Increased 1‐aminocyclopropane‐1‐carboxylate deaminase activity enhances Agrobacterium tumefaciens‐mediated gene delivery into plant cells

Abstract

Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants.

An Agrobacterium tumefaciens Strain with Gamma-Aminobutyric Acid Transaminase Activity Shows an Enhanced Genetic Transformation Ability in Plants

“…The uidA gene was used as an indicator of T-DNA transfer. After 3 days of co-cultivation, tomato segments were stained with the GUS substrate, and the stained area was analyzed with ImageJ 24 ( https://imagej.nih.gov/ij/ ). The degree of staining was categorized into four classes, and the frequency of each class was calculated.…”

“…For instance, the phytohormone ethylene is a negative factor in Agrobacterium -plant interactions 17 18 19 20 . The ability of A. tumefaciens strains to reduce ethylene evolution from plants increases the transformation frequency 21 22 23 24 .…”

Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

“…The strain has been designated as super- Agrobacterium . It showed increased transient gene delivery into melon cotyledons ( Nonaka et al, 2008b ), tomatoes, and E. ravennae ( Someya et al, 2013 ). In addition, the super- Agrobacterium also increased stable transformation in Egusi melon and three canola species ( Ntui et al, 2009 ; Hao et al, 2010 ).…”

“…Panels (A,B) are cited from Nonaka et al (2008b) . Panels (C,D) are cited from Someya et al (2013) .…”

“…This newly developed A. tumefaciens strain showed higher T-DNA transformation efficiency in tomatoes (Figures 2C,D ) and higher biomass production than Erianthus ravennae , which shows very low transformation frequency. Therefore, A. tumefaciens with higher ACC deaminase activity is a powerful tool for the Agrobacterium -mediated genetic engineering of plants ( Someya et al, 2013 ). Introducing ACC deaminase into A. tumefaciens is effective at increasing the stable transformation frequency.…”