Abstract
Endocytosis is an intracellular trafficking pathway that occurs in nutrient uptake, signal transduction and reconstruction of cell polarity and is conserved in eukaryotic cells. In fungi, endocytosis plays crucial roles in the physiology of hyphal growth and pathogenicity. vidence for endocytosis in filamentous fungi is detected by the membrane-selective dyes FM4-64. Cells of a range of filamentous fungal species readily take up these dyes. However, the method for endocytosis detection has not been well established in Magnaporthe oryzae. Here, we provide a protocol for tracking endocytosis in Magnaporthe oryzae.
Keywords: Endocytosis, Intracellular trafficking, Fungi, FM4-64, Hyphal growth, Pathogenicity, Magnaporthe oryzae
Background
Endocytosis is an important cellular process that internalizes extracellular materials and retrieves membrane and proteins from the plasma membrane. Endocytosis starts from the plasma membrane and transports proteins, lipids and molecules from the external environment to degradative organelles, or recycled back to the plasma membrane by vesicles and endosomes (Kaksonen et al., 2003). Several studies have demonstrated the existence of endocytosis with the fluorescent dye FM4-64, a specific tracer of endocytosis, suggesting this organelle may be involved in endocytic membrane recycling in filamentous fungi (Fischer-Parton et al., 2000; Wedlich-Soldner et al., 2000; Penalva, 2005; Li et al., 2017). FM4-64 is also used for detecting membrane traffic from endosomes toward the vacuole and autophagy in yeast (Hayden et al., 2013; Journo et al., 2008). Many protocols for detecting endocytosis have been established in Aspergillus oryzae and yeast (Higuchi et al., 2011; Higuchi et al., 2009; Kamble et al., 2011). However, there is no specific protocol established for Magnaporthe oryzae, which is the causal agent of rice blast, the most serious fungal disease in the world. Here, we describe a reliable and simple protocol for detecting the endocytic pathway of M. oryzae.
Materials and Reagents
Equipment
Procedure
Data analysis
For endocytosis defect analysis, culture the strains Guy11 and the gene knock-out mutants in the background of Guy11 for 2 days on microscope slides overlaid with complete medium (CM) before staining with FM4-64 dye and take photographs at various times after FM4-64 exposure, and the signal appeared on the plasma membrane and endomembrane compartments in Guy11, but did not occur or delayed until 20-30 min after staining in the mutant cells (Li et al., 2017). Each result is presented at least three replicated measurements. The significance of differences between treatments is statistically evaluated using SDs and one-way analysis of variance (ANOVA) in SPSS 2.0 (https://spss.en.softonic.com/, Chicago, IL, USA). Data for two specific different treatments are compared statistically using ANOVA, followed by an F-test if the ANOVA result is significant at P < 0.05 or P < 0.01.
Recipes
Acknowledgments
This research was supported by the Fundamental Research Funds for the Central Universities (grant number KYT201805) and Innovation Team Program for Jiangsu Universities (2017).
Competing interests
No conflicts of interest or competing interests.
References
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