Function Characterization Of AREA Gene In Fusarium Graminearum

In the yeast and filament fungal, nitrogen metabolite is mediated by transcription factors belonging to the GATA family. GATA transcription factors are a family of transcription factors that have one or two Cys2/Cys2-type zinc fingers in their DNA-binding domain and recognize the consensus DNA sequence 5’-HGATAR-3’(where H stands for A, T, or C and R stands for A or G) in the promoter of target genes. Most of the structural genes involved in nitrogen metabolism can be recognized by GATA transcription factors to ensure their transcriptional activation for scavenging and degradation of energetically less favored nitrogen sources.In this study, we functionally characterized the GATA transfactor gene Are A in Fusarium graminearum the causal agent of wheat and barley head blight. Despite of its normal conidia and colony morphology, the Δare A mutant was reduced in conidiation and growth rate, indicating that AREA is not important for morphological maintenance but for development. Deletion of AREA also resulted in the loss of perithecium and ascospores production, which suggests that AREA plays important roles in sexual reproduction regulation. The Δare A mutant showed increased sensitivities to oxidative stress. In infection assays with flowering wheat heads, corn stalks, and corn silks, the Δare A mutant was significantly reduced in plant infection and DON production.The Are A-GFP fusion construct was generated by gap-repair and transformed into the Δare A mutant. The resulting transformant was normal in growth, conidiation, sexual reproduction, DON production, and virulence. GFP signals were observed in the nucleus in conidia and hyphae of the Δare A/AREA transformant. We deleted three of the putative nuclear localization signal(NLS) sequences, respectively, and found NLS3 is important for Are A location and function.Unlike the wild type, the Δare A mutant is unable to assimilate the secondary nitrogen source such as ammonium and glutamine, and hard to induce the transcription of nitrate and nitrite reductase. The q RT-PCR result also showed that ammonium repressed AREA expression and in contrast, nitrate induced AREA expression, indicating that AREA positively regulates nitrogen metabolism.DON production of the wild type strain was significant decreased by adding ammonium into the rice media, which was not observed in the Δare A mutant. On contrast with the wild type, ammonium failed to repress the expression level of TRI5 and TRI6(DON biosynthesis gene) in the Δare A mutant when added in DON inducing media., suggesting that the suppression of DON production and TRI gene expression by ammonium may be regulated by AREA. Moreover, Are A interacted with Tri10 in co-immunoprecipitation assays, further proving the involvement of AREA in DON biosynthesis.In PKA activities assay and TEY phosphorylation assay, the Δare A mutant reduced in PKA activities and Pmk1 phosphorylation. When S874, the putative PKA phosphorylation site in Are A, was mutated, the mutant lost virulence and Are A activities. In the meantime, transcription level of the ammonium permease(MEP) was significantly decreased in Δare A mutant. All these showed that AREA participates c AMP-PKA and MAPK signal pathway probably by regulating MEP expression and further regulates DON biosynthesis.Overall our data indicate that GATA transfactor Are A plays a critical role in the regulation of development, sexual and asexual reproduction, pathogenesis, nitrogen metabolism, and DON biosynthesis in F. graminearum.