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Title: Dothistromin genes at multiple separate loci are regulated by AflR

item CHETTRI, PRANAV - Massey University
item Ehrlich, Kenneth
item Cary, Jeffrey
item COLLEMARE, JEROME - Massey University
item COX, MURRAY - Massey University
item GRIFFITHS, SCOTT - Massey University
item OLSON, MALIN - Massey University
item DE WIT, PIERRE - Wageningen University
item BRADSHAW, ROSIE - Massey University

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/13/2012
Publication Date: 2/15/2013
Citation: Chettri, P., Ehrlich, K., Cary, J.W., Collemare, J., Cox, M.P., Griffiths, S.A., Olson, M.A., De Wit, P.J., Bradshaw, R.E. 2013. Dothistromin genes at multiple separate loci are regulated by AflR. Fungal Genetics and Biology. 51:12-20.

Interpretive Summary: Dothistroma septosporum, is a fungus that infects pine needles eventually leading to tree death. During growth it produces dothistromin, a polyketide toxin very closely related to aflatoxin (AF) and sterigmatocystin (ST) synthesized by Aspergillus spp. This work describes experiments that have been conducted in an effort to better understand the genetic mechanisms that control dothistomin production and evolution of the D. septosporum toxin biosynthetic genes. Inactivation of the key pathway regulatory gene, aflR, resulted in significant reduction in dothistromin production. Interestingly, the closely related tomato pathogen, Cladosporium fulvum, also harbored genes necessary for dothistromin production but it does not make the toxin. Analysis of the genes demonstrated that key biosynthetic genes were mutated such that they were non-functional explaining the lack of dothistromin production in C. fulvum. This work indicates that the presence or absence of a functional dothistromin biosynthetic pathway may be related to the particular pathogenic lifestyles of D. septosporum and C. fulvum.

Technical Abstract: In fungi, genes involved in the production of secondary metabolites are generally clustered at one location. There are some exceptions, such as genes required for synthesis of dothistromin, a toxin that is a chemical analog of the aflatoxin precursor versicolorin A and made by the pine needle pathogen Dothistroma septosporum. The availability of the D. septosporum genome sequence enabled identification of putative dothistromin genes, including an ortholog of the aflatoxin regulatory gene AflR, and revealed that most of the genes are spread over six separate regions (mini-clusters) on chromosome 12 (1.3 Mb). Here we show that levels of expression of the widely dispersed genes in D. septosporum are not correlated with gene location with respect to their distance from a telomere, but that AflR regulates them. The production of dothistromin by D. septosporum in which the AflR gene was knocked out ('DsAflR) was drastically reduced, but still detectable. This is in contrast to orthologous 'AflR mutants in Aspergillus species that lack any aflatoxin production. Expression patterns in 'DsAflR mutants helped to predict the complete set of genes involved in dothistromin production. This included a short-chain aryl alcohol dehydrogenase (NorB), which is located on chromosome 11 rather than chromosome 12, but was 24-fold down regulated in 'DsAflR. An orthologous set of dothistromin genes, organized in a similar fragmented cluster arrangement to that seen in D. septosporum, was found in the closely related tomato pathogen Cladosporium fulvum even though this species does not produce dothistromin. In C. fulvum, pseudogenization of key biosynthetic genes explains the lack of dothistromin production. However, the remarkable conservation of some of the predicted proteins (e.g. 97% amino acid identity between DsVer1 and CfVer1) and partial complementation of the 'DsAflR mutant by CfAflR suggest only recent loss of the capacity to produce dothistromin in C. fulvum. The fragmented dothistromin cluster provides an example of coordinated control of a dispersed set of secondary metabolite genes; it also provides an example where loss of dothistromin production might have allowed adaptation to a new pathogenic lifestyle.