Conservation of the Pal/Rim Pathway in Ustilaginomycetes.

The ability of fungi to effectively sense and internalize signals related to extracellular changing environments is essential for survival. This adaptability is particularly important for fungal pathogens of humans and plants that must sense and respond to drastic environmental changes when colonizing their hosts. One of the most important physicochemical factors affecting fungal growth and development is the pH. Ascomycota fungal species possess mechanisms such as the Pal/Rim pathway for external pH sensing and adaptation. However, the conservation of this mechanism in other fungi, such as Ustilaginomycetes is still little studied. To overcome this knowledge gap, we used a comparative genomic approach to explore the conservation of the Pal/Rim pathway in the 13 best sequenced and annotated Ustilaginomycetes. Our findings reveal that the Rim proteins and the Endosomal Sorting Complex Required for Transport (ESCRT) proteins are conserved in Ustilaginomycetes. They conserve the canonical domains present in Pal/Rim and ESCRT proteins of Ascomycota. This study sheds light on the molecular mechanisms used by these fungi for responding to extracellular stresses such as the pH, and open the door to further experimentations for understanding the molecular bases of the signaling in Ustilaginomycetes.

Fungal volatile organic compounds: mechanisms involved in their sensing and dynamic communication with plants.

Microbial volatile organic compounds (MVOCs) are mixtures of gas-phase hydrophobic carbon-based molecules produced by microorganisms such as bacteria and fungi. They can act as airborne signals sensed by plants being crucial players in triggering signaling cascades influencing their secondary metabolism, development, and growth. The role of fungal volatile organic compounds (FVOCs) from beneficial or detrimental species to influence the physiology and priming effect of plants has been well studied. However, the plants mechanisms to discern between FVOCs from friend or foe remains significantly understudied. Under this outlook, we present an overview of the VOCs produced by plant-associate fungal species, with a particular focus on the challenges faced in VOCs research: i) understanding how plants could perceive FVOCs, ii) investigating the differential responses of plants to VOCs from beneficial or detrimental fungal strains, and finally, iii) exploring practical aspects related to the collection of VOCs and their eco-friendly application in agriculture.

Molecular Mechanisms Involved in the Multicellular Growth of Ustilaginomycetes.

Multicellularity is defined as the developmental process by which unicellular organisms became pluricellular during the evolution of complex organisms on Earth. This process requires the convergence of genetic, ecological, and environmental factors. In fungi, mycelial and pseudomycelium growth, snowflake phenotype (where daughter cells remain attached to their stem cells after mitosis), and fruiting bodies have been described as models of multicellular structures. Ustilaginomycetes are Basidiomycota fungi, many of which are pathogens of economically important plant species. These fungi usually grow unicellularly as yeasts (sporidia), but also as simple multicellular forms, such as pseudomycelium, multicellular clusters, or mycelium during plant infection and under different environmental conditions: Nitrogen starvation, nutrient starvation, acid culture media, or with fatty acids as a carbon source. Even under specific conditions, Ustilago maydis can form basidiocarps or fruiting bodies that are complex multicellular structures. These fungi conserve an important set of genes and molecular mechanisms involved in their multicellular growth. In this review, we will discuss in-depth the signaling pathways, epigenetic regulation, required polyamines, cell wall synthesis/degradation, polarized cell growth, and other cellular-genetic processes involved in the different types of Ustilaginomycetes multicellular growth. Finally, considering their short life cycle, easy handling in the laboratory and great morphological plasticity, Ustilaginomycetes can be considered as model organisms for studying fungal multicellularity.

Cell wall glucans of fungi. A review.

Glucans are the most abundant polysaccharides in the cell walls of fungi, and their structures are highly variable. Accordingly, their glucose moieties may be joined through either or both alpha (α) or beta (ß) linkages, they are either lineal or branched, and amorphous or microfibrillar. Alpha 1,3 glucans sensu strictu (pseudonigerans) are the most abundant alpha glucans present in the cell walls of fungi, being restricted to dikarya. They exist in the form of structural microfibrils that provide resistance to the cell wall. The structure of beta glucans is more complex. They are linear or branched, and contain mostly ß 1,3 and ß 1,6 linkages, existing in the form of microfibrils. Together with chitin they constitute the most important structural components of fungal cell walls. They are the most abundant components of the cell walls in members of all fungal phyla, with the exception of Microsporidia, where they are absent. Taking into consideration the importance of glucans in the structure and physiology of the fungi, in the present review we describe the following aspects of these polysaccharides: i) types and distribution of fungal glucans, ii) their structure, iii) their roles, iv) the mechanism of synthesis of the most important ones, and v) the phylogentic relationships of the enzymes involved in their synthesis.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

Phylogenetic relationships of the wall-synthesizing enzymes of Basidiomycota confirm the phylogeny of their subphyla.

Basidiomycota is one of the phyla of kingdom Fungi. This phylum contains besides non-pathogenic species and mushrooms, the important plant pathogens, smuts and rusts, and has been recently divided into three subphyla: Ustilaginomycotina, Pucciniomycotina, and Agaricomycotina (James et al. Nature 443:818-822, 2006; Hibbert et al. Mycological Research 111:509-547, 2007). Although the monophyletic origin of Basidiomycota appears practically undisputed, the phylogenetic relationships of the three subphyla have been considered somewhat uncertain (James et al. Nature 443:818-822, 2006). Previously, we described a hypothetical evolutionary scheme of the fungal cell wall (Ruiz-Herrera and Ortiz-Castellanos FEMS Yeast Research 10:225-243, 2010) that coincided with the accepted evolution tree of kingdom fungi (Cavalier-Smith Proceedings of the Royal Society of London B 271:1251-1262, 2004; James et al. Nature 443:818-822, 2006; Hibbert et al. Mycological Research 111:509-547, 2007). Based on the results of that study, we have now made an analysis of the phylogenetic relationships of the enzymes involved in the synthesis of the cell wall polysaccharides in Basidiomycota. According to our data, there is a close relationship of the wall-synthesizing enzymes with the accepted taxonomy of the group, with a few exceptions, noticeably the absence of chitin synthase IIb subclass in Pucciniomycotina, the duplication of chitin synthase class III in the same group, and the duplication of the gene encoding ß-1,3-glucan synthase (Gls) in Agaricomycotina. These results give some clues on the evolution of the cell wall in Basidiomycota.

A molecular probe for Basidiomycota: the spermidine synthase-saccharopine dehydrogenase chimeric gene.

By means of an in silico analysis, we demonstrated that a previously described chimeric gene (Spe-Sdh) encoding spermidine synthase, a key enzyme involved in the synthesis of polyamines, and saccharopine dehydrogenase, an enzyme involved in lysine synthesis in fungi, were present exclusively in members of all Basidiomycota subphyla, but not in any other group of living organisms. We used this feature to design degenerated primers to amplify a specific fragment of the Spe-Sdh gene by PCR, as a tool to unequivocally identify Basidiomycota isolates. The specificity of this procedure was tested using different fungal species. As expected, positive results were obtained only with Basidiomycota species, whereas no amplification was achieved with species belonging to other fungal phyla.

Functional analysis of the pH responsive pathway Pal/Rim in the phytopathogenic basidiomycete Ustilago maydis.

The most important mechanism for fungal response to the environmental pH is the Rim or Pal pathway. Details on its operation are known through the analysis of ascomycete fungi. In this study we analyzed whether this pathway is conserved in a basidiomycete, Ustilago maydis. We could identify only five homologues of the seven known components of the pathway in the U. maydis as well as in other basidiomycete genomes. We determined that only genes encoding Rim20/PalA, Rim13/PalB and Rim23/PalC, that constitute the endosomal membrane complex, and Rim9/PalI of the complex located at the plasma membrane are conserved, but this latter lacked a detectable role in signal transduction. Mutants in this pathway showed a pleiotropic phenotype, but dimorphism and virulence were not affected. Our data reveal that the Rim/Pal pathway is conserved in basidiomycetes, but with notable differences to the ascomycete systems.

Analysis of the phylogenetic relationships and evolution of the cell walls from yeasts and fungi.

The fungal cell wall is a coherent structure formed by microfibrillar polysaccharides and amorphous material made of other polysaccharides and proteins. We performed a phylogenetic analysis of covalent proteins and enzymes that synthesize fungal wall polysaccharides to determine the possible evolution of the wall structure. It is suggested that the components that made up the archaic walls were structural ones, forming a primitive girdle that retained noncovalently bound proteins in the periplasm and allowed cell growth in hypotonic media. The following hypothetical series of events in fungal wall evolution is suggested: (1) Construction of a primitive wall made of chitin and chitosan by division 2 chitin synthases and chitin deacetylases, respectively. (2) Appearance of class II chitin synthase genes (CHS) after separation of Microsporidia. (3) Capture of a gene encoding beta-1,3-glucan synthase from an organism related to Plantae or Chromista by horizontal transfer after separation of Chytridiomycota. (4) Appearance or horizontal capture from Chromista of genes involved in beta-1,6-glucan synthesis after separation of Zygomycota. (5). Appearance of class III CHS genes. (6) After split of Dikarya phyla, appearance in Ascomycota of class I CHS genes and the capacity to synthesize covalently bound wall proteins.

Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication.

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called "zygomycetes," R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.

Analysis of the proteins involved in the structure and synthesis of the cell wall of Ustilago maydis.

A study of the proteins involved in the synthesis and structure of the cell wall of Ustilago maydis was made by in silico analysis of the fungal genome, with reference to supporting experimental evidence. The composition of the cell wall of U. maydis shows similarities with the structural composition of the walls of Ascomycetes, but also shows important differential features. Accordingly, the enzymes involved in the synthesis of the U. maydis wall polysaccharides chitin and beta-1,6 glucans displayed some differential characteristics. The most salient difference in protein composition was the predicted absence of Pir proteins, an important class of proteins present in the Ascomycetes. Other classes of proteins that are covalently-linked to the wall in Ascomycetes, including those bound through disulfide linkages, joined by alkali-labile bonds, and GPI proteins, were predicted to be present in the U. maydis walls. The main characteristic of the exo-cellular, non-covalently-bound proteins was their relative low number, especially for hydrolytic enzymes.

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.

The ambient pH response Rim pathway in Yarrowia lipolytica: identification of YlRIM9 and characterization of its role in dimorphism.

Yarrowia lipolytica is a dimorphic fungus that secretes either an acidic or an alkaline protease depending on the environmental pH. Previous results have indicated that secretion of the alkaline protease is under control of the pH signaling Pal/Rim pathway originally described in Aspergillus nidulans. Several Y. lipolytica mutants defective in some Rim components of this pathway have been previously isolated and the RIM genes characterized. In the present study, Y. lipolytica RIM9 (palI) gene (YlRIM9) was sequenced from a plasmid (AL414126) of the Genolevures project (the DNA sequence data for YlRIM9 gene has been deposited at EMBL with accession number AJ566902). The derived translation product contains 724 amino acids with a predicted signal peptide and four transmembrane domains in its N-terminal region. We demonstrated that mutation in YlRIM9, as well as in other genes encoding members of the Pal/Rim pathway, did not affect the pH-dependent dimorphic transition of Y. lipolytica. A different pathway must exist in this fungus that controls the effect of pH on dimorphism.