Fungal model systems and the elucidation of pathogenicity determinants

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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

Fungal model systems and the elucidation of pathogenicity determinants

This is the final version of the article. Available from Elsevier via the DOI in this record.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.This review was carried out by members of the EU-Initial Training Network Ariadne (PITN-GA-2009-237936), which provided financial support for C.B., S.D., M.E.G., E.G., E.M., P.V.M., M.M., V.N., M.F.A.N., T.O., M.O.R., K.S. and L.W

The General Transcriptional Repressor Tup1 Is Required for Dimorphism and Virulence in a Fungal Plant Pathogen

A critical step in the life cycle of many fungal pathogens is the transition between yeast-like growth and the formation of filamentous structures, a process known as dimorphism. This morphological shift, typically triggered by multiple environmental signals, is tightly controlled by complex genetic pathways to ensure successful pathogenic development. In animal pathogenic fungi, one of the best known regulators of dimorphism is the general transcriptional repressor, Tup1. However, the role of Tup1 in fungal dimorphism is completely unknown in plant pathogens. Here we show that Tup1 plays a key role in orchestrating the yeast to hypha transition in the maize pathogen Ustilago maydis. Deletion of the tup1 gene causes a drastic reduction in the mating and filamentation capacity of the fungus, in turn leading to a reduced virulence phenotype. In U. maydis, these processes are controlled by the a and b mating-type loci, whose expression depends on the Prf1 transcription factor. Interestingly, Δtup1 strains show a critical reduction in the expression of prf1 and that of Prf1 target genes at both loci. Moreover, we observed that Tup1 appears to regulate Prf1 activity by controlling the expression of the prf1 transcriptional activators, rop1 and hap2. Additionally, we describe a putative novel prf1 repressor, named Pac2, which seems to be an important target of Tup1 in the control of dimorphism and virulence. Furthermore, we show that Tup1 is required for full pathogenic development since tup1 deletion mutants are unable to complete the sexual cycle. Our findings establish Tup1 as a key factor coordinating dimorphism in the phytopathogen U. maydis and support a conserved role for Tup1 in the control of hypha-specific genes among animal and plant fungal pathogens

Relationships between the cell cycle and the differentiation programs associated to virulence in the phytopathogen fungus Ustilago maydis

[ES]Ustilago maydis es el agente responsable del carbón del maíz. El desarrollo patógeno en este hongo está íntimamente vinculado a la diferenciación sexual y se acompaña de numerosas transiciones morfológicas asociadas a un control preciso del ciclo celular. Esto hace que este hongo sea un excelente modelo para identificar dianas del ciclo celular que jueguen papeles esenciales en las enfermedades causadas por hongos. Dado que en la mayoría de las situaciones, los fungicidas no son efectivos en el control de la enfermedad si el patógeno ya ha penetrado en los tejidos de la planta, la enfermedad debe impedirse durante las primeras etapas de la infección. U. maydis debe tomar dos decisiones cruciales de desarrollo, que ocurren en la superficie de la planta, antes de penetrar el tejido vegetal. La primera tiene lugar cuando dos células haploides son capaces de reconocerse mediante un sistema feromona-receptor. Las células detienen su ciclo celular, forman cada una un tubo de conjugación que crece a favor de gradiente de feromona hasta aparearse. La segunda decisión es la formación de la hifa dicariótica, que implica tanto una detención específica del ciclo celular en G2 como una activación de un fuerte crecimiento polar (Perez-Martin and Castillo-Lluva, 2008). Por lo tanto, entender los mecanismos moleculares implicados en el apareamiento y en la formación del filamento infectivo es crucial en el diseño de antifúngicos. Nuestro objetivo ha sido dilucidar los mecanismos responsables de estas paradas de ciclo celular con el fin de desacoplar la detención del ciclo del resto de los procesos durante la formación de las hifas infectivas y de este modo averiguar sus consecuencias en el proceso infectiv

Fungal model systems and the elucidation of pathogenicity determinants

Under a Creative Commons license.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. © 2014 The Authors.This review was carried out by members of the EU-Initial Training Network Ariadne (PITN-GA-2009-237936), which provided financial support for C.B., S.D., M.E.G., E.G., E.M., P.V.M., M.M., V.N., M.F.A.N., T.O., M.O.R., K.S. and L.W. Open Access funded by Wellcome TrustPeer Reviewe

The fungus Ustilago maydis, from the aztec cuisine to the research laboratory

Ustilago maydis is a plant pathogen fungus responsible for corn smut. It has a complex life cycle. In its saprophitic stage, it grows as haploid yeast cells, while in the invasive stage it grows as a mycelium formed by diploid cells. Thus, a correlation exists between genetic ploidy, pathogenicity and morphogenesis. Dimorphism can be modulated in vitro by changing environmental parameters such as pH. Studies with auxotrophic mutants have shown that polyamines play a central role in regulating dimorphism. Molecular biology approaches are being employed for the analysis of fundamental aspects of the biology of this fungus, such as mating type regulation, dimorphism or cell wall biogenesis

Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis

Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens

Programmed cell cycle arrest is required for infection of corn plants by the fungus Ustilago maydis

Ustilago maydis is a plant pathogen that requires a specific structure called infective filament to penetrate the plant tissue. Although able to grow, this filament is cell cycle arrested on the plant surface. This cell cycle arrest is released once the filament penetrates the plant tissue.The reasons and mechanisms for this cell cycle arrest are unknown. Here, we have tried to address these questions. We reached three conclusions from our studies. First, the observed cell cycle arrest is the result of the cooperation of at least two distinct mechanisms: one involving the activation of the DNA damage response (DDR) cascade; and the other relying on the transcriptional downregulation of Hsl1, a kinase that modulates the G2/M transition. Second, a sustained cell cycle arrest during the infective filament step is necessary for thevirulence in U. maydis, as a strain unable to arrest the cell cycle was severely impaired in its ability to infect corn plants. Third, production of the appressorium, a structure required for plant penetration, is incompatible with an active cell cycle. The inability to infect plants bystrains defective in cell cycle arrest seems to be caused by their failure to induce the appressorium formation process. In summary, our findings uncover genetic circuits to arrest the cell cycle during the growth of this fungus on the plant surface, thus allowing the penetration into plant tissue.Fil: Castanheira, Sónia. Consejo Superior de Investigaciones Científicas. Instituto de Biología Funcional y Genómica; EspañaFil: Mielnichuk, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. Consejo Superior de Investigaciones Científicas. Instituto de Biología Funcional y Genómica; EspañaFil: Pérez Martín, José. Consejo Superior de Investigaciones Científicas. Instituto de Biología Funcional y Genómica; Españ