Key message We demonstrate non-immunogenic circRNA as a tool for targeted gene regulation in plants, where it acts in an isoform- and sequence-specific manner, enabling future agronomic applications. Abstract Circular RNAs (circRNAs) are single-stranded RNA molecules characterized by their covalently closed structure and are emerging as key regulators of cellular processes in mammals, including gene expression, protein function and immune responses. Recent evidence suggests that circRNAs also play significant roles in plants, influencing development, nutrition, biotic stress resistance, and abiotic stress tolerance. However, the potential of circRNAs to modulate target protein abundance in plants remains largely unexplored. In this study, we investigated the potential of designer circRNAs to modulate target protein abundance in plants using Arabidopsis protoplasts as a model system. We show that PEG-mediated transfection with a 50-nt circRNAGFP containing a 30-nt GFP-antisense sequence results in a dose- and sequence-dependent reduction of GFP reporter target protein abundance. Notably, a single-stranded open isoform of circRNAGFP had little effect on protein abundance, indicating the importance of the closed circular structure. Additionally, circRNAGFP also reduced GFP abundance in Arabidopsis mutants defective in RNA interference (RNAi), suggesting that circRNA activity is independent of the RNAi pathway. We also show that circRNA, unlike dsRNA, does not induce pattern-triggered immunity (PTI) in plants. Findings of this proof-of-principle study together are crucial first steps in understanding the potential of circRNAs as versatile tools for modulating gene expression and offer exciting prospects for their application in agronomy, particularly for enhancing crop traits through metabolic pathway manipulation. Supplementary Information The online version contains supplementary material available at 10.1007/s00299-025-03512-y.

Abstract Bidirectional communication between pathogenic microbes and their plant hosts via small RNA (sRNA)-mediated cross-kingdom RNAi (ckRNAi) is a key element for successful host colonization. Whether mutualistic fungi of the Serendipitaceae family, known for their extremely broad host range, use sRNAs to colonize plant roots is still under debate. To address this question, we developed a pipeline to validate the accumulation, translocation, and activity of fungal sRNAs in post-transcriptional silencing of Arabidopsis thaliana genes. Using stem–loop quantitative reverse transcription–PCR, we detected the expression of a specific set of Serendipita indica (Si) sRNAs, targeting host genes involved in cell wall organization, hormonal signalling regulation, immunity, and gene regulation. To confirm the gene silencing activity of these sRNAs in plant cells, SisRNAs were transiently expressed in protoplasts. Stem–loop PCR confirmed sRNA expression and accumulation, while qPCR validated post-transcriptional gene silencing of their predicted target genes. Furthermore, Arabidopsis ARGONAUTE 1 immunoprecipitation revealed the loading of fungal SisRNAs into the plant RNAi machinery, suggesting the translocation of SisRNA from the fungus into root cells. In conclusion, this study provides a blueprint for rapid selection and analysis of sRNA effectors and further supports the model of cross-kingdom communication in the Sebacinoid symbiosis.

RNA interference (RNAi) is a crucial mechanism that can contribute to immunity against infectious microbes through the action of DICER-LIKE (DCL) and ARGONAUTE (AGO) proteins. In the case of the fungal pathogen Botrytis cinerea and the oomycete Hyaloperonospora arabidopsidis, plant DCL and AGO proteins have proven roles as negative regulators of immunity, suggesting functional specialization of these proteins. To address this aspect in a broader taxonomic context, we characterized the colonization pattern of an informative set of DCL and AGO loss-of-function mutants in Arabidopsis thaliana upon infection with a panel of pathogenic microbes with different lifestyles, and a fungal mutualist. Our results revealed that AGO1 and AGO4 function as positive regulators of immunity to a bacterial and a fungal pathogen, respectively. Additionally, AGO2 and AGO10 positively modulated the colonization by a fungal mutualist. Therefore, analysing the role of RNAi across a broader range of plant-microbe interactions has identified previously unknown functions for AGO proteins. For some pathogen interactions, however, all tested mutants exhibited wild type-like infection phenotypes, suggesting that the roles of AGO and DCL proteins in these interactions may be more complex to elucidate.

Cross-kingdom RNA interference (ckRNAi), the bidirectional communication between microbes and their host counterparts, is a key element in the outcome of host colonization. Whether mutualistic fungi of the Serendipitaceae family with their broad host range use small RNA effectors (sRNAs) to colonize plant roots is still under debate. To investigate if ckRNAi is a factor in the symbiosis of Serendipita indica (Si) with Arabidopsis thaliana (Ath), we established a pipeline to validate expression, translocation and post-transcriptional gene silencing of host genes by Si-derived sRNAs (SisRNAs). First, we confirmed the expression of SisRNAs both in axenic fungal culture and during Ath root colonization using stem-loop PCR. Then, to verify the translocation of putative SisRNA effectors, an ARGONAUTE 1 immuno-purification assay (AtAGO1-IP) was employed, detecting fungal SisRNAs being loaded into the plant RNAi machinery in Si-colonised roots. Subsequently, SisRNAs and artificial sRNAs (amiRNAs), were transiently expressed in Ath protoplasts to test their gene silencing activity. Stem-loop PCR confirmed expression of sRNA effectors and qPCR validated post-transcriptional gene silencing of their predicted target genes involved in cell wall organization, hormonal signalling regulation, plant immunity and gene expression. Moreover, 5’-RLM-RACE analysis revealed amiRNA-mediated canonical cleavage in Arabidopsis targets. In conclusion, this study provides a blueprint for rapid selection and analysis of sRNA effectors in plant-microbe interactions in general and suggests cross-kingdom communication in the Sebacinoid symbiosis.

Circular RNAs (circRNAs) are single-stranded molecules that have attracted increasing attention in recent years due to their covalently closed structure and their diverse functional roles in mammalian cells, where they are involved in the regulation of gene expression and protein function. Increasing evidence suggests that circRNAs have similar functions in plants, where they play a role in plant development, resistance to biotic stress, and abiotic stress tolerance. Here, we investigated the agronomically relevant question of whether synthetic designer circRNAs can be used to modulate in a sequence-specific manner gene expression in plants. We show that treatment of GFP-expressing Arabidopsis protoplasts with designer 50 nt GFP antisense circRNA (circRNAGFP) reduces the cellular accumulation of the reporter protein in a sequence-specific and dose-dependent manner. This inhibitory activity of circRNAGFP was not abolished in various Arabidopsis ago and dcl mutants with defective RNAi pathways. Moreover, and in contrast to other types of RNA such as double-stranded (ds)RNA, circRNAs did not induce a PTI response in plant leaves. We discuss the possibility that circRNA may be applied to regulate endogenous plant genes and thus may have future potential as a novel bioherbicide.

Background Beneficial associations between plants and microbes are widespread in nature and have been studied extensively in the microbial-dominant environment of the rhizosphere. Such associations are highly advantageous for the organisms involved, benefiting soil microbes by providing them access to plant metabolites, while plant growth and development are enhanced through the promotion of nutrient uptake and/or protection against (a)biotic stresses. While the establishment and maintenance of mutualistic associations have been shown to require genetic and epigenetic reprogramming, as well as an exchange of effector molecules between microbes and plants, whether short RNAs are able to effect such changes is currently unknown. Here, we established an interaction between the model grass species Brachypodium distachyon ( Bd , Pooideae) and the beneficial fungal root endophyte Serendipita indica ( Si , syn. Piriformospora indica , Sebacinales) to elucidate RNA interference-based regulatory changes in gene expression and small (s)RNA profiles that occurred during establishment of a Sebacinalean symbiosis. Results Colonization of Bd roots with Si resulted in higher grain yield, confirming the mutualistic character of this interaction. Resequencing of the Si genome using the Oxford Nanopore technique, followed by de novo assembly yielded in 57 contigs and 9441 predicted genes, including putative members of several families involved in sRNA production. Transcriptome analysis at an early stage of the mutualistic interaction identified 2963 differentially expressed genes (DEG) in Si and 317 in Bd line 21-3. The fungal DEGs were largely associated with carbohydrate metabolism, cell wall degradation, and nutrient uptake, while plant DEGs indicated modulation of (a)biotic stress responses and defense pathways. Additionally, 10% of the upregulated fungal DEGs encode candidate protein effectors, including six DELD proteins typical for Sebacinales. Analysis of the global changes in the sRNA profiles of both associated organisms revealed several putative endogenous plant sRNAs expressed during colonization belonging to known micro (mi)RNA families involved in growth and developmental regulation. Among Bd - and Si -generated sRNAs with putative functions in the interacting organism, we identified transcripts for proteins involved in circadian clock and flowering regulation as well as immunity as potential targets of fungal sRNAs, reflecting the beneficial activity of Si . Conclusions We detected beneficial effects of Si colonization on Bd growth and development, and established a novel plant-mutualist interaction model between these organisms. Together, the changes in gene expression and identification of interaction-induced sRNAs in both organisms support sRNA-based regulation of defense responses and plant development in Bd , as well as nutrient acquisition and cell growth in Si . Our data suggests that a Sebacinalean symbiosis involves reciprocal sRNA targeting of genes during the interaction.