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The present invention relates to a root knot nematode responsive promoter isolated from Arabidopsis thaliana elongation specific endo-1, 4-beta-glucanase (cel1) gene, and a method of producing transgenic nematode and insect resistant plants and cells using cel1 promoter. Kim, et al., A 20 nucleotide upstream element is essential for the nopaline synthase (nos) promoter activity, Plant Molecular Biology, vol. Shani, Ziv, et al., Cloning and characterization of elongation specific endo-1,4-?-glucanase (cel1) from Arabidopsis thaliana, Plant Molecular Biology, vol.
Zane, et al., Cloning and characterization of elongation specific endo-1,5-B-glucanase (cel1) from Arabidopsis thaliana, Plant Molecular Biology, vol. Capital City Steppers is a social networking and dance organization dedicated to promoting the art of Chicago-Style Steppin in North Carolina and abroad. The DNA construct according to claim 1, wherein said heterologous nucleic acid encodes an insecticidal protein.
The DNA construct according to claim 1, wherein said heterologous DNA encodes a Bacillus thuringiensis crystal protein toxic to insects.
A method of producing a transformed plant, comprising regenerating a plant from the plant cell according to claim 6. A method of producing a transformed plant, comprising infecting a plant cell with the Agrobacterium tumefaciens cell according to claim 8 to produce a transformed plant cell, and regenerating a plant from said transformed plant cell.
A method of making a transformed plant, comprising propelling the microparticle according to claim 10 into a plant cell to produce a transformed plant cell, and regenerating a plant from said transformed plant cell. A method of producing a transformed plant, comprising regenerating a plant from the plant cell protoplast according to claim 12.
A transformed plant according to claim 14, wherein said plant is a tobacco (Nicotiana tabacum) plant.
A plant seed from the plant according to claim 14, wherein the seed comprises the DNA construct. Our organization will be committed to giving back to the community by conducting workshops, fundraisers for local charities and volunteering our time through community service.
Our members are expected to be respectful of the different styles of individuals, instructors and steppin' groups.
We strive to create an excitement for Stepping and encourage our members to contribute to its future. A promoter may consist of a number of different regulatory elements which affect a structural gene operationally associated with the promoter in different ways. Capital City Steppers is the newest Chicago Style Steppin' organization in the Triangle area. For example, a regulatory gene may enhance or repress expression of an associated structural gene, subject that gene to developmental regulation, or contribute to the tissue-specific regulation of that gene. We are committed to participating in and perfecting the dance art form of Chicago Style Steppin. Modifications to promoters can make possible optional patterns of gene expression, using recombinant DNA procedures. We encourage our members to keep the integrity of the dance form while enhancing relationships through fellowship. If these are qualities that help to define your character, then you should be a member of Capital City Steppers! 5,459,252 to Conkling and Yamamoto describes a root specific promoter designated RB7, which was identified in tobacco. 5,750,386 to Conkling, Opperman and Taylor describes pathogen resistant transgenic plants in which a nematode-responsive element is operatively associated with a nucleotide of interest (in this case, a gene encoding a product toxic to plant cells). Plant parasitic nematodes cause approximately 100 billion dollars annually in crop loses worldwide. These embodiments are not intended to provide a detailed catalog of all manner in which the instant invention may be carried out, as numerous variations will be apparent to persons skilled in the arts to which the invention pertains.



Accordingly, the following is set forth for illustrative purposes, and is not intended to be limiting of the invention.1. Root-knot nematodes are sedentary endoparasites with an extremely intimate and complex relationship to the host plant. Upon location of a host root, the J2 penetrates the root intercellularly in the region just posterior to the root cap and migrates to the developing vascular cylinder.
The nematode then orients itself parallel to the cylinder and injects glandular secretions into the plant cells surrounding its head, resulting in the initiation of nematode feeding cells. These 5–7 cells undergo rapid nuclear divisions, increase tremendously in size, and become filled with pores and cell wall invaginations.
The feeding site cells, or “giant cells”, function as super transfer cells to provide nourishment to the developing nematode.
During this time, the nematode loses the ability to move and swells from the normal eel shaped J2 to a large, pear shaped adult female. As the nematode feeds on the giant cells, parthenogenic reproduction results in the disposition of 300–400 eggs. This entire process occurs over the span of 20–30 days, and root-knot nematodes may complete as many as 7 generations during a cropping season. Thus, in addition to delivering at the feeding site a product that is toxic to the nematode, it will be seen that, by causing the plant itself to kill or disable the cells upon which the pathogen must feed, the pathogen will be much less successful at infecting the plant.2. Promoters.As used herein, a nematode responsive (or “nematode inducible”) promoter refers to a promoter that (a) does not normally drive transcription in a plant cell except when that cell resides in tissue infected by a root knot nematode, or (b) normally drives transcription in a plant cell, and which drives increased levels of transcription when that cell resides in tissue infected by a root knot nematode. The promoter may be a naturally occurring promoter, may comprise a nematode responsive element isolated from a naturally occurring promoter, or may be a synthetic promoter.A preferred promoter for use in the present invention is the elongation specific endo-1,4-?-glucanase (cel1) promoter of Arabidopsis thaliana, has been described by Z. Shoseyov, Plant Molecular Biology 34, 837–842 (1997), and has been assigned EMBL, GenBank and DDBJ Nucleotide Sequence Database accession number X98543. This promoter is referred to herein as an Arabidopsis cel1 promoter, and is set forth herein as SEQ ID NO: 1. The Arabidopsis cel1 promoter and other promoters that may be used to carry out the present invention is also disclosed in U.S. Heterologous DNAs and Expression Cassettes.DNA constructs, or “expression cassettes,” of the present invention include, 5?-3? in the direction of transcription, a nematode responsive promoter of the present invention, a heterologous DNA segment operatively associated with the promoter, and, optionally, transcriptional and translational termination regions such as a termination signal and a polyadenylation region.
The 3? termination region may be derived from the same gene as the transcriptional initiation region or from a different gene.The term “operatively associated,” as used herein, refers to DNA sequences contained within a single DNA molecule which are associated so that the function of one is affected by the other. The promoter is said to be “upstream” from the gene, which is in turn said to be “downstream” from the promoter.Heterologous DNAs used to carry out the present invention may encode any product that is disruptive of nematode attack when that DNA is transcribed (and, where applicable, translated) in a plant cell, including but not limited to proteins, peptides, and non-protein products such as antisense RNAs, ribozymes, other nucleic acids that suppress expression by sense strand suppression or triplex formation, etc.
Proteins to which such antibodies can be directed include, but are not limited to, RNA polymerase, respiratory enzymes, cytochrome oxidase, Krebs cycle enzymes, protein kinases, aminocyclopropane-1-carboxylic acid synthase, and enzymes involved in the shikimic acid pathway such as enolpyruvyl shikimic acid-5-phosphate synthase.One preferred heterologous DNA is a structural gene encoding mature Bacillus amyloliquefaciens RNase (or Barnase).
Hartley, Gene 40, 231–39 (1985).Note that the toxic product may either kill the plant cell in which it is expressed or simply disable the cell so that it is less capable of supporting the pathogen.
Thus the heterologous DNA may encode a Bacillus thuringiensis crystal protein toxic to insects.
For example, the heterologous DNA may encode a peptide, antibody or the like that disrupts feeding by interacting with the ingestion or digestion of food such as one of the antibodies described for soybean cyst nematode including that against the dorsal pharyngeal gland (Atkinson et al, 1988 Annals of Applied Biology 112, 459–469), modified as necessary for specificity to the root knot nematode, using the procedures for transgenic expression of antibodies in plants described by Hiatt, A. Various secretory leaders, membrane integration sequences, and translocation sequences for directing the peptide expression product to a particular site are described in the literature. In this manner, at each stage after each manipulation, the resulting construct may be cloned, sequenced, and the correctness of the manipulation determined. In addition to the replication system, there may be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts.
That is, one marker may be employed for selection in a prokaryotic host while another marker may be employed for selection in a eukaryotic host, particularly the plant host. Exemplary genes which may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrilase, and the gentamicin resistance gene.


For plant host selection, non-limiting examples of suitable markers are beta-glucuronidase (GUS) (providing indigo production), luciferase (providing visible light production), NPTII (providing kanamycin resistance or G418 resistance), HPT (providing hygromycin resistance), and the mutated aroA gene (providing glyphosate resistance).An advantage of the present invention is that two or more promoters can be “daisychained” to a single structural gene. Where each promoter is responsive to a different pathogen, the plant is then provided with resistance to a plurality of promoters. For example, a second promoter may be positioned upstream from the structural gene and operatively associated therewith so that the structural gene is associated with a plurality of promoters, with each of the promoters activated by a different plant pathogen. Other promoters that may be used in conjunction with the instant promoter are described in U.S.
5,750,386 to Conkling et al.The various fragments comprising the various constructs, expression cassettes, markers, and the like may be introduced consecutively by restriction enzyme cleavage of an appropriate replication system and insertion of the particular construct or fragment into the available site. Vectors which may be used to transform plant tissue with DNA constructs of the present invention include both Agrobacterium vectors and ballistic vectors, as well as vectors suitable for DNA-mediated transformation. Agrobacterium tumefaciens cells containing a DNA construct of the present invention, wherein the DNA construct comprises a Ti plasmid, are useful in methods of making transformed plants. Plant cells are infected with an Agrobacterium tumefaciens to produce a transformed plant cell, and then a plant is regenerated from the transformed plant cell.Numerous Agrobacterium vector systems useful in carrying out the present invention are known. 4,459,355 discloses a method for transforming susceptible plants, including dicots, with an Agrobacterium strain containing the Ti plasmid. The microparticle is propelled into a plant cell to produce a transformed plant cell and a plant is regenerated from the transformed plant cell.
Any suitable ballistic cell transformation methodology and apparatus can be used in practicing the present invention. When using ballistic transformation procedures, the expression cassette may be incorporated into a plasmid capable of replicating in the cell to be transformed. Examples of microparticles suitable for use in such systems include 1 to 5 ?m gold spheres. Examples of plant species suitable for transformation with the DNA constructs of the present invention include both monocots and dicots, and include but are not limited to tobacco, soybean, potato, cotton, sugarbeet, sunflower, carrot, celery, flax, cabbage and other cruciferous plants, pepper, tomato, citrus trees, bean, strawberry, lettuce, maize, alfalfa, oat, wheat, rice, barley, sorghum and canola.
Thus an illustrative category of plants which may be transformed with the DNA constructs of the present invention are the dicots, and a more particular category of plants which may be transformed using the DNA constructs of the present invention are members of the family Solanacae.Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector of the present invention.
The term “organogenesis,” as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers; the term “embryogenesis,” as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Thus, the present invention provides a method of controlling nematodes, comprising: (a) providing a root knot nematode-responsive promoter as described above, (b) preparing a construct as described above by combining said promoter with a further region which codes for a product disruptive of nematode attack, and (c) transforming plants with the construct to obtain plants which are root knot nematode resistant.
The plants employed may be as described above, and transformation may be carried out as described above. Once a first generation (Fo generation) of transformed plants are obtained, plant seed that contains the aforesaid construct, and that germinates into a root knot nematode resistant transgenic plant, may be be produced from the Fo plants by conventional breeding procedures. The cel1-GUS transgenic tobacco roots did not exhibit GUS staining other than in the elongation zone of the root tips. However, it was also found that plants infected with the root knot nematode (Meloidogyne incognita), which also parasitizes tobacco, specifically upregulated cel1-GUS in specialized feeding sites called giant cells around which galls form on the roots.
A time course study was carried out from the time of infection through the root knot nematode life cycle, and it was found that expression correlates with the onset of giant cell formation and is maintained throughout the nematode life cycle (up to about 2 months). Roots were sectioned and it was found that GUS staining was specifically localized in the giant cells.The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.



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