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

2020 ◽  
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.

scholarly journals Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess Their Role in RNA Spray-Based Crop Protection

2021 ◽  
Vol 22 (13) ◽  
pp. 7212
Author(s):  
Timo Schlemmer ◽  
Patrick Barth ◽  
Lisa Weipert ◽  
Christian Preußer ◽  
Martin Hardt ◽  
...  
Keyword(s):  
Hordeum Vulgare ◽  
Gene Silencing ◽  
Extracellular Vesicles ◽  
Fungal Pathogens ◽  
Target Genes ◽  
Crop Protection ◽  
Transgenic Expression ◽  
Isolation And Characterization ◽  
Sequence Complementarity

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.

Host-induced gene silencing and spray-induced gene silencing for crop protection against viruses.

2021 ◽  
pp. 72-85
Author(s):  
Angela Ricci ◽  
Silvia Sabbadini ◽  
Laura Miozzi ◽  
Bruno Mezzetti ◽  
Emanuela Noris
Keyword(s):  
Gene Silencing ◽  
Plant Pathogens ◽  
Crop Protection ◽  
Plant Defence ◽  
Transcriptional Gene Silencing ◽  
Target Sequence ◽  
Sequence Specificity ◽  
Rna Molecules ◽  
Political Concern ◽  
Post Transcriptional Gene Silencing

Abstract Since the beginning of agriculture, plant virus diseases have been a strong challenge for farming. Following its discovery at the very beginning of the 1990s, the RNA interference (RNAi) mechanism has been widely studied and exploited as an integrative tool to obtain resistance to viruses in several plant species, with high target-sequence specificity. In this chapter, we describe and review the major aspects of host-induced gene silencing (HIGS), as one of the possible plant defence methods, using genetic engineering techniques. In particular, we focus our attention on the use of RNAi-based gene constructs to introduce stable resistance in host plants against viral diseases, by triggering post-transcriptional gene silencing (PTGS). Recently, spray-induced gene silencing (SIGS), consisting of the topical application of small RNA molecules to plants, has been explored as an alternative tool to the stable integration of RNAi-based gene constructs in plants. SIGS has great and innovative potential for crop defence against different plant pathogens and pests and is expected to raise less public and political concern, as it does not alter the genetic structure of the plant.

scholarly journals Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

2019 ◽  
Author(s):  
Celine Everaert ◽  
Hetty Helsmoortel ◽  
Anneleen Decock ◽  
Eva Hulstaert ◽  
Ruben Van Paemel ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Extracellular Vesicles ◽  
Platelet Rich Plasma ◽  
Rna Seq ◽  
Total Rna ◽  
Rna Molecules ◽  
Rna Profiling ◽  
Wide Range ◽  
Read Distribution ◽  
Free Plasma

AbstractRNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

scholarly journals Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Celine Everaert ◽  
Hetty Helsmoortel ◽  
Anneleen Decock ◽  
Eva Hulstaert ◽  
Ruben Van Paemel ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Extracellular Vesicles ◽  
Platelet Rich Plasma ◽  
Rna Seq ◽  
Total Rna ◽  
Rna Molecules ◽  
Rna Profiling ◽  
Wide Range ◽  
Read Distribution ◽  
Free Plasma

AbstractRNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

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

2021 ◽  
Author(s):  
Timo Schlemmer ◽  
Richard Lischka ◽  
Dagmar Biedenkopf ◽  
Aline Koch
Keyword(s):  
Gene Silencing ◽  
Fusarium Graminearum ◽  
Extracellular Vesicles ◽  
Fungal Growth ◽  
Host Cells ◽  
Double Stranded Rna ◽  
Microtiter Plates ◽  
Barley Leaves ◽  
Or Gene

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.

Host-induced gene silencing and spray-induced gene silencing for crop protection against viruses.

2021 ◽  
pp. 72-85
Author(s):  
Angela Ricci ◽  
Silvia Sabbadini ◽  
Laura Miozzi ◽  
Bruno Mezzetti ◽  
Emanuela Noris
Keyword(s):  
Gene Silencing ◽  
Plant Pathogens ◽  
Crop Protection ◽  
Plant Defence ◽  
Transcriptional Gene Silencing ◽  
Target Sequence ◽  
Sequence Specificity ◽  
Rna Molecules ◽  
Political Concern ◽  
Post Transcriptional Gene Silencing

Abstract Since the beginning of agriculture, plant virus diseases have been a strong challenge for farming. Following its discovery at the very beginning of the 1990s, the RNA interference (RNAi) mechanism has been widely studied and exploited as an integrative tool to obtain resistance to viruses in several plant species, with high target-sequence specificity. In this chapter, we describe and review the major aspects of host-induced gene silencing (HIGS), as one of the possible plant defence methods, using genetic engineering techniques. In particular, we focus our attention on the use of RNAi-based gene constructs to introduce stable resistance in host plants against viral diseases, by triggering post-transcriptional gene silencing (PTGS). Recently, spray-induced gene silencing (SIGS), consisting of the topical application of small RNA molecules to plants, has been explored as an alternative tool to the stable integration of RNAi-based gene constructs in plants. SIGS has great and innovative potential for crop defence against different plant pathogens and pests and is expected to raise less public and political concern, as it does not alter the genetic structure of the plant.

scholarly journals Isolation and characterization of barley (Hordeum vulgare) extracellular vesicles and their role in RNAi-based crop protection

2021 ◽  
Author(s):  
T Schlemmer ◽  
L Weipert ◽  
C Preußer ◽  
M Hardt ◽  
A Möbus ◽  
...  
Keyword(s):  
Hordeum Vulgare ◽  
Gene Silencing ◽  
Extracellular Vesicles ◽  
Crop Protection ◽  
Minor Role ◽  
Transgenic Expression ◽  
Isolation And Characterization ◽  
A Minor

AbstractThe demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce gene silencing of pathogenic targets was groundbreaking. However, future field applications will require fundamental mechanistic knowledge on dsRNA uptake, processing, and its transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. Here, we examined the role of EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to the target fungusFusarium graminearum. We found barley EVs with 156 nm in size containing predominantly 21 and 19 nucleotide (nt) siRNAs starting with a 5’-terminal Adenine. Notably, barley EVs contain less siRNA compared to EVs isolated from transgenic HIGS Arabidopsis plants. Together our results further underpin mechanistic differences between HIGS and SIGS applications and a minor role of EVs in SIGS.

scholarly journals Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

2021 ◽  
Vol 22 (9) ◽  
pp. 4823
Author(s):  
María Fernanda González ◽  
Paula Díaz ◽  
Alejandra Sandoval-Bórquez ◽  
Daniela Herrera ◽  
Andrew F. G. Quest
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Outer Membrane ◽  
Extracellular Vesicles ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Gastric Epithelium ◽  
Infected Cells ◽  
H Pylori

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.

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

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.

scholarly journals The Role of Extracellular Vesicles as Shuttles of RNA and Their Clinical Significance as Biomarkers in Hepatocellular Carcinoma

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 902
Author(s):  
Eva Costanzi ◽  
Carolina Simioni ◽  
Gabriele Varano ◽  
Cinzia Brenna ◽  
Ilaria Conti ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Extracellular Vesicles ◽  
Tumor Metastasis ◽  
Biological Processes ◽  
Rna Molecules ◽  
Expression Of Genes ◽  
Non Coding Rna ◽  
Donor Cells ◽  
Relevant Role

Extracellular vesicles (EVs) have attracted interest as mediators of intercellular communication following the discovery that EVs contain RNA molecules, including non-coding RNA (ncRNA). Growing evidence for the enrichment of peculiar RNA species in specific EV subtypes has been demonstrated. ncRNAs, transferred from donor cells to recipient cells, confer to EVs the feature to regulate the expression of genes involved in differentiation, proliferation, apoptosis, and other biological processes. These multiple actions require accuracy in the isolation of RNA content from EVs and the methodologies used play a relevant role. In liver, EVs play a crucial role in regulating cell–cell communications and several pathophysiological events in the heterogeneous liver class of cells via horizontal transfer of their cargo. This review aims to discuss the rising role of EVs and their ncRNAs content in regulating specific aspects of hepatocellular carcinoma development, including tumorigenesis, angiogenesis, and tumor metastasis. We analyze the progress in EV-ncRNAs’ potential clinical applications as important diagnostic and prognostic biomarkers for liver conditions.

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