Plant Physiol. (1990) 921, 276-2800032-0889/90/92/0276/05/$01 .00/0

Received for publication August 31, 1989and in revised form October 12, 1989

Communication

Differential Activation of Expression of a Suberization-Associated Anionic Peroxidase Gene in Near-IsogenicResistant and Susceptible Tomato Lines by Elicitors of

Verticillium albo-atratrum'

Royce Mohan and Pappachan E. Kolattukudy*

The Ohio State Biotechnology Center and Biochemistry Program, The Ohio State University,Columbus, Ohio 43210

ABSTRACT

We tested whether the expression of the suberization-associ-ated anionic peroxidase gene is involved in the timely appearanceof the vascular suberized coating involved in the resistance of atomato line to Verticillium albo-atrum. The mRNA for this peroxi-dase appeared at a higher level one day earlier in wound-healingfruits of the resistant tomato line than in a near-isogenic suscep-tible line. Cell cultures from the resistant line, when treated withlow levels (nanograms per milliliter) of fungal elicitor, generatedthe peroxidase mRNA and this apparent activation of the peroxi-dase gene expression could be detected in minutes, whereas thecells from the susceptible line hardly responded.

possess inherent differences in their ability to respond to thefungal attack. Resistance, which was found to be from a singledominant Mendelian gene (25), was shown to be associatedwith a timely deposition of vascular coating on the walls ofthe infected cells (27). The chemical composition of thisvascular coating showed it to be suberin ( 18). Since suberiza-tion has been found to be regulated by a highly anionicperoxidase (17, 24), it appeared possible that the resistanceresponse might involve induction of this suberization-associ-ated peroxidase, and the two cell lines might differ in theirability to respond to fungal signals by triggering the peroxidasegene expression. In this report, we show that wounding andfungal elicitors induce the peroxidase mRNA earlier and to ahigher level in the resistant tomato line than in the susceptibleline.

Peroxidases have been implicated in many diverse physio-logical processes such as dormancy, organogenesis, aging,lignification and suberization (12). Such enzymes probablycatalyze the polymerization of the phenolic components oflignin (13, 20) and suberin (2, 17). The deposition of suberinmonomers in wound-healing potato (Solanum tuberosum)tuber slices was shown to be associated with induction of ahighly anionic peroxidase (2, 7). Immunocytochemical local-ization showed that this peroxidase was induced only duringthe time of suberization and was located exclusively in thewalls ofthe suberizing cells ofthe wound-healing potato tissue(8). The cDNA for this peroxidase from potato was clonedand sequenced (24), and it was found to be highly homologousto the tomato peroxidase gene (23). The peroxidase mRNAwas shown to be induced in wound-healing potato tubers andtomato fruits (24).

Suberization has been suggested to be a defense responsethat reinforces the cell walls and thus limits ingress of patho-gens into the host (5, 16, 19, 21, 28). However, conclusiveevidence for this hypothesis is not available. The fungalpathogen, Verticillium sp., causes vascular wilts in a varietyof plants. Two near isogenic tomato lines that differed greatlyin their sensitivity to Verticillium albo-atrum were shown to

'This work was supported in part by the National Science Foun-dation grant DCB-88 19008.

MATERIALS AND METHODS

Isolation of Elicitor from Culture Filtrate of Verticilliumalbo-atrum

Verticillium albo-atrum was maintained on potato dextroseagar plates at 2 1°C. For liquid cultures, 100 mL ofT8 mineralmedia (15), supplemented with 2% dextrose (w/v) and 0.1%yeast extract (w/v), was inoculated with the fungal spores, andgrown in an Erlenmeyer flask shaking at 100 rpm for 24 h at21°C. This culture was used to inoculate 1 L of the samemedium, and the culture was grown for 48 h as mentionedabove. Elicitor fraction was prepared from the culture filtrateas described (1). The carbohydrate content of the elicitorpreparation was quantitated as hexose equivalents by theanthrone method (6).

Plant Materials and Wounding Experiment

The tomato plants (Lycopersicon esculentum) were grownfrom seed in a green house. Young green tomato fruits fromwhich the epidermal layer was excised were incubated in amoist chamber at 26°C (24). The fruits were allowed towound-heal for 0, 1, 2, and 3 d and, the wound-periderm wasused to prepare RNA.

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ANIONIC PEROXIDASE GENE EXPRESSION IN FUNGAL RESISTANCE

Tomato Cell Suspension Cultures

Cultured cells of near-isogenic tomato (L. esculentum) Crai-gella susceptible (S-GCR 26) and Craigella resistant (R-GCR218) were obtained from Dr. Brian E. Ellis (University ofGuelph, Ontario, Canada) as callus cultures on agar platescontaining Gamborg's B-5 medium supplemented with 3%sucrose (w/v), 1.0 mg/L 2,4-D, and 0.1 mg/L kinetin. Thecallus cultures were transferred into 125 mL Erlenmeyer flaskscontaining the same growth medium and shaken at 120 rpmunder fluorescent lights at 250C. The fine suspension cellswere transferred every 12 d to fresh media and were thussubcultured through 8 to 10 cycles. For the induction exper-iments, the cells were subcultured for different periods oftimeprior to incubation with the fungal elicitor to determine theinfluence of culture age on peroxidase induction; all of thedata presented in this communication were from 4-d-oldcultures.

Induction Experiment

Cultured tomato cells (5 mL containing 2 g wet weight ofcells) were transferred into 45 mL fresh medium in 250 mLErlenmeyer flasks and allowed to grow for 24 h, and subse-quently they were incubated with different amounts of thefungal elicitor for different periods of time. The amount ofelicitor used is expressed in hexose equivalents per millilitercell culture volume. The cells were recovered by centrifugationat 6000g for 5 min in a swinging bucket rotor (HS-4, Sorvall)and immediately frozen in liquid nitrogen.

Isolation of RNA and Northern Analysis

Total RNA from the frozen tomato cells (2 g) was isolatedusing the single-step method of Chomczynski and Sacchi (3).Total RNA (10 yg) was heated at 65°C for 10 min andelectrophoresed on a 1% agarose gel containing 0.67 M form-aldehyde (1 1). The RNA was blotted onto a Nytran mem-brane (Schleicher & Schuell), and hybridized to labeled potatoperoxidase cDNA. The blots were subjected to autoradiogra-phy at -80°C.

RESULTS

Induction of Anionic Peroxidase mRNA in Wound-HealingTomato Fruits

RNA from tomato wound periderm, isolated 0, 1, 2, and 3d after wounding, was blotted onto a Nytran membrane andhybridized with a potato peroxidase cDNA that is highlyhomologous to the tomato peroxidase genes. A slot-blot ofRNA from unwounded and 2-d wound-healed fruits fromresistant and susceptible tomato lines (Fig. 1A) revealed thatthe resistant line had a higher level of RNA that hybridizedwith the peroxidase probe. Densitometric scanning of theautoradiogram ofRNA from fruits wound-healed for variousperiods of time showed the induction profile of peroxidasemessage from the resistant and susceptible tomato lines (Fig.1 B). Unwounded tissues from both lines showed little perox-idase mRNA. After 24 h ofwound-healing the mRNA for theanionic peroxidase appeared in both lines, but the tissues

ARc

Sc

R2

S2

B

CC ,. _

lpg 4pag 20nl

E0 Susceptible

M Resistant

0 1 2Days after wounding

7nn* WUU

-100

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Figure 1. A, Slot blots showing peroxidase message induction inwound-healing fruits of tomato lines resistant (R) and susceptible (S)to V. albo-atrum. The blot of the indicated amounts of total RNA fromunwounded (top) and periderm from 2-d wound-healed fruits (bottom)was probed with a 32P-labeled homologous potato peroxidase cDNA.B, Time-course of appearance of the peroxidase message in wound-healing tomato fruits. Slot blots were hybridized with 32P-labeledcDNA and the autoradiograms were subjected to densitometricscanning.

from the resistant tomato line had much more peroxidasemessage than the susceptible line. By the third day of wound-healing, the message level in the resistant line decreased to alevel that was reached by the susceptible line.

Induction of Peroxidase mRNA in Tomato Cell-Suspension Cultures by Fungal Elicitor

In a series of experiments the effect of adding an elicitorpreparation from V. albo-atrum cultures into the cell suspen-sion culture of the two tomato lines was examined. It wasfound that the peroxidase mRNA level was barely detectablein actively growing young cultures but the peroxidase messageappeared in cultures older than 10 d. Therefore, we chosecells from 4-d-old cultures to test the effect of the fungalelicitor.

Cell suspension cultures from the resistant and susceptibletomato lines were incubated with different levels of the fungal

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MOHAN AND KOLATTUKUDY

elicitor, and the cells were harvested after 15 min or 3 h ofcontact with the elicitor. Total RNA was electrophoresed,blotted, and the Northern blots were probed with the 800 bp2potato peroxidase cDNA (Fig. 2). The probe hybridized to amRNA species of approximately 1600 bp, which was of thesame size as the peroxidase message detected in wound-healing potato and tomato tissues (24). The resistant cell lineresponded to the elicitor by producing the peroxidase message(Fig. 2, bottom). In this cell line as little as 8 ng/mL elicitorinduced peroxidase mRNA which was detected by 3 h. Athigher concentrations of the elicitor (1.0 ,ug/mL), the peroxi-dase mRNA could be detected in the resistant line within 15min after treatment with the elicitor. On the other hand, thesusceptible cells (Fig. 2, top) showed barely detectableamounts ofperoxidase message up to 3 h after treatment evenwith up to 5 gg/mL of the elicitor. Ribosomal RNA contentwas used to monitor recovery ofRNA and actin mRNA level,that was used as a control, did not change with elicitortreatment.

Temporal Induction of Peroxidase mRNA in Tomato CellCultures

Cell cultures from resistant and susceptible tomato lines,treated with 1.0 ,ug/mL elicitor for 0, 4, 16, 24, and 48 h,were harvested and total RNA isolated from the cells weresubjected to Northern analysis using the 800 bp anionicperoxidase cDNA probe (Fig. 3). The resistant cell line re-sponded to the elicitor by producing high levels of peroxidasemessage, which reached a maximum by 16 h and then thelevel decreased. The susceptible line showed very little induc-tion that reached a maximum by 4 h.

Since only the cell cultures from the resistant line showeda major induction of anionic peroxidase mRNA in response

8' ,___________, _ _ ... _ $ __

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0.:1

N,$`J i xa"'*IN r*2 ;,', IsI

0008 0.04 0.2Elicitor pgmI

.0 i5.0

Figure 2. Autoradiogram of a Northern blot of 10 Aig total RNA fromsusceptible (top) and resistant (bottom) tomato cell suspension cul-tures treated with different concentrations of elicitor from V. albo-atrum. The cells were harvested 15 min and 3 h after elicitor treatmentand the blots were probed with 32P-labeled peroxidase cDNA.

2 Abbreviation: bp, base pair.

kb7<.5

4.4

2.4-

1.4-

Resistant

Oh 4h 16h 24h 48h

Susceptibke

4 .

Oh 4h '16 24)' 4Ar

Figure 3. Autoradiogram of a Northern blot showing the induction ofperoxidase message in elicitor treated cell suspension cultures fromtomato lines resistant and susceptible to V. albo-atrum. Total RNA(20 Asg) isolated from cultures that were incubated with 1.0 Mg/mLelicitor for 0, 4, 16, 24, and 48 h were loaded per lane, and the RNAgel blots were probed as in Figure 1.

z

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co

00~

0-

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Time (min)Figure 4. Time-course of induction of peroxidase message in resist-ant tomato cell suspension cultures treated with 1.0 Ag/mL elicitorfrom V. albo-atrum. Video densitometric scanning of the autoradi-ogram was used to quantitate the amount of peroxidase messagethat hybridized to the probe on the RNA gel blot.

to low levels of the fungal elicitor, we attempted to determinehow soon after the elicitor treatment we could detect theperoxidase message in this cell line. The induction profile ofperoxidase mRNA was determined by densitometric scanningof a RNA gel blot of resistant cells that were treated with 1.0ug/mL elicitor for 0, 5, 15, 60, 240, and 720 min. (Fig. 4).Induction of peroxidase message could be detected as early as5 min after elicitor addition and continued to increase steadilywith time.

DISCUSSION

Deposition of polymeric materials containing phenoliccomponents on the cell wall is a common response to patho-gen attack of plants. In most cases such deposits have beenexamined only by microscopic techniques and have beendesignated variously as lignin, suberin, lignin-suberin-like, etc.(16). Lignin and suberin contain aromatic domains that havemany structural features in common. Suberin contains ali-

I" 4..

..l... ..l.

278 Plant Physiol. Vol. 92,1990

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ANIONIC PEROXIDASE GENE EXPRESSION IN FUNGAL RESISTANCE

phatic components that are not found in lignin (17). Suberi-zation of the cell walls is a response to injury and pathogenattack that reinforces the walls and makes a barrier to diffu-sion and pathogen ingress (5, 19, 28). Chemical compositionof pathogen-induced deposits on the cell walls have not beendetermined in most cases. The vascular coating induced byVerticillium albo-atrum in the resistant tomato line not onlystains for phenolic components (27), but also contains ali-phatic monomers characteristic of suberin (18). Thus, thishost-pathogen system provides a model for examining themolecular basis of cell wall reinforcement by suberization,induced by a pathogenic fungus.A highly anionic peroxidase is most probably involved in

the polymerization that generates the aromatic matrix ofsuberin (17). Suberization induction and inhibition caused bysuch factors as wounding, hormones, and mineral deficiencywere found to be associated with induction or inhibition ofproduction of the anionic peroxidase (19). Therefore, it ap-peared likely that a key factor in the fungus-induced deposi-tion of suberized vascular coating might be the induction ofthis enzyme. In fact, the two near isogenic tomato linesshowed inherent difference in the inducibility of the anionicperoxidase gene. The wound-healing fruits of the resistantline showed a faster and higher induction of the peroxidasemRNA that reached a maximal level one day earlier than thefruits from the susceptible line.

If the expression of the gene for the suberization-associatedperoxidase is a key factor in resistance against V. albo-atrum,a signal from the fungus might be expected to trigger theexpression of this gene in the resistant tomato line. In fact,suspension cultures from the resistant tomato line were ableto respond to as little as 8.0 ng/mL of the fungal elicitor, aknown class of fungal signals (4). The susceptible cell line onthe other hand did not respond to such low levels of elicitor,and required as much as 1.0 gg/mL of elicitor before peroxi-dase mRNA was detectable. Older (about 10-d old) cell cul-tures tended to show high basal levels of peroxidase message,possibly because of elicitors produced from the plant cellwalls.The cell cultures were used only as an experimental model

to test the responses and the results obtained with suchcultures might not be directly applicable to the whole plant.However, the present response is certainly consistent with thepostulated role for the peroxidase in the formation of thevascular coating triggered by the infection process in theresistant tomato line. Furthermore, Northern blot analysis ofmRNA from inoculated petioles from the resistant tomatoline showed higher amounts of peroxidase mRNA than thatfound in the susceptible line (R. Mohan, J. Robb, P. E.Kolattukudy, unpublished data), suggesting that the observa-tions made with the suspension culture might reflect theevents that occur when the fungus attacks the plant. Theelicitor-treated cell cultures had about 8- to 10-fold higherlevels of peroxidase message than the tomato petioles thatwere challenged with fungal spores. This difference might bein part due to the fact that all of the cells in the cultures wereexposed to the elicitor, whereas in the petioles probably onlythe few cells localized around the fungal spore would beinduced. Similar differences between cell cultures treated with

fungal elicitors and plant tissues inoculated with fungal sporeshave been observed ( 14).The cell cultures from the resistant tomato line were able

to perceive the elicitor and they responded by producingperoxidase mRNA within 5 min after treatment. The resistantcell cultures also responded to very low levels of the elicitor,whereas the susceptible cell line did not. This observationsuggests that the major difference between the two lines mightbe in some step from the perception of the signal to the actualenhancement of gene expression. The mechanism by whichelicitors trigger gene expression is not understood. One pos-sibility is that the susceptible line has low amounts of receptoror a defective receptor for the elicitor. It is also possible thatsome other protein factor involved in the cascade of signaltransduction resulting in gene expression is defective in thesusceptible line. Yet another possibility is that both the resist-ant and susceptible cell lines have the same trans-actingfactors, but differ in their cis-acting elements, making theregulatory region of the gene from the susceptible line lessresponsive to the trans-acting factors.

Infection of plants depends on the ability of pathogenicfungi to gain entry into the internal plant tissues, eitherthrough wounds or by penetration through the intact barriers.Our studies indicate that suberization might be important inresistance. Higher levels of peroxidase activities have beenassociated with resistance to other pathogens (10, 12, 22, 26,29). The nature of the reactions such peroxidases catalyze arenot known. In all such cases suberization might not be in-volved. Deposition of phenolic polymers on walls or cross-linking of wall components catalyzed by such peroxidases (9)could make the cell walls less susceptible to fungal enzymesand thus help to limit the ingress of the pathogen. Thus, suchperoxidases could be at least in part responsible for theresistance to pathogen attack. Transfer of the peroxidase genefrom resistant plants to the susceptible line would help inunderstanding the molecular mechanisms of resistance andprovide us with a means to genetically engineer crop plantsresistant to fungi.

ACKNOWLEDGMENTS

We thank Prof. Brian E. Ellis for providing us the tomato cellcultures, Dr. Jane Robb for the Verticilliuim albo-atruim culture, andJim Marquart for growing the tomato plants in the green house.

LITERATURE CITED

1. Anderson-Prouty AJ, Albersheim P (1975) Host-Pathogen Inter-actions. Isolation of a pathogen-synthesized fraction rich inglucan that elicits a defense response in the pathogen's host.Plant Physiol 56: 286-291

2. Borchert R (1978) Time course and spatial distribution of phen-ylalanine ammonia-lyase and peroxidase activity in woundedpotato tuber tissue. Plant Physiol 62: 789-793

3. Chomczynski P, Sacchi N (1987) Single-step method for RNAisolation by acid-guanidium thiocyanate-phenol-chloroformextraction. Anal Biochem 162: 156-159

4. Darvill AG, Albersheim P (1984) Phytoalexins and their elici-tors-a defense against microbial infection in plants. AnnuRev Plant Physiol 35: 243-275

5. DeLeeuw GTN (1985) Deposition of lignin, suberin and callosein relation to the restriction of infection by Botrvtis cinerea inghost spots of tomato fruits. Phytopathol Z 112: 143-152

279

www.plantphysiol.orgon April 6, 2019 - Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved.

MOHAN AND KOLATTUKUDY

6. Dische Z (1962) Color reaction ofcarbohydrates. In RL Whistler,ML Wolfrom, eds, Methods in Carbohydrate Chemistry, Vol1. Academic Press, New York, pp 477-512

7. Espelie KE, Kolattukudy PE (1985) Purification and character-ization of an abscisic acid-inducible peroxidase associated withsuberization in potato (Solanum tuberosum). Arch BiochemBiophys 240: 539-545

8. Espelie KE, Franceschi VR, Kolattukudy PE (1986) Immuno-cytochemical localization and time course of appearance of ananionic peroxidase associated with suberization in wound-healing potato tuber tissue. Plant Physiol 81: 487-492

9. Everdeen DS, Kiefer S, Willard JJ, Muldoon EP, Dey PM, LiXB, Lamport DTA (1988) Enzymatic cross-linkage of mono-meric extensin precursors in vitro. Plant Physiol 87: 616-621

10. Flott BE, Moerschbacher BM, Reisener HJ (1989) Peroxidaseisozyme patterns of resistant and susceptible wheat leavesfollowing stem rust infection. New Phytol 111: 413-421

11. Fourney RM, Miyakoshi J, Day III RS, Paterson MC (1988)Northern blotting: efficient RNA staining and transfer. Focus10: 5-7

12. Gaspar T, Penel CL, Thorpe T, Greppin H (1982) Peroxidases1970-1980. A Survey of their Biochemical and PhysiologicalRoles in Higher Plants. Universite de Geneve, Geneve, pp 89-112

13. Grisebach H (1981) Lignins. In PK Stumpf, EE Conn, eds, TheBiochemistry of Plants, Vol 7. Academic Press, New York, pp457-478

14. Habereder H, Schroder G, Ebel J (1989) Rapid induction ofphenylalanine ammonia-lyase and chalcone synthase mRNAsduring fungus infection of soyabean (Glycine max L.) roots orelicitor treatment of soyabean cell cultures at the onset ofphytoalexin synthesis. Planta 177: 58-65

15. Hankin L, Kolattukudy PE (1968) Metabolism of a plant waxparaffin (n-nonacosane) by a soil bacterium (Microccus cerifi-cans). J Gen Microbiol 51: 457-463

16. Kolattukudy PE (1980) Biopolyester membranes of plants: cutinand suberin. Science 208: 990-1000

17. Kolattukudy PE (1987) Lipid-derived defensive polymers andwaxes and their role in plant-microbe interaction. In PK

Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol 9.Academic Press, New York, pp 291-314

18. Kolattukudy PE, Podila GK, Mohan R (1989) Molecular basis ofthe early events in plant-fungus interaction. Genome 31: 342-349

19. Kolattukudy PE, Podila GK, Roberts E, Dickman MD (1989)Gene expression resulting from the early signals in plant-fungusinteraction. In B Staskawicz, P Ahlquist, 0 Yoder, eds, Molec-ular Biology of Plant-Pathogen Interactions. Alan R Liss, Inc,New York, pp 87-102

20. Lagrimini LM, Rothstein S (1987) Tissue specificity of tobaccoperoxidase isozymes and their induction by wounding andtobacco mosaic virus infection. Plant Physiol 84: 438-442

21. Pearce RB, Rutherford J (1981) A wound-associated suberizedbarrier to the spread of decay in the sapwood of oak (Quercusrobur L.). Physiol Plant Pathol 19: 359-369

22. Retig N (1974) Changes in peroxidase and polyphenoloxidaseassociated with natural and induced resistance of tomato toFusarium wilt. Physiol Plant Pathol 4: 145-150

23. Roberts E, Kolattukudy PE (1989) Molecular cloning, nucleotidesequence and abscisic acid induction of a suberization-associ-ated highly anionic peroxidase. Mol Gen Genet 217: 223-231

24. Roberts E, Kutchan T, Kolattukudy PE (1988) Cloning andsequencing of cDNA for a highly anionic peroxidase frompotato and the induction of its mRNA in suberizing potatotubers and tomato fruits. Plant Mol Biol 11: 15-26

25. Schaible L, Cannon OS, Waddoups V (1951) Inheritance ofresistance to Verticillium wilt in a tomato cross. Phytopathol-ogy 41: 986-990

26. Stahmann MA, Demorest DM (1973) Changes in enzymes ofhost and pathogen with special reference to peroxidase inter-action. In R Byrde, C Cutting, eds, Fungal Pathogenicity andthe Plant's Response. Academic Press, New York, pp 405-420

27. Street PFS, Robb J, Ellis BE (1986) Secretion of vascular coatingcomponents by xylem parenchyma cells of tomatoes infectedwith Verticillium albo-atrum. Protoplasma 132: 1-11

28. Tippett JT, Hill TC (1984) Role of periderm in resistance ofEucalyptus marginata roots against Phytophthora cinnamomi.Eur J For Pathol 14: 431-439

2.Y. venere KJ (1980) Role ot1 peroxidase in cotton resistant tobacterial blight. Plant Sci Lett 20: 47-56

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