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

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.

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.