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Fungal pathogens pose escalating challenges to global food security, as resistance has emerged against nearly all major fungicides used in agriculture. RNA-based antifungals offer a sustainable and environmentally friendly alternative for disease control, but their deployment is hindered by RNA instability under environmental conditions, especially in soil. In this study, we engineered two plant-beneficial soil bacteria-Bacillus subtilis (Gram-positive) and Pseudomonas putida (Gram-negative)-to produce double-stranded RNAs (dsRNAs) targeting fungal genes in the foliar and postharvest pathogen Botrytis cinerea and the soilborne pathogen Verticillium dahliae. We found that both bacterial species secrete RNA through extracellular vesicles (EVs) and that these RNAs are transported into fungal cells, demonstrating cross-kingdom RNA trafficking from bacteria to fungi. Application of dsRNA-containing bacterial EVs to plant leaves suppressed B. cinerea infection. In addition, direct treatment with dsRNA-producing bacteria protected both Arabidopsis thaliana and tomato plants from infections by B. cinerea and V. dahliae. Our findings establish beneficial bacteria as a scalable platform for continuous production and delivery of antifungal RNAs, enabling a cost-effective strategy for sustainable crop protection. 

Abstract Small RNAs (sRNAs) of the fungal pathogen Botrytis cinerea can enter plant cells and hijack host Argonaute protein 1 (AGO1) to silence host immunity genes. However, the mechanism by which these fungal sRNAs are secreted and enter host cells remains unclear. Here, we demonstrate that B. cinerea utilizes extracellular vesicles (EVs) to secrete Bc-sRNAs, which are then internalized by plant cells through clathrin-mediated endocytosis (CME). The B. cinerea tetraspanin protein, Punchless 1 (BcPLS1), serves as an EV biomarker and plays an essential role in fungal pathogenicity. We observe numerous Arabidopsis clathrin-coated vesicles (CCVs) around B. cinerea infection sites and the colocalization of B. cinerea EV marker BcPLS1 and Arabidopsis CLATHRIN LIGHT CHAIN 1 , one of the core components of CCV. Meanwhile, BcPLS1 and the B. cinerea- secreted sRNAs are detected in purified CCVs after infection. Arabidopsis knockout mutants and inducible dominant-negative mutants of key components of the CME pathway exhibit increased resistance to B. cinerea infection. Furthermore, Bc-sRNA loading into Arabidopsis AGO1 and host target gene suppression are attenuated in those CME mutants. Together, our results demonstrate that fungi secrete sRNAs via EVs, which then enter host plant cells mainly through CME. 

Summary Spray‐induced gene silencing (SIGS) is an innovative and eco‐friendly technology where topical application of pathogen gene‐targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of RNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross‐kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for RNA encapsulation and control against the fungal pathogen, Botrytis cinerea . AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP and DODMA, and examined for their ability to protect and deliver double stranded RNA (dsRNA). All three formulations enabled dsRNA delivery and uptake by B . cinerea . Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi‐mediated protection against B . cinerea both on pre‐ and post‐harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome RNA instability in SIGS for crop protection. 

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. 

Abstract 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. 

ABSTRACT One of the most promising tools for the control of fungal plant diseases is spray‐induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double‐stranded RNA (dsRNA) targeting essential or virulence‐related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea , a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco‐friendly alternative to traditional fungicides. 

Plants have evolved variable phenotypic plasticity to counteract different pathogens and pests during immobile life. Microbial infection invokes multiple layers of host immune responses, and plant gene expression is swiftly and precisely reprogramed at both the transcriptional level and post-transcriptional level. Recently, the importance of epigenetic regulation in response to biotic stresses has been recognized. Changes in DNA methylation, histone modification, and chromatin structures have been observed after microbial infection. In addition, epigenetic modifications may be preserved as transgenerational memories to allow the progeny to better adapt to similar environments. Epigenetic regulation involves various regulatory components, including non-coding small RNAs, DNA methylation, histone modification, and chromatin remodelers. The crosstalk between these components allows precise fine-tuning of gene expression, giving plants the capability to fight infections and tolerant drastic environmental changes in nature. Fully unraveling epigenetic regulatory mechanisms could aid in the development of more efficient and eco-friendly strategies for crop protection in agricultural systems. In this review, we discuss the recent advances on the roles of epigenetic regulation in plant biotic stress responses.