Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, 130033, China.
Transgenic Res. 2019 Feb;28(1):103-114. doi: 10.1007/s11248-018-0106-x. Epub 2018 Nov 26.
Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71-82.73% reduction of lesion length in a detached stem assay (T and T generations) and 76.67-82.0% reduction of lesion area in a detached leaf assay (T generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T and T transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (HO) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of HO at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing HO levels, and eliciting defense responses mediated by multiple signaling pathways.
草酸降解酶 OxO 基因过表达提高大豆对菌核病的抗性
草酸（OA）分泌型坏死型真菌病原菌核盘菌（Sclerotinia sclerotiorum）引起的菌核茎腐病是大豆生产上的毁灭性病害之一，目前可用商业大豆品种缺乏强抗性，严重限制了病害的有效防治。本研究利用小麦草酸氧化酶基因（OXO）转化大豆，获得了过表达 OxO 的转基因植株，并对其抗核盘菌能力进行了评估。通过 Southern 和 Western blot 分析证实了转基因的整合和表达。与非转化（NT）对照植株相比，过表达 OxO 活性的转基因系在离体茎段和离体叶片试验中，表现出明显较小的病斑，即病斑长度分别降低了 58.71-82.73%（T 和 T 代）和 76.67-82.0%（T 代），对 OA（60mM）的耐受性也显著提高。连续的抗性评价进一步证实了 T 和 T 代转基因系对核盘菌的增强和稳定抗性。同样，在接种核盘菌后，转基因叶片中的 OA 含量降低，过氧化氢（HO）水平升高。定量实时聚合酶链反应（qRT-PCR）分析显示，在接种核盘菌后 1 h 和 4 h，OXO 的表达水平达到峰值。同时，与过敏反应相关的基因 GmNPR1-1、GmNPR1-2、GmSGT1 和 GmRAR 的表达水平也显著上调，最终导致感染部位 HO 的释放增加。有趣的是，水杨酸依赖基因（GmPR1、GmPR2、GmPR3、GmPR5、GmPR12 和 GmPAL）和乙烯/茉莉酸依赖基因（GmAOS、GmPPO）等其他防御相关基因在转基因植株中的表达水平也高于 NT 对照。研究结果表明，过表达 OxO 通过降解核盘菌分泌的 OA 和增加 HO 水平，增强了 SSR 抗性，并通过多条信号通路诱导防御反应。
