WO2001088168A2 - Mise en correspondance d'interactions moleculaires dans des plantes et d'analyses de complementation de fragments de proteine - Google Patents

Mise en correspondance d'interactions moleculaires dans des plantes et d'analyses de complementation de fragments de proteine Download PDF

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WO2001088168A2
WO2001088168A2 PCT/US2001/015170 US0115170W WO0188168A2 WO 2001088168 A2 WO2001088168 A2 WO 2001088168A2 US 0115170 W US0115170 W US 0115170W WO 0188168 A2 WO0188168 A2 WO 0188168A2
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pca
plant
protein
molecule
interaction
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PCT/US2001/015170
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WO2001088168A3 (fr
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Stephen W. Michnick
Normand Brisson
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Odyssey Pharmaceuticals, Inc.
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Publication of WO2001088168A3 publication Critical patent/WO2001088168A3/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
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection

Definitions

  • a first step in defining the function of a novel gene is to determine its interactions with other gene products in an appropriate context; that is, since proteins make
  • Fusion proteins are constructed with two proteins that are thought to bind to each other, fused to either of the two probe fragments. Folding of the probe protein from its fragments is catalyzed by the binding of the test proteins to each other, and is detected as reconstitution of enzyme activity.
  • the most advanced of these PCAs is one based on murine dihydrofolate reductase (mDHFR) (see Fig. 1 and discussion below).
  • PCA is "complete"; no other cellular activity is necessary and as a result a PCA can be done in any prokaryotic or eukaryotic cell type, or the PCA can be directed to a specific cellular compartment, organelle or membrane surface with the inclusion of appropriate signal sequences.
  • the portability of PCAs also means that induced versus constitutive protein-protein interactions can be distinguished by doing the PCA in a cell type where specific protein-protein interactions are thought to be induced by, for example a specific signal transduction pathway.
  • PCAs are not a single assay but a series of assays.
  • PCA strategy therefore has the added flexibility that an assay can be chosen because it works in a specific cell type appropriate for studying interactions of some class of proteins. 4) PCAs are inexpensive, requiring no specialized reagents beyond those necessary for a particular assay and off the shelf materials and technology. 5) PCAs can be automated and high-throughput screening could be done with little human intervention. 6) PCAs are designed at the level of the atomic structure of the enzymes used; because of this, there is additional flexibility in designing the probe fragments to control the sensitivity and stringencies of the assays. 7) PCAs can be based on enzymes for which the detection of protein-protein interactions can be determined differently.
  • the DHFR PCA was the first we developed and is the most advanced in refinement and application 12.
  • the instant application describes in some detail the design principles and experimental strategy of the DHFR PCA as a selection strategy in E. coli, with particular emphasis on necessary controls to assure that the PCA detects protein-protein interactions and not some non-specific response of living cells to expression of the enzyme fragments.
  • a number of mutants are studied as well as detailed kinetic studies of one of the reconstituted mutant enzymes. It also describes tliree specific examples of protein assembly that illustrate general uses of the assay strategy.
  • DHFR PCA survival Assay CHO DUKX-B11 (DHFR) cells were co-transfected with DHFR complementary fragments F[l,2] or F[3] (Fig. 1, left) fused to two partner proteins. Co- transfectants were selected for survival in nucleotide-free medium (selection for DHFR activity).
  • the assay has been demonstrated with GCN4 leucine zippers, the ras-raf complex, FKBP- rapamycin-FRB and the Erythropoietin (Epo) receptor and Epo Receptor- JAK2 kinase complexes.
  • the 'DHFR fluorescence PCA' the high-affinity fluorescein- conjugated DHFR inhibitor methotrexate (fMTX) passively diffuses into cells where it binds in a 1 : 1 complex with DHFR. Free fiuorecein-methotrexate is actively transported from the cells leaving only DHFR-bound fMTX.
  • DHFR PCA Fig. 1, right
  • two proteins are fused to one of the two complementary fragments of DHFR (F[l,2] or F[3]) and coexpressed in a cell. If the two proteins interact, the DHFR fragments are brought into proximity and can fold/reassemble, rendering them capable of binding to fMTX.
  • the first test system for the mammalian DHFR PCA was the pharmacologically well characterized rapamycin-induced association of FK506 binding protein (FKBP) to its target the FKBP- rapamycin binding domain of FRAP (FRB).
  • FKBP FK506 binding protein
  • FRAP FKBP-rapamycin binding domain of FRAP
  • Co-transfectants were selected for survival in nucleotide-free medium (selection for DHFR activity) and in the presence of rapamycin. Only cells grown in the presence of rapamycin underwent normal cell division and colony formation (Fig. 2 A). Survival was dependent only on the number of molecules of DHFR reassembled, and we determined that this number is approximately 25 molecules of DHFR per cell. 14
  • FKBP-rapamycin-FRB complex Formation of the FKBP-rapamycin-FRB complex was also detected in stably and transiently transfected cells with the fluorescence assay described above, based on stoichiometric binding of fluorescein-methotrexate to reconstituted DHFR in vivo. Fluorescence microscopy of unfixed co-transfected cells that had been incubated with fMTX showed high levels of fluorescence when cells were treated with rapamycin at saturating concentrations 14 . The fluorescence response of cell populations was quantified by FACS (Fig. 2 B). The rapamycin- induced formation of FKBP/FRB was monitored by the shift in mean cell population fluorescence compared to non-induced cells.
  • a primary object of the present invention is to develop a PCA strategy to study interactions in plant cells.
  • Another object of the present invention is the use of PCA for the detection of protein-protein interactions in vivo in plant cells.
  • a further object of the invention is the use of PCA for the detection of protein-protein interactions in appropriate contexts, such as within a specific species, organ, cell type, cellular compartment, or organelle.
  • Still another object of the invention is the use of PCA for the detection of induced- versus constitutive protein-protein interactions such as by environmental factors (light, cold, draught, pest and pathogens, etc.), developmental or hormonal signals.
  • An additional object of the invention is the use of PCA for the detection of the kinetic and equilibrium aspects of protein assembly in plant cells.
  • a still further object of the invention is the use of PCA for screening of cDNA libraries for protein-protein interactions.
  • Figure 1 illustrates a schematic representation of the strategy used to study protein-protein interactions in mammalian cells with the DHFR PCA.
  • Figure 2 shows the application of the DHFR PCA to the Rapamycin-induced interaction of FKBP with FRB.
  • Figure 3 describes DHFR PCA in potato protoplast.
  • Figure 4 shows the constitutive interaction of GCN4 leucine zippers.
  • Figure 5 illustrates how Rapamycin induces the interaction between FKBP and FRB in plant cells.
  • Figure 6 describes the flow cytometric analysis of the induced interaction between NPR1 and TGA2.
  • Figure 7 illustrates the dose-response of NPR1-TGA2 in tobacco leaf protoplasts treated with SA.
  • Figure 8 illustrates the dose-response of NPR1-TGA2 in potato leaf protoplasts treated with SA.
  • Figure 9 describes that the nprl-1 mutant fails to interact with TGA2.
  • Figure 10 describes the nuclear Localization of NPR1-TGA2 Interaction by Fluorescence Microscopy.
  • the instant invention is directed to a method of expressing PCA interacting partners in plant material comprising: (A) transforming said plant material with: (1) a first construct coding for a first fusion product comprising (a) a first fragment of a first molecule whose fragments can exhibit a detectable activity when associated and (b) a first protein-protein interacting domain; and (2) a second construct coding for a second fusion product comprising(a) a second fragment of said first molecule and (b) a second protein-protein interacting domain that can bind (l)(b) and (B) culturing said material under conditions allowing expression of said PCA interacting partners, and (C) detecting said activity.
  • the present invention is also directed to a system for use as a standard or control in a PCA assay or for use in validating a PCA assay comprising: (a) a first fusion product comprising a fragment of a first molecule whose fragments can exhibit a detectable activity when associated and a first protein-protein interacting domain; and (b) a second fusion product comprising a second fragment of said first molecule and a second protein-protein interaction domain that interacts with said first protein-protein interaction domain.
  • the invention is also directed to a plant transgenic for one or more genes, each independently selected from the group consisting of: (A) 1 or more genes coding for 1 or more interacting partners able to participate in a PCA assay, and (B) 1 more more genes which result, either directly or indirectly, in the presence of 1 or more interacting partners able to participate in a PCA assay.
  • the invention also relates to a method of determining whether a mutated gene acts upstream in a pathway affecting a constitutive or inducible interaction comprising performing a PCA assay in a mutated plant and correlating a change in PCA activity, relative to that measured in a non-mutated control plant, with the presence of one or more genes acting upstream in said pathway.
  • the instant invention further describes a method of identifying 1 or more genes involved in a pathway controlling an inducible interaction which results in a monitorable activity comprising: (1) mutagenizing a seed from a transgenic plant expressing an interacting partner involved in PCA, (2) germinating the seed, (3) treating with an inducer that controls the interaction of any interacting partners present, and (4) monitoring said activity, and (5) correlating said acitivity with 1 or more genes involved in a pathway controlling an inducible interaction .
  • the invention also describes a method comprising mutating a plant or plant material that exhibits a first level of interaction between PCA interacting partners and selecting for a resultant plant or plant material that exhibits a lower level of said interaction.
  • the invention further describes a method of identifying plant molecule that functions as a PCA interacting partner in a PCA assay comprising : (1) reacting (A) a library of plant molecules which are fused to a first fragment of a reporter molecule, said first fragment exhibiting low or no activity, with (B) a bait molecule fused to a second fragment of said reporter molecule, said second fragment also exhibiting low or no activity and (2) correlating reconstitution of reporter molecule activity with the presence of a PCA interacting partner.
  • the present invention describes the use of PCA to detect protein-protein interactions in plants as well as the DHFR PCA strategy in plant protoplasts with examples of extension to broad applications in plant and agricultural problems.
  • the invention includes the. development of a system to detect inducible and non-inducible protein-protein interactions in plant cells. The development of such a system would be useful to determine whether two proteins interact together and to characterize the structural requirements for this interaction (e.g. protein domains and specific amino acids), especially in the case where the interaction is observed only under inducible conditions.
  • the instant invention describes three approaches and the realization of the first of these approaches: 1) a transient expression approach in protoplasts; 2) transient expression in tissue explants and, 3) expression in transgenic plants.
  • applicants' have developed a cDNA screening strategy for the identification of novel interacting proteins. This would be particularly useful to identify proteins that interact only in specific organelles or compartments or when the interaction is part of a biochemical pathway specific to plants.
  • the PCA strategy can be conducted on plant material selected from the group consisting of whole plants and plant-derived organs, tissues, cells, subcellular parts, and protoplasts.
  • the plant material may also be derived from a transgenic plant.
  • an inducer is added to facilitate the interaction of the protein-protein interaction domains.
  • a fluorescent substrate is added and said activity is detected using fluorescence microscopy, spectrofluorometry, FACS analysis, or a fluorescence-detecting video system.
  • the invention also provides a system for use as a standard or control in a PCA assay or for use in validating a PCA assay comprising: (a) a first fusion product comprising a fragment of a first molecule whose fragments can exhibit a detectable activity when associated and a first protein-protein interacting domain; and (b) a second fusion product comprising a second fragment of said first molecule and a second protein-protein interaction domain that interacts with said first protein-protein interaction domain.
  • the first and second protein-protein interaction domains are selected from the group consisting of: 1) NPR1 + TGA2, 2) FKBP + FRB, 3) and leucine zippers.
  • the invention also describes a plant transgenic for one or more genes, each independently selected from the group consisting of: (A) 1 or more genes coding for 1 or more interacting partners able to participate in a PCA assay, and (B) 1 or more genes which result, either directly or indirectly, in the presence of 1 or more interacting partners able to participate in a PCA assay.
  • the transgenic plant is selected form the group consisting of the genus Arabidopsis and more specifically the plant is Arabidopsis thaliana.
  • the interacting partners comprise one or more of a leucine zipper/reporter molecule fusion, a NPR1 /reporter molecule fusion, a TGA2/reporter molecule fusion, a FKBP/reporter molecule fusion or a FRB/reporter molecule fusion.
  • the instant invention also provides a method for determining whether a mutated gene acts upstream in a pathway affecting an interaction comprising performing a PCA assay in a mutated plant and correlating a change in PCA activity, relative to that measured in a non- mutated control plant, with the presence of one or more genes acting upstream in said pathway.
  • the invention directed to a method of identifying one or more genes involved in a pathway controlling an interaction which results in a monitorable activity comprising: (1) mutagenizing a seed from a transgenic plant expressing an interacting partner involved in PCA, (2) germinating the seed,_ (3) treating with an inducer that controls the interaction of any interacting partners present, and (4) monitoring said activity, and (5) correlating said activity with one or more genes involved in a pathway controlling an interaction .
  • This method could be used for cloning a gene and making products therefrom.
  • the invention involves a method comprising mutating a plant or plant material that exhibits a first level of interaction between PCA interacting partners and selecting for a resultant plant or plant material that exhibits a lower level of said interaction.
  • the instant invention also provides a method of identifying a plant molecule that functions as a PCA interacting partner in a PCA assay comprising : (1) reacting (A) a library of plant molecules which are fused to a first fragment of a reporter molecule, said first fragment exhibiting low or no activity, with (B) a bait molecule fused to a second fragment of said reporter molecule, said second fragment also exhibiting low or no activity and (2) correlating reconstitution of reporter molecule activity with the presence of a PCA interacting partner.
  • the invention is carried out by the following but not limiting Examples.
  • EXAMPLE 1 DNA Constructs: Genes were expressed in plant protoplasts under the control of a promoter containing two Cauliflower Mosaic Virus (CaMV) 35S enhancer elements. A plasmid containing this promoter was derived from the vector pBI221 (Clontech) by replacing the single 35S enhancer element of this vector by the double 35S enhancer element (tandem repeat of the single 35S element) of plasmid pBIN35 S (gift of Dr. Daniel Matton, Universite de Montreal). The resulting vector, called pBl223D, was used to construct all the plasmids required for the PCA.
  • CaMV Cauliflower Mosaic Virus
  • NPRl mutant nprl-f was created by using the ExSite PCR-based site directed mutagenesis kit (Stratagene) and cloned as a N-terminal fusion to mDHFR fragment F[l,2:Phe31Ser].
  • EXAMPLE 2 Protoplasts isolation and electroporation Leaf mesophyll protoplasts were isolated from 6-week-old in vitro grown potato plants cv. Kennebec or Nicotiana tabacum cv. Xanthi. The protoplast isolation procedure and culture media are as described, except for the enzymatic solution which contained 0.8% w/v cellulysin and 0.1% w/v macerase. 18,19 Electroporation of protoplasts was conducted with a home-made capacitor discharge system, using the disposable electroporation chambers (0.4 cm) of the Cell-Porator System of Gibco-BRL (Gaithersburg, MD). The electrical pulse was delivered from a 1000 ⁇ F capacitor charged at 125 V.
  • Pulses from the elecfroporator were delivered to 320 ⁇ l of protoplasts (6 x 10 5 protoplasts/ml) to which was added 80 ⁇ l of a solution containing 20 mM HEPES, 300 mM NaCl, 10 mM CaCl 2 , 770 mM mannitol and 15 ug CsCl purified supercoiled plasmid DNA for each of the DHFR fusion constructs, plus 10 ug of the plasmid pBI221.
  • This plasmid contains theE. coli uidA gene, encoding the ⁇ -glucuronidase (GUS) enzyme, under the control of the CaMV 35S promoter and is used to correct for variations in electroporation efficiency.
  • GUS ⁇ -glucuronidase
  • DHFR PCA Following electroporation, the protoplasts were left on ice for 10 min and then transferred to petri dishes containing 3.5 ml of culture media supplemented with 50 uM 3,4-dehydro L-proline (DHP). A 1 ml aliquot was set aside for measuring GUS activity. 20 The rest of the protoplasts were centrifuged 10 min at 1500 g and resuspended in 1 ml culture media containing 50 uM DHP and 10 uM fiuorescem-methotrexate (Molecular Probes). When present, rapamycin was added at a 10 nM final concentration. Salicylic acid (SA) was added at concentrations varying from 0 to 500 uM.
  • SA Salicylic acid
  • Protoplasts were incubated at room temperature for 18 h in the dark and harvested by centrifugation as above. They were resuspended in 1 ml culture medium, collected again by centrifugation and washed twice at 37°C for 30 min in the same culture medium containing, when required, rapamycin or SA at the appropriate concentrations. Protoplasts were then washed once in 100 mM sodium phosphate buffer (pH 7.5) containing 8 % mamiitol.
  • EXAMPLE 4 Spectrofluorometer measurements: Protoplasts were lysed in 600 uL of 100 mM sodium phosphate buffer (pH 7.9) for 30 min in the dark. The lysed cells were centrifuged at 16,000 g at room temperature for 10 min. Aliquots (150 uL) were transferred into 96 well Microfluor Microtiter plates (Dynex Technologies) for fluorimeter reading using a Packard Fluorocount fluorimeter (excitation at 485 nm and emission at 530 run). All readings were corrected for protein concentration and GUS activity.
  • FACS Flow Cytometric Analysis
  • the protoplasts were incubated in 125 uM SA and the final wash of the protoplasts were done in W5 media (154 mM NaCl, 125 mM calcium chloride, 5 mM KC1, 5 mM glucose, pH 7.9). The protoplasts were then resuspended in 600 uL of W5 media for analysis.
  • DHFR fragments in protoplasts were obtained using a fluorescence plate reader.
  • Figure 4 shows that a 2.2 fold increase in fluorescence is detected in potato protoplasts transfected with the two recombinant plasmids as compared to protoplasts transformed only with the GCN4-F[1,2] plasmid.
  • Figure 5 shows a 1.5 and 2.3 fold increase in fluorescence when the transfected cells are incubated in the presence of 5 nM or 10 nM of rapamycin, respectively.
  • SAR systemic acquired resistance
  • SA salicylic acid
  • nprl also known as niml
  • yeast two-hybrid screens have shown that tomato and Arabidopsis nprl interacts with _TGA bZIP transcription factors, some of which have been shown to bind to sequence elements in the promoter of pr genes. 25,' 26,' 27
  • NPR1 and TGA2 bZIP transcription factor were obtained from Dr. Pierre
  • DHFR activity was measured after electroporation of the constructs into tobacco or potato leaf protoplasts. Following coelectroporation of the protoplasts with the NPRl and TGA2 DHFR plasmids (see Methods), the fluorescence response of cell populations in the presence or absence of SA was first quantified by FACS. The SA-induced formation of NPR1/TGA2 was monitored by the shift in mean cell population fluorescence compared with non-induced cells (Fig. 6). This shift corresponds to a 4- fold increase of fluorescence in the cells population.
  • the histogram at the right (green) corresponds to cells expressing NPR1-F[1,2] and TGA2-F[3] and that have been treated with 125 uM SA.
  • the histogram at the left (purple) corresponds to cells transfected with the same plasmids but untreated.
  • Figure 7 shows a dose-response histogram for SA with tobacco cells based on fluorescence measurements with the fluorimeter.
  • a low level of fluorescence is detected in the absence of SA, reflecting some constitutive interaction between NPRl and TGA2.
  • this weak interaction could be attributed to an endogenous level of S A.
  • a steady increase in fluorescence intensity is detected with increasing concentrations of SA.
  • the fluorometric readings obtained from the cells transformed with the single construct were subtracted from the reading obtained from the protoplasts transformed with both constructs. These relative fluorescence units (RFU) have been adjusted to both protein and GUS values. This is representative of at least five independent experiments and performed in triplicates.
  • the Agrobacterium tumefasciens leaf infiltration technique can be used. Vacuum infiltration of detached leaves with a culture of Agrobacterium containing foreign genes on a binary plasmid has been shown to lead to strong transient expression of the foreign genes in leaf tissue .
  • the recombinant plasmid is introduced into Agrobacterium tumefasciens strain
  • EHA105 by electroporation. Leaves from in vitro grown potato plants are then vacuum infiltrated with this Agrobacterium. To assay for DHFR activity, leaves are placed in a solution containing fMTX and a mild vacuum is applied for 1 min, as described for the assay of ⁇ -glucuronidase with
  • An alternative procedure is to select for stably transformed cells with methotrexate.
  • the reconstituted mDHFR enzyme that we used is resistant to methotrexate ⁇ 4 , only leaf cells that have integrated the Agrobacterium T-DNA will be able to grow in the presence of the drug. Transformed, green calluses should be detectable after 10 days of selection with methotrexate 31. Then a few plants are regenerated from calluses and Southern blots are performed to confirm the integration of the DHFR constructs. Tissue explants can also be treated with fMTX and fluorescence monitored by microscopy to confirm the expression of the mDHFR fusion proteins. Control transformations are done with a Ti plasmid containing only one of the fusion proteins or only the mDHFR fragments.
  • the PCA could be developed in transgenic plants. This would provide a useful system to study inducible interactions that occur only in a whole plant, as for the component of the systemic acquired resistance that occurs in distant leaves from the site of infection, and to study the effect of specific abiotic stresses on whole plants, such as cold or draught.
  • the ideal PCA for such a high-throughput screening procedure would be based on a reporter enzyme whose activity can be rapidly monitored, such as luciferase.
  • a reporter enzyme whose activity can be rapidly monitored, such as luciferase.
  • Such PCAs are presently being developed in S. Michnick's laboratory (see below), h the mean time, the feasibility of the transgenic plant PCA approach could be demonstrated using the GCN4-mDHFR constructs and the
  • Seeds showing retarded germination could be rescued by transfer on normal media containing a folate analog.
  • Transgenic Arabidopsis plants are produced by the simplified in planta transformation protocol 35 , which allows the production of a large number of transgenic seeds. Seeds are vapor-sterilized and grown on MS-Agar plates containing 50 ⁇ g/ml kanamycin. Leaves from growing plants are tested for survival on methotrexate- Agar plates and examined by the fMTX fluorescence assay.
  • the invention is also directed to the elucidation of the mechanisms that control the activation of specific genes during the defense response to pathogens in potato.
  • a nuclear factor, PBF-2 containing a 30 kD protein (p30), that binds to a DNA element controlling the expression of the PR-lOa defense gene of potato.
  • the DNA binding activity of this factor is regulated by a protein kinase C homolog and data suggest that protein-protein interactions may negatively regulate the activity of the factor 37.
  • We will construct a cDNA library in the vector pBI222 used to express the DHFR fusion proteins described in previous sections and use the p30 coding sequence as a bait to isolate putative interaction partners to this protein.
  • cDNA will be synthesized using the directional cDNA synthesis kit from Stratagene.
  • the library will be constructed as a C-terminal fusion to mDHFR F[3] in vector pBI222, placing the fusion protein under the control of the CaMV 35S promoter.
  • the cDNA fragments will be directionally inserted in the vector.
  • Short oligonucleotides will be introduced in the vector to allow amplification by PCR of whole inserts, including the 35S promoter, for retesting clones that give a positive signal (see below).
  • the p30 cDNA will be subcloned and inserted 3' to the mDHFR F[l,2] coding sequence in plasmid pBI222. This will put the fusion protein under the control of the 35S promoter.
  • FACS Fluorescence-activated Cell Sorting
  • electroporated protoplasts incubated with fMET as above, are aliquoted into 384 wells microplates and fluorescence monitored using a microplate fluorimeter.
  • Total DNA is isolated from protoplasts in wells giving a positive signal, the inserts amplified by PCR and subcloned in the mDHFR vector and the experiment repeated until a clonal population of fluorescent protoplasts is obtained.
  • cDNA inserts from these cells can be reamplified as described above, cloned and sequenced. iii) Particle bombardment.
  • Bombardment by DNA-coated microparticles offers a convenient way to introduce and express a foreign gene in tissue explants 39.
  • Pools of plasmid DNA from the cDNA library is adsorbed on gold particles and introduced, using a helium-driven particle delivery system into detached potato leaves that have been vacuum-infiltrated with fMET. Leaves are processed as described in section 1.A and fluorescence detected using a dissection microscope.
  • fluorescent spots can be excised frorm the leaf, the DNA extracted and amplified with the specific primers described in section B.l, and bombardment repeated until a single clone is isolated.
  • an autonomously replicating vector such as PVX
  • This vector would only need to undergo a few rounds of replication, therefore permitting the selection of micro colonies from protoplasts that have been immobilized in agarose.
  • a differential display 41
  • DNA replication was achieved. Following electroporation, methotrexate would be added directly to the protoplasts in liquid culture. After 5-10 days, DNA would be extracted from these cells and cDNAs in the vector amplified by PCR using the primers specific for the library vector. By comparing on a high resolution gel the pattern of amplified fragments with that obtained using control cells electroporated with the cDNA library and the vector carrying only the mDHFR fragment (without the bait protein), it should be possible to detect fragments that are more abundant due to the selective replication of the corresponding cDNAs in protoplasts in the presence of methotrexate. These fragments would then be reamplified, cloned, analyzed by restriction enzyme fingerprinting and sequenced.
  • Applicant is also developing other PCAs in which a fluorescent product is produced from reconstitution of an enzyme, including assays based on ⁇ -lactamase and firefly and renilla luciferase. These assays may prove useful to the long term objectives of this project and as alternative approaches to the DHFR assays.
  • the invention has broad applications of the PCA strategy to plant biology and agriculture. They include the following: A. Gene delivery.
  • the gene delivery system can be electroporation of plasmids containing the PCA fragments; PEG or any chemical mean of transformation, Agrobacterium tumefasciens or any other bacteria; particle bombardment; etc.
  • the PCA fragments can be inserted into a vector able to autoreplicate in plant cells, such as a vector derived from a plant virus (ex. PVX). This could be an autonomously replicating artificial chromosome.
  • the two PCA fragments can be introduced in separate cells that are then fused to produce single cells harboring the two fragments.
  • Transgenic plants containing the individual fragments can also be mated such that the progeny contains the two PCA fragments.
  • This interaction can be detected and analyzed in appropriate contexts, such as within a specific species, cell type, cellular compartment, or organelle.
  • the interaction could be monitored in isolated cells, such as protoplasts and cell suspension cultures, and in organized tissues, grown in vitro or not, such as a callus, a tissue explant (ex. detached roots, leaves, flowers, pollen, fruit, stem, etc.) and in whole plant (any plant species).
  • isolated cells such as protoplasts and cell suspension cultures
  • organized tissues grown in vitro or not, such as a callus, a tissue explant (ex. detached roots, leaves, flowers, pollen, fruit, stem, etc.) and in whole plant (any plant species).
  • the interaction can be monitored in transient expression assays or in stably transformed cells and whole plants.
  • the interaction could be monitored extracellularly, such as in intercellular spaces in differentiated plant tissues.
  • the interaction could be monitored in isolated organelles, such as in nuclei, chloroplasts and other plastids, in mitochondria, vacuoles, etc.
  • the interaction can be studied whether it occurs constitutively or only in the presence of an inducing factor.
  • inducing factors examples include environmental factors such as light, cold, draught, water, air pollution, wind, rain, wounding, pest and pathogens, deficient soils (ex. salinity, high aluminum levels, extreme pH, nitrogen and other nutrient imbalances), developmental signals and signals linked to fertilization, hormonal signals, chemical signals (ex. pesticides, herbicides, bioregulators, etc.), etc.
  • the kinetic and equilibrium aspects of protein assembly in plant cells can be determined by the PCA, including effect of small molecules on these parameters.
  • the PCA could be coupled to a mutagenesis approach to identify genes controlling the interaction between protein partners.
  • C Screening of gene libraries for the identification of novel interacting proteins.
  • the PCA could be used to identify genes encoding proteins able to interact with a bait protein expressed in a plant cell.
  • the technique could be used to identify proteins that interact only in specific plant organelles or cell compartments.
  • the technique would allow the identification of gene products that interact only in biochemical pathways specific to plants.
  • the screen can be performed in transient expression assays in plant protoplasts, in cell suspension culture, in tissue explants, in plant organs or in whole plants.
  • the gene delivery system can be as described in IV-A.
  • HTS High throughput screening
  • the screening can be performed with any plant material as long as protein-protein interactions can be monitored by the PCA.
  • This includes protoplasts, cell suspension culture, callus, tissue explants, pollen grain, plant organs, whole plant.
  • the screening can be done with stably transformed cells, tissues or whole plants, or with cells that transiently express the reporter genes (see section II).
  • Herbicides Genetic evidences indicate that the products of the Arabidopsis clavata 3 and clavata 1 gene interacts and that this interaction is required for stem meristem formation. If such an interaction could be demonstrated in a plant PCA system, this could form the basis for an HTS for molecules acting as herbicide. This strategy could be used to isolate molecules interfering with the assembly of rubisco (small and large subunits) or any other interacting proteins known to be involved in photosynthesis.
  • Example of applications could be: b) Herbicide resistance: An herbicide that leads to the disruption of a protein-protein interaction vital for the survival of the plant is used to select, by the PCA (as described above), mutations in one of the interacting partner that renders the interaction insensitive to the effect of the herbicide. The mutated gene is then introduced into a crop plant. Weeds can then be controlled by application of the herbicide, without fear for the crop, thus allowing a limited use of herbicides for the control of weeds.
  • Control of flowering When the interaction between two proteins is known to control flowering, the PCA can be used to isolate a small molecule that disrupt this interaction. Tins molecule can then be used to retard flowering, which might be useful, especially for production of ornamental plants, and for crop plants where this could allow the plant to increase its bio mass before harvesting. The same can be done for the isolation of small molecules that would induce flower development by inducing the interaction of two proteins. Then the small molecule could be used to obtain early flowering. This could have some applications for example in crops grown in colder climates where it can be useful to induce flowering before early frosts destroy the crop.
  • Hybrid plants are largely used in agriculture (ex. maize) as these plants usually show increased vigor and other enhanced characteristics. These plants are normally produced using male sterile plants, such that female flowers on a plant can only be fertilized by the pollen of another species, leading to the production of hybrid seeds. However, a way must be provided to restore the fertility of the male sterile plant, such that seeds for these plants can be obtained.
  • two interactors are known to be required for the production of male gametes, it is possible to use the PCA (or any other methods) to select for small molecules that disrupt this interaction. It is then possible to treat the plants with this small molecule to inhibit male flower development, such that these plants can only be fertilized by non-treated plants, thus allowing the production of hybrid seeds. Progenitor seeds are produced from untreated plants.
  • a similar mutagenesis strategy coupled to the PCA could be used to isolate genes encoding for proteins resistant to the action of a small molecule. This would be especially useful when the target protein does not have intrinsic activity easily measurable in vitro.
  • An example would be a protein that controls directly, or indirectly, the interaction between two proteins. Mutant proteins that are not sensitive to inhibition by the small molecule could be isolated by the PCA. The genes could then be used to produce transgenic plants resistant to the small molecule.
  • the PCA could be used to identify new intermediates in signal transduction pathways.
  • protoplasts would be cotransfected with the NPRl and TGA2 DHFR plasmids, and with a plant cDNA expression library.
  • Overexpression of a cDNA encoding a protein in the NPR1/TGA2 pathway could lead to activation of the pathway, and thus of the interaction between NPRl and TGA2, even in the absence of SA.
  • the fluorescence assay would allow the isolation of the protoplast(s) transformed with such cDNAs, and thus isolation of the cDNA by PCR.
  • a cDNA expression library under the expression of a constitutive plant promoter, is constructed into a viral vector derived form PVX. The vector is inserted into the tDNA region of Agrobacterium tumefasciens and colonies are isolated.
  • leaves of a plant for example Nicotiana benthamiana are infiltrated with a culture from a single colony, the cDNA is integrated into the plant genome, and its expression leads to cosuppression of the gene homologous to the sequence of the cDNA. If this gene is important for a pathway, this can be detected by inducing the pathway (for ex.

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Abstract

La présente invention concerne une méthode permettant d'exprimer des partenaires d'interaction pour essais de complémentation de fragments de protéines (PCA) dans un matériau végétal. Cette méthode consiste à : (A) transformer ledit matériau avec (1) une première construction codant pour un premier produit de fusion qui comprend (a) un premier fragment d'une première molécule dont les fragments peuvent présenter une activité détectable lorsqu'ils sont associés et (b) un premier domaine d'interaction protéine-protéine ; et (2) une seconde construction codant pour un second produit de fusion qui comprend (a) un second fragment de ladite première molécule et (b) un second domaine d'interaction protéine-protéine qui peut se lier avec (1)(b) ; (B) cultiver ledit matériau dans des conditions permettent l'expression desdits partenaires d'interaction PCA ; et (C) détecter ladite activité.
PCT/US2001/015170 2000-05-12 2001-05-11 Mise en correspondance d'interactions moleculaires dans des plantes et d'analyses de complementation de fragments de proteine WO2001088168A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
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WO2002029417A2 (fr) * 2000-10-05 2002-04-11 Odyssey Pharmaceuticals, Inc. Visualisation dynamique de reseaux de genes exprimes dans des cellules vivantes
EP1601576A2 (fr) * 2003-03-03 2005-12-07 The Trustees of Columbia University in the City of New York Systemes de biomarquage ligand/partenaire de liaison
JP2014516553A (ja) * 2011-06-07 2014-07-17 フラウンホーファー−ゲゼルシャフト ツル フェルデルング デル アンゲヴァンテン フォルシュング エー ファウ 単クローン性植物細胞系を生成させるための方法

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002029417A2 (fr) * 2000-10-05 2002-04-11 Odyssey Pharmaceuticals, Inc. Visualisation dynamique de reseaux de genes exprimes dans des cellules vivantes
WO2002029417A3 (fr) * 2000-10-05 2004-02-26 Odyssey Pharmaceuticals Inc Visualisation dynamique de reseaux de genes exprimes dans des cellules vivantes
EP1601576A2 (fr) * 2003-03-03 2005-12-07 The Trustees of Columbia University in the City of New York Systemes de biomarquage ligand/partenaire de liaison
EP1601576A4 (fr) * 2003-03-03 2007-12-05 Univ Columbia Systemes de biomarquage ligand/partenaire de liaison
US7575866B2 (en) 2003-03-03 2009-08-18 Virginia Cornish Ligand/binding partner bio-labeling systems
JP2014516553A (ja) * 2011-06-07 2014-07-17 フラウンホーファー−ゲゼルシャフト ツル フェルデルング デル アンゲヴァンテン フォルシュング エー ファウ 単クローン性植物細胞系を生成させるための方法

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