生物資源暨農學院: 植物病理與微生物學研究所指導教授: 陳昭瑩劉芳瑋Liu, Fang-WeiFang-WeiLiu2017-03-032018-06-292017-03-032018-06-292016http://ntur.lib.ntu.edu.tw//handle/246246/274970LsGRP1為植物第二型富含甘胺酸防禦相關蛋白，存在於百合葉片之表皮細胞及維管束組織，且位於植物細胞之表面，在百合葉片之含量會因百合灰黴病菌Botrytis elliptica感染而改變。已知在生體外處理高濃度N端區段 (LsGRP1N) 可輕微促進B. elliptica的孢子發芽，但卻抑制或不影響其他受試真菌發芽及細菌生長 (Lin et al., 2014)，且LsGRP1對B. elliptica之抗菌效果較LsGRP1C為佳 (潘，2015)，故推測LsGRP1N可能有助於LsGRP1C對B. elliptica之殺菌作用，於本研究擬解析LsGRP1N對B. elliptica的影響，以增進對LsGRP1抗菌功能之瞭解。研究發現LsGRP1N能促使B. elliptica孢子提早發芽及促進菌絲生長，當以SUMO-LsGRP1N融合蛋白注射於百合葉片後接種B. elliptica，可增加B. elliptica族群量與病徵嚴重度；由於LsGRP1對百合葉片不具毒殺作用，SUMO-LsGRP1N引起較為嚴重之病徵可能因其導致B. elliptica菌量增加所致。再者，胺基酸組成與LsGRP1N相同但排序不同之等長胜肽 (LsGRP1N identical amino acid composition, LsGRP1N-iaac) 對B. elliptica孢子發芽與菌絲生長的促進效果較LsGRP1N為低，因此排除LsGRP1N單純作為B. elliptica營養源的可能性。由於B. elliptica孢子在發芽階段較未發芽孢子對LsGRP1C更為敏感，且LsGRP1N與LsGRP1C共處理較單獨處理LsGRP1C之抑制B. elliptica孢子發芽的IC50為低，推測百合葉片上之LsGRP1可藉其N端區段促使B. elliptica孢子提早發芽及活躍生長而對C端毒殺區更為敏感，故能有效減少侵染百合葉片之B. elliptica菌量，抑制病斑之發展。LsGRP1 is a plant class II glycine-rich defense-related protein located in the cell surface of epidermis and vascular tissue of lily leaves. The LsGRP1 content appears fluctuated during Botrytis elliptica infection. Previous study demonstrated that in vitro treatment with high concentration of the N-terminal region of LsGRP1 (LsGRP1N) slightly enhanced the spore germination of B. elliptica but inhibited or did not alter the spore germination of other tested pathogenic fungi and bacterial growth (Lin et al., 2014). Moreover, LsGRP1 exhibited greater inhibitory activity on B. elliptica than LsGRP1C did (Pan, 2015), suggesting that LsGRP1N may facilitate the anti-B. elliptica effect of LsGRP1C. Thus, in this study, the effect of LsGRP1N on B. elliptica was dissected to improve the knowledge of antimicrobial activity of LsGRP1. In this research, in vitro assay showed that LsGRP1N conducted earlier spore germination and enhanced early hyphal growth of B. elliptica. When SUMO-LsGRP1N fusion protein was infiltrated into lily leaves before inoculation with B. elliptica, higher fungal biomass and more severe host cell death occurred as compared with the control treatment of SUMO partner of fusion protein. Further assay revealed that LsGRP1 did not conduct plant cell death, the enhanced growth of B. elliptica triggered by SUMO-LsGRP1N was likely a cause of higher extent of plant cell death. In addition, in vitro treatment of equal-length peptide with identical amino acid composition of LsGRP1N (LsGRP1N-iaac) did not affect spore germination and early hyphal growth of B. elliptica, the possibility of LsGRP1N simply as a nutrient supplement was excluded. On the other hand, spores of B. elliptica in germinating stage were more sensitive to LsGRP1C as compared with that of un-germinated ones, and a combination of LsGRP1N and LsGRP1C conducted stronger inhibitory effect than the sole treatment of LsGRP1C did. Thus, the N-terminal region of LsGRP1 on cell surface of lily leaf triggering earlier spore germination of B. elliptica and conducting germinating spores vulnerable to LsGRP1C, which then capable of reducing the population of B. elliptica and diminishing symptom development were persumed.論文使用權限: 不同意授權LsGRP1LsGRP1NLsGRP1C百合灰黴病菌B. ellipticathesis10.6342/NTU201603114