乙烯反應因子96藉由直接結合茉莉酸及乙烯相關防禦
基因之GCC區段正向調控阿拉伯芥對腐生型病菌抗性

阿拉伯芥 Apetalla2/Ethylene response factor (AP2/ERF)家族是一群參與調節生物性及非生物性逆境的重要轉錄因子(Transcription factors, TFs)。先前研究已報導在AP2/ERF 第九群組中的某些成員，例如 ERF1 、 ERF6 及 ORA59 參與阿拉伯芥抵抗灰黴病菌(Botrytis cinerea)之反應中。在此研究旨在探討發現同為第九群族成員的ERF96 在阿拉伯芥中抵抗腐生性病原菌反應之角色。在過量表現 ERF96 的阿拉伯芥中所得結果顯示 ERF96 對茉莉酸(Jasmonate, JA)及乙烯(Ethelene, ET)反應相關之防禦基因 PDF1.2a 、PR-3 與 PR-4 之表現有正向調控之功能，且在植物對抗腐生性真菌灰黴病菌及腐生性細菌軟腐病菌(Pectobacterium carotovorum)上亦為正向調控者，與此觀察一致的是 ERF96 之表現也受到茉莉酸及乙烯之調控。除此之外，在茉莉酸鈍感突變株 coil-16 與乙烯訊號突變株 ein2-1 中 ERF96 之轉錄是缺失的。ERF96 為位在細胞核內的轉錄增強子，並在試管反應中可與基因啟動子之 GCC 區段結合。利用過量表現 ERF96 阿拉伯芥進行微陣列分析，並搭配染色質共免疫沉澱-聚合酶連鎖反應(chromatin immunoprecipitation-PCR)所得結果顯示 ERF96 是藉由結合基因啟動子之 GCC 區段直接而調控防禦基因 ORA59、PDF1.2a、PR-3 及PR-4 之表現。藉由 RNA 干擾技術使 ERF96 表現靜默並未使阿拉伯芥對灰黴病菌及軟腐病菌更加感病，猜測是因為 ERF 家族中的轉錄因子有功能重複性。綜合以上結果，本研究揭示了 ERF96 是作用在茉莉酸與乙烯訊號傳遞途徑下游之調控因子，可藉由直接調控防禦基因與轉錄因子之表現而增強了阿拉伯芥對腐生性病原菌的抵抗性。

關鍵字

腐生性病原菌；阿拉伯芥；防禦反應；乙烯反應因子

並列摘要

The AP2/ERF family is composed of transcription factors (TFs) that are involved in regulating the Arabidopsis response to biotic and abiotic stresses. Some of the members of AP2/ERF group IX, like ERF1, ERF6 and ORA59 have been shown to participate in the defense against the pathogen Botrytis cinerea. In this study we identified and characterized ERF96, also a member of group IX, in Arabidopsis resistance against necrotrophic pathogens. Arabidopsis overexpressing ERF96 demonstrated enhanced upregulation of jasmonate (JA)- and ethylene (ET)-responsive PDF1.2a, PR-3 and PR-4 defense genes and were more resistant to the necrotrophic fungal pathogen B. cinerea and bacterial pathogen Pectobacterium carotovorum (Pcc) than wild-type (WT) plants. Consistent with these observation, ERF96 was shown to be JA- and ET -responsive. In addition, transcription of ERF96 was defective in JA insensitive coi1-16 and ET signaling ein2-1 mutants. Furthermore, characterization of ERF96 revealed that ERF96 acts as a transcriptional activator localized mainly in the nucleus that can bind to GCC box in vitro. Moreover, microarray analysis coupled to chromatin immunoprecipitation-PCR (ChIPPCR) of Arabidopsis overexpressing ERF96 revealed that this TF directly regulates the expression of ORA59, as well as PDF1.2a, PR-3 and PR-4 by binding GCC elements of their promoters. ERF96 silencing by RNA interference did not trigger an increase of Arabidopsis susceptibility to B. cinerea and Pcc, suggesting a redundancy of function between TFs of the ERF family. Altogether, our results indicate that ERF96 is acting downstream of the JA and ET signaling pathways and is positively regulating Arabidopsis defenses against necrotrophic pathogens by directly modulating defense genes as well as TF expressions.