only specific tobacco (nicotiana tabacum) chitinases and ... · plant physiol. (1993) 101: 857-863...

Plant Physiol. (1993) 101: 857-863

Only Specific Tobacco (Nicotiana tabacum) Chitinases and ,8-1,3-Clucanases Exhibit Antifungal Activity

Marianne B. Sela-Buurlage*, Anne S. Ponstein, Sandra A. Bres-Vloemans, Leo S. Melchers, Peter J. M. van den Elzen, and Ben J. C. Cornelissen'

MOGEN lnternational NV, Einsteinweg 97, 2333 CB Leiden, The Netherlands

Different isoforms of chitinases and @-1,3-glucanases of tobacco (Nicotiana fabacum cv Samsun NN) were tested for their antifungal activities. l h e class I, vacuolar chitinase and @-1,3-gIucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In addition, we observed that the class I chitinase and @-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I @-1,3-glucanase. The class I I @-1,3- glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I @-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C- terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and @-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on f. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.

Plant-pathogen interactions leading to a hypersensitive response result in the induction of resistance both locally around the sites of infection and systemically in noninfected parts of the plant. Resistance is induced against a broad range of pathogens, irrespective of which pathogen triggered the hypersensitive response. For example, inoculation of tobacco (Nicotiana tabacum cv Samsun NN) with TMV leads to a systemic induction of resistance against the fungi Phyto- phthora parasitica var nicotianae and Peronospora tabacina (McIntyre et al., 1981).

Induced resistance in tobacco is accompanied by the induced synthesis of PR proteins, including Chi and Glu (for reviews, see Bol et al., 1990; Bowles, 1990; Linthorst, 1991). In tobacco, three classes of Chi have been identified based on the structural analysis of their genes (Shinshi et al., 1990). Class I contains vacuolar isoforms with an N-terminal domain homologous to hevein. The Chi-11, known in the liter-

' Present address: University of Amsterdam, Department of Mo- lecular Cell Biology, Section of Plant Pathology, Kruislaan 318, 1098 SM Amsterdam, The Netherlands.

* Corresponding author; fax 31-71-221471. 857

ature as PR-3a and PR-3b (formerly PR-P and PR-Q), are very homologous to Chi-I, but lack the hevein domain (Lin- thorst et al., 1990b; Payne et al., 1990a) and are located extracellularly. Very recently, a third class of tobacco Chi genes was described (Lawton et al., 1992) that encodes proteins homologous to Chi in cucumber and Arabidopsis (Me- traux et al., 1989; Samac et al., 1990) but that are not related to class I or I1 isoforms. Based on their primary structures, three major classes of Glu have been identified (Payne et al., 1990b; Ward et al., 1991a). Class I contains basic, vacuolar isoforms (Glu-I), whereas class I1 consists of acidic, extracellular isoforms. In the literature, the latter proteins are also known as the PR proteins PR-2a, PR-2b, and PR-2c (formerly PR-2, PR-N, and PR-O). Glu-I and Glu-I1 are serologically related and show an identity in primary structure of approximately 50% (Linthorst et al., 1990a). To date only one class I11 enzyme has been identified, an acidic extracellular protein showing 54 to 59% identity with the class I1 isoforms (Payne et al., 1990b).

The major components of the cell walls of many fungi are the polysaccharides chitin and /3-1,3-glucan, substrates for Chi and Glu, respectively (Wessels and Sietsma, 1981). In vitro, growth of a number of fungi is inhibited by a Chi-I from bean (Schlumbaum et al., 1986) and combinations of Chi-I and Glu-I from pea (Mauch et al., 1988). These observations, together with the notion of an apparent lack of chitin in plants, and the concomitant induction of Chi and Glu and funga1 resistance have led to speculations about a role for these hydrolytic enzymes in systemically induced resistance (Mauch and Staehelin, 1989).

Here we report the antifungal activity of the Chi and Glu from tobacco. To this end, the various Chi and Glu were purified to homogeneity and tested, either alone or in combination, for their ability to inhibit in vitro growth of Fusarium solani. In addition, tobacco was transformed with modified gene constructs of the Chi-I and Glu-I and constructs containing both modified genes, resulting in extracellular targeting of these enzymes. It appeared that these proteins retained their enzymic activity and that EF harvested from these transgenic plants caused lysis and growth inhibition of F. solani germlings in vitro in the same manner as was observed with the purified proteins.

Abbreviations: Chi, chitinase(s); CTPP, carboxyl-terminal propeptide; EF, extracellular washing fluid; Glu, ~-1,3-glucanase(s); I(I1, III), class I (11, 111); PR, pathogenesis-related; TMV, tobacco mosaic virus.

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858 Sela-Buurlage et al. Plant Physiol. Vol. 101, 1993

MATERIALS AND METHODS

Biological Materials

Tobacco plants (Nicotiana tabacum cv Samsun NN) were grown in pots in a greenhouse according to standard methods. For induction of PR proteins, leaves of 5- to 6-week-old plants were inoculated with TMV as described previously (Woloshuk et al., 1991). Leaves were harvested and stored at -8OOC 7 d after inoculation.

Fusarium solani was kindly provided by Dr. P.J.G.M. de Wit, Department of Plant Pathology at the University of Wageningen, The Netherlands. The fungus was maintained on potato dextrose agar at 25OC in the light. Spores were harvested from 3- to 6-week-old plates by flooding the plate with water. The spore concentration was adjusted to 10,000 spores/mL.

Protein Purification

Proteins were extracted and purified by a modification of the method described by Woloshuk et al. (1991). TMV- infected tobacco leaves (400 g) were homogenized at 4OC in a Waring blender with 500 mL of 0.5 M sodium acetate, pH 5.2, 0.1% (v/v) P-mercaptoethanol, and active charcoal (1 g per 100 g leaves). The homogenate was filtered through four layers of cheesecloth and centrifuged at 3000g for 15 min. The supematant was centrifuged for 50 min at 20,OOOg. The final supematant was passed over a Sephadex G-25 (medium coarse; Pharmacia) column (12 X 60 cm) equilibrated in 40 mM sodium acetate, pH 5.2. The eluted protein solution was incubated overnight on ice before centrifuging for 50 min at 20,OOOg. The resulting supematant was loaded onto an S- Sepharose (Fast Flow; Pharmacia) column (5 X 5 cm), equilibrated in 40 mM sodium acetate, pH 5.2, and the adsorbed proteins were eluted with 500 mL of a linear gradient from O to 0.4 M NaCl in buffer. Fractions exhibiting Chi activity were pooled and concentrated by ultrafiltration through YMlO membranes (Amicon). The same was done for the fractions containing glucanase activity.

The Chi pool was brought to 20 mM sodium bicarbonate and further dialyzed against 20 mM sodium bicarbonate, pH 8.3. Chi-I were allowed to adsorb for 1 h to a matrix of 50 mL of regenerated chitin (Molano et al., 1977) equilibrated in 20 mM sodium bicarbonate, pH 8.3. The chitin matrix was then filtered, and a column was poured and washed with 100 mL of 20 mM sodium bicarbonate and 100 mL of 20 mM sodium acetate, pH 5.2. Bound proteins were eluted with 20 II~M acetic acid, pH 3.5. The eluate was further purified by a Superdex-75 gel-filtration column (HR 10/30; Pharmacia). Gel filtration was camed out in 50 mM potassium phosphate buffer, pH 7.0, containing 0.2 M NaCl at a flow rate of O-.5 mL min-'. The fractions showing Chi activity were pooled and dialyzed to 20 mM sodium acetate, pH 5.2, and applied to a Mono S column (HR 5/5; Pharmacia), equilibrated to the same buffer. Bound proteins were eluted from the Mono S column by a linear gradient (20 mL) of 30 to 60 mM NaCl in the above buffer (1 mL min-'). Fractions were separated on 12.5% SDS gels, and the 32- and 34-kD Chi were pooled accordingly .

The pool exhibiting Glu activity was loaded onto a gel-

filtration column as above. Glu activity eluted at an apparent molecular mass of 5 to 10 kD.

Acidic proteins running through the S-Sepharose column were dialyzed to 20 mM Tris-HC1, pH 8.0, and loaded onto a Q-Sepharose (Fast Flow; Pharmacia) column (5 X 5 cm) equilibrated in 20 mM Tris-HC1 buffer, pH 8.0. Bound proteins were eluted with 500 mL of a O to 0.3 M NaCl gradient in 20 mM Tris-HCI, pH 8.0. Fractions were analyzed by 10% native polyacrylamide gels. Pools of the class I1 proteins were prepared based on the electrophoretic pattem, except PR-~c, which was pooled based on Glu activity measurements. In some instances Mono Q (FPLC) ion exchange chromatography was performed to enhance the separation of PR-3a and PR-3b. A11 of the class I1 proteins were further purified by gel-filtration chromatography as described above.

Protein Analysis

Protein concentrations were determined by the method of Bradford (1976) using BSA as the standard. Electrophoretic analysis was performed by SDS-PAGE on 12.5% polyacrylamide gels according to the method of Laemmli (1970). In the case of native gels, SDS was omitted.

Chi and Glu activity measurements were performed using [3H]chitin and laminarin as substrates, respectively (Molano et al., 1977; Kauffmann et al., 1987). Chi activity was expressed in cpm pg-' of protein. The activity of Glu was expressed in nkat mg-' of protein, for which a nkat was the number of nmol of Glc released per s.

Transformation and Analysis of Transgenic Plants

Transgenic tobacco plants were obtained as described by Melchers et al. (1993). Isolation of EF was done as described by de Wit and Spikman (1982). Extracellularly targeted enzymes were purified from the EF as described for the proteins isolated from TMV-inoculated leaf material (see "Protein Purification"). Specific activities of Glu and Chi were determined as above.

In Vitro Antifungal Assay

The protocol described by Woloshuk et al. (1991) was modified as follows. The assay was performed in a 24-well microtiter dish (Greiner). Potato dextrose agar (250 bL) was pipetted into each well. Five hundred F. solani spores in 50 p L were added per well. The spores were pregerminated for 6 to 7 h at 25OC. Protein samples were dialyzed for 2 to 4 h against 50 lll~ potassium phosphate buffer, pH 6, at 4OC and subsequently filter sterilized through 0.22-pm filters. Protein concentrations were determined and adjusted appropriately. One hundred microliters of protein sample was pipetted into each well, resulting in a total volume of 150 pL. Denaturation of enzymes was done by boiling the samples for 10 min. Two hours after the initiation of incubation, the fungus was mon- itored microscopically for possible effects of the added proteins. After 3 d funga1 growth was stopped by staining the mycelium with lactophenol cotton blue. Subsequently, the plates were destained with water.



Antifungal Chitinases and /?-1,3-Glucanases from Tobacco 859

RESULTS

Purification of Chi and Glu

The 32- and 34-kD Chi-I, the Chi-II PR-3a and PR-3b, the33-kD Glu-I, and the Glu-II PR-2a, PR-2b, and PR-2c werepurified to homogeneity from TMV-infected Samsun NNtobacco plants. The identity of the class II proteins wasconfirmed by their mobility on a native polyacrylamide gel(Fig. 1A). In the outer lanes proteins from the EF of TMV-infected leaves were loaded and used as markers. The purityof the proteins was checked by SDS-PAGE (Fig. IB). Thespecific activity of each of the Chi after purification wasdetermined on tritiated chitin. For the determination of thespecific activity of Glu, laminarin was used as a substrate.The results are summarized in Table I. As reported by Le-grand et al. (1987), the specific activity of the Chi-I (10,000-11,000 cpm Mg'1 for the 32-kD Chi-I and 17,000-19,000 cpmMg'1 for the 34-kD Chi-I) was 10 to 15 times higher than thatof the Chi-II (1000-1100 cpm Mg"1)- In agreement withKauffmann et al. (1987), the specific activities of the Glu-Iand the extracellular PR-2c were comparable (300-500 nkatmg'1). The specific activities of PR-2a and PR-2b were 50- to70-fold lower (5-10 nkat mg~]).

Table I. Specific activities of purified Chi and Clu of tobacco

5•4

3b3a2c

2b2a

• 1 c

• 1 b

- 11

EF 3b 3a 2c 2b 2a EF

B

. 97- 66

- 40

- 25

- 1 8

2a 2b 2c 3a 3b Glu-I Chi-I Chi-I34kD 32kD

Figure 1. Native (A) and SDS-polyacrylamide (B) gel electrophoresisof proteins purified from TMV-infected Samsun NN tobacco. Mo-lecular masses are indicated in kD.

Enzyme Specific Activity"

Chi-I (32 kD)Chi-I (34 kD)Chi-II (PR-3a)Chi-II (PR-3b)Clu-lGlu-II (PR-2a)Glu-II (PR-2b)Glu-II (PR-2c)

10,000-11,00017,000-19,000 cpm1,000-1,100 cpm ̂1,000-1,100 cpm M

400-500 nkat mg5-10 nkat trig'1

5-10 nkat mg"1

400-500 nkat mg1 Chi and Glu activities were determined as described in "Mate-

rials and Methods." Ranges from three independent experimentsare listed.

In Vitro Antifungal Effect of Purified Enzymes

A microtiter dish assay was used to determine the effectsof the purified proteins on the growth in vitro of F. solani.Proteins were added, either alone or in combination, to asuspension with pregerminated spores on a layer of potatodextrose agar in wells of a microtiter dish. After an incubationperiod of 2 h, the germling suspensions were microscopicallymonitored for lysis. Up to 5 Mg per well for Chi-I, 50 Mg forPR-3a, and 30 Mg for PR-3b was tested for the Chi and up to10 Mg for each of the Glu. Of the Chi, only the Chi-I werecapable of causing lysis of F. solani germlings. Application of5 Mg of Chi-I per well showed strong lysis activity (50% for5 Mg of 32-kD Chi-I; 80% for 5 Mg of 34-kD Chi-I). Smalleramounts of these proteins showed intermediate levels of lysis.The Chi-II PR-3a and PR-3b exhibited no lysis activity at all,even when up to 50 and 30 Mg per well, respectively, wasadded. Testing of the Glu revealed that only the Glu-I isoformdisplayed lysis activity. Complete lysis was observed with 1Mg per well, but with 0.5 Mg of Glu-I no lysis was observed.The Glu-II showed no lysis activity even at the highestconcentration of 10 Mg per well.

Three days after addition of the proteins, the growingmycelium was stained with lactophenol cotton blue. Subse-quently, the amount of mycelium was taken as a measure ofgrowth. Typical results are shown in the left panel of Figure2A for the Chi and in the right panel for the Glu. Intermediategrowth inhibition of the fungus was observed upon incuba-tion of the fungus with 5 Mg per well of Chi-I. Chi-II had noeffect on growth even at the highest concentration of 50 Mgper well. Addition of 0.5 Mg of Glu-I showed intermediategrowth inhibition, and with 1 Mg of Glu-I per well no fungalgrowth was observed. The Glu-II PR-2c (Fig. 2A), PR-2a, orPR-2b (data not shown) had no effect on the growth of F.solani, even at the highest concentrations tested (10 Mg)- Asa control, enzymes were denatured by boiling for 10 min. Byusing this treatment, the antifungal activities were completelyabolished.

Synergism between Chi-I and Glu-I

Mauch and coworkers (1988) have shown that combina-tions of Chi and Glu are capable of synergistically inhibitingfungal growth in vitro. Here, we tested which specific com-binations of enzymes exhibited synergistic antifungal activity.




Chi-l Chi-ll Glu-l Glu-II

0.5

2.5

B

Glu-l Glu-II

0.1 0.5 0.1 0.5

Chi-l

0.5

Chi-ll

Figure 2. Effect of purified Chi and Clu (A) and combinationsthereof (B) on in vitro growth of F. so/am. Amounts of protein (in/ig) per well are indicated. Chi-l, 32-kD Chi-l; Chi-ll, PR-3a; Glu-II,PR-2c; C, highest amount of protein after boiling for 10 min.

2c this is striking because the specific activity of this enzymeon laminarin is comparable to that of Glu-I (Table I). MixingChi-II with 0.1 /tg of Glu-I showed no effect. However, anenhanced growth inhibition was observed in combinationwith 0.5 ;*g of Glu-I, as compared to 0.5 Mg of Glu-I alone.There was no difference in level of growth inhibition whether1 or 5 Mg of the Chi-II PR-3a was added to 0.5 ^g of Glu-I.The effect was significantly less than with Chi-I, and no lysiswas observed. The Chi-II PR-3b exhibited an effect that wassimilar but slightly more pronounced than that of PR-3a:only in combination with Glu-I was growth inhibition ob-served without lysis activity, and the antifungal effect wasfar less than that of the combination of Chi-I and Glu-I.

Antifungal Effect of Extracellularly TargetedChi-l and Glu-l

Chi-I and Glu-I are synthesized as preproproteins fromwhich an amino-terminal signal peptide and a CTPP arecleaved off during posttranslational processing (Shinshi etal., 1988; Neuhaus et al., 1991). To study the role of theCTPP in the vacuolar targeting of the enzymes, transgenicplants have been created that constitutively express either achimeric, modified Chi-I gene or a chimeric, modified Glu-Igene, or both. The modifications involved the introductionof a stop codon in the 3'-terminal coding regions of thegenes, thereby specifically excluding the CTPP-encoding se-quence from the open reading frame (Fig. 3). In these trans-genic plants, the transgene products are targeted extracellu-larly (Neuhaus et al., 1991; Melchers et al., 1993). Purificationof these proteins from the EF of the transgenic plants showedthat the specific activity of the extracellularly targeted Chi-Iand of the Glu-I was not affected (data not shown).

To determine the antifungal activity of the secreted pro-teins, in vitro assays were done on F. solani germlings usingEF from the transgenic plants. As a control, EF was harvestedfrom nontransformed, regenerated tobacco plants. Five mi-

PMOG189

pMOG549

(a 7 a.a. CTPP)

(A 22 I . CTPP)

In Figure 2B, results are shown of combinations of specificChi and Glu. As Chi-I, the 32-kD isoform was used. Neither0.1 nor 0.5 Mg of Chi-I and Chi-II showed any effect ifapplied separately (Fig. 2A). The same was true for 0.1 or 0.5Mg per well of Glu-II. No effect or an intermediate effect wasobserved with 0.1 or 0.5 /xg Glu-I, respectively. However, thecombination of 0.1 /tig of Chi-I with 0.1 ng of Glu-I causedcomplete tip lysis of germlings of F. solani 2 h after additionof the enzymes and complete growth inhibition after 3 d.The Glu-II, PR-2a, PR-2b (data not shown), and PR-2c (Fig.2B) were not able to substitute for Glu-I. Especially with PR-

pMOG556

Figure 3. Schematic drawing of the gene constructs. Expressionconstructs of the carboxyl-terminal mutants of Chi-l cDNA (pMOC189) and Clu-l gDNA (pMOC 549) and a construct containing both(pMOC 556). All genes are under the control of the 35S promoter(hatched box). The black box represents the nos terminator region.Construct pMOC 549 contains the genomic Clu-l gene with itsown terminator. The asterisk (*) indicates the presence of an addi-tional stop codon, which results in deletion of a CTPP from theprotein. Chimeric gene constructs are present in the binary vectorpMOG402 next to the neomycine phosphotransferase gene, a.a.,Aminoacid.



Antifungal Chitinases and /3-1,3-Glucanases from Tobacco 861

crograms of EF protein per well was added to F. solanigermlings. Typical results are shown in Figure 4A. The EFfrom control plants did not show any effect on the F. solanigermlings. In contrast, the EF from plants having both en-zymes targeted extracellularly exhibited almost complete lysisafter 2 h and almost full growth inhibition of the fungusafter 3 d. The EF from plants containing secreted Chi-I orGlu-1 alone had an intermediate effect on F. solani. Combi-nations of EFs were also tested to determine whether theeffect of the double construct could be mimicked by mixingthe EF of both single constructs. Results are shown in Figure4B. Mixing 2.5 ^g of protein of the EF fraction of controlplants with an equal amount of EF from either a retargetedChi-I or a GIu-I transgenic plant had a minor inhibitoryeffect. However, the combined EFs of these transgenic plantsshowed clear inhibition of fungal growth. The denatured EFfractions had no effect on the fungus.

DISCUSSION

To assess the in vitro antifungal activity of various Chi andGlu of tobacco, the class I and class II isoforms of theseenzymes were purified to homogeneity. In accordance withLegrand et al. (1987), the specific activities of the 32- and 34-kD Chi-I were found to be approximately 10- to 15-foldhigher than those of the class II PR-3a and PR-3b proteinswhen tested on tritiated chitin as substrate. When tested onlaminarin the specific activities of the 33-kD Glu-I and theclass II PR-2c protein were found to be very similar. Incontrast, the class II enzymes PR-2a and PR-2b were shownto have a 50- to 70-fold lower specific activity, despite their>90% identity in primary structure with PR-2c (Linthorst et

B

189

549

556

189 + C

549 + C

189 + 549

denatured EF

Figure 4. Effect of EF fractions (A) and mixtures of EF fractions (B)harvested from transgenic plants on in vitro growth of F. solani. Atotal of 5 /jg of protein was added per well. pMOG 189, Extracel-lularly targeted Chi-I; pMOC 549, extracellularly targeted Glu-I;pMOG 556, extracellularly targeted Chi-I and extracellularly tar-geted Glu-I; C (control), regenerated nontransformed tobacco plant;denatured EF, EF (from a pMOG 556 plant) boiled for 10 min.

al., 1990a; Ward et al., 1991a). Similar results have beenreported by Kauffmann et al. (1987).

To determine the effects of the purified proteins on thegrowth in vitro of F. solani, a microtiter dish assay was used.Both Glu-I and Chi-I exhibited high antifungal activities.Combinations of Glu-I and Chi-I revealed that these twopurified enzymes acted synergistically to inhibit fungalgrowth. The Chi-II PR-3a and PR-3b are not inhibitory bythemselves, even when amounts of protein up to 50 jtg wereadded to the wells; in combination with Glu-I, synergismwas observed, although to a much lesser extent than betweenChi-I and Glu-I and without lysis activity. In contrast to Glu-I, none of the three Glu-II showed any antifungal activity,either alone or in combination with Chi. In view of thespecific activity of the Glu-II PR-2c and its approximately50% identity in primary structure with Glu-I, this result isunexpected. Apparently, hydrolytic activities as determinedon the artificial substrate laminarin are not inhibitory per sefor fungal growth in vitro. Clearly, the classification of en-zymes based solely on their ability to hydrolyze a certainsubstrate is an oversimplification. Here we demonstrated thatthere is a clear distinction between enzymic and antifungalactivities of the different Chi and Glu. This finding contrib-utes to our understanding of why so many different kinds ofhydrolytic proteins are produced in plants.

A hypersensitive reaction of plants to pathogenic infectionsleads to the induction of Chi and Glu activities, both locallyand systemically. At the same time, resistance is induced,directed toward a broad range of pathogens, including fungi.The observation that Chi and Glu exhibit antifungal activitiesin vitro has led to speculations regarding a direct antifungalrole of these hydrolytic enzymes in systemically inducedfungal resistance (Boiler, 1988; Mauch and Staehelin, 1989).

Recently, it was shown that in TMV-infected tobacco themessengers for Glu-II and Chi-II are induced both locallyaround the site of infection and systemically in the nonin-fected parts of the plants. In contrast, the class I isoforms ofboth enzymes are induced to a high level locally, but theyare not induced systemically (Brederode et al., 1991; Ward etal., 1991b). Here, we have shown that only the locallyinduced, class I hydrolytic enzymes exhibit antifungal activityin vitro, These observations together make it very unlikelythat either class I or class II Chi and Glu fulfill a directantifungal role in systemically induced fungal resistance. Thisdoes not rule out the possibility that the enzymes have anindirect role in plant defense or that they act synergisticallywith other types of antifungal proteins. They might be in-volved in the generation of signal molecules, which in turntrigger the defense system. Indeed, Keen and Yoshikawa(1983) have shown that Glu from soybean are capable ofreleasing elicitor-active carbohydrates from fungal cell walls.

In addition to their possible role in defense, Glu and Chimay have functions in a number of developmental processesin plants as well. Glu's are very likely involved in cell wallmetabolism and cell expansion in seedlings (Wong andMacLachlan, 1980) and in microsporogenesis (Worall et al.,1992). Recently, a Chi was shown to have the capability torescue a temperature-sensitive carrot somatic embryo mutant,suggesting that the enzyme might be involved in somaticembryogenesis (De Jong et al., 1992). In this context it is www.plantphysiol.orgon June 7, 2020 - Published by Downloaded from

Copyright © 1993 American Society of Plant Biologists. All rights reserved.



noteworthy that during the early period of imbibition of barley seeds, two embryo-associated Chi are selectively released (Swegle et al., 1992) and that in Saccharomyces cerevisiae a Chi is required for cell separation during growth (Kuranda and Robbins, 1991).

Transgenic plants have been made that constitutively express a modified Chi-I, a Glu-I, or both. In these plants the modified gene products were targeted extracellularly. The specific activities of the targeted Chi and Glu were not decreased as determined by incubation on their respective artificial substrates, tritiated chitin and laminarin (our unpub- lished data).

The EF from transgenic plants expressing either the modified Chi-I or the modified Glu-I gene exhibited intermediate inhibitory activity on F. solani. The amount of Glu and Chi in the various EF fractions was calculated, and it appeared that the same amount of EF enzyme was needed to demon- strate antifungal activity as in the assays using the purified proteins. Clearly, the extracellularly targeted class I proteins have retained their antifungal activity. Mixed EFs of the plants containing the single constructs showed synergism similar to that observed with the purified proteins. Almost complete lysis of the germling tips and growth inhibition of the fungus were demonstrated. In agreement herewith was the drastic antifungal effect found in the EF harvested from transgenic plants containing the construct of both modified genes. It can be concluded that targeting of these enzymes can be done successfully without any significant loss of antifungal activity.

Recently, Broglie and coworkers (1991) demonstrated in- creased resistance to Rhizoctonia solani in transgenic tobacco plants constitutively expressing a bean Chi-I. Many fungi penetrate their host through the stomates and never actually enter the plant cell but reside in the extracellular space. Future experiments will show whether targeting of Chi-I and Glu-I to the extracellular space is a n effective way of increasing fungal resistance in plants.

ACKNOWLEDCMENTS

The authors would like to thank Drs. C.P. Woloshuk, E.J.S. Meu- lenhoff, and J.S.C. Van Roekel for technical assistance, and Drs. J.B. Bade and A. Hoekema for critica1 review of this manuscript.

Received September 30, 1992; accepted November 19, 1992. Copyright Clearance Center: 0032-0889/93/101/0857/07.

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