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In vertebrates and plants, dsRNA plays crucial roles as PAMP and as a mediator of RNAi. How higher fungi respond to dsRNA is not known. We demonstrate that Magnaporthe oryzae ( Mo ), a globally significant crop pathogen, internalizes dsRNA across a broad size range of 21 to about 3000 bp. Incubation of fungal conidia with 10 ng/µL dsRNA, regardless of size or sequence, induced aberrant germ tube elongation, revealing a strong sequence-unspecific effect of dsRNA in this fungus. Accordingly, the synthetic dsRNA analogue poly(I:C) and dsRNA of various sizes and sequences elicited canonical fungal stress pathways, including nuclear accumulation of the stress marker mitogen-activated protein kinase Hog1p and production of ROS. Leaf application of dsRNA to the cereal model species Brachypodium distachyon suppressed the progression of leaf blast disease. Notably, the sequence-unspecific effect of dsRNA depends on higher doses, while pure sequence-specific effects were observed at low concentrations of dsRNA ( < 0.03 ng/µL). The protective effects of dsRNA were further enhanced by maintaining a gap of at least seven days between dsRNA application and inoculation, and by stabilising the dsRNA in alginate-chitosan nanoparticles. Overall, our study opens up additional possibilities for the development and use of dsRNA pesticides in agriculture. Exogenous dsRNA controls the rice blast fungus Magnaporthe oryzae via stress responses and RNAi. Plant protection improves with dsRNA delivery in chitosan-alginate nanoparticles, offering new possibilities for dsRNA-based pesticides.

Fusarium fungi are a pervasive threat to global agricultural productivity. They cause a spectrum of plant diseases that result in significant yield losses and threaten food safety by producing mycotoxins that are harmful to human and animal health. In recent years, the exploitation of the RNA interference (RNAi) mechanism has emerged as a promising avenue for the control of Fusarium‐induced diseases, providing both a mechanistic understanding of Fusarium gene function and a potential strategy for environmentally sustainable disease management. However, despite significant progress in elucidating the presence and function of the RNAi pathway in different Fusarium species, a comprehensive understanding of its individual protein components and underlying silencing mechanisms remains elusive. Accordingly, while a considerable number of RNAi‐based approaches to Fusarium control have been developed and many reports of RNAi applications in Fusarium control under laboratory conditions have been published, the applicability of this knowledge in agronomic settings remains an open question, and few convincing data on RNAi‐based disease control under field conditions have been published. This review aims to consolidate the current knowledge on the role of RNAi in Fusarium disease control by evaluating current research and highlighting important avenues for future investigation. We review biotechnology‐based crop protection against Fusarium diseases with novel RNA delivery technologies and RNA active ingredients.

Small RNAs regulate various biological process involved in genome stability, development, and adaptive responses to biotic or abiotic stresses. Small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs). MicroRNAs (miRNAs) are regulators of gene expression that affect the transcriptional and post-transcriptional regulation in plants and animals through RNA interference (RNAi). miRNAs are endogenous small RNAs that originate from the processing of non-coding primary miRNA transcripts folding into hairpin-like structures. The mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) and drive the Argonaute (AGO) proteins towards their mRNA targets. siRNAs are generated from a double-stranded RNA (dsRNA) of cellular or exogenous origin. siRNAs are also involved in the adaptive response to biotic or abiotic stresses. The response of plants to hypoxia includes a genome-wide transcription reprogramming. However, little is known about the involvement of RNA signaling in gene regulation under low oxygen availability. Interestingly, miRNAs have been shown to play a role in the responses to hypoxia in animals, and recent evidence suggests that hypoxia modulates the expression of various miRNAs in plant systems. In this review, we describe recent discoveries on the impact of RNAi on plant responses to hypoxic stress in plants.

Plants seem to take up exogenous RNA that was artificially designed to target specific genes, followed by activation of the RNA interference (RNAi) machinery. It is, however, not known whether plants use RNAs themselves as signalling molecules in plant-to-plant communication, other than evidence that an exchange of small RNAs occurs between parasitic plants and their hosts. Exogenous RNAs from the environment, if taken up by some living organisms, can indeed induce RNAi. This phenomenon has been observed in nematodes and insects, and host Arabidopsis cells secrete exosome-like extracellular vesicles to deliver plant small RNAs into Botrytis cinerea . Here we show that micro-RNAs (miRNAs) produced by plants act as signalling molecules affecting gene expression in other, nearby plants. Exogenous miRNAs, such as miR156 and miR399 , trigger RNAi via a mechanism requiring both AGO1 and RDR6. This emphasizes that the production of secondary small interfering RNAs is required. This evidence highlights the existence of a mechanism in which miRNAs represent signalling molecules that enable communication between plants. This study shows that miRNAs produced by plants act as signalling molecules that affect gene expression in nearby plants. This RNAi induced by exogenous miRNAs enables communication between plants and requires the production of secondary siRNAs.

GET3 is an ATPase protein that plays a pivotal role in the guided entry of the tail-anchored (GET) pathway. The protein facilitates the targeting and inserting of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) by interacting with a receptor protein complex on the ER. The role of GET3 in various biological processes has been established in yeast, plants, and mammals but not in filamentous fungi. Fusarium graminearum is the major causal agent of Fusarium head blight (FHB), posing a threat to the yield and quality of wheat. In this study, we found that FgGET3 exhibits a high degree of sequence and structural conservation with its homologs across a wide range of organisms. Ectopic expression of FgGET3 in yeast restored the growth defects of the Saccharomyces cerevisiae ScGET3 knock-out mutant. Furthermore, FgGET3 was found to dimerize and localize to the cytoplasm, similar to its homologs in other species. Deletion of FgGET3 in F. graminearum results in decreased fungal growth, fragmented vacuoles, altered abiotic stress responses, reduced conidia production, delayed conidial germination, weakened virulence on wheat spikes and reduced DON production. Collectively, these findings underscore the critical role of FgGET3 in regulating diverse cellular and biological functions essential for the growth and virulence of F. graminearum.

In vertebrates and plants, dsRNA plays crucial roles as PAMP and as a mediator of RNAi. How higher fungi respond to dsRNA is not known. We demonstrate that Magnaporthe oryzae (Mo), a globally significant crop pathogen, internalizes dsRNA across a broad size range of 21 to about 3000 bp. Incubation of fungal conidia with 10 ng/µL dsRNA, regardless of size or sequence, induced aberrant germ tube elongation, revealing a strong sequence-unspecific effect of dsRNA in this fungus. Accordingly, the synthetic dsRNA analogue poly(I:C) and dsRNA of various sizes and sequences elicited canonical fungal stress pathways, including nuclear accumulation of the stress marker mitogen-activated protein kinase Hog1p and production of ROS. Leaf application of dsRNA to the cereal model species Brachypodium distachyon suppressed the progression of leaf blast disease. Notably, the sequence-unspecific effect of dsRNA depends on higher doses, while pure sequence-specific effects were observed at low concentrations of dsRNA ( < 0.03 ng/µL). The protective effects of dsRNA were further enhanced by maintaining a gap of at least seven days between dsRNA application and inoculation, and by stabilising the dsRNA in alginate-chitosan nanoparticles. Overall, our study opens up additional possibilities for the development and use of dsRNA pesticides in agriculture.

Macrophage migration inhibitory factors (MIFs) are multifunctional proteins regulating major processes in mammals, including activation of innate immune responses. In invertebrates, MIF proteins participate in the modulation of host immune responses when secreted by parasitic organisms, such as aphids. In this study, we assessed the possibility to use MIF genes as targets for RNA interference (RNAi)-based control of the grain aphid Sitobion avenae (Sa) on barley (Hordeum vulgare). When nymphs were fed on artificial diet containing double-stranded (ds) RNAs (SaMIF-dsRNAs) that target sequences of the three MIF genes SaMIF1, SaMIF2 and SaMIF3, they showed higher mortality rates and these rates correlated with reduced MIF transcript levels as compared to the aphids feeding on artificial diet containing a control dsRNA (GFP-dsRNA). Comparison of different feeding strategies showed that nymphs’ survival was not altered when they fed from barley seedlings sprayed with naked SaMIF-dsRNAs, suggesting they did not effectively take up dsRNA from the sieve tubes of these plants. Furthermore, aphids’ survival was also not affected when the nymphs fed on leaves supplied with dsRNA via basal cut ends of barley leaves. Consistent with this finding, the use of sieve tube-specific YFP-labeled Arabidopsis reporter lines confirmed that fluorescent 21 nt dsRNACy3, when supplied via petioles or spraying, co-localized with xylem structures, but not with phloem tissue. Our results suggest that MIF genes are a potential target for insect control and also imply that application of naked dsRNA to plants for aphid control is inefficient. More efforts should be put into the development of effective dsRNA formulations.

Abstract Macrophage migration inhibitory factors (MIF) are multifunctional proteins regulating major processes in mammals, including activation of innate immune responses. In invertebrates, MIF proteins participate in the modulation of host immune responses when secreted by parasitic organisms, such as aphids. In this study, we assessed the possibility to use MIF genes as targets for RNA interference (RNAi)-based control of the grain aphid Sitobion avenae (Sa) on barley (Hordeum vulgare). When nymphs were fed on artificial diet containing double-stranded (ds)RNAs (SaMIF-dsRNAs) that target sequences of the three MIF genes SaMIF1, SaMIF2 and SaMIF3, they showed higher mortality rates and these rates correlated with reduced MIF transcript levels as compared to the aphids feeding on artificial diet containing a control dsRNA (GFP-dsRNA). Comparison of different feeding strategies showed that nymphs’ survival was not altered when they fed from barley seedlings sprayed with SaMIF-dsRNAs, suggesting they did not effectively take up dsRNA from the sieve tubes of these plants. Furthermore, aphids’ survival was also not affected when the nymphs fed on leaves supplied with dsRNA via basal cut ends of barley leaves. Consistent with this finding, the use of sieve-tube-specific YFP-labeled Arabidopsis reporter lines confirmed that fluorescent 21 nt dsRNACy3 supplied via petioles co-localized with xylem structures, but not with phloem tissue. Our results suggest that MIF genes are a potential target for insect control and also imply that application of naked dsRNA to plants for aphid control is inefficient. More efforts should be put into the development of effective dsRNA formulations. Competing Interest Statement The authors have declared no competing interest. Footnotes * Shaoshuai.Liu{at}agrar.uni-giessen.de, Maria.Ladera-Carmona{at}agrar.uni-giessen.de, minna.poranen{at}helsinki.fi, Aart.v.Bel{at}bot1.bio.uni-giessen.de, Karl-Heinz.Kogel{at}agrar.uni-giessen.de, Jafargholi.Imani{at}agrar.uni-giessen.de