The high degree of specificity that many pathogens have with their planthosts is well-documented from field observations and genetic analysis.Hundreds of disease-specificity genes have been described in plants (resis-tance genes) and in pathogens (pathogenicity genes). Significant progresshas been made in the genetics of specificity, in Mendelian and, morerecently, molecular terms. However, a major challenge confronting molec-ular plant pathologists today is to understand the biochemical and physio-logical mechanisms controlled by specificity genes. Mendelian genetics islimited in the information it can supply about mechanisms, and althoughmolecular genetics supplies detailed information about a gene, including itspattern of expression and the size and amino acid sequence of the product,there have been many cases, both in plant/microbe interactions and in otherfields of biology, in which having a cloned gene in hand has not led toinsights into the function of that gene.
For decades, the only known agents of specificity in any plant/microbeinteraction were the host-selective toxins (HSTs). The known HSTs are withone exception (94a) low molecular weight secondary metabolites (51). are known only from fungal pathogens, especially the genera Alternariaand Cochliobolus (Helminthosporium). The most important attribute of HSTsis that they~ are agents of virulence or pathogenicity: a fungus that makesan HST causes more disease on its host than one that is otherwise identicalbut does not make the HST. Plant insensitivity to an HST confers increasedresistance to the producing organism. Aspects of the biology and chemistryof selective and nonselective phytotoxins have been reviewed in recent years(25, 50, 118).
Although most HSTs are secondary metabolites, they show great diversityin their chemistry and in their biological effects. Known HSTs includecyclic peptides, terpenoids, oligosaccharides, polyketides, and compoundsof unknown biogenesis. Most HSTs from any single pathogen occur asfamilies of closely related compounds, and some HSTs from differentspecies, especially those from Alternaria, are structurally related (51).However, it is sometimes overlooked that there is no single genetic patternof host response to HSTs and to the fungi that produce them (9, 74).Genetic analysis has shown that sensitivity to particular HSTs is controlledmonogenically in the host plants, but sensitivity can be dominant, semi-dominant, recessive, or cytoplasmically inherited (29, 118). HSTs differconsiderably in their biological effects on sensitive host tissues (51). Thebasis of specificity is known for two HSTs. Specific response to T-toxinfrom Cochliobolus heterostrophus is mediated by a novel protein, URFI3,encoded by the mitochondrial genome of maize and located in the innermitochondrial membrane of sensitive plants (22). The specificity of HC-toxin, produced by the maize pathogen C. carbonum, is not related to itssite of action but to differential detoxification; resistance is dominant (40,62). It has been proposed that the specificity of victorin, the HST producedby C. victoriae, is related to differential binding to a membrane-localizedprotein (114), but other researchers came to a different conclusion (1).Regardless, sensitivity to victorin is clearly a dominant trait.
The fact that HSTs were for a long period the only known agents ofspecificity in any plant/microbe interaction has produced a degree of con-ceptual polarization among plant pathologists. On the one hand, HSTs havebeen granted mythic properties or, on the other, dismissed as oddities. Theformer point of view can be understood in light of the unusual propertiesof the most famous HST, victorin. Victorin, the first HST to gain widespreadattention, is the most phytotoxic and most selective compound known, being
active against sensitive oats at 10 pg/ml (13 pM), yet not affecting resistantoats or any other plant even at a one millionfold higher concentration (e.g.see ref. 105). The single Mendelian locus, called Hv-1, that confers dominantsensitivity to victorin and susceptibility to C. victoriae is either the sameor is very tightly linked to Pc-2, which confers dominant resistance to manyraces of the unrelated pathogen Puccinia coronata, the cause of crown rust.It has long been hoped (57, 76) that study of victorin might open an avenueto understanding the basis of resistance to the important but experimentallyintractable rust fungi. Contributing to the HST mythology, victorin was fora long time recalcitrant to purification and characterization, this beingaccomplished only 40 years after its discovery (105, 115). Aptly, thestructure of victorin is unusual (115).
Recent progress on the chemistry and biology of HSTs indicates thatHSTs are ordinary natural products. The chemical structures, biosyntheticpathways, and molecular genetics of HSTs are similar to those of othermicrobial secondary metabolites, whether they be called antibiotics, anti-metabolites, nonspecific toxins, xenobiotics, growth regulators, teratogens,mutagens, pigments, etc. The vast majority of secondary metabolites haveno known biological function; it is as agents of virulence and specificity inthe interactions between microbes and plants that HSTs are special to naturalproducts biology and, of course, to plant pathology.
The belief that HSTs are "atypical" agents of specificity was unaddressablefor many years since no other agents of specificity were known. However,we can now point out that HSTs share key attributes with recently discoveredagents of specificity in symbiotic plant/microbe interactions and with bothhost-selective and nonselective "elicitors". Clearly, the charge of abnormalityagainst HSTs is not justified.
Even in the days when specificity in other diseases was a completemystery, scientific objections were raised against the possible universal oreven widespread involvement of HSTs. As a result, HSTs are not centralto most models of specificity that have been proposed over the years. Inthe first part of this review we discuss several of these objections and arguethat they do not constitute serious reasons to exclude HSTs or HST-likemolecules as possible widespread agents of specificity.
Toxins Appear Incompatible with Monogenic Inheritance ofSpecificityConsidering the complexity of host/pathogen interactions, there are surpris-ingly numerous instances in which pathogenicity and resistance are controlledby single Mendelian loci. Single gene resistance to particular species, races,pathovars, and strains of viruses, nematodes, bacteria, and fungi has beenidentified in most major crop plants. It has also been shown in a number
of fungi and some bacteria that specific pathogenicity is inherited mono-genically.
Monogenic inheritance of resistance and pathogenicity is the major con-straint on all attempts to build models of the underlying biochemical eventscontrolled by those genes. It has been argued that monogenic inheritanceexcludes complex molecules as mediators of specificity, and that thereforeprimary gene products, i.e. proteins, must do so. This is because complexmolecules require many enzymes for their synthesis, and therefore it shouldbe possible to identify many genes contributing to the specificity of aparticular disease interaction. Such genetic interactions are observed onlyrarely. Ellingboe (26) has discussed the difficulty of explaining the geneticsof host/pathogen interactions on the basis of complex glycoprotein orcarbohydrate elicitors, and similar arguments apply to secondary metabolites.Monogenic specificity mediated directly by the gene product is known inonly one disease interaction to date (100).
Plant secondary metabolites such as flavonoid phytoalexins (101) gibberellins (30) clearly do require many individual enzymatic steps andgenes. In microorganisms, however, it is now known that tight clusteringof secondary metabolite genes is not only common but is the rule. Geneclusters control biosynthesis of actinorhodin (19), tetracenomycin C (11),erythromycin (24), graniticin (86), penicillins in both bacteria and fungi(e.g. 88), and others (36). The genes necessary for synthesis of nonspecific bacterial toxins made by different pathovars of Pseudomonassyringae, namely, syringomycin, syringotoxin, phaseolotoxin, tabtoxin, andcoronatine, are clustered (32, 111).
A different kind of clustering is exemplified by multifunctional enzymes.The cyclic hexadepsipeptide enniatin from Fusarium oxysporum is syn-thesized by a single 250-kd polypeptide (123). The undecapeptidecyclosporin is synthesized by a 1.4-MDa protein that is apparently a singlepolypeptide (80). Although molecular genetic studies of bacterial toxinshave not yet been complemented by enzymatic ones, the association ofvery large polypeptides with the syringotoxin and syringomycin biosyn-thetic genes (66, 116) suggests that these peptide secondary metabolitesare synthesized by multifunctional enzymes similar to other cyclic peptidesynthetases (48).
There are at least two additional ways, at least in fungi, in which multiplegenes can segregate as a single Mendelian locus. The first is due to thefact that secondary metabolite genes and gene clusters can be completelymissing in nonproducing strains, e.g. the penicillin cluster in Aspergillusnidulans (59), and TOX2 in C. carbonum (70). There is no physical limitto the size of a Mendelian "gene" if the alternate "allele" on the homologouschromosome is a deletion. Second, some, perhaps most, pathogenic fungi
have heterogeneous chromosome numbers and sizes. Genes present onchromosomes that are absent in one parent in a cross (for example,dispensable "B" chromosomes) are always linked (47, 64).
An intriguing aspect of the biology of the HSTs has been the geneticsof the control of their production. In three species of Cochliobolus (Alterna-ria, unfortunately, has no known perfect stage), single but different genescontrol production of their respective host-selective toxins. These genes arecalled TOX1, TOX2, and TOX3 and control production of T-toxin, HC-toxin,and victorin in C. heterostrophus, C. carbonum, and C. victoriae, respec-tively (12). It has long been a puzzle how single genes apparently single-handedly control production of these complex secondary metabolites.Elucidation of the structure of TOX genes could be relevant to the manyother situations in which pathogenic specificity is under monogenic control.
Various hypotheses are consistent with single gene control of the pro-duction of the HSTs of Cochliobolus. For example, a toxin could be afortuitously phytotoxic intermediary metabolite that accumulates in thepresence of the nonfunctional allele of the TOX gene. This possibilitypredicts that toxin production would be recessive, but using forcedheterokaryons between TOX1 and toxl strains of C. heterostrophus Leachet al (54) concluded that T-toxin production is dominant to nonproduction(but see ref. 12). Furthermore, the structures of most other HSTs (51) argueagainst them being metabolic intermediates. More likely, the TOX genesencode multifunctional enzymes or are gene clusters. The Mendelian locusTOX2 in C. carbonum that controls HC-toxin biosynthesis is a cluster ofat least 54 kb encoding in part two copies of a large multifunctional enzyme.Since nontoxin-producing isolates of C. carbonum lack DNA homologousto TOX2, the entire region segregates as a single Mendelian locus (70).Our studies on the structure of TOX2 are discussed in more detail below.
Genes controlling synthesis of complex secondary metabolites are notnecessarily larger than "normal" genes. There is no reason why an HST ora host-selective elicitor could not be a single enzymatic step from a primarymetabolite, or a single step from a secondary pathway common to all racesof a pathogen (e.g. Rhizobium nod factors, see below). The avrD gene ofP. syringae pv. tomato encodes a protein of deduced Mr 34 kd (49). Thisprotein is not secreted and has no elicitor activity itself. When expressedin E. coli, the avrD gene induces the production of two related host-selectiveelicitors of low molecular weight. These compounds, tentatively namedsyringolide 1 and 2, could plausibly be synthesized by condensation andsubsequent rearrangement of two E. coli metabolites, xylulose and either aC-8 or C-10 13-hydroxy fatty acid (45, 89a).
The conclusion from recent results on antibiotic production in bacteriaand fungi as well as from work on TOX2 and avrD is that mediation of
specificity by complex secondary metabolites is completely compatible withmonogenic inheritance of specificity.
Many models invoke specific "receptors" as key elements of the plantside of plant/pathogen interactions. These receptors are hypothesized to bethe products of plant resistance or susceptibility genes. For HSTs, it ishypothesized that the presence of the appropriate receptor is required forthe toxin to act, and, since HSTs are agents of pathogenicity, in the absenceof the receptor the plant is resistant (28). This model is easily compatiblewith dominant toxin sensitivity, for example, to victorin. In models ofdiseases in which pathogen avirulence is dominant to virulence, it is proposedthat active host defenses are triggered when a pathogen signal (a specific"elicitor") binds to a specific receptor. In the absence of the receptor, norecognition occurs and disease results.
The use of the word receptor in models of plant pathology deservescomment. The concept of receptors in plant pathology is often constrainedto that of the canonical receptors in mammals, which are involved in internalhomeostasis of the organism, linked to signal transduction pathways, andfrequently membrane-localized (e.g. 34). But it is important to keep open mind about what biochemical properties a "receptor" involved in aplant/pathogen interaction might have. Plant receptors are postulated torecognize and bind specificity factors, but enzymes also recognize and bindtheir substrates. The theoretical treatment of receptor/ligand interactions isclosely related to the analysis of enzyme/substrate interactions (84). Theenzyme that is the site of action of a biologically active compound can beconsidered its receptor, e.g. the chloroplast ATPase for tentoxin (4), evenif the binding is biologically fortuitous.
A gene that encodes a protein that, for example, inactivates an HST ortransduces an elicitor of plant defenses would give dominant resistance. Agene that encodes a toxin-activating protein (consider, for example, activa-tion of the nonspecific bacterial toxin tabtoxin by plant peptidases (98)) a receptor that transduces a suppressor of plant defenses would give recessiveresistance. If the two alleles of a plant resistance gene encode two functionalforms of an essential enzyme, one of which is sensitive to a toxin and theother insensitive (for example, the ornithine carbamoyl transferases of P.syringae pv. phaseolicola (67)), then resistance could be either dominant,semi-dominant, or recessive. This would depend on whether the insensitiveenzyme could compensate completely or only partially for the inhibitedenzyme in the heterozygote, or whether inhibition of the sensitive enzymecaused accumulation of a toxic metabolite. Sensitivity to AL-toxin issemidominant (29). Despite the ubiquity of receptors in plant disease models,to date there is no strong evidence for or against specific receptors analogousto mammalian receptors in any plant/pathogen system. The only disease-re-
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HOST-SELECTIVE TOXINS 281
sistance gene cloned to date encodes an enzyme that detoxifies the HSTHC-toxin (40). Specificity of HC-toxin does not require specificity in thesite of action of HC-toxin.
HSTs Have Been Searched For but Never Found in ManyPlant Diseases
Inability to find an HST in any particular plant disease does not, of course,mean that it is not there. It is impossible to know from the scientificliterature how many searches have been unsuccessful, but HSTs have notbeen found even in some cases where they seem likely to be present, eitherby consideration of symptoms or because the pathogens are related to knownHST-producers. For example, resistance to Setosphaeria turcica (H.turcicum), the cause of Northern corn leaf blight, is monogenic and thefungus is related to a number of HST-producing fungi, yet no HST hasever been found. There is rather strong circumstantial evidence for theinvolvement of HSTs in wilt diseases caused by Fusarium species (76a,91). Consideration of pathogen and host variation suggests that severaladditional Alternaria species or pathotypes produce HSTs (77). Preliminaryor unconfirmed reports of other HSTs are generally not included in reviewsof HSTs (51).
Finding, characterizing, and proving the pathogenic relevance of an HSTcan require serendipity and perseverance. Without wishing to depreciate thesignificant accomplishments of the discoverers of the known HSTs, thesewere probably the easy ones to find. One problem can be an over-abundanceof candidates: many cellular pathogens make many phytotoxic compounds,which distract the investigator or interfere with analysis of the importantones. Culture filtrate components can be toxic and obscure the presence ofa toxin that is actually involved in pathogenesis. The most powerfultechnique for demonstrating the importance of HSTs in disease has beengenetic correlation using variation in sensitivity in the plant or variation inproduction by the pathogen, but this is not possible with some plants andmost pathogens.
Two facets of phytotoxicology are particularly critical: getting the patho-gen to make the HST in sufficient quantitites to detect and purify, andchoice of bioassay (85, 119). Species of Alternaria and Cochliobolusconstitutively make relatively large amounts of their HSTs in culture, butspecial media are sometimes required. PC-toxin production by Periconiacircinata and HC-toxin production by C. carbonum require the addition ofyeast extract to the medium (76a). Zinc ions are required for effectiveproduction of ACR and ACT toxins, specific for rough lemon and tangerine,respectively, from Alternaria citri (53). Ferric ions are required for produc-tion of the peptidic nonspecific toxin syringotoxin by Pseudomonas syringae
pv. syringae (66). Phenolic 13-glucosides induce the genes responsible forthe synthesis of syringomycin (65), and flavonoids induce the nodgenesof Rhizobium (see below).
It is likely that there are other HSTs that have not been discovered becausethe correct culture conditions have not been found. Tight developmentaland/or nutritional control of secondary metabolism is the rule in mostmicroorganisms (58). Other HSTs might be made only in planta, eitherbecause the toxins are made only at a particular developmental stage of thepathogen that occurs only in the plant, or because their synthesis is inducedby a physical or chemical signal from the plant. Few HSTs have beensuccessfully extracted from infected tissues. The histories of the character-ization of the two known toxic host-selective elicitors, the avr9 peptidefrom C. fulvum and syringolide from P. syringae pv. tomato, are instructive.The avr9 peptide was originally isolated from leaves of cfl)/cf9 tomato leavesinfected with Cladosporium fulvum containing the putative avr9 gene foravirulence. Subsequent to the cloning of the gene it was found that avr9is also expressed in culture, but only when nitrogen is limiting (99). Hence,the avr9 peptide could have been discovered in axenic culture, like classicHSTs, if the proper growth conditions had been known. Likewise, thehost-selective elicitor, syringolide, whose synthesis is controlled by the avrDgene of P. syringae pv. tomato, was found only when avrD was over-ex-pressed in E. coli; expression of the avrD gene is enhanced 100-fold inplanta and, like other bacterial avr genes (38), under appropriate culturalconditions (45). In principle it, too, could have been discovered before thedevelopment of molecular cloning.
An inappropriate bioassay will not manifest a toxin even if it is there.For example, a chloroplast-specific toxin will not inhibit root growth. Toxinaction in leaf assays can be suppressed or retarded by light (52). Someassays are much more sensitive or quantitative than others. Furthermore,even toxins that are important in disease are not necessarily toxic at thecellular level. Toxins such as victorin and T-toxin, as well as manynonspecific cellular poisons, cause cells to collapse and die, but other toxinsare more subtle. HC-toxin, for example, which is a certified HST andbeyond any doubt the critical specificity factor in leaf spot disease of maize(62, 70), inhibits root growth but not only does not kill nondividing leafmesophyll protoplasts but actually promotes their survival (112). While mosttoxins stimulate ion leakage, indicative of a malfunction of the plasmamembrane, HC-toxin stimulates uptake of certain ions (120). HC-toxin andits analog chlamydocin inhibit cell division but do not directly kill culturedmouse mastocytoma ceils; these compounds are more properly called cyto-static and not cytotoxic (107). Among other possibilities, a nontoxic toxincould be required for disease by inhibiting a constitutive enzyme required
only for defense or by inhibiting the induction of a defense response. Anontoxic HST of this nature could be called a host-selective suppressor (e.g.52).
It" the agent of specificity is known, rational and rapid progress can bemade towards both the cloning and functional analysis of pathogenicitygenes and resistance genes. Direct identification of the agents of specificityin plant diseases should be a high priority. Whether the agent is called anHST, an elicitor, or some other name, the experimental obstacles are similar:(a) induction of production, (b) assay, (c) purification, (d) structural dation, and (e) evaluation of its role in disease. Good biology is necessaryto overcome the first two; advances in chemistry have helped considerablyin the third and fourth; molecular genetics is the most powerful tool toaddress the fifth (45, 70, 73, 100).
Cell-Autonomous Resistance and Localized HypersensitivityAppear to Preclude "’Diffusible" Substances
The rubric "diffusible substance" is applied to small molecules such astoxins to distinguish them from macromolecules such as proteins. By Fick’sand Graham’s laws, and from empirical measurements of diffusion coeffi-cients, a molecule 100 times larger than another diffuses 10,000 to 100,000times more slowly (15, 69). Hence, small molecules such as toxins canmove in plant tissues and cause symptoms at a distance from the site ofsynthesis. Physiological perturbations caused by proteins or large polysac-charides are likely to be localized. Diseases in which toxins are involvedtypically show pathological symptoms some distance from the site ofinfection. Symptoms at a distance are prima facie evidence for the involve-ment of a toxin. For example, almond trees infected with Fusicoccumamygdali wilt due to the movement of fusicoccin to the stomata via thetranspiration stream (8).
In contrast, the hypersensitive response (HR), considered by many to the archetypal resistance response, is by definition localized to the site ofinoculation (96). In genetically chimeric plants and in tissue sandwichexperiments in which tissues expressing HR (incompatible) are immediatelyadjacent to susceptible (compatible) tissue, there appears to be no influenceof the compatible reaction on adjacent incompatible tissue, nor of incompati-ble tissue on adjacent compatible tissue (10, 16, 17). Hence, it has beenconcluded that, at least in rust diseases, no "diffusible" substances (i.e.toxins) are involved. "Cell-autonomy" of HR is consistent with models inwhich the pathogen elicitors and the host receptors are large molecules(polysaccharides or proteins) or are membrane-bound (43).
However, small molecules are not necessarily physically or functionallycapable of moving in a plant. Toxins can have limited solubility either in
the hydrophobic cellular membrane or in the intra- or extracellular aqueousapoplast; they might be metabolized; and they might become immobilizedlocally by specific or nonspecific binding. In C. carbonum race 1 infectionsof maize, symptoms are localized to the infected tissue and there are nosymptoms at a distance, even though an HST is involved in this disease.This is perhaps because HC-toxin moves but does not cause any overtsymptoms, or because the epoxide group reacts rapidly with omnipresentnucleophiles (87). Although metabolic degradation of HC-toxin is the basisof Hm-mediated resistance to C. carbonum race 1, HC-toxin is metabolizednonspecifically in the vascular tissues (62, 63). As a result, the half-life HC-toxin even in sensitive maize leaves might be too short to allow anysignificant movement and hence symptoms at a distance.
Potentially diffusible substances can elicit localized HR. The HR in tomatoinfected with C. fulvum (81) and in bean infected with P. syringae pv.tomato (45) is mediated by compounds small enough to be considereddiffusible. Both disease interactions have been claimed to conform to Flor’sgene-for-gene hypothesis (49, 100). E. coli cells overexpressing avrD fromP. syringae pv. tomato cause "systemic, spreading necrosis" (45), a symptommore characteristic of a toxin than of HR.
Furthermore, some large elicitors are capable of movement in plants. Theendoxylanase of Trichoderma viride, a 22-kd protein that elicits "’defenseresponses" such as ethylene biosynthesis, electrolyte leakage, and necrosis,moves in the xylem (6). Overall, apparent cell autonomy of resistance doesnot preclude the involvement of HSTs or other small molecules, nor dosymptoms at a distance preclude the involvement of macromolecules.
Although not all certified "toxins", e.g. HC-toxin, are toxic, it appears thatall known elicitors are toxic. Nonspecific biotic elicitors such as pectinase,xylanase, and pectic and other cell wall fragments cause necrosis, stimulateelectrolyte leakage, inhibit protein synthesis, and induce ethylene biosyn-thesis (e.g. 5, 18, 117). Abiotic elicitors such as UV irradiation, detergents,and heavy metals are detrimental to all cells. The two known host-selectiveelicitors, the avr9 peptide from C. fulvum and syringolide from P. syringaepv. tomato, cause host-selective necrosis (45, 100). This situation is para-doxical since by definition a toxin should be toxic, whereas there is no apriori reason why an elicitor must be toxic to induce plant defenses.
One of the hrp genes of Erwinia amylovora encodes a 44-kd protein("harpin") that causes necrosis and associated symptoms on the nonhosttobacco similar to that induced by the bacteria themselves (109). Other toxic
proteins from plant pathogens have been reported (e.g. 94) but theirbiological significance is less clear. Harpin is required for pathogenicity,like a toxin, but is also required for HR, like an elicitor. Should harpin becalled an elicitor or a toxin? It will be interesting to see if the gene forharpin, hrpN, is found in the hrp clusters of all pathogenic bacteria, and,if so, how the phytotoxicity of harpin relates to the toxicity of the productsof avrD and of other avr genes.
We do not know the modes of action of most HSTs (which, as discussedabove, is not necessarily the same as the basis of their specificity), but wedo know that they have different effects. Some toxins cause slow cellularcollapse, some act rapidly. Some toxins cause chlorosis, or membraneleakiness, or accumulation of toxic metabolic intermediates, or ultrastructuralalterations in organelles. Regardless of their action, the simple (and perhapssimplistic) raison d’etre of toxins is that they kill or metabolically compro-mise sensitive cells and thereby turn the plant tissue into a nutrient medium.To rationalize the selective advantage of HSTs, it is not necessary to postulateadditional steps in the process, although this does not mean that additionalsteps are not required.
For elicitors, however, simple cell death is inadequate to account for theultimate resistance phenotype, at least in diseases caused by nonobligatepathogens. Associated with or as a result of HR-associated cell death theremust also be the induction of "active defense responses", since there is noevidence that a dead cell per se is inhibitory to any potential pathogen.What these defenses actually are is still controversial, but candidates includelignification, synthesis of pathogenesis-related proteins such as chitinase and131,3-glucanase, and accumulation of phytoalexins. But how do elicitors killcells, and is the means by which they do so fundamentally different fromhow HSTs kill cells? Despite the description of HR-associated phenomenasuch as activated oxygen production (92) and K+/H+ exchange (3), we not know the mechanism of HR-associated cell death. Much of the descrip-tive work on HR can be seen as a list of the symptoms of a particular wayof dying. Electrolyte leakage, for example, is a commonly reported "defense"response to specific and nonspecific elicitors (e.g. 5, 46, 71); it is also theoldest and most widely used assay for HSTs (110). Because stimulation ion leakage is such a general response of injured cells, it has never beena very useful clue to the mode of action of any toxic agent.
It has been proposed that in HR the plant cell is triggered by the elicitorto commit suicide (e.g. 20). Support for this idea comes from the frequentobservation that elicitation of HR and the induction of phytoalexins by fungiand bacteria are blocked by inhibitors of plant protein synthesis and by heatpretreatment (44, 58a, 93). Significantly, the toxic effects of some HSTsalso require protein and/or RNA synthesis. Pretreatment of oat tissues or
protoplasts with various inhibitors of protein or RNA synthesis protects themfully, and reversibly, against the effects of victorin (28, 106). Cycloheximidepretreatment protects sorghum tissues against PC-toxin (113). Inhibitors protein synthesis as well as light suppress the action of some AlternariaHSTs (52). Heat treatment of tissues induces insensitivity to several HSTs,including PC-toxin, victorin, AM-toxin, AK-toxin, and helminthosporoside(13, 77). In at least three cases, heat-induced insensitivity to an HST correlated with the pattern of seasonal field resistance to the producingpathogen (77). If elicitors induce plant suicide, we must consider thepossibility that HSTs do so also (106).
The fundamental question for distinguishing between a toxin and anelicitor is how can cell death be associated in some diseases with suscep-tibility and in others with resistance? For obligate pathogens, host death atthe cellular level must perforce lead to resistance of the plant as a whole.For facultative saprophytes, which include most imperfect and ascomycetousfungi and bacteria, the relation between HR-induced cell death and resistanceis obscure. If cell morbidity is sufficient to trigger effective defense re-sponses, then HSTs must somehow kill cells without allowing those defensesto be triggered, or else the fungi that make HSTs must be resistant to thosedefenses. Victorin induces avenalumin production in oats, and is thereforean elicitor as well as an HST, but the producing fungus is resistant to thisphytoalexin (60). The ability of P. circinata to infect sorghum is unaffectedby prior induction of "defense" responses with an elicitor (75). Perhaps pathogen must be specialized to cope with all of the inducible defenses ofits host (whatever these might be) before an HST will confer any selectiveadvantage.
Overall, there appear to be no chemical or biochemical criteria by whichone can distinguish, as biologically active molecules, elicitors from toxins.If a pathogen-produced toxic compound has not been or cannot be shownto elicit either a susceptible or resistant reaction in the host, then whetherto call it a toxin or an elicitor is completely arbitrary. Many describedcompounds fall into this category. Furthermore, for neither elicitors norHSTs can we distinguish between cell death as a symptom and cell deathas a determinant of the ultimate pathogenic phenotype. If cell death is adeterminant, then why does cell death in some cases lead to successfulcolonization and in other cases the opposite? Bailey & O’Connell (7) proposethat it is simply a matter of timing: if only moribt~nd cells can mount aneffective active defense response, then susceptibility will result if plant cellsdie quickly and resistance will result if the cells die slowly. Therefore, afast-acting elicitor would be a toxin, and a slow-acting toxin would be anelicitor. The question of whether elicitors are triggers of a process by whichplant cells kill themselves can also be asked of toxins. The importance of
the answer supports the importance of the study of the mode of action ofboth elicitors and toxins.
SECONDARY METABOLITES ARE OF PRIMARYIMPORTANCE IN THE LIVES OF MICROORGANISMSAND PLANTS
The past few years have seen the discovery of the first peptide hormone inplants (61), the first proteinaceous HST (94a), and the first ribosomallysynthesized specific (81, 100) and nonspecific (e.g. 109) peptide elicitors.At the same time, however, the number and types of secondary metabolitesshown to be involved in plant/microbe interactions is expanding at a greatrate. Tens of thousands of secondary metabolites, with the vast majoritybeing from plants and microorganisms, have been described (58). There a growing consensus that secondary metabolites are important above all inthe interactions between organisms, including herbivory, pathogenesis, sym-biosis, and competition (90). Many animal hormones are ribosomally syn-thesized polypeptides, but with the exception of systemin (61), all knownplant growth regulators are small molecules, synthesized by enzymes inpathways that are distinct from those of primary metabolism. In animalpathology "toxins" are, except for the relatively minor endotoxins, allbacterial proteins. These toxins are important virulence factors in foodpoisoning, botulism, cholera, diphtheria, gangrene, dysentery, and whoopingcough. Again in contrast to mammalian toxins, all phytotoxins are with fewexceptions secondary metabolites.
In addition to HSTs and elicitors, secondary metabolites that are knownor suspected to be important in plant/pathogen interactions include phyto-alexins, produced by plants in reponse to biotic and abiotic stresses (23),and nonspecific toxins (8, 111). Biological control of pathogenic microor-ganisms by other organisms can involve competition, parasitism and preda-tion, or antibiosis (27); the evidence for antibiosis in some situations particularly strong. For example, suppression of black root rot of tobaccoby Pseudomonas fluorescens is due to 2,4-diacetylphloroglucinol (42). 2.8-kb region of DNA from P. aureofaciens controls both the synthesis ofthree phenazine antibiotics and the suppression of take-all disease of wheat(73). Suppression by P. fluorescens of damping-off of cotton is associatedwith the production of the antibiotic oomycin A (37). That biological controlcan actually be a variety of "microchemical" control epitomizes the import-ance of secondary metabolites in the ecological relationships of microorga-nisms with each other and with plants.
Both specific and nonspecific secondary metabolites also play an importantrole in symbiotic plant/microbe interactions. Various plant flavonoids are
the first known signal to move between Rhizobium and its hosts, setting inmotion the complex series of developmental events leading to nodulationand nitrogen fixation (72). (Other compounds of the phenylpropanoid path-way induce the vir genes of Agrobacterium necessary for infection of itsdicotyledonous hosts (122)). Discrimination among various flavonoids the product of the nodD gene of Rhizobium contributes to host-rangespecificity (72). NodD activates the other nod genes, both the "common"ones and the "host-specific" ones determining host-range. The structuralnod genes encode enzymes that catalyze the various steps in the synthesisof small lipo-oligosaccharides known as Nod factors. The discovery of theNod factors (55) ended the monopoly of HSTs as the only known agentsof specificity in plant/microbe interactions. The common nod genes nodABCdetermine a core Nod-factor structure, which is an acylated tetramer ofN-acetylglucosamine. The deduced nodC protein sequence is highly similarto chitin synthetases from yeast, and therefore nodC likely encodes anenzyme that synthesizes the tetrameric backbone (2, 21). The core of theNod factors is modified by the gene products of the host-specific nod genessuch as nodFEG, nodH, nodPQ, and nodSU. By analysis of the differencesbetween Nod factors, and by amino acid sequence similarity to genes ofknown function, the functions of the host-specificity nodP and nodE genes,for example, have been deduced to encode the biosynthetic enzymes ATPsulfurylase and [~-ketoacyl synthase, respectively (cited in (2)).
The characterization of Rhizobium nod genes makes an interesting para-digm that might also occur in plant/pathogen interactions. A biosyntheticpathway in which a nonspecific core compound is modified to one withspecificity is a way in which genes for "basic compatibility" (35) couldintermesh with pathogen genes determining specificity. The Rhizobium nodgene situation is reminiscent of isoflavonoid phytoalexin biosynthesis, whereenzymes early in the pathway such as phenylalanine ammonia lyase andchalcone synthase are common to many plant species, but later enzymessynthesize the spectrum of flavonoids unique to various species. Althoughphytoalexins are generally considered to be part of a general defense responseof plants, there is no reason that genes encoding enzymes late in thephytoalexin biosynthetic pathway could not behave as specific, monogenicresistance genes. Tolerance ("resistance") to particular phytoalexins madeby the host plant by detoxificative metabolism is important in virulence ofthe pea pathogen Nectria haematococca and probably other fungal pathogens(101). This is the converse of detoxification of HC-toxin as the basis resistance of maize to C. carbonum race 1 (40, 62). Toxic secondarymetabolites and means of neutralizing them are demonstrated successfulstrategies of both plants and pathogens.
MOLECULAR BIOLOGY OF HOST-SELECTIVE TOXINBIOSYNTHESIS
As discussed above, it is important to understand the modes of action ofmolecules such as HSTs and host-selective elicitors that have a known rolein disease. Likewise, it is important to understand the nature of the pathogengenes that control the synthesis of these compounds. This might lead toinsights into the evolution of host-range of plant pathogens, which, in turn,might allow plant pathologists and plant breeders to develop more effectivedisease-control strategies.
The molecular genetics of the biosynthesis of a number of bacterialnonspecific toxins have been studied (32, 111), but to the best of ourknowledge only HC-toxin biosynthesis by C. carbonum has been studiedenzymologically. The goal of this research has been to understand howHC-toxin is synthesized, and to use the enzymes as a means of cloning theTOX2 gene. Research on TOX2 can also serve as a model for understandingother TOX genes in Cochliobolus and Alternaria.
In 1961, Nelson & Ullstrup (68) demonstrated that races 1 and 2 of carbonum differ by a single Mendelian gene with respect to pathogenicityon maize lacking the Hrn gene for resistance. This was before the discoveryof HC-toxin and no function other than determination of race-specificpathogenicity could be assigned to the unnamed gene in the fungus. Afterthe discovery that race 1 of C. carbonum uniquely produces the host-selectivemetabolite, HC-toxin, in culture (79), Scheffer et al (78) showed that ability to produce HC-toxin segregated with the previously identified patho-genicity gene. This gene is now called TOX2 (121).
Enzymology of HC-toxin Production
The elucidation of the structure of HC-toxin as a cyclic tetrapeptide,cyclo(D-prolyl-L-alanyl-D-alanyl-L-Aeo), where Aeo stands for 2-amino-9,10-epoxy-8-oxodecanoic acid (33, 41, 104), suggested that the compoundmight be synthesized like other small microbial peptides such as gramicidinand tyrocidine. Peptide antibiotics are produced on large, multifunctionalenzymes that catalyze several sequential steps. The amino acids are firstactivated by adenylation (amino acid + ATP ~- aminoacyl-AMP + PPi),then covalently bound to the enzyme by a thioester linkage with the releaseof AMP. Peptide bonds are then formed between the bound amino acidsand the peptide is released. The amino acid-adenylation step is reversibleat equilibrium and serves as the basis of the convenient ATP/PPi exchangeassay for nonribosomal peptide synthetases (48).
Based on this assay, amino acid-dependent ATP/PPi exchange activities
predicted for an "HC-toxin synthetase" were detected in C. carbonum race1 (102). Activities dependent on the amino acids D-alanine, L-alanine, andL-proline (but not D-proline) were found. These enzyme activities aredetectable only in race 1 isolates of C. carbonum, and genetically segregatewith TOX2 (102). All three activities co-precipitate at a low ammoniumsulfate concentration, but the L-proline activity is separable from the twoalanine activities on several chromatographic media. Therefore, our initialconclusion was that at least two enzymes participate in the synthesis ofHC-toxin, and that therefore TOX2 is a gene cluster, perhaps similar toother antibiotic gene clusters (36).
These enzymes were purified. HC-toxin synthetase 1 (HTS-1), has an rof 220 kd, catalyzes ATP/PPi exchange dependent on L-proline, and epi-merlzes L-proline to D-proline, the isomer found in HC-toxin. The secondenzyme, HC-toxin synthetase 2 (HTS-2), has an r of 1 60 kd, c atalyzesATP/PPi exchange with either D-alanine or L-alanine, and epimerizes L-al-anine to D-alanine, but not vice versa. Both enzymes bind their amino acidsubstrates covalently as thioesters (108).
The discovery that HC-toxin synthetase would recognize and incorporateD-alanine into HC-toxin (102) suggested a method to prepare radiolabeledHC-toxin. D-alanine should be a relatively specific precursor, and in factup to 3% of radiolabeled D-alanine added to the culture medium is incor-porated into HC-toxin (63). As expected, little of the radiolabeled D-alanine,unlike L-alanine, is diverted to protein (63). Radiolabeled HC-toxin preparedin this way was used to study metabolism of HC-toxin by maize (62, 63).
Cloning of HTS1
Research on the gene(s) encoding HTS-1 and HTS-2 began with the isolationof a cDNA clone from a lambda gtll expression library screened withmurine polyclonal antibodies raised against HTS-1. One immunopositivecDNA was found to encode a peptide obtained by direct sequencing ofHTS-1 (83). This cDNA was used as a probe to identify genomic clonescontaining the HTS-l-encoding gene, called HTS1 (70).
Without considering the molecular details of HTS1 or the function of itsproduct, there are three striking features of the chromosomal locus encodingthis gene. First, HTS1 is part of a contiguous region of DNA, 22 kb inlength, that has absolutely no homology to DNA from any non-HC-toxin-producing race of C. carbonum or species of Cochliobolus tested (70).HTSl-specific primers amplify a fragment of the right size by PCR withDNA from all race 1 isolates, but never from isolates of other races orspecies (40a). Second, this 22 kb of race l-unique DNA is flanked on eitherside by moderately reiterated DNA (70). Elements from the 5’ and 3’ flanks
have homology to one another as well as to repeated sequences from otherraces of C. carbonum and C. victoriae (70; D. G. Panaccione & J. D.Walton, unpublished results). Third, the entire 22-kb race l-unique region,as well as at least 6 kb of repeated DNA on the 5’ flank and 2 kb on the3’ flank, are duplicated (70). The duplication has been found in all race isolates examined, and the duplicate copies are genetically linked.
These findings have interesting implications with respect to the evolutionof race-specific pathogenicity in C. carbonum. The finding that the DNAfrom toxin-nonproducing races of C. carbonum has no homology with theDNA required for HC-toxin production and pathogenicity indicates that theevolution of races in C. carbonum involved the deletion or acquisition ofa substantial region of DNA. HC-toxin biosynthetic capability might at onetime have been common to the species and was subsequently deleted incertain isolates, to create race 2, or it might have been recently acquiredby the species, to create race 1. This question has practical implications.If HC-toxin production is an ancestral trait lost by some races, then theappearance of new toxin-producing races of Cochliobolus pathogens isunlikely. However, if Cochliobolus species can acquire novel toxin biosyn-thetic genes, by horizontal gene transfer for example (58), then isolateswith the ability to produce new toxins might appear periodically.
The presence of repeated DNA flanking the 22-kb race 1-unique DNAis curious given the relative rarity of repeated sequences in filamentousfungi. Limited sequence analysis has failed to find significant similaritiesbetween the repeated elements and any known gene or transposable element.Although this repeated DNA is common to tox+ and tox- isolates, it is notknown if it is present in the same region on the homologous tox- chromo-some or whether it is scattered throughout the genome. If in the samelocation, then crossing over within the repeated DNA of the TOX2 locusshould be possible, resulting in aberrant segregation ratios for the tox+
phenotype. This has not been observed (68). Physical mapping of TOX2using pulsed-field gel electrophoresis should resolve the nature and locationof the TOX2-associated repeated DNA (J. Ahn & J. D. Walton, researchin progress).
The significance of the duplication of the HTS1 locus, with respect toevolution, is in its precision. No RFLPs were detected with any of 22 six-bprecognition sequence restriction enzymes tested (70), suggesting recentevolution of the duplication. If strains with a single copy of the HTS1 locuscan be found, then the precision of the duplication merely indicates thatHTS1 was duplicated recently, and no inferences to the evolutionary ageof race 1 can be drawn. However, if the duplicate form is the original stateof race 1, then race 1 is probably of recent origin. Six isolates of C.
carbonum race 1 of broad geographical distribution (South America, Europe,and North America) have been examined, and all contain the duplicationof HTS1 and flanking DNA (D. G. Panaccione & J. D. Walton, unpublishedresults).
Disruption of HTS1
There were three compelling reasons to create mutants of HTSI: first, toconfirm the identity of the cloned gene as encoding the L-proline activatingenzyme, HTS-1; second, to test definitively the role of HTS-1 in HC-toxinbiosynthesis; and third, to test further the essential role of HC-toxin as apathogenicity determinant. The linked duplication of the HTS1 gene pre-sented an unusual problem for the creation of mutants by transformation-mediated gene disruption. The problem was overcome by disrupting singlecopies of HTS1 in two successive transformation experiments with twodifferent selection systems (70).
Disruption of one copy of HTSI near the 5’ end of the gene results inan approximately 50% reduction in HTS-1 activity, measured as L-proline-dependent ATP/PPi exchange. Mutants created in this way still produceHC-toxin and cause lesions identical to those caused by wild-type race 1isolates. When the second copy of HTS1 in these strains is disrupted at thesame site, the mutants completely lose HTS-1 activity (70). These strains,now with no functional copies of HTS1, are unable to produce HC-toxinand cause only small chlorotic flecks, identical to those produced by race2 isolates, on susceptible corn. This constitutes the strongest evidence todate for the essential role of HC-toxin in leaf spot disease. Unexpectedly,HTS1 mutant strains also have a reduction in HTS-2 activity (i.e. D- andL-alanine-dependent ATP/PPi exchange) proportional to the correspondingreduction in HTS-1 activity (70). Until this experiment, the biochemicalevidence had indicated that I-ITS-1 and I-ITS-2 were separate enzymes thatfunctioned independently (70, 108).
In a second set of disruption experiments, both copies of HTS1 weredisrupted near the 3’ end (70). Mutants created in this way are unable produce HC-toxin and lack race 1 pathogenicity. However, these strainsretain 60% of their HTS-1 and HTS-2 activities. As demonstrated withstrains that have one copy of the 5’ end of HTS1 disrupted, these levels ofHTS-1 and HTS-2 activities are sufficient for HC-toxin production (70).Thus, mutants created by disruption near the 3’ end of HTS1 are unable toproduce HC-toxin due to the loss of some activity other than those associatedwith HTS-1 and HTS-2. These data indicate that the product of the HTS1gene catalyzes multiple steps in the synthesis of HC-toxin.
All mutants created by disruption of HTS1 accumulate in culture a novelepoxide-containing metabolite that is apparently related to the amino acid
Aeo found in HC-toxin (D. G. Panaccione, K. Akimitsu, & J. D. Walton,unpublished results). This molecule is made in trace amounts by wild-typerace 1, in high amounts by HTS1 mutants, but not at all by race 2 isolates.Therefore, the epoxide-containing compound is linked to HC-toxin, perhapsas a precursor or shunt metabolite of Aeo. If so, this indicates that, first,this molecule is not synthesized by the product of the HTS1 gene, and,second, that race 2 lacks the enzymes to make it. Since, to the best of ourknowledge, only a single gene, TOX2, is necessary for HC-toxin production,the presence of this epoxide-containing compound indicates that TOX2includes novel genetic information necessary for HC-toxin production inaddition to HTS1.
Sequence of HTS1
The resolution to the problem of coordinate reduction in HTS-2 activityupon disruption of the gene for HTS-1 lies in the sequence of ttTS1 (83).HTS1 is an enormous, 15.7-kb open reading frame (ORF), capable encoding a protein of 5215 amino acids and a Mr of 570 kd (Figure 1).This is to the best of our knowledge the largest known ORF from anyorganism, and it encodes the second largest known polypeptide of thosewhose size has been confirmed by DNA sequencing (83). The deducedproduct of this ORF contains stretches of peptide sequence obtained bydirectly sequencing HTS-1, and also a 20-amino acid peptide obtained fromsequencing HTS-2. Thus, HTS-1 and HTS-2 are potentially part of a singlepolypeptide. In support of the hypothesis that HTS-1 and HTS-2 exist asa single, large polypeptide in the cell, anti-HTS-2 antiserum recognizes apolypeptide of Mr greater than 480 kd in some HTS preparations (83).Furthermore, the predicted product of HTS1 has a primary structure verysimilar to other large peptide antibiotic synthetases (Figure 1).
The deduced product of the HTS1 ORF contains four domains, each ofabout 600 amino acids, that are similar to each other as well as to similardomains found in other eukaryotic and prokaryotic peptide synthetases(Figure 1; 83). These include the aminoadipyl-cysteinyl-valine (ACV) thetases of various prokaryotes and filamentous fungi (e.g. 89) and gram-icidin synthetase 2 of Bacillus brevis (95). In the multifunctional peptidesynthetases studied thus far, the number of domains in a particular enzymecorresponds to the number of amino acids activated by that enzyme. Thiscorrespondence appears to be true also with HC-toxin synthetase, sinceHTS1 of C. carbonum encodes four domains and HC-toxin is a tetrapeptide.However, the ability of the HTS1 product to activate Aeo has not yet beendemonstrated due to its unavailability.
The sizes of the regions between domains are also quite similar amongthe three multifunctional cyclic peptide synthetases (Figure 1). Although
Figure 1 Comparison of three multifunctional peptide synthetases. Left: deduced amino acidsequence of the tripartite ACV synthetase encoded by acvA from Penicilliurn chrysogenurn (89).Center: the tetrapartite gramicidin synthetase 2 encoded by grsB from Bacillus brevis ATCC 9999(95). Right: the tetrapartite HTS encoded by HTS1 from Cochliobolus carbonum race 1 (83).Domains that are similar to each other within and between the different enzymes are shaded (83).The sequences for ACV synthetase and gramicidin synthetase 2 start at the known translationalstart sites, and the sequence of HTS starts at the beginning of the HTS1 ORF. The presumedtranslational start of HTS is the methionine at position 16 or 18 (83).
both gramicidin synthetase 2 and HC-toxin synthetase activate four aminoacids, HC-toxin synthetase is larger because of a larger region betweendomains 1 and 2 (Figure 1). The enzymatic significance of this region unknown (83).
There is less overall amino acid identity among the four domains of theHTS1 product than there is among the domains of ACV synthetase orgramicidin synthetase (83). If one assumes that multifunctional peptide
synthetase genes arose by repeated duplication of one such domain, then,based on similarity among domains in individual proteins, HC-toxin syn-thetase is older than the ACV synthetases or gramicidin synthetase.
A striking feature of HTS1 is its apparent lack of introns (83). All fourother known C. carbonum genes, although only a fraction of the size ofHTS1, contain one or two introns (82; Walton et al, unpublished results).The other eukaryotic peptide synthetase genes sequenced thus far (the ACVsynthetase genes of P. chrysogenum, C. acremonium, and A. nidulans) alsocontain no introns (see (83)).
Is TOX2 a precedent for other TOX loci? First, exhaustive efforts toobtain TOX1 mutants of C. heterostrophus by classical mutagenesis havebeen unsuccessful (14). One possible explanation for this failure is thatsome or all of TOX1, like TOX2, is duplicated. Second, TOX1 and TOX3of C. victoriae could also encode biosynthetic enzymes, for T-toxin andvictorin, respectively. Victorin is also a cyclic peptide, so "victorinsynthetase" is undoubtedly functionally and structurally related to HC-toxinsynthetase and other cyclic peptide synthetases. T-toxin is a polyketide,and all known polyketides are synthesized by large, multifunctionalenzymes encoded by gene clusters (36). Polyketide and cyclic peptidesynthetases are functionally related (56). Using PCR with primers basedon amino acid regions conserved in all peptide synthetases, we havecloned fragments of peptide synthetase genes from C. victoriae and arein the process of testing by targeted gene disruption their involvement invictorin biosynthesis as well as their relationship to TOX3 (D. G.Panaccione & J. D. Walton, unpublished results). In contrast to TOX2,the DNA encoding the putative victorin synthetase is found in allCochliobolus species tested. Therefore, TOX3 might differ from TOX2 inthis fundamental aspect.
What is TOX2?
Despite extensive structural and functional analysis of HTS1, we do not yetunderstand all of TOX2. Including the known repetitive DNA flanking the22-kb tox÷-unique DNA (70), which is also duplicated and shows tox÷-
unique RFLPs, we estimate that TOX2 is at least 56 kb in size (70).However, there is no theoretical reason why TOX2 could not be even tentimes larger. We are currently mapping the distance between the two copiesof HTS1 using pulsed-field gel electrophoresis. If TOX2 is large enoughrelative to the size of the chromosome it is on, there might be detectablechromosomal size polymorphisms between race 1 and race 2 isolates of C.carbonum. Between races T and O of C. heterostrophus, there is achromosomal polymorphism resulting from a translocation associated withthe TOX1 locus (12).
HTS1 is the core of TOX2, but it appears likely that additional enzymaticactivities are necessary for the synthesis of Aeo. If those additional enzymeactivities are restricted to race 1 of C. carbonum, as appears to be the case,then they must be linked to HTS1 as part of TOX2. DNA linked to HTS1and unique to race 1 of C. carbonum has been identified by chromosomewalking from the 22-kb race 1-unique region (Walton et al, unpublished).These sequences may contain additional genes involved in HC-toxin bio-synthesis.
The complexity of TOX2 was implicit from earlier genetic studies. Schefferet al (78) discussed evidence that the locus governing HC-toxin biosynthesismay be a complex locus. Progeny of crosses between race 1 isolates ofdifferent geographical origin differed in the amount of HC-toxin theyproduced, leading to the conclusion that "the amount of toxin produced isconditioned by several genes" (78). Based on what we now know of theTOX2 locus, recombination within this locus in a cross between a race 1isolate and a toxin-nonproducing isolate is unlikely because of the absenceof a recessive allele for HTS1. However, in crosses between race 1 isolatesmeiotic recombination within the TOX2 locus could occur (the ratio ofphysical to genetic distance in C. heterostrophus has been estimated at 23kb/cM (97)) and result in progeny that produce increased or decreasedquantities of HC-toxin.If race 1 did evolve recently through the acquisition of HC-toxin biosyntheticcapability, from where did that DNA originate? Cyclic tetrapeptides con-taining Aeo are found in four other, unrelated fungi. These are Cylindroclad-ium scoparium, which produces Cyl-2, Diheterospora chlamydosporia,which produces chlamydocin, Petriella guttalata, which produces WF-3161,and Helicoma ambiens, which produces trapoxin (39, 103). The gene forHTS1 does not hybridize to DNA from these other species, indicating thatthe other cyclic peptide synthetases are not highly similar to HTS1, butsequences encoding peptide synthetases from several of these fungi havebeen cloned by PCR using primers based on amino acid sequences highlyconserved among the cyclic peptide synthetase genes that have been se-quenced (83; A. Nikolskaya & J. D. Walton, unpublished results). Com-parison of the sequences of these other cyclic peptide synthetase sequencesto HTS1 will reveal their evolutionary relatedness.
The mechanism and distribution of plant resistance to HC-toxin counterthe hypothesis that HC-toxin synthesis might have evolved recently in C.carbonum race 1. Resistance to C. carbonum race 1 in maize occurs byenzymatic detoxification of HC-toxin (40, 62). The responsible enzyme found in resistant maize but also wheat, barley, oats, and sorghum (63a).Maize of genotype hm/hm lacks a functional gene and lacks the enzyme,
yet is phenotypically normal, indicating that this enzyme has no essentialhousekeeping function (40). If HC-toxin reductase exists only to protectplants against HC-toxin or its Aeo-containing relatives, then it would appearthat these cyclic peptides have been a significant factor in the evolution ofthe Poaceae.
CONCLUSION
Plant pathologists have for many years discussed the possibility of a modeldisease upon which a community of researchers could focus. The reality,however, is that viruses, bacteria, nematodes, and nectrotrophic andbiotrophic fungi are so different from each other that it is unlikely that theirinteractions with plants are governed by the same processes and agents.Therefore, although HSTs are critically involved in many diseases, andalthough many HSTs undoubtedly remain to be discovered, we would notwant to propose that all or even the majority of diseases will prove toinvolve HSTs or HST-like molecules such as secondary metabolite elicitors.Recent research on diseases other than those traditionally associated withHSTs demonstrates that HSTs and the diseases in which they occur do sharemany key attributes with other agents of specificity and other diseases.However, despite the emergence of this theme, the only currently safegeneralization is that speculation about diseases for which there is noexperimental evidence is hazardous, all the more so in view of the accel-erating pace of experimental progress in plant pathology.
The only answer to the question of whether HSTs are typical or atypicalspecificity factors are further questions. Is C. fulvum a typical fungalpathogen? Is syringolide a typical bacterial elicitor? Do leaf mold andbacterial speck of tomato represent typical gene-for-gene interactions? Thesequestions are unanswerable and will remain so until many more diseaseinteractions are understood in detail. It is exciting that recent years haveseen so much definitive progress. The next few years should see furthertremendous progress and even, as has long been the not-so-secret hope ofresearchers in the field of plant/pathogen interactions, the emergence ofnovel ideas of interest to all biologists.
ACKNOW~.ED~MErCr
Research in J. D. W.’s laboratory has been supported by the US Departmentof Energy Division of Energy Biosciences, the US National Science Foun-dation, and the US Department of Agriculture. The authors thank RobertScheffer for helpful discussions of some of the ideas expressed in thisreview, but they alone take responsibility for the contents.
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