Abstract
Nuclear protein LaeA is known as the global regulator of secondary metabolism in Aspergillus. LaeA connects with VeA and VelB to form a heterotrimeric complex, which coordinates fungal development and secondary metabolism. Here, we describe a new interaction partner of LaeA, the kinetochore protein Spc105, from the aflatoxin-producing fungus Aspergillus flavus. We showed that in addition to involvement in nuclear division, Spc105 is required for normal conidiophore development and sclerotia production of A. flavus. Moreover, Spc105 positively regulates the production of secondary metabolites such as aflatoxin and kojic acid, and negatively regulates the production of cyclopiazonic acid. Transcriptome analysis of the Δspc105 strain revealed that 23 backbone genes were differentially expressed, corresponding to 19 of the predicted 56 secondary metabolite gene clusters, suggesting a broad regulatory role of Spc105 in secondary metabolism. Notably, the reduced expression of laeA in our transcriptome data led to the discovery of the correlation between Spc105 and LaeA, and double mutant analysis indicated a functional interdependence between Spc105 and LaeA. Further, in vitro and in vivo protein interaction assays revealed that Spc105 interacts directly with the S-adenosylmethionine (SAM)-binding domain of LaeA, and that the leucine zipper motif in Spc105 is required for this interaction. The Spc105-LaeA interaction identified in our study indicates a cooperative interplay of distinct regulators in A. flavus, providing new insights into fungal secondary metabolism regulation networks.
Highlights
Aspergillus flavus is a ubiquitous saprophytic filamentous fungus that infects economically important crops such as peanuts, maize, and many other seed crops during preharvest or storage (Amaike and Keller, 2011)
The structural analysis of Spc105 proteins from several species showed that all analyzed fungi and bacteria share a conserved Spc7 domain (Figure 1B), and Spc105 proteins from Aspergillus most closely resemble their orthologs in Penicillium subrubescens (Figure 1C)
Through green fluorescent protein (GFP) labeling we found that the Spc105 protein is localized in the nucleus of A. flavus (Figure 1D). quantitative RT-PCR (qRT-PCR) analysis of spc105 gene expression in a wild-type (WT) strain showed that it is a low-expressionlevel gene, and its expression was almost constant during the vegetative growth phase, implying that spc105 is a constitutively expressed gene in A. flavus
Summary
Aspergillus flavus is a ubiquitous saprophytic filamentous fungus that infects economically important crops such as peanuts, maize, and many other seed crops during preharvest or storage (Amaike and Keller, 2011). A series of regulatory factors outside of the AF cluster have been proved to control AF biosynthesis, including the velvet complex (Bayram et al, 2008), transcription factors such as NsdC, NsdD, MeaB, and mtfA (Cary et al, 2012; Amaike et al, 2013; Zhuang et al, 2016), oxidative stress response-related genes (Linz et al, 2013; Zhao et al, 2018), and epigenetic modifications including chromatin remodeling and histone acetylation (Lan et al, 2016; Pfannenstiel et al, 2018). We demonstrate for the first time that the conserved kinetochore protein Spc105 in A. flavus regulates developmental differentiation, including conidia development and sclerotia production, and secondary metabolism. The relationship between Spc105 and LaeA highlights the coordinated interplay between distinct regulators, ensuring precise fungal development and secondary metabolism
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