Expression of a Structural Protein of the Mycovirus FgV-ch9 Negatively Affects the Transcript Level of a Novel Symptom Alleviation Factor and Causes Virus Infection-Like Symptoms in Fusarium graminearum

Abstract

Infections of fungi by mycoviruses are often symptomless but sometimes also fatal, as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a double-stranded RNA (dsRNA) chrysovirus-like mycovirus, debilitates Fusarium graminearum, the causal agent of fusarium head blight. In search for potential symptom alleviation or aggravation factors in F. graminearum, we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring large amounts of virus. Deletion of this gene, named virus response 1 (vr1), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus infection-like symptoms when expressed in the wild type but not in the vr1 overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1 This was confirmed by comparative transcriptome analysis, showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant, and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps us understand the underlying mechanism of hypovirulence.IMPORTANCE Virus infections of phytopathogenic fungi occasionally impair growth, reproduction, and virulence, a phenomenon referred to as hypovirulence. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. However, the poor understanding of the molecular basis of hypovirulence induction limits their application. Using the devastating fungal pathogen on cereal crops, Fusarium graminearum, we identified an mRNA binding protein (named virus response 1, vr1) which is involved in symptom expression. Downregulation of vr1 in the virus-infected fungus and vr1 deletion evoke virus infection-like symptoms, while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein is sufficient to trigger the fungal response, i.e., vr1 downregulation, and symptom development similar to virus infection. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics.

Keywords: Chrysoviridae; FgV-ch9; Fusarium graminearum; fungal response; hypovirulence; mycovirus.

FIG 1

Detection of viral dsRNA. Preparation of total RNA from mycelium of the virus-free wild type (PH1-vf), the virus-infected wild type (PH1-vi), a vr1 overexpression mutant (vr1^oe-vi), a P3-expressing mutant (P3-vi), and a vr1 deletion strain (Δvr1-vi) grown in liquid complete medium for 3 days. Virus-infected strains harbor viral dsRNA at high molecular weight.

FIG 2

FgV-ch9 and vr1-related phenotypes in Fusarium graminearum. The virus-free (PH1-vf) and virus-infected (PH1-vi) wild-type PH1, the vr1 deletion mutant (Δvr1), and the vr1 overexpression mutants (vr1^oe-vf and vr1^oe-vi) were tested for colony growth (3 dpi) (A), infection structure development (10 days on wheat glumes and stained with fluorescein isothiocyanate conjugated Triticum vulgare lectin [scale bar, 10 mm]) (B), virulence on wheat (21 dpi) (C), conidium production (D), perithecium formation (scale bar, 10 mm) (E), anastomosis formation (scale bar, 10 μm) (F), and relative vr1 gene expression (G). Virus infection triggers transcriptional downregulation of vr1 and causes phenotypes similar to those observed upon vr1 deletion. Overexpression of vr1 overrules the devastating effects of the virus infection, leading to a symptomless virus infection. The results for all strains are significantly different (P ≤ 0.05 by Student's t test) from those for the Δvr1 strain.

FIG 3

Subcellular localization of vr1. The open reading frame of Vr1 was fused to the red fluorescent protein mCherry and expressed in mycelia of Fusarium graminearum. Fluorescence microscopy reveals an even distribution of Vr1 in the cytosol. Scale bar, 20 μm.

FIG 4

Biolayer interferometry for binding affinities of purified, recombinant Vr1 and GFP to mRNA. Native, His₆-tag-purified Vr1 and GFP were assayed for binding capacities for poly(A)-oligo(dT)-purified mRNA using biolayer interferometry. In the association phase, Vr1, but not GFP, strongly binds mRNA. The association is partially released in the dissociation phase. The measurement was repeated twice.

FIG 5

Fusarium graminearum expressing the viral coat protein P3. (A) Colony morphology on minimal agar medium supplemented with CuSO₄ for repression or bathocuproinedisulfonic acid disodium salt (BCS) for induction of P3 expression. Radial growth is reduced in the mutant expressing P3 (P3-vf) compared to that of the virus-free wild type (PH1-vf). Virus infection of P3 mutants enhances the phenotype. Constitutive expression of vr1 (vr1^oe-vf) abolishes symptom development. (B) Conidium production assay. P3 expression and additional virus infection reduces the ability of the fungus to propagate vegetatively. (C) Virulence assay on wheat (21 dpi). The mutants expressing P3 are reduced in virulence compared to PH1-vf. (D) Assay for anastomosis formation. Expression of P3 and additional virus infection reduces the ability of F. graminearum to form anastomoses. (E and F) vr1 gene expression analysis. (E) Expression of P3 triggers the transcriptional downregulation of vr1 in 2 independent mutants (P3-1 and P3-3). Virus infection further enhances the downregulation of vr1. (G) Colony morphology on complete agar medium inoculated with PH1-vf and mutants expressing a P3 gene carrying a frameshift mutation. Expression of a nonsense transcript does not cause symptoms. Error bars indicate standard deviations. The dotted line refers to the respective reference conditions set to 1. The results for all strains are significantly different (P ≤ 0.05 by Student's t test) compared to those for PH1-vf and P3-vf, respectively.

FIG 6

Heterologous expression of potential coat protein orthologues. The impact of the expression of potential coat proteins on vegetative growth on minimal medium (3 dpi) supplemented with 150 μM CuSO₄ or 150 μM bathocuproinedisulfonic acid disodium salt (BCS) was assayed. None of the coat proteins showed adverse phenotypes compared to that of the wild type.

FIG 7

Venn diagram summarizing the global changes of transcription in F. graminearum due to virus infection (PH1-vi), vr1 deletion (Δvr1), and P3-expression (P3-vf). Transcriptomic profiling by RNA sequencing was performed using three biological replicates of each strain. Numbers represent genes that were significantly (P_adj value of < 0.05) differentially (log₂ >2, log₂ <−2) regulated compared to PH1-vf.

FIG 8

Gene replacement, Southern hybridization, and diagnostic PCRs for vr1 deletion. (A) Replacement and Southern hybridization strategy for vr1. Deletion of vr1 (2) by homologous recombination using a replacement fragment excised from pRS426:deltavr1 using restriction enzyme SpeI and XhoI (1) (3, genotype of disrupted strains). Flanking regions are indicated as boldface black lines. The gene flanks were fused to a nourseothricin (nat) resistance cassette, consisting of the nat acetyltransferase gene fused to the A. nidulans oliC promoter (P-oliC). Primer binding sites for PCR are indicated as small arrows (numbering refers to that in Table 4). The region used as probe for Southern analysis is represented by the dashed line. Scheme not to scale. (B) PCR analysis of three independent Δvr1 strains, one ectopic mutant, and the wild type. Deletion of vr1 was verified in the Δvr1 mutants (analyzed after single-spore purification) using primers 11 and 12. The wild type and the ectopic strain (ECT) were PCR positive for the gene internal fragment (987 bp). (C) Southern analysis of Δvr1 and the wild type. DNA of the mutants and wild-type strain was digested using SphI, separated on agarose gels, blotted on membranes, and probed with a DIG-labeled probe for a fragment of the flanking region of vr1. The probe hybridized with the DNA of the disruption mutants (2,413 bp) and the wild type (1,181 bp).