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

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.