WO2014006057A1 - Tolérance au stress chez les plantes - Google Patents

Tolérance au stress chez les plantes Download PDF

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WO2014006057A1
WO2014006057A1 PCT/EP2013/063956 EP2013063956W WO2014006057A1 WO 2014006057 A1 WO2014006057 A1 WO 2014006057A1 EP 2013063956 W EP2013063956 W EP 2013063956W WO 2014006057 A1 WO2014006057 A1 WO 2014006057A1
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aska
g6pd6
plant
activity
stress
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PCT/EP2013/063956
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English (en)
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Claudia Jonak
Silvia DALSANTOS
Hansjörg STAMPFL
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Gregor Mendel Institute Of Molecular Plant Biology Gmbh
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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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance

Definitions

  • the invention refers to a method for producing a plant with increased tolerance against abiotic and biotic stress by engineering a recombinant plant cell for elevating the level of phosphorylated cytosolic G6PD in said plant cell, and a method of producing genetically transformed plants which are tolerant toward stress conditions including abiotic stress conditions, such as those generating reactive oxygen species (ROS), and/ or biotic stress conditions, such as to render the plants more resistant to pathogens.
  • abiotic stress conditions such as those generating reactive oxygen species (ROS)
  • ROS reactive oxygen species
  • High soil salinity is a major environmental constraint for plant growth and development and negatively affects agricultural productivity.
  • Salinity imposes a water-deficit and ion stress, which cause effects such as inhibition of essential enzymes, destabilization of cell membranes, a decrease in nutrient supply, and overproduction of reactive oxygen species (ROS).
  • ROS production is a universal feature of aerobic metabolism, but excess levels of ROS, generated as a consequence of an insult, can lead to oxidative damage of cells.
  • G6PD Glucose-6-phosphate dehydrogenase
  • OPPP oxidative pentose phosphate pathway
  • G6PD is the principal source of NADPH and is of central importance for cellular redox regulation in response to stress.
  • Mouse embryonic stem cells in which G6PD activity is disrupted are extremely sensitive to oxidative stress.
  • Pathogen infection, certain drugs, or ingestion of fava beans can trigger hemolytic anemia in humans with a G6PD deficiency, the most common enzymopathy in humans.
  • yeast mutants defective in G6PD show an increased susceptibility, and fail to induce adaptation, to oxidative stress.
  • G6PD activity is present in the plastids and in the cytosol. G6PD activity has been positively correlated with environmental stresses such as salt stress (Valderrama et al., 2006; Wang et al., 2008), aluminium-stress (Slaski et al., 2006), viral infection (Sindelar and Sindelarova, 2002), and resistance to fungal pathogens (Scharte et a!., 2009). In contrast, a decrease in chloroplastic G6PD activity has been shown to be beneficial for withstanding oxidative stress (Debnam et al., 2004).
  • G6PD The activity of G6PD is tightly controlled. In addition to regulation at the transcriptional level, redox-regulation and the cellular NADPH/NADP+ ratio modulate the activity of the different G6PD isoforms.
  • G6PD is phosphorylated in animals and plants, which has been correlated with both enhanced (Ramnanan and Storey, 2006; Dieni and Storey, 2010; Gupte et al., 2011 ) and reduced activity (Hauschild & von Schaewen, 2003; Xu et al, 2005; Zhang et al, 2000). While different signaling have been implicated in regulating G6PD phosphorylation in animals under a variety of different conditions, the upstream regulator(s) of G6PD phosphorylation in plants have not been identified before.
  • Glycogen synthase kinase 3 constitutes a class of evolutionarily conserved serine/threonine protein kinases. Originally identified in mammals as a cytoplasmic modulator of glycogen metabolism, GSK3 is now recognized as a central regulator of an array of cellular events including cell fate determination, microtubule function, cell cycle regulation, apoptosis, and inflammatory responses.
  • GSK3/shaggy-like kinases are encoded by a gene family that directs different physiological responses (Jonak and Hirt, 2002). In Medicago sativa, several GSKs have emerged as regulatory components in stress signaling. WIG (wound-induced GSK) is post-translationally activated by wounding (Jonak et al., 2000). MsK1 is important in innate immunity that limits the severity of virulent bacterial infections (Wrzaczek et al., 2007). MsK4 regulates high salt tolerance by adjusting carbohydrate metabolism in response to environmental stress (Kempa et al., 2007).
  • WO2010/025936A1 describes plants with enhanced pathogen resistance and/or resistance to stress. A kinetically superior isozyme enhanced stress- resistance in the progeny of a susceptible plant.
  • EP2327770A1 describes the use of Group I shaggy-like kinases for increasing salt-stress resistance and yield of plants, and transgenic plants with increased expression of a Group I shaggy-like kinase of Oryza sativa and homologues thereof.
  • WO2010/037714A1 describes a transgenic plant cell with increased resistance to biotic stress by increasing a variety of activities in the plant, inter alia disclosing glucose-6-phosphate 1 -dehydrogenase.
  • recombinant plant cell for elevating the level of phosphorylated cytosolic G6PD in said plant cell and regenerating a transgenic plant from said plant cell, which has increased tolerance to abiotic and biotic stress conditions.
  • said cytosolic G6PD is G6PD6 and/or G6PD5, preferably G6PD6 encoded by a nucleotide sequence of SEQ ID 2, or a functionally active variant thereof comprising the T467 phosphorylation site.
  • the level of phosphorylated cytosolic G6PD is elevated by increasing the phosphorylation or the level of G6PD6 and/or G6PD5, and/or by increasing the level of ASKalpha.
  • a recombinant, double-stranded DNA molecule is incorporated into the genome of said plant cell which comprises:
  • a DNA sequence preferably a promoter or enhancer
  • a DNA sequence which encodes eukaryotic ASKalpha which comprises the nucleic acid sequence of SEQ ID NO:1 or functional equivalents thereof that are capable of phosphorylating G6PD6 and/or G6PD5, and optionally
  • a transcription termination signal operably linked to said DNA sequence wherein the regulatory element is operably linked to said DNA sequence and capable of directing the expression of said DNA sequence in a plant cell transformed with said DNA sequence.
  • Said regulatory element may be heterologous with respect to said DNA sequence, e.g. an ASKalpha promoter operatively linked to said DNA sequence, wherein the promoter is an artificial sequence or wherein both sequences are originating from different species or organisms.
  • the regulatory element may be homologous, e.g. an ASKalpha promoter operatively linked to said DNA sequence, wherein both are originating from the same species or organism.
  • said DNA sequence is an exogenous or endogenous DNA sequence.
  • said DNA sequence may specifically be an
  • endogenous DNA sequence e.g. by incorporating a regulatory element or enhancer into the plant genome to mediate effective expression of endogenous ASKalpha in the plant cell.
  • an exogenous DNA sequence may be used to be incorporated into the plant genome, e.g. supplementing or substituting endogenous ASKalpha activity.
  • said DNA sequence is overexpressed in said plant.
  • the ASKalpha is a plant ASKalpha.
  • said DNA sequence is selected from the group consisting of SEQ ID NO: 1 or functional equivalents thereof that are capable of phosphorylating G6PD, in particular G6PD6 and/or G6PD5.
  • said DNA sequence is inducibly or constitutively expressed in said transgenic plant.
  • a regulatory element which is a promoter or enhancer, such as a constitutive or inducible promoter, e.g. plant DNA virus promoter, including CaMV35S and FMV35S.
  • a regulatory element is provided which is a transcription termination signal operably linked to said DNA sequence, in particular a poly A addition site.
  • DNA molecule is cloned into an expression cassette, such as a plasmid or vector, e.g. a viral vector.
  • an expression cassette such as a plasmid or vector, e.g. a viral vector.
  • the stress tolerance refers to abiotic and biotic stress conditions, including resistance to infections by pathogens.
  • said abiotic stress is any of dehydration, excess salinity, or other exposure to physical or chemical conditions generating reactive oxygen species (ROS), and said biotic stress is any one induced by pathogen infection.
  • the plant cell is rendered more pathogen resistant, such as to protect the plant from infection by bacterial or fungal pathogens to a higher extent as the wild-type.
  • the stress tolerant plant is selected from the group consisting of crops, including all kinds of vegetables, and bioenergy crops. This includes in particular crops for marginal arable lands and salty soils.
  • crops including all kinds of vegetables, and bioenergy crops.
  • crops for marginal arable lands and salty soils are commonly used plants.
  • the commonly used plants are Miscanthus, Poplar, Willow, Millet, Elephant Grass, Sunflower, Jatropha, Cassava, and further Arabidopsis, brassica, cotton, potato, soya, sugar beet, sugar cane, an ornamental plant, rice, maize, tomato, barley or wheat.
  • At least one of seeds, seedlings or plant derived tissue comprising a plant cell according to the invention.
  • tissue culture of the tissue according to the invention in particular for testing specific properties or functions of the plant cells.
  • a method for selectively controlling weeds in a field containing a crop having planted crop plants or seeds comprising the steps of:
  • Said reference may be an activity derived from a normal population of plants.
  • the kinase assay is an immunokinase assay employing a reagent capable of binding an N-terminal sequence of ASKalpha.
  • the G6PD6 and/or G6PD5 activity of G6PD6 and/or G6PD5, or a functional equivalent thereof, which is phosphorylated by ASKalfa is enhanced by an altered expression or sequence of G6PD6 and/or G6PD5 and/or ASKalpha, such as a sequence expressing a functional equivalent thereof with improved properties, like activity, expression level, phosphorylating properties, etc, e.g. a G6PD6 sequence including at least the T467E mutation.
  • the activity may be enhanced through addition or expression of co -factors or enhancers.
  • FIG. ASKa is activated by high salinity and modulates Arabidopsis salt stress resistance.
  • A Salt tolerance of ASKa activity mutants during early seedling development.
  • B Germination efficiency on 1 ⁇ 2 MS plates and on plates
  • ASKa kinase activity was determined in kinase assays with [ ⁇ -32 ⁇ ] ATP and MBP as a substrate.
  • Threonine 467 is necessary for G6PD6 activation by ASKa in vitro.
  • ASKa activates G6PD6 in vivo.
  • A Phosphorylation of G6PD6 by immunoprecipitated ASKa. Immunokinase assay using antimyc antibodies (Santa Cruz) to purify ASKa-myc from non-stressed and high salinity-stressed ASKa-myc-expressing plants (same plant material as in Figure 1 D) using GST-G6PD6 as a substrate.
  • B G6PD6 is an in vivo
  • A. thaliana protoplasts were transformed with G6PD6-HA or co- transformed with G6PD6-HA and ASKa-myc or ASKa K98R-myc and labeled with 32P-orthophosphate.
  • the autoradiogram shows G6PD6 immunoprecipitated from 200 pg of protein extracts with HA antibodies (Santa Cruz).
  • ASKa K98R-myc and untransformed protoplasts were used as specificity controls for phosphorylation and immunoprecipitation.
  • C In vivo activity of G6PD6, G6PD6 T467A and G6PD6 T467E.
  • the assay was repeated three times with similar results. See also Figure S4.
  • FIG. g6pd6.2 shows an altered redox status and enhanced sensitivity to salt stress.
  • FIG. 7 ASKa regulates salt stress tolerance by phosphorylating G6PD6.
  • A Phosphorylation of G6PD6 is important for salt stress tolerance. Germination efficiency of Col 0, aska and aska mutants transformed with either G6PD6 or G6PD6 T467E on 1 ⁇ 2 MS plates supplemented with 100 mM NaCI Data are means of three independent biological replicates with at least 75 plants each. Asterisks indicate a significant difference compared to aska under stress conditions ( * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.005) using Student ' s t-test for pairwise comparison.
  • B ASKa and G6PD6 function in the same signaling pathway.
  • ASKa is a positive regulator of Pseudomonas resistance.
  • ASKa Overexpression of ASKa enhances resistance to Pseudomonas syringae. Bacterial growth count of virulent Pseudomonas syringae pv. tomato DC3000 after spray inoculation of four-week-old Arabidopsis thaliana wild-type (WT), aska and ASKa overexpressing plants grown at 21 °C at a 12/12 hour light regime .
  • ASKa is transcriptionally induced by Pseudomonas infection.
  • ASKa relative gene expression after infection of four-week-old Arabidopsis thaliana wild-type plants with Pseudomonas syringae pv. maculicola ES4326 monitored by quantitative RT- PCR and normalized to EF1 -a (AT5G60390).
  • ASKa kinase activity is induced by Pseudomonas infection.
  • ASKa kinase activity after infection of four-week-old Arabidopsis thaliana wild-type plants with Pseudomonas syringae pv. maculicola ES4326.
  • Figure 10 ASKalpha cDNA sequence of Arabidopsis thaliana (SEQ ID 1 )
  • Figure 11 G6PD6 cDNA sequence of Arabidopsis thaliana (SEQ ID 2)
  • FIG. 12 G6PD5 cDNA sequence of Arabidopsis thaliana (SEQ ID 3)
  • Supplementary Figure 1 aska T-DNA insertion mutant and specificity of the anti-ASKa antibody.
  • A Schematic diagram of ASKa showing the site of T-DNA insertion in aska. Black boxes indicate coding and white boxes intron regions.
  • B qRT-PCR analysis of wild-type Col 0, aska and ASKa overexpressor lines OE#4 and Oe#5 showing the absence of a full length ASKa transcript in aska plants and enhanced ASKa transcript levels in ASKa overexpressor lines.
  • C Four-week old A. thaliana wild-type Col 0, aska and ASKa-overexpressor lines OE#4 and OE#5 grown under control conditions in soil.
  • Arabidopsis leaf protein extract was immunoprecipitated with ASKa-specific antibodies from 00 pg of total protein extract. Subsequent western analysis was performed with an anti-ASKa antibody with (+) or without (-) prior blocking of the antibody with the N-terminal ASKa peptide.
  • FIG. 1 ASKa and G6PD6 function in the same signaling pathway.
  • A, B g6pd6.2 T-DNA insertion mutant. Schematic diagram of G6PD6 showing the site of TDNA insertion in the g6pd6.2 mutant (A). Black boxes indicate the coding region. The position of Thr467 is depicted.
  • FIG. 1 Characterization of Thr467 of G6PD5.
  • A In vitro kinase assay with GST-ASKa and GST-G6PD5, GST-G6PD5 T467A or GSTG6PD5 T467E. The experiment was repeated three times showing comparable results.
  • B G6PD5 T467A displays enhanced enzymatic activity. G6PD activity was quantified of GSTG6PD5, GST-G6PD5 T467A or GST-G6PD5 T467E with or without prior kinase reaction with GST-ASKa or with GST-ASKa K98R. The assay was performed three times.
  • ASKalpha (also referred to as ASKa) as used herein shall mean any of the ASKalpha polypeptides, functional equivalents or functionally equivalent homologues thereof, in particular capable of phosphorylating G6PD.
  • G6PD6 as used herein shall mean any of the G6PD6 polypeptides, such as encoded by SEQ ID 2, any isoforms, functional equivalents or functionally equivalent homologues thereof.
  • G6PD5 as used herein shall mean any of the G6PD5 polypeptides, such as encoded by SEQ ID 3, any isoforms, functional equivalents or functionally equivalent homologues thereof.
  • G6PD activity as used herein shall mean activity of phosphorylated G6PD6 and/or G6PD5.
  • stress as used herein shall mean abiotic and biotic stress.
  • abiotic stress shall mean any adverse effect on metabolism, growth, biomass, reproduction yield and/or viability of a eukaryotic organism, such as a plant, which is caused by directly imposed physiological stress, for example, heat, cold, salt, wounding, toxicity through organic or anorganic chemicals, ultraviolet-B, desiccation or water stress.
  • physiological stress for example, heat, cold, salt, wounding, toxicity through organic or anorganic chemicals, ultraviolet-B, desiccation or water stress.
  • ROS are physical or chemical conditions mediating or generating the formation of ROS.
  • ROS are highly reactive and increase dramatically upon abiotic stress, which may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress or oxidative stress conditions.
  • crop protectants such as herbicides are grouped into different classes based on their mode-of-action, or the manner in which the herbicide affects a plant.
  • the ROS producing herbicides are chlorogenic herbicides or other herbicides such as paraquat, having an activity dependent on its effect generating ROS.
  • biotic stress shall mean any adverse effect on metabolism, growth, biomass, reproduction yield and/or viability of a eukaryotic organism, such as a plant, which is caused by pathogens, specifically
  • Curvularia Colletotrichum, Magnaporthe, Puccinia, Aspergillus, Ustilago, Septoria, Erisyphe, Rhizoctonia, Fusarium, Blumeria, Saccharomyces, Penicillium, Neurospora, Alternaria, Sclerotina, Chaetomium, Phycomyces, Erysiphe, Peronospora, Phytophthora, Pythium, Plasmopara and Albugo: or
  • Pseudomonas Pseudomonas (Pseudomonas syringae), Ralstonia, Erwinia, Pectobacterium, Pantoea, Agrobacterium, Acidovorax, Clavibacter, Streptomyces, Xylella, Spiroplasma and Phytoplasma; or nematodes, including Meloidogyne, Globodera and Heterodera, or
  • biotrophic or necrotrophic pathogens or semibiotrophic pathogens such as Pseudomonas syringae.
  • a plant being tolerant to biotic stress exerts an increased resistance to infection by pathogens, e.g. as measured by the titer or degree of infection. This may be determined by the reduced growth of a virulent strain of a pathogen in the plant as compared to the wild-type pathogen.
  • expression or "expression system” or “expression cassette” refers to nucleic acid molecules containing a desired coding sequence and control sequences in operable linkage, so that hosts transformed or transfected with these sequences are capable of producing the encoded proteins or host cell metabolites.
  • the expression system may be included in a vector; however, the relevant regulatory sequences or DNA may also be integrated into the host chromosome.
  • Expression may refer to products, including polypeptides or metabolites, in either compartment of a cell or secreted to the outside of the cell.
  • Expression constructs "cassettes” or “vectors” used herein are defined as DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e. of recombinant genes and the translation of their mRNA in a suitable host organism.
  • Expression vectors usually comprise an origin for autonomous replication in the host cells, selectable markers well-known in the art, e.g. GOX, NPTII, PAT etc. but also GFP or derivatives thereof (Miki et al. Journal of Biotechnology 107 (2004) 193-232), a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, which components are operably linked together.
  • the terms “plasmid” and “vector” as used herein include autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences.
  • the "functional equivalent" of a protein such as ASKalpha or G6PD6 or G6PD5, as used herein means an isoform, a variant or fragment of a polypeptide encoded by an ASKalpha or G6PD6 or G6PD5 sequence as defined by the SEQ ID 1 , 2 and 3, respectively, or any sequence derived from such sequences encoding a polypeptide exhibiting the same function, e.g. a functional equivalent of ASKalpha having a sequence derived from SEQ ID 1 , in particular those effecting
  • G6PD6 and/or G6PD5 phosphorylation of G6PD6 and/or G6PD5; or a functional equivalent of G6PD6 having a sequence derived from SEQ ID 2, in particular those effecting enzymatic G6PD activity, or a functional equivalent of G6PD5 having a sequence derived from SEQ ID 3 effecting enzymatic G6PD activity.
  • sequence may be derived from the parent sequence by modification, e.g. point mutations, including insertion, deletion and/or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, and which modification does not affect (in particular impair but optionally enhance) the activity of this sequence.
  • the functional equivalents may be analogue of a species other than the parent sequence, or homologues having a certain homology.
  • homologous sequence indicates that two or more nucleotide sequences have the same or conserved base pairs at a corresponding position, to a certain degree, up to a degree close to 00%.
  • a homologous sequence typically has at least about 50% nucleotide sequence identity, preferably at least about 60% identity, more preferably at least about 70% identity, more preferably at least about 80% identity, more preferably at least about 90% identity, more preferably at least about 95% identity.
  • the functionally equivalent ASKalpha homologue typically comprises a high percentage of sequence identity of the kinase domain, which is highly conserved, possibly with a degree of homology between 95 and 100%. Yet, the N-terminal or C- terminal region of the ASKalpha homologue may have some variability, e.g. within the range of 50 to 99%, preferably at least 60%, or at least 70%, or at least 80% or at least 90%.
  • the functionally equivalent G6PD6 or G6PD5 homologue typically comprises a high percentage of sequence identity, in particular in the phosphorylated region, e.g. within the range of 50 to 99%, preferably at least 60%, or at least 70%, or at least 80% or at least 90%.
  • Percent (%) identity with respect to the nucleotide sequence of a gene is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • kinase assay shall mean methods for detecting the level of functional ASKalpha in a sample, as well as for detecting ASKalpha kinase activity in a sample.
  • the level of ASKalpha, or nucleic acid encoding ASKalpha may generally be determined using a reagent that binds to the N-terminal sequence of ASKalpha which determines its phorsphorylation activity or potency, and the respective, DNA or RNA.
  • standard hybridization and/or PCR techniques may be employed using a nucleic acid probe or a PCR primer.
  • Suitable probes and primers may be designed by those of ordinary skill in the art based on the ASKalpha cDNA sequence provided in SEQ ID NO: 1 .
  • the reagent is typically an antibody or another affinity ligand including specifically binding peptides, which may be prepared according to the state of the art.
  • affinity ligand may be immobilized on a solid support such that it can bind to and remove the polypeptide from the sample.
  • the bound polypeptide may then be detected using a second antibody that binds to the ligand/peptide complex and contains a detectable reporter group.
  • a competitive assay may be utilized, in which
  • polypeptide that binds to the immobilized ligand is labeled with a reporter group and allowed to bind to the immobilized ligand after incubation of the ligand with the sample.
  • the extent to which components of the sample inhibit the binding of the labeled polypeptide to the ligand is indicative of the level of polypeptide within the sample.
  • Suitable reporter groups for use in these methods include, but are not limited to, enzymes, substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • an immunokinase assay For detecting active ASKalpha in a sample, an immunokinase assay may be employed.
  • immunokinase assay means a specific type of biochemical test that measures the presence or concentration of a substance by its binding to an immunoligand, such as an antibody or other affinity ligand including peptides, which immunoligand is specifically binding to the region of the harboring the kinase activity.
  • an immunoligand such as an antibody or other affinity ligand including peptides, which immunoligand is specifically binding to the region of the harboring the kinase activity.
  • polyclonal or monoclonal antibodies may be raised against a unique sequence of ASKalpha, such as the amino-terminus, e.g. spanning from amino acid 7 to 22, using standard techniques.
  • a sample to be tested such as a cellular extract, is incubated with the anti- ASKalpha antibodies to immunoprecipitate ASKalpha, and the immunoprecipitated material is then incubated with a substrate, e.g. MBP (myeline basic protein) under suitable conditions for substrate
  • a substrate e.g. MBP (myeline basic protein) under suitable conditions for substrate
  • the level of substrate phosphorylation may generally be determined using any of a variety of assays, as described herein.
  • ASKalpha kinase assays may as well be used to determine the functionality of functional equivalents or functionally equivalent ASKalpha homologues, including any assays that evaluate a compound's ability to phosphorylate G6PD6 and/or G6PD5 or other substrates indicating the phosphorylating activity or potency.
  • G6PD6 and/or G6PD5 for use in such methods may be endogenous, purified or recombinant, and may be prepared using any of a variety of techniques that are well-known to those of ordinary skill in the art.
  • An ASKalpha kinase assay may be performed substantially as described in Jonak et al. 2000 and as described in the Examples section below.
  • the kinase activity of ASKalpha being the average activity of a normal population of plants or traits of the same species may be used, e.g. a plant population grown under normal growth condition without abiotic stress or biotic stress, such as pathogen infection.
  • a differential value indicating a stress tolerance of the plant, as determined according to the invention means an increase in ASKalpha activity by an abnormal magnitude, wherein the ASKalpha activity is outside the standard deviation for the reference, the ASKalpha activity is upregulated relative to the reference value by at least 1.5-, 2-, 3-, or 4-fold of the standard deviation. It is not necessary, that all of the individual plants show significantly upregulated ASKalpha activity, but a significant proportion in a population, e.g. at least 50%, preferably at least 60%, 70%, 80%, 90% or more.
  • polypeptides as used herein always includes proteins or smaller amino acid sequences, including variants (functional equivalents) or fragments or proteins.
  • a recombinant organism or plant comprises at least one recombinant nucleic acid, e.g. a genetically transformed plant or a transgenic plant.
  • a recombinant plant cell specifically comprises an expression vector or cloning vector, or it has been genetically engineered to contain a recombinant nucleic acid sequence.
  • a plant cell or plant comprising the recombinant DNA molecule according to some embodiments of the invention is also referred to as recombinant ASKalpha overexpressor.
  • regulatory element shall mean at least one of the elements employed in genetically engineering a recombinant DNA molecule in support of expressing a polypeptide of interest, such as ASKalpha.
  • the regulatory elements are e.g. promoter, including inducible, constitutive or stress- related promoter, enhancer, and auxiliary sequences, such as signal sequences or transcription termination signals.
  • the term "tolerance” or “resistance” to abiotic and/or biotic stress as used herein refers to the ability of a plant to endure the stress without suffering a substantial alteration in metabolism, growth, productivity and/or viability.
  • the genetically transformed plant of the some embodiments of the invention exhibits at least 10% more, 20% more, 30% more, 40% more, 50% more, 60% more, 70% more, 80% more, 90% more or even higher tolerance to stress than the reference, e.g. a normal population of plants of the same species.
  • the stress tolerance is specifically determined by the yield, biomass and growth rate, or else the germination tolerance of seeds and seedlings.
  • a low tolerance of e.g. less than 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less or even lower than the reference is understood as an increased sensitivity or susceptibility to stress.
  • G6PD was identified as an in vivo target of ASKa, and a novel molecular mechanism for the regulation of G6PD activity is presented.
  • ASKalpha activity could also effectively improve the plants resistance to herbicides, in particular to ROS-producing herbicides.
  • the protection of crops from weeds and other vegetation which inhibit crop growth is a constantly recurring problem in agriculture.
  • ROS producing chemicals are used to effectively control such unwanted growth.
  • Paraquat is one of the most widely used ROS-producing herbicides, e.g. marketed under the brand name Gramoxone by Syngenta. The activity of paraquat as a herbicide is dependent on its ROS-producing activity for weed control.
  • ROS producing herbicides are specifically used to control broad-leaved weeds and grasses and are widely used for weed control in fruit orchards and plantation crops, including coffee, cocoa, coconut, oil palms, rubber, bananas, vines, olives and tea, ornamental trees and shrubs and in forestry. Other uses include weed control in alfalfa, onion, leeks, sugar beet or asparagus. ROS producing herbicides are increasingly used to destroy weeds in preparing land for planting in combination with no-till agricultural practices which minimise ploughing and help prevent soil erosion.
  • ASKa is involved in regulating pathogen resistance in plants overexpressing ASKa.
  • the growth of a virulent strain of Pseudomonas syringae pv. maculicola ES4326 containing the luxCDAEB operon was determined in leaves of aska knock-out, ASKa overexpressor and wild-type plants showing that plants overexpressing ASKa were more resistant to this bacterial pathogen.
  • ASKa is involved in Arabidopsis salt stress resistance
  • ASK activity mutants were screened (i.e., ask knock-out and ASK overexpressor lines) for their performance under various stress conditions.
  • This systematic study revealed that ASKa activity mutants resembled wild-type under normal growth conditions (Supplemental Figure 1 C) but displayed altered tolerance to high salt conditions.
  • High soil salinity stress imposed by watering soil-grown plants with 150 mM NaCI solution over three weeks, showed that aska T-DNA insertion knock-out plants (Figure S1A, B) are highly sensitive to salt stress (Figure 1A).
  • ASKa is activated by high salinity stress
  • ASKa protein kinase activity was first determined from long-term salt stressed and non-stressed plants expressing ASKa-myc.
  • ASKa immunokinase assays were performed on plants exposed to salt stress for 6h, 12h, 1 d, or 3d.
  • ASKa activity was low.
  • high soil salinity induced ASKa activity rapidly and persistently over an experimental period of 3 days ( Figure 1 F, upper panel).
  • the ASKa steady state protein levels remained constant ( Figure 1 F, lower panel), suggesting a posttranslational activation mechanism.
  • G6PD activity also provides reducing power important for ROS detoxification.
  • H2O2 hydrogen peroxide
  • ASKa phosphorylates and activates G6PD6 in vitro
  • Threonine 467 is necessary for G6PD6 activation by ASKa
  • Human Thr466 is located remotely from the dimer and tetramer interfaces of the G6PD molecule, suggesting that this residue does not affect the oligomeric state (Figure 4A). Interestingly, human Thr466 is in close proximity (3 A) to the ⁇ -ae loop, which is part of the NADP binding site ( Figure 4B). Phosphorylation of Thr467 in G6PD6 might thus introduce structural changes in the coenzyme binding site connected with an enhanced G6PD activity.
  • Thr467 modification for G6PD6 activity was mutated, either to alanine (T467A), which cannot be phosphorylated, or to glutamic acid (T467E) mimicking the phosphorylation event.
  • T467A alanine
  • T467E glutamic acid
  • G6PD6 is phosphorylated and activated by ASKa in vivo
  • G6PD activity is modified in ASKa activity mutants upon salt stress. To assess whether G6PD6 might be a direct target of ASKa in vivo, it was first tested whether ASKa activity immunoprecipitated from control and stressed plants could
  • G6PD6 exists as an in vivo phosphoprotein.
  • HA-tagged G6PD6 was transiently expressed in radiolabeled Arabidopsis protoplast cells and immunoprecipitated with anti-HA antibodies.
  • FIG. 5B G6PD6 is phosphorylated in vivo.
  • Co-transformation of G6PD6-HA with ASKa further enhanced G6PD6 in vivo phosphorylation.
  • Co-expression of ASKa K98R did not significantly increase G6PD6 phosphorylation.
  • G6PD activity was quantified in protoplast cells transformed with G6PD6 in the presence or absence of ASKa ( Figure 5C).
  • G6PD activity could be enhanced by ASKa but not by ASKa K98R in cells expressing G6PD6.
  • ASKa was unable to stimulate G6PD activity.
  • Cells expressing G6PD6 T467E showed constitutively high G6PD activity, which could not be further stimulated by ASKa, indicating that Thr467 phosphorylation of G6PD6 is necessary and sufficient for activation.
  • G6PD6 Loss of G6PD6 alters the cellular redox state and renders plants more sensitive to high salt stress
  • G6PD activity is modulated by high salt conditions.
  • g6pd6.2 knock-out plants ( Figure S2A, B) were assayed for their stress sensitivity. Wild-type seeds germinated nearly as well on medium supplemented with 100 mM NaCI as on normal growth medium. However, the germination efficiency of g6pd6.2 was significantly reduced under high salinity conditions ( Figure 6A). Similarly, root growth of g6pd6.2 was more strongly affected by NaCI compared to wild-type ( Figure 6B), indicating that G6PD6 is important for tolerance to high salt.
  • Glutathione is a key marker of the intracellular redox state. Consistent with a role of G6PD6 in redox regulation, levels of oxidized glutathione (GSSG) were elevated ( Figure 6C) and the ratio of reduced (GSH) to oxidized glutathione was altered in g6pd6.2 under salt stress conditions ( Figure 6D). Early seedling development and the glutathione redox status of aska plants were more strongly affected by high salinity conditions than that in g6pd6.2 plants, suggesting that ASKa also regulates other targets in addition to G6PD6 for successful acclimation to stress.
  • ROS ROS
  • GSK3 ASKa
  • G6PD6 activity G6PD6 activity
  • glutathione redox status G6PD6 activity
  • ROS levels ROS levels
  • ASKa is a novel component regulating the high salinity response. Loss of ASKa activity rendered plants very sensitive to high soil salinity, whereas plants overexpressing ASKa were more able to tolerate this stressful condition. ASKa has previously been implicated in flower development (Dornelas et a!., 2000), however, using aska knock-out and ASKa-overexpressing lines, the flower phenotype described in ASKa antisense plants were not observed here. The involvement of ASKa in stress responses was corroborated by the stimulation of ASKa activity by high salinity. ASKa appears to be activated by salt stress at the post-translational level. While salt stress rapidly enhanced ASKa activity, ASKa protein levels remained constant.
  • GSK3 The activity of mammalian GSK3 is modulated by various means including phosphorylation and differential protein complex formation. Recently, it has been shown that ASKs are phosphorylated in vivo and that tyrosine phosphorylation is necessary for ASKa activity (de la Fuente van Bentem et al., 2008). In mammals, GSK3 is a central regulatory component controlling a multitude of cellular targets. Similarly, the localization of ASKa to the cytoplasm and the nucleus implies that ASKa might act on different organelle specific targets. G6PD6 appears to be one of the cytosolic in vivo targets of ASKa.
  • G6PD6 occurs as a phosphoprotein in vivo and that ASKa is able to directly phosphorylate G6PD6 on Thr467, thereby enhancing its enzymatic activity.
  • Mutational analyses of G6PD6 indicated that Thr467 phosphorylation is necessary and sufficient for stimulation of basal G6PD activity in cells.
  • ASKy the closest homologue of ASKa, was unable to enhance G6PD6 activity in cells, thus emphasizing the specificity of ASKa on G6PD6 in vivo regulation.
  • G6PD is subject to complex control and regulation of G6PD transcript and protein levels has been reported (Salvemini et al., 1999; Nemoto and Sasakuma, 2000; Scharte et al., 2009). Additionally, redox-regulation and product inhibition participate in modulating G6PD activity (Wendt et al., 2000; Debnam et al., 2004; Schurmann and Buchanan, 2008). Previous studies in plants suggested a negative regulation of G6PD by phosphorylation (Zhang et al., 2000; Hauschild and von Schaewen, 2003; Xu et al., 2005). However, we provide direct evidence that G6PD6 activity can be enhanced by phosphorylation.
  • Thr467 The ASKa phosphorylation site Thr467 of G6PD6 is conserved in eukaryotes. Interestingly, structural analyses show that Thr467 is in close proximity to the ⁇ -ae loop that is part of the NADP binding region. Phosphorylation of Thr467 might thus introduce defined structural
  • High salinity activates ASKa which in turn, phosphorylates G6PD6 on Thr467, thereby stimulating its activity.
  • Enhanced G6PD activity provides NADPH for the antioxidant system to remove excess ROS.
  • Reduction of H 2 O 2 to H 2 O can be mediated by the glutathione peroxidase cycle or by the ascorbate-glutathione cycle.
  • Example 1 Plant growth and stress treatments
  • Arabidopsis thaliana ecotype Columbia (Col 0) was germinated on 1 ⁇ 2 MS medium (Duchefa). After 10 days, seedlings were transferred to soil and cultivated in a 16 h light/8 h dark regime at 150 E.m "2 .s "1 light intensity and 60% relative humidity. For high soil salinity stress, plants were watered with a 150 mM NaCI solution. For germination and root length assay under high salt conditions, seeds were allowed to germinate on 1 ⁇ 2 MS or 1 ⁇ 2 MS supplemented with 100 mM NaCI in vertical plates in a 16 h light/8 h dark regime. Seeds used for one experiment were propagated in the same growth chamber at the same time.
  • Example 2 Plant material and plasmid constructs
  • the aska mutant (SAIL_ 055_F02) was genotyped using primers ASKa 5' and ASKa 9ex 3' (Table S1 , see Supplemental Figure S4)
  • the g6pd6.2 mutant (Gabi_KAT_142G07) was genotyped using primers G6PD6 9ex and G6PD6 13ex (Table S1 ).
  • ASKa-Myc and ASKaK98R-Myc were cloned into the expression vector pGreenll0029 under the control of the 35S promoter.
  • G6PD6-HA and its mutated variants were cloned into pGWR8.
  • a bidopsis Col 0 plants were transformed using the floral dippingmethod (Clough and Bent, 1998).
  • RT-PCR experiments were performed with cDNAs generated from 2 pg of total RNA using oligo (dT) primers and M-MuLV Reverse Transcriptase (Q-BIOgene). The RT- PCR exponential phase was determined on 22-30 cycles to allow semi-quantitative comparisons of cDNAs developed from identical reactions.
  • Oligonucleotide primers used for semi-quantitative RT-PCR were designed based on the 3' UTR of control and selected genes (Table S1 ).
  • 50 mg of plant material was extracted in 0.5 ml of enzyme extraction buffer (10% glycerol, 0.25% BSA, 0.1 % Triton X-100, 50 mM HEPES-KOH pH7.5, 10 mM MgCI2, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 1 mM 6-aminocaproic acid, 1 mM PMSF, 10 ⁇ leupeptin).
  • enzyme extraction buffer (10% glycerol, 0.25% BSA, 0.1 % Triton X-100, 50 mM HEPES-KOH pH7.5, 10 mM MgCI2, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 1 mM 6-aminocaproic acid, 1 mM PMSF, 10 ⁇ leupeptin).
  • G6PD total activity was determined as described in (Stanton et al., 1991 ) with some modifications. 5 ⁇ of enzyme extract was used in a 100 ⁇ total reaction volume. Absorbance was measured with a micro plate reader spectrophotometer (GeniosPro, TECAN). In vivo G6PD activities were calculated in nmol
  • Hydrogen peroxide levels were measured according to Wolff, 1994. Briefly, 50 mg of freshly pulverized plant material was extracted with 0.5 ml of 25 mM H2SO4. Samples were centrifuged at 4°C and 100 ⁇ of clear supernatant was added to 900 ⁇ of reagent solution (0.1 mM Xylenol Orange tetrasodium salt, 0.25 mM ammonium-iron(ll)sulphate, 100 mM sorbitol, 25 mM H2SO4; all reagents were purchased from Sigma). Reactions were incubated for 1 hour at RT in the dark.
  • reagent solution 0.1 mM Xylenol Orange tetrasodium salt, 0.25 mM ammonium-iron(ll)sulphate, 100 mM sorbitol, 25 mM H2SO4; all reagents were purchased from Sigma.
  • Oxidized and reduced forms of glutathione were determined with a
  • Protoplasts from an A. thaliana cell suspension culture were transformed as described (Cardinale et al., 2002).
  • HA-tagged G6PD6 was transiently expressed in Arabidopsis protoplasts.
  • [32P] orthophosphoric acid 0.1 mCi/ml
  • carrier free Hartmann Analytic
  • Immunoprecipitation was performed using HA antibodies (Santa-Cruz).
  • ASKa cDNA was cloned into pGEX5x3 (GE Healthcare), cDNAs from G6PD5 and G6PD6 were cloned into pGEX4T1 (GE Healthcare) . Recombinant proteins were expressed as GST fusion proteins in the E. coli BI_21 codon plus strain
  • Proteins were purified using the Sepharose beads affinity method (Glutathione SepharoseTM 4B, GE Healthcare).
  • PGR was performed using 2.5 U of Pfu Ultra (Stratagene) with the primers listed in Suppl. Table S1 . PGR mixtures were digested with the enzyme Dpnl for 2 hours at 37°C and transformed into E. coli. In vitro mutagenesis was verified by sequencing.
  • a rabbit polyclonal ASKa-specific antibody was raised against the synthetic peptide (PNPGARDSTGVDKL, SEQ ID 4) from the N-terminus of ASKa. Crude serum antibody was used for immunoprecipitation of the kinase. An antibody specificity test for immunoprecipitation was performed using ASKa, ASKy and ASKE in v/iro-translated with the T3/T7 coupled translation kit (Amersham Biosciences). Kinases were immunoprecipitated with the ASKa antibody as described for immunokinase assays.
  • Immunokinase assays were performed as described (Jonak et a!., 2000). G6PD or MBP was used as specific or general substrate, respectively (De Rybel et al., 2009; Jonak et al., 2000).
  • In vitro kinase assays using recombinant proteins were carried out in a total volume of 20 ⁇ of kinase buffer (20 mM HEPES, pH7.5, 15 mM MgCI 2 , 5 mM EGTA). The reaction was started with 2 ⁇ [ ⁇ -32 ⁇ ]- ⁇ and incubated at room temperature for 30 minutes. The reaction was stopped by addition of 5 ⁇ of 4x SDS loading buffer. Proteins were resolved by 8% SDS-PAGE. The gel was dried and exposed overnight to a phosphoimager screen.
  • phosphorylated peptides were evaluated manually.
  • An in-house generated FASTA database was used for the search containing the sequences of the target proteins, common contaminants and proteolytic enzymes. Peptides were filtered according to the XCorr/Charge state values; false positive rate was set to 5%.
  • Example 15 Enhanced Tolerance against Chlorogenic ROSs of plants with elevateed ASKa activity
  • ASKa is a regulator of ROS detoxification, the tolerance of ASKa
  • Paraquat or methyl viologen dichloride
  • the typical symptom of a paraquat-treated plant is the formation of wide areas of leaf chlorosis.
  • ASKa overexpressor lines exhibit an altered resistance towards this herbicide
  • seeds from selected homozygous lines were germinated on 1 ⁇ 2 MS plates supplemented with 1 ⁇ of paraquat.
  • three main phenotypes were observed: seeds that gave rise to green and healthy seedlings, seeds that stopped their development after the first protruding of the root tip or that produced unhealthy and bleached seedlings and seeds that failed to germinate.
  • ASKa overexpressor lines showed anincrease in tolerance to paraquat. The percentages of both germinated but stunted seedlings and of normally developing green seedlings were higher as compared to WT.
  • Example 16 Enhanced resistance of plants overexpressing ASKa to Pseudomonas syringae.
  • Floral dip a simplified method for Floral dip
  • Glucose-6-phosphate dehydrogenase is a regulator of vascular smooth muscle contraction. Antioxid Redox Signal 14, 543-558.
  • G6PDH glucose-6- phosphate dehydrogenase
  • G6PD glucose 6-phosphate dehydrogenase
  • Meiotic chromosome homology search involves modifications of the nuclear envelope protein Matefin/SUN-1 . Cell 139, 920-933.
  • AtGSKI Constitutive over-expression of AtGSKI induces NaCl stress responses in the absence of NaCl stress and results in enhanced NaCl tolerance in Arabidopsis. Plant J 27, 305-314.
  • Glucose-6- phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress.
  • Diabetes causes inhibition of glucose-6-phosphate dehydrogenase via activation of PKA, which contributes to oxidative stress in rat kidney cortex.
  • PKA glucose-6-phosphate dehydrogenase

Abstract

L'invention concerne un procédé de production d'une plante présentant une tolérance augmentée au stress abiotique et biotique par conception par génie génétique d'une cellule végétale recombinante pour augmenter le niveau de G6PD cytosolique phosphorylé dans ladite cellule végétale et régénération d'une plante transgénique à partir de ladite cellule végétale, qui présente une tolérance augmentée aux conditions de stress abiotique et biotique, et une plante tolérante au stress pouvant être obtenue par le procédé.
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