Increased duration of pollen and mold exposure are linked to climate change.

Pollen and molds are environmental allergens that are affected by climate change. As pollen and molds exhibit geographical variations, we sought to understand the impact of climate change (temperature, carbon dioxide (CO2), precipitation, smoke exposure) on common pollen and molds in the San Francisco Bay Area, one of the largest urban areas in the United States. When using time-series regression models between 2002 and 2019, the annual average number of weeks with pollen concentrations higher than zero increased over time. For tree pollens, the average increase in this duration was 0.47 weeks and 0.51 weeks for mold spores. Associations between mold, pollen and meteorological data (e.g., precipitation, temperature, atmospheric CO2, and area covered by wildfire smoke) were analyzed using the autoregressive integrated moving average model. We found that peak concentrations of weed and tree pollens were positively associated with temperature (p < 0.05 at lag 0-1, 0-4, and 0-12 weeks) and precipitation (p < 0.05 at lag 0-4, 0-12, and 0-24 weeks) changes, respectively. We did not find clear associations between pollen concentrations and CO2 levels or wildfire smoke exposure. This study's findings suggest that spore and pollen activities are related to changes in observed climate change variables.

Lifestyle Characteristics and Gene Expression Analysis of Colletotrichum camelliae Isolated from Tea Plant [Camellia sinensis (L.) O. Kuntze] Based on Transcriptome.

Colletotrichum camelliae is one of the most serious pathogens causing anthracnose in tea plants, but the interactive relationship between C. camelliae and tea plants has not been fully elucidated. This study investigated the gene expression changes in five different growth stages of C. camelliae based on transcriptome analysis to explain the lifestyle characteristics during the infection. On the basis of gene ontology (GO) enrichment analyses of differentially expressed genes (DEGs) in comparisons of germ tube (GT)/conidium (Con), appressoria (App)/Con, and cellophane infectious hyphae (CIH)/Con groups, the cellular process in the biological process category and intracellular, intracellular part, cell, and cell part in the cellular component category were significantly enriched. Hydrolase activity, catalytic activity, and molecular_function in the molecular function category were particularly enriched in the infection leaves (IL)/Con group. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the DEGs were enriched in the genetic information processing pathway (ribosome) at the GT stage and the metabolism pathway (metabolic pathways and biosynthesis of secondary metabolism) in the rest of the stages. Interestingly, the genes associated with melanin biosynthesis and carbohydrate-active enzymes (CAZys), which are vital for penetration and cell wall degradation, were significantly upregulated at the App, CIH and IL stages. Subcellular localization results further showed that the selected non-annotated secreted proteins based on transcriptome data were majorly located in the cytoplasm and nucleus, predicted as new candidate effectors. The results of this study may establish a foundation and provide innovative ideas for subsequent research on C. camelliae.

Control of the microsporidian parasite Nosema ceranae in honey bees (Apis mellifera) using nutraceutical and immuno-stimulatory compounds.

Nosema ceranae is a microsporidian parasite that causes nosemosis in the honey bee (Apis mellifera). As alternatives to the antibiotic fumagillin, ten nutraceuticals (oregano oil, thymol, carvacrol, trans-cinnmaldehyde, tetrahydrocurcumin, sulforaphane, naringenin, embelin, allyl sulfide, hydroxytyrosol) and two immuno-stimulatory compounds (chitosan, poly I:C) were examined for controlling N. ceranae infections. Caged bees were inoculated with N. ceranae spores, and treatments were administered in sugar syrup. Only two compounds did not significantly reduce N. ceranae spore counts compared to the infected positive control, but the most effective were sulforaphane from cruciferous vegetables, carvacrol from oregano oil, and naringenin from citrus fruit. When tested at several concentrations, the highest sulforaphane concentration reduced spore counts by 100%, but also caused 100% bee mortality. For carvacrol, the maximum reduction in spore counts was 57% with an intermediate concentration and the maximum bee mortality was 23% with the highest concentration. For naringenin, the maximum reduction in spore counts was 64% with the highest concentration, and the maximum bee mortality was only 15% with an intermediate concentration. In the longevity experiment, naringenin-fed bees lived as long as Nosema-free control bees, both of which lived significantly longer than infected positive control bees. While its antimicrobial properties may be promising, reducing sulforaphane toxicity to bees is necessary before it can be considered as a candidate for controlling N. ceranae. Although further work on formulation is needed with naringenin, its effect on extending longevity in infected bees may give it an additional value as a potential additive for bee feed in honey bee colonies.

Understanding interaction effect of arbuscular mycorrhizal fungi in rice under elevated carbon dioxide conditions.

Arbuscular mycorrhizal fungi (AMF), particularly the Glomerales group, play a paramount role in plant nutrient uptake, and abiotic and biotic stress management in rice, but recent evidence revealed that elevated CO2 concentration considerably reduces the Glomerales group in soil. In view of this, the present study was initiated to understand the interaction effect of native Glomerales species application in rice plants (cv. Naveen) under elevated CO2 concentrations (400 ± 10, 550 ± 20, and 700 ± 20 ppm) in open-top chambers. Three different modes of application of the AMF inoculum were evaluated, of which, combined application of AMF at the seedling production and transplanting stages showed increased AMF colonization, which significantly improved grain yield by 25.08% and also increased uptake of phosphorus by 18.2% and nitrogen by 49.5%, as observed at 700-ppm CO2 concentration. Organic acids secretion in rice root increased in AMF-inoculated plants exposed to 700-ppm CO2 concentration. To understand the overall effect of CO2 elevation on AMF interaction with the rice plant, principal component and partial least square regression analysis were performed, which found both positive and negative responses under elevated CO2 concentration.

The role of in vitro cultivation on symbiotic trait and function variation in a single species of arbuscular mycorrhizal fungus.

In vitro propagation of AM fungi using transformed root cultures (TRC) is commonly used to obtain pure AM fungal propagules for use in research and industry. Early observations indicate that such an artificial environment can alter traits and function of AM fungi over time. We hypothesized that increased in vitro cultivation may promote ruderal strategies in fungi by enhancing propagule production and reducing mutualistic quality. To examine the effect of in vitro cultivation on the trait and function of AM fungi, we inoculated plants with 11 Rhizoglomus irregulare isolates which fell along a cultivation gradient spanning 80 generations. We harvested plants at 10, 20 and 30 d post inoculation to observe differences in fungal and plant traits post infection. In vitro cultivation led to increased spore production but reduced plant shoot phosphorus. Our results indicate that in vitro propagation may indirectly select for traits that affect symbiotic quality.

Inactivation of bacterial and fungal spores by UV irradiation and gaseous iodine treatment applied to air handling filters.

Exposure to viable bacterial and fungal spores re-aerosolized from air handling filters may create a major health risk. Assessing and controlling this exposure have been of interest to the bio-defense and indoor air quality communities. Methods are being developed for inactivating stress-resistant viable microorganisms collected on ventilation filters. Here we investigated the inactivation of spores of Bacillus thuringiensis var. kurstaki (Btk), a recognized simulant for B. antracis, and Aspergillus fumigatus, a common opportunistic pathogen used as an indicator for indoor air quality. The viability change was measured on filters treated with ultraviolet (UV) irradiation and gaseous iodine. The spores were collected on high-efficiency particulate air (HEPA) and non-HEPA filters, both flattened for testing purposes to represent "surface" filters. A mixed cellulose ester (MCE) membrane filter was also tested as a reference. Additionally, a commercial HEPA unit with a deep-bed (non-flattened) filter was tested. Combined treatments of Btk spores with UV and iodine on MCE filter produced a synergistic inactivation effect. No similar synergy was observed for A. fumigatus. For spores collected on an MCE filter, the inactivation effect was about an order of magnitude greater for Btk compared to A. fumigatus. The filter type was found to be an important factor affecting the inactivation of Btk spores while it was not as influential for A. fumigatus. Overall, the combined effect of UV irradiation and gaseous iodine on viable bacterial and fungal spores collected on flat filters was found to be potent. The benefit of either simultaneous or sequential treatment was much lower for Btk spores embedded inside the deep-bed (non-flattened) HEPA filter, but for A. fumigatus the inactivation on flattened and non-flattened HEPA filters was comparable. For both species, applying UV first and gaseous iodine second produced significantly higher inactivation than when applying them simultaneously or in an opposite sequence.

Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

Mucormycosis is a life-threatening respiratory fungal infection predominantly caused by Rhizopus species. Mucormycosis has incompletely understood pathogenesis, particularly how abnormalities in iron metabolism compromise immune responses. Here we show how, as opposed to other filamentous fungi, Rhizopus spp. establish intracellular persistence inside alveolar macrophages (AMs). Mechanistically, lack of intracellular swelling of Rhizopus conidia results in surface retention of melanin, which induces phagosome maturation arrest through inhibition of LC3-associated phagocytosis. Intracellular inhibition of Rhizopus is an important effector mechanism, as infection of immunocompetent mice with swollen conidia, which evade phagocytosis, results in acute lethality. Concordantly, AM depletion markedly increases susceptibility to mucormycosis. Host and pathogen transcriptomics, iron supplementation studies, and genetic manipulation of iron assimilation of fungal pathways demonstrate that iron restriction inside macrophages regulates immunity against Rhizopus. Our findings shed light on the pathogenetic mechanisms of mucormycosis and reveal the role of macrophage-mediated nutritional immunity against filamentous fungi.

A novel photosensitization treatment for the inactivation of fungal spores and cells mediated by curcumin.

The global concerns regarding the emergence of fungicide-resistant strains and the impact of the excessive use of fungicidal practises on our health, food, and environment have increased, leading to a demand for alternative clean green technologies as treatments. Photosensitization is a treatment that utilises a photosensitiser, light and oxygen to cause cell damage to microorganisms. The effect of photosensitization mediated by curcumin on Aspergillus niger, Aspergillus flavus, Penicillium griseofulvum, Penicillium chrysogenum, Fusarium oxysporum, Candida albicans and Zygosaccharomyces bailii was investigated using three methods. The viability of spores/cells suspended in aqueous buffer using different concentrations of curcumin solution (100-1000µM) and light dose (0, 24, 48, 72 and 96J/cm2) were determined. Spraying curcumin solution on inoculated surfaces of agar plates followed by irradiation and soaking spores/cells in curcumin solution prior to irradiation was also investigated. In aqueous mixtures, photosensitised spores/cells of F. oxysporum and C. albicans were inhibited at all light doses and curcumin concentrations, while inactivation of A. niger, A. flavus P. griseofulvum, P. chrysogenum and Z. bailii were highly significant (P<0.001) reduced by 99%, 88.9%, 78%, 99.7% and 99.2% respectively. On the surface of agar plates, spores/cells exposed to a light dose of 360J/cm2 sprayed with curcumin at 800µM showed complete inhibition for A. niger, F. oxysporum, C. albicans and Z. bailii, while A. flavus P. griseofulvum, and P. chrysogenum reduced by 75%, 80.4% and 88.5% respectively. Soaking spores/cells with curcumin solution prior to irradiation did not have a significant effect on the percentage reduction. These observations suggest that a novel photosensitization mediated curcumin treatment is effective against fungal spores/cells and the variation of percentage reduction was dependent on curcumin concentration, light dosage and fungal species.

Estimating the abundance of airborne pollen and fungal spores at variable elevations using an aircraft: how high can they fly?

Airborne pollen and fungal spores are monitored mainly in highly populated, urban environments, for allergy prevention purposes. However, their sources can frequently be located outside cities' fringes with more vegetation. So as to shed light to this paradox, we investigated the diversity and abundance of airborne pollen and fungal spores at various environmental regimes. We monitored pollen and spores using an aircraft and a car, at elevations from sea level to 2,000 m above ground, in the region of Thesssaloniki, Greece. We found a total of 24 pollen types and more than 15 spore types. Pollen and spores were detected throughout the elevational transect. Lower elevations exhibited higher pollen concentrations in only half of plant taxa and higher fungal spore concentrations in only Ustilago. Pinaceae and Quercus pollen were the most abundant recorded by airplane (>54% of the total). Poaceae pollen were the most abundant via car measurements (>77% of the total). Cladosporium and Alternaria spores were the most abundant in all cases (aircraft: >69% and >17%, car: >45% and >27%, respectively). We conclude that pollen and fungal spores can be diverse and abundant even outside the main source area, evidently because of long-distance transport incidents.

Cation-Stress-Responsive Transcription Factors SltA and CrzA Regulate Morphogenetic Processes and Pathogenicity of Colletotrichum gloeosporioides.

Growth of Colletotrichum gloeosporioides in the presence of cation salts NaCl and KCl inhibited fungal growth and anthracnose symptom of colonization. Previous reports indicate that adaptation of Aspergillus nidulans to salt- and osmotic-stress conditions revealed the role of zinc-finger transcription factors SltA and CrzA in cation homeostasis. Homologs of A. nidulans SltA and CrzA were identified in C. gloeosporioides. The C. gloeosporioides CrzA homolog is a 682-amino acid protein, which contains a C2H2 zinc finger DNA-binding domain that is highly conserved among CrzA proteins from yeast and filamentous fungi. The C. gloeosporioides SltA homolog encodes a 775-amino acid protein with strong similarity to A. nidulans SltA and Trichoderma reesei ACE1, and highest conservation in the three zinc-finger regions with almost no changes compared to ACE1 sequences. Knockout of C. gloeosporioides crzA (ΔcrzA) resulted in a phenotype with inhibited growth, sporulation, germination and appressorium formation, indicating the importance of this calciu006D-activated transcription factor in regulating these morphogenetic processes. In contrast, knockout of C. gloeosporioides sltA (ΔsltA) mainly inhibited appressorium formation. Both mutants had reduced pathogenicity on mango and avocado fruit. Inhibition of the different morphogenetic stages in the ΔcrzA mutant was accompanied by drastic inhibition of chitin synthase A and B and glucan synthase, which was partially restored with Ca2+ supplementation. Inhibition of appressorium formation in ΔsltA mutants was accompanied by downregulation of the MAP kinase pmk1 and carnitine acetyl transferase (cat1), genes involved in appressorium formation and colonization, which was restored by Ca2+ supplementation. Furthermore, exposure of C. gloeosporioides ΔcrzA or ΔsltA mutants to cations such as Na+, K+ and Li+ at concentrations that the wild type C. gloeosporioides is not affected had further adverse morphogenetic effects on C. gloeosporioides which were partially or fully restored by Ca2+. Overall results suggest that both genes modulating alkali cation homeostasis have significant morphogenetic effects that reduce C. gloeosporioides colonization.

Sporoderm-Broken Spores of Ganoderma lucidum Inhibit the Growth of Lung Cancer: Involvement of the Akt/mTOR Signaling Pathway.

The sporoderm-broken spores of Ganoderma lucidum (SBGS) and their extracts exhibited a wide range of biological activities. In the present study, we prepare ethanol/ethanol extract (E/E-SBGS) and ethanol/aqueous extract (E/A-SBGS) from SBGS and examine their antitumor activities against human lung cancer. Our results showed that E/E-SBGS, not E/A-SBGS, inhibited the survival and migration of lung cancer cells in a dose-dependent manner. E/E-SBGS arrested cell cycle at G2/M phase and triggered apoptosis by decreasing the expression and activity of cell cycle regulators, cyclin B1 and cdc2, as well as anti-apoptotic proteins, Bcl-2 and Bcl-xl. Consequently, colony formation of lung cancer cells was markedly blocked by E/E-SBGS at subtoxic concentrations. Oral administration of both E/E-SBGS and SBGS significantly suppressed tumor volume and tumor weight without gross toxicity in mice. Mechanism study showed that E/E-SBGS dose-dependently suppressed the activation of Akt, the mammalian target of rapamycin (mTOR) and their downstream molecules S6 kinase and 4E-BP1 in treated tumor cells. Taken together, these results indicate that the ethanol extract of sporoderm-broken spores of G. lucidum suppresses the growth of human lung cancer, at least in part, through inhibition of the Akt/mTOR signaling pathway, suggesting its potential role in cancer treatments.

Effect on tomato plant and fruit of the application of biopolymer-oregano essential oil coatings.

BACKGROUND: Oregano essential oil (EO) was incorporated into film-forming dispersions (FFDs) based on biopolymers (chitosan and/or methylcellulose) at two different concentrations. The effect of the application of the FFDs was evaluated on tomato plants (cultivar Micro-Tom) at three different stages of development, and on pre-harvest and postharvest applications on tomato fruit. RESULTS: The application of the FFDs at '3 Leaves' stage caused phytotoxic problems, which were lethal when the EO was applied without biopolymers. Even though plant growth and development were delayed, the total biomass and the crop yield were not affected by biopolymer-EO treatments. When the FFDs were applied in the 'Fruit' stage the pre-harvest application of FFDs had no negative effects. All FFDs containing EO significantly reduced the respiration rate of tomato fruit and diminished weight loss during storage. Moreover, biopolymer-EO FFDs led to a decrease in the fungal decay of tomato fruit inoculated with Rhizopus stolonifer spores, as compared with non-treated tomato fruit and those coated with FFDs without EO. CONCLUSION: The application of biopolymer-oregano essential oil coatings has been proven to be an effective treatment to control R. stolonifer in tomato fruit. © 2016 Society of Chemical Industry.

N-Chlorotaurine Exhibits Fungicidal Activity against Therapy-Refractory Scedosporium Species and Lomentospora prolificans.

N-Chlorotaurine (NCT), a well-tolerated endogenous long-lived oxidant that can be applied topically as an antiseptic, was tested on its fungicidal activity against Scedosporium and Lomentospora, opportunistic fungi that cause severe infections with limited treatment options, mainly in immunocompromised patients. In quantitative killing assays, both hyphae and conidia of Scedosporium apiospermum, Scedosporium boydii, and Lomentospora prolificans (formerly Scedosporium prolificans) were killed by 55 mM (1.0%) NCT at pH 7.1 and 37°C, with a 1- to 4-log10 reduction in CFU after 4 h and a 4- to >6-log10 reduction after 24 h. The addition of ammonium chloride to NCT markedly increased this activity. LIVE/DEAD staining of conidia treated with 1.0% NCT for 0.5 to 3 h increased the permeability of the cell wall and membrane. Preincubation of the test fungi in 1.0% NCT for 10 to 60 min delayed the time to germination of conidia by 2 h to >12 h and reduced their germination rate by 10.0 to 100.0%. Larvae of Galleria mellonella infected with 1.0 × 10(7) conidia of S. apiospermum and S. boydii died at a rate of 90.0 to 100% after 8 to 12 days. The mortality rate was reduced to 20 to 50.0% if conidia were preincubated in 1.0% NCT for 0.5 h or if heat-inactivated conidia were used. Our study demonstrates the fungicidal activity of NCT against different Scedosporium and Lomentospora species. A postantifungal effect connected with a loss of virulence occurs after sublethal incubation times. The augmenting effect of ammonium chloride can be explained by the formation of monochloramine.

Yeast m6A Methylated mRNAs Are Enriched on Translating Ribosomes during Meiosis, and under Rapamycin Treatment.

Interest in mRNA methylation has exploded in recent years. The sudden interest in a 40 year old discovery was due in part to the finding of FTO's (Fat Mass Obesity) N6-methyl-adenosine (m6A) deaminase activity, thus suggesting a link between obesity-associated diseases and the presence of m6A in mRNA. Another catalyst of the sudden rise in mRNA methylation research was the release of mRNA methylomes for human, mouse and Saccharomyces cerevisiae. However, the molecular function, or functions of this mRNA 'epimark' remain to be discovered. There is supportive evidence that m6A could be a mark for mRNA degradation due to its binding to YTH domain proteins, and consequently being chaperoned to P bodies. Nonetheless, only a subpopulation of the methylome was found binding to YTHDF2 in HeLa cells.The model organism Saccharomyces cerevisiae, has only one YTH domain protein (Pho92, Mrb1), which targets PHO4 transcripts for degradation under phosphate starvation. However, mRNA methylation is only found under meiosis inducing conditions, and PHO4 transcripts are apparently non-methylated. In this paper we set out to investigate if m6A could function alternatively to being a degradation mark in S. cerevisiae; we also sought to test whether it can be induced under non-standard sporulation conditions. We find a positive association between the presence of m6A and message translatability. We also find m6A induction following prolonged rapamycin treatment.

Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f.sp. lycopersici in tomato via 12-oxo-phytodienoic acid, salicylic acid, and flavonol accumulation.

Methyl jasmonate (MeJA) was tested by seed treatment for its ability to protect tomato seedlings against fusarium wilt caused by the soil-borne fungal pathogen Fusarium oxysporum f.sp. lycopersici. Isolated from Solanum lycopersicon L. seeds, cv. Beta fungus was identified as F. oxysporum f.sp. lycopersici Race 3 fungus by using phytopathological and molecular methods. MeJA applied at 0.01, 0.1 and 1 mM reduced spore germination and mycelial growth in vitro. Soaking of tomato seeds in MeJA solution at 0.1 mM for 1 h significantly enhanced the resistance level against the tested fungus in tomato seedlings 4 weeks after inoculation. The extracts from leaves of 15-day-old seedlings obtained from previously MeJA soaked seeds had the ability to inhibit in vitro spore germination of tested fungus. In these seedlings a significant increase in the levels phenolic compounds such as salicylic acid (SA), kaempferol and quercetin was observed. Up-regulation of phenylalanine ammonia-lyase (PAL5) and benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) genes and down-regulation of the isochorysmate synthase (ICS) gene in response to exogenous MeJA application indicate that the phenylalanine ammonia-lyase (PAL), not the isochorismate (IC) pathway, is the primary route for SA production in tomato. Moreover, the increased accumulation of the flavonols quercetin and kaempferol appears closely related to the increase of PAL5, chalcone synthase (CHS) and flavonol synthase/flavanone 3-hydroxylase-like (FLS) genes. Elevated levels of salicylic acid in seedlings raised from MeJA-soaked seeds were simultaneously accompanied by a decrease of jasmonic acid, the precursor of MeJA, and an increase of 12-oxo-phytodienoic acid (OPDA), the precursor of jasmonic acid. The present results indicate that the priming of tomato seeds with 0.1mM MeJA before sowing enables the seedlings grown from these seeds to reduce the attack of the soil-borne fungal pathogen F. oxysporum f.sp. lycopersici, so it can be applied in practice.

[Research of chemotaxis response of Botrytis cinerea and Alternaria panax on total ginsenosides].

In this paper, three kinds of chemotactic parameters (concentration, temperature and pH) were determined by plate assay and spore germination method to research the chemotactic response of Botrytis cinerea and Alternaria panax, and their spores on total ginsenosides. The results showed that Botrytis cinerea had strong chemotactic response at the mid-concentration of total ginsenosides (cultivation temperature was 20 degrees C and pH value was 6), and the data of chemotactic migration index (CMI) was 1.293 0, chemotactic growth rate (CGR) was 0.476 0, spore germination rate (SGR) was 53%, and dry weight of mycelial (DWM) was 0.452 6 g x L(-1); however, Alternaria panax had strong chemotactic response at the low-concentration of total ginsenosides (cultivation temperature was 25 degrees C and pH value was 6), and the data of chemotactic migration index (CMI) was 1.235 4, chemotactic growth rate (CGR) was 0.537 0, spore germination rate (SGR) was 67%, and dry weight of mycelial (DWM) was 0.494 8 g x L(-1). The results indicated that the low and middle concentration (2, 20 mg x L(-1)) of total ginsenosides had significant promoting effect on chemotactic response of these two pathogens, and the spore germination, mycelial growth rate, dry weight of mycelial of them were also significantly improved by this chemotactic response, whereas it decreased as the increase of total ginsenosides concentration.

Effect of 5-n-alkylresorcinol extracts from durum wheat whole grain on the growth of fusarium head blight (FHB) causal agents.

In an approach toward the identification of ecofriendly compounds for fusarium head blight biocontrol, the in vitro antifungal activity of 5-n-alkylresorcinol (AR) extracts, obtained from durum wheat intact kernels, was tested. In comparison with ethyl acetate and acetone extracts containing AR, total inhibition of Fusarium graminearum spore germination was observed with cyclohexane extract, which also exhibited a significant fungistatic activity against F. graminearum, Fusarium culmorum, Fusarium avenaceum, and Fusarium poae. Additionally, the study of the influence of such variables as predrying of seeds and durum wheat genotype on AR cyclohexane extract properties allowed the association of its highest antifungal activity with the AR homologue composition and, in particular, with the presence of a higher C21:0/C23:0 ratio. The interesting finding of this study suggests a potential application of the AR homologues in crop protection systems and could be an important step toward the development of commercial formulations suitable to the prevention of fungal diseases.

A method for sporulating budding yeast cells that allows for unbiased identification of kinase substrates using stable isotope labeling by amino acids in cell culture.

Quantitative proteomics has been widely used to elucidate many cellular processes. In particular, stable isotope labeling by amino acids in cell culture (SILAC) has been instrumental in improving the quality of data generated from quantitative high-throughput proteomic studies. SILAC uses the cell's natural metabolic pathways to label proteins with isotopically heavy amino acids. Incorporation of these heavy amino acids effectively labels a cell's proteome, allowing the comparison of cell cultures treated under different conditions. SILAC has been successfully applied to a variety of model organisms including yeast, fruit flies, plants, and mice to look for kinase substrates as well as protein-protein interactions. In budding yeast, several kinases are known to play critical roles in different aspects of meiosis. Therefore, the use of SILAC to identify potential kinase substrates would be helpful in the understanding the specific mechanisms by which these kinases act. Previously, it has not been possible to use SILAC to quantitatively study the phosphoproteome of meiotic Saccharomyces cerevisiae cells, because yeast cells sporulate inefficiently after pregrowth in standard synthetic medium. In this study we report the development of a synthetic, SILAC-compatible, pre-sporulation medium (RPS) that allows for efficient sporulation of S. cerevisiae SK1 diploids. Pre-growth in RPS supplemented with heavy amino acids efficiently labels the proteome, after which cells proceed relatively synchronously through meiosis, producing highly viable spores. As proof of principle, SILAC experiments were able to identify known targets of the meiosis-specific kinase Mek1.