Discovery of interaction-related sRNAs and their targets in theBrachypodium distachyonandMagnaporthe oryzaepathosystem
Authors: Zanini Silvia , Šečić Ena , Busche Tobias , Kalinowski Jörn , Kogel Karl-Heinz 
Published: May 2019

AbstractMicrobial pathogens secrete small RNA (sRNA) effectors into plant hosts to aid infection by silencing transcripts of immunity and signaling-related genes through RNA interference (RNAi). Similarly, sRNAs from plant hosts have been shown to contribute to plant defense against microbial pathogens by targeting transcripts involved in virulence. This phenomenon is called bidirectional RNA communication or cross kingdom RNAi (ckRNAi). How far this RNAi-mediated mechanism is evolutionarily conserved is the subject of controversial discussions. We examined the bidirectional accumulation of sRNAs in the interaction of the hemibiotrophic rice blast fungusMagnaporthe oryzae(Mo) with the grass model plantBrachypodium distachyon(Bd). By comparative deep sequencing of sRNAs and mRNAs from axenic fungal cultures and infected leaves and roots, we found a wide range of fungal sRNAs that accumulated exclusively in infected tissues. Amongst those, 20-21 nt candidate sRNA effectors were predictedin silicoby selecting thoseMoreads that had complementary mRNA targets inBd. Many of those mRNAs predicted to be targeted byMosRNAs were differentially expressed, particularly in the necrotrophic infection phase, including gene transcripts involved in plant defense responses and signaling. Vice versa, by applying the same strategy to identifyBdsRNA effectors, we found thatBdproduced sRNAs targeting a variety of fungal transcripts, encoding fungal cell wall components, virulence genes and transcription factors. Consistent with function as effectors of theseBdsRNAs, their predicted fungal targets were significantly down-regulated in the infected tissues compared to axenic cultures, and deletion mutants for some of these target genes showed heavily impaired virulence phenotypes. Overall, this study provides the first experimentally-based evidence for bidirectional ckRNAi in a grass-fungal pathosystem, paving the way for further validation of identified sRNA-target duplexes and contributing to the emerging research on naturally occurring cross-kingdom communication and its implications for agriculture on staple crops.Author SummaryIn the present work, we provide first experimental evidence for bidirectional RNA communication in a grass-fungal pathosystem. We deployed the monocotyledonous plantBrachypodium distachyon, which is a genetic model for the staple crops wheat and rice, to investigate the interaction-related sRNAs for their role in RNA communication. By applying a previously published bioinformatics pipeline for the detection of sRNA effectors we identified potential plant targets for fungal sRNAs and vice versa, fungal targets for plant sRNAs. Inspection of the respective targets confirmed their downregulation in infected relative to uninfected tissues and fungal axenic cultures, respectively. By focusing on potential fungal targets, we identified several genes encoding fungal cell wall components, virulence proteins and transcription factors. The deletion of those fungal targets has already been shown to produce disordered virulence phenotypes. Our findings establish the basis for further validation of identified sRNA-mRNA target duplexes and contribute to the emerging research on naturally occurring cross-kingdom communication and its implications for agriculture.