植物青枯病菌在胞外多醣體、生物膜、游動性或阿拉伯芥致病性有差異表現的突變株之特性研究

Characterization of Ralstonia solanacearum mutants with altered phenotypes on exopolysaccharide production, motility, biofilm formation or Arabidopsis pathogenesis

黃培城(Pei-Cheng Huang)

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(2008年)

https://doi.org/10.6342/NTU.2008.02091

摘要

青枯病菌(R. solanacearum)能在許多重要的作物上引發致死的萎凋病徵。此病菌具備了許多致病機制，例如移動能力(motility, MO，包括swimming及twitching)、生物膜的產生 (biofilm formation, BF)、胞外多醣的產生(exopolysaccharide, EPS)以及避開或抑制寄主植物的抵抗反應等。本研究的整體目標是針對青枯病菌參與毒力及適應性相關候選基因之突變株進行特性分析。在第一部分中，實驗室前人篩選出一群因跳躍子插入而導致缺乏或降低移動能力、產生生物膜及分泌胞外多醣的突變株，本研究發現其同源突變株(allelic mutant)也具有相同的外表型改變，故確認跳躍子的插入確實是造成外表型改變的原因。針對兩個與BF及MO有關的新穎基因突變株特性研究結果顯示：RSc0727與BF及twitching motility有關，而RSp0199則與swimming motility能力有關。進一步對此群突變株交互詰問之後初步發現：(1) EPS是產生BF所必需的因子，但與swimming motility的關係尚未有定論；(2) BF似乎與 swimming motility無直接的相關性，但是twitching motility對於BF卻是十分重要。在第二部份的研究中，在先前實驗室系統性篩選跳躍子插入突變株時，發現兩個對番茄具有正常毒力但對阿拉伯芥毒力卻下降的突變株，分別是RSc1206::Tn5及18A7。此兩突變株的同源突變株也具有相同的外表型改變，本研究也發現此兩株突變株在菸草及番茄中的繁殖能力與野生型無顯著性差異，然而在阿拉伯芥中的增殖則有顯著性的下降，此結果與其在番茄與阿拉伯芥中具有不同毒力應有密切的相關性。進一步互補實驗更證實RSc1206基因與細胞膜或外套蛋白的完整性相關。綜合目前所得結果推測RSc1206基因對青枯病菌在適應不同寄主環境當中是必須的，而其病原性的下降可能是由於對營養源利用的改變、逆境耐受性、生物膜的形成、swimming motility能力的下降。另一突變株18A7具有與野生型幾乎相同的生長外表型，即便是處理了逆境的藥劑後仍然不變，然而swimming motility能力的下降可能與18A7病原性的降低有關，但是否有其他參與在18A7對阿拉伯芥毒力下降的因子仍尚未被確認。

關鍵字

青枯病菌 ； 胞外多醣 ； 生物膜 ； 移動能力 ； 寄主適應性

並列摘要

Ralstonia solanacearum causes a deadly wilting disease on a wide range of crops. To achieve a successful infection, R. solanacearum is well equipped with many pathogenesis machineries, including swimming and twitching motility (MO), biofilm formation (BF), production of exopolysaccharides (EPS) and avoidance or suppression of host defenses. The general objective of this study is to characterize mutants of candidate genes involved in R. solanacearum virulence and fitness. Firstly, a group of transposon (Tn5)-insertional R. solanacearum mutants defective in EPS, MO or BF were characterized. Allelic mutants of these mutants displayed consistent phenotypic changes, confirming that the transposon insertions are responsible for the altered phenotypes. Studies on two novel genes related to BF and MO revealed that RSc0727 is related to BF and twitching motility, and that RSp0199 is related to swimming motility. Further cross-examination of a range of mutants suggested that (1) EPS production is required for BF, but its role in swimming and twitching motility is inconclusive; (2) BF and swimming motility seem to be independent of each other, but twitching motility is important to BF. Secondly, previous systematic screens of Tn5-insertional R. solanacearum mutants led to the identification of two mutants displaying normal pathogenesis on tomato but decreased pathogenesis on Arabidopsis. Allelic mutants exhibited similar in vitro characteristics and differential pathogenesis on tomato and Arabidopsis, verifying the transposon insertions are responsible for the altered phenotypes. Multiplication of these mutants in Nicotiana benthamiana and tomato was not significantly different from that of the wild-type strains. However, the significantly decreased mutants’ colonization in Arabidopsis was found to be correlated with differential pathogenesis on tomato and Arabidopsis. Complementation tests further evidenced RSc1206 is essential for cell membrane/envelope integrity. Results gathered from this study suggested that RSc1206 is required for adaptation of this bacterium in different host environments, and the decreased pathogenesis might be due to the alterations in nutrient utilization, reduced stress tolerance, biofilm formation and swimming motility. On the other hand, the mutant 18A7 displayed mostly normal phenotypes under normal and stress conditions, except for its reduced swimming motility which might be related to the decreased pathogenesis on Arabidopsis. However, whether other factors and how they may contribute to the decreased pathogenesis of 18A7 on Arabidopsis still remains to be determined in the future.

並列關鍵字

Ralstonia ； exopolysaccharide ； biofilm ； motility ； host adaptation

參考文獻

Arlat, M., Gough, C.L., Zischek, C., Barberis, P.A., Trigalet, A., and Boucher, C.A. (1992). Transcriptional organization and expression of the large hrp gene cluster of Pseudomonas solanacearum. Mol Plant Microbe Interact 5, 187-193.

Blair, D.F. (1995). How bacteria sense and swim. Annu Rev Microbiol 49, 489-522.

Brown, D.G., and Allen, C. (2004). Ralstonia solanacearum genes induced during growth in tomato: an inside view of bacterial wilt. Mol Microbiol 53, 1641-1660.

Brown, D.G., Swanson, J.K., and Allen, C. (2007). Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence. Appl Environ Microbiol 73, 2777-2786.

Brumbley, S.M., Carney, B.F., and Denny, T.P. (1993). Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator. J Bacteriol 175, 5477-5487.