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. 1998 Mar 17;95(6):3306-11.
doi: 10.1073/pnas.95.6.3306.

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

C Ji  1 , C Boyd, D Slaymaker, Y Okinaka, Y Takeuchi, S L Midland, J J Sims, E Herman, N Keen
Affiliations

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

C Ji et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Figure 1
Figure 1
Isolation of syringolide binding protein(s) by affinity chromatography. (A) Specific binding activity of 125I-syringolide 1 to soybean leaf soluble proteins recovered from a syringolide affinity column. (B) Detection of syringolide binding proteins using the native gel binding assay as described in Materials and Methods, with specific bound radioactivity shown by the arrows. CR denotes crude extracts. Unbound 125I-syringolide 1 occurs at the bottom of the gel.
Figure 2
Figure 2
12% SDS acrylamide gel of proteins in fractions from a syringolide affinity column. Lane 1, protein size markers; lane 2, crude soybean leaf extract; lane 3, fraction 6; lane 4, fraction 12; lane 5, fraction 15; lane 6, fraction 16; lane 7, fraction 18. Arrows denote 34-, 31-, and 28-kDa proteins eluted.
Figure 3
Figure 3
Purification of syringolide binding protein. (A) Gel filtration purified 28-, 31-, and 34-kDa proteins were electrophoresed on a 12% SDS acrylamide gel. Lane 1, fraction 15 from the affinity column run in Fig. 1; lane 2, 28-kDa protein; lane 3, 31-kDa protein; lane 4, 34-kDa protein. (B) Syringolide binding activity of purified 28-, 31-, and 34-kDa proteins. Lanes are as in A.
Figure 4
Figure 4
Detection of P34 in soluble protein fractions of soybean seeds, cotyledons, and leaves. Equal amounts of protein (20 μg) were loaded in each lane.
Figure 5
Figure 5
Expression of His-tagged 46-, 34-, and 32-kDa soybean proteins in baculovirus expression vector pVL1392. Lane 1, protein size markers; lane 2, protein extract from cells expressing pVL1392–46M1; lane 3, Ni-purified 46-kDa protein; lane 4, protein extract from cells expressing pVL1392–34M2; lane 5, Ni-purified 34-kDa protein; lane 6, protein extract from cells expressing pVL1392–32M3; lane 7, Ni-purified 32-kDa protein.
Figure 6
Figure 6
125I-syringolide 1 binding activity of soybean protein produced as His-tagged fusions (Fig. 5). (A) Binding activity in the native gel assay. Four micrograms of protein was loaded in each lane. (B) Quantitation of A of the protein–125I-syringolide complexes by scintillation counting. −, absence of competitive cold syringolide 1; +, presence of 1,500-fold cold syringolide 1.
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