疫病菌外泌蛋白OPEL在植物反應所扮演的角色

The role of OPEL, a secretory protein of Phytophthora parasitica, involved in induction of plant immunity

張怡萱(Yi-Hsuan Chang)

指導教授 ： 劉瑞芬

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10966

摘要

疫病菌屬 (Phytophthora) 中有許多種類 (species) 皆屬於重要經濟作物的病原菌，往往造成農業重大損失。Phytophthora與植物交互作用過程所涉及的分子機制非常複雜，近年來的研究發現Phytophthora含有可引發植物免疫反應的分子如pathogen-associated molecular patterns (PAMPs) 及avirulence effectors等，這些分子被植物辨識到後所引發之植物免疫反應分別為PAMP-triggered immunity (PTI) 以及effector-triggered immunity (ETI)，但仍有許多putative effectors的功能尚待研究釐清，本研究的目的即是探討一未知功能的疫病菌 (Phytophthora parasitica)外泌性蛋白OPEL的功能與特性。在OPEL N端具有一段訊號肽，並有三個保守性區域分別為thaumatin-like domain (Thau)、glycine-rich domain (GR) 及glycosyl hydrolase domain (GH) 。OPEL為卵菌特有之基因，其同源性序列僅在Phytophthora spp.、Albugo、Hyaloperonospora arabidopsidis及Pythium被發現。即時定量聚合酶鏈鎖反應 (real-time quantitative reverse transcriptase-PCR) 分析顯示，OPEL在疫病菌不同生長階段皆有表現，但在感染植物後表現量顯著提升。為了瞭解其在疫病菌與植物交互作用所扮演的角色，我們利用E. coli表現OPEL重組蛋白以進行後續分析，結果顯示將OPEL注射入菸草葉片會引發癒傷葡聚醣堆積 (callose deposition)、產生活性氧分子 (reactive oxygen species, ROS) 以及誘導水楊酸 (salicylic acid, SA) 和PTI防禦反應相關基因表現。除此之外，經由OPEL處理之菸草可增加系統葉對Tobacco mosaic virus (TMV) 之抗性。而為了進一步了解是OPEL中哪個功能性區域可引發植物抗病反應，我們同樣利用E. coli表現Thau、GR、GH及GR+GH重組蛋白，研究結果顯示GH及GR+GH與OPEL結果相似，皆可引發植物累積癒傷葡聚醣及產生H2O2。綜合上述結果顯示OPEL具有既可引發植物基礎防禦反應PTI，也可誘導植物產生後天系統性抗性 (systemic acquired resistance, SAR) 之獨特性質，而glycosyl hydrolase domain則是引發抗病反應所必須。

關鍵字

OPEL ； PAMP-triggered immunity (PTI) ； Phytophthora parasitica ； Systemic acquired resistance (SAR)

並列摘要

The genus Phytophthora includes more than 100 species, with many of them notorious pathogens of economically important crops. Interaction between Phytophthora pathogens and plant hosts involves the exchange of complex molecular signals from both sides. Recent studies in Phytophthora have led to the identification of some pathogen-associated molecular patterns (PAMPs) as well as many avirulence effectors, which are known to elicit PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. However, there are still numerous putative effectors with unknown function. In this study, we report the functional characterization of OPEL, a secretory protein of Phytophthora parasitica, which contains a signal peptide in the N-terminus and three other conserved domains including a thaumatin-like domain (Thau), a glycine-rich domain (GR), and a glycosyl hydrolase domain (GH). Homologs of OPEL were identified only in Oomycetes, including Phytophthora spp., Albugo, Hyaloperonospora arabidopsidis, and Pythium but not fungi and other organisms. Analysis by quantitative reverse transcriptase-PCR indicated that OPEL is expressed throughout development of P. parasitica, but is especially highly induced following the infection on plant. To characterize its function, we obtained OPEL recombinant proteins from E. coli. Infiltration of the recombinant protein into leaves of tobacco (Nicotiana tabacum) resulted in callose deposition, production of reactive oxygen species (ROS), and induced expression of salicylic acid-responsive defense genes. Moreover, when the systemic leaves of the OPEL-treated tobacco plants were challenged with Tobacco mosaic virus (TMV), the number of local lesions caused by TMV infection was significantly reduced compared to the control plants. To identify conserved domain(s) which is involved in elicitation of plant immunity, we obtained recombinant proteins representing Thau, GR, GH, or GR+GH, respectively, from E. coli and analyze their activity. The results showed that GH and GR+GH could induce callose deposition and ROS production in a level similar to those induced by OPEL recombinant protein. These results demonstrate the unique characteristics of OPEL which can induce plant defense response and systemic resistance in tobacco. Moreover, the glycosyl hydrolase domain plays an essential role for OPEL to elicit plant immunity.

並列關鍵字

OPEL ； PAMP-triggered immunity (PTI) ； Phytophthora parasitica ； Systemic acquired resistance (SAR)

參考文獻

3. 顏豪志. 2001. 疫病菌分泌性蛋白質OPEL選殖與特性研究. 國立臺灣大學植物病理學硏究所碩士論文.

64. Vandenabeele, S., Van Der Kelen K., Dat, J., Gadjev, I., Boonefaes, T., Morsa, S., Rottiers, P., Slooten L., Van Montagu M., Zabeau, M., Inze D., and Van Breusegem F. 2003. A comprehensive analysis of hydrogen peroxide-induced gene expression in tobacco. Proceedings of the National Academy of Sciences of the United States of America 100: 16113-16118.

5. Bernard, H., and Gideon, D. 1997. Structural and sequence-based classification of glycoside hydrolases. Current Opinion in Structural Biology 7: 637-644.

6. Bisson, M. M., Bleckmann, A., Allekotte, S., and Groth, G. 2009. EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1. Biochemical Journal 424: 1-6.

7. Bonshtien, A., Lev, A., Gibly, A., Debbie, P., Avni, A., and Sessa, G. 2005. Molecular properties of the Xanthomonas AvrRxv effector and global transcriptional changes determined by its expression in resistant tomato plants. Molecular Plant-Microbe Interactions 18: 300-310.