Fungal small RNA hijacking: a new layer of cross-kingdom communications in arbuscular mycorrhizal symbiosis

Abstract

A complex hierarchy of cross-kingdom communications controls mutualistic and pathogenic interactions between bacteria, fungi, and plant hosts (Cai et al., 2018; Jimenez-Jimenez et al., 2019; Betz et al., 2024; Wang et al., 2024). Among them, the inter-kingdom interactions between mutualistic fungi, such as mycorrhizal fungi and host plants during mycorrhiza formation, are characterized by the exchange of molecular signals. These facilitate mineral nutrition assimilation and contribute to abiotic stress tolerance (Plett et al., 2014; Lanfranco et al., 2018; Kang et al., 2020; Wong-Bajracharya et al., 2022). In previous studies, researchers found that some mycorrhizal fungi exported effector molecules (similar to pathogenic effectors) into roots to reprogram plant cells or suppress host immunity (Kloppholz et al., 2011; Zeng et al., 2020; Betz et al., 2024). For example, the mycorrhizal fungal effectors SP7 and SP7-like regulate symbioses at the protein level (Kloppholz et al., 2011; Betz et al., 2024). There is also emerging evidence from studies of plant symbiotic systems that suggests effector-like small RNAs (sRNAs) can travel between fungi and host plants to trigger cross-kingdom RNA interference (ckRNAi) in recipient cells and facilitate symbiosis (Wong-Bajracharya et al., 2022; Nasfi et al., 2024). For example, it was found that Pmic_miR-8, a microRNA (miRNA) encoded by the ectomycorrhizal fungus Pisolithus microcarpus, was transported into Eucalyptus grandis roots during a mutualistic interaction. Experimental analysis suggests that Pmic_miR-8 may target host transcripts containing the NB-ARC domain, which in turn stabilizes mycorrhizal symbiosis in E. grandis by subverting host immunity signals (Wong-Bajracharya et al., 2022). However, until now the role of fungal sRNAs in arbuscular mycorrhizal (AM) symbiosis has remained unknown. In a priority report recently published in New Phytologist, Silvestri et al. (2024, doi: 10.1111/nph.20273) use an in silico prediction and molecular analyses to present biochemical and reverse genetics evidence that Rir2216, an sRNA from the model AM fungus Rhizophagus irregularis, acts as an sRNA effector when delivered to Medicago truncatula root cells. Once delivered, Rir2216 hijacks the host Argonaute (AGO) protein, MtAGO1, and silences the host gene MtWRKY69, giving rise to a successful AM symbiosis.

… AM fungal small RNAs just entered the ‘chat', and a new layer of cross-kingdom molecular signals enables AM symbiosis.

Eukaryotic sRNAs are short noncoding regulatory elements (usually 20–25 nucleotides in length) that trigger the RNAi process in cells and act as big players in microbe–plant interactions (Huang et al., 2019). Arbuscular mycorrhizal fungal sRNAs are emerging as crucial molecules in the symbiotic regulatory networks at the RNA level (Silvestri et al., 2019; Ledford et al., 2024). Recently, ckRNAi was revealed to form an essential component of bidirectional interactions between AM fungi and host plants, regulating crosstalk between mycorrhizal symbiosis and host immunity, indicating that sRNA translocation occurs in AM symbiosis (Qiao et al., 2023). Furthermore, it has been observed that arbuscular cell invasion coincides with the formation of extracellular vesicles (EVs) and membrane tubules (Roth et al., 2019). These findings are interesting, as EVs have been shown to represent transfer pathways for cross-kingdom molecular signals (such as double-strand RNAs (dsRNAs), sRNAs, mRNAs, and proteins) in ckRNAi during plant–pathogen interactions (Cai et al., 2018; He et al., 2023; Wang et al., 2024; Zhang et al., 2024). In addition, host- and virus-induced gene silencing approaches are suitable tools for silencing AM fungal genes in mycorrhizal roots, supporting the movement of sRNAs from root cells to AM fungi (Helber et al., 2011; Kikuchi et al., 2016).

In AM fungi, the potential role of sRNAs in fungus-to-plant transfer and ckRNAi processes remained elusive until Silvestri et al. (2019) revealed that the AM fungus R. irregularis possesses RNAi machinery and generates many sRNAs, some of which were predicted to target mRNAs from the host M. truncatula. Silvestri et al. (2019) also proposed that fungal sRNAs possibly participate in a ckRNAi process during AM symbiosis, similar to the role of fungal sRNAs in cross-kingdom interactions at the pathogen–plant interface.

In this study, Silvestri et al. (2024) build on their previous work by conducting in silico prediction analysis to identify an sRNA from R. irregularis and its target gene in the host plant M. truncatula. Through multiple assays, they have achieved the first experimental evidence that an AM fungal sRNA mediates plant gene silencing through ckRNAi (Fig. 1a–d), which results in the promotion of the AM symbiosis. In the field of AM fungal molecular biology, this provides valuable insight into a new layer of fungus–plant communication during AM symbiosis and inspires further research into the molecular mechanisms underlying AM fungal symbionts.

Fig. 1

Open in figure viewerPowerPoint

Cross-kingdom small RNA (sRNA) Rir2216 trafficking between Rhizophagus irregularis and host plant Medicago truncatula. (a) Schematic representation of the M. truncatula seedling colonized with arbuscular mycorrhizal (AM) fungus R. irregularis. (b) Rhizophagus irregularis-derived sRNA Rir2216 is delivered from the extraradical mycelium (ERM) into the intraradical mycelium (IRM) and arbuscule-containing cortical cells. (c) A simplified scheme of an arbuscule-containing cell showing the fungal membrane, periarbuscular space, periarbuscular membrane, and Rir2216 transfer from the intraradical hyphae into the arbuscule. (d) Cross-kingdom RNA interference (RNAi) between R. irregularis and M. truncatula root. In this process, the R. irregularis-derived sRNA molecule Rir2216 can serve as an effector that is translocated into host plant cells to hijack the M. truncatula Argonaute (AGO) protein MtAGO1 to silence the host defense gene MtWRKY69 for successful AM colonization. On the other hand, host-derived double-strand RNAs (dsRNAs) or sRNAs are delivered into the cells of the symbiont R. irregularis and induce target gene silencing in trans. While the delivery mechanisms remain unknown, it is proposed that extracellular vesicles (EVs) may play a crucial role in the cross-kingdom sRNA trafficking and that cross-kingdom RNAi may be bidirectional between AM fungi and host plants.

The authors use a sequence complementarity approach to show that the AM fungal sRNA Rir2216 is predicted to target the mRNA of WRKY69 from the host M. truncatula. Heterologous co-expression, 5′ RACE reactions, and AGO1-immunoprecipitation assays demonstrate the direct interaction between Rir2216 and MtWRKY69. The assays revealed that R. irregularis sends the sRNA, Rir2216, into Medicago root cells by hijacking the MtAGO1 protein-equipped RNAi machinery to silence MtWRKY69 at the post-transcriptional level (Fig. 1d). The implications of this finding are exciting. As Nasfi et al. (2024) reported, the beneficial Serendipita indica SisRNAs are translocated from the fungus into Arabidopsis root cells to hijack AtAGO1 and induce plant RNAi machinery, suggesting that the model of cross-kingdom sRNA transfer is conserved in the fungus–plant interaction.

The genetic manipulation of AM fungi has been hampered by the fact that they are obligate biotrophs and have multi-nuclei. Therefore, it is impossible to directly knockout the Rir2216 gene from the genome of R. irregularis at the present time. However, Silvestri et al. (2024) use both constitutive and conditional expression strategies to overexpress MtWRKY69. MtWRKY69 overexpression resulted in reduced mycorrhiza formation.

These findings are very timely. In a recent review, Ledford et al. (2024) also describe ‘the characteristics of AM fungal-derived sRNAs and emerging evidence for the bidirectional transfer of functional sRNAs between the two partners to mutually modulate gene expression and control the symbiosis’. Together, the authors provide direct evidence of previously undescribed sRNA movement from AM fungi into host roots to increase colonization levels. A proposed working model by Silvestri et al. (2024) would be that the Rir2216-mediated knockdown of MtWRKY69 in arbuscule-containing cells could contribute to the suppression of host immunity in roots, enabling AM symbiosis since many WRKY transcription factors are expressed in response to plant pathogens (Jiang et al., 2017).

Silvestri et al.'s (2024) multiple assays represent a rigorous dissection of a molecular pathway in AM fungi and provide a better methodological framework for addressing mechanistic issues. This framework can be used to validate the involvement of crucial fungal miRNAs during AM symbiosis. In this report, Silvestri et al. (2024) highlight the key roles of sRNAs in AM fungi and RNAi machinery in symbiosis, suggesting that AM fungal sRNAs just entered the ‘chat’, and a new layer of cross-kingdom molecular signals enables AM symbiosis. Understanding the molecular mechanisms of fungal sRNA trafficking and RNAi machinery will help us develop novel approaches for effectively promoting AM symbiosis and plant nutrition.

Despite the breakthrough, many mysteries remain. Primarily, whether in vitro synthetic Rir2216 artificially increases the miRNA Rir2216 level in R. irregularis during AM fungal colonization via the sRNA treatment (Wang et al., 2016), leading to the accelerated degradation of MtWRKY69 transcripts. Further investigations into whether R. irregularis delivers Rir2216 and other sRNAs into root cells via EVs are needed (Roth et al., 2019; He et al., 2023; Fig. 1d). Additionally, the precise functions of MtWRKY69 in mycorrhizal roots are largely unknown so far; indeed, it would be interesting to create CRISPR mutant lines in order to elucidate whether the loss of function of this gene efficiently promotes AM symbiosis. Finally, it remains unknown whether ckRNAi in the AM symbiosis is a bidirectional process (Fig. 1d), as has been extensively reported in other microbe–plant interactions (Huang et al., 2019; He et al., 2023).

In summary, the publication by Silvestri et al. (2024) demonstrates that AM fungi can regulate host plant gene expression to promote symbiosis via functional sRNAs and sheds light on how mycorrhizal fungi have evolved mechanisms to colonize plant roots. Moreover, the authors open new avenues to effectively promote AM symbiosis in the future.