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.

Plants and animals utilize various regulatory mechanisms for control of gene expression during development in different tissues and cell types. About 30 years ago, a new mechanism of gene regulation, termed RNA interference (RNAi), was discovered and proved revolutionary for the mechanistic understanding of gene regulation. Noncoding RNAs, including short, 21-24 nucleotide (nt) long microRNAs (miRNAs), endogenously-generated from MIR genes, are key components of RNAi processes, by post-transcriptionally controlling transcripts with antisense complementarity through either translational repression or mRNA degradation. Since their discovery, important roles in regulation of ontogenetic development, cell differentiation, proliferation, and apoptosis in eukaryotes have been elucidated. In plants, miRNAs are known regulatory elements of basic endogenous functions and responses to the environmental stimuli. While the role of miRNAs in regulation of nutrient uptake, circadian clock and general response to abiotic stress is already well understood, a comprehensive understanding of their immune-regulatory roles in response to various biotic stress factors has not yet been achieved. This review summarizes the current understanding of the function of miRNAs and their targets in plants during interaction with microbial pathogens and symbionts. Additionally, we provide a consensus conclusion regarding the typical induction or repression response of conserved miRNA families to pathogenic and beneficial fungi, bacteria, and oomycetes, as well as an outlook of agronomic application of miRNAs in plants. Further investigation of plant miRNAs responsive to microbes, aided with novel sequencing and bioinformatics approaches for discovery and prediction in non-model organisms holds great potential for development of new forms of plant protection.

In plants, hypoxia can be induced by submergence, and the lack of oxygen impairs mitochondrial respiration, thus affecting the plant’s energy status. Hypoxia has major effects on gene expression; these changes induce key responses that help meet the needs of the stressed plant. However, little is known about the possible role of RNA signaling in the regulation of gene expression under limited oxygen availability. Here, we report the contribution of ARGONAUTE1 (AGO1) to hypoxia-induced gene regulation in Arabidopsis (Arabidopsis thaliana). Submergence induced changes in levels of the microRNAs miR2936 and miR398, but this had no obvious effects on their putative target mRNA levels. However, we found that ago1-27 plants are intolerant to submergence and transcriptome analysis identified genes whose regulation requires functional AGO1. Analysis of mutants affected in various branches of RNA signaling highlighted the convergence of AGO1 signaling with the AGO4-dependent RNA-directed DNA methylation (RdDM) pathway. AGO4-dependent RdDM represses the expression of HOMOLOG OF RPW8 4 (HR4) and alters its response to submergence. Remarkably, methylation of the second exon of HR4 is not only reduced in ago4-1 but also in plants overexpressing a constitutively stable version of the oxygen sensor RELATED TO APETALA2 12 (RAP2.12), indicating convergence of oxygen signaling with epigenetic regulation of gene expression. Therefore, our results identify a role for AGO1 and AGO4 RNA-silencing pathways in low-oxygen signaling in Arabidopsis.

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 dsRNA_Cy3, 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.

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.