Fluorescent markers of the endocytic pathway in Zymoseptoria tritici.
Fluorescent markers of the endocytic pathway in Zymoseptoria tritici.
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DOI：
10.1016/j.fgb.2015.03.019
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发表时间：
2015-06
期刊：
影响因子：
--
通讯作者：
Steinberg G
中科院分区：
文献类型：
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作者：
Kilaru S;Schuster M;Latz M;Guo M;Steinberg G
我们建立了小麦纤维蛋白（ztfiim1）和小gtp酶（ZtRab5、ZtRab7）作为内吞标记。通过活细胞成像、共染色和药物研究，所有标记物都能正确定位。我们提供了3个抗碳毒素传递载体，用于将所有标记整合到sdi1位点。我们提供了3个耐潮霉素b的载体，用于随机整合所有标记物。丝状真菌的菌丝生长是由生长尖端的膜和相关蛋白质的摄取（内吞作用）和再循环支持的。在各种真菌中发表的越来越多的证据表明，这一过程对真菌生长和致病性至关重要。在这里，我们引入荧光标记来可视化小麦病原菌酵母的内吞途径。我们将增强的绿色荧光蛋白（eGFP）与肌动蛋白结合蛋白纤蛋白（ZtFim1）融合，该蛋白位于肌动蛋白斑块中，该斑块在质膜上形成，并参与细胞表面的内吞摄取。此外，我们通过egfp标记rab5同源物（ZtRab5）标记早期核内体，通过表达eGFP-Rab7同源物（ZtRab7）标记晚期核内体和液泡。利用荧光染料和活细胞成像，我们证实了这些标记物的动态行为和定位。这一套分子工具能够对小麦小麦小偃麦草突变株进行深入的表型分析，从而为控制小麦小麦小偃麦草的目标提供新的策略支持。
We establish Z. tritici fimbrin (ZtFim1) and small GTPases (ZtRab5, ZtRab7) as endocytic markers. All markers localize correctly, proven by live cell imaging and co-staining and pharmaceutical studies. We provide 3 carboxin-resistance conveying vectors for integration of all markers into the sdi1 locus. We provide 3 hygromycin B-resistance conveying vectors for random integration of all markers. Hyphal growth in filamentous fungi is supported by the uptake (endocytosis) and recycling of membranes and associated proteins at the growing tip. An increasing body of published evidence in various fungi demonstrates that this process is of essential importance for fungal growth and pathogenicity. Here, we introduce fluorescent markers to visualize the endocytic pathway in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein (eGFP) to the actin-binding protein fimbrin (ZtFim1), which is located in actin patches that are formed at the plasma membrane and are participating in endocytic uptake at the cell surface. In addition, we tagged early endosomes by eGFP-labelling a Rab5-homologue (ZtRab5) and late endosomes and vacuoles by expressing eGFP-Rab7 homologue (ZtRab7). Using fluorescent dyes and live cell imaging we confirmed the dynamic behavior and localization of these markers. This set of molecular tools enables an in-depth phenotypic analysis of Z. tritici mutant strains thereby supporting new strategies towards the goal of controlling wheat against Z. tritici.