效應蛋白RSp0213與PopP3在青枯病菌致病力之功能研究

Roles of Effectors RSp0213 and PopP3 in Virulence of Ralstonia solanacearum

鞠泰翔(Tai-Hsiang Chu)

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(2014年)

https://doi.org/10.6342/NTU.2014.00199

摘要

青枯病菌（Ralstonia solanacearum）引發全球許多作物之致死萎凋病害，常造成全球作物巨大經濟損失。隸屬於phylotype I的青枯病菌菌株可根據在抗感病品系番茄上引起的致死率，進一步定義其毒力高低，但目前關於決定青枯病菌致病力之分子機制所知極少。為瞭解青枯病菌之致病力因子，我們已完整定序四個不同毒力的青枯病菌株，發現效應蛋白（effector）RSp0213只存在中毒力菌株（如Pss4）與低毒力菌株（如Pss216）中，而高毒力菌株（如Pss190）則無此effector。本研究之研究目標為探討RSp0213在青枯病菌致病力及與寄主交互作用中之角色。結果發現在具有RSp0213的中毒力菌株Pss4中將RSp0213剔除會提高Pss4在番茄之致病力，而在無RSp0213的高毒力菌株Pss190中大量表現此effector基因則可降低Pss190在番茄之致病力，故RSp0213確實是決定青枯病菌致病力之關鍵因子。此外，在菸草葉片中表現RSp0213會造成細胞死亡的現象；在番茄中過量表現RSp0213亦導致細胞死亡與植株矮化，且會誘導水楊酸、乙烯及過敏性反應訊息傳導相關基因之表現，進而增強番茄對高毒力青枯病菌菌株Pss190之抗性。另外，序列分析預測與GFP融合蛋白檢測確認RSp0213座落在菸草細胞膜上，且此座落特性為執行其功能之必要條件。以上結果顯示，RSp0213可能被番茄細胞辨認並進而誘發防禦機制，導致含有此效應蛋白之青枯病菌菌株的致病力降低，而高毒力菌株則因無RSp0213而較不會誘發寄主之強烈抗病機制。 論文的第二部分由於源自蕃茄的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在青枯病菌致病力之功能與其相關機制。本研究室的先前研究證實此T3E會受T3SS調控，研究結果亦顯示基因轉移表現至中毒力菌株Pss4可增強其在番茄上的致病力，而將PopP3表現於菸草葉片無病徵產生但有利於青枯病菌之生長，並推測其在植物粒線體的座落位置與其功能有關，可能藉此影響番茄PTI指標之一的癒傷葡聚糖累積 (callose deposition)，以抑制植物PTI反應。本研究旨在進一步證實PopP3粒線體座落位置對其功能之重要性，並檢測此T3E在基因轉移表現下，青枯病菌在番茄上的致病力表現，和其功能與HR之相關性，以闡明PopP3如何抑制植物的相關防禦反應機制。

關鍵字

青枯病菌 ； 效應蛋白 ； RSp0213 ； PopP3 ； 致病力 ； effector-triggered immunity ； effector-triggered susceptibility

並列摘要

Ralstonia solanacearum, the causal bacterium of bacteria wilt (BW), causes severe production losses on many important crops worldwide. 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. To uncover virulence-related factors, genomes of four R. solanacearum phylotype I strains with different virulence were sequenced. A putative effector protein RSp0213 is present in medium-virulence and low-virulence strain such as Pss4 and Pss216, but absent in high-virulnece strain such as Pss190. This study aimed to elucidate roles of RSp0213 in R. solanacearum virulence and plant-bacterium interaction. Diruption of RSp0213 in medium-virulence strain Pss4 resulted in increased bacterial virulence in tomato, while RSp0213 overexpression in high-virulence strain Pss190 leads to decreased bacterial virulence, evidencing a decisive role of RSp0213 in R. solanacearum virulence. Additionally, transient local overexpression of RSp0213 in Nicotiana benthamiana leaves led to cell death. Virus-mediated overexpression of RSp0213 in tomato also caused cell death, reduced plant growth, increased expression of marker genes involved in defense hormones and hypersensitive-response signaling pathways, and enhanced resistance to the high-virulence strain Pss190. Furthermore, GFP-tagged RSp0213 colocalizes with cell membrane of N. benthamiana and membrane localization is required for its functions. These results suggest that RSp0213 can be recognized by host cell, leading to induced defense response and thus reduced virulence of RSp0213-containing R. solanacearum strains. The absence of this effector enables high-virulence strains escape from host recognition, leading to the high virulence. The second part of this thesis aimed to uncover a YopJ-type effector protein Pseudomonas outer protein P 3 (PopP3) unique to a high-virulence strain Pss190. Our previous study showed that overexpression of popP3 in Rs medium-virulence strain Pss4 led to increased bacterial virulence in tomato. Transient overexpression of popP3 in tomato suppresses pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) related callose deposition. popP3 transcripts was positively regulated by the type III secretion system (T3SS) regulator HrpG. Furthemore, This study proved the mitochondria localization of PopP3 in plant cell is critical to it function on tomato. These results together suggest that PopP3 is a T3SS effector and contributes to the virulence of R. solanacearum by suppressing plant defense response which related to mitochondrial functions.

並列關鍵字

Ralstonia solanacearum ； effector ； RSp0213 ； PopP3 ； virulence ； effector-triggered immunity ； effector-triggered susceptibility

參考文獻

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