Investigating the role of small RNAs in the interaction between the model grass Brachypodium distachyon and the endosymbiont Serendipita indica (syn. Piriformospora indica)

Abstract

Plants and microbes readily establish interactions resulting in varying outcomes, ranging from antagonistic to beneficial for both organisms. Mutualistic interactions with reciprocal benefits are called symbioses, and they include an exchange of nutrients between plant and microbe, as well as growth promotion and priming against biotic and abiotic stresses within the host. The rhizosphere is a microbe-rich environment in which roots come in contact with many organisms, including beneficial endophytes that can colonize and proliferate inside plants. Recent discoveries emphasized the role of small RNAs (sRNAs) and RNA interference (RNAi) in modulating gene expression by cross-kingdom (ck) communication. In plant-pathogen ck communication, sRNAs are exchanged between a plant and a microbe in a bidirectional fashion, targeting virulence genes of the pathogen and defense-related transcripts in the plant. Beyond this exogenous role of sRNAs, it is known that both plants and microbes reprogram their transcriptional landscape in response to their interactions, by deploying endogenous gene silencing via micro RNAs (miRNAs). RNAi-associated proteins, which facilitate both endogenous and ck-guided gene silencing, are encoded by Argonaute (AGO) and Dicer-like (DCL) gene families. Since these gene families are expanded in many plant species, we analyzed the genome of the model grass plant Brachypodium distachyon to detect its putative AGOs and DCLs and elucidate on their structures. Subsequently, a novel model system for plant-endophyte research, between B. distachyon and the endosymbiont Serendipita indica (syn. Piriformospora indica) was established. Concurrent mRNA and sRNA sequencing of the colonized roots and control samples within this interaction was performed, and a previously established bioinformatics pipeline applied to predict putative sRNAs with ck and endogenous roles. Target prediction and analysis of downregulation indicate that sRNA- mediated silencing might be involved in growth, development, modulation of plant immunity and fungal nutrient acquisition during the colonization. Thus, RNAi-based regulation in this system provides novel insight into sRNAs in mutualistic interactions, and also promises target discovery and translation to crop species that can be colonized by S. indica. The role of sRNA-mediated silencing during infection of B. distachyon tissues with the fungal pathogens Magnaporthe oryzae and Fusarium graminearum also indicated control of plant immune responses. Evidently, sRNAs are widely utilized to navigate the transcriptional landscape in plant- microbe interactions. Conserved plant miRNA families are also variably responsive to pathogens and mutualists, reinforcing both the parallels and differences in plant responses. RNAi-based plant protection, which utilizes this pathway to silence pathogenic virulence genes, is an attractive addition to existing plant protection strategies. We concluded that the requirements for efficient RNAi-based application against fungal pathogens converge on stability of applied sRNA, uptake and expression of fungal RNAi proteins.