Co-immunoprecipitation for Assessing Protein–Protein Interactions in Agrobacterium-Mediated Transient Expression System in Nicotiana benthamiana

Nam, Ji Chul; Bhatt, Puneet; Kim, Sung-Il; Kang, Hong-Gu

doi:10.1007/978-1-0716-3327-4_9

Cited by 4 publications

(1 citation statement)

References 25 publications

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“…Transient gene expression obtained in plant is sufficient for RNA and protein analysis, with potential for laboratory-scale protein production in a short period of time [ 54 , 55 ]. Tobacco is the most widely used species to study the gene function using the transient expression method [ 56 , 57 ]. However, expression of heterologous gene has limited applications for validating gene function.…”

Section: Discussionmentioning

confidence: 99%

A high-efficiency transient expression system mediated by Agrobacterium tumefaciens in Spinacia oleracea leaves

Zhang,

Qiu,

Zhang

et al. 2024

Plant Methods

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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 tumefaciens‑mediated 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.

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Section: Discussionmentioning

confidence: 99%

A high-efficiency transient expression system mediated by Agrobacterium tumefaciens in Spinacia oleracea leaves

Zhang,

Qiu,

Zhang

et al. 2024

Plant Methods

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Transient Gene Expression in Plants Is an Efficient Experimental Platform for Functional Genomics

Goldenkova-Pavlova,

Pavlenko,

Demyanchuk

et al. 2024

Russ J Plant Physiol

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

Zhang,

Qiu,

Zhang

et al. 2023

Preprint

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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.

show abstract

Co-immunoprecipitation for Assessing Protein–Protein Interactions in Agrobacterium-Mediated Transient Expression System in Nicotiana benthamiana

Cited by 4 publications

References 25 publications

A high-efficiency transient expression system mediated by Agrobacterium tumefaciens in Spinacia oleracea leaves

A high-efficiency transient expression system mediated by Agrobacterium tumefaciens in Spinacia oleracea leaves

Transient Gene Expression in Plants Is an Efficient Experimental Platform for Functional Genomics

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

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