FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici
FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici
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DOI：
10.1016/j.fgb.2015.03.015
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发表时间：
2015-06-01
影响因子：
3
通讯作者：
Kema, Gerrit H. J.
中科院分区：
文献类型：
--
作者：
Ben M'Barek, Sarrah;Cordewener, Jan H. G.;Kema, Gerrit H. J.
研究了小麦真菌致病菌小麦酵母菌（Zymoseptoria tritici）的培养滤液在不同小麦品种叶片中渗透后的致坏死活性。对活性组分进行了部分纯化和表征。CFs中的坏死诱导因子是蛋白质，热稳定的，其诱导坏死活性依赖于温度和光。通过蛋白酶K （PK）共浸润时间序列检测CF的植物内活性，在CF浸润30 min后不影响其活性。这表明坏死诱导蛋白（NIPs）要么在外质体中缺失，可能被主动转运到叶肉细胞中，要么通过与受体结合而免受蛋白酶的影响。另外，植物细胞死亡信号通路可能在前30分钟内完全参与，即使在PK处理后也不能逆转。采用快速液相色谱法、SDS-PAGE和质谱法对诱导坏死活性最高的cf进行进一步分离。这表明，大多数存在于馏分中的蛋白质以前没有被描述过。两个最突出的ZtNIP编码候选基因在毕赤酵母中异源表达，随后的浸润试验显示它们在一系列小麦品种中的差异活性。(C) 2015爱思唯尔公司版权所有。
Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30 min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30 min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars. (C) 2015 Elsevier Inc. All rights reserved.