Photorhabdus luminescens is an enterobacterium that is symbiotic with soil entomopathogenic nematodes and pathogenic to a wide range of insects. P. luminescens promotes its own transmission among susceptible insect populations using its nematode host as vector 1 . Its life cycle comprises a symbiotic stage in the nematode's gut and a virulent stage in the insect larvae, which it kills through toxemia and septicemia. After the nematode attacks a prey insect and P. luminescens is released, the bacterium produces a wide variety of virulence factors ensuring rapid insect killing. Bioconversion of the insect cadaver by exoenzymes produced by the bacteria allows the bacteria to multiply and the nematode to reproduce. During this process P. luminescens produces antibiotics to prevent invasion of the insect cadaver by bacterial or fungal competitors. Finally, elimination of competitors allows P. luminescens and the nematode to reassociate specifically before leaving the insect cadaver 2,3 .To better understand this complex life style, we determined the genome sequence of P. luminescens subspecies laumondii strain TT01 4 , a symbiont of the nematode Heterorhabditis bacteriophora isolated on Trinidad and Tobago. RESULTS General featuresStrain TT01 possesses a single circular chromosome of 5,688,987 bp with an average GC content of 42.8%. No plasmid replicon was found.A total of 4,839 protein-coding genes, including 157 pseudogenes, seven complete sets (23S, 5S and 16S) of ribosomal RNA operons and 85 tRNA genes, were predicted ( Fig. 1; Supplementary Table 1 online). Toxins against insectsMore toxin genes were predicted in the P. luminescens genome than in any other bacterial genome sequenced yet. A large number of these toxins may be involved in the killing of a wide variety of insects. Some may act synergistically or use redundancy for 'overkill' 5 , ensuring a quick death of the host. In addition, some may kill insects by interfering with their development. In the TT01 genome, two paralogs, plu4092 and plu4436, encode proteins similar to juvenile hormone esterases (JHEs) of the insect Leptinotarsa decemlineata 6 . Juvenile hormone maintains the insect in a larval state. Its inactivation by JHE allows metamorphosis to proceed. JHEs may be used to trigger the insect endocrine machinery at an inappropriate time and thus represents a promising approach for insect control 7 . These genes are located downstream of highly related orphan genes (plu4093 and plu4437), suggesting a locus duplication.The toxicity of the proteins encoded by these two loci was verified experimentally. Two Escherichia coli clones, containing the recombinant BAC1A02 and BAC8C11, were shown to be toxic toward insects. BAC1A02, which contains the locus plu4093-plu4092, exhibited substantial oral toxicity toward three mosquito species, Aedes aegypti,

The reduced form of glutathione (GSH), when supplied to suspension cultured cells of bean (Phaseolus vulgaris L.) at concentrations in the range 0.01 to 1.0 millimolar, stimulates transcription of defense genes including those that encode cel wall hydroxyproline-rich glycoproteins and the phenylpropanoid biosynthetic enzymes phenylalanine ammonialyase (PAL) and chalcone synthase (CHS) involved in lignin (PAL) and phytoalexin (PAL, CHS) production. Transcriptional activation of these genes leads to marked accumulation of the corresponding transcripts, contributing to a massive change in the overall pattern of protein synthesis which closely resembles that previously observed in response to fungal elicitor. GSH causes a marked increase in extractable PAL activity, whereas the oxidized form of glutathione, constituent amino acids, or other reducing agents are inactive. Possible roles of GSH in signaling biological stress are discussed.Glutathione (y-L-glutamyl-L-cysteinyl-glycine) is a low mol wt thiol implicated in a wide range of metabolic processes (16). Functions proposed for glutathione in higher plants include: storage and transport of reduced sulfur; protein reductant; destruction of H202 in chloroplasts, and detoxification of xenobiotics including certain herbicides and pesticides (8,20). Overall, glutathione appears to play a key role in protection against oxidative damage arising from a number of stresses such as irradiation (16), heat (18), and exposure to heavy metals (10).Redox perturbations including generation of superoxide anions and lipid peroxidation appear to be a characteristic response to mechanical damage and microbial infection (4). Moreover, certain sulfhydryl reagents stimulate the production of phytoalexins and the activation of other defense responses associated with the expression of disease resistance (11,24). Taken

Secretion is a fundamental process providing plants with the means for disposal of solutes, improvement of nutrient acquisition, and attraction of other organisms. Specific secretory organs, such as nectaries, hydathodes, and trichomes, use a combination of secretory and retrieval mechanisms, which are poorly understood at present. To study the mechanisms involved, an Arabidopsis thaliana activation tagged mutant, glutamine dumper1 (gdu1), was identified that accumulates salt crystals at the hydathodes. Chemical analysis demonstrated that, in contrast with the amino acid mixture normally present in guttation droplets, the crystals mainly contain Gln. GDU1 was cloned and found to encode a novel 17-kD protein containing a single putative transmembrane span. GDU1 is expressed in the vascular tissues and in hydathodes. Gln content is specifically increased in xylem sap and leaf apoplasm, whereas the content of several amino acids is increased in leaves and phloem sap. Selective secretion of Gln by the leaves may be explained by an enhanced release of this amino acid from cells. GDU1 study may help to shed light on the secretory mechanisms for amino acids in plants.

Phenylalanine ammonia-lyase and chalcone synthase catalyze the first reaction of phenylpropanoid biosynthesis and the first reaction of a branch pathway specific for flavonoid-isoflavonoid biosynthesis, respectively. These enzymes are key control elements in the synthesis of kievitone, phaseollin, and related isoflavonoidderived phytoalexins. RNA blot hybridization with 32P-labeled cDNA sequences was used to demonstrate marked accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs in excision-wounded hypocotyls of Phaseolus vulgans L. (dwarf French bean) and during race-cultivar-specific interactions between hypocotybls of P. vulgans and the partialiy biotrophic fungus CoUetotrichum lindemudtianum, the causal agent of anthracnose. In an incompatible interaction (host resistant), early concomitant accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs, loalized malnly but not entirely in tissue adjacent to the site of infection, was observed prior to the onset of phytoalexin accumulation and expression of localized, hypersensitive resitance. In contrast, in a compatible interaction (host susceptible) there was no early accumulation of these transcripts; instead, there was a delayed widespread response associated with phytoalexin accumulation during attempted lesion limitation. Two-dimensional gel electrophoresis of [S]methionine-labeled polypeptides synthesized in vitro by translation of isolated polysomal RNA demonstrated stimulation of the synthesis of characteristic sets of phenylalanine ammonia-lyase and chalcone synthase isopolypeptides in directly infected tissue and distant, hitherto uninfected tissue in both compatible and incompatible interactions. Our data show that specific accumulation of plant defense gene tanscripts is a key early component in the sequence of events leading to expression of defense responses in wounded tissue and in infected tissue during race-cultivar-specific interactions and that an elicitation signal is transmitted intercellularly in response to infection.

Murine monoclonal antibodies to membrane antigens were generated by immunization with a crude cellular membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. From a panel of thirteen monoclonal antibodies, seven were found to be directed against antigens present on the plasma-membrane by immunofluorescence visualization of antibody binding to the surface of isolated protoplasts. The corresponding set of plasma-membrane antigen(s) were present in root, shoot and leaf tissue and some but not all of these antigens were of wide species distribution, being found in Nicotiana tabacum L., N. plumbaginifolia L., Glycine max L., Phaseolus vulgaris L. and Triticum aestivum L. Topologically specific labeling of intact protoplasts with a monoclonal antibody reactive with an epitope present on the plasma-membrane specifically labeled a membrane fraction which equilibrated at a density of 1.14 kg/l following centrifugation in a sucrose gradient. In addition to use as biochemical markers for fractionation and molecular characterization of plasma-membranes, these monoclonal antibodies provide the basis for new selection tools in plant cell and gene manipulations.