病菌誘發之番茄轉錄因子ERF68調控過敏性細胞死亡及防禦反應

Ethylene responsive factor (ERF)為一群植物特有之轉錄因子家族，於植物生長發育與逆境反應中，扮演相當重要之角色，但在番茄中仍有許多ERF之功能尚未明。於本研究中，我們針對一群屬於ERF-IX次族群但功能未知之ERF進行研究。其中，ERF68能被不同之細菌病原菌、防禦相關賀爾蒙乙烯及水楊酸大量誘導其基因表現；但是在第三型分泌系統缺失之突變株與非寄主性病原菌之處理下，誘導表現量卻大量下降，顯示病菌第三型效應蛋白可能為誘發ERF68大量表現之主要因子。利用螢光蛋白質融合定位法發現ERF68座落於細胞核內，同時利用Transactivation assay與electrophoretic mobility shift assays (EMSA)分析確認其具有轉錄因子之活性與功能。再者，利用病毒誘導基因過量表現也發現，大量表達ERF68基因可於番茄及菸草中誘發自發性細胞死亡並加強參與在乙烯、水楊酸、茉莉酸、抗病性過敏反應路徑之相關標誌基因表現。同時利用病毒誘發基因靜默法觀察ERF68靜默後之番茄對病原菌抗性反應，也顯示ERF68會降低對低致病力的茄科細菌性斑點病菌(bacterial spots)之抗性，但是對細菌性葉斑病菌(bacterial speck)或萎凋病(bacterial wilting)之抗性則無影響或影響輕微。此結果顯示ERF68應參與在番茄之effector-triggered immunity (ETI)抗病反應上。更進一步，為鑑定受ERF68所調控之下游標的基因，我們也利用染色質免疫沉澱法搭配高通量次世代定序法(ChIP-seq)，解析可能之受調控標的基因。而此結果也顯示一些可能調控細胞死亡與植物防禦反應上的基因可直接受ERF68調控，進而參與在抗病反應中。總和上述結果，本研究證明番茄ERF68經由不同的反應路徑而正向調控過敏性細胞死亡與防禦反應，並提供ERF轉錄因子調控之訊息傳導網路研究一些新的資訊。

關鍵字

番茄； Ethylene responsive factor ；細胞死亡；防禦反應；細菌性斑點病

並列摘要

Ethylene response factors (ERFs) are a large plant-specific transcription factor family and play diverse important roles in various plant functions. However, most tomato ERFs are not characterized. In this study, we showed that expression of an uncharacterized member of tomato ERF-IX subgroup, ERF68, was significantly induced by treatments of different bacterial pathogens, ethylene (ET) and salicylic acid (SA), but only slightly induced by bacterial mutants defective in the type III secretion system (T3SS) or non-host pathogens. ERF68-GFP fusion protein localized in the nucleus. Transactivation and electrophoretic mobility shift assays (EMSA) further showed that ERF68 was a functional transcriptional activator and bound to the GCC-box. Moreover, transient overexpression of ERF68 led to spontaneous lesions in tomato and tobacco leaves and enhanced expression of genes involved in ET, SA, jasmonic acid (JA) and hypersensitive response (HR) pathways, while silencing of ERF68 increased tomato susceptibility to two incompatible Xanthomonas spp. These results reveal the involvement of ERF68 in effector-triggered immunity (ETI) pathway. To identify ERF68 target genes, chromatin-immunopreciptation combined high-throughput sequencing (ChIP-seq) was performed. Among the confirmed target genes, a few genes involved in cell death or disease defense were differentially regulated by ERF68. Our study demonstrates function of ERF68 in positively regulating hypersensitive cell death and disease defense by modulating multiple signaling pathways, and provides important new information on the complex regulatory function of ERFs.

並列關鍵字

Tomato ； ERF ； cell death ； disease ； defense

參考文獻

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病菌誘發之番茄轉錄因子ERF68調控過敏性細胞死亡及防禦反應

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