Cross-kingdom RNA interference promotes arbuscular mycorrhiza development

nature plants Brief Communication https://doi.org/10.1038/s41477-026-02247-2 Cross-kingdom RNA interference promotes arbuscular mycorrhiza development Received: 16 April 2024 Accepted: 10 February 2026 Annika Usländer 1,2,5, Manisha V. Haag 1,5, An-Po Cheng 1,3,5, Bernhard Lederer1,3, Jin Yan Khoo 3, Florian Dunker 3, Ivan F. Acosta Arne Weiberg 3,4 & Caroline Gutjahr 1,2 , 1 Published online: xx xx xxxx Check for updates Cross-kingdom RNA interference is an emerging concept in plant– pathogen interactions. Here we provide evidence that cross-kingdom RNA interference also occurs in a beneficial plant symbiosis called arbuscular mycorrhiza. The arbuscular mycorrhizal fungus Rhizophagus irregularis transfers small RNAs into plant cells, promoting the colonization of host roots. This finding establishes inter-organismal RNA communication as a new regulatory mechanism of this ancient and widespread symbiosis. The roots of about 80% of land plants are colonized by fungi of the Glomeromycotina to form arbuscular mycorrhiza (AM) symbioses, which increase plant nutrition and performance and therefore raise interest for application in sustainable agricultural practices1,2. The fungi collect mineral nutrients from the soil, transport them into the roots and release them to plants via highly branched hyphal structures, the arbuscules, which form inside root cortex cells; in turn, they receive organic carbon in the form of sugars and lipids from plant hosts3. A plant signalling network encoded by so-called common symbiosis genes ensures a compatible interaction and accommodation of the fungus inside root cells4. Still, it appears that AM fungi need to additionally overcome plant defences at the initial stages of colonization and in arbuscule-containing cells, which may be elicited through chitin and/ or β-glucan fragments peeling off their cell walls5–7. Cross-kingdom RNA interference (ckRNAi) is an emerging concept in plant–microbe interactions in which fungal and oomycete pathogens deliver small RNAs (sRNAs) into plants to promote infection8,9. These pathogen sRNAs load into the plant’s own Argonaute(AGO)/ RNA-induced silencing complex to silence host mRNAs with functions in plant defence. ckRNAi is bidirectional, as plants too transfer sRNAs into attacking pathogens for defence10. Rhizobial bacteria engaging in root nodule symbiosis and ectomycorrhizal fungi have also been found to release sRNAs to host roots to support colonization and symbiosis11,12, and one sRNA from Rhizophagus irregularis (Rir2216) was observed to transfer into Medicago truncatula roots, silencing a WRKY transcription factor to promote symbiosis13. It is thus an appealing hypothesis that inter-organismal RNA communication occurred in ancient AM symbiosis, which evolved about 450 million years ago, although AM fungi belong to a different clade (the Glomeromycotina) and have a different lifestyle than pathogenic fungi. Here we probed for evidence of ckRNAi in AM using the model fungus R. irregularis during root colonization of the model legume Lotus japonicus. To identify candidate R. irregularis sRNAs that potentially induce ckRNAi in roots, we analysed the sRNA load of L. japonicus AGO1 during root colonization via AGO1 co-immunoprecipitation (co-IP). We used the Agrisera anti-AGO1 antibody raised against the amino-terminal part of Arabidopsis thaliana AGO1. A phylogenetic tree displayed a close relationship between AtAGO1 and LjAGO1 and a large distance to AGO proteins from R. irregularis (Supplementary Fig. 1 and Supplementary Data 1). To evaluate cross-reactivity with LjAGO and RiAGOs, we performed a multiple sequence alignment of the N-terminal part of AGOs, including AtAGO1 (At1G48410), LjAGO1 (LotjaGi2g1v0183100), the two Lotus AGOs LotjaGi5g1v0309200 and LotjaGi6g1v0002100 that cluster with AtAGO10, and three RiAGOs (UniProt entries A0A2H5TEZ5, A0A2H5R841 and A0A2H5TEY9). The PIWI domains are highly conserved across AGOs from all three organisms. However, while the N-terminal region of AGO1 is conserved across A. thaliana and L. japonicus, it is not for RiAGOs, thus allowing for specific pull-down of LjAGO (Supplementary Fig. 2). Nevertheless, to probe for potential cross-reactivity with fungal AGO proteins, we conducted LjAGO-IP from either non-colonized (mock) or R. irregularis-colonized (AM) L. japonicus roots followed by mass spectrometry (MS)-based proteomics (Supplementary Fig. 3 and Supplementary Data 2). In two co-IP experiments with independently grown plants that were Max-Planck-Institute of Molecular Plant Physiology, Potsdam-Golm, Germany. 2Plant Genetics, TUM School of Life Sciences, Technical University of Munich, Freising, Germany. 3Faculty of Biology, Genetics, University of Munich, Martinsried, Germany. 4Institute of Plant Science and Microbiology, Department of Biology, University of Hamburg, Hamburg, Germany. 5These authors contributed equally: Annika Usländer, Manisha V. Haag, An-Po Cheng. e-mail: ; 1 Nature Plants Brief Communication processed on different days, we detected a number of L. japonicus and R. irregularis proteins, but only one AGO protein, LjAGO1. This confirms that the LjAGO co-IP was successful and the Agrisera anti-AGO1 antibody is specific, at least within the limits of detection by MS. We then successfully performed LjAGO co-IP from non-colonized (mock) or R. irregularis-colonized (AM) L. japonicus roots for the detection of sRNAs8,14, as verified via LjAGO1 immunoblot analysis and by detecting two L. japonicus microRNAs, LjmiR166 and LjmiR396, via stem–loop reverse transcription (RT)-PCR (Supplementary Fig. 4a,b). To generate high-confidence results, we conducted four entirely independent experiments, for which the plants were grown in four different periods, in two different locations (TUM, Freising, experiments 1 and 2; and MPI of Molecular Plant Physiology, Potsdam, experiments 3 and 4) and by two different persons. The AGO1 pulldown, library constructions and sequencing were performed separately and also by two different persons. The fourth experiment was represented by two biological replicates (Supplementary Fig. 4a). Upon cloning and sequencing of sRNAs, we achieved depths of 10–49 million reads per sample using Illumina-based deep sequencing (Supplementary Fig. 5a). We found a size enrichment of 21-nucleotide reads with 5′-terminal uracil (U) preference for sRNAs that mapped to a L. japonicus reference genome (Gifu_v1.2) (Fig. 1a and Supplementary Fig. 5b). This was in accordance with sRNA sequencing data obtained from AtAGO1 co-IP experiments in previous studies15. Interestingly, while some variation in the size profile of sRNAs was detected among the four independently grown experiments (Fig. 1a and Supplementary Fig. 5b), the two replicates of experiment 4, which were grown and processed in parallel, showed the exact same size profile, indicating that the common practice of growing and processing samples for omics experiments in paral (...truncated)