A pipeline for validation of <i>Serendipita indica</i> effector-like sRNA suggests cross-kingdom communication in the symbiosis with Arabidopsis

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.