LecRK-V.2及LecRK-VII.1在阿拉伯芥氣孔防禦反應中的功能性分析

Abstract

植物會藉由細胞膜上的模式辨識受體 (PRR) 來辨識病原菌相關分子模式 (PAMPs) ，並啟動下游的防禦反應。透過分析微陣列資訊及T-DNA 插入型突變的表型，Zimmerli實驗室選了L型凝集素類受體激酶V.2 (LecRK-V.2) 及VII.1 (LecRK-VII.1) 做更深入的研究。LecRK包含了胞外的豆科植物凝集素結構域、穿膜結構域以及胞內的激酶結構域。這兩個基因剔除突變種對於由細菌、PAMP或茉莉酸所引起的氣孔關閉皆不敏感。在此篇碩士論文中，透過重複lecrk-V.2及lecrk-VII.1的感染及氣孔實驗，我證實了先前的研究結果。由於只有一個lecrk-V.2的突變株，我將由原生啟動子所引導的LecRK-V.2表現在lecrk-V.2突變株中，用以確認在lecrk-V.2突變株種中受損的防禦反應是咎因於LecRK-V.2的失能。透過磷酸化染劑發現LecRK蛋白是有活性的激酶，其中，轉殖了失去激酶活性LecRK的lecrk回復株對於細菌和PAMP的反應是一樣的，表示這些失去激酶活性的LecRK無法補償突變種中受損的氣孔反應。雙分子螢光互補法顯示這兩個LecRK分別只會在處理PAMP之後與PRR─FLS2進行交互作用，反之，不論有否PAMP處理，LecRK都會跟FLS2的共同受體BAK1交互作用。此外，在互相的lecrk突變種中，LecRK都不能跟FLS2進行交互作用，而失去激酶活性的LecRK則可以在沒有PAMP處理之下就跟FLS2接觸。總體而言，這些實驗結果指出，LecRK-V.2和LecRK-VII.1都參與在阿拉伯芥氣孔的免疫系統中，而且他們的激酶活性對功能非常重要。

Plants recognize pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) by cell-membrane localized pattern recognition receptors (PRRs) to activate downstream PAMP-triggered immunity (PTI) responses. Analyzing microarray profiles and T-DNA insertion mutant phenotypes, the Zimmerli laboratory selected two legume-like lectin receptor kinases (LecRKs), LecRK-V.2 and LecRK-VII.1 for further characterization. LecRKs possess an extracellular legume-like lectin domain, a transmembrane domain and an intracellular kinase domain. Both lecrk-V.2 and lecrk-VII.1 knock-out mutants were insensitive to bacteria-, PAMP- and MeJA-induced stomatal closure. In this master thesis, I confirmed preliminary data by repeating infection and stomatal aperture assays in lecrk-V.2 and lecrk-VII.1 mutants. Since only one T-DNA insertion knock-out mutant could be isolated for LecRK-V.2, lecrk-V.2-1 / NP: LecRK-V.2-GFP lines were generated to confirm that impaired defense responses in the lecrk-V.2 mutant were caused by loss-of-function of LecRK-V.2. In addition, phosphorylation analyses revealed that both LecRKs are active kinases. Notably, stomata of lecrk mutants transformed with LecRK dead kinase version, lecrk-V.2-1 / 35S: LecRK-V.2D459A-GFP and lecrk-VII.1-1 / 35S: LecRK-VII.1D475A-GFP responded to bacteria and PAMPs as mutant lines, suggesting that these constructs could not complement the defective stomatal closure of lecrk mutants. Furthermore, interaction analyses with the bimolecular fluorescence complementation technique suggested that LecRK-V.2 and LecRK-VII.1 only directly interacted with the PRR FLS2 after treatment with the PAMP flg22. By contrast, both LecRKs interacted with the FLS2 co-receptor BAK1 independently of flg22 treatment. In addition, LecRK-V.2 and LecRK-VII.1 failed to interact with FLS2 in the other mutant background and dead kinase version of both LecRKs directly interacted with FLS2, even before flg22 treatment. Taken together, these data suggest that LecRK-V.2 and LecRK-VII.1 are both involved in stomatal immunity at the PRR FLS2 complex, and their kinase activities are critical for their functions.