Abstract
GATA-type transcription factors (TFs) such as the nitrogen regulators AreA and AreB, or the light-responsive TFs WC-1 and WC-2, play global roles in fungal growth and development. The conserved GATA TF NsdD is known as an activator of sexual development and key repressor of conidiation in Aspergillus nidulans, and as light-regulated repressor of macroconidia formation in Botrytis cinerea. In the present study, we functionally characterized the NsdD ortholog in Fusarium fujikuroi, named Csm1. Deletion of this gene resulted in elevated microconidia formation in the wild-type (WT) and restoration of conidiation in the non-sporulating velvet mutant Δvel1 demonstrating that Csm1 also plays a role as repressor of conidiation in F. fujikuroi. Furthermore, biosynthesis of the PKS-derived red pigments, bikaverin and fusarubins, is de-regulated under otherwise repressing conditions. Cross-species complementation of the Δcsm1 mutant with the B. cinerea ortholog LTF1 led to full restoration of WT-like growth, conidiation and pigment formation. In contrast, the F. fujikuroi CSM1 rescued only the defects in growth, the tolerance to H2O2 and virulence, but did not restore the light-dependent differentiation when expressed in the B. cinerea Δltf1 mutant. Microarray analysis comparing the expression profiles of the F. fujikuroi WT and the Δcsm1 mutant under different nitrogen conditions revealed a strong impact of this GATA TF on 19 of the 47 gene clusters in the genome of F. fujikuroi. One of the up-regulated silent gene clusters is the one containing the sesquiterpene cyclase-encoding key gene STC1. Heterologous expression of STC1 in Escherichia coli enabled us to identify the product as the volatile bioactive compound (–)-germacrene D.
Highlights
Filamentous fungi produce a diverse array of low-molecular-mass compounds known as secondary metabolites (SMs)
Phylogenetic analysis confirmed the closer relationship of Csm1 to the orthologs of two other members of the peritheciumforming class Sordariomycetes, N. crassa, and S. macrospora, compared to Ltf1 from B. cinerea, a member of the apotheciumforming class Leotiomycetes, and NsdD from A. nidulans, a member of the cleistothecium-forming class Eurotiomycetes (Figure 1B)
To show which of the pigments is de-regulated in the mutant, we studied the expression of bikaverin and fusarubin biosynthetic genes under the different nitrogen conditions (6 and 60 mM glutamine, and 6 mM NaNO3, respectively) in the WT, the CSM1 deletion mutant and the complemented strain CSM1C CSM1 (Figure 3B)
Summary
Filamentous fungi produce a diverse array of low-molecular-mass compounds known as secondary metabolites (SMs). Clusters containing modular polyketide synthase (PKS) or non-ribosomal peptide synthetase (NRPS) genes are easy to detect by scanning the genome for genes that encode enzymes with conserved characteristic domains, e.g., the “adenylation (A)” and “condensation (C)” domains of NRPSs (Weber and Kim, 2016). Many of these SM gene clusters either remain “silent” or weakly expressed under standard experimental conditions and many of their products are still unknown (Brakhage, 2013). A better understanding of the complex regulatory network that modulates the expression of SM biosynthetic genes is of great importance to overcome the silencing of cryptic gene clusters and to discover new bioactive compounds
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