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Apr 2019

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Endocytosis Detection in Magnaporthe oryzae
稻瘟病菌内吞作用的检测   

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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), Fungi (真菌), FM4-64 (FM4-64), Hyphal growth (菌丝生长), Pathogenicity (致病性), 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

  1. Microscope slide (Sail brand, catalog number: 7105)
  2. Microscope cover glass (Fisher brand, catalog number: 12-540-A)
  3. Hyphae of Magnaporthe oryzae 
  4. 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.)
  5. N-(3-triethylammoniumpropyl)-4-(pdiethyl-aminophenyl-hexatrienyl) pyridinium dibromide FM4-64 (Invitrogen, catalog number: T13320)
  6. Latrunculin B (Cayman, catalog number: 10010631)
  7. Double-distilled H2O (ddH2O)
  8. DMSO (Sigma, catalog number: D2650)
  9. D-glucose (Sigma, catalog number: 47829)
  10. Peptone (Sangon biotech, catalog number: A505247)
  11. Yeast extract (Oxoid, catalog number: LP0021)
  12. Casamino acid (Sangon biotech, catalog number: A603060)
  13. Biotin (Sigma, catalog number: B4501)
  14. Pyridoxin (Sigma, catalog number: P9755)
  15. Thiamine (Sangon biotech, catalog number: A600939)
  16. Riboflavin (Sangon biotech, catalog number: A600470)
  17. p-aminobenzoic acid (Amresco, catalog number: 0779)
  18. Nicotinic Acid (Sigma, catalog number: 72309)
  19. Liquid complete medium (Liquid CM medium) (see Recipes)
  20. Vitamin solution (see Recipes)

Equipment

  1. Eppendorf micropipette (1,000 μl, 100 μl, 10 μl)
  2. Confocal fluorescence microscope (Zeiss LSM710, 63x oil)

Procedure

  1. Culture the wild type strain Guy11 and ∆Moark1 on solid CM medium for 4 days at 28 °C. Cut the agar culture into 1 mm x 1 mm squares and culture the squares in liquid CM for 2 more days.
  2. Prepare a stock solution of FM4-64 (1.3 mg/ml in DMSO) and dilute in distilled H2O at a final concentration of 5 μg/ml, keep this staining solution on ice.
  3. Wash the hyphae with distilled water and stain with FM4-64 on glass slide, about 20 μl working solution dropped on the hyphae and stain for 2 min at room temperature. 
  4. Before observation, wash the hyphae with ddH2O.
  5. Take photographs under a confocal fluorescence microscope (63x oil, 570/620 nm excitation, 590 nm dichroic, 630/660 nm emission is used to observe the fluorescence of FM4-64) after exposure to FM4-64. A representative picture of the plasma membrane and the inner cellular membrane components after staining 2 min with FM4-64 is shown in Figure 1.


    Figure. 1 Endocytosis assay in M. oryzae. A. The wild type strain Guy11 and the ∆Moark1 were grown in liquid CM for 48 h before the addition of FM4-64 and photographs were taken under a confocal fluorescence microscope (Zeiss LSM710, 63x oil) after 2 min of exposure to FM4-64. Bars = 10 μm. B. The calculation of relative fluorescent density. Error bars represent SD and double asterisks indicate statistically significant differences (**, P < 0.01).

  6. Prepare a stock solution of Latrunculin B (Lat B) in DMSO at a concentration of 25 mg/ml and dilute in distilled H2O at a final concentration of 0.1 μg/ml. Lat B is used as an actin polymerization inhibitor to disrupt endocytosis (Kaksonen et al., 2003).
  7. For Lat B staining, pretreat the hyphae with Lat B when cultured in Step 1 for 30 min prior to the addition of FM4-64.
  8. Repeated Steps 2-5.

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

  1. Liquid CM medium
    10 g D-glucose
    2 g peptone
    1 g yeast extract
    1 g casamino acid
    1 ml Vitamin Solution
    Hygrothermal high pressure sterilization at 121 °C for 20 min.
  2. Vitamin Solution
    0.01 g Biotin
    0.01 g Pyridoxin
    0.01 g Thiamine
    0.01 g Riboflavin
    0.01 g p-aminobenzoic acid
    0.01 g Nicotinic Acid
    Add ddH2O to 100 ml and store in a dark glass bottle at 4 °C

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

  1. Fischer-Parton, S., Parton, R.M., Hickey, P.C., Dijksterhuis, J., Atkinson, H.A. and Read, N.D. (2000). Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae. J Microsc 198(3): 246-259.
  2. Hayden, J., Williams, M., Granich, A., Ahn, H., Tenay, B., Lukehart, J., Highfill, C., Dobard, S. and Kim, K. (2013). Vps1 in the late endosome-to-vacuole traffic. J Biosci 38(1): 73-83.
  3. Higuchi, Y., Arioka, M. and Kitamoto, K. (2011). Functional analysis of the putative AAA ATPase AipA localizing at the endocytic sites in the filamentous fungus Aspergillus oryzae. FEMS Microbiol Lett 320(1): 63-71.
  4. Higuchi, Y., Shoji, J.Y., Arioka, M. and Kitamoto, K. (2009). Endocytosis is crucial for cell polarity and apical membrane recycling in the filamentous fungus Aspergillus oryzae. Eukaryot Cell 8(1): 37-46.
  5. Journo, D., Winter, G. and Abeliovich, H. (2008). Monitoring Autophagy in Yeast Using Fm 4-64 Fluorescence. Methods Enzymol 451: 79-88.
  6. Kaksonen, M., Sun, Y. and Drubin, D. G. (2003). A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell 115(4): 475-487.
  7. Kamble, C., Jain, S., Murphy, E. and Kim, K. (2011). Requirements of Slm proteins for proper eisosome organization, endocytic trafficking and recycling in the yeast Saccharomyces cerevisiae. J Biosci 36(1): 79-96.
  8. Li, L., Chen, X., Zhang, S., Yang, J., Chen, D., Liu, M., Zhang, H., Zheng, X., Wang, P., Peng, Y. and Zhang, Z. (2017). MoCAP proteins regulated by Moark1-mediated phosphorylation coordinate endocytosis and actin dynamics to govern development and virulence of Magnaporthe oryzae. PLoS Genet 13(5): e1006814.
  9. Peñalva, M.A. (2005). Tracing the endocytic pathway of Aspergillus nidulans with FM4-64. Fungal Genet Biol 42: 963-975.
  10. Wedlich-Soldner, R., Bolker, M., Kahmann, R. and Steinberg, G. (2000). A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis. EMBO J 19(9): 1974-1986.

简介

内吞作用是细胞内运输途径,其发生在营养摄取,信号转导和细胞极性的重建中,并且在真核细胞中是保守的。 在真菌中,胞吞作用在菌丝生长和致病性的生理学中起着至关重要的作用。 通过膜选择性染料FM4-64检测丝状真菌内吞作用的证据。 一系列丝状真菌物种的细胞容易吸收这些染料。 然而,在 Magnaporthe oryzae 中尚未完全确定内吞作用检测的方法。 在这里,我们提供了跟踪 Magnaporthe oryzae 内吞作用的方案。
【背景】内吞作用是一种重要的细胞过程,其内化细胞外物质并从质膜中回收膜和蛋白质。内吞作用从质膜开始,将蛋白质,脂质和分子从外部环境转运到降解细胞器,或通过囊泡和内体再循环回到质膜(Kaksonen et al。,2003)。一些研究表明,荧光染料FM4-64(内吞作用的特异性示踪剂)存在内吞作用,这表明这种细胞器可能参与丝状真菌的内吞膜再循环(Fischer-Parton et al。, 2000; Wedlich-Soldner et al。,2000; Penalva,2005; Li et al。,2017)。 FM4-64还用于检测从内体到膜泡的膜通路和酵母中的自噬(Hayden 等人,<2013; Journo 等人,2008)。已经在米曲霉和酵母中建立了许多检测内吞作用的方案(Higuchi et al。,2011; Higuchi et al。,2009; Kamble et al。,2011)。然而,没有为 Magnaporthe oryzae 建立特定的方案,它是稻瘟病的致病因子,稻瘟病是世界上最严重的真菌病。在这里,我们描述了一种可靠而简单的方法来检测 M的内吞途径。米曲霉。

关键字:内吞, 胞内运输, 真菌, FM4-64, 菌丝生长, 致病性, 稻瘟病菌

材料和试剂

  1. 显微镜载玻片(Sail品牌,目录号:7105)
  2. 显微镜盖玻片(Fisher品牌,目录号:12-540-A)
  3. Magnaporthe oryzae的菌丝&nbsp;
  4. 微米。 oryzae Guy11,用作亲本野生型菌株。 ( MoARK1 基因敲除突变体Δ Moark1 在我们以前的工作中获得。所有菌株都在完全培养基[CM]琼脂平板上培养。液体CM培养基用于准备菌丝体用于胞吞作用测定。菌株Guy11储存在我们的实验室中。)
  5. N-(3-三乙基铵丙基)-4-(pdiethyl-aminophenyl-hexatrienyl)吡啶鎓二溴化物FM4-64(Invitrogen,目录号:T13320)
  6. Latrunculin B(Cayman,目录号:10010631)
  7. 双蒸H 2 O(ddH 2 O)
  8. DMSO(Sigma,目录号:D2650)
  9. D-葡萄糖(西格玛,目录号:47829)
  10. 蛋白胨(Sangon biotech,目录号:A505247)
  11. 酵母提取物(Oxoid,目录号:LP0021)
  12. 酪蛋白氨基酸(Sangon biotech,目录号:A603060)
  13. 生物素(Sigma,目录号:B4501)
  14. 吡哆醇(Sigma,目录号:P9755)
  15. 硫胺素(Sangon biotech,目录号:A600939)
  16. 核黄素(Sangon biotech,目录号:A600470)
  17. 对氨基苯甲酸(Amresco,目录号:0779)
  18. 烟酸(西格玛,目录号:72309)
  19. 液体完全培养基(液体CM培养基)(见食谱)
  20. 维生素溶液(见食谱)

设备

  1. Eppendorf微量移液管(1,000μl,100μl,10μl)
  2. 共聚焦荧光显微镜(蔡司LSM710,63x油)

程序

  1. 在28℃下在固体CM培养基上培养野生型菌株Guy11和Δ Moark1 4天。将琼脂培养物切成1mm×1mm的正方形,并将方块在液体CM中再培养2天。
  2. 制备FM4-64的储备溶液(1.3mg / ml,在DMSO中)并在蒸馏的H 2 O中以5μg/ ml的终浓度稀释,将该染色溶液保持在冰上。
  3. 用蒸馏水清洗菌丝,用载玻片上的FM4-64染色,将约20μl工作溶液滴在菌丝上,在室温下染色2分钟。&nbsp;
  4. 在观察之前,用ddH 2 O洗涤菌丝。
  5. 在共聚焦荧光显微镜下(63x油,570 / 620nm激发,590nm二向色,630 / 630nm发射用于观察FM4-64的荧光)在暴露于FM4-64后拍照。用FM4-64染色2分钟后质膜和内部细胞膜组分的代表性图片如图1所示。


    的图。 1 M中的胞吞作用测定。 A.野生型菌株Guy11和Δ Moark1 在液体CM中生长48小时,然后加入FM4-64并在共聚焦下拍摄照片暴露于FM4-64 2分钟后的荧光显微镜(Zeiss LSM710,63x油)。条=10μm。 B.相对荧光密度的计算。误差条表示SD,双星号表示统计学上显着的差异(**, P <0.01)。

  6. 制备Latrunculin B(Lat B)在DMSO中的浓度为25mg / ml的储备溶液,并在蒸馏的H 2 O中稀释,终浓度为0.1μg/ ml。 Lat B用作肌动蛋白聚合抑制剂来破坏内吞作用(Kaksonen et al。,2003)。
  7. 对于Lat B染色,在加入FM4-64之前,在步骤1中培养30分钟,用Lat B预处理菌丝。
  8. 重复步骤2-5。

数据分析

对于胞吞作用缺陷分析,在用FM4-64染料染色之前,在显微镜载玻片上覆盖菌株Guy11和Guy11背景中的基因敲除突变体2天,覆盖完全培养基(CM)并在FM4-后不同时间拍照暴露64,并且信号出现在Guy11中的质膜和内膜隔室上,但是在突变细胞染色后20-30分钟没有发生或延迟(Li et al。,2017)。每个结果至少呈现三次重复测量。在SPSS 2.0中使用SD和单因素方差分析(ANOVA)统计评估治疗之间差异的显着性( https ://spss.en.softonic.com/ ,芝加哥,IL,美国)。使用ANOVA对两种特定不同处理的数据进行统计学比较,然后如果ANOVA结果在 P <1时显着,则进行F检验。 0.05或 P &lt; 0.01。

食谱

  1. 液体CM培养基
    10克D-葡萄糖
    2克蛋白胨
    1克酵母提取物
    1克酪蛋白氨酸
    1毫升维生素溶液
    在121°C下进行湿热高压灭菌20分钟。
  2. 维生素溶液
    0.01克生物素
    0.01克吡哆醇
    0.01克硫胺素
    0.01克核黄素
    0.01克对氨基苯甲酸
    0.01克烟酸
    将ddH 2 O加入100 ml并储存在4°C的深色玻璃瓶中

致谢

该研究得到了中央大学基础研究基金(拨款号KYT201805)和江苏大学创新团队计划(2017)的支持。

利益争夺

没有利益冲突或竞争利益。

参考

  1. Fischer-Parton,S.,Parton,R.M.,Hickey,P.C.,Dijksterhuis,J.,Atkinson,H.A。和阅读,N.D。(2000)。 FM4-64共聚焦显微镜作为分析内吞作用的工具囊泡运输活的真菌菌丝。 J Microsc 198(3):246-259。
  2. Hayden,J.,Williams,M.,Granich,A.,Ahn,H.,Tenay,B.,Lukehart,J.,Highfill,C.,Dobard,S。和Kim,K。(2013)。 Vps1在晚期内体到液泡的交通中。 J Biosci 38(1):73-83。
  3. Higuchi,Y.,Arioka,M。和Kitamoto,K。(2011)。 推定的AAA ATPase AipA的功能分析定位于丝状真菌的内吞位点米曲霉(Aspergillus oryzae)。 FEMS Microbiol Lett 320(1):63-71。
  4. Higuchi,Y.,Shoji,J.Y.,Arioka,M。和Kitamoto,K。(2009)。 内吞作用对细胞极性和丝状真菌中的顶膜回收至关重要 Aspergillus oryzae 。 Eukaryot Cell 8(1):37-46。
  5. Journo,D.,Winter,G。和Abeliovich,H。(2008)。 使用Fm 4-64荧光监测酵母中的自噬。 方法Enzymol 451:79-88。
  6. Kaksonen,M.,Sun,Y。和Drubin,D。G.(2003)。 内吞内化过程中受体,衔接子和肌动蛋白结合的途径。 Cell 115(4):475-487。
  7. Kamble,C.,Jain,S.,Murphy,E。和Kim,K。(2011)。 Slm蛋白对酵母中正确的eisosome组织,内吞运输和再循环的要求 Saccharomyces cerevisiae 。 J Biosci 36(1):79-96。
  8. Li,L.,Chen,X.,Zhang,S.,Yang,J.,Chen,D.,Liu,M.,Zhang,H.,Zheng,X.,Wang,P.,Peng,Y。and张,Z。(2017)。 由 Moark1 介导的磷酸化协调内吞作用和肌动蛋白动力学调节的MoCAP蛋白治疗 Magnaporthe oryzae 的发育和毒力。 PLoS Genet 13(5):e1006814。
  9. Peñalva,M.A。(2005)。 使用FM4-64追踪 Aspergillus nidulans 的内吞途径。 Fungal Genet Biol 42:963-975。
  10. Wedlich-Soldner,R.,Bolker,M.,Kahmann,R。和Steinberg,G。(2000)。 推定的内体t-SNARE将植物病原真菌Ustilago maydis中的外部和内吞作用联系起来。 EMBO J 19(9):1974-1986。
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引用:Liu, M. and Zhang, Z. (2019). Endocytosis Detection in Magnaporthe oryzae. Bio-protocol 9(15): e3322. DOI: 10.21769/BioProtoc.3322.
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