Conidiation under illumination enhances conidial tolerance of insect-pathogenic fungi to environmental stresses

Light is an important signal for fungi in the environment and induces many genes with roles in stress and virulence responses. Conidia of the entomopathogenic fungi Aschersonia aleyrodis, Beauveria bassiana, Cordyceps fumosorosea, Lecanicillium aphanocladii, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium robertsii, Simplicillium lanosoniveum, Tolypocladium cylindrosporum, and Tolypocladium inflatum were produced on potato dextrose agar (PDA) medium under continuous white light, on PDA medium in the dark, or under nutritional stress (= Czapek medium without sucrose = MM) in the dark. The conidial tolerance of these species produced under these different conditions were evaluated in relation to heat stress, oxidative stress (menadione), osmotic stress (KCl), UV radiation, and genotoxic stress caused by 4-nitroquinoline 1-oxide (4-NQO). Several fungal species demonstrated greater stress tolerance when conidia were produced under white light than in the dark; for instance white light induced higher tolerance of A. aleyrodis to KCl and 4-NQO; B. bassiana to KCl and 4-NQO; C. fumosorosea to UV radiation; M. anisopliae to heat and menadione; M. brunneum to menadione, KCl, UV radiation, and 4-NQO; M. robertsii to heat, menadione, KCl, and UV radiation; and T. cylindrosporum to menadione and KCl. However, conidia of L. aphanocladii, S. lanosoniveum, and T. inflatum produced under white light exhibited similar tolerance as conidia produced in the dark. When conidia were produced on MM, a much stronger stress tolerance was found for B. bassiana to menadione, KCl, UV radiation, and 4-NQO; C. fumosorosea to KCl and 4-NQO; Metarhizium species to heat, menadione, KCl, and UV radiation; T. cylindrosporum to menadione and UV radiation; and T. inflatum to heat and UV radiation. Again, conidia of L. aphanocladii and S. lanosoniveum produced on MM had similar tolerance to conidia produced on PDA medium in the dark. Therefore, white light is an important factor that induces higher stress tolerance in some insect-pathogenic fungi, but growth in nutritional stress always provides in conidia with stronger stress tolerance than conidia produced under white light.     

Superoxide dismutase production by Isaria fumosorosea on metals and its role in stress tolerance and fungal virulence

Superoxide dismutase (SOD) is an anti-oxidant enzyme which also plays a role in fungal virulence. The present study was conducted to elucidate its role in fungal infection and stress tolerance of entomopathogenic fungi, Isaria fumosorosea. SOD activity of I. fumosorosea conidia differed significantly on the growth medium supplemented with different metal compounds. The use of Cu + Zn proved to be the most active inducer of SOD activity. Cu + Zn treatment enhanced the fungal tolerance to oxidative stress generated by menadione in the medium (0–3 mM) as evaluated by colony growth. The conidial tolerance to UV-B radiation and heat was evaluated by assays of spore germination. Conidia produced on cultures with Cu + Zn were more tolerant to UV-B and thermal stress as well as exhibiting a higher rate of virulence against P. xylostella larvae. Our study highlights that SOD contributes significantly to the virulence and stress tolerance of I. fumosorosea and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of fungal pathogens.     

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione–GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

球孢白僵菌响应氧化胁迫的分子机制研究进展

球孢白僵菌作为模式丝状真菌,以分生孢子、菌丝体、虫菌体等多种形态存在,在真菌孢子发育、寄主与宿主互作的研究中具有重要意义。同时,球孢白僵菌又是一类广泛应用的真菌杀虫剂,对森林防护和农业生产具有实际应用价值。球孢白僵菌的相关基因被敲除后,突变体响应氧化胁迫,孢子发育和毒力会发生改变。本文综述了近年来球孢白僵菌在响应氧化胁迫方面的研究进展,为丝状真菌氧化胁迫信号途径的研究提供参考。     

Cytotoxicity Mechanism of Two Naphthoquinones (Menadione and Plumbagin) in Saccharomyces cerevisiae

Background

Quinones are compounds extensively used in studies of oxidative stress due to their role in plants as chemicals for defense. These compounds are of great interest for pharmacologists and scientists, in general, because several cancer chemotherapeutic agents contain the quinone nucleus. However, due to differences in structures and diverse pharmacological effects, the exact toxicity mechanisms exerted by quinones are far from elucidatation.

Methodology/Principal Findings

Using Saccharomyces cerevisiae, we evaluated the main mechanisms of toxicity of two naphthoquinones, menadione and plumbagin, by determining tolerance and oxidative stress biomarkers such as GSH and GSSG, lipid peroxidation levels, as well as aconitase activity. The importance of glutathione transferases (GST) in quinone detoxification was also addressed. The GSSG/GSH ratio showed that menadione seemed to exert its toxicity mainly through the generation of ROS while plumbagin acted as an electrophile reacting with GSH. However, the results showed that, even by different pathways, both drugs were capable of generating oxidative stress through their toxic effects. Our results showed that the control strain, BY4741, and the glutathione transferase deficient strains (gtt1Δ and gtt2Δ) were sensitive to both compounds. With respect to the role of GST isoforms in cellular protection against quinone toxicity, we observed that the Gtt2 deficient strain was unable to overcome lipid peroxidation, even after a plumbagin pre-treatment, indicating that this treatment did not improve tolerance when compared with the wild type strain. Cross-tolerance experiments confirmed distinct cytotoxicity mechanisms for these naphthoquinones since only a pre-treatment with menadione was able to induce acquisition of tolerance against stress with plumbagin.

Conclusions/Significance

These results suggest different responses to menadione and plumbagin which could be due to the fact that these compounds use different mechanisms to exert their toxicity. In addition, the Gtt2 isoform seemed to act as a general protective factor involved in quinone detoxification.     

A conidial protein (CP15) of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses

Aerial conidia are central dispersing structures for most fungi and represent the infectious propagule for entomopathogenic fungus Beauveria bassiana, thus the active ingredients of commercial mycoinsecticides. Although a number of formic-acid-extractable (FAE) cell wall proteins from conidia have been characterized, the functions of many such proteins remain obscure. We report that a conidial FAE protein, termed CP15, isolated from B. bassiana is related to fungal tolerance to thermal and oxidative stresses. The full-length genomic sequence of CP15 was shown to lack introns, encoding for a 131 amino acid protein (15.0 kDa) with no sequence identity to any known proteins in the NCBI database. The function of this new gene with two genomic copies was examined using the antisense-RNA method. Five transgenic strains displayed various degrees of silenced CP15 expression, resulting in significantly reduced conidial FAE protein profiles. The FAE protein contents of the strains were linearly correlated to the survival indices of their conidia when exposed to 30-min wet stress at 48°C (r ² = 0.93). Under prolonged 75-min heat stress, the median lethal times (LT₅₀s) of their conidia were significantly reduced by 13.6–29.5%. The CP15 silenced strains were also 20–50% less resistant to oxidative stress but were not affected with respect to UV-B or hyperosmotic stress. Our data indicate that discrete conidial proteins may mediate resistance to some abiotic stresses, and that manipulation of such proteins may be a viable approach to enhancing the environmental fitness of B. bassiana for more persisting control of insect pests in warmer climates.     

A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogen

A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn²⁺, suppressed by Cu²⁺ and Zn²⁺ but inhibited by Fe³⁺. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 ± 6.1 U mg⁻¹ protein) by 4–10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 ± 74.7 U mg⁻¹ was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC₅₀ = 2.41 ± 0.03 versus 1.25 ± 0.01 mM) and conidial germination (EC₅₀ = 0.89 ± 0.06 versus 0.55 ± 0.07 mM), and 23% more tolerant to UV-B irradiation (LD₅₀ = 0.49 ± 0.02 versus 0.39 ± 0.01 J cm⁻²). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT₅₀ = 4.5 (4.2–4.8) versus 5.7 (5.2–6.4) days]. Our study highlights for the first time that the Mn²⁺-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.     

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

Application of a YHB1-GFP reporter to detect nitrosative stress in yeast

Abstract

Yeast flavohemoglobin (Yhb1p) plays a pivotal role in NO^? detoxification and has also been implicated in oxidative/reductive stress responses. In this study, we have used a YHB1-GFP reporter expressing a full-length chromosome-tagged Yhb1-GFP fusion protein to monitor changes in flavohemoglobin levels after cell treatment with oxidants, antioxidants and nitric oxide donors. Only nitric oxide donors were found to induce a dose-dependent increase in Yhb1-GFP expression while hydrogen peroxide, menadione and diamide caused a moderate diminution of YHB1-GFP fluorescence. Additionally, the levels of endogenous and hydroperoxide-induced ROS production in the Δyhb1 mutant were comparable to those in the isogenic wild-type strain. Although peroxides increased NO^? generation while nitrite and nitric oxide donors augmented ROS levels in yeast cells, their effects were generally not more pronounced in Δyhb1 than in wild-type cells. Taken together, these data suggest that yeast flavohemoglobin does not contribute to cross-protection against oxidative and nitrosative stress.     

Sulfonylurea resistance as a new selectable marker for the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a filamentous ascomycete that is pathogenic towards a broad host range of insect targets and is increasingly serving as a model for examining fungal development and host-pathogen interactions. B. bassiana displays a prohibitive level of resistance against many current fungal and/or yeast selection markers including hygromycin, neomycin, and zeocin. A genetic transformation system for B. bassiana based upon the use of a sulfonylurea resistance cassette derived from the Magnaporthe grisea, acetolactate synthase gene (sur) was developed. The transformation frequency ranged from 100–150 transformants per microgram DNA/10⁸ cells and Southern blot analysis indicated that the plasmid vector was randomly integrated into the genome of B. bassiana. In addition, a construct bearing the sur gene and the enhanced green fluorescent protein gene egfp as a visual marker was used to successfully transform B. bassiana. Over 95% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. The described transformation method increases opportunities for the genetic manipulation of B. bassiana.     

Rice ASR1 protein with reactive oxygen species scavenging and chaperone-like activities enhances acquired tolerance to abiotic stresses in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Abscisic acid stress ripening (ASR1) protein is a small hydrophilic, low molecular weight, and stress-specific plant protein. The gene coding region of ASR1 protein, which is induced under high salinity in rice (Oryza sativa Ilmi), was cloned into a yeast expression vector pVTU260 and transformed into yeast cells. Heterologous expression of ASR1 protein in transgenic yeast cells improved tolerance to abiotic stresses including hydrogen peroxide (H₂O₂), high salinity (NaCl), heat shock, menadione, copper sulfate, sulfuric acid, lactic acid, salicylic acid, and also high concentration of ethanol. In particular, the expression of metabolic enzymes (Fba1p, Pgk1p, Eno2p, Tpi1p, and Adh1p), antioxidant enzyme (Ahp1p), molecular chaperone (Ssb1p), and pyrimidine biosynthesis-related enzyme (Ura1p) was up-regulated in the transgenic yeast cells under oxidative stress when compared with wild-type cells. All of these enzymes contribute to an alleviated redox state to H2O2-induced oxidative stress. In the in vitro assay, the purified ASR1 protein was able to scavenge ROS by converting H₂O₂ to H₂O. Taken together, these results suggest that the ASR1 protein could function as an effective ROS scavenger and its expression could enhance acquired tolerance of ROS-induced oxidative stress through induction of various cell rescue proteins in yeast cells.     

Characterization of Beauveria bassiana neutral trehalase (BbNTH1) and recognition of crucial stress-responsive elements to control its expression in response to multiple stresses

A neutral trehalase (NTH1) of fungal entomopathogen Beauveria bassiana was characterized for the first time as a 743-aa enzyme (84.4 kDa). To identify crucial stress-responsive elements (STREs) to control the expression of the NTH-coding gene (BbNTH1) in response to different stresses, the full-length promoter (−2713 bp) upstream of its open reading frame and three upstream-truncated fragments (−1912, −1060 and −560 bp) were fused to the reporter gene eGFP and then transformed into B. bassiana, respectively. Consequently, eGFP was well expressed as intensive fluorescence in mycelia, conidiogenic cells and forming conidia controlled by the full-length promoter with five STREs. Surprisingly, transformants controlled by the shortest fragment with last two STREs at −315 and −274 bp exhibited consistently brightest fluorescence in mycelia under 3-h oxidative adaption of 0.3-1.2 mM menadione, and in colonies under 6-day osmotic stress of 0.5-1 M NaCl and thermal stress of 15-540 min at 40 °C after 3-day growth at 25 °C. Single or dual site-directed mutations of the two STREs from CCCCT to CATCT significantly altered the gene response to the multiple stresses. Thus, the two STREs in the downstream 560-bp region of the promoter are crucial to regulating not only constitutive but stress-inducible expression of the target gene.     

鸭疫里氏杆菌B739＿RS00825基因缺失株抗血红素毒性和氧化应激损伤以及定殖能力分析

【目的】血红素可作为细菌重要的铁离子来源,然而转运过多的血红素也会对细菌造成毒性。细菌通过调节、外排、螯合等多种方式减轻血红素毒性作用。鸭疫里氏杆菌(Riemerella anatipestifer, RA)是一种感染鸭及其他禽类的革兰氏阴性病原菌。前期研究表明,该菌编码血红素转运系统,且能够从血红蛋白获取血红素,然而该菌是否编码血红素解毒蛋白未知。本研究以编码一氧化氮合成酶的基因B739＿RS00825为研究对象,分析其在抗血红素毒性和氧化应激损伤以及定殖能力中的功能。【方法】构建B739＿RS00825缺失株,并通过测定生长曲线、细菌存活率、毒力及定殖等试验方法鉴定其在抗血红素毒性、抗氧化应激损伤、宿主致病中的功能。【结果】与RA CH-1相比,RA CH-1ΔB739＿RS00825在添加过量血红素的培养基中生长不受影响;然而与RACH-1Δfur相比,RACH-1ΔfurΔB739＿RS00825在含血红素培养基中的生长明显受到抑制且对H₂O₂的抵抗力降低;B739＿RS00825基因在氧化应激条件下及fur缺失株中明显上调;与RA ...     

Multifunctional role of a fungal pathogen-secreted laccase 2 in evasion of insect immune defense

Laccases are widely present in bacteria, fungi, plants and invertebrates and involved in a variety of physiological functions. Here, we report that Beauveria bassiana, an economic important entomopathogenic fungus, secretes a laccase 2 (BbLac2) during infection that detoxifies insect immune response-generated reactive oxygen species (ROS) and interferes with host immune phenoloxidase (PO) activation. BbLac2 is expressed in fungal cells during proliferation in the insect haemocoel and can be found to distribute on the surface of haemolymph-derived in vivo fungal hyphal bodies or be secreted. Targeted gene-knockout of BbLac2 increased fungal sensitivity to oxidative stress, decreased virulence to insect, and increased host PO activity. Strains overexpressing BbLac2 showed increased virulence, with reduced host PO activity and lowered ROS levels in infected insects. In vitro assays revealed that BbLac2 could eliminate ROS and oxidize PO substrates (phenols), verifying the enzymatic functioning of the protein in detoxification of cytotoxic ROS and interference with the PO cascade. Moreover, BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns (PAMPs), enabling the pathogen to evade immune recognition. Our data suggest a multifunctional role for fungal pathogen-secreted laccase 2 in evasion of insect immune defenses.