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Protein- O-mannosyltransferases (Pmt proteins) catalyse the addition of mannose to serine or threonine residues of secretory proteins. This modification was described first for yeast and later for other fungi, mammals, insects and recently also for bacteria. O-mannosylation depends on specific isoforms of the three Pmt1, 2 and 4 subfamilies. In fungi, O-mannosylation determines the structure and integrity of cell walls, as well as cellular differentiation and virulence. O-mannosylation of specific secretory proteins of the human fungal pathogen Candida albicans and of the bacterial pathogen Mycobacterium tuberculosis contributes significantly to virulence. In mammals and insects, Pmt proteins are essential for cellular differentiation and development, while lack of Pmt activity causes Walker-Warburg syndrome (muscular dystrophy) in humans. The susceptibility of human cells to certain viruses may also depend on O-mannosyl chains. This review focuses on the various roles of Pmt proteins in cellular differentiation, development and virulence. [ABSTRACT FROM AUTHOR]

The association of Cryptococcus gattii with Eucalyptus trees has been well established. Here we report the isolation of both C. gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India. We investigated a total of 233 samples of Eucalyptus trees: 120 flowers, 81 fragments of bark, and 32 leaves. C. gattii was isolated from two samples of flowers of Eucalyptus terreticornis. C . neoformans var . grubii was recovered twice from the bark of Eucalyptus camaldulensis , initially from one of three samples, and again 2 months later, from one of four samples collected beneath the canopy of the tree. The primary isolation medium was Nigerseed agar, and brown colonies were presumptively identified as C. gattii or C . neoformans . The species identification was confirmed by morphological and biochemical characteristics. Using the Crypto–Check kit (Iatron, Tokyo, Japan), the first two isolates were identified as serotype B ( C . gattii ) and the other two were serotype A ( C . neoformans var. grubii ). PCR analysis of the isolates of C . neoformans var. grubii revealed that they possessed the MATα mating type allele. Molecular typing by amplified fragment length polymorphism markers indicated that both isolates of C . neoformans var. grubii possessed the same genotype. This study demonstrates that C . neoformans var. grubii , as well as C . gattii , may be associated with Eucalyptus trees. [ABSTRACT FROM AUTHOR]

Improved agricultural and industrial production organisms are required to meet the future global food demands and minimize the effects of climate change. A new resource for crop and microbe improvement, designated FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal PCR), provides ultrafast identification and isolation of predetermined, targeted genetic variants in a screening cycle of less than 10 days. Using large-scale sample pooling in combination with droplet digital PCR (ddPCR) greatly increases the size of low-mutation density and screenable variant libraries and the probability of identifying the variant of interest. The method is validated by screening variant libraries totaling 500,000 barley ( Hordeum vulgare ) individuals and isolating more than 125 targeted barley gene knockout lines and miRNA or promoter variants enabling functional gene analysis. FIND-IT variants are directly applicable to elite breeding pipelines and minimize time-consuming technical steps to accelerate the evolution of germplasm.

Extensive 5' untranslated regions (UTR) are a hallmark of transcripts determining hyphal morphogenesis in Candida albicans The major transcripts of the EFG1 gene, which are responsible for cellular morphogenesis and metabolism, contain a 5' UTR of up to 1,170 nucleotides (nt). Deletion analyses of the 5' UTR revealed a 218-nt sequence that is required for production of the Efg1 protein and its functions in filamentation, without lowering the level and integrity of the EFG1 transcript. Polysomal analyses revealed that the 218-nt 5' UTR sequence is required for efficient translation of the Efg1 protein. Replacement of the EFG1 open reading frame (ORF) by the heterologous reporter gene CaCBGluc confirmed the positive regulatory importance of the identified 5' UTR sequence. In contrast to other reported transcripts containing extensive 5' UTR sequences, these results indicate the positive translational function of the 5' UTR sequence in the EFG1 transcript, which is observed in the context of the native EFG1 promoter. It is proposed that the 5' UTR recruits regulatory factors, possibly during emergence of the native transcript, which aid in translation of the EFG1 transcript. IMPORTANCE Many of the virulence traits that make Candida albicans an important human fungal pathogen are regulated on a transcriptional level. Here, we report an important regulatory contribution of translation, which is exerted by the extensive 5' untranslated regulatory sequence (5' UTR) of the transcript for the protein Efg1, which determines growth, metabolism, and filamentation in the fungus. The presence of the 5' UTR is required for efficient translation of Efg1, to promote filamentation. Because transcripts for many relevant regulators contain extensive 5' UTR sequences, it appears that the virulence of C. albicans depends on the combination of transcriptional and translational regulatory mechanisms., (Copyright © 2018 Desai et al.)

Proteotoxic stress may occur upon exposure of yeast cells to different stress conditions. The induction of stress response mechanisms is important for cells to adapt to changes in the environment and ensure survival. For example, during exposure to elevated temperatures the expression of heat shock proteins such as Hsp104 is induced in yeast. Hsp104 extracts misfolded proteins from aggregates to promote their refolding. We used an Hsp104-GFP reporter to analyze the stress profiles of Saccharomyces species hybrids. To this end a haploid S. cerevisiae strain, harboring a chromosomal HSP104-GFP under control of its endogenous promoter, was mated with stable haploids of S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. uvarum. Stress response behaviors in these hybrids were followed over time by monitoring the appearance and dissolution of Hsp104-GFP foci upon heat shock. General stress tolerance of these hybrids was related to the growth rate detected during exposure to e.g. ethanol and oxidizing agents. We observed that hybrids were generally more resistant to high temperature and ethanol stress compared to their parental strains. Amongst the hybrids differential responses regarding the appearance of Hsp104-foci and the time required for dissolving these aggregates were observed. The S. cerevisiae/S. paradoxus hybrid, combining the two most closely related strains, performed best under these conditions., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Sporulation in Ashbya gossypii is induced by nutrient-limited conditions and leads to the formation of haploid spores. Using RNA-seq, we have determined a gene set induced upon sporulation, which bears considerable overlap with that of Saccharomyces cerevisiae but also contains A. gossypii-specific genes. Addition of cyclic AMP (cAMP) to nutrient-limited media blocks sporulation and represses the induction of sporulation specific genes. Deletion of the protein kinase A (PKA) catalytic subunits encoded by TPK1 and TPK2 showed reduced growth in tpk1 but enhanced growth in the tpk2 strain; however, both mutants sporulated well. Sporulation can be blocked by cAMP in tpk1 but not in tpk2 strains. Similarly, TPK2 acts at a second developmental switch promoting the break in spore dormancy. In S. cerevisiae, PKA phosphorylates and inhibits Msn2/4. The transcript profiles of the tpk1 and msn2/4 mutants were very similar to that of the wild type under sporulation conditions. However, deletion of the single A. gossypii MSN2/4 homolog generated a specific sporulation defect. We identified a set of genes involved in spore wall assembly that was downregulated in the msn2/4 mutant, particularly DIT2, suggesting that poor spore viability may be due to lysis of spores. Our results reveal specific functional differences between the two catalytic PKA subunits in A. gossypii and identified Tpk2 as the key A kinase that transduces developmental decisions of growth. Our data also suggest that Msn2/4 is involved only at a late step of sporulation in A. gossypii and is not a major regulator of IME1., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Efg1p is a key transcriptional regulator in Candida albicans which controls various aspects of morphogenesis and metabolism in this organism. Efg1p contains a central basic helix-loop-helix (bHLH) domain, flanked by sequences highly conserved in fungal APSES proteins, as well as polyglutamine stretches at the N- and C-terminal ends. A systematic deletion approach to specify functional domains of Efg1p revealed that the APSES domain is essential for morphogenesis of the normal yeast and true hyphal cell forms and that bHLH flanking sequences are needed for Efg1p stability. Additional C-terminal sequences were required for hyphal formation on some inducing media, and most Efg1p sequences were needed for chlamydospore morphogenesis. Overexpression of EFG1 led to pseudohypha formation only if a functional APSES domain was present, while a switch from the opaque to the white cell type in addition depended on the presence of certain N- and C-terminal segments. Yeast two-hybrid experiments revealed that binding of Efg1p to its antagonist Czf1p required two regions outside of the APSES domain, which did not coincide with Efg1p sequences needed for its transcriptional repressor activity. Binding of the Flo8 transcription factor to Efg1p did not require the APSES domain but appeared to occur at two or more redundant domains. In contrast, DNA binding of Efg1p to an MluI cell cycle box (MCB) element solely required the APSES domain. Overall, these results suggest that functional domains of Efg1p are spread throughout most of its sequences, including the central APSES domain involved in DNA binding, as well as flanking regions required for various protein interactions and regulatory activities.

Cryptococcus neoformans is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state. C. neoformans occurs in four predominant serotypes (A, B, C, and D), which represent different varieties or species. Rare clinical and environmental isolates with an unusual AD serotype have been reported and suggested to be diploid. We found by fluorescence-activated cell sorter analysis that serotype AD strains are aneuploid or diploid. PCR analysis with primers specific for serotype A or D alleles of the CNA1, CLA4, and GPA1 genes revealed that both alleles are often present in serotype AD strains. PCR analysis with primers specific for genes in the MATa or MATalpha mating-type loci revealed that serotype AD strains are heterozygous for the mating-type locus. Interestingly, in several serotype AD strains, the MATalpha locus was derived from the serotype D parent and the MATa locus was inherited from a serotype A parent that has been thought to be extinct. Basidiospores from a self-fertile serotype AD strain bearing the putative serotype A MATa locus showed a very low viability ( approximately 5%), and no fertile serotype A MATa strain could be recovered. Serotype AD strains were virulent in a murine model. Hybrid AD strains could readily be isolated following a laboratory cross between a serotype A strain and a serotype D strain. In summary, serotype AD strains of C. neoformans are unusual aneuploid or diploid strains that result from matings between serotype A and D strains. Self-fertile isolates fail to undergo normal meiosis because of genetic divergence. Our findings further suggest that serotype A MATa strains may exist in nature.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving mating between haploid MATa and MATalpha cells. Here we describe the isolation of part of the MATa mating-type locus encoding a Ste20 kinase homolog, Ste20a. We show that the STE20a gene cosegregates with the MATa mating type in genetic crosses, maps within the mating-type locus on a 1.8-Mb chromosome, and is allelic with the MATalpha locus. We identify the first MATa isolate of the most common pathogenic variety of C. neoformans (serotype A, variety grubii) which had been thought to be extinct. This serotype A MATa strain is sterile, fails to produce mating pheromone, and is less virulent than a serotype A MATalpha strain in an animal model. Our studies illustrate an association of mating type with virulence and suggest that, like Candida albicans, pathogenic isolates of C. neoformans may be largely asexual.

Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.

Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATalpha and MATa strains. At 37 degrees C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATalpha and MATa loci. At 24 degrees C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii., (Copyright 2000 Academic Press.)

Using arbitrary primers for a PCR reaction on cDNA from different mating interactions, genes differentially expressed in a mating-type dependent manner have been identified. The cDNAs were prepared from mRNA extracted from liquid grown cultures of the fungus in order to prevent further steps in development and increase the possibility of identifying genes directly under control of the mating type system. A reaction using a monokaryotic strain was compared to a semicompatible mating reaction induced for B-regulated development and a reaction using a fully compatible mating. In the comparison of silver-stained, non-denaturing polyacrylamide gel separation of the PCR products bands differentially detected between the samples were cloned and subjected to further analysis. Bands were characterized which were present in both mating reactions but absent in the monokaryon while the third band was specifically absent in the fully compatible mating interaction. Sequence analysis allowed design of specific primers for verification of differential expression in a semi-quantitative PCR. Thus, use of radioactivity was not necessary for the fast and reproducible method identifying genes specifically regulated by mating reactions.

A gene that exhibits sexually induced transcript accumulation, mtd1, was identified physically linked to a locus that is also under control of the mating-type genes. Genomic and cDNA sequence analyses were performed to determine a large open reading frame which starts with the amino-acid sequence M-A-T-E-D. The ORF showed similarities to a novel family of peptide transporters with at least 12 putative transmembrane structures. A gene disruption resulted in a growth-phenotype with slow aerial mycelium formation that could be complemented with peptone or uracil added to the growth medium.

To identify genes regulated by the B mating-type genes a differential RNA display was established for the homobasidiomycete Schizophyllum commune and a gene, brt1, was identified. The mRNA concerned is highly abundant in monokaryotic mycelia and is down-regulated in matings in which either the B genes alone or else both the A and B genes are different between the mates. This places the gene brt1 under the control of the pheromone response system of S. commune encoded by the B mating-type loci. Sequence analysis revealed similarity to a novel protein family, two members of which have been shown to inhibit translational re-initiation.