EXPRESSION CONSTRUCTS, VIRD2 MUTANT AGROBACTERIUM STRAINS, AND METHODS OF USE THEREOF
Agrobacterium strains with mutations in the VirD2 gene and protein and expression constructs and genome-editing systems for leveraging the same. Methods are also provided for Agrobacterium -mediated incorporation of exogenous expressible nucleic acids into a host plant material using the Agrobacterium strains, expression constructs, and/or genome-editing systems.
1 . An Agrobacterium strain comprising a mutant VirD2 gene that effects transient transformation of a plant, the mutant VirD2 strain encoding a wild-type VirD2 protein except for:
(i) a mutation at a threonine at position 319 of the VirD2 protein;
(ii) a mutation at a threonine at position 319 of the VirD2 protein, a mutation at a histidine at position 402 of the VirD2 protein, and an insertion of 30 to 40 amino acid residues at a terminal end of the VirD2 protein resulting from a truncation mutation at a glutamine at position 425; or
(iii) an insertion of 1 to 20 amino acid residues after position 326 of the VirD2 protein, which affects a downstream frameshift.
2 . The Agrobacterium strain of claim 1 , wherein the wild-type VirD2 protein is or comprises SEQ ID NO: 16 or SEQ ID NO: 17.
3 . The Agrobacterium strain of claim 1 , wherein the mutant VirD2 gene is preceded by an enhanced Shine-Dalgarno sequence such as SEQ ID NO: 31 or a functional variant of SEQ ID NO: 31.
4 . The Agrobacterium strain of claim 1 , wherein:
the mutation at position 319 of the VirD2 protein comprises substitution of threonine with an amino acid other than threonine;
the mutation at position 402 of the VirD2 protein comprises substitution of histidine with an amino acid other than histidine; and/or
the truncation of position 425 of the VirD2 protein results from a mutation of a final stop codon encoded by a wild-type VirD2 gene.
5 . The Agrobacterium strain of claim 1 , wherein the insertion of 1 to 20 amino acid residues after position 326 of the VirD2 protein is or comprises SEQ ID NO: 30 or a functional variant thereof.
6 . The Agrobacterium strain of claim 1 , wherein the insertion of 30 to 40 amino acid residues is or comprises SEQ ID NO: 32 or a functional variant thereof.
7 . The Agrobacterium strain of claim 1 , wherein the VirD2 protein is or comprises SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, or a functional variant of SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 25.
8 . The Agrobacterium strain of claim 1 , wherein the mutant VirD2 gene is or comprises SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, or a functional variant of SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 26.
9 . An expression construct comprising a mutant VirD2 gene that encodes a wild-type Agrobacterium VirD2 protein except for:
(i) a mutation at a threonine at position 319 of the VirD2 protein;
(ii) a mutation at a threonine at position 319 of the VirD2 protein, a mutation at a histidine at position 402 of the VirD2 protein, and an insertion of 30 to 40 amino acid residues at a terminal end of the VirD2 protein due to a truncation mutation at a glutamine at position 425; or
(iii) an insertion of 1 to 22 amino acid residues after position 326 of the VirD2 protein, which affects a downstream frameshift.
10 . The expression construct of claim 9 , comprising a nucleic acid construct such as a plasmid or a replicating plasmid.
11 . The expression construct of claim 9 , wherein the expression construct comprises a root-inducing plasmid (Ri-plasmid) or a tumor-inducing plasmid (Ti-plasmid).
12 . The expression construct of claim 11 , wherein the Ti-plasmid is a pTiEHA105 plasmid.
13 . The expression construct of claim 9 , wherein a wild-type VirD2 gene of the expression construct is inactive, deleted, disrupted, and/or replaced by the mutant VirD2 gene.
14 . The expression construct of claim 9 , wherein the mutant VirD2 gene is preceded by an enhanced Shine-Dalgarno sequence such as SEQ ID NO: 31 or a functional variant of SEQ ID NO: 31.
15 . The expression construct of claim 9 , wherein:
the VirD2 protein is or comprises SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, or a functional variant of SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 25; or
the mutant VirD2 gene is or comprises SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, or a functional variant of SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 26.
16 . The expression construct of claim 9 , further comprising one or more operating regulatory segments.
17 . The expression construct of claim 9 , further comprising a sequence-specific nuclease, a DNA polymerase, and/or a DNA polymerase recruitment protein, or an expression construct comprising a nucleotide sequence encoding the sequence-specific nuclease, the DNA polymerase and/or the DNA polymerase recruitment protein.
18 . A method for Agrobacterium -mediated incorporation of exogenous expressible nucleic acids into a host plant material, the method comprising infecting a target host plant material with the Agrobacterium strain comprising a mutant VirD2 gene that effects transient transformation of a plant, the mutant VirD2 strain encoding a wild-type VirD2 protein except for:
(i) a mutation at a threonine at position 319 of the VirD2 protein;
(ii) a mutation at a threonine at position 319 of the VirD2 protein, a mutation at a histidine at position 402 of the VirD2 protein, and an insertion of 30 to 40 amino acid residues at a terminal end of the VirD2 protein resulting from a truncation mutation at a glutamine at position 425; or
(iii) an insertion of 1 to 20 amino acid residues after position 326 of the VirD2 protein, which affects a downstream frameshift.
19 . The method of claim 18 , wherein infecting comprises inoculating the target host plant material with the Agrobacterium strain at a dose of at or between about 10 6 cfu/ml to about 10 9 cfu/ml.
20 . The method of claim 19 , wherein the dose is at or about 10 8 cfu/ml.