1-Aminocyclopropane-1-Carboxylate Deaminase Enhances Agrobacterium tumefaciens -Mediated Gene Transfer into Plant Cells

Abstract

Agrobacterium-mediated gene transfer is widely used for plant molecular genetics, and efficient techniques are required. Recent studies show that ethylene inhibits the gene transfer. To suppress ethylene evolution, we introduced 1-aminocyclopropane-1-carboxylate (ACC) deaminase into Agrobacterium tumefaciens. The ACC deaminase enhanced A. tumefaciens-mediated gene transfer into plants.Agrobacterium-mediated gene transfer is widely used for plant molecular genetics and its applications (14). In particular, efficient systems of genetic transformation are required for plant functional genomics and molecular breeding to improve traits (20,21). Recent studies showed that ethylene is one of the factors that inhibits Agrobacterium-mediated gene transfer (1, 3, 5). Therefore, if Agrobacterium tumefaciens has the ability to decrease the ethylene level in the host plant, the frequency of gene transfer will increase. To suppress ethylene evolution in plant cells during cocultivation, we introduced the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Pseudomonas sp. strain ACP (7, 18) into A. tumefaciens. ACC deaminase cleaves ACC (the immediate ethylene precursor) into ␣-ketobutyrate and ammonia, and as a result, the ethylene level is decreased (4, 12, 16).The ACC deaminase gene was amplified and cloned into pBBR1MCS-5 (10), a broad-host-range plasmid, to generate a lacZ::acdS translational fusion (Fig. 1A). The resulting plasmid was designated pBBRacdS and was introduced into A. tumefaciens C58 (17) or C58C1Rif R (2) by electroporation (19). The binary vector pIG121-Hm, involved in T-DNA transfer (6), was also harbored in A. tumefaciens C58C1Rif R (pBBRacdS, pIG121-Hm). The ACC deaminase activity in A. tumefaciens C58C1Rif R (pBBRacdS, pIG121-Hm) was assayed according to the method of Honma and Shimomura (7). The amount of ␣-ketobutyrate in the reaction buffer was estimated from a standard curve based on a dilution of 10 to 400 M (detected at 340 nm). The controls for this experiment were C58C1Rif R (pBBRMCS-5, pIG121-Hm) and samples in reaction buffer without the substrate 200 M ACC. The accumulation of ␣-ketobutyrate was observed only in the lysate from A. tumefaciens C58C1Rif R (pBBRacdS, pIG121-Hm) in the presence of the substrate (Fig. 1B). Therefore, we succeeded in conferring ACC deaminase activity on A. tumefaciens.Surface-sterilized melon (Cucumis melo L. var. cantaloupensis cv. Vedrantais) seeds were sown on a half-strength preparation of Murashige and Skoog's medium (MS) (13) and incubated at 25°C with 16 h of light per day for 5 days. Cotyledons from the germinated seedlings were transversely sectioned by hand into five pieces, and among these five, three internal pieces were inoculated. The segments were soaked in an A. tumefaciens cell suspension of 10 7 cells ml Ϫ1 for 20 min and then placed on cocultivation medium (MS containing 1.0 mg of 6-benzylamino-purine liter Ϫ1 , 2% glucose, and 0.4% Gelrite [Wako, Tokyo, Japan], pH 5.5) in a gas vial with 16 h of light per day. Thirty melon cotyledon seg...

An efficient Agrobacterium tumefaciens-mediated genetic transformation of “Egusi” melon (Colocynthis citrullus L.)

“…It might be that EHA101(pIG121-Hm, pBBRacdS) with ACC deaminase gene reduced ethylene evolution in ''Egusi'' cotyledons and increased their ability for stable transformation, as indicated previously (Nonaka et al 2008a, b). It has been shown that ACC deaminase cleaves ACC (the immediate ethylene precursor) into a-ketobutyrate and ammonia, and as a result decreases ethylene level and enhances the ability of gene transfer from A. tumefaciens into plant tissues (Nonaka et al 2008a).…”

“…The plasmid pBBRacdS was kindly provided by Dr. Horoshi Ezura, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai1-1-1, Tsukuba, Japan, and was introduced into EHA101(pIG121-Hm) using the heat-shock method. Binary vector pIG121-Hm harboured hygromycin phosphotransferase (hpt), neomycin phosphotransferase II (nptII), and b-glucuronidase (gus) as reporter genes in T-DAN region, while pBBRacdS contained 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene, which was reported to increase transformation efficiency by inhibiting ethylene production of explants (Nonaka et al 2008a, Fig. 1b).…”

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

“…Ethylene is the primary cause of vitrification (Kevers et al 1984), and also inhibits vir gene induction of Agrobacterium (Nonaka et al 2008b). pSuperAgro (Inplanta Innovations Inc. Kanagawa, Japan) is a plasmid for Agrobacterium containing a 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene that suppresses ethylene evolution during co-cultivation (Nonaka et al 2008a). Selective agent is another factor in efficient transformation.…”

Agrobacterium-mediated transformation of protocorm-like bodies in Cattleya

“…Agrobacterium tumefaciens strain EHA101 (Hood et al 1986) carrying either plasmid pIG121-Hm (Ohta et al 1990), harboring genes coding for neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt), and b-glucuronidase (GUS) in the T-DNA region or plasmid pBBRacdS harboring the same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene, which was reported to increase the frequency of gene transfer by decreasing the ethylene level in the host plant (Nonaka et al 2008), was used.…”