植物保護用菌種 Pseudomonas taiwanensis BCRC 17751 之功能分析

Functional Analysis of Biocontrol Bacterium Pseudomonas taiwanensis BCRC 17751

陳文仁(Wen-Jen Chen)

指導教授 ： 施明哲

共同指導教授 ： 劉?睿(Je-Ruei Liu)

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2016年)

https://doi.org/10.6342/NTU201610355

摘要

Pseudomonas taiwanensis 為台灣北部篩選出的新型假單胞菌屬的土壤菌，經實驗結果我們發現P. taiwanensis 對植物致病菌及昆蟲病害展現出強烈的對峙活性與致病性。在我的論文第一部分以全基因體定序為主。結合次世代定、Sanger 定序、全基因體限制酵素切位圖譜資訊，得到P. taiwanensis 基因體全長為 5.08 百萬鹼基對並含有 4665 編碼序列。根據全基因體的編碼資訊，展現出可能參與抗菌及昆蟲致病特性的相關基因。在論文第二部分，藉由P. taiwanensis 抗水稻白葉枯病病菌(Xoo) 的特性，我們找出抗性因子及它所調節的功能。結合基因體與Tn5 突變庫的資訊，得到了參與抗水稻白葉枯病菌相關基因。進而得知，螯鐵素(pyoverdine) 在抗菌上扮演了重要的角色。我們並發現螯鐵素會被 EnvZ/OmpR 雙分子系統(two-component system) 正向調節，相反的螯鐵素會被 Sigma factor RpoS 負向調節。有趣的是，第六型蛋白質分泌系統參與了螯鐵素的分泌。在論文的第三部分我們探討了P. taiwanensis 在昆蟲致病上的活性分析。我們使用十字花科病蟲害小菜蛾 (Plutella xylostella) 幼蟲當目標防治病害。我們探討 P. taiwanensis 的致病機制，發現其在自然的口腔餵食中如何在小菜蛾腸道中避免宿主的免疫反應攻擊進而形成菌落。 P. taiwanensis 在經由口腔進入小菜蛾腸胃道後，會引起腸道細胞嚴重的傷害且破壞腸道上皮組織的完整性。機制上，經實驗證明了昆蟲毒性蛋白C 元件 (C component of insecticidal toxin protein complex, TccC) 在致病上是一個很重要的元素。 TccC 蛋白參與抗氧化機制和抑制宿主細胞的吞噬作用，最後誘導宿主的腸道細胞產生細胞凋亡。綜合以上結果，我們得知了 P. taiwanensis 在抗植物致病菌及病蟲害上的機制。

關鍵字

假單胞菌屬 ； 水稻白葉枯病病菌 ； EnvZ/OmpR 雙分子系統 ； 螯鐵素 ； 第六型蛋白質分泌系統 ； 蟲毒性蛋白C 元件

並列摘要

A novel bacterial species, Pseudomonas taiwanensis, which was isolated from soil in northern Taiwan, was found to display strong antagonistic activity and pathogenicity against plant pathogens and insect pests. The first part of my thesis focuses on whole genome sequencing of P. taiwanensis. The complete sequence of the 5.08-Mb genome sequence and 4665 CDS were determined by a combination of Roche 454, Illumina Solexa, Sanger sequencing and Optical restriction-mapping. According to whole-genome sequence, genes encoding potential anti-microbial and entomopathogenic properties were revealed. The second part of my thesis focuses on the identification of the synthesis and regulation of toxin factors in P. taiwanensis that have antagonistic activity against rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We combined whole genome sequencing and Tn5-transponson mutagenesis to identify related genes that are involved in the anti-Xoo activity. Our results show that the siderophore pyoverdine has an important role in antagonistic activity against Xoo. Our results also show that synthesis of pyoverdine is positively and negatively regulated by EnvZ/OmpR two-component system and sigma factor RpoS, respectively. Interestingly, we found that Type VI secretion system (T6SS) is involoved in secretion of pyoverdine. The third part of my thesis focuses on entomopathogeic activity of P. taiwanensis. Here, we used insect larva of the diamond back moth (Plutella xylostella), a cruciferous crop pest, to study the pathogenic mechanism of infection by novel bacterium P. taiwanensis. We examined how P. taiwanensis colonizes and escapes the immune response in the gut of P. xylostella and the mechanism of pathogenesis after oral ingestion of P. taiwanensis. Oral ingestion of P. taiwanensis induced severe damage in intestinal cells of P. xylostella and disrupted intestinal epithelial integrity. A toxin protein, the C component of insecticidal toxin protein complex (TccC) contributed to the pathogenicity of P. taiwanensis by thwarting oxidative stress and phagocytosis and inducing apoptosis in the gut cells of the host. Taken together our results shed light on the bacterial toxicity processes in plant pathogens and insect pests, particularly the mechanism of competition and pathogenesis of P. taiwanensis.

並列關鍵字

Pseudomonas taiwanensis ； Xanthomonas oryzae pv. oryzae ； EnvZ/OmpR two-component system ； pyoverdine ； Type VI secretion system ； insecticidal toxin protein complex

參考文獻

1. South Pacific Commission., Food and Agriculture Organization of the United Nations. Implementation of the International code of conduct on the distribution and use of pesticides : report. s.n. (1989).

2. United Nations Environment Programme. World Health Organization. Public Health Impact of Pesticides Use in Agriculture (1990).

3. Pretty J. Agricultural sustainability: concepts, principles and evidence. Philosophical Transactions of the Royal Society London. Series B Biological Sciences 363, 447-465 (2008).

4. Donaldson D, Kiely, T, Grube, A. Pesticide's industry sales and usage 1998-1999 market estimates. US Environmental Protection Agency; Washington (DC): Report No EPA-733-R-02-OOI, (1999).

5. Zhang WJ, Jiang FB, Ou JF. Global pesticide consumption and pollution: with China as a focus. Proceedings of the International Academy of Ecology and Environmental Sciences 1, 125-144 (2011).