Cis-regulation of toxin clusters in Fusarium.

Fusarium species are able to produce an array of known and unknown metabolites. Fusarium plant pathogens produce toxins that accumulate in the edible plant parts contaminating food and feed. Current European regulations determine limits for some fusariotoxins such as deoxynivalenol and fumonisins. These two toxins are produced by F. graminearum and F. verticillioides by two main gene clusters (the tri and fum cluster, respectively) conferring ability to synthesize, intracellularly detoxify and export the toxin. Understanding the mechanisms driving toxin synthesis and regulation is therefore important for further developing containment measures. Methods Here the two key regulatory Transcription Factor Binding Sites (TFBSs) specific for the tri cluster and the fum cluster were identified by overrepresentation analyses. Moreover for F. graminearum full genome expression studies were used to identify genes controlled by the main transcriptional regulator of the cluster. In F. verticillioides in vivo and in planta experiments were carried out to confirm the biological function of the newly identified binding site. Results In F. graminearum, the TFBS identified confirmed a previous EMSA study. The TFBS also was found upstream of the genes of the primary metabolism necessary for toxin synthesis. Comparative genomics showed that this is true only for the species carrying the cluster therefore suggesting that co-evolution of the genome and the cluster has occurred. In F. verticillioides a new cis-regulatory sequence was identified. When experimentally modified, the newly discovered TFBS resulted in a decreased transcriptional activity of the fumonisin cluster PKS gene (FUM1), suggesting a direct role of the predicted binding site in fumonisin regulation. The TFBS is partially conserved in F. oxysporum and also in the phylogenetically distant Aspergillus niger (both able to produce fumonisins), suggesting the sequence is bound by a transcriptional factor specific for the cluster. Structural and bioinformatics data suggest it may be the zinc-finger protein FUM21 that has a slight modification of the DNA-binding site between F. verticillioides, F. oxysporum and A. niger which could account for the slight different preferences in the TFBS sequence. Conclusion After acquisition of a toxin biosynthetic cluster two events were observed. In F. graminearum the genome appears to adapt to the cluster specific transcription factor while in the fumonisin producing species (A. niger-F. verticillioides-F. oxysporum). the TFBS has coevolved with the zinc finger protein binding it.