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THE CHARACTERIZATION OF PEROXIDASE ASSOCIATED WITH INDUCED
SYSTEMIC RESISTANCE IN CUCUMBER AND THE ISOLATION OF TN5
MUTANTS OF PSEUDOMONAS SXBINQAE PV. SXBINGAE NO LONGER ABLE

TO INDUCE RESISTANCE

BY

Jennifer Alice Smith

A DISSERTATION

Submitted to

Michigan State University

in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1988

(.. J

‘ s
' l
l

\r’\

ABSTRACT
THE CHARACTERIZATION OF PEROXIDASE ASSOCIATED WITH INDUCED
SYSTEMIC RESISTANCE IN CUCUMBER AND THE ISOLATION OF TN5

MUTANTS OF PSEUDOMONAS SYRINGAE PM SYRINGAE NO LONGER ABLE

 

TO INDUCE RESISTANCE

BY

Jennifer Alice Smith

Induced systemic resistance in cucumber, muskmelon and
watermelon was accompanied by a systemic increase in
peroxidase activity. The increased activity was due to an
increase in an acidic isozyme localized to the extracellar
spaces of leaves. Peroxidase levels reached approximately
6% of total soluble protein in induced cucumber leaves as
determined by radial immunodiffusion.

The anodic peroxidase isozymes of cucumber, muskmelon
and watermelon appeared as a cluster of three bands (30-33
kd) on native and denaturing gels. The three forms of the
cucumber isozyme ( 33,31 and 30 Rd) were purified and found
to be differentially glycosylated as determined by their
differential affinity for Con A-Sepharose and
susceptibility to czmannosidase. The amino acid sequence of
three fragments from the 33 Rd peroxidase shared homology
with sequences in tobacco, turnip and horseradish
peroxidase.

Peroxidase levels began to increase systemically

in cucumber seedlings 24-30 hr after inoculation of the

first leaf with the HR inducing pathogen Pseudomonas

 

syrinae pv syringae. The rapid increase in peroxidase
activity was accompanied by a rapid increase in systemic
resistance. Induced resistance was expressed within 2 days
of the HR inducing inoculation as measured by a 70%

decrease in Colletotrichum lagenarium lesion diameter. Tn5

 

mutants of §.§. syringae selected for their inability to
induce resistance in cucumber had also lost their ability
to cause disease on the host plant wheat. In two mutants
isolated, Tn5 had inserted into a region of the genomic DNA
which shares homology to a functionally similar region

cloned from Pseudomonas syringae pv phaseolicola. A

 

 

genomic clone was isolated from a pLAFR3 library of P.§.
syringae which restored the ability of both mutants to

induce HR, peroxidase activity and resistance.

In memory of my father, Howard

iv

ACKNOWLEDGMENTS

I thank Dr. Ray Hammerschmidt for allowing me the freedom
to explore and for his enthusiastic encouragement, support
and guidance. I also thank the members of my guidance
committee; Dr. Dennis Fulbright, Dr. Karen Klomparens and
Dr. Christopher Somerville, for their valuable suggestions
and critical review of this dissertation.

I am particularly grateful to the members of Dr.
Barbara Sears', Dr. Dennis Fulbright's and Dr. Shauna
Somerville's labs from whom I begged and borrowed (but
never stole) everything from bad jokes to Good buffers.
Special acknowledgments are in order for Dr. Robert
Creelman, for serving as ambassador to the PRL; for Therese
Best, for preserving and promoting an ecologically sound
way of life; and for Jun Tsuji, for raising new questions
about old answers.

My years as a graduate student were deeply enriched by
the friendship of Dr. Alice Bonnen, who bailed me out of
more than a few tight spots and who generously shared her
family, friends and cookies. Finally, I thank my mother
June, and my sister Rebecca, for their confidence, patience
and sense of humor.

TABLE OF CONTENTS

List of Tables ......................................... viii
List of Figures .......................................... ix
General Introduction ...................................... 1

References ........................................... 6

Chapter I. Comparative study of acidic peroxidases

associated with induced systemic resistance in cucumber,

muskmelon and watermelon .................................. 8
Introduction .......................................... 9
Experimental and Results .............................. 9
Conclusions... ...................................... 14
References ...... ' ..................................... 15

Chapter II. Further characterization of anodic peroxidase

triplet associated with induced resistance in cucumber...16

Introduction ......................................... 17
Materials and Methods ................................ 22
Results .............................................. 27
Conclusions .......................................... 40
References ........................................... 42

Chapter III. Isolation and characterization of Tn5 mutants

of Pseudomonas syringae pv syringae no longer able to

 

induce resistance ........................................ 45
Introduction ......................................... 46
Materials and Methods ................................ 48
Results .............................................. 54

vi

Discussion ........................................... 69

References ........................................... 73
Future directions ........................................ 77
References ........................................... 81

vii

LIST OF TABLES

Table Page

1. Fractionation of soluble. peroxidase activity from
control and induced cucumber, muskmelon, and

watermelon leaves ................................. “n.11

2. Results of peroxidase immunodiffusion assay of
protected leaves .................................. ...30

3. Purification of peroxidase from systemically
protected leaves .................................. ...31

4. Quantification and specific activity of individual
peroxidases isolated by electroelution from a
polyacrylamide gel ................................ ....32

5. Bacterial strains and plasmids .................... ”n.49

viii

LIST OF FIGURES

Figure Page
1. Native polyacrlamide gel electrophoresis of
purified peroxidases from cucumber (C),
muskmelon(M), and watermelon (W) .................. ...12
2. SDS polyacrylamide gel electrophoresis and
molecular weight determination of purified
peroxidases from watermelon (W), muskmelon (M), and
cucumber (C) ...................................... ...13
3. Cross-reactivity of anti-cucumber peroxidase
antisera with muskmelon and watermelon
peroxidase ......................................... ...U+
4. Radial immunodiffusion of peroxidase from
systemically protected (P) and control (C) leaf
homogenates ...................................... .fl...28
5. Standard curve for radial immunoassay of
peroxidase ........................................ ....29
6. SDS polyacrylamide gel electrophoresis of isolated
high (H), medium (M), and low (L) molecular weight
forms of the anodic peroxidase
triplet ........................................... ....33
7. Isoelectric focusing of peroxidase from
intercellular extracts and total leaf
homogenates ....................................... . . . .31+
8. Affinity of anodic peroxidases for con A-
sepharose ........................................ .....36
9. Susceptibility of high (H), medium (M), and low (L)
molecular weight forms of peroxidase to
a mannosidase ...................................... . . . . 37
10. Affinity of anti-cucumber peroxidase for high (H),

medium (M), and low (L) molecular weight forms of
anodic cucumber isozyme ........................... “n.38

ix

Funum

11.

12.

14.

15.

16

17.

18.

19.

20.

 

 

 

 

 

 

 

 

 

Page
Comparison of amino acid sequence of three
fragments of cucumber peroxidase with horseradish,
turnip, and tobacco peroxidase .................... .“u39
Systemic increases in peroxidase activity in
response to inoculation of leaf 1 with Pseudomonas
syringae pv.syringae (Pssozo) or Pseudomonas
syringae pv.lachrymans
(P.S.I.) ooooooooooooooooooooooooooooooo 00000000000000.0055
Time course for the induction of peroxidase
activity in the second leaf of plants inoculated on
the first leaf with Pseudomonas syringae pv
syringae ( 4r ) or control plants ( 4p) ........... .HH56
Time course for the expression of induced systemic
resistance in plants inoculated on leaf 1 with
Pseudomonas syringae pv syringae ( 4r ) or
Pseudomonas syringae pv lachryman5(.o.) ......... .Hu5y
Comparative ability of wild type, Tn5 mutants, and
restored mutants of §.§. syringae to induce
resistance ........................................ ....59

.A,B In planta growth of bacteria ................. ....60,61

Location of Tn5 insertion sites ( l ) in EcoRI (E)
fragments from PSSD21 and PSSD22 .................. ....63
Hybridization of 10 kb HindIII fragment from
pPSSDZl to EcoRI digested genomic DNA from PSSDZO,
PSSDZl and PSSD22 (lanes 1,2 and 3,
respectively) .................................... .....6l+
Comparative ability of wild type, TnS mutants, and
restored mutants of g. s. syringae to induce
systemic increases in peroxidase activity ......... .uu66
Homology between Pseudomonas syringae pv
phaseolicola hrp region and a portion of the
functionally similar region from
PSSDZO ............................................ ....68

GENE RAL INTRODUCT I ON

GENERAL INTRODUCTION

In contrast to the circulatory immune system of animals,
plants frequently sequester invading bacterial and fungal
pathogens at the sites of attemped infection.
Compartmentalization is achieved by the rapid localized
activation of defense related metabolism in response to
pathogen invasion. Several examples of defense related
responses which have been reviewed recently include
hypersensitive cell death (11), phytoalexin production (1,2),
and lignin biosynthesis (19). Rapid activation of one or more
of these processes resulting in compartmentalization of
pathogens is determined by the presence or absense of disease
resistance genes in the host plant. The mechanism by which
disease resistance genes mediate the expression of defensive
responses is unknown.

In contrast to genetic resistance, induced resistance
involves the activation of defense mechanisms in the absence
of specific genes for disease resistance. Resistance may be
induced at the site of the resistance inducing treatment
(locally) or in tissues distant to the site of treatment
(systemically) (12,13,16,18). Resistance can be locally
induced by prior inoculation of tissue with non-host
pathogens or by treatment with elicitors of defense
responses. Elicitors are constituents of pathogens or hosts
which appear to act as signal molecules for the elicitation

of resistance related metabolism. They have been

characterized as oligosaccharides (17,20,21), glycoproteins
(3,21) and lipids (6). With the exception of the
proteinase inhibitor inducing factor isolated from tomato,
a pectic fragment of MW 5000 (4,17), no elicitor has been
identified in induced systemic responses.

Systemic induced resistance has generated considerable
interest because of its phenomenological similarity to the
process of immunization in animals. The majority of
research on induced systemic resistance employs cucumber
or tobacco as the host plant. In cucumber, inoculation of
the first leaf (inducing inoculation) of seedling plants
with necrosis inducing fungal, bacterial, or viral
pathogens induces systemic resistance to an array of
pathogens (13). Resistance is maximal several days after
the inducing inoculation and may be enhanced by additional
inoculations. Induced resistance in cucumber persists
until flowering. Jenns and Kuc (10) found that

resistance to Colletotrichum lagenarium could be induced in

 

susceptible watermelon and muskmelon by grafting stems to
the stem of an induced cucumber plant. Further evidence
for the vascular transport of a 'signal' for induced
resistance was provided by Guedes et a1 (7). They found
that steam girdling of petioles prevented the expression of
induced resistance in leaves distal to the induced leaf.
Although the induction of systemic resistance in plants
appears to involve the transport of signal through the

vascular tissue, the mechanism of resistance is the same

as for other plant disease resistance reactions; enhanced
ability to compartmentalize invading pathogens. Thus
induced resistance is expressed as a decrease in the size
and number of fungal lesions or as a decrease in the number
of lesions induced by bacteria and viruses (12). This
decrease in successful infection by pathogens has been
correlated with an enhanced ability of the epidermal cells
to lignify at infection sites (8).

Prior to the expression of enhanced lignification,
biochemical changes resulting from the inducing inoculation
include systemic increases in extracellular chitinase and
peroxidase. Both enzymes are similar with respect to their
anionic charge, molecular weight (approx. 30,000) and their
regulation in response to pathogens (5,8,14,15).
Hammerschmidt and Kuc determined that increases in
peroxidase activity occur systemically in cucumber tissue
as soon as pathogen induced necrotic lesions appear on the
inducing leaf (9). They associated the increased peroxidase
activity with an anodic triplet localized to the
extracellular spaces of leaf tissue. Chapters 1 and 2 are
devoted to the further characterization of this peroxidase
triplet.

Considerably more is known about the biochemical
events that follow resistance inducing inoculations than
about the sensing mechanism that the triggers the response.

The involvement of elicitors in local induced resistance

responses suggests that they may play a role in induced

l‘il

O

D)

systemic resistance. A systematic search for elicitor
induced resistance should take into consideration the
criteria which have been established for the response by
Kuc et al. (7-10,12,13):

1. Resistance can only be induced by living pathogens;

abiotic damage to tissue has no resistance inducing
effect.

fx’

The resistance inducing organism must cause a
necrotic lesion.

3. The level of resistance induced is directly
correlated to the amount of necrosis produced by the
inducing organism.

The requirement for a living organism to induce
systemic resistance suggests that the triggering mechanism
is some process unique to pathogen/host interactions rather
than a specific component of either organism alone.
Resistance can be induced by viruses, bacteria, and fungi;
yet it is unlikely that these diverse organisms share
preformed constituents which activate induced resistance.
Likewise, the inability to induce resistance through
wounding or other abiotic damage indicates that the active
factor is not solely of plant origin. Chapter 3 proposes a
system in which to study the components of the

pathogen/host interaction and their importance to induced

systemic resistance.

GENERAL INTRODUCTION REFERENCES

Albersheim, P. and 8.5. Valent. 1978. Host-Pathogen
interactions in plants. J. Cell Biol. 78:627-643.

Bailey, J.A., and J.W. Mansfield Eds.1982. Phytoalexins.
Blackie, Glasgow and London.

Bell, A.A. 1981. Biochemical mechanisms of disease
resistance. Ann. Rev. Plant Physiol. 32:21-81.

Bishop, P.D., Makus, D., Pearce, G., and C.A. Ryan.
1981.Proteinase inhibitor-inducing factor activity in
tomato leaves resides in oligosaccharaides
enzymatically released from cell walls. Proc. Natl.

Acad. SCi. (USA). 78:3536-3540.

Boller, T., and J.P. Metraux. 1988. Extracellular
localization of chitinase in cucumber. Physiol. Molec
Plant Pathol. 33:11-16.

Bostock, R.J., Kuc, J., and R. Laine. 1981.
Eicosapentaenoic and arachidonic acids from Phytophthora
infestans elicit fungitoxic sesquiterpenes in potato.
Science. 212:67-69.

Guedes, M.E.M., Richmond, S. and J. Kuc. 1980. Induced
Systemic resistance to anthracnose in cucumber as
influenced by the location of the inducer inoculation
with Colletotrichum lagenarium and the onset of
flowering and fruiting. Physiol. Plant Path. 17:229-
233.

Hammerschmidt, R., and J. Kuc.1982. Lignification as a
mechanism for induced systemic resistance in cucumber.
Physiol. Molec. Plant Pathol. 20:61-71.

Hammerschmidt, R., Nuckles, E.M., and J. Kuc. 1982.
Association of enhanced peroxidase activity with induced
systemic resistance of cucumber to Colletotrichum
lagenarium. Physiol. Plant Pathol. 20:73-82.

Jenns, A. and J. Kuc. 1979. Graft transmission of
systemic resistance of cucumber to anthracnose induced
by Colletotrichum lagenarium and tobacco necrosis virus.
Phytopath. 69:753-756.

 

Klement, Z. 1982. Hypersensitivity. In: Phytopathogenic
Prokaryotes, Vol.II, Ed. by M.S. Mount and G.H. Lacy, pp
149-177. Academic Press, New York and London.

13.

14.

15.

16.

17.

18.

20.

21.

Kuc, J. 1987. Plant immunization and its applicability
for disease control. In: Innovative approaches to plant
disease control. Ed. by Ilan Chet, pp 255-274. John Wiley
& Sons, New York.

Kuc, J. 1983. Induced systemic resistance in plants to
diseases caused by fungi and bacteria. In: The dynamics
of host defense. Ed. by J. Bailey and B. Deverall.
Academic, Sydney, pp. 191-221.

Metraux, J.P., and T. Boller.1986. Local and systemic
induction of chitinase in cucumber plants in response to
viral, bacterial and fungal infections. Physiol. Molec.
Plant Pathol. 28:161-169.

Metraux, J.P., Streit, L., and T. Staub. 1988. A
pathogenesis-related protein in cucumber is a chitinase.
Physiol. Molec. Plant Pathol. 33:1-9.

Ross, F.A. 1961. Systemic acquired resistance induced by
localized virus infections in plants. Virology 14:340-
358.

Ryan, C.A. 1974. Assay and biochemical properties of the
proteinase inhibitor-inducing factor, a wound hormone.
Plant Physiol. 54:328-332.

Sequeira, L. 1983. Mechanisms of induced resistance in
plants. Ann. Rev. Microbiol. 37:51-79.

Vance, C.P., Sherwood, R.T. and T.K. Kirk. 1980.
Lignification as a mechanism of disease resistance. Ann.
Rev. Phytopath. 81:259-288.

West, C.A., Bruce, R.J., and D.F. Jin. 1984. Pectic
fragments of plant cell walls as mediators of stress
responses. In: Structure, function, and biosynthesis of
plant cell walls. Ed. by W.M. Dugger and S.B.-Garcia.
American Society of Plant Physiologists. Waverly press,
Baltimore, Maryland.

West, C.A., 1981. Fungal elicitors of the phytoalexin
response in higher plants. Naturwissenschaften. 68:447-
457.

CHAPTER I.

COMPARATIVE STUDY OF ACIDIC PEROXIDASES ASSOCIATED WITH
INDUCED SYSTEMIC RESISTANCE IN CUCUMBER, MUSKMELON AND

WATERMELON

l’lrm'ulngrml and .Ilnln'u/ar Plant Pathology ( 1988) 33, 000-000

Comparative study of acidic peroxidases associated With
induced resistance in cucumber, muskmelon and
watermelon

Jsxxtrsa A. 5mm and RAYMOND Hannsnscmtto'r'l’
Dr/mrtnmtl q/ Botany and Plant Pathology, .Ilirltigan State University, East Lansing, M l 48824-1312, U.S.A.

. Irrrptrdfar publication February 1988)

 

Inoculation of one leaf of cucumber, muskmelon or watermelon with Colletotrirltum lagenarium
induces a systemic increase in soluble peroxidase activity. Increased t0tal activity is reflected in an
increase in activity in intercellular wash fluids of leaf tissue. The specific activity of peroxidase
extracted from intercellular wash fluids frotn systemically induced leaves was at least two-fold
higher than specific activity extracted from control leaves. Intercellular peroxidase migrates as a
cluster of three hands on high pH native polyacrylamide gels, and the patterns are similar for
cucumber. muskmelon and watermelon. 'l'hc peroxidases are r. 30 ItD as determined by SDS
polyacrylamide gel electrophoresis. Antibodies raised against cucumber intercellular peroxidase
reacted with muskmelon and watermelon peroxidases in patterns of partial identity in the
Ouchterloncy double dill'usion assay.

INTRODUCTION

Inoculation ofone leafofcucumbcr with any ofseveral different pathogens results in the
systemic induCtion of resistance against subsequent attack by several pathogens [13].
Similarly, inoculation ofthe first leaf of muskmelon and watermelon with C. lagenarium
also results in the induction of systemic resistance to that fungus [3, 13, 18]. Induced
resistance in cucumber and muskmelon is associated with an enhanced ability of the hOSt
plants to lignify in response to infection by pathogenic fungi [5, 8, 10, 18]. Rapid lignin
deposition may provide a physical and/or chemical barrier to the invading pathogen
[19]. Induced systemic resistance in cucumber is also associated with enhanced levels of a
group of acidic, extracellular peroxidases which accumulate systemically in plant tissues
as induced resistance develops [I I]. Peroxidascs catalyse the final polymerization step of
lignin synthesis, and may therefore be directly associated with the increased ability of
systemically prOtected tissue to lignify [9]. The purpose of this study was to further
characterize the peroxidases induced in cucumber and to determine whether similar
peroxidases are induced in watermelon and muskmelon seedlings as a result of resismnce
inducing inoculations. '

EXPERIMENTAL AND RESULTS

Pat/roger: culture and hosts

(.‘ollctotrt'rlmm lagmarium (Pass) El 1 and Halst. race I was maintained on V-8 juice agar
at I8 °C in the dark. Conidial suspensions for use as inoculum were prepared from 7- to
IO-dayoold cultures as described previously [14].

l"l'o whom correslmndence should be atltlreswtl.

10

‘-

Muskmelon ;(.'m'nmi.t mrlo l.. cv. Iroquois), watermelon ((ntrullis vulgaris L. cv. sugar
baby). and cucumber l(.'m'ttmi.t salient l.. cv. S.“ R 58) plants were grown from seed in the
greenhouse.

Inoculation (if/10513 jor peroxidase induction ‘
Peroxidase was induced in l-i-day-old plants by infiltrating approximately 500 pl of a
(I. lagenarium spore suspension (l x 105 spores ml") into l0 sites on the first true leaf
using a 3-ml disposable syringe appresscd to the underside of the leaf. Control plants
received no treatment. as it has been determined previously that water injections have no
resistance or peroxidase inducing ell'ect [11]. Seven days after inoculation, the second
true leaves of induced inoculated) attd control plants were collected for peroxidase
assays.

Extraction ercroxir/axcjtorn Ira/tissue
'l‘wenty-livc to fifty grants of leaves were rinsed with distilled water and placed into a
large desiccator one-hall'filled with ice cold distilled water. Leaves were held under the
airfare ol‘the water with a ceramic plate and infiltrated under reduced pressure for IS
min. lnliltrated leaves were blotted on cheesecloth, and four to five leaves were rolled
together and placed tips down into a 30-ml plastic centrifuge tube one-fifth filled with
B-mm diameter glass beads. Intercellular fluids were collected by centrifugation at
“2000 r min" for IS min in a Sorvall SS-34 roror. Extracts were pooled, centrifuged at
In 000 min"I for ‘20 min and the supernatant lyophilized prior to storage at — 20 °C.
Lcal'tissuc previously extracted by vacuum infiltration was frozen in liquid nitrogen
and ground to a powder with mortar and pestel. Powdered leaf tissue was suspended in
0.01 M Nal’O4 buffer pH 6-0 (4 ml g" original fresh weight). The homogenate was then
vacuum filtered through Whatman No. 4 filter paper. Homogenates were repeatedly
rinsed with the same volume of buffer until no peroxidase activity was detected in the
filtrates. The same procedure was used to extract ionically bound peroxidase from the
buffer extracted homogenate by using 021 M CaCl, in 001 M NaPO4 buffer pH 6-0.
Samples of all washes were reserved for determination of total soluble peroxidase
extracted.

Peroxidase octit'itr and protein determination

Peroxidase activity of leaf extracts was determined using guaiacol as a substrate as
described previously [ll]. Protein concentrations were determined according to
Bradford [2) using bovine serum albumin (BSA) as a standard. The fractionation of
soluble peroxidase from leaf 2 of induced or control tissue of cucumber, muskmelon and
watermelon is'summarizcd in Table 1. Total soluble peroxidase activity from induced
tissue was higher than activity from control tissue for all three plant species. Induced
cucumber, muskmelon and watermelon leaves had a six-fold, three-fold attd 35",,
increase in total soluble peroxidase activity, respectively, over control tissue aetivity. The
amount of peroxidase activity extracted by vacuum infiltration varied from 8 to 3706 of
the t0tal soluble peroxidase activity. The specific activity of peroxidase extracted by
vacuum infiltration of induced leaves from cucumber, muskmelon and watermelon was
7-, 2- and 4-fold higher, respectively, than the specific aetivity of peroxidase extracted by
vacuum infiltration of control leaves.

11

 

 

 

'l'AaLs l
Fractionation of soluble peroxidase actieitr from control and induced cucumber. muskmelon and watermelon
leave:
Total "1, Total soluble Specific activity‘
soluble activity present
activity in intercellular lntercellular Bulfer+salt
Species 'l'reatmem‘ extracted“ extract extract extract
Cucumber C 8‘8 20 71 l '3
1 49-2 37 +78 6-4»
Muskmelon C 23-l 24 - 150 l-7
l 24 298 2-2
Watermelon C 27-5 8 48 l—7
1 64-4 25 174 -l

 

‘(L t'nmrul: l. lmlltt't'd.

"Activity from intercellular extract-Factivity from boiler and salt washings. Acuvity
expressed as .‘\,_-,, min gfresh wt" '.

.\,._. min 'mg protein.

I’ut racrrlamide gel electrophoresis ( PACE)

l’tilyacrylamide slab gels. 1-5 mm thick, were prepared for native or denaturing gel
systems. For native gels. an anionic :’pH 9-3) discontinuous system was used [12]. .-\n
acrylamidc concentration of7-5”., provided optimal separation of peroxidase isozymes.
Gels were stained for peroxidase activity or prOtein upon completion ofeleCtrophoresis.
l’or denaturing gels. the SDS system of Laemmli was used with a 150,, acrylamide gel
[I5]. Gels were stained for protein following electrophoresis.

I ’of t'acrr/mnide .ch stains

a Peroxidase actiritr. .\fter eleCtrophoresis, gels were placed in a solution containing
30 mg of 3-aminci.9-ethylcarbamlc. 10ml of.N",-\’,dimethylformamide, 200 pl of 30°”
l‘l._.O._, and 190 ml ofsoditnn acetate l)ulfer pH 5-0 [7]. Bands were visualized within 10
min. Gels were rinsed and stored in a solution of7"0 acetic acid in 50",, methanol.

b I’rotein. The silver nitrate staining procedure of .\lorrissey [16] was modified as
follows. After electrophoresis. gels were placed in 509;, methanol. 10".“ acetic acid for
l-l2 h. Gels were then rinsed in many changes ofdistilled water for one hr followed by
30 min in 200 ml of?) ug ml'l dithimhreitol. Gels were incubated in O- 1 °,, aqueous silver
nitrate for l h and protein bands visualized by placing the gel in 200 ml of'Ii-O"u NaQCOa
containing 50 pl of 37"., formaldehyde. Colour development was stopped by the
addition of 1‘2 g ofcitric acid.

I ’i toxic/use purification

llammerschmidt et al. have shown previously that increased total peroxidase aetivity in
induced cucumber leaves is reflected in the increased activity of three bands of acidic
peroxidase readily extracted liom intercellular spaces [11]. For each cucurbit species

12

Protein Activity
M C M

4‘i‘r- 4..

 

Flo. l. Native tmlyacrylatnidt- gel electrophoresis ofpurifted acidic peroxidases trout cucumber
C . muskmelon (.\l) and watermelon (W). Gels were stained for protein using silver nitrate or for
peroxidase activity using 3-aminu,9-cthylcarhazole as described in the text.

in this study, lyophilized intercellular extract was resuspended in 2—3 ml of 0-05 M
Tris—HCI pH 8-0 and applied to a Sephadex G-75 column (l-5 x 25-0 cm) equilibrated
in the same buffer. The column was eluted with 0-05 M Tris—HCl pH 8-0 and 2-ml
fractions containing peroxidase activity were pooled and applied to a DEAE-Sephadex
column (2 x 10 cm) equilibrated with 005 M Tris—HCl pH 8-0. Peroxidase was eluted
from the column with a linear salt gradient of0-0-0-2 M NaCl in a total volume of l 50 ml.
Fractions ( l-3 ml each) containing peroxidase aCtivity were pooled, dialysed extensively
in distilled water, and lyophilized. Lyophilized samples were re-chromatographed on
the DEAE column, dialysed and lyophilized. Peroxidase activity eluted from DEAE as
single peaks at 0-l3, 0-14 and 016 M NaCl for cucumber, muskmelon and watermelon
peroxidase, respectively.

Peroxidase eluted two times from DEAE was essentially pure as indicated by gel
electrophoresis followed by the silver stain for protein (Fig. l). Peroxidases purified from
cucumber, muskmelon and watermelon were similar with respect to their migration on
native and denaturing acrylamide gels (Figs. 1, 2). The peroxidases from each species
migrated as three bands ofdecreasing relative protein and peroxidase staining intensity
front the slowest to fastest migrating. The peroxidases have molecular weights ranging
from 30 to 33 kD as determined by SDS gel electrophoresis with standards of known
molecular weight (Fig. 2).

Purification afcncumber peroxidase for antibody production

Cucumber peroxidase purified by DEAE chromatography was further purified by
preparative gel electrophoresis followed by electroelution of peroxidase bands.
Lyophilizcd samples were resuspended to a concentration of l-O mg ml" protein in
electrophoresis sample buffer containing 0-5 M Tris-HCI pH 6-8. 10“,, glycerol and

 

13

 

 

Fm. 2. SDS pilyaerylamidt- gel electrophoresis and molecular weight determination of purified
pt'roxidasn fmtn watermelon (W). muskmelon (M) and cucumber (C).

0-05"u bromphenol blue. One millilitre of sample containing l-O mg of prOtein was
applied to a 3 mm thick native slab gel. Peroxidase bands were detected in the gel
directly by their brown colour or by staining a 5 mm strip ofgel for peroxidase aCtivity.
Bands corresponding to peroxidase were excised with a razor blade and electroeluted
using an lSCO electroeluter with 005 M Tris-glycine pH 8-9 as the elution buffer [1].

Production qfantibadic: to cucumber peroxind

For the initial injection, 200 pg of PAGE purified native peroxidase was emulsified in
1 ml of Freund‘s complete adjuvent (Difco). Two New Zealand white female rabbits
were injected intramuscularly with equal volumes of the emulsified peroxidase (100 ug
each). Subsequent injections were given every 3—4 weeks using 100 pg of native peroxi-
dase per rabbit emulsified in Freund's incomplete adjuvent. Sera was collected 7-10 days
after each injection.

Cross reactivity of cucumber, mus/tinclon and watermelon peroxidase wit/t anti-cucumber peroxidm‘r
317a .

Ouchterloney double diffusion was carried out on glass microscope slides coated with a
3 mm layer of l ‘33 agarose in phosphate buffered saline (PBS). The centre well was filled
with 10 pl of undiluted anti-cucumber peroxidase sera and outer wells were filled with
ID pg each of cucumber, muskmelon or watermelon peroxidase. Antigen and antibody
were allowed to dilfuse towards each other at room temperature in a moist Petri dish
until precipitate formation was maximal. Unreacted proteins were washed from agarose
by placing slides in 3 l of0-3 M NaCl at 4 °C for 24 h. Slides were allowed to dry at room

11+

 

l"It;. Ii. (lum-tcactivity nfuntt-t Ill'llllllN‘l' pt'tn.\l(l.l.\(' antisera with muskmelon and watermelon
peroxidase. (It-lute well contains HI tll nfanti-t ucumbcr peroxidase whole sera. Outer wells were
loaded with ll) )1: in lll til each ol'cucttmber (I .\\.Ilt'rmt'llln -\\" or muskmelon t.\ll peroxidase.

temperature and precipitation battds visualized by staining with Coomassie blue [6].
.\luskmelon and watermelon peroxidase reacted with anti-cucumber peroxidase serum
in patterns of partial identity with cucumber peroxidase (Fig. 3). The anti-cucumber
peroxidase serum showed a slightly higher degree of cross-reactivity with muskmelon
than with watermelon as evidenced by the shorter and less distinct “spur" produced.

CONCLUSIONS

lntluced resistance itt cucumber, muskmelon and watermelon is accompanied by a
systemic increase in peroxidase activity. Although the acidic peroxidases induced in each
ofthe three plant species migrate as three bands on polyacrylamide gels, it is unclear
whether they are the products ofdilferent genes or result from post-translational modifi-
cations of a single gene product. Genetic studies ofthis peroxidase triplet in cucumber
ltave shown that the three bands are inlterited as a single locus [4]. Factors. such as
dill'erential glycosylation and interactions with phenolics may alter the mobility of a
single protein on polyacrylamide gels [17.20].

Peroxidases indttced in the three plant species are similar with respect to charge and
molecular weight and show a high degree ofimmunological cross-reactivity. The struc-
tttral and regulatory similarity of peroxidases induced in cucumber, muskmelon and

15

watermelon may rellect a similar role for the enzymes in the systemic tnduccd resmtancc

response.

This work was supported in part by a grant from The Rackham Foundation and USDA]
CRGO grant 85-CRCR-l-l535. Michigan Agricultural Experiment Station Journal
Article No. -

REFERENCES

I.

DO

l9.

2t).

Buowx. A. 5.. Moqu‘... Hl'N‘t'LR. F. & Bexxs't'r, C.J. (1980;. High-sensitivity sequence determination
ofproteins quantitatively recovered for sodium dodecyl sulfate gels using an improved electrodialysis
procedure. .-lnalrtiml Blur/titttlslp‘ 103, 134490. .

. Btuoroao. .\l. .\l. . l976). A rapid and sensitive method for the quantitation ufmicrogram quantities of

protein using the principle of prOtein-dye binding. Analytical Biochemistry 72, 248-259.

. (IAttt‘so. F. L. 8; Kt't"..J. ; l977i. Protection of watermelon and muskmelon against Colletotrichum lagrnarium

by (.‘ullrtotriclmm lagmarium. l’ltt'tu/iatlwlogr57, ”85- ”89.

. sze. F. . I983). Cucurbits. ln Imam: in Plant Genetic: and Breeding, Part 8. Ed. by S. D. Tanksley & 'lJ.

Orton. pp. 369 390. lilscvier Science Publishers 8. \'.. Amsterdam.

. . l)t.A.\'. R. A. & Kta’aJ. mum. Rapid lignification in restxntse to wounding and infection as a mechanism

for induced systemic protection in cucumber. l’ltt'xiolugiml and Molecular l’lant l’atlmlagr 31, 69—8].

. CARVEYJ. 3.. (Issues. X. 1'2. 8; Sessoottr. D. H. f l977). .llrllmdi in lmmunnlngr. Third Edition. Addison-

\\'csley. Reading Massat‘hUsclts.

. Gaunt. R. C.. chonout. L'. & Karts-cvsxv. .\l.J. (1965). Cytochemical demonstration ofperoxidase

activity with 3-amin-9-ethylcarbazole. Jaunml uth'storltemistg and Qrtocliemistrr 13, 150-152.

. Crust). C. 8; RosstoxOL. .\l. t1982). Changes in the lignification process induced by localized infection of

muskmelons by Colletotrichum lagenarium. Plant Scimcr Letters 28, 103—] l0.

. Cnoss. G. G. 1979.. Recent advances in the chemistry and biochemistry of lignin. Recent Advances in

I’itt'lm‘lirmlslfr 12, l 77- 220.

. HAMMLRSCHASIDT. R. 8; Kt‘¢.J. il982). Lignification as a mechanism for induced systemic response in

cucumber. I’lnztialngz‘ml Plant Pathology 20, 61—7].

. HAMMERSCHMIDT. R.. Nt‘tzxtes. E. M. 8: Kt‘é.J. t1982). Association of enhanced peroxidase activity

with induced systemic resistance of cucumber to Colletotrichum lagenarium. Pltrsiologiral Plant Pathologt 20,
73-82.

‘ . KELETI G. 8: Leona. W. H. ; 1974i. .Ulcramrtlmdsforlltr Biologt'ralSdmru. \’an..\'ostrand Rheinhold Co..

New York.

. Kt‘e.J. l983 . induced systemic resistance in plants to diseases caused by fungi and bacteria. In The

[humans q/‘Hnst l)(/t'”.‘(. Ed. byJ. A. Bailey 82 B.J. Deverall. pp. 19l-22 l. Academic Press, New York.

. Kt't’:.J. 5: Ritatstoxn S. t l977‘.. Aspects of the protection of cucumber against Colletotrichum lagenarium by

(.‘ulltlutrtclmm lag-martian. l’lp'lopatholog) 67, 533—536.

. LAEMMLI. L'. K. 1970:. Cleavage of structural proteins during the assembly of the head of bacteriophage

T4. . \atun 227, (580 4385.

. Morons“. J. .lSlHI . Silver stain for proteins in polyacrylamide gels: a modified procedure with

enhanced uniform sensitivity. .‘lnalt'timl Birirltrmislp 117, 307-310.

. Srivastava. O. l’. & \'att Huystee. R. B. ~,1977i. Interactions among phenolia and peroxidase isozymes.

lintaniml (iayltc 138, 457’ 454.

'l'ot'ze. A. & Rosstoxot. .\l. t1977i. Lignification and the onset of premonition in muskmelon plants.
In Ilimlmuislrr Relatrrl Io S/arrj/iritr in Host—Plant l’atlmgm Interactions, Ed. by E. B. Solmer & J. Raa,
pp. 227‘230. 'l'runtso L'nivenitie. Forlagcn.

\'.\xta;. (2. l’.. Smmwoon. R. 'l'. & Knot. 'l‘. K. .' l980). Lignification as a mechanism ofdisease resistance.
.lmmal Rcrica' ofl’ltt'Iu/oatlmlag) 3], 259-288.

\'.sx Ht'yn'ue. R. B. 1987‘. Some molecular aspects of plant peroxidase biosynthetic studies. Annual
Rn'lfi.‘ u/il’ltml Plinth/0g] 38, 205-2”).

PLEASE NOTE:

Copyrighted materials in this document have
not been filmed at the request of the author.
They are available for consultation, however.
in the author's university library.

These consist of pages:
9 — 1 5

U-M-I

CHAPTER II.

FURTHER CHARACTERIZATION OF ANODIC PEROXIDASE TRIPLET

ASSOCIATED WITH INDUCED RESISTANCE IN CUCUMBER

16

17

CHAPTER II. INTRODUCTION

Peroxidase is ubiquitous in higher plants and has been the
subject of thousands of journal articles during the past
century. Theorell crystallized horseradish peroxidase in
1942 (cited in 24), and biochemists and biophysisists have
subsequently documented virtually every nuance of its
spectroscopic, catalytic, sequence and structural properties
(24,29). In spite of the quantity of data available on the
reactions catalyzed by peroxidase in yitrg, its function in
plants has remained obscure due, in part, to its broad
substrate specificity and the presence of numerous isozymes.
Researchers have, nevertheless, assigned various functions to
plant peroxidases based on their tissue localization and
activities on plant derived substrates. Several functions
suggested for peroxidase include hydrogen peroxide formation
(22), ethylene biosynthesis (20,26), IAA oxidation (10,17,28)
and lignin biosynthesis (11,13,21).

Although a number of model systems have been proposed
for the study of peroxidase regulation and function, two
which hold considerable promise are the genetic analysis of
peroxidase isozymes in petunia and the molecular analysis of
isozymes in tobacco. Berg et al. have greatly simplified the
interpretation of complex peroxidase isozyme profiles by
providing genetic evidence for post-translational
modifications of peroxidase isozymes (1-5,16). They have

focused on three structural genes designated prxA, ppr, and

18

per which code for the 10 major peroxidase isozymes found in
petunia. The prxA locus is of particular interest, as it
codes for an acidic cell wall associated cluster of 3-4 bands
similar to those that have also been observed in tomato (25),
tobacco (23) and cucumber (9,14). Five alleles of the prxA
locus have been identified based on shifts in the.
electrophoretic mobility of the peroxidase cluster. The prxA
alleles are co-dominant, indicating that variations in
electrophoretic mobility of the enzymes are due to mutations
in the structural gene rather than a modifying enzyme.
Several lines of evidence support the hypothesis that a
single gene product of the prxA locus is post-translationally
modified to produce the 3-4 bands observed on starch gels.
First, only the slowest migrating band of the PRXa cluster is
seen in young leaf tissue. The two faster migrating bands
appear gradually as the leaf matures and are suggested to be
processed forms of the primary band. These secondary bands
have been termed 'mozymes' for 'modified enzymes'. Second,
in crosses of plants with different alleles of prxA, only the
original triplets and combined hexaplets were observed in the
progeny. The authors concede that it is possible, though
unlikely, that the observed segregation is due to three
tightly linked genes. Finally, internal site mutations at
prxA that affect - temporal expression of the gene
simultaneously affect the expression of the entire PRXa
cluster. The PRXa clusters were purified from plants

homozygous for each of the 5 alleles and tested for cross-

19

reactivity to antibodies prepared to one cluster.
Peroxidases encoded by the 5 alleles were immunologically
identical as determined by Ouchterlony double diffusion
assays. Antibodies raised to the PRXa cluster did not cross-
react with PRXb or PRXc peroxidases. Future work in this
system will focus on the differential expression of the PRXa
cluster using the antibodies specific for that isozyme.
Lagrimini et al. (18,19) have adopted a molecular rather
than genetic approach in their study of the regulation and
function of peroxidase isozymes in tobacco. In their initial
report, they characterized tissue specific, wound inducible,
and virus inducible expression of 12 isozymes by isoelectric
focusing. They subsequently isolated a cDNA clone for the
acidic (pI 3.5, pI 3.75) isozymes and used Northern blot
analysis to determine tissue specific message levels for the
isozymes. Message levels for the anionic isozymes were
greatest in stem tissue, where they are thought to function
in the lignification of xylem. Southern blots of genomic DNA
from three species of tobacco probed with the peroxidase
cDNA indicate that the acidic isozymes are the products of
allelic genes. Similar experiments are planned for the study
of cationic and moderately anionic isozyme expression.
Lagrimini has continued the study of isozyme function by
transforming tobacco with anionic peroxidase clones under the
control of a promoter which allows constitutively high levels
of expression (personal communication). He has also

introduced an ’antisense' sequence, a sequence encoding an

20

mRNA complementary to the the peroxidase message, which
effectively reduced levels of peroxidase activity in the
tissue. The physiological effects of such transformations
promise to reveal valuable information on the regulation and
function of peroxidase isozymes.

The model systems described above are useful because
they have simplified the complex problem of assigning
function to numerous bands on a gel. The first system
simplifies the study of peroxidase isozymes conceptually, by
providing conclusive evidence that groups of 'isozymes' are
actually the products of a single structural gene. The
second study provides another tool with which to study the
regulation of peroxidase isozymes without relying on their
electrophoretic mobility. The use of specific probes for
each isozyme message will allow greater sensitivity in
determining the factors that affect their regulation.

The purpose of this chapter is to propose pathogen
mediated expression of peroxidase in cucumber as a model
system for the study of peroxidase regulation in

Cucurbitaceae. Preliminary evidence indicates that

 

peroxidase is regulated similarly in response to pathogens

among species of Cucumis, Citrullus (Chapter 1) and Cucurbita

 

 

 

(unpublished). The study of peroxidase regulation in
cucumber is inherently simplified by the presence of only one
anodic and two cathodic isozymes in leaf tissue. The anodic
isozyme, Px2, appears as a triplet in starch gels and is

similar to PRXa in petunia in that the three bands may be

21

attributed to a single structural gene (9) . In addition, a
specific function for this isozyme in lignification
associated with disease resistance has been proposed (15).
The systemic increase in peroxidase activity associated with
induced systemic resistance in cucumber is due to increases
in the anodic isozyme (14). An understanding of factors
important in the regulation of this isozyme in response to
disease will necessarily contribute to our understanding of

the regulation of induced systemic resistance.

CHAPTER II. MATERIALS AND METHODS

Peroxidase purification

Acidic intercellular peroxidase was purified from 200g of
the second and third leaves of cucumber seedlings 7 days
after the first leaf had been inoculated at 10 sites with a

1x10 spore/ml suspension of Colletotrichum lagenarium as

 

described in Chapter 1. The three forms of the cucumber
peroxidase triplet were separated by excising them
individually from a preparative native polyacrylamide gel
prior to electro-elution. Prior to extraction, one cm leaf
discs were removed from leaves for use in radial
immunoassays of peroxidase. Native and denaturing gels were

prepared and stained as described in Chapter 1.

Radial immunodiffusion

The quantity of native peroxidase protein in leaf extracts
was estimated by radial immunodiffusion. Immunodiffusion
plates were prepared by diluting crude anti-peroxidase sera
1:80 in 10 ml 1% melted agarose (50C),50mM Barbital buffer
pH 8.6. The agarose-antibody dilution was poured out onto
an 8X8 cm glass slide and allowed to solidify. Two mm
diameter wells were formed with a brass borer and agarose
plugs removed by aspiration. Wells were loaded with 5 ml
supernatant (2 mg protein/ml) of leaf homogenates from
systemically protected or control leaves, or with dilutions

of purified peroxidase. Proteins were allowed to diffuse

23

from wells in a moist petri dish for 5 days at room
temperature. Unprecipitated proteins were washed from the
agarose and precipitin rings were stained as described in
Chapter 1. A standard curve of peroxidase concentration
vs. the square of precipitin ring diameter was prepared
using the known dilutions of purified peroxidase, and
peroxidase content of leaf samples was calculated using

this curve.

Isoelectric focusing

Pre-poured ultra-thin (0.15 mm) isoelectric focusing gels
(pH 3-10) were obtained from Serva. Electrophoresis was
carried out on a horizontal electrophoresis unit (Bio-
Rad) to a final voltage of 1700V with a maximum power of 4
watts. Starting current was adjusted to obtain an intial
voltage of 200V. Electrode buffers were obtained from
Serva. Ten ml of leaf homogenate supernatants containing
10 mg protein were loaded into 20 ul capacity wells placed
on the center of the isoelectric focussing gel. A total of
1 ug of intercelluar extract protein was used for IEF gels.
Following electrophoresis, gels were stained for peroxidase

activity as described in Chapter 1.

Concanavalin A column chromatography
Peroxidase was chromatographed on ConA sepharose according
to manufacturers instructions (Pharmacia). Five micrograms

of pure peroxidase were applied to a 0.5x 1 cm column

24

prepared in a 3ml syringe stoppered with glass wool. The
column was washed with 0.02 M Tris-HCl,0.5M NaCl pH 7.4
until no peroxidase activity eluted. Bound peroxidase was
eluted with 0.5 M a-D-methylmannoside. Peroxidase in
column fractions was characterized by native gel

electrophoresis.

Treatment of isozymes with cxmannosidase

The purified bands of the cucumber peroxidase triplet were
treated individually with -mannosidase as described by
Gaudreault and Tyson (12). Twenty ug of each peroxidase was
incubated in SOmM Na Acetate (pH 4.5), 5mM Znso4 containing
crmannosidase (Sigma). The mixtures were placed at 37C for

72 hr prior to analysis by native PAGE.

Affinity purification of anti-cucumber peroxidase antibody
Five mg of DEAE purified peroxidase was coupled to 1 g
CNBr- activated sepharose according to manufacturers

instructions (Pharmacia). The freeze-dried sepharose was

washed in 1 mM HCl prior to the addition of peroxidase

dissolved in 5 ml coupling buffer ( 0.1M NaHC03 pH 8.3,
0.5M NaCl). The mixture was incubated overnight at 4C with
occasional stirring. Remaining active groups on the CNBr-

Sepharose were blocked with 0.2M glycine, pH 8.0 for 2 h at
room temperature. Excess peroxidase was removed with
sequential washes in coupling buffer followed by acetate

buffer (0.1M, pH 4) containing 500 mM NaCl, followed again

25

by coupling buffer. The gel was washed until no peroxidase
activity was detected in the wash fluid. For affinity
purification, 2 ml crude sera were diluted 1:1 in 2X PBS-
Tween ( 0.2% Tween 20,20mM NaH2P04, 300 mM NaCl, adjusted
to pH 7.2 with NaOH) and added to the peroxidase-sepharose
gel material. The mixture was incubated overnight at room
temperature with occasional mixing. Unreacted antibody was
washed from the gel with PBS-Tween. Bound antibody was
eluted from the gel using a modified procedure for antibody
elution from nitrocellulose (27). The sepharose mixture
was poured into a 3ml syringe stoppered with glass wool.
The syringe was suspended into a 15 ml disposable
centrifuge tube containing 100 .ml of 0.5M Na2PO4. One ml
of antibody elution buffer ( 5 mM glycine-HCl, pH 2.3, 500
mM NaCl, 0.1% Tween 20, 100 mg BSA/ml) was added at the top
of the syringe prior to centrifugation at 1600g for 2 min.
Rapid neutralization of eluted antibody was necessary for

the preservation of activity.

Western blots

The individual bands of the peroxidase triplet were tested
for their affinity to anti-cucumber peroxidase in a Western
blot. Equal quantities (500 ng) of each peroxidase were
electrophoresed in SDS polyacrylamide gels as described in
Chapter 1. After electrophoresis, the peroxidases were
blotted to nitrocellulose and probed with affinity purified

anti-cucumber peroxidase (1:100), and stained with alkaline

26

phosphatase according to the method of Blake et al. (6).
Attempts to blot native gels of peroxidase were

unsuccessful.

Peroxidase amino acid sequence analysis

The electroeluted peroxidase band with the highest apparent
molecular weight was selected for sequence analysis because
it was available in the greatest quantity. Peroxidase
sequence analysis of endoproteinase Lys-C (Boehringer-
Mannheim) fragments was conducted by William Burkhart
(Ciba- Geigy Agricultural Biotechnology Unit, Research
Triangle Park, NC). Digestion fragments were separated by
HPLC and sequenced by automated Edman degradations on the
Applied Biosystems 470A gas-phase sequencer.
Phenylthiohydantoin amino acids were analyzed using the

Applied Biosystems 120A PTH Analyzer.

27
CHAPTER II. RESULTS

Quantitation of leaf peroxidase by Radial immunodiffusion

Radial immunodiffusion results are summarized in Figs. 4 and
5 and Table 2. Peroxidase accounts for approximately 6% of
total soluble protein in homogenates of induced leaves
according to this assay. No precipitin rings developed
around extracts from control leaves, indicating that
constitutive levels of peroxidase are less than 1% of total

soluble protein

Purification of acidic peroxidase triplet

The purification scheme for peroxidase is shown in Tables 3
and 4. The high (H), medium (M), and low (L) molecular weight
bands of the peroxidase triplet were successfully separated
from one another as determined by SDS polyacrylamide gel
electrophoresis (Fig. 6 ). The three bands were eluted in
quantities that reflect the intensity of their staining on
native and denaturing polyacrylamide gels. The H, M, and L
bands represented 65%, 25%, and 10% of the total peroxidase
eluted, respectively. The three bands migrate to a single
isoelectric point of approximately 3.0 on isoelectric
focusing gels (Fig. 7 ), indicating that differences in
mobility on native polyacrylamide gels are due to variations

in molecular weight rather than charge.

28

 

“-71"
VLi

and
saw-:5
4;;

x.
....

4'.
'J.
*6

 

 

 

Radial immunodiffusion of peroxidase from

Fig.

leaf

control (C)

and

(P)

protected

systemically

50, and 25

of 200, 100,

#1

are 5

Standards

homogenates.

ug/ml peroxidase.

29

 

 

 

 

60
Q) q
E 50
(I
g 1
t:
e 4o-
(J
LU
l:
a.
‘Y: 30-
LIJ
l-
LU
22
S
O 20- l

10 . - , ,

0 100 200 300
[PEROXIDASE] (pg/ml)

Fig. 5. Standard curve for radial immunoassay

peroxidase.

0

Hi

30

Table 2. Results of peroxidase immunodiffusion assay of

protected leaves.

 

 

Sample Precipitin ring (diameter )2 Peroxidase %Total
diameter (mm) concentration (ug/ml) Protein
Standards (ug/ml)
200 7.5 56.3
100 5.5 30.3
50 4.5 20.3
25 3.5 12.3

Protected Leaves

1 5.0 25 72 3.6
2 8.5 72 266 13.3
3 6.0 36 1 16 5.3
4 5.5 30.3 92 4.6
5 7.5 56.3 202 10.1
6 6.0 36 1 16 5.3
7 4.5 20.3 52 2.6
8 7.5 56.3 202 10.1
9 6.0 36 1 16 5.3
10 6.0 36 1 16 5.3

 

AVERAGE 6.6 + 1.0

31

 

 

2 89. 3:. 85 85.82.8m

9. 8a.: 3 88 mfio

om com. 8 ad. 89 xocmzqmm
8. 83m 92 8: sera
6.2.8.25

6.5
2.52% 3 298.... 6535552 3
9m :3. > :>:o< 559 $5» E>fio< o_“__omam 20.551“.

 

>Hamofiamum>m Scum

mmmcfixoumm mo

.mm>mmH cmuomuoua

aowumoflMHusm

.m manna

32

 

S 8 . 83 8

 

mm O—N 85 Pa
8 0mm 80m mm
8:5 2...: 85d 5:03. A 022.2 2.. << . 5: mw<oxomma
.zsohew >:s=o<nueoyew

 

.me mUHEMa>uom>Hon
m souw ceausamouuomam an cmumaomfi mommcaxoumm

Heaea>auae mo >5a>aaee eeueeedm use aoaeeeameoamao .e manna

33

.umadfiuu mmmcfixoumd oflcocm one
we mau0w usage: hoazomaoa Aqv 30H can .2. asficms .A=. now:

Umumflomfl mo mfimwuonmouuomam Hmm wcfiEmH>uom>Hoa mam .m .ufim

 

Us pal Ill

Ohm A I I

+y ; l
- i I ' E Total
( i . - f - ~ ’ ~ Intercellular
, .._ 1 l _ - -
we“
Fig. 7. Isoelectric focusing of peroxidase from
intercellular extracts and total leaf homogenate. The gel

was stained for peroxidase activity and major bands of
activity are indicated by arrows ( l ). The anodic end of

the gel is indicated by (+) and the cathodic end by (-).

35

Concanavalin A-sepharose chromatography

Figure 8 shows the results of Con A-sepharose chromatography
of the acidic peroxidase triplet. The lowest molecular weight
peroxidase had no affinity for Con A while the band with
intermediate mobility bound somewhat and the slowest

migrating band bound tightly.

aMannosidase treatment

The effect of crmannosidase treatment on mobility of the
three peroxidases is illustrated in Fig. 9 . Treatment of
the highest and intermediate molecular weight peroxidases
with crmannosidase increased their mobility slightly on
native gels while mobility of the fastest migrating band was

unaffected.

Western blot

The three peroxidases reacted differentially with antibodies
prepared to all three (Fig. 10 ). The antibody reacted
strongly to the highest molecular weight peroxidase, slightly
to the intermediate peroxidase, and weakly to the lowest
molecular weight band. Smearing of the peroxidase bands is
possibly due to the incomplete reduction of disulfide bonds
in the presence of B-mercaptoethanol. This problem was
eliminated by using dithiothreitol in the sample buffer as

the reducing agent (Fig. 6 )

ConA
Total Bound

   

~.- .
. '
L—... A‘ L—“A u A AAA-
. x..—

Fig. 8. Affinity of anodic peroxidases for Con A-sepharose.

37

.ucwEummuu 0: Al. .pmummuu mmmpfimozcmse A+V

ou mmmcfixoumn mo mau0m usage: umaaomaos Aqv

. OWQHVHWOZCME U
00“.“

Bed nzt .sz szfivms .AIV :UH: mo >Hfiafinfiuamomzm .m

38

Fig. 10. Affinity of anti-cucumber peroxidase for high
(H), medium (M), and low (L) molecular weight forms of

anodic cucumber isozyme. Equal quantities (500 ng) of each

form are present.

39

Amino acid sequence of peroxidase

The sequence of three fragments of cucumber peroxidase is
compared to the corresponding sequences from horseradish,
turnip, and tobacco in Fig. 11. The cucumber peroxidase

fragments showed a slightly higher degree of homology to

tobacco peroxidase than to horseradish and turnip.

36 62
Tobacco IIRLHFHDCFVNGCDGSILLD-TDGTQT
Horseradish L A N TSFR
Turnip L F D SSFTG
Cucumber D V EDQ IT

83 122
Tobacco TALENVCPGVVSCADILALASEIGVVLAKGPSWQVLFGRK
Horseradish A v SA RT L TI AQQS T G R PL R
Turnip S V KA I ARDS QLG N N KV R
Cucumber V E RDA T S QG T QL

288 302
Tobacco TGTNGQIRTDCKRVN*
Horseradish Q LN RV SNS*
Turnip SS E KV GKT *
Cucumber T E N R L *

Fig. 11. Comparison of the amino acid sequence of three
fragments of cucumber peroxidase with horseradish, turnip,
tobacco peroxidase. Amino acids are designated by the
single letter code and numbered from the amino terminal of
tobacco peroxidase. Blank spaces indicate homology to

tobacco peroxidase.

#0

CHAPTER II. CONCLUSIONS

Peroxidase accumulates intercellularly in leaves during
induced systemic resistance and may comprise approximately
6% of the total soluble protein in the leaf. Constitutive
levels of the enzyme are much lower than this, indicating
that peroxidase is probably synthesized Q; 3212 in response
to stress signals associated with pathogen aggression. In
order to determine the involvement of dg‘ngyg transcription
and translation for peroxidase, it will be useful to have
antibodies which are specific for the peptide moiety of the
peroxidase glycoprotein.

Antibodies raised against the native acidic peroxidase
isozyme, which appears as three bands differing in size by
1-2 kd on SDS gels, react preferentially with the largest
of the three forms of the isozyme. The basis for this
differential reactivity may lie in the observed
differential glycosylation of the three bands. Clarke and
Shannon (8) have determined that the predominant
carbohydrate residues on horseradish peroxidase isozyme C
are mannose and glucosamine. Interestingly, Boller and
Metraux (7) have recently reported that significant levels
of a-mannosidase and 0-N-acetyl-glucosaminidase are
present in intercellular extracts of cucumber leaves. The
two faster migrating forms of peroxidase presumably result

from the post-translational modification of the larger

41

form, as has been suggested for the PRXa isozyme in Petunia
(1). Alternatively, the peroxidases differ in the extent
to which they are glycosylated initially. It is possible
that some peroxidase is exported from the cell before the
completion of glycosylation processes. A complete amino
acid sequence of the peroxidase isozyme will reveal the
potential sites for glycosylation.

The sequencing and subsequent cloning of the gene for
this peroxidase isozyme will facilitate definitive
experiments designed to answer questions regarding gene
copy number, time-course of mRNA synthesis in response to
pathogen stress, and the nature of a signal sequence which
may initially target the protein for export. Manipulation
of peroxidase expression through transformation experiments
similar to those being conducted with tobacco may
ultimately reveal the function of peroxidase in induced

systemic resistance.

10.

42

CHAPTER II. REFERENCES

Berg, B.M. van den, Bianchi, F., and H.J.W. Wijsman.
1984. Genetics of the peroxidase isoenzymes in Petunia.
Part 7: the alleles prxA6 and prxA7. Theor. Appl.
Genet.68:25-28.

Berg, B.M. van den, Hartings, H., Bianchi, F., and
H.J.W. Wijsman. 1984. Genetics of peroxidase isoenzymes
in Petunia. Part 9: immunological investigation into
differential expression of prxA alleles. Theor. Appl.
Genet. 68:265-268.

Berg, B.M. van den, and H.J.W. Wijsman. 1981. Genetics
of the peroxidase isoenzymes in Petunia. Part 1: organ
specificity and general genetic aspects of the
peroxidase isoenzymes. Theor. Appl. Genet. 60:71-76.

Berg, B.M. van den, Bianchi, F., and H.J.W. Wijsman.
1983. Genetics of the peroxidase isoenzymes in Petunia.
Part 5: differential temporal expression of prxA
alleles. Theor. Appl. Genet. 65:1-8.

Berg, B.M. van den, and H.J.W. Wijsman. 1982. Genetics
of the peroxidase isoenzymes in Petunia. Part 3:
location and developmental expression of the structural
gene prxA. Theor. Appl. Genet. 63:33-38.

Blake, M.S., Johnston, K.H., Russel-Jones, G.J., and
E.C. Gotschlich. 1984. A rapid, sensitive method for
detection of alkaline phosphatase-conjugated anti-
antibody on Western blots. Anal. Bioch. 136:175-179.

Boller, T., and J.P. Metraux. 1988. Extracellular
localization of chitinase in cucumber. Physiol. Molec.
Plant Pathol. 33:11-16.

Clarke, J. and L. M. Shannon. 1976. The isolation and
characterization of the glycopeptides from horseradish
peroxidase isoenzyme C. Biochim. Biophys. Acta 427:428-
442.

Dane, F. 1983. Cucurbits. In: Isozymes in Plant Genetics
and Breeding. Part B. Elsevier, Amsterdam. S.D. Tanksley
and T.J. Orton eds. pp. 369-390.

Dencheva, A., and D. Klisurska. 1982. Interaction
between peroxidase and IAA-oxidase in the course of
growth and differentiation of plant cells. Physiol.
Veg. 20:385-394.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

43

Fukuda, Komamine A. 1982. Lignin synthesis and its
related enzymes as markers of tracheary-element
differentiation in single cells isolated from the
mesophyll of Zinnea elegans. Planta 155:423-430

Gaudreault, P.R., and H. Tyson. 1988. Elimination of
differences in the mobility of flax isoperoxidases on
PAGE by digestion with a -Mannosidase. Plant Physiol.
86:288-292.

Halliwell, B. 1978. Lignin synthesis: the generation of
hydrogen peroxide and superoxide by horseradish
peroxidase and its stimulation by manganese (II) and
phenols. Planta 140:81-88.

Hammerschmidt, R. and J. Kuc. 1982. Association of
enhanced peroxidase activity with induced systemic
resistance of cucumber to Colletotrichum lagenarium.
Physiol. Plant Pathol. 20:73-82.

Hammerschmidt, R., Nuckles, E., and J. Kuc. 1982.
Lignification as a mechanism for induced systemic
resistance in cucumber. Physiol. Plant Pathol. 20:61-71.

Hendriks,T., B.M. van den Berg, A.W. Schram. 1985.
Cellular location of peroxidase isozymes in leaf tissue
of Petunia and their affinity for concanavalin A-
Sepharose.P1anta 164:89-95

Hinman, R.L., and J. Lang. 1965. Peroxidase catalyzed
oxidation of indole-3-acetic acid. Bioch. 4:144-158.

Lagrimini, L.M., Burkhart, W., Moyer, M., and S.
Rothstein. 1987. Molecular cloning of complementary DNA
encoding the lignin-forming peroxidase form tobacco:
molecular analysis and tissue-specific expression.

Proc. Natl. Acad. Sci.(USA). 84:7542-7546.

Lagrimini, L.M., and S. Rothstein. 1987. Tissue
specificity of tobacco peroxidase isozymes and their
induction by wounding and tobacco mosaic virus
infection. Plant Physiol. 84:438-442.

Machackova, I., and z. Zmrhal. 1981. Is peroxidase
involved in ethylene biosynthesis? Physiol. Plant.
53:479-482.

Mader M., and V. Amberg-Fisher. 1982. Role of
peroxidase in lignification of tobacco cells. Plant
Physiol.70:1128-1131.

23.

24.

25.

26.

27.

28.

29.

an

Mader, M., Ungemach, J., and P. Schloss. 1980. The role
of peroxidase isozyme groups of Nicotiana tabacum in
hydrogen peroxide formation. Planta 147:467-470.

Mader, M. 1976. Localization of peroxidase isozyme group
G1 in the wall of tobacco tissues. Planta 131:11-15.

Paul, K.G. 1986. Peroxidases: historical backround. In:
Molecular and Physiological Aspects of Plant Peroxidases.
H. Greppin, C. Pennel, Th. Gaspar, eds. Imprimerie
Nationale, Geneva. pp. 1-14. ‘

Rick, C.M., Tanksley, S.D., and J. Fobes. 1979. A pseudo-
duplication in Lycopersicon piminellifolium. Proc. Natl.
Acad. Sci.(USA) 76:3435-3439.

Rohwer, F. and M. Mader. 1981. the role of peroxidase in
ethylene formation from 1-aminocyclopropane-1-carboxylic
acid. 2. Pflanzenphysiol. 104:363-372.

Smith, D.E., and P. A. Fisher. 1984. Identification,
developmental regulation, and response to heat shock of
two antigenically related forms of a major nuclear
envelope protein in Drosophila embryos: application of an
improved method for affininty purification of antibodies
using polypeptides immobilized on nitrocellulose blots. J
Cell Biol. 99:20-28.

Srivastava, C.P., and R.B. van Huystee. 1973. Evidence
for close association of peroxidase, polyphenyl oxidase
and IAA oxidase isozymes of peanut cell suspension
culture medium. Can J. Bot 51:2207-2215.

Welinder, K.G. 1985. Plant peroxidases: their primary,
secondary and tertiary structures, and relation to
cytochrome c peroxidase. Eur. J. Biochem. 151:497-503.

CHAPTER III.

ISOLATION AND CHARACTERIZATION OF TN5 MUTANTS OF

PSEUDOMONAS SYRINGAE PV. SYRINGAE NO LONGER ABLE TO INDUCE

 

RESISTANCE

45

45

CHAPTER III. INTRODUCTION

Induced systemic resistance in cucurbits was first
described by Kuc et a1 (1975). They reported that

inoculation of one leaf of cucumber with Colletotrichum

 

lagenarium induced systemic resistance against subsequent

 

challenge by Colletotrichum lagenarium. Since that initial

 

report, more than 12 pathogens, including viruses, bacteria
and fungi have been shown to induce resistance (Kuc 1983).
Resistance is nonspecific with respect to the inducing
pathogen and the challenge pathogen. Histochemical and
ultrastructural studies of infection sites on protected
leaves indicate that fungal penetration is arrested in the
epidermal cell layer (Richmond et a1. 1979, Xuei et a1
1987). The inability of pathogens to penetrate the
epidermis is associated with an enhanced ability of the
outer epidermal walls to lignify at infection sites
(Hammerschmidt and Kuc 1982, Basham and Cohen 1983, Dean
and Kuc 1987).

Biochemical changes which occur prior to challenge
inoculations include systemic increases in extracellular
chitinase and peroxidase ( Metraux and Boller 1986, Metraux
et al. 1988, Hammerschmidt et al. 1982, Smith and
Hammerschmidt 1988). Peroxidase has provided a convenient
marker for induced resistance, as increases in peroxidase
activity precede the expression of induced resistance

(Hammerschmidt and Kuc 1982, Stermer and Hammerschmidt

47
1984). The suggested role for peroxidase in lignin
biosynthesis and the potential role of chitinases in the
release of elicitors from fungal cell walls (Boller, 1985)
indicates that these enzymes may be important components of
the induced resistance response.

A fundamental question remaining in the study of
induced systemic resistance is the nature of the 'signal'
that has been hypothesized to move from the leaf on which
the inducing inoculum is applied to other tissues (Jenns
and Kuc 1979, Guedes et a1. 1980, Dean and Kuc 1986). We
have chosen a genetic approach to look for specific
signals generated in the pathogen-host interaction which
are involved in the induced resistance response.
Specifically, we hoped to identify gene products of a
pathogen which would potentially have in vitro resistance
inducing activity. A bacterial system was chosen for this
study because of the relative ease of genetic manipulation.
In the initial experiments, we found that the
hypersensitive response (HR) elicited by a wheat isolate of

Pseudomonas syringae pv syringae was more effective than

 

the disease reaction caused by Pseudomonas syringae pv

 

lachrymans at inducing systemic resistance in cucumber. A

 

Tn5 mediated mutational analysis of P.§. syringae was

conducted in order to identify bacterial genes responsible

for the efficient induction of resistance.

48

CHAPTER III. MATERIALS AND METHODS

Media and bacterial strains
Luria-Bertani media was used for culturing E. coli and

Pseudomonas spp. overnight cultures. Davis minimal agar

 

plates (Lederberg 1950) were used for growing strains of

Pseudomonas spp. Antibiotics were used in the following

 

concentrations: naladixic acid (Nal) 200 ug/ml; kanamycin
(Kan) 50 ug/ml; tetracycline (Tc) 15 ug/ml and rifampicin
(Rif) 100 ug/ml. Table 1 lists bacteria, plasmids and

their relevant characteristics.

Culture of plants
Cucumber seedlings var SMR-58 were grown from seed in 6
inch clay pots in the greenhouse as described previously

(Hammerschmidt et al. 1982).

Challenge inoculations

The fungal pathogen Colletotrichum lagenarium, causal agent

 

of anthracnose on cucurbits, was used for all challenge
inoculations. Culture of the pathogen was as described
previously ( Kuc and Richmond 1977). For challenge
inoculations, ten 10 pl drops of a 1 x losspore/ml
suspension were placed on leaf 2 of induced or control
plants at various times after the inducing inoculation.
High relative humidity necessary for spore germination was

maintained by placing the plants individually into plastic

49

 

scene mans

scene mans
sesum mags

Aommfi. .He no muuao
Ahmma. .Hm um NUHmemmum
Imam“. .He no cesam
Lemma. .Hm um cmuocafiq

oflno .cmmuo ocfiazom

.OU Ncfiwx .=.h .GLOH .<.U

>Usum macs

Amman.
unopenfizm one bcmeafi>

<29 owsocmo
omommm acficfioucoo mmm<aa “Doe

mmommm scum acmeoouu Hmoom
mce ocficfimucou mmméqm «Dex .uoh

Hmammm sown ucmsomuw Hmoom
mcb ocficfioucoo mmmdqm «wax .uok

+Hmowu .+~xmmue .moCH .usx
+~xmomu +moo mocH pub

HEU .uex umce

 

Afimfimmmz. eHoofiHommmnd .>a
mmacapaw .m mo :ofiomu on: mzu
ocfi>uumo paamoo mmm<qn “no?

HEX

:moocumo umnssoso “mafia

mCEHHONDmmm uufix .HHMZ

cwoosumo been: unamz

 

ammo

Nmommmm

HNQmme

mHoNme
mmmdqn

“Hofiasmd

moan
Ammo

mcmahusomd >o
wmocfiu>m mocosopsmmm

NNQmma .Hmomma

.omnmmm. mmocfip>m >o
mmocfiu>m mucosopzmmm

 

OUH§OW HO OOCGHQHOK

mouufimapouomnmso

cfiEmmHo no :Hmnum

 

mpfiammao paw mcflmuum Hmfiumuomm

.m mHQMB

50

bags for 24 hr. Lesion diameter was measured to the

nearest millimeter 7 days after the challenge inoculation.

Transposon mutagenesis

The suicide plasmid vector of Tn5, pSUP1011, was used to
generate mutants of PSSD20 as described by Anderson and
Mills (1985). Colonies of PSSD20 containing Tn5 insertions
were selected on minimal media supplemented with 50 ug/ml
kanamycin. Prototrophic transconjugates from two separate
matings were screened for HR and peroxidase inducing
ability. Attempts to select for Tn5 insertions in g.

syringae pv lachrymans were unsuccessful.

 

Screening of Tn5 mutants for loss of ability to induce
peroxidase activity

Single colonies of prototrophic kanamycin resistant
bacteria were aseptically transfered into sterile test
tubes containing 2 ml LB media. The tubes were placed in a
test tube rack on a rotory shaker at room temperature.
Overnight cultures were diluted to 10 ml with dH20 and
infiltrated individually at ten sites into the first leaves
of cucumber seedlings. Three days after infiltration, 1 cm
leaf discs were removed from each plant and homogenized
separately in 1.9 ml microfuge tubes using a modified drill
and drill bit. Leaf homogenates were resuspended in 1 ml of
0.01 M phosphate buffer (pH 6), centrifuged in a microfuge

for 5 min and the supernatants assayed for peroxidase

51

activity.

Peroxidase assay

Peroxidase activity in leaf disc homogenates was assayed by
adding 2.5 ul of each supernatant to 25 ul dH20 in
microtiter plate wells (Nunc) followed by the addition of
100 ul substrate solution containing 220 ug ABTS/ml ( 2-2'-
Azino-di(3-ethyl benzthiazoline sulfonic acid), 50 mM
citrate buffer pH 4.0 and 0.02% H202. Color development
was allowed to proceed for 15 min followed by the addition
of 100 nl stopping reagent containing 0.17% HF, 6 mM NaOH,
and 5th EDTA. An ELISA plate reader (Dynatech) was used
to quantitate color development ( A.A405) in wells. Ten
control samples and 10 samples from induced plants were
included in each plate. Mutants from inoculated plants
which gave absorbance values below the average of the

positive controls were retested.

In give growth of bacteria

Overnight cultures of bacteria were washed in saline and
diluted to an OD600 of 0.05 for inoculation (approximately
104 cfu/ml). Approximately 200,ul of each bacterial strain
was infiltrated into 5 separate plants for each time point.
One cm discs containing the infiltration site were removed
at various times after infiltration, surface sterilized in
20% bleach, and homogenized in saline. Homogenates were

serially diluted in saline and total cfu/infiltration site

52

determined for each disc.

Cloning of Tn5 insertion sites from PSSD21 and PSSD22 .

Southern blots revealed that both mutants contained Tn5 in
a 17 kb EcoRI fragment. Genomic DNA from mutants was
digested to completion with EcoRI and size fractionated on
a linear 5-40% sucrose gradient for 5 hr at 25,000K in a
Sorvall TH641 rotor. Fractions containing fragments in the
10-20 kb range were pooled, ligated to pLAFR3 and packaged
into phage using a commercially available packaging extract
(Promega). Phage from the packaging mixture were used to
transduce E. coli H3101 according to manufacturers
recommendations. Colonies containing Tn5 were selected on
LB containing 50.ng/ml kanamycin. Twenty km colonies were
selected from each packaging reaction and screened in mini-
preps for cosmids containing only the 17 kb EcoRI genomic
fragments from PSSDZl and PSSD22. The resulting cosmids,
pPSSD21 and pPSSD22, were used for mapping of Tn5 insertion

sites.

Construction of PSSD20 genomic library in pLAFR3

A genomic library of PSSD20 was constructed as described by
Lindgren and Panopoulos (1986). Total genomic DNA was
partially digested with Sau3A and size fractionated on a
linear 5-40% sucrose gradient as described above.
Fragments of MW 15-30 kb were ligated to the BamHI site of

pLAFR3. The cosmid ligation mixture was packaged in vitro

53

and the resulting phage were transduced into E. coli HB101.
Transconjugants were selected on LB containing tetracyline

and streptomycin.

Tri-parental matings
Ten ml overnight cultures of recipient E.§. syringae

strains, donor E. coli containing pLAFR3 clones and E. coli

 

HB101 containing the helper plasmid pRK2013 were pelleted

at 4000g for 10 min in a Sorval SS-34 rotor. Cells were
washed twice in 10 ml 0.85% sterile saline prior to
mating. Twenty ul of each bacteria was pipetted onto a
sterile 45 pm. millipore filter on complete agar plates.
After incubation in a 27C incubator for 8 hr, filters were
washed in 10 ml sterile saline and serial dilutions were

plated on selective media.

Basic molecular techniques

Nick translations were performed using a commercially
available kit (BRL). DNA fragments for use as probes were
electrophoretically eluted from gels onto DEAE membranes
(Schleicher & Schull) according to manufacturers
recommendations. Standard procedures, including isolation
of genomic and plasmid DNA, agarose gel electrophoresis,
ligation, restriction endonuclease digestions and Southern
blotting were performed as described by Maniatis et

al.(1982).

54

CHAPTER III. RESULTS

HR vs. disease induced peroxidase activity and resistance

The hypersensitive response elicited by Pseudomonas syringae

 

pv. syringae was more effective than disease caused by

Pseudomonas syringae pv. lachrymans at inducing systemic

 

 

increases in peroxidase activity. Peroxidase induction was
maximal after only 2 days for plants induced with E. syringae
pv. syringae while those induced with the pathogen E.

syringae pv lachrymans required 6 days for maximal peroxidase

 

induction (Fig. 12, A, B). Further time course experiments
indicate that peroxidase activity begins to increase at 24-36
hr after inoculation with E.§.syringae, precisely the time HR
symptoms begin to appear (Fig. 13). Consistent with the
rapid increase in peroxidase activity, near maximal
resistance in HR induced plants is expressed 2 days after the
inducing inoculation as demonstrated by a decrease in C.

lagenarium lesion diameter on challenged leaves (Fig 14)

 

Plants induced with the pathogen P.§.lachrymans required 5-6

 

days after the inducing inoculation to acquire the same level

of resistance.

Mutant isolation

Approximately 2000 prototrophic kanamycin resistant mutants
were initially screened, and two were isolated which did not
induce peroxidase activity. These mutants were designated

PSSD21 and PSSD22. Both mutants had lost the ability to

PEROXIDASE ACTIVITY AA 405

55

 

0.4 'I

0.3 "

0.2“

0.1 -‘

 

 

 

 

0.0 "

8
D
0)
°-' 82
BACTERIA I‘NFILTRATED INTO LEAFl

a. a -
e o ,,,
l‘ f

Fig. 12. Systemic increases in peroxidase activity in
response to inoculation of' leaf 1 with Pseudomonas syringae

pv. syringae (PSSD20) or Pseudomonas syringae pv. lachrymans

 

 

 

(P.s.l.). Peroxidase activity in leaf 2 of plants was
determined at 2 days (A), or 6 days (B) after inoculation of

leaf 1. Control plants received no treatment.

PEROXIDASE ACTIVITY AA 405

56

 

 

 

 

 

 

02
0.1 -
.l
4
F
0.0 . r r , r
20 30 40 50
TIME (HOURS AFTER INFILTRATION)
Fig. 13. Time course for the induction of peroxidase

activity in the second leaf of plants inoculated on the first
leaf with Pseudomonas syringae pv syringae ( -.- ) or control

plants ( 4? ).

57

120

 

% CONTROL LESION DIAMETER

 

 

 

f r t I r r f I ' I ' fl

0 1 2 3 4 ' 5 6 v 7
TIME (DAYS AFTER INOCULATION)

Fig. 14. Time course for the expression of induced systemic

resistance in plants inoculated on leaf 1 with Pseudomonas

 

syringae pv syringae ( 4P ) or Pseudomonas syringae pv.
lachrymans ('0'). Leaf 2 of inoculated or control plants was
challenged with 10 drops of a 10s spore/ml suspension of
Colletotrichum lagenarium at various times after inoculation.
Lesion diameter was measured 7 days after challenge

inoculations and reported as % of control lesion diameter for

each time point.

58

produce a hypersensitive reaction (HR‘ ) and had
simultaneously lost the ability to cause disease on the host
plant wheat. Mutant PSSD21 produced chlorosis at the site of
infiltration when greater than 10 cfu/ml were infiltrated
while PSSD22 produced no reaction at any concentration
infiltrated. Neither mutant was able to induce resistance in

cucumber (Fig. 15).

In vivo growth of bacteria

59 vivo growth of PSSD21 was compared to that of the wild

type E.§. syringae and the pathogen E.§. lachrymans. After an

 

initial 10-fold increase the population of mutant remained
relatively stable. (Fig. 16 A,B). Wild type E-E' syringae
multiplied 10 - 10 during the first 2 days after

infiltration while the pathogen E.§. lachrymans multiplied

 

10 during the same time period (Fig. 16A). Mutant PSSD22
did not multiply in leaf tissue as well as the wild type as
determined by subsequent 53 .XEXS growth studies (Fig. 16B).
The genomic clone pPs1 which restored the ability of the
mutants to produce an HR slightly enhanced the ability of the
mutants to multiply in leaf tissue (Fig168) This enhancement
was only observed during the first 2 days after infiltration,
however, possibly due to the instability of the large cosmid
in the absence of antibiotic selection. The loss of ability

to induce an HR was not solely due to the inability of
mutants to grow in leaf tissue, as infiltration of up to 10

cfu/ml still did not produce a hypersensitive response.

59

 

LESION DIAMETER (mm)
0|

 

 

 

 

PSSDQI
PSSD22
PSSDZJ

PSSDZHpPSI)
PSSDZ2(pPSI)

BACTERIA INFILTRATED INTO LEAF 1

Fig. 15. Comparative ability of wild type, Tn5 mutants, and
restored mutants of §,§, syringae to induce systemic
resistance. Bacteria were infiltrated at 10 sites into leaf
1 at a concentration of 10 CFU/ml and leaf 2 was challenged
4 days later with 10 drops of a 105 spore/ml suspension of
Colletotrichum lagenarium. E. lagenarium lesion diameter was

determined 7 days after the challenge inoculation.

60

10

 

LOG CFU/ LEAF DISC
0')

 

 

 

 

 

‘ + I I
4 ' ‘ *3:
‘ at PSSDZO
4r PSSDZ1
+ P. s. pv lachrymans l
2 r F r T f I T 1 fi
0 1 2 3 4 5
TIME (DAYS)

Fig. 16. A,B. £2 planta growth of bacteria. Each data point
represents the mean of the log of total cfu/disc from 5

plants. Standard error bars are shown.

LOG CFU/ LEAF DISC

61

 

 

   

 

5 -l 4:- PSSDZ1

 

 

+ PSSDZZ
-l- PSSDZ1(pPSt)
+ P88022(pPS1)
-II- PSSDZO
4 . . , l , , I
o 1 2 3

TIME (DAYS)

62

Physical characterization of Tn5 insertion sites

Southern blots of Eco R1 digested genomic DNA from the two
mutants probed with pRKC7, which contains the Km gene
present within Tn5, indicated that Tn5 was present in a 17 kb
fragment in each of the two mutants (blot not shown). Tn5
insertion sites were deduced from a restriction map of the
cloned fragments using BamHI, and HindIII which out within
Tn5 but did not cut within flanking regions of genomic DNA
(Fig. 17). A 10 kb EcoRI HindIII fragment from pPSSD21
indicated in Fig 17, was used as a probe in a Southern blot
of EcoRl digested DNA from PSSD20, PSSD21, and PSSD22 (Fig.
18). The probe hybridized to one 11 kb fragment of PSSD20 and
to one 17 kb fragment in each of the two mutants, indicating
that Tn5 had inserted into different sites of the same Eco R1

fragment in the mutants.

Isolation of clone from library and complementation of
mutants
The 10 kb HindIII fragment from PSSD21 (Fig 17) was used to

probe a genomic pLAFR3 library of the wild type E.

[In

syringae. Approximately 1000 colonies were screened and two
were isolated which hybridized strongly to the probe; pPSl
and pPSZ. The cosmids were mobilized separately into PSSD21
and PSSD22 and the resulting transconjugates were tested for

their ability to induce an HR on cucumber. One cosmid, pPSl

63

 

.czonm an Hmommm scum mnoud HHHceem ax oH one .mmommao

can ”Newman an A a. mouse coauummefi.mae mo coaumooq .AH .ofim

e:

T:

JLm
1T”

IZOSSd -'
ZZOSSd "’

..>Hm>auemdeeu m can N .H meson .NNQmme
one Hmamma .omomma seem aza eeeoaeo nonmemea Hmoem on Hwnmmac

acne caeaeeum HHH caem mg as no aoeueueenunsm .me .ofim

Aux

65

restored the ability to induce HR to both mutants while the
second cosmid, pPSZ restored function to PSSD22 but not
PSSD21. As Tn5 was inserted into different sites in the
same EcoR1 fragment in the two mutants, we tested the ability
of pPSSD21 and pPSSD22 to restore function to PSSDZZ and
PSSD21, respectively. Introduction of pPSSD21 into PSSD22
restored the ability of PSSD22 to cause HR on cucumber and
disease on wheat. However introduction of pPSSD22 into PSSD21

did not restore function to that mutant.

Restoration of Resistance Inducing Activity to Mutants

The genomic clone pPSl restored the ability of PSSD21 and
PSSD22 to induce resistance and peroxidase activity, although
the restored mutants were somewhat less effective than

PSSD20 (Figs. 15,19).

Homology to HRP cluster from E. sygingae pv. phaseolicola

 

The simultaneous loss of ability to cause disease on wheat
and HR on cucumber indicates that mutants PSSD21 and PSSD22
belong to a class termed 'HRP' mutants by Lindgren et

al.(1986). The E. syringae pv. phaseolicola hrp gene cluster

 

 

necessary for disease and HR has been cloned and
characterized (Lindgren et. al. 1986; Lindgren et. al. 1988).
We obtained plasmid pPL6 containing the entire hrp cluster

from E.§. phaseolicola to study functional and sequence

 

homology to the P.§. syringae region. Plasmid pPL6 restored

the ability of both PSSD21 and PSSD22 to cause HR on

PEROXIDASE ACTIVITY AA405

 

02

 

 

 

PSSDQO

E 8
2,3 :3
O.

PSSD21(pPSI)
P88022(pP81)

Fig. 19. Comparative ability of wild type, Tn5 mutants, and
restored mutants of E.§. sygingae to induce systemic
increases in peroxidase activity. Plants were inoculated
with bacteria as described in Fig. 15. One cm leaf discs
were removed for peroxidase determination prior to challenge

with Colletotrichum lagenarium.

67

cucumber. A southern blot of BglII/EcoRi digested pPL6 was
probed with the 10 kb EcoRI HindIII fragment from pPSSD21
and the probe hybridized strongly to the 7.4 kb fragment of

pPL6, and weakly to the 4.3 and 4.0 kb bands (Fig. 20).

68

kb

23-'

9A-

40‘—

 

Fig. 20. Homology between Pseudomonas syringae pv.

 

phaseolicola HRP region and a portion of the functionally

 

similar region from PSSD20. A, pPL6 digested with BglII and
EcoRI. B, hybridization of 10 kb HindIII fragment from
PSSD21 to BglII/EcoRI digested pPL6. The blot was

hybridized and washed in the presence of 1.0 M NaCl and 1%

SDS at 68C.

 

69

CHAPTER III. DISCUSSION

In all cases of induced sytemic resistance in cucumber
reported, the level of resistance induced systemically has
been quantitatively correlated to the amount of disease
necrosis on the inducing leaf (Hammerschmidt et al. 1982, Kuc
1983). Similarly, the expression of induced systemic
resistance is related temporally to the appearance of
necrosis on the inducer leaf. These observations are
supported in our study, in which the HR induced by E.
syringae pv. syringae induces resistance more rapidly than
the more slowly developing necrotic lesion induced by E.

syringae pv. lachrymans. The requirement for necrotic lesion

 

formation has been supported genetically through the
generation of Tn5 mutants of E. syringae pv. syringae that no
longer induced the HR or systemic resistance.
Complementation of the mutations demonstrated that
restoration of HR also resulted in the restoration of
resistance inducing activity.
The mutants isolated in this study are characteristic

of a class of mutants described in other pathovars of

Pseudomonas syringae that exhibit a simultaneous loss in the

 

ability to cause an HR on nonhosts and the ablility to cause
disease on host plants. The region of the genome necessary
for these functions, known as the 'HRP' region, has been
shown by Lindgren and Panopoulos (1987) to be conserved among

Pseudomonas syringae pathovars. The genomic region of E.

 

7O

syringae pv syringae that we have identified as important in
the induced resistance response shares homology with the

cloned HRP region of E. syringae pv. phaseolicola (pPL6)

 

based on complementation tests and DNA hybridization. A
functionally similar hrp cluster in Pseudomonas solanacearum
has been shown by Boucher et al (1987) to share homology with

other strains of P. solanacearum and eight pathovars of

 

Xanthomonas campestris.

The observation that common bacterial genes are
involved in pathogenesis and the hypersensitive response
suggests that the two reactions may produce common 'signal'
molecules in bacteria-plant interactions. Thus with respect
to induced systemic resistance, the difference in reponse to
disease and HR causing pathogens is quantitative rather that
qualitative. Lyon and Wood (1976) have observed similar
quantitative differences in the ability of HR and disease

causing races of E. phaseolicola to cause electrolyte leakage

 

in bean leaves. Both races induced significant electrolyte
leakage, but the HR reaction induced high levels of leakage
after only 24 hr while the pathogen required three to four
days to induce a similar response. The saprophyte E.

fluorescens did not induce any electrolyte leakage. Klement

 

(1982) has also suggested that hypersensitive and disease
reactions differ primarily in the speed and magnitude of the
response.

A better understanding of the physiology and biochemistry

of the HR may provide insight into the nature of the signal

71

generated in response to pathogens. Physiological changes
which have been associated with the HR include lipid
peroxidation (Keppler and Novacky 1986,1987) and loss in
membrane potential accompanied by a rapid efflux of K+ ions
(Pavlovkin et a1 1986, Atkinson et al. 1985). Baker et al.
(1987) demonstrated that Tn5 mutants of E. syringae pv. Elil
that have lost the ability to cause a hypersensitive response
on tobacco, also fail to induce the K+ ion efflux. Atkinson
and Baker (1987) have also shown that the enhanced K+ efflux,
H+ influx associated with the HR helps to promote the growth
of bacteria in intercellular spaces of leaves. How these
physiological changes associated with the HR may relate to
the induction of systemic resistance is unknown and area for
future research.

In conclusion, we have demonstrated that rapid
coordinated cell death associated with the HR induced by E.
syringae pv. syringae induces systemic resistance and
peroxidase activity more efficiently than does the necrotic

lesion caused by E. syringae pv. lachrymans. In addition,

 

Tn5 mutagenesis and complementation studies have provided the
first genetic evidence for the requirement of pathogen
induced necrosis for induced resistance expression. We have
also demonstrated that the region of the E. syringae pv.
syringae genome necessary for inducing resistance is
functionally homologous to the hrp region of E. syringae pv.

phaseolicola and shares at least some sequence homology. The

 

mutants generated in this study will provide a new tool to

72

study the physiological and biochemical events underlying

induced systemic resistance in cucumber.

73

CHAPTER III. REFERENCES

Anderson, D. M. and Mills, D. 1985. The use of transposon
mutagenesis in the isolation of nutritional and virulence
mutants in two pathovars of Pseudomonas syringae.
Phytopathology 75:104-108.

 

Atkinson, M.M., Huang, J.-S., and Knopp J. A. 1985. The
hypersensitive reaction of tobacco to Pseudomonas syringae pv
pisi: Activation of a plasmalemma K+/H+ exchange mechanism.
PIant Physiol. 79:843-847.

Atkinson, M.M., and Baker, C.J. 1987. Association of host
plasma membrane K+/H+ exchange with multiplication of
Pseudomonas syringae pv. syringae in Phaseolus vulgaris.
PhytopathSlogy 77:1273-1279.

 

 

Baker, C.J., Atkinson, M.M., and Collmer, A. 1987.
Concurrent loss in Tn5 mutants of Pseudgmonas syringae pv.
syringae of the ability to induce the HR afid host plasma
memBrane K+/H+ exchange in tobacco. Phytopathology 77:1268-
1272.

 

Bashan, B., and Cohen, Y. 1983. Tobacco necrosis virus
induces systemic resistance in cucumbers against Sphaerotheca
fuliginea. Physiol. Plant Pathol. 23:137-144.

 

 

Boller, T. 1985. Induction of hydrolases as a defense
reaction against pathogens. In: Cellular and Molecular
Biology of Plant Stress. J.L. Key and T. Kosuge eds. A. R.
Liss. New York. pp. 247-262.

Boucher, C.A., Ban Gijesegem, F., Barberis, F.A., Arlat, M.
and Zischek C. 1987. Pseudomonas solanacearum genes
controlling both pathogenicity on tomato and hypersensitivity
on tobacco are clustered. J. Bacteriol. 169:5626-5632.

 

 

Dean, R.A., and Kuc, J. 1986. Induced systemic protection in
cucumber: the source of the signal. Physiol. Mol. Plant Path.
28:227-233.

Dean, R.A., and Kuc, J. 1987. Rapid lignification in response
to wounding and infection as a mechanism for induced systemic
protection in cucumber. Physiol. Mol. Plant Pathol. 31:69-
81.

Ditta, G., Stanfield 8., Dorbin, D., and Helinski, D.R. 1980.
Broad host range cloning system for gram-negative bacteria:
construction of a gene bank of Rhizobium melilota. Proc.
Natl. Acad. Sci. USA. 27:7347-7351.

 

 

74

Guedes, M.E.M., Richmond, S., and Kuc, J.1980. Induced
systemic resistance to antracnose in cucumber as influenced
by the location of the inducer inoculation with
Colletotrichum lagenarium and the onset of flowering and
fruiting. Physiol. Plant Pathol. 17:229-233.

Hammerschmidt, R. and Kuc, J. 1982. Lignification as a
mechanism for induced systemic resistance in cucumber.
Physiol. Plant Pathol. 20:61-71

Hammerschmidt, R., Nuckles, E., and Kuc, J. 1982. Association
of enhanced peroxidase activity with induced systemic
resistance of cucumber to Colletotrichum lagenarium.
Physiol. Plant Pathol. 20:73-82.

 

 

Jenns, A., and Kuc, J. 1979. Graft transmission of systemic
resistance of cucumber to anthracnose induced 'by
Colletotrichum lagenarium and tobacco necrosis virus.
Phytopathology. 69:753-756.

 

 

Keppler, L.D., and Novacky, A. 1987. The initiation of
membrane lipid peroxidation during bacteria induced HR.
Physiol. Mol. Plant Pathol. 30:233-245.

Keppler, L.D., and Novacky, A. 1986. Involvement of membrane
lipid peroxidation in the development of bacterially induced
hypersensitive reaction. Phytopath. 76:104-108.

Klement, Z. 1982. Hypersensitivity. In: Phytopathogenic
Prokaryotes. Vol. 2. M.S. Mount and G.H. Lacy eds. American
Press. pp 149-177.

Kuc, J. 1983. Induced Systemic resistance inplants to
diseases caused by fungi and bacteria. In: The Dynamics of
Host Defense. J. Bailey and B. Deverall, eds. Academic,
Sydney. pp. 191-221.

Kuc, J. and Richmond S. 1977. Aspects of the protection of
cucumber against Colletotrichum lagenarium by Colletotrichum
lagenarium Phytopathology 67:533-536.

 

 

 

Kuc, J., Schockley, E., and Kearney, K. 1975. Protection of
cucmber against Colletotrichum lagenarium by Colletotrichum
lagenarium. Physiol. Plant Pathol. 7:195-199.

 

 

 

Lederberg, J. 1950. Isoalation and characterization of
biochemical mutants of bacteria. Methods Med. Res. 3:5-22.

Lindgren, P.B., Peet, R.C, and Panopoulos, N.J. 1986. Gene
cluster of Pseudomonas syringae pv. phaseolicola controls
pathogenicity of bean plants and hypersensitivity on nonhost
plants. J. Bacteriol. 168:512-522.

 

 

Lindgren, P.B., Panopoulos, N.J., Staskawicz, B.J., and

 

75

Dahlbeck, D. 1988. Genes required for pathogenicity and
hypersensitivity are conserved and interchangeable among
pathovars of Pseudomonas syringae. Mol. Gen. Genet. 211:499-
506.

 

Lyon, F., and R.K.S. Wood. 1976. The hypersensitive reaction
and other responses of bean leaves to bacteria. Ann. Bot.
40:479-491.

Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular
cloning: a laboratory manual. Cold Spring Harbor Laboratory
Press, New York.

Metraux, J.P., and Boller, Th. 1986. Local and systemic
induction of chitinase in cucumber plants in response to
viral, bacterial and fungal infections. Physiol. Mol. Plant
Pathol. 28:161-169.

Pavlovkin, J., Novacky, A., and Ullrich-Eberius, C.I. 1986.
Membrane potential changes during bacteria-induced
hypersensitive reaction. Physiol. Mol. Plant Pathol. 28:125-
135.

Richmond, S.J., Kuc, J., and Elliston, J. 1979. Penetration
of cucumber leaves by Colletotrichum lagenarium is reduced in
plants sytemically protected by previous infection with the
pathogen. Physiol. Plant Pathol. 14:329-338.

 

Simon, R., Priefer, U., and Puhler, A. 1983. A broad host
range mobilization system for in vivo genetic engineering:
Transposon mutagenesis in gram negative bacteria.

Biotechnology 1:784-791.

Smith, J.A., and Hammerschmidt, R. 1988. Comparative study of
acidic peroxidases asociated with induced resistance in
cucumber, muskmelon and watermelon. Physiol. Mol. Plant
Pathol. 33:(in press).

Staskawicz, B., Dahlbeck, D., Keen, N., and Napoli, C. 1987.
Molecular characterization of cloned avirulence genes from
race 0 and race 1 of Pseudomonas syringae pv. glycinea. J.
Bacteriology 169:5789-5794.

 

Stermer, B., and Hammerschmidt, R. 1984. Heat shock induces
resistance to Cladosporium cucumerinum and enhanced
peroxidase activity in cucumbers. Physiol. Plant Pathol.
25:239-249.

 

 

Vincent, J.R., and Fulbright, D.W. 1983. Transfer of pRDl to
Pseudomonas syringae and evidence for its integration into
the chromosome. J. Bacteriol. 156:1349-1351.

 

 

76

 

Xuei, X.L., Jarlfor, U., and Kuc, J. 1988. Ultrastructural
changes associated with induced systemic resistance of
cucumber to disease: host response and development of
Colletotrichum lagenarium in systemically protected leaves.
Can. J. Bot. 66:1028-1038.

 

FUTURE DIRE CTIONS

77

78

FUTURE DIRECTIONS

The plant pathologist's search for a resistance inducing
signal has proved as elusive as the plant physiologist‘s
search for florigen. In retrospect, the inability to isolate
a 'signal' is of minor significance considering the valuable
information induced systemic resistance may contribute to our
understanding of disease resistance, peroxidase function, and
pathogen mediated plant gene regulation. The following two
paragraphs suggest 'the next experiment' to follow those
presented in Chapters 1-3. The concluding paragraphs propose
several ways in which induced systemic resistance in cucumber
may be used to explore more fundamental questions pertaining
to plant cell biology.

The use of HR causing bacteria to induce resistance has
several advantages over the use of fungal pathogens: bacteria
are easier to maintain, they induce resistance more quickly,
and the genome is readily accessible for study. Although

Pseudomonas syringae pv syringae may be considered 'typical'

 

of HR inducing bacteria, other strains should be tested for
their ability to induce systemic resistance in order to
ensure that the response is non-specific. Many E. syringae
pathovars produce a toxin, syringomycin, and the role such a
toxin might play in induced systemic resistance should be
assessed. Finally, Grimm and Panopoulos (1988) have suggested
that the hrp region regulates the expression of genes

involved specifically in disease processes. The premise for

79

this hypothesis lies in the observation that several genes in
the hrp region share homology to regulatory proteins in

Rhizobium spp and Klebsiella pneumoniae which mediate plant-

 

 

bacteria interactions. Sule and Klement (1971), and Lyon and
Wood (1976) have reported that viable cells from old cultures

of E. phaseolicola have a decreased capacity to elicit an

 

HR. Plant pathogens frequently lose their virulence when they
are subcultured for long periods of time in the absence of a
plant host, though no molecular explanation for this
phenomenon has been presented. One easily tested
possibility, at least for bacterial, pathogens is that the
functional 'hrp' locus is lost from cultures in the absence
of selective pressure. If this is the case, it should be
possible to complement bacteria which have lost virulence in
culture with the cloned hrp region.

A systemic increase in peroxidase activity is the first
detectable evidence that a cucumber plant has responded to a
resistance inducing inoculation. This response can by quite
rapid, as demonstrated in Chapter 3. The next question to
address in this respect is: how do the levels of peroxidase
mRNA vary temporally and spatially during induced resistance?
Specific probes for the anodic peroxidase may be used in a
Northern blot analysis of mRNA prepared from tissue taken at
various times in order to determine when and where peroxidase
transcription is activated. The gene for cucumber chitinase
has also been cloned (Metraux et al., in preparation) and a

comparison of the message levels for these two proteins in

 

80

response to pathogen stress should indicate whether their
regulation is influenced by the same factors.

A recent review has emphasized the need for a more
thorough investigation into the processes of protein sorting
in plant cells (Della-Cioppa et al. 1987). Both chitinase
and peroxidase accumulate in the extracellular spaces of
leaves, making them suitable proteins for the study of
sorting mechanisms which target proteins for export. Both
proteins are specifically regulated by biological stress and
are similar with respect to size and charge. Characterization
of the respective genomic clones for these proteins should
reveal whether they share common regulatory and signal
sequences. A comparison of the glycosylation profiles of
chitinase and peroxidase will indicate whether this
modification is conserved. The size and structure of
extracellular proteins must presumably be limited by their
need to pass from the cytoplasm through the cell wall.
Recent studies on the conformation of cell-cell channels may
help to delimit the structural constraints on extracellular

-roteins (Meiners et al. 1988).

'U

A thorough understanding of the regulation and
processing of peroxidase and chitinase may, in the future,

provide a variety of useful applications. Both chitinase and

peroxidase are putative defense related proteins. The
possibility exists that novel, more effective defense
proteins may be introduced into plants under the specific

regulation of pathogen stress. These compounds would

81

preferably be designed to meet the criteria which allow their
selective regulation and cellular localization. Finally, the
use of plant cells in biotechnology for the production and
export of commercially valuable products may be facilitated
by an understanding of the characteristics of a protein
which result in its export from the plant cell.

The 'signal' released by pathogen induced stress governs
fundamental processes in plant cell biology. Investigation
into the nature of this signal is perhaps best approached

through the study of these processes and the elements of

their regulation.

REFERENCES

Della-Cioppa, G., Kishore, G., Beachy, R. and R. Fraley.
1987. Protein trafficking in plant cells. Plant Physiol.
84:965-968.

Grimm, C-, and N.J. Panopoulos. 1988. The predicted protein
product of a pathogenicity locus from Pseudomonas syringae pv
phaseolicola is homologous to a highly conserved domain of
several prokaryotic regulatory proteins. (submitted, EMBO J)

 

 

Lyon, F. and R.K.S. Wood. 1976. The hypersensitive reaction
and other responses of bean leaves to bacteria. Ann. Bot.
40:479-491.

Meiners, S., Baron-Epel, O., and M. Schindler. 1988.
Intercellular communication-filling in the gaps. Plant
Physiol. 88:791-793.

Sule, S. and z. Klement. 1971. Effect of high temperature and
the age of bacteria on the hypersensitive reaction of
tobacco. Acta Phytopath. Acad. Sci. Hungar. 6:119-122.