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Alternative oxidase activity in Metarhizium anisopliae and Neurospora crassa when grown in yeast extract-peptonedextrose (YPD) or YPD + chloramphenicol (Cam) for 10 and 6 days for M. anisopliae and N. crassa, respectively. The respiration rate of the treatments without inhibitors for YPD and YPD-Cam were 842 and 1290 mmol O2Á(mg dry mass)-1 Ámin-1 , respectively. The bars above the columns represent the standard deviation (n = 4). KCN, potassium cyanide; SHAM, salicylhydroxamic acid.
Source publication
Mitochondria of Metarhizium anisopliae contain an alternative oxidase (AOX), which reduces oxygen to water by accepting electrons directly from ubiquinol. AOX activity is demonstrated in situ as a constitutive enzyme. Greatest activity of AOX appears at the beginning and at the end of the fungal developmental cycle, germination of aerial conidia an...
Contexts in source publication
Context 1
... of KCN to M. anisopliae cultures incubated in YPD and YPD+Cam media maintained approximately 80% of respiration capacity compared with 20% and 93% main- tained with N. crassa incubated in YPD and YPD+Cam, re- spectively ( Fig. 1). This result demonstrates the presence of AOX activity in M. anisopliae, which drops to approxi- mately 26% after addition of KCN plus SHAM (the specific AOX inhibitor). The data show constitutive expression of AOX in M. anisopliae, as compared with N. crassa, which shows differences in expression depending on the presence or absence ...
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... has virtually no effect on the respiration rate, indicat- ing a high level of alternative oxidase. It may also explain the lower oxygen consumption of M. anisopliae in the treat- ment with Cam when SHAM was added first to the respira- tion chamber. Together, these data suggest an even higher reliance of M. anisopliae on alternative respiration (Fig. 1). Interestingly, the treatment with YPD+Cam presented 18% more oxygen consumption than the treatment with YPD in the presence of SHAM+KCN, which we suspect may be due to the induction of yet another terminal oxidase system reported for the phytopathogenic fungus Gaeumannomyces graminis var. tritici (Joseph- Horne et al. 1998; ...
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... results presented in Fig. 1 are dependent on the as- sumption that both respiratory inhibitors, KCN and SHAM, are taken up by M. anisopliae cells regardless of whether mycelia or conidia arise from submerged growth, as both were present in the cultures. We used permeabilization of conidia using the method of Chelico and Khachatourians (2003). A solution of 10% ...
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... B. The asterisk (*) indicates conserved residues, differing from those in the boxes. The closed arrowheads indicate the invariant tyrosine, which may have a relevant functional role. The open arrowheads indicate the proposed metal ligands binding sites of the Andersson and Nordlund (1999) model. chamber of a 10-day-old culture of M. anisopliae (Fig. 1) despite the presence of a functional AOX throughout growth. Therefore, the AOX is likely to be produced in the cells during the entire growth but activated when the fungal cells are under stress conditions such as the presence of in- hibitors of the cytochrome pathway (Medentsev and Akimenko 1999). It is clear that in YPD alone, the ...
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... hibitors of the cytochrome pathway (Medentsev and Akimenko 1999). It is clear that in YPD alone, the presence of KCN did not interrupt respiration in M. anisopliae as it did in N. crassa. It may suggest that AOX is not only constitu- tively produced in M. anisopliae, but also functional, at least after 10 days of culture, as was the evaluation in Fig. 1. The low and lack of reduction in respiration when KCN and SHAM, respectively, were added first to the respiration chamber of a 10-day-old culture of M. anisopliae (Fig. 1) suggest that here, as compared with N. crassa, the cyto- chrome pathway and AOX can accommodate the entire flow of electrons even when one pathway is impaired. This ...
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... in N. crassa. It may suggest that AOX is not only constitu- tively produced in M. anisopliae, but also functional, at least after 10 days of culture, as was the evaluation in Fig. 1. The low and lack of reduction in respiration when KCN and SHAM, respectively, were added first to the respiration chamber of a 10-day-old culture of M. anisopliae (Fig. 1) suggest that here, as compared with N. crassa, the cyto- chrome pathway and AOX can accommodate the entire flow of electrons even when one pathway is impaired. This explanation is consistent with the data of Medentsev and Akimenko (1999), who indicated that in Yarrowia lipolytica both systems are equally functional to accommodate the ...
Citations
... and Metarhizium spp. (Khachatourians et al. 2008;Leclerque et al. 2004). Conidia are the main virulence determinants for fungal pathogenicity to insects; once attached to the cuticle, conidia germinate rapidly to produce appressoria and convert to penetration pegs to penetrate into insects. ...
Objectives
Microsclerotia (MS), anti-stress structures produced by many filamentous fungi, have been proven to be a great substitute for conidia in the production of insecticides within entomogenous fungi. NADPH oxidase (Nox) is a highly conserved ROS-response protein family that is widespread in eukaryotes and plays distinct roles in environmental fitness among various filamentous fungi. However, it is not clear whether the formation of MS and pathogenicity in entomogenous fungi is regulated by the Nox inside. In this study, we reported the presence of NADPH oxidase homologs in a great potential biocontrol fungus, Metarhizium rileyi, and further showed multiple biological functions.
Results
Three Nox homologous genes in M. rileyi showed high expression throughout the entire process of MS formation. Targeted deletion of MrNoxA, MrNoxB and MrNoxR all led to a decrease in MS yield and impaired morphology. Moreover, the anti-adversity assay showed that they are indispensable for growth, osmotic pressure and oxidative stress regulation in Metarhizium rileyi. Most importantly, △MrNoxR and △MrNoxA but not △MrNoxB showed a dramatic reduction in virulence via inoculation. The normality of appressoria might be unaffected in mutants since there are no striking differences in virulence compared with WT by topical injections.
Conclusion
Our results revealed that NADPH oxidase plays important roles in growth regulation, MS formation and pathogenicity in M. rileyi, perhaps in the ROS response and hyphal polarity.
... and Metarhizium spp. (Khachatourians et al. 2008; Leclerque et al. 2004). Conidia are the main virulent determinants of fungal pathogenicity; once attaching to cuticle, conidia will germinate rapidly to produce appressoria, convert to penetration pegs to penetrate and enter into insects, then intrusive hyphal body began to proliferate sharply, compete and consume host nutrients, secrete toxins into insect hemocoel, and result in the death of insect eventually. ...
Microsclerotia (MS), the anti-stress structures produced by many filamentous fungi, have been proved to be a great substitute for conidia in production of insecticides in entomogenous fungi. NADPH oxidase (Nox) constitutes ROS-response protein family widespread in eukaryotes and plays distinct roles in environmental fitness among various filamentous fungi. However, it is not clear whether Nox is required in the pathogenicity and formation of MS in insect pathogenic fungi. In this study, we reported the presence of NADPH oxidase homologs, which contain three different subunits (NoxAp/NoxBp catalytic subunit and NoxRp regulatory subunit), and further showed biological function in the entomopathogenic fungus, Metarhizium rileyi. Three Nox genes all showed high expression levels in the entire process of MS formation. Targeted deletion of MrNoxA , MrNoxB and MrNoxR all led to a decrease in MS yield and impaired morphology. Moreover, our anti-adversity results showed that they are indispensable to regulate the growth, tolerate the osmotic pressure and oxidative stress in Metarhizium rileyi . Most importantly, △MrNoxR and △MrNoxA but not △MrNoxB showed a dramatic reduction in virulence via inoculation. The normality of appressoria was impervious since there are no striking differences in virulence compared with WT and all mutants by topical injections. In general, our results revealed that NADPH oxidase may be involved in growth regulation, MS formation and pathogenicity by regulating the ROS metabolism and hyphal polarity.
... The number of nuclear genes encoding AOX varies, but typically, as in Arabidopsis, the various AOX isoforms are functionally distinct, differentially expressed in the tissues and life cycle, and respond differently to environmental cues [92,[134][135][136][137][138]. Similarly in fungi, except where AOX is absent altogether [2,133], its expression can be constitutive [133,139], variable according to life cycle stages [127,140], or regulated by environmental factors and substrates [14,139,[141][142][143]. Plant AOX is regulated chiefly by organic acids [144], whereas both fungal [141,[144][145][146][147] and protistan [148,149] AOXs are modulated by purine nucleotides, with a fungal-specific N-terminal tract of 20-25 amino acids suggested to confer this sensitivity [91]. Excellent reviews dealing extensively with AOX regulation are available [92,126,133] . ...
In a large number of organisms covering all phyla, the mitochondrial respiratory chain harbors, in addition to the conventional elements, auxiliary proteins that confer adaptive metabolic plasticity. The alternative oxidase (AOX) represents one of the most studied auxiliary proteins, initially identified in plants. In contrast to the standard respiratory chain, the AOX mediates a thermogenic cyanide-resistant respiration; a phenomenon that has been of great interest for over 2 centuries in that energy is not conserved when electrons flow through it. Here we summarize centuries of studies starting from the early observations of thermogenicity in plants and the identification of cyanide resistant respiration, to the fascinating discovery of the AOX and its current applications in animals under normal and pathological conditions.
... SDH is present in most of the species and additional enzymes with quinone reductase activity such as ETFQO, GlpD, DHOQO and SQR are also expected to be present, all of them being non-energy conserving enzymes. In addition, quinol may be oxidized directly by AOX or by the Complexes III and IV, in the latter case with contribution to the formation of membrane potential (Uribe & Khachatourians, 2008). S. cerevisiae may also conserve energy through V-ATPase by the hydrolysis of ATP to ADP (Zhou et al., 2015). ...
The diversity of microbial cells is reflected in differences in cell size and shape, motility, mechanisms of cell division, pathogenicity or adaptation to different environmental niches. All these variations are achieved by the distinct metabolic strategies adopted by the organisms. The respiratory chains are integral parts of those strategies especially because they perform the most or, at least, most efficient energy conservation in the cell. Respiratory chains are composed of several membrane proteins, which perform a stepwise oxidation of metabolites toward the reduction of terminal electron acceptors. Many of these membrane proteins use the energy released from the oxidoreduction reaction they catalyze to translocate charges across the membrane and thus contribute to the establishment of the membrane potential, i.e. they conserve energy. In this work we illustrate and discuss the composition of the respiratory chains of different taxonomic clades, based on bioinformatic analyses and on biochemical data available in the literature. We explore the diversity of the respiratory chains of Animals, Plants, Fungi and Protists kingdoms as well as of Prokaryotes, including Bacteria and Archaea. The prokaryotic phyla studied in this work are Gammaproteobacteria, Betaproteobacteria, Epsilonproteobacteria, Deltaproteobacteria, Alphaproteobacteria, Firmicutes, Actinobacteria, Chlamydiae, Verrucomicrobia, Acidobacteria, Planctomycetes, Cyanobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Aquificae, Thermotogae, Deferribacteres, Nitrospirae, Euryarchaeota, Crenarchaeota and Thaumarchaeota.
... AOX activity is known to be consistently present during aerobic metabolism in all plants, several yeast species, and fungi (Cabrera-Orefice et al. 2014;Van Aken et al. 1998), and it is regulated at the transcriptional level in the process of CA production (Hattori et al. 2009). Equivalent findings were reported for Yarrowia lipolytica (Medentsev et al. 2002) and Pichia pastoris (Kern et al. 2007), as well as Metarhizium anisopliae (Uribe and Khachatourians 2009) and Paracoccidioides brasiliensis (Martins et al. 2011). The results were essentially the same in A. niger. ...
In this work, we constructed the aox1 disruption strains 3–4 and 4–10, as well as the aox1 overexpression strains 72 and 102 in Aspergillus niger. The energy metabolism, EMP, TCA pathways, and flux were investigated for the citric acid (CA) overproduction via the aox1 overexpression among them. As expected, the overexpression of the aox1 gene enabled a higher growth rate than that of the rate of its parent strain in medium with respiratory chain inhibitors. In liquefied corn starch medium supplemented with 0.2 μg/mL antimycin A, the CA production of the overexpression strain 102 reached up to 169.1 g/L, whereas the highest value of the parent strain was 158.9 g/L. For the perspective of the aox1 disruption strain 4–10, the yield of CA dropped to 125.6 g/L, and the loose mycelial pellets forming in the medium also revealed that the fundamentally important role of AOX in A. niger lies in the resistance to oxidative stress under fully aerobic conditions. Based on real-time qPCR gene expression analysis and measurement of intracellular ATP and NADH levels, we came to a conclusion that the higher NADH oxidation rate resulting from the overexpression of the aox1 gene mainly contributed to rate-limited step’s acceleration and strengthened metabolic flow via mycelia and led to the CA yield in these strains increased by 13.5 and 10.8%, respectively. Subsequently, it was found that overexpression strains had higher AOX relative content and more oxygen consumption at different fermentation stages, which fully confirmed the close relationship between aox1 gene and energy metabolism, and comprehensively revealed aox1 gene function through the combination with the above conclusions.
... In fungi, AOX plays a role in growth regulation and development, resistance, pathogenesis and pathogenicity, and may contribute to fungal ecological fitness (Umbach and Siedow, 2000;Uribe and Khachatourians, 2008;Ruiz et al., 2011;Grahl et al., 2012;Thomazella et al., 2012;Xu T. et al., 2012). Unlike plants, in which AOX form small multigenic families, the analysis of the fungal genomes currently available reveals that a majority of fungal species possessing the AOX pathway have only one gene sequence, with a maximum of three sequences per genome (Mercy et al., 2015). ...
Arbuscular mycorrhizal fungi (AMF) are crucial components of fertile soils, able to provide several ecosystem services for crop production. Current economic, social and legislative contexts should drive the so-called “second green revolution” by better exploiting these beneficial microorganisms. Many challenges still need to be overcome to better understand the mycorrhizal symbiosis, among which (i) the biotrophic nature of AMF, constraining their production, while (ii) phosphate acts as a limiting factor for the optimal mycorrhizal inoculum application and effectiveness. Organism fitness and adaptation to the changing environment can be driven by the modulation of mitochondrial respiratory chain, strongly connected to the phosphorus processing. Nevertheless, the role of the respiratory function in mycorrhiza remains largely unexplored. We hypothesized that the two mitochondrial respiratory chain components, alternative oxidase (AOX) and cytochrome oxidase (COX), are involved in specific mycorrhizal behavior. For this, a complex approach was developed. At the pre-symbiotic phase (axenic conditions), we studied phenotypic responses of Rhizoglomus irregulare spores with two AOX and COX inhibitors [respectively, salicylhydroxamic acid (SHAM) and potassium cyanide (KCN)] and two growth regulators (abscisic acid – ABA and gibberellic acid – Ga3). At the symbiotic phase, we analyzed phenotypic and transcriptomic (genes involved in respiration, transport, and fermentation) responses in Solanum tuberosum/Rhizoglomus irregulare biosystem (glasshouse conditions): we monitored the effects driven by ABA, and explored the modulations induced by SHAM and KCN under five phosphorus concentrations. KCN and SHAM inhibited in vitro spore germination while ABA and Ga3 induced differential spore germination and hyphal patterns. ABA promoted mycorrhizal colonization, strong arbuscule intensity and positive mycorrhizal growth dependency (MGD). In ABA treated plants, R. irregulare induced down-regulation of StAOX gene isoforms and up-regulation of genes involved in plant COX pathway. In all phosphorus (P) concentrations, blocking AOX or COX induced opposite mycorrhizal patterns in planta: KCN induced higher Arum-type arbuscule density, positive MGD but lower root colonization compared to SHAM, which favored Paris-type formation and negative MGD. Following our results and current state-of-the-art knowledge, we discuss metabolic functions linked to respiration that may occur within mycorrhizal behavior. We highlight potential connections between AOX pathways and fermentation, and we propose new research and mycorrhizal application perspectives.
... Instead, these fungi have genes encoding NDH-2s [16]. Also usually, fungi have additional components, such as AOX [17][18]. Fungal parasites, like obligate intracellular Microsporidians, lack genes for energy metabolism and are strictly dependent on the host for energy. ...
... These genes are present in the three domains of life, Eukarya, Bacteria and Archaea, in 5%, 27% and 9% of the species, respectively (Fig. 3). The protists kingdom has two phyla with MQO, specifically 77% and 100% of Alveolates (17) and Haptophyta (1) species, respectively. Moreover, also in Eukarya, genes coding for MQO are observed in 50% of Cnidarians phyla (2 species). ...
Acquisition of energy is central to life. In addition to the synthesis of ATP, organisms need energy for the establishment and maintenance of a transmembrane difference in electrochemical potential allowing cells to import and export metabolites or to their motility. The membrane potential is established by a variety of membrane bound respiratory complexes. In this work we explored the diversity of membrane respiratory chains and the presence of the different enzyme complexes in the several phyla of life. We performed taxonomic profiles of the several membrane bound respiratory proteins and complexes evaluating the presence of their respective coding genes in all species deposited in KEGG database. We evaluated 26 quinone reductases, 5 quinol:electron carriers oxidoreductases and 18 terminal electron acceptor reductases. We further included in the analyses enzymes performing redox or decarboxylation driven ion translocation, ATP synthase and transhydrogenase and we also investigated the electron carriers that perform functional connection between the membrane complexes, quinones or soluble proteins. Our results bring a novel, broad and integrated perspective of membrane bound respiratory complexes and thus of the several energetic metabolisms of living systems. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
... Information on the biology and ecology of entomopathogenic fungi can be obtained from Steinhaus (1949Steinhaus ( , 1964, Evans (1989), and Balazy (1993). Uribe and Khachatourians (2008) have also described the life cycle of M. anisopliae under liquid culture conditions. 6.3.6 Isolation of Entomopathogenic Fungi 6.3.6.1 From Insects Entomopathogenic fungi have been isolated from infected insects and soil from around the world. ...
Lepidopterans are major pests of economically important crops with extensive host ranges. Entomopathogenic fungi can provide an alternative and more environment friendly approach for the control of Spodoptera spp. These fungi have wide host range and have been isolated from a variety of insect orders. In the following compilation, data on natural occurrence and distribution, general biology, mode of action, host range, isolation, multiplication, and bioassay of entomopathogenic fungi against lepidopteran pests are summarized. Use of entomopathogenic fungi as insect biological control agent, their current status as mycoinsecticides, and compatibility of fungi with other crop protection techniques along with control measures of Spodopteran spp. are also mentioned.
... Cellular antioxidants are also represented by such diverse molecules as the nonenzymatic vitamins C and E (Halliwell & Whiteman 2004), melanin (Nosanchuk & Casadevall 2003), and carotenoids (Luque et al. 2012). In addition, fungal mitochondria contain a constitutive enzyme alternative oxidase (AOX), which reduces oxygen to water by accepting electrons directly from ubiquinol (Uribe & Khachatourians 2008). ...
Entomopathogenic fungi are predisposed to ROS induced by heat and UV-A radiation when outside the insect host. When inside the host, they are subject to phagocytic cells that generate ROS to eliminate invading pathogens. The oxidative stress tolerance of the entomopathogenic fungi Aschersonia aleyrodis (ARSEF 430 and 10276), Aschersonia placenta (ARSEF 7637), Beauveria bassiana (ARSEF 252), Isaria fumosorosea (ARSEF 3889), Lecanicillium aphanocladii (ARSEF 6433), Metarhizium acridum (ARSEF 324), Metarhizium anisopliae (ARSEF 5749), Metarhizium brunneum (ARSEF 1187 and ARSEF 5626), Metarhizium robertsii (ARSEF 2575), Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), and Simplicillium lanosoniveum (ARSEF 6430 and ARSEF 6651) was studied based on conidial germination on a medium supplemented with menadione. Conidial germination was evaluated 24 h after inoculation on potato dextrose agar (PDA) (control) or PDA supplemented with menadione. The two Aschersonia species (ARSEF 430, 7637, and 10276) were the most susceptible fungi, followed by the two Tolypocladium species (ARSEF 3392 and 4877) and the M. acridum (ARSEF 324). Metarhizium brunneum (ARSEF 5626) and M. anisopliae (ARSEF 5749) were the most tolerant isolates with MIC 0.28 mM. All fungal isolates, except ARSEF 5626 and ARSEF 5749, were not able to germinate at 0.20 mM.
Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
... The yeast-like, dimorphic mode of growth in Beauveria bassiana was described by Alves et al (2002); and the production of oblong blastosporelike propagules in M. flavoviride was described by Fargues et al (2002). The life cycle of M. anisopliae under liquid culture conditions has also been described (Uribe and Khachatourians, 2008). ...
An attractive alternative method to chemical pesticides is the microbial biocontrol (MBCAs) agents. They are the natural enemies devastating the pest population with no hazard effects on human health and the environment. Entomopathogenic fungi has an important position among all the biocontrol agents because of its route of pathogenicity, broad host rang and its ability to control both sap sucking pests such as mosquitoes and aphids as well as pests with chewing mouthparts, yet they only cover a small percentage of the total insecticide market. Improvements are needed to fulfill the requirements for high market share. Entomopathogenic fungi differ from other microorganisms in their infection process: they directly breach the cuticle to enter the insect hemocoel, while other microorganisms enter by ingestion through mouth and then cause disease. Insect cuticle is mainly composed of chitin and protein surrounded by wax, lipid layer or fatty acids. Fungal pathogenesis mainly starts with the secretion of cuticle degrading enzymes. Some important and well known cuticle degrading enzymes are chitinase, protease and lipase which can degrade chitin, protein and lipid of the cuticle, respectively. In this review we collected literatures from different sources and we arranged them in a such a way to better to understand the process of insect pathogenicity of entomopathogenic fungi and to find ways to improve the virulence of wild strain fungi to shorten the killing time of the pest and to commercialize the entomopathogenic fungi. In this way, the market share of the fungal entomopathogenic fungi will increase and a decrease in the usage of synthetic chemical pesticides will automatically follow.
