Fast-suppressor screening for new components in protein trafficking, organelle biogenesis and silencing pathway in Arabidopsis thaliana using DEX-inducible FREE1-RNAi plants.
Fast-suppressor screening for new components in protein trafficking, organelle biogenesis and silencing pathway in Arabidopsis thaliana using DEX-inducible FREE1-RNAi plants.
复制标题
使用 DEX 诱导的 FREE1-RNAi 植物快速抑制子筛选拟南芥中蛋白质运输、细胞器生物发生和沉默途径中的新成分
DOI：
10.1016/j.jgg.2015.03.012
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发表时间：
2015-06-20
期刊：
影响因子：
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通讯作者：
Jiang L
中科院分区：
文献类型：
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作者：
Zhao Q;Gao C;Lee P;Liu L;Li S;Hu T;Shen J;Pan S;Ye H;Chen Y;Cao W;Cui Y;Zeng P;Yu S;Gao Y;Chen L;Mo B;Liu X;Xiao S;Zhao Y;Zhong S;Chen X;Jiang L
膜运输对于植物生长和对外部信号的反应至关重要。植物特有的含有 FYVE 结构域的蛋白质 FREE1 是 ESCRT 复合物（运输所需的内体分选复合物）的组成部分。 FREE1 在调节蛋白质运输和细胞器生物发生中发挥多种作用，包括多泡体 (MVB) 腔内囊泡的形成、液泡蛋白运输和液泡生物发生以及自噬降解。 FREE1 敲除植物表现出 MVB 形成缺陷、液泡运输异常、液泡破碎、自噬体积累和幼苗致死。为了进一步揭示FREE1在植物中功能的潜在机制，我们对抑制FREE1-RNAi转基因植物幼苗致死表型的突变体进行了正向遗传筛选。获得的突变体被称为 free1 (sof) 的抑制子。迄今为止，已鉴定出 229 个假定的 sof 突变体。利用 M3 植物勉强检测到 FREE1 蛋白，进一步鉴定出 84 个 FREE1 相关抑制因子。另外，145 个没有显示 FREE1 蛋白减少的突变体被称为 RNAi 相关突变体。通过对两个 RNAi 相关 sof 突变体的 F2 作图群体的大量 DNA 进行下一代测序 (NGS)，鉴定出插入到 1 号染色体上的 FREE1-RNAi T-DNA，并类似地鉴定了假定的 sof 突变体的因果突变。这些FREE1和RNAi相关的sof突变体将成为有用的工具和资源，用于阐明FREE1在细胞内运输和细胞器生物发生中的功能的潜在机制，以及揭示参与植物沉默途径调节的新成分。
Membrane trafficking is essential for plant growth and responses to external signals. The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex (endosomal sorting complex required for transport). FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body (MVB), vacuolar protein transport and vacuole biogenesis, and autophagic degradation. FREE1 knockout plants show defective MVB formation, abnormal vacuolar transport, fragmented vacuoles, accumulated autophagosomes, and seedling lethality. To further uncover the underlying mechanisms of FREE1 function in plants, we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants. The obtained mutants are termed as suppressors of free1 (sof). To date, 229 putative sof mutants have been identified. Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors. Also 145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants. Through next-generation sequencing (NGS) of bulked DNA from F2 mapping population of two RNAi-related sof mutants, FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly. These FREE1- and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis, as well as for uncovering the new components involved in the regulation of silencing pathways in plants.