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標題: 蘭花褐斑病菌HrpWAaca蛋白質之特性分析與Acidovorax spp.第三型分泌系統誘導培養基之改良
Characterization of HrpWAaca protein of Acidovorax avenae subsp. cattleyae OAC1 and improvement of hrp gene inducing medium for Acidovorax spp.
作者: 陳羿君
Chen, Yi-Jyun
關鍵字: HrpWAaca
Acidovorax avenae subsp. cattleyae
Type III secretion system
出版社: 生物科技學研究所
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摘要: 植物病原細菌 Acidovorax avenae subsp. cattleyae (Aaca) OAC1能在蘭花引起細菌性褐斑病 (Brown spot),而A. avenae subsp. citrulli (Aac) 則引起瓜類細菌性斑點病 (Bacterial fruit blotch disease, BFB), 此些病原菌藉由第三型分泌系統 (type three secretion system, TTSS),分泌致病相關蛋白質 (effector) 至植物細胞內,以干擾寄主正常的生理生化反應及防禦機制,進而在寄主植物引發病害,或引起非寄主植物產生過敏性反應 (Hypersensitivity response, HR)。在此研究以loss-of-function策略分析蘭花菌株AacaOAC1的hrpWAaca基因缺失菌株之生物特性,在菸草葉片誘發延遲之過敏性反應,但在寄主蝴蝶蘭和文心蘭所呈現的病原性與野生菌株無明顯差異,顯示HrpWAaca似乎在病原性上並非為必要之蛋白質。經由序列比對發現hrpWAaca序列中含有17.58%的甘胺酸 (glycine)而不含半胱胺酸 (cysteine),且以E. coli BL21 (λDE3)大量表現HrpWAaca-His6融合蛋白之粗萃取溶液及經由Ni-NTA親合性層析管柱純化之HrpWAaca-His6融合蛋白,無論加熱與否皆可於菸草葉片上引發過敏性反應,顯示HrpWAaca-His6蛋白N端harpin domain有生物活性且具熱穩定性。又以HrpWAac156的抗體進行西方雜合分析,結果顯示HrpWAaca經XVM2培養液誘導表現後可被分泌至菌體外,將上述HrpWAaca分析結果與已知harpin蛋白特性相比較,可將HrpWAaca視為一個harpin-like蛋白。HrpWAaca蛋白於C端之Pel domain經PGA果膠分解酵素活性測定方法,證實其具有果膠分解酵素活性。此外以gel filtraction分析顯示,HrpWAaca-His6能以雙聚體 (120 kDa,dimer)、六聚體 (400 kDa,hexamer)和多聚體 (>2000 kDa, oligomer)存在。而以HrpWAaca-His6蛋白溶液施用於番茄植株四天後,可使植株對番茄細菌性斑點病較具有抗病性。此外,以gfp為報導基因尋找合適Aac第三型分泌系統之誘導因子 (induction factor),發現以XVM2為基礎加入西瓜細胞萃取液及絲瓜水之培養基,可以有效誘導第三型分泌系統相關基因hrcC promoter之表現。
Acidovorax avenae subsp. cattleyae (Aaca) OAC1 causes a brown spot disease on Cattleyae, while A. a. subsp. citrulli (Aac) causes Bacterial fruit blotch disease (BFB) on cucurbitaceae. These bacteria causing diseases in host plants or eliciting hypersensitive response (HR) on nonhost plants depend on type III secretion system (T3SS), encoded by hrp/hrc gene cluster. In this study, we analyzed the biological function of hrpWAaca of Aaca by loss-of-function strategy. The hrpWAaca mutant elicited a delayed HR on Nicotiana tabacum, but still maintained the same virulence as its wild type on its host Oncidium and Phalaenopsis. Hence, HrpWAaca might be not play an essential role in pathogenicity. The purified or crude extracted HrpWAaca-His6 proteins could elicit HR with or without heat treatment. Moreover, HrpWAaca-His6 protein contains 17.58% glycine but no cysteine, and HrpWAaca could be secreted into bacterial milieu under T3SS inducing condition. Therefore, HrpWAaca is a harpin-like protein. The Pel domain in C-terminus of HrpWAaca has the pectate lyase activity based on the pel assay with polyglacturonic acid as a substrate. Besides, Gel filtration assay showed the purified HrpWAaca-His6 could be detected as dimer (120 kDa)、hexamer (400 kDa) and oligomer (>2000 kDa) in size. To control the tomato bacterial spot caused by Xanthomonas vesicatoria, HrpWAaca-His6 solution was sprayed onto tomato leaves 1 or 4 days before inoculated with X. vesicatoria. The result showed that HrpWAaca-His6 could reduce bacterial spot severity after 4 days harpin treatment. To improve the T3SS induction medium for Aac, extracts of watermelon suspension cell and loofah were total using gfp as a reporter gene under the control of hrcC promoter. The results indicated that both extracts significantly improved induction efficiency of the XVM2 medium.
其他識別: U0005-1808201018301400
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