SummaryIn eukaryotes, the retromer is an endosome-localized complex involved in protein retrograde transport. However, the role of such intracellular trafficking events in pathogenic fungal development and pathogenicity remains unclear.The role of the retromer complex in Fusarium graminearum was investigated using cell biological and genetic methods.We observed the retromer core component FgVps35 (Vacuolar Protein Sorting 35) in the cytoplasm as fast-moving puncta. FgVps35-GFP co-localized with both early and late endosomes, and associated with the trans-Golgi network (TGN), suggesting that FgVps35 functions at the donor endosome membrane to mediate TGN trafficking. Disruption of microtubules with nocodazole significantly restricted the transportation of FgVps35-GFP and resulted in severe germination and growth defects. Mutation of FgVPS35 not only mimicked growth defects induced by pharmacological treatment, but also affected conidiation, ascospore formation and pathogenicity. Using yeast two-hybrid assays, we determined the interactions among FgVps35, FgVps26, FgVps29, FgVps17 and FgVps5 which are analogous to the yeast retromer complex components. Deletion of any one of these genes resulted in similar phenotypic defects to those of the DFgvps35 mutant and disrupted the stability of the complex.Overall, our results provide the first clear evidence of linkage between the retrograde transport mediated by the retromer complex and virulence in F. graminearum.
Fusarium graminearum is a fungal pathogen that causes Fusarium head blight (FHB) in wheat and barley. Autophagy is a highly conserved vacuolar degradation pathway essential for cellular homeostasis in which Atg9 serves as a multispanning membrane protein important for generating membranes for the formation of phagophore assembly site. However, the mechanism of autophagy or autophagosome formation in phytopathogens awaits further clarifications. In this study, we identified and characterized the Atg9 homolog (FgAtg9) in F. graminearum by live cell imaging, biochemical and genetic analyses. We find that GFP-FgAtg9 localizes to late endosomes and trans-Golgi network under both nutrient-rich and nitrogen starvation conditions and also show its dynamic actin-dependent trafficking in the cell. Further targeted gene deletion of FgATG9 demonstrates that it is important for growth, aerial hyphae development, and pathogenicity in F. graminearum. Furthermore, the deletion mutant (ΔFgatg9) shows severe defects in autophagy and lipid metabolism in response to carbon starvation. Interestingly, small GTPase FgRab7 is found to be required for the dynamic trafficking of FgAtg9, and co-immunoprecipitation (Co-IP) assays show that FgAtg9 associates with FgRab7 in vivo. Finally, heterologous complementation assay shows that Atg9 is functionally conserved in F. graminearum and Magnaporthe oryzae. Taken together, we conclude that FgAtg9 is essential for autophagy-dependent development and pathogenicity of F. graminearum, which may be regulated by the small GTPase FgRab7.
Plants generate multitude of aldehydes under abiotic and biotic stress conditions. Ample demonstrations have shown that rice-derived aldehydes enhance the resistance of rice against the rice-blast fungus Magnaporthe oryzae. However, how the fungal pathogen nullifies the inhibitory effects of host aldehydes to establish compatible interaction remains unknown. Here we identified and evaluated the in vivo transcriptional activities of M. oryzae aldehyde dehydrogenase (ALDH) genes. Transcriptional analysis of M. oryzae ALDH genes revealed that the acetylating enzyme Methylmalonate-Semialdehyde Dehydrogenase (MoMsdh/MoMmsdh) elevated activities during host invasion and colonization of the fungus. We further examined the pathophysiological importance of MoMSDH by deploying integrated functional genetics, and biochemical approaches. MoMSDH deletion mutant ΔMomsdh exhibited germination defect, hyper-branching of germ tube and failed to form appressoria on hydrophobic and hydrophilic surface. The MoMSDH disruption caused accumulation of small branch-chain amino acids, pyridoxine and AMP/cAMP in the ΔMomsdh mutant and altered Spitzenkörper organization in the conidia. We concluded that MoMSDH contribute significantly to the pathogenesis of M. oryzae by regulating the mobilization of Spitzenkörper during germ tube morphogenesis, appressoria formation by acting as metabolic switch regulating small branch-chain amino acids, inositol, pyridoxine and AMP/cAMP homeostasis.
Cr(VI) is a type of dangerous effluent that has caused great harm to human health and the environment. Recognition and perception of Cr(VI) by artificial receptors has attracted extensive attention. A novel fluorescent chemical sensor based on the 5,7‐dihydroxyflavone skeleton was designed and synthesized for the selective recognition of Cr(VI). As confirmed by fluorescence technology, the fluorescent probe 4‐dimethylaminobenzyl chrysin ester–Zn (DBC–Zn) showed high sensitivity and selectivity for dichromate and a fast response (less than 30 sec) recognition. The fluorescence intensity of DBC–Zn varies linearly with the concentration of Cr(VI) in the range 0.1–1 μM. The detection limit of Cr2O72− by DBC–Zn is 2.3 nM, which is far lower than the national safe drinking water standard stipulated by the US Environmental Protection Agency (1.9 μM). The quenching mechanism of the probe can be attributed to the interaction of the dynamic quenching effect and the fluorescence internal filtration effect. In addition, the probe has good stability in both neutral and alkaline environments, and the accuracy of quantitative analysis of Cr2O72− in lake water or tap water is more than 80%. The test paper based on DBC–Zn can effectively detect Cr2O72− at the concentration of 100 ppb. This shows that the probe has a certain practical application value.
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