MXPA01004411A - Plants having enhanced gall resistance and methods and compositions for producing same. - Google Patents

Abstract

Methods and compositions for suppressing gall formation in plant cells induced by Agrobacterium infection are disclosed. The methods involve introducing at least one DNA construct, encoding at least one untranslatable sense-strand RNA and/or double-stranded RNA, into a plant cell. Introduction of these molecules into the plant cell causes the plant cells to become resistant to gall formation.

Description

PLANTS WITH INCREASED RESISTANCE TO FORMATION OF WRINKLES AND METHODS AND COMPOSITIONS TO PRODUCE THEM DESCRIPTION OF THE INVENTION This invention is generally directed to compositions and methods for producing plants resistant to gall disease. The methods involve transforming the plants with nucleic acid constructs encoding meaningless chain non-translatable RNA molecules and / or double-stranded RNA molecules, molecules, RNAs that are similar (but not identical) to certain genes that induce the Agrobacterium tumor. Several different recombinant DNA technologies have been employed to interfere with the expression of genes in plants, including technologies based on ribosome molecules, molecules, antisense RNA, and cosuppression constructions. For a general discussion of ribozyme technology, antisense technology and cosuppression technology, see Castanotto, Crit. Rev. Eukaryot. Gen. Expr. 2: 331-357, 1997; Simons, Gene, 72: 35-44, 1988; and Jorgensen, Biotechnol. 8: 340-344, 1990, respectively. In addition to standard suppression techniques, researchers have studied the suppression of a target gene triggered by the introduction of a nontranslatable chain transgene. "(Dougherty and Parks, Curr Opin, Cell Biol. 7: 399-405 , 1995, Jorgensen et al., Scienqe, FR- 279: 1486-1487, 1998). This method has been used to silence a number of endogenous plant genes, as well as viral genes (Maraño and Baulcombe, Plant J. 13: 537 546, 1998; Conner et al., Plant J. 11: 809-823, 1997, Dougherty et al., Mol Plant-Microbe Interact 7: 544-552, 1994; and Smith et al., Plant Cell 6: 1441-1453, 1994). The bacterial gene of Agrobacterium contains several species that cause diseases in economically important plants. For example, Agrobacterium tumefacien causes gall tumors when it infects the wounded tissue of certain dicotyledonous plants such as apple trees and grape vineyards (DeCleene and DeLey, Bot. Rev. 42: 389-466, 1976). Pathogenic strains of Agrobacterium harbor a plasmid that induces the tumor (Ti) that carries the essential genes for gall tumorigenesis (Watson et al., J. Bacteriol., 123: 255-264, 1975; Van Larebeke et al., Nature 252: 169-170, 1974). The transferred DNA portion ("T-DNA"; Figure 1) of the Ti plasmid enters the cells of the susceptible plants and is integrated into the nuclear DNA (Chilton et al., Cell 1: 263-271, 1977; Willmitzer et al. al., Nature 287: 359-361, 1980; Chilton et al., Proc. Nati, Acad. Sci. USA 77: 4060-4064, 1980). After the integration of T-DNA into the cellular AD, the expression of three T-DNA genes, iaaM, iaaH and ipt, leads to the overproduction of the plant growth hormones auxin and cytokinin, resulting in tumorous growths referred to as " guts". (Ream, Annual Rev. Phytopathol., 27: 583-618, 1989; Winans, Micro., Rev. 56: 12-31, 1992; and Zambryski, Annu., Rev. Plant Physiol. Plant Mol. Biol. 43: 465-490. , 1992) (Figure 2). The iaaM, iaaH I ipt genes are well characterized, and the sequences of these genes are available for example in the Genbank database under accession numbers X56185, M25805, and X17428, respectively. M yA and M aaH are required for auxin production; the enzyme codißjL-each by iaaM converts the triftofan to acetamide indole, which is converted by the enzyme encoded by iaaH into acetic acid indole, an auxin (Schroeder et al., Eur. J. Biochem. 138: 387-391, 1984; Thomashow et al., Proc. Nati Acad. Sci. 81: 5071-5075, 1985; Thomashow et al., Science 231: 616-618, 1986; and Inze et al., Mol. Gen. Genet. 194: 265-274, -1984). The loss of any enzyme prevents the production of auxin. Ipt is required for cytokinin production; the enzyme produced by this gene converts the monophosphate adenosine into monophosphate adenosine isopentenyl, a site called aiinin (Akiyoshi et al., Proc. Nati, Sci. USA 81: 5994-5998, 1984; - Bárry et al., Proc. Nati. Acad. Sci. USA 81: 4776-4780, 1984 and Buchman et al., EMBO J. 4: 853-859, 1985). Inactivation of ipt and any of the auxin biosynthesis genes in the Ti plasmid will reduce gall formation (Ream et al., Proc Nati Acad Sci USA 80: 1660-1664, 1983).

Additionally, galls are formed in cases in which the cytokinin pathway, or only the auxine biosynthesis pathway, is functional. In these cases the gills are visually distinguishable based on their different morphological characteristics. For example, plants infected with an Agrobacterium strain that carries' * < - Only the gene necessary for the production of auxin develop "necrotic" gills. In contrast, plants infected with strains of the bacteria that induce cytokinin-only production produce shoot-out gills. At -date, no "there is an adequate means to control a disease of gall formation in vineyards of grapes, trees" fruit trees, - trees of nuts, chrysanthemums, roses, strawberries of woody stem, ornamental shrubs and ofcrps plantings of The inoculation of the plants with the strain of Agrobacterium radiobacter K84 produces some protection against specific strains of A. tumefaciens (Moore, Microbiol.Sci. 5: 92-95, 1988), however, the disease of gall formation remains As a global multimillion dollar problem, Arabidopsis thalianq plants resistant to gall disease have been produced by a traditional genetic procedure (Nam et al., Plant Cell 9: 317-733, 1997), but The strategy is not currently applicable to plants in which galls are a problem.

This inction is directed towards a new, more effective method of producing plants that are substantially resistant to gall-forming disease caused by bacteria. -Á The present invention provides compositions that can be used to prevent the formation of tumors (galls) in plants infected with bacterial pathogens such as Agrobacterium tumefaciens and other * Agrobacterium species. These compositions comprise nucleic acid molecules which, when introduced into plants, can produce transgenic plants that have increased resistance to tumors caused by such pathogens. The invention also "comprises transgenic plants that contain these nucleic acid molecules. In general terms, the invention involves the use of nucleic acid constructs encoding RNA molecules of a non-translatable strand, double-stranded RNA, and / or non-translatable double-stranded RNA that share specific high levels of sequence identity with target genes in the, - bacterial pathogen. For example, constructs can encode one or more types of non-translatable RNA molecules that share high levels of sequence identity with one or more tumor genes iaaM, iaaH or Agrobacterium ipt. The invention also provides nucleic acid constructs that encode multiple different RNA molecules from a non-translatable chain, double-stranded RNA and / or non-translatable double-stranded RNA, including both sense and antisense RNA molecules. For example, a construct provided by the invention encodes "non-translatable RNA" forms of both ipt and iaaM genes of Agrobacterium tumefaciens, this construction can suppress both shoot and necrotic gill formation in plants caused by a pathogenic strain of A. tumefaciens that infects The nontranslatable RNA molecules appear to inhibit the formation of galls by specific destruction of activating sequences of RNA molecules encoded by certain "target genes" of the pathogen of infection in the plant. which is not completely understood, for optimal efficiency, the RNA molecules of a non-translatable chain, double-stranded RNA and / or non-translatable double-stranded RNA must be of sufficient length and share a minimum degree of sequence identity. with 'the target bacterial gene in such a way that resistance to the disease is conferred on the host cell d) The level of disease resistance conferred to the host cell by the RNA molecule can be determined by using the experiments described below. Thus, the RNA molecules encoded by the constructs provided herein are typically at least 25 nucleotides in length and share at least 60% sequence identity with the target gene of the pathogen. Increased tumor suppression is obtained by increasing the length of the RNA molecule of a non-translatable chain, double-stranded RNA, and / or double-stranded non-translatable RNA (eg, at least 100, or at least 200 nucleotides) and / or increasing the level of sequence identity with the target gene (for example for at least 70%, 75%, 80%, or 90% sequence identity) The invention also provides transgenic plants that contain one or more of the nucleic acid constructs described above.These constructions can be introduced into any plant species, but it is expected that plants will be particularly benefited by this technology. commercial clutch for which the Agrobacterium is a significant pathogen. The invention also provides chimeric plants that include both transformed and transformed cells. These plants exhibit "conferred resistance" since the transgenic portion confers resistance to the disease for the non-transgenic portion. Chimeric plants are, particularly. useful for generating resistance, without being transgenic, fruits and other consumable portions of plants that are not transgenic.

SEQUENCE LISTING The nucleic acid and amino acid sequences listed in the appended sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids.

Only one chain of each acid sequence is shown. *. nucleic, but s; and understands that the complementary chain is included by any reference to the displayed chain. SEQ ID NO: 1 is a primer used to produce the iaaM mutant gene. SEQ ID NO: 2 is a primer used to produce the mutant iaaM gene. SEQ ID'NO: 3 is a primer used to produce the > * • mutant ipt gene. "., - SEQ ID NO: 4 is a primer used to produce the ipt mutant gene - SEQ ID NO: '5 is the sequence of the DNA encoding an RNA-non-translatable molecule having a high sequence identity with the gene ipírde Agrobacterium SEQ ID NO: 6 is an amino acid sequence that is encoded by the nontranslatable RNA molecule represented in SEQ ID NO: 5.. '[^ - • SEQ ID NO: .7 is a sequence of the DNA encoding the non-translatable RNA molecule having a high sequence identity with the Agrobacterium iaaM gene SEQ ID. * NO: 8 is an amino acid sequence encoded by the non-translatable RNA molecule depicted in FIG. SEQ ID NO: 7, • SEQ ID NO: 9 is the nucleic acid sequence of the ipt / iaaM non-translatable construct SEQ ID NO: 10 is the nucleic acid sequence of the iaaM gene SEQ ID NO: 11 is the sequence of nucleic acid of the iaaH gene SEQ ID NO: 12 is the nucleic acid sequence of the ipt gene SHORT DESCRIPTION OF DRAWINGS Figure i. is a diagram of the DNA transferred, from A. tumefaciens CT-'DNA ") that contains three oncogenes: iaaM, iaaH and ipt.The triangles represent the T-DNA boundary sequences that delimit the ends of the T-DNA at the beginning and end the transfer, del-T-DNA (Peralta and Ream Proc.

Nati Acad. Sci. 82": S1.12-5116, 1985. Arrows indicate the direction and length of messenger RNAs (mRNA) produced in gall tumors, Figure 2 is a diagram of the biosynthetic pathways of" auxin and Cytokinin encoded by the T-DNA oncogenes of A. tumefaciens Figure 3 is a diagram of the binary yegetal transformation vector (pPEV6) (panel A) and of mutant transgenes that can be used to produce cosuppression of oncogenes T-DNA. "RB" and "LB" denotes J-limit sequence of right and left T-DNA, respectively., NOS prom / NPTII denotes the nopaline belt promoter fused to the neomycin phosphotransferase II gene (which confers resistance to kanamycin)., 5'UTR and 3 'UTR denote the 5"and 3" untranslated regions, respectively, associated with the cauliflower mosaic viruide promoter (CaMV 35S Pro). The heads of the 4 arrows denote sites of respective restriction for Xbal, BamHl and EcoRI, the numbers syndicate coordinates respective in relation to the sequence of the vector. Bold premature stop codons are shown, while natural start codons are shown in plain text. Figure 4 illustrates the most likely scenario for the creation of double-stranded RNA. The sense strand is transcribed from the CaMV 35S promoter and the antisense strand of the NOS promoter is transcribed. * Figures 5 (A) -5 (F) represent the various representative illustrations. of bacterial resistance constructions (BR constructions) according to the invention. The promoters are designated by the capital letter "p" and the direction of the expression is denoted by the respective arrows The DNA sequences encoding non-translatable RNA molecules that target specific target genes are indicated, however, an ordinary expert in the relevant technique will appreciate that those non-translatable RNA molecules that target other genes can be substituted.Figure 5 (A) shows a BR construct in which a promoter (designated Px) is used to express (the direction i. expression, indicated by the arrows) a DNA sequence that encodes a chain with sense of a sequence that targets the ipt gene; and the chain with sense of a sequence that targets the gene 'iaaM. This same construct also contains a second promoter (designated P2) that drives the expression of the antisense strand of the iaaM gene and the antisense strand of the ip ^ gene. In this way, the combination of both promoters will generate a double-stranded RNA that contains a section that targets the ipt gene and a section that targets the iaaM gene. Figure 5 (B) shows a construct containing a promoter (Px) expressing a DNA sequence encoding several different non-translatable RNA sequences that target both the ipt gene and the iaaM gene. When starting at the 5 'end of the construct, the Pi promoter triggers the expression-of a sense-targeting strand of the ipt gene, a sense strand that targets the iaaM gene, an antisense strand that targets the iaaM gene, and a chain with sense that objectifies the gene. This sequence can be duplicated again by itself, and thus generate a double-stranded RNA molecule comprising regions homologous to both ipt and iaaM genes Figure 5 (C) shows another variation of a BR construction This construct comprises independent promoters, Px and P2, to drive the expression of a "non-translatable RNA molecule in both sense and antisense orientations, respectively. Two additional promoters (P3 and P4) are used for the expression of the sense and antisense expression of a second non-translatable RNA. Figure 5 (D) shows another variation of the construction 'Bit; This construct comprises a promoter yt (Pi) to first express the sense strand and then the antisense strand of a nontranslatable RNA molecule that targets the ipt gene. This construct also comprises a second promoter to express both the sense and antisense strand of u 'n r-.? RNA not translatable that targets the iaaM gene. Figures' 5 (E) and 5 (F) illustrate additional variations that can be used to express the non-translatable RNA molecules of a chain. I. Definitions and abbreviations: Non-translatable RNA molecule: An RNA molecule, either a chain or a chain, which is based on an RNA molecule that "is present in a natural form but which, as a result of. a genetic manipulation, it is unable to be translated to produce the full-length protein encoded by its naturally occurring counterpart. For example, such RNA may be brought to be non-translatable through the introduction of one or more codons, premature termination (TAA, TAG or TGA), or by introduction of a mutation that changes the reading structure. Alternatively, for example, the RNA may be rendered untranslatable by the removal of the AUG traducation start codon, or from the AAUAAA polyadenylation signal. The RNA can also be taken to not be translated by simply removing a portion of the cDNA that encodes the full-length protein. In this way, even if the sequence is translated, it can produce a truncated version of its counterpart which is found naturally. Antisense strand RNA: An RNA molecule that is complementary to a messenger RNA (which represents a corresponding "senfido" chain). Transgenic plant resistant to disease: An i- "transgenic plant resistance to disease" as used herein is% a transgenic plant that exhibits more resistance to gall disease than a corresponding non-transgenic plant. Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence at any time the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence * if the promoter affects the transcription or expression of the coding sequence. Usually, the operably linked DNA sequences are contiguous. Recombinant: A recombinant nucleic acid is one that has a sequence that does not occur naturally in the organism in which it is expressed, or has a sequence made by an artificial combination of two sequences separated in some way, shorter. This artificial combination is often carried out by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by geriatric modified techniques. "Recombinant" is also used to describe nucleic acid molecules that have been manipulated-artificially, but that contain the same control sequences and t coding regions that are found in the organism from which the gene is isolated. Chain-sense RNA: An RNA molecule that can directly serve as messenger RNA, that is, that can be translated by ribosomes. Double-stranded RNA: A "double-stranded RNA" is an RNA molecule that contains at least a 25 bp sequence that includes both a sense strand and an antisense strand. The double-stranded RNA molecules are further characterized by the level of complementarity between the chain with. sense and the antisense chain. Complementarity refers to the amount of base matching between the two sequences. For example, the sequence 5'-AUCGGAUAGU-3"is '1005 complementary to the sequence S" -ACUAUCCGAU-3'. The double-stranded RNA may be of sufficient length and have a sufficient level of - r complementarity in such a way as to confer disease resistance to the host cell (resistance to the disease can be determined by using the assays described below). For example, double-stranded RNA can have as little as 60% complementarity. Generally, however, the double-stranded RNA sequences will have at least 70%, 75%, 80% or 90% complementarity. Vector: A "vector" is a nucleic acid molecule as it is introduced into a host cell, which thereby produces a transformed host cell. A vector can include nucleic acid sequences, such as an origin of replication, which allows the vector to replicate in a host cell. A vector may also include one or more screening markers, selectable markers, or reporter genes and other genetic elements known in the art.

Sequence Identity: The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. The sequence identity is typically expressed in terms of percent identity, the higher the percentage, the greater the similarity in the two sequences. Methods for aligning sequences for purposes of comparison in the art are well known. Various programs, programs and alignment algorithms are described in: Smith & Waterman '.Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970; Pearson and Lipman, Proc. Nati Acad. Sci. USA 85: 2444-2448, 1988; Higgins and Sharp, Gene 73: 237-244, '1988; Higgins and Sharp, CABIOS 5: 151-153, 1989; Corpet et al., Nucleic Acids Research 16: 10881-10890, 1988; Huang, et al., Computer Applications in the Bioscieties 8: 155-165, 1992; And Pearson et al., Methods in Molecular Biology 24: 307-331, 1994. 'tschul et al., J. Mol. Biol .., 215: 403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The alignment research tool NCBI (BLASTMR, Ateschul et al., J. Mol. Biol., 215: 403-410, 1990) is available from. several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blagtx, tblastn and tblastx. You can have access to BLAST M1R at http: // www. ncbi. nlm. nih gov / BLAST /. A description of how to determine sequence identity using this program is available at http: // ww. ncbi. nlm. nih gov / BLAST / blast help. html For comparisons of the T amino acid sequences U of more than approximately 30 amino acids, the "Blast 2 sequences" function is used in the BLASTMR program using the BLOSUM62 fixed-start matrix in the start parameters, (space existence cost of 11). , and a space cost per residue of 1). - When short peptides are aligned (less than approximately 30 amino acids), the alignment must be done using the Blast 2 sequence function, by using the fixed PAM30 matrix in the starting parameters (aperture space surcharges 9, extension space 1) ). The proteins that have even greater similarity to the reference sequences will show increased percentages of identities when evaluated by this method, such as at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least'- 90%, or at least 95% sequence identity. 1 A first nucleic acid "is" substantially similar "to a second nucleic acid if, when aligned optimally (with appropriate insertions or deletions of nucleotides) with the other # nucleic acid (or its complementary strand), sequence identity occurs of nucleotides in at least about 60%, 75%, 80%, 85%, 90% or 95% of the ytucleotide bases (As used herein, "optimally aligned" sequences exhibit a maximum possible sequence identity) Sequence similarity can be determined by comparing the nucleotide sequences of two nucleic acids using the BLAST1 MR (blastn) sequence analysis software available from the National Center for Biotechnology Information .. Such comparisons can be made using the # / fixed software in start parameters (expected = 10, pairs forms, alignments = 500, alignment view = standard, space existence cost = ll, existence per residue = l, space cost of per residue = 0.85). Similarly, a first polypeptide is substantially similar to a second polypeptide if it shows sequence identity of at least about 75% -90% or more when it is optimally aligned and compared using the BLASTMR software (blaqtp) using start parameters. Construction of . resistance to bacteria (BR construction): A "bacteria resistance construct" (abbreviated as "BR construction") is a DNA sequence that incorporates at least one promoter and at least one DNA sequence encoding a nontranslatable RNA (either Double-stranded RNA, one-stranded RNA or a combination thereof). The arrangement of the promoter (s) in relation to the DNA sequence (s) may vary. Representative configurations are illustrated in Figures 5 (A) -5 (G). Figure 5 (A) shows a BR construct in which a promoter (designated Px) is used to express, * •• - '(the address d' expression is indicated by the arrows) a DNA sequence encoding 1-a chain with sense of a sequence that targets the ipt gene and the chain with sense of a sequence that objectifies the iaaM gene. This same construct also contains a second promoter (designated P2) that drives the expression of the antisense strand of the iaaM gene and the anti-sense strand of the ipt gene. In this way, the combination of arabos promoters will generate a double-stranded RNA that contains a section that targets the ipt gene and a section that targets iaaM el- gene. Figure 5 (B) shows a construct containing a promoter (Pi) expressing a DNA sequence encoding several different non-translatable RNA sequences that both target the ipt gene and the iaaM gene. When starting at the 5"end of the construct, the PI promoter triggers the expression of a meaningful strand that targets the ipt gene, a sense strand that targets the iaaM gene, a strand, antisense that targets the iaaM gene, and a sense chain that targets the ipt gene This sequence can duplicate itself again, and in this way generates a double-stranded RNA molecule comprising regions homologous to both the ipt and iaaM gene, Figure 5 (C) shows Another variation of a BR construction This construct comprises independent promoters, Px and P2 to drive the expression of a non-translatable RNA molecule in both sense and antisense orientations, respectively Two additional promoters (P3 and P) are used for the expression of the sense and antisense strands of a second nontranslatable RNA Figure 5 (D) shows another Varyation of the BR construct This construct comprises a promoter (Px) to express first the Sense chain and then the antisense strand of a nontranslatable RNA molecule that targets the ipt gene. This construct also comprises a second promoter to express both the sense and antisense strand of a nontranslatable RNA that targets the iaaM gene. Figures 5 (E) and 5 (F) illustrate additional variations that can be used to express nontranslatable RNA molecules of a chain. Figures 5 (A) - (F) are proposed to illustrate a few different promoter / sequence DNA combinations that are possible. A person of ordinary skill in the art will appreciate that many additional combinations can be used in the BR constructions according to the invention.

II. General Description The present method provides new and effective methods to suppress the expression of oncogenes from Agrobacterium In particular, this invention is directed, inter alia, to the production of plants that are capable of substantially inhibiting. the formation of galls induced by bacteria. These plants are produced by selecting a target gene of Agrobacterium origin, designing a BR construction (typically DNA) 'of nucleic acid encoding molecules, RNA of a "non-translatable" chain, double-stranded RNA, and / or translatable n -vo double-stranded RNA having a high sequence identity V for the target gene , and introducing at least one of the BR constructs into a host cell a) Selection of the target gene 5 v There are many species of Agrobacterium, for example Agrobacterium tumefaciens, Agrobacterium vi tis, Agrobacterium rubi and Agrobacterium rhizogenes. The nucleic acid sequences of the respective oncogenes contained within these species are highly conserved. Therefore, a -BR construct designed to encode at least one RNA molecule having high sequence identity to one or more Agrobacterium target genes that induce the tumor will similarly confer resistance to at least one (typically multiple) a species of Agrobacterium. b. Producing a nucleic acid construct coding for a non-translatable RNA. Practicing the present invention requires the manipulation of a DNA sequence by using molecular biological techniques to produce the desired construction of nucleic acids. The DNA sequence can be manipulated by using standard procedures such as restriction enzyme digestion, DNA polymerase filling, elimination by etXonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence alteration via V intermediary of bacteriophage of a chain or with the use of specific oligonucleotides in combination with PCR. The details of these techniques are provided in standard laboratory manuals such as Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2 * ed., Cold Spring Harbor Laboratories, Cold Spring. Harbor, N.Y., 1989, and Ausubel et al., Short Protocols in Molecular Biology, 2nd ed. , John Wiley and Sons, 1992.. Deletion of an oncogene is achieved by using a method according to this invention by expressing in a host cell, molecules of a BR construction having high sequence identity to the target gene (s). At least one molecule of the BR construct having sequence identity substantial to the target gene is introduced into a host cell. The BR construction can be supplied in the. genome of the host cell by supplying in the host a vector cell containing the BR construct. The application of the present invention for the construction of a Br construct desirably involves the sectioning of appropriate control sequences such as promoters, enhancers, and 3'-non-translated regions. The choice of the control sequence tip for use in a BR construct will depend on the particular tissue in which the BR construct and the particular sequences of the target gene will be expressed. The plant variety that is selected for transformation will also affect the choice of the control sequences, the vector, and the transformation method. The construction of a suitable Br construct involves the selection of a DNA sequence that will be used to objectify at least one specific bacterial gene.

Generally, the DNA sequence will encode a non-translatable RNA having at least 70% sequence identity with the target bacterial gene (i.e. the gene to be deleted). By producing the BR construct, a DNA molecule homologous to a target bacterial gene can be manipulated to carry a chain or double-stranded RNA molecule encoded by the DNA molecule to be untranslated. This can be done, for example, by introducing any of several termination signals into the open reading structure of the DNA molecule to produce the BR construct. For example, the BR construction can be configured to contain one or more premature termination codons. A "stop codon" is a codon of the genetic code that signals ribosomes to complete the translation of a messenger RNA molecule. There are three stop codons in the genetic code: TAA, TAG and TGA, as illustrated in Table 1 below. : Z TABLE 1 •, The genetic code First Second position Third position position (end T C A G (extreme ') 3') Phe Ser Tyr Cys C Leu Ser • Termination (och) Termination A Leu Ser Terminaciórl (amb) Trp G c Leu Pro His Arg T Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G A lie Thr Asn Ser T He Thr Asn Ser C He Thr Lys Arg A 9 Met Thr Lys Arg G Val Wing Asp Gly T Val *. '' Ala Asp Gly C Val Ala Glu Gly A Val (Met) f .Ala Glu gly G In the context of the above discussion, a "premature" termination codon is located in a position transcriptionally upstream of a stop codon that is in natural form. Generally, the premature termination codon is placed within 100 nucleotides of the translation start codon, and typically U within 50 nucleotides of the translation initiation codon. They can > to be inserted one or more stop codons in the • DNA sequence to produce the BR construct. Alternatively, the DNA sequence can be mutated to convert an existing triplet codon (encoding an amino acid) - of the sequence into a stop codon. For example, the hypothetical DNA sequence: .. CTT AAC TGG TCC. It can normally produce a corresponding RNA translated by ribosomes to produce the polypeptide: f-z Leu Asn Trp Ser The corresponding RNA can be taken to be non-translatable by introducing a stop codon, 'such as TAA, in the DNA sequence. For example, the modified DNA sequence: CTT TAA AAC TGG TCC ... * Encodes:, - \ ... Leu TERMINATION Alternatively, in the above hypothetical DNA sequence, the substitution of the nine nucleotides (guanosine (G )) by adenosine (A) will result • in the creation of a premature stop codon, leading to the corresponding RNA not being translatable, that is, the following DNA sequence is produced: ... CTT AAC TGA TCC ... The which encodes: .... Leu Asn TERMINATION Alternatively, the addition or X can be used. • elimination of. ^ • one or more bases in or of, respectively, the sequence "of DNA (except multiple v of three bases) to displace the reading structure 'of the DNA sequence * which may lead to non-translatable The corresponding RNA molecule For example, the introduction of a single nucleotide (G) into the first codon of the hypothetical DNA sequence above creates a new reading structure containing (a premature termination codon: - * - ... CTG TAA CTG GTC C ... • which codes: ... Leu TERMINATION An alternative way to make the RNA non-translatable is to eliminate the start codon of translation (usually ATG) of the corresponding DNA sequence. However, this method may result in use by ribosomes of an ATG codon anywhere in the DNA sequence to initiate translation. The removal of the ATG start codon is, therefore, desirably used in conjunction with the insertion of one or more premature stop codons into a DNA sequence. Another method for introducing a premature termination codon is by truncating the subject DNA sequence. This can be done, for example, by using a restriction enzyme to cleave a portion of a target gene and then cloning only the excised portion of the gene in a vector. Transcription of the vector will produce a segment of effective homologous RNA to produce a phenomenon of co-repression, but it will not produce a fully processed protein. Currently, the mechanism by which cosuppression works is not known. However, the phenomenon has been previously in detail (Lindbo and Dougherty, Virology 189: 725-733, 1992). One or more of the above procedures may be used to carry an RNA molecule encoded by a BR construct to be non-translatable. It will be appreciated that a large number of sequence changes can be made in a suitable form, the precise nature of which can depend on the current DNA sequence being manipulated, to bring the corresponding RNA sequence to be non-translatable. In certain embodiments, more than one stop codon can be introduced into the DNA sequence. For example, the resulting BR construct may contain a DNA sequence encoding nontranslatable RNA that has been altered to contain at least one stop codon located initially in the open reading frame; the open reading structure is thus "truncated". Many standard DNA mutagenesis techniques are available to modify DNA sequences in a manner as described above to produce the BR construct. Mutagenesis techniques include, for example, mutagenesis of M13 primer. Details of such techniques are provided in Sambrook et al., Chápter 15, Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring 'Harbor Laboratories, Cold Spring Harbor, N.Y.', 1989; and in Ausubel et al., Chapter 8, Short Protocols in Molecular Biology, 2nd ed. , John Wiley and Sons, 1992. According to another aspect of the invention, double-stranded RNA molecules that are homologous to a target bacterial gene are provided. Each double-stranded RNA strand of separate BR constructs can be produced, wherein a first BR construct encodes the antisense strand and a second BR construct encodes the strand. ^ *., chain with sense. Upon transcription of the first and second BR constructs, the resulting sense strand and the corresponding antisense strand form dqble strand RNA. Alternatively, the sense chain and the antisense cache can be produced from a simple BR construction. The simple BR construct can use two different promoters to initiate the expression of the antisense strand and the sense strand, or a promoter to drive the expression of both strands. When a single promoter is used, both the sense and antisense chain are normally produced as a simple transcript, where the RNA sequence will duplicate itself again to create a double-stranded RNA. Similarly, multiple combinations of promoters, antisense chains, and sense strings can be encoded in the same BR construction.

"Double-stranded RNA can also be produced by transforming cells with a BR construct that encodes the sense strand, a self-dividing ribozyme, and the antisense strand all in a single transcript." After the transcription of such a BR construct, produces the sense chain, the self division ribozyme and the antisense strand.The self-dividing ribozyme sequence is duplicated again in the resulting transcript and assembles the transcription between sense and antisense strands, generating two complementary independent strands of RNA A proposed mechanism for suppressing a bacterial gene was to introduce a BR constriction that has one or more sequences homologous to at least a portion of the ge-target typically into the activity of an inducible, inducible cellular activity in host cell molecules. specific RNA sequences.This mechanism requires that the RNA of a chain, not t The double-stranded RNA and / or the non-translatable double-stranded RNA encoded by the BR construct is recognizable by the RNA-specific cellular activity. For this purpose, the target RNA sequence and the DNA sequence of the BR construct desirably share a sequence identity. In the Examples presented below, the DNA sequences of the respective BR constructs are derived directly from the target bacterial nucleotide sequences. The sequences of the respective BR constructs differ from the main corresponding target sequences and only by the presence (in the BR constructs sequences) of premature termination codons inserted by mutagenesis involving one or more structure change mutations. Thus, in these examples, the BR constructions have more than 90% of < ? sequence identity for the target DNA sequences corresponds however, a BR construct needs to have only sufficient sequence identity to the target gene in such a way that the BR construct confers disease resistance to the host cell. Therefore, a BR construct encoding an RNA molecule having less than 90% "sequence identity to the corresponding target DNA sequence can be used, In any case, the BR '* construct has" typically at least 60". % sequence identity to the corresponding target DNA molecule, and in some cases at least 70%, 75%, or 80% sequence identity to the target DNA molecule. The following examples also describe BR constructs in which the DNA sequence corresponds to 700 base pairs of the ELA (open reading frame) of the target gene. It will be understood, however, that the untranslated RNA provided by the BR construct need not correspond specifically to 700 base pairs of the Ela of the target gene; shorter sequences of nontranslatable RNA may also be effective. The BR construct must be sufficiently large and share a sufficient level of sequence identity to the target gene of *% such that the BR construct confers disease resistance to the host cell. The capacity of a BR construction. Particular to confer disease resistance to a host cell can be easily determined by using a test described below, in which a transgenic plant is infected with a bacterial pathogen. Typically, the ELA of the DNA sequence of the construct is at least 25-100 nucleotides in length, although larger sequences, such as 100-250, 250-500, or more than 500 nucleotides may be used and may provide results superiors In embodiments where shorter sequences (ie, less than 250 nucleotides) are used * the respective BR constructs will similarly share a high degree of sequence identity (eg, at least 60% sequence identity) with the gene sequence. objective. The present invention can be used to suppress the expression of a bacterial tumor gene in a plant cell. For this purpose, the plant cell is transformed with a BR construction having a high sequence identity to a target bacterial gene. The construction molecule BR encodes one or more RNA molecules of a non-translatable chain and / or double-stranded RNA. The BR construct may comprise a sequence of at least 25 nucleotides in length having at least 60% sequence identity to the target bacterial gene. Alternatively, the BR construct may comprise a sequence of at least 100 nucleotides in length that has at least 60% sequence identity to the target bacterial gene. Additionally,;. the BR construct may comprise a nucleic acid sequence of at least 250 nucleotides in length having at least 60% sequence identity to the target bacterial gene. The present invention is not limited to the suppression of a single target bacterial gene. But, it is possible to suppress, according to the invention, the expression of multiple target genes using one or more BR constructs comprising DNA sequences corresponding to several respective genes expressed in the target cell. This procedure can be used to increase resistance to a single bacterial strain or to produce resistance to multiple strains of bacteria in a single plant. As illustrated in the Examples below, the formation of a gill in a plant by a pathogenic bacterium that infects the plant by the introduction into the cells of the plant of a BR construct having a high molecular identity can be reduced or eliminated. sequence to at least one gene of the infection bacterium. This procedure allows the production of transgenic plants resistant to the bacteria, c. Vectors It will be appreciated that the efficacy of the present invention depends on the transcription of the DNA sequence of the BR construct introduced into a host plant cell. The transcription of a DNA sequence into messenger RNA is preferably carried out by including a promoter region at the 5"end of the DNA sequence of the BR construct together with regulatory sequences at the 3 'end of the DNA sequence. Many sequences identified for promoter and 3 'regulatory sequences and characterized in a wide range of viral, bacterial, fungal, plant and animal genes, therefore, the selection of the appropriate promoter and a suitable 3"regulatory sequence is within the ability of a person of ordinary experience in the relevant technique. After the choice and incorporation of the appropriate regulatory DNA sequence in the BR construct, the BR construct is incorporated into a vector suitable for introduction into the host cell. The choice of regulatory sequences, as well as the choice of a suitable transformation vector, will depend on the type of host cell into which the BR construct will be introduced and after the desired transformation method for the host cell. Suitable vectors are subsequently described for transforming plant cells (derived from both monocotyledonous or dicotyledonous plants). The transformation of both monocotyledonous and dicotyledonous plant cells is now routine and the selection of the most appropriate transformation technique will be determined by an expert practitioner. A number of promoters that are active in plant cells have been described in the literature. Promoters that are known to elicit DNA transcription in plant cells can be used to suppress gene expression in plants. Such promoters can be obtained from plant or plant virus cells and include, but are not limited to, the CaMV 35S promoter (cauliflower mosaic viruq). It is desirable that the chosen promoter be capable of eliciting sufficient expression of the BR construct to produce sufficient RNA to suppress expression of the target gene. The amount of RNA necessary to suppress expression of a particular target gene can vary from one target gene to another, level of expression of the particular target gene, and the type of plant. Therefore, while A that the CaMV promoter is employed in the Examples presented below, it will be understood that. this promoter may not be optimal for all embodiments of the present invention. The vector can be configured to optimally exploit the promoter and the termination sequences in the same target gene. Used in this way, the promoter of the target gene is not altered and subsequent termination signals are introduced into the ELA of the target gene. Additionally, the termination signals of the target gene can be left intact. This configuration allows to increase the transcription of non-translatable RNA and / or double-stranded RNA when the necessary factors for transcription of the target gene are present. The promoter used in a BR construct according to the invention can be modified, if desired, to include additional regulatory sequences. Such additional regulatory sequences may confer, for example, specificity to the region of the respective promoter. For example, the small subunit of ribulose bis-phosphate carboxylase ("ss RUBISCO") is expressed in plant leaves but not in root tissues. A sequence motif that represses the expression of the ss RUBISCQ gene in the absence of light has been identified (to create a promoter which is active in leaves but not in root tissue). This and / or other regulatory sequences such as the petunia HSP70 leader sequence, can be linked to promoters such as the CaMV 35S promoter, or fig mosaic virus (VMH) promoter, to modify the expression patterns of a gene . The 3"untranslated region (which functions as a polyadenylation site for RNA in plant cells) of a gene in the present invention may also be used, such 3" untranslated regions include, but are not limited to, the transcribed 3 '(but not translated) of the CaMV 35S gene, region 3"pea-rbcS E9 untranslated, region 3" untranslated NOS and one or more untranslated regions, transcribed 3' (but not translated) containing the signals of polyadenylation, of the genes that induce tumors (Ti) of Agroabacterium, such as the large gene of tumor morphology (tml). d. Plant species The formation of galls in plants can be induced by any of several organisms. For example, galls may be induced by bacteria of the genus Agrobacterium, and certain spices of Pseudomonas, Rhizobacter and Rhodococcus. . • '' In the case of Agrobacterium, the formation of galls is caused by genes introduced into the plant cells in the Ti plasmid provided by an infection bacterium.There are a variety of Ti plasmids that occur in a natural form which can give rise to Different types of galls are usually caused by A. tumefaciens that infect more than 391 genera of plants that include shrubs, fruit trees, nut trees, tubers, conifers, grape vines, chrysanthemums, roses, woody stem strawberries, nursery plantings, and commercially important plants (Bradbury, JF Guide to Pathogenic Bacteria, CAB International, Slough, Brítain, 1986) The hairy root is another type of gall induced by A. rhjzogenes, which mainly affects roots of apple trees Finally, Agrobacterium rubi can infect raspberries and blackberries and give rise to galls of canes. (Citrus, Plant Pathology , Academic Press, 1997). * '; e. Transformation A BR construct according to the invention is introduced into a plant cell using a suitable vector and transformation method as described below. The resulting transformed cells can be allowed to regenerate in whole plants. Any of several conventional methods used to transform plant cells to generate transgenic plants, including stably transformed plants, can be used in this invention. The choice of method will typically vary with the type of plant to be transformed; persons skilled in the art will recognize the suitability of particular methods for given types of plants. Suitable methods may include, but are not limited to: electroporation of plant protoplasts, liposome-mediated transformation; Polyethylene glycol mediated transformation; transformation using virus; micsoinjection of plant cells; bombardment of microprojectile plant cells and transformation mediated by Agrobacterium tumefaciens (AT). The DNA sequences comprising the CaMV 35S promoter and 3"untranslated region of CaMV 35S and the mutated cDNA encoding a non-translatable RNA can be combined into a simple cloning vector.This vector can be subsequently transformed into, for example, cultured tobacco cells, F. conferred resistance The mechanism of cosuppression is unknown, however, transgenic tissue containing a cosuprtesion gene in a non-transgenic plant can be grafted in. The graft then confers resistance to the non-transgenic plant ( Jorgensen et al., Science, 279: 1486-87, 1998.) Additionally, this "conferred resistance" can be established by either grafting a cut (superior vegetative tissue) into a transgenic rootstock pattern (lower vegetative tissue) or by grafting a transgenic cutting in a non-transgenic rootstock pattern, this conferred resistance can be transmitted through at least 30 cm of non-transgenic tissue It is possible that the phenomenon of * conferred resistance may allow resistance to gall formation to be introduced in existing plants. In this way, a mature plant resistant to gill formation can be made by grafting transgenic tissue into the plant.

Additionally, several grafts can be placed independently on the plant to produce resistance in the plant to several different bacterial pathogens. * -S • There may be a more flexible graft protocol «^ when using cross-species grafting. In this way, a graft of, for example, almonds (Prunus amigdalus) can be placed in a peach rootstock pattern (Prunus pérsica). Such flexibility increases the range of plants for which a given transgenic tissue can be used. For example, after almond tissue having the desired BR construction has been generated, the tissue can be used to confer resistance not only to other almond trees, but also to peach trees. The use of conferred resistance also decreases the possibility of a transgene made in accordance with the invention being introduced into the environment. This is because the resistance is derived from the same graft and the rest of the plant remains l * Lhre of the transgene. Therefore, the fruits and seeds of the plant can be left as non-transgenic, so as not to transmit the gene. By preventing a transgene from being systematically introduced into the plant, products for human consumption can be produced by the plant that is not transgenic. III. Examples a. Construction of BR constructs that encode nontranslatable RNA molecule To produce an ADB construct capable of producing cosuppression, se. modify the iaaM and ipt oncogenes by changing the third codon of each gene to a stop codon and introducing a current structure change mutation below each respective introduced stop codon, thereby creating additional termination codons in the reading structure . (Figure 3). The oligonucleotides of the polymerase chain reaction (PCR) primer used to create these mutations in the DNA construct are described in Table 2. The primers used to modify the ipt gene result in the removal of C 5 'from the third codon and switch to G 3' of the third codon by A. the modifications of iaaM introduce the sequence of termination i. TGA in the third codon and the two bases following the third codon are eliminated. A second DNA construct is made containing a hybrid transgene configured to produce cosuppression of both ipt and iaaM. The second construction of ADItf- is made by digesting a vector (containing both the ipt and iaaM mutation) and a vector containing the mutation terminating iaaM with BamHl. The fragment containing the iaaM termination mutation is then ligated to the fragment containing the ipt-termination mutation, thereby producing a BR construct containing 1 mutant iaaM gene inserted into the mutant ipt gene (Figure 3). The sequence of each DNA construct is verified, and the BR construct is inserted into a plant transformation vector (pPEV6; Lindbo and Dougherty, Mol.Plant-Microbe Interact., 5: 144-153, 1992) oriented in such a way that the sense chain of each mutant is expressed from the cauliflower mosaic virus promoter (CaMV 35S) (Figure 3). Table 2 PCR primers used to produce iaaM and ipt mutant genes Sequence primers - * I- Primer 5 '5'-CGGGATCCATGTCATGAACCTCTCCTTGATAAC-3' of iaaM (SEQ ID NO: 1) Primer 3 '5"-CGGGATCCTGCGACTCATAGT-3' (SEQ ID NO: 2) Primer 5 '5'-GAAGATCTGATCATGGACTGAATCTAA- of ipt TTTTCGGTCC-3' (SEQ ID NO: 3) 3 '5' Primer -GAAGATCTGATCACTAATACATTCCGAACGG-3 '(SEQ ID NO: 4) b. Construction of double-stranded RNA and non-translatable double-stranded RNA vectors A vector encoding an iaaM-terminating mutation (ie, a DNA construct modified to incorporate a stop codon in the third codon, followed by an elimination of two base pairs) exhibits evidence of bacterial resistance. This resistance is derived from the production of double-stranded RNA by transcription of ^ ^ Vector (Figure 4). The chain with the RNA sense is transcribed. double chain of the CaMV 35S promoter and probably transcribed the antisense chain of the nopaline ribbon (NOS) promoter. Thus, this vector can produce a large double-stranded RNA that is approximately '* ^ 000 bp in length. Evidence for such double-stranded RNA is found in northern blots showing partially degraded - aritisenid chains in the size range of 3.4 Kb '* a_ less than 0.24 Kb. As depicted in Figure 4, the double RNA The chain seems to include a region of approximately 2,000 bp. is 100% homologous to the sequence of iaaM.,. Therefore, bacterial resistance observed in transgenic plants of iaaM is probably attributable to the presence of double-stranded RNA. This resistance does not appear to have been solely due to the presence of the introduced stop codon. The double-stranded RNA is able to suppress genes from endogenous plants, as well as suppress viral infection (Ruiz et al., Plant Cell 10: 937-946, 1998). However, the deletion, via double-stranded RNA and / or non-translatable RNA, of disease symptoms caused by bacteria, such as Agrobacterium, has not been previously shown. c. Production of plant homozygotes for non-translatable RNA transgenes Se genes of iaaM (SEQ ID NO: 7), ipt (SEQ ID NO: 5) and ipt / iaaM (SEQ ID NO: 9) in haploid tobacco (N. tabacum, crops "Burley" and "Kentucky"). The chromosomes of kanamycin-resistant transformants of these plants are duplicated to produce homozygous plants for each transgene. These plants are the subject of initial tests, which are repeated in the Fl progenies of auto pollinations. - * d. Phenotypes of plants resistant to gall formation Putative iaaM suppression plants are infected (designated lines "TDP1") with mutant ipt of A. tumefaciens (strain 338, Garfinkel et al., Cell 27: 143-153, 1981). From 63 tested lines, 35 lines, including TDP1-B7 & TDP1-B17, suppress the wild-type, ± aaM gene in T-DNA of A. tumefaciens intreducido in such plants, so it prevents tumor growth in the respective plants. When they are inoculated with A. tumefaciens of the wild type (strain A348; Garfinkel et al., Cell 27: 143-153, 1981), each of the 35 lines produces "recannable" galls typical of cytokinin-activated tumors, such as those induced in wild-type plants inoculated with mutant iaaM of A. tumefaciens (Ream et al., Proc Nati. Acad. Sci USA 80: 1660-1664, 1983; and Garf-inkel et al., Cell 27: 143-1.53, 1981) . In this way, the plants that produce shootable gills exhibit the expected phenotype of a transgenic plant capable of suppressing iaaM. í Putative ipt suppression plants are infected (designated "CW1" lines) with iaaM mutant of A. tumefacjens (strain 338; Garfinkel et al., Cell 27: 143-153, 1981). From 45 investigated lines, one clearly deleted (designated "CW1-K27") the wild-type ipt gene in introduced T-DNA from A. tumefaciens. Another 6 lines exhibit similar results in initial tests on the original transgenic plants, but these lines are lost before the seeds are obtained. The plant that suppresses ipt (CW1-K27), when inoculated with A. tumefaciens of the wild type, produces small necrotic gills typical of auxin-activated tumors, such as those induced in wild type plants inoculated with mutant of ipt of A. tumefaciens (Ream et al., Proc. Nati, Acad Sci USA 80: 1660-1664, 1983, Garfinkel et al., Cell 27: 143-153, 1981). There are 30 tested lines (designated "CW4" lines) that contain the hybrid ipt-iaaM transgene. One line (designated "CW4-B30") exhibits a significant reduction in tumorigenesis when inoculated with either mutant of iaaM, or mutant of ipt of A. tumef hundreds of the wild type. Although the CW4-B30 line exhibits markedly reduced gill size and absence of gall formation at some inoculation sites, gall tumorigenesis is not totally absent. The controls are plant lines resistant to anamicin (designated "PEV6" lines) that contain the T-DNA transformation vector (of pPEV6) without a transgene located downstream of the CaMV 35S promoter.

As expected, none of these lines exhibits any '-' 'deletion of oncogenes from "T-DNA before infection with A. tumefaciens Molecular analysis of transgenic plants To examine the transgene copy number and structural integrity, Southern blot analyzes on plant-representative lines of each type Most of the lines contain several copies of the transgene, and all lines contain one or more intact copies (see Tables 3, 4 and 5 below). they contain redispatched copies of the transgene, which are identified by the presence of multiple bands in the Southern blot analysis .. Table 3 Southern Blot of genomic ADB digested with BamHl Table 4 Southern blot of genomic ADB digested with gcoHl Table 5 Southern blot of genomic DNA digested with either Hinf1 or EcoRl digests genomic DNA The results mentioned above show that cosupresium drastically reduces the accumulation of mRNA encoded by it transgene production and mRNA encoded by other genes that contain sufficient (approximately 0% or more) sequence similarity (Maraño and Baulcombe, Plant J. 13: 537 -546, 1998). In fact, Northern blot analysis was used to measure mRNA accumulation (but no transcription ratio). In all the deletion lines of iaaM. which are examined, the transgene mRNA is not detectable, as expected, using a chain-specific hybridization probe. However, the full-length, degraded antisense RNA molecules that include the transgene sequences are detected using a double-stranded hybridization probe.,. These transcripts appear to have initiated from the nopaline ribbon vector promoter and continue through both the neomycin phosphotransferase gene (in the sense direction) and the iaaM transgene (in the antisense direction). In the successful ipt deletion line, trace amounts of mRNA encoded by transgene are detected. Another line containing the ipt transgene accumulates a large amount of transgene mRNA, but this line fails to cosuppress an ipt gene originating from T-DNA of A. incoming tumefaciens. Plants containing the hybrid ipt-iaaM transgene accumulate several amounts of mRNA encoded by transgene. As expected, most of these lines produce no cosuppression and only one line produces partial cosuppression, f. Discussion In studying the mutant capacity of iaaM or ipt of A. tumefaciens and of wild type to induce tumors, in several lines of transgenic plants, it has been shown that: 1) many lines suppress an incoming T-DNA iaaM gene, 2) a line suppresses an incoming T-DNA ipt gene, and 3) a line partially suppresses both ipt and iaaM genes simultaneously. By combining successful iaaH or iaaM deletion transgenes with a ipt suppression transgene in a single plant line, a transgenic plant totally resistant to gall formation can be produced. This can be done through standard seeding techniques, in which a cross between homozygous lines for individual genes is performed. Generation F2 will include plant homozygotes for both transgenes; it is expected that such plants will substantially resist the formation of galls. This expectation is supported by data from a plant that contains the hybrid ipt-iaaM transgene. This plant almost prevents the formation of galls, indicating that a single hybrid gene can produce cosuppression against two target oncogenes. The transgenes used in this study are likely to produce co-suppression against a wide variety of Agrobacterium species, as well as several strains of such species. This allows the production of plants (such as grape vines, fruit trees, tree nuts and chrysanthemums) that are resistant to gall formation.

Other studies have established that approximately 70% sequence identity is required to produce cosuppression (Maraño and Baulcombe, Plant J. 13: 537-546, 1998). Fortunately, the target T-DNA oncogenes (iaaM and ipt) are highly conserved among various strains of A. tumefaciens. Even strains of A. vi tis specific to grapes of limited host range have more than 90% sequence identity with the oncogenes of the octopine type used in our study (Otten and DeRuffray, Mol.Gen. Genet, 245: 493-505, 1994). Having illustrated and described the principles of the invention in multiple modalities and examples, it may be apparent to those skilled in the art that the invention can be modified in detail and in detail without departing from such principles. Therefore, the invention encompasses all modifications that are within the spirit and scope of the following claims.

Claims (28)

1. CLAIMS 1. A method for producing a veget cell that is resistant to gall disease, the method characterized in that it comprises transforming a plant tag with at least one nucleic acid molecule that is homologous to at least one responsible gene for causing the disease of "gill formation", the nucleic acid molecule encoding an RNA molecule selected from the group consisting of non-translatable sense RNA molecules, double-stranded RNA and untranslated double-stranded RNA molecules.
2. 2. The method according to claim 1, characterized in that the nucleic acid molecule contains at least one sequence that is homologous to at least one gene selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and fragments thereof. ,.
3. 3. A plant transformation vector, characterized in that it comprises the nucleic acid molecule according to claim 2.
4. 4. A plant cell characterized in that it is transformed with the plant transformation vector according to claim 3.
5. 5. A differentiated plant, characterized in that it comprises plant cells produced in accordance with the method according to claim 1.
6. 6. A method for producing a plant exhibiting reduced susceptibility to gall disease caused by Agrobacterium, characterized in that it comprises: transforming at least one plant cell with at least one nucleic acid molecule that is homologous to at least one gene responsible for causing the gall disease, or fragment thereof, the nucleic acid molecule encodes a selected RNA molecule of the group that cons-ifete of RNA molecules with sense, not translated bles, double-stranded RNA molecules, and molecules, of non-translatable double-stranded RNA. grow plants of the transformed plant cells; and selecting a plant showing reduced susceptibility to gall formation disease caused by Agrobacterium.
7. 7. A plant characterized because it exhibits reduced susceptibility to disease caused by Agrobacterium, produced by the method according to claim 6.
8. 8. A chimeric plant, characterized in that it comprises at least one untransformed plant cell grafted to the plant according to claim 7.
9. 9. A plant, characterized in that it is produced by sexual or asexual reproduction of the plant in accordance with claim 7.
10. 10. A seed characterized in that it is produced by itself or by crossing the plant in accordance with claim 7. *.
11. 11. A recombinant nucleic acid molecule characterized in that it comprises a nucleic acid sequence having at least 60% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, and fragments thereof, the recombinant nucleic acid molecule, when introduced into and expressed in a plant, reduces susceptibility of the plant to disease caused by Agrobacterium.
12. 12. A vector, characterized in that it comprises the recombinant nucleic acid molecule according to claim 11.
13. 13. A transgenic plant cell characterized in that it is transformed with the vector according to claim 12.
14. 14. A transgenic plant, characterized in that ** comprises at least one transgenic cell transformed with a recombinant nucleic acid molecule, according to claim 13.
15. 15. The transgenic plant according to claim 14, characterized in that the plant is selected from the group consisting of apricot, blackberry, pear, peach *, lime, cranberry, cherry, kiwi, quince, raspberry and rose.
16. 16. A chimeric plant, characterized in that it comprises at least one transgenic plant cell according to claim 13.
17. 17. A BR construction.
18. 18. A composition, characterized in that it comprises the BR construction according to claim 17.
19. 19. A / RNA transcribed from the BR construction according to claim 17.
20. 20. A vector, characterized in that it comprises the BR construction according to claim 17. 21 A plant cell transformed with the vector according to claim 20. 22. A plant, characterized in that it comprises at least one"cell": plant according to claim
21. 21. 23. A method for producing a plant cell that is resistant to gall disease, the method characterized in that it comprises: providing a BR construction; introduce the BR construction in a plant cell; and evaluate the level of gall resistance exhibited by the plant cell. 24. A plant, characterized in that it comprises at least one plant cell produced in accordance with the method according to claim 23. 25. The transgenic plant according to claim 14, characterized in that the plant is chrysanthemum. 26. The transgenic plant according to claim 1, characterized in that the plant is selected from the group consisting of conifers and poplars. 27. The transgenic plant according to claim 14, characterized in that the plant is an ornamental shrub. • 28. The transgenic plant according to claim 14, characterized in that the plant is selected from the group consisting of almonds, apples, grapes and walnut of Castilla.