The dual role of a yeast metacaspase: What doesn't kill you makes you stronger

Recent reports suggest that the yeast Saccharomyces cerevisiae caspase‐related metacaspase, Mca1, is required for cell‐autonomous cytoprotective functions that slow cellular aging. Because the Mca1 protease has previously been suggested to be responsible for programmed cell death (PCD) upon stress and aging, these reports raise the question of how the opposing roles of Mca1 as a protector and executioner are regulated. One reconciling perspective could be that executioner activation may be restricted to situations where the death of part of the population would be beneficial, for example during colony growth or adaptation into specialized survival forms. Another possibility is that metacaspases primarily harbor beneficial functions and that the increased survival observed upon metacaspase removal is due to compensatory responses. Herein, we summarize data on the role of Mca1 in cell death and survival and approach the question of how a metacaspase involved in protein quality control may act as killer protein.     

14-3-3 regulates the G2/M transition in the basidiomycete Ustilago maydis

14-3-3 proteins are a family of highly conserved polypeptides that function as small adaptors that facilitate a diverse array of cellular processes by binding phosphorylated target proteins. One of these processes is the regulation of the cell cycle. Here we characterized the role of Bmh1, a 14-3-3 protein, in the cell cycle regulation of the fungus Ustilago maydis. We found that this protein is essential in U. maydis and that it has roles during the G2/M transition in this organism. The function of 14-3-3 in U. maydis seems to mirror the proposed role for this protein during Schizosaccharomyces pombe cell cycle regulation. We provided evidence that in U. maydis 14-3-3 protein binds to the mitotic regulator Cdc25. Comparison of the roles of 14-3-3 during cell cycle regulation in other fungal system let us to discuss the connections between morphogenesis, cell cycle regulation and the evolutionary role of 14-3-3 proteins in fungi.     

Insight into the biochemical and cell biological function of an intrinsically unstructured heat shock protein,Hsp12 of Ustilago maydis

Small heat shock proteins (sHsps) play diverse roles in the stress response and maintenance of cellular functions. The Ustilago maydis genome codes for few sHsps. Among these, Hsp12 has previously been demonstrated to be involved in the pathogenesis of the fungus by our group. In the present study we further investigated the biological function of the protein in the pathogenic development of U. maydis. Analysis of the primary amino acid sequence of Hsp12 in combination with spectroscopic methods to analyse secondary protein structures revealed an intrinsically disordered nature of the protein. We also carried out detailed analysis on the protein aggregation prevention activity associated with Hsp12. Our data suggest Hsp12 has trehalose-dependent protein aggregation prevention activity. Through assaying the interaction of Hsp12 with lipid membranes in vitro we also showed the ability of U. maydis Hsp12 to induce stability in lipid vesicles. U. maydis hsp12 deletion mutants exhibited defects in the endocytosis process and delayed completion of the pathogenic life cycle. Therefore, U. maydis Hsp12 contributes to the pathogenic development of the fungus through its ability to relieve proteotoxic stress during infection as well as its membrane-stabilizing function.     

Extra N-Terminal Residues Have a Profound Effect on the Aggregation Properties of the Potential Yeast Prion Protein Mca1

The metacaspase Mca1 from Saccharomyces cerevisiae displays a Q/N-rich region at its N-terminus reminiscent of yeast prion proteins. In this study, we show that the ability of Mca1 to form insoluble aggregates is modulated by a peptide stretch preceding its putative prion-forming domain. Based on its genomic locus, three potential translational start sites of Mca1 can give rise to two slightly different long Mca1 proteins or a short version, Mca1_451/453 and Mca1_432, respectively, although under normal physiological conditions Mca1₄₃₂ is the predominant form expressed. All Mca1 variants exhibit the Q/N-rich regions, while only the long variants Mca1_451/453 share an extra stretch of 19 amino acids at their N-terminal end. Strikingly, only long versions of Mca1 but not Mca1₄₃₂ revealed pronounced aggregation in vivo and displayed prion-like properties when fused to the C-terminal domain of Sup35 suggesting that the N-terminal peptide element promotes the conformational switch of Mca1 protein into an insoluble state. Transfer of the 19 N-terminal amino acid stretch of Mca1₄₅₁ to the N-terminus of firefly luciferase resulted in increased aggregation of luciferase, suggesting a protein destabilizing function of the peptide element. We conclude that the aggregation propensity of the potential yeast prion protein Mca1 in vivo is strongly accelerated by a short peptide segment preceding its Q/N-rich region and we speculate that such a conformational switch might occur in vivo via the usage of alternative translational start sites.     

Stress‐related Responses in Alexandrium tamarense Cells Exposed to Environmental Changes

Organisms tend to be sensitive to drastic changes in environmental conditions. For unicellular microorganisms, variations in physico‐chemical conditions are particularly challenging and may result in acclimation, entrance into quiescence, or death through necrotic or autocatalytic pathways. This study focuses on the thecate dinoflagellate Alexandrium tamarense. Cellular responses to oxidative, thermal, and nutrient stress were characterized using stress indicators, such as pigment content, efficiency of photosystem II or production of reactive oxygen species (ROS), as well as hallmarks of apoptosis including activity of caspase‐like enzymes and expression of a metacaspase gene homolog. The formation of temporary cysts, a survival strategy of short‐term quiescence, was also monitored. Cellular responses appeared to depend on multifactorial influences where type and intensity of stimulus as well as position in cell cycle may act in combination. Sequences of events observed implicate ROS production as a key determinant of stress‐related pathways, playing potential roles in intracellular signaling, formation of temporary cysts, or cellular damage. Variations observed in caspase‐like activities and metacaspase gene expression did not appear to be associated with programmed cell death pathways; our results suggest a wider range of functions for these proteases in phytoplankton cells, including roles in survival pathways and cell cycle progression.     

Establishment of compatibility in the Ustilago maydis/maize pathosystem

The fungus Ustilago maydis is a biotrophic pathogen parasitizing on maize. The most prominent symptoms of the disease are large tumors in which fungal proliferation and spore differentiation occur. In this study, we have analyzed early and late tumor stages by confocal microscopy. We show that fungal differentiation occurs both within plant cells as well as in cavities where huge aggregates of fungal mycelium develop. U. maydis is poorly equipped with plant CWDEs and we demonstrate by array analysis that the respective genes follow distinct expression profiles at early and late stages of tumor development. For the set of three genes coding for pectinolytic enzymes, deletion mutants were generated by gene replacement. Neither single nor triple mutants were affected in pathogenic development. Based on our studies, we consider it unlikely that U. maydis feeds on carbohydrates derived from the digestion of plant cell wall material, but uses its set of plant CWDEs for softening the cell wall structure as a prerequisite for in planta growth.     

Ustilago maydis accumulates β-carotene at levels determined by a retinal-forming carotenoid oxygenase

The basidiomycete Ustilago maydis, the causative agent of corn smut disease, has emerged as a model organism for dimorphism and fungal phytopathogenicity. In this work, we line out the key conserved enzymes for β-carotene biosynthesis encoded by the U. maydis genome and show that this biotrophic fungus accumulates β-carotene. The amount of this pigment depended on culture pH and aeration but was not affected by light and was not increased by oxidative stress. Moreover, we identified the U. maydis gene, cco1, encoding a putative β-carotene cleavage oxygenase. Heterologous overexpression and in vitro analyses of purified enzyme demonstrated that Cco1 catalyzes the symmetrical cleavage of β-carotene to yield two molecules of retinal. Analyses of β-carotene and retinal contents in U. maydis cco1 deletion and over-expression strains confirmed the enzymatic function of Cco1, and revealed that Cco1 determines the β-carotene content. Our data indicate that carotenoid biosynthesis in U. maydis is carried out to provide retinal rather than to deliver protective pigments. The U. maydis genome also encodes three potential opsins, a family of photoactive proteins that use retinal as chromophore. Two opsin genes showed different light-regulated expression patterns, suggesting specialized roles in photobiology, while no mRNA was detected for the third opsin gene in the same experiments. However, deletion of the cco1 gene, which should abolish function of all the retinal-dependent opsins, did not affect growth, morphology or pathogenicity, suggesting that retinal and opsin proteins play no relevant role in U. maydis under the tested conditions.     

Response to osmotic stress and temperature of the fungus <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Ustilago maydis is a fungal pathogen which is exposed during its life cycle to both abiotic and biotic stresses before and after the infection of maize. To cope with extreme environmental changes, microorganisms usually accumulate the disaccharide trehalose. We have investigated both the accumulation of trehalose and the activity of trehalase during the adaptation of U. maydis haploid cells to thermal, sorbitol, and NaCl stresses. Sorbitol and sodium chloride induced sustained accumulation of trehalose, while a transient increase was observed under heat stress. Sorbitol stressed cells showed higher trehalase activity compared with control cells and to those stressed by NaCl and high temperature. Addition of cycloheximide, a protein synthesis inhibitor, did not affect the trehalose accumulation during the first 15 min, but basal levels of trehalose were reached after the second period of 15 min. The proteomic analysis of the response of U. maydis to temperature, sorbitol, and salt stresses indicated a complex pattern which highlights the change of 18 proteins involved in carbohydrate and amino acid metabolism, protein folding, redox regulation, ion homeostasis, and stress response. We hypothesize that trehalose accumulation during sorbitol stress in U. maydis might be related to the adaptation of this organism during plant infection.     

Genome‐wide assessment of tandem repeat markers for biogeographical analyses of the corn smut fungus,Ustilago maydis

It has been postulated that the fungus Ustilago maydis followed its host maize around the world. In order to understand the biogeography of this fungus relative to its host, we used a bioinformatics approach to develop 86 tandem repeat markers useful for population studies of U. maydis. We characterized repeat motifs between two and 155 nucleotides using 36 isolates from USA, Mexico and South America. Our data suggested that (i) repeat motif length does not predict the number of alleles for that locus, and (ii) that the more polymorphic markers can resolve differences in a small geographical area.     

Septins from the phytopathogenic fungus Ustilago maydis are required for proper morphogenesis but dispensable for virulence

Background

Septins are a highly conserved family of GTP-binding proteins involved in multiple cellular functions, including cell division and morphogenesis. Studies of septins in fungal cells underpin a clear correlation between septin-based structures and fungal morphology, providing clues to understand the molecular frame behind the varied morphologies found in fungal world.

Methodology/Principal Findings

Ustilago maydis genome has the ability to encode four septins. Here, using loss-of-function as well as GFP-tagged alleles of these septin genes, we investigated the roles of septins in the morphogenesis of this basidiomycete fungus. We described that septins in U. maydis could assemble into at least three different structures coexisting in the same cell: bud neck collars, band-like structures at the growing tip, and long septin fibers that run from pole to pole near the cell cortex. We also found that in the absence of septins, U. maydis cells lost their elongated shape, became wider at the central region and ended up losing their polarity, pointing to an important role of septins in the morphogenesis of this fungus. These morphological defects were alleviated in the presence of an osmotic stabilizer suggesting that absence of septins affected the proper formation of the cell wall, which was coherent with a higher sensitivity of septin defective cells to drugs that affect cell wall construction as well as exocytosis. As U. maydis is a phytopathogen, we analyzed the role of septins in virulence and found that in spite of the described morphological defects, septin mutants were virulent in corn plants.

Conclusions/Significance

Our results indicated a major role of septins in morphogenesis in U. maydis. However, in contrast to studies in other fungal pathogens, in which septins were reported to be necessary during the infection process, we found a minor role of septins during corn infection by U. maydis.     

MEK/ERK inhibitor U0126 enhanced salt stress-induced programmed cell death in <Emphasis Type="Italic">Thellungiella halophila</Emphasis> suspension-cultured cells

Programmed cell death (PCD) is an active cellular suicide that occurs both in animals and plants throughout development and in response to abiotic or biotic stress. In contrast to plant hypersensitive response-like cell death, little is known about the molecular machinery that regulates the halophyte plant PCD under high salinity stress. Since mitogen-activated protein kinases (MAPKs) are involved in plant response/tolerance to salt stress, and plant MAPK genes belong to the extracellular signal-regulated kinase (ERK) subfamily, we have investigated the role of ERK-like enzymes in high salinity stress-induced cell death in Thellungiella halophila. The data showed that ERK-like enzymes were early (10 min) and transiently activated under 300 mM NaCl stress. Pretreatment with 10 μM U0126, a special MEK/ERK inhibitor, resulted in a small but statistically significant increase of the percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive nuclei in contrast to salt alone. The effects of U0126 on H₂O₂ production and cytochrome c (cyt c) release were also investigated. We found that the pretreatment with U0126 accelerated H₂O₂ production as well as cyt c release, and eventually enhanced cell death. The results suggest that ERK-like enzymes in Thellungiella halophila may act as a positive regulator of salt tolerance, as illustrated by pretreatment with U0126 which enhanced cell death under high salinity stress.     

MicroRNA‐183‐5p is stress‐inducible and protects neurons against cell death in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons. A fundamental pathogenesis of ALS is the prolonged cell stress in neurons, which is caused by either accumulation of protein aggregates or reactive oxygen species. However, the mechanistic link between stress sensing and cell death is unsettled. Here, we identify that miR‐183‐5p, a neuron‐enriched miRNA, couples stress sensing and cell death programming in ALS. miR‐183‐5p is immediately induced by hydrogen peroxide, tunicamycin or TNF‐α in neurons. The overexpression of miR‐183‐5p increases neuron survival under stress conditions, whereas its knockdown causes neuron death. miR‐183‐5p coordinates apoptosis and necroptosis pathways by directly targeting PDCD4 and RIPK3, and thus protects neurons against cell death under stress conditions. The consistent reduction of miR‐183‐5p in ALS patients and mouse models enhances the notion that miR‐183‐5p is a central regulator of motor neuron survival under stress conditions. Our study supplements current understanding of the mechanistic link between cell stress and death/survival, and provides novel targets for clinical interventions of ALS.     

Ustilago maydis Produces Cytokinins and Abscisic Acid for Potential Regulation of Tumor Formation in Maize

The infection of maize (Zea mays) by the basidiomycete fungus Ustilago maydis leads to common smut of corn characterized by the production of tumors in susceptible aboveground plant tissues. LC-(ES)MS/MS profiles of abscisic acid (ABA) and 12 different cytokinins (CKs) were determined for infected and uninfected maize tissues over a time course following fungal exposure. Samples were taken at points corresponding to the appearance of disease symptoms. Axenic cultures of haploid and dikaryon forms of U. maydis were also profiled. This study confirmed the capability of Ustilago maydis to synthesize CKs, ABA, and auxin (IAA). It also provided evidence for the involvement of CK and ABA in the U. maydis-maize infection process. Significant quantities of CKs and ABA were detected from axenic cultures of U. maydis as was IAA. CKs and ABA levels were elevated in leaves and stems of maize after infection; notable was the high level of cis-zeatin 9-riboside. Variation among hormone profiles of maize tissues was observed at different time points during infection and between infections with nonpathogenic haploid and pathogenic dikaryon strains. This suggested that CKs and ABA accumulate and are likely metabolized in maize tissue infected with U. maydis. Because U. maydis produced these phytohormones at significant levels, it is possible that the fungal pathogen is a source of these compounds in infected tissue. This is the first study to confirm the production of CKs and document the production of ABA by U. maydis. This study also established an involvement of these phytohormones and a possible functional role for ABA in U. maydis infection of maize.