- 學位論文
LsGRP1C對十字花科炭疽病之抑病功能及抑菌機制探討
Exploration of disease suppression function of LsGRP1C on crucifer anthracnose and its antifungal mechanism
摘要
植物為固著生物體，無法移動躲避病原菌的侵襲，因此常藉由產生具有抗菌活性的生化產物，達到阻止病害發生的效果。LsGRP1(Lilium ‘Stargazer’ glycine-rich protein 1) 為葵百合上一富含甘胺酸的防禦相關蛋白，其C端的38個胺基酸 (LsGRP1C) 組成富含半胱胺酸區段。十字花科炭疽病菌Colletotrichum higginsianum為能夠造成十字花科作物炭疽病的半活物寄生型病原真菌。利用人工合成LsGRP1C進行生體外抑菌試驗確定LsGRP1C對C. higginsianum的孢子發芽、壓器形成與菌絲生長都具有良好的抑制作用。免疫螢光染色的結果顯示LsGRP1C主要結合於C. higginsianum菌絲表面，利用SYTOX Green染色分析證明，LsGRP1C會造成C. higginsianum細胞膜透化，添加陽離子則會抑制LsGRP1C所導致的細胞膜透化。這些結果顯示LsGRP1C可能藉其帶淨正電之特性破壞C. higginsianum細胞膜之完整性，表現抑菌之活性。另一方面，DAPI、H2DCFDA與TUNEL染色分析指出，LsGRP1C會誘發C. higginsianum產生細胞質皺縮、胞內活性氧物質累積、染色質凝集、細胞核DNA斷裂等類細胞凋亡之計畫性細胞死亡現象。進一步在阿拉伯芥上以農桿菌注入法短暫表現LsGRP1、LsGRP1ΔNΔG、LsGRP1ΔC及LsGRP1C，接種C. higginsianum的結果發現，表現LsGRP1、LsGRP1ΔNΔG或LsGRP1C均顯著抑制病徵之發展及產孢量。染色及顯微觀察指出，阿拉伯芥上表現LsGRP1、LsGRP1ΔNΔG或LsGRP1C均可抑制C. higginsianum次生菌絲之形成，因此推測植體上表現LsGRP1、LsGRP1ΔNΔG及LsGRP1C可能導致C. higginsianum初生菌絲之細胞死亡。
並列摘要
Plants are sessile organisms and unable to avoid pathogen attack by moving ability. Plants may protect themselves by generating biochemical products with antimicrobial activity. LsGRP1 is a glycine-rich defense-related protein of Lilium cv. Stargazer, with a C-terminal portion (LsGRP1C) comprised of 38 amino acids rich in cysteine residue. Colletotrichum higginsianum causing crucifer anthracnose is a hemibiotrophic fungal pathogen. In vitro assays with the synthetic peptide of LsGRP1C showed inhibition of spore germination, appressorium formation and mycelial growth of C. higginsianum. Immunofluorescence staining showed that LsGRP1C bound to the outer layer of C. higginsianum mycelia. SYTOX Green staining assay demonstrated that LsGRP1C caused membrane permeabilization of C. higginsianum and its suppression by cations. Accordingly, disruption of fungal membrane integrity by LsGRP1C due to its net positive charge was presumed. On the other hand, DAPI, H2DCFDA and TUNEL staining assays showed that LsGRP1C caused apoptosis-like programmed cell death phenomena in C. higginsianum, including cytoplasm shrinkage, intracellular accumulation of reactive oxygen species, chromatin condensation and nuclear DNA cleavage etc. Transient expression of LsGRP1, LsGRP1ΔNΔG, LsGRP1ΔC or LsGRP1C in the leaves of Arabidopsis thaliana followed by inoculation with C. higginsianum demonstrated that expression of LsGRP1, LsGRP1ΔNΔG, and LsGRP1C could suppress symptom development and sporulation, and the secondary hyphae were not present as indicated by staining and microscopic examinations. Thus, the primary hyphal cell death of C. higginsianum caused by the expression of LsGRP1, LsGRP1ΔNΔG and LsGRP1C in planta was presumed.