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Salicylic acid: a systemic signal in induced plant disease resistance
R E V I E W S
Salicylic acid: a systemic signal in induced plant disease resistance Nasser Yalpani and Ilya Raskin
p
lants face a constant barSome plants respond to infection by tobacco PR-2 and PR-3 famirage of microbial organpathogens with both localized and lies of proteins have in vitro isms, but infection and systemic resistance responses. These ~-l,3-glucanase or chitinase disease rarely develop from prevent the spread of the disease-causing activity, respectively, and can these contacts. In addition to organism and reduce the severity of a hydrolyse fungal and bacterial chemical and physical barriers subsequent infection. Recent evidence cell wallsL Chitinase, alone or such as the cuticle, cell wall suggests that systemic increases in the in combination with 15-1,3-gluand antimicrobial chemicals host's salicylic acid levels act as a signal canase, has been shown to that are constitutively present, for the activation of at least some of degrade fungal or bacterial plants frequently activate a these induced defenses. cell wall preparations and to range of defense-related proinhibit fungal growth in vitro N. Yalpani and I. Raskm are in the AgBiotecb teins upon attack by pathoand in vivo 9,~°. PR-S is strucCenter, Cook College, Rutgers University, New gens. These inducible defenses turally similar to osmotin and Brunswick, NJ 08903-0231, USA. often accompany the developzeamatin, both of which are ment of a hypersensitive reknown to have antifungal sponse (HR). In this process, host cells in the im- activity in vitro 11. The function of other PR proteins, mediate vicinity of the site of pathogen penetration such as those in the PR-1 family, is unknown. The 'commit coordinated suicide', possibly killing the close association between PR protein accumulation pathogen or restricting its spreadL and resistance and the in vitro antimicrobial activity Activation of defenses may not only be localized of some PR proteins suggests that they function around the HR site but can also extend to tissue un- in defense. However, the extent to which these exposed to the inducing pathogen. Ross 2'3 described proteins contribute to resistance against pathogens, the development of such systemic acquired resistance particularly viruses, is not clear. (SAR) in tobacco showing an HR to tobacco mosaic SAR is likely to result from the synthesis and revirus (TMV). SAR is not only effective against the lease of a signal compound that moves from the site inducing pathogen but also against some taxonomi- of initial pathogen penetration to other parts of the cally distant disease agents. For instance, inoculation plant 12,13. Considerable attention has recently been of tobacco with HR-inducing viruses can 'immunize' focused on salicylic acid (Fig. 1), an ubiquitous plant the whole plant against attack by bacteria, fungi or phenolic, as a candidate signal molecule in SAR. other viruses (reviewed in Ref. 4). Thus SAR covers Salicylates from plant sources have been used in a much broader spectrum of pathogens than viral medicine since antiquity, and aspirin (Fig. 1), a close coat protein mediated resistances or even many com- analog of salicylic acid, has become one of the most mercial pesticides. SAR may be effective for weeks to popular pharmaceutical preparations in the world. months under field conditions4. Understanding the mechanisms that underlie the induction of SAR may have profound implications for the development of (a) COOH crop varieties resistant to a broad spectrum of pathogens. Because inoculation with one pathogen induces protection against unrelated organisms, it is likely that SAR results from the activation of a combination of diverse biochemical processes in the plant. Among the known systemic responses are a rise in peroxi(b) COOH (C) COOH dase activity, which may increase polymerization of 'lignin-like' cell wall phenolics6, and the coordinated de novo synthesis of so-called pathogenesis-related (PR) proteins (reviewed in Ref. 7). At least five families of host-encoded, low molecular mass PR proteins are induced in tobacco. Homologous proteins have been detected in at least 20 dicotyledonous and Rg. 1. The structures of (a) salicylic acid, (b) 2,6-dihydroxymonocotyledonous plant species after treatment with benzoic acid and (¢) acetylsalicylic acid (aspirin). pathogens or specific chemicalss. Members of the
oh.o
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A regulatory role of salicylic acid in plants was uncovered when it was identified as the natural inducer of alternative oxidase activity and heat and odor production in the inflorescences of thermogenic species TM. Salicylic acid affects a number of other physiological processes, ranging from promotion of flowering in some species to inhibition of stomatal closure and ion uptake in others (see Ref. 15). Several lines of evidence suggest that salicylic acid is the endogenous signal involved in induction of PR protein synthesis and SAR in tobacco and cucumber.
25-
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induction of resistance by salicylic acid In tobacco, resistance to TMV is linked to the presence of the dominant N gene 16. Treatment of the NNgenotype tobacco Nicotiana tabacum cv. Xanthi-nc with salicylic acid results in the coordinate expression of all known PR protein genes 17and reduced development of HR lesions to TMV TM. Salicylic acid also induces an acidic peroxidase, a manganese superoxide dismutase and a glycine-rich wall protein ~9,2°, which are not classified as PR proteins but may also function in defense. In nn-genotype tobacco, in which a lack of HR to TMV results in a systemic TMV infection, salicylic acid also inhibits virus proliferation 21. In addition to its effects in tobacco, salicylic acid treatment induces PR protein accumulation and protects cucumber and a number of other species against viral, bacterial and fungal pathogens (summarized in Ref. 22). Among salicylic acid analogs, only salicylic acid, acetylsalicylic acid (aspirin) which is rapidly hydrolyzed to salicylic acid in biological tissues - and 2,6-dihydroxybenzoic acid (Fig. 1) are effective direct inducers of resistance and PR proteins 8. Support for the role of salicylic acid in SAR comes from measurements of endogenous salicylic acid. Systemic levels of salicylic acid increase dramatically following inoculation of tobacco or cucumber with SAR-inducing pathogens. Tobacco carrying the N gene develops an HR by approximately 48 h after TMV inoculation. As HR lesions appear, the level of salicylic acid in the inoculated leaf and in pathogenfree leaves increases (Fig. 2) 23,24. Salicylic acid levels continue to increase over six days and may exceed preinfection levels more than 20-fold in the inoculated leaves and up to fivefold in untreated leaves of the same plant (Fig. 2). The pathogen-induced rise in salicylic acid levels parallels or precedes the accumulation of PR-1 mRNA 23. The increases in salicylic acid are always associated with the resistance response. Neither salicylic acid nor PR-1 mRNA accumulate in mock-inoculated plants or TMV-infected nn-genotype tobacco, which do not form HR lesions23. The rise in salicylic acid is highest in the immediate vicinity of HR lesions 24. Acquired resistance develops in this zone four days after TMV inoculation 2, correlating with the spatial and temporal accumulation of PR proteins 2s. Salicylic acid treatment experiments demonstrate that the levels of salicylic acid observed in untreated leaves of TMV-inoculated NN tobacco are sufficient to result
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Fig. 2. Accumulation of salicylic acid in TMV-inoculated tobacco. (a) Inoculated leaf tissue expressing a hypersensitive response. (b) The basal uninoculated portion of an inoculated leaf (squares), and the untreated leaf immediately above the inoculated leaf (triangles). Data adapted from Ref. 24.
in accumulation of PR proteins and resistance to a subsequent challenge with TMV 24'26. Dramatic increases in salicylic acid levels are not unique to the tobacco-TMV system but appear to be part of a generalized resistance response. Similar increases in salicylic acid levels have been observed in tobacco and soybean upon induction of HR with bacterial and fungal pathogens (P. Silverman and I. Raskin, unpublished), and in cucumber inoculated with viruses, bacteria or fungi27,28. Stem girdling and grafting experiments with cucumber and tobacco suggest that the SAR signal moves through the phloem 29-31. Studies with cucumber and tobacco show that the development of SAR is correlated with increases of salicylic acid levels in the phloem sap of hypersensitively responding plants, indicating a systemic role for salicylic acid in resistance 26. However, it remains uncertain whether the
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phloem export of salicylic acid from the inoculated leaf rises early enough, and to sufficient levels, to account for the salicylic acid increases in pathogen-free tissues 2s. It is still possible that another systemically mobile molecule triggers salicylic acid accumulation and at least some component of pathogen resistance.
on levels of resistance. A number of correlative results support this prediction. In NN-genotype tobacco, salicylic acid accumulation is inhibited if TMVinoculated plants are kept at 32°C (Ref. 26). Under these conditions, the HR and associated accumulation of PR proteins is also blocked 32,33. Synthesis of PR proteins at this temperature can be restored by spraying plants with salicylic acid2°,26. An amphidiploid hybrid generated from Nicotiana glutinosa and N. debneyi is known for its greater constitutive levels of PR proteins and high resistance to viral, bacterial and fungal pathogens when compared to its parental species or N. tabacum cv. Xanthi-nc 34,3s. Healthy hybrid plants have 30-times greater levels of salicylic acid than the less-resistant Xanthi-nc plants 36, which supports an involvement of salicylic acid in resistance. PR proteins and increased TMV resistance can be detected in the leaves of healthy, flowering Xanthinc tobacco 37. These developmentally induced increases in resistance are also correlated with elevated leaf levels of salicylic acid ~6.
Correlation between salicylic acid levels and resistance No mutants blocked in salicylic acid biosynthesis or metabolism are available to demonstrate conclusively a messenger role for salicylic acid in resistance. However, if salicylic acid is the natural signal for induced resistance, then treatments that inhibit or enhance its accumulation should have a corresponding effect
COOH '~NH2
L-Phenylalanine
PAL
Regulation of salicylic acid accumulation It is generally believed that, in plants, salicylic acid is derived from the phenylpropanoid pathway and is most likely synthesized from transcinnamic acid, a product of phenylalanine ammonia lyase (PAL). The activity of PAL, and some other enzymes of this pathway, is strongly induced in tobacco with the development of TMV-induced necrosis38 and also other biotic and abiotic stresses 39. The phenylpropanoid pathway produces a variety of structural and defense-related phenolics such as lignin and phytoalexins. The formation of hydroxybenzoic acids from cinnamic acid in plants may proceed by two pathways (Fig. 3). Establishment of the hydroxylation pattern of hydroxybenzoic acids can occur before or after the decarboxylation step. This chain-shortening decarboxylation may proceed via coenzyme A intermediates, analogous to the ~5-0xidation of fatty acids as suggested by Zenk 4°, or it may proceed via other, possibly nonoxidative, mechanisms41-44. For example, [14C]benzoic acid can serve as a precursor for labeled salicylic acid in Helianthus annuus, Pisum sativum and Solanum tuberosum 4s. Several reports implicate both o-coumaric acid and
COOH ~
.-""
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~OH
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~ O-o-Giucosylsalicylicacid
Local ancl systemic nductlon of PR protelns J and resistance
)
Fig. 3. Salicylic acid biosynthesis and metabolism in plants. Plus signs indicate steps induced in the vicinity of hypersensitive response lesions. PAL, phenylalanine ammonia lyase.
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benzoic acid as intermediates in the synthesis of salicylic acid 46-4s. In TMV-inoculated tobacco exhibiting SAR, o-coumaric acid does not serve as a salicylic acid precursor (N. Yalpani, J. Leon and I. Raskin, unpublished). Instead, as suggested in Fig. 3, increased salicylic acid synthesis is associated with a large increase in leaf benzoic acid and induction of the activity o f a benzoic acid-o-hydroxylase (J. Leon, N. Yalpani and I. Raskin, unpublished). It should be noted, however, that salicylic acid accumulation is specifically associated with H R and SAR and that it is not a wound response, whereas PAL activity is induced by wounding and a variety of abiotic and biotic stresses 49. Hence PAL activity does not appear to be the only determinant of salicylic acid accumulation. It is possible that, analogous to the induction of lignin and phytoalexin biosynthesis, stimulation of salicylic acid synthesis is a result of the coordinate activation of a number of enzymes of the phenylpropanoid pathway. Conjugation and/or further hydroxylation reactions appear to result in metabolic inactivation of salicylic acid. l~-O-D-Glucosylsalicylic acid (GSA; Fig. 3) is the major metabolite of salicylic acid in tobacco 24 and other plants s°-53. This conjugate accumulates rapidly in tobacco tissues treated with salicylic acid or in the immediate vicinity of TMV-induced lesions 24. GSA is formed by the action of a UDPglucose:salicylic acid glucosyltransferase, which is induced by the high levels of salicylic acid present in TMV-inoculated N tobacco leaves s4. No conjugates of salicylic acid are detected in healthy plants, in the phloem, or in pathogen-free tissues of TMV-inoculated N tobacco 24. Thus GSA does not seem to be directly involved in the induction of PR proteins and SAR. Other metabolites of salicylic acid may arise from additional hydroxylation of the aromatic ring. Thus 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid have been detected in Astilbe sinensis and tomato plants fed [14C]-labeled cinnamic or benzoic acid 46,55. Future directions
Present evidence strongly supports the theory that salicylic acid is a signal molecule in induced PR protein synthesis and some of the resistance responses of plants to pathogens. While salicylic acid has been detected in a large number of plant taxa s6, it is unlikely that salicylic acid is the only messenger or is an universal signal in induced resistance. Moreover, it still needs to be established conclusively that salicylic acid is the primary trigger for development of SAR. Thus it is unclear whether phloem export of salicylic acid from hypersensitively responding tissues is sufficient to account for the increases in salicylic acid and resistance in pathogen-free portions of plants. It is possible that salicylic acid stimulates its own biosynthesis in pathogen-free tissues, in a manner similar to the autocatalytic effects of ethylene on its own biosynthesis sT, or that salicylic acid synthesis throughout the plant is induced by other transmissible signals as suggested by Rasmussen et al. 28. Conclusive evidence for the messenger role of salicylic acid could be estab-
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lished with genetically engineered plants or mutants with altered salicylic acid accumulation or sensitivity. Rapidly developing research on SAR in A r a b i d o p s i s 68 may be a promising approach to obtaining direct evidence for a role of salicylic acid in SAR. Little is known about the transduction pathway preceding and following the increases in salicylic acid during development of SAR. Hence the role of a salicylic acid binding protein with putative receptor function is of great interest 59. Research on the processes involved in inducible plant defense responses may generate novel strategies for improving disease resistance in crops. Acknowledgements
The authors' work is supported by grants from the US Dept of Agriculture (Competitive Research Grants Office), the Division of Energy Biosciences of the US Dept of Energy, the Rockefeller Foundation, the New JerseyCommissionfor Scienceand Technology, and the New JerseyAgricultural Experiment Station. References
1 Klement, Z. (1982) in Phytopathogenic Prokaryotes (Vol.2) (Mount, M.S. and Lacey,G.H., eds), pp. 150-170, Academic Press 2 Ross, A.F. (1961) Virology 13,329-339 3 Ross,A.F. (1961) Virology 13,340-358 4 Madamanchi, N.R. and Kuc,J. (1991) in The Fungal Spore and Disease Initiation in Plants and Animals (Cole, G.T. and Hoch, H.C., eds), pp. 347-362, Plenum Press 5 Beachy,R.N., Loesch-Fries,S. and Turner, N.E. (1990) Annu. Rev. Phytopathol. 28,451-474 6 Hammerschmidt,R., Nuckles, E.M. and Kuc,J. (1982) Physiol. Plant Pathol. 20, 73-82 7 Linthorst, H.J.M. (1991) Crit. Rev. Plant Sci. 10, 123-150 8 Van Loon, L.C. (1983) Neth. J. Plant Pathol. 89, 265-273 9 Broglie,K. et al. (1991 ) Science 254, 1194-1197 10 Mauch, F., Mauch-Mani, B. and Boiler,T. (1988) Plant Physiol. 88,936-942 11 Woloshuk, C.P. et al. (1991) Plant Cell 3, 619-628 12 Ross, A.F. (1966) in Viruses of Plants (Beemster,A.B.R.and Dijkstra J., eds), pp. 127-150, North-Holland 13 Kuc,J. (1982) Bioscience 32, 854-860 14 Raskin, I. et al. (1987) Science 237, 1545-1546 15 Raskin, I. (1992) Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 439--463 16 Holmes,F.O. (1938) Phytopatbology 28,553-561 17 Ward, E.R. et al. (1991) Plant Cell 3, 1085-1094 18 White, R.F. (1979) Virology 99, 410--412 19 Bowler,C. et al. (1989) EMBO J. 8, 31-38 20 Van de Rhee, M.D. et al. (1990) Plant Cell 2, 357-366 21 White, R.F. et al. (1983) Phytopatbol. Z. 107, 224-232 22 Malamy,J. and Klessig,D.F. (1992) Plant]. 2,643-654 23 Malamy,J. et al. (1990) Science 250, 1002-1004 24 Enyedi,A.J. et al. (1992) Proc. Natl Acad. USA 89, 2480-2484 25 Antoniw,J.F. and White, R.F. (1986) Plant Mol. Biol. 6, 145-149 26 Yalpani, N. et al. (1991) Plant Cell 3, 809-818 27 M&raux,J.P. et al. (1990) Science 250, 1004-1006 28 Rasmussen,J.B., Hammerschmidt, R. and Zook, M.N. (1991) Plant Physiol. 97, 1342-1347 29 Jenns, A. and Kuc,J. (1979) Phytopathology 69, 753-756 30 Guedes,M.E.M., Richmond, S. and Kuc,J. (1980) Physiol. Plant Patbol. 17, 229-233 31 Tuzun, S. and Kuc,J. (1985) Phytopatbology 26, 321-330 32 Kassanis, B. (1952) Ann. Appl. Biol. 39, 358-369 33 Van Loon, L.C. (1975) Physiol. Plant Patbol. 6,289-300 34 Ahl, P. and Gianinazzi, S. (1982) Plant Sci. Lett. 26, 173-181 35 Ahl Goy, P. etal. (1992) Physiol. Mol. Plant Pathol. 41, 11-21
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36 Yalpani, N., Shulaev,V. and Raskin Phytopathology (in press) 37 Fraser, R.S.S. (1981) Physiol. Plant Pathol. 19, 69-76 38 Legrand, M., Fritig, B. and Hirth, L. (1976) Phytochemistry 15, 1353-1359 39 Boiler,T. (1991) in The Plant Hormone Ethylene (Mattoo, A.K. and Suttle,J.C., eds), pp. 293-313, CRC Press 40 Zenk, M.H. (1979) in Recent Advances in Phytochemistry (Vol. 12) (Swain,T., Harborne, J.B. and Van Sumere, C.F., eds), pp. 139-176, Plenum Press 41 French, C.J., Vance, C.P. and Towers, G.H.N. (1976) Phytochemistry 15, 564-566 42 Funk, C. and Brodelius,P.E. (1990) Plant Physiol. 94, 95-101 43 Funk, C. and Brodelius,P.E. (1990) Plant Physiol. 94, 102-108 44 Schnitzler,J.P. et aL (1992) Planta 188, 594-600 45 Kl~imbt,H.D. (1962) Nature 196, 491 46 Chadha, K.C. and Brown, S.A. (1974) Can. J. Bot. 52, 2041-2046
47 E1-Basyouni,S. et al. (1964) Phytochemistry 3,485-492 48 Ellis, B.E. and Amrhein, N. (1971) Phytochemistry 10, 3069-3072 49 Liang, X. et al. (1989)]. Biol. Chem. 264, 14486-14492 50 Cooper-Driver,G., Corner-Zamodits,J. and Swain,T. (1972) Z. Naturforsch. B 27, 943-946 51 Tanaka, S. et al. (1990) Phytochemistry 29, 1555-1558 52 Comer,J.J. and Swain, T. (1965) Nature 207, 634-635 53 Yalpani, N. et al. (1992)Plant Physiol. 100, 1114-1119 54 Enyedi,A.J. and Raskin I. (1993) Plant Physiol. 101, 1375-1380 55 Billek,G. and Schmook,F.P. (1967) Monatsh. Chem. 98, 1651-1664 56 Raskin, I. etal. (1990) Ann. Bot. 66, 369-373 57 Kende,H. (1976) Nova Acta Leopold. Suppl. 7, 165-174 58 Uknes,S. et al. (1992) Plant Cell 4, 645-656 59 Chen, Z. and Klessig,D.F. (1991) Proc. Natl Acad. Sci. USA 88, 8179-8183
DNA topology and bacterial virulence gene regulation Charles J. Dorman and Niamh Ni Bhriain he ability to infect hosts The topology of bacterial DNA varies in regulators, which use phosresponse to extracellular environmental affords bacteria the photransfer to transmit sigopportunity to inhabit stimuli, providing a possible mechanism nals concerning the state of privileged environmental niches. for environmental control of gene the environment to the reTo profit from such an asexpression during bacterial pathogenesis. sponse machinery; the latter is sociation, the bacterium must The contribution of DNA topology to the usually (but not exclusively) adapt to the changing environ- control of transcription is complex, but an the genome 1. Others involve mental circumstances that acappreciation of the distinction between yet further protein 'families' company the transition from local and global DNA topological effects named after their prototypic a free-living state to a hostis helping to clarify this complexity. examples (the arabinose operassociated niche. This transition on regulatory protein, AraC; cd. Dorman and N. Ni Bhriain are in the Molecular the cyclic-AMP receptor procan involve severe fluctuations Genetics Laboratory, Dept of Biochemistry, in physical or chemical partein, Crp; the anaerobic gene University of Dundee, Dundee, UK DDI 4HN. ameters such as temperature, activator protein, Fnr; the lyosmolarity, oxygen availability, sine regulatory protein, LysR, pH, nutrition, host defences, and the activities of etc.), whose members regulate transcription in response to specific environmental signals 2-5. These reguother members of the micro flora. It is not surprising to discover that much of the adaptation to their latory proteins exert specific effects because the genes dynamic surroundings involves changes in the tranthat they regulate possess a particular DNA sequence scriptional profile of the bacterial genome. Conseat which they bind. Genes subject to such collective quently, much research has been devoted to the control by a common regulator are said to constitute a question of how changes in the environment elicit 'regulon', and mutations in the regulatory gene are modulations in gene expression. In the case of pathousually pleiotropic for all of the structural genes within genic bacteria, the fact that the success of these its subservient regulon 6. For this reason, many workers organisms in achieving such modulations frequently describe such genes as 'global' regulators. However, has dire consequences for ourselves has given added this title is difficult to reconcile with the fact that the interest to the inquiry. expression of only a subset of cellular genes is affected Before the bacterium can mount a transcriptional by such mutations. Searches for regulators with truly response to an environmental stimulus, it must first global control potential have led to an investigation of detect that stimulus and communicate it to the the structure of the bacterial genome itself. Since this genome. It has become clear that there is no single represents a genuinely common denominator in gene mechanism by which this is achieved, although expression, it is reasonable to anticipate that factors certain themes recur. One of these concerns the everaltering it are likely to have widespread influence on growing family of histidine-protein-kinase response the expression of the genetic material.
T
© 1993 Elsevier Science Publishers Ltd (UK) 0966 842X/93/$06.00
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Salicylic acid: a systemic signal in induced plant disease resistance Nasser Yalpani and Ilya Raskin
p
lants face a constant barSome plants respond to infection by tobacco PR-2 and PR-3 famirage of microbial organpathogens with both localized and lies of proteins have in vitro isms, but infection and systemic resistance responses. These ~-l,3-glucanase or chitinase disease rarely develop from prevent the spread of the disease-causing activity, respectively, and can these contacts. In addition to organism and reduce the severity of a hydrolyse fungal and bacterial chemical and physical barriers subsequent infection. Recent evidence cell wallsL Chitinase, alone or such as the cuticle, cell wall suggests that systemic increases in the in combination with 15-1,3-gluand antimicrobial chemicals host's salicylic acid levels act as a signal canase, has been shown to that are constitutively present, for the activation of at least some of degrade fungal or bacterial plants frequently activate a these induced defenses. cell wall preparations and to range of defense-related proinhibit fungal growth in vitro N. Yalpani and I. Raskm are in the AgBiotecb teins upon attack by pathoand in vivo 9,~°. PR-S is strucCenter, Cook College, Rutgers University, New gens. These inducible defenses turally similar to osmotin and Brunswick, NJ 08903-0231, USA. often accompany the developzeamatin, both of which are ment of a hypersensitive reknown to have antifungal sponse (HR). In this process, host cells in the im- activity in vitro 11. The function of other PR proteins, mediate vicinity of the site of pathogen penetration such as those in the PR-1 family, is unknown. The 'commit coordinated suicide', possibly killing the close association between PR protein accumulation pathogen or restricting its spreadL and resistance and the in vitro antimicrobial activity Activation of defenses may not only be localized of some PR proteins suggests that they function around the HR site but can also extend to tissue un- in defense. However, the extent to which these exposed to the inducing pathogen. Ross 2'3 described proteins contribute to resistance against pathogens, the development of such systemic acquired resistance particularly viruses, is not clear. (SAR) in tobacco showing an HR to tobacco mosaic SAR is likely to result from the synthesis and revirus (TMV). SAR is not only effective against the lease of a signal compound that moves from the site inducing pathogen but also against some taxonomi- of initial pathogen penetration to other parts of the cally distant disease agents. For instance, inoculation plant 12,13. Considerable attention has recently been of tobacco with HR-inducing viruses can 'immunize' focused on salicylic acid (Fig. 1), an ubiquitous plant the whole plant against attack by bacteria, fungi or phenolic, as a candidate signal molecule in SAR. other viruses (reviewed in Ref. 4). Thus SAR covers Salicylates from plant sources have been used in a much broader spectrum of pathogens than viral medicine since antiquity, and aspirin (Fig. 1), a close coat protein mediated resistances or even many com- analog of salicylic acid, has become one of the most mercial pesticides. SAR may be effective for weeks to popular pharmaceutical preparations in the world. months under field conditions4. Understanding the mechanisms that underlie the induction of SAR may have profound implications for the development of (a) COOH crop varieties resistant to a broad spectrum of pathogens. Because inoculation with one pathogen induces protection against unrelated organisms, it is likely that SAR results from the activation of a combination of diverse biochemical processes in the plant. Among the known systemic responses are a rise in peroxi(b) COOH (C) COOH dase activity, which may increase polymerization of 'lignin-like' cell wall phenolics6, and the coordinated de novo synthesis of so-called pathogenesis-related (PR) proteins (reviewed in Ref. 7). At least five families of host-encoded, low molecular mass PR proteins are induced in tobacco. Homologous proteins have been detected in at least 20 dicotyledonous and Rg. 1. The structures of (a) salicylic acid, (b) 2,6-dihydroxymonocotyledonous plant species after treatment with benzoic acid and (¢) acetylsalicylic acid (aspirin). pathogens or specific chemicalss. Members of the
oh.o
© 1993 Elsevier Science Publishers Ltd (UK) 0966 842X/93/$06.00 TRENDS IN MICROBIOLOGY
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A regulatory role of salicylic acid in plants was uncovered when it was identified as the natural inducer of alternative oxidase activity and heat and odor production in the inflorescences of thermogenic species TM. Salicylic acid affects a number of other physiological processes, ranging from promotion of flowering in some species to inhibition of stomatal closure and ion uptake in others (see Ref. 15). Several lines of evidence suggest that salicylic acid is the endogenous signal involved in induction of PR protein synthesis and SAR in tobacco and cucumber.
25-
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induction of resistance by salicylic acid In tobacco, resistance to TMV is linked to the presence of the dominant N gene 16. Treatment of the NNgenotype tobacco Nicotiana tabacum cv. Xanthi-nc with salicylic acid results in the coordinate expression of all known PR protein genes 17and reduced development of HR lesions to TMV TM. Salicylic acid also induces an acidic peroxidase, a manganese superoxide dismutase and a glycine-rich wall protein ~9,2°, which are not classified as PR proteins but may also function in defense. In nn-genotype tobacco, in which a lack of HR to TMV results in a systemic TMV infection, salicylic acid also inhibits virus proliferation 21. In addition to its effects in tobacco, salicylic acid treatment induces PR protein accumulation and protects cucumber and a number of other species against viral, bacterial and fungal pathogens (summarized in Ref. 22). Among salicylic acid analogs, only salicylic acid, acetylsalicylic acid (aspirin) which is rapidly hydrolyzed to salicylic acid in biological tissues - and 2,6-dihydroxybenzoic acid (Fig. 1) are effective direct inducers of resistance and PR proteins 8. Support for the role of salicylic acid in SAR comes from measurements of endogenous salicylic acid. Systemic levels of salicylic acid increase dramatically following inoculation of tobacco or cucumber with SAR-inducing pathogens. Tobacco carrying the N gene develops an HR by approximately 48 h after TMV inoculation. As HR lesions appear, the level of salicylic acid in the inoculated leaf and in pathogenfree leaves increases (Fig. 2) 23,24. Salicylic acid levels continue to increase over six days and may exceed preinfection levels more than 20-fold in the inoculated leaves and up to fivefold in untreated leaves of the same plant (Fig. 2). The pathogen-induced rise in salicylic acid levels parallels or precedes the accumulation of PR-1 mRNA 23. The increases in salicylic acid are always associated with the resistance response. Neither salicylic acid nor PR-1 mRNA accumulate in mock-inoculated plants or TMV-infected nn-genotype tobacco, which do not form HR lesions23. The rise in salicylic acid is highest in the immediate vicinity of HR lesions 24. Acquired resistance develops in this zone four days after TMV inoculation 2, correlating with the spatial and temporal accumulation of PR proteins 2s. Salicylic acid treatment experiments demonstrate that the levels of salicylic acid observed in untreated leaves of TMV-inoculated NN tobacco are sufficient to result
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Fig. 2. Accumulation of salicylic acid in TMV-inoculated tobacco. (a) Inoculated leaf tissue expressing a hypersensitive response. (b) The basal uninoculated portion of an inoculated leaf (squares), and the untreated leaf immediately above the inoculated leaf (triangles). Data adapted from Ref. 24.
in accumulation of PR proteins and resistance to a subsequent challenge with TMV 24'26. Dramatic increases in salicylic acid levels are not unique to the tobacco-TMV system but appear to be part of a generalized resistance response. Similar increases in salicylic acid levels have been observed in tobacco and soybean upon induction of HR with bacterial and fungal pathogens (P. Silverman and I. Raskin, unpublished), and in cucumber inoculated with viruses, bacteria or fungi27,28. Stem girdling and grafting experiments with cucumber and tobacco suggest that the SAR signal moves through the phloem 29-31. Studies with cucumber and tobacco show that the development of SAR is correlated with increases of salicylic acid levels in the phloem sap of hypersensitively responding plants, indicating a systemic role for salicylic acid in resistance 26. However, it remains uncertain whether the
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phloem export of salicylic acid from the inoculated leaf rises early enough, and to sufficient levels, to account for the salicylic acid increases in pathogen-free tissues 2s. It is still possible that another systemically mobile molecule triggers salicylic acid accumulation and at least some component of pathogen resistance.
on levels of resistance. A number of correlative results support this prediction. In NN-genotype tobacco, salicylic acid accumulation is inhibited if TMVinoculated plants are kept at 32°C (Ref. 26). Under these conditions, the HR and associated accumulation of PR proteins is also blocked 32,33. Synthesis of PR proteins at this temperature can be restored by spraying plants with salicylic acid2°,26. An amphidiploid hybrid generated from Nicotiana glutinosa and N. debneyi is known for its greater constitutive levels of PR proteins and high resistance to viral, bacterial and fungal pathogens when compared to its parental species or N. tabacum cv. Xanthi-nc 34,3s. Healthy hybrid plants have 30-times greater levels of salicylic acid than the less-resistant Xanthi-nc plants 36, which supports an involvement of salicylic acid in resistance. PR proteins and increased TMV resistance can be detected in the leaves of healthy, flowering Xanthinc tobacco 37. These developmentally induced increases in resistance are also correlated with elevated leaf levels of salicylic acid ~6.
Correlation between salicylic acid levels and resistance No mutants blocked in salicylic acid biosynthesis or metabolism are available to demonstrate conclusively a messenger role for salicylic acid in resistance. However, if salicylic acid is the natural signal for induced resistance, then treatments that inhibit or enhance its accumulation should have a corresponding effect
COOH '~NH2
L-Phenylalanine
PAL
Regulation of salicylic acid accumulation It is generally believed that, in plants, salicylic acid is derived from the phenylpropanoid pathway and is most likely synthesized from transcinnamic acid, a product of phenylalanine ammonia lyase (PAL). The activity of PAL, and some other enzymes of this pathway, is strongly induced in tobacco with the development of TMV-induced necrosis38 and also other biotic and abiotic stresses 39. The phenylpropanoid pathway produces a variety of structural and defense-related phenolics such as lignin and phytoalexins. The formation of hydroxybenzoic acids from cinnamic acid in plants may proceed by two pathways (Fig. 3). Establishment of the hydroxylation pattern of hydroxybenzoic acids can occur before or after the decarboxylation step. This chain-shortening decarboxylation may proceed via coenzyme A intermediates, analogous to the ~5-0xidation of fatty acids as suggested by Zenk 4°, or it may proceed via other, possibly nonoxidative, mechanisms41-44. For example, [14C]benzoic acid can serve as a precursor for labeled salicylic acid in Helianthus annuus, Pisum sativum and Solanum tuberosum 4s. Several reports implicate both o-coumaric acid and
COOH ~
.-""
trans-Cinnamic
J
acid
I
COOH
÷
J ¢'-'
~OH
~jOH/ '
o - C o u m a d c acicl
Pathogen
Benzoic acicl
• "'-
C O O H OH
Salicylicacid U
~ O-o-Giucosylsalicylicacid
Local ancl systemic nductlon of PR protelns J and resistance
)
Fig. 3. Salicylic acid biosynthesis and metabolism in plants. Plus signs indicate steps induced in the vicinity of hypersensitive response lesions. PAL, phenylalanine ammonia lyase.
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benzoic acid as intermediates in the synthesis of salicylic acid 46-4s. In TMV-inoculated tobacco exhibiting SAR, o-coumaric acid does not serve as a salicylic acid precursor (N. Yalpani, J. Leon and I. Raskin, unpublished). Instead, as suggested in Fig. 3, increased salicylic acid synthesis is associated with a large increase in leaf benzoic acid and induction of the activity o f a benzoic acid-o-hydroxylase (J. Leon, N. Yalpani and I. Raskin, unpublished). It should be noted, however, that salicylic acid accumulation is specifically associated with H R and SAR and that it is not a wound response, whereas PAL activity is induced by wounding and a variety of abiotic and biotic stresses 49. Hence PAL activity does not appear to be the only determinant of salicylic acid accumulation. It is possible that, analogous to the induction of lignin and phytoalexin biosynthesis, stimulation of salicylic acid synthesis is a result of the coordinate activation of a number of enzymes of the phenylpropanoid pathway. Conjugation and/or further hydroxylation reactions appear to result in metabolic inactivation of salicylic acid. l~-O-D-Glucosylsalicylic acid (GSA; Fig. 3) is the major metabolite of salicylic acid in tobacco 24 and other plants s°-53. This conjugate accumulates rapidly in tobacco tissues treated with salicylic acid or in the immediate vicinity of TMV-induced lesions 24. GSA is formed by the action of a UDPglucose:salicylic acid glucosyltransferase, which is induced by the high levels of salicylic acid present in TMV-inoculated N tobacco leaves s4. No conjugates of salicylic acid are detected in healthy plants, in the phloem, or in pathogen-free tissues of TMV-inoculated N tobacco 24. Thus GSA does not seem to be directly involved in the induction of PR proteins and SAR. Other metabolites of salicylic acid may arise from additional hydroxylation of the aromatic ring. Thus 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid have been detected in Astilbe sinensis and tomato plants fed [14C]-labeled cinnamic or benzoic acid 46,55. Future directions
Present evidence strongly supports the theory that salicylic acid is a signal molecule in induced PR protein synthesis and some of the resistance responses of plants to pathogens. While salicylic acid has been detected in a large number of plant taxa s6, it is unlikely that salicylic acid is the only messenger or is an universal signal in induced resistance. Moreover, it still needs to be established conclusively that salicylic acid is the primary trigger for development of SAR. Thus it is unclear whether phloem export of salicylic acid from hypersensitively responding tissues is sufficient to account for the increases in salicylic acid and resistance in pathogen-free portions of plants. It is possible that salicylic acid stimulates its own biosynthesis in pathogen-free tissues, in a manner similar to the autocatalytic effects of ethylene on its own biosynthesis sT, or that salicylic acid synthesis throughout the plant is induced by other transmissible signals as suggested by Rasmussen et al. 28. Conclusive evidence for the messenger role of salicylic acid could be estab-
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lished with genetically engineered plants or mutants with altered salicylic acid accumulation or sensitivity. Rapidly developing research on SAR in A r a b i d o p s i s 68 may be a promising approach to obtaining direct evidence for a role of salicylic acid in SAR. Little is known about the transduction pathway preceding and following the increases in salicylic acid during development of SAR. Hence the role of a salicylic acid binding protein with putative receptor function is of great interest 59. Research on the processes involved in inducible plant defense responses may generate novel strategies for improving disease resistance in crops. Acknowledgements
The authors' work is supported by grants from the US Dept of Agriculture (Competitive Research Grants Office), the Division of Energy Biosciences of the US Dept of Energy, the Rockefeller Foundation, the New JerseyCommissionfor Scienceand Technology, and the New JerseyAgricultural Experiment Station. References
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DNA topology and bacterial virulence gene regulation Charles J. Dorman and Niamh Ni Bhriain he ability to infect hosts The topology of bacterial DNA varies in regulators, which use phosresponse to extracellular environmental affords bacteria the photransfer to transmit sigopportunity to inhabit stimuli, providing a possible mechanism nals concerning the state of privileged environmental niches. for environmental control of gene the environment to the reTo profit from such an asexpression during bacterial pathogenesis. sponse machinery; the latter is sociation, the bacterium must The contribution of DNA topology to the usually (but not exclusively) adapt to the changing environ- control of transcription is complex, but an the genome 1. Others involve mental circumstances that acappreciation of the distinction between yet further protein 'families' company the transition from local and global DNA topological effects named after their prototypic a free-living state to a hostis helping to clarify this complexity. examples (the arabinose operassociated niche. This transition on regulatory protein, AraC; cd. Dorman and N. Ni Bhriain are in the Molecular the cyclic-AMP receptor procan involve severe fluctuations Genetics Laboratory, Dept of Biochemistry, in physical or chemical partein, Crp; the anaerobic gene University of Dundee, Dundee, UK DDI 4HN. ameters such as temperature, activator protein, Fnr; the lyosmolarity, oxygen availability, sine regulatory protein, LysR, pH, nutrition, host defences, and the activities of etc.), whose members regulate transcription in response to specific environmental signals 2-5. These reguother members of the micro flora. It is not surprising to discover that much of the adaptation to their latory proteins exert specific effects because the genes dynamic surroundings involves changes in the tranthat they regulate possess a particular DNA sequence scriptional profile of the bacterial genome. Conseat which they bind. Genes subject to such collective quently, much research has been devoted to the control by a common regulator are said to constitute a question of how changes in the environment elicit 'regulon', and mutations in the regulatory gene are modulations in gene expression. In the case of pathousually pleiotropic for all of the structural genes within genic bacteria, the fact that the success of these its subservient regulon 6. For this reason, many workers organisms in achieving such modulations frequently describe such genes as 'global' regulators. However, has dire consequences for ourselves has given added this title is difficult to reconcile with the fact that the interest to the inquiry. expression of only a subset of cellular genes is affected Before the bacterium can mount a transcriptional by such mutations. Searches for regulators with truly response to an environmental stimulus, it must first global control potential have led to an investigation of detect that stimulus and communicate it to the the structure of the bacterial genome itself. Since this genome. It has become clear that there is no single represents a genuinely common denominator in gene mechanism by which this is achieved, although expression, it is reasonable to anticipate that factors certain themes recur. One of these concerns the everaltering it are likely to have widespread influence on growing family of histidine-protein-kinase response the expression of the genetic material.
T
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