Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase
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 (T3 and T4 generations) and 76.67–82.0% reduction of lesion area in a detached leaf assay (T4 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 T3 and T4 transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H2O2) 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 H2O2 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 H2O2 levels, and eliciting defense responses mediated by multiple signaling pathways.
KeywordsSclerotinia sclerotiorum Oxalic acid Hydrogen peroxide Defense response
This work was supported by grants from China National Novel Transgenic Organisms Breeding Project (2016ZX08004-004), Jilin Provincial Agricultural Science & Technology Innovation Project (CXGC2017JQ013), and National Natural Science foundation of China (31671764). We would also like to thank Editage (www.editage.cn) for English language editing.
YD and QL designed the experiments. XY and JY conducted the experiments and drafted the manuscript. YW and LS conducted the S. sclerotiorum inoculation experiments. LN, HH, and XZ performed the western blot, OA, and H2O2 analyses. DG, GX, and QZ conducted Agrobacterium-mediated transformation experiments. All authors participated in the manuscript revision.
Conflict of interest
The authors declare that they have no competing interests.