效應蛋白PopP3 在青枯病菌毒力之功能研究與青枯病菌噬菌體裂解蛋白之分析

Role of Effector PopP3 in Virulence of Ralstonia solanacearum and Characterization of Phage Lysis Proteins

吳紅葳(Hung-Wei Wu)

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(2013年)

https://doi.org/10.6342/NTU.2013.10883

摘要

由青枯病菌 (Ralstonia solanacearum, Rs) 所引發的青枯病為重要且極複雜之土壤傳播性細菌性植物病害，其寄主範圍甚廣，可造成全球嚴重作物經濟損失。屬於phylotype I的青枯病菌菌株在番茄上呈現明顯的毒力變異性，但其相關的分子機制訊息極少。源自蕃茄的Pss190菌株具由極高毒力，根據其基因體序列顯示 Pss190特有效應蛋白Pseudomonas outer protein P 3 (PopP3)，故本研究旨在探討PopP3在青枯病菌毒力之功能與其相關機制。結果發現在原本缺少PopP3的中毒力青枯病菌菌株Pss4中大量表現PopP3可提高其在番茄之毒力；在菸草中短暫表現PopP3也可使Pss4菌量提高；同時，在番茄中過量表現PopP3則會壓制植物與pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) 相關的癒傷葡聚醣之累積 (callose deposition)，也使番茄對軟腐病菌Pectobacterium carotovorum subsp. carotovorum (Pcc) 更感病。另外，啟動子分析試驗顯示popP3受到第三型分泌蛋白系統HrpG所管控，PopP3-GFP重組蛋白座落在菸草原生質體之粒線體上。以上結果顯示PopP3是會壓制植物免疫反應以提高青枯病菌的毒力之第三型分泌蛋白系統之效應蛋白，且可能干擾植物粒線體功能。 論文的第二部分針對青枯病菌噬菌體與其相關蛋白進行研究，發現可裂解中毒力青枯病菌Pss4之T7-like噬菌體RsφP29的基因組有功能未知且可能與寄主交互作用相關的蛋白。RsφP29的裂解蛋白中的L蛋白之GST重組蛋白在in vitro外加試驗下不具有裂解青枯病菌的效果，而H蛋白則無法在大腸桿菌表現，推測H蛋白可能具有裂解細菌的能力。此外，短暫表現H蛋白之菸草會造成物細胞死亡，此現象在植物感染不同病原細菌或其第三型分泌系統壞損之突變菌株後更加明顯，而全株短暫表現H蛋白之番茄對Rs與Pcc之抗性則會提升，依據目前結果推測H 蛋白對植物細胞產生的傷害可能引發類似damage-associated molecular patterns (DAMPs)-triggered immunity (DTI) 反應。此外，我們也進一步分離出本土可裂解高毒力青枯病菌的九類特性不同的噬菌體，其中噬菌體Rsφ34具有最廣的寄主範圍，而噬菌體 Rsφ4則具有最優異之殺菌效果，同時也發現高毒力青枯病菌較抗這些噬菌體。 本論文所得之重要資訊與基礎，應有助於未來對青枯病菌毒力關鍵決定因子、青枯病菌與植物/噬菌體之交互作用及病害防治更深入的研究。

關鍵字

青枯病菌 ； 效應蛋白 ； PopP3 ； 毒力 ； 裂解蛋白 ； DAMPs-triggered immunity

並列摘要

Ralstonia solanacearum (Rs), the causal agent of bacteria wilt (BW), has an unusual wide host range, including many important crops. R. solanacearum phylotype I strains confer varied degrees of virulence/aggressiveness on tomato plants; however, genetic information on pathogen virulence-related factors is still very limited. Our comparative genome sequence analysis identified a YopJ-type effector protein Pseudomonas outer protein P 3 (popP3) unique to a high-virulence strain Pss190. The first part of this thesis aimed to study function and property of PopP3 in Rs virulence. The results showed that overexpression of PopP3 in Rs medium-virulence strain Pss4 led to increased bacterial virulence in tomato. Transient local overexpression of PopP3 in Nicotiana benthamiana caused enhanced propagation of Rs. Transient overexpression of PopP3 in tomato suppresses pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) related callose deposition and increases the susceptibility to the Pectobacterium carotovorum subsp. carotovorum (Pcc). popP3 transcription was positively regulated by the type III secretion system (T3SS) regulator HrpG. In addition, GFP-tagged PopP3 colocalized with the mitochondria of N. benthamiana protoplast. These results together suggest that PopP3 is a T3SS effector and contributes to the virulence of R. solanacearum by suppressing plant defense response which probably is related to mitochondrial functions. The second part of this thesis aimed to collect information on R. solanacearum phages and their lysis proteins. Genome annotation of a T7-like bacteriophage RsφP29 capable of lysing the medium-virulence Rs strain Pss4 revealed the existence of a group of novel proteins which may involve in host specificity. In addition, characterization of its lysis proteins showed that GST-L recombinant protein might not have in vitro anti-Rs activity and that the failure of expressing the H protein in E. coli might be due to its antibacterial activity. Interestingly, transient overexpression of RsφP29 H protein in tobacco caused cell death and this effect can be further promoted by the infection of different phytopathogenic bacteria or their T3SS-defective mutants. Furthermore, tomato plants systemically overexpressing H protein were more resistant to Rs and Pcc. These results enable us to propose an effect for RsφP29 H protein in eliciting damage-associated molecular patterns (DAMPs)-triggered immunity (DTI) in plant. In addition, nine groups of local phages with varied capability of lysing local and imported high-virulence Rs strains were isolated, and Rs strains with higher virulence generally were tolerant to these phages. The information collected from this thesis, regarding Rs virulence determinants, Rs-plant/phage interactions and related resources, would pave the way to further studies of these important aspects.

並列關鍵字

Ralstonia solanacearum ； effector ； PopP3 ； virulence ； lysis protein ； DAMPs-triggered immunity

參考文獻

Able, A.J., Guest, D.I., and Sutherland, M.W. (2000). Hydrogen peroxide yields during the incompatible interaction of tobacco suspension cells inoculated with Phytophthora nicotianae. Plant Physiology 124, 899-910.

Afzal, A.J., da Cunha, L., and Mackey, D. (2011). Separable fragments and membrane tethering of Arabidopsis RIN4 regulate its suppression of PAMP-triggered immunity. The Plant Cell 23, 3798-3811.

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. Molecular plant-microbe interactions 5, 187-193.

Bai, J., Choi, S.H., Ponciano, G., Leung, H., and Leach, J.E. (2000). Xanthomonas oryzae pv. oryzae avirulence genes contribute differently and specifically to pathogen aggressiveness. Molecular Plant-Microbe Interactions 13, 1322-1329.

Bernoux, M., Timmers, T., Jauneau, A., Briere, C., de Wit, P.J., Marco, Y., and Deslandes, L. (2008). RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector. The Plant Cell 20, 2252-2264.