Dual-Luciferase Reporter Assay for Prescreening CRISPR (d)Cas9-Mediated Epigenetic Editing on a Plant Promoter Using Human Cells.

Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing.

Improving RNA-based crop protection through nanotechnology and insights from cross-kingdom RNA trafficking.

Spray-induced gene silencing (SIGS) is a powerful and eco-friendly method for crop protection. Based off the discovery of RNA uptake ability in many fungal pathogens, the application of exogenous RNAs targeting pathogen/pest genes results in gene silencing and infection inhibition. However, SIGS remains hindered by the rapid degradation of RNA in the environment.

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

Numerous reports have shown that incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto their leaves successfully protects them against invading pathogens exploiting the mechanism of 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 pathogens.

Lab-to-Field Transition of RNA Spray Applications - How Far Are We?

The drastic loss of biodiversity has alarmed the public and raised sociopolitical demand for chemical pesticide-free plant production, which is now treated by governments worldwide as a top priority. Given this global challenge, RNAi-based technologies are rapidly evolving as a promising substitute to conventional chemical pesticides. Primarily, genetically modified (GM) crops expressing double-stranded (ds)RNA-mediating gene silencing of foreign transcripts have been developed.

CRISPR/SpCas9-mediated double knockout of barley Microrchidia MORC1 and MORC6a reveals their strong involvement in plant immunity, transcriptional gene silencing and plant growth.

The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs.

Fusarium graminearum DICER-like-dependent sRNAs are required for the suppression of host immune genes and full virulence.

In filamentous fungi, gene silencing by RNA interference (RNAi) shapes many biological processes, including pathogenicity. Recently, fungal small RNAs (sRNAs) have been shown to act as effectors that disrupt gene activity in interacting plant hosts, thereby undermining their defence responses. We show here that the devastating mycotoxin-producing ascomycete Fusarium graminearum (Fg) utilizes DICER-like (DCL)-dependent sRNAs to target defence genes in two Poaceae hosts, barley (Hordeum vulgare, Hv) and Brachypodium distachyon (Bd).

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

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.

Host-induced gene silencing - mechanisms and applications.

Host-induced gene silencing (HIGS) technology has emerged as a powerful alternative to chemical treatments for protecting plants from pathogens or pests. More than 170 HIGS studies have been published so far, and HIGS products have been launched. First, we discuss the strengths and limitations of this technology in a pathosystem-specific context.

RNA-Spray-Mediated Silencing of and Genes Improve Barley Disease Resistance.

Over the last decade, several studies have revealed the enormous potential of RNA-silencing strategies as a potential alternative to conventional pesticides for plant protection. We have previously shown that targeted gene silencing mediated by an expression of non-coding inhibitory double-stranded RNAs (dsRNAs) can protect host plants against various diseases with unprecedented efficiency. In addition to the generation of RNA-silencing (RNAi) signals , plants can be protected from pathogens, and pests by spray-applied RNA-based biopesticides.

Different Components of the RNA Interference Machinery Are Required for Conidiation, Ascosporogenesis, Virulence, Deoxynivalenol Production, and Fungal Inhibition by Exogenous Double-Stranded RNA in the Head Blight Pathogen .

In filamentous fungi, gene silencing through RNA interference (RNAi) shapes many biological processes, including pathogenicity. We explored the requirement of key components of fungal RNAi machineries, including DICER-like 1 and 2 (DCL1, DCL2), ARGONAUTE 1 and 2 (AGO1, AGO2), AGO-interacting protein QIP (QDE2-interacting protein), RecQ helicase (QDE3), and four RNA-dependent RNA polymerases (RdRP1, RdRP2, RdRP3, RdRP4), in the ascomycete mycotoxin-producing fungal pathogen () for sexual and asexual multiplication, pathogenicity, and its sensitivity to double-stranded (ds)RNA. We corroborate and extend earlier findings that conidiation, ascosporogenesis, and Fusarium head blight (FHB) symptom development require an operable RNAi machinery.

SIGS vs HIGS: a study on the efficacy of two dsRNA delivery strategies to silence Fusarium FgCYP51 genes in infected host and non-host plants.

CYP3RNA, a double-stranded (ds)RNA designed to concomitantly target the two sterol 14α-demethylase genes FgCYP51A and FgCYP51B and the fungal virulence factor FgCYP51C, inhibits the growth of the ascomycete fungus Fusarium graminearum (Fg) in vitro and in planta. Here we compare two different methods (setups) of dsRNA delivery, viz. transgene expression (host-induced gene silencing, HIGS) and spray application (spray-induced gene silencing, SIGS), to assess the activity of CYP3RNA and novel dsRNA species designed to target one or two FgCYP51 genes.

Catch Me If You Can! RNA Silencing-Based Improvement of Antiviral Plant Immunity.

Viruses are obligate parasites which cause a range of severe plant diseases that affect farm productivity around the world, resulting in immense annual losses of yield. Therefore, control of viral pathogens continues to be an agronomic and scientific challenge requiring innovative and ground-breaking strategies to meet the demands of a growing world population. Over the last decade, RNA silencing has been employed to develop plants with an improved resistance to biotic stresses based on their function to provide protection from invasion by foreign nucleic acids, such as viruses.

MORC Proteins: Novel Players in Plant and Animal Health.

Microrchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL-type ATPase and an S5 fold. MORC proteins in plants were first discovered via a genetic screen for Arabidopsis mutants compromised for resistance to a viral pathogen.

An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery.

Meeting the increasing food and energy demands of a growing population will require the development of ground-breaking strategies that promote sustainable plant production. Host-induced gene silencing has shown great potential for controlling pest and diseases in crop plants. However, while delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species.

Conserved nematode signalling molecules elicit plant defenses and pathogen resistance.

Plant-defense responses are triggered by perception of conserved microbe-associated molecular patterns (MAMPs), for example, flagellin or peptidoglycan. However, it remained unknown whether plants can detect conserved molecular patterns derived from plant-parasitic animals, including nematodes. Here we show that several genera of plant-parasitic nematodes produce small molecules called ascarosides, an evolutionarily conserved family of nematode pheromones.

Silencing the expression of the salivary sheath protein causes transgenerational feeding suppression in the aphid Sitobion avenae.

Aphids produce gel saliva during feeding which forms a sheath around the stylet as it penetrates through the apoplast. The sheath is required for the sustained ingestion of phloem sap from sieve elements and is thought to form when the structural sheath protein (SHP) is cross-linked by intermolecular disulphide bridges. We investigated the possibility of controlling aphid infestation by host-induced gene silencing (HIGS) targeting shp expression in the grain aphid Sitobion avenae.

New wind in the sails: improving the agronomic value of crop plants through RNAi-mediated gene silencing.

RNA interference (RNAi) has emerged as a powerful genetic tool for scientific research over the past several years. It has been utilized not only in fundamental research for the assessment of gene function, but also in various fields of applied research, such as human and veterinary medicine and agriculture. In plants, RNAi strategies have the potential to allow manipulation of various aspects of food quality and nutritional content.

Lucimycin, an antifungal peptide from the therapeutic maggot of the common green bottle fly Lucilia sericata.

We report the identification, cloning, heterologous expression and functional characterization of a novel antifungal peptide named lucimycin from the common green bottle fly Lucilia sericata. The lucimycin cDNA was isolated from a library of genes induced during the innate immune response in L. sericata larvae, which are used as therapeutic maggots.

The compromised recognition of turnip crinkle virus1 subfamily of microrchidia ATPases regulates disease resistance in barley to biotrophic and necrotrophic pathogens.

MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1.

Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase-encoding genes confers strong resistance to Fusarium species.

Head blight, which is caused by mycotoxin-producing fungi of the genus Fusarium, is an economically important crop disease. We assessed the potential of host-induced gene silencing targeting the fungal cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes, which are essential for ergosterol biosynthesis, to restrict fungal infection. In axenic cultures of Fusarium graminearum, in vitro feeding of CYP3RNA, a 791-nt double-stranded (ds)RNA complementary to CYP51A, CYP51B, and CYP51C, resulted in growth inhibition [half-maximum growth inhibition (IC50) = 1.