ChLae1 and ChVel1 Regulate T-toxin Production, Virulence, Oxidative Stress Response, and Development of the Maize Pathogen Cochliobolus heterostrophus

LaeA and VeA coordinate secondary metabolism and differentiation in response to light signals in Aspergillus spp. Their orthologs, ChLae1 and ChVel1, were identified in the maize pathogen Cochliobolus heterostrophus, known to produce a wealth of secondary metabolites, including the host selective toxin, T-toxin. Produced by race T, T-toxin promotes high virulence to maize carrying Texas male sterile cytoplasm (T-cms). T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT. Correspondingly, expression of T-toxin biosynthetic genes (Tox1) is up-regulated in the dark in WT, while dark-induced expression is much reduced/minimal in Chvel1 and Chlae1 mutants. Toxin production and Tox1 gene expression are increased in ChVEL1 overexpression (OE) strains grown in the dark and in ChLAE1 strains grown in either light or dark, compared to WT. These observations establish ChLae1 and ChVel1 as the first factors known to regulate host selective toxin production. Virulence of Chlae1 and Chvel1 mutants and OE strains is altered on both T-cms and normal cytoplasm maize, indicating that both T-toxin mediated super virulence and basic pathogenic ability are affected. Deletion of ChLAE1 or ChVEL1 reduces tolerance to H2O2. Expression of CAT3, one of the three catalase genes, is reduced in the Chvel1 mutant. Chlae1 and Chvel1 mutants also show decreased aerial hyphal growth, increased asexual sporulation and female sterility. ChLAE1 OE strains are female sterile, while ChVEL1 OE strains are more fertile than WT. ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains. Thus, ChLae1 and ChVel1 positively regulate T-toxin biosynthesis, pathogenicity and super virulence, oxidative stress responses, sexual development, and aerial hyphal growth, and negatively control melanin biosynthesis and asexual differentiation

Regulation of <i>Aspergillus nidulans</i> CreA-Mediated Catabolite Repression by the F-Box Proteins Fbx23 and Fbx47

ABSTRACT The attachment of one or more ubiquitin molecules by SCF ( S kp– C ullin– F -box) complexes to protein substrates targets them for subsequent degradation by the 26S proteasome, allowing the control of numerous cellular processes. Glucose-mediated signaling and subsequent carbon catabolite repression (CCR) are processes relying on the functional regulation of target proteins, ultimately controlling the utilization of this carbon source. In the filamentous fungus Aspergillus nidulans , CCR is mediated by the transcription factor CreA, which modulates the expression of genes encoding biotechnologically relevant enzymes. Although CreA-mediated repression of target genes has been extensively studied, less is known about the regulatory pathways governing CCR and this work aimed at further unravelling these events. The Fbx23 F-box protein was identified as being involved in CCR and the Δ fbx23 mutant presented impaired xylanase production under repressing (glucose) and derepressing (xylan) conditions. Mass spectrometry showed that Fbx23 is part of an SCF ubiquitin ligase complex that is bridged via the GskA protein kinase to the CreA-SsnF-RcoA repressor complex, resulting in the degradation of the latter under derepressing conditions. Upon the addition of glucose, CreA dissociates from the ubiquitin ligase complex and is transported into the nucleus. Furthermore, casein kinase is important for CreA function during glucose signaling, although the exact role of phosphorylation in CCR remains to be determined. In summary, this study unraveled novel mechanistic details underlying CreA-mediated CCR and provided a solid basis for studying additional factors involved in carbon source utilization which could prove useful for biotechnological applications. IMPORTANCE The production of biofuels from plant biomass has gained interest in recent years as an environmentally friendly alternative to production from petroleum-based energy sources. Filamentous fungi, which naturally thrive on decaying plant matter, are of particular interest for this process due to their ability to secrete enzymes required for the deconstruction of lignocellulosic material. A major drawback in fungal hydrolytic enzyme production is the repression of the corresponding genes in the presence of glucose, a process known as carbon catabolite repression (CCR). This report provides previously unknown mechanistic insights into CCR through elucidating part of the protein-protein interaction regulatory system that governs the CreA transcriptional regulator in the reference organism Aspergillus nidulans in the presence of glucose and the biotechnologically relevant plant polysaccharide xylan. </jats:p

The Aspergillus nidulans MAPK module AnSte11-Ste50-Ste7-Fus3 controls development and secondary metabolism.

The sexual Fus3 MAP kinase module of yeast is highly conserved in eukaryotes and transmits external signals from the plasma membrane to the nucleus. We show here that the module of the filamentous fungus Aspergillus nidulans (An) consists of the AnFus3 MAP kinase, the upstream kinases AnSte7 and AnSte11, and the AnSte50 adaptor. The fungal MAPK module controls the coordination of fungal development and secondary metabolite production. It lacks the membrane docking yeast Ste5 scaffold homolog; but, similar to yeast, the entire MAPK module's proteins interact with each other at the plasma membrane. AnFus3 is the only subunit with the potential to enter the nucleus from the nuclear envelope. AnFus3 interacts with the conserved nuclear transcription factor AnSte12 to initiate sexual development and phosphorylates VeA, which is a major regulatory protein required for sexual development and coordinated secondary metabolite production. Our data suggest that not only Fus3, but even the entire MAPK module complex of four physically interacting proteins, can migrate from plasma membrane to nuclear envelope

The Aspergillus nidulans MAPK Module AnSte11-Ste50- Ste7-Fus3 Controls Development and Secondary Metabolism

The sexual Fus3 MAP kinase module of yeast is highly conserved in eukaryotes and transmits external signals from the plasma membrane to the nucleus. We show here that the module of the filamentous fungus Aspergillus nidulans (An) consists of the AnFus3 MAP kinase, the upstream kinases AnSte7 and AnSte11, and the AnSte50 adaptor. The fungal MAPK module controls the coordination of fungal development and secondary metabolite production. It lacks the membrane docking yeast Ste5 scaffold homolog; but, similar to yeast, the entire MAPK module’s proteins interact with each other at the plasma membrane. AnFus3 is the only subunit with the potential to enter the nucleus from the nuclear envelope. AnFus3 interacts with the conserved nuclear transcription factor AnSte12 to initiate sexual development and phosphorylates VeA, which is a major regulatory protein required for sexual development and coordinated secondary metabolite production. Our data suggest that not only Fus3, but even the entire MAPK module complex of four physically interacting proteins, can migrate from plasma membrane to nuclear envelope

The Aspergillus nidulans MAPK Module AnSte11-Ste50-Ste7-Fus3 Controls Development and Secondary Metabolism

The sexual Fus3 MAP kinase module of yeast is highly conserved in eukaryotes and transmits external signals from the plasma membrane to the nucleus. We show here that the module of the filamentous fungus Aspergillus nidulans (An) consists of the AnFus3 MAP kinase, the upstream kinases AnSte7 and AnSte11, and the AnSte50 adaptor. The fungal MAPK module controls the coordination of fungal development and secondary metabolite production. It lacks the membrane docking yeast Ste5 scaffold homolog; but, similar to yeast, the entire MAPK module's proteins interact with each other at the plasma membrane. AnFus3 is the only subunit with the potential to enter the nucleus from the nuclear envelope. AnFus3 interacts with the conserved nuclear transcription factor AnSte12 to initiate sexual development and phosphorylates VeA, which is a major regulatory protein required for sexual development and coordinated secondary metabolite production. Our data suggest that not only Fus3, but even the entire MAPK module complex of four physically interacting proteins, can migrate from plasma membrane to nuclear envelope

The Aspergillus nidulans MAPK Module AnSte11-Ste50-Ste7-Fus3 Controls Development and Secondary Metabolism

The sexual Fus3 MAP kinase module of yeast is highly conserved in eukaryotes and transmits external signals from the plasma membrane to the nucleus. We show here that the module of the filamentous fungus Aspergillus nidulans (An) consists of the AnFus3 MAP kinase, the upstream kinases AnSte7 and AnSte11, and the AnSte50 adaptor. The fungal MAPK module controls the coordination of fungal development and secondary metabolite production. It lacks the membrane docking yeast Ste5 scaffold homolog; but, similar to yeast, the entire MAPK module's proteins interact with each other at the plasma membrane. AnFus3 is the only subunit with the potential to enter the nucleus from the nuclear envelope. AnFus3 interacts with the conserved nuclear transcription factor AnSte12 to initiate sexual development and phosphorylates VeA, which is a major regulatory protein required for sexual development and coordinated secondary metabolite production. Our data suggest that not only Fus3, but even the entire MAPK module complex of four physically interacting proteins, can migrate from plasma membrane to nuclear envelope

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.</jats:p

Loss of sexual fruiting bodies and heterokaryon formation in <i>mkkB</i>Δ strain lacking AnSte7.

<p>(A) Sexual developments of a wild type, <i>mkkB</i>Δ, <i>mkkB</i> complementation (comp+), <i>steC</i>Δ [lacking AnSte11] strains, which were point inoculated (1×10⁴) and grown on minimal medium in dark conditions (5 days at 37°C). Small squares are the close-up stereomicroscopic images of the strains. Red arrows indicate the mature black fruiting bodies of the wild type and complementation strains and yellow arrows denote the premature nests produced by <i>mkkB</i><b>Δ</b> and <i>steC</i><b>Δ</b> strains. (B) Raster electron microscopy (REM) image of the strains from (A). The wild type fruiting body (cleistothecium: Cl) is surrounded by the globose Hülle cells. <i>steC</i> and <i>mkkB</i> mutants produce only dispersed groups of Hülle cells (yellow arrows) instead of mature fruiting bodies. (C) Monitoring hyphal fusions and heterokaryons via fluorescence microscopy. Strains bearing either cytoplasmic synthetic green fluorescent protein (sGFP) or nuclear red fluorescent protein fused histone 2A (mRFP) were used in different combinations. Only two wild types form green and red fluorescent combinations through hyphal fusions. (D) Isolated protoplasts from two wild types (yellow and green), <i>steC</i>Δ (green), <i>mkkB</i>Δ (yellow or green) were used for protoplast fusions as shown in combinations and plated on selective medium. <i>wt</i>/<i>wt</i>, <i>wt</i>/<i>steC</i>Δ, <i>wt</i>/<i>mkkB</i>Δ combinations produce fruiting bodies after 7–8 days, whereas <i>mkkB</i><b>Δ</b>/<i>mkkB</i><b>Δ</b> combination only produces nests.</p

Subcellular locations of the AnSte50-Ste11, AnSte50-Ste7, and AnSte50-Fus3 complexes <i>in vivo</i>.

<p>(A) Interactions of N-EYFP::AnSte11 [SteC] and C-EYFP::AnSte50 [SteD] proteins in the fungal cells. Upper panel shows the nuclear mRFP signal and lower panel FM4-64 in comparison to the split YFP signal. (B) Interactions of AnSte50 [SteD] protein with AnSte7 [MkkB]. (C) Interactions of AnSte50 protein with AnFus3 [MpkB]. (D–F) Measurement of the subcellular locations of the AnSte50-Ste11, -Ste7, and Fus3 complexes that are frequently found in the nuclear envelope, plasma membrane, at the hypal tip and less often in the septal locations. Quantification was performed and analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002816#pgen-1002816-g007" target="_blank">Figure 7</a>. (G) Detection of the AnSte50-Ste11, AnSte50-Ste7, and AnSte50-Fus3 complexes on the vesicle (V) of the asexual conidiophore structures. S; stalk. Scale bars represent 10 µm.</p