Characterization of a 34-kDa soybean binding protein for the syringolide elicitors.

Syringolides are water-soluble, low-molecular-weight elicitors that trigger defense responses in soybean cultivars carrying the Rpg4 disease-resistance gene but not in rpg4 cultivars. 125I-syringolide 1 previously was shown to bind to a soluble protein(s) in extracts from soybean leaves. A 34-kDa protein that accounted for 125I-syringolide 1 binding activity was isolated with a syringolide affinity-gel column. Partial sequences of internal peptides of the 34-kDa protein were identical to P34, a previously described soybean seed allergen. In soybean seeds, P34 is processed from a 46-kDa precursor protein and was shown to have homology with thiol proteases. P34 is a moderately abundant protein in soybean seeds and cotyledons but its level in leaves is low. cDNAs encoding 46-, 34-, and 32-kDa forms of the soybean protein were cloned into the baculovirus vector, pVL1392, and expressed in insect cells. The resulting 32- and 34-kDa proteins, but not the 46-kDa protein, exhibited ligand-specific 125I-syringolide binding activity. These results suggest that P34 may be the receptor that mediates syringolide signaling.

Specific Binding of the Syringolide Elicitors to a Soluble Protein Fraction from Soybean Leaves.

Syringolides are glycolipid elicitors produced by Gram-negative bacteria expressing Pseudomonas syringae avirulence gene D. The syringolides mediate gene-for-gene complementarity, inducing the hypersensitive response only in soybean plants carrying the Rpg4 disease resistance gene. A site(s) for 125I-syringolide 1 was detected in the soluble protein fraction from soybean leaves, but no evidence for ligand-specific binding to the microsomal fraction was obtained. The Kd value for syringolide 1 binding with the soluble fraction was 8.7 nM, and binding was greatly reduced by prior protease treatment or heating. A native gel assay was also used to demonstrate ligand-specific binding of labeled syringolide 1 with a soluble protein(s). Competition studies with 125I-syringolide 1 and several structural derivatives demonstrated a direct correlation between binding affinity to the soluble fraction and elicitor activity. However, differential competition binding studies disclosed no differences in syringolide binding to soluble fractions from Rpg4/Rpg4 or rpg4/rpg4 soybean leaves. Thus, the observed binding site fulfills several criteria expected of an intracellular receptor for the syringolides, but it is most likely not encoded by the Rpg4 gene. Instead, the Rpg4 gene product may function subsequent to elicitor binding, possibly in intracellular signal transduction.

Prevention of skeletal muscle catabolism in sepsis does not impair visceral protein metabolism.

We investigated whether the preservation of gastrocnemius proteins by interleukin-1 receptor antagonist (IL-1ra) during sepsis altered protein metabolism in visceral tissues. Sepsis was induced by creation of an abdominal abscess followed by infusion of saline of IL-1ra. Five days later, the tissue protein content and rate of protein synthesis were measured. IL-1ra did not significantly alter hepatic protein metabolism in septic or control animals. In kidney, the protein content and rate of protein synthesis were both decreased by sepsis and significantly ameliorated by the infusion of IL-1ra. Sepsis decreased the rate of protein synthesis in the small intestine. IL-1ra increased intestinal protein synthesis in both control and septic animals; however, the effects were localized to the seromuscular layer. The preservation of muscle protein by IL-1ra in sepsis did not adversely affect protein synthesis in any of the visceral tissues examined. IL-1 appears to mediate the sepsis-induced changes in protein synthesis in kidney and small intestine but not in liver or spleen. Protein synthesis in each visceral organ responds differently to the septic insult and modulation of IL-1 bioactivity.

Avirulence gene avrPphC from Pseudomonas syringae pv. phaseolicola 3121: a plasmid-borne homologue of avrC closely linked to an avrD allele.

Cosmid clone pPsp01 from race 1 Pseudomonas syringae pv. phaseolicola isolate 3121 conferred a unique pattern of soybean cultivar reactions when expressed in P. s. pv. glycinea R4. The avirulence phenotype was shown to result from the presence in clone pPsp01 of an avrD allele as well as an additional avirulence gene located approximately 5-kb upstream. The new gene, called avrPphC, shows high identity to and is phenotypically identical to avrC, previously cloned from P. s. pv. glycinea race 0. avrD and avrPphC occur on an approximately 120-kb indigenous plasmid in P. s. pv. phaseolicola 3121. Although commonly observed in Xanthomonas campestris, this is the first noted occurrence of multiple avirulence genes on a single plasmid in Pseudomonas syringae. Unlike avrD, however, avrPphC does not appear to occur widely in pathovars of Pseudomonas syringae.

Evidence of a small hydrophobic domain in the core protein of the heparan sulfate proteoglycan from human colon carcinoma cells.

We demonstrate that the cell surface heparan sulfate proteoglycan of human colon carcinoma cells has an affinity for a hydrophobic matrix. This property is mediated by sequences in the core protein, since papain-or alkaline borohydride-released heparan sulfate chains do not bind to the matrix. Trypsin releases a [3H]leucine-rich, unsulfated, hydrophobic peptide, with Mr approximately 5000. This domain is present in neither the proteoglycan released into the medium nor in the intracellular degradation products. It is proposed that this peptide may represent the portion of the core protein intercalated into the plasma membrane.