EP0971579A1 - Gene de regulation de la reponse de plantes aux pathogenes - Google Patents

Gene de regulation de la reponse de plantes aux pathogenes

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Publication number
EP0971579A1
EP0971579A1 EP98908868A EP98908868A EP0971579A1 EP 0971579 A1 EP0971579 A1 EP 0971579A1 EP 98908868 A EP98908868 A EP 98908868A EP 98908868 A EP98908868 A EP 98908868A EP 0971579 A1 EP0971579 A1 EP 0971579A1
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European Patent Office
Prior art keywords
seq
dna sequence
isolated dna
lsdl
sequence
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EP98908868A
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German (de)
English (en)
Inventor
Jeffery L. Dangl
Robert A. Dietrich
Michael H. Richberg
Petra M. Epple
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University of North Carolina at Chapel Hill
University of North Carolina System
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University of North Carolina at Chapel Hill
University of North Carolina System
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Publication of EP0971579A1 publication Critical patent/EP0971579A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance

Definitions

  • This invention relates to a novel DNA molecule that encodes a novel polypeptide, LSDl, which has an effect in regulating the initial response of plants to pathogens and the subsequent spread of plant cell death engendered by infection, the protein encoded by the gene, and transgenic plants comprising the DNA molecule.
  • This invention also relates to novel DNA molecules encoding LSDl related proteins LOLl and LOL2. In addition, it relates to novel DNA molecules encoding proteins which directly interact with LSDl .
  • R genes and their predicted products (reviewed by Dangl, 1995; Staskawicz et al, 1995; Bent, 1996). These molecules function to recognize avr dependent signals and trigger the plant cell to begin the chain of signal transduction events culminating in a halt of pathogen growth.
  • the simplest mechanistic interpretation of allele-specific disease resistance is that the R gene product recognizes the avr gene product directly.
  • non-host resistance The second mode of genetic control of disease resistance and describes in essence those interactions which lack genetic variability in either host or pathogen such that no virulent pathogen and no susceptible host line have been identified.
  • HR cell death during the HR may be a direct consequence of ROI toxicity, or it may be a secondary consequence of signals derived from ROI. It is not known whether HR is required to halt pathogen growth. Nonetheless, HR is correlated with the onset of systemic acquired resistance (SAR) to secondary infection in distal tissue (reviewed by Ryals et al., 1996). In at least tobacco and Arabidopsis, enzymatic blocking of salicylic acid (SA) accumulation subsequent to infection alters disease resistance responses, and SA in distal tissues is required for SAR (Gaffney et al., 1993; Delaney et al., 1994; Vernooij et al., 1994). SA accumulates following the oxidative burst to high levels locally at infection sites.
  • SAR systemic acquired resistance
  • the Isdl mutant is exceptional. In conditions permissive for wild type plant growth and in the absence of detectable microscopic lesions, the Isdl mutant is hyper-responsive to challenge by a variety of stimuli including pathogens and low doses of chemicals which trigger the onset of SAR (Dietrich et al, 1994). Mutant Isdl plants are resistant to otherwise virulent pathogens in conditions where no spontaneous cell death lesions form. Following initiation of cell death in a local spot on a leaf, lesions propagate throughout the leaf and kill it 2-4 days later. Propagation of locally initiated cell death is confined to the inoculated leaf. Thus, LSDl functions to negatively regulate both the initial response to pathogens and the subsequent spread of cell death.
  • the invention herein includes the LSDl gene, which encodes the first member of a new subclass of zinc- finger proteins in Arabidopsis.
  • LSDl novel DNA molecule isolated from Arabidopsis which works to protect plant cells in response to pathogens, and DNA molecules encoding LSDl related proteins LOLl and LOL2. It is a further object of the invention to provide the protein encoded by LSDl, and transgenic plants comprising LSDl.
  • Knowledge of the structure of the LSDl gene allows accurate creation of particular mutants (e.g., deletion and point mutations), for example, mutants having a dominant negative phenotype, analogous to the mutants of Drosophila PANNIER gene (Ramain et al., 1993), using methods known in the art. This in turn allows engineering of transgenic crop plants which do not suffer cell death, but are still resistant to infection.
  • expression of the dominant negative LSDl protein may be refined so that it is expressed very quickly after infection.
  • the LSDl protein is also a useful target for herbicide development.
  • Transgenic plants may be made in which LSDl mutant genes are expressed which are resistant to herbicidal compounds which normally result in cell death in combination with the wild-type LSDl. Mutants of the LSDl gene are tested in a Isdl background to determine if the mutant has a normal or novel function, and in a wild-type background to determine the existence of a dominant negative function.
  • the invention herein comprises the DNA molecule of the wild-type LSDl, which functions to monitor levels of a superoxide-dependent signal and negatively regulates a plant cell death pathway.
  • the predicted LSDl protein contains three zinc-finger domains, defined by CxxCxRxxLMYxxGASxVxCxxC (SEQ ID NO.54).
  • the invention further comprises a protein encoded by LSDl, and transgenic plants comprising LSDl, and mutations thereof.
  • the preferred embodiments of the invention herein include the following: an isolated DNA molecule, encoding the LSDl polypeptide sequence, selected from the group consisting of SEQ ID NOS: 13- 15; the LSDl DNA molecule having the nucleotide sequence as set forth in SEQ ID NO: 13; the DNA molecule that is cDNA; the DNA molecule which is genomic DNA; a chimeric construction comprising a promoter sequence and the LSDl DNA molecule or portions of the LSDl DNA molecule; a recombinant plant transformed with the LSDl DNA molecule; a transformed plant comprising a DNA molecule encoding a protein as set out in SEQ ID NO: 16 or SEQ ID NO:17; an isolated protein molecule comprising the protein set out in SEQ ID NO:16 or SEQ ID NO:17; a transformation vector comprising a LSDl DNA molecule as set forth herein; an isolated DNA molecule encoding the zinc finger consensus sequence shown in SEQ ID NOS: 1-3; and anything that hybrid
  • Figures 1A-C show the physical delineation of the Isdl mutation.
  • Figure 1A shows YAC clones at Isdl.
  • the arrowheads imply the YAC clone extending in the direction given, solid vertical black bars denote YAC ends used to isolate genomic phage clones and subsequently converted into CAPS RFLP markers as described (refer to Figure 2 for their map position and to Tables 1 and 2, Examples II and III, for their definition).
  • Figure IB shows the three BAC clones which contained the CAPS markers listed above BAC1G5.
  • the arrowheads imply extension of the BAC clone in the direction shown.
  • the scale in Figures 1A and IB are the same.
  • Figure 1C shows the genomic phage clones positioned under an expansion of three of the BACs.
  • the diamond-filled bar represents the 8A6-1.3 clone, which co-segregated with Isdl, used to isolate these phage.
  • the Isdl deletion is noted at the bottom.
  • Figure 2 is a genetic linkage map of the Isdl region.
  • the vertical line at the left represents the section of Arabidopsis chromosome 4 between CH42 and B9-1.8 (telomeric toward bottom).
  • CAPS-based RFLP markers discussed in the text intersect the chromosome, and their relative recombination frequencies in the F mapping population are placed in the center.
  • the number of meioses identified among the total number of F 's scored is at the right.
  • the arrowhead denotes the co-segregating marker.
  • Figures 3A-C show molecular fine mapping of the Isdl locus.
  • Figures 3A and 3B show genomic DNA blots demonstrating the presence of a 0.8 kb deletion om the Isdl mutant.
  • Genomic DNA (5 g) from wild type Ws-0 or Isdl was digested with (for each pair of lanes from left to right) EcoRI, Hindlll, a double digest of Hindlll and Xbal. or Kpnl.
  • the blot was probed with the 0.8 kb EcoRI-Xbal.
  • Figure 3B a duplicate blot was probed with the 4.5 kb Pstl-Xhol fragment.
  • the probes are depicted in Figure 3C, and were isolated from phage clones depicted in Figure lC.
  • Molecular weight markers are the Gibco-BRl 1 kb ladder.
  • Figure 3C shows the restriction map in and around the Isdlgene. The extent of the deletion of this locus is shown as are the extent of the hybridization of the various restriction fragments with Isdl cDNAs. Genomic restriction fragments used in complementation experiments are underlined. The asterisk refers to an Xhol site derived from the phage lambda cloning junction.
  • Figure 4 shows that the Isdl mutation is an mRNA null allele.
  • RNA blots (1 g of polyA+ RNA) from leaf tissue of 5 week old plants kept in short days (permissive for Isdl growth) 3 days after spraying with either IN A (0.3 mg/ml powder containing 25% active ingredient, or 4 mM), or wettable powder control.
  • IN A 0.3 mg/ml powder containing 25% active ingredient, or 4 mM
  • wettable powder control wettable powder control.
  • Probes were purified inserts from the LSDl cDNA as represented by EST 82D11T7 (top), a PR-1 cDNA (Uknes et al, 1993b), and an actin cDNA. The blot was probed successively in the order displayed.
  • Figure 5 shows the zinc finger domains (SEQ ID NOS:l-3) of the predicted LSDl protein and the alignment of the three zinc finger domains.
  • the numbers at the left and right refer to amino acid residue position in the deduced LSDl protein.
  • Vertical lines indicate conservation in pairwise comparison, and a colon indicates conservative substitution.
  • a consensus sequence is listed below, with conservative substitutions noted in the second line of consensus where "+” is basic, plus charged; and "@” is amide, polar, uncharged, hydrophilic.
  • Figure 6 shows how the carboxyl portion of the deduced LSDl protein is related to known DNA-binding and transcription factors. Vertical lines indicate conservation in pairwise comparison, and a colon indicates conservative substitution.
  • Figure 6A shows homology of a slightly longer portion of the deduced LSDl protein with mammalian insulin receptor substrate proteins.
  • the LSDl translation product (SEQ ID NO:4) is shown on the top, aligned with the mouse insulin receptor substrate (SEQ ID NO:5). In this region, all mammalian insulin receptor substrates are identical.
  • Figure 6B shows the homology of LSDl, on each top line, with four known transcription factors.
  • the LSDl translation product (SEQ ID NO:6) is shown on top, and below it are the related domains from a human early growth response (EGR) Zn-fmger protein (SEQ ID NO: 7, a human TGF —early induced Zn-fmger protein (SEQ ID NO: 8), a Xenopus laevis H-L-H transcription factor (SEQ ID NO:9), and the human ELK-1 protein (SEQ ID NO: 10).
  • Figure 6C shows the homology of a LSDl transcription product (SEQ ID NO: 11) with a putative maize transcription initiator binding protein (SEQ ID NO: 12). GenBank accession numbers of each protein are listed at the right.
  • Figure 7 shows the consensus sequence of the zinc finger domains (SEQ ID NOS:63-65, respectively) of LSDl (A), LOLl (B) and LOL2 (C).
  • Figure 8 shows the homologies between the first (A), second (B) and third (C) zinc finger domains of LSDl, LOLl and LOL2
  • the present invention provides a genomic DNA sequence (SEQ ID NO: 13) and a cDNA sequence (SEQ ID NOS: 14- 15) or the LSDl gene which is required for the regulation of initial plant response to pathogens, and cDNA proteins deduced (from short form, MG7-SEQ ID NO: 16; from long form, MG, SEQ ID NO: 17).
  • the invention herein provides functional protein domain sequences involved in regulating genes controlling cell death. Gene expression can be regulated by attaching a promoter to the LSDl gene, which may be either the native promoter or any other promoter.
  • the invention herein includes the DNA molecule having the nucleotide sequence as set forth in SEQ ID NOS: 13, 14 and 15, encoding either of two LSDl polypeptides, which are preferably the LSDl polypeptides set forth in SEQ ID NOS: 16 and 17.
  • This DNA molecule may be cDNA or genomic.
  • the invention also includes as the open reading frame any chimeric construction comprising a promoter sequence and the DNA molecule of the invention, a recombinant plant transformed with the DNA molecule, and any transformation vector comprising the DNA of the invention.
  • DNA sequence of either the full-length SEQ ID NO: 13, or a shortened or otherwise modified version thereof may be modified to optimize its expression in plants, with codons chosen for production of the same or a similar protein as encoded by the wild type LSDl gene.
  • Other modifications of the LSDl gene that yield a protein having essentially the same properties as the LSDl gene are included within the invention herein.
  • the invention herein also includes anything that hybridizes to the LSDl DNA (SEQ ID NO: 13) of the invention as discussed above, under hybridization conditions, which are defined as: 7% Na dodecyl sulfate (SDS), 0.5 M sodium phosphate, pH 7.0, 1 mM EDTA at 50C, and wash in 2X SSC buffer, 1% SDS, at 50C (Church and Gilbert, 1984). Proc. Natl. Acad. Sci. USA 81 :1991-1995 (1984)).
  • novel LSDl gene of the present invention it its wild type form or as mutated by selected mutations and genetically engineered derivatives obtained as is known in the art, and proteins encoded thereby, are included in the invention herein, and may be transferred into any plant host using methodology known in the art for purposes of altering the extent and type of plant resistance to pathogens, and to change resistance to particular herbicides.
  • the mutant phenotype of the null Isdl allele suggests that the wild type product is a negative regulator of cell death.
  • Isdl reacts to both nominally virulent pathogens, and to chemicals which trigger the onset SAR, with an HR-like response.
  • Isdl expresses wild type timing of R gene driven HR (Dietrich et al, 1994) ⁇ it is the subsequent spread of cell death which distinguishes the mutant.
  • cell autonomous signals required for R gene function are intact in an Isdl null, but the response to cell non-autonomous signals emanating from cells undergoing HR is perturbed.
  • LSDl functions to limit both the initiation of defense responses and the subsequent extent of the HR.
  • the fact that an Isdl null is hyper-responsive to signals initiating the defense response and HR-like cell death additionally suggests that these pathways are functionally intact in the wild type cell, but require a threshold level of signal for full activation.
  • LSDl appears to act as a transcription factor (or as a protein which sequesters a transcription factor).
  • the oxidative burst in an infected cell generates a superoxide-dependent signal up-regulating the HR pathway. This signal overcomes the negative regulatory function of the available LSDl, and drives primary responding cells into the HR pathway. Additionally, the cells undergoing HR amplify the signal, probably via a sustained extracellular oxidative burst, to neighboring cells.
  • the primary signal molecule may be diffusible over short ranges (Levine et al., 1994), could act as an autocrine signal, and could lead to the accumulation of a secondary signal molecule in a steep spatial gradient from the infection site.
  • LSDl could act as a transcriptional repressor directly on genes in the pro-death effector pathway. This scenario differs from the first only in that the set of target genes would be different. The availability of extragenic suppressors of Isdl will aid in identifying LSDl targets (Jabs et al., 1996).
  • the A. thaliana Isdl mutant phenotype is characterized by enhanced disease resistance, spontaneous formation of lesions in the absence of cell death initiators and failure to limit the extent of cell death.
  • the wildtype LSDl protein therefore negatively regulates a cell death pathway involved in plant defense responses.
  • the LSDl gene encodes a protein containing a novel zinc finger protein, which is included in the invention herein and is defined by its three consensus zinc fingers: CxxCRxxLMYxxGASxRxVxCxxC (SEQ ID NO:52). These three zinc finger domains have not been observed before in the range of zinc finger proteins. As shown in Dietrich et al., 1997, the LSDl gene is a key negative regulator of hypersensitive cell death in plants. We sought other versions of this consensus zinc finger sequence in other plant proteins.
  • LSDl and LOL2 zinc finger domains The data on homologies between the LSDl and LOLl and LOL2 zinc finger domains indicates that LSDl as well as LOLl and LOL2 are members of a novel subclass of zinc finger proteins that are involved in plant cell death pathways.
  • LOLl and LOL2 might function in cell death phenomena leading to hypersensitive response and disease resistance as has been shown for LSDl .
  • the homologues may also be involved in programmed cell death (PCD) pathways occurring in plants. Examples of PCD n plants include lateral root development, tracheary element differentiation, and abscission of leafs. Preliminary expression studies suggest that LOL2 is expressed in flowers and siliques.
  • LOL2 in PCD pathways leading to petal senescence, anther dihiscence or PCD of nucellar cells is not unlikely. It is also possible that LOL2 is involved in the hypersensitive response and disease resistance in flowers, thus protecting seeds and ultimately the following generations from pathogen. Alternatively, LOL2 could be up- regulated during the hypersensitive response. Use of LOLl and LOLl should allow prediction of the protein's function with respect to protection from programmed cell death.
  • Such plants will likely be more resistant to pathogen attack, if, in the first case, the LOL genes function to repress defense response (as does LSDl). Alternatively, if the LOL genes function to activate defense mechanisms, then overexpression will lead to a more effective pathogen response. Because zinc fmger proteins featuring other non-LSD 1 type DNA binding domains function to either activate or repress gene transcription, we cannot distinguish at present between these two models.
  • the invention also includes plant proteins, and the genes which encode them, which directly interact with LSDl protein. Gene regulation in response to pathogen attack is controlled, in part, by the repression and activation of genes.
  • the LSDl, LOLl and LOL2 proteins encode a novel branch of the zinc-finger DNA binding protein superfamily with roles in controlling plant cell death. As such, they are expected to interact with other proteins. Paradigms of gene activation currently demonstrate that DNA binding proteins can have two classes of "partners". The first class sequesters the DNA binding protein in the cell's cytosol. These partner proteins hold the DNA binding protein out of the nucleus until the correct cellular stimulus is received. This stimulus disrupts the physical interaction, and the DNA binding protein is free to migrate into the nucleus and activate or repress transcription.
  • the second class of protein which interacts with DNA binding protein is made up of proteins which are partners having the role of "enhancing" the gene activation or repression encoded by the DNA binding protein. These partners are termed “co- activators” or “co-repressors” and they may or may not have intrinsic DNA binding activity.
  • co- activators or “co-repressors” and they may or may not have intrinsic DNA binding activity.
  • a common assay called a "yeast two-hybrid interaction trap” to detect such interactions genetically (Fields and Sternglanz, 1994; Finley and Brent, 1996). Because the inactivation of LSDl by mutation leads to enhanced disease resistance, the LSDl partner proteins represent novel targets for engineering plants with enhanced resistance to pathogens.
  • this invention includes all proteins which interact with the cell death regulator LSDl (SEQ ID NOS: 66-91 (includes sequential pairs of nucleic acids and corresponding amino acid sequences).
  • This primer set amplified a rapid amplified polymorphic DNA (RAPD) marker (size difference in Ws-0 versus Col-0 without restriction digestion), and map data generated using this primer allowed us to place Isdl below (telomeric to) it.
  • Probe B9-1.8 isolated as a 1.8 kb Sstl-EcoRI fragment from the JGB9 genomic phage clone (RI map position -75; gift of Dr. George Coupland, Cambridge Laboratories, Norwich U.K.) was converted into a CAPS marker. Mapping of this polymorphism placed Isdl above (centromeric to) it (Fig. 2).
  • Recombinants were identified as homozygous for one of these CAPS markers, and heterozygous for the other using DNA from F2 individuals. F3 progeny from these recombinants were then scored as either homozygous Isdl, segregating Isdl, or homozygous wild-type for lesion spread. All CAPS markers we developed are described in Table 1 (below).
  • YACs were defined (Schmidt et al., 1995; Schmidt et al, 1996, http://genom.e- www.stanford.edu/Arabidopsis/JIC-contigs.html), confirmed by DNA blotting to establish a contig and their ends were isolated by vectorette PCR as described (Matallana et al., 1992; Grant et al., 1995). These ends were also used to isolate genomic phage from a Ws-0 genomic library (Fig. 1). Insert fragments of 1-3 kb were cloned into PBS and end sequenced for derivation of primers identifying new CAPS.
  • PCR conditions DNA Engine MJ Research
  • Isdl deletion primers are: 92°C, 3'; 35 cycles of (denature 92°C, 30"; anneal 50°C, 30"; extend 72°C, 2'30”); 72°C, 3'.
  • 8A6-1.3 and the Isdl deletion primer pairs we used 53°C annealing.
  • Table 2 shows the primer sequences used to identify new CAPS markers.
  • pSGCGF was made by restricting pGPTV-Hyg with
  • Hindlll and Sad and replacing this fragment with a Hindlll-Sacl fragment containing the polylinker from pIC20H (GenBank accession L08912; provided by Steve Goff, Novartis,
  • the EST 82D11 cDNA sequence was isolated as a Sall-Xbal fragment from pZLl (Newman et al., 1994) and cloned into Xhol-Xbal digested pHyg35S.
  • the genomic Ws-0 library in 1GEM11 was a gift of Dr. Kenneth A. Feldmann (Univ. of Arizona).
  • the cDNA library is an oligo-dT primed library prepared from polyA+ Col-0 mRNA from leaves cloned into IZAPII (Stratagene, La Jolla, CA) according to the manufacturer's instructions (gift of Dr. Douglas C. Boyes and Dr. Murray R. Grant).
  • IZAPII Stratagene, La Jolla, CA
  • the sequences of the LSDl cDNA (SEQ ID NOS: 14 and 15) and the 4.5 kb LSDl Xhol-Pstl genomic fragment (SEQ ID NO:13; the longest 5'LSDl cDNA starts at base 1892 of this sequence) are deposited in GenBank as accessions U 87833 and U 87834, respectively. Endpoints of the various LSDl cDNAs isolated are shown in Table3A and examples are provided by SEQ ID NO: 14 (short form from cDNA MG7 as shown in Table 3) and SEQ ID NO: 15 (long form, from cDNA MG8). The polypeptides deduced from these are shown in Fig. 11-12, respectively.
  • Table 3B shows the sizes of each intron deduced from comparison of the sequence shown in SEQ ID NO: 13.
  • Numbers in parentheses refer to the number of isolates of the same clone.
  • Nucleotide numbers at the 5' and 3' ends refer to nucleotide positions from SEQ ID NO: 13.
  • An A at the 3' endpoint can be either an A in the genomic sequence or the first A of the polyA tail. The endpoint marked with an * had no polyA tail.
  • Intron splice junction positions are located at bses 198-199, 260-261, 447-448, 552-553, 692-693, 764-765, and 836-837 in SEQ ID NO: 13.
  • the Isdl mutation segregates as a monogenic recessive (Dietrich et al., 1994).
  • F2 progeny of a cross between Isdl (Ws-0 background) and Col-0 (LSDl) were analyzed using the co-dominant amplified polymorphic sequences (CAPS) mapping procedure (Konieczny and Ausubel, 1993) to first establish linkage to the AG marker on chromosome 4.
  • CAS co-dominant amplified polymorphic sequences
  • the closely linked gl3838 probe (3 recombinants in 1632 meioses) was used to identify YAC (yeast artificial chromosome) clones (Schmidt et al., 1995; Schmidt et al., 1996).
  • FIG. 1A We constructed a physical contig of these YACs, shown in figure 1A.
  • YAC ends CIC1H1L, yUP5F7R and EG20B4L to isolate genomic phage clones, subcloned fragments form each of these, end-sequenced the subclones, derived primer sequences and developed new CAPS markers (see Tables 1 and 2).
  • the CAPS markers 1H1L-1.6 and 5F7R-1.5 mapped closest to Isdl (1 and 3 recombinants, respectively from 2054 meioses); see Tables 1 and 2 for new CAPS markers).
  • BAC clone 1G5 should contain the gene.
  • transgenic plants were treated with droplets of 2,6-dichloroisonicotinic acid (IN A); 0.3 mg/ml wettable powder containing 25% active ingredient, Uknes et al., 1993a) a potent inducer of SAR and the Isdl phenotype (Dietrich et al., 1994). If the mutation were complemented, then INA treatment should not lead to spreading cell death.
  • Table 4 shows that transgenic plants carrying either the 7kb Xhol fragment or the 4.5 kb Pstl-Xhol ( Figure 3C) all survived this treatment, and are thus complemented for the Isdl mutation.
  • Selfed progeny from a complemented F individual (homozygous Ws-0 alleles through the Isdl interval) were screened by PCR at F for presence of the hygromycin resistance gene and then INA tested, c F parents were identified as hygromycin resistant and heterozygous through the Isdl interval, then selfed and re-screened as hygromycin resistant and homozygous Ws-0 through the Isdl interval at F before INA testing.
  • SEQ ID NO: 13 eight independent cDNAs (Example VII) and completed the sequence of the full 82D11T7 EST sequence.
  • cDNAs we identified two classes expressing open reading frames of either 184 or 189 amino acids (SEQ ID NO: 16 and 17).
  • An alternate splice which adds 61bp to the 5' region of some cDNAs also provides an alternate translation start, hence, the extra five amino acids in SEQ ID NO: 17.
  • the sequences of both cDNA classes matched exactly the genomic sequence except at the positions of 7 introns (see Table 3). Nucleotide 1 of the longest cDNA is at position 1892 in the 4.5 kb Pstl-Xhol genomic sequence (SEQ ID NO: 13 ).
  • the Isdl phenotype can be observed in all cell types examined after initiation of lesion formation (Dietrich et al., 1994).
  • RNA blot analysis of seedlings, stems, leaves and flowers demonstrated that the LSDl gene is expressed constitutively in each of these Arabidopsis tissues (data not shown).
  • the requirement for LSDl activity in these tissues is consistent with the gene's expression pattern.
  • Example XI The LSDl mRNA encodes a novel zinc-finger domain
  • the plant members of this sub-family described to date include the CO gene, which controls transition to flowering (Putterill et al., 1995), a set of related DNA binding proteins (Yanagisawa, 1995; De Paolis et al., 1996) and a gene whose transcription is salt stress- induced (Lippuner et al., 1996). None of these proteins shares with LSDl the consensus homology within the Zn-fingers.
  • the second homology domain is derived from the carboxy 1 portion of LSDl, from residues 129 to 180 ( Figure 6-SEQ ID NO:4). This region of LSDl exhibits homology to three broad classes of regulatory proteins. First, all mammalian insulin receptor substrates; second, a set of animal transcription factors; and third, a maize transcription initiator binding protein.
  • the conceptual LSDl translation product also identified two additional Arabidopsis ESTs via their predicted amino acid homology. Importantly, each has at least one C-x-x-C Zn-fmger and most of the associated consensus residues found in the LSDl internal homologies. They are ESTs 172A7T7 (GenBank R6552)(SEQ ID NO: 58 and 132J21T7 (GenBank T45809). Thus, it is probable that LSDl is the first member of a widely distributed Zn-fmger sub-family in plants, defined by the internal homology within each zinc-finger. The other amino acids in the consensus section are not known to be found in any other zinc finger proteins.
  • Example XII Identification of expressed target sequence tags (EST) and cDNAs containing LSDl -type zinc finger domains was used to search the GenBank database (NCBI). Two Arabidopsis thaliana ESTs (EST132J21T7 and EST 172A7T7) were identified, each of which contains at least two zinc finger domains and most of the associated consensus residues found in the LSDl internal homologies (Dietrich, 1997). These ESTs were ordered from Ohio State University Arabidopsis Biological Resource Stock Center and resequenced. Sequences were analyzed with the Genetics Computer Group programs (Devereaux et al.,1994).
  • a specific probe isolated from EST172A7T7 was subsequently used for screening of cDNA and genomic libraries.
  • the bacterial strain carrying EST132J21T7 was not viable. Therefore, degenerated primers were designed based on the EST132J21T7 sequence.
  • Genomic Arabidopsis thaliana Ws-0 DNA was used in the PCR reaction and gave rise to a specific PCR product of approximately 400 bp. This fragment was subcloned via the TA Cloning Kit (Invitrogen, Carlsbad, CA) into pBluescript KS(+). Two new genes were identified as described here. Their predicted protein products are highly related to that of LSDl indicating an involvement in the control of cell death in plants
  • RNA isolated from uninduced and P. syringae DC3000 induced Arabidopsis thaliana Col-0 leaf tissue was reverse transcribed.
  • the resulting cDNA population was subcloned unidirectionally into the EcoRI/Xhol - sites of a lambda-Zap II vector using the cDNA-synthesis Kit (Stratagene, La Jolla, CA) according to the
  • EST172A7T7 With the probe specific for EST132J21T7, four cDNA clones were identified and subcloned via the Stratagene excision system. One clone contained an insert of less than 100 bp in length and was not further analyzed. The three remaining clones were sequenced by standard protocol (primers: M13F, M13R, PE6, and PE7); for primer sequences refer to Table 5, below). Clones 2 and 3 contained identical open reading frames (ORFs) and were homologous to EST132J21T7 and to another identical and overlapping EST clone, EST119C9T7.
  • ORFs open reading frames
  • the fourth clone consisted of a chimeric cDNA of approximately 1500 bp, with approximately 400 bp similarity to EST132J21T7, EST119C9T7, and clones 2 and 3. It was also not analyzed further.
  • PE8 5'-AGAGGAAGGTCCGCCTCCGG-3' 41
  • PE9 5'-CTCTGCTCTCCTGAGACTGCTT-3' 42
  • Example XIV LOL2 cDNA By screening the MG-cDNA-library, no clones homologous to EST172A7T7 could be obtained. Therefore, the AB-cDNA-library (derived from RNA isolated from different tissues of sterile grown plants, available at the European .Arabidopsis Stock Center,
  • the deduced protein (SEQ ID NO:55) consisting of two LSDl -type zinc fmger domains extending from bases 130-195 and 244- 309 of SEQ ID NO:54 (SEQ ID NOS:56-57, respectively).
  • Comparison to EST172A7T7 shows that the EST (SEQ ID NO:58) contains a 124 bp insertion (bases 386-509 after the second zinc finger of SEQ ID NO:58), leading to a different C-terminal.
  • Comparison of these two partial cDNA sequences with the genomic LOLl sequence (see below) demonstrates that they are alternate splice forms from the same gene encoding two related proteins.
  • the genomic LOL2 sequence has a length of 3060 bp. Promoter and 5' untranslated regions consist of approximately 1200 bp. The translation products are encoded by three exons, which are interrupted by two introns of 182 bp and 458 bp length, respectively. The overall length of the coding sequence is 1232 bp. Due to alternative splice sites, two proteins which differ in their C-terminal regions are encoded by the LOLl gene (SEQ ID NO:59). A first protein, of 155 amino acids (SEQ ID NO:60), is identical to the LOLl cDNA and contains two zinc finger domains of the LSDl -type.
  • the other translation product corresponds to EST172A7T7, consists of 147 amino acids, and contains two and a half zinc finger domains (SEQ ID NO:61).
  • the consensus sequence of the two zinc finger domains of LOL2 is CxxCxxLLxYxxGxxxVxCSSC (SEQ ID NO:62).
  • TM commonly available LexA yeast two-hybrid system (Matchmaker , Clonetech, Palo Alto, CA) to generate plasmids pEG202-L and pEG202-S.
  • Yeast strain EGY48 is transformed with this plasmid, and appropriate controls performed to ascertain the LSDl fusion protein encoded by plasmids pEG202-L and pEG202-S did not intrinsically activate expression of the yeast markers used in this system.
  • a yeast gene expression library was constructed in plasmid pJG4-5 using RNA from Arabidopsis leaves infected with Pseudomonas syringae. This library encodes fusion proteins of expressed Arabidopsis genes and the B42 transcriptional activation domain. The library was transformed en masse into the yeast strain EGY48 carrying either plasmid pEG202-L or -S. From an equivalent of 6 million clones screened, 122 were isolated. The longest insert of a member from each of these classes was sequenced using standard DNA sequencing methods. Because the novel Arabidopsis gene so identified is produced as an active translation fusion in this system, one is immediately able to identify the deduced protein sequence. The most interesting sequences thus defined, and their deduced protein sequences, are set forth herein as SEQ ID NOS: 66-91. The first main class of LSDl -interacting proteins has no database homologues.
  • These proteins encode putative "sequestration" proteins for LSDl whose function is to inhibit LSDl function until the correct pathogen signal is received. Their utility lies in manipulation of the interaction with LSDl in plant cells such that LSDl is altered in its ability to regulate the response to pathogen. Alternatively, these novel LSDl -interacting proteins may encode new components of the gene regulation machinery working together with LSDl to control transcription in response to pathogen infection. These proteins are valuable because of the knowledge that LSDl is a key regulator of cell death in plants in response to pathogens. Proteins which physically interact with LSDl share in this cellular function.
  • the second class defines proteins having database homologies to other proteins, strongly suggesting a role in control of gene transcription (e.g., CAAT box binding proteins which are known to bind the common CAAT regulatory unit in DNA preceding nearly all genes encoding eukaryotic mRNA).
  • CAAT box binding proteins which are known to bind the common CAAT regulatory unit in DNA preceding nearly all genes encoding eukaryotic mRNA.
  • LSDl partner proteins identify other components of the gene regulatory machinery required for response to pathogens.
  • Manipulation of the expression of, for example, CAAT box binding proteins will result in altered response to pathogen infection.
  • a rolB regulatory factor belongs to a new class of single zinc fmger plant proteins. Plant J. 70, 215- 224.
  • the erytroid-specific transcription factor EryFl a new finger protein. Cell 58, 877-885.
  • H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583-593.
  • Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell 84, 61-11.
  • the CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors. Cell 80, 847-857.
  • pannier a negative regulator of achaete and scute in Drosophila, encodes a zinc finger protein with homology to the vertebrate transcription factor GATA-1. Development 119, 1277-1291.
  • Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance, but is required in signal transduction. Plant Cell 6, 959-965.
  • TNF- and cancer therapy- induced apoptosis potentiation by inhibition of NF- ⁇ B. Science 174, 784-787.
  • Apoptosis A functional paradigm for programmed plant cell death induced by a host-selective phytotoxin and invoked during development. Plant Cell 8, 375-391.

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Abstract

La présente invention concerne des molécules d'ADN codant pour une famille de domaines à doigts de zinc de fixation sur ADN, qui paraît fonctionner de façon à contrôler des niveaux d'un signal dépendant d'un superoxyde et réguler négativement un mécanisme de mort cellulaire de plante comprenant le gène LSD1 de type sauvage, et les protéines LOL1 et LOL2 qui interagissent physiquement avec LSD1, indiquant une fonction qui avec LSD1 régule la réponse des cellules des plantes aux pathogènes.
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JP3331367B2 (ja) 1997-03-11 2002-10-07 独立行政法人農業生物資源研究所 細胞死抑制遺伝子が導入されたストレス抵抗性植物およびその作出方法
US7858848B2 (en) 1999-11-17 2010-12-28 Mendel Biotechnology Inc. Transcription factors for increasing yield
US7868229B2 (en) 1999-03-23 2011-01-11 Mendel Biotechnology, Inc. Early flowering in genetically modified plants
US7345217B2 (en) 1998-09-22 2008-03-18 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
FR2796653B1 (fr) * 1999-07-23 2003-11-28 Agronomique Inst Nat Rech Acides nucleiques codant pour la proteine svn1, application a la modulation des mecanismes de mort cellulaire programmee, notamment la reponse hypersensible (hr), chez les plantes
BR0015634A (pt) * 1999-11-17 2002-12-31 Mendel Biotechnology Inc Planta, polinucleotìdeo, vetor, célula, composição, polipeptìdeo, métodos para produzir uma planta e para identificar um fator que é modulado por, ou interage com, um polipeptìdeo codificado por um polinucleotìdeo, uma molécula que module a atividade ou a expressão de um polinucleotìdeo ou polipeptìdeo de interesse e uma sequência similar ou homóloga a um ou mais polinucleotìdeos e sistema integrado, meio computadorizado ou legìvel em computador
US7939715B2 (en) 2000-11-16 2011-05-10 Mendel Biotechnology, Inc. Plants with improved yield and stress tolerance
US8426678B2 (en) 2002-09-18 2013-04-23 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
US8106253B2 (en) * 2006-11-15 2012-01-31 Agrigenetics, Inc. Generation of plants with altered protein, fiber, or oil content
CN101130777B (zh) * 2007-01-24 2011-03-23 中国科学院微生物研究所 与水稻生长发育和抗病性相关的编码锌指蛋白的水稻新基因

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