The KdmB-EcoA-RpdA-SntB (KERS) chromatin regulatory complex controls development, secondary metabolism and pathogenicity in Aspergillus flavus.

The filamentous fungus Aspergillus flavus is a plant and human pathogen predominantly found in the soil as spores or sclerotia and is capable of producing various secondary metabolites (SM) such as the carcinogenic mycotoxin aflatoxin. Recently, we have discovered a novel nuclear chromatin binding complex (KERS) that contains the JARID1-type histone demethylase KdmB, a putative cohesion acetyl transferase EcoA, a class I type histone deacetylase RpdA and the PHD ring finger reader protein SntB in the model filamentous fungus Aspergillus nidulans. Here, we show the presence of the KERS complex in A. flavus by immunoprecipitation-coupled mass spectrometry and constructed kdmBΔ and rpdAΔ strains to study their roles in fungal development, SM production and histone post-translational modifications (HPTMs). We found that KdmB and RpdA couple the regulation of SM gene clusters with fungal light-responses and HPTMs. KdmB and RpdA have opposing roles in light-induced asexual conidiation, while both factors are positive regulators of sclerotia development through the nsdC and nsdD pathway. KdmB and RpdA are essential for the productions of aflatoxin (similar to findings for SntB) as well as cyclopiazonic acid, ditryptophenaline and leporin B through controlling the respective SM biosynthetic gene clusters. We further show that both KdmB and RpdA regulate H3K4me3 and H3K9me3 levels, while RpdA also acts on H3K14ac levels in nuclear extracts. Therefore, the chromatin modifiers KdmB and RpdA of the KERS complex are key regulators for fungal development and SM metabolism in A. flavus.

F-box receptor mediated control of substrate stability and subcellular location organizes cellular development of Aspergillus nidulans.

Fungal growth and development are coordinated with specific secondary metabolism. This coordination requires 8 of 74 F-box proteins of the filamentous fungus Aspergillus nidulans. F-box proteins recognize primed substrates for ubiquitination by Skp1-Cul1-Fbx (SCF) E3 ubiquitin RING ligases and degradation by the 26S proteasome. 24 F-box proteins are found in the nuclear fraction as part of SCFs during vegetative growth. 43 F-box proteins interact with SCF proteins during growth, development or stress. 45 F-box proteins are associated with more than 700 proteins that have mainly regulatory roles. This corroborates that accurate surveillance of protein stability is prerequisite for organizing multicellular fungal development. Fbx23 combines subcellular location and protein stability control, illustrating the complexity of F-box mediated regulation during fungal development. Fbx23 interacts with epigenetic methyltransferase VipC which interacts with fungal NF-κB-like velvet domain regulator VeA that coordinates fungal development with secondary metabolism. Fbx23 prevents nuclear accumulation of methyltransferase VipC during early development. These results suggest that in addition to their role in protein degradation, F-box proteins also control subcellular accumulations of key regulatory proteins for fungal development.

The KdmB-EcoA-RpdA-SntB chromatin complex binds regulatory genes and coordinates fungal development with mycotoxin synthesis.

Chromatin complexes control a vast number of epigenetic developmental processes. Filamentous fungi present an important clade of microbes with poor understanding of underlying epigenetic mechanisms. Here, we describe a chromatin binding complex in the fungus Aspergillus nidulans composing of a H3K4 histone demethylase KdmB, a cohesin acetyltransferase (EcoA), a histone deacetylase (RpdA) and a histone reader/E3 ligase protein (SntB). In vitro and in vivo evidence demonstrate that this KERS complex is assembled from the EcoA-KdmB and SntB-RpdA heterodimers. KdmB and SntB play opposing roles in regulating the cellular levels and stability of EcoA, as KdmB prevents SntB-mediated degradation of EcoA. The KERS complex is recruited to transcription initiation start sites at active core promoters exerting promoter-specific transcriptional effects. Interestingly, deletion of any one of the KERS subunits results in a common negative effect on morphogenesis and production of secondary metabolites, molecules important for niche securement in filamentous fungi. Consequently, the entire mycotoxin sterigmatocystin gene cluster is downregulated and asexual development is reduced in the four KERS mutants. The elucidation of the recruitment of epigenetic regulators to chromatin via the KERS complex provides the first mechanistic, chromatin-based understanding of how development is connected with small molecule synthesis in fungi.

Novel Fus3- and Ste12-interacting protein FsiA activates cell fusion-related genes in both Ste12-dependent and -independent manners in Ascomycete filamentous fungi.

Filamentous fungal cells, unlike yeasts, fuse during vegetative growth. The orthologs of mitogen-activated protein (MAP) kinase Fus3 and transcription factor Ste12 are commonly involved in the regulation of cell fusion. However, the specific regulatory mechanisms underlying cell fusion in filamentous fungi have not been revealed. In the present study, we identified the novel protein FsiA as an AoFus3- and AoSte12-interacting protein in the filamentous fungus Aspergillus oryzae. The expression of AonosA and cell fusion-related genes decreased upon fsiA deletion and increased with fsiA overexpression, indicating that FsiA is a positive regulator of cell fusion. In addition, the induction of cell fusion-related genes by fsiA overexpression was also observed in the Aoste12 deletion mutant, indicating that FsiA can induce the cell fusion-related genes in an AoSte12-independent manner. Surprisingly, the fsiA and Aoste12 double deletion mutant exhibited higher cell fusion efficiency and increased mRNA levels of the cell fusion-related genes as compared to the fsiA single deletion mutant, which revealed that AoSte12 represses the cell fusion-related genes in the fsiA deletion mutant. Taken together, our data demonstrate that FsiA activates the cell fusion-related genes by suppressing the negative function of AoSte12 as well as by an AoSte12-independent mechanism.

RcLS2F - A Novel Fungal Class 1 KDAC Co-repressor Complex in Aspergillus nidulans.

The fungal class 1 lysine deacetylase (KDAC) RpdA is a promising target for prevention and treatment of invasive fungal infection. RpdA is essential for survival of the most common air-borne mold pathogen Aspergillus fumigatus and the model organism Aspergillus nidulans. In A. nidulans, RpdA depletion induced production of previously unknown small bioactive substances. As known from yeasts and mammals, class 1 KDACs act as components of multimeric protein complexes, which previously was indicated also for A. nidulans. Composition of these complexes, however, remained obscure. In this study, we used tandem affinity purification to characterize different RpdA complexes and their composition in A. nidulans. In addition to known class 1 KDAC interactors, we identified a novel RpdA complex, which was termed RcLS2F. It contains ScrC, previously described as suppressor of the transcription factor CrzA, as well as the uncharacterized protein FscA. We show that recruitment of FscA depends on ScrC and we provide clear evidence that ΔcrzA suppression by ScrC depletion is due to a lack of transcriptional repression caused by loss of the novel RcLS2F complex. Moreover, RcLS2F is essential for sexual development and engaged in an autoregulatory feed-back loop.

Control of Development, Secondary Metabolism and Light-Dependent Carotenoid Biosynthesis by the Velvet Complex of Neurospora crassa.

Neurospora crassa is an established reference organism to investigate carotene biosynthesis and light regulation. However, there is little evidence of its capacity to produce secondary metabolites. Here, we report the role of the fungal-specific regulatory velvet complexes in development and secondary metabolism (SM) in N. crassa Three velvet proteins VE-1, VE-2, VOS-1, and a putative methyltransferase LAE-1 show light-independent nucleocytoplasmic localization. Two distinct velvet complexes, a heterotrimeric VE-1/VE-2/LAE-1 and a heterodimeric VE-2/VOS-1 are found in vivo The heterotrimer-complex, which positively regulates sexual development and represses asexual sporulation, suppresses siderophore coprogen production under iron starvation conditions. The VE-1/VE-2 heterodimer controls carotene production. VE-1 regulates the expression of >15% of the whole genome, comprising mainly regulatory and developmental features. We also studied intergenera functions of the velvet complex through complementation of Aspergillus nidulans veA, velB, laeA, vosA mutants with their N. crassa orthologs ve-1, ve-2, lae-1, and vos-1, respectively. Expression of VE-1 and VE-2 in A. nidulans successfully substitutes the developmental and SM functions of VeA and VelB by forming two functional chimeric velvet complexes in vivo, VelB/VE-1/LaeA and VE-2/VeA/LaeA, respectively. Reciprocally, expression of veA restores the phenotypes of the N. crassa ve-1 mutant. All N. crassa velvet proteins heterologously expressed in A. nidulans are localized to the nuclear fraction independent of light. These data highlight the conservation of the complex formation in N. crassa and A. nidulans However, they also underline the intergenera similarities and differences of velvet roles according to different life styles, niches and ontogenetic processes.

Assembly of a heptameric STRIPAK complex is required for coordination of light-dependent multicellular fungal development with secondary metabolism in Aspergillus nidulans.

Eukaryotic striatin forms striatin-interacting phosphatase and kinase (STRIPAK) complexes that control many cellular processes including development, cellular transport, signal transduction, stem cell differentiation and cardiac functions. However, detailed knowledge of complex assembly and its roles in stress responses are currently poorly understood. Here, we discovered six striatin (StrA) interacting proteins (Sips), which form a heptameric complex in the filamentous fungus Aspergillus nidulans. The complex consists of the striatin scaffold StrA, the Mob3-type kinase coactivator SipA, the SIKE-like protein SipB, the STRIP1/2 homolog SipC, the SLMAP-related protein SipD and the catalytic and regulatory phosphatase 2A subunits SipE (PpgA), and SipF, respectively. Single and double deletions of the complex components result in loss of multicellular light-dependent fungal development, secondary metabolite production (e.g. mycotoxin Sterigmatocystin) and reduced stress responses. sipA (Mob3) deletion is epistatic to strA deletion by supressing all the defects caused by the lack of striatin. The STRIPAK complex, which is established during vegetative growth and maintained during the early hours of light and dark development, is mainly formed on the nuclear envelope in the presence of the scaffold StrA. The loss of the scaffold revealed three STRIPAK subcomplexes: (I) SipA only interacts with StrA, (II) SipB-SipD is found as a heterodimer, (III) SipC, SipE and SipF exist as a heterotrimeric complex. The STRIPAK complex is required for proper expression of the heterotrimeric VeA-VelB-LaeA complex which coordinates fungal development and secondary metabolism. Furthermore, the STRIPAK complex modulates two important MAPK pathways by promoting phosphorylation of MpkB and restricting nuclear shuttling of MpkC in the absence of stress conditions. SipB in A. nidulans is similar to human suppressor of IKK-ε(SIKE) protein which supresses antiviral responses in mammals, while velvet family proteins show strong similarity to mammalian proinflammatory NF-KB proteins. The presence of these proteins in A. nidulans further strengthens the hypothesis that mammals and fungi use similar proteins for their immune response and secondary metabolite production, respectively.

The HamE scaffold positively regulates MpkB phosphorylation to promote development and secondary metabolism in Aspergillus nidulans.

Mitogen-activated protein kinase (MAPK) pathways are conserved signalling cascades in eukaryotes which regulate a myriad of processes in fungi from sexual reproduction to stress responses. These pathways rely on recruitment of three kinases on a scaffold protein to facilitate efficient kinase phosphorylation and subsequent downstream signalling to the nucleus. The model filamentous fungus Aspergillus nidulans utilises a MAPK pathway termed the pheromone module to regulate both development and secondary metabolism. This complex consists of the MAP3K (SteC), MAP2K (MkkB), MAPK (MpkB) and adaptor protein SteD. To date, there has been no scaffold protein identified for this MAPK pathway. In this study, we characterised a protein termed HamE, which we propose as a scaffold that regulates kinase phosphorylation and signalling in the pheromone module. Mass spectrometry analysis and BIFC experiments revealed that HamE physically interacts with both MkkB and MpkB and transiently interacts with SteC. Deletion of hamE or any of the pheromone module kinases results in reduced sporulation and complete abolishment of cleistothecia production. Mutants also exhibited reductions in expression of secondary metabolite gene clusters, including the velvet complex and sterigmatocystin genes. HamE acts as a positive regulator of MpkB phosphorylation, allowing for HamE to subsequently regulate development and secondary metabolism.