Mitsubishi Electric MXZ-18TV -E1 User manual

Construction of Plant Transformation Cassettes and
Heterologous Expression of Genes Encoding Cyanamide
Hydratase and Antifungal Proteins
Thesis submittedto the Pondicherry University for the Degree of
Doctor of Philosophy
by
S. ISAAC KlRUBAKARAN
Departmentof Biotechnology
School of Life Sciences
Pondicherry University
Puducherry
- 605 014
INDIA
MAY -m

PRY %,,L PONDICHERRY UNIVERSITY
DEPARTMENT OF BIOTECHNOLOGY
SCHOOL OF LIFE SCIENCES
PUDUCHERRY
605 014, INDIA
Dr. N. Sakthivel
Reader
& Head
CERTIFICATE
Cen~fiedthat th~srhesik entltlcd "Construction of plant trvnsformation
cassettes and heterologous expression of genes encoding cyanamide hydrahue
and antifungal proteins" ir ;I record CISresearch work done by ~hccandldulc,
Mr. S. Isaac Kirubakaran, durlng the perlod of h~rrtudy In lhc Dcpalinlent of
Blchechnolopy. Pondicherry llni\err~ty,Puduchcrry, under my aupervlrion atid thnl it
has not formed prevlourly the bakik lor the awud
of any degree, dlplorna.
asrnciateship or fellowrh~p.
Puducherry
Date: I I I eq I b Y
w LL-/
(N. Sakthivel)
Phone:[phone] (off.) Fax: [phone]
Etnnil:[email protected]

I hereby declare that the work prescirted in his Ihesis has ken c:~rriedour by
me under the guidance and supervision of Dr. N. SAKTHIVEI.. Dcpann~cnl of
Biotechnology. School of Life Sciences, Pondicherry Clniversily,Puducherry b0.5 014
and this work ha%not been whrnitted elsewhere for any other degrce.
Puducherry
Date:
11 O? C 2

DEDICATED
TO
MY BELOVED PARENTS

ACKNOWLEDGEMENT
During the years as a Ph.D. btudent, rirany ~xoplehaye contributed to\vanls ;I
111e:lnin~fill:lnd succeshflll ourccvnr of I~IS rcsc:~rcl~l)~oje~.t.tlo\vevc.r :I \,cry I'cw Ii;~v'
stood :~pi~rtby sheer virtue ol'cxccllrnce ill ~hcirlieltl nritl I have Iwcn fortuli:~lcto
bcncfit iron] their guidance.
I hereby express my deep sense of gratitudr to niy guide Dr. N. Sakthivcl, !'or his
expert guidance. constant encour:lgement and ide;~swhich rchulted in the culrnini~tion
of this thesis.
I reserve my special thanks fro111the hottom of my he:lrt to Prof. S. Jayarhandran
for the support and encouragement.
I am grateful to Dr. V. Arul, Dr. S. Sudharani, Dr. N. Arurnugarn,
Dr. Lata Shukla,
and Dr. K. Prashant for their co-oprition.
I wish to record my slncere thanks to the doctoral committee members
Pruf. S. Jayachandran and Dr. N. Parthasarathy.
I express my deep sense of gratitude to Prof. S. Muthukrishnan. Kansas State
University. Department of Biochemistry. Kansa~,USA for providing me the harley
and wheat cDNA clones and cnh and chitinase antiserum and
Prol. Tranquet Olivier, Scientific Leader. Antibody production unit, Institut
National de la Recherche Agronomique (INRA). Unite Biopolymeres, Interactions,
Assemblages, Nantes. Cedex, France for providing me wheat LTP antiserum.
I am indebted to Dr. K. Ulaganathan, Associate Professor. Centre for Plant
Molecular Biology, Osmania University. Hyderahad, for helping me with the plant
transformation work.

I am really grateful to Ms. Mubarak Begum. Ms. Snehalatha Rani and
Mr.
M. Shashikanth Research Scholm. Centre for Plant Molecular Bicioloyy.
Osnlania University. tlyderah:~d,for helping Ine in le;~rning;\lid prcweetling with plant
tr;~nsforlnation.rtuJies.
My ainccrt tli;lnks to hIr. 11. Iialachantlran. Dioi~lfor~~i;~tirsCcntre. llnivcrsity of
h1adr.d~:11iJ >IS. N. Shanthi, Bicli~~formatlcrC'entrc. Pt~ndiclicrryll~~ivcrsityfor
helping me in molecular modeling studics.
I thank all my fellow collengues, seniors and juniors, whoni I might have left out due
to oversight. hut whose best wishes have ;~lwaysencclur~gednlc.
I would like to thank our office staffs Mr. ('. Iiul~~krishnan,Mr. P. Rnn~alinguni,
Mr. Rarnarnuorlhy, Mr. S. Kannuyiram, hlrs. S. Valli and Mrs. Kalivnnl, for their
co-operation and help. My speci:~lthanks goes to all the non-teaching staff menlhcrs
of Pondicherry University who helped me throughout the study.
I gratefully acknowledge Department of Science and Technology (1)ST). New
Delh~for providing me with Junior Research Fellowship.
Last and definitely by no means the least.
I would like to rnake a mention of my
parents, all my family members and well wishers, who have ken eagerly looking
forward to this personal milestone. However, they have alstr been very patient with
my failures
and have always extended their support providing me with a driving force
for the successful completion of this research work.
Date:
jl,i,l LS
j)ph*
(S. ISAAC KIRUBAKAKAN)

CONTENTS
CHAPTER
1. INTRODUCTION
2. REVIEW OFLITERATURE
3. MATERIALS ANDMETHODS
4. RESULTS
5. DISCUSSION
6. SUMMARY
7. REFERENCES
Page No.

LIST OFABBREVIATIONS
3D
2 D
BAP
BClP
BLAST
hP
BSA
cDNA
cm
DNA
DTT
dNTP
EDTA
EPSPS
ER
GlcNAc
GLP
h
IAA
IPTG
J A
kb
kDa
kHz
kV
LB
LTP
mA
MEGA
mg
three-dimenaion:~l
two-dimension:~l
6-henzyl-a11iirio-pur1ne
5-bromo-4-clilorc)-3-indolyl pli~ispl~;rtc
basic local ;ilignmcnt s'n~ch t<r>l
base pair
bovine scrum albumin
complementmy DNA
centimeter
deoxyrihonucleic acid
Dithiothreitol
2'deoxynucleotide S'trlphosphate
ethylenediaminetetraacetic arid
5-enol pyruvyl shikimate-.%phosphate synthase
endoplasmic reticulum
N-acetyl-D-glucosamine
gerrnin-like protein
hour
hygromycin phosphotransferase
indole-3-aceticacid
isopropyl P-D-thiogalactoside
jasmonic acid
kilo
base
kilo Dalton
kilo hertz
kilo volt
Luria Bertani
lipid transfer protein
milli Ampere
molecular evolutionary genetics and sequence alignment tool
milligram

min
ml
mm
mhl
%IS
NAA
NBT
ng
NJ
nni
nmol
NMR
npt
ns-LTP
OD
par
PBS
PCR
PDA
PR
RMSD
ROS
rl' m
S A
SARE
SDS
SDS-PAGE
SEM
SSC
TAE
T-DNA
TE
TEMED
Ti
minutes
milliliter
niilli~iieter
niilli~iiolar
Murilhhige Skwg
naphthalene acetic :wid
nitrohlue terr~zuliuln
nanogram
neighhour-jolning
nanometer
nanornole
nuclear magnetic resonance
neornycin phosphorri~n~icr;~~
non-specific lipid rr;in,lcr protein
optical density
phosphinothricin N-acetyl-rr:insferase
phosphate huffer saline
polynierase chain reaction
potato dextrose agar
pathogenesis-related
root mean square dev~alion
reactive oxygen species
revolulions per mlnute
salicylic acid
SA rehponsive element
sodium dodecyl sulphate
sodium dodecyl sulphate-polyacrylamidc gel electrophoresis
scanning electron microscope
saline sodium citr:~te
Tris acetate EDTA
transfer DNA
Tris EDTA
tetmethylelhylenediamine
tumor inducing

TLP
UPGMA
U v
v
\\,/v
WGA
YEB
thaumatin-like protein
unweighted pair gmup
of cuithl~lcticmean analysis
lllt~l~i~ll~t
vcrlt
volil~nc by vc>lumc
weight hy volume
\vhc;rt gcrm :~:glutiriin
yeast extract hrorh
microgram
microlitre

INTRODUCTION

CHAPTER 1
INTRODUCTION
Plant diseases are one of the major concerns to n$ricultur;~lproduction. It has
heen estimated that total loss as a consequencc of plant dise:ises reaches 25% of the
yield in western countries and almost
50% in developing countries. Of this, one third
yield loss is due to fungal infections (Bowyer, 1999). There is
a pressing need to
control fungal diseases to ensure a steady and constant food supply to feed the ever
increasing population of the world. Conventional practice to control fungal diseases
has been the use of chemical fungicides which have adverse environmental effects
causing health hazards to humans and other non-target organisms, including
beneficial life forms. Due to the increasing concern towards the toxicity and
biomagnification potential of chemical fungicides in agriculture. current practices
based on molecular biology techniques which involve the development of transgenic
resistance in crop plants merit attention (Schickler and Chet, 1997). In response to
developmental signnls and pathogen attack plants evolve a number of defense
responses (Bell, 1981) and synthesize an array of proteins and peptides such
as
chitinase, glucanase, a-defensins, lipid transfer proteins, thionins, hevein- and knottin-
type peptides, cyclopeptides. alkaloids and other unique peptide groups as part of their
natural defense systems (Broekaett
er al., 1997; Yun et 01.. 1997; Somssich and
Halbrock, 1998).
Efforts have been made for isolation and characterization of pathogenesis-
related (PR) proteins (Carmona et al.. 1993; Terras et ul.. 1995; Molina and Garcia-
Olmedo. 1997; Datta et al., 1999). It is reported that not all the PR-proteins are

antimicrobial and therefore, in virro screening of proteins for their antimicrobial
potential is important to use them for developing transgenic resistance in crop plants.
Selectable markers such
as phosphinothricin acetyl trnnsfcrnsc (Ixrr),
dihydrofolnte reductase (dhfr), hyyromycin phosphotransferase (hph), neomycin
phosphotransferase (nprlf), glyphosate-tolerant CP4 and COX genes have heen used
in plant transformation work for screening of transgenic plants. Each of these
selectable markers has its particular advantages and disadvantages. The use of
herbicide resistance genes including bar. CP4 and GOX concerns the outcrossing
through sexual transmission to wild relatives producing herbicide-resistant weeds
(Vasil, 1994; Arriola and Ellstrand. 1996). In comparison to other selectable markers.
cyanamide hydratose (cah) gene is being considered as a unique marker because of its
ability to convert calcium cyanamide into urea, a useful nitrogen compound, which
can be considered
as a fertilizer source to plants (Maier-Greiner et ul.. 1991a;
Sahrawat et al., 2003).
Recombinant DNA technology has paved the way for obtaining and
combining genes from a variety of sources and the possibility of expressing genes in
different host cells. Escherichia coli has been successfully used for the production of
relatively complex proteins in large quantities (Rudolph, 1996). Considering the
number of proteins produced during biological stress in plants, only few of them have
been characterized and utilized for developing transgenic resistance against fungal
pathogens.

Present investigation was aimed for construction of plant transfomntion
cassettes and heterologous expression of cyanamide hydratase (cah) marker gene and
antifungal genes encoding chitinase and lipid transfer protein (LTP).
Objectives of the present study have been formulated as follows:
I. Cloning and expression of chitinase and LTP genes in E. coli and evaluation
of their antimicrobial potential towards phytopathogens.
2. Site-directed mutagenesis, cloning and expression of cuh gene in E. coli
3. Construction of gene cassettes with cah marker and antifungal gents encoding
chitinase and LTP.
4. Transgenic expression of cah marker and antifungal genes in tobacco

REVIEW OF LITERATURE

CHAPTER 2
REVIEW OF LITERATURE
2.1. Importance of transgenic resistance
Although fungicides, pesticides and insecticides have offered protection
against crops, reluctance to rely on chemicals is increasing because of environmental
and health concerns. In recent years, with the advent of
recombinant DNA and tissue
culture technologies transgenic resistance against phytopathogens using antimicrobial
genes has been achieved. Development of transgenic resistance has the promise of
providing season-long protection against specific pathogens. Plant transformation
represents a powerful supplemental tool to conventional breeding programs that can
accelerate the development of elite varieties with disease resistance and good
agronomic characteristics (Muthukrishnan er ol., 2001).
2.2. Pathogenesis-related proteins: Biological activity and function
Plants synthesize a variety of compounds when exposed to biological stress
viz., fungi, bacteria, viruses, insects and herbivores (Collinge and Slusarenko, 1987;
Linthorst, 1991). The inducible defense responses include the production of reactive
oxygen species (ROS), phytoalexins, cell wall components (callose, glycine or
hydroxyproline-rich proteins) and other specific group of proteins called
pathogenesis-related (PR) proteins. The PR-proteins were identified initially
as
inducible proteins in leaf extracts of tobacco plants reacting hypersensitively to
tobacco mosaic virus infection
(Van Loon, 1985; Van Loon, 1999). Five distinct

bands with different electrophoretic mobilities wen identified in inoculated plants
that were absent in water-inoculated plants and were referred to as PR-I through PR-5
proteins. High resolution gels, column chrolnntogmphy, i~nmunologic;~lre;~ctivityof
the induced proteins and tinally, sequencing d:rta of PR-proteins as well ;ISgencs from
different plants have led to the designation of sever:~ldistinct groups of PR-proteins
(Van Loon and Van Strien. 1999). It is now well established that proteins with
immunological and amino-acid sequence similarities to tobacco PR-proteins are
induced in a variety of plants and their induction only in pathological situations
suggests a role for these proteins in plant defense.
The major clue regarding the precise biological role of PR-proteins surfaced
when the biochemical activities of the PR-2 and PR-3 classes of proteins were
determined. The PR-2 proteins has an endo P-1,3 glucanase activity (Kauffmann
et
01.. 1987). whereas the PR-3. PR-4, PR-8 and PR-I I proteins were shown to have
endochitinase activity (Legrand
el ul., 1987). Most fungi contain P-1,3-glucans or
chitin in their cell walls (Bartnicki-Garcia, 1968).Thus, exposure of fungal pathogens
to these hydrolytic enzymes could result into the release of cell wall fragments
containing oligosaccharides, which have been shown to elicit defense responses
(including the induction of the phenylpropanoid pathway enzymes and PR-proteins)
in a variety of plants (Mauch and Staehelin, 1989; Takeuchi
er ul., 1990; Ham et 01..
1991). High concentrations of lytic enzymes may result in the dissolution of cell wall.
if their substrates are exposed. Testing of purified chitinases and P-1.3-glucanases for
antifungal activity showed that they are inhibitory against
a variety of fungi especially
in combinations (Mauch et 01.. 1988). Similarly, purified PR-I and PR-5 proteins also
were shown to inhibit the growth of specific fungi (Roberts md Selitrennikoff, 1990;

Niderman er al.. 1995). The PR-5 proteins, which also nre referred to as Ihaumatin-
like proteins (TLP), because of their sequence similarity to the intensely sweet protein
thaumatin from Tho~rrnurucorc~rs dnnielti (Daniell, 1852). PR-5 proteins ci~~~sed
leakage of cytoplasmic material from the fung~ and hyphal rupture.
Purified PR-I protein from tobacco and tomato inhibited growth of
oomycetous fungi (Vigers
et ul., 1991; Woloshuk et al.. 1991). The PR-6 proteins
were shown to be the inhibitors of nematodes, insects and microbial proteases (Green
and Ryan, 1972). The PR-7 are endoproteases (Vera and Conejero, 1988) and the
PR-8 protein can disrupt Gram-positive bacteria due to lysozyme activity (Van Loon
and Van Strien. 1999; Van Loon. 2001; Selitrennikoff, 2001).The PR-9 proteins are
lignin-forming peroxidases (Reimers el al., 1992; Baga et dl., 1995; Johansson and
Nyman, 1996) and some PR-10 proteins have been shown to have an RNAse activity
which is important to cleave viral RNA's in antiviral pathway (Somssich
et ul.. 1986;
Moiseyev
et al., 1994). The PR-protein classificution has been extended to include
other inducible proteins namely defensins (PR-12). thionins (PR-13) and lipid transfer
protein (PR-14) (Van Lmn and Van Strien, 1999). Germins and germin-like proteins
(GLPs) have been classified as PR-15 (Zhang et a/., 1995) and PR-16 (Wei et a[.,
1998). The PR-16 proteins have been identified from hot pepper during resistance
response to bacterial and viral infection (Park
et ul., 2004a). Multifacited functionality
of PR-15 and PR-16, including cell wall remodeling ability, can be directed against
pathogens and may have protective role (Park
et ul., 2004b). The PR-17 group
contains two protein families, WCI-5 from wheat and HvPR-17 and HvPR-17b from
barley, with antifungal properties (Gorlach
et al., 1996; Christensen et ul., 2002). The
two PR-17 proteins from barley are extracellular and may affect cell wall metabolism

or signal transduction and are active against the fungus Blirmeria graminis which
causes powdery mildew on barley. Recognized families of PR proteins are listed in
Table
I.
2.2.1. Biochemical and structural characteristics of PR-proteins
PR-proteins are distinguished by specific biochemical properties. They are
low-molecular weight proteins (6-43
kDa), extractable and stahle at low pH (c 3).
thermostable and highly resistant to proteases (Van Loon, 1999). The structure of
a
PR-1 family member (tomato PRl-b) was solved by nuclear magnetic resonance
(NMR) and was found to represent a unique molecular architecture. The protein
contains four a-helices and four P-strands arranged antiparallel between helices. The
tight packing of the a-helices on both sides of the central P-sheet (a-@-asandwich
structure) results in a compact, bipartite molecular core, which is stabilized by
hydrophobic interactions and multiple hydrogen bonds (Fernsndez
er ol., 1997). This
compact structure probably determines the high stability of PR-proteins and their
insensitivity to proteases.

Table 1 Recognized familiesof pathogenesis-related proteins
-
Families Type member Properties Reference
PR-1
PR-2
PR-3
PR-4
PR-5
PR-6
PR-7
PR-8
PR-9
PR-I0
PR-I1
PR-I2
PR-13
PR-14
PR-I5
PR-16
PR-17
Tobacco PR-la Antifung;~l
Tobacco PR-2 P-1.3-glucanase
Tobacco P, Q Chitinase type I, 11, IV,V,VI.Vii
Tobacco 'R' Chitinase type I. I1
Tobacco S Thaumatin-like
Tomato Inhibitor 1 Proteinase-inhibitor
Tomato P69 Endopmteinase
Cucumber chitinase Chitinase type Ill
Tobacco peroxidase Lignin-forming peroxydase
Parsley 'PRI' Ribonuclease-like
Tobacco chitinase Chitinase,type I
Radish Rs-AFP3 Defensin
Arabidopsis THI2.1 Thionin
Barley LTP4 Lipid-transfer protein
Barley OxOa (germin) Oxalate oxidase
Barley OxOLP Oxalate oxidase-like
Tobacco PRp27 Unknown
Antoniw er 111.. 1980
Antoniw rr 111.. 1980
Van Loon. 1982
Van Loon, I982
Van Loon. 1982
Green and Ryan, 1972
Vero and Conejero. 1988
Metraux
ef al., 1988
Lagrimini et 01.. 1987
Somssich
et ul.. 1986
Melchers
et ul.. 1994
Terras et 01.. 1992
Epple
el ol., 1995
Garcia-Olmedo et 01.. 1995
Zhang er ul., 1995
Wei
el al., 1998
Okushima
el ol.. 2000