bioRxiv2021
MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein-coding gene expression primarily found in plants and animals. Fungi produce microRNA-like RNAs (milRNAs) that are structurally similar to miRNAs and functionally important in various biological processes. The fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Panama disease that threatens global banana production. It remains uncharacterized about the biosynthesis and functions of milRNAs in Foc. In this study, we investigated the biological function of milRNAs contributing to Foc pathogenesis. Within 24 hours post infecting the host, the Argonaute coding gene FoQDE2, and two Dicer coding genes FoDCL1 and FoDCL2, all of which are involved in milRNA biosynthesis, were significantly induced. FoQDE2 deletion mutant exhibited decreased virulence and hypersensitivity to hydrogen peroxide (H2O2). These results indicate that milRNA biosynthesis is crucial for Foc pathogenesis. By small RNA sequencing, we identified 364 small RNA-producing loci in the Foc genome, 25 of which were significantly downregulated in the FoQDE2 deletion mutant, from which milR-87 was verified as a FoQDE2-depedent milRNA based on qRT-PCR analysis. Through deletion and overexpression of milR-87 in the wild-type Foc strain, functions of milR-87 were studied. The results showed that milR-87 is crucial for Foc virulence in infection process. We furthermore identified a glycosyl hydrolase-coding gene, FOIG_15013, as the direct target of milR-87. The FOIG_15013 deletion mutant displayed a dramatic increase in the growth, conidiation and virulence. Transient expression of FOIG_15013 in Nicotiana benthamiana leaves activates the host defense responses. Collectively, this study documents the involvement of milRNAs in the manifestation of the devastating fungal disease in banana, and demonstrates the importance of milRNAs in the pathogenesis and other biological processes. Further analyses of the biosynthesis and expression regulation of fungal milRNAs may offer a novel strategy to combat devastating fungal diseases. Author summaryThe fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Panama disease that threatens global banana production. As a typical representative of F. oxysporum species complex, the pathogen has been widely concerned. However, pathogenesis of Foc is not fully elucidated. In particular, pathogenic regulatory mechanism of the microRNA like small RNAs (milRNAs) found in Foc is unknown. Here, we found that FoQDE2, one Argonaute coding gene, and two Dicer coding genes FoDCL1 and FoDCL2, which are involved in milRNA biosynthesis, are significantly induced during the early infection stage of Foc. The results suggested that the milRNAs biosynthesis mediated by these genes may play an active role in the infection process of Foc. Based on this assumption, we subsequently found a FoQDE2-dependent milRNA (milR-87) and identified its target gene. Functional analysis showed that FoQDE2, miR-87 and its target gene were involved in the pathogenicity of Foc in different degree. The studies help us gain insight into the pathogenesis with FoQDE2, milR-87, and its target gene as central axis in Foc. The identified pathogenicity-involved milRNA provides an active target for developing novel and efficient biocontrol agents against Panama disease.