Constitutive expression of clathrin hub hinders elicitor-induced clathrin-mediated endocytosis and defense gene expression in plant cells.

Endocytosis has been recently implicated in the signaling network associated with the recognition of microbes by plants. In a previous study, we showed that the elicitor cryptogein was able to induce clathrin-mediated endocytosis (CME) in tobacco suspension cells. Herein, we investigate further the induced CME by means of a GFP-tagged clathrin light chain and a CME inhibitor, the hub domain of clathrin heavy chain. Hub constitutive expression does affect neither cell growth nor constitutive endocytosis but abolishes cryptogein-induced CME. Such an inhibition has no impact on early events in the cryptogein signaling pathway but reduces the expression of defense-associated genes.

The fungal elicitor cryptogein induces cell wall modifications on tobacco cell suspension.

Upon addition of the fungal elicitor cryptogein, suspension cells of tobacco (Nicotiana tabacum cv. Xanthi) aggregated in clusters. Cytochemical experiments indicated that elicited cells displayed fibrillar expansions of pectin along the primary cell wall. Immunocytochemical detection of pectin epitopes indicated that the fibrillar material surrounding the treated cells was mostly composed of low methylated galacturonan sequences, but the use of the cationic probe did not reveal the presence of negatively charged carboxyl groups: the presence of important amounts of calcium ions in these pectic fibrillar expansions accounts for these observations. These data indicate that tobacco cells treated with cryptogein show a cell wall altered by the presence of a calcium pectate gel, resulting from the reorganization of pectin in the middle lamellae. These results are consistent with a drastic reduction in wall digestibility, partially reversed by increasing the pectolyase concentration in the hydrolytic solution. Diphenylene iodonium, an inhibitor of the oxidative burst triggered by cryptogein on tobacco cells, partially prevents elicited cell walls from this loss of digestibility, suggesting a possible role of active oxygen species in the cell wall strengthening. This work represents a new element of the signal transduction cascade triggered on tobacco cells by cryptogein.

Cloning of Rac and Rho-GDI from tobacco using an heterologous two-hybrid screen.

To examine whether molecular similarities exist between the animal and plant Rho GTPase signaling pathways, we have developed a heterologous two-hybrid screening method. By this technique, we have cloned a cDNA encoding a tobacco Rac-like protein able to interact with a mammalian Rho-GDI. In a second screen this tobacco Rac was used as a bait and a tobacco homologue of Rho-GDI was identified. These results show that some components of the animal and plant Rac signaling pathways are similar enough to allow their interaction in an heterologous approach. Moreover these data suggest a similar regulation of Rho GTPases in animals and plants.

Tobacco cells contain a protein, immunologically related to the neutrophil small G protein Rac2 and involved in elicitor-induced oxidative burst.

Suspension-cultured cells of Nicotiana tabacum generated active oxygen species (AOS) when they were treated with the proteinaceous elicitor, cryptogein. This response was blocked by diphenylene iodonium, an inhibitor of the neutrophil NADPH oxidase. When microsomal extracts of tobacco cells were probed with an antibody directed against the human small G protein Rac2, two immunoreactive proteins were detected at 18.5 and 20.5 kDa. The same experiment performed with cytosolic extracts of tobacco cells led to the observation of a strong immunoreactive protein at 21.5 kDa only in the cryptogein-treated cells. The appearance of this cytosolic protein was related to the production of AOS by the elicited cells. These results provide evidence for the possible involvement of small G proteins, homologous to the neutrophil Rac2 protein, in the regulation of the elicitor-induced oxidative burst in plant.

Activation of the plant plasma membrane H+-ATPase. Is there a direct interaction between lysophosphatidylcholine and the C-terminal part of the enzyme?

The antagonistic effects of the fungal toxin beticolin-1 and of L-alpha-lysophosphatidylcholine (lysoPC) were investigated on the plasma membrane H+-ATPase of the plant Arabidopsis thaliana (isoform 2) expressed in yeast, using both wild-type enzyme (AHA2) and C-terminal truncated enzyme (aha2delta92). Phosphohydrolytic activities of both enzymes were inhibited by beticolin-1, with very similar 50% inhibitory concentrations, indicating that the toxin action does not involve the C-terminal located autoinhibitory domain of the proton pump. Egg lysoPC, a compound that activates the H+-ATPase by a mechanism involving the C-terminal part of the protein, was found to be able to reverse the inhibition of AHA2 by beticolin-1. The lack of effect of other detergents and the comparison of different carbon chain length lysoPCs show that the capacity to reverse the enzyme inhibition is clearly related to their ability to activate the pump. Long chain length lysoPC was also shown to reverse the inhibition of aha2delta92 by beticolin-1, which strongly suggests that lysoPC binds to the H+-ATPase on site(s) not located on its autoinhibitory domain.

Cercospora beticola toxins. Part XVII. The role of the beticolin/Mg2+ complexes in their biological activity. Study of plasma membrane H(+)-ATPase, vacuolar H(+)-PPase, alkaline and acid phosphatases.

Beticolin-1 and beticolin-2, yellow toxins produced by the phytopathogenic fungus Cercospora beticola, inhibit the plasma membrane H(+)-ATPase. Firstly, since beticolins are able to form complexes with Mg2+, the role of the beticolin/Mg2+ complexes in the inhibition of the plasma membrane proton pump has been investigated. Calculations indicate that beticolins could exist under several forms, in the H(+)-ATPase assay mixture, both free or complexed with Mg2+. However, the percentage inhibition of the H(+)-ATPase activity is correlated to the concentration of one single form of beticolin, the dimeric neutral complex Mg2H2B2, which appears to be the active form involved in the H(+)-ATPase inhibition. Secondly, since previous data suggested that beticolins could also be active against other Mg2(+)-dependent enzymes, we tested beticolin-1 on the vacuolar H(+)-PPase, which requires Mg2+ as co-substrate, and on the alkaline and acid phosphatases, which do not use Mg2+ as co-substrate. Only vacuolar H(+)-PPase is sensitive to beticolin-1, which suggests that beticolins are specific to enzymes that use a complex of Mg2+ as the substrate. The same Mg2H2B2 complex which is responsible of the plasma membrane H(+)-ATPase inhibition appears to be also involved in the inhibition of the vacuolar H(+)-PPase.

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1).

Beticolin-1 is a toxin produced by the fungus Cercospora beticola. The chemical structure of this toxin was previously elucidated. The effects of beticolin-1 on purified corn root plasma membrane H+-ATPase were studied in a solubilized form or were reconstituted into liposome membranes. The ATP hydrolysis activity of the purified solubilized enzyme was inhibited by micromolar concentrations of beticolin-1, and this inhibition was noncompetitive with respect to ATP. When this purified enzyme was inserted into liposome membranes, a competitive inhibition of the H+-ATPase hydrolysis activity by beticolin-1 was observed. The effect of beticolin-1 on the formation of H+-ATPase-phosphorylated intermediate was also studied. With the purified enzyme in its solubilized form, the level of phosphorylated intermediate was not affected by the presence of beticolin-1, whereas micromolar concentrations of the toxin led to a marked inhibition of its formation when the enzyme was reconstituted into liposomes. These data suggest that (a) the plasma membrane H+-ATPase is a direct target for beticolin-1, and (b) the kinetics of inhibition and the effect on the phosphorylated intermediate are linked and both depend on the lipid environment of the enzyme.

A test for screening monoclonal antibodies to membrane proteins based on their ability to inhibit protein reconstitution into vesicles.

The hypothesis that the binding of an antibody to a membrane protein is likely to prevent the reconstitution of the protein into liposomes was checked, by using the plant plasma membrane H(+)-ATPase (EC 3.6.1.35) as a model system, and two reconstitution procedures: spontaneous insertion (SI) of purified H(+)-ATPase into preformed liposomes, and a detergent-mediated reconstitution (DMR) procedure allowing the reconstitution of the whole membrane protein content. Nine monoclonal antibodies (MABs) raised against H(+)-ATPase were tested. None affected the functioning of the enzyme reconstituted in liposomes, suggesting that the probability to obtain an inhibitory MAB is low. Five MABs inhibited its SI, and seven inhibited its reconstitution in the DMR procedure. These results indicate that it is possible to screen antibodies directed against membrane protein, by making use of their ability to inhibit the reconstitution of these proteins.

Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer.

The purified (H+)ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H(+)-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H+)ATPase molecules were closely associated with liposomes, exhibiting a high H(+)-pumping activity (150,000% quenching min-1.mg-1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H+)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H+)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles ("inside-out" orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H+)ATPase could also functionally insert into bilayer from PC:PE:PG or PC:PE:PI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H+)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H+)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation.