Molecular Genetic Characterization of the Mating Pair Formation and Vegetative Replication Regions on the Agrobacterium Tumefaciens Ti Plasmid pTiC58
Li, Pei-Li
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https://hdl.handle.net/2142/85064
Description
Title
Molecular Genetic Characterization of the Mating Pair Formation and Vegetative Replication Regions on the Agrobacterium Tumefaciens Ti Plasmid pTiC58
Author(s)
Li, Pei-Li
Issue Date
2000
Doctoral Committee Chair(s)
Farrand, Stephen K.
Department of Study
Crop Sciences
Discipline
Crop Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture, Plant Pathology
Language
eng
Abstract
Conjugal transfer of pTiC58 requires the tra region and an undefined region located at the 2-o'clock position of this plasmid. This region, now called trb, and the adjacent vegetative replication (rep) region have been the focus of this dissertation research. Nucleotide sequence analysis of the 11-kb trb region identified 13 significant open reading frames (ORFs). The first, encoding traI, is responsible for the synthesis of Agrobacterium autoinducer (AAI) which is the co-inducer of the transcriptional activator, TraR. The next 11 ORFs showed similarities with the trb genes of RP4, which encode mating pair formation (Mpf) functions essential for conjugation, as well as with other genes from the type IV secretion family. Expression of the Ti trb genes required the traI promoter, TraR, and AAI. Genetic studies showed that non-polar insertion mutations in 10 of the 11 trb genes abolished conjugal transfer, whereas the trbI mutant transferred at very low but detectable levels. Transfer of the trbK mutant was unaffected. Transfer of each of the mutants could be restored by a clone expressing only the corresponding trb gene. The rep locus contained three ORFs that are homologous to repA, repB, and repC of the RepABC-type replicator of several large plasmids found in the family Rhizobiaceae. The TraR recognition sites located in the traI-repA intergenic region affected expression of both traI-trb operon and repA. Copy number of a minimal rep plasmid and the Ti plasmid increased in the presence of TraR and AAI. We concluded that, (A) The Trb system of the Ti plasmid is responsible for the Mpf functions and belongs to the type IV secretion family; (B) With the exception of trbI and trbK all of the genes in the traI-trb operon are essential for conjugal transfer of pTiC58; (C) Copy number of pTiC58 is influenced by the quorum-sensing system, suggesting a connection between conjugation and replication of this virulence element. Finally, we proposed that the large plasmids from the family Rhizobiaceae evolved from a basic rep-tra-trb replicon. Other loci encoding various functions were incorporated into this hypothetical replicon in a modular fashion to give each plasmid its distinct characteristics.
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