PFLP之磷酸化為強化阿拉伯芥之PTI免疫反應所必需

Phosphorylation of plant ferredoxin-like protein is required for intensifying PAMP-triggered immunity in Arabidopsis thaliana

陳姿誼

Plant ferredoxin-like protein (PFLP) ； PAMPs-triggered immunity ； casein kinase II ； mitogen-activated protein kinase ； Plant ferredoxin-like protein (PFLP) ； PAMPs-triggered immunity ； casein kinase II ； mitogen-activated protein kinase

屏東科技大學植物醫學系所學位論文

2016年

碩士

林宜賢

繁體中文

植物病原菌小分子被植物細胞表面受器辨識後所觸發之免疫反應稱為PAMPs-triggered immunity (PTI)。已有多篇文獻證明PTI之訊號可被外源的plant ferredoxin-like protein (PFLP) 所提升，使多種植物產生抵抗細菌性病害之能力。目前亦已發現PFLP所含有之casein kinase II磷酸化區域為強化植物抗病性所必需，但其磷酸化反應之發生與增強抗病是否相關則仍未知。因此，本研究利用胺基酸點突變方式，將PFLP經單一胺基酸置換所獲得之無法磷酸化的PFLPT90A 與模擬磷酸化的PFLPT90D分別進行分析。結果顯示，無法磷酸化與模擬磷酸化之重組蛋白均無法提升HrpZ蛋白所誘導的過敏性反應及對Pectobacterium carotovorum subsp. carotovorum ECC17的抗病性。由此可推測PFLP蛋白在植物中的實際磷酸化可能為提升抗病所必需。為進一步了解PFLP磷酸化與植物細胞內casein kinase II (CK2) 是否相關，本研究分別利用阿拉伯芥CK2插入突變株cka2及恢復突變株CKA2R於flg22Pst存在下探討PFLP在過敏性反應、激活化氧的快速產生、癒傷葡聚醣的累積及軟腐病菌之罹病度上之影響。結果證明，CK2均為 PFLP 在上述抗病反應及對軟腐病菌之抗病性上所必需。為了解PFLP在強化PTI的過程中是否參與mitogen-activated protein kinase (MAPK) 之相關訊號傳遞路徑，以MAPK路徑上之相關基因FRK1與WRKY22/29之表現進行探討的結果顯示，PFLP可提升flg22Pst誘導之MAPK訊號傳遞路徑且需要CK2之參與。另一方面，本研究亦證明阿拉伯芥於flg22Pst誘導下，PFLP不論在野生型植株或cka2突變株中均能提升水楊酸及茉莉酸之抗病路徑。說明PFLP在此兩條路徑上之提升可能不需要CK2之參與。由此推測PFLP在提升對軟腐病之抗病性上，水楊酸及茉莉酸路徑可能不是主要的途徑。此外，本研究利用酵母菌雙融和系統分析顯示PFLP可與CK2發生蛋白質交互作用，且此交互作用之發生可能與PFLP的C端較為有關。綜合以上之結果可推測，PFLP在提升阿拉伯芥對細菌軟腐病之抗病機制可能主要是藉由提升PAMPs被植物細胞辨識後所啟動的MAPK訊號傳遞路徑，而此防禦路徑之強化需要PFLP在阿拉伯芥中被CK2磷酸化。

Defense responses triggered by pathogen-associated molecular patterns (PAMPs) recognition on plasma membrane is called PAMPs-triggered immunity (PTI). It has been demonstrated that PTI can be intensified by plant ferredoxin-like protein (PFLP) to improve plant disease resistance against bacterial pathogens. The improvement of this disease resistance requires the casein kinase II phosphorylation (CK2P) site of PFLP. However, whether the phosphorylation of CK2P site was required for intensifying disease resistance is still unknown. In this study, un-phosphorylated and mimic phosphorylated recombinant proteins were generated from PFLP. Result revealed that these proteins could not intensify PAMPs-triggered hypersensitive reaction (HR) and disease resistance against bacterial soft rot. It suggested that the actual phosphorylation of PFLP in plant may be necessary to enhance disease resistance. To understand whether PFLP phosphorylated is related to the extracellular casein kinase II (CK2) in Arabidopsis thaliana, the cka2 mutant and its recovery mutant CKA2R was used for further analysis. Results demonstrated that the PFLP-intensified PTI responses and disease resistance were abolished in cka2 mutant and restored in CKA2R mutant. To gain more insight on CK2 is required for PFLP-intensified PTI signaling, expression of marker genes in mitogen-activated protein kinase (MAPK) was assayed. Results exhibited that the CK2 was required for PFLP to enhance expression on flg22Pst-induced FRK1 and WRKY22/29 in MAPK pathway. Moreover, we provided evidences that PFLP was interacted with CK2 at C-terminal by yeast two-hybrid assay. Therefore, we concluded that extracellular casein kinase II was required for PFLP to activate MAPK pathway in PTI for further enhancing disease resistance against bacterial soft rot in Arabidopsis thaliana.

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