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1.
FIG. 5.

FIG. 5. From: The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea.

(A) Southern blot analysis of DNA from wild-type strain ATCC 58025; replacement mutants ΔBccpr1-3, ΔBccpr1-8, and ΔBccpr1-16; and complemented mutant Bccpr1C5. DNA was digested with XbaI, blotted, and hybridized with the 3′ flank of the replacement vector pCPR1Rep. (B) Northern analysis. Total RNA from ATCC 58025, replacement mutants ΔBccpr1-8 and ΔBccpr1-16, and complemented mutant Bccpr1C5 was blotted and also probed with the 3′ flank of pCPR1Rep. Equal loading of the lanes was checked by hybridization with rDNA.

Verena Siewers, et al. Appl Environ Microbiol. 2004 July;70(7):3868-3876.
2.
FIG. 8.

FIG. 8. From: The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea.

(A) Southern blot analysis of wild-type strain ATCC 58025; replacement mutants ΔBcaba1-2, ΔBcaba1-27, and ΔBcaba1-36; ectopic transformant T35; and complemented mutants Bcaba1C4 and Bcaba1C5. DNA was digested with HindIII and hybridized with the 5′ flank of the replacement vector pABA1Rep. (B) Northern analysis of total RNA from ATCC 58025, replacement mutants ΔBcaba1-2 and ΔBcaba1-27, and complemented mutant Bcaba1C5 also probed with the 5′ flank of pABA1Rep. Equal loading of the lanes was checked by hybridization with rDNA.

Verena Siewers, et al. Appl Environ Microbiol. 2004 July;70(7):3868-3876.
3.
FIG. 1.

FIG. 1. From: The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea.

Postulated biosynthetic pathways of ABA in higher plants and in C. pini-densiflorae (19, 22), respectively. Intermediates identified in C. pini-densiflorae are α-ionylideneacetic acid (structure 1), 4′S-OH-α-ionylideneethanol (structure 2), 1′-OH-α-ionylideneethanol (structure 3), (1′R)-4′R-OH-α-ionylideneacetic acid (structure 4), (1′R)-4′S-OH-α-ionylideneacetic acid (structure 5), 1′-OH-α-ionylideneacetic acid (structure 6), and 1′-deoxy-ABA (structure 7). The site of action of DPA is indicated.

Verena Siewers, et al. Appl Environ Microbiol. 2004 July;70(7):3868-3876.
4.
FIG. 6.

FIG. 6. From: The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea.

LC-MS analyses. (A) Mass spectrum of ABA standard showing a predominant peak at m/z 247.1335 (M+H+-H2O). (B) Ion traces for m/z 247.1335 with a width of 30 ppm of metabolites extracted from wild-type ATCC 58025 (dotted line) and replacement mutants ΔBccpr1-16 (dashed line) and ΔBcaba1-27 (full line). (C) Ion traces for m/z 235.172 with a width of 30 ppm of metabolites extracted from wild-type ATCC 58025 (dotted line) and replacement mutant ΔBcaba1-27 (full line). (D) Mass spectrum of the unknown metabolite from replacement mutant ΔBcaba1-27 eluting at 4.854 min.

Verena Siewers, et al. Appl Environ Microbiol. 2004 July;70(7):3868-3876.
5.
FIG. 2.

FIG. 2. From: The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea.

Cladogram of fungal cytochrome P450 oxidoreductases based on amino acid sequences. The accession numbers are as follows: Aspergillus niger CprA, CAA81550; Candida maltosa NCP1, P50126; Candida tropicalis NCP1, P37201; Coriolus versicolor CPR, BAB83588; Cunninghamella echinulata CPR, AAF89959; Cunninghamella elegans CPR, AAF89958; Gibberella fujikuroi CPRGf, AJ576025; Phanerochaete chrysosporium CPR, AAG31350; Rhizopus stolonifer CPR isoenzyme 1, AAG23833; Rhizopus stolonifer CPR isoenzyme 2, AAG23834; Saccharomyces cerevisiae NCP1, P16603; and Schizosaccharomyces pombe CCR1, P36587.

Verena Siewers, et al. Appl Environ Microbiol. 2004 July;70(7):3868-3876.
6.
FIG. 4.

FIG. 4. From: The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea.

Scheme of gene replacement approach for bccpr1. Physical maps of bccpr1 from wild-type strain ATCC 58025 (A), the gene replacement fragment pCPR1Rep (B), and bccpr1 from a ΔBccpr1 replacement mutant (C) showing the organization of exons (□), introns (), the hygromycin resistance cassette (▨), and flanking regions of bccpr1 (heavy lines). Orientations of the genes are indicated by arrowheads. Binding sites of primers 1 to 9 used for PCR analysis of replacement mutants (see Materials and Methods) and 3′ flank of pCPR1Rep (dotted line) used as a probe for Southern and Northern analyses are indicated.

Verena Siewers, et al. Appl Environ Microbiol. 2004 July;70(7):3868-3876.
7.
FIG. 7.

FIG. 7. From: The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea.

Scheme of gene replacement approach for bcaba1. Physical maps of bcaba1 from wild-type strain ATCC 58025 (A), the gene replacement fragment pABA1Rep (B), and bcaba1 from a ΔBccpr1 replacement mutant (C) showing organization of exons (□), introns (), the hygromycin resistance cassette (▨), and flanking regions of bcaba1 (heavy lines). Orientations of the genes are indicated by arrowheads. The binding sites of primers 6, 7, and 10 to 16 used for PCR analysis of replacement mutants (see Materials and Methods) and the 5′ flank of pABA1Rep (dotted line) used as a probe for Southern and Northern analysis are indicated.

Verena Siewers, et al. Appl Environ Microbiol. 2004 July;70(7):3868-3876.
8.
FIG. 3.

FIG. 3. From: The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea.

Expresssion analysis of genes bccpr1, bcaba1, and P450-12. (A) Total RNA was extracted from ABA-producing B. cinerea strain ATCC 58025 (lanes +) and nonproducing strain B05.10 grown for 4 days (bccpr1 and bcaba1) or 11 days (P450-12) in Sprecher medium. (B) Total RNA was extracted from strain ATCC 58025 grown for 3 days in Sprecher medium. Mycelia were harvested 30, 60, 90, 120, and 180 min after the addition of 3.8 mM mevalonic acid lactone. Fragments of the 3′ region of bccpr1 (see Fig. 4), the 5′ region of bcaba1 (see Fig. 7), and an internal 720-bp PCR fragment of P450-12 were used for probing. Loading of lanes with RNA was checked by probing with ribosomal DNA.

Verena Siewers, et al. Appl Environ Microbiol. 2004 July;70(7):3868-3876.

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