Extracellular vesicles isolated from dsRNA sprayed barley plants exhibit no growth inhibition or gene silencing in Fusarium graminearum

Abstract Incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto plant leaves successfully protects plants against invading pathogens with RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their interacting pathogens. Recently, we found that EVs isolated from HIGS or SIGS plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from RNA-sprayed barley ( Hordeum vulgare ) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum ( Fg ). Encouraged by our previous finding that dropping barley-derived EVs on Fg cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated Fg with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of Fg macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect Fg growth and showed no gene silencing activity on FgCYP51 genes. We conclude that either the amount of spray-derived sRNA was insufficient to induce target gene silencing (SIGS) in Fg or Fg uptake of plant EVs from liquid cultures was inefficient or impossible.

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Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

International Journal of Molecular Sciences ◽

10.3390/ijms22137099 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7099

Author(s):

Pradeep Kumar Kopparapu ◽

Meghshree Deshmukh ◽

Zhicheng Hu ◽

Majd Mohammad ◽

Marco Maugeri ◽

...

Keyword(s):

Extracellular Vesicles ◽

Inflammatory Responses ◽

Surface Proteins ◽

Host Cells ◽

Immune Stimulation ◽

Domains Of Life ◽

Major Surface ◽

Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

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Cinnamic-Derived Acids Significantly Affect Fusarium graminearum Growth and In Vitro Synthesis of Type B Trichothecenes

Phytopathology ◽

10.1094/phyto-09-10-0230 ◽

2011 ◽

Vol 101 (8) ◽

pp. 929-934 ◽

Cited By ~ 49

Author(s):

Nadia Ponts ◽

Laetitia Pinson-Gadais ◽

Anne-Laure Boutigny ◽

Christian Barreau ◽

Florence Richard-Forget

Keyword(s):

Fusarium Graminearum ◽

Phenolic Acids ◽

Antioxidant Properties ◽

Fungal Growth ◽

Type B ◽

In Planta ◽

In Vitro Synthesis ◽

The Impact ◽

Toxin Accumulation

The impact of five phenolic acids (ferulic, coumaric, caffeic, syringic, and p-hydroxybenzoic acids) on fungal growth and type B trichothecene production by four strains of Fusarium graminearum was investigated. All five phenolic acids inhibited growth but the degree of inhibition varied between strains. Our results suggested that the more lipophilic phenolic acids are, the higher is the effect they have on growth. Toxin accumulation in phenolic acid-supplemented liquid glucose, yeast extract, and peptone cultures was enhanced in the presence of ferulic and coumaric acids but was reduced in the presence of p-hydroxybenzoic acid. This modulation was shown to correlate with a regulation of TRI5 transcription. In this study, addition of phenolic acids with greater antioxidant properties resulted in a higher toxin accumulation, indicating that the modulation of toxin accumulation may be linked to the antioxidant properties of the phenolic acids. These data suggest that, in planta, different compositions in phenolic acids of kernels from various cultivars may reflect different degrees of sensitivity to “mycotoxinogenesis.”

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Investigating Useful Properties of Four Streptomyces Strains Active against Fusarium graminearum Growth and Deoxynivalenol Production on Wheat Grains by qPCR

Toxins ◽

10.3390/toxins12090560 ◽

2020 ◽

Vol 12 (9) ◽

pp. 560

Author(s):

Elena Maria Colombo ◽

Andrea Kunova ◽

Claudio Gardana ◽

Cristina Pizzatti ◽

Paolo Simonetti ◽

...

Keyword(s):

Fusarium Graminearum ◽

Plant Pathogens ◽

Fungal Growth ◽

Toxin Production ◽

Fungal Mycelium ◽

Head Blight ◽

Wheat Grains ◽

Streptomyces Spp ◽

Dual Plate

Streptomyces spp. can be exploited as biocontrol agents (BCAs) against plant pathogens such as Fusarium graminearum, the main causal agent of Fusarium head blight (FHB) and against the contamination of grains with deoxynivalenol (DON). In the present research, four Streptomyces strains active against F. graminearum in dual plate assays were characterized for their ability to colonize detached wheat grains in the presence of F. graminearum and to limit DON production. The pathogen and BCA abundance were assessed by a quantitative real-time PCR, while DON production was assessed by HPLC quantification and compared to ergosterol to correlate the toxin production to the amount of fungal mycelium. Fungal growth and mycotoxin production were assessed with both co-inoculation and late inoculation of the BCAs in vitro (three days post-Fusarium inoculation) to test the interaction between the fungus and the bacteria. The level of inhibition of the pathogen and the toxin production were strain-specific. Overall, a higher level of DON inhibition (up to 99%) and a strong reduction in fungal biomass (up to 71%) were achieved when streptomycetes were co-inoculated with the fungus. This research enabled studying the antifungal efficacy of the four Streptomyces strains and monitoring their development in DON-inducing conditions.

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Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage Functions

Infection and Immunity ◽

10.1128/iai.01171-09 ◽

2010 ◽

Vol 78 (4) ◽

pp. 1601-1609 ◽

Cited By ~ 131

Author(s):

Débora L. Oliveira ◽

Célio G. Freire-de-Lima ◽

Joshua D. Nosanchuk ◽

Arturo Casadevall ◽

Marcio L. Rodrigues ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Extracellular Vesicles ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Interleukin 10 ◽

Biologically Active ◽

Host Cells ◽

Phagocytic Cells ◽

Necrosis Factor Alpha

ABSTRACT Cryptococcus neoformans and distantly related fungal species release extracellular vesicles that traverse the cell wall and contain a varied assortment of components, some of which have been associated with virulence. Previous studies have suggested that these extracellular vesicles are produced in vitro and during animal infection, but the role of vesicular secretion during the interaction of fungi with host cells remains unknown. In this report, we demonstrate by fluorescence microscopy that mammalian macrophages can incorporate extracellular vesicles produced by C. neoformans. Incubation of cryptococcal vesicles with murine macrophages resulted in increased levels of extracellular tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), and transforming growth factor β (TGF-β). Vesicle preparations also resulted in a dose-dependent stimulation of nitric oxide production by phagocytes, suggesting that vesicle components stimulate macrophages to produce antimicrobial compounds. Treated macrophages were more effective at killing C. neoformans yeast. Our results indicate that the extracellular vesicles of C. neoformans can stimulate macrophage function, apparently activating these phagocytic cells to enhance their antimicrobial activity. These results establish that cryptococcal vesicles are biologically active.

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RNA-Mediated Gene Silencing in Hematopoietic Cells

Journal of Biomedicine and Biotechnology ◽

10.1155/jbb/2006/87340 ◽

2006 ◽

Vol 2006 ◽

pp. 1-13 ◽

Cited By ~ 3

Author(s):

Letizia Venturini ◽

Matthias Eder ◽

Michaela Scherr

Keyword(s):

Gene Silencing ◽

Mrna Translation ◽

Therapeutic Approaches ◽

Double Stranded Rna ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene ◽

Specific Degradation

In the past few years, the discovery of RNA-mediated gene silencing mechanisms, like RNA interference (RNAi), has revolutionized our understanding of eukaryotic gene expression. These mechanisms are activated by double-stranded RNA (dsRNA) and mediate gene silencing either by inducing the sequence-specific degradation of complementary mRNA or by inhibiting mRNA translation. RNAi now provides a powerful experimental tool to elucidate gene function in vitro and in vivo, thereby opening new exciting perspectives in the fields of molecular analysis and eventually therapy of several diseases such as infections and cancer. In hematology, numerous studies have described the successful application of RNAi to better define the role of oncogenic fusion proteins in leukemogenesis and to explore therapeutic approaches in hematological malignancies. In this review, we highlight recent advances and caveats relating to the application of this powerful new methodology to hematopoiesis.

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Host-induced gene silencing involves transfer of dsRNA-derived siRNA via extracellular vesicles

10.1101/2020.02.12.945154 ◽

2020 ◽

Cited By ~ 1

Author(s):

A Koch ◽

T Schlemmer ◽

L Höfle ◽

BT Werner ◽

C Preußer ◽

...

Keyword(s):

Gene Silencing ◽

Extracellular Vesicles ◽

Fungal Pathogens ◽

Host Cells ◽

Target Sequence ◽

Rna Seq ◽

Leaf Extracts ◽

Transgenic Expression ◽

Rna Molecules ◽

Expression Host

AbstractSmall (s)RNA molecules are crucial factors in the communication between hosts and their interacting pathogens, where they function as effectors that can modulate both host defense and microbial virulence/pathogenicity through a mechanism termed cross-kingdom RNA interference (ckRNAi). Consistent with this recent knowledge, sRNAs and their double-stranded (ds)RNA precursors have been adopted to control diseases in crop plants through transgenic expression (host-induced gene silencing, HIGS) or exogenous application (spray-induced gene silencing, SIGS). While these strategies proved to be effective, the mechanism of RNA transfer at the plant - pathogen interface is widely unresolved. Here we show that extracellular vesicles (EVs) purified from Arabidopsis (Arabidopsis thaliana) leaf extracts and apoplastic fluids contain transgene-derived sRNAs. EVs from plants expressing CYP3RNA, a 791 nt long dsRNA, which was originally designed to target the three CYP51 genes of the fungal pathogen Fusarium graminearum, contain CYP3RNA-derived small interfering (si)RNAs as shown by RNA sequencing (RNA-seq) analysis. Notably, the EVs cargo retained the same CYP3RNA-derived siRNA profile as the respective leaf extracts, suggesting that there was no selective uptake of specific artificial sRNAs into EVs. In addition, mutants of the ESCRT-III complex were impaired in HIGS further indicating that endosomal vesicle trafficking supports transfer of transgene-derived siRNAs between donor host cells and recipient fungal cells. Further supporting the relevance of EV-mediated transport of sRNA, we demonstrate that HIGS plants, expressing a 100 nt dsRNA-target-sequence identified via EV-sRNA-seq of CYP3RNA Arabidopsis, confers strong resistance to F. graminearum. Together, these findings support the view that EVs are key mediators in the transport of HIGS-related sRNAs to reduce the virulence of interacting fungal pathogens during host-pathogen interaction.

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Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome

Wellcome Open Research ◽

10.12688/wellcomeopenres.11910.2 ◽

2017 ◽

Vol 2 ◽

pp. 50 ◽

Cited By ~ 7

Author(s):

Abdirahman Abdi ◽

Lu Yu ◽

David Goulding ◽

Martin K. Rono ◽

Philip Bejon ◽

...

Keyword(s):

Clinical Isolate ◽

Extracellular Vesicles ◽

Genetic Material ◽

Host Cells ◽

Mass Spectrometry Data ◽

Host Interactions ◽

Effector Molecules ◽

Host Immune Responses ◽

Maurer's Clefts

Background: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. Methods: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate that had been adapted to in vitro culture for a relatively shorter period, and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). Results: We show that P. falciparum extracellular vesicles (PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer’s clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that PfEVs may play a role in parasite-host interactions. Comparison of this dataset with previously published datasets helps to define a core secretome present in PfEVs. Conclusions: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify new targets for interventions or diagnostics.

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Peptidylarginine Deiminase inhibition abolishes the production of large extracellular vesicles fromGiardia intestinalis, affecting host-pathogen interactions by hindering adhesion to host cells

10.1101/586438 ◽

2019 ◽

Cited By ~ 8

Author(s):

Bruno Gavinho ◽

Izadora Volpato Rossi ◽

Ingrid Evans-Osses ◽

Sigrun Lange ◽

Marcel Ivan Ramirez

Keyword(s):

Extracellular Vesicles ◽

Mammalian Cells ◽

Deep Understanding ◽

Arginine Deiminase ◽

Host Cells ◽

Peptidylarginine Deiminase ◽

Host Pathogen Interactions ◽

Host Pathogen ◽

Pad Inhibitor

AbstractGiardia intestinalisis an anaerobic protozoan that is an important etiologic agent of inflammation-driven diarrhea worldwide. Although self-limiting, a deep understanding of the factors involved in the pathogenicity that produces the disruption of the intestinal barrier remains unknown. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which could modulate the physiopathology of giardiasis. Here we describe new insights ofG. intestinalisEV production, revealing its capacity to shed two different enriched EV populations (large and small extracellular vesicles) and identified a relevant adhesion function associated only with the larger population. Our work also aimed at assessing the influences of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release fromGiardiaand their putative effects on host-pathogen interactions. PAD-inhibitor Cl-amidine and CBD were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of large extracellular vesicles and interfering within vitrohost-pathogen interactions. The strong efficacy of the PAD-inhibitor onGiardiaEV release indicates a phylogenetically conserved pathway of PAD-mediated EV release, most likely affecting theGiardiaarginine deiminase (GiADI) homolog of mammalian PADs. While there is still much to learn aboutG. intestinalisinteraction with its host, our results suggest that large and small EVs may be differently involved in protozoa communication, and that EV-inhibitor treatment may be a novel strategy for recurrent giardiasis treatment.

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The Triad Hsp60-miRNAs-Extracellular Vesicles in Brain Tumors: Assessing Its Components for Understanding Tumorigenesis and Monitoring Patients

Applied Sciences ◽

10.3390/app11062867 ◽

2021 ◽

Vol 11 (6) ◽

pp. 2867

Author(s):

Francesca Graziano ◽

Domenico Gerardo Iacopino ◽

Giacomo Cammarata ◽

Gianluca Scalia ◽

Claudia Campanella ◽

...

Keyword(s):

Brain Tumors ◽

Extracellular Vesicles ◽

Current Knowledge ◽

Disease Status ◽

Experimental Models ◽

Host Cells ◽

Target Cells ◽

Original Cell

Brain tumors have a poor prognosis and progress must be made for developing efficacious treatments, but for this to occur their biology and interaction with the host must be elucidated beyond current knowledge. What has been learned from other tumors may be applied to study brain tumors, for example, the role of Hsp60, miRNAs, and extracellular vesicles (EVs) in the mechanisms of cell proliferation and dissemination, and resistance to immune attack and anticancer drugs. It has been established that Hsp60 increases in cancer cells, in which it occurs not only in the mitochondria but also in the cytosol and plasma-cell membrane and it is released in EVs into the extracellular space and in circulation. There is evidence suggesting that these EVs interact with cells near and far from their original cell and that this interaction has an impact on the functions of the target cell. It is assumed that this crosstalk between cancer and host cells favors carcinogenesis in various ways. We, therefore, propose to study the triad Hsp60-related miRNAs-EVs in brain tumors and have standardized methods for the purpose. These revealed that EVs with Hsp60 and related miRNAs increase in patients’ blood in a manner that reflects disease status. The means are now available to monitor brain tumor patients by measuring the triad and to dissect its effects on target cells in vitro, and in experimental models in vivo.

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Silencing of the Slt2-Type MAP Kinase Bmp3 in Botrytis cinerea by Application of Exogenous dsRNA Affects Fungal Growth and Virulence on Lactuca sativa

International Journal of Molecular Sciences ◽

10.3390/ijms22105362 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5362

Author(s):

Maria Spada ◽

Claudio Pugliesi ◽

Marco Fambrini ◽

Susanna Pecchia

Keyword(s):

Map Kinase ◽

Botrytis Cinerea ◽

Crop Protection ◽

Fungal Growth ◽

Small Interfering Rnas ◽

Sustainable Technologies ◽

Microtiter Plates ◽

Topical Applications

Botrytis cinerea can attack over 500 genera of vascular plants and is considered the second phytopathogen in the ‘top ten’ for its economic importance. Traditional fungicides can be ineffective and with increasing fungicide resistance, new sustainable technologies are required. Lately, RNA interference-based fungicides are emerging for their potential uses in crop protection. Therefore, we assessed the potential of this innovative approach targeting the MAP kinase Bmp3 in B. cinerea, a gene involved in saprophytic growth, response to low osmolarity, conidiation, surface sensing, host penetration and lesion formation. After performing a prediction analysis of small interfering RNAs, a 427 nucleotides long dsRNA was selected as construct. We tested the effect of topical applications of dsRNA construct both in vitro by a fungal growth assay in microtiter plates and in vivo on detached lettuce leaves artificially inoculated. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination, an evident growth retardation and a strong reduction of necrotic lesions on leaves. These results correlated with a strongly reduced expression of Bmp3 gene. In accordance to these findings, the Bmp3 gene could be a promising target for the development of an RNAi-based fungicide against B. cinerea.

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