Polarization and cell-fate decision facilitated by the adaptor Ste50 in Saccharomyces cerevisiae

Polarization or directional growth is a major morphological change that occurs in yeast cells during pheromone response to mate with the opposite partner. In the pheromone signaling pathway, the adaptor Ste50 is required to bind MAP3K Ste11 for proper polarization; cells lacking Ste50 are impaired in polarization. Direct involvement of Ste50 in the polarization process has not been explored systematically. Here, we used single-cell fluorescent time-lapse microscopy to characterize Ste50 involvement in the establishment of cell polarity. We found early localization of Ste50 patches on the cell cortex that mark the point of shmoo initiation, these polarity sites move, and patches remain associated with the growing shmoo tip in a pheromone concentration-dependent manner until shmoo maturation. By quantitative analysis we show that polarization corelates with the rising levels of Ste50 enabling rapid individual cell responses to pheromone that corresponds to a critical level of Ste50 at the initial G1 phase. Suggesting Ste50 to be a pheromone responsive gene. We exploited the quantitative differences in the pattern of Ste50 expression to corelate with the cell-cell phenotypic heterogeneity showing Ste50 involvement in the cellular differentiation choices. Taken together, these findings present spatiotemporal localization of Ste50 during yeast polarization, suggesting that Ste50 is a component of the polarisome, and plays a critical role in regulating the polarized growth of shmoo during pheromone response.

Cdc42-Specific GTPase-Activating Protein Rga1 Squelches Crosstalk between the High-Osmolarity Glycerol (HOG) and Mating Pheromone Response MAPK Pathways

Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5′-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of “kinetic proofreading”. Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.

Dynamic Phosphorylation of RGS Provides Spatial Regulation of G-alpha And Promotes Completion of Cytokinesis

Yeast use a G-protein coupled receptor (GPCR) signaling pathway to detect mating pheromone, arrest in G1, and direct polarized growth towards the potential mating partner. The primary negative regulator of this pathway is the regulator of G-protein signaling (RGS), Sst2, which induces Gα GTPase activity and subsequent inactivation of all downstream signaling. MAPK phosphorylates the RGS in response to pheromone, but the role of this modification is unknown. We set out to examine the role of RGS phosphorylation during the pheromone response. We found that phosphorylation of the RGS peaks early in the pheromone response and diminishes RGS localization to the polarization site and focuses Gα/MAPK complexes there. At later time points, RGS is predominantly unphosphorylated, which promotes RGS localization to the polar cap and broadens the distribution of Gα/MAPK complexes relative to the Cdc42 polarity machinery. Surprisingly, we found that phosphorylation of the RGS is required for the completion of cytokinesis prior to pheromone induced growth. The completion of cytokinesis in the presence of pheromone is promoted by the formin Bnr1 and the kelch-repeat protein, Kel1, both proteins previously found to interact with the RGS.

Actin Cytoskeleton Regulation by the Yeast NADPH Oxidase Yno1p Impacts Processes Controlled by MAPK Pathways

Reactive oxygen species (ROS) that exceed the antioxidative capacity of the cell can be harmful and are termed oxidative stress. Increasing evidence suggests that ROS are not exclusively detrimental, but can fulfill important signaling functions. Recently, we have been able to demonstrate that a NADPH oxidase-like enzyme (termed Yno1p) exists in the single-celled organism Saccharomyces cerevisiae. This enzyme resides in the peripheral and perinuclear endoplasmic reticulum and functions in close proximity to the plasma membrane. Its product, hydrogen peroxide, which is also produced by the action of the superoxide dismutase, Sod1p, influences signaling of key regulatory proteins Ras2p and Yck1p/2p. In the present work, we demonstrate that Yno1p-derived H2O2 regulates outputs controlled by three MAP kinase pathways that can share components: the filamentous growth (filamentous growth MAPK (fMAPK)), pheromone response, and osmotic stress response (hyperosmolarity glycerol response, HOG) pathways. A key structural component and regulator in this process is the actin cytoskeleton. The nucleation and stabilization of actin are regulated by Yno1p. Cells lacking YNO1 showed reduced invasive growth, which could be reversed by stimulation of actin nucleation. Additionally, under osmotic stress, the vacuoles of a ∆yno1 strain show an enhanced fragmentation. During pheromone response induced by the addition of alpha-factor, Yno1p is responsible for a burst of ROS. Collectively, these results broaden the roles of ROS to encompass microbial differentiation responses and stress responses controlled by MAPK pathways.

Signal-mediated localization of Candida albicans pheromone response pathway components

Candida albicans opaque cells release pheromones to stimulate cells of opposite mating type to activate their pheromone response pathway. Although this fungal pathogen shares orthologous proteins involved in the process with Saccharomyces cerevisiae, the pathway in each organism has unique characteristics. We have used GFP-tagged fusion proteins to investigate the localization of the scaffold protein Cst5, as well as the MAP kinases Cek1 and Cek2, during pheromone response in C. albicans. In wild-type cells, pheromone treatment directed Cst5-GFP to surface puncta concentrated at the tips of mating projections. These puncta failed to form in cells defective in either the Gα or β subunits. However, they still formed in response to pheromone in cells missing Ste11, but with the puncta distributed around the cell periphery in the absence of mating projections. These puncta were absent from hst7Δ/Δ cells, but could be detected in the ste11Δ/Δ hst7Δ/Δ double mutant. Cek2-GFP showed a strong nuclear localization late in the response, consistent with a role in adaptation, while Cek1-GFP showed a weaker, but early increase in nuclear localization after pheromone treatment. Activation loop phosphorylation of both Cek1 and Cek2 required the presence of Ste11. In contrast to Cek2-GFP, which showed no localization signal in ste11Δ/Δ cells, Cek1-GFP showed enhanced nuclear localization that was pheromone independent in the ste11Δ/Δ mutant. The results are consistent with CaSte11 facilitating Hst7-mediated MAP kinase phosphorylation and also playing a potentially critical role in both MAP kinase and Cst5 scaffold localization.

Prey sensing and response in a nematode-trapping fungus is governed by the MAPK pheromone response pathway

Detection of surrounding organisms in the environment plays a major role in the evolution of interspecies interactions, such as predator–prey relationships. Nematode-trapping fungi (NTF) are predators that develop specialized trap structures to capture, kill, and consume nematodes when food sources are limited. Despite the identification of various factors that induce trap morphogenesis, the mechanisms underlying the differentiation process have remained largely unclear. Here, we demonstrate that the highly conserved pheromone-response MAPK pathway is essential for sensing ascarosides, a conserved molecular signature of nemaotdes, and is required for the predatory lifestyle switch in the NTF Arthrobotrys oligospora. Gene deletion of STE7 (MAPKK) and FUS3 (MAPK) abolished nematode-induced trap morphogenesis and conidiation and impaired the growth of hyphae. The conserved transcription factor Ste12 acting downstream of the pheromone-response pathway also plays a vital role in the predation of A. oligospora. Transcriptional profiling of a ste12 mutant identified a small subset of genes with diverse functions that are Ste12 dependent and could trigger trap differentiation. Our work has revealed that A. oligospora perceives and interprets the ascarosides produced by nematodes via the conserved pheromone signaling pathway in fungi, providing molecular insights into the mechanisms of communication between a fungal predator and its nematode prey.

Sulcatol: Enantiospecific Attractant for Monarthrum mali (Coleoptera: Curculionidae: Scolytinae), Leptostylus asperatus (Coleoptera: Cerambycidae) and Associated Predators

In 2014–2019, we conducted six experiments in north-central Georgia in an attempt to verify the aggregation pheromone response of the ambrosia beetle Gnathotrichus materiarius (Fitch) (Coleoptera: Curculionidae: Scolytinae: Scolytini: Corthylina) to sulcatol known to be produced by male G. materiarius; we failed to catch any G. materiarius. However, we did find that another corthyline ambrosia beetle species Monarthrum mali (Fitch) was attracted to (R)-(–)-sulcatol, whereas the longhorn beetle Leptostylus asperatus (Haldeman) (Coleoptera: Cerambycidae: Lamiinae) was attracted to (S)-(+)-sulcatol. Attraction of both species was unaffected by the respective antipodes. Ethanol enhanced attraction of both species to traps baited with sulcatol. In at least one experiment, attraction to ethanol-baited traps was enhanced by sulcatol for Xylosandrus crassiusculus (Motschulsky), Xyleborus spp., and Hypothenemus spp. but reduced for Cnestus mutilatus (Blandford) (Coleoptera: Curculionidae: Scolytinae). Additionally, traps baited with ethanol and racemic sulcatol [50% (S)-(+): 50% (R)-(-)] caught the greatest numbers of four species of beetle predators: Coptodera aerata Dejean (Coleoptera: Carabidae), Colydium lineola Say (Coleoptera: Zopheridae), Madoniella dislocata (Say), and Pyticeroides laticornis (Say) (Coleoptera: Cleridae). Ethanol but not sulcatol attracted Temnoscheila virescens (F.) (Coleoptera: Trogossitidae). Information on interspecific relationships within forested communities may help us to better determine the roles of these species in maintaining stable and resilient forested ecosystems.

Unfolded protein response and scaffold independent pheromone MAP kinase signalling control Verticillium dahliae growth, development and plant pathogenesis

SummaryDevelopment and virulence of the vascular plant pathogen Verticillium dahliae are connected and depend on a complex interplay between the unfolded protein response, a Ham5 independent pheromone MAP kinase module and formation of precursors for oxylipin signal molecules.Genes coding for the unfolded protein response regulator Hac1, the Ham5 MAPK scaffold protein, and the oleate Δ12-fatty acid desaturase Ode1 were deleted and their functions in growth, differentiation, and virulence on plants were studied using genetic, cell biology, and plant infection experiments.The unfolded protein response transcription factor Hac1 is required for initial root colonization, fungal conidiation and propagation inside the host and is essential for resting structure formation. Microsclerotia development, growth and virulence require the pheromone response MAPK pathway, but without the Ham5 scaffold function. Single ER-associated enzymes for linoleic acid production make important contributions to fungal growth but have only a minor impact on the pathogenicity of V. dahliae.Fungal growth, sporulation, dormant structure formation and plant infection require a network of the Hac1-regulated unfolded protein response, a scaffold-independent pheromone response MAPK pathway and formation of precursors for signalling. This network includes interesting targets for disease management of the vascular pathogen V. dahliae.