ras2 Controls morphogenesis,pheromone response,and pathogenicity in the fungal pathogen Ustilago maydis

Ustilago maydis, a pathogen of maize, is a useful model for the analysis of mating, pathogenicity, and the morphological transition between budding and filamentous growth in fungi. As in other fungi, these processes are regulated by conserved signaling mechanisms, including the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and at least one mitogen-activated protein kinase (MAP kinase) pathway. A current challenge is to identify additional factors that lie downstream of the cAMP pathway and that influence morphogenesis in U. maydis. In this study, we identified suppressor mutations that restored budding growth to a constitutively filamentous mutant with a defect in the gene encoding a catalytic subunit of PKA. Complementation of one suppressor mutation unexpectedly identified the ras2 gene, which is predicted to encode a member of the well-conserved ras family of small GTP-binding proteins. Deletion of the ras2 gene in haploid cells altered cell morphology, eliminated pathogenicity on maize seedlings, and revealed a role in the production of aerial hyphae during mating. We also used an activated ras2 allele to demonstrate that Ras2 promotes pseudohyphal growth via a MAP kinase cascade involving the MAP kinase kinase Fuz7 and the MAP kinase Ubc3. Overall, our results reveal an additional level of crosstalk between the cAMP signaling pathway and a MAP kinase pathway influenced by Ras2.     

Pathocycles: Ustilago maydis as a model to study the relationships between cell cycle and virulence in pathogenic fungi

Activation of virulence in pathogenic fungi often involves differentiation processes that need the reset of the cell cycle and induction of a new morphogenetic program. Therefore, the fungal capability to modify its cell cycle constitutes an important determinant in carrying out a successful infection. The dimorphic fungus Ustilago maydis is the causative agent of corn smut disease and has lately become a highly attractive model in addressing fundamental questions about development in pathogenic fungi. The different morphological and genetic changes of U. maydis cells during the pathogenic process advocate an accurate control of the cell cycle in these transitions. This is why this model pathogen deserves attention as a powerful tool in analyzing the relationships between cell cycle, morphogenesis, and pathogenicity. The aim of this review is to summarize recent advances in the unveiling of cell cycle regulation in U. maydis. We also discuss the connection between cell cycle and virulence and how cell cycle control is an important downstream target in the fungus-plant interaction.     

Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

Background  

Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.     

An adaptor protein BmSte50 interacts with BmSte11 MAPKKK and is involved in host infection,conidiation, melanization,and sexual development in Bipolaris maydis

A mitogen-activated protein kinase (MAPK) signaling pathway regulates specialized cellular responses to external stimuli. In Bipolaris maydis, a Chk1 MAPK orthologous to Fus3/Kss1 MAPKs of Saccharomyces cerevisiae is known to regulate various developmental processes, including the formation of appressoria. However, upstream factors that regulate the Chk1 cascade have not been well clarified. In this study, we identified and characterized the BmSte50 gene, an ortholog of the yeast Ste50 in B. maydis. Our yeast two-hybrid assay indicated that BmSte50 interacts with a MAPK kinase kinase BmSte11, a component of the Chk1 cascade. ΔBmSte50 strains exhibited a loss of pathogenicity due to a lack of appressorial formation. The mutants also showed a reduction in melanization, conidial production, and aerial-mycelial and sexual development. Such phenotypes of the mutants were consistent with those of the Chk1 cascade gene mutants previously reported. In addition, ΔBmSte50 strains indicated lower conidial germination efficiency than the wild type. Notably, a significant number of ΔBmSte50 conidia could be germinated, while the Chk1 cascade gene mutants were reported to lack conidial germination ability. Our results suggested that BmSte50 may act as an adaptor protein for the Chk1 cascade and is involved in the regulation of various cellular processes.     

Introducing fluorescence resonance energy transfer‐based biosensors for the analysis of cAMP‐PKA signalling in the fungal pathogen Candida glabrata

The cyclic adenosine monophosphate‐protein kinase A (cAMP‐PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP‐PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP‐PKA activity in this pathogen, we here present the usage of two FRET‐based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time‐resolved manner, as we exemplify by glucose‐induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the cAMP‐PKA signalling cascade to downstream processes, such as virulence, possibly in a host environment.     

A Ras1-Cdc24 signal transduction pathway mediates thermotolerance in the fungal pathogen Cryptococcus neoformans

Pathogenic microorganisms must precisely regulate morphogenesis to survive and proliferate within an infected host. This regulation is often controlled by conserved signal transduction pathways that direct morphological changes in varied species. One such pathway, whose components include Ras proteins and the PAK kinase Ste20, allows the human fungal pathogen Cryptococcus neoformans to grow at high temperature. Previously, we found that Ras1 signalling is required for differentiation, thermotolerance and pathogenesis in C. neoformans. We show here that the guanine nucleotide exchange factor Cdc24 is a Ras1 effector in C. neoformans to mediate the ability of this fungus to grow at high temperature and to cause disease. In addition, we provide evidence that the Ras1-Cdc24 signalling cascade functions specifically through one of the three Cdc42/Rac1 homologues in C. neoformans. In conclusion, our studies illustrate how components of conserved signalling cascades can be specialized for different downstream functions, such as pathogenesis.     

Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts

Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65(Pak)). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho.     

The Colletotrichum graminicola striatin orthologue Str1 is necessary for anastomosis and is a virulence factor

Striatin family proteins are key regulators in signalling pathways in fungi and animals. These scaffold proteins contain four conserved domains: a caveolin‐binding domain, a coiled‐coil motif and a calmodulin‐binding domain at the N‐terminus, and a WD‐repeat domain at the C‐terminus. Fungal striatin orthologues are associated with sexual development, hyphal growth and plant pathogenesis. In Fusarium verticillioides, the striatin orthologue Fsr1 promotes virulence in the maize stalk. The relationship between fungal striatins and pathogenicity remains largely unexplored. In this study, we demonstrate that the Colletotrichum graminicola striatin orthologue Str1 is required for full stalk rot and leaf blight virulence in maize. Pathogenicity assays show that the striatin mutant strain (Δstr1) produces functional appressoria, but infection and colonization are attenuated. Additional phenotypes of the Δstr1 mutant include reduced radial growth and compromised hyphal fusion. In comparison with the wild‐type, Δstr1 also shows a defect in sexual development and produces fewer and shorter conidia. Together with the fact that F. verticillioides fsr1 can complement Δstr1, our results indicate that C. graminicola Str1 shares five phenotypes with striatin orthologues in other fungal species: hyphal growth, hyphal fusion, conidiation, sexual development and virulence. We propose that fungal striatins, like mammalian striatins, act as scaffolding molecules that cross‐link multiple signal transduction pathways.     

Interaction of the heterotrimeric G protein alpha subunit SSG-1 of <Emphasis Type="Italic">Sporothrix schenckii</Emphasis> with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay

Background  

Important biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins.     

A translocated protein of Bartonella henselae interferes with endocytic uptake of individual bacteria and triggers uptake of large bacterial aggregates via the invasome

Bartonella henselae enters human endothelial cells (ECs) by two alternative routes: either by endocytosis, giving rise to Bartonella‐containing vacuoles or by invasome‐mediated internalization. Only the latter process depends on the type IV secretion system VirB/VirD4 and involves the formation of cell surface‐associated bacterial aggregates, which get engulfed by EC membranes in an F‐actin‐dependent manner, eventually resulting in their complete internalization. Here, we report that among the VirB/VirD4‐translocated effector proteins BepA‐BepG only BepG is required for triggering invasome‐mediated internalization. Expression of BepG in the Bep‐deficient ΔbepA–G mutant restored invasome‐mediated internalization. Likewise, ectopic expression of BepG in ECs also restored invasome‐mediated internalization of the ΔbepA–G mutant, while no discernable cytoskeletal rearrangements were triggered in uninfected cells. Rather, BepG inhibited endocytic uptake of B. henselae into Bartonella‐containing vacuoles and other endocytic processes, that is, invasin‐mediated uptake of Yersinia enterocolitica and uptake of inert microspheres. BepG thus triggers invasome‐mediated internalization primarily by inhibiting bacterial endocytosis. Bacteria accumulating on the cell surface then induce locally the F‐actin rearrangements characteristic for the invasome. These cytoskeletal changes encompass both the rearrangement of pre‐existing F‐actin fibres and the de novo polymerization of cortical F‐actin in the periphery of the invasome by Rac1/Scar1/WAVE‐ and Cdc42/WASP‐dependent pathways that involve the recruitment of the Arp2/3 complex.     

The two‐component histidine kinase Fhk1 controls stress adaptation and virulence of Fusarium oxysporum

Fungal histidine kinases (HKs) have been implicated in different processes, such as the osmostress response, hyphal development, sensitivity to fungicides and virulence. Members of HK class III are known to signal through the HOG mitogen‐activated protein kinase (MAPK), but possible interactions with other MAPKs have not been explored. In this study, we have characterized fhk1, encoding a putative class III HK from the soil‐borne vascular wilt pathogen Fusarium oxysporum. Inactivation of fhk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides, as well as increased sensitivity to hyperosmotic stress and menadione‐induced oxidative stress. The osmosensitivity of Δfhk1 mutants was associated with a striking and previously unreported change in colony morphology. The Δfhk1 strains showed a significant decrease in virulence on tomato plants. Epistatic analysis between Fhk1 and the Fmk1 MAPK cascade indicated that Fhk1 does not function upstream of Fmk1, but that the two pathways may interact to control the response to menadione‐induced oxidative stress.     

Scaffold protein GhMORG1 enhances the resistance of cotton to Fusarium oxysporum by facilitating the MKK6‐MPK4 cascade

In eukaryotes, MAPK scaffold proteins are crucial for regulating the function of MAPK cascades. However, only a few MAPK scaffold proteins have been reported in plants, and the molecular mechanism through which scaffold proteins regulate the function of the MAPK cascade remains poorly understood. Here, we identified GhMORG1, a GhMKK6‐GhMPK4 cascade scaffold protein that positively regulates the resistance of cotton to Fusarium oxysporum. GhMORG1 interacted with GhMKK6 and GhMPK4, and the overexpression of GhMORG1 in cotton protoplasts dramatically increased the activity of the GhMKK6‐GhMPK4 cascade. Quantitative phosphoproteomics was used to clarify the mechanism of GhMORG1 in regulating disease resistance, and thirty‐two proteins were considered as the putative substrates of the GhMORG1‐dependent GhMKK6‐GhMPK4 cascade. These putative substrates were involved in multiple disease resistance processes, such as cellular amino acid metabolic processes, calcium ion binding and RNA binding. The kinase assays verified that most of the putative substrates were phosphorylated by the GhMKK6‐GhMPK4 cascade. For functional analysis, nine putative substrates were silenced in cotton, respectively. The resistance of cotton to F. oxysporum was decreased in the substrate‐silenced cottons. These results suggest that GhMORG1 regulates several different disease resistance processes by facilitating the phosphorylation of GhMKK6‐GhMPK4 cascade substrates. Taken together, these findings reveal a new plant MAPK scaffold protein and provide insights into the mechanism of plant resistance to pathogens.     

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.     

The pep4 gene encoding proteinase A is involved in dimorphism and pathogenesis of Ustilago maydis

Vacuole proteases have important functions in different physiological processes in fungi. Taking this aspect into consideration, and as a continuation of our studies on the analysis of the proteolytic system of Ustilago maydis, a phytopathogenic member of the Basidiomycota, we have analysed the role of the pep4 gene encoding the vacuolar acid proteinase PrA in the pathogenesis and morphogenesis of the fungus. After confirmation of the location of the protease in the vacuole using fluorescent probes, we obtained deletion mutants of the gene in sexually compatible strains of U. maydis (FB1 and FB2), and analysed their phenotypes. It was observed that the yeast to mycelium dimorphic transition induced by a pH change in the medium, or the use of a fatty acid as sole carbon source, was severely reduced in Δpep4 mutants. In addition, the virulence of the mutants in maize seedlings was reduced, as revealed by the lower proportion of plants infected and the reduction in size of the tumours induced by the pathogen, when compared with wild‐type strains. All of these phenotypic alterations were reversed by complementation of the mutant strains with the wild‐type gene. These results provide evidence of the importance of the pep4 gene for the morphogenesis and virulence of U. maydis.