Właściwości antybiotyczne związków w infekcji roślin, Publikacje naukowe
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//-->Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite inplant defenceFrancisco García-Olmedo*, Pablo Rodríguez-Palenzuela, Antonio Molina, Josefa M. Alamillo,Emilia López-Solanilla, Marta Berrocal-Lobo, César Poza-CarriónDepartamento de Biotecnología - UPM, ETS Ingenieros Agrónomos, E-28040 Madrid, SpainAbstract Genes encoding plant antibiotic peptides show ex-pression patterns that are consistent with a defence role.Transgenic over-expression of defence peptide genes is poten-tially useful to engineer resistance of plants to relevantpathogens. Pathogen mutants that are sensitive to plant peptidesin vitro have been obtained and a decrease of their virulence inplanta has been observed, which is consistent with theirhypothetical defence role. A similar approach has been followedto elucídate the potential direct anti-microbial role of hydrogenperoxide. Additionally, a scavenger of peroxynitrite has beenused to investígate its involvement in plant defence. © 2001Published by Elsevier Science B.V. on behalf of the Federationof European Biochemical Societíes.Key words:Innate immunity; Active oxygen;Nitrogen species; Plant disease resistance; Cell death; Urate;Plant-microbe interactionvations of diverse nature, which include the finding of a cor-relation between expression levéis and the severity of symp-toms and/or between pathogen resistance to the agent andvirulence. This review will focus on recent evidence concerningthe possible in vivo antibiotic activities of peptides, hydrogenperoxide and peroxynitrite, as well as on the pathogen re-sponse to these challenges (Fig. 1).2. Novel families of plant anti-microbial peptidesA number of antibiotic peptide families have been describedin plants (Table 1). Until recently, only globular peptides,stabilised by disulphide bonds, had been identified in plants[4,6]. Thionins were the first whose activity against plantpathogens was demonstrated in vitro [7]. Subsequently, sev-eral families of cysteine-rich peptides have been characterised,including defensins [4], lipid transfer proteins (LTPs) [4,6],hevein-type peptides [4] and knottin-type peptides [4], aswell as peptide MBP-1 from maize [8] and a group of 20-residue peptides (Ib-AMPs) isolated from the seeds ofImpa-tiens balsamina[9,10]. Novel plant antibiotic peptides includethe following types: the snakin/GASA family of 12-cysteinepeptides ([11], Berrocal-Lobo et al., unpublished), which havebeen first isolated from potato and are ubiquitous, as judgedfrom the multiplicity of homologous cDNAs that have beenreported [12,13]; the shepherdins, which are linear glycine/his-tidine-rich peptides isolated from the roots of shepherd'spurse(Capsella bursa-pastoris)[14]; and the macrocyclic cys-teine-knot peptides that have been purified from differentplants of the Rubiaceae family (coffee and other tropicalplants) in a screening for anti-HIV compounds [15].3. Expression of peptide-encoding genes and disease toleranceCorrelation of altered peptide levéis in planta with variationof disease tolerance is indicative of the possible defence role ofthe peptides [16-21]. Observations concerning thionins, defen-sins and LTPs are consistent with the defence hypothesis.Thionin mRNA is transiently induced in barley upon infec-tion withErysiphe graminisin both susceptible and resistantcultivars [22,23]. Transgenic tobáceo plants expressing a bar-ley thionin gene showed reduced lesión size when the plantswere challenged with two strains ofPseudomonas syringae[23], whereas other strains did not seem to be affected [24].Subsequently, over-expression of an endogenous thionin was1. IntroductionPlants have complex defence mechanisms that are eitherpre-formed or activated in response to pathogen attack [1,2].Resistance or susceptibility to a particular pathogen dependson various factors, including pathogen recognition, activationof signal transduction pathways and elicitation of active andpassive defence molecules [2]. In vitro anti-microbial activityhas been demonstrated for the following types of molecules:(i) some of the so-called pathogenesis-related proteins, whichwere originally identified as pathogen-elicited proteins of un-known function [2]; (ii) a considerable variety of plant organiccompounds, classified into phytoanticipins and phytoalexins,which include phenols and phenolic glycosides, unsaturatedlactones, sulphur compounds, saponins, cyanogenetic glyco-sides, glucosinolates, 5-alkylated resorcinols and dienes [3];(iii) a number of plant anti-microbial peptide families [4];and (iv) active oxygen and nitrogen species, such as hydrogenperoxide and peroxynitrite [5]. Demonstration of a possible invivo defence role for a given antibiotic agent involves obser-Plant CellRgenes •SignalTransductionPathwaysRecognitionElicitationPathogenr genesPASSIVE/DEFENCEACTIVEInducible/PR proteinsConstitutivePhytoalexinsPhytoanticipinsAntibiotic PeptidesI ,Active O& N ?JRESISTANCE/SUSCEPTIBILITYsapA-FRfaFIPT1oxyRmsrAgene(Rh3);and infection with the compatible bacterialpathogenP. syringaepv.japónicaswitches offLTPgene ex-pression [6,30]. Also, induction ofLTPgenes by cauliflowermosaic virus infection inArabidopsis[31], byXanthomonassp.in pepper [32] and by arbuscular mycorrhiza in rice [33] havebeen reported. The possible defence role of LTPs is furthersupported by the observation that transgenic tobáceo andArabidopsisplants over-expressing a barley LTP showed dras-tic reduction of disease symptoms after infection of the leaveswith the bacterial pathogenP. syringae[21].4. Pathogen sensitivity to plant peptides and virulenceIn agreement with the defence hypothesis, peptide-sensitivemutants of the pathogens show significantly decreased viru-lence towards plant tissues in which these peptides are present[34,35]. Furthermore, the latter type of evidence indicates thatboth plant and animal pathogens deal in a similar way withhost defences, as the equivalent mutants of animal pathogensshow also decreased virulence [35,36]. The possibility that thepathogen defence system against anti-microbial peptides mayshow specificity towards the peptide type has been suggestedand might be highly relevant in plant-pathogen interactions[35].A first type of mutant with increased sensitivity to thioninsand LTPs was obtained by insertion of transposon Tn5 inRalstonia solanacearum.This mutation interrupted therfaFgene, which encodes a heptosyl transferase involved in thesynthesis of lipopolysaccharide (LPS). Consequently, LPS ofthe mutant lacked heptose and the phosphate groups thatreside in this sugar, and the mutant was avirulent in planta[34].It seems that phosphate groups in the LPS act as traps forthe peptides and prevent their interaction with target sites [34].Also, a defensin-resistant mutation in geneIPT1ofSaccha-romyees cerevisiae,which prevents phosphorylation of a mem-brane sphingolipid, mannose-(inositol-phosphate)2-ceramide,has been recently reported [37].A second type of peptide-sensitive mutants are affected inthesapoperon (for sensitive to anti-microbial peptides), whichhas been well studied inSalmonella typhimurium,where it isrequired for peptide resistance and for virulence in mice [38].Thesapgenes are organised in a single operon and exhibitsequence similarity with ABC transporters described in pro-karyotes and eukaryotes. The proposed mechanism of actionfor the Sap system includes binding of the periplasmic com-ponent SapA to the anti-microbial peptide, followed by pep-tide transport to the cytoplasm, where peptide degradationand/or activation of resistance determinants oceur. Thesap AFig. 1. Simplified scheme of plant-pathogen interactions. Aspectsdiscussed in this review are highlighted in bold letters (see text forexplanations).reported to enhance resistance ofArabidopsis thalianaagainstFusarium oxysporum[17] andPlasmodiophora brassicae[20].Experiments with radish seeds have demonstrated that de-fensins represent over 30% of the proteins released duringgermination (about 1 ug/seed) and that the released defensinis sufhcient for fungal inhibition, an effect that may contributeto the enhancement of seedling survival rate [18]. DefensinPDF 1.2gene fromArabidopsisis upregulated by jasmonateand ethylene, as well as by infection with the fungal pathogensAlternaría alternataandBotrytis cinérea[25]. Mutants im-paired in the jasmonate and ethylene signal transduction path-ways, which do not express genePDF1.2,show enhanced sus-ceptibility to these necrotrophic fungal pathogens [26,27]. Amore direct evidence of an in vivo role for defensins is pro-vided by over-expression experiments. Thus, transgenic ex-pression in tobáceo of the Rs-AFP2 defensin from radish(up to 0.2% of leaf proteins) resulted in a seven-fold reductionin lesión size with respect to the non-transformed control,upon infection with the foliar fungal pathogenAlternaríalongipes[18], and over-expression of an anti-fungal defensinfromMedicago sativain potato conferred robust resistanceunder field conditions [28].It has been shown thatLTPgenes respond to pathogeninfection in a complex manner, as they can be induced abovebasal levéis or be switched off by different plant pathogensthat infect barley [6,29,30]. Thus, infection by the fungalpathogenRhynchosporium secalisincreasesLTPgene expres-sion only in the incompatible interaction, not in the compat-ible one, and this induction is under the control of a resistanceTable 1Plant anti-microbial peptidesPeptide familyNumber of residuesLTPsSnakins (GASA)DefensinsThioninsHevein-likeKnottin-likeShepherdinsMBP-1Macrocyclic peptidesIb-AMPs90-9561-7045-5445^74336-3728-383329-3120Disulphide bridges3^643^430 (linear)232Types/subfamiliesIIII-IIII-IVI-IVIIIIIII-IIIIActive againstbacteria and fungibacteria and fungibacteria and fungibacteria and fungiGram(+) bacteria and fungiGram(+) bacteria and fungibacteria and fungibacteria and fungiGram(+) bacteriaGram(+) bacteria and fungitosapFoperon from the pathogenic bacteriumErwinia chrys-anthemihas five open reading frames that are closely related(71% overall amino acid identity) and are in the same order asthose of thesapAtosapFoperon fromS. typhimurium.AnE. chrysanthemi sapmutant was more sensitive to wheat a-thionin and to snakin-1, and also less virulent than the wild-type strain in potato tubers. These results indícate that theinteraction of anti-microbial peptides from the host with thesap AtosapFoperon from the pathogen plays a similar role inanimal and in plant bacterial pathogenesis [35] and, indeed,thesapoperons fromErwiniaandSalmonellashowed recip-rocal functional complementation (López-Solanilla et al., un-published results). Moreover, the mutation in thesaplocushad a greater effect on virulence than those in other well-characterised gene systems involved in plant-pathogen inter-actions [39], such as the PelABCE locus [40], which codes forpectate-lyases, and the Hrp locus [41], which codes for a typeIII secretion system.5. Active oxygen and nitrogen species versus virulenceA similar approach to that followed with the antibioticpeptides has been used to ascertain the possible in vivo anti-microbial properties of active oxygen species (AOS), whichmay play a dual role in defence: a direct antibiotic activityand an indirect effect as mediators of the activation of otherdefence components [42]. Although the in vitro activity of O-Tand H2O2 against phytopathogenic bacteria has been reported[43], its role in vivo remains controversial. Thus, a mutationof theoxyRgene, which controls several enzymes involved inAOS detoxification, had no effect on virulence ofE. chrysan-themi[44]. OxyR is a transcriptional activator of genes encod-ing several enzymes, including catalase and glutathione reduc-íase [45]. TheE. chrysanthemi oxyRmutant strain, which wasmore sensitive to H2O2 and was unable to form individualcolonies on solid médium unless catalase was added exoge-nously, retained full virulence in potato tubers and tobáceoleaves. Moreover, both the wild-type strain and theoxyRmu-tant were insensitive to exogenously added H2O2 when inoc-ulated into the plant. These data point towards a lack ofdirect anti-microbial effect of H2O2 in the plant defenceagainstErwiniainvasión, possibly because the combined ef-fects of anti-oxidant enzymes and reductant molecules fromthe plant prevent H2O2 from reaching concentrations that arelethal to the bacteria. In contrast, El Hassouni et al. [46]reported that themsrAmutant ofE. chrysanthemi,whichaffeets an enzyme that repairs oxidised proteins, was moresensitive to oxidative stress and had diminished virulence inChicorium intibus(chicory) andSaintpaulia ionantha.The in-terpretation of these results is complicated by the pleiotropyof themsrAmutation, since the diminished virulence could bedue to either increased sensitivity to oxidative stress, alteredmotility or other unknown effeets of the mutation.Nitric oxide (NO) has been recently demonstrated to play aprominent role during plant hypersensitive response and celldeath [47^9]. One likely role for NO and AOS is to promoteplant cell death and pathogen killing, as in the mammalianinflammatory response, probably by reaction of NO with O-Tto produce peroxynitrite [50-53]. However, it is unclearwhether NO or its activated derivatives are directly toxic topathogens in plants. In vitro growth of both a virulent(P. syringae pv. phaseolicola110; avrRPMl~) and an aviru-SYMPTOMSAUTOFLUORESCENCETRYPAN-BLUE STAINUrate-^+-+MückP.s.ph411 (üvrRPM 1 +)Fig. 2. Effeets of urate, a scavenger of peroxynitrite, on the plantresponse to bacterial pathogens [5]. A: Col-0Arabidopsisplantswere inoculated with 106cfu/ml of avirulent,avrRPM1+,P. syrin-gae pv. phaseolicola411(P.s.ph411); or virulent, avrRPMl~,P. sy-ringae pv. phaseolicola110(P.s.ph110), with or without 1 mg/mluric acid. After 24 h, leaves were examined for visible symptoms(left) or ultraviolet-stimulated autofluorescence (right). B: Micro-scope photographs of control andP.s.ph411 inoculated leaves, withand without urate stained with the Trypan-blue dye, at 24 h postinfection.lent(P. syringae pv. phaseolicola411; avrRPMl+) bacterialstrain was inhibited by NO, as well as by the peroxynitritegenerating system sodium nitroprusside+hypoxanthine/xan-thine oxidase, and direct application of peroxynitrite inducesplant cell death, which is prevented by the peroxynitrite scav-enger urate [5]. Using urate, it has been shown that althoughperoxynitrite was responsible for most of the host cell death ofArabidopsisin response to the avirulentP. syringaestrain(Fig. 2), scavenging of peroxynitrite did not compromise theeffective defence against this avirulent pathogen, in spite ofthe reduction in plant cell death [5]. Although peroxynitritehas been suggested as being responsible for direct pathogenkilling [52,53], urate scavenging of toxic peroxynitrite did notlead to a higher growth of either the virulent or the avirulentstrains, which indicated that peroxynitrite toxicity was notlimiting bacterial growth in planta [5]. On the contrary, theuse of the urate promoted discrete death of plant tissue chal-lenged with the virulent strainP. syringaepv.phaseolicola110(avrRPMl~) (Fig. 2) and resulted in a severe growth restric-tion of the pathogen [5]. This complex situation may parallelthat in animal systems where protective a n d toxic effects havebeen suggested for nitric oxide-related compounds [54-56].Acknowledgements:Financial support from the Spanish DirecciónGeneral de Investigación Científica y Técnica (Grants PB92-0325and P98-0734), the Comunidad de Madrid (Grant 07B/0002/1999)and the European Commission (Grant BIO 4-CT97-2120) is gratefullyacknowledged.ReferencesMcDowell, J.M. and Dangl, J.L. 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Nati. Acad. Sci. USA 90,9813-9817. [ Pobierz całość w formacie PDF ] |