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. Guide RNAs are used to deliver the dCas9-epigenetic modifier fusion protein to the target gene sequence, where it induces modification of MLO gene expression. We perform preliminary experiments using a plant promoter cloned into the luciferase reporter system, which is transfected into a human system and analyzed using the dual-luciferase reporter assay. The results suggest that this approach may be useful in the early stages of plant epigenome editing, as it can aid in the selection of appropriate modifications to the plant promoter prior to conducting in vivo experiments under plant system conditions. Overall, the results demonstrate the potential of CRISPR (d)Cas9-based EpiEdit for precise and controlled regulation of gene expression.

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. As extracellular vesicles are used by plants, animals, and microbes in nature to transport RNAs for cross-kingdom/species RNA interference between hosts and microbes/pests, nanovesicles and other nanoparticles have been used to prevent RNA degradation. Efforts examining the effect of nanoparticles on RNA stability and internalization have identified key attributes that can inform better nanocarrier designs for SIGS. Understanding sRNA biogenesis, cross-kingdom/species RNAi, and how plants and pathogens/pests naturally interact are paramount for the design of SIGS strategies. Here, we focus on nanotechnology advancements for the engineering of innovative RNA-based disease control strategies against eukaryotic pathogens and pests.

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. Recently, we found that EVs isolated from host-induced gene silencing (HIGS) or spray-induced gene silencing (SIGS) plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from dsRNA-sprayed barley (Hordeum vulgare) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum. Encouraged by our previous finding that dropping barley-derived EVs on F. graminearum cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated F. graminearum with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of F. graminearum macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect F. graminearum growth and showed no gene silencing activity on F. graminearum CYP51 genes. Based on our findings, we concluded that either the amount of SIGS-derived siRNA was insufficient to induce target gene silencing in F. graminearum, indicating that the role of EVs in SIGS is minor, or that F. graminearum uptake of plant EVs from liquid cultures was inefficient or impossible.

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. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9-mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium-mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9-induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo-/heteromers with other HvMORCs and interact with components of the RNA-directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.

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. However, since the cultivation of GM RNAi crops is viewed negatively in numerous countries, GM-free exogenous RNA spray applications attract tremendous scientific and political interest. The sudden rise in demand for pesticide alternatives has boosted research on sprayable RNA biopesticides, generating significant technological developments and advancing the potential for field applications in the near future. Here we review the latest advances that could pave the way for a quick lab-to-field transition for RNA sprays, which, as safe, selective, broadly applicable, and cost-effective biopesticides, represent an innovation in sustainable crop production. Given these latest advances, we further discuss technological limitations, knowledge gaps in the research, safety concerns and regulatory requirements that need to be considered and addressed before RNA sprays can become a reliable and realistic agricultural approach.

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). We identified 104 Fg-sRNAs with sequence homology to host genes that were repressed during interactions of Fg and Hv, while they accumulated in plants infected by the DCL double knock-out (dKO) mutant PH1-dcl1/2. The strength of target gene expression correlated with the abundance of the corresponding Fg-sRNA. Specifically, the abundance of three tRNA-derived fragments (tRFs) targeting immunity-related Ethylene overproducer 1-like 1 (HvEOL1) and three Poaceae orthologues of Arabidopsis thaliana BRI1-associated receptor kinase 1 (HvBAK1, HvSERK2 and BdSERK2) was dependent on fungal DCL. Additionally, RNA-ligase-mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified infection-specific degradation products for the three barley gene transcripts, consistent with the possibility that tRFs contribute to fungal virulence via targeted gene silencing.

Isolation and Characterization of Barley (Hordeum vulgare) 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. 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 - 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. Next, we highlight the requirement for fundamental knowledge on the molecular mechanisms (i.e. uptake, processing and translocation of transgene-expressed double-stranded RNAs) that determine the efficacy and specificity of HIGS. Additionally, we speculate on the contribution of host and target RNA interference machineries, which may be incompatible depending on the lifestyle of the pathogen or pest. Finally, we predict that closing these gaps in knowledge will lead to the development of novel integrative concepts, precise risk assessment and tailor-made HIGS therapy for plant diseases.

RNA-Spray-Mediated Silencing of Fusarium graminearum AGO and DCL 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 in planta 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 in planta, plants can be protected from pathogens, and pests by spray-applied RNA-based biopesticides. Despite the striking efficiency of RNA-silencing-based technologies holds for agriculture, the molecular mechanisms underlying spray-induced gene silencing (SIGS) strategies are virtually unresolved, a requirement for successful future application in the field. Based on our previous work, we predict that the molecular mechanism of SIGS is controlled by the fungal-silencing machinery. In this study, we used SIGS to compare the silencing efficiencies of computationally-designed vs. manually-designed dsRNA constructs targeting ARGONAUTE and DICER genes of Fusarium graminearum (Fg). We found that targeting key components of the fungal RNAi machinery via SIGS could protect barley leaves from Fg infection and that the manual design of dsRNAs resulted in higher gene-silencing efficiencies than the tool-based design. Moreover, our results indicate the possibility of cross-kingdom RNA silencing in the Fg-barley interaction, a phenomenon in which sRNAs operate as effector molecules to induce gene silencing between species from different kingdoms, such as a plant host and their interacting pathogens.

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 Fusarium graminearum.

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 (FgDCL1, FgDCL2), ARGONAUTE 1 and 2 (FgAGO1, FgAGO2), AGO-interacting protein FgQIP (QDE2-interacting protein), RecQ helicase (FgQDE3), and four RNA-dependent RNA polymerases (FgRdRP1, FgRdRP2, FgRdRP3, FgRdRP4), in the ascomycete mycotoxin-producing fungal pathogen Fusarium graminearum (Fg) 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. The involvement of RNAi in conidiation is dependent on environmental conditions as it is detectable only under low light (<2 µmol m-2 s-1). Although both DCLs and AGOs partially share their functions, the sexual ascosporogenesis is mediated primarily by FgDCL1 and FgAGO2, while FgDCL2 and FgAGO1 contribute to asexual conidia formation and germination. FgDCL1 and FgAGO2 also account for pathogenesis as their knockout (KO) results in reduced FHB development. Apart from KO mutants Δdcl2 and Δago1, mutants Δrdrp2, Δrdrp3, Δrdrp4, Δqde3, and Δqip are strongly compromised for conidiation, while KO mutations in all RdPRs, QDE3, and QIP strongly affect ascosporogenesis. Analysis of trichothecenes mycotoxins in wheat kernels showed that the relative amount of deoxynivalenol (DON), calculated as [DON] per amount of fungal genomic DNA was reduced in all spikes infected with RNAi mutants, suggesting the possibility that the fungal RNAi pathways affect Fg's DON production. Moreover, silencing of fungal genes by exogenous target gene-specific double-stranded RNA (dsRNA) (spray-induced gene silencing, SIGS) is dependent on DCLs, AGOs, and QIP, but not on QDE3. Together these data show that in F. graminearum, different key components of the RNAi machinery are crucial in different steps of fungal development and pathogenicity.

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. Using Arabidopsis and barley, we found that dsRNA designed to target two FgCYP51 genes inhibited fungal growth more efficiently than dsRNA targeting a single gene, although both dsRNA species reduced fungal infection. Either dsRNA delivery method reduced fungal growth stronger than anticipated from previous mutational knock-out (KO) strategies, where single gene KO had no significant effect on fungal viability. Consistent with the strong inhibitory effects of the dsRNAs on fungal development in both setups, we detected to a large extent dsRNA-mediated co-silencing of respective non-target FgCYP51 genes. Together, our data further support the valuation that dsRNA applications have an interesting potential for pesticide target validation and gene function studies, apart from their potential for crop protection.

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. This natural phenomenon can be exploited to control agronomically relevant plant diseases. Recent evidence argues that this biotechnological method, called host-induced gene silencing, is effective against sucking insects, nematodes, and pathogenic fungi, as well as bacteria and viruses on their plant hosts. Here, we review recent studies which reveal the enormous potential that RNA-silencing strategies hold for providing an environmentally friendly mechanism to protect crop plants from viral diseases.

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. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and transposable element repression. Emerging data suggest that MORC proteins also participate in pathogen-induced chromatin remodeling and epigenetic gene regulation. In addition, biochemical analyses recently demonstrated that plant MORCs have topoisomerase II (topo II)-like DNA modifying activities that may be important for their function. Interestingly, animal MORC proteins exhibit many parallels with their plant counterparts, as they have been implicated in disease development and gene silencing. In addition, human MORCs, like plant MORCs, bind salicylic acid and this inhibits some of their topo II-like activities. In this review, we will focus primarily on plant MORCs, although relevant comparisons with animal MORCs will be provided.

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. Using the agronomically important barley-Fusarium graminearum pathosystem, we alternatively demonstrate that a spray application of a long noncoding dsRNA (791 nt CYP3-dsRNA), which targets the three fungal cytochrome P450 lanosterol C-14α-demethylases, required for biosynthesis of fungal ergosterol, inhibits fungal growth in the directly sprayed (local) as well as the non-sprayed (distal) parts of detached leaves. Unexpectedly, efficient spray-induced control of fungal infections in the distal tissue involved passage of CYP3-dsRNA via the plant vascular system and processing into small interfering (si)RNAs by fungal DICER-LIKE 1 (FgDCL-1) after uptake by the pathogen. We discuss important consequences of this new finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential as a new plant protection strategy.

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. Picomolar to micromolar concentrations of ascr#18, the major ascaroside in plant-parasitic nematodes, induce hallmark defense responses including the expression of genes associated with MAMP-triggered immunity, activation of mitogen-activated protein kinases, as well as salicylic acid- and jasmonic acid-mediated defense signalling pathways. Ascr#18 perception increases resistance in Arabidopsis, tomato, potato and barley to viral, bacterial, oomycete, fungal and nematode infections. These results indicate that plants recognize ascarosides as a conserved molecular signature of nematodes. Using small-molecule signals such as ascarosides to activate plant immune responses has potential utility to improve economic and environmental sustainability of agriculture.

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. When aphids were fed on transgenic barley expressing shp double-stranded RNA (shp-dsRNA), they produced significantly lower levels of shp mRNA compared to aphids feeding on wild-type plants, suggesting that the transfer of inhibitory RNA from the plant to the insect was successful. shp expression remained low when aphids were transferred from transgenic plants and fed for 1 or 2 weeks, respectively, on wild-type plants, confirming that silencing had a prolonged impact. Reduced shp expression correlated with a decline in growth, reproduction and survival rates. Remarkably, morphological and physiological aberrations such as winged adults and delayed maturation were maintained over seven aphid generations feeding on wild-type plants. Targeting shp expression therefore appears to cause strong transgenerational effects on feeding, development and survival in S. avenae, suggesting that the HIGS technology has a realistic potential for the control of aphid pests in agriculture.

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. In addition, the demonstration that agricultural pests, such as insects and nematodes, can be killed by exogenously supplied RNAi targeting their essential genes has raised the possibility that plant predation can be controlled by lethal RNAi signals generated in planta. Indeed, recent evidence argues that this strategy, called host-induced gene silencing (HIGS), is effective against sucking insects and nematodes; it also has been shown to compromise the growth and development of pathogenic fungi, as well as bacteria and viruses, on their plant hosts. Here, we review recent studies that reveal the enormous potential RNAi strategies hold not only for improving the nutritive value and safety of the food supply, but also for providing an environmentally friendly mechanism for plant protection.

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 peptide comprises 77 amino acid residues with a molecular mass of 8.2 kDa and a pI of 6.6. It is predicted to contain a zinc-binding motif and to form a random coil, lacking ß-sheets or other secondary structures. Lucimycin was active against fungi from the phyla Ascomycota, Basidiomycota and Zygomycota, in addition to the oomycete Phytophtora parasitica, but it was inactive against bacteria. A mutant version of lucimycin, lacking the four C-terminal amino acid residues, displayed 40-fold lower activity. The activity of lucimycin against a number of highly-destructive plant pathogens could be exploited to produce transgenic crops that are resistant against fungal diseases.

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. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn2+-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other's function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.

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) = 1.2 nM] as well as altered fungal morphology, similar to that observed after treatment with the azole fungicide tebuconazole, for which the CYP51 enzyme is a target. Expression of the same dsRNA in Arabidopsis and barley rendered susceptible plants highly resistant to fungal infection. Microscopic analysis revealed that mycelium formation on CYP3RNA-expressing leaves was restricted to the inoculation sites, and that inoculated barley caryopses were virtually free of fungal hyphae. This inhibition of fungal growth correlated with in planta production of siRNAs corresponding to the targeted CYP51 sequences, as well as highly efficient silencing of the fungal CYP51 genes. The high efficiency of fungal inhibition suggests that host-induced gene-silencing targeting of the CYP51 genes is an alternative to chemical treatments for the control of devastating fungal diseases.