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Department of Plant Pathology and Microbiology
The Robert H. Smith Faculty of Agriculture, Food & Environment
The Hebrew University of Jerusalem

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Rehovot 76100 
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Publications

2019
Druseikis, M. ; Ben-Ari, J. ; Covo, S. The Goldilocks effect of respiration on canavanine tolerance in Saccharomyces cerevisiae. Current Genetics 2019, 65, 1199-1215. Publisher's VersionAbstract
When glucose is available, Saccharomyces cerevisiae prefers fermentation to respiration. In fact, it can live without respiration at all. Here, we study the role of respiration in stress tolerance in yeast. We found that colony growth of respiratory-deficient yeast (petite) is greatly inhibited by canavanine, the toxic analog of arginine that causes proteotoxic stress. We found lower amounts of the amino acids involved in arginine biosynthesis in petites compared with WT. This finding may be explained by the fact that petite cells exposed to canavanine show reduction in the efficiency of targeting of proteins required for arginine biosynthesis. The retrograde (RTG) pathway signals mitochondrial stress. It positively controls production of arginine precursors. We show that canavanine abrogates RTG signaling especially in petite cells, and mutants in the RTG pathway are extremely sensitive to canavanine. We suggest that petite cells are naturally ineffective in production of some amino acids; combination of this fact with the effect of canavanine on the RTG pathway is the simplest explanation why petite cells are inhibited by canavanine. Surprisingly, we found that canavanine greatly inhibits colony formation when WT cells are forced to respire. Our research proposes a novel connection between respiration and proteotoxic stress. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature.
Anand, G. ; Waiger, D. ; Vital, N. ; Maman, J. ; Ma, L. J. ; Covo, S. How does Fusarium oxysporum sense and respond to nicotinaldehyde, an inhibitor of the NAD+ salvage biosynthesis pathway?. Frontiers in Microbiology 2019, 10. Publisher's VersionAbstract
Plant pathogenic fungi are a major threat to food security and impose a severe economic burden, thus there is a continuous need to develop new strategies to manage them. NAD+ is a co-factor in numerous enzymatic activities and determines the metabolic fate of the cell. Therefore, maintenance of NAD+ concentration is important for cellular viability. Consequently, the NAD+ biosynthetic pathway and redox homeostasis was suggested as a target for antifungal development. We aimed to study how Fusarium oxysporum senses and responds to nicotinaldehyde (NA), an inhibitor of Pnc1, a key enzyme in the salvage pathway of NAD+ biosynthesis. We were able to show that NA was inhibitory in high concentrations to several fungal plant pathogens, with much milder effects on tomato growth. Under low nutrient conditions NA reduced the total amounts of NAD+ in the fungal cell, a trend that was also observed in rich media, although without statistical significance. In low and high nutrient availability NA dramatically reduced the NAD+/NADH ratio. After exposure to NA, NADH levels were increased and NAD+ levels and the biomass were greatly reduced. Cells responded to NA by up-regulation of oxidoreductases, with hardly any up-regulation of the classic response to oxidative stress. Direct measurement of oxidative stress response showed that unlike formaldehyde and hydrogen peroxide, NA caused reductive rather than oxidative stress. Surprisingly, alcohol dehydrogenases were significantly up-regulated more than any other dehydrogenases, including aldehyde dehydrogenases. We propose that conidia of F. oxysporum efficiently detoxified the aldehyde group of NA by reducing NAD+ to NADH; the high concentrations of the latter provoked the expression of alcohol dehydrogenases that in yeast can act to reduce NADH and increase NAD+ amounts, respectively. Overall, the results suggest that targeting NAD+ biosynthesis pathway and redox homeostasis can be a potential approach to manage fungal plant pathogens. Many of the natural antifungal compounds produced by bio-control agents or even the natural biome are aldehydes, and thus the results presented here predict the possible response of Fusarium to wide sources of toxicity in the environment. © 2019 Frontiers Media S.A. All Rights Reserved.
Milo, S. ; Harari-Misgav, R. ; Hazkani-Covo, E. ; Covo, S. Limited DNA Repair Gene Repertoire in Ascomycete Yeast Revealed by Comparative Genomics. Genome biology and evolution 2019, 11, 3409-3423. Publisher's VersionAbstract
Ascomycota is the largest phylogenetic group of fungi that includes species important to human health and wellbeing. DNA repair is important for fungal survival and genome evolution. Here, we describe a detailed comparative genomic analysis of DNA repair genes in Ascomycota. We determined the DNA repair gene repertoire in Taphrinomycotina, Saccharomycotina, Leotiomycetes, Sordariomycetes, Dothideomycetes, and Eurotiomycetes. The subphyla of yeasts, Saccharomycotina and Taphrinomycotina, have a smaller DNA repair gene repertoire comparing to Pezizomycotina. Some genes were absent from most, if not all, yeast species. To study the conservation of these genes in Pezizomycotina, we used the Gain Loss Mapping Engine algorithm that provides the expectations of gain or loss of genes given the tree topology. Genes that were absent from most of the species of Taphrinomycotina or Saccharomycotina showed lower conservation in Pezizomycotina. This suggests that the absence of some DNA repair in yeasts is not random; genes with a tendency to be lost in other classes are missing. We ranked the conservation of DNA repair genes in Ascomycota. We found that Rad51 and its paralogs were less conserved than other recombinational proteins, suggesting that there is a redundancy between Rad51 and its paralogs, at least in some species. Finally, based on the repertoire of UV repair genes, we found conditions that differentially kill the wine pathogen Brettanomyces bruxellensis and not Saccharomyces cerevisiae. In summary, our analysis provides testable hypotheses to the role of DNA repair proteins in the genome evolution of Ascomycota. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Cohen, R. ; Milo, S. ; Sharma, S. ; Savidor, A. ; Covo, S. Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress. DNA Repair 2019, 83. Publisher's VersionAbstract
Ribonucleotide reductase (RNR) catalyzes the rate limiting step in dNTP biosynthesis and is tightly regulated at the transcription and activity levels. One of the best characterized responses of yeast to DNA damage is up-regulation of RNR transcription and activity and consequently, elevation of the dNTP pools. Hydroxyurea is a universal inhibitor of RNR that causes S phase arrest. It is used in the clinic to treat certain types of cancers. Here we studied the response of the fungal plant pathogen Fusarium oxysporum to hydroxyurea in order to generate hypotheses that can be used in the future in development of a new class of pesticides. F. oxysporum causes severe damage to more than 100 agricultural crops and specifically threatens banana cultivation world-wide. Although the recovery of F. oxysporum from transient hydroxyurea exposure was similar to the one of Saccharomyces cerevisiae, colony formation was strongly inhibited in F. oxysporum in comparison with S. cerevisiae. As expected, genomic and phosphoproteomic analyses of F. oxysporum conidia (spores) exposed to hydroxyurea showed hallmarks of DNA replication perturbation and activation of recombination. Unexpectedly and strikingly, RNR was not induced by either hydroxyurea or the DNA-damaging agent methyl methanesulfonate as determined at the RNA and protein levels. Consequently, dNTP concentrations were significantly reduced, even in response to a low dose of hydroxyurea. Methyl methanesulfonate treatment did not induce dNTP pools in F. oxysporum, in contrast to the response of RNR and dNTP pools to DNA damage and hydroxyurea in several tested organisms. Our results are important because the lack of a feedback mechanism to increase RNR expression in F. oxysporum is expected to sensitize the pathogen to a fungal-specific ribonucleotide inhibitor. The potential impact of our observations on F. oxysporum genome stability and genome evolution is discussed. © 2019 Elsevier B.V.
Vela-Corcía, D. ; Aditya Srivastava, D. ; Dafa-Berger, A. ; Rotem, N. ; Barda, O. ; Levy, M. MFS transporter from Botrytis cinerea provides tolerance to glucosinolate-breakdown products and is required for pathogenicity. 2019, 10, 2886. Publisher's VersionAbstract
Glucosinolates accumulate mainly in cruciferous plants and their hydrolysis-derived products play important roles in plant resistance against pathogens. The pathogen Botrytis cinerea has variable sensitivity to glucosinolates, but the mechanisms by which it responds to them are mostly unknown. Exposure of B. cinerea to glucosinolate-breakdown products induces expression of the Major Facilitator Superfamily transporter, mfsG, which functions in fungitoxic compound efflux. Inoculation of B. cinerea on wild-type Arabidopsis thaliana plants induces mfsG expression to higher levels than on glucosinolate-deficient A. thaliana mutants. A B. cinerea strain lacking functional mfsG transporter is deficient in efflux ability. It accumulates more isothiocyanates (ITCs) and is therefore more sensitive to this compound in vitro; it is also less virulent to glucosinolates-containing plants. Moreover, mfsG mediates ITC efflux in Saccharomyces cerevisiae cells, thereby conferring tolerance to ITCs in the yeast. These findings suggest that mfsG transporter is a virulence factor that increases tolerance to glucosinolates.
Herold, I. ; Kowbel, D. ; Delgado-Álvarez, D. L. ; Garduño-Rosales, M. ; Mouriño-Pérez, R. R. ; Yarden, O. Transcriptional profiling and localization of GUL-1, a COT-1 pathway component, in Neurospora crassa. Fungal Genetics and Biology 2019, 126, 1 - 11. Publisher's VersionAbstract
Impairment of theNeurospora crassaCOT-1 kinase results in defects in hyphal polarity. Some of these effects are partially suppressed by inactivation of gul-1 (encoding an mRNA-binding protein involved in translational regulation). Here, we report on the transcriptional profiling of cot-1 inactivation and demonstrate that gul-1 affects transcript abundance of multiple genes in the COT-1 pathway, including processes such as cell wall remodeling, nitrogen and amino acid metabolism. The GUL-1 protein itself was found to be distributed within the entire hyphal cell, along with a clear presence of aggregates that traffic within the cytoplasm. Live imaging of GUL-1-GFP demonstrated that GUL-1 transport is microtubule-dependent. Cellular stress, as imposed by the presence of the cell wall biosynthesis inhibitor Nikkomycin Z or by nitrogen limitation, resulted in a 2–3-fold increase of GUL-1 aggregate association with nuclei. Taken together, this study demonstrates that GUL-1 affects multiple processes, its function is stress-related and linked with cellular traffic and nuclear association.
Amend, A. ; Burgaud, G. ; Cunliffe, M. ; Edgcomb, V. P. ; Ettinger, C. L. ; Gutiérrez, M. H. ; Heitman, J. ; Hom, E. F. Y. ; Ianiri, G. ; Jones, A. C. ; et al. Fungi in the Marine Environment: Open Questions and Unsolved Problems. mBio 2019, 10, e01189-18. Publisher's VersionAbstract
Terrestrial fungi play critical roles in nutrient cycling and food webs and can shape macroorganism communities as parasites and mutualists. Although estimates for the number of fungal species on the planet range from 1.5 to over 5 million, likely fewer than 10% of fungi have been identified so far. To date, a relatively small percentage of described species are associated with marine environments, with ∼1,100 species retrieved exclusively from the marine environment. Nevertheless, fungi have been found in nearly every marine habitat explored, from the surface of the ocean to kilometers below ocean sediments. Fungi are hypothesized to contribute to phytoplankton population cycles and the biological carbon pump and are active in the chemistry of marine sediments. Many fungi have been identified as commensals or pathogens of marine animals (e.g., corals and sponges), plants, and algae. Despite their varied roles, remarkably little is known about the diversity of this major branch of eukaryotic life in marine ecosystems or their ecological functions. This perspective emerges from a Marine Fungi Workshop held in May 2018 at the Marine Biological Laboratory in Woods Hole, MA. We present the state of knowledge as well as the multitude of open questions regarding the diversity and function of fungi in the marine biosphere and geochemical cycles.
Wang, Z. ; Miguel-Rojas, C. ; Lopez-Giraldez, F. ; Yarden, O. ; Trail, F. ; Townsend, J. P. Metabolism and Development during Conidial Germination in Response to a Carbon-Nitrogen-Rich Synthetic or a Natural Source of Nutrition in  Neurospora crassa . mBio 2019, 10, e00192-19. Publisher's VersionAbstract
Fungal spores germinate and undergo vegetative growth, leading to either asexual or sexual reproductive dispersal. Previous research has indicated that among developmental regulatory genes, expression is conserved across nutritional environments, whereas pathways for carbon and nitrogen metabolism appear highly responsive—perhaps to accommodate differential nutritive processing. To comprehensively investigate conidial germination and the adaptive life history decision-making underlying these two modes of reproduction, we profiled transcription of Neurospora crassa germinating on two media: synthetic Bird medium, designed to promote asexual reproduction; and a natural maple sap medium, on which both asexual reproduction and sexual reproduction manifest. A later start to germination but faster development was observed on synthetic medium. Metabolic genes exhibited altered expression in response to nutrients—at least 34% of the genes in the genome were significantly downregulated during the first two stages of conidial germination on synthetic medium. Knockouts of genes exhibiting differential expression across development altered germination and growth rates, as well as in one case causing abnormal germination. A consensus Bayesian network of these genes indicated especially tight integration of environmental sensing, asexual and sexual development, and nitrogen metabolism on a natural medium, suggesting that in natural environments, a more dynamic and tentative balance of asexual and sexual development may be typical of N. crassa colonies.IMPORTANCE One of the most remarkable successes of life is its ability to flourish in response to temporally and spatially varying environments. Fungi occupy diverse ecosystems, and their sensitivity to these environmental changes often drives major fungal life history decisions, including the major switch from vegetative growth to asexual or sexual reproduction. Spore germination comprises the first and simplest stage of vegetative growth. We examined the dependence of this early life history on the nutritional environment using genome-wide transcriptomics. We demonstrated that for developmental regulatory genes, expression was generally conserved across nutritional environments, whereas metabolic gene expression was highly labile. The level of activation of developmental genes did depend on current nutrient conditions, as did the modularity of metabolic and developmental response network interactions. This knowledge is critical to the development of future technologies that could manipulate fungal growth for medical, agricultural, or industrial purposes.
Calderón, C. E. ; Rotem, N. ; Harris, R. ; Vela-Corcía, D. ; Levy, M. Pseudozyma aphidis activates reactive oxygen species production, programmed cell death and morphological alterations in the necrotrophic fungus Botrytis cinerea. Molecular Plant Pathology 2019, 20, 562 - 574. Publisher's VersionAbstract
Summary Many types of yeast have been studied in the last few years as potential biocontrol agents against different phytopathogenic fungi. Their ability to control plant diseases is mainly through combined modes of action. Among them, antibiosis, competition for nutrients and niches, induction of systemic resistance in plants and mycoparasitism have been the most studied. In previous work, we have established that the epiphytic yeast Pseudozyma aphidis inhibits Botrytis cinerea through induced resistance and antibiosis. Here, we demonstrate that P. aphidis adheres to B. cinerea hyphae and competes with them for nutrients. We further show that the secreted antifungal compounds activate the production of reactive oxygen species and programmed cell death in B. cinerea mycelium. Finally, P. aphidis and its secreted compounds negatively affect B. cinerea hyphae, leading to morphological alterations, including hyphal curliness, vacuolization and branching, which presumably affects the colonization ability and infectivity of B. cinerea. This study demonstrates additional modes of action for P. aphidis and its antifungal compounds against the plant pathogen B. cinerea.
Habtom, H. ; Pasternak, Z. ; Matan, O. ; Azulay, C. ; Gafny, R. ; Jurkevitch, E. Applying microbial biogeography in soil forensics. Forensic Science International: Genetics 2019, 38, 195 - 203. Publisher's VersionAbstract
The ubiquity, heterogeneity and transferability of soil makes it useful as evidence in criminal investigations, especially using new methods that survey the microbial DNA it contains. However, to be used effectively and reliably, more needs to be learned about the natural distribution patterns of microbial communities in soil. In this study we examine these patterns in detail, at local to regional scales (2 m–260 km), across an environmental gradient in three different soil types. Geographic location was found to be more important than soil type in determining the microbial community composition: communities from the same site but different soil types, although significantly different from each other, were still much more similar to each other than were communities from the same soil type but from different sites. At a local scale (25–1000 m), distance-decay relationships were observed in all soil types: the farther apart two soil communities were located, even in the same soil type, the more they differed. At regional-scale distances (1–260 km), differences between communities did not increase with increased geographic distance between them, and the dominant factor determining the community profile was the physico-chemical environment, most notably annual precipitation (R2 = 0.69), soil sodium (R2 = 0.49) and soil ammonium (R2 = 0.47) levels. We introduce a likelihood-ratio framework for quantitative evaluation of soil microbial DNA profile evidence in casework. In conclusion, these profiles, along with detailed knowledge of natural soil microbial biogeography, provide valuable forensic information on soil sample comparison and allow the determination of approximate source location on large (hundreds of km) spatial scales. Moreover, at small spatial scales it may enable pinpointing the source location of a sample to within at least 25 m, regardless of soil type and environmental conditions.
Gatica, J. ; Jurkevitch, E. ; Cytryn, E. Comparative Metagenomics and Network Analyses Provide Novel Insights Into the Scope and Distribution of β-Lactamase Homologs in the Environment. Frontiers in Microbiology 2019, 10, 146. Publisher's VersionAbstract
The β-lactams are the largest group of clinically applied antibiotics, and resistance to these is primarily associated with β-lactamases. There is increasing understanding that these enzymes are ubiquitous in natural environments and henceforth, elucidating the global diversity, distribution and mobility of β-lactamase-encoding genes is crucial for holistically understanding resistance to these antibiotics. In this study, we screened 232 shotgun metagenomes from ten different environments against a custom-designed β-lactamase database, and subsequently analyzed β-lactamase homologues with a suite of bioinformatic platforms including cluster and network analyses. Three interrelated β-lactamase clusters encompassed all of the human and bovine feces metagenomes, while β-lactamases from soil, freshwater, glacier, marine and wastewater grouped within a separate “environmental” cluster that displayed high levels of inter-network connectivity. Interestingly, almost no connectivity occurred between the “feces” and “environmental” clusters. We attributed this in part to the divergence in microbial community composition (dominance of Bacteroidetes and Firmicutes vs. Proteobacteria, respectively). The β-lactamase diversity in the “environmental” cluster was significantly higher than in human and bovine feces microbiomes. Several class A, B, C and D β-lactamase homologues (blaCTX-M, blaKPC, blaGES) were ubiquitous in the “environmental” cluster, whereas bovine and human feces metagenomes were dominated by class A (primarily cfxA) β-lactamases. Collectively, this study highlights the ubiquitous presence and broad diversity of β-lactamase gene precursors in non-clinical environments. Furthermore, it suggests that horizontal transfer of β-lactamases to human-associated bacteria may be more plausible from animals than from terrestrial and aquatic microbes, seemingly due to phylogenetic similarities.
Chefetz, B. ; Marom, R. ; Salton, O. ; Oliferovsky, M. ; Mordehay, V. ; Ben-Ari, J. ; Hadar, Y. Transformation of lamotrigine by white-rot fungus Pleurotus ostreatus. Environmental Pollution 2019, 250, 546 - 553. Publisher's VersionAbstract
One of the most persistent pharmaceutical compounds commonly found in treated wastewater is lamotrigine (LTG). It has also been detected in soils and crops irrigated with treated wastewater. Here we focused on the ability of the white-rot edible mushroom Pleurotus ostreatus to remove and transform LTG in liquid cultures. At concentrations of environmental relevance (1 and 10 μg L−1) LTG was almost completely removed from the culture medium within 20 days. To elucidate the mechanism of LTG removal and transformation, we applied a physiological-based approach using inhibitors and a competing agent. These experiments were conducted at a higher concentration for metabolites detection. Based on identification of sulfur-containing metabolites and LTG N2-oxide and the effect of specific inhibitors, cytochrome P450 oxidation is suggested as one of the reaction mechanisms leading to LTG transformation. The variety and number of transformation products (i.e., conjugates) found in the current study were larger than reported in mammals. Moreover, known conjugates with glucuronide, glutathione, or cysteine/glycine, were not found in our system. Since the majority of the identified transformation products were conjugates of LTG, this study highlights the persistence of LTG as an organic pollutant in ecosystems exposed to wastewater.
Usyskin-Tonne, A. ; Hadar, Y. ; Minz, D. Altering N2O emissions by manipulating wheat root bacterial community. Scientific Reports 2019, 9 7613. Publisher's VersionAbstract
Nitrous oxide (N2O) is a greenhouse gas and a potent ozone-depleting substance in the stratosphere. Agricultural soils are one of the main global sources of N2O emissions, particularly from cereal fields due to their high areal coverage. The aim of this study was to isolate N2O-reducing bacteria able to mitigate N2O emissions from the soil after inoculation. We isolated several bacteria from wheat roots that were capable of N2O reduction in vitro and studied their genetic potential and activity under different environmental conditions. Three of these isolates- all carrying the nitrous oxide reductase-encoding clade I nosZ, able to reduce N2O in vitro, and efficient colonizers of wheat roots- presented different N2O-reduction strategies when growing in the root zone, possibly due to the different conditions in situ and their metabolic preferences. Each isolate seemed to prefer to operate at different altered oxygen levels. Isolate AU243 (related to Agrobacterium/Rhizobium) could reduce both nitrate and N2O and operated better at lower oxygen levels. Isolate AU14 (related to Alcaligenes faecalis), lacking nitrate reductases, operated better under less anoxic conditions. Isolate NT128 (related to Pseudomonas stutzeri) caused slightly increased N2O emissions under both anoxic and ambient conditions. These results therefore emphasize the importance of a deep understanding of soil–plant–microbe interactions when environmental application is being considered.
Zolti, A. ; Green, S. J. ; Ben Mordechay, E. ; Hadar, Y. ; Minz, D. Root microbiome response to treated wastewater irrigation. Science of The Total Environment 2019, 655, 899 - 907. Publisher's VersionAbstract
With increasing fresh water (FW) scarcity, the use of treated wastewater (TWW) for crop irrigation is expanding globally. Besides clear benefits, some undesired long-term effects of irrigation with this low quality water on plant performance have been reported. As the rhizosphere microbiome can mediate plant-soil interactions, an examination of the response of these organisms to TWW is necessary to understand the full effects of water quality. In the current study, the effects of irrigation water quality on the microbial community structure of soil and roots as well as edaphic properties and plant performance were evaluated. We compared soil and roots microbiomes of two different plant species (tomato and lettuce), each grown in two distinct soils, and irrigated with either FW or TWW. Irrigation with TWW significantly increase soil pH, EC, K, Na and DOC, and decrease plant fruit and shoot weight, relatively to samples irrigated with FW. We calculated the effect size of plant species, soil type, and irrigation water quality on microbial community structure in soil and root. In the roots, plant species and irrigation water were the dominant factors in shaping both total (DNA based) and active (RNA based) microbial communities, with both factors contributing similarly to the observed microbial population. Soil type and irrigation water were the dominant factors shaping the total microbial community in the soil and were of similar magnitude. Irrigation water quality is demonstrated to be a major force in shaping root-associated microbiome, leading to altered microbial community structure in the critical juncture between plant and soil.
Vetvicka, V. ; Gover, O. ; Karpovsky, M. ; Hayby, H. ; Danay, O. ; Ezov, N. ; Hadar, Y. ; Schwartz, B. Immune-modulating activities of glucans extracted from Pleurotus ostreatus and Pleurotus eryngii. Journal of Functional Foods 2019, 54, 81 - 91. Publisher's VersionAbstract
We compared the immune-modulating activity of glucans extracted from P. ostreatus and P. eryngii on phagocytosis of peripheral blood neutrophils, and superoxide release from HL-60 cells. The results suggest that the anti-inflammatory properties of these glucans are partially mediated through modulation of neutrophil effector functions (P. eryngii was more effective). Additionally, both glucans dose-dependently competed for the anti-Dectin-1 and anti-CR3 antibody binding. We then tested the putative anti-inflammatory effects of the extracted glucans in inflammatory bowel disease (IBD) using the dextran sulfate sodium (DSS)–induced model in mice. The clinical symptoms of IBD were efficiently relieved by the treatment with two different doses of the glucan from both fungi. Glucan fractions, from either P. ostreatus or P. eryngii, markedly prevented TNF-α mediated inflammation in the DSS–induced inflamed intestine. These results suggest that there are variations in glucan preparations from different fungi in their anti-inflammatory ability.
Milo-Cochavi, S. ; Pareek, M. ; Delulio, G. ; Almog, Y. ; Anand, G. ; Ma, L. - J. ; Covo, S. The response to the DNA damaging agent methyl methanesulfonate in a fungal plant pathogen. Fungal Biol 2019, 123, 408-422.Abstract
DNA damage can cause mutations that in fungal plant pathogens lead to hypervirulence and resistance to pesticides. Almost nothing is known about the response of these fungi to DNA damage. We performed transcriptomic and phosphoproteomic analyses of Fusarium oxysporum exposed to methyl methanesulfonate (MMS). At the RNA level we observe massive induction of DNA repair pathways including the global genome nucleotide excision. Cul3, Cul4, several Ubiquitin-like ligases and components of the proteasome are significantly induced. In agreement, we observed drug synergism between a proteasome inhibitor and MMS. While our data suggest that Yap1 and Xbp1 networks are similarly activated in response to damage in yeast and F. oxysporum we were able to observe modules that were MMS-responsive in F. oxysporum and not in yeast. These include transcription/splicing modules that are upregulated and respiration that is down-regulated. In agreement, MMS treated cells are much more sensitive to a respiration inhibitor. At the phosphoproteomic level, Adenylate cyclase, which generates cAMP, is phosphorylated in response to MMS and forms a network of phosphorylated proteins that include cell cycle regulators and several MAPKs. Our analysis provides a starting point in understanding how genomic changes in response to DNA damage occur in Fusarium species.
Anand, G. ; Waiger, D. ; Vital, N. ; Maman, J. ; Ma, L. J. ; Covo, S. How Does Sense and Respond to Nicotinaldehyde, an Inhibitor of the NAD Salvage Biosynthesis Pathway?. Front Microbiol 2019, 10, 329.Abstract
Plant pathogenic fungi are a major threat to food security and impose a severe economic burden, thus there is a continuous need to develop new strategies to manage them. NAD is a co-factor in numerous enzymatic activities and determines the metabolic fate of the cell. Therefore, maintenance of NAD concentration is important for cellular viability. Consequently, the NAD biosynthetic pathway and redox homeostasis was suggested as a target for antifungal development. We aimed to study how senses and responds to nicotinaldehyde (NA), an inhibitor of Pnc1, a key enzyme in the salvage pathway of NAD biosynthesis. We were able to show that NA was inhibitory in high concentrations to several fungal plant pathogens, with much milder effects on tomato growth. Under low nutrient conditions NA reduced the total amounts of NAD in the fungal cell, a trend that was also observed in rich media, although without statistical significance. In low and high nutrient availability NA dramatically reduced the NAD/NADH ratio. After exposure to NA, NADH levels were increased and NAD levels and the biomass were greatly reduced. Cells responded to NA by up-regulation of oxidoreductases, with hardly any up-regulation of the classic response to oxidative stress. Direct measurement of oxidative stress response showed that unlike formaldehyde and hydrogen peroxide, NA caused reductive rather than oxidative stress. Surprisingly, alcohol dehydrogenases were significantly up-regulated more than any other dehydrogenases, including aldehyde dehydrogenases. We propose that conidia of efficiently detoxified the aldehyde group of NA by reducing NAD to NADH; the high concentrations of the latter provoked the expression of alcohol dehydrogenases that in yeast can act to reduce NADH and increase NAD amounts, respectively. Overall, the results suggest that targeting NAD biosynthesis pathway and redox homeostasis can be a potential approach to manage fungal plant pathogens. Many of the natural antifungal compounds produced by bio-control agents or even the natural biome are aldehydes, and thus the results presented here predict the possible response of to wide sources of toxicity in the environment.
Kan, Y. ; Jiang, N. ; Xu, X. ; Lyu, Q. ; Gopalakrishnan, V. ; Walcott, R. ; Burdman, S. ; Li, J. ; Luo, L. Induction and Resuscitation of the Viable but Non-culturable (VBNC) State in Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch of Cucurbitaceous Crops. Frontiers in Microbiology 2019, 10, 1081. Publisher's VersionAbstract
Acidovorax citrulli is a gram-negative bacterium that infects a wide range of cucurbits causing bacterial fruit blotch (BFB) disease. Copper-based compounds are the most widely-used chemicals for managing BFB and other bacterial diseases in the field. Many bacteria can enter a viable but nonculturable (VBNC) state in response to stress, including exposure to copper, and recover the culturability when favorable conditions return. The present study demonstrates that A. citrulli strain AAC00-1 is able to enter into the VBNC state by treatment with different concentrations of copper sulfate. It took 3 h, 5 d and 15 d for all viable cells to lose culturability upon exposure to copper sulfate concentrations of 50 μM, 10 μM and 5 μM, respectively. The VBNC A. citrulli cells regained culturability when the Cu2+ ions were removed by chelation with EDTA or by transfer of cells to LB broth, a cell-free supernatant from a suspension of AAC00-1, oligotrophic media amended with casein hydrolysate or watermelon seedling juice. We also found that the VBNC cells induced by Cu2+ were unable to colonize or infect watermelon seedlings directly, but that resuscitated cells recovered full virulence equivalent to untreated bacterial cells in the log phase. To the best of our knowledge this is the first report on the VBNC state in A. citrulli and the factors that facilitate resuscitation and restoration of pathogenicity.
2018
Hu, C. ; Zhou, M. ; Wang, W. ; Sun, X. ; Yarden, O. ; Li, S. Abnormal Ergosterol Biosynthesis Activates Transcriptional Responses to Antifungal Azoles. Front Microbiol 2018, 9 9.Abstract
Fungi transcriptionally upregulate expression of azole efflux pumps and ergosterol biosynthesis pathway genes when exposed to antifungal agents that target ergosterol biosynthesis. To date, these transcriptional responses have been shown to be dependent on the presence of the azoles and/or depletion of ergosterol. Using an inducible promoter to regulate , which encodes the major azole target, sterol 14α-demethylase, we were able to demonstrate that the CDR4 azole efflux pump can be transcriptionally activated by ergosterol biosynthesis inhibition even in the absence of azoles. By analyzing ergosterol deficient mutants, we demonstrate that the transcriptional responses by and, unexpectedly, genes encoding ergosterol biosynthesis enzymes ( genes) that are responsive to azoles, are not dependent on ergosterol depletion. Nonetheless, deletion of , which encodes C-8 sterol isomerase, also induced expression of . Deletion of also induced the expression of , the gene encoding C-14 sterol reductase, but not other tested genes which were responsive to inactivation. This indicates that inhibition of specific steps of ergosterol biosynthesis can result in different transcriptional responses, which is further supported by our results obtained using different ergosterol biosynthesis inhibitors. Together with the sterol profiles, these results suggest that the transcriptional responses by and genes are associated with accumulation of specific sterol intermediate(s). This was further supported by the fact that when the mutant was treated with ketoconazole, upstream inhibition overrode the effects by downstream inhibition on ergosterol biosynthesis pathway. Even though expression is associated with the accumulation of sterol intermediates, intra- and extracellular sterol analysis by HPLC-MS indicated that the transcriptional induction of did not result in efflux of the accumulated intermediate(s). This study demonstrates, by detailed genetic and chemical analysis, that transcriptional responses by a major efflux pump and genes of the ergosterol biosynthesis pathway to ergosterol biosynthesis inhibitors can be independent of the presence of the drugs and are linked with the accumulation of ergosterol intermediate(s).
du Plessis, I. L. ; Druzhinina, I. S. ; Atanasova, L. ; Yarden, O. ; Jacobs, K. The diversity of Trichoderma species from soil in South Africa, with five new additions. Mycologia 2018, 110, 559-583.Abstract
Fourteen Trichoderma (Hypocreales) species were identified during a survey of the genus in South Africa. These include T. afroharzianum, T. asperelloides, T. asperellum, T. atrobrunneum, T. atroviride, T. camerunense, T. gamsii, T. hamatum, T. koningii, T. koningiopsis, T. saturnisporum, T. spirale, T. virens, and T. viride. Ten of these species were not known to occur in South Africa prior to this investigation. Five additional species were novel and are described here as T. beinartii, T. caeruleimontis, T. chetii, T. restrictum, and T. undulatum. These novel Trichoderma species display morphological traits that are typical of the genus. Based on molecular identification using calmodulin, endochitinase, nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2), RNA polymerase II subunit B, and translation elongation factor 1-α gene sequence data, T. beinartii, T. caeruleimontis, and T. chetii were found to belong to the Longibrachiatum clade, whereas T. restrictum is a member of the Hamatum clade. Trichoderma undulatum occupies a distinct lineage distantly related to other Trichoderma species. Strains of T. beinartii and T. chetii were isolated previously in Hawaii and Israel; however, T. caeruleimontis, T. restrictum, and T. undulatum are so far known only from South Africa.