百合灰黴病菌BeSerp參與致病及植物防禦之功能探討

Abstract

百合灰黴病菌 (Botrytis elliptica) 為百合屬 (Lilium) 植物的專一性死體營養型病原真菌，在低溫高濕環境下感染百合組織，造成葉片焦枯、花苞畸形、花瓣枯萎等病徵，嚴重影響百合切花及種球的產量與品質。東方型葵百合 (Lilium ‘Stargazer’) 防禦蛋白LsGRP1之N端區段 (LsGRP1N)，在體外試驗標定於百合灰黴病菌之菌絲表面，推測LsGRP1N與百合灰黴病菌蛋白具有交互作用，並經免疫共沉澱法獲得百合灰黴病菌推定類枯草桿菌蛋白酶 (putative subtilisin-like proteases (subtilases), BeSerp)。本研究以雙分子螢光互補法 (bimolecular fluorescence complementation) 觀察到LsGRP1與BeSerp會在菸草細胞表面產生交互作用；以RT-qPCR分析百合灰黴病菌感染感病品種亞洲型百合Lilium ‘Tresor’之BeSerp表現時間圖譜，於感染後24小時，BeSerp表現量即提高5.5倍隨後微幅下降，並於接種後72小時增加表現量至20倍。續以噴灑誘導基因靜默 (spray-induced gene silencing) 方式降低百合灰黴病菌之BeSerp表現量，罹病面積有下降的趨勢。然而以農桿菌浸潤法於Lilium ‘Tresor’上預表現BeSerp基因三天，卻可降低百合灰黴病之罹病面積，故推測BeSerp可能具有毒力與激發子角色。進一步以大腸桿菌系統表現BeSepΔSS融合蛋白並自包涵體進行純化，所得BeSepΔSS融合蛋白在脫脂奶粉培養基分析及蛋白酶螢光檢測法均未測得酵素活性。為了解BeSerp是否經由直接或間接殺滅百合細胞以促進感染，將BeSepΔSS融合蛋白單獨或輔以能夠毒殺百合細胞的百合灰黴病菌外泌液浸潤於百合葉圓盤組織中，經由偵測細胞電解質滲漏以量化百合細胞死亡程度，發現BeSerpΔSS融合蛋白不會造成百合細胞死亡，反而能抑制由百合灰黴病菌外泌液誘發的百合細胞死亡。而以BeSerpΔSS融合蛋白預處理百合葉片時，也可減少百合灰黴病菌接種後所造成的病斑面積，並能增加百合灰黴病菌外泌液所誘發的癒傷葡聚醣沉積量，說明BeSerpΔSS融合蛋白之激發子功能。當以BeSerp基因靜默之百合灰黴病菌孢子生產外泌液並浸潤至百合葉圓盤組織時，葉組織電解質滲漏程度降低，癒傷葡聚醣沉積量則減少。綜合上述結果得知，百合灰黴病菌BeSerp在毒力及寄主防禦誘發上扮演重要角色。

Botrytis elliptica is a Lilium-specific necrotrophic fungal pathogen that infects lily tissues under low temperature and high humidity environments, resulting in symptom development such as leaf blight, flower bud deformation and petal blight, which seriously affects the yield and quality of lily cut flowers and bulbs. In an in vitro assay, the N-terminal region (LsGRP1N) of the defense protein LsGRP1 from Lilium ‘Stargazer’ was marked on the mycelial surface of B. elliptica. LsGRP1N was proposed to interact with the protein of B. elliptica, which was identify to be a putative subtilisin-like proteases (subtilases), BeSerp, by immune-coprecipitation and mass spectrometry. In this study, the bimolecular fluorescence complementation assay showed that LsGRP1 and BeSerp colocalized to the surface of tobacco cells. RT-qPCR analysis showed the BeSerp expression pattern in B. elliptica-infected susceptible Asiatic lily cultivar ‘Tresor’ increasing 5.5-fold at 24 hours post inoculation, then slightly reduced, followed by an increase to 20-fold at 72 hours post inoculation. By spray-induced gene silencing (SIGS) assay, reduced BeSerp gene expression and lesion area caused by B. elliptica were observed. However, pre-expression of BeSerp gene in Lilium ‘Tresor’ by Agrobacterium tumefaciens-mediated plant transformation reduced the lesion area caused by B. elliptica. Thus, BeSerp might have dual functions as virulence factor and elicitor. To further investigate the function of BeSerp, the BeSepΔSS fusion protein was expressed in Escherichia coli system and purified from inclusion bodies. However, BeSepΔSS fusion protein did not show enzyme activity in skim milk agar assay and protease fluorescent detection method. To investigate whether BeSerp could promote B. elliptica infection via directly or indirectly killing lily cells, the BeSepΔSS fusion protein was infiltrated into lily leaf disc tissues alone or with B. elliptica secretion which could kill lily cells, and the cell electrolyte leakage was detected to quantify the level of lily cell death. The results showed that BeSerpΔSS fusion protein did not casue lily cell death but inhibit the cell death induced by B. elliptica secretion. Moreover, infiltration of BeSerpΔSS fusion protein in lily leaf disc tissues reduced lesion area caused by B. elliptica and enhanced callose deposition, indicating the elicitor function of BeSerpΔSS fusion protein. Further examination by infiltrating lily leaf disc tissues with the secretion of BeSerp gene-silenced B. elliptica spore culture showed that the reduction of BeSerp expression not only reduced the lethality of B. elliptica secretion but also decreased the callose deposition. In summary, BeSerp of B. elliptica plays important roles in the virulence and plant defense induction.