百合受到灰黴病菌 (Botrytis elliptica) 感染後,由防禦相關基因 LsGRP1 所編碼之第二型富含甘胺酸蛋白表現量會提高;基於兩種植物第二型富含甘胺酸蛋白 (菸草之 cdiGRP、阿拉伯芥之 AtGRP-3) 會藉由蛋白–蛋白交互作用活化植物防禦反應,因此 LsGRP1 很可能利用類似的機制介導百合的防禦反應。本研究利用免疫共沉澱法得到數個 LsGRP1 可能的交互作用蛋白,再利用液相色譜串聯質譜儀鑑定其身分,結果顯示這些蛋白參與葉綠體電子傳遞鏈與卡爾文循環。由於這些蛋白在光合作用中扮演重要的角色,推測百合之光照情形會影響 LsGRP1 的表現或是功能。將百合葉片避光處理後,LsGRP1 的累積量會下降,且會在光照後得到補償。此外於百合原生質體中觀察 LsGRP1 之分布,顯示 LsGRP1 會累積在葉綠體,這些結果顯示 LsGRP1 介導的防禦反應與光合作用相關。此外,利用病毒基因靜默系統於百合植株上靜默 LsGRP1 與其假定交互作用蛋白 Rubisco small subunit (RbcS) 之基因,接種試驗顯示 LsGRP1 及 RbcS 基因靜默植株對灰黴病的抗性皆降低。接續測定正常百合與基因靜默植株,於灰黴病菌接種前後光合作用速率與葉綠體相關基因表現之變化,結果顯示灰黴病菌所導致的光合作用效率下降與氣孔關閉有關,而 LsGRP1 與 RbcS 會影響 Photosystem I reaction center subunit N 與 Photosystem II subunit O-2 之基因表現;由於此二基因編碼之蛋白在光合作用反應中扮演重要的角色,推測 LsGRP1 可能會與葉綠體蛋白交互作用共同調控百合的生理生化反應。本研究嘗試闡述 LsGRP1 與其他百合蛋白間的交互作用關係,以及此交互作用關係對於百合抗病性的影響,以促進對於植物第二型富含甘胺酸蛋白參與植物防禦反應機制之瞭解。
The defense-related gene LsGRP1 encoding a plant class II glycine-rich protein (GRP) exhibits an increased level of expression in Lilium spp. after being infected by Botrytis elliptica, the fungal pathogen of lily leaf blight. Referred to two characterized plant class II GRPs (i.e., tobacco cdiGRP and Arabidopsis AtGRP-3) which activate plant defense via protein-protein interactions, LsGRP1 is proposed to implement a similar mechanism. In this study, a number of putative LsGRP1-interacting lily proteins taking a part in electron transport chain and Calvin cycle were identified using co-immunoprecipitation and liquid chromatography-electrospray ionization-tandem mass spectrometry. Due to these putative LsGRP1-interacting proteins playing roles in photosynthesis process, light exposure was proposed affecting the expression and/or function of LsGRP1. Preliminary assay showed that dark treatment could decrease LsGRP1 content which re-built up after light exposure. Fluorescent microscopic examination indicated that LsGRP1 present in the chloroplasts of lily protoplasts. These findings revealed that LsGRP1-mediated defense in lily would be photosynthesis-related. While using virus-induced gene silencing (VIGS) to analyze the functions of LsGRP1 and putative LsGRP1-interacting protein, Rubisco small subunit (RbcS), silenced LsGRP1 and RbcS conducted a more susceptibility to B. elliptica. Changes of photosynthesis rate and chloroplast-related gene expressions before and after fungal inoculation were examined. The results showed that a decrease of photosynthesis rate caused by B. elliptica was related to stomatal closure, and LsGRP1 and RbcS involved in the gene expression of Photosystem I reaction center subunit N and Photosystem II subunit O-2 which playing importants roles in photosynthesis. Thus, LsGRP1 interacting with chloroplast proteins to regulate the physiological and biochemical responses of lily was proposed. This study will elaborate the knowledge on the interactions between LsGRP1 and other lily proteins, and the following effects on disease resistance to get better understanding on the defensive mechanism of plant class II GRPs.
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