百合灰黴病菌分泌蛋白BePME1為引發寄主程序性細胞死亡之毒力因子

Abstract

百合灰黴病菌Botrytis elliptica為死體營養型病原真菌，藉由外泌性蛋白質成分誘導植物組織發生程序性細胞死亡，並自死亡之寄主組織獲得養份，造成壞疽病徵，嚴重危害百合之栽培與生產，然導致此程序性細胞死亡的蛋白質身分與特性仍待鑑定與分析。實驗室前人透過電解質滲漏分析發現百合灰黴病菌之果膠甲基酯酶蛋白(pectin methyl esterase 1, BePME1) 會引起百合細胞死亡，並可透過外源添加促進病原菌的感染；本研究進一步闡明BePME1對百合灰黴病菌之毒力與引起寄主程序性細胞死亡的貢獻。首先建立百合灰黴病菌之聚乙二醇介導原生質體轉型法以建構BePME1剔除突變株，獲得兩個突變株bepme1-1及bepme1-2，所造成之病徵明顯地較野生型菌株弱，但外源添加BePME1蛋白可挽救之，證實BePME1為一毒力因子。以BePME1蛋白處理百合會引起PCD典型徵狀DNA片段化，處理程序性細胞死亡抑制物會抵消BePME1對百合細胞的致死效應，而BePME1突變株之外泌液引起百合細胞死亡及DNA片段化程度較野生型菌株為低，故認為BePME1為百合灰黴病菌引起寄主程序性細胞死亡之因子。已知百合防禦蛋白LsGRP1可抑制百合灰黴病菌外泌液引起之寄主細胞死亡，而BePME1在LsGRP1基因靜默的百合葉組織會造成更嚴重的寄主細胞死亡，說明BePME1是百合防禦系統的干擾目標。此外，BePME1也能引發阿拉伯芥葉組織的死亡，顯示BePME1的致死能力應無寄主選擇性。另一方面，以源自不同植物的果膠作為基質均無法測得BePME1的酵素活性。本研究首次證明百合灰黴病菌之分泌蛋白BePME1對病原菌毒力的貢獻以及誘發寄主程序性細胞死亡的功能。

Botrytis elliptica, a necrotrophic fungal pathogen causing lily gray mold disease, induces host programmed cell death (PCD) via secretory proteinaceous compounds to obtain nutrients from dead host cells, resulting in necrosis symptom and severely damaging lily cultivation and production. However, the host PCD-eliciting compounds of B. elliptica have not been identified and characterized. A secretory pectin methyl esterase protein of B. elliptica, named BePME1, had been previously demonstrated capable of causing lily leaf cell death and promoting B. elliptica infection via exogenous application. In this study, the contributions of BePME1 in the virulence and PCD-eliciting activity of B. elliptica were clarified. Firstly, two BePME1-deleted mutant lines, bepme1-1 and bepme1-2, were generated using a polyethylene glycol-mediated B. elliptica protoplast transformation procedure established in this study. The two mutant lines caused smaller necrotic lesions as compared to wild-type B. elliptica, but this phenomenon could be rescued by exogenous application of BePME1, revealing BePME1 is a virulence factor. BePME1 treatment triggered a PCD-specific characteristic DNA fragmentation of lily cells, which could be rescued by the application of PCD inhibitors. The secretions of BePME1-deleted mutant lines conducted weaker DNA fragmentations of the host plant. Accordingly, BePME1 is a host PCD inducer. Since plant defense protein LsGRP1 had been known to prevent lily cell death caused by B. elliptica secretion, more severe host cell death in LsGRP1-silenced lily caused by BePME1 indicated that BePME1 is the interfering target of lily defense system. Besides, BePME1 caused the cell death of Arabidopsis leaves, revealing the cell death-inducing ability of BePME1 is non-host selective. Besides, BePME1 did not show detectable enzymatic activity as assayed using pectin substrates from different plants. This study firstly proves that the secretory protein BePME1 contributes to the virulence and functions as a host PCD inducer of B. elliptica.