Cytosolic pH Controls Fungal MAPK Signaling and Pathogenicity.

Mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes in eukaryotes. In fungal pathogens, conserved MAPK pathways control key virulence functions such as infection-related development, invasive hyphal growth, or cell wall remodeling. Recent findings suggest that ambient pH acts as a key regulator of MAPK-mediated pathogenicity, but the underlying molecular events are unknown. Here, we found that in the fungal pathogen Fusarium oxysporum, pH controls another infection-related process, hyphal chemotropism. Using the ratiometric pH sensor pHluorin we show that fluctuations in cytosolic pH (pHc) induce rapid reprogramming of the three conserved MAPKs in F. oxysporum, and that this response is conserved in the fungal model organism Saccharomyces cerevisiae. Screening of a subset of S. cerevisiae mutants identified the sphingolipid-regulated AGC kinase Ypk1/2 as a key upstream component of pHc-modulated MAPK responses. We further show that acidification of the cytosol in F. oxysporum leads to an increase of the long-chain base (LCB) sphingolipid dihydrosphingosine (dhSph) and that exogenous addition of dhSph activates Mpk1 phosphorylation and chemotropic growth. Our results reveal a pivotal role of pHc in the regulation of MAPK signaling and suggest new ways to target fungal growth and pathogenicity. IMPORTANCE Fungal phytopathogens cause devastating losses in global agriculture. All plant-infecting fungi use conserved MAPK signaling pathways to successfully locate, enter, and colonize their hosts. In addition, many pathogens also manipulate the pH of the host tissue to increase their virulence. Here, we establish a functional link between cytosolic pH (pHc) and MAPK signaling in the control of pathogenicity in the vascular wilt fungal pathogen Fusarium oxysporum. We demonstrate that fluctuations in pHc cause rapid reprogramming of MAPK phosphorylation, which directly impacts key processes required for infection, such as hyphal chemotropism and invasive growth. Targeting pHc homeostasis and MAPK signaling can thus open new ways to combat fungal infection.

Fusarium oxysporum Casein Kinase 1, a Negative Regulator of the Plasma Membrane H+-ATPase Pma1, Is Required for Development and Pathogenicity.

Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pHc) and mitogen-activated protein kinase (MAPK) signaling. In fungi, pHc is mainly controlled by the essential plasma membrane H+-ATPase Pma1. Here we created mutants of F. oxysporum lacking casein kinase 1 (Ck1), a known negative regulator of Pma1. We found that the ck1Δ mutants have constitutively high Pma1 activity and exhibit reduced alkalinization of the surrounding medium as well as decreased hyphal growth and conidiation. Importantly, the ck1Δ mutants exhibit defects in hyphal chemotropism towards plant roots and in pathogenicity on tomato plants. Thus, Ck1 is a key regulator of the development and virulence of F. oxysporum.

Localisation of phosphoinositides in the grass endophyte Epichloë festucae and genetic and functional analysis of key components of their biosynthetic pathway in E. festucae symbiosis and Fusarium oxysporum pathogenesis.

Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signaling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localize the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloë festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homolog of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did enable the detection of PI[3,4,5]P3 in septa and mycelium of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P3 in ΔtepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 and that fungal PTEN homologs are functional lipid phosphatases. The F. oxysporum chemotropism defect suggests a conserved role of PTEN homologs in chemotaxis across protists, fungi and mammals.

In Vivo Monitoring of Cytosolic pH Using the Ratiometric pH Sensor pHluorin.

Cytosolic pH (pHcyt) is a key factor controlling cell fate. The genetically encoded pH-sensor pHluorin has proven highly valuable for studies on pHcyt in many living organisms. pHluorin displays a bimodal excitation spectrum with peaks at 395 nm and 475 nm, which is dependent on pH. Here we describe two different protocols for determining pHcyt in the soil-borne fungal pathogen Fusarium oxysporum, based either on population or single-cell analysis.

Dual-specificity protein phosphatase Msg5 controls cell wall integrity and virulence in Fusarium oxysporum.

Mitogen-activated protein kinase (MAPK) cascades are key signaling modules controlling development and virulence in fungal pathogens. Down-regulation of MAPK activity by protein phosphatases provides a critical layer of control during desensitization or adaptation to stimuli. In Saccharomyces cerevisiae, the dual-specificity phosphatase Msg5 dephosphorylates target threonine and tyrosine residues in the two MAPKs Mpk1 and Fus3, which regulate the cell wall integrity (CWI) and pheromone responses, respectively. Here we studied the role of the Msg5 ortholog in Fusarium oxysporum, a soilborne phytopathogen that infects host plants through the roots to cause vascular wilt and plant death. F. oxysporum mutants lacking Msg5 showed constitutively high levels of Mpk1 phosphorylation and increased sensitivity to the cell wall targeting compound Calcofluor White. Moreover, these mutants displayed reduced colony growth and conidiation. Importantly, msg5Δ mutants were impaired in hyphal chemotropism towards host plant roots and in virulence on tomato plants. These results reveal a key role of Msg5 in regulation of the CWI MAPK cascade of F. oxysporum as well as in infection-related signaling of this important fungal pathogen.

NADPH oxidase regulates chemotropic growth of the fungal pathogen Fusarium oxysporum towards the host plant.

Soil-inhabiting fungal pathogens use chemical signals to locate and colonise the host plant. In the vascular wilt fungus Fusarium oxysporum, hyphal chemotropism towards tomato roots is triggered by secreted plant peroxidases (Prx), which catalyse the reductive cleavage of reactive oxygen species (ROS). Here we show that this chemotropic response requires the regulated synthesis of ROS by the conserved fungal NADPH oxidase B (NoxB) complex, and their transformation into hydrogen peroxide (H2 O2 ) by superoxide dismutase (SOD). Deletion of NoxB or the regulatory subunit NoxR, or pharmacological inhibition of SOD, specifically abolished chemotropism of F. oxysporum towards Prx gradients. Addition of isotropic concentrations of H2 O2 rescued chemotropic growth in the noxBΔ and noxRΔ mutants, but not in a mutant lacking the G protein-coupled receptor Ste2. Prx-triggered rapid Nox- and Ste2-dependent phosphorylation of the cell wall integrity mitogen-activated protein kinase (CWI MAPK) Mpk1, an essential component of the chemotropic response. These results suggest that Ste2 and the CWI MAPK cascade function downstream of NoxB in Prx chemosensing. Our findings reveal a new role for Nox enzymes in directed hyphal growth of a filamentous pathogen towards its host and might be of broad interest for chemotropic interactions between plants and root-colonising fungi.

Contacts in Death: The Role of the ER-Mitochondria Axis in Acetic Acid-Induced Apoptosis in Yeast.

Endoplasmic reticulum-mitochondria contact sites have been a subject of increasing scientific interest since the discovery that these structures are disrupted in several pathologies. Due to the emerging data that correlate endoplasmic reticulum-mitochondria contact sites function with known events of the apoptotic program, we aimed to dissect this interplay using our well-established model of acetic acid-induced apoptosis in Saccharomyces cerevisiae. Until recently, the only known tethering complex between ER and mitochondria in this organism was the ER-mitochondria encounter structure (ERMES). Following our results from a screening designed to identify genes whose deletion rendered cells with an altered sensitivity to acetic acid, we hypothesized that the ERMES complex could be involved in cell death mediated by this stressor. Herein we demonstrate that single ablation of the ERMES components Mdm10p, Mdm12p and Mdm34p increases the resistance of S. cerevisiae to acetic acid-induced apoptosis, which is associated with a prominent delay in the appearance of several apoptotic markers. Moreover, abrogation of Mdm10p or Mdm34p abolished cytochrome c release from mitochondria. Since these two proteins are embedded in the mitochondrial outer membrane, we propose that the ERMES complex plays a part in cytochrome c release, a key event of the apoptotic cascade. In all, these findings will aid in targeted therapies for diseases where apoptosis is disrupted, as well as assist in the development of acetic acid-resistant strains for industrial processes.

Effects of thyroid hormones on aortic tissue after myocardial infarction in rats.

Studies have shown a cardioprotective role of thyroid hormones (THs) in cardiac remodeling after acute myocardial infarction (MI). However, there is no data in the literature examining the influence of TH administration on the aortic tissue in an animal model of MI. This study aimed to evaluate the effects of thyroid hormones on the aorta after MI. Male Wistar rats were divided into a sham group (SHAM), infarcted group (AMI), sham+TH (SHAMT) and AMI+TH (AMIT). After MI, the animals received T3 and T4 (2 and 8µg/100g/day, respectively) by oral gavage for 12 days. Later, the animals underwent echocardiography and euthanasia and the aorta was collected for molecular and biochemical analysis. T3 and T4 administration increased the expression of the pro-angiogenic proteins vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1α (HIF-1α) in the aorta of AMIT rats when compared with AMI. With respect to TH receptors, AMI rats presented a decrease in TRß levels, which was prevented by the hormonal administration. In AMIT rats, both TRα and TRß levels were increased when compared with the AMI group. Reactive oxygen species levels and NADPH oxidase activity were decreased in both treated groups when compared with the non-treated animals. TH administration after MI may improve angiogenic signaling in the aorta as well as the responsiveness of this vessel to T3 and T4. These positive effects in the aorta may result in additional protection for the cardiovascular system in the context of cardiac ischaemic injury.

Thyroid hormones improve cardiac function and decrease expression of pro-apoptotic proteins in the heart of rats 14 days after infarction.

Apoptosis is a key process associated with pathological cardiac remodelling in early-phase post-myocardial infarction. In this context, several studies have demonstrated an anti-apoptotic effect of thyroid hormones (TH). The aim of this study was to evaluate the effects of TH on the expression of proteins associated with the apoptotic process 14 days after infarction. Male Wistar rats (300-350 g) (n = 8/group) were divided into four groups: Sham-operated (SHAM), infarcted (AMI), sham-operated + TH (SHAMT) and infarcted + TH (AMIT). For 12 days, the animals received T3 and T4 [2 and 8 µg/(100 g day)] by gavage. After this, the rats were submitted to haemodynamic and echocardiographic analysis, and then were sacrificed and the heart tissue was collected for molecular analysis. Statistical analyses included two-way ANOVA with Student-Newman-Keuls post test. Ethics Committee number: 23262. TH administration prevented the loss of ventricular wall thickness and improved cardiac function in the infarcted rats 14 days after the injury. AMI rats presented an increase in the pro-apoptotic proteins p53 and JNK. The hormonal treatment prevented this increase in AMIT rats. In addition, TH administration decreased the Bax:Bcl-2 ratio in the infarcted rats. TH administration improved cardiac functional parameters, and decreased the expression of pro-apoptotic proteins 14 days after myocardial infarction.

Catalase influence in the regulation of coronary resistance by estrogen: joint action of nitric oxide and hydrogen peroxide.

We tested the influence of estrogen on coronary resistance regulation by modulating nitric oxide (NO) and hydrogen peroxide (H2O2) levels in female rats. For this, estrogen levels were manipulated and the hearts were immediately excised and perfused at a constant flow using a Langendorff's apparatus. Higher estrogen levels were associated with a lower coronary resistance, increased nitric oxide bioavailability, and higher levels of H2O2. When oxide nitric synthase blockade by L-NAME was performed, no significant changes were found in coronary resistance of ovariectomized rats. Additionally, we found an inverse association between NO levels and catalase activity. Taken together, our data suggest that, in the absence of estrogen influence and, therefore, reduced NO bioavailability, coronary resistance regulation seems to be more dependent on the H2O2 that is maintained at low levels by increased catalase activity.

Genome-wide identification of genes involved in the positive and negative regulation of acetic acid-induced programmed cell death in Saccharomyces cerevisiae.

BACKGROUND: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by Saccharomyces cerevisiae, which it frequently impairs. The more recent finding that acetic acid triggers apoptotic programmed cell death (PCD) in yeast sparked an interest to develop strategies to modulate this process, to improve several biotechnological applications, but also for biomedical research. Indeed, acetate can trigger apoptosis in cancer cells, suggesting its exploitation as an anticancer compound. Therefore, we aimed to identify genes involved in the positive and negative regulation of acetic acid-induced PCD by optimizing a functional analysis of a yeast Euroscarf knock-out mutant collection. RESULTS: The screen consisted of exposing the mutant strains to acetic acid in YPD medium, pH 3.0, in 96-well plates, and subsequently evaluating the presence of culturable cells at different time points. Several functional categories emerged as greatly relevant for modulation of acetic acid-induced PCD (e.g.: mitochondrial function, transcription of glucose-repressed genes, protein synthesis and modifications, and vesicular traffic for protection, or amino acid transport and biosynthesis, oxidative stress response, cell growth and differentiation, protein phosphorylation and histone deacetylation for its execution). Known pro-apoptotic and anti-apoptotic genes were found, validating the approach developed. Metabolism stood out as a main regulator of this process, since impairment of major carbohydrate metabolic pathways conferred resistance to acetic acid-induced PCD. Among these, lipid catabolism arose as one of the most significant new functions identified. The results also showed that many of the cellular and metabolic features that constitute hallmarks of tumour cells (such as higher glycolytic energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer sensitivity to acetic acid-induced PCD, potentially explaining why tumour cells are more susceptible to acetate than untransformed cells and reinforcing the interest in exploiting this acid in cancer therapy. Furthermore, our results clearly establish a connection between cell proliferation and cell death regulation, evidencing a conserved developmental role of programmed cell death in unicellular eukaryotes. CONCLUSIONS: This work advanced the characterization of acetic acid-induced PCD, providing a wealth of new information on putative molecular targets for its control with impact both in biotechnology and biomedicine.