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WO2009015505A1 - Interactions entre des espèces végétales séparées par des barrières d'espèce et utilisations de celles-ci - Google Patents

Interactions entre des espèces végétales séparées par des barrières d'espèce et utilisations de celles-ci Download PDF

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WO2009015505A1
WO2009015505A1 PCT/CH2008/000334 CH2008000334W WO2009015505A1 WO 2009015505 A1 WO2009015505 A1 WO 2009015505A1 CH 2008000334 W CH2008000334 W CH 2008000334W WO 2009015505 A1 WO2009015505 A1 WO 2009015505A1
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seq
nucleotides
plant species
amino acids
fer
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Ueli Grossniklaus
Juan-Miguel Escobar-Restrepo
Norbert Huck
Sharon Kessler
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Zurich Universitaet Institut fuer Medizinische Virologie
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Zurich Universitaet Institut fuer Medizinische Virologie
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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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis

Definitions

  • the present invention relates to tools for overcoming species barriers in eukaryotic plants.
  • the tools in particular vectors, make use of all functional elements of a FER gene of a donor plant species or, alternatively, nucleic acids encoding the extracellular (ECD) of the FER protein of the donor plant species and the intracellular domain (ICD) of the FER protein of the recipient plant species.
  • ECD extracellular
  • ICD intracellular domain
  • Plant breeding strategies focus on the introgression of desirable traits (e.g., disease resistance, nutritional values, flavors, scents, and many more) from different individuals into a single one.
  • desirable traits e.g., disease resistance, nutritional values, flavors, scents, and many more
  • species barriers prevent the combination of desirable traits of species separated by species barriers. Therefore, there is a need for breaking the species barriers to obtain plants with enhanced characteristics over the parental plants.
  • a pollen tube germinates out of the pollen grain and grows into the female transmitting tissues towards the ovules.
  • the embryo sac Inside the ovules lies a structure known as the embryo sac.
  • the embryo sac is composed of two synergid cells, an egg cell, a central cell, and three antipodal cells.
  • contact between the synergid and the approaching pollen tube causes the pollen tube to arrest its growth after penetration of one of the synergids. After this the rupture of the pollen tube inside this synergid cell releases the sperm cells. These events are collectively called pollen tube reception.
  • one sperm cell fertilizes the egg to give rise to the embryo and the second sperm cell fertilizes the central cell to form the endosperm, which nutures the developing embryo.
  • the female gametophyte Proper delivery of the sperms depends on signals from the female gametophyte (H ⁇ lskamp et al., 1995; Ray et al., 1997). These chemotactic signals guide the pollen tube into the micropylar opening of the ovule, the reproductive structure that harbors the female gametophyte.
  • the female gametophyte has seven cells: the egg cell flanked by the two synergids, which lie just inside the micropylar opening of the ovule, the central cell, and the three antipodals (Grossniklaus et al., 1998) (Fig. 1A).
  • Presented herein are tools e.g., compositions, methods that enable overcoming ("crossing" of) species barriers/performance of crosses across species barriers in plants.
  • pre-zygotic species barriers are crossed, in particularly preferred embodiments barriers at the level of pollen tube reception.
  • the invention relates to compositions and methods for overcoming interspecific crossing barriers, e.g. by manipulating such barriers, in plants.
  • the invention relates to the use of the FERONIA (FER) gene, or portions thereof, such as a portion encoding the extracellular domain (ECD) of FER, for manipulating barriers, including those relating to pollen tube reception.
  • FERONIA FERONIA
  • ECD extracellular domain
  • the present invention offers, in certain embodiments, a number of advantages, including, but not limited to, allowing for the creation of new varieties of plants, including ones with novel properties. For example, new varieties of fruits, cereals, vegetables, and flowers may be produced, which may allow for, e.g., new flavors and textures, but also for higher yield and/or tolerance to biotic and/or abiotic stresses.
  • the invention is directed towards an isolated nucleic acid comprising a sequence encoding a bi-functional protein comprising:
  • ICD intracellular domain
  • RLK receptor- like kinase
  • the invention is directed towards an isolated nucleic acid comprising a sequence encoding a bi-functional protein comprising:
  • ICD intracellular domain
  • RLK receptor- like kinase
  • the ICD preferably comprises a sequence encoding a kinase, e.g., residues 1594 to 2418 Of SEQ ID No. 10.
  • the first nucleotide sequence and said second nucleotide sequence are of said first plant species.
  • the first nucleotide sequence in (a) and said second nucleotide sequence in (b) may be separated by a nucleotide sequence encoding a transmembrane domain (TMD) and/or may further comprising a nucleotide sequence encoding a signal peptide (SP).
  • TMD transmembrane domain
  • SP signal peptide
  • the nucleic acid including the first nucleotide sequence in (a), the second nucleotide sequence in (b), and optionally the nucleotide sequence encoding a TMD sequence and/or the nucleotide sequence encoding a SP may correspond to or be based on a nucleotide sequence of one FER gene of a species of interest.
  • the isolated nucleic acid may be integrated into a vector and/or may be part of a composition.
  • a nucleic acid may be part of a kit for plant transformation.
  • the kit may also include, in a separate container, instructions as to how to use such a kit.
  • the invention is directed towards a bi-functional protein comprising:
  • ECD extracelluar kinase domain
  • RLK receptor-like kinase
  • ICD intracellular kinase domain
  • RLK receptor-like kinase
  • the invention is directed towards a bi-functional protein comprising:
  • ICD intracellular kinase domain
  • RLK receptor-like kinase
  • the bi-functional protein may be a fusion protein.
  • the present invention is also directed at uses of the above nucleic acids, nucleotide sequences, proteins, amino acid sequences, compositions, vectors and/or kits.
  • the present invention is also directed at a method of fertilizing an egg of a second plant species (recipient plant species) with a sperm of a first plant species (donor plant species) comprising: introducing into said second plant species (recipient plant species) a composition from a first plant species at least in part associated with pollen tube reception, and allowing sperm cells of said first plant species (donor plant species) to contact an egg of said second plant species, wherein a wild-type of said second plant species is separated from said first plant species by a species barrier.
  • the present invention is also directed at a transgenic plant cell of said second plant species (recipient plant species) transformed with the isolated nucleic acid comprising a sequence encoding said bi-functional protein.
  • the present invention is also directed at a transgenic plant of said second (recipient) plant species transformed with the isolated nucleic acid comprising a sequence encoding said bi-functional protein.
  • the present invention is also directed at a transgenic plant of said second (recipient) plant species produced via the methods disclosed herein and at progeny plants, in particular non-transgenic progeny plants, of such a transgenic plants as well as seeds obtained from the transgenic plant and/or their progenies.
  • the progeny plants and their seeds of said transgenic plant of said second (recipient) plant species may comprise a trait, gene(s) for a trait of the first plant species (donor) not present in the wild-type of the second (recipient) plant species and said progeny plant may be non-transgenic and devoid of the nucleic acid encoding the bi-functional protein from said first (donor) plant species.
  • the invention is also directed at a plant seed of said second (recipient) plant species which is true breeding for the trait, gene(s) for a trait of the first (donor) plant species.
  • transgenic plant and any of its progeny may be a monocot or a dicot.
  • FIGURE 1 Pollen tube reception is mediated by the FERONIA RLK.
  • A Diagram of a mature female gametophyte.
  • B 1 C Epifluorescence micrographs of aniline blue stained ovules. Fluorescent signal indicates the presence of callose, a major component of the cell wall of the pollen tube.
  • B A fertilized wild-type ovule, white arrow indicates the site of pollen tube arrest.
  • C An unfertilized fer gametophyte. The white arrow indicates the site of pollen tube arrest in wild-type ovules. The darker arrow shows the pollen tube growing inside the female gametophyte. Arrowheads indicate two pollen tubes entering the same ovule.
  • FIGURE 2 FERONIA, a plant-specific, widely expressed RLK phosphorylates itself.
  • A Unrooted tree showing FER and homologues from different species.
  • the FER homologues whose clade is supported by a 100% bootstrap value, are indicated in gray.
  • the other plant species together with A. thaliana in this clade are: Arabidopsis lyrata, Brassica oleracea, Brassica rapa and Cardamine flexuosa homologues 1 , 2, 3 and 4 belonging to the Brassicaceae, Oryza sativa (Os., Poaceae), and Populus trichocarpa (Salicaceae).
  • the rest of the tree contains the A. thaliana members of the CrRLKI L-1 subfamily of kinases.
  • the branch length scale bar represents 0.1 substitutions per site.
  • C Quantification of FER transcripts in leaves, closed flower buds, open flowers, siliques collected 1 to 4 days after hand pollination (1 -4 dap) and mature pollen grains. Transcript levels were normalized to 18s ribosomal RNA; average and standard deviation of three quantification replicas are shown.
  • FIGURE 3 FERONIA is expressed in developing primordia and synergid cells.
  • FP floral apex
  • OP ovule primordia
  • CW carpel wall
  • MP microspore cells
  • TP tapetum cells
  • PG pollen grain
  • CC central cell
  • EC egg cell
  • SC synergid cell
  • GE globular stage embryo.
  • FIGURE 4 FERONIA is a cell membrane-localized RLK targeted to the filiform apparatus and sequence divergence in its extracellularar domain correlates with fero ⁇ /a-like phenotypes in inter-specific crosses.
  • A Transmission image of a plasmolyzed onion epidermal cell transiently expressing FER-GFP under FER promoter (pFER::FER-GFP). Black arrow identifies the cell wall; white arrow identifies the cell membrane.
  • B Confocal Laser Scanning Microscopy (CLSM) single optical section of (A) with FER-GFP localized at the periphery of the cell membrane.
  • CLSM Confocal Laser Scanning Microscopy
  • C Epifluorescence micrograph of an onion cell transiently expressing GFP (35S::GFF).
  • D CLSM single optical section of leaf epidermis of an A. thaliana plant stably transformed with pFER "FER-GFP.
  • E A. thaliana leaf epidermal cell transiently expressing 35S::GFP.
  • F Ovule from the same plant as in (D) under CLSM. GFP signal in green, chlorophyll autofluorescence in red.
  • G Maximum projection of several sections of the micropyle area of ovule in (F) showing FER-GFP accumulation in the filiform apparatus of the synergids.
  • the least divergent homologue of C. flexuosa is shown.
  • Scale bars represent: 30 ⁇ m (A,B,F,I,J); 60 ⁇ m (C); 10 ⁇ m (D 1 E); and 20 ⁇ m (G,H).
  • SC synergid cell
  • FA filiform apparatus
  • EC egg cell.
  • FIGURE 5 Mapping and complementation of the fer mutation, and analyses of interspecific crosses.
  • A Diagram of the FER and PP2C loci showing the position of the Ds element and the 4-bp insertion. The positions of primers G1 , G2, and Ds3-3 (used in (B) for genotyping PP2C) as well as the intron spanning primers RT1 and RT2 (used in (C) for FER RT-PCR) are indicated.
  • B Genotyping PCR for complementation. A single 634-bp fragment indicates a wild-type plant, a 634, 460-bp doublet indicates a heterozygous plant, and a single 460-bp band indicates a homozygous mutant.
  • FIGURE 6 Sequence Alignment Amino acid alignment performed with ClustalX program (Pairwise parameters set to protein weight matrix Gonnet 250 with gap opening penalty of 35.00 and gap extension penalty of 0.75 and for multiple alignments gap opening penalty of 15.00 and gap extension penalty of 0.30) and protein features of FER relatives.
  • FIGURE 7 shows a Confocal Laser Scanning Micrograph of leaf epidermal cells expressing FER-GFP chimeric constructs. This image is representative of plants expressing either pJME3 or pJME4. FER-GFP is shown at the border of the cell confirming membrane localization.
  • Fig. 7b shows Confocal Laser Scanning Micrograph of an ovule expressing FER-GFP chimeric constructs in an A. thaliana ovule. Signal is strong in the synergids and ovule integuments.
  • the FERONIA (FER) gene of the plant system Arabidopsis thaliana (gene number At3g51550) (see also US Patent Publication 20030014776 to Grossniklaus et al., which is specifically incorporated herein by reference) encodes a receptor-like kinase (RLK).
  • RLKs are transmembrane proteins that receive signals through an extracellular domain (ECD) and subsequently activate signaling cascades via their intracellular kinase domain (or intracellular domain, ICD).
  • ECD extracellular domain
  • ICD intracellular domain
  • the pollen tube In embryo sacs carrying a mutated feronia (fer) allele, the pollen tube neither arrests its growth inside the synergid cell, nor ruptures to release sperm cells. Instead, the pollen tube continues to grow inside the fer embryo sac and fertilization is not achieved.
  • the FER gene generally comprises sequences encoding: a signal peptide (SP), an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD), the latter of which contains a kinase active domain.
  • SP signal peptide
  • ECD extracellular domain
  • ICD intracellular domain
  • gene refers to any segment of nucleic acid associated with a biological function, in particular with an advantageous trait of an organism, in particular a plant. Generally, such a gene encodes protein, however, genes encoding, e.g., functional RNA are also within the scope of the present invention. Genes may, in certain embodiments, also include non-expressed DNA segments that, for example, form recognition sequences for other proteins.
  • mutant or wild type plant species refers to a plant species prior to transformation.
  • ICD intracellular domain of a receptor-like kinase (RLK) according to the present invention is any ICD or a functional fragment or functional variant thereof that provides for the stated function of said ICD, namely actively signaling a cascade that leads to pollen tube arrest.
  • a functional fragment can, for example, comprise terminal and/or internal omissions compared to the wild type, as long as the stated function is still fulfilled.
  • a functional variant can, for example, contain one or more point mutations compared to the wild type.
  • a functional variant may also be a functional fragment, that is, comprise one or more point mutations as well as terminal and/or internal omissions as long as the functional variant maintains the stated function.
  • functional fragments have at least about 90% of the length of the wild type (wt) ICD, more preferably about 95% or even 98 or 99% of the length of the wt ICD.
  • Functional fragments and functional variants having the stated function, but whose functionality is reduced to about 70%, about 80% or about 90% of the wild type, or enhanced by about 10%, about 20% or about 30% compared to the wild type, are also within the scope of the present invention.
  • An "ECD” (extracellular domain) of a RLK, in particular of the FER RLK, according to the present invention is any ECD or a functional fragment or functional variant thereof that provides for the stated function of said ECD, namely to interact with the molecular ligand. This interaction preferably results in the signaling cascade mediated by the ICD.
  • a functional fragment can, for example, comprise terminal and/or internal omissions compared to the wild type, as long as the stated function is still fulfilled.
  • a functional variant can, for example, contain one or more point mutations compared to the wild type.
  • a functional variant may also be a functional fragment, that is, comprise one or more point mutations as well as terminal and/or internal omissions as long as the functional variant maintains the stated function.
  • Functional fragments and functional variants having the stated function, but whose functionality is reduced to about 70%, about 80% or about 90% of the wild type, or are enhanced by about 10%, about 20% or about 30% compared to the wild type, are also within the scope of the
  • a 'TMD" and "SP", respectively (TMD/SP) of a RLK according to the present invention is any TMD/SP or a functional fragment or functional variant thereof that provides for the stated function of said TMD/SP, which is that of a transmembrane domain located between the ECD and IDC and a signaling domain attached to the ECD, respectively.
  • a functional fragment can, for example, comprise terminal and/or internal omissions compared to the wild type, as long as the stated function is still fulfilled.
  • a functional variant can, for example, contain one or more point mutations compared to the wild type.
  • a functional variant may also be a functional fragment, that is, comprise one or more point mutations as well as terminal and/or internal omissions as long as the functional variant maintains the stated function.
  • Functional fragments and functional variants having the stated function, but whose functionality is reduced to about 70%, about 80% or about 90% of the wild type, or are enhanced by about 10%, about 20% or about 30% compared to the wild type, are also within the scope of the present invention.
  • Two species are, in the context of the present invention, said to be separated by a "species barrier” (also referred to herein as an interspecies, interspecific or crossing barrier) if, for instance, sperm cells of one species cannot interact with egg cells from another species to form viable embryos.
  • this species barrier results from a failed pollen tube reception. What is considered a species at a given point may vary considerably as knowledge about a certain plant changes. However, in the context of the present invention, two plants are said to be different species if these two plants are in fact separated by such a pre-zygotic species barrier.
  • a plant/seed is considered "true breeding" for a particular trait if it is genetically homozygous for that trait to the extent that, when the true-breeding plant is self-pollinated, a significant amount of independent segregation of the trait among the progeny is not observed.
  • the trait arises from the expression of one or more DNA sequences introduced into a plant via interspecific cross(es).
  • a "bi-functional protein” has, as the name implies, at least two functionalities. In the context of the present invention, one functionality may be that of an extracellular domain (ECD) and the other may be that of an intracellular domain (ICD).
  • the bi-functional protein may be encoded by a nucleic acid sequence of a first plant species, but may nonetheless function in a second plant species or vice versa. This is particularly true if the respective nucleic acid sequence is under the control of a promoter of the second plant species or first plant species, respectively.
  • the bi-functional protein may also be a fusion protein. In a preferred embodimient such a fusion protein comprise a ECD of a first plant species and an ICD of a second plant species.
  • the bi-functional protein is preferably a receptor like kinase (RLK), in particular FER RLK, and may also comprise TMD of varying length and composition and, at least temporarily, a SP.
  • a "transgenic plant” according to the present invention is a plant that possesses gene(s) that have been transferred by, e.g. the transformation methods described herein, from a different plant species.
  • a non-transgenic plant according to the present invention does not comprise such transferred gene(s).
  • a non- transgenic progeny plant is devoid of such transferred gene(s), but may retain genes obtained by interspecific crosses.
  • compositions consisting essentially of certain components, such as a vector(s), means in the context of the present invention that the composition consists of the specified vector(s) and any additional materials or components that do not materially affect the basic characteristics of the vector(s).
  • FER encodes a kinase-active RLK, which is localized at the plasma membrane and, in particular, is asymmetrically localized to the filiform apparatus of the synergid cells. Interspecific crosses indicated that the interaction of the FER RLK with a putative ligand on the pollen tube may be involved in reproductive isolation barriers.
  • FER of A. thaliana was molecularly cloned using positional methods.
  • the mutation was mapped to At3g51550, which in the fer mutant contains a 4-bp insertion corresponding to the footprint left after excision of a Ds transposon (Fig. 1 D).
  • Fig. 1 E the coding region of At3g51550 in the mutant was sequenced and a single base pair deletion was found (Fig. 1 E). Both mutations lead to frame-shifts that result in premature stop codons, therefore FER and SIR are allelic.
  • the FER open reading frame (ORF) contains a single 175 bp intron in the 5' untranslated region and produces a transcript of 2,682 bp, which encodes a putative receptor-like serine/threonine kinase (RLK) (Fig. 1 F).
  • FER which is, in A. thaliana, an unique gene, and belongs to the CrRLKI L-1 subfamily of kinases, whose members have no known function (Fig. 2A) (Shiu et al., 2003).
  • Plant RLKs belong to a monophyletic gene family with over 600 members (Shiu et al., 2001 ).
  • RLKs are transmembrane proteins that receive signals through an extracellular domain and subsequently activate signaling cascades via their intracellular kinase domain, a molecular function consistent with the role of FER in a signaling process.
  • the predicted intracellular domain (GST-FERwt) and a kinase-inactive translational fusion (GST-FERKR) were fused to glutathione S-transferase (GST). GST and GST-FERKR have no kinase activity, while GST-FERwt is autophosphorylated (Fig. 2B).
  • GST and GST-FERKR have no kinase activity, while GST-FERwt is autophosphorylated (Fig. 2B).
  • experimentally verified phosphorylation sites are present in the FER kinase domain (Nuhse et al., 2004) (Fig. 1 F, see asterisks).
  • FER encodes a kinase-active RLK involved in a novel signaling pathway that plays a critical role in the last stages of the communication between female and male gametophytes required for fertilization.
  • the temporal and spatial expression patterns of FER were examined.
  • FER mRNA was detected throughout the mature plant: specifically in leaves, buds, flowers and siliques, but it was not detected in mature pollen (Fig. 2C).
  • FER transcripts were detected in floral apices, young ovule primordia, and in young anthers with immature pollen (Fig. 3, A to C).
  • FER message was not detected (Fig. 3D), consistent with the quantitative real-time RT-PCR experiments and the role of FER in fertilization being female-specific.
  • a very weak FER signal was detected throughout mature unfertilized ovules, and a stronger signal could be detected in the synergid cells (Fig. 3E).
  • Fig. 3G After fertilization FER transcripts were detected in globular embryos (Fig. 3G) consistent with the finding that, in rare cases where fertilization of fer gametophytes is achieved, the resulting homozygous embryos abort (Huck et al., 2003).
  • FER::FER-GFP a FER-GFP construct driven by the FER promoter
  • the FER-GFP protein was localized to the plasma membrane (Fig. 4, A and B) in contrast to 35S::GFP, which is detected throughout the cell (Fig. 4, C and E).
  • the pFER::FER-GFP construct was also stably transformed into A. thaliana, fully complemented the fer mutation (Table 1 ), and the fusion protein was localized at the plasma membrane in leaf epidermal cells (Fig. 4D, see also Fig. 7A).
  • Unfertilized ovules accumulated high levels of GFP signal in the lower part of the synergids (Fig. 4F) where the filiform apparatus is located.
  • Weaker GFP fluorescence was detected in the membranes of the synergid cells, the surrounding maternal sporophytic cells, as well as faintly in the egg cell (Fig. 4G). Since the filiform apparatus is a structure rich in plasma membrane, it was tested whether the asymmetric distribution of FER may simply be due to an enrichment of plasma membrane in the area. Therefore, the distribution of FER was compared to that of another GFP fusion construct that has a plasma membrane localization motif and is expressed in the female gametophyte (pAtD123::EGFP- AtROP ⁇ C).
  • FER-GFP levels were much higher in the filiform apparatus than in the rest of the synergids' cell membranes when compared to EGFP- AtROP ⁇ C (Fig. 4H). This finding suggests that FER is polarly transported to the filiform apparatus. Taken together, the data appear to show that FER, localized in the filiform apparatus, binds a putative ligand on the approaching male gametophyte, which then triggers the molecular events involved in pollen tube reception.
  • failed receptions are either caused by a divergent ligand that is inefficiently recognized by the receptor domain of the A. thaliana FER-RLK, or due to the presence of two polymorphic forms of the putative ligand, one which is and one which is not recognized by the A. thaliana FER-RLK.
  • the ligand or ligands are predicted to have diverged to a degree that they cannot be recognized by the A. thaliana FER-RLK, thereby causing the fer-like phenotype in A. thaliana ovules.
  • FER homologues in these species were isolated and the ratio between the number of non-synonymous substitutions per non-synonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) (Nei et al., 1986) were calculated as an indication for their divergence in pair-wise comparisons between A. thaliana and A. lyrata (one FER homologue) or C. flexuosa (4 FER homologues) (Fig. 4K, Fig. 5D).
  • Figure 6 shows amino acid alignments performed with ClustalX program (Pairwise parameters set to protein weight matrix Gonnet 250 with gap opening penalty of 35.00 and gap extension penalty of 0.75 and for multiple alignments gap opening penalty of 15.00 and gap extension penalty of 0.30) and protein features of FER relatives.
  • accession numbers all of which are specifically incorporated herein by reference in their entirety: "Boleracea” EF681 131 Brassica oleracea; “Alyrata” EF681133 Arabidopsis lyrata; A. Thaliana L-er (“L-ER”), EF681137 A.
  • the sequence starts with a 20 to 40 base pair long signal sequence.
  • the ECD follows the signal sequence cleavage site (grey) and is about 430 to 440 amino acid long.
  • grey signal sequence cleavage site
  • the ECD shows high variability between species and, according to the present invention, serves, according to one aspect of the invention, as the primary tool to overcome species barriers.
  • certain amino acids of the ECD are also conserved between the shown species.
  • the TMD (dark grey), which is also highly variable and generally does not play any part in the species barriers, is about 20 amino acids long and is followed by the ICD, which displays, relative to the ECD, a considerably higher degree of sequence identities between species, especially in the kinase domain (italics), and has about the same length as the ECD. Modifications of the FER protein
  • the FER protein as well as individual domains of the FER protein, in particular the ECD and the ICD, can be modified using site-directed mutagenesis.
  • the changes to the nucleotides will result in amino acid changes in the translated protein.
  • primers with nucleotide changes are used to amplify fragments of the FER gene. Restriction digestion, ligation and/or fusion PCR can be used to replace a wild type domain with the modified domain.
  • Functionality of the modified FER gene can then be tested by biochemical assays (for changes in the kinase domain) and/or by introducing the modified version of FER into fer mutant plants using Agrobacterium transformation followed by a determination of whether or not the modified protein can complement the fer mutant phenotype.
  • the biochemical assay of FER kinase activity is performed by expressing normal and modified versions of the FER intracellular domain (ICD) as glutathione S-transferase (GST) fusion proteins in E. coli.
  • the fusion proteins are purified on glutathione-coupled resin and then used for in vitro kinase assays.
  • the purified GST-FER fusion proteins are incubated with radiolabeled ATP and putative phosphorylation acceptor proteins, then subjected to PAGE analysis and autoradiography to detect phosphorylated proteins.
  • the assays have shown that FER is capable of auto-phosphorylation in vitro. Mutation of the lysine 565 in the CoI-O ecotoype (at position 593 in Fig.
  • the FER protein has two serines (S695 and S701- at positions 724 and 730 in Fig. 6, which also includes the Signal Sequence) and a threonine (T696- at position 725 in Fig. 6) in the predicted activation loop that have been shown to be phosphorylated in a phosphoproteomic analysis of membrane proteins in Arabidopsis (Nuhse et al., 2004). Changes of these amino acids singly or in pairs to aspartic acid had no effect on the ability to complement the mutant phenotype. However, when all three amino acids were changed, the construct was no longer able to complement the fer mutation. These results indicate that at least one Ser or Thr in the activation loop is necessary for FER function.
  • the extracellular domain of FER is highly divergent in different species (while the kinase domain is highly conserved) suggesting that the extracellular domain provides specificity for a ligand, but does not affect the core biochemical function of the protein.
  • the extracellular domain of FER is modified or substituted to change ligand-binding specificities.
  • An ECD of a first species having an advantageous trait e.g. X
  • X an advantageous trait
  • the FER protein of the first organism can be expressed in the second organism under the regulatory elements of the second organism.
  • the first and second organisms are, however, in any case, separated by a species barrier.
  • the amino acid sequence of FER identified from, e.g., A. thaliana can be used to identify related amino acid sequences from other species. The amino acid sequences can then be tested as to whether they perform a role similar or identical to the amino acid sequence of FER. In a preferred embodiment, only the ECD domain of FER of, e.g., A. thaliana is used for identification purposes since the ICD is shared among several proteins including non-RLK that perform functions not related to pollen tube explosion.
  • the amino acid sequence can be used in the online available BLAST program (Basic Local Alignment Search Tool). The BLAST searches nucleotide and protein databases and detects regions of similarity in otherwise unrelated proteins.
  • results of the searches can be used to design primers to isolate the corresponding FER amino acid sequences from other species when enough similarity between the two proteins exists.
  • Similarity is the extent to which nucleotide or protein sequences are related. In BLAST, particularly, similarity refers to a positive matrix score. The extent of similarity between two sequences can, in the context of BLAST, be based on percent of so called “sequence identity” and/or “conservation” according to BLAST. In the context of BLAST “identity” refers to the extent to which two (nucleotide or amino acid) sequences are invariant.
  • BLAST refers to changes at a specific position of an amino acid or, less commonly, DNA sequences, that preserve the physico-chemical properties of the original residue.
  • BLAST user decides which sequences are relevant.
  • SP-ECD SP-ECD of FER from A. thaliana:
  • BLAST analysis using any other known SP-ECD or ECD of FER such as, but not limited to, the ECD of FER of A. lyrata, can be performed. Sequences with low e-values (expectancy values) are selected.
  • the so identified potential FERs can be tested for functionality as a whole or, alternative, the identified ECDs can be fused to the ICD of an appropriate plant, preferably via a TMD and then be tested for its functionality as further discussed below.
  • the FER gene from A.lyrata (one sequence) and from C. flexuosa (4 homologous sequences that are likely alleles as C. flexuosa is a tetraploid plant; "Cflexuosa 4, 3, 2 and V), obtained by isolation, are shown in Fig. 6. Comparison of the sequences isolated from these species revealed that the ECD is more variable while the ICD is more conserved. This correlation and the results from the interspecific crosses strongly suggest that a lack of recognition between ECD of FER from A. thaliana and its putative male ligand in A. lyrata and C. flexuosa is responsible for the failure of pollen tube reception in crosses between these species.
  • Certain embodiments of the present invention comprise isolated nucleotide sequences, which might be integrated into, e.g., vectors, for the production of a bi-specific protein.
  • a bi-specific protein may, in certain embodiments, be a fusion protein comprising the ECD of the FER gene of a first plant species (e.g., C. flexuosa) and the TMD and ICD of the FER gene of a second plant species (e.g., A. thaliana).
  • the production of the bi-specific protein may be, e.g., driven by the FER promoter sequence of the second species (e.g., A. thaliana).
  • the bi-specific protein is in certain embodiments encoded by the nucleotide sequence of one species (e.g., the entire coding sequence of the A. lyrata FER gene).
  • the resulting protein will display ECD activity of a first species (e.g. A. lyrata) and ICD activity that is compatible with that of a second species (e.g., A. thaliana).
  • a first species e.g. A. lyrata
  • ICD activity that is compatible with that of a second species
  • A. thaliana e.g., A. thaliana
  • Examples 17 and 18 reflect results obtained with vectors pJME3 and pMJE4, which contain the A. lyrata FER open reading frame/genomic piece (for details see Examples) and were used to transform Hyg resistant progeny of fer/FER and FER/FER plants, which upon transformation, showed expression of the GFP in synergids, ovule integuments and leaf epidermal cells (Fig. 7) (see also, Escobar- Restrepo et al. (2007)). pJME3 rescued the fer mutation in fer/+ plants.
  • Ds/Ds plants showed an increase of fertilized ovules to 68%.
  • pJME4 rescued the fertilization defect in fer/+ plants.
  • Seed counts showed an increase from the expected 50% to 72% fertilized ovules.
  • bi-specificity of the protein is accomplished by the introduction of certain mutations, e.g., point mutations or insertion and/or deletion mutations into preferably the ICD sequence.
  • a vector encoding such a bi- specific protein into a second plant species (e.g., A. thaliana), and, e.g., placing the respective isolated nucleotide sequence under the control of a promoter, such as a FER gene promoter of the second species (e.g., A. thaliana)
  • these plants (second species) become compatible female partners in interspecific crosses with pollen from the first species.
  • the reception of the pollen tube is achieved via the introduced ECD or, e.g., the full length sequence of the FER of the first plant species.
  • the ECD of FER is used, in one embodiment, to break the species barriers between two otherwise incompatible species.
  • the person skilled in the art will readily appreciate, while certain embodiments of the present invention are designed to break species barriers between species of Brassicacea, barriers between any species, in particular species in which the crossing barrier results from of an incompatibility at pollen tube reception, can be broken with the tools provided herein.
  • FER homologues from a wide variety of species.
  • homologues of FER are found in the Oryza (Rice) public database.
  • Fig. 6 two sequences (see Fig. 6) have been identified, namely Os03g21540 and Os01g56330.
  • the ECD is less conserved than the ICD:
  • sequences, in particular the ECD, of FER serve as templates for the design of primers that allow the isolation of FER homologues in Monocotyledonous or Dicotyledonous species.
  • sequence identities In the context of the present invention, how two sequences are related will be expressed in terms of "sequence identities,” a term that will be further explained below.
  • the first and/or second species may be a species of an angiosperm, including a monocotyledon and dicotyledon.
  • angiosperms which are contemplated by the present invention: Amaryllidaceae, Anacardiaceae, Asteraceae, Araceae, Arecaceae, Brassicaceae, Caricaceae, Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Dioscoreaceae, Euphorbiaceae, Fabaceae, Lauraceae, Malvaceae, Moraceae, Musaceae, Oleaceae, Poaceae, Rosaceae, Rubiaceae, Rutaceae, Solanaceae, Umbelliferae, Vitaceae.
  • the nucleotide sequence encoding a FER protein of a first plant species is identified and isolated (referred to herein as O. yield).
  • this isolated nucleotide sequence is cloned into an appropriate plant vector, here, pMDC1 11 , and a second plant species, such as another species of rice, having another advantageous trait, here pest resistance (referred to herein as O. resist), but being separated from O. yield by a species barrier, is transformed with this vector.
  • a pollen tube germinates out of the pollen grain of the first plant species (e.g., O. yield), after being transported to the stigma of the second plant species (e.g., O.
  • the synergid of the second plant species e.g., O. resist
  • the approaching pollen tube e.g., O. yield
  • the pollen tube causes the pollen tube to arrest its growth after penetration of one of the synergids and to rupture inside the synergid cell to release sperm cells.
  • One of the released sperm cells ferilizes an egg of the second plant species (e.g., O. resist) to give rise to an embryo comprising genetic material of O. yield.
  • the nucleotide sequence encoding an ECD of the FER protein of a first plant species called herein O. yield, is identified and isolated.
  • the nucleotide sequence encoding an ICD as well as the TMD and SP of the FER protein of a second plant species O. resist is also identified. Identification is performed via primers having sequence identities/homologies to the ECD of the FER protein of another plant species, e.g., A. thaliana.
  • the nucleic acid sequence encoding the ECD of O. yield and the nucleic acid sequence encoding the ICD, TMD and SP of the O. resist are cloned into a vector, here, pMDC111 , under the control of a promoter. O. resist is transformed with this vector. A pollen tube germinates out of the pollen grain of a O. yield plant, after being transported to the stigma of the transformed O.
  • a first plant species e.g., jimson weed
  • has an advantageous property e.g., resistance against apple scab fungus.
  • the region "RESIST" of the genome of jimson weed which causes the resistance is well known and can be readily identified with methods known in molecular biology.
  • a second plant species e.g., malus vulgaris (apple)
  • Such a resistance is lacking, but is highly desirable.
  • Crosses are contemplated between the first plant and the second plant to introduce the desirable trait, e.g. resistance into the second species.
  • the plants are separated by a species barrier.
  • a transgenic plant based on apple is produced via, e.g., a vector carrying, e.g., the ICD of the FER protein of apple and the ECD of the FER protein of jimson weed.
  • the vector is introduced into calli of an apple plant via, e.g., particle bombardment and a transgenic apple plant is produced that expresses this fusion protein in all cells.
  • the first plant (jimson weed) can now be crossed with the transgenic apple plant and progeny plants are grown. Out of these progeny plants, those are selected that carry the region "RESIST" or a functional variant thereof.
  • sequence identity refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has recognized meaning in the art and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • nucleic acid molecule is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, a wild type ICD nucleic acid sequence, or a subportion thereof, can be determined conventionally using known computer programs such as DNAsis software (Hitachi Software, San Bruno, Calif.) for initial sequence alignment followed by ESEE version 3.0 DNA/protein sequence software (cabot@trog.mbb.sfu.ca) for multiple sequence alignments.
  • DNAsis software Haitachi Software, San Bruno, Calif.
  • ESEE version 3.0 DNA/protein sequence software cabot@trog.mbb.sfu.ca
  • Whether the amino acid sequence is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to, for instance a wild-type ICD amino acid sequences, or a subportion thereof, can be determined conventionally using known computer programs such the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711).
  • BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleic acid sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • a nucleotide sequence is said to be based on nucleotide sequences of at least one FER genes if it is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleic acid sequence of a wild type FER gene.
  • Fusion proteins can be generated by, e.g., methods involving restriction digest followed by DNA ligation and/or fusion PCR. If natural restriction sites are not present in the desired DNA fragments, then restriction sites can be introduced into PCR primers. These primers are used to amplify the fragments, the products are digested with the fitting restriction enzymes, and finally the two fragments are ligated together. Alternatively, PCR primers may be used which contain tails that are homologous to the other fragment. Sequential PCR reactions are then used to fuse the fragments together.
  • the complete fusion gene is introduced into a vector containing promoter and terminator sequences necessary for plant expression, along with an antibiotic or herbicide resistance gene that allows for selection of transformed plants.
  • the Agrobacterium binary vector is transformed into, e.g, Agrobacterium tumafaciens, which is then used to infect plant cells in order to achieve stable integration of the DNA into the plant genome.
  • Agrobacterium transformation can be performed using the floral dip method (in receptive plants such as Arabidopsis thaliana (see,e g., Clough et al. (1998)) or by tissue culture techniques in which whole plants are regenerated from transformed undifferentiated cells (calli).
  • heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line.
  • it may be useful to target heterologous DNA insertion in order to achieve site-specific integration e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression.
  • site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.
  • Transformation methods of this invention are in one preferred embodiment practiced in tissue culture on media and in a controlled environment.
  • Media refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism.
  • Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells.
  • Practical transformation methods and materials for making transgenic plants of this invention, e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. patent application Ser. No. 09/757,089, which are incorporated herein by reference.
  • Marker genes are used to provide an efficient system for identification of those cells with nuclei that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes.
  • Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.
  • GFP green fluorescent protein
  • GUS beta-glucuronidase or uidA gene
  • Other marker genes confer resistance to a selective agent, such as an antibiotic or herbicide.
  • telomeres are useful selective marker genes.
  • useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptll), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable markers are illustrated in U.S. Pat. Nos.
  • Cells that have been scored positive in a screening assay may be cultured in regeneration media and allowed to mature into plants.
  • Developing plantlets can be transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO 2 , and 25-250 microeinsteins m ⁇ 2 s- 1 of light, prior to transfer to a greenhouse or growth chamber for maturation.
  • Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue.
  • cells are grown to plants on solid media at about 19 to 28 5 C. After regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.
  • Plants may be pollinated using conventional plant breeding methods known to those skilled in the art and seed may be produced.
  • Progeny may be recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide.
  • useful assays include, for example, "molecular biological” assays, such as Southern and Northern blotting and PCR; "biochemical” assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
  • Arabidopsis growth conditions were as previously described (Huck et al., 2003) and equally applied to Arabidopsis lyrata (a gift from Detlef Weigel, Max Planck Institute f ⁇ r Ecksbiologie, Tubingen), Brassica oleracea (NASC line N29002) and Cardamine flexuosa plants (collected at the Botanical Garden of the University of Zurich).
  • the fer mutant was isolated from a collection of transposants containing enhancer detector Ds transposable elements in Ler Arabidopsis plants (Huck et al., 2003). Isolation of flanking sequences indicated that the Ds element is inserted in PP2C, a protein phosphatase gene (At3g51470) on BAC F26O13. However, excision of the Ds element failed to produce phenotypically wild-type revertants and a genomic fragment containing PP2C failed to complement the mutation, indicating that FER was not disrupted by the Ds element (Huck et al., 2003).
  • a mapping population comprising about 1 ,200 F2 plants was generated from two parental crosses between the Columbia (CoI-O) accession as pollen acceptor and fer/FER plant as pollen donor.
  • the F1 plants were selected for both the resistance to kanamycin (conferred by the Ds element) and the fer phenotype (semi-sterility, where half the ovules remain unfertilized).
  • the F2 progeny were grown without selection.
  • PCR markers were generated based on InDeI (Insertion-Deletion) polymorphisms between Ler and CoI-O (data not shown).
  • the InDeI sequences are available at the Cereon database (www.arabidopsis.org/Cereon/index.html).
  • a starting population of 46 plants was used to identify markers both proximal and distal to the Ds insertion that resulted in no more than 4 recombination events (roughly 2-8 centiMorgans or 0.25-2 Mb away from FER).
  • the InDeI markers BAC F11C1 and BAC F18021 fit this criterion, and were used to identify recombination events in this region from the whole mapping population. All plants were also screened for semi-sterility.
  • the identified recombinant ferIFER plants were further tested with a series of additional markers to narrow down the interval containing the fer mutation.
  • no recombinant was isolated that separated the semi-sterility caused by the fer mutation from the Ds element. It was therefore not surprising that fer mapped close to the Ds insertion, between coordinates 32 kb and 88 kb of BAC F26O13.
  • the identified interval contains 19 annotated genes, with nearly half of the genes encoding proteins of unknown function.
  • the number of candidate genes was further reduced based on gene expression data from Affymetrix GeneChip® experiments (Henning et al., 2004). Genes whose steady- state transcript levels are higher in reproductive tissue were analyzed using denaturing high performance liquid chromatography (dHPLC, see below).
  • Samples for dHPLC analysis were obtained using genomic DNA from fer/FER, Ler, and F1 plants of a cross between CoI-O and fer/FER (CoI x fer) as a template to generate 500-700 bp PCR fragments with overlaps of ca. 100 bp from the predicted transcripts: At3g51470, At3g51480, At3g51500, At3g51510, At3g51520, At3g51530, At3g51550 and At3g51580.
  • PCR amplification was performed on an MJ PTC-200 thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA) with initial denaturation at 94 °C for 2 minutes and 40 cycles, each with denaturation at 94 °C for 15 seconds, annealing at 5O 0 C for 15 seconds and extension at 72°C for 30 seconds, and a final extension at 72 0 C for 3 minutes. All PCR fragments were analyzed with an automated Wave® dHPLC instrument (Transgenomic, Crewe, UK) equipped with a DNASep® column (Transgenomic). The optimal temperature and solvent gradients for dHPLC analysis were calculated with the WAVEMAKER® software (version 4.1.42; Transgenomic).
  • DNA extracted from leaves of sir/SIR plants was used for PCR amplification and sequencing of the genomic region that comprises the FER gene in overlapping fragments of 600 to 800 bp.
  • Analysis of sequence chromatograms from heterozygous plants revealed a 1 bp deletion in a 640 bp PCR fragment amplified with primers 5'-GTG AAA CCA GAG TCA ATG CTG-3' and 5'-CCC TGT AGC ATC AGG TCG-3'.
  • This PCR product was subsequently cloned into pDRIVE vector and several clones were sequenced. The deletion was confirmed in about half of the sequenced clones while the rest showed the wild-type sequence.
  • At3g51550 a 6.4 kb fragment spanning 2.5 kb upstream of the initiation codon to 1.2 kb downstream of the stop codon of At3g51550 was PCR amplified from Lerwith Expand High Fidelity PCR (Roche Diagnostics, Mannheim, Germany) using primers 5'-CTT CAA CAT CTT CCA ATG GAG-3' and 5'-TGT TGC GGA TCC AAC TAG GCC AGG-3' (with an introduced BamHI site). The product was cloned into pTOPO vector (Invitrogen).
  • Positive clones were digested with BamHI and cloned into pCAMBIA-3300 linearized with BamHI. Plasmids containing the gene were sequenced and introduced into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method, and used for transformation of Arabidopsis fer/FER plants using the floral dip method (Clough et al., 1998). The progeny were collected, grown, and sprayed with BAST A ® (Oyma, Oftringen, Switzerland) to select transformants.
  • the Ds element is tightly linked to the fer mutation (Huck et al., 2003) and in the mutant no homozygous plant for the Ds element can be recovered because of female gametophytic and embryonic lethality. Therefore, if lines containing the complementation construct are also homozygous for the Ds element, we can conclude that At3g51550 complements the fer mutation.
  • DNA was extracted from BAST A ® (Oyma, Oftringen, Switzerland) resistant plants to genotype with primers G1 5'-AAG CAC TCC TTG TTG CCT TC-3 1 and G2 5'-CAC ATT GGA AGT CGC AGA TG-3' specific for the PP2C gene and primer Ds3-3 5'-GTT ACC GAC CGT TTT CAT CC-3' specific for 3' end of Ds element (Fig. S1 A).
  • FER transcripts were amplified with the intron spanning primers RT1 5'-CTC TCT CCG ATT TCA TCG CTT AGG-3 1 and RT2 5"- GGA TCT TGT GTT AAC GCT GG-3' (Fig. SIA).
  • a 1.7 kb fragment of FER was amplified from genomic DNA with primers 5'-CTA ATC TAG ACG ACA CAG ATA ACC G-3' (with an introduced Xbal site) and 5'-CAA TCA AGG ACA CAA GAT GAC G-3'.
  • the fragment was cut with Hindlll and Xbal and inserted into pBluescript KS.
  • the plasmid was digested with Hindlll and Spel, the single-stranded overhangs filled in with T4 DNA polymerase, gel purified and religated to obtain a plasmid containing a 941 -bp FER specific sequence.
  • the antisense probe was obtained by in vitro transcription of the Xbal linearized plasmid with T3 RNA polymerase in the presence of digoxigenin-labeled rUTP (Roche Diagnostics, Mannheim, Germany).
  • the sense probe was generated identically, except that Sail was used to linearize the vector and T7 RNA polymerase was used for in vitro transcription (Roche Diagnostics, Mannheim, Germany). These probes were hydrolyzed with alkaline carbonate buffer (60 mM Na 2 CO 3 , 40 mM NaHCO 3 ; pH 10.2) to a size of approximately 150-200 bp according to the manufacturer's instructions (Roche Diagnostics).
  • alkaline carbonate buffer 60 mM Na 2 CO 3 , 40 mM NaHCO 3 ; pH 10.2
  • 1.3 kb of putative upstream regulatory sequence was amplified with Long Expand PCR (Roche Diagnostics, Mannheim, Germany) from genomic DNA using primers 5'-AGG CTT CAA CAT CTT CCA ATG GAG-3' and 5'-GAG ACG GAA TCG TCC CAT GGT GAT CTT CAT CGA TC-3' (with an introduced Ncol site).
  • the PCR product was digested with Ncol and BamHI, ligated into pCAMBIA-1391 z linearized with Ncol and BamHI, transformed into Escherichia coli strain DH5 ⁇ . Transgenic plant lines were created as described above.
  • More than 20 primary transformants were isolated on 0.5X MS medium agar plates containing hygromycin (20 ⁇ g/ml) and subsequently transferred to soil. After flowering, ovules were dissected and GUS assays were performed as described previously (Vielle- Calzada et al., 2000).
  • the amplified product was cloned into pDONR207 by the BP recombination reaction via the GATEWAY system (Invitrogen, Carlsbad, U.S.A) according to the manufacturer's instructions.
  • This construct was used to transfer the promoter and coding region upstream of a GFP reporter gene (mGFP6) in the plant transformation vector pMDC1 11 (Curtis et al., 2003) (a gift of Dr, Mark Curtis) by the LR recombination reaction (Invitrogen, Carlsbad, U.S.A), resulting in the pFER::FER-GFP construct.
  • mGFP6 GFP reporter gene
  • This vector was used both for transient expression assays (by biolistic bombardment of onion skin epidermal cells and Arabidopsis leaves, as described previously (Varagona et al., 1992)) and for generation of transgenic plant lines (as described above).
  • plasmolysis was induced by incubation of the onion cells in 0.8M mannitol.
  • Non-specific localization in the transient expression assays was demonstrated using the plasmid ppkl OO, in which the GFP is under the control of a double Cauliflower mosaic virus 35S RNA promoter (35S::GFP) (a gift of Drs. Robert Blanvillain and Patrick Gallois, University of Manchester, UK).
  • the pAtD123::EGFP- AtROP ⁇ C line used as control was constructed as follows: The 1857bp promoter region (-1852 to +5) and 585bp 3'-UTR of the AtD123 gene (At4g05440) was obtained by PCR with primer combinations 5'- ⁇ aaqCTTTAAACCTACCACACTATA (Hindlll) / 5'- ⁇ CT ⁇ CA ⁇ CGCGATT AACGAATTCGT (Pstl) and 5'-
  • the EGFP-AtROP6C fusion was made by combining the EGFP gene and the DNA sequence coding for the C-terminal 20 amino acid of AtROP6 ( 10) (Bischoff et al., 2000), then cloned into the p1300AtD123 cassette and used to transform A.thaliana plants as previously described.
  • Specimens were analyzed with a TCS SP2 confocal laser-scanning microscope (Leica, Bensheim, Germany) using excitation at 488 nm and recording the emission from 495 to 525 nm.
  • Single-focusing-plane images of 1024x1024 pixels were recorded with a scan speed of 400 Hz.
  • 3D stacks were recorded as images of 1024x1024, using 2OX and 40X glycerol-immersion objectives.
  • amino acids 476-922 of FER (the predicted intracellular domain) from the CoI-O ecotype were fused to glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • EcoR1 and SaM sites introduced with the PCR primers 5'-TCA CGA ATT CCG TGG TGA TTA CCA GCC TGC-3' and 5'-GAG TCG ACG TCC CTT TGG ATT CAT G-3' were used to insert the FER intracellular domain into digested pGEX-4T-1 (Amersham Biosciences).
  • a kinase-inactive translational fusion (GST-FERKR) was prepared by site- directed mutagenesis with primers 5'-CAG GAG AGG CAA CCC AAT GTC C-3' and 5'- TTG GGT TGC CTC TCC TGA TGG CTA CCT TTG-3' to change the essential lysine 565 (In CoI-O) in the predicted phosphotransfer domain ( 77) (Hanks et al., 1988) to an arginine.
  • the expression plasmids were transformed into E. coli strain BL21(DE3) for induction.
  • the in vitro kinase assay was modified from that described in Peck, 2006. GST and GST-fusion proteins immobilized on glutathione Sepharose 4B beads were incubated in kinase buffer (5OmM HEPES-KOH, pH7.5; 15OmM NaCL; 0.1 mM EGTA; 10 mM MgCI2; 10% glycerol; 1 mM DTT; 0.1 mM ATP and 37 MBq of [ ⁇ - 32 P]ATP) for 15 minutes at room temperature. The kinase assay was stopped by adding SDS/PAGE sample buffer and heating the reaction to 65°C for 5 min. The samples were run on SDS/PAGE and the gel was stained with Coomassie brilliant blue for visualization of the proteins.
  • kinase buffer 5OmM HEPES-KOH, pH7.5; 15OmM NaCL; 0.1 mM EGTA; 10 mM MgCI2; 10% glycerol; 1 mM DTT;
  • DNA was extracted by the phenol :chloroform method (Sambrook et al., 1989) from A. lyrata, A. thaliana (Le ⁇ , B. oleracea and C. flexuosa.
  • primers that amplify the genomic region of FER were designed based on the deposited sequences in the A. thaliana public database (Rhee et al., 2003). Nucleotide sequences from the putative extracellular domain of Arabidopsis were used for BLAST searches in the TIGR B.
  • thaliana were used with the DNA Walking Kit (Seegene, Inc. Seoul, Korea). After amplification, fragments were cloned and sequenced to design primers: 5'-GAG CTG CTC CGA TCG ATG-3' and 5'-CGG TCT CCT TTG GAT GGA GC-3' that amplify the complete coding region of FER. All PCR products were cloned into pDRIVE and several clones were sequenced. 4 different sequences were found for C. flexuosa, indicating the presence of several homologues in this species. The PROSITE motif search (Bairoch et al., 1997) and CBS Prediction Servers (Bendtsen et al., 2004, Sonnhammer et al., 1998) were used to predict the protein domains.
  • GenBank numbers (Genbank submission bankit numbers) for FER homologues (as of August 2, 2007): B. oleracea (EF681131 (bankit91556)), A. lyrata (EF681133 (bankit921006)), A. thaliana L-er(EF681137 (bankit921012)), C .flexuosa 1 (EF681134 (bankit920059)), C .flexuosa 2 (EF681135 (bankit920061)), C .flexuosa 3 (EF681136 (bankit920065)), C .flexuosa 4 (EF681132 (bankit920057)), all of which are incorporated herein by reference in their entirety.
  • Pairwise parameters set to protein weight matrix Gonnet 250 with gap opening penalty of 35.00 and gap extension penalty of 0.75 and for multiple alignments gap opening penalty of 15.00 and gap extension penalty of 0.30 (Hall, 2001).
  • the phytogeny of the aligned sequences was generated using the neighbor-joining method and bootstrap analysis was conducted with 1 ,000 replicates.
  • the software Treeview (Page, 1996) was used for the display of the unrooted tree. Pairwise alignments of nucleotide and amino acid sequences were processed with BioEdit Sequence Alignment Editor v 5.0.9 to improve the alignment quality (Hall, 1999).
  • the amplified product was cloned into pDONR207 by the BP recombination reaction via the GATEWAY system (Invitrogen, Carlsbad, U.S.A) according to the manufacturer's instructions.
  • This construct was used to transfer the promoter and coding region upstream of a GFP reporter gene (mGFP6) in the plant transformation vector pMDC111 (a gift of Dr, Mark Curtis) by the LR recombination reaction (Invitrogen, Carlsbad, U.S.A), resulting in the pFER::FER-GFP construct.
  • Subsequent sequencing confirmed the reading frame of the fusion.
  • Plasmids containing the gene were sequenced and introduced into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method, and used for transformation of Arabidopsis plants using the floral dip method.
  • Primary transformants were isolated on 0.5X MS medium agar plates containing hygromycin (20 ⁇ g/ml) and subsequently transferred to soil.
  • hygromycin 20 ⁇ g/ml
  • Table 1 At3g51550 encoding a receptor-like kinase complements the feronia mutant. Representative data for 4 of the 41 complementation lines are shown, either carrying a genomic (Tf ⁇ FEfl]) or a FER-GFP fusion protein (T ⁇ pFER::FER-GFP ⁇ ) complementation construct. Lines carrying a single, unlinked complementation construct were chosen. Homozygosity for ferwas determined by PCR (see above online material and methods).
  • FERONIA fusion proteins containing extracellular and intracellular domains from different species are produced via PCR and restriction digest-based methods using the respective nucleotide sequences .
  • the fusion proteins are introduced into binary vectors for Agrobacterium tumafaciens transformation, and the resulting Agrobacterium strains were used to transform plants by the floral dip method. Transformants were selected on media with antibiotics, and verified with PCR.
  • Construct pJME3 and pJME4 serve as examples (Example 17 and 18). In these tow constructs, the entire FER protein of A. lyrata was introduced into A. thaliana.
  • Example 17 Construct pJME3 pJME3 is a fusion of a A. thaliana promoter to the A. lyrata FER (SEQ ID No. 21) open reading frame (ORF).
  • A. lyrata FER SEQ ID No. 21
  • ORF open reading frame
  • the difference between the ICD of A. thaliana and A. lyrata is 1 amino acid. This amino acid was not expected to and did not influence the outcome of experiments, in particular as it is not inside a conserved domain, e.g., the kinase domain.
  • a genomic piece of FER from A. lyrata (SEQ ID No. 10) spanning 2 Kb of the putative promoter upstream of the ATG plus 2.7 Kb of the open reading frame without the stop codon, was cloned in frame to the GFP sequence in PMDC111.
  • constructs were introduced into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method, and used for transformation of Arabidopsis fer/FER and wild type plants (FER/FER) of the L-er accession using the floral dip method (Clough et al. (1998)).
  • Transformants were selected in MS (Murashige and Shoog media for plant culture) plates supplemented with Hygromycin (Hyg). F1 Hyg resistant plants were analyzed with the following techniques:
  • Fig. 7a shows a Confocal Laser Scanning Micrograph of leaf epidermal cells expressing FER-GFP chimeric constructs. This image is representative of plants expressing either pJME3 or pJME4. FER-GFP is shown at the border of the cell confirming membrane localization.
  • Fig. 7b shows Confocal Laser Scanning Micrograph of an ovule expressing FER-GFP chimeric constructs in an A. thaliana ovule. Signal is strong in the synergids and ovule integuments.
  • F1 transformants were emasculated and used as female partners in crosses with A. lyrata pollen and analyzed through aniline blue staining of callose followed by fluorescence microscopy analysis.
  • plants that are homozygous for both the transgene and the fer mutation will be obtained.
  • the only functional FER protein is encoded by the introduced transgene.
  • These plants are crossed to A. lyrata pollen and are analyzed for a reduction of the pollen tube overgrowth phenotype.

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Abstract

La présente invention concerne des outils, notamment des procédés et des vecteurs qui permettent de franchir des barrières d'espèce dans des végétaux supérieurs. Elle concerne des vecteurs comprenant tous les éléments fonctionnels du gène FER d'une espèce de végétaux donneurs ou, alternativement, des acides nucléiques codant le domaine extracellulaire (ECD) d'une protéine de FER de l'espèce de végétaux donneurs et le domaine intracellulaire (ICD) d'une protéine de FER de l'espèce de végétaux receveurs. Un transfert de ces vecteurs dans des végétaux receveurs permet de produire des végétaux receveurs qui, avant le transfert, étaient séparés des végétaux donneurs par une barrière d'espèce, mais qui peuvent désormais franchir ces barrières.
PCT/CH2008/000334 2007-08-01 2008-07-31 Interactions entre des espèces végétales séparées par des barrières d'espèce et utilisations de celles-ci Ceased WO2009015505A1 (fr)

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CN107964548A (zh) * 2016-10-20 2018-04-27 中南林业科技大学 一种水稻OsFLRs基因及其应用
CN115152622A (zh) * 2022-08-11 2022-10-11 山东农业大学 抑制柱头fer表达量促进远缘杂交受精的用法和应用

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DATABASE EMBL [online] 13 June 2001 (2001-06-13), "Arabidopsis thaliana putative receptor-protein kinase (At3g51550) mRNA, complete cds.", XP002506431, retrieved from EBI accession no. EMBL:AY035053 Database accession no. AY035053 *
ESCOBAR-RESTREPO JUAN-MIGUEL ET AL: "The FERONIA receptor-like kinase mediates male-female interactions during pollen tube reception", SCIENCE (WASHINGTON D C), vol. 317, no. 5838, 3 August 2007 (2007-08-03), pages 656 - 660, XP009109567, ISSN: 0036-8075 *
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HUCK NORBERT ET AL: "The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception.", DEVELOPMENT (CAMBRIDGE), vol. 130, no. 10, May 2003 (2003-05-01), pages 2149 - 2159, XP009109570, ISSN: 0950-1991 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107964548A (zh) * 2016-10-20 2018-04-27 中南林业科技大学 一种水稻OsFLRs基因及其应用
CN107964548B (zh) * 2016-10-20 2021-03-23 中南林业科技大学 一种水稻OsFLRs基因及其应用
CN115152622A (zh) * 2022-08-11 2022-10-11 山东农业大学 抑制柱头fer表达量促进远缘杂交受精的用法和应用

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