應用TurboID鄰近標記方法探討馬鈴薯Y病毒與寄主因子之交互作用

Abstract

植物病毒所編碼的蛋白常具有多功能性，能透過與寄主蛋白相互作用以促進病毒感染。馬鈴薯 Y 病毒 (potato virus Y, PVY) 同樣會利用自身編碼之病毒蛋白抑制植物免疫反應以達到感染之目的，是造成全球茄科作物產量損失的主要病毒之一。然而，目前 PVY 與寄主蛋白之間的相互作用仍然有許多未知，因此若能了解兩者間的相互作用關係，將有助於開發針對 PVY 的有效抗病毒策略。本研究藉由 TurboID 鄰近標定技術於圓葉菸草 (Nicotiana benthamiana) 中進行全面性分析，以探討與 PVY 各病毒蛋白具有相互作用之潛在寄主蛋白，TurboID 鄰近標定技術可於植物細胞中標定與目標蛋白之間具有強、弱或短暫相互作用的寄主蛋白。本研究首先將 PVY 各病毒蛋白分別構築於 TurboID 載體中，並於圓葉菸草上優化 TurboID 鄰近標定技術之條件。透過TurboID 鄰近標定技術將各病毒交互作用蛋白標定後，再以液相層析串聯質譜儀 (liquid chromatography–tandem mass spectrometry, LC-MS/MS) 鑑定標定之交互作用蛋白。本研究首先針對 PVY 的外鞘蛋白 (coat protein, CP) 及其交互作用蛋白進行探討，主要原因爲 potyvirus 的 CP 可能作為效應子 (effector) 抑制植物免疫反應。我們驗證 PVY CP 作為效應子之結果顯示，瞬時表達 PVY CP 於圓葉菸草會導致圓葉菸草中水楊酸 (salicylic acid, SA) 相關免疫基因與模式誘導免疫 (pattern-triggered immunity, PTI) 相關免疫基因之表現量下降，證實 PVY CP 作為效應子之可能性。而透過 TurboID 鄰近標定分析，本研究於候選蛋白中發現水楊酸生合成中的關鍵酵素苯丙胺酸裂解酶 (phenylalanine ammonia-lyase, PAL) 為 PVY CP 之潛在相互作用對象，故後續以螢光素酶互補試驗 (split-luciferase complementation assay) 與雙分子螢光互補試驗 (bimolecular fluorescence complementation, BiFC) 進一步驗證 PAL 確實能與 CP相互作用。最後，PAL 之基因功能性分析亦顯示，於圓葉菸草中瞬時靜默 PAL 之基因表現會導致 PVY 病毒累積量顯著上升，證實 PAL 在圓葉菸草對 PVY 的抗性中具有正向調控作用。本研究結果提供了有關 PVY 如何透過與寄主蛋白的交互作用來抑制植物免疫機制的重要觀點，為未來發展有效的抗病毒策略提供良好基礎。

Plant virus-encoded proteins perform multiple functions that facilitate infection by interacting with host proteins. Like many other viruses, potato virus Y (PVY), a member of the Potyvirus genus, also employs its various proteins to suppress plant immunity, causing significant damage to the plants in Solanaceae family worldwide. However, the mechanisms underlying the interactions between PVY and its host remain largely unknown. Addressing this knowledge gap is critical for developing effective antiviral strategies. In this study, the TurboID-based proximity labeling (PL) approach was performed to identify the interacting proteins of PVY in N. benthamiana. The TurboID-based PL approach enables the identification of interacting proteins exhibiting strong, weak, or transient interactions under native cellular conditions. Each PVY-encoding proteins were cloned into TurboID vector, and the procedures for performing TurboID-based PL in Nicotiana benthamiana was optimized. After performing TurboID-based PL, the putative PVY interacting proteins (PVY-IPs) were identified using liquid chromatography–tandem mass spectrometry (LC-MS/MS). To further investigate PVY–host interactions, this study first focused on the PVY coat protein (CP), as previous research has demonstrated that potyviral CPs can act as effectors that suppress plant immune responses. Our results showed that transient expression of PVY CP can decreased the expression of salicylic acid marker genes as well as pattern-triggered immunity maker genes supporting its role as an immune-suppressing effector. Therefore, identifying host proteins targeted by PVY CP is crucial to understanding how this viral effector suppresses plant immunity. Among the candidate host proteins identified through TurboID-based PL, phenylalanine ammonia-lyase (PAL) was found to interact with CP. PAL is a key enzyme in salicylic acid (SA) biosynthesis, which plays important role in SA-mediated antiviral immunity. The interaction of PAL with CP was further validated using split-luciferase complementation and bimolecular fluorescence complementation assays, confirming the interaction between PAL and CP in N. benthamiana. Furthermore, gene function analysis revealed that transient silencing of PAL led to a significant increase in PVY accumulation, indicating that PAL acts as a positive regulator against PVY. These findings offer valuable insights into how PVY suppresses plant immunity through interactions with host proteins. Bridging this knowledge gap is essential for the development of effective antiviral strategies.