阿拉伯芥逆境相關蛋白AtSAP5為硫氧化還原蛋白TRX-h3之還原酶

Abstract

植物荷爾蒙水楊酸(salicylic acid)所調節的免疫反應，在對抗非生物與生物逆境中扮演重要角色。在阿拉伯芥水楊酸免疫反應路徑中，NPR1調控約90%下游防禦基因的表現。在正常的狀況下，NPR1 是以多聚體的形式存在於細胞質中，當病菌侵染時，NPR1 會被硫氧化還原蛋白 TRX-h3或h5 還原成單體，單體的 NPR1 可入細胞核調控下游的防禦基因表現。先前的研究中顯示，NPR1表現與逆境相關蛋白AtSAP5有關；另外，初步研究發現，AtSAP5與TRX-h3有交互作用，但其交互作用後啟動的功能則未知。本研究發現AtSAP5具有結合並還原TRX-h3的能力且AtSAP5序列刪減分析之結果顯示 AtSAP5 的 A20 domain 與 TRX-h3 具有最佳結合能力。在還原力的測試顯示，A20 domain 具還原TRX-h3的能力，且在cysteine突變分析發現A20 domain中四個在不同植物物種中保守的cysteine在與TRX-h3之交互作用與保有還原酶活性皆十分重要。本研究對AtSAP5 如何參與 SA調控的免疫途徑提出新的見解。

Phytohormone salicylic acid (SA) plays a vital role in the plant defense responses against abiotic and biotic stresses. In SA-mediated immune pathway, NONEXPRESSOR OF PATHOGENESIS RELATED GENES1 (NPR1) involved in regulation of 90% of downstream SA-dependent immune related genes in Arabidopsis thaliana. Under normal conditions, NPR1 mainly exists as oligomeric form and is located in the cytosol. Upon pathogen infection, NPR1 is reduced from oligomer to monomer through thioredoxin (TRX)-h3 or –h5. Previous result indicates that the expression of NPR1 was regulated by stress-associated protein 5 (AtSAP5). Preliminary data also suggests that AtSAP5 could interact with TRX-h3. However, the function of interaction between AtSAP5 and TRX-h3 is unclear. In this study, the interaction between AtSAP5 and TRX3 is further confirmed and AtSAP5 function as a reductase to reduce TRX-h3 is demonstrated. To identify the region of AtSAP5 that interacts with TRX-h3, I generated several deletion clones of AtSAP5. The results show that the A20 domain exhibits the highest interacting ability with TRX-h3 and also confers the reductase activity. Four conserved cysteines were identified from SAP family in different plant species. Mutational analysis demonstrates each conserved cysteine is important for A20 domain to binds to TRX-h3, and also important for conferring reductase activity. This finding brings a new insight regarding how AtSAP5 regulates the SA-mediated immune pathway.