Abstract
ABSTRACTDicer-like proteins (DCLs) play a vital role in RNA interference (RNAi), by cleaving RNA filament into small RNAs. Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in mycoparasitic interactions is yet to be investigated. In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and characterize the functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced secondary metabolite production, antagonism toward the plant-pathogenic fungus Botrytis cinerea, and reduced ability to control Fusarium foot rot disease on wheat, caused by Fusarium graminearum. Transcriptome sequencing of the in vitro interaction between the C. rosea Δdcl2 strain and B. cinerea or F. graminearum identified the downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes, and secondary metabolites biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild-type interaction. A total of 61 putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea, and 11 were downregulated in the Δdcl2 mutant. In addition to putative endogenous gene targets, these milRNAs were predicted to target B. cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the Δdcl2 mutant incapable of producing the targeting milRNAs. In summary, this study constitutes the first step in elucidating the role of RNAi in mycoparasitic interactions, with important implications for biological control of plant diseases, and poses the base for future studies focusing on the role of cross-species RNAi regulating mycoparasitic interactions.IMPORTANCE Small RNAs mediated RNA interference (RNAi) known to regulate several biological processes. Dicer-like endoribonucleases (DCLs) play a vital role in the RNAi pathway by generating sRNAs. In this study, we investigated a role of DCL-mediated RNAi in interference interactions between mycoparasitic fungus Clonostachys rosea and the two fungal pathogens Botrytis cinerea and Fusarium graminearum (here called mycohosts). We found that the dcl mutants were not able to produce 11 sRNAs predicted to finetune the regulatory network of genes known to be involved in production of hydrolytic enzymes, antifungal compounds, and membrane transporters needed for antagonistic action of C. rosea. We also found C. rosea sRNAs putatively targeting known virulence factors in the mycohosts, indicating RNAi-mediated cross-species communication. Our study expanded the understanding of underlying mechanisms of cross-species communication during interference interactions and poses a base for future works studying the role of DCL-based cross-species RNAi in fungal interactions.
Highlights
Dicer-like proteins (DCLs) play a vital role in RNA interference (RNAi), by cleaving RNA filament into small RNAs
Phylogenetic analyses using DCL, AGO, and RNA dependent RNA polymerase (RDR) amino acid sequences revealed that C. rosea putative DCLs were most closely related to their homologs in Acremonium chrysogenum, with around 57% sequence identity, and the same was true for C. rosea homologs of AGO1 and AGO2, but with an identity around 51%
The putative DCLs of C. rosea diverged in two clusters separating the DCL1 and DCL2 from the analyzed species, and the same was evident for AGO1 and AGO2
Summary
Dicer-like proteins (DCLs) play a vital role in RNA interference (RNAi), by cleaving RNA filament into small RNAs. DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in mycoparasitic interactions is yet to be investigated. Dicer-like endoribonucleases (DCLs) play a vital role in the RNAi pathway by generating sRNAs. In this study, we investigated a role of DCL-mediated RNAi in interference interactions between mycoparasitic fungus Clonostachys rosea and the two fungal pathogens Botrytis cinerea and Fusarium graminearum (here called mycohosts). SRNAs can move bidirectionally between the species and modulate cellular functions of recipient cells by hijacking their RNAi machinery They play an important role in interspecies communication between closely interacting symbiotic organisms, including parasitic and mutualistic interactions [35,36,37,38,39,40]. The role of secreted proteins/enzymes, secondary metabolites, and membrane transporters in antibiosis and mycoparasitism in C. rosea is proven [42,43,44, 50, 53, 54]; the role of RNAi in regulating the cellular regulatory network during such interactions has not yet been investigated
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