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A Fully Functional ROP Fluorescent Fusion Protein Reveals Roles for this GTPase in Subcellular and Tissue-level Patterning.
The Plant Cell ( IF 11.6 ) Pub Date : 2020-11-01 , DOI: 10.1105/tpc.20.00440
Xiaohang Cheng 1 , Bethany W Mwaura 1 , Sophia R Chang Stauffer 1 , Magdalena Bezanilla 2
Affiliation  

Rho of Plants (ROPs) are GTPases that regulate polarity and patterned wall deposition in plants. As these small, globular proteins have many interactors, it has been difficult to ensure that methods to visualize ROP in live cells do not affect ROP function. Here, motivated by work in fission yeast (Schizosaccharomyces pombe), we generated a fluorescent moss (Physcomitrium [Physcomitrella] patens) ROP4 fusion protein by inserting mNeonGreen after Gly-134. Plants harboring tagged ROP4 and no other ROP genes were phenotypically normal. Plants lacking all four ROP genes comprised an unpatterned clump of spherical cells that were unable to form gametophores, demonstrating that ROP is essentially for spatial patterning at the cellular and tissue levels. The functional ROP fusion protein formed a steep gradient at the apical plasma membranes of growing tip cells. ROP also predicted the site of branch formation in the apical cell at the onset of mitosis, which occurs one to two cell cycles before a branch cell emerges. While fluorescence recovery after photobleaching studies demonstrated that ROP dynamics do not depend on the cytoskeleton, acute depolymerization of the cytoskeleton removed ROP from the membrane only in recently divided cells, pointing to a feedback mechanism between the cell cycle, cytoskeleton, and ROP.



中文翻译:

全功能 ROP 荧光融合蛋白揭示了该 GTP 酶在亚细胞和组织水平模式形成中的作用。

植物 Rho (ROP) 是调节植物极性和图案壁沉积的 GTP 酶。由于这些小球状蛋白具有许多相互作用子,因此很难确保在活细胞中可视化 ROP 的方法不会影响 ROP 功能。在这里,受裂殖酵母 ( Schizosaccharomyces pombe ) 工作的启发,我们通过在 Gly-134 后插入 mNeonGreen生成了荧光苔藓 ( Physcomitrella [ Physcomitrella ] patens ) ROP4 融合蛋白。含有标记的ROP4且没有其他ROP基因的植物表型正常。缺乏全部四种ROP 的植物基因由一团无图案的球形细胞组成,无法形成配子体,这表明 ROP 本质上是细胞和组织水平上的空间图案。功能性 ROP 融合蛋白在生长的尖端细胞的顶端质膜处形成陡峭的梯度。ROP 还预测了顶端细胞在有丝分裂开始时分支形成的位置,有丝分裂发生在分支细胞出现之前的一到两个细胞周期。虽然光漂白研究后的荧光恢复表明 ROP 动力学不依赖于细胞骨架,但细胞骨架的急性解聚仅在最近分裂的细胞中从膜上去除 ROP,这表明细胞周期、细胞骨架和 ROP 之间存在反馈机制。

更新日期:2020-11-03
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