Component Interaction of ESCRT Complexes Is Essential for Endocytosis-Dependent Growth, Reproduction, DON Production and Full Virulence in Fusarium graminearum

Abstract

Multivesicular bodies (MVBs) are critical intermediates in the trafficking of ubiquitinated endocytosed surface proteins to the lysosome/vacuole for destruction. Recognizing and packaging ubiquitin modified cargoes to the MVB pathway require ESCRT (Endosomal sorting complexes required for transport) machinery, which consists of four core subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. Our previous results showed that ESCRT-0 is essential for fungal development and pathogenicity in Fusarium graminearum. We then, in this study, systemically studied the protein-protein interactions within F. graminearum ESCRT-I, -II or -III complex, as well as between ESCRT-0 and ESCRT-I, ESCRT-I and ESCRT-II, and ESCRT-II and ESCRT-III complexes and found that loss of any ESCRT component resulted in abnormal function in endocytosis. In addition, ESCRT deletion mutants displayed severe defects in growth, deoxynivalenol (DON) production, virulence, sexual, and asexual reproduction. Importantly genetic complementation with corresponding ESCRT genes fully rescued all these defective phenotypes, indicating the essential role of ESCRT machinery in fungal development and plant infection in F. graminearum. Taken together, the protein-protein interactome and biological functions of the ESCRT machinery is first profoundly characterized in F. graminearum, providing a foundation for further exploration of ESCRT machinery in filamentous fungi.

Keywords: ESCRT complexes; Fusarium graminearum; endocytosis; interactome; pathogenicity.

Figure 1

Interactions of the ESCRT machinery based on the yeast two-hybrid assay. (A) The interaction of pGBKT7-53/pGADT7-T and pGBKT7-Lam/pGADT7-T were used as the positive and negative control, respectively. Interactions within (B) and between (C) ESCRT subcomplexes. Yeast transformants carrying the indicated constructs were diluted into different concentrations and plated onto the selective plates supplemented with X-gal and without Leu/Trp/His/Ade for assaying the growth and β-galactosidase (LacZ) activities. (D) A model depicting the protein-protein interactions of ESCRT machinery. Lines indicate the interactions that we have showed in this study.

Figure 2

Deletion of ESCRT-I, -II, and -III components perturb the transport of FM4-64 from the plasma membrane to the vacuolar membrane. Cells of indicated strains were stained with 8 mM FM4-64 and the internalization of FM4-64 was observed under Nikon A1R laser scanning confocal fluorescence microscope. Photographs were taken at indicated periods. Bar = 10 μm.

Figure 3

The loss of ESCRT-I, -II, and -III components cause a significant reduction in growth and hydrophobicity of aerial hyphae. (A) Colony of the wild-type strain PH-1, ESCRT gene deletion mutants and corresponding complementary strains grown on complete medium. (B) Microscopy images of mycelial morphology and branching patterns of PH-1 and ESCRT gene deletion mutant strains on CM agar. Bar = 100 μm. (C,D) Colony diameters of indicated strains were measured after incubation at 25°C for 3 days. Line bars in each column represent the standard deviation (SD) from three independent experiments. ^***p < 0.001; Student's T test was used. (E) Hydrophobic character of the wild-type strain PH-1, ESCRT gene deletion mutants and corresponding complementary strains, measured 15 s after deposition of the 2.5% bromophenol blue droplets.

Figure 4

Defects of the ESCRT-I, -II and -III gene deletion mutants in response to various stressors. (A) The wild-type strain PH-1 and ESCRT gene deletion mutants were cultured on complete medium with hyperosmotic and oxidative stressors. (B) The indicated strains were cultured on complete medium supplemented with cell wall and cell membrane damaging agents. (C,D) The growth inhibition rate of the indicated strains under different stress conditions. Line bars in each column represent the standard deviation (SD) from three independent experiments. ^**p < 0.01; ^***p < 0.001; Student's T test was used.

Figure 5

The ESCRT gene deletion mutants were defective in sexual reproduction. Mating cultures of the wild-type strain PH-1, ESCRT gene deletion mutants and corresponding complementary strains. The photographs were taken 2 weeks after sexual induction. Perithecia were only produced by PH-1 and complementary strains.

Figure 6

ESCRT-I, -II and -III components are required for plant infection and DON production. (A) Wheat heads were point inoculated with wild-type strain, ESCRT gene deletion mutants and corresponding complementary strains. Photographs were taken 14 days after inoculation. Black dots indicate the inoculated spikelet in each head. Wheat heads inoculated with water was used as the negative control. The bottom right hand corner of each wheat head shown the disease symptom of wheat head rachis. (B) DON production in 7-day-old TBI cultures of the wide type strain PH-1, ESCRT gene deletion mutants and corresponding complementary strains. (C) The relative expression level of DON synthesis-related gene TRI5, TRI6, and TRI12 in wide type strain PH-1 and ESCRT gene deletion mutants. Their expression in wild-type stain was set to 1. Line bars in each column represent the standard deviation (SD) from three independent experiments. ^***p < 0.001; Student's T test was used.

Figure 7

Subcellular localization of ESCRTs in F. graminearum. Green fluorescent protein (GFP) was fused to the C-terminus of each ESCRT gene, which was driven by native promoter. The resulting constructs were transformed into corresponding ESCRT gene deletion mutants. The conidium of indicated strains were stained with 8 mM FM4-64 followed by imaging with a Nikon A1R laser scanning confocal fluorescence microscope. GFP, FM46-4, and differential interference contrast (DIC), GFP and Fm4-64 overlay images of the same field are shown. Bar = 20 μm.