Pathogen-induced Programmed Cell Deathin Plants, a Possible Defense MechanismRON MITTLER, OLGA DEL POZO, LEE MEISEL, AND ERIC LAMCenter for Agricultural Molecular Biology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey

ABSTRACT As much as the definition of life maybe controversial, the definition of death also may proveproblematic. In recent years it became apparent that thedeath of a living cell may follow more than one possiblescenario: it may result from an externally applied physi-cal injury (an accidental death), or it may be the outcomeof activating an internal pathway for cell suicide (aprogrammed death). That cells can participate in theirown execution may indicate that certain types of celldeaths that were previously considered to be caused byforeign agents such as pathogens or drugs may actuallyresult from the activation of a programmed cell deathpathway that is normally latent in cells. Here, wedescribe the activation of such a cell suicide pathway inplant cells upon the recognition of an invading patho-gen. We discuss the possible use of this pathway as adefense mechanism against infection and the possibilitythat in many ways the use of this type of cell death inplants is functionally analogous to that used by mamma-lian cells in response to infection by pathogens. Dev.Genet. 21:279–289, 1997. r 1997 Wiley-Liss, Inc.

Key words: apoptosis; hypersensitive response; patho-gen; plant; programmed cell death

INTRODUCTIONThe term ‘‘programmed cell death’’ (pcd) is used to

describe cell death that results from the activation of acell suicide pathway that is encoded by the genome ofthe dying cell [Vaux, 1993; Martin et al., 1994]. As thisterm implies, living cells contain a battery of cellularcomponents aimed at their own self-execution anddismantling. These mechanisms include lytic enzymessuch as proteases, nucleases, and lipases that functionduring or following the execution phase and a set ofregulatory proteins that control the activation of thislethal pathway [Korsmeyer, 1995]. It has been demon-strated that in at least some cases the essential path-way for pcd is always present, albeit suppressed by aparticular signal. Therefore, in these examples death ofcells is the immediate default option for the removal ofthis signal [Raff, 1992]. In other cases, the induction ofpcd requires the de novo synthesis of proteins, indicatingthat not all components of the cell death signaling orexecution machinery are present in the cell prior to theactivation of pcd [Schwartzman and Cidlowski, 1993].

Pcd is used as a mechanism to control the develop-ment and morphogenesis of multicellular organisms[Ellis and Horvitz, 1984]. This control is achieved byactivating pcd in unwanted or misplaced cells and inparticular structures or organs. However, pcd is alsothought to occur in unicellular organisms such asDictyostelium [Cornillon et al., 1994] and even bacteria[Yarmolinsky, 1995]. Therefore, the presence of a ge-netic program for a cell suicide pathway may be univer-sal to all organisms. A variety of genes that are thoughtto be involved in pcd have been isolated from animals.These include genes that are required to activate pcdsuch as Ced-3, Ced-4, and Bax and genes that inhibitpcd such as Ced-9 and Bcl-2 [Ellis and Horvitz, 1984;Korsmeyer, 1995].

In addition to its function during development andmorphogenesis, pcd is also used as a defense mecha-nism against infectious pathogens, cancer, and environ-mental insults [Schwartzman and Cidlowski, 1993].Thus in animals pcd may be activated in virus-infectedcells in order to prevent further proliferation of thepathogen. As a consequence some animal viruses ac-quired genes such as the adenovirus E1B-19 gene, thebaculovirus P35 and IAP genes, and the cowpox CrmAgene that inhibit pcd in the host cells [Henderson et al.,1991; Lakshmi et al., 1992; Bump et al., 1995; Ray andPickup, 1996]. Pcd also may be activated in trans-formed cells in order to prevent the development ofcancer. A classic example for this process was discov-ered when it was found that in B-cell lymphoma lines,the overexpression of a particular gene (Bcl-2) pro-tected cells against pcd [Korsmeyer, 1995]. Environmen-tal insults such as drugs and high energy radiation that

Contract grant sponsors: New Jersey Agricultural Experiment Sta-tion, The New Jersey Commission of Science and Technology, and TheU.S. Department of Agriculture.

Ron Mittler is now at the Department of Plant Sciences, HebrewUniversity of Jerusalem, Jerusalem 91904, Israel.

*Correspondence to: Eric Lam, Center for Agricultural MolecularBiology, Rutgers University, Foran Hall, Dudley Rd., Cook College, Bx231, New Brunswick, NJ 08903-0231. E-mail: lam@aesop.rutgers.edu

Received for publication 1 October 1996; accepted 5 June 1997.

DEVELOPMENTAL GENETICS 21:279–289 (1997)

r 1997 WILEY-LISS, INC.

induce DNA damage also were shown to activate pcd ina pathway that involves the p53 protein [Clarke et al.,1993]. It was suggested that this mechanism alsoprevents cancer that may result from DNA damage.However, despite the extensive research aimed at eluci-dating the mechanisms underlying pcd, the exact causeof cell death during pcd remains to be defined.

The distinction between ‘‘accidental’’ or ‘‘programmed’’death is not always clear since external insults thatwere typically considered to induce accidental deathmay in fact cause the activation of the internal cellsuicide pathway. Also, the activation of pcd may resultin cell death that in some cases is almost phenotypicallyindistinguishable from accidental cell death. Althoughit seems that the use of specific molecular and morpho-logical markers may make it possible to distinguishbetween ‘‘programmed’’ or ‘‘accidental’’ cell death, at-tempts to use such markers to define cell death in avariety of different experimental systems revealed thatthere are no such universal markers at present. Someof the best studied markers for pcd include the fragmen-tation of nuclear DNA and the specific ultrastructuralchanges that accompany pcd. Based on these character-istics, a particular type of pcd was defined and namedapoptosis [Kerr et al., 1972; Wyllie et al., 1984]. Apopto-sis is characterized by internucleosomal cleavage ofnuclear DNA (the formation of fragments that aremultimers of ,180 bp), the condensation of nuclearmaterial and fragmentation of the nuclei, and theformation of vesicles that contain cellular material(apoptotic bodies). These characteristics can be distin-guished from cell death that is caused by a physicalinjury in which the cell looses its capability to maintainproper compartmentalization (sometimes referred to as‘‘necrosis’’). This type of cell death is typically accompa-nied by swelling of cells and rapid leakage of cellularcontent [Kerr, 1995]. However, recent studies revealedthat the definition of apoptosis may not suite all typesof pcd [Schwartz et al., 1993]. Thus it appears that thecontext of cell death may determine the way by which acell dies and hence its cell death characteristics. Forexample, pcd of a neuron during development shouldnot induce an inflammatory response and thereforeshould not result in leakage of cellular content. There-fore, this type of cell death is likely to utilize anapoptotic-type mechanism for cell death in which thecellular content is packaged into apoptotic bodies untilit is engulfed by neighboring cells or macrophages.However, the death of a cell in an organism that doesnot have an immune system such as a plant may notrequire the formation of apoptotic bodies. Moreover, thepresence of the plant cell wall will prevent the engulf-ment of apoptotic bodies by other plant cells. It there-fore appears that the cause and requirements of death,and not the presence or absence of specific morphologi-cal or biochemical markers, should be used as the maincriteria for defining pcd.

In plants, pcd is thought to be activated during thecourse of many developmental processes and in re-sponse to attack by pathogens. The involvement of pcdin plant development and morphogenesis has been thesubject of several recent reviews [Greenberg, 1996;Jones and Dangl, 1996; Fukuda, 1996] and is notaddressed here. Instead, we address the possible in-volvement of pcd in the defense of plants againstinvading pathogens as well as present data demonstrat-ing that the activation of pathogen-induced cell deathcan be uncoupled from the induction of other defensemechanisms during the interaction of Arabidopsisplants with a bacterial pathogen.

MATERIALS AND METHODS

Plant Material and Infection With Pathogens

Fully expanded leaves of 3- to 4-week-old Arabidopsisthaliana plants (ecotype Colombia) were infiltratedwith the bacterial pathogen Pseudomonas syringae pvmaculicola strain ES4326 (Psm ES4326; 105 CFU/cm2)that does not induce an HR, Pseudomonas syringae pvmaculicola strain ES4326 that contains the avrRpt2gene (Psm ES4326/avrRpt2; 105 CFU/cm2) and inducesan HR, or mock infected with 10 mM MgSO4 asdescribed by Greenberg et al. [1994]. Plants were keptat 16°C or 22°C under continuous illumination pro-vided by cool-white fluorescent lamps (100 µmol m22

sec21). Sixteen hours after inoculation, leaves weresampled, photographed, and analyzed. Bacteria weregrown in King’s Broth medium as described by Mindri-nos et al. [1994]. Bacteria was also infiltrated intoleaves in the presence of 1 mM lanthanum chloride(LaCl3), 1.4 3 1024 M cycloheximide, or 1–5 mMphenylmethylsulfonyl fluoride (PMSF; freshly pre-pared immediately prior to infiltration).

RNA Isolation and Analysis

Total RNA was isolated as previously described [Mit-tler et al., 1995] and subjected to RNA gel blot analysis.RNA gel blots were first hybridized with the various PRprobes (PR-1, PR-2, and PR-5) [Glazebrook et al., 1996]and then with a probe for the 18S rRNA [Mittler andZilinskas, 1994]. RNA gel blot hybridization and mem-brane washing were performed using Duralose-UVmembranes and QuickHyb solution (Stratagene,LaJolla, CA), as suggested by the manufacturer.

Protein Isolation and Immunodetection

Immunodetection of tubulin was performed by pro-tein blot analysis of total leaf protein with a chemilumi-nescence detection system (Renaissance kit from Du-Pont, Wilmington, DE) as previously described [Mittleret al., 1995]. Anti-tubulin antibodies were purchasedfrom Sigma (St. Louis, MO) (T-4026).

Field Inversion Gel Electrophoresis

Field inversion gel electrophoresis was performed asdescribed by Mittler et al. [1997]. Briefly, tissue samples

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(0.2 g) were ground to a fine powder in liquid nitrogen.The powder was resuspended in 1 mL 1 3 ET buffer (10mM Tris-HCl, pH 8.0, 50 mM EDTA) preheated to 50°Cand mixed with 1 mL 1.4% (w/v) low-melting-pointagarose (Bethesda Research Laboratories, Bethesda,MD) in 1 3 ET preheated to 50°C. Agarose plugs werepoured in a Bio-Rad (Richmond, CA) standard mold.Plugs were incubated for 3 hours in 10 mM Tris-HCl,pH 8.0, 0.5 M EDTA, 1% sarcosyl, and 1 mg/mLproteinase K (Promega) at 52–54°C with gentle agita-tion. Plugs were then washed with 1 3 ET and stored at4°C in 1 3 ET. Field inversion gel electrophoresis wasperformed with a Bio-Rad CHEF Mapper apparatususing 1% agarose (Bethesda Research Laboratories)gels and 0.5 3 TBE buffer (45 mM Tris-borate, 1 mMEDTA, pH 8.0). Electrophoretic separation was per-formed with a 6 V/cm voltage gradient at an angle of120° and a linear ramping factor with alternating pulsetime of 5.3–20.5 seconds over the course of 18 hours.Following electrophoretic separation, gels were depuri-nated with UV light or 0.25 N HCl, washed withdistilled water, and incubated in 0.4 N NaOH, 1.5 MNaCl for 20 minutes. DNA was transferred to a nylonmembrane (Zeta-Probe GT, Bio-Rad) by the capillarytransfer method using 0.4 N NaOH, 1.5 M NaCl for 48hours. The membrane was briefly washed in 2 3 SSC(300 mM NaCl, 30 mM Na-citrate, pH 7.0) and baked ina vacuum oven at 80°C for 45 minutes. Hybridizationwith nuclear specific probes was performed as previ-ously described [Mittler et al., 1997].

RESULTS AND DISCUSSION

Pathogen-induced ProgrammedCell Death in Plants

Plants contain an arsenal of defense mechanismsaimed at combating pathogen infection. These includepreexisting structural and chemical barriers as well aspathogen-induced antimicrobial defenses. Although theaction of the preexisting barriers is sufficient to blockinfection by most pathogens, the inhibition of patho-gens that penetrate these barriers requires the timelyactivation of induced defense mechanisms. However,the induction of these mechanisms depends in turnupon the recognition of the invading pathogen by theplant cell. Thus a plant that can recognize the pathogenwill mount a rapid defense response that will preventpathogen proliferation (a resistant plant), whereas aplant that is incapable of recognizing the pathogen willnot mount a defense response, or will do so only slowly,and will be unsuccessful in preventing pathogen prolif-eration (a susceptible plant). This crucial step of recog-nition is traditionally described by the ‘‘gene-for-gene’’or ‘‘corresponding gene’’ model [Keen, 1990; de Wit,1992]. In this model, an avirulence factor encoded, orproduced, by an Avr gene in the pathogen can bespecifically recognized by a resistance (R) gene productin the host plant. When a pathogen carrying a particu-

lar Avr gene invades a host plant carrying the corre-sponding R gene, its recognition triggers the rapidactivation of defense mechanisms and the proliferationof the microbe within the plant is inhibited. This istermed an ‘‘incompatible’’ interaction. In the absence ofeither the Avr or its corresponding R gene, the patho-gen is apparently not recognized by the host and canproliferate within the plant leading to the appearanceof disease symptoms. This is termed a ‘‘compatible’’interaction and usually signifies susceptibility of aplant to the particular pathogen. Recently, a variety ofbacterial, viral and fungal Avr genes, as well as someplant encoded R genes, were cloned and characterized[Bent et al., 1994; Hammond-Kosack et al., 1994; Joneset al., 1994; Mindrinos et al., 1994; Whitham et al.,1994; Boyes et al., 1996). However, their modes of actionremain to be elucidated.

During an incompatible interaction, the recognitionof an invading pathogen activates a signal transductionpathway that results in the rapid induction of manyplant defense mechanisms. These include the accumu-lation of salicylic acid (SA), a key mediator of defenseresponse activation, synthesis of pathogenesis-related(PR) proteins that exhibit antimicrobial activity such asglucanase and chitinase, thickening and hardening ofcell walls, and an increased production of antimicrobialcompounds called phytoalexins [Bowles, 1990; Bradleyet al., 1992; Glazebrook and Ausubel, 1994; Linthorst,1991; Malamy et al., 1990; Metraux et al., 1990].Although some of these defense mechanisms also maybe activated during a compatible interaction, their rateof activation is typically slower, thus rendering themineffective in pathogen growth inhibition.

In some incompatible interactions, the activation ofdefense mechanisms is accompanied by the induction ofa rapid cell death process that results in the formationof a zone of dead cells around the infection site. Thiszone of dead cells, also called a ‘‘hypersensitive re-sponse (HR) lesion,’’ is thought to provide a physicalbarrier against further spread of pathogens. The deadcells surrounding the infection site also may serve as asink for the deposition of antimicrobial compounds thatare produced in response to pathogen infection. Sincecell death occurring during the HR is very rapid, it mayfunction to limit or delay the spread of the pathogenduring early stages of the defense response in order toallow the timely induction of other defense mechanismssuch as synthesis of PR proteins and production ofphytoalexins [Goodman and Novacky, 1994].

The compatible/incompatible interaction betweenPseudomonas syringae and Arabidopsis thaliana isillustrated in Figure 1. Arabidopsis plants of the ec-otype Colombia contain the resistance (R) gene Rps2[Bent et al., 1994; Mindrinos et al., 1994]. These plantscan recognize Pseudomonas strains that contain theavirulance gene avrRpt2 and mount an HR (Fig. 1C).However, they are unable to recognize a Pseudomonasstrain that does not contain the avrRpt2 gene and are

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therefore unable to mount a defense response in theform of an HR (Fig. 1B). In addition, as shown in Figure2, infection of Arabidopsis plants that contain the Rgene with a Pseudomonas strain that contains the

avrRpt2 gene results in an increase in the steady-statelevel of transcripts encoding the PR proteins PR-1,PR-2, and PR-5. These PR proteins were shown to haveantimicrobial activity and are part of the defense

Fig. 1. Activation of pcd in Arabidopsis thaliana plants thatcontain the RPS2 gene by bacteria containing the avrRpt2 gene. A.Control Arabidopsis leaf mock-infiltrated with 10 mM MgSO4. B.Arabidopsis leaf infiltrated with the bacterial pathogen Pseudomonassyringae pv maculicola strain ES4326 (Psm ES4326), that does notcontain the avrRpt2 gene, showing that this pathogen does not inducepcd. C. Infiltration of an Arabidopsis leaf with the bacterial pathogenPsm E4326 that contains the avrRpt2 gene (Psm ES4326/avrRpt2)showing that the recognition of the avrRpt2 gene by Arabidopsisplants, which contain the RPS2 gene, results in the activation of pcd.D. Infiltration of Arabidopsis with Psm ES4326/avrRpt2 in the

presence of 1 mM lanthanum chloride (LaCl3) showing that inhibitionof Ca21 flux results in the suppression of bacteria-induced pcd. E.Infiltration of Arabidopsis with Psm ES4326/avrRpt2 in the presenceof 1.4 3 1024 M cycloheximide showing that de novo protein synthesisis required for the activation of bacteria-induced pcd. F. Infiltration ofArabidopsis with Psm ES4326/avrRpt2 in the presence of 1 mMphenylmethylsulfonyl fluoride (PMSF) indicating that bacteria-induced pcd requires the activity of serine proteases. Arabidopsisleaves (ecotype Colombia) were mock infiltrated with MgSO4 orinfected with the different bacteria in the presence or absence of thedifferent inhibitors, sampled and photographed 16 h postinoculation.

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response activated upon recognition of pathogens [Law-ton et al., 1993; Dietrich et al., 1994; Greenberg et al.,1994]. Thus the recognition of the invading pathogen,which is governed by the genetic interaction betweenthe plant Rps2 gene and the bacterial avrRpt2 gene,triggers the coordinated activation of cell death anddefense mechanisms. In contrast, infection of plantswith a Pseudomonas strain that does not contain theavrRpt2 gene does not result in a coordinated activationof pcd and defense mechanisms and only leads to theinduction of PR-proteins (Figs. 1B, 2).

Several lines of evidence suggest that cell death thatoccurs during the HR results from the activation of anintrinsic cell death program that is encoded by theplant genome [Greenberg, 1996; Jones and Dangl,1996; Mittler and Lam, 1996]. These include the activa-tion of cell death in the absence of pathogens by certainelicitors [He et al., 1993; Hammond-Kosack et al., 1994;Levine et al., 1994], by expression of different foreigngenes [Takahashi et al., 1989; Becker et al., 1993;Mittler et al., 1995] and as a result of mutations incertain genes that are thought to be involved in the celldeath pathway [Walbot et al., 1983; Wolter et al., 1993;

Dietrich et al., 1994; Greenberg et al., 1994]. That celldeath resembling the HR can be activated in theabsence of a pathogen strongly suggests that this typeof cell death is not directly caused by the invadingpathogen, but rather results from the activation of ahost-encoded pathway for cell death (i.e., pcd). More-over, the HR was shown to require active plant metabo-lism and to depend on the activity of the host transcrip-tion and translation machinery [He et al., 1993, 1994].These findings further imply that genes that are en-coded by the plant genome are actively involved in thedeath of plant cells during the HR. In accordance withthe possible role of plant genes in bacteria-induced pcdin Arabidopsis, cyclohexamide, an inhibitor of eukary-otic protein synthesis is shown in Figure 1E to inhibitthis pcd process.

The isolation and characterization of mutants thatdevelop HR lesions in the absence of a pathogen is ofparticular interest to the study of pcd in plants. Thesemutants, also referred to as ‘‘disease lesion mimics,’’provide strong evidence for the existence of genes thatregulate pcd in plants. Thus mutations in these genesresult in the abnormal activation or suppression ofpathogen-induced pcd [Greenberg and Ausubel, 1993;Dietrich et al., 1994; Greenberg et al., 1994]. Diseaselesion mimic mutants were isolated from maize, barley,and Arabidopsis and were classified into two majorgroups: initiation mutants and feedback or propagationmutants. Initiation mutants form lesions with a definedborder. They are thought to be defective in regulatingthe trigger of the HR and may either lack a negativeregulator of cell death activation (recessive mutations),or constitutively activate a cell death signal (dominantmutations). Propagation mutants form lesions thatspread indeterminately. They are presumed to be defec-tive in down-regulating the process of cell death in cellssurrounding a developing lesion (recessive mutations).

The cloning and characterization of the genes respon-sible for these different lesion-mimic phenotypes areperhaps one of the most important steps toward under-standing the process of pcd in plants. Recently thecloning of three plant genes that regulate pathogen-induced programmed cell death was reported. Dietrichet al. [1997] reported the cloning of the LSD1 gene fromArabidopsis, Buschges et al. [1997] reported the cloningof the Mlo gene from barley, and Gray et al. [1997]reported the cloning of the Lls1 gene from maize. TheLSD1 gene, which is a negative regulator of cell death,was found to encode a novel zinc finger protein. It wassuggested that LSD1 regulates transcription and eitherrepresses a prodeath pathway or activates an antideathpathway, in response to signals such as superoxide thatare produced by cells undergoing HR cell death. TheMlo gene, which is also thought to be a negativeregulator of HR cell death, was found to encode a novelprotein that contains six membrane spanning helices.The Lls1 gene, which limits the spread of cell death inmature leaves (i.e., a suppressor of cell death), encodesa novel protein that is thought to degrade phenolic

Fig. 2. Induction of pathogenesis-related proteins (PR) proteins canbe uncoupled from bacteria-induced pcd in Arabidopsis by the inhibi-tion of Ca21 flux or serine protease activity. Total RNA was isolatedfrom Arabidopsis leaves infiltrated with MgCl2, Psm ES4326 (Psm),Psm ES4326/avrRpt2 (Psm/avrRpt2), or Psm ES4326/avrRpt2 in thepresence of lanthanum chloride (LaCl3), cycloheximide (CHX), orphenylmethylsulfonyl fluoride (PMSF) as described in the legend forFigure 1. Total RNA was also isolated from Arabidopsis plants treatedfor 24 h with 1 mM salicylic acid (SA), which causes the induction ofdefense mechanisms, and from Arabidopsis leaves subjected to afreeze-thaw cycle (i.e., necrotic cell death control). Total RNA from thedifferent treatments was subjected to RNA gel blot analysis withprobes for Arabidopsis PR-1, PR-2, and PR-5. A probe for ribosomalRNA (18S) was used to demonstrate equal loading of RNA. Infiltrationof control leaves with 1 mM LaCl3 did not result in a significantincrease in the level of transcripts encoding PR proteins (data notshown).

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compounds that may function as mediators of celldeath. The cloning of the LSD1, Mlo, and Lls1 genes islikely to provide researchers with a first entry pointinto the cell death pathway of plants. It also present thefirst solid evidence for the existence of genes thatregulate cell death in plants.

An alternative approach to the study of genes thatmay be involved in the process of pcd in plants is tostudy the effect of overexpressing particular animalgenes that are known to be involved in regulating pcd.Transgenic plants can be made to express these genes,and the effect of their expression can be studied onseveral developmental processes such as the differentia-tion of xylem [Fukuda, 1996; Mittler and Lam, 1995b],or on the response of these plants to pathogens [Mittlerand Lam, 1996]. Of interest are genes that belong tothree different families. The first are genes that belongto the Bcl-2 family. These genes are thought to functionas key regulators of pcd in many animal cells [Boise etal., 1993; Korsmeyer, 1995]. However, the study ofgenes that belong to this family (i.e., Bcl-XL, or Bcl-2) intobacco plants revealed that their ectopic expressiondoes not cause the inhibition of pathogen-induced pcd[Mittler et al., 1996a; Chen and Klessig, pers. comm.].

The second group of animal genes that suppress pcdencode products that can inhibit ICE-like proteases.This family includes the p35 protein from baculovirusand the CrmA gene from cowpox virus [Bump et al.,1995; Ray and Pickup, 1996]. Studies on the effect ofoverexpressing these genes in plants and on the re-sponse of plants expressing these genes to pathogenattack are underway [del Pozo and Lam, unpubl. re-sults].

The third group of animal genes that inhibit pcdincludes homologs of the Defender Against Death 1(Dad1) gene [Nakashima et al., 1993; Sugimoto et al.,1995]. Dad1 was initially identified as a gene that canrescue a temperature-sensitive hamster cell line fromapoptosis. Highly conserved homologs of Dad1 werefound in human, mice, Yeast, Xenopus, Caenorhabditiselegans, and plants. Dad1 is thought to block cell deathat a step downstream from or independent of Bcl-2.However, recently it was reported that the Dad1 genemay encode one of the subunits of oligosaccharyltrans-ferase [Silberstein et al., 1995]. Further studies maytherefore be required in order to elucidate the role ofthis gene in inhibiting pcd. Studies on the effect ofoverexpressing or suppressing Dad1 in Arabidopsis areunderway in our laboratory [Meisel and Lam, unpubl.data]. The studies described above may reveal if plantsutilize pcd mechanisms that are similar to those usedby animal cells.

SIGNAL TRANSDUCTION PATHWAYS ANDPROGRAMMED CELL DEATH IN PLANTS

The signal transduction events that lead to inductionof the HR in plants are of particular interest to the

study of pathogen-induced pcd [Godiard et al., 1994;Mittler and Lam, 1996]. Some of the early eventsassociated with the HR involve rapid generation ofreactive oxygen species (ROS) in the form of H2O2, theso-called oxidative burst [Goodman and Novacky, 1994;Baker and Orlandi, 1995; Levine et al., 1994; Tenhakenet al., 1995], and a rapid flux of ions across the plasmamembrane, the so-called XR [Atkinson and Baker,1989; He et al., 1994; Nurnberger et al., 1994]. Althoughthe role of the XR in inducing the HR is not clear [He etal., 1994], the accumulation of H2O2 during early stagesof the HR was suggested to activate defense mecha-nisms and to induce pcd [Levine et al., 1994, 1996]. ROSalso have been implicated during late stages of the HR,and the HR was associated with an increase in lipidperoxidation [Goodman and Novacky, 1994]. We re-cently examined the effect of low oxygen pressure onthe HR [Mittler et al., 1996]. Under conditions of lowoxygen pressure, the production of ROS is expected tobe suppressed [Jacobson and Raff, 1995; Shimizu et al.,1995]. Indeed, under these conditions we found thatpathogen-induced pcd was inhibited. Our findings arein agreement with a recent study that shows inhibitionof thymocyte apoptosis at low oxygen pressure[McLaughlin et al., 1996], but in contrast to otherstudies performed with animal cells in which apoptosiswas not inhibited at low oxygen pressure [Jacobson andRaff, 1995; Shimizu et al., 1995]. Interestingly, theinhibition of pcd in our assay system did not result inthe inhibition of other defense mechanisms such as theinduction of PR proteins. These results suggest that pcdcan be uncoupled from the activation of other defensemechanisms during the HR. This finding is supportedby previous studies that suggested that activation ofdefense mechanisms can occur in the absence of pcd[Gross et al., 1993; Jakobek and Lindgren, 1993; Law-ton et al., 1993; Bowling et al., 1994; Hammond-Kosacket al., 1996]. Despite the strong evidence supporting therole of H2O2 in inducing pcd in plants, a recent studydemonstrated that bacteria that is incapable of induc-ing the HR can still cause the induction of the oxidative-burst [Glazener et al., 1996]. Therefore, ROS alone maybe insufficient for HR induction per se. Further studiesare required to address the role of H2O2 as a possiblemediator of the HR [Baker and Orlandi, 1995].

The signal transduction pathway that leads to theactivation of the HR was also suggested to involveincreases in Ca21 flux and protein phosphorylation[Felix et al., 1991; Levine et al., 1996]. We examined thepossible involvement of Ca21 flux in HR induction inArabidopsis. As shown in Figure 1D, lanthanum chlo-ride, a Ca21 channel blocker can inhibit bacteria-induced pcd in Arabidopsis. However, this inhibitordoes not suppress the induction of other defense mecha-nisms in the form of PR proteins (Fig. 2). Therefore, itappears that the changes in Ca21 flux that are associ-ated with the HR [Levine et al., 1996] only may beinvolved in signaling the activation of pcd, whereas the

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induction of PR proteins is mediated via a pathway thatis not inhibited by lanthanum chloride.

Although the signal transduction events that lead tothe activation of pcd and defense mechanisms duringthe HR are controlled by a single gene-for-gene interac-tion (R in the plant and Avr in the pathogen), it appearsas if two distinct pathways may be set into motion bythis recognition event. One pathway controls the activa-tion of pcd, whereas the other controls the induction ofPR proteins and perhaps other defense mechanisms.Although the pathway that controls the activation ofpcd is inhibited by lanthanum chloride and low oxygenpressure (Figs. 1 and 3) [Mittler et al., 1996], thepathway that controls the induction of PR proteins isinsensitive to these treatments (Fig. 2) [Mittler et al.,1996a]. Interestingly, de novo protein synthesis oncytoplasmic ribosomes appears to be required for bothpathways.

MOLECULAR CHARACTERISTICS OFPATHOGEN-INDUCED PROGRAMMED CELL

DEATH IN PLANTSBecause different types of pcd may be required in

different tissues or organisms, characterization of themolecular and biochemical events that accompanypathogen-induced pcd in plants may contribute to ourunderstanding of the nature and context of this process.Ultrastructural studies of plant tissues undergoingpathogen-induced pcd revealed that in general plantcells do not form apoptotic bodies [Mittler and Lam,1996]. In addition, although the nuclear material ofplant cells condenses during pcd, the nuclei do notfragment [Mittler et al., 1997; Mittler and Lam, 1996].An exception to this is a report by Wang et al. [1996] inwhich the nuclei of tomato protoplasts undergoingtoxin-induced death were shown to fragment in amanner that may be similar to the fragmentation ofnuclei during apoptosis. In addition, these authors, aswell as Levine et al. [1996], reported the detection ofapoptotic bodies in plant cells undergoing pcd. How-ever, the significance of these bodies is not clear atpresent since they were not shown to be engulfed byneighboring cells. We found that virus-induced pcd intobacco is accompanied by condensation and vacuoliza-tion of the cytoplasm and condensation of nuclearmaterial [Mittler et al., 1997]. Because it appears thatdifferent plant cells may undergo different ultrastruc-tural changes in response to different pathogens, fur-ther studies of the morphological events that accom-pany pathogen-induced pcd in plants are needed toextend our understanding of this process and thesignificance of the particular morphological changes tothe specific plant and pathogen [Jones and Dangl, 1996;Mittler and Lam, 1996].

Studies on the biochemical processes that accompanypathogen-induced pcd in plants are currently inprogress. Two aspects of pcd that have been extensively

studied in animals are also being investigated in plants.These are the changes in the integrity of nuclear DNAthat may be mediated by specific nucleases and theprocessing of certain proteins by specific proteases (seebelow). In animals, the degradation of nuclear DNA byparticular nucleases is considered a late and irrevers-ible stage of pcd [Peitsch et al., 1994; Bortner et al.,1995]. The degradation of nuclear DNA during pcd inanimals is thought to occur via two independent mecha-nisms: (1) cleavage of chromatin into large DNA frag-ments of ,300 and/or 50 kb, (2) digestion of DNA tosmaller fragments. Digestion of DNA into small frag-ments may occur by specific cleavage between nucleo-somes that yield fragments that are multimers of ,180bp, or by a nonspecific degradation that typically re-sults in a random pattern of DNA fragments. Thedifference between these two mechanisms may resultfrom the timing of the proteolytic digestion of proteinsthat comprise the nucleosomes. If these proteins aredigested before the nucleases are activated, then thepattern of DNA degradation will be random. However,if the nucleases are activated before the proteolyticdigestion of nucleosomes, then they will only cleave theDNA between nucleosomes, and the resulting DNAfragments will be multimers of ,180 bp. In plants, bothrandom and ordered DNA degradation were reported.The degradation of DNA during virus-induced pcd intobacco and bacteria-induced pcd in soybean resulted inthe formation of large DNA fragments of ,50 kb[Levine et al., 1996; Mittler et al., 1997]. However,further degradation of DNA occurred in a randommanner that did not result in the formation of DNAladders [Mittler and Lam, 1995a; Levine et al., 1996].In contrast, toxin- and fungal-induced cell death intomato and cowpea was accompanied by the formationof fragments that are multimers of ,180 bp [Reyersonand Heath, 1996; Wang et al., 1996]. An interesting, butas yet little studied observation, is the fate of DNA,which is localized in organelles. In plants the chloro-plast DNA is thought to exist as a mixture of multimers(the monomeric size of the tobacco genome being 155kb). We found that during virus-induced pcd in tobacco,the chloroplast genome undergoes structural changesthat resulted in a dramatic increase in the number ofmonomeric chloroplast DNA, which can be visualizedas a fragment of ,155 kb that specifically hybridizedwith chloroplast-specific probes [Mittler et al., 1997].

Virus-induced pcd in tobacco is accompanied by induc-tion of Ca21-dependent nucleases that may mediate thedegradation of nuclear DNA during this process [Mit-tler and Lam, 1995a]. These nucleases were found innuclei isolated from cells undergoing pcd as well as inthe intercellular fluid of leaves that contain HR lesions[Mittler and Lam, 1997]. Although the relationshipbetween the increase in nuclease activities and thedegradation of nuclear DNA was not directly demon-strated, it is possible that nuclear localized nucleasesparticipate in the initial degradation of nuclear DNA

PATHOGEN-INDUCED PCD IN PLANTS 285

during early stages of the HR (detected by TUNELstaining) [Mittler et al., 1995]. The secreted nucleasesthat are found in the intercellular fluid of leaves mayparticipate in the more extensive degradation of DNAthat occurs late during pcd when the integrity of the cellmembrane is compromised [Mittler et al., 1997]. Asshown in Figure 3A, bacteria-induced pcd in Arabidop-sis is accompanied by the formation of DNA fragmentsof ,50 kb. Cleavage of DNA is not observed followinginfection with a bacterial strain that does not inducethe HR (Figs. 1B, 3A) and in leaves that were infectedwith bacteria in the presence of lanthanum chloridewhich inhibits pcd (Figs. 1D, 3A). These results supportthe correlation between pathogen-induced pcd and thecleavage of nuclear DNA into fragments of ,50 kb.

During the process of pcd in animals, a cascade ofproteases is activated [Fraser and Evan, 1996]. Theseare substrate-specific proteases that are thought to beinvolved in mediating the activation of pcd. Thus asignal for cell death that is perceived by the cell causesthe activation of a specific initiator protease, whichcleaves and activates a set of amplifier proteases that inturn cleave and activate another set of machineryexecution proteases. The activation of this cascaderesults in the proteolytic cleavage of many cellularproteins and the induction of pcd. Some of the knownsubstrates of this cascade include poly(ADP-ribosyl)polymerase (PARP), actin, tubulin, fodrin, and lamin.The possible involvement of protease activity in patho-

gen-induced pcd in plants was suggested by Levine etal. [1996], who demonstrated that treatment of soybeancells with a serine proteinase inhibitor (AEBSF) sup-pressed pcd that is induced by H2O2, or a calciumionophore. Here we show that phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor, inhibitsbacteria-induced pcd in Arabidopsis (Fig. 1F). Interest-ingly, PMSF does not suppress the induction of PRproteins (Fig. 2). This finding further suggests that thepathway for pcd may be distinct from the pathway thatcontrols induction of PR proteins (described above). Wealso examined the proteolytic degradation of tubulinduring bacteria-induced pcd in Arabidopsis. As shownin Figure 3B, pcd induced by bacteria that contains theavrRpt2 gene is accompanied by proteolytic digestion oftubulin, as visualized by the appearance of a smallmolecular weight peptide that cross-reacted with ananti-tubulin monoclonal antibody. A similar digestion isnot observed following necrotic cell death, which isinduced by freezing and thawing plant tissue (freeze-thaw, Fig. 3B). Thus the proteolytic digestion of tubulinappears to be specific to pcd, which is activated uponpathogen recognition.

That protease inhibitors can inhibit pathogen-induced pcd in plants suggests that the activation ofproteases is an important step in the induction of thisprocess in plants as well. It is not known, however,whether a cascade of proteases that is similar to thatactivated during pcd in animal cells is also involved in

Fig. 3. Fragmentation of nuclear DNA and proteolytic degradationof tubulin during bacteria-induced pcd in Arabidopsis. A. A DNA blotof a field inversion gel electrophoresis (FIGE) gel probed with amixture of nuclear gene probes showing that bacteria (Psm/avrRpt2)-induced pcd is accompanied by the cleavage of nuclear DNA intofragments of ,50 kb. This cleavage is specific for the recognition of theavrRp52 gene and is inhibited by lanthanum chloride (LaCl3) which

suppresses pcd (Fig. 1). B. A protein blot probed with an anti-tubulinserum showing that bacteria (Psm/avrRpt2)-induced pcd is accompa-nied by the degradation of tubulin (arrow head). Arabidopsis plantswere treated as described in the legends for Figure 1 and 2 andsampled and processed for FIGE, DNA blots, and protein blots asdescribed in Materials and Methods.

286 MITTLER ET AL.

pathogen-induced pcd in plants. Our studies with thebaculovirus p35 protein may help to address this ques-tion in the near future.

SUMMARYEvidence supporting the involvement of a pcd path-

way in the response of plants to infection by pathogens(i.e., the HR) is mounting. These include the isolation ofmutants that correspond to genes that are responsiblefor pathogen-induced pcd, as well as biochemical andultrastructural characterizations. Although it appearsthat the HR may use a pcd machinery that shares somebiochemical and ultrastructural characteristics withpcd in animals, it is not clear whether this process issimilar to that used by animals. With the exception ofthe Dad1 gene, no plant homologs of other animal genesthat are involved in pcd (i.e., Bcl-2, Ced-3, Ced-4, . . .)have been found yet. It is therefore possible that plantsevolved a pcd machinery that is distinct from that usedby animals. This may not be surprising since therequirements from a pcd pathway in plants may bedifferent from that of a pcd pathway in animals.However, although the specific requirements for the celldeath machinery may be different between plants andanimals, functionally the process of pathogen-inducedpcd appears to be used in a similar manner. Thus pcdmay be activated in infected plant and animal cells inorder to prevent further proliferation of the invadingpathogen by killing and dismantling the infected cellprior to the successful multiplication of the pathogen[Zychlinsky et al., 1992; Mittler and Lam, 1996].

Although it is becoming apparent that the HR is atype of pcd, elucidating the role of this response in thedefense of plants against pathogens will require furtherstudies. For example, it is not known whether pathogen-induced activation of defense mechanisms in the ab-sence of pcd (i.e., when pcd is suppressed) will result ininhibition of pathogen growth. In addition, it is notknown whether a reverse situation in which pcd isactivated in the absence of defense mechanisms willresult in inhibition of pathogen growth. Two mutants ofArabidopsis that may correspond to this reverse situa-tion are acd1 and NDR1 [Greenberg and Ausubel, 1993;Century et al., 1995]. In these mutants, cell death isactivated in response to pathogen infection. However,this activation is not sufficient to block the proliferationof the pathogen. Drawing on this limited amount ofinformation regarding the role of pcd in the defense ofplants against invading pathogens, it appears that thecoordinated activation of pcd and defense mechanismsis required in order to fully optimize the effectiveness ofthe resistance response.

The use of Arabidopsis plants for the study of patho-gen-induced pcd may open the way to the rapid identifi-cation, characterization, and cloning of genes involvedin this process [Buschges et al., 1997; Dietrich et al.,1997]. Thus in addition to the cloning of genes respon-

sible for the ‘‘disease lesion mimic’’ phenotypes, screen-ing for mutations that result in the suppression of pcdalso may lead to the identification of genes that areresponsible for the activation or suppression of thisprocess. For example, a mutated population of Arabidop-sis plants can be infected with a pathogen that inducespcd and screened for mutations that result in thesuppression of pathogen-induced cell death. Alterna-tively, Arabidopsis plants can be made to express a genethat activates pcd [Mittler et al., 1995]. A homozygouspopulation of these plants can then be mutated andscreened for suppression of pcd, thus identifying extra-genic suppressor loci that may be required for pcdactivation or execution. Eventually, through the cross-ing of different Arabidopsis mutants, an ordered path-way for pathogen-induced pcd in plants may be con-structed and the genetic relationships between differentcell death phenomena elucidated. Therefore, due totheir small and well-mapped genome, Arabidopsisplants should significantly contribute to the study ofpcd in plants, perhaps as much as C. elegans hascontributed to the study of pcd in animals [Ellis andHorvitz, 1986].

ACKNOWLEDGMENTSWe thank Drs. Daniel Klessig, Frederick Ausubel,

Jane Glazebrook, Fumiaki Katagiri, and John Ryals forgifts of plasmids and bacterial strains. This work wassupported in part by the New Jersey AgriculturalExperiment Station, the New Jersey Commission ofScience and Technology, and the U.S. Department ofAgriculture.

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