Exploration of disease suppression function of LsGRP1C on crucifer anthracnose and its antifungal mechanism

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。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.壹、中文摘要............................................. I 貳、英文摘要............................................. II 參、前言................................................. 1 肆、前人研究............................................. 3 一、植物富含甘胺酸蛋白之分類及功能....................... 3 二、植物富含半胱胺酸胜肽之生物功能....................... 4 三、抗菌胜肽之理化特性................................... 5 四、植物抗菌胜肽之抗菌機制............................... 5 五、絲狀真菌的類細胞凋亡之計畫性細胞死亡現象............. 6 六、十字花科炭疽病菌及其與寄主之親和性反應............... 7 伍、材料與方法........................................... 10 一、供試菌株之培養與保存以及接種源之製備................. 10 1. 菌株鑑定及培養........................................ 10 2. 菌株保存.............................................. 10 3. 接種源製備............................................ 10 二、胜肽合成............................................. 10 三、植物材料............................................. 11 1. 芥菜種植.............................................. 11 2. 阿拉伯芥種植.......................................... 11 四、生體外抑菌試驗....................................... 11 1. 抑制孢子發芽能力...................................... 11 2. 抑制生長能力.......................................... 12 3. 菌絲形態觀察及死亡測試................................ 12 五、芥菜上之LsGRP1C抑病試驗.............................. 13 六、免疫螢光染色分析..................................... 13 七、SYTOX Green螢光染色觀察.............................. 14 1. SYTOX Green染色....................................... 14 2. CHXL1-4菌絲內部SYTOX Green螢光定量.................... 14 八、不同螢光染色方式觀察CHXL1-4菌絲的類細胞凋亡之計畫性細胞死亡現象................................................. 15 1. CHXL1-4菌絲細胞氧化壓力偵測........................... 15 2. CHXL1-4菌絲染色質凝集之觀察........................... 16 3. CHXL1-4菌絲細胞核DNA斷裂之觀察........................ 16 九、農桿菌載體構築....................................... 17 1. 目標基因的增幅........................................ 17 2. DNA片段的回收與純化................................... 18 3. TA選殖................................................ 18 4. T-DNA載體構築......................................... 18 5. 大腸桿菌勝任細胞製備.................................. 19 6. 大腸桿菌轉形.......................................... 19 7. 大腸桿菌轉形株之篩選.................................. 19 8. 大腸桿菌質體抽取...................................... 20 9. 農桿菌電穿孔法........................................ 20 十、農桿菌注入法......................................... 21 十一、阿拉伯芥葉片全RNA萃取及反轉錄...................... 21 十二、阿拉伯芥離葉接種................................... 21 十三、真菌侵入構造及菌絲死亡之觀察....................... 22 十四、CHXL1-4於阿拉伯芥葉片產孢量計算.................... 22 陸、結果................................................. 23 一、LsGRP1C於生體外對CHXL1-4之抑制作用................... 23 二、LsGRP1C對CHXL1-4菌絲形態造成之影響................... 23 三、LsGRP1C於生體外造成CHXL1-4細胞死亡................... 24 四、LsGRP1C對芥菜炭疽病之抑病效果........................ 24 五、LsGRP1C主要分布在CHXL1-4細胞外層..................... 24 六、LsGRP1C改變CHXL1-4菌絲之細胞膜通透性................. 25 七、陽離子會降低LsGRP1C對CHXL1-4的抑菌活性............... 25 八、LsGRP1C增加CHXL1-4菌絲細胞內之氧化壓力............... 26 九、LsGRP1C改變CHXL1-4菌絲染色質狀態..................... 26 十、LsGRP1C引起CHXL1-4菌絲細胞核DNA斷裂.................. 27 十一、於阿拉伯芥葉片短暫表現LsGRP1、LsGRP1ΔC、LsGRP1ΔNΔG與LsGRP1C ................................................27 十二、表現LsGRP1、LsGRP1ΔC、LsGRP1ΔNΔG與LsGRP1C對阿拉伯芥葉片罹病度之影響......................................... 27 十三、表現LsGRP1、LsGRP1ΔC、LsGRP1ΔNΔG與LsGRP1C對阿拉伯芥葉片內CHXL1-4侵染構造之影響.............................. 28 十四、染色觀察阿拉伯芥葉片內之植物與CHXL1-4菌絲細胞死亡.. 29 十五、表現LsGRP1、LsGRP1ΔC、LsGRP1ΔNΔG與LsGRP1C對阿拉伯芥葉片上CHXL1-4產孢量之影響................................ 29 柒、討論................................................. 30 捌、參考文獻............................................. 34 玖、圖表集............................................... 44 表一、引子............................................... 45 圖一、LsGRP1C對CHXL1-4孢子發芽的影響..................... 46 圖二、LsGRP1C對CHXL1-4壓器形成的影響..................... 47 圖三、LsGRP1C對CHXL1-4生長的影響......................... 48 圖四、利用光學顯微鏡觀察LsGRP1C對CHXL1-4菌絲形態的影響... 49 圖五、Evans blue染色觀察CHXL1-4細胞死亡.................. 50 圖六、LsGRP1C對芥菜炭疽病病徵發展之影響.................. 51 圖七、LsGRP1C於CHXL1-4細胞之分布......................... 52 圖八、LsGRP1C對CHXL1-4菌絲細胞膜通透性的影響............. 53 圖九、LsGRP1C造成之CHXL1-4菌絲細胞膜透化與生長抑制之關聯性....................................................... 54 圖十、陽離子對LsGRP1C造成之菌絲細胞膜透化與其抑菌活性之影響....................................................... 56 圖十一、LsGRP1C增加CHXL1-4菌絲細胞內之氧化壓力........... 57 圖十二、LsGRP1C造成CHXL1-4菌絲染色質凝集................. 58 圖十三、LsGRP1C造成CHXL1-4菌絲細胞核DNA斷裂.............. 59 圖十四、引子於LsGRP1開放讀碼框架位置..................... 60 圖十五、建構重組載體pBI-LsGRP1、pBI-LsGRP1ΔC、pBI-LsGRP1ΔNΔG與pBI-LsGRP1C......................................... 62 圖十六、以反轉錄PCR分析A. tumefaciens C58C1轉形株注入阿拉伯芥Col-0葉片之基因表現.................................... 63 圖十七、十字花科炭疽病於阿拉伯芥Col-0葉片之發病指數...... 64 圖十八、十字花科炭疽病於各農桿菌注射處理阿拉伯芥葉片之病徵比較....................................................... 65 圖十九、各農桿菌注射處理阿拉伯芥葉片內CHXL1-4侵染構造之顯微觀察..................................................... 66 圖二十、染色觀察阿拉伯芥葉片內植物與CHXL1-4菌絲細胞死亡.. 67 圖二十一、各農桿菌注射處理阿拉伯芥葉片上CHXL1-4產孢量.... 6