褐根病菌 Phellinus noxius 候選效應蛋白 cerato-platanins 之探討

Abstract

褐根病菌 (Phellinus noxius) 是危害樹木根莖部的重要壞死性病原菌，其引起的樹木褐根病 (brown root rot disease) 近年來嚴重危害果樹、觀賞樹木、森林及都市林木，造成經濟損失並影響公共安全，而P. noxius的致病機制仍有諸多未知。本研究以基因組預測並比對轉錄組數據，篩選出 79 個候選效應蛋白 (effectors)，其中包含 26 個 hypothetical proteins、29 個碳水化合物活性酶 (carbohydrate-active enzymes, CAZymes)、4 個 cerato-platanins (CPs) 及 3 個疏水蛋白 (hydrophobins)。進一步針對 P. noxius 基因體中 5 個 cerato-platanins (PnCPs) 的核苷酸多樣性、親緣關係及蛋白質預測結構進行分析，發現其可能受純化選擇作用，具有 CPs 特徵序列與保守結構，並具有幾丁質四聚體結合潛力。檢測不同環境條件下 PnCPs 之基因表現，發現 PnCP1 及 PnCP4 在馬鈴薯葡萄醣瓊脂培養基 (potato dextrose agar, PDA) 上菌絲生長旺盛時表現量較高，但液態完全培養基 (liquid complete medium, LCM) 下僅 PnCP4 有較高表現量。感染毛果楊 (Populus trichocarpa) 離體莖段時，PnCP1於 1 dpi (day post-infection) 之後表現量即顯著降低直至 4 dpi，可能與避免誘導植物啟動免疫反應有關；而 PnCP4 與 PnCP5 在 2 dpi 表現量顯著上調，其中 PnCP4 上調 14 倍以上，顯示兩者可能與 P. noxius 毒力有相關性。利用農桿菌浸潤法初步檢測5個 PnCPs 及其他 10 個候選效應蛋白基因之功能，在圓葉菸草 (Nicotiana benthamiana) 葉片短暫表達後皆未能誘導細胞死亡，推測這些候選效應蛋白可能有寄主或組織特異性，或具有誘導細胞死亡之外的生物活性。本研究為 P. noxius 效應蛋白的功能性研究建立重要的基礎資料，可作為未來功能性驗證與致病機制研究之參考。

Phellinus noxius, the causal of brown root rot disease, is a significant necrotrophic pathogen that damages the roots and stems of trees. In recent years, brown root rot disease has severely affected fruit, ornamental, forest, and urban trees, causing economic losses and posing risks to public safety. However, the pathogenic mechanisms of P. noxius remain largely unknown. This study utilized genome predictions and transcriptomic data to identify 79 candidate effectors, including 26 hypothetical proteins, 29 carbohydrate-active enzymes (CAZymes), four cerato-platanins (CPs), and three hydrophobins. Further analysis of five P. noxius cerato-platanins (PnCPs) focused on the nucleotide diversity, phylogenetic relationships, and predicted protein structures. PnCPs are likely under purifying selection. PnCPs possess CPs signature sequences and conserved structures, and have potential chitin tetramer-binding ability. Gene expression analysis under different conditions showed that PnCP1 and PnCP4 had higher expression levels during vigorous mycelial growth on potato dextrose agar (PDA), while only PnCP4 exhibited high expression in liquid complete medium (LCM). When infecting the detached stem segment of Populus trichocarpa, PnCP1 expression was significantly down-regulated after 1 dpi (day post-infection) and remained low until 4 dpi, potentially associated with avoiding the triggering of plant immune response. In contrast, PnCP4 and PnCP5 expression were significantly up-regulated at 2 dpi, with PnCP4 increasing by over 14-fold, suggesting their potential role in virulence. Preliminary functional tests using agroinfiltration-based transient expression in Nicotiana benthamiana leaves showed that five PnCPs and another 10 candidate effector genes did not induce cell death. These candidate effectors may be host- or tissue-specific, or have biological activities other than inducing cell death. This study provides crucial foundational data for functional studies of P. noxius effectors and offers valuable references for future research on their pathogenic mechanisms.