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.
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.
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 (IC ₅₀) = 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.
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.
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.
Summary
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.
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 (
Fg
DCL1,
Fg
DCL2), ARGONAUTE 1 and 2 (
Fg
AGO1,
Fg
AGO2), AGO-interacting protein
Fg
QIP (QDE2-interacting protein), RecQ helicase (
Fg
QDE3), and four RNA-dependent RNA polymerases (
Fg
RdRP1,
Fg
RdRP2,
Fg
RdRP3,
Fg
RdRP4), 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
Fg
DCL1 and
Fg
AGO2, while
Fg
DCL2 and
Fg
AGO1 contribute to asexual conidia formation and germination.
Fg
DCL1 and
Fg
AGO2 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.
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.
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. 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
and
genes of
(
). We found that targeting key components of the fungal RNAi machinery via SIGS could protect barley leaves from
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
-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.
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