鑑定參與在水楊酸調節免疫路徑之水稻逆境相關蛋白

Abstract

生物型及半生物營養型病原菌連年造成水稻產量的嚴重損失，水楊酸 (Salicylic acid, SA) 為調節植物對抗生物及半生物營養性病原菌防禦反應的重要植物荷爾蒙，而過去單子葉和雙子葉作物的研究中，發現植物中特定的逆境相關蛋白 (Stress Associated Proteins, SAPs) 在水楊酸所調節的防禦路徑中扮演重要的調控樞紐。為了提升水稻抗性，本研究擬尋找在水稻中調節水楊酸免疫反應的SAPs。親緣演化分析顯示在水稻18個SAPs (OsSAPs) 中，有兩個SAPs ― OsSAP1與OsSAP11與已知能誘導強抗病能力的阿拉伯芥AtSAP5親緣關係最為相近，且只有上述兩者能在水楊酸處理與稻熱病菌接種後都能被誘導表現。另外分析過去發表的稻熱病菌接種後水稻轉錄組資料也發現OsSAP1與OsSAP11只能在帶有Pi9抗稻熱病基因的抗病近等基因系 (Near Isogenic Line，NIL) 中被誘導基因表現，隨後利用水稻原生植體的瞬時表達功能性分析也顯示過量表現OsSAP1與OsSAP11能誘導水稻水楊酸相關基因包含PR1a (PATHOGENESIS-RELATED PROTEIN 1a) 的表現。本研究也建立了OsSAP1與OsSAP11的Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)、RNA干擾，以及過表達轉基因水稻以利後續分析。總結本研究鑑定參與在水稻水楊酸調控免疫路徑中的逆境相關蛋白，並對未來利用水稻SAPs提升對生物營養或半生物營養型病害的抗性建立了良好的研究基礎。

Biotrophic and hemi-biotrophic pathogens cause serious yield loss on rice. Salicylic acid (SA) is an important phytohormone that regulates the defense responses of plants against biotrophic and hemi-biotrophic pathogens. Previous studies have found that Stress-associated proteins (SAPs) serve as an important regulatory hub in the SA-mediated immune pathway in both monocot and dicot plants. To improve rice disease resistance, this study aimed to identify SAPs that regulate SA mediated immune response in rice. Phylogenetic analysis revealed that among 18 rice SAPs (OsSAPs), OsSAP1 and OsSAP11, are the most closely related to Arabidopsis AtSAP5, which can induce strong disease resistance. In addition, only OsSAP1 and OsSAP11 could be induced after SA treatment and Pyricularia oryzae inoculation. Analysis of published transcriptome data of P. oryzae inoculated rice also showed that the expression of OsSAP1 and OsSAP11 can only be induced in resistance near isogenic line (NIL) which carrying the Pi9 resistance gene after P. oryzae inoculation. Moreover, overexpression of OsSAP1 and OsSAP11 in rice protoplast induced the expression of the rice SA-related marker genes, PATHOGENESIS-RELATED PROTEIN 1a (PR1a). In addition, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), RNA interference and overexpression transgenic rice lines were generated for detailed functional analysis of OsSAP1 and OsSAP11. In conclusion, this study identified SAPs involved in SA-mediated immunity in rice and can be further used to enhance the resistance of rice against biotrophic or hemi-biotrophic pathogens.