Interactions between pectic fragments and extracellular components from the fungal pathogen Colletotrichum lindemuthianum

147

Physiological and Molecular Plant Pathology (1990) 36, 147-158

Interactions between pectic fragments and extracellular components from the fungal pathogen

Colletotrichum lindemuthianum CRAIG S . TEPPERt

and

ANNE J . ANDERSON+

Department of Biology, Utah State University, Logan, UT 84322-5305, U .S.A . (Accepted for publication October 1989)

Extracellular components of the a and ß races of Collelotrichum lindemuthianum and citrus pectic fragments had synergistic effects on the accumulation of phenolics and phytoalexins in cotyledons of bean, Phaseolus vulgaris . Treatment of the cultivar Dark Red Kidney with crude, partially purified and purified elicitor preparations isolated from an incompatible a race of the fungus and pectic fragments resulted in strong synergism for the production of soluble phenolics, phytoalexins and condensed phenolics . However, crude a race elicitor and pectic fragments had little additive effect on the accumulation of mRNAs for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), enzymes involved in the early stages of phenolic and phytoalexin biosynthesis . A purified carbohydrate-rich elicitor, a galactoglucomannan, isolated from the incompatible a race, displayed complex patterns of synergism with pectic fragments for the accumulation of phenolics and phytoalexins on Dark Red Kidney . Synergism and additive effects on compatible cultivars between preparations from a and ß races, which alone had little elicitor activity, and pectic fragments were observed for accumulations of mRNAs and phenolics . These data suggest that several fungal components interact with pectic fragments to stimulate the accumulation of defence related products in bean .

INTRODUCTION

Elicitors are microbial or plant components that trigger metabolic changes in plants associated with resistance reactions . Elicitor active fungal components include glucans and lipids derived from cell walls, as well as extracellular glycoproteins and more complex polysaccharides [1, 13] . Plant cell wall fragments also have elicitor activity [3-6, 8, 18] . These fragments are released when plant cell walls are incubated with the microbial pectic-degrading enzymes, polygalacturonase and pectate lyase . Oligomers between 9 and 12 galacturonic acid units in length have optimal activity . Cervone et al . [2] recently demonstrated that the presence of a polygalacturonase inhibitor in incubations of a polygalacturonase with plant cell walls alters the release time for the breakdown products . Production of larger elicitor-active fragments increased, whereas fewer smaller oligomers that were inactive as elicitor were released in the same incubation time . The contribution of elicitors to plant pathogen specificity is unresolved . Synergism t Present address : Department of Biology, Cornell College, Mt . Vernon, IA 52314, U .S .A . + To whom all correspondence should be addressed .

0885--5765/90/020147+12 $03 .00/0

© 1990 Academic Press Limited



148

C . S . Tepper and A . J . Anderson

between elicitors is possible . Synergism has been reported between fungal glucans and lipid components from Phytophthora infestans [14] in potato . Synergism also occurred in soybean and parsley tissues with plant pectic fragments and glucans derived from the cell wall of P. megasperma [5, 7] . We have demonstrated that extracellular products from the a and 13 races of Colletotrichum lindemuthianum differed in elicitor activity when assayed on three bean cultivars [19, 20] . One of the extracellular products from the a race, a galactoglucomannan, was active as an elicitor on a race-incompatible cultivar Dark Red Kidney but was inactive on a race-compatible Great Northern [19, 201 . The studies reported in this paper were conducted to determine if pectic fragments interacted with the elicitor potential of crude, partially purified and purified extracellular components from the a and (3 races of C . lindemuthianum . Recently, Dixon et al . [91 indicated that there was no synergism in bean suspension cells between pectic fragments and a mixture of elicitors derived from the cell wall of C. lindemuthianum . Synergism was determined by the activity of an enzyme required for phenolic and phytoalexin synthesis, phenylalanine ammonia-lyase (PAL) . In our studies, elicitor activity was assessed by measuring the accumulation of soluble and condensed phenolics as well as phytoalexins in elicitor-treated bean cotyledons . Altered accumulation of mRNAs for PAL and another enzyme involved in phenolic biosynthesis, chalcone synthase (CHS), was also monitored .

MATERIALS AND METHODS

Plant material Dark Red Kidney and Great Northern bean seeds were grown for 5 days in darkness as previously described [19] . Cotyledons were removed, sterilized in I "' , hypochlorite for 15 min, rinsed in sterile distilled water and sliced for use in the elicitor assay [191 . Virulence patterns on Dark Red Kidney and Great Northern with a and (3 races of were determined as described previously [19] .

C . lindemuthianum Sources of elicitors

Pectic fractions with elicitor activity were prepared from citrus pectin by procedures described by Stekoll & West [18] . The size of these fragments was between 9 and 15 galacturonic acid units . The C . lindemuthianum extracellular elicitors were obtained from culture filtrates of the a and ß races as described by Tepper & Anderson [20] . Crude culture filtrate and DEAE-Sephadex non-adsorbed and adsorbed fractions were used . The fractions (1-8) or the a race corresponded to materials that eluted with KCl concentrations : 1, 0-0 . 001 M ; 2, 0. 001-0 . 06 M ; 3, 0 . 06-0 •09 M ; 4, 0 . 09-0 . 12 M ; 5, 0-12-0-15 M ; 6, 0. 150 . 28 M ; 7, 0 . 28-1 . 3 M ; 8, 1 . 4-1 . 9 M, and fractions (1-6) for the [3 race : 1, 0 . 00-0 . 04 M ; 2, 0 . 04-0. 08 M ; 3, 0 . 08-0 . 11 M ; 4, 0. 11-0. 15 M ; 5, 0 . 15-0 . 27 M ; 6, 0 . 28-1 . 8 M . A galactoglucomannan, obtained by further purification [20] of the a race DEAESephadex non-adsorbed fraction, was also used .



Interactions with

Colletotrichum lindemuthianum

149

Determination of elicitor activity

Elicitor activity was determined on cotyledons of Dark Red Kidney and Great Northern bean cultivars by procedures described by Tepper & Anderson [19, 20] . Water was the control treatment . The accumulation of low molecular weight phenolics after 24 h of treatment was measured by extracting cotyledons with ethanol and measuring absorbance at 280 nm using salicylic acid as as standard . One unit of elicitor activity at 280 nm is defined as the increase in pg salicyclic acid equivalents per cotyledon in ethanol extracts from elicitor-treated cotyledons above that from cotyledons treated with water . Phytoalexin concentrations in these ethanol extracts were determined by HPLC on a C-18 column by the method described in Rogers et al . [17] . Condensed phenolics were assayed by extracting ethanol-treated tissues with 0 . 5 M NaOH at 70 °C for 15 min . The absorbance of the extract was recorded at 400 nm and compared to data obtained using Kraft lignin . One unit of elicitor activity at 400 nm represents an increase in equivalents of Kraft lignin (µg per cotyledon) in extracts prepared from treated cotyledons above that in extracts from control, watertreated cotyledons . Elicitor activity was assayed qualitatively by observing the degree of browning of the cotyledons . A+indicates slight browning and + + + + maximum browning . The accumulation of mRNAs specific for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) in treated cotyledons were measured by total RNA extraction from 3 . 3 g of tissue and Northern blot analysis by following the procedures described by Tepper et al. [19] . Cotyledons were harvested at 0 to 8 h after treatments with water, crude a or ß race elicitors and/or pectic fragments . The procedure was modified by using labelled RNA probes . The cDNA clones for PAL and CHS were obtained from Dr C . Lamb, (Salk Institute, San Diego, CA) and were subcloned into p-Bluescript KS (Stratagene, La Jolla, CA) . RNA probes labeled with 32 P-UTP were prepared by transcription of the phagemid from the T 3 promoter following linearization with Bam HI . These RNA probes were hybridized to blots (Gene Screen Plus, New England Nuclear, Boston, MA) of total RNA (15 µg) extracted from treated cotyledons, which was glyoxated following procedures described by Tepper et al. [19] . Filters were prehybridized and hybridized at 65 °C in buffer containing 50 0, 0 formamide . Following hybridization, the filters were washed at 65 ° C in 0. 1 x SSC and 0. 1 % SDS . Hybridization was quantified by methods described by Tepper et al . [19] . Messenger RNA accumulations in cotyledons were monitored within single blots for all time courses . RESULTS Virulence

The a race was compatible on Great Northern and produced small lesions on hypocotyls, whereas on Dark Red Kidney only limited flecks characteristic of the incompatible hypersensitive response were observed . The ß race was compatible on both cultivars ; lesions on hypocotyls were large and spreading on Dark Red Kidney but more limited lesions were formed on Great Northern .



1 50

C . S . Tepper and A . J . Anderson TABLE 1

Concentration effects of elicitors on Dark Red Kidney cotyledons

Treatment pg carbohydrate equivalents/cotyledon Pectic fragments

500 100 50 5 0. 5

Browning + + + + + + +

a Race factors

22 . 5 2.5 0 . 25 0 . 025

+ + + + -

ß Race factors

12 . 5 1 . 25 0 . 125 0 .025

+ -

Elicitor activity' units 280 nm 400 nm

K

Phytoalexins' pg/cotyledon PI

Ph

78 26 10 0 0 180 54 28 16

25 15 15 10 0

20 0 0 0

0 0 0 0

15 0 0 0

48 20 10 0

120 10 0 0

10 0 0 0

60 0 0 0

12 0 0 0

30 8 8 0

20 0 0 0

0 0 0 0

8 0 0 0

a

Pectic fragments were obtained as described in Materials and Methods, and were used to treat Dark Red Kidney cotyledons at the designated concentrations . Crude filtrate preparations were obtained from a and ß races as described in Tepper & Anderson [20] and were used at the designated concentrations. ° Elicitor activity was assayed as described in Materials and Methods . Data are results typical of five distinct studies . K, kievitone ; PI, phaseollinisoflavan ; Ph, phaseollin.

Effect of pectic fragments on phenolic accumulations : responses on cultivar Dark Red Kidney

Pectic fragments were active as an elicitor on Dark Red Kidney cotyledons (Table 1) . The tissue browned within 9 h of treatment with pectic fragments and darkened with time . Low molecular weight phenolics, phytoalexins and condensed phenolics accumulated in the tissues treated with higher concentrations of pectic fragments . Preparations from the ß race induced only low accumulations of soluble phenolics and phytoalexins ; however, browning occurred and condensed phenolics were produced (Table 1) . The a race fractions were the most active elicitors in promoting the accumulations of soluble phenolics and phytoalexins . Cotyledons treated with a race products produced more kievitone, phaseollinisoflavan and phaseollin than water treatments . Treating cotyledons with either pectic fragments or a and ß race C . lindemuthianum products did not enhance the accumulation of material that stained red with phloroglucinol, a test indicating the presence of lignin . The presence of pectic fragments enhanced the elicitor activity of both the a and ß race preparations on Dark Red Kidney (Table 2) . Data shown in Table 2 involved treatment of cotyledons with concentrations of a race components and pectic fragments that alone had weak elicitor activity ; the ß race preparation had no elicitor activity . The addition of pectic fragments to the a race preparation enhanced production of low molecular weight phenolics nearly 2-fold and increased the synthesis of condensed phenolics about 4-fold . There were higher levels of the phytoalexins kievitone and phaseollin in cotyledons treated with both pectic fragments and a race products than with either alone . Production of phytoalexins and condensed phenolics also increased



Interactions with Colletotrichum lindemuthianum

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TABLE 2

Elicitor activity on Dark Red Kidney cotyledons following treatment with C . lindemuthianum fractions and pectic fragments

Treatments

Browning

280

Elicitor units' nm 400 nm

Pectic fragments + 15 (±5) 15 (±2) a Race factors + 46 (±523 (f5) a Race factors +pectic + + + 82 (± 5) 93 (± 10) fragments 0(±0) 3(±1) 0 Race factors ß Race factors+ pectic + + 20 (±2) 25 (±0) fragments

Phytoalexins° µg/cotyledon K PI Ph 2 0 50

2 10 40

0 0 0

0 33

0 0 0 17

a Pectic fragments were obtained as described in Materials and Methods, and were used at a concentration of 50 µg per cotyledon . Crude filtrate preparations were obtained from a and 3 races as described in Tepper & Anderson [20] and were used at a concentration of 2 . 3 and 1 .3 µg carbohydrate equivalents per cotyledon respectively . ' Elicitor activity was assayed as described in Materials and Methods . Data are the means of five assays and the standard error of the mean is provided . e K, kievitone ; PI, phaseollinisoflavan ; Ph, phaseollin .

following treatment with (3 race preparations and pectic fragments . These synergistic effects were seen with three different preparations of culture filtrate from the a and ß races . To investigate the nature of the fungal components that could act synergistically with the pectic fragments, interactions and fractionated culture filtrate products were examined (Table 3) . The fractions were obtained by DEAE-Sephadex chromatography in 20 mm Tris-HCI pH 8 . 2 [20] . Adsorbed fractions (1-8 for a race products and 1-6 for (3 race products) were eluted with a gradient of KCI (0-2 . 0 M) in 20 mm Tris-HCI (pH 8. 2) and pooled according to protein and carbohydrate peaks [20] . The phenolic responses varied with each a race fraction (Table 3) . Adsorbed fraction 1 and pectic fragments acted synergistically for the production of soluble phenolics, phytoalexins and condensed phenolics . Adding other a race-adsorbed fractions (3, 4 and 5) with pectic fragments also enhanced phytoalexin production, and fractions 2 and 3 interacted with pectic fragments to increase the production of condensed phenolics . Pectic fragments with ß race non-adsorbed material and adsorbed fractions 2, 3 and 4 increased the production of soluble phenolics and phytoalexins, but did not affect the accumulation of condensed phenolics . Galactoglucomannan, (GGM), exhibited a complex pattern of stimulation depending on the concentration of both fungal product and pectic fragments (Table 4) . At the higher concentration of GGM, there was little stimulation in the production of condensed phenolics but some increase in the production of soluble phenolics and phytoalexins . At lower GGM concentrations, pectic fragments had a greater effect on the accumulation of soluble and condensed phenolics including phytoalexins . Responses on cultivar Great Northern Elicitor activity in ß race preparations in the presence of pectic-fragments on the compatible cultivar Dark Red Kidney prompted the attempt to detect synergism in



1 52

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Interactions with Colletotrichum lindemuthianum

153

TABLE 4 Concentration effects on interactions between an elicitor from C . lindemuthianum [a purled galactoglucomannan (GGM)] and pectic fragments in Dark Red Kidney cotyledons

Elicitor activity units" 400 nm 280 nm

Trcatmenta

GG Ni (5 pg) GGM (5 pg)+P (50 pg) GG %1 (1 pg) GG M ( 1 pg) + (50 pg) GUM (1 pg) + P (5 pg) GG .N-1 (I pg)+P (0. 5 pg) P 1 50 p1n ) P (5 pin) P (0 . 5 pmj

340 500 20 134 52 40 10 0 0

105 120 10 83 38 10 15 10 0

Phvtoalexins' jig/cotyledon PI Ph

K 130 188 0 33 10 (1 2 0 NI)

11 12 0 0 0 0 0 0 ND

63 93 fl 43 0 (t 2 0 ND

a Galactoglucomannan (GGM) was prepared from the a race of C. lindemuthianum as described by Tepper & Anderson [20] . GGM was used at concentrations of 5 or I pg per cotyledon as designated . The pectic fragments (P) were prepared as described in Materials and Methods and were used at concentrations ranging from 50 to 0. 5 pg per cotyledon . " Elicitor activity was quantified by assays described in Materials and Methods . Data are representative of three replicate studies . K, kievitone ; P1, phaseollinisoflavan ; Ph, phascollin .

TABLE 5 Effect of pectic fragments on elicitor activity of fungal components in Great Vorthern cotyledons

Treatment``

a a +P ß (3+1' P

Browning"

++ ++ +

Elicitor Units' 280 nm 400 nm

11 (±3) 15 ("±3) 0 0 0

12 ;±3) 34 (±51 0 20 ±5 ; 7 ;+2)

Phytoalexinse Vg/cotyledon K PI Ph

14 14 0 0 7

0 0 0 0 0

0 0 0 0 0

a Great Northern cotyledons were treated with a or 13 race crude extracellular products at 2 .3 and 1 . 3 pg carbohydrate equivalents per cotyledons and pectic fragments at 50 jig per cotyledons as indicated using procedures described in the Materials and Methods . " Elicitor activity was assayed as described in Materials and Methods . K, kievitone ; PI, phaseollinisollavan ; Ph, phaseollin .

cultivar Great Northern, which is weakly compatible with both a and (3 races . Results (Table 5) demonstrated that pectic fragments alone had a limited ability to induce the production of condensed phenolis . The a and ß race preparations were also weak elicitors . Production of the condensed phenolics but not of the soluble phenolics or phytoalexins was enhanced by either a or (3 race components and pectic fragments (Table 5) . Effects of pectic fragments on mRNA accumulations : responses on Dark Red Kidney Hybridization to CHS and PAL mRNA occurred in total RNA preparations from the control cotyledons treated with water . Hybridization for CHS mRNA was enhanced in preparations from Dark Red Kidney cotyledons treated with pectic fragments,



1 54

C. S . Tepper and A . J . Anderson 6000

5000

4000

o 0

00 .c

3000

f a U 2000

1000

0

2

3

4

5

6

7

8

Time (h) FIG . 1 . Accumulation of hybridizable mRNA for chalcone synthase from Dark Red Kidney cotyledons treated with elicitors. Cotyledons were treated with either a race crude elicitor (2 . 2 µg glucose equivalents per cotyledon) ® ; pectic fragments (50 µg per cotyledon) N ; a crude elicitor and pectic fragments (at the above concentrations) ®, or distilled water (®) . Hybridization were performed as previously described [19] . Data represent a single experiment that was repeated three times .

a race crude elicitor, or a mixture of fungal and plant elicitors . However, accumulation showed no synergism upon treatment with the mixed fungal and plant elicitors . Accumulation varied over time (Fig . 1) . Peaks occurred at 2 and 6-8 h of treatment . Each treatment had a distinct pattern of maximum accumulations : 2 and 6 h for the a race elicitor, 8 h for the pectic fragments and 6 and 7 h for the mix . Similar results were obtained when RNA was hybridized with a PAL specific probe . Mixed elicitors did not have an additive effect on the accumulation of PAL mRNA (data not shown) . Potential synergism in a compatible system was investigated by examining the effects of ß race elicitor and pectic fragments on Dark Red Kidney (Fig. 2a and b) . Unlike the interaction using the incompatible a race products (Fig . 1), the combination of 3 race products and pectic fragments increased the accumulations of both CHS (Fig . 2 a) and PAL (Fig . 2b) mRNAs . The effects varied with time . Synergism was apparent only at 6 h whereas additive effects were observed at other time points . Cotyledons treated with 3 race material accumulated less PAL mRNA than water-treated control cotyledons (Fig. 2b) . Responses on Great Northern

The additive effects of pectic fragments and preparations from a compatible fungal race in Dark Red Kidney cotyledons prompted studies in which Great Northern was

Interactions with Colletotrichum lindemuthianum

155

2

3

4

5

6

7

Time (h) FIG . 2 . Accumulation of hybridizable mRNA for chalcone synthesis or phenylalanine ammonia lyase mRNA from Dark Red Kidney cotyledons treated with elicitors . Cotyledons were treated with either ß race crude elicitor (1 . 2 pg glucose equivalents per cotyledon) / ; pectic fragments (50 gg per cotyledon) / ; a combination of ß crude elicitor and pectic fragments (at the above concentrations) ®, or distilled . water (2 b only) (/) . Hybridization for CHS (2 a) or PAL (2 b) RNAs were performed as described [19] . Data represent a single experiment that was repeated twice .

treated with preparations from the a race which is compatible with this cultivar (Fig . 3a and b) . Pectic fragments alone enhanced mRNA accumulation more than the fungal preparations (Fig. 3a) . The mixture of elicitors had an additive effect, especially at the 6, 7 and 8 h. the level of PAL mRNA was only slightly higher than in water-treated cotyledons except at 2 h, following treatment with the pectic fragments and the mixture of elicitors (Fig . 3 b) . DISCUSSION Pectic fragments have been demonstrated to possess elicitor activity in bean by promoting accumulation of low molecular weight phenolics, including phytoalexins and condensed phenolics . The alkali lability of the condensed phenolics suggests that they have an ester linkage . Esterification of phenolics onto polysaccharides in the cell wall has been reported [10, 21] . The condensed product that accumulated in cotyledons treated with pectic fragments did not appear tcshave a typical lignin-like reaction with phloroglucinol . Thus, this response in bean is distinct from that activated in cucumber cotyledons, where enhanced lignin formation occurred following exposure to pectic

8



1 56

C. S . Tepper and A . J . Anderson

o o o

4

5 Time (h)

6

4

5 Time (h)

FIG . 3 . Accumulation of hybridizable mRNA for chalcone synthase phenylalanine ammonia lyase in Great Northern cotyledons treated with elicitors . Cotyledons were treated with either a crude elicitor (2 . 2 pg glucose equivalents per cotyledon) ; pectic fragments (50 pg per cotyledon) / ; a combination of a crude elicitor and pectic fragments (,at the above concentrations) ®, or distilled water ® . Hybridization for CHS (3 a) or PAL (3b) RNAs were performed as previously described [19] . Data represent a single experiment that was repeated twice .

fragments [16] . The increase in phytoalexins in bean is similar to responses in soybean and parsley treated with pectic fragments [5, 7] . On a weight basis, pectic fragments were less effective than fungal elicitors from C . lindemuthianum . Pectic fragments acted synergistically with fungal components from C . lindemuthianum to promote elicitor activity, based on phenolic accumulations . Synergism was observed with pectic fragments and a purified polysaccharide-rich elicitor, a galactoglucomannan, from the incompatible a race as well as a crude mixture of fungal elicitors on Dark Red Kidney . Components from the compatible [3 race which possessed little autonomous elicitor activity had a synergistic effect on the production of phenolics in Dark Red Kidney . One of these components was the DEAE-Sephadex non-adsorbed, polysaccharide-enriched fraction with a neutral carbohydrate composition of 8 . 5 % mannose, 2 . 7 % galactose, and 88 . 7 % glucose. This interaction may be compared to the synergism in potato between arachidonic acid and glucans from P . infestans [14], although these glucans alone possessed suppressor activity [15] . Glucans and pectic fragments also had a synergistic effect on the production of soluble phenolics in soybean, a tissue which is sensitive to glucans as elicitors [51 . These observations suggest that there are multiple components of diverse structure from pathogenic fungi that can act synergistically with pectic fragments to stimulate the accumulation of phenolic defence-related products . The cellular mechanisms of this



Interactions with Colletotrichum lindemuthianum

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process are unclear . Our data demonstrate very limited additive or synergistic effects between the products of an incompatible race and pectic fragments upon the accumulation of mRNAs for PAL and CHS, although both of these enzymes are involved in early stages of phenolic and phytoalexin biosynthesis . Our lack of additive effects upon PAL mRNA accumulation agrees with the finding of Dixon et al. [9], who did not detect synergism in PAL activity in bean cell suspension cultures treated with a mixture of elicitors from the cell wall of C. lindemuthianum and pectic fragments isolated from bean . Consequently, the regulation of the synthesis or activity of other enzymes in the biosynthetic pathways may be the key to enhanced production of phenolics . Our studies on the effects of pectic fragments and fungal components, alone or as mixtures, show the complexity of the plant's response at the mRNA level . When cotyledons are treated with elicitors, CHS and PAL mRNA accumulate in a wave-like pattern [19], and the patterns for different elicitors are not identical . When Dark Red Kidney cotyledons are treated with preparations from compatible (3 race of C . lindemuthianum, less mRNA for PAL accumulates than in water-treated controls . This depression may be related to a suppressor in the fungal preparation . Several other fungal pathogens are reported to produce suppressors . For example a glycoprotein from Ascochyta rabiei inhibited elicitor-induced isoflavonoid production in chick pea cotyledons [12] . Treatment with pectic fragments and preparations from compatible (3 race products on Dark Red Kidney and a race components on Great Northern had additive effects on the mRNA level for PAL and CHS . The enhanced accumulations of mRNA for these enzymes that are involved with phenolic biosynthesis may be directly related to the increase in phenolic depositions . Pectic fragments markedly enhanced the production of phenolics and phytoalexins in Dark Red Kidney treated with [3 race preparations . In Great Northern the pectic fragments increased browning and accumulation of condensed phenolics in the presence of of and (3 race products . Pectic fragments might be generated in the interaction of C. lindemuthianum and bean because this fungus produces pectic-degrading enzymes [11, 22] . Consequently, both the fungal products and pectic fragments may be present in vivo to stimulate phenolic defences in the host . In the compatible interaction these responses may help delimit the lesion areas . Results of our studies suggest that both plant and fungal factors interact with each other to regulate the expression of defence response in compatible as well as the incompatible plant-microbe challenges . The authors are grateful to Dr R . Whetton for discussion and preparation of the p-Bluescript clones, M . Copeland for technical assistance and Dr J . Bowman and R . Buell for suggestions . This work was supported by grants from the USU Biotechnology Center and from the National Science Foundation to Anne J . Anderson . Utah State Agricultural Experiment Station Paper Number 3838 .

REFERENCES J . (1988) . Elicitors, the hypersensitive response and phytoalexins . In Physiology and Ed . by N . T . Keen, T. Kosuge & L . L. Walling, pp . 103-110 . American Society of Plant Physiologists, Rockville, Maryland . CERVONE, F ., DE LORENZO, G ., DEGRA, L., GIOVANNI, S . & BERGAMI, M . (1987) . Purification and

1 . ANDERSON, A .

Biochemistry of Plant-Microbial Interactions,

2.



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