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WO1999013094A2 - Genes de pathogenicite fongique - Google Patents

Genes de pathogenicite fongique Download PDF

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Publication number
WO1999013094A2
WO1999013094A2 PCT/US1998/018730 US9818730W WO9913094A2 WO 1999013094 A2 WO1999013094 A2 WO 1999013094A2 US 9818730 W US9818730 W US 9818730W WO 9913094 A2 WO9913094 A2 WO 9913094A2
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WIPO (PCT)
Prior art keywords
nucleic acid
acid fragment
seq
fungal
isolated nucleic
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PCT/US1998/018730
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English (en)
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WO1999013094A3 (fr
Inventor
James A. Sweigard
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E.I. Du Pont De Nemours And Company
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Priority to AU95673/98A priority Critical patent/AU9567398A/en
Publication of WO1999013094A2 publication Critical patent/WO1999013094A2/fr
Publication of WO1999013094A3 publication Critical patent/WO1999013094A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • This invention is in the field of molecular biology. More specifically, the invention pertains to nucleic acid fragments encoding key proteins that regulate fungal pathogenicity.
  • the pathogen cycle involves (minimally) the following developmental sequence: (1) deposition of a conidium on the plant surface, (2) germination of the conidium to form a germ tube, (3) differentiation of the germ tube into a specialized infection structure called an appressorium, (4) penetration of the plant leaf surface by the melanized appressorium via a penetration peg, (5) differentiation of the penetration peg into a secondary hypha that then grows throughout the invaded plant cell, (6) escape from the first infected plant cell and growth throughout the surrounding plant tissue, (7) production of aerial conidiophores, (8) production of conidia and, finally, (9) release of the conidia to complete the cycle (Howard and Valent, 1996, Annu.
  • Rice is a vital food crop in developing as well as industrial countries. Pathogenic rice blast fungi attack and destroy rice plants in the way described above. The effects of the fungi create an annual worldwide blast fungicide market of approximately $500 million.
  • Plant-Microbe Interact., 1996, 9(6), 450-456) teach the cloning of the MPG1 gene, expressed during appressoria formation and encoding a hydrophobic protein necessary for infectivity. Additionally, NPR1 and NPR2, genes involved in the regulation of MPG1 have also been identified. (Lau and Hamer, Plant Cell, 1996, 8(5), 771 -781 ). Other genes that play a role in appressorium development include APP1 (Zhu et al., Mol.
  • Plant-Microbe Interact., 1996, 9(9), 161-114) and cpkA encoding a cAMP-dependant kinase subunit (Mitchell et al., Plant Cell, 1995, 7(11), 1869-78).
  • Genes not involved in appressorium formation but implicated in rice blast pathogenicity include the PWL genes (PWL ⁇ , PWL2, PWL3, PWL4) involved in host specificity (Sweigard et al., Plant Cell 1995, 7(8), 1221-33; Kang et al, Mol. Plant-Microbe Interact., 1995, 8(6), 939-48).
  • Applicant's invention relates to new genes involved in rice blast pathogenicity that either encode enzymes or proteins not heretofore implicated in the pathogenic process. These novel genes expand the understanding of rice blast pathogenesis and will lead to the development of screens for compounds able to inhibit these newly discovered pathogenic targets.
  • the instant invention relates to isolated genes encoding proteins implicated in the pathogenicity of rice blast.
  • this invention relates to nucleic acid fragments that are complementary to the pathogenicity genes.
  • the invention further includes the gene products of isolated fungal pathogen genes.
  • An additional embodiment of the instant invention concerns a method for obtaining all or a portion of the instant fungal pathogenic genes by using the sequence of the genes to design oligonucleotide probes or PCR primers.
  • the invention provides a method for evaluating at least one compound for its ability to inhibit the activity of a fungal pathogenicity gene product, the genes selected from the group consisting O ⁇ PTH2, and PTH3, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a fungal pathogenicity gene selected from the group consisting of PTH2, and PTH3, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the protein encoded by the operably linked nucleic acid fragment in the transformed host cell; (c) optionally purifying the fungal pathogenicity protein expressed by the transformed host cell; (d) treating the fungal pathogenicity protein with a compound to be tested; and (e) comparing the activity of the protein that has been treated with a test compound to the activity of an untreated fungal pathogenicity protein, thereby selecting compounds with potential for inhibitor
  • the invention provides a method for evaluating at least one compound for its ability to inhibit the activity of the PTH11 gene product by transforming a suitable host with the PTH11 gene wherein expression of the PTH11 gene permits the host to grow and inhibition of the gene will be lethal to the host; contacting the transformed host with a compound suspected of being a PTH11 inhibitor and monitoring the growth of the transformed host to measure the efficacy of the inhibitor compound.
  • the invention provides a method for evaluating at least one compound for its ability to inhibit the activity of the PTHI2 gene product by transforming a suitable host cell with the PTH12 gene such that the gene is expressed; further engineering the host cell such that the expression of a reporter gene is dependent on the binding of the PTH12 gene product to the reporter gene; contacting the transformed host with a compound suspected of inhibiting the activity of the PTH12 gene product and monitoring the expression of reporter gene to determine the efficacy of the inhibitory compound.
  • Figs, la-e shows leaf sections from barley plants inoculated with wild-type strain 4091-5-8(2 x 10 5 conidia/mL) and pth2. pth3. pthll and pth!2 strains (1 x IO 6 conidia/mL). Wild type causes coalescing Type 5 lesions while pth2 and pthl 2 strains (Figs, lb and le) never cause disease lesions (Type 0). pthll (Fig. Id) strains cause rare lesions. pth3 (Fig. lc) strains cause numerous slowly expanding lesions (Type 2-3) that produce limited conidia compared to wild-type.
  • Figures la-e are a set of photographs showing leaf sections from barley plants with varying degrees of lesions cause by pathogenic strains of rice blast.
  • Figure la shows type 5 lesion seen in wildtype plants.
  • Figures lb and le show a type 0 lesion, indicating strong blast resistance.
  • Figure lc shows a rare intermediate lesion of type 1 to type 2.
  • Figure Id shows slowly expanding lesions of type 2 to 3.
  • Figure 2 is a hydrophobicity plot illustrating the secondary structure of the protein encoded by pthll (SEQ ID NO:9).
  • the host strain Magnaporthe grisea 4091-5-8 was deposited on February 7, 1992 with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA (ATCC) under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purpose of Patent Procedure. The deposit is designated as ATCC 74134.
  • sequence descriptions and sequences listings attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. ⁇ 1.821-1.825.
  • the Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in Nucleic Acids Research 75:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2 ⁇ :345-373 (1984) which are herein incorporated by reference.
  • the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1.822.
  • SEQ ID NO:l is the full length nucleotide sequence comprising PTH1.
  • SEQ ID NO:2 is the nucleotide sequence for the coding region of PTH2.
  • SEQ ID NO: 3 is the deduced amino acid sequence encoded by the coding region ofPTHl demonstrating 41% sequence identity to yeast carnitine acetyl transferase.
  • SEQ ID NO:4 is the nucleotide sequence of the full length gene comprising PTH3.
  • SEQ ID NO:5 is the nucleotide sequence of the coding region for PTH3.
  • SEQ ID NO:6 is the deduced amino acid sequence encoded by the coding region ofPTH3 demonstrating 63% sequence identity to a Saccharomyces imidazole glycerol phosphate dehydratase (IGPD).
  • IGPD Saccharomyces imidazole glycerol phosphate dehydratase
  • SEQ ID NO: 7 is the nucleotide sequence of the full length gene comprising PTH11, a gene encoding a membrane associate protein.
  • SEQ ID NO:8 is the nucleotide sequence of the coding region ofPTHll.
  • SEQ ID NO:9 is the deduced amino acid sequence encoded by the coding region of PTH11.
  • SEQ ID NO: 10 is the nucleotide sequence for the full length gene comprising PTH12, a gene encoding a homeodomain transcription factor.
  • SEQ ID NO: 11 is the nucleotide sequence for the coding region of PTH12.
  • SEQ ID NO: 12 is the deduced amino acid sequence encoded by the coding region ofPTH12. DETAILED DESCRIPTION OF THE INVENTION Inhibition of any of the genes PTH2, PTH3, PTH1 and PTH12 results in the reduction or elimination of the pathogenic phenotype of the fungus.
  • the isolated genes are useful in the design of screens to identify inhibitors of the fungal pathogenic gene products.
  • Isolation of the genes of the present invention proceeded by the transformation of pathogenic M. grisea strains with plasmids containing a gene for hygromycin resistance and the selection of hygromycin-resistant strains that demonstrated reduced or no pathogenicity.
  • the plasmid inserts were recovered by plasmid rescue and confirmation of the gene's pathogenic function was obtained by complementation of the wildtype strain with the mutant gene.
  • the genes have been sequenced and their function putatively identified on the basis of BLAST analysis of the genetic databases.
  • PTH refers to a wildtype gene.
  • pth refers to a mutant gene.
  • Pthp refers to the protein encoded by the gene.
  • PTH2 is a wildtype gene encoding carnitine acetyl transferase;
  • pth2 is the mutant gene encoding a mutation in that gene resulting in a phenotype of lessened pathogenicity;
  • Pth2p refers to the carnitine acetyl transferase protein.
  • PTH3 is a wildtype gene encoding imidazole glycerol phosphate dehydratase (IGPD); pth3 is the mutant gene encoding a mutation in that gene resulting in a phenotype of lessened pathogenicity; and Pth3p refers to the imidazole glycerol phosphate dehydratase protein.
  • PTH 11 is a wildtype gene encoding a membrane associated protein involved in fungal pathogenicity; pthl 1 is the mutant gene encoding a mutation in that gene resulting in a phenotype of lessened pathogenicity; and Pthllp refers to the protein encoded by that gene.
  • PTH12 is a wildtype gene encoding a homeodomain transcription factor involved in fungal pathogenicity
  • pthl 2 is the mutant gene encoding a mutation in that gene resulting in a phenotype of lessened pathogenicity
  • Pthl2p refers to the protein encoded by that gene.
  • isolated nucleic acid fragment is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
  • fungal pathogenicity gene refers to a gene which encodes a rice blast fungal protein necessary for the fungus to successfully penetrate a plant host cell and cause disease.
  • the gene may be implicated in any system associated with pathogenicity, including but not limited to, formation of the appressorium, melanaization of the appressorium, formation or development of the penetration peg or development of secondary hypha.
  • fungal pathogenicity protein refers to any protein encoded by a fungal pathogenicity gene. Fungal pathogenicity proteins may have enzymatic function or be of unknown function but expression of the protein, either individually or in concert with the expression of other proteins, will be necessary to convey a pathogenic phenotype on the fungal organism.
  • plasmid rescue refers to a technique for circularizing restriction enzyme-digested fungal genomic DNA that carries DNA fragments bearing a bacterial origin of replication and antibiotic resistance such that this circularized fragment can be propagated as a plasmid in a bacterial host cell such as E. coli.
  • Substantially similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but the functional properties of the protein encoded by the DNA sequence are not affected. "Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript vis-a-vis the alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary sequences.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine).
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid
  • one positively charged residue for another such as lysine for arginine
  • Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.
  • substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65°C), with the sequences exemplified herein.
  • Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are at least 90% identical to the identical to the DNA sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein.
  • a "substantial portion" of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. 1993, J Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).
  • BLAST Basic Local Alignment Search Tool
  • a sequence often or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
  • gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
  • short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
  • a "substantial portion" of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence.
  • the instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular fungal proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
  • identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk. A. M.. ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D.
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual. Altschul et al., Natl. Cent. Biotechnol. Inf., Natl.
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO:l for example, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1 , for example.
  • nucleotide having a nucleotide sequence at least 95%) identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO:3, for example is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO:3, for example.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • Codon degeneracy refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the pathogenicity proteins as set forth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO: 12.
  • Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
  • “Native gene” refers to a gene as found in nature with its own regulatory sequences.
  • Chimeric gene refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • Coding sequence refers to a DNA sequence that codes for a specific amino acid sequence.
  • regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
  • suitable regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences as discussed infra.
  • Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence.
  • the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.
  • Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters".
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
  • Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of fungal transformation are given in Lemke et al.. Genetic manipulation of fungi by DNA-mediated transformation. Mycota, 1995, Volume 2, 109-139. Editor(s): Kueck, Ulrich. Publisher: Springer, Berlin, Germany; Riach et al., Mycol. Ser., 1996, 13(Fungal Genetics), 209-233.
  • Nucleic acid fragments have been isolated that encode all or a substantial portion of genes that encode proteins important in affecting fungal pathogenicity in rice blast disease. Fungal Strains
  • the present fungal pathogenicity genes were isolated from a wildtype strain (4091-5-8; Valent et al, 1986, Iowa State J. Res., 60:569-594) that can infect weeping lovegrass and barley. As a species Magnaporthe grisea infects many different grass hosts, though individual isolates have a limited host range.
  • the present wildtype strains of M. grisea are particularly useful in the isolation of these pathogenicity genes since they are easily and efficiently transformed and may be used in high volume infectivity assays.
  • Insertional mutagenesis was chosen in the present case. Insertional mutagenesis was originally employed in Aspergillus nidulans (Diallinas et al., 1989, Genetics, 122:341-350) and Neurospora crassa (Kang et al., 1993, Genetics, 133:193-202). Subsequently, methods for insertional mutagenesis were extended to include Restriction Enzyme- Mediated Integration (REMI). REMI transformations differ from normal transformations only in that restriction enzymes are added to the transformation milieu. The purpose of the restriction enzymes in REMI is to cut the genomic DNA of the transformation recipient to provide sites for the integration of the transforming DNA.
  • REMI Restriction Enzyme- Mediated Integration
  • REMI has been extensively used in Dictyostelium discoideum (Kupsa et al., 1992, Proc. Natl. Acad. Sci. USA, 89:8803-8807).
  • REMI has been used to tag the Toxl locus in Cochlibolous heterostrophus (Lu et al, 1994, Proc. Natl. Acad. Sci. USA, 91 :12649-12653), conidiation genes in M. grisea (Shi et al., 1995, Mol. Plant-Microbe Interact., 8:949-959)and genes required for pathogenicity in Ustilago maydis (Bolker et al., 1995, Mol. Gen. Genet., 248:547-552).
  • wildtype pathogenic strains of M. grisea were transformed by REMI using plasmids carrying a hygromycin resistance gene.
  • Transformants were selected for hygromycin resistance on a complete medium and single conidial isolates were tested for lessened pathogenicity. Screening For Hygromycin Resistance And Pathogenicity Infection Assays Methods of determining fungal pathogenicity are common (see, for example,
  • the assays used within the context of the present invention involved contacting an effective amount of fungal conidia with the host plant, either weeping lovegrass, barley, or rice.
  • the exposed hosts were examined over time for the appearance of disease lesions as indicated in Figure 1. The degree of infectivity was determined visually as described in Example 2.
  • Plasmid rescue is a common technique and is employed in plant cell as well as fungal genetics (see, for example, Behringer et al., 1992, Plant Mol. Biol. Rep., 10, 190).
  • the vectors used to transform the fungal strains contain a gene for hygromycin resistance and carry sequences required for autonomous replication of DNA in bacteria. Once this DNA is inserted in the fungal genome, specific restriction endonuclease digests can be used to generate fragments that can be circularized, ligated, and transformed into E. coli. Circularized DNA from the T-DNA will generate functional plasmids that confer antibiotic resistance to their bacterial hosts such that they can be identified by growth on selective media.
  • the cloning of mutated genes proceeded by utilizing the insert flanking DNA of the rescued plasmids to obtain a plasmid or cosmid with the wildtype DNA sequence from wildtype strains. Wildtype cosmids or plasmids were then transfected into a non-pathogenic or lessened pathogenic mutant and infectivity was measured. Restoration of the pathogenic phenotype provided strong evidence that the isolated genes were implicated in fungal pathogenicity. Further confirmation was obtained by subcloning smaller pieces from the original complementing plasmid that could still complement the mutant and sequencing the smallest pieces that were still able to alter pathogenicity. In this fashion, putative open reading frames (ORF) were identified. Final confirmation was obtained by introducing mutations (by restriction digests) in the ORF of the pathogenic genes and assaying for lessened pathogenicity. The sequenced genes were compared with the Gen ⁇ MBL database using the
  • BLAST algorithm Basic Local Alignment Search Tool; Altschul et al., 1993. J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) to determine and similarity to known sequences.
  • BLAST analysis revealed that PTH2 had on the order of 41% identity to a yeast carnitine acetyl transferase at the amino acid level and that PTH3 has about 63% identity to a Saccharomyces imidazole glycerol phosphate dehydratase, an enzyme implicated in histidine biosynthesis.
  • the hydropathy plot of Pthl Ip of Figure 2 shows at least 7 trans-membrane domains in the N-terminal region of the protein.
  • BLAST analysis of the genetic databases revealed a significant homology between 55 amino acids of PTH12 and the homeobox of homeodomain transcription factors. Microscopic examination of the development of PTH12 mutants on the leaf surface shows that these mutants form pseudo-appressoria (swellings that resemble appressoria which fail to mature into the full blown appressorial structure). Although PTH12 mutants give rise to a germ tube from the pseudo-appressoria, no other infectivity structures develop.
  • the instant fungal pathogenicity genes can be used as targets to facilitate design and/or identification of inhibitors of the proteins that may be useful as fungicides. This is desirable because the proteins encoded by these genes are necessary for the fungus to attain pathogenic phenotype. Accordingly, inhibition of the activity of one or more of the proteins could lead to inhibition or eradication of pathogenicity.
  • Inhibitor screens are preferably facile and rapid. Screens that rely on whole plant systems, such as infectivity assays, are time consuming. Microbial-based expression systems that can over-express the desired gene products offer a more rapid method of designing gene inhibitor screens.
  • Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for the overexpression of the instant pathogenicity genes. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high level expression of the proteins.
  • the coding sequence may be inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced.
  • the choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the chosen host is within the level of skill of the routineer in the art.
  • the resultant molecule, containing the individual elements linked in proper reading frame may be inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli (see, e.g., Studier and Moffatt, 1986. J.
  • plasmids such as pBluescript (Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Haven, CT), pTrcHis (Invitrogen, La Jolla, CA), and baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV).
  • a preferred baculovirus/insect system is pVl 11392/Sf21 cells (Invitrogen, La Jolla, CA).
  • Recombinantly produced fungal pathogenicity proteins can be isolated and purified using a variety of standard techniques. The specific techniques will vary depending upon the host organism used, whether the protein is designed for secretion, and other such factors familiar to the skilled artisan (see, e.g., chapter 16 of Ausubel, F. et al., "Current Protocols in Molecular Biology", pub. by John Wiley & Sons, Inc. (1994)). Where the fungal pathogenicity protein is an enzyme, as with PTH2 and
  • PTH3 standard enzyme assays may be used to screen for compounds that have fungicidal potential.
  • a typical screen will proceed, for example, by the production and purification of the protein as described above, followed by contacting the enzyme with an appropriate substrate either in the presence or absence of a fungicide candidate. Comparison of the activity of the enzyme alone against the activity of the enzyme contacted with the fungicide candidate will indicate the candidate's efficacy.
  • Enzyme assays for carnitine acetyl transferase (the enzyme encoded by PTH2) are known, (see, for example, Bergstrom et al., 1980, Arch. Biochem.
  • pathogenicity protein is not an enzyme
  • screens must be developed around the characteristics of the protein.
  • the characteristics of the protein suggest that it is membrane-associated and may be upstream of a cAMP-dependent step. These aspects of the protein must therefore form the basis for any assay.
  • mutants of the rice blast strain 4091-5-8 that lack a functional PTHI1 gene form few appressoria compared to wild-type strains.
  • Addition of cAMP (10 mM) reverses the appressorial formation defect of pthl i ( " ) mutants.
  • PTH11 is upstream of cAMP in the signaling pathway that leads to appressorial formation and therefore also upstream of adenylyl cyclase, the enzyme that forms cAMP.
  • grisea appressorial signaling where the GTP-binding protein ras transmits the signal from Pthllp to adenylyl cyclase. It is expected that inhibition of this signaling would yield to poor appressorial formation and, therefore, control of disease.
  • PTH12 encodes a transcription factor, responsible for general fungal appressorial formation. It is contemplated that routine methods may be used to identify and isolate the genes that are the target of the transcription factor encoded by PTH12. For example, such an identification might be accomplished by differential expression of PTH 12 followed by subtraction. Two pools of mRNA could be prepared. One pool from a strain where PTH 12 is not present and the other from a strain where PTH12 expression is under the control of highly inductive promoter.
  • a screen could be designed to search for compounds that inhibit the binding of the PTH 12 protein to its DNA recognition site.
  • a strain of an organism suitable for high throughput screening e.g., Saccharomyces cerevisiae
  • a gene required for growth by this organism could be engineered so that binding sites for the protein are upstream of the gene such that binding of the PTH12 protein is required for expression of this gene.
  • a leu2- auxotroph of S. cerevisiae could have the LEU2 gene engineered in such a fashion such that LEU2 expression was dependent on binding of the PTH12 protein to its DNA binding site.
  • This strain could be screened for inhibition of growth in the presence of test compounds.
  • General inhibitors of the organism could be distinguished from compounds that were directly interfering with the binding of Pthl 2p to its cognate binding site by reversal of inhibition by leucine. Mapping It is contemplated that the instant genes will also be useful in the mapping of the pathogenic fungal genome.
  • the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention.
  • the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et at, 1987, Genomics 7:174-181) in order to construct a genetic map.
  • the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein. D. et al, (l9S0)Am.J.Hum.Genet.32:314-331).
  • Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel, et al., In: Nonmammalian Genomic Analysis: A Practical Guide, Academic Press, 1996, pp. 319-346, and references cited therein).
  • nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping.
  • FISH direct fluorescence in situ hybridization
  • nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification, polymorphism of PCR-amplified fragments (CAPS), allele-specific ligation, nucleotide extension reactions, Radiation Hybrid Mappings and Happy Mapping.
  • the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art.
  • Mycelium for protoplast formation was produced by macerating about 25 cm 2 of mycelium from oatmeal agar plates in a blender containing about 50 mL of sterile complete medium. All subsequent manipulations were performed aseptically. This macerated mycelium was added to 100-200 mL of complete medium and grown with swirling at room temperature for 1-3 d with or without one or two additional cycles of blender maceration. The resulting mycelium was harvested by filtration, washed with distilled water, weighed, and resuspended in 30 mL of 1 M sorbitol.
  • Novozym 234 (20 mg/mL in 1 M sorbitol) was then added at a rate of 1.75 mL of Novozym 234 solution per 3 grams of mycelium.
  • the enzyme/mycelium mixture was gently swirled at room temperature.
  • protoplasts were harvested by filtering sequentially through cheesecloth and a nylon membrane (Nytex, 25 ⁇ m pore size, from Tetko Co., Briarcliff Manor, NY).
  • the protoplast suspension was centrifuged in a swinging bucket rotor (4100 X g, 10 min), and the pellet was resuspended in 10 mL of 1 M sorbitol.
  • restriction enzymes (10-50 units) were added to the transformation mix followed by the addition of 1.25 mL restriction PTC (40% PEG 8000, 20% sucrose, 50 mM KC1, 50 mM NaCl, 10 mM MgCl 2 , 50 mM Tris-HCl, pH 8.0). After an additional 20 min, 3 mL of TB3 (complete medium with 1 M sorbitol) was added, and the protoplasts were gently swirled for 3-6 h. The protoplast suspension was then centrifuged as before, and the pellet was resuspended in 0.1 mL of STC.
  • restriction PTC 50% PEG 8000, 20% sucrose, 50 mM KC1, 50 mM NaCl, 10 mM MgCl 2 , 50 mM Tris-HCl, pH 8.0.
  • 3 mL of TB3 complete medium with 1 M sorbitol
  • Molten regeneration medium (10-15 mL TB3 with 2%> low melting point agarose (Bethesda Research Labs, Gaithersburg, MD) at 50°C) was added, and the protoplasts were poured onto TB3 agar plates containing 200 ⁇ g hyg B/mL. After 5-7 d transformants were picked to oatmeal plates and allowed to sporulate. Single conidia were then isolated from each transformant to insure that the strain was derived from a single nucleus.
  • hygromycin-resistant strains were selected on complete medium (Crawford et al, 1986, Genetics 114:1111-1129) with 200 ug hygromycin/mL. After 5 d hygromycin-resistant strains were transferred to oatmeal medium (Crawford et al., supra) and allowed to conidiate. Single conidial isolates were then picked for each transformant. Selection for hygromycin-resistance resulted in 4800 hygromycin resistant conidial isolates.
  • Hygromycin resistance clones were selected for lessened pathogenicity according to a two part infectivity screen. All conidial isolates were first subjected to a rapid infectivity assay. Those isolates demonstrating reduced pathogenicity using the rapid screen were further tested using the standard infectivity assay. Rapid Assay
  • Figs, la-e shows leaf sections from barley plants inoculated with wild-type strain 4091-5-8 (2 x IO 5 conidia/mL) and pth2,pth3, pthll and pthl 2 strains (1 x IO 6 conidia/mL).
  • Wild type causes coalescing Type 5 lesions while pth2 andpth!2 strains (Figs, lb and le) never cause disease lesions (Type 0).
  • pthll (Fig. Id) strains cause rare lesions.
  • pth3 (Fig. lc) strains cause numerous slowly expanding lesions (Type 2-3) that produce limited conidia compared to wild-type. This rating system does not measure the potential of these mutants to cause epidemics. The system does not measure the ability of the mutants to produce inoculum and to initiate multiple sounds of infection.
  • the digested DNA was ethanol precipitated (Maniatis) and ligated in a 100 uL ligation reaction using a composition of ligation reaction solutions (Maniatis).
  • Maniatis a composition of ligation reaction solutions
  • Four uL of this ligation mix was transformed into E. coli strain DH5 ⁇ (Hanahan, 19??, J Mol. Biol. 166:557-580). Chemical transformation was used for pth2 as described in Maniatis.
  • Electroporation into DH10B (Bethesda Research Labs. Gaithersburg, MD) was used for pthll as described in Maniatis. Positive transformants were selected for chloramphenicol resistance. Genomic DNA from positive transformants was isolated and subjected to southern blots (Southern, A75, J.
  • EXAMPLE 4 Identification of Wildtype Plasmid and Complementation of Mutant Strains Plasmids isolated in Example 3 were used to identify wildtype plasmids in strain 4091-8-5. The wildtype plasmids were then used to complement the DNA of the mutant strains, restoring pathogenicity.
  • a plasmid was rescued from pth2 genomic DNA by digestion with Hindlll, ligation, transformation of the ligation into E. coli, and selection for chloramphenicol resistant plasmids.
  • This plasmid was designated pCB880.
  • This plasmid was used to probe a cosmid library (Sweigard et. al., 1995, Plant Cell 7:1221-1233, 1995)
  • Cosmids homologous to pCB880 were transformed into pth2 protoplasts and the transformants were tested for pathogenicity.
  • Cosmid A1F3 could transform the non- pathogenic pth2 mutant to pathogenicity.
  • DNA fragments from this cosmid that were homologous to the rescued plasmid (pCB880) were subcloned and tested for the ability to complement the pth2 mutant.
  • a 6 kb Hindlll fragment from this cosmid could also complement the mutant (pCB897).
  • This plasmid was further subcloned to a 4 kb BamH I/SstII fragment (pCB913), and then a 3.2 kb EcoRI/Sstll fragment (pCB914). Genomic sequence from this region suggested that there was a large gene with homology to many members of a general class of enzymes called acyl transferases.
  • the fungal pathogenicity nucleic acid sequences were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTX algorithm provided by the National Center for Biotechnology Information (NBC). Amino acid sequences were compared using the BLASTP program [Altschul, S.F., et al., Nucleic Acids Res. 25:3389-3402].
  • DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "NR" database using the BLAST algorithm (Gist et al., 1993, Nature Genetics 3:266-272) provided by the NBC or the Wisconsin Genetics Computer Group package (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI) represent homologous proteins. Amino acid sequences were compared against the SWISS-PROT protein sequence database.
  • the insert from clone PTH2 was sequenced.
  • the strongest homology (41% identity, 52% similarity) was to carnitine acetyl transferase of S. cerevisiae (P32796) [Kispal,G.
  • Example 6 illustrates the use of the gene product of PTH2 in a method for the identification of antifungal compounds.
  • the PTH2 gene may be first expressed recombinantly and the expressed gene may be purified or partially purified for use in the assay. Enzyme activity is measured both in the presence and the absence of an inhibitor candidate and a comparison of these activities indicates the efficacy of a particular candidate as an antifungal compound.
  • PTH2 encodes for the enzyme carnitine acetyl transferase (CAT), PTH2, or any portion of PTH2 that contains the coding region of the gene may be inserted into an appropriate expression vector and used to transform a suitable expression host under the control of either an inducible or constitutive promoter.
  • CAT carnitine acetyl transferase
  • plasmid DNA containing PTH2 may be purified QIAFilter cartridges (Qiagen, Inc., 9600 De Soto Ave, Chatsworth, CA) according to the manufacturer's instructions, and inserted into ligation independent cloning (LIC) pET30 vector (Novagen, Inc., 597 Science Dr., Madison, WI) under the control of the T7 promoter, according to the manufacturer's instructions (see Novagen publications "LIC Vector Kits", publication number TB163 and US 4952496, herein incorporated by reference).
  • the vector may be used to transform competent E. coli hosts such as BL21(DE3).
  • primers with a specific 3' extension designed for ligation-independent cloning are designed to amplify the PTH2 gene (Maniatis). Amplification products may be gel-purified and annealed into the LIC vector after treatment with T4 DNA polymerase (Novagen). Insert containing vectors are then used to transform NovaBlue competent E. coli cells and transformants may be screened for the presence of viable inserts. Clones in the correct orientation with respect to the T7 promoter are transformed into BL21(DE3) competent cells (Novagen) and selected on LB agar plates containing 50 ug/ml kanamycin.
  • the culture may be harvested, resuspended in binding buffer, lysed with a French Press and cleared by centrifugation.
  • the expressed carnitine acetyl transferase can be purified or partially purified according to standard methods and used in an assay.
  • the purified or partially purified CAT enzyme may be assayed in a 1 mL reaction solution that contains 900 uL of 0.1 M KHPO4, 0.1 mM 5,5'dithiobis-(2- nitrobenzoic acid), pH 8.0; 70 uL water; 10 uL 20 mM acetyl coenzyme A; 10 uL 80 mM L-carnitine; and 10 uL enzyme.
  • Two reaction solutions are prepared, one with an inhibitor candidate and the other lacking the candidate.
  • the reaction mixtures are incubated at about 30°C and absorbance is measured on a spectrophotometer where an increase in absorbance was measured at 412 nM and using an extinction coefficient of 13.6.
  • Example 7 illustrates the use of the gene product of PTH3 in a method for the identification of antifungal compounds.
  • the PTH3 gene may be first expressed recombinantly and the expressed gene may be purified or partially purified for use in the assay, according to techniques essentially described in Example 6. As in
  • Example 6 enzyme activity may be measured both in the presence and the absence of an inhibitor candidate and a comparison of these activities indicates the efficacy of a particular candidate as an antifungal compound.
  • Imidazoleglycerolphosphate dehydratase (IGPD).
  • IGPD Imidazoleglycerolphosphate dehydratase
  • Expressed enzyme may be purified or partially purified according to standard methods.
  • the purified or partially purified IPGD enzyme may be assayed at 37°C in a 100 uL reaction consisting of 0.1 M triethalonamine hydrochloride (pH 7.2), 0.2 mM MnCl 2 , 85 mM B-mercaptoethanol, 3 mM imidazolglycerol phosphate.
  • Two reaction solutions are prepared, one with an inhibitor candidate and the other lacking the candidate.
  • the reaction is initiated by addition of enzyme and terminated by addition of 175 uL of 1.43 N NaOH. After further incubation at 37°C for 30 min, the absorbance at 290 nm is determined using 5100 as the molar extinction coefficient for imidazolacetol phosphate, the product of the dehydratase reaction.
  • a comparison of the absorbance measurements from the reaction solution containing the inhibitor candidate and the reaction solution lacking the candidate give an indication of the efficacy of the candidate as an antifungal compound.

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Abstract

Cette invention concerne des fragments d'acide nucléique isolés codant tout ou une partie substantielle de gènes codant des protéines importantes en ce qu'elles sont impliquées dans la pathogénicité fongique dans la puriculariose du riz. Les gènes de pathogénicité peuvent être exprimés sous forme de gènes chimères liés à des éléments régulateurs appropriés et ils sont utiles dans le développement de cribles pour identifier des inhibiteurs des produits géniques.
PCT/US1998/018730 1997-09-10 1998-09-08 Genes de pathogenicite fongique WO1999013094A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077036A2 (fr) * 1999-06-16 2000-12-21 Aventis Cropscience S.A. Gene pls1 (ou gene 421) du champignon pathogene du riz magnaporthe grisea indispensable a la pathogenie du champignon
FR2795092A1 (fr) * 1999-06-16 2000-12-22 Rhone Poulenc Agrochimie Gene 421 du champignon pathogene du riz magnaporthe grisea indispensable a la pathogenie du champignon
FR2807064A1 (fr) * 2000-03-31 2001-10-05 Aventis Cropscience Sa Gene 763 de champignon phytopatogene et son utilisation pour l'identification de composes fongicides
FR2815356A1 (fr) * 2000-10-16 2002-04-19 Aventis Cropscience Sa Gene 77 de champignon phytopathogene et son utilisation pour l'identification de composes fongicides
US7270977B2 (en) 2001-12-11 2007-09-18 Bayer Cropscience Ag Polypeptides for identifying fungicidally active compounds
US9249460B2 (en) 2011-09-09 2016-02-02 The Board Of Trustees Of The Leland Stanford Junior University Methods for obtaining a sequence
CN111979244A (zh) * 2020-08-25 2020-11-24 南京农业大学 一种抑制稻瘟病菌致病性的小分子rna及应用

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WO1998044135A2 (fr) * 1997-04-02 1998-10-08 Hoechst Aktiengesellschaft Procede de depistage de substances a action antimycosique

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077036A2 (fr) * 1999-06-16 2000-12-21 Aventis Cropscience S.A. Gene pls1 (ou gene 421) du champignon pathogene du riz magnaporthe grisea indispensable a la pathogenie du champignon
FR2795092A1 (fr) * 1999-06-16 2000-12-22 Rhone Poulenc Agrochimie Gene 421 du champignon pathogene du riz magnaporthe grisea indispensable a la pathogenie du champignon
WO2000077036A3 (fr) * 1999-06-16 2001-06-28 Aventis Cropscience Sa Gene pls1 (ou gene 421) du champignon pathogene du riz magnaporthe grisea indispensable a la pathogenie du champignon
FR2807064A1 (fr) * 2000-03-31 2001-10-05 Aventis Cropscience Sa Gene 763 de champignon phytopatogene et son utilisation pour l'identification de composes fongicides
WO2001075115A1 (fr) * 2000-03-31 2001-10-11 Aventis Cropscience S.A. Gene 763 de champignon phytopathogene magnaporthe grisea et son utilisation pour l'identification de composes fongicides
US7070981B2 (en) 2000-03-31 2006-07-04 Bayer Cropscience S.A. Gene 763 of phytopathogenic fungus Magnaporthe grisea and use thereof for identifying fungicidal compounds
US7566547B2 (en) 2000-03-31 2009-07-28 Bayer Cropscience S.A. Gene 763 of phytopathogenic fungus Magnaporthe grisea and use thereof for identifying fungicidal compounds
FR2815356A1 (fr) * 2000-10-16 2002-04-19 Aventis Cropscience Sa Gene 77 de champignon phytopathogene et son utilisation pour l'identification de composes fongicides
US7270977B2 (en) 2001-12-11 2007-09-18 Bayer Cropscience Ag Polypeptides for identifying fungicidally active compounds
US9249460B2 (en) 2011-09-09 2016-02-02 The Board Of Trustees Of The Leland Stanford Junior University Methods for obtaining a sequence
US9725765B2 (en) 2011-09-09 2017-08-08 The Board Of Trustees Of The Leland Stanford Junior University Methods for obtaining a sequence
CN111979244A (zh) * 2020-08-25 2020-11-24 南京农业大学 一种抑制稻瘟病菌致病性的小分子rna及应用

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