| Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin
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Author:
Date:
2016-08-20
[Abstract] Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI) including the generation of reactive oxygen species, callose deposition, and expression of several PTI-related genes. Acidovorax avenae is a Gam-negative bacterium that causes a seedling disease characterized by the deposition of brown stripes on the sheaths of infected plants. We previously reported that flagellin isolated from the rice avirulent A. avenae N1141 strain induces PTI, while flagellin isolated from the rice virulent A. ...
[摘要] 植物通过识别引起PAMP触发的免疫（PTI）的保守的病原体相关分子模式（PAMP）来感测潜在的病原体，包括活性氧的产生，胼lose质沉积和几个PTI相关基因的表达。 酸性弧菌是一种革兰氏阴性细菌，其导致幼苗疾病，其特征在于在感染的植物的鞘上沉积棕色条纹。 我们以前报告了从无毒力的大米中分离的鞭毛蛋白。 avenae N1141菌株诱导PTI，而从水稻毒力分离的鞭毛蛋白。 avenae K1株不诱导PTI。 为了检查N1141鞭毛蛋白的特异性PTI诱导的分子机制，需要来自N1141或K1菌株的高度纯化的鞭毛蛋白。 在这里，我们描述了一种高质量的纯化方法。 avenae鞭毛蛋白，并用于PTI诱导研究。
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| Vacuole Structure Analysis during Cell Death Subsequent to Application of Erwinia carotovora Culture Filtrates to Cell Cultures of Nicotiana tabacum
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Author:
Date:
2015-10-20
[Abstract] We recently established an experimental model system for efficient defense-related cell death using tobacco BY-2 cultured cells treated with culture filtrates of the pathogenic bacterium Erwinia carotovora (E. carotovora) (Hirakawa et al., 2015). Applying this experimental system to transgenic BY-2 cells stably expressing the vacuolar membrane marker GFP-VAM3 (Kutsuna and Hasezawa, 2002) allowed us to monitor changes in vacuolar membrane structures including a decrease of transvacuolar strands during cell death (Hirakawa et al., 2015). Our model system can ...
[摘要] 我们最近建立了使用烟草BY-2培养细胞的有效防御相关细胞死亡的实验模型系统，所述培养细胞用致病性细菌欧文氏菌（Erwinia carotovora）（ E。carotovora （Hirakawa ，，2015）。将该实验系统应用于稳定表达液泡膜标记物GFP-VAM3的转基因BY-2细胞（Kutsuna和Hasezawa，2002），使我们能够监测液泡膜结构的变化，包括细胞死亡期间跨血管链的减少（Hirakawa et al。 al。，2015）。我们的模型系统可以帮助调查细胞器动力学在国防相关的细胞死亡。在这里，我们显示应用 E的协议。 carotovora过滤到BY-2细胞和共聚焦观察液泡膜动力学和随后的细胞死亡。我们使用细胞周期同步的BY-2细胞来有效地监测内陷的液泡膜，例如在我们最近的报告中的跨血管链（Hirakawa等人，2015）;然而，我们不描述本文中的细胞周期同步的协议。对于BY-2细胞同步的逐步方案，请参考先前的方案论文（Nagata和Kumagai，1999; Kumagai-Sano等人，2006）。
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| Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040
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Author:
Date:
2015-10-20
[Abstract] Prominent accumulation of D-leucine, D-allo-isoleucine and D-valine was observed in the culture medium of the heterofermentative bacterial species, Lactobacillus otakiensis (L. otakiensis) JCM 15040. The racemase enzyme that resulted in this accumulation, isoleucine 2-epimerase, was purified from the bacterial cells. This is the first reported observation of such production of D-branched chain amino acids in lactic acid bacteria, and the first example of a racemase with isoleucine 2-epimerase activity in any organisms. In the described protocol, we introduce methods ...
[摘要] 在异质发酵细菌物种，例如otakiensis的乳酸杆菌（Lactobacillus otakiensis）的培养基中观察到D-亮氨酸，D-异亮氨酸 - 异亮氨酸和D-缬氨酸的显着积累。 otakiensis）JCM 15040.从细菌细胞中纯化导致这种积累的消旋酶，异亮氨酸2-差向异构酶。这是首次报道的在乳酸菌中这种D-支链氨基酸的产生的观察结果，以及在任何生物体中具有异亮氨酸2-差向异构酶活性的消旋酶的第一个实例。在所述的方案中，我们介绍从L中纯化该蛋白质的方法。因为没有鉴定到对该酶具有高亲和力的特异性配体，所以使用硫酸铵级分，四种类型的柱层析和制备型Native-PAGE，不使用亲和柱层析进行纯化。我们希望协议将提供有用的信息用于纯化不能容易地使用亲和柱层析纯化的酶。
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