分析多重複半胱胺酸受體激酶是否參與阿拉伯芥中的抗菌反應

Abstract

在阿拉伯芥的類受體蛋白激酶中，凝集素受體激酶VI.2(LecRK-VI.2)是先天免疫的正向調控者，阿拉伯芥凝集素受體激酶VI.2的過度表現轉殖株的轉錄組學分析中許多基因被正向調控，而在這些基因當中，有13個基因同屬於一類受體蛋白激酶中的亞家族成員，此亞家族叫多重複半胱胺酸受體激酶(CRKs)。CRKs在細胞外的區域包含了1-4個C-X8-C-X2-C模組的重複序列，而且目前已經知道，CRKs可以被細菌性斑點病病原菌(Pst DC3000)所誘導表現。根據這些線索，我們有個假設便是這13個CRKs在調控病原菌的防禦上扮演著重要的角色。在使用T-DNA插入突變株的分析中，在感染Pst DC3000後，除了crk18突變株以外，我們並沒有看到任何相較於野生株顯著不同的病徵。這些結果表示這剩餘的12個CRKs與植物免疫反應無相關，或是這些CRKs基因在功能上可能扮演著類似的角色。為了進一步闡明所選擇的12 CRKs是否涉及在植物的免疫，我們決定分析這12個基因在野生型(Col-0)中大量表現後的改變。我們目前的結果顯示，相較於野生型，大量表現CRK4、CRK6、CRK12、CRK24和CRK36對Pst DC3000有較大的抗性。此外，在處理過鞭毛蛋白(flg22)後，相較於野生型，這五個轉殖株展現出較高的活性氧類和免疫相關的基因FRK1的表現，且增強了氣孔相關的免疫能力。經這些結果我們推論CRKs可能與植物的防禦有關。

Of the receptor-like kinases (RLKs) in Arabidopsis, LECTIN RECEPTOR KINASE-VI.2 (LecRK-VI.2) is a positive regulator of innate immunity. Transcriptome analysis of LecRK-VI.2 overexpression line revealed up-regulation of numerous genes. Among the up-regulated genes, 13 genes which belong to the same sub-family of RLKs which named CYSTEINE-RICH REPEAT KINASES (CRKs). CRKs contain 1-4 copies of the C-X8-C-X2-C motif in their extracellular domains and CRKs expression can be induced by pathogenic bacteria such as Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). According to these clues, we have a hypothesis that these 13 CRKs may play an important roles in the regulation of pathogen defense. Using a loss-of-function approach, most crk T-DNA insertion mutant lines tested did not demonstrate an altered resistance phenotype after Pst DC3000 inoculation except crk18. This observation suggests that the remaining 12 CRKs are functionally redundant or are not involved in plant defense. To further clarify whether the selected 12 CRKs are involved in plant immunity, a gain-of-function approach by over-expression in wild-type Col-0 was performed. Our results indicate that CRK4, CRK6, CRK12, CRK24 and CRK36 over-expression lines are more resistant to Pst DC3000 than Col-0 wild-type plants. In addition, after flg22 treatment, these five CRKs over-expression lines showed enhanced reactive oxygen species (ROS) production, increased up-regulation of the plant immunity marker gene FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1), and a reinforced stomatal immunity. These results suggest that CRK4, CRK6, CRK12, CRK24 and CRK36 are involved in the plant innate immunity response.