臘狀芽孢桿菌C1L增強對灰黴病抗性之百合轉錄體分析

Transcriptome analysis of lily exhibiting enhanced resistance against gray mold triggered by Bacillus cereus C1L

吳亭利(Ting-Li Wu)

指導教授 ： 陳昭瑩

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201600350

摘要

本研究以次世代定序及基因微陣技術全面性分析百合灰黴病菌Botrytis elliptica侵襲百合葉片過程中，臘狀芽孢桿菌Bacillus cereus C1L透過影響那些防禦相關荷爾蒙傳訊區塊(sector)調節植物的抗病反應，以幫助鑑定單子葉植物受根圈細菌誘發的防禦荷爾蒙調控路徑，增進對單子葉作物抗病生理的瞭解。收取僅處理B. cereus C1L、僅接種百合灰黴病菌、處理B. cereus C1L後接種百合灰黴病菌、無處理無接種對照等四組百合的全RNA進行次世代定序分析，共獲得78,674個contigs，其中能比對到參照序列者占總數之35%。為鑑定B. cereus C1L誘發百合抗灰黴病過程中特有的基因差異性表現，選出各處理間變異度超過0.5，且於次世代定序與基因微陣列分析中表現趨勢一致的contigs進行文氏圖分析，發現有383個contigs表現量上升，113個contigs表現量下降，其防禦相關功能包括參與離層酸訊息路徑、調控氣孔開閉、過氧化氫相關反應、表現總穀胱甘肽硫轉移酶等，暗示離層酸傳訊路徑可能在B. cereus C1L誘導百合系統性抗灰黴病過程中扮演重要角色。為進一步探討預處理B. cereus C1L可能通過影響那些防禦傳訊區塊以強化百合對灰黴病的抗性，選取與阿拉伯芥防禦訊息路徑中關鍵基因具有最高相似度的百合同源contigs，並以其基因微陣列分析數據進行熱圖譜分析，結果顯示B. cereus C1L可能經由調控百合之茉莉酸、乙烯、離層酸傳訊區塊增進植物之系統抗病性；即時定量聚合酶連鎖反應的結果亦證實百合在抵抗灰黴病期間，B. cereus C1L預處理會促使茉莉酸傳訊區塊更加活化。此外，預處理B. cereus C1L能促進百合葉部氣孔關閉以減少百合灰黴病菌的感染，暗示離層酸傳訊區塊應參與此抗病性調控。在葉盤接種試驗中同步處理防禦相關荷爾蒙，發現同步處理茉莉酸、乙烯或離層酸可降低百合灰黴病的病徵發展，但同步處理水楊酸會使病徵輕微加劇，點明在百合灰黴病菌攻擊當下即時增加茉莉酸、乙烯或離層酸的含量可能促進百合的抗病性，暗示活化茉莉酸/乙烯及離層酸傳訊區塊在百合防禦灰黴病上具有正面貢獻。綜合文氏圖、熱圖譜、葉盤試驗及氣孔關閉比例分析之結果，推斷B. cereus C1L經由調控茉莉酸/乙烯及離層酸傳訊區塊，誘發百合對灰黴病之系統性抗病。

關鍵字

臘狀芽孢桿菌 C1L ； 百合灰黴病 ； 轉錄體分析 ； 基因微陣技術 ； 茉莉酸/乙烯傳訊區塊 ； 離層酸傳訊區塊

並列摘要

This research is to analysis the influence of Bacillus cereus C1L on what hormone signaling sectors to regulates defense response of Botrytis elliptica-infected lily by next-generation sequencing (NGS) and microarray analyses. The result may help to identify the regulation of monocot defense related hormone signaling pathway triggered by rhizobacteria and promotes the understanding of resistance physiology of monocot. Total RNAs were collected from B. cereus C1L-drenched leaves, B. elliptica-inoculated leaves, B. elliptica-inoculated leaves pre-drenched with B. cereus C1L and untreated control of lily plants for NGS analysis. Among 78,674contigs, 35% of contigs hit the reference sequences. To identify unique gene differentially expressed while triggered by B. cereus C1L on B. elliptica-inoculated lily, a criteria of variety over 0.5 of each treatment was set for selecting those contigs with expression pattern similar between NGS and customized array to create venn diagram. In this venn diagram analysis, expression of 383 contigs exhibited up-regulation while 113 contigs were down-regulated. Their functions related to plant defenses involved abscisic acid (ABA) signaling, stomatal closure regulation, hydrogen peroxide response and glutathione S-transferase, indicating that ABA signaling may play a role in lily systemic resistance against B. elliptica as triggered by B. cereus C1L. To further study about which hormone signaling pathway B. cereus C1L may utilize to strengthen resistance of lily against B. elliptica, the most similar contig homologs to the selected key gene sequences in different defense signaling pathway of Arabidopsis were selected for the analysis of heat map. Results showed that B. cereus C1L probably increase lily resistance through jasmonic acid (JA), ethylene (ET) and ABA signaling pathway. Quantitative real-time polymerase chain reaction analysis verified that pre-drench with B. cereus C1L could activate JA signaling sector upon B. elliptica attack. Besides, ABA treatment could increase stomatal closure rate and reduce the infection by B. elliptica, indicating an involvement of ABA signaling in C1L-conducted defense response. In the leaf disc assay, treatments with JA, ET or ABA at the same time of B. elliptica inoculation decreased symptom development; however, treatment with salicylic acid did not show significant effect on symptom development. These results collectively suggested that activations of JA/ET and ABA signaling sectors had positive contribution to the defense against B. elliptica. As a whole, according to results of venn diagram, heat map, leaf disc test and stomatal closure rate, B. cereus C1L–induced systemic resistance against B. elliptica in lily regulated by JA/ET and ABA signaling sectors was presumed.

並列關鍵字

Bacillus cereus C1L ； Botrytis elliptica ； transcriptome analysis ； microarray ； JA/ET signaling sector ； ABA signaling sector

參考文獻

Agrawal, G.K., Jwa, N.S., Rakwal, R. 2000. A novel rice (Oryza sativa L.) acidic PR1 gene highly responsive to cut, phytohormones, and protein phosphatase inhibitors. Biochem. Biophys. Res. Commun. 274, 157-65.

An, C., Mou, Z. 2011. Salicylic acid and its function in plant immunity. J. Integr. Plant Biol. 53, 412-28.

Appel, H.M., Cocroft, R.B. 2014. Plants respond to leaf vibrations caused by insect herbivore chewing. Oecologia 175, 1257-66.

Asselbergh, B., De Vleesschauwer, D., Hofte, M. 2008. Global switches and fine-tuning-ABA modulates plant pathogen defense. Mol. Plant-Microbe Interact. 21, 709-19.

Balmer, D., Planchamp, C., Mauch-Mani, B. 2013. On the move: induced resistance in monocots. J. Exp. Bot. 64, 1249-61.