Alain Stintzi

Thierry Heitz

Vivek Prasad

Sabine Wiedemann-Merdinoglu

Serge Kauffmann

Pierrette Geoffroy

Michel Legrand

Bernard Fritig

A Stintzi3

T Heitz3

V Prasadb

S Wiedemann-Merdinogluc

S Kauffmann3

P Geoffroy3

M Legrand3

B Fritig3

Plant 'pathogenesis-related' proteins and their role in defense against pathogens

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pathogenesis-related (PR) proteins were frst detected in the early 1970s [START_REF] Payne | Isolation and sequence of a genomic clone encoding the basic form of pathogenesis related protein I from Nicotiana tahacum[END_REF][START_REF] Van Loon | Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var 'Samsun' and 'Samsun NN'. Changes in protein constitution after infection with TMV[END_REF] in tobacco leaves reacting hypersensitively to tobacco mosaic virus (TMV). This is the biological system that we are also using and *Correspondence and reprints from which the highest number of PR-proteins have been characterized so far.

The hypersensitive reaction to pathogen attack is considered as a very strong defense response induced in plants by the pathogen itself. It follows the pheno menon of gene-fr-gene complementarity [START_REF] Flor | Inheritance of pathogenicity in Melamp sora lini[END_REF][START_REF] Ellingboe | Changing concepts in host pathogen genetics[END_REF][START_REF] Ellingboe | Genetical aspects of active defence[END_REF][START_REF] Crute | The genetic bases of relationships be tween microbial parasites and their hosts[END_REF] be tween plant and pathogen. In the presence of domi nant. matching alleles of a pathogen avirulence gene and of a plant disease resistance gene, recognition occurs and the plant responds to infection by produ � ina the hypersensitive response [START_REF] Keen | Gene-for-gene complementarity in plant pathogen interactions[END_REF][START_REF] Wit | Molecular characterization of gene for-gene systems in plant-fungus interactions and the application of avirulence genes in control of plant patho gens[END_REF][START_REF] Keen | The molecular biology of disease resis tance[END_REF]. Necrotic le�ons are fonned around the initial sites of patho genic attack . The pathogen wil� be r � stricted to a zone of cells surrounding the necrotic les1 � n � , as a re � ult � f a very intense defense response that 1s mduc � d . m this ring of cells [ 10]. In the case of the hypersens1t1ve res ponse of tobacco to TMV, the zones of highly-induced defense responses can easi�y . �e det � cted under UV light as rings of cells exh1b1tmg bnght blue fluor escence and surrounding the necrotic lesions. These cells have accumulated derivatives of the phenyl propanoid pathway [11][START_REF] Legrand | Enzymes of the phe nylpropunoid pathway and the necrotic reaction of hyper sensitive tobacco to tobacco mosaic virus[END_REF][START_REF] Legr | ind M (l 983 J Phenylpropanoid metabolism and its regulation in disease[END_REF], some of which are fluo rescent, and thus represent good indicators of defense responsive cells. Other low molecular mass compo nents accumulate equally well in these celJs and have antimicrobial activity, the so-called 'phytoalexins' [START_REF] Bailey | Pliytoalexi11s[END_REF][15][START_REF] Darvill | !"hytoalexins and their elicitors-a defense against microbial infection in plants[END_REF][START_REF] Ebel | Phytoalexin synthesis: the biochemical ana lysis of the induction process[END_REF]. But many other changes occur in t�e sa!11e cells [10,[START_REF] Fritig | Mechanism of the hypersensitive reac tion of plants[END_REF][START_REF] Collinge | Plant gene expres sion in response to pathogens[END_REF]. For instance, celJ wall thickening and reinforcement by deposition of various mac � o molecules such as carbohydrates (callose), proteins [START_REF] Bradley | Elicitor-and wound-induced oxidative cross-linking of a proline-rich 38[END_REF] and hydroxyproline-�ich. gl>'c � pr�teins (2 ! ],

aromatic polymers such as hgnm, hgnm-hke matenal or other yet undefined cell wall bou. nd phenolic. s [22,23). Another important change which occurs m the fluorescent cells is the production of the numerous PR-proteins which, after a few days post-inoculation, may account for 10% of the content of the leaves in soluble proteins [24][25][26][27][28][29].

Some of these changes are strictly localized around the infection sites, for instance the accumulation of Huorescem phenylpropanoid metabolites of tobacco I 12) or generally in most plant species the production of low molecular mass antimicrobial substances ( l 0, 17). However, some of the changes are also induced at distance from the infection sites and even in uninocu lated and uninfected pans of partially-infected plants, though to a much lower extent than locally. This is the case for some of the PR-proteins, whose production in uninoculated tobacco leaves represents 5-10% of their production in inoculated hypersensitively reacting leaves. Even though this 'systemic' defense response is of much lower cellular intensity than the local response it still represents a massive amplification in the response since it con\!ems the whole plant. Further more, this systemic defense response is accompanied by the phenomenon of •systemic acquired resistance'. If part of a plant has already reacted hypersensitively to a pathogen, the uninoculated parts of this plant develop an increased state of resistance evidenced by smaller lesions and greater restriction of the infection upon challenge inoculation by the same or any other unrelated but necrotizing pathogen [30][31][32][33][34][35][36][37]. Since the PR-proteins are induced in parallel with the appear. ance of systemic acquired resistance they are believed to participate in its efficiency, but a causal relationship has still not bee n clearly proven. They represent also very interesting and sensitive markers �or the search of the signals involve� . tha� are . movmg . from _the primary sites of necrotJZJng mfection to distant sites where both systemic acquired resistance and PR proteins are induced (38).

Occurrence, induction and general properties of PR-proteins

Since the discovery of the first four PR-proteins in tobacco [ l, 2), many other proteins with similar ph � si cochemical and induction properties have been iso lated from tobacco (see below) as well as from many other plant species, including dicots and monocots.

It has been reported that PRs are produced during the response of plants to infection by viruses, viroids, bacteria or fungi (for reviews see [2�, �9-43p. _ Yery interestingly. they are produced constuut1vely m mter specific hybrids which exhibit high constitutive resist ance to viral infection [START_REF] Ahl | b-Protein as a constitutive component in highly (TMV) resistant interspecific hybrids of Nicotia11a gl1tti11osa x Nicotiana deb11eyi[END_REF]. However, at least some members of the different PR families have been found to be induced under treatments with chemicals such as polyacrylic acid [START_REF] Dumas | Genetic aspects of polyacrylic-acid-induced resistance to tobacco mosaic virus and tobacco ne crosis virus in Nicotia11a plants[END_REF], amino acid derivatives [START_REF] Asselin | Light-influenced extracellular accumulation of b (pathogenesis-related) proteins in Nicotiana green tissue induced by various chemicals or prolonged c.Moating on water[END_REF], heavy metal salts [START_REF] Ta Pia | Analysis of acid extractable bean leaf proteins induced by mercuric chloride treatment and alfalc.G mosaic virus infection. Partial purification and characterization[END_REF][START_REF] Nasser | Maize pathoge nesis-related proteins: (:huracterization and c.:ellular distri but ion of P-1.3-glucanases und chitinuses induced by bmme mosaic virus infoction or mercuric chloride treat ment[END_REF], aspirin and salicylic acid [START_REF] Bol | Plant pathogenesis-related proteins induced by virus infection[END_REF]42,[49][START_REF] Van De Rhee | Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treat ment and virus infection[END_REF][START_REF] Ward | Coordinate gene activity in response to agents that induce systemic acquired resistance[END_REF][START_REF] Ya | Sahcylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco[END_REF][START_REF] Malamy | Salicylic acid and plant disease resistance[END_REF][START_REF] Uknes | Acquired resistance in Arabidopsis[END_REF] and air pollutants [START_REF] Ec | Ozone induced changes of mRNA levels of �-1,3glucanase, chitinase and 'pathogenesis-related' protein lb in tobacco plants[END_REF][START_REF] Didierjean | Plant genes induced by chemicals and pollutants[END_REF]. Some PRs are also induced by treatments with phytohormones.

Even though cases of induction by auxins [57, �8] or cytokinins [59, 60} are known, the pattern of mduc• tion by these two honnones is unclear, except under conditions of strong disturbance in the balance be• tween them which lead to high induction of various families of PRs [START_REF] Mohnen | Hor monal regulation of (3-1,3-glucanase messenger RNA levels in cultured tobacco tissues[END_REF][START_REF] Felix | Developmental and honnonal regulation of j}-1,3-glucanase in tobacco[END_REF][START_REF] Leguay | Auxin (2,4-dichiorophe noxyacetic acid) starvation and treatment with glucan eli citor isolated from Pllytophtlwra megasperma induces similar responses in soybean-cultured cell suspensions[END_REF][START_REF] Shinshi | Regulation of a plant pathogenesis-related enzyme: inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin[END_REF][START_REF] Sauter | The mycorrhizal fungus Ama• ,;ta nmscaria induces chitinase ac;tivity in roots and in suspension-cultured cells of its host Picea abies[END_REF]. Some PRs are induced under osmotic stress or salt stress conditions [START_REF] King | A prote in induced by NaCl in suspension cultures of Nicotia11a tahac11m ac cumulates in whole plant roots[END_REF]671 with a possible involvement of abscisic acid [START_REF] Nelson | Analysis of structure and transcrip tional activation of an osmotin gene[END_REF]. Another phytohonnone, ethylene, considered as a 'stress' hor mone, is a potent inducer of PR proteins [58-60, 69-77J. PR production has also been shown to be deve lopmentally regulated in healthy plants since high levels of some PRs have been reported in roots and senescent leaves [START_REF] Memelink | To bacco genes encoding acidic and basic isofonns of pathogenesis-related proteins display different expres sion patterns[END_REF][START_REF] Felix | Developmental and honnonal regulation of j}-1,3-glucanase in tobacco[END_REF][START_REF] Shinshi | Regulation of a plant pathogenesis-related enzyme: inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin[END_REF][START_REF] King | A prote in induced by NaCl in suspension cultures of Nicotia11a tahac11m ac cumulates in whole plant roots[END_REF][START_REF] Castresana | Tissue-specific and pathogen-induced regulation of a Nicotiana plumbagini fo lia (3-1.3-glucanase gene[END_REF][START_REF] Cote | Pathoge nesis-related acidic (3-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals[END_REF], and during flowering [80][START_REF] Lotan | Pathogenesis-r�lated pro teins are developmentally regulated in tobacco flowers[END_REF][START_REF] Neale | Chi tinase. (3-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower fonnation[END_REF][START_REF] Leung | Involvement of plant chitinase in sexual reproduction of higher plants[END_REF][START_REF] Richard | Immunological evidence of thau matin-like proteins during tobacco flural differentiation[END_REF].

Since PRs are induced under so many different conditions where pathogens are not involved, should they still be called •pathogenesis•related'? In fact, the widest range of PRs in a given plant species is induced in response to pathogens (see below) and, therefore, this name appears still justified. Further• more, it is known that during response of plants to pathogens, and particularly in necrotic infections, metabolic alterations include the production of the stress hormones ethylene and possibly abscisic acid, the synthesis and migration of the strong PR inducer salicylic acid [5 1-53, 85-871 and changes in the balance between auxins and cytokinins. As mentioned above, all these individual changes have the potential to induce the production of a given set of PRs. and, hence, it is not surprising that the response to necrotic infections which produces a combination of these inducers is accompanied by the induction of the widest an-ay of PRs.

PR-proteins display very characteristic physico chemical properties which aid in their detection and isolation: 1) they are very stable at low pH and remain soluble (for instance in an extraction buffer of pH 2.8) whereas most other plant proteins are denatured; 2) they are relatively resistant to the action of proteo lytic enzymes of endogenous but also exogenous origin; 3) they are monomers (with a few exceptions. see below) of rather low molecular mass (8-50 kDa); 4) they are localized in compartments such as the vacuole, the cell wall and/or the apoplast. Properties 1). 2), and 4) are tightly related, ie the high level of resistance of PRs to acidic pH and to protease action appear to be well adapted to these harsh environments where they occur. Resistance to proteases also suggests that the PRs have a low turnover. There has been a report, however, indicating a degradation of PRs by an extracellular aspartyl protease which might be responsible for PR turnover in tomato [START_REF] Rodrigo | Degradation of tobacco pathogenesis-related prote ins. Evidence for conserved mechanisms of degradation of pathogenesis-related prote ins in plants[END_REF].

One can wonder whether the conditions of induc tion or these physicochemical properties should be taken into account to define a protein as pathogen related. In the opinion of the authors. rather than givi;)g priority to the induction conditions which are common to many other defense-related proteins and en!:Jmes, one should consider high resistance to very acidic medium and to proteases as an additional property of PRs, since they enable these proteins to survive in very hostile environments to which they are targeted: the vacuolar compartment, the intercellular spaces and the cell wall.

Classifi cation of PR• proteins

The first four discovered PRs [START_REF] Payne | Isolation and sequence of a genomic clone encoding the basic form of pathogenesis related protein I from Nicotiana tahacum[END_REF][START_REF] Van Loon | Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var 'Samsun' and 'Samsun NN'. Changes in protein constitution after infection with TMV[END_REF] could easily be detected because of their very high mobility in native basic polyacrylamide gels and because they migrated to a zone of the gel containing very few other cellular proteins. Other, less mobile PRs were present in the same gels but could not be resolved from the nume rous proteins with similar mobilities. The procedure of extraction at low pH introduced in the late 1970s eliminated most of the less mobile proteins, and a comparison of the gels loaded with extracts from tobacco leaves reacting hypersensitively to TMV or from healthy leaves revealed six other bands of PRs [24]. These 10 major acidic tobacco PRs were well resolved upon electrophoresis on these native basic gels and their nomenclature was based on their relative electrophoretic mobility. They have been referred to as PR-la, -lb, -le. -2, -N , -0, -P, -Q, -R and -S in order of decreasing mobility (fig 1) [START_REF] Antoniw | Comparison of three pathogenesis-related prote ins from plants of two cultivars of tobacco infected with TMV[END_REF]. PR-la, -l b and -le were studied extensively and shown to be serologically related. We purified to homogeneity the seven other PRs [27,[START_REF] Kauffmann | Les proteines PR (pathogenesis related) du tabac: des proteines impliquees dans les reac tions de defense aux agents phytopathogenes. lsolement, proprietes serologiques et activites biologiques[END_REF] and demonstrated that they were members of three other distinct serological families containing PR-2, -N and -0, PR-P and -Q, PR-R and -S, respectively [START_REF] Kauffmann | Les proteines PR (pathogenesis related) du tabac: des proteines impliquees dans les reac tions de defense aux agents phytopathogenes. lsolement, proprietes serologiques et activites biologiques[END_REF]. The availability of these proteins in their native form enabled us to assign biological functions to five of these proteins which have remained mysterious for 15 years: we demonstrated that PR-2, -N and -0 had endo-1,3-�-glucanase activity [91] and that the major proteins PR-P and -Q had endo-chitinasc activity [START_REF] Legrand | Biological function of pathogenesis-related proteins: four tobacco pathogenesis-related proteins are chitinases[END_REF].

This finding could then be extended to other plant species from which PRs were also identified as endo-1,3-l}-glucanases or endo-chitinases [START_REF] Kombrink | Several 'pathogenesis-related' prote ins in potato are 1,3-�-gluca nases and chilinases[END_REF][START_REF] Nasser | Identification and character ization of maize pathogenesis-related proleins. Four maize PR proteins are chitinases[END_REF][START_REF] Awade | Biological function of bean pathogenesis-related (PR 3 and PR 4) proteins[END_REF][START_REF] Fischer | De Wit PJGM ( 1%9)' ldenti l kation of several puthogencsis-relat<!d proteins in 1omalo leaves inoculated with C"'do.�pori11m fi1lr11111 {syn F11Ma .filim) as 1.3-�•glucmrnscs uml chitinuses[END_REF][97][START_REF] Vo Gelsang | Elicitation of �-1 ,3-gluca• mtse and chitinasc ;1c1ivitics in cell suspension cultures of A.mwhyta 1'(( 1'iei rcsistum und suscepliblc cuhivurs of chickpea (Ciel'/' <Jl'i<•ti1111111 )[END_REF].

In the late 1980s, proteins similar to those of tobacco were detected or characterized in many plant species. their cDNAs and gene(s) cloned, so that many sequence data became available. They con firmed sequence similarities between the PRs men tioned above having chitinase or glucanase activity $ -+ 'l.;•h ... resistance to acidic pH and to protease cleavage [24]. A mixture of the IO proteins purified according to [START_REF] Kauffmann | Les proteines PR (pathogenesis related) du tabac: des proteines impliquees dans les reac tions de defense aux agents phytopathogenes. lsolement, proprietes serologiques et activites biologiques[END_REF][START_REF] Pritig | Virus-induced glyclmhydro luscs m1d effects ot' oligosucchuride signals on plant-virus intcructions, Ill: Si�11a/ Molecules 111 Plants and Plalll• MicmhC' /11ft'l'rlctio11s[END_REF] were submitted to slab gel electrophoresis under non-dena turing conditions, as described in [START_REF] Legrand | Biological function of pathogenesis-related proteins: four tobacco pathogenesis-related proteins are chitinases[END_REF]. After electrophoretic tran sfer onto nitrocellulose sheets, the proteins were stained with amido black (lane NA) or immunodetected with sera raised against proteins PR-S, -R. -Q, -P, -0, -N, -2 and -lb (lanes S5, SR, SQ, Sp. S0, SN, S2 and S1b, respectively), or incubated with a preimmune serum (Jane S1). Note that the proteins are well resolved from each other under the native conditions of electrophoresis used and that, in addition to the known PR-1 serological group containing three proteins [26,[START_REF] Antoniw | Comparison of three pathogenesis-related prote ins from plants of two cultivars of tobacco infected with TMV[END_REF] there are three other distinct serological groups including two, two and three proteins. respectively. ext

? •---------•--------•----�--•----•
and well known constitutive plant chitinases and glu canases, respectively. However, they usually did not give any clue to the biological activity or function of the other families of PRs.

We have extended our studies in tobacco by both molecular biology and biochemical approaches. Concerning the biochemistry, we used a combination of conventional and HPLC means of chromatography involving a cascade of steps based on various physi cochemical properties of the proteins [START_REF] Pritig | Virus-induced glyclmhydro luscs m1d effects ot' oligosucchuride signals on plant-virus intcructions, Ill: Si�11a/ Molecules 111 Plants and Plalll• MicmhC' /11ft'l'rlctio11s[END_REF]. These steps include cation or anion exchange chromato gmphy, chromatofocusing, hydrophobic interaction and molecular sieving. By combining these steps we were able to isolate up to now from tobacco, that is still considered as a prototype system for PR-proteins, more than 30 PR-proteins. Isolation of these proteins in their native form allowed us to raise antibodies, but the major advantage of having them in the native fonn was the possibility to determine and compare their specific biological activities.

Among the newly-isolated proteins some are related to the 10 acidic extracellular proteins already described in tobacco. Some are obviously members of new families in tobacco, but are related to proteins of other plant species. At the 3rd International Workshop on PR-proteins (Arolla, Switzerland, August 16-20, 1992) it was agreed that to enable an easier compa rison of related PR-proteins members between dif ferent plant species, a common classification and way of nomenclature should be used. It was particularly agreed and recognized that five families or groups of PR-proteins have been found in most studied plant species and, thus, appear to be ubiquit � us.

. The following paragraphs will de�cnbe these di�er ent groups and their possible role m defense, taking the tobacco PRs as the model system.

The PR-1 group

The present stage of knowledge concerning tobacco proteins of group 1 is summarized in table I. To bacco acidic PRs of group l (PR-la, -lb and -le) were the subject of most of the earlier work. They were the first to be purified [27,[START_REF] Antoniw | Comparison of three pathogenesis-related prote ins from plants of two cultivars of tobacco infected with TMV[END_REF]100] and were shown to belon . g to a serological group in tobacco and to be serologi cally related to PRs from other plant species [START_REF] Nasser | Identification and character ization of maize pathogenesis-related proleins. Four maize PR proteins are chitinases[END_REF][101][102][103]. cDNA clones encoding the acidic tobacco PRs of group I were isolated from cDNA librai: ies derived from mRNA extracted from leaves reactmg hypersensitively to T�V [104-108}. It wa � s . ho � n that these three proteins have sequence simtlanty higher than 90% and that their increased synthesis is under transcriptional control. They are encoded by a small family of at least eight genes [l 091 and several of these genes have been isolated from genomic libr � ries (for reviews see [START_REF] Bol | Plant pathogenesis-related proteins induced by virus infection[END_REF]42]). cDNAs and genomic clones corresponding to a basic protein which has approximately 65% similarity to PR-la, -lb and -le have also been characterized from TMV-infected leaves [ 109, 11 O]. Using the antibodies raised against tobacco acidic PR�l proteins, it was not possible to detect basic PR-I protein(s). Recently, we were able to isolate a basic 17 kDa protein and partial micro sequencing indicated almost complete identity to the protein sequences deduced from the cDNA � nd genomic clones (Frilig ct al, uniJablished). Acco�mg to the rules proposed by a nomenclature committee for PR-proteins, this basic member should be called .

Comparison of the amino acid sequences of all known PRs-1 with those available in data banks did not give any clue about the potential biological . ac tivity or function of these proteins [105,112]. Smee they were the first discovered PRs in response to viruses, they have been believed to participate in resistance to viruses or in their localization. Consist ent with this hypothesis several treatments that induced PRs-1 in tobacco were also efficient inducers of resistance to viruses. Another strong argument was provided by the interspecific hybrids of Nicotiana glutinosa x Nic:otiana debneyi that produced consti tutively PRs-1 and were highly resistant to TMV [�]. This question was addressed further by producing transgenic tobacco plants expressing PR�la or PR-lb genes constitutively [113,114]. In transfonned plants the protein encoded by the chimeric gene was pro duced and targeted to the extracellular spaces, as expected. However, these plants did not show higher levels of resistance to virus infection, and apparently represent an argument that breaks the correlation be tween high levels of resistance to vims and high levels of PRs-1. Some data. are already available, suggesting that some PR-1 members have antifungal activity. It has been shown recently [115) that three members of the PR-1 group from tomato had antifungal activity in in vitro assays against Phytophthora infestans. In the same assays, PR-la and PR-lb from tobacco showed lower but still significant activity of inhibiting Phyto phthora infestans (Niderman and Fritig, unpublished). This activity observed in vitro for PR-la is consistent with recent results obtained in vivo by the group of John Ryals [38): they observed that transgenic tobacco plants expressing constitutively high levels of PR-I a exhibited a significant protection from infec tion by Peronospora tabacina that causes the blue mold disease of tobacco.

It will be now interesting to assay other PRs-1 of various plant species for antimicrobial activity and to investigate the molecular mechanism(s) involved.

The PR-2 (1,3-J3-glucanase) and PR-3 (chitinase) groups

Since the discovery that major tobacco PR-proteins had endo-chitinase [START_REF] Legrand | Biological function of pathogenesis-related proteins: four tobacco pathogenesis-related proteins are chitinases[END_REF] and endo-1,3-13-glucanase activity (91 ], the literature on plant chitinases and 1,3-13-glucanases has grown to such an extent that a com prehensive analysis is beyond the limitations of this review which will not deal with the structure and the regulation of their ¥enes, but will focus merely on their biological activities and their role in defense. From tobacco we have isolated five distinct mem bers (table II): four acidic enzymes and a major basic enzyme (that consists perhaps of several closely related isoforms, as suggested by two-dimensional polyacrylamide gel electrophoresis [6 71). The latter is a class I enzyme, PR-2, -N and -0 are of class II, and another extracellular enzyme, PR-Q' has to be con sidered in the separate class III according to sequence data [118). They all are serologically related [START_REF] Kauffmann | Les proteines PR (pathogenesis related) du tabac: des proteines impliquees dans les reac tions de defense aux agents phytopathogenes. lsolement, proprietes serologiques et activites biologiques[END_REF]91].

A protein called O', migrating like PR-0 in native basic gels [START_REF] Pritig | Virus-induced glyclmhydro luscs m1d effects ot' oligosucchuride signals on plant-virus intcructions, Ill: Si�11a/ Molecules 111 Plants and Plalll• MicmhC' /11ft'l'rlctio11s[END_REF], did not cross-react with anti-glucanase antibodies, has a dimeric structure, and was found to possess no glucanase activity after several successive chromatographic steps aimed to remove any contami nation by PR-0. The acidic enzymes are extracellular [START_REF] Kauffmann | Les proteines PR (pathogenesis related) du tabac: des proteines impliquees dans les reac tions de defense aux agents phytopathogenes. lsolement, proprietes serologiques et activites biologiques[END_REF]91] whereas the basic counterpart occurs in the vacuoles [START_REF] Keefe | The effect of ethylene on the cell-type-specific and intracellular localization of the 13-1,3-glucanase and chitinase in tobacco leaves[END_REF]121 ]. Another 1,3-(3-glucanase called sp41 has been found in large amounts in specific flower organs [START_REF] Lotan | Pathogenesis-r�lated pro teins are developmentally regulated in tobacco flowers[END_REF]. Unlike other PRs of group 2, sp4 l is glycosylated and has not been found after TMV infection.

We have measured the specific activity of these enzymes towards Jaminarin as the substrate, and. sur prisingly found pronounced differences, exceeding two orders of magnitude (table II). It is noteworthy that there is no correlation between the level of sequence similarity and the relative catalytic activity of the enzymes: PR-proteins 2 and N show 99% amino acid similarity but N was found five times Table II. The tobacco PR-2 group: 1,3-Jl-glucanases. The proteins were isolated � ccording to . [90, ?I .• 99). The different clas � es are based on differences in amino acid sequence as described in [117][118][119][120]. Specific enzymauc act1\ 1ty has been i:n easured "". t!h laminarin as the substrate as described in [91). Relative specific activity is expressed as the percemuge of the htghe . .;t specific activity. For all other items, see legend to table I. Table III. The tobacco PR-3 group: chitinases -lysozymes. Proteins chi 34, chi 32, P and Q were i�olated according to {_ 90, 92, 99). Proteins chi 28, tys 28a, lys bl and Jys _ b2 have been isolat � d re � ently by the authors (unp��hshed results). The d1�fe_r � nt classes are based on differences in amino acid sequen7e as descnbed m [_ 117, 123, 131-133]. Chltmase and lysozyme acttvtt1es were measured as described in ( 92) and [ 126), respectively. For all other items, see legend to table I Ill 28 more active than 2; likewise PR-0 is 93% similar to PR-2 in amino acid sequence but is 250 times more active. It has an activity similar to that of the basic glucanase, even though the two amino acid sequences are very different so that they fa ll into distinct classes (table II). This raises the problem of differe nces in substratP-specific�ty between glucanases of a given plant species and also between glucanases of different plant species.

Clear evidence fþr such differences in substrate specificity was obtained in a collaborative work with P Albersheim and A Darv ill (Complex Carbohydrate Research Center, Athens, Georgia, USA). They purified to homogfŇl' leity a soybeun 1.3-�-gh .. ci�nase 1122) that shows also high specific activity towards laminarin, like PR•proteins 0 and glue b of tobacco (table II). PreparaH011s of pure soybean glucanase and of the most active tobae;co glucanases were adj usted to the same activity towards laminarin and faced with another substrate consisting of a preparation of cell walls from the fungus Phytophthora megasperma, a pathogen of soybean. Very striking differences in ac tivity were observed for the different glucanases: the tobacco enzymes had no significant activity towards this substrate, whereas the soybean glucanase was very active (Fritig et al, unpublished). In conclusion, it appears that 1,3-�-glucanases may differ very much in specific activity hJwards a given substrate and more generally in substrate specificity. In re sponse to TMV inføction, tobacco plants pro duce PR-P <md PR-Q as the major acidic PRs. We showed J92J that they hydrolysed efficiently chitin, a polymer of fÏ-1,4-N-acetylglucosamine, that is a major component of the exoskeleton of insects and of the cell wall of most fu ngi. This was very surprising since the inducing pathogen, TMV, does not contain chitin. Furthennore, two other chitinases of basic isoelectric point (chi 32 and chi 34) were also found induced by TMV infection. Chi 32 and chi 34 are the most active enzymes and account for about 70% of the total chiti nase activity in TMV-infected tobacco extracts. These fonns have been detected in other situations, for in stance in tobacco tissue and protoplast cultures [START_REF] Shinshi | Regulation of a plant pathogenesis-related enzyme: inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin[END_REF][START_REF] Grosset | To bacco mesophyll protoplasts synthesize t ,3-ji glucanase, chitinases, and 'osmotins' during i11 ''itro culture[END_REF] and, like other basic members of other groups of PRs, in roots and old leaves of tobacco [START_REF] Shinshi | Regulation of a plant pathogenesis-related enzyme: inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin[END_REF]. The basic chi tinases chi 32 and chi 34 have also lysozyme activity, in contrast to chitinases P and Q (table Ill). Recently a third basic chitinase, chi 28, closely relate� to chi 32 and 34 has been isolated (Stintzi et al, unpublished). Furthennore, two other basic proteins, lys b 1 and lys b2, have been isolated and demonstrated to have high lysozyme activity on Micrococcus luteus cells (Stintzi et al. unpublished} and a significant chitinase activity. Antibodies raised against lys bl recognized lys b2 and a novel acidic protein called lys 28a. The latter was purified and, surprisingly showed no significant lyso zyme activity in the assay with Micrococcus luteus cells. Since at the pH of incubation there could be repulsion effec ts of negative charges between the enzyme and the substrate, we used another classical substrate of lysozymes, 4-methylumbelliferyl-chito triose, and found that all three lysozymes had high ac tivity towards this substrate (Stintzi et al, unpublished).

ConcefĘing the localization of the different members of tobacco chitinases-lysozymes, some are vacuolar proteins while others are extracellular. Till recently, all acidic tobacco proteins had been found to be extra cellular and all their basic counterparts localized in the vacuoles [START_REF] Bol | Plant pathogenesis-related proteins induced by virus infection[END_REF]. Now, in addition to PR-lg (table I) which is an extracellular basic protein, table III reveals three other basic proteins that are targeted to the extracellular spaces: chi 28, lys bl and lys b2.

Comparison of deduced amino acid sequences of cDNA and genomic clones indicates that there are at least three classes of chitinases in tobacco [ 123]. Class I contains the basic chitinases with a N-terminal cysteine-rich domain of approximately 40 amino acids and a highly conserved main structure, separated by a variable hinge region. Class II chitinases include PR-P and PR-Q and lack the N-tenninal cysteine-rich domain but have a high amino acid sequence identity to the main structufÕ: of class I chitinases [130,225,226]. In contrast, class III chitinases of tobacco have no similarity with class I and class II but are very similar to a cucumber chitinase having lysozyme ac tivity [131][132][133].

Like for glucanases, chitinase structures appear very conserved in the plant kingdom. This was evi denced by serological studies (48, 94-97, 134, 135) and from comparison of the decoded cDNA or gene sequences (for reviews see [ 117. 123]). Thus, class I, class II and class III chitinases have been found in various plant species. Recent studies have shown that a short C-tefØinal extension of a few amino acids in a tobacco class I chitinase was both necessary and suffi cient for vacuolar localization [ 136). Such sequences are also found in other vacuolar chitinases from tobacco, bean, potato, poplar and Arabidopsis [START_REF] Samac | Isolation and characterization of the genes encoding basic and acidic chitinase in Arabidopsis thaliana[END_REF](137)(138)(139)(140) and these chitinascs are designated as class Ia to distinguish them from class lb found in Brassica napus, pea, rice and barley [123,[141][142][143] and lacking this C-terminal extension, suggesting extracellular localization. In tobacco, chi 28 has an N-terminal sequence identical to chi 32 and chi 34 (Stintzi et al, unpublished); it is extracellular since it probably lacks also the C-terminal end present in chi 32 and chi 34.

A fourth distinct class of chitinases, class IV, still not detected in tobacco, has fâen proposed to include chitinases present in sugar beet, rape and bean (123,144]. These class IV chitinases contain a cysteine-rich domain and a conserved main structure which are similar to those of class I chitinases but smaller due to several deletions.

The antimicrobial activities of glycanhydrolases of groups PR-2 and PR-3

It was suggested already in 1971 [ 145] that chitinases and 1,3-fÏ-glucanases may fjnction as a defense against fjngal pathogens, but support for this sugges tion came much later when several plant chitinases were purified to homogeneity and shown to digest cell walls of pathogenic fungi [START_REF] Boller | Chitinase in bean leaves: induction by ethylene, purification, proper ties, and possible function[END_REF]146] and when some of the extracellularly targeted PR-proteins were found to possess chit�nase [START_REF] Legrand | Biological function of pathogenesis-related proteins: four tobacco pathogenesis-related proteins are chitinases[END_REF] or l ,3-fć-glucanase (9 I] activity.

Direct antimicrobial activity of chitinases and 1,3-� glucanases Antifjngal activities in vitro of plant chitinases and 1,3-�-glucanases have been studied with various bio assays (for reviews see (116,147]). Antifjngal ac tivity of plant chitinases was first shown in a bioassay on agar plates with a saprophyte, Trichoderma viride (148]. A similar bioassay was used by several groups to demonstrate a significant inhibition of growth of rapidly growing saprophytes [124,134,149), but the results were rather disappointing with a number of phytopathogenic fungi examined, with some excep tions (150]. Using a modification of this bioassay with small filter disks laid on the agar and enzyme sol utions ap p lied to the disks it was shown that chitinases and 1,3-f § -glucanases purified from elicited pea pods, taken individually, were inhibitory only in exceptional cases to fungal growth around the filter disks [ 1511. But combinations of the two purified enzymes in hibited growth of most of the fungi studied and more or less mimicked the antifungal activity observed with the crude pea pod extracts [ 151 }. Using a bioassay in microtiter plates that allows to observe the growth of fungi in liquid media and the same plant hydrolases, it was found that inhibition of growth of Nectria hae matococca was only transient [ 152], suggesting that the fungus apparently had the capacity to adapt to the chitinases and the l ,3-fć-glucanases in its growth medium. However, the bioassay confirmed the syner gistic effects of chitinase and 1,3-�-glucanase, that were also found for several of the tobacco hydrolases [153, 154}. A detailed analysis of the in vitro anti fungal activities of individual hydrolases and of various combinations was made. Class I chitina�>es (table Ill) and class I glucanase (table 11) were the most active in the lysis of hyphal tips and in inhibiting the growth of Fusarium solani gennlings. The class II isoforms had no effect if assayed individually. This again underlines the striking differences in substrate specifkcity between glucanases (fÿr instance PR-0 and glue b of table II) even if they exhibit similar capaci ties of hydrolysing laminarin. Furthennore, class II chitinases showed limited inhibitory activity in any combination. Finally the most efficient system was shown to be a combination of class I chitinases and class I glucanases that provided strong synergistic effects. Synergistic antifungal activities were also observed for combinations of basic chitinases and basic glucanases purifled from sugar beet and assayed against Trichoderma viride and Cercospora beticola [ 155]. Since chitin and l ,3-IJ-glucan fübrec; are synthe sized simultaneously in the apex of growing hyphae of fúlamentous fu ngi, and since these polysaccharides may cross-link and be overlaid with other material in cell wall distant from the apex, these results observed in ritm are not really surprising: nascent chitin of the hyphal apex is tf most accessible to hydrolysis by chitinase [ 123) and the effectiveness of a hydrolase may depend on the simultaneous action of another one to hydrolyse mixed chitin-glucan fübres.

What about antimicrobial activity in vivo of glycan hydrolases? First there are several lines of evidence obtained fsom immunocytochemical approaches that hydrolases are indeed produced in the vicinity of infection sites and accumulate around fu ngal ce11 walls in planta [ 156-1 58 ]. Furthermore, there is gen erally a spatio-temporal regulation so that glucanases and chitinases are produced simultaneously at the same sites, suggesting that synergistic effects required fr optimal antifungal activity in vitro are likely to occur in \'il'o.

Since many chitinase and 1,3-fŁ -glucanase cDNAs or genes have been cloned, the prospects for geneti cally engineering plants with impfďved resistance by tfČnsformation with chitinase or/and glucanase genes are reasonably good, and this approach is now being utilized in many laboratories all over the world (for reviews see 1159, 160]}. Several successful cases have already been reported. Tobacco and canola plants constitutively expressing high levels of a class I bean chitinase were more resistant against the root patho gen, Rlti=octonia solani ( 150,161]. whose sensitivity in virro to this chitinase was well documented by an immunocytochemical approach showing a progressive breakdown of labelled chitin [ 162]. Transgenic tobacco expressing high levels of either the tobacco class I basic chitinase, the tobacco acidic class Ill chi tinase or the cucumber class III chitinasc showed signifmcant resistance against disease caused by Rhi zoctonia solani 138]. Reduction in disease symptoms caused by the same fungus was also reported in trans genic tobacco constitutively expressing a chitinase of bacterial origin [ 163 ]. In contrast, Nicotiana sy/\lestris transgenic plants expressing high levels of the tobacco class I chitinase showed unaltered susceptibility to the leaf pathogen, Cercospora nicotianae. It is fòssible that the highly expressed chitinase, since it is located intracellularly, did not come to contact with the inter cellular developing fungus. Since many fungi grow intercellularly, the manipulated enzymes should be targeted to the extracellular spaces. Such attempts are already under way, and the most potent antifungal tobacco hydrolases of class I have been modifned at their C-terminus to be targeted to the extracellular spaces of transgenic plants. It was found [ 153) that the extracellular washing fpuids indeed contained the modifoed proteins that had retained full antifungal activity when assayed individually in vitro and their synergistic potential when used in combination. Ex periments are now in progress to see whether targe ting of the tobacco class I hydrolases to the extracel lular space is an effective way of increasing fungal resistance. There is an example, however, that re moval of the targeting sequence of the class I tobacco chitinase suppressed resistance against Rhizoctonia solani in transgenic plants [ 1641. There is also a report of an antibacterial resistance obtained in transgenic potato expressing constitutively phage T4 lysozyme [ J 65). It will be interesting to test antibacterial resis tance by constitutive expression of plant lysozyme genes, fr instance those of class III chitinases-lyso zymes.

In conclusion, direct antifungal activities of plant chitinases and 1,3-f¡-glucanases have been clearly demonstrated with various bioassays. It is very likely that plants use induction of these hydrolases as an ef ficient defense response. Fufĕher results obtained from transgenic plants expressing high constitutive levels of hydrolases have to be awaited befÿre it can f concluded that the strategy is valuable and mimics, at least partially, a pathogen-induced defense response. In this respect, the observations in [152] are important showing that ;,, vitro fungi are onay temporarily in hibited by chitinases and 1,3-f¡-glucanases and can adapt to even high levels of these hydrolases. Thus, a constitutively expressed high level may not be very effective in genetically-engineered defense, and it would be preferable to increase thefr levels suddenly in the vicinity of approaching hyphae. In natural induced defense, such as the hypersensitive reaction, levels of hydrolases are highly induced within a short time and operate in two successive lines: a frst line of extracellularly-targeted enzymes and a second line of the vacuolar and very active enzymes released during hypersensitive cell death and collapse (7 1].

Indirect a1llimicrobial activity of chitinases and glu canases

It has been known for some time that plant chitinases and 1,3-fł-glucanases have a potential to partially degrade fungal cell walls [START_REF] Boller | Chitinase in bean leaves: induction by ethylene, purification, proper ties, and possible function[END_REF]146,151 ]. The products released are oligosaccharides, some of which have been shown to be perceived by the plant cell as signals, so-called 'elicitors', and to induce active defense responses in the absence of any infection (for reviews see [START_REF] Darvill | !"hytoalexins and their elicitors-a defense against microbial infection in plants[END_REF]167,168]). Indeed, chitin and chi tosan oligomers and 1,3-P-glucans with some bran ching have been shown to induce a set of metabolic alterations typical of defense responses [START_REF] Darvill | !"hytoalexins and their elicitors-a defense against microbial infection in plants[END_REF][167][168][169][170][171]. The biologically active oligochitins and oligochito sans are thought to be released during the plant pathogen interaction by the action of plant chitinases and chitosanases [168,170]. This might lead to an amplification of the defense responses, some of the induced proteins being again chitinases [ 171 ]. ConcefĘ ing the occurrence and activity of glucan elicitors (for a review see [167]), many cases have probably to be re-examined since the discovery that the activity of fungal elicitor fractions believed to contain mainly glucans was due to proteinaceous or glycoprotein elici tors [172][173][174][175]227]. Indeed the elicitors active on soybean are glucans derived from the fungal cell wall and their structure has been clearly established [ 176,177]. It has also been shown that soybean endo-1,3-P glucanases were indeed able to release elicitor active fāagments from the fungal cell wall [122,178-1 80].

In conclusion, plant chitinases and 1,3-P-glucanases are likely to play a dual role: they are directly antimi crobial by hydrolysing structural components of the cell wall of microbes, furthermore they are also in directly antimicrobial by releasing oligosaccharides that are active e1icitors of defense responses and, in tum, may be responsible for the amplification of the defense responses [IO,[START_REF] Fritig | Mechanism of the hypersensitive reac tion of plants[END_REF]167].

The PR•4 group

In tobacco four proteins of the PR-4 group have been isolated fāom TMV-infected leaves and named rl, r2, sl, s2 [181]. They are all acidic proteins (table IV), with yet no known basic counterpart, and migrate on Nothing is known about the biological function or activity of these proteins which have not been studied extensively. They represent also the less ubiquitous of the five PR-groups since only one similar protein has been described so f÷r, a 15-kDa tomato protein [ 184].

The PR-5 group: 'thaumatin-like' proteins

In tobacco Samson NN this group includes two almost neutral proteins named R and S (table V). These extracellular proteins were among the 10 initially detected on the native basic gel system [24] and we demonstrated {90, 181] that they belonged to a dis tinct serological group (see also fig Table V. The tobacco PR-5 group: 'thaumatin-like' proteins. The proteins were isolated as described in [181,186].

For all other items, see legend to table I.

Name Molecular Elution pH Locali:ation Biological mass (chromato-ac•tivity (kDa) fo £'11Sillg)

-��----R 24

6.9 int anti fu ngal

--�---• ••----- Table VI.
Anti-fungal activity of PR-5 proteins estimated from inhibition of spore gennination (S) or of hyphal growth (H). Fusarium oxyspor:m1

Hyphomycetes H

[224]

Altemaria so/a11i

Ascomycetes H

(224]

Trimatin (wheat)

Candida albica11s

Hyphomycetes s [199] Tr iclwderma reesei Hyphomycetes H, S

Neum.�pora crassa Ascomycetes H, S

[199]

Protei n R (barley) [204). For basic PR-5 a study of tissue-specifi c expression indicates high expression in roots and stems and a possible involvement of abscisic acid in promoter activation during the response to salt stress. cDNAs encoding PRs-5 from several other plant species have been cloned [START_REF] Uknes | Acquired resistance in Arabidopsis[END_REF]197,[205][206][207][208], Concerning the search fþr a possible biological ac tivity of proteins of the PR-5 group, the fjrst attempts were based on sequence similarity to a bifunctional inhibitor of maize, an inhibitor of both trypsin and insect a-amylase ( 193 ]. We did not fnd this bifunc tional activity in any of the isolated tobacco proteins of table V, and it is noteworthy that this activity has also not be reported for any of the numerous thau matin-1 ike proteins isolated by others (see table VI), including the maize protein zeamatin [199,209] that wus found highly similar, if not identical, to the maize bifunctional inhibitor (Selitrennikoff, perso11al com� munication). Transgenic plants expressing constitut ively acidic PR-5 were not protected from eating by Spodoptera exiga and H virescens larvae, as well as not being more resistant to inføction by TMV [113).

The frst repofĔ on biological activity of a PR-5 pro tein was the demonstration that fŚamatin, a protein from maize, was antifungal with membrane per meabilizing activity [210). Other cereals apparently contained related proteins with similar antifungal ac tivity [199]. Then another group found that tobacco osmotin and the tomato analog was active against Phytophtlwra i11festa11s, responsible for late blight disease of potato (200]. We confifċed and extended these results by showing that several memfrs of the PR-5 group had differential antifungal activities with specificity for different fungal species [209]. There are now many lines of evidence that proteins of the PR-5 group from various plant species have direct antifungal activity against several classes of fungi (table VI). They have been shown to inhibit hyphal growth and mediate hyphal and spore lysis in in vitro assays [197. 199, 200, 209, 210]. It has been proposed that the antifungal activity of the maize PR-5, zea matin, is the result of permeabilization of fungal membranes [210] thfčugh a mechanism that is still operative at 4°C. This capacity may be related to formation of amphipathic domains resulting from secondary structure mediated by disulfi de linkages analogous to another type of plant defense proteins, thionins [211]. These amphipathic domains interact with membranes resulting in pore or channel forma tion, and in extreme conditions membrane lysis.

More information has to be obtained on the mol ecular mechanisms of antifungal activity of PRs-5. If the effects on fungal membrane permeabilization were confifėed, then a combination of an hydrolytic enzyme of group PR-2 or PR-3 and a PR-5 protein expressed constitutively by genetic engineefĊng would be very likely to confer a high level of protection against infection by ftngi. since the two types of pro teins would be acting on two different and essential targets of the pathogen.

Other tobacco PR-proteins

Several tobacco PRs have been characterized recently which cannot be classified into the five major groups (table VII).

We have isolated a 45-kDa basic protein [START_REF] Kauffmann | Les proteines PR (pathogenesis related) du tabac: des proteines impliquees dans les reac tions de defense aux agents phytopathogenes. lsolement, proprietes serologiques et activites biologiques[END_REF] which cross-reacts with antisera raised against proteins of groups PR-2, PR-3 and PR-5 (Stintzi, unpublished). The significance of these observations is not under-stood yet, since the antibodies used have been shown to be very specific to proteins of the cofđesponding groups in tobacco [67. 91, 92, 181, 186] or in other plant species [97,135,212], and no hydrolase activity has been found associated to the purified protein even by using very sensitive enzymatic assays . cDNA cloning is presently in progress.

Another basic protein with a molecular mass of 16 kDa has been found to accumulate in TMV infected tobacco leaves and its biological activity is also unknown. It does not cross-react with antibodies raised against the acidic PRs-1 (-la, -1 b and -le) or against the basic counterpafĚ PR-lg (table I). These data and pafĚial microsequencing demonstrate that it does not belong to the PR-1 group in spite of a similar molecular mass. It is presently under investigation.

Two a-amylases are strongly induced in tobacco leaves upon infection by TMV [213]. They are endo type enzymes and have many fe atures in common with other typical acidic PRs. for instance an apo plastic localization and a high degree of resistance to proteolytic cleavage. Both proteins have an apparent molecular mass of 44-kDa. Like glycanhydrolases of groups PR-2 and PR-3 they can be considered as direct antifungal enzymes since their substrates, 1.4a-glucans, occur in the cell wall of many fungi.

We have also shown a strong induction of a protei nase inhibitofė activity in tobacco during the hyper sensitive reaction to TMV [214 ]. The active inhibitor was purified and shown to be a small polypeptide of the inhibitor I type which displays a strict specificity for serine proteinases of microbial origin, such as sub tilisin or proteinase K. Thus, the tobacco inhibitor appears as a good candidate frr re ducing proteolytic activities of phytopathogenic bacteria or fungi in planta. It is noteworthy that the active inhibitors are not monomers, in contrast with most of the other PRs.

Tuble VII.

Other not yet classified tobacco PR-proteins. Protcios T!MPa and TI!\1Pb, amyl I and amyl 2, 45 � and 16 K, were isolated and assayed as described in [2 14, 215), (213), [START_REF] Kauffmann | Les proteines PR (pathogenesis related) du tabac: des proteines impliquees dans les reac tions de defense aux agents phytopathogenes. lsolement, proprietes serologiques et activites biologiques[END_REF], respectively. Protems P39a and P40a have been isolated rec � ntly by the authors (unpublished results) and partial amino acid sequencing has shown identity to a cloned tobacco peroxidase (218). For all other items, see legend to table I.

Name

Molecular lsoelectric

Localization Biological

TIMPa TIM Pb t�e presence of a glutamic acid residue at the Pl p�s1�1on of t�e active site, known to determine the spec1fúc1ty of this type of inhibitors. However. the V8 proteinase from Staphylo cocc•us aureus. an enzyme that cleaves polypeptides after glutamic residues �as not affected by the tobacco inhibitor. We have demonstrated a strong accumulation of both mRNAs and proteins in re sponse to TMV. messengers and products of the two genes being present in a 3/2 ratio in infected leaves as weJI as in the upper uninfected leaves. the induction being markedly lower, however, at distance _fĀom the infection sites. We found that the transcnpts also accumulated in sepals and petals of healthy plants, indicating that these genes are developmentally regu lated. Unlike the tomato and potato I inhibitors [2l6l, the tobacco inhibitor was only weakly induced by wounding, but was expressed upon treatment with salicylic acid and ethylene, like many other PR-proteins.

We have also isolated two acidic extracelluf ar pro teins with peroxidase activity. Peroxidase isozyme induction by TMV infection of tobacco had been stud ied by others 1217] and indicated a preferential induc tion of two moderately anionic peroxidases called P56 and P6 1, whereas the anionic isozymes P35 and P37 were already present in uninfected material and only weakly induced during iufection. In fact the isofonns P39a and P40a that we have isolated are strongly induced in TMV-infected Samsun NN (Stintzi, un published). Surprisingly, partial amino acid sequence analysis indicates identity to P35 whose cDNA has been cloned 1218) and which has been described as a lignin-forming enzyme, based on its high expression in the lignified stem and on its affinity for coniferyl alcohol. In our group we are also studying the induc tion of aromatic metabolism around the virus lesions and already have evidence for an increased incorpor ation of phenolics into the cell wall [219, 2201 that is perhaps catalysed by P39a and P40a. Further studies on these peroxidases are under way to understand their function and role in defense.

Conclusion

This special issue is dedicated to Professor Leon Hifęh who initiated part of the work described above and encouraged three of us, P Geoffroy, M Legrand and B Fritig already in the early 1970s to develop a research project on the response of plants to exposure to pathogens, namely his fövourite pathogens, viruses. Endowed with an outstanding scientific intuition, he suggested already at that time that the hypersensitive response to viruses would be a particularly good model to study.

In föct the hypersensitive response (HR) of plants to pathogens is one of the most efficient natural mechanisms of induced defense of plants against pathogens. This is also true in the case of virus infec tions, viruses being the inducers in our model system. In hypersensitivity to viruses the same characteristics are found as in incompatible interactions between plants and fungi or bacteria [START_REF] Keen | The molecular biology of disease resis tance[END_REF]lOJ: i) necrosis at and around each point at which the leaf tissue was infected; ii) localization of the pathogen to the region of each initiated infection; and iii) induction of marked metabolic changes in the cells surrounding the necrotic area, these changes being believed to cause, or at least to contribute to, the resistance observed. Furthermore, the hypersensitive response to viruses involves a cascade of events and signals [START_REF] Fritig | Mechanism of the hypersensitive reac tion of plants[END_REF] similar to that propo�ed fqr active defense against fungi and bacteria: the response would be initiated by a specifc gene-for-gene recognition between the plant and the virus that would lead to cell damage (death of a number of cells) and the fªlease of intermediary signals which in tum would enter a reception-trans duction pathway leading to major changes in gene expression (usually gene activation) responsible for the resistance observed:

Under at first the tutelage of Professor Hirth and later his suppofĒ through stimulating discussions and encouragements, our group has studied particularly the downstream pafē of this cascade, le the changes in host gene expression and their possible contribution to tlie defense response. For some time we have invested efùorts to understand the role of plant phenolics in disease resistance, and these efforts are still conti nuing with some focus on the role of cell wall bound phenolics [221 ]. However, since 1985, major emphasis has been put on a group of strongly induced proteins with typical properties, the PR-proteins also referred to as 'stress proteins•. Tobacco responding hypersensitively to TMV proved to be a particularly useful experimental system. as anticipated by Pro fessor Leon Hirth, and this model is still recognized as the prototype system from which we have characte rized biochemically the highest number and widest array of PRs. Among the 33 proteins isolated, 31 have been characterized and 25 fall into five groups of ubi quitous proteins. We have identifûed the enzymatic activities and functions of proteins of groups PR-2 and PR-3 as the hydrolytic erzymes 1,3-�-glucanases and chitinases-lysozymes, fªspectively. We have also pafęicipated in the discovery that PR-5 proteins had antifungal activity, and presently, are participating in a collaborative work aiming to study the antifungal pro perties of proteins of group PR-I, the initially disco vered group of PRs [I, 2). The PR-4 group is still without kno� n biological activity. Other proteins cha racterized in tobacco are the hydrolases a-amylases, inhibitors of microbial proteases, and peroxidases. With the exception of the latter, all activities characterized appear to be direct antimicrobial activities (antifungal and/or antibacterial). Some of the proteins also exhibit indirect antimicrobial activities, for instance the eli citor-releasing hydrolases and peroxidases that are likely to catalyse cell wall cross-linking of phenolics.

Very sufĐrisingly, nof× of the characterized pfĎ teins appears to have direct antiviral activity, even though all of them afĈ induced in response to the virus and this response obviously includes the induction of an antiviral state that finally localizes the vifĖs. It has been reported that two antiviral proteins called gp35 and gp22 were purified from tobacco and characte rized by monoclonal antibodies to human fŃ-interferon (222). Later it was shown [223) that gp35 corres ponded to the basic, vacuolar form of 1,3-f¡-glucanase (glue b of table II) and gp22 was related to boih the acidic and basic forms of PR-5 from tobacco. We have tested glue b (table II) and osmotin (table V) using exactly the bioassay of reference [222) and could not fûnd any antiviral activity associated to these proteins. In f÷ct the bioassay itself is not really repre sentative of what happens in hypersensitively-induced fªsistance to the virus. The mechanism to f unra velled is not an inhibition of establishment of virus infection, but rather a mechanism responsible for inhi bition of virus spread. This mechanism might rely on yet undetected component(s), it might also result from the wide range and superposition of metabolic altera tions, fýr instance the cell wall thickening that could affect the viral •movement' prote:ins(s).

As described above, the prospects for genetically engineefĉd plants with improved resistance against fungi and bacteria by transfýfėation with PR genes are promising, especially if combinations of PRs are expressed with potential synergistic efùects or with different target components in the microbe. This still does mimic only partially natural defense which involves a wide array of direct and indirect antimicro bial compounds. The natural defense system could fâ better • approached by manipulating the signals that trigger a battery of defense components, and the characterization of such signals and their manipula tion is now under study in many laboratories all over the world.

Fig 1 .

 1 Fig 1. Serological relationships between the 10 acidic extra cellular PR-proteins initially detected in tobacco by their

  Properties and biologic:al activities of PRs-2 (I ,3-p gluc:anases) Many 1,3-�glucanases have been purified and cha racterized (for a review see [116)). Like most of the PR-proteins, they usually are monomers with a mole cular mass in the 25-35-kDa range. Most are endoglu canases, producing oligomers of chain lengths of 2-6 glucose units from the classical substrate laminarin, an almost unbranched 1,3-(3-glucan, or from other 1,3-13-glucans. Many glucanases from various plant species have been cloned and sequenced (for a review see [ 117)). On the basis of a comparison of the deduced amino acid sequences of tobacco 1,3-(3-glu canases, ihey have been grouped into three classes [118-120].

  Properties and biological activities of PRs-3 (chi ti nase s-lysozymes)A large number of chitinases have been purified and characterized (for reviews see[ l 16, 123]}. Like the glucanases they are usually monomers of 25-35-kDa molecular mass. Most plant chitinases characterized far are endochitinases, producing chito-oligosac charides of 2-6 N-acetylglucosamine units. Many purified plant endochitinase-s also possess some lyso zyme activity, ie they can hydrolyse J3-l ,4-1inkages between N-acetylmuramic acid and N-acetylglucos amine in the bacterial peptidoglycan[116,[124][125][126][127].Likewise, most plant lysozymes show signifcant levels of chitinase activity[126][127][128]. Some chitinases also act as chitosanases but, in addition, specific chito sanases are induced in plants in response to pathogens[ 129).

  l ). They corres pond probably to proteins R-minor and R-major found by others { 185] in the cultivar Xanthi nc of Nicotiana tabacum. Two basic serologically-related counterparts which accumulate also in infected leaves (table V) have been identified as osmotins (67, 186}. Osmotin had been previously found to accumulate in tobacco cells in response to salt stress [ 187]. The basic pro teins of group PR-5 are localized in the vacuolar com partment [67, 187} whereas the slightly acidic-neutral members R and S are apoplastic [I 88, 189]. cDNAs have been cloned which correspond to acidic 1190, 191] and basic [192} tobacco PRs-5. There is more

  ----••--••----•----•--•--------... -----------than 90% sequence identity between PR-R and PR-S and more than 60% between the acidic nnd the basic counterpm1s. There is also 6()0/o sequence similarity between the acidic PRs-5 and thaumatin. a sweet-tas ting prote in extracted from the african shrub, Thau matoc•occus da11it•IU: therefore. the proteins of group PR-5 are also culled the 'thaumatin-like PRs'. Thau matin•like proteins have now been characterized fro m various plant species l 193-201 J. Genes encoding acidic and basic forms of tobacco PR-5 proteins have been isolated {202, 2031 and do not contain introns. At least two domains of the promoter are involved in the induction of acidic PR-5 upon TMV infection

  plant cell wall protein: A novel. rapid defense response. Cell 70. 21-30 21 Varner JE. Lin LS ( 1989) Plant cell wall architecture. Ce/1 56. 231-239 22 Vam•e CP. Kirk TK. Shervood RT ( 1980) Lignification as a mt�chanism of disease resistance. A111111 Rev Plrytopa thol 18. 259-288 23 Matec.v U. Kneusel RE (\988) Phenolic compounds in plant disease resistanc�. Pllytoparasitica 16. 153-170 24 Van Loon LC (1982) Regulation of changes in prote ins and enzymes associated with active defence against virus infection. /11: Acrii•e Defl'llSc? Meclianisms in Plams (Wood RKS. ed). Plenum Press. London, 37, 247-273 25 Jamet E. Kopp M, Fritig B (1985) The pathogenesis related proteins of tobacco: their labelling from ! 14C] amino acids in leaves reacting hypersensitively to infec tion by tobacco mosaic virus. Physiol Plant Pathol 27. 29-41 26 Van Loon LC ( 1985) Pathogenesis-related proteins. Plant Mo/ Biol 4. I 1 1-116 27 Jamet E, Fritig B ( 1986) Purification and characterization of 8 of the pathogenesis-related proteins in tobacco leaves reacting hypersensitively to tobacco mosaic virus. Plant Mo/ Biol 6. 69-80 28 Pierpoint WS (1986) The pathogenesis-related proteins of tobacco leaves. f>hV1ochm1i.w•v 25. 1595-160 I 29 Van Loon LC. Gerritsen YA M, Ritter CE ( 1987) Identifi cation. purilication, and characterization of pathogencsis rclated proteins from virus-infected Samsun NN tobacco leaves. Plant Mol Biol 9, 593-609 30 Ross AF ( 1961) Systemic acquired resistance induced by localized virus infection in plants. ViroloRY 14, 340-358 31 Russ AF ( 1966) Systemic effects of local lesion formu tion. /11: \1i1 11ses <!f' Plam�• (Bccmster ABR. Dijk:itrn J, eds), North-Holland Publishing Compuny. Amsterdam. 127-150 . n Lochcnstcin G ( 19721 Luculization und induced rcsis• tuncc in virus-infected plants. A111111 /? <'I• Pllytopatlrol 10, 177-206 33 Mdntyrn .IL. Dodds JA ( 1979) lmluction of loculized and sysh.Hllk prnlcctiun against Phytoplltlwm 11cm1.�itica var nimriwwe by tobucco mosaic virus inl'cction ot' tobacco hypersensitive to the virus. Pfly.1•iol Plalll Par/101 15. 32 1-330 J4 Ahl P. Bcnjanrn A. Sumson R, Gianinazzi S ( 1981) Induc tion dtez le tabuc par Pse11domo11e1s syri11ga<' de nou vellcs protcines (proteines 'b') associees au developpe mcnt d'une rt: sis!ance non spedfiquc a une deuxieme infection. Phy topathol Z I02, 201-212 35 Bergstrom CTC. Johnson MC, Kuc J (1982) Effects of IC!cHI intCctiun of cucumber by Co/l,•torridmm lage11a mm1, P.,•c•1ulo111one1s /achnww11.� or 1obacco necrosis virus on systemic re sistance to 'cucumber mosaic virus. Phwof't.'tlwlogy 72, 922-926 • . 16 Mcintyre JL, Dodds JA, Hare JD ( 1981) Effects of loca lized infection» of Nicoticma taharnm by tobacco mosaic virus on systemic 1esistance against diverse pathogens and an insect. Pl1ytopathofo.�y 71. 297-10 1 37 Y� XS. Pan SQ •. Kuc � ( 1989) Pathogenesis-related pro teins und systemic resistance to blue mold and tobacco 1r.osaic virus induced by tobacco mosaic virus, Pcnmo spom :e1hc1cirw and aspirin. Plivsiol Mot Ple111t Parlwl 35 161-175 .

Table I .

 I The

	tobacco PR-1 group: antifungal . teins PR-I a, -1 b and -I c w!!re isolated according to (27, 90). proteins. Pro Protein PR-lg has been isolated recently by th � authors (unpublished results) and its amino � id sequen � e 1s almost identical to that deduced from previously descnbed �DNA and genomic clones [ 109, l 10). The . no. menclature 1s that proposed in [29 • . 11 l 1: Similar boxes md1cate demonst � ated serological relat1onsh1ps. Molecular mass values are either deduced from corresponding cloned cDl�A or calcula!ed from relative mobility upon electrophoresis of the proteins under denaturing conditions. Values of . p/ are calculated from the amino acid sequences or elution pH have been measured in chromatofocusing experiments ; . 1:he 1 . ocal ization is referred to 'ext' for extracellular and mt for intra cellular occurrence of the proteins.
	Name Molernlar mass E/11tio11 pH (kDaJ (chmmatojiJcusi11g}	Localization Biological acri l'ity
	� c lg	15.5 15.5 15 17	<7 4.S 4.7 >7	ext ext ext	anti fungal anti fungal ?

  .

	Name	Class	Molecular mass Jsoe/ectric point (kDa) (elution pH)	Localization	Relative specific activities(%) Chitinase Lysozyme
	chi34 j p ! Q lys 28a	la Ia lb Ua lla m	34 32 28 27 28 28
	ly.> bl	m	28
	lys b2

Table IV .

 IV The tobacco PR-4 grou p: 'win-like' proteins. The proteins were isolated according to[181]. For all other items, see legend to Lable i.

					the native basic gel system (see fig 1) in the vicinity of acidic PRs-5. In fact the couples rl/r2, sl/s2 migrate close to PR-R and PR-S, respectively. However, in denaturing gels, PRs of group 4 migrate as small 13-14.5-kDa proteins and are clearly distinguishable from other PRs (181 ]. They all are extracellular and serologically related to each other but do not show a serological relationship to other PRs or PR-groups. Primary structure deduced from cDNA clones is known for two of these proteins [182, 183] and indi cates 50% sequence similarity to Wini and Win2 that are induced in potato in response to wounding. They show also significant similarity to the C-terminal sequence of hevein, a protein fĀom latex of Hevea brasiliensis.
	Name Molecular Elution pH Localization Biological mass (chromato-activity
		(kDa)	fo cusing)
	r2 rl s2 sl	13 14.S 13 14.S	7.0 7.0 7.4 7.4	ext ext ext ext	? ? ? ?

Ward ER, Payne GB, Moyer MB, Williams SC, Dincher SS, Sharkey KC, Beck JJ, Tay lor HP, Ahl-Goy P, Meins F,

Acknowledgments

The authors are indebted to D Bersuder and I Genetet for their contribution to the isolation of some of the PR-proteins and express their thanks to J Vo nesch for typing the manuscript.