- 學位論文
鏈黴菌誘導阿拉伯芥抗病性探討
Studies on Streptomyces-induced disease resistance in Arabidopsis thaliana
摘要
為了避免化學肥料和農藥過度使用導致環境汙染的狀況，近年來許多研究都希望找出替代方法，其中一種替代方法便是使用促進植物生長根棲細菌 (plant growth promoting rhizobacteria, PGPR)。PGPR 是指可以藉由直接或間接機制來促進植物生長的土壤益生菌，本研究在探討土壤中常見之兩種鏈黴菌 (Streptomyces coelicolor 和 antibioticus)，對於模式植物阿拉伯芥 (Arabidopsis thaliana) 在生長以及抗病能力上的影響。結果發現與對照組相比，施加任一種鏈黴菌的阿拉伯芥地上部鮮重有顯著增加；進一步研究發現，阿拉伯芥生長素突變株 aux1-7 與離層酸突變株 abi4-1 和 abi5 施加任一種鏈黴菌後，地上部鮮重和控制組皆沒有顯著差異，顯示生長素和離層酸調控根部的生長發育可能是鏈黴菌影響阿拉伯芥地上部鮮重增加的關鍵之一。另外，在感染丁香假單胞菌 (Pseudomonas syringae pv. tomato DC3000, Pst DC3000) 三天後，有先經過鏈黴菌處理的阿拉伯芥，其地上部的菌數會顯著下降，顯示兩種鏈黴菌均擁有增強阿拉伯芥對於 PstDC3000的抗病能力。以反轉錄聚合酶鏈鎖反應偵測已知的阿拉伯芥抗病相關基因 PDF1.2 和 PR1，發現此二基因在阿拉伯芥與任一種鏈黴菌共培養的情形下，於感染 Pst DC3000 後的 24 至 72 小時其地上部基因表現量都高於未共培養者，顯示此二鏈黴菌可能是透過類似誘導植物的系統獲得性抗性 (Systemic acquired resistance, SAR) 與誘導系統型抗性 (Induced systemic resistance, ISR) 來加強阿拉伯芥的抗病能力。同時藉由地上部菌數以及此二抗病相關基因表現量的實驗結果推論兩種鏈黴菌都能對阿拉伯芥產生誘發效應 (The priming effect)。阿拉伯芥乙烯 (ethylene, ET) 相關基因的突變株 ein2-1 和 eto1-1 以及茉莉酸 (jasmonic acid, JA) 相關基因突變株 jar1-1 與水楊酸 (salicylic acid, SA) 相關基因突變株sid2-1 和 pad4-1 施加任一種鏈黴菌後，抗病能力和控制組皆沒有顯著差異，表示 ET 相關訊息傳導途徑、JA 相關訊息傳導途徑和 SA 相關訊息傳導途徑是鏈黴菌影響阿拉伯芥抗病性的關鍵之一。進一步以反轉錄聚合酶鏈鎖反應分析由阿拉伯芥與任一種鏈黴菌共培養再感染 Pst DC3000 後，ET、JA 以及SA 相關基因的表現量，並以未與鏈黴菌共培養者作為對照組。結果顯示若植株與鏈黴菌共培養，ET 相關基因 GST2在感病後 72 小時的表現量高於對照組；JA 相關基因 LOX2 及 VSP2 在感病後 48 小時的表現量高於對照組；SA 相關基因 PR2 和 PR5則於感病後 24 小時呈現高於對照組的表現量。這些結果印證先前利用突變株進行的實驗，說明 ET、JA 和 SA相關訊息傳導途徑皆參與鏈黴菌調控阿拉伯芥的抗病性；而由基因的表現情形來看，不同種的訊息傳導途徑作用時間也不一樣，SA 訊息傳導途徑較早參與，JA 及 ET 訊息傳導途徑則較晚。此外，hdt3 (histone deacetylase 3) 、hta11 (histone H2A 11) 以及 hda6 (histone deacetylase 6) 阿拉伯芥突變株處理任一種鏈黴菌後，抗病能力和對照組也沒有顯著差異。由於這些突變參與在組蛋白修飾上，因此這兩種鏈黴菌可能是透過誘導組蛋白修飾以對後續病原菌攻擊有較快或較強的免疫反應。
並列摘要
To avoid the situation that excessive use of chemical fertilizers and pesticides lead to environmental contamination, many studies focused on finding alternatives for agricultural practice in recent years, one of which is use of plant growth-promoting rhizobacteria (PGPR). PGPR can promote plant growth either directly or indirectly. In this study, two soil habitants, Streptomyces coelicolor strain M145 and Streptomyces antibioticus were used to explore their effects on enhancing plant growth and inducing resistance of the model plant Arabidopsis thaliana. The results showed that compared to the control, application of either S. coelicolor or S. antibioticus to Arabidopsis led to a significant increase in fresh weight. Further studies found that no significant change on fresh weight of the Arabidopsis mutant aux1-7 and abscisic acid mutants abi4-1 and abi5 after treatment with either Streptomyces spp., indicating that auxin and abscisic acid play roles in Streptomyces-induced Arabidopsis growth enhancement. On the other hand, bacterial numbers were lower in the Streptomyces-treated Arabidopsis shoots three days after infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), and this means both Streptomyces spp. are able to enhanced disease resistance of Arabidopsis against Pst DC3000. The data of reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression of disease-related genes PDF1.2 and PR1 was higher 24 to 72 hours after Pst DC3000 infection in the Arabidopsis shoots when plants co-cultivated with either Streptomyces spp. These data also revealed that S. coelicolor and S. antibioticus may enhance plant defense of Arabidopsis through induction of systemic acquired resistance (SAR)-like and induced systemic resistance (ISR). The results of Pst DC3000 population and these two gene expression from shoots also showed that Streptomyces are able to prime Arabisopsis. The ethylene (ET)-related gene mutants ein2-1 and eto1-1, jasmonic acid (JA)-responsive mutant jar1-1 and salicylic acid (SA)-related mutants sid2-1 and pad4-1 did not show significant difference in population of Pst DC3000 in the plants treated with or without either Streptomyces spp., indicating that ET, JA and SA signaling pathways are required for Streptomyces-induced Arabidopsis resistance against Pst DC3000. Furthermore, the expression of ET, JA and SA-related genes in Arabidopsis co-cultured with Streptomyces after Pst DC3000 infection was analyzed by RT-PCR. ET-related gene GST2 and JA-related gene LOX2, VSP2 showed higher expression at 72 hour after infection in co-cultured plants. On the other hands, SA-related genes PR2 and PR5 showed higher expression 24 hour after infection in Streptomyces-treated Arabidopsis. These results are consistent with previous discoveries that ET, JA and SA signaling pathways are involved in Streptomyces-induced Arabidopsis resistance against Pst DC3000, and these hormones may work in concert at different timeline. Additionally, hdt3 (histone deacetylase 3), hta11 (histone H2A 11) and hda6 (histone deacetylase 6) mutants showed no significant difference in disease resistance against Pst DC3000 when either S. coelicolor or S. antibioticus was applied. These genes participate in histone modification, indicating that both Streptomyces may prime the Arabidopsis defense responses through epigenetic manners.
參考文獻
延伸閱讀
- 黃博洋（2021）。LsGRP1誘導阿拉伯芥系統性抗病之傳訊路徑探討〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU202102427
- 沈育年（2014）。探討阿拉伯芥中DEAD-box RNA 7解旋酶之功能〔碩士論文，國立中央大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0031-0412201512014873
- 梁詩婕（2008）。酵母菌BRE1同源基因HUB1於阿拉伯芥開花生理調控路徑之功能研究〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2008.10308
- Chen, P. W. (2012). 阿拉伯芥透過誘發反應調控先天性免疫作用抵抗死體寄生型之軟腐病原細菌 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2012.00774
- Jen, C. Y. (2021). Analysis of AtNPF6.4 transport activity and its role in Arabidopsis response to Pseudomonas syringae [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU202103588