A pipeline for validation of sRNA effectors suggests cross-kingdom communication in the symbiosis of Arabidopsis with Serendipita indica

Bidirectional communication between pathogenic microbes and their plant hosts via small (s)RNA-mediated cross-kingdom RNA interference (ckRNAi) is one key element for successful host colonisation. However, whether mutualistic fungi from the Serendipitaceae family, known for their extremely broad host range, employ sRNAs to colonize plant roots is still under discussion. 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 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 sRNA in plant cells, SisRNAs were transiently expressed in protoplasts. Stem-loop PCR proved the expression of sRNAs, while qPCR validated post-transcriptional gene silencing of their predicted target genes. Furthermore, ARGONAUTE 1 immunoprecipitation (AtAGO1-IP) 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 in plant-microbe interactions and further suggests cross-kingdom communication in the Sebacinoid symbiosis. Highlight Small RNAs of the beneficial fungus Serendipita indica are translocated and silence Arabidopsis genes at the onset of the interaction, revealing cross-kingdom communication in sebacinoid symbiosis.