NMT是基因功能的活體檢測技術，已被31位諾貝爾獎得主所在單位，及北大、清華、中科院使用。
北京林業大學林金星團隊發表植物胞吐與胞吞協調轉運方面的綜述論文
期刊：Development
主題：固醇調控FLS2蛋白胞吞的新機制（鈣信號）
標題：Sterols regulate endocytic pathways during flg22-induced defense responses in Arabidopsis
影響因子：5.413
檢測指標：Ca2+流速
檢測樣品：擬南芥葉片
Ca2+流速流實驗處理方法：
7日齡擬南芥，0.1 μM flg22處理
Ca2+流速流實驗測試液成份：無
推薦測試液：0.1mM CaCl2，pH 6.0
作者：北京林業大學林金星、李曉娟
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（谷歌機翻）
植物跨膜受體激酶FLAGELLIN SENSING 2（FLS2）對于先天免疫至關重要。盡管以前的研究已經報道了通過網格蛋白介導的途徑介導的FLS2介導的信號轉導和胞吞作用，但尚不清楚其他內吞途徑是否會影響FLS2介導的防御反應。
在這里，我們顯示擬南芥的甾醇缺乏突變體類固醇甲基轉移酶1在鞭毛蛋白衍生的肽flg22誘導的免疫反應中顯示缺陷。可變角度全內反射熒光顯微鏡（VA-TIRFM）結合單粒子跟蹤顯示，在處理的細胞中，FLS2-GFP的時空動態在毫秒級變化，質膜上的FLS2-GFP停留時間增加與未經處理的對應物相比，含固醇提取劑。
我們進一步證明，flg22誘導的FLS2聚集和內吞涉及與固醇相關的內吞途徑，這不同于網格蛋白介導的途徑。此外，flg22通過固醇相關途徑增強了FLS2-GFP與膜微區標記Flot 1-mCherry和FLS2內吞的共定位。這表明植物可以通過調節兩種不同的內吞途徑來響應病原體侵襲。
兩者合計，我們的結果表明固醇穩態在flg22誘導的植物防御反應中的關鍵作用。
結果表明：相比野生型，smt1（甾醇甲基轉移酶1）突變體表現出迅速且強烈的Ca2+吸收速率高于根冠（R0和R1），低于根伸長區（R2）。增加，說明突變體對于flg22更為敏感。smt1突變沒有改變FLS2的同源寡聚狀態，但影響FLS2簇形成。smt1突變體中FLS2的內吞功能受到損傷。由上述結果得到一個假設，即甾醇相關的內吞途徑對于flg22誘導的FLS2動力學和植物防御至關重要。