利用 EXO70 抑制剂 Endosidin2 进行化学遗传筛选,发现拟南芥中潜在的外泌调节剂。

IF 2.3 3区 生物学 Q2 PLANT SCIENCES Plant Direct Pub Date : 2024-06-14 eCollection Date: 2024-06-01 DOI:10.1002/pld3.592
Xiaohui Li, Diwen Wang, Xianglin Yin, Mingji Dai, Christopher J Staiger, Chunhua Zhang
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引用次数: 0

摘要

外泌在将蛋白质、脂质和细胞壁多糖输送到质膜和细胞外空间方面发挥着重要作用。通过外泌作用进行准确分泌是植物正常发育以及应对生物和非生物胁迫的关键。在外分泌过程中,一种名为外囊的八聚体蛋白质复合物有助于将分泌囊泡拴系到质膜上。尽管通过反向遗传学和直接相互作用测定对外囊功能的分子和细胞方面有了一定的了解,但对上游调节因子和基因相互作用因子的了解仍然有限。传统的遗传筛选在外显子胞亚基突变背景下会遇到一些实际问题,如某些基因敲除突变体的致死率和/或EXO70同源物的潜在冗余。为了应对这些挑战,本研究利用化学遗传学的可调控性和可逆性,采用Endosidin2(ES2)--一种EXO70的合成抑制剂--在拟南芥中进行大规模化学遗传突变体筛选。这种方法鉴定出了 70 个 ES2 超敏感突变体,命名为 es2s。通过基于全基因组测序的图谱绘制策略,绘制出了 14 个非等位的 es2s 突变体,并在此报告了候选突变。此外,还测试了作为替代等位基因的 T-DNA 插入系,以确定因果突变。我们发现,DCP5、VAS1/ISS1、ArgJ 和 MEF11 的 T-DNA 插入等位基因对 ES2 的根生长抑制作用不敏感。这项研究不仅为系统鉴定与拟南芥外囊相互作用的分子角色提供了新的遗传资源,而且加深了对外囊分泌调控的理解。
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A chemical genetic screen with the EXO70 inhibitor Endosidin2 uncovers potential modulators of exocytosis in Arabidopsis.

Exocytosis plays an essential role in delivering proteins, lipids, and cell wall polysaccharides to the plasma membrane and extracellular spaces. Accurate secretion through exocytosis is key to normal plant development as well as responses to biotic and abiotic stresses. During exocytosis, an octameric protein complex named the exocyst facilitates the tethering of secretory vesicles to the plasma membrane. Despite some understanding of molecular and cellular aspects of exocyst function obtained through reverse genetics and direct interaction assays, knowledge about upstream modulators and genetic interactors remains limited. Traditional genetic screens encounter practical issues in exocyst subunit mutant backgrounds, such as lethality of certain knockout mutants and/or potential redundancy of EXO70 homologs. To address these challenges, this study leverages the tunable and reversible nature of chemical genetics, employing Endosidin2 (ES2)-a synthetic inhibitor of EXO70-for a large-scale chemical genetic mutant screen in Arabidopsis. This approach led to the identification of 70 ES2-hypersensitive mutants, named es2s. Through a whole-genome sequencing-based mapping strategy, 14 nonallelic es2s mutants were mapped and the candidate mutations reported here. In addition, T-DNA insertion lines were tested as alternative alleles to identify causal mutations. We found that T-DNA insertion alleles for DCP5, VAS1/ISS1, ArgJ, and MEF11 were hypersensitive to ES2 for root growth inhibition. This research not only offers new genetic resources for systematically identifying molecular players interacting with the exocyst in Arabidopsis but also enhances understanding of the regulation of exocytosis.

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来源期刊
Plant Direct
Plant Direct Environmental Science-Ecology
CiteScore
5.00
自引率
3.30%
发文量
101
审稿时长
14 weeks
期刊介绍: Plant Direct is a monthly, sound science journal for the plant sciences that gives prompt and equal consideration to papers reporting work dealing with a variety of subjects. Topics include but are not limited to genetics, biochemistry, development, cell biology, biotic stress, abiotic stress, genomics, phenomics, bioinformatics, physiology, molecular biology, and evolution. A collaborative journal launched by the American Society of Plant Biologists, the Society for Experimental Biology and Wiley, Plant Direct publishes papers submitted directly to the journal as well as those referred from a select group of the societies’ journals.
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