RESUMEN
BACKGROUND: Optimization of a highly efficient transient expression system is critical for the study of gene function, particularly in those plants in which stable transformation methods are not widely available. Agrobacterium tumefaciensmediated transient transformation is a simple and low-cost method that has been developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported. RESULTS: We developed a transient expression system in spinach leaves of the Sp75 and Sp73 varieties. 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 efficient than AGL1 in expressing recombinant protein in spinach leaves. In general, Sp75 leaves were more suitable than Sp73 leaves, regardless of grow stage. At four-leaf stage, higher intensity and efficiency of transient expression were observed in group 1 (G1) of Sp75 at 53 h after injection (HAI) and in G1 of 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 method of highly efficient transient expression mediated by Agrobacterium in spinach leaves. The transient expression system will facilitate the analysis of gene function and lay a solid foundation for molecular design breeding of spinach.
RESUMEN
Pto interaction (Pti) proteins are a group of proteins that can be phosphorylated by serine/threonine protein kinase Pto, which have diverse functions in plant development and stress response. In this study, we analyzed the phylogenetic relationship, gene structure, and conserved motifs of Pti1s and predicted the potential cis-elements in the promoters of Pti1 genes using bioinformatics methods. Importantly, we systematically summarized the diverse functions of Pti1s in tomato, rice, Arabidopsis, potato, apple, and cucumber. The potential cis-elements in promoters of Pti1s decide their functional diversity in response to various biotic and abiotic stresses. The protein kinase Pti1 was phosphorylated by Pto and then modulated the downstream signaling pathways for PTI and ETI in the disease insistence process. In addition, some transcription factors have been defined as Ptis (e.g., Pti4, Pti5, and Pti6) originally, which actually were ethylene-response factors (ERFs). Pti4, Pti5, and Pti6 were modulated by salicylic acid (SA), jasmonate (JA), and ethylene signaling pathways and regulated diverse defense-related gene expression to cope with Pst infection and insect wounding.
