Pathway-specific regulation of the botcinic acid biosynthetic gene cluster in the grey mould fungus Botrytis cinerea

Antoine Porquier 1 Bérengère Dalmais 1 Guillaume Morgant 1 Javier Moraga 2 Adeline Simon 1 Hind Sghyer 1 Jean-Marc Pradier 1 Isidro G. Collado 2 Muriel Viaud 1
1Bioger, INRA, Grignon, France
2Departamento de Quimica Organica, Universidad de Cadiz, Cadiz, Spain

Botcinic acid (BOA) is a non-host specific phytotoxin produced by Botrytis cinerea. Its biosynthesis relies on the two PolyKetide Synthase encoding genes Bcboa6 and Bcboa9 which are clustered together with co-regulated genes putatively also involved in the pathway. In order to understand how BOA biosynthesis is regulated, we investigated the genomic environment of the BOA cluster and searched for putative regulator encoding genes.

Amongst the clustered genes, Bcboa13 was predicted to encode a Zn(II)2Cys6 transcription factor (TF). Inactivation of the BcBoa13 gene resulted in a drastic diminution of the expression of the Bcboa genes and in the absence of BOA. These data revealed a major positive role of BOA13 in the regulation of the cluster. Fusion of BcBoa13 with GFP further indicated that it localizes into nuclear foci. Heterochromatin and telomeric markers are currently developed to investigate the nature of these foci.

In addition to Bcboa13, another gene (Bcboa1) encodes a putative regulator: the predicted protein has a NmrA-like domain that may be involved in protein-protein interactions. The impact of BcBoa1 on BOA production is investigated by gene inactivation, while its possible interaction with BcBoa13 is tested by Bimolecular Fluorescence Complementation (BiFC).

Finally, the BOA cluster is localized in a subtelomeric region in which the A+T/G+C-equilibrated regions that contain 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 BOA synthesis. Several histone methyl transferases are under studies to test this hypothesis.

Identification of BcBoa13 as the major regulator of BOA synthesis is the first step toward a comprehensive understanding of the regulation network of toxin synthesis in B. cinerea. Ongoing work may point out the respective role of pathway-specific transcriptional regulators and chromatin structure modifications.