Nonthermal Plasma Effects on Fungi: Applications, Fungal Responses, and Future Perspectives.

The kingdom of Fungi is rich in species that live in various environments and exhibit different lifestyles. Many are beneficial and indispensable for the environment and industries, but some can threaten plants, animals, and humans as pathogens. Various strategies have been applied to eliminate fungal pathogens by relying on chemical and nonchemical antifungal agents and tools. Nonthermal plasma (NTP) is a potential tool to inactivate pathogenic and food-contaminating fungi and genetically improve fungal strains used in industry as enzyme and metabolite producers. The NTP mode of action is due to many highly reactive species and their interactions with biological molecules. The interaction of the NTP with living cells is believed to be synergistic yet not well understood. This review aims to summarize the current NTP designs, applications, and challenges that involve fungi, as well as provide brief descriptions of underlying mechanisms employed by fungi in interactions with the NTP components.

Non-thermal plasma induces changes in aflatoxin production, devitalization, and surface chemistry of Aspergillus parasiticus.

Non-thermal plasma (NTP) represents the fourth state of matter composed of neutral molecules, atoms, ions, radicals, and electrons. It has been used by various industries for several decades, but only recently NTPs have emerged in fields such as medicine, agriculture, and the food industry. In this work, we studied the effect of NTP exposure on aflatoxin production, conidial germination and mycelial vitality, morphological and surface changes of conidia and mycelium. When compared with colonies grown from untreated conidia, the colonies from NTP-treated conidia produced significantly higher levels of aflatoxins much earlier during development than colonies from untreated conidia. However, at the end of cultivation, both types of cultures yielded similar aflatoxin concentrations. The increase in the accumulation of aflatoxins was supported by high transcription levels of aflatoxin biosynthetic genes, which indicated a possibility that NTP treatment of conidia was having a longer-lasting effect on colony development and aflatoxins accumulation. NTP generated in the air at atmospheric pressure effectively devitalized Aspergillus parasiticus in conidia and hyphae within a few minutes of treatment. To describe devitalization kinetics, we applied Weibull and Hill models on sets of data collected at different exposure times during NTP treatment. The damage caused by NTP to hyphal cell wall structures was displayed by raptures visualized by scanning electron microscopy. Fourier transform infrared spectroscopy demonstrated that changes in cell envelope correlated with shifts in characteristic chemical bonds indicating dehydration, oxidation of lipids, proteins, and polysaccharides. Key points • Non-thermal plasma increases aflatoxin production shortly after treatment. • Non-thermal plasma rapidly devitalizes Aspergillus parasiticus. • Non-thermal plasma disrupts the cell surface and oxidizes biological components.

The effect of cold atmospheric pressure plasma on Aspergillus ochraceus and ochratoxin A production.

Aspergillus ochraceus is a soil fungus known to produce ochratoxin A, a harmful secondary metabolite. Prevention and control of fungal pathogens mostly rely on chemical fungicides, which is one of the contributing factors in the emergence of the fungal resistance, hence novel methods for fungal eradication have been extensively researched. The cold atmospheric pressure (CAP) plasma generated in ambient air has been recently applied in microbial decontamination. Here we used the diffuse coplanar surface barrier discharge in inactivation of a toxigenic strain A. ochraceus. The plasma-treated conidia and mycelium exhibited morphological changes such as ruptures and desiccation. Mycelium dehydration and changes in the chemical composition of hyphal surface accompanied plasma treatment. The growth of 26 h old mycelia were significantly restricted after 30 s of plasma treatment. The conidial vitality declined 4 logs after 180 s of plasma exposure leading to almost complete decontamination. After shorter plasma treatment of conidia, the ochratoxin A (OTA) production increased at the early stage of cultivation, but the overall level was significantly reduced compared to untreated samples after longer cultivation. Our results indicated that the fungal growth and the OTA production were significantly changed by plasma treatment and underscored CAP plasma as a promising method in the decontamination of A. ochraceus without a risk to generate strains with increased OTA production.

Functional dissection of gamma-aminobutyric acid metabolism in Neurospora crassa.

The GABA shunt is one of the metabolic pathways that is ubiquitous in prokaryotes and eukaryotes. γ-aminobutyric acid (GABA) in fungi is required in the stress responses, virulence and development. The number of genes encoding glutamate decarboxylase (gad), GABA transaminase (gta) and succinic semialdehyde dehydrogenase (ssadh) varies between fungal species. The genome-wide analysis in Neurospora crassa resulted in the identification of a gta and a ssadh. Disruption of either gta or ssadh decreased respiration rate and biomass accumulation, reduced growth on GABA and beta-alanine. The gta and ssadh mutants exhibited aberrant hyphal morphology and displayed differential transcription of the GABA shunt genes. In the gta mutant, protoperithecia and perithecia formation was almost completely suppressed in the presence of GABA and beta-alanine, indicating GTA requirement for the turnover of these amino acids. The strains displayed differential metabolic dysregulations in response to different nitrogen sources. The phenotypic differences between the gta and ssadh mutants could be contributed to accumulation of intermediates of the GABA shunt and/or GABA shunt-independent functions. Together, our data suggest that the GABA shunt could function as a moderate modulator of multiple biological events, including respiration, energy metabolism, carbon and nitrogen metabolism, growth, as well as sexual development in N. crassa.

Technical applications of plasma treatments: current state and perspectives.

Rapidly evolving cold atmospheric pressure plasma (CAPP)-based technology has been actively used not only in bioresearch but also in biotechnology, food safety and processing, agriculture, and medicine. High variability in plasma device configurations and electrode layouts has accelerated non-thermal plasma applications in treatment of various biomaterials and surfaces of all sizes. Mode of cold plasma action is likely associated with synergistic effect of biologically active plasma components, such as UV radiation or reactive species. CAPP has been employed in inactivation of viruses, to combat resistant microorganisms (antibiotic resistant bacteria, spores, biofilms, fungi) and tumors, to degrade toxins, to modify surfaces and their properties, to increase microbial production of compounds, and to facilitate wound healing, blood coagulation, and teeth whitening. The mini-review provides a brief overview of non-thermal plasma sources and recent achievements in biological sciences. We have also included pros and cons of CAPP technologies as well as future directions in biosciences and their respective industrial fields.

Cold plasma treatment triggers antioxidative defense system and induces changes in hyphal surface and subcellular structures of Aspergillus flavus.

The cold atmospheric-pressure plasma (CAPP) has become one of the recent effective decontamination technologies, but CAPP interactions with biological material remain the subject of many studies. The CAPP generates numerous types of particles and radiations that synergistically affect cells and tissues differently depending on their structure. In this study, we investigated the effect of CAPP generated by diffuse coplanar surface barrier discharge on hyphae of Aspergillus flavus. Hyphae underwent massive structural changes after plasma treatment. Scanning electron microscopy showed drying hyphae that were forming creases on the hyphal surface. ATR-FTIR analysis demonstrated an increase of signal intensity for C=O and C-O stretching vibrations indicating chemical changes in molecular structures located on hyphal surface. The increase in membrane permeability was detected by the fluorescent dye, propidium iodide. Biomass dry weight determination and increase in permeability indicated leakage of cell content and subsequent death. Disintegration of nuclei and DNA degradation confirmed cell death after plasma treatment. Damage of plasma membrane was related to lipoperoxidation that was determined by higher levels of thiobarbituric acid reactive species after plasma treatment. The CAPP treatment led to rise of intracellular ROS levels detected by fluorescent microscopy using 2',7'-dichlorodihydrofluorescein diacetate. At the same time, antioxidant enzyme activities increased, and level of reduced glutathione decreased. The results in this study indicated that the CAPP treatment in A. flavus targeted both cell surface structures, cell wall, and plasma membrane, inflicting injury on hyphal cells which led to subsequent oxidative stress and finally cell death at higher CAPP doses.

Light-induced conidiation of Trichoderma spp. strains is accompanied by development-dependent changes in the Ca2+ binding to cell walls.

The effect of light on the binding of Ca2+ to mycelia and to cell walls isolated from aerial mycelia of three strains of Trichoderma spp. was studied. Two independent methods were used to measure the total Ca2+ content in mycelia and the Ca2+ bound to cell walls isolated from aerial mycelia. The results of these methods showed that the light-induced formation and maturation of conidia in Trichoderma spp. is accompanied by increased Ca2+ deposition in mycelia and cell walls. Moreover, the cultivation of Trichoderma atroviride F-534 in the presence of 45Ca2+ under circadian illumination showed that radioactivity was exclusively localized in the light-induced conidial rings of aerial mycelia. The fluorescence microscopy of chlortetracycline-stained mycelia showed that the major fraction of Ca2+ was accumulated in conidia and fructification structures, or some intracellular compartments in T. atroviride F-534 grown under circadian illumination, while only a limited amount of Ca2+ was associated with hyphal surfaces. In addition, the study of 45Ca2+ binding to cell walls revealed that T. atroviride F-534 displays both increased 45Ca2+ binding capacity and elevated affinity to 45Ca2+ binding upon illumination. The results indicate that conidia formation and (or) maturation is associated with changes in Ca2+ homeostasis.

Development and Optimization of a High-Throughput Screening Assay for Rapid Evaluation of Lipstatin Production by Streptomyces Strains.

Pancreatic lipase inhibitors, such as tetrahydrolipstatin (orlistat), are used in anti-obesity treatments. Orlistat is the only anti-obesity drug approved by the European Medicines Agency (EMA). The drug is synthesized by saturation of lipstatin, a ß-lactone compound, isolated from Streptomyces toxytricini and S. virginiae. To identify producers of novel pancreatic lipase inhibitors or microbial strains with improved lipstatin production and higher chemical purity remains still a priority. In this study, a high-throughput screening method to identify Streptomyces strains producing potent pancreatic lipase inhibitors was established. The assay was optimized and validated using S. toxytricini NRRL 15443 and its mutants. Strains grew in 24-well titer plates. Lipstatin levels were assessed directly in culture medium at the end of cultivation by monitoring lipolytic activity in the presence of a chromogenic substrate, 1,2-Di-O-lauryl-rac-glycero-3-glutaric acid 6-methylresorufin ester (DGGR). The lipase activity decreased in response to lipstatin production, and this was demonstrated by accumulation of red-purple methylresorufin, a product of DGGR digestion. The sensitivity of the assay was achieved by adding a lipase of high lipolytic activity and sensitivity to lipstatin to the reaction mixture. In the assay, the fungal lipase from Mucor javanicus was used as an alternative to the human pancreatic lipase. Many fungal lipases preserve high lipolytic activity in extreme conditions and are not colipase dependent. The assay proved to be reliable in differentiation of strains with high and low lipstatin productivity.

Global Analysis of Predicted G Protein-Coupled Receptor Genes in the Filamentous Fungus, Neurospora crassa.

G protein-coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization.

Glutamic acid decarboxylase gene disruption reveals signalling pathway(s) governing complex morphogenic and metabolic events in Trichoderma atroviride.

Glutamate decarboxylase (GAD) catalyses decarboxylation of glutamate to gamma-aminobutyrate (GABA) in a metabolic pathway connected to citrate cycle and known as GABA shunt. The gene (gad) was disrupted in Trichoderma atroviride CCM F-534 and viable mutants were characterized. Two of them were found to arise by homologous recombination and were devoid of both GAD activity and GABA. Mutants grew slower as compared to the wild type (F534). In the submerged culture, mutants developed less CO2 and consumed less O2 than the F534 without changing their respiratory quotients. Hyphae of mutants were more ramified than those of F534. Their ramification, in contrast to F534, was not increased by cyclosporin A, a drug causing hyphae ramification of several fungi and which is a calcineurin/cyclophilin inhibitor, or by FK506. Rapamycin, which is a cyclophilin but not calcineurin inhibitor, had a different effect on hyphae ramification in F534 and mutants. To examine the presence of GABA receptors in the fungus the effect of mammalian GABA-receptor modulators, such as bicuculline, gabapentin or carbamazepine on fungal morphology were investigated. Conidia of mutants germinated in a multipolar manner more frequently (up to 80 %) than those of F534. This trait was modified with cyclosporine A, FK506 and GABA receptor modulators in a different manner. Transport of chlorides, an intimate feature of GABA-regulated receptors/channels in animal cells, was measured in vegetative mycelia by means (36)Cl(-) uptake. It was significantly reduced in gad mutants. The results suggest that T. atroviride possesses a signalling pathway that involves GABA, putative GABA receptor(s), calcineurin, target of rapamycin and chloride transporter(s) to regulate physiological functions.

Genetic and physical interactions between Gα subunits and components of the Gßγ dimer of heterotrimeric G proteins in Neurospora crassa.

Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus Neurospora crassa. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gß subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gß and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the Gα binding partners for the Gßγ dimer is currently lacking. In this study, the three N. crassa Gα subunits are analyzed for genetic epistasis with gnb-1 and for physical interaction with the Gßγ dimer. We created double mutants lacking one Gα gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each Gα gene into the Δgnb-1 background. Genetic analysis revealed that gna-3 is epistatic to gnb-1 with regard to negative control of submerged conidiation. gnb-1 is epistatic to gna-2 and gna-3 for aerial hyphal height, while gnb-1 appears to act upstream of gna-1 and gna-2 during aerial conidiation. None of the activated Gα alleles restored female fertility to Δgnb-1 mutants, and the gna-3(Q208L) allele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from the gng-1-FLAG strain demonstrated that the three Gα proteins interact with the Gßγ dimer. The finding that the Gßγ dimer interacts with all three Gα proteins is supported by epistasis between gnb-1 and gna-1, gna-2, and gna-3 for at least one function.

Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa.

Here we characterize the relationship between the PRE-2 pheromone receptor and its ligand, CCG-4, and the general requirements for receptors, pheromones, G proteins, and mating type genes during fusion of opposite mating-type cells and sexual sporulation in the multicellular fungus Neurospora crassa. PRE-2 is highly expressed in mat a cells and is localized in male and female reproductive structures. Δpre-2 mat a females do not respond chemotropically to mat A males (conidia) or form mature fruiting bodies (perithecia) or meiotic progeny (ascospores). Strains with swapped identity due to heterologous expression of pre-2 or ccg-4 behave normally in crosses with opposite mating-type strains. Coexpression of pre-2 and ccg-4 in the mat A background leads to self-attraction and development of barren perithecia without ascospores. Further perithecial development is achieved by inactivation of Sad-1, a gene required for meiotic gene silencing. Findings from studies involving forced heterokaryons of opposite mating-type strains show that presence of one receptor and its compatible pheromone is necessary and sufficient for perithecial development and ascospore production. Taken together, the results demonstrate that although receptors and pheromones control sexual identity, the mating-type genes (mat A and mat a) must be in two different nuclei to allow meiosis and sexual sporulation to occur.

RIC8 is a guanine-nucleotide exchange factor for Galpha subunits that regulates growth and development in Neurospora crassa.

Heterotrimeric (αßγ) G proteins are crucial components of eukaryotic signal transduction pathways. G-protein-coupled receptors (GPCRs) act as guanine nucleotide exchange factors (GEFs) for Gα subunits. Recently, facilitated GDP/GTP exchange by non-GPCR GEFs, such as RIC8, has emerged as an important mechanism for Gα regulation in animals. RIC8 is present in animals and filamentous fungi, such as the model eukaryote Neurospora crassa, but is absent from the genomes of baker's yeast and plants. In Neurospora, deletion of ric8 leads to profound defects in growth and asexual and sexual development, similar to those observed for a mutant lacking the Gα genes gna-1 and gna-3. In addition, constitutively activated alleles of gna-1 and gna-3 rescue many defects of Δric8 mutants. Similar to reports in Drosophila, Neurospora Δric8 strains have greatly reduced levels of G-protein subunits. Effects on cAMP signaling are suggested by low levels of adenylyl cyclase protein in Δric8 mutants and suppression of Δric8 by a mutation in the protein kinase A regulatory subunit. RIC8 acts as a GEF for GNA-1 and GNA-3 in vitro, with the strongest effect on GNA-3. Our results support a role for RIC8 in regulating GNA-1 and GNA-3 in Neurospora.

High-throughput production of gene replacement mutants in Neurospora crassa.

The model filamentous fungus Neurospora crassa has been the focus of functional genomics studies for the past several years. A high-throughput gene knockout procedure has been developed and used to generate mutants for more than two-thirds of the ∼10,000 annotated N. crassa genes. Yeast recombinational cloning was incorporated as an efficient procedure to produce all knockout cassettes. N. crassa strains with the Δmus-51 or Δmus-52 deletion mutations were used as transformation recipients in order to reduce the incidence of ectopic integration and increase homologous recombination of knockout cassettes into the genome. A 96-well format was used for many steps of the procedure, including fungal transformation, isolation of homokaryons, and verification of mutants. In addition, development of software programs for primer design and restriction enzyme selection facilitated the high-throughput aspects of the overall protocol.

Transient excretion of succinate from Trichoderma atroviride submerged mycelia reveals the complex movements and metabolism of carboxylates.

Submerged growth of Trichoderma atroviride CCM F 534 on glucose-containing medium was accompanied by the excretion of organic acids (succinate, citrate, alpha-ketoglutarate, fumarate, aconitate). The excretion of succinate was transient. After 48-72 h cultivation, millimolar amounts of succinate disappeared from the medium. We studied the mechanism of the removal of succinate from the medium and demonstrated the activation of the inward transport of succinate by submerged mycelia. This transport was carrier-mediated, had a low solute specificity, and was driven by proton-motive force. The last aspect was provided by the activation of the H(+)-ATPase, as documented by measurements of ATPase activity and expression of the pma gene. The disruption of the pma gene abolished the capacity of the mycelia to re-uptake succinate but not its production. Results show that excreted carboxylates could serve as alternative nutrients in the late phase(s) of submerged growth, explain why inward transport system(s) for carboxylates are induced, and indicate that the inward-directed transport could interfere with the production of carboxylic acids by fungi.

Heterotrimeric G protein signaling in filamentous fungi.

Filamentous fungi are multicellular eukaryotic organisms known for nutrient recycling as well as for antibiotic and food production. This group of organisms also contains the most devastating plant pathogens and several important human pathogens. Since the first report of heterotrimeric G proteins in filamentous fungi in 1993, it has been demonstrated that G proteins are essential for growth, asexual and sexual development, and virulence in both animal and plant pathogenic filamentous species. Numerous G protein subunit and G protein-coupled receptor genes have been identified, many from whole-genome sequences. Several regulatory pathways have now been delineated, including those for nutrient sensing, pheromone response and mating, and pathogenesis. This review provides a comparative analysis of G protein pathways in several filamentous species, with discussion of both unifying themes and important unique signaling paradigms.

The predicted G-protein-coupled receptor GPR-1 is required for female sexual development in the multicellular fungus Neurospora crassa.

G-protein-coupled receptors (GPCRs) control important aspects of asexual and sexual development in eukaryotic organisms. We have identified a predicted GPCR in the filamentous fungus Neurospora crassa with similarity to cyclic AMP-receptor like GPCRs from Dictyostelium discoideum and GCR1 from Arabidopsis thaliana. Expression of gpr-1 is highest in female reproductive structures, and deletion of gpr-1 leads to defects during sexual development. Unfertilized female structures (protoperithecia) from Deltagpr-1 strains are weakly pigmented, small, and submerged in the agar. The perithecia produced after fertilization have deformed beaks that lack ostioles, the openings through which ascospores are discharged. Localization studies using a GPR-1-green fluorescent protein fusion protein showed that GPR-1 is targeted to female reproductive structures. Genetic epistasis experiments with the three Galpha genes were inconclusive due to the early block in mating exhibited by Deltagna-1 strains. Phenotypic analysis of mutants from a high-throughput N. crassa knockout project allowed identification of BEK-1, a homeodomain transcription factor that is a potential target of GPR-1. The perithecial defects of Deltabek-1 strains are similar to those of the Deltagpr-1 strain, and epistasis analysis indicates that bek-1 could function downstream of gpr-1 during postfertilization events. The effect must be posttranscriptional, as bek-1 transcript levels are not affected in Deltagpr-1 strains. The lack of ostioles in Deltagpr-1 and Deltabek-1 mutants has an undesirable effect on the ability to spread progeny (ascospores) by the normal ejection mechanism and would severely compromise the fitness of these strains in nature.

The heterotrimeric G-protein subunits GNG-1 and GNB-1 form a Gbetagamma dimer required for normal female fertility, asexual development, and galpha protein levels in Neurospora crassa.

We have identified a gene encoding a heterotrimeric G protein gamma subunit, gng-1, from the filamentous fungus Neurospora crassa. gng-1 possesses a gene structure similar to that of mammalian Ggamma genes, consisting of three exons and two introns, with introns present in both the open reading frame and 5'-untranslated region. The GNG-1 amino acid sequence displays high identity to predicted Ggamma subunits from other filamentous fungi, including Giberella zeae, Cryphonectria parasitica, Trichoderma harzianum, and Magnaporthe grisea. Deletion of gng-1 leads to developmental defects similar to those previously characterized for Deltagnb-1 (Gbeta) mutants. Deltagng-1, Deltagnb-1, and Deltagng-1 Deltagnb-1 strains conidiate inappropriately in submerged cultures and are female sterile, producing aberrant female reproductive structures. Similar to previous results obtained with Deltagnb-1 mutants, loss of gng-1 negatively influences levels of Galpha proteins (GNA-1, GNA-2, and GNA-3) in plasma membrane fractions isolated from various tissues of N. crassa and leads to a significant reduction in the amount of intracellular cyclic AMP. In addition, we show that GNB-1 is essential for maintenance of normal steady-state levels of GNG-1, suggesting a functional interaction between GNB-1 and GNG-1. Direct evidence for a physical association between GNB-1 and GNG-1 in vivo was provided by coimmunoprecipitation.

The genome sequence of the filamentous fungus Neurospora crassa.

Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes--more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca2+ signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes.

Phenylethylamine induces an increase in cytosolic Ca2+ in yeast.

Beta-phenylethylamine (PEA) induced an increase in cytosolic free calcium ion concentration ([Ca2+]c) in Saccharomyces cerevisiae cells monitored with transgenic aequorin, a Ca2+-dependent photoprotein. The PEA-induced [Ca2+]c increase was dependent on the concentrations of PEA applied, and the Ca2+ mostly originated from an extracellular source. Preceding the Ca2+ influx, H2O2 was generated in the cells by the addition of PEA. Externally added H2O2 also induced a [Ca2+]c increase. These results suggest that PEA induces the [Ca2+]c increase via H2O2 generation. The PEA-induced [Ca2+]c increase occurred in the mid1 mutant with a slightly smaller peak than in the wild-type strain, indicating that Mid1, a stretch-activated nonselective cation channel, may not be mainly involved in the PEA-induced Ca2+ influx. When PEA was applied, the MATa mid1 mutant was rescued from alpha-factor-induced death in a Ca2+-limited medium, suggesting that the PEA-induced [Ca2+]c increase can reinforce calcium signaling in the mating pheromone response pathway.