整合轉錄體學與活性氧抑制實驗以探討炭腐病菌微菌核之形成

Integration of RNA-Seq and Reactive Oxygen Species Inhibition Assay to Study Microsclerotia Development of Macrophomina phaseolina

劉軒豪(Hsien-Hao Liu)

指導教授 ： 張皓巽

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(2021年)

https://doi.org/10.6342/NTU202103947

摘要

微菌核 (microsclerotia) 為真菌的特殊殘存構造之一，具繁殖能力且可承受多樣環境壓力。前人研究指出微菌核與菌核之結構和發展模式相似，並認為活性氧 (Reactive oxygen species, ROS) 為刺激微菌核與菌核分化的重要因子。然而目前活性氧刺激菌核與微菌核分化的相關研究，多以子囊菌 (Ascomycetes) 糞殼菌綱(Sordariomycetes) 中的菌核病菌 (Sclerotinia sclerotium) 、黃萎病菌 (Verticillium dahliae) 與綠殭菌 (Nomuraea rileyi) 為主，且文獻多數支持過氧化氫 (hydrogen peroxide) 為主要刺激分化的活性氧種類。關於活性氧是否在演化分歧後仍參與座殼菌綱 (Dothideomycetes) 炭腐病菌之微菌核形成，目前尚無實驗探究。本研究進行核糖核酸測序 (RNA-Sequencing)，分析炭腐病菌由菌絲至微菌核形成的四個時期 (MS0-MS3) 基因表現差異，並以基因表現趨勢分析 (cluster analysis) 與功能富集分析 (functional enrichment analysis) 發現六個基因群中的四個基因群與活性氧代謝具相關性，包括過氧化體 (peroxisome)、單加氧酶活性 (monooxygenase activity)、麩胱甘肽轉移酶 (glutathione transferase) 等。另於菌絲 (MS0) 與微菌核形成階段 (MS1-MS3) 差異表現基因分析，發現1683個上調基因與1552個下調基因之中，氧化還原和抗氧化物活性相關功能之比例皆顯著提升，其中MS1階段主要以超氧化物 (superoxide) 代謝，MS2與MS3階段則以過氧化氫代謝為主。總觀基因表現，分析結果支持活性氧參與炭腐病菌微菌核形成。抗氧化物抑制測試與活性氧染色試驗結果顯示，添加抗壞血酸 (ascorbic acid)、二硫蘇糖醇 (dithiothreitol) 與麩胱甘肽 (glutathione) 皆可抑制微菌核形成，且抑制率隨濃度上升而增加。基於50%微菌核抑制率之濃度下進行活性氧染色，發現NBT染色程度於三種抗氧化物處理相似，然而於二硫蘇糖醇處理組，DAB的染色程度較低與其他兩種抗氧化物處理具顯著差異，推測超氧化物可能為刺激微菌核形成的主要因子。另於培養基添加DETC與過氧化氫實驗中，僅於添加誘導超氧化物累積之DETC具有效刺激微菌核合成。本研究整合轉錄體與活性氧相關試驗結果，證實活性氧參與炭腐病菌微菌核形成，且發現超氧化物為刺激炭腐病菌之菌絲分化成微菌核的主要活性氧種類。

關鍵字

炭腐病菌 ； 微菌核 ； 活性氧 ； 超氧化物 ； 轉錄體

並列摘要

Microsclerotia are specialized survival structures which can serve as propagules and resist various environmental pressures for different fungi. Previous studies showed that microsclerotia have similarity in histology and development to sclerotia-forming fungi. Reactive oxygen species (ROS) has been shown to be the key to induce microsclerotia or sclerotia development, and these studies were mostly based on the Sordariomycetes such as Sclerotinia sclerotiorum, Verticillium dahliae and Nomuraea rileyi. While literature mostly suggested hydrogen peroxide (H2O2) as the main ROS to induce microsclerotia and sclerotia development, it remains unclear whether ROS stimulation remains evolutionary conserved in the microsclerotia development of Dothideomycetes Macrophomina phaseolina. In this study, RNA-Seq was used to investigate transcriptomic dynamics of four microsclerotia development stages (MS0-MS3) in M. phaseolina. Clustering and functional enrichment analyses showed that four expression clusters were related to ROS metabolism, including peroxisome, monooxygenase and glutathione transferase activity. Compared the three microsclerotia development stages (MS1-3) to the hyphae stage (MS0), 1683 and 1552 consensus transcripts were up-regulated and down-regulated, respectively; and ROS-related activities were functional-enriched in these transcripts. Transcripts for ROS metabolism were tended to detoxify superoxide (O2-) in MS1 and detoxify H2O2 in MS2-3. ROS inhibition assay and ROS staining were applied to validate these observations. Exogenous antioxidants (ascorbic acid, DTT, and glutathione) caused a concentration-dependent inhibition of microsclerotia development. Based on ROS staining at the 50% microsclerotia inhibition rate, the degree of NBT staining was similar among three antioxidants. However, the degree of DAB staining was significantly lower in the DTT treatments compared to other two antioxidants, implying O2- may be the main stimulus to induce microsclerotia development. Moreover, M. phasolina grew on the superoxide dismutase inhibitor DETC-amended plates resulted in more microsclerotia, but not the H2O2-amended plates. Therefore, the integration of transcriptomics and ROS assays supported that ROS involves in the microsclerotia development, and O2- is the key stimulus to initiate hyphae development into microsclerotia of M. phasolina.

並列關鍵字

Macrophomina phaseolina ； Microsclerotia ； Reactive Oxygen Species (ROS) ； Superoxide ； Transcriptomics

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