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標題: Xanthomonas第三型致病性蛋白之細胞及生化功能分析
Cellular and biochemical characterization of Xanthomonas type III effectors
作者: 林鴻
Lin, Hong
關鍵字: Xanthomonas campestris pv. campestris;十字花科黑腐菌
出版社: 生物化學研究所
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Xanthomonas為革蘭氏陰性植物病原細菌,其寄主範圍廣泛,可以感染多種的重要經濟作物,也可以感染模式植物擬南芥。Xanthomonas致病力依賴於第三型分泌系統,會注射致病性蛋白分子進入植物細胞中,來幫助病原菌繁殖,然而絕大多數的致病性蛋白分子,其致病的分子機制仍然是未知。藉由基因轉殖實驗發現X. campestris pv. campestris的xopd8004轉基因植株,在誘導劑處理下會形成胚軸延長的白化苗,這種外表型跟光型態發育的缺失相似。目前XopD的相關研究主要侷限在X. campestris pv. vescatoria 85-10中。相較於XopD85-10的760個胺基酸,XopD8004只有442個胺基酸,但經由in vitro和in vivo的SUMO蛋白水解酶活性測試可知XopD8004和XopD85-10一樣能夠專一性辨識鍵結在受質蛋白上的
SUMO並進行水解。在in vitro pull-down分析中得知XopD8004會和植物光訊息傳導的轉錄因子HFR1有交互作用。而且在共軛焦顯微鏡的觀察下,發現XopD8004-YFP和HFR1-CFP一樣會在細胞核中聚集成小點,並且座落於相同位置。因此推測HFR1是XopD8004在植物細胞中的目標蛋白,而且可能參與植物的免疫反應。同時本實驗室也建立起ADP ribosyltransferases (ADP-RTs)的酵素活性鑑定法,並且偵測到P. syringae pv. tomato DC3000 HopU1的ADP-RTs活性。雖然X. axonopodis pv. citri 306的XopAI其蛋白C端被推測可能具有ADP-RTs的酵素活性,但實驗中並無法發現,推論XopAI306可能需要其他蛋白的幫助才能進行ADP-RTs的酵素反應。

Xanthomonas is a Gram-negative bacterial pathogens. It can infect not only a variety of important economic crops, but also the model plant Arabidopsis thaliana. Xanthomonas use the type III secretion system to deliver the type III effector proteins into the plant cells to help bacteria multiply. However, the functions of most effector proteins are still unknown. Here XVE:xopd8004 transgenic plants showed etiolated phenotypes with long hypocotyls and chlorophyll-less cotyledons. This phenomenon is the same with the deficiency of photomorphogenesis. So far, the related researches of XopD largely confined to that of X. campestris pv. vescatoria 85-10. The protein size of XopD85-10 is 760 amino acids. Although XopD8004 only has 442 amino acids,
Nevertheless XopD8004 can specifically recognize and hydrolyze the SUMO-modified proteins in vitro and in vivo. Using a pull-down assay, XopD8004 was found to interact with HFR1, a transcription factor in light signaling transduction pathway. Moreover, using confocal laser scanning, the fluorescent signal of XopD8004-YFP was observed in the nucleus as foci and co-localized with HFR1-CFP. As a result, we proposed that HFR1 is the cellular target protein of XopD8004 and may be involved in the plant immune response. Here, we also established a protocol to identify ADP ribosyltransferases (ADP-RTs) activity of type III effectors. In P. syringae, three type III effectors have been suggested to have ADP-RTs activities including HopO1-1, HopO1-2 and HopU1. According to the method, we have observed ADP-RTs activity of HopU1. Although X. axonopodis XopAI306 has been suggested to have similar domain as HopO1-1, no enzyme activity was observed using our method.
其他識別: U0005-2908201111531500
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