Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

An Unconventional Melanin Biosynthesis Pathway in Ustilago maydis.

Ustilago maydis is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well-established model organism for the study of plant-microbe interactions, its potential to produce specialized metabolites, which might contribute to this interaction, has not been studied in detail. By analyzing the U. maydis genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (pks3, pks4, and pks5), a cytochrome P450 monooxygenase (cyp4), and a protein with similarity to versicolorin B synthase (vbs1). Metabolic profiles of deletion mutants in this gene cluster suggested that Pks3 and Pks4 act in concert as heterodimers to generate orsellinic acid (OA), which is reduced to the corresponding aldehyde by Pks5. The OA-aldehyde can then react with triacetic acid lactone (TAL), also derived from Pks3/Pks4 heterodimers to form larger molecules, including novel coumarin derivatives. Our findings suggest that U. maydis synthesizes a novel type of melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or l-3,4-dihydroxyphenylalanine (l-DOPA). Along with these observations, this work also provides insight into the mechanisms of polyketide synthases in this filamentous fungus.IMPORTANCE The fungus Ustilago maydis represents one of the major threats to maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by U. maydis to protect itself against harsh environments, such as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in an unconventional way, requiring more than one polyketide synthase for producing melanin precursors, gives a different perspective on the complexity of these multidomain enzymes and their evolution in the fungal kingdom.

Exploring molecular tools for transformation and gene expression in the cultivated edible mushroom Agrocybe aegerita.

Agrocybe aegerita is a cultivated edible mushroom in numerous countries, which also serves as a model basidiomycete to study fruiting body formation. Aiming to create an easily expandable customised molecular toolset for transformation and constitutive gene of interest expression, we first created a homologous dominant marker for transformant selection. Progeny monokaryons of the genome-sequenced dikaryon A. aegerita AAE-3 used here were identified as sensitive to the systemic fungicide carboxin. We cloned the wild-type gene encoding the iron-sulphur protein subunit of succinate dehydrogenase AaeSdi1 including its up- and downstream regions, and introduced a single-point mutation (His237 to Leu) to make it confer carboxin resistance. PEG-mediated transformation of protoplasts derived from either oidia or vegetative monokaryotic mycelium with the resulting carboxin resistance marker (CbxR) plasmid pSDI1E3 yielded carboxin-resistant transformants in both cases. Plasmid DNA linearised within the selection marker resulted in transformants with ectopic multiple insertions of plasmid DNA in a head-to-tail repeat-like fashion. When circular plasmid was used, ectopic single integration into the fungal genome was favoured, but also gene conversion at the homologous locus was seen in 1 out of 11 analysed transformants. Employing CbxR as selection marker, two versions of a reporter gene construct were assembled via Golden Gate cloning which allows easy recombination of its modules. These consisted of an eGFP expression cassette controlled by the native promoter PAaeGPDII and the heterologous terminator Tnos, once with and once without an intron in front of the eGFP start codon. After protoplast transformation with either construct as circular plasmid DNA, GFP fluorescence was detected with either transformants, indicating that expression of eGFP is intron-independent in A. aegerita. This paves the way for functional genetics approaches to A. aegerita, e.g., via constitutive expression of fruiting-related genes.

Elucidation of substrate specificities of decorating enzymes involved in mannosylerythritol lipid production by cross-species complementation.

Mannosylerythritol lipids (MELs) are surface active molecules produced by many basidiomycetous fungi. MELs consist of a mannosylerythritol disaccharide, which is acylated with short and medium chain fatty acids at the mannosyl moiety. A gene cluster composed of five genes is required for MEL biosynthesis. Here we show that the plant pathogenic fungus Ustilago hordei secretes these glycolipids under nitrogen starvation conditions. In contrast to MELs produced by the closely related fungus Ustilago maydis those secreted by U. hordei are mostly mono-acetylated and contain a different mixture of acyl groups. Cross-species complementation between these fungi revealed that these differences result from different catalytic activities of the acetyltransferase Mat1 and the acyltransferases Mac1 and Mac2. U. maydis mat1 mutants expressing the homologous mat1 gene from U. hordei produced mostly mono-acetylated variants and lack di-acetylated MELs normally produced by U. maydis. Furthermore, we determined that the acyltransferase Mac1 acylates the mannosylerythritol moiety at position C2 while Mac2 acylates C3. The identification of decorating enzymes with different substrate specificities will allow the tailor-made production of novel subsets of MELs.

The Obvious and the Hidden: Prediction and Function of Fungal Peroxisomal Matrix Proteins.

Fungal peroxisomes are characterized by a number of specific biological functions. To understand the physiology and biochemistry of these organelles knowledge of the proteome content is crucial. Here, we address different strategies to predict peroxisomal proteins by bioinformatics approaches. These tools range from simple text searches to network based learning strategies. A complication of this analysis is the existence of cryptic peroxisomal proteins, which are overlooked in conventional bioinformatics queries. These include proteins where targeting information results from transcriptional and posttranscriptional alterations. But also proteins with low efficiency targeting motifs that are predominantly localized in the cytosol, and proteins lacking any canonical targeting information, can play important roles within peroxisomes. Many of these proteins are so far unpredictable. Detection and characterization of these cryptic peroxisomal proteins revealed the presence of novel peroxisomal enzymatic reaction networks in fungi.

Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi.

Peroxisomes are eukaryotic organelles important for the metabolism of long-chain fatty acids. Here we show that in numerous fungal species, several core enzymes of glycolysis, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK), reside in both the cytoplasm and peroxisomes. We detected in these enzymes cryptic type 1 peroxisomal targeting signals (PTS1), which are activated by post-transcriptional processes. Notably, the molecular mechanisms that generate the peroxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis, peroxisomal targeting of Pgk1 results from ribosomal read-through, whereas alternative splicing generates the PTS1 of Gapdh. In the filamentous ascomycete Aspergillus nidulans, peroxisomal targeting of these enzymes is achieved by exactly the opposite mechanisms. We also detected PTS1 motifs in the glycolytic enzymes triose-phosphate isomerase and fructose-bisphosphate aldolase. U. maydis mutants lacking the peroxisomal isoforms of Gapdh or Pgk1 showed reduced virulence. In addition, mutational analysis suggests that GAPDH, together with other peroxisomal NADH-dependent dehydrogenases, has a role in redox homeostasis. Owing to its hidden nature, partial peroxisomal targeting of well-studied cytoplasmic enzymes has remained undetected. Thus, we anticipate that further bona fide cytoplasmic proteins exhibit similar dual targeting.

Ribosomal readthrough at a short UGA stop codon context triggers dual localization of metabolic enzymes in Fungi and animals.

Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated.

Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production.

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis' itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5-fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical.

Crosstalk between the unfolded protein response and pathways that regulate pathogenic development in Ustilago maydis.

The unfolded protein response (UPR) is a conserved eukaryotic signaling pathway regulating endoplasmic reticulum (ER) homeostasis during ER stress, which results, for example, from an increased demand for protein secretion. Here, we characterize the homologs of the central UPR regulatory proteins Hac1 (for Homologous to ATF/CREB1) and Inositol Requiring Enzyme1 in the plant pathogenic fungus Ustilago maydis and demonstrate that the UPR is tightly interlinked with the b mating-type-dependent signaling pathway that regulates pathogenic development. Exact timing of UPR is required for virulence, since premature activation interferes with the b-dependent switch from budding to filamentous growth. In addition, we found crosstalk between UPR and the b target Clampless1 (Clp1), which is essential for cell cycle release and proliferation in planta. The unusual C-terminal extension of the U. maydis Hac1 homolog, Cib1 (for Clp1 interacting bZIP1), mediates direct interaction with Clp1. The interaction between Clp1 and Cib1 promotes stabilization of Clp1, resulting in enhanced ER stress tolerance that prevents deleterious UPR hyperactivation. Thus, the interaction between Cib1 and Clp1 constitutes a checkpoint to time developmental progression and increased secretion of effector proteins at the onset of biotrophic development. Crosstalk between UPR and the b mating-type regulated developmental program adapts ER homeostasis to the changing demands during biotrophy.

Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi.

Many microorganisms secrete surface-active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis-targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross-talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell.

Small-GTPase-associated signaling by the guanine nucleotide exchange factors CpDock180 and CpCdc24, the GTPase effector CpSte20, and the scaffold protein CpBem1 in Claviceps purpurea.

Monomeric GTPases of the Rho subfamily are important mediators of polar growth and NADPH (Nox) signaling in a variety of organisms. These pathways influence the ability of Claviceps purpurea to infect host plants. GTPase regulators contribute to the nucleotide loading cycle that is essential for proper functionality of the GTPases. Scaffold proteins gather GTPase complexes to facilitate proper function. The guanine nucleotide exchange factors (GEFs) CpCdc24 and CpDock180 activate GTPase signaling by triggering nucleotide exchange of the GTPases. Here we show that CpCdc24 harbors nucleotide exchange activity for both Rac and Cdc42 homologues. The GEFs partly share the cellular distribution of the GTPases and interact with the putative upstream GTPase CpRas1. Interaction studies show the formation of higher-order protein complexes, mediated by the scaffold protein CpBem1. Besides the GTPases and GEFs, these complexes also contain the GTPase effectors CpSte20 and CpCla4, as well as the regulatory protein CpNoxR. Functional characterizations suggest a role of CpCdc24 mainly in polarity, whereas CpDock180 is involved in stress tolerance mechanisms. These findings indicate the dynamic formation of small GTPase complexes and improve the model for GTPase-associated signaling in C. purpurea.

Septation of infectious hyphae is critical for appressoria formation and virulence in the smut fungus Ustilago maydis.

Differentiation of hyphae into specialized infection structures, known as appressoria, is a common feature of plant pathogenic fungi that penetrate the plant cuticle. Appressorium formation in U. maydis is triggered by environmental signals but the molecular mechanism of this hyphal differentiation is largely unknown. Infectious hyphae grow on the leaf surface by inserting regularly spaced retraction septa at the distal end of the tip cell leaving empty sections of collapsed hyphae behind. Here we show that formation of retraction septa is critical for appressorium formation and virulence in U. maydis. We demonstrate that the diaphanous-related formin Drf1 is necessary for actomyosin ring formation during septation of infectious hyphae. Drf1 acts as an effector of a Cdc42 GTPase signaling module, which also consists of the Cdc42-specific guanine nucleotide exchange factor Don1 and the Ste20-like kinase Don3. Deletion of drf1, don1 or don3 abolished formation of retraction septa resulting in reduced virulence. Appressorium formation in these mutants was not completely blocked but infection structures were found only at the tip of short filaments indicating that retraction septa are necessary for appressorium formation in extended infectious hyphae. In addition, appressoria of drf1 mutants penetrated the plant tissue less frequently.

Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa.

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O-glycosidically linked to 3,15,16-trihydroxypalmitic acid. The sugar moiety is acylated with 2-hydroxy-octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl-transferase and is lacking a gene homologous to the α-hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl-transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl-transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Two Pex5 Proteins With Different Cargo Specificity Are Critical for Peroxisome Function in Ustilago maydis.

Peroxisomes are dynamic multipurpose organelles with a major function in fatty acid oxidation and breakdown of hydrogen peroxide. Many proteins destined for the peroxisomal matrix contain a C-terminal peroxisomal targeting signal type 1 (PTS1), which is recognized by tetratricopeptide repeat (TPR) proteins of the Pex5 family. Various species express at least two different Pex5 proteins, but how this contributes to protein import and organelle function is not fully understood. Here, we analyzed truncated and chimeric variants of two Pex5 proteins, Pex5a and Pex5b, from the fungus Ustilago maydis. Both proteins are required for optimal growth on oleic acid-containing medium. The N-terminal domain (NTD) of Pex5b is critical for import of all investigated peroxisomal matrix proteins including PTS2 proteins and at least one protein without a canonical PTS. In contrast, the NTD of Pex5a is not sufficient for translocation of peroxisomal matrix proteins. In the presence of Pex5b, however, specific cargo can be imported via this domain of Pex5a. The TPR domains of Pex5a and Pex5b differ in their affinity to variations of the PTS1 motif and thus can mediate import of different subsets of matrix proteins. Together, our data reveal that U. maydis employs versatile targeting modules to control peroxisome function. These findings will promote our understanding of peroxisomal protein import also in other biological systems.

Import and Export of Mannosylerythritol Lipids by Ustilago maydis.

Upon nitrogen starvation, the basidiomycete Ustilago maydis, which causes smut disease on corn, secretes amphipathic glycolipids, including mannosylerythritol lipids (MELs). MELs consist of a carbohydrate core whose mannosyl moiety is both acylated with fatty acids of different lengths and acetylated. Here, we report the transport of MELs into and out of the cell depending on the transport protein Mmf1, which belongs to the major facilitator superfamily. Analysis of mmf1 mutants and mutants lacking the acetyltransferase Mat1 revealed that Mmf1 is necessary for the export of acetylated MELs, while MELs without an acetyl group are secreted independently of this transporter. Upon deletion of mmf1, we detected novel MEL species lacking the acyl side chain at C-3'. With the help of feeding experiments, we demonstrate that MELs are taken up by U. maydis in an mmf1-independent manner. This leads to catabolism or rearrangement of acetyl and acyl side groups and subsequent secretion. The catabolism of MELs involves the presence of Mac2, an enzyme required for MEL biosynthesis. In cocultivation experiments, mutual exchange of MELs between different mutants was observed. Thus, we propose a novel function for fungal glycolipids as an external carbon storage. IMPORTANCE Fungi produce and secrete various secondary metabolites that can act as weapons against competitors, help in accessing nutrients, or assist in development and communication. One group of secondary metabolites are surface-active glycolipids, which have significant biotechnological potential as biodegradable detergents. While the biosynthesis of several fungal biosurfactants is well characterized, their biological functions and transport routes are less understood. We developed a cocultivation assay to show that a class of glycolipids from Ustilago maydis called mannosylerythritol lipids (MELs) can be exchanged between cells and modified or even degraded by recipient cells. Feeding assays with purified MELs led to similar results. These data provide insight into the surprising biological role of MELs as putative external carbon sources. Applying feeding and cocultivation experiments on MEL biosynthesis mutants turned out to be a valuable strategy for systematically studying the import routes and degradation pathways of glycolipids. By using these assays, we demonstrate the function of the transport protein Mmf1 as a specific exporter of acetylated MELs. We propose that these assays may be applied more generally, thereby opening novel areas of research.

Beta hydroxylation of glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-dependent ß-hydroxylase.

Flocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced by Pseudozyma flocculosa, a biocontrol agent, and Ustilago maydis, a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the ß position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the ß position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of the P. flocculosa fhd1 gene and its homolog from U. maydis, uhd1, have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted the uhd1 gene in U. maydis and analyzed the secreted UA. uhd1 deletion strains produced UA lacking the ß-hydroxyl group of the short-chain fatty acid. To analyze the function of P. flocculosa Fhd1, the corresponding gene was used to complement U. maydis Δuhd1 mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for ß hydroxylation of the flocculosin short-chain fatty acid. We also investigated a P. flocculosa homolog of the U. maydis long-chain fatty-acid alpha hydroxylase Ahd1. The P. flocculosa ahd1 gene, which does not reside in the flocculosin gene cluster, was introduced into U. maydis Δahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis Δahd1 phenotype nor resulted in the production of ß-hydroxylated UA. This suggests that P. flocculosa Ahd1 is not involved in flocculosin hydroxylation.

Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis.

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

Engineering Ustilago maydis for production of tailor-made mannosylerythritol lipids.

Mannosylerythritol lipids (MELs) are surface active glycolipids secreted by various fungi. MELs can be used as biosurfactants and are a biodegradable resource for the production of detergents or pharmaceuticals. Different fungal species synthesize a unique mixture of MELs differing in acetyl- and acyl-groups attached to the sugar moiety. Here, we report the construction of a toolbox for production of glycolipids with predictable fatty acid side chains in the basidiomycete Ustilago maydis. Genes coding for acyl-transferases involved in MEL production (Mac1 and Mac2) from different fungal species were combined to obtain altered MEL variants with distinct physical properties and altered antimicrobial activity. We also demonstrate that a U. maydis paralog of the acyltransferase Mac2 with a different substrate specificity can be employed for the biosynthesis of modified MEL variants. In summary, our data showcase how the fungal repertoire of Mac enzymes can be used to engineer tailor-made MELs according to specific biotechnological or pharmaceutical requirements.

The germinal centre kinase Don3 triggers the dynamic rearrangement of higher-order septin structures during cytokinesis in Ustilago maydis.

The dimorphic phytopathogenic fungus Ustilago maydis grows in its haploid phase by budding. Cytokinesis and separation of daughter cells are accomplished by the consecutive formation of two distinct septa. Here, we show that both septation events involve the dynamic rearrangement of septin assemblies from hourglass-shaped collars into ring-like structures. Using a chemical genetic approach we demonstrate that the germinal centre kinase Don3 triggers this septin reorganization during secondary septum formation. Although chemical inhibition of an analogue-sensitive version of Don3 prevented septation, a stable septin collar was assembled at the presumptive septation site. Interestingly, the essential light chain of type II myosin, Cdc4, was already associated with this septin collar. Release of Don3 kinase inhibition triggered immediate dispersal of septin filaments and concomitant incorporation of Cdc4 into a contractile actomyosin ring, which also contained the F-BAR domain protein Cdc15. Inhibition of actin polymerization or deletion of the cdc15 gene, did not affect assembly of the initial collar consisting of septin and myosin light chain. However, reassembly of septin filaments into a ring-like structure was prevented in the absence of either F-actin or Cdc15, indicating that septin ring formation in U. maydis depends on a functional contractile actomyosin ring.