The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development. While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized. We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains. We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs. Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant. Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress. We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.

Although phosphatidylinositol transfer proteins (PITPs) are known to serve critical functions in regulating a varied array of signal transduction processes in animals and yeast, the discovery of a similar class of proteins in plants occurred only recently. Here, we report the participation of Ssh1p, a soybean PITP-like protein, in the early events of osmosensory signal transduction in plants, a function not attributed previously to animal or yeast PITPs. Exposure of plant tissues to hyperosmotic stress led to the rapid phosphorylation of Ssh1p, a modification that decreased its ability to associate with membranes. An osmotic stress-activated Ssh1p kinase activity was detected in several plant species by presenting recombinant Ssh1p as a substrate in in-gel kinase assays. Elements of a similar osmosensory signaling pathway also were conserved in yeast, an observation that facilitated the identification of soybean protein kinases SPK1 and SPK2 as stress-activated Ssh1p kinases. This study reveals the activation of SPK1 and/or SPK2 and the subsequent phosphorylation of Ssh1p as two early successive events in a hyperosmotic stress-induced signaling cascade in plants. Furthermore, Ssh1p is shown to enhance the activities of a plant phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, an observation that suggests that the ultimate function of Ssh1p in cellular signaling is to alter the plant's capacity to synthesize phosphoinositides during periods of hyperosmotic stress. INTRODUCTIONPhosphatidylinositol transfer proteins (PITPs) were identified originally by their ability to serve as diffusible carriers of phosphatidylinositol (PtdIns) and to a lesser extent phosphatidylcholine (PtdCho) from one distinct membrane compartment to another by using an in vitro assay (Wirtz, 1991). In recent years, several intriguing and critical biological roles beyond the transfer of phospholipids have been attributed to yeast and animal PITPs. The yeast PITP (Sec14p) is an essential protein that is required for cells to properly execute the formation of secretory vesicles from the Golgi complex (Bankaitis et al., 1990). A considerable body of evidence suggests that Sec14p serves as a "molecular sensor" to monitor and regulate the levels of PtdIns, PtdCho, and potentially diacylglycerol in the Golgi complex of yeast (Skinner et al., 1995;Kearns et al., 1997). In addition, Sec14p has been implicated in modulating the activity of a PtdIns 4-kinase that regulates protein secretion (Hama et al., 1999).An essential role for PITPs also is observed in mammals and Drosophila, in which the loss of PITP function leads to specific neurodegenerative diseases (Hamilton et al., 1997;Milligan et al., 1997). At the cellular level, the mammalian PITP is known to be required for inositol lipid signaling, secretory vesicle formation from the trans -Golgi network, and the fusion of secretory vesicles to the plasma membrane (Hay and Martin, 1993;Cunningham et al., 1995;Kauffmann-Zeh et al., 1995). Although yeast and animal PITPs are very similar...

The distribution of the strA-strB streptomycin-resistance (Smr) genes associated with Tn5393 was examined in bacteria isolated from the phylloplane and soil of ornamental pear and tomato. Two ornamental pear nurseries received previous foliar applications of streptomycin, whereas the tomato fields had no prior exposure to streptomycin bactericides. Although the recovery of culturable Smr bacteria was generally higher from soil, the highest occurrence of Smr was observed in phylloplane bacteria of an ornamental pear nursery that received 15 annual applications of streptomycin during the previous 2 years. Twenty-two and 12% of 143 Gram-negative phylloplane and 163 Gram-negative soil isolates, respectively, contained sequences that hybridized to probes specific for the strA-strB Smr genes and for the transposase and resolvase genes of Tn5393. These sequences were located on large plasmids (> 60 kb) in 74% of the isolates. The 77 Smr Gram-positive bacteria isolated in the present study showed no homology to the Tn5393-derived probes. Although the repeated use of a single antibiotic in clinical situations is known to favor the development of strains with resistance to other antibiotics, we found no evidence that intensive streptomycin usage in agricultural habitats favors the development of resistance to tetracycline, an antibiotic also registered for disease control on plants. The detection of Tn5393 in bacteria with no prior exposure to streptomycin suggests that this transposon is indigenous to both phylloplane and soil microbial communities.

The bacterium Agrobacterium tumefaciens transforms eukaryotic hosts by transferring DNA to the recipient cell where it is integrated and expressed. Bacterial factors involved in this interkingdom gene transfer have been described, but less is known about host-cell factors. Using the yeast Saccharomyces cerevisiae as a model host, we devised a genetic screen to identify yeast mutants with altered transformation sensitivities. Twenty-four adenine auxotrophs were identified that exhibited supersensitivity to A. tumefaciens-mediated transformation when deprived of adenine. We extended these results to plants by showing that purine synthesis inhibitors cause supersensitivity to A. tumefaciens transformation in three plant species. The magnitude of this effect is large and does not depend on prior genetic manipulations of host cells. These data indicate the utility of yeast as a model for the transformation process and identify purine biosynthesis as a key determinant of transformation efficiency. These findings should increase the utility of A. tumefaciens in genetic engineering.A grobacterium tumefaciens, a Gram-negative soil bacterium, genetically transforms plants by transferring DNA to the host cell where it is integrated into the host chromosome and expressed. Exogenous DNA sequences introduced into transferred DNA (T-DNA) vectors can be delivered to plants, making A. tumefaciens a cornerstone of plant genetic engineering. Under controlled conditions, A. tumefaciens can also transform mammalian cells and a variety of fungi, including the yeast Saccharomyces cerevisiae (1-6).Understanding the cellular factors influencing transformation will provide broader insights into the mechanisms underlying interkingdom DNA transfer and should increase the utility of A. tumefaciens in genetic engineering. Bacterial factors that control virulence gene induction as well as processing and delivery of the T-DNA have been studied extensively (7,8). Recently, a few host-cell factors have been identified that participate in A. tumefaciens-mediated transformation. Studies in Arabidopsis thaliana have implicated histone H2A in chromosomal integration of the T-DNA (9). Studies in S. cerevisiae have implicated a nuclear pore protein in T-DNA nuclear import (10) and nonhomologous end-joining proteins in T-DNA chromosomal integration (11). To date, however, the facile yeast system has not been used to perform a large-scale screen to identify host factors that influence transformation sensitivity. Consequently, we devised a genetic screen to isolate yeast mutants with altered sensitivity to A. tumefaciens-mediated transformation. This approach revealed an unexpected link between transformation efficiency and de novo biosynthesis of adenine, an essential purine precursor of DNA, RNA, and ATP. Materials and MethodsStrains and Plasmids. The supervirulent A. tumefaciens strain EHA105 harboring pKP506 served as the bacterial donor strain in yeast-transformation experiments (1). The pKP506 plasmid contains the yeast TRP1 marker and the ARS1 rep...