Chitinase mRNA and enzyme activity in Phaseolus vulgaris (L.) increase more rapidly in response to avirulent than to virulent cells of Pseudomonas syringae pv. phaseolicola

Physiological and Molecular Plant Pathology (1989) 35, 403-412

403

Chitinase mRNA and enzyme activity in Phaseolus vulgaris (L .) increase more rapidly in response to avirulent than to virulent cells of Pseudomonas syringae pv. phaseolicola CHRISTINE R . VOISEY Department

of Applied

and A . J . SLUSARENKOt

Biology, University

of Hull,

Hull, HU6 7RX, U. K.

(Accepted,/or publùalion May 1989)

Changes in chitinase mRNA concentration in leaves of French bean cultivar Red Mexican, after inoculation with virulent or avirulent races of Pseudomonas syringae pv . phaseolicola, were estimated by probing Northern and dot blots with radiolabelled cDNA for bean chitinase . In leaves inoculated with the avirulent race 1 isolate of the pathogen, chitinase mRNA levels had begun to increase by 6 h after inoculation . In contrast, chitinase mRNA levels first began to increase between 20 to 24 h after inoculation of leaves with a virulent race 3 isolate of the pathogen . Chitinase mRNA activity was estimated by immunoprecipitation of in vitro translation products using antiserum raised against bean chitinase . Changes in chitinase mRNA activity reflected closely, changes in mRNA concentration . Increases in extractable chitinase enzyme activity occurred 3-6 h after increases in mRNA concentration and activity . As far as we are aware this is the first report of the differential induction of chitinase mRNA and enzyme activity in a host plant by virulent and avirulent races of a homologous bacterial plant pathogen . Heat-killed and UV-killed cells of both avirulent and virulent races of P .s . pv . phaseolicola induced chitinase enzyme activity . This shows that chitinase is induced separately from the hypersensitive reaction and phytoalexin synthesis ; both of which are induced only by metabolically active, living bacteria .

INTRODUCTION

Chitinase hydrolyses the ß-(1,4)-glycosidic bond in chitin, a polymer of Nacetylglucosamine . Chitinase activity increases in several plants after inoculation with fungal, bacterial and viral pathogens [6, 16, 24, 26-28, 32, 33], and after treatment with ethylene or elicitors [5, 9, 12, 24, 34] . Chitin has not thus far been found in plants [4], although, it is a common constituent of insect cuticles and of most fungal cell walls [2] . The breakdown products of chitin can serve as elicitors of plant defence reactions [14] . Chitin is deposited in fungi predominantly at the growing hyphal tip [13] and it has been shown recently that plant chitinases are potent inhibitors of hyphal extension and growth [31, 35] . Chitin has features in common with the glycan portion ofpeptidoglycan, a major constituent of Gram-positive and Gram-negative bacterial cell walls . +To whom correspondence should be addressed at : Institut h r Pflanzenbiologie der Universität Zürich, Zollikerstr. 107, CH-8008 Zürich, Switzerland . 0885--5765/89/110403 + 10 $03 .00/0

© 1989 Academic Press Limited



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Peptidoglycan comprises alternate ß-(1,4)-linked N-acetylglucosamine and N-acetylmuramic acid residues and is hydrolysed by lysozyme . Chitinase has lysozyme activity under certain conditions [5, 31] . Chitinase is widely distributed amongst higher plants [4], including a variety of seeds [20, 29, 31], and the production of this enzyme may be an active defence mechanism of plants against invading microorganisms [4, 5] . The French bean (Phaseolus vulgaris)/Pseudomonas syringae pv . phaseolicola combination provides a good experimental system in which to study the interaction between host cultivars and pathogen races . The French bean cultivar Red Mexican is resistant to race 1 isolates of the pathogen but is susceptible to races 2 and 3 . A coordinated set of specific changes in host gene expression occurs during the resistance response to an avirulent race of the pathogen [36] . The purpose of this work was to investigate changes in bean chitinase mRNA and enzyme activity upon inoculation with isolates of virulent or avirulent races of P.s. pv . phaseolicola, to assess the possible contribution of induced chitinase to the spectrum of defence responses in bean plants .

MATERIALS AND METHODS

Plant material Seedlings of Phaseolus vulgaris (L .) cv . Red Mexican were grown, and inoculated by vacuum infiltration, as described previously [36] . Inoculated leaves were harvested from the plants at the desired sampling time, frozen in liquid nitrogen and stored at - 75 ° C .

Bacteria Race 1 isolate 31A and race 3 isolate 1301A were cultured and maintained as described previously [36] with the exception that the bacteria were suspended in 0 . 0014 M KH 2 PO 4 and 0 . 006 M Na 2 HPO4 (pH 7 . 3) for inoculation, and adjusted to OD,,,, = 0 .6 corresponding to a viable count of approx . I x 108 cfu ml -1 . Heat-killed cells were prepared by autoclaving the inoculum for 15 min at 120 °C . UV-killed cells were prepared by exposure to UV light at 254 nm at 0 . 65 Wm -2 for 10 min (total UV dose = 3 .9 x 10 2 Jm -2 ) . Fifty µl samples of irradiated inoculum were placed on to nutrient agar (Oxoid) supplemented with 1 % glycerol and incubated at 25 °C to test for viability . Invariably, no colonies were detected, indicating that viability had been reduced from 1 x 108 cfu ml -r to < 20 cfu ml -1 .

Northern and dot blot analysis of chitinase mRNA Total RNA was extracted from plants at various times after inoculation as described previously [36] . For Northern blots, 7 . 5 gg of RNA was denatured by glyoxalation and electrophoresed on a 1-2% (w/v) agarose gel after the method of Carmichael & McMaster [11] . The buffer used throughout was 0. 01 M sodium phosphate (pH 7 . 0) . After electrophoresis, RNA was transferred by capillary blotting on to filters (Hybond-N, Amersham) according to the manufacturers instructions . For RNA dot blots, 1 . 8 gg of glyoxalated RNA was applied directly to Hybond-N . All filters were then vacuum baked for 2 h at 80 ° C to reverse the glyoxalation . The bean chitinase clone pCHT12 .3 [15] was kindly donated by C . Lamb . Filters were prehybridized in a mixture containing 5 x SSPE (1 x SSPE is 0 . 18 M NaCl, 0.01 M sodium phosphate



Differential chitinase activity in response to bacteria

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(pH 7 . 7) and 0 . 0001 M sodium EDTA), 50% (v/v) freshly deionized formamide, 5 x Denhardt's solution (1 x Denhardt's is 0 . 02 % (w/v) BSA, 0 . 02 % (w/v) ficoll and 0 . 02 % (w/v) polyvinylpyrollidone), 0 . 5 % (w/v) SDS and 20 gg ml -1 denatured salmon sperm DNA at 42 °C for 2 h . The hybridization conditions were the same as for prehybridization except that the mixture contained in addition the chitinase insert released from pCHT12 .3 by EcoRI-HindIIl digestion and labelled with 32 P using an oligolabelling kit (Pharmacia) according to the manufacturer's instructions ; incubation was overnight . The filters were washed at 42 °C in 2 x SSPE containing 0 . 1 % (w/v) SDS for 30 min, then 1 x SSPE, 0 .1 % (w/v) SDS at 42 °C for 2 x 15 min, followed by 0 . 1 x SSPE, 0 . 1 % (w/v) SDS at 42 ° C for 2 x 15 min . After drying, the filters were exposed at -75 ° C to preflashed X-ray film (Hyperfilm-MP, Amersham) . The autoradiographs were scanned using a Chromoscan-3 Joyce-Loebl) . Immunoprecipitation of in vitro translation products In vitro translation was carried out as previously described [36] . Incorporation of [35 S]methionine into trichloroacetic acid (TCA) precipitable protein was determined by liquid scintillation spectrometry as previously described [36] . Quantities of in vitro translation mix containing equivalent counts per minute incorporated into protein were taken for immunoprecipitation . The translation products were diluted to 70 µl with 1 M NaCl containing 1 % v/v Triton X-100 . Five microlitres of anti- (chitinase) serum (a kind gift from T. Boller) were added and the mixture incubated at 26 °C for 30 min . After incubation, 50 pl of protein-A-Sepharose (Pharmacia), 652 . 5 mg ml -1 in 25 mm Tris/HCl pH 7 . 5, was added and the mixture was incubated overnight at 4 °C with continuous gentle shaking . The protein-A-Sepharose was pelleted by centrifugation (5 min at top speed in an MSE Micro Centaur) and washed at least five times with 0 . 75 M NaCl containing 1 % (v/v) Triton X-100 . The pellets were resuspended in 20 µl of sample buffer (0.0625 M Tris/HCl pH 6.8, 10 % (v/v) glycerol, 2 %, (w/v) SDS, 0 . 1 M dithiothreitol, 0 . 0025 % (w/v) bromophenol blue) and heated at 100 °C for 4 min . Insoluble material was pelleted by centrifugation (5 min at top speed in the Micro Centaur) and the supernatant was loaded directly into the sample wells for electrophoresis . Electrophoresis Samples were electrophoresed on 10% (w/v) polyacrylamide slab gels with a 41'/0 (w/v) stacking gel [19] . [ 14 C]-labelled molecular weight standards were prepared by the method of Anderson [1] from bovine plasma albumin (Mr = 66000), ovalbumin (,W, = 45000), glyceraldehyde-3-phosphate dehydrogenase (Mr = 36000) and bovine pancreas trypsinogen (Mr = 24000) purchased from Sigma . Gels were fixed overnight in ethanol-glacial acetic acid-water (15 :6 :29 by volume), then washed in distilled water for 30 min and immersed in at least 10 x the gel volume of 1 M sodium salicylate (pH 6) for 30 min . Gels were dried under vacuum at 60 °C and fluorography carried out at -75 ° C with pre-flashed Hyperfilm-MP (Amersham) . Fluorographs were scanned using a Chromoscan-3 (Joyce-Loebl) . Despite attempts to standardize the total cpm loaded into each well, some variation was always apparent on the exposed fluorographs . Thus, the integral of the peak corresponding to chitinase, as a percentage of the integral for a band at M, about 50000, the intensity of which



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did not appear to vary on infection, was used as the estimate of chitinase mRNA activity . Crude enzyme preparation Crude enzyme was extracted according to :Much et al . [24] from leaves frozen in liquid nitrogen and stored at - 75 ° C . The leaf tissue was ground in liquid nitrogen, then homogenized in a Potter homogenizer in 0 . 1 M sodium citrate pH 5-0 (1 :2 w/v) and centrifuged at 10000 x g for 10 min at 2 °C . The supernatant was collected and 2 ml samples desalted on a Sephadex fine G-25 (Pharmacia) 10 x 2 cm column equilibrated with an 85 mm phosphate buffer pH 6 . 4 . The extract was stored in glycerol (10% v/v at -75 ° C . Total protein was determined using Bradford's reagent [81 (Bio-Rad protein assay kit) with bovine serum albumin standards . Chitinase assay Colloidal chitin was prepared according to the method of Berger et al . [3] . Chitinase was assayed by the colorimetric method of :Much et al . [24] . The 0 . 5 ml reaction mixture contained 1 mg colloidal chitin and 50 gl diluted crude enzyme preparation in 85 mm phosphate buffer pH 6 . 4 . Samples were incubated at 37 ° C in an orbital shaker for 1 h . The reaction was stopped by boiling, the mixture centrifuged and 0 . 3 ml of the supernatant was incubated at 30 °C for 30 min with 10 gl of snail gut juice (Sigma) which had been desalted on a Pharmacia PD-10 column [l0] . The reaction was stopped by boiling and N-acetyl-D-glucosamine was determined by the method of Reissig et al . [30] . The rate of release of N-acetyl-D-glucosamine from colloidal chitin by chitinase is not directly proportional to enzyme concentration [5, 34] . Therefore, a dilution series of each crude enzyme preparation was assayed and activity plotted against concentration . The most linear portion of the curve was invariably where the enzyme was most dilute . Therefore, before calculating specific activities, enzyme samples were diluted so that their activity fell within the range of the initial slope [5] . Specific activity is expressed as µmoles of N-acetyl-n-glucosamine released per mg protein per h at 37 ° C . Each value is the mean of four replicate assays from one representative inoculation with at least two plants sampled per time point . The experiment was repeated in three independent inoculations . For the sake of clarity, error bars have been omitted from the graphs but the average standard error was 12 ° ;, of the mean .

RESULTS Induction of chitinase enzyme activity Low levels of chitinase activity were observed in healthy, buffer-inoculated leaves of Phaseolus vulgaris cv . Red Mexican (Fig . 1) . In leaves of Red Mexican inoculated with cells of the avirulent race I isolate (31A) of P .s . pv . phaseolicola (incompatible combination) an increase in chitinase activity was detected between 6-9 h after inoculation . Chitinase activity was well established by 12 h, and by 48 h after inoculation was elevated almost nineteen-fold over basal levels (Fig . 1) . In contrast, in leaves of Red Mexican inoculated with cells of the virulent race 3 isolate (1301A) of P .s . pv . phaseolicola (compatible combination) chitinase activity was still minimal by



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FIG . 1 . Timing of the appearance of mRNA activity (I---0) and mRNA concentration (RNA dot blots) for bean chitinase in relation to measurable chitinase enzyme activity (Q-Q) in leaves of P. vulgaris cv . Red Mexican after inoculation with avirulent race 1 cells of P.s . pv . phaseolicola . Chitinase enzyme activity in buffer inoculated control leaves is also shown (/ /) .

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Timing of the appearance of mRNA activity (00) and mRNA concentration (RNA dot blots) for bean chitinase in relation to measurable chitinase enzyme activity (Q-Q) in leaves of P. vulgaris cv . Red Mexican after inoculation with virulent race 3 cells of P.s . pv . phaseolicola . FIG . 2 .

24 h after inoculation but had risen to approx . 75 % of that in the resistant reaction by 48 h (Fig . 2) . Induction of chitinase mRNA

Northern (Fig . 3) and RNA dot blot analysis (Figs 1 and 2) of total RNA revealed an increase in hybridizable chitinase mRNA in the incompatible combination beginning between 3 and 6 h after inoculation . In the susceptible response chitinase mRNA increased between 18-24 h after inoculation . Thus, increase in chitinase mRNA preceded the increase in extractable chitinase enzyme activity in both the resistant and susceptible reactions by 3-6 h (Figs 1 and 2) . There was no detectable hydridizable chitinase mRNA in a Northern blot of total RNA extracted from bufferinoculated leaf tissue (Fig . 3) .



40 8

C . R . Voisey and A . J . Slusarenko HYPERSENSITIVE

RESPONSE

BUFFER CONTROL C

0

3

6

9

12

15

18

24

30

4

10 .5

Time after inoculation (h )

SUSCEPTIBLE RESPONSE

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Time after inoculation (h )

FIG. 3 . Induction of chitinase mRNA in P. vulgaris cv . Red Mexican leaves after inoculation with avirulent (A) and virulent (B) isolates of P .s . pv . phaseolicola or buffer (C) respectively . Total RNA was extracted at different time points after inoculation, electrophoresed in an agarose gel, blotted onto nylon filters and hybridized to a 32P-labelled probe prepared from the pCHT12 .3 chitinase clone .

Chitinase mRNA activity, estimated by immunoprecipitation of in vitro translation products of total RNA and scanning densitometry of fluorographs after electrophoresis, increased at the same time as mRNA concentration increased on Northern and RNA dot blots (Figs 1, 2 and 3) . This suggests that induction of chitinase enzyme activity may be controlled, at least in part, at the level of transcription . Chitinase elicitation by heat- and UV-killed cells

Heat-killed and UV-killed cells of the avirulent (race 1) isolate of P .s. pv . phaseolicola induced chitinase activity but at a lower level than did inoculation with living cells . By



Differential chitinase activity in response to bacteria

409

HEAT-KILLED CELLS

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FIG . 4 . Elicitation of chitinase activity in bean leaves inoculated with heat-killed cells of an avirulent race 1 isolate (A--A) and a virulent race 3 isolate (,L-A) of P .s. pv . phaseolicola .

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FIG . 5 . Elicitation of chitinase activity in bean leaves inoculated with UV-killed cells of an avirulent race I isolate (A-A) and a virulent race 3 isolate (Q-Q) of P.s. pv . phaseolicola .

48 h after inoculation, heat-killed race 1 cells had elicited 75 %, and UV-killed race 1 cells only 45 % of the activity elicited by living race I cells (cf . Figs 1, 4 and 5) . Similarly, heat-killed and UV-killed cells of the virulent race 3 isolate induced less chitinase activity after 48 h than did living cells (cf. Figs 2, 4 and 5) . However, induction of chitinase by heat-killed and UV-killed cells was earlier than with living, virulent race 3 cells . This was most noticeable with UV-killed race 3 cells which elicited chitinase activity at about the same time as did race 1 cells (living or dead) . The response to autoclaved race 3 cells more closely resembled the response to living race 3 cells and led us initially to suspect a race-specific elicitation of chitinase by dead bacteria . However, the results with UV-killed cells and corresponding experiments (unpublished work) with cv . Tendergreen (resistant to race 3 isolates but susceptible to race 1 isolates) did not support this hypothesis .



41 0

C . R . Voisey and A. J . Slusarenko

DISCUSSION

Differential induction of chitinase mRNA and enzyme activity has been reported in Phaseolus vulgaris hypocotyls upon infection with spores of avirulent and virulent races of the anthracnose-causing pathogen Colletolrichum lindemuthianum [15] . It has also been observed that Yicotiana tabacum cv . Samsun plants carrying the V allele are resistant to TMV and accumulate chitinase within 3 days following inoculation . Susceptible Samsun nn plants do not show elevated chitinase activity upon infection up to 6 days after inoculation [38] . As far as we are aware this is the first report of the differential induction of chitinase mRNA and enzyme activity in a host plant by virulent and avirulent races of a homologous bacterial plant pathogen . No evidence of chitinase activity could be found in fluids from axenic cultures of either race of P .s . pv . phaseolicola tested, and we have assumed that chitinase of bacterial origin is not produced in planta . The enzyme is evidently of plant origin because autoclaved cells of races 1 and 3 induce chitinase and bean chitinase mRNA was found to increase in response to living bacteria . Increase in chitinase mRNA concentration and activity (Figs 1, 2 and 3), suggests that increases in enzyme activity may be regulated, at least in part, by elevated transcription of the gene(s) involved . At least two of the genes in the multigene family for chitinase in bean were shown to be expressed in response to ethylene [9] and it is possible that other members of this family are expressed in response to pathogens . Indeed, different isozymes of chitinase have been reported from several sources [16, 18, 32, 37], and in pea pods chitinase isozymes appear to be differentially regulated with respect to stage of development and response to fungal pathogens [25] . The resistance of French bean to avirulent isolates of P .s . pv . phaseolicola is associated with a hypersensitive reaction and accumulation of phytoalexins [22] . Living bacterial cells are needed to induce the hypersensitive response and a period of host protein synthesis is required for hypersensitive cell collapse to occur [17, 23] . Chitinase was induced in Red Mexican leaves by heat- and UV-killed cells of the avirulent race I isolate (Figs 4 and 5) in the absence of a hypersensitive reaction . Neither the hypersensitive response nor antibacterial isoflavonoids are induced by heat-killed [21] or UV-killed (Slusarenko, unpublished work) cells of P .s . pv . phaseolicola . Further work to characterize the elicitor of chitinase from P .s . pv . phaseolicola is in progress . Chitinase activity has been reported to increase in the intercellular spaces of leaves in response to pathogen attack [6, 14, 18, 27] . However, it has been shown that ethylene induced chitinase accumulates in the vacuoles of bean cells [7] . Bacteria remain in the intercellular spaces throughout the course of an infection . We have not attempted to assay for chitinase activity in intercellular washing fluid . However, upon cell collapse at the manifestation of the separately-induced hypersensitive reaction, the contents of the vacuole would, presumably, be released into the intercellular spaces . Thus, in the hypersensitive reaction, the pathogen would be exposed to a concentrated burst of chitinase at the time of cell collapse . Purified bean chitinase shows lysozyme activity against the Gram-positive cell walls of Micrococcus lysodeikticus [5] . Lysozyme activity is induced coordinately with chitinase activity in crude enzyme preparations from Red Mexican leaves . However, crude enzyme preparations with high chitinase activity fail to lyse cells of Gram-negative P .s . pv . phaseolicola (Voisey & Slusarenko, unpublished work) ; presumably because the



Differential chitinase activity in response to bacteria

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outer membrane restricts access of the enzyme to the peptidoglycan substrate . Thus, although there is a clear differential induction of chitinase in French bean leaves by avirulent and virulent isolates of P .s. pv . phaseolicola respectively, there is no clear role for chitinase in resistance to that particular pathogen . It is possible that chitinase is induced simply as a component of the whole spectrum of plant responses to an incompatible pathogen . Many plants, when treated with elicitors or inoculated with pathogens, produce both ethylene and chitinase . It has been suggested that ethylene may act as a second messenger for chitinase induction . In melon seedlings (Cumumis melo) treated with elicitor from Collelotrichum lagenarium, inhibition of ethylene biosynthesis by treatment with aminoethoxyvinylglycine (AVG) also inhibited chitinase induction [34] . However, in pea (Pisum sativum) pods inoculated with Fusarium solani f .sp . phaseoli or treated with elicitors, ethylene biosynthesis could be suppressed without inhibiting the induction of chitinase [24] . Thus, different control mechanisms may exist regulating the induction of chitinase in different plant species . We have investigated changes in ethylene levels in inoculated bean leaves and are now exploring the possibility that ethylene might be acting as a second message for chitinase induction in this host-pathogen interaction . Warmest thanks are due to T . Boller and U . Vögeli for a generous gift of antiserum to bean chitinase, and likewise to C . J . Lamb for a bean chitinase clone . This research was supported by an SERC appeal studentship (C .V .) and an award from the Gatsbv Charitable foundation (A .J .S .) .

REFERENCES I.

J . (1979) . The structure and amount of actin in IMR-90 fibroblasts . Biochemical Journal 179, 425--430 . 2 . BARTNICKI-GARCIA, S . (1970, . Cell wall composition and other biochemical markers in fungal phylogeny . In Phytochemical Phylogeny, Ed . by J . B. Harborne, pp. 81-103 . Academic Press, London . 3 . BERGER, L . R . & REYNOLDS, D. M . (1958) . The chitinase system of a strain of Streptomyces griseus . Biochimica et Biophysica Acta 29, 522-534 . 4 . BOLLER, T . (1985) . Induction of hydrolases as a defense reaction against pathogens . In Cellular and Molecular Biology of Plant Stress, Ed . by J . L. Key & T . Kosuge, pp . 247-262 . Liss, New York . 5 . BOLLER, T ., GEHRI, A., MAUCH . F . & VOEGELI, U . 19831 . Chitinase in bean leaves : Induction he ethylene, purification, properties, and possible function . Planta 157, 22-31 . 6 . BOLLER, T . & METRAUX, J . P . (1988) . Extracellular localization of chitinase in cucumber . Physiological and Molecular Plant Pathology 33, 11- 16 . 7 . BOLLER, T . & VOEGELI, U . ( 1984) . Vacuolar localization of ethylene-induced chitinase in bean leaves . Plant Physiology 74, 442-444 . 8 . BRADFORD, M . M . (1976) . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding . Analytical Biochemistry 72, 248-254 . 9 . BROGLIE, K . E ., GAYNOR, T . J . & BROGLIE, R . M . 1986) . Ethylene-regulated gene expression : Molecular cloning of the genes encoding an endochitinase from Phaseolus rulgaris . Proceedings of the .9Rtional .-Icademv o! Sciences of the G.S.A . 83, 6820-6824 . 10 . CABIB, E . & BowERS, B . (1971) . Chitin and yeast budding. Localization of chitin in yeast bud scar, . ,7ournal of Biological Chemistry 246, 152-159 . 11 . CARMICHAEL, G . G . & MCMASTER, G . K . (1980) . The analysis of nucleic acids in gels using glyoxal and acridine orange . In Alethods in En,rmologs' 65, Ed . by h . Grossman & K . Moldave . pp. 380-391 . Academic Press, London . 12 . GoMEZ Lim, M . A., KELLY, P ., SEXTON, R . & TREWAVAS, A . J . (1987) . Identification ofchitinase mRNA in abscission zones from bean (Phaseolus vulgaris Red Kidney) during ethylene-induced ahcission . Plant . Cell and Environment 10, 741-746 . ANDERSON, P .



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C . R . Voisey and A . J . Slusarenko

13 . GOODAY, G . W . (1977) . Biosynthesis of the fungal wall - mechanisms and implications . Journal of General Microbiology 99, 1-11 . 14. HADWIGER, L. A . & LOSCHKE, D . C . (1981) . Molecular communication in host parasite interactions : Hexosamine polymers (chitosan) as regulatory compounds in race specific and other interactions . Phytopathology 71, 756-762 . 15 . HEDRICK, S ., BELL, J . N ., BOLLER, T . & LAMB, C . J . 1988) . Chitinase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection . Plant Physiology 86, 182-186 . 16 . HOOFT VAN HUIJSDUIJNEN, R . A . M ., KAUFMANN, S ., BREDERODE, F. TH ., CORNELISSEN, B . J . C., LEGRAND, M ., FRITIG, B . & BoL, J . F . (1987) . Homology between chitinases that are induced by TMV infection of tobacco . Plant Molecular Biology 9, 411-420 . 17 . KEEN, N . T ., ERSEK, T ., LONG, M ., BRUEGGER, B . & HOLLIDAY, M . (1981) . Inhibition of the hypersensitive reaction of soybeans to incompatible Pseudomonas s pp . by blasticidin S, streptomycin or elevated temperature . Physiological Plant Pathology 18, 325-337 . 18 . KOMBRINK, E ., SCHROEDER, M . & HAHLBROCK, K . (1988) . Several "pathogenesis-related" proteins in potato are 1,3-ß-glucanases and chitinases . Proceedings of the National Academy ofSciences of the U.S .A . 85, 782-786 . 19 . LAEMMLI, U . K . (1970) . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 . 20 . LEAH, R ., MIKKELSEN, J . D ., MUNDY, J . & SVENDSEN, I . B . (1987) . Identification of a 28000 Dalton endochitinase in barley endosperm . Carlsberg Research Communications 52, 31-37 . 21 . LYON, F . & WOOD, R . K . S . (1976) . The hypersensitive reaction and other responses of bean leaves to bacteria . Annals of Botany 40, 479-491 . 22 . LYON, F. & WOOD, R . K . S . (1975) . Production of phaseollin, coumestrol and related compounds in bean leaves inoculated with Pseudomonas spp . Physiological Plant Pathology 6, 117-124 . 23 . LYON, F . & WOOD, R . K . S . (1977) . Alteration of response of bean leaves to compatible and incompatible bacteria . Annals of Botany 41, 359-367 . 24 . MAUCH, F ., HADWIGER, L . A. & BOLLER, T . (1984) . Ethylene : Symptom, not signal for induction of chitinase and ß-1,3-glucanase in pea pods by pathogens and elicitors . Plant Physiology 76, 607-611 . 25 . MAUCH, F., HADWIGER, L . A . & BOLLER, T . (1988) . Antifungal hydrolases in pea tissue . I . Purification and characterization of two chitinases and two ß-1,3-glucanases differentially regulated during development and in response to fungal infection . Plant Physiology 87, 325-333 . 26 . METRAUX, J . P. & BOLLER, T . (1986) . Local and systemic induction of chitinase in cucumber plants in response to viral, bacterial and fungal infections . Physiological and Molecular Plant Pathology 28, 161-169 . 27 . METRAUX, J . P., STREIT, L . & STAUB, TH . (1988) . A pathogenesis-related protein in cucumber is a chitinase . Physiological and Molecular Plant Pathology 33, 1-9 . 28 . PEGG, G . F . & YOUNG, D . H . (1982) . Purification and characterization of chitinase enzymes from healthy and Verticillium albo-atrum-infected tomato plants, and from V. albo-atrum . Physiological Plant Pathology 21, 389-409 . 29 . POWNING, R . F . & IRZYKIEWICZ, H . (1965) . Studies on the chitinase system in bean and other seeds . Comparative Biochemistry and Physiology 14, 127-133 . 30 . REISSIG, J . L ., STROMINGER, J . L . & LELOIR, L . F. (1955) . A modified colorimetric method for the estimation of N-acetylamino sugars . Journal of Biological Chemistry 217, 959-966 . 31 . ROBERTS, W. K . & SELITRENNIKOFF, C . P. (1988) . Plant and bacterial chitinases differ in antifungal activity . Journal of General Microbiology 134, 169-176 . 32 . ROBY, D . & ESQUERRE-TUGAYE, M-T . (1987) . Purification and some properties of chitinases from melon plants infected by Colletotrichum lagenarium . Carbohydrate Research 165, 93-104. 33 . ROBY, D. & ESQUERRE-TUGAYE, M-T . (1987) . Induction of chitinases and of translatable mRNA for these enzymes in melon plants infected with Colletotrichum lagenarium . Plant Science 52, 175-185 . 34 . ROBY, D ., TOPPAN, A . & EsQuERRE-TUGAYE, M-T . (1986) . Cell surfaces in plant microorganism interactions. VI . Elicitors of ethylene from Colletotrichum lagenarium trigger chitinase activity in melon plants . Plant Physiology 81, 228-233 . 35 . SCHLUMBAUM, A ., MAUCH, F ., VOEGELI, U . & BOLLER, T . (1986) . Plant chitinases are potent inhibitors of fungal growth . Nature 324, 365-367 . 36 . SLUSARENKO, A . J . & LONGLAND, A. (1986) . Changes in gene activity during expression of the hypersensitive response of Phaseolus vulgaris cv . Red Mexican to an avirulent race I isolate of Pseudomonas syringae pv . phaseolicola . Physiological and Molecular Plant Pathology 29, 79-94 . 37 . SHINSH, H ., MOHNEN, D . & MEINS JR, F. (1987) . Regulation of a plant pathogenesis-related enzyme : Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin . Proceedings of the National Academy of Sciences of the U.S.A . 84, 89-93 . 38 . VOEGELI-LANGE, R ., HANSEN-GEHRI, A ., BOLLER, T . & MEINS JR, F . (1988) . Induction of the defenserelated glucanohydrolases, fl- 1,3-glucanase and chitinase, by tobacco mosaic virus infection of tobacco leaves . Plant Science 54, 171-176 .