NOVEL AGROBACTERIUM WITH NO OVERGROWTH IN PLANT TRANSFORMATION
The instant disclosure provides a genetically modified bacterial cell, i.e., modified Agrobacterium strain, which expresses a levansucrase and has reduced expression of endogenous CysK. As a result, the growth of the modified bacterial cell on medium or tissue culture with sucrose and deficient in cysteine is inhibited. The two-action growth control of the modified Agrobacterium strain can overcome the unmet issue of Agrobacterium overgrowth during plant transformation, and therefore increase the efficiency of plant transformation.
1 . A modified bacterial cell comprising a heterologous polynucleotide comprising a nucleic acid sequence encoding a levansucrase enzyme, wherein an endogenous CysK gene has reduced expression when compared to a proper control bacterium.
2 . (canceled)
3 . The modified bacterial cell of claim 1 , wherein an endogenous CysK gene expression is reduced due to a mutation in the CysK gene or regulatory region of the CysK gene, wherein the mutation is an insertion.
4 . (canceled)
5 . The modified bacterial cell of claim 3 , wherein the insertion is an insertion of a heterologous polynucleotide comprising a nucleic acid sequence encoding the levansucrase enzyme, wherein the nucleic acid molecule encoding the levansucrase enzyme is a SacB/SacR gene.
6 . (canceled)
7 . The modified bacterial cell of claim 5 , wherein the SacB/SacR gene comprises
(i) at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with SEQ ID NO: 2, and retains levansucrase activity; or
(ii) the nucleotide sequence of SEQ ID NO: 2.
8 . The modified bacterial cell of claim 1 , wherein the levansucrase enzyme comprises
(i) at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% amino acid sequence identity with SEQ ID NO: 1, and retains levansucrase activity; or
(ii) the amino acid sequence of SEQ ID NO: 1.
9 . The modified bacterial cell of claim 1 , wherein the endogenous CysK coding sequence comprises
(i) at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9, and retains CysK activity; or
(ii) the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9; or wherein the endogenous CysK protein comprises
(iii) at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10, and retains CysK activity; or
(iv) the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10.
10 .- 11 . (canceled)
12 . The modified bacterial cell of claim 1 , wherein the modified bacterial cell is cysteine auxotrophic.
13 . (canceled)
14 . The modified bacterial cell of claim 1 , wherein the modified bacterial cell is an Agrobacterium.
15 . The modified bacterial cell of claim 1 , wherein the modified bacterial cell further comprises one or more heterologous polynucleotide of interest.
16 . The modified bacterial cell of claim 15 , wherein the one or more heterologous polynucleotide of interest encodes an editing reagent and a repair template, and wherein homology-directed repair (HDR) is increased, an HDR to non-homologous end-joining (NHEJ) ratio is increased, chimerism is reduced, mosaicism is reduced, and/or uniform editing of a plant genome is increased relative to a control plant or plant part, when the DNA construct is introduced in a plant or plant part.
17 . (canceled)
18 . The modified bacterial cell of claim 1 , wherein the growth of the modified bacterial cell in a plant tissue culture medium deficient in cysteine is inhibited; or
wherein the growth of the modified bacterial cell in a plant tissue culture medium comprising sucrose is inhibited.
19 .- 20 . (canceled)
21 . The modified bacterial cell of claim 1 , wherein the growth of the modified bacterial cell in a plant tissue culture medium comprising sucrose and deficient in cysteine is inhibited when compared to a control cell.
22 . (canceled)
23 . A population of the modified bacterial cell of claim 1 .
24 .- 28 . (canceled)
29 . A polynucleotide comprising from the 5′ to 3′:
(a) a 5′ fragment of a CysK coding sequence;
(b) a levansucrase coding sequence; and
(c) a 3′ fragment of the CysK coding sequence.
30 . The polynucleotide of claim 29 , wherein the levansucrase enzyme coding sequence is a SacB/SacR gene.
31 . The polynucleotide of claim 30 , wherein the SacB/SacR gene comprises
(i) at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with SEQ ID NO: 2, and retains levansucrase activity; or
(ii) the polynucleotide sequence of SEQ ID NO: 2.
32 . The polynucleotide of claim 29 , wherein the levansucrase comprises
(i) at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with SEQ ID NO: 1, and retains levansucrase activity; or
(ii) the amino acid sequence SEQ ID NO: 1.
33 . The polynucleotide of claim 29 , wherein the CysK coding sequence comprises
(i) at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9, and retains CysK activity; or
(ii) the polynucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9; or wherein the CysK protein comprises
(i) at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10, and retains CysK activity; or
(ii) the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10.
34 . (canceled)
35 . The polynucleotide of claim 29 , further comprising a RecA gene.
36 .- 37 . (canceled)
38 . A method of transforming a cell, comprising the steps of:
(a) introducing the polynucleotide of claim 29 into a cell;
(b) culturing the cell;
(c) selecting for the cells from step (b) which express levansucrase and have reduced expression of CysK.
39 . The method of claim 38 , wherein a SacB/SacR gene is inserted into the CysK coding sequence, wherein insertion of the SacB/SacR gene into the endogenous CysK coding sequence results the expression of the SacB/SacR gene and reduces the expression of the endogenous CysK gene.
40 .- 41 . (canceled)
42 . The method of claim 38 , further comprising introducing at least one heterologous polynucleotide of interest into the cell in step (a).
43 . The method of claim 42 , wherein the at least one heterologous polynucleotide of interest encodes an editing reagent and a repair template, and wherein homology-directed repair (HDR) is increased, an HDR to non-homologous end-joining (NHEJ) ratio is increased, chimerism is reduced, mosaicism is reduced, and/or uniform editing of a plant genome is increased relative to a control plant or plant part, when the DNA construct is introduced in a plant or plant part.
44 .- 45 . (canceled)
46 . The method of claim 38 , wherein the cell is an Agrobacterium.
47 . A method of transforming a plant or plant part using the cell of claim 1 , wherein the transformation efficiency is increased when compared to transforming a plant or plant part with a control cell, wherein the plant is an explant or seed; or
wherein the plant is a soybean plant.
48 .- 51 . (canceled)