白胺酸重複受體激酶與半胱胺酸重複受體激酶在阿拉伯芥抗病反應之功能性分析

Abstract

摘要一 植物可藉由處理β- aminobutyric acid (BABA) 而增強對病原的抗性，我們在先的研究指出，leucine-rich repeat protein kinase (LRRPK) 可提供植物對抗細菌性斑點病原菌 (Pseudomonas syringae pv. tomato DC3000) 的耐受性，同時，lrrpk對細菌性斑點病原菌的突變株hrcC-也呈現較易感染的情況，因此，LRRPK在植物的免疫反應上是被需要的，lrrpk喪失了BABA在植物被細菌性斑點病原菌感染後對氣孔的調節，lrrpk在處理flg22後也被證實免疫反應的相關基因表現量降低和醣類堆積下降。然而卻沒有改變對Reactive oxygen species 的調控，本研究之目標是尋找LRRPK是否能對抗另外一種病原和分析大量表現LRRPK後植物的表徵，我發現了lrrpk對Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) 也表現出較易感染的現象，此外，大量表現後的植株對Pst DC3000較有抗性，且在處理flg22後也較多的醣類累積，細菌也無法在3小時後將氣孔重新開啟，結合突變株和大量表現後的植株的實驗，可推論LRRPK參與植物的免疫反應和BABA誘發的防禦反應。 摘要二 DNA微陣列分析，發現了12個多重複半胱胺酸受體激酶，相較於野生株，這12個基因在大量表現凝集素受體激6.2後，至少被誘導了四倍以上，在阿拉伯芥中，多重複半胱胺酸受體激酶(CRK)被歸類成一類的蛋白質受體激酶，在細胞外的區域包含了兩個重複序列C-X8-C-X2-C的模組，目前已經知道，這類的蛋白質受體激酶可以被參與防禦反應的賀爾蒙水楊酸和細菌性斑點病病原菌所誘導表現，在大量表現阿拉伯芥凝集素受體激酶6.2 (LecRK-VI.2) 的在感染細菌性斑點病病原菌 (Pseudomonas syringae pv. tomato DC3000) 後，在插入突變株的分析中，我們並沒有看到任何病徵的改變，這暗示著這些基因在功能上，可能扮演著類似的角色，因此，我們決定分析這12個基因在大量表現後的改變，我們目前的結果顯示，大量表現CRK-H 和CRK-K使植物對細菌性斑點病病原菌有較大之抗性提升，且持續性醣類堆積和免疫相關的基因FRK1與PR1的表現，並使氣孔關閉，由此可以推論CRKs可能與植物的防禦反應有關。

Abstract I Plants can acquire enhanced resistance to pathogens after treatment with beta- aminobutyric acid (BABA). BABA primes plant’s defense responses. Previously, we showed that a leucine-rich repeat protein kinase (LRRPK) contributes to disease resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. Indeed, lrrpk mutants were more susceptible to Pst DC3000 hrcC-, suggesting this LRRPK is required for pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We have demonstrated that lrrpk mutants were defective in BABA-mediated blockage of stomatal reopening after Pst DC3000 infection. lrrpk mutants also demonstrated reduced PTI marker gene induction and impaired callose deposition after flg22 treatment. However, lrrpk mutants demonstrated a normal reactive oxygen species burst. The aim of this study was to identify whether LRRPK can enhance disease resistance to another bacterial pathogen and to analyze the phenotype of LRRPK over-expression (OE) lines. lrrpk mutants were found to be more susceptible to virulent Pseudomonas syringae pv. maculicola (Psm) ES4326 and defective in BABA-mediated plant defense. Moreover, OE lines were more resistant to Pst DC3000 and accumulated more callose after treatment with the PAMP flg22. In addition, stomata of OE lines did not reopen 3 hours post inoculation with bacteria. Knock-out mutants and OE lines phenotypes suggest that LRRPK plays a role in PTI and BABA-induced resistance. Abstract II Genome-wide microarray analysis of an Arabidopsis Lectin Receptor Kinase-VI.2 (LecRK-VI.2) over-expression line shows that 12 cysteine-rich repeat like kinases (Cysteine-rich receptor like kinases, CRK-A, -B, -C, -D. –E, -F, -G, -H, -I , -J, -K, and -L) were up-regulated at least 4-fold when compared to WT. In Arabidopsis, CRKs are a sub-family of receptor-like protein kinases that contain two copies of the C-X8-C-X2-C motif in their extracellular domains. It has been shown that CRK genes can be induced by the defense hormone salicylic acid and bacteria Pst DC3000 (Pseudomonas syringae pv. tomato DC3000) bacteria. Using a T-DNA knock-out approach, we did not observe any altered phenotype after Pst DC3000 inoculation, suggesting that these genes are functionally redundant. Therefore, we decided to use a gain-of-function approach by over-expressing these CRKs in wild-type Col-0 background. Our preliminary results indicate that CRK-H and CRK-K over-expression lines are more resistant to Pst DC3000. In addition, these two CRKs over-expression lines showed constitutive callose deposition and expression of PTI marker gene PR1 (PATHOGENESIS-RELATIVE 1) and FRK1 (FLG22-INDUCED RECEPTOR-LIKE 1), and an altered stomatal immunity. We suggest that CRK-H and CRK-K are involved in plant defense.