A high‑efficiency Agrobacterium tumefaciens‑mediated transient expression system in the leaves of Spinacia oleracea

Abstract

Abstract Background The establishment of a highly efficient transient expression system is critical for the study of gene function, particularly in plants for which stable transformation methods are not widely available. Agrobacterium tumefaciens‑mediated transient transformation is a simple and low-cost method that has been extensively developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported. Results Here, we performed a transient expression system in leaves of spinach variety Sp75 and Sp73. Several factors influencing the transformation efficiency were optimized such as Agrobacterium strain, spinach seedling stage, leaf position, and the expression time after injection. Agrobacterium strain GV3101 (pSoup-p19) was more effective than AGL1 in expressing recombinant protein in spinach leaves. In general, the leaves of Sp75 were more suitable than those of Sp73 no matter at four-leaf stage or at six-leaf stage. At four-leaf stage, higher transient expression intensity and efficiency were observed in group 1 (G1) of Sp75 at 53 hours after injection (HAI) and in G1of Sp73 at 64 HAI. At six-leaf stage of Sp75, group 3 (G3) at 72 HAI were the most effective condition for transient expression. Using the optimized expression system, we detected the subcellular localization of a transcriptional co-activator SoMBF1c and a NADPH oxidase SoRbohF. We also detected the interaction of the protein kinase SoCRK10 and the NADPH oxidase SoRbohB. Conclusion This study established a high‑efficiency Agrobacterium‑mediated transient expression method using spinach leaves. The transient expression system will facilitate the gene function analysis and lay a solid foundation for molecular design breeding of spinach.