Effects of the deletion and over-expression of Fusarium graminearum gene FgHal2 on host response to mycovirus Fusarium graminearum virus 1

Abstract

The mycovirus Fusarium graminearum virus 1 (FgV1) is associated with reduced virulence (hypovirulence) of Fusarium graminearum. Transcriptomic and proteomic expression profiling have shown that many F. graminearum genes are differentially expressed as a consequence of FgV1 infection. Several of these genes may be related to the maintenance of the virus life cycle. The host gene, FgHal2, which has a highly conserved 3'-phosphoadenosine 5'-phosphatase (PAP phosphatase-like) domain or inositol monophosphatase (IMPase) superfamily domain, shows reduced expression in response to FgV1 infection. We generated targeted gene deletion and over-expression mutants to clarify the possible function(s) of FgHal2 and its relationship to FgV1. The gene deletion mutant showed retarded growth, reduced aerial mycelia formation and reduced pigmentation, whereas over-expression mutants were morphologically similar to the wild-type (WT). Furthermore, compared with the WT, the gene deletion mutant produced fewer conidia and these showed abnormal morphology. The FgHal2 expression level was decreased by FgV1 infection at 120 h post-inoculation (hpi), whereas the levels were nine-fold greater for both the virus-free and virus-infected over-expression mutant than for the WT. FgV1 RNA accumulation was decreased in the deletion mutant at 48, 72 and 120 hpi. FgV1 RNA accumulation in the over-expression mutant was reduced relative to that of the WT at 48 and 120 hpi, but was similar to that of the WT at 72 hpi. The vertical transmission rate of FgV1 in the gene deletion mutant was low, suggesting that FgHal2 may be required for the maintenance of FgV1 in the host cell. Together, these results indicate that the putative 3'(2'),5'-bisphosphate nucleotidase gene, FgHal2, has diverse biological functions in the host fungus and may affect the viral RNA accumulation and transmission of FgV1.

Keywords: 3′(2′); 5′-bisphosphate nucleotidase; FgHal2; Fusarium graminearum; Fusarium graminearum virus 1; secondary metabolism; virus-host interaction.

Figure 1

Amino acid sequence alignment of FgHal2 corresponding to the conserved 3′‐phosphoadenosine 5′‐phosphatase (PAP phosphatase‐like) domain of the predicted FgHal2 protein and orthologues. The positions of the amino acid sequences are indicated on the right. Species and GenBank accession numbers are as follows: Arabidopsis thaliana (NP201205.1); Aspergillus clavatus (XP001268064.1); Magnaporthe oryzae (XP003719499.1); Fusarium graminearum (XP389708.1); Fusarium oxysporum (EGU82355.1); Fusarium verticillioides (EWG42162); Neurospora crassa (EAA28448.2); Saccharomyces cerevisiae (NP014577.1) The conserved amino acids are shaded. The alignment was generated with GeneDoc software. The PAP phosphatase‐like domain [cd01517] is boxed; the conserved active site residues are indicated by a bar above the sequence; the substrate binding site residues are indicated by arrowheads; and the putative Li⁺/Na⁺ binding sites are indicated by black arrows.

Figure 2

Generation of FgHal2 gene deletion, complementation and over‐expression mutants of Fusarium graminearum. (A) Schematic representation of the homologous gene recombination strategy used to generate the FgHal2 deletion mutants (left), FgHal2 complementation mutant (middle) and FgHal2 over‐expression mutant (right). The FgHal2 complementation mutant strain was fused with green fluorescent protein (GFP). The promoter was replaced with the elongation factor 1α (EF1α) promoter in the FGHAL2  OE strain. WT, F. graminearum wild‐type strain PH‐1; ΔFgHal2, FgHal2 gene deletion mutant; FgHal2C, FgHal2 complementation and GFP‐fused mutant; FGHAL2  OE, FgHal2 over‐expression mutant. (B) Southern blot hybridization of the F. graminearum mutant strains. The sizes of the DNA standards are indicated to the left of the blot. Lanes 1–6 indicate Southern blot images of BamHI‐digested genomic DNA of each strain. Lanes 1 and 2 represent the WT, lanes 3 and 4 represent ΔFgHal2, and lanes 5 and 6 represent the complementary strain (FgHal2C). A ³²P‐labelled DNA fragment of the 5′ flanking region of the FgHal2 gene was used as a probe for Southern blot hybridization (lanes 1–6). Lanes 7–10 show Southern blot images of StuI‐digested genomic DNA of each strain. Lanes 7 and 8 represent the WT, and lanes 9 and 10 represent FGHAL2  OE. A ³² P‐labelled DNA fragment of the 3′ flanking region of the FgHal2 gene was used as a probe (lanes 7–10). In lanes 1–10, odd‐numbered lanes indicate virus‐free strains and even‐numbered lanes indicate virus‐infected strains. (C) Relative FgHal2  mRNA transcript levels in the WT and mutant strains. Relative transcript levels were normalized to elongation factor 1α and ubiquitin C‐terminal hydrolase. cDNAs were generated from total RNA extracts from mycelia harvested after 120 h of incubation. Error bars indicate standard deviations. Values with different letters are significantly different (P < 0.05) based on Tukey's test. VF, virus‐free; VI, Fusarium graminearum virus 1 (FgV1)‐infected strains.

Figure 3

Colony morphology and mycelial growth of virus‐free and Fusarium graminearum virus 1 (FgV1)‐infected strains [wild‐type (WT) and FgHal2 gene deletion, complementation and over‐expression mutants] of F. graminearum. (A) Colony morphology on complete medium (CM) agar. Colonies were photographed after 5 days of incubation. (B) Radial growth after 5 days of incubation on CM agar. Means and standard deviations were calculated from three repeated experiments. Bars with different letters are significantly different (P < 0.05) based on Tukey's test, which was conducted using SPSS 12.0 (SPSS, Inc., Chicago, IL, USA).

Figure 4

Conidial production by virus‐free and Fusarium graminearum virus 1 (FgV1)‐infected strains [wild‐type (WT) and FgHal2 gene deletion, complementation and over‐expression mutants] of F. graminearum. (A) Conidia production after 5 days on carboxymethyl cellulose (CMC). Values are means and standard deviations of three repeated experiments. Means with different letters are significantly different (P < 0.05) based on Tukey's test. (B) Conidial morphology. Representative conidia of virus‐free (VF) and virus‐infected (VI) strains were photographed with differential interference contrast optics. Bar, 20 μm.

Figure 5

Virulence of virus‐free and Fusarium graminearum virus 1 (FgV1)‐infected F. graminearum strains [wild‐type (WT) and FgHal2 gene deletion, complementation and over‐expression mutants] on wheat. (A) Representative wheat heads 14 days after they were inoculated with conidia. (B) The percentage of symptomatic wheat spikelets 14 days after inoculation. Means and standard deviations were calculated from independent repeated experiments. Error bars indicate standard deviation. Bars with different letters are significantly different (P < 0.05).

Figure 6

Accumulation of Fusarium graminearum virus 1 (FgV1) viral RNA in FgV1‐infected wild‐type (WT), ΔFgHal2, FgHal2C and FGHAL2  OE strains of F. graminearum. Quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) was used to quantify FgV1 viral RNA at 48, 72 and 120 h post‐inoculation (hpi). Error bars indicate standard deviations. Values with different letters are significantly different (P < 0.05) based on Tukey's test.