US20030084478A1

US20030084478A1 - Plants having enhanced gall resistance and methods and ocmpositions for producing same

Info

Publication number: US20030084478A1
Application number: US10/187,339
Authority: US
Inventors: Lloyd Ream; Machteld Mok; Hyewon Lee
Original assignee: Oregon State University
Current assignee: Oregon State University
Priority date: 1998-11-05
Filing date: 2002-06-28
Publication date: 2003-05-01
Also published as: US6759574B1

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

CROSS REFERENCE TO RELATED CASES

This is a continuation of U.S. application Ser. No. 09/434,837, filed Nov. 4, 1999, which claims the benefit of U.S. Provisional Application No. 60/107,185, filed Nov. 5, 1998, which is incorporated herein by reference.[0001]

STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with United States government support pursuant to at least one grant from the USDA; the United States government has certain rights in the invention.

FIELD OF THE INVENTION

This invention is directed generally to compositions and methods for producing plants resistant to gall disease. The methods involve transforming plants with nucleic acid constructs encoding sense-strand untranslatable RNA molecules and/or double-stranded RNA molecules, RNA molecules that are similar to (but not identical to) certain Agrobacterium tumor-inducing genes.

BACKGROUND OF THE INVENTION

Several different recombinant DNA technologies have been employed to interfere with gene expression in plants including technologies based on ribozyme molecules, antisense RNA molecules, and co-suppression constructs. For a general discussion of ribozyme technology, antisense technology and co-suppression 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 co-suppression techniques, researchers have studied the suppression of a target gene caused by the introduction of an untranslatable sense-strand transgene. (Dougherty and Parks, Curr. Opin. Cell Biol. 7:399-405, 1995; Jorgensen et al., Science, 279:1486-1487, 1998). This method has been used to silence a number of endogenous plant genes, as well as viral genes. (Marano 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 genus Agrobacterium contains several species that cause disease in economically important plants. For example, Agrobacterium tumefaciens causes gall tumors when it infects the wounded tissue of certain dicotyledonous plants such as apple trees and grape vines (DeCleene and DeLey, Bot. Rev. 42:389-466, 1976). Pathogenic strains of Agrobacterium harbor a tumor-inducing (Ti) plasmid that carries genes essential for gall tumorigenesis (Watson et al., J. Bacteriol. 123:255-264, 1975; Van Larebeke et al., Nature 252:169-170, 1974). The transferred DNA (“T-DNA”; FIG. 1) portion of the Ti plasmid enters the cells of susceptible plants and integrates into nuclear DNA (Chilton et al., Cell 11:263-271, 1977; Willmitzer et al., Nature 287:359-361, 1980; Chilton et al., Proc. Natl. Acad. Sci. USA 77:4060-4064, 1980). Following T-DNA integration in cellular DNA, expression of three T-DNA genes, iaaM, iaaH, and ipt, leads to overproduction of the plant growth hormones auxin and cytokinin, resulting in tumorous growths referred to as “galls.” (Ream, Annual Rev. Phytopathol. 27:583-618, 1989; Winans, Micro. Revs. 56:12-31, 1992; and Zambryski, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:465-490, 1992) (FIG. 2). The iaaM, iaaH, and ipt genes have been well characterized, and the sequences of these genes are available for example in the Genbank database under accession numbers X56185, M25805, and X17428, respectively.

iaaM and iaaH are required for auxin production: the enzyme encoded by iaaM converts tryptophan into indole acetamide, which the enzyme encoded by iaaH converts into indole acetic acid, an auxin (Schroeder et al., Eur. J. Biochem. 138:387-391, 1984; Thomashow et al., Proc. Natl 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). Loss-of either enzyme prevents auxin production. ipt is required for cytokinin production; the enzyme encoded by this gene converts adenosine monophosphate into isopentenyl adenosine monophosphate, a cytokinin (Akiyoshi et al. Proc. Natl. Acad. Sci. USA 81:5994-5998, 1984; Barry et al., Proc. Natl. Acad. Sci. USA 81:4776-4780, 1984; and Buchmann et al., EMBO J 4:853-859, 1985). Inactivation of ipt and either one of the two auxin-biosynthesis genes on the Ti plasmid will reduce gall formation (Ream et al., Proc. Natl. Acad. Sci. USA 80:1660-1664, 1983).

Additionally, galls can be formed in cases in which just the cytokinin pathway, or only the auxin biosynthesis pathway, is functional. In these cases the galls 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 galls. In contrast, plants infected with strains of the bacteria that induce only the production of cytokinin produce shooty galls.

To date, adequate means do not exist to control gall disease on grape vines, fruit trees, nut trees, chrysanthemums, roses, cane berries, ornamental shrubs, and other nursery crops. Inoculation of plants with Agrobacterium radiobacter strain K84 affords some protection against specific strains of A. tumefaciens (Moore, Microbiol. Sci. 5:92-95, 1988); however, gall disease remains a multimillion dollar worldwide problem. Arabidopsis thaliana plants resistant to gall disease have been produced by a traditional genetic approach (Nam et al., Plant Cell 9:317-333, 1997), but this strategy currently is not applicable to plants in which gall is a problem.

This invention is also directed towards a new, more effective method of producing plants that are substantially resistant to gall disease caused by bacteria.

SUMMARY OF THE INVENTION

The present invention provides compositions that can be used to prevent tumor (gall) formation in plants infected with bacterial pathogens such as Agrobacterium tumefaciens and other species of Agrobacterium. These compositions comprise nucleic acid molecules that, when introduced into plants, can produce transgenic plants having enhanced resistance to tumors caused by such pathogens. The invention also encompasses transgenic plants that contain these nucleic acid molecules.

In general terms, the invention involves the use of nucleic acid constructs that encode untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA molecules that share specified high levels of sequence identity with target genes in the bacterial pathogen. For example, the constructs may encode one or more types of untranslatable RNA molecules sharing high levels of sequence identity with one or more of the iaaM, iaaH, or ipt tumor genes of Agrobacterium. The invention also provides nucleic acid constructs that encode multiple different untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA molecules, including both sense and antisense RNA molecules. For example, one construct provided by the invention encodes untranslatable RNA forms of both the ipt and iaaM genes of Agrobacterium tumefaciens; this construct can suppress both shooty and necrotic gall formation in plants caused by a pathogenic strain of A. tumefaciens infecting the plant.

The untranslatable RNA molecules appear to inhibit gall formation by triggering sequence-specific destruction of RNA molecules encoded by certain “target genes” of the infecting pathogen in the plant. However, the mechanism by which this occurs is not fully understood. For optimal efficacy, the untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA molecules should be of a sufficient length and share a minimum degree of sequence identity with the target bacterial gene such that disease resistance is conferred to the host cell. The level of disease resistance conferred to the host cell by the RNA molecule can be determined 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. However, enhanced tumor suppression may be obtained by increasing the length of the untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA molecules (e.g., to at least 100, or at least 200 nucleotides) and/or by enhancing the level of sequence identity with the target gene (e.g., to at least 70%, 75%, 80%, or 90% sequence identity).

The invention also provides transgenic plants containing one or more of the above-described nucleic acid constructs. These constructs may be introduced into any plant species, but commercial crop plants for which Agrobacterium is a significant pathogen are expected to be particularly benefited by this technology.

The invention also provides chimeric plants that include both untransformed cells and transformed cells. These plants display “conferred resistance” in that the transgenic portion confers disease resistance to the non-transgenic portion. The chimeric plants are particularly useful for generating resistant, but not transgenic, fruits and other consumable portions of plants that are not transgenic.

SEQUENCE LISTINGS

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NO: 1 is a primer used to produce the mutant iaaM 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 mutant ipt gene.

SEQ ID NO: 5 is the DNA sequence that encodes an untranslatable RNA molecule having a high sequence identity with the Agrobacterium ipt gene.

SEQ ID NO: 6 is an amino acid sequence that is encoded by the untranslatable RNA molecule depicted in SEQ ID NO: 5.

SEQ ID NO: 7 is a DNA sequence encoding the untranslatable RNA molecule having a high sequence identity with the Agrobacterium iaaM gene.

SEQ ID NO: 8 is an amino acid sequence encoded by the untranslatable RNA molecule depicted in SEQ ID NO: 7.

SEQ ID NO: 9 is the nucleic acid sequence of the ipt/iaaMuntranslatable construct.

SEQ ID NO: 10 is the nucleic acid sequence of the iaaM gene.

SEQ ID NO: 11 is the nucleic acid sequence of the iaaH gene.

SEQ ID NO: 12 is the nucleic acid sequence of the ipt gene.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of the [0029] A. tumefaciens transferred DNA (“T-DNA”) that contains three oncogenes: iaaM, iaaH, and ipt. Triangles represent T-DNA border sequences which delimit T-DNA ends by initiating and terminating T-DNA transfer (Peralta and Ream Proc. Natl. Acad Sci. 82:5112-5116, 1985). Arrows show direction and length of messenger RNAs (mRNAs) produced in gall tumors.
FIG. 2 is a diagram of the auxin and cytokinin biosynthetic pathways encoded by the [0030] A. tumefaciens T-DNA oncogenes.
FIG. 3 is a diagram of the binary plant transformation vector (pPEV6) (panel A) and of mutant transgenes that can be used to elicit co-suppression of T-DNA oncogenes. “RB” and “LB” denote right and left T-DNA border sequences, respectively. NOS prom/NPTII denotes the nopaline synthase promoter fused to the neomycin phosphotransferase II gene (conferring kanamycin resistance). 5′ UTR and 3′ UTR denote the 5′ and 3′ untranslated regions, respectively, associated with the cauliflower mosaic virus promoter ([0031] CaMV 35S Pro). Arrowheads denote respective restriction sites for XbaI, BamHI, and EcoRI; numbers indicate respective coordinates relative to the vector sequence. Premature stop codons are shown in bold, whereas natural start codons are shown in plain text.
FIG. 4 illustrates the most likely scenario for the creation of double-stranded RNA. The sense strand is transcribed from the [0032] CaMV 35S promoter and the anti-sense strand is transcribed from the NOS promoter.
FIGS. [0033] 5(A)-5(F) depict several representative illustrations of bacterial-resistance constructs (BR construct) according to the invention. Promoters are designated by the capital letter “P” and the direction of expression is denoted by respective arrows. DNA sequences encoding untranslatable RNA molecules that target specific genes are indicated; however, one of ordinary skill in the relevant art will appreciate that untranslatable RNA molecules targeting other genes can be substituted. FIG. 5(A) shows a BR construct in which one promoter (designated P₁) is used to express (direction of expression is indicated by the arrows) a DNA sequence encoding the sense-strand of a sequence that targets the ipt gene and the sense-strand of a sequence targeting 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 antisense-strand of the ipt gene. Thus, the combination of both promoters will generate a double-stranded RNA that contains a section targeting the ipt gene and a section targeting the iaaMgene. FIG. 5(B) shows a BR construct that contains one promoter (P₁) that expresses a DNA sequence encoding several different untranslatable RNA sequences that target both the ipt gene and the iaaM gene. Starting at the 5′ end of the construct, the promoter P₁drives the expression of a sense-strand targeting the ipt gene, a sense-strand targeting the iaaM gene, an antisense-strand targeting the iaaM gene, and a sense-strand targeting the ipt gene. This sequence can fold back on itself, and thus generate a double-stranded RNA molecule comprising regions homologous to both the ipt and iaaM gene. FIG. 5(C) shows another variation of a BR construct. This construct comprises independent promoters, P₁and P₂, to drive the expression of an untranslatable RNA molecule in both the sense and anti-sense orientations, respectively. Two additional promoters (P₃and P₄) are utilized for the expression of the sense and anti-sense expression of a second untranslatable RNA. FIG. 5(D) shows another variation of the BR construct. This construct comprises one promoter (P₁) to express first the sense-strand and then the antisense-strand of an untranslatable RNA molecule targeting the ipt gene. This construct also comprises a second promoter to express both the sense-strand and antisense-strand of an untranslatable RNA targeting the iaaM gene. FIGS. 5(E) and 5(F) illustrate additional variations that can be used to express single-stranded untranslatable RNA molecules.

DETAILED DESCRIPTION

I. Definitions and Abbreviations: [0034]
Untranslatable RNA molecule: An RNA molecule, either single-stranded or double-stranded, that is based on a naturally occurring RNA molecule but which, as a result of genetic manipulation, is incapable of being translated to yield the full-length protein encoded by its naturally occurring counterpart. For example, such RNA can be rendered untranslatable through the introduction of one or more premature termination codons (TAA, TAG, or TGA), or by introduction of a mutation that shifts the reading frame. Alternatively, for example, the RNA may be rendered untranslatable by the deletion of the AUG translation-initiation codon, or of the AAUAAA polyadenylation signal. The RNA may also be rendered untranslatable by simply removing a portion of the cDNA encoding the full-length protein. Thus, even if the sequence were translated, it would produce a truncated version of its naturally occurring counterpart. [0035]
Antisense-strand RNA: An RNA molecule that is complementary to a messenger RNA (which represents a corresponding “sense” strand). [0036]
Disease-resistant transgenic plant: A “disease-resistant transgenic plant” as used herein is a transgenic plant exhibiting more resistance to gall disease than a corresponding non-transgenic plant. [0037]
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence whenever the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous. [0038]
Recombinant: A recombinant nucleic acid is one having a sequence that is not naturally occurring in the organism in which it is expressed, or has a sequence made by an artificial combination of two otherwise-separated, shorter sequences. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. “Recombinant” is also used to describe nucleic acid molecules that have been artificially manipulated, but that contain the same control sequences and coding regions that are found in the organism from which the gene was isolated. [0039]
Sense-strand RNA: An RNA molecule that can serve directly as messenger RNA, i.e., that can be translated by ribosomes. [0040]
Double-stranded RNA: A “double-stranded RNA” is an RNA molecule that contains at least a 25-bp sequence including both a sense-strand and an antisense-strand. Double-stranded RNA molecules are additionally characterized by the level of complementarity between the sense-strand and the antisense-strand. Complementarity refers to the amount of base pairing between the two sequences. For example, the [0041] sequence 5′-AUCGGAUAGU-3′ is 100% complementary to the sequence 5′-ACUAUCCGAU-3′ and 70% complementary to the sequence 5′-GAAAUCCGAU-3′. The double-stranded RNA should be of a sufficient length and have a sufficient level of complementarity such that it confers disease resistance to the host cell (disease resistance can be determined using the assays described below). For example, the double-stranded RNA may have as little as 60% complementarity. Generally, however, double-stranded RNA sequences will have at least 70%, 75%, 80%, or 90% complementarity.
Vector: A “vector” is a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences, such as an origin of replication, that permit the vector to replicate in a host cell. A vector may also include one or more screenable markers, selectable markers, or reporter genes and other genetic elements known in the art. [0042]
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. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences. [0043]
Methods for aligning sequences for comparison purposes are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, [0044] Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. 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 Biosciences 8:155-165, 1992; and Pearson et al., Methods in Molecular Biology 24:307-331, 1994. Altschul et al., J. Mol. Biol., 215:403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST™, Altschul et al. [0045] 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, blastx, tblastn and tblastx. BLAST™ and a description of how to determine sequence identity using this program can also be accessed on the Internet.
For comparisons of amino acid sequences of greater than about 30 amino acids, the “[0046] Blast 2 sequences” function in the BLAST™ program is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per-residue gap cost of 1). When aligning short peptides (fewer than about 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins having even greater similarity to the reference sequences will show increasing percentage identities when assessed 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.
A first nucleic acid is “substantially similar” to a second nucleic acid if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), nucleotide sequence identity occurs in at least about 60%, 75%, 80%, 85%, 90% or 95% of the nucleotide bases (As used herein, “optimally aligned” sequences exhibit a maximal possible sequence identity). Sequence similarity can be determined by comparing the nucleotide sequences of two nucleic acids using the BLAST™ sequence analysis software (blastn) available from The National Center for Biotechnology Information. Such comparisons may be made using the software set to default settings (expect=10, filter=default, descriptions=500 pairwise, alignments=500, alignment view=standard, gap existence cost=11, per residue existence=1, per residue gap cost=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 greater when optimally aligned and compared using BLAST™ software (blastp) using default settings. [0047]
Bacterial resistance construct (BR construct): A “bacterial resistance construct” (abbreviated “BR construct”) is a sequence of DNA that incorporates at least one promoter and at least one DNA sequence encoding an untranslatable RNA (either double-stranded RNA, single-stranded RNA or a combination thereof). The arrangement of the promoter(s) in relationship to the DNA sequence(s) can vary. Representative configurations are illustrated in FIGS. [0048] 5(A)-5(G). FIG. 5(A) shows a BR construct in which one promoter (designated P₁) is used to express (direction of expression is indicated by the arrows) a DNA sequence encoding the sense-strand of a sequence that targets the ipt gene and the sense-strand of a sequence targeting 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 antisense-strand of the ipt gene. Thus, the combination of both promoters will generate a double-stranded RNA that contains a section targeting the ipt gene and a section targeting the iaaM gene. FIG. 5(B) shows a construct that contains one promoter (P₁) that expresses a DNA sequence that encodes several different untranslatable RNA sequences targeting both the ipt gene and the iaaM gene. Starting at the 5′ end of the construct, the promoter P₁drives the expression of a sense-strand targeting the ipt gene, a sense-strand targeting the iaaM gene, an antisense-strand targeting the iaaM gene, and a sense-strand targeting the ipt gene. This sequence can fold back on itself, and thus generate a double-stranded RNA molecule comprising regions homologous to both the ipt and iaaMgene. FIG. 5(C) shows another variation of a BR construct. This construct comprises independent promoters, P₁and P₂, to drive the expression of an untranslatable RNA molecule in both the sense and anti-sense orientations, respectively. Two additional promoters (P₃and P₄) are utilized for the expression of the sense and anti-sense strands of a second untranslatable RNA. FIG. 5(D) shows another variation of the BR construct. This construct comprises one promoter (P₁) to express first the sense-strand and then the antisense-strand of an untranslatable RNA molecule targeting the ipt gene. This construct also comprises a second promoter to express both the sense-strand and antisense-strand of an untranslatable RNA targeting the iaaM gene. FIGS. 5(E) and 5(F) illustrate additional variations that can be used to express single-stranded untranslatable RNA molecules. FIGS. 5(A)-(F) are intended to illustrate a few of various different promoter/DNA sequence combinations that are possible. A person of ordinary skill in the art will appreciate that many additional combinations can be used in BR constructs according to the invention.
II. General Description [0049]
The present method provides new and effective methods for suppressing the expression of Agrobacterium oncogenes. In particular, this invention is directed at, inter alia, producing plants that are capable of substantially inhibiting the formation of bacterially induced galls. These plants are produced by selecting a target gene of Agrobacterium origin, designing a nucleic acid (typically DNA) BR construct encoding untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA molecules having a high sequence identity to the target gene, and introducing at least one of these BR constructs into a host cell. [0050]
a. Selection of the Target Gene [0051]
There are many species of Agrobacterium, for example [0052] Agrobacterium tumefaciens, Agrobacterium vitis, 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 target tumor-inducing Agrobacterium genes will likely confer resistance to at least one (typically multiple) species of Agrobacterium.
b. Producing a Nucleic Acid Construct Encoding an Untranslatable RNA [0053]
Practicing the present invention requires the manipulation of a DNA sequence using molecular biological techniques to produce the desired nucleic acid construct. The DNA sequence can be manipulated using standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence-alteration via single-stranded bacteriophage intermediate or with the use of specific oligonucleotides in combination with PCR. Details of these techniques are provided in standard laboratory manuals such as Sambrook et al., [0054] Molecular Cloning: A Laboratory Manual, 2^nded., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., Short Protocols in Molecular Biology, 2^nded., John Wiley and Sons, 1992.
Suppression of an oncogene using a method according to this invention is achieved by expressing, in a host cell, molecules of a BR construct having high sequence identity to the target gene(s). At least one molecule of the BR construct having substantial sequence identity to the target gene(s) is introduced into a host cell. The BR construct can be delivered into the host cell genome by delivering into the host a vector cell containing the BR construct. [0055]
Application of the present invention to construction of a BR construct desirably involves the selection of appropriate control sequences such as promoters, enhancers, and 3′-untranslated regions. The choice of which type of control sequence(s) to use in a BR construct will depend upon the particular tissue in which the BR construct will be expressed and the particular sequence(s) of the target gene(s). The variety of plant that is selected for transformation will also affect the choice of the control sequence(s), the vector, and the transformation method. [0056]
Constructing a suitable BR construct involves the selection of a DNA sequence that will be used to target at least one specific bacterial gene. Generally, the DNA sequence will encode an untranslatable RNA having at least 70% sequence identity with the target bacterial gene(s) (i.e., the gene(s) that will be suppressed). [0057]

In producing the BR construct, a DNA molecule homologous to a target bacterial gene can be manipulated to render a single-stranded RNA or double-stranded RNA molecule encoded by the DNA molecule untranslatable. This can be done by, e.g., introducing any of various stop signals into the open reading frame of the DNA molecule to produce the BR construct. For example, the BR construct can be configured to contain one or more premature stop codons. A “stop codon” is a codon of the genetic code that signals ribosomes to terminate 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.

TABLE 1


The Genetic Code

First		Third
Position	Second position	Position

(5′ end)	T	C	A	G	(3′ end)

T	Phe	Ser	Tyr	Cys	T
	Phe	Ser	Tyr	Cys	C
	Leu	Ser	STOP (och)	STOP	A
	Leu	Ser	STOP (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	Ile	Thr	Asn	Ser	T
	Ile	Thr	Asn	Ser	C
	Ile	Thr	Lys	Arg	A
	Met	Thr	Lys	Arg	G
G	Val	Ala	Asp	Gly	T
	Val	Ala	Asp	Gly	C
	Val	Ala	Glu	Gly	A
	Val (Met)	Ala	Glu	Gly	G

In the context of the foregoing discussion, a “premature” stop codon is located at a position transcriptionally upstream of a naturally occurring stop codon. Generally, the premature stop codon is positioned within 100 nucleotides of the translation-start codon, and typically within 50 nucleotides of the translation-start codon. [0059]
One or more stop codons can be inserted into 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: [0060]
. . . CTT AAC TGG TCC . . . [0061]
would normally produce a corresponding RNA translated by ribosomes to produce the polypeptide: [0062]
. . . Leu Asn Trp Ser . . . [0063]
The corresponding RNA can be rendered untranslatable by introducing a stop codon, such as TAA, into the DNA sequence. For example, the modified DNA sequence: [0064]
. . . CTT TAA AAC TGG TCC . . . [0065]
encodes: [0066]
. . . Leu STOP [0067]
Alternatively, in the foregoing hypothetical DNA sequence, substitution of the ninth nucleotide (guanosine (G)) by adenosine (A) will result in the creation of a premature stop codon, rendering the corresponding RNA untranslatable, i.e., the following DNA sequence is produced: [0068]
. . . CTT AAC TGA TCC . . . [0069]
which encodes: [0070]
. . . Leu Asn STOP [0071]
Alternatively, the addition or deletion of one or more bases into or from, respectively, the DNA sequence (except multiples of three bases) can be used to displace the reading frame of the DNA sequence which can render the corresponding RNA molecule untranslatable. For example, the introduction of a single nucleotide (G) into the first codon of the foregoing hypothetical DNA sequence creates a new reading frame containing a premature stop codon: [0072]
. . . CTG TAA CTG GTC C . . . [0073]
which encodes: [0074]
. . . Leu STOP [0075]
An alternative manner of rendering an RNA molecule untranslatable is to remove the translation-initiation codon (usually ATG) from the corresponding DNA sequence. However, this method may result in use by ribosomes of an ATG codon elsewhere in the DNA sequence to initiate translation. Removal of the initiation codon ATG is, therefore, desirably used in conjunction with inserting one or more premature stop codons into a DNA sequence. [0076]
Another method of introducing a premature stop codon is by truncating the subject DNA sequence. This can be done, for example, by using a restriction enzyme to excise a portion of a target gene and then cloning only the excised portion of the gene into a vector. Transcription of the vector will produce a segment of homologous RNA effective to produce a co-suppression phenomenon, but will not produce a fully processed protein. Currently, the mechanism by which co-suppression works is not known. However, the phenomenon has been previously described in detail (Lindbo and Dougherty, [0077] Virology 189:725-733, 1992).
One or more of the above approaches may be used to render an RNA molecule encoded by a BR construct untranslatable. It will be appreciated that a very large number of sequence changes can be made in a suitable manner, the precise nature of which may depend on the actual DNA sequence being manipulated, to render the sequence of the corresponding RNA untranslatable. In certain embodiments, more than one stop codon may be introduced into the DNA sequence. For example, the resulting BR construct may contain a DNA sequence encoding untranslatable RNA that has been altered to contain at least one stop codon situated early in the open reading frame; the open reading frame is thus “truncated.”[0078]
Many standard DNA mutagenesis techniques are available to modify DNA sequences in a manner as described above to produce the BR construct. The mutagenesis techniques include, for example, M13 primer mutagenesis. Details of such techniques are provided in Sambrook et al., Chapter 15, [0079] Molecular Cloning: A Laboratory Manual, 2^nded., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989; and in Ausubel et al., Chapter 8, Short Protocols in Molecular Biology, 2^nded., John Wiley and Sons, 1992.
According to another aspect of the invention, double-stranded RNA molecules are provided that are homologous to a target bacterial gene. Each strand of the double-stranded RNA can be produced from separate BR constructs, wherein a first BR construct encodes the antisense-strand and a second BR construct encodes the sense-strand. Upon transcription of the first and second BR constructs, the resulting sense-strand and corresponding antisense-strand form double-stranded RNA. [0080]
Alternatively, the sense-strand and antisense-strand can be produced from a single BR construct. The single BR construct can utilize two distinct promoters to initiate the expression of the antisense-strand and the sense-strand, or one promoter to drive expression of both strands. When a single promoter is utilized, both the sense-strand and the antisense-strand are normally produced as a single transcript, wherein the RNA sequence will fold back on itself to create a double-stranded RNA. Similarly, multiple combinations of promoters, antisense-strands, and sense-strands can be encoded on the same BR construct. [0081]
Double-stranded RNA can also be produced by transforming cells with a BR construct that encodes the sense-strand, a self-cleaving ribozyme, and the antisense-strand all on a single transcript. Upon transcription of such a BR construct, the sense-strand, the self-cleaving ribozyme, and the antisense-strand are produced. The self-cleaving ribozyme sequence folds back on the resulting transcript and splices the transcript between the sense- and antisense-strands, generating two independent complementary strands of RNA. [0082]
A proposed mechanism for suppressing a target bacterial gene by introducing into a host cell molecules of a BR construct having one or more sequences homologous to at least a portion of the target gene typically involves the activity of an inducible, cellular activity that degrades specific RNA sequences. This mechanism requires that the untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA encoded by the BR construct be recognizable by the RNA-specific cellular activity. To such end, the target DNA sequence and the DNA sequence of the BR construct desirably share a sequence identity. [0083]
In the Examples presented below, the DNA sequences of the respective BR constructs are derived directly from corresponding target bacterial nucleotide sequences. The sequences of the respective BR constructs differ from the corresponding target sequences primarily only by the presence (in the sequences of the BR constructs) of premature stop codons inserted by mutagenesis involving one or more frame-shift mutations. Thus, in these examples, the BR constructs have over 90% sequence identity to the corresponding target DNA sequences. However, a BR construct needs only to have sufficient sequence identity to the target gene so that the BR construct confers disease resistance to the host cell. Therefore, a BR construct that encodes an RNA molecule having less than 90% sequence identity to the corresponding target DNA sequence can be used. In any event, the BR construct typically has 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. [0084]
The following examples also describe BR constructs in which the DNA sequence corresponds to 700 base pairs of the ORF (open reading frame) of the target gene. It will be understood, however, that the untranslatable RNA provided by the BR construct need not correspond specifically to 700 base pairs of the ORF of the target gene; substantially shorter sequences of untranslatable RNA may also be effective. [0085]
The BR construct should be sufficiently long and share a sufficient level of sequence identity to the target gene such that the BR construct confers disease resistance to the host cell. The ability of a particular BR construct to confer disease resistance to a host cell can readily be determined by using the assay described below, in which a transgenic plant is challenged with a bacterial pathogen. Typically, the ORF of the DNA sequence of the BR construct is at least 25-100 nucleotides in length, although longer sequences, such as 100-250, 250-500, or greater than 500 nucleotides may be used and may provide superior results. In embodiments where shorter sequences (i.e., less than 250 nucleotides) are utilized, the respective BR constructs will likely share a high degree of sequence identity (e.g., at least 60% sequence identity) with the target gene sequence. [0086]
The present invention can be used to suppress the expression of a bacterial tumor gene in a plant cell. To such end, the plant cell is transformed with a BR construct having a high sequence identity to a target bacterial gene. The molecule of the BR construct encodes one or more untranslatable single-stranded RNA and/or double-stranded RNA molecules. The BR construct can 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 can comprise a sequence of at least 100 nucleotides in length having at least 60% sequence identity to the target bacterial gene. Furthermore, the BR construct can comprise a nucleic acid sequence of at least 250 nucleotides in length having at least 60% sequence identity to the target bacterial gene. [0087]
The present invention is not limited to the suppression of a single target bacterial gene. Rather, it is possible to suppress, according to the invention, the expression of multiple target genes using one or more BR constructs that comprise DNA sequences corresponding to several respective genes expressed in the target cell. This approach can be used to enhance 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 gall on a plant by a pathogenic bacterium infecting the plant can be reduced or eliminated by the introduction into the cells of the plant, a BR construct having a high sequence identity to at least one gene of the infecting bacterium. This approach allows the production of transgenic plants resistant to the bacterium. [0088]
C. Vectors [0089]
It will be appreciated that the efficacy of the present invention depends on transcription of the DNA sequence of the BR construct introduced into a host plant cell. Transcription of a DNA sequence into messenger RNA is preferably effected 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 promoter sequences and 3′-regulatory sequences have been identified and characterized in a wide range of viral, bacterial, fungal, plant, and animal genes. Therefore, the selection of suitable promoter and a suitable 3′-regulatory sequence is within the capability of a person of ordinary skill in the relevant art. [0090]
After choosing and incorporating the appropriate regulatory DNA sequence(s) into the BR construct, the BR construct is incorporated into a suitable vector for introduction into the host cell. The choice of regulatory sequences, as well as the choice of a suitable transformation vector will depend upon the type of host cell into which the BR construct is to be introduced and upon the desired transformation method for the host cell. Vectors suitable for transforming plant cells (derived from either monocotyledonous or dicotyledonous plants) are described below. Transformation of both monocotyledonous and dicotyledonous plant cells is now routine and the selection of the most appropriate transformation technique will be determined by the skilled practitioner. [0091]
A number of promoters that are active in plant cells have been described in the literature. Promoters that are known to cause transcription of DNA in plant cells can be used to suppress gene expression in plants. Such promoters can be obtained from plant cells or from plant viruses and include, but are not limited to, the CaMV (cauliflower mosaic virus) 35S promoter. It is desirable that the chosen promoter be capable of causing sufficient expression of the BR construct to produce sufficient RNA to suppress expression of the target gene. The amount of RNA needed to suppress expression of a particular target gene may vary from one target gene to another, expression level of the particular target gene, and the plant type. Accordingly, whereas 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. [0092]
The vector can be configured to optimally exploit the promoter and termination sequences in the target gene itself. Used in this way, the target-gene promoter is not altered and the subsequent stop signals are introduced into the ORF of the target gene. Additionally, the termination signals of the target gene can be left intact. This configuration permits increasing the transcription of untranslatable RNA and/or double-stranded RNA whenever the factors necessary for transcription of the target gene are present. [0093]
The promoter(s) used in a BR construct according to the invention may be modified, if desired, to include additional regulatory sequence(s). Such additional regulatory sequences can confer, e.g., specificity to the respective promoter region. For example, the small subunit of the ribulose bis-phosphate carboxylase (“ss RUBISCO”) gene is expressed in plant leaves but not in root tissues. A sequence motif that represses the expression of the ss RUBISCO gene in the absence of light (to create a promoter which is active in leaves but not in root tissue), has been identified. This and/or other regulatory sequences such as the HSP70 leader sequence from petunia, may be ligated to promoters such as the [0094] CaMV 35S, or figwort mosaic virus (FMV) promoter, to modify the expression patterns of a gene.
The 3′-nontranslated region (that functions as a polyadenylation site for RNA in plant cells) of a gene can also be used in the present invention. Such 3′-non-translated regions include, but are not limited to, the 3′-transcribed (but non-translated) region of the [0095] CaMV 35S gene, the pea-rbcS E9 3′-non-translated region, the NOS 3′-non-translated region, and one or more of the 3′-transcribed, non-translated (but non-translated) regions containing the polyadenylation signals of the tumor-inducing (Ti) genes of Agrobacterium, such as the tumor morphology large (tml) gene.
d. Plant Species [0096]
Gall formation in plants can be induced by any of various organisms. For example, galls can be induced by bacteria from the genus Agrobacterium, and certain species of Pseudomonas, Rhizobacter, and Rhodococcus. [0097]
In the case of Agrobacterium, gall formation is caused by genes introduced into the plant cells on the Ti plasmid provided by an infecting bacterium. There are a variety of naturally occurring Ti plasmids that can give rise to different types of galls. Galls are normally caused by [0098] A. tumefaciens which infects over 391 genera of plants including shrubs, fruit trees, nut trees, tuber-producing plants, conifers, grape vines, chrysanthemums, roses, cane berries, nursery crops, and commercially important plants (Bradbury, J. F. Guide to Pathogenic Bacteria. CAB International, Slough, Britain, 1986). Hairy root is another type of gall induced by A. rhizogenes, that primarily affects the roots of apple trees. Finally, Agrobacterium rubi can infect both raspberries and black berries, and give rise to cane galls. (Agrios, Plant Pathology, Academic Press, 1997).
e. Transformation [0099]
A BR construct according to the present invention is introduced into a plant cell using a suitable vector and transformation method as described below. The resulting transformed cells can then be allowed to regenerate into whole plants. Any of various conventional methods employed for transforming 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 plant types. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome mediated transformation; polyethylene glycol-mediated transformation; transformation using viruses; microinjection of plant cells; microprojectile bombardment of plant cells and [0100] Agrobacterium tumefaciens (AT)-mediated transformation. The DNA sequences comprising the CaMV 35S promoter and CaMV 35S non-translated 3′ region and the mutated cDNA encoding a untranslatable RNA can be combined in a single cloning vector. This vector can be transformed subsequently into, e.g., cultured tobacco cells.
f. Conferred Resistance [0101]
The mechanism of co-suppression is unknown. However, transgenic tissue containing a co-suppression gene can be grafted onto a non-transgenic plant. The graft then confers resistance to the non-transgenic plant (Jorgensen et al., [0102] Science, 279:1486-87, 1998). Furthermore, this “conferred resistance” can be established by either grafting a scion (upper vegetative tissue) onto a transgenic stock (lower vegetative tissue), or by grafting a transgenic scion onto a non-transgenic stock. 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 into existing plants. Thus, a mature plant can be made resistant to gall formation by grafting transgenic tissue onto the plant. Furthermore, several grafts can be placed independently on the plant to produce resistance in the plant to several different bacterial pathogens. [0103]
A grafting protocol can be made more flexible by utilizing cross-species grafting. Thus, a graft from, e.g., almond ([0104] Prunus amygdalus) can be placed on stock from peach (Prunus persica). Such flexibility increases the range of plants for which a given transgenic tissue can be used. For example, after almond tissue containing the desired BR construct 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 that a transgene made according to the invention will be introduced into the environment. This is because the resistance is derived from the graft itself and the rest of the plant remains free of the transgene. Therefore, the fruits and seeds of the plant can be left non-transgenic, so as not to transmit the gene. [0105]
By preventing a transgene from being systemically introduced into the plant, products for human consumption can be produced by the plant that are not transgenic. [0106]

III. EXAMPLES

a. Construction of BR Constructs Encoding Untranslatable RNA molecules [0107]

To produce a DNA construct capable of eliciting co-suppression, the iaaM and ipt oncogenes were modified by changing the third codon of each gene to a stop codon and introducing a frameshift mutation downstream of each respective introduced stop codon, thereby creating additional stop codons in the reading frame (FIG. 3). The polymerase chain reaction (PCR) primer oligonucleotides used to create these mutations in the DNA construct are described in Table 2. The primers used to modify the ipt gene resulted in the deletion of the 5′ C of the third codon and changed the 3′ G of the third codon to an A. The iaaM modifications introduced the stop sequence TGA at the third codon and the two bases following the third codon were deleted. A second DNA construct was made that contained a hybrid transgene configured to elicit co-suppression of both ipt and iaaM. The second DNA construct was made by digesting a vector (containing the ipt-stop mutation) and a vector containing the iaaM-stop mutation with BamH1. The fragment containing the iaaM-stop mutation was then ligated into the fragment containing the ipt-stop mutation, thereby yielding a BR construct containing the mutant iaaM gene inserted into the mutant ipt gene (FIG. 3). The sequence of each DNA construct was verified, and the BR construct was inserted into a plant-transformation vector (pPEV6; Lindbo and Dougherty, Mol. Plant-Microbe Interact. 5:144-153, 1992) oriented such that the sense-strand of each mutant was expressed from the cauliflower mosaic virus (CaMV 35S) promoter (FIG. 3).

TABLE 2


PCR Primers Used to Produce Mutant iaaM and ipt Genes

Primers	Sequence

iaaM stop

	5′ primer	5′-CGGGATCCATGTCATGAACCTCTCCTTG
		ATAAC-3′
		(SEQ ID NO: 1)
	3′ primer	5′-CGGGATCCTGCGACTCATAGT-3′
		(SEQ ID NO: 2)
ipt stop	5′ primer	5′-GAAGATCTGATCATGGACTGAATCTAAT
		TTTCGGTCC-3′
		(SEQ ID NO: 3)
	3′ primer	5′-GAAGATCTGATCACTAATACATTCCGAA
		CGG-3′
		(SEQ ID NO: 4)

b. Construction of Double-Stranded RNA and Untranslatable Double-Stranded RNA Vectors [0109]
A vector encoding an iaaM-stop mutation (i.e., a DNA construct modified to incorporate a stop codon at the third codon, followed by a two base pair deletion) exhibited evidence of bacterial resistance. This resistance was derived from the production of double-stranded RNA by transcription of the vector (FIG. 4). The sense-strand of the double-stranded RNA was transcribed from the [0110] CaMV 35S promoter and the antisense-strand was probably transcribed from the nopaline synthase promoter (NOS). Thus, this vector can produce a long double-stranded RNA that is approximately 2,000 bp long. Evidence of such a double-stranded RNA was found in northern blots that showed partially degraded antisense-strands ranging in size from 3.4 Kb to less than 0.24 Kb. As is depicted in FIG. 4, the double-stranded RNA appeared to include an approximately 2,000 bp long region that was 100% homologous to the iaaM sequence. Therefore, bacterial resistance observed in iaaM-transgenic plants is probably attributable to the presence of the double-stranded RNA. This resistance does not appear to have been due solely to the presence of the introduced stop codon.
Double-stranded RNA is capable of suppressing endogenous plant genes, as well as suppressing viral infection (Ruiz et al., [0111] Plant Cell 10:937-946, 1998). However, the suppression, via double-stranded RNA and/or untranslatable RNA, of disease symptoms caused by bacteria, such as Agrobacterium, has previously not been shown.
C. Production of Plants Homozygous for Untranslatable RNA Transgenes [0112]
Mutant iaaM (SEQ ID NO: 7), ipt (SEQ ID NO: 5), and ipt/iaaM genes (SEQ ID NO: 9) were introduced separately into haploid tobacco ([0113] N. tabacum, “Burley” and “Kentucky” cultivars). The chromosomes of kanamycin-resistant transformants of these plants were doubled to produce plants homozygous for each transgene. These plants were the subjects of initial tests, which were repeated on the F1 progeny of self pollinations.
d. Gall-resistant Plant Phenotypes [0114]
Putative iaaM-suppressing plants (designated “TDP1” lines) were challenged with ipt-mutant [0115] A. tumefaciens (strain 338; Garfinkel et al., Cell 27:143-153, 1981). Of 63 lines tested, 35 lines, including TDP1-B7 & TDP1-B17, suppressed the wild-type iaaM gene on A. tumefaciens T-DNA introduced into such plants, thereby preventing tumorous growth in the respective plants. When inoculated with wild-type A. tumefaciens (strain A348; Garfinkel et al., Cell 27:143-153, 1981), each of the 35 lines produced “shooty” galls typical of cytokinin-driven tumors, such as those induced on wild-type plants inoculated with iaaM-mutant A. tumefaciens (Ream et al., Proc. Natl Acad. Sci. USA 80:1660-1664, 1983; and Garfinkel et al., Cell 27:143-153, 1981). Thus, the plants that produced shooty galls exhibited the phenotype expected of a transgenic plant capable of suppressing iaaM.
Putative ipt-suppressing plants (designated “CW1” lines) were challenged with iaaM-mutant [0116] A. tumefaciens (strain 328; Garfinkel et al., Cell 27:143-153, 1981). Of 45 lines investigated, one (designated “CW1-K27”) clearly suppressed the wild-type ipt gene on introduced A. tumefaciens T-DNA. Another 6 lines exhibited similar results in initial tests on the original transgenic plants, but these lines were lost before seeds were obtained. The ipt-suppressing plant (CW1-K27), when inoculated with wild-type A. tumefaciens, produced small necrotic galls typical of auxin-driven tumors, such as those induced on wild-type plants inoculated with ipt-mutant A. tumefaciens (Ream et al., Proc. Natl. Acad. Sci. USA 80:1660-1664, 1983; Garfinkel et al., Cell 27:143-153, 1981). There were 30 lines tested (designated “CW4” lines) that contained the hybrid ipt-iaaMtransgene. One line (designated “CW4-B30”) exhibited a significant reduction in tumorigenesis when inoculated with either wild-type, iaaM-mutant, or ipt-mutant A. tumefaciens. Although the CW4-B30 line exhibited markedly reduced gall size and absence of gall formation at some inoculation sites, gall tumorigenesis was not entirely absent. The controls were kanamycin-resistant plant lines (designated “PEV6” lines) that contained the transformation vector T-DNA (from pPEV6) without a transgene located downstream of the CaMV 35S promoter. As expected, none of these lines exhibited any suppression of T-DNA oncogenes upon infection with A. tumefaciens.
e. Molecular Analysis of Transgenic Plants [0117]

To examine transgene copy number and structural integrity, Southern blot analyses were perforrned on representative plant lines of each type. Most lines contained several copies of the transgene, and all lines contained one or more intact copies (see Tables 3, 4, and 5, below). Some lines also contained rearranged copies of the transgene, which were identified by the presence of multiple bands in the Southern blots.

TABLE 3


Southern Blot of Genomic DNA Digested with BamH1

	1799-bp Transgene	Oncogene
Transgenic Plant	Fragment (+/−)*	Suppression (+/−)*

CW4 B1 (ipt-stop/iaaM-stop)	+	−
CW4 B11 (ipt-stop/iaaM-stop)	+	−
CW4 B22 (ipt-stop/iaaM-stop)	+	−
CW4 B30 (ipt-stop/iaaM-stop)	+	+/−
TDP1 B7 (iaaM-stop)	+	+
TDP1 B17 (iaaM-stop)	+	+
TDP1 B27 (iaaM-stop)	+	+
TDP1 B31 (iaaM-stop)	+	−
PEV6 B2 (vector alone)	−	−
PEV6 B14 (vector alone)	−	−

TABLE 4


Southern Blot of Genomic DNA Digested with EcoR1

	Transgene Copy	Oncogene
Transgenic Plant	Number	Suppression (+/−)*

TDP1 B7 (iaaM-stop)	3-4	+
CW4 B1 (ipt-stop/iaaM-stop)	2	−
CW4 B11 (ipt-stop/iaaM-stop)	2	−
CW4 B22 (ipt-stop/iaaM-stop)	2	−
CW4 B30 (ipt-stop/iaaM-stop)	4	+/−
TDP1 B17 (iaaM-stop)	2	+
TDP1 B27 (iaaM-stop)	1	+
TDP1 B31 (iaaM-stop)	4<	−
PEV6 B2 (vector alone)	0	−
PEV6 B14 (vector alone)	0	−

TABLE 5


Southern Blot of Genomic DNA Digested with
either Hinf1 or EcoR1 Digested Genomic DNA

	Presence of
	Restriction	Restriction	Suppression of
Transgenic Plant	Fragment (+/−)*	Enzyme	Oncogenes (+/−)*

CW1 K27 (ipt-stop)	+	Hinf1	+
CW1 K27 (ipt-stop)	+	EcoR1	+
CW1 K52 (ipt-stop)	+	Hinf1	−
CW1 K52 (ipt-stop)	+	EcoR1	−

The foregoing results show that co-suppression drastically reduces accumulation of mRNA encoded by the eliciting transgene and mRNA encoded by other genes that contain sufficient (approximately 70% or more) sequence similarity (Marano and Baulcombe, [0121] Plant J. 13:537-546, 1998). Hence, Northern blot analysis was used to measure mRNA accumulation (but not transcription rate). In all iaaM-suppressing lines that were examined, transgene mRNA was undetectable, as expected, using a strand-specific hybridization probe. However, full-length and degraded antisense RNA molecules that included the transgene sequences were detected using a double-stranded hybridization probe. These transcripts appear to have initiated from the vector's nopaline synthase promoter and continued through both the neomycin phosphotransferase gene (in the sense direction) and the iaaM transgene (in the antisense direction). In the successful ipt-suppressing line, a trace amount of transgene-encoded mRNA was detected. Another line that contained the ipt transgene accumulated a large amount of transgene mRNA, but this line failed to co-suppress an incoming A. tumefaciens T-DNA-borne ipt gene. Plants that contained the hybrid ipt-iaaM transgene accumulated varying quantities of transgene-encoded mRNA. As expected, most of these lines did not elicit any co-suppression and only one line elicited partial co-suppression.
f. Discussion [0122]
By studying the ability of wild-type and iaaM- or ipt-mutant [0123] A. tumefaciens to induce tumors on various lines of transgenic plants, it has been shown that: 1) many lines suppressed an incoming T-DNA iaaM gene, 2) one line suppressed an incoming T-DNA ipt gene, and 3) one line partially suppressed both ipt and iaaM genes simultaneously.
By combining successful iaaH- or iaaM-suppressing transgenes with an ipt-suppressing transgene in a single plant line, a transgenic plant fully resistant to gall formation can be produced. This can be done through standard breeding techniques, in which a cross between lines homozygous for the individual genes is performed. The F2 generation will include plants homozygous for both transgenes; such plants are expected to substantially resistant to gall formation. This expectation is supported by data from a plant that contained the hybrid ipt-iaaM transgene. This plant nearly prevented gall formation, indicating that a single hybrid gene can elicit co-suppression against two target oncogenes. [0124]
The transgenes used in this study will likely elicit co-suppression against a broad variety of Agrobacterium species, as well as various strains of such species. This allows the production of plants (such as grape vines, fruit trees, nut trees, and chrysanthemums) that are resistant to gall formation. Other studies have established that approximately 70% sequence identity is required to elicit co-suppression (Marano and Baulcombe, [0125] Plant J. 13:537-546, 1998). Fortunately, the target T-DNA oncogenes (iaaM and ipt) are highly conserved among diverse A. tumefaciens strains. Even limited-host-range grape-specific A. vitis strains have greater than 90% sequence identity with the octopine-type oncogenes 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 embodiments and examples, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. Therefore, the invention encompasses all modifications coming within the spirit and scope of the following claims. [0126]
1 12 1 33 DNA Artificial Sequence Description of Artificial Sequence PCR Primer 1 cgggatccat gtcatgaacc tctccttgat aac 33 2 19 DNA Artificial Sequence Description of Artificial Sequence PCR Primer 2 cgggatcctg cgactcata 19 3 37 DNA Artificial Sequence Description of Artificial Sequence PCR Primer 3 gaagatctga tcatggactg aatctaattt tcggtcc 37 4 31 DNA Artificial Sequence Description of Artificial Sequence PCR Primer 4 gaagatctga tcactaatac attccgaacg g 31 5 747 DNA Agrobacterium tumefaciens 5 gaagatctga tcatggactg aatctaattt tcggtccaac ttgcacagga aagacgacga 60 ccgcgatagc tcttgcccag cagacagggc ttccagtcct ttcgcttgat cgggtccaat 120 gctgtcctca actatcaacc ggaagcggac gaccaacagt ggaagaactg aaaggaacga 180 cgcgtctcta ccttgatgat cggcctctgg tggagggtat catcgcagcc aagcaagctc 240 atcataggct gatcgaggag gtgtataatc atgaggccaa cggcgggctt attcttgagg 300 gaggatccac ctcgttgctc aactgcatgg cgcgaaacag ctattggagt gcagattttc 360 gttggcatat tattcgccac aagttacccg accaagagac cttcatgaaa gcggccaagg 420 ccagagttaa gcagatgttg caccccgctg caggccattc tattattcaa gagttggttt 480 atctttggaa tgaacctcgg ctgaggccca ttctgaaaga gatcgatgga tatcgatatg 540 ccatgttgtt tgctagccag aaccagatca cggcagatat gctattgcag cttgacgcaa 600 atatggaagg taagttgatt aatgggatcg ctcaggagta tttcatccat gcgcgccaac 660 aggaacagaa attcccccaa gttaacgcag ccgctttcga cggattcgaa ggtcatccgt 720 tcggaatgta ttagtgatca gatcttc 747 6 2 PRT Agrobacterium tumefaciens 6 Met Asp 1 7 1807 DNA Agrobacterium tumefaciens 7 cgggatccat gtcatgaacc tctccttgat aaccagtgcg atcatctccc aaccaaaatg 60 gtggatctga caatggtcga taaggcggat gaattggacc gcagggtttc cgatgccttc 120 ttagaacgag aagcttctag gggaaggagg attactcaaa gctccaccga gtgcagcgct 180 gggttagctt gcaaaaggct ggccgatggt cgcttccccg agatctcagc tggtggaaag 240 gtagcagttc tctccgctta tatctatatt ggcaaagaaa ttctggggcg gatacttgaa 300 tcgaaacctt gggcgcgggc aacagtgagt ggtctcgttg ccatcgactt ggacaccatt 360 tgcatggatt tctccgaagc acaactaatc caagccctgt ttttgctgag cggtaaaaga 420 tgtgcaccga ttgatcttag tcatttcgtg gccatttcaa tctctaagac tgccggcttt 480 cgaaccctgc caatgccgct gtacgagaat ggcacgatga aatgcgttac cgggtttacc 540 ataacccttg aaggggccgt gccatttgac atggtagctt atggtcgaaa cctgatgctg 600 aagggttcgg caggttcctt tccaacaatc gacttgctct acgactgcag accgtttttt 660 gaccaatgtt ccgatagtgg acggatcggc ttctttccgg aggatgttcc taagccgaaa 720 gtggcggtca ttggcgctgg catttccgga ctcgtggtgg caaacgaact gcttcatgct 780 ggggtagacg atgttacaat atatgaagca agtgatcgtg ttggaggcaa gctttggtca 840 catgctttca gggacgctcc tagtgtcgtg gccgaaatgg gggcgatgcg atttcctcct 900 gctgcattct gcttgttttt cttcctcgag cgttacggcc tgtcttcgat gaggccgttc 960 ccaaatcccg gcacagtcga cacttacttg gtctaccaag gcgtccaata catgtggaaa 1020 gccgggcagc tgccaccgaa gctgttccat cgcgtttaca acggttggcg tgcgttcttg 1080 aaggacggtt ttcatgagcg agatattgtg ttggcttcgc ctgtcgctat tactcaggcc 1140 ttgaaatcag gacacattag gtgggctcat gactcctggc aaatttggct gaaccgtttc 1200 gggagggagt ccttctcttc agggatagag aggatctttc tgggcacaca tcctcctggt 1260 ggtaaacatg gagttttcct catgattggg acctattcaa gctaatggga ataggatctg 1320 gcggggtttg gtccagtttt tgaaagcggg tttattgaga tcctccgctt ggtcatcaac 1380 ggatatgaag aaaatcagcg gatgtgccct gaaggaatct cagaacttcc acgtcggatc 1440 gcatctgaag tggttaacgg tgtgtctgtg agccagcgca tatgccatgt tcaagtcagg 1500 gcgattcaga aggaaaagac aaaaataaag ataaggctta agagcgggat atctgaactt 1560 tatgataagg tggtggtcac atctggactc gcaaatatcc aactcaggca ttgcctgaca 1620 tgcgatacca atatttttca ggcaccagtg aaccaagcgg ttgataacag ccatatgaca 1680 ggatcgtcaa aactcttcct gatgactgaa cgaaaattct ggttagacca tatcctcccg 1740 tcttgtgtcc tcatggacgg gatcgcaaaa gcagtgtatt gcctggacta tgagtcgcag 1800 gatcccg 1807 8 2 PRT Agrobacterium tumefaciens 8 Met Ser 1 9 2544 DNA Agrobacterium tumefaciens 9 gaagatctga tcatggactg aatctaattt tcggtccaac ttgcacagga aagacgacga 60 ccgcgatagc tcttgcccag cagacagggc ttccagtcct ttcgcttgat cgggtccaat 120 gctgtcctca actatcaacc ggaagcggac gaccaacagt ggaagaactg aaaggaacga 180 cgcgtctcta ccttgatgat cggcctctgg tggagggtat catcgcagcc aagcaagctc 240 atcataggct gatcgaggag gtgtataatc atgaggccaa cggcgggctt attcttgagg 300 gaggatccat gtcatgaacc tctccttgat aaccagtgcg atcatctccc aaccaaaatg 360 gtggatctga caatggtcga taaggcggat gaattggacc gcagggtttc cgatgccttc 420 ttagaacgag aagcttctag gggaaggagg attactcaaa gctccaccga gtgcagcgct 480 gggttagctt gcaaaaggct ggccgatggt cgcttccccg agatctcagc tggtggaaag 540 gtagcagttc tctccgctta tatctatatt ggcaaagaaa ttctggggcg gatacttgaa 600 tcgaaacctt gggcgcgggc aacagtgagt ggtctcgttg ccatcgactt ggacaccatt 660 tgcatggatt tctccgaagc acaactaatc caagccctgt ttttgctgag cggtaaaaga 720 tgtgcaccga ttgatcttag tcatttcgtg gccatttcaa tctctaagac tgccggcttt 780 cgaaccctgc caatgccgct gtacgagaat ggcacgatga aatgcgttac cgggtttacc 840 ataacccttg aaggggccgt gccatttgac atggtagctt atggtcgaaa cctgatgctg 900 aagggttcgg caggttcctt tccaacaatc gacttgctct acgactgcag accgtttttt 960 gaccaatgtt ccgatagtgg acggatcggc ttctttccgg aggatgttcc taagccgaaa 1020 gtggcggtca ttggcgctgg catttccgga ctcgtggtgg caaacgaact gcttcatgct 1080 ggggtagacg atgttacaat atatgaagca agtgatcgtg ttggaggcaa gctttggtca 1140 catgctttca gggacgctcc tagtgtcgtg gccgaaatgg gggcgatgcg atttcctcct 1200 gctgcattct gcttgttttt cttcctcgag cgttacggcc tgtcttcgat gaggccgttc 1260 ccaaatcccg gcacagtcga cacttacttg gtctaccaag gcgtccaata catgtggaaa 1320 gccgggcagc tgccaccgaa gctgttccat cgcgtttaca acggttggcg tgcgttcttg 1380 aaggacggtt ttcatgagcg agatattgtg ttggcttcgc ctgtcgctat tactcaggcc 1440 ttgaaatcag gacacattag gtgggctcat gactcctggc aaatttggct gaaccgtttc 1500 gggagggagt ccttctcttc agggatagag aggatctttc tgggcacaca tcctcctggt 1560 ggtaaacatg gagttttcct catgattggg acctattcaa gctaatggga ataggatctg 1620 gcggggtttg gtccagtttt tgaaagcggg tttattgaga tcctccgctt ggtcatcaac 1680 ggatatgaag aaaatcagcg gatgtgccct gaaggaatct cagaacttcc acgtcggatc 1740 gcatctgaag tggttaacgg tgtgtctgtg agccagcgca tatgccatgt tcaagtcagg 1800 gcgattcaga aggaaaagac aaaaataaag ataaggctta agagcgggat atctgaactt 1860 tatgataagg tggtggtcac atctggactc gcaaatatcc aactcaggca ttgcctgaca 1920 tgcgatacca atatttttca ggcaccagtg aaccaagcgg ttgataacag ccatatgaca 1980 ggatcgtcaa aactcttcct gatgactgaa cgaaaattct ggttagacca tatcctcccg 2040 tcttgtgtcc tcatggacgg gatcgcaaaa gcagtgtatt gcctggacta tgagtcgcag 2100 gatccacctc gttgctcaac tgcatggcgc gaaacagcta ttggagtgca gattttcgtt 2160 ggcatattat tcgccacaag ttacccgacc aagagacctt catgaaagcg gccaaggcca 2220 gagttaagca gatgttgcac cccgctgcag gccattctat tattcaagag ttggtttatc 2280 tttggaatga acctcggctg aggcccattc tgaaagagat cgatggatat cgatatgcca 2340 tgttgtttgc tagccagaac cagatcacgg cagatatgct attgcagctt gacgcaaata 2400 tggaaggtaa gttgattaat gggatcgctc aggagtattt catccatgcg cgccaacagg 2460 aacagaaatt cccccaagtt aacgcagccg ctttcgacgg attcgaaggt catccgttcg 2520 gaatgtatta gtgatcagat cttc 2544 10 2268 DNA Agrobacterium tumefaciens 10 atgtcagctt cacctctcct tgataaccag tgcgatcatc tcccaaccaa aatggtggat 60 ctgacaatgg tcgataaggc ggatgaattg gaccgcaggg tttccgatgc cttcttagaa 120 cgagaagctt ctaggggaag gaggattact caaagctcca ccgagtgcag cgctgggtta 180 gcttgcaaaa ggctggccga tggtcgcttc cccgagatct cagctggtgg aaaggtagca 240 gttctctccg cttatatcta tattggcaaa gaaattctgg ggcggatact tgaatcgaaa 300 ccttgggcgc gggcaacagt gagtggtctc gttgccatcg acttggacac catttgcatg 360 gatttctccg aagcacaact aatccaagcc ctgtttttgc tgagcggtaa aagatgtgca 420 ccgattgatc ttagtcattt cgtggccatt tcaatctcta agactgccgg ctttcgaacc 480 ctgccaatgc cgctgtacga gaatggcacg atgaaatgcg ttaccgggtt taccataacc 540 cttgaagggg ccgtgccatt tgacatggta gcttatggtc gaaacctgat gctgaagggt 600 tcggcaggtt cctttccaac aatcgacttg ctctacgact gcagaccgtt ttttgaccaa 660 tgttccgata gtggacggat cggcttcttt ccggaggatg ttcctaagcc gaaagtggcg 720 gtcattggcg ctggcatttc cggactcgtg gtggcaaacg aactgcttca tgctggggta 780 gacgatgtta caatatatga agcaagtgat cgtgttggag gcaagctttg gtcacatgct 840 ttcagggacg ctcctagtgt cgtggccgaa atgggggcga tgcgatttcc tcctgctgca 900 ttctgcttgt ttttcttcct cgagcgttac ggcctgtctt cgatgaggcc gttcccaaat 960 cccggcacag tcgacactta cttggtctac caaggcgtcc aatacatgtg gaaagccggg 1020 cagctgccac cgaagctgtt ccatcgcgtt tacaacggtt ggcgtgcgtt cttgaaggac 1080 ggttttcatg agcgagatat tgtgttggct tcgcctgtcg ctattactca ggccttgaaa 1140 tcaggacaca ttaggtgggc tcatgactcc tggcaaattt ggctgaaccg tttcgggagg 1200 gagtccttct cttcagggat agagaggatc tttctgggca cacatcctcc tggtggtaaa 1260 catggagttt tcctcatgat tgggacctat tcaagctaat gggaatagga tctggcgggg 1320 tttggtccag tttttgaaag cgggtttatt gagatcctcc gcttggtcat caacggatat 1380 gaagaaaatc agcggatgtg ccctgaagga atctcagaac ttccacgtcg gatcgcatct 1440 gaagtggtta acggtgtgtc tgtgagccag cgcatatgcc atgttcaagt cagggcgatt 1500 cagaaggaaa agacaaaaat aaagataagg cttaagagcg ggatatctga actttatgat 1560 aaggtggtgg tcacatctgg actcgcaaat atccaactca ggcattgcct gacatgcgat 1620 accaatattt ttcaggcacc agtgaaccaa gcggttgata acagccatat gacaggatcg 1680 tcaaaactct tcctgatgac tgaacgaaaa ttctggttag accatatcct cccgtcttgt 1740 gtcctcatgg acgggatcgc aaaagcagtg tattgcctgg actatgagtc gcaggatccg 1800 aatggtaaag gtctagtgct catcagttat acatgggagg acgactccca caagctgttg 1860 gcggtccccg acaaaaaaga gcgattatgt ctgctgcggg acgcaatttc gagatctttc 1920 ccggcgtttg cccagcacct atttcctgcc tgcgctgatt acgaccaaaa tgttattcaa 1980 catgattggc ttacagacga gaatgccggg ggagctttca aactcaaccg gcgtggtgag 2040 gatttttatt ctgaagaact tttctttcaa gcactggaca cggctaatga taccggagtt 2100 tacttggcgg gttgcagttg ttccttcaca ggtggatggg tggagggtgc tattcagacc 2160 gcgtgtaacg ccgtctgtgc aattatccac aattgtggag gcattttggc aaagggcaat 2220 cctctcgaac actcttggaa gagatataac taccgcagta gaaattag 2268 11 1404 DNA Agrobacterium tumefaciens 11 atggtggcca ttacctcgtt agcccaaagc ctagaacacc tgaaacggaa agactactcc 60 tgcttagaac tagtagaaac tctgatagcg cgttgtgaag ctgcaaaatc attaaacgcc 120 cttctggcta cagactggga tggtttgcgg cgaagcgcca aaaaaattga tcgccatgga 180 aacgccggag taggtctttg cggcattcca ctctgtttta aggcgaacat cgctaccggc 240 gtatttccca caagcgccgc tacgccggcg ctgataaacc acttgccaaa gataccatcc 300 cgcgtcgcag aaagactttt ttcagctgga gcactgccgg gtgcctcggg aaatatgcat 360 gagttatcgt ttggaattac aagcaacaac tatgccaccg gggcggtgcg aaacccgtgg 420 aatccagatc tgataccagg gggctcaagc ggtggtgtgg ctgctgcggt agcaagccga 480 ttgatgttag gcggcatagg caccgatacc ggtgcatctg ttcgcctacc cgcagccctg 540 tgtggcgtag taggatttcg accgacgctt ggtagatatc cgggagatcg gataataccg 600 gttagcccta cccgggacac tcccggaatc atagcgcagt gcgtagccga tgttgtaatc 660 ctcgaccgga taatttccgg cacaccggag agaataccac ccgtgccgct gaaggggcta 720 aggatcggcc tccctacaac ctacttttat gatgaccttg atgctgatgt ggccctagca 780 gctgaaacaa cgattcgcct gctagcaaac aaaggcgtaa cttttgttga agctaacatt 840 ccccaacttg acgaactgaa taaaggggcc agcttcccag ttgcactcta tgaatttcca 900 cacgctctaa aacagtatct cgacgacttt gtaaaaactg tttctttttc tgacgtcatc 960 aaaggaattc gtagccctga tgtagccaac attgccaatg cgcaaattga tggacatcaa 1020 atttccaaag ctgaatatga actggcccgc cactccttca gaccaagact tcaagccacc 1080 tatcgcaact acttcaaact gaatagatta gatgctattc tcttcccaac agcacccttg 1140 gtggccagac ccataggtca ggattcctca gttatccaca atggcacgat gctggacaca 1200 ttcaagatct acgtgcgaaa tgtggaccca agcagcaacg caggcctacc tggcttgagc 1260 attcctgttt gcctgacacc tgatcgcttg cctgttggaa tggagatcga tggattagcg 1320 gattcagacc aacgtctgtt agcaatcggg ggggcattgg aagaagccat tggattccga 1380 tattttgccg gtttacccaa ttaa 1404 12 723 DNA Agrobacterium tumefaciens 12 atggacctgc atctaatttt cggtccaact tgcacaggaa agacgacgac cgcgatagct 60 cttgcccagc agacagggct tccagtcctt tcgcttgatc gggtccaatg ctgtcctcaa 120 ctatcaaccg gaagcggacg accaacagtg gaagaactga aaggaacgac gcgtctctac 180 cttgatgatc ggcctctggt ggagggtatc atcgcagcca agcaagctca tcataggctg 240 atcgaggagg tgtataatca tgaggccaac ggcgggctta ttcttgaggg aggatccacc 300 tcgttgctca actgcatggc gcgaaacagc tattggagtg cagattttcg ttggcatatt 360 attcgccaca agttacccga ccaagagacc ttcatgaaag cggccaaggc cagagttaag 420 cagatgttgc accccgctgc aggccattct attattcaag agttggttta tctttggaat 480 gaacctcggc tgaggcccat tctgaaagag atcgatggat atcgatatgc catgttgttt 540 gctagccaga accagatcac ggcagatatg ctattgcagc ttgacgcaaa tatggaaggt 600 aagttgatta atgggatcgc tcaggagtat ttcatccatg cgcgccaaca ggaacagaaa 660 ttcccccaag ttaacgcagc cgctttcgac ggattcgaag gtcatccgtt cggaatgtat 720 tag 723

Claims

We claim:

1. A method of producing a plant cell that is resistant to gall disease, the method comprising transforming a plant cell with at least one nucleic acid molecule that is homologous to at least one gene responsible for causing gall disease, the nucleic acid molecule encoding an RNA molecule selected from the group consisting of untranslatable plus-sense RNA molecules, double-stranded RNA molecules, and untranslatable double-stranded RNA molecules.

2. The method of claim 1, wherein 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. A plant-transformation vector, comprising the nucleic acid molecule of claim 2.

4. A plant cell transformed with the plant-transformation vector of claim 3.

5. A differentiated plant, comprising plant cells produced according to the method of claim 1.

6. A method of producing a plant exhibiting a reduced susceptibility to gall disease caused by Agrobacterium, comprising:

transforming at least one plant cell with at least one nucleic acid molecule that is homologous to at least one gene responsible for causing gall disease, or fragment thereof, the nucleic acid molecule encoding an RNA molecule selected from the group consisting of untranslatable plus-sense RNA molecules, double-stranded RNA molecules, and untranslatable double-stranded RNA molecules;

growing plants from the transformed plant cells; and

selecting a plant that shows a reduced susceptibility to gall disease caused by Agrobacterium.

7. A plant exhibiting a reduced susceptibility to disease caused by Agrobacterium, produced by the method of claim 6.

8. A chimeric plant, comprising at least one non-transformed plant cell grafted to the plant of claim 7.

9. A plant produced by sexual or asexual reproduction of the plant of claim 7.

10. A seed produced by selfing or outcrossing the plant of claim 7.

11. A recombinant nucleic acid molecule comprising 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. A vector, comprising the recombinant nucleic acid molecule of claim 11.

13. A transgenic plant cell transformed with the vector of claim 12.

14. A transgenic plant, comprising at least one transgenic cell transformed with a recombinant nucleic acid molecule, as recited in claim 13.

15. The transgenic plant of claim 14, wherein the plant is selected from the group consisting of apricot, blackberry, pear, peach, plum, blueberry, cherry, kiwi, quince, raspberry, and rose.

16. A chimeric plant, comprising at least one transgenic plant cell as recited in claim 13.

17. A BR construct.

18. A composition, comprising the BR construct of claim 17.

19. An RNA transcribed from the BR construct of claim 17.

20. A vector, comprising the BR construct of claim 17.

21. A plant cell transformed with the vector of claim 20.

22. A plant, comprising at least one plant cell according to claim 21.

23. A method of producing a plant cell that is resistant to gall disease, the method comprising:

providing a BR construct;

introducing the BR construct into a plant cell; and

assessing the level of gall resistance exhibited by the plant cell.

24. A plant, comprising at least one plant cell produced according to the method of claim 23.

25. The transgenic plant of claim 14, wherein the plant is chrysanthemum.

26. The transgenic plant of claim 14, wherein the plant is selected from the group consisting of conifers and poplars.

27. The transgenic plant of claim 14, wherein the plant is an ornamental shrub.

28. The transgenic plant of claim 14, wherein the plant is selected from the group consisting of almond, apple, grape, and walnut.

29. A method of producing a plant root cell that is resistant to crown gall disease, the method comprising transforming a root cell with at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or fragments thereof, thereby producing the root cell that is resistant to crown gall disease, as compared to an untransformed control root cell.

30. The method of claim 29, wherein the method comprises more than one nucleic acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 12, or fragments thereof, thereby producing the root cell that is resistant to crown gall disease, as compared to an untransformed control root cell.

31. The method of claim 30, wherein the plant root cell is a fruit tree root cell or a nut tree root cell.

32. The method of claim 31, wherein the fruit tree root cell is an apple tree root cell.

33. The method of claim 31, wherein the nut tree root cell is a walnut tree root cell or an almond tree root cell.

34. The method of claim 30, wherein the nucleic acid sequence fragments are at least about 25 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 250 nucleotides, at least about 500 nucleotides, or at least about 700 nucleotides in length.

35. The method of claim 30, wherein at least one nucleic acid sequence encodes an untranslatable double-stranded RNA molecule.

36. The method of claim 35, wherein the untranslatable double-stranded RNA molecule results from the introduction of a mutation in the nucleic acid sequence encoding the double-stranded RNA molecule.

37. The method of claim 36, wherein the mutation in the nucleic acid sequence creates a stop codon.

38. The method of claim 37, wherein the stop codon is created transcriptionally upstream from a naturally occurring stop codon.

39. The method of claim 37, wherein the stop codon is positioned about 50 nucleotides transcriptionally downstream from a translation start codon.

40. The method of claim 37, wherein the stop codon is positioned about 100 nucleotides transcriptionally downstream from a translation-start codon.

41. The method of claim 37, wherein the stop codon is positioned at a third codon of the nucleic acid sequence.

42. The method of claim 30, wherein the more than one nucleic acid sequences are contained in a single construct.

43. A root cell transformation vector, comprising the more than one nucleic acid molecules of claim 30.

44. The root cell transformation vector of claim 43, wherein at least one of the nucleic acid sequences is operably linked to at least one promoter, and wherein the promoter drives the transcription of one or more of the nucleic acid sequences.

45. The root cell transformation vector of claim 44, wherein the at least one promoter is the cauliflower mosaic virus 35S promoter or the figwort mosaic virus promoter.

46. The root cell transformation vector of claim 44, wherein the at least one promoter drives transcription of a sense strand and an antisense strand of a nucleic acid molecule, as set forth in SEQ ID NO: 10, SEQ ID NO: 12, or fragments thereof, wherein the sense strand and the anti-sense strand are comprised in a single transcript, and wherein the transcript can fold back on itself to create a double-stranded RNA.

47. The root cell transformation vector of claim 45, wherein the cauliflower mosaic virus 35S promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10, SEQ ID NO: 12, or fragments thereof, and the figwort mosaic virus promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10, SEQ ID NO: 12, or fragments thereof.

48. The root cell transformation vector of claim 45, wherein the cauliflower mosaic virus 35S promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10 and the figwort mosaic virus promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10, and wherein the cauliflower mosaic virus 35S promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 12 and the figwort mosaic virus promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 12.

49. The root cell transformation vector of claim 45, wherein the figwort mosaic virus promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10, SEQ ID NO: 12, or fragments thereof, and the cauliflower mosaic virus 35S promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10, SEQ ID NO: 12, or fragments thereof.

50. The root cell transformation vector of claim 45, wherein the figwort mosaic virus promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10 and the cauliflower mosaic virus 35S promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10, and wherein the figwort mosaic virus promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 12 and the cauliflower mosaic virus 35S promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 12.

51. A root cell transformed with the transformation vector of claim 44.

52. A fruit tree, comprising a fruit tree root cell produced according to the method of claim 31.

53. A nut tree, comprising a nut tree root cell produced according to the method of claim 31.

54. A method of producing a plant that is resistant to crown gall disease caused by Agrobacterium tumefaciens, comprising:

transforming at least one plant root cell with more than one nucleic acid molecule wherein the more than one nucleic acid sequence is homologous to a nucleic acid molecule as set forth in SEQ ID NO: 10, SEQ ID NO: 12, or fragments thereof, the more than one nucleic acid molecule encoding at least one untranslatable double-stranded RNA molecule;

growing plants from the transformed plant root cell;

contacting the plants with wild-type Agrobacterium tumefaciens; and

selecting a plant that shows resistance to crown gall disease caused by Agrobacterium tumefaciens.

55. The method of claim 55, wherein the plant is a fruit tree or a nut tree.

56. The method of claim 54, wherein the fruit tree is an apple tree.

57. The method of claim 55, wherein the nut tree is a walnut tree or an almond tree.

58. A tree that is resistant to crown gall disease caused by Agrobacterium tumefaciens, produced by the method of claim 55.

59. A chimeric tree, comprising at least one non-transformed tree cell grafted to the fruit tree of claim 58.

60. A tree produced by sexual or asexual reproduction of the tree of claim 58.

61. A seed produced by the tree of claim 58.

62. A recombinant nucleic acid molecule comprising a nucleic acid sequence having at least 90% sequence identity with a nucleic acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:12, or fragments thereof, the recombinant nucleic acid molecule, when introduced into and expressed in a plant, reduces the susceptibility of the plant to crown gall disease caused by Agrobacterium tumefaciens.

63. A vector, comprising the recombinant nucleic acid molecule of claim 62.

64. The vector of claim 63, wherein the recombinant nucleic acid molecule is operably linked to at least one promoter.

65. The vector of claim 64, wherein the at least one promoter drives the transcription of a sense strand and an antisense strand of a nucleic acid molecule, as set forth in SEQ ID NO: 10, SEQ ID NO: 12, or fragments thereof, wherein the sense strand and the anti-sense strand are comprised in a single transcript, and wherein the transcript can fold back on itself to create a double-stranded RNA.

66. The vector of claim 64, wherein the at least one promoter is the cauliflower mosaic virus 35S promoter or the figwort mosaic virus promoter.

67. The vector of claim 66, wherein the cauliflower mosaic virus 35S promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10 and SEQ ID NO: 12, and the figwort mosaic virus promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10 and SEQ ID NO: 12.

68. The vector of claim 66, wherein the cauliflower mosaic virus 35S promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10 and the figwort mosaic virus promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10, and wherein the cauliflower mosaic virus 35S promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 12 and the figwort mosaic virus promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 12.

69. The vector of claim 66, wherein the figwort mosaic virus promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10 and SEQ ID NO: 12, and the cauliflower mosaic virus 35S promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10 and SEQ ID NO: 12.

70. The vector of claim 66, wherein the figwort mosaic virus promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10 and the cauliflower mosaic virus 35S promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 10, and wherein the figwort mosaic virus promoter drives the transcription of a sense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 12 and the cauliflower mosaic virus 35S promoter drives the transcription of an antisense-strand of a nucleic acid molecule as set forth in SEQ ID NO: 12.

71. A transgenic tree root cell transformed with the vector of claim 67.

72. A transgenic tree root cell transformed with the vector of claim 68.

73. A transgenic tree root cell transformed with the vector of claim 69.

74. A transgenic tree root cell transformed with the vector of claim 70.

75. A transgenic tree, comprising at least one transgenic cell transformed with the vector of claim 67.

76. A transgenic tree, comprising at least one transgenic cell transformed with the vector of claim 67.

77. A transgenic tree, comprising at least one transgenic cell transformed with the vector of claim 69.

78. A transgenic tree, comprising at least one transgenic cell transformed with the vector of claim 70.

79. A chimeric tree, comprising at least one transgenic tree root cell of claim 75.

80. A chimeric tree, comprising at least one transgenic tree root cell of claim 76.

81. A chimeric tree, comprising at least one transgenic tree root cell of claim 77.

82. A chimeric tree, comprising at least one transgenic tree root cell of claim 78.