the mature protein was amplified in two steps. As the 5' coding sequence was lacking from our cDNA clone, a primer was designed to add a 5' codon-optimized sequence stretch based on the N-terminal protein sequence (Ecp7NtermF) and used in combination with the reverse primer Ecp7OE-R (Table S2). The resulting PCR product was used as template for a second PCR with primer Ecp7OE-F that also contained the sequence encoding the C. fulvum Avr4 signal peptide for extracellular targeting and a HindIII restriction site in combination with the reverse primer Ecp7OE-R that contained a Xmal restriction site (Table S2). All PCR reactions were carried out under the following conditions: an initial denaturation step for 2 min followed by denaturation for 15 s at 94°C, annealing for 30 s at 56°C and extension for 1 min at 72°C for 30 cycles, followed by a final elongation step at 72°C for 5 min. PCR products were separated on 1% agarose gels and purified using the DNeasy kit (Qiagen, Valencia, CA). Subsequently, PCR products were cloned into the pGemT-Easy vector and sequenced. A correct clone was digested with EcoRI (for Ecp6) or HindIII and Xmal (for Ecp7), cleaned from gel, and ligated into the EcoRI- (for Ecp6) or HindIII- and Xmal- (for Ecp7) digested plasmid pFBT004. The constructs were transformed into A. tumefaciens strain LBAlIOO [containing the binary vector pSoup (Hellens et al., supra)] by electroporation essentially as described by Mersereau M. et al. (1990, Gene 90:149-151).
Agrobacterium-mediated transformation of F. oxysporum f.sp. lycopersici was performed as described (Mullins, E.D. et al.,2001, Phytopathol. 91:173-180).
Ecp6 gene walking
Three primers designed on the region encoding the mature Ecp6 protein (TSPl, TSP2 and TSP3; Table S2) were used to amplify the genomic DNA sequence upstream of the region that encodes the mature Ecp6 protein using the DNAWalking SpeedUpTM Premix Kit (Seegene, Rockville, MD) according to the manufacturer's instructions. Amplified products were cloned in the pGEM-T Easy vector (Promega, Madison, WI) and sequenced. Putative open reading frames (ORFs) were predicted using the FGENESH program (Salamov, A.A. and Solovyev, V.V., 2000, Genome Res. 10:516-522) of the MOLQUEST software package (available at http://softberry.com/berry.phtml; Softberry, NY, USA) using the genetic codes of several fungi present in the database as models. ORFs were verified by cloning Ecp6 cDNA. For this purpose, total RNA was isolated from leaves of MM-Cf-O plants inoculated with a race 5 strain of C. fulυum at 11 dpi and used for cDNA synthesis using an oligo-(dT) primer (Table S 2) and the Superscript II reverse transcriptase kit (Invitrogen, Carsbad, CA) as described previ- ously (van Esse et al., supra). The generated cDNA was used as template for the primers Ecp6_ChrWal_Fl and Ecp6_R (Table S2) to amplify the predicted Ecp6 ORF. The primers Ecp6_F3, Ecp6_F2, Ecp6_R3, Ecp6_R2 (Table S2) that hybridized outside the predicted Ecp6 ORF were used as negative controls. The 50 ml PCR reaction mixes contained 5.0 ml of 1Ox SuperTaq PCR reaction buffer, 10 mM of each dNTP (Promega Benelux bv, Leiden, the Netherlands), 20 mM of each primer, 1 unit of SuperTaq DNA polymerase (HT Biotechnology, Cambridge, UK) and approximately 100 ng of cDNA as template. The PCR programme consisted of an initial 5 min denaturation step at 94°C, followed by 35 cycles of denaturation at 94°C (30 s), annealing at 55°C (30 s) and extension at 72°C (60 s). A final extension step at 72°C (7 min) concluded the reaction. Amplified products were cloned in the pGEM-T Easy vector (Promega, Madison, WI) and sequenced.
Ecp6 allelic variation
Allelic variation in Ecp6 was determined for 50 C. fulvum strains (Table Sl) that are part of a previously described collection (Stergiopoulos, I. et al., 2007, MoI. Plant Microbe Interact. 20:1271- 1283; Stergiopoulos, I et al., 2007, Fungal Genet. Biol. 44:415-429). Strains were cultured on half-strength PDA (Oxoid, Hampshire, England) at 22°C. Conidia were harvested from 15-day-old cultures and freeze-dried prior to DNA extraction. Genomic DNA isolations were performed using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The forward primer Ecp6_F3, located 424 bp upstream of the Ecpβ translation start codon, and the reverse primer Ecp6_R3, located 99 bp downstream of the Ecpβ stop codon, were used to amplify Ecpβ (Table S2). The 50 ml PCR reaction mixes contained 5.0 ml of 10¥ SuperTaq PCR reaction buffer, 10 mM of each dNTP (Promega Benelux bv, Leiden, the Netherlands), 20 mM of each primer, 1 unit of SuperTaq DNA polymerase (HT
Biotechnology, Cambridge, UK) and approximately 100 ng of genomic DNA as template. The PCR programme consisted of an initial 5 min denaturation step at 94°C, followed by 35 cycles of denaturation at 94°C (30 s), annealing at 55°C (30 s) and extension at 72°C (60 s). A final extension step at 72°C (7 min) concluded the reaction. Amplified PCR products were excised from 0.8% agarose gels, purified using the GFXTM PCR DNA and Gel Band Purification Kit (Amersham Biosciences UK limited, Buckinghamshire, England), and sequenced using the forward primers Ecp6_F2 and Ecp6_F in combination with the reverse primer Ecp6_R3 (Table S2).
Bioinformatical analysis of Ecp6-like proteins
EST sequences from various fungal pathogens were downloaded from the COGEME Phytopathogenic Fungi and Oomycete EST Database version 1.6 (http://cogeme.ex.ac.uk) (Soanes, D.M. and Talbot, N.J., 2006, MoI. Plant Pathol. 7:61-70). The genome sequences of various fungi listed in Table 2 were consulted at the website of Fungal Genome Initiative of the Broad Institute of MIT and Harvard (http://www.broad.mit.edu/annotation/fgi/) or at the website of the USA Department of Energy Joint Genome Institute (http://genome.jgi- psf.org). The mining of Ecp6-like proteins was performed using NCBI BLAST, and the Standalone-BLAST version 2.2.3 (Altschul, S.F. et al., 1997, Nucleic Acids Res. 25:338-3402; Schaffer, A.A. et al., 2001, Nucleic Acids Res. 29:2994- 3005). Hmmpfam analysis of each identified candidate was performed by running a customized Perl script for Pfam HMM detection, available at ftp://ftp.sanger.ac.uk/pub/databases/Pfam, using Bioperl version 1.4 (http://bioperl.org) and HMMER version 2.3.2 (http://hmmer.janelia.org), which was loaded with the current Pfam Is and fs models (02.10.2007), for whole domain and fragment models respectively. An E- value of 0.001 was used as cut-off. The retained sequences were analysed in BioEdit version 7.0.5.3 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Multiple sequence alignment was performed by CLUSTALW version 1.83 and for phylogenetic tree construction Molecular Evolutionary Genetic Analysis 4.0 (MEGA) was used (Kumar, S. et αZ.,2001, Bioinformatics 17:1244-1245; Tamura, K. et al, 2007, MoI. Biol. Evol. 24:1596-1599). Phylogeny construction of fungal Ecp6-like proteins was performed by neighbour- joining analysis. We used p-distance as the distance parameter as specified in the program MEGA. The inferred phylogeny was tested by 500 bootstrap replicates (Felsenstein J., 1985, Evolution 39:783-791).
Three-dimensional modelling was performed using the Protein Homology/analogY Recognition Engine (Phyre), a Protein fold recognition server (http://www.sbg.bio.ic.ac.uk/~phyre/; Kelley L.A. et al., 2000, J. MoI. Biol. 299:501-522; Bennett-Lovsey R.M. et al., 2008, Proteins: Structure, Function, Bioinformatics 70:611-625). Estimated precision of generated models was used as an indication of significance. Subsequent analyses, visualization and preparation of 3D figures were performed in the Swiss- Pdb Viewer version 3.7 (http://www.expasy.org/spdbv).
Results
Quantification of C. fulvum biomass in infected tomato leaves In a compatible interaction involving the susceptible MoneyMaker Cf-O (MM- Cf-O) tomato cultivar which lacks resistance genes against this pathogen, the fungus colonizes the apoplast around leaf mesophyll cells. Conidiophores emerge from stomata 7 days post inoculation (dpi) to produce conidia (Fig. IA). Using real-time PCR to quantify fungal biomass in the plant tissue it is evident that fungal biomass gradually increases until the fungus is extensively sporulating (Fig. IB). In the incompatible interaction, such as with resistant Money-Maker Cf-4 (MM-Cf-4) tomato plants that recognize C. fulυum strains expressing wild- type Aυr4 (Joosten, M.H.A.J. et al., 1994, Nature 367:384-386), no disease symptoms are visible (not shown). Real-time PCR confirms that in such an incompatible interaction no significant increase in fungal biomass occurs when compared with the compatible interaction (Fig. IB).
Characterization of the C. fulvum-infected tomato apoplast proteome In previous analyses, the protein composition of the apoplastic space of C. fulvum-infected tomato leaves has mainly focused on identification of effectors that are secreted by the fungus during infection and that invoke a resistance response in tomato. For an inventory of the apoplast proteome of C. fulvum- infected tomato and to identify secreted fungal proteins that might play a role in virulence, 2D-PAGE was utilized that allowed the comparison of MM-Cf-O and MM-Cf-4 plants infected by a race 5 C. fulvum strain (compatible and incompatible interaction respectively). At 2 weeks post inoculation of susceptible MM-Cf-O plants, the fungus has generated considerable biomass and has extensively colonized the host tissue (Fig. 1), likely resulting in a large quantity of fungal proteins in the apoplast as compared with resistant MM-Cf- 4 plants. Therefore, this time point was chosen for detailed analysis of fungal proteins (Fig. 2). Proteins present in 2 ml of apoplastic fluid isolated from the two different interactions were analysed with 2D-PAGE. Separation of the proteins in the first dimension was carried out on Immobiline DryStrips (pH 4—7) and for the second dimension 12.5% polyacrylamide gels were used. After Coomassie brilliant blue staining, 16 protein spots specific for, or highly induced during, the compatible interaction were excised from the gel (Fig. 2). Subsequently, the proteins were digested with trypsin and the generated peptides were analysed with matrix-assisted laser desorption ionization time- of-flight (MALDI-TOF) MS and peptide fragment spectra were obtained with liquid chromatography (LC) MS/MS. Peptide mass fingerprints and peptide sequence information were used to search for protein identity in databases. This resulted in the identification of a tomato endochitinase and the C. fulυum proteins Ecpl, Ecp2 and Ecp5. Proteins present in the other spots could not be identified solely based on the data obtained in the MS analysis.
Six of these non-identified protein spots (5-10; Fig. 2) resulted in a comparable peptide mass fingerprint and are therefore likely to be derived from the same protein. One of the protein spots (5; Fig. 2) was subsequently subjected to N- terminal sequencing, resulting in a 46-amino acid sequence that was found to harbour the previously identified MS/MS tags. As the obtained sequence showed homology to a structural Aspergillus nidulans phialide protein, this protein was designated CfPhiA. This is a protein that typically occurs on phialides, which are sporogenous cells that release conidia from their apex by budding (Melin, P. et al., 2003, Fungal Genet. Biol. 40:234-241). Three other protein spots (11-13; Fig. 2) also generated a comparable mass fingerprint, implicating that also these spots may be derived from the same protein. N- terminal sequencing of spot 12 resulted in a 26-amino-acid sequence harbouring the identified MS/MS sequence tags and the corresponding protein was designated Ecp6. The remaining protein spots (14, 15; Fig. 2), of which we obtained peptide mass fingerprints as well as peptide fragment spectra, were also subjected to N-terminal sequencing. For protein spot 14, the 25-amino-acid sequence that was obtained matched the corresponding MS/MS sequence tags and the protein was designated Ecp7. Although sequence information based on MS/MS was available for protein spot 15, this protein was not considered for further study because N-terminal sequence failed repeatedly.
Cloning of extracellular protein genes Degenerate primers were designed based on the N-terminal protein sequences of CfPhiA and Ecp6 and were used in combination with an oligo-(dT) primer to amplify the coding regions of the corresponding genes using a cDNA library from C. fulvum-iniected tomato leaves as template. For Ecp7, a degenerate primer based on an MS sequence tag was used because the N-terminal sequence was not yet available when the cloning was initiated. In all cases, a cDNA sequence was successfully amplified which corresponded to MS/MS and N-terminal peptide sequences. For CfPhiA, a 720 bp fragment encoding the mature protein and part of the 3'UTR was cloned (Fig. Sl). The predicted mature CfPhiA protein contains 175 amino acids and has a predicted molecular mass of about 19 kDa and an isoelectric point (pi) of 5.0. BLASTP analysis of the amino acid sequence showed that this protein shares similarity to putative proteins of several fungal species including A. nidulans, A. fumigatus and Neurospora crassa. Of these orthologues, the PhiA protein from A. nidulans has been functionally characterized (Melin et al., supra), and was found to be essential for growth and sporulation of the fungus as phiA mutants were found to be impaired in phialide development. Therefore, it is likely that the C. fulvum putative orthologue CfPhiA has a similar function. A 742 bp fragment with the coding region for the mature Ecp6 protein and the 3'UTR was cloned. Ecp6 encodes a mature protein of 199 amino acids, including eight cysteines, and has a predicted molecular mass of 21 kDa and a pi of 4.6. Furthermore, Ecp6 contains five predicted N-glycosylation sites, explaining the location of the Ecp6 protein spots on the 2D-gel. Based on BLASTP analysis, Ecp6 was found to share significant homology to the glycoprotein CIHl identified in the plant pathogenic fungus Colletotrichum lindemuthianum (Perfect, S. E. et α/.,1998, Plant J. 15:273-279). Although the contribution of CIHl to pathogenicity is unknown, it has been shown in this reference to accumulate during infection on bean in the walls of intracellular hyphae and the interfacial matrix which separates the hyphae from the invaginated host plasma membrane. For Ecp7, a 464 bp cDNA fragment was cloned containing the coding region for 84 amino acids of the mature Ecp7 protein. N-terminal sequencing of Ecp7 revealed that a stretch of 16 amino acids precedes the peptide that was identified as an MS tag, and based on which the degenerate primer for cloning the cDNA was designed. Therefore it should be concluded that Ecp 7 encodes a mature protein of 100 amino acids which includes six cysteines and has a predicted molecular mass of 11 kDa and a pi of 6.0. BLASTP analysis of the amino acid sequence revealed no significant homology of Ecp7 to other protein sequences deposited in public databases.
CfPhiA, Ecpβ and Ecp7 are expressed during infection
With real-time PCR assays using genomic DNA from C. fulυum as a template and Avr2 as a single-copy reference gene (Luderer, R. et al., 2002, MoI. Microbiol. 45:875-884), it was determined that the C. fulvum genome contains only one copy of the CfPhiA, Ecpβ and Ecp 7 genes (results not shown). Furthermore, real-time PCR analysis of CfPhiA, Ecpβ and Ecp 7 transcripts, using the constitutively expressed C. fulvum actin gene as an endogenous control, revealed that all genes are expressed in both compatible and incompatible interactions (Fig. 3). CfPhiA expression is induced already early in the compatible interaction, at 6 dpi, and maintains this level of expression for all time points analysed. In the incompatible interaction, CfPhiA is also induced, although its expression level is approximately half of that found in the compatible interaction (Fig. 3). Both Ecp6 and Ecp7 show a low but steady level of expression in the incompatible interaction when compared with that of the C. fulvum actin gene, while the genes are clearly induced in the compatible interaction. While Ecp7 peaks at 9 dpi (Fig. 3), Ecp6 is maximally expressed at 13 dpi (Fig. 3). In contrast to the expression pattern of the CfPhiA gene, the patterns of Ecp6 and Ecp 7 typically resemble those of other genes encoding secreted C. fulvum effectors. For example, C. fulvum Avr9 is highly expressed throughout the compatible interaction, with maximum expression at 9 dpi, whereas its expression in the incompatible interaction remains low (Fig. 3). Nevertheless, the expression level of the Avr9 gene is much higher than those of Ecp6 and Ecp7 (Fig. 3).
Heterologous expression of Ecp6 in Fusariumoxysporum f. sp. lycopersici enhances virulence on tomato
To investigate whether C. fulvum Ecp6 and Ecp7 may act as fungal virulence factors, we overexpressed these Ecps in F. oxysporum f. sp. lycopersici. To this end, the sequences encoding the mature proteins were fused in frame with the sequence encoding the C. fulvum Avr4 signal peptide for extracellular targeting (Joosten, M. H.A.J, et al., 1997, Plant Cell 9:367-379) into a binary vector under control of the fungal constitutive ToxA promoter. Using Agrobacterium-mediated transformation a large number of transformants were obtained, and presence of the transgene was confirmed by PCR (data not shown). Four transformants were randomly picked for each of the C. fulvum Ecps and tested in an inoculation assay on tomato. Upon inoculation of tomato plants with transformants that overexpress Ecp7, disease development was indistinguishable from disease caused by the nontransformed progenitor strain (data not shown). In contrast, on tomato plants that were inoculated with each of the four transformants that overexpress Ecp6, disease symptoms developed earlier and were more severe compared with the inoculation with the non-transformed progenitor F. oxysporum f. sp. lycopersici strain (Fig. 4A and B) or the transformants that overexpress Ecp7 (data not shown). With reverse transcription PCR it was confirmed that in each of the transformants, but not in the progenitor F. oxysporum f. sp. lycopersici strain, Ecp6 was expressed (Fig. 4C).
RNAi-mediated silencing of Ecp6 compromises C. fulvum virulence on tomato.
RNAi has been successfully employed for gene functional analysis in filamentous fungi (Nakayashiki, H. et al., 2005, Fungal Genet. Biol. 42:275- 283). This is particularly relevant for fungi like C. fulvum for which homologous recombination is not straightforward. Recent evidence has shown that PEG-mediated transformation may generate somaclonal variation that may be circumvented by Agrobacterium-mediated transformation which is, however, significantly less efficient (van Esse et αl., supra). Therefore, RNAi was recently successfully implemented to silence the expression of C. fulvum effector genes (van Esse et al., supra). Based on the results obtained with heterologous expression of C. fulvum Ecp6 in F. oxysporum f. sp. lycopersici, we applied RNAi-mediated silencing for functional analysis of the C. fulvum Ecp 6 gene using Agrobacterium-mediated transformation with constructs aimed at generating double- stranded RNA that targets these genes (RNAi). A pGREEN-based binary vector, carrying transfer DNA (T-DNA) that contains either a nourseothricin resistance cassette or a hygromycin resistance cassette, and an inverted-repeat fragment of the target gene under control of the fungal constitutive ToxA promoter, was used to provoke RNAi-mediated gene silencing. To target the expression of the Ecp6 gene, two RNAi constructs were generated based on different sections of the Ecp6 coding region. Agrobacterium-mediated transformation of the RNAi constructs generated several antibiotic-resistant transformants for each construct. Analysis of the transformants indicated that their growth in vitro was indistinguishable from that of the progenitor race 5 isolate (data not shown). As C. fulvum effector genes show variable expression when cultured in vitro (Thomma et αl., 2006), 4-week-old MM-Cf-O tomato plants were inoculated with three transgenic C. fulvum strains to determine whether the introduc- tion of the inverted-repeat construct resulted in Ecp6 silencing. Utilizing real-time PCR, a strong reduction in transcription of the target gene was found when compared with the progenitor isolate in several transformed isolates using expression of the C. fulvum actin gene as a reference (Fig. 5A). At 10 dpi, transformants Ecp6i-1 and Ecp6i-4 of the first construct, and Ecp6i2-1 of the second construct, showed a reduction to 36%, 27% and 48% of the wild-type Ecp6 expression level respectively (Fig. 5A). At later time points, the level oi Ecpβ reduction increased for the Ecp6i2-1 transformant, while the reduction in the Ecp6i-1 and Ecp6i-4 remained rather consistent, which may possibly be attributed to different regions of the transcript that are targeted for gene silencing (data not shown).
Visual inspection of the inoculated MM-Cf-O tomato plants showed a clearly delayed progression of disease for the Ecp6 RNAi transformants (Fig. 6). While conidiophores were emerging from the stomata on the lower surface of tomato leaves inoculated with the wild-type progenitor strain at 10 dpi, the leaves inoculated with transformant Ecp6i-4 were devoid of these structures (Fig. 6). Although leaves inoculated with transformants Ecp6i-1 (Fig. 6) and Ecp6i2-1 (data not shown) showed some fungal growth, the extent of leaf colonization was significantly less than that observed for the wild-type strain. To measure the extent of fungal growth of RNAi transformants compared with the parental wild-type strain, the constitutively expressed C. fulvum actin gene was used as a marker in real-time PCR analyses (Fig. 5B). The constitutively expressed tomato chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a reference for the ratio of fungal biomass to plant biomass to determine the degree of colonization. After inoculation of MM-Cf-O tomato lines, all Ecp6 RNAi transformants showed significant reduction in growth compared with the parental race 5 isolate (Fig. 5B). Ecp6 sequence analysis from a worldwide collection of C. fulvum strains
As our results showed that Ecp6 is a virulence factor of C. fulvum, we assessed sequence variation of Ecp6 in a worldwide collection of strains. We first obtained 691 bp of genomic sequence upstream of the region that encodes the mature Ecp6 protein by gene walking. Sequence analysis using the gene prediction algorithm FGENESH identified a putative start codon and predicted intron/exon boundaries using the genetic codes of several fungi present as models in the database. These were confirmed by cloning the Ecp6 cDNA from infected plant material, showing that the Ecp6 ORF is 669 bp, interrupted by two introns of 68 and 111 bp, respectively, and encodes a protein of 222 amino acids (Fig. 7). The full-length sequence of Ecp6 was obtained from a collection of 50 C. fulvum strains (Table Sl). Analysis of the sequence 62 bp upstream of the start codon to 91 bp downstream of the stop codon revealed that variation within Ecp6 was very limited, resulting in a total of five single-nucleotide polymorphisms (SNPs) within these strains (Fig. 7). One SNP (G > A at 494 bp downstream of the putative start codon) occurred inside the second intron oiEcpβ, and was only detected in one Canadian strain (#34; Table Sl). The other four SNPs all occurred in seven strains originating from North America (#31, #34, #40, #41; Table Sl), and Japan (#67, #71, #74; Table Sl).
While one SNP (G > A at 128 bp) occurred in the first intron, two other SNPs are silent mutations (C >T at 335 bp and G > A at 662 bp). Only one SNP (C > A at 142 bp) is predicted to result in an amino acid substitution (Thr25 > Asn; Fig. 7).
Orthologues o/"Ecp6 are found in several fungal species
Interrogation of the C. fulvum Ecp6 protein sequence using BLASTP and Pfam analysis indicates that the Ecp6 protein contains three lysin motif (LysM) domains. These domains are widespread protein modules of approximately 40 amino acids, originally identified in a bacterial autolysin that degrades bacterial cell walls (Joris, B. et ah, 1992, FEMS Microbiol. Lett. 70:257-264). LysM domains are also found in eukaryotic proteins, and presently LysM domains are implicated in binding of diverse carbohydrates that occur in bacterial peptidoglycan, fungal chitin and Nod factor signals that are produced by Rhizobium bacteria during the initiation of root nodules on legumes.We queried all available fungal genome sequences and expressed sequence tag (EST) libraries (Table 2) for the presence of Ecp6-like proteins using BLASTP or TBLASTN respectively. The retrieved sequences were subsequently analysed for predicted protein domains using HMMER
(http://hmmer.janelia.org/) loaded with the current Pfam HMM library (http://pfam.sanger.ac.uk). Prediction of significant LysM domains (E- value cut-off 0.001) was used as a selection criterion for further analysis. Subsequently, all sequences containing predicted LysM domains were aligned, permitting for the selection of fungal proteins with high overall similarity to C. fulυum Ecp6. In this way, a list of 16 putative C. fulυum Ecp6-like proteins was generated, containing five Aspergillus niger proteins, two Magnaporthe grisea proteins, and one from each Mycosphaerella fijiensis, M. graminicola, Botrytis cinerea, Sclerotinia sclerotiorum, A. nidulans, A. oryzae, A. flavus, C. lindemuthianum and Leptosphaeria maculans. For these 17 proteins, using CLUSTALW, a multiple sequence alignment analysis was performed (Fig. S2). In addition to the LysM domains, the positions of the cysteine residues that flank the LysM domains, and the high abundance of proline, serine and threonine residues in the LysM linker regions appear to be conserved (Fig. S2). Subsequently a neighbour-joining tree) was constructed to reveal evolutionary relationships (Fig. 8).
Based on this tree, the 16 Ecp6-like proteins can be divided into three groups. C. fulυum Ecp6 clusters with three Ecp6-like proteins of M. graminicola, M. fijiensis and L. maculans that all contain three LysM domains (Group 1, Fig. 8). The second group of Ecp6-like proteins encompasses the two M. grisea Ecp6-like proteins and CIHl from C. lindemuthianum that are shorter than other Ecp6-like proteins and have only two LysM domains (Group 2, Fig. 8). The largest group of Ecp6-like proteins, encompassing the five A. niger proteins in addition to those of A. nidulans, A. oryzae, S. sclerotiorum and B. cinerea, contain two LysM domains and a weak, but not significant, signature of a third LysM domain (Group 3, Fig. 8).
Homology modelling of Ecpβ LysM domains
Although LysM domains have been identified in over 1500 proteins, the three- dimensional (3D) structure of only three LysM domains has been reported. Two of these are of bacterial origin, the 3D structure of a LysM domain of the Escherichia coli membrane-bound lytic murein transglycosylase D (MItD; PDB code: IEOG; Bateman, A. and Bycroft M., 2000, J. MoI. Biol. 299:1113-1119) and the LysM domain of the Bacillus subtilis spore protein ykuD of unknown function (PDB code: 1Y7M; Bielnicki, J. et al., 2006, Proteins 62:144-151). Recently, the 3D structure of the LysM domain of the human hypothetical protein SB145 was determined using nuclear magnetic reso- nance (NMR) imaging (PDB code: 2DJP). The structural organization of the three LysM domains from these different proteins is highly similar, and characterized by a βααβ fold, with the two helices stacking on one side of the plate generated by a double-stranded antiparallel β-sheet. Recently, the first characterization of an interaction of a LysM domain with its ligand was reported (Ohnuma, T. et al., 2008, J. Biol. Chem. 283:5178-5187). Binding of oligomers of N- acetylglucosamine [(GlcNAc)n], a monosaccharide derivative of glucose that is a building block for bacterial peptidoglycan and fungal chitin, to the LysM domains of a chitinase from Pteris ryukyuensis was monitored with NMR spectroscopy. The stoichiometry of (GIcNAc) n/LysM binding was found to occur in a 1:1 ratio. Furthermore, using (GIcNAc) 5 it was shown that binding of this oligomer to the LysM domain occurs at a shallow groove formed by the N-terminal part of helix 1, the loop between strand 1 and helix 1, the C- terminal part of helix 2, and the loop between helix 2 and strand 2. To predict the ligand binding site with corresponding binding specificities of the C. fulvum Ecp6 LysM domains, homology-based modelling based on the 3D structure of the LysM domain of the MItD structure was performed. The MItD and Ecp6 LysM domains show moderate but significant overall sequence similarity (53%, 47% and 33%, respectively, for LysM domains 1, 2 and 3; Fig. 9A). Moreover, by assessing local Kyte-Doolittle (KD) hydrophobicity values (Kyte, J. and Doolittle, R.F., 1982, J. MoI. Biol. 157:105-132), the conserved secondary structure could be predicted, which was subsequently used to predict the 3D structure. The predicted 3D structure of the three individual Ecp6 LysM domains is highly similar, with small changes in the position of the second loop of the third LysM domain (Fig. 9B). Moreover, due to sufficient similarity (52%) of LysM domain 1 of C. fulvum Ecp6 to LysM domains 1 and 2 of P. ryukyuensis PrChi-A (Fig. 9A), ligand binding can be modelled according to the interaction between chitin oligomers and PrChi-A LysM domains. The molecular surface of the first LysM domain of Ecp6 (Fig. 9B, panel 1) was computed and is shown in panel 4 of Fig. 9B. In the surface of this LysM domain, a cavity is observed that fulfils the requirements to act as binding site of chitin oligomers, based on the structural homology with PrChi- A.
EXAMPLE 2
MATERIALS AND METHODS
Heterologous production of Cladosporium fulvum Ecp6 in Pichia pastoris
The C. fulvum sequence coding for the mature part of Ecp6 was fused to the Avr4 signal peptide and a His6-FLAG-tag by overlap extension PCR, and transformed into Pichia pastoris. Subsequently, Ecp6 was produced and purified using a Ni-NTA column. Finally the fractions containing Ecp6 were isolated and dialyzed against ddH20. The protein concentration was measured using BCA, and determined to be approximately 5.5 mg/ml.
Ecp6 protein injections
Fully expanded leafs of various wild tomato varieties of approximately 4 weeks old were injected with 0.11 mg/ml of Ecp6 using a Hamilton syringe. Using this approach on average 40-60 μl of protein solution was injected in each infiltration site. Leaves were inspected regularly for visual appearance of symptoms, and pictures were taken at 10 days post injection.
PVX expression of Ecp6 orthologs
The binary PVX vector pGrlO6 (Jones, L. et al., 1999, Plant Cell 11:2291-2301) was used as a backbone for all PVX expression constructs. The coding sequences of the Ecp6 orthologs were fused to the tobacco PR- Ia signal sequence, for extracellular targeting, using overlap extension PCR and directionally cloned into the Clal-Notl restriction sites of pGrlOδ. The resulting plasmids were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. The A. tumefaciens strains were cultured on plates containing modified LB medium (10 g 1/1 bacto-peptone; 5 g 1/1 yeast extract; 2.5 g 1/1 NaCl; 10 g 1/1 mannitol) for 48 h at 28 degrees Celcius. Subsequently, colonies were selected and inoculated on 2-week-old tomato plants by toothpick inoculation.
RESULTS
Identification of tomato lines that respond with an HR towards Ecp6
Pichia pastoris expressed Ecp6 was purified from the culture medium and used to screen a collection of 28 tomato lines for the occurrence of a hypersensitive response upon injection with Ecp6 (Table S3). Ecp6 injection triggered the development of strong chlorotic and necrotic lesions in the center of the injected area within 5 days in leaves of 7 lines (Figure 10; Table S3). In another 6 lines a less strong response was observed. Inoculation of these lines with a similar concentration of Avr4 caused no symptoms. The remaining 15 lines and the control cultivars MoneyMaker and Motelle, that both lack functional C. fulvum resistance genes, exhibited no symptoms upon injection of Ecp6.
These results indicate that Ecp6 is both necessary and sufficient to induce HR on the responding lines.
Recognition of Ecp6 is sufficient to confer resistance against C. fulvum
We used isogenic strains of C. fulvum, containing or lacking the Ecp6 gene, to prove that resistance of the responding lines is solely caused by the fact that the fungus produces Ecp6 during infection. We used one line of each of the wild species (S. pimpinelli folium, S. cheesmanii, and S. parυiflorum) as a representative of the responding lines. The set of fungal strains tested comprised a wild-type, Ecp6-producing strain (race 5), and a corresponding isogenic Ecp6-disruption mutant in which the Ecp6 gene is replaced by an antibiotic resistance cassette. Whereas susceptible MoneyMaker and Motelle plants showed disease symptoms when inoculated with either of the two strains, only the Ecp6-lacking mutant caused disease on the three Ecp6- responding lines. Plants of the three Ecp6-responding lines showed full resistance without any visible disease symptoms after inoculation with the wild-type Ecp6-producing strain. Microscopic examination of these resistant plants confirmed that fungal growth is arrested early after penetration of the tomato leaf. Thus, resistance towards C. fulvum of the three Ecp6-responding lines solely depends on recognition of the Ecp6 protein.
Resistance is based on a single dominant gene, designated Cf-Ecp6 We used one line of each of the wild species (S. pimpinellifolium, S. cheesmanii, and S. parυiflorum) as a representative of the responding lines. Ecp6-responsive plants were crossed with MoneyMaker Cf-O plants that lack functional Cf resistance genes. The progeny of the cross (Fl) was tested for Ecp6 responsiveness by injection of P. pastoris produced Ecp6 and tested for C. fulυum resistance by challenge inoculation with various strains of C. fulυum that all possess Ecp6. All Fl plants obtained in the diverse crosses showed Ecp6-responsiveness as well as resistance against all C. fulυum strains that were tested. F2 progenies were generated to study the heritability of HR upon exposure to Ecp6. The three F2 populations exhibited a 3:1 ratio for Ecp6- responsiveness as well as for C. fulυum resistance, showing that both traits are conferred by a single dominant gene, designated Cf-Ecp6, in the three wild tomato species. To test whether Ecp6-responsiveness in the three wild tomato species is conferred by alleles of the same gene or by different genes, pairwise crosses were made and F2 progeny was obtained. Since all individuals of the diverse F2 populations exhibited Ecp6-responsiveness, it is concluded that Ecp6 responsiveness in the three wild tomato species is conferred by different alleles of the same gene.
Cf-Ecp6 recognizes Ecp6 orthologs of multiple fungal species To assess whether Ecp6-responsiveness in the three wild tomato species is confined to Ecp6 of C. fulυum, or also concerns Ecp6 orthologs from other fungal species, responsiveness towards various Ecp6 orthologs was tested. Screenings were carried out by using Potato Virus X (PVX) for systemic production of the Ecp6 orthologs that were targeted to the apoplast of virus- infected plants. To this end, the Ecp6 orthologs were cloned from Mycosphaerella fijiensis, M. graminicola, Cercospora beticola and Septoria lycopersici that, similar to C. fulυum, belong to the Mycosphaerellaceae. In addition, Ecp6 orthologs were cloned from the distantly related tomato pathogens Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Verticillium dahliae.
Inoculation with wild-type PVX caused normal mosaic symptoms. These results demonstrate that Cf-Ecp6 is not solely activated by C. fulvum Ecp6, but also recognizes Ecp6 orthologs of other tomato pathogens as well as pathogens of other plant species.
Cf-Ecp6 plants are resistant to multiple, Ecp6 expressing, 2 fungal pathogens
To assess whether Ecp6-responsiveness in the three wild tomato species is correlated with resistance to fungal pathogens other than C. fulvum that contain Ecp6 orthologs, the resistance of these species to the fungal pathogen species Verticillium dahliae was tested. These analyses showed that the three species showed enhanced resistance towards V. dahliae. The progeny of the cross (Fl and F2) of Ecp6-responsive plants with MoneyMaker Cf-O was tested for resistance towards this pathogen, showing that resistance is correlated with the presence Cf-Ecp6 in the three wild tomato species. These results show that Cf-Ecp6 acts as a broad- spectrum resistance gene that is not only effective against C. fulvum, but also against V. dahliae.
Example 3
Ecp6 recognition also occurs in Arabidopsis In total, 280 Arabidopsis accessions were screened for responsiveness to C. fulvum Ecp6 in the greenhouse. Even though C. fulvum is not a tomato pathogen, two accessions that clearly responded with an HR to Ecp6 injection were identified. In addition, the screen of 280 Arabidopsis accessions was performed in the climate chamber under controlled conditions. This time, three different accessions were retained. So, in total five Arabidopsis accessions were retained that respond with an HR upon injection with C. fulvum Ecp6. These same five accessions respond with an HR upon injection with Ecp6 orthologs from other fungal pathogens. This demonstrates that Ecp6 recognition is conserved across plant families.
Table Sl. Cladosporium fulυum isolates used in this study.
Co Accession Origin Code Accession Origin de
1 0 Netherlands 42 Can 84 Canada
2 2 Netherlands 52 IMI Day5 054977 UK
3 4E Netherlands 57 IPO 2459 (30787)
Netherlands
4 2 4 Netherlands 58 IPO 2459 (50381)
Netherlands
5 2 4 11 Poland 59 IPO 2459 (60787)
Netherlands
6 2 4 5 Netherlands 60 IPO 248911 Polen Poland
7 2 4 5 11 Netherlands 61 IPO 249 France France
8 2 4 5 7 Netherlands 62 IPO 2679 New Zealand
SECRET
10 2 4 5 9 11 Netherlands 65 IPO 5 (15104)
IPO Netherlands
11 2 4 8 11 Netherlands 66 IPO 80379
Netherlands
12 2 4 9 11 Poland 67 Jap 12 Japan
15 2 5 9 France 69 Jap 15 Japan
16 4 Netherlands 71 Jap Cf32 Japan
17 4 (2) Netherlands 73 Jap Cf5 Japan
19 5 Kim France 74 Jap Cf 56 Japan
20 5 Marmeisee France 75 Jap Cf9 Japan
22 Alenya B France 78 MUCL723 Belgium
24 Brest 84 France 80 MUCL725 Belgium
25 Brest Rianto France 82 Nantes 89 France
85
26 BuI 20 Bulgaria 84 Pons 89
Netherlands
31 Can USA USA 87 T Hijwegen
Amherst Netherlands
34 Can 38 USA 111 VKM 1437 Former
USSR
35 Can 43 Canada 112 Z. Am 1 South America
40 Can 62 Canada 117 Turk Ia Turkey
41 Can 69 Canada 122 Turk 3c Turkey Table S2. Primers used in this study.
Primer name Sequence (5 '-3 ' ) Description
Deg-PhiA ATGGAYCCNATHGAYGTNGTNTGGAA Degenerate primer for
CfPhiA cloning
Deg-Ecp6 GARACNAARGCNACNGAYTGYGG Degenerate primer for Ecp6 cloning
Deg-Ecp7 CARATHACNACNCARGAYTTYGG Degenerate primer for Ecp7 cloning oligo-dT TTGGATCCTCGAGTTTTTTTTTTTTTTTT PoIy-T primer with Ncol TT (bold) and Sad
(underlined)
Avr2RQ-F ACCGCATCCGAAGTAATAGCA Avr2 qRT-PCR expression fwd
Avr2RQ-R CCAGACTTCTCCTTCACTTTGCA Avr2 qRT-PCR expression rev
Avr9RQ-F GAGCTTGCTCTCCTAATTGCTACTACT Avr9 qRT-PCR expression fwd
Avr9RQ-R GTAGTCTAGCCCGACTCCCAATC Avr9 qRT-PCR expression rev
CfPhiARQ-F TGAGGACCAGAAGTGGACTCTTTC CfPhiA qRT-PCR expression fwd
CfPhiARQ-R ATCTCGCACAAATGCCTTGAG CfPhiA qRT-PCR expression rev
Ecp6RQ-F GCTCAAGGTTGGTCAGCAGAT Ecpβ qRT-PCR expression fwd
Ecp6RQ-R TTCACACCTGACAGATCACTTATGC Ecp6 qRT-PCR expression reve
Ecp7RQ-F TGGTTTTTCTTCTTTCTATAGTCGAGTCT Ecp7 qRT-PCR expression
A fwd
Ecp7RQ-R TTCTTAGCCCCTGCGTTCTGT Ecp7 qRT-PCR expression rev
CfPhiAi-F CCATGGAGCACCCAAGGTCGGCGACA CfPhiA RNAi fwd with Ncol
(bold)
CfPhiAi-R GAATTCGCGGCCGCACACTGCAGTATCTC CfPhiA RNAi rev with EcoRl GCACA (bold) and JVofl
(underlined)
Ecp6i-F CCATGGAGATCGAGAACCCAGATGCC Ecp6 RNAi forward with Ncol (bold) Ecp6i-R GAATTCGCGGCCGCCCCGACCATCTTCAC Ecp6 RNAi reverse with ACCTG EcoRl (bold) and JVofl
(underlined)
Ecp6i2-F GAATTCGAAGGCGACGGATTGCGGTT Ecpβ RNAi2 forward with EcoRl (bold)
Ecp6i2k-R GCGGCCGCTGGAAGACCTGGCACGCAAG Ecp6 RNAi2 reverse with JVofl (bold)
Ecp6i21-R GCGGCCGCTCGAGCGTGATGTTGAAGTC Ecp6 RNAi2 reverse with JVofl (bold) Ecp6-RNAi-RQ-F GTCAGATTAAGGCTCTCAAC Ecpβ qRT-PCR RNAi expression fwd
Ecp6-RNAi-RQ-R GTTTAAGTACAAGACCATTC Ecpβ qRT-PCR RNAi expression rev
Ecp6-RNAi2-RQ-F GTCAGATTAAGGCTCTCAAC Ecpβ qRT-PCR RNAi expression fwd
Ecp6-RNAi2-RQ-R GTTTAAGTACAAGACCATTC Ecpβ qRT-PCR RNAi expression rev
Ecp6OE-F AAGCTTATGGGATTTGTTCTCTTTTCACA Ecpβ over-expression with ATTGCCTTCATTTCTTCTTGTCTCTACAC coding sequence for C. TTCTCTTATTCCTAGTAATATCCCACTCT fulvum Avr4 signal peptide TGCCGTGCCCAAAATGAAACCAAAGCGAC (bold) and Hm&lll GGAC
(underlined)
Ecp6OE-R TTATGCCACAGCAGTAGTGA Ecpβ over-expression Ecp7NtermF CACTACTTGACCATCTACAGCAACATCGG Ecp7 over-expression CTGCCGCAAGGGCAGCCAGATTACGACGC primer to obtain coding AGGATTTTGGTCACGAG sequence for mature protein (bold)
Ecp7OE-F AAGCTTATGGGATTTGTTCTCTTTTCACA Ecp7 over-expression with ATTGCCTTCTTTCTTCTTGTCTCTACACT coding sequence for C. TCTCTTATTCCTAGTAATATCCCACTCTT fulvum Avr4 signal peptide GCCGTGCCCAAAATCACTACTTGACCATC (bold) and Hmdlll TAC
(underlined)
Ecp7OE-R CCCGGGAATTCTTAACAATCAACTCTG Ecp7 over-expression with Xmal site (bold)
TSPl TTGACGGATACGATGTTG Gene wlaking
TSP2 TTGGCAATGGAGGTGAGG Gene wlaking
TSP3 CCTTGACGACAGTGTATTTGATG Gene walking
Ecpδ ChromWal CCATGCAGTCGATGATTC cDNA cloning, start codon Fl in bold Ecpδ R ACAGCAGTAGTGACGTTCTTG cDNA cloning
Ecpδ F2 ACTCTCGTTAGATTGCATTC Allelic variation
Ecpδ R2 GTTACTCTCAACACGCTG Allelic variation
Ecpδ F3 CCTCGCTGCTATCACATC Allelic variation
Ecpδ_R3 GTTGTCGAATAGCTGATG Allelic variation
Ecpδ Fl AAATACACTGTCGTCAAGGG Allelic variation Table S3
UNICODE Species Ecp6 Verticillium response resistance
PRJ- \ S. peruvianum LA2172 ? No
PRI-2 No No
PRI-3 S- pimpindlifolim Gl.1554 Yes Yes
PRJ.4 S. glabratum Gl.1561 No No
PRJ . g S. habrochaites Gl .1560 No No
PRI- 6 ^' fycoPersic°ides No No
PRJ- 7 S. habrochaites Gl.1290 Yes
PRJ. g S. pennellii LA716 No No
PRJ- \Q L.parviflorum G1.1601 No No
PRI- 13 LMrsutum PI127826 Gl.1607 No No
PRI- 14 LMrsutum Gl.1257 Yes No
PRI- 17 L.chilense G1.1556 Yes Yes
PRI- 18 L.chilense G1.1558 No No
PRI-20 L.chees.v.typ.PI266375 Gl.1615 Yes Yes
PRI-21 L.pimpinelUfolium Gl.1596 Yes Yes pRJ.22 L.pimpinelUfolium Gl.1310 Yes Yes
PRI - 23 L- cheesmanii PI266375 Gl.1615 Yes Yes
PRI-24 L.parviflorum G 1.1604 Yes Yes
PRI-25 L.parviflorum LA1045 Gl.1603 Yes No pRJ.27 LMrsutum Gl.1378 No No
PRI-28 LMrsutum var.glabr. Gl.1562 No No
PRI - 30 L- hirsutum Gl ■ 17°8 No No
PRI- 35 L.pennellii Gl.1608 No No
PRI-39 L.pimpinel.G1.562 No No
PRI-40 L.pimpinel.G1.563 No No
PRI-41 L.pimpinel.G1.564 No Yes PRI-42 L.pimpinel. Gl.565 No No
PRI-43 L.pimpinel.G1.704 No No
PRI-44 L.pimpinel.PIl 87002 Gl.1703 No Yes
PRI-45 L.pimpinel.PI126915 Gl.1704 No No
PRI-47 L.pimpinel. Gl.1416 No No
PRI-48 L.pimpinel. Gl.1555 No No
PRI-49 L.pimpinel. PI126933 No Yes
PRI- 50 L.pimpinel. P1126947 Gl.1589 No No
PRI-51 L.pimpinel. Gl.1590 Yes Yes
PRI- 55 L.pimpinel. PI344102 Gl.1594 Yes Yes
PRI- 56 L.pimpinel. Gl.1595 ? Yes
PRI- 63 L.pimpinel. Gl.1914 Yes Yes
MoneyMaker L. esculentum No No
Motella L. esculentum No No

Claims

Claims
1. A fungal effector protein comprising at least one LysM domain according to the sequence of SEQ ID NO: 4 or a sequence which is 95% or more identical thereto.
2. A fungal effector according to claim 1 comprising the amino acid sequence as depicted in SEQ ID NO:3 or a protein that is more than 95% identical thereto.
3. A fungal effector according to claim 1, wherein said effector is an ortholog of Ecp6, selected from the orthologs of Fig. 11 or a protein that is more than 95% identical thereto.
4. A method for detecting the presence of c/Ecp6 orthologs in a plant comprising a. Introducing a fungal effector protein according to any of claims 1-
3 to a plant or a plant part; and b. Detecting whether a resistance reaction in said plant or plant part occurs.
5. Method according to claim 4, wherein said presence of c/Ecp6 orthologs confers resistance to fungal pathogens.
6. Method according to claim 4 or 5, wherein introduction of said fungal effector protein is established by infecting the plant or plant part with a fungus capable of expressing said protein.
7. Method according to claim 4 or 5, wherein introduction of said fungal effector protein is established by application of said protein to the plant or a plant.
8. Method according to claim 7, wherein said application is application into the apoplastic space.
9. Method according to claim 4 or claim 5, wherein introduction of said fungal effector protein is by transformation of a plant with a construct encoding said protein.
10. Method according to claim 9, wherein said effector protein is transiently expressed.
11. Method according to claim 9 or 10, wherein introduction of said fungal effector protein is achieved by transient Agrobacterium transformation (ATTA).
12. Method according to claim 9, wherein said construct is a viral construct, preferably a potatoviral construct.
13. Use of an effector protein according to any of claims 1-3 for assaying the presence of c/Ecp6 orthologs in plants.
14. Method for providing pathogen resistant plants, comprising: a. Selecting a plant having pathogen resistance and containing a resistance gene cognate for an effector protein according to any of claims 1-3; b. Crossing said plant with a plant that needs to be provided with pathogen resistance; c. Assaying offspring of said crossing by testing for the presence of the resistance gene cognate for an effector protein according to any of claims 1-3; d. Selecting those plants that contain said resistance gene.
15. Method according to claim 14, wherein said assaying of step (c) is performed with a method according to any of claims 4-11.
PCT/NL2010/050275 2009-05-12 2010-05-12 New fungal elicitor protein and its use as resistance marker WO2010131960A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NLPCT/NL2009/050250 2009-05-12
PCT/NL2009/050250 WO2010131946A1 (en) 2009-05-12 2009-05-12 New fungal elicitor protein and its use as resistance marker

Publications (2)

Publication Number Publication Date
WO2010131960A2 true WO2010131960A2 (en) 2010-11-18
WO2010131960A3 WO2010131960A3 (en) 2011-02-03

Family

ID=41429439

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/NL2009/050250 WO2010131946A1 (en) 2009-05-12 2009-05-12 New fungal elicitor protein and its use as resistance marker
PCT/NL2010/050275 WO2010131960A2 (en) 2009-05-12 2010-05-12 New fungal elicitor protein and its use as resistance marker

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/NL2009/050250 WO2010131946A1 (en) 2009-05-12 2009-05-12 New fungal elicitor protein and its use as resistance marker

Country Status (1)

Country Link
WO (2) WO2010131946A1 (en)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030192075A1 (en) * 2000-07-03 2003-10-09 Frank Takken Elicitor from cladosporium

Non-Patent Citations (62)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL, S.F. ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 338 - 3402
ARLAT, M. ET AL., EMBO J., vol. 13, 1994, pages 543 - 553
BAKER, C.J. ET AL., PLANT PHYSIOL., vol. 102, 1993, pages 1341 - 1344
BALLAS ET AL., NUCLEIC ACIDS RES., vol. 17, 1989, pages 7891 - 7903
BATEMAN, A.; BYCROFT M., J. MOL. BIOL., vol. 299, 2000, pages 1113 - 1119
BENNETT-LOVSEY R.M. ET AL.: "Proteins: Structure, Function", BIOINFORMATICS, vol. 70, 2008, pages 611 - 625
BIELNICKI, J. ET AL., PROTEINS, vol. 62, 2006, pages 144 - 151
CHANG, J.H. ET AL., PROC. NATL. ACAD. SCI USA, vol. 102, 2005, pages 2549 - 2554
CIUFFETTI L.M. ET AL., PLANT CELL, vol. 9, 1997, pages 135 - 144
DE WIT, P.J.G.M., NETH. J. PLANT PATHOL., vol. 44, 1977, pages 337 - 366
FELSENSTEIN J., EVOLUTION, vol. 39, 1985, pages 783 - 791
GUERINEAU ET AL., MOL. GEN. GENET., vol. 262, 1991, pages 141 - 144
HAANSTRA, J.P.W. ET AL., THEOR. APPL. GENET., vol. 101, 2000, pages 661 - 668
HAMMOND-KOSACK, K.E. ET AL., PLANT PHYSIOL., vol. 110, 1996, pages 1381 - 1394
HELLENS R.P. ET AL., PLANT MOL. BIOL., vol. 42, 2000, pages 819 - 832
HONEE G. ET AL., PLANT MOL. BIOL., vol. 29, 1995, pages 909 - 920
JONES, L. ET AL., PLANT CELL, vol. 11, 1999, pages 2291 - 2301
JOOSTEN, M.H.A.J. ET AL., NATURE, vol. 367, 1994, pages 384 - 386
JOOSTEN, M.H.A.J. ET AL., PLANT CELL, vol. 9, 1997, pages 367 - 379
JORIS, B. ET AL., FEMS MICROBIOL. LETT., vol. 70, 1992, pages 257 - 264
JOSHI ET AL., NUCLEIC ACID RES., vol. 15, 1987, pages 9627 - 9639
KAMOUN, S., ANN. REV. PHYTOPATHOL., vol. 44, 2006, pages 41 - 60
KEEN, N.T., ANN. REV. GEN., vol. 24, 1990, pages 447 - 463
KELLEY L.A. ET AL., J. MOL. BIOL., vol. 299, 2000, pages 501 - 522
KOBE, B.; DEISENHOFER, J., TRENDS BIOCHEM. SCI., vol. 19, 1994, pages 415
KRUIJT, M. ET AL., MOL. PLANT PATHOL., vol. 6, 2005, pages 85 - 97
KUMAR, S. ET AL., BIOINFORMATICS, vol. 17, 2001, pages 1244 - 1245
KYTE, J.; DOOLITTLE, R.F., J. MOL. BIOL., vol. 157, 1982, pages 105 - 132
LAUGE, R. ET AL., MOL. PLANT MICROBE INTERACT., vol. 10, 1997, pages 725 - 734
LUDERER, R. ET AL., MOL. MICROBIOL., vol. 45, 2002, pages 875 - 884
MALONEK S. ET AL., J. BIOL. CHEM., vol. 279, 2004, pages 25075 - 25084
MELIN, P. ET AL., FUNGAL GENET. BIOL., vol. 40, 2003, pages 234 - 241
MERSEREAU M. ET AL., GENE, vol. 90, 1990, pages 149 - 151
MOGEN ET AL., PLANT CELL, vol. 2, 1990, pages 1261 - 1272
MULLINS, E.D. ET AL., PHYTOPATHOL., vol. 91, 2001, pages 173 - 180
MUNROE ET AL., GENE, vol. 91, 1990, pages 151 - 158
NAKAYASHIKI, H. ET AL., FUNGAL GENET. BIOL., vol. 42, 2005, pages 275 - 283
OHNUMA, T. ET AL., J. BIOL. CHEM., vol. 283, 2008, pages 5178 - 5187
PERFECT, S.E. ET AL., PLANT J., vol. 15, 1998, pages 273 - 279
PROUDFOOT, CELL, vol. 64, 1991, pages 671 - 674
PUNT P.J. ET AL., GENE, vol. 56, 1987, pages 117 - 124
RONALD, P.C. ET AL., J. BACTERIOL., vol. 174, 1992, pages 1604 - 1611
SALAMOV, A.A.; SOLOVYEV, V.V., GENOME RES., vol. 10, 2000, pages 516 - 522
SANFACON ET AL., GENES DEV., vol. 5, 1991, pages 141 - 149
SCHAFFER, A.A. ET AL., NUCLEIC ACIDS RES., vol. 29, 2001, pages 2994 - 3005
SHEVCHENKO A. ET AL., ANAL. CHEM., vol. 69, 1996, pages 850 - 858
SMALL, I. ET AL., PROTEOMICS, vol. 4, 2004, pages 1581 - 1590
SOANES, D.M.; TALBOT, N.J., MOL. PLANT PATHOL., vol. 7, 2006, pages 61 - 70
STASKAWICZ, B.J. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6024 - 6028
STERGIOPOULOS, I ET AL., FUNGAL GENET. BIOL., vol. 44, 2007, pages 415 - 429
STERGIOPOULOS, I. ET AL., PLANT MICROBE INTERACT., vol. 20, 2007, pages 1271 - 1283
TAMURA, K. ET AL., MOL. BIOL. EVOL., vol. 24, 2007, pages 1596 - 1599
TANG, X. ET AL., MOL. PLANT MICROBE INTERACT., vol. 19, 2006, pages 1159 - 1166
THOMMA, B.P.H.J. ET AL., MOL. PLANT PATHOL., vol. 6, 2005, pages 379 - 393
VAN DEN ACKERVEKEN, G. ET AL., CELL, vol. 87, 1996, pages 1307 - 1316
VAN ESSE, H.P. ET AL., MOL. PLANT MICROBE INTERACT., vol. 20, 2006, pages 1092 - 1101
VAN KAN, J.A.L. ET AL., MOL. PLANT MICROBE INTERACT, vol. 4, 1991, pages 52 - 59
VIVIAN, A. ET AL., PHYSIOL. MOL. PLANT PATHOL., vol. 35, 1989, pages 335 - 344
WESTERINK, N. ET AL., MOL. MICROBIOL., vol. 54, 2004, pages 533 - 545
WHISSON, S.C. ET AL., NATURE, vol. 450, 2007, pages 115 - 118
WHITHAM ,S. ET AL., CELL, vol. 78, 1994, pages 1 - 20
WUBBEN, J.P. ET AL., MOL. PLANT MICROBE INTERACT., vol. 7, 1994, pages 516 - 524

Also Published As

Publication number Publication date
WO2010131960A3 (en) 2011-02-03
WO2010131946A1 (en) 2010-11-18

Similar Documents

Publication Publication Date Title
Westerink et al. Cladosporium fulvum circumvents the second functional resistance gene homologue at the Cf‐4 locus (Hcr9‐4E) by secretion of a stable avr4E isoform
Wang et al. A novel wheat NAC transcription factor, TaNAC30, negatively regulates resistance of wheat to stripe rust
Franco‐Orozco et al. A new proteinaceous pathogen‐associated molecular pattern (PAMP) identified in Ascomycete fungi induces cell death in Solanaceae
AU2016206158B2 (en) Protein associated with disease resistance and encoding gene thereof, and use thereof in regulation of plant disease resistance
DK1472281T3 (en) GENES RESISTING TO PHYTOPHTHERA INFESTANS (POTATO FARM)
US10676756B2 (en) Plant pattern recognition receptor and chimeras thereof for use against bacterial infections
AU697247B2 (en) Plant pathogen resistance genes and uses thereof
Zhang et al. A truncated CC-NB-ARC gene TaRPP13L1-3D positively regulates powdery mildew resistance in wheat via the RanGAP-WPP complex-mediated nucleocytoplasmic shuttle
CN110066811A (en) A kind of rice sheath blight disease effector gene RsIA_SCR28 and its application
Westerink et al. The AVR4 elicitor protein of Cladosporium fulvum binds to fungal components with high affinity
US20160319301A1 (en) Methods for Increasing Resistance to Soybean Cyst Nematode in Soybean Plants
EP3656784A1 (en) Chimeric pattern recognition receptor kinases
WO2006042145A2 (en) THE RICE BACTERIAL BLIGHT DISEASE RESISTANCE GENE xa5
Lian et al. ShNPSN11, a vesicle-transport-related gene, confers disease resistance in tomato to Oidium neolycopersici
JP5173847B2 (en) Disease resistant plant
EP2911517B1 (en) A novel immunogenic fungal extract and pattern recognition receptor in plants
WO2010131960A2 (en) New fungal elicitor protein and its use as resistance marker
AU2001270611B2 (en) Elicitor from cladosporium
KR101028113B1 (en) CaH1 gene and its use in red pepper involved in growth growth, flame resistance and aging regulation
WO2008100112A1 (en) Transgenic plants exhibiting increased resistance to biotic and abiotic stresses or accelerated flowering time and methods for producing the same
EP2283034B1 (en) Genes and methods for increasing disease resistance in plants
CN116064463B (en) Application of tomato gene SlPKG and its encoded protein in disease resistance
EP2451830B1 (en) Method for increasing the resistance of a plant or a part thereof to a pathogen, method for screening the resistance of a plant or part thereof to a pathogen, and use thereof
AU709028B2 (en) Plant pathogen resistance genes and uses thereof
WO2006068481A2 (en) Novel necrosis and ethylene inducing proteins from botrytis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10722436

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10722436

Country of ref document: EP

Kind code of ref document: A2