|Title of Invention||
METHOD FOR IMPROVING GROWTH CHARACTERISTICS OF A PLANT
|Abstract||The present invention concerns a method for improving growth characteristics of plants by increasing expression and/or activity in a plant of an LRR receptor kinase or a homologue thereof. One such method comprises introducing into a plant an RLK827 nucleic acid molecule or functional variant thereof. The invention also relates to transgenic plants having improved growth characteristics, which plants have modulated expression of a nucleic acid encoding an LRR receptor kinase. The present invention also concerns constructs useful in the methods of the invention.|
Plants having improved growth characteristics and method
for making the same
The present invention relates generally to the field of molecular biology and concerns a method for improving plant growth characteristics. More specifically, the present invention concerns a method for increasing yield and/or biomass of a plant by increasing the expression and/or activity of an LRR receptor kinase (RLK827) or a homologue thereof in a plant. The present invention also concerns plants having increased expression of a nucleic acid encoding an LRR receptor kinase or a homologue thereof, which plants have improved growth characteristics relative to corresponding wild type plants. The invention also provides constructs useful in the methods of the invention.
Given the ever-increasing world population, and the dwindling area of land available for agriculture, it remains a major goal of agricultural research to improve the efficiency of agriculture and to increase the diversity of plants in horticulture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic complements that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to manipulate the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has led to the development of plants having various improved economic, agronomic or horticultural traits. Traits of particular economic interest are growth characteristics such as high yield. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production and more. Root development, nutrient uptake and stress tolerance may also be important factors in determining yield. Crop yield may therefore be increased by optimising one of the abovementioned factors.
Growth and development of plants is determined by environmental and internal signals, such as hormone mediated signalling, stress and nutrient signalling, cell cycle control or developmental signalling. Cells perceive these signals via cell surface receptors, which
transduce the signal to the inside of the cell. Many of these receptors are protein kinases. Protein kinases comprise a large family of enzymes that mediate the response of eukaryotic cells to stimuli by phosphorylation of hydroxyamino acids. The enzymes fall into two broad classes with respect to their substrate specificity: serine/threonine specific or tyrosine specific enzymes. Kinases involved in signal transduction may be classified into different families which are mostly made up of tyrosine kinases. Receptor Tyrosine Kinases (RTK) in animals have a uniform structure and are composed of an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic tyrosine kinase domain. Among the plant tyrosine kinases, the Receptor-Like Kinase (RLK) proteins take a prominent place. More than 600 different RLKs are known in plants. They have a similar structure as the animal RTKs, a classification is given in Fig. 1 (Shiu and Bleecker, Proc. Natl. Acad. Sci USA 98, 10763-10768, 2001). Several plant RLK proteins have been characterised, for example BRI1 (brassinoid signalling), CLV1 (meristem differentiation), HAESA (abscission of floral organs), XA21 (fungal detection) CR4 (leaf and endosperm development), FLS2 (flagellin/pathogen detection), SRK (self-incompatibility), among others (Becraft, Annu. Rev. Cell Dev. Biol. 18, 163-192, 2002; Di6vart and Clark, Curr. Opin. Plant Biol. 6, 507-516). About 200 of the plant RLKs possess a Leucine Rich Repeat (LRR). LRRs are sequence motifs of 23 to 25 residues, which comprise a consensus sequence LxxLxLxxN/CxL wherein x may be any amino acid. These LRRs are present in proteins with diverse functions, such as hormone receptor interactions, enzyme inhibition, cell adhesion and cellular trafficking and frequently the LRR domains are organised in tandem arrays. It was shown that LRRs may be critical for the morphology and dynamics of the cytoskeleton. The primary function of these motifs appears to be providing a versatile structural framework for the formation of protein-protein interactions" (Kobe and Kajava, Curr. Opin. Struct. Biol. 11, 725-732, 2001).
The combination of Leucine Rich Repeats and kinase domains is characteristic for receptor proteins that mediate external signals into the cell. They are thought to act by a mechanism in which the LRR domain(s), mostly extracellular, act as a sensor for an extracellular signal whereas the kinase domain is usually internal and participates in the transduction of the signal by phosphorylating intracellular targets and thus initiating the signal transduction. RLKs have been implicated in plants in a variety of process like plant development, disease resistance or self-incompatibility. It is shown in this invention that plant growth characteristics, and in particular yield, may be improved by modulating expression in a plant of a nucleic acid encoding an RLK.
International patent application WO 03/072763 disclosed a receptor like kinase which, when overexpressed in plants, resulted in increased plant growth and seed production. However,
the subject RLK protein did not comprise any LRR domains in its non-cytoplasmic domain, but instead this domain was Proline rich. Another disclosure (WO 00/04761) reported that upon overexpression of the RKN receptor kinase, root growth was enhanced. Similarly, it was suggested, but not shown, in WO 98/59039 that overexpression of the BRI1 receptor kinase would result in modulated yield. However the RLK used in the latter two cases comprised 22 LRR domains in the non-cytoplasmic domain, typical for the LRR-X subfamily of receptor like kinases. So far there have been no reports to show or even suggest that receptor like kinases of the LRR-I subfamily may be useful for improving plant growth characteristics, and in particular in increasing yield.
It has now surprisingly been found that increasing expression and/or activity, relative to corresponding wild type plants, of an RLK827 protein in plants gives plants having improved growth characteristics, and in particular increased yield.
RLK827 is a receptor like kinase that is structurally related to LRRPK, which is a member of the LRR-I subfamily of receptor like kinases (Shiu and Bleecker, 2001). The mature RLK827 protein has, starting from the N-terminus, a long putative non-cytoplasmic domain, a single transmembrane domain and a kinase domain in the C-terminal cytoplasmic part. The receptor like kinases are classified according to the composition of their non-cytoplasmic domain, which may comprise proline rich sequences, lectin domains, LRR domains, EGF repeats, TNFR repeats, thaumatin or agglutinin domains etc. A large group of receptor like kinases have Leucine Rich Repeats (LRR) in the non-cytoplasmic domain. The various LRR subfamilies differ from each other'in the number and position of these leucine rich repeats (fd? an overview, see Shiu and Bleecker, 2001). The putative non-cytoplasmic domain of RLK827 is characterised by the presence of one up to three tandem leucine rich repeat domains in its C-terminal part; RLK827 therefore belongs in the subfamily of LRR-I receptor kinases. The various LRR subfamilies of receptor kinases also differ from one other in their chromosomal distribution. Often they are arranged in tandem repeats. Tandem duplications, large-scale duplications and rearrangements of chromosomes are, at least in part, responsible for the evolution and expansion of the LRR receptor like kinases in plants. Accordingly, with a few exceptions, LRR-I receptor kinases are distributed on chromosome I, II and III in Arabidopsis.
According to one embodiment of the present invention there is provided a method for improving growth characteristics of a plant comprising increasing expression and/or activity of an RLK827 polypeptide or a homologue thereof and optionally selecting for plants having improved growth characteristics.
Advantageously, performance of the methods according to the present invention result in plants having a variety of improved growth characteristics, such as improved growth, improved yield, improved biomass, modified architecture or improved cell division, each relative to corresponding wild type plants. Preferably, the improved growth characteristics comprise at least increased yield relative to corresponding wild type plants. Preferably, the increased yield is increased seed yield, which includes increased number of (filled) seeds, increased total weight of seeds and increased harvest index.
The term "increased yield" as defined herein is taken to mean an increase in any one or more of the following, each relative to corresponding wild type plants: (i) increased biomass (weight) of one or more parts of a plant, particularly aboveground (harvestable) parts, increased root biomass or increased biomass of any other harvestable part; (ii) increased total seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant (total seed weight) or on an individual seed basis; (iii) increased number of (filled) seeds; (iv) increased seed size; (v) increased seed volume; (vi) increased individual seed area; (vii) increased individual seed length; (viii) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass; (ix) increased number of florets per panicle which is extrapolated from the total number of seeds counted and the number of primary panicles; and (x) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size (length, width or both) and/or seed weight. An increased TKW may result from an increase in embryo size and/or endosperm size.
Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, TKW, ear length/diameter, among others. Taking rice as an example, a yield increase may be manifested by an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in the seed filling rate, increase in TKW, among others. An increase in yield may also result in modified architecture, or may occur as a result of modified architecture.
Preferably, performance of the methods of the present invention results in plants having increased yield and/or increased biomass. More particularly, performance of the methods according to the present invention results in plants having increased seed yield. Preferably, the increased seed yield comprises an increase in one or more of number of filled seeds, total
seed weight, and harvest index, each relative to control plants. Therefore, according to the present invention, there is provided a method for increasing plant yield, which method comprises increasing expression and/or activity in a plant of an RLK827 polypeptide or a homologue thereof.
Since the modified plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of corresponding wild type plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts or cell types of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, flowering time and speed of seed maturation. An increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible. If the growth rate is sufficiently increased, it may allow for the sowing of further seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the sowing of further seeds of different plants species (for example the sowing and harvesting of rice plants followed by, for example, the sowing and optional harvesting of soy bean, potatoes or any other suitable plant). Harvesting additional times from the same rootstock in the case of some plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves plotting growth experiments, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
' Performance of the methods of the invention gives plants having an increased growth rate. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises increasing expression and/or activity in a plant of an RLK827 polypeptide or a homologue thereof.
An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Rants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the typical stresses to which a plant may be exposed. These stresses may be the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Typical abiotic or environmental stresses include temperature stresses caused by atypical hot or cold/freezing temperatures; salt stress; water stress (drought or excess water). Abiotic stresses may also be caused by chemicals. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
The abovementioned growth characteristics may advantageously be modified in any plant.
The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest or the specific modification in the gene/nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, i gametophytes, sporophytes, pollen, and microspores, again wherein each of the aforementioned comprise the gene/nucleic acid of interest.
Plants that are particularly useful in the methods of the invention include algae, ferns, and all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants, including fodder or forage legumes, ornamental plants, food crops, trees, or shrubs selected from the list comprising Abelmoschus spp., Acer spp., Actinidia spp., Agropyron spp., Allium spp., Amaranthus spp., Ananas comosus, Annona spp., Apium graveolens, Arabidopsis thaliana, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena
saliva, Averrhoa carambola, Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp-, Cadaba farinosa, Camellia sinensis, Canna indica, Capsicum spp., Carica papaya, Carissa macrocarpa, Carthamus tinctorius, Carya spp., Castanea spp., Cichorium endivia, Cinnamomum spp.t C'rtrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Cola spp., Colocasia esculenta, Corylus spp., Crataegus spp., Cucumis spp., Cucurbita spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscor&a spp., Diospyros spp., Echinochloa spp., Beusine coracana, Eriobotrya japonica, Eugenia uniflora, Fagopymm spp., Fagus spp., F/cas carica, Fortunella spp.r Fragaria spp., Ginkgo biloba, Glycine spp., Gossypium hirsutum, Helianthus spp., Hibiscus spp., Hordeum spp., Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyms spp., Lemna spp., Lens culinaris, Unum usitatissimum, Utchi chinensis, Lotus spp., Lu/fe acutangula, Lupinus spp., Macrotyloma spp., Malpighia emarginata, Malus spp., Mammea americana, Mang'rfera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Momordica spp., Morus n/gra, Musa spp., Nicotiana spp., O/ea spp., Opuntia spp., Ornithopus spp., Oryza spp., Panicum miliaceum, Passiflora eduiis, Pastinaca sativa, Persea spp., Petroselinum crispum, Phaseolus spp., Phoenix spp., Physalis spp., P/nus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pynjs communis, Quercus spp., Raphanus sativus, Rheum rhabarbamm, Ribes spp., Rubus spp., Sacchamm spp., Sambucus spp., Seca/e cereale, Sesamum spp., Solanum spp., Sorghum bicolor, Spinacia spp., Syzygium spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp., Vaccinium spp., Wc/a spp., V7gna spp., Vrt/s spp., Zea mays, Zizania palustrist Ziziphus spp., amongst others.
According to a preferred feature of the present invention, the plant is a crop plant comprising soybean, sunflower, canola, alfalfa, rapeseed or cotton. Further preferably, the plant according to the present invention is a monocotyledonous plant such as sugarcane, most preferably a cereal, such as rice, maize, wheat, millet, barley, oats or sorghum.
: The activity of an RLK827 protein may be increased by increasing levels of the RLK827 polypeptide. Alternatively, activity may also be increased when there is no change in levels of an RLK827, or even when there is a reduction in levels of an RLK827. This may occur when the intrinsic properties of the polypeptide are altered, for example, by making a mutant or selecting a variant that is more active that the wild type.
The term "RLK827 or homologue thereor as defined herein refers to a Receptor Like Kinase (RLK) having kinase activity and comprising in its mature form a non-cytoplasmic domain (extracellular domain), a single transmembrane domain and a putative cytoplasmic kinase
domain. The non-cytoplasmic domain or RLK827 comprises at least 1 but no more than 3 Leucine Rich Repeat (LRR) domains, preferably two LRR domains are present, more preferably three LRR domains. Further preferably, the length of the non-cytoplasmic domain ranges between 250 and 550 amino acids. The non-cytoplasmic domain preferably comprises the amino acid sequence motif LRxFP(E/D)GxRNC(Y/F) (SEQ ID NO: 33), wherein x may be any amino acid and where up to 2 other amino acids may be replaced by a conserved substitution as listed in Table 2. Preferably, the first x in this motif is Y or A, and the second x preferably is one of V, F, E and A.
The term "RLK827 or homologue thereof also refers to amino acid sequences having in increasing order of preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% overall sequence identity to the amino acid represented by SEQ ID NO: 2.
The term "RLK827 or homologue thereof comprises RLK827 (SEQ ID NO 2), its paralogues and orthologues. The overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), using default parameter settings.
The various structural domains in an RLK827 protein may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; http://smart.embl-heidelberg.de/) or Pfem (Bateman et a/., Nucleic Acids Research 30(1):276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/).
The kinase domain is of a STYKc type (SMART accession number SM00221, Interpro accession number IPR004040) and has possibly dual-specificity Ser/Thr/Tyr kinase activity. In the N-terminal extremity of the catalytic domain there is a glycine-rich stretch of residues in the vicinity of a lysine residue, which has been shown to be involved in ATP binding. In the central part of the catalytic domain there is a conserved aspartic acid residue, which is important for the catalytic activity of the enzyme.
Furthermore, LRR domains are well known in the art and are defined in Pfam (accession PF00560) as 20 to 29-residue sequence motifs present in tandem arrays in a number of
proteins with diverse functions, such as hormone receptor interactions, enzyme inhibition, cell adhesion and cellular trafficking. Recent studies revealed the involvement of LRR proteins in early mammalian development, neural development, cell polarization, regulation of gene expression and apoptosis signalling. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions-Sequence analyses of LRR proteins suggested the existence of several different subfamilies of LRRs. Apparently the repeats from different subfamilies never occur simultaneously and most probably evolved independently. However, all major classes of LRR seem to have curved horseshoe structures with a parallel beta sheet on the concave side and mostly helical elements on the convex side. At least six families of LRR proteins, characterised by different lengths and consensus sequences of the repeats, have been identified. Eleven-residue segments of the LRRs (LxxLxLxxN/CxL), corresponding to the ft-strand and adjacent loop regions, are usually conserved in LRR proteins, whereas the remaining parts of the repeats may be very different. Despite the differences, each of these variable parts contains two half-turns at both ends and a "linear"* segment (as the chain follows a linear path overall), usually formed by a helix, in the middle. The concave face and the adjacent loops are the most common protein interaction surfaces on LRR proteins. 3D structures of some LRR protein-ligand complexes show that the concave surface of LRR domain is ideal for interaction with alpha-helices, thus supporting earlier conclusions that the elongated and curved LRR structure provides an outstanding framework for achieving diverse protein-protein interactions. Molecular modelling suggests that the pattern LxxLxL, which is often conserved and which is shorter than the previously proposed LxxLxLxxN/CxL, is sufficient to impart the characteristic horseshoe curvature to proteins with 20- to 30-residue repeats. LRR domains of an LRK827 protein may differ from the canonical LRR domains known in the art but may be identified by suitable computer algorithms, preferably those used in the Pfam database.
Transmembrane domains are about 15 to 30 amino acids long and are usually composed of hydrophobic residues that form an alpha helix. They are usually predicted on the basis of hydrophobicity (for example Klein et a/., Biochim. Biophys. Acta 815, 468, 1985; or Sonnhammer et a/., In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen, editors, Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-182, Menlo Park, CA, 1998. AAAI Press.).
Methods for the search and identification of RLK827 homologues would be well within the realm of persons skilled in the art. Such methods comprise comparison of the sequences represented by SEQ ID NO 1 or 2, in a computer readable format, with sequences that are available in public databases such as MIPS (http://mips.gsf.de/), GenBank
(http://www.ncbi.nlm.nih.gov/Genbank/index.html) or EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/index.html), using algorithms well known in the art for the alignment or comparison of sequences, such as GAP (Needleman and Wunsch, J. Mol. Biol. 48; 443-453 (1970)), BESTFIT (using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2; 482^89 (1981))), BLAST (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J., J. Mol. Biol. 215:403-410 (1990)), FASTA and TFASTA (W. R. Pearson and D. J. Lipman Proc.Natl.Acad.Sci. USA 85:2444- 2448 (1988)). The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). The homologues mentioned below were identified using BLAST default parameters (BLOSUM62 matrix, gap opening penalty 11 and gap extension penalty 1) and preferably the full-length sequences are used for analysis.
Examples of proteins falling under the definition of "RLK827 polypeptide or a homologue thereof include the Arabidopsis proteins At1g51850, At1g51805, At1g51810, At2g04300, At3g21340, At1g49100. It should be noted that a cluster of related putative receptor like kinases are located in tandem on chromosome 1 of Arabidopsis thaliana, including At1g51800, At1g51805, At1g51810, At1g51820, At1g51830, At1g51840, At1g51850, At1g51860, At1g51870, At1g51880 and At1g51890, of which at least four of them are highly related to RLK827.
It is to be understood that the term RLK827 polypeptide or a homologue thereof is not to be limited to the sequences represented by SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 and SEQ ID NO: 19, but that'any polypeptide meeting the criteria of (i) having a cytoplasmic kinase domain and (ii) having at least one but no more than three LRR domains and preferably the consensus sequence of SEQ ID NO: 33 in the putative non-cytoplasmic part of the protein, separated from the kinase domain by a transmembrane region, and which kinase domain comprises the STYKc consensus sequence and/or (iii) being a paralogue or orthologue of RLK827 and having at least 25% sequence identity to the sequence of SEQ ID NO: 2, may be suitable for use in the methods of the invention. Preferably, the kinase domain is functional, meaning that the RLK827 polypeptide or its homologue has kinase activity.
To determine the kinase activity of RLK827, several assays are available and well known in the art (for example Current Protocols in Molecular Biology, Volumes 1 and 2, Ausubel et al. (1994), Current Protocols; or online such as http://www.protocol-online.org). In brief, the kinase assay generally involves (1) bringing the kinase protein into contact with a substrate polypeptide containing the target site to be phosphoryiated; (2) allowing phosphorylation of the
target site in an appropriate kinase buffer under appropriate conditions; (3) separating phosphorylated products from non-phosphorylated substrate after a suitable reaction period. The presence or absence of kinase activity is determined by the presence or absence of a phosphorylated target. In addition, quantitative measurements can be performed. Purified RLK827 protein, or cell extracts containing or enriched in the RLK827 protein could be used as source for the kinase protein. Alternatively, the approach of Zhao et a/. (Plant Mol. Biol. 26, 791-803, 1994) could be used, where the cytoplasmic domain of a rice receptor like kinase was expressed in Escherichia coli and assayed for kinase activity. As a substrate, small peptides are particularly well suited. The peptide must comprise one or more serine, threonine or tyrosine residues in a phosphorylation site motif. A compilation of phosphorylation sites can be found in Biochimica et Biophysica Acta 1314, 191-225, (1996). In addition, the peptide substrates may advantageously have a net positive charge to facilitate binding to phosphocellulose filters, (allowing to separate the phosphorylated from non-phosphorylated peptides and to detect the phosphorylated peptides). If a phosphorylation site motif is not known, a general tyrosine kinase substrate can be used. For example, "Src-related peptide" (RRLIEDAEYAARG) is a substrate for many receptor and non-receptor tyrosine kinases). To determine the kinetic parameters for phosphorylation of the synthetic peptide, a range of peptide concentrations is required. For initial reactions, a peptide concentration of 0.7-1.5 mM could be used. For each kinase enzyme, it is important to determine the optimal buffer, ionic strength, and pH for activity. A standard 5x Kinase Buffer generally contains 5 mg/ml BSA (Bovine Serum Albumin preventing kinase adsorption to the assay tube), 150 mM Tris-CI (pH 7.5), 100 mM MgCI2. Divalent cations are required for most tyrosine kinases, although some tyrosine kinases (for example, insulin-, IGF-1-, and PDGF receptor kinases) require MnCI2 instead of MgCI2 (or in addition to MgCI2). The optimal concentrations of divalent cations must be determined empirically for each protein kinase. A commonly used donor for the phophoryl group is radio-labelled [gamma-32P]ATP (normally at 0.2 mM final concentration). The amount of 32P incorporated in the peptides may be determined by measuring activity on the nitrocellulose dry pads in a scintillation counter.
Alternatively, the activity of an RLK827 polypeptide or of a homologue thereof may be assayed by expressing the RLK827 polypeptide or of a homologue thereof under control of a rice GOS2 promoter in rice plants, and in particular in the rice variety Nipponbare, which results in plants with increased yield compared to corresponding wild type plants. This increase in yield may for example be measured as one or more of an increase in number of filled seeds, in total weight of seeds and/or in harvest index.
The nucleic acid encoding an RLK827 polypeptide or a homologue thereof may be any natural or synthetic nucleic acid. An RLK827 polypeptide or a homologue thereof as defined herein is encoded by an RLK827 nucleic acid molecule. Therefore the term "RLK827 nucleic acid molecule" or "RLK827 gene" as defined herein is any nucleic acid molecule encoding an RLK827 polypeptide or a homologue thereof as defined above. Examples of RLK827 nucleic acid molecules include those represented by any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31. RLK827 nucleic acids and functional variants thereof may be suitable in practising the methods of the invention. Functional variant RLK827 nucleic acids include portions of an RLK827 nucleic acid molecule and/or nucleic acids capable of hybridising with an RLK827 nucleic acid molecule or with a nucleic acid molecule encoding a homologue of RLK827. The term "functional" in the context of a functional variant refers to a variant RLK827 nucleic acid molecule (i.e. a portion or a hybridising sequence), which encodes a polypeptide having kinase activity and comprising a non-cytoplasmic (extracellular) domain, which non-cytoplasmic domain comprises at least 1 but no more than 3 LRR motifs and preferably also the amino acid sequence motif of SEQ ID NO: 33 as defined above, and a C-terminal kinase domain that is separated from the non-cytoplasmic domain by a transmembrane domain.
The LRR-I type of receptor like kinases in plants have a modular structure, and it has been shown that one LRR protein is able to bind different ligands, for example the tomato SR160 receptor and its tomato homologue tBRH are able to bind brassinolide hormones and systemin, a long distance signalling peptide. Brassinolide and systemin do not compete for binding, suggesting they bind to different sites. Therefore, it is envisaged that engineering of LRR domains (e.g. by altering the number of LRR domains, or by performing domain stacking (binding to same or different ligand(s)), or domain shuffling), in such a way that the activity of the LRR is retained or modified, is useful in generating variant RLK827 nucleic acid molecules for performing the methods of the invention. In a similar way, the kinase domain may be engineered to improve kinase activity. A preferred type of variant includes those generated by domain deletion, stacking or shuffling (see for example He et a/., Science 288, 2360-2363, 2000, or US patents 5,811,238 and 6,395,547).
The term portion as defined herein refers to a piece of DNA comprising at least 150 nucleotides. A portion may be prepared, for example, by making one or more deletions to an RLK827 nucleic acid. The portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities, one of them being protein kinase activity. When fused to other coding
sequences, the resulting polypeptide produced upon translation could be bigger than that predicted for the RLK827 portion. The portion useful in the methods of the present invention comprises at least the kinase domain, preferably also a non-cytoplasmic LRR domain and a transmembrane domain located N-terminally of the kinase domain, more preferably the portion comprises in the non-cytoplasmic domain at least 1 but no more than 3 LRR domains, most preferably, the portion comprises in the non-cytoplasmic domain at least 1 but no more than 3 LRR domains and the amino acid sequence motif of SEQ ID NO: 33 as defined above. Preferably, the functional portion is a portion of a nucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.
The term "hybridisation" as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids. The stringency of hybridisation is influenced by conditions such as r temperature, salt concentration, ionic strength and hybridisation buffer composition.
"Stringent hybridisation conditions" and "stringent hybridisation wash conditions" in the context of nucleic acid hybridisation experiments such as Southern and Northern hybridisations are sequence dependent and may differ depending on environmental parameters. The skilled artisan is aware of various parameters which may be altered during hybridisation and washing and which will either maintain or change the stringency conditions.
The Tm is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16°C up to 32°C below Tm. The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands
' thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M. Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7°C for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45°C, though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1°C per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:
♦ DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138:267-284, 1984):
Tm= 81.5°C + 16.6xlog[NaT + 0.41x%[G/e*l - 500x[LT - 0.61x% formamide
♦ DNA-RNA or RNA-RNA hybrids:
Tm= 79.8 + 18.5 (logi0[NaT) + 0.58 (%G/Cb) +11.8 (%Gf(?)2 - 820/Lc
♦ oligo-DNA or oligo-RNAd hybrids:
For For 20-35 nucleotides: Tm= 22 + 1.46 (/n )
a or for other monovalent cation, but only accurate in the 0.01-0.4 M range.
b only accurate for %GC in the 30% to 75% range.
c L = length of duplex in base pairs.
d Oligo, oligonucleotide; /„, effective length of primer = 2*(no. of G/C)+(no. of A/T).
Note: for each 1% formamide, the Tm is reduced by about 0.6 to 0.7°C, while the presence of 6M urea reduces the Tm by about 30°C
Specificity of hybridisation is typically the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. Generally, low stringency conditions are selected to be about 50°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20°C below Tm, and high stringency conditions are when the temperature is 10°C below Tm. For example, stringent conditions are those that are at least as stringent as, for example, conditions A-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R. Non-
* The "hybrid length" is the anticipated length for the hybridising nucleic acid. When nucleic
acids of known sequence are hybridised, the hybrid length may be determined by aligning the
sequences and identifying the conserved regions described herein.
f SSPE (1*SSPE is 0.15M NaCI, 10mM NaFfePO*, and 1.25mM EDTA, pH7.4) may be substituted for SSC (1*SSC & 0.15M NaCI and 15mM sodium citrate) in the hybridisation and wash buffers; washes are performed for 15 minutes after hybridisation is complete. The hybridisations and washes may additionally include 5 * Denhardt's reagent, 0.5-1.0% SDS, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.
* Tb-Tr: The hybridisation temperature for hybrids anticipated to be less than 50 base pairs in
length should be 5-10°C less than the melting temperature Tm of the hybrids; the Tm is
determined according to the above-mentioned equations.
* The present invention also encompasses the substitution ofany one, or more DNA or RNA
hybrid partners with either a PNA, or a modified nucleic acid.
For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et ai (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).
For example, a nucleic acid encoding SEQ ID NO: 2 or a homologue thereof may be used in a hybridisation experiment. Alternatively fragments thereof may be used as probes. Depending on the starting pool of sequences from which the RLK is to be identified, different fragments for hybridization can be selected. For example, when a limited number of homologues with a high sequence identity to RLK827 are desired, a less conserved fragment may be used for hybridisation such as GGTAGACTCGCCAAAGAATTTGAACCACTCGTTGAT (nucleotides 184 to 219 of SEQ ID NO: 1). By aligning SEQ ID NO 2 and homologues thereof it is possible to design equivalent nucleic acid fragments useful as probes for hybridisation. Preferably the
hybridising sequence comprises at least the kinase domain, preferably also a non-cytopiasmic LRR domain and a transmembrane domain located N-terminally of the kinase domain, more preferably the portion comprises in the non-cytoplasmic domain at least 1 but no more than 3 LRR domains, most preferably, the portion comprises in the non-cytoplasmic domain at least 1 but no more than 3 LRR domains and the amino acid sequence motif of SEQ ID NO: 33 as defined above.
After hybridisation and washing, the duplexes may be detected by autoradiography (when radiolabeled probes were used) or by chemiluminescence, immunodetection, by fluorescent or chromogenic detection, depending on the type of probe labelling. Alternatively, a ribonuclease protection assay may be performed for detection of RNA:RNA hybrids
The RLK827 nucleic acid molecule or variant thereof may be derived from any natural or
artificial source. The nucleic acid/gene or variant thereof may be isolated from a microbial
source, such as bacteria, yeast or fungi, or from a plant, alga or animal (including human)
source. This nucleic acid may be modified from its native form in composition and/or genomic
environment through deliberate human manipulation. The nucleic acid is preferably of plant
origin, whether from the same plant species (for example to the one in which rt is to be
introduced) or whether from a different plant species. The nucleic acid may be isolated from a
dicotyledonous species, preferably from the family Brassicaceae, further preferably from
Arabidopsis thaliana. More preferably, the RLK827 isolated from Arabidopsis thaiiana is
represented by SEQ ID NO: 1 and the RLK827 amino acid sequence is as represented by
SEQ ID NO: 2. '"""
Functional variants useful in the methods of the present invention also include alternative splice variants of an RLK827 nucleic acid molecule or gene. The term "alternative splice variant" as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced or added. Such variants will be ones in which the biological activity of the protein is retained, which may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for making such splice variants are well known in the art. Preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 1. Further preferred are splice variants encoding a polypeptide retaining kinase activity and having at least one but no more than three LRR domains in the putative non-cytoplasmic part of the protein, separated from the kinase domain by a transmembrane region. More preferred splice variants comprise in addition also the amino acid sequence motif of SEQ ID NO: 33 in
the putative non-cytoplasmic domain. Most preferred splice variants of an RLK827 nucleic acid molecule are those that encode an RLK827 polypeptide as defined above.
Functional variants useful in the methods of the present invention furthermore include allelic variants of a nucleic acid encoding an RLK827 polypeptide or a homologue thereof, preferably an allelic variant of the nucleic acid represented by SEQ ID NO 1. Further preferably, the polypeptide encoded by the allelic variant has kinase activity and retains at least one but no more than three LRR domains in the putative non-cytoplasmic part of the protein, separated from the kinase domain by a transmembrane region. More preferred allelic variants comprise in addition also the amino acid sequence motif of SEQ ID NO: 33 in the putative non-cytoplasmic domain. Most preferred allelic variants of an RLK827 nucleic acid molecule are those that encode an RLK827 polypeptide as defined above. Allelic variants exist in nature and encompassed within the methods of the present invention is the use of these natural alleles. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
The expression and/or activity of an RLK827 polypeptide or a homologue thereof may also be increased by introducing a genetic modification (preferably in the locus of an RLK827 gene). The locus of a gene as defined herein is taken to mean a genomic region which includes the gene of interest and 10 kb up- or downstream of the coding region.
The genetic modification may be introduced, for example, by any one (or more) of the following methods: TDNA activation, TILLING, site-directed mutagenesis, homologous recombination, directed evolution or by introducing and expressing in a plant a nucleic acid encoding an RLK827 polypeptide or a homologue thereof. Following introduction of the genetic modification there follows a step of selecting for increased expression and/or activity of an RLK827 polypeptide, which increase in expression and/or activity gives plants having improved growth characteristics.
T-DNA activation tagging (Hayashi et a/. Science 258, 1350-1353, 1992) involves insertion of T-DNA usually containing a promoter (may also be a translation enhancer cr an intron), in the genomic region of the gene of interest or 10KB up- or down stream of the coding region of a gene in a configuration such that such promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter. The promoter is typically
embedded in a T-DNA. This T-DNA is randomly inserted into a plant genome, for example, through Agrobacterium infection and leads to overexpression of genes near to the inserted T-DNA. The resulting transgenic plants show dominant phenotypes due to overexpression of genes close to the introduced promoter. The promoter to be introduced may be any promoter capable of directing expression of a gene in the desired organism, in this case a plant. For example, constitutive, tissue-preferred, cell type-preferred and inducible promoters are all suitable for use in T-DNA activation.
A genetic modification may also be introduced in the locus of an RLK827 gene using the technique of TILLING (Targeted Induced Local Lesions IN Genomes). This is a mutagenesis technology useful to generate and/or identify, and to eventually isolate mutagenised variants of an RLK827 nucleic acid molecule capable of exhibiting RLK827 activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may even exhibit higher RLK827 activity than that exhibited by the gene in its natural form. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei and Koncz (1992), In: C Koncz, N-H Chua, J Schell, eds, Methods in Arabidopsis Research. World Scientific, Singapore, pp 16-82; Feldmann et a/., (1994) In: EM Meyerowitz, CR Somerville, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 137-172; Lightner and Caspar (1998), In: J Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa, NJ, pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods for TILLING are well known in the art (McCallum Nature Biotechnol. 18, 455-457, 2000, Stemple Nature Rev. Genet. 5, 145-150, 2004).
Site-directed mutagenesis may be used to generated variants of RLK827 nucleic acids or portions thereof that retain activity, namely, protein kinase activity. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (See for example Ausubel et a/., Current Protocols in Molecular Biology. Wiley Eds. http://www.4ulr.com/products/cun-entprotocoIs/index.html).
Directed evolution may be used to generate variants of RLK827 nucleic acid molecules or portions thereof encoding RKS11 or RKS4 polypeptides or orthologues or portions thereof having an increased biological activity. Directed evolution consists of iterations of DNA
shuffling followed by appropriate screening and/or selection (Castle et al., (2004) Science 304(5674): 1151-4; US patents 5.811,238 and 6,395,547).
TDNA activation, TILLING, site-directed mutagenesis and directed evolution are examples of technologies that enable the generation novel alleles and variants of RLK827 that retain RLK827 function and which are therefore useful in the methods of the invention.
Homologous recombination allows introduction in a genome of a selected nucleic acid at a defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organism such as yeast or the moss Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offringa et al (1990) EMBO J. 9, 3077-3084) but also for crop plants, for example rice (Terada et a/., (2002) Nature Biotechnol. 20, 1030-1034; or lida and Terada (2004) Curr. Opin. Biotechnol. 15, 132-138). The nucleic acid to be targeted (which may be an RLK827 nucleic acid molecule or variant thereof as hereinbefore defined) need not be targeted to the locus of an RLK827 gene, but may be introduced in, for example, regions of high expression. The nucleic acid to be targeted may be an improved allele used to replace the endogenous gene or may be introduced in addition to the endogenous gene.
According to a preferred embodiment of the invention, plant growth characteristics may be improved by introducing and expressing in a plant a nucleic acid encoding an RLK827 polypeptide or a homologue thereof.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of an RLK827 gene) is to introduce and express in a plant a nucleic acid encoding an RLK827 polypeptide or a homologue thereof. An RLK827 polypeptide or homologue thereof as mentioned above is one having kinase activity and comprising a non-cytoplasmic (extracellular) domain, which non-cytoplasmic domain comprises at least 1 but no more than 3 LRR motifs and preferably also the amino acid sequence motif of SEQ ID NO: 33 as defined above, and a C-terminal kinase domain that is separated from the non-cytoplasmic domain by a transmembrane domain. Preferably, the RLK827 polypeptide or homologue thereof has in increasing order of preference, at least 25%, 26%. 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%. 54%, 55%. 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%,
98% or 99% overall sequence identity to the amino acid sequence represented by SEQ ID NO: 2.
"Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
Encompassed by the term "homologues" are orthologous sequences and paralogous sequences, two special forms of homology which encompass evolutionary concepts used to describe ancestral relationships of genes.
The term "paralogous" relates to gene-duplications within the genome of a species leading to paralogous genes. Paralogues of RLK827 may easily be identified by performing a BLAST analysis against a set of sequences from the same species as the query sequence.
The term "orthologous" relates to homologous genes in different organisms due to speciation. Orthologues in, for example, monocot plant species may easily be found by performing a so-called reciprocal blast search. This may be done by a first blast involving blasting the sequence in question (for example, SEQ ID NO: 1 or SEQ ID NO: 2) against any sequence database, such as the publicly available NCBI database which may be found at: http://www.ncbi.nlm.nih.gov. If orthologues in rice were sought, the sequence in question would be blasted against, for example, the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI. BLASTn or tBLASTX may be used when starting from nucleotides or BLASTP or TBLASTN when starting from the protein, with standard default values. The blast results may be filtered. The full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence in question is derived. The results of the first and second blasts are then compared. An orthologue is found when the results of the second blast give as hits with the highest similarity an RLK827 nucleic acid or RLK827 polypeptide, for example, if one of the organisms is Arabidopsis thaliana then a paralogue is found. In the case of large families, ClustalW may be used, followed by the construction of a neighbour joining tree, to help visualize the clustering. Using a reciprocal BLAST procedure a rice orthologue (Unigene accession number Os.26918) was identified represented by the ESTs CB631540, CB628137.1 and CB31541.1. Preferred orthologues are those having the highest similarity to RLK827 or to a paralogue thereof in a reciprocal BLAST search. Other examples of rice orthologues are given in SEQ ID NOs 24, 26, 28, 30 and 32.
A homologue may be in the form of a "substitutional variant" of a protein, i.e. where at least one residue in an amino acid sequence has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1 to 10 amino acid residues. Preferably, amino acid substitutions comprise conservative amino acid substitutions (Table 2). To produce such homologues, amino acids of the protein may be replaced by other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break o-helical structures or p-sheet structures). Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company).
Less conserved substitutions can be made in case the above-mentioned amino acid properties are not so critical.
A homologue may also be in the form of an "insertiorial variant" of a protein, i.e. where one or more amino acid residues are introduced into a predetermined site in a protein. Insertions may comprise amino-terminal and/or carboxy-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino- or carboxy-terminal fusions, of the order of about 1 to 10 residues. Examples of amino- or carboxy-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)6-tag, glutathione S-transferase-tag, protein A, maltose-binding
protein, dihydrofolate reductase, Tag 100 epitope, c-myc epitope, FLAG -epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
Homologues in the form of "deletion variants" of a protein are characterised by the removal of one or more amino acids from a protein.
Amino acid variants of a protein may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
The RLK827 polypeptide or homologue thereof may be a derivative. "Derivatives" include peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise substitutions, deletions or additions of naturally and non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of the protein, for example, as presented in SEQ ID NO 2. "Derivatives" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise naturally occurring altered, glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
According to a preferred aspect of the present invention, enhanced or increased expression of an RLK827 nucleic acid molecule or variant thereof is envisaged. Methods for obtaining enhanced or increased expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of an RLK827 nucleic acid or variant thereof. For example, endogenous promoters may be altered
in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et a/., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region may be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg, Mol. Cell Biol. 8, 4395-4405 (1988); Callis et a/., Genes Dev. 1, 1183-1200 (1987)). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, MY. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:
(i) an RLK827 nucleic acid molecule or functional variant thereof;
(ii) one or more control sequence capable of driving expression of the nucleic acid
sequence of (i); and optionally (iii) a transcription termination sequence.
Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells.
Plants are transformed with a vector comprising the sequence of interest (i.e., an RLK827 nucleic acid or functional variant thereof). The sequence of interest is operably linked to one or more control sequences (at least to a promoter). The terms "regulatory element", "control sequence" and "promoter" are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences. The term "regulatory element" also encompasses a synthetic fusion molecule or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ. The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence. The promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus. An example of an inducible promoter being a stress-inducible promoter, i.e. a promoter activated when a plant is exposed to various stress conditions, is the water stress induced promoter WSI18. Additionally or alternatively, the promoter may be a tissue-specific promoter, i.e. one that is capable of preferentially initiating transcription in certain tissues, such as the leaves, roots, seed tissue etc. An example of a seed-specific promoter is the rice oleosin 18 kDa promoter (Wu etal. (1998) J Biochem 123(3): 386-91).
Preferably, the RLK827 nucleic acid or functional variant thereof is operably linked to a constitutive promoter. A constitutive promoter is transcriptionally active during most, but not necessarily all, phases of its growth and development and is substantially ubiquitously expressed. Preferably, the constitutive promoter is a GOS2 promoter (from rice) (nucleotides 1 to 2193 in SEQ ID NO: 3). It should be clear that the applicability of the present invention is not restricted to the RLK827 nucleic acid represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of an RLK827 nucleic acid when driven by a GOS2
promoter. Examples of other constitutive promoters that may also be used to drive expression of a RLK827 nucleic acid are shown in Table 3 below.
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term "terminator* encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences which may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
An example of an expression cassette comprising the RLK827 nucleic acid operably linked to the GOS2 promoter and further comprising a terminator sequence is given in SEQ ID NO: 3.
The genetic constructs of the invention may further include an origin of replication sequence, which is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
The genetic construct may optionally comprise a selectable marker gene. As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a nucleic acid construct of the invention. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as npt\\ that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin), to herbicides (for example bar which provides resistance to Basta; aroA or gox providing resistance against glyphosate), or genes that provide a metabolic trait (such as rnanA that allows plants to use mannose as sole carbon source). Visual marker genes result in the formation of colour (for example ^-glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof).
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants obtainable by the methods according to the present invention, which plants have introduced therein an RLK827 nucleic acid or a functional variant thereof, or which plants have introduced therein a genetic modification, preferably in the locus of an RLK827 gene.
The invention also provides a method for the production of transgenic plants having improved growth characteristics, comprising introduction and expression in a plant of an RLK827 nucleic acid or a functional variant thereof.
More specifically, the present invention provides a method for the production of transgenic plants having improved growth characteristics, which method comprises:
(i) introducing into a plant or plant cell an RLK827 nucleic acid or a functional
variant thereof; and
(ii) cultivating the plant cell under conditions promoting plant growth and
The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
The term Transformation" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens et a/. (1982) Nature 296, 72-74; Negrutiu et aL (1987) Plant Mol. Biol. 8, 363-373); electroporation of protoplasts (Shillito et aL (1985) Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway et aL (1986) Mol. Gen. Genet. 202, 179-185); DNA or RNA-coated particle bombardment (Klein et aL (1987) Nature 327, 70) infection with (non-integrative) viruses and the like. Transgenic rice plants expressing an RLK827 gene are preferably produced via Agrobacterium-mediated transformation using any of the well known methods for rice transformation, such as described in any of the following: published European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199, 612-617, 1996); Chan et aL (Plant Mol. Biol. 22, 491-506, 1993), Hiei et aL (Plant J. 6, 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of com transformation, the preferred method is as described in either Ishida et aL (Nature Biotechnol. 14, 745-50; 1996) or Frame et aL (Plant Physiol. 129, 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art. The cultivation of transformed plant cells into mature plants may thus encompass steps of selection and/or regeneration and/or growing to maturity.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The present invention clearly extends to any plant ceil or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced in the parent by the methods according to the invention. The invention also includes host cells containing an isolated RLK827 nucleic acid or a functional variant thereof. Preferred host cells according to the invention are plant cells. The invention also extends to harvestable parts of a plant according to the invention such as but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products directly derived from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
The present invention also encompasses the use of RLK827 nucleic acids or functional variants thereof and to the use of RLK827 polypeptides or homologues thereof.
One such use relates to improving the growth characteristics of plants. A preferred use relates to improving yield of plants, a more preferred use relates to increasing seed yield. The seed yield may include one or more of the following: increased number of (filled) seeds, increased seed weight, increased harvest index, among others.
RLK827 nucleic acids or functional variants thereof or RLK827 polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to an RLK827 gene or variant thereof. The RLK827 or variants thereof or RLK827 or homologues thereof may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programs to select plants having altered growth characteristics. The RLK827 gene or variant thereof may. for example, be a nucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31.
Allelic variants of an RLK827 may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called "natural" origin caused unintentionally. Identification of allelic variants then takes place by, for example, PCR. This is followed by a selection step for selection of superior allelic variants of the sequence in question and which give rise improved growth characteristics in a plant. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question, for example, different allelic variants of any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants, in which the superior allelic variant was identified, with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
An RLK827 nucleic acid or variant thereof may also be used as a probe for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of RLK827 nucleic acids or variants thereof requires only a nucleic acid
sequence of at ieast 15 nucleotides in length. The RLK827 nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots of restriction-digested plant genomic DNA may be probed with the RLK827 nucleic acids or variants thereof. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1, 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the RLK827 nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32, 314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bematzky and Tanksley (Plant Mol. Biol. Reporter 4f 37-41, 1986). Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid probes may also be used for physical mapping (i.e., for the placing of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7, 149-154). Although current methods of FISH mapping favour use of large clones (several to several hundred KB; see Laan etal. (1995) Genome Res. 5, 13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11, 95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et a/. (1993) Genomics 16, 325-332), allele-specific ligation (Landegren et al. (1988) Science 241, 1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18, 3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7, 22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17, 6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the
amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic add sequence. This, however, is generally not necessary for mapping methods.
In this way, generation, identification and/or isolation of modified plants with altered RLK827 expression and/or activity displaying improved growth characteristics can be performed.
RLK827 nucleic acids or functional variants thereof or RLK827 polypeptides or homologues thereof may also find use as growth regulators. Since these molecules have been shown to be useful in improving the growth characteristics of plants, they would also be useful growth regulators, such as herbicides or growth stimulators. The present invention therefore provides a composition comprising an RLK827 or a functional variant thereof or an RLK827 polypeptide or homologue thereof, together with a suitable carrier, diluent or excipient, for use as a growth regulator.
The methods according to the present invention result in plants having improved growth characteristics, as described hereinbefore. These advantageous growth characteristics may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to various stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Description of figures
The present invention will now be described with reference to the following figures in which:
Fig. 1 gives a graphical overview of plant receptor like kinase structures (adapted from Shiu & Bleecker, 2001). The arrow indicates the structure of RLK827 and the subfamily to which RLK827 belongs.
Fig. 2 shows a schematic representation of the structure of SEQ ID NO: 2. The triangle indicates sequence with an ATP-binding site signature.
Fig. 3 shows the binary vector p031 for transformation and expression in Oryza sativa of an Arabidopsis thalianan RLK827 (internal reference CDS0827) under the control of a rice GOS2 promoter (internal reference PRO0129).
Fig. 4 details examples of sequences useful in performing the methods according to the present invention. The "At" number refers to the MIPs Accession number (http://mips.gsf.de/); other identifiers refer to GenBank accession numbers.
The present invention will now be described with reference to the following examples, which are by way of illustration alone.
DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel ef al (1994), Current Protocols in Molecular Biology, Current Protocols (http://www.4ulr.com/products/currentprotocols/index.html). Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1: Gene Cloning
The Arabidopsis AIRLK827 (internal code CDS0827) was amplified by PCR using as template an Arabidopsis thaliana seedling cDNA library (Invitrogen, Paisley, UK). After reverse transcription of RNA extracted from seedlings, the cDNAs were cloned into pCMV Sport 6.0. Average insert size of the bank was 1.5 kb, and the original number of clones was 1.59x107 cfu. Original titer was determined to be 9.6x105 cfu/ml, and after a first amplification of 6x1011 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 pi PCR mix. Primers prm00405 (SEQ ID NO: 4, sense) and prm00406 (SEQ ID NO: 5, reverse complementary), which include the AttB sites for Gateway recombination, were used for PCR amplification. PCR was performed using Hifi Taq DNA polymerase in standard conditions. A PCR fragment of 2750 bp was amplified and purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway® terminology, an "entry done", p3080. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 2: Vector Construction and Rice Transformation
The entry clone p3080 was subsequently used in an LR reaction with a destination vector used for Oryza sativa transformation. This vector contained as functional elements within the T-DNA borders: a plant selectable marker; a visual marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the sequence of interest already cloned in the entry clone. A rice GOS2 promoter for constitutive expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector p031 (Figure 3) was transformed into the Agrobacterium strain LBA4404 and subsequently to Oryza sativa plants. Transformed rice plants were allowed to grow and were then examined for the parameters described in Example 3.
Example 3: Evaluation of Transformants: Growth Measurements
Approximately 15 to 20 independent TO transformants were generated. The primary transformants were transferred from tissue culture chambers to a greenhouse for growing and harvest of T1 seed. Five events of which the T1 progeny segregated 3:1 for presence/absence of the transgene were retained. For each of these events, 10 T1 seedlings containing the transgene (hetero- and homo-zygotes), and 10 T1 seedlings lacking the transgene (nullizygotes), were selected by visual marker screening. The selected T1 plants were transferred to a greenhouse. Each plant received a unique barcode label to link unambiguously the phenotyping data to the corresponding plant The selected T1 plants were grown on soil in 10 cm diameter pots under the following environmental settings: photoperiod= 11.5 h, daylight intensity^ 30,000 lux or more, daytime temperature= 28°C or higher, night time temperature= 22°C, relative humidity= 60-70%. Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048x1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
The mature primary panicles were harvested, bagged, barcode-labelled and then dried for three days in the oven at 37°C. The panicles were then threshed and all the seeds collected. The filled husks were separated from the empty ones using an air-blowing device. After separation, both seed lots were then counted using a commercially available counting machine. The empty husks were discarded. The filled husks were weighed on an analytical
balance and the cross-sectional area of the seeds was measured using digital imaging. This procedure resulted in the set of seed-related parameters described below.
These parameters were derived in an automated way from the digital images using image analysis software and were analysed statistically. A two factor ANOVA (analyses of variance) corrected for the unbalanced design was used as statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured of all the plants of all the events transformed with that gene. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also named herein "global gene effect". If the value of the F test shows that the data are significant, than it is concluded that there is a "gene" effect, meaning that not only presence or the position of the gene is causing the effect. The threshold for significance for a true global gene effect is set at 5% probability level for the F test.
To check for an effect of the genes within an event, i.e., for a line-specific effect, a West was performed within each event using data sets from the transgenic plants and the corresponding null plants. "Null plants" or "null segregants" or "nullizygotes" are the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test is set at 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also named herein a "line effect of the gene". The p-value is obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
The data obtained in the first experiment were confirmed in a second experiment with T2 plants. Three lines that had the correct expression pattern were selected for further analysis. Seed batches from the positive plants (both hetero- and homozygotes) in T1, were screened by monitoring marker expression. For each chosen event, the heterorygote seed batches were then retained for T2 evaluation. Within each seed batch an equal number of positive and negative plants were grown in the greenhouse for evaluation.
A total number of 120 RLK827 transformed plants were evaluated in the T2 generation, that is 40 plants per event of which 20 positives for the transgene, and 20 negatives.
Because two experiments with overlapping events have been carried out, a combined analysis was performed. This is useful to check consistency of the effects over the two experiments, and if this is the case, to accumulate evidence from both experiments in order to increase confidence in the conclusion. The method used was a mixed-model approach that takes into account the multilevel structure of the data (i.e. experiment - event - segregants). P-values are obtained by comparing likelihood ratio test to chi square distributions.
Example 4: Evaluation of Transformants: Measurement of Seed-Related
Upon analysis of the seeds as described above, the inventors found that plants transformed with the RLK827 gene construct had a higher number of filled seeds, a higher total weight of seeds and an increased harvest index compared to plants lacking the RLK827 transgene. Positive results obtained for plants in the T1 generation were again obtained in the T2 generation. As an example, data for line OS2 are given (Table 4).
Line OS2 T1 generation T2 generation Combined analysis
% difference p-value % difference p-value p-value
Nr filled seeds 41 0.0047 54 0.0726 0.0079
Total weight seeds 43 0.0051 60 0.0655 0.0065
Harvest Index 48 0.0007 57 0.0527 0.0003
Number of filled seeds:
The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. Line OS2 showed a significant increase in filled seed number of 41% for the T1 generation. This increase was also observed in the T2 generation (+54%). The combined analysis of T1 and T2 data confirmed that the effect on the number of filled seeds was highly significant (p-value of 0.0079).
Total seed yield:
The total seed yield (total weight of seeds) per plant was measured by weighing all filled husks harvested from a plant. Not only the number of filled seeds was increased, but also the total seed weight. In the first generation there was an increase of 43%, which increase was statistically significant. This increase was confirmed in the T2 generation and the combined analysis showed that the increases in seed yield were significant (p-value of 0.0065).
Line 0S2 furthermore had an increased harvest index. The harvest index in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. Both in T1 and T2 a positive effect on harvest index was observed (increase of respectively 48 and 57% with p-values of 0.0007 and 0.0527). Here too, the combined analysis of the T1 and T2 data showed a significant effect (p-value 0.0003).
1) Method for improving growth characteristics of a plant relative to corresponding wild type plants, comprising increasing activity of an RLK827 polypeptide or a homologue thereof and/or by increasing expression of an RLK827 encoding nudeic acid molecule, and optionally selecting for plants having improved growth characteristics.
2) Method of daim 1, wherein said increased activity and/or increased expression is effected by introducing a genetic modification preferably in the locus of a gene encoding an RLK827 polypeptide or a homologue thereof.
3) Method of daim 2, wherein said genetic modification is effected by one of site-directed mutagenesis, homologous recombination, TILLING, direded evolution and T-DNA activation.
4) Method for improving plant growth characteristics relative to corresponding wild type plants, comprising introducing and expressing in a plant an RLK827 nudeic acid molecule or a functional variant thereof.
5) Method of daim 4, wherein said functional variant is a portion of an RLK827 nudeic acid molecule or a sequence capable of hybridising to an RLK827 nudeic acid molecule and wherein said functional variant comprises a non-cytoplasmic domain with at least 1 but no more than 3 LRR domains and the amino acid sequence motif of SEQ ID NO: 33, a transmembrane domain and a kinase domain.
6) Method of daim 4 or 5, wherein said RLK827 nudeic acid molecule or functional variant thereof is overexpressed in a plant.
7) Method according to any one of daims 4 to 6, wherein said RLK827 nudeic acid molecule or functional variant thereof is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Brassicaceae, more preferably the nudeic add is from Arabidopsis thaliana.
8) Method according to any one of daims 4 to 7, wherein said functional variant encodes an orthologue or paralogue of RLK827.
9) Method according to any one of claims 4 to 8, wherein said RLK827 nucleic acid molecule or functional variant thereof is operably linked to a constitutive promoter.
10) Method according to claim 9, wherein said constitutive promoter is a GOS2 promoter.
11) Method according to any one of claims 1 to 10, wherein said improved plant growth characteristic is increased yield.
12) Method according to claim 11, wherein said increased yield is increased seed yield.
13) Method according to claim 12, wherein said increased seed yield is selected from any one or more of (i) increased seed biomass; (ii) increased number of (filled) seeds; (iii) increased seed size; (iv) increased seed volume; (v) increased harvest index (HI); and (vi) increased thousand kernel weight (TKW).
14) Plant or plant cell obtainable by a method according to any of claims 1 to 13.
15) Construct comprising:
(i) an RLK827 nucleic acid molecule or functional variant thereof;
(ii) one or more control sequence capable of driving expression of the nucleic acid
sequence of (i); and optionally (iii) a transcription termination sequence.
16) Construct according to claim 15, wherein said control sequence is a constitutive promoter.
17) Construct according to claim 16, wherein said constitutive promoter is a GOS2 promoter.
18) Plant or plant ceil transformed with a construct according to claim 15 or 17.
19) Method for the production of a transgenic plant having improved growth characteristics, which method comprises:
(i) introducing into a plant an RLK827 nucleic acid molecule or functional variant
thereof; (ii) cultivating the plant cell under conditions promoting plant growth and
20) Transgenic plant or plant cell having improved growth characteristics relative to
corresponding wild type plants resulting from an RLK827 nucleic acid molecule or
functional variant thereof introduced into said plant or plant cell, or resulting from a genetic
modification preferably in the locus of a gene encoding an RLK827 polypeptide or a
21) Transgenic plant or plant cell according to claim 14, 18 or 20, wherein said plant is a
monocotyledonous plant, such as sugar cane or wherein the plant is a cereal, such as rice,
maize, wheat, barley, millet, rye oats or sorghum; or wherein said plant cell is derived from
a monocotyledonous plant, such as sugar cane or wherein said plant cell is derived from a
cereal, such as rice, maize, wheat, barley, millet, rye oats or sorghum.
22)Harvestable parts, and/or products directly derived therefrom, of a plant according to any one of claims 14, 18, 20 or 21.
23) Harvestable parts according to claim 22, wherein said harvestable parts are seeds.
24) Use of an RLK827 nucleic acid molecule or functional variant thereof or use of an RLK827
polypeptide or homologue thereof in improving the growth characteristics of plants, in
particular in improving yield, especially seed yield.
25) Use according to claim 24, wherein said seed yield includes one or more of the following:
increased number of (filled) seeds, increased seed weight, increased harvest index and
increased thousand kernel weight.
26) Use of an RLK827 nucleic acid molecule or functional variant thereof as a molecular
27) A composition comprising an RLK827 nucleic acid molecule or functional variant thereof for
improving growth characteristics of plants, preferably for use as a growth regulator.
28) A composition comprising an RLK827 protein or a homologue thereof for improving growth
characteristics of plants, preferably for use as a growth regulator.
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|Indian Patent Application Number||142/CHENP/2007|
|PG Journal Number||40/2010|
|Date of Filing||12-Jan-2007|
|Name of Patentee||CropDesign N.V.|
|Applicant Address||TECHNOLOGIEPARK 3, B-9052 ZWIJNAARDE,|
|PCT International Classification Number||C12N 15/82|
|PCT International Application Number||PCT/EP05/53397|
|PCT International Filing date||2005-07-14|