稻瘟病菌内吞作用的检测

Muxing Liu; Zhengguang  Zhang

doi:10.21769/BioProtoc.3322

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Peer-reviewed

Endocytosis Detection in Magnaporthe oryzae

稻瘟病菌内吞作用的检测

ML Muxing Liu

Zhengguang Zhang email

发布: 2019年08月05日第9卷第15期 DOI: 10.21769/BioProtoc.3322 浏览次数: 5017

评审: LAXMI NARAYAN MISHRAAshish RanjanAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Molecular Plant Microbe Interactions, featuring study using the protocol.

Apr 2019

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

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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 (内吞)

Intracellular trafficking (胞内运输)

Magnaporthe oryzae (稻瘟病菌)

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

Microscope slide (Sail brand, catalog number: 7105)
Microscope cover glass (Fisher brand, catalog number: 12-540-A)
Hyphae of Magnaporthe oryzae
M. oryzae Guy11, used as the parental wild type strain. (MoARK1 gene knock-out mutant ∆Moark1 was obtained in our previous work. All the strains were cultured on complete medium [CM] agar plates. Liquid CM medium was used to prepare the mycelia for endocytosis assay. The strain Guy11 was stored in our laboratory.)
N-(3-triethylammoniumpropyl)-4-(pdiethyl-aminophenyl-hexatrienyl) pyridinium dibromide FM4-64 (Invitrogen, catalog number: T13320)
Latrunculin B (Cayman, catalog number: 10010631)
Double-distilled H₂O (ddH₂O)
DMSO (Sigma, catalog number: D2650)
D-glucose (Sigma, catalog number: 47829)
Peptone (Sangon biotech, catalog number: A505247)
Yeast extract (Oxoid, catalog number: LP0021)
Casamino acid (Sangon biotech, catalog number: A603060)
Biotin (Sigma, catalog number: B4501)
Pyridoxin (Sigma, catalog number: P9755)
Thiamine (Sangon biotech, catalog number: A600939)
Riboflavin (Sangon biotech, catalog number: A600470)
p-aminobenzoic acid (Amresco, catalog number: 0779)
Nicotinic Acid (Sigma, catalog number: 72309)
Liquid complete medium (Liquid CM medium) (see Recipes)
Vitamin solution (see Recipes)