Monitoring Autophagy in Rice With GFP-ATG8 Marker Lines.
Monitoring Autophagy in Rice With GFP-ATG8 Marker Lines.
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使用 GFP-ATG8 标记线监测水稻的自噬
DOI：
10.3389/fpls.2022.866367
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发表时间：
2022
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
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作者：
自噬是真核生物大量降解和回收细胞成分的一种保守的细胞内运输途径。自噬的特征是形成被称为自噬小体的双膜囊，它选择性或非选择性地包装各种大分子和细胞器，并将这些货物运送到液泡/溶酶体中。像所有其他膜运输途径一样，自噬的观察在很大程度上依赖于标记线。ATG8/LC3是唯一一种与磷脂酰乙醇胺(PE)共价结合的自噬相关(ATG)蛋白，它与隔离膜/自噬前结构(PAS)、生长的吞噬体、成熟的自噬小体和自噬小体紧密结合。因此，荧光蛋白(FP)标记的ATG8已被广泛用于监测自噬小体的形成和自噬通量。在水稻中，利用花椰菜花叶病毒(CaMV)35S启动子驱动的FP-OsATG8对自噬小体和自噬小体进行了成像。在此，我们构建了三个携带GFP-OsATG8a的载体，分别由35S、泛素和内源性Atg8a启动子驱动。然后，我们通过观察瞬时转化水稻原生质体中GFP-Atg8a点的形成，以及用GFP-ATG8裂解试验追踪水稻稳定转基因系中的自噬通量，比较了它们在监测自噬方面的适用性。三个标记系还进行了GFP-Trap免疫沉淀和质谱分析，表明它们可以可靠地用于蛋白质组学研究。我们发现，泛素启动子是最适合原生质体成像的。利用GFP-ATG8裂解试验，三个标记系的转基因水稻幼苗在自噬通量测定中表现出相似的表现。令人惊讶的是，所有标记系的GFP-Atg8a转录本水平和蛋白质含量都是相似的，表明转基因表达的转录后调控机制尚不清楚。这些标记系可作为研究水稻自噬的有用工具。
Autophagy is a conserved intracellular trafficking pathway for bulk degradation and recycling of cellular components in eukaryotes. The hallmark of autophagy is the formation of double-membraned vesicles termed autophagosomes, which selectively or non-selectively pack up various macromolecules and organelles and deliver these cargoes into the vacuole/lysosome. Like all other membrane trafficking pathways, the observation of autophagy is largely dependent on marker lines. ATG8/LC3 is the only autophagy-related (ATG) protein that, through a covalent bond to phosphatidylethanolamine (PE), associates tightly with the isolation membrane/pre-autophagosomal structure (PAS), the growing phagophore, the mature autophagosome, and the autophagic bodies. Therefore, fluorescent protein (FP)-tagged ATG8 had been widely used for monitoring autophagosome formation and autophagic flux. In rice (Oryza sativa), FP-OsATG8 driven by Cauliflower mosaic virus (CaMV) 35S promoter had been used for imaging autophagosome and autophagic bodies. Here, we constructed three vectors carrying GFP-OsATG8a, driven by 35S, ubiquitin, and the endogenous ATG8a promoter, individually. Then, we compared them for their suitability in monitoring autophagy, by observing GFP-ATG8a puncta formation in transiently transformed rice protoplasts, and by tracking the autophagic flux with GFP-ATG8 cleavage assay in rice stable transgenic lines. GFP-Trap immunoprecipitation and mass spectrometry were also performed with the three marker lines to show that they can be used reliably for proteomic studies. We found out that the ubiquitin promoter is the best for protoplast imaging. Transgenic rice seedlings of the three marker lines showed comparable performance in autophagic flux measurement using the GFP-ATG8 cleavage assay. Surprisingly, the levels of GFP-ATG8a transcripts and protein contents were similar in all marker lines, indicating post-transcriptional regulation of the transgene expression by a yet unknown mechanism. These marker lines can serve as useful tools for autophagy studies in rice.