THE POTENTIAL OF HYPERSENSITIVE RESPONSE ASSISTING ...

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Oct 23, 2013 (3 years and 11 months ago)

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THE POTENTIAL OF HYPERSENSITIVE RESPONSE ASSISTING
PROTEIN (HRAP) TO TRANSFORM BANANA AGAINST
XANTHOMONAS WILT DISEASE



Henry Mwaka

(B.Sc. Agric, Mak)

(2005/HD02/2073U)





Department of Crop Science, Faculty of Agriculture, Makerere

University








ATHESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES IN PARTIAL
FULFILMENT FOR THE REQUIREMENTS OF THE AWARD OF A MASTER OF
SCIENCE IN CROP SCIENCE DEGREE OF MAKERERE UNIVERSITY KAMPALA

D
eclaration


I, Henry Mwaka, certify that this is m
y original work and that it has not and never been presented
for examination for any award in any University.

DATE…………………………………………..

Signature………………………………………

Henry Mwaka





This thesis has been submitted for examination with our approval as his
supervisors.


Signed……………………………………….Date……………………………………..

Dr. Tusiime Geof
f
rey



Signed……………………………………….Date……………………………………..

Dr. Leena Tripathi


Dedication


I would like to dedicate this work to my mother, Jessica, my dad Dr. Ojhara B., my supportive
br
others Peter, Denis, Paul, Emmanuel and David, as well as my lovely sister Gloria. You are my
brilliant stars.


Acknowledgment


I hereby express my sincere gratitude to The Rockefeller Foundation, which provided the funds
for this study through National

Banana Research Program of NARO.I am grateful to my
supervisors Dr.Tusiime Geoffrey, Dept. of Crop Science, Makerere University, Dr. Leena
Tripathi of the International Institute of Tropical Agriculture (IITA
-
Uganda), Dr. Arinaitwe
Geofrey and Dr. Kiggund
u Andrew of NARO who provided expert guidance at every stage of
this research. I am also indebted to Dr. W .K. Tushemereirwe, Head of National Banana
Research Program, who had confidence in me and whose leadership and supervision provided an
enabling envir
onment for the successful completion of this work. Your guidance and valuable
criticism right from the inception of the study to the end are highly appreciated.


It is impossible to forget the input of my colleagues at the National
B
anana
R
esearch
P
rogram

at
Kawanda
,
especially the
B
iotechnology and
Breeding

team
.

I would in a very special way like to
appreciate Alice

Bukenya, Kumakech Alfred, Lamwaka Pamela, Betty

Magambo, Muwonge
Abubaker, Natukunda Loice, Ssali R. Tendo, Tindamanyire Jimmy
,
, Rebecca
Nakachwa, Roger

Serruga
, Moses Matovu, Nayiti Sarah, Bo
n
ny

Oloka
, Susan

Akello
,

Grace Bwengye, Jackie
Teko, Marg
a
ret Atim

and

Basheija Henry
.

Great appreciation also goes to my family for their encouragement and support throughout this
study, Mum, Dad, Pe
ter, Denis, Paul, Emmanuel, David and Gloria
.


Finally, I will forever be grateful to the almighty God, who has enabled me successfully
complete this study.



Abstract

The Banana Xanthomonas Wilt (BXW)

caused by
Xanthomonas campestris
pv.
musacearum
(
Xcm
)

is currently the most de
vastating

banana
disease, causing
near 100%
plantation loss where
no
preemptive
control measures are
engaged
.
According to germplasm screening conducted
since 2001, all banana types are susceptible to the BXW disease, and no resista
nce has been
identified in bananas
.

This study therefore aimed at developing transgenic bananas with resistance to the Banana
Xanthomonas wilt and its objectives were: 1) to investigate the potential for genetic
transformation of banana embryogenic cell s
uspensions (ECS) with the
hrap

gene from Sweet
Pepper, and 2) to challenge the transformed lines with
Xcm

so as to determine, and monitor
disease development in the transgenic banana. Transformation of ECS of the banana cultivar
‘Sukali Ndiizi’ (AAB) was d
one using
Agrobacterium tumefaciens
, AGL1 strain possessing the
binary vector PBI
-
HRAP, containing Neomycin phosphotransferase (
nptII
) as the plant cell
selectable marker and the
hrap

gene. Transformed cells were selected on Kanamycin
-
supplemented growth m
edia and the surviving lines were regenerated into plantlets.
The
presence of
hrap

and its expression in the transgenic banana was detected by PCR
(polymerase chain reaction) and Southern blot analysis. Preliminary
testing assays

show
that some of the transgenic lines are resistant showing full recovery after inoculation with the
Xanthomonas campestris musacearum
.


R
esults stemming from this study offers a ray of hope to
wards the improvement of resistance of
banana to BXW.

The
hra
p

gene is
probably
an excellent candidate that could play a pivotal role
in as the last line of defence against the Banana
Xanthomonas

wilt through genetic
transformation.

Table of contents

Declaration
................................
................................
................................
...........................

2

Dedication

................................
................................
................................
............................

3

Acknowledgment

................................
................................
................................
.................

4

Abstract
................................
................................
................................
................................

5

Table of contents

................................
................................
................................
.................

6

List of Tables

................................
................................
................................
.....................

10

List of Figure
s
................................
................................
................................
....................

11

List of acronyms and abbreviations

................................
................................
................

12

Chapter 1

................................
................................
.................
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INTRODUCTION
................................
................................
...
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1.1 Importance of Bananas in the East African region

....

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1.2 Banana Xanthomonas wilt and economic impact

......

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1.5 Aim of the study
................................
.........................

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1.6 Specific Objective of study

................................
........

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1.7 Hypothesis
................................
................................
..

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Chapter 2: Literature Review

................................
................
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2.1.1 BXW in Uganda and economic impact

..................

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2.1.2 Distribution of BXW in East and Central Africa

....

Error! Bookmark not defined.

2.2.1 Characteristics of
Xanthomon
as

campestris

pv.
musacearum
Error! Bookmark not
defined.

2.2.2 Diagnosis of BXW

................................
..................

Error! Bookmark not defined.

2.3.1
Aetiology and epidemiology of BXW

....................

Erro
r! Bookmark not defined.

2.3.2 Pathogenicity of
Xanthomonas campestris

pv.
musacearum
Error! Bookmark not
defined.

2.3.3 Control and Management Strategies

.......................

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2.4.1 Plant defense responses
................................
...........

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2.4.2 Jasmonic Acid

................................
.........................

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2.4.3 Salicylic acid

................................
...........................

Error! Bookmark not defined.

2.5.3 Mode of R
-

Avr
interaction
................................
.....

Error! Bookmark not defined.

2.5.3a Receptor
-
elicitor theory
................................
.........

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2.5.3b Guard theory

................................
.........................

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2.6 Protein secretion apparatus in bacteria
.......................

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ookmark not defined.

2.6.1 Elicitation of hypersensitive response (HR) by bacteria
Error! Bookmark not
defined.

2.6.2 Molecular mechanism of HR

................................
..

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2.6.3 Role of Harpins in HR

................................
............

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2.6 Defense genes inducing harpin
-
mediated hypersensitive response
Error! Bookmark not
defined.

2.6.4 Action of the hypersen
sitive response assisting protein (HRAP)
Error! Bookmark not
defined.

2.7 Use of
hrap

gene in plants

................................
.........

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2.8 Control of bacterial diseases using genetic engineering
Error! Bookmark not defined.

2.9 Genetic Modification of banana
................................
.

Error! Bookmark not defined.

Chapter 3:

................................
................................
................
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ark not defined.

Transformation of
banana embryogenic cell suspensions with the
hrap

transgene and
validation for stable integration

................................
............
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3.0 Introduction

................................
................................

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3.1 Materials and Methods
................................
...............

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3.1.1 Plant material

................................
..........................

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3.1.2 Ge
ne construct
................................
.........................

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3.1.3 Rejuvenation of
hrap
-

E.coli

from a stab culture

..

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3.1.4 Extraction of plasmid DNA

................................
....

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3.1.5. Restriction
Analysis of plasmid DNA

...................

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3. 1.6 Transformation of Agrobacterium

........................

Error! Bookmark
not defined.

A. Preparation of competent cells of
Agrobacterium tumefaciens
Error! Bookmark not
defined.

B. Transformation of
Agrobacterium
, selection and purification
Error! Bookmark not
defined.

3.1.7 Isolation of plasmid DNA from engineered Agrobacteri
um
Error! Bookmark not
defined.

3.1.9 Preparation of
Agrobacterium

for plant transformation
Error! Bookmark not defined.

3.2 Preparation of banana cell suspensions for genetic transformation
Error! Bookmark not
defined.

3.3 Transformation of embryogenic cell suspensions, selection and regeneration
...

Error!
Bookmark not defined.

3.4
Histochemical GUS assay

................................
..........

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3.5 Genomic DNA Isolation

................................
............

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3.6 PCR analysis of transgenic plants

..............................

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defined.

3.6.3 Southern Blot analysis

................................
............

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3.7 Results

................................
................................
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3.7.1 Analysis of plasmid DNA

................................
......

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3.7.2 Confirmation of the presence of the
hrap

gene in the plasmid DNA of the
Agrobacterium strain AGL1

................................
............

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3.7.2 Histochemical GUS assay

................................
.......

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3.7.3 Selection and regeneration of transformants
...........

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3.7.4 Confirmatory PCR Analysis of transgenic lines

.....

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3.8 Southern blot analysis

................................
................

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3.9 Discussion

................................
................................
..

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ned.

Chapter 4: Evaluation of
hrap
transgenic banana plants for resistance against
X
anthomonas
campestris

pv
musacearum

.............................
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4.0 Introduction

................................
................................

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4.1 Materials and Method
s
................................
...............

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4.1.1 Source of inoculum

................................
.................

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4.1.2 Preservation of
Xcm

................................
................

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4.
3 Screening of
in vitro

plants

................................
........

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4.4 Disease assessment due to BXW

...............................

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4.5 Data collection

................................
...........................

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4.7.1 Evaluation of transgenic banana lines for resistance against
Xcm
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not defined.

4.8 Discussion

................................
................................
..

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Chapter 5: General Discussion, Conclusion and Recommendations
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defined.

5.1 General Discussion

................................
....................

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5.2 Conclusion and recommendations

.............................

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References

................................
................................
................
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Appendix

................................
................................
..................
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List of Tables

Table 1: Classification of R genes on the basis of structural features
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defined.

Table 2: Restriction digestions reaction setup

.........................

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Table 3 Gus Assay Results confirming transformation success
Error! Bookmark not defined.

Table 4: Summary of data collected on incidence and severity post inoculation

.............

Erro
r!
Bookmark not defined.





List of Figures

Figure 1: Banana Xanthomonas Wilt


................................
......

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Figure 2: Graph showing conservative loss if BXW is not controlled
Error! Bookmark not
defined.

Figure 3 Hypersensitve response aided by genetic engineering. .
Error! Bookmark not defined.

Figure 4: Schematic
diagram of pBI
-
HRAP gene construct;.
..

Error! Bookmark not defined.

Figure 5 restriction sites in the
hrap

gene construct

................

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Figure 6 Restriction of Plasmid DNA from
E.coli
:

...............

Error! Bookmark not defined.

Figure 7: Restriction of plasmid DNA from transformed Agrobacterium
Error! Bookmark not
defined.

Figure 8: Histochemical gus assay..
................................
.........

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Figure 9:
S

................................
................................
................

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ookmark not defined.

selection pressure

................................
................................
.....

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Figure 11:

different stages of the regeneration process from developing embryos up to a
complete transgenic plantlet
................................
.....................

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Figure 12:
In vitro

plantlets in the the growth room being subjected to micropropagation for
replication purposes: 5reps were generated per line

................

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Figure 13: PCR analysis of
hrap

transgenic banana

plants.

..

Error! Bookmark not defined.

Figure 14:
G
ene
-
specific PCR
analysis of the
hrap

transgenic banan
a plants.
Error! Bookmark
not defined.

Figure 15: Southern Blotting results a varying number of integration sites estimated by counting
the number of band
signals

................................
......................

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Figure 16:
O
bservation after 6 weeks post inoculation

...........

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List of acronyms and abbreviations



A
-
mT

Agrobacterium
-
mediated transformation

AS

Acetosyringone

Bp

Base pair

BXW

Banana Xanthomonas Wilt

CaMV

Cauliflower Mosaic Virus

DNA

Deoxyribonucleic acid

EAHBs

East Africa highland bananas

ECS

E
mbryogenic
C
ell
S
uspensions

GDP

Gross domestic product

HR

Hypersensitive response

HRAP

hypersensitive response assisting protein

IITA

Institute of
I
nternational
T
ropical
A
griculture

K50

Kanamycin at a concentration of 50mg/ml

LB
-


Luria Bertoni

MA2

Cell suspension maintenance medium

MA3

Selection and embryo initiation medium

MA4

shoot germination medium

MAAIF

Ministry of Agriculture Animal industry and fisheries

NABC

National

Agricultural Biotechnology Centre


NARL

National Agricultural Research Laboratories

NARO


National Agricultural Research Organisation

NABC

National Biotechnology Centre

NBRP


National Banana
R
esearch
P
rogram

nptII

Neomycin
phosphotransfererase gene

PCR

Polymerase Chain Reaction

RB

Right T
-
DNA border sequence

RD1

Embryo development medium

SCV

Settled Cell Volume

Taq

Thermus aquaticus

T
-
DNA

Transferred DNA

TE

Tris

EDTA

TMA1

Transformation medium

Xcm

Xanthomonas campestris

pv

musacearum

YPG:

Yeast peptone Glucose

Xcm

Xanthomonas vasicolar
pv

musacearum