RESISTANCE GENES AND THE PERCEPTION AND TRANSDUCTION OF ELICITOR SIGNALS
II. General elicitors and their perception
An essential condition for the activation of defense responses is recognition, i.e., perception of the presence of a potential pathogen. Although such recognition can be based on physical contact, e.g. when a pathogen exerts pressure during penetration, it is likely that the primary mode of recognition is through chemical signals produced by invading microbes. All microbial signals that are perceived by plant cells and induce defensive responses are considered elicitors (Keen and Bruegger, 1977), and the concept of elicitor perception has been studied extensively at the biochemical and molecular level (for recent reviews see Boller, 1995; Hahn, 1996). Often, plant cell cultures have been used as models, as they offer the advantage of reacting rapidly and uniformly to elicitors (Boller and Felix, 1996). However, in such model systems it is difficult to link the observed reactions to plant-pathogen interactions, and students of elicitor perception should be aware that many of the general elicitors studied in cell culture systems are produced not only by pathogens, but also by harmless saprophytes and even by symbionts. It has been found that general elicitors comprise very different types of compounds which appear to be non-specifically recognized by plants and can elicit from only some of the responses associated with active defense up to cell death similar to the HR. Their actual contributions to the initiation of defense reactions may also differ depending on plant species and the rate at which defense develops upon pathogen attack, as well as on the type of assay used.
The biochemistry of elicitor perception has been studied extensively, starting from the hypothesis that the selective and highly sensitive perception of elicitors is based on recognition by specific receptors, and that these receptors are located at the outer surface of the plasma membrane, at least for the hydrophilic elicitors. The putative receptors are thus identified in plant cell membranes as high-affinity binding sites,
Perception and Transduction of Elicitor Signals in Host-Pathogen Interactions 193
using radioactively labeled elicitors for binding studies. It is important to verify that the selectivity of the binding site for various structural analogues of the elicitor corresponds to the biological activity of the analogues. Subsequently, the binding site can be solubilized and purified, although this procedure is rendered difficult by the fact that such binding sites have a low abundance and often represent less than 0.001 % of the membrane protein.
Using these approaches, several studies have identified high-affinity binding sites with the expected selectivity and specificity for some of the elicitors. It is probable that these binding sites represent the receptors although functional proof for this is still lacking.
In the following sections, we discuss the most important general elicitors derived from fungi and summarize current information on the putative receptors for general elicitors.
A. CELL WALL POLYSACCHARIDES
1. Glucans
A classic elicitor is the branched ~-1,3,~-1,6-heptaglucoside (Fig. 1a), isolated as the smallest elicitor-active compound from cell walls of Phytophthora megasperma f.sp. glycinea, a fungal pathogen of soybean (Darvill and Albersheim, 1984).
As demonstrated in the early studies with oligosaccharides derived from Phytophthora cell walls, many seemingly similar oligomers of glucose, with slightly different linkage patterns, are inactive as elicitors, attesting to the high selectivity and specificity of the perception system (Darvill and Albersheim, 1984; Cheong et aI., 1991; Ebel and Cosio, 1994; Hahn, 1996). Based on the initial finding of a high-affinity binding site for Phytophthora glucans in soybean plasma membranes (Schmidt and Ebel, 1987), this putative receptor was investigated by several groups. Extensive studies with structural analogues showed a close correlation between elicitor activity in vivo and the capacity to compete with elicitor binding in vitro (Cheong et aI., 1991), indicating that the binding site functions indeed as a receptor. Also, the presence of related binding sites in membranes of various legumes and their affinities correlated with the responsiveness of the legumes to the glucan elicitors (Cosio et al., 1996). The binding site has been solubilized (Cosio et al., 1990; Cheong et al., 1993) and partially purified (Frey et aI., 1993), and a cDNA encoding the binding site has recently been cloned (Umemoto et al., 1997), allowing proof of its receptor function in the near future.
While soybean and other related legumes are sensitive to the glucan elicitors, many other plants are insensitive, indicating a diversity of recognition systems in different plants (Ebel and Cosio, 1994; Hahn, 1996). It is worth noting that glucans occur in fungal cell walls in insoluble form, and that elicitor-active fragments must first be liberated before recognition can occur. It is likely that ~-1 ,3-glucanases, in addition to their direct role as antifungal enzymes, also function in the release of elicitors (see Hahn, 1996 for a recent review).
2. Chitin fragments
Chitin, a ~-I,4-linked linear polymer of N-acetylglucosamine, is a major constituent of the cell walls of most higher fungi, and it also occurs in arthropods and many other invertebrates but is not present in plants. Plant cells have a highly sensitive perception
system for chitin fragments (Fig. la), as studied in some detail in tomato (Felix et al., 1993). Chitin oligomers with four or more N-acetylglucosamine units are recognized by the plant cells at threshold concentrations of about 1 pM; the trisaccharide is about WOO-fold less active, and the dimer and monomer, which potentially could be present in plants, are essentially inactive (Felix et aI., 1993).
Tomato cells and membranes possess a high affinity binding site for chitin oligomers with four or more N-acetylglucosamine units (Kn of - 1-3 x 10-9 M for intact tomato cells) but a - 300-fold and - 300,OOO-fold lower affinity for the trimer and dimer, respectively (Baureithel et aI., 1994). The relative affinities of these compounds for the binding site are ,similar to their relative biological activities, and distinguish the binding site from chitin-binding lectins (Baureithel et aI., 1994). A high-affinity binding site for chitin oligomers has also been found in rice cells (Shibuya et aI., 1993). Compared to the chitin-binding site of tomato, it has a lower affinity for the smaller chitin fragments and reaches its highest affinity only with oligomers of eight N-acetylglucosamine units (Shibuya et al., 1996). Chitin perception appears to be a typical non-self recognition system (Boller, 1995). As in the case of glucans, chitin itself is highly insoluble, and for recognition to take place, it is necessary for the plant to secrete chitinases that release chitin fragments from invading fungi (Felix et aI., 1993). Thus, as in the case of glucanases, chitinases may function in the release of elicitors in addition to their direct antifungal activity (Boller, 1995).
In some groups of fungi, chitin occurs in association with chitosan, a deacetylated form of chitin. Also chitosan oligosaccharides (Fig. la) can act as elicitors, but in contrast to chitin, at least seven residues are required for biological activity (Cote and Hahn, 1994).
B. SECRETED PROTEINS AND GLYCOPEPTIDES
1. Pectolytic enzymes
Pectolytic enzymes had early been found to induce defense responses, and it became clear that the elicitor-active principle was not in the pectolytic enzymes themselves, but in fragments that they released from the plant cell wall (see West, 1981 for an early review). Oligogalacturonides (Fig. Ib) with a degree of polymerization between 10 and 20 residues have been found to be biologically active. Because they are plant-derived, they are called endogenous elicitors (Hahn et aI., 1981). Recognition of such elicitors is an example of an indirect mode for perception of microbial attack. One could say that the primary recognition event is actually the interaction of polygalacturonase or pectate lyase with the pectic component of plant cell walls, and that the degradation products generated, the endogenous elicitors, represent already second messengers. Interestingly, many plants produce inhibitors of the pectic enzymes of microbes, and these inhibitors not only reduce the enzymatic activity but also cause a shift in the chain length of the fragments generated, so that the proportion of highly active endogenous elicitor molecules is enhanced (Cervone and Albersheim, 1989).
2. Xylanase
Xylanase from the saprophyte Trichoderma viride acts as a potent elicitor in tobacco leaves (Sharon et al., 1993), as well as in tobacco and tomato cells (reviewed in
Perception and Transduction of Elicitor Signals in Host-Pathogen Interactions 195
a Fungal elicitors
GI ucan heptasaccharide
Chitin oligosaccharides
Chitosan oligosaccharides
protein
N-linked side chain of yeast glycopeptide
b Endogenous elicitors
up to 20 Pectin oligosaccharides (oligogalacturonides)
Figure 1. Schematic structures of carbohydrate elicitors of defense reactions derived from (a) fungal components and (b) plant cell walls. The types of linkage between individual residues are indicated.
Gle, glucose; GleNac, N-acetylglucosamine; GleN, glucosamine; Man, mannose; GalA, galacturonic acid.
Boller and Felix, 1996). Although it was initially thought that xylanase acts through the liberation of xylan fragments acting as endogenous elicitors, current evidence indicates that this is not the case (Sharon et ai., 1993). Hence, plants are able to detect the presence of microbial enzymes either indirectly, through the products formed, as in the case of pectic enzymes, or by direct recognition of the protein, as in the case of xylanase.
3. Extracellular proteins from Phytophthora species
Crude cell wall preparations and partially purified glucan preparations from several species of Phytophthora act as elicitors in various plant model systems, and it had initially been thought that this elicitor activity was based on the cell wall glucans contained in the preparation. However, it was subsequently found that upon application of an elicitor preparation from P. megasperma f.sp. gZycinea parsley cells do not react to the glucans but to the protein fraction (Parker et ai., 1988). Interestingly, soybean tissue reacts only to the glucans but not to the proteins, the opposite of the parsley pattern (Parker et ai., 1988). Subsequent analyses showed that a single glycoprotein was responsible for most of the elicitor activity, and this elicitor-active protein was later purified and cloned (Sacks et ai., 1995). It is not known whether the protein has an enzymatic function and why it is secreted by the fungus. The protein retains elicitor activity after boiling and can be fragmented. A careful study demonstrated that the elicitor-active principle resided in a short non-glycosylated peptide, and that a synthetic peptide with this sequence carried full elicitor activity (NUrnberger et ai., 1994). The principle of recognition of a short peptide sequence is curiously similar to the recognition of proteins by the animal immune system, but it is presently unknown whether fragmentation of the glycoprotein and the release of the short peptide is a precondition for elicitor activity in parsley cells.
Attempts to search in parsley cell membranes for an elicitor-binding site were initially made with the whole purified, labeled elicitor-active protein of 42 kDa molecular mass, but this proved to be difficult because of high nonspecific binding.
This is not uncommon when working with large elicitors. However, because the small peptide derived from the protein carries the whole elicitor activity, it could be used instead. In elegant work, it was shown that a high-affinity binding site for this elicitor- active peptide existed in parsley membranes (KD of - 2 x 10-9 M). The ability of similar peptides to compete with the radioligand for the binding site correlated with their biological activity, strongly indicating that the binding site represents the receptor (NUrnberger et aZ., 1994).
Extracellular proteins from Phytophthora species have also been characterized as elicitors in other systems. For example, the elicitins are a group of highly related, non-glycosylated 10 kDa proteins that are produced by most Phytophthora species and have been identified as elicitors of necrosis in tobacco leaves (Ricci et ai., 1989).
These proteins have a compact structure and are quite stable. They are highly conserved among Phytophthora species but their function is unknown. In addition, a 32 kDa elicitin-related glycoprotein has been purified from P. megasperma that likewise elicits necrosis in tobacco leaves (Baillieul et aZ., 1995). Both proteins are active at nanomolar levels in appropriate bioassays, documenting the very high sensitivity of tobacco plants to these particular stimuli. Interestingly, tomato cells recognize neither the glycoprotein with elicitor activity in parsley, nor elicitins, but are sensitive to another glycoprotein in a crude P. megasperma cell wall preparation (Boller and Felix, 1996).
Thus, different plant species have different capabilities to recognize extracellular proteins of Phytophthora, each species apparently recognizing one specific protein with exceedingly high sensitivity. It is characteristic that isolates of Phytophthora that are able to colonize tobacco as pathogens do not express the elicitins recognized by
Perception and Transduction of Elicitor Signals in Host-Pathogen Interactions 197
tobacco. This implies that specific recognition of such proteins is an important factor in the resistance of tobacco against other Phytophthora species (Bonnet et aI., 1994).
4. Gtycopeptides
Also acting as general elicitors are glycopeptides derived from extracellular proteins of yeast which were found to be recognized at nanomolar concentrations by tomato cells (Basse and Boller, 1992; Basse et at., 1992). In this case, the structure of the N-linked glycan side chains is decisive for activity: glycopeptides with 8 mannosyl residues have little elicitor activity but those with 9-11 residues are potent elicitors (Fig. la).
To study rare high-affinity binding sites such as elicitor receptors, it is necessary to obtain ligands with very high specific activity, such that binding can be studied in the presence of 10-10 M concentrations of the ligand or less. To achieve this, chemically defined glycopeptides were labeled with 35S, yielding radioligands with specific activities of 1000 Ci/mmol. Using this material, it was possible to demonstrate a high-affinity binding site for the yeast glycopeptides, both on intact tomato cells and in tomato membranes (Basse et aI., 1993). Analysis of the binding data yielded a KD of about 3 x 10-9 M, a value which corresponds to the concentration required for half-maximal elicitor activity (Basse et at., 1993). Glycopeptides with N-linked side chains of 9-11 mannosyl residues have a high affinity for the binding site while those with side chains of 8 mannosyl residues have low affinity, as would be expected for a binding site representing the biological receptor (Basse et at., 1993). This binding site has been solubilized and partially purified (Fath and Boller, 1996), using various chromatographic techniques. The binding site appeared to be stable after solubilization and maintained its binding characteristics, but further progress was rendered difficult because after binding to an affinity column containing the elicitor as a ligand, the binding protein could not be released in a functional form.
Interestingly, the structure with 8 mannosyl residues, which are present in the inactive glycopeptides, forms a core which is present in all eukaryotes, including plants.
However, all the larger chains, which are present in the active glycopeptides, have their nineth mannosyl residue attached to this core at a position unique for fungi, indicating that this perception system is specific for non-self molecules (Basse et at., 1992). One particularly intriguing aspect of this recognition system is the fact that the oligosaccharides present in the active elicitors act as competitive antagonists of elicitation when they are liberated from the glycopeptide. Thus, the free N-linked glycans, after cleavage from the peptide, counteract the activity of the corresponding elicitors and functionally act as suppressors (Basse et aI., 1992; Basse and Boller, 1992). Suppressors may be important in modulating the action of elicitors, and plant pathogens may accordingly need suppressors of this or other kinds to avoid recognition by the plant's general surveillance system (see Boller, 1995, for discussion).
C. MEMBRANE CONSTITUENTS
1. Arachidonic acid
Arachidonic acid, a fatty acid not normally present in plants but a major constituent of membrane lipids of Phytophthora infestans, acts as an elicitor in potato (Bostock