探討天麻在與蜜環菌共生關係中的細胞壁變化

Abstract

天麻 (Gastrodia elata) 是一種完全真菌異營性的蘭科植物，其塊莖發育完全依賴與一種木材腐朽菌-蜜環菌 (Armillaria spp.) 的共生關係來獲取養分。在共生培養系統中，我們觀察到蜜環菌感染的塊莖基部細胞結構發生了顯著變化，細胞分化為三種不同的類型，包括感染細胞、大細胞和內部皮層細胞。其中，菌絲侵入主要局限於大細胞，而大細胞會明顯膨大，並在細胞壁上出現不均勻的加厚現象。因此，我們致力於探索天麻與蜜環菌共生過程中細胞壁成分的變化，特別關注果膠修飾相關的蛋白質、伸展蛋白 (extensin) 及阿拉伯半乳聚糖蛋白 (AGPs) 。我們使用定量即時聚合酶鏈反應 (quantitative real time polymerase chain reaction, qPCR) 分析與雷射切割 (laser microdissection, LMD) 技術來收集不同的細胞類型，以測定基因相對表達量；使用綜合微陣列聚合物分析 (comprehensive microarray polymer profiling, CoMPP) 來分析細胞壁抗原決定位；並透過免疫膠粒金標誌技術比較塊莖共生前後不同細胞層中的抗原決定位分布。研究結果顯示，果膠甲基酯酶 (pectin methylesterase, PME) 表現於共生後下調，顯示同型半乳糖醛酸聚糖 (homogalacturonan, HG) 為高度甲基酯化的狀態。JIM5和JIM7在大細胞的細胞壁上表現出密集的膠粒金標誌，而 JIM20 在由宿主形成的共生界面上顯示出密集的膠粒金標誌，並且在CoMPP分析中感染後的塊莖有強烈的JIM20訊號。根據基因表達量的變化及細胞壁成分的累積位置，推測細胞壁保護植物避免受蜜環菌病害入侵的同時，也在共生界面處進行與真菌的營養與訊息交流。這種共生關係在天麻和蜜環菌之間形成了一種「亦敵亦友」的狀態，其相關的分子調控機制仍有許多未解之謎值得探索，深入研究感染蜜環菌前後的細胞壁組成變化將有助於更全面地了解它們的共生關係。

Gastrodia elata, a fully myco-heterotrophic orchid, relies entirely on a symbiotic relationship with a wood-decaying fungus, Armillaria for its nutrient supply during tuber development. In the symbiotic culture system, notable changes were observed in the cell differentiation of the basal mycorrhizal tuber. Upon fungal infection, cells differentiate into three distinct types: the infected cell, the large boundary cell, and the inner cortical cell. Fungal invasion is restricted by the large boundary cell. Simultaneously, the large boundary cells enlarge significantly, with some uneven thickening on its wall. Thus, this study explores the cell wall remodeling during symbiosis with Armillaria, focusing on proteins related to pectin modification, extensin, and arabinogalactan proteins (AGPs). We used quantitative real time polymerase chain reaction (qPCR) analysis and laser microdissection (LMD) to dissect and capture different cell layers to determine gene expression levels in specific cell types, comprehensive microarray polymer profiling (CoMPP) to analyze cell wall epitopes, and immunogold localization to detect the distribution of epitopes across different cell layers before and after infection. Our findings reveal that pectin methylesterase (PME) expression was downregulated after symbiosis, indicating that homogalacturonan (HG) was highly methyl esterified. Abundant labeling of JIM5 and JIM7 was observed on the large boundary cell wall, while JIM20 showed abundant labeling on the interfacial matrix and a strong signal in the mycorrhizal tuber in CoMPP analysis. Based on the changes in gene expression levels and the specific accumulation of these cell wall components, it is proposed that the cell wall serves as a barrier to protect against Armillaria invasion while simultaneously regulating nutrient transport and signal exchange with the fungus at the interfacial matrix. The symbiotic relationship between G. elata and Armillaria forms a "frenemy" situation, with the molecular regulatory mechanisms still holding many mysteries to explore. Understanding cell wall remodeling will provide deeper insights into their symbiotic relationship.