Patent Application
20110030095
The present invention relates to means for enhancing the resistance of plants to nematodes.
Plant endoparasitic nematodes are small microscopic roundworms which live in the soil and infect plant roots. They can affect a broad range of species, including vegetables, cereals, fruits, flowers, and woody plants, causing important pre- and post-harvest losses. Plant endoparasitic nematodes include for instance Hirchmaniella spp, Pratylenchus spp, Radopholus spp, Ditylenchus spp, Anguina spp, Aphelenchoides spp, Bursaphelenchus spp, Rhadinaphelenchus spp, Heterodera spp, Rotylenchus spp, Tylenchulus spp, Nacobbus and Globodera spp. Among the must-damaging nematodes, one can cite the root-knot nematodes Meloidogyne spp. These nematodes establish and maintain permanent multinucleate plant feeding cells. These giant cells are essential for this obligate biotrophic pathogen growth and reproduction. After root penetration, nematode larva intercellularly migrates to induce the dedifferentiation of five to seven vascular root cells. Selected cells tremendously enlarge and become multinucleate through synchronous nuclear divisions without complete cytokinesis (Jones and Payne, 1978). Hypertrophied mature giant cell contains more than a hundred polyploid nuclei, which have also undergone extensive endoreduplication (Wiggers et al., 1990) and presents dense granular cytoplasm with numerous organelles (Jones, 1981). Surrounding giant cells, parenchyma cells hyperplasia and hypertrophy leads to typical root gall formation, the most visible infection symptom. It is not yet understood how these nematodes cause such alterations, but it is suspected that secreted parasitism proteins may have direct effects on host cells (Davis et al., 2004). Nematode feeding site ontogenesis complexity is reflected by extensive gene expression modification in infected root cells (Gheysen and Fenoll, 2002; Jammes et al., 2005). Genes involved in diverse processes such as cell cycle activation (de Almeida-Engler et al., 1999), cell wall modification (Goellner et al., 2001), hormone and defense response (Lohar et al., 2004; Jammes et al., 2005) have been identified. However unique example of gene function essential for giant cell formation has been demonstrated by knock out of the rpe gene that encodes a key enzyme in the pentose phosphate pathway (Favery et al., 1998). Giant cell cytoskeleton rearrangements have also been described to be a plant key component in compatible plant-nematode interactions (de Almeida-Engler et al., 2004; Favery et al., 2004).
Plant microtubule associated proteins MAP65 were first purified from tobacco (Nicotiana tabacum) and carrot (Daucus carota) microtubule (MT) preparation as 65-kDa proteins (Jiang and Sonobe, 1993; Chan et al., 1996). In Arabidopsis, nine genes encoding MAP65 have been identified (Hussey et al., 2002). Biochemical experiments demonstrated that MAP65s bind and bundle microtubules (MTs) in vitro (Jiang and Sonobe, 1993; Chan et al., 1996; Wicker-Planquart et al., 2004). The ability to bind MTs is dependent on a conserved motif located at the protein C-terminal half (Smertenko et al., 2004). Plant MAP65s share a large conserved domain with the yeast Anaphase spindle elongation factor 1 (Ase1p) and the human Protein Regulation Cytokinesis 1 (PRC1). Ase1p and PRC1 are involved in central spindle formation and cytokinesis (Pellman et al., 1995; Mollinari et al., 2002; Schuyler et al., 2003). Different plant MAP65s may have distinct activities and functions in relation to the different MTs arrays. Transitory expression experiments in tobacco cells revealed that several members localized to the phragmoplast, but also to other MT-based structures such as the cortical MTs, preprophase band (PPB) and mitotic spindles (Van Damme et al., 2004).
AtMAP65-3/PLEIADE, an AtMAP65 member, has been isolated by a genetic screen for root morphogenesis mutants (Müller et al., 2002). AtMAP65-3 binds to the MTs during cell division. The atmap65-3/ple mutants display cytokinesis defects in the root meristem, presumably due to compromised phragmoplast organization (Müller et al., 2004). The amino acid sequence of the AtMAP65-3 protein is available under accession number NP—199973 (or gi:15242132) in the GENBANK database. It is reproduced herein as SEQ ID NO: 2. The corresponding nucleotide sequence is also available in the GENBANK database under accession number NM—124539 (gi:30696073). It is reproduced herein as SEQ ID NO: 1. AtMAP65-3 comprises a MAP65_ASE1 domain (accession number pfam03999 in the CDD database, Marchler-Bauer and Bryant, 2004, or accession number IPR007145 in the InterPro database, Mulder et al., 2007) corresponding to the amino acids 36-625 of AtMAP65-3, and also comprises an AAA (ATPase) sub-domain (accession number SM00382 in the SMART database (Schultz et al., 1998 and Letunic et al., 2006) corresponding to the amino acids 6-449 of AtMAP65-3.
The Inventors have investigated the molecular mechanisms underlying giant cell formation. Using a promoter trap strategy in Arabidopsis, they identified AtMAP65-3 gene to be expressed at early stages of nematode feeding site formation. Promoter-GFP fusion analysis showed that AtMAP65-3 was expressed in all tissues enriched in dividing cells. Dwarf map65-3 mutants microscopical analysis revealed polynucleate and hypertrophied plant cells. In AtMAP65-3 absence, giant cells were induced but failed to fully differentiate and finally decayed. These giant cell defects lead to improper maturation of the infecting nematodes that depend on the nutrient supply from fully developed giant cell. Thus, the Inventors have shown that AtMAP65-3 is an essential host susceptibility gene, playing a key role in plant-nematode interaction by its requirement for giant cell development.
The present invention thus provides a method for increasing the resistance of a plant to an endoparasitic nematode, wherein said method comprises the inhibition in said plant of a microtubule associated protein of the MAP65 family, hereinafter designated as MAP65-3 protein, said MAP65-3 protein having at least 50%, preferably at least 55% and by order of increasing preference, at least 58%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or at least 65%, preferably 70% and by order of increasing preference, at least 71%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity with the AtMAP65-3 protein (SEQ ID NO: 2).
According to a preferred embodiment, said MAP65-3 protein comprises a MAP65_ASE1 domain having at least 55%, and by order of increasing preference, at least 60%, 63%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or at least 70%, and by order of increasing preference, at least 75%, 76%, 77%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity with the MAP65_ASE1 domain of the AtMAP65-3 protein as defined above.
According to a further preferred embodiment, said MAP65-3 protein comprises an AAA sub-domain having at least 55%, and by order of increasing preference, at least 60%, 65%, 67%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or at least 75%, and by order of increasing preference, at least 80%, 85%, 90%, 95%, 98% or 99% sequence similarity with the AAA sub-domain of the AtMAP65-3 protein as defined above.
Unless otherwise specified, the protein sequence identity and similarity values provided herein are calculated using the BLASTP program under default parameters, on a comparison window including the whole sequence of the proteins or of the domain to be compared. Similarity calculations are performed using the scoring matrix BLOSUM62. By way of example of MAP65-3 proteins, one can cite the putative microtubule-associated protein of Solanum demissum (amino acid sequence available under accession number AAT40494 (gi:11320514) in the GENBANK database), the microtubule-associated protein-like of Oryza sativa (japonica cultivar group) (amino acid sequence available under accession number NP—001043901 (gi:115439243) in the GENBANK database), or the protein of Vitis vinifera (amino acid sequence is available under accession number CAO49743 (gi:157355886) in the GENBANK database).
If necessary, a MAP65-3 protein can further be identified by determining its localization in planta (MAP65-3 locates to all microtubule arrays in dividing cells only such as the midline of the anaphase spindle and the cytokinetic phragmoplast), e.g. by the methods described in Müller et al., 2004, Van Damme et al., 2004 and/or Caillaud et al., 2008. A MAP65-3 protein can also be identified by showing that said protein enables the complementation of the Arabidopsis thaliana map65-3 mutant phenotype, e.g., by expressing said protein in the dyc283 mutant (see Example 1 below).
The inhibition of a MAP65-3 protein can be obtained either by abolishing, blocking or decreasing its function (i.e. binding to the MTs during cell division), or advantageously, by preventing or down-regulating the expression of its gene.
By way of example, inhibition of said MAP65-3 protein can be obtained by mutagenesis of the corresponding gene or of its promoter, and selection of the mutants having partially or totally lost the MAP65-3 protein activity. For instance, a mutation within the coding sequence can induce, depending on the nature of the mutation, the expression of an inactive protein, or of a protein with impaired activity; in the same way, a mutation within the promoter sequence can induce a lack of expression of said MAP65-3 protein, or decrease thereof.
Mutagenesis can be performed for instance by targeted deletion of the MAP65-3 coding sequence or promoter, or of a portion thereof, or by targeted insertion of an exogenous sequence within said coding sequence or said promoter. It can also be performed by random chemical or physical mutagenesis, followed by screening of the mutants within the MAP65-3 gene. Methods for high throughput mutagenesis and screening are available in the art. By way of example, one can mention TILLING (Targeting Induced Local Lesions IN Genomes, described by McCallum et al., 2000).
Advantageously, the inhibition of said MAP65-3 protein is obtained by silencing of the corresponding gene. Methods for gene silencing in plants are known in themselves in the art. For instance, one can mention by antisense inhibition or co-suppression, as described by way of example in U.S. Pat. Nos. 5,190,065 and 5,283,323. It is also possible to use ribozymes targeting the mRNA of said MAP65-3 protein.
Preferred methods are those wherein post transcriptional gene silencing is induced by means of RNA interference (RNAi) targeting the MAP65-3 gene to be silenced. Various methods and DNA constructs for delivery of RNAi are available in the art (for review, cf. Watson et al., 2005). Typically, DNA constructs for delivering RNAi in a plant include at least a fragment of 300 by or more (generally 300-800 bp, although shorter sequences may sometime induce efficient silencing) of the cDNA of the target gene, under transcriptional control of a promoter active in said plant. Currently, the more widely used DNA constructs are those that encode hairpin RNA (hpRNA). In these constructs, the fragment of the target gene is inversely repeated, with generally a spacer region between the repeats.
The present invention provides chimeric DNA constructs for silencing a MAP65-3 gene.
A chimeric DNA construct of the invention comprises:
According to a preferred embodiment of the invention, said chimeric DNA construct comprises:
The spacer can be a random fragment of DNA. However, preferably, one will use an intron which is spliceable by the target plant cell. Its size is generally 400 to 2000 nucleotides in length.
A large choice of promoters suitable for expression of heterologous genes in plants is available in the art.
They can be obtained for instance from plants, plant viruses, or bacteria such as Agrobacterium. They include constitutive promoters, i.e. promoters which are active in most tissues and cells and under most environmental conditions, tissue or cell specific promoters which are active only or mainly in certain tissues (e.g. root cells) or certain cell types (e.g. giant cells), and inducible promoters that are activated by physical or chemical stimuli, such as those resulting from nematode infection.
One can use constitutive promoters that are commonly used in plant cells are the cauliflower mosaic virus (CaMV) 35S promoter, the Nos promoter, the rubisco promoter.
However, the most suitable promoters for silencing the MAP65-3 gene in a plant in order to increase its resistance to an endoparasitic nematode are promoters that are tissue-specific, having a preferential or exclusive expression in the tissues infected by the nematodes, in particular the giant cells, and/or promoters that are inducible by nematode infection, i.e. promoters that drive an increased level of expression in cells infected by nematodes, when compared to non-infected cells. Said promoters may be naturally occurring promoter, may comprise a nematode responsive element isolated from a naturally occurring promoter, or may be a synthetic promoter.
Non-limitative examples of preferred promoters for carrying out the invention include:
The DNA constructs of the invention generally also include a transcriptional terminator (for instance the 35S transcriptional terminator, or the nopaline synthase (Nos) transcriptional terminator).
These DNA constructs can be obtained and introduced in a host cell or organism by the well-known techniques of recombinant DNA and genetic engineering. The choice of the recombinant vector depends on the intended host and on the intended method of transformation of said host. A variety of methods for genetic transformation of plant cells or plants are available in the art for many plant species, dicotyledons or monocotyledons. By way of non-limitative examples, one can mention virus mediated transformation, transformation by microinjection, by electroporation, microprojectile mediated transformation, Agrobacterium mediated transformation, and the like.
The invention also comprises plant cells or plants genetically modified by a construct of the invention. The polynucleotide may be transiently expressed; it can also be incorporated in a stable extrachromosomal replicon, or integrated in the chromosome.
In particular the invention relates to a transgenic plant containing a transgene comprising a chimeric DNA construct of the invention. The expression of said chimeric DNA constructs, resulting in a down regulation of the MAP65-3 protein, provides to said transgenic plant an increased resistance to endoparasitic nematodes when compared with a plant devoid of said transgene.
The present invention applies to monocot- or dicotyledon plants of agronomical interest, such as wheat, maize, rice, oats, barley, rye, sorghum, triticale, sugar cane, tomato, potato, sugarbeet, rape, flax, tobacco, sunflower, cotton, peas, beans, soybean or alfalfa.
The invention also encompasses isolated organs or tissues of said plants (such as seeds, leafs, flowers, roots).
Foregoing and other objects and advantages of the invention will become more apparent from the following detailed description and accompanying drawings. It is to be understood however that this foregoing detailed description is exemplary only and is not restrictive of the invention.
The T-DNA mutagenized Arabidopsis thaliana line collection (ecotype Wassilewskija, Wis.) was generated at Institut National de la Recherche Agronomique Versailles for promoter trap and gene tagging (Bechtold et al., 1993). The lines were screened individually for GUS expression after Meloidogyne incognita infection as previously described (Favery et al., 1998). For in vitro analyses, seeds were surface sterilized and grown on MS medium containing 1% sucrose, 0.7% plant cell culture tested agar (Sigma, St. Louis, Mo., USA), and 50 μg/mL of kanamycin. Plates were inclined at an angle of 60° to allow the roots to grow along the surface. Kanamycin resistance was scored in 2-week-old seedlings. For nematode infection in vitro, 100 surface-sterilized freshly hatched M. incognita J2 were added on each 2-week-old seedling. The plates were kept at 20° C. with a 16-h photoperiod.
GUS activity was assayed histochemically with 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid as described by Favery et al. (1998). Galls, root apex, and shoot apical meristems were dissected from GUS-stained plants, fixed in 1% glutaraldehyde and 4% formaldehyde in 50 mM sodium phosphate buffer, pH 7.2, dehydrated, and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) as described by the manufacturer. Sections (4 μm) were stained with 0.05% ruthenium red or toluidine blue and mounted in DPX (BDH Laboratory Supplies, VWR International, Fontenay-sous-Bois, France). Sections were observed with a Zeiss Axioplan 2 microscope (Jena, Germany).
T-DNA Insertion Site Analysis and Isolation of Homozygous dyc283 and ebj96 Plants
The insertion site was characterized by sequencing the genomic regions flanking the inserted T-DNA as described by Samson et al. (2002). To isolate homozygous dyc283/dyc283 and ebj96/ebj96 plants, the segregation of the kanamycin marker carried by the T-DNA on progenies resulting from each of 20 plants was analyzed. Progenies of five plants segregated 100% kanamycin-resistant plants, indicating that they were homozygous for the T-DNA tagged allele. To confirm these results, PCR experiments were done with the AtMAP65-3 primers which span the T-DNA insertion site, and a third primer GUS (5′-tcc aga ctg aat gcc cac ag-3′, SEQ ID NO: 3) specific for the sequence of the T-DNA. The primers DYCR (5′-gca gtt cag aag ctg atg gag g-3′, SEQ ID NO: 4) and DYC5RC (5′-cct gcc tga gta tgt tat act cc-3′, SEQ ID NO: 5) were used for DYC283, and DYC6 (5′-gga gta taa cat act cag gca gg-3′, SEQ ID NO: 6) and DYC9RC (5′-gat gat caa acc aaa cga cat tca g-3′, SEQ ID NO: 7) for EBJ96. When genomic DNA from homozygous plants was used as a template, a single PCR product was obtained from amplifications with all three primers.
For AtMAP65-3 promoter GFP:GUS fusion, a fragment of 1240 by upstream of the start codon was amplified by PCR using the primers Gw5pdyc (5′-aaa aag cag get tca cac tct tee cta cac aaa acc gc-3′, SEQ ID NO: 8) and Gw3pdyc (5′-aga aag ctg ggt gtt cga aat get taa gcc tgt aac agg g-3′, SEQ ID NO: 9). The PCR fragment was inserted into the pDON207 donor vector and then in the plant expression vectors pKGWFS7 (Karimi et al., 2002) using Gateway Technology (Invitrogen). For the subcellular localisation of MAP65-3, the Pro35S HindIII/SpeI fragment of the pK7WGF2, pK7FWG2, pH7WGY2, pH7YWG2 vectors (Karimi et al., 2002) was replaced by ProMAP65-3. The coding sequence of MAP65-3 was amplified by PCR, using wild-type plant cDNAs as the template and the primers Gw5dycB (5′-aaa aag cag get tca cca tgg caa gtg ttc aaa aag atc cg-3′, SEQ ID NO: 10) and Gw3dycK (5′-aga aag ctg ggt gtc aaa cca aac gac att cag act g-3′, SEQ ID NO: 11) or Gw3dycL (5′-aga aag ctg ggt gaa cca aac gac att cag act g-3′, SEQ ID NO: 12) for GFP or YFP:AtMAP65-3 or AtMAP65-3:GFP or YFP fusion respectively. These sequences were inserted into the pDON207 donor vector and then in the ProMAP65-3 plant expression vectors using Gateway Technology (Invitrogen). These constructs were sequenced by Genome Express (Grenoble, France) and transformed into Agrobacterium tumefaciens strain GV3101. Wild type WS, homozygous dyc283 or ebj96 Arabidopsis plants were transformed using the dipping method (Clough and Bent, 1998) and selected on MS medium 0.7% agar plates containing 50 μg/ml kanamycin or hygromycin. Transformed plants were transferred to soil and seeds collected. Transgenic plants expressing ProMAP65-3:AtMAP65-3:GFP or the N-terminal domain of the MT-binding domain of MAP4 fused to the GFP (Pro35S:MBD:GFP) were crossed with Pro35S:H2B:YFP Arabidopsis plants. Plants expressing the two constructions were obtained and homozygous progeny was used for microscopy analysis.
Optical sections were obtained on fresh roots by using an inverted confocal microscope (model LSM510; Zeiss, Jena, Germany). To visualize the MT cytoskeleton and nuclei in nematode feeding site, galls of 7 and 14 dpi were excised and embedded in agar 7%. Vibroslices of 100 μm diameter—for galls 7 dpi—or 300 μm diameter—for galls 14 dpi—were obtained using a vibratome HM560V (Microm). Fresh roots and vibroslices were observed with a ×63 water immersion apochromat objective (Zeiss, Jena, Germany). YFP and GFP fluorescence were monitored in Lambda mode with a 499-550-nm beam path (488-nm excitation line). Fluorescent dye FM4-64 (Molecular probes) was used at 1 μM final concentration. GFP or YFP and FM4-64 fluorescence were monitored in Lambda mode with a 499-620-nm beam path (488-nm excitation line).
To isolate genes involved in giant cell formation induced by Meloidogyne incognita, a promoter trap strategy was developed. 20,000 T-DNA-tagged Arabidopsis lines were screened by GUS assay after root-knot nematode infection, and lines showing GUS induction in root galls (Favery et al., 2004) were identified. One of these lines, DYC283, displayed early GUS activity in galls (
During plant development, the GUS gene was expressed in the root meristem and in the root elongation zone, in which cells divide and expand (
Plants homozygous for the T-DNA mutation (dyc283 mutant) showed a strong reduction of the shoot part of the plant (
dyc283 mutant cellular organization was characterized by performing sections through different tissues. dyc283 mutant SAM presented irregular cell outer layers. Hypocotyl and leaf primordia exhibit polynucleate cells and aberrant cell wall stubs (
dyc283 mutant response to the nematode M. incognita was examined. The infective second stage juvenile (J2) was able to invade the dyc283 mutant root tissue by penetrating the elongation zone, migrating along the vascular cylinder and induce a gall, as well as in wild type plant. Observation of gall sections 7 dpi revealed that the nematode could initiate its feeding site composed of 5 to 7 giant cells (
Searching Arabidopsis mutant collections for loss-of-function insertion mutants, an additional map65-3 allele was identified from INRA Versailles (Samson et al., 2002). The EBJ96 line carried a single T-DNA insertion in the AtMAP65-3 eleventh exon. As expected, plants homozygous for the ebj96 mutation exhibited similar mutant phenotype to dyc283 mutant. To complement the mutant phenotype, two AtMAP65-3:GFP fusions, GFP:AtMAP65-3 and AtMAP65-3:GFP, were constructed under the ProMAP65-3 control. When introduced into dyc283 or ebj96 mutants both constructs restored wild type phenotype. These results demonstrated that the map65-3 recessive mutation was responsible to the observed phenotype.
For AtMAP65-3 specific silencing in giant cells, the Pro35S Sad/SpeI fragment of the pH7GWIWG2 (II) vectors (VIB Gent, Karimi et al., 2002) was replaced by the promoter of AtFH6. The 449 by 5′UTR-exonI sequence of MAP65-3 (SEQ ID NO: 13) was amplified by PCR, using wild-type plant genomic DNA as the template and the primers MAP_RNAiGW5 (5′-aaa aag cag get tcc cca aaa cct ttt act tct tcg-3′, SEQ ID NO: 14) and MAP_RNAiGW3 (5′-aga aag ctg ggt ggg agt tcg aat aaa aga gat cca-3′, SEQ ID NO: 15)—AtMAP65-3 specific sequence underlined. This sequence was inserted into the pDON207 donor vector and then in the ProAtFH6-pH7GWIWG2 (II) vector using Gateway Technology (Invitrogen). The ProAtFH6-RNAi AtMAP65-3 construct (
T2 plants from 19 T1 transformants obtained with the ProAtFH6-RNAi AtMAP65-3 construct showed a significant reduction of the GUS expression limited to the galls induced by the nematodes. As expected, the AtFH6 promoter being not activated in the root meristems, no reduction of the GUS activity was observed in the root meristems and no developmental phenotype was detected. The first test of infection by the root knot nematodes performed on the T2 plants coming from 10 T1 plants bearing the ProMAP65-3:GFP-GUS and ProAtFH6-RNAi AtMAP65-3 constructs allowed identifying a line with a reduction of 60% of the number of nematodes able to carry out their development cycle (2 months after infection).
| Number | Date | Country | Kind |
|---|---|---|---|
| 07290610 | May 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2008/002459 | 5/15/2008 | WO | 00 | 10/18/2010 |
