青枯病菌效應蛋白 RipBJ 誘發植物防禦反應之機制研究

青枯病菌 (Ralstonia solanacearum, Rs) 之寄主範圍廣泛，常造成全球大規模農作物經濟損失，然而目前關於青枯病菌致病力 (virulence) 之分子機制瞭解仍有限。本研究室先前發現 Rs 效應蛋白基因 ripBJ 僅存於低/中致病力菌株；在中致病力菌株Pss4中將 ripBJ 剔除會提高 Pss4 之致病力，而在原本無 ripBJ 的高致病力菌株Pss190中大量表現 ripBJ 基因則可降低 Pss190 之致病力，故 RipBJ 之存在會導致 Rs 致病力下降；另外，在植物大量表現 ripBJ 後會造成細胞凋亡，並且提升植物對 Rs 之抗性。本研究旨在探討 RipBJ 誘發植物防禦反應之作用機制。研究結果顯示在 Pss4 中將 ripBJ 剔除雖會提升病菌致病力但並未影響病菌在植物體內之增殖能力，且互補試驗證實 RipBJ 之存在確實會使 Rs 之致病力下降。另外，RipBJ 蛋白質會座落於菸草細胞膜上；在番茄表現 RipBJ 會造成 H2O2 大量累積，並誘導水楊酸、乙烯及抗病過敏性反應代表性防禦基因之表現，且提升番茄對多種特性迥異之重要病菌的防禦力。此外，RipBJ 與植物細胞膜上之 NADPH oxidase SlWfi1 (SlRbohB) 有直接交互作用且提升 SlWfi1 (SlRbohB) 之轉錄表現，而 RipBJ 所誘導之H2O2 大量累積反應則需依賴 SlWfi1 (SlRbohB) 與其菸草同源基因 NbRbohB 之正常轉錄表現與作用。為進一步探討 RipBJ 是否有其他作用途徑，透過共免疫沈澱與奈流液相層析串連二次質譜 (Co-IP-nanoUPLC-MS/MS) 之技術，已篩選出其他 RipBJ 之可能交互作用蛋白質，需待後續驗證。本研究所得之資訊應有助於未來對青枯病菌致病力、植物與青枯病菌交互作用及病害防治之深入研究。

關鍵字

青枯病菌；效應蛋白； RipBJ ；致病力；細胞死亡；過氧化氫； NADPH oxidase

並列摘要

Bacteria wilt (BW) caused by Ralstonia solanacearum (Rs) causes global severe crop losses. However, mechanisms about how virulence-related factors function are still limited. Our previous studies showed that Rs effector gene ripBJ is unique to low/medium virulence strains and it has a crucial negative role in virulence. In addition, ripBJ expression in tomato led to cell death and enhanced tolerance to Rs. This study aims to elucidate the mechanism underlying RipBJ-induced plant defense responses. The results showed that RipBJ deletion did not affect in planta Rs multiplication. The deletion mutant restored low virulence after complementation with a functional ripBJ, confirming that RipBJ is a key negative factor for Rs virulence. Furthermore, RipBJ may localize at plant cell membrane. RipBJ expression in tomato led to increased H2O2 accumulation and enhanced expression of marker genes involved in salicylic acid, ethylene and hypersensitive response, and promoted defense against multiple distinct pathogens with economic importance. In addition, RipBJ interacts with plasma-membrane-located NADPH oxidase SlWfi1 (SlRbohB). In planta ripBJ expression augmented SlWfi1 (SlRbohB) expression, while RipBJ-mediated H2O2 accumulation relied on SlWfi1 (SlRbohB) and its tobacco homolog NbRbohB. Furthermore, Co-IP-nanoUPLC-MS/MS was used to search for additional candidate targets of RipBJ’s effects on tomato. Further validation is required to confirm the interaction between RipBJ and the identified candidate proteins. The information collected from this study would pave the way to further studies on Rs virulence mechanisms, Rs-plant interactions and disease control.

並列關鍵字

Ralstonia solanacearum ； effector ； RipBJ ； virulence ； cell death ； H2O2 ； NADPH oxidase

參考文獻

李千惠。2009。番茄青枯病菌毒力相關因子之研究。國立台灣大學植物科學研究所碩士論文。

鞠泰翔。2014。效應蛋白 RSp0213 與 PopP3 在青枯病菌致病力之功能研究。國立台灣大學植物科學研究所碩士論文。

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Asai, S., and Shirasu, K. (2015). Plant cells under siege: plant immune system versus pathogen effectors. Curr Opin Plant Biol 28, 1-8.

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青枯病菌效應蛋白 RipBJ 誘發植物防禦反應之機制研究

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