The botrydial (BOT) and botcinic acid (BOA) biosynthetic gene clusters dispolay a bipartite genomic structure and are controlled by putatiave pathway-specific ZN(II)2CYS6 Transcription factors

The sesquiterpene Botrydial (BOT) and the polyketide botcinic acid (BOA) are non-host specific phytotoxins produced by Botrytis cinerea. In addition, their botryanes and botcinines derivatives were shown to have biological activities against bacteria and fungi. Both BOT and BOA biosynthetic gene clusters were previously identified. Inactivation of the key enzymes-encoding genes, i.e. the sesquiterpene cyclase-encoding gene Bcbot2 and the polyketide synthase-encoding gene Bcboa6, revealed that they may have a redundant role in virulence. In order to understand how BOT, BOA and their derivatives are regulated during the life cycle of B. cinerea, we investigated the genomic environment of the BOT and BOA clusters and searched for putative transcription factor (TF) encoding genes. Thanks to the recent release of the gapless genome of B05.10 strain (J. van Kan, Univ. Wageningen, NL), the BOT and BOA clusters were localized in two A+T-rich regions, one being subtelomeric (BOA). Our bioinformatics analyses revealed that the A+T/G+C-equilibrated regions that contain the Bcbot and Bcboa genes alternate with A+T-rich regions (>85%) made of relics of transposable elements that have undergone repeat-induced point (RIP) mutations. The occurrence of RIP raises questions about possible chromatin-based regulation of BOT and BOA synthesis. Several chromatin modifiers (KMT1, KMT6, HP1) are under studies to test this hypothesis. Amongst the previously identified Bcboa genes, Bcboa13 was predicted to encode a Zn(II)2Cys6 TF. In addition, the gapless version of the genome allowed us to identify a similar gene (Bcbot6) flanking the previously identified BOT cluster. Gene inactivation demonstrated that BcBot6 and BcBoa13 are the major positive regulators of BOT and BOA biosynthesis, respectively. Interestingly, the functional fusion protein BcBot6-GFP and BcBoa13-GFP localize into nuclear foci. Identification of BcBot6 as the major regulators of BOT and BOA synthesis is the first step toward a comprehensive understanding of the complete regulation network of toxin synthesis in B. cinerea. We are currently using a Yeast-One-Hybrid approach to search the upstream TF(s) that are interacting with the promoters of Bcbot6 and Bcboa13 genes.