探討OPEL引發植物免疫反應之機制

Investigating the mechanism underlying OPEL-induced plant immune responses

賴沛宜(Pei-Yi Lai)

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(2022年)

https://doi.org/10.6342/NTU202200490

摘要

OPEL為疫病菌 (Phytophthora parasitica) 的外泌蛋白，序列預測顯示其具有signal peptide、thaumatin-like domain、glycine-rich domain及GH16 domain，GH16 domain包含β-1,3-glucanase活性區的保守性序列。以OPEL重組蛋白處理菸草 (Nicotiana tabacum cv. Samsun-NN) 葉片能引發細胞死亡、癒傷葡聚醣沉積、活性氧分子累積及誘導水楊酸和PTI相關防禦基因表現。對處理OPEL重組蛋白之菸草葉片所進行的轉錄體分析發現多個編碼receptor-like proteins (RLPs) 或receptor-like kinases (RLKs) 的差異表現基因 (FC ≥ 2, p-value < 0.01)，為瞭解其在OPEL誘發植物免疫的重要性，本研究挑選具研究潛力者，包括被高度誘導表現的NtLYM2、涉及多種醣分子辨識的adaptor RLKs 基因NtCERK1-like以及參與蛋白質辨識的NtSOBIR1和NtBAK1等；RT-qPCR分析顯示這些基因的表現都會因應OPEL之處理顯著提升。進一步以cucumber mosaic virus (CMV) 於N. tabacum cv. Samsun-NN誘導基因靜默，再分析其對OPEL誘發癒傷葡聚醣沉積和細胞離子滲漏的影響，發現靜默NtLYM2顯著減少OPEL介導癒傷葡聚醣累積的數量，靜默NtBAK1降低OPEL引發的細胞離子滲漏量，而靜默NtCERK-like或NtSOBIR1對二項觀測指標皆無顯著影響。此外，疫病菌感染誘導NtLYM2大量表現，而且暫表現NtLYM2顯著提升疫病菌在菸草的感染面積。這些研究顯示NtLYM2和NtBAK1在OPEL引發的植物免疫反應扮演重要角色，至於NtLYM2如何參與菸草和疫病菌的交互作用則待更多研究探討。

關鍵字

植物免疫反應 ； 菸草 ； 疫病菌 ； CMV誘導基因靜默

並列摘要

OPEL, an apoplastic effector from Phytophthora parasitica, contains signal peptide, thaumatin-like domain, glycine-rich domain, and glycoside hydrolase 16 (GH16) domain with β-1,3-glucanase active site consensus. Upon infiltration on the tobacco (Nicotiana tabacum cv. Samsun-NN) leaves, OPEL recombinant protein caused cell death, callose deposition, and reactive oxygen species (ROS) production, as well as induced the expression of PTI marker genes and salicylic acid-responsive genes. Previous transcriptome analysis identified a variety of differentially expressed genes (FC ≥ 2, p-value < 0.01) from tobacco leaves pretreated with OPEL, including those encoding receptor-like proteins (RLPs) or receptor-like kinases (RLKs). In this study, genes of interest were further investigated for their roles in OPEL-induced plant immune responses, including the highly induced NtLYM2 and genes encoding adaptor RLKs NtCERK1-like, NtSOBIR1, and NtBAK1. Their induced expression in response to OPEL treatment was verified by RT-qPCR. Down-regulation by employing cucumber mosaic virus (CMV)-mediated gene silencing on N. tabacum cv. Samsun-NN, followed by analyses of OPEL-induced callose deposition and cell ion leakage, indicated that NtLYM2 silencing compromised callose deposition and NtBAK1 silencing compromised cell ion leakage, whereas silencing of NtCERK1-like or NtSOBIR1 had no effect. Notably, NtLYM2 was up-regulated by P. parasitica infection as well. Nonetheless, when overexpressed in Nicotiana benthamiana, it enhanced P. parasitica infection. These results indicate the important roles of NtLYM2 and NtBAK1 in OPEL-induced plant immunity. How NtLYM2 is involved in tobacco-P. parasitica interaction awaits further investigation.

並列關鍵字

OPEL ； receptor-like proteins (RLPs) ； receptor-like kinases (RLKs) ； CMV-mediated gene silencing ； Nicotiana tabacum ； pattern-triggered immunity ； Phytophthora parasitica,

參考文獻

Albert, I., Böhm, H., Albert, M., Feiler, C. E., Imkampe, J., Wallmeroth, N., et al. 2015. An RLP23–SOBIR1–BAK1 complex mediates NLP-triggered immunity. Nat. Plants 1:1-9.

Appella, E., Weber, I. T., and Blasi, F. 1988. Structure and function of epidermal growth factor‐like regions in proteins. FEBS Lett. 231:1-4.

Aziz, A., Gauthier, A., Bézier, A., Poinssot, B., Joubert, J. M., Pugin, A., et al. 2007. Elicitor and resistance-inducing activities of β-1, 4-cellodextrins in grapevine, comparison with β-1, 3 glucans and α-1, 4-oligogalacturonides. J. Exp. Bot. 58:1463-1472.

Barghahn, S., Arnal, G., Jain, N., Petutschnig, E., Brumer, H., and Lipka, V. 2021. Mixed linkage β-1, 3/1, 4-glucan oligosaccharides induce defense responses in Hordeum vulgare and Arabidopsis thaliana. Front. Plant Sci. 12:1201.

Bartels, S., and Boller, T. 2015. Quo vadis, Pep? Plant elicitor peptides at the crossroads of immunity, stress, and development. J. Exp. Bot. 66:5183-5193.