Identification of SclB, a Zn(II)2Cys6 transcription factor involved in sclerotium formation in Aspergillus niger

Thomas R Jørgensen,Anne-Marie Burggraaf,Mark Arentshorst,Tabea Schutze,Gerda Lamers,Jing Niu,Min Jin Kwon,Joohae Park,Jens C Frisvad,Kristian F Nielsen,Vera Meyer,Cees A.M.J.J Van Den Hondel,Paul S Dyer,Arthur F.J Ram

doi:10.1016/j.fgb.2020.103377

Abstract

Certain Aspergillus species such as Aspergillus flavus and A. parasiticus are well known for the formation of sclerotia. These developmental structures are thought to act as survival structures during adverse environmental conditions but are also a prerequisite for sexual reproduction. We previously described an A. niger mutant (scl-2) which formed sclerotium-like structures, suggesting a possible first stage of sexual development in this species. Several lines of evidence presented in this study support the previous conclusion that the sclerotium-like structures of scl-2 are indeed sclerotia. These included the observations that: (i) safranin staining of the sclerotia-like structures produced by the scl-2 mutant showed the typical cellular structure of a sclerotium; (ii) metabolite analysis revealed specific production of indoloterpenes, which have previously been connected to sclerotium formation; (iii) formation of the sclerotium-like structures is dependent on a functional NADPH complex, as shown for other fungi forming sclerotia. The mutation in scl-2 responsible for sclerotium formation was identified using parasexual crossing and bulk segregant analysis followed by high throughput sequencing and subsequent complementation analysis. The scl-2 strain contains a mutation that introduces a stop codon in the putative DNA binding domain of a previously uncharacterized Zn(II)2Cys6 type transcription factor (An08g07710). Targeted deletion of this transcription factor (sclB) confirmed its role as a repressor of sclerotial formation and in the promotion of asexual reproduction in A. niger. Finally, a genome-wide transcriptomic comparison of RNA extracted from sclerotia versus mycelia revealed major differences in gene expression. Induction of genes related to indoloterpene synthesis was confirmed and also let to the identification of a gene cluster essential for the production of aurasperones during sclerotium formation. Expression analysis of genes encoding other secondary metabolites, cell wall related genes, transcription factors, and genes related to reproductive processes identified many interesting candidate genes to further understand the regulation and biosynthesis of sclerotia in A. niger. The newly identified SclB transcription factor acts as a repressor of sclerotium formation and manipulation of sclB may represent a first prerequisite step towards engineering A. niger strains capable of sexual reproduction. This will provide exciting opportunities for further strain improvement in relation to protein or metabolite production in A. niger.

Highlights

Aspergillus niger is an industrially important filamentous fungus which is only known to reproduce asexually through production of conidiospores
In the case of the genus Aspergillus, the formation of sclerotia is required for sexual reproduction in members of the section Flavi and section Nigri, in which fruit bodies known as cleistothecia develop in a stroma within sclerotia (Dyer and O'Gorman, 2011, 2012)
The scl-2 mutant was isolated in a fwnA mutant background in which the fwnA gene, encoding a polyketide synthase required for the biosynthesis of conidial melanin and naphtho-γ-pyrones (Jørgensen et al, 2011b), was deleted

Summary

Introduction

Aspergillus niger is an industrially important filamentous fungus which is only known to reproduce asexually through production of conidiospores. In the case of the genus Aspergillus, the formation of sclerotia is required for sexual reproduction in members of the section Flavi (which includes economically important species such as A. flavus, A. parasiticus and A. oryzae) and section Nigri (which includes A. niger), in which fruit bodies known as cleistothecia develop in a stroma (a mass or matrix of vegetative hyphae) within sclerotia (Dyer and O'Gorman, 2011, 2012). This differs from the majority of aspergilli (such as the model organism A. nidulans), where cleistothecia develop direct without the prior need for formation of sclerotia

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