No abstract

Agrobacterium tumefaciens transfers its Ti‐plasmid T‐DNA to plant cells. This process is initiated by plant‐induced activation of the Ti‐plasmid virulence loci, resulting in the generation of single stranded (ss) cleavages of the Ti‐plasmid T‐DNA border sequences (border nicks) and ss linear unipolar T‐DNA molecules (T‐strands). A single T‐strand is produced from the two‐border T‐region of the pGV3850 nopaline plasmid. In this paper the induced molecular events for the complex T‐region of the pTiA6 octopine plasmid are analyzed. This T‐region carries four T‐DNA borders delimiting three T‐DNA elements (TR, TC and TL). Induction of pTiA6 generates cleavages independently at its border repeats, and six distinct T‐strand species corresponding to TR, TR/TC, TR/TC/TL, TC, TC/TL and TL. These T‐strand molecules are linear and correspond to the bottom strand of the pTiA6 T‐region. Thus, borders can function for both initiation and termination of T‐strand synthesis. We propose that the different pTiA6 T‐strands are independently generated, and that the distribution of border nicks within the parental T‐region determines which T‐strand is produced. To identify genes involved in T‐strand production, pTiA6 virulence (vir) and chromosomal virulence (chv) mutant strains were analyzed. VirA and VirG, the vir regulatory loci are required. Furthermore, the two 5′ cistrons of virD are required for both border nicks and T‐strands, suggesting that these genes encode the border endonuclease, and that T‐strand production is dependent on border nicks. That no mutants are defective for T‐strands alone suggests that functions encoded outside of vir and chv might mediate some of the later reactions of T‐strand synthesis.

Detection, quantification, separation and characterization of T and B cells reactive to specific antigens are important tasks in both basic and clinical immunology. Here, we describe an approach allowing the performance of all four tasks on a functional basis by flow cytometry. The assay is based on the property of lymphocytes to capture membrane components from the cells they interact with, in a process we call trogocytosis. Working with CD8 + CTL and target cells labeled with membrane markers, we describe the conditions allowing reactive lymphocytes to be detected rapidly and inexpensively within mixed populations. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this method to the detection of antigen-specific CD4 + T and B cells. While our method is, for the time being, not as sensitive as staining of CTL with MHC class I multimers, it allows the simultaneous quantitative identification of reactive CD8 + , CD4 + and B cells. Altogether, our method offers a simple and general alternative to other methods previously described to detect and quantify lymphocyte reactivity, and it can also be used in combination with those.

The T-DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the expression of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. The vir gene products act in trans to mobilize the T-DNA element from the bacterial Ti plasmid. The T-DNA is bounded by 25-base pair direct repeat sequences, which are the only sequences on the element essential for transfer. Thus, specific reactions must occur at the border sites to generate a transferable T-DNA copy. The T-DNA border sequences were shown in this study to be specifically nicked after vir gene activation. Border nicks were detected on the bottom strand just after the third or fourth base (+/- one or two nucleotides) of the 25-base pair transferpromoting sequence. Naturally occurring and base-substituted derivatives of the 25-base pair sequences are effective substrates for acetosyringone-induced border cleavage, whereas derivatives carrying only the first 15 or last 19 base pairs of the 25-base pair sequence are not. Site-specific border cleavages occur within 12 hours after acetosyringone induction and probably represent an early step in the T-DNA transfer process.

In an attempt to elucidate the transfer and integration mechanism of Agrobacterium DNA upon crown gall induction, we translocated a borderless T‐DNA to different sites of the C58 Ti plasmid. As a result of the physical linkage of the T‐DNA onc genes with other Ti plasmid functions, the concerned strain retained tumor‐inducing capacity. However, when the borderless T‐DNA is separated on an independent replicon while all other pTi functions are provided in trans, the strain can no longer induce tumors on plants. We provide evidence that the right T‐DNA border region harbors one or more in cis active functions essential in the transfer and/or stabilization of the T‐DNA into plant cells. The strains used in these experiments allowed us to conclude that some function(s) of the Ti plasmid can induce plant cell proliferations independently of the T‐DNA transformation event. The results described here indicate that other Ti plasmid sequences than solely the T‐region can be transferred to plant cells.