Sesquiterpene synthase from the botrydial biosynthetic gene cluster of the phytopathogen Botrytis cinerea

Abstract

The fungus Botrytis cinerea is the causal agent of the economically important gray mold disease that affects more than 200 ornamental and agriculturally important plant species. B. cinerea is a necrotrophic plant pathogen that secretes nonspecific phytotoxins, including the sesquiterpene botrydial and the polyketide botcinic acid. The region surrounding the previously characterized BcBOT1 gene has now been identified as the botrydial biosynthetic gene cluster.Five genes including BcBOT1 and BcBOT2 were shown by quantitative reverse transcription-PCR to be co-regulated through the calcineurin signaling pathway. Inactivation of the BcBOT2 gene, encoding a putative sesquiterpene cyclase, abolished botrydial biosynthesis, which could be restored by in trans complementation.Inactivation of BcBOT2 also resulted in overproduction of botcinic acid that was observed to be strain-dependent. Recombinant BcBOT2 protein converted farnesyl diphosphate to the parent sesquiterpene of the botrydial biosynthetic pathway, the tricyclic alcohol presilphiperfolan-8beta-ol.

Figure 1

Metabolites isolated from B. cinerea strains and mutants.

Figure 2

Biosynthesis of botrydial by Botrytis cinerea. a. Cyclization of farnesyl diphosphate (FPP) to presilphiperfolan-8β-ol (10) catalyzed by the BcBOT2 protein and biosynthesis of botrydial from 10. B. cinerea mutant bcbot1 does not form botrydial and other late stage intermediates but does accumulate 9 and other probotryanes, while mutant bcbot2 is blocked in the entire pathway. b. Cyclization of [1,1-²H₂]farnesyl diphosphate to [5,5-²H₂]presilphiperfolan-8β-ol.

Figure 3

Physical cluster of co-regulated genes of Botrytis cinerea flanking the botrydial biosynthesis gene BcBOT1. a. Five open reading frames of the botrydial biosynthetic gene cluster (accession number at NCBI: AY277723). Putative functions predicted by protein sequence similarity are indicated under each gene. b. Down-regulation of the clustered genes in the presence of the calcineurin inhibitor cyclosporin A (10 μg/ml) and in the bcg1Δ mutant (18). The expression level of each of the five clustered genes was analysed by qRT-PCR with the constitutively expressed elongation factor gene EF1b serving as a control (35).

Figure 4

Pathogenicity assays of Botrytis cinerea. Wild-type T4 strains, recipient strains (ku mutants) and botrydial mutants on (a) bean leaves, (b) tomato leaves, and (c) grape berries. Leaves were inoculated with plugs of mycelium while the tops of the berries were inoculated with 10³ conidia.

Figure 5

The presilphiperfolan-8-yl cation 11 is the likely precursor of a wide variety of triquinane and related sesquiterpenes from fungi and higher plants.

Figure 5

The presilphiperfolan-8-yl cation 11 is the likely precursor of a wide variety of triquinane and related sesquiterpenes from fungi and higher plants.