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Title: | NbRLP1 在植物防禦反應的角色 The role of NbRLP1 in plant immunity |
Authors: | Yi-Hua Li 李怡樺 |
Advisor: | 劉瑞芬 |
Keyword: | elicitin,EPCS,NbRLP1,ParA1,SOBIR1,疫病菌,植物防禦反應, elicitin,EPCS,NbRLP1,ParA1,SOBIR1,Phytophthora,plant immunity, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | Elicitin 為卵菌特有的外泌蛋白,被植物細胞膜上的受體辨識後會誘發植物細胞的基礎防禦反應,包括累積活性氧分子、活化 MAP kinase cascade、誘導防禦基因的表現、甚至造成細胞死亡的現象。Peng et al. (2015) 發現 SlSOBIR1 參與 ParA1 elicitin 所引起的植物防禦反應及細胞壞疽。黃 (2014)利用蛋白共免疫沉澱法尋找可能與 SlSOBIR1 交互作用的蛋白,施(2017) 針對其中的 NbRLP1 深入研究,得知過量表現 NbRLP1 會加強 ParA1 引起的細胞壞疽;且因應 ParA1 處理,原本位於菸草細胞內質網膜之NbRLP1-GFP位移至內質網與細胞膜的接觸點(endoplasmic reticulum–plasma membrane contact sites; EPCS),同時環繞於因應 ParA1 處理也進入細胞質的SlSOBIR1-mCherry,顯示 NbRLP1-GFP 很可能參與 ParA1 誘發的植物防禦反應。為進一步探究 NbRLP1 在植物基礎防禦反應中扮演的角色,本研究首先以蛋白共免疫沉澱與雙分子螢光實驗證實 NbRLP1 與 NbSOBIR1 的交互作用,且發現其交互作用的發生可能在內質網與細胞膜的接觸點 (EPCS)。NbRLP1 過表現除了增強 ParA1 重組蛋白引起的細胞壞疽,也增加因應 ParA1 處理而生成之NbSOBIR1 胞內體數量,顯示 NbRLP1 很可能藉由與 NbSOBIR1 在 EPCS 的交互作用影響 NbSOBIR1 的胞吞作用,以參與在 ParA1 引起的植物防禦反應當中。進一步利用蛋白共免疫沉澱與液態層相串連質譜分析法分析可能與 NbRLP1 具有交互作用的蛋白,發現ERdj3b只出現在 ParA1處理組,其為ER quality control (ERQC)相關的蛋白;將蛋白序列以 GO terms 分析,發現在只處理 ParA1 組相較 MES 對照組的蛋白參與許多內質網與高基式體的傳送。在植物遭遇逆境會產生大量防禦相關蛋白,很可能促使 ERQC 的啟動 (Deng et al., 2013),NbRLP1 可能參與在這樣的植物反應當中。但 NbRLP1 與 NbSOBIR1 在 EPCS 的交互作用究竟參與在哪個途徑,尚待深入探討。 Elicitins are structurally conserved secretory proteins identified solely in Phytophthora and Pythium, both oomycetes. Treatment with elicitins activates plant defense responses, including reactive oxygen species (ROS) burst, MAP kinase cascade activation, induction of defense genes expression, and necrosis. Recent studies indicates that SlSOBIR1 participates in plant defense responses towards Phytophthora parasitica as well as ParA1 (elicitin of P. parasitica) - induced necrosis (Peng et al. 2015). To understand how is SlSOBIR1 involved in these processes, various putative SlSOBIR1-interacting proteins, including NbRLP1, were identified by co-immunoprecipitation (Co-IP) coupled to LC- MS/MS (Huang, 2014). Further analysis demonstrated that NbRLP1 overexpression enhanced ParA1-induced necrosis in Nicotiana benthamiana. Moreover, NbRLP1-GFP is located in the membrane of endoplasmic reticulum. Interestingly, in response to ParA1 treatment, NbRLP1-GFP translocates to the contact sites between endoplasmic reticulum and plasma membrane (EPCS), and is surrounded by SlSOBIR1-mcherry which moves from plasma membrane to endosomes following ParA1 treatment (Shih, 2017). In this study, the role of NbRLP1 in plant immunity was further explored. First, using co- immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) confirmed the interaction between NbRLP1 and NbSOBIR1 and their interaction is on EPCS. NbRLP1 overexpression not only enhances cell necrosis caused by ParA1 recombinant protein treatment, but also increases the number of NbSOBIR1 endosomes induced by ParA1 treatment. This showed NbRLP1 impact the endocytosis of NbSOBIR1 by interacting with NbSOBIR1 on EPCS to participate in ParA1 induced plant immunity. Further, we found a protein –Erdj3b, protein included in ER quality control pathway, only appeared in ParA1 treatment by Co-IP and LC/MS/MS analysis. Using GO terms analysis, the proteins appeared in ParA1 treatment associated with ER to Golgi or Golgi to ER transport. In reponse of stress, plant produced a large number of defense-related proteins, which is likely to trigger the initiation of ERQC (Deng et al., 2013). NbRLP1 may be involved in such plant responses. However, the function of NbRLP1 and NbSOBIR1 interaction in EPCS is still in the process. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73896 |
DOI: | 10.6342/NTU201903582 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 植物病理與微生物學系 |
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