Characterization of Fusarium viruses and host factor fghal2 required for host defense against Fusarium graminearum virus 1 infection

Abstract

Many mycoviruses have been identified from diverse plant-pathogenic Fusarium species including F. graminearum. Infections with mycoviruses in F. graminearum mostly remain persistently and asymptomatically in their host, however, some mycoviruses causing altered phenotype, such as reduced growth, pigmentation, sporulation or virulence. Among the Fusarium graminearum viruses (FgVs), FgV1 or FgV2 infections caused several phenotypic alterations in F. graminearum. In contrast, FgV3 and FgV4 infections did not cause any phenotypic change. In this study, we determined complete genome sequences of FgV2, FgV3, and FgV4 and analyzed its phylogenetic relationship with other mycoviruses. The three mycoviruses consist of one to five different segments of dsRNA ranging in size from approximately 1.7 to 9.3 kb. FgV2 consist moinocistronic dsRNA segments, denoted as dsRNA-1 to dsRNA-5. The lengths of FgV2 dsRNAs 1–5 ranged from 2414 to 3580 base pairs (bp). The viral genome of FgV3 is 9098 bp long and contains open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase and a protein of unknown function. The FgV4 genome is composed of two dsRNA genome segments of 2383bp and 1739bp. According to phylogenetic analysis, the FgV2, FgV3, and FgV4 is closed to Chrysoviridae, Totiviridae, and Partitiviridae, respectively, but consist a distinct virus clade within the Family. Transcriptomic and protein expression profiling have shown that many F. graminearum genes are differentially expressed as a consequence of FgVs infection. Of the identified host genes involved in the interaction between mycovirus and fungus host, FgHal2 shows reduced expression in response to FgV1 infection. FgHal2 has a highly conserved 3'-phosphoadenosine 5'-phosphatase (PAP phosphatase-like) domain or inositol monophosphatase (IMPase) superfamily domain. We generated targeted gene deletion and over-expression mutants to clarify the role(s) of FgHal2 in FgV1 infection. The FgHal2 deletion led to decrease in mycelial growth, aerial mycelia formation, and pigmentation whereas over-expression mutants were morphologically similar to the wild type (WT). Furthermore, compared to 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 9-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 the WT at 48 and 72 hpi but was similar to that of the WT at 72 hpi. Furthermore, the low vertical transmission rate of FgV1 and continually growing of virus-free sectors in the colonies of FgV1-infected gene deletion mutant suggesting that FgHal2 might be required for stable maintenance of FgV1 infection. Together, these results indicate that the putative 3'(2'), 5'-bisphosphate nucleotidase gene, FgHal2, has diverse biological functions in the host fungus and might affect the viral RNA accumulation and transmission of FgV1.