NMT是基因功能的活体检测技术，已被31位诺贝尔奖得主所在单位，及北大、清华、中科院使用。
北京林业大学林金星团队发表植物胞吐与胞吞协调转运方面的综述论文
期刊：Development
主题：固醇调控FLS2蛋白胞吞的新机制（钙信号）
标题：Sterols regulate endocytic pathways during flg22-induced defense responses in Arabidopsis
影响因子：5.413
检测指标：Ca2+流速
检测样品：拟南芥叶片
Ca2+流速流实验处理方法：
7日龄拟南芥，0.1 μM flg22处理
Ca2+流速流实验测试液成份：无
推荐测试液：0.1mM CaCl2，pH 6.0
作者：北京林业大学林金星、李晓娟
英文摘要
The plant transmembrane receptor kinase FLAGELLIN SENSING 2 (FLS2) is crucial for innate immunity. Although previous studies have reported FLS2-mediated signal transduction and endocytosis via the clathrin-mediated pathway, whether additional endocytic pathways affect FLS2-mediated defense responses remains unclear.
Here, we show that the Arabidopsis thaliana sterol-deficient mutant steroid methyltransferase 1 displays defects in immune responses induced by the flagellin-derived peptide flg22. Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) coupled with single-particle tracking showed that the spatiotemporal dynamics of FLS2-GFP changed on a millisecond time scale and that the FLS2-GFP dwell time at the plasma membrane increased in cells treated with a sterol-extracting reagent when compared with untreated counterparts.
We further demonstrate that flg22-induced FLS2 clustering and endocytosis involves the sterol-associated endocytic pathway, which is distinct from the clathrin-mediated pathway. Moreover, flg22 enhanced the colocalization of FLS2-GFP with the membrane microdomain marker Flot 1-mCherry and FLS2 endocytosis via the sterol-associated pathway. This indicates that plants may respond to pathogen attacks by regulating two different endocytic pathways.
Taken together, our results suggest the key role of sterol homeostasis in flg22-induced plant defense responses.
中文摘要（谷歌机翻）
植物跨膜受体激酶FLAGELLIN SENSING 2（FLS2）对于先天免疫至关重要。尽管以前的研究已经报道了通过网格蛋白介导的途径介导的FLS2介导的信号转导和胞吞作用，但尚不清楚其他内吞途径是否会影响FLS2介导的防御反应。
在这里，我们显示拟南芥的甾醇缺乏突变体类固醇甲基转移酶1在鞭毛蛋白衍生的肽flg22诱导的免疫反应中显示缺陷。可变角度全内反射荧光显微镜（VA-TIRFM）结合单粒子跟踪显示，在处理的细胞中，FLS2-GFP的时空动态在毫秒级变化，质膜上的FLS2-GFP停留时间增加与未经处理的对应物相比，含固醇提取剂。
我们进一步证明，flg22诱导的FLS2聚集和内吞涉及与固醇相关的内吞途径，这不同于网格蛋白介导的途径。此外，flg22通过固醇相关途径增强了FLS2-GFP与膜微区标记Flot 1-mCherry和FLS2内吞的共定位。这表明植物可以通过调节两种不同的内吞途径来响应病原体侵袭。
两者合计，我们的结果表明固醇稳态在flg22诱导的植物防御反应中的关键作用。
结果表明：相比野生型，smt1（甾醇甲基转移酶1）突变体表现出迅速且强烈的Ca2+吸收速率高于根冠（R0和R1），低于根伸长区（R2）。增加，说明突变体对于flg22更为敏感。smt1突变没有改变FLS2的同源寡聚状态，但影响FLS2簇形成。smt1突变体中FLS2的内吞功能受到损伤。由上述结果得到一个假设，即甾醇相关的内吞途径对于flg22诱导的FLS2动力学和植物防御至关重要。