Functional analysis of HEX1 gene in interaction between Fusarium graminearum and Fusarium graminearum virus 1

Abstract

Fusarium graminearum virus 1 strain DK21 (FgV1) is fungal virus (mycovirus) isolated from the devastating plant pathogenic fungi, Fusarium graminearum. The FgV1 genome consists of 6,624 nucleotides, excluding the 3ʹ-terminal poly (A) tail. The viral genome has 53- and 46-nucleotide 5ʹ-and 3ʹ-untranslated regions (UTRs), respectively, and four putative open reading frames. The FgV1 infection perturbs in various aspects of host fungus biology such as morphology, growth, development, metabolism, and even virulence to plant. To identify a certain host factor interacting with FgV1 which involves in such alteration of host, proteome and transcriptome based approaches have been tried previously. Among them, the hexagonal peroxisome (Hex1) protein is fungal protein that is highly expressed when FgV1 infects its host. The Hex1 protein constitutes Woronin body (WB), which is peroxisome derived dense-core micro organelle for sealing the septal pore in response to hyphal damage. In the present study, cellular functions of Hex1 in F. graminearum and particular function for FgV1 were characterized. First, in order to investigate roles of Hex1, HEX1 gene deletion, over-expression and complementation mutants were generated and then infected by FgV1. Deletion and over-expression of Hex1 did not affect to vegetative growth in virus-free (VF) strains, while both changes resulted in reduced production of conidia and reduced virulence. Additionally, fungal cells could not maintain cellular integrity after hyphal wounding in the absence of HEX1 gene and even they showed cytoplasmic bleeding. Taken together, combined results suggested that HEX1 gene is required for asexual reproduction and pathogenesis and maintaining cellular integrity. Although deletion and over-expression did not affect vegetative growth at all in VF strains, both genetic alterations substantially affected vegetative growth in virus-infected (VI) strains. Vegetative growth was increased in deletion strains and decreased in over-expression strains comparing with WT-VI strain, while viral RNA accumulation was decreased in deletion strains and increased in over-expression strain. To clarify the cause of these different RNA accumulations in different HEX1 strains, FgV1 and Hex1 interaction studies were carried out. Homology based protein tertiary structure prediction analysis demonstrated that the structure of Hex1 is similar to that of eukaryotic translational initiation factor 5A (IF-5A) which have RNA-binding folds on its surface. Therefore, I carried out electrophoretic mobility shift assays (EMSAs) and the analysis revealed Hex1 protein bind to both 5ʹ-and 3ʹ-untranslated regions (UTRs) of plus-strand viral RNA. Strand specific Northern blot and qRT-PCR analyses were conducted to determine the effect of Hex1 on FgV1 (+)- and (-)-strand RNA accumulation. Both analyses exhibited that Hex1 protein affected both (+)- and (-)-strand RNA accumulations.