果膠甲基酯酶 BePME1 在百合灰黴病菌感染過程扮演的角色

百合灰黴病是百合重要病害之一，主要由Botrytis elliptica造成，可發生於全株地上部，產生黃褐色壞疽病斑，影響光合作用及花器觀賞價值，目前仍不清楚B. elliptica的致病因子。前人研究指出B. elliptica在孢子萌芽時會產生酯酶，並且分泌蛋白，造成百合細胞死亡；本研究以真菌外泌性蛋白為研究對象，期了解B. elliptica毒力因子及其於灰黴病菌致病過程中之角色。本研究發現相較於僅除去菌體之B. elliptica培養液及經聚偏氟乙烯膜(polyvinylidene difluoride)過濾之濾液，混合纖維膜(mixed cellulose ester)之濾液明顯減少對百合細胞的致死能力。以質譜分析差異性蛋白質條帶，找到與灰黴病菌B. cinerea之BcPME1序列高度相似的外泌蛋白(BePME1)，預測其帶有一訊息胜肽及果膠甲基酯酶酵素活性區段。百合灰黴病菌感染72小時內，在中度抗病性百合‘Star Gazer’上，接種B. elliptica後24小時，bepme1表現量上升，並在接種後60小時有最高相對表現量；在高度感受性百合‘Tresor’上，接種B. elliptica後12小時，表現量上升並隨即下降，接種後36小時後的表現量再度上升，並在接種後48小時達到最高相對表現量，指出BePME1參與B. elliptica與寄主的早期交互作用以及B. elliptica感染後的病徵擴展。以dsRNA進行bepme1基因靜默，反轉錄-即時定量聚合酶連鎖反應偵測較少的bepme1表現，百合灰黴病菌感染導致的病斑擴展速率較慢，並且延遲植體上的菌體增加量；以高濃度菌量接種感病寄主時，bepme1基因靜默則不會影響灰黴病病徵發展。以Pichia pastoris生產移除訊息胜肽的BePME1 (BePME1ΔS)未能檢測出具果膠甲基酯酶活性；然將BePME1ΔS導入百合葉盤，則可使百合細胞內離子外漏，以Evans blue染色後亦可檢測出死亡的百合細胞。將B. elliptica接種於處理1 µM BePME1ΔS或以農桿菌浸潤法短暫表現bepme1之百合葉盤，均可造成較大的病斑。本研究結果指出BePME1可做為毒力因子，引起百合細胞死亡，有助於B. elliptica侵染寄主後的病徵發展。

關鍵字

Botrytis elliptica ；果膠甲基酯酶；毒力因子；基因靜默；農桿菌介導轉形；百合細胞死亡

並列摘要

Gray mold disease, caused by Botrytis elliptica, as one of the severe diseases on lily causes brown necrotic spots on above-ground part of the plant, influencing photosynthesis and reducing flower value. It is still unclear what virulence facters B. elliptica owns. As known, B. elliptica produces esterase while spores germinate and secretes protein(s) which inducing lily cell death. This study targeted the secreted proteins and aimed to figure out the role of B. elliptica virulence factor during the infection of lily. Firstly, the lily cell-lethal ability of the culture filtrate of B. elliptica further filtrated through polyvinylidene difluoride membrane and mixed cellulose ester membrane were compared and showed that the former had higher lethal effect. Selected protein bands were analyzed by LC-MS/MS. Among them, a secreted protein, named BePME1, was found highly similar to BcPME1 of B. cinerea, which was predicted presence of a signal peptide and a pectin methylesterase catalytic domain. RT-qPCR detected higher bepme1 expression in medium resistant lily ‘Star Gazer’ at 24 hours post inoculation (hpi) and attained highest relative expression level at 60 hpi while in highly susceptible lily ‘Tresor’, an increase of bepme1 expression was showed at 12 hpi, then dropped the expression and rose later at 36 hpi, getting the highest relative expression level at 48 hpi, implying BePME1 is involved in the early interaction between B. elliptica and lily, and symptom development. Bepme1 silencing via dsRNA-mediated interference showed reduced symptom development and delayed the increase of fungal mass in planta, indicating bepme1 affects the host infection of B. elliptica. However, symptom development was not affected as challenge with fungal inoculum of high spore concentration on susceptible lily cultivar. Signal peptide-deleted BePME1 (BePME1ΔS) produced in Pichia pastoris system did not show pectin methylesterase activity. However, infiltrating BePME1ΔS into lily leaf discs caused electrolyte leakage and plant cell death as detected by Evans blue staining. Lesions caused by B. elliptica infection on lily leaf discs infiltrated with BePME1ΔS and lily leaves post Agrobacterium-mediated bepme1 transient expression appeared larger. These results indicate that BePME1 is a virulence factor, causing lily cell death and facilitating symptom development caused by B. elliptica.