A Second T-Region of the Soybean-Supervirulent Chrysopine-Type Ti Plasmid pTiChry5, and Construction of a Fully Disarmed vir Helper Plasmid

Abstract

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

RecombinantAgrobacteriumAgaE-like Protein with Fructosyl Amino Acid Oxidase Activity

“…Recently, the genome structures of A. tumefaciens and Masorhizobium loti were analyzed. A putative reductase from A. tumefaciens (GenBank AAK08613) 20) and a hypothetical protein from M. loti (DDBJ The amino acid sequence of the putative reductase deduced from the database sequence was composed of 299 amino acid residues. The primary structure presumed from the sequence of the open reading frame was similar to the AgaE-like protein.…”

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…In addition, regeneration of whole plants from transformed somatic cells sometimes leads to somatic mutations. The presence of phytohormones in the tissue culture during regeneration also increases the chance of somatic mutations (Bent, 2000;Bent, 2006;Tague and Mantis, 2006). In order to resolve these difficulties, several plant transformation methods have been established and improved (Feldmann and Marks, 1987;Bechtold et al, 1993;Chang et al, 1994;Katavic et al, 1994;Clough and Bent, 1998;Richardson et al, 1998;Martinez-Trujillo et al, 2004;Zhang et al, 2006).…”

“…It was later discovered that submergence of inflorescences in the early flowering stages in a A. tumefaciens suspension is sufficient to obtain successful transformants (Clough and Bent, 1998). Furthermore, the vacuum infiltration process can be eliminated; simple dipping of developing floral tissues into the bacterial suspension is enough to acquire transformants in the next generation (Clough and Bent, 1998;Richardson et al, 1998;Bent, 2000). A. tumefaciens strains are first cultured in rich media with appropriate antibiotic selection until stationary phase (OD 600 = 0.8-1.0).…”

“…A. tumefaciens strains are first cultured in rich media with appropriate antibiotic selection until stationary phase (OD 600 = 0.8-1.0). The A. tumefaciens cultures are then collected by centrifugation and the pellet is resuspended in a solution containing 5% sucrose and the surfactant Silwet L-77 (Clough and Bent, 1998;Bent, 2000). Both sucrose and surfactant 0.05% Silwet L-77 are important for the success of the floral dip method (Clough and Bent, 1998;Richardson et al, 1998;Bent, 2000;Bent, 2006).…”

Sorghum Transformation: Overview and Utility

“…The inoculum is an A. tumefaciens strain NTL 4 /pTiPKPSF2 (Palanichelvam et al 2000), suspension in cocultivation medium (OD 660 0.3-0.5). Inoculation time is 5 min.…”