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US 2023/0203512 A1Published application
Compositions and methods for agrobacterium mediated transformation of chloroplasts in seed plants
AssigneeRutgers, The State University of New Jersey
InventorsPal Maliga, Aki Matsuoka
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Abstract
Compositions and methods for agrobacterium-mediated chloroplast transgene expression are provided.
Claims34
A method for delivering a polynucleotide of interest to plant cell chloroplasts, said method comprising
- a) providing an Agrobacterium variant strain lacking VirD2 in the Vir operon,
- b) introducing a binary vector plasmid encoding a polynucleotide fusion construct encoding a VirD2 variant protein with a chloroplast transit peptide operably linked to a promoter and terminator which drives expression in said Agrobacterium variant strain and a T DNA comprising a chloroplast sequence mediating homologous recombination into the chloroplast genome, a nucleic acid encoding a polypeptide of interest, and a nucleic acid encoding a selectable marker, wherein said VirD2 variant protein forms a complex with said T-DNA and delivers said complex to the chloroplast,
- c) selecting infected plant cells which survive in the presence of a selection agent, thereby identifying cells expressing said polynucleotide of interest.
An isolated Agrobacterium strain XYA105Pt variant selected from XYA105Pt45, XYA105Pt46, and XYA105Pt47, comprising a reconstituted virD operon encoding a VirD2 variant selected from SEQ ID NO:
- 45, SEQ ID NO:
- 46 and SEQ ID NO:
- 47, said variant Agrobacterium strain optionally comprising a binary vector harboring a T-DNA encoding a nucleic acid of interest and a selectable marker, said T-DNA comprising a sequence which mediates homologous recombination in a chloroplast genome in said plant cell.
A method for delivering a polynucleotide of interest to plant cell chloroplasts, said method comprising
- a) provide an Agrobacterium variant strain with a wild type Vir operon,
- b) introducing a binary vector plasmid encoding a polynucleotide fusion construct encoding a VirD2 variant protein operably linked to a promoter and terminator which drives expression in said Agrobacterium variant strain, and
- a T DNA comprising a chloroplast sequence mediating homologous recombination into the chloroplast genome, a nucleic acid encoding a polypeptide of interest, and a nucleic acid encoding a selectable marker, wherein said VirD2 variant protein forms a complex with said T-DNA and delivers said complex to the chloroplast,
- c) selecting infected plant cells which survive in the presence of a selection agent, thereby identifying cells expressing said polynucleotide of interest.
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Dependencies are inferred from the claim text.