百合防禦蛋白LsGRP1抗灰黴病之關鍵區段探討

Abstract

百合灰黴病由Botrytis elliptica (Berk.) Cooke所引起，為台灣重要的病害之一。東方型雜交品種葵百合經澆灌水楊酸後，能誘導百合系統抗病性，得以抵抗灰黴病菌的感染，伴隨著累積植物第二型富含甘胺酸蛋白LsGRP1，但關於LsGRP1各區段如何參與在防禦反應之誘發則尚未明白，因此本研究深入探討LsGRP1蛋白片段在植物中所扮演的功能及角色，本研究藉由在百合內源性地表現與綠色螢光蛋白融合之LsGRP1及其片段缺失型，發現百合過量表現LsGRP1時，除了增強表現葉的抗病性之外，亦誘導百合系統性抗病力。LsGRP1片段缺失型中，以表現LsGRP1∆N-EGFP的百合葉片相較於表現LsGRP1-EGFP失去抗性程度最多。在無LsGRP1背景的圓葉菸草中再次驗證LsGRP1N對LsGRP1誘發防禦反應的重要性，顯示LsGRP1N為LsGRP1啟動防禦反應的關鍵區段。水楊酸生合成基因LsICS1的表現，在表現LsGRP1∆N-EGFP的百合葉片有抑制作用；乙烯路徑轉錄因子基因LsEIN3的表現在灰黴病菌接種後24小時提升，推測LsGRP1N缺乏可導致ICS1水楊酸生合成路徑受到影響，並且改變百合葉的抗病性。本研究並證明表現LsGRP1N區段上泛素化潛能位點突變之LsGRP1K32, 37, 48R-EGFP與LsGRP1∆N-EGFP的百合葉片有相近的感病程度，說明泛素化潛能對位點對LsGRP1誘發抗灰黴病的重要性。另一方面，藉由綠色螢光影像觀察到於百合葉片表現LsGRP1-EGFP後接種灰黴病菌，LsGRP1蛋白分佈位置從百合葉肉細胞的細胞膜移動到細胞基質中，細胞核周圍出現少數點狀構造，推測LsGRP1的移動與防禦訊息傳導有關。LsGRP1∆N-EGFP和LsGRP1K32, 37, 48R-EGFP在百合葉細胞中有相近的分布變化，但與LsGRP1-EGFP不同，推測LsGRP1N區段與LsGRP1在百合細胞的正確移位有關。以上研究結果指出，LsGRP1N區段與百合LsICS1基因表現有關，LsGRP1N泛素化位點影響LsGRP1次細胞定位，進而能調控對灰黴病菌的防禦反應。

Lily gray mold caused by Botrytis elliptica (Berk.) Cooke is a severe disease in Taiwan. Drench with salicylic acid could systemically enhance gray mold resistance in Lilium oriental hybrid cv. Stargazer accompanied with accumulation of a leaf-specific plant class II glycine-rich protein LsGRP1. How each region of LsGRP1 to playing role in triggering immunity is not know yet. In this research, agroinfiltration with enhanced green florescence protein (EGFP) fusions of LsGRP1 and its partial deletions showed the best local and systemic protection ability in full length-LsGRP1-overexpressed lily leaves, but lowest in the deletion of LsGRP1N. Similar results were shown in Nicotiana benthamiana, indicating that LsGRP1N is the key region of LsGRP1 for plant defense activation. In addition, expression of salicylic acid synthase gene (LsICS1) was examined and the result showed that LsICS1 expression was supressed in LsGRP1∆N-EGFP-expressed lily leaves as compared with that in LsGRP1-EGFP-expressed leaves. Expression of LsEIN3 encoding for a transcription factor of ethylene signaling pathway increased at 24 hours post inoculation with B. elliptica. Howener, lack of LsGRP1N interfering ICS1 salicylic acid synthesis pathway and altering disease resistance ability of lily leaves was presumed. Lily leaves expressing LsGRP1K32, 37, 48R with triple point mutation in LsGRP1N showed similar disease level as that expressing LsGRP1∆N, indicating that ubiquitination is important for LsGRP1 to trigger resistance against gray mold disease. On the other hand, subcellular localization of LsGRP1-EGFP showed moving from the cell membrane to cytoplasm and around nucleus with particles in lily leaf cells after inoculation with B. elliptica inoculation, suggesting that LsGRP1 is related to defense signaling transduction. The alternation of subcellular location of LsGRP1∆N-EGFP and LsGRP1K32, 37, 48R-EGFP was similar but different from that of LsGRP1-EGFP; thus LsGRP1N related to accurate translocation of LsGRP1 in lily cells was presumed. According to these results, LsGRP1N would be involved in the expression of salicylic acid synthase gene LsICS1 and playing a role in the subcellular localization of LsGRP1 via the function of ubiquitnation site of LsGRP1N to trigger defense against gray mold disease.