Gerhard H. Braus

Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans , light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of secondary metabolites.

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.

Filamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkörper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkörper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established.

The EUROFUNG network is a virtual centre of multidisciplinary expertise in the field of fungal biotechnology. The first academic-industry Think Tank was hosted by EUROFUNG to summarise the state of the art and future challenges in fungal biology and biotechnology in the coming decade. Currently, fungal cell factories are important for bulk manufacturing of organic acids, proteins, enzymes, secondary metabolites and active pharmaceutical ingredients in white and red biotechnology. In contrast, fungal pathogens of humans kill more people than malaria or tuberculosis. Fungi are significantly impacting on global food security, damaging global crop production, causing disease in domesticated animals, and spoiling an estimated 10 % of harvested crops. A number of challenges now need to be addressed to improve our strategies to control fungal pathogenicity and to optimise the use of fungi as sources for novel compounds and as cell factories for large scale manufacture of bio-based products. This white paper reports on the discussions of the Think Tank meeting and the suggestions made for moving fungal bio(techno)logy forward.

The two highly conserved RAS genes of the budding yeast Saccharomyces cerevisiae are redundant for viability. Here we show that haploid invasive growth development depends on RAS2 but not RAS1. Ras1p is not sufficiently expressed to induce invasive growth. Ras2p activates invasive growth using either of two downstream signaling pathways, the filamentation MAPK (Cdc42p/Ste20p/MAPK) cascade or the cAMP-dependent protein kinase (Cyr1p/cAMP/PKA) pathway. This signal branch point can be uncoupled in cells expressing Ras2p mutant proteins that carry amino acid substitutions in the adenylyl cyclase interaction domain and therefore activate invasive growth solely dependent on the MAPK cascade. Both Ras2p-controlled signaling pathways stimulate expression of the filamentation response element-driven reporter gene depending on the transcription factors Ste12p and Tec1p, indicating a crosstalk between the MAPK and the cAMP signaling pathways in haploid cells during invasive growth.

This review focuses on the gene-enzyme relationships and the regulation of different levels of the aromatic amino acid biosynthetic pathway in a simple eukaryotic system, the unicellular yeast Saccharomyces cerevisiae. Most reactions of this branched pathway are common to all organisms which are able to synthesize tryptophan, phenylalanine, and tyrosine. The current knowledge about the two main control mechanisms of the yeast aromatic amino acid biosynthesis is reviewed. (i) At the transcriptional level, most structural genes are regulated by the transcriptional activator GCN4, the regulator of the general amino acid control network, which couples transcriptional derepression to amino acid starvation of numerous structural genes in multiple amino acid biosynthetic pathways. (ii) At the enzyme level, the carbon flow is controlled mainly by modulating the enzyme activities at the first step of the pathway and at the branch points by feedback action of the three aromatic amino acid end products. Implications of these findings for the relationship of S. cerevisiae to prokaryotic as well as to higher eukaryotic organisms and for general regulatory mechanisms occurring in a living cell such as initiation of transcription, enzyme regulation, and the regulation of a metabolic branch point are discussed.

The soil-borne fungal pathogen Verticillium longisporum causes vascular disease on Brassicaceae host plants such as oilseed rape. The fungus colonizes the root xylem and moves upwards to the foliage where disease symptoms become visible. Using Arabidopsis as a model for early gene induction, we performed root transcriptome analyses in response to hyphal growth immediately after spore germination and during penetration of the root cortex, respectively. Infected roots showed a rapid reprogramming of gene expression such as activation of transcription factors, stress-, and defense-related genes. Here, we focused on the highly coordinated gene induction resulting in the production of tryptophan-derived secondary metabolites. Previous studies in leaves showed that enzymes encoded by CYP81F2 and PEN2 (PENETRATION2) execute the formation of antifungal indole glucosinolate (IGS) metabolites. In Verticillium-infected roots, we found transcriptional activation of CYP81F2 and the PEN2 homolog PEL1 (PEN2-LIKE1), but no increase in antifungal IGS breakdown products. In contrast, indole-3-carboxylic acid (I3CA) and the phytoalexin camalexin accumulated in infected roots but only camalexin inhibited Verticillium growth in vitro. Whereas genetic disruption of the individual metabolic pathways leading to either camalexin or CYP81F2-dependent IGS metabolites did not alter Verticillium-induced disease symptoms, a cyp79b2 cyp79b3 mutant impaired in both branches resulted in significantly enhanced susceptibility. Hence, our data provide an insight into root-specific early defenses and suggest tryptophan-derived metabolites as active antifungal compounds against a vascular pathogen.

Morphological development of fungi and their combined production of secondary metabolites are both acting in defence and protection. These processes are mainly coordinated by velvet regulators, which contain a yet functionally and structurally uncharacterized velvet domain. Here we demonstrate that the velvet domain of VosA is a novel DNA-binding motif that specifically recognizes an 11-nucleotide consensus sequence consisting of two motifs in the promoters of key developmental regulatory genes. The crystal structure analysis of the VosA velvet domain revealed an unforeseen structural similarity with the Rel homology domain (RHD) of the mammalian transcription factor NF-κB. Based on this structural similarity several conserved amino acid residues present in all velvet domains have been identified and shown to be essential for the DNA binding ability of VosA. The velvet domain is also involved in dimer formation as seen in the solved crystal structures of the VosA homodimer and the VosA-VelB heterodimer. These findings suggest that defence mechanisms of both fungi and animals might be governed by structurally related DNA-binding transcription factors.

The corn pathogen Ustilago maydis is a well-studied fungal model organism. Along with a broad set of experimental tools, versatile strategies for the generation of gene replacement mutants by homologous recombination in U. maydis have been developed. Nevertheless, the production of corresponding linear DNA constructs still constitutes a time-limiting step. To overcome this bottleneck, various resistance cassette modules were adopted for use with the so-called Golden Gate cloning strategy. These modules allow not only simple gene deletions but also more sophisticated genetic manipulations like inserting sequences for C-terminal protein tagging. The type IIs restriction enzyme BsaI was selected for this novel approach as its recognition sites are comparatively rare in the U. maydis genome. To test the efficiency of the new strategy it was used to test the influence of varying flank lengths as well as the effect of non-homologous flank ends on homologous recombination. Importantly, to proof a broad applicability in other fungi the same strategy was used to generate mutants in the filamentous ascomycete Aspergillus nidulans. Hence, we present a highly efficient and economic cloning strategy that speeds up reverse genetic approaches in fungi.

In search of Botrytis cinerea cell death-inducing proteins, we found a xyloglucanase (BcXYG1) that induced strong necrosis and a resistance response in dicot plants. Expression of the BcXYG1 gene was strongly induced during the first 12 h post inoculation, and analysis of disease dynamics using PathTrack showed that a B. cinerea strain overexpressing BcXYG1 produced early local necrosis, supporting a role of BcXYG1 as an early cell death-inducing factor. The xyloglucanase activity of BcXYG1 was not necessary for the induction of necrosis and plant resistance, as a mutant of BcXYG1 lacking the xyloglucanase enzymatic activity retained both functions. Residues in two exposed loops on the surface of BcXYG1 were found to be necessary for the induction of cell death but not to induce plant resistance. Further analyses showed that BcXYG1 is apoplastic and possibly interacts with the proteins of the plant cell membrane and also that the BcXYG1 cell death-promoting signal is mediated by the leucine-rich repeat receptor-like kinases BAK1 and SOBIR1. Our findings support the role of cell death-inducing proteins in establishing the infection of necrotrophic pathogens and highlight the recognition of fungal apoplastic proteins by the plant immune system as an important mechanism of resistance against this class of pathogens.

Cytoplasmic lipid droplets (LDs) are found in all types of plant cells; they are derived from the endoplasmic reticulum and function as a repository for neutral lipids, as well as serving in lipid remodelling and signalling. However, the mechanisms underlying the formation, steady-state maintenance and turnover of plant LDs, particularly in non-seed tissues, are relatively unknown. Previously, we showed that the LD-associated proteins (LDAPs) are a family of plant-specific, LD surface-associated coat proteins that are required for proper biogenesis of LDs and neutral lipid homeostasis in vegetative tissues. Here, we screened a yeast two-hybrid library using the Arabidopsis LDAP3 isoform as 'bait' in an effort to identify other novel LD protein constituents. One of the candidate LDAP3-interacting proteins was Arabidopsis At5g16550, which is a plant-specific protein of unknown function that we termed LDIP (LDAP-interacting protein). Using a combination of biochemical and cellular approaches, we show that LDIP targets specifically to the LD surface, contains a discrete amphipathic α-helical targeting sequence, and participates in both homotypic and heterotypic associations with itself and LDAP3, respectively. Analysis of LDIP T-DNA knockdown and knockout mutants showed a decrease in LD abundance and an increase in variability of LD size in leaves, with concomitant increases in total neutral lipid content. Similar phenotypes were observed in plant seeds, which showed enlarged LDs and increases in total amounts of seed oil. Collectively, these data identify LDIP as a new player in LD biology that modulates both LD size and cellular neutral lipid homeostasis in both leaves and seeds.

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. Previous work suggested that the principal activation domain of GCN4 is a highly acidic segment of approximately 40 amino acids located in the center of the protein. We conducted a mutational analysis of GCN4 with a single-copy allele expressed under the control of the native promoter and translational control elements. Our results indicate that GCN4 contains two activation domains of similar potency that can function independently to promote high-level transcription of the target genes HIS3 and HIS4. One of these domains is coincident with the acidic activation domain defined previously; the other extends over the N-terminal one-third of the protein. Both domains are partially dependent on the coactivator protein ADA2. Each domain appears to be composed of two or more small subdomains that have additive effects on transcription and that can cooperate in different combinations to promote high-level expression of HIS3 and HIS4. At least three of these subdomains are critically dependent on bulky hydrophobic amino acids for their function. Five of the important hydrophobic residues, Phe-97, Phe-98, Met-107, Tyr-110, and Leu-113, fall within a region of proposed sequence homology between GCN4 and the herpesvirus acidic activator VP16. The remaining three residues, Trp-120, Leu-123, and Phe-124, are highly conserved between GCN4 and its Neurospora counterpart, cpc-1. Because of the functional redundancy in the activation domain, mutations at positions 97 and 98 must be combined with mutations at positions 120 to 124 to observe a substantial reduction in activation by full-length GCN4, and substitution of all eight hydrophobic residues was required to inactivate full-length GCN4. These hydrophobic residues may mediate important interactions between GCN4 and one or more of its target proteins in the transcription initiation complex.

Detailed evaluation of gene functions in an asexual fungus requires advanced methods of molecular biology. For the generation of targeted gene deletions in the opportunistic pathogen Aspergillus fumigatus we designed a novel blaster module allowing dominant selection of transformants due to resistance to phleomycin as well as dominant (counter)selection of a Cre recombinase-mediated marker excision event. For validation purposes we have deleted the A. fumigatus pabaA gene in a wild-type isolate by making use of this cassette. The resulting pabaA::loxP strain served as the recipient for subsequent targeting of the velvet locus. Homologous reconstitution of the deleted gene was performed by an allele whose expression is driven in a nitrogen source-dependent manner, as validated by Northern analyses. Overexpression of the veA locus in A. fumigatus does not result in any obvious phenotype, whereas the sporulation capacities of the veA null mutant are reduced on nitrate-containing medium, a phenotype that is completely restored in the reconstituted strain.

The velvet factor of the homothallic fungus Aspergillus nidulans promotes sexual fruiting body formation. The encoding veA gene is conserved among fungi, including the ascomycete Neurospora crassa. There, the orthologous ve-1 gene encodes a deduced protein with high similarity to A. nidulans VeA. Cross-complementation experiments suggest that both the promoter and the coding sequence of N. crassa ve-1 are functional to complement the phenotype of an A. nidulans deletion mutant. Moreover, ve-1 expression in the heterologous host A. nidulans results in development of reproductive structures in a light-dependent manner, promoting sexual development in the darkness while stimulating asexual sporulation under illumination. Deletion of the N. crassa ve-1 locus by homologous gene replacement causes formation of shortened aerial hyphae accompanied by a significant increase in asexual conidiation, which is not light-dependent. Our data suggest that the conserved velvet proteins of A. nidulans and N. crassa exhibit both similar and different functions to influence development of these two ascomycetes.

Summary Six transcription regulatory genes of the V erticillium plant pathogen, which reprogrammed nonadherent budding yeasts for adhesion, were isolated by a genetic screen to identify control elements for early plant infection. V erticillium transcription activator of adhesion V ta2 is highly conserved in filamentous fungi but not present in yeasts. The M agnaporthe grisea ortholog conidiation regulator C on7 controls the formation of appressoria which are absent in V erticillium species. V ta2 was analyzed by using genetics, cell biology, transcriptomics, secretome proteomics and plant pathogenicity assays. Nuclear V ta2 activates the expression of the adhesin‐encoding yeast flocculin genes FLO 1 and FLO 11 . V ta2 is required for fungal growth of V erticillium where it is a positive regulator of conidiation. V ta2 is mandatory for accurate timing and suppression of microsclerotia as resting structures. V ta2 controls expression of 270 transcripts, including 10 putative genes for adhesins and 57 for secreted proteins. V ta2 controls the level of 125 secreted proteins, including putative adhesins or effector molecules and a secreted catalase‐peroxidase. V ta2 is a major regulator of fungal pathogenesis, and controls host‐plant root infection and H 2 O 2 detoxification. V erticillium impaired in V ta2 is unable to colonize plants and induce disease symptoms. V ta2 represents an interesting target for controlling the growth and development of these vascular pathogens.

The Asc1 protein of <i>Saccharomyces cerevisiae</i> is a scaffold protein at the head region of ribosomal 40S that links mRNA translation to cellular signaling. In this study, proteins that colocalize with Asc1p were identified with proximity-dependent <i>Bio</i>tin <i>ID</i>entification (BioID), an <i>in vivo</i> labeling technique described here for the first time for yeast. Biotinylated Asc1p-birA*-proximal proteins were identified and quantitatively verified against controls applying SILAC and mass spectrometry. The mRNA-binding proteins Sro9p and Gis2p appeared together with Scp160p, each providing ribosomes with nuclear transcripts. The cap-binding protein eIF4E (Cdc33p) and the eIF3/a-subunit (Rpg1p) were identified reflecting the encounter of proteins involved in the initiation of mRNA translation at the head region of ribosomal 40S. Unexpectedly, a protein involved in ribosome preservation (the clamping factor Stm1p), the deubiquitylation complex Ubp3p-Bre5p, the RNA polymerase II degradation factor 1 (Def1p), and transcription factors (Spt5p, Mbf1p) colocalize with Asc1p in exponentially growing cells. For Asc1^{R38D, K40E}p, a variant considered to be deficient in binding to ribosomes, BioID revealed its predominant ribosome localization. Glucose depletion replaced most of the Asc1p colocalizing proteins for additional ribosomal proteins, suggesting a ribosome aggregation process during early nutrient limitation, possibly concomitant with ribosomal subunit clamping. Overall, the characterization of the Asc1p microenvironment with BioID confirmed and substantiated our recent findings that the β-propeller broadly contributes to signal transduction influencing phosphorylation of colocalizing proteins (<i>e.g.</i> of Bre5p), and by that might affect nuclear gene transcription and the fate of ribosomes.

Epigenetic and transcriptional control of gene expression must be coordinated in response to external signals to promote alternative multicellular developmental programs. The membrane-associated trimeric complex VapA-VipC-VapB controls a signal transduction pathway for fungal differentiation. The VipC-VapB methyltransferases are tethered to the membrane by the FYVE-like zinc finger protein VapA, allowing the nuclear VelB-VeA-LaeA complex to activate transcription for sexual development. Once the release from VapA is triggered, VipC-VapB is transported into the nucleus. VipC-VapB physically interacts with VeA and reduces its nuclear import and protein stability, thereby reducing the nuclear VelB-VeA-LaeA complex. Nuclear VapB methyltransferase diminishes the establishment of facultative heterochromatin by decreasing histone 3 lysine 9 trimethylation (H3K9me3). This favors activation of the regulatory genes brlA and abaA, which promote the asexual program. The VapA-VipC-VapB methyltransferase pathway combines control of nuclear import and stability of transcription factors with histone modification to foster appropriate differentiation responses.

Verticillium wilt causes severe yield losses in a broad range of economically important crops worldwide. As many soil fumigants have a severe environmental impact, new biocontrol strategies are needed. Members of the genus Bacillus are known as plant growth-promoting bacteria (PGPB) as well as biocontrol agents of pests and diseases. In this study, we isolated 267 Bacillus strains from root-associated soil of field-grown tomato plants. We evaluated the antifungal potential of 20 phenotypically diverse strains according to their antagonistic activity against the two phytopathogenic fungi Verticillium dahliae and Verticillium longisporum. In addition, the 20 strains were sequenced and phylogenetically characterized by multi-locus sequence typing (MLST) resulting in 7 different Bacillus thuringiensis and 13 Bacillus weihenstephanensis strains. All B. thuringiensis isolates inhibited in vitro the tomato pathogen V. dahliae JR2, but had only low efficacy against the tomato-foreign pathogen V. longisporum 43. All B. weihenstephanensis isolates exhibited no fungicidal activity whereas three B. weihenstephanensis isolates showed antagonistic effects on both phytopathogens. These strains had a rhizoid colony morphology, which has not been described for B. weihenstephanensis strains previously. Genome analysis of all isolates revealed putative genes encoding fungicidal substances and resulted in identification of 304 secondary metabolite gene clusters including 101 non-ribosomal polypeptide synthetases and 203 ribosomal-synthesized and post-translationally modified peptides. All genomes encoded genes for the synthesis of the antifungal siderophore bacillibactin. In the genome of one B. thuringiensis strain, a gene cluster for zwittermicin A was detected. Isolates which either exhibited an inhibitory or an interfering effect on the growth of the phytopathogens carried one or two genes encoding putative mycolitic chitinases, which might contribute to antifungal activities. This indicates that chitinases contribute to antifungal activities. The present study identified B. thuringiensis isolates from tomato roots which exhibited in vitro antifungal activity against Verticillium species.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

The CPCA protein of the filamentous fungus Aspergillus nidulans is a member of the c-Jun-like transcriptional activator family. It acts as central transcription factor of the cross-pathway regulatory network of amino acid biosynthesis and is functionally exchangeable for the general control transcriptional activator Gcn4p of Saccharomyces cerevisiae. In contrast to GCN4, expression of cpcA is strongly regulated by two equally important mechanisms with additive effects that lead to a fivefold increased CPCA protein amount under amino acid starvation conditions. One component of cpcA regulation involves a transcriptional autoregulatory mechanism via a CPCA recognition element (CPRE) in the cpcA promoter that causes a sevenfold increased cpcA mRNA level when cells are starved for amino acids. Point mutations in the CPRE cause a constitutively low mRNA level of cpcA and a halved protein level when amino acids are limited. Moreover, two upstream open reading frames (uORFs) in the 5' region of the cpcA mRNA are important for a translational regulatory mechanism. Destruction of both short uORFs results in a sixfold increased CPCA protein level under nonstarvation conditions and a 10-fold increase under starvation conditions. Mutations in both the CPRE and uORF regulatory elements lead to an intermediate effect, with a low cpcA mRNA level but a threefold increased CPCA protein level independent of amino acid availability. These data argue for a combined regulation of cpcA that includes a translational regulation like that of yeast GCN4 as well as a transcriptional regulation like that of the mammalian jun and fos genes.

The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.

Lobosphaera incisa (L. incisa) is an oleaginous microalga that stores triacylglycerol (TAG) rich in arachidonic acid in lipid bodies (LBs). This organelle is gaining attention in algal research, since evidence is accumulating that proteins attached to its surface fulfill important functions in TAG storage and metabolism. Here, the composition of the LB proteome in L incisa was investigated by comparing different cell fractions in a semiquantitative proteomics approach. After applying stringent filters to the proteomics data in order to remove contaminating proteins from the list of possible LB proteins (LBPs), heterologous expression of candidate proteins in tobacco pollen tubes, allowed us to confirm 3 true LBPs: A member of the algal Major Lipid Droplet Protein family, a small protein of unknown function and a putative lipase. In addition, a TAG lipase that belongs to the SUGAR DEPENDENT 1 family of TAG lipases known from oilseed plants was identified. Its activity was verified by functional complementation of an Arabidopsis thaliana mutant lacking the major seed TAG lipases. Here we describe 3 LBPs as well as a TAG lipase from the oleaginous microalga L. incisa and discuss their possible involvement in LB metabolism. This study highlights the importance of filtering LB proteome datasets and verifying the subcellular localization one by one, so that contaminating proteins can be recognized as such. Our dataset can serve as a valuable resource in the identification of additional LBPs, shedding more light on the intriguing roles of LBs in microalgae.

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.

Trehalose on both sides of the bilayer is a requirement for full protection of membranes against stress. It was not known yet how trehalose, synthesized in the cytosol when dividing Saccharomyces cerevisiae cells are shifted from 28°C to 40°C, is transported to the outside and degraded when cells return to 28°C. According to our results, the lack of Agt1, a trehalose transporter, although had not affected trehalose synthesis, reduced cell tolerance to 51°C and increased lipid peroxidation. The damage was reversed when external trehalose was added during 40°C adaptation, confirming that the reason for the agt1Δ sensitivity is the absence of trehalose at the outside of the lipid bilayer. The 40-28°C condition caused cytosolic trehalase (Nth1) activation, reducing intracellular trehalose and, consequently, the survival rates after 51°C. Although lower than nth1Δ strain, cells deficient in acid trehalase (ath1Δ) maintained increased trehalose levels after 40°C-28°C shift, which conferred protection against 51°C. Both Ath1 and Agt1 were found into vesicles near to plasma membrane in response to stress. This suggests that Agt1 containing vesicles would fuse with the membrane under 40°C to transport part of the cytosolic trehalose to the outside. By a similar mechanism, Ath1 would reach the cell surface to hydrolyze the external trehalose but only when the stress would be over. Corroborating this conclusion, Ath1 activity in soluble cell-free extracts increased after 40°C adaptation but decreased when cells returned to 28°C. During 40°C, Ath1 is confined into vesicles, avoiding the cleavage of the outside trehalose.

The yeast PROPPIN Atg18 folds as a β-propeller with two binding sites for phosphatidylinositol-3-phosphate (PtdIns3P) and PtdIns(3,5)P2 at its circumference. Membrane insertion of an amphipathic loop of Atg18 leads to membrane tubulation and fission. Atg18 has known functions at the PAS during macroautophagy, but the functional relevance of its endosomal and vacuolar pool is not well understood. Here we show in a proximity-dependent labeling approach and by co-immunoprecipitations that Atg18 interacts with Vps35, a central component of the retromer complex. The binding of Atg18 to Vps35 is competitive with the sorting nexin dimer Vps5 and Vps17. This suggests that Atg18 within the retromer can substitute for both the phosphoinositide binding and the membrane bending capabilities of these sorting nexins. Indeed, we found that Atg18-retromer is required for PtdIns(3,5)P2-dependent vacuolar fragmentation during hyperosmotic stress. The Atg18-retromer is further involved in the normal sorting of the integral membrane protein Atg9. However, PtdIns3P-dependent macroautophagy and the selective cytoplasm-to-vacuole targeting (Cvt) pathway are only partially affected by the Atg18-retromer. We expect that this is due to the plasticity of the different sorting pathways within the endovacuolar system.Abbreviations: BAR: bin/amphiphysin/Rvs; FOA: 5-fluoroorotic acid; PAS: phagophore assembly site; PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol-3-phosphate; PX: phox homology.

Xylem sap is the major transport route for nutrients from roots to shoots. In the present study, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap and the growth of the xylem endophyte Brennaria salicis, and we also report transcriptional re-wiring of leaf defenses in poplar (Populus × canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. Approximately 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, whereas nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased, whereas salicylic acid and jasmonoyl-isoleucine decreased, with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 124 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N, except coumarins. Brennaria salicis growth was reduced in nutrient-supplemented xylem sap of low- and high- NO3- -fed plants compared to that of NH4+ -fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defenses related to systemic acquired and induced systemic resistance. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses.

This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae. These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene. The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes. We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency. The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes. Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT. Both sequence elements have previously been proposed to be involved in 3' end formation. Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation. The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites. Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.

Amino acid limitation results in impaired sexual fruit body formation in filamentous fungi such as Aspergillus nidulans. The starvation signal is perceived by the cross-pathway regulatory network controlling the biosynthesis of translational precursors and results in increased expression of a transcriptional activator encoded by a c-Jun homologue. In the presence of amino acids, the gene product of the mammalian RACK1 homologue cpcB is required to repress the network. Growth under amino acid starvation conditions permits the initiation of the sexual developmental programme of the fungus, but blocks fruit body formation before completion of meiosis. Accordingly, arrest at this defined control point results in microcleistothecia filled with hyphae. Addition of amino acids results in release of the block and completion of development to mature ascospores. The same developmental block is induced by either overexpression of c-Jun homologues or deletion of the RACK1 homologue cpcB of A. nidulans in the presence of amino acids. Therefore, the amino acid starvation signal regulates sexual development through the network that also controls the amino acid biosynthetic genes. Expression of the RACK1 gene suppresses the block in development caused by a deletion of cpcB. These data illuminate a connection between metabolism and sexual development in filamentous fungi.

We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA-proximal positions in mammalian cells, e.g. the acidic-type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline-rich domains, as e.g. in AP-2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine-rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters.

Two regulatory proteins, PHO2 and the general control regulator GCN4, bind in vitro to the promoter of the tryptophan biosynthetic TRP4 gene; the TRP4 gene product catalyses the phosphoribosylation of anthranilate. PHO2 binds specifically to the TRP4 promoter, but does not bind to any other TRP promoter. PHO2 and GCN4 proteins bind in a mutually exclusive manner to the same sequence, UAS1, one of two GCN4 binding sites in the TRP4 promoter. UAS1 is the major site for GCN4-dependent TRP4 activation. The second GCN4 binding site, UAS2, interacts with GCN4 alone. PHO2 binding interferes with the general control response of TRP4 under low phosphate conditions and simultaneous amino acid starvation and thus the PHO2 regulatory protein connects phosphate metabolism and amino acid biosynthesis in yeast. The GCN4 protein mediates the response of the transcriptional apparatus to the environmental signal 'amino acid limitation', while PHO2 seems to be the phosphate sensor that adjusts the response to the availability of phosphate precursors.

The role of the complex network of the ubiquitin-like modifier SumO in fungal development was analysed. SumO is not only required for sexual development but also for accurate induction and light stimulation of asexual development. The Aspergillus nidulans COMPASS complex including its subunits CclA and the methyltransferase SetA connects the SumO network to histone modification. SetA is required for correct positioning of aerial hyphae for conidiophore and asexual spore formation. Multicellular fungal development requires sumoylation and desumoylation. This includes the SumO processing enzyme UlpB, the E1 SumO activating enzyme AosA/UbaB, the E2 conjugation enzyme UbcN and UlpA as major SumO isopeptidase. Genetic suppression analysis suggests a connection between the genes for the Nedd8 isopeptidase DenA and the SumO isopeptidase UlpA and therefore a developmental interplay between neddylation and sumoylation in fungi. Biochemical evidence suggests an additional connection of the fungal SumO network with ubiquitination. Members of the cellular SumO network include histone modifiers, components of the transcription, RNA maturation and stress response machinery, or metabolic enzymes. Our data suggest that the SumO network controls specific temporal and spatial steps in fungal differentiation.

The plant-pathogenic fungus Verticillium longisporum is a causal agent of early senescence and ripening in cruciferous crops like Brassica napus. Verticillium wilts have become serious agricultural threats in recent decades. Verticillium species infect host plants through the roots and colonize xylem vessels of the host plant. The xylem fluid provides an environment with limited carbon sources and unbalanced amino acid supply, which requires V. longisporum to induce the cross-pathway control of amino acid biosynthesis. RNA-mediated gene silencing reduced the expression of the two CPC1 isogenes (VlCPC1-1 and VlCPC1-2) of the allodiploid V. longisporum up to 85%. VlCPC1 encodes the conserved transcription factor of the cross-pathway control. The silenced mutants were highly sensitive to amino-acid starvation, and the infected plants showed significantly fewer symptoms such as stunting or early senescence in oilseed rape plant infection assays. Consistently, deletion of single CPC1 of the haploid V. dahliae resulted in strains that are sensitive to amino-acid starvation and cause strongly reduced symptoms in the plant-host tomato (Solanum lycopersicum). The allodiploid V. longisporum and the haploid V. dahliae are the first phytopathogenic fungi that were shown to require CPC1 for infection and colonization of their respective host plants, oilseed rape and tomato.

The vascular plant pathogen Verticillium dahliae colonizes the xylem fluid where only low nutrient concentrations are provided. Biosynthesis of the vitamin thiamine is connected to oxidative stress. The highly conserved VdThi4 protein is localized in fungal mitochondria and is required under vitamin B1 limiting conditions. Deletion of the corresponding VdTHI4 gene by Agrobacterium-mediated transformation resulted in strains which were impaired in growth on thiamine-free medium and could be rescued by additional vitamin supply or by complementation with the original gene after protoplastation. Furthermore, we show that VdThi4 increases fungal stress tolerance such as UV-damage or oxidative stress. The orthologous sti35 gene of Fusarium oxysporum, another vascular wilt fungus, was shown to be involved in stress response, however to be dispensable for pathogenicity on tomato. In contrast, VdTHI4 is required for fungal-induced tomato disease demonstrated by infection assays with a V. dahliae ΔVdTHI4 deletion strain which is still able to invade plants through the roots but is asymptomatic. Our results suggest remarkable differences between two vascular tomato pathogens where VdThi4 is required for pathogenicity of V. dahliae, whereas F. oxysporum still causes disease when the corresponding Sti35 protein is absent.

Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in autoimmune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope-tagged MOS6 from pathogen-challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR-NBS-type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co-immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN-α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N-terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.

Summary Verticillium dahliae nuclear transcription factors Som1 and Vta3 can rescue adhesion in a FLO 8‐ deficient Saccharomyces cerevisiae strain. Som1 and Vta3 induce the expression of the yeast FLO 1 and FLO 11 genes encoding adhesins. Som1 and Vta3 are sequentially required for root penetration and colonisation of the plant host by V. dahliae . The SOM 1 and VTA 3 genes were deleted and their functions in fungus‐induced plant pathogenesis were studied using genetic, cell biology, proteomic and plant pathogenicity experiments. Som1 supports fungal adhesion and root penetration and is required earlier than Vta3 in the colonisation of plant root surfaces and tomato plant infection. Som1 controls septa positioning and the size of vacuoles, and subsequently hyphal development including aerial hyphae formation and normal hyphal branching. Som1 and Vta3 control conidiation, microsclerotia formation, and antagonise in oxidative stress responses. The molecular function of Som1 is conserved between the plant pathogen V. dahliae and the opportunistic human pathogen Aspergillus fumigatus . Som1 controls genes for initial steps of plant root penetration, adhesion, oxidative stress response and VTA 3 expression to allow subsequent root colonisation. Both Som1 and Vta3 regulate developmental genetic networks required for conidiation, microsclerotia formation and pathogenicity of V. dahliae .

The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. SclB function is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.

Monoterpenoids, such as the plant metabolite geraniol, are of high industrial relevance since they are important fragrance materials for perfumes, cosmetics, and household products. Chemical synthesis or extraction from plant material for industry purposes are complex, environmentally harmful or expensive and depend on seasonal variations. Heterologous microbial production offers a cost-efficient and sustainable alternative but suffers from low metabolic flux of the precursors and toxicity of the monoterpenoid to the cells. In this study, we evaluated two approaches to counteract both issues by compartmentalizing the biosynthetic enzymes for geraniol to the peroxisomes of Saccharomyces cerevisiae as production sites and by improving the geraniol tolerance of the yeast cells. The combination of both approaches led to an 80% increase in the geraniol titers. In the future, the inclusion of product tolerance and peroxisomal compartmentalization into the general chassis engineering toolbox for monoterpenoids or other host-damaging, industrially relevant metabolites may lead to an efficient, low-cost, and eco-friendly microbial production for industrial purposes.

The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae . Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V . dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V . dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta .

Parkinson’s disease is a neurodegenerative disorder associated with misfolding and aggregation of α-synuclein as a hallmark protein. Two yeast strain collections comprising conditional alleles of essential genes were screened for the ability of each allele to reduce or improve yeast growth upon α-synuclein expression. The resulting 98 novel modulators of α-synuclein toxicity clustered in several major categories including transcription, rRNA processing and ribosome biogenesis, RNA metabolism and protein degradation. Furthermore, expression of α-synuclein caused alterations in pre-rRNA transcript levels in yeast and in human cells. We identified the nucleolar DEAD-box helicase Dbp4 as a prominent modulator of α-synuclein toxicity. Downregulation of DBP4 rescued cells from α-synuclein toxicity, whereas overexpression led to a synthetic lethal phenotype. We discovered that α-synuclein interacts with Dbp4 or its human ortholog DDX10, sequesters the protein outside the nucleolus in yeast and in human cells, and stabilizes a fraction of α-synuclein oligomeric species. These findings provide a novel link between nucleolar processes and α-synuclein mediated toxicity with DDX10 emerging as a promising drug target.

Membrane contact sites (MCSs) are interorganellar connections that allow for the direct exchange of molecules, such as lipids or Ca2+ between organelles, but can also serve to tether organelles at specific locations within cells. Here, we identified and characterized three proteins of Arabidopsis thaliana that form a lipid droplet (LD)-plasma membrane (PM) tethering complex in plant cells, namely LD-localized SEED LD PROTEIN (SLDP) 1 and SLDP2 and PM-localized LD-PLASMA MEMBRANE ADAPTOR (LIPA). Using proteomics and different protein-protein interaction assays, we show that both SLDPs associate with LIPA. Disruption of either SLDP1 and SLDP2 expression, or that of LIPA, leads to an aberrant clustering of LDs in Arabidopsis seedlings. Ectopic co-expression of one of the SLDPs with LIPA is sufficient to reconstitute LD-PM tethering in Nicotiana tabacum pollen tubes, a cell type characterized by dynamically moving LDs in the cytosolic streaming. Furthermore, confocal laser scanning microscopy revealed both SLDP2.1 and LIPA to be enriched at LD-PM contact sites in seedlings. These and other results suggest that SLDP and LIPA interact to form a tethering complex that anchors a subset of LDs to the PM during post-germinative seedling growth in Arabidopsis.

Ubiquitin-mediated proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit. Like ubiquitylation, protein modification with the small ubiquitin-related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO-conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation. Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C-mediated proteolysis in Saccharomyces cerevisiae. We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1. This metaphase block was partially relieved by a deletion of PDS1. The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation. Temperature-sensitive ubc9-2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis. The requirement of SUMOylation for APC/C-mediated degradation was tested more directly in G1-arrested cells. Both ubc9-2 and smt3-331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected. We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.

The general control transcriptional regulator gene cpcA of Aspergillus niger was cloned by complementation of a Saccharomyces cerevisiae delta gcn4 mutant strain. The encoded protein conferred resistance to amino acid analogues when expressed in yeast. Disruption of cpcA in A. niger resulted in a strain which is sensitive towards 3-aminotriazole and fails to respond to amino acid starvation, cpcA encodes a transcript of approximately 2400 nucleotides in length that includes a 5' leader region of 900 nucleotides. The 5' leader region contains two small open reading frames, suggesting translational control of gene expression. Steady-state mRNA levels of cpcA increase by a factor of three upon amino acid starvation. The coding region of cpcA is interrupted by a 57 bp intron and the deduced amino acid sequence displays an approximately 30% overall identity to yeast GCN4p and Neurospora crassa cpc1p. Critical amino acid residues of the transcriptional activation domains of GCN4p are conserved in cpcAp. The basic DNA-binding domain shows up to 70% amino acid sequence identity to other basic zipper (bZIP)-type transcriptional activators. cpcAp binds specifically to a GCN4p recognition element in gel retardation experiments. The C-terminal dimerization domain encodes a leucine zipper with only a single leucine residue.

In Saccharomyces cerevisiae, the transcription factors Tec1p and Ste12p are required for haploid invasive and diploid pseudohyphal growth. Tec1p and Ste12p have been postulated to regulate these developmental processes primarily by cooperative binding to filamentous and invasion-responsive elements (FREs), which are combined enhancer elements that consist of a Tec1p-binding site (TCS) and an Stel2p-binding site (PRE). They are present in the promoter regions of target genes, e.g., FLO11. Here, we show that Tec1p efficiently activates target gene expression and cellular development in the absence of Stel2p. We further demonstrate that TCS elements alone are sufficient to mediate Tec1p-driven gene expression by a mechanism termed TCS control that is operative even when Stel2p is absent. Mutational analysis of TEC1 revealed that TCS control, FLO11 expression, and haploid invasive growth require the C terminus of Tec1p. In contrast, the Ste12p-dependent FRE control mechanism is sufficiently executed by the N-terminal portion of Tec1p, which contains the TEA/ATTS DNA-binding domain. Our study suggests that regulation of haploid invasive and diploid pseudohyphal growth by Stel2p and Tec1p is not only executed by combinatorial control but involves additional control mechanisms in which Stel2p activates TEC1 expression via clustered PREs and where Tec1p regulates expression of target genes, e.g., FLO11, by TCS control.

The Saccharomyces cerevisiae ARO7 gene product chorismate mutase, a single-branch-point enzyme in the aromatic amino acid biosynthetic pathway, is activated by tryptophan and subject to feedback inhibition by tyrosine. The ARO7 gene was cloned on a 2.05-kilobase EcoRI fragment. Northern (RNA) analysis revealed a 0.95-kilobase poly(A)+ RNA, and DNA sequencing determined a 771-base-pair open reading frame capable of encoding a protein 256 amino acids. In addition, three mutant alleles of ARO7 were cloned and sequenced. These encoded chorismate mutases which were unresponsive to tyrosine and tryptophan and were locked in the on state, exhibiting a 10-fold-increased basal enzyme activity. A single base pair exchange resulting in a threonine-to-isoleucine amino acid substitution in the C-terminal part of the chorismate mutase was found in all mutant strains. In contrast to other enzymes in this pathway, no significant homology between the monofunctional yeast chorismate mutase and the corresponding domains of the two bifunctional Escherichia coli enzymes was found.

Abstract The yeast transcriptional regulator protein GCN4 harbors the bZIP DNA binding motif, which is common to a family of DNA-binding proteins in eukaryotic organisms from yeast to man. GCN4 and the mammalian activator protein AP-1 (jun/fos) regulate transcription by binding the same consensus DNA sequence ATGA (C/G)TCAT. GCN4 positively regulates the production of precursors of protein synthesis in yeast cells in response to the environmental signal amino acid starvation. We find three GCN4 responsive elements (GCREs) in the 5'-flanking region of the purine biosynthetic gene ADE4 and demonstrate that GCN4 efficiently activates transcription of ADE4. Two GCREs are essential to synergistically activate ADE4 transcription by binding GCN4. The distal GCRE1 is also required for basal transcription of ADE4. Therefore, transcription factor GCN4 affects, in addition to protein biosynthesis, also nucleotide biosynthesis and, comparable to its mammalian counterpart AP-1, has a more general function within the yeast cell than previously assumed.

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.

The devastating soilborne fungal pathogen Verticillium longisporum is host specific to members of the family Brassicaceae, including oilseed rape (Brassica napus) as the economically most important crop. The fungus infects through the roots and causes stunting and early senescence of susceptible host plants and a marked decrease in crop yield. We show here that V. longisporum reacts to the presence of B. napus xylem sap with the production of six distinct upregulated and eight downregulated proteins visualized by two-dimensional gel electrophoresis. Identification of 10 proteins by mass spectrometry revealed that all upregulated proteins are involved in oxidative stress response. The V. longisporum catalase peroxidase (VlCPEA) was the most upregulated protein and is encoded by two isogenes, VlcpeA-1 and VlcpeA-2. Both genes are 98% identical, corroborating the diploid or “amphihaploid” status of the fungus. Knock downs of both VlcpeA genes reduced protein expression by 80% and resulted in sensitivity against reactive oxygen species. Whereas saprophytic growth and the initial phase of the plant infection were phenotypically unaffected, the mutants were not able to perform the late phases of disease. We propose that the catalase peroxidase plays a role in protecting the fungus from the oxidative stress generated by the host plant at an advanced phase of the disease.

Verticillium longisporum and Verticillium dahliae are soil borne vascular pathogens. Under standard laboratory growth conditions both fungi infect Arabidopsis thaliana ecotype Col-0 plants via primary and lateral (secondary) root tips and colonize the vascular cylinder including proto- and metaxylem. After entry into the hypocotyl both Verticillium species spread into petioles and leaves, but with different efficiency and distinct disease symptom patterns. V. longisporum typically induces early senescence whereas V. dahliae infections cause wilting. In both cases, these symptoms coincide with the switch from a biotrophic to a necrotrophic life style. Fungal proliferation analyses monitored by quantitative real-time PCR and anatomical studies show that V. dahliae is able to colonize aerial plant parts more efficiently compared to V. longisporum. Moreover, V. longisporum-infection triggers hyperplastic xylem formation and cellular transdifferentiation in Arabidopsis, whereas V. dahliae-infection induces enhanced xylem lignification. We have recently shown that V. longisporum induced de novo xylem formation results in enhanced drought stress tolerance, suggesting a conditionally mutualistic interaction. Here we show that on the contrary V. dahliae infections do not provide protection of the plant against drought. Together, our results demonstrate that V. dahliae and V. longisporum induce distinct and species-specific developmental and structural alterations in Arabidopsis, which differently affect plant fitness under concomitant abiotic drought stress conditions.

Summary Aspergillus fumigatus , a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA , vosA and velB , the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the Δ mybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA‐sequencing and biochemical studies of the Δ mybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane‐associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus .

Most of the metal transporters in Aspergillus fumigatus are yet uncharacterized. Their role in fungal metabolism and virulence remains unclear. This paper describes the novel PIB-type cation ATPase PcaA, which links metal homeostasis and heavy metal tolerance in the opportunistic human pathogen A. fumigatus. The protein possesses conserved ATPase motif and shares 51% amino acid sequence identity with the Saccharomyces cerevisiae cadmium exporter Pca1p. A pcaA deletion, an overexpression and a gfp-pcaA complementation strain of A. fumigatus were constructed and their heavy metal susceptibilities were studied. The pcaA knock out strain showed drastically decreased cadmium tolerance, however, its growth was not affected by the exposure to high concentrations of copper, iron, zinc or silver ions. Although the lack of PcaA had no effect on copper adaption, we demonstrated that not only cadmium but also copper is able to induce the transcription of pcaA in A. fumigatus wild type Af293. Similarly, cadmium and copper could induce the copper exporting ATPase crpA. These data imply a general response on the transcriptomic level to heavy metals in A. fumigatus through the induction of detoxification systems. Confocal microscopy of the gfp-pcaA complementation strain expressing functional GFP-PcaA supports the predicted membrane localization of PcaA. The GFP-PcaA fusion protein is located in the plasma membrane of A. fumigatus in the presence of cadmium. Virulence assays support a function of PcaA for virulence of A. fumigatus in the Galleria mellonella wax moth larvae model, which might be linked to the elimination of reactive oxygen species.

Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unkown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase of Neurospora crassa affect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 minute) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The differential phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation.

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteomes of the allodiploid Verticillium longisporum upon cultivation in different media or xylem sap extracted from its host plant Brassica napus were compared. Secreted fungal proteins were identified by label free liquid chromatography-tandem mass spectrometry screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 221 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 143 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known pathogenicity factors, metallopeptidases and carbohydrate-active enzymes. The most abundant proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the carbohydrate-active enzymes Gla1, Amy1 and Cbd1. Their pathogenicity contribution was analyzed in the haploid parental strain V. dahliae. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the MEP1, NLP2 and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization.

Lipid droplets (LDs) are neutral-lipid-containing organelles found in all kingdoms of life and are coated with proteins that carry out a vast array of functions. Compared to mammals and yeast, relatively few LD proteins have been identified in plants, particularly those associated with LDs in vegetative (non-seed) cell types. Thus, to better understand the cellular roles of LDs in plants, a more comprehensive inventory and characterization of LD proteins is required. Here, we performed a proteomics analysis of LDs isolated from drought-stressed Arabidopsis leaves and identified EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) as a putative LD protein. mCherry-tagged ERD7 localized to both LDs and the cytosol when ectopically expressed in plant cells, and the protein’s C-terminal senescence domain (SD) was both necessary and sufficient for LD targeting. Phylogenetic analysis revealed that ERD7 belongs to a six-member family in Arabidopsis that, along with homologs in other plant species, is separated into two distinct subfamilies. Notably, the SDs of proteins from each subfamily conferred targeting to either LDs or mitochondria. Further, the SD from the ERD7 homolog in humans, spartin, localized to LDs in plant cells, similar to its localization in mammals; although, in mammalian cells, spartin also conditionally localizes to other subcellular compartments, including mitochondria. Disruption of ERD7 gene expression in Arabidopsis revealed no obvious changes in LD numbers or morphology under normal growth conditions, although this does not preclude a role for ERD7 in stress-induced LD dynamics. Consistent with this possibility, a yeast two-hybrid screen using ERD7 as bait identified numerous proteins involved in stress responses, including some that have been identified in other LD proteomes. Collectively, these observations provide new insight to ERD7 and the SD-containing family of proteins in plants and suggest that ERD7 may be involved in functional aspects of plant stress response that also include localization to the LD surface.

The crystal structure of an allosteric chorismate mutase, the Thr-226--&gt;Ile mutant, from yeast Saccharomyces cerevisiae has been determined to 2.2-A resolution by using the multiple isomorphous replacement method. Solvent-flattening and electron-density modification were applied for phase improvement. The current crystallographic R factor is 0.196. The final model includes 504 of the 512 residues and 97 water molecules. In addition, two tryptophan molecules were identified in the interface between monomers. The overall structure is completely different from the reported structure of chorismate mutase from Bacillus subtilis. This structure showed 71% helices with essentially no beta-sheet structures.

The cruciferous fungal pathogen Verticillium longisporum represents an allodiploid hybrid with long spores and almost double the amount of nuclear DNA compared to other Verticillium species. V. longisporum evolved at least three times by hybridization. In Europe, virulent A1xD1 and avirulent A1xD3 hybrids were isolated from the oilseed crop Brassica napus. Parental A1 or D1 species are yet unknown whereas the D3 lineage represents Verticillium dahliae. Eleven V. longisporum isolates from Europe or California corresponding to hybrids A1xD1 or A1xD3 were compared. A single characteristic type of nuclear ribosomal DNA could be assigned to each hybrid lineage. The two avirulent A1xD3 isolates carried exclusively D3 ribosomal DNA (rDNA) which corresponds to V. dahliae. The rDNA of all nine A1xD1 isolates is identical but distinct from D3 and presumably originates from A1. Both hybrid lineages carry two distinct isogene pairs of four conserved regulatory genes corresponding to either A1 or D1/D3. D1 and D3 paralogues differ in several single nucleotide polymorphisms. Southern hybridization patterns confirmed differences between the A1 and D1/D3 isogenes and resulted in similar patterns for D1 and D3. Distinct signatures of the Verticillium transcription activator (VTA)2 regulatory isogene pair allow identification of V. longisporum hybrids by a single polymerase chain reaction and the separation from haploid species as V. dahliae or Verticillium albo-atrum. The combination between VTA2 signature and rDNA type identification represents an attractive diagnostic tool to discriminate allodiploid from haploid Verticillia and to distinguish between A1xD1 and A1xD3 hybrids which differ in their virulence towards B. napus.

Fungal molecular biology has benefited from the enormous advances in understanding protein–protein interactions in prokaryotic or eukaryotic organisms of the past decade. Tandem affinity purification (TAP) allows the enrichment of native protein complexes from cell extracts under mild conditions. We codon-optimized tags and established TAP, previously not applicable to filamentous fungi, for the model organism Aspergillus nidulans. We could identify by this method the trimeric Velvet complex VelB/VeA/LaeA or the eight subunit COP9 signalosome. Here, we describe an optimized protocol for A. nidulans which can also be adapted to other filamentous fungi.

Aspergillus spp. comprises a very diverse group of lower eukaryotes with a high relevance for industrial applications and clinical implications. These multinucleate species are often cultured for many generations in the laboratory, which can unknowingly propagate hidden genetic mutations. To assess the likelihood of such events, we studied the genome stability of aspergilli by using a combination of mutation accumulation (MA) lines and whole genome sequencing.We sequenced the whole genomes of 30 asexual and 10 sexual MA lines of three Aspergillus species (A. flavus, A. fumigatus and A. nidulans) and estimated that each MA line accumulated mutations for over 4000 mitoses during asexual cycles. We estimated mutation rates of 4.2 × 10-11 (A. flavus), 1.1 × 10-11 (A. fumigatus) and 4.1 × 10-11 (A. nidulans) per site per mitosis, suggesting that the genomes are very robust. Unexpectedly, we found a very high rate of GC → TA transversions only in A. flavus. In parallel, 30 asexual lines of the non-homologous end-joining (NHEJ) mutants of the three species were also allowed to accumulate mutations for the same number of mitoses. Sequencing of these NHEJ MA lines gave an estimated mutation rate of 5.1 × 10-11 (A. flavus), 2.2 × 10-11 (A. fumigatus) and 4.5 × 10-11 (A. nidulans) per base per mitosis, which is slightly higher than in the wild-type strains and some ~ 5-6 times lower than in the yeasts. Additionally, in A. nidulans, we found a NHEJ-dependent interference of the sexual cycle that is independent of the accumulation of mutations.We present for the first time direct counts of the mutation rate of filamentous fungal species and find that Aspergillus genomes are very robust. Deletion of the NHEJ machinery results in a slight increase in the mutation rate, but at a rate we suggest is still safe to use for biotechnology purposes. Unexpectedly, we found GC→TA transversions predominated only in the species A. flavus, which could be generated by the hepatocarcinogen secondary metabolite aflatoxin. Lastly, a strong effect of the NHEJ mutation in self-crossing was observed and an increase in the mutations of the asexual lines was quantified.

Acetobacterium woodii, Acetohalobium arabaticum, Clostridium formicoaceticum, and Sporomusa silvacetica were found to contain carbonic anhydrase (CA). Minimal to no CA activity was detected in Moorella thermoautotrophica, Moorella thermoacetica subsp. "pratumsolum," Sporomusa termitida, and Thermoanaerobacter kivui. Of the acetogens tested, A. woodii had the highest CA specific activity, approximately 14 U mg of protein(-1), in extracts of either glucose- or H2-CO2-cultivated cells. CA of A. woodii was cytoplasmic and was purified approximately 300-fold to a specific activity of 5,236 U mg of protein(-1). Intracellular acetate concentrations inhibited CA activity of A. woodii by 50 to 85%, indicating that intracellular acetate may affect in situ CA activity.

DNA sequences of single-copy genes coding for chitin synthases (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase; EC 2.4.1.16) were used to characterize ectomycorrhizal fungi. Degenerate primers deduced from short, completely conserved amino acid stretches flanking a region of about 200 amino acids of zymogenic chitin synthases allowed the amplification of DNA fragments of several members of this gene family. Different DNA band patterns were obtained from basidiomycetes because of variation in the number and length of amplified fragments. Cloning and sequencing of the most prominent DNA fragments revealed that these differences were due to various introns at conserved positions. The presence of introns in basidiomycetous fungi therefore has a potential use in identification of genera by analyzing PCR-generated DNA fragment patterns. Analyses of the nucleotide sequences of cloned fragments revealed variations in nucleotide sequences from 4 to 45%. By comparison of the deduced amino acid sequences, the majority of the DNA fragments were identified as members of genes for chitin synthase class II. The deduced amino acid sequences from species of the same genus differed only in one amino acid residue, whereas identity between the amino acid sequences of ascomycetous and basidiomycetous fungi within the same taxonomic class was found to be approximately 43 to 66%. Phylogenetic analysis of the amino acid sequence of class II chitin synthase-encoding gene fragments by using parsimony confirmed the current taxonomic groupings. In addition, our data revealed a fourth class of putative zymogenic chitin synthesis.

The crystal structure of the tyrosine-bound T state of allosteric yeast Saccharomyces cerevisiae chorismate mutase was solved by molecular replacement at a resolution of 2.8 angstroms using a monomer of the R-state structure as the search model. The allosteric inhibitor tyrosine was found to bind in the T state at the same binding site as the allosteric activator tryptophan binds in the R state, thus defining one regulatory binding site for each monomer. Activation by tryptophan is caused by the larger steric size of its side chain, thereby pushing apart the allosteric domain of one monomer and helix H8 of the catalytic domain of the other monomer. Inhibition is caused by polar contacts of tyrosine with Arg-75 and Arg-76 of one monomer and with Gly-141, Ser-142, and Thr-145 of the other monomer, thereby bringing the allosteric and catalytic domains closer together. The allosteric transition includes an 8 degree rotation of each of the two catalytic domains relative to the allosteric domains of each monomer (domain closure). Alternatively, this transition can be described as a 15 degree rotation of the catalytic domains of the dimer relative to each other.

The soil microbiome comprises numerous filamentous fungi and bacteria that mutually react and challenge each other by the production of bioactive secondary metabolites. Herein, we show in liquid co-cultures that the presence of filamentous Streptomycetes producing antifungal glycopeptide antibiotics induces the production of the antibacterial and iron-chelating tropolones anhydrosepedonin (1) and antibiotic C (2) in the mold Aspergillus nidulans. Additionally, the biosynthesis of the related polyketide tripyrnidone (5) was induced, whose novel tricyclic scaffold we elucidated by NMR and HRESIMS data. The corresponding biosynthetic polyketide synthase-encoding gene cluster responsible for the production of these compounds was identified. The tropolones as well as tripyrnidone (5) are produced by genes that belong to the broad reservoir of the fungal genome for the synthesis of different secondary metabolites, which are usually silenced under standard laboratory conditions. These molecules might be part of the bacterium-fungus competition in the complex soil environment, with the bacterial glycopeptide antibiotic as specific environmental trigger for fungal induction of this cluster.

Abstract In budding yeast, the Ras/cAMP pathway is involved in the coordination of cell growth and cell division. Glucose-rich medium stimulates Ras/cAMP signaling, which causes an increase in the critical cell size for cell cycle entry. Here we show that glucose and activated Ras proteins also influence the function of the anaphase-promoting complex (APC/C), a ubiquitin-protein ligase required for sister chromatid separation and mitotic exit. We found that apc10-22 and other mutants defective in the APC/C are suppressed by reduced Ras signaling activity, by a deletion of the RAS2 gene, by a cdc25 mutation, by elevated levels of PDE2, or by growth without glucose. Viability of these mutants is also enhanced by decreased Cdk1 activity. In contrast, a constitutively activated RAS2Val19 allele or shifts to glucose medium are deleterious to apc10-22 mutants. Remarkably, cdc34-2 mutants, which are impaired in SCF function, are differently affected with respect to Ras activity. Viability of cdc34-2 mutants at elevated temperatures is dependent on glucose and the RAS2 gene. We conclude that glucose and Ras proteins influence the APC/C and the SCF complex in an opposite manner. These ubiquitin ligases might represent novel targets for modulating cell division in response to growth conditions.

The initial step of tryptophan biosynthesis is catalyzed by the enzyme anthranilate synthase, which in most microorganisms is subject to feedback inhibition by the end product of the pathway. We have characterized the TRP2 gene from a mutant Saccharomyces cerevisiae strain coding for an anthranilate synthase that is unresponsive to tryptophan. Sequence analysis of this TRP2(Fbr) (feedback-resistant) allele revealed numerous differences from a previously published TRP2 sequence. However, TRP2(Fbr) was found to differ in only one single-point mutation from its own parent wild type, a C-to-T transition resulting in a serine 76-to-leucine 76 amino acid substitution. Therefore, serine 76 is a crucial amino acid for proper regulation of the yeast enzyme. We constructed additional feedback-resistant enzyme forms of the yeast anthranilate synthase by site-directed mutagenesis of the conserved LLES sequence in the TRP2 gene. From analysis of these variants, we propose an extended sequence, LLESX10S, as the regulatory element in tryptophan-responsive anthranilate synthases from prokaryotic and eukaryotic organisms.

The Saccharomyces cerevisiae HIS7 gene was cloned by its location immediately downstream of the previously isolated and characterized ARO4 gene. The two genes have the same orientation with a distance of only 416 bp between the two open reading frames. The yeast HIS7 gene represents the first isolated eukaryotic gene encoding the enzymatic activities which catalyze the fifth and sixth step in histidine biosynthesis. The open reading frame of the HIS7 gene has a length of 1,656 bp resulting in a gene product of 552 amino acids with a calculated molecular weight of 61,082. Two findings implicate a bifunctional nature of the HIS7 gene product. First, the N-terminal and C-terminal segments of the deduced HIS7 amino acid sequence show significant homology to prokaryotic monofunctional glutamine amidotransferases and cyclases, respectively, involved in histidine biosynthesis. Second, the yeast HIS7 gene is able to suppress His auxotrophy of corresponding Escherichia coli hisH and hisF mutants. HIS7 gene expression is regulated by the general control system of amino acid biosynthesis. GCN4-dependent and GCN4-independent (basal) transcription use different initiator elements in the HIS7 promoter.

The HIS7 gene of Saccharomyces cerevisiae encodes a bifunctional glutamine amidotransferase:cyclase catalyzing two reactions that lead to the formation of biosynthetic intermediates of the amino acid histidine and the purine adenine. The HIS7 gene is activated by GCN4p under environmental conditions of amino acid starvation through two synergistic upstream sites GCRE1 and GCRE2. The BAS1p-BAS2p complex activates the HIS7 gene in response to adenine limitation. For this activation the proximal GCN4p-binding site GCRE2 is required. GCN4p and BAS1p bind to GCRE2 in vitro. Under conditions of simultaneous amino acid starvation and adenine limitation the effects of GCN4p and BAS1/2p are additive and both factors are necessary for maximal HIS7 transcription. These results suggest that GCN4p and BAS1/2p are able to act simultaneously through the same DNA sequence in vivo and use this site independently from each other in a non-exclusive manner. The HIS7 gene of Saccharomyces cerevisiae encodes a bifunctional glutamine amidotransferase:cyclase catalyzing two reactions that lead to the formation of biosynthetic intermediates of the amino acid histidine and the purine adenine. The HIS7 gene is activated by GCN4p under environmental conditions of amino acid starvation through two synergistic upstream sites GCRE1 and GCRE2. The BAS1p-BAS2p complex activates the HIS7 gene in response to adenine limitation. For this activation the proximal GCN4p-binding site GCRE2 is required. GCN4p and BAS1p bind to GCRE2 in vitro. Under conditions of simultaneous amino acid starvation and adenine limitation the effects of GCN4p and BAS1/2p are additive and both factors are necessary for maximal HIS7 transcription. These results suggest that GCN4p and BAS1/2p are able to act simultaneously through the same DNA sequence in vivo and use this site independently from each other in a non-exclusive manner.

Abstract Transcription of the gene for phosphoribosyl-anthranilate isomerase (TRP1) from the TRP1 promoter is initiated only approximately half as frequently as, for example, from the TRP3 promoter, but TRP1 mRNA is approximately twice as stable as TRP3 mRNA. Therefore, the steady state amount of TRP1 mRNA in yeast cells, grown without amino acid limitation, is similar to the steady-state amount of TRP3 mRNA. The protein concentration of both enzymes in yeast cells is about the same, but the basal specific enzyme activity in permeabilized cells of the TRP1 gene product N-(5'-phosphoribosyl-1)-anthranilate isomerase is about 2-3 times higher than that of any of the other TRP enzymes. According to the kinetic parameters of the purified isomerase protein, the enzyme is more active than, for example, the purified TRP3 enzyme indoleglycerol-phosphate synthase. It is suggested that the TRP1 gene of Saccharomyces cerevisiae might be the result of a rearrangement event, separating the N-(5'-phosphoribosyl-1)-anthranilate isomerase domain from the indoleglycerol-phosphate synthase domain and putting the catalytically more active isomerase domain behind a weak and nonregulated constitutive promoter.

Many fungi are able to produce resting structures, which ensure survival and protect them against various stresses in their habitat such as exposure to UV light, temperature variations, drought as well as changing pH and nutrient conditions. Verticillium dahliae is a plant pathogenic fungus that forms melanized resting structures, called microsclerotia, for survival of time periods without a host. These highly stress resistant microsclerotia persist in the soil for many years and are therefore problematic for an effective treatment of the fungus. The Verticillium transcription activator of adhesion 1 (Vta1) was initially identified as one of several transcriptional regulators that rescue adhesion in non-adhesive Saccharomyces cerevisiae cells. Vta2 and Vta3 are required for early steps in plant infection and colonization and additionally control microsclerotia formation. Here, we show that Vta1 function is different, because it is dispensable for root colonization and infection. Vta1 is produced in the fungal cell during microsclerotia development. Analysis of the deletion mutant revealed that the absence of Vta1 allows microsclerotia production, but they are colorless and no more melanized. Vta1 is required for melanin production and activates transcription of melanin biosynthesis genes including the polyketide synthase encoding PKS1 and the laccase LAC1. The primary function of Vta1 in melanin production is important for survival of microsclerotia as resting structures of V. dahliae.

ABSTRACT The c-Jun-like transcriptional activator Gcn4p controls biosynthesis of translational precursors in the yeast Saccharomyces cerevisiae . Protein stability is dependent on amino acid limitation and cis signals within Gcn4p which are recognized by cyclin-dependent protein kinases, including Pho85p. The Gcn4p population within unstarved yeast consists of a small relatively stable cytoplasmic fraction and a larger less stable nuclear fraction. Gcn4p contains two nuclear localization signals (NLS) which function independently of the presence or absence of amino acids. Expression of NLS-truncated Gcn4p results in an increased cytoplasmic fraction and an overall stabilization of the protein. The same effect is achieved for the entire Gcn4p in a yrb1 yeast mutant strain impaired in the nuclear import machinery. In the presence of amino acids, controlled destabilization of Gcn4p is triggered by the phosphorylation activity of Pho85p. A pho85Δ mutation stabilizes Gcn4p without affecting nuclear import. Pho85p is localized within the nucleus in the presence or absence of amino acids. Therefore, there is a strict spatial separation of protein synthesis and degradation of Gcn4p in yeast. Control of protein stabilization which antagonizes Gcn4p function is restricted to the nucleus.

Two genes (tetC and tetD) were identified and located on transposon Tn10 between gene tetA and insertion sequence IS10R. Genes tetC and tetD encode proteins of apparent subunit molecular weights of 23,000 and 18,000, respectively. The TetD protein was found to be membrane associated. Tetracycline resistance levels promoted by transposon Tn10 were found to be unaffected in Escherichia coli K-12 when mutants lacking tetC or tetC and tetD were tested. The nucleotide sequence of genes tetC and tetD is reported in the accompanying article (K. Schollmeier and W. Hillen, J. Bacteriol. 160:499-503, 1984).

Abstract The sexual stage of Aspergillus (Emericella) nidulans consists of cleistothecia containing asci, each with eight ascospores. The fungus completes the sexual cycle in a homokaryotic or a heterokaryotic mycelium, respectively. The common assumption for the last 50 years was that different nuclear types are not distinguishable when sexual development is initiated. When cultured on a medium limited for glucose supplemented with 2% sorbitol, sexual development of A. nidulans is slowed and intact tetrads can be isolated. Through tetrad analysis we found that unlike haploid nuclei fuse preferentially to the prezygotic diploid nucleus. When heterokaryons are formed between nuclei of different genetic backgrounds, then recombinant asci derived from opposite nuclei are formed exclusively. Strains in the same heterokaryon compatibility group with moderate differences in their genetic backgrounds can discriminate between the nuclei of a heterokaryon and preferentially form a hybrid diploid nucleus, resulting in 85% recombinant tetrads. A. nidulans strains that differ at only a single genetic marker fuse the haploid nuclei at random for formation of diploid nuclei during meiosis. These results argue for a genetically determined “relative heterothallism” of nuclear recognition within a heterokaryon and a specific recruitment of different nuclei for karyogamy when available.

The ARO3 gene encodes one of two 3-deoxy-D-arabino-heptulosonate-7-phosphate isoenzymes in Saccharomyces cerevisiae catalyzing the first step in the biosynthesis of aromatic amino acids. The ARO3-encoded 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) is feedback inhibited by phenylalanine; its isoenzyme, the ARO4 gene product, is inhibited by tyrosine. Both genes ARO3 and ARO4 are strongly regulated under the general control regulatory system. Cells carrying only one intact isogene are phenotypically indistinguishable from a wild-type strain when grown on minimal medium. The complete functional ARO3 promoter comprises 231 base pairs and contains only one TGACTA binding site for the general control activator protein GCN4. Mutating this element to TTACTA inhibits binding of GCN4 and results in a decreased basal level of ARO3 gene product and slow growth of a strain defective in its isogene ARO4. In addition, ARO3 gene expression cannot be elevated under amino acid starvation conditions. An ARO3 aro4 strain with gcn4 genetic background has the same phenotype of low ARO3 gene expression and slow growth. The amount of GCN4 protein present in repressed wild-type cells therefore seems to contribute to a basal level of ARO3 gene expression. The general control activator GCN4 has thus two functions: (i) to maintain a basal level of ARO3 transcription (basal control) in the presence of amino acids and (ii) to derepress the ARO3 gene to a higher transcription rate under amino acid starvation (general control).

Antifungal resistance is an emerging problem and one of the reasons for treatment failure of invasive aspergillosis (IA). Voriconazole has become a standard therapeutic for the treatment of this often fatal infection. We studied the differentially expressed proteins as a response of Aspergillus fumigatus to voriconazole by employing the two-dimensional difference gel electrophoresis (DIGE) technique. Due to addition of drug, a total of 135 differentially synthesized proteins were identified by MALDI-TOF/TOF-mass spectrometry. In particular, the level of proteins involved in the general stress response and cell detoxification increased prominently. In contrast, cell metabolism and energy proteins were down-regulated, which suggests the cellular effort to maintain balance in energy utilization while trying to combat the cellular stress exerted by the drug. We detected several so-far uncharacterized proteins which may play a role in stress response and drug metabolism and which could be future targets for antifungal treatment. A mutant strain, which is deleted in the cross-pathway control gene cpcA, was treated with voriconazole to investigate the contribution of the general control of amino acid biosynthesis to drug resistance. We compared the mutant strain’s protein expression profile with the wild-type strain. The absence of CpcA led to an increased resistance to voriconazole and a reduced activation of some general stress response proteins, while the transcript level of the triazole target gene erg11A (cyp51A) remained unchanged. In contrast, the sensitivity of strain ΔcpcA to terbinafine and amphotericin B was slightly increased. These findings imply a role of CpcA in the cellular stress response to azole drugs at the post transcriptional level.

The COP9 signalosome (CSN) is a conserved eukaryotic complex, essential for vitality in all multicellular organisms and critical for the turnover of key cellular proteins through catalytic and non-catalytic activities. Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of the CSN complex, since it includes a conserved enzymatic core but lacks non-catalytic activities, probably explaining its non-essentiality for life. A previous transcriptomic analysis of an S. cerevisiae strain deleted in the CSN5/RRI1 gene, encoding to the CSN catalytic subunit, revealed a downregulation of genes involved in lipid metabolism. We now show that the S. cerevisiae CSN holocomplex is essential for cellular lipid homeostasis. Defects in CSN assembly or activity lead to decreased quantities of ergosterol and unsaturated fatty acids (UFA); vacuole defects; diminished lipid droplets (LDs) size; and to accumulation of endoplasmic reticulum (ER) stress. The molecular mechanism behind these findings depends on CSN involvement in upregulating mRNA expression of SPT23. Spt23 is a novel activator of lipid desaturation and ergosterol biosynthesis. Our data reveal for the first time a functional link between the CSN holocomplex and Spt23. Moreover, CSN-dependent upregulation of SPT23 transcription is necessary for the fine-tuning of lipid homeostasis and for cellular health.

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.

The topic of ‘fungal stress’ is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.

Aggregation of α-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD). The budding yeast Saccharomyces cerevisiae serves as reference cell to study the interplay between αSyn misfolding, cytotoxicity and post-translational modifications (PTMs). The synuclein family includes α, β and γ isoforms. β-synuclein (βSyn) and αSyn are found at presynaptic terminals and both proteins are presumably involved in disease pathogenesis. Similar to αSyn, expression of βSyn leads to growth deficiency and formation of intracellular aggregates in yeast. Co-expression of αSyn and βSyn exacerbates the cytotoxicity. This suggests an important role of βSyn homeostasis in PD pathology. We show here that the small ubiquitin-like modifier SUMO is an important determinant of protein stability and βSyn-induced toxicity in eukaryotic cells. Downregulation of sumoylation in a yeast strain, defective for the SUMO-encoding gene resulted in reduced yeast growth, whereas upregulation of sumoylation rescued growth of yeast cell expressing βSyn. This corroborates a protective role of the cellular sumoylation machinery against βSyn-induced toxicity. Upregulation of sumoylation significantly reduced βSyn aggregate formation. This is an indirect molecular process, which is not directly linked to βSyn sumoylation because amino acid substitutions in the lysine residues required for βSyn sumoylation decreased aggregation without changing yeast cellular toxicity. αSyn aggregates are more predominantly degraded by the autophagy/vacuole than by the 26S ubiquitin proteasome system. We demonstrate a vice versa situation for βSyn, which is mainly degraded in the 26S proteasome. Downregulation of sumoylation significantly compromised the clearance of βSyn by the 26S proteasome and increased protein stability. This effect is specific, because depletion of functional SUMO did neither affect βSyn aggregate formation nor its degradation by the autophagy/vacuolar pathway. Our data support that cellular βSyn toxicity and aggregation do not correlate in their cellular impact as for αSyn but rather represent two distinct independent molecular functions and molecular mechanisms. These insights into the relationship between βSyn-induced toxicity, aggregate formation and degradation demonstrate a significant distinction between the impact of αSyn compared to βSyn on eukaryotic cells.

Ultrananocrystalline diamond (UNCD) layers exhibit excellent mechanical properties and combine chemical inertness with good biological compatibility. Therefore, UNCD is considered a promising material for coating of implants. In this work we present the preparation of thin UNCD films with embedded silver nanodroplets that provide antimicrobial property, addressing another important topic concerning implant surgery, namely the risk of a life threatening bacterial infection. UNCD layers were prepared by microwave plasma-assisted chemical vapor deposition on a silicon substrate. Afterwards, a thin film of silver was deposited on top and treated by rapid thermal annealing (RTA) leading to dewetting and formation of silver nanodroplets on the surface. A second UNCD deposition with a short duration between 5 and 30 min was applied for capping the silver nanodroplets with a thin layer. The sample surfaces were characterized after each step by atomic force microscopy and scanning electron microscopy. The composition of the final samples, including the depth of the incorporated Ag nanodroplets, was analyzed by Auger electron spectroscopy. The impact of the silver layer thickness and the RTA temperature on the nanodroplet morphology was investigated. It was found that after 10 min of capping deposition the silver particles were completely covered with UNCD. In order to study the release of silver ions, the UNCD/Ag/UNCD samples were submerged in deionized water for 7 days at 37 °C, followed by detection of the silver concentration in the aqueous samples by inductively coupled plasma mass spectrometry. The determined concentration was strongly dependent on the thickness of the capping UNCD layer, exhibiting the highest silver content for the sample with the thinnest capping layer. Thus, the UNCD layer thickness can be utilized to control the amount of Ag ions released into the surrounding environment. The antibacterial properties were investigated with bacterial assays of the Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria that were exposed to the samples. All silver containing samples showed significant antimicrobial propensity, whereas the different capping thicknesses affected the time-course dependent antibacterial efficiency. Thin UNCD films were provided with supreme antibacterial propensity by embedding of silver nanodroplets. The thickness of the UNCD capping layer was varied and its influence on the release of silver in water was investigated. The UNCD/Ag/UNCD samples showed significant and controlled by the capping thicknesses time-dependent antibacterial efficiency against Gram-negative E. coli and Gram-positive B. subtilis bacteria.

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.

Aggregation of α-synuclein (αSyn) into proteinaceous deposits is a pathological hallmark of a range of neurodegenerative diseases including Parkinson’s disease (PD). Numerous lines of evidence indicate that the accumulation of toxic oligomeric and prefibrillar αSyn species may underpin the cellular toxicity and spread of pathology between cells. Therefore, aggregation of αSyn is considered a priority target for drug development, as aggregation inhibitors are expected to reduce αSyn toxicity and serve as therapeutic agents. Here, we used the budding yeast S. cerevisiae as a platform for the identification of short peptides that inhibit αSyn aggregation and toxicity. A library consisting of approximately one million peptide variants was utilized in two high-throughput screening approaches for isolation of library representatives that reduce αSyn-associated toxicity and aggregation. Seven peptides were isolated that were able to suppress specifically αSyn toxicity and aggregation in living cells. Expression of the peptides in yeast reduced the accumulation of αSyn-induced reactive oxygen species and increased cell viability. Next, the peptides were chemically synthesized and probed for their ability to modulate αSyn aggregation in vitro . Two synthetic peptides, K84s and K102s, of 25 and 19 amino acids, respectively, significantly inhibited αSyn oligomerization and aggregation at sub-stoichiometric molar ratios. Importantly, K84s reduced αSyn aggregation in human cells. These peptides represent promising αSyn aggregation antagonists for the development of future therapeutic interventions.

Phytopathogenic Verticillia cause Verticillium wilt on numerous economically important crops. Plant infection begins at the roots, where the fungus is confronted with rhizosphere inhabiting bacteria. The effects of different fluorescent pseudomonads, including some known biocontrol agents of other plant pathogens, on fungal growth of the haploid Verticillium dahliae and/or the amphidiploid Verticillium longisporum were compared on pectin-rich medium, in microfluidic interaction channels, allowing visualization of single hyphae, or on Arabidopsis thaliana roots. We found that the potential for formation of bacterial lipopeptide syringomycin resulted in stronger growth reduction effects on saprophytic Aspergillus nidulans compared to Verticillium spp. A more detailed analyses on bacterial-fungal co-cultivation in narrow interaction channels of microfluidic devices revealed that the strongest inhibitory potential was found for Pseudomonas protegens CHA0, with its inhibitory potential depending on the presence of the GacS/GacA system controlling several bacterial metabolites. Hyphal tip polarity was altered when V. longisporum was confronted with pseudomonads in narrow interaction channels, resulting in a curly morphology instead of straight hyphal tip growth. These results support the hypothesis that the fungus attempts to evade the bacterial confrontation. Alterations due to co-cultivation with bacteria could not only be observed in fungal morphology but also in fungal transcriptome. P. protegens CHA0 alters transcriptional profiles of V. longisporum during 2 h liquid media co-cultivation in pectin-rich medium. Genes required for degradation of and growth on the carbon source pectin were down-regulated, whereas transcripts involved in redox processes were up-regulated. Thus, the secondary metabolite mediated effect of Pseudomonas isolates on Verticillium species results in a complex transcriptional response, leading to decreased growth with precautions for self-protection combined with the initiation of a change in fungal growth direction. This interplay of bacterial effects on the pathogen can be beneficial to protect plants from infection, as shown with A . thaliana root experiments. Treatment of the roots with bacteria prior to infection with V. dahliae resulted in a significant reduction of fungal root colonization. Taken together we demonstrate how pseudomonads interfere with the growth of Verticillium spp. and show that these bacteria could serve in plant protection.

Fungal Hülle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hülle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hülle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones accumulate emodins and are delayed in maturation and growth of fruiting bodies. Emodin represses fruiting body and resting structure formation in other fungi. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. Our findings reveal a novel role of Hülle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis.

The yeast TRP4 promoter contains three responsive elements (GCREs) for the 'general control' transcriptional activator GCN4, which are arranged in two upstream elements, UAS1 (GCRE1) and UAS2 (GCRE2 and GCRE3). A point mutation analysis of these elements revealed that all three GCREs are required for GCN4-dependent transcription, but none are involved in basal transcription. Basal transcription and GCN4-dependent transcription use completely different initiator elements in the TRP4 promoter. UAS1 acts synergistically with UAS2 to activate the GCN4-dependent transcription of TRP4. A consensus TATA box can functionally replace the UAS2 element to allow normal GCN4-dependent transcription, suggesting that UAS2 is analogous to the TATA element of other promoters. GCN4 might therefore activate transcription by exhibiting two alternative functions within the natural TRP4 promoter.

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same-sex mating-type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage-specific (LS) region apparently originating from the Verticillium dahliae-related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence-reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.

Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) acts as a negative regulator of autophagy by interacting with Beclin 1 at Golgi membranes in mammalian cells. The molecular mechanism of this interaction is largely unknown. We recently showed that human GAPR-1 (hGAPR-1) has amyloidogenic properties resulting in the formation of protein condensates upon overexpression in Saccharomyces cerevisiae. Here we show that human Beclin 1 (hBeclin 1) has several predicted amyloidogenic regions and that overexpression of hBeclin 1-mCherry in yeast also results in the formation of fluorescent protein condensates. Surprisingly, co-expression of hGAPR-1-GFP and hBeclin 1-mCherry results in a strong reduction of hBeclin 1 condensates. Mutations of the known interaction site on the hGAPR-1 and hBeclin 1 surface abolished the effect on condensate formation during co-expression without affecting the condensate formation properties of the individual proteins. Similarly, a hBeclin 1-derived B18 peptide that is known to bind hGAPR-1 and to interfere with the interaction between hGAPR-1 and hBeclin 1, abolished the reduction of hBeclin 1 condensates by co-expression of hGAPR-1. These results indicate that the same type of protein-protein interactions interfere with condensate formation during co-expression of hGAPR-1 and hBeclin 1 as previously described for their interaction at Golgi membranes. The amyloidogenic properties of the B18 peptide were, however, important for the interaction with hGAPR-1, as mutant peptides with reduced amyloidogenic properties also showed reduced interaction with hGAPR-1 and reduced interference with hGAPR-1/hBeclin 1 condensate formation. We propose that amyloidogenic interactions take place between hGAPR-1 and hBeclin 1 prior to condensate formation.

The ARO3 gene encodes one of two 3-deoxy-D-arabino-heptulosonate-7-phosphate isoenzymes in Saccharomyces cerevisiae catalyzing the first step in the biosynthesis of aromatic amino acids. The ARO3-encoded 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) is feedback inhibited by phenylalanine; its isoenzyme, the ARO4 gene product, is inhibited by tyrosine. Both genes ARO3 and ARO4 are strongly regulated under the general control regulatory system. Cells carrying only one intact isogene are phenotypically indistinguishable from a wild-type strain when grown on minimal medium. The complete functional ARO3 promoter comprises 231 base pairs and contains only one TGACTA binding site for the general control activator protein GCN4. Mutating this element to TTACTA inhibits binding of GCN4 and results in a decreased basal level of ARO3 gene product and slow growth of a strain defective in its isogene ARO4. In addition, ARO3 gene expression cannot be elevated under amino acid starvation conditions. An ARO3 aro4 strain with gcn4 genetic background has the same phenotype of low ARO3 gene expression and slow growth. The amount of GCN4 protein present in repressed wild-type cells therefore seems to contribute to a basal level of ARO3 gene expression. The general control activator GCN4 has thus two functions: (i) to maintain a basal level of ARO3 transcription (basal control) in the presence of amino acids and (ii) to derepress the ARO3 gene to a higher transcription rate under amino acid starvation (general control).

Research Article1 December 1992free access Sequence-specific initiator elements focus initiation of transcription to distinct sites in the yeast TRP4 promoter. H.U. Mösch H.U. Mösch Institute of Microbiology, Swiss Federal Institute of Technology, (ETH), Zürich. Search for more papers by this author R. Graf R. Graf Institute of Microbiology, Swiss Federal Institute of Technology, (ETH), Zürich. Search for more papers by this author G.H. Braus G.H. Braus Institute of Microbiology, Swiss Federal Institute of Technology, (ETH), Zürich. Search for more papers by this author H.U. Mösch H.U. Mösch Institute of Microbiology, Swiss Federal Institute of Technology, (ETH), Zürich. Search for more papers by this author R. Graf R. Graf Institute of Microbiology, Swiss Federal Institute of Technology, (ETH), Zürich. Search for more papers by this author G.H. Braus G.H. Braus Institute of Microbiology, Swiss Federal Institute of Technology, (ETH), Zürich. Search for more papers by this author Author Information H.U. Mösch1, R. Graf1 and G.H. Braus1 1Institute of Microbiology, Swiss Federal Institute of Technology, (ETH), Zürich. The EMBO Journal (1992)11:4583-4590https://doi.org/10.1002/j.1460-2075.1992.tb05560.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Transcription from the yeast TRP4 promoter initiates at two basal (i127 and i76) and three GCN4 dependent (i31, i25 and i12) initiator elements. All of these elements contain not more than one deviation from the earlier proposed initiator consensus sequence PuPuPyPuPu, a pyrimidine nucleotide flanked on either side by two purine nucleotides. A point mutation analysis of these elements in various combinations was performed and revealed that the central pyrimidine nucleotide and at least one of the 3′ flanking purine nucleotides of the PuPuPyPuPu consensus sequence are essential but alone not sufficient to define a functional initiator element. Multiple cryptic transcription start sites, which function independently whether they are located on the coding or the non-coding strand, can replace the function of mutated initiator elements and therefore the overall level of transcription initiation is not affected. The sequence specificity is identical for basal and GCN4 dependent initiator elements demonstrating that they are functionally homologous. These findings imply that the role of initiator elements is to ‘focus’ the start point(s) of transcription to distinct sites located in the region between the site(s) of the assembly of the transcriptional complex and the start codon of translation. Previous ArticleNext Article Volume 11Issue 121 December 1992In this issue RelatedDetailsLoading ...

In the fungus Ustilago maydis, sexual pheromones elicit mating resulting in an infective filament able to infect corn plants. Along this process a G2 cell cycle arrest is mandatory. Such as cell cycle arrest is initiated upon the pheromone recognition in each mating partner, and sustained once cell fusion occurred until the fungus enter the plant tissue. We describe that the initial cell cycle arrest resulted from inhibition of the nuclear transport of the mitotic inducer Cdc25 by targeting its importin, Kap123. Near cell fusion to take place, the increase on pheromone signaling promotes Cdc25 degradation, which seems to be important to ensure the maintenance of the G2 cell cycle arrest to lead the formation of the infective filament. This way, premating cell cycle arrest is linked to the subsequent steps required for establishment of the infection. Disabling this connection resulted in the inability of fungal cells to infect plants.

Chorismate mutase (EC 5.4.99.5) from the yeast Saccharomyces cerevisiae is an allosteric enzyme which can be locked in its active R (relaxed) state by a single threonine-to-isoleucine exchange at position 226. Seven new replacements of residue 226 reveal that this position is able to direct the enzyme's allosteric equilibrium, without interfering with the catalytic constant or the affinity for the activator.

ABSTRACT Pseudomonas fluorescens strain 2-79, a natural isolate of the rhizosphere of wheat ( Triticum aestivum L.), possesses antagonistic potential toward several fungal pathogens. We report the draft genome sequence of strain 2-79, which comprises 5,674 protein-coding sequences.