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Development：北京林業(yè)大學(xué)丨固醇調(diào)控FLS2蛋白胞吞的新機制
NMT是基因功能的活體檢測技術(shù)，已被31位諾貝爾獎得主所在單位，及北大、清華、中科院使用。
期刊：Development
主題：固醇調(diào)控FLS2蛋白胞吞的新機制（鈣信號）
標(biāo)題：Sterols regulate endocytic pathways during flg22-induced defense responses in Arabidopsis
影響因子：5.413
檢測指標(biāo)：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(yè)大學(xué)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(guān)重要。盡管以前的研究已經(jīng)報道了通過網(wǎng)格蛋白介導(dǎo)的途徑介導(dǎo)的FLS2介導(dǎo)的信號轉(zhuǎn)導(dǎo)和胞吞作用，但尚不清楚其他內(nèi)吞途徑是否會影響FLS2介導(dǎo)的防御反應(yīng)。
在這里，我們顯示擬南芥的甾醇缺乏突變體類固醇甲基轉(zhuǎn)移酶1在鞭毛蛋白衍生的肽flg22誘導(dǎo)的免疫反應(yīng)中顯示缺陷。可變角度全內(nèi)反射熒光顯微鏡（VA-TIRFM）結(jié)合單粒子跟蹤顯示，在處理的細胞中，F(xiàn)LS2-GFP的時空動態(tài)在毫秒級變化，質(zhì)膜上的FLS2-GFP停留時間增加與未經(jīng)處理的對應(yīng)物相比，含固醇提取劑。
我們進一步證明，flg22誘導(dǎo)的FLS2聚集和內(nèi)吞涉及與固醇相關(guān)的內(nèi)吞途徑，這不同于網(wǎng)格蛋白介導(dǎo)的途徑。此外，flg22通過固醇相關(guān)途徑增強了FLS2-GFP與膜微區(qū)標(biāo)記Flot 1-mCherry和FLS2內(nèi)吞的共定位。這表明植物可以通過調(diào)節(jié)兩種不同的內(nèi)吞途徑來響應(yīng)病原體侵襲。
兩者合計，我們的結(jié)果表明固醇穩(wěn)態(tài)在flg22誘導(dǎo)的植物防御反應(yīng)中的關(guān)鍵作用。
結(jié)果表明：相比野生型，smt1（甾醇甲基轉(zhuǎn)移酶1）突變體表現(xiàn)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(qū)（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(tài)，但影響FLS2簇形成。smt1突變體中FLS2的內(nèi)吞功能受到損傷。由上述結(jié)果得到一個假設(shè)，即甾醇相關(guān)的內(nèi)吞途徑對于flg22誘導(dǎo)的FLS2動力學(xué)和植物防御至關(guān)重要。