探討疫病菌質外體效應蛋白OPEL對於菸草轉錄體的影響

Transcriptome analysis to investigate the effect of OPEL, an apoplastic effector from Phytophthora parasitica, on Nicotiana tabacum

柯岱妤(Tai-Yu Ke)

指導教授 ： 劉瑞芬

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(2019年)

https://doi.org/10.6342/NTU201903584

摘要

疫病菌外泌蛋白 OPEL 為質外體效應蛋白，處理菸草葉片後會引發活性氧分子產生、癒傷葡聚醣沉積及細胞死亡。此外，OPEL 會誘導系統葉 PAMP-triggered immunity (PTI) 指標基因及水楊酸相關防禦基因的表現，並引發植物對於多種病原的系統性抗性。OPEL 引發植物免疫反應的關鍵構造為其 glycoside hydrolase 16 (GH16) domain；該 GH16 domain 含有 beta-1,3-glucanase 保守性胺基酸序列，將預測的酵素活性位點分別進行單點突變皆會大幅降低 OPEL 激發植物防禦反應的能力，推測 OPEL 可能藉由其水解產物引發植物防禦反應。本研究利用 RNA-seq 分析菸草以OPEL (wt) 重組蛋白處理 6 小時後的轉錄體，發現相對於處理 MES 緩衝液的對照組，總計有 8,102 個差異表現基因 (differentially expressed genes；DEGs)。GO 富集分析顯示這些 DEGs 主要涉及逆境反應或多醣的辨識，其產物包含膜蛋白及類囊體蛋白，分子功能以激酶活性、氧化還原活性，和碳水化合物的結合為主。KEGG 路徑富集分析發現，OPEL 引發的反應涉及植物－病原交互作用、MAPK 訊號傳遞、次級代謝物的生合成、胺基酸代謝、脂質代謝及光合作用相關路徑等，但光合作用相關基因主要為負調控，顯示 OPEL 可能抑制光合作用。這些 DEGs 所編碼的轉錄因子以 WRKY、AP2-EREBP (APETALA2/ethylene-responsive element binding protein) 及 MYB 為大宗，可能與誘導植物防禦基因的表現有關。此外，OPEL 也會擾動生長素、水楊酸、茉莉酸及乙烯等植物荷爾蒙的調控。另一方面，為釐清預測的酵素活性位點在 OPEL 引發植物防禦反應的角色，本研究另表現將預測酵素活性位雙點突變之 OPEL 重組蛋白 (dm)；該蛋白幾乎無法引發植物產生活性氧分子及沉積癒傷葡聚醣，但在處理菸草後 4 或 6 小時都能誘導處理葉表現 PTI 指標基因 Pti5 及 Gras2。以 dm 處理菸草再進行轉錄體分析，發現相對於處理 MES 緩衝液的對照組，總計有 1,565 個 DEGs，其中 1,538 個基因也是 wt 處理後具有表現差異的基因；GO 及 KEGG 路徑富集分析發現 dm 處理引發的植物反應與 wt 相當類似，顯示 dm 很可能也會引發植物 PTI。但值得注意的是，dm 二次重複的基因表現情形非常不同，一組與 wt 相近，另一組與 MES 對照組較為相近。本研究的轉錄體分析結果除了為 OPEL 引發的 PTI 提供強力支持，還揭示其引發的 PTI 反應可能涉及 RLCKs 及 MAPK cascade 訊號傳遞路徑，並透過 WRKYs、AP2-EREBP 及 MYBs 調控植物防禦反應相關基因的表現。至於 OPEL 酵素活性位點在引發植物防禦反應的重要性，仍待進一步探究。

關鍵字

OPEL ； pattern-triggered immunity (PTI) ； 疫病菌 ； 菸草 ； 轉錄體分析

並列摘要

OPEL, an apoplastic effector of Phytophthora parasitica, is known to induce reactive oxygen species (ROS) accumulation, callose deposition, and cell death in Nicotiana tabacum cv. Samsun NN. In addition, it induces the expression of PAMP-triggered immunity (PTI) marker genes and SA-responsive genes on the systemic leaves of tobacco, and confers systemic acquired resistance against variable pathogens. The glycoside hydrolase 16 (GH16) domain of OPEL containing conserved amino acid sequence signature of beta-1,3-glucanase is indispensable for the elicitor activity of OPEL. Interestingly, single point mutation on the putative enzymatic active site reduced the elicitor activity, which suggests the possibility that degradation products of OPEL may serve as damage-associated molecular pattern (DAMP) to trigger plant immunity. In this study, RNA-seq was conducted to analyze the transcriptomes of tobacco at 6 hours post infiltration with OPEL (wt) recombinant protein, resulting in the identification of in total 8,102 differentially expressed genes (DEGs) when compared with RNA-seq data obtained from the MES buffer control. Gene ontology (GO) enrichment analysis revealed that these DEGs, mostly involved in stress responses or recognition of polysaccharides, encode membrane proteins and thylakoid proteins among others, which show molecular functions encompassing mainly kinase activity, oxidoreductase activity, and carbohydrate binding. KEGG pathway enrichment analysis indicated that OPEL-triggered responses involved in plant-pathogen interaction, MAPK signaling, biosynthesis of secondary metabolites, amino acid metabolism, lipid metabolism, photosynthesis-related pathways, and so on. Notably, DEGs involved in photosynthesis were mostly down-regulated, suggesting that OPEL might inhibit photosynthesis. The top three transcription factor families encoded by DEGs were WRKY, APETALA2/ethylene-responsive element binding protein (AP2-EREBP) and MYB, which may be involved in transcriptional reprogramming of plant defense genes. As well, OPEL regulates the signaling of plant hormones such as auxin, salicylic acid, jasmonic acid, and ethylene. On the other hand, to clarify the role of the putative enzymatic active site in OPEL-triggered immunity, recombinant protein harboring double mutation in the putative active site (dm) were expressed, which barely induced ROS production or callose deposition on tobacco, but induced the expression of Pti5 and Gras2, both PTI marker genes. Comparison of RNA-seq data from dm-treated tobacco leaves with those from the MES control identified in total 1,565 DEGs, of which 1,538 genes were also differentially expressed in the OPEL (wt)-treated plants. GO and KEGG pathway enrichment analysis showed that the responses induced by dm was quite similar to those elicited by wt, suggesting that dm may also trigger PTI. It is worth of noting, however, that RNA-seq data obtained from two biological repeats of dm deviate from each other, with one close to wt while the other to the MES control. These results not only provide strong support for OPEL to elicit PTI, but also reveal molecular mechanisms underlying OPEL-induced plant immunity, which involves signal transduction through RLCKs and MAPK cascade, as well as transcriptional reprogramming of plant defense genes through the interplay of various transcriptional factors such as WRKYs, AP2-EREBPs, and MYBs. The role of the putative enzymatic active sites of OPEL in OPEL-triggered immunity, however, awaits further investigation.

並列關鍵字

Nicotiana tabacum ； OPEL ； pattern-triggered immunity (PTI) ； Phytophthora parasitica ； transcriptome

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