# Patent

## Title
Compositions and methods of use of mitogen-activated protein kinase kinase kinase

## Abstract

Methods and compositions for modulating plant response, development and yield under varying environmental conditions are provided. Methods employing MAPKKK are provided. The MAPKKK sequences are used in a variety of methods including modulating root development, modulating leaf and/or shoot development, modulating tolerance under abiotic stress and modulating yield. Transformed plants, plant cell, tissues, seed and expression vectors are also provided.

## Background
CROSS-REFERENCE
This utility application claims the benefit U.S. Provisional Application Ser. No. 61/060,249, filed Jun. 10, 2008 which is incorporated herein by reference.


FIELD OF THE INVENTION
The invention relates to the field of the genetic manipulation of plants, particularly the modulation of gene activity and development in plants.
BACKGROUND OF THE INVENTION
The mitogen-activated protein kinases (MAPKs) integrate multiple intracellular signals transmitted by various second messengers in a complex signal transduction mechanism. MAPKs phosphorylate and regulate the activity of a variety of enzymes and transcription factors. The activity of MAPKs is triggered by a series of cascades that result in the phosphorylation of the MAPK on both a threonine and tyrosine by a MAPK kinase (MAPKK). The MAPKK is activated by a MAPKKK that becomes active by phosphorylation on its serine/threonine.
The MAPK phosphorylation cascade is highly conserved in eukaryotes. Indeed, homologs in yeast, Drosophila, mammalian cells and plants have been identified. As of 2002, more than 60 MAPKKK genes have been identified in Arabidopsis alone. (Ichimura, et al., (2002) Trends Plant Sci 7:301-308). Because of the large number of proteins involved in the cascade, it is not apparent which proteins are essential, cause lethality if absent, or are functionally redundant.
MAPKKKs and their targets have been implicated in eukaryotic organisms' growth and development. For example, in plants, MAPKKK cascades have been associated with embryonic development, cell division, disease defense responses and abiotic stress responses (Tena, et al., (2001) Curr Opin Plant Biol 4:392-400.).
It has recently been discovered that loss of function mutations in a MAPKKK gene called YODA (YDA) produce Arabidopsis plant embryos lacking a suspensor, a tissue that functions to provide nutrients from the endosperm to the growing embryo. Not all yda plants developed into mature plants and those that did exhibited delayed root development and were smaller than wild-type plants. Known plant hormones were not able to rescue the yda phenotype, suggesting a novel developmental signaling pathway. (Lukowitz, et al., (2004) Sci. STKE 2004 tw21).
Several MAPKKKs in the Arabidopsis ANP family have been identified and implicated in regulating cell division. (Krysan, et al., (2002) Plant Cell 14:1109-1120). A MAPKKK has also been identified in N. benthamiana leaves and found to play a role in the hypersensitive response and resistance against Pseudomonas syringae. (Pozo, et al., (2004) The EMBO Journal 23:3072-3082). The same MAPKKK was found to regulate cell death in susceptible leaves undergoing P. syringae infection. (Pozo, et al., (2004) The EMBO Journal 23:3072-3082).
Transgenic tobacco lines expressing different levels of the constitutively active Arabidopsis ortholog of Tobacco NPK1 were found to grow more vigorously than did the wild type plants in the presence of elevated salt levels, cold temperatures and heat shock, but phenotypically did not differ from wild type plants under normal growth conditions (U.S. Pat. No. 6,613,959). The manipulation of this oxidative stress signaling regulator can protect plant cells from diverse environmental stresses, such as heat and high salt. See, U.S. Pat. No. 6,613,959 (Kovtun, et al., (2000) Proc. Natl. Acad. Sci. USA 97:2940-2945).
Thus, MAPKKKs are involved in a variety of aspects of plant growth and development. Given the important role of members of the MAPKKK signal transduction cascade, in particular the MAPKKK signal transduction molecules, in regulating plant cellular processes ranging from cellular proliferation and differentiation to cellular apoptosis, there exists a need for identifying plant MAPKKK polynucleotides and polypeptides as well as for modulators of such molecules for use in regulating a variety of responses and development. For these and other reasons, there is a need for the present invention.
BRIEF SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide polynucleotides and polypeptides relating to MAPKKK. It is an object of the present invention to provide transgenic plants comprising the polynucleotides and polypeptides of the present invention. Additionally, it is an object of the present invention to provide methods of modulating, in a plant cell or in a transgenic plant, the expression of the polynucleotides and polypeptides of the present invention. Yet another object of the present invention is to provide methods of increasing abiotic stress resistance or tolerance in a plant.
Therefore, in one aspect, the present invention relates to an isolated MAPKKK polynucleotide that encodes the polypeptide of SEQ ID NO: 2, 5, 8 or 10; a polynucleotide having the sequence of SEQ ID NO: 1, 4, 7 or 9; a polynucleotide having at least 30 nucleotides in length which hybridizes under stringent conditions to any of the former polynucleotides. In another aspect, the present invention includes a polynucleotide having at least 60% sequence identity to SEQ ID NOS: 1, 4, 7 or 9. Also included are isolated polynucleotides amplified from a nucleic acid library using primers based on sequences of the present invention, for example, ZmNPK1b—Forward Primer and ZmNPK1b—Reverse Primer as set forth in SEQ ID NOS: 12 and 13 respectively. In one aspect, the nucleic acid library is a Zea mays (maize) library. In another aspect, the nucleic acid library is a cDNA library. Provided herein in another aspect of the invention are isolated polynucleotides degenerate as a result of the genetic code for any of the MAPKKKs of the present invention. In another aspect, an isolated polynucleotide is complementary to a polynucleotide of any one of the MAPKKKs of the present invention. In another aspect, the present invention relates to an isolated polynucleotide that encodes a MAPKKK polypeptide that confers resistance or tolerance to dehydration, salinity, temperature stress, environmental stress or a pathogen.
In yet another aspect, the present invention relates to a transgenic plant including a recombinant expression cassette comprising a plant promoter operably linked to any of the isolated polynucleotides of the present invention. The present invention also provides for transgenic seed from the transgenic plant. In another aspect, the present invention is directed to a host cell transfected with the recombinant expression cassette comprising a plant promoter operably linked to any of the isolated polynucleotides of the present invention. In one aspect, the host cell is a soybean, rice or maize cell.
In a further aspect, the present invention relates to an isolated polypeptide having an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 10 and MAPKKK activity. In yet another aspect, the present invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to an isolated polynucleotide encoding a polypeptide that has an amino acid sequence that has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 10 and has MAPKKK activity. The present invention also provides for transgenic seed from the transgenic plant. In another aspect, the present invention is directed to a host cell transfected with the recombinant expression cassette comprising a plant promoter operably linked to any of the isolated polynucleotides encoding polypeptides of the present invention.
In a further aspect, the present invention relates to a method of modulating the level of MAPKKK protein in a plant cell. In one aspect, the method includes transforming a plant cell with a MAPKKK polynucleotide operably linked to a promoter. The polynucleotide may be in sense or antisense orientation. The method further includes expressing the polynucleotide for an amount of time sufficient to modulate the MAPKKK protein in the plant cell.
In another aspect, the present invention provides a method of modulating the level of MAPKKK protein in a plant. The method includes stably transforming a plant cell with a MAPKKK polynucleotide, in sense or antisense orientation, operably linked to a promoter functional in a plant cell. The method includes regenerating the transformed plant cell into a transformed plant that expresses the MAPKKK polynucleotide in an amount sufficient to modulate the level of MAPKKK protein in the plant.
In another aspect, the present invention relates to a method of increasing abiotic stress resistance or tolerance in a plant. In one aspect, the method includes introducing into plant cells a construct comprising a polynucleotide encoding a MAPKKK of the present invention. The polynucleotide may be operably linked to a promoter functional in plant cells to yield transformed plant cells. The transformed plant cells are regenerated into a transgenic plant. The MAPKKK is expressed in at least some of the cells of the transgenic plant at levels sufficient to induce abiotic stress resistance or tolerance. In one aspect, the abiotic stress is drought, cold temperatures, salt, osmotic stress, frost or freeze, high temperatures, oxidative stress or chemical stress. The method may provide tolerance to other environmental stresses, such as UV-B, ozone, photooxidation, herbicide, pathogen or other stresses that also involve oxidative stress damage and the like.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.



BRIEF DESCRIPTION OF THE FIGURES
The invention can be more fully understood from the following detailed description and the accompanying figures and Sequence Listing which form a part of this application.
FIG. 1. Differences in stress-induced gene expression of ZmNPK1b between B73 and CML349 under cold stress.
FIG. 2. CLUSTAL X (1.83) multiple sequence alignment of MAPKKKs. ZmNPK1a (SEQ ID NO: 2), ZmNPK1b (SEQ ID NO: 5) and ZmNPK1d (SEQ ID NO: 10) were aligned against the rice sequences, OsNPK1-like proteins, NP—917084 (SEQ ID NO: 14) NP—917080 (SEQ ID NO: 15) and BAF24980 (SEQ ID NO: 16), as well as the Arabidopsis ANP1 sequence, O22040 (SEQ ID NO: 17). The partial protein, ZmNPK1c (SEQ ID NO: 8), was excluded from this alignment. Perfectly conserved residues, which are primarily located at the N-terminal half of the protein, are marked by an asterisk.
FIG. 3. The phylogenetic tree constructed from the maize, rice and Arabidopsis NPK1-like sequences on the basis of their amino acid sequences. The dendrogram was constructed using the multiple alignment tool, CLUSTAL.



BRIEF DESCRIPTION OF THE SEQUENCES
The application provides details of MAPKKK sequences as shown in Table 1 below.










TABLE 1





SEQ ID





NO:
pnt or ppt
Length
Identification


















1
pnt
1396
ZmNPK1a complete CDS represented by





PCO644860


2
ppt
441
Zm NPK1a protein sequence


3
pnt
6053
ZmNPK1a genomic sequence predicted by





Pioneer proprietary gene modeling





algorithms


4
pnt
1864
ZmNPK1b complete CDS represented by





PCO644861


5
ppt
514
ZmNPK1b complete protein sequence


6
pnt
4019
ZmNPK1b genomic sequence from BAC





clone p1.bacb.pk191.e03


7
pnt
1662
ZmNPK1c partial CDS represented by





PCO622918


8
ppt
366
ZmNPK1c partial protein sequence


9
pnt
1375
ZmNPK1d complete CDS represented by





PCO638212


10
ppt
392
ZmNPK1d complete protein sequence


11
pnt
2923
ZmNPK1d genomic sequence predicted by





Pioneer proprietary gene modeling





algorithms


12
pnt
21
ZmNPK1b - Forward Primer


13
pnt
22
ZmNPK1b - Reverse Primer


14
ppt
404
OsNPK1-like NP_917084


15
ppt
474
OsNPK1-like NP_917080


16
ppt
653
OsNPK1-like BAF24980


17
ppt
666
Arabidopsis ANP1 O22040


18
pnt
615
Zm rab17 promoter


19
pnt
1625
Arabidopsis rd29a promoter


20
pnt
811
eep5 promoter









DETAILED DESCRIPTION OF THE INVENTION
The present invention now will be described more fully hereinafter with reference to the accompanying examples, in which some, but not all embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the invention set forth herein will come to mind to one skilled in the art to which this invention pertains, having the benefit of the teachings presented in the descriptions and the drawings herein. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more than one element.
Overview
The present invention provides novel compositions and methods for modulating, for example, increasing or decreasing, the level of MAPKKK protein in a plant cell or plant. In particular, the polynucleotides and polypeptides of the present invention can be used to generate transgenic plants expressing MAPKKKs of the present invention.
The present inventors have discovered four novel MAPKKKs, one of which (ZmNPK1b) was found to be expressed at high levels in CML349, a tropical highland line known to be cold tolerant, relative to expression levels in B73, a corn-belt dent inbred relatively less tolerant to cold. Modulation of the MAPKKKs of the present invention would provide a mechanism for manipulating a plant's response to abiotic stresses, including but not limited to drought, cold temperatures, salt, osmotic stress, frost or freeze, high temperatures, oxidative stress and chemical stress as well as stress caused by other environmental factors, such as UV-B, ozone, photooxidation, herbicide, pathogen or other stresses that also involve oxidative stress damage. Thus, the present invention provides methods for modulating, for example, increasing or decreasing, a plant's resistance or tolerance to stresses, particularly abiotic stresses, using MAPKKK polynucleotides and polypeptides of the present invention.
Compositions
Compositions include plants having altered levels and/or activities of mitogen-activated protein kinase kinase kinase (MAPKKK). As used herein, the term mitogen-activated protein kinase kinase kinase (MAPKKK) includes but is not limited to the sequences disclosed herein, such as MAPKKK, their conservatively modified variants, regardless of source and any other variants which retain the biological properties of the MAPKKK, for example, MAPKKK activity as disclosed herein.
In specific compositions, the plants have an altered level and/or activity of a MAPKKK polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 10 or an active variant or fragment thereof. Further provided are plants having an altered level and/or activity of the MAPKKK polypeptides encoded by a polynucleotide set forth in SEQ ID NO: 1, 4, 7 or 9 or an active variant or fragment thereof. The plants of the invention may exhibit modulation in stress tolerance, seed set, plant yield, plant vigor, shoot growth, leaf senescence, shoot regeneration or root growth.
In specific embodiments, the plants of the invention have stably incorporated into their genomes a MAPKKK sequence. In further embodiments, the MAPKKK sequence is operably linked to a tissue-preferred promoter active in the plant.
Other embodiments provide plants which have been genetically modified at a native genomic locus encoding a MAPKKK polypeptide. By “native genomic locus” is intended a naturally occurring genomic sequence. In some embodiments, the native genomic locus is set forth in SEQ ID NOS: 3, 6 and 11 respectively for ZmNPK1a, ZmNPK1b and ZmNPK1d. Genomic sequences for ZmNPK1a and ZmNPK1d are provided using Pioneer proprietary gene modeling algorithms. The gene modeling algorithm reconciles both public and proprietary information on maize sequences to generate gene structure and as new maize sequence information becomes available the gene structures may be revised. Genomic sequence information for ZmNPK1b, is provided using the information from the sequenced BAC clone, p1.bacb.pk191.e03.
Genetic modification encompasses either introduction of a MAPKKK sequence or modification of a native genomic locus encoding a MAPKKK or both and may result in phenotypic change. By “phenotypic change” is intended a measurable change in one or more cell functions. For example, plants having genetic modification at a genomic locus encoding a MAPKKK polypeptide may show reduced or eliminated expression or activity of the MAPKKK polypeptide. Certain phenotypic changes may be observed at the tissue or whole-plant level, for example modified root development or enhanced seedling growth. Various methods of genetic modification are described in more detail elsewhere herein, as are examples of phenotypes that can result from modification affecting the level and/or activity of a MAPKKK sequence of the invention.
Phenotypic changes may include but are not limited to a modulation in root development, stress tolerance, shoot development, leaf development, leaf senescence, photosynthesis, callus regeneration, seed set, plant yield or plant vigor.
Modified plants are of interest, as are modified plant cells, plant protoplasts, plant cell tissue cultures from which a plant can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, grain and the like. As used herein, “grain” means the mature seed produced by commercial growers for purposes other than advancing or reproducing the species, e.g. for such end uses as feed, food, or fiber. Progeny, variants and mutants of the regenerated plants are also included within the scope of the invention, provided that such plants or plant parts comprise the genetic modification.
The MAPKKK polypeptides employed in the invention share sequence identity with members of the MAPKKK family of proteins. Changes in MAPKKK activity alter the intracellular signaling process involving Mitogen Activated Protein kinase kinase kinase (MAPKKK) cascades. These include the cascades of MAPK kinase kinase (MAP3K, also called MAPKKK or MEKK), MAPK kinase (MAP2K, also called MKK or MEK) and MAPK or extracellular signal-regulated kinase (ERK). MAPKKK/MEKK phosphorylates and activates its downstream protein kinase, MAPKK/MEK, which in turn activates MAPK.
As described herein, the inventors have identified four novel MAPKKK cDNAs in maize that are homologs to rice NPK1. The maize MAPKKKs polynucleotides of this invention are 1396 (ZmNPK1a), 1864 (ZmNPK1b), 1662 (ZmNPK1c) and 1375 (ZmNPK1d) nucleotides in length encoding polypeptides with calculated molecular weight of 46 KDa (ZmNPK1a), 54 KDa (ZmNPK1b), 41 KDa (partial ZmNPK1c) and 40 KDa (ZmNPK1d). The polypeptides ZmNPK1a, ZmNPK1b and ZmNPK1d share approximately 53% amino acid consensus between ZmNPK1a and ZmNPK1b, 65% between ZmNPK1a and ZmNPK1d, 61% between ZmNPK1b and ZmNPK1d using GAP (BLOSUM 62). The maize cDNAs for Zm NPK1a, ZmNPK1b and ZmNPK1d encode polypeptides with approximately 58%, 73%, 63% overall amino acid identity respectively to the rice NPK1-like gene (dbj|BAB64165.1| (AP003254) NPK1-related protein kinase-like protein [Oryza sativa]. The maize MAPKKKs were checked for possible chromosomal position by BLAST search against public and proprietary BAC sequences. Three of the maize MAPKKKS, ZmNPK1a, ZmNPK1b and ZmNPK1d, mapped to chromosome 3, while the fourth, namely, ZmNPK1c (PCO622918) localized to chromosome 2. Potential association with any known QTLs for drought stress was checked using a Pioneer proprietary association tool. Thus, ZmNPK1a and ZmNPK1d were observed to be potentially associated with a QTL related to the Staygreen phenotype (Thomas and Howarth, (2000) J Exp Bot 51 Spec No: 329-337) and ZmNPK1c was observed to be potentially associated with yield QTLs and also with the drought QTLs for Anthesis Silking Interval, Staygreen and Barrencount. These potential associations with QTLs predicted by this proprietary association tool does not prove that these specific genes are controlling these traits, rather it merely hints at which trait associations exist. To further characterize the MAPKKKs, the expression of all four MAPKKK sequences were analyzed in Lynx Massively Parallel Signature Sequencing (MPSS) libraries (Table 4). Brenner, et al., (2000) Nat Biotechnol. 18:630-634). The expression of ZmNPK1a was found to be high in stalk, root and pulvinus tissue of B73 stalks. Expression of ZmNPK1b was found to be high in kernel and root tissue, maize kernels at 0 days after pollination, and in maize primary roots. Expression of the ZmNPK1c was not detected in specific tissues most likely to very little representation in the Lynx libraries. Finally, the expression of ZmNPK1d was uniformly distributed in all tissues, the highest level in maize kernels at 0 days after pollination. As shown herein in Example 10, ZmNPK1b and ZmNPK1d were also found to be specifically induced by drought stress and treatment with the stress-hormones, ABA and ethylene as described herein.
Without wishing to be bound by this theory, the present inventors believe that MAPKKKs of the present invention will be useful for increasing stress resistance or tolerance to a number of abiotic stresses. As used herein, the term “abiotic stress” includes but is not limited to drought, cold temperatures, salt, osmotic stress, frost or freeze, high heat temperatures, oxidative stress and chemical stress as well as stress by other environmental stresses, such as UV-B, ozone, photooxidation, herbicide, pathogen, or other stresses that also involve oxidative stress damage (Green and Fluhr, (1995) Plant Cell 7:203-212; Prasad, (1996) Plant J. 10:1017-1026; Willekens, et al., (1997) EMBO J. 16:4806-4816; Chamnongpol, et al., (1998) Proc. Natl. Acad. Sci USA 95:5818-5823; Schraudner, et al., (1998) Plant J. 16:235-245; Karpinski, et al., (1999) Science 284:654-657).
Resistance or tolerance to one or more abiotic stresses may be achieved directly through activation of targets of the MAPKKKs or indirectly through MAPKKK signal transduction cascades, including downstream targets of the MAPKKKs of the present invention. Thus, modulation of MAPKKK activity of the MAPKKKs of the present invention in a plant cell provides a novel strategy for cross protection from one or more abiotic stresses in plants.
Also contemplated is the activation or expression of genes that lie upstream of the ZmNPK MAPKKKs in the regulatory cascade so that activation of a MAPKKK target is achieved. Targets or substrates of the MAPKKKs include but are not limited to transcription factors, other protein kinases and cytoskeleton-associated proteins. Targets or substrates may be identified using techniques common to one skilled in the art including in gel kinase assays, yeast-two hybrid assays, protoplast transient expression assays using stress-responsive promoters linked to a reporter gene, for example, a promoter that is activated during oxidative stress, heat, cold or drought and the like. (Kovtun, et al., (2000). Proc. Natl. Acad. Sci. USA 97:2940-2945; Machida, et al., (1997) Critic. Rev. Plant Sci. 16:481-496; Mazoguchi, et al., (1997) Trends Biotechnol. 15:15-19; Zhang and Klessig, (1997) Plant Cell 9:809-824; Jonak, et al., (1999) Cell. Mol. Life. Sci. 55:204-231).
Compounds that modulate the activity of MAPKKKs of the present invention may be determined by assessing the interaction between a transcription factor and a regulatory element in a MAPKKK promoter, for example, a hormone- or stress-responsive regulatory element. For example, within the promoter sequence of the ZmNPK1b, there is at least one Abscissic Acid—Responsive Element (ABRE), specifically the ABREAT consensus, YACGTGGC and also the C-repeat/Dehydration Response Element (CRT/DRE) consensus, CCGAC. Assays are known in the art that detect the interaction of a DNA binding protein with a target DNA sequence (e.g., electrophoretic mobility shift assays, DNAse I footprinting assays and the like). By performing such assays in the presence and absence of test compounds, for example, auxin or hydrogen peroxide, these assays can be used to identify compounds that modulate (e.g., inhibit or enhance) the interaction of the DNA binding protein with its target DNA sequence.
As used herein, “wherein the stress resistance or tolerance of a plant is increased relative to the stress resistance or tolerance of a control plant that is non-transgenic for a MAPKKK of the present invention”, refers to an increase in plant growth and/or yield and/or improved resistance to stress compared to the control plant. For example, stress resistance or tolerance of a plant to a particular abiotic stress, for example, salinity, may be assessed by comparing physical features and characteristics of plant growth, such as, plant height and weight, leaf area, plant water relations, ability to flower, ability to generate seeds, yield/productivity and sugar content of transgenic plants and non-transgenic control plants. (Shou, et al., (2004) J Exp Bot. 55(399):1013-9). In another aspect, physical features and characteristics of plant growth of the transgenic MAPKKK plant observed under abiotic stress may also be compared to the physical features and characteristics of a transgenic MAPKKK plant or a control plant non-transgenic for MAPKKKs of the invention that are not exposed to the abiotic stress so that “normal” plant growth and characteristics may be further evaluated.
By “increasing stress resistance or tolerance” is meant mediating a level of endurance, adaptability, or durability to a stress (e.g., a man-made stress, such as pollution or an environmental stress, such as drought, salinity and oxidative and temperature stresses) in a transgenic plant which is greater than that exhibited by a control plant (for example, a non-transgenic plant). Preferably, the level of stress resistance or tolerance in a transgenic plant (or transformed plant cell, plant component, plant tissue, or plant organ) of the invention is at least 5%, 10% or 20% (and preferably 30% or 40%) greater than the tolerance to a stress exhibited in a non-transgenic control plant (or control plant cell, plant component, plant tissue or plant organ). In other preferred embodiments, the level of stress resistance or tolerance to a stress is 50% greater, 60% greater and more preferably even 75% or 90% greater than a control plant, with up to 100% above the level of tolerance as compared to a control plant being most preferred. The level of stress resistance or tolerance is measured by conventional methods used to determine plant growth and response to stress. For example, the level of stress tolerance to salinity may be determined by comparing physical features and characteristics (for example, plant height and weight, leaf area, plant water relations, ability to flower, ability to generate seeds and yield/productivity) of transgenic plants and non-transgenic control plants.
Fragments and variants of the MAPKKK polynucleotides and proteins encoded thereby can be employed in the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and hence retain MAPKKK activity, for example, a constitutively active MAPKKK created by deletion of its putative regulatory domains.
As used interchangeably herein, a “MAPKKK activity”, “biological activity of MAPKKK” or “functional activity of MAPKKK”, refers to an activity exerted by a MAPKKK protein, polypeptide or portion thereof as determined in vivo or in vitro, according to standard techniques. In one embodiment, a MAPKKK activity is a direct activity, such as an association with a MAPKKK-target molecule. As used herein, a “target molecule” is a molecule with which a MAPKKK protein binds or interacts in nature, such that MAPKKK-mediated function is achieved. A MAPKKK target molecule can be a non-MAPKK molecule or a MAPKKK protein or polypeptide of the present invention or a MAPKK protein or polypeptide. In an exemplary embodiment, a MAPKKK target molecule is a MAPKKK substrate (including, for example, but not limited to ZmMPK4 (MAP kinase 4, Genbank Accession Number BAA74733) or ZmMPK5 (MAP kinase 5, Genbank Accession Number BAA74734.1). In a preferred embodiment, a MAPKKK activity is at least one or more of the following activities: (i) interaction of a MAPKKK protein with soluble MAPKKK ligand (e.g., but not limited to ZmMPK4 or ZmMPK5 and the like); (ii) modulation of the activity of a MAPKKK substrate; (iii) activation of a MAPKKK substrate; (iv) indirect modulation of a downstream signaling molecule (e.g., MAPKK.) In yet another preferred embodiment, a MAPKKK activity is at least one or more of the following activities: (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of gene transcription in a cell expressing a MAPKKK protein; (3) regulation of gene transcription in a cell expressing a MAPKKK protein, wherein said cell is involved in abiotic stress resistance or tolerance; (4) regulation of cellular proliferation; (5) regulation of cellular differentiation; (6) regulation of development and (7) regulation of cell death.
Alternatively, fragments of a polynucleotide that are useful as hybridization probes or PCR primers generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, up to the full-length polynucleotide encoding the proteins employed in the invention.
A fragment of a MAPKKK polynucleotide that encodes a biologically active portion of a MAPKKK protein employed in the invention will encode at least 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 220 or 225 contiguous amino acids, or up to the total number of amino acids present in a full-length or partial MAPKKK protein of the invention (for example, 441, 514, 366 or 392 amino acids for SEQ ID NOS: 2, 5, 8 and 10, respectively).
A biologically active portion of a MAPKKK protein can be prepared by isolating a portion of one of the MAPKKK polynucleotides employed in the invention, expressing the encoded portion of the MAPKKK protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the MAPKKK protein. Polynucleotides that are fragments of a MAPKKK nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 500, 550, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100 nucleotides or up to the number of nucleotides present in a full-length MAPKKK polynucleotide disclosed herein (for example, 1396, 1864, 1662 and 1375 nucleotides for SEQ ID NOS: 1, 4, 7 and 9 respectively).
“Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the MAPKKK polypeptides of the invention. Naturally occurring variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a MAPKKK protein employed in the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 50%, 55%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
Variants of a particular polynucleotide employed in the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, an isolated polynucleotide that encodes a polypeptide with a given percent sequence identity to any one of the polypeptides of SEQ ID NO: 2, 5, 8 or 10 is encompassed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 50%, 55%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
“Variant” protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, MAPKKK activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native MAPKKK protein of the invention will have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 5, 3, 2 or even 1 amino acid residue.
The proteins employed in the methods of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the MAPKKK proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:588-592; Kunkel, et al., (1987) Methods in Enzymol. 155:367-382; U.S. Pat. No. 5,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal. Variants of MAPKKK polypeptides can also include isolating natural variants from plants cells that exist in nature or creating recombinant MAPKKKs.
Thus, the genes and polynucleotides employed in the invention include both the naturally-occurring sequences as well as mutant forms. Likewise, the proteins employed in the invention encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired MAPKKK activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and optimally will not create complementary regions that could produce secondary mRNA structure.
The deletions, insertions and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity and/or expression can be evaluated by in gel kinase assays, real time RT-PCR analysis, Northern, Westerns, electrophoretic mobility shift assays, DNAse I footprinting assays and the like. (Shou, et al., (2004) J Exp Bot. 55(399):1013-9). Assays for detecting such activity or expression are known to one skilled in the art. Alternately, they are described in detail elsewhere herein. For example, an oligonucleotide of at least 15, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides in length and sufficient to specifically hybridize under stringent conditions to MAPKKK mRNA may be used in Northern blot analysis. MAPKKK proteins may be detected using a labeled antibody capable of binding to MAPKKKs proteins of the present invention. Antibodies can be polyclonal, or more preferably, monoclonal. An isolated MAPKKK protein, or fragment thereof, can be used as an immunogen to generate antibodies that bind specifically to MAPKKKs of the present invention using standard techniques for polyclonal and monoclonal antibody preparation. Techniques for detection of MAPKKK protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different MAPKKK coding sequences can be manipulated to create a new MAPKKK possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the MAPKKK gene of the invention and other known MAPKKK genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased Km in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1995) Proc. Natl. Acad. Sci. USA 91:10757-10751; Stemmer, (1995) Nature 370:389-391; Crameri, et al., (1997) Nature Biotech. 15:536-538; Moore, et al., (1997) J. Mol. Biol. 272:336-357; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 95:5505-5509; Crameri, et al., (1998) Nature 391:288-291 and U.S. Pat. Nos. 5,605,793 and 5,837,558.
The polynucleotides employed in the invention can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly other monocots. In this manner, methods such as PCR, hybridization and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire MAPKKK sequences set forth in SEQ ID NO: 1, 4, 7 or 9 or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. “Orthologs” is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode a MAPKKK protein and which hybridize under stringent conditions to the sequence of SEQ ID NO: 1, 4, 7 or 9 or to complements, variants or fragments thereof, are encompassed by the present invention.
In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York) and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers and the like.
In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides, and may be labeled with a detectable group such as 32P or another detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the MAPKKK polynucleotides of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
For example, the entire MAPKKK polynucleotide disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding MAPKKK polynucleotide and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among MAPKKK polynucleotide sequences and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length. Such probes may be used to amplify corresponding MAPKKK polynucleotide from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optimally less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 50 to 55% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 25 hours, usually about 5 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, (1985) Anal. Biochem. 138:267-285: Tm=81.5° C.+16.6 (log M)+0.51 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 5° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 15, 15 or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 55° C. (aqueous solution) or 32° C. (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York) and Ausubel, et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity” and (d) “percentage of sequence identity.”
(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 50, 100 or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, (1988) CABIOS 5:11-17; the local alignment algorithm of Smith, et al., (1981) Adv. Appl. Math. 2:582; the global alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 58:553-553; the search-for-local alignment method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2555-2558; the algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA 872265, modified as in Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA Accelrys® GCG® (Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins, et al., (1988) Gene 73:237-255 (1988); Higgins, et al., (1989) CABIOS 5:151-153; Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992) CABIOS 8:155-65 and Pearson, et al., (1995) Meth. Mol. Biol. 25:307-331. The ALIGN program is based on the algorithm of Myers and Miller, (1988) supra. A PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 5 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul, et al., (1990) J. Mol. Biol. 215:503 are based on the algorithm of Karlin and Altschul, (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul, et al., (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See, Altschul, et al., (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The United States' National Center for Biotechnology Information and the European Bioinformatics Institute of the European Molecular Biology Laboratory provide such tools, as do various commercial entities known to those of skill in the art. Alignment may also be performed manually by inspection.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix. GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 58:553-553, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG® Wisconsin Genetics Software Package® for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, 55, 50, 55, 60, 65 or greater.
(c) As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
An “isolated” or “purified” polynucleotide or protein or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 5 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
Methods
I. Providing Sequences
The sequences of the present invention can be introduced/expressed in a host cell such as bacteria, yeast, insect, mammalian or optimally plant cells. It is expected that those of skill in the art are knowledgeable in the numerous systems available for the introduction of a polypeptide or a nucleotide sequence of the present invention into a host cell. No attempt to describe in detail the various methods known for providing proteins in prokaryotes or eukaryotes will be made.
By “host cell” is meant a cell which comprises a heterologous nucleic acid sequence of the invention. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian or mammalian cells. Host cells can also be monocotyledonous or dicotyledonous plant cells. In one embodiment, the monocotyledonous host cell is a maize host cell.
The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally-occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.
A MAPKKK polynucleotide employed of the invention can be provided in expression cassettes for expression in the plant of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to a MAPKKK polynucleotide. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a promoter is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, operably linked means that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the MAPKKK polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
The expression cassette will include, in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a MAPKKK polynucleotide of the invention and a transcriptional and translational termination region (i.e., termination region) functional in plants. The regulatory regions (including promoters, transcriptional regulatory regions and translational termination regions) and/or the MAPKKK polynucleotide of the invention may be native/analogous to the host cell and/or to each other. Alternatively, the regulatory regions and/or the MAPKKK polynucleotide of the invention may be foreign/heterologous to the host cell and/or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a promoter that is heterologous to the coding sequence.
While it may be optimal to express the sequences using heterologous promoters, the native promoter sequences may be used. Such constructs can change the expression levels of the MAPKKK in the plant or plant cell. Thus, the phenotype of the plant or plant cell can be altered.
The termination region may be native with the transcriptional initiation region, may be native with the operably linked MAPKKK polynucleotide of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the MAPKKK polynucleotide of interest, the plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991) Mol. Gen. Genet. 262:151-155; Proudfoot, (1991) Cell 65:671-675; Sanfacon, et al., (1991) Genes Dev. 5:151-159; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) Nucleic Acids Res. 15:9627-9639.
Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant by using plant-preferred codons. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831 and 5,536,391 and Murray, et al., (1989) Nucleic Acids Res. 17:577-598, herein incorporated by reference.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
The expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 155:9-20) and human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-95); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 5) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, N.Y.), pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 85:965-968. Other methods known to enhance translation can also be utilized, for example, introns and the like.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones and 2,5-dichlorophenoxyacetate (2,5-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su, et al., (2005) Biotechnol Bioeng 85:610-9 and Fetter, et al., (2005) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte, et al., (2005) J. Cell Science 117:953-55 and Kato, et al., (2002) Plant Physiol 129:913-52) and yellow florescent protein (PhiYFPTM from Evrogen, see, Bolte, et al., (2005) J. Cell Science 117:953-55). For additional selectable markers, see generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6315-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2519-2522; Barkley, et al., (1980) in The Operon, pp.177-220; Hu, et al., (1987) Cell 58:555-566; Brown, et al., (1987) Cell 59:603-612; Figge, et al., (1988) Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA 86:5500-5505; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86:2559-2553; Deuschle, et al., (1990) Science 258:580-583; Gossen, (1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol. 10:3353-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:5657-5653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:153-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1095-1105; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5557-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill, et al., (1988) Nature 335:721-725. Such disclosures are herein incorporated by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.
A number of promoters can be used in the practice of the invention, including the native promoter of the polynucleotide sequence of interest. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible, or other promoters for expression in plants.
Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/53838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1985) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and the like. Other constitutive promoters include, for example, those disclosed in U.S. Pat. Nos. 5,608,159; 5,608,155; 5,605,121; 5,569,597; 5,566,785; 5,399,680; 5,268,563; 5,608,152 and 6,177,611.
Tissue-preferred promoters can be utilized to target enhanced type A RR expression within a particular plant tissue. By “tissue-preferred” is intended to mean that expression is predominantly in a particular tissue, albeit not necessarily exclusively in that tissue. Tissue-preferred promoters include Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet. 255(3):337-353; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1351; Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-525; Yamamoto, et al., (1995) Plant Cell Physiol. 35(5):773-778; Lam, (1995) Results Probl. Cell Differ. 20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J. 5(3):595-505. Such promoters can be modified, if necessary, for weak expression. See, also, US Patent Application Publication Number 2003/0074698, herein incorporated by reference.
Leaf-preferred promoters are known in the art. See, for example, Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1995) Plant Physiol. 105:357-67; Yamamoto, et al., (1995) Plant Cell Physiol. 35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138; Baszczynski, et al., (1988) Nucl. Acid Res. 16:5732; Mitra, et al., (1995) Plant Molecular Biology 26:35-93; Kayaya, et al., (1995) Molecular and General Genetics 258:668-675 and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590. Senescence regulated promoters are also of use, such as, SAM22 (Crowell, et al., (1992) Plant Mol. Biol. 18:559-566). See also, U.S. Pat. No. 5,589,052, herein incorporated by reference.
Shoot-preferred promoters include, shoot meristem-preferred promoters such as promoters disclosed in Weigal, et al., (1992) Cell 69:853-859; Accession Number AJ131822; Accession Number Z71981; Accession Number AF059870, the ZAP promoter (U.S. patent application Ser. No. 10/387,937), the maize tb1 promoter (Wang, et al., (1999) Nature 398:236-239 and shoot-preferred promoters disclosed in McAvoy, et al., (2003) Acta Hort. (ISHS) 625:379-385.
Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire, et al., (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. Biol. 15(3):533-553 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633-651, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus and in both instances root-specific promoter activity was preserved. Leach and Aoyagi, (1991) describe their analysis of the promoters of the highly expressed roIC and roID root-inducing genes of Agrobacterium rhizogenes (see, Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri, et al., (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2′ gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see, EMBO J. 8(2):353-350). The TR1′ gene, fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(5):759-772); roIB promoter (Capana, et al., (1995) Plant Mol. Biol. 25(5):681-691; and the CRWAQ81 root-preferred promoter with the ADH first intron (U.S. Provisional Patent Application Ser. No. 60/509,878, filed Oct. 9, 2003, herein incorporated by reference). See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,559,252; 5,501,836; 5,110,732 and 5,023,179.
“Seed-preferred” promoters include “seed-specific” promoters (those promoters active only in seed tissues, such as promoters of seed storage proteins). Seed-preferred promoters include those that are active either before or after pollination, or those that are active independent of pollination. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase) (see, WO 00/11177 and U.S. Pat. No. 6,225,529; herein incorporated by reference); PCNA2 (U.S. patent application Ser. No. 10/388,359, filed Mar. 13, 2003) and CKX1-2 (US Patent Application Publication Number 2002/0152500). Gamma-zein is an endosperm-specific promoter. Globulin-1 (Glob-1) is a representative embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also, WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed and WO 01/21783 and U.S. Pat. No. 6,403,862, where the Zm40 promoter is disclosed; both herein incorporated by reference. Embryo-specific promoters include ESR (US Patent Application Publication Number 2004/0210960) and lec1 (U.S. patent application Ser. No. 09/718,754, filed Nov. 22, 2000). Additional embryo specific promoters are disclosed in Sato, et al., (1996) Proc. Natl. Acad. Sci. 93:8117-8122; Nakase, et al., (1997) Plant J 12:235-56 and Postma-Haarsma, et al., (1999) Plant Mol. Biol. 39:257-71. Endosperm-preferred promoters include eep1 and eep2 as disclosed in US Patent Application Publication Number 2004/0237147. Additional endosperm specific promoters are disclosed in Albani, et al., (1985) EMBO 3:1505-15; Albani, et al., (1999) Theor. Appl. Gen. 98:1253-62; Albani, et al., (1993) Plant J. 5:353-55; Mena, et al., (1998) The Plant Journal 116:53-62 and Wu, et al., (1998) Plant Cell Physiology 39:885-889. Also of interest is the maize eep5 promoter (for example, see SEQ ID NO: 20). In maize, immature-ear tissue-preferred promoters can also be employed; for example, the ADF4 promoter (US Patent Application Publication Number 2009/0094713)
Dividing cell or meristematic tissue-preferred promoters have been disclosed in Ito, et al., (1995) Plant Mol. Biol. 25:863-878; Reyad, et al., (1995) Mol. Gen. Genet. 258:703-711; Shaul, et al., (1996) Proc. Natl. Acad. Sci. 93:5868-5872; Ito, et al., (1997) Plant J. 11:983-992; Trehin, et al., (1997) Plant Molecular Biology 35:667-672; Zag1 (Schmidt, et al., (1993) The Plant Cell 5:729-37) and Zag2 from maize (Theissen, et al., (1995) Gene 156:155-166) GenBank Accession Number X80206 and Hubbard, et al., (2002) Genetics 162:1927-1935, all of which are herein incorporated by reference. Certain promoters are active during the time of germination; see, Thompson, et al., (1989) BioEssays 10:108.
Inflorescence-preferred promoters include the promoter of chalcone synthase (Van der Meer, et al., (1990) Plant Mol. Biol. 15:95-109), LAT52 (Twell, et al., (1989) Mol. Gen. Genet. 217:250-255), pollen specific genes (Albani, et al., (1990) Plant Mol Biol. 15:605, Zm13 (Buerrero, et al., (1993) Mol. Gen. Genet. 225:161-168), maize pollen-specific gene (Hamilton, et al., (1992) Plant Mol. Biol. 18:211-218), sunflower pollen expressed gene (Baltz, et al., (1992) The Plant Journal 2:713-721), B. napus pollen specific genes (Arnoldo, et al., (1992) J. Cell. Biochem, Abstract Number Y101205). Immature ear tissue-preferred promoters can also be employed.
Stress inducible promoters include salt/water stress-inducible promoters such as P5CS (Zang, et al., (1997) Plant Sciences 129:81-89); cold-inducible promoters, such as, cor15a (Hajela, et al., (1990) Plant Physiol. 93:1256-1252), cor15b (Wilhelm, et al., (1993) Plant Mol Biol 23:1073-1077), wsc120 (Ouellet, et al., (1998) FEBS Lett. 523:325-328), ci7 (Kirch, et al., (1997) Plant Mol Biol. 33:897-909), ci21A (Schneider, et al., (1997) Plant Physiol. 113:335-55); and MLIP15 (U.S. Pat. No. 6,479,734) drought-inducible promoters, such as, Trg-31 (Chaudhary, et al., (1996) Plant Mol. Biol. 30:1257-57), rd29 (Kasuga, et al., (1999) Nature Biotechnology 18:287-291); osmotic inducible promoters, such as, Rab17 (Vilardell, et al., (1991) Plant Mol. Biol. 17:985-93; see also, SEQ ID NO: 18), also inducible by abscisic acid and osmotin (Raghothama, et al., (1993) Plant Mol Biol 23:1117-28) and heat inducible promoters, such as, heat shock proteins (Barros, et al., (1992) Plant Mol. 19:665-75; Marrs, et al., (1993) Dev. Genet. 15:27-51), senescence inducible promoters, such as SEE1 (GB_AJ494982) and smHSP (Waters, et al., (1996) J. Experimental Botany 57:325-338). Other stress-inducible promoters include rip2 (U.S. Pat. Nos. 5,332,808 and 7,074,985) and RD29A (Yamaguchi-Shinozaki, et al., (1993) Mol. Gen. Genetics 236:331-340; see also SEQ ID NO: 19).
Nitrogen-responsive promoters can also be used in the methods of the invention. Such promoters include, but are not limited to, the 22 kDa Zein promoter (Spena, et al., (1982) EMBO J 1:1589-1594 and Muller, et al., (1995) J. Plant Physiol 145:606-613); the 19 kDa zein promoter (Pedersen, et al., (1982) Cell 29:1019-1025); the 14 kDa zein promoter (Pedersen, et al., (1986) J. Biol. Chem. 261:6279-6284), the b-32 promoter (Lohmer, et al., (1991) EMBO J 10:617-624) and the nitrite reductase (NiR) promoter (Rastogi, et al., (1997) Plant Mol Biol. 34(3):465-76 and Sander, et al., (1995) Plant Mol Biol. 27(1):165-77). For a review of consensus sequences found in nitrogen-induced promoters, see for example, Muller, et al., (1997) The Plant Journal 12:281-291.
Other useful promoters include F3.7 (U.S. Pat. No. 5,850,018) and the maize thioredoxin H promoter (Nu, et al., MGCNL 2004; U.S. Provisional Patent Application Ser. No. 60/514,123). A promoter may fall into none, one, or more of the above groupings and may have utility in the present invention with respect to its tissue-specificity or timing or other characteristic, or with respect to a combination of such characteristics.
In addition, the constructs may contain control regions that regulate as well as engender expression. Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcription, such as transcription factors, repressor binding sites and termination signals, among others. For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancers useful in the invention to increase transcription of the introduced DNA segment, include, inter alia, viral enhancers like those within the 35S promoter, as shown by Odell, et al., (1988) Plant Mol. Biol. 10:263-72, and an enhancer from an opine gene as described by Fromm, et al., (1989) Plant Cell 1:977. The enhancer may affect the tissue-specificity and/or temporal specificity of expression of sequences included in the vector.
Termination regions also facilitate effective expression by ending transcription at appropriate points. Useful terminators for practicing this invention include, but are not limited to, pinII (see, An, et al., (1989) Plant Cell 1(1):115-122), glb1 (see, Genbank Accession Number L22345), gz (see, gzw64a terminator, Genbank Accession Number S78780) and the nos terminator from Agrobacterium. 
The methods of the invention involve introducing a polypeptide or polynucleotide into a plant. “Introducing” is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods and virus-mediated methods.
“Stable transformation” is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway, et al., (1986) Biotechniques 5:320-335), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (Townsend, et al., U.S. Pat. No. 5,563,055; Zhao, et al., U.S. Pat. No. 5,981,850), direct gene transfer (Paszkowski, et al., (1985) EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for example, Sanford, et al., U.S. Pat. No. 5,955,050; Tomes, et al., U.S. Pat. No. 5,879,918; Tomes, et al., U.S. Pat. No. 5,886,255; Bidney, et al., U.S. Pat. No. 5,932,782; Tomes, et al., (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Berlin); McCabe, et al., (1988) Biotechnology 6:923-926) and Lec1 transformation (WO 00/28058). Also see, Weissinger, et al., (1988) Ann. Rev. Genet. 22:521-577; Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-675 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor. Appl. Genet. 96:319-325 (soybean); Datta, et al., (1990) Biotechnology 8:736-750 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:5305-5309 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,250,855; Buising, et al., U.S. Pat. Nos. 5,322,783 and 5,325,656; Tomes, et al., (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg, (Springer-Verlag, Berlin) (maize); Klein, et al., (1988) Plant Physiol. 91:550-555 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1985) Nature (London) 311:763-765; Bowen, et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 85:5355-5359 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:515-518 and Kaeppler, et al., (1992) Theor. Appl. Genet. 85:560-566 (whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell 5:1595-1505 (electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:507-513 (rice); Osjoda, et al., (1996) Nature Biotechnology 15:755-750 (maize via Agrobacterium tumefaciens); Leelavathi, et al., (2004) Plant Cell Reports 22:465-470 (cotton via Agrobacterium tumefaciens); Kumar, et al., (2004) Plant Molecular Biology 56:203-216 (cotton plastid via bombardment), all of which are herein incorporated by reference.
In specific embodiments, the MAPKKK sequences employed in the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the MAPKKK protein or variants and fragments thereof directly into the plant or the introduction of the MAPKKK transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al., (1986) Plant Sci. 55:53-58; Hepler, et al., (1995) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush, et al., (1995) The Journal of Cell Science 107:775-785, all of which are herein incorporated by reference. Alternatively, the MAPKKK polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use particles coated with polyethylimine (PEI; Sigma #P3153).
In other embodiments, the polynucleotide of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that a MAPKKK of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931 and Porta, et al., (1996) Molecular Biotechnology 5:209-221, herein incorporated by reference.
Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99/25821, WO99/25855, WO99/25850, WO99/25855 and WO99/25853, all of which are herein incorporated by reference. Briefly, the polynucleotide of the invention can be contained in transfer cassette flanked by two non-identical recombination sites. The transfer cassette is introduced into a plant have stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-85. These plants may then be pollinated with either the same transformed strain or different strains, and the resulting progeny having desired expression of the phenotypic characteristic of interest can be identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds can be harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides a transformed seed (also referred to as a “transgenic seed”) having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into its genome.
Pedigree breeding generally starts with the crossing of two genotypes, such as an elite line of interest and one other line having one or more desirable characteristics (e.g., having stably incorporated a polynucleotide of the invention, having a modulated activity and/or level of the polypeptide of the invention) which complements the elite line of interest. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive filial generations. In the succeeding filial generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding, five or more successive filial generations of selfing and selection are practiced: F1→F2; F2→F3; F3→F5; F5→F5, etc. After a sufficient amount of inbreeding, successive filial generations will serve to increase seed of the developed inbred. Preferably, the inbred line comprises homozygous alleles at about 95% or more of its loci.
In addition to being used to create a backcross conversion, backcrossing can also be used in combination with pedigree breeding to modify an elite line of interest and a hybrid that is made using the modified elite line. As discussed previously, backcrossing can be used to transfer one or more specifically desirable traits from one line, the donor parent, to an inbred called the recurrent parent, which has overall good agronomic characteristics yet lacks that desirable trait or traits. However, the same procedure can be used to move the progeny toward the genotype of the recurrent parent but at the same time retain many components of the non-recurrent parent by stopping the backcrossing at an early stage and proceeding with selfing and selection. For example, an F1, such as a commercial hybrid, is created. This commercial hybrid may be backcrossed to one of its parent lines to create a BC1 or BC2. Progeny are selfed and selected so that the newly developed inbred has many of the attributes of the recurrent parent and yet several of the desired attributes of the non-recurrent parent. This approach leverages the value and strengths of the recurrent parent for use in new hybrids and breeding.
Therefore, an embodiment of this invention is a method of making a backcross conversion of maize inbred line of interest, comprising the steps of crossing a plant of maize inbred line of interest with a donor plant comprising a mutant gene or transgene conferring a desired trait (i.e., increased root growth, increased yield, increased tolerance to drought, increased or maintained seed set during abiotic conditions, increased shoot growth, delayed senescence or increased photosynthesis), selecting an F1 progeny plant comprising the mutant gene or transgene conferring the desired trait and backcrossing the selected F1 progeny plant to the plant of maize inbred line of interest. This method may further comprise the step of obtaining a molecular marker profile of maize inbred line of interest and using the molecular marker profile to select for a progeny plant with the desired trait and the molecular marker profile of the inbred line of interest. In the same manner, this method may be used to produce an F1 hybrid seed by adding a final step of crossing the desired trait conversion of maize inbred line of interest with a different maize plant to make F1 hybrid maize seed comprising a mutant gene or transgene conferring the desired trait.
Recurrent selection is a method used in a plant breeding program to improve a population of plants. The method entails individual plants cross pollinating with each other to form progeny. The progeny are grown and the superior progeny selected by any number of selection methods, which include individual plant, half-sib progeny, full-sib progeny, selfed progeny and topcrossing. The selected progeny are cross-pollinated with each other to form progeny for another population. This population is planted and again superior plants are selected to cross pollinate with each other. Recurrent selection is a cyclical process and therefore can be repeated as many times as desired. The objective of recurrent selection is to improve the traits of a population. The improved population can then be used as a source of breeding material to obtain inbred lines to be used in hybrids or used as parents for a synthetic cultivar. A synthetic cultivar is the resultant progeny formed by the intercrossing of several selected inbreds.
Mass selection is a useful technique when used in conjunction with molecular marker enhanced selection. In mass selection seeds from individuals are selected based on phenotype and/or genotype. These selected seeds are then bulked and used to grow the next generation. Bulk selection requires growing a population of plants in a bulk plot, allowing the plants to self-pollinate, harvesting the seed in bulk and then using a sample of the seed harvested in bulk to plant the next generation. Instead of self pollination, directed pollination could be used as part of the breeding program.
Mutation breeding is one of many methods that could be used to introduce new traits into an elite line. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means including temperature, long-term seed storage, tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (emitted from radioisotopes such as phosphorus 32 or carbon 15) or ultraviolet radiation (preferably from 2500 to 2900 nm) or chemical mutagens (such as base analogues (5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid or acridines. Once a desired trait is observed through mutagenesis the trait may then be incorporated into existing germplasm by traditional breeding techniques, such as backcrossing. Details of mutation breeding can be found in “Principles of Cultivar Development” Fehr, 1993, Macmillan Publishing Company, the disclosure of which is incorporated herein by reference. In addition, mutations created in other lines may be used to produce a backcross conversion of elite lines that comprise such mutations.
The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays, also known as maize), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals and conifers.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.) and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis) and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum.
Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta) and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea) and cedars such as Western red cedar (Thuja pilicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean plants are optimal and in yet other embodiments corn plants are optimal.
Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
Typically, an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the nucleic acid of interest can be isolated in significant quantities for introduction into the desired plant cells. In one embodiment, plant promoters that do not cause expression of the polypeptide in bacteria are employed.
Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:5057) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake, et al., (1981) Nature 292:128). The inclusion of selection markers in DNA vectors transfected in E coli. is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline or chloramphenicol.
The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene 22:229-235); Mosbach, et al., (1983) Nature 302:553-555).
A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, a polynucleotide of the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.
Synthesis of heterologous polynucleotides in yeast is well known (Sherman, et al., (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory). Two widely utilized yeasts for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
A protein of the present invention, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lists. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
The sequences of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect or plant origin. Illustrative cell cultures useful for the production of the peptides are mammalian cells. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21 and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen, et al., (1986) Immunol. Rev. 89:59) and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV50 large T Ag poly A addition site) and transcriptional terminator sequences. Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection.
Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (see, Schneider, (1987) J. Embryol. Exp. Morphol. 27:353-365).
As with yeast, when higher animal or plant host cells are employed, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV50 (Sprague, et al., (1983) J. Virol. 55:773-781). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors (Saveria-Campo, (1985) DNA Cloning Vol. II a Practical Approach, Glover, Ed., IRL Press, Arlington, Va., pp. 213-238).
Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextrin, electroporation, biolistics and micro-injection of the DNA directly into the cells. The transfected cells are cultured by means well known in the art (Kuchler, (1997) Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc.).
In certain embodiments the nucleic acid sequences of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. The combinations generated may include multiple copies of any one of the polynucleotides of interest. For example, a polynucleotide of the present invention may be stacked with any other polynucleotide(s) of the present invention. The polynucleotides of the present invention can also be stacked with any other gene or combination of genes involved in abiotic stress tolerance including for example, polynucleotides involved in osmoprotection, antioxidant responses, and/or membrane stability. One such polynucleotide includes but is not limited to C-repeat Binding Factor (CBF) a transcription factor that is known to bind to C-repeat element (CRT), otherwise known as Dehydration Response Element (DRE) element. (See, US Patent Application Publication Number 2006/0026716 and U.S. Pat. Nos. 6,706,866; 6,417,428; 5,965,705; 5,929,305; 5,892,009 and 5,891,859, the disclosures of which are herein incorporated by reference). DRE's are present in the promoters of several genes induced by abiotic stress in the form of drought or cold. CRT/DRE elements are known to be present in the promoters of several dehydrin or LEA (late embryonic abundant) genes. Rodriguez, et al., (2005) Theor. Appl Genet. 110(5):852-858; Kobayashi, (2004), Regulation of cold-responsive Cor/Lea genes and their transcription factors by the major freezing tolerance locus Fr-1 in wheat, In Recent research developments in plant science, Vol. 2, pages 249-266. Transgenic plants overexpressing CBF is known to accumulate higher levels, than non-transgenic plants, of sugars and proline which are considered to be osmoprotectants. See, for example, Yamada, et al., (2005) J. Exp. Botany 56(417):1975-1981. Thus, in one aspect of the invention, a MAPKKK of the present invention is stacked with a CBF. In one aspect, the MAPKKK polynucleotide is ZmNPK1a. In one aspect, the CBF is CBF1. (See, U.S. Pat. Nos. 6,706,866; 6,417,428; 5 965,705; 5,929,305; 5,892,009 and 5,891,859, the disclosures of which are herein incorporated by reference). In one aspect, the CBF1 is from maize. (See, SEQ ID NO: 94 of U.S. Pat. No. 6,417,428). In one aspect, the CBF is driven by the same promoter as the MAPKKK polynucleotide. In one aspect, the CBF is driven by a promoter different than the MAPKKK polynucleotide. In one aspect, the promoter is a stress-inducible promoter. In another aspect, the promoter is RAB17. (Vilardell, et al., (1990) Plant Mol Biol 14:423432).
The polynucleotides of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including but not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802 and 5,703,409); barley high lysine (Williamson, et al., (1987) Eur. J. Biochem. 165:99-106 and WO 98/20122) and high methionine proteins (Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene 71:359 and Musumura, et al., (1989) Plant Mol. Biol. 12:123)); increased digestibility (e.g., modified storage proteins (U.S. patent application Ser. No. 10/053,410, filed Nov. 7, 2001) and thioredoxins (U.S. patent application Ser. No. 10/005,429, filed Dec. 3, 2001)), the disclosures of which are herein incorporated by reference. The polynucleotides of the present invention can also be stacked with traits desirable for insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser, et al., (1986) Gene 48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol. 24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones, et al., (1994) Science 266:789; Martin, et al., (1993) Science 262:1432; Mindrinos, et al., (1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene) and glyphosate resistance (EPSPS gene)) and traits desirable for processing or process products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)) and polymers or bioplastics (e.g., U.S. Pat. No. 5.602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert, et al., (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the present invention with polynucleotides affecting agronomic traits such as male sterility, stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364; WO 99/25821).
These stacked combinations can be created by any method including but not limited to cross breeding plants by any conventional or TopCross methodology or genetic transformation. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant.
II. Modulating the Concentration and/or Activity of a MAPKKK Polypeptide A method for modulating the concentration and/or activity of a polypeptide of the present invention in a plant is provided. In general, concentration and/or activity is increased or decreased by at least 1%, 5%, 10%, 20%, 30%, 50%, 50%, 60%, 70%, 80% or 90% relative to a native control plant, plant part, or cell. Modulation in the present invention may occur at any desired stage of development. In specific embodiments, the polypeptides of the present invention are modulated in monocots, particularly maize.
A “subject plant or plant cell” is one in which genetic alteration, such as transformation, has been effected as to a gene of interest or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of the subject plant or plant cell.
A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.
The expression level of the MAPKKK polypeptide may be measured directly, for example, by assaying for the level of the MAPKKK polypeptide in the plant, or indirectly, for example, by measuring the MAPKKK activity of the MAPKKK polypeptide in the plant. Methods for determining the MAPKKK activity are described elsewhere herein and include evaluation of phenotypic changes, such as increased abiotic stress resistance or tolerance.
In specific embodiments, the MAPKKK polypeptide or polynucleotide of the invention is introduced into the plant cell. Subsequently, a plant cell having the introduced sequence is selected using methods known to those of skill in the art such as, but not limited to, Southern blot analysis, DNA sequencing, PCR analysis or phenotypic analysis. A plant or plant part altered by the foregoing embodiments is grown under plant forming conditions for a time sufficient to allow modulation of the concentration and/or activity of polypeptides of the present invention in the plant. Plant forming conditions are well known in the art and are discussed briefly elsewhere herein.
It is also recognized that the level and/or activity of the polypeptide may be modulated by employing a polynucleotide that is not capable of directing, in a transformed plant, the expression of a protein or an RNA. For example, the polynucleotides of the invention may be used to design polynucleotide constructs that can be employed in methods for altering or mutating a genomic nucleotide sequence in an organism. Such polynucleotide constructs include, but are not limited to, RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases. Such nucleotide constructs and methods of use are known in the art. See, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,985; all of which are herein incorporated by reference. See also, WO 98/59350, WO 99/07865, WO 99/25821 and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA 96:8775-8778; herein incorporated by reference.
It is therefore recognized that methods of the present invention do not depend on the incorporation of the entire polynucleotide into the genome, only that the plant or cell thereof is altered as a result of the introduction of the polynucleotide into a cell. In one embodiment of the invention, the genome may be altered following the introduction of the polynucleotide into a cell. For example, the polynucleotide, or any part thereof, may be incorporated into the genome of the plant. Alterations to the genome of the present invention include, but are not limited to, additions, deletions and substitutions of nucleotides into the genome. While the methods of the present invention do not depend on additions, deletions and substitutions of any particular number of nucleotides, it is recognized that such additions, deletions or substitutions comprise at least one nucleotide.
A. Increasing the Activity and/or Level of a MAPKKK Polypeptide
Methods are provided to increase the activity and/or level of a MAPKKK polypeptide. An increase in the level and/or activity of the MAPKKK polypeptide of the invention can be achieved by providing to the plant a MAPKKK polypeptide. The MAPKKK polypeptide can be provided by introducing the amino acid sequence encoding the MAPKKK polypeptide into the plant, introducing into the plant a nucleotide sequence encoding a MAPKKK polypeptide, or alternatively, by modifying a genomic locus encoding the MAPKKK polypeptide. In one aspect, a polynucleotide encoding a MAPKKK polypeptide of the invention is over-expressed in a plant cell or plant. As used herein, the term “over-express”, “over-expressed”, “over-expressing” or “over-expression” refers to the production of a MAPKKK polynucleotide and/or polypeptide in a plant cell in amounts exceeding that normally produced in the plant cell. The MAPKKK polynucleotides and/or polypeptides of the invention may be over-expressed in the plant cell at a specified point in time or at particular stage of plant development.
As discussed elsewhere herein, many methods are known in the art for providing a polypeptide to a plant including, but not limited to, direct introduction of the polypeptide into the plant, introducing into the plant (transiently or stably) a polynucleotide construct encoding a polypeptide having MAPKKK activity. It is also recognized that the methods of the invention may employ a polynucleotide that is not capable of directing, in the transformed plant, the expression of a protein or an RNA. Thus, the level and/or activity of a MAPKKK polypeptide may be increased by altering the gene encoding the MAPKKK polypeptide or its promoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868. Therefore mutagenized plants that carry mutations in MAPKKK genes, where the mutations increase expression of the MAPKKK gene or increase the MAPKKK activity of the encoded MAPKKK polypeptide are provided. As used herein, the term “over-express”, “over-expressing” or “over-expression” refers to the production of a MAPKKK polynucleotide and/or polypeptide in a plant cell in amounts exceeding that normally produced in the plant cell.
B. Reducing the Activity and/or Level of a MAPKKK Polypeptide
Methods are provided to reduce or eliminate the level and/or the activity of a MAPKKK polypeptide by transforming a plant cell with an expression cassette that expresses a polynucleotide that inhibits the expression of the MAPKKK polypeptide. The polynucleotide may inhibit the expression of one or more MAPKKK polypeptides directly, by preventing translation of the MAPKKK messenger RNA or indirectly, by encoding a polypeptide that inhibits the transcription or translation of a plant gene encoding a MAPKKK polypeptide. Methods for inhibiting or eliminating the expression of a gene in a plant are well known in the art, and any such method may be used in the present invention to inhibit the expression of one or more MAPKKK polypeptide.
In accordance with the present invention, the expression of a MAPKKK polypeptide is inhibited if the protein level of the MAPKKK polypeptide is statistically significantly lower than the protein level of the same MAPKKK polypeptide in a plant that has not been genetically modified or mutagenized to inhibit the expression of that protein. In particular embodiments of the invention, the protein level of the MAPKKK polypeptide in a modified plant according to the invention is less than 96%, less than 90%, less than 80%, less than 75%, less than 60%, less than 50%, less than 50%, less than 30%, less than 20%, less than 10% or less than 5% of the protein level of the same MAPKKK polypeptide in a plant that is not a mutant or that has not been genetically modified to inhibit the expression of that MAPKKK polypeptide. The expression level of the MAPKKK polypeptide may be measured directly, for example, by assaying for the level of MAPKKK polypeptide expressed in the plant cell or plant or indirectly, for example, by measuring the activity of the MAPKKK polypeptide in the plant cell or plant. Methods for determining the MAPKKK activity of MAPKKK polypeptide are described elsewhere herein.
In other embodiments of the invention, the activity of one or more MAPKKK is reduced or eliminated by transforming a plant cell with an expression cassette comprising a polynucleotide encoding a polypeptide that inhibits the activity of one or more MAPKKK. The MAPKKK activity of a MAPKKK is inhibited according to the present invention if the MAPKKK activity of the MAPKKK is statistically significantly lower than the activity of the same MAPKKK in a plant that has not been genetically modified to inhibit the MAPKKK activity of that. In particular embodiments of the invention, the MAPKKK activity of the MAPKKK in a modified plant according to the invention is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 50%, less than 30%, less than 20%, less than 10% or less than 5% of the MAPKKK activity of the same MAPKKK in a plant that that has not been genetically modified to inhibit the expression of that MAPKKK. The MAPKKK activity of a MAPKKK is “eliminated” according to the invention when it is not detectable by the assay methods described elsewhere herein. Methods of determining the MAPKKK activity of a MAPKKK are described elsewhere herein.
In other embodiments, the activity of a MAPKKK may be reduced or eliminated by disrupting the gene encoding the MAPKKK. The invention encompasses mutagenized plants that carry mutations in MAPKKK genes, where the mutations reduce expression of the MAPKKK gene or inhibit the MAPKKK activity of the encoded MAPKKK.
Thus, many methods may be used to reduce or eliminate the activity of a MAPKKK. More than one method may be used to reduce the activity of a single MAPKKK. In addition, combinations of methods may be employed to reduce or eliminate the activity of two or more different MAPKKK polypeptides.
Non-limiting examples of methods of reducing or eliminating the expression of a MAPKKK are given below.
1. Polynucleotide-Based Methods
In some embodiments of the present invention, a plant cell is transformed with an expression cassette that is capable of expressing a polynucleotide that inhibits the expression of MAPKKK polypeptides. The term “expression” as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. For example, for the purposes of the present invention, an expression cassette capable of expressing a polynucleotide that inhibits the expression of at least one MAPKKK polypeptide is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of at least one MAPKKK polypeptide. The “expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while the “expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide.
Examples of polynucleotides that inhibit the expression of a MAPKKK polypeptide are given below.
i. Sense Suppression/Cosuppression
In some embodiments of the invention, inhibition of the expression of a MAPKKK polypeptide may be obtained by sense suppression or cosuppression. For cosuppression, an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding a MAPKKK polypeptide in the “sense” orientation. Over-expression of the RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the cosuppression expression cassette are screened to identify those that show the greatest inhibition of MAPKKK polypeptide expression.
The polynucleotide used for cosuppression may correspond to all or part of the sequence encoding the MAPKKK polypeptide, all or part of the 5′ and/or 3′ untranslated region of a MAPKKK transcript or all or part of both the coding sequence and the untranslated regions of a transcript encoding MAPKKK polypeptide. In some embodiments where the polynucleotide comprises all or part of the coding region for the MAPKKK polypeptide, the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be transcribed.
Cosuppression may be used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes. See, for example, Broin, et al., (2002) Plant Cell 15:1517-1532. Cosuppression may also be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,952,657. Methods for using cosuppression to inhibit the expression of endogenous genes in plants are described in Flavell, et al., (1995) Proc. Natl. Acad. Sci. USA 91:3590-3596; Jorgensen, et al., (1996) Plant Mol. Biol. 31:957-973; Johansen and Carrington, (2001) Plant Physiol. 126:930-938; Broin, et al., (2002) Plant Cell 15:1517-1532; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Yu, et al., (2003) Phytochemistry 63:753-763 and U.S. Pat. Nos. 5,035,323, 5,283,185 and 5,952,657; each of which is herein incorporated by reference. The efficiency of cosuppression may be increased by including a poly-dT region in the expression cassette at a position 3′ to the sense sequence and 5′ of the polyadenylation signal. See, US Patent Application Publication Number 2002/0058815, herein incorporated by reference. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, optimally greater than about 65% sequence identity, more optimally greater than about 85% sequence identity, most optimally greater than about 95% sequence identity. See, U.S. Pat. Nos. 5,283,185 and 5,035,323; herein incorporated by reference.
Transcriptional gene silencing (TGS) may be accomplished through use of hpRNA constructs wherein the inverted repeat of the hairpin shares sequence identity with the promoter region of a gene to be silenced. Processing of the hpRNA into short RNAs which can interact with the homologous promoter region may trigger degradation or methylation to result in silencing. (Aufsatz, et al., (2002) PNAS 99(4):16499-16506; Mette, et al., (2000) EMBO J 19(19):5194-5201) See also, US Patent Application Publication Number 2005/0246796.
ii. Antisense Suppression
In some embodiments of the invention, inhibition of the expression of the MAPKKK polypeptide may be obtained by antisense suppression. For antisense suppression, the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the MAPKKK polypeptide. Over-expression of the antisense RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the antisense suppression expression cassette are screened to identify those that show the greatest inhibition of MAPKKK polypeptide expression.
The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the MAPKKK polypeptide, all or part of the complement of the 5′ and/or 3′ untranslated region of the MAPKKK polypeptide transcript or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the MAPKKK polypeptide. In addition, the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,952,657. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 500, 550, 500, 550 or greater may be used. Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, for example, in Liu, et al., (2002) Plant Physiol. 129:1732-1753 and U.S. Pat. Nos. 5,759,829 and 5,952,657. Efficiency of antisense suppression may be increased by including a poly-dT region in the expression cassette at a position 3′ to the antisense sequence and 5′ of the polyadenylation signal. See, US Patent Application Publication Number 2002/0058815.
iii. Double-Stranded RNA Interference
In some embodiments of the invention, inhibition of the expression of a MAPKKK polypeptide may be obtained by double-stranded RNA (dsRNA) interference. For dsRNA interference, a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.
Expression of the sense and antisense molecules can be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the greatest inhibition of MAPKKK polypeptide expression. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA 95:13959-13965, Liu, et al., (2002) Plant Physiol. 129:1732-1753 and WO 99/59029, WO 99/53050, WO 99/61631 and WO 00/59035.
iv. Hairpin RNA Interference and Intron-Containing Hairpin RNA Interference
In some embodiments of the invention, inhibition of the expression of one or more type A RR polypeptide may be obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference. These methods are highly efficient at inhibiting the expression of endogenous genes. See, Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 5:29-38 and the references cited therein.
For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem. The base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence. Thus, the base-paired stem region of the molecule generally determines the specificity of the RNA interference. hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:5985-5990; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731 and Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 5:29-38. Methods for using hpRNA interference to inhibit or silence the expression of genes are described, for example, in Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:5985-5990; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 5:29-38; Pandolfini, et al., BMC Biotechnology 3:7 and US Patent Application Publication Number 2003/0175965; each of which is herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga, et al., (2003) Mol. Biol. Rep. 30:135-150.
For ihpRNA, the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron that is capable of being spliced in the cell in which the ihpRNA is expressed. The use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith, et al., (2000) Nature 507:319-320. In fact, Smith, et al., show 100% suppression of endogenous gene expression using ihpRNA-mediated interference. Methods for using ihpRNA interference to inhibit the expression of endogenous plant genes are described, for example, in Smith, et al., (2000) Nature 507:319-320; Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001) Curr. Opin. Plant Biol. 5:156-150; Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 5:29-38; Helliwell and Waterhouse, (2003) Methods 30:289-295 and US Patent Application Publication Number 2003/0180955.
The expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, it is the loop region that determines the specificity of the RNA interference. See, for example, WO 02/00905.
v. Amplicon-Mediated Interference
Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus. The viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication. The transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for MAPKKK polypeptide). Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in Angell and Baulcombe, (1997) EMBO J. 16:3675-3685, Angell and Baulcombe, (1999) Plant J. 20:357-362 and U.S. Pat. No. 6,656,805.
vi. Ribozymes
In some embodiments, the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of MAPKKK polypeptide. Thus, the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the MAPKKK polypeptide. This method is described, for example, in U.S. Pat. No. 5,987,071.
vii. Small Interfering RNA or Micro RNA
In some embodiments of the invention, inhibition of the expression of one or more MAPKKK polypeptides may be obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA). miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNA are highly efficient at inhibiting the expression of endogenous genes. See, for example Javier, et al., (2003) Nature 525:257-263, herein incorporated by reference.
For miRNA interference, the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene. The miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence). For suppression of MAPKKK polypeptide expression, the 22-nucleotide sequence is selected from a MAPKKK transcript sequence and contains 22 nucleotides of said MAPKKK polypeptide sequence in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.
2. Polypeptide-Based Inhibition of Gene Expression
In one embodiment, the polynucleotide encodes a zinc finger protein that binds to a gene encoding a MAPKKK polypeptide, resulting in reduced expression of the gene. In particular embodiments, the zinc finger protein binds to a regulatory region of a MAPKKK polypeptide gene. In other embodiments, the zinc finger protein binds to a messenger RNA encoding a MAPKKK polypeptide and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,553,252 and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in US Patent Application Publication Number 2003/0037355.
3. Polypeptide-Based Inhibition of Protein Activity
In some embodiments of the invention, the polynucleotide encodes an antibody that binds to at least one MAPKKK polypeptide and reduces the MAPKKK activity of the MAPKKK polypeptide. In another embodiment, the binding of the antibody results in increased turnover of the antibody-MAPKKK polypeptide complex by cellular quality control mechanisms. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald, (2003) Nature Biotech. 21 :35-36, incorporated herein by reference.
4. Gene Disruption
In some embodiments of the present invention, the activity of a MAPKKK polypeptide is reduced or eliminated by disrupting the gene encoding the MAPKKK polypeptide. The gene encoding the MAPKKK polypeptide may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing plants using random or targeted mutagenesis, and selecting for plants that have reduced MAPKKK activity.
i. Transposon Tagging
In one embodiment of the invention, transposon tagging is used to reduce or eliminate the MAPKKK activity of one or more MAPKKK polypeptides. Transposon tagging comprises inserting a transposon within an endogenous MAPKKK polypeptide gene to reduce or eliminate expression of the MAPKKK polypeptide. “MAPKKK gene” is intended to mean the gene that encodes a MAPKKK polypeptide according to the invention.
In this embodiment, the expression of one or more MAPKKK polypeptides is reduced or eliminated by inserting a transposon within a regulatory region or coding region of the gene encoding the MAPKKK polypeptide. A transposon that is within an exon, intron, 5′ or 3′ untranslated sequence, a promoter or any other regulatory sequence of a MAPKKK gene may be used to reduce or eliminate the expression and/or activity of the encoded MAPKKK polypeptide.
Methods for the transposon tagging of specific genes in plants are well known in the art. See, for example, Maes, et al., (1999) Trends Plant Sci. 5:90-96; Dharmapuri and Sonti, (1999) FEMS Microbiol. Lett. 179:53-59; Meissner, et al., (2000) Plant J. 22:265-275; Phogat, et al., (2000) J. Biosci. 25:57-63; Walbot, (2000) Curr. Opin. Plant Biol. 2:103-107; Gai, et al., (2000) Nucleic Acids Res. 28:95-96; Fitzmaurice, et al., (1999) Genetics 153:1919-1928). In addition, the TUSC process for selecting Mu insertions in selected genes has been described in Bensen, et al., (1995) Plant Cell 7:75-85; Mena, et al., (1996) Science 275:1537-1550 and US Patent Number 5,962,765.
ii. Mutant Plants with Reduced Activity
Additional methods for decreasing or eliminating the expression of endogenous genes in plants are also known in the art and can be similarly applied to the instant invention. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted. For examples of these methods see, Ohshima, et al., (1998) Virology 253:572-581; Okubara, et al., (1995) Genetics 137:867-875 and Quesada, et al., (2000) Genetics 155:521-536. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention. See, McCallum, et al., (2000) Nat. Biotechnol. 18:555-557.
Mutations that impact gene expression or that interfere with the function (MAPKKK activity) of the encoded protein are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the MAPKKK activity of the encoded protein. Such mutants can be isolated according to well-known procedures, and mutations in different type A RR loci can be stacked by genetic crossing. See, for example, Gruis, et al., (2002) Plant Cell 15:2863-2882.
In another embodiment of this invention, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of a duplicated gene locus. See, for example, Kusaba, et al., (2003) Plant Cell 15:1555-1567.
The invention encompasses additional methods for reducing or eliminating the activity of one or more MAPKKK polypeptides. Examples of other methods for altering or mutating a genomic nucleotide sequence in a plant are known in the art and include, but are not limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases. Such vectors and methods of use are known in the art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,985. See also, WO 98/59350, WO 99/07865, WO 99/25821 and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA 96:8775-8778.
iii. Modulating the Stress Tolerance of a Plant Methods are provided for the use of the MAPKKK sequences of the invention to modulate the tolerance of a plant to abiotic stress. In specific embodiments, methods are provided to increase or maintain plant growth and development during abiotic stress episodes. During periods of stress (i.e., drought, salt, heavy metals, temperature extremes, etc.) plant development is often delayed or reduced. Modulating the level and/or activity of a MAPKKK sequence of the invention can maintain or improve plant growth, even under stress. Particularly vulnerable developmental periods include early seedling development and flowering. In one method, a MAPKKK nucleotide sequence is introduced into the plant and the level and/or activity of the MAPKKK polypeptide is modulated, thereby improving the tolerance of the plant to stress conditions and maintaining growth, which may be reflected in, for example, the rate of shoot growth, the extent of root development, the success of anthesis and seed set or the number or size of seed produced. Often the introduced MAPKKK nucleotide construct is stably incorporated into the genome of the plant and transmitted to progeny.
Methods to assay for a modulation in seed set during abiotic stress are known in the art. For example, plants having the modulated MAPKKK activity can be monitored under various stress conditions and compared to controls plants. For instance, the plant having the modulated MAPKKK activity and/or level can be subjected to various degrees of stress during flowering and seed set. Under identical conditions, the genetically modified plant having the modulated level and/or activity of MAPKKK polypeptide will have a higher number and/or mass of developing seed than a wild type (non-transformed) plant.
Accordingly, the present invention further provides plants having increased yield or a maintained yield during periods of abiotic stress (i.e. drought, salt, heavy metals, temperature extremes, etc). In some embodiments, the plants having an increased or maintained yield during abiotic stress have a modulated level/activity of a MAPKKK polypeptide of the invention. In other embodiments, the plant comprises a MAPKKK nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell. In certain embodiments, such plants have stably incorporated into their genome a nucleic acid molecule comprising a MAPKKK nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell.
iv. Modulating Shoot and Leaf Development
Methods are also provided for modulating shoot and leaf development in a plant. By “modulating shoot development” and/or “modulating leaf development” is intended any alteration in the development of the plant shoot and/or leaf. Such alterations in shoot and/or leaf development include, but are not limited to, alterations in shoot meristem development, in leaf number, leaf size, leaf and stem vasculature, internode length and leaf senescence. As used herein, “leaf development” and “shoot development” encompass all aspects of growth of the different parts that make up the leaf system and the shoot system, respectively, at different stages of their development, both in monocotyledonous and dicotyledonous plants. Methods for measuring such developmental alterations in the shoot and leaf system are known in the art. See, for example, Werner, et al., (2001) PNAS 98:10587-10592 and US Patent Application Publication Number 2003/0075698, each of which is herein incorporated by reference.
The method for modulating shoot and/or leaf development in a plant comprises modulating the activity and/or level of a MAPKKK polypeptide of the invention. In one embodiment, a MAPKKK sequence of the invention is provided. In other embodiments, the MAPKKK nucleotide sequence can be provided by introducing into the plant a polynucleotide comprising a MAPKKK nucleotide sequence of the invention, expressing the MAPKKK sequence and thereby modifying shoot and/or leaf development. In other embodiments, the MAPKKK nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
In specific embodiments, shoot and/or leaf development is modulated by modulating the level and/or activity of the MAPKKK in the plant. A modulation in MAPKKK activity can result in at least one or more of the following alterations in shoot and/leaf development including, but not limited to, altered (increased or decreased) shoot growth, altered photosynthesis, modulated leaf number, altered leaf surface, altered length of internodes and modulated leaf senescence. Modulating the level of the MAPKKK polypeptide in the plant can thereby increase plant yields.
As discussed above, one of skill will recognize the appropriate promoter to use to modulate shoot and leaf development of the plant. Exemplary promoters for this embodiment include constitutive promoters or promoters that are preferentially active in photosynthetic tissues including, for example, shoot-preferred promoters, shoot meristem-preferred promoters and leaf-preferred promoters. Exemplary promoters have been disclosed elsewhere herein.
Accordingly, the present invention further provides plants having a modulated shoot and/or leaf development when compared to a control plant. In some embodiments, the plant of the invention has an increased level/activity or a decreased level/activity of a MAPKKK polypeptide of the invention.
Methods for establishing callus from explants are known. For example, roots, stems, buds, immature embryos and aseptically germinated seedlings are just a few of the sources of tissue that can be used to induce callus formation. Generally, young and actively growing tissues (i.e. young leaves, roots, meristems) are used, but are not required. Callus formation is controlled by growth regulating substances present in the medium (auxins and cytokinins). The specific concentrations of plant regulators needed to induce callus formation vary from species to species and can even depend on the source of explant. In some instances, it is advised to use different growth substances (i.e. 2,5-D or NAA) or a combination of them during tests, since some species may not respond to a specific growth regulator. In addition, culture conditions (i.e. light, temperature, etc.) can also influence the establishment of callus. Once established, callus cultures can be used to initiate shoot regeneration. See, for example, Gurel, et al., (2001) Turk J. Bot. 25:25-33; Dodds, et al., (1995). Experiments in Plant Tissue Culture, Cambridge University Press; Gamborg (1995) Plant Cell, Tissue and Organ Culture, eds. Phillips and US Patent Application Publication Number 2003/0180952, all of which are herein incorporated by reference.
It is further recognized that increasing seed size and/or weight can be accompanied by an increase in the rate of growth of seedlings or an increase in early vigor. In addition, modulating the plant's tolerance to stress, as discussed above, along with modulation of root, shoot and leaf development can increase plant yield and vigor. As used herein, the term “vigor” refers to the relative health, productivity and rate of growth of the plant and/or of certain plant parts, and may be reflected in one or more various developmental attributes, such as concentration of chlorophyll, photosynthetic rate, total biomass and root biomass. Of particular relevance is the ability of a plant to grow rapidly during early development, and relates to the successful establishment, after germination, of a well-developed root system and a well-developed photosynthetic apparatus. Improvements in vigor are measured with reference to a control as defined elsewhere herein.
v. Modulating Root Development
Methods for modulating root development in a plant are provided. By “modulating root development” is intended any alteration in the development of the plant root when compared to a control plant. Such alterations in root development include, but are not limited to, alterations in the growth rate of the primary root, the fresh root weight, the extent of lateral and adventitious root formation, the vasculature system, meristem development or radial expansion.
The methods for modulating root development comprise modulating (reducing or increasing) the level and/or activity of the MAPKKK polypeptide in the plant. In one method, a MAPKKK nucleotide sequence is introduced into the plant and the level and/or activity of the MAPKKK polypeptide is modulated. In other methods, the MAPKKK nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
A modulation in MAPKKK activity can result in at least one or more of the following alterations to root development, including, but not limited to, larger root meristems, increased root growth, enhanced radial expansion, an enhanced vasculature system, increased root branching, more adventitious roots and/or increased fresh root weight when compared to a control plant.
As used herein, “root growth” encompasses all aspects of growth of the different parts that make up the root system at different stages of its development in both monocotyledonous and dicotyledonous plants. It is to be understood that enhanced root growth can result from enhanced growth of one or more of its parts including the primary root, lateral roots, adventitious roots, etc. Methods of measuring such developmental alterations in the root system are known in the art. See, for example, US Patent Application Publication Number 2003/0075698 and Werner, et al., (2001) PNAS 18:10587-10592, both of which are herein incorporated by reference.
As discussed above, one of skill will recognize the appropriate promoter to use to modulate root development in the plant. Exemplary promoters for this embodiment include root-preferred promoters, which have been disclosed elsewhere herein.
Stimulating root growth and increasing root mass by modulating the activity and/or level of the polypeptide also finds use in improving the standability of a plant. The term “resistance to lodging” or “standability” refers to the ability of a plant to fix itself to the soil. For plants with an erect or semi-erect growth habit, this term also refers to the ability to maintain an upright position under adverse (environmental) conditions. This trait relates to the size, depth and morphology of the root system. In addition, stimulating root growth and increasing root mass by modulating the level and/or activity of the MAPKKK polypeptide also finds use in promoting in vitro propagation of explants.
Accordingly, the present invention further provides plants having modulated root development when compared to the root development of a control plant. In some embodiments, the plant of the invention has a modulated level/activity of the MAPKKK polypeptide of the invention and has enhanced root growth and/or root biomass. In other embodiments, such plants have stably incorporated into their genome a nucleic acid molecule comprising a MAPKKK nucleotide sequence of the invention operably linked to a root-preferred promoter that drives expression in the plant cell, wherein expression of the sequence modulates the level and/or activity of the MAPKKK polypeptide.
The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES
Example 1
Maize Transformation with the Sequences of the Invention
Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing an expression cassette MAPKKK, as detailed in methods described elsewhere herein. The MAPKKK polynucleotide is operably linked to a MAPKKK—promoter and the selectable marker gene PAT (Wohlleben, et al., (1988) Gene 70:25-37), which confers resistance to the herbicide Bialaphos. Alternatively, the selectable marker gene is provided on a separate plasmid. Transformation is performed as follows. Media recipes follow below.
The ears are husked and surface sterilized in 30% Clorox® bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 5 hours and then aligned within the 2.5 cm target zone in preparation for bombardment.
A plasmid vector comprising the maize RR5 sequence operably linked to a Zea mays RAB17 promoter is made. This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl2 precipitation procedure as follows: 100 μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA); 100 μl 2.5 M CaCl2 and 10 μl 0.1 M spermidine.
Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol and centrifuged for 30 seconds. Again the liquid is removed and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
The sample plates are bombarded at level #5 in particle gun #HE35-1 or #HE35-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-5 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5′′ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored under various stress conditions and compared to control plants. Alterations in phenotype, such as improved tolerance to stress, will be monitored.
Bombardment medium (560Y) comprises 5.0 g/l N6 basal salts (SIGMA C-1516), 1.0 ml/I Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,5-D and 2.88 g/l L-proline (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite® (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 5.0 g/l N6 basal salts (SIGMA C-1516), 1.0 ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose and 2.0 mg/l 2,5-D (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite® (added after bringing to volume with D-I H2O) and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).
Plant regeneration medium (288J) comprises 5.3 g/l MS salts (GIBCO 11117-075), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.50 g/l glycine brought to volume with polished D-I H2O) (Murashige and Skoog, (1962) Physiol. Plant. 15:573),100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H2O after adjusting to pH 5.6); 3.0 g/l Gelrite® (added after bringing to volume with D-I H2O) and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 5.3 g/l MS salts (GIBCO 11117-075), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.50 g/l glycine brought to volume with polished D-I H2O), 0.1 g/l myo-inositol and 50.0 g/l sucrose (brought to volume with polished D-I H2O after adjusting pH to 5.6) and 6 g/l BactoTM-agar solidifying agent (added after bringing to volume with polished D-I H2O), sterilized and cooled to 60° C.
Example 2
Modulating Plant Yields
For Agrobacterium-mediated transformation of maize with the MAPKKK nucleotide sequence (SEQ ID NO: 1, 4, 7 or 9) operably linked to a Zea mays ubiquitin promoter, or a stress-inducible promoter, the method of Zhao is employed (U.S. Pat. No. 5,981,850 and PCT Patent Publication Number WO098/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the MAPKKK nucleotide sequence to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos are immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional “resting” step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 5: the selection step). The immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium are cultured on solid medium to regenerate the plants.
The plants are monitored for a modulation in shoot growth, leaf senescence, and/or photosynthesis when compared to an appropriate control plant. A modulation in plant yield is also monitored.
Example 3
Soybean Transformation
Soybean embryos are bombarded with a plasmid containing the MAPKKK sequence operably linked to a Zea mays ubiquitin promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface-sterilized, immature seeds of the soybean cultivar A2872, are cultured in the light or dark at 26° C. on an appropriate agar medium for six to ten weeks. Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below.
Soybean embryogenic suspension cultures can be maintained in 35 ml liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium.
Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein, et al., (1987) Nature (London) 327:70-73, U.S. Pat. No. 5,955,050). A DuPont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.
A selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz, et al., (1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The expression cassette comprising the MAPKKK operably linked to the Zea mays ubiquitin promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (in order): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 500 μl 70% ethanol and resuspended in 50 μl of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded on each macro carrier disk.
Approximately 300-500 mg of a two-week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/ml hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post-bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
Example 4
Sunflower Meristem Tissue Transformation
Sunflower meristem tissues are transformed with an expression cassette containing the MAPKKK (SEQ ID NO: 1, 4, 7 or 9) operably linked to a Zea mays ubiquitin promoter or a stress-inducible promoter as follows (see also, European Patent Number EP 0 586233, herein incorporated by reference and Malone-Schoneberg, et al., (1995) Plant Science 103:199-207). Mature sunflower seed (Helianthus annuus L.) are dehulled using a single wheat-head thresher. Seeds are surface sterilized for 30 minutes in a 20% Clorox® bleach solution with the addition of two drops of Tween20® per 50 ml of solution. The seeds are rinsed twice with sterile distilled water.
Split embryonic axis explants are prepared by a modification of procedures described by Schrammeijer, et al., (Schrammeijer, et al., (1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled water for 60 minutes following the surface sterilization procedure. The cotyledons of each seed are then broken off, producing a clean fracture at the plane of the embryonic axis. Following excision of the root tip, the explants are bisected longitudinally between the primordial leaves. The two halves are placed, cut surface up, on GBA medium consisting of Murashige and Skoog mineral elements (Murashige, et al., (1962) Physiol. Plant., 15:573-597), Shepard's vitamin additions (Shepard, (1980) in Emergent Techniques for the Genetic Improvement of Crops (University of Minnesota Press, St. Paul, Minn.), 50 mg/l adenine sulfate, 30 g/l sucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-acetic acid (IAA), 0.1 mg/l gibberellic acid (GA3), pH 5.6, and 8 g/l Phytagar.
The explants are subjected to microprojectile bombardment prior to Agrobacterium treatment (Bidney, et al., (1992) Plant Mol. Biol. 18:301-313). Thirty to forty explants are placed in a circle at the center of a 60×20 mm plate for this treatment. Approximately 5.7 mg of 1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are used per bombardment. Each plate is bombarded twice through a 150 mm nytex screen placed 2 cm above the samples in a PDS 1000® particle acceleration device.
Disarmed Agrobacterium tumefaciens strain EHA105 is used in all transformation experiments. A binary plasmid vector comprising the expression cassette that contains the RR6 gene operably linked to the Zea mays ubiquitin promoter is introduced into Agrobacterium strain EHA105 via freeze-thawing as described by Holsters, et al., (1978) Mol. Gen. Genet. 163:181-187. This plasmid further comprises a kanamycin selectable marker gene (i.e., nptII). Bacteria for plant transformation experiments are grown overnight (28° C. and 100 RPM continuous agitation) in liquid YEP medium (10 gm/l yeast extract, 10 gm/l Bacto®peptone and 5 gm/l NaCl, pH 7.0) with the appropriate antibiotics required for bacterial strain and binary plasmid maintenance. The suspension is used when it reaches an OD600 of about 0.5 to 0.8. The Agrobacterium cells are pelleted and resuspended at a final OD600 of 0.5 in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH5Cl, and 0.3 gm/l MgSO5.
Freshly bombarded explants are placed in an Agrobacterium suspension, mixed, and left undisturbed for 30 minutes. The explants are then transferred to GBA medium and co-cultivated, cut surface down, at 26° C. and 18-hour days. After three days of co-cultivation, the explants are transferred to 375B (GBA medium lacking growth regulators and a reduced sucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/l kanamycin sulfate. The explants are cultured for two to five weeks on selection and then transferred to fresh 375B medium lacking kanamycin for one to two weeks of continued development. Explants with differentiating, antibiotic-resistant areas of growth that have not produced shoots suitable for excision are transferred to GBA medium containing 250 mg/l cefotaxime for a second 3-day phytohormone treatment. Leaf samples from green, kanamycin-resistant shoots are assayed for the presence of NPTII by ELISA and for the presence of transgene expression by assaying for MAPKKK activity.
NPTII-positive shoots are grafted to PIONEER® hybrid 6550 in vitro-grown sunflower seedling rootstock. Surface sterilized seeds are germinated in 58-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3% Gelrite® pH 5.6) and grown under conditions described for explant culture. The upper portion of the seedling is removed, a 1 cm vertical slice is made in the hypocotyl, and the transformed shoot inserted into the cut. The entire area is wrapped with PARA film® to secure the shoot. Grafted plants can be transferred to soil following one week of in vitro culture. Grafts in soil are maintained under high humidity conditions followed by a slow acclimatization to the greenhouse environment. Transformed sectors of To plants (parental generation) maturing in the greenhouse are identified by NPTII ELISA and/or by MAPKKK activity analysis of leaf extracts while transgenic seeds harvested from NPTII-positive To plants are identified by MAPKKK activity analysis of small portions of dry seed cotyledon.
Example 5
Rice Transformation
One method for transforming DNA into cells of higher plants that is available to those skilled in the art is high-velocity ballistic bombardment using metal particles coated with the nucleic acid constructs of interest (see, Klein, et al., (1987) Nature (London) 327:70-73 and see, U.S. Pat. No. 4,945,050). A Biolistic PDS-1000/He (BioRAD Laboratories, Hercules, Calif.) is used for these complementation experiments.
The bacterial hygromycin B phosphotransferase (Hpt II) gene from Streptomyces hygroscopicus that confers resistance to the antibiotic may be used as the selectable marker for rice transformation. In the vector, the Hpt II gene may be engineered with the 35S promoter from Cauliflower Mosaic Virus and the termination and polyadenylation signals from the octopine synthase gene of Agrobacterium tumefaciens. For example, see the description of vector pML18 in WO97/47731, published on Dec. 18, 1997, the disclosure of which is hereby incorporated by reference.
Embryogenic callus cultures derived from the scutellum of germinating rice seeds serve as source material for transformation experiments. This material is generated by germinating sterile rice seeds on a callus initiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-D and 10 μM AgNO3) in the dark at 27-28° C. Embryogenic callus proliferating from the scutellum of the embryos is transferred to CM media (N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D, Chu, et al., (1985) Sci. Sinica 18:659-668). Callus cultures are maintained on CM by routine sub-culture at two-week intervals and used for transformation within 10 weeks of initiation.
Callus is prepared for transformation by subculturing 0.5-1.0 mm pieces approximately 1 mm apart, arranged in a circular area of about 4 cm in diameter, in the center of a circle of Whatman® #541 paper placed on CM media. The plates with callus are incubated in the dark at 27-28° C. for 3-5 days. Prior to bombardment, the filters with callus are transferred to CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr in the dark. The petri dish lids are then left ajar for 20-45 minutes in a sterile hood to allow moisture on tissue to dissipate.
Each genomic DNA fragment is co-precipitated with pML18 (containing the selectable marker for rice transformation) onto the surface of gold particles. To accomplish this, a total of 10 μg of DNA at a 2:1 ratio of trait:selectable marker DNAs are added to 50 μl aliquot of gold particles that have been resuspended at a concentration of 60 mg ml−1. Calcium chloride (50 μl of a 2.5 M solution) and spermidine (20 μl of a 0.1 M solution) are then added to the gold-DNA suspension as the tube is vortexing for 3 min. The gold particles are centrifuged in a microfuge for 1 sec and the supernatant removed. The gold particles are washed twice with 1 ml of absolute ethanol and then resuspended in 50 μl of absolute ethanol and sonicated (bath sonicator) for one second to disperse the gold particles. The gold suspension is incubated at −70° C. for five minutes and sonicated (bath sonicator) if needed to disperse the particles. Six μl of the DNA-coated gold particles are then loaded onto Mylar® macrocarrier disks and the ethanol is allowed to evaporate.
At the end of the drying period, a petri dish containing the tissue is placed in the chamber of the PDS-1000/He. The air in the chamber is then evacuated to a vacuum of 28-29 inches Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1080-1100 psi. The tissue is placed approximately 8 cm from the stopping screen and the callus is bombarded two times. Two to four plates of tissue are bombarded in this way with the DNA-coated gold particles. Following bombardment, the callus tissue is transferred to CM media without supplemental sorbitol or mannitol.
Within 3-5 days after bombardment the callus tissue is transferred to SM media (CM medium containing 50 mg/l hygromycin). To accomplish this, callus tissue is transferred from plates to sterile 50 ml conical tubes and weighed. Molten top-agar at 40° C. is added using 2.5 ml of top agar/100 mg of callus. Callus clumps are broken into fragments of less than 2 mm diameter by repeated dispensing through a 10 ml pipette. Three ml aliquots of the callus suspension are plated onto fresh SM media and the plates are incubated in the dark for 4 weeks at 27-28° C. After 4 weeks, transgenic callus events are identified, transferred to fresh SM plates and grown for an additional 2 weeks in the dark at 27-28° C.
Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitsch vitamins, 2% sucrose, 3% sorbitol, 0.4% Gelrite®+50 ppm hyg B) for 2 weeks in the dark at 25° C. After 2 weeks the callus is transferred to RM2 media (MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4% Gelrite®+50 ppm hyg B) and placed under cool white light ( ̃40 μEm−2s−1) with a 12 hr photoperiod at 25° C. and 30-40% humidity. After 2-4 weeks in the light, callus begin to organize and form shoots. Shoots are removed from surrounding callus/media and gently transferred to RM3 media (1⁄2×MS salts, Nitsch and Nitsch vitamins, 1% sucrose+50 ppm hygromycin B) in PHYTATRAYTM disposable plant cell culture vessels (Sigma Chemical Co., St. Louis, Mo.) and incubation is continued using the same conditions as described in the previous step.
Plants are transferred from RM3 to 4′′ pots containing Metro mix 350 after 2-3 weeks, when sufficient root and shoot growth have occurred.
Example 6
Variants of MAPKKK
A. Variant Nucleotide Sequences of MAPKKK (SEQ ID NOS: 1, 4, 7, 9) That Do Not Alter the Encoded Amino Acid Sequence
The MAPKKK nucleotide sequences set forth in SEQ ID NOS: 1, 4, 7, 9 may be used to generate variant nucleotide sequences having 0%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity when compared to the starting unaltered ORF nucleotide sequence of SEQ ID NOS:1, 4, 7, 9. These functional variants are generated using a standard codon table. While the nucleotide sequence of the variant is altered, the amino acid sequence encoded by the open reading frame does not change.
B. Variant Amino Acid Sequences of MAPKKK
Variant amino acid sequences of MAPKKK may be generated. In this example, one amino acid may be altered. Specifically, the sequences set forth in SEQ ID NO: 2, 5, 8 or 10 may be reviewed to determine the appropriate amino acid alteration. The selection of the amino acid to change may be made by consulting the protein alignment. See, FIG. 2. An amino acid may be selected that is deemed not to be under high selection pressure (not highly conserved) and which is rather easily substituted by an amino acid with similar chemical characteristics (i.e., similar functional side-chain). Using the protein alignment set forth in FIG. 2 an appropriate amino acid can be changed. Amino acid residues that show a low percentage of sequence identity among the Zea mays MAPKKK proteins are not highlighted. Additional conserved residues can be found in FIG. 2. Once the targeted amino acid is identified, the procedure outlined in Example 5A is followed. Variants having about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NOS: 2, 5, 8 or 10 may be generated using this method.
C. Additional Variant Amino Acid Sequences of MAPKKK
In this example, artificial protein sequences are created having 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity relative to the reference protein sequence. This latter effort requires identifying conserved and variable regions from the alignment set forth in FIG. 2 and then the judicious application of an amino acid substitutions table. These parts will be discussed in more detail below.
Largely, the determination of which amino acid sequences may be altered is made based on the conserved regions among MAPKKK proteins. See, FIG. 2. Based on the sequence alignment, the various regions of the MAPKKK that can likely be altered can be determined. It is recognized that conservative substitutions can be made in the conserved regions without altering function. In addition, one of skill will understand that functional variants of the MAPKKK sequences of the invention may also have minor amino acid alterations in the conserved domain.
Artificial protein sequences are then created that are different from the original in the intervals of 80-85%, 85-90%, 90-95% and 95-100% identity. Midpoints of these intervals are targeted, with liberal latitude of plus or minus 1%, for example. The amino acids substitutions will be effected by a custom Perl script. The substitution table is provided below in Table 2.







TABLE 2







Substitution Table











Strongly Similar and
Rank of Order to



Amino Acid
Optimal Substitution
Change
Comment













I
L, V
1
50:50 substitution


L
I, V
2
50:50 substitution


V
I, L
3
50:50 substitution


A
G
5



G
A
5



D
E
6



E
D
7



W
Y
8



Y
W
9



S
T
10



T
S
11



K
R
12



R
K
13



N
Q
15



Q
N
15



F
Y
16



M
L
17
First methionine





cannot change


H

Na
No good





substitutes


C

Na
No good





substitutes


P

Na
No good





substitutes









First, any conserved amino acid in the protein that should not be changed is identified and “marked off” for insulation from the substitution. The start methionine will of course be added to this list automatically. Next, the changes are made.
H, C and P are not changed in any circumstance. The changes will occur with isoleucine first, sweeping N-terminal to C-terminal; then leucine and so on down the list until the desired target is reached. Interim number substitutions can be made so as not to cause reversal of changes. The list is ordered 1-17, so as many isoleucine changes are made as needed before leucine and so on down to methionine. Clearly many amino acids will in this manner not need to be changed. L, I and V will involve a 50:50 substitution of the two alternate optimal substitutions.
The variant amino acid sequences are written as output. Perl script is used to calculate the percent identities. Using this procedure, variants of MAPKKK are generated having about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the starting unaltered ORF sequence of SEQ ID NO: 3 or 6.
Example 7
Identification of the maize NPK1-related sequences
A maize MAPKKK that was orthologous to the rice NPK1-like gene (dbj|BAB64165.1| (AP003254) NPK1-related protein kinase-like protein [Oryza sativa] E=0.094 [5′(1),3′(0) PCL253028(1) cds3f.pk005.d19) was first identified from two expression profiling Agilent experiments, namely, ‘Cold-induced gene expression in B73 seedling shoots with time of exposure to low-temperature stress’, and ‘Stress-induced gene expression in CML349 seedling shoots with time of exposure to stress’.
The objective of these experiments was to develop a picture of the changing gene expression pattern under increasing times of exposure to dehydration stress, applied in the form of low-temperature stress. The first experiment involved growing of B73 seedlings for 14 days from planting, followed by imposition of a cold stress of 10° C., and then collecting the entire shoot tissue above the coleoptile at 0 h, 0.5 h, 1 h, 4 h, 8 h and 24 h of exposure to low temperature and also after 48 h of recovery at 25° C. following the last low-temperature stress treatment, namely, 24 h of cold stress. Pairwise comparisons were made to determine the nature of gene-expression changes at each time-point after exposure to cold, relative to the zero time control. This experiment gave an understanding of the timed induction of various genes and pathways in B73 that were turned on from the early to the later times of exposure to cold stress. It also helped to identify candidate genes and promoters for both drought and cold, by (1) determining if the genes with critical induction behavior under cold stress were reported in the literature as having relevance to drought stress and (2) if the expression of the genes with critical induction behavior under cold stress showed drought or stress-hormone (ABA, ethylene) related induction in Lynx MPSS experiments.
The above experiment was repeated using CML349 in place of B73. CML349 is a tropical highland maize line from CIMMYT that is known to have improved tolerance to cold temperatures. Following the second experiment, comparisons were made between timed induction of genes in CML349, the Mexican highland line that has tolerance to cold, and B73, the corn-belt dent line that has less tolerance to cold than CML349.
A maize EST with homology to a rice NPK1-like kinase exhibited an interesting behavior in these gene-expression profiling experiments. We found that the maize EST, cds3f.pk005.d19, showed highest expression levels at the early time point of 1 hour after cold exposure in CML349, while it was not induced to the same extent in B73 (Table 3, FIG. 1). The intensities of Agilent expression indicated that B73 had high expression levels of the gene under normal temperatures, which was further increased two-fold 4 h following exposure to cold-temperature stress. CML349, on the other hand, had low levels of expression of this gene under normal temperatures, but was induced almost 36-fold very early (1 h) after exposure to the cold stress. The induced level at the early time point of 1 h after stress exposure was greater than the level in B73 at the same time point, and also greater than the induced level in B73 4 h after exposure to stress. Our previous experiments with protein profiling (collaboration with Oxford GlycoSciences) had shown that the protein levels of several genes involved in combating oxidative stress were either normally high or induced to higher levels in CML349 relative to B73. This is consistent with reported action of the Arabidopsis ortholog of NPK1, namely ANP1, which itself is induced by hydrogen peroxide mimicking oxidative stress, and which when constitutively active increased the promoter activity of specific stress-responsive genes including GST6 (Kovtun, et al., (2000) Proc Natl Acad Sci 97:2940-2945).







TABLE 3







Stress-induced expression levels of ZmNPK1b in B73 and CML349.










B73











Time after stress
Intensity 1

CML349











exposure
(0 h)
Intensity 2
Intensity 1 (0 h)
Intensity 2














0.5 h at 10° C.
1247.9409
1030.0294
242.1342
645.7405


  1 h at 10° C.
1103.7903
2178.2920
110.8528
4462.0635


  4 h at 10° C.
1485.4978
3222.3875
117.6474
251.4074


  8 h at 10° C.
1205.4165
1020.0490
84.3675
124.7931


 24 h at 10° C.
1398.2936
1198.3543
219.4813
207.8984


 48 h Recovery
2025.9680
319.3025
399.1231
309.4664





ZmNPK1b expression levels in B73 and CML349 at different time periods after exposure to low-temperature stress, as measured by color intensity in microarray analysis (Agilent Technologies, Santa Clara, CA).


Intensity 2 denotes the gene expression at any given time point after exposure to stress.






Example 8
Isolation of the Maize NPK1-Related Sequences
Upon full-insert sequencing of EST cds3f.pk005.d19, it was revealed that it contained a partial clone. The sequence of cds3f.pk005.d19 was used in two ways to obtain full sequence of NPK1.
First, blast searches were done for homologs of the cds3f.pk005.d19 partial sequence against PHI contigs. Based on the results, PCO527001 (UC5.1) or PCO644860 (UC6.0) was identified as a close homolog and a representative EST, ctst1s.pk017.e17 was submitted to full-insert sequencing and subsequently found to be full-length.
The sequence of cds3f.pk005.d19 was also used for BAC-library screening. A BAC clone was identified (bacb.pk191.e03) and sequenced. Coding sequence information pieced together from genomic information in the BAC sequence was used to PCR out the full coding sequence (CDS). The full CDS sequence is represented in PCO0644861 (Top Blast UPI00000AA4AE NPK1-related protein kinase-like protein [Oryza sativa (japonica cultivar-group)] E=0.0 (Reference Proteins MAR-31-2006) [Members=20, ORFCode=5NOCXX] UC6.0). Consequently, full-insert sequencing of several component ESTs was requested and completed. Primers as set forth in SEQ ID NO: 12 and 13 were used to amplify Zm NPK1b complementary DNA sequence from the BAC clone, p1.bacb.pk191.e03.
The rice NPK1-related protein kinase that is identified as being orthologous to ctst1s.pk017.e17 (PCO644860) is NP—917080, and that homologous to cds3f.pk005.d19 (PCO644861) is NP—917084/BAB64165.1. The maize NPK1 sequence derived from the former was named ZmNPK1 a and that from the latter was named ZmNPK1b.
Subsequent searches were done using the tobacco NPK1 protein to identify rice sequences in the public domain with the closest homology and this yielded the rice protein BAF24980. The rice protein had 57.5% consensus and 48.2% identity with the tobacco protein. The rice protein was used to search Unicorn 6.0 and this search identified the closest maize ortholog to be PCO622918. This sequence is partial with an incomplete 5′ end and was named ZmNPK1c. In addition, while checking possible map location, it was identified that ZmNPK1a co-localized with an NPK1-related kinase represented by PCO638212, which was named ZmNPK1d. Sequence information for ZmNPK1a, ZmNPK1b, ZmNPK1c and ZmNPK1d are provided as SEQ ID NOS: 1-3, 4-6, 7-8, and 9-11, respectively.
Example 9
Chromosome Localization, Expression Information and Cell Specificity of the Identified Sequences:
The contigs pertinent to ZmNPK1a, ZmNPK1b, ZmNPK1c and ZmNPK1d (PCO644860, PCO644861, PCO622918 and PCO638212 respectively) were checked for possible chromosomal position using BLAST searches against public and proprietary BAC sequences. ZmNPK1a (PCO644860), ZmNPK1b (PCO644861) and ZmNPK1d (PCO638212) were located to chromosome 3, while ZmNPK1c was located to chromosome 2. ZmNPK1a and ZmNPK1d were found to be potentially associated with QTL for the Staygreen phenotype with a proprietary association tool. With this same tool it was observed that ZmNPK1c (PCO622918) shows potential association with yield QTLs and also with the drought QTLs for Anthesis Silking Interval, Staygreen and Barrencount. It should be noted that the proprietary association tool uses the proprietary QTL data that involves low-resolution phenotypic associations to chromosomal regions that may be as large as 75 cM for some traits. Accordingly, associations presented here do not verify that a specific gene is controlling these traits, rather they suggest which trait associations exist for consideration.
The native expression of all four sequences was analyzed in Lynx MPSS libraries. The expression of ZmNPK1a was found to be high in stalk and root tissue, with the highest expression of 204 ppm observed in pulvinus tissue of B73 stalks. Expression of ZmNPK1b was found to be high in kernel and root tissue, with the highest expression of 984 ppm in maize kernels at 0 days after pollination, closely followed by 928 ppm in maize primary roots. ZmNPK1c had very little representation in the Lynx libraries and thus has extremely low expression levels of the gene. Finally, the expression of ZmNPK1d was uniformly distributed in all tissues, the highest being 263 ppm in maize kernels at 0 days after pollination.
Cellular localization of all four sequences was checked scanning the protein sequences for typical target peptides using the commercially or publicly available research tools of TargetP, ChloroP, SIGNALP and PSORT. (Emanuelsson et al. (2007) Nature Protocols 2:953-971; Nakai and Horton (1999) Trends Biochem. Sci. 24(1):34-36) The results indicated that ZmNPK1a, ZmNPK1b and ZmNPK1d localize to the mitochondria in the plant cell. The localization for ZmNPK1c could not be predicted with any degree of confidence, likely due to its incomplete N-terminal region.
Example 10
Induction of Native MAPKKKs by Stress or Hormone Treatments
Stress-induced expression profiling of maize genes was conducted using Massively Parallel Signature Sequencing in Lynx (MPSSTM) (see, Brenner, et al., (2000) Nature Biotechnology 18:630-634, Brenner, et al., (2000) Proc Natl Acad Sci USA 97:1665-1670).
Maize hybrid 3245 was subjected to a severe water stress aimed to reduce yield by approximately 55-70% relative to the same hybrid under well-watered conditions. The stress treatment was imposed 5 weeks prior to anthesis, at about 650-700 GDU (growing-degree units), and continued for 2 weeks or over 300 GDU, post-anthesis. Each plot contained 4 rows of plants. During this stress period, samples were collected from ear leaves, immature ears at first silk and ear-base and ear-tip kernels at seven days after pollination, in both the well-watered and drought-stressed treatments. Samples were ground under liquid nitrogen, RNA extracted and subjected to expression profiling.
For cold-stress induction studies, maize seedlings of the inbred B73 or CML349 were germinated and grown under optimum temperature conditions in the greenhouse. Leaves of ten-day-old seedlings constituted the sample material. The experiment included three treatments, namely, control or optimum temperature, chilling treatment and recovery from freezing. Control seedlings that did not receive any cold stress were moved to a growth chamber maintained at the same temperature regime as the greenhouse, while seedlings intended for the chilling treatment were moved to a growth chamber maintained at 10° C. Seedlings intended for recovery from freezing were subjected to −2° C. for 2 hours and then allowed to recover at optimal temperature for 6 hours. Chilling and recovery from freezing were continued for a period of 6 hours. All three treatments were harvested at the end of the required time period, ground in liquid nitrogen and subjected to RNA extraction followed by expression profiling.
For hormone-induction studies, maize seedlings of the inbred B73 were grown in the greenhouse up to the V5 stage. At this time, the plants were treated with 0.1 mM ABA (abscisic acid) or 1 mM Ethephon plant growth regulator. Leaves from six plants thus treated were harvested at 0, 24 and 48 hours of ABA or Ethephon treatment, ground in liquid nitrogen, and subjected to RNA extraction followed by expression profiling. The plants harvested at 0 h of hormone treatment constituted the control.
As indicated in Table 4, ZmNPK1b and ZmNPK1d were found to be specifically induced by drought stress and treatment with the stress hormones ABA and ethylene. ZmNPK1b was induced by chilling.







TABLE 4







Stress-related expression of ZmNPK1b and ZmNPK1d in Lynx MPSS


libraries.











Name
PPM
Tissue
Genotype
Treatment










Stress-related expression of ZmNPK1b in Lynx MPSS libraries (Tag: GATCAGCGGATGCTTCG)











Cla24lm-sig
104
leaf
B73
Corn, B73, v5 ABA treated leaves, 24 hr


Cla48lm-sig
38
leaf
B73
Corn, B73, v5 ABA treated leaves, 48 hr


Cle24lm-sig
55
leaf
B73
Corn, B73, v5 Ethephon treated leaves, 24 hr


Cle48lm-sig
45
leaf
B73
Corn, B73, v5 Ethephon treated leaves, 48 hr


Cl0lm-sig
0
leaf
B73
Corn, B73, v5 leaves


Cktsslm-sig
34
seed
3245
Seed, 3245, drought stressed 7-DAP Apical Kernels


Ckbsslm-sig
108
seed
3245
Seed, 3245, drought stressed 7-DAP Basal Kernels


Cktwwlm-sig
7
seed
3245
Seed, 3245, well-watered 7-DAP Apical Kernels


Ckbwwlm-sig
0
seed
3245
Seed, 3245, well-watered 7-DAP Basal Kernels


Csdl1lm-chil
88
seedling
B73
seedling subjected to chilling treatment at 10° C.


Csdl1lm-ctr
8
seedling
B73
seedling, control







Stress-related expression of ZmNPK1d in Lynx MPSS libraries (Tag: GATCCCGGGTGTTGTGT)











Cla24lm-sig
99
leaf
B73
Corn, B73, v5 ABA treated leaves, 24 hr


Cla48lm-sig
205
leaf
B73
Corn, B73, v5 ABA treated leaves, 48 hr


Cle24lm-sig
75
leaf
B73
Corn, B73, v5 Ethephon treated leaves, 24 hr


Cle48lm-sig
19
leaf
B73
Corn, B73, v5 Ethephon treated leaves, 48 hr


Cl0lm-sig
52
leaf
B73
Corn, B73, v5 leaves


Cktsslm-sig
114
seed
3245
Seed, 3245, drought stressed 7-DAP Apical Kernels


Ckbsslm-sig
29
seed
3245
Seed, 3245, drought stressed 7-DAP Basal Kernels


Cktwwlm-sig
74
seed
3245
Seed, 3245, well watered 7-DAP Apical Kernels


Ckbwwlm-sig
155
seed
3245
Seed, 3245, well watered 7-DAP Basal Kernels


Csdl1lm-chil
16
seedling
B73
seedling subjected to chilling treatment at 10° C.


Csdl1lm-fro
39
seedling
B73
seedling recovering from freezing treatment at −2° C.









Example 11
Stress-Induced Expression of Transgenic MAPKKKs
The sequence of ZmNPK1a was incorporated into the maize transformation vector PHP29013 (RAB17::ZmNPK1a+RAB17::ZmCBF1) to test for efficacy under abiotic stress. Additional constructs have been made, as follows: RAB17::ZmNPK1b (PHP32420); RD29A::ZmNPK1a (PHP32647); RD29A::ZmNPK1b (PHP32984) and ZmEEP5::ZmNPK1a (PHP36818).
As previously indicated, the RAB17 and RD29A promoters are stress-induced. To test stress-induced expression of the MAPKKK transgenes, T1 plants comprising the RAB17::ZmNPK1b, RD29A::ZmNPK1a or RD29A::ZmNPK1b construct were grown under drought stress in the greenhouse, as described in Example 12. Leaf punches were taken from individual plants and stored at −80° C. for RNA extraction using the RNeasy 96 Kit from QIAGEN, Inc. (Valencia, Calif.). The cDNA was produced from total RNA using the QuantiTect Reverse Transcription Kit from QIAGEN as directed by the manufacturer and expression of the transgene of interest was analyzed by Q-PCR. Events of all three constructs have shown stress-inducible expression. Field testing under drought conditions is further used to evaluate effect of each of the transgenes. Field evaluation will include assessment for vivipary, which may be associated with use of rab17 and is generally an undesirable trait.
Example 12
Testing of Transgenic Plants Under Stress Conditions
Transgenic plants, for example those created using the methods of Example 1 or 2, expressing a recombinant MAPKKK of the present invention, may be subjected to artificial environments to simulate abiotic stresses, for example, cold, drought or limited water conditions, a combination of drought and heat, or salinity stress. Prior to applying the stress, plants are established in controlled growing conditions, as follows:
Nutrient Solution: Nutrient solution is prepared using a 20:10:20 NPK fertilizer mix, at a concentration of 3.7 ounces per 5 gallons of water. This stock solution is further diluted to 1/16th concentration with water and applied to plants.
It is useful to add half a teaspoon of Osmocote® (NPK 15:9:12) to the pot at the time of transplanting or after emergence (The Scotts Miracle-Gro Company, Ohio, USA).
Border plants: Place a row of border plants on bench-edges adjacent to the glass walls of the greenhouse or adjacent to other potential causes of microenvironment variability such as a cooler fan.
Automation: Watering can be done using PVC pipes with drilled holes to supply water to systematically positioned pots using a siphoning device. Irrigation scheduling can be done using timers.
Replications: Eight to ten individual plants are used per treatment per event.
Following treatment and data collection, mean values for plant size, color and chlorophyll fluorescence recorded on transgenic events under different stress treatments are exported to Spotfire (Spotfire, Inc., MA, USA). Treatment means are evaluated using Analysis of Variance.
A. Low-temperature Tolerance
To demonstrate whether expression of MAPKKKs of the present invention can confer increased germination ability under cool conditions, transgenic seeds expressing a MAPKKK polypeptide of the present invention may be germinated under conditions similar to the standard cold germination tests used in the seed industry. Alternatively, transgenic seeds expressing such a MAPKKK may be planted under seed bed conditions made cool by artificial environments or naturally cool seed beds in the field. Additionally, plants expressing the MAPKKK may be challenged during the seed development period by cool night time temperatures to demonstrate decreased inhibition of leaf or canopy activity as a result of cold stress during this time of crop development.
Young transgenic seedlings may be grown at a low temperature, such as about 13° C., during the light and 13° C. during the dark period. Plant seeds in 96-pod flats containing greenhouse soil medium. Water initially with Seplex (Blackmore Company, Belleville, Mich.) water for the first day after planting and germinate seedlings in the greenhouse. After the initial watering, continue watering seedlings with 85 ppm 20:10:20 fertilizer water. Once plants reach the V3 stage (approximately 10-14 days), move them to a growth chamber and subject to a chilling regimen of 16/8 hour light/dark cycle, where day/night temperatures are maintained at 15° C. light/13° C. dark, under constant humidity. The pots should be placed in flats that do not have the slit openings in the flats. Bottom water the seedlings with 85 ppm 20:10:20 fertilizer water, keeping the seedlings well-watered. Subject seedlings to chilling conditions for the next 16 days. Score for visual yellowing at 4, 8, 12 and 16 days into the stress period and also record chlorophyll fluorescence using the Hansatech FMS2 chlorophyll fluorescence meter (Hansatech Instruments Ltd). Leaf discs can be used to determine ROS (reactive oxygen species) accumulation due to photo-oxidative damage under low temperature combined with high light. At the end of the stress period, harvest plants at soil level and record fresh weight or biomass accumulation. Inclusion of check varieties that are tolerant and susceptible to low temperature growth is important for normalization of observations across experiments. Tolerance of the transgenic plant to the stress may be assessed on the basis of increased plant growth, fresh or dry weight of the seedlings and/or increased photosynthetic activity or chlorophyll fluorescence in the transgenic plant relative to control non-transgenic plants. Physical characteristics of the transgenic and control plants' growth may be assessed as described herein.
Transgenic plants expressing a MAPKKK of the present invention may also be assayed for increased freezing tolerance at the seedling stage as well as late season periods. These assays are preferably done in artificial environments to simulate frost or freeze events. In addition, transgenic seeds may be planted outside during times when the natural environment would impose the stress, e.g., at times when frost is present.
B. Drought Tolerance
Transgenic plants expressing a MAPKKK of the present invention may also be assayed in artificial drought-stress environments in pot-based studies or under managed drought-stress conditions in the field in order to demonstrate that the transgene confers resistance or tolerance to drought. Transgenic maize seedlings containing the candidate gene can be screened for tolerance to drought stress by a pot-based screen in the following manner. Transgenic maize plants are subjected to well-watered conditions (control) and to drought-stressed conditions. Transgenic maize plants are screened at the T1 generation or later. Stress is imposed starting at 10 to 14 days after sowing (DAS) or 7 days after transplanting, and is continued through to silking. Pots are watered by an automated system fitted to timers to provide watering at 25 or 50% of field capacity during the entire period of drought-stress treatment. The intensity and duration of this stress will allow identification of the impact on vegetative growth as well as on the anthesis-silking interval (ASI).
Potting mixture: A mixture of 1⁄3 turface (Profile Products LLC, IL, USA), 1⁄3 sand and 1⁄3 SB300 (Sun Gro Horticulture, WA, USA) can be used. The SB300 can be replaced with Fafard Fine-Germ (Conrad Fafard, Inc., MA, USA) and the proportion of sand in the mixture can be reduced. Thus, a final potting mixture can be 3⁄8 (37.5%) turface, 3⁄8 (37.5%) Fafard and 1⁄4 (25%) sand.
Field Capacity Determination: The weight of the soil mixture (w1) to be used in one S200 pot (minus the pot weight) is measured. If all components of the soil mix are not dry, the soil is dried at 100° C. to constant weight before determining w1. The soil in the pot is watered to full saturation and all the gravitational water is allowed to drain out. The weight of the soil (w2) after all gravitational water has seeped out (minus the pot weight) is determined. Field capacity is the weight of the water remaining in the soil obtained as w2-w1. It can be written as a percentage of the oven-dry soil weight.
Stress Treatment: Plants are allowed to grow for an initial period of 12 to 14 days under well-watered conditions, following which the soil moisture content is reduced to  ̃30% field capacity to administer a chronic drought stress. Measurements on chlorophyll fluorescence are recorded during this early growth period (well-watered observations) and during recovery from drought stress upon rewatering during the chronic drought stress period (drought-stressed observations). After the chronic drought-stress treatment, water is completely withheld to allow the plants to come very close to permanent wilting point ( ̃8% field capacity), at which point they are watered to saturation. The recovery of plants from this severe drought is recorded as hours to 50% recovery or as the number of plants recovered after 48 hours of drought stress. At the end of the experiments, shoots are collected for fresh and dry weight measurements.
Observations Made: Observations are recorded on chlorophyll fluorescence as PhiPSII (which is indicative of the operating quantum efficiency of photosystem II photochemistry) and Fv′/Fm′ (which is the maximum efficiency of photosystem II), during the well-watered growth, as well as during recovery from drought stress upon re-watering. These measurements are recorded using the Hansatech FMS2 instrument (Hansatech Instruments Ltd. Norfolk, England). Measurements are recorded for the youngest fully expanded leaf. Observations are also recorded on the recovery of plants after a severe drought stress, and also on the fresh and dry weights of the shoots at the end of the experimental period.
Six events of the rd29a:ZmNPK1b construct were tested for improved drought tolerance. Under well-watered conditions, Fv′/Fm′ for two of the six events was significantly higher than that of the control. Notably, under drought conditions as described above, five of the six events showed significantly higher Fv′/Fm′ than the control. Four of those five also scored significantly better than the control for PSII fluorescence under drought.
Recovery score indicates a plant's ability to recover after the drought stress is relieved as described above. Early recovery or reduced time required for recovery is sought in a plant that has improved drought tolerance. Two of the events with improved fluorescence scores also showed significantly reduced recovery time compared to the control.
To investigate whether these positive data reflected a bias toward smaller plants, wet and dry mass of the seedling shoots was measured. Only one event (Event 3.1) had an average wet shoot mass smaller than the control. All others were equivalent or larger (Event 3.39). Five of the six events were unchanged in dry shoot mass relative to the control; one event had a reduced mass.







TABLE 7







Seedling drought screen of PHP32984 transgenic maize















WW
WW
DRT
DRT

Wet




Fluorescence
Fluorescence
Fluorescence
Fluorescence
Recovery
Shoot
Dry Shoot


Event
Fv′/Fm′
?PSII
Fv′/Fm′
?PSII
hour
gram
gram

















3.39
0.530a
0.420a
0.687a
0.511a
12.3b
2.90a
0.3999a


3.32
0.488b
0.380a
0.668ab
0.505a
18.7a
2.34b
0.3683a


3.19
0.464b
0.366a
0.640ab
0.484a
12.8b
2.43b
0.3441b


2.9
0.489b
0.392a
0.603c
0.452b
21.3a
2.28b
0.3657a


1.3
0.476b
0.415a
0.615b
0.459b
18.4a
2.32b
0.4004a


3.1
0.497a
0.420a
0.661ab
0.485a
20.2a
1.99c
0.3946a


control
0.475b
0.400a
0.605c
0.460b
19.9a
2.09b
0.3697a









In summary, 83% of rd29a::ZmNPK1b events tested showed significantly improved Fv′/Fm′ under drought conditions, relative to the control.
Oxidative stress is a major cause of damage in plants exposed to stressful environmental conditions. Oxidative stress results from the cellular damage caused by reactive oxygen species that are generated in cells. These reactive oxygen molecules can damage membranes, proteins, and nucleic acids. Transgenic plants that express a MAPKKK of the present invention may be analyzed for improved resistance to oxidative stress.
Transgenic plants expressing a MAPKKK of the present invention may be assayed in artificial environments or in the field to demonstrate that the transgene confers resistance or tolerance to chemicals (e.g., herbicides, ozone or pollutants) or metals (e.g., copper or zinc). Transgenic plants having an increased ability to grow in the presence of higher concentrations of the toxic compound, as compared to non-transgenic plants, are useful in the invention.
In another aspect, a MAPKKK of the present invention described herein may improve crop yield or productivity. Seeds of transgenic plants expressing a MAPKKK of the present invention may be planted in test plots and their agronomic performance compared to standard plants using techniques familiar to those of skill in the art. Optionally included in this comparison are plants of similar genetic background without the transgene. A yield benefit may be observed and plants exhibiting the increased yield are advanced for commercialization.
In addition, transgenic plants expressing a MAPKKK of the present invention may be field tested for agronomic performance under conditions, including, but not limited to, limited or inadequate water availability to simulate drought. When compared to nontransgenic plants, transgenic plants expressing a MAPKKK of the present invention may exhibit higher yield than their nontransgenic counterparts under non-optimal growing conditions.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. An isolated mitogen-activated protein kinase kinase kinase (MAPKKK) polynucleotide selected from the group consisting of:
(a) a polynucleotide that encodes the polypeptide of SEQ ID NO: 2;
(b) a polynucleotide comprising the sequence set forth in SEQ ID NO: 1;
(c) a polynucleotide encoding a polypeptide having at least 95% sequence identity to the entire length of SEQ ID NO: 2, wherein the % sequence identity is determined by GAP Version 10 under default parameters; and
(d) an isolated polynucleotide degenerate from any of (a) to (c) as a result of the genetic code.

2. A vector comprising at least one polynucleotide of claim 1.

3. An isolated polypeptide with MAPKKK activity, selected from the group consisting of:
(a) an isolated polypeptide comprising SEQ ID NO: 2;
(b) a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2;
(c) a polypeptide that is encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the full length of SEQ ID NO: 1; and
(d) a fragment comprising at least 200 consecutive amino acids of SEQ ID NO: 2.

4. A recombinant expression cassette comprising a polynucleotide operably linked to a promoter, wherein the polynucleotide encodes the polypeptide of claim 3.

5. A transformed host cell comprising the isolated polypeptide of claim 3.

6. The host cell of claim 5, wherein the host cell is a transformed plant cell.

7. The plant cell of claim 6, wherein the plant cell is selected from the group consisting of sorghum, maize, rice, wheat, soybean, sunflower, canola, alfalfa, barley and millet.

8. A transformed plant regenerated from the plant cell of claim 6.

9. A transformed seed of the plant of claim 8.