# Patent

## Title
Anti-apoptosis genes and methods of use thereof

## Abstract

The invention provides isolated nucleic acids and their encoded proteins that act as cell death inhibitors and methods of use thereof. The invention further provides expression cassettes, transformed host cells, transgenic plants and plant parts, and antibody compositions.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. application Ser. No. 60/297,478 filed Jun. 12, 2001, the disclosure of which is incorporated herein by reference.


TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
BACKGROUND OF THE INVENTION
Cell division plays a crucial role during all phases of plant development. The continuation of organogenesis and growth responses to a changing environment requires precise spatial, temporal and developmental regulation of cell division activity in meristems (and in cells with the capability to form new meristems such as in lateral root formation). Such control of cell division is also important in organs themselves (i.e. separate from meristems per se), for example, in leaf expansion, secondary growth, and endoreduplication.
A complex network controls cell proliferation in eukaryotes. Various regulatory pathways communicate environmental constraints, such as nutrient availability, mitogenic signals such as growth factors or hormones, or developmental cues such as the transition from vegetative to reproductive stages. Ultimately, these regulatory pathways control the timing, frequency (rate), plane and position of cell divisions.
Plants have unique developmental features that distinguish them from other eukaryotes. Plant cells do not migrate, and thus only cell division, expansion and programmed cell death determine morphogenesis. Organs are formed throughout the entire life span of the plant from specialized regions called meristems. In addition, many differentiated cells have the potential to both dedifferentiate and to reenter the cell cycle. There are also numerous examples of plant cell types that undergo endoreduplication, a process involving nuclear multiplication without cytokinesis. The study of plant cell cycle control genes is expected to contribute to the understanding of these unique phenomena. O. Shaul et al., Regulation of Cell Division in Arabidopsis, Critical Reviews in Plant Sciences 15 (2):97–112 (1996).
Current transformation technology provides an opportunity to engineer plants with desired traits. Major advances in plant transformation have occurred over the last few years. However, in many major crop plants, serious genotype limitations still exist. Transformation of some agronomically important crop plants continues to be both difficult and time consuming. For example, it is difficult to obtain a culture response from some maize varieties. Typically, a suitable culture response has been obtained by optimizing medium components and/or explant material and source. This has led to success in some, but not all, genotypes. While, transformation of model genotypes is efficient, the process of introgressing transgenes into production inbreds is laborious, expensive and time consuming. It would save considerable time and money if genes could be introduced into and evaluated directly in commercial hybrids.
Current methods for genetic engineering in maize require a specific cell type as the recipient of new DNA. These cells are found in relatively undifferentiated, rapidly growing callus cells or on the scutellar surface of the immature embryo (which gives rise to callus). Irrespective of the delivery method currently used, DNA is introduced into literally thousands of cells, yet transformants are recovered at frequencies of 10−5 relative to transiently-expressing cells. Exacerbating this problem, the trauma that accompanies DNA introduction directs recipient cells into cell cycle arrest and accumulating evidence suggests that many of these cells are directed into apoptosis or programmed cell death. (Reference Bowen et al., Tucson International Mol. Biol. Meetings). Therefore, it would be desirable to provide improved methods capable of increasing transformation efficiency by reducing cell death in a number of cell types.
Despite the number of identified plant diseases and stresses, the understanding of the molecular and cellular events that are responsible for plant disease and stress resistance remains rudimentary. This is especially true of the events controlling the earliest steps of active plant defense, recognition of a potential pathogen and transfer of the cognitive signal throughout the cell, surrounding tissue and cell death within that tissue.
Diseases are particularly destructive processes resulting from specific causes and characterized by specific symptoms. Generally the symptoms can be related to a specific cause, usually a pathogenic organism. In plants, a variety of pathogenic organisms cause a wide variety of disease symptoms. It would therefore be valuable to develop new methods that contribute to the increase in crop yield to protect plants against cell death associated with pathogen and stress.
In spite of increases in yield and harvested area worldwide, it is predicted that over the next ten years, meeting the demand for corn will require an additional 20% increase over current production (Dowswell, C. R., Paliwal, R. L., Cantrell, R. P. (1996) Maize in the Third World, Westview Press, Boulder, Colo.).
The components most often associated with maize productivity are grain yield or whole-plant harvest for animal feed (in the forms of silage, fodder, or stover). Thus the relative growth of the vegetative or reproductive organs might be increased or decreased, depending on the ultimate use of the crop. Whether the whole plant or the ear are harvested, overall yield will depend strongly on vigor and growth rate. It would therefore be valuable to develop new methods that contribute to the increase in crop yield.
SUMMARY OF THE INVENTION
The invention provides isolated BI nucleic acids and their encoded proteins that act as cell death inhibitors and methods of using to improve transformation, improve stress resistance, disease resistance, change the architecture of a plant and affect male sterility. The invention further provides expression cassettes, transformed host cells, transgenic plants and plant parts, and antibody compositions.
DETAILED DESCRIPTION OF THE INVENTION
Apoptosis is an evolutionarily conserved form of cell death that plays crucial roles in the development and homeostasis of multicellular animals. It is brought about by the action of a group of cysteine proteases, called caspases. Modulation of the BI gene may affect apoptosis. Introducing BI into plants can improve transformation, increase disease and stress resistance and increase agronomic advantage.
Definitions
The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material.
As used herein, “nucleic acid” means a polynucleotide and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides.
As used herein, “polynucleotide” includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof, that have the essential nature of a natural ribonucleotide in that they hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
As used herein, “BI polynucleotide” means a nucleic acid sequence of BI.
As used herein, “polypeptide” means proteins, protein fragments, proteins, amino acid sequences and synthetic amino acid sequences. The polypeptide may be glycosylated or not.
As used herein, “BI polypeptide” means a polypeptide comprising at least 200 bp of the BI amino acid coding sequence, including fusions with other polypeptides such as VirE. The BI polypeptide may inhibit Bax-induced cell death or not.
As used herein, “BI activity” or “Bax inhibitor activity” means the BI polypeptides or fragments or fusions thereof that inhibit Bax-induced cell death and may be measurable by standard methods such as DNA fragmentation, visual necrosis or genetic assays. “Bax inhibitor activity” may be measurable or detectable through increased transformation efficiency, increased co- or re-transformation efficiency, increased identification of transgenic events, improved disease resistance, improved stress resistance, modulation of plant architecture, and/or modulation of sterility.
As used herein, “plant” includes plants and plant parts including but not limited to plant cells, plant tissue such as leaves, stems, roots, flowers, and seeds.
As used herein, “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
By “fragment” is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native nucleic acid. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes may not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence are generally greater than 25, 50, 100, 200, 300, 400, 500, 600, or 700 nucleotides and up to and including the entire nucleotide sequence encoding the proteins of the invention. Generally the probes are less than 1000 nucleotides and preferably less than 500 nucleotides. Fragments of the invention include antisense sequences used to decrease expression of the inventive polynucleotides. Such antisense fragments may vary in length ranging from greater than 25, 50, 100, 200, 300, 400, 500, 600, or 700 nucleotides and up to and including the entire coding sequence.
By “functional equivalent” as applied to a polynucleotide or a protein is intended a polynucleotide or a protein of sufficient length to modulate the level of BI activity in a plant cell. A polynucleotide functional equivalent can be in sense or antisense orientation.
By “variants” is intended substantially similar sequences. Generally, nucleic acid sequence variants of the invention will have at least 51%, 55%, 57%, 60%, 63%, 65%, 67%, 69%, 70%, 72%, 73%, 75%, 77%, 78%, 80%, 81%, 83%, 84%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the native nucleotide sequence, wherein the % sequence identity is based on the amino acid coding region available for the inventive sequence and is determined by GAP 10 analysis using default parameters. Generally, polypeptide sequence variants of the invention will have at least about 60%, 63%, 65%, 67%, 70%, 73%, 75%, 78%, 80%, 83%, 85%, 87%, 88%, 90%, 93%, 95%, 98% or 99% sequence identity to the native protein, wherein the % sequence identity is based on the entire sequence and is determined by GAP 10 analysis using default parameters and is any integer. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443–453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
The public databases were searched by BLAST using default parameters for the closest related sequences to each of the seven maize or ten soybean BI homolog genes in question. The comparison was done, wherever possible, using the nucleotide coding regions of the maize or soybean BI genes in a global alignment (GAP) with the closest public counterpart. The closest ‘hit’ was determined by a combination of the percent identity between the two sequences times the ratio of the amino acid coding region that that public sequences represented relative to the BI genes of the present invention. So, for example if a public BI EST is 100% identical at the nucleotide level, but it only represents 30% of the coding region of the proprietary BI gene, then it is expressed as 30% related. Conversely, if a public full-length cDNA represents 100% of the coding region, but is only 70% identical at the nucleotide level, then it is expressed as only 70% related. In this way the closest public relative to each of the seven maize and ten soybean BI genes was determined.
As used herein a “responsive cell” refers to a cell that exhibits a positive response to the introduction of BI polypeptide or BI polynucleotide compared to a cell that has not been introduced with BI polypeptide or BI polynucleotide. The response can be to enhance tissue culture response, induce somatic embryogenesis, increase transformation efficiency or increase recovery of regenerated plants.
As used herein a “recalcitrant plant cell” is a plant cell that exhibits unsatisfactory tissue culture response, transformation efficiency or recovery of regenerated plants compared to model systems. In maize such a model system is Hi-II. Elite maize inbreds are typically recalcitrant. In soybeans such model systems are Peking or Jack.
As used herein “Transformation” includes stable transformation and transient transformation unless indicated otherwise.
As used herein “Stable Transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism (this includes both nuclear and organelle genomes) resulting in genetically stable and heritable inheritance. In addition to traditional methods, stable transformation includes the alteration of gene expression by any means including chimerplasty or transposon insertion.
As used herein “Transient Transformation” refers to the transfer of a nucleic acid fragment or protein into the nucleus (or DNA-containing organelle) of a host organism resulting in gene expression without integration and stable inheritance.
As used herein “Transformation Efficiency” refers to parameters involved in improving transformation favorably, that influence transformation and/or regeneration and thus result in an increased recovery of transformed resultants (i.e. callus, shoots, plants) from a transformation attempt. Transformation efficiency may be calculated as the number of transformed resultants divided by the number of targets having DNA introduced times 100. For a review related to callus formation see, for example, Duncan et al, (Planta 165:322–332, 1985).
As used herein, “Co-introduced” refers to situations where 2 or more nucleic acids, proteins or combinations thereof, are introduced into the target at the same time.
As used herein, a “BI-DR” (BI-Down Regulated) construct as an expression cassette whose transcribed mRNA or translated protein will diminish the functional expression of active BI in the cell. Such silencing can be achieved through expression of an antisense construct targeted against the BI structural gene, a vector in which the BI structural gene or a portion of this sequence is used to make a silencing hairpin (or where silencing hairpin is conjoined to the BI sequence in some fashion), or where a BI-overexpression cassette is used to co-suppress endogenous BI levels. Reducing activity of endogenous BI protein can also be achieved through expression of a transgene encoding an antibody (including single chain antibodies) directed against a functional domain within the BI molecule, introduction of such an antibody or through introduction of an “aptamer”.
As used herein, “aptamers” are DNA or RNA molecules that have been artificially evolved and selected to bind other proteins (such as BI), molecules, viruses, etc. They have many potential uses in medicine and technology. For example see J. Feigon, T. Dieckmann, and F. W. Smith: “Aptamer structures from A to zeta”, Chem. and Biol. 3:611–617 (1996).
As used herin, “architecture” refers to the structural organization, placement, relative shape and/or relative size (e.g. organ placement/presence/size, such as ear).

    Seq. ID No. 1-ZmBI-1 nucleotide sequence
    Seq. ID No. 2-ZmBI-1 protein sequence
    Seq. ID No. 3-ZmBI-2 nucleotide sequence
    Seq. ID No. 4-ZmBI-2 protein sequence
    Seq. ID No. 5-ZmBI-3 nucleotide sequence
    Seq. ID No. 6-ZmBI-3 protein sequence
    Seq. ID No. 7-ZmBI-4 nucleotide sequence
    Seq. ID No. 8-ZmBI-4 protein sequence
    Seq. ID No. 9-ZmBI-5 nucleotide sequence
    Seq. ID No. 10-ZmBI-5 protein sequence
    Seq. ID No. 11-ZmBI-6 nucleotide sequence
    Seq. ID No. 12-ZmBI-6 protein sequence
    Seq. ID No. 13-ZmBI-7 nucleotide sequence
    Seq. ID No. 14-ZmBI-7 protein sequence
    Seq. ID No. 15-GmBI-1 nucleotide sequence
    Seq. ID No. 16-GmBI-1 protein sequence
    Seq. ID No. 17-GmBI-2 nucleotide sequence
    Seq. ID No. 18-GmBI-2 protein sequence
    Seq. ID No. 19-GmBI-3 nucleotide sequence
    Seq. ID No. 20-GmBI-3 protein sequence
    Seq. ID No. 21-GmBI-4 nucleotide sequence
    Seq. ID No. 22-GmBI-5 nucleotide sequence
    Seq. ID No. 23-GmBI-6 nucleotide sequence
    Seq. ID No. 24-GmBI-7 nucleotide sequence
    Seq. ID No. 25-GmBI-7 protein sequence
    Seq. ID No. 26-GmBI-8 nucleotide sequence
    Seq. ID No. 27-GmBI-8 protein sequence
    Seq. ID No. 28-GmBI-9 nucleotide sequence
    Seq. ID No. 29-GmBI-9 protein sequence
    Seq. ID No. 30-GmBI-10 nucleotide sequence
    Seq. ID No. 31-ZmBI-2 amended nucleotide sequence
    Seq. ID No. 32-ZmBI-2 amended protein sequence
    Seq. ID No. 33-ZmBI-3 amended nucleotide sequence
    Seq. ID No. 34-ZmBI-3 amended protein sequence


Nucleic Acids
Plant cell death occurs through the course of normal plant development, and helps sculpt the plants life-cycle, from embryo development, to scutellum and aleurone degeneration during germination, to leaf and fruit abscission, to plant senescence, as in annual crop plants. In addition plant cell death can occur in stressful situations such as mechanical wounding, as from wind-blown sand, hail, and insect foraging, and from other biotic stresses such as fungal, bacterial and viral infection. Methods of introducing DNA into cells, cell culture and selection techniques involved in transformation can also increase cell death.
Crop plants are vulnerable to various abiotic stresses, which can cause tissue damage and necrosis. These stresses include water stress, temperature stress, light stress, and mechanical stress. Water stress can be insufficient water or drought stress, but also flooding or stress. Temperature stress can be excessive or prolonged heat or cold. Light stress can occur as well, and can be exasperated by temperature and water stress. For example, light reflected off standing water can cause hyper-exposure to light, as on the underside of leaves, and ‘burn’ the crop plants. This is more commonly a problem for young soybean and maize plants where foliage cover between the rows has not yet occurred. Mechanical stress can be caused by factors such as wind and hail, the former causing aggravated damage should wind-blown particles, such as sand, pock the plant tissues. Because the BI genes are inhibitors of cell death, their appropriate (usually ectopic) expression, as in a transgenic plant, could cause suppression of death brought by these abiotic stresses. As death is a primary adverse symptom of these stresses, this suppression of the death response would reduce the severity of the abiotic stress symptoms, and thus the crop plants would be more ‘tolerant’ to these stresses.
The BI genes of this invention then may find a number of applications for agronomic advantages and transformation improvement of a plant via methods to control plant cell death. In general ectopic expression of BI would retard cell death and promote viable tissue. Conversely reducing BI expression would promote cell death. By “expression” we mean ultimately the capacity of the function of the gene product, a protein, that may be controlled via a number of means, including mRNA levels, protein levels, and modifications of amino acids sites on the protein to effect altered functional capacity of the protein itself.
Increasingly various genetic engineering strategies are being put forth to create enhanced disease resistance using recombinant DNA technology and transgenic plants. Sometimes this involves isolation of a resistance gene and then discreetly inserting it into a susceptible plant by transformation. For example, this was done for the Xa21 gene of rice. Other strategies involve engineering elevated expression of antimicrobial compounds or genes, such as PR or pathogenesis-related proteins like chitinase and beta-glucanase, or genes which alter reactive oxygen species, which are known to be antimicrobial and/or stimulators of plant defense systems. Other strategies are also being tried.
These genetic engineering strategies are meeting with varied success. No one strategy or gene has proven to be a panacea, although some show limited promise. Successful broad improvement of crop resistance will likely require multiple strategies. This is so for several reasons. One is that no one strategy seems to work for every pathogen; in fact, often the resistance created is either specific for particular pathogens or small groups of pathogens. Another reason is that the resistance created is often not robust enough or limited to tissue or genetic background. Yet another reason is that given the continuing evolution of the pathogen, no one strategy is likely to work for long.
This invention provides novel genes and novel descriptions of how they can be used, as in a transgenic plant, to effect enhancement transformation, and disease or stress resistance of that crop plant.
This invention overcomes the limitations of previous related genetic engineering strategies for crop plants by providing seven novel maize genes and ten novel soybean genes for such purposes. These genes are called Zea mays and Glycine max BI genes, or Zm-BI and Gm-BI for short, based on their structural similarity to the bax inhibitor genes of animals, and likewise based on their presumed related function to these animal bax inhibitor genes. It is recognized that no single gene will work in all crops against all pathogens. This invention can complement conventional breeding strategies and other genetic engineering methods to enhance disease/stress resistance and increase transformation frequencies in plants.
Other Possible Applications for these BI Genes
While the examples described herewithin are limited to the areas of plant transformation and disease/stress resistance and agronomic traits, other applications can be envisioned.
First, plants can be wounded abiotically, as by drought stress, wind stress (which includes damage by wind-blown soil particles), and chemical and nutrient stress. Such stresses can precipitate cell death that can affect plant yield. To the extent that BI may retard cell death, they may be able to retard the symptom development of necrosis results from these stresses, for example with a death-inducible promoter. As such, BI might prove agronomically advantageous.
Second, the BI genes may have application in the development and implementation of herbicide resistance mechanisms in crop plants. Ectopic expression of the BI genes, as in leaves, may result in a retardation of cell death that could occur following application of herbicides. We recognize that this would be subject of the kind of herbicide and its mode of action, but it is a possible area of use for these genes. Herbicides and herbicide resistance systems are often used as selectable markers in plant transformation experiments. So in a way similar to the herbicide resistance application, these BI genes could figure in as selectable markers—only cells expressing the BI genes (sic ectopically) would grow or stay alive in the face of an antibiotic/herbicide medium. This application of course starts to overlap with the examples given above for improving plant transformation.
Third, the BI genes might be useful for affecting the architecture (organ placement/presence and/or structural organization) of a plant. This may be accomplished, for example, by controlling the senescence of crop plants, whole plants or special tissues. It is recognized that maturity and dry-down are important agronomic traits in maize and other crop plants. While the biology is undoubtedly complex in senescence, in the extent of their ability to control cell death, BI may be able to control the timing and onset of senescence. For certain crops particular tissue or organs are desired to senesce. This includes controlled dropping of cotton leaves to facilitate cotton ball harvesting. Sometimes organs are desired not to senesce, as in the petioles of fruit; premature fruit drop can cause loss of yield. Modulation of BI may provide agronomic advantages by promoting or delaying senescence and other developmental signals.
The isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof. In some embodiments, the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a monocot or dicot. Typical examples of monocots are corn, sorghum, barley, wheat, millet, rice, or turf grass. Typical dicots include soybeans, sunflower, canola, alfalfa, potato, or cassava.
Functional fragments included in the invention can be obtained using primers that selectively hybridize under stringent conditions or through enzyme restriction. Primers are generally at least 12 bases in length and can be as high as 200 bases, but will generally be from 15 to 75, preferably from 15 to 50 bases. Functional fragments can be identified using a variety of techniques such as restriction analysis, Southern analysis, primer extension analysis, and DNA sequence analysis.
The present invention includes a plurality of polynucleotides that encode for the identical amino acid sequence. The degeneracy of the genetic code allows for such “silent variations” which can be used, for example, to selectively hybridize and detect allelic variants of polynucleotides of the present invention. Additionally, the present invention includes isolated nucleic acids comprising allelic variants. The term “allele” as used herein refers to a related nucleic acid of the same gene.
Variants of nucleic acids included in the invention can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like. See, for example, Ausubel, pages 8.0.3–8.5.9. Also, see generally, McPherson (ed.), DIRECTED MUTAGENESIS: A Practical Approach, (IRL Press, 1991). Thus, the present invention also encompasses DNA molecules comprising nucleotide sequences that have substantial sequence similarity with the inventive sequences.
Variants included in the invention may contain individual substitutions, deletions or additions to the nucleic acid or polypeptide sequences which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host.
The present invention also includes “shufflents” produced by sequence shuffling of the inventive polynucleotides to obtain a desired characteristic. Sequence shuffling is described in PCT publication No. 96/19256. See also, Zhang, J. H., et al., Proc. Natl. Acad. Sci. USA 94:4504–4509 (1997).
The present invention also includes the use of 5′ and/or 3′ UTR regions for modulation of translation of heterologous coding sequences. Positive sequence motifs include translational initiation consensus sequences (Kozak, Nucleic Acids Res.15:8125 (1987)) and the 7-methylguanosine cap structure (Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negative elements include stable intramolecular 5′ UTR stem-loop structures (Muesing et al., Cell 48:691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284 (1988)).
Further, the polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as “Codon Preference” available from the University of Wisconsin Genetics Computer Group (see Devereaux et al., Nucleic Acids Res. 12:387–395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).
For example, the inventive nucleic acids can be optimized for enhanced expression in plants of interest. See, for example, EPA0359472; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324–3328; and Murray et al. (1989) Nucleic Acids Res. 17:477–498. In this manner, the polynucleotides can be synthesized utilizing plant-preferred codons. See, for example, Murray et al. (1989) Nucleic Acids Res. 17:477–498, the disclosure of which is incorporated herein by reference.
The present invention provides subsequences comprising isolated nucleic acids containing at least 20 contiguous bases of the inventive sequences. For example the isolated nucleic acid includes those comprising at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, or 600 contiguous nucleotides of the inventive sequences. Subsequences of the isolated nucleic acid can be used to modulate or detect gene expression by introducing into the subsequences compounds which bind, intercalate, cleave and/or crosslink to nucleic acids.
The nucleic acids of the invention may conveniently comprise a multi-cloning site comprising one or more endonuclease restriction sites inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention.
A polynucleotide of the present invention can be attached to a vector, adapter, promoter, transit peptide or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known and extensively described in the art. For a description of such nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).
The isolated nucleic acid compositions of this invention, such as RNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plant biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library.
Exemplary total RNA and mRNA isolation protocols are described in Plant Molecular biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Total RNA and mRNA isolation kits are commercially available from vendors such as Stratagene (La Jolla, Calif.), Clonetech (Palo Alto, Calif.), Pharmacia (Piscataway, N.J.), and 5′–3′ (Paoli, Pa.). See also, U.S. Pat. Nos. 5,614,391; and, 5,459,253.
Typical cDNA synthesis protocols are well known to the skilled artisan and are described in such standard references as: Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). cDNA synthesis kits are available from a variety of commercial vendors such as Stratagene or Pharmacia.
An exemplary method of constructing a greater than 95% pure full-length cDNA library is described by Carninci et al., Genomics 37:327–336 (1996). Other methods for producing full-length libraries are known in the art. See, e.g., Edery et al., Mol. Cell Biol. 15(6):3363–3371 (1995); and PCT Application WO 96/34981.
It is often convenient to normalize a cDNA library to create a library in which each clone is more equally represented. A number of approaches to normalize cDNA libraries are known in the art. Construction of normalized libraries is described in Ko, Nucl. Acids. Res. 18(19):5705–5711 (1990); Patanjali et al., Proc. Natl. Acad. U.S.A. 88:1943–1947 (1991); U.S. Pat. Nos. 5,482,685 and 5,637,685; and Soares et al., Proc. Natl. Acad. Sci. USA 91:9228–9232 (1994).
Subtracted cDNA libraries are another means to increase the proportion of less abundant cDNA species. See, Foote et al. in, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, Technique 3(2):58–63 (1991); Sive and St. John, Nucl. Acids Res. 16(22):10937 (1988); Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); and, Swaroop et al., Nucl. Acids Res. 19(8):1954 (1991). cDNA subtraction kits are commercially available. See, e.g., PCR-Select (Clontech).
To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation. Examples of appropriate molecular biological techniques and instructions are found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Vols. 1–3 (1989), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits for construction of genomic libraries are also commercially available.
The cDNA or genomic library can be screened using a probe based upon the sequence of a nucleic acid of the present invention such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous polynucleotides in the same or different plant species. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. The degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide.
Typically, stringent hybridization 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 sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55° 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° C. Typically the time of hybridization is from 4 to 16 hours.
An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Often, cDNA libraries will be normalized to increase the representation of relatively rare cDNAs.
The nucleic acids of the invention can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the present invention and related polynucleotides directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
Examples of techniques useful for in vitro amplification methods are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., U.S. Pat. No. 4,683,202 (1987); and, PCR Protocols A Guide to Methods and Applications, Innis et al., Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products. PCR-based screening methods have also been described. Wilfinger et al. describe a PCR-based method in which the longest cDNA is identified in the first step so that incomplete clones can be eliminated from study. BioTechniques 22(3):481–486 (1997).
In one aspect of the invention, nucleic acids can be amplified from a plant nucleic acid library. The nucleic acid library may be a cDNA library, a genomic library, or a library generally constructed from nuclear transcripts at any stage of intron processing. Libraries can be made from a variety of plant tissues. Good results have been obtained using mitotically active tissues such as shoot meristems, shoot meristem cultures, embryos, callus and suspension cultures, immature ears and tassels, and young seedlings. The cDNAs of the present invention were obtained from immature zygotic embryo and regenerating callus libraries.
Alternatively, the sequences of the invention can be used to isolate corresponding sequences in 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 having substantial sequence similarity to the sequences of the invention. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). and Innis et al. (1990), PCR Protocols: A Guide to Methods and Applications (Academic Press, New York). Coding sequences isolated based on their sequence identity to the entire inventive coding sequences set forth herein or to fragments thereof are encompassed by the present invention.
The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90–99 (1979); the phosphodiester method of Brown et al., Meth. Enzymol. 68:109–151 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett 22:1859–1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts. 22(20):1859–1862 (1981), e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter et al., Nucleic Acids Res. 12:6159–6168 (1984); and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
Expression Cassettes
In another embodiment expression cassettes comprising isolated nucleic acids of the present invention are provided. An expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.
The construction of such expression cassettes which can be employed in conjunction with the present invention is well known to those of skill in the art in light of the present disclosure. See, e.g., Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor, N.Y.; (1989); Gelvin et al.; Plant Molecular Biology Manual (1990); Plant Biotechnology: Commercial Prospects and Problems, eds. Prakash et al.; Oxford & IBH Publishing Co.; New Delhi, India; (1993); and Heslot et al.; Molecular Biology and Genetic Engineering of Yeasts; CRC Press, Inc., USA; (1992); each incorporated herein in its entirety by reference.
For example, plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5′ and 3′ regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
Constitutive, tissue-preferred or inducible promoters can be employed. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, the actin promoter, the ubiquitin promoter, the histone H2B promoter (Nakayama et al., 1992, FEBS Left 30:167–170), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1–8 promoter, and other transcription initiation regions from various plant genes known in the art.
Examples of inducible promoters are the Adh1 promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter which is inducible by light, the In2 promoter which is safener induced, the ERE promoter which is estrogen induced and the Pepcarboxylase promoter which is light induced.
Examples of promoters under developmental control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers. An exemplary promoter is the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051). Examples of seed-preferred promoters include, but are not limited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A., Martinez, M. C., Reina, M., Puigdomenech, P. and Palau, J.; Isolation and sequencing of a 28 kD glutelin-2 gene from maize: Common elements in the 5′ flanking regions among zein and glutelin genes; Plant Sci. 47:95–102 (1986) and Reina, M., Ponte, I., Guillen, P., Boronat, A. and Palau, J., Sequence analysis of a genomic clone encoding a Zc2 protein from Zea mays W64 A, Nucleic Acids Res. 18(21):6426 (1990). See the following site relating to the waxy promoter: Kloesgen, R. B., Gierl, A., Schwarz-Sommer, Z. S. and Saedler, H., Molecular analysis of the waxy locus of Zea mays, Mol. Gen. Genet. 203:237–244 (1986). The disclosures of each of these are incorporated herein by reference in their entirety.
The barley or maize Nuc1 promoter, the maize Cim 1 promoter or the maize LTP2 promoter can be used to preferentially express in the nucellus. See for example WO00/11177 and U.S. Pat. No. 6,225,529, issued May 1, 2001, the disclosures of which are incorporated herein by reference.
Either heterologous or non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter concentration and/or composition of the proteins of the present invention in a desired tissue.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence can be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates. See for example Buchman and Berg, Mol. Cell Biol. 8:4395–4405 (1988); Callis et al., Genes Dev. 1:1183–1200 (1987). Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).
The vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene which confers a selectable phenotype on plant cells. Usually, the selectable marker gene will encode antibiotic or herbicide resistance. Suitable genes include those coding for resistance to the antibiotics spectinomycin and streptomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance.
Suitable genes coding for resistance to herbicides include those which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), those which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta and the ALS gene encodes resistance to the herbicide chlorsulfuron.
While useful in conjunction with the above antibiotic and herbicide-resistance selective markers (i.e. use of the BI gene can increase transformation frequencies when using chemical selection), use of the BI gene confers a growth advantage to transformed cells without the need for inhibitory compounds to retard non-transformed growth. Thus, BI transformants are recovered based solely on their differential growth advantage.
Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. In Enzymol. 153:253–277 (1987). Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al., Gene 61:1–11 (1987) and Berger et al., Proc. Natl. Acad. Sci. USA 86:8402–8406 (1989). Another useful vector herein is plasmid pBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto, Calif.).
A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
A polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired. In plant cells, it has been shown that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy et al., Proc. Natl. Acad. Sci. USA 85:8805–8809 (1988); and Hiatt et al., U.S. Pat. No. 4,801,340.
Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2:279–289 (1990) and U.S. Pat. No. 5,034,323. Recent work has shown suppression with the use of double stranded RNA. Such work is described in Tabara et al., Science 282:5388:430–431 (1998). Hairpin approaches of gene suppression are disclosed in WO 98/53083 and WO 99/53050.
Catalytic RNA molecules or ribozymes can also be used to inhibit expression of plant genes. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585–591 (1988).
A variety of cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986) 14:4065–4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. A report of similar work by the same group is that by Knorre, D. G., et al., Biochimie (1985) 67:785–789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (J. Am. Chem. Soc. (1987) 109:1241–1243). Meyer, R. B., et al., J. Am. Chem. Soc. (1989) 111:8517–8519, effect covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. A photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197–3203. Use of crosslinking in triple-helix forming probes was also disclosed by Home et al., J. Am. Chem. Soc. (1990) 112:2435–2437. Use of N4, N4-ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides has also been described by Webb and Matteucci, J. Am. Chem. Soc. (1986) 108:2764–2765; Nucleic Acids Res (1986) 14:7661–7674; Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991). Various compounds to bind, detect, label, and/or cleave nucleic acids are known in the art. See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and, 5,681941.
Proteins
Proteins of the present invention include proteins having the disclosed sequences as well as proteins coded by the disclosed polynucleotides. In addition proteins of the present invention include proteins derived from the native protein by deletion (so-called truncation), addition or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
For example, amino acid sequence variants of the polypeptide can be prepared by mutations in the cloned DNA sequence encoding the native protein of interest. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488–492; Kunkel et al. (1987) Methods Enzymol. 154:367–382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference. 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 preferred.
In constructing variants of the proteins of interest, modifications to the nucleotide sequences encoding the variants will generally be made such that variants continue to possess the desired activity.
The Zea mays Bax inhibitor proteins of the instant invention are aligned below (see Table 4). The seven genes appear to be divided into two groups; BI-1, -2, -3 and -4 and BI-5, -6 and -7. Areas of homology are indicated and a consensus sequence is also shown.
The Glycine max Bax inhibitor proteins of the instant invention are aligned below (see Table 5). The ten genes appear to be divided into several groups; one of Gm-BI-1 and -5; Gm-BI-2 and -3; and another containing closely related Gm-BI-6, -9, -7 and -8. Gm-BI-4 and and Gm-BI-10 appear to be in between the -1, 2, 3, 5 and -6, 9, 7, 8 groupings. Areas of homology are indicated and a consensus sequence is also shown.







TABLE 4





Protein alignment of Maize Bax Inhibitor genes





















1                                               50


(SeqIDNo2)
BI-1
(1)
--------------MESLFG--------QSQRRRRAGGSGFESLKRLG--


(SeqIDNo32)
BI-2
(1)
--------------MDAFFS----ASSASAPYGYGAGGWSYDSLKNFR--


(SeqIDNo34)
BI-3
(1)
--------------MDAFYSTTASSSTSSAPYGGGGEGWGYDSMKNFR--


(SeqIDNo8)
BI-4
(1)
--------------------------------------------------


(SeqIDNo10)
BI-5
(1)
-----------MFGYRKADP----------DLEAGGSSLLYPGMTESPE-


(SeqIDNo12)
BI-6
(1)
MASVAEMQPLAPAGYRRAPEMKEKVEASVVDLEAGTGETLYPGISRGESA


(SeqIDNo14)
BI-7
(1)
-----------MFGYQKGLDVEAGTSG---AAATGGARQLYPGMQESPE-


Consensus

(1)
                                  GGG   Y  M


 





51                                             100



BI-1
(27)
---HISPAVQSHLKHVYLTLCSALAFSALGAYLH---ILLNVGGALTTVG



BI-2
(31)
---QITPAVQTHLKLVYLTLCAALASSAVGAYLH---VVWNIGGTLTMLG



BI-3
(35)
---QISPAVQTHLKLVYLTLCVALASSAVGAYLH---VVWNIGGMLTMLG



BI-4
(1)
--------------------------------------------------



BI-5
(29)
LRWAFVRKIYVILAVQLAMTAAVSAFVVKVPAVSNFFVFSNAGVALYIFL



BI-6
(51)
LRWGFVRKVYGILAAQLLLTTAVSALTVLHPTLN---ATLSDSPGLALVL



BI-7
(36)
LRWALIRKIYVILSLQLLLTAVVAAVVVKVRAIPHFFTTTSAGLGLYIFL


Consensus

(51)
    I   V   L L  L   A  A S L   L    V  N G AL IL


 





101                                            150



BI-1
(71)
CVASIAFLISLPASRDQERNRLALLMSAALLQGASVGPLVDLVIDLDSRI



BI-2
(75)
CVGSIAWLFSVPVYEE--RKRYGLLMAAALLEGASVGPLVKLAVEFDPSI



BI-3
(79)
CVGSIAWLFSVPVYEE--RKRYWLLMAAALLEGASVGPLIKLAVEFDPSI



BI-4
(1)
---------------------------------------MTNGCFFSLSI



BI-5
(79)
IILPFLVLCPLRYYHQKHPVNLLLLGLFTVAISFAVG---MTCAFTSGKI



BI-6
(98)
AVLPFILMIPLYHYQHKHPHNFVFLGLFTLCLSFSIG--VAC-ANTQGKI



BI-7
(86)
IILPFIVLCPLYFYHEKHPVNLILLGLFTVAISFAVG---MTCAFTSGKV


Consensus

(101)
 V     L  L  Y E       LL    L    SVG  V        KI


 





151                                            200



BI-1
(121)
LVTAFVGTAVAFACFSGAAIIAKRR--EYLYLGGLLSSGLSILLWLQFAT



BI-2
(123)
LVTAFVGTAIAFACFTGAAMVARRR--EYLYLGGLLSSGLSILLWLQLAG



BI-3
(127)
LVTAFVGTAIAFACFSCAAMVAKRR--EYLYLGGLLSSGLSILLWLQFAA



BI-4
(12)
LVTAFVGTAIAFACFTGAAMVARRR--EYLYLGGLLSSGLSILLWLQLAG



BI-5
(126)
ILEAAILTAVVVISLTAYTFWAAKRGHDFNFLGPFLFAAIMVLMVFSLIQ



BI-6
(145)
VLEALVLTAGVVVSLTAYAFWASKKGKEFGYLGPILSSALTILVLTSFLQ



BI-7
(133)
ILESAILTTVVVLSLTAYTFWAVNRGKDFSFLGPFLFAAIIVLLVFALIQ


Consensus

(151)
LVTAFVGTAIAFACFTAAAMVAKRR--EYLYLGGLLSSGLSILLWLQLA





201                                            250


 



BI-1
(169)
SIFGHTSAT-FMFELYFGLLVFLGYMVFDTQEIIERAHRGDMDYIKHALT



BI-2
(171)
SIFGHSATS-FMFEVYFGLLIFLGYVVYDTQEIIERAHRGDMDHVKHALT



BI-3
(175)
SIFGHQSTSSFMFEVYFGLLIFLGYMVYDTQEVIERAHHGDMDYIKHALT



BI-4
(60)
SIFGHSATS-FMFEVYFGLLIFLGYVVYDTQEIIERAHRGDMDHVKHALT



BI-5
(176)
IFFPLGKIS-VMIYGGLASLIFCGYIIYDTDNVIKRYTY--DEYIWAAVS



BI-6
(195)
VFFPLGPVS-VGLFGGLGALVFSGFILYDTENLIKRHTY--DEYIWASVG



BI-7
(183)
ILFPLGKLS-QMIYGGLASLIFSGYIVYDTNNIIKRYTY--DQYVWAAVS


Consensus

(201)
SIFGH A S FMFEVYFGLLIFLGYIVYDTQEIIERAHYGDMDYIKHALT


 





251                                            300



BI-1
(218)
LFTDFVAVLVRILVIMMKNAQEKSQDEKKRKKR-----------------



BI-2
(220)
LFTDFVAVLVRVLVIMLKNGADKSEDKKRKKRS-----------------



BI-3
(225)
LFTDFVAVLVRILVIMLKNAADKSEDKRRKRRSW----------------



BI-4
(109)
LFTDFVAVLVRVLVIMLKNGADKSEDKKRKKRS-----------------



BI-5
(223)
LYLDVINLFLSLLQLLRAADS-----------------------------



BI-6
(242)
LYLDILNLFLSILNMLRSMQSDN---------------------------



BI-7
(230)
LYLDVINLFLSLMTLFRAAD------------------------------


Consensus

(251)
LFTDFVAVLVRILVIMLK  ADKSEDKKRKKRS





301     311
















TABLE 5





Protein alignment of Glycine max Bax Inhibitor genes





















1                                               50


(SeqIDno16)
Gm-BI1-1
(1)
---ARAFNSFFDSRNRWNYDTLKNFRQISPVVQNHLKQVYFTLCFAVVAA


(SeqIDno18)
Gm-BI1-2
(1)
MDTFFNSQSSSSSRSRWSYDTLKNFREISPLVQNHIKRVYFTLCCAVVAA


(SeqIDno20)
Gm-BI1-3
(1)
--------------------------------------------------


(trnslofSeqIDno21)
Gm-BI1-4
(1)
--------------------------------------------------


(trnslofSeqIDno22)
Gm-BI1-5
(1)
--------------------------------------------------


(trnslofSeqIDno23)
Gm-BI1-6
(1)
---------MFEPQQLYTRAKTEEFDLESG-------ETLYPGLSVGENQ


(SeqIDno25)
Gm-BI1-7
(1)
----------------MWNQPFGKTDLESG------SRPLYPMMLESP-E


(SeqIDno27)
Gm-BI1-8
(1)
----------------MWNQPLGKTDLESG------SRPLYPMMLESP-E


(SeqIDno29)
Gm-BI1-9
(1)
---------MFEPQQLYTRAKTEEFDLESG-------ETLYPGLSVGENQ


(trnslofSeqIDno30)
Gm-1-10
(1)
-----------------------KXDVESGGDGNANPRPLYPAMLEXP-Q


Consensus

(1)
                         DLESG         LYP M


 





51                                             100



Gm-BI1-1
(48)
AVGAYLHVLLNIGGFLTTVACMGSSFWLLSTPPFEERKRVT-----LLMA



Gm-BI1-2
(51)
AVGAFLHVLWNIGGFLTTLASIGSMVWLLSTPPVEEQKRLS-----LLMA



Gm-BI1-3
(1)
--------------------------------------------------



Gm-BI1-4
(1)
--------------------------------------------------



Gm-BI1-5
(1)
----------------TTVACVGSSVWLLSTPPFEERKRVT-----LLMA



Gm-BI1-6
(35)
LRWGFIRKVYGILSAQIVLTTLVSVTTVFYTPINDLLKGNS----TLLLI



Gm-BI1-7
(28)
LRWSFIRKVYSIIAIQLLVTIVVGAVVVTVRPISVFFATTG-AGLALYIV



Gm-BI1-8
(28)
LRWSFIRKVYSIIAIQLLVTIVVGAVVVTVRPISVFFATTG-AGLALYIV



Gm-BI1-9
(35)
LRWGFIRKVYGILSAQIVLTTLVSVTTVFYTPINDLLKGNS----TLLLI



Gm-BI1-10
(27)
LRWAFIRKXYTILTIQLLLTIAVASVVRLRSAPSLFSSVSSPGGLALYIV


Consensus

(51)
LRWAFIRKVY IL  QLLLT VVSSV V  TP  E  K  S     LLIV


 





101                                            150



Gm-BI1-1
(93)
ASLFQGSSIGPLIDLAIHIDPSLIFSAFVGTALAFACFS-----------



Gm-BI1-2
(96)
SALFQGASIGPLIDLAIAIDPSLIVSAFVATSLAFACFS-----------



Gm-BI1-3
(1)
--------------------------------------------------



Gm-BI1-4
(1)
--------------------------------------------------



Gm-BI1-5
(30)
ASLFQGASIGPLIDLAIQIDPSLIFSAFVGTSLAFACFS-----------



Gm-BI1-6
(81)
LLFLPFIFLIPLLKYQQKHPHNYILLALFTVSISSTVRSQLAPTPTGKLC



Gm-BI1-7
(77)
LIFVPFITLCPLYYYSQKHPVNYLLLGVFTVSLGFVVG------------



Gm-BI1-8
(77)
LIFVPFITLCPLYYYSQKHPVNYLLLAVFTVSLGFVVG------------



Gm-BI1-9
(81)
LLFLPFIFLIPLLKYQQKHPHNYILLALFTVSISSTIG------------



Gm-BI1-10
(77)
LLXAPLILVCPLYYYHQETPLNYILLFXFTVTLAXA--------------


Consensus

(101)
LL  P ISL PLI YAQ  P NYILLA FTVSLAF


 





151                                            200



Gm-BI1-1
(132)
----------------------------GAALVARRREYLYLGGLVSSGL



Gm-BI1-2
(135)
----------------------------AAALVARRREYLYLGGLLSSGL



Gm-BI1-3
(1)
--------------------------------------------------



Gm-BI1-4
(1)
--------------------------------------------------



Gm-BI1-5
(69)
----------------------------GAALVARRREYLYLGGLVSSGL



Gm-BI1-6
(131)
LTCANTDGKIVLEALILTSAVVSSLTGYAFWASKKGKDFSFLGPXLFTSL



Gm-BI1-7
(115)
LSCAFTSEKVILEAVILTAVVVIGLTLYTFWAARRGHDFNFLGPFLFGAV



Gm-BI1-8
(115)
LSCAFTSEKVILEAVILTAVVVIALTLYTFWAARRGHDFNFLGPFLFGAV



Gm-BI1-9
(119)
VTCANTDGKIVLEALILTSAVVSSLTGYAFWASKKGKDFSFLGPILFTSL



Gm-BI1-10
(113)
--------------------------------------------------


Consensus

(151)
                            A     R RDF FLG  L SGL


 





201                                            250



Gm-BI1-1
(154)
SILLWLHFASSIFGGSTALFKFELYFGLLVFVGYIVVDTQEIVERAHLGD



Gm-BI1-2
(157)
SILMWLHFASSLFGGSIALFKFELYFGLLVFVGYVFVDTQEIIERAHFGD



Gm-BI1-3
(1)
-------------GGSIALFKFELYFGLLVFVGYVIVDTQEIIERAHFGD



Gm-BI1-4
(1)
----------NISSGGTYLQFLQLYFGLLVFVGYVIVDTQEIIERAHFGD



Gm-BI1-5
(91)
SILLWLHFASSIFGGSTALFKFELYFGLLVFVGYIVVDTQEIVEXAHLGD



Gm-BI1-6
(181)
FTLILTGMMQMFFPLGPTAHAIYGAIGAMIFSGYIVYDTDNLIKRFTY--



Gm-BI1-7
(165)
LVLMVFALIQVLFPLGKLSVMIYGCLAAIIFCGYIIYDTDNLIKRYSY--



Gm-BI1-8
(165)
LVLMVFALIQVLFPLGKLSVMIYGCLAAIIFCGYIIYDTDNLIKRYSY--



Gm-BI1-9
(169)
ITLILTGMMQMFFPLGPTAHAIYGAIGAMIFSGYIVYDTDNLIKRFTY--



Gm-BI1-10
(113)



Consensus

(201)
 ILM       IF GG  L  I LYFGLLVFVGYIIVDTQEIIERAHYGD


 





251                                            300



Gm-BI1-1
(204)
LDYVKHALTLFTDLVAVFVRILVIMLKNSTE-------------------



Gm-BI1-2
(207)
LDYVKHALTLFTDLAAIFVRILIIMLKNSFG-------------------



Gm-BI1-3
(38)
LDYVKHALTLFTDLAAIFVRILIIMLK-----------------------



Gm-BI1-4
(41)
LDYVKHALTLFTDLAAIFVRILIIMVSWTSSYWCSFFFVSSR-IGIHKVL



Gm-BI1-5
(141)
LDYVKHALTLFTDLXAI---------------------------------



Gm-BI1-6
(229)
DEYIGASVTLYLDILNLFLSILRILREA----------------------



Gm-BI1-7
(213)
DEYIWASISLYLDIINLFLSLLTIFRAADS--------------------



Gm-BI1-8
(213)
DEYIWASISLYLDIINLFLSLLTIFRAADS--------------------



Gm-BI1-9
(217)
DEYIGASVTLYLDILNLFLSILRILREANN--------------------



Gm-BI1-10
(113)
--------------------------------------------------


Consensus

(251)
LDYVKHALTLFTDLIAIFL IL IM


 





301                            334



Gm-BI1-1
(235)
----------RNEKKKKRRD--------------



Gm-BI1-2
(238)
----------GNGKKKKRGGLLADRPTRAQASLP



Gm-BI1-3
(65)
------NSSERNEKKKKRRD--------------



Gm-BI1-4
(90)
ILSQLKNSSERNEKKKKRRD--------------



Gm-BI1-5
(158)
----------------------------------



Gm-BI1-6
(257)
----------------------------------



Gm-BI1-7
(243)
----------------------------------



Gm-BI1-8
(243)
----------------------------------



Gm-BI1-9
(247)
----------------------------------



Gm-BI1-10
(113)
----------------------------------


Consensus

(301)









The consensus sequence of each of the Zea mays and Glycine max BI polypeptide describes polypeptides of the invention and illustrates more conserved amino acid residues of the genus.
The isolated proteins of the present invention include a polypeptide comprising at least 30 contiguous amino acids encoded by any one of the nucleic acids of the present invention, or polypeptides that are conservatively modified variants thereof. The proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 30 to the number of residues in a full-length polypeptide of the present invention. Optionally, this subsequence of contiguous amino acids is at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 220, 240, 250, 270, 290, 300, 320, 340, 350, 370, 390, 400, 420, 440, 450, 470, 490 or 500 amino acids in length.
The present invention includes catalytically active polypeptides (i.e., enzymes). Catalytically active polypeptides will generally have a specific activity of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% that of the native (non-synthetic), endogenous polypeptide. Further, the substrate specificity (kcat/Km) is optionally substantially similar to the native (non-synthetic), endogenous polypeptide. Typically, the Km will be at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% that of the native (non-synthetic), endogenous polypeptide. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity (kcat/Km), are well known to those of skill in the art.
The present invention includes modifications that can be made to an inventive protein. In particular, it may be desirable to diminish the activity of the gene. Other modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
Using the nucleic acids of the present invention, one may express a protein of the present invention in recombinantly engineered cells such as bacteria, yeast, insect, mammalian, or plant cells. The cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.
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 gene of interest can be isolated in significant quantities for introduction into the desired plant cells.
Host cells that can be used in the practice of this invention include prokaryotes and eukaryotes. Prokaryotes include bacterial hosts such as Eschericia coli, Salmonella typhimurium, and Serratia marcescens. Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention. Since these hosts are also microorganisms, it will be essential to ensure that plant promoters which do not cause expression of the polypeptide in bacteria are used in the vector.
Commonly used prokaryotic control sequences include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake et al., Nature 292:128 (1981)). 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. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva et al., Gene 22:229–235 (1983); Mosbach et al., Nature 302:543–545 (1983)).
Synthesis of heterologous proteins in yeast is well known. See Sherman, F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982). Two widely utilized yeast 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 lysates. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
The proteins of the present invention can also be constructed using non-cellular synthetic methods. Solid phase synthesis of proteins of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3–284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al., J. Am. Chem. Soc. 85:2149–2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end (e.g., by the use of the coupling reagent N,N′-dicycylohexylcarbodiimide)) is known to those of skill.
The proteins of this invention, recombinant or synthetic, may be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: New York (1982); Deutscher, Guide to Protein Purification, Academic Press (1990). For example, antibodies may be raised to the proteins as described herein. Purification from E. coli can be achieved following procedures described in U.S. Pat. No. 4,511,503. Detection of the expressed protein is achieved by methods known in the art and include, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation.
The present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or composition of the polypeptides of the present invention in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the composition (i.e., the ratio of the polypeptides of the present invention) in a plant.
The method comprises transforming a plant cell with an expression cassette comprising a polynucleotide of the present invention to obtain a transformed plant cell, growing the transformed plant cell under conditions allowing expression of the polynucleotide in the plant cell in an amount sufficient to modulate concentration and/or composition in the plant cell.
In some embodiments, the content and/or composition of polypeptides of the present invention in a plant may be modulated by altering, in vivo or in vitro, the promoter of a non-isolated gene of the present invention to up- or down-regulate gene expression. In some embodiments, the coding regions of native genes of the present invention can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868. One method of down-regulation of the protein involves using PEST sequences that provide a target for degradation of the protein.
In some embodiments, an isolated nucleic acid (e.g., a vector) comprising a promoter sequence is transfected into a plant cell. Subsequently, a plant cell comprising the promoter operably linked to a polynucleotide of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom. A plant or plant part altered or modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to modulate the concentration and/or composition of polypeptides of the present invention in the plant. Plant forming conditions are well known in the art.
In general, content of the polypeptide is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a native control plant, plant part, or cell lacking the aforementioned expression cassette. Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation as discussed in greater detail, supra. Induction of expression of a polynucleotide of the present invention can also be controlled by exogenous administration of an effective amount of inducing compound. Inducible promoters and inducing compounds which activate expression from these promoters are well known in the art. In another embodiment, the polypeptides of the present invention are modulated in monocots or dicots, preferably maize, soybeans, sunflower, sorghum, canola, wheat, alfalfa, rice, barley and millet.
Means of detecting the proteins of the present invention are not critical aspects of the present invention. In another embodiment, the proteins are detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Methods in Cell Biology, Vol. 37: Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, Eds. (1991). Moreover, the immunoassays of the present invention can be performed in any of several configurations, e.g., those reviewed in Enzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Fla. (1980); Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B. V., Amsterdam (1985); Harlow and Lane, supra; Immunoassay: A Practical Guide, Chan, Ed., Academic Press, Orlando, Fla. (1987); Principles and Practice of Immunoassays, Price and Newman Eds., Stockton Press, NY (1991); and Non-isotopic Immunoassays, Ngo, Ed., Plenum Press, NY (1988).
Typical methods include Western blot (immunoblot) analysis, analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems which may be used, see, U.S. Pat. No. 4,391,904, which is incorporated herein by reference.
Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
The proteins of the present invention can be used for identifying compounds that bind to (e.g., substrates), and/or increase or decrease (i.e., modulate) the enzymatic activity of, catalytically active polypeptides of the present invention. The method comprises contacting a polypeptide of the present invention with a compound whose ability to bind to or modulate enzyme activity is to be determined. The polypeptide employed will have at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the specific activity of the native, full-length polypeptide of the present invention (e.g., enzyme). Methods of measuring enzyme kinetics are well known in the art. See, e.g., Segel, Biochemical Calculations, 2nd ed., John Wiley and Sons, New York (1976).
Antibodies can be raised to a protein of the present invention, including individual, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these proteins in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons of skill.
In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in, e.g., Basic and Clinical Immunology, 4th ed., Stites et al., Eds., Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane, Supra; Goding, Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press, New York, N.Y. (1986); and Kohler and Milstein, Nature 256:495–497 (1975).
Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors (see, e.g., Huse et al., Science 246:1275–1281 (1989); and Ward et al., Nature 341:544–546 (1989); and Vaughan et al. Nature Biotechnology, 14:309–314 (1996)). Alternatively, high avidity human monoclonal antibodies can be obtained from transgenic mice comprising fragments of the unrearranged human heavy and light chain Ig loci (i.e., minilocus transgenic mice). Fishwild et al., Nature Biotech. 14:845–851 (1996). Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen et al., Proc. Natl. Acad. Sci. 86:10029–10033 (1989).
The antibodies of this invention can be used for affinity chromatography in isolating proteins of the present invention, for screening expression libraries for particular expression products such as normal or abnormal protein or for raising anti-idiotypic antibodies which are useful for detecting or diagnosing various pathological conditions related to the presence of the respective antigens.
Frequently, the proteins and antibodies of the present invention will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.
Transformation of Cells
The method of transformation is not critical to the present invention; various methods of transformation are currently available. As newer methods are available to transform crops or other host cells they may be directly applied. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a host cell to obtain the transcription and/or translation of the sequence to effect phenotypic changes in the organism. Thus, any method which provides for efficient transformation/transfection may be employed.
A DNA sequence coding for the desired polynucleotide of the present invention, for example a cDNA or a genomic sequence encoding a full length protein, can be used to construct an expression cassette which can be introduced into the desired plant. Isolated nucleic acid acids of the present invention can be introduced into plants according techniques known in the art. Generally, expression cassettes as described above and suitable for transformation of plant cells are prepared.
Techniques for transforming a wide variety of higher plant species are well known and described in the technical, scientific, and patent literature. See, for example, Weising et al., Ann. Rev. Genet. 22:421–477 (1988). For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, PEG poration, particle bombardment, silicon fiber delivery, or microinjection of plant cell protoplasts or embryogenic callus. See, e.g., Tomes et al., Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment. pp. 197–213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods. eds. O. L. Gamborg and G. C. Phillips. Springer-Verlag Berlin Heidelberg New York, 1995. Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. See, U.S. Pat. No. 5,591,616.
The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al., Embo J. 3:2717–2722 (1984). Electroporation techniques are described in Fromm et al., Proc. Natl. Acad. Sci. 82:5824 (1985). Ballistic transformation techniques are described in Klein et al., Nature 327:70–73 (1987).
Agrobacterium tumefaciens—meditated transformation techniques are well described in the scientific literature. See, for example Horsch et al., Science 233:496–498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. 80:4803 (1983). For instance, Agrobacterium transformation of maize is described in U.S. Pat. No. 5,981,840. Agrobacterium transformation of soybean is described in U.S. Pat. No. 5,563,055.
Other methods of transformation include (1) Agrobacterium rhizogenes—mediated transformation (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, Vol. 6, P W J Rigby, Ed., London, Academic Press, 1987; and Lichtenstein, C. P., and Draper, J,. In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985), Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988) describes the use of A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al., Plant Cell Physiol. 25:1353, (1984)), (3) the vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci. USA 87:1228, (1990)).
DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al., Methods in Enzymology 101:433 (1983); D. Hess, Intern Rev. Cytol. 107:367 (1987); Luo et al., Plane Mol. Biol. Reporter 6:165 (1988). Expression of polypeptide coding polynucleotides can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al., Nature 325:274 (1987). DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaus et al., Theor. Appl. Genet. 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27–54 (1986).
Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transformation 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 dextran, 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, R. J., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977).
Transgenic Plant Regeneration
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with a polynucleotide of the present invention. For transformation and regeneration of maize see, Gordon-Kamm et al., The Plant Cell 2:603–618 (1990).
Plants cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pp. 124–176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21–73 (1985).
The regeneration of plants containing the foreign gene introduced by Agrobacterium can be achieved as described by Horsch et al., Science 227:1229–1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. U. S. A. 80:4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.
Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys. 38:467–486 (1987). The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis. (1988).
One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings, via production of apomictic seed, or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype.
Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.
Transgenic plants expressing a selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then be analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.
Another embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non-transgenic). Still another embodiment involves back-crossing to a parental plant and/or out-crossing with a non-transgenic plant.
Seeds derived from plants regenerated from transformed plant cells, plant parts or plant tissues, or progeny derived from the regenerated transformed plants, may be used directly as feed or food, or further processing may occur.
The present invention may be used for transformation of any plant species, monocotyledonous and dicotyledonous, including, but not limited to, corn (Zea mays), 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), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), millet (Pennisetum glaucum, Panicum miliaceum, Eleusine coracana, Setaria italica), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea 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), oats, barley (Hordeum vulgare), 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 plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
Plants of the present invention include crop plants (for example, corn, alfalfa, sunflower, safflower, canola, soybean, casava, cotton, peanut, sorghum, rice, wheat, millet, tobacco, rye, oats, barley, turf grass, etc.). In one embodiment plants of the present invention include corn, soybean, canola, rice, sunflower, wheat and sorghum plants, and in another corn and soybean plants.

Insect Pests
The compositions of the present invention may be effective against a variety of plant pests including but not limited to insects of the order Lepidoptera, e.g. Achoroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integeffima, Dendrolimus sibericus, Desmia feneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loffini, Esphestia elutella, Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis, Paleacrita vemata, Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota stultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp., Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia hi, Udea rubigalis, Xylomyges curiails, and Yponomeuta padella. 
Also, the compositions of the present invention may be effective against insect pests including insects selected from the orders Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera, especially Diabrotica virgifera and Lepidoptera. Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicomis, corn bloth leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, two spotted spider mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, leser cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissus leucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, plae western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabrotica undecimpunctata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower: Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens, cotton boll worm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; Anthonomus grandis, bool weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper; Blissus leucopterus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, cotton boll worm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, twospotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Jylemya platura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid.
Furthermore, compositions of the present invention may be effective against Hemiptera such as Lygus hesperus, Lygus lineolaris, Lygus pratensis, Lygus rugulipennis Popp, Lygus pabulinus, Calocoris norvegicus, Orthops compestris, Plesiocoris rugicollis, Cyrtopeltis modestus, Cyrtopeltis notatus, Spanagonicus albofasciatus, Diaphnocoris chlorinonis, Labopidicola allii, Pseudatomoscelis seriatus, Adelphocoris rapidus, Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae, Nysiusraphanus, Euschistus servus, Nezara viridula, Eurygaster, Coreidae, Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and Cimicidae.
The preferred stage of experimental organism for testing for pesticidal activity is larvae or immature forms of these above mentioned insect pest. Experimental organisms may be reared in total darkness at from about 20° C. to about 30° C. and from about 30% to about 70% relative humidity. Bioassays may be performed as described in Czapla T. H. and Lang B. A. Effect of Plant Lectins on the Larval Development of European Corn Borer (Lepidoptera: Pyralidae) and Southern Corn Rootworm (Coleoptera: Chrysomelidae), J. Econ. Entomol. 83(6): 2480–2485 (1990). Methods of rearing insect pest larvae and performing bioassays are well known to one skilled in the art.
A wide variety of bioassay techniques are known to one skilled in the art. General procedures include addition of experimental compound to the diet source in an enclosed container. Pesticidal activity can be measured by, but is not limited to, mortality, weight loss, attraction, repellency and other behavioral and physical changes after feeding and exposure for an appropriate length of time. Bioassays described herein, can be used with any feeding insect pest in the larval or adult stage.
Disease
The methods of the invention can be used with other methods available in the art for enhancing disease resistance in plants. Similarly, the plant defense mechanisms described herein may be used alone or in combination with other proteins or agents to protect against plant diseases and pathogens.
Pathogens of the invention include, but are not limited to, viruses or viroids, bacteria, insects, nematodes, fungi, and the like. Viruses include any plant virus, for example, tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specific fungal and viral pathogens for the major crops include: Soybeans: Phytophthora megasperma f.sp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchiil, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Scierotinia scierotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibater michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae; Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, High Plains Virus, European wheat striate virus; Sunflower: Orobanche, Plasmophora halstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis helianthi, Altemaria helianthi, Altemaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Corn: Fusarium moniliforme var. subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O,T (Cochliobolus heterostrophus), Helminthosporium carbonum I, II & IlI (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella-maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora, Com stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum, Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria altemata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronoscierospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola, etc.
Nematodes include parasitic nematodes such as root-knot, cyst, lesion, and renniform nematodes, etc.
All publications cited in this application 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.
The present invention will be further described by reference to the following detailed examples. It is understood, however, that there are many extensions, variations, and modifications on the basic theme of the present invention beyond that shown in the examples and description, which are within the spirit and scope of the present invention.


EXAMPLES
Example 1
Library Construction used for Maize and Soybean BI EST's
A. Total RNA Isolation
Total RNA was isolated from various maize or soybean tissues with TRIzol Reagent (Life Technology Inc. Gaithersburg, Md.) using a modification of the guanidine isothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi (Chomczynski, P., and Sacchi, N. Anal. Biochem. 162, 156 (1987)). In brief, plant tissue samples were pulverized in liquid nitrogen before the addition of the TRIzoI Reagent, and then were further homogenized with a mortar and pestle.
Addition of chloroform followed by centrifugation was conducted for separation of an aqueous phase and an organic phase. The total RNA was recovered by precipitation with isopropyl alcohol from the aqueous phase.
B. Poly(A)+ RNA Isolation
The selection of poly(A)+ RNA from total RNA was performed using PolyATact system (Promega Corporation. Madison, Wis.). In brief, biotinylated oligo(dT) primers were used to hybridize to the 3′ poly(A) tails on mRNA. The hybrids were captured using streptavidin coupled to paramagnetic particles and a magnetic separation stand. The mRNA was washed at high stringent condition and eluted by RNase-free deionized water.
C. cDNA Library Construction
cDNA synthesis was performed and unidirectional cDNA libraries were constructed using the SuperScript Plasmid System (Life Technology Inc. Gaithersburg, Md.). The first stand of cDNA was synthesized by priming an oligo(dT) primer containing a Not I site. The reaction was catalyzed by SuperScript Reverse Transcriptase II at 45° C. The second strand of cDNA was labeled with alpha-32P-dCTP and a portion of the reaction was analyzed by agarose gel electrophoresis to determine cDNA sizes. cDNA molecules smaller than 500 base pairs and unligated adapters were removed by Sephacryl-S400 chromatography. The selected cDNA molecules were ligated into pSPORT1 vector in between of Not I and Sal I sites.
Example 2
Sequencing and cDNA Subtraction Procedures used for Maize and Soybean BI EST's
A. Sequencing Template Preparation
Individual colonies were picked and DNA was prepared either by PCR with M13 forward primers and M13 reverse primers, or by plasmid isolation. All the cDNA clones were sequenced using M13 reverse primers.
B. Q-bot Subtraction Procedure
cDNA libraries subjected to the subtraction procedure were plated out on 22×22 cm2 agar plate at density of about 3,000 colonies per plate. The plates were incubated in a 37° C. incubator for 12–24 hours. Colonies were picked into 384-well plates by a robot colony picker, Q-bot (GENETIX Limited). These plates were incubated overnight at 37° C.
Once sufficient colonies were picked, they were pinned onto 22×22 cm2 nylon membranes using Q-bot. Each membrane contained 9,216 colonies or 36,864 colonies. These membranes were placed onto agar plate with appropriate antibiotic. The plates were incubated at 37° C. for overnight.
After colonies were recovered on the second day, these filters were placed on filter paper prewetted with denaturing solution for four minutes, then were incubated on top of a boiling water bath for additional four minutes. The filters were then placed on filter paper prewetted with neutralizing solution for four minutes. After excess solution was removed by placing the filters on dry filter papers for one minute, the colony side of the filters were place into Proteinase K solution, incubated at 37° C. for 40–50 minutes. The filters were placed on dry filter papers to dry overnight. DNA was then cross-linked to nylon membrane by UV light treatment.
Colony hybridization was conducted as described by Sambrook, J., Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A laboratory Manual, 2nd Edition). The following probes were used in colony hybridization:

    1. First strand cDNA from the same tissue from which the library was made to remove the most redundant clones.
    2. 48–192 most redundant cDNA clones from the same library based on previous sequencing data.
    3. 192 most redundant cDNA clones in the entire corn sequence database.
    4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GM AAA AAA AAA AAA AAA AAA, removes clones containing a poly A tail but no cDNA.
    5. cDNA clones derived from rRNA.


The image of the autoradiography was scanned into computer and the signal intensity and cold colony addresses of each colony was analyzed. Re-arraying of cold-colonies from 384 well plates to 96 well plates was conducted using Q-bot.
Example 3
Identification of Maize and Soybean BI EST's from a Computer Homology Search
Gene identities were determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403–410; see also www.ncbi.nlm.nih.gov/BLAST/) searches under default parameters for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases. For the NCBI Nonredundant (NR) database last release of which was Feb. 17, 2001 at 4:52 AM, containing 197,782,823 letters and 625,274 sequences. For the NCBI Nucleotide (NT) database last release of which was Feb. 17, 2001 at 4:52 AM, containing 2,752,804,350 letters and 775,058 sequences. The cDNA sequences were analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm. The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX algorithm (Gish, W. and States, D. J. (1993) Nature Genetics 3:266–272) provided by the NCBI. In some cases, the sequencing data from two or more clones containing overlapping segments of DNA were used to construct contiguous DNA sequences.
Example 4
Composition of cDNA Libraries Used to Isolate and Sequence Additional cDNA Clones
cDNA libraries representing mRNAs from various maize (genotype B73) tissues were generated Based on the distribution of BI EST's, the tissue sources from which the various maize BI genes can be derived are presented in Table 1 below.







TABLE 1







Maize tissues used for RNA extraction and construction of cDNA


libraries, from which the indicated ZmBl-related ESTs (and ultimately


the full-length genes) can be derived (as indicated).








Tissue source for
ZmBl Gene














library*
1-1
1-2
1-3
1-4
1-5
1-6
1-7





leaf1
X

X


X



stress2

X







kernel



X





embryo



X





stalk (stem)






X


whorl






X


seedling




X







1= RNA extracted from seedling leaves


2= RNA extracted from stress-induced seedlings


*These libraries were normalized essentially as described in U.S. Pat. No. 5,482,845, incorporated herein by reference.






In a similar fashion, cDNA libraries representing mRNAs from various soybean tissues were generated, and used to identify soybean BI genes.







TABLE 2







Identity of Glycine max soybean clone, reference EST,


line and tissue from which genes were identified.










Gene
Reference EST
Line
Tissue





Gm-Bl1-1
sls1c.pk024.d8 or
Wye genotype
Embryo



sls1c.pk011.p17


Gm-Bl1-2
ses4d.pk0036.d1
Wye genotype
Cells, culture


Gm-Bl1-3
sl2.pk0091.d2
Wye genotype
Pods


Gm-Bl1-4
sdp4c.pk036.m11
Wye genotype
Pods


Gm-Bl1-5
sgs4c.pk002.g3
Wye genotype
Seeds, flower


Gm-Bl1-6
sdp2c.pk032.l20
Wye genotype
Pods


Gm-Bl1-7
sfl1n.pk0001.b6
Wye genotype
Pods


Gm-Bl1-8
sl2.pk0031.c3
Williams 82 genotype
Seedling,





shoot, leaf


Gm-Bl1-9
sls2c.pk010.d12
Wye genotype
Pods


Gm-Bl1-10
src2c.pk015.h4
Soy/437654
Root









cDNA libraries were prepared in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). Conversion of the Uni-ZAPTM libraries into plasmid libraries was accomplished according to the protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasmid vector pBluescript. cDNA inserts from randomly picked bacterial colonies containing recombinant pBluescript plasmids were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences or plasmid DNA was prepared from cultured bacterial cells. Amplified insert DNAs or plasmid DNAs were sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”; see Adams, M. D. et al., (1991) Science 252:1651). The resulting ESTs were analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
Example 5
Transformation and Regeneration of Maize Callus
Immature maize embryos from greenhouse or field grown High type 11 donor plants are bombarded with a plasmid containing a polynucleotide of the invention (BI). The BI polynucleotide is operably linked to a constitutive promoter such as nos, or an inducible promoter, such as In2, plus a plasmid containing the selectable marker gene PAT (Wohileben et al. (1988) Gene 70:25–37) that confers resistance to the herbicide Bialaphos, fused to the Green Fluorescence protein. Transformation is performed as follows.
The ears are surface sterilized in 50% Chlorox 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. These were cultured on 560 L medium 4 days prior to bombardment in the dark. Medium 560 L is an N6-based medium containing Eriksson's vitamins, thiamine, sucrose, 2,4-D, and silver nitrate. The day of bombardment, the embryos are transferred to 560 Y medium for 4 hours and are arranged within the 2.5-cm target zone. Medium 560Y is a high osmoticum medium (560L with high sucrose concentration).











TABLE 3







(560L)
(560Y)
(560P)






















CHU(N6) BASAL SALTS
4.0
g/l
4.0
g/l
4.0
g/l


ERIKSSON'S VITAMIN
1.0
ml/l
1.0
ml/l
1.0
ml/l


MIX (1000X)


THIAMINE HCL
0.5
mg/l
0.5
mg/l
0.5
mg/l


2,4-D
1.0
mg/l
2.0
mg/l
2.0
mg/l


L-PROLINE
2.88
g/l
0.69
g/l
0.69
g/l


SILVER NITRATE
4.25
mg/l
0.85
mg/l
0.85
mg/l


SUCROSE
20.0
g/l
120.0
g/l
30.0
g/l


GELRITE
2.0
g/l
3.0
g/l
3.0
g/l


pH 5.8









A plasmid vector comprising a polynucleotide of the invention operably linked to the selected promoter is constructed. 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 (0.6 mg) in water, 20 μl (2 μg) DNA in Tris-EDTA buffer (1 μg total), 100 μl 2.5 M CaCl2, 40 μl 0.1 M spermidine.
Each reagent is added sequentially to the tungsten particle suspension. The final mixture is sonicated briefly. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 μl 100% ethanol, and centrifuged again for 30 seconds. Again the liquid is removed, and 60 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 5 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
The sample plates are bombarded at a distance of 8 cm from the stopping screen to the tissue, using a Dupont biolistics helium particle gun. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
Four to twelve hours post bombardment, the embryos are moved to 560P (a low osmoticum callus initiation medium similar to 560L but with lower silver nitrate), for 3–7 days, then transferred to 560R selection medium, an N6 based medium similar to 560P containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. Multicellular GFP cell clusters become visible after two weeks and their numbers are periodically recorded. After approximately 10 weeks of selection, selection-resistant GFP positive callus clones are sampled for PCR and activity of the polynucleotide of interest (see example 7). Positive lines are transferred to 288J medium, an MS-based medium with lower sucrose and hormone levels (0.5 mg/l zeatin, 1.0 mg/l IAA, 0.1 mg/l ABA, 0.6% sucrose and 3 mg/l bialophos, to initiate plant regeneration. Following somatic embryo maturation (2–4 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 medium in tubes for 7–10 days until plantlets were 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 ClassicTM 600 pots (1.6 gallon)(#14–9674–9; Hummert International, Earth City, Mo.) and grown to maturity. Plants are monitored for expression of the polynucleotide of interest.
It is noted that any suitable method of transformation can be used, such as sonication, electroporation, microinjection, and others, as well as the more established methods for maize which include particle delivery (described above) and Agrobacterium-mediated transformation. Numerous varieties of maize germplasm including publicly-available and proprietary hybrids and inbreds can be transformed using the Agrobacterium mediated DNA delivery method, as described by U.S. Pat. No. 5,981,840, which we follow for this example with the following modifications. Agrobacteria are grown to the log phase in liquid minimal A medium containing 100 μM spectinomycin. Embryos are immersed in a log phase suspension of Agrobacteria adjusted to obtain an effective concentration of 5×108 cfu/ml. Embryos are infected for 5 minutes and then co-cultured on culture medium containing acetosyringone for 7 days at 20° C. in the dark. After 7 days, the embryos are transferred to standard culture medium (MS salts with N6 macronutrients, 1 mg/L 2,4-D, 1 mg/L Dicamba, 20 g/L sucrose, 0.6 g/L glucose, 1 mg/L silver nitrate, and 100 mg/L carbenicillin) with 3 mg/L Bialaphos® as the selective agent. Plates are maintained at 28° C. in the dark and are observed for colony recovery with transfers to fresh medium every two to three weeks.
The problem of inefficient production of transgenic crop plants, especially, but not limited to maize and soybean, is addressed in the three examples below. These examples illustrate how the BI genes can be used to improve plant transformation efficiency.
Example 6
Transient Zm-BI Expression Stimulates DNA Replication and Enhances Transgene Integration
Regardless of the method of DNA delivery, cells competent for the integration of foreign DNA must be actively dividing. There is a growing body of evidence suggesting that integration of foreign DNA occurs in dividing cells (this includes both Agrobacterium and direct DNA delivery methods). It has long been observed that dividing transformed cells represent only a fraction of cells that transiently express a transgene. It is well known (in non-plant systems) that the delivery of damaged DNA, (similar to what we introduce by particle gun delivery methods) induces an immediate cell cycle arrest and often triggers events leading to apoptosis. This cessation of the cell cycle and stimulation of apoptosis can be reversed by ectopic transient over-expression of positive cell cycle regulators, or by inhibitors of apoptosis. The overall result will be a stimulation of the cell cycle which will increase integration frequencies.
To demonstrate this, a Zm-BI or Gm-BI gene is cloned into a cassette with a constitutive promoter (i.e. either a strong maize promoter such as the ubiquitin promoter including the first ubiquitin intron, or a weak constitutive promoter such as nos). Delivery of the BI gene in an appropriate plant expression cassette (for example, in a UBI::BI::pinII-containing plasmid) along with UBI::bar::pinII can be accomplished through particle bombardment or Agrobacterium-mediated transformation. DNA is introduced into maize cells capable of growth on suitable maize culture medium. Such competent cells can be from maize suspension culture, callus culture on solid medium, freshly isolated immature embryos or meristem cells. Immature embryos of the PHP38 genotype are used as the target for co-delivery of these two plasmids. Transient expression of the BI gene inhibits apoptosis and increases the proportion of cells that can progress through the cell cycle, thus increasing the proportion of recipient-cells (i.e. into which DNA was introduced) that enter S-phase.
This stimulation through the G1/S transition in cells harboring transgenic plasmid DNA provides an optimal cellular environment for integration of the introduced genes. Cytological methods can be used to verify increased frequencies of progression through S-phase and mitosis (i.e. for cells in which a visual marker such as GFP was transformed alongside BI, the green fluorescent cells will exhibit a higher mitotic index). Cells in S-phase (undergoing DNA replication) can be monitored by detecting nucleotide analog incorporation. For example, following incubation of cells with bromodeoxyuridine (BrdU) incorporation of this thymadine analog can be detected by methods such as antiBrdU immunocytochemistry or through enhancement of Topro3 fluorescence following BrdU labeling. BI expression will increase the proportion of cells incorporating BrdU (i.e. a higher percentage of transformed cells will incorporate BrdU relative to untransformed cells). Increased DNA synthesis can also be monitored using such methods as fluorescence activated cell sorting (FACS) of protoplasts (or nuclei), in conjunction with appropriate BrdU-insensitive fluorescent DNA labels such as propidium iodide and DAPI or BrdU-detecting methods described above. For example, tissue is homogenized to release nuclei that are analyzed using the FACS for both green fluorescence (from our accompanying GFP marker) and DNA content. Such analysis can not only determine stages of the cell cycle but can be used to quantify the proportion of the cell population undergoing apoptosis. Such FACS analysis demonstrates that expression of a co-transformed GFP reporter correlates with BI induced reduction in the proportion of cells exhibiting signs of apoptosis such as chromosomal DNA fragmentation and changes in mitochondrial permeability. Similar experiments can be run using the fluorescently labeled anti-BrdU antisera to demonstrate that BI expression increased the percentage of cells in S-phase. Cell cycle stage-specific probes can also be used to monitor cell cycle progression. For example, numerous spindle-associated proteins are expressed during a fairly narrow window during mitosis, and antibodies or nucleic acid probes to cyclins, histones, or DNA synthesis enzymes can be used as positive markers for the G1/S transition. For cells that have received the BI gene cassette, changes in the cell population receiving DNA is manifested in a decrease in apoptotic cells (which can be verified by flow cytometry) and an increased mitotic index (detected by staining for mitotic figures using a DNA dye such as DAPI or Hoechst 33258).
To assess the effect on transgene integration, growth of bialaphos-resistant colonies on selective medium is a reliable assay. Within 1–7 days after DNA introduction, the embryos are moved onto culture medium containing 3 mg/l of the selective agent bialaphos. Embryos, and later callus, are transferred to fresh selection plates every 2 weeks. After 6–8 weeks, transformed calli are recovered. Transgenic callus containing the introduced genes can be verified using PCR and Southern analysis. Northern analysis can also be used to verify which calli are expressing the bar gene, and whether the BI gene is being expressed at levels above normal wild-type cells (based on hybridization of probes to freshly isolated mRNA population from the cells). In immature embryos that had transient, elevated BI expression, higher numbers of stable transformants are recovered (likely a direct result of increased integration frequencies). Increased transgene intregration frequency can also be assessed using such well-established labeling methods such as in situ hybridization.
For this specific application (using transient BI-mediated cell cycle stimulation to increase transient integration frequencies), it may be desirable to reduce the likelihood of ectopic stable expression of the BI gene. Strategies for transient-only expression can be used. This includes delivery of RNA (transcribed from the BI gene) or BI protein along with the transgene cassettes to be integrated to enhance transgene integration by transient stimulation of cell division. Using well-established methods to produce in vitro BI-RNA, this can then be purified and introduced into maize cells using physical methods such as microinjection, bombardment, electroporation or silica fiber methods. For protein delivery, the gene is first expressed in a bacterial or baculoviral system, the protein purified and then introduced into maize cells using physical methods such as microinjection, bombardment, electroporation or silica fiber methods. Alternatively, BI proteins are delivered from Agrobacterium tumefaciens into plant cells in the form of fusions to Agrobacterium virulence proteins. Fusions are constructed between BI and bacterial virulence proteins such as VirE2, VirD2, or VirF which are known to be delivered directly into plant cells. Fusions are constructed to retain both those properties of bacterial virulence proteins required to mediate delivery into plant cells and the BI activity required for enhancing transgene integration. This method ensures a high frequency of simultaneous co-delivery of T-DNA and functional BI protein into the same host cell. The methods above represent various means of using the BI gene or its encoded product to transiently stimulate DNA replication and cell division, which in turn enhances transgene integration by providing an improved cellular/molecular environment for this event to occur.
Example 7
Altering BI Expression Stimulates the Cell Cycle and Growth
Based on results in other eukaryotes, expression of BI genes inhibits apoptosis and thus increases the proportion of cells that can continue to divide. This increase in division rate is assessed in a number of different manners, being reflected in smaller cell size, more rapid incorporation of radiolabeled nucleotides, and faster growth (i.e. more biomass accumulation). Delivery of the BI in an appropriate plant expression cassette is accomplished through particle bombardment or Agrobacterium-mediated transformation. Through inhibition of apoptosis, BI gene expression improves the cellular environment for integration of introduced genes (as per Example 1). This will trigger a tissue culture response (cell divisions) in genotypes that typically do not respond to conventional culture techniques, or stimulate growth of transgenic tissue beyond the normal rates observed in wild-type (non-transgenic) tissues. This will also improve the culture response during selection protocols (i.e. the application of chemical selection to favor growth of transformants). The inhibition of apoptosis during this stressful period will stimulate growth beyond levels normally observed during the selection process.
To demonstrate this, the BI gene is cloned into a cassette with a constitutive promoter (i.e. either a strong maize promoter such as the ubiquitin promoter including the first ubiquitin intron, or a weak constitutive promoter such as nos). Either particle-mediated 93 DNA delivery or Agrobacterium-mediated delivery are used to introduce the UBI::BI::pinII-containing plasmid along with a UBI::bar::pinII-containing plasmid into maize cells capable of growth on suitable maize culture medium. Such competent cells can be from maize suspension culture, callus culture on solid medium, freshly isolated immature embryos or meristem cells. Immature embryos of the PHP38 genotype are used as the target for co-delivery of these two plasmids, and within 1–7 days the embryos are moved onto culture medium containing 3 mg/l of the selective agent bialaphos. Alternately, instead of using chemical selection to identify transformants, a more vigorous growth pattern identifies the transformants (see Example 9).
Embryos, and later callus, are transferred to fresh selection plates every 2 weeks. After 6–8 weeks, transformed calli are recovered. In treatments where both the bargene and BI gene have been transformed into immature embryos, a higher number of growing calli are recovered on the selective medium and callus growth is stimulated (relative to treatments with the bar gene alone). Transgenic callus can be verified using PCR and Southern analysis. Northern analysis can also be used to verify which calli are expressing the bar gene, and which are expressing the BI gene at levels above normal wild-type cells (based on hybridization of probes to freshly isolated mRNA population from the cells).
Inducible Expression Variation. The BI gene can also be cloned into a cassette with an inducible promoter such as the benzenesulfonamide-inducible promoter. The BI expression vector is co-introduced into plant cells with a marker containing vector and after selection on bialaphos, the transformed cells are exposed to the safener (inducer). This chemical induction of BI expression results in decreased apoptosis and a overall stimulation of growth. The cells are screened for the presence of BI RNA by northern, or RT-PCR (using transgene specific probes/oligo pairs), for BI-encoded protein using BI-specific antibodies in Westerns or using hybridization. Increased DNA replication is detected using BrdU labeling followed by antibody detection of cells that incorporated this thymidine analogue. Likewise, other cell cycle division assays could be employed, as described above.
Example 8
BI Expression Resulted in Increased Growth Rates, Which Could be used as a Screening Criterion for Positive Selection of Transformants
Using two promoters of increasing strength to drive BI expression in maize, BI stimulates more rapid callus growth over control treatments, and the stronger promoter driving BI results in faster growth than with the low-level promoter. For example, an experiment is performed to compare the nos and UBI promoters. As noted above, based on our experience with these two promoters driving other genes, the In2 promoter (in the absence of an inducer other than auxin from the medium) would drive expression at very low levels. The nos promoter has been shown to drive moderately-low levels of transgene expression (approximately 10- to 30-fold lower than the maize ubiquitin promoter). One control treatment is used in this experiment, the UBI:PAT ̃GFPmo:pinII construct by itself (with no BI). PHP38 immature embryos are bombarded as previously described, and transgenic, growing events are scored at 3 and 6 weeks. The control treatment results in a transformation frequency of 0.5 to 3.0%. In2: BI, nos:BI and UBI:BI treatments result in substantially higher transformation frequencies, with the improvement (relative to the control) being greatest in the treatment with the stronger promoter (UBI).
Within these treatments we also expect there to be an increase in the overall frequency of vigorously growing calli, relative to the control treatment, again with the stronger promoter results in the greatest increase in growth rate.
Example 9
Re-transformation of BI-transgenic Progeny Results in Elevated Transformation Frequency of PHP38
Agrobacterium mediated transformation As the starting point for Agrobacterium-mediated re-transformation experiments, regenerated PHP38 T0 transformants are produced containing maize BI-1 expression cassettes and UBI::moPAT ̃GFP::pinII. The BI-1 expression cassette with the nopaline synthase promoter from Agrobacterium tumefaciens (Shaw et al., Nucl. Acids Res. 12:7831–7846, 1984) or modified nos promoters is described below. The PAT ̃GFP cassette contains a maize-optimized gene encoding phosphinothricin acetyltransferase (moPAT, see WO 98/30701) followed by a sequence encoding 4×(GSSS), a flexible polypeptide linker of GLY-SER-SER-SER, and then a maize-optimized nucleic acid sequence encoding Green Fluorescence Protein (GFP; see WO 98/01575). This PAT ̃GFP fusion construct is driven by the maize ubiquitin promoter (Christensen et al., Plant Mol. Biol. 18:675–689,1992) and contains a potato proteinase inhibitor II3′ sequence (An et al., Plant Cell 1:115–122, 1989).
Transgenic PHP38 plants containing a co-segregating BI-1 expression cassette and the UBI::PAT ̃GFP expression cassette are crossed to wild-type (non-transformed) PHP38 plants (using the non-transformed parent as the pollen donor). As expected from such a cross, the developing embryos on these ears segregate either for transgene expression or wild-type. Immature embryos are harvested 12 days after pollination and transformed with an Agrobacterium binary plasmid containing UBI::moCAH::pinII (moCAH is a maize optimized [for codon usage] gene that encodes for the Myrothecium verrucaria cyanamide hydratase protein [CAH] that can hydrate cyanamide to non-toxic urea). A standard Agrobacterium-mediated transformation protocol (U.S. Pat. No. 5,981,840) adapted for cyanamide selection (see WO 98/30701) is used, with additional modifications listed below. Agrobacterium is grown to log phase in liquid minimal-A medium containing 100 μM acetosyringone and spectinomycin. Embryos are immersed in a log phase suspension of Agrobacterium adjusted to obtain 3×108 CFU's/ml. Embryos are then co-cultured on culture medium with acetosyringone for 3 days at 20° C. After 3 days the embryos are returned to standard culture medium with 100 mg/l carbenicillin added to kill residual Agrobacterium. After an additional 4 days the segregating embryos are divided into GFP positive and GFP negative populations and moved to fresh culture medium with 50 mg/l cyanamide for selection. After 8 weeks the numbers of transformed colonies are determined.
Since the PAT ̃GFP and BI-1 expression cassettes are co-segregating, GFP expression is used to separate segregating transgenic (PAT ̃GFP+/BI-1+) and non-transgenic (wild-type) embryos after Agrobacterium-mediated transformation, and then these separate populations are cultured and selected as independent groups. Using embryos from different ears co-segregating for GFP and BI-1, we expect the BI-1-containing embryos to exhibit a much higher transformation frequency demonstrating that ectopic BI-1 expression improves re-transformation frequencies. For ears from which the wild-type embryos (non-transgenic segregants) produce very low levels (or no) transformants, we expect the GFP+/BI-1-containing embryos from the same ears to produce cyanamide-resistant transformants at approximately a 5–10% frequency. In ears in which the wild-type, non-transformed embryos produce higher levels of transformants (for example, upwards of 10%), we expect the transformation frequencies from the BI-1 expressing embryos to be elevated to even greater levels, i.e. upwards of 30–40%.
Particle gun transformation re-transformations. As the starting point for particle gun-mediated re-transformation experiments, regenerated PHP38 T0 transformants are produced containing maize BI-1 expression cassettes and UBI::moPAT ̃GFP::pinII. Transformants containing UBI::moPAT ̃GFP::pinII and BI-1 expression cassettes are tested; with BI-1 being driven by a nos promoter. As a control, a non-functional version of BI-1 is used, in which the BI-1 coding sequence is frame-shifted by 1 position after the START codon, resulting in essentially the same mRNA species but producing a non-functional protein. Expression of this frame-shifted sequence (abbreviated “f-shift” below) is driven by the nos promoter. As mentioned above for the functional BI-1 genes, this f-shift BI-1 cassette co-segregates with GFP in the T1 progeny embryos.
Transgenic PHP38 plants containing a co-segregating BI-1 expression cassette and the UBI::PAT ̃GFP expression cassette are crossed to wild-type (non-transformed) PHP38 plants (using the non-transformed parent as the pollen donor). As expected from such a cross, the developing embryos on these ears segregate either for transgene expression or wild-type. Embryos co-segregating for GFP and BI-1 (functional and frame-shift (fs) versions) are transformed using a particle gun using the standard immature embryo bombardment transformation protocol (Songstad D. D. et al., In Vitro Cell Dev. Biol. Plant 32:179–183, 1996). Cells are transformed by culturing maize immature embryos (approximately 1–1.5 mm in length) onto 560P medium containing N6 salts, Erikkson's vitamins, 0,69 g/l proline, 2 mg/l 2,4-D and 3% sucrose. After 4–5 days of incubation in the dark at 28° C., embryos are removed from 560P medium and cultured, scutellum up, onto 560Y medium which is equivalent to 560P but contains 12% sucrose. Embryos are allowed to acclimate to this medium for 3 h prior to transformation. The scutellar surface of the immature embryos is targeted using particle bombardment with a ubi:moCAH:pinII plasmid. Embryos are transformed using the PDS-1000 Helium Gun from Bio-Rad at one shot per sample using 650 P.S.I. rupture disks. DNA delivered per shot averages at 0.1667 ug. Following bombardment, all embryos are maintained on 560L medium (N6 salts, Eriksson's vitamins, 0.5 mg/l thiamine, 20 g/l sucrose, 1 mg/l 2,4-D, 2.88 g/l proline, 2.0 g/l gelrite, and 8.5 mg/l silver nitrate). After 2–7 days post-bombardment, all the embryos from both treatments are transferred onto N6-based medium containing 50 mg/l cyanamide (Pioneer 560P medium described above, with 50 mg/l cyanamide). Plates are maintained at 28° C. in the dark and are observed for colony recovery with transfers to fresh medium occurring every two to three weeks. Early in the sub-culture regime, GFP+ and GFP− embryos are separated. These two sub-populations are subsequently cultured and analyzed as separate treatments. The PAT ̃GFP expression cassette and the BI-1 expression cassette co-segregate together, and thus the presence of GFP expression is used to separate BI-1+ and BI-1− progeny for analysis.
Comparing PAT ̃GFP+/BI-1+transgenic embryos with wild-type (non-transgenic) embryos from the same ear we expect will show that the overall recovery of cyanimide-resistant transformants is much higher for the BI-1 transgenic embryos. For ears from PAT ̃GFP+/BI-1fs transgenic plants (containing the frame-shift control) we expect there to be no significant improvement in transformation frequencies over segregating wild-type embryos.
Example 10
Using the GmBI1 Gene to Improve Soybean Transformation
Delivery of the GmBI (the soybean BI) gene can be accomplished through numerous well-established methods for plant cells, including for example particle bombardment, sonication, PEG treatment or electroporation of protoplasts, electroporation of intact tissue, silica-fiber methods, microinjection or Agrobacterium-mediated transformation. Using one of the above methods, DNA is introduced into soybean cells capable of growth on suitable soybean culture medium. The BI gene (GmBI1) is cloned into a cassette with a constitutive promoter (for example, the SCP-1 promoter which confers constitutive expression in soybean, see PHI Patent application WO 99/43838) and a 3′ sequence such as the nos 3′ region. Particle bombardment is used to introduce the SCP1::GmBI1:: nos-containing plasmid along with a SCP1::HYG::nos-containing plasmid (which, when expressed produces a protein which confers hygromycin resistance) into soybean cells capable of growth on suitable soybean culture medium. Such competent cells can be from soybean suspension culture, cell culture on solid medium, freshly isolated cotyledonary nodes or meristem cells. Suspension-cultured somatic embryos of Jack, a Glycine max (I.) Merrill cultivar, are used as the target for co-delivery of a BI1 and a HYG-expressing plasmid. For target tissues receiving the BI1 expression cassette, transformation frequency is improved. Media for induction of cell cultures with high somatic embryogenic capacity, for establishing suspensions, and for maintenance and regeneration of somatic embryos are described (Bailey M A, Boerma H R, Parrott W A, 1993 Genotype effects on proliferative embryogenesis and plant regeneration of soybean, In Vitro Cell Dev Biol 29P: 102–108). Likewise, methods for particle-mediated transformation of soybean are well established in the literature, see for example Stewart N C, Adang M J, All J N, Boerma H R, Cardineau G, Tucker D, Parrott W A, 1996, Genetic transformation, recovery and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis crylAc gene, Plant Physiol 112:121–129.
Maintenance of Soybean Embryogenic Suspension Cultures
Soybean embryogenic suspension cultures are maintained in 35 ml liquid media SB196 or SB172 in 250 ml Erlenmeyer flasks on a rotary shaker, 150 rpm, 26C with cool white fluorescent lights on 16:8 hr day/night photoperiod at light intensity of 30–35 uE/m2 s.
Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of fresh liquid media. Alternatively, cultures are initiated and maintained in 6-well Costar plates.
SB 172 media is prepared as follows: (per liter), 1 bottle Murashige and Skoog Medium (Duchefa # M 0240), 1 ml B5 vitamins 1000×stock, 1 ml 2,4-D stock (Gibco 11215–019), 60 g sucrose, 2 g MES, 0.667 g L-Asparagine anhydrous (GibcoBRL 11013–026), pH 5.7
SB 196 media is prepared as follows: (per liter) 10 ml MS FeEDTA,10 ml MS Sulfate, 10 ml FN-Lite Halides, 10 ml FN-Lite P, B, Mo, 1 ml B5 vitamins 1000×stock, 1 ml 2,4-D, (Gibco 11215–019), 2.83 g KNO3, 0.463 g (NH4)2SO4, 2 g MES, 1 g Asparagine Anhydrous, Powder (Gibco 11013–026), 10 g Sucrose, pH 5.8.
2,4-D stock concentration 10 mg/ml is prepared as follows: 2,4-D is solubilized in 0.1 N NaOH, filter-sterilized, and stored at −20° C.
B5 vitamins 1000×stock is prepared as follows: (per 100 ml)—store aliquots at −20° C., 10 g myo-inositol, 100 mg nicotinic acid, 100 mg pyridoxine HCl, 1 g thiamine.
Particle Bombardment
Soybean embryogenic suspension cultures are transformed with various plasmids by the method of particle gun bombardment (Klein et al. 1987; Nature 327:70.
To prepare tissue for bombardment, approximately two flasks of suspension culture tissue that has had approximately 1 to 2 weeks to recover since its most recent subculture is placed in a sterile 60×20 mm petri dish containing 1 sterile filter paper in the bottom to help absorb moisture. Tissue (i.e suspension clusters approximately 3–5 mm in size) is spread evenly across each petri plate. Residual liquid is removed from the tissue with a pipette, or allowed to evaporate to remove excess moisture prior to bombardment. Per experiment, 4–6 plates of tissue are bombarded. Each plate is made from two flasks.
To prepare gold particles for bombardment, 30 mg gold is washed in ethanol, centrifuged and resuspended in 0.5 ml of sterile water. For each plasmid combination (treatments) to be used for bombardment, a separate micro-centrifuge tube is prepared, starting with 50 μl of the gold particles prepared above. Into each tube, the following are also added; 5 μl of plasmid DNA (at 1 μg/μl), 50μl CaCl2, and 20μl 0.1 M spermidine. This mixture is agitated on a vortex shaker for 3 minutes, and then centrifuged using a microcentrifuge set at 14,000 RPM for 10 seconds. The supernatant is decanted and the gold particles with attached, precipitated DNA are washed twice with 400 μl aliquots of ethanol (with a brief centrifugation as above between each washing). The final volume of 100% ethanol per each tube is adjusted to 40 ul, and this particle/DNA suspension is kept on ice until being used for bombardment.
Immediately before applying the particle/DNA suspension, the tube is briefly dipped into a sonicator bath to disperse the particles, and then 5 μg of DNA prep is pipetted onto each macro-carrier and allowed to dry. The macro-carrier is then placed into the DuPont® Biolistics PDS1000/HE gun. Using the DuPont® Biolistic PDS1000/HE instrument for particle-mediated DNA delivery into soybean suspension clusters, the following settings are used. The membrane rupture pressure is 1100 psi. The chamber is evacuated to a vacuum of 27–28 inches of mercury. The tissue is placed approximately 3.5 inches from the retaining/stopping screen (3rd shelf from the bottom). Each plate is bombarded twice, and the tissue clusters are rearranged using a sterile spatula between shots.
Following bombardment, the tissue is re-suspended in liquid culture medium, each plate being divided between 2 flasks with fresh SB196 or SB172 media and cultured as described above. Four to seven days post-bombardment, the medium is replaced with fresh medium containing 25 mg/L hygromycin (selection media). The selection media is refreshed weekly for 4 weeks and once again at 6 weeks. Weekly replacement after 4 weeks may be necessary if cell density and media turbidity is high.
Four 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 6-well microtiter plates with liquid medium to generate clonally-propagated, transformed embryogenic suspension cultures.
Each embryogenic cluster is placed into one well of a Costar 6-well plate with 5 mls fresh SB196 media with 25 mg/L hygromycin. Cultures are maintained for 2–6 weeks with fresh media changes every 2 weeks. When enough tissue is available, a portion of surviving transformed clones are subcultured to a second 6-well plate as a back-up to protect against contamination.
In treatments where both the HYG and BI1 expression cassettes are transformed into immature embryos, a higher number of growing embryogenic cultures are expected on the selective medium and growth of embryogenic cultures is stimulated (relative to treatments with the HYG gene alone).
Regeneration of Soybean Somatic Embryos
To promote in vitro maturation, transformed embryogenic clusters are removed from liquid SB196 and placed on solid agar media, SB 166, for 2 weeks. Tissue clumps of 2–4 mm size are plated at a tissue density of 10 to 15 clusters per plate. Plates are incubated in diffuse, low light (<10 μE) at 26+/−1° C. After two weeks, clusters are subcultured to SB 103 media for 3–4 weeks.
SB 166 is prepared as follows: (per liter), 1 pkg. MS salts (Gibco/BRL—Cat# 11117–017), 1 ml B5 vitamins 1000×stock, 60 g maltose, 750 mg MgCl2 hexahydrate, 5 g activated charcoal, pH 5.7, 2 g gelrite.
SB 103 media is prepared as follows: (per liter), 1 pkg. MS salts (Gibco/BRL—Cat# 11117–017), 1 ml B5 vitamins 1000× stock, 60 g maltose, 750 mg MgCl2 hexahydrate, pH 5.7, 2 g gelrite.
After 5–6 week maturation, individual embryos are desiccated by placing embryos into a 100×15 petri dish with a 1 cm2 portion of the SB103 media to create a chamber with enough humidity to promote partial desiccation, but not death.
Approximately 25 embryos are desiccated per plate. Plates are sealed with several layers of parafilm and again are placed in a lower light condition. The duration of the desiccation step is best determined empirically, and depends on size and quantity of embryos placed per plate. For example, small embryos or few embryos/plate require a shorter drying period, while large embryos or many embryos/plate require a longer drying period. It is best to check on the embryos after about 3 days, but proper desiccation will most likely take 5 to 7 days. Embryos will decrease in size during this process.
Desiccated embryos are planted in SB 71–1 or MSO medium where they are left to germinate under the same culture conditions described for the suspension cultures. When the plantlets have two fully-expanded trifoliolate leaves, germinated and rooted embryos are transferred to sterile soil and watered with a half-strength MS-salt solution. Plants are grown to maturity for seed collection and analysis. Embryogenic cultures from the BI1 treatment are expected to regenerate easily. Healthy, fertile transgenic plants are grown in the greenhouse. Seed-set on BI1 transgenic plants is expected to be similar to control plants, and transgenic progeny are recovered.
SB 71–1 is prepared as follows: 1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL—Cat# 21153–036), 10 g sucrose, 750 mg MgCl2 hexahydrate, pH 5.7, 2 g gelrite.
MSO media is prepared as follows: 1 pkg Murashige and Skoog salts (Gibco 11117–066), 1 ml B5 vitamins 1000× stock, 30 g sucrose, pH 5.8, 2 g Gelrite.
It is expected that higher BI1-transgene expression levels improve transformation. For this bombardment experiment (to be performed in a similar manner to that described above), soybean suspension cultures are used as the target tissue for bombardment. The treatments include a no-BI control (SCP1:: HYG::nos), or the SCP1:: HYG::nos marker plus one of two BI-expressing plasmids (SCP1:: BI1::nos or nos::BI::nos). For this experiment high levels of BI expression (SCP1) are compared to low levels (nos) of expression. When the SCP1 promoter drives the expression of BI, the transformation frequencies are expected increase. Placing the BI1 gene behind the nos promoter is expected to produce a transformation frequency intermediate to the SCP1:: BI1 frequency and that of the control treatment. It is expected that higher expression levels result in correspondingly higher recovery of transformants.
Example 11
Engineering Resistance Against Maize Ear Mold Disease
A number of fungal pathogens, such as Fusarium moniliforme, cause ear mold in maize. Fusarium moniliforme growth in maize appears dependent on the presence of dead, senescing or decaying tissues. Among the dead or decaying tissue that are often so exploited by Fusarium are silks, husks, pericarp or the cob. Tissues that are still alive are somehow recalcitrant to Fusarium ingress. Exactly why this is so remains unknown at this time. However, this dependency of Fusarium upon dead tissue availability, could be turned into an advantage for improving maize resistance to it, if death or senescence of tissues can be delayed.
Consequently, to the extent that the inventive maize BI genes claimed herein can control cell death in maize, they can be used to enhance ear mold resistance. To that end, any one of these BI genes, or other related inventive genes of this patent, could be used in crop plants, especially maize, to retard cell death and senescence. Ideally this would be done by driving the expression of the BI genes with tissue-preferred promoters in a transgenic plant—especially promoters specific to the tissue most accounting for ear mold ingress, namely silks, husks, pericarp or the cob. Work had already occurred and is continuing to identify and characterize such promoters. Additionally, other promoters, such as those for senescence-induced genes, such as a cysteine protease, could be used to bump up the expression of the BI when senescence begins. Upon elevation of the BI expression by this promoter, the senescence process and cell death will be retarded. Accordingly, ear mold resistance will be gained.
Example 12
Developing General Increased Tolerance to Diseases in Maize and other Crop Plants
Plant disease symptoms usually result from cell death in infected tissues. The cause of this death can be two fold: a) direct death-inflicting activity by the pathogens, such as by their production of antibiotic (here anti-plant) compounds or proteins; and b) cell death resulting from activation of the plants' own cell death mechanisms—something that is intrinsic to many of the plants' responses to pathogens. Some pathogens (such as Sclerotinia, Helminthosporium sp., Botrytis etc.) first actively kill the plant tissue and then colonize it, and they can also take advantage of any cell death resulting from activation of the plants' own defense system.
For these reasons, genes which would suppress the initiation and/or the spread of cell death following infection, would help alleviate the damage done by pathogenic organisms, regardless of these two sources. The BI presented here are one such class of genes. A promoter that could drive the BI gene expression in the tissue that is infected by the pathogens would be of choice. In some situations a constitutive promoters such as ubiquitin could be used to drive BI expression constitutively, and therefore keep the plant on guard against cell death and cover many possible tissues that may become infected. In another strategy, promoters for genes known to be expressed in particular tissues can be chosen where the pathogen of interest is known to target particular tissues. Alternatively, or in addition to the tissue-specificity of the promoter, a defense-inducible or death-inducible promoter would be chosen. The expression of a number of maize genes is induced following pathogen attack and many of these are induced in association with necrosis that results from such infection. The promoters for these genes could be used to drive the expression of the BI genes. The expected outcome of this is that upon pathogen attack, the death process is turned on. This then will result in the elevated (ectopic) expression of the BI genes. The expression of these BI genes will then result in a retardation of the initiation of death, or if initiated already, it will retard the spread of the death. In this way, the Bax-inhibitor (BI) gene expression will result in a lessening of the disease symptoms, for after all disease symptoms are in many cases largely a function of the extent of death. It is this death that causes, as in the case of leaf blights, a loss of photosynthetic capacity of the plant, and a corresponding decrease in yield.
Example 13
Use of the Maize BI-DR to Induce Male Sterility
Expression vectors useful for promoting apoptosis through the modulation of BI expression are those that down-regulate BI levels or activity (abbreviated hereafter as BI-DR constructs). A BI-DR construct is an expression cassette in which the transcribed RNA results in decreased levels of BI protein in the cell. Examples would include expressing antisense, expressing an inverted-repeat sequence (which will form a hairpin) constructed from a portion of the BI sequence, expressing the BI sequence fused to another such “hairpin” forming sequence, or expressing BI in a manner that will favor co-suppression of endogenous BI.
Maize expression cassettes directing BI-DR expression to tapetum can be constructed. An expression cassette directing expression of the BI-DR polynucleotide to the tapetum during microsporogenesis is made using the maize MS45 promoter (U.S. Pat. No. 6,037,523 issued Mar. 14, 2000). Embryos are co-bombarded with the selectable marker PAT fused to the GFP gene along with the tapetum-specific BI-DR expression cassette described above. Both inbred and Hi-II transformants are obtained and regenerated as described in examples 6 and 7 above.
It is anticipated that in the regenerated plants (and progeny obtained through the ear of such transgenic plants), induction of apoptosis in the tapetum during the microsporogenesis process will result in male sterility. Upon microscopic examination of the developing anthers it will be apparent that apoptosis has occurred by the death of the tapetal cell layer and abortion of microspores.
Example 14
Cell Death Assays
Gene-directed cell death can be detected using numerous methods. These include methods used to recognize characteristic patterns of morphological, biochemical and molecular changes. These are typically grouped into two broad categories. The first group is morphological changes such as nuclear fragmentation, condensation of cytoplasm, appearance of apoptotic bodies and ultimately phagocytosis of remains. The second group is functional or biochemical changes such as an increase in free calcium, cell dehydration, loss of mitochondrial membrane potential, proteolysis, phosphatidylserine externalization, DNA denaturation and fragmentation, intranucleosomal cleavage and protein cross-linkage.
Commonly used methods for detecting DNA fragmentation include ELISA methods, the TUNEL assay, gel electrophoresis methods, and flow cytometry. Methods are also well known for detecting increased apoptosis-associated proteins such as capsases and PARP. Cell integrity can be assessed using vital staining methods such as propidium iodide or Evan's Blue exclusion. One of the first sub-cellular changes that appear to trigger the cell death cascade is altered ionic balance and membrane potential across the mitochondrial membrane. Mitochondrial membrane potential can be measured using three cationic fluorescent probes, DiOC(6), JC-1, and TMRM. Commercial kits for the above assays currently exist for all of the above (see online catalog & information from CLONTECHTM or from ROCHETM)“both of which are incorporated by reference.
Example 15
Transformation and Regeneration of Maize Callus
Immature maize embryos from greenhouse grown High type II donor plants were bombarded with a plasmid containing a polynucleotide of the invention, ZmBI1–3. The ZmBI1–3 polynucleotide was operably linked to the constitutive promoter nos and the potato proteinase inhibitor 3′ sequence (pinII). The plasmid containing nos::ZmBI1–3:: pinII was co-transformed along with a plasmid containing a fusion between a maize-optimized PAT selectable marker gene that confers resistance to the herbicide Bialaphos and a maize-optimized Green Fluorescent Protein (GFP). Transformation was performed as follows.
The ears were surface sterilized in 50% Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos were excised and placed embryo axis side down (scutellum side up), 25 embryos per plate. These were cultured on 560 L medium (see Table 3 for medium formulations) 4 days prior to bombardment in the dark. Medium 560 L was an N6-based medium containing Eriksson's vitamins, thiamine, sucrose, 2,4-D, and silver nitrate. The day of bombardment, the embryos were transferred to 560 Y medium for 4 hours and were arranged within the 2.5-cm target zone. Medium 560Y was a high osmoticum medium (560L with high sucrose concentration).
The plasmid DNA containing the nos:BI1–3:: pinII expression cassette plus plasmid DNA containing the PAT ̃GFP fusion marker were precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl2 precipitation procedure as follows: 100 μl prepared tungsten particles (0.6 mg) in water, 20 μl (2 μg) DNA in Tris-EDTA buffer (1 μg total), 100 μl 2.5 M CaC12, 40 μl 0.1 M spermidine. As a control treatment, the Ubi::PAT ̃GFP::pinII plasmid was co-precipitated with a second plasmid containing a Ubi::firefly luciferase::pinII cassette (keeping the ratios of PAT ̃GFP plasmid/total DNA consistent in both the control and BI-gene treatments).
Each reagent was added sequentially to the tungsten particle suspension. The final mixture was sonicated briefly. After the precipitation period, the tubes were centrifuged briefly, liquid removed, washed with 500 μl 100% ethanol, and centrifuged again for 30 seconds. Again the liquid was removed, and 60 μl 100% ethanol was added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles were briefly sonicated and 5 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
The sample plates were bombarded at a distance of 8 cm from the stopping screen to the tissue, using a DupontTM biolistics helium particle gun. All samples received a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
Four to twelve hours post bombardment, the embryos were moved to 560P (a low osmoticum callus initiation medium similar to 560L but with lower silver nitrate), for 3–7 days, then transferred to 560R selection medium, an N6 based medium similar to 560P containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. Multicellular GFP cell clusters became visible after two weeks and their numbers were periodically recorded. After approximately 10 weeks of selection, selection-resistant GFP positive callus clones were sampled and PCR analysis was performed to confirm the presence of the nos::ZmBaxl1–3:: pinII cassette. As seen in Table 3 below, including the nos::BI1–3:: pinII plasmid resulted in a significant increase in transformation frequency (Student's T-test; p<0.05)
Table 3. Transformation results for immature embryos harvested from four ears and split equally between the control treatment (A) and the treatment including the BI gene (B). Transformation frequencies were calculated based on the number of bialaphos-resistant, GFP+ calli recovered per total number of embryos bombarded for a given treatment within an ear.


















Total
Tx Freq



Ear
Tx Events
Embryos
(%)
















A. Control












1
1
75
1.3



2
5
75
6.7



3
9
100
9.0



4
13
100
13.0





Mean
7.5





SD
4.9







B. nos::Bl::pinII












1
12
75
16.0



2
8
75
10.7



3
17
100
17.0



4
13
100
13.0





Mean
14.2





SD
2.9










Positive lines were transferred to 288J medium, an MS-based medium with lower sucrose and hormone levels, to initiate plant regeneration. Following somatic embryo maturation (2–4 weeks), well-developed somatic embryos were transferred to medium for germination and transferred to the lighted culture room. Approximately 7–10 days later, developing plantlets were transferred to medium in tubes for 7–10 days until plantlets were well established. Plants were 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 ClassicTM 600 pots (1.6 gallon)(#14–9674–9; Hummert International, Earth City, Mo.) and grown to maturity. Mature, normal-phenotype plants containing the nos::ZmBI1–3:: pinII cassette integrated into the genome were recovered.

## Claims

1. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide of SEQ ID NO: 34;
(b) the polynucleotide of SEQ ID NO:33; and
(c) the polynucleotide which is complementary to a polynucleotide of (a) or
(b);
wherein the polynucleotide of (a), (b) or (c), modulates Bax inhibitor activity.

2. An expression cassette comprising at least one polynucleotide of claim 1.

3. A transgenic plant cell comprising at least one expression cassette of claim 2.