Mutations that Affect Agrobacterium vitis-Induced Grape Necrosis also Alter Its Ability to Cause a Hypersensitive Response on Tobacco.

ABSTRACT Tn5-induced mutations in Agrobacterium vitis F2/5 resulted in both altered grape necrosis and tobacco leaf panel collapse phenotypes, suggesting that the underlying mechanisms of the reactions are related. The reaction on tobacco resembles the classical hypersensitive response (HR) caused by several plant pathogenic bacteria in that it is observable within 14 h, is inhibited by treatment of plants with metabolic inhibitors, and results in the inability to recover the pathogen from the necrotic zone. Strains of A. vitis differ with regard to their efficiency of causing the reaction on tobacco. An EcoRI fragment from one mutant, M6, which is necrosis-altered and HR-minus, was cloned and sequenced. Sequence analysis revealed that the Tn5 insertion occurred in a region that shares significant homology with genes involved in long chain fatty acid production by the marine bacteria Shewanella spp. and Moritella marina. Complementation of M6 with a cosmid clone from an F2/5 DNA library restored the tobacco HR and grape necrosis phenotypes.

Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization.

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24 h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40-50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kb Hind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells of Escherichia coli, and colonies were recovered that carried pGEM with Hind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transform E. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning into E. coli.

Variation in host fruit volatiles attractive to apple maggot fly,Rhagoletis pomonella.

We conducted a comparative study of volatiles produced by wholeCrataegus hawthorn fruit and four cultivars of apple (Royal Red Delicious, Red Astrachan, McIntosh, and Wealthy) and determined quantitative and qualitative changes of volatiles associated with fruit ripening. Within the approximate range of the GLC fraction known to elicit behavioral activity in the apple maggot fly,Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), 52 esters were identified.

Excretion and tissue disposition of dichloroacetonitrile in rats and mice.

The excretion and tissue distribution of [1-14C]dichloroacetonitrile and [2-14C]dichloroacetonitrile were studied in male Fischer 344 rats and male B6C3F1 mice. Three dose levels of dichloroacetonitrile (DCAN) (0.2, 2, or 15 mg/kg) were administered to rats and two dose levels of DCAN (2 or 15 mg/kg) to mice. Daily excreta including exhaled volatiles and radiolabeled carbon dioxide (14CO2) were analyzed for radiolabeled carbon (14C) until greater than 70% of the radioactivity was excreted. At that time the animals were sacrificed and tissues were collected. Tissues and excreta were analyzed for 14C by combustion and liquid scintillation counting. Rats administered [1-14C]DCAN excreted 62 to 73% of the 14C in 6 days, with 42 to 45% in urine, 14 to 20% in feces, and 3 to 8% as CO2. Rats administered [2-14C]DCAN excreted 82 to 86% of the 14C in 48 hr, with 35 to 40% in urine, 33 to 34% as CO2, and 10 to 13% in feces. Mice administered [1-14C]DCAN excreted 83 to 85% of the 14C in 24 hr, with 64 to 70% in urine, 9 to 13% in feces, and 5 to 6% as CO2. Mice administered [2-14C]DCAN excreted 84 to 88% of the 14C in 24 hr with 42 to 43% in urine, 8 to 11% in feces, and 31 to 37% as CO2. Liver tissues retained the most 14C in all studies except the study of [1-14C]DCAN in rats, where blood contained the most 14C. These results indicate that DCAN was absorbed rapidly after oral administration in water. The differences in the route of excretion of [1-14C]DCAN compared to [2-14C]DCAN indicated that the molecule was being cleaved in the body and metabolized by different mechanisms.