Fusicoccin-binding Protein Research Articles

Isolation and biochemical characterization of fusicoccin-insensitive variant lines of Arabidopsis thaliana (L.) Heynh

Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H+-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a 3H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.

A Fusicoccin Binding Protein Belongs to the Family of 14-3-3 Brain Protein Homologs

A Fusicoccin Binding Protein Belongs to the Family of 14-3-3 Brain Protein Homologs

Purification of the Fusicoccin-Binding Protein from Oat Root Plasma Membrane by Affinity Chromatography with Biotinylated Fusicoccin.

Fusicoccin (FC), a fungal phytotoxin, evokes a number of physiological responses after binding to the FC-binding protein (FCBP). For characterization of this plasma membrane protein and elucidation of the signal transduction pathway, we purified active FCBP from oat (Avena sativa L. cv Valiant) root plasma membranes using avidin-biotin affinity chromatography. For the binding of FCBP to immobilized avidin, a bifunctional FC derivative (FC-biotin, FCBio) was synthesized. FCBio retained high binding affinity for the FCBP (KD = 70 nM), it elicited a biological response comparable to FC, and it was bound by avidin. The purification of the FCBP involved three important steps. First, FCBio was bound to the FCBP in purified plasma membrane vesicles. Next, plasma membrane proteins were solubilized in detergent, and part of the solubilized proteins was precipitated by decreasing the detergent concentration below the critical micelle concentration. The FCBP remained in the soluble fraction, and this fraction was loaded on a "low-affinity" avidin column. Proteins, bound through a biotin moiety to the column, were specifically eluted with excess biotin. This resulted in fractions active in [3H]FC binding and two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 31 and 30 kD. The nonhydrophobic behavior of the FCBP was confirmed by means of phase separation with Triton X-114, wherein the FCBP migrated to the hydrophilic phase. Purification of the FCBP in active form using this novel affinity technique opens the possibility to study other features of the FCBP necessary for inducing physiological responses in plant cells.

Aluminium fluoride and magnesium, activators of heterotrimeric GTP‐binding proteins, affect high‐affinity binding of the fungal toxin fusicoccin to the fusicoccin‐binding protein in oat root plasma membranes

The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [3H]fusicoccin binding to almost zero in a concentration-dependent way, with an optimum at approximately 20 mM sodium fluoride. The presence of magnesium (> 100 microM) was required for the inhibitory action of fluoride, whereas addition of low amounts of aluminum (25 microM) shifted the fluoride optimum to lower concentrations. Fluoride changes the biochemical properties of the binding protein in a reversible manner, because the inhibition was both prevented and reversed by 1 M ammonium sulphate. The combined effects of aluminium, fluoride and magnesium are reminiscent of the action of activated GTP-binding proteins. Since no functional assay for GTP-binding-protein activity in plants is available yet, GTP-binding-protein activation by fluoride and magnesium was deduced from competition with binding of [gamma-35S]GTP[S] to purified plasma membranes. Indeed, fluoride (20 mM) completely blocked the specific binding of [gamma-35S]GTP[S]. It is concluded that the inhibitory effect of fluoride upon the binding of fusicoccin is indirect and mediated through activated GTP-binding proteins. A hypothesis on the mechanism of fusicoccin action is presented wherein the fusicoccin-binding protein is one component of a signal-transduction chain, two or more steps downstream of a heterotrimeric GTP-binding protein.

Modulation of Fusicoccin-Binding Protein Activity in Mung Bean (Vigna radiata L.) Hypocotyls by Tissue Maturation and by Fusicoccin.

The phytotoxin fusicoccin (FC), after binding to a plasma membrane-localized receptor, causes higher plant cells to excrete protons. Ligand-binding analysis has been used to show that the plasma membrane of mung bean (Vigna radiata L.) hypocotyls contains both high-affinity (HA) and low-affinity (LA) binding sites for FC. The effect of tissue maturation on these sites was determined on isolated membrane vesicles from the meristematic region (hook) and the elongation zone and from mature hypocotyl tissues. In the meristematic region the HA:LA ratio was 1:20. As hypocotyl tissues matured, the site density of HA increased and there was no change in LA density, so that the HA:LA ratio increased to 1:2 in maturet issues. FC-induced proton excretion correlates with the HA density, not the LA density. When sections isolated from each region were incubated with FC prior to isolation of membranes, there was an apparent conversion of LA to HA sites during the first 90 min in all regions. During the next 1 to 3 h there was a further 2.5- to 3- fold increase in binding sites in all regions, accompanied by a slight decline in dissociation constant. The increase in binding sites, but not the apparent conversion of LA to HA, was partly blocked by cycloheximide. These data suggest that FC alters FC-binding protein activity in two ways: first, by causing an increase in affinity for FC of preexisting LA receptors, and second by inducing the synthesis of additional FC receptors. This apparent up-regulation of a phytotoxin receptor by its ligand in plants has not previously been reported.

Survey of the taxonomic and tissue distribution of microsomal binding sites for the non-host selective fungal phytotoxin, fusicoccin.

The recent identification of the fusicoccin-binding protein (FCBP) in plasma membranes from monocotyledonous and dicotyledonous angiosperms has opened the basis for an elucidation of the toxin's mechanism(s) of action and indicated a widespread occurrence of the FCBP in plants. Results of a detailed taxonomic survey of fusicoccin-binding sites are reported. Binding sites were not found in prokaryotes, animal tissues, fungi and algae including the most direct extant ancestors of the land plants (Coleochaete). From the Psilotales (Psilophytatae) to the monocotyledonous angiosperms, all taxa analyzed possessed high-affinity microsomal fusicoccin-binding sites. A heterogeneous picture emerged for the Bryophyta. Anthoceros crispulus (Anthocerotae), the only hornwort available to study, lacked fusicoccin binding. Within the Hepaticae as well as the Musci, species lacking and species exhibiting toxin binding were found. The binding site thus seems to have emerged very early in the evolution of the land plants. The tissue distribution of fusicoccin-binding sites was studied in Vicia faba L. shoots. All tissues analyzed showed fusicoccin binding, although not to the same extent. On a per-cell basis, guard cells were found to contain, compared to mesophyll cells, a nine-fold higher number of binding sites. Based on cell surface area, the site density is by a factor of 32 higher in guard cells than in mesophyll cells. Tissue specific expression of the binding sites is suggested by these findings.

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin, fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60-70% of the complexes can be solubilized with 50-60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml(-1) soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9'-nor-fusicoccin-8'-alcohol-[(3)H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4-10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (Mr) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an Mr of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9'-nor-8'[(3,5-[(3)H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([(3)H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an Mr of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [(3)H]ABE-FC in the presence of 0.1-1 μM fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh.

Using the novel radioligand, [(3)H]-9'-nor-fusicoccin-8'-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K(+), Mg(2+)-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [(3)H]-9'-nor-fusicoccin-8'-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (K(D)), K(D1) = 1.5 nanomolar and K(D2) = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 +/- 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin.

Purification and identification of the fusicoccin binding protein from oat root plasma membrane.

Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.