Effector discovery in the fungal wheat pathogen Zymoseptoria tritici

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up‐regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici–wheat pathosystem.     

Quantitative trait locus mapping reveals complex genetic architecture of quantitative virulence in the wheat pathogen Zymoseptoria tritici

We conducted a comprehensive analysis of virulence in the fungal wheat pathogen Zymoseptoria tritici using quantitative trait locus (QTL) mapping. High‐throughput phenotyping based on automated image analysis allowed the measurement of pathogen virulence on a scale and with a precision that was not previously possible. Across two mapping populations encompassing more than 520 progeny, 540 710 pycnidia were counted and their sizes and grey values were measured. A significant correlation was found between pycnidia size and both spore size and number. Precise measurements of percentage leaf area covered by lesions provided a quantitative measure of host damage. Combining these large and accurate phenotypic datasets with a dense panel of restriction site‐associated DNA sequencing (RADseq) genetic markers enabled us to genetically dissect pathogen virulence into components related to host damage and those related to pathogen reproduction. We showed that different components of virulence can be under separate genetic control. Large‐ and small‐effect QTLs were identified for all traits, with some QTLs specific to mapping populations, cultivars and traits and other QTLs shared among traits within the same mapping population. We associated the presence of four accessory chromosomes with small, but significant, increases in several virulence traits, providing the first evidence for a meaningful function associated with accessory chromosomes in this organism. A large‐effect QTL involved in host specialization was identified on chromosome 7, leading to the identification of candidate genes having a large effect on virulence.     

tRNA cleavage is a conserved response to oxidative stress in eukaryotes

Recent results have identified a diversity of small RNAs in a wide range of organisms. In this work, we demonstrate that Saccharomyces cerevisiae contains a small RNA population consisting primarily of tRNA halves and rRNA fragments. Both 5′ and 3′ fragments of tRNAs are detectable by Northern blot analysis, suggesting a process of endonucleolytic cleavage. tRNA and rRNA fragment production in yeast is most pronounced during oxidative stress conditions, especially during entry into stationary phase. Similar tRNA fragments are also observed in human cell lines and in plants during oxidative stress. These results demonstrate that tRNA cleavage is a conserved aspect of the response to oxidative stress.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Altered growth characteristics of skin fibroblasts from wild-derived mice, and genetic loci regulating fibroblast clone size

Mouse fibroblast senescence in vitro is an important model for the study of aging at cellular level. However, common laboratory mouse strains may have lost some important allele variations related to aging processes. In this study, growth in vitro of tail skin fibroblasts (TSFs) derived from a wild-derived stock, Pohnpei (Pohn) mice, differed from growth of control C57BL/6 J (B6) TSFs. Pohn TSFs exhibited higher proliferative ability, fewer apoptotic cells, decreased expression of Cip1, smaller surface areas, fewer cells positive for senescence associated-beta-galactosidase (SA-beta-gal) and greater resistance to H(2)O(2)-induced SA-beta-gal staining and Cip1 expression. These data suggest that TSFs from Pohn mice resist cellular senescence-like changes. Using large clone ratio (LCR) as the phenotype, a quantitative trait locus (QTL) analysis in a Pohn/B6 backcross population found four QTLs for LCR: Fcs1 on Chr 3 at 55 CM; Fcs2 on Chr X at 50 CM; Fcs3 on Chr 4 at 51 CM and Fcs4 on Chr 10 at 25 CM. Together, these four QTLs explain 26.1% of the variations in LCRs in the N2 population. These are the first QTLs reported that regulate fibroblast growth. Glutathione S transferase mu (GST-mu) genes are overrepresented in the 95% confidence interval of Fcs1, and Pohn TSFs have higher H(2)O(2)-induced GST-mu 4, 5 and 7 mRNA levels than B6 TSFs. These enzymes may protect Pohn TSFs from oxidation.     

Adaptation of neuronal cells to chronic oxidative stress is associated with altered cholesterol and sphingolipid homeostasis and lysosomal function

Chronic oxidative stress has been causally linked to several neurodegenerative disorders. As sensitivity for oxidative stress greatly differs between brain regions and neuronal cell types, specific cellular mechanisms of adaptation to chronic oxidative stress should exist. Our objective was to identify molecular mechanisms of adaptation of neuronal cells after applying chronic sublethal oxidative stress. We demonstrate that cells resistant to oxidative stress exhibit altered cholesterol and sphingomyelin metabolisms. Stress-resistant cells showed reduced levels of molecules involved in cholesterol trafficking and intracellular accumulation of cholesterol, cholesterol precursors, and metabolites. Moreover, stress-resistant cells exhibited reduced SMase activity. The altered lipid metabolism was associated with enhanced autophagy. Treatment of stress-resistant cells with neutral SMase reversed the stress-resistant phenotype, whereas it could be mimicked by treatment of neuronal cells with a specific inhibitor of neutral SMase. Analysis of hippocampal and cerebellar tissue of mouse brains revealed that the obtained cell culture data reflect the in vivo situation. Stress-resistant cells in vitro showed similar features as the less vulnerable cerebellum in mice, whereas stress-sensitive cells resembled the highly sensitive hippocampal area. These findings suggest an important role of the cell type-specific lipid profile for differential vulnerabilities of different brain areas toward chronic oxidative stress.     

Detection of quantitative trait loci for growth and fatness in pigs

A quantitative trait locus (QTL) analysis of growth and fatness data from a three-generation experimental cross between Meishan (MS) and Large White (LW) pig breeds is presented. Six boars and 23 F1 sows, the progeny of six LW boars and six MS sows, produced 530 F2 males and 573 F2 females. Nine growth traits, i.e. body weight at birth and at 3, 10, 13, 17 and 22 weeks of age, average daily gain from birth to 3 weeks, from 3 to 10 weeks and from 10 to 22 weeks of age, as well as backfat thickness at 13, 17 and 22 weeks of age and at 40 and 60 kg live weight were analysed. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using two interval mapping methods: a line-cross (LC) regression method where founder lines were assumed to be fixed for different QTL alleles and a half-/full-sib (HFS) maximum likelihood method where allele substitution effects were estimated within each half-/full-sib family. Both methods revealed highly significant gene effects for growth on chromosomes 1, 4 and 7 and for backfat thickness on chromosomes 1, 4, 5, 7 and X, and significant gene effects on chromosome 6 for growth and backfat thickness. Suggestive QTLs were also revealed by both methods on chromosomes 2 and 3 for growth and 2 for backfat thickness. Significant gene effects were detected for growth on chromosomes 11, 13, 14, 16 and 18 and for backfat thickness on chromosome 8, 10, 13 and 14. LW alleles were associated with high growth rate and low backfat thickness, except for those of chromosome 7 and to a lesser extent early-growth alleles on chromosomes 1 and 2 and backfat thickness alleles on chromosome 6.     

Microarray expression profiling of Spodoptera litura in response to oxidative stress

To examine the expression profile of oxidative stress responsive genes in Spodoptera litura, we constructed a cDNA library from S. litura injected with hydrogen peroxide (H(2)O(2)). Using a microarray chip composed of 2,964 cDNAs, we screened gene expression at 1, 3, 5, 7, and 9 h post H(2)O(2) injection. Data were clustered into 15 groups of genes that behave similarly across each time course. Seventy-three genes were identified as being at least twofold up- or downregulated after treatment with H(2)O(2) in S. litura. We constructed expressed sequence tags (ESTs) for genes that changed at least twofold after treatment with H(2)O(2) . The functional classification of these ESTs based on Gene Ontology showed that the ESTs are rich in genes involved in oxidoreductase activity (5.7%), defense (14.3%), cellular process (22.9%), and development (17.1%).     

酿酒酵母氧化胁迫应答反应机制

氧化胁迫是生物体面对逆境时的重要反应。在与逆境和活性氧做斗争的过程中,细胞进化出一套完整的应答调控机制,通过调节体内活性氧的代谢平衡,来保护DNA、脂质和蛋白质等免受氧化攻击。本文以酿酒酵母为例,根据近年来国内外研究的进展,围绕其在氧化胁迫应答过程中的三道保护屏障,即抗氧化物质和防御酶系统、转录调节和氧化物降解以及细胞器自噬,综述了其抗氧化代谢机理,为深入认识细胞的抗氧化应答机制奠定基础。     

Defences against oxidative stress in vibrios associated with corals

Bacteria colonizing healthy coral tissue may produce enzymes capable of overcoming the toxic effects of reactive oxygen species, including superoxide dismutase (SOD) and catalase. Significant differences in the activities of these enzymes were observed in cultures of Vibrio campbellii, Vibrio coralliilyticus, Vibrio harveyi, Vibrio mediterranei, Vibrio pelagius, Vibrio rotiferanus, Vibrio tasmaniensis, and Photobacterium eurosenbergii isolated from healthy, bleached or necrotic tropical and cold water corals. Levels of SOD in exponential phase cultures of V. coralliilyticus grown at 28 degrees C were only slightly higher than those grown at 16 degrees C whereas the levels in stationary phase cultures at 28 degrees C were 7.3 x higher than those at 16 degrees C. The increase in catalase activity of V. coralliilyticus and V. harveyi upon entry to stationary phase conferred protection against killing by oxidative stress. Increased temperature affected up-regulation of enzymes in stationary phase cultures, but pretreatment of cultures with hydrogen peroxide had no significant effect on induction of catalase or SOD. The increased activities appear to be due to up-regulation of gene expression rather than induction of different forms of the enzymes. We suggest that SOD and catalase are unlikely to be major factors in the virulence of these bacteria for corals and that their main function may be to protect against endogenous superoxide.     

Chronic exposure to U18666A is associated with oxidative stress in cultured murine cortical neurons

Findings that antioxidant treatment may be beneficial in Alzheimer's disease indicate that oxidative stress is an important factor in its pathogenesis. Studies have also suggested that cholesterol imbalance in the brain might be related to the development of neurological disorders. Previously, we have reported that U18666A, a cholesterol transport-inhibiting agent, leads to apoptosis and intracellular cholesterol accumulation in primary cortical neurons. In this study, we found that neuronal apoptosis mediated by U18666A is associated with oxidative stress in the treated cortical neurons. Cortical neurons treated with U18666A also showed decreased secretion and increased intraneuronal accumulation of beta-amyloid. The association of neuronal apoptosis with oxidative stress and Abeta accumulation may provide clues to the pathogenesis of Alzheimer's disease, as well as the role oxidative stress plays in other neurodegenerative diseases.     

Environmental stress increases selection against and dominance of deleterious mutations in inbred families of the Pacific oyster Crassostrea gigas

The deleterious effects of inbreeding are well documented and of major concern in conservation biology. Stressful environments have generally been shown to increase inbreeding depression; however, little is known about the underlying genetic mechanisms of the inbreeding-by-stress interaction and to what extent the fitness of individual deleterious mutations is altered under stress. Using microsatellite marker segregation data and quantitative trait locus (QTL) mapping methods, I performed a genome scan for deleterious mutations affecting viability (viability or vQTL) in two inbred families of the Pacific oyster Crassostrea gigas, reared in a stressful, nutrient-poor diet and a favourable, nutrient-rich diet, which had significant effects on growth and survival. Twice as many vQTL were detected in the stressful diet compared with the favourable diet, resulting primarily from substantially greater mortality of homozygous genotypes. At vQTL, estimates of selection (s) and dominance (h) were greater in the stressful environment (= 0.86 vs. 0.54 and = 0.35 vs. 0.18, in stressful and nonstressful diets, respectively). There was no evidence of interaction between vQTL. Individual vQTL differed across diets in selection only, or in both selection and dominance, and some vQTL were not affected by diet. These results suggest that stress-associated increases in selection against individual deleterious alleles underlie greater inbreeding depression with stress. Furthermore, the finding that inbreeding-by-environment interaction appears, to some extent, to be locus specific, helps to explain previous observations of lineage-specific expression of inbreeding depression and environment-specific purging, which have important implications for conservation and evolutionary biology.     

Compensatory growth and oxidative stress in a damselfly

Physiological costs of compensatory growth are poorly understood, yet may be the key components in explaining why growth rates are typically submaximal. Here we tested the hypothesized direct costs of compensatory growth in terms of oxidative stress. We assessed oxidative stress in a study where we generated compensatory growth in body mass by exposing larvae of the damselfly Lestes viridis to a transient starvation period followed by ad libitum food. Compensatory growth in the larval stage was associated with higher oxidative stress (as measured by induction of superoxide dismutase and catalase) in the adult stage. Our results challenge two traditional views of life-history theory. First, they indicate that age and mass at metamorphosis not necessarily completely translate larval stress into adult fitness and that the observed physiological cost may explain hidden carry-over effects. Second, they support the notion that costs of compensatory growth may be associated with free-radical-mediated trade-offs and not necessarily with resource-mediated trade-offs.     

Telomerase activity in response to mild oxidative stress

We have analysed telomerase activity to determine whether it can be modified when BCL-2 is endogenously overexpressed in response to a mild oxidative stress treatment as part of a survival mechanism, in contrast with an exogenous bcl-2 overexpression due to a retroviral infection. Endogenous bcl-2 overexpression was induced after a low oxidative insult of H2O2 in mice primary lung fibroblasts and L929 cell, whereas bcl-2 exogenous overexpression was performed using a retroviral infection in L929 cells. Telomerase activity was quantified in Bcl-2 overexpressing cells by the TRAP assay. When the cells were treated with different H2O2 concentrations, only those exposed to 50 μM showed increased telomerase activity. This correlates with BCL-2 expression as part of the endogenous response to mild oxidative stress. Oxidative stress generated during the toxic mechanism of chemotherapeutic drugs might induce BCL-2 increment, enhancing telomerase activity and reactivating the oncogenic process. Clinical trials should take into consideration the possibility of telomerase activation following increased BCL-2 expression when treating patients with ROS (reactive oxygen species) generation by anti-cancer drugs.     

Upregulation of Daxx mediates apoptosis in response to oxidative stress

Oxidative stress induces apoptosis in a variety of cell types by as yet unclear signaling mechanisms. The Daxx protein is reportedly involved in apoptosis through its interactions with Fas, transforming growth factor-beta receptor, and promyelocytic leukemia protein (PML). Here, we explored the possible roles of Daxx in oxidative stress-induced apoptosis. We found that both the mRNA and protein levels of Daxx markedly increased when cells underwent apoptosis after H2O2 treatment. Pretreatment with the cell-permeable antioxidant, N-acetyl cysteine, prevented cells from H2O2-induced Daxx upregulation and subsequent apoptosis, indicating that the endogenous oxidant regulated Daxx expression. Furthermore, suppression of endogenous Daxx expression by antisense oligonucleotide technology inhibited oxidative stress-induced apoptosis in HeLa cells. Taken together, these results suggest that Daxx acts as an intermediary messenger of pro-apoptotic signals triggered by oxidative stress.     

Tolerance,arsenic uptake,and oxidative stress in Acacia farnesiana under arsenate-stress

Acacia farnesiana is a shrub widely distributed in soils heavily polluted with arsenic in Mexico. However, the mechanisms by which this species tolerates the phytotoxic effects of arsenic are unknown. This study aimed to investigate the tolerance and bioaccumulation of As by A. farnesiana seedlings exposed to high doses of arsenate (AsV) and the role of peroxidases (POX) and glutathione S-transferases (GST) in alleviating As-stress. For that, long-period tests were performed in vitro under different AsV treatments. A. farnesiana showed a remarkable tolerance to AsV, achieving a half-inhibitory concentration (IC₅₀) of about 2.8 mM. Bioaccumulation reached about 940 and 4380 mg As·kg^?1 of dry weight in shoots and roots, respectively, exposed for 60 days to 0.58 mM AsV. Seedlings exposed to such conditions registered a growth delay during the first 15 days, when the fastest As uptake rate (117 mg kg^?1 day^?1) occurred, coinciding with both the highest rate of lipid peroxidation and the strongest up-regulation of enzyme activities. GST activity showed a strong correlation with the As bioaccumulated, suggesting its role in imparting AsV tolerance. This study demonstrated that besides tolerance to AsV, A. farnesiana bioaccumulates considerable amounts of As, suggesting that it may be useful for phytostabilization purposes.     

Nonylphenol exposure is associated with oxidative and nitrative stress in pregnant women

ABSTRACT

Animal studies have shown that exposure to nonylphenol (NP) increases oxidative/nitrative stress, but whether it does so in humans is unknown. This study examines prenatal exposure to NP and its effects on oxidatively/nitratively damaged DNA, lipid peroxidation, and the activities of antioxidants. A total of 146 urine and blood specimens were collected during gestational weeks 27–38 and hospital admission for delivery, respectively. Urinary NP was analyzed by high-performance liquid chromatography (HPLC). Urinary biomarkers of oxidatively/nitratively damaged DNA and lipid peroxidation, including 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG), 8-nitroguanine (8-NO₂Gua), 8-iso-prostaglandin F_2α (8-isoPF_2α) and 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA), were simultaneously analyzed using isotope-dilution liquid-chromatography/electron spray ionization tandem mass spectrometry. The activities of maternal plasma superoxide dismutase and glutathione peroxidase were analyzed by enzyme-linked immunosorbent assay. Urinary NP level was significantly associated with 8-oxodG and 8-NO₂Gua levels in late pregnancy, suggesting that NP may enhance oxidatively and nitratively damaged DNA. The adjusted odds ratios for high 8-oxodG level exhibited a significantly dose–response relationship with NP levels, stratified into four quartiles. 8-oxodG appears to be a more sensitive and effective biomarker of NP exposure than 8-NO₂Gua. These relationships suggest NP may play a role in the pregnancy complications.     

Embryonic exposure to corticosterone modifies the juvenile stress response, oxidative stress and telomere length

Early embryonic exposure to maternal glucocorticoids can broadly impact physiology and behaviour across phylogenetically diverse taxa. The transfer of maternal glucocorticoids to offspring may be an inevitable cost associated with poor environmental conditions, or serve as a maternal effect that alters offspring phenotype in preparation for a stressful environment. Regardless, maternal glucocorticoids are likely to have both costs and benefits that are paid and collected over different developmental time periods. We manipulated yolk corticosterone (cort) in domestic chickens (Gallus domesticus) to examine the potential impacts of embryonic exposure to maternal stress on the juvenile stress response and cellular ageing. Here, we report that juveniles exposed to experimentally increased cort in ovo had a protracted decline in cort during the recovery phase of the stress response. All birds, regardless of treatment group, shifted to oxidative stress during an acute stress response. In addition, embryonic exposure to cort resulted in higher levels of reactive oxygen metabolites and an over-representation of short telomeres compared with the control birds. In many species, individuals with higher levels of oxidative stress and shorter telomeres have the poorest survival prospects. Given this, long-term costs of glucocorticoid-induced phenotypes may include accelerated ageing and increased mortality.