| Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae
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Author:
Date:
2017-02-20
[Abstract] Verticillium dahliae is a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues. The great loss of economically important crop caused by V. dahliae has raised worldwide concern, however, little is known about the mechanism of its pathogenicity (Klosterman et al., 2011; Yadeta and Thomma, 2013). Our recent work has shown that V. dahliae develops hyphopodium as an infection structure to breach plant root cell wall (Zhao et al., 2016). Here, we provide a detailed protocol to analyze the penetration ability and ...
[摘要] 大丽轮枝菌属是土壤传播的植物病原真菌，其感染宿主根并在血管组织中增殖。经济上重要的作物造成的巨大损失。大丽花已经引起了全世界的关注，然而，其致病性机制知之甚少（Klosterman等人，2011; Yadeta和Thomma，2013）。我们最近的工作表明，大丽花开发hyphopodium作为感染结构以破坏植物根细胞壁（Zhao等人，2016）。在这里，我们提供了一个详细的方案来分析渗透能力和致病性。大丽花，以及从以前的研究（Zhang等人，2016a和2016b; Zhao等人，2016年）开发的感染棉花茎中恢复真菌菌丝）。玻璃纸膜已被用于诱导叶面病原体的发育，而不是根系病原体（Bourett和Howard，1990）。我们采用玻璃纸膜的方法诱导和评估hyphopodium的发展。希望能大力促进调控传染性发育的宿主的分子生物学研究。该协议还有助于识别控制V的致病性的关键成分。大丽花，并扩大了我们对植物与微生物相互作用机理的理解。
背景 玻璃纸膜被广泛用于研究叶病原体感染结构的发展（Bourett和Howard，1990; Kleemann等人，2012; Gu等人， ...
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| In vitro Cell Wall Stress Assay for Fusarium oxysporum
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Author:
Date:
2016-09-05
[Abstract] In this protocol we describe a cell wall stress assay for the fungal pathogen F. oxysporum, based on exposure to the two anionic dyes Calcofluor White (CFW) and Congo Red (CR). Both compounds have been used to exert stress upon the fungal cell wall in vitro (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Leach et al., 2012; Heilmann et al., 2013; Garcia et al., 2015). CFW perturbs chitin assembly, whereas CR interferes with β-glucan synthesis, resulting in cell wall-weakening and activation of the cell wall stress response ...
[摘要] 在该协议中，我们描述了真菌病原体F的细胞壁应激测定。 ，基于暴露于两种阴离子染料Calcofluor White（CFW）和刚果红（CR）。两种化合物都已经用于在体外对真菌细胞壁施加应激（Perez-Nadales和Di Pietro，2015; Perez-Nadales和Di Pietro，2011; Leach等人， 2012; Heilmann等人，2013; Garcia等人，2015）。 CFW干扰几丁质装配，而CR干扰β-葡聚糖合成，导致细胞壁弱化和细胞壁应激反应的激活（Ram和Klis，2006; Kopecka和Gabriel，1992; Roncero和Duran，1985）。据推测，与该反应相关的信号通路和细胞壁变化反映了正常生长期间的细胞壁稳态以及响应于真菌 - 宿主相互作用期间遇到的应力的细胞壁重塑事件。本方案中规定的CFW和CR培养基的制备条件是基于Ram和Klis的论文"Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red"，published in Nature protocols（Ram and ...
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| Quantification of Chitinase Activity in Fusarium oxysporum
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Author:
Date:
2016-08-05
[Abstract] Fungal morphogenetic development requires modification and plasticity of the cell wall, which implies synthesis and remodelling of its components, including chitin and glucan. Thus chitinase and glucanase activities are crucial for cell-wall biogenesis and cell division. Quantification of chitinase activity might be useful to identify structural defects that could negatively influence growth and morphogenesis of some filamentous fungi like Fusarium oxysporum, which produces both intracellular and secreted chitinases. The chitinolytic enzymes are categorized based on their enzymatic ...
[摘要] 真菌形态发生需要细胞壁的修饰和可塑性，这意味着其组分（包括壳多糖和葡聚糖）的合成和重塑。因此几丁质酶和葡聚糖酶活性对于细胞壁生物发生和细胞分裂是至关重要的。几丁质酶活性的定量可能有助于鉴定可能负面影响一些丝状真菌如产生细胞内和分泌型几丁质酶的尖孢镰刀菌的生长和形态发生的结构缺陷。基于它们对壳多糖底物的酶作用将壳聚糖分解酶分类。内酯酶被定义为催化几丁质链中内部点的随机裂解的酶。外切球蛋白酶催化乙酰壳寡糖或N-乙酰葡糖胺从几丁质的非还原端逐步释放，因此分别称为壳聚糖酶和β-N-乙酰氨基葡糖苷酶。在这里，我们描述了一种简单的方法来轻易地纯化几丁质酶，以便比较不同的F的内切几丁质酶活性和外切几丁质酶活性。尖孢镰孢菌株。该方案可适用于任何真菌物种。
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