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

Abstract

l h e 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 1.) 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 HALA 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 HALA ratio increased to 1:2 in mature tissues. FC-induced proton excretion correlates with the HA density, not the LA density. When sedions 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. l h e increase in binding sites, but not the apparent conversion of LA to HA, was partly blocked by cycloheximide. lhese 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.The phytotoxin FC, produced by the fungus Fusicoccum amygdali Del., has the ability to cause proton excretion from a11 higher plants so far tested (Marré, 1979). After binding to a 30.5-to 32.5-kD receptor (FCBP) (de Boer et al., 1989;Feyerabend and Weiler, 1989) on the PM, it activates the PM H+-ATPase, apparently by relieving the inhibition caused by the autoinhibitory C-terminal domain of the ATPase (Pugliarello et al., 1992;Johansson et al., 1993).Our previous study (Basel and Cleland, 1992) , 1982;Meyer et al., 1989;Schulz et al., 1990; Abramycheva et al., 1993). Because our previous FCBP assay would have measured only HA sites, this study was undertaken to determine whether mung bean hypocotyls possess both HA and LA FC-binding sites and, if so, whether the distributions of HA and LA sites were the same. Preliminary experiments indicated that, when hook sections from mung bean hypocotyls were treated with FC, there was no detectable inaease in proton excretion during the 1st h, as expected from the apparent lack of FCBP in this region. But after the 1st h, FC-enhanced H+ excretion commenced, suggesting that FC might have up-regulated the ability of these cells to bind FC. To test these possibilities, ligandbinding analysis (Scatchard, 1949) has been used with microsoma1 and PM vesicles prepared from freshly cut sections of the mung bean hypocotyl or prepared after preincubation of sections for up to 6 h with or without FC. The Scatchard ana...

A High-Affinity Binding Site for the AVR9 Peptide Elicitor of Cladosporium fulvum Is Present on Plasma Membranes of Tomato and Other Solanaceous Plants.

“…The apparent discrepancy between the in vitro and in vivo results may be due to the fact that the appearance of necrosis can be seen as a final step in the active defense response, which depends on physiological and environmental conditions. A similar phenomenon has been observed for fusicoccin, which invokes proton excretion in higher plants at a concentration of ~10~7 M, whereas its high-affinity receptors have reported K d values of ~10~9 M (Basel et al, 1994). It is possible that the observed discrepancy occurs because binding of AVR9 to membranes is highly influenced by salt concentration and is optimal at low ionic strength.…”

A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs.

“…Could it be that the amount of FCBPs (after all, one of the best-characterized targets for fungal attack in plants) is also modified during fungal infection (i.e., during binding of FC) as suggested recently by Basel et al (1994)? We addressed this question by in vivo incubation of oat roots with 10~5 M FC for different times (0,5,10,20,40, and 60 min).…”

“…and is controlled by the developmental stage of the tissue (Basel and Cleland, 1992). Modulation of the receptor affinity for FC by binding of its own ligand (Basel et al, 1994) further accentuates the role of the FCBP in the physiology of plants.…”

A Single Binding Site Mediates Resistance- and Disease-Associated Activities of the Effector Protein NIP1 from the Barley Pathogen Rhynchosporium secalis

“…Similar apparent discrepancies between binding and response have also been described for other receptor-ligand interactions. For the fungal phytotoxin fusicoccin, a concentration 100-fold higher than the K d of 1 nM was necessary to stimulate the plant plasma membrane H 1 -ATPase (Basel et al, 1994). An even larger difference (approximately 4,000-fold) between K d value and the minimal elicitor concentration required for inducing a hypersensitive response in tomato leaves was described for the AVR9 elicitor from C. fulvum (Kooman-Gersmann et al, 1996).…”