探討疫病菌質外體效應蛋白OPEL如何引發植物免疫反應

Abstract

OPEL為Phytophthora parasitica所分泌的質外體效應蛋白 (apoplastic effector)，序列分析顯示其具有signal peptide, thaumatin-like domain, glycine-rich domain及glycosyl hydrolase (GH) 16 domain，後者包含β-1,3-glucanase的保守性序列。以OPEL重組蛋白處理Nicotiana tabacum cv. Samsun-NN可引發明顯之壞疽斑 (necrosis)、癒傷葡聚醣 (callose) 沉積、活性氧分子 (reactive oxygen species, ROS) 累積及誘導防禦相關基因表現。此外，OPEL引發植物免疫反應的關鍵構造為GH16 domain，將其預測的酵素活性位點進行單點突變後，即大幅降低OPEL激發植物防禦反應的能力，因此OPEL很可能藉由分解植物細胞壁產生DAMP而引發免疫反應。為探討這個可能性，本研究製備OPEL重組蛋白及其酵素活性區雙點突變蛋白 (OPEL-dm)，用以處理菸草後，收集質外體液 (分別稱為AF-OPEL及AF-OPEL-dm)，防禦反應分析結果顯示AF-OPEL可引發菸草細胞壞疽、誘導防禦相關基因表現及增加對P. parasitica的抗性，但AF-OPEL-dm僅在菸草葉片引發微弱黃化。以95 oC加熱15分鐘後，AF-OPEL仍具有引發菸草細胞壞疽的活性；除此之外，AF-OPEL也較AF-OPEL-dm含有較多的還原糖。為了進一步找出AF-OPEL內引發植物免疫反應的活性物質，先後以正己烷及乙酸乙酯萃取，將AF-OPEL的成分分成低極性、中極性以及高極性等三個部份，續以the luminol-based chemiluminescence分析方法檢測各極性層萃取物引發活性氧分子累積的活性。結果顯示AF-OPEL的低極性層 (AF-OPEL/H)、中極性層 (AF-OPEL/EA) 及高極性層 (AF-OPEL-A) 萃取物均具有誘導活性氧分子累積的能力，但AF-OPEL/EA展現的活性明顯高於其他二者。進一步以高效液相層析法分析，發現三個AF-OPEL/EA特有的波峰，分別稱為peak I, peak II與peak III，其中僅peak II具有誘導活性氧分子累積的能力。這些結果顯示OPEL引發植物免疫反應之機制很可能是藉由辨識DAMPs而不是PAMPs且與其酵素活性密切相關。未來研究目標將著重於找出peak II所代表之物質，以瞭解其對於OPEL及植物間的交互作用。

OPEL is an elicitor protein identified in Phytophthora parasitica, which contains signal peptide in the N-terminus, followed by a thaumatin-like domain, a glycine rich domain and a GH16 domain. GH16 domain is characterized by the presence of a conserved active site for beta-1,3-glucanase. It has been shown previously that infiltration of OPEL recombinant protein into leaves of Nicotiana tabacum cv. Samsun-NN resulted in necrosis, callose deposition, ROS production as well as the induction of defense gene expression. Moreover, domain analysis indicated the elicitor activity of OPEL depends solely on the GH16 domain. Interestingly, the elicitor activity is compromised with OPEL recombinant protein harboring a single-point mutation in the predicted active site. It suggests an essential role of the enzymatic activity of OPEL in inducing plant defense responses, which might involve the generation of damage/danger-associated molecular pattern in the apoplast. To investigate this possibility, we prepared OPEL recombinant protein and OPEL mutant (OPEL-dm) with mutations at two key residues in the putative active site of the GH16 domain, infiltrated them individually into N. tabacum leaves, and then collected apoplastic fluid from the treated leaves, named AF-OPEL and AF-OPEL-dm, respectively. When infiltrated into tobacco leaves, AF-OPEL caused necrosis as well as induced defense gene expression and resistance against P. parasitica. In contrast, the activity of AF-OPEL-dm was significantly compromised. After heat treatment at 95 oC for 15 mins, AF-OPEL still retained most of its necrosis-inducing activity. Moreover, AF-OPEL contained more reducing sugars than AF-OPEL-dm. To identify the PTI-inducing substances, AF-OPEL was extracted by using n-hexane (H) and then ethyl acetate (EA) as the solvent, resulting in the collection of three fractions: AF-OPEL/H (low polar), AF-OPEL/EA (medium polar), and AF-OPEL/A (high polar). All three fractions induced ROS production as shown by the luminol-based chemiluminescence assay. However, the activity of AF-OPEL/EA was much higher than the other two.Further purification by high performance liquid chromatography (HPLC) identified three peaks (known as peak I, peak II, and peak III), which were found only in AF-OPEL/EA but not in the control experiment.Notably, only peak II induced ROS production. Based on these results, the mechanism for OPEL to induce PTI is through recognition of DAMP rather than PAMP and related to its putative site. In the future, we hope to identify the peak II-represented substance to understand the interaction between OPEL and plants.