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THE PATHOGENESIS RELATED PROTEIN, CHITINASE, AND ITS
ROLE IN THE SYSTEMIC ACQUIRED RESISTANCE PHENOTYPE
IN CUCUMBER PLANTS (Cucumis sativus L.)

presented by
Luis Velasquez

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6/01 cJCIRCJDatoDuopes-ms

THOGENESIS RELATED PROTEIN, CHITINASE, AND ITS ROLE IN THE
SYSTEMIC ACQUIRED RESISTANCE PHENOTYPE IN CUCUMBER PLANTS
(Cucumis sativus L.)

By
LUIS A VELASQUEZ

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY

Botany and Plant Pathology
Department of Botany and Plant Pathology

2002

ABSTRACT
THE PATHOGENESIS RELATED PROTEIN, CHITINASE, AND ITS ROLE IN
THE SYSTEMIC ACQUIRED RESISTANCE PHENOTYPE IN CUCUMBER
PLANTS (Cucumis sativus L.)
By
Luis A Velasquez

Systemic Acquired Resistance (SAR) refers to a distinct plant response
that systemically confers broad-spectrum disease resistance after an induction of
biotic or abiotic nature. Accumulation of Pathogenesis-Related (PR) proteins
such as chitinase (PR-8) takes place during the induction of SAR.

In order to study chitinase activity in cucumbers, a new method for
chitinase detection and quantification was developed. In addition to determine if
SAR requires active metabolism after challenge, leaf disks of cucumber plants
eXpressing SAR were treated with the protein synthesis inhibitor cycloheximide
and then challenged with the fungus Colletotn’chum orbiculare. Cyclohexamide
treatment in SAR expressing plants caused an increase in penetration of the
fungus and symptom development was comparable to non-induced plants. This
result suggests that the induced state of cucumber plants is dependant of protein
synthesis.

In field and greenhouse experiments, no correlation between the amount
of chitinase induction and the degree of resistance acquired through SAR was
found. In addition, a differential cultigen response to two pathogens with different
infections strategy was found. Cucumber cultivars expressing SAR are generally

resistant to Colletotn'chum . orbiculare but are not always resistant to Didimella

bryoniae. Several cultivars expressing SAR developed more severe symptoms
to D. bryoniae despite the high chitinase activity detected. This suggests a
differential resistance pathway and questions the ability of chitinase to decrease
pathogen growth within the tissue.

Transgenic cucumber plants bearing the cDNA sequence of chitinase type
III in the sense and antisense directions were generated. Treatment of the
transformed plants with the resistance activator acybenzolar-S-Methyl (ASM)
demonstrated no significant differences in their ability to withstand pathogen
attack or express induced resistance. Results obtained suggest that the acidic
chitinase is important for the basal levels of resistance prior to induction. The
amount of acidic chitinase activity was suppressed in plants bearing the sense
and antisense constructs possible due to gene silencing.

The NPR1 gene regulates SAR and ISR and could be a good molecular
SAR marker. Constitutive levels of expression can be correlated with resistance
due to SAR. A partial sequence of a cDNA fragment of the NPR1 of cucumber
plants was isolated.

The present study indicates that chitinase does not play a significant role
in the SAR phenotype. It is possible that the timely activation of the entire set of
SAR genes is more important than the effect of a single gene even if it is over

expressed.

Copyright by
LUIS A VELASQUEZ

2002

DEDICATION

A mis padres, Don ObduIio Velasquez y dona Bertita de Velasquez por
darme vida y darme Ia oportunidad de seguir mis suefios. A mis hermanos y
hermanas por su apoyo moral. A mi adorada esposa, Sara, por su apoyo, su
amor y su comprension. A mi pueblo, San Pedro Sacatepequez, San Marcos,
en la tierra de la eterna primavera, Guatemala, Centro America. Pedacito de mi

corazon que jamas olvido.

ACKNOWLEDGMENTS

I would like to thank everybody who has helped me throughout my
studies at Michigan State. Specially, to my advisor Dr. Ray Hammerschmidt, a
source of inspiration and financial support. To my committee members, Dr.
Irvin Widders, Dr. Frances Trail and Dr. Mary Hausbeck for their words of
encouragement and advice. I also would like to thank the undergraduate
workers, without them I would have never been able to finish this work; Koji
Sunazaki, and Ashley Fontana for their hard work in the beginning stages of my
Ph.D. program. Meg Goecker, and Teresa Taylor, whose hard work and
dedication made my progress possible.

I also would like to acknowledge the members of the Hammerschmidt
lab, for their help and friendship. To my best friend Dr. Dilson Bisognin, for the
countless hours we spent studying and discussing our work. To Monica
Guhamajumdar and lffa Gaffoor for their help with my molecular biology blues.

Last, but definitely not least, thanks to my wife Sara, for her love,
support, understanding, encouragement and because she is always so proud of

me. I would marry her a thousand times over.

vi

TABLE OF CONTENTS

LIST OF TABLES .............................................................................................. XI
LIST OF FIGURES .......................................................................................... Xll
CHAPTER I:

GENERAL INTRODUCTION ............................................................................. 1
Historical perspective of SAR .................................................................. 3
Biochemistry of SAR ............................................................................... 4

Induction of SAR ........................................................................... 4
The Signaling nature of SAR ........................................................ 5
Changes in planta after SAR induction ......................................... 6
The Pathogenesis Related (PR) Proteins ............................................... 7
Definition: ..................................................................................... 7
Classification of the PR proteins: .................................................. 7
Possible Functions: ...................................................................... 8
Chitinase: ................................................................................................ 9
Classification of chitinases: ........................................................ 10
PR-8/Class III chitinases ............................................................ 11
The Connection of Chitinase and SAR .................................................. 11
Chitinase as a biochemical marker for SAR ............................... 12
Over expression experiments ..................................................... 13
NPR1: A regulator gene for the expression of Induced Resistance ...... 14
Npr1 gene regulation .................................................................. 15
Bibliography .......................................................................................... 18
CHAPTER II:

DEVELOPMENT OF A METHOD FOR THE DETECTION AND
QUANTIFICATION OF TOTAL CHITINASE ACTIVITY FROM PLANT

EXTRACTS ...................................................................................................... 25
ABSTRACT ........................................................................................... 26
INTRODUCTION ................................................................................... 27
MATERIALS AND METHODS .............................................................. 30

Protein Extraction ....................................................................... 30
Preparation of the substrate and enzyme activity assay ............. 31
Chitinase Detection and Standard Curve Production ................. 32
Image Analysis ........................................................................... 32
RESULTS .............................................................................................. 34
Standard Assay. ......................................................................... 34
Detecting commercial chitinase. ................................................. 34
Detection of Cucumber Chitinases ............................................. 35

vii

DISCUSSION ........................................................................................ 37
BIBLIOGRAPHY ................................................................................... 46

CHAPTER III:

THE EXPRESSION OF INDUCED SYSTEMIC RESISTANCE TO PATHOGEN

CHALLENGE IS DEPENDANT ON PROTEIN SYNTHESIS ........................... 50
ABSTRACT. ........................................................................................ 51
INTRODUCTION ................................................................................... 52
MATERIALS AND METHODS .............................................................. 55

Plant Material and Fungal cultures. ............................................ 55
Preparation of fungal cell walls ................................................... 56
Histological observations ............................................................ 56
Protein Extraction ....................................................................... 56
Assay of Peroxidase and Chitinase Activities ............................. 57
PAGE and gel enzyme detection ................................................ 57
Detection of H202 in cucumber leaf disks. .................................. 58
RESULTS .................................................................................... 60
The effect of CHX in fungal growth and development: ............... 6O
Cytological observations: ........................................................... 60
Papillae formation and lignin deposition in leaf disks treated
with CHX. ................................................................................... 61
Effect of challenge inoculation and CHX treatment on PR
protein expression ...................................................................... 62
The effect of CHX in the generation of Reactive
Oxygen Species ............................................................................................ 63
The effect of treating levels of CHX in combination with a
SAR Inducer. .............................................................................. 64
DISCUSSION ........................................................................................ 65
BIBLIOGRAPHY ................................................................................... 89
CHAPTER IV

THE RESPONSE OF CUCUMBER CULTIVARS TO INDUCTION OF SAR IN
GREENHOUSE AND FIELD CONDITIONS AND THE SCREENING OF
RESISTANCE AGAINST Colletotn'chum orbicu/are AND Dydimella bryoniae.94

ABSTRACT ...................................................................................................... 95
INTRODUCTION ............................................................................................. 96
MATERIALS AND METHODS ......................................................................... 99

viii

Culture of host and pathogens. ............................................................. 99

Induction of SAR ................................................................................. 100
Detached leaf resistance bioassay ...................................................... 100
Cucumber fruit bioassay ..................................................................... 101
Assay of Peroxidase and Chitinase Activities ...................................... 101
Experimental design. ........................................................................... 102
RESULTS ...................................................................................................... 103
There is a differential response of plants expressing SAR against pathogens
with different mode of invasion .................................................................... 104

There is a genotypic difference between cultigens to express natural
resistance against Colletotn’chum orbiculare and Dydimella bryioniae in
cucumber 106

Cucumber Cultigens present little genetic difference in resistance against
Rhyzoctonia solani ...................................................................................... 107
ASM application does not enhance the resistance of cucumber fruits against
R. solani. 107

Levels of Resistance against a challenge inoculation of C. orbiculare in

cucumber treated with ASM. ....................................................................... 108
Correlation between the chitinase expressed and the resistance acquired. 109
DISCUSION ................................................................................................... 1 10
BIBLIOGRAPHY ............................................................................................ 135
APPENDIX I
THE TRANSFORMATION OF CUCUMBER EXPRESSING SENSE AND
ANTISENSE cDNA SEQUENCE OF ACIDIC CHITINASE TYPE III ............ 139
ABSTRACT .................................................................................................... 140
INTRODUCTION ........................................................................................... 141
MATERIALS AND METHODS ....................................................................... 143
Plant Material ...................................................................................... 143
Vector construction ............................................................................. 143
Transformation procedure ................................................................... 144
Confirmation of transformation ............................................................ 145
Detection of resistance against Colletotn'chum orbicu/are in transgenic
cucumber plants .......................................................................................... 146
Chitinase detection .............................................................................. 147
RESULTS ................................... ' ................................................................... 149
Development of kanamycin resistant plantlets. ................................... 149
Resistance to Colletotrichum orbicu/are ............................................. 149
The levels of chitinase activity in transgenic plants. ............................ 150
DISCUSSION ................................................................................................. 155
BIBLIOGRAPHY ............................................................................................ 161

APPENDIX ll
ISOLATION OF A CDNA FRAGMENT OF THE NPR1 GENE FROM

CUCUMBER PLANTS ................................................................................... 164
ABSTRACT .................................................................................... 165
INTRODUCTION .............................................................................. 166
MATERIALS AND METHODS ....................................................................... 169
Cucumber cDNA: ................................................................................ 169
Primer selection: ................................................................................. 169
PCR optimization: ............................................................................... 170
Cloning of the PCR product: ................................................................ 170
RESULTS ...................................................................................................... 173
DISCUSSION ................................................................................................. 178
BIBLIOGRAPHY ............................................................................................ 181
FUTURE DIRECTIONS ...................................................................... 182

LIST OF TABLES

Table 2.1: Results of three different experiments using commercial chitinase
and five different concentrations ............................................................ 45

Table 4.1: Greenhouse screening of cucumber cultigens expressing SAR
induced with ASM and challenged with the fungi Colletotrichum orbiculare. .120

Table 4.2: Statistical analysis of the cucumber leaf lesion size data obtained
from the field test in 1998. The leaves were challenged with C. orbiculare.126

Table 4.3. Analysis of cucumber cultivars grown in field conditions. A
detached leaf bioassay was performed by challenging the leaves with a spore
suspension of C. orbiculare. .......................................................................... 129

Table 4.4. Analysis of cucumber cultivars grown in field conditions. A
detached leaf bioassay was performed by challenging the leaves with a spore
suspension of D. bryoniae. ............................................................................ 130

Table 4.5: Results of a field experiment cucumber cultivars treated with ASM
and control plants ........................................................................................... 132

Table 4.6: Chitinase levels of cucumber plants from a field experiment with
cucumber plants treated with ASM ................................................................ 133

xi

LIST OF FIGURES

Figure 1.1: Signal-transduction pathways leading to pathogen-induced systemic
acquired resistance (SAR) and rhizobacteria-mediated induced systemic
resistance (ISR) in Arabidopsis thaliana. Adapted from L. C. van Loon et al

Annu. Rev. Phytopathol. 1998. 36:453-483 ..................................................... 17
Figure 2.1: Sample of an activity reaction agarose plate viewed

under UV light. ................................................................................................. 39
Figure 2-2: Regression analysis of commercial chitinase activity expressed in
number of pixels in relation to time of incubation ............................................. 40

Figure 2-3. Regression analysis of chitinase activity expressed in number of
pixels in relation to different chitinase concentrations for different times of

incubation. ....................................................................................................... 42
Figure 2-4: Graph shows the resulting chitinase detection from cucumber
plants, (control and induced) ................................................................ 44

Figure 3.1A: Leaf discs from induced plants non treated with CHX (left panel)
and CHX treated (right panel). Leaf discs were challenged with C. orbiculare
and treated with phloroglucinol to detect lignin deposition ............................... 72

Figure 318: Leaf Discs from induced plants non treated with CHX (left panel)
and CHX treated (right panel). Leaf discs were challenged with C. orbiculare
and stained with lacmoid blue to detect callus deposition at ............................ 73

Figure 3.1C: Leaf disks from induced plants 48 hours after challenge with C.
orbiculare stained with aniline blue to detect papillae formation. ..................... 74

Figure 3.2. The effect of cycloheximide in the penetration rate of C. orbiculare.75

Figure 3.3: The effect of cycloheximide in the growth rate of the fungal hyphae
within the tissue. .............................................................................................. 76

Figure 3.4: Micrographs of cucumber leaf disks under visible light taken with a
Leica microscope. Arrows indicate the hypha within the tissue. Leaf discs

were viewed 48 hours after inoculation. ........................................................... 77
Figure 3.5. Detection of chitinase activity of leaf disks treated with
cycloheximide during the process of infection by C. orbiculare ........................ 78
Figure 3.6: The effect of the treatment with CHX to cucumber leaf disks
inoculated with C. orbiculare in peroxidase activity. ........................................ 79
Figure 3.7: Peroxidase activity from leaf disks of induced and control plants in
the presence of a fungal cell wall preparation. . .............................................. 81
Figure 3.8. Effect of CHX on Catalase Activity (A) and SOD activity (B)
detected via PAGE activity staining. ................................................................ 82

xii

Figure 3.9: Inhibition by cycloheximide of the elicitation of H202 from leaf disks
. ........................................................................................................................ 84

Figure 3.10: Gel electrophoresis detection acidic peroxidase (A) and chitinase
(B) of plants sprayed with Water, BTH (20ppm) BTH+CHX and CHX (10ug/ml)
and (C) Glucanase activity detection. ............................................................. 85

Figure 3.11. The systemic effect of induction of cucumber plants with BTH and
BTH/CHX. Second leaf was harvested and challenged with a spore
suspension of C. orbiculare the disease assessment was done 7 days after
inoculation, lesion size (A) and lesion number (B) were measured. ................ 87

Figure 4.1: Lesion size and lesion number analysis of greenhouse experiment
of cucumber plants treated with ASM and challenged with Colletotn'chum
orbiculare ....................................................................................................... 118

Figure 4.2: Chitinase activity (A) and peroxidase activity (C) analysis of 5
commercial cultivars of cucumber grown in greenhouse conditions. B is the
statistical analysis of chitinase activity comparing the basal levels and induced
levels of the enzyme. ..................................................................................... 119

Figure 4.3: Differential response of plants expressing SAR and challenged with
two different pathogens .................................................................................. 122

Figure 4.4: Analysis of lesion size and lesion number of cucumber cultivars
induced to express SAR by ASM Leaves were collected and challenged against

C. orbiculare and D. bryonieae. ..................................................................... 124
Figure 4.5: Analysis of chitinase activity at seven days after induction with ASM.
. ...................................................................................................................... 125
Figure 4.6. Data analysis of the lesion size of the field trail conducted during
1998. .............................................................................................................. 126
Figure 4.7: Statistical analysis of the data from the detached bioassay for
Colletotn'chum orbiculare and Dydimella bryionieae (1999 field traial) .......... 127

Figure 4.8: Data analysis of the average of the means of the cultivars tested in
a field experiment in 1999. ............................................................................. 128

Figure 4.9: Field experiment of three cultivars of cucumber treated with ASM
(20 ppm). Leaves were harvested and challenged with a spore suspension of

Colletotn'chum orbiculare. ............................................................................. 131
Figure 4.10 Correlation between the Lesion size and the chitinase activity of
induced plants in the field. No correlation was found. ................................... 134
Figure 5.1: Vector construction for transformation of cucumber plants. ........ 148

Figure 5.2: Transgenic shoots and transgenic plants regenerated from
cotyledon explants. ........................................................................................ 152

Figure 5.3: PCR analysis of regenerated shoots after Agrobacterium co
cultivation. .................................................................................................... 1 53

xiii

Figure 5.4: Resistance to Colletotn'chum orbiculare of non ASM treated
transgenic plants bearing the sense and the antisense construct of chitinase

type III under the control of the S35 promoter. ............................................... 154
Fig 5.5: Acidic chitinase of transgenic and control plants ............................... 155
Figure 6.1: Alignment of Amino acid sequences of the NPR1 gene. ........... 172
Figure 6.2. Agarose gel electrophoresis of a PCR product using degenerate
primers to amplify the NPR1 gene of cucumber ............................................ 174
Figure 6.3: Clustal alignment of the Cucumber NPR1 translated sequence and
the Arabidopsis NPR1 sequence ................................................................... 175
Figure 6.4: Alignment of the NPR1 cDNA fragment of cucumber and
Arabidopsis . .................................................................................................. 176
Figure 6.5: Sequence similarities of NPR1 amino acid sequence with mouse
ankyrin 3 (ANK3) ............................................................................................ 177

xiv

CHAPTER I

GENERAL INTRODUCTION

INTRODUCTION

Plants are subjected to various adverse conditions throughout their life
from stresses caused by both, abiotic and biotic agents such as plant pathogenic
microorganisms (Agrios 1997). The challenge of producing an adequate supply
of food becomes harder as the world population grows, the amount of land for
agriculture shrinks and the pressure of new or emerging and pesticide resistant
plant pathogens intensifies.

Resistance, as defined by Agrios (Agrios 1997), is the ability of an
organism to exclude or overcome, completely or in some degree the effect of a
pathogen or other damaging factor. Disease resistance in plants is expressed in
two forms; a constitutively expressed resistance and resistance that is induced to
be expressed after initial infection. The preexisting structural and chemical
structure is the first line of defense, which the pathogen must penetrate to cause
disease. Such structures include, amount and quality of wax and cuticle over the
epidermal cells, the size, location and shapes of stomata and lenticels, and the
presence on the plant tissue made of thick-walled cells that hinder the advance
of the pathogen. In addition there are also the preexisting chemical defenses.
These include among others, fungi-toxic exudates, inhibitors present in the cell
before infection such as phenolic compounds, tannins and some fatty acid-like
substances.

The Induced form of resistance must have a recognition event of the
pathogen by plants. The speed by which the plant recognizes the pathogen,

sends out alarm message(s), and mobilize its defenses will determine whether

Ix)

an infection will be successful or not, and/or how severe the disease will be. The
induced form of resistance can be segregated into two different groups, induced
structural defenses and induced biochemical defenses. It has been accepted
that plants develop a broad type of resistance known as systemic acquired
resistance (SAR) (Kuc 1982). The SAR phenomenon has been marked by the
systemic protection of plants against pathogens. SAR activation starts with a
local infection of plants by pathogens, which result in the spreading of the
resistance to the vicinity of the lesion and to distant plant organs (Kuc 1995;

Ryals et al. 1996).

Historical perspective of SAR

SAR was recognized by the description of the initial observations since the
turn of the century compiled by Chester in (Chester 1933). Such observations
described the heightened resistance to pathogen attack that developed by plants
surviving an initial infection. Ross (Ross 1961)coined the term “Systemic
Acquired Resistance” and made extensive characterizations of the phenomenon.
After inoculating Ngene tobacco with TMV, Ross observed that the area around
the inoculation became more resistant to TMV, a phenomenon now recognized
as localized acquired resistance. In addition, other plant parts remote from the
initial inoculation site became more resistant, as expressed by the fewer number
of lesions developed after a challenge inoculation to TMV. In the 1970's, Kuc
and coworkers, demonstrated that induction of the first leaf of cucumber plants

with Colletotrichum orbiculare primed the rest of the plant to become more

resistant against Colletotn'chum orbiculare, and other fungi, bacteria and viruses
(Kuc 1982). Overall, the symptom severity caused by the challenging pathogen
was substantially reduced (Hammerschmidt 1999). These observations indicate
that SAR is a mechanism through which the level of general resistance to

pathogens is greatly increased (Van Loon 1997).

Biochemistry of SAR
Induction of SAR

The induction of systemic acquired resistance can be accomplished by an
array of biological and chemical inducers (Oostendorp et al. 2001). In all cases,
the induction is characterized by an overall decrease of pathogen damage in
parts remote from the site of primary inoculation/induction.

The activation of SAR can be achieved by several means. This usually
refers to macromolecules, originating either from the host plant or from the plant
pathogens, which are capable of inducing SAR such as chitosan and chitin
oligomers (Barber et al. 1989; Barber and Ride 1994; Benhamou and Nicole
1999). Microbial-mediated SAR as in the case of fungi (Roby et al. 1988
Hammerschmidt, 1981 #169) bacteria (Kuc and Hammerschmidt 1978), viruses
(Ross 1961). In addition to the biotic inducers, the chemically induction of SAR is
also possible by the application of plant disease resistance activators (Friedrich
et al. 1996; Kastner et al. 1998; Narusaka et al. 1999). Among these plant
disease resistance activators are exogenous applications of salicylic acid, 2,6-
dichloroisonicotinic acid (DCIA) (Kessmann et al. 1994), the novel plant

protection substance acibenzolar S-methyl (ASM) formerly known as BTH

(Friedrich et al. 1996; Lawton et al. 1996); (Dann et al. 1998) and ethylene (Boller

etaL,1983)

The Signaling nature of SAR

Evidence indicates that the primary inoculation/induction results in the
translocation of a still unknown signal produced at the site of the initial inoculation
(Metraux 2001). This signal seems to trigger a complex array of defense
mechanisms that in turn make the plant more resistant to pathogen attack. The
nature of the signal triggering this response has been object of intense review
and research. Early experimental data indicated that exogenous application of
Salicylic acid (SA) might play a role in the establishment of SAR (White 1979).
Moreover, in cucumber, it was showed that SA concentrations become elevated
after pathogen infection of leaves (Metraux et al. 1990) providing further
evidence of the possible role of SA in SAR expression and establishment. SA
was then, regarded as the direct signaling mechanism to trigger SAR . However,
the findings of Vernooij et al (Vernooij et al. 1994) employing NahG tobacco in
grafting experiments showed that SA might not be the systemic signal. In
addition, the analysis of phloem exudates from cucumber petioles prior during
and after the setting of SAR suggest that SA and is synthesized de novo in stems
and petioles in response to a mobile signal from the inoculated leaves (Smith
Becker et al. 1998). This supports the findings of Rassmussen and
Hammerschmidt (Rasmussen et al. 1991). Their work endorse a role for salicylic

acid as an endogenous inducer of resistance, but their data also suggest that

salicylic acid is not the primary systemic signal of induced resistance in

cucumber.

Changes in planta after SAR induction

Once the SAR signal is produced, an array of responses are generated in the
areas surrounding the original inoculation as well as in areas remote from the
initial inoculation point (Sticher et al. 1997). Activation of several gene families
and changes in the biochemistry of the plant is usually the norm. Among the
initial responses, Salicylic acid is synthesized (Smith Becker et al. 1998), lignin
deposition is increased (Hammerschmidt and Kuc 1982; Stein et al. 1993). In
addition, an enhanced peroxidase activity that has been associated with systemic
acquired resistance of cucumber against Colletotrichum orbiculare, also takes
place (Hammerschmidt et al. 1982; Rasmussen et al. 1995). After infection,
strengthening of the cell wall can also occur by the action of peroxidase
catalyzed cross-linking of hydroproline-rich structural cell wall glycoproteins
(Bradley et al. 1992). In addition to cell wall depositions, the accumulation of the
proteins commonly known as Pathogenesis-Related (PR) proteins also takes
place during the induction of systemic acquired resistance (van Loon and van
Kammen 1970). Among these PR proteins, the presence of B-1,3-glucanase
(PR-2) and chitinase (PR-8) among others are a characteristic of cucumber
plants expressing SAR. The expression of the SAR genes and the changes in
the biochemistry of the plant after induction may contribute to the enhanced
resistance of the plant against pathogens. This resistance might be expressed

by either stopping the pathogen from infecting the plant and or by limiting the

spread of the disease, which would allow the plant to respond to the disease
more effectively by activating other defense mechanisms (Metraux 2001 ). Among
the most studied responses of the SAR phenomenon, is the expression increase
of the pathogenesis related (PR) proteins after induction and their possible role in

plant resistance.

The Pathogenesis Related (PR) Proteins

Definition:
PR- proteins were originally described in the 1070’s independently by Van

Loon and Gianinazzi (Gianinazzi et al. 1970; van Loon and van Kammen 1970).
These proteins were first observed as new protein components induced by the
interaction between tobacco mosaic virus (TMV) in hypersensitivity reacting
tobacco (van Loon and Cornelis 1999) detected by protein electrophoresis
analysis. Originally they were defined as acid-soluble protease-resistant, acidic
proteins localized in the extracellular space (Sticher et al. 1997). Later, the name
PR-proteins was coined to refer to proteins coded by the host plant, but induced
only in pathological related situations (van Loon and Cornelis 1999). These
pathological related situations were not only limited to plant pathogen interaction,
but also includes nematodes, insects or herbivores, treatments with certain

chemicals, and other types of stress (Sticher et al. 1997).

Classification of the PR proteins:
As research on the PR proteins continued, the proteins have been

grouped into families. Initially, the 10 major acidic PRs of tobacco were grouped

into five families, designated PR-1 to PR—5 (van Loon et al. 1994; van Loon and
Cornelis 1999). This classification sets a convenient protocol to follow for
classification of other PR proteins from other species. Eleven families are now
known in tobacco, and the existing nomenclature is being used to classify PR
proteins in other plant species such as maize, cucumber, Arabidopsis (van Loon
et al. 1994). The proposed nomenclature calls for the families to be numbered,
and the different members within each family are assigned letters according of
the order in which they are described. They must belong to the same PR famil,
but the lettering only reflects how many proteins of that family had been identified
within the plant species previous to their discovery (van Loon and Cornelis 1999;

Van Loon and Van Strien 1999).

Possible Functions:

Phytoalexin accumulation and cell wall modifications are local reactions,
accumulation of PR proteins extends into non-inoculated plant parts, that upon
challenge, exhibit SAR (Ryals et al. 1996). The demonstration that some PR
proteins such as 8-1,3-glucanase and chitinases have lysozyme activity
(Kauffmann et al. 1987; Legrand et al. 1987) and anti-fungal activity in vitro (Ji
and Kuc 1996) immediately suggest that these enzymes might have a direct role
against cell walls of bacteria and fungi. In addition, their localization, apoplast,
(Boiler and Metraux 1988) seems to guarantee contact with invading pathogens
before these are able to colonize plant tissue. This fact suggests that these PR
proteins are indeed playing a direct role in the defense mechanism of the SAR

expressing plants (Boller and Metraux 1988; Lee and Hwang 1996). Another

possible role suggested is the release of elicitors from the walls of the invading
pathogens (van Loon and Cornelis 1999). These elicitors might trigger or
enhance the expression other defense mechanisms. This role might be possible
since chitinases and glucanases do release chitin and glucan oligomers from the
cell wall of invading pathogens and their effects on the defense of plants has
been somewhat assessed (Roby et al. 1987; Roby et al. 1988). In addition to
their possible antimicrobial characteristics, most PR proteins are also found in
various floral tissues suggesting specific physiological functions during flower
development rather than a role in general defense against pathogen infection

(van Loon and Cornelis 1999).

Chitinase:

Chitinase occurs naturally in a wide variety of higher plants and catalyzes
the hydrolysis of chitin, a linear polymer of (3(1,4)-N-acetyglocosamine
(Muzzarelli 1977), a constituent of fungal cell walls and insect cuticles. Chitinase
from plant tissue has been found to be partially responsible in the process of
dissolution of hyphal walls in plants in vitro (Skujins et al. 1965; Ji and Kuc 1996).
Increase in chitinase activity has been correlated with the increase in disease
resistance observed in plants expressing induced resistance (Roby and Esquerre
Tugaye 1987; Roby et al. 1988; Dalisay and Kuc 1995; Hwang et al. 1997; Xue
et al. 1998). SAR inducible acidic chitinase is found in very low amounts in
healthy tissue, but its quantity and activity is increased in response to pathogen
attack by fungi (Roby and Esquerre Tugaye 1987; Dalisay and Kuc 1995; Dann

et al. 1996; Kastner et al. 1998), bacteria, (Lee and Hwang 1996; Gerhardt et al.

1997) viruses, nematodes (Rahimi et al. 1998) and even insects (van der
Westhuizen et al. 1998). The enzyme can be induced by wounding (Cabello et al.
1994; Zhang and Punja 1994), heat shock (Margispinheiro et al. 1994) and the
application of plant disease resistance activators (Friedrich et al. 1996; Kastner
et al. 1998; Narusaka et al. 1999) or ethylene (Boller et al. 1983). The systemic
induction of PR proteins in SAR is associated with an overall decrease of
pathogen damage in parts remote from the site of primary inoculation/induction.
In addition, their localization within the tissue in association with fungal structures

in resistant cultivars has been also demonstrated (Boller and Metraux 1988).

Classification of chitinases:

As of today a total of 189 chitinases have been purified and/or cloned from many
plants and reported in the EMBL database. Originally chitinases were classified:
as glycosyl hydrolases, as pathogenesis-related proteins, chitinases classes, and
gene families. A classification was introduced which was compossed of three
classes, later it was extended to six classes based on sequence comparisons.
To better understand this nomenclature the following chart shows the current

classification of plant chitinases (Neuhaus 1999)

 

 

 

 

 

 

 

 

 

Family of Gene Chitin- C atal tic Number of
PR Proteins glycosyl Class binding y known
hydrolases name domain domain sequences
Chial I 1 l 50
ChiaZ llb - l 11
Chia3 Ila - ll 16
PR'3 19 Chia4 IV 1 IV 24
Chia5 V 2 C 1
Chia6 VI 1/2 + Pro 1

 

 

 

 

 

 

 

 

 

 

Chia7 vn - IV 1

PR-8 18 Chib/ m - 19
PR-11 18 Chic I l - 3
Unassign Chid1 I 7
PR'4 ed Chid2 ll ' 5

 

 

 

 

 

PR-B/Class Ill chitinases

These chitinases were originally purified and cloned from cucumber (Metraux et
al. 1989) and soon many others follow. The chitinases in this class have similar
amino acid sequences. However, they vary in isoelectric points (Boller and
Metraux 1988). The cucumber class III chitinase is acidic, and is localized in the
intercellular spaces of plant expressing SAR. (Boiler and Metraux 1988).
Chitinases from cucumber of this class has been found to have antimicrobial

activity in vitro (Ji and Kuc 1996).

The Connection of Chitinase and SAR

During the last years of chitinase research in cucumber, most of the data has
come from the use of a limited number of varieties. In addition to SMR-58, which
has been the primary cultivar used for induced resistance research in
cucumbers, two other cucumber cultivars had been examined for this enzyme. In
all cases, chitinase activity has been correlated with the amount of enhanced
resistance after SAR induction. In other words, the activation of SAR in
cucumbers via biotic or abiotic inducers follows an activation of SAR genes
including acidic chitinases and an enhanced resistance against a series of

pathogens. However, these correlations present several problems. First, as

mentioned before, the majority of the experiments had been done with a limited
amount of cucumber genotypes, and most of the experiments had been done in
controlled greenhouse conditions. Therefore, the lack of diversity in the
genotypes used for SAR activation and correlation studies linking chitinase
expression with the enhanced state of resistance after SAR activation questions
the validity of these correlations. In addition experiments have also been done in
which the state of resistance is long gone, however the chitinase activity still high

in induced plants.

Chitinase as a biochemical marker for SAR

The chitinase in cucumber, and in many other species has been frequently
used as a marker for systemic acquired resistance, and other forms of resistance
against pathogens (Roby et al. 1988; Roberts et al. 1994; Salzer et al. 2000). In
addition, the localization of the chitinase in the apoplast (Boller and Metraux
1988), and its in vitro antifungal activity (Skujins et al. 1965) has provided
additional arguments to support the role for the acidic chitinases in plant defense.
However, there is inconclusive evidence in vivo for the role of the induced
chitinase in stopping the spread of the pathogen in host tissue. As for the marker
theory, it is limited to speculations since no many experiments had been done in
order to quantify the induction of chitinase and its possible use as marker for
plant resistance. It was not until recently, when it was reported that the basal
amounts of PR proteins might serve as markers for resistance (Vleeshouwers et
al. 2000). Finally, there is a lack of information on the interaction of the chitinase

and the pathogen in vivo.

Over expression experiments

Transgenic plants overexpressing chitinase had been use to generate
evidence for the role of chitinase in resistance (Sticher et al. 1997);(Bauer et al.
1998; Tabei et al. 1998; Tabaeizadeh et al. 1999), However, the results have not
been as clear as expected. There are several reasons why these experiments
might have shown inconsistent results. For example, once the chitinase gene is
being overexpressed, there are reports where this overexpression might undergo
plant host gene silencing (Chareonpornwattana et al. 1999). The gene silence
mechanism of the plant avoids the accumulation/over expression of the chitinase,
which would be limited to perform its function. Despite the success in the

generation of positive transformants, the chitinase might not be expressed fully.

NPR1: A regulator gene for the expression of Induced Resistance

The Arabidopsis NPR1 gene controls the onset of systemic acquired
resistance (Cao et al. 1994). The npr1 (nonexpresser of PR genes) mutant failed
to respond to various SAR-inducing treatments (Cao et al. 1997), displaying little
expression of pathogenesis-related (PR) genes and exhibiting increased
susceptibility to infection (van Loon et al. 1998). NPR1 was cloned using a map-
based approach and was found to encode a novel protein containing ankyrin
repeats (Cao et al. 1997). The lesion in one npr1 mutant allele disrupted the
ankyrin consensus sequence, suggesting that these repeats are important for
NPR1 function. Furthermore, transformation of the cloned wild-type NPR1 gene
into npr1 mutants not only complemented the mutations, restoring the
responsiveness to SAR induction with respect to PR-gene expression and
resistance to infections, but also rendered the transgenic plants more resistant to
infection by P. syringae in the absence of SAR induction (Cao et al. 1997; Cao et
al. 1998). During an experiment in which enhancement of induced resistance by
simultaneously activation of salicylate and jasmonate—dependent defense
pathways in Arabidopsis was found several key findings were reported (fig 1.1).
First, plants expressing both types of induced resistance (SAR, ISR) did not
show elevated Npr1 transcript levels indicating that the constitutive level of NPR1
is sufficient to facilitate the expression of both SAR and ISR (Saskia et al 2000).
The finding is interesting because the basal levels of NPR1 transcription might
not be the same across species or cultivars expressing different levels of SAR

potentiation. If this is the case, the basal NPR1 expression can be investigated

and associated/correlated with the ability of the cultivar to express disease

resistance.

Npr1 gene regulation

Arabidopsis mutant npr1 does not express PR-genes and does not exhibit
SAR (Cao et al. 1994) Because rhizobacteria-mediated ISR was found to be
independent of SA and not associated with PRs (Pieterse et al. 1998), it was
expected that ISR would still be expressed in this mutant. However, the npr1
mutant of Arabidopsis did not express ISR mediated by the rhyzobacterium
WCS417 (Pieterse et al. 1998). This finding implies that NPR1 functions beyond
the expression of PR—genes and is required for both pathogen and rhizobacteria-
mediated systemic induced resistance. The theory in that Salicylic or Jasmonic
acid pathways might be controlled or regulated by one gene implies that SAR
and ISR converge at the last part of the signaling pathway. This can explain why
SAR and ISR are phenotypically similar. In both the defensive capacity of the
plant against a broad spectrum of pathogens is enhanced after an initial
induction. Yet, PRs are induced concomitant with SAR expresion, whereas
activation of PR genes is not part of the pathway leading to ISR in Arabidopsis.
Apparently, NPR1differentially regulates defense responses mediated by different
signaling pathways. Despite the source of induction, the NPR1 gene seems to
potentiate the defense mechanisms typical of SAR and ISR. However, the study
of the NPR1 basal levels and its correlation with SAR expression ability is
worthwhile aiming to provide a marker for SAR potentiation since SAR levels

positively correlate with pathogen resistance. This hypothesis is supported by

the overexpression of the NIM1 (NPR1) gene in Arabidopsis. In this study,
transgenic lines expressing the NPR1 gene exhibited heightened responsiveness
to SAR-inducing compounds. Activation of SAR in transgenic plants was
detected when treated with SA concentrations that would not activate SAR in wild

plants (Friedrich et al 2001).

 

 

Plant Rhyzobacterium P'a'“ Pathogen

 

 

 

Interaction . ~ Interaction .
(Necrosrs)
JA Response
(jar1)

 

t

Ethylene Response Salicylic Acid
(9W1) (NahG)

L J

PR's

   

Enhanced Defensive
Capacity

Figure 1.1: Signal-transduction pathways leading to pathogen-induced systemic
acquired resistance (SAR) and rhizobacteria-mediated induced systemic
resistance (ISR) in Arabidopsis thaliana. In plant species other than Arabidopsis,
rhizobacteriaIIy-produced salicylic acid can trigger the SAR pathway as well as
ISR. In parenthesis, mutations that compromise the normal pathway. (Modiefied

from L. C. van Loon et alAnnu. Rev. Phytopathol. 1998. 36:453-483

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Chapter II

DEVELOPMENT OF A METHOD FOR THE DETECTION AND
QUANTIFICATION OF TOTAL CHITINASE ACTIVITY FROM PLANT

EXTRACTS.

25

ABSTRACT

A method for the quantitative assessment of chitinase activity from crude
plant extracts has been developed. Dilution series of commercial chitinase
extracts were assayed under different conditions using glycochitln as enzyme
substrate. The assay is based on the affinity of fluorescent brightener 28 with
undigested glycochitln. An agarose plate is used to support the substrate and the
reaction plate is viewed under UV translumination. Results obtained revealed
that the linearity between chitinase activity values measured using this method
and enzyme concentration was reproducible and reliable and most importantly

faster allowing analysis of large number of samples.

26

INTRODUCTION

Chitinase occurs naturally in a wide variety of higher plants and catalyzes
the hydrolysis of chitin, a linear polymer of [3(1, 4)-N-acetyglocosamine
(Muzzarelli 1977), found in fungal cell walls and insect cuticles. Chitinase from
plant tissue has been reported to be antifungal and cause partial dissolution of
hyphal walls. (Skujins et al. 1965). These results suggest a role for chitinase in
plant defense. Some chitinases function as pathogenesis related (PR) proteins.
PR proteins are found in very low concentration in plant tissues until after
pathogen attack. (Roby and Esquerre Tugaye 1987; Dalisay and Kuc 1995; Dann
et al. 1996; Kastner et al. 1998) bacteria, (Lee and Hwang 1996; Gerhardt et al.
1997), viruses, nematodes (Rahimi et al. 1998) and insects (van der Westhuizen
et al. 1998). The enzyme can also be induced by wounding (Cabello et al. 1994;
Zhang and Punja 1994), heat shock (Margispinheiro et al. 1994), and the
application of plant disease resistance activators (Friedrich et al. 1996; Kastner
et al. 1998; Narusaka et al. 1999) or ethylene (Boller et al. 1983). The induction
of chitinase after pathogen or pest attack provided further correlative evidence for

the role of this enzyme in pathogen resistance.

Because of their potential role in plant defense, chitinases have received
wide research attention. Increase in chitinase activity has been correlated with
an increase in disease resistance observed in plants expressing induced

resistance (Roby and Esquerre Tugaye 1987; Roby et al. 1988; Dalisay and Kuc

27

1995; Hwang et al. 1997; Xue et al. 1998). In addition, their localization within the
tissue in association with fungal structures in resistant cultivars has also been
demonstrated (Manocha and Zhonghua 1997). Therefore, chitinase has been
used as a molecular marker for Systemic Acquired Resistance (SAR), and in
experiments involving plant microbe interactions at the molecular and
biochemical level. Over the last few years much research effort has been made
in finding improved methods of identifying promising germplasm for disease
resistance breeding. The success of plant breeding depends on the ability to
identify this promising germplasm and to recognize and select effectively
between segregating populations. Among the traits that are highly desirable is
the ability of a plant to withstand a pathogen attack by rapidly deploying its
defense mechanisms. In addition to being useful as a marker for such
deployment, chitinases have been shown to have antifungal activity in vitro. The
possible correlation between their expression and increased plant resistance
makes chitinase a potential biochemical marker to screen for resistant cultivars
(Roberts et al. 1994). PR protein basal expression was found to be correlated to
non-specific resistance in Solanum species to Phytophtora infestans and it has
been suggested that these levels might be used as biochemical markers for

germplasm selection for resistance (Vleeshouwers et al. 2000)

The potential use of chitinase as a biochemical marker for resistance
would involve screening large population samples in an effective and efficient

way. The current detection methods for activity include colorimetric assays

28

(Reissig et al. 1955), radioactive assays (Molano et al. 1977), direct detection of
activity after polyacrylamide gel electrophoresis (Trudel and Asselin 1989; Pan et
al. 1991), HPLC (Koga et al. 1998), and even reflectance spectroscopy (Roberts
et al. 1994). These methods albeit satisfactory, require large sample sizes, use
of hazardous chemicals, and expensive equipment and can be very time
consuming. In addition, some of these methods use colloidal chitin as a substrate
with one serious disadvantage; colloidal chitin is not always dispersed
homogeneously in the substrate. This decreases sensitivity and reliability for
some of the techniques (Nitoda et al. 1999). In this chapter, a rapid and simple
method to detect and quantify chitinase activity in plant extracts is described.
This method is based on the affinity of Calcoflour white M2R for chitin (Maeda et
al., 1967) using glycol chitin as a substrate for endochitinases (Pan et al. 1991).
Glycol chitin is embedded in an agarose gel providing a homogeneous substrate
for the reaction to take place. The principle of the detection is as follows; glycol
chitin, which serves as a substrate for chitinases (Koga and J. 1983), binds to
fluorescent brightener 28 by affinity (Maeda and lshida 1967). After proper
incubation and enzymatic activity, the brightener is bound only to undigested
glycol chitin (Trudel and Asselin 1989). The result is a well defined dark area on

a fluorescent background viewed under UV light translumination.

MATERIALS AND METHODS

Glycol chitin, deacetylated (C7753), pure chitinase enzyme from
Streptomices gn'ceus (C6137), and Calcoflour white M2R (F3543) were obtained
from Sigma (St Louis, MO). High melting point, analytical grade agarose was
obtained from Gibco Corp. The benzothiadiazole derivative, CGA-245704 (BTH)
was obtained from Syngenta Corporation. All other chemicals used for buffers
were obtained from Sigma (St. Louis, MO) unless otherwise specified. Glycol
chitin, a soluble modified form of chitin was prepared as previously described
(Trudel and Asselin 1989). Protein extracts were obtained from cucumber plants

expressing Systemic Acquired Resistance (SAR) and from control plants.

Cucumber plants (Cucumis sativus L.) cv. Wisconsin SMR58 were grown
under standard greenhouse conditions. The cucumber plants were induced to
express SAR with the benzothiadiazole derivative, acibenzolar-S-methyl (CGA-
245704, ASM) (Friedrich et al. 1996) at a rate of 20 ppm of active ingredient in
water. Treatments were applied to plants when the first true leaf (leaf 1) was fully

expanded and the leaf above (leaf 2) was one-third to one-half expanded.

Protein Extraction

Leaf tissues from cucumber plants expressing SAR and control plants
were collected seven days after induction with ASM, immediately weighed and
frozen in liquid nitrogen and stored at -20 °C. Frozen leaf tissues were ground

using a mortar and pestle and homogenized in 0.01 M sodium acetate (pH 5.0) in

30

a ratio of 3 ml of buffer for 1 g of fresh tissue. The homogenate was filtered
through 2 layers of cheesecloth, centrifuged at 12000xg for 15 min at 4 °C and
the supernatant was decanted into a clean tube. The protein content of the
supernatant was measured by the method of Bradford (Bradford 1976) and was

used as crude enzyme extract.

Preparation of the substrate and enzyme activity assay

A 1% (w/v) agarose solution was prepared in sodium phosphate (0.01M
pH 5.5). The solution was melted and cooled to a temperature of 56 °C. When
the agarose gel reached the desired temperature, 1 ml of a 1% glycol chitin
solution was added to 100 ml of the agarose solution. The resulting suspension
was stirred to ensure homogeneous distribution of the substrate and, 30 ml
aliquots were poured into polypropylene petri dishes (15 cm diameter). The
agarose was allowed to cool and solidify for 20-25 min. Small wells (3mm
diameter) were carved in the agarose gels at a distance of 1.5 cm from each
other to form a grid. The agarose plugs were removed by suction with a small
Pasteur pipette attached to a vacuum. Five pl samples of the enzyme solution
were loaded into each well. The plate was incubated for different times at 37 °C
by floating the petri dishes in a water bath . After incubation, 50 ml of 0.5 M
solution of Tris-HCI (pH 8.9) with 0.01% calcoflour brightener 28 were added to

the plate and incubated for 10 min to the petri dish to stop the reaction and stain

31

the plate. The gel was rinsed twice and flooded with distilled water followed by

overnight color development in the dark.

Chitinase Detection and Standard Curve Production

Dilutions of commercial chitinase were prepared to contain 12.5, 25, 50,
100 and 200 uunits ml'1 in sodium phosphate buffer (0.01M pH 7.0). Five pl of
each concentration were placed in the wells of the reaction plate. Each
concentration was assessed five times per experiment. Total protein extracts
from cucumber plants expressing SAR as well as control plants were measured
using Biorad’s protein quantification kit. The protein concentration was equalized
among all samples and 5 pl of each sample were loaded in the wells of the
reaction plate. After the wells were loaded with the protein extracts, the reaction
plates were placed in a water bath at 37 0C for different lengths of time to

optimize the assay as described above.

Image Analysis

The day after the assay was performed, the gel was photographed under
long wave UV light (356 nm) and the resulting image was analyzed using a
personal computer with software for image processing developed by Jandel
Scientific (Sigma Scan). The area of activity, observed as a dark area against a
white background was measured in number of pixels. The ability of the software
to discriminate areas of different color intensities made the analysis very easy to
perform. The data was subjected to statistical analysis. Each sample in every

test was measured at least five times, and the average area of activity expressed

32

in number of pixels was statistically analyzed. In addition, similar results were
obtained when the area of activity was measured by other means, such as

diameter of the area of activity or total area of activity expressed in mmz.

33

RESULTS

Standard Assay.

The solubility of the chitinase substrate glycol chitin allowed us to devise a
simple assay for total chitinase activity by uniformly distributing the substrate in
an agarose matrix. Because glycol chitin binds the fluorescent brightener
calcoflour 28, glycol chitin that is hydrolyzed by chitinase will lose the ability the
bind calcoflor and thus not fluoresce. The reaction mixture contained, in a total
volume of 30 ml per 15 cm diameter petri dish, a 1% (w/v) agarose prepared in
buffer, and 1% (v/v) of the prepared glycolchitin. Areas of activity were easily
observed under UV translumination as non-fluorescent dark regions in a
fluorescent background (Fig 2.1). The reaction gel as observed under
translumination revealed the potential to detect as little as 12.5 pUnits ml'1 of

commercial chitinase.

Detecting commercial chitinase.

The generation of a linear relationship for the quantification of chitinase
through this method depends on two variables: the time of incubation and the
amount of enzyme added. The data collected (square pixels) were plotted as a
function of time (Fig 2.2) and as a function of enzyme added (Fig 2.3). The
regressions analysis in the figures were calculated from at least 3 independent

experiments and each experiment contained at least 5 data points per

34

concentration. In all experiments, there was a slight departure from linearity as a
function of time. This deviation was partially explained by Algranati (Algranati
1963) for enzymatic reactions that progress in a non-linear fashion with time in
which the product of the reaction might interfere with the ongoing reaction. The
statistical analysis for the maximum time of incubation for this experiment, 6
hours, yielded an r‘2 of 0.8911, similar results had been used for germplasm
screening using a different chitinase activity test (Roberts et al., 1994). The
differences between readings from experiment to experiment were not
significantly different at any level (Table 1) and the standard errors were near
10% of the mean. After 6 hours of incubation, the edges of the areas of activity
of the higher concentrations started to merge thus making the analysis of the
gels difficult, nevertheless, the r2 was 0.8911. The highest linearity was
observed at 1 hour of incubation, (r2 = 0.94), however the area of activity for the
lower chitinase concentrations were difficult to analyzed due to its small size.
Incubation for 4 hours yielded the best resolution and size of areas of activity for

easy analysis yielding an r2 of 0.8990.

Detection of Cucumber Chitinases

The treatment of cucumber plants with ASM induces accumulation of
chitinases. Chitinase has been correlated with the activation of SAR thus; a large
increase in chitinase activity throughout the whole plant was expected. When

assessed, using this method, chitinase activity from induced plants versus control

35

plants was always significantly higher than extracts from control plants (Fig 2.4).
In addition, the degree of chitinase induction measured using this method is also
consistent with results obtained elsewhere.

The preparation of the substrate gels with agarose and glycol chitin is
relatively easy, and can be accomplished in a very short time. However, due to
thinness, the gels are fragile and must be handled carefully. In addition when
preparing the wells, care must be taken when removing the plugs to ensure
uniform well size othenivise skewed data might be produced. A Pasteur pipette
attached to a vacuum hose gave the best results to remove the previously carved
agarose plugs. The casting of the gels must also be done in level surfaces to
avoid areas of different gel thickness which also could distort the results since
the wells in areas of uneven thickness would be of different volumes. After the
proper incubation time, the agarose gel once attached to the plate becomes
loose in the plate and if not handled carefully would break easily. In addition, it is
important to mark the gel according to the direction in which the samples were
loaded in order to avoid confusion, since the gel becomes detached from the
plate and easily misread. The area of activity, a black circular area on a
fluorescent blue background can easily be seen immediately after the removal of
the Tris-HCI + Calcoflour solution and the first rinse with distilled water.
However, the maximum color development was attained after a minimum of 6

hours of incubation at room temperature in the dark.

DISCUSSION

We have developed an assay to detect and quantify total chitinase
enzyme activity, which has the potential to simplify and speed its analysis.
Because very few chitinases seem unable to digest glycol chitin (Molano et al
1979) this method of detection and quantification of total chitinase activity from
plant extracts can be used widely. A basal expression of PR proteins has been
identified as a possible marker for resistance in Solanum sp against Phytophtora
infestans (Vleeshouwers et al. 2000). Therefore, plant breeders, for example,
could potentially screen germplasm using this method. It can also be utilized in
the investigation of the induction of the enzyme in plant-pathogen interactions,
SAR and plant defense deployment.

It is important to mention that total protein extracts include in addition to
the acidic inducible chitinase, basic chitinases and other enzymes that might
express partial endochitinolitic activity. However, it is likely that the increase of
activity due to SAR is directly linked to the newly synthesized inducible PR
chitinase. In cucumber plants, the acidic chitinase type III is exported to the
apoplast immediately after synthesis. Potentially, the intercellular fluids can be
harvested via infiItration/centrifugation and their chitinase activity assessed.

Overall, there are some critical steps to be considered when doing this
assay; the time of incubation and the well distance are key factors. Thus, when
dealing with samples of unknown chitinase activity, initial tests should be

performed in order to optimize the distance between wells as well as the time of

37

incubation and protein concentration. Moreover, documentation of the gel should
be done after the maximum color development has taken place usually the next
morning. Failure to do so usually results in the loss of color and contrast in the
picture. This method has been used successfully with cucumber, potato, and
soybean extracts (data now shown). Other groups are also using it for detection
of chitinase activity in root extracts of sugar beet and Arabidopsis .

In conclusion, this procedure provides a simple, rapid and reliable method
for detection chitinolytic activity from plant protein extracts. It avoids the hassle
of handling hazardous chemicals, it saves time and materials and most

importantly, it can be performed with small sample quantity.

 

Figure 2.1: Sample of an activity reaction agarose plate viewed and
photographed under UV light (365 nm). Increasing amounts of enzyme were
placed in the wells A-G (0-250 uUnits ml‘I), and each concentration was
repeated 5 times as indicated in the gel. Notice the consistence of the color,

shape and size of the chitinase activity areas.

39

Figure 2-1: Regression analysis of commercial chitinase activity expressed in
number of pixels in relation to time of incubation for 5 different chitinase
concentrations. Inserted are the r2 values of the regression analysis equation,

bars represent standard deviation.

4O

 

 

.m.N_. mm cm 2:.
a. l- l J 7 III la ml Ill IL Ll Ill
2 i- E - I ll I
: .. E g
. I : i g l... I
_4w : M _ , 8
5w. .1»... l
2 wt I..l ,. ‘ at :l .. 0.1 II. , ,,

...:ll.l L 2 IEM
mmmmmmmmmm mmmmmmmmmmmmmmmmm
<0

79:53 32:20

 

 

 

 

 

_.___..._____q—_ 1

 

 

 

E

e. 1
«We llz.
nP.\ Io»

.- .. I
39.3%“

Incubation Time (Hrs)

41

Figure 2-2. Regression analysis of chitinase activity expressed in number of
pixels in relation to different chitinase concentrations for different times of
incubation. Inserted are the r2 values of the regression analysis equation, bars

represent the standard deviation.

42

 

 

Average Area (Square Pixels)

 

1 HOUR

 

 

 

 

 

 

 

 

4 HOURS

 

6 HOURS

-M » -rfiiefi- *7 - - 4

50 100 150 200 250

Chitinase (pUnits mL'I)

43

2HOURS

Incubation Time

Expression of Chitinase in Control and Induced Plants

 

 

 

 

 

 

SMR58
1600
- Control

1400 ‘ 1::1 Induced __
g 1200 -
.5
D.
g 1000 -
.4?
g 800 1
O
<
a 600 -
(U
.E
g 400 —

200 -

0 .

 

 

 

 

   

 

 

 

Trial l Trial lI

Figure 2-3: Graph shows the resulting chitinase detection from cucumber plants,
(control and induced). Two different extractions were made from the same group
of plants and detected in different reaction plates labeled Trial I and II. No

significant differences were observed between trials (P=<0.01).

44

Table 2.1: Results of three different experiments using commercial chitinase and

five different concentrations.

 

 

 

Chitinase Average Area of Activity, **
pUnits/ml After 4 hours of incubation
*ir'k *** *** TOtal
run1 run 2 run3 Average* St Dev
12.5 844.33 872.14 870.50 862.33 1“ 15.60
25 1013.00 1049.57 1111.57 1058.05 i 49.83
50 1099.86 1147.14 1142.00 1129.67 i 25.94
100 1269.43 1292.86 1404.67 1322.32 i 72.27
200 1300.20 1332.71 1688.00 1440.30 3‘. 215.13

 

* No significant difference was detected between experimental trials

** Data taken in number of pixels in the area of activity

***Each data point in each trial for every chitinase concentration was the average
of 5 data points.

45

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Boller, T., A. Gehri, F. Mauch and U. Vogeli (1983). Chitinase in bean leaves:
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Bradford, Marion M (1976). A rapid and sensitive method for the quantitation of
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Dalisay, R. F. and J. A. Kuc (1995). Persistence of reduced penetration by
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Dann, E. K., P. Meuwly, J. P. Metraux and B. J. Deverall (1996). The effect of
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Kastner, B., R. Tenhaken and H. Kauss (1998). Chitinase in cucumber
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Lee, Yeon Kyeong and Byung Kook Hwang (1996). Differential induction and
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Maeda, H and N lshida (1967). Specificity of binding of hexopyranosyl
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Manocha, M. S. and 2. Zhonghua (1997). Immunocytochemical and
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Margispinheiro, M., J. Marivet and G. Burkard (1994). Bean class-iv chitinase
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47

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Roby, D. and M. T. Esquerre Tugaye (1987). Purification and some properties of
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Roby, D., A. Toppan and M. T. Esquerretugaye (1988). Systemic induction of
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Trudel, J. and A. Asselin (1989). Detection of chitinase activity after
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van der Westhuizen, A. J., X. M. Qian and A. M. Botha (1998). Differential
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48

Vleeshouwers, Vgaa, W. Van Dooijeweert, F. Govers, S. Kamoun and L. T.
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Xue, L., P. M. Charest and S. H. Jabaji-Hare (1998). Systemic induction of
peroxidases, 1,3-beta-glucanases, chitinases, and resistance in bean
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Zhang, Y. Y. and Z. K. Punja (1994). Induction and characterization of chitinase
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49

CHAPTER III
THE EXPRESSION OF INDUCED SYSTEMIC RESISTANCE TO PATHOGEN

CHALLENGE IS DEPENDANT ON PROTEIN SYNTHESIS

50

ABSTRACT

During the development of SAR in cucumber, there is systemic expression
of chitinase, glucanase and peroxidase as well as the enhanced ability to block
subsequent challenge infection by fungi through rapidly induced-cell wall
changes. However, it is not know if new translation/transcription beyond that
expressed during the resistance induction phase are needed to block fungal
penetration into SAR-expressing tissues. Leaf disks of cucumber plants
expressing SAR were treated with the protein inhibitor cycloheximide and
challenged with the fungus Colletotrichum orbiculare. The effect of cycloheximide
on induced plants was an increase in penetration of the fungus into the tissue,
and symptom development was comparable to non-induced plants. The effect of
the protein synthesis inhibitor in induced plants suggest that the induced stated

of cucumber plants is dependant of protein synthesis.

SI

INTRODUCTION

Systemic Acquired Resistance (SAR) generally is associated with a
variety of induced defense mechanisms in plants. These defense mechanisms
include the production of phytoalexins (Hammerschmidt 1999), hydroxyprolyne-
rich glycoproteins (Garcia-Muniz et al. 1998; Raggi 1998), callose and silicon
deposition (Stein et al. 1993), lignin (Hammerschmidt and Kuc 1982) and a
variety of proteins known as pathogenesis related proteins (PR). Some PR
proteins are known to by lytic enzymes such as B-1,3-glucanases and the acidic
type III chitinase (van Loon and Cornelis 1999). In cucumber plants, PR protein
research in relation to SAR has focus on the acidic type III chitinase (Dalisay and
Kuc 1995) and 8-1,3-glucanase (Narusaka et al. 1999). Both of these enzymes
have been shown to accumulate in plants tissues following resistance-inducing
treatments. They have also shown to have antifungal activity in vitro (Skujins et
al. 1965; Ji and Kuc 1996). These authors purified an acidic 8-1 ,2 glucanase and
three isozomes of chitinase isolated from cucumber associated with SAR, and
reported antifungal activity. In tomato, these PR proteins have also been found to
have Iysosome activity (Kauffmann et al. 1987; Legrand et al. 1987). Finally, in
cucumber, their extracellular localization places them in the immediate vicinity of

tissue penetrated by fungi (BoIIer and Metraux 1988).

In addition to the characteristics of the PR proteins above mentioned,
some have suggested that these PR proteins due to their hydrolytic activity are

also capable of releasing elicitors from the invading pathogen in the form of chitin

and glucan oligomers (Roby et al. 1987; Barber and Ride 1994); (Keen and
Yoshikawa 1983). Consequently, studies have correlated the increase of

chitinase activity to the ability of the plant to inhibit infection.

In cucumber plants, the expression of systemic acquired resistance to
Colletotrichum orbiculare is characterized by the ability of the plant to block
penetration by the formation and rapid deployment of structures such as papillae
that contain callose (Kovats et al. 1991), lignin (Hammerschmit and Kuc 1982)
and silicon (Stein et al 1993). These structures and their timely deployment are
believed to be important for the SAR phenotype to be expressed fully in
cucumber plants against pathogens. However, it is not known if new
translation/transcription beyond that expressed during the SAR induction phase
are needed to block fungal penetration into SAR expressing tissues. In other
words, is protein synthesis required to maintain the state of induction? Would PR
proteins and their antifungal activities restrict the pathogen from spreading into

the induced tissue in the absence of newly synthesized proteins?

Cycloheximide (CHX), a protein synthesis inhibitor, compromises the
ability of plants to form papilla after inoculation with fungal pathogens (Vance and
Shenlvood 1976; Skalamera et al. 1997) Based on these studies, it seemed
possible that use of synthetic protein synthesis inhibitor could be a tool to help
determine the role of papilla formation in cucumber induced resistance. In this
study, we tested the effect of cycloheximide on the expression of induced
resistance in cucumber that was induced with the synthetic plant activator

acybenzolar—S-methyl (“ASM", “Actigard”).

53

In previous experiments it was found that spraying low levels of
cycloheximide induced mRNA expression of PR-related proteins in tomato
(Uknes et al. 1993). Why this response to partial protein synthesis inhibition
occurred is not clear. It has been suggested that the plant cell could detect some
sort of protein imbalance, which would lead to expression of defense genes
(Lawton et al. 1994). The treatment of plants with a resistance activator in
combination with low levels of cycloheximide yielded a super induction of
chitinase in tobacco (Hovarth and Chua 1996). In the present study the super-
induction of chitinase, (3-1,3-g|ucanase and peroxidase caused by cycloheximide
in combination with the SAR activator and its effect on the expression of
resistance in cucumber plants was investigated. Despite the higher induction of
chitinase, glucanase and peroxidase in plants induced with the CHX/BTH

combination vs. BTH alone, the levels of resistance were not heightened.

54

MATERIALS AND METHODS

Plant Material and Fungal cultures.

Cucumber plants (Cucumis sativus L.) cv. Wisconsin SMR58 were grown under
standard greenhouse conditions. The cucumber plants were induced to express
SAR when the plants’ first true leaf (leaf 1) was fully expanded and the leaf
above (leaf 2) one-third to one-half expanded. The induction was achieved by
spraying the plants with the benzothiadiazole derivative, CGA-245704 (ASM)
(Friedrich et al. 1996) at a rate of 20 PPM of active ingredient in water. Controls
were sprayed with water. Seven days after induction, the second leaf of control
and induced plants were harvested and brought into the laboratory. Ten mm
diameter leaf-disks were cut from the leaves with a cork borer and then floated
on water or aqueous solutions of cycloheximide (10 ug/ml) in petri dishes (Vance

and Sherwood 1976).

Cultures of Colletotrichum orbiculare were maintained on V-8 juice agar at
room temperature in the dark. Conidia were harvested from 7-10 days cultures
by flooding the dish with sterile water. Following filtration through cheesecloth,
spore concentration was adjusted to 10 5 spores ml". A 10 pl drop of inoculum
suspension was placed on the upper surface of the leaf disks. After inoculation,

the petri dishes containing the leaf disks were maintained at room temperature.

Preparation of fungal cell walls

A conidia suspension of Colletotrichum orbiculare was added to Czapec-
Dox media (Bonnen and Hammerschmidt 1989) and grown for five to seven
days. The mycelium was harvested by suction filtration, washed with double
distilled water to remove the media and crude fungal cell wall preparations were
made as previously reported (Ren and West 1992). The crude cell wall
preparations were freeze dried and ground with a mortar and pestle. One

preparation was made for all of the experiments described within.
Histological observations

For histological observations, leaf disks were removed from the Petri
dishes and cleared in a solution of glacial acetic acidzethanol (1:3 v/v). After
clearing, the disks were hydrated and mounted in glass slides with sterile
glycerol. The following stains were used: for callose deposition, lacmoid blue and
aniline blue; for lignin, pholoroglucinoI-HCI (Krishnamurthy 1999); cotton blue

was used for fungal hyphae (Nicole and Gianinazzi-Pearson 1996).
Protein Extraction

Leaf disks harvested at different times of the infection process were frozen
in liquid nitrogen ground using a small plastic mortar in 1.5 ml eppendorf tube
and homogenized in 0.01 M sodium acetate (pH 5.0) in a ration of 3:1 (v/w). The
homogenate was centrifuged at 12000g for 15min at 4°C and the supernatant

decanted into a clean tube. The protein content of the supernatant was

56

measured by the method of Bradford (ref here) and used for enzyme

determinations.
Assay of Peroxidase and Chitinase Activities

Peroxidase activity of leaf disks extracts was determined using guaiacol
as a substrate as described previously (Hammerschmidt et al. 1982). Chitinase
activity was determined as follows. A 1% agarose in Potassium phosphate buffer
10 mM (pH 5.0) was prepared with a 1% (v/v) of glycol chitin which serves as
chitinase substrate (Trudel and Asselin 1989). After polymerization, 3mm
diameter plugs were carved using a small cork borer in the agarose. A 5 pl
aliquot of the total protein extract were placed in the wells (3 wells per sample),
and incubated at 37 °C for 4 hours. After incubation, the reaction plate was
rinsed and flooded for 10 min in 0.5 M solution of Tris-HCI (pH 8.9) with 0.01%
(w/v) of calcoflour brightener 28 in order to stop the reaction and stain the plate.
When viewed with UV translumination, the areas of activity appeared as a well-
defined dark area on a fluorescent background (Velasquez, Unpublished).
Glucanase activity was determined using Iaminarin as substrate as previously

reported (Biely et al. 1985).
PAGE and gel enzyme detection

Polyacrylamide minigels 1.5 mm thick, were prepared. For native gels, an
anionic (pH 9.3) discontinuous system was used. An acrylamide concentration of

7% provided optimal separation of cucumber proteins.

57

Chitinase activity in PAGE was visualized as previously described (Trudel
and Asselin 1989) with the following modification. The glycol chitin, substrate for
chitinase, was added directly to the components of the gel before polymerization.
Therefore, an overlay gel was not needed since the detection was done in the
separation gel. Peroxidase activity was visualized in PAGE as previously
reported (Smith and Hammerschmidt 1988). After electrophoresis, the gels were
placed in a solution of 50 mg of 3-amino,9-ethylacarbazole, 10 ml of MN,
dimethylformamide, 200 pl of 30% H202 and 190 ml of sodium acetate buffer (pH

5.0). Bands were visible within 10 min of incubation.

SOD and Catalase activity were visualized in gels by the method of
Milosevic (Milosevic and Slusarenko 1996). The gels were scanned and
analyzed by the software package Quantity-One (Bio-Rad Laboratories,

Hercules, CA).

Detection of H202 in cucumber leaf disks.

To study H202 production by leaf disks, the Phenol red assay was used
(Svalheim and Robertsen 1993). In this assay, the increase in absorbance at
610 nm resulting from the peroxidase-catalyzed oxidation of phenol red by H202
is measured. Ten leaf disks from SAR expressing plants were incubated in glass
vials containing 1 ml 10 mM MES-KOH, pH 6.5, and 10 pg of chloroamphenicol
with or without fungal cell wall preparations from C. orbiculare that served as a

source of elicitor. The cell wall preparation was added to the leaf discs in buffer.

58

Since the cell wall preparations were not soluble in water, the leaf disks
containing the cell wall preparations (10 pg/ml) were placed in a shaker to
maintain the cell well preparation in solution. The segments were incubated at
room temperature in the dark for 2 hours. Ten pL phenol red solution (10 g L‘1 in
distilled H2O) was added and the mixtures were incubated for an additional two
hours. The reaction was terminated by the addition of 20 pL of 1 M NaOH to
each vial. The A620 of the incubation solution was read against a blank prepared

in the same manner, but in the absence of leaf disks.

59

RESULTS

The effect of CHX in fungal growth and development:

Disease symptoms. The first anthracnose symptoms on untreated
cucumber leaf disks were visible 48 hours after inoculation. The lesions in the
area of inoculation appeared first as green-brown, water soaked areas that
become necrotic and sometimes developed a chlorotic halo. Symptoms on
induced cucumber leaf disks developed more slowly and less severely. SAR
expressing leaf disks treated with CHX were infected by C. orbiculare to the
same extent as the non-induced control plants. There were no noticeable
differences in terms of lesion development between the control disks and the

induced disks treated with CHX.
Cytological observations:

Twenty-four hours after inoculation, conidia had germinated and formed
appresoria on leaf disks from all treatments. Penetration pegs were visible
piercing the outer cell wall of the epidermal layer by forty-eight hours after
inoculation. The rate of penetration was assessed for each of the treatments
(Fig 3.2). Two hundred fully formed appresorium were observed in each of the
treatments in up to ten different leaf disks. The results obtained show that the
number of penetrations between the induced leaf disks treated with CHX was
comparable to the number of penetrations observed in the non-induced leaf

disks.

60

To determine CHX had an effect on the development of the fungus in
induced tissue the fungal development was observed. Hyphal length was
measured 72 hours after inoculation in all treatments. The results show that the
growth rate of fungi in induced plants is greatly reduced (fig 3.3). In contrast,
non-induced leaf disks allowed a considerable higher fungal growth within the
tissue. The hyphal length at 72 hours after inoculation of induced CHX-treated
leaf disks was similar than those in non-induced leaf disks. Therefore, leaf disks
treated with CHX did not only allow more penetrations, but also tolerated a higher

rate of fungal growth within the tissue (fig 3.4).

Papillae formation and lignin deposition in leaf disks treated with CHX.

Induced leaf disks floated in water developed normal papilla and lignin
deposition associated with the site of penetration (fig 3.1A, 3.1C). The
association of lignification at the site of penetration and resistance in cucumber
plants has been reported (Hammerschmidt and Kuc 1982). Leaf disks of induced
plants were treated with CHX and inoculated with a droplet of a spore
suspension of conidia from C. orbiculare. In such leaf disks, papillae formation
was drastically reduced and when present, it was unable to stop the pathogen
from penetrating the plant tissue (fig 3.1 B). Lignification in leaf disks treated with
CHX was not primarily associated with areas of penetration. When present, the
areas stained with the method of pholoroglucinol-HCI were more generally

associated with damage post penetration linked with cell damage. (Fig 3.1A)

61

Effect of challenge inoculation and CHX treatment on PR protein activity

In non-induced leaf disks, a sharp increase of chitinase activity was
detected at 48 hours after inoculation with C. orbiculare. This is consistent with
the observations that the defense mechanisms are further deployed after a
pathogen attack. Non-induced, non-challenged leaf disks also had an increase
of chitinase activity, however, the levels are far below the expressed by
challenged leaf disks. Therefore, wounding and/or CHX treatment can also
induce chitinase. Cycloheximide untreated and treated leaf disks and
challenged with the fungus, also had a small increase of chitinase activity, but
their levels were already higher due to SAR expression. Overall, chitinase activity
is not hindered by CHX treatment. Glucanase activity, just as chitinase was not
greatly reduced and its activity remain constant in induced leaf disks treated with

CHX (data not shown).

The levels of peroxidase activity in induced plants compared to non-
induced plants were higher at all times before and after inoculation as previously
reported (Hammerschmidt et al. 1982). On the other hand, the treatment of leaf
disks with CHX yielded a drastic reduction in POX activity. However the kinetics
and in PAGE detection activity clearly show that peroxidase activity was greatly
reduced in leaf disks treated with CHX (Fig 3.6A). However, CHX does not affect
the activity of POX directly since the enzyme kinetics show that in the presence
of CHX up to a concentration of 10 mM did not affect the activity of cucumber

POX (Fig 3.68).

62

In order to examine the levels of activity of POX in the presence of an
elicitor, leaf disks of induced and non-induced plants were incubated in the
presence of a fungal cell wall preparation (FCP) and its peroxidase activity
determined at different times during the experiment. The results show that POX
activity increases greatly in presence of the FCP. Changes on the activity of
POX are detectable by 4 hours after exposure to the elicitor. The induced leaf
disks exposed to the crude fungal cell wall preparations expressed the greatest

peroxidase activity change (Fig 3.7)
The effect of CHX in the generation of Reactive Oxygen Species

The levels of production of the highly reactive oxygen species (ROS) and
the activity of the primary enzymes that catalyze the production of these ROS
were studied. At 96 hours after treatment with CHX, in PAGE detection of
catalase and SOD was performed (Fig 3.8). The activity of both enzymes,
catalase and superoxidase dismutase (SOD), were detected at he same levels
before and after CHX treatment, therefore, establishing that CHX did not affect
their activity. In this work, the effect of CHX clearly compromises the amount of
H202 produced. In an experiment with leaf disks of induced plants, the detection
of H202 was assessed in the presence of CHX. Data shows that the ability to
elicit and increase the production levels of H202 in induced material is

compromised by the protein synthesis inhibitor. (Fig 3.9).

The effect of treating levels of CHX in combination with a SAR Inducer.

As reported before the application of low concentrations of CHX in
combination with SA produced a super induction of chitinase in tobacco (Horvath
and Chua 1996). The effect of CHX in combination with the SA analog BTH also
produced a systemic “super induction” of chitinase, B-1,3, glucanase and
peroxidase in cucumber plants (Fig 3.10). After induction in leaf #1, leaf #2 was
harvested and a detached bioassay for resistance was performed in addition to
biochemical analysis of the induced plants. Ten micro-liter droplets of a spore
suspension of C. orbiculare were placed on the leaves in a sterile Petri dish with
a moist filter paper. The assessment of the disease progress was performed
seven days after the inoculation. The number of lesions was greatly reduced in
plants treated with BTH as well as with plants treated with the combination of
BTH and CHX. Control plants allowed a larger number of lesions and a larger
lesion size just as the plants treated with CHX alone (Fig. 3.11). However, there
were no statistically significant differences between the lesion size or lesion
number among the plants treated with BTH and plants treated with the

combination BTH-CHX.

64

DISCUSSION

Hammerschmidt and Kuc have shown that the phenotype of SAR against
Colletotrichum orbiculare is closely related to the ability of the epidermal cells to
respond rapidly with deposition of lignified structures around the areas of
possible penetration (Hammerschmidt and Kuc 1982). Lignification, in their study,
was found to occur sooner and to be more intense beneath appressorium and
around sites of penetration in induced compared to control leaves. This was also
demonstrated in potato tissues where pre-treatment of the tissue with a-
aminooxiacetic acid, a competitive inhibitor of PAL, induced a state of
“susceptibility” associated with the lack of lignin deposition (Hammerschmidt

1984).

Penetration of Colletrotn'chum orbiculare into cucumber plants expressing
SAR was found to be markedly reduced after challenge with conidia of the
fungus. However, the treatment of induced leaf disks with CHX increased fungal
penetration and did not affect fungal growth within the induced tissue. The SAR
phenotype against C. orbiculare has been shown to be expressed in the
epidermal cells (Xuei et al. 1988; Kovats et al. 1991; Stein et al. 1993). Thus the
initial line of defense against pathogen attack is being compromised by the
inhibition of protein synthesis. In studies elsewhere it has been found that the
induction of phenylalanine ammonia lyase (PAL) activity was blocked by protein
synthesis inhibitor, CHX or actinomycin and largely prevented by a protein kinase

inhibitor (Mackintosh et al. 1994). PAL catalyses the formation of cinnamc acid

65

from phenylalanine, and the first step leading to the synthesis of
phenylpropanoid phenolics such as lignin precursors. Thus, the inhibition of PAL
expression or other enzymes leading to lignin biosynthesis is a possible
explanation for the results described above. Hammerschmidt and Kuc have
shown that the phenotype of SAR against Colletotrichum orbiculare is closely
related to the ability of the epidermal cells to respond rapidly with deposition of
lignified structures around the areas of possible penetration (Hammerschmidt
and Kuc 1982). Lignification, in their study, was found to occur sooner and to be
more intense beneath apresorium and around sites of penetration in induced
compared to control leaves. This was also demonstrated in potato tissues where
pre-treatment of the tissue with a-aminooxiacetic acid, a competitive inhibitor of
PAL, induced a state of “susceptibility” associated with the lack of lignin

deposition (Hammerschmidt 1984).

In addition to lignin deposition, papillae formation was also altered.
For the most part papillae like structures were formed however, they were unable
to restrict fungal penetration in induced leaf disks treated with CHX. In
experiments with the defense-compromised Arabidopsis nim1-1 and salicylate
hydroxylase -expressing plants suggest that extrahaustorial callose production is
enhanced by treatments that activate SAR, and that this induction may involve
the NIM1/NPR1 pathway (Dempsey et al. 1999). In mutant plants npr1 the
callose deposition could not be rescued by applications of INA. In contrast, nahg
plants, the ability to deposit callose was rescued by the application of INA. The

authors noted that that callose deposition must occur for the phenotype of SAR

66

to be expressed but also noted the dependability on the NPR1 gene and SA.
The observations made in this work suggest that protein synthesis might also

drive papillae formation.

In this work, the induced plants treated with CHX were able to produce
papillae, but these did not effectively stop pathogen penetration. SAR primes the
plant to deploy callose deposition in a timely manner, but its ability to stop the
fungi is protein synthesis dependent and the synchronized availability of the
building blocks for its synthesis. It has not escaped our attention that a series of
experiments have been done in order to assess the value of callose in stopping
the pathogen penetration. Smart et 3! (Smart et al. 1986) concluded that callose
deposition alone is not sufficient to stop pathogen attack. However, inhibition of
callose synthesis using 2-deoxy-d glucose reduced papillae formation and led to
enhanced penetration of powdery mildew Erishaphe graminus in barley
suggesting that callose formation is important for resistance. (Bayles et al. 1990).
Vance and Shenivood however, showed that the deployment of papillae is
dependant of protein synthesis since CHX inhibited the production of papillae
formation and allowed fungal penetration by spores of normally non-infective
pathogens of reed canary grass (Vance and Shen~ood 1976). From these
experiments, it can be concluded that papillae formation is a key factor in the
resistance mechanism, and the timely deposition of papillae and its proper
lignification is a key factor of SAR. Since CHX also compromises PAL activity, it
is also possible that the combination of callose depostion and lignification must

be present in order for the phenotype of SAR in epidermal cells to occur.

67

H202 is believed to be important in lignin biosynthesis (Gonzalez et al.
1999), and is also antifungal (Joseph et al. 1998). The SAR-enhanced production
of H202 was found to be sensitive to CHX treatment and was fully inhibited at 5
pM concentration. This is consistent with an earlier report in which the same
results were obtained in cucumber hypocotyls (Fauth et al. 1996). These authors
concluded that CHX did not inhibit the H202 producing enzyme system. Our
results show that indeed, catalase and SOD were not greatly affected by CHX
treatment. This finding is important since it indicates that protein synthesis is
required for the full production of H202 in SAR expressing cucumber plants. In
tobacco, a catalase has been identified as a SA binding protein (Chen et al
1993). Since binding of SA to catalase inhibits its activity, increased amounts of
SA could indirectly result in increased quantities of H202 in the tissue. Therefore
the hypothesis that the mechanism of action of SA is to elevate the levels of
H202, which then serves as an intermediate in the SA signaling pathway was
born (Chen et al 1993). It was later found that BTH and INA acted similarly, they
both block the activity of catalase (Yang et al. 1997). It is important to mention
that H202 from the oxidative burst drives several plant defense responses
including cross-linking of cell wall proteins (Levine et al. 1994; Brown et al. 1998).
H202 production has been regarded as a key factor in the early defense
response against pathogen attack in SAR expressing plants. Therefore when
CHX compromises its production, it also compromises the full expression of

SAR.

68

Induction of SAR by SA or its analogs (BTH, INA) is usually associated
with the accumulation of Pathogenesis Related (PR) proteins. These are usually
found in the intercellular fluids (IWF) of leaves of plants expressing SAR.
Chitinases and B-glucanases and peroxidases are the most abundant in the
apoplast. These are expressed at very low levels in non-induced plants, but their
expression is highly increased when plants are expressing SAR. CHX did not
affect the activity of the chitinases and glucanases. However, peroxidase activity

is compromised by the presence of CHX.

The results of this work show that peroxidase activity is hindered by CHX
treatment. The levels of H202 are also greatly reduced and the ability of induced
plants to hinder fungal penetration and fungal growth is compromised. These
findings in addition to previous evidence show that CHX also inhibits PAL activity
(Mackintosh et al. 1994) thus limiting the availability of phenolic and lignin
precursors have one thing in common. They are all involved directly in the early
response stages against pathogen attack. Peroxidase activity has a function in
the generation and utilization of H202 for cross-linking of protein and phenolics
during the construction of papillae (Brown et al. 1998). Papillae are often Iignified,
and at the sites of potential penetration a higher concentration of H202 has also
been found (Huckelhoven et al. 1999). Since peroxidase induction and H202
production are blocked by CHX these results indicate that de novo synthesis of
protein is required for H202 generation, peroxidase activity, lignin synthesis and

the construction of effective structures to stop fungal penetrations. The blockage

69

of induction of peroxidase by CHX has also been reported in cucumber

hypocotyls (Svalheim and Robertsen 1990)

Chitinase and glucanase are thought to be a key component in the SAR
mechanism. Chitinase mRNA increase slightly following cycloheximide
application; however, its potent induction by salicylic acid was inhibited by
cycloheximide treatment suggesting that protein synthesis is required for
chitinase mRNA induction to take place. (Lawton et al. 1994). However, using a
transgenic plant expressing a rice chitinase, it was found that CHX did not inhibit
chitinase activity, (Kim et al. 1998). The fact that the growth of the fungal hyphae
within the tissue of induced plants treated with CHX was comparable to non-
induced plants questions the role of chitinase and glucanase in effectively
restricting fungal growth. The higher activity of both enzymes was not a factor in

the degree of fungal growth within the induced tissue.

When low levels of CHX are sprayed in combination with a SAR
activator, the systemic “super induction” of the PR proteins glucanase and
chitinase in addition to peroxidase takes place. This phenomenon although not
totally understood, theoretically should prove useful to investigate the role of
these PR proteins in the defense response of the plant. Although the levels of
activity were statistically significant between the BTH induced plants and the
CHX/BTH induced plants. The higher levels of PR expression of the CHX/BTH
induced plants failed to decrease the development of the pathogen within the

fissue.

70

Our experiments have show that the CHX treatment of SAR expressing
cucumber leaf disks enhanced the susceptibility against the pathogen C.
orbiculare. The lesions were comparable to the developed by leaf disks not
expressing SAR. This suggests that protein synthesis is needed in order to keep
the state of induction in cucumber plants expressing SAR. We are not
suggesting that protein synthesis inhibition is the only factor responsible for
breaking down of the resistance; CHX might have other unknown effects in the
plant metabolism. Molecular approaches and mutation analysis might prove
useful to disclose the specificities and the contributions of individual building

blocks of the Systemic Acquired Resistance phenotype.

71

 

 

 

 

 

 

Figure 3.1A: Leaf discs from induced plants non treated with CHX (left panel)
and CHX treated (right panel). Leaf discs were challenged with C. orbiculare and
treated with phloroglucinol to detect lignin depostion72 hours after inoculation.
Induced plants non-treated with CHX were successful in stopping fungal
penetration. Lignin deposition was observed directly under and around areas of
possible penetration. In CHX treated leaf discs the areas of lignification were
more spread, but the fungus was able to break through the lignified areas.
Arrowheads indicate the appresorium, arrows indicate hyphae growing within the

tissue. Pictures taken at magnification 100X

72

 

 

 

 

 

 

Figure 313: Leaf Discs from induced plants non treated with CHX (left panel)
and CHX treated (right panel). Leaf discs were challenged with C. orbiculare and
stained with lacmoid blue to detect callus deposition at 72 hours after inoculation.
Leaf disks from induced plants treated with CHX had callus deposition but were

unable to stop penetration of the pathogen. Pictures taken at magnification 100x

73

 

Figure 3.10: Leaf disks from induced plants 48 hours after challenge with C.
orbiculare stained with aniline blue to detect papillae formation. Left panel.
picture taken under visible light. right panel taken with UV light. Note the small

ring (papillae) directly under the appresorium. Pictures taken at magnification

100X

74

 

100

80~

60*

4o-

Penetration Percentage

20-

 

 

 

Control Induced Cont+CHX lnd+CHX

Figure 3.2. The effect of cycloheximide in the penetration rate of C. orbiculare 24
hours after inoculation with a 10 pl droplet of a conidia suspension (106 spores
ml"). Cleared leaf disks were observed under a Leica microscope and up to 200
fully formed appresoria were examined in 10 leaf disks for each treatment. A
successful penetration was regarded as the visible presence of a penetration peg

and a visible primary hyphae within the tissue.

75

Fungal Lentht 72 Hours After Inoculation

 

 

40
30—

E

1

E

O)

c

2 20‘

To

U)

c

3

U.
10‘
o-

 

 

Ind + CHX Control + CHX Induced Non Induced

Figure 3.3: The effect of cycloheximide in the growth rate of the fungal hyphae
within the tissue. Measurements were taken from the base of the appresorium to
the hyphal tip of the invading pathogen. The measurements were taken 72 hours
after inoculation with C. orbiculare and treatment with CHX. Statistical
differences were only observed in the leaf disk from SAR expressing plants

(p=<0.01).

76

Non-Induced Induced

Water I

CHX

 

Figure 3.4: Micrographs of cucumber leaf disks under visible light taken with a
Leica microscope. Arrows indicate the hypha within the tissue. Leaf discs were

viewed 48 hours after inoculation, pictures taken at magnification 100x.

Chitinase and CHX

3000

 

2500 -

2000 ~

1500 a

1000 -

Chitinase Activity Pixels2

500 -

 

 

0 I l I I I
0 10 20 30 40 50

 

Hours After Inoculation

Figure 3.5. Detection of chitinase activity of leaf disks treated with
cycloheximide during the process of infection by C. orbiculare. Leaf disks of
induced plants treated with (O) or without CHX (0), and control plants treated
with (V) and without CHX (V) were collected and their chitinase activity was
assayed. It is apparent that chitinase activity was not affected by the presence of

cycloheximide

78

Figure 3.6: The effect of the treatment with CHX to cucumber leaf disks
inoculated with C. orbiculare Induced leaf disks treated with (V) and without (V)
CHX. Control plants treated with (O) and without (0) CHX. B: Cucumber total
protein extracts from induced plants were assayed for POX activity using guiacol
as a substrate in the presence of CHX. No differences were detected in the

initial enzyme kinetics up to the concentration used in the experiment (10 mM).

79

 

digs
IIV}

Iota

 

 

POX activity
Change'in Absorbance (470nm) min‘1 gr"I fresh Tissue

Absorption (470 nm)

POX Activity in Leaf disks treated with CHX

 

 

 

 

 

 

   

 

 

400
300 <
200 -
100 —
L /
o . . a .
0 1o 20 30 4o 50
Hours After Inoculation
1.0
0.8 —
0.6 -
0,4 —
0.2 j o CHX10mM
0 Control
7 CHX 5 mM
0 o , . . . i
o o o 5 1 0 1 5 2 0

Time (min)

25

 

80

 

 

250 _

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CD
3
$2 - Control
l: [:3 Induced
g 200 _ - controI+FCP T“ __
i [:1 Induced+FCP
a!” .
.3 '5 150
8 E
X 8
83 § 100 -
‘5
8
(U
C
O)
C
(B
.C
O
0
0 4 8 24 48
Time After Exposure to the Fungal Cell Wall Preparations (FCP)
(Hours)

Figure 3.7: Peroxidase activity from leaf disks of induced and control plants in
the presence of a fungal cell wall preparation. Leaf disks were collected and
placed in Sodium buffer with or without the fungal cell wall preparation from C.
orbiculare. Leaf disks were collected during the course of the treatment, their
total protein extracted and the activity of POX measured using guiacol as a
substrate. Note the major differences of the cucumber leaf disks in the presence
of FCP. This suggests that the accumulation and or activity of peroxidase

increase beyond the induction phase of SAR.

81

Figure 3.8. Catalase Activity (A) and SOD activity (8) detected via PAGE activity
staining. After staining, the gels were photographed and analyzed to detect the
average intensity of the areas of enzymatic activity. Samples were collected 96
hours after inoculation, and 20 ug of total protein were loaded per well. In
addition, samples from induced and non-induced plants at 0 hours of inoculation

were also loaded (0 HAI).

82

Average Intensity

 

160
140 H
120 - A l—
100 -

80-

 

60-

4o-

20‘l

 

 

 

 

 

 

 

 

 

 

 

100- l-
B r

80 1

l

60

40a

 

 

 

 

 

 

 

 

 

 

T

CHX No CHX

83

0 HAI

- BTH
EZI CONTROL

Effect of CHX in the generation of A03 in
leaf disks of cucumber

 

0.28

0.26 -

0.24 -

0.22 1

0.20 -*

H202 Generation
A600

0.18l
0.16 -

0.14 -

 

 

 

0.12

d

I I I I

0 1 2 3 4 5 6

-l

Cycloheximide Concentration ( uM )

Figure 3.9: Inhibition by cycloheximide of the elicitation of H202 from leaf disks.
The induced leaf disks were conditioned with the indicated concentrations of
cycloheximide followed by elicitation with 10 ug ml'1 of crude fungal cell wall
preparations for 30 min. Results are given from one representative experiment of

three performed.

84

Figure 3.10: Gel electrophoresis detection acidic peroxidase (A) and chitinase
(B) of plants sprayed with Water, BTH (20ppm) BTH+CHX and CHX (mug/ml).
30 ug of total protein was loaded in each well. Protein samples came from 2"cl
leaves of sprayed plants. (C) Gluoanase activity detection. Detection of

glucanase was done twice, graph above are the results of one of two different

experiments.

85

m units/gr FT

 

C BTfi BE‘CHX
CHX

Water BTH+CHX

86

Figure 3.11. The systemic effect of induction of cucumber plants with BTH and
BTH/CHX. CHX was sprayed in combination with an application of 20 PPM of
BTH and the application of CHX was done at a rate of about 10 uM solution.
Second leaf was harvested and challenged with a spore suspension of C.
orbiculare the disease assessment was done 7 days after inoculation and lesion

size (A) and lesion number (B) were measured.

87

 

 

 

223.. Go 33:52

 

ASE. 2.2mm... ho LeuoEmE

Water control BTH BTH+CHX CHX

Treatment

88

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CHAPTER IV

THE RESPONSE OF CUCUMBER CULTIVARS TO INDUCTION OF SAR IN
GREENHOUSE AND FIELD CONDITIONS AND THE SCREENING OF

RESISTANCE AGAINST Colletotrichum orbiculare AND Dydimella bryoniae.

94

ABSTRACT

Increase in activity of chitinase, a pathogenesis related (PR) protein, is
associated with systemic acquired resistance induced in cucumber by plant
activators and biological agents. Field and greenhouse experiments were
conducted in order to correlate the amount of chitinase in different SAR
expressing cucumber cultivars and the degree of resistance induced against
Colletotrichum Iagenan'um and Didyme/Ia bryoniae. The results suggest that
there is little correlation between the amount of chitinase induction and the
degree of resistance acquired through SAR. However, the screening of these
cultivars yielded a differential cultigen response to these two pathogens.
Cucumber cultivars expressing SAR are generally resistant to C. orbiculare
but are not always resistant to D. bryioniae. In addition, several cultivars
expressing SAR developed more severe symptoms despite the high chitinase
activity detected. Lesions number development by C. Iagenan'um varied
significantly among cultivars, yet the size of the lesions did not. The variation
in number of lesions generated by D. bryoniae was not as significantly
different among cultivars whereas the size of the lesions varied greatly. This

suggests different defense strategies against these two pathogens.

95

INTRODUCTION

Systemic acquired resistance (SAR) is a general defense response in
plants that is characterized by the expression of pathogenesis-related (PR)
genes (van Loon and van Kammen 1970). SAR can be induced after a
necrotic response to an avirulent or virulent pathogen or by treatment with
chemical SAR inducers such as the benzothiadiazole derivative, CGA-245704
(ASM), INA and salicylic acid (Lawton et al. 1996; Benhamou and Belanger
1998), The ground-breaking experiments of Kuc and coworkers have shown
that plants expressing SAR are 'sensitized' to better withstand the infection of
pathogens (Kuc 1982). Its action is nonspecific throughout the plant and
reduces severity of diseases caused by all classes of pathogens. In
cucumber plants, the expression of systemic acquired resistance against
Colletotrichum orbiculare is characterized by the ability of the plant to block
penetration by the formation of papillae, lignin, silicon deposition (Stein et al.
1993), and by an increase in peroxidase activity (Hammerschmidt and Kuc
1980).

Most of the research on cucumber SAR has studied the interaction
with C. orbiculare (Kuc 1987). However, no two-pathogen species are
completely similar in terms of their strategies for invading plants and causing
disease. Colletotrichum orbiculare, relies on the formation of appresorium
and mechanical penetration in order to infect plant material (Kovats et al.
1991; Stein et al. 1993). Alternatively, another pathogen of cucurbits,

Didymella bryoniae has been reported to actively use pectolytic enzymes

96

during the colonization of plant tissue (Chilosi and Magro 1998), as well as
penetration through the stomata (da Rocha, Personal communication).
Didymella bryoniae (anamorph Phoma cucurbitacearum), the cause of
gummy stem blight of cucurbits, and Colletotrichum orbiculare, which causes
anthracnose of cucurbits, occur throughout the Eastern United States and are
two of the most important diseases to attack cucurbits (Agrios 1997). The
need to find control strategies that will be effective against pathogens
attacking the cucurbit industry is very important and, SAR presents itself as a
good potential control method against these diseases (Hammerschmidt and
Dam 1997; Benhamou and Nicole 1999). Under field conditions, SAR has
been reported to be effective in some cucurbits including cucumber (Caruso
and Kuc 1977).

In cucumber, SAR has been mainly studied in a small number of
cultigens (mostly Wisconsin SMR 58 and Straight 8). However, there are
many others commercial varieties and breeding lines (Dijkhuizen et al. 1996;
Staub et al. 1999). Therefore, it is easy to hypothesize that their resistance
and SAR expression might vary significantly because of their genetic
differences. To our knowledge there has not been an extensive study of a
broad collection of cucumber cultigens for their SAR response. Consequently,
a comprehensive genotype evaluation was performed.

The purpose of this work is to screen a collection of cucumber
cultigens for their genetic differences in resistance against C. orbiculare and

D. bryionieae. In addition, to evaluate their SAR expression levels under

97

greenhouse and field conditions. The correlation between the induction of
resistance due to SAR and the amount of the PR protein expressed was also

evaluated.

98

MATERIALS AND METHODS

Culture of host and pathogens.

In greenhouse, cucumber plants (Cucumis sativus L.) were planted in
clay pots filled with Pro-Mix BX (Premier Peat Moss Corporation Marketing,
New York, NY). The pots were grown under daylight supplemented with 14 h
of fluorescent and incandescent light.

Field experiments were made during the growing seasons of 1998,
1999, and 2000 at the Plant Pathology research farm and at the Horticulture
farm, both located in East Lansing, MI. Growing cultural practices were
standardized throughout the experiments.

Cultures of Colletotrichum orbiculare were maintained on V-8 juice
agar at room temperature in the dark. Conidia were harvested from 7-10
days cultures by flooding the dish with sterile water. Following filtration
through cheesecloth, the spore concentration was adjusted to 106 spores mL’
1. Dydimella bryoniae was grown on % strength Potato Dextrose Agar (PDA)
at a temperature of 25° C in the dark. lnoculum preparations were done by
flooding plates with a nutrient solution of Casamino acids (0.05 g, Difco Co.)
and sucrose (1.0g, EM Science Co.) in distilled water (100 ml). The surface
of the agar was scraped with a rubber spatula, and the mix was filtered
through two layers of cheesecloth. The spore suspension was standardized to

a concentration of 106 spores mL". Rhizoctonia solani was maintained in

PDA. lnoculum was prepared from 3-4 day old cultures, agar plugs (5 mm

99

diameter) from the edge of the growing mycelia were taken and placed on
small wounds done to the cucumber fruit.

Induction of SAR

In Greenhouse experiments, the cucumber plants were induced to
express SAR when the first true leaf (leaf 1) was fully expanded and the leaf
above (leaf 2) one-third to one-half expanded. The induction was achieved
by spraying the plants with the benzothiadiazole derivative, CGA-245704
(ASM) (Friedrich et al. 1996) at a rate of 20 PPM of active ingredient in water.
Induction of SAR in field experiments was done using a solution of ASM with
water (20 ppm) and sprayed using a compressed air sprayer. The induction

was done when the majority of the plants had two fully expanded true leaves.

Detached leaf resistance bioassay

For both, greenhouse and field experiments, leaves were collected and
brought into the lab. They were out along the midrib and inoculated with 10
droplets of 10 pl of a spore suspension of either C. orbiculare (106/ml) in
water or D. bryoniae (106 spore/ml) in a casamino acids/glucose solution.
The half leaves were placed in sterile Petri dishes underlined with moist filter
paper. Seven days after challenge, the number of lesions allowed per half

leaf was counted and the lesion size diameter (mm) assessed.

100

Cucumber fruit bioassay

Cucumber fruits were harvested when fruit 1% to 2 inches in diameter
were first observed. The fruit was harvested into clean, plastic containers,
washed in chlorinated water and placed in trays underlined with moist paper
towels. A small wound was done in each cucumber fruit using a razor blade
and a 5 mm agar plug of Rhizoctonia solani was placed on top of the wound.
The cucumber fruits were kept under high humidity conditions. About 8-10
days after inoculation, the fruit was cut in half directly over the wound and the

damage was measured in two dimensions, lesion diameter and lesion depth.

Assay of Peroxidase and Chitinase Activities

Peroxidase activity of leaf extracts was determined using guaiacol as a
substrate as described previously (Hammerschmidt et al. 1982). Chitinase
activity was determined as follows. A 1% agarose in potassium phosphate
buffer 10 mM (pH 5.0) was prepared with a 1% (v/v) of glycol chitin which
serves as chitinase substrate (Trudel and Asselin 1989) and poured in a 15
cm diameter Petri dish. After solidification, 3mm diameter plugs were carved
using a small cork borer in the agarose. Five pl of the total protein extract
were placed in the wells (3 wells per sample), and incubated at 37 °C for 4
hours. Following incubation, the reaction plate was rinsed and flooded for 10
min in 0.5 M solution of Tris-HCI (pH 8.9) with 0.01% (w/v) of calcoflour
brightener 28 in order to stop the reaction and stain the plate. When viewed

with UV translumination, the areas of activity appeared as a well-defined dark

10]

areas on a fluorescent background. The diameter of the circular area of

activity was measured and used for data analysis.

Experimental design.

Greenhouse experiments were done based on a complete randomized
block design. In comparing induced plants and control plants, 3 blocks per

treatment of at least 5 plants per block were analyzed.

In 1998 and 1999, two field experiments were done to study genetic
resistance differences of a cucumber collection at the Horticulture research
farm. In addition an experiment to study the effect of ASM was done in the
Botany and Plant pathology research farm. These experiments were based
on a randomized complete block design. In 2000, one main experiment was
performed to study ASM effect on cucumber plants. The experiment was
designed as a split plot with cultigens as main plots and treatments (control

and ASM) as subplots.

RESULTS

Greenhouse screening of cucumber cultigens revealed different levels
of resistance after treatment with the synthetic plant activator acybenzolar-S-
methyl (“ASM”, “Actigard”). Initial observation using five cultigens revealed
that the levels of expression of SAR significantly enhanced the ability of the
treated plants to hinder fungal invasion as well as fungal spread within the
tissue (fig 4.1). In both, lesion size and lesion number, statistical analysis
revealed that comparison within varieties (control vs. treatment) were
statistical significant at all levels.

When the basal levels of resistance in control plants was analyzed, the
levels expressed in control plants did not present differences among the
varieties tested in terms of lesion number. In terms of lesion size and the
ability of the plant to hinder fungal growth within the tissue, two groups were
observed. Pioneer and National Pickling had a slightly higher resistance than
SMR 58, Straight 8 and MM 80 (fig 4.1).

The induced levels of resistance were found to behave similarly in both
measurements, lesion size and lesion number reduction. Pioneer being the
more resistant and Straight 8 being the most susceptible.

In addition, all five cultigens had expressed an increase in chitinase as
well as peroxidase activity after ASM treatment (fig 4.2). Additionally, it was
observed that the basal levels of chitinase activity were non statistically
different. However, when the plants were induced with ASM, the levels of

chitinase were increased and three statistical significant groups were

103

observed. (fig 4.2). However, when the number of cucumber cultigens was
increased, the results became more complex. Although the majority of the
cultigens treated with ASM had fewer lesions and reduced lesion size, the
reductions were not always at significant levels. The majority of the cultigens
were also more successful stopping the initial infection than actually reducing
fungal growth within the tissue as shown by the number of lesions observed
(table 4.1). The bulk of the cultigens had a significant reduction of lesion
number even though some of the same cultigens had no differences between
the control and treated plants in terms of lesion size. Nonetheless, Pl
390240 and PI 390259 were more successful reducing fungal growth within
the tissue by limiting the lesion size than stopping the pathogen from initial

infection.

There is a differential response of plants expressing SAR against
pathogens with different mode of invasion.

In order to study the response of SAR against two different pathogens
with different modes of infection, plants were induced with ASM and
challenged with two different pathogens. Colletotrichum orbiculare, uses the
formation of appresorium and mechanical penetration to infect the plant
material (Kovats et al. 1991; Stein et al. 1993). Alternatively, Didymella
bryoniae has been reported to penetrate through stomata (da Rocha,
Personal communication) and actively use pectolytic enzymes during the

colonization of plant tissue (Chilosi and Magro 1998).

104

Seven days after induction with ASM the second true leaf was
harvested in brought into the lab. The leaf was out along the center vein and
one half was challenged with C. orbiculare and the other half was challenged
with D. bryoniae. The treated plants expressed an increase in resistance
against C. orbiculare.

Conversely, the plants treated with ASM and challenged with D.
bryoniae gave results that were unexpected. In almost half of the cultigens
tested against this pathogen, the treated plants allowed higher incidences of
disease caused by this organism when compared to the control plants. Higher
amount of disease in ASM treated plants occurred even in lines that
expressed good induced resistance to C. orbiculare (fig 4.3). The treatment
of cucumber plants with ASM appears to predispose some the cultigens to
become more susceptible to D. bryoniae. However, there were some
cultigens that did express a higher degree of resistance to D. bryoniae after
ASM treatment. A second experiment was done to corroborate the findings
described above, the results shown (fig 4.4) clearly demonstrates that there is
a differential expression of SAR induced by ASM against C. orbiculare and D.
bryoniae. Chitinase activity in this experiment shows that the plants treated
with ASM have higher expressions of the enzyme. The differences in

expression of resistance are statistically significant.

105

There is a genotypic difference between cultigens to express natural
resistance against Colletotrichum orbiculare and Dydimella bryioniae.

Experiments performed at the Horticulture farm at Michigan State
University during the 1998 and 1999 growing seasons were structured to
observe the genotype genetic response to C orbiculare and D. bryionieae.

When the leaves were infected with C. orbiculare, all of the cultigens
tested, supported the same number of lesions (1998 trail). However, the
cultigens were different in their ability to restric lesion development after initial
infection (fig 4.6, tables 4.2, 4.3) In this trial, the lesion numbers between
cultigens vary somewhat. However, the trend is still the same. Most cultigens
were not successful in stopping the initial penetration.

When plants were challenged with D bryonieae, the cultigens also
varied in their response (table 4.4). There was a significant variation in lesion
number, but no size among the cultigens. Thus, the response of the plants
against D. bryoniae seems to be more effective in stopping the initial
penetration rather than the spread of the fungus within the tissue (table 4.4).

While most of the cultigens were more successful reducing the size of
the lesions against C. orbiculare, the opposite occurs when challenging the
plant with D. bryonieae (fig 4.7). On the other hand, the cultigens were more
successful in reducing the lesion number against D. bryoniae, as opposed to

C. orbiculare. This variation is easily seen in figure 4.8.)

106

Cucumber Cultigens present little genetic difference in resistance
against Rhyzoctonia solani

During the 1998 and 1999 growing seasons, cucumber fruits were
collected from field experiments performed at the experimental farms of
horticulture and plant Pathology farms at Michigan State University. The
results obtained indicate that there was no genotypic variation between the
genotypes (Data no shown). In fact, the results imply that there is no

significant genetic resistance variation among all of the cultigens.

ASM application does not enhance the resistance of cucumber fruits
against R. solani.

Cucumber fruits were obtained from an experiment in which the plants
were induced to express SAR with ASM. Results indicate SAR, is not
effective against R solani in the fruit. Since, the levels of resistance between
ASM treated plants, and control plants were not statistically different, data
from both samples were combined for analysis of variance comparing the
cucumber genotypes. No getonotypic differences were obtained; all of the
cultigens were infected at the same rate. A random sampling of cucumber
fruits were analyzed and their chitinase activity measured, although there was
not enough data to perform a full statistically analysis, the observations seem
to suggest that the amount of chitinase induction between cucumber fruits

obtained from control and treated plants was different. There was an

107

increase of chitinase activity from cucumber fruits from plants treated with

ASM (data no shown).

Levels of Resistance against a challenge inoculation of C. orbiculare in
cucumber treated with ASM.

In 1999, a pilot experiment was performed with three varieties of
cucumber, SMR-58, MM86 and Straight 8 in order to investigate the effect of
treating plants with ASM. At seven days of the treatment, the leaves were
collected and challenged with C. orbiculare. The data obtained in this
preliminary experiment shows that a treatment with ASM does not provide the
level of protection observed under greenhouse conditions. In fact, there were
no measurable differences between the cultigens as well as between control
plants and induced plants. Two leaf harvests were analyzed seven days after
induction (fig 4.9).

A more comprehensive experiment was performed in 2000. Data
obtained this year suggests that the levels of protection obtained from ASM
applications in the field do provide some resistance to the fungus.
Interestingly, the levels of acquired resistance is more pronounced in the
ability of the plant to stop pathogen spreading rather than the ability to stop
initial penetration. Of the sixteen varieties tested, ten had significant
differences between the ASM treated plants and their controls (table 4.4).

However data obtained from three cultigens, (County fair, H19 and M21),

108

suggests that their control plants had higher levels of resistance than the
ASM treated plants under field conditions.

In terms of lesion number, as observed in the genotypic screenings,
the differences between the control and induced plants did not yield well-
defined differences. However, in this particular experiment four out of sixteen
varieties had lower number of lesions after the induction. Interestingly, one of
those varieties, H-19 was one of the varieties that allowed a larger lesion size
after induction (table 4.5).

As another assessment of the level of SAR induced in the plants,
chitinase activity was measured (table 4.6). These results suggest that the
levels of chitinase activity is significant different between the control and the
ASM treated plants. However, it is important to note that 3 of the 16 varieties
(Anka, Sumpter and PI 231054), had levels of chitinase higher in control
plants than in the treated plants. M-21 did not express an increase of
chitinase and the other 12 did express higher levels of chitinase after ASM

treatment.

Correlation between the chitinase expressed and the resistance
acquired.

Analysis of levels of induced chitinase and resitance show that they are not
significantly correlated. Figure 4.10 shows the distribution of one of those
correlations for field grown plants. In greenhouse conditions the correlations

are also non-significant.

109

DISCUSION

Systemic Acquired Resistance has been researched over the last
years as a potential control method for a broad spectrum of diseases in many
species. A compound [azibenzolar-S-methyl (ASM)] that activates the plant's
natural defense has been released commercially. This activator has been
registered for a variety of cultigens. The manufacturer’s web site
(http:/Mwwsyngentacom) cites bananas, tomatoes and tobacco as main
crops, but cucurbits and other species are also included. Most of the
experiments and the development of the SAR technology have been done
under greenhouse conditions and a few field experiments have been
published, in some cases negative results have been reported. In one study,
the authors state that the treatment with acibenzolar-S-methyl alone was
effective in reduction of the disease severity in many but not all situations
(Huang et al. 2000). This statement suggests that SAR is not effective
against all diseases or under all conditions, which is also demonstrated in this
work. The fact that the levels of SAR induction are mostly effective against
Colletotrichum orbiculare but not, in many cases, to Didymella bryonieae
supports Huang et al.

Several conclusions can be drawn from my results. First, the response
of induced cucumber plants against Colletotrichum orbiculare in greenhouse
conditions seems to be consistent across the collection of cultigens tested.
According to the literature, the phenotype of SAR in cucumber is expressed in

the epidermal layer by decreasing the number of successful penetrations

IIO

(Kovats et al. 1991). In the epidermis, enhanced formation of papillae
beneath appressoria, and increase lignification (Hammerschmidt and Kuc
1982) are correlated with reduced development of infection hyphae. The data
in this work supports this observation, since most cultigens treated with ASM
were more effective in reducing the number of successful penetrations than
reducing the spread of the fungus within the tissue. However, five out of 40
varieties tested did not have a significant reduction of lesion number and two
out of those five were more successful in reducing the fungal growth within
the tissue. When it comes reducing the growth of the fungus within the
tissue, the majority of the cultigens were successful in reducing the fungal
growth. The fact that the majority of the cultigens behaved in a similar way in
terms for their response to SAR induction against Colletotrichum orbiculare
penetration suggests that the differences in terms of the SAR phenotype
expression is generalized across the specie. On the other hand, in reducing
the lesion size, thus reducing the fungal growth within the tissue, cultigens
presented some differences. Not all cultigens were able to decrease the
levels of disease in ASM treated plants.

The increased resistance associated with SAR has been thought to be
linked to the action of PR proteins. The function of some PR proteins is
unknown, while others have been found to be p-glucanases and chitinases
(Van. Loon 1997). Thus, given that B-glucans and chitin are major constituents

of the fungal cell wall, such enzymes have an obvious anti-microbial role.

Ill

Therefore, since no all of the varieties were able to decrease the fungal
growth within the tissue questions the role of these enzymes in resitance.

When the plants were challenged with Dydimella bryoniae, a fungus
with a different infection strategy, a variation in response among cultigens
was observed. The ability of the ASM treatment to enhance susceptibility to
some of the cultigens to infection by Didymella bryoniae seems to suggest
that there is a negative physiological effect caused by ASM in the plants.
However, in some varieties the opposite occurs: treatment with ASM actually
reduced the infection of D. bryoniae. Since some cucumber varieties were
more resistant after ASM induction seems to suggest that there is a genetic
difference in such plants that allows them to increase their defenses against
D. bryonieae. On the other hand, a published experiment reports that SAR is
effective against D. bryoniae (Mucharromah and Kuc 1991) in cucumbers.
However, the inducers in this particular experiment were C. orbiculare and
aqueous solutions (20 or 50 mM) of oxalate, potassium phosphate dibasic
(KzHPO4) or tribasic (K3PO4). This brings up a question, does the type of
induction plays a factor in the resistance against Dydimella bryoniae?

It is also possible that there is a morphological difference among the
cultigens that can explain the difference (eg. stomata or hydathodes). ASM
may cause these natural openings to behave differently by opening longer
time periods, or increasing their opening size (da Rocha personal

communication).

Additionally, the strategy of infection of the fungus and the effect of the
induction in the plant might play a role in this differetial display of resistance
agains D. bryoniae. For instance, C. orbiculare relies on the formation of an
appresorium to penetrate, and the phenotype of SAR has been clearly
demonstrated to be expressed in the epidermal layer (Kovats et al. 1991;
Stein et al. 1992). On the other hand, D. bryoniae has the ability to penetrate
through natural openings such as stomata (da Rocha personal
communication) or by using pectolytic enzymes to help in their initial
penetration (Chilosi and Magro 1998). It also happens that Dydimella
bryonieae produces a polygalacturonase (PG) when infecting cucumber, in
fact, it is the first enzyme produced followed by other cell-wall degrading
enzymes (Chilosi and Magro 1998; Zhang et al. 1999). Because many plant
species produce polygaractuonase-inhibiting proteins (pgip) (De Lorenzo et
al. 2001), the possible role of these proteins in cucumber is also worth
investigating. Interestingly, transcripts of pgip’s accumulate in P. vulgaris
hypocotyls in response to wounding or treatment with salicylic acid
(Bergmann et al. 1994).

SA and JA dependant defense pathways interact, for example the SA
analog INA and ASM can suppress the JA dependant defense gene
expression. (Penacortes et al. 1993; van Wees et al. 2000), possibly through
the inhibition of JA biosynthesis and action (Penacortes et al. 1993). Thus,
over expressing the SA dependant pathway by ASM application could have

detrimental effects on the Jasmonic acid/Ethylene pathway that confers

113

resistance against insects and certain group of pathogens. It is possible that
because of the D. bryoniae mode of infection (pectolictic enzyme production)
the JA dependant pathway might be a better fit for expression of resistance
against this pathogen.

Screening of a cucumber collection under field conditions for
resistance to D. bryoniae and C. orbiculare has provided interesting results.
In the 1998 trial, the levels of basal resistance were clearly expressed in the
ability of the plants to stop fungal growth within the tissue, however there
were no differences between the cultigens in reducing the number of lesions.
In the 1999 trial, the results were consistent with the findings of 1998. The
cucumber cultigens show little variation in reducing the lesion number, but the
ability of the plants to stop fungal growth within the tissue and reduce lesion
size is evident. In contrast, resistance against Dydimella bryoniae was
expressed in reducing the initial penetration of the fungus. This dissimilarity
is very interesting and suggests that the levels of basal resistance against
both pathogens come from different defense responses in the plants. As
mentioned before, the initial penetration by C. orbiculare is via an
appressorium. Therefore, The fact that there is little variation in the number of
lesion across the cultigens clearly establishes the argument that SAR
activates the defense response at the epidermal layer, since in greenhouse
conditions the number of lesions is reduced when plants are expressing SAR.

In the case of D. bryoniae, the lesion number varies significantly across the

114

cultigens, suggesting that there is a genetic control of this part of the host
ressitance.

SAR induction in the field with a biotic inducer such as Colletotrichum
orbiculare in cucumber or by plant growth-promoting rhizobacteria (pgpr) has
been reported in a variety of species including cucumber (Wei et al. 1996);
(Caruso and Kuc 1977; Caruso and Kuc 1977). ASM has also been reported
to increase levels of resistance against a variety of pathogens in a variety of
species (Cole 1999; Pappu et al. 2000; Csinos et al. 2001; Romero et al.
2001) including the post harvest protection of cucurbits against some fungi
(Huang et al. 2000). During the 1999 trials, an experiment was design to
observe the levels of resistance acquired after ASM induction in field
conditions. Cucumber fruits were no protected against R. solani by ASM
application. There has been evidence that the there is an antagonism of the
and ISR pathways (Pieterse et al. 2001). An application of ASM is enough to
induce in the plant the higher levels of SAR, and suppress the JA pathway
and ISR. It could be possible that ISR is actually effective against R. solani
as it is against other pathogens of cucurbit fruits as recently shown (Huang et
al. 2000).

The results of the 1998 trial in which the basal resistance of the
cucumber cultigens was tested against R. solani supports the data obtained
in 1999. Both years, there were no differences among cultigens and as in the
case of the 1999 trial, no differences between control and induced plants

were observed. It has not escaped our attention that the resistance might be

115

expressed in the epidermal layer of the fruit. By causing the small wound in
the epidermis in order to cause infection the fungus might be surpassing the
actual site of resistance.

The data obtained in the field in 1999 suggests that under field
conditions, SAR is not as effective as under greenhouse conditions. In 2000,
the experiment performed spraying the plants with ASM, shows that the levels
of resistance against C. orbiculare seems to be more effective in reducing the
lesion size rather than reducing the number of lesions. As stated before, SAR
phenotype is expressed in the epidermal layers, which reduce the number of
successful penetrations. In this particular experiment and in the 1998 trial, no
dramatic differences were found in reduction of lesion number. Interestingly
using chitinase activity as marker for SAR suggests that SAR activation was
successful. The majority of the cultigens had a higher chitinase activity than
plants from the control plots. However, this marker is not generalized in all
cultivars since some did not express an enhanced resistance, yet their
chitinase activity was higher in ASM treated plants.

The levels of chitinase induction in all experiments, field and
greenhouse did not correlate with the reduction of lesion size at any statistical
level. This finding is rather controversial since the induction of SAR has been
correlated with disease suppression. However, the correlation is more of a
qualitative marker, rather than a quantitative marker. As shown in this work,
the levels of chitinase in induced plants are usually higher than in control

plants yet, the ability of the plant to defend itself seems to be unrelated to the

116

chitinase activity level. So far there is no real evidence that chitinase
induction has a direct involvement in the suppression of disease other, than
serving as a marker for induction of SAR. The basal levels of PR proteins
have been suggested to be used as a marker for resistance (Vleeshouwers et
al. 2000). However, before any recommendations are to be made, a

comprehensive study needs to be done in order to validate these claims.

117

 

Lesion Number
M b 0) co 5
I 4 I l 4
—
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l—l

 

 

 

 

- Control 0 —
[:1 Treatment

 

 

 

Lesion Diameter (mm)
b O)
I 1

 

 

P onner

Nat Pickling
SMR 58
Straight 8

Marketmore 80

Figure 4.1: Lesion size and lesion number analysis of greenhouse
experiment of cucumber plants treated with ASM and challenged with
droplets of 10 pl of a spore suspension of Colletotrichum orbiculare (106
spores/ml). Measurement disease severity was made seven days after
challenge. Basal levels of resistance (black bars) no statistical differences
were observed in lesion number between cultivars. Lesion diameter,
however, pioneer and Nat Pickling were a statistical different group from

SMR58, Straight 8, and MM80.

118

1400

 

 

- Control
II: I ASM

1200 a

1000 4 %

800 a

600 - FL _I_

400 -*

 

 

 

EL

Chitinase Activity

200 ‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pionner Pickling SMR58 Straight 8 MM80

 

 

 

 

 

 

 

 

 

 

 

 

SMR58 Pioneer MM80 Picklirm Straight8
Control
A A A A A
ASM A B B C C
140
g 120 C l 533'”
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Pionner Pickling SMR58 Straight 8 MM80

Figure 4.2: Chitinase activity (A) and peroxidase activity (C) analysis of 5
commercial cultivars of cucumber grown in greenhouse conditions. B is the
statistical analysis of chitinase activity comparing the basal levels and
induced levels of the enzyme.

119

Table 4.1: Greenhouse screening of cucumber cultigens expressing SAR
induced with ASM and challenged with the fungi Colletotrichum orbiculare. The
experiment was performed in three different phases; SMR58 was used as a
reference in each of the experiments. Notice the SMR58 results, Despite the fact
that the numbers vary the statistical analysis outcome was the same. The
symbol * represents the presence of a significant difference between the control
and the plants treated with ASM. A larger number of cultigens expressed higher
resistance by suppressing the number of lesions than reducing the actually

spread of the pathogen within the tissue.

120

Lesion size

Lesion Number

 

 

 

 

121

 

I ” Cultigen Control Treatment 5* Control Treatment 8*
1 1160 Dwarf 2.896 2.179 8.600 4.000 '
2 Ames 4.233 1.965 ‘ 9.000 0.500 *
3 C. hardwickii 3.598 2.714 8.200 1.000 *
4 Calypso 1.918 3.885 4.250 0.400 *
5 Carolina 2.824 2.246 6.000 3.200 ‘
6 Castle 3.1 1 1.779 ‘ 5.800 4.500 *
7 Collet 3.419 1.074 ' 7.400 1.333 ‘
8 Coolgreen 4.177 1 .134 * 9. 500 0.600 ‘
9 County Fair 3.984 1.56 6.250 0.200 "
10 Early Pick 5.348 7.393 ' 7.200 1.750 '
11 . Freemont . 3.549 2.33- ' 8.250 0.400 ‘
12 GY14 3.757 3.534 8.750 5.500 ‘
13 H19 3.082 -7.77E-15 * 9.333 0.000 ‘
14 M21 3.047 ‘ 2.505 7.000 ~ 2.500 ‘_ *
15 Marbel 3.359 2.803 9.250 3.600 '
16 MM 76 4.356 1.816 " 10.000 1.500 ‘
17 Model 6.026 4.194 " 9.200 4.000 *
18 Poinssette 3.296 -1.52E-14 ‘ 10.000 0.000 *
19 Regal 2.834 1.564 6.000 1.750 *

~20 SMR 58 5.471 - 4.234 ' 7.750 5.400 ‘
21 SMR 58 3.53 2.414 * 9.500 2.667 *
22 - SMR58 7.118 2.505 * 10.000 2.500 ‘
23 Straight 8 3.909 3.27 ‘ 9.750 5.750 *
24 Sumpter 3.543 2.753 ‘ 8.750 3.800 '
25 . TC 52 M 5.687 3.78 * 9.667 3.500 ‘
26 W 1983 3.247 2.153 7.500 3.667 *
27 W 5096 2.847 2.853 4.600 1.400 ‘
28 Wautoma 5.933 2.133 * 10.000 . - ‘ 2.000 _ ‘
29 P76 ' ' 2.791 ‘ 1.44 ' 7.750 0.750 *
30 PI 288238 4.832 4.961 5.444 6.400

31 Pl 234517 1.689 , 1.993 7.250 2.333 ‘
32 PI 267942 6.141 3.081 ‘

33 Pl 279468 5.077 4.923 2.000 2.800

34 PI 330628 3.596 2.699 "8.000 4.000 *
35 PI 390240 6.956 5.709 ' 8.000 6.333

36 Pl 390244 5.714 6.63 8.500 6.000

37 PI 390259 6.939 5.329 ' 6.000 5.200

38 PI 391570 3.531 3.152 * 5.600 3.250 '
39 Pl 426170 5.194 3.166 7.000 3.200

40 Pl 432865 5.95 3.513 ' 7.250 2.000 *
41 Pl 432865 4.627 3.326 9.750 1.333 *
42 PI 432867 3.982 0.99 * 8.200 0.250 *
43 PI 432890 3.754 0.546 ' 7.800 0.500 *
43 Pioneer 3.381 1 .67 ' 7.200 1.000 '
44 Pl 466922 4.848 2.772 " 8.600 2.200 '

Figure 4.3: Differential response of plants expressing SAR and challenged with
two different pathogens. Plants were grown under greenhouse conditions and
induced with ASM. Seven days after induction, the second leaves were collected
and brought into the lab. The leaves were cut along the midrib and half of each
leaf was challenged with one pathogeniand the other half with the second
pathogen. Upper panels represent the leaves challenged with D. bryioniae.
Lower panels are leaves challenged with C. orbiculare. Black bars represent
control leaves and gray bars represent the leaves treated with ASM. Only plants

with statistical significance are depicted in the graph.

122

 

 

 

 

 

 

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Figure 4.4: Analysis of lesion size and lesion number of cucumber cultivars

grown under greenhouse conditions and induced to express SAR by ASM

Leaves were collected and challenged against C. orbiculare and D.

bryonieae.

124

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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ASM. The chitinase activity was measured using the agar plate diffusion

method and expressed in diameter of the area of activity in mm.

125

 

10-1

Lesion size (mm)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4.6. Data analysis of the lesion size of the field trail conducted in

White Russian 4

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1998.
Variety Mean Stdev
M-21 4.057 1.151 A
SMR 18 4.082 1.021 A8
Wi 2757 4.086 2.353 A8
Slice 29 4.128 1.622 A8
TMG-1 4.245 1.726 AB
Poinsette 76 4.659 1.552 AB
Sumpter 18 5.378 2.004 B
Wautoma 5.66 1.612 B
M-27 5.737 1.835 B
GY-14 5.897 1.777 B
White Russian 7.61 2.632 C
SMR 58 8.919 2.483 D
Stwt 8 9.262 2.087 D

 

 

 

 

 

Table 4.2: Statistical analysis of the cucumber leaf lesion size data obtained
from the field test in 1998. The leaves were challenged with C. orbiculare and

measured seven days after inoculation.

 

 

 

 

Dydimella bryoniae

 

 

Colletotrichum orbiculare

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 4.7: Statistical analysis of the data from the detached bioassay for

Colletotrichum orbiculare and Dydimella bryionieae (1999 field traial). The

youngest fully expanded leaf were brought into the lab and challenged. The

measurement of the size of the lesions was done 7 days after inoculation

127

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 4.8: Data analysis of the average of the means of the cultivars tested
in a field experiment in 1999. Lesion size and lesion numbers means of the
varieties tested were used to perform this analysis. Note the differences of

variation between lesion size and lesion number for each challenge pathogen.

Field Data 1999
Challenge pathogen Colletotrichum orbiculare

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cultigen Lesion Number Significance Culti gen Lesion size Significance
Average mean (mm)
P=<0.001 P=<0-001
H 19 7.423 B Lemon 7.11 A
M 21 7.526 A8 M 21 3.86 G
Slice 7.538 A8 Slice 4.175 G
WI 2757 7.76 AB Pointssett 76 4.2 G
Pointssett 76 7.808 AB WI 2757 4.665 F G
TMG1 7.808 A8 H 19 4.7 FG
Chipper 8.28 AB TMG 1 4.97 F
Clinton 8.5 AB M-27 5.065
Sumpter 8.6 A Clinton 5.18
M-27 8.846 AB Anka 5.315 EF
CY 14 9.077 A Sumpter 5.36 EF
Marketmore 86 9.208 A CY 14 5.53 EF
Anka 9.231 A Chipper 5.56 DEF
County Fair 9.24 A Marketmore 86 5.76 CDEF
Straight 8 9.462 A Pioneer 5.79 CDEF
SMR 58 9.538 A Producer 5.795 CDEF
Tablegreen 72 9.615 A Early Russian 5.82 CDE
Pioneer 9.667 A Little leaf 5.9 CDE
WI SMR 18 9.708 A County Fair 5.91 CDE
Lemon 9.75 A Straight 8 6.325 800
Little leaf 9.765 A Wautoma 6.435 ABCD
Early Russian 9.852 A Delcrow 6.52 ABCD
Wautoma 9.857 A SMR 58 6.52 ABCD
Delcrow 9.9 A WI SMR 18 6.68 ABCD
Model 9.929 A Model 6.72 ABC
Tablegreen 72 6.885 A8

 

 

 

 

 

 

 

 

Table 4.3. Analysis of cucumber cultivars grown in field conditions. A
detached leaf bioassay was performed by challenging the leaves with a spore

suspension of C. orbiculare.

Field Data 1999
Challenge pathogen D. bryonieae

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cultigen Lesion number Significance Cultigen mean. lesion Significance
average srze
P=<0.001 P=<0.001
Anka 8.7 A WI SMR 18 2.937 E
Little Leaf 8.633 A8 Anka 3.279 E
Delcrow 7.5 ABC Model 3.498 E
Early Russian 7.025 ABC Producer 3.99 DE
WI 2757 6.7 ABC M21 4.025 DE
TMG 1 6 ABCD Pointssett 76 4.134 DE
County Fair 4.5 ABCDE TMG 1 4.151 DE
H19 5.7 ABCDE H19 4.313 D
Lemon 5.1 ABCDE GY14 4.318 D
M27 5.2 ABCDE Sumpter 4.342 D
Pioneer 5 ABCDE Early Russian 4.485 D
Poinssett 76 4.4 ABCDE Straight 8 4.509 D
Producer 6 ABCDE Slice 4.521 D
Slice 4.5 ABCDE WI 2757 4.657 D
Tablegreen 72 5.2 ABCDE Clinton 4.715 D
GY 14 3.45 CDE County Fair 4.73 CD
Model 3.3 CDE Tablegreen 72 4.865 CD
SMR 58 3.325 CDE Wautoma 4.876 CD
Straight 8 3.6 CDE SMR 58 5.103 BCD
Wautoma 3.8 CDE M27 5.189 BCD
WI SMR 18 2.575 CDE Chipper 5.379 BCD
Clinton 2 DE Lemon 5.905 BC
M21 2.5 DE Little leaf 6.094 AB
Sumpter 1.6 DE Pioneer 7.862 A
Chipper 0.4 E
Marketmore 86 1.1 E

 

 

 

 

 

 

 

 

Table 4.4. Analysis of cucumber cultivars grown in field conditions. A
detached leaf bioassay was performed by challenging the leaves with a spore

suspension of D. bryoniae.

I30

 

- Control

. . Lesion Number
Lesron Size II)? Induced

 

 

 

 

Lesion diameter (mm)
0
Lesron Number

 

 

l
l
l
J
l
I

 

 

 

 

 

 

 

 

 

 

 

 

MM 86 Slralght 8 WI SMR58 Stralght 8 WI SMR58

 

 

 

Figure 4.9: Field experiment of three cultivars of cucumber treated with ASM
(20 ppm). Leaves were harvested and challenged with a spore suspension of
Colletotrichum orbiculare (105 spores/ml) Lesions were assessed seven days
after challenge. Upper panels indicate the analysis the control and the
induced plants. Since no statistical differences were found between the
controls and treated plants, the data was combined and analyzed for each
cultivar. Lower panels are the results of the analysis. No statistical

differences were found between the tree cultivars tested.

13]

 

 

 

LesionSizea Lesion Number
Si nificance" Si nificancer
Control ASM‘ gp<005 Control ASM" 9P<0.05
Anka 6.45 6.91 7.33 7.00
Clinton (male) 7.06 5.57 * 7.42 7.63
County fair 4.77 5.72 * 7.55 6.96
GY14 5.50 5.56 5.87 7.83
H19 4.63 5.43 * 8.36 8.31 *
Lemon 6.20 6.55 9.35 8.69
M21 5.68 6.62 * 8.23 6.34
M-27 6.01 6.50
MM 86 8.46 7.38 * 9.73 8.37 *
Pl 231054 5.30 5.74 7.38 8.25
Pioneer 6.68 5.86 * 9.70 8.12
Poinssette 5.38 4.70 * 8.60 7.78 *
Slice 7.92 5.07 * 8.39 7.86
SMR58 7.52 5.94 * 9.23 9.27 *
Straight 8 7.51 6.48 * 9.68 9.86
Sumpter 6.89 6.38 8.83 8.20

 

 

 

 

 

 

a: Lesion size is the diameter of the lesion measured in millimeters.

b: Significance column marked (*), there is a statistical difference between the control and the
ASM treated plants.

c: Plots were treated with a solution of ASM diluted in water at a concentration of 20 ppm.

Table 4.5: Results of a field experiment cucumber cultivars treated with ASM
and control plants. Plants were treated with ASM. Seven days after
treatment, leaves were brought into the lab for analysis. They were
challenged with a spore suspension of C. orbiculare and the lesion

assessment was done seven days after the challenge

 

 

Chitinase Activitya
Significance15
Control ASM P<0.05

Ankal 1.370 1.205 *
Clinton (male) 1.277 1.394 *
County Fair 1.085 1.216 *
GY14 1.165 1.481 *
H19 1.258 1.622 *
Lemon 1 .087 1.252 *

M21 1.310 1.343
M27 1.390 1.467 *
MM86 1.090 1.295 *
Pl 2310541 1.426 1.213 *
Pioneer 1.083 1.218 *
Poinssette 1.201 1.428 *
Slice 1.200 1.381 *
SMR58 1.010 1.168 *
Straight 8 1.097 1.246 *
Sumptel 1.396 1.345 *

 

 

 

a: chitinase activity measured by the agar diffusion plate expressed in
diameter of the area of activity.

b: statistical analysis was made combining the two harvests and the
two replicates per harvest. Each sample was measure in triplicates

Table 4.6: Chitinase levels of cucumber plants from a field experiment at the
Horticultural farm, Michigan State University. Plants were induced to express
SAR with a treatment of ASM and leaves were collected seven days after
induction. Leaves were frozen and ground in liquid nitrogen. The chitinase

activity levels were determined using the agar plate diffusion test.

 

14

 

 

 

 

o
12 -
10 - . o '
8
'0') o '.
5 8 ‘ o
'7) 0.. Q ~
3 ‘. . O . ” .. . C
. . . .. ‘ ‘ . .
5 .——————-rW——1—;{—r
3 .o o '0’. . O
0 ~ ..
4f . 9 .o . o
o . . 9
o
2 I T I I r
1.0 12 1.4 16 18
Chitinase Activity

Figure 4.10 Correlation between the Lesion size and the chitinase activity of

induced plants in the field. No correlation was found.

134

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van Wees, S. C. M., E. A. M. de Swart, J. A. van Pelt, L. C. van Loon and C.
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1032.

138

APPENDIX I

THE TRANSFORMATION OF CUCUMBER EXPRESSING SENSE AND

ANTISENSE CDNA SEQUENCE OF ACIDIC CHITINASE TYPE III.

139

ABSTRACT

The cDNA chitinase sequence of acidic chitinase type III from
cucumber was cloned in the Agrobacterium vector pGA643 in the sense and
antisense directions. Transgenic cucumber plants (Cucumis sativus L.) were
produced by inoculating cotyledon explants with Agrobacterium tumefaciens
bearing a Ti plasmid with NPT II gene for kanamycin resistance. Kanamacin
resistant plantlets, (resulting from self-pollination of transgenic plants) were
evaluated for their tolerance to C. orbiculare under greenhouse conditions.
ASM treated transformed plants did not demonstrate significant differences in
their ability to withstand pathogen attack. Non-ASM treated plants bearing
the antisense construct were more susceptible to pathogen spread within the
tissue after initial penetration. Thus it suggests that the levels of acidic
chitinase are important for the basal levels of resistance prior to induction.
The amount of acidic chitinase activity was suppressed in plants bearing the

sense and antisense constructs possibly due to gene silencing.

I40

INTRODUCTION

Induced resistance has been recognized as an inducible defense
response mechanism to pathogens since the turn of the century (Chester
1933). Recently, studies have been undertaken to quantify the intensity of
the defense response in the induced plant (Ryals et al. 1996). The discovery
that proteins, known as Pathogenesis Related (PR) proteins, were associated
with the induction of resistance (Gianinazzi et al. 1970; van Loon and van
Kammen 1970) suggested that these could be used as markers for SAR. In
cucumber, the acidic type III chitinase has been associated with the induction
of SAR (Lawton et al. 1994; Narusaka et al. 1999) and has been used
extensively as a SAR marker. In addition to serving as a biochemical marker,
chitinase has also been hypothesized to have an active role in the defense of
plants expressing SAR. Among the reasons behind this line of thought is the
fact that acidic chitinase has been found to have antifungal activity in vitro (Ji
and Kuc 1996). It accumulates in the apoplast (Boller and Metraux 1988),
thus placing it in the path of any invading fungi. The time of induction of
acidic chitinase also correlates with the onset of resistance in plants
expressing SAR (Kastner et al. 1998). Using molecular techniques, the
acidic chitinase type III cDNA sequence has been elucidated (Metraux et al.
1989), and its regulation and gene structure has also been reported (Lawton

et al. 1994).

I41

The discovery of DNA delivery and transfer by Agrobacterium
tumefaciens into plants has resulted in the production of several transgenic
lines bearing chitinase of various types. For instance, cucumber plants have
been transformed to express a rice chitinase and reported to have enhanced
resistance to gray mold (Botn'tis cinerea) (Tabei et al. 1998). Raharjo et a/
(Raharjo et al. 1996), has also transformed cucumber with chitinase encoding
genes from petunia, tobacco and bean. They report that, in both growth
chamber studies using whole plants and in vitro inoculations conducted with
detached leaves, no differences in disease development (rate and final levels)
were detected between transgenic and non-transgenic plants (Punja and
Raharjo 1996). These transgenic plants were made to enhance resistance
based on the circumstantial evidence that chitinase might play an active role
in the defense mechanism of the plant. These findings have provided little
evidence concerning the role of chitinase in cucumber plants. Therefore, the
circumstantial evidence in which chitinase is an active player in the defense
mechanism is still questionable.

In this work using molecular techniques, constructs bearing the
antisense and sense strands of the chitinase type III from cucumber regulated
by the cauliflower mosaic virus S35 promoter were used to transform
cucumbers. The transgenic lines were used to assess the role of chitinase in

the defense response against pathogens.

MATERIALS AND METHODS

Plant Material

Cucumber plants cultivar Wisconsin SMR58 were used. The seeds
were washed in distilled water and soaked for 30 min. Seed coats were
carefully removed, following the removal of the coats; the seeds were
surfaced sterilized as described elsewhere (Raharjo et al. 1996). The
uncoated seeds were treated with a 10% solution of commercial bleach in
distilled water for 10 minutes and rinsed 3 times with distilled water. Following
the bleach treatment, the seeds were treated with a solution of 70% ethanol
for 30 seconds and washed with distilled water a minimum of three times.
The seeds were placed on germination media consisting of 4.4 9 MS basal
media (Murashige and Skoog 1962) amended with Sucrose 30 g/L, BAP 2
mg/L, ABA 1mg/L and Gelrite 2.5 g/L for 24 hours in the dark. All chemicals
used were tissue culture grade obtained from Sigma Inc. (St. Louis MO) or
otherwise specified. Explants for co-cultivation were pieces of 1 day old

cotyledons of about 1-2 mmz.

Vector construction

The complete cDNA chitinase type III sequence was isolated via PCR
from cDNA previously prepared by RT-PCR. The primers used to amplify the
genomic DNA were synthesized to contain one of two restriction enzyme sites

Xba I and Ole I. Two primer sets were synthesized and after a PCR reaction

I43

the product contained the restriction sites at 3’ and at the 5’ of the sequence.

 

In order to make the sense construct the primers used were 5’-lT AC ATC GAT]

 

 

AAG AAA GCT crr TAA GCA ATG G-3’ and 5’-[TAT CTA GA]T GGA GAA

 

GAT GAA GTC TCA CTT T-3’. To make the antisense construct, the primers

 

used were 5’-[TAT CTA GAJA AGA AAG CTC TI'T AAG CAA TGG-3’ and 5'-

 

 

fiAc ATC GAT free AGA AGA TGA AGT CTC ACT TT-3’ (fig 5.1). The

 

boxed partial sequence denotes the restriction sites introduced to the PCR
product to allow directional cloning. The primers amplified from position 2 to
position 986 of the cDNA sequence (Metraux et al. 1989). Directional cloning
of the sense and antisense sequences of the cDNA was done into the
Agrobacterium tumefaciens plasmid pGA643 (kindly provided Grumet’s
group). The antisense and sense sequences were cloned in the plasmid to
be constitutively expressed by the S35 promoter of the cauliflower mosaic
virus. Agrobacterium tumefaciens strain LBA4404 was transformed with the

construct and used for explant transformation.

Transformation procedure

The Agrobacterium was grown in LB media with 100 mg L'1 of the
antibiotic kanamycin. A single bacterial colony was inoculated into 15 ml of
liquid medium with 100 mg L'1of kanamycin and incubated at 29 °C on shaker
at 250 rpm for 24 hours. The Agrobacterium culture was diluted 100 fold. A
small amount of the bacterial suspension was placed in a Petri dish and the

partially germinated seedlings were added. The cotyledon pairs were

144

separate and sliced in the Agrobacterium suspension. The edges were cut
and discarded and the remaining piece of cotyledon was sliced into 4-6
pieces and co-cultivated in the Agrobacterium for 10 min. Following
incubation, the explants were blotted onto sterile filter paper and placed on
shoot induction media consisting of 4.4 9 MS basal media (Murashige and
Skoog 1962) amended with Sucrose 30 glL, BAP 2 mg/L, ABA 1mg/L and
Gelrite 2.5 g/L and 0.4 g L‘1 of Timetin and cultured in the dark for 3 days.
After the proper incubation, the explants were retrieved and rinsed with
distilled water and placed in selection media, shoot induction media amended
with kanamycin 25 mg L". The explants were cultured for seven days at
room temperature with amended fluorescent lighting.

When shoots regenerated in the kanamycin amended media, they
were excised and transferred to MS media with kanamycin but without growth
regulators. Roots developed after 2-3 transfers onto the same medium. The
plantlets were transferred to sterile soil and maintained in a growth chamber

at 26 °C and 16-h/day photoperiod.

Confirmation of transformation

Total nucleic acids were extracted from putatively transgenic plants and from
untransformed plants (negative) controls. A small piece of tissue (100 mg)
was frozen in liquid nitrogen and ground using a mortar and pestle in 1 ml
eppedorf tubes. The resulting powdered tissue was suspended in a mixture

of 200 pl TE buffer (10 mM Tris-HCI and 1 mM EDTA, pH 8.0) and 200 pl

145

saturated phenol solution and vortexed vigorously for 1 min. Following
centrifugation, (14,000 g, 5 min room temperature), the isolated DNA was
purified by ethanol precipitation. Precipitated DNA was dissolved in 50 pl of
sterile distilled water, and 1 pl was used for PCR reaction.

Two specific primer sequences for the NPT// (kanamycin resistance
gene) coding region were synthesized, forward primer 5’-TCG GGA GCG
GCG ATA CCG—3’ and reverse primer 5’-GGT TCT CCG GCC GCT TGG—3’.
The isolated DNA in combination with the primers were used to set up a PCR
reaction (25 pl ) consisting of 1x Taq buffer (MgClz-free), 1 mM MgCl2, 200
uM of dNTP’s 0.5 uM of each oligo-nucleotide primer, 1 pl of the isolated
genomic DNA and 2 units of Taq polymerase (lnvitrogen Inc. Carlsbad CA).
The volume was brought up to 25 pl with distilled water. PCR reactions was
carried out in a thermocycler (Perkin Elmer, Boston MA) under the following
conditions: the reaction was incubated at 94 °C for 6 minutes followed by 25
cycles of 94 °C for 45 sec, 62 °C for 1 min and 72 °C for 2 min. The last cycle
was the same with the exception that the DNA extension at 72 °C was carried
out for 10 min. The PCR products were analyzed by electrophoresis on a

1.0% agarose gels and detected using ethidium bromide staining.

Detection of resistance against Colletotrichgm oLbiculare in transgenic
cucumber plants.

Leaves from transformed and control seedlings (untransformed plants) were

collected and brought into the lab. They were out along the midrib and

I46

inoculated with 10 droplets of 10 pl of a spore suspension of C. orbiculare
(106 spores/ml) in water. The half leaves were placed in sterile Petri dishes
underlined with moist filter paper. Seven days after challenge, the number of
lesions allowed per half leaf was counted and the lesion size diameter (mm)

measured.

Chitinase detection

Chitinase activity was determined as follows. A 1% solution of agarose in
potassium phosphate buffer 10 mM (pH 5.0) was prepared with a 1% (v/v) of
glycol chitin, the chitinase substrate (Trudel and Asselin 1989). Thirty ml of
agarose solution were poured in a 15 cm Petri dish. After solidification, 3 mm
diameter plugs were carved using a small cork borer in the agarose. Five pl
of the total protein extract were placed in the wells (3 wells per sample) and
incubated at 37 °C for 4 hours. Following incubation, the reaction plate was
rinsed and flooded for 10 min in 0.5 M solution of Tris-HCI (pH 8.9) with
0.01% (w/v) of calcoflour brightener 28 in order to stop the reaction and stain
the plate. When viewed with UV translumination, the areas of activity
appeared as a well-defined dark area on a fluorescent background. The
diameter of the circular area of activity was measured and used for data
analysis. The acidic chitinase from intra cellular fluids was detected by using
a modified method previously reported (Trudel and Asselin 1989). The
substrate, glycol chitin was added to a non-denatured polyacrilamide gel

electrophoresis instead of using an overlay gel as the authors suggested.

I47

CIaI X bal

Chitinase Sense'cDNA g» Terminator

-<Chitinase Antisense CDNA" * I Terminato>

 

 

 

 

 

 

I

 

 

 

pGA643

Figure 5.1: Vector construction for transformation of cucumber plants. The
chitinase cDNA sequence was amplified using two sets of primers that
introuduced the la! and Xba I restriction sites. The plasmid pGA643 was cut
with the same enzymes that allowed directional cloning to produce the sense

and the antisense constructs.

148

RESULTS

Development of kanamycin resistant plantlets.

Multiple shoots were induced from both explants infected by
Agrobacterium and control explants (uninfected with Agrobacterium) in basal
MS media without kanamycin. When the shoots were exposed to
kanamycinn (25 pg mi") amended media and Timentin (400 pg ml"),
untransformed shoots exhibited slow growth and eventually were bleached.
Shoots that survived this selection were subcultured to a fresh medium of the
same composition where they further developed (Fig 5.2). These kanamycin-
resistant shoots were assumed to have neomycin phototranspherase (NPT
II). The integration of the NPT gene was confirmed via PCR (fig 5.3). All
kanamycin positive plantlets from PCR were acclimatized in growth chamber
and transferred to soil and placed in the greenhouse to be selfed. After
acclimatization and growth under greenhouse conditions, there was no
difference between transgenic and non-transgenic cucumber plants from
viewpoints of growth and morphological characters. The plants were selfed

and seeds obtained and used for resistance experiments.

Resistance to Colletotrichum orbiculare.
Sense and antisense seedlings (T1 generation) were screened for the
NPTII gene, which confirmed the insertion of the transformation construct.

Transformed seedlings were transplanted into sterile soil and grown under

149

greenhouse conditions. The cucumber plants were induced to express SAR '
when the plants' first true leaf (leaf 1) was fully expanded and the leaf above
(leaf 2) one-third to one-half expanded. The induction was achieved by
spraying the plants with the benzothiadiazole derivative, GGA-245704 (ASM)
(Friedrich et al. 1996) at a rate of 20 ppm of active ingredient in water.
Leaves were harvested and challenged with a spore suspension of C.
orbiculare seven days after induction. Statistical analysis of the data
collected revealed that there were no significant changes in terms of lesion
size or number between the ASM treated sense, antisense and control plants.
On the other hand, when non ASM treated plants (controls, sense and
antisense) were compared, the data obtained shows significant differences
(fig 5.4). Statistical analysis shows that the number of lesions did not vary
between the transgenic and control plants. However, the development of the
fungi, determined by the lesion size, varied between constructs. Transgenic
plants expressing the antisense construct allowed a larger lesion size when
compared with the sense and the control plants. Interestingly, the control
plants and the plants expressing the sense construct were not significantly

different in terms of lesion development.
The levels of chitinase activity in transgenic plants.

The levels of the acidic type III chitinase activity in transgenic plants were
monitored by gel electrophoresis with protein samples extracted from

intracellular fluids. The levels of acidic chitinase activity were similar in sense

150

and antisense plants, but the levels were higher in control plants (Fig 5.5).
Additionally, the levels of total chitinase activity measured by the agarose gel
diffusion activity assay presented no differences at any level between the

transgenic plants and the control plants.

 

Figure 5.2: Transgenic shoots and transgenic plants regenerated from cotyledon
explants. Left picture, Petri dish containing regenerated shoots in kanamycin
amended media. T: kanamycin resistant shoot, AR: adventicious root, U:
untransformed, non kanamycin resistant shoot. Right picture, kanamycin

resistant shoots in elongation media.

152

123456789NCEL

 

Figure 5.3: PCR analysis of regenerated shoots after Agrobacterium co-
cultivation. Lanes from 1—9 DNA samples from different adventitious shoots, L:
DNA ladder, N: non transformed plant, C: vector containing the kanamycin
resistant gene, E is negative control, no template. Plants 2, 4 and 7 were kept

and used further.

153

 

10

 

 

 

 

 

 

 

 

 

 

 

 

g T
'6 6 y T
a)
_I
0.—
O
E
2 4‘ A B B
.9
D
2 ..
0 I I I
Antisense Sense Control

Construct

Figure 5.4: Resistance to Colletotrichum orbiculare of non ASM treated
transgenic plants bearing the sense and the antisense construct of chitinase
type III under the control of the S35 promoter. Three groups of at least 15
plants for each treatment were used data analysis. Control plants are non-
transforrned plants. The differences in the mean values among the treatment
groups are greater than would be expected by chance; there is a statistically

significant difference (P = <0.001).

154

160

 

140 4

 

120 4

100 J

80- T I

 

 

60-

Average Intensity

40-

20~

 

 

 

 

 

 

 

 

0 I I I
Antisense Sense Control

 

Figure 5.5: Acidic chitinase of transgenic and control plants. Three different
extractions were made from intracellular fluids of transgenic seedlings. Each
extraction was loaded the combination of at least 5 transgenic seedlings. 30
pg of total protein was loaded in an acrylamide gel electrophoresis in non-
denatured conditions. The chitinase was detected using the method of Trudel
and Asselin. The bands generated were calculated using a software package
(Quantity one, Bio Rad Inc). The average intensity of the bands was
calculated. No significant changes were observed between the transgenic

plants, but they did had less activity as compared to the control plants.

155

DISCUSSION

This work demonstrates the introduction of the cucumber chitinase
type III cDNA sequence was introduced in the sense and antisense directions
in cucumber. Our transformation efficiency was not higher than 5% other
reports of transformation of cucumber plants have also been successful, with
a low transformation efficiency (Broglie and Broglie 1993; Raharjo et al. 1996;
Tabei et al. 1998). In the regeneration system employed in this experiment,
one-day-old cotyledons used as explants proved novel and efficient as
opposed to current cucumber transformation methods. This method, recently
developed (Tabei et al. 1998), and adapted by the Grumet (personal
communication) will contribute to the more efficient production of transgenic
cucumbers.

When treating the transgenic plants with ASM, the levels of expression
of resistance in plants (sense, antisense and control plants) were to the same
degree. In other words, there were no statistically measurable differences in
terms of lesion development or number of lesions allowed. In the systemic
acquired resistance phenotype, the levels of chitinase activity rises with the
process of induction and with increased levels of resistance. The
observations shown here demonstrate that the levels of lesion development
and lesion size are not affected by the construct present in the plants. This is
rather interesting since it has been suggested that the levels of resistance
due to SAR are tightly correlated to the increase of the PR proteins in

particular, chitinase and B-glucanase. The fact that the level of resistance

156

was unchanged in the transgenic plants suggests that chitinase suppression
or overexpression does not affect the normal levels of resistance in control
plants after SAR induction. This fact, contests the argument that chitinase is
an active player in the defense of plants due to SAR.

An interesting finding is the basal levels of resistance in transgenic and
control plants. The non-induced plants expressing the antisense construct
allowed for a larger lesion size when compared to controls and plants
expressing the sense construct (fig 5.4). No differences in resistance were
found between the control plants and the plants expressing the sense
construct. Interestingly, no effect in disease development caused by
Colletotrichum orbiculare was found in transgenic cucumbers expressing
petunia and tobacco chitinases (Punja and Raharjo 1996). However the
chitinase expressed in this experiment was of basic nature. There is only one
report of transgenic plants expressing an acidic chitinase from Lycopersicum
chilense (Tabaeizadeh et al. 1999). The data obtained from transformed
plants (tomato) in this report suggestted that the levels of resistance in
transgenic plants demonstrated improved resistance to Verticr'llum dahliae
race 2. However, these experiments only suggest that acidic chitinase could
potentially be able to increase the level of resistance in tomato, in cucumber
however it could be different. Additionally antisense experiments with p-
glucanase , another PR protein, did not exhibit increased susceptibility to C.
nicotianae infection. These results suggest that expression of the beta—1,3-

glucanase isoforrn blocked by antisense transformation is not necessary for

157

'housekeeping' functions of N. sylvestris nor defense against the fungal
pathogen tested. Chitinase, and glucanase, have been topics of heated
debate over their role in the plant. Even with the antisense insertion of
glucanase, it failed to become “less resistant” than control plants. The plants
did not exhibit increased susceptibility to Cercospora nicotianae infection
(Beffa and Meins 1996). These results suggest that expression of the beta-
1,3-glucanase isoforrn blocked by antisense transformation is not necessary
for 'housekeeping' functions of N. sylvestris nor defense against the fungal
pathogen tested.

The fact that only in non-induced plants, the antisense construct was
able to make a significant reduction in the levels of disease by allowing the
fungi to spread further into the tissue suggest that constitutive levels of
chitinase could potentially be of importance for housekeeping of the
resistance functions in cucumber. This suggests that the constitutive levels of
chitinase are important for the basal resistance in plants, and the over
expression of the gene does not enhance the ability of the plants to withstand
pathogen attack.

The difference in the level of total chitinase activity among the
transgenic and control plants was not significant. However, the acidic type III
chitinase expression varied between constructs. Interestingly, the levels of
acidic chitinase expressed in the transgenic plants were lower than in control
plants. Experiments have shown that transgenes introduced into plants can

interect with homologus host genes resulting in the inactivation of both genes

158

(Jorgensen 1991; Matzke and Matzke 1995). A phenomenon called co-
supression or plant gene silencing. As reported, some plants transformed
with chitinase genes failed to overexpress (Tabaeizadeh et al. 1999), and in
some cases it reduces the total chitinase expression (Neuhaus et al. 1991).
In this study, the lack of over expression of chitinase activity in plants
expressing the sense construct might explain the fact that no detectable
differences were found in the resistance against C. orbiculare. On the other
hand, despite the fact that no significant differences were obtained in acidic
chitinase expression between the sense and the antisense transgenes, the
levels of non-transgenic plants were considerably higher. The transgenic
cucumber plants expressing the antisense construct, were successful at
reducing the translation of the chitinase transcripts. This fact, could be
responsible for the increase of susceptibility to the spread of the fungus within
the tissue.

The most widely used approach to enhance resistance in plants
against pathogens, has been the overexpression of hydrolytic enzymes,
mainly glucanases and chitinases which belong to the group of the PR
proteins (Neuhaus 1999; Punja 2001). Following expression of several
chitinases in a range of transgenic plant species, has proven that the levels of
chitinase induction does not necessarily is translated to an enhanced
resistance. This might indicate that differences exist in sensitivity of fungi to
chitinase. But most importantly, the levels of induction in most experiments

do not correlate with the levels of resistance acquired. These results

159

questions the effort and the potential to express hydrolytic enzymes in plants
to enhace resistance. Additionally, it has been also suggested that
pathogen-inducble PR proteins do not play a major role in the resistance (Van
Loon 1997). Thus the idea of generating transgenic plants with PR proteins to

enhance the resistance against pathogens could be challenged.

160

BIBLIOGRAPHY

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1,3-glucanases investigated by antisense transformation - a review.
Gene 179: 97-103.

Boller, T. and J. P. Metraux (1988). Extracellular localization of chitinase in
cucumber. Physiological and Molecular Plant Pathology 33: 11-16.

Broglie, R. and K. Broglie (1993). Chitinase gene-expression in transgenic
plants - a molecular approach to understanding plant defense
responses. Philosophical Transactions of the Royal Society of London
Series B-Biological Sciences 342: 265-270.

Chester, Keneth (1933). The problem of acquired physiological immunity in
plants. Quarterly Review of Biology 8: 275-324.

Friedrich, L., K. Lawton, W. Ruess, P. Masner, N. Specker, M. G. Rella, B.
Meier, S. Dincher, T. Staub, S. Uknes, J. P. Metraux, H. Kessmann
and J. Ryals (1996). A benzothiadiazole derivative induces systemic
acquired resistance in tobacco. Plant Journal 10: 61-70.

Gianinazzi, S, C. Martin and JC Vallee (1970). Hypersensibilite aux virus,
temperatue et proteines solubles chez le Nicotiana xhanti n.C.
Apparition de nouvelles machomolecules losr de la represion de la
synthase virale. C. R. Acad. Sci. Paris 270: 2383-2386.

Ji, C. and J. Kuc (1996). Antifungal activity of cucumber beta-1,3-glucanase
and chitinase. Physiological and Molecular Plant Pathology 49: 257-
265.

Jorgensen, R. (1991). Beyond antisense - how do transgenes interact with
homologous plant genes. Trends in Biotechnology 9: 266-267.

Kastner, B., R. Tenhaken and H. Kauss (1998). Chitinase in cucumber
hypocotyls is induced by germinating fungal spores and by fungal
elicitor in synergism with inducers of acquired resistance. Plant Journal
13: 447-454.

Lawton, K. A., J. Beck, S. Potter, E. Ward and J. Ryals (1994). Regulation of
cucumber class-iii chitinase gene-expression. Molecular Plant-Microbe
Interactions 7: 48-57.

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Matzke, A. J. M. and M. A. Matzke (1995). Trans-inactivation of homologous
sequences in Nicotiana-tabacum. Gene SilencingLin Higher Plants and
Related Phenomena in Other Eukagrotes Salzburg,Austria 197: 1-14.

Metraux, J. P., W. Burkhart, M. Moyer, S. Dincher, W. Middlesteadt, S.
Williams, G. Payne, M. Carnes and J. Ryals (1989). Isolation of a
complementary-dna encoding a chitinase with structural homology to a
bifunctional lysozyme chitinase. Proceedings of the National Academy
of Sciences of the United States of America 86: 896-900.

Murashige, T and F Skoog (1962). A revised medium for rapid growth and
biossays with tobacco tissue culture. Physiologia Plantarum 15: 473-
497.

Narusaka, Y., M. Narusaka, T. Horio and H. Ishii (1999). Comparison of local
and systemic induction of acquired disease resistance in cucumber
plants treated with benzothiadiazoles or salicylic acid. Plant an4d Cell
Physiology 40: 388-395.

Neuhaus, J. M., P. Ahlgoy, U. Hinz, S. Flores and F. Meins (1991). High-level
expression of a tobacco chitinase gene in Nicotiana sylvestris
susceptibility of transgenic plants to cercospora- nicotianae infection.
Plant Molecular Biology 16: 141-151.

Neuhaus, Jean-Marc (1999). Plant chitinases (pr-3, pr-4, pr-8, pr-11).
Pathogenesis-related proteins in plants. Swapan K Datta. Boca Raton,
Florida, CRC Press: 77-105.

Punja, 2. K. (2001). Genetic engineering of plants to enhance resistance to
fungal pathogens - a review of progress and future prospects.
Canadian Journal of Plant Pathology-Revue Canadienne De
Phytopathologie 23: 216-235.

Punja, Z. K. and S. H. T. Raharjo (1996). Response of transgenic cucumber
and carrot plants expressing different chitinase enzymes to inoculation
with fungal pathogens. Plant Disease 80: 999-1005.

Raharjo, S. H. T., M. O. Hernandez, Y. Y. Zhang and 2. K. Punja (1996).
Transformation of pickling cucumber with chitinase-encoding genes
using Agrobacterium tumefaciens. Plant Cell Reports 15: 591-596.

Ryals, J. A., U. H. Neuenschwander, M. G. Willits, A. Molina, H. Y. Steiner
and M. D. Hunt (1996). Systemic acquired resistance. Plant Cell 8:
1809-1819.

162

Tabaeizadeh, Z., Z. Agharbaoui, H. Harrak and V. Poysa (1999). Transgenic
tomato plants expressing a Lycopersicon chilense chitinase gene
demonstrate improved resistance to Verticillium dahliae race 2. Plant
Cell Reports 19: 197-202.

Tabei, Y., S. Kitade, Y. Nishizawa, N. Kikuchi, T. Kayano, T. Hibi and K.
Akutsu (1998). Transgenic cucumber plants harboring a rice chitinase
gene exhibit enhanced resistance to gray mold (Botrytis cinerea). Plant
Cell Reports 17: 159-164.

Trudel, J. and A. Asselin (1989). Detection of chitinase activity after
polyacrylamide-gel electrophoresis. Analytical Biochemistry 178: 362-
366.

Van Loon, L. C. (1997). Induced resistance in plants and the role of
pathogenesis- related proteins. European Journal of Plant Pathology
103: 753-765.

van Loon, L. C. and A van Kammen (1970). Polyacrylamide disk

electrophoresis of the soluble leaf proteins from Nicotiana tabacum
var. "samsum" and "samsum nn". Virology 40: 199-211.

163

APPENDIX II
ISOLATION OF A CDNA FRAGMENT OF THE NPR1 GENE FROM

CUCUMBER PLANTS

164

ABSTRACT

Systemic Acquired Resistance and Induced Systemic Resistance are
regulated by NPR1 gene. The present work reports the isolation and partial
sequence of a cDNA fragment of the NPR1 of cucumber plants. cDNA was
prepared via reverse transcription. Degenerate primers were designed from
the alignment of NPR1 cDNA sequences from rice, Arabidopsis and tobacco.
The PCR product obtained was cloned and sequenced, results show that the
NPR1 cDNA partial sequence from cucumber is highly homologous to the
reported Arabidopsis sequence. Its translated sequence also shows ankyrin

repeats a hallmark of the NPR1 genes reported in the literature.

165

INTRODUCTION

Plants are in constant contact with pathogens. Thus, they have
evolved complex mechanisms of defense. These include, among others, a
mechanism that is activated by exposure to pathogens. Systemic Acquired
Resistance (SAR) is a state of enhanced resistance to pathogens, and it is
correlated to the deployment of a battery of a several Pathogenesis Related
(PR) proteins (Van Loon 1997; Metraux 2001). From the whole battery of
genes deployed during the SAR response, there has not been one conclusive
report of showing a single gene to be essential for SAR. This suggests that
the coordination of deployment of these genes might be more important than
a single gene deployed.

Regulation of the Systemic Acquired Resistance has been shown to be
dependant on the NPR1 gene. The Arabidopsis NPR1 gene controls the
onset of systemic acquired resistance (Cao et al. 1994). Using mutagenesis,
a mutant, npr1 (nonexpressor of PR genes), was discovered to be non
responsive the SA application and other SAR-inducing treatments (Cao et al.
1994). Further characterization showed that the mutant displays little
expression of pathogenesis-related (PR) genes and exhibiting increased
susceptibility to infection (van Loon et al. 1998). Characterization of the
NPR1 gene has shown that it has ankyrin repeats (Cao et al. 1997),
suggesting that these repeats are important for NPR1 function. Other key
experiments with the NPR1 gene include the transformation of the cloned

wild-type NPR1 gene into npr1 mutants. The transgenic gene

166

complementation not only restored the mutation but also rendered the
transgenic plants more resistant to infection by P. syringae in the absence of
SAR induction (Cao et al. 1997; Cao et al. 1998). Another complementation
experiment also showed similar results in addition to reporting that
overexpression of NPR1 gene prones the plants to be more responsive to SA
or a SA-dependent signal (Friedrich et al. 2001).

The NPR1 has also been shown to regulate Induced Systemic
Resistance (ISR). This resistance pathway relies on the activation of the
Jasmonic acid pathway (van Loon et al. 1998). Because rhizobacteria-
mediated ISR was found to be independent of SA and not associated with
PRs (Pieterse et al. 1998), it was expected that ISR would still be expressed
in this mutant. However, the npr1 mutant of Arabidopsis did not express ISR
mediated by the rhyzobacterium WCS417 (Pieterse et al. 1998). This implies
that NPR1 functions beyond the expression of PR-genes and is required for
both pathogen and rhizobacteria-mediated systemic induced resistance. This
can possibly explain why SAR and ISR are phenotypically similar. In both, the
defensive capacity of the plant against a broad spectrum of pathogens is
enhanced after an initial induction. Yet, PRs are induced concomitant with
SAR expresion, whereas activation of PR genes is not part of the pathway
leading to ISR in Arabidopsis. Apparently, NPR1 differentially regulates
defense responses mediated by different signaling pathways. Despite the
source of induction, the NPR1 gene seems to potentiate the defense

mechanisms typical of SAR and ISR (van Loon et al. 1998).

167

Cucumber plants have been used extensively for the study of SAR,
and their importance as model has been increasingly noted. The SAR
mechanism was initially characterized using cucumber plants, and to our
knowledge no NPR1 sequence has been isolated from cucumber. This work

describes the isolation of the NPR1 cDNA sequence from cucumber.

168

MATERIALS AND METHODS

Cucumber cDNA:

Cucumber plants var. Wisconsin SMR 58 were grown in a growth
chamber. The cucumber plants were induced to express SAR with the
benzothiadiazole derivative, acibenzolar-S-methyl (CGA—245704, ASM)
(Friedrich et al. 1996) at a rate of 20 ppm of active ingredient in water. Total
RNA was isolated from leaves as previously described. mRNA was isolated
using mRNA isolation kit using paramagnetic beads (Promega Inc). cDNA
was made from mRNA using reverse transcriptase kit (Roche Inc). The

cDNA obtained was used for downstream applications.

Primer selection:

Alignment of the published amino acid sequences in the database of
NPR1 was performed (fig 6.1). Degenerate primers were designed from the
conserved regions of the amino acid alignment of the sequences of
Arabidopsis, rice and tobacco NPR1. The primer combination AAR GCI YTN
GAY WSN GAY GAY (forward), and NGC NAR NBC NAC NCK RTT YTC
(reverse) was the one primer pair that amplified a distinct band from
cucumber cDNA. The primer combination corresponds to the amino acid
sequences KALDSDD and ENRVALA which are present in the three NPR1

amino acid sequences from the species above mentioned.

169

PCR optimization:

The optimization of the PCR reaction was performed in a Perkin Elmer
Thermocycler machine. The successful amplification of the NPR1 fragment
of cucumber cDNA was done using the following parameters. The PCR
reaction (25 pl ) consisting of 1x Taq buffer (MgClz-free), 1 mM MgClz, 200
uM of dNTP’s 0.5 uM of each oligo—nucleotide primer, 1 pl of a 1:100 dilution
of the cDNA made and 2 units of High fidelity Taq polymerase (lnvitrogen Inc.
Carlsbad CA). The volume was brought up to 25 pl with distilled water. PCR
reactions were carried out in a thermocycler (Perkin Elmer Inc.) under the
following conditions: The reaction was incubated at 94 °C for 6 minutes
followed by 30 cycles of 94 °C for 45 sec, 55 °C for 1 min and 72 °C for 2 min.
After each cycle the annealing temperature was lowered by 0.5 °C. The last
cycle was the same with the exception that the DNA extension at 72 °C was
carried out for 10 min. The PCR products were analyzed by electrophoresis

on a 1.0% agarose gels and detected using ethidium bromide staining.

Cloning of the PCR product:

After optimization of the PCR reaction, a product was obtained (fig
6.2). The bands were extracted from the gel using a PCR cleaning kit
(Promega Inc), the end product was cloned into a plasmid (Topo II) which
allows the cloning of PCR products with an adenine overhang (lnvitrogen Inc).
The ligation reaction was done according to the manufacturers

recommendations. The resulting ligation product was used for the

170

transformation of E. co/i competent cells as previously described (Sambrook
et al. 1998). The cells were plated on a kanamycin amended media (50
pg/ml) and incubated at 37 °C overnight. The transformed cells formed white
colonies due to the disruption of the IacZ gene by the insert. Plasmid DNA

was obtained quantified and sequenced.

I71

Arabidopsis
Tobacco
Rica

Arabidopsis
Tobacco
Rice

Arabidopsis
Tobacco
Rice

Arabidopsis
Tobacco
Rice

Arabidopsis
Tobacco
Rice

Arabidopsis
Tobacco
Rice

Arabidopsis
Tobacco
Rice

Arabidopsis
Tobacco
Rice

Arabidopsis
Tobacco
Rice

Arabidopsis
Tobacco
Rice

Arabidopsis
Tobacco
Rice

1 TIDGE

  

  

 

464 LEPDRLTGT
463 IG-KKMAN
468 GA-NPPPE
524 ACGE
522 I.YMG
525 VSLG
584 'FSHRRR 593
581 PFRK-- 568
580 ------ 582

Figure 6.1: Alignment of Amino acid sequences of the NPR1 gene. Arrows

indicate the position of the degenerate primers used in this work.

sequences used were accession

BAB12719.

The

numbers AAM65726, AAM62410,

172

RESULTS

After PCR optimization a faint, yet well-defined band was observed in
the PCR reaction (fig 6.2). This band was gel purified and cloned into the
Topo 2 plasmid (lnvitrogen Inc). A series of clones that contained the right
size fragment were sent for sequencing. The obtained sequences were
blasted for homology to the published database. A group of clones was
observed to have very strong homology to the Arabidospsis NPR1 amino acid
sequence (fig 6.3) and when comparing the nucleic acid sequence the
homology was even higher (fig 6.4)

A BLAST search of gene bank showed that the NPR1 gene cDNA
sequence from cucumber also encodes a sequence which highly homologous
to ankryn (fig 6.5) The ankyrin repeat concesus has been identified in diverse
group or proteins involved in cell structure transcription and regulation, cell
differentiation and enzymatic and toxic activities (Cao et al. 1997) In short, the
amino acid sequence deduced from the partial cDNA sequence from
cucumber shares a very high similarity with the NPR1 cDNA sequence from

Arabidopsis.

173

 

Figure 6.2. Agarose gel electrophoresis of a PCR product using degenerate
primers to amplify the NPR1 gene of cucumber. Three distinct bands (A, B
and C) were purified from the gel and cloned. Band A encoded the NPR1
partial sequence of cucumber. Lanes (1-11) represent the PCR product of
several PCR reactions under different parameters. Bands 6 and 7 showed

distinct bands that could be isolated and cloned.

174

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

MDTTIDGFADSYEISSTSFVATDNTDSSIVYLAAEQVLTGPDVSALQLLSNSFESVFDSP

L0 81
TL RS

  

AFSED
KRHRR

Ziggfliééfi

MKGTCEF
RE-

 

TSSTS
RLLPH

GG IRK
PV TV

RRR 593

IX-

 

 

CE — GE
SRAV S JRT

308

300
29

360
89

420
149

480
193

538
252

Figure 6.3: Clustal alignment of the Cucumber NPR1 translated sequence

and the Arabidopsis NPR1 sequence (Accesion # AAM65726)

175

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsi-
Cucumber

Arabidopeia
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsi-
Cucumber

Arabidopeie
Cucumber

Arabidopeia
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

Arabidopsis
Cucumber

TACCTAAAGTAAAGAAACATGTCTCGAATGTACAT‘EC‘EG_
----------------------------------- 'I‘ T

116-:me}. if“? 1p.-.
..-._.....- z».

2‘!

l

 

AAGAGGTCTAACCGTAAACTCTCTCATCGT

Figure 6.4: Alignment of the NPR1 cDNA fragment of cucumber and

Arabidopsis (Acc # U76707). Note the high homology between both

sequences.

176

NPRICucumber:

ANK3:

NPRlCucumber:
.ANKB:
NPRlCucumber:

ANK3:

NPRICucumber:

ANK3:

Figure 6. 5:

154

740

172

614

NHRNPRGYTVLHVAAMRKEPOLILSLLEKGAS SEATLVGRTALMIAHQ 300
N + GYT LH AA + +1 LL+ AS +E T+ G TAL IA++
NAKTKNGYTALHQAAQQGHTHIINVLLQNNASPNELTVNGNTALAIARR 788

DDIELVKLLLK ----- EDHTNLDDACALHFAVAYCNXXXKKXXXXXXIXXXNHRNPRGYT 180
D + V+LLL+ +D TN D ALH A A+C N + G+T
DHLNCVQLLLQHNVPVDDVTN-DYLTALHVA-AHCGHYKVAKVLLDKKASPNAKALNGFT 385

VLHVAAMRKEPQLILSLLEKGASASEATLVGRTALMIA 294
LH+A + +++ LL+ GAS T G T + +A
PLHIACKKNRIRVMELLLHHGASIQAVTESGLTPIHVA 423

GYTVLHVAAHRHEPQLILSLLEHGASASEATLVGRTALHIA7Q 300
GYT LH+AA + + + SLLE GA A+ T G ++ +A Q
GYTPLHIAAKKNQMDIATSLLEYGADANAVTRQGIASVHLAAQ 656

Sequence similarities of NPR1 amino acid sequence with mouse

ankyrin 3 (ANK3). Three regions produced the highest scoring pairs. The

sequence similarities are 42% 32% and 48%. The Identical and similar (+)

amino acids are highlighted in bold letters.

I77

DISCUSSION

In the search for novel methods of disease control, Systemic Acquired
Resistance (SAR) is a potential candidate. However, the search of genes that
would serve as markers and as targets for disease resistance improvement
has been difficult. The NPR1 is important because it regulates the
deployment of the battery of SAR and ISR genes. In Arabidopsis, the NPR1
has been characterized and has served to find other genes in relationship to
SAR. For example, The npr1 Arabidopsis mutant could be rescued by
another mutation, sni1 (Li et al. 1999). The npr1-sni1 double mutant was then
able to induce PR expression when treated with INA as inducer (Li et al.
1999). It has been proposed that the NPR1 gene is a positive regulator of
SAR, which is repressed in wild type plants by SNI1 gene. It has been
suggested that the role of NPR1 is to remove the SN|1 repression (Li et al.
1999). If this is correct and the fact that the NPR1 basal levels remain the
same in induced and control plants, these levels might be an clue for the level
of SAR protection among cultivars. However, the fact that the npr1-sni1
mutant is able to respond to exogenous applications of SA and INA, it also
opens the possibility of a pathway independent of NPR1 (Li et al. 1999).
Nevertheless, the study of the NPR1 basal levels and its correlation with SAR
expression ability is worthwhile aiming to provide a marker for SAR
potentiation since SAR levels positively correlate with pathogen resistance.
This hypothesis is supported by the overexpression of the NIM1 (NPR1) gene

in Arabidopsis. Transgenic lines expressing the NPR1 gene exhibited

178

heightened responsiveness to SAR-inducing compounds. Activation of SAR
in transgenic plants was detected when treated with SA concentrations that
would not activate SAR in wild plants (Friedrich et al. 2001) . In addition,
NPR1 has also been found to differentially interact with members of the
TGA/OBF family which is implicated in the activation of SA-responsive genes,
including PR-1 (Zhou et al. 2000). Increased levels of PR-1 activation were
found in Arabidopsis plants overexpressing the NPR1 gene (Friedrich et al.
2001). In addition, npr1 mutants presented an abnormal callose response
and hypersusceptibility to Peronospera parasitica thus compromising
resistance (Donofrio and Delaney 2001). The phenotype was rescued by
exogenous applications of SA, but it seems dependant on the NPR1 pathway.
It seems then, that the NPR1 gene does play an important role in the
expression and strength of the SAR response.

PR protein basal expression was found to be correlated to non-
specific resistance ins Solarium species to Phytophtora infestans and it has
been suggested that these levels might be used as biochemical markers for
germplasm selection for resistance (Vleeshouwers et al. 2000). This finding
not only provides partial evidence of the importance of measuring basal levels
of enzyme activity, but also encourages further studies at the level of mRNA
via RT-PCR and northern analysis.

In short, the cucumber NPR1 partial sequence isolated in this work is
almost identical to the cDNA NPR1 sequence of Arabidopsis. Because NPR1

overexpression can result in reduced pathogen growth on the plants with the

17‘)

use of less fungicide (Friedrich et al. 2001), not only will reduce increase
pathogen resistance to pesticides by exposing the pathogen to less of the
active ingredient. Thus the screening for plants with higher NPR1 expression

might be a strategy to look for the “magic” biochemical marker for SAR.

180

BIBLIOGRAPHY

Cao, H., S. A. Bowling, A. S. Gordon and X. N. Dong (1994). Characterization
of an arabidopsis mutant that is nonresponsive to inducers of systemic
acquired-resistance. Plant Cell 6: 1583-1592.

Cao, H., J. Glazebrook, J. D. Clarke, S. Volko and X. N. Dong (1997). The
arabidopsis npr1 gene that controls systemic acquired resistance
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Cao, H., X. Li and X. N. Dong (1998). Generation of broad-spectrum disease
resistance by overexpression of an essential regulatory gene in
systemic acquired resistance. Proceedipqs of the National Academy of
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Donofrio, N. M. and T. P. Delaney (2001). Abnormal callose response
phenotype and hypersusceptibility to Peronospora parasitica in
defense-compromised Arabidopsis nim1- 1 and salicylate hydroxylase-
expressing plants. Molecular Plant-Microbe Interactions 14: 439-450.

Friedrich, L., K. Lawton, R. Dietrich, M. Willits, R. Cade and J. Ryals (2001).
Nim1 overexpression in arabidopsis potentiates plant disease
resistance and results in enhanced effectiveness of fungicides.
Molecular Plant-Microbe Interactions 14: 1114-1124.

 

Friedrich, L., K. Lawton, W. Ruess, P. Masner, N. Specker, M. G. Rella, B.
Meier, S. Dincher, T. Staub, S. Uknes, J. P. Metraux, H. Kessmann
and J. Ryals (1996). A benzothiadiazole derivative induces systemic
acquired resistance in tobacco. Plant Journal 10: 61-70.

Li, X., Y. L. Zhang, J. D. Clarke, Y. Li and X. N. Dong (1999). Identification
and cloning of a negative regulator of systemic acquired resistance,
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Metraux, J. P. (2001). Systemic acquired resistance and salicylic acid:
Current state of knowledge. European Journal of Plant Pathology 107:
13-18.

Pieterse, C. M. J., S. C. M. van Wees, J. A. van Pelt, M. Knoester, R. Laan,
N. Gerrits, P. J. Weisbeek and L. C. van Loon (1998). A novel
signaling pathway controlling induced systemic resistance in
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Sambrook, J., E.F. Fritsch and T. Maniatis (1998). Molecular cloning: A
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Van Loon, L. C. (1997). Induced resistance in plants and the role of
pathogenesis- related proteins. European Journal of Plant Pathology
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van Loon, L. C., Pahm Bakker and C. M. J. Pieterse (1998). Systemic
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Colon (2000). Does basal pr gene expression in solanum species
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Zhou, J. M., Y. Trifa, H. Silva, D. Pontier, E. Lam, J. Shah and D. F. Klessig
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182

FUTURE DIRECTIONS

It is well established that a large number of genes are expressed in plants
upon induction of Systemic Acquired Resistance (SAR). Some of those genes
encode proteins for which a function has been hypothesized based on
correlational and in vitro studies. The enhanced expression of acidic chitinase in
cucumber plants expressing SAR is one of these genes. However, mounting
evidence has challenged the hypothesis of a direct role of chitinase against the
invading pathogen.

The overall results of the present study suggests that the role of the
pathogenesis related (PR) protein does not directly affect the outcome of the
resistance conferred by SAR in cucumber plants. Direct evidence shows that the
presence of high levels of acidic chitinase and other PR proteins in the tissue do
not hamper the growth of the invading pathogen. Additionally, no correlation was
found between the levels of resistance by SAR and levels of expression of acidic
chitinase in the SAR expressing plants. Many comparisons were made between
the levels of chitinase between SAR-expressing plants and control plants.
Although, the data were statistically significant, their biological significance is
questionable. On the other hand, chitin oligomers have been shown to elicit
lignification and other defense responses in plants, (Roby et al. 1987; Barber et
al. 1989; Barber and Ride 1994). Hence, that does not exclude the possibility
that acidic chitinase in SAR expressing plant might release elicitors from the
invading pathogen that trigger defenses (Hammerschmidt 1999). In order to more

fully understand the role of chitinase or other PR proteins, the study of

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genetically modified plants engineered to silence or under express chitinase or
other PR proteins may help to understand their role in resistance. In this work,
preliminary experiments have been performed, and transgenic lines under
expressing chitinase have been generated. The further study of these transgenic
plants is needed in order to enhance the understanding of the role of acidic
chitinase in SAR expressing plants.

Evidence is provided to support the idea that the state of resistance by
SAR is protein synthesis dependant. Blocking protein synthesis in SAR
expressing cucumber plants leads to the breakdown of the resistance
accompanied by a higher rate of fungal growth within the tissue. Additionally,
peroxidase (POX) activity and H202 production are hindered. The link between
the decrease of POX activity, the low H202 production and the breakdown of
resistance is unclear thus the investigation of such relationship is important.

In greenhouse and field experiments, a differential expression of SAR
against pathogens with different mode of entry was found. Some cucumber
cultigens expressing SAR are resistant to Colletotrichum orbiculare but not to
Dydimella bryonieae. On the other hand, several other cultigens expressing SAR
are resistant to both pathogens. The morphological, biochemical or genetic
differences that make this differential response to SAR induction could potentially
lead to the discovery of new resistant genes or defense
mechanisms/interactions. For instance, because many plant species produce
polygaractuonase-inhibiting proteins (pgip) (De Lorenzo et al. 2001), the possible

role of these proteins in cucumber plant defense against Dydimella bryoniae is

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worth investigating. Interestingly, transcripts of pgip’s accumulate in P. vulgaris
hypocotyls in response to wounding or treatment with salicylic acid (Bergmann et
al. 1994). It is possible to hypothesize that some cultigens might express the
pgip's in response to SAR induction, making the cultigens resistant to Dydimella
bryonieae in addition to C. orbiculare.

The access to collections of cucumber cultigens and crossing lines, in
addition to this differential expression of resistance is a goldmine yet to be
explored. Crosses between cultigens that express this differential resistance and
the generation of molecular markers to track and pinpoint the source of this
response can provide more information of how plants expressing SAR restrict
differentially pathogen development.

The search for genes that would serve as markers and as targets for
disease resistance improvement has been difficult. The NPR1 gene is important
because it regulates the deployment of the battery of SAR and ISR genes. In
this work a fragment of the NPR1 gene has been isolated, which is almost
identical to the cDNA NPR1 sequence of Arabidopsis. NPR1 over expression
results in reduced pathogen growth (Cao et al. 1994; Friedrich et al. 2001).
Screening of cucumber cultigens levels of expression of NPR1 and their
correlation with resistance induced by SAR could yield a potential marker for

SAR which is worth investigating.

I85

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