Compositions affecting programmed cell death and their use in the modification of plant development

Abstract

Novel isolated polynucleotides associated with programmed cell death and various plant developmental mechanisms are provided, together with genetic constructs comprising such sequences. Methods for the modulation of the content, structure and metabolism of plants, and particularly for the modulation of PCD and various plant developmental mechanisms in plants, are also disclosed, the methods comprising incorporating one or more of the polynucleotides or genetic constructs of the present invention into the genome of a plant.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 09/325,932, filed Jun. 4, 1999.

FIELD OF INVENTION

[0002] This invention involves the modification of plant developmental responses. Specifically, this invention relates to polynucleotides and polypeptides that affect programmed cell death. These polynucleotides and polypeptides, and genetic constructs comprising such polynucleotides and polypeptides, may be used to modulate programmed cell death and thereby alter the developmental cycle of plant cells, hence altering plant development.

REFERENCE TO SEQUENCE LISTING SUBMITTED ON COMPACT DISC

[0003] This application incorporates by reference in its entirety the Sequence Listing contained in the accompanying two compact discs, one of which is a duplicate copy. Each CD contains the following file: 1022c1 Seqlist.txt, having a date of creation of Aug. 8, 2002.

BACKGROUND OF THE INVENTION

[0004] Programmed cell death (PCD) refers to an active process, in which gene expression is intimately associated with the events leading to cell death. The plant life cycle contains many instances of such cell death. During plant reproduction and early embryogenesis, events such as organ ablation during unisexual flower development, tapetum degeneration during pollen development and suspensor degeneration during embryo development all involve an active cell death process. During plant morphogenesis and maturation, aleurone cell degradation, the terminal phase of tracheary element differentiation in xylem, leaf blade development in some plants (e.g. genus Monstera), leaf/organ senescence, root cap cell differentiation and the hypersensitive response in plant/pathogen interactions provide further examples of the role of cell death programs in plant developmental cycles.

[0005] Most of the scientific investigation relating to programmed cell death to date has involved PCD in mammalian cells. PCD in these cells is evidenced by distinct morphological characteristics, such as cytoplasmic condensation, membrane blebbing, DNA fragmentation, condensation and fragmentation of the nucleus, and finally cell corpse engulfment. In mammalian cells, PCD provides a mechanism for removing unwanted cells, as well as for removing pathogens or pathogen-infected cells. It is also believed that a breakdown in normal PCD mechanisms plays an important role in many disease states, including many malignancies.

[0006] The role of PCD in plant systems has not been studied extensively. Preliminary comparisons between plant and mammalian PCD mechanisms suggest some similarities in the mechanisms. The potential similarities include: an oxygen requirement; activation by hydrogen peroxide; a role for calcium in the activation process; a transcription requirement; a dephosphorylation requirement; proteolytic and nucleolytic enzyme involvement and cell condensation and shrinkage. Modulation of the PCD mechanism in any one or more of these areas may affect plant development.

SUMMARY OF THE INVENTION

[0007] Briefly, the present invention provides isolated polypeptides having activity in PCD pathways and various developmental pathways in plant species, together with isolated polynucleotides encoding such polypeptides. Genetic constructs comprising such polynucleotides and methods for the use of such genetic constructs to modulate PCD and various developmental pathways in plants, such as forestry plants, are also provided. Transgenic cells and plants incorporating such genetic constructs and exhibiting a modified content of the polynucleotides and/or polypeptides of the present invention compared to a wild-type plant, are also provided. Methods for modulating plant cell death, as well as for modulating various plant species developmental pathways, using the polynucleotides and/or polypeptides of the present invention, are disclosed.

[0008] In a first aspect, the present invention provides isolated polynucleotide sequences identified in the attached Sequence Listing as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; variants of those sequences; extended sequences comprising the sequences set out in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, and their variants; oligonucleotide probes and primers corresponding to the sequences set out in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, and their variants; isolated polynucleotides comprising at least a specified number of contiguous residues of any of the polynucleotides identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248 (x-mers); and extended sequences comprising portions of the sequences set out in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; all of which are referred to herein, collectively, as “polynucleotides of the present invention.” The present invention also provides isolated polypeptide sequences identified in the attached Sequence Listing as SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290; variants of those sequences; and polypeptides comprising the isolated polypeptide sequences and variants of those sequences.

[0009] In one embodiment, isolated polynucleotides of the present invention comprise a sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; (b) complements of the sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; (c) reverse complements of the sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; (d) reverse sequences of the sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; and (e) sequences having at least 75%, 90%, 95% or 98% identity, as defined herein, to a sequence of (a)-(d) or a specified region of a sequence of (a)-(d).

[0010] In a further aspect, isolated polypeptides encoded by the polynucleotides of the present invention are provided. In one embodiment, such polypeptides comprise an amino acid sequence recited in SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290, and variants thereof, as well as polypeptides expressed by polynucleotides of the present invention, including polynucleotides comprising a sequence of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248.

[0011] In another aspect, the invention provides genetic constructs comprising a polynucleotide of the present invention, either alone, in combination with one or more additional polynucleotides of the present invention, or in combination with one or more known polynucleotides, together with cells and target organisms, such as plants, comprising such constructs.

[0012] In a related aspect, the present invention provides genetic constructs comprising, in the 5′-3′ direction, a gene promoter sequence, an open reading frame coding for at least a functional portion of a polypeptide encoded by a polynucleotide of the present invention, and a gene termination sequence. The open reading frame may be oriented in either a sense or antisense direction. Genetic constructs comprising a gene promoter sequence, a polynucleotide of the present invention, and a gene termination sequence are also contemplated, as are genetic constructs comprising a gene promoter sequence, an untranslated region of a polynucleotide of the present invention, or a nucleotide sequence complementary to an untranslated region, and a gene termination sequence. The genetic construct may further include a marker for the identification of transformed cells.

[0013] The gene promoter and termination sequences are preferably functional in a host plant and, most preferably, are those native to the host plant. Promoter and termination sequences that are generally used in the art, such as the Cauliflower Mosaic Virus (CMV) promoter, with or without enhancers such as the Kozak sequence or Omega enhancer, and Agrobacterium tumefaciens nopaline synthase terminator, are useful. Tissue-specific promoters may be employed in order to target expression to one or more desired tissues.

[0014] In a further aspect, methods for producing plants, including forestry plants, having a modified content of a polynucleotide or polypeptide of the present invention compared to a native organism are provided. The methods involve transforming a target plant with a genetic construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth. Cells comprising the genetic constructs of the present invention are also provided, together with tissues and plants comprising such transgenic cells, and fruits, seeds and other products, derivatives, or progeny of such plants.

[0015] In yet another aspect of the present invention, methods for modulating PCD, and for modulating various developmental pathways of plants are provided, such methods including stably incorporating into the genome of a plant a genetic construct of the present invention. More specifically, methods for modulating developmental pathways, including wood development, senescence and reproductive development, as well as methods for modulating stress responses in plants, are provided. Preferred plants include woody plants, preferably selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species, more preferably from the group consisting of pine and eucalyptus species, and most preferably from the group consisting of Eucalyptus grandis and Pinus radiata.

[0016] The isolated polynucleotides of the present invention also have utility in genome mapping, in physical mapping, and in positional cloning of genes. Additionally, the polynucleotide sequences identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206-248 and their variants, may be used to design oligonucleotide probes and primers. Oligonucleotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion of the polynucleotide. Oligonucleotide probes designed using the polynucleotides of the present invention may be used to detect the presence and examine the expression patterns of genes in any organism having sufficiently similar DNA and RNA sequences in their cells using techniques that are well known in the art, such as slot blot DNA hybridization techniques. Oligonucleotide primers designed using the polynucleotides of the present invention may be used for PCR amplifications. Oligonucleotide probes and primers designed using the polynucleotides of the present invention may also be used in connection with various microarray technologies, including the microarray technology used by Affymetrix (Santa Clara, Calif.).

[0017] Polypeptides encoded by the polynucleotides of the present invention may be expressed and used in various assays to determine their biological activity. Such polypeptides may be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively determine levels of interacting proteins or other compounds.

[0018] The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows a Southern blot analysis of tobacco plants transformed with an antisense sequence of a Pinus radiata DAD1 gene (SEQ ID NO: 8).

[0020] FIG. 2 shows a Northern blot analysis of tobacco plants transformed with an antisense sequence of Pinus radiata DAD1 gene (SEQ ID NO: 8).

[0021] FIG. 3 illustrates detection of a Pinus unique sequence identifier in transformed tobacco plants. Lanes A and B show the hybridization of a probe from SEQ ID NO: 202 to the genomic DNA of tobacco plants which lack the Pinus unique sequence identifier (empty-vector transformed control plants or wild type). Lanes C-E show the hybridization of the probe to the genomic DNA of tobacco plants containing one to three copies of the Pinus unique sequence identifier.

[0022] FIG. 4 illustrates detection of a Eucalyptus unique sequence identifier in transformed tobacco plants. Lanes A and B show the hybridization of a probe from SEQ ID NO: 203 to the genomic DNA of tobacco plants which lack the Eucalyptus unique sequence identifier (empty-vector transformed control plants or wild type). Lanes C-E show the hybridization of the probe to the genomic DNA of tobacco plants containing one to two copies of the Eucalyptus unique sequence identifier.

[0023] FIG. 5 shows the amino acid sequence of SEQ ID NO: 62 encoding an annexin homologue. The annexin conserved domains (residue 13 to 80, 85 to 152, 243 to 310 and 168 to 235) are marked in bold and the annexin consensus pattern (residue 258 to 311) is underlined.

[0024] FIG. 6 shows the amino acid sequence of SEQ ID NO: 67 encoding a prohibitin homologue. The signal peptide is underlined and the conserved prohibitin SPFH domain/Band 7 family (residue 27 to 219) is marked in bold.

[0025] FIG. 7 shows the amino acid sequence of SEQ ID NO: 73 encoding a Rac-like GTP binding protein homologue. The ATP/GTP-binding site motif A is marked in bold (residue 13 to 19).

[0026] FIG. 8 shows the amino acid sequence of SEQ ID NO: 143 encoding a cysteine protease homologue. The signal peptide at the start of the ORF is double underlined. The papain family cysteine protease region (residue 145 to 361) is marked in bold, the Thiol Protease Asn region (residue 320 to 340), the Thiol Protease His region (residue 303 to 314) and the Thiol Protease Cys region (residue 163 to 175) are boxed.

[0027] FIG. 9 shows the amino acid sequence of SEQ ID NO: 149 encoding a cysteine protease homologue. The signal peptide at the start of the ORF is double underlined. The papain family cysteine protease region (residue 171 to 387) is marked in bold, the granulin region (residue 421 to 469) is marked bold and underlined, the ERFNIN region (residue 98 to 117), the Thiol Protease Asn region (residue 346 to 366), the Thiol Protease His region (residue 329 to 340), the Thiol Protease Cys region (residue 189 to 201) and the PA2 His region (residue 445 to 453) are boxed.

[0028] FIG. 10 shows the amino acid sequence of SEQ ID NO: 152 encoding a senescence-specific SAG12 proteinase homologue. The signal peptide at the start of the ORF is double underlined. The papain family cysteine protease region (residue 163 to 380) is marked in bold, the ERFNIN region (residue 91 to 110), the Thiol Protease Asn region (residue 340 to 360), the Thiol Protease His region (residue 320 to 331), the Thiol Protease Cys region (residue 181 to 193) and the DNAJ NTER region (residue 117 to 137) are boxed.

[0029] FIG. 11 shows the amino acid sequence of SEQ ID NO: 153 encoding a cysteine proteinase homologue (vignain precursor). The signal peptide at the start of the ORF is double underlined. The papain family cysteine protease region (residue 160 to 377) is marked in bold, the aldehyde dehydrogenases glutamic acid active site (residue 296 to 304), the ERFNIN region (residue 86 to 105), the Thiol Protease Asn region (residue 337 to 357), the Thiol Protease His region (residue 319 to 330) and the Thiol Protease Cys region (residue 178 to 190) are boxed.

[0030] FIG. 12 Amino acid sequence of SEQ ID NO: 158 encoding a cysteine protease homologue. The signal peptide at the start of the ORF is double underlined. The papain family cysteine protease region (residue 161 to 376) is marked in bold, the ERFNIN region (residue 95 to 114), the Thiol Protease Asn region (residue 340 to 360), the Thiol Protease His region (residue 323 to 334) and the Thiol Protease Cys region (residue 179 to 191) are boxed.

[0031] FIG. 13 Amino acid sequence of SEQ ID NO: 169 encoding a cysteine protease homologue. The signal peptide at the start of the ORF is double underlined. The papain family cysteine protease region (residue 143 to 368) is marked in bold, the Thiol Protease Asn region (residue 332 to 352), the Thiol Protease His region (residue 308 to 319) and the Thiol Protease Cys region (residue 161 to 173) are boxed.

[0032] FIG. 14 shows the amino acid sequence of SEQ ID NO: 201 encoding a Seven in Absentia protein homologue. The carbamoyl-phosphate synthase subdomain signature 2 region (residue 310 to 318) is boxed and labeled, the Seven in Absentia protein family region (residue 119 to 322) is marked in bold and the TRAF-type zinc finger (residue 154 to 210) is boxed.

[0033] FIG. 15 shows the amino acid sequence of SEQ ID NO: 205 encoding a BAG-1 Bcl-2-associated athano protein homologue. The ORF start methionine residue is boxed. The BAG domain (residue 143 to 221) is in bold and the ubiquitin family region (residue 50 to 123) is in bold and underlined.

[0034] FIG. 16 shows the amino acid sequence of SEQ ID NO: 249 encoding a Ring zinc finger protein homologue. The C3HC4 type (RING finger) zinc finger region (residue 161 to 202) is marked in bold with residues involved in binding zinc boxed and the PHD-finger region (residue 163 to 205) underlined.

[0035] FIG. 17 shows the amino acid sequence of SEQ ID NO: 250 encoding a Ring zinc finger protein homologue. The C3HC4 type (RING finger) zinc finger region (residue 130 to 171) is in bold with residues involved in binding zinc boxed and the PHD-finger region (residue 129 to 174) underlined.

[0036] FIG. 18 shows the amino acid sequence of SEQ ID NO: 251 encoding a Ring zinc finger protein homologue. The C3HC4 type (RING finger) zinc finger region (residue 111 to 152) is marked in bold with residues involved in binding zinc boxed and the PHD-finger region (residue 113 to 155) underlined.

[0037] FIG. 19 shows the amino acid sequence of SEQ ID NO: 252 encoding a defender against apoptotic cell death (Dad-1, Dad-2) protein homologue. The conserved DAD family region (residue 4 to 115) is in bold.

[0038] FIG. 20 shows the amino acid sequence of SEQ ID NO: 253 encoding a defender against apoptotic cell death (Dad-1, Dad-2) protein homologue. The conserved DAD family region (residue 4 to 115) is in bold.

[0039] FIG. 21 shows the amino acid sequence of SEQ ID NO: 254 encoding a defender against apoptotic cell death (Dad-1, Dad-2) protein homologue. The conserved DAD family region (residue 4 to 115) is in bold.

[0040] FIG. 22 shows the amino acid sequence of SEQ ID NO: 255 encoding a bifunctional nuclease homologue. The S1/P1 Nuclease region (residue 1 to 148) is marked in bold, the DNase active site (Pro-Leu-His) is underlined and residues involved in binding of zinc atoms are boxed.

[0041] FIG. 23 shows the amino acid sequence of SEQ ID NO: 256 encoding a lethal leaf spot protein (Lls1) homologue. The Rieske [2Fe-2S] domain region (residue 96 to 204) is in bold and the zinc binding residues in both the Rieske-type iron-sulfur binding site and the mononuclear iron-binding site are boxed.

[0042] FIG. 24 shows the amino acid sequence of SEQ ID NO: 257 encoding a lesion simulating cell death (lsd1) zinc finger protein homologue. Residues involved in the three conserved zinc finger domains are boxed and additional residues conserved in lsd1 genes are in bold.

[0043] FIG. 25 shows the amino acid sequence of SEQ ID NO: 258 encoding a lesion simulating cell death (lsd1) zinc finger protein homologue. Residues involved in the three conserved zinc finger domains are boxed and additional residues conserved in lsd1 genes are in bold.

[0044] FIG. 26 shows the amino acid sequence of SEQ ID NO: 259 encoding a nucellin homologue. The two eukaryotic and viral aspartyl proteases active sites (residues 412 to 424 and 201 to 213) are in bold and the leucine zipper (residue 79 to 101) is bold and underlined.

[0045] FIG. 27 shows the amino acid sequence of SEQ ID NO: 260 encoding an annexin homologue. The four conserved annexin regions (residue 13 to 80, 85 to 152, 168 to 235 and 243 to 310) are in bold and the annexin region (residue 258 to 311) is underlined.

[0046] FIG. 28 shows the amino acid sequence of SEQ ID NO: 263 encoding a protein kinase-like protein homologue. The conserved protein kinase domain region (residue 71 to 351) is marked in bold, the tyrosine protein kinases specific active-site signature (residue 199 to 212) is underlined and the Protein kinases ATP-binding region signature (residue 77 to 101) is double underlined.

[0047] FIG. 29 shows the amino acid sequence of SEQ ID NO: 265 encoding a RAC 1 protein homologue (fragment). The Ras family region (residue 8 to 196) is marked in bold and the ATP_GTP_A region (residue 13 to 21) is boxed.

[0048] FIG. 30 shows the amino acid sequence of SEQ ID NO: 267 encoding a ntrb1 protein homologue. The retinoblastoma-associated protein A domain (residue 298 to 499) is marked in bold and the retinoblastoma-associated protein B domain (residue 622 to 756) is marked in bold and underlined.

[0049] FIG. 31 shows the amino acid sequence of SEQ ID NO: 271 encoding a transcription initiation factor TFIID (TATA-box factor) homologue. The TFIID conserved domains (residues 20 to 105 and 110 to 196) are in bold and the two TFIID repeat signature domains (residues 52 to 102 and 143 to 193) are underlined.

[0050] FIG. 32 shows the amino acid sequence of SEQ ID NO: 272 encoding transcription initiation factor TFIID (TATA-box factor) homologue. The TFIID conserved domains (residues 20 to 105 and 110 to 196) are in bold and the two TFIID repeat signature domains (residues 52 to 102 and from 143 to 193) are underlined.

[0051] FIG. 33 shows the amino acid sequence of SEQ ID NO: 276 encoding a cysteine protease homologue. The signal peptide is double underlined. The papain family cysteine protease region (residue 129 to 345) is in bold, the granulin region (residue 379 to 427) is bold and underlined, the ERFNIN region (residue 58 to 77), the Thiol Protease Asn region (residue 304 to 324), the Thiol Protease His region (residue 287 to 298), the Thiol Protease Cys region (residue 147 to 159) and the PA2 His region (residue 403 to 411) are boxed.

[0052] FIG. 34 shows the amino acid sequence of SEQ ID NO: 277 encoding a cysteine protease homologue. The signal peptide is double underlined. The papain family cysteine protease region (residue 132 to 348) is in bold, the granulin region (residue 382 to 436) is in bold and underlined, the ERFNIN region (residue 60 to 79), the Thiol Protease Asn region (residue 307 to 327), the Thiol Protease His region (residue 290 to 301), the Thiol Protease Cys region (residue 150 to 162) and the PA2 His region (residue 406 to 414) are boxed.

[0053] FIG. 35 shows the amino acid sequence of SEQ ID NO: 278 encoding a cysteine protease homologue. The signal peptide is double underlined. The papain family cysteine protease region (residue 141 to 357) is in bold, the granulin region (residue 391 to 439) is in bold and underlined, the ERFNIN region (residue 69 to 88), the Thiol Protease Asn region (residue 316 to 336), the Thiol Protease His region (residue 299 to 310), the Thiol Protease Cys region (residue 159 to 171) and the PA2 His region (residue 415 to 423) are boxed.

[0054] FIG. 36 shows the amino acid sequence of SEQ ID NO: 279 encoding a thiol protease homologue. The signal peptide is double underlined. The papain family cysteine protease region (residue 151 to 371) is marked in bold, the granulin region (residue 426 to 474) is marked in bold and underlined, the Thiol Protease Asn region (residue 331 to 351), the Thiol Protease His region (residue 314 to 325) and the Thiol Protease Cys region (residue 169 to 181) are boxed.

[0055] FIG. 37 shows the amino acid sequence of SEQ ID NO: 281 encoding a cysteine protease homologue. The signal peptide is double underlined. The papain family cysteine protease region (residue 135 to 360) is in bold, the Thiol Protease Asn region (residue 324 to 344), the Thiol Protease His region (residue 300 to 311) and the Thiol Protease Cys region (residue 153 to 165) are boxed.

[0056] FIG. 38 shows the amino acid sequence of SEQ ID NO: 282 encoding a cysteine protease homologue. The signal peptide is double underlined. The papain family cysteine protease region (residue 127 to 344) is in bold, the ERFNIN region (residue 55 to 74), the Thiol Protease Asn region (residue 304 to 324), the Thiol Protease His region (residue 286 to 297), the Thiol Protease Cys region (residue 145 to 157) and the DnaJ ‘J’ domain signature (residue 81 to 101) are boxed.

[0057] FIG. 39 shows the amino acid sequence of SEQ ID NO: 283 encoding a cysteine protease homologue. The signal peptide is double underlined. The papain family cysteine protease region (residue 139 to 355) is in bold, the Thiol Protease Asn region (residue 319 to 339), the Thiol Protease His region (residue 301 to 312) and the Thiol Protease Cys region (residue 157 to 169) are boxed.

[0058] FIG. 40 shows the amino acid sequence of SEQ ID NO: 285 encoding a cysteine protease homologue. The signal peptide is double underlined. The papain family cysteine protease region (residue 137 to 354) is in bold, the aldehyde dehydrogenases glutamic acid active site (residue 273 to 281), the ERFNIN region (residue 63 to 82), the Thiol Protease Asn region (residue 314 to 334), the Thiol Protease His region (residue 296 to 307) and the Thiol Protease Cys region (residue 155 to 167) are boxed.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0059] Using the methods and materials of the present invention, PCD and/or specific developmental pathways may be modulated in a plant by modifying the polynucleotide and/or polypeptide content of the target organism, for example, by incorporating sense or antisense copies of polynucleotides of the present invention that encode polypeptides involved in the PCD and/or specific developmental pathways into the genome of the plant. In addition, the number of copies and combination of polynucleotides of the present invention may be manipulated in a plant to modify the relative amounts of polypeptides synthesized, thereby producing biological materials having an altered composition and/or developmental metabolism.

[0060] According to one embodiment, the present invention provides isolated polynucleotides encoding, or partially encoding, polypeptides involved in PCD and/or specific developmental pathways in plants. The polynucleotides of the present invention were isolated from eucalyptus and pine species, but they may alternatively be synthesized using conventional synthesis techniques. Specifically, isolated polynucleotides of the present invention include polynucleotides comprising a sequence selected from the group consisting of sequences identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; complements of the sequences identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; reverse sequences of the sequences identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; reverse complements of the sequences identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; at least a specified number of contiguous residues (x-mers) of any of the above-mentioned polynucleotides; antisense sequences corresponding to any of the above polynucleotides; and variants of any of the above polynucleotides, as that term is described in this specification.

[0061] The isolated polynucleotides recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 207-248, encode, or partially encode, polypeptides recited in SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290, respectively, that are involved in PCD and/or developmental processes, as identified in Tables 1 and 2 below. Using methods and materials of the present invention, the polynucleotide and/or polypeptide content of a target organism, such as a plant, may be increased or reduced, thereby modulating PCD in the organism or in a tissue of the organism, or modulating a developmental pathway in the organism or a tissue by incorporating various polynucleotides of the present invention, including untranslated portions of such polynucleotides and antisense copies of such polynucleotides.

[0062] In another embodiment, the present invention provides isolated polypeptides encoded by the DNA sequences of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 207-248. As noted above, the amino acid sequences corresponding to the polynucleotides set out in SEQ ID NOS: 1-28, 30-45, 90-140, 192-195, 200, 204 and 207-248 are provided in SEQ ID NOS: 46-73, 74-89, 141-191, 196-199, 201, 205 and 249-290, respectively.

[0063] The polypeptides of the present invention and the polynucleotides encoding the polypeptides have activity in PCD and various developmental pathways in plants. The polynucleotides were putatively identified by DNA and polypeptide similarity searches. In the attached Sequence Listing, SEQ ID NOS. 1-28, 30-45, 90-140, 192-195, 200, 204 and 207-248 are polynucleotide sequences that encode the polypeptides listed in SEQ ID NOS. 46-73, 74-89, 141-191, 196-199, 201, 205 and 249-290, respectively. The polynucleotides and polypeptides of the present invention have demonstrated similarity to polypeptides that are known to be involved in PCD and/or plant developmental processes as indicated below in Tables 1 and 2. 1 TABLE 1 Polynucleotide Polypeptide POLYPEPTIDE IDENTITY SEQ ID NO. SEQ ID NO. ATL2 1-5, 207-209 46-50, 249-251 DAD1 (Defender Against Cell 6-9, 210-212 51-54, 252-254 Death) DNase 10, 213 55, 255 lls (lethal leaf spot) 11, 12, 214 56, 57, 256 lsd1 (lesion stimulating death) 13, 14, 215, 216 58, 59, 257, 258 Nucellin-like aspartic protease 15, 16, 217 60, 61, 259 Annexin 17-21, 218 62-66, 260 Prohibitin 22-26, 219, 220 67-71, 261, 262 Fen-like protein 27, 221 72, 263 Rac2 28-35, 222-224 73, 74-79, 264- 266 Retinoblastoma-related Protein 36, 37, 225, 226 80, 81, 267, 268 SINA (Seven in absentia) 38-40, 200, 227, 82-84, 201, 269, 228 270 TFIID (Transcription Initiation 41, 42, 229, 230 85, 86, 271, 272 Factor) TEGT (Testis Enhanced Gene 43, 44, 231, 232 87, 88, 273, 274 Transcript) Xylogenic RNase 45 89 Pur-alpha 90, 91, 233 141, 142, 275 Cysteine proteases 92-125, 234-243 143-176, 276- 285 RPP5-like proteins 126-140, 244- 177-191, 128- 247 289 gp 91 NADPH oxidase subunit 192 196 NPR-like proteins 193-195, 248 197-199, 290 BAG-1 204 205

[0064] 2 TABLE 2 SEQ ID NO: SEQ ID NO: Polynucleotide Polypeptide Description 207-209 249-251 The RING-finger is a zinc binding domain of 40 to 60 residues that binds two atoms of zinc and is found in a wide range of regulatory proteins including viral transcription factors, oncoproteins and components of signal transduction pathways, and in proteins involved in DNA repair and recombination. Proteins carrying this domain have been implicated in a range of diverse biological processes such as development, apoptosis, cell- cycle control and ubiquitination. (Grishin, Nucleic Acids Res. 29:1703-1714, 2001). 210-212 252-254 DAD1 (Defender Against Cell Death 1) is associated with cellular housekeeping functions that are necessary for cell health and survival, and their loss may lead to cell death. Cells in temperature-sensitive mutant hamster cell lines undergo PCD at restrictive temperatures, and it has been shown that the Arabidopsis DAD1 can rescue the hamster temperature-sensitive mutant. DAD1 is a component of oligosaccharyltransferase, involved in N-linked glycosylation. DAD1 is involved in induction of cell death and reduction of PCD during development. 214 256 Homologue of lls1 (lethal leaf spot protein) that was identified from a maize mutant, and that is required to limit the spread of cell death in a developmental manner in leaves. 215-216 257-258 Homologue of Lsd1 (lesion simulating cell death) that is involved in superoxide-dependent signalling and acts as a negative regulator of a plant cell death pathway. Lsd1 encodes a zinc finger protein with homology with GATA- type transcription factors, and the LSD1 protein functions either to negatively regulate a pro-death pathway component or to activate a repressor of plant cell death (Dietrich et al., Cell 88: 685-694, 1997). This homologue contains the three zinc finger domains conserved in Arabidopsis thaliana lsd1. 217 259 Homologue encoding nucellin that is involved in cell death. The actual process of cell death involves the degradation of proteins and nucleic acids, mediated by proteases and nucleases. In addition, an aspartic nuclease, nucellin, is specifically associated with nucellar cell death (Chen and Foolad, Plant Mol. Biol. 35: 821-831, 1997). 218 260 Homologue of the plant protein oxy5 from Arabidopsis that is a member of the annexin family and protect bacterial cells from oxidative stress. Oxy5 protects mammalian cells from tumor necrosis factor-induced cell death (Kush and Sabapathy, Int. J. Biochem. Cell. Biol. 33: 591-602, 2001). The involvement of oxidative stress in the various instances of programmed cell death in plants suggests that oxy5 plays a protective role. 219-220 261-262 Homologue of the tumor suppressor gene, prohibitin, that is involved in cell cycle arrest. In rat B lymphocytes, the association of prohibitin with membrane-bound IgM is a mediator of programmed cell death in these cells. Furthermore, in yeast, the deletion of prohibitin homologs resulted in a decreased replicative lifespan, leading to successive decreases in cell cycle time, ageing and cellular senescence. 221 263 Protein kinases have a conserved catalytic core common to both serine/threonine and tyrosine protein kinases which play critical roles in regulating cell development and PCD. 222-223 264-265 The Ras branch of the Ras superfamily consists of small GTPases, most closely related to Ras and includes the R- Ras, Rap, Ral, Rheb, Rin and Rit proteins. Studies have shown that treatments that induce programmed cell death (PCD) also cause oxidative stress, suggesting a role for oxidative stress in PCD. NADPH oxidase and rac2 are present in plant cells and interact during hypersensitive response PCD. Furthermore, the NADPH oxidase is active during osmotic stress-mediated cell death and during the terminal phase of tracheary element differentiation. 224 266 Homologue of rac2, a small cytosolic protein that is required for activation of oxidases that plays a role in PCD. When a constitutively active rac2 mutant was inserted into mice, a significant enhancement of PCD occurred compared to wild type mice. Biochemical and immunochemical studies have shown that rac2 is present in plant cells and interact during hypersensitive response PCD. 225-226 267-268 Homologue of the retinablastoma gene (RB) from mammals that is involved in p45 mediated responses. P45 is a known tumor repressor that can mediate cell cycle arrest and trigger PCD. This tumor suppressor can bind and inhibit the transcription factors that initiate entry into the cell cycle. In addition, RB plays a regulatory role in the cell death process, depending on its phosphorylation status. The regulation of RB proteolysis by phosphorylation status, and the consequent RB levels in the cells are important in the determination of cellular fates. 227-228 269-270 Homologue of the mammalian gene SINA (Seven In Absentia) that is activated during PCD. Human homologs to the Drosophila SINA gene are activated during PCD and target specific proteins for ubiquitination and degradation in both humans and Drosophila. In addition to actual protease activity, targeting of proteins for proteolytic degradation via the ubiquitin-proteosome pathway is up- regulated during PCD. 229-230 271-272 Homologue of the TATA Box Binding Protein (TFIID). TFIID is a housekeeping gene that may be used to control cell survival and cell death and is the most important general factor required for gene transcription by RNA Polymerase II. TFIID binds to the TATA box and participates in the first steps of transcription factor assembly, which is important for the control of gene expression (Martinez et al., Proc. Natl. Acad. Sci. USA 92:11864-11868, 1995). The ability to developmentally or tissue-specifically knock-out TFIID activity provides a method of specifically inducing cell death. Attempts at TFIID knock-out have not been reported for plants. 231-232 273-274 Homologue of Bax inhibitor (BI-1) that inhibits Bax- induced cell death. This gene is identical to a previously identified human gene identified as TEGT (Testis Enhanced Gene Transcript). In mammalian systems, caspase activation can be inhibited by proteins such as Bcl- 2, providing protection against cell death. However, other members of the Bcl-2 family, such as Bax, are antagonistic towards the protective effect of Bcl-2 and promote cell death, due to their ability to interact with Bcl-2 and inhibit its protective ability. 233 275 Homologue of the mammalian transcription factor Pur- alpha that is involved in the control of mammalian cell death. Pur-alpha is a single-stranded DNA binding protein, which has plays a role in both DNA replication and transcriptional regulation. Pur-alpha is able to suppress PCD of mammalian cells by two mechanisms. The first is the transcriptional repression of Fas (CD-95), a receptor which transduces a cell death signal by interaction with its ligand, and the second is the protection of mammalian cells against cell death mediated by p53. Pur alpha contains a region with limited homology to the simian virus 40 large tumor antigen, and this region is implicated in the binding of each of these proteins to Rb, the retinoblastoma tumor suppressor gene product. 234-243 276-285 Eukaryotic thiol proteases (EC 3.4.22.-) are a family of proteolytic enzymes which contain an active site cysteine. Families C1, C2 and C10 are loosely termed papain-like. The papain family has a wide variety of activities, including broad-range (papain) and narrow-range endo- peptidases, aminopeptidases, dipeptidyl peptidases and enzymes with both exo- and endo-peptidase activity (such as cathepsins B and H). The papain family is generally synthesized with signal peptides and propeptides at the N- terminus and therefore proteolytic cleavage of the propeptide is required for enzyme activation. The majority of the propeptides are similar to that of papain, the first 5 amino acids being part of the ‘ERFNTN’ motif used to identify papain-related propeptides (Karrer et al., Proc. Natl. Acad. Sci. USA 90:3063-3067, 1993).

[0065] In mammalian PCD, regulation of cell cycle entry appears to be important, and it has been suggested that cell cycle checkpoint regulators may be involved in the commitment of a cell to death. For example, the known tumor suppressor p45 is capable of mediating cell cycle arrest and can trigger PCD. One of the key genes involved in p45 mediated responses is the retinoblastoma gene (RB). This tumor suppressor can bind and inhibit the transcription factors that initiate entry into the cell cycle. In addition, RB plays a regulatory role in the cell death process, depending on its phosphorylation status. The regulation of RB proteolysis by phosphorylation status, and the consequent RB levels in the cells are important in the determination of cellular fates. Two polynucleotides encoding retinoblastoma-related polypeptides (SEQ ID NOS: 36 and 37) have been isolated from forestry species. Retinoblastoma-related polypeptides encoded by these polynucleotides are identified as SEQ ID NOS: 80 and 81, respectively.

[0066] Another tumor suppressor gene, prohibitin, can also arrest the cell cycle. In rat B lymphocytes, the association of prohibitin with membrane-bound IgM has been suggested as a mediator of PCD in these cells. Furthermore, in yeast, the deletion of prohibitin homologs resulted in a decreased replicative lifespan, leading to successive decreases in cell cycle time, ageing and cellular senescence. While the above studies have been conducted in non-plant systems, it is likely that similar cell cycle modulators are effective in plant systems. Several polynucleotides encoding prohibitin-related polypeptides (SEQ ID NOS: 22-26) have been isolated from forestry species. Prohibitin-related polypeptides encoded by these polynucleotides are identified as SEQ ID NOS: 67-71, respectively.

[0067] Polynucleotides associated with cellular housekeeping functions are necessary for cell health and survival, and their loss may lead to cell death. One such polynucleotide, initially identified in temperature-sensitive mutant hamster cell lines, is DAD1 (Defender Against Cell Death 1). Cells in temperature-sensitive mutant hamster cell lines undergo PCD at restrictive temperatures, and it has been shown that the Arabidopsis DAD1 can rescue the hamster temperature-sensitive mutant. The presence of DAD1 can also reduce cell death in the developing embryo of the worm Caenorhabditis elegans, which undergoes developmentally-regulated cell death. DAD1 has been shown to be a component of oligosaccharyltransferase, involved in N-linked glycosylation. The induction of cell death by DAD1 inactivation, as well as the ability of DAD1 to reduce PCD during development illustrates the essential role of this housekeeping gene. Several polynucleotides encoding DAD1-related polypeptides (SEQ ID NOS: 6-9) have been isolated from forestry species. DAD1-related polypeptides encoded by these polynucleotides are identified as SEQ ID NOS: 51-54, respectively.

[0068] Another housekeeping polynucleotide which may be used to control cell survival and cell death is the TATA Box Binding Protein (TFIID). TFIID is the most important general factor required for gene transcription by RNA Polymerase II. TFIID binds to the TATA box and participates in the first steps of transcription factor assembly, which is important for the control of gene expression. The ability to developmentally or tissue-specifically knock-out TFIID activity provides a method of specifically inducing cell death. Attempts at TFIID knock-out have not been reported for plants. Polynucleotides encoding TFIID-related transcription initiation factors (SEQ ID NOS: 41 and 42) have been isolated from forestry species. TFIID-related transcription initiation factors encoded by these polynucleotides are identified as SEQ ID NOS: 85 and 86, respectively.

[0069] Another transcription factor involved in the control of mammalian cell death is pur-alpha. Pur-alpha is a single-stranded DNA binding protein, which has been shown to play a role in both DNA replication and transcriptional regulation. Pur-alpha is able to suppress PCD of mammalian cells by two mechanisms. The first is the transcriptional repression of Fas (CD-95), a receptor which transduces a cell death signal by interaction with its ligand, and the second is the protection of mammalian cells against cell death mediated by p53. Polynucleotides encoding allelic variants of plant pur-alpha have been isolated (SEQ ID NOS: 90 and 91) from forestry species. The corresponding amino acid sequences of the pur-alpha polypeptides encoded by these polynucleotides are identified as SEQ ID NOS: 141 and 142.

[0070] The actual process of cell death involves the degradation of proteins and nucleic acids, mediated by proteases and nucleases. Experimental work done with mammalian systems suggests that proteases may be an important trigger of cell death. In animals, the caspase family of cysteine proteases are major effectors of this process. Cysteine proteases have been identified in plants which are up-regulated and specifically associated with aleurone and tracheary element cell death. Polynucleotides encoding cysteine proteases in forestry species have been identified as SEQ ID NOS: 92-125. The corresponding amino acid sequences of polypeptides encoded by these polynucleotides are identified by SEQ ID NOS: 143-176, respectively. In addition, an aspartic nuclease, nucellin, has been shown to be specifically associated with nucellar cell death. Polynucleotides encoding a nucellin-like aspartic protease (SEQ ID NOS: 15 and 16) have been isolated from forestry species. The corresponding amino acid sequences of the aspartic nuclease encoded by these polynucleotides are identified in SEQ ID NOS: 60 and 61, respectively.

[0071] In addition to actual protease activity, targeting of proteins for proteolytic degradation via the ubiquitin-proteosome pathway is up-regulated during PCD. Human homologs to the Drosophila SINA (Seven In Absentia) gene are activated during PCD. SINA has been shown to target specific proteins for ubiquitination and degradation in both humans and Drosophila. Polynucleotides encoding SINA-related polypeptides (SEQ ID NOS: 38-40 and 200) have been isolated from forestry species. SINA-related polypeptides encoded by these polynucleotides are identified as SEQ ID NOS: 82-84 and 201, respectively.

[0072] Nuclear DNA cleavage, nuclear fragmentation and RNA degradation are active processes that occur during PCD in animals and plants. Specific plant DNases and RNases have been identified during PCD in plant aleurone cells, tracheary elements, cells undergoing a hypersensitive response to a pathogen, as well as during salt stress-induced cell death. Polynucleotides encoding a plant DNase (SEQ ID NO: 10) and xylogenic RNase (SEQ ID NO: 45) have been isolated from forestry species. The corresponding amino acid sequences of the DNase and RNase encoded by these polynucleotides are identified in SEQ ID NOS: 55 and 89, respectively.

[0073] In mammalian systems, caspase activation can be inhibited by proteins such as Bcl-2, providing protection against cell death. However, other members of the Bcl-2 family, such as Bax, are antagonistic towards the protective effect of Bcl-2 and promote cell death, due to their ability to interact with Bcl-2 and inhibit its protective ability. A recently discovered gene, BI-1 (Bax Inhibitor-1), was found to inhibit Bax-induced cell death. This gene is identical to a previously identified human gene identified as TEGT (Testis Enhanced Gene Transcript). Polynucleotides encoding TEGT polypeptides isolated from forestry species are identified as SEQ ID NOS: 43-44. The corresponding amino acid sequences of the TEGT polypeptides encoded by these polynucleotides are identified as SEQ ID NOS: 87-88, respectively.

[0074] Another protein involved in inhibition of PCD is BAG-1 (Bcl-2-Associated-athanoGene), a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hormone receptors in mammalian cells. It is identical to a hormone-receptor binding protein RAP46. BAG-1 binds to and potentiates the effect of the anti-apoptotic protein Bcl-2, to make cells more resistant to apoptosis. Human BAG-1 is overexpressed in human leukemias, colon, cervical, breast, prostate and lung cancer cell lines. A polynucleotide encoding a BAG-1 polypeptide isolated from forestry species is identified as SEQ ID NO: 204. The corresponding amino acid sequence of the BAG-1 polypeptide encoded by the polynucleotide is identified as SEQ ID NO: 205. The isolated polynucleotide sequence encoding BAG-1 contains a PROSITE motif for a ubiquitin-like domain that is also present in the human and mouse BAG-1 proteins.

[0075] Numerous studies of mammalian systems have shown that treatments that induce PCD also cause oxidative stress, suggesting a role for oxidative stress in PCD. This has been confirmed by observations that the addition of ROS (Reactive Oxygen Species) or a depletion of cellular antioxidants can cause PCD. PCD can be associated with ROS induction, and PCD can be blocked by the addition of compounds with antioxidant properties. Reactive oxygen species such as superoxide, the hydroxyl radical and hydrogen peroxide can react with and damage cell macromolecules. Additionally, they may set in motion chain reactions in which free radicals are passed from one molecule to another, resulting in extensive cell damage and toxicity.

[0076] Plants also exhibit ROS induction during PCD, such as during osmotic stress-mediated death, the hypersensitive response and the terminal stages of tracheary element differentiation. In animal cells, the membrane bound NADPH oxidase complex leads to the generation of superoxide, which is then converted to other ROS. In addition, the small cytosolic protein rac2 is required for activation of the oxidase. When a constitutively active rac2 mutant was inserted into mice, a significant enhancement of PCD occurred compared to wild type mice. Biochemical and immunochemical studies have shown that NADPH oxidase and rac2 are present in plant cells and interact during hypersensitive response PCD. Furthermore, the NADPH oxidase is active during osmotic stress-mediated cell death and during the terminal phase of tracheary element differentiation. The gp 91 NADPH oxidase subunit has been cloned from rice and Arabidopsis. Polynucleotides encoding polypeptides relating to Rac2 (SEQ ID NOS: 28-35) and the gp 91 NADPH oxidase subunit (SEQ ID NO: 192) have been isolated from forestry species. The corresponding amino acid sequences for the Rac2-related polypeptides encoded by the polynucleotides given in SEQ ID NOS: 28 and 30-45 are given in SEQ ID NOS: 73 and 74-79, respectively. The corresponding amino acid sequence for the gp 91 NADPH oxidase subunit related polypeptide is given in SEQ ID NO: 196.

[0077] The role of superoxide compounds in plant cell death was illustrated with the discovery of the lesion simulating cell death (lsd1) mutant in Arabidopsis. In this mutant, superoxide was necessary and sufficient to induce and propagate cell death. Lsd1 in wild type plants is believed to serve as a monitor to a superoxide-dependent signal and to act as a negative regulator of a plant cell death pathway. Polynucleotides encoding lsd1-related polypeptides (SEQ ID NOS: 13 and 14) have been isolated from forestry species. Lsd1-related polypeptides encoded by the polynucleotides are identified as SEQ ID NOS: 58 and 59, respectively.

[0078] ATL2 was identified as an Arabidopsis cDNA which was toxic when overexpressed in yeast. The nucleotide sequences of five ATL2 variants isolated from forestry species are given in SEQ ID NOS: 1-5 and the corresponding amino acid sequences in SEQ ID NOS: 46-50, respectively.

[0079] Another gene, lls1, identified from a maize mutant, is also required to limit the spread of cell death in a developmental manner in leaves. Polynucleotides encoding lethal leaf spot protein lls1-related polypeptides (SEQ ID NOS: 11 and 12) have been isolated from forestry species. Polypeptides encoded by these polynucleotides are identified as SEQ ID NOS: 56 and -57, respectively.

[0080] Another plant protein from Arabidopsis (oxy5) has been shown to be a member of the annexin family of proteins and protect bacterial cells from oxidative stress. Oxy5 has also been shown to protect mammalian cells from tumor necrosis factor-induced cell death. The involvement of oxidative stress in the various instances of PCD in plants suggests that oxy5 plays a protective role. The annexin sequences show good homology to oxy5, and hence are expected to provide the same function or similar function. The nucleotide sequences of annexin-like proteins isolated from forestry species are given in SEQ ID NOS: 17-21 and the corresponding amino acid sequences in SEQ ID NOS: 62-66, respectively.

[0081] The most actively investigated example of PCD in plants concerns the hypersensitive response (HR) to pathogens. The HR is found in most responses mediated by disease resistance (R) genes. The HR is invoked by the association of a pathogen avirulence gene product with a receptor. This sets in motion a cascade of events involving ion fluxes, kinase/phosphatase actions and an oxidative burst leading to localized cell death and the induction of systemic acquired resistance (SAR), in which other parts of the plant develop an acquired resistance to the pathogen. A wide range of plant disease receptors have been identified, including polypeptides that span the cell membrane and contain an extracellular and cytoplasmic domain, as well as polypeptides that are strictly cytoplasmic and do not contain an extracellular domain.

[0082] Of the cytoplasmic polypeptide receptors involved in the HR, three families are of primary interest. The first is the RPS2-like polypeptide family, in which the polypeptides include an amino-terminal leucine zipper region, a nucleotide-binding site, an internal hydrophobic domain and a carboxy-terminal leucine-rich repeat. The second is the RPP5-like polypeptide family, in which the polypeptides include an amino-terminal Toll-like domain, a nucleotide-binding site, an internal hydrophobic domain and a carboxy-terminal leucine-rich repeat region. The nucleotide sequences of RPP5-like proteins isolated from forestry species are given in SEQ ID NOS:126-140, and the corresponding amino acid sequences in SEQ ID NOS: 177-191, respectively.

[0083] The third family of cytoplasmic receptors involved in the HR is the PTO-like family, in which the polypeptides include a serine-threonine kinase domain. The exact mechanisms by which the HR cell death signals are transduced are not known, although protein-protein interactions and kinase reactions have been shown to be involved in the PTO-like family, with several PTO-interacting protein genes identified.

[0084] Downstream of the initial avirulence/receptor interaction, the development of SAR occurs, which involves the NPR1 gene. Mutations in the NPR1 gene increase the susceptibility of plants to pathogen infection and prevent the development of HR PCD and SAR. The expression of R genes in transgenic plants has allowed the development of HR PCD and resistance to specific pathogens. In addition, the expression of PTO-like family members, such as Fen, can lead to PCD in the absence of a pathogen. Nucleotide sequences encoding NPR1-like proteins isolated from forestry species are given in SEQ ID NOS: 193-195 and the corresponding amino acid sequences in SEQ ID NOS: 197-199, respectively. The nucleotide sequence encoding a Fen-like protein isolated from forestry species is given in SEQ ID NO: 27, and the corresponding amino acid sequence in SEQ ID NO: 72. Little is known about the roles of these genes in other cases of plant PCD. An interesting point comes from the realisation that members of the plant R gene families and NPR1 show similarity to several proteins that are involved in animal development and defense. The discovery of a shared pathway linking developmental processes and disease resistance suggests that there may be roles for HR-associated genes in other plant PCD and developmental pathways.

[0085] The term “polynucleotide(s),” as used herein, means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and antisense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides. An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner. An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised. A polynucleotide may consist of an entire gene, or any portion thereof. A gene is a DNA sequence that codes for a functional protein or RNA molecule. Operable antisense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of “polynucleotide” therefore includes all such operable antisense fragments. Antisense polynucleotides and techniques involving antisense polynucleotides are well known in the art and are described, for example, in Robinson-Benion, et al., “Antisense techniques,” Methods in Enzymol. 254(23): 363-375 (1995); and Kawasaki, et al., Artific. Organs 20(8):836-848 (1996). Polynucleotides of the present invention also encompass polynucleotide sequences that differ from the disclosed sequences but which, as a result of the degeneracy of the genetic code, encode a polypeptide which is the same as that encoded by a DNA sequence disclosed herein.

[0086] The definitions of the terms “complement”, “reverse complement” and “reverse sequence”, as used herein, are best illustrated by the following examples. For the sequence 5′ AGGACC 3′, the complement, reverse complement and reverse sequences are as follows:

[0087] complement 3′ TCCTGG 5′

[0088] reverse complement 3′ GGTCCT 5′

[0089] reverse sequence 5′ CCAGGA 3′.

[0090] Identification of genomic DNA and heterologous species DNAs can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of a cDNA sequence as a probe to screen an appropriate library. Alternatively, PCR techniques using oligonucleotide primers that are designed based on known genomic DNA, cDNA and protein sequences can be used to amplify and identify genomic and cDNA sequences. Synthetic DNA corresponding to the identified sequences and variants may be produced by conventional synthesis methods. All of the polynucleotides described herein are isolated and purified, as those terms are commonly used in the art.

[0091] Some of the polynucleotides of the present invention may be “partial” sequences, in that they do not represent a full length gene encoding a full length polypeptide. Such partial sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well known hybridization and/or PCR techniques. Partial sequences may be extended until an open reading frame encoding a polypeptide, a full length polynucleotide and/or gene capable of expressing a polypeptide, or another useful portion of the genome is identified. Such extended sequences, including full length polynucleotides and genes, are described as “corresponding to” a sequence identified as one of the sequences of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, or a variant thereof, or a portion of one of the sequences of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, or a variant thereof, when the extended polynucleotide comprises an identified sequence or its variant, or an identified contiguous portion (x-mer) of one of the sequences of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, or a variant thereof. Similarly, RNA sequences, reverse sequences, complementary sequences, antisense sequences, and the like, corresponding to the polynucleotides of the present invention, may be routinely ascertained and obtained using the cDNA sequences identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248.

[0092] Certain of the polynucleotides identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248 contain open reading frames (“ORFs”) or partial open reading frames encoding polypeptides. Additionally, open reading frames encoding polypeptides may be identified in extended or full length sequences corresponding to the sequences set out as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248. Open reading frames may be identified using techniques that are well known in the art. These techniques include, for example, analysis for the location of known start and stop codons, most likely reading frame identification based on codon frequencies, etc. Tools and software suitable for ORF analysis include GeneWise (The Sanger Center, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom), Diogenes (Computational Biology Centers, University of Minnesota, Academic Health Center, UMHG Box 43 Minneapolis Minn. 55455), and GRAIL (Informatics Group, Oak Ridge National Laboratories, Oak Ridge, Tenn., Tenn.). Open reading frames and portions of open reading frames may be identified in the polynucleotides of the present invention. Once a partial open reading frame is identified, the polynucleotide may be extended in the area of the partial open reading frame using techniques that are well known in the art until the polynucleotide for the full open reading frame is identified. Thus, open reading frames encoding polypeptides may be identified using the polynucleotides of the present invention.

[0093] Several of the sequences provided in SEQ ID NO: 1-45, 90-140, 192-195, 200, 204 and 206-248 were found to be full-length and to contain open reading frames (ORFs). These full-length sequences, the location of ORFs (by nucleotide position) contained within these sequences, and the corresponding amino acid sequences are provided in Table 3 below. 3 TABLE 3 Polynucleotide Polypeptide SEQ ID NO: ORF SEQ ID NO: 17  95-1045 62 22 97-924 67 28 109-699  73 90 80-913 141 92  69-1163 143 98  68-1504 149 101 109-1146 152 102  70-1191 153 107  58-1137 158 118  13-1113 169 200  24-1001 201 204 23-733 205 207 269-1021 249 209 97-996 251 210 68-415 252 211 107-454  253 212 69-416 254 213 17-466 255 215 308-829  257 216 212-754  258 217  74-1744 259 218  72-1022 260 221 508-1608 263 223 197-787  265 229 286-888  271 230 261-863  272 231 96-859 273 232 123-866  274 234 213-1571 276 235 172-1554 277 236  84-1496 278 237  35-1600 279 239  38-1150 281 240  68-1105 282 241 264-1337 283 243 506-1627 285 244  1-1663 286 245  1-710 287 246  1-473 288 247 125-423  289 248 15-781 290

[0094] Once open reading frames are identified in the polynucleotides of the present invention, the open reading frames may be isolated and/or synthesized. Expressible genetic constructs comprising the open reading frames and suitable promoters, initiators, terminators, etc., which are well known in the art, may then be constructed. Such genetic constructs may be introduced into a host cell to express the polypeptide encoded by the open reading frame. Suitable host cells may include various prokaryotic and eukaryotic cells, including plant cells, mammalian cells, bacterial cells, algae and the like. In another aspect, the present invention provides isolated polypeptides encoded, or partially encoded, by the above polynucleotides. As used herein, the term “polypeptide” encompasses amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds. The term “polypeptide encoded by a polynucleotide” as used herein, includes polypeptides encoded by a polynucleotide which comprises an isolated DNA sequence or variant provided herein. In specific embodiments, the inventive polypeptides comprise an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290, as well as variants of such sequences.

[0095] Polypeptides of the present invention may be produced recombinantly by inserting a DNA sequence that encodes the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, insect, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner may encode naturally occurring polypeptides, portions of naturally occurring polypeptides, or other variants thereof.

[0096] In a related aspect, polypeptides are provided that comprise at least a functional portion of a polypeptide having an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290, and variants thereof. As used herein, the “functional portion” of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the portion of the molecule that is capable of binding one or more reactants. A functional portion comprising an active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high binding affinity.

[0097] Functional portions of a polypeptide may be identified by first preparing fragments of the polypeptide by either chemical or enzymatic digestion of the polypeptide, or by mutation analysis of the polynucleotide that encodes the polypeptide and subsequent expression of the resulting mutant polypeptides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain biological activity, using, for example, the representative assays provided below.

[0098] The term “polypeptide encoded by a polynucleotide” as used herein, includes polypeptides encoded by a polynucleotide comprising a partial isolated polynucleotide of the present invention.

[0099] Portions and other variants of the inventive polypeptides may also be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied Biosystems, Inc. (Foster City, Calif.), and may be operated according to the manufacturer's instructions. Variants of a native polypeptide may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492, 1985). Sections of DNA sequences may also be removed using standard techniques to permit preparation of truncated polypeptides.

[0100] In general, the polypeptides disclosed herein are prepared in an isolated, substantially pure form. Preferably, the polypeptides are at least about 80% pure; more preferably at least about 90% pure; and most preferably, at least about 99% pure. In certain preferred embodiments, described in detail below, the isolated polypeptides are incorporated into pharmaceutical compositions or vaccines for use in the treatment of skin disorders.

[0101] As used herein, the term “variant” comprehends nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 75%; more preferably, at least 90%; and most preferably, at least 95% or 98% identity to a sequence of the present invention. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.

[0102] Polynucleotide and polypeptide sequences may be aligned, and percentage of identical nucleotides in a specified region may be determined against another polynucleotide, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. Polynucleotides may also be analyzed using the BLASTX algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The similarity of polypeptide sequences may be examined using the BLASTP algorithm. The BLASTN, BLASTX and BLASTP programs are available on the NCBI anonymous FTP server. The BLASTN algorithm version 2.0.4 [Feb. 24, 1998] and version 2.0.6 [Sep. 16, 1998], set to the default parameters described in the documentation and distributed with the algorithm, are preferred for use in the determination of polynucleotide variants according to the present invention. The BLASTP algorithm, set to the default parameters described in the documentation and distributed with the program, is preferred for use in the determination of polypeptide variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described at NCBI's website and in the publication of Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25: 3389-3402, 1997.

[0103] The computer algorithm FASTA is available on the Internet and from the University of Virginia by contacting David Hudson, Assistant Provost for Research, University of Virginia, PO Box 9025, Charlottesville, Va. 22906-9025 USA. Version 2.0u4, February 1996, set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of variants according to the present invention. The use of the FASTA algorithm is described in Pearson, W R and Lipman, D J, “Improved Tools for Biological Sequence Analysis,” Proc. Natl. Acad. Sci. USA 85: 2444-2448, 1988; and W. R. Pearson, “Rapid and Sensitive Sequence Comparison with FASTP and FASTA,” Methods in Enzymology 183: 63-98, 1990.

[0104] The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotide sequences: Unix running command: blastall -p blastn -d embldb -e 10 -G0 -E0 -r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (blastn only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [File In]; and -o BLAST report Output File [File Out] Optional. The following running parameters are preferred for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity of polypeptide sequences: blastall -p blastp -d swissprotdb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional. The “hits” to one or more database sequences by a queried sequence produced by BLASTN, FASTA, BLASTP or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.

[0105] The BLASTN, FASTA, and BLASTP algorithms also produce “Expect” (E) values for alignments. The Expect value (E) indicates the number of hits one can “expect” to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. By this criterion, the aligned and matched portions of the polynucleotide sequences then have a probability of 90% of being the same. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.

[0106] According to one embodiment, “variant” polynucleotides and polypeptides, with reference to each of the polynucleotides and polypeptides of the present invention, preferably comprise sequences having the same number or fewer nucleic or amino acids than each of the polynucleotides or polypeptides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99% probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described above. According to a preferred embodiment, a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at parameters described above. Similarly, according to a preferred embodiment, a variant polypeptide is a sequence having the same number or fewer amino acids than a polypeptide of the present invention that has at least a 99% probability of being the same as a polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the parameters described above.

[0107] Polynucleotides having a specified degree of identity to, or capable of hybridizing to, a polynucleotide of the present invention preferably additionally have at least one of the following features: (1) they contain an open reading frame or partial open reading frame encoding a polypeptide, or a functional portion of a polypeptide, having substantially the same functional properties as the polypeptide, or functional portion thereof, encoded by a polynucleotide in a recited SEQ ID NO.; or (2) they contain identifiable domains in common. Similarly, polypeptides, or functional portions of polypeptides, having a specified degree of identity to a polypeptide of the present invention shares a high degree of identity in their primary structure and have substantially similar functional properties.

[0108] As noted above, the percentage identity is determined by aligning sequences using one of the BLASTN, FASTA, or BLASTP algorithms, set at the running parameters described above, and identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percentage identity. For example, a polynucleotide of the present invention having 220 nucleic acids has a hit to a polynucleotide sequence in the EMBL database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the parameters described above. The 23 nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide. The percentage identity of the polynucleotide of the present invention to the hit in the EMBL library is thus 21/220 times 100, or 9.5%. The polynucleotide sequence in the EMBL database is thus not a variant of a polynucleotide of the present invention.

[0109] Alternatively, variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, or complements, reverse sequences, or reverse complements of those sequences under stringent conditions. As used herein, “stringent conditions” refers to prewashing in a solution of 6× SSC, 0.2% SDS; hybridizing at 65° C., 6× SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1× SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2× SSC, 0.1% SDS at 65° C.

[0110] The present invention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the discrepancy of the genetic code, encode a polypeptide having similar enzymatic activity as a polypeptide encoded by a polynucleotide of the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, or complements, reverse sequences, or reverse complements of those sequences as a result of conservative substitutions are contemplated by and encompassed within the present invention. Additionally, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, or complements, reverse complements, or reverse sequences as a result of deletions and/or insertions totaling less than 10% of the total sequence length are also contemplated by and encompassed within the present invention. Similarly, polypeptides comprising sequences that differ from the polypeptide sequences recited in SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290 as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by an encompassed within the present invention, provided the variant polypeptide has activity in a PCD or plant developmental pathway.

[0111] The polynucleotides of the present invention may be isolated from various libraries, or may be synthesized using techniques that are well known in the art. The polynucleotides may be synthesized, for example, using automated oligonucleotide synthesizers (e.g., Beckman Oligo 1000M DNA Synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids. A plurality of such polynucleotide segments may then be ligated using standard DNA manipulation techniques that are well known in the art of molecular biology. One conventional and exemplary polynucleotide synthesis technique involves synthesis of a single stranded polynucleotide segment having, for example, 80 nucleic acids, and hybridizing that segment to a synthesized complementary 85 nucleic acid segment to produce a 5 nucleotide overhang. The next segment may then be synthesized in a similar fashion, with a 5 nucleotide overhang on the opposite strand. The “sticky” ends ensure proper ligation when the two portions are hybridized. In this way, a complete polynucleotide of the present invention may be synthesized entirely in vitro.

[0112] Some of the polynucleotides identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, are referred to as “partial” sequences, in that they may not represent the full coding portion of a gene encoding a naturally occurring polypeptide. The partial polynucleotide sequences disclosed herein may be employed to obtain the corresponding full length genes for various species and organisms by, for example, screening DNA expression libraries using hybridization probes based on the polynucleotides of the present invention, or using PCR amplification with primers based upon the polynucleotides of the present invention. In this way one can, using methods well known in the art, extend a polynucleotide of the present invention upstream and downstream of the corresponding mRNA, as well as identify the corresponding genomic DNA, including the promoter and enhancer regions, of the complete gene. The present invention thus comprehends isolated polynucleotides comprising a sequence identified in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, or a variant of one of the specified sequences, that encode a functional polypeptide, including full-length genes. Such extended polynucleotides may have a length of from about 50 to about 4,000 nucleic acids or base pairs, and preferably have a length of less than about 4,000 nucleic acids or base pairs, more preferably yet a length of less than about 3,000 nucleic acids or base pairs, more preferably yet a length of less than about 2,000 nucleic acids or base pairs. Under some circumstances, extended polynucleotides of the present invention may have a length of less than about 1,800 nucleic acids or base pairs, preferably less than about 1,600 nucleic acids or base pairs, more preferably less than about 1,400 nucleic acids or base pairs, more preferably yet less than about 1,200 nucleic acids or base pairs, and most preferably less than about 1,000 nucleic acids or base pairs.

[0113] Polynucleotides of the present invention also comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NO: 1-45, 90-140, 192-195, 200, 204 and 206-248, complements, reverse sequences, and reverse complements of such sequences, and their variants. Similarly, polypeptides of the present invention comprehend polypeptides comprising at least a specified number of contiguous residues (x-mers) of any of the polypeptides identified as SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290, and their variants. As used herein, the term “x-mer,” with reference to a specific value of “x,” refers to a sequence comprising at least a specified number (“x”) of contiguous residues of any of the polynucleotides identified as SEQ ID NO: 1-45, 90-140, 192-195, 200, 204 and 206-248, or the polypeptides identified as SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290. According to preferred embodiments, the value of x is preferably at least 20; more preferably, at least 40; more preferably yet, at least 60; and most preferably, at least 80. Thus, polynucleotides and polypeptides of the present invention comprise a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250-mer, a 300-mer, a 400-mer, a 500-mer or a 600-mer of a polynucleotide or polypeptide identified as SEQ ID NOS: 1-201 and 204-290, and variants thereof.

[0114] Oligonucleotide probes and primers complementary to and/or corresponding to SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248, and variants of those sequences, are also comprehended by the present invention. Such oligonucleotide probes and primers are substantially complementary to the polynucleotide of interest. As used herein, the term “oligonucleotide” refers to a relatively short segment of a polynucleotide sequence, generally comprising between 6 and 60 nucleotides, and comprehends both probes for use in hybridization assays and primers for use in the amplification of DNA by polymerase chain reaction.

[0115] An oligonucleotide probe or primer is described as “corresponding to” a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248 or a variant thereof, if the oligonucleotide probe or primer, or its complement, is contained within one of the sequences set out as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248 or a variant of one of the specified sequences.

[0116] Two single stranded sequences are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared, with the appropriate nucleotide insertions and/or deletions, pair with at least 80%, preferably at least 90% to 95%, and more preferably at least 98% to 100%, of the nucleotides of the other strand. Alternatively, substantial complementarity exists when a first DNA strand will selectively hybridize to a second DNA strand under stringent hybridization conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM, and preferably less than about 200 mM. Hybridization temperatures can be as low as 5° C., but are generally greater than about 22° C., more preferably greater than about 30° C., and most preferably greater than about 37° C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. The DNA from plants, or samples or products containing plant material, can be either genomic DNA or DNA derived by preparing cDNA from the RNA present in the sample.

[0117] In addition to DNA-DNA hybridization, DNA-RNA or RNA-RNA hybridization assays are also possible. In the first case, the mRNA from expressed genes would then be detected instead of genomic DNA or cDNA derived from mRNA of the sample. In the second case, RNA probes could be used. In addition, artificial analogs of DNA hybridizing specifically to target sequences could also be used.

[0118] In specific embodiments, the oligonucleotide probes and/or primers comprise at least about 6 contiguous residues, more preferably at least about 10 contiguous residues, and most preferably at least about 20 contiguous residues complementary to a polynucleotide sequence of the present invention. Probes and primers of the present invention may be from about 8 to 100 base pairs in length or, preferably from about 10 to 50 base pairs in length or, more preferably from about 15 to 40 base pairs in length. The probes can be easily selected using procedures well known in the art, taking into account DNA-DNA hybridization stringencies, annealing and melting temperatures, and potential for formation of loops and other factors, which are well known in the art. Tools and software suitable for designing probes, and especially suitable for designing PCR primers, are available on the Internet, for example. Preferred techniques for designing PCR primers are also disclosed in Dieffenbach, C W and Dyksler, G S, PCR Primer: a laboratory manual, CSHL Press: Cold Spring Harbor, N.Y., 1995. A software program suitable for designing probes, and especially for designing PCR primers, is available from Premier Biosoft International, 3786 Corina Way, Palo Alto, Calif. 94303-4504.

[0119] A plurality of oligonucleotide probes or primers corresponding to a polynucleotide of the present invention may be provided in a kit form. Such kits generally comprise multiple DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence. Kits of the present invention may comprise one or more probes or primers corresponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248.

[0120] In one embodiment useful for high-throughput assays, the oligonucleotide probe kits of the present invention comprise multiple probes in an array format, wherein each probe is immobilized at a predefined, spatially addressable location on the surface of a solid substrate. Array formats which may be usefully employed in the present invention are disclosed, for example, in U.S. Pat. Nos. 5,412,087, 5,545,451, and PCT Publication No. WO 95/00450, the disclosures of which are hereby incorporated by reference.

[0121] Probes, preferably in the form of an array, may be employed to screen for differences in organisms or samples or products containing genetic material using high-throughput screening techniques that are well known in the art. The significance of using probes in high-throughput screening systems is apparent for applications such as plant breeding and quality control operations in which there is a need to identify large numbers of seed lots and plant seedlings, to examine samples or products for unwanted plant materials, to identify plants or samples or products containing plant material for quarantine purposes, etc., or to ascertain the true origin of plants or samples or products containing plant material. Screening for the presence or absence of polynucleotides of the present invention used as identifiers for tagging plants is valuable for later detecting the amount of gene flow in plant breeding, introgression of genes via dispersed pollen, etc.

[0122] In this manner, oligonucleotide probe kits of the present invention may be employed to examine the presence/absence (or relative amounts in case of mixtures) of polynucleotides in different samples or products containing different materials rapidly and in a cost-effective manner. Examples of plant species, which may be examined using the present invention include forestry species, such as pine and eucalyptus species, other tree species, and even agricultural and horticultural plants.

[0123] For applications where modulation of PCD and/or a developmental pathway is desired, an open reading frame encoding a polypeptide of the present invention may be inserted into a genetic construct in a sense or antisense orientation, such that transformation of a plant with the genetic construct produces a change in the copy number of a polynucleotide or the expression level of a polypeptide compared to the polynucleotide copy number and/or polypeptide expression level in a wild-type organism. Transformation with a genetic construct comprising an open reading frame in a sense orientation will generally result in an increased expression level of the polypeptide encoded by the selected polynucleotide, while transformation with a genetic construct comprising an open reading frame in an antisense orientation will generally result in reduced expression of the polypeptide encoded by the selected polynucleotide. A plant transformed with a genetic construct comprising an open reading frame of the present invention in either a sense or antisense orientation may be screened for increased or reduced copy numbers of the selected polynucleotide, or increased or reduced expression of the polypeptide of interest using techniques well known to those of skill in the art. Plants having the desired alterations may thus be identified and isolated. In general, an increase or reduction in the expression level of a polypeptide of interest of at least 25%, compared to expression levels in a corresponding wild-type organism, is significant.

[0124] Transformation of a target organism with a genetic construct of the present invention results in a modification in the polypeptide synthesis or content or structure in the target organism, thereby producing a modification from the wild-type plant in the area of PCD or in a developmental pathway. Methods of the present invention involve modulating, generally promoting or inhibiting, PCD in a plant species. For example, transformation of a target organism with a genetic construct having an open reading frame coding for a polypeptide encoded by a polynucleotide of the present invention wherein the open reading frame is in a sense orientation and the polypeptide contributes to an increased level of PCD, generally produces a significant increase in the amount or expression of the polypeptide in the target organism and, consequently, a significant increase in PCD. Similarly, transformation of a target organism with a genetic construct having an open reading frame coding for a polypeptide encoded by a polynucleotide of the present invention wherein the open reading frame is in a sense orientation and the polypeptide contributes to a reduced level of PCD, generally produces a significant reduction in the amount or expression of the polypeptide in the target organism and, consequently, a significant reduction in PCD. Transformation of a target organism with a genetic construct comprising an open reading frame in an antisense orientation or an untranslated region of a polynucleotide generally produces a decrease in the level of the corresponding polypeptide, thereby producing a corresponding increase or reduction in PCD, depending on the role of the specific polypeptide. It will be recognized that transformation with other genetic constructs of the present invention will produce changes in the content, composition and/or metabolism of various polypeptides that play a role in PCD and/or plant developmental pathways, thereby producing changes in the content, composition, and/or metabolism of the plant.

[0125] Expression of a polynucleotide involved in PCD or a selected developmental pathway may be inhibited by inserting a portion of an open reading frame of the present invention, in either sense or antisense orientation, in the genetic construct. Such portions need not be full-length but preferably comprise at least 25 and more preferably at least 50 residues of a polynucleotide of the present invention. A much longer portion, or even the full length polynucleotide corresponding to the complete open reading frame, may be employed. The portion of the open reading frame does not need to be precisely the same as the endogenous sequence, provided that there is sufficient sequence similarity to achieve inhibition of the target polynucleotide. Thus a sequence derived from one species may be used to inhibit expression of a polypeptide in a different species.

[0126] According to another embodiment, the genetic constructs of the present invention comprise a polynucleotide including a non-coding, or untranslated, region of a polynucleotide coding for a polypeptide encoded by a polynucleotide of the present invention, or a polynucleotide complementary to such a non-coding region. As used herein the term “non-coding region” includes both transcribed sequences which are not translated, and non-transcribed sequences within about 2000 base pairs 5′ or 3′ of the translated sequences or open reading frames. Examples of non-coding regions which may be usefully employed in the inventive constructs include introns and 5′-non-coding leader sequences. Transformation of a target plant with such a DNA construct may lead to a reduction in the amount of lignin synthesized by the plant by the process of co-suppression, in a manner similar to that discussed, for example, by Napoli et al., Plant Cell 2:279-290, 1990; and de Carvalho Niebel et al., Plant Cell 7:347-358, 1995.

[0127] Alternatively, regulation may be achieved by inserting appropriate sequences or subsequences (e.g. DNA or RNA) in ribozyme constructs (McIntyre C L, Manners J M, “Strategies for the suppression of peroxidase gene expression in tobacco: designing efficient ribozymes,” Transgenic Res. 5(4): 257-262, 1966). Ribozymes are synthetic RNA molecules that comprise a hybridizing region complementary to two regions, each of which comprises at least 5 contiguous nucleotides in a mRNA molecule encoded by one of the inventive polynucleotides. Ribozymes possess highly specific endonuclease activity, which autocatalytically cleaves the mRNA.

[0128] The genetic constructs of the present invention may further comprise a gene promoter sequence and a gene termination sequence, operably linked to the polynucleotide and capable of controlling expression of the polypeptide. The gene promoter sequence is generally positioned at the 5′ end of a polynucleotide to be transcribed, and is employed to initiate transcription of the polynucleotide. Gene promoter sequences are generally found in the 5′ non-coding region of a gene but they may exist in introns (Luehrsen K R, Mol. Gen. Genet. 225:81-93, 1991, or in the coding region, as for example in PAL of tomato (Bloksberg, Studies on the Biology of Phenylalanine Ammonia Lyase and Plant Pathogen Interaction, Ph.D. Thesis, University of California, Davis, 1991, University Microfilms International Order No. 9217564). When the construct includes an open reading frame in a sense orientation, the gene promoter sequence also initiates translation of the open reading frame. For genetic constructs comprising either an open reading frame in an antisense orientation or a non-coding region, the gene promoter sequence consists only of a transcription initiation site having a RNA polymerase binding site.

[0129] Numerous gene promoter sequences that may be usefully employed in genetic constructs of the present invention are well known in the art. The gene promoter sequence, and also the gene termination sequence, may be endogenous to the target host or may be exogenous, provided the promoter is functional in the target host. For example, the promoter and termination sequences used when the target organism is a plant, may be from other plant species, plant viruses, bacterial plasmids and the like. In preferred embodiments, the gene promoter and termination sequences are common to those of the polynucleotide being introduced.

[0130] Factors influencing the choice of promoter include the desired tissue specificity of the construct, and the timing of transcription and translation. For example, constitutive promoters, such as the 35S Cauliflower Mosaic Virus (CaMV 35S) promoter with or without enhancers, such as the Kozak sequence or the Omega enhancer, and Agrobacterium tumefaciens nopaline synthase terminator, may be usefully employed in the present invention. Use of a tissue specific promoter will result in production of the desired sense or antisense RNA only in the tissue of interest. With genetic constructs employing inducible gene promoter sequences, the rate of RNA polymerase binding and initiation can be modulated by external stimuli, such as light, heat, anaerobic stress, alteration in nutrient conditions and the like. Temporally regulated promoters can be employed to effect modulation of the rate of RNA polymerase binding and initiation at a specific time during development of a transformed cell. Preferably, the original promoters from the gene in question, or promoters from a specific tissue-targeted gene in the organism to be transformed, such as eucalyptus or pine are used. Other examples of gene promoters which may be usefully employed in the present invention include mannopine synthase (mas), octopine synthase (ocs) and those reviewed by Chua et al. (Science 244: 174-181, 1989).

[0131] The gene termination sequence, which is located 3′ to the polynucleotide to be transcribed, may come from the same gene as the gene promoter sequence or may be from a different gene. Many gene termination sequences known in the art may be usefully employed in the present invention, such as the 3′ end of the Agrobacterium tumefaciens nopaline synthase gene. However, preferred gene terminator sequences are those from the original polynucleotide, or from the target species to be transformed.

[0132] The genetic constructs of the present invention may also contain a selection marker that is effective in target cells, such as plant cells, to facilitate the detection of transformed cells containing the genetic construct. Such markers, which are well known in the art, typically confer resistance to one or more toxins. One example of such a marker is the NPTII gene, whose expression results in resistance to kanamycin or hygromycin, antibiotics which are usually toxic to plant cells at a moderate concentration (Rogers et al. in Weissbach, A and Weissbach, H, eds., Methods for Plant Molecular Biology, Academic Press: San Diego, Calif., 1988). Transformed cells can thus be identified by their ability to grow in media containing the antibiotic in question. Alternatively, the presence of the desired construct in transformed cells can be determined by means of other techniques that are well known in the art, such as Southern and Western blots.

[0133] Techniques for operatively linking the components of the genetic constructs of the present invention are well known in the art and include the use of synthetic linkers containing one or more restriction endonuclease sites as described, for example, by Sambrook, et al., Molecular Cloning: A Laboratory Manual, CSHL: Cold Spring Harbor, N.Y., 1989). Genetic constructs of the present invention may be linked to a vector having at least one replication system, for example E. coli, whereby after each manipulation, the resulting construct can be cloned and sequenced, and the correctness of the manipulation determined.

[0134] The genetic constructs of the present invention may be used to transform a variety of plants, both monocotyledonous (e.g., grasses, corn, grains, oat, wheat and barley), dicotyledonous (e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple), and Gymnosperms (e.g., Scots pine; see Aronen, Finnish Forest Res. Papers, Vol. 595, 1996), white spruce (Ellis et al., Biotechnology 11:94-92, 1993), and larch (Huang et al., In Vitro Cell 27:201-207, 1991). In a preferred embodiment, the inventive genetic constructs are employed to transform woody plants, herein defined as a tree or shrub whose stem lives for a number of years and increases in diameter each year by the addition of woody tissue. Preferably the target plant is selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. Other species which may be usefully transformed with the DNA constructs of the present invention include, but are not limited to, any of the species in the following list:

[0135] Pines: Pinus banksiana, Pinus brutia, Pinus caribaea, Pinus clausa, Pinus contorta, Pinus coulteri, Pinus echinata, Pinus eldarica, Pinus ellioti, Pinus jeffreyi, Pinus lambertiana, Pinus monticola, Pinus nigra, Pinus palustrus, Pinus pinaster, Pinus ponderosa, Pinus resinosa, Pinus rigida, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, Pinus virginiana.

[0136] Other gymnosperms: Abies amabilis, Abies balsamea, Abies concolor, Abies grandis, Abies lasiocarpa, Abies magnifica, Abies procera, Chamaecyparis lawsoniona, Chamaecyparis nootkatensis, Chamaecyparis thyoides, Juniperus virginiana, Larix decidua, Larix laricina, Larix leptolepis, Larix occidentalis, Larix siberica, Libocedrus decurrens, Picea abies, Picea engelmanni, Picea glauca, Picea mariana, Picea pungens, Picea rubens, Picea sitchensis, Pseudotsuga menziesii, Sequoia gigantea, Sequoia sempervirens, Taxodium distichum, Tsuga canadensis, Tsuga heterophylla, Tsuga mertensiana, Thuja occidentalis, Thuja plicata.

[0137] Eucalypts: Eucalyptus alba, Eucalyptus bancroftii, Eucalyptus botyroides, Eucalyptus bridgesiana, Eucalyptus calophylla, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus coccifera, Eucalyptus curtisii, Eucalyptus dalrympleana, Eucalyptus deglupta, Eucalyptus delagatensis, Eucalyptus diversicolor, Eucalyptus dunnii, Eucalyptus ficifolia, Eucalyptus globulus, Eucalyptus gomphocephala, Eucalyptus gunnii, Eucalyptus henryi, Eucalyptus laevopinea, Eucalyptus macarthurii, Eucalyptus macrorhyncha, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus megacarpa, Eucalyptus melliodora, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus nova-anglica, Eucalyptus obliqua, Eucalyptus obtusiflora, Eucalyptus oreades, Eucalyptus pauciflora, Eucalyptus polybractea, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus robusta, Eucalyptus rudis, Eucalyptus saligna, Eucalyptus sideroxylon, Eucalyptus stuartiana, Eucalyptus tereticornis, Eucalyptus torelliana, Eucalyptus urnigera, Eucalyptus urophylla, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo, Eucalyptus youmanni; and hybrids of any of the above species. As discussed above, transformation of a plant with a genetic construct including an open reading frame coding for an enzyme encoded by an inventive polynucleotide wherein the open reading frame is orientated in a sense direction will produce a modified PCD or developmental pathway in the plant. Transformation of a plant with a genetic construct comprising an open reading frame in an antisense orientation or a non-coding (untranslated) region of a gene will also produced a modification in PCD or a developmental pathway of the transformed plant.

[0138] Polynucleotides of the present invention may also be used to specifically suppress gene expression by methods that operate post-transcriptionally to block the synthesis of products of targeted genes, such as RNA interference (RNAi) and quelling. Briefly, traditional methods of gene suppression, employing anti-sense RNA or DNA, operate by binding to the reverse sequence of a gene of interest such that binding interferes with subsequent cellular processes and therefore blocks synthesis of the corresponding protein. RNAi also operates on a post-translational level and is sequence specific, but suppresses gene expression far more efficiently. Exemplary methods for controlling or modifying gene expression using RNAi are provided in WO 99/49029 and WO 99/53050. In these methods, post-transcriptional gene silencing is brought about by a sequence-specific RNA degradation process which results in the rapid degradation of transcripts of sequence-related genes. Studies have shown that double-stranded RNA may act as a mediator of sequence-specific gene silencing (see, for example, Montgomery and Fire, Trends in Genetics, 14:255-258, 1998). Gene constructs that produce transcripts with self-complementary regions are particularly efficient at gene silencing. A unique feature of this post-transcriptional gene silencing pathway is that silencing is not limited to the cells where it is initiated. The gene-silencing effects may be disseminated to other parts of an organism and even transmitted through the germ line to several generations.

[0139] The polynucleotides of the present invention may thus be employed to generate gene silencing constructs and/or gene-specific self-complementary RNA sequences that can be delivered by conventional art-known methods to plant tissues, such as forage grass tissues. Within genetic constructs, sense and antisense sequences can be placed in regions flanking an intron sequence in proper splicing orientation with donor and acceptor splicing sites, such that intron sequences are removed during processing of the transcript and sense and antisense sequences, as well as splice junction sequences, bind together to form double-stranded RNA. Alternatively, spacer sequences of various lengths may be employed to separate self-complementary regions of sequence in the construct. During processing of the gene construct transcript, intron sequences are spliced-out, allowing sense and anti-sense sequences, as well as splice junction sequences, to bind forming double-stranded RNA. Select ribonucleases then bind to and cleave the double-stranded RNA, thereby initiating the cascade of events leading to degradation of specific mRNA gene sequences, and silencing specific genes. Alternatively, rather than using a gene construct to express the self-complementary RNA sequences, the gene-specific double-stranded RNA segments are delivered to one or more targeted areas to be internalized into the cell cytoplasm to exert a gene silencing effect. The double-stranded RNA must have sufficient homology to the targeted gene to mediate RNAi and is preferably at least 25 nucleotides in length. Preferably, the double-stranded RNA corresponds specifically to a polynucleotide of the present invention. Gene silencing RNA sequences comprising the polynucleotides of the present invention are useful for creating genetically modified plants with desired phenotypes as well as for characterizing genes (for example, in high-throughput screening of sequences), and studying their functions in intact organisms.

[0140] The production of RNA in target cells may be controlled by choice of the promoter sequence, or by selecting the number of functional copies or the site of integration of the polynucleotides incorporated into the genome of the target organism. A target plant may be transformed with more than one construct of the present invention, thereby modulating the lignin biosynthetic pathway for the activity of more than one enzyme, affecting enzyme activity in more than one tissue or affecting enzyme activity at more than one expression time. Similarly, a construct may be assembled containing more than one open reading frame coding for an enzyme encoded by a polynucleotide of the present invention or more than one non-coding region of a gene coding for such an enzyme. The polynucleotides of the present invention may also be employed in combination with other known sequences encoding enzymes involved in PCD or developmental pathways.

[0141] Techniques for stably incorporating genetic constructs into the genome of specific target organisms are well known in the art. Techniques that are suitable for transforming plants include Agrobacterium tumefaciens mediated introduction, electroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction and the like. The choice of technique will depend upon the target plant to be transformed. For example, dicotyledonous plants and certain monocots and gymnosperms may be transformed by Agrobacterium Ti plasmid technology, as described, for example by Bevan, Nucl. Acid Res. 12:8711-8721, 1984. Targets for the introduction of the genetic constructs of the present invention include tissues, such as leaf tissue, dissociated cells, protoplasts, seeds, embryos, meristematic regions, cotyledons, hypocotyls, and the like. One preferred method for transforming eucalyptus and pine is a biolistic method using pollen (see, for example, Aronen, Finnish Forest Res. Papers, Vol. 595:53, 1996) or easily regenerable embryonic tissues. Other transformation techniques which may be usefully employed in the inventive methods include those taught by Ellis et al., (Plant Cell Reports, 8:16-20, 1989), Wilson et al., (Plant Cell Reports 7:704-707, 1989) and Tautorus et al., (Theor. Appl. Genet. 78:531-536, 1989).

[0142] Target cells having non-native genetic constructs incorporated in their genome may be selected by means of a marker, such as the kanamycin resistance marker discussed above. Transgenic cells may then be cultured in an appropriate medium to regenerate whole plants, using techniques that are well known in the art. In the case of protoplasts, the cell wall is allowed to reform under appropriate osmotic conditions. In the case of seeds or embryos, an appropriate germination or callus initiation medium is employed. For explants, an appropriate regeneration medium is used. Regeneration of plants is well established for many species. For a review of regeneration of forest trees see Dunstan et al., “Somatic embryogenesis in woody plants,” pp. 471-450 in Thorpe, T A, ed., “In vitro embryogenesis of plants,” (Vol. 20 of Current Plant Science and Biotechnology in Agriculture). Specific protocols for the regeneration of spruce are discussed by Roberts et al., “Somatic Embryogenesis of Spruce,” pp. 427-449, in Redenbaugh, K, ed., Synseed: applications of synthetic seed to crop improvement, CRC Press, 1993). The resulting transformed plants may be reproduced sexually or asexually, using methods well known in the art, to give successive generations of transgenic plants.

[0143] As discussed above, the production of RNA in target plant cells may be controlled by the choice of an appropriate promoter sequence, or by selecting the number of functional opies or the site of integration of the polynucleotides incorporated into the genome of the target host. A target host organism may be transformed with more than one genetic constructs of the present invention, thereby modulating the concentration or activity of more than one polypeptide, affecting more than one tissue, or affecting more than one expression time. Similarly, a genetic construct may be assembled containing more than one open reading frame encoded by a polynucleotide of the present invention or more than one untranslated region of a polynucleotide. The polynucleotides of the present inventive may also be employed in combination with other known sequences encoding various polypeptides.

[0144] Additionally, the polynucleotides of the present invention may be used as non-disruptive tags for marking organisms, particularly plants. Genetic constructs comprising polynucleotides of the present invention may be stably introduced into an organism as heterologous, non-functional, non-disruptive tags. It is then possible to identify the origin or source of the organism at a later date by determining the presence or absence of the tag(s) in a sample of material. Organisms other than plants may also be tagged with the polynucleotides of the present invention, including commercially valuable animals, fish, bacteria and yeasts.

[0145] Detection of the tag(s) may be accomplished using a variety of conventional techniques, and generally involves the use of nucleic acid probes. Sensitivity in assaying for the presence of probe may be usefully increased by using branched oligonucleotides, as described by Horn, T, Chang, C A, and Urdea, M S, “Chemical synthesis and characterization of branched oligo-deoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays,” Nucleic Acids Res. 25(23): 4842-4849, 1997, enabling detection of as few as 50 DNA molecules in the sample.

[0146] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

Isolation and Characterization of cDNA Clones from Pinus radiata and Eucalyptus grandis

[0147] Pinus radiata and Eucalyptus grandis cDNA libraries were constructed using non-subtracted or subtracted methods and screened as follows. Total RNA was extracted from the plant tissue using the protocol of Chang et al., Plant Molecular Biology Reporter 11:113-116, 1993. mRNA was isolated from the total RNA preparation using a Poly(A) Quik mRNA Isolation Kit (Stratagene, La Jolla, Calif.) or Dynal Oligo (dT)25 Beads (Dynal, Skogen, Norway). Non-subtracted cDNA libraries were constructed from the purified mRNA by reverse transcriptase synthesis followed by insertion of the resulting cDNA clones in Lambda ZAP using a ZAP Express cDNA Synthesis Kit (Stratagene, La Jolla Calif.) or a SuperScript Choice System (Gibco BRL Life Technologies, Gaithersburg Md.), according to the manufacturer's protocol. The resulting cDNAs were packaged using a Gigapack II Packaging Extract (Stratagene) employing 1 &mgr;l of sample DNA from the 5 &mgr;l ligation mix. Mass excision of the library was done using XL1-Blue MRF' cells and XLOLR cells (Stratagene) with ExAssist helper phage (Stratagene). The excised phagemids were diluted with NZY broth (Gibco BRL, Gaithersburg, Md.) and plated out onto LB-kanamycin agar plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) and isopropylthio-beta-galactoside (IPTG).

[0148] Subtracted Pinus radiata cDNA libraries (developed using early wood xylem [tester DNA] subtracted against late wood xylem [driver DNA] or late wood xylem [tester DNA] subtracted against early wood xylem [driver DNA] were constructed as follows. mRNA isolated using Dynal Beads (see above) was used to generate cDNA using manufacturer's instructions from the Clontech PCR-Select™ cDNA Subtraction Kit (Clontech Laboratories Inc, Palo Alto, Calif.). Both the tester and driver double-stranded cDNA preparations were digested with restriction endonuclease RsaI and then only the digested tester cDNA population used to generate two distinct tester populations each with different adaptors ligated. The first round of hybridization, using both tester cDNA populations and the driver cDNA population combined, was performed to allow for equalization and enrichment of differentially expressed sequences. This was followed by a second round of hybridization to generate the templates for PCR amplification. Using suppression PCR, differentially expressed sequences were favorably amplified exponentially. This resultant population of cDNAs was then used in a second round of PCR amplification to remove background and further enrich for differentially expressed sequences. PCR products were ligated to T-tailed pBluescript II SK+ (constructed according to the method of Khan et al., Trends in Genetics 10:7, July 1994; or Hadjeb and Berkowitz, Biotechniques, January 1996). Electro-competent XL1-Blue E. coli cells were electroporated with recombinant plasmids and cells plated onto LB-ampicillin plates containing X-gal and IPTG.

[0149] Colonies containing cDNA inserts were cultured in NZY broth with the appropriate antibiotic and cDNA was purified by means of alkaline lysis and polyethylene glycol (PEG) precipitation. Agarose gel at 1% was used to screen sequencing templates for chromosomal contamination. Dye primer sequences were prepared using a Turbo Catalyst 800 machine (Perkin Elmer/Applied Biosystems Division, Foster City, Calif.) according to the manufacturer's protocol.

[0150] Polynucleotides for positive clones were obtained using a Perkin Elmer/Applied Biosystems Division Prism 377 sequencer. cDNA clones were sequenced first from the 5′ end and, in some cases, also from the 3′ end. For some clones, internal sequences were obtained using subcloned fragments, exonuclease III deletions, or by direct sequencing using gene-specific primers designed to identified regions of the gene of interest. Subcloning was performed using standard procedures of restriction mapping and subcloning to pBluescript II SK+ vector (Stratagene) and other standard sequencing vectors.

[0151] The determined cDNA sequences, including the polynucleotides of the present invention, were compared to and aligned with known sequences in the EMBL database (as updated to end of August, 1998). Specifically, the polynucleotides identified in SEQ ID NOS. 1-45, 90-140, 192-195, 200, 204 and 206, were compared to polynucleotides in the EMBL database using the BLASTN algorithm version 2.0.4 [Feb. 24, 1998] and version 2.0.6 [Sep. 16, 1998] set to the preferred parameters described above. Running parameters used for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity were as follows: Unix running command: blastall -p blastn -d embldb -e 10 -G0 -E0 -r 1 -v 30 -b 30 -i queryseq -o results. Multiple alignments of redundant sequences were used to build up reliable consensus sequences. Based on similarity to known sequences from other plant or non-plant species, the isolated polynucleotides of the present invention identified as SEQ ID NOS. 1-45, 90-140, 192-195, 200 and 204 were putatively identified as encoding polypeptides having similarity to the polypeptides shown in Tables 1 and 2, above.

[0152] The isolated cDNA sequences were compared to sequences in the EMBL DNA database using the computer algorithm BLASTN. The corresponding protein sequences (DNA translated to protein in each of six reading frames) were compared to sequences in the SwissProt database using the computer algorithm BLASTP. Comparisons of DNA sequences provided in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206 to sequences in the EMBL DNA database (using BLASTN), and amino acid sequences provided in SEQ ID NOS: 46-89, 141-191, 196-199, 201 and 205 to sequences in the SwissProt database (using BLASTP) were made as of March, 1999. Analysis of the amino acid sequences against the EMBL DNA database dynamically translated in all six reading frames (both strands) was conducted using the TBLASTN algorithm. Analysis of six-frame translations of the polynucleotides of SEQ ID NOS: 1-45, 90-140, 192-195 200, 204 and 206, were also compared to and aligned with the six-frame translations of polynucleotides in the EMBL database using the TBLASTX program.

[0153] BLASTN Polynucleotide Analysis

[0154] The cDNA sequences of SEQ ID NOS: 1-7, 10-18, 20-31, 35-40, 43, 44, 90-94, 98-107, 109, 110, 112-118, 120, 121, 123-140, 192-195, 200, 204 and 206, were determined to have less than 50% identity, determined as described above, to sequences in the EMBL database using the computer algorithm BLASTN. The cDNA sequences of SEQ ID NOS: 8, 9, 33, 34, 41, 42, 96, 108 and 111 were determined to have less than 75% identity, determined as described above, to sequences in the EMBL database using BLASTN. The cDNA sequences of SEQ ID NOS: 32, 95, 97, 119 and 122, were determined to have less than 90% identity, determined as described above, to sequences in the EMBL database using BLASTN. The cDNA sequence of SEQ ID NO: 9 was determined to have less than 98% identity, determined as described above, to sequences in the EMBL database using BLASTN.

[0155] BLASTP Amino Acid Analysis

[0156] The amino acid sequences of SEQ ID NOS: 46, 48-50, 55, 58, 60, 61, 80, 81, 87-89, 141, 142, 144, 156, 157, 160, 161, 173, 174, 177, 179, 180, 182-191, 198 and 205, were determined to have less than 50% identity, determined as described above, to sequences in the SwissProt database using the BLASTP computer algorithm. The amino acid sequences of SEQ ID NOS: 49, 57, 59, 66, 67, 72, 82-84, 143, 145, 149, 150, 152-155, 159, 162-173, 178, 181, 196, 197, 199 and 201, were determined to have less than 75% identity, determined as described above, to sequences in the SwissProt database using the computer algorithm BLASTP. The amino acid sequences of SEQ ID NOS: 51-54, 56, 62-65, 68-71, 73, 74, 79, 146, 147, 151, 158 and 176, were determined to have less than 90% identity, determined as described above, to sequences in the SwissProt database using the computer algorithm BLASTP. The amino acid sequences of SEQ ID NOS: 75, 78, 85, 86 and 148, were determined to have less than 98% identity, determined as described above, to sequences in the SwissProt database using the computer algorithm BLASTP.

[0157] TBLASTN and TBLASTX Analysis

[0158] The amino acid sequences of SEQ ID NOS: 46, 47, 49, 50, 58, 60, 61, 80, 83, 87, 89, 141, 157, 160, 161, 174, 175, 177, 179-180, 182-191 and 197-199 were determined to have less than 50% identity, determined as described above, to amino acids dynamically translated in all six reading frames (both strands of polynucleotides) in the EMBL database. The amino acid sequences of SEQ ID NOS: 48, 55, 57, 59, 66, 67, 69, 71, 72, 81, 82, 88, 142-145, 149, 150, 152, 153, 155, 156, 162-165, 167-172, 176, 178, 181 and 205 were determined to have less than 75% identity, determined as described above, to amino acids dynamically translated in all six reading frames (both strands of polynucleotides) in the EMBL database. The amino acid sequences of SEQ ID NOS: 51, 52, 56, 62-65, 68, 73, 74, 84, 146, 147, 151, 154, 158, 159, 166, 173 and 196 were determined to have less than 90% identity; and the amino acid sequences of SEQ ID NOS: 53, 75, 78, 79, 85, 86 and 148 were determined to have less than 98% identity; all to dynamic translations in all six reading frames of sequences in the EMBL DNA database using the TBLASTN algorithm version 2.0.6 [Sep. 16, 1998] set to the following parameters: Unix running command: blastall -p blastn -d embldb -e10 -G0-E0-v30-b30-i queryseq -o results.

[0159] Finally, the six-frame translations of the polynucleotide sequences of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206 were compared to and aligned with six-frame translations of polynucleotides in the EMBL database using the TBLASTX program version 2.0.6 [Sep. 16, 1998] set to the following running parameters: Unix running command: blastall -p blastn -d embldb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results. The translations of the polynucleotides of SEQ ID NOS: 1-8, 10, 12-16, 18, 20-22, 24-27, 31, 34-40, 42-45, 90-140, 192-195, 204 and 206 were determined to have less than 50% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm TBLASTX. The translations of the polynucleotides of SEQ ID NOS: 17, 28-30 and 41 were determined to have less than 75% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm TBLASTX. The translations of the polynucleotide sequences of SEQ ID NOS: 11, 19 and 23 were determined to have less than 90% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm TBLASTX. The translations of the polynucleotide sequence of SEQ ID NO: 9 were determined to have less than 98% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm TBLASTX.

[0160] Further studies led to the isolation of extended sequences for the DNA sequences of SEQ ID NO: 1, 2, 5, 6, 8-10, 12-15, 20, 23, 26, 27, 31, 34-39, 41-44, 91, 95, 97, 103, 110, 112, 114, 117, 120, 121, 123, 129, 130, 136, 138 and 193. These extended sequences are provided in SEQ ID NO: 207-248, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 249-290, respectively.

EXAMPLE 2

Use of a DAD1 Gene to Modify Tobacco

[0161] Transformation of Tobacco Plants with a Pinus radiata DAD1 Gene

[0162] A genetic construct comprising the antisense sequence of a polynucleotide comprising the coding region of DAD1 (SEQ ID NO: 8) from P. radiata was constructed and inserted into Agrobacterium tumefaciens by direct transformation using published methods (See An G, Ebert P R, Mitra A, Ha S B, “Binary Vectors,” in Gelvin S B, Schilperoort R A (eds). Plant Molecular Biology Manual, Kluwer Academic Publishers: Dordrecht, 1988). The nucleotide sequence of the antisense sequence is given in SEQ ID NO: 206. General methods for plant transformation are described in Horsch et al., Science 227:1229-1231, 1985. The antisense DNA construct was made by PCR amplification of the open reading frame for the cDNA, followed by purification and cloning of the PCR product into pART7 plasmid. The plasmid was then digested with restriction endonuclease NotI and the 35S promoter-Insert-OCS 3′UTR cloned into the pART27 plant expression vector (See Gleave A, “A versatile binary vector system with a T-DNA organizational structure conducive to efficient integration of cloned DNA into the plant genome,” Plant Mol. Biol. 20:1203-1207, 1992). The presence, integrity and orientation of the transgenic construct was verified by restriction digestion and DNA sequencing.

[0163] Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed with the antisense construct using the method of Horsch et al. Science 227:1229-1231, 1985. Multiple independent transformed plant lines were established for the sense construct. Transformed plants containing the appropriate gene construct were verified using Southern blot experiments.

[0164] FIG. 1 illustrates genomic DNA isolated from seven DAD1 transgenic tobacco lines (lanes 1-7) and from a non-DAD1 control tobacco plant (lane 8). As can be seen, plants 1-7 contain DNA which hybridizes with the pine DAD1 sequence (final wash conditions: 1× SSC, 0.1% SDS at 65° C.), while the control tobacco plant does not. This demonstrates that the antisense polynucleotides corresponding to DAD1 were successfully transformed into target plants.

[0165] Total RNA was isolated from each of the seven DAD1 antisense transformed tobacco plant lines, and from a control (non DAD 1-containing) tobacco line. The RNA samples were analysed in a Northern blot experiment to determine the level of expression in each line. mRNA was hybridized with a pine DAD1 probe (final wash conditions: 0.1x SSC, 0.1% SDS at 65° C.). FIG. 2 illustrates the presence of the pine DAD1 antisense mRNA in all seven transgenic tobacco lines (lanes 1-7), but not in the control tobacco line (lane 8).

EXAMPLE 3

Demonstration of the Presence/Absence of Unique Sequence Identifiers in Plants

[0166] Transgenic tobacco plants were created using unique identifier sequences which are not found in tobacco. The unique identifier sequences inserted were isolated from Pinus radiata, SEQ ID NO: 202, and Eucalyptus grandis, SEQ ID NO: 203. The unique identifier sequences were inserted into Agrobacterium tumefaciens LBA4301 (provided as a gift by Dr. C. Kado, University of California, Davis, Calif.) by direct transformation using published methods (See, An G, Ebert P R, Mitra A, Ha S B, “Binary Vectors,” in Gelvin S B, Schilperoort R A (eds), Plant Molecular Biology Manual, Kluwer Academic Publishers: Dordrecht, 1988). The presence and integrity of the unique identifier sequences in the Agrobacterium transgenic constructs were verified by restriction digestion and DNA sequencing.

[0167] Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed using the method of Horsch et al., Science, 227:1229-1231, 1985. Three independent transformed plant lines were established for each unique sequence identifier used. Two empty-vector control plant lines were established using an empty gene transfer vector that lacked a unique sequence identifier.

[0168] The uniqueness of the sequence identifiers was assayed using Southern blot analyses to test for the presence of the sequence identifier in the genome of the plants. If the sequence identifier is unique and therefore useful as a tag, then the sequence identifier should be clearly absent in plants which have not been tagged and it should be clearly present in plants which have been tagged. In the present example, the unique identifiers would be expected to be absent in the empty-vector transformed control plants. The unique identifier would be expected to be present in the transgenic plants transformed with the unique sequence identifiers.

[0169] Genomic DNA was prepared from empty-vector transformed control plants and plants transformed with unique sequence identifiers using the cetyltrimethyl-ammonium bromide (CTAB) extraction method of Murray and Thompson, Nucleic Acids Res. 8:4321-4325, 1980. The DNA samples were digested with the restriction enzyme EcoRI in the case of the plants transformed with the Pinus unique sequence identifier (SEQ ID NO: 202) and the restriction enzyme XbaI in the case of the plants transformed with the Eucalyptus unique sequence identifier (SEQ ID NO: 203). The DNA fragments produced in the restriction digests were resolved on a 1% agarose gel.

[0170] After the agarose gel electrophoresis step, the DNA samples were transferred to Hybond-N+ brand nylon membranes (Amersham Life Science, Little Chalfont, Buckinghamshire, England) using methods established by Southern, J. Mol. Biol. 98: 503-517, 1975. The nylon membranes were probed with radioactively-labeled probes for the unique sequence identifiers identified above and washed at high stringency (final wash: 0.5× salt sodium citrate buffer (SSC) plus 0.1% sodium dodecyl sulfate (SDS), 15 minutes at 65° C.). The hybridization of the probes to complementary sequences in the genomic DNA samples was detected using auto-radiography.

[0171] The results are shown in FIGS. 3 and 4.

[0172] FIG. 3 shows the hybridization pattern detected in the Southern blot analysis using a probe derived from the Pinus sequence identifier (SEQ ID NO: 202). Lanes A-B contain DNA samples from empty-vector transformed control plants and lanes C-E contain DNA from plants transformed with SEQ ID NO: 202. There is no hybridization in lanes A-B indicating that SEQ ID NO: 202 is not present in empty-vector transformed tobacco plants; that is, SEQ ID NO: 202 is a unique tag suitable for unambiguous marking of tobacco plants. There is strong hybridization in lanes C-E, indicating that the plants which received SEQ ID NO: 202 via transformation have been clearly and unambiguously tagged with the unique sequence contained in SEQ ID NO: 202.

[0173] FIG. 4 shows the hybridization pattern detected in the Southern blot analysis using a probe derived from the Eucalyptus sequence identifier (SEQ ID NO: 203). Lanes A-B contain DNA samples from empty-vector transformed control plants and lanes C-E contain DNA from plants transformed with SEQ ID NO: 203. There is no hybridization in lanes A-B indicating that SEQ ID NO: 203 is not present in empty-vector transformed tobacco plants; that is, SEQ ID NO: 203 is a unique tag suitable for unambiguous marking of tobacco plants. There is strong hybridization in lanes C-E indicating that the plants which received SEQ ID NO: 203 via transformation have been clearly and unambiguously tagged with the unique sequence contained in SEQ ID NO: 203.

[0174] The data clearly demonstrates the utility of the sequences disclosed in this specification for the purposes of unambiguously tagging transgenic materials. A unique sequence was selected from a large number of potential tags and shown to be absent in the genome of the organism to be tagged. The tag was inserted into the genome of the organism to be tagged and a well-established DNA detection method was used to clearly detect the unique sequence identifier used as the tag.

[0175] Because of the sequence-specific detection methods used in the example, a user of the invention disclosed in this specification has both a high likelihood of finding a sequence identifier, among the list which has been disclosed, which will be useful for tagging any given organism and an unequivocal method for demonstrating that a tagged organism could only have acquired a given tag through the deliberate addition of the unique sequence to the genome of the organism to be tagged. If the user of this invention maintains the precise sequence of the tag used in a given organism as a secret, then any disputes as to the origin and history of the organism can be unambiguously resolved using the tag detection techniques demonstrated in the present example.

[0176] SEQ ID NOS: 1-290 are set out in the attached Sequence Listing. The codes for nucleotide sequences used in the attached Sequence Listing, including the symbol “n,” conform to WIPO Standard ST.25 (1998), Appendix 2, Table 1.

[0177] All references cited herein, including patent references and non-patent publications, are hereby incorporated by reference in their entireties. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248.

2. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) complements of the sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248;

(b) reverse complements of the sequences recited in SEQ ID NOS: 1-45, 90-140; 192-195, 200, 204 and 206-248; and

(c) reverse sequences of the sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248.

3. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) sequences having at least 75% identity to a sequence of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248;

(b) sequences having at least 90% identity to a sequence of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248;

(c) sequences having at least 95% identity to a sequence of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; and

(d) sequences that hybridize to a sequence of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248 under stringent hybridization conditions.

4. The isolated polynucleotide of claim 3, wherein the polynucleotide encodes a polypeptide having activity in a plant cell death pathway.

5. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) sequences that are 200-mers of a sequence of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248; and

(b) sequences that are 100-mers of a sequence of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206-248.

6. An isolated oligonucleotide probe or primer comprising at least 10 contiguous resides complementary to 10 contiguous residues of a nucleotide sequence of SEQ ID NO: 1-45, 90-140, 192-195, 200, 204 and 206-248.

7. A genetic construct comprising at least one polynucleotide according to any one of claims 1-3.

8. A transgenic plant cell comprising at least one genetic construct according to claim 7.

9. A genetic construct comprising, in the 5′-3′ direction:

(a) a gene promoter sequence;

(b) a polynucleotide sequence comprising at least one of the following: (1) a sequence comprising a coding region of a polynucleotide of any one of claims 1-3; and (2) a sequence comprising a coding region of a polynucleotide of any one of claims 1-3; and

(c) a gene termination sequence.

10. The construct of claim 9, wherein the polynucleotide sequence is in a sense orientation.

11. The construct of claim 9, wherein the polynucleotide sequence is in an antisense orientation.

12. The construct of claim 9, wherein the gene promoter sequence and gene termination sequences are functional in a plant host.

13. A transgenic plant cell comprising a construct of claim 9.

14. A plant comprising a transgenic plant cell according to claim 13, or fruit or seeds or progeny thereof.

15. The plant of claim 14, wherein the plant is a woody plant.

16. The plant of claim 15, wherein the plant is selected from the group consisting of eucalyptus and pine species.

17. A method for modulating a plant cell death pathway in a plant, comprising stably incorporating into the genome of the plant a genetic construct of claim 9.

18. The method of claim 17, wherein the genetic construct incorporated into the genome of the plant provides a plant cell death pathway that is not present in a native form of the plant.

19. A method for producing a plant having an altered cell death pathway, comprising:

(a) transforming a plant cell with a genetic construct of claim 9 to provide a transgenic plant cell; and

(b) cultivating the transgenic plant cell under conditions conducive to regeneration and mature plant growth.

20. An isolated polypeptide encoded by a polynucleotide of any one of claims 1-3.

21. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290.

22. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) sequences having at least 75% identity to a sequence of SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290;

(b) sequences having at least 90% identity to a sequence of SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290; and

(c) sequences having at least 95% identity to a sequence of SEQ ID NOS: 46-89, 141-191, 196-199, 201, 205 and 249-290,

wherein the polypeptide is active in a plant cell death pathway.

23. A method for modifying the activity of a polypeptide involved in a plant cell death pathway in a plant, comprising introducing into cells of the plant RNA corresponding to a polynucleotide of any one of claims 1-3, thereby inhibiting expression of a polypeptide encoded by the polynucleotide.

24. A method for modifying the activity of a polypeptide involved in a plant cell death pathway in a plant, comprising introducing into cells of the plant double stranded RNA corresponding to a polynucleotide of any one of claims 1-3, thereby inhibiting expression of a polypeptide encoded by the polynucleotide.