Publications by authors named "Omer Barda"

12 Publications

  • Page 1 of 1

Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by .

J Fungi (Basel) 2020 Dec 28;7(1). Epub 2020 Dec 28.

Institute of Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion 7528809, Israel.

is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by , both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of to produce and accumulate OTA in grapes.
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http://dx.doi.org/10.3390/jof7010010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823970PMC
December 2020

Functional roles of LaeA, polyketide synthase, and glucose oxidase in the regulation of ochratoxin A biosynthesis and virulence in Aspergillus carbonarius.

Mol Plant Pathol 2021 01 10;22(1):117-129. Epub 2020 Nov 10.

Institute of Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion, Israel.

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks-knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.
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http://dx.doi.org/10.1111/mpp.13013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749749PMC
January 2021

New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Through Deletion of the Epigenetic Reader SntB.

Front Microbiol 2020 9;11:610. Epub 2020 Apr 9.

Department of Medical Microbiology and Immunology, University of Wisconsin - Madison, Madison, WI, United States.

is one of the most harmful post-harvest pathogens of pomaceous fruits and the causal agent of blue rot disease. During infection, produces the toxic secondary metabolites patulin and citrinin that can impact virulence and, further, render the fruit inedible. Several studies have shown that epigenetic machinery controls synthesis of secondary metabolites in fungi. In this regard, the epigenetic reader, SntB, has been reported to govern the production of multiple toxins in species, and impact virulence of plant pathogenic fungi. Here we show that deletion of in results in several phenotypic changes in the fungus including stunted vegetative growth, reduced conidiation, but enhanced germination rates as well as decreased virulence on Golden Delicious apples. In addition, a decrease in both patulin and citrinin biosynthesis and patulin in apples, was observed. SntB positively regulates expression of three global regulators of virulence and secondary metabolism (LaeA, CreA, and PacC) which may explain in part some of the phenotypic and virulence defects of the PeΔ strain. Lastly, results from this study revealed that the controlled environmental factors (low temperatures and high CO levels) to which is commonly exposed during fruit storage, resulted in a significant reduction of expression and consequent patulin and citrinin reduction. These data identify the epigenetic reader SntB as critical factor regulated in post-harvest pathogens under storage conditions and a potential target to control fungal colonization and decaying of stored fruit.
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http://dx.doi.org/10.3389/fmicb.2020.00610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160234PMC
April 2020

The pH-Responsive Transcription Factor PacC Governs Pathogenicity and Ochratoxin A Biosynthesis in .

Front Microbiol 2020 13;11:210. Epub 2020 Feb 13.

Institute of Postharvest and Food Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in pathogenicity using designed gene knockout mutant. Δ mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The Δ mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of resulted in a complete inhibition of OTA production at pH 7.0. Additionally, Δ mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of gene into Δ mutant restored the WT phenotype. Our results demonstrate important roles of PacC of in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.
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http://dx.doi.org/10.3389/fmicb.2020.00210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031272PMC
February 2020

Synergistic Inhibition of Mycotoxigenic Fungi and Mycotoxin Production by Combination of Pomegranate Peel Extract and Azole Fungicide.

Front Microbiol 2019 20;10:1919. Epub 2019 Aug 20.

Department of Food Quality and Safety, Institute for Postharvest and Food Sciences, Agricultural Research Organization, The Volcani Center, Rishon LeZion, Israel.

Fungal plant pathogens cause considerable losses in yield and quality of field crops worldwide. In addition, under specific environmental conditions, many fungi, including such as some and spp., are further able to produce mycotoxins while colonizing their host, which accumulate in human and animal tissues, posing a serious threat to consumer health. Extensive use of azole fungicides in crop protection stimulated the emergence of acquired azole resistance in some plant and human fungal pathogens. Combination treatments, which become popular in clinical practice, offer an alternative strategy for managing potentially resistant toxigenic fungi and reducing the required dosage of specific drugs. In the current study we tested the effect of pomegranate peel extract (PPE) on the growth and toxin production of the mycotoxigenic fungi and , both alone and in combination with the azole fungicide prochloraz (PRZ). Using time-lapse microscopy and quantitative image analysis we demonstrate significant delay of conidial germination and hyphal elongation rate in both fungi following PPE treatment in combination with PRZ. Moreover, PPE treatment reduced aflatoxin production by up to 97%, while a combined treatment with sub-inhibitory doses of PPE and PRZ resulted in complete inhibition of toxin production over a 72 h treatment. These findings were supported by qRT-PCR analysis, showing down-regulation of key genes involved in the aflatoxin biosynthetic pathway under combined PPE/PRZ treatment al low concentrations. Our results provide first evidence for synergistic effects between the commercial drug PRZ and natural compound PPE. Future application of these findings may allow to reduce the required dosage of PRZ, and possibly additional azole drugs, to inhibit mycotoxigenic fungi, ultimately reducing potential concerns over exposure to high doses of these potentially harmful fungicides.
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http://dx.doi.org/10.3389/fmicb.2019.01919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710344PMC
August 2019

MFS transporter from Botrytis cinerea provides tolerance to glucosinolate-breakdown products and is required for pathogenicity.

Nat Commun 2019 06 28;10(1):2886. Epub 2019 Jun 28.

Department of Plant Pathology and Microbiology, the Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, 76100, Israel.

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.
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http://dx.doi.org/10.1038/s41467-019-10860-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599007PMC
June 2019

Pseudozyma aphidis Induces Salicylic-Acid-Independent Resistance to Clavibacter michiganensis in Tomato Plants.

Plant Dis 2015 May 30;99(5):621-626. Epub 2015 Apr 30.

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

The ability of plant pathogens to rapidly develop resistance to commonly used pesticides challenges efforts to maximize crop production. Fungal biocontrol agents have become an important alternative to chemical fungicides as a result of environmental concerns regarding conventional pesticides, including resistance issues. The complex mode of action of biocontrol agents reduces the likelihood that pathogens will develop resistance to them. We recently isolated a unique, biologically active isolate of the epiphytic fungus Pseudozyma aphidis. We show that the extracellular metabolites secreted by our P. aphidis isolate can inhibit Xanthomonas campestris pv. vesicatoria, X. campestris pv. campestris, Pseudomonas syringae pv. tomato, Erwinia amylovora, Clavibacter michiganensis, and Agrobacterium tumefaciens in vitro. Moreover, application of Pseudozyma aphidis spores on tomato plants in the greenhouse significantly reduced (by 60%) the incidence of bacterial wilt and canker disease caused by C. michiganensis subsp. michiganensis on those plants as well as disease severity by 35%. Furthermore, infected plants treated with P. aphidis were 25% taller than control infected plants. We found that P. aphidis activates PR1a-and other pathogenesis-related genes in tomato plants-and can trigger an induced-resistance response against C. michiganensis that proceeds in a salicylic-acid-independent manner, as shown using NahG-transgenic tomato plants.
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http://dx.doi.org/10.1094/PDIS-04-14-0377-REDOI Listing
May 2015

The transcription factor SlSHINE3 modulates defense responses in tomato plants.

Plant Mol Biol 2014 Jan 13;84(1-2):37-47. Epub 2013 Aug 13.

Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, 76100, Rehovot, Israel.

The cuticle plays an important role in plant interactions with pathogens and with their surroundings. The cuticle acts as both a physical barrier against physical stresses and pathogens and a chemical deterrent and activator of the plant defense response. Cuticle production in tomato plants is regulated by several transcription factors, including SlSHINE3, an ortholog of the Arabidopsis WIN/SHN3. Here we used a SlSHINE3-overexpressing (SlSHN3-OE) and silenced (Slshn3-RNAi) lines and a mutant in SlCYP86A69 (Slcyp86A69)--a direct target of SlSHN3--to analyze the roles of the leaf cuticle and cutin content and composition in the tomato plant's defense response to the necrotrophic foliar pathogen Botrytis cinerea and the biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria. We showed that SlSHN3, which is predominantly expressed in tomato fruit epidermis, also affects tomato leaf cuticle, as morphological alterations in the SlSHN3-OE leaf tissue resulted in shiny, stunted and permeable leaves. SlSHN3-OE leaves accumulated 38% more cutin monomers than wild-type leaves, while Slshn3-RNAi and Slcyp86A69 plants showed a 40 and 70% decrease in leaf cutin monomers, respectively. Overexpression of SlSHN3 resulted in resistance to B. cinerea infection and to X. campestris pv. vesicatoria, correlated with cuticle permeability and elevated expression of pathogenesis-related genes PR1a and AOS. Further analysis revealed that B. cinerea-infected Slshn3-RNAi plants are more sensitive to B. cinerea and produce more hydrogen peroxide than wild-type plants. Cutin monomer content and composition differed between SlSHN3-OE, Slcyp86A69, Slshn3-RNAi and wild-type plants, and cutin monomer extracted from SlSHN3-OE plants altered the expression of pathogenesis-related genes in wild-type plants.
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http://dx.doi.org/10.1007/s11103-013-0117-1DOI Listing
January 2014

The effects of glucosinolates and their breakdown products on necrotrophic fungi.

PLoS One 2013 5;8(8):e70771. Epub 2013 Aug 5.

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

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070771PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733641PMC
March 2014

Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA.

Nucleic Acids Res 2010 Jun 16;38(10):e114. Epub 2010 Feb 16.

The Mina & Everard Goodman Faculty of Life Sciences, and Advanced Materials and Nanotechnology Institute Bar-Ilan University, Ramat-Gan 52900, Israel.

In trypanosomes a 39 nucleotide exon, the spliced leader (SL) is donated to all mRNAs from a small RNA, the SL RNA, by trans-splicing. Since the discovery of trans-splicing in trypanosomes two decades ago, numerous attempts failed to reconstitute the reaction in vitro. In this study, a crude whole-cell extract utilizing the endogenous SL RNA and synthetic tubulin pre-mRNA were used to reconstitute the trans-splicing reaction. An RNase protection assay was used to detect the trans-spliced product. The reaction was optimized and shown to depend on ATP and intact U2 and U6 snRNPs. Mutations introduced at the polypyrimidine tract and the AG splice site reduced the reaction efficiency. To simplify the assay, RT-PCR and quantitative real-time PCR assays were established. The system was used to examine the structural requirements for SL RNA as a substrate in the reaction. Interestingly, synthetic SL RNA assembled poorly to its cognate particle and was not utilized in the reaction. However, SL RNA synthesized in cells lacking Sm proteins, which is defective in cap-4 modification, was active in the reaction. This study is the first step towards further elucidating the mechanism of trans-splicing, an essential reaction which determines the trypanosome transcriptome.
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http://dx.doi.org/10.1093/nar/gkq065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879531PMC
June 2010

Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1.

Mol Biochem Parasitol 2009 Apr;164(2):137-46

Laboratorio de Biología Molecular de la Enfermedad de Chagas, INGEBI-CONICET, Buenos Aires, Argentina.

Early in the assembly of eukaryotes the branch-point binding protein (BBP, also called SF1) recognizes the branch point sequence, whereas the heterodimer U2AF, consisting of a 65 and a 35 kDa subunit, contacts the polypyrimidine tract and the AG splice site, respectively. Herein, we identified, cloned and expressed the Trypanosoma cruzi and Trypanosoma brucei U2AF35, U2AF65 and SF1. Trypanosomatid U2AF65 strongly diverged from yeast and human homologues. On the contrary, trypanosomatid SF1 was conserved but lacked the C-terminal sequence present in the mammalian protein. Yeast two hybrid approaches were used to assess their interactions. The interaction between U2AF35 and U2AF65 was very weak or not detectable. However, as in other eukaryotes, the interaction between U2AF65 and SF1 was strong. At the cellular level, these results were confirmed by fractionation and affinity-selection experiments in which SF1 and U2AF65 were affinity-selected with TAP tagged SF1, but not with TAP tagged U2AF35. Silencing one of the three factors affected growth and trans-splicing in the first step of this reaction. Trypanosomes are the first described example of eukaryotic cells in which the interaction of two expressed U2AF factors seemed to be very weak, or not detectable.
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http://dx.doi.org/10.1016/j.molbiopara.2008.12.009DOI Listing
April 2009

Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism.

RNA 2009 Apr 13;15(4):648-65. Epub 2009 Feb 13.

Bar-Ilan University, Ramat-Gan, Israel.

Trypanosomatid genomes encode for numerous proteins containing an RNA recognition motif (RRM), but the function of most of these proteins in mRNA metabolism is currently unknown. Here, we report the function of two such proteins that we have named PTB1 and PTB2, which resemble the mammalian polypyrimidine tract binding proteins (PTB). RNAi silencing of these factors indicates that both are essential for life. PTB1 and PTB2 reside mostly in the nucleus, but are found in the cytoplasm, as well. Microarray analysis performed on PTB1 and PTB2 RNAi silenced cells indicates that each of these factors differentially affects the transcriptome, thus regulating a different subset of mRNAs. PTB1 and PTB2 substrates were categorized bioinformatically, based on the presence of PTB binding sites in their 5' and 3' flanking sequences. Both proteins were shown to regulate mRNA stability. Interestingly, PTB proteins are essential for trans-splicing of genes containing C-rich polypyrimidine tracts. PTB1, but not PTB2, also affects cis-splicing. The specificity of binding of PTB1 was established in vivo and in vitro using a model substrate. This study demonstrates for the first time that trans-splicing of only certain substrates requires specific factors such as PTB proteins for their splicing. The trypanosome PTB proteins, like their mammalian homologs, represent multivalent RNA binding proteins that regulate mRNAs from their synthesis to degradation.
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http://dx.doi.org/10.1261/rna.1230209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661826PMC
April 2009