Cross-kingdom RNA Interference Research Articles

Cross-kingdom RNA interference mediated by insect salivary microRNAs may suppress plant immunity

Communication between insects and plants relies on the exchange of bioactive molecules that traverse the species interface. Although proteinic effectors have been extensively studied, our knowledge of other molecules involved in this process remains limited. In this study, we investigate the role of salivary microRNAs (miRNAs) from the rice planthopper Nilaparvata lugens in suppressing plant immunity. A total of three miRNAs were confirmed to be secreted into host plants during insect feeding. Notably, the sequence-conserved miR-7-5P is specifically expressed in the salivary glands of N. lugens and is secreted into saliva, distinguishing it significantly from homologues found in other insects. Silencing miR-7-5P negatively affects N. lugens feeding on rice plants, but not on artificial diets. The impaired feeding performance of miR-7-5P-silenced insects can be rescued by transgenic plants overexpressing miR-7-5P. Through target prediction and experimental testing, we demonstrate that miR-7-5P targets multiple plant genes, including the immune-associated bZIP transcription factor 43 (OsbZIP43). Infestation of rice plants by miR-7-5P-silenced insects leads to the increased expression of OsbZIP43, while the presence of miR-7-5P counteracts this upregulation effect. Furthermore, overexpressing OsbZIP43 confers plant resistance against insects which can be subverted by miR-7-5P. Our findings suggest a mechanism by which herbivorous insects have evolved salivary miRNAs to suppress plant immunity, expanding our understanding of cross-kingdom RNA interference between interacting organisms.

Host-induced gene silencing in wild apple germplasm Malus hupehensis confers resistance to the fungal pathogen Botryosphaeria dothidea.

Host-induced gene silencing (HIGS) is an inherent mechanism of plant resistance to fungal pathogens, resulting from cross-kingdom RNA interference (RNAi) mediated by small RNAs (sRNAs) delivered from plants into invading fungi. Introducing artificial sRNA precursors into crops can trigger HIGS of selected fungal genes, and thus has potential applications in agricultural disease control. To investigate the HIGS of apple (Malus sp.) during the interaction with Botryosphaeria dothidea, the pathogenic fungus causing apple ring rot disease, we evaluated whether apple miRNAs can be transported into and target genes in B. dothidea. Indeed, miR159a from Malus hupehensis, a wild apple germplasm with B. dothidea resistance, silenced the fungal sugar transporter gene BdSTP. The accumulation of miR159a in extracellular vesicles (EVs) of both infected M. hupehensis and invading B. dothidea suggests that this miRNA of the host is transported into the fungus via the EV pathway. Knockout of BdSTP caused defects in fungal growth and proliferation, whereas knockin of a miR159a-insensitive version of BdSTP resulted in increased pathogenicity. Inhibition of miR159a in M. hupehensis substantially enhanced plant sensitivity to B. dothidea, indicating miR159a-mediated HIGS against BdSTP being integral to apple immunity. Introducing artificial sRNA precursors targeting BdSTP and BdALS, an acetolactate synthase gene, into M. hupehensis revealed that double-stranded RNAs were more potent than engineered MIRNAs in triggering HIGS alternative to those natural of apple and inhibiting infection. These results provide preliminary evidence for cross-kingdom RNAi in the apple-B. dothidea interaction and establish HIGS as a potential disease control strategy in apple.

A fungal RNA-dependent RNA polymerase is a novel player in plant infection and cross-kingdom RNA interference.

Small RNAs act as fungal pathogen effectors that silence host target genes to promote infection, a virulence mechanism termed cross-kingdom RNA interference (RNAi). The essential pathogen factors of cross-kingdom small RNA production are largely unknown. We here characterized the RNA-dependent RNA polymerase (RDR)1 in the fungal plant pathogen Botrytis cinerea that is required for pathogenicity and cross-kingdom RNAi. B. cinerea bcrdr1 knockout (ko) mutants exhibited reduced pathogenicity and loss of cross-kingdom small RNAs. We developed a "switch-on" GFP reporter to study cross-kingdom RNAi in real-time within the living plant tissue which highlighted that bcrdr1 ko mutants were compromised in cross-kingdom RNAi. Moreover, blocking seven pathogen cross-kingdom small RNAs by expressing a short-tandem target mimic RNA in transgenic Arabidopsis thaliana led to reduced infection levels of the fungal pathogen B. cinerea and the oomycete pathogen Hyaloperonospora arabidopsidis. These results demonstrate that cross-kingdom RNAi is significant to promote host infection and making pathogen small RNAs an effective target for crop protection.

The roles of small RNAs in rice-brown planthopper interactions

Interactions between rice plants (Oryza sativa L.) and brown planthoppers (Nilaparvata lugens Stål, BPHs) are used as a model system to study the molecular mechanisms underlying plant-insect interactions. Small RNAs (sRNAs) regulate growth, development, immunity, and environmental responses in eukaryotic organisms, including plants and insects. Recent research suggests that sRNAs play significant roles in rice-BPH interactions by mediating post-transcriptional gene silencing. The focus of this review is to explore the roles of sRNAs in rice-BPH interactions and to highlight recent research progress in unraveling the mechanism of cross-kingdom RNA interference (ckRNAi) between host plants and insects and the application of ckRNAi in pest management of crops including rice. The research summarized here will aid in the development of safe and effective BPH control strategies.

A perspective on cross-kingdom RNA interference in mutualistic symbioses.

RNA interference (RNAi) is arguably one of the more versatile mechanisms in cell biology, facilitating the fine regulation of gene expression and protection against mobile genomic elements, whilst also constituting a key aspect of induced plant immunity. More recently, the use of this mechanism to regulate gene expression in heterospecific partners - cross-kingdom RNAi (ckRNAi) - has been shown to form a critical part of bidirectional interactions between hosts and endosymbionts, regulating the interplay between microbial infection mechanisms and host immunity. Here, we review the current understanding of ckRNAi as it relates to interactions between plants and their pathogenic and mutualistic endosymbionts, with particular emphasis on evidence in support of ckRNAi in the arbuscular mycorrhizal symbiosis.

Fusarium oxysporum f. sp. cepae small RNAs (Foc-sRNAs) promote disease susceptibility in onion (Allium cepa L.) through cross kingdom RNA interference

Fusarium basal rot (FBR) caused by Fusarium oxysporum f. sp. cepae (Foc), is one of the most vicious fungal phytopathogen infecting onion and allied crops across the world. Small RNAs (sRNAs) are important pathogenicity factor in various fungal phytopathogens that impairs host immune responses. However, the sRNA repertoire from Foc and their roles in onion-Foc interaction is not clear yet. Here, we performed genome-wide small-RNA profiling of Foc post infection of onion and detected three Foc-sRNAs (Fx-sR3, Fx-sR4 and Fx-sR9) that were significantly induced in Foc sensitive onion genotype. Co-transformation assay showed that Foc-sRNAs facilitate cross-kingdom RNA interference (RNAi) cleaving the onion defense genes encoding NLR class R protein, LRR-receptor kinase and F-box transcription factor. Inhibition of the Foc-sRNAs resulted in an increased onion resistance, while knockdown of the targeted defense genes increased the susceptibility of onion to FBR infection. This suggest that Foc-sRNAs represses the host immune response by suppressing the expression of the three targeted immunity specific genes. All together, we propose that Foc-sRNAs are central factors of Foc pathogenicity, and could suppress host immunity by acting as effectors. Our results revealed significant understandings into the pathogenicity of FBR infection in onion.

Site-specific analysis reveals candidate cross-kingdom small RNAs, tRNA and rRNA fragments, and signs of fungal RNA phasing in the barley-powdery mildew interaction.

The establishment of host-microbe interactions requires molecular communication between both partners, which may involve the mutual transfer of noncoding small RNAs. Previous evidence suggests that this is also true for powdery mildew disease in barley, which is caused by the fungal pathogen Blumeria hordei. However, previous studies lacked spatial resolution regarding the accumulation of small RNAs upon host infection by B. hordei. Here, we analysed site-specific small RNA repertoires in the context of the barley-B. hordei interaction. To this end, we dissected infected leaves into separate fractions representing different sites that are key to the pathogenic process: epiphytic fungal mycelium, infected plant epidermis, isolated haustoria, a vesicle-enriched fraction from infected epidermis, and extracellular vesicles. Unexpectedly, we discovered enrichment of specific 31-33-base 5'-terminal fragments of barley 5.8S ribosomal RNA in extracellular vesicles and infected epidermis, as well as particular B. hordei transfer RNA fragments in haustoria. We describe canonical small RNAs from both the plant host and the fungal pathogen that may confer cross-kingdom RNA interference activity. Interestingly, we found first evidence of phased small interfering RNAs in B. hordei, a feature usually attributed to plants, which may be associated with the posttranscriptional control of fungal coding genes, pseudogenes, and transposable elements. Our data suggest a key and possibly site-specific role for cross-kingdom RNA interference and noncoding RNA fragments in the host-pathogen communication between B. hordei and its host barley.

Molecular characterization of cross-kingdom RNA interference in Botrytis cinerea by tomato small RNAs.

Previous studies have suggested that plants can modulate gene expression in pathogenic fungi by producing small RNAs (sRNAs) that can be translocated into the fungus and mediate gene silencing, which may interfere with the infection mechanism of the intruder. We sequenced sRNAs and mRNAs in early phases of the Solanum lycopersicum (tomato)-Botrytis cinerea interaction and examined the potential of plant sRNAs to silence their predicted mRNA targets in the fungus. Almost a million unique plant sRNAs were identified that could potentially target 97% of all fungal genes. We selected three fungal genes for detailed RT-qPCR analysis of the correlation between the abundance of specific plant sRNAs and their target mRNAs in the fungus. The fungal Bcspl1 gene, which had been reported to be important for the fungal virulence, showed transient down-regulation around 20 hours post inoculation and contained a unique target site for a single plant sRNA that was present at high levels. In order to study the functionality of this plant sRNA in reducing the Bcspl1 transcript level, we generated a fungal mutant that contained a 5-nucleotide substitution that would abolish the interaction between the transcript and the sRNA without changing the encoded protein sequence. The level of the mutant Bcspl1 transcript showed a transient decrease similar to wild type transcript, indicating that the tomato sRNA was not responsible for the downregulation of the Bcspl1 transcript. The virulence of the Bcspl1 target site mutant was identical to the wild type fungus.

Extracellular vesicles: cross-organismal RNA trafficking in plants, microbes, and mammalian cells.

Extracellular vesicles (EVs) are membrane-enclosed nanometer-scale particles that transport biological materials such as RNAs, proteins, and metabolites. EVs have been discovered in nearly all kingdoms of life as a form of cellular communication across different cells and between interacting organisms. EV research has primarily focused on EV-mediated intra-organismal transport in mammals, which has led to the characterization of a plethora of EV contents from diverse cell types with distinct and impactful physiological effects. In contrast, research into EV-mediated transport in plants has focused on inter-organismal interactions between plants and interacting microbes. However, the overall molecular content and functions of plant and microbial EVs remain largely unknown. Recent studies into the plant-pathogen interface have demonstrated that plants produce and secrete EVs that transport small RNAs into pathogen cells to silence virulence-related genes. Plant-interacting microbes such as bacteria and fungi also secrete EVs which transport proteins, metabolites, and potentially RNAs into plant cells to enhance their virulence. This review will focus on recent advances in EV-mediated communications in plant-pathogen interactions compared to the current state of knowledge of mammalian EV capabilities and highlight the role of EVs in cross-kingdom RNA interference.

Fungal Secondary Metabolites and Small RNAs Enhance Pathogenicity during Plant-Fungal Pathogen Interactions.

Fungal plant pathogens use proteinaceous effectors as well as newly identified secondary metabolites (SMs) and small non-coding RNA (sRNA) effectors to manipulate the host plant's defense system via diverse plant cell compartments, distinct organelles, and many host genes. However, most molecular studies of plant-fungal interactions have focused on secreted effector proteins without exploring the possibly equivalent functions performed by fungal (SMs) and sRNAs, which are collectively known as "non-proteinaceous effectors". Fungal SMs have been shown to be generated throughout the plant colonization process, particularly in the early biotrophic stages of infection. The fungal repertoire of non-proteinaceous effectors has been broadened by the discovery of fungal sRNAs that specifically target plant genes involved in resistance and defense responses. Many RNAs, particularly sRNAs involved in gene silencing, have been shown to transmit bidirectionally between fungal pathogens and their hosts. However, there are no clear functional approaches to study the role of these SM and sRNA effectors. Undoubtedly, fungal SM and sRNA effectors are now a treasured land to seek. Therefore, understanding the role of fungal SM and sRNA effectors may provide insights into the infection process and identification of the interacting host genes that are targeted by these effectors. This review discusses the role of fungal SMs and sRNAs during plant-fungal interactions. It will also focus on the translocation of sRNA effectors across kingdoms, the application of cross-kingdom RNA interference in managing plant diseases and the tools that can be used to predict and study these non-proteinaceous effectors.

Beyond transcription: compelling open questions in plant RNA biology.

The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader.

Mycovirus-encoded suppressors of RNA silencing: Possible allies or enemies in the use of RNAi to control fungal disease in crops.

Plants, fungi, and many other eukaryotes have evolved an RNA interference (RNAi) mechanism that is key for regulating gene expression and the control of pathogens. RNAi inhibits gene expression, in a sequence-specific manner, by recognizing and deploying cognate double-stranded RNA (dsRNA) either from endogenous sources (e.g. pre-micro RNAs) or exogenous origin (e.g. viruses, dsRNA, or small interfering RNAs, siRNAs). Recent studies have demonstrated that fungal pathogens can transfer siRNAs into plant cells to suppress host immunity and aid infection, in a mechanism termed cross-kingdom RNAi. New technologies, based on RNAi are being developed for crop protection against insect pests, viruses, and more recently against fungal pathogens. One example, is host-induced gene silencing (HIGS), which is a mechanism whereby transgenic plants are modified to produce siRNAs or dsRNAs targeting key transcripts of plants, or their pathogens or pests. An alternative gene regulation strategy that also co-opts the silencing machinery is spray-induced gene silencing (SIGS), in which dsRNAs or single-stranded RNAs (ssRNAs) are applied to target genes within a pathogen or pest. Fungi also use their RNA silencing machinery against mycoviruses (fungal viruses) and mycoviruses can deploy virus-encoded suppressors of RNAi (myco-VSRs) as a counter-defence. We propose that myco-VSRs may impact new dsRNA-based management methods, resulting in unintended outcomes, including suppression of management by HIGS or SIGS. Despite a large diversity of mycoviruses being discovered using high throughput sequencing, their biology is poorly understood. In particular, the prevalence of mycoviruses and the cellular effect of their encoded VSRs are under-appreciated when considering the deployment of HIGS and SIGS strategies. This review focuses on mycoviruses, their VSR activities in fungi, and the implications for control of pathogenic fungi using RNAi.

Expression of mosquito miRNAs in entomopathogenic fungus induces pathogen-mediated host RNA interference and increases fungal efficacy

The growing threat of insecticide resistance prompts the urgent need to develop additional tools for mosquito control. Entomopathogenic fungi provide an eco-friendly alternative to chemical insecticides. One limitation to the use of mycoinsecticides is their relatively low virulence. Here, we report an approach for suppressing mosquito immunity and increasing fungal virulence. We engineered Beauveria bassiana to express Aedes immunosuppressive microRNAs (miRNAs) to induce host RNA interference (RNAi) immune responses. We show that engineered strains can produce and deliver the miRNAs into host cells to activate cross-kingdom RNAi during infection and suppress mosquito immunity by targeting multiple host genes, thereby dramatically increasing fungal virulence against Aedes aegypti and Galleria mellonella larvae. Importantly, expressing host miRNAs also significantly increases fungal virulence against insecticide-resistant mosquitoes, creating potential for insecticide-resistance management. This pathogen-mediated RNAi (pmRNAi)-based approach provides an innovative strategy to enhance the efficacy of fungal insecticides and eliminate the likelihood of resistance development.

New Insights on the Integrated Management of Plant Diseases by RNA Strategies: Mycoviruses and RNA Interference.

RNA-based strategies for plant disease management offer an attractive alternative to agrochemicals that negatively impact human and ecosystem health and lead to pathogen resistance. There has been recent interest in using mycoviruses for fungal disease control after it was discovered that some cause hypovirulence in fungal pathogens, which refers to a decline in the ability of a pathogen to cause disease. Cryphonectria parasitica, the causal agent of chestnut blight, has set an ideal model of management through the release of hypovirulent strains. However, mycovirus-based management of plant diseases is still restricted by limited approaches to search for viruses causing hypovirulence and the lack of protocols allowing effective and systemic virus infection in pathogens. RNA interference (RNAi), the eukaryotic cell system that recognizes RNA sequences and specifically degrades them, represents a promising. RNA-based disease management method. The natural occurrence of cross-kingdom RNAi provides a basis for host-induced gene silencing, while the ability of most pathogens to uptake exogenous small RNAs enables the use of spray-induced gene silencing techniques. This review describes the mechanisms behind and the potential of two RNA-based strategies, mycoviruses and RNAi, for plant disease management. Successful applications are discussed, as well as the research gaps and limitations that remain to be addressed.

Regulatory Roles of Small Non-coding RNAs in Sugar Beet Resistance Against Beet curly top virus

Beet curly top virus (BCTV) mediated yield loss in sugar beets is a major problem worldwide. The circular single-stranded DNA virus is transmitted by the beet leafhopper. Genetic sources of BCTV resistance in sugar beet are limited and commercial cultivars rely on chemical treatments versus durable genetic resistance. Phenotypic selection and double haploid production have resulted in sugar beet germplasm (KDH13; 13 and KDH4-9; 4) that are highly resistant to BCTV. The molecular mechanism of resistance to the virus is unknown, especially the role of small non-coding RNAs (sncRNAs) during early plant–viral interaction. Using the resistant lines along with a susceptible line (KDH19-17; 19), we demonstrate the role of sugar beet microRNAs (miRNAs) in BCTV resistance during early infection stages when symptoms are not yet visible. The differentially expressed miRNAs altered the expression of their corresponding target genes such as pyruvate dehydrogenase (EL10Ac1g02046), carboxylesterase (EL10Ac1g01087), serine/threonine protein phosphatase (EL10Ac1g01374), and leucine-rich repeats (LRR) receptor-like (EL10Ac7g17778), that were highly expressed in the resistant lines versus susceptible lines. Pathway enrichment analysis of the miRNA target genes showed an enrichment of genes involved in glycolysis/gluconeogenesis, galactose metabolism, starch, and sucrose metabolism to name a few. Carbohydrate analysis revealed altered glucose, galactose, fructose, and sucrose concentrations in the infected leaves of resistant versus susceptible lines. We also demonstrate differential regulation of BCTV derived sncRNAs in the resistant versus susceptible lines that target sugar beet genes such as LRR (EL10Ac1g01206), 7-deoxyloganetic acid glucosyltransferase (EL10Ac5g12605), and transmembrane emp24 domain containing (EL10Ac6g14074) and altered their expression. In response to viral infection, we found that plant derived miRNAs targeted BCTV capsid protein/replication related genes and showed differences in expression among resistant and susceptible lines. The data presented here demonstrate the contribution of miRNA mediated regulation of metabolic pathways and cross-kingdom RNA interference (RNAi) in sugar beet BCTV resistance.

Overexpression of an Osa-miR162a Derivative in Rice Confers Cross-Kingdom RNA Interference-Mediated Brown Planthopper Resistance without Perturbing Host Development.

Rice is a main food crop for more than half of the global population. The brown planthopper (BPH, Nilaparvata lugens) is one of the most destructive insect pests of rice. Currently, repeated overuse of chemical insecticides represents a common practice in agriculture for BPH control, which can induce insect tolerance and provoke environmental concerns. This situation calls for innovative and widely applicable strategies for rice protection against BPH. Here we report that the rice osa-miR162a can mediate cross-kingdom RNA interference (RNAi) by targeting the NlTOR (Target of rapamycin) gene of BPH that regulates the reproduction process. Through artificial diet or injection, osa-miR162a mimics repressed the NlTOR expression and impaired the oviposition of BPH adults. Consistently, overproduced osa-miR162a in transgenic rice plants compromised the fecundity of BPH adults fed with these plants, but meanwhile perturbed root and grain development. To circumvent this issue, we generated osa-miR162a-m1, a sequence-optimized osa-miR162a, by decreasing base complementarity to rice endogenous target genes while increasing base complementarity to NlTOR. Transgenic overexpression of osa-miR162a-m1 conferred rice resistance to BPH without detectable developmental penalty. This work reveals the first cross-kingdom RNAi mechanism in rice-BPH interactions and inspires a potentially useful approach for improving rice resistance to BPH. We also introduce an effective strategy to uncouple unwanted host developmental perturbation from desirable cross-kingdom RNAi benefits for overexpressed plant miRNAs.

Establishment of a DFG-funded research group on the topic of plant-microbe communication through extracellular RNA

The exRNA consortium, a team of researchers funded by the Deutsche Forschungsgemeinschaft (DFG), addresses crucial aspects of cross-kingdom RNA interference (ckRNAi) and RNA application in plant protection, mainly focusing on mechanistic considerations and application efficiencies.

Plant extracellular vesicles: Trojan horses of cross-kingdom warfare.

Plants communicate with their interacting microorganisms through the exchange of functional molecules. This communication is critical for plant immunity, for pathogen virulence, and for establishing and maintaining symbioses. Extracellular vesicles (EVs) are lipid bilayer‐enclosed spheres that are released by both the host and the microbe into the extracellular environment. Emerging evidence has shown that EVs play a prominent role in plant–microbe interactions by safely transporting functional molecules, such as proteins and RNAs to interacting organisms. Recent studies revealed that plant EVs deliver fungal gene‐targeting small RNAs into fungal pathogens to suppress infection via cross‐kingdom RNA interference (RNAi). In this review, we focus on the recent advances in our understanding of plant EVs and their role in plant–microbe interactions.

Message in a Bubble: Shuttling Small RNAs and Proteins Between Cells and Interacting Organisms Using Extracellular Vesicles.

Communication between plant cells and interacting microorganisms requires the secretion and uptake of functional molecules to and from the extracellular environment and is essential for the survival of both plants and their pathogens. Extracellular vesicles (EVs) are lipid bilayer-enclosed spheres that deliver RNA, protein, and metabolite cargos from donor to recipient cells and participate in many cellular processes. Emerging evidencehas shown that both plant and microbial EVs play important roles in cross-kingdom molecular exchange between hosts and interacting microbes to modulate host immunity and pathogen virulence. Recent studies revealed that plant EVs function as a defense system by encasing and delivering small RNAs (sRNAs) into pathogens, thereby mediating cross-species and cross-kingdom RNA interference to silence virulence-related genes. This review focuses on the latest advances in our understanding of plant and microbial EVs and their roles in transporting regulatory molecules, especially sRNAs, between hosts and pathogens. EV biogenesis and secretion are also discussed, as EV function relies on these important processes.

Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis.

Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex to post-transcriptionally silence host target genes. We hereby describe the methodological details of how we recovered cross kingdom sRNA effectors of the oomycete pathogen Hyaloperonospora arabidopsidis during infection of its host plant Arabidopsis thaliana. This Bio-protocol contains two parts: first, a detailed description on the procedure of plant AGO/sRNA co-immunopurification and sRNA recovery for Illumina high throughput sequencing analysis. Second, we explain how to perform bioinformatics analysis of sRNA sequence reads using a Galaxy server. In principle, this protocol is suitable to investigate AGO-bound sRNAs from diverse host plants and plant-interacting (micro)organisms.