PLANT YIELD IMPROVEMENT BY STE20-LIKE GENE EXPRESSION

Abstract

The present invention concerns a method for increasing plant yield by modulating expression in a plant of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof. One such method comprises introducing into a plant a Ste20-like nucleic acid or variant thereof. The invention also relates to transgenic plants having introduced therein a Ste20-like nucleic acid or variant thereof, which plants have increased yield relative to control plants. The present invention also concerns constructs useful in the methods of the invention.

Description

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 11/988,254 filed Jan. 23, 2008, which is a national stage application (under 35 U.S.C. 371) of PCT/EP2006/063976 filed Jul. 6, 2006, which claims benefit of European application 05106135.6 filed Jul. 6, 2005 and U.S. Provisional application 60/697,338 filed Jul. 8, 2005. The entire content of each above-mentioned application is hereby incorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequence_List_--32279_--00043. The size of the text file is 112 KB, and the text file was created on Jan. 16, 2012.

FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of molecular biology and concerns a method for increasing plant yield relative to control plants. More specifically, the present invention concerns a method for increasing plant yield comprising modulating expression in a plant of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof. The present invention also concerns plants having modulated expression of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof, which plants have increased yield relative to control plants. The invention also provides constructs useful in the methods of the invention.

BRIEF SUMMARY OF THE INVENTION

[0004] The ever-increasing world population and the dwindling supply of arable land available for agriculture fuels research towards improving the efficiency of agriculture. 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 components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify 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 the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits. A trait of particular economic interest is yield, necessarily related to a specified crop, area and/or period of time. 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. Optimizing one of the abovementioned factors may therefore contribute to increasing crop yield.

[0005] Plant biomass is yield for forage crops like alfalfa, silage corn and hay. Many proxies for yield have been used in grain crops. Chief amongst these are estimates of plant size. Plant size can be measured in many ways depending on species and developmental stage, but include total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number and leaf number. Many species maintain a conservative ratio between the size of different parts of the plant at a given developmental stage. These allometric relationships are used to extrapolate from one of these measures of size to another (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105: 213). Plant size at an early developmental stage will typically correlate with plant size later in development. A larger plant with a greater leaf area can typically absorb more light and carbon dioxide than a smaller plant and therefore will likely gain a greater weight during the same period (Fasoula & Tollenaar 2005 Maydica 50:39). This is in addition to the potential continuation of the micro-environmental or genetic advantage that the plant had to achieve the larger size initially. There is a strong genetic component to plant size and growth rate (e.g. ter Steege et al 2005 Plant Physiology 139:1078), and so for a range of diverse genotypes plant size under one environmental condition is likely to correlate with size under another (Hittalmani et al 2003 Theoretical Applied Genetics 107:679). In this way a standard environment is used as a proxy for the diverse and dynamic environments encountered at different locations and times by crops in the field.

[0006] Harvest index, the ratio of seed yield to above-ground dry weight, is relatively stable under many environmental conditions and so a robust correlation between plant size and grain yield can often be obtained (e.g. Rebetzke et al 2002 Crop Science 42:739). These processes are intrinsically linked because the majority of grain biomass is dependent on current or stored photosynthetic productivity by the leaves and stem of the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa State University Press, pp 68-73) Therefore, selecting for plant size, even at early stages of development, has been used as an indicator for future potential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105: 213). When testing for the impact of genetic differences on stress tolerance, the ability to standardize soil properties, temperature, water and nutrient availability and light intensity is an intrinsic advantage of greenhouse or plant growth chamber environments compared to the field. However, artificial limitations on yield due to poor pollination due to the absence of wind or insects, or insufficient space for mature root or canopy growth, can restrict the use of these controlled environments for testing yield differences. Therefore, measurements of plant size in early development, under standardized conditions in a growth chamber or greenhouse, are standard practices to provide indication of potential genetic yield advantages.

[0007] Seed yield is a particularly important trait since the seeds of many plants are important for human and animal nutrition. Crops such as, corn, rice, wheat, canola and soybean account for over half the total human caloric intake, whether through direct consumption of the seeds themselves or through consumption of meat products raised on processed seeds. They are also a source of sugars, oils and many kinds of metabolites used in industrial processes. Seeds contain an embryo (the source of new shoots and roots) and an endosperm (the source of nutrients for embryo growth during germination and during early growth of seedlings). The development of a seed involves many genes, and requires the transfer of metabolites from the roots, leaves and stems into the growing seed. The endosperm, in particular, assimilates the metabolic precursors of carbohydrates, oils and proteins and synthesizes them into storage macromolecules to fill out the grain. The ability to increase plant seed yield, whether through seed number, seed biomass, seed development, seed filling, or any other seed-related trait would have many applications in agriculture, and even many non-agricultural uses such as in the biotechnological production of substances such as pharmaceuticals, antibodies or vaccines.

[0008] Ste20 is a Ser/Thr kinase belonging to the group of MAP4 kinases (MAP4Ks, MAP kinase kinase kinase kinases, or MAP3K kinases), and was for the first time isolated from yeast. MAP4K are kinases that activate MAP kinase cascades by directly phosphorylating MAP3Ks. A recent phylogenetic study discriminated 6 major groups of MAP4Ks, among which the STE20/PAK group of MAP4Ks (Champion et al., Trends Plant Sci. 9, 123-129, 2004). Most of the MAP4Ks have an N-terminal catalytic domain, although plant proteins homologous to Ste20 may have a different organisation. Members of the Ste20 group of kinases are believed to act as regulators of MAP kinase cascades (Dan et al., Trends Cell Biol. 11, 220-230, 2001), and are believed to act in particular upon MAP3 Kinases of the MEKK and Raf types, downstream of G-proteins (Champion et al., 2004). Yeast Ste20 plays a role in various signalling pathways, for example Candida Ste20 was shown to be involved in pheromone signalling, invasive growth, hypertonic stress response, cell wall integrity and in binding of CDC42, required for polarized morphogenesis (Calcagno et al., Yeast 21, 557-568, 2004). In Drosophila, the Ste20 homologue Hippo is reported to be involved in cell cycle progression (Udan et al., Nat. Cell Biol. 5, 853-855, 2003). In general, the effects of STE20/PAK directed signalling appear to be nuclear events that influence gene expression on the one hand, and cytoskeletal events that impact upon cellular dynamics (Bagrodia and Cerione, Trends Cell Biol. 9, 350-355, 1999). Although Ste20 and related proteins are relatively well studied in yeast, Drosophila and in mammalian cells, little or nothing is known about the plant homologues of yeast Ste20. Leprince et al, (Biochim. Biophys. Acta 1444, 1-13, 1999) have characterised a MAP4K from Brassica napus. Its expression seemed regulated by the cell cycle and transcripts were reported to most abundant in roots, siliques and flower buds. However, no mutants or transgenic plants were described.

[0009] Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof gives plants having increased yield relative to control plants. Preferably, the Ste20-like polypeptide or a homologue thereof is of plant origin.

[0010] Therefore, the invention provides a method for increasing plant yield, comprising modulating expression in a plant of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof.

[0011] Advantageously, performance of the methods according to the present invention results in plants having increased yield, particularly seed yield, relative to control plants.

[0012] The choice of control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or even of the same variety as the plant to be compared. The control plant may also be a nullizygote of the plant to be compared. Nullizygotes are individuals missing the transgene by segregation. A "control plant" as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.

[0013] A "reference", "reference plant", "control", "control plant", "wild type" or "wild type plant" is in particular a cell, a tissue, an organ, a plant, or a part thereof, which was not produced according to the method of the invention. Accordingly, the terms "wild type", "control" or "reference" are exchangeable and can be a cell or a part of the plant such as an organelle or tissue, or a plant, which was not modified or treated according to the herein described method according to the invention. Accordingly, the cell or a part of the plant such as an organelle or a plant used as wild type, control or reference corresponds to the cell, plant or part thereof as much as possible and is in any other property but in the result of the process of the invention as identical to the subject matter of the invention as possible. Thus, the wild type, control or reference is treated identically or as identical as possible, saying that only conditions or properties might be different which do not influence the quality of the tested property. That means in other words that the wild type denotes (1) a plant, which carries the unaltered or not modulated form of a gene or allele or (2) the starting material/plant from which the plants produced by the process or method of the invention are derived.

[0014] Preferably, any comparison between the wild type plants and the plants produced by the method of the invention is carried out under analogous conditions. The term "analogous conditions" means that all conditions such as, for example, culture or growing conditions, assay conditions (such as buffer composition, temperature, substrates, pathogen strain, concentrations and the like) are kept identical between the experiments to be compared.

[0015] The "reference", "control", or "wild type" is preferably a subject, e.g. an organelle, a cell, a tissue, a plant, which was not modulated, modified or treated according to the herein described process of the invention and is in any other property as similar to the subject matter of the invention as possible. The reference, control or wild type is in its genome, transcriptome, proteome or metabolome as similar as possible to the subject of the present invention. Preferably, the term "reference-" "control-" or "wild type-"-organelle, -cell, -tissue or plant, relates to an organelle, cell, tissue or plant, which is nearly genetically identical to the organelle, cell, tissue or plant, of the present invention or a part thereof preferably 95%, more preferred are 98%, even more preferred are 99.00%, in particular 99.10%, 99.30%, 99.50%, 99.70%, 99.90%, 99.99%, 99.999% or more. Most preferable the "reference", "control", or "wild type" is preferably a subject, e.g. an organelle, a cell, a tissue, a plant, which is genetically identical to the plant, cell organelle used according to the method of the invention except that nucleic acid molecules or the gene product encoded by them are changed, modulated or modified according to the inventive method.

[0016] In case, a control, reference or wild type differing from the subject of the present invention only by not being subject of the method of the invention can not be provided, a control, reference or wild type can be a plant in which the cause for the modulation of the activity conferring the increase of the metabolites as described under examples.

[0017] The term "yield" in general means a measurable produce of economic value, necessarily related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight. Whereas the actual yield is the yield per acre for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted acres.

[0018] The terms "increase", "improving" or "improve" are interchangeable and shall mean in the sense of the application at least a 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield and/or growth in comparison to the wild type plant as defined herein.

[0019] The increase referred to the activity of the polypeptide amounts in a cell, a tissue, a organelle, an organ or an organism or a part thereof preferably to at least 5%, preferably to at least 10% or at to least 15%, especially preferably to at least 20%, 25%, 30% or more, very especially preferably are to at least 40%, 50% or 60%, most preferably are to at least 70% or more in comparison to the control, reference or wild type.

[0020] The term "increased yield" as defined herein is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground.

[0021] In particular, such harvestable parts are seeds and leafy biomass, and performance of the methods of the invention results in plants having increased leafy biomass and increased seed yield relative to the seed yield of control plants.

[0022] Increased seed yield may manifest itself as one or more of the following: a) an increase in seed biomass (total seed weight) which may be on an individual seed basis and/or per plant and/or per hectare or acre; b) increased number of flowers per plant; c) increased number of (filled) seeds; d) increased seed filling rate (which is expressed as the ratio between the number of filled seeds divided by the total number of seeds; e) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, divided by the total biomass; and f) 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 and/or seed weight, and may also result from an increase in embryo and/or endosperm size.

[0023] The term "expression" or "gene expression" means the appearance of a phenotypic trait as a consequence of the transcription of a specific gene or specific genes. The term "expression" or "gene expression" in particular means the transcription of a gene or genes into structural RNA (rRNA, tRNA) or mRNA with subsequent translation of the latter into a protein. The process includes transcription of DNA, processing of the resulting mRNA product and its translation into an active protein.

[0024] The term "modulation" means in relation to expression or gene expression, a process in which the expression level is changed by said gene expression in comparison to the control plant, preferably the expression level is increased. The original, unmodulated expression may be of any kind of expression of a structural RNA (rRNA, tRNA) or mRNA with subsequent translation. The term "modulating the activity" shall mean any change of the expression of the inventive nucleic acid sequences or encoded proteins, which leads to increased yield and/or increased growth of the plants.

[0025] An increase in seed yield may also be manifested as an increase in seed size and/or seed volume, which may also influence the composition of seeds (including oil, protein and carbohydrate total content and composition). Furthermore, an increase in seed yield may also manifest itself as an increase in seed area and/or seed length and/or seed width and/or seed perimeter. Increased yield may also result in modified architecture, or may occur because of modified architecture.

[0026] 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, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as 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 (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others. An increase in yield may also result in modified architecture, or may occur as a result of modified architecture.

[0027] According to a preferred feature, performance of the methods of the invention result in plants having increased yield, particularly seed yield. Therefore, according to the present invention, there is provided a method for increasing plant yield, which method comprises modulating expression in a plant of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof.

[0028] Since the transgenic 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 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 (a similar effect may be obtained with earlier flowering time). If the growth rate is sufficiently increased, it may allow for the further sowing of 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 further sowing of 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, potato or any other plant). Harvesting additional times from the same rootstock in the case of some crop 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, 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.

[0029] According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate or increased yield in comparison to control plants. Therefore, according to the present invention, there is provided a method for increasing yield and/or the growth rate of plants, which method comprises modulating, preferably increasing, expression in a plant of a nucleic acid encoding a Ste20-like protein.

[0030] 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. Plants 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. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. 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). Chemicals may also cause abiotic stresses. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects. In another preferred embodiment of the invention an increase in yield and/or growth rate occurs according to the method of invention under non-stress or mild abiotic or biotic stress conditions, preferably on non-stress or mild abiotic stress conditions.

[0031] The abovementioned growth characteristics may advantageously be modified in any plant.

[0032] The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants, plant cells 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. The term "plant" also encompasses suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprise the gene/nucleic acid of interest.

[0033] Plants that are particularly useful in the methods of the invention include 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 Acer spp., Actinidia spp., Abelmoschus spp., Agropyron spp., Allium spp., Amaranthus spp., Ananas comosus, Annona spp., Apium graveolens, Arabidopsis thaliana, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena sativa, Averrhoa carambola, Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp., Cadaba farinosa, Camellia sinensis, Canna indica, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Eleusine coracana, Eriobotrya japonica, Eugenia uniflora, Fagopyrum spp., Fagus spp., Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp., Gossypium hirsutum, Helianthus spp., Hemerocallis fulva, Hibiscus spp., Hordeum spp., Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp., Panicum miliaceum, Passiflora edulis, Pastinaca sativa, Persea spp., Petroselinum crispum, Phaseolus spp., Phoenix spp., Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Rubus spp., Saccharum spp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp., Sorghum bicolor, Spinacia spp., Syzygium spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp., Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others. According to a preferred embodiment of the present invention, the plant is a crop plant such as soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. Further preferably, the plant is a monocotyledonous plant, such as sugar cane. More preferably the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.

[0034] Other advantageous plants are selected from the group consisting of Asteraceae such as the genera Helianthus, Tagetes e.g. the species Helianthus annus [sunflower], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [Marigold], Brassicaceae such as the genera Brassica, Arabadopsis e.g. the species Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape] or Arabidopsis thaliana. Fabaceae such as the genera Glycine e.g. the species Glycine max, Soja hispida or Soja max [soybean]. Linaceae such as the genera Linum e.g. the species Linum usitatissimum, [flax, linseed]; Poaceae such as the genera Hordeum, Secale, Avena, Sorghum, Oryza, Zea, Triticum e.g. the species Hordeum vulgare [barley]; Secale cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida [oat], Sorghum bicolor [Sorghum, millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn, maize] Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare [wheat, bread wheat, common wheat]; Solanaceae such as the genera Solanum, Lycopersicon e.g. the species Solanum tuberosum [potato], Lycopersicon esculentum, Lycopersicon lycopersicum., Lycopersicon pyriforme, Solanum integrifolium or Solanum lycopersicum [tomato].

[0035] The term "Ste20-like polypeptide or homologue thereof" as defined herein refers to a MAP4K polypeptide, preferably of plant origin, comprising an N-terminal Ser/Thr kinase domain (matching the SMART database entry SM00220, InterPro accession IPR002290). The kinase domain in SEQ ID NO: 2 starts at Y15 and ends at F293, and comprises the ProSite Ser/Thr protein kinase pattern PS00108:

[0036] [LIVMFYC]-x-[HY]-x-D-[LIVMFY]-K-x(2)-N-[LIVMFYCT](3), wherein the first x is missing. Preferably, the Ste20-like polypeptide or homologue thereof comprises the Ste20 signature sequence:

TABLE-US-00001 (SEQ ID NO: 6; Dan et al., 2001) G(T/N)P(Y/C/R)(W/R)MAPE(V/K),

more preferably it comprises the sequence motif:

TABLE-US-00002 (SEQ ID NO: 7) (S/H/N)(I/L)(V/I/L/M)(S/K)(S/H/T/A/I/V)(S/G/V/A) (F/Y)(P/Q)(S/N/D/E)G,

most preferably the Ste20-like polypeptide or homologue thereof comprises at least one of the following sequence motifs:

TABLE-US-00003 SEQ ID NO: 8 (V/I)HSH(T/N/V)GY(G/S)(F/I), SEQ ID NO: 9 RPPLSHLPP(L/S)KS, SEQ ID NO: 10 RRISGWNF,

At the C-terminal end of the protein, a coiled coil motif may be present (K450 to T477 in SEQ ID NO: 2).

[0037] The term "domain" refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family (in this case, the family of Ste20-like proteins). The term "motif" refers to a short conserved region in a protein sequence. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).

[0038] Specialist databases exist for the identification of domains. The kinase domain in a Ste20-like protein may be identified using, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of tools for in silico analysis of protein sequences is available on the ExPASY proteomics server (hosted by the Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788 (2003)).

[0039] By aligning other protein sequences with SEQ ID NO: 2, the corresponding Ste20 signature sequence, the kinase domain and other sequence motifs detailed above may easily be identified. In this way, Ste20-like polypeptides or homologues thereof (encompassing orthologues and paralogues) may readily be identified, using routine techniques well known in the art, such as by sequence alignment. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full length sequences for the identification of homologues, specific domains (such as the kinase domain) may be used as well. The sequence identity values, which are indicated above as a percentage were determined over the entire conserved domain or nucleic acid or amino acid sequence using the programs mentioned above using the default parameters.

[0040] Examples of Ste20-like polypeptides or homologues thereof include the Arabidopsis sequences SEQ ID NO: 12 (corresponding to At5g14720, encoded by GenBank accession number AAL38867), SEQ ID NO: 14 (At1g70430, GenBank NP_--177200), SEQ ID NO: 16 (At1g23700, GenBank NP_--173782), SEQ ID NO: 18 (At1g79640, GenBank NP_--178082), SEQ ID NO: 20 (At4g10730, GenBank NP_--192811), SEQ ID NO: 22 (At4g24100, GenBank NP_--194141); the rice sequences SEQ ID NO: 24 (GenBank XP_--469286, the rice orthologue of SEQ ID NO: 2), SEQ ID NO: 26 (GenBank BAD37346), SEQ ID NO: 28 (GenBank XP_--468215), SEQ ID NO: 30 (GenBank AAL54869), SEQ ID NO: 32 (GenBank NP_--912431) and the Medicago sequence SEQ ID NO: 34 (orthologue of SEQ ID NO: 2).

[0041] It is to be understood that sequences falling under the definition of "Ste20-like polypeptide or homologue thereof" are not to be limited to the sequences represented by SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34, but that any polypeptide comprising a N-terminal kinase domain as defined above, the Ste20 signature sequence and preferably also one or more of the sequence motifs detailed in SEQ ID NO: 7, 8, 9 and 10, may be suitable for use in the methods of the invention. In a preferred embodiment, the homologue used in the methods of the present invention is an othologue of SEQ ID NO: 2.

[0042] An assay may be carried out to determine Ste20-like activity. For example to determine the kinase activity, 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). The Ste20-like protein is a MAP4K kinase involved in signal transduction. For several organisms, the substrate of Ste20 was identified as Ste11p (Drogen et al., Current Biology 10, 630-639, 2000). Besides in vitro phosphorylation of the Ste11p protein, Ste20 was also shown to phosphorylate histone H2B (Ahn et al., Cell 120, 25-36, 2005). Buffer composition, ionic strength, and pH may be optimized starting from a standard kinase assay mixture. A standard 5× 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 MgCl₂. Divalent cations are required for most tyrosine kinases, although some tyrosine kinases (for example, insulin-, IGF-1-, and PDGF receptor kinases) require MnCl₂ instead of MgCl₂ (or in addition to MgCl₂). 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.

[0043] Furthermore, expression of the Ste20-like protein or of a homologue thereof in plants, and in particular in rice, has the effect of increasing yield of the transgenic plant when compared to control plants, wherein increased yield comprises at least one of: total weight of seeds, number of filled seeds and harvest index.

[0044] "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.

[0045] 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. Paralogues are genes within the same species that have originated through duplication of an ancestral gene and orthologues are genes from different organisms that have originated through speciation.

[0046] Orthologues and paralogues may easily be found by performing a so-called reciprocal blast search. This may be done by a first BLAST involving BLASTing a query sequence (for example, SEQ ID NO: 1 or SEQ ID NO: 2) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) may be used when starting from a nucleotide sequence and BLASTP or TBLASTN (using standard default values) may be used when starting from a protein sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 1 or SEQ ID NO: 2, the second BLAST would therefore be against Arabidopsis sequences). The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the second BLAST is from the same species as from which the query sequence is derived; an orthologue is identified if a high-ranking hit is not from the same species as from which the query sequence is derived. Preferred orthologues are orthologues of SEQ ID NO: 1 or SEQ ID NO: 2. High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. Preferably the score is greater than 50, more preferably greater than 100; and preferably the E-value is less than e-5, more preferably less than e-6. In the case of large families, ClustalW may be used, followed by the generation of a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues. Examples of sequences orthologous to SEQ ID NO: 2 include SEQ ID NO: 24 and SEQ ID NO: 34. Examples of paralogues of SEQ ID NO: 2 include SEQ ID NO: 12 (At5g14720) and SEQ ID NO: 20 (At4g10730).

[0047] Preferably, the kinase domains of Ste20-like proteins useful in the methods of the present invention have, in increasing order of preference, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the kinase domain of the Ste20 protein of SEQ ID NO: 2. An example detailing the identification of homologues is given in Example 1. The matrix shown in Example 1 (Table 4) shows similarities and identities (in bold) over the full-length of the protein. In case only specific domains are compared, the identity or similarity may be higher among the different proteins (Table 5: comparison of the kinase domains only).

[0048] A Ste20-like polypeptide or homologue thereof is encoded by a Ste20-like nucleic acid/gene. Therefore the term "Ste20-like nucleic acid/gene" as defined herein is any nucleic acid/gene encoding a Ste20-like polypeptide or a homologue thereof as defined above.

[0049] Examples of Ste20-like nucleic acids include but are not limited to those represented by any one of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33.

[0050] Ste20-like nucleic acids/genes and variants thereof may be suitable in practising the methods of the invention. Variant Ste20-like nucleic acid/genes include portions of a Ste20-like nucleic acid/gene, splice variants, allelic variants and/or nucleic acids capable of hybridising with a Ste20-like nucleic acid/gene.

[0051] The term portion as defined herein refers to a piece of DNA encoding a polypeptide comprising the Ste20 signature sequence G(T/N)P(Y/C/R)(W/R)MAPE(V/K) (SEQ ID NO: 6) and a N-terminal Ser/Thr kinase domain as defined above. A portion may be prepared, for example, by making one or more deletions to a Ste20-like 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. When fused to other coding sequences, the resulting polypeptide produced upon translation may be bigger than that predicted for the Ste20-like fragment. The portion is typically at least 300, 400, 500, 600 or 700 nucleotides in length, preferably at least 750, 900, 850, 900 or 950 nucleotides in length, more preferably at least 1000, 1100, 1200 or 1300 nucleotides in length and most preferably at least 1350, 1400 or 1450 nucleotides in length. Preferably, the portion is a portion of a nucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33. Most preferably the portion of a nucleic acid is as represented by SEQ ID NO: 1.

[0052] The terms "fragment", "fragment of a sequence" or "part of a sequence" "portion" or "portion thereof" mean a truncated sequence of the original sequence referred to. The truncated sequence (nucleic acid or protein sequence) can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity of the original sequence referred to or hybidizing with the nucleic acid molecule of the invention or used in the process of the invention under stringend conditions, while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence. A comparable function means at least 40%, 45% or 50%, preferably at least 60%, 70%, 80% or 90% or more of the original sequence.

[0053] Another variant of a Ste20-like nucleic acid/gene is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a Ste20-like nucleic acid/gene as hereinbefore defined or with a portion as defined hereinabove.

[0054] Hybridising sequences useful in the methods of the present invention encode a polypeptide having a Ste20 signature sequence G(T/N)P(Y/C/R)(W/R)MAPE(V/K) (SEQ ID NO: 6) and a N-terminal Ser/Thr kinase domain as defined above and having substantially the same biological activity as the Ste20-like protein represented by SEQ ID NO: 2 or homologues thereof. The hybridizing sequence is typically at least 800 nucleotides in length, preferably at least 1000 nucleotides in length, more preferably at least 1200 nucleotides in length and most preferably at least 1400 nucleotides in length.

[0055] Preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by (or to probes derived from) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33, or to a portion of any of the aforementioned sequences, a portion being as defined above. Most preferably the hybridising sequence is capable of hybridising to SEQ ID NO: 1, or to portions (or probes) thereof. Methods for designing probes are well known in the art. Probes are generally less than 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp in length, preferably less than 500 bp, 400 bp, 300 bp 200 bp or 100 bp in length. Commonly, probe lengths for DNA-DNA hybridisations such as Southern blotting, vary between 100 and 500 bp, whereas the hybridising region in probes for DNA-DNA hybridisations such as in PCR amplification generally are shorter than 50 but longer than 10 nucleotides, preferably they are 15, 20, 25, 30, 35, 40, 45 or 50 bp in length.

[0056] 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 micro-arrays 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.

[0057] The term "stringency" refers to the conditions under which a hybridisation takes place. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30° C. lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20° C. below T_m, and high stringency conditions are when the temperature is 10° C. below T_m. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid molecules.

[0058] The T_m is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The T_m 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 T_m. 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 (for higher concentrations, this effect may be ignored). 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 T_m decreases about 1° C. per % base mismatch. The T_m may be calculated using the following equations, depending on the types of hybrids:

1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):

T_m=81.5° C.+16.6×log₁₀[Na+]^a+0.41×%[G/C^b]-500×[L1- ^c]-0.61×% formamide

2) DNA-RNA or RNA-RNA hybrids:

Tm=79.8+18.5(log₁₀[Na+]^a)+0.58(% G/C^b)+11.8(% G/C^b)²-820/L^c

3) oligo-DNA or oligo-RNA^d hybrids:

[0059] For <20 nucleotides: Tm=2 (l_a)

[0060] For 20-35 nucleotides: Tm=22+1.46 (I_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; I_n, effective length of primer=2×(no. of G/C)+(no. of NT).

[0061] Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase. For non-homologous probes, a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68° C. to 42° C.) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%). The skilled artisan is aware of various parameters which may be altered during hybridisation and which will either maintain or change the stringency conditions.

[0062] Besides the hybridisation conditions, specificity of hybridisation typically also depends on 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. A positive hybridisation gives a signal that is at least twice of that of the background. 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. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions.

[0063] For example, typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65° C. in 1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at 65° C. in 0.3×SSC. Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed by washing at 50° C. in 2×SSC. The length of the hybrid 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. 1×SSC is 0.15M NaCl and 15 mM sodium citrate; the hybridisations and washes may additionally include 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.

[0064] For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et al. (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 and yearly updates).

[0065] Also useful in the methods of the invention are nucleic acids encoding homologues of the amino acid sequence represented by SEQ ID NO 2.

[0066] 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. 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 α-helical structures or β-sheet structures). Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company and Table 1 below).

TABLE-US-00004 TABLE 1 Examples of conserved amino acid substitutions Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Gln Asn Cys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0067] A homologue may also be in the form of an "insertional 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 N-terminal and/or C-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 N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-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, Tag100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.

[0068] Homologues in the form of "deletion variants" of a protein are characterised by the removal of one or more amino acids from a protein.

[0069] Amino acid variants of a protein (substitution-, deletion- and/or insertion-variants) 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 substitution 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, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.

[0070] The Ste20-like polypeptide or homologue thereof may also 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" include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the one presented in SEQ ID NO: 2, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. "Derivatives" of a protein also encompass peptides, oligopeptides, polypeptides which may comprise naturally occurring altered (glycosylated, acylated, ubiquinated, prenylated, phosphorylated, myristoylated, sulphated etc) or non-naturally altered 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 or additions 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. Derivatives of orthologues or paralogues of SEQ ID NO: 2 are further examples which may be suitable for use in the methods of the invention.

[0071] The Ste20-like polypeptide or homologue thereof may be encoded by a splice variant of a Ste20-like nucleic acid/gene. The term "splice variant" as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the biological activity of the protein is substantially retained, this 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 known in the art. Preferred splice variants are splice variants of the nucleic acid encoding a polypeptide comprising the Ste20 signature sequence (SEQ ID NO: 6) and a N-terminal Ser/Thr kinase domain as defined above. Preferably, the Ste20-like polypeptide or a homologue thereof additionally comprises SEQ ID NO: 7, more preferably the Ste20-like polypeptide or a homologue thereof comprises one or more of the following: SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. Further preferred are splice variants of nucleic acids represented by SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 33. Most preferred is a splice variant of the nucleic acid represented by SEQ ID NO: 1.

[0072] Another nucleic acid variant useful in the methods of the invention is an allelic variant of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof as defined above, preferably an allelic variant of a nucleic acid encoding a Ste20-like polypeptide comprising the Ste20 signature sequence (SEQ ID NO: 6) and a N-terminal Ser/Thr kinase domain. Preferably, the Ste20-like polypeptide or a homologue thereof additionally comprises SEQ ID NO: 7, more preferably the Ste20-like polypeptide or a homologue thereof comprises one or more of the following: SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. Further preferred are allelic variants of nucleic acids represented by SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 33. Most preferred is an allelic variant of a nucleic acid as represented by SEQ ID NO: 1. 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.

[0073] A further nucleic acid variant useful in the methods of the invention is a nucleic acid variant obtained by gene shuffling. Gene shuffling or directed evolution may also be used to generate variants of Ste20-like nucleic acids. This consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of Ste20-like nucleic acids or portions thereof having a modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).

[0074] Furthermore, site-directed mutagenesis may be used to generate variants of Ste20-like nucleic acids. Several methods are available to achieve site-directed mutagenesis; the most common being PCR based methods (Current Protocols in Molecular Biology. Wiley Eds.).

[0075] The Ste20-like nucleic acid 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 yeast or fungi, or from a plant, algae 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 it 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 Ste20-like nucleic acid is isolated from Arabidopsis thaliana and is represented by SEQ ID NO: 1, and the Ste20-like amino acid sequences is as represented by SEQ ID NO: 2.

[0076] According to a preferred aspect of the present invention, modulated, preferably increased expression of the Ste20-like nucleic acid or variant thereof is envisaged. Methods for increasing 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 a Ste20-like 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 al., 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. Methods for reducing the expression of genes or gene products are well documented in the art.

[0077] The expression of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof may be modulated by introducing a genetic modification (preferably in the locus of a Ste20-like 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 down stream of the coding region.

[0078] The genetic modification may be introduced by, for example, T-DNA activation, TILLING, or homologous recombination. Following introduction of the genetic modification, there follows a step of selecting for modified expression of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof, which modification in expression gives plants having increased yield.

T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353) involves insertion of T-DNA, usually containing a promoter (may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10 kb up- or down stream of the coding region of a gene in a configuration such that the 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 the plant genome, for example, through Agrobacterium infection and leads to overexpression of genes near 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.

[0079] A genetic modification may also be introduced in the locus of a Ste20-like 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 a Ste20-like nucleic acid with modulated expression and/or activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may exhibit modified expression, either in strength or in location or in timing (if the mutations affect the promoter for example). These mutant variants may even exhibit higher Ste20-like 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 GP and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) In J Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa, N.J., 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 et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet 5(2): 145-50).

T-DNA activation and TILLING are examples of technologies that enable the generation of novel alleles and Ste20-like variants.

[0080] The effects of the invention may also be reproduced using homologous recombination, which 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 organisms such as yeast or the moss Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offring a et al. (1990) EMBO J 9(10): 3077-84) but also for crop plants, for example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; lida and Terada (2004) Curr Opin Biotech 15(2):132-8). The nucleic acid to be targeted (which may be a Ste20-like nucleic acid or variant thereof as hereinbefore defined) need not be targeted to the locus of a Ste20-like 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.

[0081] A preferred method for introducing a genetic modification (which in this case need not be in the locus of a Ste20-like gene) is to introduce and express in a plant a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof, as defined above. The nucleic acid to be introduced into a plant may be a full-length nucleic acid or may be a portion or a hybridising sequence as hereinbefore defined.

[0082] 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.

[0083] Therefore, there is provided a gene construct comprising: [0084] (i) a Ste20-like nucleic acid or variant thereof, as defined hereinabove; [0085] (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally [0086] (iii) a transcription termination sequence.

[0087] 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.

[0088] Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a Ste20-like polypeptide or homologue thereof). The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. 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. The term "promoter" typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in recognising and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. 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 that 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.

[0089] Suitable promoters, which are functional in plants, are generally known. They may take the form of constitutive or inducible promoters. Suitable promoters can enable the development- and/or tissue-specific expression in multi-celled eukaryotes; thus, leaf-, root-, flower-, seed-, stomata-, tuber- or fruit-specific promoters may advantageously be used in plants.

[0090] Different plant promoters usable in plants are promoters such as, for example, the USP, the LegB4-, the DC3 promoter or the ubiquitin promoter from parsley.

[0091] A "plant" promoter comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or microorganisms, in particular for example from viruses which attack plant cells.

[0092] The "plant" promoter can also originates from a plant cell, e.g. from the plant, which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other "plant" regulatory signals, for example in "plant" terminators.

[0093] For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and in a cell- or tissue-specific manner. Usable promoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those which originate from plant viruses, such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913), 34S FMV (Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443), the parsley ubiquitin promoter, or plant promoters such as the Rubisco small subunit promoter described in U.S. Pat. No. 4,962,028 or the plant promoters PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, PGEL1, OCS [Leisner (1988) Proc Natl Acad Sci USA 85(5): 2553-2557], lib4, usp, mas [Comai (1990) Plant Mol Biol 15 (3):373-381], STLS1, ScBV (Schenk (1999) Plant Mol Biol 39(6):1221-1230), B33, SAD1 or SAD2 (flax promoters, Jain et al., Crop Science, 39 (6), 1999: 1696-1701) or nos [Shaw et al. (1984) Nucleic Acids Res. 12(20):7831-7846]. Further examples of constitutive plant promoters are the sugarbeet V-ATPase promoters (WO 01/14572). Examples of synthetic constitutive promoters are the Super promoter (WO 95/14098) and promoters derived from G-boxes (WO 94/12015). If appropriate, chemical inducible promoters may furthermore also be used, compare EP-A 388186, EP-A 335528, WO 97/06268. Stable, constitutive expression of the proteins according to the invention a plant can be advantageous. However, inducible expression of the polypeptide of the invention is advantageous, if a late expression before the harvest is of advantage, as metabolic manipulation may lead to plant growth retardation.

[0094] The expression of plant genes can also be facilitated via a chemical inducible promoter (for a review, see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108). Chemically inducible promoters are particularly suitable when it is desired to express the gene in a time-specific manner. Examples of such promoters are a salicylic acid inducible promoter (WO 95/19443), and abscisic acid-inducible promoter (EP 335 528), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404), a cyclohexanol- or ethanol-inducible promoter (WO 93/21334) or others as described herein.

[0095] Other suitable promoters are those which react to biotic or abiotic stress conditions, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the tomato heat-inducible hsp80 promoter (U.S. Pat. No. 5,187,267), the potato chill-inducible alpha-amylase promoter (WO 96/12814) or the wound-inducible pinll promoter (EP-A-0 375 091) or others as described herein.

[0096] Preferred promoters are in particular those which bring gene expression in tissues and organs, in seed cells, such as endosperm cells and cells of the developing embryo. Suitable promoters are the oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), the Arabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter (WO 91/13980), the bean arc5 promoter, the carrot DcG3 promoter, or the Legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9), and promoters which bring about the seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like. Advantageous seed-specific promoters are the sucrose binding protein promoter (WO 00/26388), the phaseolin promoter and the napin promoter. Suitable promoters which must be considered are the barley Ipt2 or Ipt1 gene promoter (WO 95/15389 and WO 95/23230), and the promoters described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene and the rye secalin gene). Further suitable promoters are Amy32b, Amy 6-6 and Aleurain [U.S. Pat. No. 5,677,474], Bce4 (oilseed rape) [U.S. Pat. No. 5,530,149], glycinin (soya) [EP 571 741], phosphoenolpyruvate carboxylase (soya) [JP 06/62870], ADR12-2 (soya) [WO 98/08962], isocitrate lyase (oilseed rape) [U.S. Pat. No. 5,689,040] or α-amylase (barley) [EP 781 849]. Other promoters which are available for the expression of genes in plants are leaf-specific promoters such as those described in DE-A 19644478 or light-regulated promoters such as, for example, the pea petE promoter.

[0097] Further suitable plant promoters are the cytosolic FBPase promoter or the potato ST-LSI promoter (Stockhaus et al., EMBO J. 8, 1989, 2445), the Glycine max phosphoribosylpyrophosphate amidotransferase promoter (GenBank Accession No. U87999) or the node-specific promoter described in EP-A-0 249 676.

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 chemical, environmental or physical stimulus. An example of an inducible promoter is a stress-inducible promoter, i.e. a promoter activated when a plant is exposed to various stress conditions, or a pathogen-induced promoter. Additionally or alternatively, the promoter may be a tissue-preferred promoter, i.e. one that is capable of preferentially initiating transcription in certain tissues, such as the leaves, roots, seed tissue etc; or may be a ubiquitous promoter, which is active in substantially all tissues or cells of an organism, or the promoter may be developmentally regulated, thereby being active during certain developmental stages or in parts of the plant that undergo developmental changes. Promoters able to initiate transcription in certain tissues only are referred to herein as "tissue-specific", similarly, promoters able to initiate transcription in certain cells only are referred to herein as "cell-specific".

[0098] Preferably, the Ste20-like nucleic acid or 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 under most environmental conditions in at least one cell, tissue or organ. A preferred constitutive promoter is a constitutive promoter that is also substantially ubiquitously expressed. Further preferably the promoter is derived from a plant, more preferably a monocotyledonous plant. Most preferred is use of a GOS2 promoter (from rice) (as used in the expression cassette of SEQ ID NO: 5). It should be clear that the applicability of the present invention is not restricted to the Ste20-like nucleic acid represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of a nucleic acid encoding a Ste20-like protein when driven by a GOS2 promoter. Examples of other constitutive promoters which may also be used to drive expression of a nucleic acid encoding a Ste20-like protein are shown in Table 2 below.

TABLE-US-00005 TABLE 2 Examples of constitutive promoters Expression Gene Source Pattern Reference Actin Constitutive McElroy et al, Plant Cell, 2: 163-171, 1990 CAMV 35S Constitutive Odell et al, Nature, 313: 810-812, 1985 CaMV 19S Constitutive Nilsson et al., Physiol. Plant. 100: 456-462, 1997 GOS2 Constitutive de Pater et al, Plant J Nov; 2(6): 837-44, 1992 Ubiquitin Constitutive Christensen et al, Plant Mol. Biol. 18: 675-689, 1992 Rice Constitutive Buchholz et al, Plant Mol Biol. 25(5): cyclophilin 837-43, 1994 Maize H3 Constitutive Lepetit et al, Mol. Gen. Genet. 231: histone 276-285, 1992 Actin 2 Constitutive An et al, Plant J. 10(1); 107-121, 1996

[0099] Optionally, one or more terminator sequences (also a control sequence) may 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. The terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, less preferably from any other eukaryotic gene. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that 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.

[0100] 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 al., 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, N.Y. (1994).

[0101] Other control sequences (besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or 5'UTR regions) may be protein and/or RNA stabilizing elements. Such sequences would be known or may readily be obtained by a person skilled in the art.

[0102] The genetic constructs of the invention may further include an origin of replication sequence that 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.

[0103] For the detection and/or selection of the successful transfer of the nucleic acid sequences as depicted in the sequence protocol and used in the process of the invention, it is advantageous to use marker genes (=reporter genes). These marker genes enable the identification of a successful transfer of the nucleic acid molecules via a series of different principles, for example via visual identification with the aid of fluorescence, luminescence or in the wavelength range of light which is discernible for the human eye, by a resistance to herbicides or antibiotics, via what are known as nutritive markers (auxotrophism markers) or antinutritive markers, via enzyme assays or via phytohormones. Examples of such markers which may be mentioned are GFP (=green fluorescent protein); the luciferin/luceferase system, the β-galactosidase with its colored substrates, for example X-Gal, the herbicide resistances to, for example, imidazolinone, glyphosate, phosphinothricin or sulfonylurea, the antibiotic resistances to, for example, bleomycin, hygromycin, streptomycin, kanamycin, tetracyclin, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycin or blasticidin, to mention only a few, nutritive markers such as the utilization of mannose or xylose, or antinutritive markers such as the resistance to 2-deoxyglucose. This list is a small number of possible markers. The skilled worker is very familiar with such markers. Different markers are preferred, depending on the organism and the selection method.

[0104] Therefore the genetic construct may optionally comprise a selectable marker gene. As used herein, the term "selectable marker" or "selectable marker gene" includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that 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 nptll 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 manA that allows plants to use mannose as sole carbon source). Visual marker genes result in the formation of colour (for example β-glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof).

[0105] It is known of the stable or transient integration of nucleic acids into plant cells that only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene encoding for a selectable marker (as described above, for example resistance to antibiotics) is usually introduced into the host cells together with the gene of interest. Preferred selectable markers in plants comprise those, which confer resistance to an herbicide such as glyphosate or gluphosinate. Other suitable markers are, for example, markers, which encode genes involved in biosynthetic pathways of, for example, sugars or amino acids, such as β-galactosidase, ura3 or ilv2. Markers, which encode genes such as luciferase, gfp or other fluorescence genes, are likewise suitable. These markers and the aforementioned markers can be used in mutants in whom these genes are not functional since, for example, they have been deleted by conventional methods. Furthermore, nucleic acid molecules, which encode a selectable marker, can be introduced into a host cell on the same vector as those, which encode the polypeptides of the invention or used in the process or else in a separate vector. Cells which have been transfected stably with the nucleic acid introduced can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).

[0106] Since the marker genes, as a rule specifically the gene for resistance to antibiotics and herbicides, are no longer required or are undesired in the transgenic host cell once the nucleic acids have been introduced successfully, the process according to the invention for introducing the nucleic acids advantageously employs techniques which enable the removal, or excision, of these marker genes. One such a method is what is known as cotransformation. The cotransformation method employs two vectors simultaneously for the transformation, one vector bearing the nucleic acid according to the invention and a second bearing the marker gene(s). A large proportion of transformants receives or, in the case of plants, comprises (up to 40% of the transformants and above), both vectors. In case of transformation with Agrobacteria, the transformants usually receive only a part of the vector, the sequence flanked by the T-DNA, which usually represents the expression cassette. The marker genes can subsequently be removed from the transformed plant by performing crosses. In another method, marker genes integrated into a transposon are used for the transformation together with desired nucleic acid (known as the Ac/Ds technology). The transformants can be crossed with a transposase resource or the transformants are transformed with a nucleic acid construct conferring expression of a transposase, transiently or stable. In some cases (approx. 10%), the transposon jumps out of the genome of the host cell once transformation has taken place successfully and is lost. In a further number of cases, the transposon jumps to a different location. In these cases, the marker gene must be eliminated by performing crosses. In microbiology, techniques were developed which make possible, or facilitate, the detection of such events. A further advantageous method relies on what are known as recombination systems; whose advantage is that elimination by crossing can be dispensed with. The best-known system of this type is what is known as the Cre/lox system. Cre1 is a recombinase, which removes the sequences located between the loxP sequences. If the marker gene is integrated between the loxP sequences, it is removed, once transformation has taken place successfully, by expression of the recombinase. Further recombination systems are the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000: 553-566). A site-specific integration into the plant genome of the nucleic acid sequences according to the invention is possible. Naturally, these methods can also be applied to microorganisms such as yeast, fungi or bacteria.

[0107] The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants obtainable by the method according to the present invention, which plants have introduced therein a Ste20-like nucleic acid or variant thereof.

[0108] The invention also provides a method for the production of transgenic plants having increased yield, comprising introduction and expression in a plant of a Ste20-like nucleic acid or a variant thereof as defined above.

[0109] For the purposes of the invention, "transgenic", "transgene" or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette (=gene construct) or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either [0110] a) the nucleic acid sequences according to the invention, or [0111] b) genetic control sequences which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or [0112] c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette--for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide having kinase domains or a homologue of such polypeptide--becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.

[0113] A transgenic plant for the purposes of the invention is therefore understood as meaning, as above, that the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein.

Host plants for the nucleic acids or the vector used in the method according to the invention, the expression cassette or construct or vector are, in principle, advantageously all plants, which are capable of synthesizing the polypeptides used in the inventive method.

[0114] More specifically, the present invention provides a method for the production of transgenic plants having increased yield, which method comprises: [0115] introducing and expressing in a plant cell a Ste20-like nucleic acid or variant thereof; and [0116] (ii) cultivating the plant cell under conditions promoting plant growth and development. 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.

[0117] The term "introduction" or "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 there from. 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.

[0118] The transfer of foreign genes into the genome of a plant is called transformation. In doing this the methods described for the transformation and regeneration of plants from plant tissues or plant cells are utilized for transient or stable transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Further advantageous transformation methods, in particular for plants, are known to the skilled worker and are described herein below.

[0119] 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, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R. D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein T M et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic rice plants expressing a Ste20-like nucleic acid/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 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, in particular of crop plants such as by way of example tobacco plants, for example by bathing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0120] 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.

[0121] As mentioned Agrobacteria transformed with an expression vector according to the invention may also be used in the manner known per se for the transformation of plants such as experimental plants like Arabidopsis or crop plants, such as, for example, cereals, maize, oats, rye, barley, wheat, soya, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, carrot, bell peppers, oilseed rape, tapioca, cassaya, arrow root, tagetes, alfalfa, lettuce and the various tree, nut, and grapevine species, in particular oil-containing crop plants such as soya, peanut, castor-oil plant, sunflower, maize, cotton, flax, oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa beans, for example by bathing scarified leaves or leaf segments in an agrobacterial solution and subsequently growing them in suitable media.

[0122] In addition to the transformation of somatic cells, which then has to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternative methods are based on the repeated removal of the influorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the"floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, SJ and Bent, AF (1998). The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from nontransgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process, which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview can be taken from Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient cointegrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).

[0123] The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the above-mentioned publications by S. D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

[0124] 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, or quantitiative PCR, all techniques being well known to persons having ordinary skill in the art.

[0125] 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 and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.

[0126] 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).

[0127] The present invention clearly extends to any plant cell 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 by the parent in the methods according to the invention. The invention also includes host cells containing an isolated Ste20-like nucleic acid or variant thereof. Preferred host cells according to the invention are plant cells. The invention also extends to harvestable parts of a plant 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.

[0128] The present invention also encompasses use of Ste20-like nucleic acids or variants thereof and use of Ste20-like polypeptides or homologues thereof.

[0129] One such use relates to improving the growth characteristics of plants, in particular in improving yield, especially seed yield. The seed yield may include one or more of the following: increased total weight of seeds, increased number of filled seeds and increased Harvest Index.

[0130] Ste20-like nucleic acids or variants thereof, or Ste20-like polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a Ste20-like gene or variant thereof. The Ste20-like nucleic acids/genes or variants thereof, or Ste20-like polypeptides or homologues thereof may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having increased yield. The Ste20-like gene or variant thereof may, for example, be a nucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 33.

[0131] Allelic variants of a Ste20-like nucleic acid/gene 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, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. 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: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 33. 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.

[0132] A Ste20-like nucleic acid or variant thereof may also be used as probes 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 Ste20-like nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The Ste20-like nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the Ste20-like 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 Ste20-like 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).

[0133] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. 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., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

[0134] 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 kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

[0135] A variety of nucleic acid amplification-based methods for 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 al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren at 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 acid sequence. This, however, is generally not necessary for mapping methods.

[0136] The methods according to the present invention result in plants having increased yield, 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

[0137] The present invention will now be described with reference to the following figures in which:

[0138] FIG. 1 shows the typical domain structure of Ste20-like polypeptides. The N-terminal end of the protein comprises a Ser/Thr kinase domain. The most C-terminal domain (in light grey) has a coiled coil structure, which is usually but not always present.

[0139] FIG. 2 shows a binary vector p070, for expression in Oryza sativa of an Arabidopsis thaliana Ste20-like coding sequence under the control of a GOS2 promoter (internal reference PRO0129).

[0140] FIG. 3 details examples of sequences useful in performing the methods according to the present invention.

EXAMPLES

[0141] The present invention will now be described with reference to the following examples, which are by way of illustration alone. 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 et al. (1994), Current Protocols in Molecular Biology, Current Protocols. 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

Identification of Homologues of the Ste20-Like Protein of SEQ ID NO: 2 and Determination of their Similarity/Identity

[0142] Sequences (full length cDNA, ESTs or genomic) related to the nucleic acid sequence used in the methods of the present invention were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). This program is typically used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. The polypeptide encoded by the nucleic acid of the present invention was used with the TBLASTN algorithm, with default settings and the filter for ignoring low complexity sequences was set off. The output of the analysis was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflect the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In some instances, the default parameters may be adjusted to modify the stringency of the search.

[0143] Rice sequences and EST sequences from various plant species may also be obtained from other databases, such as KOME (Knowledge-based Oryza Molecular biological Encyclopedia; Kikuchi et al., Science 301, 376-379, 2003), Sputnik (Rudd, S., Nucleic Acids Res., 33: D622-D627, 2005) or the Eukaryotic Gene Orthologs database (EGO, hosted by The Institute for Genomic Research). These databases are searchable with the BLAST tool. SEQ ID NO: 11 to SEQ ID NO: 34 are nucleic acid and protein sequences of homologues of SEQ ID NO: 2 and were obtained from the above-mentioned databases using SEQ ID NO: 2 as a query sequence.

[0144] Percentages of similarity and identity between the full-length sequences and the sequences of the kinase domains of Ste20-like proteins were determined using MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pair-wise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 11, and a gap extension penalty of 1), calculates similarity and identity using for example Blosum 62 (for polypeptides), and then places the results in a distance matrix. Sequence similarity is shown in the bottom half of the dividing line and sequence identity is shown in the top half of the diagonal dividing line. The sequence of SEQ ID NO: 2 is indicated as number 1 in the matrix.

[0145] The kinase domains of the Ste20-like proteins were delineated using the SMART tool and the obtained sequences are listed in Table 3.

TABLE-US-00006 TABLE 3 list of the kinase domains in the various SEQ ID Nos: SEQ ID NO: 2 YEIICKIGVGVSASVYKAICIPMNSMVVAIKAIDLDQSRADFDSLRRETKTMSLLS HPNILNAYCSFTVDRCLWVVMPFMSCGSLHSIVSSSFPSGLPENCISVFLKETLNA ISYLHDQGHLHRDIKAGNILVDSDGSVKLADFGVSASIYEPVTSSSGTTSSSLRLT DIAGTPYWMAPEVVHSHTGYGFKADIWSFGITALELAHGRPPLSHLPPLKSLLMKI TKRFHFSDYEINTSGSSKKGNKKFSKAFREMVGLCLEQDPTKRPSAEKLLKHPFF SEQ ID NO: 12 YKLYEEIGDGVSATVHRALCIPLNVVVAIKVLDLEKCNNDLDGIRREVQTMSLINH PNVLQAHCSFTTGHQLWVVMPYMAGGSCLHIIKSSYPDGFEEPVIATLLRETLKAL VYLHAHGHIHRDVKAGNILLDSNGAVKLADFGVSACMFDTGDRQRSRNTFVGTPCW MAPEVMQQLHGYDFKADVWSFGITALELAHGHAPFSKYPPMKVLLMTLQNAPPGLD YERDKRFSKAFKEMVGTCLVKDPKKRPTSEKLLKHPFF SEQ ID NO: 14 YELFEEVGEGVSATVYRARCIALNEIVAVKILDLEKCRNDLETIRKEVHIMSLIDH PNLLKAHCSFIDSSSLWIVMPYMSGGSCFHLMKSVYPEGLEQPIIATLLREVLKAL VYLHRQGHIHRDVKAGNILIHSKGVVKLGDFGVSACMFDSGERMQTRNTFVGTPCW MAPEVMQQLDGYDFKYLAHGHAPFSKYPPMKVLLMTLQNAPPRLDYDRDKKFSKSF RELIAACLVKDPKKRPTAAKLLKHPFF SEQ ID NO: 16 YEILEEIGDGVYRARCILLDEIVAIKIWNLEKCTNDLETIRKEVHRLSLIDHPNLL RVHCSFIDSSSLWIVMPFMSCGSSLNIMKSVYPNGLEEPVIAILLREILKALVYLH GLGHIHRNVKAGNVLVDSEGTVKLGDFEVSASMFDSVERMRTSSENTFVGNPRRMA PEKDMQQVDGYDFKVDIWSFGMTALELAHGHSPTTVLPLNLQNSPFPNYEEDTKFS KSFRELVAACLIEDPEKRPTASQLLEYPFL SEQ ID NO: 18 YTLYEFIGQGVSALVHRALCIPFDEVVAIKILDFERDNCDLNNISREAQTMMLVDH PNVLKSHCSFVSDHNLWVIMPYMSGGSCLHILKAAYPDGFEEAIIATILREALKGL DYLHQHGHIHRDVKAGNILLGARGAVKLGDFGVSACLFDSGDRQRTRNTFVGTPCW MAPEVMEQLHGYDFKADIWSFGITGLELAHGHAPFSKYPPMKVLLMTLQNAPPGLD YERDKKFSRSFKQMIASCLVKDPSKRPSAKKLLKHSFF SEQ ID NO: 20 YKLMEEVGYGASAVVHRAIYLPTNEVVAIKSLDLDRCNSNLDDIRREAQTMTLIDH PNVIKSFCSFAVDHHLWVVMPFMAQGSCLHLMKAAYPDGFEEAAICSMLKETLKAL DYLHRQGHIHRDVKAGNILLDDTGEIKLGDFGVSACLFDNGDRQRARNTFVGTPCW MAPEVLQPGSGYNSKADIWSFGITALELAHGHAPFSKYPPMKVLLMTIQNAPPGLD YDRDKKFSKSFKELVALCLVKDQTKRPTAEKLLKHSFF SEQ ID NO: 22 YKLMEEIGHGASAVVYRAIYLPTNEVVAIKCLDLDRCNSNLDDIRRESQTMSLIDH PNVIKSFCSFSVDHSLWVVMPFMAQGSCLHLMKTAYSDGFEESAICCVLKETLKAL DYLHRQGHIHRDVKAGNILLDDNGEIKLGDFGVSACLFDNGDRQRARNTFVGTPCW MAPEVLQPGNGYNSKADIWSFGITALELAHGHAPFSKYPPMKVLLMTIQNAPPGLD YDRDKKFSKSFKEMVAMCLVKDQTKRPTAEKLLKHSCF SEQ ID NO: 24 YRLLCKIGSGVSAVVYKAACVPLGSAVVAIKAIDLERSRANLDEVWREAKAMALLS HRNVLRAHCSFTVGSHLWVVMPFMAAGSLHSILSHGFPDGLPEQCIAVVLRDTLRA LCYLHEQGRIHRDIKAGNILVDSDGSVKLADFGVSASIYETAPSTSSAFSGPINHA PPPSGAALSSSCFNDMAGTPYWMAPEVIHSHVGYGIKADIWSFGITALELAHGRPP LSHLPPSKSMLMRITSRVRLEVDASSSSSEGSSSAARKKKKFSKAFKDMVSSCLCQ EPAKRPSAEKLLRHPFF SEQ ID NO: 26 YKLCEEVGDGVSATVYKALCIPLNIEVAIKVLDLEKCSNDLDGIRREVQTMSLIDH PNLLRAYCSFTNGHQLWVIMPYMAAGSALHIMKTSFPDGFEEPVIATLLREVLKAL VYLHSQGHIHRDVKAGNILIDTNGAVKLGDFGVSACMFDTGNRQRARNTFVGTPCW MAPEVMQQLHGYDYKADIWSFGITALELAHGHAPFSKYPPMKVLLMTLQNAPPGLD YERDKRFSKSFKDLVATCLVKDPRKRPSSEKLLKHSFF SEQ ID NO: 28 YELYEEIGQGVSAIVYRSLCKPLDEIVAVKVLDFERTNSDLWLVVMQVGYTRIVAI YVPPLDLSKMIVTRICLTQNNIMREAQTMILIDQPNVMKAHCSFTNNHSLWVVMPY MAGGSCLHIMKSVYPDGFEEAVIATVLREVLKGLEYLHHHGHIHRDVKAGNILVDS RGVVKLGDFGVSACLFDSGDRQRARNTFVGTPCWMAPEVMEQLHGYDFKADIWSFG ITALELAHGHAPFSKFPPMKVLLMTLQNAPPGLDYERDKKFSRHFKQMVAMCLVKD PSKRPTAKKLLKQPFF SEQ ID NO: 30 YQLMEEVGYGAHAVVYRALFVPRNDVVAVKCLDLDQLNNNIDEIQREAQIMSLIEH PNVIRAYCSFVVEHSLWVVMPFMTEGSCLHLMKIAYPDGFEEPVIGSILKETLKAL EYLHRQGQIHRDVKAGNILVDNAGIVKLGDFGVSACMFDRGDRQRSRNTFVGTPCW MAPEVLQPGTGYNFKADIWSFGITALELAHGHAPFSKYPPMKVLLMTLQNAPPGLD YDRDRRFSKSFKEMVAMCLVKDQTKRPTAEKLLKHSFF SEQ ID NO: 32 YRLLEEVGYGANAVVYRAVFLPSNRTVAVKCLDLDRVNSNLDDIRKEAQTMSLIDH PNVIRAYCSFVVDHNLWVIMPFMSEGSCLHLMKVAYPDGFEEPVIASILKETLKAL EYLHRQGHIHRDVKRNIIQAGNILMDSPGIVKLGDFGVSACMFDRGDRQRSRNTFV GTPCWMAPEVLQPGAGYNFKKYVSNHLFTNLIWLFKISLRGKNSNYHKNTGNKVLL MTLQNAPPGLDYDRDKRFSKSFKEMVAMCLVKDQTKRPTAEKLLKHSFF SEQ ID NO: 34 YKIVDEIGAGNSAVVYKAICIPINSTPVAIKSIDLDRSRPDLDDVRREAKTLSLLS HPNILKAHCSFTVDNRLWVVMPFMAGGSLQSIISHSFQNGLTEQSIAVILKDTLNA LSYLHGQGHLHRDIKSGNILVDSNGLVKLADFGVSASIYESNNSVGACSSYSSSSS NSSSSHIFTDFAGTPYWMAPEVIHSHNGYSFKADIWSFGITALELAHGRPPLSHLP PSKSLMLNITKRFKFSDFDKHSYKGHGGSNKFSKAFKDMVALCLNQDPTKRPSAEK LLKHSFF

[0146] Results of the MATGAT analysis are shown in Table 4 for the full-length sequences and in Table 5 for the kinase domains of the Ste20-like polypeptides. Percentage identity is given above the diagonal (in bold) and percentage similarity is given below the diagonal (normal font). Percentage identity between kinase domains of Ste20-like paralogues and orthologues of SEQ ID NO: 2 ranges between 44% (for SEQ ID NO: 28) and 71% (for SEQ ID NO: 34).

TABLE-US-00007 TABLE 4 Sequence similarity\identity for the full-length sequences 1 2 3 4 5 6 7 8 9 10 11 12 13 1. SEQIDNO2 30.7 30.2 32.6 29.3 28.8 28.9 46.5 29.0 26.8 35.5 28.9 53.5 2. SEQIDNO12 45.8 45.3 33.9 45.2 42.8 43.1 28.6 55.1 42.7 41.5 38.3 32.3 3. SEQIDNO14 48.3 62.3 41.2 39.6 37.9 38.7 28.1 44.3 37.2 40.3 35.7 30.1 4. SEQIDNO16 55.2 48.1 56.4 28.9 27.4 27.8 30.7 33.7 29.2 35.8 30.1 32.8 5. SEQIDNO18 43.9 63.4 56.8 43.7 41.5 40.5 27.1 42.3 48.9 37.9 36.4 28.7 6. SEQIDNO20 42.1 61.2 53.8 41.9 61.4 73.9 26.8 41.2 40.2 45.2 47.7 29.8 7. SEQIDNO22 42.3 60.8 53.2 41.9 60.8 84.9 25.7 41.2 39.7 44.1 48.5 29.2 8. SEQIDNO24 59.9 43.9 48.1 47.8 41.6 42.7 41.9 28.3 26.3 32.2 26.9 47.1 9. SEQIDNO26 44.2 72.4 58.2 47.5 62.8 61.0 60.4 44.3 41.1 39.8 35.9 29.2 10. SEQIDNO28 39.3 57.7 51.5 41.7 64.9 58.6 56.4 39.3 58.6 36.8 34.2 25.9 11. SEQIDNO30 51.6 54.5 58.1 52.5 52.2 57.5 56.4 50.8 52.4 49.4 48.0 33.0 12. SEQIDNO32 42.8 54.9 51.8 42.9 53.9 64.1 63.0 43.9 54.5 51.1 60.9 29.3 13. SEQIDNO34 69.3 48.2 50.5 53.9 44.8 44.6 45.3 63.5 45.0 41.5 53.4 48.2

TABLE-US-00008 TABLE 5 Sequence similarity\identity for the kinase domain sequences of Table 3 1 2 3 4 5 6 7 8 9 10 11 12 13 1. SEQIDNO2 54.5 46.6 46.5 48.7 50.9 51.3 66.0 52.3 44.1 51.3 43.4 70.8 2. SEQIDNO12 69.9 72.5 60.5 77.5 74.0 74.4 50.2 86.6 70.3 74.4 64.5 52.3 3. SEQIDNO14 62.4 84.7 65.8 67.6 64.9 64.5 41.9 72.1 61.8 65.3 60.1 43.2 4. SEQIDNO16 65.2 77.9 82.3 54.5 52.3 52.6 40.9 59.8 51.3 53.8 49.1 44.8 5. SEQIDNO18 65.2 89.3 81.7 73.3 73.7 72.1 45.8 74.4 73.3 69.1 61.6 47.7 6. SEQIDNO20 67.4 86.3 78.2 72.9 87.0 93.1 47.0 73.3 63.9 81.3 72.5 51.9 7. SEQIDNO22 67.4 86.3 77.9 72.9 85.9 97.3 47.0 73.3 64.5 81.3 72.1 52.6 8. SEQIDNO24 77.1 64.6 59.3 58.9 60.9 62.3 62.0 49.2 43.2 47.0 39.9 64.4 9. SEQIDNO26 68.5 96.2 84.0 78.6 87.0 86.6 87.0 64.0 67.2 73.3 64.1 51.6 10. SEQIDNO28 62.8 79.1 73.0 66.2 81.8 76.7 76.7 61.3 78.0 63.2 55.5 43.0 11. SEQIDNO30 66.3 85.5 80.2 74.0 86.3 93.5 92.7 62.0 85.9 75.7 77.2 49.1 12. SEQIDNO32 62.0 78.0 72.9 66.7 78.8 85.0 83.9 57.9 78.8 71.6 87.2 44.4 13. SEQIDNO34 80.5 69.0 61.3 64.5 65.9 69.3 69.3 76.4 68.6 64.9 67.9 66.2

Example 2

Gene Cloning of Ste20-Like

[0147] The Arabidopsis thaliana Ste20-like gene 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 of the order of 1.59×10⁷ cfu. Original titer was determined to be 9.6×10⁵ cfu/ml after first amplification of 6×10¹¹ cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers prm03186 (SEQ ID NO: 3; sense; start codon in bold, AttB1 site in italic: 5'-ggggacaagtttgtacaaaaaagcaggcttca caatggctcggaacaagctc 3') and prm03187 (SEQ ID NO: 4; reverse, complementary, AttB2 site in italic: 5' ggggaccactttgtacaagaaagctgggtaatagttaacccaaaacactatcttta 3'), 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 1532 bp (including attB sites) 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 clone", p068. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.

Example 3

Vector Construction

[0148] The entry clone p068 were subsequently used in an LR reaction with p00640, a destination vector used for Oryza sativa transformation. This vector contains as functional elements within the T-DNA borders: a plant selectable marker; a screenable 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 (nucleotides 1 to 2193 of SEQ ID NO: 5) for constitutive expression (PRO0129) was located upstream of this Gateway cassette.

[0149] After the LR recombination step, the resulting expression vector, p070 for Ste20-like (FIG. 2) was transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants. Transformed rice plants were allowed to grow and were then examined for the parameters described in Example 4.

Example 4

Evaluation and Results of Ste20-Like Under the Control of the Rice GOS2 Promoter

[0150] Approximately 15 to 20 independent TO rice transformants were generated. The primary transformants were transferred from a tissue culture chamber 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, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. 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., 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 (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.

[0151] 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 the following seed-related parameters:

[0152] The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. The total seed yield was measured by weighing all filled husks harvested from a plant. Total seed number per plant was measured by counting the number of husks harvested from a plant. Thousand Kernel Weight (TKW) is extrapolated from the number of filled seeds counted and their total weight. Harvest index is defined as the ratio between the total seed weight and the above-ground area (mm²), multiplied by a factor 10⁶. These parameters were derived in an automated way from the digital images using image analysis software and were analysed statistically. Individual seed parameters (including width, length, area, weight) were measured using a custom-made device consisting of two main components, a weighing and imaging device, coupled to software for image analysis.

[0153] 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.

[0154] To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test 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.

[0155] The data obtained for Ste20 in the first experiment were confirmed in a second experiment with T2 plants. Four 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 heterozygote 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.

[0156] A total number of 120 Ste20 transformed plants were evaluated in the T2 generation, that is 30 plants per event of which 15 positives for the transgene, and 15 negatives.

[0157] Because two experiments with overlapping events had 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 5

Evaluation of Ste20 Transformants: Measurement of Yield-Related Parameters

[0158] Upon analysis of the seeds as described above, the inventors found that plants transformed with the Ste20 gene construct had a higher seed yield, expressed as number of filled seeds, total weight of seeds and harvest index, compared to plants lacking the Ste20 transgene.

The results obtained for plants in the T1 generation are summarised in Table 6:

TABLE-US-00009 TABLE 6 % difference p-value Nr filled seeds +38 0.0003 Total weight seeds +38 0.0004 Harvest Index +42 0.0001

[0159] These positive results were again obtained in the T2 generation. In Table 7, data show the overall % increases for the number of filled seeds, total weight of seeds and harvest index, calculated from the data of the individual lines of the T2 generation, and the respective p-values. These T2 data were re-evaluated in a combined analysis with the results for the T1 generation, and the obtained p-values show that the observed effects were highly significant.

TABLE-US-00010 TABLE 7 T2 generation Combined analysis % difference p-value p-value Nr filled seeds +30 0.0004 0.0000 Total weight seeds +29 0.0008 0.0000 Harvest Index +33 0.0001 0.0000

Sequence CWU 1

3411554DNAArabidopsis thaliana 1cataacaatt caataagcaa gagtgtactc atcttctttc tatttatggc tcggaacaag 60ctcgagttcc ctcttgatgc tgaagcctac gagatcatct gcaagatagg cgttggtgtt 120agtgcttcgg tctacaaggc catatgcatt ccgatgaact caatggtagt tgctatcaaa 180gccatcgatc ttgatcagtc gcgggctgac tttgacagtc ttcgccgtga aaccaagacg 240atgtctctgc tttctcatcc gaatattctc aatgcttatt gttcattcac cgttgatcga 300tgtctctggg tggttatgcc attcatgtct tgtggctctc ttcattcgat cgtctcttcg 360tcttttccaa gtgggttacc agaaaactgc atttccgtct tcctcaagga aactctgaat 420gcaatctcgt atcttcacga tcagggtcat ttgcaccgtg acatcaaggc aggtaacatt 480ctggtagatt ctgatggatc cgtgaagctc gctgatttcg gagtatctgc atccatctat 540gaacccgtga catcttcctc tggaacaaca tcttcttctt taaggttaac tgatatagcg 600ggaacaccgt attggatggc tccggaagtg gttcattccc acacagggta tggtttcaaa 660gcagacattt ggtctttcgg gataacagcg ttggagttag cacatggaag acctccgtta 720tctcacttac cgccgttgaa gagtctgctc atgaagatca ccaaaaggtt tcatttttct 780gattacgaga tcaatacgag cggaagcagc aaaaagggta acaagaagtt ctcaaaagct 840tttagagaaa tggttggttt gtgtctagag caagatccta ctaaaagacc atcggcagag 900aagttgttga agcatccttt tttcaagaac tgtaaaggac tcgactttgt ggtcaagaac 960gtgttgcata gcttgtcaaa cgcagagcag atgtttatgg agagtcagat tttgatcaag 1020agtgttggag atgatgatga agaagaagaa gaagaagacg aagagatagt gaagaataga 1080agaatcagtg ggtggaattt ccgtgaagac gatctccaac ttagtccagt gttcccagct 1140actgaatcag actcttctga gtccagtcca cgtgaagaag atcaatcaaa agacaaaaag 1200gaagacgata acgtcacaat aacggggtat gaactcggtt taggtttgtc gaacgaggaa 1260gctaagaacc aagaaggtga ggttgttggg tttgataaag atttggtgtt agagaaactg 1320aaagtgttga agaaaagttt agagcatcaa agagcaagag tgtcgattat aatcgaagca 1380ttgagtgggg acaaggaaga gaagagcaga gaagaagagc ttctagagat ggtggagaag 1440ttaaagattg aattggaaac tgagaagcta aagaccttgc gtgctgataa agatagtgtt 1500ttgggttaac tattctaaac ttgttaatat tttttttcta tatgctaaaa ttat 15542487PRTArabidopsis thaliana 2Met Ala Arg Asn Lys Leu Glu Phe Pro Leu Asp Ala Glu Ala Tyr Glu1 5 10 15Ile Ile Cys Lys Ile Gly Val Gly Val Ser Ala Ser Val Tyr Lys Ala 20 25 30Ile Cys Ile Pro Met Asn Ser Met Val Val Ala Ile Lys Ala Ile Asp 35 40 45Leu Asp Gln Ser Arg Ala Asp Phe Asp Ser Leu Arg Arg Glu Thr Lys 50 55 60Thr Met Ser Leu Leu Ser His Pro Asn Ile Leu Asn Ala Tyr Cys Ser65 70 75 80Phe Thr Val Asp Arg Cys Leu Trp Val Val Met Pro Phe Met Ser Cys 85 90 95Gly Ser Leu His Ser Ile Val Ser Ser Ser Phe Pro Ser Gly Leu Pro 100 105 110Glu Asn Cys Ile Ser Val Phe Leu Lys Glu Thr Leu Asn Ala Ile Ser 115 120 125Tyr Leu His Asp Gln Gly His Leu His Arg Asp Ile Lys Ala Gly Asn 130 135 140Ile Leu Val Asp Ser Asp Gly Ser Val Lys Leu Ala Asp Phe Gly Val145 150 155 160Ser Ala Ser Ile Tyr Glu Pro Val Thr Ser Ser Ser Gly Thr Thr Ser 165 170 175Ser Ser Leu Arg Leu Thr Asp Ile Ala Gly Thr Pro Tyr Trp Met Ala 180 185 190Pro Glu Val Val His Ser His Thr Gly Tyr Gly Phe Lys Ala Asp Ile 195 200 205Trp Ser Phe Gly Ile Thr Ala Leu Glu Leu Ala His Gly Arg Pro Pro 210 215 220Leu Ser His Leu Pro Pro Leu Lys Ser Leu Leu Met Lys Ile Thr Lys225 230 235 240Arg Phe His Phe Ser Asp Tyr Glu Ile Asn Thr Ser Gly Ser Ser Lys 245 250 255Lys Gly Asn Lys Lys Phe Ser Lys Ala Phe Arg Glu Met Val Gly Leu 260 265 270Cys Leu Glu Gln Asp Pro Thr Lys Arg Pro Ser Ala Glu Lys Leu Leu 275 280 285Lys His Pro Phe Phe Lys Asn Cys Lys Gly Leu Asp Phe Val Val Lys 290 295 300Asn Val Leu His Ser Leu Ser Asn Ala Glu Gln Met Phe Met Glu Ser305 310 315 320Gln Ile Leu Ile Lys Ser Val Gly Asp Asp Asp Glu Glu Glu Glu Glu 325 330 335Glu Asp Glu Glu Ile Val Lys Asn Arg Arg Ile Ser Gly Trp Asn Phe 340 345 350Arg Glu Asp Asp Leu Gln Leu Ser Pro Val Phe Pro Ala Thr Glu Ser 355 360 365Asp Ser Ser Glu Ser Ser Pro Arg Glu Glu Asp Gln Ser Lys Asp Lys 370 375 380Lys Glu Asp Asp Asn Val Thr Ile Thr Gly Tyr Glu Leu Gly Leu Gly385 390 395 400Leu Ser Asn Glu Glu Ala Lys Asn Gln Glu Gly Glu Val Val Gly Phe 405 410 415Asp Lys Asp Leu Val Leu Glu Lys Leu Lys Val Leu Lys Lys Ser Leu 420 425 430Glu His Gln Arg Ala Arg Val Ser Ile Ile Ile Glu Ala Leu Ser Gly 435 440 445Asp Lys Glu Glu Lys Ser Arg Glu Glu Glu Leu Leu Glu Met Val Glu 450 455 460Lys Leu Lys Ile Glu Leu Glu Thr Glu Lys Leu Lys Thr Leu Arg Ala465 470 475 480Asp Lys Asp Ser Val Leu Gly 485352DNAArtificial sequenceprimer prm03186 3ggggacaagt ttgtacaaaa aagcaggctt cacaatggct cggaacaagc tc 52456DNAArtificial sequenceprimer prm03187 4ggggaccact ttgtacaaga aagctgggta atagttaacc caaaacacta tcttta 5653710DNAArtificial sequenceexpression cassette 5aatccgaaaa gtttctgcac cgttttcacc ccctaactaa caatataggg aacgtgtgct 60aaatataaaa tgagacctta tatatgtagc gctgataact agaactatgc aagaaaaact 120catccaccta ctttagtggc aatcgggcta aataaaaaag agtcgctaca ctagtttcgt 180tttccttagt aattaagtgg gaaaatgaaa tcattattgc ttagaatata cgttcacatc 240tctgtcatga agttaaatta ttcgaggtag ccataattgt catcaaactc ttcttgaata 300aaaaaatctt tctagctgaa ctcaatgggt aaagagagag atttttttta aaaaaataga 360atgaagatat tctgaacgta ttggcaaaga tttaaacata taattatata attttatagt 420ttgtgcattc gtcatatcgc acatcattaa ggacatgtct tactccatcc caatttttat 480ttagtaatta aagacaattg acttattttt attatttatc ttttttcgat tagatgcaag 540gtacttacgc acacactttg tgctcatgtg catgtgtgag tgcacctcct caatacacgt 600tcaactagca acacatctct aatatcactc gcctatttaa tacatttagg tagcaatatc 660tgaattcaag cactccacca tcaccagacc acttttaata atatctaaaa tacaaaaaat 720aattttacag aatagcatga aaagtatgaa acgaactatt taggtttttc acatacaaaa 780aaaaaaagaa ttttgctcgt gcgcgagcgc caatctccca tattgggcac acaggcaaca 840acagagtggc tgcccacaga acaacccaca aaaaacgatg atctaacgga ggacagcaag 900tccgcaacaa ccttttaaca gcaggctttg cggccaggag agaggaggag aggcaaagaa 960aaccaagcat cctcctcctc ccatctataa attcctcccc ccttttcccc tctctatata 1020ggaggcatcc aagccaagaa gagggagagc accaaggaca cgcgactagc agaagccgag 1080cgaccgcctt cttcgatcca tatcttccgg tcgagttctt ggtcgatctc ttccctcctc 1140cacctcctcc tcacagggta tgtgcccttc ggttgttctt ggatttattg ttctaggttg 1200tgtagtacgg gcgttgatgt taggaaaggg gatctgtatc tgtgatgatt cctgttcttg 1260gatttgggat agaggggttc ttgatgttgc atgttatcgg ttcggtttga ttagtagtat 1320ggttttcaat cgtctggaga gctctatgga aatgaaatgg tttagggtac ggaatcttgc 1380gattttgtga gtaccttttg tttgaggtaa aatcagagca ccggtgattt tgcttggtgt 1440aataaaagta cggttgtttg gtcctcgatt ctggtagtga tgcttctcga tttgacgaag 1500ctatcctttg tttattccct attgaacaaa aataatccaa ctttgaagac ggtcccgttg 1560atgagattga atgattgatt cttaagcctg tccaaaattt cgcagctggc ttgtttagat 1620acagtagtcc ccatcacgaa attcatggaa acagttataa tcctcaggaa caggggattc 1680cctgttcttc cgatttgctt tagtcccaga attttttttc ccaaatatct taaaaagtca 1740ctttctggtt cagttcaatg aattgattgc tacaaataat gcttttatag cgttatccta 1800gctgtagttc agttaatagg taatacccct atagtttagt caggagaaga acttatccga 1860tttctgatct ccatttttaa ttatatgaaa tgaactgtag cataagcagt attcatttgg 1920attatttttt ttattagctc tcaccccttc attattctga gctgaaagtc tggcatgaac 1980tgtcctcaat tttgttttca aattcacatc gattatctat gcattatcct cttgtatcta 2040cctgtagaag tttctttttg gttattcctt gactgcttga ttacagaaag aaatttatga 2100agctgtaatc gggatagtta tactgcttgt tcttatgatt catttccttt gtgcagttct 2160tggtgtagct tgccactttc accagcaaag ttcatttaaa tcaactaggg atatcacaag 2220tttgtacaaa aaagcaggct tcacaatggc tcggaacaag ctcgagttcc ctcttgatgc 2280tgaagcctac gagatcatct gcaagatagg cgttggtgtt agtgcttcgg tctacaaggc 2340catatgcatt ccgatgaact caatggtagt tgctatcaaa gccatcgatc ttgatcagtc 2400gcgggctgac tttgacagtc ttcgccgtga aaccaagacg atgtctctgc tttctcatcc 2460gaatattctc aatgcttatt gttcattcac cgttgatcga tgtctctggg tggttatgcc 2520attcatgtct tgtggctctc ttcattcgat cgtctcttcg tcttttccaa gtgggttacc 2580agaaaactgc atttccgtct tcctcaagga aactctgaat gcaatctcgt atcttcacga 2640tcagggtcat ttgcaccgtg acatcaaggc aggtaacatt ctggtagatt ctgatggatc 2700cgtgaagctc gctgatttcg gagtatctgc atccatctat gaacccgtga catcttcctc 2760tggaacaaca tcttcttctt taaggttaac tgatatagcg ggaacaccgt attggatggc 2820tccggaagtg gttcattccc acacagggta tggtttcaaa gcagacattt ggtctttcgg 2880gataacagcg ttggagttag cacatggaag acctccgtta tctcacttac cgccgttgaa 2940gagtctgctc atgaagatca ccaaaaggtt tcatttttct gattacgaga tcaatacgag 3000cggaagcagc aaaaagggta acaagaagtt ctcaaaagct tttagagaaa tggttggttt 3060gtgtctagag caagatccta ctaaaagacc atcggcagag aagttgttga agcatccttt 3120tttcaagaac tgtaaaggac tcgactttgt ggtcaagaac gtgttgcata gcttgtcaaa 3180cgcagagcag atgtttatgg agagtcagat tttgatcaag agtgttggag atgatgatga 3240agaagaagaa gaagaagacg aagagatagt gaagaataga agaatcagtg ggtggaattt 3300ccgtgaagac gatctccaac ttagtccagt gttcccagct actgaatcag actcttctga 3360gtccagtcca cgtgaagaag atcaatcaaa agacaaaaag gaagacgata acgtcacaat 3420aacggggtat gaactcggtt taggtttgtc gaacgaggaa gctaagaacc aagaaggtga 3480ggttgttggg tttgataaag atttggtgtt agagaaactg aaagtgttga agaaaagttt 3540agagcatcaa agagcaagag tgtcgattat aatcgaagca ttgagtgggg acaaggaaga 3600gaagagcaga gaagaagagc ttctagagat ggtggagaag ttaaagattg aattggaaac 3660tgagaagcta aagaccttgc gtgctgataa agatagtgtt ttgggttaac 3710610PRTArtificial sequenceste20 signature sequence 6Gly Xaa Pro Xaa Xaa Met Ala Pro Glu Xaa1 5 10710PRTArtificial sequenceconsensus sequence 7Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly1 5 1089PRTArtificial sequenceconsensus sequence 8Xaa His Ser His Xaa Gly Tyr Xaa Xaa1 5912PRTArtificial sequenceconsensus sequence 9Arg Pro Pro Leu Ser His Leu Pro Pro Xaa Lys Ser1 5 10108PRTArtificial sequenceconsensus sequence 10Arg Arg Ile Ser Gly Trp Asn Phe1 5112772DNAArabidopsis thaliana 11cagacgacag aaaagctaac cacaagagga ggagagaaac tcgataacaa acaagagaaa 60gagaaagcga gattctaaaa tctaatctcg tgcttccaat tcaaataatt ttgtctcctt 120agcggatcga tcgtagatta taaagctccg ccgtcgcctc cgccgcaatc gacggcggtg 180tctacgtcgc tttcgtttcg tgcgtaacag gaggagcagc agcaaaataa gtcagcttaa 240gtaacgccgt ctttgatttg acttgagata agtattttgg tgatatggca ttgatgatgt 300ttccgcattt gctcgacgtt gacgaaaagt aaaatgctgg cgaattggaa gaaaccacat 360acagattgat gctctcttca gtcgacctct tttgtaaatt tgttgaaact tacggggtcg 420aaggtgtgta gcatatatgc tataagaaga ttataaagta aaaattatgg aatcgggttc 480agagaaaaag ttccctctca atgcaaaaga ctacaagtta tatgaagaaa ttggagatgg 540tgtcagtgcg actgtgcata gagctttgtg tataccgctt aatgtggtag ttgctatcaa 600ggttcttgat ctggaaaagt gcaacaacga tctggatggg atccggagag aggtgcaaac 660aatgagtctg atcaaccatc caaatgtgtt gcaagctcat tgctcattta ccaccggaca 720ccagctttgg gttgtgatgc cttacatggc tggaggatct tgtctccata taattaagtc 780ttcctatcca gatggatttg aggaacctgt tatcgctact ttacttcgtg agactctgaa 840agctcttgta tatcttcatg ctcatgggca tatccacagg gatgtgaagg ctggaaacat 900tttattggat tccaatggtg ccgttaagtt agcagacttt ggagtatcag cttgcatgtt 960tgatacggga gatagacaac gttccagaaa tacatttgtt gggactccat gctggatggc 1020tcctgaagtc atgcagcaac tacatggata tgatttcaaa gcagatgtat ggtcatttgg 1080aataacagca cttgaattgg cacatggtca tgccccattt tccaaatatc cgccaatgaa 1140ggttttgctg atgaccttac aaaatgcacc tcctggactt gactacgaga gagacaaaag 1200attctcgaaa gccttcaagg aaatggtggg tacatgcctg gtgaaggacc caaagaagcg 1260tccaacttca gaaaagcttt tgaaacaccc tttcttcaaa catgcacgtc cagctgatta 1320cctggttaaa acaattctaa atggtcttcc tccattaggt gatcgctata gacaaataaa 1380gtcgaaggaa gctgatctcc taatgcaaaa caaatctgaa tatgaagcgc acttatcaca 1440gcaagagtat ataaggggaa taagcgcttg gaatttcaat ctcgaggacc taaaaactca 1500agctgccctt atttcagatg atgatacttc acatgctgaa gagcccgatt tcaaccaaaa 1560gcaatgtgaa agacaggatg aatctgctct ttcccctgaa agggctagca gctcagcaac 1620agctcctagt caagatgacg aactgaatga tattcatgat ttagagagtt ctttcgcctc 1680atttccaatc aaacctcttc aagcactaaa aggctgcttt gatatcagtg aggacgagga 1740taatgcaact actcctgatt ggaaagatgc taatgtaaat tctggacaac agcttttaac 1800aaaggcttcc attggatctt tggccgaaac cacgaaagaa gaggacactg cagcacaaaa 1860cacttcttta ccacgtcatg tcatttctga acagaaaaaa tatttgagcg gttcaattat 1920accagagagt actttctctc caaaaagaat cacatctgat gctgataggg agtttcaaca 1980gcgtagatat caaacagagc ggagctacag cggatcatta taccgcacca agagagattc 2040cgtggacgag acgtcagaag tcccgcatgt ggagcacaag ggacggttta aggtcacatc 2100agcagatctg agtcccaagg gatctacaaa ctctacattc acaccattta gtggtggtac 2160aagcagccct agttgcctca atgctacaac cgcctcaatc ctcccatcaa ttcagtcgat 2220tttgcagcaa aatgctatgc aacgggaaga gattttgaga ctaatcaaat acttggagca 2280aacctctgcc aagcaacctg gatcgcctga gacgaacgtc gatgacctat tgcagacgcc 2340tcctgcaacc tcacgagaga gagaacttca gtctcaagtc atgctactac aacaaagctt 2400ttccagccta acagaagaac taaagaaaca gaagcagaaa aatgggcagt tggagaatca 2460gttgaacgca ttaacacaca gaaatgattg agtctcaaaa gccatcgaga caaggctgag 2520agatacaact ggggatcttg agttaaaaaa acacaaaatt ccctttcaag gcaaaaagaa 2580gaaatagaga agatttgtgt gctttatatt tctattgggt gtaatttgtt tgacaggttt 2640atattatgtg acaactacta cagtgatttt cttatttttg gggaagtttt ccccactttt 2700cttttttact tatttgtgtt ttatgatatg ctatgtaaac aaaatactat tgtttaatta 2760tgtttctgtg tg 277212674PRTArabidopsis thaliana 12Met Glu Ser Ser Ser Glu Lys Lys Phe Pro Leu Asn Ala Lys Asp Tyr1 5 10 15Lys Leu Tyr Glu Glu Ile Gly Asp Gly Val Ser Ala Thr Val His Arg 20 25 30Ala Leu Cys Ile Pro Leu Asn Val Val Val Ala Ile Lys Val Leu Asp 35 40 45Leu Glu Lys Cys Asn Asn Asp Leu Asp Gly Ile Arg Arg Glu Val Gln 50 55 60Thr Met Ser Leu Ile Asn His Pro Asn Val Leu Gln Ala His Cys Ser65 70 75 80Phe Thr Thr Gly His Gln Leu Trp Val Val Met Pro Tyr Met Ala Gly 85 90 95Gly Ser Cys Leu His Ile Ile Lys Ser Ser Tyr Pro Asp Gly Phe Glu 100 105 110Glu Pro Val Ile Ala Thr Leu Leu Arg Glu Thr Leu Lys Ala Leu Val 115 120 125Tyr Leu His Ala His Gly His Ile His Arg Asp Val Lys Ala Gly Asn 130 135 140Ile Leu Leu Asp Ser Asn Gly Ala Val Lys Leu Ala Asp Phe Gly Val145 150 155 160Ser Ala Cys Met Phe Asp Thr Gly Asp Arg Gln Arg Ser Arg Asn Thr 165 170 175Phe Val Gly Thr Pro Cys Trp Met Ala Pro Glu Val Met Gln Gln Leu 180 185 190His Gly Tyr Asp Phe Lys Ala Asp Val Trp Ser Phe Gly Ile Thr Ala 195 200 205Leu Glu Leu Ala His Gly His Ala Pro Phe Ser Lys Tyr Pro Pro Met 210 215 220Lys Val Leu Leu Met Thr Leu Gln Asn Ala Pro Pro Gly Leu Asp Tyr225 230 235 240Glu Arg Asp Lys Arg Phe Ser Lys Ala Phe Lys Glu Met Val Gly Thr 245 250 255Cys Leu Val Lys Asp Pro Lys Lys Arg Pro Thr Ser Glu Lys Leu Leu 260 265 270Lys His Pro Phe Phe Lys His Ala Arg Pro Ala Asp Tyr Leu Val Lys 275 280 285Thr Ile Leu Asn Gly Leu Pro Pro Leu Gly Asp Arg Tyr Arg Gln Ile 290 295 300Lys Ser Lys Glu Ala Asp Leu Leu Met Gln Asn Lys Ser Glu Tyr Glu305 310 315 320Ala His Leu Ser Gln Gln Glu Tyr Ile Arg Gly Ile Ser Ala Trp Asn 325 330 335Phe Asn Leu Glu Asp Leu Lys Thr Gln Ala Ala Leu Ile Ser Asp Asp 340 345 350Asp Thr Ser His Ala Glu Glu Pro Asp Phe Asn Gln Lys Gln Cys Glu 355 360 365Arg Gln Asp Glu Ser Ala Leu Ser Pro Glu Arg Ala Ser Ser Ser Ala 370 375 380Thr Ala Pro Ser Gln Asp Asp Glu Leu Asn Asp Ile His Asp Leu Glu385 390 395 400Ser Ser Phe Ala Ser Phe Pro Ile Lys Pro Leu Gln Ala Leu Lys Gly 405 410 415Cys Phe Asp Ile Ser Glu Asp Glu Asp Asn Ala Thr Thr Pro Asp Trp 420 425 430Lys Asp Ala Asn Val Asn Ser Gly Gln Gln Leu Leu Thr Lys Ala Ser 435 440 445Ile Gly Ser Leu Ala Glu Thr Thr Lys Glu Glu Asp Thr Ala Ala Gln 450 455 460Asn Thr Ser Leu Pro Arg His Val Ile Ser Glu Gln Lys Lys Tyr Leu465 470 475 480Ser Gly Ser Ile Ile Pro Glu Ser Thr Phe Ser Pro Lys Arg Ile Thr 485 490 495Ser Asp Ala Asp Arg Glu Phe Gln Gln Arg Arg Tyr Gln Thr Glu Arg 500 505 510Ser Tyr Ser Gly Ser Leu Tyr Arg Thr Lys Arg

Asp Ser Val Asp Glu 515 520 525Thr Ser Glu Val Pro His Val Glu His Lys Gly Arg Phe Lys Val Thr 530 535 540Ser Ala Asp Leu Ser Pro Lys Gly Ser Thr Asn Ser Thr Phe Thr Pro545 550 555 560Phe Ser Gly Gly Thr Ser Ser Pro Ser Cys Leu Asn Ala Thr Thr Ala 565 570 575Ser Ile Leu Pro Ser Ile Gln Ser Ile Leu Gln Gln Asn Ala Met Gln 580 585 590Arg Glu Glu Ile Leu Arg Leu Ile Lys Tyr Leu Glu Gln Thr Ser Ala 595 600 605Lys Gln Pro Gly Ser Pro Glu Thr Asn Val Asp Asp Leu Leu Gln Thr 610 615 620Pro Pro Ala Thr Ser Arg Glu Arg Glu Leu Gln Ser Gln Val Met Leu625 630 635 640Leu Gln Gln Ser Phe Ser Ser Leu Thr Glu Glu Leu Lys Lys Gln Lys 645 650 655Gln Lys Asn Gly Gln Leu Glu Asn Gln Leu Asn Ala Leu Thr His Arg 660 665 670Asn Asp 131785DNAArabidopsis thaliana 13atggctggtt catcaacgaa acgatttcct ctatatgcta aagattatga gctctttgaa 60gaggtaggag aaggtgttag tgctactgtg tatagagctc gttgcattgc tcttaacgag 120attgtcgctg ttaaaatctt ggatctcgaa aaatgcagga atgatttgga aacaatacgc 180aaggaagttc atataatgag tttgattgat catccgaatt tattgaaagc gcattgttcg 240tttatcgaca gtagtagttt gtggattgta atgccttata tgtcgggtgg ttcttgtttt 300catttaatga aatctgtata tccggaaggt cttgagcaac ctataattgc tactttgttg 360agggaagtgc ttaaagctct tgtttatctt catagacaag gtcacatcca tagagatgtt 420aaggctggga atatattgat tcactcaaaa ggcgtagtta aacttggaga ctttggagtt 480tcagcatgta tgtttgatag tggagaaaga atgcaaacaa ggaatacatt cgttgggact 540ccttgttgga tggcacctga ggttatgcag caactagatg gatatgattt caagtatctt 600gctcatggtc atgccccatt ttccaaatat ccacctatga aggtgctact aatgacatta 660caaaatgcac ctcctcgtct agactatgac agagataaga aattctcaaa gtcatttaga 720gagttaatcg cagcgtgctt agttaaagat ccgaaaaagc gtccaactgc agcaaaactt 780ctgaaacatc ctttcttcaa acatgctcgg tctacagatt atttgtcccg taaaattctt 840catggtcttt ctccacttgg tgaacgtttt aaaaagctca aggaggcaga ggctgagttg 900ttcaaaggca taaatggtga caaagaacag ttgtctcagc atgagtatat gagaggaatt 960agtgcttgga actttgatct tgaagcattg agaaggcagg catcacttgt aattattcca 1020aatgaagaaa tctataattc agagatacag gaactgaaca gaaatggaga tgtaccaaaa 1080ggaaaaccag tgatacaaag gtcacagact atgcctttgg aatatttctc agaaaaggca 1140agtgatatgg tgagtgagag tagcagtcaa ttaaccggtt cattacttcc ttcgtttcat 1200cgcaaattcc tcccggctct tggcaatgca tgtaactcga gcgatagagc agcagagaag 1260ctcgcttttg aagagccacg tcaagtacta cacccattag cggatacaaa gaaaattaga 1320aaagcaggaa gtgatcagca ggagaaacca aaaaatggtt acgcagatag tcctgtgaac 1380cgtgaatctt ccacattatc aaaggaacca ttagcggata caaagcaagt tagaaaacca 1440ggaaatgagc aggagaaacc aaaaaacggc tatatagtta gtcatgtgaa ccgtgaatct 1500tccacatcag aggaaatcct cccactgttg cagagtctcc tggttcagaa tgacattcaa 1560agggcgcaag taatcaggtt aattagattt tttgatcgaa ctgcgaaaac ggaaaatcca 1620atctcaaaaa ccgaaggagt gcaggagaaa gatctgcaat ctcaagttca gtttttggag 1680caaagtgttg agaagcttgt agaggaagtt cagagaagaa aagatataaa tagtcagcta 1740gagcaacaga tcagctctct gattagcagc aacaacatct cttaa 178514594PRTArabidopsis thaliana 14Met Ala Gly Ser Ser Thr Lys Arg Phe Pro Leu Tyr Ala Lys Asp Tyr1 5 10 15Glu Leu Phe Glu Glu Val Gly Glu Gly Val Ser Ala Thr Val Tyr Arg 20 25 30Ala Arg Cys Ile Ala Leu Asn Glu Ile Val Ala Val Lys Ile Leu Asp 35 40 45Leu Glu Lys Cys Arg Asn Asp Leu Glu Thr Ile Arg Lys Glu Val His 50 55 60Ile Met Ser Leu Ile Asp His Pro Asn Leu Leu Lys Ala His Cys Ser65 70 75 80Phe Ile Asp Ser Ser Ser Leu Trp Ile Val Met Pro Tyr Met Ser Gly 85 90 95Gly Ser Cys Phe His Leu Met Lys Ser Val Tyr Pro Glu Gly Leu Glu 100 105 110Gln Pro Ile Ile Ala Thr Leu Leu Arg Glu Val Leu Lys Ala Leu Val 115 120 125Tyr Leu His Arg Gln Gly His Ile His Arg Asp Val Lys Ala Gly Asn 130 135 140Ile Leu Ile His Ser Lys Gly Val Val Lys Leu Gly Asp Phe Gly Val145 150 155 160Ser Ala Cys Met Phe Asp Ser Gly Glu Arg Met Gln Thr Arg Asn Thr 165 170 175Phe Val Gly Thr Pro Cys Trp Met Ala Pro Glu Val Met Gln Gln Leu 180 185 190Asp Gly Tyr Asp Phe Lys Tyr Leu Ala His Gly His Ala Pro Phe Ser 195 200 205Lys Tyr Pro Pro Met Lys Val Leu Leu Met Thr Leu Gln Asn Ala Pro 210 215 220Pro Arg Leu Asp Tyr Asp Arg Asp Lys Lys Phe Ser Lys Ser Phe Arg225 230 235 240Glu Leu Ile Ala Ala Cys Leu Val Lys Asp Pro Lys Lys Arg Pro Thr 245 250 255Ala Ala Lys Leu Leu Lys His Pro Phe Phe Lys His Ala Arg Ser Thr 260 265 270Asp Tyr Leu Ser Arg Lys Ile Leu His Gly Leu Ser Pro Leu Gly Glu 275 280 285Arg Phe Lys Lys Leu Lys Glu Ala Glu Ala Glu Leu Phe Lys Gly Ile 290 295 300Asn Gly Asp Lys Glu Gln Leu Ser Gln His Glu Tyr Met Arg Gly Ile305 310 315 320Ser Ala Trp Asn Phe Asp Leu Glu Ala Leu Arg Arg Gln Ala Ser Leu 325 330 335Val Ile Ile Pro Asn Glu Glu Ile Tyr Asn Ser Glu Ile Gln Glu Leu 340 345 350Asn Arg Asn Gly Asp Val Pro Lys Gly Lys Pro Val Ile Gln Arg Ser 355 360 365Gln Thr Met Pro Leu Glu Tyr Phe Ser Glu Lys Ala Ser Asp Met Val 370 375 380Ser Glu Ser Ser Ser Gln Leu Thr Gly Ser Leu Leu Pro Ser Phe His385 390 395 400Arg Lys Phe Leu Pro Ala Leu Gly Asn Ala Cys Asn Ser Ser Asp Arg 405 410 415Ala Ala Glu Lys Leu Ala Phe Glu Glu Pro Arg Gln Val Leu His Pro 420 425 430Leu Ala Asp Thr Lys Lys Ile Arg Lys Ala Gly Ser Asp Gln Gln Glu 435 440 445Lys Pro Lys Asn Gly Tyr Ala Asp Ser Pro Val Asn Arg Glu Ser Ser 450 455 460Thr Leu Ser Lys Glu Pro Leu Ala Asp Thr Lys Gln Val Arg Lys Pro465 470 475 480Gly Asn Glu Gln Glu Lys Pro Lys Asn Gly Tyr Ile Val Ser His Val 485 490 495Asn Arg Glu Ser Ser Thr Ser Glu Glu Ile Leu Pro Leu Leu Gln Ser 500 505 510Leu Leu Val Gln Asn Asp Ile Gln Arg Ala Gln Val Ile Arg Leu Ile 515 520 525Arg Phe Phe Asp Arg Thr Ala Lys Thr Glu Asn Pro Ile Ser Lys Thr 530 535 540Glu Gly Val Gln Glu Lys Asp Leu Gln Ser Gln Val Gln Phe Leu Glu545 550 555 560Gln Ser Val Glu Lys Leu Val Glu Glu Val Gln Arg Arg Lys Asp Ile 565 570 575Asn Ser Gln Leu Glu Gln Gln Ile Ser Ser Leu Ile Ser Ser Asn Asn 580 585 590Ile Ser 151422DNAArabidopsis thaliana 15atgacgagtt caccggaaac gagatttcct ctggttgcga aagattacga gattttagaa 60gagataggcg atggtgttta cagagctcga tgcattctac ttgatgaaat tgtagccatc 120aagatctgga accttgaaaa atgcaccaac gatctggaaa ccataaggaa agaagttcat 180agattgagct taattgatca tccaaatcta ttgagggtgc attgctcttt catagatagt 240agcagcttgt ggattgtgat gccttttatg tcgtgcggct cttccttgaa cataatgaaa 300tcagtctatc caaatggtct tgaggaacct gtaattgcta tattgttgcg ggagattctt 360aaagctcttg tttaccttca tggactagga cacatccatc gaaatgttaa ggctgggaat 420gtactggttg actcagaagg aactgttaag ctcggtgact ttgaagtttc agcatccatg 480tttgatagtg tggaaaggat gcgtactagt tctgagaata cttttgttgg aaatccacgc 540cggatggcac ctgagaagga tatgcagcaa gttgatggct atgatttcaa agtggatatc 600tggtcgtttg gcatgactgc cctggaactt gcccatggtc attcacctac cacggtgcta 660ccattgaact tacaaaattc tccctttcct aactatgaag aagacacgaa attctctaag 720tcttttagag agttggtcgc agcttgcttg atagaagatc cagaaaaacg tccgaccgct 780tcacaactac tggaatatcc gttcttacag caaactcttt ctactgaata cttggctagt 840acatttcttg atggcctctc tccgcttggt gagcgttata gaaagctgaa ggaggaaaag 900gccaagttgg ttaaaggtgt agatggtaac aaggagaaag tatctcagga aaatgttgaa 960gcgctgctga tggaacctgc tagtcttgtg aaccctgttt cttgtgatac tgctcaagtc 1020ctcccaatct tacagaatat cctgatccaa aatgatatcc aaagggaaaa tgttgaagcg 1080ctgctgacgg aacctgctat tcttgtgaac cctgtttctt gtgatactgc tcaagtcctc 1140ccaatcgtac agaatatcct gatccagaat gatatccaaa ggaaaaggtt aatcggttta 1200atgcaactct gtgatccaac tgctggtaag tttgctgttc tatcactaga atttgcatct 1260tctctatgtt acaagttcca tgacctgatc ttgatttttg tacagaaatc agaattccga 1320ttggcaatac agaagttggg cagatatcaa caacagagac agatctattg tctgaggttc 1380acgttttgca gcagaggtaa tgataaattc cacaagcttt aa 142216473PRTArabidopsis thaliana 16Met Thr Ser Ser Pro Glu Thr Arg Phe Pro Leu Val Ala Lys Asp Tyr1 5 10 15Glu Ile Leu Glu Glu Ile Gly Asp Gly Val Tyr Arg Ala Arg Cys Ile 20 25 30Leu Leu Asp Glu Ile Val Ala Ile Lys Ile Trp Asn Leu Glu Lys Cys 35 40 45Thr Asn Asp Leu Glu Thr Ile Arg Lys Glu Val His Arg Leu Ser Leu 50 55 60Ile Asp His Pro Asn Leu Leu Arg Val His Cys Ser Phe Ile Asp Ser65 70 75 80Ser Ser Leu Trp Ile Val Met Pro Phe Met Ser Cys Gly Ser Ser Leu 85 90 95Asn Ile Met Lys Ser Val Tyr Pro Asn Gly Leu Glu Glu Pro Val Ile 100 105 110Ala Ile Leu Leu Arg Glu Ile Leu Lys Ala Leu Val Tyr Leu His Gly 115 120 125Leu Gly His Ile His Arg Asn Val Lys Ala Gly Asn Val Leu Val Asp 130 135 140Ser Glu Gly Thr Val Lys Leu Gly Asp Phe Glu Val Ser Ala Ser Met145 150 155 160Phe Asp Ser Val Glu Arg Met Arg Thr Ser Ser Glu Asn Thr Phe Val 165 170 175Gly Asn Pro Arg Arg Met Ala Pro Glu Lys Asp Met Gln Gln Val Asp 180 185 190Gly Tyr Asp Phe Lys Val Asp Ile Trp Ser Phe Gly Met Thr Ala Leu 195 200 205Glu Leu Ala His Gly His Ser Pro Thr Thr Val Leu Pro Leu Asn Leu 210 215 220Gln Asn Ser Pro Phe Pro Asn Tyr Glu Glu Asp Thr Lys Phe Ser Lys225 230 235 240Ser Phe Arg Glu Leu Val Ala Ala Cys Leu Ile Glu Asp Pro Glu Lys 245 250 255Arg Pro Thr Ala Ser Gln Leu Leu Glu Tyr Pro Phe Leu Gln Gln Thr 260 265 270Leu Ser Thr Glu Tyr Leu Ala Ser Thr Phe Leu Asp Gly Leu Ser Pro 275 280 285Leu Gly Glu Arg Tyr Arg Lys Leu Lys Glu Glu Lys Ala Lys Leu Val 290 295 300Lys Gly Val Asp Gly Asn Lys Glu Lys Val Ser Gln Glu Asn Val Glu305 310 315 320Ala Leu Leu Met Glu Pro Ala Ser Leu Val Asn Pro Val Ser Cys Asp 325 330 335Thr Ala Gln Val Leu Pro Ile Leu Gln Asn Ile Leu Ile Gln Asn Asp 340 345 350Ile Gln Arg Glu Asn Val Glu Ala Leu Leu Thr Glu Pro Ala Ile Leu 355 360 365Val Asn Pro Val Ser Cys Asp Thr Ala Gln Val Leu Pro Ile Val Gln 370 375 380Asn Ile Leu Ile Gln Asn Asp Ile Gln Arg Lys Arg Leu Ile Gly Leu385 390 395 400Met Gln Leu Cys Asp Pro Thr Ala Gly Lys Phe Ala Val Leu Ser Leu 405 410 415Glu Phe Ala Ser Ser Leu Cys Tyr Lys Phe His Asp Leu Ile Leu Ile 420 425 430Phe Val Gln Lys Ser Glu Phe Arg Leu Ala Ile Gln Lys Leu Gly Arg 435 440 445Tyr Gln Gln Gln Arg Gln Ile Tyr Cys Leu Arg Phe Thr Phe Cys Ser 450 455 460Arg Gly Asn Asp Lys Phe His Lys Leu465 470172085DNAArabidopsis thaliana 17atggagaaga agaagtatcc aattggacca gagcattata ctctctacga gtttattgga 60caaggtgtta gtgctctagt gcatcgtgct ttgtgcattc cgtttgatga agtcgttgct 120attaagattc ttgattttga acgcgataac tgcgatctga acaacatctc tcgtgaagcg 180cagacgatga tgcttgttga tcatcccaat gtgttgaagt cacattgttc ctttgttagt 240gatcacaatt tgtgggtcat catgccatac atgtctggtg gttcttgtct tcacattcta 300aaagctgcat atcctgatgg ttttgaagaa gctattatag ctactatatt gcgtgaagct 360ttgaagggat tagactatct ccatcagcat ggccacattc atcgcgatgt caaagctggg 420aatatattgc ttggtgctcg aggtgcagtc aagttgggag actttggtgt atctgcctgt 480ctctttgatt caggtgatag gcaacggaca aggaacacat ttgttggaac accttgctgg 540atggcacctg aagtcatgga gcagctacat ggttatgact tcaaggctga tatttggtcg 600tttggtataa ctgggctaga gcttgctcat ggtcacgctc ctttctctaa atatccacca 660atgaaggttc tgcttatgac gttgcaaaat gcaccaccag ggctggatta cgaaagagat 720aagaagtttt ccaggtcttt caagcagatg atcgccagtt gtctagttaa agacccttcc 780aaacgcccgt ctgcaaaaaa gttgttaaag cactcctttt tcaagcaagc aagatcaagc 840gattacattg cacgaaaact tctggatggg ttaccagatc ttgttaatcg tgttcaggca 900ataaagagaa aggaagaaga tatgcttgca caagagaaaa tggcagatgg agaaaaggaa 960gaattgtccc agcctttaaa cgcttgtcat agtaccatgc agaatgaata taagagaggt 1020atcagcgggt ggaatttcaa tcttgatgat atgaaagccc aggcttcatt gatccaggac 1080atggactgtg gcttttcgga cagtttatcg ggaagtgcaa cttcgttgca ggctctagat 1140tcacaggata cacaatcgga gattcaggag gatactggtc aaataactaa taagtatctc 1200caacctctga ttcaccgaag tctaagtatc gcgagggata aatctgatga tgattcaagt 1260cttgccagcc ccagttatga tagctacgta tattcctccc cccgtcatga ggatttatct 1320ttaaacaata cacatgttgg tagtacgcat gcaaacaatg ggaaaccaac ggatgccaca 1380tcaatcccaa ccaatcaacc aacagagatt atagcaggga gctctgtttt ggcagatgga 1440aatggtgctc ccaataaagg agagagtgat aaaactcaag aacagcttca aaacgggtca 1500aactgcaatg ggacacatcc tacagtggga ggagatgacg taccaacgga gatggctgtt 1560aaaccaccca aagcagcatc aagcctagat gaatctgatg acaaatcaaa gccgccagtt 1620gtgcagcaaa gagggcgttt taaagtaact tctgaaaatc tcgacatcga gaaggtggtg 1680gcgccttcgc caatactgca aaagagtcac agcatgcagg tgctctgcca acattcctct 1740gcttctctac ctcactctga tgtcacattg ccaaacctaa ccagctcata tgtttacccg 1800ctggtgtatc cagttctgca aactaatatt ttggaaaggg ataacatttt gcatatgatg 1860aaagtactca ccaacagaga gttgacagat ggacgtgcag ttgaacaagg aagtatacaa 1920caacctactg tgcccccaac tgagaaatcc atgcttgaag cagcacacga aagagagaaa 1980gaactgctcc atgacataac cgacctgcaa tggaggctca tttgtgcaga agaagagctt 2040cagaaataca aaaccgaaca cgcccaagta agtatgagta actaa 208518694PRTArabidopsis thaliana 18Met Glu Lys Lys Lys Tyr Pro Ile Gly Pro Glu His Tyr Thr Leu Tyr1 5 10 15Glu Phe Ile Gly Gln Gly Val Ser Ala Leu Val His Arg Ala Leu Cys 20 25 30Ile Pro Phe Asp Glu Val Val Ala Ile Lys Ile Leu Asp Phe Glu Arg 35 40 45Asp Asn Cys Asp Leu Asn Asn Ile Ser Arg Glu Ala Gln Thr Met Met 50 55 60Leu Val Asp His Pro Asn Val Leu Lys Ser His Cys Ser Phe Val Ser65 70 75 80Asp His Asn Leu Trp Val Ile Met Pro Tyr Met Ser Gly Gly Ser Cys 85 90 95Leu His Ile Leu Lys Ala Ala Tyr Pro Asp Gly Phe Glu Glu Ala Ile 100 105 110Ile Ala Thr Ile Leu Arg Glu Ala Leu Lys Gly Leu Asp Tyr Leu His 115 120 125Gln His Gly His Ile His Arg Asp Val Lys Ala Gly Asn Ile Leu Leu 130 135 140Gly Ala Arg Gly Ala Val Lys Leu Gly Asp Phe Gly Val Ser Ala Cys145 150 155 160Leu Phe Asp Ser Gly Asp Arg Gln Arg Thr Arg Asn Thr Phe Val Gly 165 170 175Thr Pro Cys Trp Met Ala Pro Glu Val Met Glu Gln Leu His Gly Tyr 180 185 190Asp Phe Lys Ala Asp Ile Trp Ser Phe Gly Ile Thr Gly Leu Glu Leu 195 200 205Ala His Gly His Ala Pro Phe Ser Lys Tyr Pro Pro Met Lys Val Leu 210 215 220Leu Met Thr Leu Gln Asn Ala Pro Pro Gly Leu Asp Tyr Glu Arg Asp225 230 235 240Lys Lys Phe Ser Arg Ser Phe Lys Gln Met Ile Ala Ser Cys Leu Val 245 250 255Lys Asp Pro Ser Lys Arg Pro Ser Ala Lys Lys Leu Leu Lys His Ser 260 265 270Phe Phe Lys Gln Ala Arg Ser Ser Asp Tyr Ile Ala Arg Lys Leu Leu 275 280 285Asp Gly Leu Pro Asp Leu Val Asn Arg Val Gln Ala Ile Lys Arg Lys 290 295 300Glu Glu Asp Met Leu Ala Gln Glu Lys Met Ala Asp Gly Glu Lys Glu305 310 315 320Glu Leu Ser Gln Pro Leu Asn Ala Cys His Ser Thr Met Gln Asn Glu 325 330 335Tyr Lys Arg Gly Ile Ser Gly Trp Asn Phe Asn Leu Asp Asp Met Lys

340 345 350Ala Gln Ala Ser Leu Ile Gln Asp Met Asp Cys Gly Phe Ser Asp Ser 355 360 365Leu Ser Gly Ser Ala Thr Ser Leu Gln Ala Leu Asp Ser Gln Asp Thr 370 375 380Gln Ser Glu Ile Gln Glu Asp Thr Gly Gln Ile Thr Asn Lys Tyr Leu385 390 395 400Gln Pro Leu Ile His Arg Ser Leu Ser Ile Ala Arg Asp Lys Ser Asp 405 410 415Asp Asp Ser Ser Leu Ala Ser Pro Ser Tyr Asp Ser Tyr Val Tyr Ser 420 425 430Ser Pro Arg His Glu Asp Leu Ser Leu Asn Asn Thr His Val Gly Ser 435 440 445Thr His Ala Asn Asn Gly Lys Pro Thr Asp Ala Thr Ser Ile Pro Thr 450 455 460Asn Gln Pro Thr Glu Ile Ile Ala Gly Ser Ser Val Leu Ala Asp Gly465 470 475 480Asn Gly Ala Pro Asn Lys Gly Glu Ser Asp Lys Thr Gln Glu Gln Leu 485 490 495Gln Asn Gly Ser Asn Cys Asn Gly Thr His Pro Thr Val Gly Gly Asp 500 505 510Asp Val Pro Thr Glu Met Ala Val Lys Pro Pro Lys Ala Ala Ser Ser 515 520 525Leu Asp Glu Ser Asp Asp Lys Ser Lys Pro Pro Val Val Gln Gln Arg 530 535 540Gly Arg Phe Lys Val Thr Ser Glu Asn Leu Asp Ile Glu Lys Val Val545 550 555 560Ala Pro Ser Pro Ile Leu Gln Lys Ser His Ser Met Gln Val Leu Cys 565 570 575Gln His Ser Ser Ala Ser Leu Pro His Ser Asp Val Thr Leu Pro Asn 580 585 590Leu Thr Ser Ser Tyr Val Tyr Pro Leu Val Tyr Pro Val Leu Gln Thr 595 600 605Asn Ile Leu Glu Arg Asp Asn Ile Leu His Met Met Lys Val Leu Thr 610 615 620Asn Arg Glu Leu Thr Asp Gly Arg Ala Val Glu Gln Gly Ser Ile Gln625 630 635 640Gln Pro Thr Val Pro Pro Thr Glu Lys Ser Met Leu Glu Ala Ala His 645 650 655Glu Arg Glu Lys Glu Leu Leu His Asp Ile Thr Asp Leu Gln Trp Arg 660 665 670Leu Ile Cys Ala Glu Glu Glu Leu Gln Lys Tyr Lys Thr Glu His Ala 675 680 685Gln Val Ser Met Ser Asn 690192591DNAArabidopsis thaliana 19gtcacacaag ccgaatccaa aaatgtaaca agaaaaacaa atcttcacaa ggcaaaaaat 60ccaaaattga gttttttttt tcttcatttt ttacaatggt gtctcggttt cgtcttgctc 120tagaggctgt tttgggttcg agacgacgta agaagatggc gagtactagt agtggtggtg 180gtggtggtgg tgataagaag aagaagaaag gtttctctgt aaaccctaaa gattataaac 240ttatggaaga agttggatat ggtgctagtg ctgttgttca tcgtgctatt tatcttccta 300ctaatgaagt tgttgctatc aagtctttgg atctcgatcg ctgcaatagt aatctggatg 360atataaggag ggaggctcag actatgactt tgatagacca tccgaatgtt ataaagtcgt 420tttgttcgtt tgctgttgat catcatctat gggtcgttat gccatttatg gctcagggtt 480cgtgtttgca tctaatgaaa gcagcgtatc cagatggatt tgaagaggcg gctatatgtt 540ctatgctgaa agaaacactt aaagctcttg attatcttca tagacaaggg catatccatc 600gagatgttaa ggctggaaac atacttcttg atgacactgg cgagattaag ttaggtgatt 660ttggtgtctc tgcatgtttg tttgacaatg gcgataggca acgtgcaaga aatacatttg 720ttggtactcc atgctggatg gcaccggaag tcttgcagcc agggagtgga tacaattcaa 780aggctgatat atggtctttt ggaataacgg cgctggagtt ggctcacggt catgcacctt 840tctcaaaata tccccctatg aaggtactct taatgactat ccaaaatgca ccacctggcc 900ttgattatga ccgtgataag aagttttcaa agtcctttaa agaattggta gcattgtgtc 960tggtgaaaga tcaaacaaaa aggccaactg ctgaaaaatt gttgaaacac tcatttttca 1020agaatgtgaa gcctccagag atctgtgtaa aaaaattatt tgtcgattta ccacctcttt 1080ggactcgcgt aaaagctctt caggccaagg atgctgcaca gcttgctttg aaaggaatgg 1140cctctgctga ccaggatgct atatcacaga gtgaatacca aagaggagta agtgcttgga 1200acttcaatat cgaagatttg aaagaacaag catctttgct agatgatgat gacattctaa 1260cagagagtag ggaagaagaa gaatcttttg gcgaacagtt gcataataag gctaggcaag 1320tatctggtag tcaattgcta tctgaaaaca tgaatggaaa ggaaaaagct tcagatactg 1380aggtggtaga acctatctgt gaagagaaat ccactctcaa ttcaaccact tcttctgtgg 1440aacaaccggc atcaagttca gaacaagacg ttccacaggc caagggtaag ccagtgagac 1500tccagactca tagtggacca ctttcatccg gtgtcgtgtt aatcaattca gactcagaga 1560aggttcatgg ttatgaaagg tctgagagtg aacggcaact gaaatcatca gtccggaggg 1620cacccagctt tagtggtcct ttgaatcttc caaatcgtgc ttcagcaaac agtctttcag 1680ctcctatcaa atcttctgga ggatttcgtg attctataga tgacaagtcg aaggctaatg 1740tggttcaaat caaaggaaga ttttcagtaa catcagaaaa cttggatctt gcaagggcat 1800cccctttgag aaaatctgcc agtgttggga attggatact tgattctaaa atgccaacgg 1860gccaggccat caaggagtca agtagtcatc tctcattcat tatacctcag cttcaaaatc 1920tgttccagca aaattcaatg cagcaggatc ttattatgaa tctagtgaat accttacaac 1980aagctgctga aacaacagat ggttctcaaa atggaaagtt gccgcctttg cctcgaggat 2040ctgacagcaa tggaaccgtt gtagaactta cagcagctga gcgagagagg ttactactta 2100ccaagataac cgagcttcga gctaggatga aagagttgac ggaagaactt gaagtagaaa 2160aatcaaaaca gacccaactg cagcagaaat tgaaatcagt caccggtcgc gagcaattgt 2220aatcagagac cgggaacact gaccttacta cagagaagct ttttaggagg agagaaaagt 2280atgttttgta cactaagaaa accagagagc tctctgatca tgaaagcaaa aaggacaggt 2340ttggttctgt tctgtataag tgcagaagca gagtcaccat cggccatttg tttctgacag 2400aagaagccgg aaacaaaaac agatagagag agataaatag agaagaaagc tctttggcca 2460tggaaaattg tatttgttta tttaatctaa acactacaaa actttacatt ttttattatt 2520gttagcaaca aatatagact cttccttttt tgtgtggatt gtaaatgaaa cattatttga 2580tgtatttgtt t 259120708PRTArabidopsis thaliana 20Met Val Ser Arg Phe Arg Leu Ala Leu Glu Ala Val Leu Gly Ser Arg1 5 10 15Arg Arg Lys Lys Met Ala Ser Thr Ser Ser Gly Gly Gly Gly Gly Gly 20 25 30Asp Lys Lys Lys Lys Lys Gly Phe Ser Val Asn Pro Lys Asp Tyr Lys 35 40 45Leu Met Glu Glu Val Gly Tyr Gly Ala Ser Ala Val Val His Arg Ala 50 55 60Ile Tyr Leu Pro Thr Asn Glu Val Val Ala Ile Lys Ser Leu Asp Leu65 70 75 80Asp Arg Cys Asn Ser Asn Leu Asp Asp Ile Arg Arg Glu Ala Gln Thr 85 90 95Met Thr Leu Ile Asp His Pro Asn Val Ile Lys Ser Phe Cys Ser Phe 100 105 110Ala Val Asp His His Leu Trp Val Val Met Pro Phe Met Ala Gln Gly 115 120 125Ser Cys Leu His Leu Met Lys Ala Ala Tyr Pro Asp Gly Phe Glu Glu 130 135 140Ala Ala Ile Cys Ser Met Leu Lys Glu Thr Leu Lys Ala Leu Asp Tyr145 150 155 160Leu His Arg Gln Gly His Ile His Arg Asp Val Lys Ala Gly Asn Ile 165 170 175Leu Leu Asp Asp Thr Gly Glu Ile Lys Leu Gly Asp Phe Gly Val Ser 180 185 190Ala Cys Leu Phe Asp Asn Gly Asp Arg Gln Arg Ala Arg Asn Thr Phe 195 200 205Val Gly Thr Pro Cys Trp Met Ala Pro Glu Val Leu Gln Pro Gly Ser 210 215 220Gly Tyr Asn Ser Lys Ala Asp Ile Trp Ser Phe Gly Ile Thr Ala Leu225 230 235 240Glu Leu Ala His Gly His Ala Pro Phe Ser Lys Tyr Pro Pro Met Lys 245 250 255Val Leu Leu Met Thr Ile Gln Asn Ala Pro Pro Gly Leu Asp Tyr Asp 260 265 270Arg Asp Lys Lys Phe Ser Lys Ser Phe Lys Glu Leu Val Ala Leu Cys 275 280 285Leu Val Lys Asp Gln Thr Lys Arg Pro Thr Ala Glu Lys Leu Leu Lys 290 295 300His Ser Phe Phe Lys Asn Val Lys Pro Pro Glu Ile Cys Val Lys Lys305 310 315 320Leu Phe Val Asp Leu Pro Pro Leu Trp Thr Arg Val Lys Ala Leu Gln 325 330 335Ala Lys Asp Ala Ala Gln Leu Ala Leu Lys Gly Met Ala Ser Ala Asp 340 345 350Gln Asp Ala Ile Ser Gln Ser Glu Tyr Gln Arg Gly Val Ser Ala Trp 355 360 365Asn Phe Asn Ile Glu Asp Leu Lys Glu Gln Ala Ser Leu Leu Asp Asp 370 375 380Asp Asp Ile Leu Thr Glu Ser Arg Glu Glu Glu Glu Ser Phe Gly Glu385 390 395 400Gln Leu His Asn Lys Ala Arg Gln Val Ser Gly Ser Gln Leu Leu Ser 405 410 415Glu Asn Met Asn Gly Lys Glu Lys Ala Ser Asp Thr Glu Val Val Glu 420 425 430Pro Ile Cys Glu Glu Lys Ser Thr Leu Asn Ser Thr Thr Ser Ser Val 435 440 445Glu Gln Pro Ala Ser Ser Ser Glu Gln Asp Val Pro Gln Ala Lys Gly 450 455 460Lys Pro Val Arg Leu Gln Thr His Ser Gly Pro Leu Ser Ser Gly Val465 470 475 480Val Leu Ile Asn Ser Asp Ser Glu Lys Val His Gly Tyr Glu Arg Ser 485 490 495Glu Ser Glu Arg Gln Leu Lys Ser Ser Val Arg Arg Ala Pro Ser Phe 500 505 510Ser Gly Pro Leu Asn Leu Pro Asn Arg Ala Ser Ala Asn Ser Leu Ser 515 520 525Ala Pro Ile Lys Ser Ser Gly Gly Phe Arg Asp Ser Ile Asp Asp Lys 530 535 540Ser Lys Ala Asn Val Val Gln Ile Lys Gly Arg Phe Ser Val Thr Ser545 550 555 560Glu Asn Leu Asp Leu Ala Arg Ala Ser Pro Leu Arg Lys Ser Ala Ser 565 570 575Val Gly Asn Trp Ile Leu Asp Ser Lys Met Pro Thr Gly Gln Ala Ile 580 585 590Lys Glu Ser Ser Ser His Leu Ser Phe Ile Ile Pro Gln Leu Gln Asn 595 600 605Leu Phe Gln Gln Asn Ser Met Gln Gln Asp Leu Ile Met Asn Leu Val 610 615 620Asn Thr Leu Gln Gln Ala Ala Glu Thr Thr Asp Gly Ser Gln Asn Gly625 630 635 640Lys Leu Pro Pro Leu Pro Arg Gly Ser Asp Ser Asn Gly Thr Val Val 645 650 655Glu Leu Thr Ala Ala Glu Arg Glu Arg Leu Leu Leu Thr Lys Ile Thr 660 665 670Glu Leu Arg Ala Arg Met Lys Glu Leu Thr Glu Glu Leu Glu Val Glu 675 680 685Lys Ser Lys Gln Thr Gln Leu Gln Gln Lys Leu Lys Ser Val Thr Gly 690 695 700Arg Glu Gln Leu705212782DNAArabidopsis thaliana 21aaactactag tctctatctc tctcagctcc agattttgtt tcttcttctt ctgtgttaaa 60ttcatttgat ttgttgtatc tgaaggcgaa attactggtt tctgattttt ggtggtattc 120agggcggttt taaagcgacg gaagaagatg gtgggaggag gaggaggtag tagtggtcgt 180ggtggtggta gtggtagtgg tagtagtaag cagcagagag gtttctctat gaatcctaaa 240gactataagc taatggaaga aataggccat ggagctagcg ctgttgtcta tcgagcgatc 300tatctcccta ctaatgaagt cgtcgccatc aagtgtttgg atctcgatcg ctgcaatagc 360aatctggatg atattaggag ggaatctcag actatgagtt tgatagacca tcccaacgtt 420ataaagtcgt tttgttcatt ctctgtcgac catagtcttt gggttgttat gccattcatg 480gctcaaggtt cgtgtttgca tcttatgaag actgcgtatt cagacggatt tgaagagtct 540gctatatgtt gtgtattaaa agaaactctt aaagctcttg attatcttca tagacaaggc 600catatccatc gggatgttaa ggctggaaac atacttcttg atgacaatgg tgagattaag 660cttggcgatt ttggtgtctc tgcttgcttg tttgataacg gtgataggca acgtgctaga 720aacacatttg ttggtactcc ttgctggatg gcaccggaag ttttgcagcc gggaaatgga 780tacaattcca aggctgatat ctggtcattt ggtataacag cacttgaatt ggcccatggt 840catgcacctt tctcaaaata tcctcccatg aaggtgctcc taatgactat tcaaaacgca 900cctcctggcc ttgattatga ccgtgataag aaattttcta agtcctttaa agaaatggtt 960gcaatgtgtt tggtgaaaga tcaaacaaaa aggccaactg ctgaaaaact gctgaagcac 1020tcctgtttca aacacacgaa gcctccagag caaactgtga aaattttatt ttccgattta 1080ccacctcttt ggacacgtgt aaaatctctt caggataagg atgctcaaca gcttgcatta 1140aagagaatgg ccactgctga cgaggaagct atatcacaga gcgaatacca aagaggagtg 1200agcgcttgga actttgacgt cagagacttg aaaacacaag catctttgtt aattgatgat 1260gatgatctag aagagagtaa ggaagatgaa gaaatattat gtgcacagtt taataaggtg 1320aatgacagag agcaagtatt tgatagtctg caactatatg aaaacatgaa cggaaaagaa 1380aaggtttcca atactgaggt ggaagaacca acctgcaaag agaaattcac tttcgttaca 1440actacttctt ctttagaacg aatgtcacca aattcagagc atgacattcc cgaggccaag 1500gttaagccat taagacgcca aagtcagagt ggaccactta caagcaggac tgtattaagc 1560cactcggctt cagagaaaag tcatatcttt gaaagatccg agagtgaacc gcagacggca 1620ccaacagtcc gaagagcacc cagctttagt ggtcctttga atctttcaac ccgtgcttct 1680tcaaacagtt tgtctgctcc catcaaatac tcaggaggat tccgtgattc tctggatgat 1740aagtcaaagg ctaatctggt tcagaaagga cgattttcag taacatcagg aaatgtagat 1800cttgcgaagg atgttccatt aagtatagtc cctcgtcgat ctccacaggc gacccccctg 1860agaaaatctg caagtgtggg taactggata cttgagccca aaatgccaac agctcagcct 1920cagacgatca aggagcatag tagccatcct acgtcttcct cacccatcat gcctcaactt 1980caacatctat tccagcaaaa ctcaatacaa caggatctta ttatgaattt actaaatagc 2040ttacaacccg tggaggcaac agaaggttct caatctggga agttaccacc tttgcctcgc 2100tcagacagta atggaaacgt tgaacctgtg gcttcagaga gggagaggtt acttcttagc 2160agtatctccg acctccgtgc taggctggac gacttaacgg aggaactcga tatagagaaa 2220tcaaaataca gccaactgca acagaaattg aaagcattca cgggtcgcga acactaagtg 2280taaccagagg gaaagcgaca ctggaaacac tgaactgcac agaacctgta ggagaaagag 2340tgaagtctct tttggttata acagtaataa ccagacaaga gcttagagac agtgaggcat 2400agagcatatc aatttcttta gttgggttca gtgtaggttc cagacgatga caatgacgac 2460taaaacaaga tacgaccgat gtctgcttct gatgtaaact actagttgaa gacaacagaa 2520acgaatacag aaataaaaga aaaggagaag aaagttcctt tggggggtct caaccccaca 2580tatatttgct tatatattta ttatcacacg ttttgatcat tttttgtttt attttgtttg 2640gtgtatcata atttactagt gagataaagg agaaagctct tcttttgggt tctttgtgta 2700ttgtaatttg taaatgcaaa ttgattgatg tacttttgtg ttttcatcac attcttaaac 2760attatcttct ggttttacct ta 278222709PRTArabidopsis thaliana 22Met Val Gly Gly Gly Gly Gly Ser Ser Gly Arg Gly Gly Gly Ser Gly1 5 10 15Ser Gly Ser Ser Lys Gln Gln Arg Gly Phe Ser Met Asn Pro Lys Asp 20 25 30Tyr Lys Leu Met Glu Glu Ile Gly His Gly Ala Ser Ala Val Val Tyr 35 40 45Arg Ala Ile Tyr Leu Pro Thr Asn Glu Val Val Ala Ile Lys Cys Leu 50 55 60Asp Leu Asp Arg Cys Asn Ser Asn Leu Asp Asp Ile Arg Arg Glu Ser65 70 75 80Gln Thr Met Ser Leu Ile Asp His Pro Asn Val Ile Lys Ser Phe Cys 85 90 95Ser Phe Ser Val Asp His Ser Leu Trp Val Val Met Pro Phe Met Ala 100 105 110Gln Gly Ser Cys Leu His Leu Met Lys Thr Ala Tyr Ser Asp Gly Phe 115 120 125Glu Glu Ser Ala Ile Cys Cys Val Leu Lys Glu Thr Leu Lys Ala Leu 130 135 140Asp Tyr Leu His Arg Gln Gly His Ile His Arg Asp Val Lys Ala Gly145 150 155 160Asn Ile Leu Leu Asp Asp Asn Gly Glu Ile Lys Leu Gly Asp Phe Gly 165 170 175Val Ser Ala Cys Leu Phe Asp Asn Gly Asp Arg Gln Arg Ala Arg Asn 180 185 190Thr Phe Val Gly Thr Pro Cys Trp Met Ala Pro Glu Val Leu Gln Pro 195 200 205Gly Asn Gly Tyr Asn Ser Lys Ala Asp Ile Trp Ser Phe Gly Ile Thr 210 215 220Ala Leu Glu Leu Ala His Gly His Ala Pro Phe Ser Lys Tyr Pro Pro225 230 235 240Met Lys Val Leu Leu Met Thr Ile Gln Asn Ala Pro Pro Gly Leu Asp 245 250 255Tyr Asp Arg Asp Lys Lys Phe Ser Lys Ser Phe Lys Glu Met Val Ala 260 265 270Met Cys Leu Val Lys Asp Gln Thr Lys Arg Pro Thr Ala Glu Lys Leu 275 280 285Leu Lys His Ser Cys Phe Lys His Thr Lys Pro Pro Glu Gln Thr Val 290 295 300Lys Ile Leu Phe Ser Asp Leu Pro Pro Leu Trp Thr Arg Val Lys Ser305 310 315 320Leu Gln Asp Lys Asp Ala Gln Gln Leu Ala Leu Lys Arg Met Ala Thr 325 330 335Ala Asp Glu Glu Ala Ile Ser Gln Ser Glu Tyr Gln Arg Gly Val Ser 340 345 350Ala Trp Asn Phe Asp Val Arg Asp Leu Lys Thr Gln Ala Ser Leu Leu 355 360 365Ile Asp Asp Asp Asp Leu Glu Glu Ser Lys Glu Asp Glu Glu Ile Leu 370 375 380Cys Ala Gln Phe Asn Lys Val Asn Asp Arg Glu Gln Val Phe Asp Ser385 390 395 400Leu Gln Leu Tyr Glu Asn Met Asn Gly Lys Glu Lys Val Ser Asn Thr 405 410 415Glu Val Glu Glu Pro Thr Cys Lys Glu Lys Phe Thr Phe Val Thr Thr 420 425 430Thr Ser Ser Leu Glu Arg Met Ser Pro Asn Ser Glu His Asp Ile Pro 435 440 445Glu Ala Lys Val Lys Pro Leu Arg Arg Gln Ser Gln Ser Gly Pro Leu 450 455 460Thr Ser Arg Thr Val Leu Ser His Ser Ala Ser Glu Lys Ser His Ile465 470 475 480Phe Glu Arg Ser Glu Ser Glu Pro Gln Thr Ala Pro Thr Val Arg Arg 485 490 495Ala Pro Ser Phe Ser Gly Pro Leu Asn Leu Ser Thr Arg Ala Ser Ser 500

505 510Asn Ser Leu Ser Ala Pro Ile Lys Tyr Ser Gly Gly Phe Arg Asp Ser 515 520 525Leu Asp Asp Lys Ser Lys Ala Asn Leu Val Gln Lys Gly Arg Phe Ser 530 535 540Val Thr Ser Gly Asn Val Asp Leu Ala Lys Asp Val Pro Leu Ser Ile545 550 555 560Val Pro Arg Arg Ser Pro Gln Ala Thr Pro Leu Arg Lys Ser Ala Ser 565 570 575Val Gly Asn Trp Ile Leu Glu Pro Lys Met Pro Thr Ala Gln Pro Gln 580 585 590Thr Ile Lys Glu His Ser Ser His Pro Thr Ser Ser Ser Pro Ile Met 595 600 605Pro Gln Leu Gln His Leu Phe Gln Gln Asn Ser Ile Gln Gln Asp Leu 610 615 620Ile Met Asn Leu Leu Asn Ser Leu Gln Pro Val Glu Ala Thr Glu Gly625 630 635 640Ser Gln Ser Gly Lys Leu Pro Pro Leu Pro Arg Ser Asp Ser Asn Gly 645 650 655Asn Val Glu Pro Val Ala Ser Glu Arg Glu Arg Leu Leu Leu Ser Ser 660 665 670Ile Ser Asp Leu Arg Ala Arg Leu Asp Asp Leu Thr Glu Glu Leu Asp 675 680 685Ile Glu Lys Ser Lys Tyr Ser Gln Leu Gln Gln Lys Leu Lys Ala Phe 690 695 700Thr Gly Arg Glu His705232286DNAOryza sativa 23atggccaagg cgtgggagaa ggtggcgacg gcggcggggt tgggtgggtc gggggagagg 60cgcaagtacc cgatccgcgt ggaggactac gagctgtacg aggagatcgg gcagggggtc 120agcgccatcg tgtaccgatc gctctgcaag cccctcgacg agatcgtcgc cgtcaaggtg 180ctcgacttcg agcgcacaaa cagtgacctg tggttagttg taatgcaagt aggttatact 240cggattgttg cgatttacgt accgccgctt gatctgtcta aaatgatagt aacacggata 300tgcttgacgc agaacaacat catgcgtgaa gctcagacga tgattctcat agatcagcct 360aacgtcatga aggcacattg ttcatttaca aataaccact cgttgtgggt ggtcatgcca 420tacatggctg gagggtcttg ccttcacata atgaagtcag tctatccaga tggttttgaa 480gaagctgtca ttgcaactgt acttcgtgaa gtcctgaaag gtttggagta ccttcatcat 540catgggcata tacatcgtga tgtgaaggca gggaatatac ttgttgattc acggggtgta 600gtcaagcttg gagattttgg ggtttctgct tgcctttttg attctggtga caggcaacgg 660gctaggaata ctttcgtggg aactccttgc tggatggcac cagaggttat ggagcagcta 720catggatacg atttcaaggc agacatatgg tccttcggaa ttactgcact tgaacttgcc 780catggtcatg ctcctttctc gaagttccct cccatgaagg tcttacttat gacacttcag 840aatgcccctc cgggccttga ctatgagaga gataagaaat tttcaaggca tttcaagcaa 900atggttgcta tgtgtctggt aaaagaccct tcaaaaaggc ctacagcgaa aaaattgctg 960aagcaaccct ttttcaagca agctcgctcc agtgatttca ttagtcgaaa gcttttggag 1020ggattgcctg gccttggtgc cagatattta gctctgaagg aaaaggatga agttttactt 1080tctcaaaaga aaatgcctga tggacagaag gaagaaatct cacaggatga atacaaaaga 1140ggcatcagta gctggaactt tgatatggat gacctgaagt ctcaagcttc acttattaca 1200gagtgtgatg acagtatatc gtgcaaagat tcagatgcat catgtttcta tgacttggac 1260accattttac cagagcgagc aacaggacct catatgtcaa gagttttttc aattaagtat 1320gatacggaca ccgaaaatga tgtgatgagc aatgataagt cagcagtttc atctcctgag 1380caccccattt gtttagcaag gaatacatca atgctcagga ctacaaacgg ggtacatgca 1440aatggccagg tcaggaaaca cagctccaca gaaagtagtg aactggactt gcaagagaaa 1500gattcagatg ctattccaac cagttcattc agctcatttc atgaaaggaa gttttctttc 1560agttcttgct catctgatgg atttctttca tccaaagaga gctcgaagca tcaaattaac 1620attcataacc gtgacaagtg caacggagga cccttgcaag ttgcagatga accatcccct 1680gaagctgttc caaaggtgcc taaatcatca gcagcaaatg ttgaggacca cgacgataga 1740tcgaaacctc ctcttataca gcaaagaggc cgttttaaag ttacgcctgg gcatgttgag 1800ttggataagg attttcaata tcgttcgatt caagaattga tgccatctgt tgggagcaat 1860atacaggcaa tttcgcacct tccttcgtta agtataccat cctcaattga ggctgcatca 1920accattattg gtgggtccct ttatatgcag ctgtacaatg ttctacagac aaatatgctt 1980cagagggagc aaatacttca tgcgatgaaa cagttaagtg gttgcgatat ggcaatgacg 2040tcacctgcct gcattgctcc tgcaagtcgc gcatcatctc catcatcagc attatcaatt 2100gacagatcat tgttggaagc ggcacacgaa aaggagaagg agctggtcaa tgagatcact 2160gagctgcaat ggcggttagt gtgttcgcag gacgagatac agaggctcaa agcaaaggca 2220gcccaggtga ccatatctga tcttgtggag atgctgttag atatggaaca gcacgggaag 2280gattga 228624534PRTOryza sativa 24Met Ala Ala Ala Ala Gly Ser Val Gly Gly Asp Asp His His His His1 5 10 15Gln Gln Ala Arg Tyr Pro Leu Asp Ala Gly Ser Tyr Arg Leu Leu Cys 20 25 30Lys Ile Gly Ser Gly Val Ser Ala Val Val Tyr Lys Ala Ala Cys Val 35 40 45Pro Leu Gly Ser Ala Val Val Ala Ile Lys Ala Ile Asp Leu Glu Arg 50 55 60Ser Arg Ala Asn Leu Asp Glu Val Trp Arg Glu Ala Lys Ala Met Ala65 70 75 80Leu Leu Ser His Arg Asn Val Leu Arg Ala His Cys Ser Phe Thr Val 85 90 95Gly Ser His Leu Trp Val Val Met Pro Phe Met Ala Ala Gly Ser Leu 100 105 110His Ser Ile Leu Ser His Gly Phe Pro Asp Gly Leu Pro Glu Gln Cys 115 120 125Ile Ala Val Val Leu Arg Asp Thr Leu Arg Ala Leu Cys Tyr Leu His 130 135 140Glu Gln Gly Arg Ile His Arg Asp Ile Lys Ala Gly Asn Ile Leu Val145 150 155 160Asp Ser Asp Gly Ser Val Lys Leu Ala Asp Phe Gly Val Ser Ala Ser 165 170 175Ile Tyr Glu Thr Ala Pro Ser Thr Ser Ser Ala Phe Ser Gly Pro Ile 180 185 190Asn His Ala Pro Pro Pro Ser Gly Ala Ala Leu Ser Ser Ser Cys Phe 195 200 205Asn Asp Met Ala Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile His 210 215 220Ser His Val Gly Tyr Gly Ile Lys Ala Asp Ile Trp Ser Phe Gly Ile225 230 235 240Thr Ala Leu Glu Leu Ala His Gly Arg Pro Pro Leu Ser His Leu Pro 245 250 255Pro Ser Lys Ser Met Leu Met Arg Ile Thr Ser Arg Val Arg Leu Glu 260 265 270Val Asp Ala Ser Ser Ser Ser Ser Glu Gly Ser Ser Ser Ala Ala Arg 275 280 285Lys Lys Lys Lys Phe Ser Lys Ala Phe Lys Asp Met Val Ser Ser Cys 290 295 300Leu Cys Gln Glu Pro Ala Lys Arg Pro Ser Ala Glu Lys Leu Leu Arg305 310 315 320His Pro Phe Phe Lys Gly Cys Arg Ser Arg Asp Tyr Asp Tyr Leu Val 325 330 335Arg Asn Val Leu Asp Ala Val Pro Thr Val Glu Glu Arg Cys Arg Asp 340 345 350Ser Thr Gln Leu Cys Gly Cys Ala Arg Gly Ala Arg Cys Val Ser Pro 355 360 365Cys Arg His Ala Ser Ser Gly Ser Asn Val Val Ala Ala Lys Asn Arg 370 375 380Arg Ile Ser Gly Trp Asn Phe Asn Glu Glu Ser Phe Glu Leu Asp Pro385 390 395 400Thr Asp Lys Pro Pro Glu Gln Gln Gln Gln Gln Pro Cys Phe Pro Phe 405 410 415His His Asp Asn Asp Asp Asp Met Val Glu His Glu Gln Glu Gln Arg 420 425 430Arg Arg Gln Asp Gly Asn Asp Gly Ser Ser Asp Val Ala Val Pro His 435 440 445Leu Val Thr Ile Leu Gly Ser Leu Glu Met Gln Arg Asp Met Val Met 450 455 460Gln Val Leu Glu Gly Asp Gly Gly Gly Gly Gly Glu Thr Ala Gly Arg465 470 475 480Glu Glu Met Leu Val Gly Tyr Val Arg Glu Leu Glu Lys Arg Val Gln 485 490 495Glu Leu Ser Thr Glu Val Glu Glu Glu Met Ala Arg Asn Ala His Leu 500 505 510Gln Glu Leu Leu His Glu Arg Ala Cys Glu Asn His Thr Asp Ser Ser 515 520 525His Thr Ser Gly Ser Arg 530253038DNAOryza sativa 25gaccttctct tcctccctcg acacctctcc ccacgttacg ctgcctcctc ctcctcctcg 60cctccctctc gtgggcgtcg tccccctccg ccaccgccgc cgcccgccgc agcagccgca 120gaaggggact ccacctcctc ccggatctgc tcgatcgccc ccgattctgt agcttctcct 180ctgctcagat ccgccccctt gcttttcatc cagctcgtgg cacccgagat ccgctgccgc 240cgccgccgtc tctgcggtcc tcctcccccc tcgccggtgg ggaaccccgc cgccccgaag 300cgcgttgcag cgtgtactac tgccggactg ccaaagtacg cttgctgcta gccatttcgg 360tagcttttgt ggtttctact taactatcgt gtttggcaat acaacacctt ggaccaaagg 420atgcttgaaa gatagactgg ataattaaga ctggatgcca aagtacgctt gctgctagcc 480atttcggtag cttttgtggt ttctacttaa ctatcgtgtt tggcaataca acaccttgga 540ccaaaggatg cttgaaagat agactggata attaagaact atattggact gtacattcgc 600ctatagactt agtcatgctg ctggttgttc tctgtcgctg ctacaaggtg ttcgtgctat 660tgccttctgc ttctgtgtgc tatggtaatt gtaactctgt ggttattgcg catgatgctc 720tttgaatttg agccatggag catgcaagga gatttccaac agatcccaaa gaatataaat 780tatgtgagga agttggagat ggtgtagtgc tacggtgtac aaagctcttt gtatcccact 840taatattgaa gttgccatta aagttcttga ccttgagaag tgcagtaatg acctggatgg 900gataagacga gaagtacaaa ccatgagctt gattgatcat ccaaatcttc ttcgagcata 960ttgctcgttt acaaatggtc atcagctttg ggttattatg ccttacatgg ccgctggatc 1020tgctctgcac attatgaaaa cttcttttcc agatgggttt gaggagccag tcattgcaac 1080tcttttgcgg gaggttctta aagctcttgt ctacctacac tcccaagggc atattcacag 1140agatgtaaag gctggaaata tcctaataga tacaaatgga gctgtcaagc taggagactt 1200tggagtgtca gcctgcatgt ttgatactgg gaataggcaa agagcacgaa acacttttgt 1260agggacccct tgctggatgg ctccagaagt tatgcaacaa ctgcatggtt atgattacaa 1320agctgacatt tggtcctttg gtataacggc attggaacta gcatatggtc atgctccatt 1380ttcaaagtac cctccaatga aggtattgct tatgaccttg caaaatgcac caccaggtct 1440agactatgag agggacaagc gattttcaaa gtctttcaag gatttggttg caacttgctt 1500agtcaaggat ccacgcaagc gtccatcttc agaaaagctc ttgaagcatt ctttttttaa 1560gcatgcacgt acagctgaat ttcttgcacg aagtattctt gacggcctcc ccccgctggg 1620tgaacgcttt aggacattga agggaaaaga ggctgacttg cttcttagta ataagcttgg 1680ttcagagagc aaggagcaac tatcacagaa agagtacata cgaggaatca gtggttggaa 1740cttcaatctg gaggacttga aaaatgcagc cgcccttata gacaatacaa acggaacgtg 1800ccatttagat ggtgttaaca gcaaattcaa ggatggttta caagaagcta atgaaccaga 1860aaatatttac cagggacggg ctaaccttgt tgcttctgca aggcctgagg atgagataca 1920agaggtcgaa gatctggatg gtgctctcgc ctcttctttc cccagccgcc cccttgaggc 1980actaaaatct tgctttgatg tttgtgggga tgatgatccc cctactgcta ctgatttgag 2040ggagcaacca aatatggaat ctacatcacc tatgcagcag ttccaacaaa ttgagaatca 2100taaaagtgcc aactgtaatg gtgaaagttt ggaaagaagt gcctctgtac catcaaattt 2160ggtcaatagt gggtcccaca agttcttaag tggttccctg atacctgaac atgttctttc 2220tccttacagg aatgttggca atgacccagc aaggaatgag tgtcatcaga aaaatacatg 2280caacaggaac cgcagtgggc ctttattccg ccaaatgaaa gatccacgcg cacatctgcc 2340tgttgaacct gaggagcaat ccgaaggaaa agttatccag cgaagggggc gttttcaggt 2400tacatcagat agtattgctc aaaaggtagc ttcatccgca agcagcagta ggtgctcaaa 2460tttaccaatc ggagtaacac gatcaactgt ccatccatcg acaattcttc caacactaca 2520attcatgata cagcaaaata ctatgcaaaa ggaagtgata agtagactga tttcttcaat 2580tgaggaaata tctgatgctg ctgatgcaag tacaactggt tcatctcagc catctggagt 2640gcatttcaga gagaaggaac tgcagtcgta catcgccaac ttgcagcaaa gtgtcaccga 2700acttgctgag gaagttcaga gattaaagct caaaaacact cagctcgagg agcagatcaa 2760tgcattgccc aaaaaagatg aaaggttacg aagagaggat acccgacaac aatgatatgc 2820acaatgcact tgtaaccccc gctgtaaaat cagttcccca attttgaatt tggttagcaa 2880aattatttgt attttgttcg aagtcaggcc tggtgtatct ttgtaatttg taattatttt 2940agcaaggtga aattatagtt attttcattt gtacaggata tttcaatcta taccaaagtt 3000aaaagcttgg tactagaaaa taccaaatca tctttcct 303826693PRTOryza sativa 26Met Glu His Ala Arg Arg Phe Pro Thr Asp Pro Lys Glu Tyr Lys Leu1 5 10 15Cys Glu Glu Val Gly Asp Gly Val Ser Ala Thr Val Tyr Lys Ala Leu 20 25 30Cys Ile Pro Leu Asn Ile Glu Val Ala Ile Lys Val Leu Asp Leu Glu 35 40 45Lys Cys Ser Asn Asp Leu Asp Gly Ile Arg Arg Glu Val Gln Thr Met 50 55 60Ser Leu Ile Asp His Pro Asn Leu Leu Arg Ala Tyr Cys Ser Phe Thr65 70 75 80Asn Gly His Gln Leu Trp Val Ile Met Pro Tyr Met Ala Ala Gly Ser 85 90 95Ala Leu His Ile Met Lys Thr Ser Phe Pro Asp Gly Phe Glu Glu Pro 100 105 110Val Ile Ala Thr Leu Leu Arg Glu Val Leu Lys Ala Leu Val Tyr Leu 115 120 125His Ser Gln Gly His Ile His Arg Asp Val Lys Ala Gly Asn Ile Leu 130 135 140Ile Asp Thr Asn Gly Ala Val Lys Leu Gly Asp Phe Gly Val Ser Ala145 150 155 160Cys Met Phe Asp Thr Gly Asn Arg Gln Arg Ala Arg Asn Thr Phe Val 165 170 175Gly Thr Pro Cys Trp Met Ala Pro Glu Val Met Gln Gln Leu His Gly 180 185 190Tyr Asp Tyr Lys Ala Asp Ile Trp Ser Phe Gly Ile Thr Ala Leu Glu 195 200 205Leu Ala His Gly His Ala Pro Phe Ser Lys Tyr Pro Pro Met Lys Val 210 215 220Leu Leu Met Thr Leu Gln Asn Ala Pro Pro Gly Leu Asp Tyr Glu Arg225 230 235 240Asp Lys Arg Phe Ser Lys Ser Phe Lys Asp Leu Val Ala Thr Cys Leu 245 250 255Val Lys Asp Pro Arg Lys Arg Pro Ser Ser Glu Lys Leu Leu Lys His 260 265 270Ser Phe Phe Lys His Ala Arg Thr Ala Glu Phe Leu Ala Arg Ser Ile 275 280 285Leu Asp Gly Leu Pro Pro Leu Gly Glu Arg Phe Arg Thr Leu Lys Gly 290 295 300Lys Glu Ala Asp Leu Leu Leu Ser Asn Lys Leu Gly Ser Glu Ser Lys305 310 315 320Glu Gln Leu Ser Gln Lys Glu Tyr Ile Arg Gly Ile Ser Gly Trp Asn 325 330 335Phe Asn Leu Glu Asp Leu Lys Asn Ala Ala Ala Leu Ile Asp Asn Thr 340 345 350Asn Gly Thr Cys His Leu Asp Gly Val Asn Ser Lys Phe Lys Asp Gly 355 360 365Leu Gln Glu Ala Asn Glu Pro Glu Asn Ile Tyr Gln Gly Arg Ala Asn 370 375 380Leu Val Ala Ser Ala Arg Pro Glu Asp Glu Ile Gln Glu Val Glu Asp385 390 395 400Leu Asp Gly Ala Leu Ala Ser Ser Phe Pro Ser Arg Pro Leu Glu Ala 405 410 415Leu Lys Ser Cys Phe Asp Val Cys Gly Asp Asp Asp Pro Pro Thr Ala 420 425 430Thr Asp Leu Arg Glu Gln Pro Asn Met Glu Ser Thr Ser Pro Met Gln 435 440 445Gln Phe Gln Gln Ile Glu Asn His Lys Ser Ala Asn Cys Asn Gly Glu 450 455 460Ser Leu Glu Arg Ser Ala Ser Val Pro Ser Asn Leu Val Asn Ser Gly465 470 475 480Ser His Lys Phe Leu Ser Gly Ser Leu Ile Pro Glu His Val Leu Ser 485 490 495Pro Tyr Arg Asn Val Gly Asn Asp Pro Ala Arg Asn Glu Cys His Gln 500 505 510Lys Asn Thr Cys Asn Arg Asn Arg Ser Gly Pro Leu Phe Arg Gln Met 515 520 525Lys Asp Pro Arg Ala His Leu Pro Val Glu Pro Glu Glu Gln Ser Glu 530 535 540Gly Lys Val Ile Gln Arg Arg Gly Arg Phe Gln Val Thr Ser Asp Ser545 550 555 560Ile Ala Gln Lys Val Ala Ser Ser Ala Ser Ser Ser Arg Cys Ser Asn 565 570 575Leu Pro Ile Gly Val Thr Arg Ser Thr Val His Pro Ser Thr Ile Leu 580 585 590Pro Thr Leu Gln Phe Met Ile Gln Gln Asn Thr Met Gln Lys Glu Val 595 600 605Ile Ser Arg Leu Ile Ser Ser Ile Glu Glu Ile Ser Asp Ala Ala Asp 610 615 620Ala Ser Thr Thr Gly Ser Ser Gln Pro Ser Gly Val His Phe Arg Glu625 630 635 640Lys Glu Leu Gln Ser Tyr Ile Ala Asn Leu Gln Gln Ser Val Thr Glu 645 650 655Leu Ala Glu Glu Val Gln Arg Leu Lys Leu Lys Asn Thr Gln Leu Glu 660 665 670Glu Gln Ile Asn Ala Leu Pro Lys Lys Asp Glu Arg Leu Arg Arg Glu 675 680 685Asp Thr Arg Gln Gln 690272286DNAOryza sativa 27atggccaagg cgtgggagaa ggtggcgacg gcggcggggt tgggtgggtc gggggagagg 60cgcaagtacc cgatccgcgt ggaggactac gagctgtacg aggagatcgg gcagggggtc 120agcgccatcg tgtaccgatc gctctgcaag cccctcgacg agatcgtcgc cgtcaaggtg 180ctcgacttcg agcgcacaaa cagtgacctg tggttagttg taatgcaagt aggttatact 240cggattgttg cgatttacgt accgccgctt gatctgtcta aaatgatagt aacacggata 300tgcttgacgc agaacaacat catgcgtgaa gctcagacga tgattctcat agatcagcct 360aacgtcatga aggcacattg ttcatttaca aataaccact cgttgtgggt ggtcatgcca 420tacatggctg gagggtcttg ccttcacata atgaagtcag tctatccaga tggttttgaa 480gaagctgtca ttgcaactgt acttcgtgaa gtcctgaaag gtttggagta ccttcatcat 540catgggcata tacatcgtga tgtgaaggca gggaatatac ttgttgattc acggggtgta 600gtcaagcttg gagattttgg ggtttctgct tgcctttttg attctggtga caggcaacgg 660gctaggaata ctttcgtggg aactccttgc tggatggcac cagaggttat ggagcagcta 720catggatacg atttcaaggc agacatatgg tccttcggaa ttactgcact tgaacttgcc 780catggtcatg ctcctttctc gaagttccct cccatgaagg tcttacttat gacacttcag 840aatgcccctc cgggccttga ctatgagaga gataagaaat tttcaaggca tttcaagcaa 900atggttgcta

tgtgtctggt aaaagaccct tcaaaaaggc ctacagcgaa aaaattgctg 960aagcaaccct ttttcaagca agctcgctcc agtgatttca ttagtcgaaa gcttttggag 1020ggattgcctg gccttggtgc cagatattta gctctgaagg aaaaggatga agttttactt 1080tctcaaaaga aaatgcctga tggacagaag gaagaaatct cacaggatga atacaaaaga 1140ggcatcagta gctggaactt tgatatggat gacctgaagt ctcaagcttc acttattaca 1200gagtgtgatg acagtatatc gtgcaaagat tcagatgcat catgtttcta tgacttggac 1260accattttac cagagcgagc aacaggacct catatgtcaa gagttttttc aattaagtat 1320gatacggaca ccgaaaatga tgtgatgagc aatgataagt cagcagtttc atctcctgag 1380caccccattt gtttagcaag gaatacatca atgctcagga ctacaaacgg ggtacatgca 1440aatggccagg tcaggaaaca cagctccaca gaaagtagtg aactggactt gcaagagaaa 1500gattcagatg ctattccaac cagttcattc agctcatttc atgaaaggaa gttttctttc 1560agttcttgct catctgatgg atttctttca tccaaagaga gctcgaagca tcaaattaac 1620attcataacc gtgacaagtg caacggagga cccttgcaag ttgcagatga accatcccct 1680gaagctgttc caaaggtgcc taaatcatca gcagcaaatg ttgaggacca cgacgataga 1740tcgaaacctc ctcttataca gcaaagaggc cgttttaaag ttacgcctgg gcatgttgag 1800ttggataagg attttcaata tcgttcgatt caagaattga tgccatctgt tgggagcaat 1860atacaggcaa tttcgcacct tccttcgtta agtataccat cctcaattga ggctgcatca 1920accattattg gtgggtccct ttatatgcag ctgtacaatg ttctacagac aaatatgctt 1980cagagggagc aaatacttca tgcgatgaaa cagttaagtg gttgcgatat ggcaatgacg 2040tcacctgcct gcattgctcc tgcaagtcgc gcatcatctc catcatcagc attatcaatt 2100gacagatcat tgttggaagc ggcacacgaa aaggagaagg agctggtcaa tgagatcact 2160gagctgcaat ggcggttagt gtgttcgcag gacgagatac agaggctcaa agcaaaggca 2220gcccaggtga ccatatctga tcttgtggag atgctgttag atatggaaca gcacgggaag 2280gattga 228628761PRTOryza sativa 28Met Ala Lys Ala Trp Glu Lys Val Ala Thr Ala Ala Gly Leu Gly Gly1 5 10 15Ser Gly Glu Arg Arg Lys Tyr Pro Ile Arg Val Glu Asp Tyr Glu Leu 20 25 30Tyr Glu Glu Ile Gly Gln Gly Val Ser Ala Ile Val Tyr Arg Ser Leu 35 40 45Cys Lys Pro Leu Asp Glu Ile Val Ala Val Lys Val Leu Asp Phe Glu 50 55 60Arg Thr Asn Ser Asp Leu Trp Leu Val Val Met Gln Val Gly Tyr Thr65 70 75 80Arg Ile Val Ala Ile Tyr Val Pro Pro Leu Asp Leu Ser Lys Met Ile 85 90 95Val Thr Arg Ile Cys Leu Thr Gln Asn Asn Ile Met Arg Glu Ala Gln 100 105 110Thr Met Ile Leu Ile Asp Gln Pro Asn Val Met Lys Ala His Cys Ser 115 120 125Phe Thr Asn Asn His Ser Leu Trp Val Val Met Pro Tyr Met Ala Gly 130 135 140Gly Ser Cys Leu His Ile Met Lys Ser Val Tyr Pro Asp Gly Phe Glu145 150 155 160Glu Ala Val Ile Ala Thr Val Leu Arg Glu Val Leu Lys Gly Leu Glu 165 170 175Tyr Leu His His His Gly His Ile His Arg Asp Val Lys Ala Gly Asn 180 185 190Ile Leu Val Asp Ser Arg Gly Val Val Lys Leu Gly Asp Phe Gly Val 195 200 205Ser Ala Cys Leu Phe Asp Ser Gly Asp Arg Gln Arg Ala Arg Asn Thr 210 215 220Phe Val Gly Thr Pro Cys Trp Met Ala Pro Glu Val Met Glu Gln Leu225 230 235 240His Gly Tyr Asp Phe Lys Ala Asp Ile Trp Ser Phe Gly Ile Thr Ala 245 250 255Leu Glu Leu Ala His Gly His Ala Pro Phe Ser Lys Phe Pro Pro Met 260 265 270Lys Val Leu Leu Met Thr Leu Gln Asn Ala Pro Pro Gly Leu Asp Tyr 275 280 285Glu Arg Asp Lys Lys Phe Ser Arg His Phe Lys Gln Met Val Ala Met 290 295 300Cys Leu Val Lys Asp Pro Ser Lys Arg Pro Thr Ala Lys Lys Leu Leu305 310 315 320Lys Gln Pro Phe Phe Lys Gln Ala Arg Ser Ser Asp Phe Ile Ser Arg 325 330 335Lys Leu Leu Glu Gly Leu Pro Gly Leu Gly Ala Arg Tyr Leu Ala Leu 340 345 350Lys Glu Lys Asp Glu Val Leu Leu Ser Gln Lys Lys Met Pro Asp Gly 355 360 365Gln Lys Glu Glu Ile Ser Gln Asp Glu Tyr Lys Arg Gly Ile Ser Ser 370 375 380Trp Asn Phe Asp Met Asp Asp Leu Lys Ser Gln Ala Ser Leu Ile Thr385 390 395 400Glu Cys Asp Asp Ser Ile Ser Cys Lys Asp Ser Asp Ala Ser Cys Phe 405 410 415Tyr Asp Leu Asp Thr Ile Leu Pro Glu Arg Ala Thr Gly Pro His Met 420 425 430Ser Arg Val Phe Ser Ile Lys Tyr Asp Thr Asp Thr Glu Asn Asp Val 435 440 445Met Ser Asn Asp Lys Ser Ala Val Ser Ser Pro Glu His Pro Ile Cys 450 455 460Leu Ala Arg Asn Thr Ser Met Leu Arg Thr Thr Asn Gly Val His Ala465 470 475 480Asn Gly Gln Val Arg Lys His Ser Ser Thr Glu Ser Ser Glu Leu Asp 485 490 495Leu Gln Glu Lys Asp Ser Asp Ala Ile Pro Thr Ser Ser Phe Ser Ser 500 505 510Phe His Glu Arg Lys Phe Ser Phe Ser Ser Cys Ser Ser Asp Gly Phe 515 520 525Leu Ser Ser Lys Glu Ser Ser Lys His Gln Ile Asn Ile His Asn Arg 530 535 540Asp Lys Cys Asn Gly Gly Pro Leu Gln Val Ala Asp Glu Pro Ser Pro545 550 555 560Glu Ala Val Pro Lys Val Pro Lys Ser Ser Ala Ala Asn Val Glu Asp 565 570 575His Asp Asp Arg Ser Lys Pro Pro Leu Ile Gln Gln Arg Gly Arg Phe 580 585 590Lys Val Thr Pro Gly His Val Glu Leu Asp Lys Asp Phe Gln Tyr Arg 595 600 605Ser Ile Gln Glu Leu Met Pro Ser Val Gly Ser Asn Ile Gln Ala Ile 610 615 620Ser His Leu Pro Ser Leu Ser Ile Pro Ser Ser Ile Glu Ala Ala Ser625 630 635 640Thr Ile Ile Gly Gly Ser Leu Tyr Met Gln Leu Tyr Asn Val Leu Gln 645 650 655Thr Asn Met Leu Gln Arg Glu Gln Ile Leu His Ala Met Lys Gln Leu 660 665 670Ser Gly Cys Asp Met Ala Met Thr Ser Pro Ala Cys Ile Ala Pro Ala 675 680 685Ser Arg Ala Ser Ser Pro Ser Ser Ala Leu Ser Ile Asp Arg Ser Leu 690 695 700Leu Glu Ala Ala His Glu Lys Glu Lys Glu Leu Val Asn Glu Ile Thr705 710 715 720Glu Leu Gln Trp Arg Leu Val Cys Ser Gln Asp Glu Ile Gln Arg Leu 725 730 735Lys Ala Lys Ala Ala Gln Val Thr Ile Ser Asp Leu Val Glu Met Leu 740 745 750Leu Asp Met Glu Gln His Gly Lys Asp 755 760291623DNAOryza sativa 29atggggagga acgggagcgt caagcgtacg tcgtcgtcgg gggcggcggc ggcgttcacg 60gcgaatcccc gcgactacca gctcatggag gaggtcgggt acggggcgca cgccgtcgtg 120taccgcgcgc tgttcgtccc caggaacgac gtcgtggctg tcaagtgcct ggatctcgat 180cagctcaaca acaacatcga tgaaatccaa cgggaggctc aaatcatgag cttgatagag 240catcctaatg tcatcagggc ttactgctca tttgttgttg agcacagcct ttgggtagta 300atgccattta tgactgaggg ttcatgtctg cacctaatga agattgcata tcctgatggt 360ttcgaggaac ctgttattgg ctctattcta aaggaaacac ttaaggcttt ggagtacctt 420cacaggcaag gacaaatcca tcgtgatgtc aaggccggca atatccttgt tgataatgct 480ggtatagtga agcttgggga cttcggcgtg tctgcttgta tgtttgatag aggtgatcga 540caaagatcta ggaacacatt tgtgggaaca ccgtgttgga tggctccaga agtgctccag 600ccaggcactg gatataactt caaagctgac atatggtcat ttggaatcac tgcacttgaa 660cttgcccatg gccatgcacc gttttcaaag tatcccccta tgaaggttct tctcatgacc 720ctccagaatg ctccacctgg tctcgactat gatcgagacc gaagattctc aaagtcattt 780aaggagatgg ttgcaatgtg cttggtaaaa gatcaaacaa agagaccaac agctgagaag 840ttgctaaagc attcattttt caaaaatgca aaacctccag aattgacaat gaagggtatc 900ttaactgatt tacctcctct atgggaccgt gtaaaggctc tccagcttaa agatgcagca 960cagttggcct tgaagaaaat gccttcttct gagcaggagg cactctccat gagtgaatac 1020caacgaggtg ttagtgcatg gaacttcgat gttgaagatc tcaaggccca agcatcacta 1080attcgtgatg atgaaccccc tgaaataaaa gaagacgatg atactgcaag aaccattgaa 1140gttgaaaagg attcattttc taggaatcat ttggggaagt cgtcgagtac aattgaaaat 1200ttcttcagtg gacggacctc taccactgca gcaaattcgg atggaaaagg cgatttttca 1260tttgaagctt ttgattttgg tgaaaacaac gttgatacta aaattatgcc caatgggtat 1320gaaaacgcta gatcagagaa tagctcatca ccctctacat caaagcaaga tccagagtca 1380aaatattgga gaagtacttc tggacagaaa caacaaactt ctggcactcc agctgtccat 1440tctggtgggg ttaatagctc aacaactgaa aagggccatg gtgttgaaag ggatgcaact 1500gttcaattgg catctgataa acttaggact gaaacgagaa gagcaacaaa tcttagtggt 1560ccattgtcac tgccaactcg tgcttctgca aacagtctgt cagctcctat tcgatcttca 1620gga 162330541PRTOryza sativa 30Met Gly Arg Asn Gly Ser Val Lys Arg Thr Ser Ser Ser Gly Ala Ala1 5 10 15Ala Ala Phe Thr Ala Asn Pro Arg Asp Tyr Gln Leu Met Glu Glu Val 20 25 30Gly Tyr Gly Ala His Ala Val Val Tyr Arg Ala Leu Phe Val Pro Arg 35 40 45Asn Asp Val Val Ala Val Lys Cys Leu Asp Leu Asp Gln Leu Asn Asn 50 55 60Asn Ile Asp Glu Ile Gln Arg Glu Ala Gln Ile Met Ser Leu Ile Glu65 70 75 80His Pro Asn Val Ile Arg Ala Tyr Cys Ser Phe Val Val Glu His Ser 85 90 95Leu Trp Val Val Met Pro Phe Met Thr Glu Gly Ser Cys Leu His Leu 100 105 110Met Lys Ile Ala Tyr Pro Asp Gly Phe Glu Glu Pro Val Ile Gly Ser 115 120 125Ile Leu Lys Glu Thr Leu Lys Ala Leu Glu Tyr Leu His Arg Gln Gly 130 135 140Gln Ile His Arg Asp Val Lys Ala Gly Asn Ile Leu Val Asp Asn Ala145 150 155 160Gly Ile Val Lys Leu Gly Asp Phe Gly Val Ser Ala Cys Met Phe Asp 165 170 175Arg Gly Asp Arg Gln Arg Ser Arg Asn Thr Phe Val Gly Thr Pro Cys 180 185 190Trp Met Ala Pro Glu Val Leu Gln Pro Gly Thr Gly Tyr Asn Phe Lys 195 200 205Ala Asp Ile Trp Ser Phe Gly Ile Thr Ala Leu Glu Leu Ala His Gly 210 215 220His Ala Pro Phe Ser Lys Tyr Pro Pro Met Lys Val Leu Leu Met Thr225 230 235 240Leu Gln Asn Ala Pro Pro Gly Leu Asp Tyr Asp Arg Asp Arg Arg Phe 245 250 255Ser Lys Ser Phe Lys Glu Met Val Ala Met Cys Leu Val Lys Asp Gln 260 265 270Thr Lys Arg Pro Thr Ala Glu Lys Leu Leu Lys His Ser Phe Phe Lys 275 280 285Asn Ala Lys Pro Pro Glu Leu Thr Met Lys Gly Ile Leu Thr Asp Leu 290 295 300Pro Pro Leu Trp Asp Arg Val Lys Ala Leu Gln Leu Lys Asp Ala Ala305 310 315 320Gln Leu Ala Leu Lys Lys Met Pro Ser Ser Glu Gln Glu Ala Leu Ser 325 330 335Met Ser Glu Tyr Gln Arg Gly Val Ser Ala Trp Asn Phe Asp Val Glu 340 345 350Asp Leu Lys Ala Gln Ala Ser Leu Ile Arg Asp Asp Glu Pro Pro Glu 355 360 365Ile Lys Glu Asp Asp Asp Thr Ala Arg Thr Ile Glu Val Glu Lys Asp 370 375 380Ser Phe Ser Arg Asn His Leu Gly Lys Ser Ser Ser Thr Ile Glu Asn385 390 395 400Phe Phe Ser Gly Arg Thr Ser Thr Thr Ala Ala Asn Ser Asp Gly Lys 405 410 415Gly Asp Phe Ser Phe Glu Ala Phe Asp Phe Gly Glu Asn Asn Val Asp 420 425 430Thr Lys Ile Met Pro Asn Gly Tyr Glu Asn Ala Arg Ser Glu Asn Ser 435 440 445Ser Ser Pro Ser Thr Ser Lys Gln Asp Pro Glu Ser Lys Tyr Trp Arg 450 455 460Ser Thr Ser Gly Gln Lys Gln Gln Thr Ser Gly Thr Pro Ala Val His465 470 475 480Ser Gly Gly Val Asn Ser Ser Thr Thr Glu Lys Gly His Gly Val Glu 485 490 495Arg Asp Ala Thr Val Gln Leu Ala Ser Asp Lys Leu Arg Thr Glu Thr 500 505 510Arg Arg Ala Thr Asn Leu Ser Gly Pro Leu Ser Leu Pro Thr Arg Ala 515 520 525Ser Ala Asn Ser Leu Ser Ala Pro Ile Arg Ser Ser Gly 530 535 540311938DNAOryza sativa 31atggtgagga gcgggagtgt gcggcggacg gccgcgtcgt cgtcgcccgc cgcggcggcg 60gtgccgacgg ccttcaccgc ctcgcccggc gactaccgcc ttctggagga ggtcggctac 120ggcgcgaacg ccgtcgtgta ccgggcggtg ttcctgccat ccaaccggac cgtcgccgtc 180aagtgcctgg atctcgatcg tgtcaacagt aacctcgatg atataagaaa agaggcacaa 240acgatgagct tgatagatca ccctaatgtc atcagggctt actgctcatt tgttgtggat 300cataacctct gggtgataat gccattcatg tcagagggtt catgtttaca cctgatgaag 360gttgcatatc ctgatggttt tgaggagcct gttatcgcct ctatcctaaa ggaaacactt 420aaggctctag agtacctcca tcggcaagga catatccata gggatgtcaa gcgtaatatt 480atacaggcgg gtaatatcct tatggacagt cctggtatag tgaaacttgg ggactttggt 540gtctctgctt gtatgtttga tagaggtgat agacaaagat ccaggaatac attcgtggga 600acaccatgct ggatggctcc agaagttctc cagcctggag caggatataa tttcaagaaa 660tatgtttcaa accatttgtt taccaactta atttggttat ttaaaatttc cttaaggggt 720aagaactcta actaccataa aaatactggg aataaggttc ttctcatgac ccttcaaaat 780gcaccaccag gccttgacta tgaccgtgat aaaagattct caaagtcttt caaggaaatg 840gttgcaatgt gcctggtcaa agatcaaaca aagaggccaa cggctgaaaa gttactaaag 900cactcatttt tcaagaacgc aaaacctcca gagctgactg ttaagagtat tttaactgat 960ttgccccctc tgtgggatcg tgtaaaagcg ctccagctaa aagatgcagc acaattagct 1020ttgaagaaaa tgccttcttc tgaacaggag gcactttcta tgattcatga tgatgatcca 1080cctgaaataa aggaagatgt tgacaatgat agaataaatg aagctgataa ggagccgttt 1140tctggcaatc attttggaca accaaaaatt ttgagtggaa agcacttcag gttgaatcat 1200gaacaaactt gtgtcactgc agtaagtcca ggggggaata tgcatgagac aagcagagga 1260ttggtttctg aacctggtga tgctgatagt gaaaggaaag ttgatggata tagaaaacaa 1320ggggaagcgg cagttaagtt ggcatctgat aaacaaaaga gttgtacaaa aagaaccaca 1380aatctcagtg gtcctcttgc actccctact cgtgcttctg caaatagtct gtctgctcct 1440attcggtctt ctggaggcta tgtgggctcc ttgggagata agtctaagcg tagtgtggtg 1500gagataaaag gacgtttttc agtgacatct gagaatgtgg atcttgcaaa ggttcaggaa 1560gttccaacaa gcggcatttc acgcaaatta caggagggat cttcactgag aaaatcagcc 1620agcgttggtc attggccggt ggatgctaag ccaatggatc tcatcacaaa cctcctaagt 1680agcttgcaac agaatgagaa agctgacgca acacagtata gacttggtaa tatggatggt 1740gatacagagg ttgaaacgtc tatttccgag ggagaacggt cattacttgt caaaatattt 1800gaattgcaat ctagaatgat ttcattaacc gatgaactga tcacaacaaa actgcaacat 1860gtccagctac aagaagagct aaaaatactg tactgtcacg aagaaataat cgacactagg 1920gaggtggaca atgcttga 193832645PRTOryza sativa 32Met Val Arg Ser Gly Ser Val Arg Arg Thr Ala Ala Ser Ser Ser Pro1 5 10 15Ala Ala Ala Ala Val Pro Thr Ala Phe Thr Ala Ser Pro Gly Asp Tyr 20 25 30Arg Leu Leu Glu Glu Val Gly Tyr Gly Ala Asn Ala Val Val Tyr Arg 35 40 45Ala Val Phe Leu Pro Ser Asn Arg Thr Val Ala Val Lys Cys Leu Asp 50 55 60Leu Asp Arg Val Asn Ser Asn Leu Asp Asp Ile Arg Lys Glu Ala Gln65 70 75 80Thr Met Ser Leu Ile Asp His Pro Asn Val Ile Arg Ala Tyr Cys Ser 85 90 95Phe Val Val Asp His Asn Leu Trp Val Ile Met Pro Phe Met Ser Glu 100 105 110Gly Ser Cys Leu His Leu Met Lys Val Ala Tyr Pro Asp Gly Phe Glu 115 120 125Glu Pro Val Ile Ala Ser Ile Leu Lys Glu Thr Leu Lys Ala Leu Glu 130 135 140Tyr Leu His Arg Gln Gly His Ile His Arg Asp Val Lys Arg Asn Ile145 150 155 160Ile Gln Ala Gly Asn Ile Leu Met Asp Ser Pro Gly Ile Val Lys Leu 165 170 175Gly Asp Phe Gly Val Ser Ala Cys Met Phe Asp Arg Gly Asp Arg Gln 180 185 190Arg Ser Arg Asn Thr Phe Val Gly Thr Pro Cys Trp Met Ala Pro Glu 195 200 205Val Leu Gln Pro Gly Ala Gly Tyr Asn Phe Lys Lys Tyr Val Ser Asn 210 215 220His Leu Phe Thr Asn Leu Ile Trp Leu Phe Lys Ile Ser Leu Arg Gly225 230 235 240Lys Asn Ser Asn Tyr His Lys Asn Thr Gly Asn Lys Val Leu Leu Met 245 250 255Thr Leu Gln Asn Ala Pro Pro Gly Leu Asp Tyr Asp Arg Asp Lys Arg 260 265 270Phe Ser Lys Ser Phe Lys Glu Met Val Ala Met Cys Leu Val Lys Asp 275 280 285Gln Thr Lys Arg Pro Thr Ala Glu Lys Leu Leu Lys His Ser Phe Phe 290 295 300Lys Asn Ala Lys Pro Pro Glu Leu Thr Val Lys Ser Ile Leu Thr Asp305 310 315

320Leu Pro Pro Leu Trp Asp Arg Val Lys Ala Leu Gln Leu Lys Asp Ala 325 330 335Ala Gln Leu Ala Leu Lys Lys Met Pro Ser Ser Glu Gln Glu Ala Leu 340 345 350Ser Met Ile His Asp Asp Asp Pro Pro Glu Ile Lys Glu Asp Val Asp 355 360 365Asn Asp Arg Ile Asn Glu Ala Asp Lys Glu Pro Phe Ser Gly Asn His 370 375 380Phe Gly Gln Pro Lys Ile Leu Ser Gly Lys His Phe Arg Leu Asn His385 390 395 400Glu Gln Thr Cys Val Thr Ala Val Ser Pro Gly Gly Asn Met His Glu 405 410 415Thr Ser Arg Gly Leu Val Ser Glu Pro Gly Asp Ala Asp Ser Glu Arg 420 425 430Lys Val Asp Gly Tyr Arg Lys Gln Gly Glu Ala Ala Val Lys Leu Ala 435 440 445Ser Asp Lys Gln Lys Ser Cys Thr Lys Arg Thr Thr Asn Leu Ser Gly 450 455 460Pro Leu Ala Leu Pro Thr Arg Ala Ser Ala Asn Ser Leu Ser Ala Pro465 470 475 480Ile Arg Ser Ser Gly Gly Tyr Val Gly Ser Leu Gly Asp Lys Ser Lys 485 490 495Arg Ser Val Val Glu Ile Lys Gly Arg Phe Ser Val Thr Ser Glu Asn 500 505 510Val Asp Leu Ala Lys Val Gln Glu Val Pro Thr Ser Gly Ile Ser Arg 515 520 525Lys Leu Gln Glu Gly Ser Ser Leu Arg Lys Ser Ala Ser Val Gly His 530 535 540Trp Pro Val Asp Ala Lys Pro Met Asp Leu Ile Thr Asn Leu Leu Ser545 550 555 560Ser Leu Gln Gln Asn Glu Lys Ala Asp Ala Thr Gln Tyr Arg Leu Gly 565 570 575Asn Met Asp Gly Asp Thr Glu Val Glu Thr Ser Ile Ser Glu Gly Glu 580 585 590Arg Ser Leu Leu Val Lys Ile Phe Glu Leu Gln Ser Arg Met Ile Ser 595 600 605Leu Thr Asp Glu Leu Ile Thr Thr Lys Leu Gln His Val Gln Leu Gln 610 615 620Glu Glu Leu Lys Ile Leu Tyr Cys His Glu Glu Ile Ile Asp Thr Arg625 630 635 640Glu Val Asp Asn Ala 645332040DNAMedicago sativa 33atttattaaa attgatgtga cggtctctat agggccgttt catctaaatt tattaaaatt 60tagatccaac tatcttataa tccgttgcac tgtgcaacag ttatagagaa tccaaattcc 120gtaacaggag cttaaattct ctatgattgt tgcacatctc cggtagagta tctaaattta 180agaaatacaa catatcagag atattatgta gaaacacata ttatcaagtt aattaactag 240taggactatt agcagcagag gaaattaggt gaagcttgat attctccagc tgcatctcca 300gctgcaagtt cttcttcttc tcattttcca attctgtcct caacttagag atctcttgca 360ccattttctc atcactttca gccacatgaa tctcctctcc accaattaga ttcattagaa 420acttaacttg tcccaactct tgttccaagc tctctttcaa cacattcaac gttgccaacg 480tagcttcacg gttcttaaca acaccaccga cattttcatg atcaaccaca tcagaagtat 540tggtctttgg ctccatcaca gttcctgatg ctgtaacaac agcatcctct tggatcactt 600tttcttcttc gaaccgaact tgtttcacaa cttcatcatc tttgctttga tcctttggga 660atacaggcac aagttccaat ccatcttcat tgaaattcca gccactgatt cttctctgtt 720tcacattttt cactgactcg tcatcatcgt cgtctccatc atccttgcac tttgaatctg 780gatccattat agctttgatc tctttatacc ttttctcaac actaggcaat ccattcaaca 840cattcttcac caaaacatct gatcccttgc agtttttaaa gaatgaatgc ttaagtaact 900tctcagcaga gggtcttttt gtaggatctt gattcaaaca taaagcaacc atatccttaa 960aagccttaga gaatttgtta ctaccaccat gacccttgta actatgttta tcaaaatcag 1020aaaacttaaa cctctttgta atgtttagca tcaatgactt agaaggagga agatgagaaa 1080gtggtggtct tccatgtgct aactccaaag ctgttatccc aaaagaccag atatcagctt 1140tgaaactgta accattatga gagtgaataa cctcaggagc catccaataa ggtgttccag 1200caaaatcagt aaatatatga gaagaagaag aattcgaaga cgaagatgaa taagaagaac 1260atgctcctac agagttgttt gattcataaa tggaagcaga aacaccaaaa tctgcaagtt 1320tcactaatcc atttgagtca acaaggatgt taccagattt gatatctcta tgaagatgtc 1380cttgtccatg aaggtaagaa agagcattga gagtgtcttt gagaataaca gctatggatt 1440gttctgttaa gccgttttgg aaagagtgag agataatgga ttgtaatgaa cctccagcca 1500tgaatggcat aaccacccaa agacggttgt caacggtgaa agaacagtga gctttgagga 1560tgttggggtg ggaaagaagt gataatgtct ttgcttcacg tctaacatcg tcaaggtcgg 1620ggcgcgaacg atccaagtct atggatttga tagctactgg tgtggagttt atagggatgc 1680agattgcttt gtagacgacg gcgctgttac cggcgccaat ctcgtcgacg attttatagg 1740aggaagagtc taatggatat tgcactcttt ctgctatgtt ggtagccatg gaaataatgg 1800aagttgagat ataagagtgt gtgtgtgtgt gtgtgttaga ggttgaaaat ggttgtttga 1860aaatatataa gatgaatgaa ttgcatggat tggtgatgga tggatggatg gtgaaattaa 1920ttgataaaga gagtggtagg ttgagtttga gcagttttct tgtgaaggtt gatgaaaaaa 1980gaagaaaata tcattaggca gaggtgattt atttctcaat gatctatcag tggttgtgaa 204034518PRTMedicago sativa 34Met Ala Thr Asn Ile Ala Glu Arg Val Gln Tyr Pro Leu Asp Ser Ser1 5 10 15Ser Tyr Lys Ile Val Asp Glu Ile Gly Ala Gly Asn Ser Ala Val Val 20 25 30Tyr Lys Ala Ile Cys Ile Pro Ile Asn Ser Thr Pro Val Ala Ile Lys 35 40 45Ser Ile Asp Leu Asp Arg Ser Arg Pro Asp Leu Asp Asp Val Arg Arg 50 55 60Glu Ala Lys Thr Leu Ser Leu Leu Ser His Pro Asn Ile Leu Lys Ala65 70 75 80His Cys Ser Phe Thr Val Asp Asn Arg Leu Trp Val Val Met Pro Phe 85 90 95Met Ala Gly Gly Ser Leu Gln Ser Ile Ile Ser His Ser Phe Gln Asn 100 105 110Gly Leu Thr Glu Gln Ser Ile Ala Val Ile Leu Lys Asp Thr Leu Asn 115 120 125Ala Leu Ser Tyr Leu His Gly Gln Gly His Leu His Arg Asp Ile Lys 130 135 140Ser Gly Asn Ile Leu Val Asp Ser Asn Gly Leu Val Lys Leu Ala Asp145 150 155 160Phe Gly Val Ser Ala Ser Ile Tyr Glu Ser Asn Asn Ser Val Gly Ala 165 170 175Cys Ser Ser Tyr Ser Ser Ser Ser Ser Asn Ser Ser Ser Ser His Ile 180 185 190Phe Thr Asp Phe Ala Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile 195 200 205His Ser His Asn Gly Tyr Ser Phe Lys Ala Asp Ile Trp Ser Phe Gly 210 215 220Ile Thr Ala Leu Glu Leu Ala His Gly Arg Pro Pro Leu Ser His Leu225 230 235 240Pro Pro Ser Lys Ser Leu Met Leu Asn Ile Thr Lys Arg Phe Lys Phe 245 250 255Ser Asp Phe Asp Lys His Ser Tyr Lys Gly His Gly Gly Ser Asn Lys 260 265 270Phe Ser Lys Ala Phe Lys Asp Met Val Ala Leu Cys Leu Asn Gln Asp 275 280 285Pro Thr Lys Arg Pro Ser Ala Glu Lys Leu Leu Lys His Ser Phe Phe 290 295 300Lys Asn Cys Lys Gly Ser Asp Val Leu Val Lys Asn Val Leu Asn Gly305 310 315 320Leu Pro Ser Val Glu Lys Arg Tyr Lys Glu Ile Lys Ala Ile Met Asp 325 330 335Pro Asp Ser Lys Cys Lys Asp Asp Gly Asp Asp Asp Asp Asp Glu Ser 340 345 350Val Lys Asn Val Lys Gln Arg Arg Ile Ser Gly Trp Asn Phe Asn Glu 355 360 365Asp Gly Leu Glu Leu Val Pro Val Phe Pro Lys Asp Gln Ser Lys Asp 370 375 380Asp Glu Val Val Lys Gln Val Arg Phe Glu Glu Glu Lys Val Ile Gln385 390 395 400Glu Asp Ala Val Val Thr Ala Ser Gly Thr Val Met Glu Pro Lys Thr 405 410 415Asn Thr Ser Asp Val Val Asp His Glu Asn Val Gly Gly Val Val Lys 420 425 430Asn Arg Glu Ala Thr Leu Ala Thr Leu Asn Val Leu Lys Glu Ser Leu 435 440 445Glu Gln Glu Leu Gly Gln Val Lys Phe Leu Met Asn Leu Ile Gly Gly 450 455 460Glu Glu Ile His Val Ala Glu Ser Asp Glu Lys Met Val Gln Glu Ile465 470 475 480Ser Lys Leu Arg Thr Glu Leu Glu Asn Glu Lys Lys Lys Asn Leu Gln 485 490 495Leu Glu Met Gln Leu Glu Asn Ile Lys Leu His Leu Ile Ser Ser Ala 500 505 510Ala Asn Ser Pro Thr Ser 515

Claims

1-13. (canceled)

14. A construct comprising: (i) a Ste20-like nucleic acid or variant thereof; (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally (iii) a transcription termination sequence.

15. The construct of claim 14, wherein said control sequence is a constitutive promoter.

16. The construct of claim 15, wherein said constitutive promoter is a GOS2 promoter.

17. The construct of claim 16, wherein said GOS2 promoter comprises nucleotides 1 to 2193 of SEQ ID NO: 5.

18. A plant transformed with the construct of claim 14.

19-27. (canceled)

28. A method of producing a transgenic plant, plant, or part thereof, comprising introducing the construct of claim 14 into a plant cell, plant, or part thereof.

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