Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea

Abstract

Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.

FIG. 1.

Scheme of the putative ABA gene cluster of B. cinerea. EcoRI, SacI, and HindIII restriction sites are indicated.

FIG. 2.

Expression analysis of bcaba1-4. Total RNA was extracted from strain ATCC 58025, which was 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. cDNA fragments of the respective genes were used for probing. Loading of lanes with RNA was checked by probing with rRNA genes.

FIG. 3.

Scheme of the gene replacement approach for bcaba2. Physical maps of bcaba2 from wild-type strain ATCC 58025 (A), the gene replacement fragment pABA2Rep (B), and bcaba2 from a ΔBcaba2 replacement mutant (C) showing the organization of exons (white bars), introns (gray bars), the hygromycin resistance cassette (hatched bars), and flanking regions of bcaba2 (bold lines) are shown. Arrowheads indicate orientations of the genes. Binding sites of primers 1 to 9, used for PCR analysis of replacement mutants (see Materials and Methods), as well as the 3′ flank of pABA2Rep (dotted line) used as a probe for Southern analysis, are indicated.

FIG. 4.

Southern blot analysis of wild-type strain ATCC 58025 and ΔBcaba3 (A), ΔBcaba2 (B), and ΔBcaba4 (C) replacement mutants. DNA was digested with SacI, EcoRV, and HindIII, respectively, blotted, and hybridized with the 3′ flank of the respective replacement vectors.

FIG. 5.

Expression analysis of the genes bcaba1-4 in wild-type strain ATCC 58025 as well as ΔBcaba3, ΔBcaba1, ΔBcaba2, and ΔBcaba4 mutants. Total RNA was extracted from mycelium grown for 4 days in Sprecher medium. PCR fragments of the respective genes were used for probing. Loading of lanes with RNA was checked by probing with rRNA genes. Because of the strong signals in some of the mutant lanes, exposure time had to be short; therefore, the wild-type lane shows rather weak signals (compare to Fig. 2).

FIG. 6.

Postulated biosynthetic pathway of ABA in C. pini-densiflorae (late steps, modified from a method described previously by Okamoto et al. [34]). Bold lines indicate the major route. Intermediates identified are α-ionylideneacetic acid (3), 4′-S-OH-α-ionylideneethanol (4), 1′-OH-α-ionylideneethanol (5), 4′-R-OH-α-ionylideneacetic acid (6), 4′-S-OH-α-ionylideneacetic acid (7), and 1′-OH-α-ionylideneacetic acid (8). The enzymatic steps possibly catalyzed by BcABA1, BcABA2, and BcABA4 are indicated.