Disease control of the plant bacterial pathogens causing citrus canker and rice blight
AssigneeAuxergen, Inc.
InventorTing-Yu YEH
Life of this patent
Estimating the term…
Abstract
The present invention describes applications and methods (1) to use bacteriophage Cf and its variants to prevent and treat the citrus canker pathogen, Xanthomonas citri subsp. citri; (2) to engineer recombinant Cf phages that the infectivity is controllable without being harmful to the rest of environment; (3) to engineer and produce recombinant Cf phages with longer storage shelf life; (4) to use Cf phage as a vector for the introduction and insertion of foreign genetic material into Xanthomonas citri subsp. citri. genome; (5) to use and engineer Xp12 and Xf bacteriophages to inhibit Xanthomonas oryzae pv. oryzae, the causal agent of the rice blight disease.
Claims40
An isolated or recombinant nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence, wherein said nucleotide sequence is selected from the group consisting of:
- a) the nucleotide sequence set forth in any of SEQ ID NO:1, 24, 25, 2 or full length complements thereof;
- and
- b) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:
- 1, 24, 25 or 2.
A method of expressing a recombinant gene in a Cf-type phage-infected Xanthomonas citri cell comprising:
- (a) preparing a nucleic acid molecule comprising a recombinant gene fused with a sequence or in accordance with SEQ ID NO:1, 24, 25 or 2 which encodes the amino acid sequences comprising SEQ ID NO:
- 3-12, or SEQ ID NO:
- 27-52;
- (b) transforming said molecule into a uninfected or phage-infected bacterial cell;
- and
- (c) culturing said cell under conditions appropriate for the expression of said recombinant gene.
A method of generating recombinant Cf-type phages that infect Xanthomonas citri cell comprising:
- (a) preparing a nucleic acid molecule comprising SEQ ID NO:1, 24, 25, 2 or one or more nucleotide are additions, deletions, or substitutions of SEQ ID NO:
- 1, 24, 25 or 2;
- (b) transforming said molecule into a uninfected or phage-infected bacterial cell;
- and
- (c) culturing said cell under conditions appropriate for the growth of recombinant phages.
A method of generating recombinant and controllable Cf-type phages that infect Xanthomonas citri cell comprising:
- (a) preparing a nucleic acid molecule comprising SEQ ID NO:5, 32, or 44 or one or more amino acid additions, deletions, or substitutions of SEQ ID NO:
- 5, 32, or 44;
- (b) preparing a nucleic acid molecule comprising a X. citri gene fused with a sequence or in accordance with SEQ ID NO:1, 24, 25, to replace the nucleotide sequence that encodes the amino acid sequences containing SEQ ID 5, 32, 44;
- (c) transforming said molecules in (a) and (b) into a uninfected or phage-infected bacterial cell;
- and
- (d) culturing said cell under conditions appropriate for the growth of recombinant phages.
A method of generating recombinant Cf-type phages that infect Xanthomonas citri cell with improved shelf life comprising:
- (a) preparing a nucleic acid molecule comprising a recombinant gene, which encodes the amino acid sequences containing SEQ ID 13-23, fused with a sequence or in accordance with SEQ ID NO:1, 24, or 25;
- (b) preparing a nucleic acid molecule comprising a gene sequence encoding SEQ ID NO:13-23 or one or more amino acid are additions, deletions, or substitutions of SEQ ID NO:
- 13-23;
- (c) preparing a nucleic acid molecule comprising a X. citri gene fused with a sequence or in accordance with SEQ ID NO:1, 24 or 25, to replace the nucleotide sequence that encodes the amino acid sequences containing SEQ ID 4, 30, or 45;
- (d) preparing a nucleic acid molecule comprising a sequence or in accordance with SEQ ID NO:1, 24, or 25 where one or more nucleotides are added, deleted, or substituted in nucleotides that encode the amino acid sequence in accordance with SEQ ID NO:4, 30, or 45;
- (e) transforming said molecules in (a), (b), (c) and (d) into a uninfected or phage-infected bacterial cell;
- and
- (f) culturing said cell under conditions appropriate for the growth of recombinant phages.
A method of generating recombinant Cf-type phages that infect and kill Xanthomonas citri cell comprising the steps of:
- (a) preparing a nucleic acid molecule comprising a recombinant gene able to inhibit and kill X. citri fused with a sequence or in accordance with SEQ ID NO:1, 24, 25, or 2;
- (b) preparing a nucleic acid molecule comprising a gene sequence encoding SEQ ID NO:5, 32, or 44;
- or one or more amino acid additions, deletions, or substitutions of SEQ ID NO:
- 5, 32, or 44;
- (c) preparing a nucleic acid molecule comprising a recombinant gene able to inhibit and kill X. citri, and a X. citri gene fused with a sequence or in accordance with SEQ ID NO:1, 24, 25, to replace the nucleotide sequence that encodes the amino acid sequences containing SEQ ID 5, 32, 44;
- (d) transforming said molecules in (a), (b) and (c) into a uninfected or phage-infected bacterial cell;
- and
- (e) culturing said cell under conditions appropriate for the growth of recombinant phages.
A method of generating recombinant Cf vectors and phages that can carry foreign genes and integrate into Xanthomonas citri host genome comprising:
- (a) preparing a nucleic acid molecule comprising a recombinant gene fused with a Cflt phage DNA sequence or in accordance with SEQ ID NO:1, 24, or 25, where one or more nucleotides are added, deleted, or substituted, to replace the nucleotide sequence that encodes the amino acid sequences containing SEQ ID 5, 32, or 44;
- (b) preparing a nucleic acid molecule comprising a gene sequence encoding SEQ ID NO:5, 32, or 44, where one or more nucleotides are added, deleted, or substituted;
- (c) transforming said molecules in (a) and (b) into a uninfected or phage-infected bacterial cell;
- and
- (d) culturing said cell under conditions appropriate for the growth of bacteria and recombinant phages.
An isolated or recombinant nucleic acid molecule comprising a contiguous sequence consisting of XP12 phage genome DNA.
A method of generating recombinant XP12 variant phages that infect Xanthomonas oryzae cell comprising the steps of:
- (a) preparing a nucleic acid molecule comprising XP12 phage DNA, where one or more nucleotides are added, deleted, or substituted;
- (b) preparing a nucleic acid molecule comprising one or two gene sequences encoding XP12 essential proteins;
- (c) preparing a nucleic acid molecule comprising a recombinant X. oryzae gene fused with XP12 phage DNA, to replace the nucleotide sequence that encodes the amino acid sequences in (b);
- (d) transforming said molecules in (a), (b) and (c) into a uninfected or XP12-infected bacterial cell;
- and
- (e) culturing said cell under conditions appropriate for the growth of recombinant phages.
An isolated or recombinant nucleic acid comprising a contiguous sequence consisting of Xf phage DNA sequence of SEQ ID NO:26 or its full length complement.
A method of generating recombinant Xf variant phages that infect Xanthomonas oryzae cell comprising:
- (a) preparing a nucleic acid molecule comprising Xf DNA, in accordance with SEQ ID NO:
- 26, where one or more nucleotides are added, deleted, or substituted;
- (b) preparing a nucleic acid molecule comprising a gene sequence encoding amino acid sequence SEQ ID NO:
- 52-62, 13, or one or more amino acid additions, deletions, or substitutions of SEQ ID NO:
- 52-62;
- 13,
- (c) preparing a nucleic acid molecule comprising a recombinant X. oryzae gene fused with Xf DNA, to replace the nucleotide sequence that encodes the amino acid sequences SEQ ID NO:
- 52-62;
- 13,
- (d) transforming said molecules in (a), (b) and (c) into a uninfected or Xf-infected bacterial cell;
- and
- (e) culturing said cell under conditions appropriate for the growth of recombinant phages.
Dependencies are inferred from the claim text.