Fungal xylanases fromTrichoderma spp. are potent elicitors of defense responses in various plants. To determine whether enzymatic activity is necessary for elicitor activity, we used site-directed mutagenesis to reduce the catalytic activity of xylanase II from Trichoderma reesei. For this, the glutamic acid residue at position 210, which is part of the active center in this family of enzymes, was changed to either aspartic acid (E210D) or serine (E210S). Wild-type and mutated forms of xylanase II were expressed in yeast cells and purified to homogeneity. Compared with the wild-type form of xylanase II, E210D had >100-fold and E210S 1,000-fold lower enzymatic activity. In contrast, these mutated forms showed no comparable drop in elicitor activity. They fully stimulated medium alkalinization and ethylene biosynthesis in suspension-cultured tomato (Lycopersicon esculentum) cells, as well as hypersensitive necrosis in leaves of tomato and tobacco (Nicotiana tabacum) plants. These results provide direct evidence that enzyme activity is not necessary for elicitor activity of fungal xylanase.

Suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) reacted to spores and spore exudates of the pathogen Cladosporium fulvum with a rapid, transient alkalinization of their growth medium that resembled the previously described alkalinization response elicited by chitin fragments (G. Felix, M. Regenass, T. Boller [1993] Plant J 4: 307–316) and was likewise inhibited by the protein kinase inhibitor K-252a. However, the spore factor recognized by the cells differed from chitin fragments in that it was butanol soluble and active in cells refractory to stimulation by chitin fragments. The spore factor was purified and identified as ergosterol, the main sterol of most higher fungi. With pure ergosterol, half-maximal induction was reached at about 10 pm. After treatment with ergosterol, tomato cells became refractory to a subsequent stimulation by C. fulvum and vice versa, indicating that ergosterol was the principal component of the spores recognized by the plant cells. Most other sterols were inactive, including cholesterol, a range of animal steroid hormones, and all natural plant sterols tested, except for stigmasterol, which was about 106 times less active than ergosterol. Our data demonstrate that tomato cells perceive ergosterol with a selectivity and sensitivity that resembles the perception of steroid hormones in animals.

In suspension-cultured cells of tomato (Lycopersicon esculentum Mill.), the activity of 1-aminocyclopropane-1-carboxylate synthase (ACC-S) rapidly increases in response to fungal elicitors. The effect of inhibitors of protein kinases and protein phosphatases on the regulation of ACC-S was studied. K-252a, an inhibitor of protein kinases, prevented induction of the enzyme by elicitors and promoted its apparent turnover in elicitor-stimulated cells, causing a 50% loss of activity within 4 to 8 min in both the presence and absence of cycloheximide. Calyculin A, an inhibitor of protein phosphatases, caused a rapid increase of ACC-S in the absence of elicitors and an immediate acceleration of the rate of ACC-S increase in elicitor-stimulated cells. In the presence of cycloheximide there was no such increase, indicating that the effect depended on protein synthesis. Cordycepin, an inhibitor of mRNA synthesis, did not prevent the elicitor-induced increase in ACC-S activity but strongly reduced the K-252a-induced decay and the calyculin A-induced increase of its activity. In vitro, ACC-S activity was not affected by K-252a and calyculin A or by treatments with protein phosphatases. These results suggest that protein phosphorylation/dephosphorylation is involved in the regulation of ACC-S, not by regulating the catalytic activity itself but by controlling the rate of turnover of the enzyme.

Suspension-cultured tomato cells react to microbial signals, so-called elicitors, with rapid alkalinization of the growth medium and increased biosynthesis of the stress hormone ethylene. These responses to elicitors can be blocked by staurosporine and K-252a, two specific inhibitors of protein kinases. Here we show that calyculin A, a potent inhibitor of protein phosphatases, mimics the action of elicitors and, at nanomolar concentrations, induces medium alkalinization as well as a strong increase in the activity of 1-aminocyclopropane-1-carboxylate synthase, the key enzyme of ethylene biosynthesis. Both responses were strongly inhibited by K-252a, and calyculin A induced both responses more rapidly than did a fungal elicitor, xylanase. For example, the lag phase for medium alkalinization was only 0.2-0.4 min for calyculin A, compared with 2 min for xylanase. To study changes in the dynamics of protein phosphorylation, cells were labeled with 30-sec pulses of [33P]orthophosphate. Calyculin A strongly increased phosphorylation of several polypeptide bands within 40 sec of treatment. The same phosphorylated bands also appeared in response to xylanase, but only after a lag phase of 2-3 min. These results show that the protein phosphatase inhibitor calyculin A leads to rapid hyperphosphorylation of specific proteins in cultured cells and indicate that elicitor action could be based on inhibition of a protein phosphatase as well as on activation of a protein kinase.

Suspension-cultured tomato cells have a sensitive perception system for chitin fragments with a degree of polymerization (DP) > or = 4 and react to these compounds with a transient alkalinization of their culture medium (Felix, G., Regenass, M., and Boller, T. (1993) Plant. J. 4, 307-316). A chitin fragment with DP 5 was aminated at the reducing end and coupled to t-butoxycarbonyl-L-[35S]methionine via an amidoglycine spacer. The radiolabeled chitin fragment (approximately 1000 Ci/mmol) exhibited specific, saturable, reversible binding to whole tomato cells as well as to tomato microsomal membranes with dissociation constants of 1.4 and 23 nM, respectively. Binding of the radioligand was competed by chitin fragments of different DP with IC50 values (50% inhibition of binding) that closely paralleled the concentrations inducing the alkalinization response half-maximally. Deacetylated chitooligosaccharides and N-propanoyl chitooligosaccharides were weak elicitors of the alkalinization response as well as weak competitors of radioligand binding. A lipochitooligosaccharide (Nod factor) from Rhizobium leguminosarum stimulated the alkalinization response in tomato cells half-maximally at 3 nM and competed radioligand binding to the cells with an IC50 of 8 nM. These results demonstrate the presence of a high affinity binding site for chitin fragments on the tomato cell membrane that may function as a receptor.

The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo chitooligosaccharides that induce the formation of nodules on their host plants. When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred. Lipo oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria. The activity was absent from preparations of the mutant strain Rhizobium sp. NGR234 Delta nodABC, unable to produce any Nod factors. Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp. NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations. This demonstrates that Nod factors can be perceived by tomato; a nonhost of rhizobia. Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases. Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp. NGR(pA28). This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.

The tobacco hybrid Nicotiana glutinosa × Nicotiana debneyi is much more resistant than either parental species to tobacco mosaic virus, tobacco necrosis virus, Pseudomonas syringae pv. syringae, P. syringae pv. tabaci and Peronospora tabacina infections. While N. glutinosa is very susceptible to Cercospora nicotianae, Chalara elegans and Phytophthora parasitica var. nicotianae, the hybrid is as resistant as N. debneyi to these fungi. The resistance of the hybrid is linked to high levels of chitinase, β-1,3-glucanase, peroxidase (PO) and polyphenoloxidase (PPO). The parental species contain much lower levels of these enzymes unless they are locally infected with necrotizing viruses. In those cases, chitinase, β-1,3-glucanase and PO, but not PPO, increase in the whole plant to levels comparable with those found in the hybrid, and resistance to secondary infection develops (systemic acquired resistance). All the acidic isoenzymes of PO present in both parental species after infection are constitutively expressed in the hybrid. The activity of phenylalanine ammonia-lyase was similar in all plants. These results reinforce the hypothesis that hydrolases and PO are involved in resistance against various pathogens.

Plants react to invasion by fungal pathogens with a range of biochemical responses. They recognize the presence of pathogens using a sensitive perception system for chemical signals derived from fungi, so-called elicitors. These elicitors, which in most cases have not been chemically well defined, comprise carbohydrates, peptides and glycopeptides (Dixon and Lamb, 1990).

Plant cells have an acute sense for pathogen-derived chemical stimuli, so-called elicitors, which induce the plant's defense response. To investigate the molecular basis of chemosensory transduction, elicitor-treated tomato cells were labeled with 1-min pulses of [32P] phosphate. This technique revealed drastic changes in protein phosphorylation in vivo within minutes of stimulation. The protein kinase inhibitors K-252a and staurosporine completely prevented these elicitor-induced changes in protein phosphorylation. They also blocked two early biochemical responses to elicitors, extracellular alkalinization and biosynthesis of ethylene. The ability of K-252a, staurosporine, and benzoylated staurosporine derivatives to inhibit elicitor responses in vivo correlated with their ability to inhibit tomato microsomal protein kinase in vitro. When K-252a was given to elicited cells 1 min after the[32] phosphate, the radioactivity in certain newly labeled phosphoprotein bands disappeared again within minutes. This correlated with an arrest of alkalinization within minutes when K-252a was applied in midcourse of elicitation. These data show that phosphorylation of protein substrates by K-252a-sensitive protein kinases is essential for transduction of elicitor signals in plant cells and that continuous phosphorylation of these proteins is required to maintain the elicited state.

The activity of 1-aminocyclopropane carboxylate (ACC) synthase increased rapidly in tomato (Lycopersicon esculentum Mill.) leaf discs after vacuum infiltration, reached a maximum after about 30 minutes, and subsequently decayed with an apparent half-life of about 20 minutes. Aminoethoxyvinylglycine, a known inhibitor of ACC synthase, did not alter the apparent turnover of ACC synthase in vivo although it efficiently blocked inactivation of the enzyme by its substrate S-adenosylmethionine in vitro. Similar results were obtained, using a novel assay with permeabilized cells, for ACC synthase in tomato cell cultures treated with a fungal elicitor. The results indicate that inactivation of ACC synthase in vivo differs from substrate-dependent inactivation in vitro.

Tobacco glucan endo-1,3-beta-glucosidase (beta-1,3-glucanase; 1,3-beta-D-glucan glucanohydrolase; EC 3.2.1.39) exhibits complex hormonal and developmental regulation and is induced when plants are infected with pathogens. We determined the primary structure of this enzyme from the nucleotide sequence of five partial cDNA clones and the amino acid sequence of five peptides covering a total of 70 residues. beta-1,3-Glucanase is produced as a 359-residue preproenzyme with an N-terminal hydrophobic signal peptide of 21 residues and a C-terminal extension of 22 residues containing a putative N-glycosylation site. The results of pulse-chase experiments with tunicamycin provide evidence that the first step in processing is loss of the signal peptide and addition of an oligosaccharide side chain. The glycosylated intermediate is further processed with the loss of the oligosaccharide side chain and C-terminal extension to give the mature enzyme. Heterogeneity in the sequences of cDNA clones and of mature protein and in Southern blot analysis of restriction endonuclease fragments indicates that tobacco beta-1,3-glucanase is encoded by a small gene family. Two or three members of this family appear to have their evolutionary origin in each of the progenitors of tobacco, Nicotiana sylvestris and Nicotiana tomentosiformis.