Optimization of Agrobacterium-mediated transient expression of heterologous genes in spinach

TY - JOUR

T1 - Optimization of Agrobacterium-mediated transient expression of heterologous genes in spinach

AU - Cao, Dang Viet

AU - Pamplona, Reniel S.

AU - Kim, Jiwon

AU - Oh, Young Kyoung

AU - Cho, Seok Keun

AU - Ahn, Jongcheol

AU - Yang, Seong Wook

AU - Riu, Key Zung

AU - Boo, Kyung Hwan

PY - 2017

Y1 - 2017

N2 - The Agrobacterium-mediated transient assay is a relatively rapid technique and a promising approach for assessing the expression of a gene of interest. Despite the successful application of this transient expression system in several plant species, it is not well understood in spinach. In this study, we analyzed various factors, including infiltration method, Agrobacterium strain and density, and co-infiltration of an RNA silencing suppressor (p19), that affect transient expression following agroinfiltration in spinach. To evaluate the effects of these factors on the transient expression system, we used the β-glucuronidase (GUS) reporter gene construct pB7WG2D as a positive control. The vacuum-based infiltration method was much more effective at GUS gene expression than was the syringe-based infiltration method. Among the three Agrobacterium strains examined (EHA105, LBA4404, and GV2260), infiltration with the GV2260 strain suspension at a final optical cell density (OD600) of 1.0 resulted in the highest gene expression. Furthermore, co-expression of suppressor p19 also increased the efficiency and duration of gene expression and protein accumulation. The results indicate that the use of optimized conditions for transient gene expression could be a simple, rapid, and effective tool for functional genomics in spinach.

AB - The Agrobacterium-mediated transient assay is a relatively rapid technique and a promising approach for assessing the expression of a gene of interest. Despite the successful application of this transient expression system in several plant species, it is not well understood in spinach. In this study, we analyzed various factors, including infiltration method, Agrobacterium strain and density, and co-infiltration of an RNA silencing suppressor (p19), that affect transient expression following agroinfiltration in spinach. To evaluate the effects of these factors on the transient expression system, we used the β-glucuronidase (GUS) reporter gene construct pB7WG2D as a positive control. The vacuum-based infiltration method was much more effective at GUS gene expression than was the syringe-based infiltration method. Among the three Agrobacterium strains examined (EHA105, LBA4404, and GV2260), infiltration with the GV2260 strain suspension at a final optical cell density (OD600) of 1.0 resulted in the highest gene expression. Furthermore, co-expression of suppressor p19 also increased the efficiency and duration of gene expression and protein accumulation. The results indicate that the use of optimized conditions for transient gene expression could be a simple, rapid, and effective tool for functional genomics in spinach.

KW - Agrobacterium tumefaciens

KW - Spinach

KW - Syringe

KW - Transient assay

KW - Vacuum

U2 - 10.1007/s11816-017-0457-4

DO - 10.1007/s11816-017-0457-4

M3 - Journal article

AN - SCOPUS:85032181097

SN - 1863-5466

VL - 11

SP - 397

EP - 405

JO - Plant Biotechnology Reports

JF - Plant Biotechnology Reports

IS - 6

ER -