Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8237—Externally regulated expression systems
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

• the present invention generally relates to the field of plant molecular biology, and more specifically to the regulation of gene expression in plants in response to stress, e.g., to pathogen exposure.
• Plants are capable of activating a large array of defense mechanisms in response to pathogen attack. A crucial factor determining the success of these mechanisms is the speed of their activation. Consequently, there is considerable interest in understanding how plants recognize pathogen attack and control expression of defense mechanisms.
• Some potential pathogens trigger a very rapid resistance response called gene-for-gene resistance. This occurs when the pathogen carries an avirulence (avr) gene that triggers specific recognition by a corresponding host resistance (R) gene.
• avr avirulence
• R host resistance
• R gene specificity is generally quite narrow, in most cases only pathogens carrying a particular avr gene are recognized. Recognition is thought to be mediated by ligand-receptor binding.
• R genes have been studied extensively in recent years. For a review of R genes, see Ellis et al. (1998); Jones et al. (1997); and Ronald (1998).
• One of the defense mechanisms triggered by gene-for-gene resistance is programmed cell death at the infection site. This is called the hypersensitive response, or HR.
• SA systemic acquired resistance
• PR pathogenesis related
• SAR systemic signal that triggers SAR is a subject of debate (Shulaev et al., 1995; Nernooji et al., 1994). SA clearly moves from the site of the HR to other parts of the plant, but if this is the signal, it must be effective at extremely low concentration (Willits et al., 1998). SAR is quite similar to some reactions that occur locally in response to attack by virulent (those that cause disease) or avirulent (those that trigger gene- for-gene resistance) pathogens. In general, activation of defense gene expression occurs more slowly in response to virulent pathogens than in response to avirulent pathogens. Some pathogens trigger expression of defense genes through a different signaling pathway that requires components of the jasmonic acid (JA) and ethylene signaling pathways (Creelman et al., 1997).
• JA jasmonic acid
• ethylene signaling pathways Creelman et al., 1997.
• NDR1 and EDSI are required for gene-for-gene mediated resistance to avirulent strains of the bacterial pathogen Pseudomonas syringae and the oomycete pathogen Peronospora parasitica.
• ndrl mutants are susceptible to one set of avirulent pathogens, whereas edsl mutants are susceptible to a non-overlapping set (Aarts et el., 1998).
• the five cloned R genes that require EDSI all belong to the subset of the nucleotide binding site-leucine rich repeat ( ⁇ BS-LRR) class of R genes that contain sequences similar to the cytoplasmic domains of Drosophila Toll and mammalian interleukin 1 transmembrane receptors (TIR- ⁇ BS-LRR).
• the two genes that require NDR1 belong to the leucine-zipper (LZ- ⁇ BS-LRR) subclass of ⁇ BS-LRR genes.
• LZ- ⁇ BS-LRR leucine-zipper
• RPP8 that does not require EDSI or NDR1
• the correlation between R gene structure and requirement for EDSI or NDR1 is not perfect. Nevertheless, these results show that R genes differ in their requirements for downstream factors and that these differences are correlated with R gene structural type.
• NDR1 encodes a protein with two predicted transmembrane domains
• RPM1 which requires NDR1 to mediate resistance, is membrane-associated, despite the fact that its primary sequence does not include any likely membrane-integral stretches (Boyes et al., 1998). It is possible that part of the function of NDR1 is to hold R proteins close to the membrane.
• EDSI encodes a protein with blocks of homology to triacyl glycerol lipases (Falk et al., 1999). The significance of this homology is not known, but it is believed to speculate that ⁇ DS1 is involved in synthesis or degradation of a signal molecule. ⁇ DS1 expression is inducible by SA and pathogen infection, suggesting that ⁇ DS1 maybe involved in signal amplification (Falk et al., 1999). It has been extremely difficult to isolate mutations in genes other than the
• R genes that are required for gene-for-gene resistance A selection procedure was devised (McNellis et al., 1998) on the basis of precisely controlled inducible expression of the avr gene avrRpt2 in plants carrying the corresponding resistance gene RPS2. Expression of avrRpt2 in this background is lethal, as it triggers a systemic HR. It is now possible to select for mutants with subtle defects in gene-for-gene signaling by requiring growth on a concentration of inducer slightly higher than the lethal dose.
• RPP7 and RPP8 were not significantly suppressed by mutations in either EDSI or NDR1, and that RPP7 resistance was also not compromised by mutations in EIN2, JAR1 or COI1, which affect ethyl ene or jasmonic acid signaling, or in coil/nprl or coil/NahG backgrounds.
• SA levels increase locally in response to pathogen attack, and systemically in response to the SAR-inducing signal.
• SA is necessary and sufficient for activation of PR gene expression and enhanced disease resistance.
• Physiological analyses and characterization of certain lesion-mimic mutants strongly suggest that there is a positive autoregulatory loop affecting SA concentrations (Shirasu et al., 1997; Hunt et al., 1997; Weymann et al., 1995).
• Several mutants with defects in SA signaling have been characterized.
• nprl in which expression of PR genes in response to SA is blocked
• cprl, cpr5, and cpr ⁇ which constitutively express PR genes
• the nprl suppressor ssil pad4, which has a defect in SA accumulation
• eds5 which has a defect in PR1 expression.
• NPR1 a gene called NIM1. Mutations in nprl abolish SAR, and cause enhanced susceptibility to infection by various pathogens (Cao et al., 1994; Delaney et al., 1995; Glazebrook et al., 1996; Shah et al., 1997). NPR1 appears to be a positive regulator of Pi? gene expression that acts downstream from SA.
• NPR1 encodes a novel protein that contains ankyrin repeats (which are often involved in protein-protein interactions (Cao et al., 1997; Ryals et al., 1997), and that is localized to the nucleus in the presence of SA (Dong et al., 1998). Consequently, it is unlikely that NPR1 acts as a transcription factor to directly control PR gene expression, but its nuclear localization suggests that it may interact with such transcription factors.
• PAD4 appears to act upstream from SA. mpad4 plants infected with a virulent P. syringae strain, SA levels, synthesis of the antimicrobial compound camalexin, and PR1 expression are all reduced (Zhou et al., 1998). SA is necessary, but not sufficient, for activation of camalexin synthesis (Zhou et al., 1998; Zhao et al., 1996). The camalexin defect in pad4 plants is reversible by exogenous SA (Zhou et al., 1998). Mutations in pad4 do not affect SA levels, camalexin synthesis, or PR1 when plants are infected with an avirulent P. syringae strain (Zhou et al., 1998). Taken together, these results suggest that PAD4 is required for signal amplification to activate the SA pathway in response to pathogens that do not elicit a strong defense response (Zhou et al., 1998). JA-dependent signaling
• JA signaling affects diverse processes including fruit ripening, pollen development, root growth, and response to wounding (Creelman et al., 1997). The jarl and coil mutants fail to respond to JA (Feys et al., 1994; Staswick et al., 1992). COI1 has been cloned, and found to encode protein containing leucine-rich repeats and a degenerate F-box motif (Xie et al., 1998). These features are characteristic of proteins that function in complexes that ubiquitinate protein targeted for degradation.
• ISR Induced systemic resistance
• ISR rhizosphere-associated bacteria promote disease resistance. This phenomenon, called ISR, has been studied using Pseudomonas fluorescens strain WCS417r to colonize Arabidopsis roots (Pieterse et al., 1996). Colonized plants are more resistant to infection by the fungal pathogen Fusarium oxysporum f sp raphani and P. syringae (Pieterse et al., 1996). ISR occurs in nahG plants, indicating that it is not a SA-dependent phenomenon (Pieterse et al., 1996). Rather, ISR appears to be JA- and ethylene- dependent. The observation that ethylene can induce ISR in jarl mutants led to the hypothesis that ISR requires a JA signal followed by an ethylene signal
• NPRl is interacting with different 'adapter' molecules to mediate the different signals.
• the ankyrin repeats found in NPRl could function in protein- protein interactions between NPRl and adapter proteins. Identification of proteins that interact with NPRl , and characterization of plants with loss-of- function mutations affecting those proteins, would be very helpful for understanding how NPRl acts in each pathway. It would also be worthwhile to determine if the ssil or cpr ⁇ mutations suppress the ISR defect of nprl mutants. Relevance to disease resistance Characterization of the effects of various mutations on resistance to different pathogens has revealed that there is considerable variation in the extent to which pathogens are affected by defense mechanisms.
• SAR is known to confer resistance to a wide array of pathogens, including bacteria, fungi, oomycetes, and viruses.
• JA signaling is important for limiting the growth of certain fungal pathogens.
• the SA pathway mutants nprl and pad4 show enhanced susceptibility to P. syringae and P. parasitica (Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997; Zhou et al., 1998; Glazebrook et al., 1997).
• Overexpression of rate-limiting defense response regulators may cause the signaling network to respond faster or more strongly to pathogen attack, thereby improving resistance.
• overexpression of NPRl caused increased resistance to P. syringae and P. parasitica in a dosage dependent manner (Cao et al., 1998).
• NRfiJ-overexpression had no obvious deleterious effects on plant growth, in contrast to mutations that lead to constitutive overexpression of defense responses, which generally cause dwarfism.
• the invention provides an isolated nucleic acid molecule (polynucleotide), e.g., DNA, comprising a plant or fungal nucleotide sequence, the expression of which is altered in response to stress, e.g., pathogen infection.
• polynucleotide e.g., DNA
• the invention provides a nucleic acid molecule comprising a plant or fungal nucleotide sequence comprising at least a portion of a key effector gene(s) responsible for host resistance to particular pathogens.
• this sequence may be overexpressed individually, in the sense or antisense orientation, or in combination with other sequences, to confer improved disease resistance or tolerance to a plant relative to a plant that does not comprise and/or express the sequence.
• the overexpression may be constitutive, or it may be preferable to express the effector gene(s) from an inducible promoter including a promoter which is responsive to external stimuli, such as chemical application, or to pathogen infection, e.g., so as to avoid possible deleterious effects on plant growth.
• the promoter employed to express the nucleotide sequence of the invention may be one that mediates rapid and transient transcription after pathogen infection.
• the invention also provides an isolated nucleic acid molecule comprising a plant nucleotide sequence which alters, e.g., increases or decreases, the transcription of plant genes, e.g., open reading frames, in response to stress, e.g., pathogen infection.
• genes showing strong Peronospora-mdnced expression changes clusters of genes were identified that were specifically upregulated by RPP7 or RPP8, or both.
• the expression of 184 genes (SEQ ID NOs:l-211 and 792) related to 217 probe sets was upregulated by either RPP7 or RPP8, or both, while in another analysis the expression of genes related to 194 probe sets (SEQ ID NOs:212-399 and 793) was upregulated by either RPP7 or RPP8, or both.
• promoters of genes that were rapidly and transiently transcribed after P. parasitica infection and were RPP7/S-dependent were significantly enriched with both novel sequence motifs and potential binding sites of known transcription factors.
• promoters were significantly enriched with two novel sequence motifs (referred to as "motif 1 " and “motif 2”), one of which was similar to binding sites for Myb transcription factors, sequence motifs related to WRKY binding sites, and two other novel sequence motifs ("motif 3" and "motif 4").
• motif 1 and “motif 2”
• motif 3 and "motif 4"
• comparisons between expression signatures of wild type plants as well as the RPP7 loss of function mutants, and edml, edm2 and edm3 mutants allow for predictions regarding the RPP7 pathway hierarchy.
• genes SEQ ID NOs:75, 214, 228, 301, 339, 400-684, 792-795
• SA protein biosynthesis inhibitor cycloheximide
• a set of immediate early SA responsive genes (SEQ ID NOs:150, 159, 117, 126, 208, 428, 426, 436, 430, 434, 478, 641, 609, 615, and 526) was found to consist exclusively of regulatory genes. Such genes are likely controlled by transcription factors acting closely downstream of SA.
• One highly conserved motif in the promoters of these immediate early genes was similar to binding sites of WRKY transcription factors (SEQ ID NOs:757-765). Other conserved promoter motifs appeared to be novel and may facilitate the cloning of their cognate transcription factors.
• a cluster of S A/CHX super-induced genes was found to show elevated expression ground states in the nprl mutant (SEQ ID NOs:214, 621, 71, 439, 78, 80, 264, 265, 613, 583, 594, 616, 355, 503, 168, 447 and 551) suggesting an NPRl -dependent de- repression mechanism in the control of some SA responsive immediate early genes.
• the invention provides an isolated nucleic acid molecule comprising a plant or fungal nucleotide sequence encoding a polypeptide which is substantially similar to an Arabidopsis polypeptide encoded by a gene comprising an open reading frame, the expression of which is altered in response to a pathogen that induces a response mediated by R genes, including pathogens such as bacteria, fungi, oomycetes, viruses, nematodes and insects, e.g., aphids (see Hammond-Kosack and Jones (1997), which is specifically incorporated by reference herein).
• pathogens such as bacteria, fungi, oomycetes, viruses, nematodes and insects, e.g., aphids (see Hammond-Kosack and Jones (1997), which is specifically incorporated by reference herein).
• the plant or fungal nucleotide sequence encodes a polypeptide that is substantially similar to an Arabidopsis polypeptide encoded by a gene comprising an open reading frame, the expression of which is altered in response to infection by an oomycete such as Peronospora, and is RPP4-, RPP7-, and/or ⁇ PP5-dependent.
• sequences can be identified by employing an array of nucleic acid samples, e.g., a plurality of oligonucleotides, each plurality corresponding to a different plant gene, on a solid substrate, e.g., a DNA chip, and probes corresponding to nucleic acid from pathogen-infected (e.g., wild-type) plant cells and to nucleic acid from uninfected plant cells or plant cells having a mutation which alters the response to pathogen infection.
• pathogen-infected e.g., wild-type
• the nucleotide sequence is from plant DNA, either a dicot or a monocot, which encodes a polypeptide that is substantially similar to an Arabidopsis polypeptide encoded by an open reading frame comprising any one of SEQ ID NOs: 1 -684 and 792-795, or the complement thereof. More preferably, the nucleotide sequence is from plant DNA that is substantially similar to an Arabidopsis nucleic acid segment having any one of SEQ ID NOs: 1-684 and 792-795, or the complement thereof.
• substantially similar when used herein with respect to a polypeptide means a polypeptide corresponding to a reference polypeptide, wherein the polypeptide has substantially the same structure and function as the reference polypeptide, e.g., where only changes in amino acid sequence are those which do not affect the polypeptide function.
• the percentage of identity between the substantially similar and the reference polypeptide or amino acid sequence is at least 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to at least 99%, where the reference polypeptide is an Arabidopsis polypeptide encoded by an open reading frame comprising any one of SEQ ID NOs: 1-684 and 792-795, or the complement thereof.
• the percent of identity between the substantially similar and the reference polypeptide may be less than 65% as long as the two polypeptides have the same or similar function, e.g., catalyze the same or similar reaction.
• Another indication that two polypeptides are substantially similar to each other is that an agent, e.g., an antibody, which specifically binds to one of the polypeptides, specifically binds to the other.
• the term "substantially similar”, when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence is from a gene that encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by a gene comprising the reference nucleotide sequence.
• the term “substantially similar” is specifically intended to include nucleotide sequences wherein the sequence has been modified to optimize expression in particular cells.
• the percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence is at least 65%, 66%, 61%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to at least 99%, wherein the reference sequence is preferably any one of SEQ ID NOs: 1-684 and 792-795, or the complement thereof.
• Sequence comparisons may be carried out using a Smith- Waterman sequence alignment algorithm (see e.g., Waterman (1995) or http://www hto.usc.edu/software/seqaln/index.html).
• the locals program, version 1.16 is preferably used with following parameters: match: 1 , mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2.
• nucleotide sequence that is "substantially similar" to a reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO , 1 mM
• SDS sodium dodecyl sulfate
• the isolated nucleic acid molecules of the invention also include the orthologs of ' the Arabidopsis sequences disclosed herein, i.e., the corresponding nucleic acid molecules in organisms other than Arabidopsis, including, but not limited to, plants other than Arabidopsis, preferably cereal plants, e.g., corn, wheat or rice, as well as rye, turfgrass, sorghum, millet, sugarcane, soybean, barley, alfalfa, sunflower, canola, cotton, peanut, tobacco or sugarbeet, and in fungi.
• An ortholog is a gene from a different species that encodes a product having the same function as the product encoded by a gene from a reference organism.
• the encoded ortholog products likely have at least 70% amino acid sequence identity to each other.
• the invention includes an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 70% identity to a polypeptide encoded by one or more of the Arabidopsis sequences, although it is also envisioned that orthologous genes to those disclosed herein may encode a polypeptide with less than 70%, e.g., less than 65% amino acid sequence identity, but which polypeptide has the same or similar function.
• GenBank GenBank or one found at http://bioserver.myongjiac.kr/rjce.html (for rice) may be employed to identify sequences related to the Arabidopsis sequences, e.g., orthologs in cereal crops such as rice.
• recombinant DNA techniques such as hybridization or PCR may be employed to identify sequences related to the Arabidopsis sequences.
• the invention preferably includes an isolated nucleic acid molecule comprising a plant or fungal nucleotide sequence that encodes a polypeptide that has at least 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and at least 99%, amino acid sequence identity to an Arabidopsis polypeptide encoded by an open reading frame comprising any one of SEQ ID NOs: 1-684 and 792-795, or a fragment (portion) thereof which encodes a polypeptide having substantially the same activity as a polypeptide encoded by an open reading frame comprising a corresponding sequence listed in SEQ ID NOs: 1-684 and 792-795.
• the isolated nucleic acid molecule is not SEQ ID NOs: 1-684 and 792-795.
• the invention also provides anti-sense nucleic acid molecules corresponding to the open reading frames or genes identified as responsive to pathogen infection.
• expression cassettes e.g., recombinant vectors, and host cells, comprising the nucleic acid molecule of the invention in either sense or antisense orientation.
• nucleic acid molecule comprising a plant or fungal nucleotide sequence comprising a nucleic acid sequence having at least 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and at least 99%, nucleic acid sequence similarity to an Arabidopsis open reading frame comprising any one of SEQ ID NOs: 1 -684 and 792-795, the complement thereof, or a fragment (portion) thereof which encodes a polypeptide having substantially the same activity as a polypeptide encoded by an open reading frame comprising a corresponding sequence listed in SEQ ID NOs: 1-684 and 792-795.
• nucleic acid molecules of the invention are useful to provide resistance to pathogens and as a diagnostic for the presence or absence of the pathogen by correlating the expression level or pattern of expression of one or more of the nucleic acid molecules of the invention or one or more of the polypeptides encoded thereby.
• the invention includes isolated nucleic acid molecules that have increased expression in response to pathogen infection, the invention further provides compositions and methods for enhancing resistance to pathogen infection.
• compositions of the invention include plant or fungal nucleotide sequences and the amino acid sequences for the polypeptides or partial-length polypeptides encoded thereby which are useful to provide tolerance or resistance to a plant to a pathogen, preferably by preventing or inhibiting pathogen infection.
• the resistance or tolerance may be accomplished by decreasing or eliminating expression of a plant gene necessary for pathogen infection and/or replication or by overexpressing a gene product that inhibits pathogen infection and/or replication.
• Methods of the invention involve stably transforming a plant with one or more of at least a portion of these nucleotide sequences which confer tolerance or resistance operably linked to a promoter capable of driving expression of that nucleotide sequence in a plant cell.
• portion or fragment as it relates to a nucleic acid molecule, sequence or segment of the invention, when it is linked to other sequences for expression, is meant a sequence having at least 80 nucleotides, more preferably at least 150 nucleotides, and still more preferably at least 400 nucleotides. If not employed for expressing, a "portion” or “fragment” means at least 9, preferably 12, more preferably 15, even more preferably at least 20, consecutive nucleotides, e.g., probes and primers (oligonucleotides), corresponding to the nucleotide sequence of the nucleic acid molecules of the invention.
• resistant is meant a plant which exhibits substantially no phenotypic changes as a consequence of infection with the pathogen.
• tolerant is meant a plant which, although it may exhibit some phenotypic changes as a consequence of infection, does not have a substantially decreased reproductive capacity or substantially altered metabolism.
• some of the isolated nucleic acid molecules of the invention are useful in a method of combating a pathogen in an agricultural crop.
• the method comprises introducing to a plant, plant cell, or plant tissue an expression cassette comprising a nucleic acid molecule of the invention so as to yield a transformed differentiated plant, transformed cell or transformed tissue. Transformed cells or tissue can be regenerated to provide a transformed differentiated plant.
• the transformed differentiated plant preferably expresses the nucleic acid molecule in an amount that confers resistance to the transformed plant to pathogen infection relative to a corresponding nontransformed plant.
• the present invention also provides a transformed plant prepared by the method, progeny and seed thereof.
• a transformed (transgenic) plant of the invention includes plants, the genome of which is augmented by a nucleic acid molecule of the invention, or in which the corresponding gene has been disrupted, e.g., to result in a loss, a decrease or an alteration, in the function of the product encoded by the gene, which plant may also have increased yields under conditions of pathogen infection and/or produce a better-quality product than the corresponding wild- type plant.
• the nucleic acid molecules of the invention are thus useful for targeted gene disruption, as well as markers and probes.
• the invention also includes recombinant nucleic acid molecules which have been modified so as to comprise codons other than those present in the unmodified sequence.
• the recombinant nucleic acid molecules of the invention include those in which the modified codons specify amino acids that are the same as those specified by the codons in the unmodified sequence, as well as those that specify different amino acids, i.e., they encode a variant polypeptide having one or more amino acid substitutions relative to the polypeptide encoded by the unmodified sequence.
• the invention further includes a nucleotide sequence which is complementary to one (hereinafter "test" sequence) which hybridizes under stringent conditions with the nucleic acid molecules of the invention as well as RNA which is encoded by the nucleic acid molecule.
• either the test or nucleic acid molecule of invention is preferably supported, e.g., on a membrane or DNA chip.
• a denatured test or nucleic acid molecule of the invention is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of, e.g., between 55 and 70°C, in double strength citrate buffered saline (SC) containing 0.1 % SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SC concentration.
• SC citrate buffered saline
• the present invention also provides a method to identify a gene, the expression of which is altered in response to an external stimulus, e.g., pathogen infection.
• the method comprises contacting a plurality of samples comprising portions or fragments of isolated nucleic acid molecules with a probe which corresponds to a population of a nucleic acid sequences, the expression of which is altered in response to an external stimulus, so as to form a binary complex. Each sample corresponds to a different gene. Then complex formation is detected or determined.
• the method may be employed with nucleic acid samples and probes from any organism, e.g., any prokaryotic or eukaryotic organism.
• the nucleic acid sample and probes are from a plant, such as a dicot or monocot.
• the method comprises contacting a solid substrate comprising a plurality of samples comprising portions or fragments of isolated plant nucleic acid with a probe comprising plant nucleic acid corresponding to at least a portion of RNA from a pathogen infected plant so as to form a complex.
• Preferred pathogens are those which induce an i?-gene dependent resistance response.
• Each individual sample comprises one or more nucleic acid sequences corresponding to a plant gene, e.g., a pool of oligonucleotides corresponding to the same gene or a portion of that gene.
• the plurality of samples is provided on a DNA chip.
• a second plurality of samples on a solid substrate, i.e., a DNA chip, each comprising a plurality of samples comprising portions or fragments of isolated plant nucleic acid is contacted with a probe comprising plant nucleic acid corresponding to at least a portion of RNA from an uninfected or mutant plant or plant cells so as to form a complex. Then complex formation with nucleic acid from infected cells and from uninfected or mutant cells is compared.
• the invention also provides a method for identifying a plant cell infected with a pathogen, e.g., one that induces a RPP4-, RPP7- and/or PPPS-dependent resistance response.
• the method comprises contacting nucleic acid obtained from a plant cell suspected of being infected with the pathogen with at least one, preferably at least two, oligonucleotides under conditions effective to amplify at least a portion of a nucleotide sequence in the isolated plant nucleic acid which is substantially similar to at least one of SEQ ID NOs: 1-684 and 792-795, so as to yield an amplified product. Then the presence of the amplified product is detected or determined.
• the presence of the amplified product e.g., in an amount that is different than the amount of the corresponding amplified product from an uninfected or mutant plant, corresponding to one or more of SEQ ID NOs: 1-684 and 792-795 or an ortholog thereof, is indicative of pathogen infection.
• the invention provides an additional method for identifying a plant cell infected with a pathogen.
• the method comprises hybridizing a probe selected from SEQ ID NOs: 1 -684 and 792-795 to nucleic acid obtained from a plant cell suspected of being infected with a pathogen.
• the amount of the probe hybridized to nucleic acid obtained from a cell suspected of being infected with a pathogen is compared to hybridization of the probe to nucleic acid isolated from an uninfected or mutant cell.
• a change in the amount of the hybridized probe in nucleic acid isolated from a cell suspected of being infected by a pathogen relative to the amount of hybridized probe in nucleic acid isolated from an uninfected or mutant cell is indicative of infection.
• an isolated nucleic acid molecule comprising a nucleotide sequence that directs transcription, e.g., a promoter, of a linked nucleic acid segment in a host cell, such as a plant cell, wherein transcription is altered, e.g., increased, in response to a pathogen infection.
• the pathogen is P-dependent, and more preferably, one that triggers a response that is dependent on RPP4, RPP7 and/or RPP8, such as an oomycete (for example, Peronospora).
• the nucleotide sequence is from plant genomic DNA which has at least 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, nucleotide sequence identity to a sequence of a promoter from an Arabidopsis gene comprising any one of SEQ ID NOs: 1-684 and 792-795, e.g., SEQ ID NOs:774-788, or comprising motifs such as one of SEQ ID NOs:685- 773.
• the invention also includes orthologs of Arabidopsis promoters.
• the nucleotide sequence includes the promoter region from a gene corresponding to SEQ ID NOs: 1-770 and 792-795, which region preferably includes at least one copy of at least one of the following, e.g., a nucleic acid sequence comprising one of SEQ ID NOs:685-697 ("motif 1"), SEQ ID NOs:698-709 (“motif 2"), GGT/CCCA ("motif 3"), GNCCAAA (“motif 4"), or SEQ ID NOs:710-713, 714-756, or 757-773.
• the promoter sequence is preferably about 25 to 2000, e.g., 50 to 500 or 100 to 1400, nucleotides in length.
• the isolated nucleic acid molecule comprises a plant nucleotide sequence which is the promoter region for any one of SEQ ID NOs: 1-684 and 792-795, or is structurally related to the promoter for SEQ ID NOs: 1-684 and 792-795, i.e., is an orthologous promoter, and is linked to a plant structural gene or open reading frame.
• the present invention further provides an expression cassette or a recombinant vector containing the nucleic acid molecule, and the vector may be a plasmid.
• cassettes or vectors when present in a plant, plant cell or plant tissue result in transcription of the linked nucleic acid segment in the plant, plant tissue or plant cell. Transcription of the linked segment is altered in response to pathogen infection, including Peronospora infection. For promoters with motif 1 , transcription of linked segments may be altered in response to agents or other stimuli that induce Myb-like transcription factors.
• the promoters of the invention may be employed to express a nucleic acid segment that is operably linked to the promoter, for example, an open reading frame or a portion thereof, an anti-sense construct or a transgene.
• the open reading frame may be obtained from an insect resistance gene, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance gene, a nematode disease resistance gene, a herbicide resistance gene, a gene affecting grain composition or quality, a nutrient utilization gene, a mycotoxin reduction gene, a male sterility gene, a selectable marker gene, a screenable marker gene, a negative selectable marker, a positive selectable marker, a gene affecting plant agronomic characteristics, i.e., yield, standability and the like, or an environment or stress resistance gene, i.e., one or more genes that confer herbicide resistance or tolerance, insect resistance or tolerance, disease resistance or tolerance (viral, bacterial, fungal, oomycete, or
• the promoters of the invention include a consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743, of the promoter sequences from genes comprising any one of SEQ ID NOs:685-788, 714-756, and 757-773, or the promoter orthologs thereof, which include the minimal promoter region.
• said consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743, has at least 75%, preferably 80%, more preferably 90% and most preferably 95% sequence identity with a corresponding consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743, of any one of SEQ ID NOs:685-788, 714-756, and 757- 773, or the promoter orthologs thereof, which include the minimal promoter region.
• the expression cassettes or vectors of the invention may optionally include other regulatory sequences, e.g., transcription terminator sequences, introns and/or enhancers, and may be contained in a host cell.
• the expression cassette or vector may augment the genome of a transformed plant or may be maintained extrachromosomally.
• the expression cassette or vector may further have a Ti plasmid and be contained in an Agrobacterium tumefaciens cell; it may be carried on a microparticle, wherein the microparticle is suitable for ballistic transformation of a plant cell; or it may be contained in a plant cell protoplast.
• the expression cassette can be contained in a plant, plant cell or plant tissue from a dicot or a monocot.
• the plant may be a cereal plant.
• the present invention further provides a method of augmenting a plant genome by contacting plant cells with an expression cassette or vector of the invention, i.e., one having a nucleotide sequence that directs transcription of a linked nucleic acid segment in a plant cell, wherein transcription of the linked segment is altered in response to a pathogen such as an oomycete, e.g., Peronospora, infection, and wherein the nucleic sequence is from plant DNA that has at least 65%, and more preferably at least 70%, identity to the sequence of a promoter from an Arabidopsis gene comprising any one of SEQ ID NOs: 1- 684 and 792-795, so as to yield transformed plant cells; and regenerating the transformed plant cells to provide a differentiated transformed plant, wherein the differentiated transformed plant expresses the linked fragment in the cells of the plant in response to infection.
• the present invention also provides a plant prepared by the method, progeny and seed thereof.
• a transformed (transgenic) plant of the invention includes plants, the genome of which is augmented by a nucleic acid molecule of the invention, or in which the corresponding gene has been disrupted, e.g., to result in a loss, a decrease or an alteration, in the function of the product encoded by the gene, which plant may also have increased yields and/or produce a better-quality product than the corresponding wild-type plant.
• the nucleic acid molecules of the invention are thus useful for targeted gene disruption, as well as markers and probes.
• the invention also provides a method of plant breeding, e.g., to prepare a crossed fertile transgenic plant.
• the method comprises crossing a fertile transgenic plant comprising a particular nucleic acid molecule of the invention with itself or with a second plant, e.g., one lacking the particular nucleic acid molecule, to prepare the seed of a crossed fertile transgenic plant comprising the particular nucleic acid molecule.
• the seed is then planted to obtain a crossed fertile transgenic plant.
• the plant may be a monocot or a dicot.
• the plant is a cereal plant.
• the crossed fertile transgenic plant may have the particular nucleic acid molecule inherited through a female parent or through a male parent.
• the second plant may be an inbred plant.
• the crossed fertile transgenic may be a hybrid. Also included within the present invention are seeds of any of these crossed fertile transgenic plants.
• the present invention also provides a method to identify a nucleotide sequence that directs transcription of nucleic acid in the genome of a plant cell in response to pathogen exposure, by contacting a probe comprising plant nucleic acid, e.g., cRNA, isolated from tissues of a plant contacted with the pathogen with a plurality of isolated nucleic acid samples on a plurality of solid substrates, wherein each sample is a plurality of oligonucleotides corresponding to at least a portion of a plant gene, so as to form a complex between at least a portion of the probe and a nucleic acid sample(s) having sequences that are structurally related to the sequences in the probe. Then complex formation is determined or detected to determine which samples represent genes comprising promoters that are responsive to infection with the pathogen.
• the probe and/or samples may be nucleic acid from a dicot or from a monocot.
• a method to shuffle the nucleic acid molecules of the invention involves fragmentation of a (parent) nucleic acid molecule corresponding to a nucleic acid sequence listed in SEQ ID NOs: 1-684 and 792-795, the ortholog thereof, or the corresponding gene thereof, followed by religation.
• This method allows for the production of polypeptides having altered activity relative to the polypeptide encoded by the parent nucleic acid molecule.
• the invention provides cells and transgenic plants containing nucleotide sequences produced through shuffling that encode polypeptides having altered activity relative to the polypeptide encoded by the parent nucleic acid molecule.
• a computer readable medium containing the nucleic acid sequences of the invention as well as methods of use for the computer readable medium are provided.
• This medium allows a nucleic acid molecule corresponding to a nucleic acid sequence listed in SEQ ID NOs: 1-399, 685-756 and 774-793 or 71, 78, 80, 117, 126, 150, 159, 168, 208, 214, 264-265, 355, 400-657, 659-684, 792- 795 to be used as a reference sequence to search against databases.
• This medium also allows for computer-based manipulation of a nucleic acid sequence corresponding to a nucleic acid sequence listed in SEQ ID NOs: 1-399, 685-756 and 774-793 or 71, 78, 80, 117, 126, 150, 159, 168, 208, 214, 264-265, 355, 400- 657, 659-684, 792-795, and the corresponding gene or polypeptide encoded by the nucleic acid sequence.
• Figure 1 depicts ⁇ PP-dependent defense pathways.
• Figure 2 depicts nucleotide sequences including the promoter region and motifs therein for genes, the expression of which is altered in response to pathogen infection (SEQ ID NOs: 774-788).
• Figure 3 is a schematic of the overlap in genes that are induced early after P. parasitica infection and in a _&PP#-specific manner, genes that are induced late after infection and in a i?PPS-specific manner, and genes induced early and late after infection in a _KPP7-specif ⁇ c manner.
• Figure 4 shows a schematic of i?PP -pathway, ⁇ PP7-pathway and RPP8- upregulated transcription factor genes.
• nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
• nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
• degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985; Rossolini et al., 1994).
• a "nucleic acid fragment" is a fraction of a given nucleic acid molecule.
• nucleotide sequence refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
• nucleic acid refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
• nucleic acid “nucleic acid molecule”, “nucleic acid fragment” or “nucleic acid sequence or segment” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.
• an "isolated” or “purified” DNA molecule or an “isolated” or “purified” polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
• An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
• an isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
• an isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
• nucleic acid molecule or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
• an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
• the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
• a protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein.
• culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of- interest chemicals.
• Fragments and variants of the disclosed nucleotide sequences and proteins or partial-length proteins encoded thereby are also encompassed by the present invention.
• fragment or portion is meant a full length or less than full length of the nucleotide sequence encoding, or the amino acid sequence of, a polypeptide or protein.
• fragments or portions of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity.
• fragments or portions of a nucleotide sequence may range from at least about 9 nucleotides, about 12 nucleotides, about 20 nucleotides, about 50 nucleotides, about 100 nucleotides or more.
• genes include coding sequences and/or the regulatory sequences required for their expression.
• gene refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences.
• Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins.
• Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
• Naturally occurring is used to describe an object that can be found in nature as distinct from being artificially produced by man.
• a protein or nucleotide sequence present in an organism which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
• a "marker gene” encodes a selectable or screenable trait.
• Selectable marker is a gene whose expression in a cell gives the cell a selective advantage.
• the selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non-transformed cells.
• the selective advantage possessed by the transformed cells, compared to non-transformed cells may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source.
• Selectable marker gene also refers to a gene or a combination of genes whose expression in a cell gives the cell both a negative and/or a positive selective advantage.
• chimeric refers to any gene or DNA that contains 1) DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.
• transgene refers to a gene that has been introduced into the genome by transformation and is stably maintained.
• Transgenes may include, for example, DNA that is either heterologous or homologous to the DNA of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes.
• endogenous gene refers to a native gene in its natural location in the genome of an organism.
• a “foreign” gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.
• protein protein
• variants are intended substantially similar sequences.
• variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.
• Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
• variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions.
• nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 91%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.
• DNA shuffling is a method to introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly.
• the DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule.
• the shuffled DNA preferably encodes a variant polypeptide modified with respect to the polypeptide encoded by the template DNA, and may have an altered biological activity with respect to the polypeptide encoded by the template DNA.
• the nucleic acid molecules of the invention can be optimized for enhanced expression in plants of interest. See, for example, EPA035472; WO91/16432; Perlak et al., 1991; and Murray et al., 1989. In this manner, the genes or gene fragments can be synthesized utilizing plant-preferred codons. See, for example, Campbell and Gowri, 1990 for a discussion of host-preferred codon usage.
• the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.
• Variant nucleotide sequences and proteins also encompass sequences and protein derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA shuffling are known in the art.
• Consatively modified variations of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein.
• nucleic acid variations are "silent variations" which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted.
• each codon in a nucleic acid except ATG, which is ordinarily the only codon for methionine
• each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
• Recombinant DNA molecule is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook et al., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (1989).
• heterologous DNA sequence each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
• a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
• the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
• the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
• a "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
• Wild-type refers to the normal gene, or organism found in nature without any known mutation.
• Gene refers to the complete genetic material of an organism.
• Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
• shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast or fungal cells).
• Coding vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.
• “Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
• the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction.
• the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
• the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
• the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.
• the promoter can also be specific to a particular tissue or organ or stage of development.
• Such expression cassettes will comprise the transcriptional initiation , region of the invention linked to a nucleotide sequence of interest.
• Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.
• the expression cassette may additionally contain selectable marker genes.
• the transcriptional cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
• the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A.
• tumefaciens such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al., 1991; Proudfoot, 1991; Sanfacon et al., 1991; Mogen et al., 1990; Munroe et al, 1990; Ballas et al., 1989; Joshi et al., 1987.
• An oligonucleotide corresponding to a nucleic acid molecule of the invention may be about 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21 or 24, or any number between 9 and 30).
• primers are upwards of 14 nucleotides in length.
• primers of 16-24 nucleotides in length may be preferred.
• probing can be done with entire restriction fragments of the gene disclosed herein which maybe 100's or even 1000's of nucleotides in length.
• Coding sequence refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions.
• An "intron” is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.
• open reading frame and “ORF” refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
• initiation codon and “termination codon” refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
• RNA transcript refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence.
• primary transcript When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.
• Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
• cDNA refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA.
• Regulatory sequences each refer to nucleotide sequences located upstream (5* non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. As is noted above, the term “suitable regulatory sequences” is not limited to promoters. However, some suitable regulatory sequences useful in the present invention will include, but are not limited to constitutive plant promoters, plant tissue-specific promoters, plant development specific promoters, inducible plant promoters and viral promoters.
• 5' non-coding sequence refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency (Turner et al., 1995).
• 3' non-coding sequence refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
• the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
• the use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., 1989.
• the term “translation leader sequence” refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon.
• the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
• mature protein refers to a post-translationally processed polypeptide without its signal peptide.
• Precursor protein refers to the primary product of translation of an mRNA.
• Signal peptide refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway.
• signal sequence refers to a nucleotide sequence that encodes the signal peptide.
• Intracellular localization sequence refers to a nucleotide sequence that encodes an intracellular targeting signal.
• An “intracellular targeting signal” is an amino acid sequence that is translated in conjunction with a protein and directs it to a particular sub-cellular compartment.
• Endoplasmic reticulum (ER) stop transit signal refers to a carboxy-terminal extension of a polypeptide, which is translated in conjunction with the polypeptide and causes a protein that enters the secretory pathway to be retained in the ER.
• ER stop transit sequence refers to a nucleotide sequence that encodes the ER targeting signal.
• Other intracellular targeting sequences encode targeting signals active in seeds and/or leaves and vacuolar targeting signals.
• Promoter refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
• Promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.
• Promoter also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
• an "enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
• the "initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e. further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.
• promoter elements particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters.”
• minimal or core promoters In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.
• a “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.
• Constant expression refers to expression using a constitutive or regulated promoter.
• Consditional and regulated expression refer to expression controlled by a regulated promoter.
• Constant promoter refers to a promoter that is able to express the gene that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant.
• Each of the transcription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant parts at a level of >1% of the level reached in the part of the plant in which transcription is most active.
• Regular promoter refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and include both tissue-specific and inducible promoters. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al., 1989. Since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
• Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid- inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome-inducible systems.
• tissue-specific promoter refers to regulated promoters that are not expressed in all plant cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). . These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence.
• “Inducible promoter” refers to those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogen.
• “Operably-linked” refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other.
• a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter).
• Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
• “Expression” refers to the transcription and/or translation of an endogenous gene or a transgene in plants.
• expression may refer to the transcription of the antisense DNA only.
• expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein.
• altered levels refers to the level of expression in transgenic cells or organisms that differs from that of normal or untransformed cells or organisms.
• “Overexpression” refers to the level of expression in transgenic cells or organisms that exceeds levels of expression in normal or untransformed cells or organisms.
• Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of protein from an endogenous gene or a transgene.
• Co-suppression and “transwitch” each refer to the production of sense
• RNA transcripts capable of suppressing the expression of identical or substantially similar transgene or endogenous genes (U.S. Patent No. 5,231,020).
• Gene silencing refers to homology-dependent suppression of viral genes, transgenes, or endogenous nuclear genes. Gene silencing may be transcriptional, when the suppression is due to decreased transcription of the affected genes, or post-transcriptional, when the suppression is due to increased turnover (degradation) of RNA species homologous to the affected genes. (English et al., 1996). Gene silencing includes virus-induced gene silencing (Ruiz et al., 1998). "Silencing suppressor” gene refers to a gene whose expression leads to counteracting gene silencing and enhanced expression of silenced genes. Silencing suppressor genes may be of plant, non-plant, or viral origin.
• Examples include, but are not limited to HC-Pro, Pl-HC-Pro, and 2b proteins. Other examples include one or more genes in TGMV-B genome.
• Transcription stop fragment refers to nucleotide sequences that contain one or more regulatory signals, such as polyadenylation signal sequences, capable of terminating transcription. Examples include the 3' non-regulatory regions of genes encoding nopaline synthase and the small subunit of ribulose bisphosphate carboxylase.
• Translation stop fragment refers to nucleotide sequences that contain one or more regulatory signals, such as one or more termination codons in all three frames, capable of terminating translation.
• cis-acting sequence and "cis-acting element” refer to DNA or RNA sequences whose functions require them to be on the same molecule.
• An example of a cts-acting sequence on the replicon is the viral replication origin.
• tr ⁇ ns-acting sequence and “tr ⁇ ns-acting element” refer to DNA or RNA sequences whose function does not require them to be on the same molecule.
• Chromosomally-integrated refers to the integration of a foreign gene or
• Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus.
• sequence relationships between two or more nucleic acids or polynucleotides are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.
• reference sequence is a defined sequence used as a basis for sequence comparison.
• a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence.
• comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
• a gap penalty is typically introduced and is subtracted from the number of matches.
• Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA).
• Alignments using these programs can be performed using the default parameters.
• the CLUSTAL program is well described by Higgins et al., 1988; Higgins et al., 1989; Corpet et al., 1988; Huang et al., 1992; and Pearson et al., 1994.
• the ALIGN program is based on the algorithm of Myers and Miller, supra.
• the BLAST programs of Altschul et al., 1990, are based on the algorithm of Karlin and Altschul supra.
• HSPs high scoring sequence pairs
• Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
• M forward score for a pair of matching residues
• N penalty score for mismatching residues; always ⁇ 0.
• a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
• the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
• Gapped BLAST in BLAST 2.0
• PSI-BLAST in BLAST 2.0
• the default parameters of the respective programs e.g. BLASTN for nucleotide sequences, BLASTX for proteins
• the BLASTN program for nucleotide sequences uses as defaults a wordlength (W) of 11,.
• sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection.
• percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
• sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
• Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non- conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
• percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
• polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91 %, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.
• nucleotide sequences can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
• Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, more preferably at least 80%, 90%, and most preferably at least 95%.
• Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
• stringent conditions encompass temperatures in the range of about 1 °C to about 20°C, depending upon the desired degree of stringency as otherwise qualified herein.
• Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
• One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
• substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window.
• optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, 1970.
• a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
• sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
• test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
• sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
• hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
• Bod(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
• Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures.
• the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post- hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
• T m can be approximated from the equation of Meinkoth and Wahl, 1984; T m 81.5°C + 16.6 (log M) +0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
• T m is reduced by about 1 °C for each 1 % of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity.
• the T m can be decreased 10°C.
• stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
• severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point (T m );
• moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (T m );
• low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (T m ).
• An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes.
• An example of stringent wash conditions is a 0.2X SSC wash at 65°C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
• a high stringency wash is preceded by a low stringency wash to remove background probe signal.
• An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides is IX SSC at 45°C for 15 minutes.
• An example low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4-6X SSC at 40°C for 15 minutes.
• stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C and at least about 60°C for long robes (e.g., >50 nucleotides).
• Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
• a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical.
• Very stringent conditions are selected to be equal to the T m for a particular probe.
• An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IX SSC at 60 to 65°C.
• Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
• a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecy
• variant polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
• variants may results form, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
• polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions.
• amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, 1985; Kunkel et al., 1987; U. S. Patent No. 4,873,192; Walker and Gaastra, 1983, and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., 1978, herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred.
• the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
• the polypeptides of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired activity.
• the deletions, insertions, and substitutions of the polypeptide sequence encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.
• Production tissue refers to mature, harvestable tissue consisting of non- dividing, terminally-differentiated cells. It excludes young, growing tissue consisting of germline, meristematic, and not-fully-differentiated cells.
• “Germline cells” refer to cells that are destined to be gametes and whose genetic material is heritable.
• plant refers to any plant, particularly to seed plant, and "plant cell” is a structural and physiological unit of the plant, which comprises a cell wall but may also refer to a protoplast.
• the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.
• Plant tissue includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture such as single cells, protoplast, embryos, and callus tissue.
• the plant tissue may be in plants or in organ, tissue or cell culture.
• altered plant trait means any phenotypic or genotypic change in a transgenic plant relative to the wild-type or non-transgenic plant host.
• transformation refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.
• Host cells containing the transformed nucleic acid fragments are referred to as "transgenic” cells, and organisms comprising transgenic cells are referred to as "transgenic organisms”. Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation (De Blaere et al., 1987) and particle bombardment technology (Klein et al., 1987; U.S. Patent No. 4,945,050).
• Transformed refers to a host cell or organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
• the nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook et al., 1989. See also Innis et al., 1995; and Gelfand, 1995; and Innis and Gelfand, 1999.
• PCR telomere set al.
• telomere set al. telomere set al.
• untransformed refers to normal plants that have not been through the transformation process.
• transgenic plant is a plant having one or more plant cells that contain an expression vector.
• Transiently transformed refers to cells in which transgenes and foreign
• DNA have been introduced (for example, by such methods as Agrobacterium- mediated transformation or biolistic bombardment), but not selected for stable maintenance.
• “Stably transformed” refers to cells that have been selected and regenerated on a selection media following transformation.
• Transient expression refers to transgene expression in cells, e.g., after transformation with recombinant virus or by such methods as Agrobacterium- mediated transformation, electroporation, or biolistic bombardment, but not selected for its stable maintenance.
• Genetically stable and “heritable” refer to chromosomally-integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations.
• Primary transformant and “TO generation” refer to transgenic plants that are of the same genetic generation as the tissue which was initially transformed (i.e., not having gone through meiosis and fertilization since transformation).
• Secondary transformants and the “TI, T2, T3, etc. generations” refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.
• “Significant increase” is an increase that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater. “Significantly less” means that the decrease is larger than the margin of error inherent in the measurement technique, preferably a decrease by about 2- fold or greater.
• This invention relates to isolated plant, e.g., Arabidopsis and rice, nucleic acid molecules, sequences and segments (fragments), the expression of which is altered in response to pathogen infection, as well as the endogenous plant promoters for those expressed molecules, sequences or segments.
• the expression of these genes may also be altered in response to non-pathogens, e.g., in response to environmental stimuli.
• the nucleic acid molecules can be used in pathogen control strategies, e.g., by overexpressing nucleic acid molecules which can confer tolerance to a cell, or by altering the expression of host genes which are required for pathogen infection, e.g., by "knocking out" the expression of at least one genomic copy of the gene.
• Plants having genetic disruptions in host genes may be less susceptible to infection, e.g., due to a decrease or absence of a host protein needed for infection, or, alternatively, hypersusceptible to infection. Plants that are hypersusceptible to infection may be useful to prepare transgenic plants as the expression of the gene(s) which was disrupted may be related to gene silencing.
• Preferred sources for the nucleic acid molecules of the invention include, but are 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), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco
• genus Lemna L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana
• genus Spirodela S. intermedia, S. polyrrhiza, S.punctata ⁇
• genus Wo ⁇ ffia Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. bras ⁇ liensis, Wa.
• Lemnaceae Any other genera or species of Lemnaceae, if they exist, are also aspects of the present invention. Lemna gibba, Lemna minor, and Lemna miniscula are preferred, with Lemna minor and Lemna miniscula being most preferred.
• Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen Institut ETH, founded Rubel, Zurich (1986)); vegetables including tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulga ⁇ s), lima beans (Phaseolus limensis), peas (Lathyms spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
• tomatoes Loxicon esculentum
• lettuce e.g., Lactuca sativa
• green beans Phaseeolus vulga ⁇ s
• lima beans Phaseeolus limensis
• peas Lath
• 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 cai ⁇ ophyllus), 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).
• pines such as loblolly pine (Pinus taeda), slash pine (P
• Leguminous plants include beans and peas.
• Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
• Legumes include, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo, Acacia, aneth, artichoke, arugula, blackberry, canola, cilantro, Clementines, escarole, eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange
• Preferred forage and turf grass nucleic acid sources for use in the methods of the invention include alfalfa, orchard grass, tall fescue, perennial ryegrass, creeping bent grass, and redtop.
• the nucleic acid sources are crop plants and in particular cereals (for example, com, alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), and even more preferably com and soybean.
• the present invention is directed to a nucleic acid molecule comprising a nucleotide sequence obtained or obtainable from any plant gene which encodes a polypeptide having at least 70% amino acid sequence identity to a polypeptide encoded by SEQ ID NOs. 1-684 or 789-795, or a promoter for said gene.
• a nucleic acid molecule comprising a nucleotide sequence obtained or obtainable from any plant gene which encodes a polypeptide having at least 70% amino acid sequence identity to a polypeptide encoded by SEQ ID NOs. 1-684 or 789-795, or a promoter for said gene.
• orthologs of those sequences maybe identified or isolated from the genome of any desired organism, preferably from another plant, according to well known techniques based on their sequence similarity to the Arabidopsis coding sequences, e.g., hybridization, PCR or computer generated sequence comparisons.
• genomic and cDNA libraries may be prepared from any cell or tissue of an organism.
• Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g., Sambrook et al., 1989) and amplification by PCR using oligonucleotide primers preferably corresponding to sequence domains conserved among related polypeptide or subsequences of the nucleotide sequences provided herein (see, e.g., Innis et al., 1990).
• oligonucleotide primers preferably corresponding to sequence domains conserved among related polypeptide or subsequences of the nucleotide sequences provided herein (see, e.g., Innis et al., 1990).
• These methods are particularly well suited to the isolation of gene sequences from organisms closely related to the organism from which the probe sequence is derived.
• the application of these methods using the Arabidopsis coding sequences as probes is well suited for the isolation of gene sequences from any source organism, preferably other plant species.
• oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest.
• Methods for designing PCR primers and PCR cloning are generally known in the art as discussed hereinabove.
• hybridization techniques all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.
• the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker.
• probes for hybridization can be made by labeling synthetic oligonucleotides based on the sequence of the invention.
• sequences that hybridize to the sequences disclosed herein will have at least 40% to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or more identity with the disclosed sequences. That is, the sequence similarity of sequences may range, sharing at least about 40% to 50%, about 60% to 70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.
• nucleic acid molecules of the invention can also be identified by, for example, a search of known databases for genes encoding polypeptides having a specified amino acid sequence identity. Methods of alignment of sequences for comparison are well known in the art and are described hereinabove.
• the present invention also encompasses expression cassettes, preferably in the form of a recombinant vectors comprising the nucleic acid sequences of the invention.
• the expression cassette comprises regulatory elements for expression, of the nucleotide sequences in a host cell capable of expressing the nucleotide sequences.
• regulatory elements usually comprise promoter and termination signals and preferably also comprise elements allowing efficient translation of polypeptides encoded by the nucleic acid sequences of the present invention.
• sequences adjacent to the initiating methionine may require modification. For example, they can be modified by the inclusion of sequences known to be effective in plants.
• Vectors comprising the nucleic acid sequences are usually capable of replication in particular host cells, e.g., as extrachromosomal molecules, and are therefore used to amplify the nucleic acid sequences of this invention in the host cells.
• host cells for such vectors are plant cells.
• promoters shown to be functional in plants The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. In many cases, expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleotide sequences in the desired cell.
• promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, stress- responsive, tissue-preferred and tissue-specific promoters.
• Promoter sequences are known to be strong or weak.
• a strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression.
• An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus.
• a bacterial promoter such as the P tac promoter can be induced to varying levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed bacterial cells.
• An isolated promoter sequence that is a strong promoter for heterologous nucleic acid is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.
• Preferred promoters that are expressed constitutively include promoters from genes encoding actin or ubiquitin and the CaMV 35S and 19S promoters.
• the nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated. This enables the nucleic acid sequence or encoded polypeptide to be .synthesized only when the crop plants are treated with the inducing chemicals.
• Preferred technology for chemical induction of gene expression is detailed in the published application EP 0 332 104 (to Ciba-Geigy) and U.S. Patent 5,614,395.
• a preferred promoter for chemical induction is the tobacco PR- la promoter.
• Tissue-specific or tissue-preferential promoters useful in the present invention are also useful.
• promoters which confer seed-specific expression such as those disclosed by Schemthaner et al., 1988; anther (tapetal) specific promoter B6 (Huffman et al.); and pistil-specific promoters such as a modified S13 promoter (Dzelkalns et al., 1993).
• Preferred tissue specific expression patterns include green tissue-specific, root-specific, stem-specific, and flower-specific. Promoters suitable for expression in green tissue include many which regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons.
• a preferred promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, 1989).
• a preferred promoter for root-specific expression is that described by de Framond (1991 ; EP 0452 269 to Ciba-Geigy).
• a preferred stem specific promoter is that described in U.S. Patent No. 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene.
• promoters which direct specific or enhanced expression in certain plant tissues will be known to those of skill in the art in light of the present disclosure. These include, for example, the rbcS promoter, specific for green tissue; the ocs, nos, and mas promoters which have higher activity in roots or wounded leaf tissue; a truncated (-90 to +8) 35S promoter which directs enhanced expression in roots, an tubulin gene that directs expression in roots and promoters derived from zein storage protein genes which direct expression in endosperm.
• ocs octopine synthase
• Preferred plant promoters include, but are not limited to, a promoter such as the CaMV 35S promoter, an enhanced 35S promoter or others such as CaMV 19S, nos, Adhl, sucrose synthase, -tubulin, ubiquitin, actin, cab, PEPCase or those associated with the R gene complex.
• a promoter such as the CaMV 35S promoter, an enhanced 35S promoter or others such as CaMV 19S, nos, Adhl, sucrose synthase, -tubulin, ubiquitin, actin, cab, PEPCase or those associated with the R gene complex.
• promoters include the U2 and U5 snRNA promoters from maize, the promoter from alcohol dehydrogenase, the Z4 promoter from a gene encoding the Z4 22 kD zein protein, the Z10 promoter from a gene encoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27 kD zein protein, the A20 promoter from the gene encoding a 19 kD -zein protein, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene and the actin promoter from rice; seed specific promoters, such as the phaseolin promoter from beans, may also be used.
• Other promoters useful in the practice of the invention are known to those of skill in the art.
• tissue specific promoters which have been described include the lectin (Vodkin, 1983; Lindstrom et al., 1990,) com alcohol dehydrogenase 1 (Vogel et al., 1992; Dennis et al., 1984), com light harvesting complex
• Inducible promoters that have been described include the ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene (Schwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988), the MPI proteinase inhibitor promoter (Cordero et al., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989).
• tissue-specific regulated genes and/or promoters have been reported in plants. These include genes encoding the seed storage proteins (such as napin, craciferin, beta-conglycinin, and phaseolin) zein or oil body proteins (such as oleosin), or genes involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase. and fatty acid desaturases (fad 2-1)), and other genes expressed during embryo development (such as Bce4, see, for example. EP 255378 and Kridl et al., 1991). Particularly useful for seed-specific expression is the pea vicilin promoter (Czako et al., 1992. (See also U.S. Pat. No.
• tissue-specific promoters include those that direct expression in leaf cells following damage to the leaf (for example, from chewing insects), in tubers (for example, patatin gene promoter), and in fiber cells (an example of a developmentally-regulated fiber cell protein is E6 (John et al., 1992). The E6 gene is most active in fiber, although low levels of transcripts are found in leaf, ovule and flower.
• tissue-specificity of some "tissue-specific" promoters may not be absolute and may be tested by one skilled in the art using the diphtheria toxin sequence.
• tissue-specific expression with "leaky” expression by a combination of different tissue-specific promoters (Beals et al., 1997).
• Other tissue-specific promoters can be isolated by one skilled in the art (see U.S. 5,589,379).
• Several inducible promoters (“gene switches") have been reported. Many are described in the review by Gatz (1996 and 1997).
• RNA transcript that interferes with translation of the mRNA of the native DNA sequence.
• Other elements include those that can be regulated by endogenous or exogenous agents, e.g., by DNA binding proteins such as zinc finger proteins, including naturally occurring zinc finger proteins or chimeric zinc finger proteins (see, e.g., U.S. Patent No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311) or myb-like transcription factors.
• DNA binding proteins such as zinc finger proteins, including naturally occurring zinc finger proteins or chimeric zinc finger proteins (see, e.g., U.S. Patent No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311) or myb-like transcription
• a chimeric zinc finger protein may include amino acid sequences which bind to a specific DNA sequence (the zinc finger) and amino acid sequences that activate (e.g., GAL 4 sequences) or repress the transcription of the sequences linked to the specific DNA sequence.
• the 3 ' nontranslated regulatory DNA sequence preferably includes from about 50 to about 1,000, more preferably about 100 to about 1 ,000, nucleotide base pairs and contains plant transcriptional and translational termination sequences.
• Appropriate transcriptional terminators and those which are known to function in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 * terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato, although other 3' elements known to those of skill in the art can also be employed.
• the 5' regulatory region of the expression cassette may also include other enhancing sequences. Numerous sequences have been found to enhance gene expression in transgenic plants. These include sequences which have been shown to enhance expression such as intron sequences (e.g., from Adhl, bronzel or the sucrose synthase intron) and viral leader sequences (e.g., from TMV,
• MCMV and AMV a number of non-translated leader sequences derived from viruses are known to enhance expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g., Gallie et al., 1987; Skuzeski et al., 1990).
• TMV Tobacco Mosaic Virus
• MCMV Maize Chlorotic Mottle Virus
• AMV Alfalfa Mosaic Virus
• Picornavirus leaders for example, EMCV leader (Encephalomyocarditis 5 noncoding region) (Elroy-Stein et al., 1989); Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize Dwarf Mosaic Virus); Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak et al.,
• nucleotide sequences of the present invention may be target expression of the nucleotide sequences of the present invention to different cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle, e.g., the nucleus, may be preferred. Subcellular localization of transgene encoded enzymes is undertaken using techniques well known in the art. Typically, the DNA encoding the target peptide from a known organelle-targeted gene product is manipulated and fused upstream of the nucleotide sequence. Many such target sequences are known for the chloroplast and their functioning in heterologous constructions has been shown.
• nucleotide sequences of the present invention is also targeted to the endoplasmic reticulum or to the vacuoles of the host cells. Techniques to achieve this are well-known in the art.
• Marker Genes In order to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene as, or in addition to, the preselected nucleic acid sequence or segment. "Marker genes" are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker.
• Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can 'select' for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i.e., by 'screening' (e.g., the R-locus trait).
• a selective agent e.g., a herbicide, antibiotic, or the like
• 'screening' e.g., the R-locus trait
• selectable or screenable marker genes are also genes which encode a "secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes which can be detected by their catalytic activity.
• Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; small active enzymes detectable in extracellular solution (e.g., ⁇ -amylase, ⁇ -lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).
• small, diffusible proteins detectable e.g., by ELISA
• small active enzymes detectable in extracellular solution e.g., ⁇ -amylase, ⁇ -lactamase, phosphinothricin acetyltransferase
• proteins that are inserted or trapped in the cell wall e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S.
• a gene that encodes a polypeptide that becomes sequestered in the cell wall, and which polypeptide includes a unique epitope is considered to be particularly advantageous.
• a secreted antigen marker would ideally employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that would impart efficient expression and targeting across the plasma membrane, and would produce protein that is bound in the cell wall and yet accessible to antibodies.
• a normally secreted wall protein modified to include a unique epitope would satisfy all such requirements.
• Possible selectable markers for use in connection with the present invention include, but are not limited to, a neo gene, which codes for kanamycin resistance and can be selected for using kanamycin, G418, a gene encoding resistance to bleomycin, and the like; a bar gene which codes for bialaphos resistance; a gene which encodes an altered EPSP synthase protein thus conferring glyphosate resistance; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil; a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS -inhibiting chemicals (European Patent Application 154,204, 1985); a methotrexate-resistant DHFR gene; a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase
• a selectable marker gene capable of being used in systems to select transformants is the genes that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes (U.S. Patent No. 5,550,318).
• the enzyme phosphinothricin acetyltransferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, causing rapid accumulation of ammonia and cell death.
• PPT phosphinothricin
• Screenable markers that may be employed include, but are not limited to, a ⁇ -glucuronidase or uidA gene (GUS) which encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues; a -lactamase gene, which encodes an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin); a xylE gene which encodes a catechol dioxygenase that can convert chromogenic catechols; an -amylase gene; a tyrosinase gene which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in rum condenses to form the easily detectable compound melanin; a -galactosidase gene, which encodes an enzyme for which there are chrom
• the R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue.
• Maize strains can have one, or as many as four, R alleles which combine to regulate pigmentation in a developmental and tissue specific manner.
• a gene from the R gene complex was applied to maize transformation, because the expression of this gene in transformed cells does not harm the cells. Thus, an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be visually scored as a red sector.
• a maize line carries dominant alleles for genes encoding the enzymatic intermediates in the anthocyanin biosynthetic pathway (C2, Al, A2, Bzl and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation.
• Exemplary lines include Wisconsin 22 which contains the rg-Stadler allele and TR112, a K55 derivative which is r-g, b, PI.
• any genotype of maize can be utilized if the Cl and R alleles are introduced together.
• a further screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene.
• the presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening. E. Other Sequences
• a vector of the invention can also further comprise plasmid DNA.
• Plasmid vectors include additional DNA sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUCl 19, and pUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors.
• the additional DNA sequences include origins of replication to provide for autonomous replication of the vector, additional selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the expression cassette, and sequences that enhance transformation of . prokaryotic and eukaryotic cells.
• Another vector that is useful for expression in both plant and prokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoort et al., U.S. Patent
• This binary Ti plasmid vector has been previously characterized by An, cited supra.
• This binary Ti vector can be replicated in prokaryotic bacteria such as E. coli and Agrobacterium.
• the Agrobacterium plasmid vectors can be used to transfer the expression cassette to dicot plant cells, and under certain conditions to monocot cells, such as rice cells.
• the binary Ti vectors preferably include the nopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions, the colEl replication of origin and a wide host range replicon.
• the binary Ti vectors carrying an expression cassette of the invention can be used to transform both prokaryotic and eukaryotic cells, but is preferably used to transform dicot plant cells.
• Virtually any DNA may be used for delivery to recipient cells to ultimately produce fertile transgenic plants in accordance with the present invention.
• DNA segments in the form of vectors and plasmids, or linear DNA fragments, in some instance containing only the DNA element to be expressed in the plant, and the like, may be employed.
• Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise the cDNA, gene or genes which one desires to introduce into the cells. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired.
• the DNA segment or gene chosen for cellular introduction will often encode a protein which will be expressed in the resultant recombinant cells, such as will result in a screenable or selectable trait and/or which will impart an improved phenotype to the regenerated plant. However, this may not always be the case, and the present invention also encompasses transgenic plants incorporating non-expressed transgenes.
• the expression cassettes of the present invention can be introduced into a host cell, e.g., a plant cell, in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of cell, e.g., monocotyledonous or dicotyledonous, targeted for transformation.
• Vectors which may be used to transform plant tissue with the expression cassettes of the present invention include both Agrobacterium vectors and ballistic vectors, as well as vectors suitable for DNA-mediated transformation, e.g., direct uptake or via electroporation. However, cells other than plant cells may be transformed with the expression cassettes of the invention.
• Suitable methods of transforming plant cells include, but are not limited to, microinjection (Crossway et al., 1986), direct DNA transfer to plant cells by PEG precipitation; liposomes; electroporation (Riggs et al., 1986, Agrobacterium-mediated transformation (Hinchee et al., 1988), direct gene transfer (Paszkowski et al., 1984), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wis. and BioRad, Hercules, Calif, (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; and McCabe et al., 1988).
• a nucleotide sequence of the present invention is directly transformed into the plastid genome.
• Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al, 1994.
• the basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation).
• the 1 to 1.5 kb flanking regions facilitate orthologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome.
• point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., 1990; Staub et al., 1992). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub et al., 1993).
• Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin- detoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab et al., 1993).
• selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state.
• Plastid expression in which genes are inserted by orthologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein.
• a nucleotide sequence of the present invention is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence.
• Agrobacterium tumefaciens cells containing a vector comprising an expression cassette of the present invention, wherein the vector comprises a Ti plasmid are useful in methods of making transformed plants. Plant cells are infected with an Agrobacterium tumefaciens as described above 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. For example, U.S. Pat. No. 4,459,355 discloses a method for transforming susceptible plants, including dicots, with an Agrobacterium strain containing the Ti plasmid. The transformation of woody plants with an Agrobacterium vector is disclosed in U.S. Patent No. 4,795,855.
• U.S. Patent No. 4,940,838 to Schilperoort et al. discloses a binary Agrobacterium vector (i.e., one in which the Agrobacterium contains one plasmid having the vir region of a Ti plasmid but no T region, and a second plasmid having a T region but no vir region) useful in carrying out the present invention. It is particularly preferred to use the binary type vectors of Ti and Ri plasmids of Agrobacterium spp.
• Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti et al., 1985: Byrne et al., 1987; Sukhapinda et al, 1987; Lorz et al., 1985; Potrykus, 1985; Park et al., 1985: Hiei et al., 1994.
• the use of T-DNA to transform plant cells has received extensive study and is amply described (EP 120516; Hoekema, 1985; Knauf, et al., 1983; and An. et al., 1985.
• nucleotide sequences of the invention can be inserted into binary vectors as described in the examples. Transformation of plants can be undertaken with a single DNA molecule or multiple DNA molecules (i.e., co-transformation), and both these techniques are suitable for use with the expression cassettes of the present invention. Numerous transformation vectors are available for plant transformation, and the expression cassettes of this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation.
• Preferred plant cells for transformation include, but are not limited to, cells from plant such as com (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), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco
• genus Lemna L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L.japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana
• genus Spirodela S. intermedia, S. polyrrhiza, S.punctata
• genus Woffia Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa.
• Lemnaceae Any other genera or species of Lemnaceae, if they exist, are also aspects of the present invention. Lemna gibba, Lemna minor, and Lemna miniscula are preferred, with Lemna minor and Lemna miniscula being most preferred. Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of
• 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 sempei-virens); 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).
• pines such as loblolly pine (Pinus taeda), slash pine
• Leguminous plants include beans and peas.
• Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
• Legumes include, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo, Acacia, aneth, artichoke, arugula, blackberry, canola, cilantro, Clementines, escarole, eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange
• plants of the present invention are crop plants and in particular cereals (for example, co , alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, and the like), and even more preferably rice, com and soybean.
• cereals for example, co , alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, and the like
• rice, com and soybean for example, co , alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, and the like
• the host cells are monocot or dicot cells, including, but are not limited to, wheat, com (maize), rice, oat, barley, millet, rye, rape and alfalfa, as well as asparagus, tomato, egg plant, apple, pear, quince, cherry, apricot, pepper, melon, lettuce, cauliflower, Brassica, e.g., broccoli, cabbage, brussels sprout, sugar beet, sugar cane, sweetcom, onion, carrot, leek, cucumber, tobacco, aubergine, beet, broad bean, carrot, celery, chicory, cotton, radish, pumpkin, hemp, buckwheat, orchardgrass, creeping bent top, redtop, ryegrass, tobacco, turfgrass, tall fescue, cow pea, endive, gourd, grape, raspberry, chenopodium, blueberry, pineapple, avocado, mango, banana, groundnut, nectarine, papaya, garlic, pea, peach
• organogenesis means a process by which shoots and roots are developed sequentially from meristematic centers;
• embryogenesis 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.
• tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
• existing meristematic tissue e.g., apical meristems, axillary buds, and root meristems
• induced meristem tissue e.g., cotyledon meristem and hypocotyl meristem.
• tissue source for transformation will depend on the nature of the host plant and the transformation protocol.
• Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like.
• the tissue source is selected and transformed so that it retains the ability to regenerate whole, fertile plants following transformation, i.e., contains totipotent cells.
• Type I or Type II embryonic maize callus and immature embryos are preferred Zea mays tissue sources. Selection of tissue sources for transformation of monocots is described in detail in U.S. Application Serial No. 08/112,245 and PCT publication WO 95/06128 (incorporated herein by reference).
• selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (Messing & Vierra, 1982); Bevan et al., 1983), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., 1990, Spencer et al., 1990), the hph gene which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., 1983).
• the present invention also provides a transformed (transgenic) plant cell, inplanta or explanta, including, but not limited to, a transformed plant cell from plants such as com (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), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculent
• genus Lemna L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana
• genus Spirodela S. intermedia, S. polyrrhiza, S.punctata
• genus Woffia Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa.
• Lemnaceae Any other genera or species of Lemnaceae, if they exist, are also aspects of the present invention.
• Lemna gibba, Lemna minor, and Lemna miniscula axe preferred, with Lemn ⁇ minor and Lemn ⁇ miniscula being most preferred.
• Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen Institut ETH, founded Rubel, Zurich (1986)); vegetables including 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).
• tomatoes Loxicon esculentum
• lettuce e.g., Lactuca sativa
• green beans Phaseeolus vulgaris
• lima beans Phaseeolus limensis
• peas Lathyrus spp.
• 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 pidcherrima), 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 sprace (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).
• pines such as loblolly pine (Pinus taeda), slash pine (P
• Leguminous plants include beans and peas.
• Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
• Legumes include, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo, Acacia, aneth, artichoke, arugula, blackberry, canola, cilantro, Clementines, escarole, eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange
• the transformed cells include, but are not limited to, transformed wheat, com (maize), rice, oat, barley, millet, rye, rape and alfalfa, as well as asparagus, tomato, egg plant, apple, pear, quince, cherry, apricot, pepper, melon, lettuce, cauliflower, Brassica, e.g., broccoli, cabbage, brussels sprout, sugar beet, sugar cane, sweetcom, onion, carrot, leek, cucumber, tobacco, aubergine, beet, broad bean, carrot, celery, chicory, cotton, radish, pumpkin, hemp, buckwheat, orchardgrass, creeping bent top, redtop, ryegrass, tobacco, turfgrass, tall fescue, cow pea, endive, gourd, grape, raspberry, chenopodium, blueberry, pineapple, avocado, mango, banana, groundnut, nectarine, papaya, garlic, pea, peach, peanut, pepper, pineapple
• assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, in situ hybridization and nucleic acid-based amplification methods such as PCR or RT- PCR; "biochemical” assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant, e.g., for disease or pest resistance.
• DNA may be isolated from cell lines or any plant parts to determine the presence of the preselected nucleic acid segment through the use of techniques well known to those skilled in the art. Note that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell.
• the presence of nucleic acid elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCR). Using this technique discreet fragments of nucleic acid are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a preselected nucleic acid segment is present in a stable transformant, but does not prove integration of the introduced preselected nucleic acid segment into the host cell genome.
• PCR polymerase chain reaction
• Southern hybridization provides information that is obtained using PCR, e.g., the presence of a preselected DNA segment, but also demonstrates integration into the genome and characterizes each individual transformant. It is contemplated that using the techniques of dot or slot blot hybridization which are modifications of Southern hybridization techniques one could obtain the same information that is derived from PCR, e.g., the presence of a preselected DNA segment. Both PCR and Southern hybridization techniques can be used to demonstrate transmission of a preselected DNA segment to progeny.
• RNA may only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues.
• PCR techniques may also be used for detection and quantitation of RNA produced from introduced preselected DNA segments. In this application of PCR it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species.
• Southern blotting and PCR may be used to detect the preselected DNA segment in question, they do not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced preselected DNA segments or evaluating the phenotypic changes brought about by their expression. Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focussing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography.
• the unique stractures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.
• Assay procedures may also be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions maybe followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed.
• the expression of a gene product is determined by evaluating the phenotypic results of its expression.
• assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant.
• Morphological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays.
• an expression cassette of the invention may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.
• Particularly preferred plants of the invention include the agronomically important crops listed above.
• the genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction and can thus be maintained and propagated in progeny plants.
• the present invention also relates to a transgenic plant cell, tissue, organ, seed or plant part obtained from the transgenic plant. Also included within the invention are transgenic descendants of the plant as well as transgenic plant cells, tissues, organs, seeds and plant parts obtained from the descendants.
• the expression cassette in the transgenic plant is sexually transmitted.
• the coding sequence is sexually transmitted through a complete normal sexual cycle of the R0 plant to the Rl generation.
• the expression cassette is expressed in the cells, tissues, seeds or plant of a transgenic plant in an amount that is different than the amount in the cells, tissues, seeds or plant of a plant which only differs in that the expression cassette is absent. The transgenic plants produced herein are thus expected to be useful for a variety of commercial and research purposes.
• Transgenic plants can be created for use in traditional agriculture to possess traits beneficial to the grower (e.g., agronomic traits such as resistance to water deficit, pest resistance, herbicide resistance or increased yield), beneficial to the consumer of the grain harvested from the plant (e.g., improved nutritive content in human food or animal feed), or beneficial to the food processor (e.g., improved processing traits).
• agronomic traits such as resistance to water deficit, pest resistance, herbicide resistance or increased yield
• beneficial to the consumer of the grain harvested from the plant e.g., improved nutritive content in human food or animal feed
• beneficial to the food processor e.g., improved processing traits
• the plants are generally grown for the use of their grain in human or animal foods.
• other parts of the plants including stalks, husks, vegetative parts, and the like, may also have utility, including use as part of animal silage or for ornamental purposes.
• chemical constituents e.g., oils or starches
• transgenic plants may be created which have enhanced or modified levels
• Transgenic plants may also find use in the commercial manufacture of proteins or other molecules, where the molecule of interest is extracted or purified from plant parts, seeds, and the like.
• Cells or tissue from the plants may also be cultured, grown in vitro, or fermented to manufacture such molecules.
• the transgenic plants may also be used in commercial breeding programs, or may be crossed or bred to plants of related crop species. Improvements encoded by the expression cassette may be transferred, e.g., from maize cells to cells of other species, e.g., by protoplast fusion.
• the transgenic plants may have many uses in research or breeding, including creation of new mutant plants through insertional mutagenesis, in order to identify beneficial mutants that might later be created by traditional mutation and selection.
• An example would be the introduction of a recombinant DNA sequence encoding a transposable element that may be used for generating genetic variation.
• the methods of the invention may also be used to create plants having unique "signature sequences" or other marker sequences which can be used to identify proprietary lines or varieties.
• the transgenic plants and seeds according to the invention can be used in plant breeding which aims at the development of plants with improved properties conferred by the expression cassette, such as tolerance of viruses or other pests, or other stresses.
• the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate descendant plants. Depending on the desired properties different breeding measures are taken.
• the relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
• Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means.
• Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines.
• the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pesticide treatment or allow to dispense with said methods due to their modified genetic properties.
• new crops with improved stress tolerance can be obtained which, due to their optimized genetic "equipment", yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.
• the invention also provides a computer readable medium having stored thereon a data structure containing nucleic acid sequences having at least 70% sequence identity to a nucleic acid sequence selected from those listed in SEQ ID Nos: 1-795 as well as complementary, ortholog, and variant sequences thereof.
• Storage and use of nucleic acid sequences on a computer readable medium is well known in the art. (See for example U.S. Patent Nos. 6,023,659; 5,867,402; 5,795,716)
• Examples of such medium include, but are not limited to, magnetic tape, optical disk, CD-ROM, random access memory, volatile memory, non- volatile memory and bubble memory.
• the nucleic acid sequences contained on the computer readable medium may be compared through use of a module that receives the sequence information and compares it to other sequence information.
• a module that receives the sequence information and compares it to other sequence information.
• other sequences to which the nucleic acid sequences of the invention may be compared include those maintained by the National Center for Biotechnology Information (NCBI)(http://www.ncbi.nlm.nih.gov/) and the Swiss Protein Data Bank.
• NCBI National Center for Biotechnology Information
• a computer is an example of such a module that can read and compare nucleic acid sequence information.
• the invention also provides the method of comparing a nucleic acid sequence of the invention to another sequence.
• a sequence of the invention may be submitted to the NCBI for a Blast search as described herein where the sequence is compared to sequence information contained within the NCBI database and a comparison is returned.
• the invention also provides nucleic acid sequence information in a computer readable medium that allows the encoded polypeptide to be optimized for a desired property. Examples of such properties include, but are not limited to, increased or decreased: thermal stability, chemical stability, hydrophylicity, hydrophobicity, and the like. Methods for the use of computers to model polypeptides and polynucleotides having altered activities are well known in the art and have been reviewed.
• RNA from plant tissue is extracted and quantified. 1. Quantify total RNA using GeneQuant
• T7-(dT) 2 oligonucleotides were prepared and purified by HPLC. (5'- GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT) 24 -
• Arabidopsis lines were infected with one of two different P. parasitica isolates (see Table 2).
• One Arabidopsis line was Col-0: :RPP8 which carries the cloned RPP8 genomic clone (RPP8 mediates resistance of the ecotype La-er against Emco5; McDowell et al. 1998) and is
• Emco5 resistant Wild-type Col-0 plants are Emco5 susceptible.
• RPP7 mediates resistance of Arabidopsis against the Peronospora isolate Hiksl, and occurs naturally in Col-0.
• Another Arabidopsis line had a loss of function rpp7 mutant allele (Col-0 (rppT)) (McDowell et al, 2000).
• rppT loss of function rpp7 mutant allele
• RNA samples were collected from two week old seedlings at three time points, 0, 12 and 24 hours post infection with 100,000 spores/ml. Twelve hours post-inoculation was determined to be an appropriate time point for analysis of both incompatible interactions, based on trypan blue staining of HR sites and PR-1 and senl induction. Forty-eight hours post-inoculation was determined to be a time point where both compatible interactions exhibit dense hyphal growth and both PR-1 and senl expression. Three independent sets of RNA were pooled.
• Col-0 (tgRPP8, incomp.) Emco5 0, 12, 48 hours
• RNAs were labeled as described in Example 1 and hybridized to an Affymetrix Gene Chip having sequences corresponding to about 8,200 Arabidopsis genes. The data collected from these chips was employed to determine:
• AD "average difference”
• the AD values were log transformed (base 2) and filtered to include only genes in the final data set that showed at least a 3- fold expression change (which equals a 1.6-fold change of the log transformed values). These criteria were fulfilled by 464 genes that define the final data set. With this data set Average linkage clustering was performed (uncentered correlation). The resulting clustergram was displayed using TREEVIEW (Eisen et al., 1998). A second clustergram was generated in the same way applying a cut off value of 50 on the normalized AD values. This clustergram includes 619 genes (probe sets).
• Cluster cR7/R8 contained genes that are early and transiently upregulated early and transiently after P. parasitica infection of Arabidopsis in an RPP7/8 dependent manner. Upregulation by the RPP8 pathway was generally stronger. Strong induced expression of cR7/R8 genes was detectable 12 hours after Emco5 infection in Col-O(tg-PPPS) plants (incompatible interaction), whereas no elevated expression can be observed 48 hours post infection in the same plants (as compared to untreated control plants). Only weakly elevated or no elevated expression was detected at all tested time points in Col-0 plants (compatible interaction).
• the cR7/R8 cluster was originally defined using the clustergram based on a cut off value of 75 and comprised only 9 genes. Applying a cut off value of 50, the cluster became larger, comprising 21 genes, which could be divided into two subclusters (Table 4).
• Subcluster cR7/R8 A includes genes strongly and transiently upregulated by the RPP8 pathway, but either not, or only faintly, upregulated by the RPP7 pathway.
• Members of subcluster B showed pronounced upregulation by both RPP pathways after 12 hours and also elevated expression during compatible Emco5 infections in Col-0.
• genes of the cR7/R8 cluster is more pronounced expression along with RPP7 and RPP8 mediated Peronospora resistance in comparison to the corresponding compatible interactions.
• a search with AlignAce revealed two conserved motifs within a set of 8 promoters comprising 7 from the originally defined cR7R8 using a cut off value of 75 and the promoter of a gene encoding a putative Myb-like transcription factor (Martin &Paz-Ares, 1997) (probe set 13176, SEQ ID NO:36), which shows a similar expression behavior as cR7/R8.
• the AlignAce output is shown below in Table 5:
• cR7/R8 promoters For each motif, examples from cR7/R8 promoters are listed. Nucleotides evaluated by AlignAce as being conserved are indicated by a "*" above the examples. Besides motifs 1 and 2, a variety of additional ones were found by AlignAce, which, however, consisted almost exclusively of Ts and As. These motifs probably do not constitute functional cts-elements. Promoter sequences are generally enriched in As or Ts (A/T content in cR7R8 promoters is 70%) and regulatory c/s-elements usually contain also Cs or Gs.
• Motif 1 (CAACA/TA/TTGA/TC/T, SEQ ID NO:710) resembles the type
• MFS Myb binding site consensus
• T/CAACT/GG Myb binding site consensus
• the 3' portion of the motif 1 consensus allows overlap with binding sites of WRKY (TTTGACC/T; Eulgem et al, 2000) or TGA-bZIP transcription factors (TGACG; Schindler et al. 1992).
• a gene encoding a putative Myb- like transcription factor (gene identification number 13176) is upregulated along with cR7/R8 genes in an ⁇ PP7/PPP5-dependent, early and transient manner.
• the factor encoded by this gene may be a regulator of cR7/R8 genes.
• This motif which consists of six strictly defined positions and four positions allowing two base pairs each, should occur by random roughly once per 20,000 bp in each of both possible orientations (assuming a T/A content of 70% and a G/C content of 30%.
• the motif occurs 9 times (roughly 20 times the statistically expected frequency). Consistent with the statistical expectation, motif 1 occurs only two times within 15,873 bp from a set of control promoters of genes showing no defense related expression (data not shown).
• Motif 2 (A/TTGGGNC/ANAA; SEQ ID NO:711) does not resemble any other known plant cis-element.
• the consensus sequence consists of six strictly defined positions, two positions with two alternatives and two positions with four alternatives; and, hence, should occur by random once per 10,000 bp.
• the latter two clusters may also include genes sharing expression features with cR7/R8 genes.
• cR7/R8 genes may also include genes sharing expression features with cR7/R8 genes.
• a motif 2-like sequence was also recognized by MEME. Within the extended set of cR7/R8 promoters, MEME found the following sequence stretch to be conserved:
• T-DNA lines are screened for knock out in Myb-like gene (AL031394.56_at) and selected members of cR7/R8, such as RNS 1 (atu05206_s_at) or the gene encoding a calmodulin-like protein (identification number 13217_s_at).
• yeast one hybrid screens are performed to clone transcription factors binding to motifs 1 and 2, or other identified motifs.
• Transgenic Arabidopsis plants carrying fusions of GUS with a representative promoter from cR7/R8 can be used to further evaluate the functional significance of motifs 1 and 2, and to perform a genetic screening for mutants impaired in regulation of cR7/R8 genes.
• Emco5 12 hour 30 AND Col RPP8 Emco5 12 hour/Col (rpp8) Emco5 12 hour > 2
• 12778_r_at (AC006577.16_R_AT) gene.
• AC006577_8 (AC006577) Belongs to the PF
• 12916_s_at (ATHCORl_S_AT) induced protein 1 [Arabidopsis thaliana] emb
• NAM no apical meristem
• 16578_s_at (ATHRPRP 1B_S_AT) glucanase 2 (BG2) [Arabidopsis thaliana] dbj
• RPP7 and RPP8 are independent of PAD4, NDR1, NahG, COI1, and EDSI. Consequently, it is unlikely that genes whose expression is reduced in p ⁇ d4 or N ⁇ hG backgrounds are important for RPP7- or RPP8- mediated resistance. Although data from Peronospora infection of pad4 or nahG plants was not available, data from Pseudomonas syringae (ES4326) infection of pad4 and NahG plants was available and employed to determine which genes depended on PAD4 or were interfered with by NahG. Resistance to P. syringae mediated by RPS2 requires NDR1 function.
• Genes whose expression requires RPS2 are less likely to be important for RPP7 or RPP8 mediated resistance than genes whose expression is independent of RPS2.
• Gene expression data was available for wild-type plants infected with Pseudomonas syringae pv. tomato DC3000 avrRpt2, and rps2 mutant plants infected with Pseudomonas syringae pv. tomato DC3000 avrRpt2, 6 hours after infection. EDM1 is required for resistance mediated by RPP7. Thus, genes whose expression requires EDM1 are likely to be important for RPP 7-mediated resistance. Gene expression data was available for an edml mutant (see above).
• rps2 Col DC3000 avrRpt2 6 hour/Col water control > 2 AND rps2 DC3000 avrRpt2 6 hour/Col DC3000 avrRpt2 6 hour ⁇ 0.5.
• Col Psm ES4326 30 hour/Col Mock 30 hour > 2 AND Col pad4 Psm ES4326 30 hour/Col Psm ES4326 30 hour ⁇ 0.5.
• Col Psm ES4326 30 hour/Col Mock 30 hour > 2 AND Col NahG Psm ES4326 30 hour/Col Psm ES4326 30 hour ⁇ 0.5.
• ASA1_S_AT 12889_s_at (ASA1_S_AT) synthase alpha subunit [Arabidopsis thaliana] dbj
• Lipase/Acylhydrolase with GDSL-motif family ESTs gb
• T22281 come from this gene. [Arabidopsis (AC006577.16_R_AT) thaliana] gb
• 14254 has a duplicate in set 2 (14256) Counted in set 2.
• 12778 has duplicates in set 2 (12777 and 12779) Counted in set 2.
• glucanase 2 (BG2) [Arabidopsis thaliana]
• contains similarity to sugar transporters (Pfam: sugar_tr.hmm, score: 395.39) [Arabidopsis 14116_at (AF077407.30_AT) thaliana] gb
• 16105 has a duplicate in set 4 (13273) Count in set 4
• 14110 (putative disease- resistance gene) is an incomplete probe set with very low signal. It has a duplicate (14111) in set 2. Count in set 2.
• the first set do not require EDM1 and are not affected by rps2, pad4 or NahG. These genes are less likely to be important for resistance mediated by RPP7 and RPP8 than the genes in the second set (set 3 above).
• the genes in the second set require EDM1 but are not affected by rps2, pad4 or NahG. These genes are more likely to be important for resistance mediated by RPP7 and RPP8.
• the genes in the third set (set 4 above) are affected by rps2, do not require EDM1, and are not affected by pad4 or NahG.
• genes in the fourth (set 5 above), seventh (set 8 above) and eight (set 9 above) sets include genes that are affected by pad4 or NahG, and so are less likely to be important for resistance mediated by RPP7 and RPP8.
• the genes in the sixth set (set 7 above) require RPP7 or RPP8 for increased expression after infection by Peronospora and are affected by rps2. These genes are also less likely to be important for resistance mediated by RPP7 and RPP8.
• Duplicates were identified by sorting "RPP7 or 8" by description, which put duplicated genes adjacent to one another. Most duplicates were in the same set, but there were nine cases of duplicates that had one probe set in one set, and another in another set. Examination of the data revealed that in these cases, the expression values were close to the cut-offs used to define the various sets. If one copy met the criteria, e.g., for 7y__?2-dependent, but the other copy did not, the gene was classified as not Duplicates are noted. One hundred eighty four unique genes remained (SEQ ID NOs: 1-211 and 792, Table 9 and Table 10).
• ATACS6_AT [Arabidopsis thaliana]
• cytochrome P450 [Arabidopsis thaliana] gb
• E_S_AT like protein [Arabidopsis thaliana]
• HSP176A_I_AT Protein 17.6A [Arabidopsis thaliana]
• HSP83_S_AT protein 83 [Arabidopsis thaliana]

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