WO2000071748A2 - Ers-genes, method of screening for chemical compounds capable of inducing ers in plants - Google Patents

Ers-genes, method of screening for chemical compounds capable of inducing ers in plants Download PDF

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Publication number
WO2000071748A2
WO2000071748A2 PCT/EP2000/004564 EP0004564W WO0071748A2 WO 2000071748 A2 WO2000071748 A2 WO 2000071748A2 EP 0004564 W EP0004564 W EP 0004564W WO 0071748 A2 WO0071748 A2 WO 0071748A2
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Prior art keywords
plant
ers
compounds
inducing
plants
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PCT/EP2000/004564
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French (fr)
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WO2000071748A3 (en
Inventor
Karl-Heinz Kogel
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Basf Aktiengesellschaft
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Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Priority to IL14623400A priority Critical patent/IL146234A0/en
Priority to EP00941966A priority patent/EP1179070A2/en
Priority to JP2000620125A priority patent/JP2003505013A/en
Priority to BR0010829-4A priority patent/BR0010829A/en
Priority to AU56748/00A priority patent/AU5674800A/en
Priority to CA002370332A priority patent/CA2370332A1/en
Publication of WO2000071748A2 publication Critical patent/WO2000071748A2/en
Publication of WO2000071748A3 publication Critical patent/WO2000071748A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline

Definitions

  • This invention concerns a method of testing a chemical compound for its capability to induce an enhanced resistance status (ERS) against pathogens in plants, and to the use of said method for screening for chemical compounds having the capability to induce ERS in plants.
  • the invention pertains to DNA molecules encoding polypept de or parts thereof the expression cf which, within their natural genetic environment, may be induced oy ERS in- ducing compounds and may be assayed in the method according to the invention, transgenic plants, plant parts or seeds comprising said DNA molecules, polypep- tides/proteins or parts thereof encoded by said DNA molecules and antibodies specifically recognizing and binding said polypeptides/proteins or parts thereof.
  • An enhanced resistance status (ERS) in plants is the result of biologically and/or chemically induced resistance phenomena which inter alia include systemic acquired resistance (SAR) and induced systemic resis- tance (ISR) mediated by Rhizobacteria .
  • SAR systemic acquired resistance
  • ISR induced systemic resis- tance
  • SA salicylic acid
  • SAR systemic acquired resistance
  • the US Patent 5,614,395 discloses a method for screening for SAR inducing agrochemicals w th the aid of genetically modified plants containing a chime ⁇ c gene which responds on SAR inducing compounds, which is disclosed as a wheat gene such as WCI-1. This method is limited to those plant species wnich are susceptible for the chimeric gene, n particular to wheat.
  • the International Patent application WO 98/00023 suggests a method of screening compounds for resistance-inducing activity by the detection of the expression of a plant defensin gene such as PDF 1 .
  • the genes disclosed there are induced by plant patnogens such as Al ternaria brassi ci cola or Botryti s cmera but not by SAR inducing agrochemicals such as salicylic acid or dichloroisonicotimc acid.
  • the present invention is based on tne most sur- prising finding that new genes exists apart from clas ⁇ sical pathogenesis-relate ⁇ (PR) genes the expression of which is mducible by SAR inducing compounds, and m a particular embodiment by both SAR inducing com ⁇ pounds and ISR inducing compounds.
  • This finding enables the establishment of novel methods for testing chemical compounds and natural products for their capability to induce an enhanced resistance status (ERS) , and particularly SAR or ISR, in plants and for screening a multiplicity of chemical compounds, for compounds or compositions having the capability to induce ERS m plants.
  • Tne finding may also enable the use of recombmant DNA molecules comprising these DNA molecules m transgenic plants to confer enhanced tolerance to disease and pest organ- isms.
  • the present invention relates to DNA molecules having the following sequences (A) to (H)
  • GTCAAGTTCA CGCCTGCCGA ATCGAGGATG ASTGCGCCGC CCCAAAAGCC 50 CCCGCCGGAG AGGAGAAGAA GACGTCATGG CCGGAGGTAG CGGGAAAGTC 100
  • encoding polypeptides or parts thereof the expression of which may be induced by ERS inducing compounds and a method of testing a chemical compound for its capability to induce an enhanced resistance status (ERS) in plants said method comprising: (a) contacting a plant or plant part comprising a gene fragment having the sequence (A) to (K) coding for a polypeptide/protein or part thereof the expres- sion of wnicn being inducible Py SAR inducing com ⁇ pounds, wit" the chemical compound; and (b) assaying the expression of said gene fragment, wherein tne expression of said gene fragment indicates that the chemical compound has the capability to in ⁇ cute ERS.
  • ERS enhanced resistance status
  • tne expression of tne said gene fragment (A) to (H) the expression of nich being inducible by SAR inducing compounds is not in ⁇ ducible by plant pathogens.
  • the expression of the said gene fragment is additionally inducible also by ISR inducing compounds.
  • this method allows to screen ERS inducing compounds in different plant species as for example cereals such as wheat or barley, and rice; to determine the presence of ERS-inducmg compounds in a sample; to determine the concentration of an ERS-inducmg compound in a sample; to compare two ERS- inducing compounds for their relative capabilities to induce ERS m plants; and to test compounds and compositions for their capa ⁇ bility to change the ERS inducing effect of a chemical compound.
  • a further aspect of the present invention relates to a kit for use m the methods of the present inven ⁇ tion.
  • Another aspect of the present invention relates to a process for providing compounds having the capa- bility of inducing ERS m plants which comprises the steps of
  • the invention pertains to DNA molecules encoding a polypeptide/protem tne expression of which may be assayed m the methods according to the invention, fragments of said DNA molecules which i.a. might be used as prooes or primers, transformed microorganisms and transgenic plants, plant parts or seeds comprising said DNA molecules, polypeptides and proteins encoded by said DNA molecules and antibodies specifically recognizing and binding said polypeptides or proteins.
  • a multiplicity of chemical compounds and compositions may be screene ⁇ to identify compounds and/or compositions having the capability to induce ERS in plants.
  • said multiplicity of chemical compounds or compositions may be provided, for example, form of a combinatorial library such as exemplified EP 0 53u, ⁇ ? o o_ 309 and EP 0 818 431, or in form of a -iOr r_ or col ⁇ lection of natural compoun ⁇ s and/or compositions or a library comprising both natural ana synthetic com- pounds and compositions.
  • the said method may aiso oe usec for determining the presence of compounds capable of inducing ERS in plants m a sample, wherein m the above step (a) the plant or plant part is contacted with an aliquot of the sample place of tne chemica. compound. The expression of the said gene fragment then indicates the presence of one or more compounds capable of inducing ERS m plants m tne sample.
  • tne expres- sion of the said gene fragment in the said plant wh-.cn is inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds, is not inducible by naturally occurring plant pathogens.
  • SAR inducing compounds preferably both by SAR inducing compounds and ISR inducing compounds
  • ISR inducing compounds ISR inducing compounds
  • This feature appears to be particularly valuable if it is intended to perform the methods of the present invention in an environment where plant pathogens may be present. This will be the case when using plants in a green house or on the field for per ⁇ forming the methods of the present invention.
  • said specific feature will ensure that any expression of the said gene fragment observed will only be attributable to the presence of a compound or composition having the capability to induce ERS m plants. If working the method according to the mven- tion in a cell or tissue culture system, however, said specific feature may be utilized but it is net essen ⁇ tial.
  • naturally occurring plant pathogens includes all kinds of phyto- pathogenic microorganisms, in particular phytopatnc- genic fungi, bacteria and viruses.
  • plants and plant parts as used heremabove and nerembelow include all kmcs of plants and plant parts, m particular foliage, roots and seeds.
  • the said plant or plant part is selected from the group consisting of a plant proto ⁇ plast, a plant cell, a plant tissue, callus, a developing plantlet, a plant leaf, an immature r.oie p.ant, a mature whole plant and seed.
  • said plant or plant part in particular plant ceil or plant leaf, is or is derived from a onocotyledonous plant, such as a cereal, particularly wheat or a plant of the genus Hordeu ⁇ v such latter plants being particularly preferred, rice as well as from dicotyledonous plants.
  • the gene fragment of step (a) may be any gene fragment having either the sequence (A) to (H) or part thereof, the expression of which being inducible ⁇ y SAR inducing compounds, and preferably both by SAR m- ducing compounds and ISR inducing compounds.
  • This definition comprises any naturally occurring gene fragments exhibiting these characteristics as well as not-naturally occurring gene fragments, the sequence of which has substantial homology with the sequence of any of said naturally occurring gene fragments, vari ⁇ ants thereof as well as gene fragments comprising one or more portions of the aforementioned gene fragments and variants wherein all these gene fragments and variants maintain the characteristic indicated above, i.e. an expression which is inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds.
  • the expression of the said gene fragment or variant which is inducible by SAR inducing compounds, and preferably both by SAP, inducing com ⁇ pounds and ISR inducing compounds, is not inducible by naturally occurring plant pathogens.
  • the said gene fragment (A) to (H) is a gene fragment, the nucleotide sequence of which exhibits substantial homology with the nucleotide sequence (A) to :H), or a variant thereof or comprises one or more portions of the nucleotide sequence given above, of a sequence having substantial homology thereto or a variant thereof. Accordingly, the term also includes sequences that can substan ⁇ tially hybridize to the nucleotide sequence given above, in particular under conditions of low strin- gency.
  • variant thereof means any substitution, variation, modification, insertion, deletion or addition of one or more nucleotides from or to the nucleotide sequence of a naturally occurring gene fragment (A) to (H) the expression of which being inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds, for example a gene fragment comprising the nucleotide sequence given above, provided that the expression of the variant being still inducible by application of SAR inducing compounds, and preferably both of SAR inducing compounds and ISR inducing compounds.
  • the phrase particularly includes allelic variants of naturally occurring gene fragments (A) to (H) the expression of which being inducible by SAR inducing compounds, and prefera ⁇ bly both by SAR inducing compounds and ISR inducing compounds.
  • the phrase also includes synthetic variants comprising or essentially consisting of nucleotide se- quences that can substantially hybridize to the nu- cleotioe sequence of tne naturally occurring parent gene fragment. .
  • sucn nybridisation occurs at low-stringency conditions, m a further embodiment also between low and high stringency conditions, and, m a very particular embodiment, even at hign stringency conditions.
  • low stringency conditions can be defined as 3 x SSC at ambient temperature to 65°C and high stringency conditions as C.l x SSC at 68°C.
  • SSC is the abbreviation of a 0.15>1 so ⁇ ium cn.o- ride, 0.015M trisodium citrate puffer.
  • substantially nomology as used nere - above and herembelow embraces nomology with respect to at least the essential nucleotioe/s of tne parent gene sequence (A) to (H) , provided that tne expression thereof still being inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds.
  • homology is shown when 60 % or more of the nucleotides are common with the naturally occurring parent gene fragment, more typically 65 % , preferably 70 %, more preferably 75 % , even more preferably 80 or 85 % and particularly preferred are 90 %, 95 %, 98 % or 99 % or more homology.
  • the gene fragment (A) to (H) the expression of which being inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds further comprises a nucleotide sequence coding for an indicator protein or polypeptide fused to the coding region of a gene corresponding to either DNA molecule of (A) to (H) .
  • the ex ⁇ pression of the indicator protein or polypeptide is inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing com- pounds, and, upon application of ERS inducing com- pounds, occurs concomitantly witi tne expression of the DNA molecule of (A; to (H) form of a fusion protein.
  • said nucleotide se ⁇ quence coding for an indicator protein or polypeptide codes for a glucuronidase enzyme, luciferase or for the green fluorescent protein (GFP) .
  • indicator proteins could be enzymes conferring toler ⁇ ance to herbicides sucn as acetolactate syntnase,
  • the SAR inducing compounds and ISR inducing corn- pounds which may be assayed with the methods according to the invention, may be summarized under tne generic term "ERS inducing compounds".
  • This term includes all compounds being capable to provide a plant ⁇ itn im ⁇ proved resistance characteristics against a_l types of pests, for example against harmful microorganisms, like phytopathogemc fungi, bacteria and viruses, as well as, pests such as insects mites and nematodes .
  • it includes l.a. all compounds being capable to induce systemic acquired resistance (SAR) and induced systemic resistance (ISR) including defense mechanisms independent of salicylic acid in plants.
  • ERS inducing compounds induce m the plants a resistance status by activating, for example, cell death reactions (HR) , cell wall appositions, PR-gene expression and/or phytoalexm accumulation.
  • SAR inducing cnemical compounds include Denzoic acid, salicylic acid, dichloroisonicotmic acid, poly- acrylic acid, am obuty ⁇ c acid, arachidonic acid and derivatives thereof and natural products such as har- p and other elicitors.
  • Another group of SAR inducing cnemical compounds are benzo-1 , 2 , 3-th ⁇ ad ⁇ azole derivatives as for example disclosed by US Patents US 4,931,581 and US 5,229,384.
  • the reference compound of the IS inducing chemica. compounds is represented py jasmon c acid and deriva ⁇ tives thereof such as methyl asmonate.
  • the known ERS inducing compounds are preferably used as reference compounds for the screening of novel agrochemical compounds.
  • Other chemical compounds, m particular agrochemicals, encompassed by the present invention, in par ⁇ ticular individual members of a synthetic combinato ⁇ rial library, can be determined eas ⁇ _y by assaying tne test chemical with the aid of the metnods according to this invention.
  • the ERS inducing chemical compounds may be applied pure form, m solution or suspension, as powders or dusts, or other conventional formulations used agriculturally.
  • Such formulations may include solid or liquid carriers, that is, materials with which said ERS inducing chemical compound is comoined to facilitate application to the plant, tissue, cell or tissue culture, or to improve storage, handling or transport properties.
  • suitable carriers include silicates, clays, resins, alcohols, ketones, aliphatic or aromatic hydrocarbons, and the like.
  • the formulation of tne ERS inducing chemical compound may include one or more con- ventional surfactants, either ionic or non-ionic, such as wetting agents, emulsifying or dispersing agents.
  • the ERS inducing chemical compounds singly or combinations may also be applied combination with another agent, for example with an adjuvant, a herbi- cide, a fungicide, an insecticide, a grovvtn regulator or a fertilizer.
  • tne ERS ln ⁇ ucmg cnem_ca_ compound may be applied as a spray to .eaves, stems or branches or to seeds before pla-tirg or to soil or other growing or cultivation me ⁇ _uir
  • the chemicals are applied a concentration of 0.1 to 1000 g active ingredient per liter of soil volume a so l drench process or, alternatively, as a spray a concentration of 0.1 - 100 mg active ingredient per liter of spray solution.
  • concentrations now based on the volume of the culture medium, may be used.
  • every conventional method or assaying the expression of genes can be used to monitor the response of the plant on the action of the chemical compound, such as Western blot or Nortnern plot analysis, detection or monitoring of the expression of an mdi- cator protein or polypeptide the gene thereof being included said gene fragment the expression of which being inducible by a SAR inducing compound, e.g. by detection of an enzymatic activity thereor . If tne expression which has to be detected occurs tissue- specific, the analysis will be performed on said spe ⁇ cific tissue.
  • step ⁇ c of the metno ⁇ according to the invention.
  • the ana-.ys-.s of the expression or tne said gene fragment (A) to (H) is carried out using a Northern blot type analysis, m particular with di- goxigenm labelled cDNAs of the gene fragment, e.g. a DNA with a nucleotide sequence as given (A) to (h , or fragments thereof, most preferred using a commercially available Northern blot kit sucn as the DIG- High Prime labelling Kit of Boenrmger Mannneiir, Ger- many.
  • tne present invention the analysis of the expression of the said gene fragment is carried out using reverse transcrip- tase-poly erase chain reaction (RT-PCR) .
  • RT-PCR reverse transcrip- tase-poly erase chain reaction
  • the analysis of the expression or tne said gene fragment is carried out using antioodies to detect the gene product of tne said gene fragment, or a specific indicator polypeptide or protein the gene se- quence of which representing a part of the said gene fragment, e.g. by performing a Western blot type analysis .
  • the detection of the expression of the said gene fragment is based on the detection of the expression of an "additional" nucleotide sequence comprised within the said gene fragment (A) to (H) and encoding an indicator protein or polypeptide
  • the detection may be performed also by other means than by use of nucleotide probes (e.g. for a Northern-blotting assay) or antibodies (e.g. for a Western-blotting assay), ⁇ e- pending on the type of indicator protein or polypeptide produced.
  • the indicator protein or polypeptide may be a fluorescent or cnemilummescent protein or polypeptide, such as the green fluorescent protein (GFP) , thus enabling the detection of expression or the said gene fragment oy detection of fluorescence or chemilummescence of the said plant cells.
  • the indicator protein or polypeptide may also exhibit an enzymatic activity not naturally occurring in the plant or plant part used for tne metnod of the invention. Accordingly, this enzymatic activity may be assayed for detection of the expression of tne indicator protein or polypeptide ana, accordingly, of the said gene fragment.
  • the assay may be oaseo en the measuring of the disappearance of a substrate for the enzyme from an assay medium or on the measuring of the formation of a product by the enzymatic conversion.
  • said product of the enzymatic conversion may be a compound which, as a result of the enzymatic conversion, is coloured.
  • the indicator protein could perform an enzymatic activity that for instance provides tolerance to another chemical agent such as a herbicide or phytotoxic compound. In such case, assaying for the expression is done m the presence of the herbicidal or phytotoxic compound and expression is scored based on tolerance to the chemical agent.
  • the assaying step (b) for assaying the expression of the said gene fragment (A) to (H) may be carried out m a way to produce a result which enables the mere determi- nation whetner or not an expression na ⁇ occurred; to enable the determination which of two or mon samples shows a higher degree of expression of the said gene fragment compared to the other (s; ("semi- quantitative determination”); to provide quantitative results as to tne leve. expression occurred ( "quantatitve determinatioi")
  • tne method may also be used for determining the concentration of a chemical compound which is capable of inducing ERS m plants m a sample; for determining whether a chemical compound or composition exhibits a stronger ERS inducing effect than an ERS inducing reference compound; and for determining whether and, optionally, to what degree a compound or composition is capable of changing the ERS inducing effect of a chemical compound.
  • the method for determining the concentration of a chemical compound which is capable of inducing ERS in plants a sample is characterized that it com ⁇ prises the following steps:
  • step (l) performing the method according to the invention comprising steps (a) and (b) as outlined above wherein step (a) the plant or plant part is contacted witn an aliquot of the sample;
  • step (n) determining the concentration of the ERS inducing chemical compound in the sample by comparing the result from step (I) with the results of corresponding tests performed with known concentrations of saio chemical compound.
  • Tne method for determining wnether a chemical compound exhibits a stronger ERS inducing effect tnan an ERS inducing reference compound, sucn as saiicylate, dichloroisonicot ic acid or asmonic acio is characterized in that it comprises the following steps: (l) performing the method according to the invention comprising steps (a) and (b) as outlined above wherein step (a) the plant or plant part is contacted with a specific concentration of the chemical compound; (li) performing tne method according to the -.nventior 1 comprising steps (a) and (cj as O tlined above wrere.n m step (a) tne plant or plant part is contacted with an amount of the ERS inducing reference compound resulting in an equimolar concentration compared to the concentration of the chemical compound step (l); (m) determining the relative level of the ERS inducing effect of the chemical compound by comparing the results from steps (l) and (n
  • step (m) will produce a semi- quantitative result m order to enable tne determina ⁇ tion which one of the chemical compound and the ERS inducing reference compound induces a higher expression of the said gene fragment and correspondingly will produce a stronger induction of ERS m a plant.
  • the results may also be evaluated to enable a quantitative assessment of the relative degree of expression induced by the chemical compound compared to the ERS inducing reference compound.
  • This evaluation may be performed for example by scanning of the analytical gels or films, photograpns or print- outs thereof by means of a densitometer or py analyzing relevant excised sections of analytical gels used for separating assay mixtures comprising radioactively labelled detection compounds (e.g. nucleotide probes or antibodies) m a liquid scintillation counter.
  • radioactively labelled detection compounds e.g. nucleotide probes or antibodies
  • the method for determining whether and, optionally, to what degree a compound or composition is ca ⁇ pable of changing the ERS inducing effect of an agro- chemical is characterized that it comprises the following steps:
  • step (l) performing the metnod according to the invention comprising steps (a) and (b) as outlined above wherein in step (a) the plant or plant part is contacted witn a specific concentration of tne compound or compos - tion;
  • step (n) performing the method according to the invention comprising steps (a) and (b) as outlined above wherein in step (a) the plant or plant part is contacted with the same specific concentration of the agrochemical as m step (l) in the presence of the compound or compo ⁇ sition;
  • step (m) will produce a semi-quantitative result in order to enaoie the determination whether the compound or composition leads to an enhancement, reduction or even inhibition of the expression of the said gene fragment and correspondingly will produce a stronger or reduced m- duction of ERS m a plant or will even abolish the - duction of ERS.
  • the results may be evaluated to enable a quantitative assessment of the relative degree of expression induced by a combination of the agrocnemicai and tne compound or composition compared to the agrocnemicai alone.
  • This evaluation may be performed for example by scanning of tne analytical gels or films, photographs or pr t-outs thereof by means of a densitometer or by analyzing relevant excised sections of analytical gels used for separating assay mixtures comprising radioactively labelled detection compounds (e.g. nucleotide probes or antibodies) in a liquid scintillation counter.
  • radioactively labelled detection compounds e.g. nucleotide probes or antibodies
  • the method for determining whether and, optionally, to what degree a compound or composition is ca- pable of changing the ERS inducing effect of an agro- chemical may further be utilized in a method for screening for compounds or compositions capable of changing the ERS inducing effect of an agrochemical .
  • kits for use the methods according to the present invention.
  • Said kits are characterized in that they comprise the fol ⁇ lowing components :
  • the kit may comprise one or more plants or plant parts wherein the expression of the said gene fragment (A) to (H) comprised therein is not inducible by plant pathogens.
  • the kit may comprise one or more plants or plant parts wherein the saio gene fragment additionally comprises a nucleotide sequence encoding an indicator protein or polypeptide.
  • kit comprises one or more plant parts, such as plant cells
  • plant parts may ce provided immobilized on a solid support.
  • Suitable supports may l. ⁇ . be made of glass, specific po sacchari ⁇ es, like aga- rose, or specific plastic materials sucn as polyacry-- amide, polystyrene, polyvinylalcoho-. , silicones. Meth- ods and techniques for immooilizmg cells and tissue on such types of supports are within tne skills of the skilled scientist.
  • the kit may comprise plates, such as microtiter plates, comprising a multiplicity of reaction wells which contain the said plant parts, in particular plant cells, preferably immobilized on the bottom and/or the walls of the wells.
  • the means for detecting the expression of the said gene fragment (A) to (H) may comprise one or more nucleotide probe (s) such as detectaoly lapelled cDNAs or cDNA fragments of the said gene fragment.
  • the detectable label may be e.g. a fluorescent, cnemilummescent or radioactive label, and is m particular a digoxi- genin molecule.
  • the means for detecting the expression of the said gene fragment will comprise an antibody which specifically recognizes and binds to the or a gene product of the said gene fragment. This antibody will carry a detectable label, e.g.
  • the means for detecting the expression of the said gene fragment will comprise one or more reagents for assaying the enzymatic activity of tne indicator protein or polypeptide including e.g. a sue- strate for tne indicator protein, optionally m comoi- nation with one or more necessary cofactor(s), and possibly reagents for detecting the product from the enzymatic conversion of the substrate.
  • said means may comprise one or more buffers for the assay reaction (s) and/or reagents for determining and, in particular, visualization of the assay result (e.g. in the case of detection of the enzymatic activity attached to the antibody) , which may be provided separately from each other and from the probes or antibodies or combined form or m form of combinations of some of said components.
  • the kit may comprise further components, m particular as separate components, such as one or more buffers for incubating the test compound or composition with a plant part, agents for breaking up the cellular structure of the plant or plant part for enabling the reactions for detection of the expression of the said gene fragment or instructions for
  • the invention pertains to a process for providing chemical compounds having tne capability of inducing ERS in plants which comprises the steps of
  • the invention also covers the new compounds having the capability of inducing ERS which are obtainable by this method and the use thereof as agro- chemical compounds, and m particular for tne induction of ERS in plants.
  • the new agrocnemicai compounds for use as an agrochemicai reference is ma ⁇ e to tne rele ⁇ vant explanations given earlier m this specification.
  • the invention is directed to an isolated DNA molecule which codes for a protein or polypeptide or part thereof, the expression of which being inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds, if said DNA molecule is situated within its natural genetic environment, or a nucleotide sequence complementary thereto.
  • the isolated DNA molecule comprises either of the nucleotide sequence (A) to (H),or a nucleotide se- quence complementary thereto.
  • the invention further pertains to isolated DNA molecules the nucleotide sequence of which having sub- stantial homology with the nucleotide sequence as given above, m particular hybridizes with the latter, or is a variant of the DNA molecule witn the nucleotide sequence as given above or is related to said DNA molecule by mutation.
  • substantially homology and “variant” refer- ence is ma ⁇ e to tne explanations given earlier in this specification.
  • specif _c embodiments are allelic variants to the DNA molecule having tne nucleotide sequence (A) to (H) as well as DNA mole- cules having a nucleotide sequence which hyoridizes tc the nucleotide sequence given m (A) to (K .
  • such hybridization occurs at low stringency conditions, m a furtner embodiment between low and high stringency conditions, and a very particular embodiment even at nigh stringency conditions.
  • low stringency condi ⁇ tions can be defined as 3 x SSC at ambient temperature to 65°C and high stringency conditions as 3.1 x SSC at 68°C, SSC being tne abbreviation of a 0.15M sodium chloride, 0.015M trisodium citrate buffer.
  • a further specific embodiment are DNA molecules having a nucleotide sequence which not necessarily hybridizes with one of the nucleotide sequences (A) to (H) , respectively, but, due to the degeneration of the genetic code, encodes the identical ammo acid sequence as given in sequence of (A) to (H) .
  • a DNA molecule encoding a prote /polypeptide or parts thereof tne expression of which, m its natural genetic environment within a cell, being inducible by SAR inducing compounds, ano preferably both by SAR inducing compounds and ISR in ⁇ ducing compounds, is derived from the genome of a plant and may be identified and isolated by use of nucleotide hybridization probes consisting of or com- prising fragments of the DNA molecule having the nucleotide sequence (A) to (H) . Preferred fragments of the said DNA molecules for screening of genes in other plants than Hordeum vul gare L .
  • further monocotyle- donous and also dicotyledonous plants comprise at least 15 nucleoti ⁇ es, typically between 15 dn ⁇ ⁇ ⁇ n u - cleotides, and more preferably at least 25 nuciec- tides, but may comprise, if necessary, even a nigner number of nucleotides such as 50 or more nucieotides.
  • fragments of the DNA molecules of the present invention and m particular of the isolated DNA molecule having the above given nucleotide se ⁇ quence are also covered by the present invention as well as the use thereof as hybridization probes, par- ticularly screening probes, or primers, in particular sequencing or PCR primers.
  • the invention also covers fragments which are shorter than mentioned above, e.g. comprising Between 8 and 15 nucleotides, in particular if they have utility as nucleotide probes or primers.
  • DNA molecules comprising or consisting of one or more of the fragments according to the present invention are employed in labeled form, e.g.
  • DNA molecules comprising or essentially consisting of one or more of the frag ⁇ ments according to the present invention are employed m a form which comprises modified nucleotide se- quences within the sequence (s) of the inventive fragment (s) which provide for appropriate restriction sites within said primers and any amplified products after the PCR which facilitate cloning of said amplified products.
  • PCR primers under the PCR conditions, will still hybridize specifically with tne target DNA molecule according to the present invention.
  • DNA fragments according to the present invention may be prepared, for example, on the basis of the nu- cleotide sequences (A) to (H) , possibly by use of ap- intestinalte restriction nucleases, or py cnemica-. syn ⁇ thesis.
  • Various suitable techniques for preparing DN ⁇ fragments and DNA molecules comprising one or more of these fragments according to the present invention are known to the skilled scientist.
  • nuc ⁇ eotioe fragments or DNA molecules according to the invention as screening probes, it may be interesting to determine whether the expression thereof being inducible by ERS inducing compounds, in particular SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds.
  • Plants identified by the described method to comprise a gene being inducible by ERS inducing compounds, in particular SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds, may tnen be used the methods according to the present invention.
  • the invention pertains to an isolated DNA molecule whicn comprises a DNA molecule or fragment as defined above fused with a nucleotide sequence coding for an indicator protein or polypeptide.
  • the complete encoded information of the said DNA molecule will be expressed if expressibly linked to a single expression control sequence.
  • the indicator protein or polypeptide encoded by this further nucleotide sequence portion of the DNA molecule is a protein or polypeptide unich is easily detectable, e.g. by use of specific nucleotide hybridization probes, by use of specific ant.Dodies, or due to specific characteristics of tne protein or polypeptide itself like fluorescence or enzymatic activity.
  • said further nucleotide sequence portion of tne DNA molecule may code for glucuronidase or the green fluorescent protein or a modified form of the plant AHAS enzyme.
  • Tne isolation and cloning of appropriate genes for indicator poly ⁇ peptides or proteins as well as the production of the recombmant chimeric DNA molecules defined aoove are well within the skills of a skilled scientist.
  • the invention also covers DNA constructs which comprise one or more of the DNA molecules and/or fragments defined above, and m particular DNA constructs serving as nucleotide probes or PCR primers, as well as constructs comprising the DNA molecule (s) and/or fragment (s) under the control of an expression control sequence, i.e. an expression control sequence opera- tively linked to said DNA molecule (s) and/or frag ⁇ ment (s) .
  • DNA mole- cules comprising or consisting of one or more of the DNA molecules and fragments according to the present invention will be employed a labeled form, wherein said labelling may be achieved by attachment of non- radioactive reporter or indicator molecules such as fluorescent or phosphorescent molecules, digoxigenm molecule (s), biotm molecule (s) or derivatives thereof.
  • non- radioactive reporter or indicator molecules such as fluorescent or phosphorescent molecules, digoxigenm molecule (s), biotm molecule (s) or derivatives thereof.
  • DNA constructs comprising or essentially consisting of one or more of the DNA molecules and fragments according to the pres- ent invention are employed in a form which comprises additional nucleotide sequences or modified nucleotide sequences within the sequence (s) of the inventive fragment (s) which provide for appropriate restriction sites within said primers and any amplified products after the PCR which facilitate cloning of said ampli ⁇ fied products.
  • additional nucleotide sequences or modified nucleotide sequences within the sequence (s) of the inventive fragment (s) which provide for appropriate restriction sites within said primers and any amplified products after the PCR which facilitate cloning of said ampli ⁇ fied products.
  • the DNA construct further comprises an expression control sequence wmch is operatively linked to said DNA molecule (s) and/or fragment (s) .
  • the invention further relates to vectors, plasmids, cosmids, viral and phage genomes comprising one or more of the DNA molecules and fragments according to the present invention.
  • the invention also pertains to DNA molecules com- prising a complementary sequence to the DNA molecules, fragments and constructs according to the present in ⁇ vention as well as the RNA transcription products of these DNA molecules, fragments and constructs.
  • the invention pertains to polypep- tides/proteins and parts thereof encoded by the DNA molecules, fragments and constructs according to the present invention, proteins having or comprising an ammo acid sequence exhibiting a homology of at least 65%, typically at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% and particularly preferred at least 95.
  • polypeptide in ⁇ cludes molecules which typically comprise at least 18 ammo acids, more particularly at least 25 ammo acids and particularly preferred more than 40 ammo acids.
  • polypeptides and proteins are fusion proteins of the proteins or parts thereof coded by the DNA molecules according to the invention, with an indicator protein or polypeptide as already outlined above.
  • polypeptides/protems or parts thereof are provided which comprise the polypeptides/protems or parts thereof described m the last two paragraphs.
  • These polypeptides/protems or parts tnereof may be obtained by cultivating a host organism transformed with one or more of said DNA molecules and optionally purifying these polypeptides and proteins py methods known to the art.
  • these polypeptides and proteins may l.a. also be prepared synthetically by use of known peptide and protein synthesizing techniques .
  • polypeptides and proteins of the invention may be utilized for the preparation of monoclonal or poly- cional antibodies or, if they exhibit enzymatic activ- lty, to utilize said enzymatic activity for various purposes .
  • the invention also covers monoclonal or polyclonal antibodies or antisera wnicn specifically recognize and bind a polypeptide or protein according to tne present invention.
  • Corresponding antibodies may be prepared by use of the polypeptides or proteins according to the present invention for lm- munization of a host according to techniques Known to the skilled scientist.
  • the invention includes transformed mi ⁇ croorganisms, such as bacteria, oacte iophages, vi- ruses, eucaryotic organisms like fungi, yeasts, proto- zoae, algae and human, animal and plant cells, which comprise a recombmant DNA sequence comprising at least one of the DNA molecules, fragments or constructs of the present invention.
  • transformed microorganisms may, l.a., be used as an expression system for producing the gene product (s) of the DNA molecules, fragments or constructs of the present invention.
  • Typical microorganisms which are used for this purpose are bacteria, like E. coll , or yeasts such as Saccharomyces cerevi siae .
  • agrobacteria like AgroDac t en urr t umefaci ens
  • inventive DNA molecules, fragments or constructs thus providing transgenic plants.
  • Methods for transformation of microorganism cells with the DNA molecules, fragments or constructs of the present invention are well known to tne skilled scientist, including the construction of expression vectors comprising the DNA sequences of the invention under the control of a constitutive or inducible promoter.
  • the promoter may also enable a tissue-specific expres ⁇ sion of the encoded information in particular compart ⁇ ments of an organism, such as a plant.
  • the invention pertains to transgemc plants, plant parts or seeds comprising a recombinant DNA sequence which comprises one or more DNA molecule (s) according to the present invention.
  • plants and “plant parts” include all of a plant propagation material such as a plant protoplast, a plant cell, a plant tissue, callus, a developing plantlet, a plant leaf, an immature whole plant and a mature whole plant.
  • the transgemc plants, plant parts or seed may contain the said recombinant DNA sequence integrated into the genome thereof or extrachromoso- mally located.
  • the techniques for introduction of genetic information into plant cells comprise a direct DNA transfer (e.g. into protoplasts by means of elec- troporation or by application of a high molecular weight osmotic agent as well as by biolistie methods wherein DNA-coated particles are shot into plant tis- sue), as well as the use of natural host/vector systems (e.g. of agrobacteria or plant viruses; .
  • TMV tobacco mosaic virus
  • potato virus X in the genome of which said recombinant DNA sequences then oemg inserted, may be utilized.
  • Exemplary plants for integration of tne said recombinant DNA sequences of tne invention include dicoty- ledonous plants as well as monocoty-eaonous plants, particularly cereals and crops, sucn as potato, canola, oilseed rape, soybean, sugar oeet or tobacco.
  • SSH Suppression subtractive hybridization
  • the technique is based on equalization of abundance of target cDNAs included in the tester cDNA population, and efficient subtraction of common sequences m tester and driver cDNA population by hy- bridization.
  • differentially expressed target cDNAs are amplified selectively whereas the amplification of nontarget cDNA is suppressed.
  • Plants namely tne barley ( Horaeum v l gare L.; culti- var Ingrio and the Rorl-2 mutant A89, were grown in a growth chamber at 16°C, 60 % relative humidity and a photo period of 16 h (100 ⁇ E s " m “: ) .
  • Control inoculation for chemical induction was done with 10 comdia mm "" from Erysipne gramim s DC: Fr . f . sp . nordei , isolate Ki (Hmze et al . , 1991; at tne third day after chemical treatment.
  • RNA Clean For northern analysis total RNA was isolated by using RNA Clean (AGS) with some modifications to the manual provided. Leaf material was ground in liquid nitrogen, After adding 1 , 7 ml RNA Clean extraction buffer and 200 ⁇ l chloroform to an aliquot of 200 mg leafpowder the content was thoroughly mixed and snax.en for 30 minutes. The samples were centrifuged at 20800 x g (4 °C, 30 minutes) and the aqueous phase was extracted again with an equal volume of chloroform.
  • AVS RNA Clean
  • RNA of different time points (12, 24, 48 hours after chemical induction and control treatment, respectively) was pooled with equal amounts. Pooled RNA was subjected to digestion with DNase as follows. Ali- quots of 750 ⁇ g RNA were made up to 250 ⁇ l by adding 12,5 ⁇ l 1 M Tris-HCl (pH 7,5), 50 ⁇ l 50 mM MgCl , 1 ⁇ l 25 mM EDTA (pH 7,5), 2,5 ⁇ l RNase Inhibitor (40 U/ ⁇ l), 10 ⁇ l DNasel (10 U/ ⁇ l) and the appropriate volume of water. The reaction mixture was incubated at 37 °C for 30 minutes m an air incubator.
  • RNA was made up to 1250 ⁇ l with water.
  • a equal volume of phenol (saturated with Tris-HCl (pH 8,0)) : chloroform (3:1) was added, thoroughly mixed, and centrifuged at 20800 x g for 5 minutes at room tempera- ture .
  • the aqueous phase was transferred to a fresh tube and subsequently 0, 1 volumes of 3 M sodium acetate (pH 5,2) and 2,5 volumes of cold (-20 °C) 96 % ethanol were added.
  • the assays were mixed and stored at -20 °C for 24 hours.
  • Polyadenylated RNA (poly (A) + RNA) was isolated using the DYNABEADS mRNA Purification Kit (Dynai) according to the manufacturer's recommendations. The quality of the poly(A)0RNA preparation was checked in a denaturing 6 % polyacrylamid gel and silver staining as follows. For casting and running the gels a Mim-PROTEAN II DC-. (BioRad) was used. To 12 ml of the gel solution 5,6 ⁇ l Te ed (SERVA) and 72 ⁇ l 10 % APS (BioRad) were ad ⁇ ed, the gel was poured immediately and allowed to polymerize for 2 hours.
  • SERVA ⁇ l Te ed
  • BioRad BioRad
  • the "driver-tester” hybridization assays were mixed, fresh denatured "driver” added and incubated at 68 °C for 12 hours.
  • the following PCR reactions done with the Advantage ⁇ cDNA Polymerase Mix (Clontech) , were performed according to tne manual provided with the subtraction kit. All PCR and ny- bridization steps were done in a Perkm-Elmer 2400 thermal cycler.
  • the PCR mixture now was enriched for differentially expressed cDNAs in roughly equal abundance.
  • Colony PCR reverse northern Plots and screening
  • colony PCR was performed, tne PCR-products Plotted onto membranes and hybridized with Digoxygenm-labeled first strand cDNA deriving from pooled poly(A;0RNA extracted from induced and non-induced plants as template.
  • a total of 480 white or light blue individual clones were picked and used to inoculate firstly agar plates containing 100 ⁇ g/ml ampicill and secondly 96-well microtitre plates containing 40 ⁇ l sterile water.
  • the bacteria in the microtitre plates were lysed by heat ⁇ ing to 98 °C for 5 minutes using a Perkm-Elmer 9700 thermal cycler.
  • PCR reaction mix was pipetted in 96- well microtitre plates as follows.
  • Filters A and B then were denatured for 2 x 5 minutes on whatman paper soaked with 0,5 M NaOn, 1,5 M NaCl and neutralized for 2 x 5 minutes on whatman paper soaked with 0,5 M Tris/HCl (ph 7,4,, 1,5 M NaCi . Subsequently DNA was fixed by cross linking GS Gene Linker, BioRad) with 125 mJ .
  • RNA RNaseA and RNaseH After incubation at room temperature for 2 minutes re- verse transcription was started by adding 40 U MMuLV reverse transcriptase (Boehrmger) . The components were mixed thoroughly and incubated at 37 °C for 1 hour. The reaction was stopped by heating at 95 °C for 5 minutes and subsequently chilled on ice for another 5 minutes. For digestion of RNA RNaseA and RNaseH, 1 U of each was added to the first strand synthesis followed by an incubation at 37 °C for 1,5 hours.
  • test poly(A)0RNA, respectively. Filters were washed two times with 2 x SSC/ 0,1 % SDS at room temperature and another two times with 0,1 x SSC/0,1 3 SDS at 50 °C. Detection by chemilummescence was per- formed according to "The DIG System User's Guide for Filter Hybridization” (Boehrmger) .
  • Bacteria were grown according to Sambrook et al . (1989) and plasmids isolated using the JETSTAR Plasmid Mmiprep Kit (Genomed) .
  • Sequencing was performed using the Thermo Sequenase labeled primer cycle sequencing kit (Amersham) ano Long Ranger Gel Solution (FMC) on a Licor 4200 sequencer (MWG-BIOTECH) .
  • the reactions were set up as follows. 2 pmol primer (5' labeled with IRD-800), 1,0 ⁇ g template DNA, 10 % (v/v) DMSO were made up to 21 ⁇ l with water. To 4,5 ⁇ l primer/template mix 1,5 ⁇ l of A, C, G or T reagent was added.
  • GCTCGCTAGC TAGCTCATAT CGTCCGGCCG TCATGTCAAT GTAATGGAGG 150 GTCATCCATC CAATAAAATT GTGGGCATGT GTTGAGTAAT AAAATTGGTC 200
  • AI622765 Zea mays cDNA
  • TTTCTTCCAT GTTTAGTGGA CCCGCAATGC ATGCATGGAG GTGTATCTTA 250 CGATTGATCG CAATTAATAA AGTGTTTCGG TACGATAGTA GC
  • blastn/dbest (ESTs unknown function )
  • accession Y11348 Medi cago sa ti va an- nexin-like protein responsive to osmotic stress, ab- scisic acid and water deficiency
  • blastn/dbest (EST unknown function)
  • GTCAAGTTCA CGCCTGCCGA ATCGAGGATG ASTGCGCCGC CCCAAAAGCC 50 CCCGCCGGAG AGGAGAAGAA GACGTCATGG CCGGAGGTAG CGGGAAAGTC 100
  • blastn/dbest (EST unknown function)
  • AAATTCAACT AGAGTTTATT tTCTTTtCCt CCAAGTtGTA ATtGGcTTTA 800
  • accession AC002332 Arabi dopsi s tna liana putative calcium-binding EF-hand protein
  • Rapid Amplification of cDNA Ends 5' and 3' ends of the cDNA were obtained by RACE using the MARATHON cDNA amplification kit (Clontech) .
  • RACE Rapid Amplification of cDNA Ends
  • A pooled poiy
  • A pooled poiy
  • the library served as template for the a - plification of 5' and 3' ends using gene specific primers in combination with adaptor primers provided with the kit.
  • Race products were cloneo into pT-Ad , propagated in E . coll TOP10F' and submitted to sequence analysis as described above.
  • RNA-loadmg buffer Formaldehyde (37 %) 260 ⁇ l
  • Fixing solution Methanol 45 % (v/v) Acetic acid 10 % (v/v) in water
  • Transgemc tobacco plants expressing pea chloropiast Nmdh cDNA m sense and antisense orientation Effects on NADP-MDH level, stability of transformants, and plant growth. Plant Physiol. 115, 705-715.
  • Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl "jasmonate, wounding, sugars, light and phosphate. Plant Mol. Biol . 1995, 2, 933-942

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Abstract

This patent application discloses new genes the expression of which is inducible by SAR inducing compounds, and in a particular embodiment by both SAR inducing compounds and ISR inducing compounds. These genes enable the establishment of novel methods for testing chemical compounds and natural products for their capability to induce an enhanced resistance status (ERS), and particularly SAR or ISR, in plants and for screening a multiplicity of chemical compounds, for compounds or compositions having the capability to induce ERS in plants. This disclosure also enables the use of recombinant DNA molecules comprising these DNA molecules in transgenic plants to confer enhanced tolerance to disease and pest organisms.

Description

ERS-GENES, METHOD OF SCREENING FOR CHEMICAL COMPOUNDS CAPABLE OF INDUCING ERS IN PLANTS
BACKGROUND OF THE INVENTION
This invention concerns a method of testing a chemical compound for its capability to induce an enhanced resistance status (ERS) against pathogens in plants, and to the use of said method for screening for chemical compounds having the capability to induce ERS in plants. Under further aspects, the invention pertains to DNA molecules encoding polypept de or parts thereof the expression cf which, within their natural genetic environment, may be induced oy ERS in- ducing compounds and may be assayed in the method according to the invention, transgenic plants, plant parts or seeds comprising said DNA molecules, polypep- tides/proteins or parts thereof encoded by said DNA molecules and antibodies specifically recognizing and binding said polypeptides/proteins or parts thereof. An enhanced resistance status (ERS) in plants is the result of biologically and/or chemically induced resistance phenomena which inter alia include systemic acquired resistance (SAR) and induced systemic resis- tance (ISR) mediated by Rhizobacteria .
In the last decade convincing data have been accumulated that salicylic acid (SA) is essential for the establishment of systemic acquired resistance (SAR) of dicotyledonous plants against a broad range of patho- gens (e.g. Yang et al . , Genes and Development 11,
1621-1639 (1997)) thus leading to an enhanced resistance status (ERS) in those plants.
In contrast to SAR, other plant defence reactions leading to ERS which are summarized under the term ISR, such as resistance mediated bv Rhizobacteria (Ann. Rev. Phytopathology 36, 453-483 (1998)) as well as the ~j asmonate-mediated wound response including ex¬ pression of the protemase inhibitor 1 ana 2 gene, are not inducible oy salicylic aciα or ctner SAR inducing compounds ana are sometimes even innioiteα by sali¬ cylic acid or derivatives thereof (e.g. Doares et ai . , Plant Physiol. 108, 1741-1746 (1995; , .
The US Patent 5,614,395 discloses a method for screening for SAR inducing agrochemicals w th the aid of genetically modified plants containing a chimeπc gene which responds on SAR inducing compounds, which is disclosed as a wheat gene such as WCI-1. This method is limited to those plant species wnich are susceptible for the chimeric gene, n particular to wheat.
The International Patent application WO 98/00023 suggests a method of screening compounds for resistance-inducing activity by the detection of the expression of a plant defensin gene such as PDF 1 . The genes disclosed there are induced by plant patnogens such as Al ternaria brassi ci cola or Botryti s cmera but not by SAR inducing agrochemicals such as salicylic acid or dichloroisonicotimc acid.
Up to now responsiveness on SAR inducing compounds has not been reported for the genes corresponding to the present sequences. Accordingly, there has been no motivation to use naturally occurring expression levels or products of the present genes for screening ERS inducing compounds including SAR inducing compounds .
The generation of synthetic combinatorial libraries is well known for example from EP 0 734 530, EP 0 816 309 and EP 0 818 431. However, up to now it was only possible to screen such libraries in orαer to select compounds with pharmaceutical properties. Tnerefore, it was highly desirable to develop a high tnrough-put screening method which allows to generate lead co - pounαs for tne agricultural research, it particular for ERS inducing compounds .
SUMMARY OF THE INVENTION
The present invention is based on tne most sur- prising finding that new genes exists apart from clas¬ sical pathogenesis-relateα (PR) genes the expression of which is mducible by SAR inducing compounds, and m a particular embodiment by both SAR inducing com¬ pounds and ISR inducing compounds. This finding enables the establishment of novel methods for testing chemical compounds and natural products for their capability to induce an enhanced resistance status (ERS) , and particularly SAR or ISR, in plants and for screening a multiplicity of chemical compounds, for compounds or compositions having the capability to induce ERS m plants. Tne finding may also enable the use of recombmant DNA molecules comprising these DNA molecules m transgenic plants to confer enhanced tolerance to disease and pest organ- isms.
The present invention relates to DNA molecules having the following sequences (A) to (H)
A: (346bp)
ACATGAAGAG CAGCGACGGC AAGGTCTACG ACTCCTTCAC CATCCACAGG 50
GATTACCGCG ACGTGCTCAG CTGCGTGCAC GACAGCTGCT TCCCCACCAC 100
GCTCGCTAGC TAGCTCATAT CGTCCGGCCG TCATGTCAAT GTAATGGAGG 150
GTCATCCATC CAATAAAATT GTGGGCATGT GTTGAGTAAT AAAATTGGTC 200
AGCTGCACAA TTTATATGTG CTAGTAAAAA GATCATGCAA GAGGTGGG G 250
TATGCTCGTT ATATATGCTT TGTAACTCCT TCATGTCATA TTWTTATGGG 300
TTAATAAAAA CATCCTTTAT CAAAAAAAAA AAAAAaAAAA GCTTGT 350
B (292bp)
ACAGTATTGG TTGATATGAT TGCTAATCCG GCCCTAGCTC GCGCAGTAAG 50
GGCATCTCCA ATGATTGTAT GATCATCGTT GGTAATATTG CCACATAGAT 100
GATTTTGATG ACATGACGCC TAATAAAAGA AGAAAGAGAA TGAAATCGTA 150
TGAACTTGAA GCAACGGTTC ACGCACAAGC TCCGAGGCAA AGCATGGTTA 200
TTTCTTCCAT GTTTAGTGGA CCCGCAATGC ATGCATGGAG GTGTATCTTA 250
CGATTGATCG CAATTAATAA AGTGTTTCGG TACGAT GTA GC
C: (387bp)
ACTTATCTTG AGCATGACAT TAGTCAGCAA ACATCCGGCG aTCATCAGAA 50
GaTCTTACTA GCCTATGTGG GCATTCCACG CTACGAAGGT CCAGAGGTTG 100
ATCCCACTAT AGTGACACAT GATGCGAAGG ACCTCTACAA AGCTGGTGAG 150
AAGAAGCTGG GCACAGATGA GAAAACCTTC ATCCGCATCT TCACTGAACG 200
CAGCTGGGCA CACATGGCAG CTGTTGCTTC TGCTTACCAG CACATGTATG 250
MTCGGTCATT ACAGAAGGTT GTGAAGAGTG AAACATCTGG AAACTTTGAA 300
GTTGCTCTGA TaACTATCCT CAGATGTGCT GAGAATCCAG CTAAGTaTTT 350
TGCTAAGGTG TTAAGGAAGT CCATGAAAGG TCTAGGT
E: (512bp)
ACAGAAGCTT GGACAGTTCC ACTCGGAAGC TTCGGTTCGC GCTACCGTTC 50
CCGATGCTCG CCTACCCATT CTACTTGTGG TCAAGGAGTC CAGGGAAGTC 100
AGGCTCGCAT TTCCACCCGA GCAGCGATTT GTTCCAGCCG AACGAGAAGA 150
ACGACATACT GACGTCGACG ACATGCTGGC TTGCCATGGC TGGCCTGCTC 200
GCTGGGCTCA CTGCCGTGAT GGGCCCCCTT CAGATACTCA AGCTCTACGC 250
CGTCCCCTAC TGGATTTTTG TTATGTGGCT GGACTTTGTC ACCTACCTGC 300
ACCACCACGG CCACAACGAC AAGCTTCCCT GGTATCGCGG AAAGGCATGG 350
AGCTATCTGC GCGGGGGCCT GACAACGCTC GACAGGGACT ACGGGTGGCT 400
CAACAACATC CACCACGACA TCGGGACTCA CGTGATCCGC CATCTTTTCC 450
CGCAAATCCC GCATTACCAT CTAGTGGAGG CGACCGAGGC GGCGAACAG3 500
TGCTAGGGAA GT
F: (484bp)
ACAAGCTTTT ττττχτττττ TTTTTGGTGG TAAACAACAA 150
CTGCTTCATA TGAACAACGG GCTTGACAAT CAAAATTCTT CCATATGTTG 200
TTATTATACA AAAAATTGCT TAGAGCCAGA GTGAAATTAC ATCAAAGGCC 250
TTTAAAACTT TGTTATAAAA TCTAGTCTCA AACTCCCCCT CGTCTACAGG 300
TGTTCTCCGA GATATTCTCC CCTTGCCTCC AGTGCGAAAT TTCTGCTATG 350
TCTATGCTCT ATTCACACGG ATGGTTTGTC CTTCCAATTC TGTGTTGTTG 400
AAAGTAGAAA CCACAGCTTC AACTTCCTCC TCTGAAGAAA ACGTGACAAA 450 ACCATATCCC TTGGACTTTG GGGTTCCCGG AATTCGGGAT ACCGTGGCAC 500 TGAGGACCTC CCCCTTTTCA GAGAAGAAGT TCTTGAGAAC TTCCGTCGTC 550 ACTTTCTTCG CAAGATTGCC AACATAAACC TTGT
G: (305bp)
GTCAAGTTCA CGCCTGCCGA ATCGAGGATG ASTGCGCCGC CCCAAAAGCC 50 CCCGCCGGAG AGGAGAAGAA GACGTCATGG CCGGAGGTAG CGGGAAAGTC 100
CATCGAGGAG GCCAAGGAGA TCATCCTTAA GGACATGCCT GAAGCGGA A 150 TCGTCGTCCT CCCAGCCGGC TCGCCGGTGA CCCTCGACTT CAGGACCAAC 200 CGTGTCCGCA TCTTCGTCGA CACTGTCGCG TCCACTCCCC ACATTGGCTA 250 GCTAGCTTTG CAAGCAAAGG CAACATGGAT GCATTGTGGA TGCTGATGAA 300 TAAGT
H (1035bp)
CAGGATCATA GCTACAGGCG ACAATGCCCG GTCATCGACA ATCGCTGGCA 50
GCGGCTTCTC TGAAGCTACC ATCTCTTCTG CTTCTGTGGA TCTTTAGCTG 100
GAACTGGGGG CATGCCGTGG CCAAGTTTGA TCCTGCAAAC ATGACGGAGC 150
TTCAGAAACA TGTCTCCTTT TTCGACCGGA ACAAGGATGG CTTCATCACT 200
CCTACAGAAA CCATCCAAGG GTTTGTTGCA ATCGGTTGCG AGTATGCATT 250
TGCTACTGCT GCCTCTGCCG CCATTCACGG TGCCCTTGCT CCTCAAAC.AA 300
CCCCGGCTGG TACACCACTG CCTCACTTGA CAATATACGT AGAGAATATC 350
CACAAAGCTA TGCATGGAAG TGATCCAGGT GTATATGATG CCAAAGGAAG 400
GTTTCTTCCC CAAAACTTTG AGGAATTATT CAAAACATAT GCAATACTCC 450
GACCAGATGC GTTGACTCTT GCGGAGATGC ATGTGATGCT CTTTGCAAAA 500
CGGGATCTAG ACCCTATATC ATGGGCACCA CCACAGGTTG AGTGGGGCCT 550
ATTATTCACG CTTGCAAGCG ATTGGCTTGG GTTCCTTCAC AAAGACAGTG 600
TTAGAGGTAT ATATGATGGA AGCCTGTTTA TCAAGTTGGA AAAGAAATGG 650
CACCCTTTTC AAAGTGCTAT GCGATGAACT TGGTGCTAGT TTAGAGTGAG 700
AGTTTGGATA TGGAAAGGTT TGTCCCGAAG AAGGTTTTCC TGCTATCTCC 750
AAATTCAACT AGAGTTTATT TTCTTTTCCT CCAAGTTGTA ATTGGTTTTA 800
TAAGACCTTC ATAGCCGATC AATACAACGA AGCAAGTTGG ATATATTTCC 850
CGACCTTGTA TTCTCTCTCA TGKGCCCCTT ATTATGTTTG CGCCATG GC 900
GCCTACCCAA GAKGAGCCAT AAGCATAAGG CTCATCCACC TATTGGCCAC 950
GACTACTGTT GGAAATATTC CCTACAKGCA ATATTGKG K GAWAAWATTT 1000
ATCTATAAAA AAAAAAAAAA AAAAAAAAAA AAAAA
encoding polypeptides or parts thereof the expression of which may be induced by ERS inducing compounds and a method of testing a chemical compound for its capability to induce an enhanced resistance status (ERS) in plants said method comprising: (a) contacting a plant or plant part comprising a gene fragment having the sequence (A) to (K) coding for a polypeptide/protein or part thereof the expres- sion of wnicn being inducible Py SAR inducing com¬ pounds, wit" the chemical compound; and (b) assaying the expression of said gene fragment, wherein tne expression of said gene fragment indicates that the chemical compound has the capability to in¬ duce ERS.
In a particular embodiment, tne expression of tne said gene fragment (A) to (H) the expression of nich being inducible by SAR inducing compounds is not in¬ ducible by plant pathogens. In a further preferred embodiment, the expression of the said gene fragment is additionally inducible also by ISR inducing compounds.
Furthermore, this method according to the present invention allows to screen ERS inducing compounds in different plant species as for example cereals such as wheat or barley, and rice; to determine the presence of ERS-inducmg compounds in a sample; to determine the concentration of an ERS-inducmg compound in a sample; to compare two ERS- inducing compounds for their relative capabilities to induce ERS m plants; and to test compounds and compositions for their capa¬ bility to change the ERS inducing effect of a chemical compound. A further aspect of the present invention relates to a kit for use m the methods of the present inven¬ tion.
Another aspect of the present invention relates to a process for providing compounds having the capa- bility of inducing ERS m plants which comprises the steps of
(a) generating a synthetic combinatorial library of different chemical compounds, and
(b) screening the compounds of said library with the screening method according to this invention. Further aspects of the invention are tne novel compounds wnich are obtainable with tne aid of this metncd and tne use thereof as agrochemicals.
The invention pertains to DNA molecules encoding a polypeptide/protem tne expression of which may be assayed m the methods according to the invention, fragments of said DNA molecules which i.a. might be used as prooes or primers, transformed microorganisms and transgenic plants, plant parts or seeds comprising said DNA molecules, polypeptides and proteins encoded by said DNA molecules and antibodies specifically recognizing and binding said polypeptides or proteins.
These and other objects and features of the invention will become apparent from the detailed de- scription set forth herein below.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It has now been found that the capability of chemi- cal compounds to induce ERS m plants may be efficiently tested by
(a) contacting a plant or plant part comprising a gene fragment (A) to (H) according to tne invention coding for a polypeptide/protem or part tnereof the expression of which being inducible by SAR inducing compounds, and in particular both by SAR inducing compounds and ISR inducing compounds, with the chemical compound; and
(b) assaying the expression of said gene fragment. By use of said method, a multiplicity of chemical compounds and compositions may be screeneα to identify compounds and/or compositions having the capability to induce ERS in plants. In a particular emoodiment said multiplicity of chemical compounds or compositions may be provided, for example, form of a combinatorial library such as exemplified EP 0
Figure imgf000009_0001
53u, Ξ? o o_ 309 and EP 0 818 431, or in form of a -iOr r_ or col¬ lection of natural compounαs and/or compositions or a library comprising both natural ana synthetic com- pounds and compositions.
Alternatively, the said method may aiso oe usec for determining the presence of compounds capable of inducing ERS in plants m a sample, wherein m the above step (a) the plant or plant part is contacted with an aliquot of the sample place of tne chemica. compound. The expression of the said gene fragment then indicates the presence of one or more compounds capable of inducing ERS m plants m tne sample.
In a particularly preferred embodiment tne expres- sion of the said gene fragment in the said plant wh-.cn is inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds, is not inducible by naturally occurring plant pathogens. This feature appears to be particularly valuable if it is intended to perform the methods of the present invention in an environment where plant pathogens may be present. This will be the case when using plants in a green house or on the field for per¬ forming the methods of the present invention. In tnese cases, said specific feature will ensure that any expression of the said gene fragment observed will only be attributable to the presence of a compound or composition having the capability to induce ERS m plants. If working the method according to the mven- tion in a cell or tissue culture system, however, said specific feature may be utilized but it is net essen¬ tial.
In the present context, the phrase "naturally occurring plant pathogens" includes all kinds of phyto- pathogenic microorganisms, in particular phytopatnc- genic fungi, bacteria and viruses.
The phrases "plants" and "plant parts" as used heremabove and nerembelow include all kmcs of plants and plant parts, m particular foliage, roots and seeds. Preferably the said plant or plant part is selected from the group consisting of a plant proto¬ plast, a plant cell, a plant tissue, callus, a developing plantlet, a plant leaf, an immature r.oie p.ant, a mature whole plant and seed. In particular, tne said plant or plant part, in particular plant ceil or plant leaf, is or is derived from a onocotyledonous plant, such as a cereal, particularly wheat or a plant of the genus Hordeuτv such latter plants being particularly preferred, rice as well as from dicotyledonous plants. The gene fragment of step (a) may be any gene fragment having either the sequence (A) to (H) or part thereof, the expression of which being inducible υy SAR inducing compounds, and preferably both by SAR m- ducing compounds and ISR inducing compounds. This definition comprises any naturally occurring gene fragments exhibiting these characteristics as well as not-naturally occurring gene fragments, the sequence of which has substantial homology with the sequence of any of said naturally occurring gene fragments, vari¬ ants thereof as well as gene fragments comprising one or more portions of the aforementioned gene fragments and variants wherein all these gene fragments and variants maintain the characteristic indicated above, i.e. an expression which is inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds.
As already mentioned above, in a particular emoooi- ent of the invention, the expression of the said gene fragment or variant which is inducible by SAR inducing compounds, and preferably both by SAP, inducing com¬ pounds and ISR inducing compounds, is not inducible by naturally occurring plant pathogens.
In an alternative embodiment, the said gene fragment (A) to (H) is a gene fragment, the nucleotide sequence of which exhibits substantial homology with the nucleotide sequence (A) to :H), or a variant thereof or comprises one or more portions of the nucleotide sequence given above, of a sequence having substantial homology thereto or a variant thereof. Accordingly, the term also includes sequences that can substan¬ tially hybridize to the nucleotide sequence given above, in particular under conditions of low strin- gency.
The phrase "variant thereof" as used hereinabove and hereinbelow means any substitution, variation, modification, insertion, deletion or addition of one or more nucleotides from or to the nucleotide sequence of a naturally occurring gene fragment (A) to (H) the expression of which being inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds, for example a gene fragment comprising the nucleotide sequence given above, provided that the expression of the variant being still inducible by application of SAR inducing compounds, and preferably both of SAR inducing compounds and ISR inducing compounds. The phrase particularly includes allelic variants of naturally occurring gene fragments (A) to (H) the expression of which being inducible by SAR inducing compounds, and prefera¬ bly both by SAR inducing compounds and ISR inducing compounds. The phrase also includes synthetic variants comprising or essentially consisting of nucleotide se- quences that can substantially hybridize to the nu- cleotioe sequence of tne naturally occurring parent gene fragment. . Preferably, sucn nybridisation occurs at low-stringency conditions, m a further embodiment also between low and high stringency conditions, and, m a very particular embodiment, even at hign stringency conditions. As a rule low stringency conditions can be defined as 3 x SSC at ambient temperature to 65°C and high stringency conditions as C.l x SSC at 68°C. SSC is the abbreviation of a 0.15>1 soαium cn.o- ride, 0.015M trisodium citrate puffer.
The phrase "substantial nomology" as used nere - above and herembelow embraces nomology with respect to at least the essential nucleotioe/s of tne parent gene sequence (A) to (H) , provided that tne expression thereof still being inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds. Typically, homology is shown when 60 % or more of the nucleotides are common with the naturally occurring parent gene fragment, more typically 65 % , preferably 70 %, more preferably 75 % , even more preferably 80 or 85 % and particularly preferred are 90 %, 95 %, 98 % or 99 % or more homology.
In a particular embodiment of the present method, the gene fragment (A) to (H) the expression of which being inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds, further comprises a nucleotide sequence coding for an indicator protein or polypeptide fused to the coding region of a gene corresponding to either DNA molecule of (A) to (H) . Consequently, also the ex¬ pression of the indicator protein or polypeptide is inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing com- pounds, and, upon application of ERS inducing com- pounds, occurs concomitantly witi tne expression of the DNA molecule of (A; to (H) form of a fusion protein. In specific embodiments said nucleotide se¬ quence coding for an indicator protein or polypeptide codes for a glucuronidase enzyme, luciferase or for the green fluorescent protein (GFP) . Additionally, indicator proteins could be enzymes conferring toler¬ ance to herbicides sucn as acetolactate syntnase,
The SAR inducing compounds and ISR inducing corn- pounds which may be assayed with the methods according to the invention, may be summarized under tne generic term "ERS inducing compounds". This term includes all compounds being capable to provide a plant ήitn im¬ proved resistance characteristics against a_l types of pests, for example against harmful microorganisms, like phytopathogemc fungi, bacteria and viruses, as well as, pests such as insects mites and nematodes . As already stated, it includes l.a. all compounds being capable to induce systemic acquired resistance (SAR) and induced systemic resistance (ISR) including defense mechanisms independent of salicylic acid in plants. ERS inducing compounds induce m the plants a resistance status by activating, for example, cell death reactions (HR) , cell wall appositions, PR-gene expression and/or phytoalexm accumulation.
The application of these compounds leads to a protection of the treated plants from serious infection by harmful microorganisms, for example phytopathogemc fungi, bacteria and viruses and protection against m- sects mites and nematodes.
SAR inducing cnemical compounds include Denzoic acid, salicylic acid, dichloroisonicotmic acid, poly- acrylic acid, am obutyπc acid, arachidonic acid and derivatives thereof and natural products such as har- p and other elicitors. Another group of SAR inducing cnemical compounds are benzo-1 , 2 , 3-thιadιazole derivatives as for example disclosed by US Patents US 4,931,581 and US 5,229,384. The reference compound of the IS inducing chemica. compounds is represented py jasmon c acid and deriva¬ tives thereof such as methyl asmonate.
The known ERS inducing compounds are preferably used as reference compounds for the screening of novel agrochemical compounds. Other chemical compounds, m particular agrochemicals, encompassed by the present invention, in par¬ ticular individual members of a synthetic combinato¬ rial library, can be determined easι_y by assaying tne test chemical with the aid of the metnods according to this invention.
The ERS inducing chemical compounds may be applied pure form, m solution or suspension, as powders or dusts, or other conventional formulations used agriculturally. Such formulations may include solid or liquid carriers, that is, materials with which said ERS inducing chemical compound is comoined to facilitate application to the plant, tissue, cell or tissue culture, or to improve storage, handling or transport properties. Examples of suitable carriers include silicates, clays, resins, alcohols, ketones, aliphatic or aromatic hydrocarbons, and the like. If formulated as conventional wettable powder, aqueous suspension or emulsion concentrate, the formulation of tne ERS inducing chemical compound may include one or more con- ventional surfactants, either ionic or non-ionic, such as wetting agents, emulsifying or dispersing agents. The ERS inducing chemical compounds singly or combinations may also be applied combination with another agent, for example with an adjuvant, a herbi- cide, a fungicide, an insecticide, a grovvtn regulator or a fertilizer.
As a liquid formulation tne ERS lnαucmg cnem_ca_ compound may be applied as a spray to
Figure imgf000015_0001
.eaves, stems or branches or to seeds before pla-tirg or to soil or other growing or cultivation meα_uir
Tne ERS inducing cheϋiic-.-. compound _s applied in an amount and over a period of time sufficient to induce ERS effects. If applied to whole plants, as a ruie, the chemicals are applied a concentration of 0.1 to 1000 g active ingredient per liter of soil volume a so l drench process or, alternatively, as a spray a concentration of 0.1 - 100 mg active ingredient per liter of spray solution. If working in a cell or tis- sue culture system, also lower concentrations, now based on the volume of the culture medium, may be used. As a guideline 2 to 72 hours, in particular 12 to 48 hours are sufficient to detect a response.
In principle every conventional method or assaying the expression of genes can be used to monitor the response of the plant on the action of the chemical compound, such as Western blot or Nortnern plot analysis, detection or monitoring of the expression of an mdi- cator protein or polypeptide the gene thereof being included said gene fragment the expression of which being inducible by a SAR inducing compound, e.g. by detection of an enzymatic activity thereor . If tne expression which has to be detected occurs tissue- specific, the analysis will be performed on said spe¬ cific tissue.
If there exists a constitutive low level expression of the said gene fragment or of a part of the said gene fragment which will enable the detection of the expression thereof in the plant or plant part in the absence of the chemical compound, the expression in¬ duced by the chemical compound will be determined as an increase m expression compared to said constitu¬ tive low level expression. This assaying variant is also encompassed by step < c , of the metnoα according to the invention.
Preferably, the ana-.ys-.s of the expression or tne said gene fragment (A) to (H) is carried out using a Northern blot type analysis, m particular with di- goxigenm labelled cDNAs of the gene fragment, e.g. a DNA with a nucleotide sequence as given (A) to (h , or fragments thereof, most preferred using a commercially available Northern blot kit sucn as the DIG- High Prime labelling Kit of Boenrmger Mannneiir, Ger- many.
In another preferred embodiment of tne present invention the analysis of the expression of the said gene fragment is carried out using reverse transcrip- tase-poly erase chain reaction (RT-PCR) . In another preferred embodiment of tne present invention the analysis of the expression or tne said gene fragment is carried out using antioodies to detect the gene product of tne said gene fragment, or a specific indicator polypeptide or protein the gene se- quence of which representing a part of the said gene fragment, e.g. by performing a Western blot type analysis .
If the detection of the expression of the said gene fragment is based on the detection of the expression of an "additional" nucleotide sequence comprised within the said gene fragment (A) to (H) and encoding an indicator protein or polypeptide, the detection may be performed also by other means than by use of nucleotide probes (e.g. for a Northern-blotting assay) or antibodies (e.g. for a Western-blotting assay), αe- pending on the type of indicator protein or polypeptide produced. The indicator protein or polypeptide may be a fluorescent or cnemilummescent protein or polypeptide, such as the green fluorescent protein (GFP) , thus enabling the detection of expression or the said gene fragment oy detection of fluorescence or chemilummescence of the said plant cells. Alternatively, the indicator protein or polypeptide may also exhibit an enzymatic activity not naturally occurring in the plant or plant part used for tne metnod of the invention. Accordingly, this enzymatic activity may be assayed for detection of the expression of tne indicator protein or polypeptide ana, accordingly, of the said gene fragment. The assay may be oaseo en the measuring of the disappearance of a substrate for the enzyme from an assay medium or on the measuring of the formation of a product by the enzymatic conversion. In a particular embodiment, said product of the enzymatic conversion may be a compound which, as a result of the enzymatic conversion, is coloured. An example for an indicator protein with an enzymatic activity, hicn usually will not be found in plants, is the glucuroni¬ dase enzyme. Additionally, the indicator protein could perform an enzymatic activity that for instance provides tolerance to another chemical agent such as a herbicide or phytotoxic compound. In such case, assaying for the expression is done m the presence of the herbicidal or phytotoxic compound and expression is scored based on tolerance to the chemical agent.
The assaying step (b) for assaying the expression of the said gene fragment (A) to (H) may be carried out m a way to produce a result which enables the mere determi- nation whetner or not an expression na≤ occurred; to enable the determination which of two or mon samples shows a higher degree of expression of the said gene fragment compared to the other (s; ("semi- quantitative determination"); to provide quantitative results as to tne leve. expression occurred ( "quantatitve determinatioi")
If care is taken to perform the assaying step ID) with identical or substantially identical amounts of nucleotide or protein material prepared from the step (a treated plants or plant parts, thus enabling a semi-quantitative determination or even quantitative determination of the level of expression or tne sα. gene fragment, tne method according to tne invention may also be used for determining the concentration of a chemical compound which is capable of inducing ERS m plants m a sample; for determining whether a chemical compound or composition exhibits a stronger ERS inducing effect than an ERS inducing reference compound; and for determining whether and, optionally, to what degree a compound or composition is capable of changing the ERS inducing effect of a chemical compound.
The method for determining the concentration of a chemical compound which is capable of inducing ERS in plants a sample, is characterized that it com¬ prises the following steps:
(l) performing the method according to the invention comprising steps (a) and (b) as outlined above wherein step (a) the plant or plant part is contacted witn an aliquot of the sample;
(n) determining the concentration of the ERS inducing chemical compound in the sample by comparing the result from step (I) with the results of corresponding tests performed with known concentrations of saio chemical compound.
Tne method for determining wnether a chemical compound exhibits a stronger ERS inducing effect tnan an ERS inducing reference compound, sucn as saiicylate, dichloroisonicot ic acid or asmonic acio, is characterized in that it comprises the following steps: (l) performing the method according to the invention comprising steps (a) and (b) as outlined above wherein step (a) the plant or plant part is contacted with a specific concentration of the chemical compound; (li) performing tne method according to the -.nventior1 comprising steps (a) and (cj as O tlined above wrere.n m step (a) tne plant or plant part is contacted with an amount of the ERS inducing reference compound resulting in an equimolar concentration compared to the concentration of the chemical compound step (l); (m) determining the relative level of the ERS inducing effect of the chemical compound by comparing the results from steps (l) and (n) .
Since any step (b) will be carried out using substan¬ tially equal amounts of nucleotide or protein material prepared from step (a) treated plant or plant part material for the assay, step (m) will produce a semi- quantitative result m order to enable tne determina¬ tion which one of the chemical compound and the ERS inducing reference compound induces a higher expression of the said gene fragment and correspondingly will produce a stronger induction of ERS m a plant. Alternatively, the results may also be evaluated to enable a quantitative assessment of the relative degree of expression induced by the chemical compound compared to the ERS inducing reference compound. This evaluation may be performed for example by scanning of the analytical gels or films, photograpns or print- outs thereof by means of a densitometer or py analyzing relevant excised sections of analytical gels used for separating assay mixtures comprising radioactively labelled detection compounds (e.g. nucleotide probes or antibodies) m a liquid scintillation counter.
The method for determining whether and, optionally, to what degree a compound or composition is ca¬ pable of changing the ERS inducing effect of an agro- chemical is characterized that it comprises the following steps:
(l) performing the metnod according to the invention comprising steps (a) and (b) as outlined above wherein in step (a) the plant or plant part is contacted witn a specific concentration of tne compound or compos - tion;
(n) performing the method according to the invention comprising steps (a) and (b) as outlined above wherein in step (a) the plant or plant part is contacted with the same specific concentration of the agrochemical as m step (l) in the presence of the compound or compo¬ sition;
(m) determining the capability of the compound or composition for changing the ERS inducing effect of the compound or composition by comparing the results from steps (l) and (n) .
Since also in this method any step (b) will be carried out using substantially equal amounts of nucleotide or protein material prepared from step (a) treated plant or plant part material for the assay, step (m) will produce a semi-quantitative result in order to enaoie the determination whether the compound or composition leads to an enhancement, reduction or even inhibition of the expression of the said gene fragment and correspondingly will produce a stronger or reduced m- duction of ERS m a plant or will even abolish the - duction of ERS. Alternatively, the results may be evaluated to enable a quantitative assessment of the relative degree of expression induced by a combination of the agrocnemicai and tne compound or composition compared to the agrocnemicai alone. This evaluation may be performed for example by scanning of tne analytical gels or films, photographs or pr t-outs thereof by means of a densitometer or by analyzing relevant excised sections of analytical gels used for separating assay mixtures comprising radioactively labelled detection compounds (e.g. nucleotide probes or antibodies) in a liquid scintillation counter.
The method for determining whether and, optionally, to what degree a compound or composition is ca- pable of changing the ERS inducing effect of an agro- chemical may further be utilized in a method for screening for compounds or compositions capable of changing the ERS inducing effect of an agrochemical .
The invention further pertains to a kit for use the methods according to the present invention. Said kits are characterized in that they comprise the fol¬ lowing components :
(a) one or more plants or plant parts comprising a gene fragment (A) to (H) the expression of which being inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds ; and
(b) means for detecting the expression of the said gene fragment.
In a particular embodiment the kit may comprise one or more plants or plant parts wherein the expression of the said gene fragment (A) to (H) comprised therein is not inducible by plant pathogens. In a further par- ticular embodiment the kit may comprise one or more plants or plant parts wherein the saio gene fragment additionally comprises a nucleotide sequence encoding an indicator protein or polypeptide.
If the kit comprises one or more plant parts, such as plant cells, said plant parts may ce provided immobilized on a solid support. Suitable supports may l.α. be made of glass, specific po sacchariαes, like aga- rose, or specific plastic materials sucn as polyacry-- amide, polystyrene, polyvinylalcoho-. , silicones. Meth- ods and techniques for immooilizmg cells and tissue on such types of supports are within tne skills of the skilled scientist. In a specific embodiment, in par¬ ticular for the screening processes according to the present invention, the kit may comprise plates, such as microtiter plates, comprising a multiplicity of reaction wells which contain the said plant parts, in particular plant cells, preferably immobilized on the bottom and/or the walls of the wells.
The means for detecting the expression of the said gene fragment (A) to (H) may comprise one or more nucleotide probe (s) such as detectaoly lapelled cDNAs or cDNA fragments of the said gene fragment. The detectable label may be e.g. a fluorescent, cnemilummescent or radioactive label, and is m particular a digoxi- genin molecule. Alternatively, for use m ELISA or Western Blot type analyses, the means for detecting the expression of the said gene fragment will comprise an antibody which specifically recognizes and binds to the or a gene product of the said gene fragment. This antibody will carry a detectable label, e.g. a fluorescent or chemiluminescent dye, a radioactive label or an enzymatically active moiety or structure or will be detectable by use of a secondary antibody specific for said first antibody which may also be provided, separately from said first antibody, in the kit. For assaying an enzymatic activity of an indicator protein or polypeptide in the case where tne saiα gene rrag- ment the expression of whicn being mduciole by ERS inducing compounds further comprises a nucleotide se- quence coding for a respective indicator protein or polypeptide, the means for detecting the expression of the said gene fragment will comprise one or more reagents for assaying the enzymatic activity of tne indicator protein or polypeptide including e.g. a sue- strate for tne indicator protein, optionally m comoi- nation with one or more necessary cofactor(s), and possibly reagents for detecting the product from the enzymatic conversion of the substrate.
Furthermore, said means may comprise one or more buffers for the assay reaction (s) and/or reagents for determining and, in particular, visualization of the assay result (e.g. in the case of detection of the enzymatic activity attached to the antibody) , which may be provided separately from each other and from the probes or antibodies or combined form or m form of combinations of some of said components.
Optionally, the kit may comprise further components, m particular as separate components, such as one or more buffers for incubating the test compound or composition with a plant part, agents for breaking up the cellular structure of the plant or plant part for enabling the reactions for detection of the expression of the said gene fragment or instructions for
Figure imgf000023_0001
Furthermore, the invention pertains to a process for providing chemical compounds having tne capability of inducing ERS in plants which comprises the steps of
(a) generating a synthetic combinatorial library of chemical compounds, and (b) screening tne compounds of said Iicrary witn the method of testing a chemical compound for its ca¬ pability to induce ERS m plants according to the in¬ vention comprising steps (a) and (b) as outlined above. The invention also covers the new compounds having the capability of inducing ERS which are obtainable by this method and the use thereof as agro- chemical compounds, and m particular for tne induction of ERS in plants. With respect to the formulation and application of the new agrocnemicai compounds for use as an agrochemicai , reference is maαe to tne rele¬ vant explanations given earlier m this specification.
Under a further aspect, the invention is directed to an isolated DNA molecule which codes for a protein or polypeptide or part thereof, the expression of which being inducible by SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds, if said DNA molecule is situated within its natural genetic environment, or a nucleotide sequence complementary thereto.
In a particular embodiment of the present invention the isolated DNA molecule comprises either of the nucleotide sequence (A) to (H),or a nucleotide se- quence complementary thereto.
The expression of these DNA molecules may be as¬ sayed in the method according to the invention.
The invention further pertains to isolated DNA molecules the nucleotide sequence of which having sub- stantial homology with the nucleotide sequence as given above, m particular hybridizes with the latter, or is a variant of the DNA molecule witn the nucleotide sequence as given above or is related to said DNA molecule by mutation. With respect to the meaning of the terms "substantial homology" and "variant" refer- ence is maαe to tne explanations given earlier in this specification. In this context, specif _c embodiments are allelic variants to the DNA molecule having tne nucleotide sequence (A) to (H) as well as DNA mole- cules having a nucleotide sequence which hyoridizes tc the nucleotide sequence given m (A) to (K . As already outlined above, preferably, such hybridization occurs at low stringency conditions, m a furtner embodiment between low and high stringency conditions, and a very particular embodiment even at nigh stringency conditions. As a rule low stringency condi¬ tions can be defined as 3 x SSC at ambient temperature to 65°C and high stringency conditions as 3.1 x SSC at 68°C, SSC being tne abbreviation of a 0.15M sodium chloride, 0.015M trisodium citrate buffer. A further specific embodiment are DNA molecules having a nucleotide sequence which not necessarily hybridizes with one of the nucleotide sequences (A) to (H) , respectively, but, due to the degeneration of the genetic code, encodes the identical ammo acid sequence as given in sequence of (A) to (H) .
In a specific embodiment a DNA molecule encoding a prote /polypeptide or parts thereof tne expression of which, m its natural genetic environment within a cell, being inducible by SAR inducing compounds, ano preferably both by SAR inducing compounds and ISR in¬ ducing compounds, is derived from the genome of a plant and may be identified and isolated by use of nucleotide hybridization probes consisting of or com- prising fragments of the DNA molecule having the nucleotide sequence (A) to (H) . Preferred fragments of the said DNA molecules for screening of genes in other plants than Hordeum vul gare L . , and m particular m other plants of the genus Hordeum, further monocotyle- donous and also dicotyledonous plants comprise at least 15 nucleotiαes, typically between 15 dnα ό ^ nu- cleotides, and more preferably at least 25 nuciec- tides, but may comprise, if necessary, even a nigner number of nucleotides such as 50 or more nucieotides. Accordingly, fragments of the DNA molecules of the present invention, and m particular of the isolated DNA molecule having the above given nucleotide se¬ quence are also covered by the present invention as well as the use thereof as hybridization probes, par- ticularly screening probes, or primers, in particular sequencing or PCR primers. The invention also covers fragments which are shorter than mentioned above, e.g. comprising Between 8 and 15 nucleotides, in particular if they have utility as nucleotide probes or primers. Especially for use as nucleotide probes, DNA molecules comprising or consisting of one or more of the fragments according to the present invention are employed in labeled form, e.g. radioactively labeled or labeled by attachment of non-radioactive reporter or indicator molecules, such as digoxigenm molecule (s) . Particularly for use as PCR primers, DNA molecules comprising or essentially consisting of one or more of the frag¬ ments according to the present invention are employed m a form which comprises modified nucleotide se- quences within the sequence (s) of the inventive fragment (s) which provide for appropriate restriction sites within said primers and any amplified products after the PCR which facilitate cloning of said amplified products. Despite these nucleotide sequence modi- fications the PCR primers, under the PCR conditions, will still hybridize specifically with tne target DNA molecule according to the present invention.
DNA fragments according to the present invention may be prepared, for example, on the basis of the nu- cleotide sequences (A) to (H) , possibly by use of ap- propriate restriction nucleases, or py cnemica-. syn¬ thesis. Various suitable techniques for preparing DNø fragments and DNA molecules comprising one or more of these fragments according to the present invention are known to the skilled scientist.
Having identified a corresponding gene m a different plant species p use of the nuc±eotioe fragments or DNA molecules according to the invention as screening probes, it may be interesting to determine whether the expression thereof being inducible by ERS inducing compounds, in particular SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds. This may oe performed, e.g., as outlined m the Examples, by cultivating the plant or plant parts m the presence of an ERS inducing compound, and analyzing said plant or plant parts then, for example, by Northern-blot or RNA dot blot analysis by use of hybridization probes specific for nucleotide fragments (A) top (H) used for identification of said gene, comparing the results with the results from a cultivation of said plant or plant parts without the ERS inducing compound. Plants identified by the described method to comprise a gene being inducible by ERS inducing compounds, in particular SAR inducing compounds, and preferably both by SAR inducing compounds and ISR inducing compounds, may tnen be used the methods according to the present invention.
In a further particular embodiment the invention pertains to an isolated DNA molecule whicn comprises a DNA molecule or fragment as defined above fused with a nucleotide sequence coding for an indicator protein or polypeptide. In a preferred embodiment the complete encoded information of the said DNA molecule will be expressed if expressibly linked to a single expression control sequence. The indicator protein or polypeptide encoded by this further nucleotide sequence portion of the DNA molecule is a protein or polypeptide unich is easily detectable, e.g. by use of specific nucleotide hybridization probes, by use of specific ant.Dodies, or due to specific characteristics of tne protein or polypeptide itself like fluorescence or enzymatic activity. In specific emoodiments said further nucleotide sequence portion of tne DNA molecule may code for glucuronidase or the green fluorescent protein or a modified form of the plant AHAS enzyme. Tne isolation and cloning of appropriate genes for indicator poly¬ peptides or proteins as well as the production of the recombmant chimeric DNA molecules defined aoove are well within the skills of a skilled scientist. The invention also covers DNA constructs which comprise one or more of the DNA molecules and/or fragments defined above, and m particular DNA constructs serving as nucleotide probes or PCR primers, as well as constructs comprising the DNA molecule (s) and/or fragment (s) under the control of an expression control sequence, i.e. an expression control sequence opera- tively linked to said DNA molecule (s) and/or frag¬ ment (s) .
Especially for use as nucleotide probes, DNA mole- cules comprising or consisting of one or more of the DNA molecules and fragments according to the present invention will be employed a labeled form, wherein said labelling may be achieved by attachment of non- radioactive reporter or indicator molecules such as fluorescent or phosphorescent molecules, digoxigenm molecule (s), biotm molecule (s) or derivatives thereof.
Especially for use as PCR primers, DNA constructs comprising or essentially consisting of one or more of the DNA molecules and fragments according to the pres- ent invention are employed in a form which comprises additional nucleotide sequences or modified nucleotide sequences within the sequence (s) of the inventive fragment (s) which provide for appropriate restriction sites within said primers and any amplified products after the PCR which facilitate cloning of said ampli¬ fied products. Despite these nucleotide sequence modifications the PCR primers, under the PCR conditions, will still hybridize specifically witn the target DNA molecule according to the present invention. In a further particular embodiment the DNA construct further comprises an expression control sequence wmch is operatively linked to said DNA molecule (s) and/or fragment (s) . The invention further relates to vectors, plasmids, cosmids, viral and phage genomes comprising one or more of the DNA molecules and fragments according to the present invention.
The invention also pertains to DNA molecules com- prising a complementary sequence to the DNA molecules, fragments and constructs according to the present in¬ vention as well as the RNA transcription products of these DNA molecules, fragments and constructs.
For the isolation, construction, synthesis and modification of the DNA molecules, fragments and con¬ structs according to the present invention various methods and techniques are known to the skilled scientist and are, e.g., described m Sambrook et al . (1989) , Ausebel et al ..
Furthermore, the invention pertains to polypep- tides/proteins and parts thereof encoded by the DNA molecules, fragments and constructs according to the present invention, proteins having or comprising an ammo acid sequence exhibiting a homology of at least 65%, typically at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% and particularly preferred at least 95. to the ammo acid sequence corresponding to DNA sequences (A) to (H) , as well as polypeptide fragments of said proteins with the proviso that tne ammo acid sequences of those fragments exhibit a nomology of at least 65%, typically at least 75--, preferably at least 80%, more preferably at least 85%, even more prefera- bly at least 90% and particularly preferred at least 95% to a corresponding "reference" fragment of tne polypeptide corresponding to DAN sequences \Aι to (H) . In the present context, the term "polypeptide" in¬ cludes molecules which typically comprise at least 18 ammo acids, more particularly at least 25 ammo acids and particularly preferred more than 40 ammo acids.
In a particular embodiment of the invention, the polypeptides and proteins are fusion proteins of the proteins or parts thereof coded by the DNA molecules according to the invention, with an indicator protein or polypeptide as already outlined above. In another embodiment, polypeptides/protems or parts thereof are provided which comprise the polypeptides/protems or parts thereof described m the last two paragraphs. These polypeptides/protems or parts tnereof may be obtained by cultivating a host organism transformed with one or more of said DNA molecules and optionally purifying these polypeptides and proteins py methods known to the art. Alternatively, these polypeptides and proteins may l.a. also be prepared synthetically by use of known peptide and protein synthesizing techniques .
The polypeptides and proteins of the invention may be utilized for the preparation of monoclonal or poly- cional antibodies or, if they exhibit enzymatic activ- lty, to utilize said enzymatic activity for various purposes .
Under a further aspect, the invention also covers monoclonal or polyclonal antibodies or antisera wnicn specifically recognize and bind a polypeptide or protein according to tne present invention. Corresponding antibodies may be prepared by use of the polypeptides or proteins according to the present invention for lm- munization of a host according to techniques Known to the skilled scientist.
Furthermore, the invention includes transformed mi¬ croorganisms, such as bacteria, oacte iophages, vi- ruses, eucaryotic organisms like fungi, yeasts, proto- zoae, algae and human, animal and plant cells, which comprise a recombmant DNA sequence comprising at least one of the DNA molecules, fragments or constructs of the present invention. These transformed microorganisms may, l.a., be used as an expression system for producing the gene product (s) of the DNA molecules, fragments or constructs of the present invention. Typical microorganisms which are used for this purpose are bacteria, like E. coll , or yeasts such as Saccharomyces cerevi siae . Other types of transformed microorganisms according to the present invention, such as agrobacteria, like AgroDac t en urr t umefaci ens, may be used for the transformation of plants with the inventive DNA molecules, fragments or constructs, thus providing transgenic plants.
Methods for transformation of microorganism cells with the DNA molecules, fragments or constructs of the present invention are well known to tne skilled scientist, including the construction of expression vectors comprising the DNA sequences of the invention under the control of a constitutive or inducible promoter. The promoter may also enable a tissue-specific expres¬ sion of the encoded information in particular compart¬ ments of an organism, such as a plant.
Finally, the invention pertains to transgemc plants, plant parts or seeds comprising a recombinant DNA sequence which comprises one or more DNA molecule (s) according to the present invention. The terms "plants" and "plant parts" include all of a plant propagation material such as a plant protoplast, a plant cell, a plant tissue, callus, a developing plantlet, a plant leaf, an immature whole plant and a mature whole plant. The transgemc plants, plant parts or seed may contain the said recombinant DNA sequence integrated into the genome thereof or extrachromoso- mally located. Various techiques and methods for the cloning of appropriate genes or gene fragments, construction of appropriate transformation vectors and the introduction of the genetic information contained in the vector into plant cells are known to the skilled scientist and enable the production of trans- genic plants, plant parts or seeds according to the present invention. The techniques for introduction of genetic information into plant cells comprise a direct DNA transfer (e.g. into protoplasts by means of elec- troporation or by application of a high molecular weight osmotic agent as well as by biolistie methods wherein DNA-coated particles are shot into plant tis- sue), as well as the use of natural host/vector systems (e.g. of agrobacteria or plant viruses; . For ex- trachromosomal maintenance of the recombinant DNA sequences, various specific viruses like tobacco mosaic virus (TMV) or potato virus X in the genome of which said recombinant DNA sequences then oemg inserted, may be utilized.
Exemplary plants for integration of tne said recombinant DNA sequences of tne invention include dicoty- ledonous plants as well as monocoty-eaonous plants, particularly cereals and crops, sucn as potato, canola, oilseed rape, soybean, sugar oeet or tobacco.
To illustrate tne invention, specific examples are set forth below. These examples are merely illustrative and are not to be understood as limiting the scope and underlying principles of tne invention in any way. Various modifications of the invention in ad¬ dition to those shown and described nerem will become apparent to those skilled in the art from the following examples and foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
EXAMPLES
Methods
Suppression subtractive hybridization (SSH) has been applied for the identification of relevant subsets of differentially expressed genes (Diatchenko et al.,
1996) . The technique is based on equalization of abundance of target cDNAs included in the tester cDNA population, and efficient subtraction of common sequences m tester and driver cDNA population by hy- bridization. In a specific form of PCR, differentially expressed target cDNAs are amplified selectively whereas the amplification of nontarget cDNA is suppressed. We isolated differentially expressed cDNA fragments from chemically induced barley plants ("tester") when subtracted from cDNAs of non-induced plants ("driver") .
Plants, pathogens, and inoculation
Plants, namely tne barley ( Horaeum v l gare L.; culti- var Ingrio and the Rorl-2 mutant A89, were grown in a growth chamber at 16°C, 60 % relative humidity and a photo period of 16 h (100 μE s" m":) . Control inoculation for chemical induction was done with 10 comdia mm"" from Erysipne gramim s DC: Fr . f . sp . nordei , isolate Ki (Hmze et al . , 1991; at tne third day after chemical treatment.
Plant activation
2, 6-Dichloroιsonιcotιnιc acid (DCINA, CGA 41396, Ciba Geigy AG, Basel, Switzerland) , formulated as 25% active ingredient with a wettable powder (WP) carrier (Metraux et al . 1991), was applied to 5-oays-olo Parley seedlings as a soil drench. The compound vjα≥, used with final concentration of 5 mg X soil volume. The suspensions were prepared with sterile tap water.
Isolation of total RNA
For isolation of poly(A)0RNA used for SSH and race experiments the extraction of total RNA was done according to DUDLER et al . (1991) from 5 g leaf material.
For northern analysis total RNA was isolated by using RNA Clean (AGS) with some modifications to the manual provided. Leaf material was ground in liquid nitrogen, After adding 1 , 7 ml RNA Clean extraction buffer and 200 μl chloroform to an aliquot of 200 mg leafpowder the content was thoroughly mixed and snax.en for 30 minutes. The samples were centrifuged at 20800 x g (4 °C, 30 minutes) and the aqueous phase was extracted again with an equal volume of chloroform. After cen- tπfugation (20800 x g, 4 °C, 15 minutes) RNA was pre- c pitated out of the upper, aqueous phase witn an equal volume of isopropanol, the samples were stored on ice for 1 hour and subsequently centrifuged at 20800 x g (4 °C, 30 minutes) . The supernatant was dis¬ carded and the pellet washed once with 70 % ethanol [ - 20 °C) . After spinning, the supernatant was tnoroughly removed, the pellets were dried at room temperature and RNA was solubilizeo m an appropriate volume of water .
Isolation of polyadenylated RNA (poly (A) +RNA)
Total RNA of different time points (12, 24, 48 hours after chemical induction and control treatment, respectively) was pooled with equal amounts. Pooled RNA was subjected to digestion with DNase as follows. Ali- quots of 750 μg RNA were made up to 250 μl by adding 12,5 μl 1 M Tris-HCl (pH 7,5), 50 μl 50 mM MgCl , 1 μl 25 mM EDTA (pH 7,5), 2,5 μl RNase Inhibitor (40 U/μl), 10 μl DNasel (10 U/μl) and the appropriate volume of water. The reaction mixture was incubated at 37 °C for 30 minutes m an air incubator. To extract the RNA the volume was made up to 1250 μl with water. A equal volume of phenol (saturated with Tris-HCl (pH 8,0)) : chloroform (3:1) was added, thoroughly mixed, and centrifuged at 20800 x g for 5 minutes at room tempera- ture . The aqueous phase was transferred to a fresh tube and subsequently 0, 1 volumes of 3 M sodium acetate (pH 5,2) and 2,5 volumes of cold (-20 °C) 96 % ethanol were added. The assays were mixed and stored at -20 °C for 24 hours. After centπfugation at 20800 x g for 1 hour at 4 °C the supernatant was discarded and the pellet washed with cold (-20 °C) 70 % ethano_ . The mixtures were centrifuged again at 20800 x g for 15 minutes at 4 °C and the supernatant was removed. The pellets were dried at room temperature and dissolved m an appropriate volume of water.
Polyadenylated RNA (poly (A) +RNA) was isolated using the DYNABEADS mRNA Purification Kit (Dynai) according to the manufacturer's recommendations. The quality of the poly(A)0RNA preparation was checked in a denaturing 6 % polyacrylamid gel and silver staining as follows. For casting and running the gels a Mim-PROTEAN II cei-. (BioRad) was used. To 12 ml of the gel solution 5,6 μl Te ed (SERVA) and 72 μl 10 % APS (BioRad) were adαed, the gel was poured immediately and allowed to polymerize for 2 hours. After a prerun at 100 V for 45 minutes, 100 ng of poly(A)+RNA and 250 ng of total RNA were denatured for 5 minutes m 5 x PAA-buffer at 98 °C, rapidly chilled on ice and loaded onto tne gel. The run was performed at 60 V m 1 x TBE . After elec- trophoresis the gel was incubated 3 times for 15 min¬ utes m fixing solution and rinsed with water 4 x 2 minutes and with 1,5 % (v/v) glycerol 1 x 2 minutes. 7 ml of staining solution A were mixed with 13 ml of staining solution B. The gel was submerged in this mix until bands became visible. The reaction was stopped by transferring the gel to the stop solution.
cDNA Synthesis and generation of a subtracted library by suppression subtractive hybridization (SSH) SSH was performed between A89 non-induced ("driver") and A89 chemically induced ("tester") using the PCR- Select™ cDNA Subtraction Kit (Clontech) . All procedures as first and second strand cDNA synthe¬ sis, Rsal digestion and adaptor ligation were done ac¬ cording to the manufacturer's guidelines except modifications concerning the hyondization steps. For tne first hybridization tne mixtures of one non-ligated "driver" and one of the two different adaptor ligated "tester" cDNA-populations were denatured and kept at 68 °C for exactly 8 hours. For the second hyondization step the "driver-tester" hybridization assays were mixed, fresh denatured "driver" added and incubated at 68 °C for 12 hours. The following PCR reactions, done with the Advantage^ cDNA Polymerase Mix (Clontech) , were performed according to tne manual provided with the subtraction kit. All PCR and ny- bridization steps were done in a Perkm-Elmer 2400 thermal cycler.
The PCR mixture now was enriched for differentially expressed cDNAs in roughly equal abundance.
Cloning of the subtracted cDNAs into a TA vector Although Advantage™ Polymerase Mixes only exhibit a minor amount of proofreading activity A-tailmg was performed directly after the second PCR amplification to ensure that most of the cDNA fragments contain "A- overhangs". 10 μl of the reaction mix were mcuoated with dATP (final concentration of 0,4 mM) and 1 U Taq Polymerase (Eurogentec) for 15 minutes at 70 °C. With¬ out further purification 1 and 3 μl aliquots of the subtracted cDNA were ligated into 50 ng of pT-Adv
(AdvanTAge™ PCR Cloning Kit, Clontech) . After the ligation mixture was introduced into E . col l TOP10 F' (Clontech) the library was plated onto agar plates containing 100 μg/ml ampicillin, 50 μg/ml X-Gal and 280 μM IPTG. Bacteria were grown until colonies were visible and blue/white staining could be clearly distinguished. Plates then were stored at 4 °C.
Colony PCR, reverse northern Plots and screening For evaluation of subtraction and the screening of true differentially expressed cDNAs, colony PCR was performed, tne PCR-products Plotted onto membranes and hybridized with Digoxygenm-labeled first strand cDNA deriving from pooled poly(A;0RNA extracted from induced and non-induced plants as template.
A total of 480 white or light blue individual clones were picked and used to inoculate firstly agar plates containing 100 μg/ml ampicill and secondly 96-well microtitre plates containing 40 μl sterile water. The bacteria in the microtitre plates were lysed by heat¬ ing to 98 °C for 5 minutes using a Perkm-Elmer 9700 thermal cycler. PCR reaction mix was pipetted in 96- well microtitre plates as follows. Aliquots of 10 μl lysate were used to amplify cloned inserts m 30 μl reactions using a standard 10 x PCR buffer (Eurogen- tec) provided with the enzyme, 200 uM dNTPs, 1 , 5 mM MgCl2, 0,5 U Tag polymerase (Eurogentec) and 0,4 μM of each primer (Nested 1 and Nested 2P) which flank the inserts. PCR was performed under the following conditions: one cycle of 94 °C for 3 minutes, 35 cycles each of 94 °C for 15 seconds, 68 °C for 30 seconds and 72 °C for 60 seconds, followed by one cycle of 72 °C for 5 minutes .
After amplification a pregel was run and a number of 360 clones were selected for reverse northern considering a minimum insert size of 0,1 kb . Two 10 μl aliquots were loaded onto a 1,5 % agarose/EtBr gel i x TBE parallel (gel A and gel B) . Equal loading was monitored by fluorescence under UV-lignt due to EtBr accumulation by nucleic acids. The gels were plotted onto nylon membranes (Boehrmger) 2 x SSC for 16 hours. Filters A and B then were denatured for 2 x 5 minutes on whatman paper soaked with 0,5 M NaOn, 1,5 M NaCl and neutralized for 2 x 5 minutes on whatman paper soaked with 0,5 M Tris/HCl (ph 7,4,, 1,5 M NaCi . Subsequently DNA was fixed by cross linking GS Gene Linker, BioRad) with 125 mJ .
For first strand cDNA synthesis aliquots of 2 μg poly(A)+RNA with 2,22 μg oligo (dT 0 primer a volume of 13 μl were heated to 65 °C for 10 minutes und chilled on ice for 5 minutes. 5 x first strata Puffer (Boehrmger), 7,5 μl dNTPs stock (2 mM dATP, dGTP, dCTP, 0,52 mM dTTP, 0,28 mM Dig-dUTP (Boehrmger)) and 37,5 U RNase inhibitor (Boehrmger) were made up to 15 μl with water and added to the poly (A) "RNA/primer mix. After incubation at room temperature for 2 minutes re- verse transcription was started by adding 40 U MMuLV reverse transcriptase (Boehrmger) . The components were mixed thoroughly and incubated at 37 °C for 1 hour. The reaction was stopped by heating at 95 °C for 5 minutes and subsequently chilled on ice for another 5 minutes. For digestion of RNA RNaseA and RNaseH, 1 U of each was added to the first strand synthesis followed by an incubation at 37 °C for 1,5 hours.
Control of Dig-labeled first strand cDNA For screening of differentially expressed cDNAs using the reverse northern technique it is extremely important to estimate and equalize the intensity of Dig- mcorporation into the populations of induced ("tester") and non-induced ("driver") single strand cDNAs . Aliquots of first strand synthesis were denatured in an appropriate volume of RNA-loadmg buffer for 5 min¬ utes at 95 °C, chilled on ice and loaded onto a oena- turing 1,5 % agarose gel m i x MOPS and 5 ιv/v Formaldenyd (37 % . After electrophoresis tne prooes were blotted onto a nylon memorane in 25 mM NaPC - buffer (pH 6,5) for 16 hours and then cross linked with 125 mJ (GS Gene Linker, BioRadj . The Dig-laoeled cDNAs were detected by chemilummescence according to "The DIG System User's Guide for Filter Hybridization" (Boehrmger) . Concentration of Dig-labeled "tester" and "driver" prooes was estimated py comparing signal intensities .
Screening for differentially expressed cDNAs was performed as follows. The filters (A and B) were hybrid¬ ized m Dig Easy Hyb (Boehrmger) for 16 hours at 50 °C with Dig-labeled single strand cDNAs derived from non-induced ("driver") and chemically induced
("tester") poly(A)0RNA, respectively. Filters were washed two times with 2 x SSC/ 0,1 % SDS at room temperature and another two times with 0,1 x SSC/0,1 3 SDS at 50 °C. Detection by chemilummescence was per- formed according to "The DIG System User's Guide for Filter Hybridization" (Boehrmger) .
Isolation of plasmids
Bacteria were grown according to Sambrook et al . (1989) and plasmids isolated using the JETSTAR Plasmid Mmiprep Kit (Genomed) .
Sequencing
Sequencing was performed using the Thermo Sequenase labeled primer cycle sequencing kit (Amersham) ano Long Ranger Gel Solution (FMC) on a Licor 4200 sequencer (MWG-BIOTECH) . The reactions were set up as follows. 2 pmol primer (5' labeled with IRD-800), 1,0 μg template DNA, 10 % (v/v) DMSO were made up to 21 μl with water. To 4,5 μl primer/template mix 1,5 μl of A, C, G or T reagent was added. Cycling was performed m a Perkm-Elmer 2400 thermal cycler with parameters as follows: one cycle of 95 °C for 5 minutes, 30 cycles each of 95 °C for 15 seconds, 61 °C <M13reverse -29' and 64 °C (M13forward -21, for 15 seconds and ^ °C for 15 seconds. The reaction was stepped by adding 4 μl stop buffer provided with the kit (Amersham) . After a prerun of 30 minutes aliquots of 1-2 μl were loaded onto the gel. The run was performed as recommended by MWG-guidelme to the sequencer.
Sequence analysis
Homology searches in GenBank and EMBL databases were done using the BLAST algorithm (Altschul et al . , 1990) . Predicted ammo acid sequences were aligned by using the CLUSTAL W program (Thompson et al . , 1994).
The sequence analysis revealed the following similari¬ ties : clone A : (346bp)
ACATGAAGAG CAGCGACGGC AAGGTCTACG ACTCCTTCAC CATCCACAGG 50
GATTACCGCG ACGTGCTCAG CTGCGTGCAC GACAGCTGC TCCCCACCAC 100
GCTCGCTAGC TAGCTCATAT CGTCCGGCCG TCATGTCAAT GTAATGGAGG 150 GTCATCCATC CAATAAAATT GTGGGCATGT GTTGAGTAAT AAAATTGGTC 200
AGCTGCACAA TTTATATGTG CTAGTAAAAA GATCATGCAA GAGGTGGGTG 250
TATGCTCGTT ATATATGCTT TGTAACTCCT TCATGTCATA TTWTTATGGG 300
TTAATAAAAA CATCCTTTAT CAAAAAAAAA AAAAAaAAAA GCTTGT 350 Homologies: blastn/other est (ESTs unknown function)
E value 5e-42, identities 98/102 (96%) to accession
AI622765 ( Zea mays cDNA)
E value 3e-20, identities 53/54 (98%) to accession AU033069 { Oryza sa ti va cDNA) blastx/nr E value 2e-08, identities 26/34 (761;, positives 26/34 (81%) to accession CAB36731 { Arabi dopsi s thal i ana putative protein with similarity to acid phosphatase EC 3.1.3.2)
Clone B (292bp)
ACAGTATTGG TTGATATGAT TGCTAATCCG GCCCTAGCTC GCGCAGTAAG 50 GGCATCTCCA ATGATTGTAT GATCATCGTT GGTAATATTG CCACATAGAT 100
GATTTTGATG ACATGACGCC TAATAAAAGA AGAAAGAGA^ TGAΛATCGTA 150
TGAACTTGAA GCAACGGTTC ACGCACAAGC TCCGAGGCAA AGCATGGTTA 200
TTTCTTCCAT GTTTAGTGGA CCCGCAATGC ATGCATGGAG GTGTATCTTA 250 CGATTGATCG CAATTAATAA AGTGTTTCGG TACGATAGTA GC
Homologies non
clone C : (387bp)
ACTTATCTTG AGCATGACAT TAGTCAGCAA ACATCCGGCG aTCATCAGAA 50
GaTCTTACTA GCCTATGTGG GCATTCCACG CTACGAAGGT CCAGAGGTTG 100
ATCCCACTAT AGTGACACAT GATGCGAAGG ACCTCTACAA AGCTGGTGAG 150 AAGAAGCTGG GCACAGATGA GAAAACCTTC ATCCGCATCT TCACTGAACG 200
CAGCTGGGCA CACATGGCAG CTGTTGCTTC TGCTTACCAG CACATGTATG 250
MTCGGTCATT ACAGAAGGTT GTGAAGAGTG AAACATCTGG AAACTTTGAA 300
GTTGCTCTGA TaACTATCCT CAGATGTGCT GAGAATCCAG CTAAGTaTTT 350
TGCTAAGGTG TTAAGGAAGT CCATGAAAGG TCTAGGT
Homologies : blastn/dbest (ESTs unknown function )
E value le-118 , identities 340/ 382 ( 89% ) to accession
C27470 { Oryza sa ti va cDNA) E value l e-73 , identities 241 /274 ( 87 % ) to access ion
D39787 ( Oryza sa ti va cDNA) blastx/nr
E value 4e-44, identities 88/128 (68e), positives
102/128 (78%) to accession Y11348 (Medi cago sa ti va an- nexin-like protein responsive to osmotic stress, ab- scisic acid and water deficiency)
clone E : (512bp)
ACAGAAGCTT GGACAGTTCC ACTCGGAAGC TTCGGTTCGC GCTACCGTTC 50
CCGATGCTCG CCTACCCATT CTACTTGTGG TCAAGGAGTC CAGGGAAGTC 100
AGGCTCGCAT TTCCACCCGA GCAGCGATTT GTTCCAGCCG AACGAGAAGA 150
ACGACATACT GACGTCGACG ACATGCTGGC TTGCCATGGC TGGCCTGCTC 200
GCTGGGCTCA CTGCCGTGAT GGGCCCCCTT CAGATACTCA AGCTCTACGC 250
CGTCCCCTAC TGGATTTTTG TTATGTGGCT GGACTTTGTC ACCTACCTGC 300
ACCACCACGG CCACAACGAC AAGCTTCCCT GGTATCGCGG AAAGGC TGG 350
AGCTATCTGC GCGGGGGCCT GACAACGCTC GACAGGGACT ACGGGTGGCT 400
CAACAACATC CACCACGAC TCGGGACTCA CGTGATCCGC CATCTTTTCt 450
CGCAAATCCC GCATTACCAT CTAGTGGAGG CGACCGAGGC GGCGAACAGS 500
TGCTAGGGAA GT
Homologies : blastn/nr
E value 0.0, identities 467/496(94%) to accession D43688 ( Tri ti cum aes ti vum mRNA for plastid omega-3 fatty acid desaturase in light- and dark-grown leaves [temperature-sensitive] )
Clone F 4 84bp )
ACAAGCTTTT TTTTTTTTTT TTTTTGGTGG TAAACAACAA 150
CTGCTTCATA TGAACAACGG GCTTGACAAT CAAAATTCTT CCATATGTTG 200
TTATTATACA AAAAATTGCT TAGAGCCAGA GTGAAATTAC ATCAAAGGCC 250
TTTAAAACTT TGTTATAAAA TCTAGTCTCA AACTCCCCCT CGTCTACAGG 300
TGTTCTCCGA GATATTCTCC CCTTGCCTCC AGTGCGAAAT TTCTGCTATG 350
TCTATGCTCT ATTCACACGG ATGGTTTGTC CTTCCAATTC TGTGTTGTTG 400
AAAGTAGAAA CCACAGCTTC AACTTCCTCC TCTGAAGAAA ACGTGACAAA 450
ACCATATCCC TTGGACTTTG GGGTTCCCGG AATTCGGGAT ACCGTGGCAC 500
TGAGGACCTC CCCCTTTTCA GAGAAGAAGT TCTTGAGAAC TTCCGTCGTC 550
ACTTTCTTCG CAAGATTGCC AACATAAACC TTGT
Homologies : blastn/dbest (EST unknown function)
E value le-37, identities 207/248 (83%) to accession Al 600292 ( Zea mays cDNA) blastx/nr
E value 2e-14, identities 40/73 (54%), positives 48/73 (64%) to accession CAB41335 ( Arabidopsi s thal i ana putative protein with similarity to RNA-b ding protein 30 - Ni coti ana pl umbagi ni folia )
Clone G : (305bp)
GTCAAGTTCA CGCCTGCCGA ATCGAGGATG ASTGCGCCGC CCCAAAAGCC 50 CCCGCCGGAG AGGAGAAGAA GACGTCATGG CCGGAGGTAG CGGGAAAGTC 100
CATCGAGGAG GCCAAGGAGA TCATCCTTAA GGACATGCCT GAAGCGGACA 150
TCGTCGTCCT CCCAGCCGGC TCGCCGGTGA CCCTCGACTT CAGGACCAAC 200 CGTGTCCGCA TCTTCGTCGA CACTGTCGCG TCCACTCCCG ACATTGGCTA 25 GCTAGCTTTG CAAGCAAAGG CAACATGGAT GCATTGTGGA TGCTGATGAA 300 TAAGT
Homologies : blastn/dbest (EST unknown function)
E value 2e-13, identities 103/125 (82%) to accession. C25074 ( Oryza sa ti va cDNA) blastx/nr
E value le-19, identities 44/59 (74%), positives 50/59 (84%) to accession S61830 ( Zea mays subtili- sm/chymotrypsm inhibitor [serine protemase inhibitor] in response to wounding and fungal infection)
Clone H (1035bp) caggATcATA GCTACAgGCg AcAATGCCCG GTCATCgACA ATCGcTgGCA 50
GCgGCTTCTC TGAAGcTACC ATCTCTTcTG CTTCTGTGGA TCTTTAGCTG 100
GAACTGGGgG CATGCCGTGG CCAAGTTTGA TCCTGCAAAC ATgACGGAGC 150
TTCAGAAACA TgTCTCCTTT TTCGACCGGA ACAAGGATgG CTTCATcACT 200
CCTACAGAAA CCATCCAAGG GTtTGTtGCA ATCGGTTGCG AGTATgCATT 250
TGCTACTGcT GCCTCTGCCG CCATTCACGG TGCCcTTGCT CCTcAAAcaA 300
CCCCGGCTGG TaCaCCACTg CCTCACTtGA CAATATACGT AGAGAATAtC 350
CACAAaGCTA TGCATGGAAG tGaTtCaGGt gTATATGAtg CCAAaGGAaG 400
GTTTCTtCCC CAAAACTTTG AGGAATTATT CAAAACATAT GCAATACTCC 450 GaCCAGATGC GTTGACTCTT GCGGAGATGC ATGTGATGQFGCHJQFGCHJ SDZXDXDZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZDDDDZZZZZZZCT CTTTGCAAAA 500
CGGGATCTAG ACCCTATATC ATGGGCACCA CCACAGGTTG AGTGGGGCCT 550
ATTATTCACG CTTGCAAGCG ATTGGCTTGG GTTCCTTCAC AAAGACAGTG 600
TTAGAGGTAT ATATGATGGA AGCCTGTTTA TCAAGTTGGA AAAGAAATGG 650
CaCCCTTTTC AAAGTGCTAT GCGATGAACT TGGTGCTAGT TTAGAGTGAG 700
AGTTtGGAtA tGGAAAGGtT tGtCCCGAAG AAGGTTTtCC tGCTATCtCC 750
AAATTCAACT AGAGTTTATT tTCTTTtCCt CCAAGTtGTA ATtGGcTTTA 800
TAAgACCTtC ATAGCCGATC AataCAACGA aGCAAGTtGg ATATATTTCC 850
CGACCTTGTA TTCTCTCTCA TGKGCCCCTT ATTATGTTTG CGCCATGAGC 900
GCCTACCCAA GAKGAGCCAT AAGCATAAGG CTCATCCACC TATTGGCCAC 950
GACTACTGTT GGAAATATTC CCTACAKGCA ATATTGKGAK GA AA ATTT 1000 ATCTATAAAA AAAAAAAAAA AAAAAAAAAA AAAAA
Homologies : blastx/nr
E value 7e-34, identities 73/173 (42c), positives
107/173 (61%) to accession AC002332 ( Arabi dopsi s tna liana putative calcium-binding EF-hand protein)
Rapid Amplification of cDNA Ends (RACE) 5' and 3' ends of the cDNA were obtained by RACE using the MARATHON cDNA amplification kit (Clontech) . For generating double-strandeo cDNA, pooled poiy (A) -t-RNA isolated from induced and non-induced plants, respectively (see above), served as template. A.fter ligation of adaptors the library served as template for the a - plification of 5' and 3' ends using gene specific primers in combination with adaptor primers provided with the kit. Race products were cloneo into pT-Ad , propagated in E . coll TOP10F' and submitted to sequence analysis as described above.
Northern blot analysis
To confirm differential expression northern blot analysis was performed by separating 5 to 10 μg total RNA on 1,5 % formaldehyde agarose gels (see above) ano transferring to nylon membranes (Boehrmger) . Membranes were hybridized under stringent conditions m Dig Easy Hyb at 60 °C with Dig-PCR-labeled cDNA-probes deriving from SSH or race. Washing was carried out two times in 2 x SSC/0, 1 % SDS at room temperature, then three times 0,1 x SSC/0, 1 % SDS at 60 °C. Detection was performed according to "The DIG System User's Guide for Filter Hybridization" (Boehrmger) .
Material
Sequencing primers (modified at the 5 'terminus with the fluorescent dye IRD 800)
M13forward(-21) 5--TGTAAAACGACGGCCAGT-3' Ta 57 M13reverse (-29) 5'-CAGGAAACAGCTATGACC-3' Ta 57 T7-Promotor: 5--TAATACGACTCACTATAGGG-3' Ta 56 PCR primers: Nested primer 1 5 ' -TCGAGCGGCCGCCCGGGCAGGT-3 ' Nested primer 2R: 5 ' -AGCGTGGTCGCGGCCGAGGT-3 ' API : 5 ' -CCATCCTAATACGACTCACTATAGGGC-3 ' Composition of the 6 % sequencing gel: 10 x TBE 5 ml
Long Ranger Gel Solution (50 %) 5 ml Urea 21 g Water ad 50 ml
TEMED 25 μl
10 % APS 250 μl
2 x SSC: NaCl 0, 3 M
Trisodium citrate 30 mM pH 7, 0
RNA-loadmg buffer: Formaldehyde (37 %) 260 μl
Formamide 720 μl
Glycerol 80 μl
Bromophenol blue (saturated solution m water) 80 μl
10 x MOPS 180 μl EtBr (10 mg/ml) 100 μl
Water (Milli Q) 100 μl
Fixing solution: Methanol 45 % (v/v) Acetic acid 10 % (v/v) in water
Staining solution A:
Na2C03 5 % (w/v) m water
Staining solution B: NH4N03 0,2 % (w/v)
AgN03 0,2 % (w/v)
Tungstosilicic acid 1 % (w/v) Formaldehyde 1,4 % (v/v) m water
Stop solution:
Acetic acid 10 % (v/v)
Glycerol 10 % (v/v)
5 x PAA-buffer:
Urea 7 M
Bromophenol blue 0, 25 % ( / V )
Xylene cyanol 0, 25 % (w/v)
EDTA 10 mM
Glycerol 30 % (v/v)
Gel solution: Urea 7 M
Acrylamide solution
(37,5:1 acrylamide :bιsacrylamιde) 11,625 ml 10 x TBE 7, 5 ml
Water ad 75 ml
10 x TBE:
Tπs base 0, 9 M
Boric acid 0, 9 M
EDTA 25 mM
REFERENCES :
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Ausubel, F. M., Brent, R., Kingston, R. F., Moore, D. D., Seidman, J. G., Smitn, J. A., Struhl, K. (Eos. .
Current Protocols in Molecular Biology. John Wiley and Sons Inc. 2000.
Diatchenko, L., Lau; Y.-F., Campbell, A. P., Cnenchik, A., Moqadam, B.H., Lukyanov, S., Lukyanov, K., Gur- skaya, N., Sverdlov, E.D. and Siebert, P.D. (1996) Proc. Natl. Acad. Sci . USA 93, 6025-6030.
Dudler, R., Hertig, C, Rebmann, G., Bull, J., and Mauch, F. (1991) A Pathogen-Induced Wheat Gene Encodes a Protein Homologous to Glutatione-S-Transferases . MPMI 4, 14-18.
Faske, M., Backhausen, J.E., Sendker, M., Smger- Bayrle, M., Scheibe, R., von Schaewen, A. (1997)
Transgemc tobacco plants expressing pea chloropiast Nmdh cDNA m sense and antisense orientation: Effects on NADP-MDH level, stability of transformants, and plant growth. Plant Physiol. 115, 705-715.
Gottlieb, M. and Chavko, M. (1987) Silver staining of native and denatured eucaryotic DNA agarose gels. Analyt. Biochem. 165, 33-37. Hmze, K., Thompson, R.D., Ritter, E., Salamim, F., and Schulze-Lefert, P. (1991). RFLP-mediated targeting of the mlo resistance locus in barley (Hordeum vui- gare) . Proc. Natl. Acad. Sci . USA 88, 3691-3695.
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Claims

1) A DNA molecule selected from the sequences (A) to (H) :
A
ACATGAAGAG CAGCGACGGC AAGGTCTACG ACTCCTTCAC CATCCACAGG 50
GATTACCGCG ACGTGCTCAG CTGCGTGCAC GACAGCTGCT TCCCCACCAC 100
GCTCGCTAGC TAGCTCATAT CGTCCGGCCG TCATGTCAAT GTAATGGAGG 150
GTCATCCATC CAATAAAATT GTGGGCATGT GTTGAGTAAT AAAATTGGTC 200
AGCTGCACAA TTTATATGTG CTAGTAAAAA GATCATGCAA GAGGTGGGTG 250
TATGCTCGTT ATATATGCTT TGTAACTCCT TCATGTCATA TTWTTATGGG 300
TTAATAAAAA CATCCTTTAT CAZ\AAAAAAA AAAAAaAAAA GCTTGT 350
B
ACAGTATTGG TTGATATGAT TGCTAATCCG GCCCTAGCTC GCGCAGTAAG 50
GGCATCTCCA ATGATTGTAT GATCATCGTT GGTAATATTG CCACATAGAT 100
GATTTTGATG ACATGACGCC TAATAAAAGA AGAAAGAGAA TGAAATCG A 150
TGAACTTGAA GCAACGGTTC ACGCACAAGC TCCGAGGCAA AGCATGGTTA 200
TTTCTTCCAT GTTTAGTGGA CCCGCAATGC ATGCATGGAG GTGTATCTTA 250
CGATTGATCG CAATTAATAA AGTGTTTCGG TACGATAGTA GC
C:
ACTTATCTTG AGCATGACAT TAGTCAGCAA ACATCCGGCG aTCATCAGAA 50
GaTCTTACTA GCCTATGTGG GCATTCCACG CTACGAAGGT CCAGAGGTTG 100
ATCCCACTAT AGTGACACAT GATGCGAAGG ACCTCTACAA AGCTGGTGAG 150
AAGAAGCTGG GCACAGATGA GAAAACCTTC ATCCGCATCT TCACTGAACG 200
CAGCTGGGCA CACATGGCAG CTGTTGCTTC TGCTTACCAG CACATGTATG 250
MTCGGTCATT ACAGAAGGTT GTGAAGAGTG AAACATCTGG AAACTTTGAA 300
GTTGCTCTGA TaACTATCCT CAGATGTGCT GAGAATCCAG CTAAGTaTTT 350
TGCTAAGGTG TTAAGGAAGT CCATGAAAGG TCTAGGT
ACAGAAGCTT GGACAGTTCC ACTCGGAAGC TTCGGTTCGC GCTACCGTTC 50
CCGATGCTCG CCTACCCATT CTACTTGTGG TCAAGGAGTC CAGGGAAGTC 100
AGGCTCGCAT TTCCACCCGA GCAGCGATTT GTTCCAGCCG AACGAGAAGA 150
ACGACATACT GACGTCGACG ACATGCTGGC TTGCCATGGC TGGCCTGCTC 200
GCTGGGCTCA CTGCCGTGAT GGGCCCCCTT CAGATACTCA AGCTCTACGC 250
CGTCCCCTAC TGGATTTTTG TTATGTGGCT GGACTTTGTC ACCTACCTGC 300
ACCACCACGG CCACAACGAC AAGCTTCCCT GGTATCGCGG AAAGGCATGG 350
AGCTATCTGC GCGGGGGCCT GACAACGCTC GACAGGGACT ACGGGTGGCT 400
CAACAACATC CACCACGACA TCGGGACTCA CGTGATCCGC CATCTTTTCC 450
CGCAAATCCC GCATTACCAT CTAGTGGAGG CGACCGAGGC GGCGAACAGS 500
TGCTAGGGAA GT
ACAAGCTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGTGG TAAACAACAA 150
CTGCTTCATA TGAACAACGG GCTTGACAAT CAAAATTCTT CCATATGTTG 200 TTATTATACA AAAAATTGCT TAGAGCCAGA GTGAAATTAC ATCAAAGGCC 250
TTTAAAACTT TGTTATAAAA TCTAGTCTCA AACTCCCCCT CGTCTACAGG 300
TGTTCTCCGA GATATTCTCC CCTTGCCTCC AGTGCGAAAT TTCTGCTATG 350
TCTATGCTCT ATTCACACGG ATGGTTTGTC CTTCCAATTC TGTGTTGTTG 400
AAAGTAGAAA CCACAGCTTC AACTTCCTCC TCTGAAGAAA ACGTGAC A 450
ACCATATCCC TTGGACTTTG GGGTTCCCGG AATTCGGGAT ACCGTGGCAC 500
TGAGGACCTC CCCCTTTTCA GAGAAGAAGT TCTTGAGAAC TTCCGTCGTC 550
ACTTTCTTCG CAAGATTGCC AACATAAACC TTGT
GTCAAGTTCA CGCCTGCCGA ATCGAGGATG ASTGCGCCGC CCCAAAAGCC 50
CCCGCCGGAG AGGAGAAGAA GACGTCATGG CCGGAGGTAG CGGGAAAGTC 100
CATCGAGGAG GCCAAGGAGA TCATCCTTAA GGACATGCCT GAAGCGGACA 150
TCGTCGTCCT CCCAGCCGGC TCGCCGGTGA CCCTCGACTT CAGGACCAAC 200
CGTGTCCGCA TCTTCGTCGA CACTGTCGCG TCCACTCCCC ACATTGGCTA 250
GCTAGCTTTG CAAGCAAAGG CAACATGGAT GCATTGTGGA TGCTGATGAA 300
TAAGT
H :
CAGGATCATA GCTACAGGCG ACAATGCCCG GTCATCGACA ATCGCTGGCA 50
GCGGCTTCTC TGAAGCTACC ATCTCTTCTG CTTCTGTGGA TCTTTAGCTG 100
GAACTGGGGG CATGCCGTGG CCAAGTTTGA TCCTGCAAAC ATGACGGAGC 150
TTCAGAAACA TGTCTCCTTT TTCGACCGGA ACAAGGATGG CTTCATCACT 200
CCTACAGAAA CCATCCAAGG GTTTGTTGCA ATCGGTTGCG AGTATGCATT 250
TGCTACTGCT GCCTCTGCCG CCATTCACGG TGCCCTTGCT CCTCAAACAA 300
CCCCGGCTGG TACACCACTG CCTCACTTGA CAATATACGT AGAGAATATC 350
CACAAAGCTA TGCATGGAAG TGATCCAGGT GTATATGATG CCAAAGGAAG 400
GTTTCTTCCC CAAAACTTTG AGGAATTATT CAAAACATAT GCAATACTCC 450
GACCAGATGC GTTGACTCTT GCGGAGATGC ATGTGATGCT CTTTGCAAAA 500
CGGGATCTAG ACCCTATATC ATGGGCACCA CCACAGGTTG AGTGGGGCCT 550
ATTATTCACG CTTGCAAGCG ATTGGCTTGG GTTCCTTCAC AAAGACAGTG 600
TTAGAGGTAT ATATGATGGA AGCCTGTTTA TCAAGTTGGA AAAGAAATGG 650
CACCCTTTTC AAAGTGCTAT GCGATGAACT TGGTGCTAGT TTAGAGTGAG 700
AGTTTGGATA TGGAAAGGTT TGTCCCGAAG AAGGTTTTCC TGCTATCTCC 750
AAATTCAACT AGAGTTTATT TTCTTTTCCT CCAAGTTGTA ATTGGTTTTA 800
TAAGACCTTC ATAGCCGATC AATACAACGA AGCAAGTTGG ATATATTTCC 850
CGACCTTGTA TTCTCTCTCA TGKGCCCCTT ATTATGTTTG CGCCATGAGC 900
GCCTACCCAA GAKGAGCCAT AAGCATAAGG CTCATCCACC TATTGGCCAC 950
GACTACTGTT GGAAATATTC CCTACAKGCA ATATTGKGAK GAWAAWATTT 1000
ATCTATAAAA AAAAAAAAAA AAAAAAAAAA AAAAA
or derivatives thereof.
(2) A method of testing a chemical compound, composi- tion or mixture for its capability to induce an enhan- ced resistance status (ERS) m plants said method com¬ prising :
(a) contacting a plant or plant part comprising a gene fragment of sequences (A) to (H) of claim 1 the ex- pression of which being inducible by SAR inducing compounds, with the chemical compound; and
(b) assaying the expression of said gene fragment, wherein the expression of said gene fragment indicates that the chemical compound, composition or mixture has the capability to induce ERS.
(3) The method according to claim 2 wnere the expression of the gene fragment is not lnα cible by plant pathogens.
(4) The method according to claim 2 or 3 wherein the expression of the gene fragment is further inducible by ISR inducing compounds.
(5) The method according to any one of claims 2 to 4 wherein the said gene fragment is a gene fragment having substantial homology with any of the nucleotide sequence given in claim 1, a variant of the gene fragments defined in claim 1 or a gene fragment comprising one or more portions of the aforementioned gene fragments and variants.
(6) The method according to any one of claims 2 to 5 wherein the said plant or plant part is selected from the group consisting of a plant protoplast, a plant cell, a plant tissue, callus, a developing plantlet, an immature whole plant, a mature whole plant and seed.
(7) The method according to any one of claims 2 to 6 wherein the said plant or plant part is a cereal, and in particular a plant of tne genus hordeum, or a plant part derived therefrom.
(8) The method according to any one of claims 2 to 7 wherein the gene fragment A to H of claim 1 comprises a nucleotide sequence coding for an indicator protein or polypeptide and wherein the assaying of the expres- sion of the said gene fragment is carried out by moni¬ toring the expression of the nucleotide sequence coding for the indicator protein or polypeptide.
(9)A method for screening for chemical compounds, com- positions or mixtures having the capability to induce ERS m plants wherein the method according to any one of claims 2 to 8 is used and wherein the chemical compounds, compositions or mixtures to be screened are provided form of a synthetic combinatorial library of chemical compounds and/or form of a library of natural products.
(10) A process for providing chemical compounds having the capability of inducing ERS in plants which com- prises the steps of
(a) generating a synthetic combinatorial library of chemical compounds, and
(b) screening the compounds of said library with a method as claimed in claim 9.
(11) A new compound, composition or mixture having the capability of inducing ERS obtainable by a process according to claim 10. 0/71
(12) Use of a compound, composition or mixture according to claim 11 as an agrochemical compound, and in particular for the induction of ERS in plants.
(13)A polypeptide or protein encoded by any of the DNA molecules as defined in claims 1.
(14)A monoclonal or polyclonal antibody whicn specifi¬ cally recognizes and binds a polypeptide or protein as defined claim 13.
(15)A transgemc plant, plant part or seed comprising a recombinant DNA sequence whicn comprises tne DNA molecule according to claim 1.
PCT/EP2000/004564 1999-05-21 2000-05-19 Ers-genes, method of screening for chemical compounds capable of inducing ers in plants WO2000071748A2 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
IL14623400A IL146234A0 (en) 1999-05-21 2000-05-19 Ers-genes, method of screening for chemical compounds capable of inducing ers in plants
EP00941966A EP1179070A2 (en) 1999-05-21 2000-05-19 Ers-genes, method of screening for chemical compounds capable of inducing ers in plants
JP2000620125A JP2003505013A (en) 1999-05-21 2000-05-19 ERS-gene, method for screening compounds capable of inducing ERS in plants
BR0010829-4A BR0010829A (en) 1999-05-21 2000-05-19 DNA molecule, methods of testing a chemical compound, composition or mixture for its ability to induce a state of increased resistance (ers) in plants, to select chemical compounds, compositions and mixtures having the ability to induce ers in plants, process for provide chemical compounds with the ability to induce ers in plants, compound, composition or mixture, use of a compound, composition or mixture, polypeptide or protein, monoclonal or polyclonal antibody, and, plant, part of a plant or transgenic seed
AU56748/00A AU5674800A (en) 1999-05-21 2000-05-19 Ers-genes, method of screening for chemical compounds capable of inducing ers inplants
CA002370332A CA2370332A1 (en) 1999-05-21 2000-05-19 Ers-genes, method of screening for chemical compounds capable of inducing ers in plants

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WO2003033712A1 (en) * 2001-10-17 2003-04-24 Basf Plant Science Gmbh Chemically inducible promoters from barley and use thereof
WO2009041805A1 (en) * 2007-09-28 2009-04-02 Universiteit Utrecht Holding B.V. Defense priming in plants

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WO2003004521A2 (en) * 2001-07-02 2003-01-16 Basf Plant Science Gmbh Nucleotide sequence and method for increasing the resistance to disease in plants
WO2003004521A3 (en) * 2001-07-02 2003-09-18 Basf Plant Science Gmbh Nucleotide sequence and method for increasing the resistance to disease in plants
WO2003033712A1 (en) * 2001-10-17 2003-04-24 Basf Plant Science Gmbh Chemically inducible promoters from barley and use thereof
WO2009041805A1 (en) * 2007-09-28 2009-04-02 Universiteit Utrecht Holding B.V. Defense priming in plants

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AU5674800A (en) 2000-12-12
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CN1370233A (en) 2002-09-18
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IL146234A0 (en) 2002-07-25
CA2370332A1 (en) 2000-11-30

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