Functional characterization of AtRUG8, a protein that modulates plasmodesmal movement of macromolecules, and of three 3 closely related proteins

我们已经证明，在拟南芥AtSUC2（蔗糖转运蛋白2）启动子的控制下，在拟南芥或烟草（Nicotiana tabacum）伴侣细胞（CCs）中合成的可溶性GFP从CCs进入筛元件（SEs），与光同化物一起从源到汇，并最终交感卸荷到不同的末端汇组织，如花药、幼叶或根尖。我们在植物中使用AtSUC2-promoter::GFP转基因，筛选了7000多个T-DNA插入系，发现了8个显著降低GFP细胞间迁移率的突变体。这些突变体被命名为“减少GFP卸载”（rug）。到目前为止，其中一种突变体rug8已经在分子水平上得到了部分表征。在两个独立的突变体（rug8-1和rug8-2）中，功能未表征的基因At1g17930被证明是观察到的表型的原因，与野生型序列的互补恢复了可溶性GFP的流动性。RUG8具有未知功能的n端结构域（DUF1723）和预测的c端核定位序列（NLS）。事实上，亚细胞定位分析证实，RUG8定位于细胞核，酵母2-杂交筛选显示，RUG8与AP2/ erf型转录因子ERF38相互作用，ERF38是一种调节细胞壁增厚相关基因表达的拟南芥蛋白。本课题将研究RUG8蛋白的生理作用以及RUG8突变对胞间连丝大小排除限（SEL）的影响。此外，我们将筛选更多的RUG8相互作用因子，寻找在RUG8突变体中改变的基因表达，并研究RUG8与ERF38的相互作用模式。最终，我们将把这些分析扩展到三个密切相关的拟南芥基因At1g48120， At2g04865和At2g25010。

We have shown that soluble GFP that is synthesized in Arabidopsis or tobacco (Nicotiana tabacum) companion cells (CCs) under the control of the Arabidopsis AtSUC2 (SUCROSE TRANSPORTER 2) promoter moves from CCs into sieve elements (SEs), traffics with the photoassimilates from source to sink, and is eventually symplastically unloaded into different terminal sink tissues, such as anthers, young leaves or root tips. Screening of more than 7,000 T-DNA insertion lines that we generated in plants with an AtSUC2-promoter::GFP transgene, identified eight mutants with significantly reduced cell-to-cell mobility of GFP. These mutants were named “reduced unloading of GFP” (rug). So far, one of these mutants, rug8, has been partly characterized on the molecular level. In two independent mutants (rug8-1 & rug8-2) the functionally uncharacterized gene At1g17930 was shown to be responsible for the observed phenotype, and complementation with the wild type sequence restored the mobility of soluble GFP. RUG8 has an N-terminal domain of unknown function (DUF1723) and a predicted Cterminal nuclear localization sequence (NLS). In fact, analyses of the subcellular localization confirmed that RUG8 localizes to the nucleus and a yeast 2-hybrid screen revealed interaction of RUG8 with the AP2/ERF-type transcription factor ERF38, an Arabidopsis protein that regulates the expression of genes involved in cell wall thickening. In the proposed project we will study the physiological role of the RUG8 protein and the effect of rug8 mutations on the size exclusion limit (SEL) of plasmodesmata. Moreover, we will screen for additional RUG8 interactors, search for altered gene expression in rug8 mutants, and study the mode of interaction between RUG8 and ERF38. Eventually we will extend these analyses to the three closely related Arabidopsis genes At1g48120, At2g04865 and At2g25010.