植物青枯病菌 tol-pal 基因群及 RSc0727 在致病功能之分析

Functional study of Ralstonia solanacearum tol-pal gene cluster and RSc0727 in pathogenesis

謝季穎(Chi-Ying Hsieh)

指導教授 ： 鄭秋萍

國立臺灣大學/生命科學院/植物科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.01318

摘要

由 Ralstonia solanacearum 引起的青枯病為土壤傳播性萎凋病害，在全球造成眾多重要作物的嚴重損失，但目前其致病機制相關資訊仍十分有限，本研究針對青枯病菌 tol-pal 基因群與 RSc0727 在環境適應性與致病力之功能進行研究。本研究室先前已發現含有跳躍子之 tol-pal 突變株在茄科植物的致病力與野生型菌株雷同，但對阿拉伯芥卻幾乎無病原力，且其泳動力下降、細胞結構受損、碳源氧化能力改變。本研究進一步發現 tol-pal 突變株之第三型分泌蛋白系統與在番茄以外的茄科植物上之病原性正常，但藉由逆境及互補實驗證實青枯病菌 tol-pal 基因群為維持細胞完整性與抗逆境之之必需基因；綜合所有結果證實以上種種缺失造成 tol-pal 突變株在非寄主植物阿拉伯芥中之適應能力大減，以致於無法成功造成病害。此外，本研究發現 tol-pal 基因群可分為五個轉錄單位，並隨細菌生長時期調整其基因表現，且可受青枯病菌毒力調控中樞 PhcA 與逆境個別調控，顯示 tol-pal 基因群可藉由精細的分工調控以達成青枯病菌在不同環境與宿主之最佳適應性。另經由測試各式阿拉伯芥突變株對本土高毒力之青枯病菌 Pss190 品系的耐受性，發現茉莉酸/乙烯訊息傳遞途徑不利植物對抗此病菌，而水楊酸訊息傳遞途徑與植物防禦素 camalexin 則有利植物對抗此病菌。本研究第二部分針對青枯病菌 RSc0727 進行研究， RSc0727 與被推測參與第四型纖毛 (type IV pilus, Tfp) 合成所需的 pilV 基因係同源基因，故本研究將其命名為 pilV 。本研究室先前已發現 pilV 參與 twitching motility 及生物膜生成能力，本研究進一步利用量化分析證實 pilV 跳躍子突變株之生物膜形成能力降低且細胞通透性改變；此外，互補實驗也驗證 pilV 參與青枯病菌在番茄上的病原性，其缺失可能導致細菌系統性感染寄主植物的能力降低。 pilV 的表現會被 PhcA、水楊酸及茉莉酸抑制，推測 Tfp 在青枯病菌致病過程的功能表現可受 PhcA 及植物防禦賀爾蒙調控或干擾。本研究再度驗證精細與縝密的複雜調控系統與青枯病菌能夠成功地適應許多不同環境且具高致病力密不可分。

關鍵字

青枯病菌 ； tol-pal ； PhcA ； 第四型纖毛 ； pilV ； RSc0727

並列摘要

Ralstonia solanacearum is a soil-borne bacterium causing devastating wilting diseases in many economically important crops. Gaining insight into pathogenesis mechanism of this pathogen is thus important. The aim of this work was to study function of R. solanacearum tol-pal gene cluster and RSc0727 in bacterial fitness and pathogenesis. Previously, our group found that transposon (Tn) insertional mutants of tol-pal gene cluster conferred normal virulence on tomato plant, but significantly reduced virulence on Arabidopsis. In addition, these mutants displayed reduced motility, defective cell integrity and altered capability in carbons oxidation. This study, further revealed that tol-pal mutants had normal function in Type III secretion system and displayed normal virulence on additional solanaceous plants. However, stress response and complementation assays indicated that tol-pal gene cluster was essential for cell integrity maintenance and stress tolerance. All of the defects together contributed to the reduced fitness of the tol-pal mutants in the non-host plant Arabidopsis and thus led to severely reduced virulence. Furthermore, this study showed that R. solanacearum tol-pal gene cluster could be organized as five transcription units, which were differentially modulated throughout the growth phase, by the global virulence transcriptional regulalor PhcA, or by stress factors. The multifaceted modulation in tol-pal gene cluster may contribute to R. solanacearum fitness in various environments. By analyzing response of various Arabidopsis Col-0 mutants defective in hormone signaling pathways to infection of a local highly aggressive R. solanacearum Pss190, this study also reveals that the jasmonic acid (JA)/ethylene (ET) signaling pathways play a negative role in Arabidopsis tolerance , while salicylic acid (SA) and camalexin related pathways play positive roles. In the second part of this study, RSc0727, which was a putative orthologue of pilV gene which was suggested to be involved in Type IV pilus (Tfp) biogenesis, was studied. The organization of RSc0727-related gene cluster was mostly conserved among representative gram-negative bacteria alalyzed. Therefore, RSc0727 was designated as pilV. Our previous study showed that pilV is involved in twitching motility and biofilm formation. In this study, quantitative assays further confirmed the reduced biofilm formation and altered cell permeability of pilV mutant. In addition, pathogenesis and complementation assays further evidenced that pilV contributes to full spectrum of virulence. Furthermore, pilV was negatively regulated by PhcA, SA and JA, suggesting that the function of Tfp might be controlled or interfered by PhcA and plant defense mechanisms in the course of R. solanacearum infection. The results of this study together further demonstrate that differentially regulation and sophisticated coordination of various R. solanacearum pathogenesis mechanisms and the involved factors contribute greatly to versatile fitness of R. solanacearum in various abiotic and host environments, leading to its success in infecting a broad range of plant species in wide geographic distribution.

並列關鍵字

R. solanacearum ； tol-pal ； PhcA ； Type IV pilus ； RSc0727 ； pilV

參考文獻

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