scholarly journals Synergistic Action of a Metalloprotease and a Serine Protease from Fusarium oxysporum f. sp. lycopersici Cleaves Chitin-Binding Tomato Chitinases, Reduces Their Antifungal Activity, and Enhances Fungal Virulence

2015 ◽  
Vol 28 (9) ◽  
pp. 996-1008 ◽  
Author(s):  
Mansoor Karimi Jashni ◽  
Ivo H. M. Dols ◽  
Yuichiro Iida ◽  
Sjef Boeren ◽  
Henriek G. Beenen ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Fusarium Oxysporum ◽  
Serine Protease ◽  
Fungal Pathogens ◽  
Effector Proteins ◽  
Fungal Cell ◽  
Fungal Virulence ◽  
Chitin Binding Domain ◽  
Double Deletion ◽  
Major Structural Component

As part of their defense strategy against fungal pathogens, plants secrete chitinases that degrade chitin, the major structural component of fungal cell walls. Some fungi are not sensitive to plant chitinases because they secrete chitin-binding effector proteins that protect their cell wall against these enzymes. However, it is not known how fungal pathogens that lack chitin-binding effectors overcome this plant defense barrier. Here, we investigated the ability of fungal tomato pathogens to cleave chitin-binding domain (CBD)-containing chitinases and its effect on fungal virulence. Four tomato CBD chitinases were produced in Pichia pastoris and were incubated with secreted proteins isolated from seven fungal tomato pathogens. Of these, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae, and Botrytis cinerea were able to cleave the extracellular tomato chitinases SlChi1 and SlChi13. Cleavage by F. oxysporum removed the CBD from the N-terminus, shown by mass spectrometry, and significantly reduced the chitinase and antifungal activity of both chitinases. Both secreted metalloprotease FoMep1 and serine protease FoSep1 were responsible for this cleavage. Double deletion mutants of FoMep1 and FoSep1 of F. oxysporum lacked chitinase cleavage activity on SlChi1 and SlChi13 and showed reduced virulence on tomato. These results demonstrate the importance of plant chitinase cleavage in fungal virulence.

scholarly journals The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

2021 ◽  
Vol 7 (2) ◽  
pp. 86
Author(s):  
Bilal Ökmen ◽  
Daniela Schwammbach ◽  
Guus Bakkeren ◽  
Ulla Neumann ◽  
Gunther Doehlemann
Keyword(s):  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Effector Proteins ◽  
Mating Partner ◽  
Fungal Virulence ◽  
Functional Studies ◽  
Infection Structures ◽  
Ustilago Hordei

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

scholarly journals Fusarium oxysporum as a Multihost Model for the Genetic Dissection of Fungal Virulence in Plants and Mammals

2004 ◽  
Vol 72 (3) ◽  
pp. 1760-1766 ◽  
Author(s):  
Montserrat Ortoneda ◽  
Josep Guarro ◽  
Marta P. Madrid ◽  
Zaira Caracuel ◽  
M. Isabel G. Roncero ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Fungal Pathogens ◽  
Mitogen Activated Protein Kinase ◽  
Genetic Dissection ◽  
Fungal Virulence ◽  
Single Strain ◽  
Tomato Plants ◽  
Ph Response ◽  
Multiple Organs ◽  
Opportunistic Human Pathogen

ABSTRACT Fungal pathogens cause disease in plant and animal hosts. The extent to which infection mechanisms are conserved between both classes of hosts is unknown. We present a dual plant-animal infection system based on a single strain of Fusarium oxysporum, the causal agent of vascular wilt disease in plants and an emerging opportunistic human pathogen. Injection of microconidia of a well-characterized tomato pathogenic isolate (isolate 4287) into the lateral tail vein of immunodepressed mice resulted in disseminated infection of multiple organs and death of the animals. Knockout mutants in genes encoding a mitogen-activated protein kinase, a pH response transcription factor, or a class V chitin synthase previously shown to be implicated in virulence on tomato plants were tested in the mouse model. The results indicate that some of these virulence factors play functionally distinct roles during the infection of tomato plants and mice. Thus, a single F. oxysporum strain can be used to study fungal virulence mechanisms in plant and mammalian pathogenesis.

scholarly journals Anticapsular and Antifungal Activity of α-Cyperone

Antibiotics ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 51
Author(s):  
Connor Horn ◽  
Govindsamy Vediyappan
Keyword(s):  
Antifungal Activity ◽  
Fungal Pathogens ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Cyperus Rotundus ◽  
Drug Resistant ◽  
Fungal Virulence ◽  
Medical Needs ◽  
Number Of Patients ◽  
Immunosuppressed Patients

Fungal infections affect 300 million people and cause 1.5 million deaths globally per year. With the number of immunosuppressed patients increasing steadily, there is an increasing number of patients infected with opportunistic fungal infections such as infections caused by the species of Candida and Cryptococcus. In fact, the drug-resistant Can. krusei and the emerging pan-antifungal resistant Can. auris pose a serious threat to human health as the existing limited antifungals are futile. To further complicate therapy, fungi produce capsules and spores that are resistant to most antifungal drugs/host defenses. Novel antifungal drugs are urgently needed to fill unmet medical needs. From screening a collection of medicinal plant sources for antifungal activity, we have identified an active fraction from the rhizome of Cyperus rotundus, the nut grass plant. The fraction contained α-Cyperone, an essential oil that showed fungicidal activity against different species of Candida. Interestingly, the minimal inhibitory concentration of α-Cyperone was reduced 8-fold when combined with a clinical antifungal drug, fluconazole, indicating its antifungal synergistic potential and could be useful for combination therapy. Furthermore, α-Cyperone affected the synthesis of the capsule in Cryp. neoformans, a causative agent of fungal meningitis in humans. Further work on mechanistic understanding of α-Cyperone against fungal virulence could help develop a novel antifungal agent for drug-resistant fungal pathogens.

scholarly journals Isolation, Characterization and Mode of Action of a Plant-Derived Antifungal Peptide, Cc-AFP1 Against Aspergillus Fumigatus

2021 ◽  
Author(s):  
Sima Sadat Seyedjavadi ◽  
Soghra Khani ◽  
Mehdi Goudarzi ◽  
Hadi Zare-Zardini ◽  
Masoomeh Shams-Ghahfarokhi ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Structure Prediction ◽  
Fungal Pathogens ◽  
Tertiary Structure ◽  
Fungal Infections ◽  
Cell Membrane Permeability ◽  
Random Coil ◽  
Iodide Uptake ◽  
Fungal Cell ◽  
Human Red Blood Cells

Abstract Among novel molecules with therapeutic efficacy, antifungal peptides (AFPs) have recently been attracted due to their unique ability to evade fungal pathogens. In this study, a novel AFP, Cc-AFP1 (MW ~ 3.759 kDa) isolated from Carum carvi L. was purified by precipitation and chromatography techniques. Sequence analysis by Edman degradation revealed a fragment of 36 amino acid residues as RVCFRPVATYLGVCGVSGACRDHCVKLGSCVYKGPG. The tertiary structure prediction using the I-TASSER showed α-helix and random coil structure. Cc-AFP1 had a net charge of +4 and hydrophobicity ratio of 44%. The antifungal activity of Cc-AFP1 was confirmed against Aspergillus species with MIC values in the range of 8-16 µg/mL. Cc-AFP1 had less than 5% hemolytic activity at 8-16 µg/mL on human red blood cells, while it was nontoxic for HEK293 cell lines. Stability analysis showed that Cc-AFP1 activity was maintained at different temperatures (20 to 80ºC) and pH (8 to 10). The results of a propidium iodide uptake and transmission electron microscopy showed that the antifungal activity of Cc-AFP1 could be attributed to altering the fungal cell membrane permeability. Taken together, these results indicate that Cc-AFP1 may be an attractive molecule to develop as a novel antifungal agent combating fungal infections cause by Aspergillus species.

scholarly journals Hexadecylphosphocholine (Miltefosine) Has Broad-Spectrum Fungicidal Activity and Is Efficacious in a Mouse Model of Cryptococcosis

2006 ◽  
Vol 50 (2) ◽  
pp. 414-421 ◽  
Author(s):  
Fred Widmer ◽  
Lesley C. Wright ◽  
Daniel Obando ◽  
Rosemary Handke ◽  
Ranjini Ganendren ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Mouse Model ◽  
Broad Spectrum ◽  
Fungal Pathogens ◽  
Inhibitory Effect ◽  
Fungal Virulence ◽  
Antifungal Effect ◽  
Pancreatic Phospholipase ◽  
Scedosporium Prolificans

ABSTRACT The alkyl phosphocholine drug miltefosine is structurally similar to natural substrates of the fungal virulence determinant phospholipase B1 (PLB1), which is a potential drug target. We determined the MICs of miltefosine against key fungal pathogens, correlated antifungal activity with inhibition of the PLB1 activities (PLB, lysophospholipase [LPL], and lysophospholipase-transacylase [LPTA]), and investigated its efficacy in a mouse model of disseminated cryptococcosis. Miltefosine inhibited secreted cryptococcal LPTA activity by 35% at the subhemolytic concentration of 25 μM (10.2 μg/ml) and was inactive against mammalian pancreatic phospholipase A2 (PLA2). At 250 μM, cytosolic PLB, LPL, and LPTA activities were inhibited by 25%, 51%, and 77%, respectively. The MICs at which 90% of isolates were inhibited (MIC90s) against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Cryptococcus gattii, Aspergillus fumigatus, Fusarium solani, Scedosporium prolificans, and Scedosporium apiospermum were 2 to 4 μg/ml. The MICs of miltefosine against Candida tropicalis (n = 8) were 2 to 4 μg/ml, those against Aspergillus terreus and Candida parapsilosis were 8 μg/ml (MIC90), and those against Aspergillus flavus (n = 8) were 2 to 16 μg/ml. Miltefosine was fungicidal for C. neoformans, with rates of killing of 2 log units within 4 h at 7.0 μM (2.8 μg/ml). Miltefosine given orally to mice on days 1 to 5 after intravenous infection with C. neoformans delayed the development of illness and mortality and significantly reduced the brain cryptococcal burden. We conclude that miltefosine has broad-spectrum antifungal activity and is active in vivo in a mouse model of disseminated cryptococcosis. The relatively small inhibitory effect on PLB1 enzyme activities at concentrations exceeding the MIC by 2 to 20 times suggests that PLB1 inhibition is not the only mechanism of the antifungal effect.

scholarly journals Proteinaceous necrotrophic effectors in fungal virulence

2010 ◽  
Vol 37 (10) ◽  
pp. 907 ◽  
Author(s):  
Kar-Chun Tan ◽  
Richard P. Oliver ◽  
Peter S. Solomon ◽  
Caroline S. Moffat
Keyword(s):  
Fungal Pathogens ◽  
Host Plants ◽  
Triticum Aestivum L ◽  
Effector Proteins ◽  
Fungal Virulence ◽  
Pyrenophora Tritici Repentis ◽  
Necrotrophic Fungi ◽  
Necrotrophic Effectors ◽  
Inside Out ◽  
Shed Light

The host–pathogen interface can be considered as a biological battlefront. Molecules produced by both the pathogen and the host are critical factors determining the outcome of the interaction. Recent studies have revealed that an increasing number of necrotrophic fungal pathogens produce small proteinaceous effectors that are able to function as virulence factors. These molecules can cause tissue death in host plants that possess dominant sensitivity genes, leading to subsequent pathogen colonisation. Such effectors are only found in necrotrophic fungi, yet their roles in virulence are poorly understood. However, several recent key studies of necrotrophic effectors from two wheat (Triticum aestivum L.) pathogens, Pyrenophora tritici-repentis (Died.) Drechs. and Stagonospora nodorum (Berk.) Castell. & Germano, have shed light upon how these effector proteins serve to disable the host from the inside out.

scholarly journals Fungal LysM effectors that comprise two LysM domains bind chitin through intermolecular dimerization

2020 ◽  
Author(s):  
Hui Tian ◽  
Gabriel L. Fiorin ◽  
Anja Kombrink ◽  
Jeroen R. Mesters ◽  
Bart P.H.J. Thomma
Keyword(s):  
Fungal Pathogens ◽  
Plant Immunity ◽  
Effector Proteins ◽  
Broad Host Range ◽  
Fungal Cell ◽  
Mould Fungus ◽  
Colletotrichum Higginsianum ◽  
Molecular Pattern ◽  
Wide Range ◽  
Binding Groove

SUMMARYChitin is a polymer of β-(1,4)-linked N-acetyl-D-glucosamine (GlcNAc) and a major structural component of fungal cell walls that acts as a microbe-associated molecular pattern (MAMP) that can be recognized by plant cell surface-localized pattern recognition receptors (PRRs) to activate a wide range of immune responses. In order to deregulate chitin-induced plant immunity and successfully establish their infection, many fungal pathogens secrete effector proteins with LysM domains. We previously determined that two of the three LysM domains of the LysM effector Ecp6 from the tomato leaf mould fungus Cladosporium fulvum cooperate to form a chitin-binding groove that binds chitin with ultra-high affinity, allowing to outcompete host PRRs for chitin binding. In this study, we describe functional and structural analyses aimed to investigate whether LysM effectors that contain two LysM domains bind chitin through intramolecular or intermolecular LysM dimerization. To this end, we focus on MoSlp1 from the rice blast fungus Magnaporthe oryzae, Vd2LysM from the broad host range vascular wilt fungus Verticillium dahliae, and ChElp1 and ChElp2 from the Brassicaceae anthracnose fungus Colletotrichum higginsianum. We show that these LysM effectors bind chitin through intermolecular LysM dimerization, allowing the formation of polymeric complexes that may precipitate in order to eliminate the presence of chitin oligomers at infection sites to suppress activation of chitin-induced plant immunity. In this manner, many fungal pathogens are able to subvert chitin-triggered immunity in their plant hosts.

scholarly journals Fungicidal Mechanisms of Cathelicidins LL-37 and CATH-2 Revealed by Live-Cell Imaging

2014 ◽  
Vol 58 (4) ◽  
pp. 2240-2248 ◽  
Author(s):  
Soledad R. Ordonez ◽  
Ilham H. Amarullah ◽  
Richard W. Wubbolts ◽  
Edwin J. A. Veldhuizen ◽  
Henk P. Haagsman
Keyword(s):  
Antifungal Activity ◽  
Cell Membrane ◽  
Live Cell Imaging ◽  
Fungal Pathogens ◽  
Cell Imaging ◽  
Mechanisms Of Action ◽  
Live Cell ◽  
Energy Status ◽  
Fungal Cell ◽  
Content Type

ABSTRACTAntifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5).Candida albicanswas used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides killC. albicansrapidly. CATH-2 is the most active peptide and killsC. albicanswithin 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to theC. albicanscell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreasesC. albicanscell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.

scholarly journals The Ustilago hordei-barley interaction is a versatile system to characterize fungal effectors

2020 ◽  
Author(s):  
Bilal Ökmen ◽  
Daniela Schwammbach ◽  
Guus Bakkeren ◽  
Ulla Neumann ◽  
Gunther Doehlemann
Keyword(s):  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Effector Proteins ◽  
Mating Partner ◽  
Fungal Virulence ◽  
Functional Studies ◽  
Infection Structures ◽  
Expression Platform

AbstractObligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence/pathogenicity. In this study, we present the U. hordei-barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We have generated U. hordei solopathogenic strains, which form infectious filaments without presence of compatible mating partner. Solopathogenic strains are suitable as heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization were analyzed by transmission electron microscopy, which shows that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

scholarly journals Functional Characterization of a Rice Thioredoxin Protein OsTrxm and Its Cysteine Mutant Variant with Antifungal Activity

Antioxidants ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 598
Author(s):  
Seong-Cheol Park ◽  
Il Ryong Kim ◽  
Jin-Young Kim ◽  
Yongjae Lee ◽  
Su-Hyang Yoo ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Fungal Pathogens ◽  
Redox Regulation ◽  
Crop Yields ◽  
Fungal Infections ◽  
Functional Characterization ◽  
Antioxidant Properties ◽  
Redox Balance ◽  
Fungal Cell

Although there are many antimicrobial proteins in plants, they are not well-explored. Understanding the mechanism of action of plant antifungal proteins (AFPs) may help combat fungal infections that impact crop yields. In this study, we aimed to address this gap by screening Oryza sativa leaves to isolate novel AFPs. We identified a thioredoxin protein with antioxidant properties. Being ubiquitous, thioredoxins (Trxs) function in the redox balance of all living organisms. Sequencing by Edman degradation method revealed the AFP to be O. sativa Thioredoxin m-type isoform (OsTrxm). We purified the recombinant OsTrxm and its cysteine mutant proteins (OsTrxm C/S) in Escherichia coli. The recombinant OsTrxm proteins inhibited the growth of various pathogenic fungal cells. Interestingly, OsTrxm C/S mutant showed higher antifungal activity than OsTrxm. A growth inhibitory assay against various fungal pathogens and yeasts confirmed the pertinent role of cysteine residues. The OsTrxm protein variants penetrated the fungal cell wall and membrane, accumulated in the cells and generated reactive oxygen species. Although the role of OsTrxm in chloroplast development is known, its biochemical and molecular functions have not been elucidated. These findings suggest that in addition to redox regulation, OsTrxm also functions as an antimicrobial agent.

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