The functions of ubiquitin-conjugating enzymes (E2) in plant immunity are not well understood. In this study, OsUBC26, a rice ubiquitin-conjugating enzyme, was characterized in the defence against Magnaporthe oryzae. The expression of OsUBC26 was induced by M. oryzae inoculation and methyl jasmonate treatment. Both RNA interference lines and CRISPR/Cas9 null mutants of OsUBC26 reduced rice resistance to M. oryzae. WRKY45 was down-regulated in OsUBC26 null mutants. In vitro E2 activity assay indicated that OsUBC26 is an active ubiquitin-conjugating enzyme. Yeast two-hybrid assays using OsUBC26 as bait identified the RING-type E3 ligase UCIP2 as an interacting protein. Coimmunoprecipitation assays confirmed the interaction between OsUBC26 and UCIP2. The CRISPR/Cas9 mutants of UCIP2 also showed compromised resistance to M. oryzae. Yeast two-hybrid screening using UCIP2 as bait revealed that APIP6 is a binding partner of UCIP2. Moreover, OsUBC26 working with APIP6 ubiquitinateds AvrPiz-t, an avirulence effector of M. oryzae, and OsUBC26 null mutation impaired the proteasome degradation of AvrPiz-t in rice cells. In summary, OsUBC26 plays important roles in rice disease resistance by regulating WRKY45 expression and working with E3 ligases such as APIP6 to counteract the effector protein AvrPiz-t from M. oryzae.

中文翻译：

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水稻泛素结合酶 OsUBC26 对稻瘟病菌的免疫至关重要

泛素结合酶 (E2) 在植物免疫中的作用尚不清楚。在这项研究中，OsUBC26 是一种水稻泛素结合酶，其特征在于对稻瘟病菌的防御。OsUBC26的表达是由稻瘟病菌接种和茉莉酸甲酯处理诱导的。OsUBC26的 RNA 干扰系和 CRISPR/Cas9 无效突变体均降低了水稻对米根霉的抗性。WRKY45在OsUBC26中被下调无效突变体。体外 E2 活性测定表明 OsUBC26 是一种有活性的泛素结合酶。使用 OsUBC26 作为诱饵的酵母双杂交试验将环型 E3 连接酶 UCIP2 鉴定为相互作用蛋白。共免疫沉淀测定证实了 OsUBC26 和 UCIP2 之间的相互作用。UCIP2的 CRISPR/Cas9 突变体也表现出对米分枝杆菌的抗性受损。使用 UCIP2 作为诱饵的酵母双杂交筛选显示 APIP6 是 UCIP2 的结合伙伴。此外，与 APIP6 一起使用的 OsUBC26 泛素化了 AvrPiz-t（米分枝杆菌的无毒效应子）和OsUBC26无效突变损害了水稻细胞中 AvrPiz-t 的蛋白酶体降解。综上所述，OsUBC26 通过调节WRKY45的表达以及与 APIP6 等 E3 连接酶对抗来自M. oryzae的效应蛋白 AvrPiz-t 在水稻抗病性中发挥重要作用。