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.

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 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 109 Isolation and characterization of myometrium and decidual cell-derived extracellular vesicles

2021 ◽  
Vol 224 (2) ◽  
pp. S75-S76
Author(s):  
Megan Shepherd ◽  
Enkhtuya Radnaa ◽  
Rheanna Urrabaz-Garza ◽  
Talar Kechichian ◽  
Ourlad Alzeus G. Tantengco ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Decidual Cell ◽  
Isolation And Characterization

scholarly journals Emerging techniques in the isolation and characterization of extracellular vesicles and their roles in cancer diagnostics and prognostics

The Analyst ◽  
2016 ◽  
Vol 141 (2) ◽  
pp. 371-381 ◽  
Author(s):  
Vijaya Sunkara ◽  
Hyun-Kyung Woo ◽  
Yoon-Kyoung Cho
Keyword(s):  
Extracellular Vesicles ◽  
Cancer Diagnostics ◽  
Clinical Implementation ◽  
Characterization Methods ◽  
Cancer Management ◽  
Isolation And Characterization

We present an overview of current isolation, detection, and characterization methods of extracellular vesicles and their applications and limitations as a potential emerging biomarker in cancer management and their clinical implementation.

Isolation and Characterization of Extracellular Vesicles in Stem Cell-Related Studies

2017 ◽  
pp. 205-223
Author(s):  
Zezhou Zhao ◽  
Dillon C. Muth ◽  
Vasiliki Mahairaki ◽  
Linzhao Cheng ◽  
Kenneth W. Witwer
Keyword(s):  
Stem Cell ◽  
Extracellular Vesicles ◽  
Isolation And Characterization

scholarly journals Isolation and characterization of platelet-derived extracellular vesicles

2014 ◽  
Vol 3 (1) ◽  
pp. 24692 ◽  
Author(s):  
Maria T. Aatonen ◽  
Tiina Öhman ◽  
Tuula A. Nyman ◽  
Saara Laitinen ◽  
Mikaela Grönholm ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Isolation And Characterization

scholarly journals Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis

2009 ◽  
Vol 8 (4) ◽  
pp. 595-605 ◽  
Author(s):  
Michael R. Botts ◽  
Steven S. Giles ◽  
Marcellene A. Gates ◽  
Thomas R. Kozel ◽  
Christina M. Hull
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Critical Role ◽  
Spore Formation ◽  
Yeast Cells ◽  
Isolation And Characterization ◽  
Specific Configuration ◽  
First Time

ABSTRACT Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.

scholarly journals Friend of Prmt1, a Novel Chromatin Target of Protein Arginine Methyltransferases

2009 ◽  
Vol 30 (1) ◽  
pp. 260-272 ◽  
Author(s):  
Thamar Bryn van Dijk ◽  
Nynke Gillemans ◽  
Claudia Stein ◽  
Pavlos Fanis ◽  
Jeroen Demmers ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Unknown Function ◽  
Target Genes ◽  
Arginine Methylation ◽  
Critical Event ◽  
Protein Arginine Methyltransferases ◽  
Isolation And Characterization ◽  
Promoter Occupancy

ABSTRACT We describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified by both asymmetric and symmetric arginine methylation in vivo. Furthermore, Fop plays an important role in the ligand-dependent activation of estrogen receptor target genes, including TFF1 (pS2). Fop depletion results in an almost complete block of estradiol-induced promoter occupancy by the estrogen receptor. Our data indicate that Fop recruitment to the promoter is an early critical event in the activation of estradiol-dependent transcription.

Isolation and characterization of aleurone protoplasts from a malting variety of barley (Hordeum vulgare L.)

PROTOPLASMA ◽  
1994 ◽  
Vol 177 (3-4) ◽  
pp. 132-143
Author(s):  
Lone T. Skj�dt ◽  
Belinda A. Phillipson ◽  
D. J. Simpson
Keyword(s):  
Hordeum Vulgare ◽  
Hordeum Vulgare L ◽  
Isolation And Characterization
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