Transfer and integration of a defined region (T-DNA) of the tumor-inducing (Ti) plasmid of Agrobacteriwm tulnefaciens is essential for tumor formation. We used a physical assay to study structural changes induced in Agrobacteriun T-DNA by cocultivation with plant cells. We show that nicks are introduced at unique; identical locations in each of the 24-base-pair imperfect direct repeats which flank the T-DNA and present evidence that a linear, single-stranded molecule is generated. We propose that these changes result from processing of the T-DNA for transfer and that they occur by a mechanism similar to DNA processing during conjugative DNA transfer between bacteria.Agrobacterium tumefaciens causes crown gall tumors on a wide variety of dicotyledonous plants. Many of the genes responsible for tumorigenesis are located on a large, tumorinducing (Ti) plasmid (for reviews, see references 8, 30, and 41). All Ti plasmids contain a region of DNA, the T-DNA, which is transferred from Agrobacterium cells to the plant cell. There it is integrated colinearly into the plant genome (reviewed in reference 5).Transfer of the T-DNA depends on cis-acting border elements consisting of 24-base-pair (bp) imperfect direct repeats which bound and functionally define the T-DNA (3, 44). T-DNAs located on the Ti plasmid require a rightward 24-bp repeat in its native orientation and relative position (20,32,42), while orientation and position requirements are less stringent for T-DNAs on separate replicons (binary vectors [16,18,19]). In addition, trans-acting functions are provided by a 35-kilobase region outside the T-DNA, the vir region (13,17,21,37). No transfer functions are encoded by the T-DNA itself (for example, see references 10, 22, and 47). Most analyses of the transfer process have depended on expression of either the oncogenic functions of the T-DNA or drug resistance markers which are expressed in plant cells and therefore have demanded both transfer and integration. More recent experiments attempting to separate transfer from integration by using viroid genomes cloned on small binary vectors (12) have confirmed the roles of the 24-bp repeat and vir loci in the transfer step. This paper focuses on the mechanism of transfer of the T-DNA to the plant genome, specifically, the "processing" which prepares T-DNA for transfer across the bacterial membrane. Several groups (1,25,26) intermediates which is similar to models for processing of DNA during conjugative DNA transfer between bacteria. MATERIALS AND METHODSBacterial strains and plant ceil culture. A348(pVK225) is A.tumefaciens A136 containing pTiA6NC (13), is merodiploid for virG, virC, virD, and virE, and is kanamycin resistant (23, 37). Bacteria were maintained on AB minimal agar (9) with kanamycin at 100 ,ug/ml when needed. A 1:1 mixture of L broth (28) and mannitol-glutamate broth (4) was used for growth in liquid medium, with kanamycin at 10 ,ug/ml as needed.Nicotiana tabacum suspension culture cell lines were maintained in supplemented liquid Murashige and Skoog (2...

The virulence (vir) region of Agrobacterium tumefaciens mediates the transfer of a defined segment of plasmid DNA (the T‐DNA) into the plant genome. The vir genes are specifically induced by molecules produced by wounded plant cells, and virA is required for this induction. We have determined the nucleotide sequence of virA loci from limited (pTiAg162) and wide (pTiA6) host range tumor‐inducing (Ti) plasmids, each of which encodes a single protein of 92,000 daltons. Using antibody directed against the virA gene product, we have localized the VirA protein to the bacterial inner membrane. VirA is homologous to at least four bacterial proteins which play a role in the transcriptional regulation of diverse families of genes. Based on its role in vir gene induction, homology to transcriptional regulators and membrane localization, we propose that VirA acts as an environmental sensor of plant‐derived inducer molecules and transmits this information to the level of vir gene expression. The pTiAg162 virA locus was shown to be ineffective at directing vir gene induction, suggesting that this may in part contribute to the narrow host range conferred by this plasmid.

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The maize rab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardell et al., Plant Mol Biol 14 (1990) 423-432). Here we demonstrate that 5' upstream sequences of the rab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5' upstream fragment of rab17 (-1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused to rab17 promoter deletions indicate that a 300 bp DNA fragment (-351/-102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (-219/-102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.