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

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.

Keywords: Argonaute; Fusarium graminearum; double-stranded RNA; small RNA; spray-induced gene silencing; wheat.

Figure 1

PCR verification of targeted gene replacement in Fusarium graminearum. (A) Amplification of an internal part of the targeted genes DCL1, DCL2, AGO1, AGO2, RdRP1, RdRP4, RdRP2, RdRP3, QIP, and QDE3 is positive in IFA WT and negative in corresponding mutants. (B) PCR with primer pairs in the right recombination sequence and hygromycin, showing that the antibiotic resistance gene had integrated into the target gene locus. PCR products were analyzed on 1.5% agarose gel electrophoresis. M, DNA marker; WT, wild type.

Figure 2

The RNAi pathway is required for asexual development of Fusarium graminearum in the absence of inductive light. (A) Number of conidia produced: means ± SEs of the percentage of conidia numbers from three repeated experiments. Significant differences are marked: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 (Student’s t test). (B) Percent of conidial germination: means± SEs of the percentage of germinated spores from three biological repetitions. Significant differences are marked: ^*p < 0.05 (Student’s t test). (C) Microscopic observation of germinated and non-germinated conidia of IFA WT and Δrdrp4. Imaging after 48 h of incubation in the dark, scale bar: 50 μm. Black arrow, conidia forming a bipolar germ tube. Red arrow, conidia forming multiple germ tubes.

Figure 3

Forcible ascospore discharge in Fusarium graminearum RNAi mutants and IFA WT. (A) Forcible ascospore firing. Half circular carrot agar blocks covered with mature perithecia were placed on glass slides. Photos from forcibly fired ascospores (white cloudy) were taken after 48 h of incubation in boxes under high humidity and fluorescent light. (B) Fired ascospores were washed off and counted. Means± SDs of the counted spores is presented from three biological repetitions. Significant differences are marked: ^*p < 0.05; ^***p < 0.001 (Student’s t test). (C) Ascospore germination. Discharged ascospores were incubated at 100% RT in the dark for 24 h at 23°C in SN liquid medium. The percentage of germination was assessed by examining the ascospore number in three random squares in the counting chamber. Means ± SEs of the percentage of germinated spores from three biological repetitions. Significant differences are marked: ^*p < 0.05 (Student’s t test).

Figure 4

Infection of Apogee wheat spikes with Fusarium graminearum RNAi mutants and IFA WT. (A) Representative samples of spikes at 9 dpi. One spikelet at the bottom of each spike (red arrow) was point-inoculated with 5 μl of 0.002% Tween 20 water containing 40,000 conidia/ml. The assay was repeated two times with 10 spikes per fungal genotype and experiment. (B) Wheat spikes at 13 dpi.

Figure 5

Infection symptoms of Fg RNAi mutants on barley leaves sprayed with the dsRNA CYP3RNA. (A) Detached leaves of 3-week-old barley plants were sprayed with 20 ng μl⁻¹ CYP3RNA or TE buffer, respectively. After 48 h, leaves were drop-inoculated with 5 × 10⁴ conidia ml⁻¹ of indicated Fg RNAi mutants and evaluated for infection symptoms at 5 dpi. Values show relative infection area as calculated from TE- vs. CYP3RNA-treated plants for each RNAi mutant with 10 leaves and three biological repetitions. Asterisks indicate statistical significant reduction of the infection area on CYP3RNA- vs. TE-treated plants measured by ImageJ for each mutant (^*p < 0.05; ^**p < 0.01; Student’s t test). The dcl1 dcl2 double mutant is generated in Fg strain PH1. (B) Down-regulation of the three CYP51 genes in Fg mutants upon colonization of CYP3RNA- vs. TE-treated barley leaves. Asterisks indicate statistically significant down-regulation of CYP51 genes on CYP3RNA vs. TE-treated plants. (^*p < 0.05; ^**p < 0.01; ^***p < 0.001; Student’s t test). Error bars indicate SE of three independent experiments in (A) and (B).