Different Components of the RNA Interference Machinery Are Required for Conidiation, Ascosporogenesis, Virulence, Deoxynivalenol Production, and Fungal Inhibition by Exogenous Double-Stranded RNA in the Head Blight Pathogen Fusarium graminearum.

Fatima Yousif Gaffar,Jafargholi Imani,Aline Koch,Karl-Heinz Kogel,Petr Karlovsky

doi:10.3389/fmicb.2019.01662

Fatima Yousif Gaffar, Jafargholi Imani + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2019.01662

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Abstract

In filamentous fungi, gene silencing through RNA interference (RNAi) shapes many biological processes, including pathogenicity. We explored the requirement of key components of fungal RNAi machineries, including DICER-like 1 and 2 (FgDCL1, FgDCL2), ARGONAUTE 1 and 2 (FgAGO1, FgAGO2), AGO-interacting protein FgQIP (QDE2-interacting protein), RecQ helicase (FgQDE3), and four RNA-dependent RNA polymerases (FgRdRP1, FgRdRP2, FgRdRP3, FgRdRP4), in the ascomycete mycotoxin-producing fungal pathogen Fusarium graminearum (Fg) for sexual and asexual multiplication, pathogenicity, and its sensitivity to double-stranded (ds)RNA. We corroborate and extend earlier findings that conidiation, ascosporogenesis, and Fusarium head blight (FHB) symptom development require an operable RNAi machinery. The involvement of RNAi in conidiation is dependent on environmental conditions as it is detectable only under low light (<2 μmol m−2 s−1). Although both DCLs and AGOs partially share their functions, the sexual ascosporogenesis is mediated primarily by FgDCL1 and FgAGO2, while FgDCL2 and FgAGO1 contribute to asexual conidia formation and germination. FgDCL1 and FgAGO2 also account for pathogenesis as their knockout (KO) results in reduced FHB development. Apart from KO mutants Δdcl2 and Δago1, mutants Δrdrp2, Δrdrp3, Δrdrp4, Δqde3, and Δqip are strongly compromised for conidiation, while KO mutations in all RdPRs, QDE3, and QIP strongly affect ascosporogenesis. Analysis of trichothecenes mycotoxins in wheat kernels showed that the relative amount of deoxynivalenol (DON), calculated as [DON] per amount of fungal genomic DNA was reduced in all spikes infected with RNAi mutants, suggesting the possibility that the fungal RNAi pathways affect Fg’s DON production. Moreover, silencing of fungal genes by exogenous target gene-specific double-stranded RNA (dsRNA) (spray-induced gene silencing, SIGS) is dependent on DCLs, AGOs, and QIP, but not on QDE3. Together these data show that in F. graminearum, different key components of the RNAi machinery are crucial in different steps of fungal development and pathogenicity.

Highlights

RNA interference (RNAi) is a conserved mechanism triggered by double-strandedRNA that mediates resistance to exogenous nucleic acids, regulates the expression of proteincoding genes on the transcriptional and post-transcriptional level, and preserves genome stability by transposon silencing (Fire et al, 1998; Mello and Conte, 2004; Hammond, 2005; Baulcombe, 2013)
The Fusarium graminearum (Fg) genome obtained from the Broad Institute2 contains many functional RNAi machinery components (Chen et al, 2015; Son et al, 2017)
Mutants were verified by PCR analysis with genomic DNA as template (Figure 1) and by expression analysis of the respective RNAi gene (Supplementary Figure S2)

Summary

Introduction

RNA interference (RNAi) is a conserved mechanism triggered by double-stranded (ds)RNA that mediates resistance to exogenous nucleic acids, regulates the expression of proteincoding genes on the transcriptional and post-transcriptional level, and preserves genome stability by transposon silencing (Fire et al, 1998; Mello and Conte, 2004; Hammond, 2005; Baulcombe, 2013). Core RNAi pathway components are conserved in eukaryotes, including most parasitic and beneficial fungi (Cogoni and Macino, 1999; Dang et al, 2011; Carreras-Villaseñor et al, 2013; Torres-Martínez and Ruiz-Vázquez, 2017): DICER-like (DCL) enzymes, which belong to the RNase III superfamily, generate double-stranded small interfering (si)RNAs and micro (mi)RNAs (Meng et al, 2017; Song and Rossi, 2017); ARGONAUTE (AGO) superfamily proteins bind small RNA duplexes to form an RNA-induced silencing complex (RISC) for transcriptional and posttranscriptional gene silencing (PTGS) (Zhang et al, 2015; Nguyen et al, 2018); and RNA-dependent RNA polymerases (RdRPs) are involved in the production of double-stranded RNA (dsRNA) that initiate the silencing mechanism as well as in the amplification of the silencing signals through the generation of secondary siRNAs (Calo et al, 2012). In Trichoderma atroviride, Abbreviations: AGO, ARGONAUTE; CYP51, Cytochrome P450 lanosterol C-14αdemethylase; DCL, DICER-like l; DON, deoxynivalenol; Fg, Fusarium graminearum; FHB, Fusarium head blight; HIGS, host-induced gene silencing; hpRNA, hairpin RNA; MSUD, meiotic silencing by unpaired DNA; NIV, nivalenol; PEG, potato extract glucose; QDE 2,3, Quelling defective 2,3; QIP, QDE-interacting protein; RdRp, RNA-dependent RNA polymerase; RISC, RNA-dependent silencing complex; RNAi, RNA interference; RPA, subunit of replication protein A; siRNA, small interfering RNA; SN, synthetic nutrient agar; ssDNA, single-stranded DNA; TGW, thousand grain weight

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