A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid

K. Palanichelvam, P. Oger, S. J. Clough, C. Cha, A. F. Bent, S. K. Farrand

Research output: Contribution to journalArticle

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 (T(R)), that confers production of the Amadori opines. The oncogenic T-left region (T(L)) 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 T(R) and the leftmost 9 kb of T(L). 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 Amodori opines at high frequency. All opine-positive transgenic plants also contained T(R)-DNA, while those plants that lacked T(R)-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.

Original languageEnglish (US)
Pages (from-to)1081-1091
Number of pages11
JournalMolecular Plant-Microbe Interactions
Volume13
Issue number10
DOIs
StatePublished - Jan 1 2000

Keywords

  • Binary system
  • Disarmed plasmid
  • Glycine max
  • T-DNA

ASJC Scopus subject areas

  • Physiology
  • Agronomy and Crop Science

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