Intracellular proteome expression during 4-n-nonylphenol biodegradation by the filamentous fungus Metarhizium robertsii

4-n-nonylphenol (4-n-NP) is an endocrine disrupting compound (EDC); pollutants that cause serious disturbances in the environment. This study shows the degradation pathway and initial proteome analysis in cultures of a fungus that actively degrades 4-n-NP, Metarhizium robertsii. The research revealed the presence of 14 4-n-NP metabolites formed as a result of the oxidation of the alkyl chain and benzene ring, which leads to the complete decomposition of the compound. Based on the trend and quantitative analysis of the formation of 4-n-NP derivatives, the best conditions for proteome analysis were established. The data collected allowed the formulation of an explanation of the microorganism's strategy towards the removal of 4-n-NP. The main groups of proteins engaged in the removal of the xenobiotic are: oxidation-reduction systems related to nitroreductase-like proteins, ROS defense systems (peroxiredoxin and superoxide dismutase), the TCA cycle and energy-related systems. Principal components analysis was applied to unidentified proteins, resulting in the formulation of three subgroups and initial classification of these proteins.     

Chlorimuron ethyl as a new selectable marker for disrupting genes in the insect-pathogenic fungus Metarhizium robertsii

A lack of selectable markers was a hindrance in investigating gene function in Metarhizium robertsii. A reliable Agrobacterium-mediated transformation system based on the use of chlorimuron ethyl as the selectable marker was developed which could serve as a useful tool to inactivate genes involved in insect pathogenicity.     

Overexpression of a Metarhizium robertsii HSP25 gene increases thermotolerance and survival in soil

Temperature extremes are an important adverse factor limiting the effectiveness of microbial pest control agents. They reduce virulence and persistence in the plant root-colonizing insect pathogen Metarhizium robertsii. Small heat shock proteins have been shown to confer thermotolerance in many organisms. In this study, we report on the cloning and characterization of a small heat shock protein gene hsp25 from M. robertsii. hsp25 expression was upregulated when the fungus was grown at extreme temperatures (4, 35, and 42 °C) or in the presence of oxidative or osmotic agents. Expression of hsp25 in Escherichia coli increased bacterial thermotolerance confirming that hsp25 encodes a functional heat shock protein. Overexpressing hsp25 in M. robertsii increased fungal growth under heat stress either in nutrient-rich medium or on locust wings and enhanced the tolerance of heat shock-treated conidia to osmotic stress. In addition, overexpression of hsp25 increased the persistence of M. robertsii in rhizospheric soils in outdoor microcosms, though it did not affect survival in bulk soil, indicating that M. robertsii's survival in soil is dependent on interactions with plant roots.     

罗伯茨绿僵菌中嘧啶前体合成酶基因MrThi12的功能研究

通过同源基因比对,在罗伯茨绿僵菌中找到了单拷贝的嘧啶前体合成酶基因MAA_02402,命名为MrThi12。该基因MrThi12全长1 234bp,cDNA序列全长1 029bp,编码342个氨基酸。构建同源重组载体,利用农杆菌介导的方法进行基因敲除。突变菌株在维生素B1缺乏的培养基上,生长很慢,菌丝形态异常,多分叉,完全不能产生气生菌丝和分生孢子。但是一旦有外源维生素B1时,生长状态能完全恢复,对家蚕的致死能力没有变化。     

mro-miR-33在绿僵菌产孢中的作用

罗伯茨绿僵菌Metarhizium robertsii是一种重要的广谱性杀虫真菌,在害虫的生物防治中应用广泛。研究表明microRNAs在丝状真菌的生长发育中可能发挥重要调控作用。迄今有关miRNAs在绿僵菌产孢过程中的作用还未见研究报道。本文分析了实验室前期鉴定的一个绿僵菌新miRNA(mro-miR-33)在绿僵菌产孢过程中的作用。结果表明,绿僵菌在不同培养条件下mro-miR-33的表达量有显著差异,且在孢子发育过程表达量下降。在野生菌株中过表达mro-miR-33后,绿僵菌的产孢量显著下降,qRT-PCR分析表明孢子发育关键调控基因brl A表达显著下调,而敲除mro-miR-33后,绿僵菌的产孢量显著增加。这些变化表明mro-miR-33在绿僵菌的产孢过程中发挥着重要的作用。     

Localization of the insect pathogenic fungal plant symbionts Metarhizium robertsii and Metarhizium brunneum in bean and corn roots

Metarhizium is an insect pathogenic fungus and a plant root symbiont. Here the root association patterns (rhizoplane or endophytic colonization) were analyzed in common beans (Phaseolus vulgaris) and sweet corn (Zea mays) using M. robertsii and M. brunneum under various vermiculite treatments (control, with sucrose, with an insect) at two time points of plant growth (10 and 20 days). We observed that M. brunneum and M. robertsii preferentially endophytically colonized the hypocotyl, however, greater rhizoplane colonization was observed at the regions proximal to the hypocotyl in both plants. Vermiculite amended with an infected insect resulted in greater endophytic and rhizoplane colonization at 20 days compared to 10 days, for both plants as well as for both Metarhizium species. Regardless of the vermiculite treatment, corn was preferentially colonized compared to bean. Sucrose amendment in the vermiculite and infected insect amended vermiculite only showed differences in rhizoplane colonization. The greatest root association occurred with M. brunneum with an infected insect and that in corn after 20 days.     

High frequency gene conversion among benomyl resistant transformants in the entomopathogenic fungus Metarhizium anisopliae

Abstract Three different methods, (i) PEG, (ii) electroporation and (iii) biolistic, were employed to transform Metarhizium anisopliae using benomyl resistance as a selectable marker. Transformation frequencies and mitotic stability varied for each method, from 0.8 to 6.9 transformants μg⁻¹ of DNA and 46%, respectively, by the PEG method; 1.3 to 1.8 transformants μg⁻¹ of DNA and 67% by electroporation; and 32 to 201 transformants μg⁻¹ of DNA and 90% by biolistic. We demonstrate by PCR that 60% of the transformants were generated by gene conversion.     

Pyrolysis-gas chromatography of the fungus Metarhizium anisopliae: An aid to strain identification

Six strains of the entomopathogenic fungus Matarhizium anisopliae var. anisopliae from North and South America and one strain of M. anisopliae var. major from Samoa were compared by pyrolysis-gas chromatography of conidia. Two strains established to be very similar by other methods proved 98% similar by pyrolysis-gas chromatography. Similarities of the other strains ranged from 62 to 88%. The method is proposed as a simple technique for routine identification of M. anisopliae strains.     

Detection and characterisation of pr1 virulent gene deficiencies in the insect pathogenic fungus Metarhizium anisopliae

The method of suppressive subtractive hybridization was employed to map out genomic differences between the highly pathogenic Yersinia enterocolitica (Ye) biogroup 1B, serotype O:8 strain (WA-314) and the closely related apathogenic Y. enterocolitica biogroup 1A, serotype O:5 strain (NF-O). A novel IS10-like element, IS1330, uncovered by this technique was found to be uniquely present in high copy numbers among the highly pathogenic Y. enterocolitica 1B strains, while a single copy of the element was found in the low pathogenic Ye biogroup 4 serotype O:3 strain. The 1321-bp repetitive element has 19-bp imperfect inverted terminal repeats and is bracketed by a 10-bp duplication of the target sequence. The predicted transposase shares high homology with the IS10 open reading frame of the large virulence plasmid pWR501, of Shigella flexneri, with IS10 transposase of Salmonella typhi, and with IS1999 (tnpA) of Pseudomonas aeruginosa. The IS1330 tnp gene is transcribed in vitro and in vivo in HeLa cells. At least one copy of IS1330 flanks the recently described chromosomal type III secretion cluster in Y. enterocolitica WA-314, O:8, and future studies should shed light on whether this novel transposase mediates transposition events in highly pathogenic Y. enterocolitica strains, thus enhancing the genetic plasticity of this species.     

精胺合成酶MrSPS参与调控罗伯茨绿僵菌的生长发育、抗逆和致病力

本研究以罗伯茨绿僵菌Metarhizium robertsii为研究对象,针对鉴定出的精胺合成酶基因(MAA_02088, Mrsps),利用农杆菌介导的同源重组方法获得Mrsps敲除株ΔMrsps。与野生型相比,ΔMrsps营养生长和产孢能力下降,对氯化钠和紫外照射耐受性增强。大蜡螟幼虫毒力分析表明,浸渍和注射两种情况下ΔMrsps致病力降低,半致死时间(LT₅₀：6.71和4.75 d)比野生型(LT₅₀：5.17和4.19 d)显著增加。Mrsps敲除后不影响附着胞形成率和蝉翅穿透能力,但会显著下调昆虫血腔定殖相关基因的表达量。这些结果说明精胺合成酶MrSPS参与调控罗伯茨绿僵菌的生长发育、外界胁迫应答和致病力。     

Identification of a putative vacuolar serine protease gene in the rice blast fungus,Magnaporthe grisea

We identified and cloned a gene designated SPM1, encoding a serine protease from the rice blast fungus Magnaporthe grisea. SPM1 is a single-copy gene, encoding a subtilisin-like serine protease with 536 amino acids. Analyses of the deduced amino acid sequence of SPM1 suggested that SPM1 would be localized in a vacuole, an important organelle in pathogenicity.     

Mapmi gene contributes to stress tolerance and virulence of the entomopathogenic fungus, Metarhizium acridum

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate (Fru-6-P) and mannose 6-phosphate (Man-6-P), providing a link between glycolysis and the mannose metabolic pathway. In this study, we identified pmi gene (Mapmi) from the entomopathogenic fungus, Metarhizium acridum, and analyzed its functions using RNA interference (RNAi). Amending the growth medium with cell stress chemicals significantly reduced growth, conidial production and percent germination in Mapmi-RNAi mutant strain, compared to the wild-type strain. Growth of RNAi mutant was lower than the wild type strain with glucose or fructose as sole carbon source. RNAi mutant exhibited a normal growth phenotype with mannose at low concentrations, while trace or high concentration of mannose was more negatively impacted the growth of RNAi mutant than the wild type strain. Infection with Mapmi-RNAi mutant against Locusta migratoria manilensis (Meyen) led to a significantly reduced virulence compared to infection with the wild-type strain. These results suggest that Mapmi plays essential roles in stress tolerance and pathogenicity of M. acridum.     

Energy requirement for the mobilization of vacuolar arginine in Neurospora crassa

The bulk of the intracellular arginine pool in exponentially growing mycelia of Neurospora crassa is sequestered in the vacuoles. Vacuolar arginine effluxes from the vacuoles into the cytosol and is catabolized to ornithine and urea upon nitrogen starvation. The energy requirement for mobilization has been studied by treating nitrogen-starved mycelia with inhibitors or respiration or glycolysis or an uncoupler of respiration. Mobilization was inhibited by the inhibitors or the uncoupler of respiration, but not by the inhibitors of glycolysis. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge of the treated mycelia. The inhibitors of glycolysis reduced the ATP pool but had no effect on the energy charge. The results indicate that mobilization of arginine from the vacuoles requires metabolic energy. The forms of this energy and the mode of its association with the mobilization process are discussed.     

An inactivating point mutation in the inhibitory wedge of CD45 causes lymphoproliferation and autoimmunity

A model has been proposed for the regulation of CD45, and by homology other RPTPs, in which dimerization inhibits phosphatase activity through symmetrical interactions between an inhibitory structural wedge and the catalytic site. Here, we report the phenotype of mice with a single point mutation, glutamate 613 to arginine, that inactivates the inhibitory wedge of CD45. The CD45 E613R mutation causes polyclonal lymphocyte activation leading to lymphoproliferation and severe autoimmune nephritis with autoantibody production, resulting in death. Both homozygotes and heterozygotes develop pathology, indicating genetic dominance of CD45 E613R. The dramatic phenotype of CD45 E613R mice demonstrates the in vivo importance of negative regulation of CD45 by dimerization, supporting the model for regulation of CD45, and RPTPs in general.     

Novel GTP-binding proteins in plasma membranes of the fungus Metarhizium anisopliae

We report the existence of several families of GTP-binding proteins in plasma membranes of Metarhizium anisopliae. Two proteins (18.4 and 24 kDa) resemble mammalian Gn-proteins in their being toxin insensitive, binding [alpha-32P]GTP on nitrocellulose blots of sodium dodecyl sulfate (SDS)-polyacrylamide gels, and also in their immunological properties. Four other proteins (31-38.2 kDa) were similar except that they did not bind [alpha-32P]GTP after treatment with sodium dodecyl sulfate. An 18.2 kDa cholera toxin substrate and three toxin insensitive bands (18.6, 18.8, and 24 kDa) are novel proteins antigenically related both to mammalian G-proteins and ras gene products. An additional 23 kDa pertussis toxin substrate (the major G-protein in a crude mycelial extract) reacted strongly with antisera to G-proteins but not with anti-ras serum. Other substrates ADP ribosylated by cholera toxin or botulinum D toxin were immunologically unreactive. Analysis of the structural and functional characteristics of these multiple GTP-binding proteins will promote a better understanding of signal transduction in fungi.     

金龟子绿僵菌(Metarhizium anisopliae HN1)几丁质酶基因的克隆及高效表达

几丁质酶是昆虫病原真菌金龟子绿僵菌致病力的主要因子之一。本实验用RT-PCR方法,从本实验室分离筛选到的高毒力金龟子绿僵菌Metarhizium anisopliae HN1中,扩增得到几丁质酶基因全长,此基因全长为1275bp,登录号为DQ011865,经Blastn分析此基因序列与M. anisopliae E6的chi1基因（AF02749）同源率为96% 。以pET-22b(+)为基础载体,构建pET-chi重组表达载体,在大肠杆菌（Escherichia. coli ）BL 21中进行表达。经SDS-PAGE分析,获得了42kDa大小的重组目的蛋白,目的蛋白占表达总蛋白含量的63.3%。菌体经冷冻与超声波破碎后,按DNS法可测得几丁质酶的活性。     

Identification of an arginine carrier in the vacuolar membrane of Neurospora crassa

A number of arginine derivatives were tested for their ability to inhibit arginine uptake into vacuolar membrane vesicles of Neurospora crassa. The guanido side chain and L-configuration were found to be important for recognition by the arginine carrier. Based upon the specificity of recognition, a reactive arginine derivative (N alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane) was synthesized which has an intact guanido side chain and a diazo group at the carboxyl end. The latter decomposes to a reactive carbene group. This derivative inhibited arginine uptake into vacuolar membrane vesicles at low concentrations. Radioactive N alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane was covalently bound to vacuoles. Binding was specific for a single membrane protein with an approximate molecular weight of 40,000, saturable (2 pmol/mg vacuolar membrane protein), and inhibited by 100 mM L-arginine but not by 100 mM L-lysine. The results suggest that this protein is the arginine carrier.