植物青枯病菌致病基因RSc0411及murI之功能性研究

Functional studies of Ralstonia solanacearum novel pathogenicity determinants RSc0411 and murI

楊文潔(Wen-Chien Yang)

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(2009年)

https://doi.org/10.6342/NTU.2009.03287

摘要

由Ralstonia solanacearum所引起的青枯病 (又稱為細菌性萎凋病) 係一全球重要之土壤傳播性維管束病害，但目前我們對於這個極為複雜且破壞力極強的病菌的了解仍是十分有限。先前的研究已經篩選到致病力缺失之青枯病菌突變株群，在其中兩個突變株中，跳躍子插入的基因分別為RSc0411與RSc1956，本論文之研究目標為深入探討其之確切功能。首先，LptC係一廣泛存在細菌但功能未知的DUF1239蛋白家族之一員，目前研究推測在大腸桿菌中此蛋白可能參與脂多醣(lipoplysaccharides或LPS)之生合成與運送。RSc0411之預期蛋白產物係DUF1239成員。由目前研究之結果發現，青枯病菌RSc0411突變株之粗型脂多醣(R-LPS)合成有缺失，其致病能力嚴重喪失，且游動力、生物膜(biofilm)形成能力及對番茄根部的附著力皆有明顯下降，且無法誘導菸草產生過敏反應(hypersensitive response)，而進一步的結果也驗證RSc0411突變後造成青枯病菌第三型分泌系統無法被正常誘導。此外，雖然在許多革蘭氏陰性細菌中lptC相關的基因成員組合具保守性，啟動子分析結果顯示在青枯病菌中之實際操縱子組成(operon organization)與大腸桿菌並不同，且將其他革蘭氏陰性菌的DUF1239蛋白基因互補到RSc0411突變株後發現，只有親緣近序列相似度高的同源基因可以功能互補RSc0411之缺失，推測可能是因為與Ralstonia屬親緣相近之細菌的DUF1239蛋白質已經演化出具較類似之特別且新穎的功能，同時參與細菌R-LPS之生合成和第三型分泌系統。本論文第二部份的工作係研究RSc1956 (murI)之功能，其預期蛋白產物係glutamate racemase，負責將L-glutamate轉化為D-glutamate，而D-glutamate是組成細菌細胞壁肽聚醣的重要成分，可以保護細菌的細胞壁免受細胞的蛋白質酶攻擊。由目前之研究結果發現，murI突變雖然不會明顯影響生物膜形成、根部附著力和分解植物細胞壁酵素之分泌，但是會造成致病能力嚴重喪失，且游動力明顯下降，而互補試驗亦進一步證實此基因確實為青枯病之關鍵致病基因，且瞭解到其基因啟動子對於有效地表現此基因之功能十分重要。藉由這些分子層面的試驗與分析，本研究得以確認並深入探討新穎蛋白RSc0411與RSc1956確實是青枯病菌之關鍵致病基因，且對於其相關分子機制有所領悟；希望藉由深入且全面性地了解青枯病菌的致病機制、其主導基因、各訊息傳導途徑間之交互作用與調控，未來可以研擬破壞病菌感染的管道，得以研發有效控制此嚴重病害的可能策略。

關鍵字

青枯病菌 ； 脂多醣 ； DUF1239蛋白 ； LptC ； 第三型分泌系統 ； MurI

並列摘要

Ralstonia solanacearum is a soil-borne bacterium infecting vascular system, causing lethal wilting symptoms on many economically important crops and resulting severe crop losses. Previously, we carried out transposon (Tn5) insertional mutant screens to identify genes involved in pathogenesis of R. solanacearum. Two mutants containing a transposon insertion in RSc0411 or RSc1956 (murI) were found to be avirulent both on tomato and Arabidopsis. The aims of this study are to further elucidate roles of these genes in bacterial pathogenesis and the molecular mechanisms involved. Firstly, R. solanacearum RSc0411 is a homologue of E. coli lptC. LptC, a member of the novel protein family DUF1239, is suggested to be involved in E. coli lipopolysaccharides (LPS) transport into the outer membrane. Here we showed that RSc0411 mutant was defective in cell integrity and in rough LPS production. Notably, this mutant displayed defects in various pathogenesis-related properties and the induction of the type III secretion system was attenuated. The organization of DUF1239-related gene cluster is conserved among gram-negative bacteria, while sequence homology among orthologous genes in the cluster and those involved in LPS biogenesis varied according to phylogenetic relationships. Complementation tests revealed that only DUF1239 members in bacteria phylogenetically related to R. solanacearum were functional to rescue the mutant’s defects, further suggesting certain specificity in the RSc0411-involved pathogenesis mechanism and LPS biogenesis machineries may have evolved. Collectively these results imply a novel and crucial role of RSc0411 in early pathogenesis and LPS biogenesis of R. solanacearum. Secondly, R. solanacearum RSc1956 encodes glutamate racemase (MurI) protein. MurI catalyzes the conversion of L-glutamate to D-glutamate which is an essential component of peptidoglycans in bacterial cell walls. Here we showed that, although RSc1956 mutant behaved similar to the wild-type strain in biofilm formation, root attachment and production of plant cell wall degrading enzymes, it was defective in swimming motility and avirulent in tomato plants. Complementation tests further confirmed the key role of RSc1956 in R. solanacearum pathogenesis and revealed the importance of its promoter in precisely expressing MurI function. Taken together, these detailed studies on RSc0411 and RSc1956 are expected to pave the way not only for elucidating mechanisms and determinants involved in R. solanacearum pathogenesis but also potentially establishing useful disease control means.

並列關鍵字

Ralstonia solanacearum ； DUF1239 ； LptC ； lipopolysaccharide ； type III secretion system ； MurI

參考文獻

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