- 學位論文
探討SlRLK1在番茄基礎防禦反應的角色
The Role of SlRLK1 in Basal Defense Responses of Tomato
摘要
植物leucine-rich repeat (LRR)-receptor like kinases (LRR-RLKs)不僅與植物生長分化相關，在植物對抗病原的抗病機制也扮演重要的角色。這類膜蛋白的作用機制通常是以胞外的LRR domain感受環境的刺激後，與其co-receptor形成dimer，經由交互磷酸化活化彼此胞內的激酶，最後將訊息傳入胞內的下游因子。近年來的研究指出，LRR-RLKs將胞外訊息傳入胞內時也涉及胞吞作用(endocytosis)。本實驗室先前針對亞磷酸處理之後的番茄(Solanum lycopersicum)進行生物晶片分析，發現多個表現顯著變化的基因，其中之一為LRR-RLK，本研究將其定名為SlRLK1。於番茄基因體進行序列比對分析後發現另一相同度極高的LRR-RLK，定名為SlRLK2。半定量RT-PCR分析結果顯示，接種疫病菌(Phytophthora parasitica)能顯著誘導SlRLK1表現，但對SlRLK2的影響不顯著，因此後續研究多集中在SlRLK1。SlRLK1也會被疫病菌PAMPs，包括ParA1與CBEL誘導表現，但處理疫病菌另一PAMP (pep13)及細菌PAMPflg22對SlRLK1的表現並無影響。為了探討SlRLK1之功能，本研究接著利用Tobacco rattle virus (TRV)系統引發SlRLK1基因靜默，發現SlRLK1表現量受到抑制的植株，在接種P. parasitica後明顯較為感病，顯示SlRLK1在番茄對於P. parasitica的抗性可能扮演重要角色。利用農桿菌在圓葉菸草(Nicotiana benthamiana)葉片上短暫表現SlRLK1-GFP的融和蛋白，發現此蛋白位於植物細胞膜；進而在葉片上接種P. parasitica或處理ParA1與CBEL皆能使SlRLK1-GFP產生胞吞現象，因此推測SlRLK1可能藉由胞吞作用將生物逆境的訊息傳入胞內並且引發相關的防禦反應。在不同植物抗病荷爾蒙處理下，水楊酸和乙烯皆會提昇SlRLK1的表現，但處理茉莉酸則導致基因表現些微下降。觀察在SlRLK1基因靜默的番茄植株中植物荷爾蒙相關基因的表現情形，發現部分水楊酸、乙烯、茉莉酸及離層素的相關調控基因的表現受到影響而表現量較低；其中受到最多影響的是茉莉酸上游基因Proteinase inhibitor ΙΙ (Pin2)與Lipoxygenase (LoxD)，顯示SlRLK1的作用很可能參與在植物荷爾蒙的調控途徑。未來的研究目標將著重於找出能夠與SlRLK1結合並啟動下游防禦反應之作用因子，進而找出它們辨識的訊息分子，以及確認SlRLK1之激酶活性以明確瞭解SlRLK1於番茄的防禦反應所扮演的角色。
並列摘要
Plant leucine-rich repeat (LRR)-receptor like kinases (LRR-RLKs) involved in not only in plant development but also innate immunity. LRR-RLKs generally perceive signals from the environment via LRR domain and dimerization with co- receptor, and then transduce extracellular signals into intracellular responses via trans- phosphorylation. Recent studies demonstrated that LRR-RLKs executed endocytosis to pass the messages from extracellular to intracellular. While identifying tomato (Solanum lycopersicum) genes which were differentially expressed in response to treatment with neutralized phosphorous acid (NPA) by microarrays, a variety of genes were found to be induced, including one that encoded a putative leucine-rich repeat receptor-like kinase (LRR-RLK), herein named SlRLK1. In addition, a close homolog of SlRLK1 was identified via blast search of the tomato genome database, herein named SlRLK2. Analysis by semi- quantitative reverse transcriptase-PCR showed that SlRLK1 was induced upon Phytophthora parasitica infection but not SlRLK2, suggesting a role of SlRLK1 in plant defense response against P. parasitica. Treatments of PAMPs form P. parasitica including ParA1 and CBEL also induced SlRLK1, but pep13 and flg22 (a bacterial PAMP) had no effect on the expression of SlRLK1. To characterize the function of SlRLK1 in plants, we down-regulated its expression by Tobacco rattle virus (TRV)-mediated gene silencing. Interestingly, SlRLK1-silenced tomato became more susceptible to P. parasitica. When SlRLK1-GFP fusion protein was transiently expressed on leaves of Nicotiana benthamiana by agroinfiltration, it was found to reside predominantly on the plasma membrane. However, endocytosis of SlRLK1-GFP fusion protein occurred after inoculation of P. parasitica as well as treatments with ParA1 and CBEL, suggesting its involvement in the perception of the Phytophthora pathogen. Treatment with either salicylic acid or ethylene enhanced the expression of SlRLK1. In contrast, methyl- jasmornate slightly reduced its transcript level. Furthermore, expression of some genes participating in salicylic acid-, ethylene-, jasmornic acid- and abscisic acid-mediated pathways were influenced in the SlRLK1-silencing tomato plants, especially Proteinase inhibitor ΙΙ (Pin2) and Lipoxygenase (LoxD) were significantly down-regulated, both of which are involved in jasmornic acid signaling. These results suggest an important role of SlRLK1 in tomato defense response against P. parasitica, but its regulation mechanism awaits further investigation.