Plant expression constructs comprising and uses thereof

Abstract

Methods of expressing a molecule of interest in a plant are disclosed. One method comprises contacting roots of the plant in a solution comprising at least one Geminivirus based expression construct so as to allow the at least one Geminivirus based expression construct to be absorbed by the roots, the expression construct comprising a polynucleotide encoding the molecule of interest, and further the expression construct being capable of systemic symptomless spread in a plant host, thereby expressing a molecule of interest in a plant. Expression constructs capable of systemic symptomless spread in a host plant are also disclosed.

What is claimed is: 
     
         1 . A method of expressing a molecule of interest in a plant, the method comprising contacting roots of the plant in a solution comprising at least one Geminivirus based expression construct so as to allow said at least one Geminivirus based expression construct to be absorbed by said roots, said expression construct comprising a polynucleotide encoding the molecule of interest, and further said expression construct being capable of systemic symptomless spread in a plant host, thereby expressing a molecule of interest in a plant. 
     
     
         2 . The method of  claim 1 , wherein said Geminivirus based expression construct is devoid of a polynucleotide sequence encoding a C2 and C3 coding sequence. 
     
     
         3 . A method of expressing a molecule of interest in a plant, the method comprising grafting a section of a first plant infected with at least one Geminivirus based expression construct onto a section of a second plant, said expression construct comprising a polynucleotide sequence which encodes the molecule of interest, and further said Geminivirus based expression construct being capable of systemic symptomless spread in a plant host, thereby expressing a molecule of interest in a plant. 
     
     
         4 . The method of  claim 3 , wherein said Geminivirus based expression construct is devoid of a polynucleotide sequence encoding a C2 and C3 coding sequence. 
     
     
         5 . A Geminivirus based expression construct, capable of systemic symptomless spread in a plant host, the expression construct being devoid of a polynucleotide sequence encoding a functional C2 and C3 protein, the construct comprising:
 (i) a polynucleotide sequence encoding a Geminivirus intergenic region (IR);   (ii) a polynucleotide sequence encoding a modification in a Geminivirus coat protein (CP), wherein said modification comprises a deletion in nucleotides encoding an N-terminal 100 amino acids, wherein a position of said modification results in a deletion in the C-terminal Geminivirus V2 protein.   
     
     
         6 . The expression construct of  claim 5 , being completely devoid of the C2 and C3 coding sequence. 
     
     
         7 . The expression construct of  claim 5 , further comprising a polynucletotide sequence encoding a truncated replicase protein (C1) which results in a reduced capability of rolling circle, single stranded DNA replication compared to an unmodified Geminivirus replicase, and further when said modification of said replicase results in a deletion in a C4 protein of said Geminivirus. 
     
     
         8 . The expression construct of  claim 7 , wherein said modified replicase protein comprises the sequence as set forth in SEQ ID NO: 36. 
     
     
         9 . The expression construct of  claim 5 , being capable of replication in a prokaryotic cell. 
     
     
         10 . The expression construct of  claim 5 , being incapable of plant to plant transmission by an insect vector. 
     
     
         11 . The expression construct of  claim 5 , further comprising a heterologous polynucleotide sequence. 
     
     
         12 . The expression construct of  claim 11 , wherein said heterologous polynucleotide comprises an operon. 
     
     
         13 . The expression construct of  claim 11 , wherein said heterologous polynucleotide encodes a dsRNA or an antisense RNA. 
     
     
         14 . The expression construct of  claim 5 , further comprising a bacterial polynucleotide sequence. 
     
     
         15 . The expression construct of  claim 5 , wherein said modified Geminivirus V2 protein comprises the sequence as set forth in SEQ ID NO: 6. 
     
     
         16 . The expression construct of  claim 5 , wherein said modified Geminivirus coat protein comprises the amino acid sequence as set forth in SEQ ID NO: 3. 
     
     
         17 . The expression construct of  claim 5 , comprising the polynucleotide sequence as set forth in SEQ ID NO: 10 or 11. 
     
     
         18 . The expression construct of  claim 5 , wherein the Geminivirus is a begomovirus. 
     
     
         19 . The expression construct of  claim 5 , wherein the Geminivirus is a Tomato yellow leaf curl virus (TYLCV). 
     
     
         20 . The expression construct of  claim 5 , wherein the expression construct is adapted for expression in a plant host selected from the group consisting of  Solanaceae, Cucurbitaceae, Umbelliferae, Liliacae, Gramineae  (Poaceae),  Rosaceae, Musaceae, Vitacea, and Cruciferae.    
     
     
         21 . A method of expressing a molecule of interest in a plant cell comprising introducing into the plant cell the nucleic acid construct of  claim 11 , said heterologous polypeptide being the molecule of interest, thereby expressing a molecule of interest in a plant cell. 
     
     
         22 . The method of  claim 21 , wherein the plant is selected from the group consisting of a  Solanaceae , a  Cucurbitaceae , an  Umbelliferae , a  Liliacae , a  Gramineae  (Poaceae), a  Rosaceae Musaceae, Vitacea  and a  Cruciferae.    
     
     
         23 . A construct system comprising:
 (i) the Geminivirus based expression construct of  claim 5 ; and   (ii) a construct comprising a Geminivirus IR polynucleotide sequence and a heterologous polynucleotide sequence which encodes a polypeptide or dsRNA.   
     
     
         24 . The construct system of  claim 23 , wherein said Geminivirus IR is flanked on both sides by heterologous polynucleotide sequences which are capable of hybridizing to form said dsRNA. 
     
     
         25 . The construct system of  claim 23 , wherein said heterologous polynucleotide sequence encodes a polypeptide.