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TRANSLOCATION OF VIRUS-DERIVED NUCLEIC ACIDS
TO CHLOROPLASTS AND MITOCHONDRIA IN PLANTS
by
Tauqeer Ahmad
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Department of Cell and Systems Biology
University of Toronto
© Copyright by Tauqeer Ahmad 2016
ii
TRANSLOCATION OF VIRUS-DERIVED NUCLEIC ACIDS
TO CHLOROPLASTS AND MITOCHONDRIA IN PLANTS
Tauqeer Ahmad
Degree of Doctor of Philosophy
Department of Cell and Systems Biology
University of Toronto
2016
ABSTRACT
In this study, I demonstrated that a non-coding RNA sequence from potato virus X as
small as 127 nucleotides (located near the 3´end of 8 kDa and the start of CP genes as well as the
non-coding intergenic region) is capable of translocating not only its own sequence but also a
reporter gene, fluorescent green protein mRNA into chloroplasts of the transgenic tobacco plants.
This is the first evidence showing that a small viral RNA sequence (designated “RNA tractor”) is
capable of translocating RNA sequences to the chloroplast. The chloroplast translocation
efficiency of the PVX RNA tractor was determined to be 120 X lower than that of Eggplant
latent viroid, a member of the Avsunviroidae family that replicates and accumulates in the
chloroplast. Furthermore, I investigated two begomoviruses on various Nicotiana species to
assess the effects of their ploidy level on infectivity and symptomatology. For this purpose,
infectious clones of Ageratum enation virus (AEV), a monopartite (DNA-A with Beta-satellite
DNA particle) and Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite (DNA-A and
DNA-B), begomoviruses were used. All plants inoculated with ToLCNDV were systemically
infected and showed characteristic symptoms. However, in the case of AEV, all plants except N.
tabacum cv. Xanthi were infected by the virus but remained symptomless. Taken together, these
results indicate that there is no clear relationship between infectivity and ploidy levels;
furthermore, symptomatology depends on the type of virus and/or plant species. Another key
iii
question to answer was whether or not the genomes of the begomoviruses could be isolated from
chloroplasts of the infected tobacco and tomato plants. PCR results confirmed the presence of
only DNA-A of the AEV in the chloroplasts. Preliminary studies clearly show that the “RNA
tractor” sequence and AEV genome are incapable of targeting the mitochondria. These findings
suggest that members from different viral families may be associated with the same organelle,
but that members do not necessarily target the different organelles. Thus, the present study could
be important for understanding the evolutionary importance of the interactions of viral genomes
with different organelles of plant cells and their consequential pathological effects.
iv
Acknowledgments
Thanks to Almighty Allah, the Omniscient, Omnipotent and Omnipresence who blessed me the
aptitude of accomplishing this colossal work.
I deem it a profound honor to express the depth of my gratitude to Prof. Mounir G. AbouHaidar,
my supervisor, for the continuous support of my Ph.D. study and related research, motivation, and
immense knowledge. I feel that his guidance has helped me to mature into an independent
researcher with the abilities to cope with any type of research at both the scientific and
administrative levels.
I am greatly indebted to my other committee members: Prof. Richard Collins and Prof. Maurice
Ringuette for their insightful comments, meticulous criticism, encouragement and critical review
of my thesis.
I would like to thank Dr. Eiji Nambara for being a part of my examining committee. I really
appreciate Dr. Andrew White who has devoted his valuable time to review my thesis and took part
in my final defense
My sincere thanks also go to Dr. Christendat and Dr. Guttman for providing me access to their
laboratory facilities. Many thanks are due, to Henry and Audrey for their cooperation with confocal
and electron microscopic studies. Bruce and Andrew, I do appreciate your efforts for the
programming of growth chamber and greenhouse supplies.
Many thanks are immense for the entire CSB staff (especially Ian and Tamar) helping me move
well along with the administration matters in all these years.
My profound thanks to Dr. Saleem Haider, the man who introduced me to Professor AbouHaidar.
Special thanks to Dr. Kathleen Hefferon for proof-reading parts of the thesis and publications.
Thanks to Dr. Srividhya Venkataramana for all the help and the opportunity to collaborate in
publications.
A special note of thanks to all of my colleagues; Alexander, Amanda, Kayvan, Tatyana, Liu,
Amjad, Hasan, Reem, Amira, Dang, Lingjie and other fellows. It has been a pleasure working with
you all and thanks for offering a helping hand whenever needed.
I would like to express my heartfelt gratitude to all my family members. It is through their
wholehearted prayers that enabled me to achieve one of my goals. I am also indebted to all those
who prayed for my success.
I must acknowledge my wife and best friend, Sadaf, without her love, encouragement and editing
assistance, I would not have finished this thesis. Love to my kids Ismaeel, Tayyab and Noor for
always cheering me up.
v
Table of Contents
Acknowledgments ................................................................................................... iv
Table of Contents ..................................................................................................... v
List of Tables ............................................................................................................ x
List of Figures .......................................................................................................... xi
List of Abbreviations ............................................................................................ xiv
CHAPTER 1 ............................................................................................................. 1
1 LITERATURE REVIEW .............................................................................. 1
1.1 POTEXVIRUSES ......................................................................................... 1
1.1.1 Replication .......................................................................................................................... 1
1.1.2 Intercellular Transport of Potexvirus .................................................................................. 4
1.1.3 Intracellular trafficking of viral RNA in potexviruses ........................................................ 5
1.1.4 Interaction between viral and chloroplast proteins ............................................................. 6
1.1.5 Virion and viral RNA within chloroplasts .......................................................................... 7
1.1.6 Targeting of nuclear-encoded proteins to organelles .......................................................... 8
1.1.7 mRNA-based protein targeting to different organelles ....................................................... 9
1.1.8 The accumulation of Avsunviroidae viroids within the chloroplasts ................................. 10
1.1.9 Non-coding RNAs in plastids ........................................................................................... 11
1.1.10 Translation in chloroplast ................................................................................................. 11
1.1.11 RNA transport into mitochondria ..................................................................................... 14
1.2 GEMINIVIRUSES ..................................................................................... 15
1.2.1 Genus Begomovirus .......................................................................................................... 17
1.2.2 Begomovirus infection ...................................................................................................... 20
1.2.3 Long distance movement within plants ............................................................................ 21
1.2.4 Translocation of begomoviruses into chloroplast ............................................................. 21
CHAPTER 2 ........................................................................................................... 23
2 STUDIES ON TRANSLOCATION OF RNAS FROM CYTOSOL
TO ORGANELLES ................................................................................... 23
2.1 INTRODUCTION ...................................................................................... 23
2.2 RESEARCH PLAN .................................................................................... 27
2.3 MATERIALS AND METHODS .............................................................. 28
2.3.1 Plasmid construction and transformation.......................................................................... 28
vi
2.3.2 Heat shock transformation of E.coli ................................................................................. 35
2.3.3 Isolation and purification of plasmid DNA from E.coli (mini-prep) ................................ 36
2.3.4 Gel electrophoresis............................................................................................................ 37
2.3.5 Agrobacterium transformation .......................................................................................... 37
2.3.6 Plant transformation .......................................................................................................... 38
2.3.7 Infection of N. tabacum cv. Xanthi with PVX and virus isolation ................................... 39
2.3.8 Extraction of viral genomic RNA ..................................................................................... 41
2.3.9 Chloroplast isolation ......................................................................................................... 42
2.3.10 cDNA synthesis and RT-PCR ........................................................................................... 43
2.3.11 Real-time RT-PCR ............................................................................................................ 44
2.3.12 SDS-PAGE and western blot analysis .............................................................................. 45
2.3.13 Isolation of intact mitochondria and enzymatic treatments .............................................. 46
2.4 RESULTS .................................................................................................... 47
2.4.1 Detection of PVX RNA and coat protein in chloroplast................................................... 47
2.4.2 Reconstruction control experiments ................................................................................. 50
2.4.3 Design of constructs to confirm RNA tractor activity in chloroplasts.............................. 51
2.4.4 Analyses for expression of different constructs in total cellular RNA ............................. 52
2.4.5 Translocation of RNA transcripts driven by different constructs into chloroplasts ......... 53
2.4.6 Quantitation of translocated RNA to chloroplasts by real-time RT-PCR......................... 54
2.4.7 Comparison of translocation efficiency of PVX RNA tractor (pTR:127) to
Eggplant latent viroid sequence (pCELVd-GFP) ............................................................. 57
2.4.8 Translocation of “RNA tractor” sequence to plant mitochondria ..................................... 58
2.5 DISCUSSION ............................................................................................. 60
CHAPTER 3 ........................................................................................................... 65
3 STUDIES ON INFECTIVITY AND TRANSLOCATION OF
VIRAL DNAS FROM CYTOSOL TO ORGANELLES ....................... 65
3.1 INTRODUCTION ...................................................................................... 65
3.2 RESEARCH PLAN .................................................................................... 68
3.3 MATERIALS AND METHODS .............................................................. 68
3.3.1 Plant growth conditions .................................................................................................... 68
3.3.2 Agrobacterium-mediated inoculation ............................................................................... 69
3.3.3 Extraction of total nucleic acids from plants and PCR ..................................................... 69
3.3.4 Isolation of intact chloroplast and enzymatic treatments .................................................. 70
3.3.5 Light microscopy and transmission electron microscopy (TEM) ..................................... 72
3.3.6 Isolation of intact mitochondria and enzymatic treatments .............................................. 73
vii
3.3.7 Isolation of virus ............................................................................................................... 74
3.4 RESULTS .................................................................................................... 75
3.4.1 Infectivity Assays: Inoculation of plants with AEV and ToLCNDV DNA clones .......... 75
3.4.2 Chloroplast DNA Analysis ............................................................................................... 80
3.4.3 Reconstruction control experiments ................................................................................. 80
3.4.4 Microscopic studies .......................................................................................................... 82
3.4.5 Translocation of AEV DNA in mitochondria ................................................................... 83
3.5 DISCUSSION ............................................................................................. 84
CHAPTER 4 ........................................................................................................... 89
4 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS ................ 89
4.1 GENERAL CONCLUSIONS .................................................................... 89
4.2 FUTURE DIRECTIONS ........................................................................... 90
APPENDIX A ......................................................................................................... 93
5 ATTEMPTS FOR RNA TRACTOR SEQUENCE
MODIFICATION FOR GFP EXPRESSION IN
CHLOROPLASTS ..................................................................................... 93
5.1 INTRODUCTION ...................................................................................... 93
5.2 Addition of SD-like sequence (pCrbcLSD-GFP) .................................... 94
5.3 Addition of 5´-translation control region of large sub-unit
RuBisCO gene ............................................................................................. 96
5.4 Addition of 5´-UTR of Psb A gene for translation initiation of
GFP in chloroplast ................................................................................... 100
5.5 Addition of bacterial translation initiation region (TIR) for GFP
expression .................................................................................................. 103
APPENDIX B ....................................................................................................... 107
6 STRATEGY TO FIND OUT THE CAPACITY OF CHIMERIC
EGGPLANT LATENT VIROID SEQUENCE AS A 5´-UTR FOR
GFP EXPRESSION IN CHLOROPLASTS .......................................... 107
APPENDIX C ....................................................................................................... 112
7 VIRAL AND CHLOROPLASTIC SIGNALS ESSENTIAL FOR
INITIATION AND EFFICIENCY OF TRANSLATION IN
AGROBACTERIUM TUMEFACIENS ................................................... 112
7.1 SUMMARY ............................................................................................... 112
7.2 INTRODUCTION .................................................................................... 113
7.3 MATERIALS AND METHODS ............................................................ 114
7.3.1 Construction of GFP expression plasmids: ..................................................................... 114
viii
7.3.2 Agrobacterium transformation ........................................................................................ 116
7.3.3 RNA isolation, reverse transcription and PCR ............................................................... 116
7.3.4 Detection of GFP expression .......................................................................................... 117
7.4 RESULTS AND DISCUSSION .............................................................. 118
7.4.1 Estimation of equal GFP transcript levels in A. tumefaciens harboring each of the
above constructs .............................................................................................................. 119
7.4.2 Major differences in translation initiation requirements between A. tumefaciens
and E. coli: High GFP translation levels in A. tumefaciens under the control of
phage T7 translational enhancer and RBS ...................................................................... 120
7.4.3 Effect of the AT-rich sequence from the (AIMV) upstream of the GFP gene on its
translation in A. tumefaciens ........................................................................................... 123
7.4.4 Analysis of 5´ -UTR sequences derived from some natural chloroplastic genes on
translation in A. tumefaciens. .......................................................................................... 124
7.4.5 Identification of the minimal translation initiation sequence of the rbcL gene
required for high-level expression in A. tumefaciens...................................................... 124
7.4.6 Comparison of the 5´-UTRs of both rbcL and Psb A genes for translation
initiation in A. tumefaciens ............................................................................................. 126
7.4.7 5´-UTR of the chloroplastic atp1 gene supports low GFP translation levels in A.
tumefaciens ..................................................................................................................... 127
7.5 CONCLUSION ......................................................................................... 128
APPENDIX D ....................................................................................................... 129
8 ANALYSIS OF THE INTERNAL RIBOSOME BINDING SITE
(IRBS) OF PVX ........................................................................................ 129
8.1 BACKGROUND ....................................................................................... 129
8.2 MATERIALS AND METHODS ............................................................ 130
8.2.1 Construction of GFP expression plasmids ...................................................................... 130
8.2.2 Plant transformation for stable gene expression ............................................................. 131
8.2.3 Confocal microscopy ...................................................................................................... 132
8.2.4 Western Blot ................................................................................................................... 132
8.3 RESULTS AND DISCUSSION .............................................................. 133
8.3.1 Expression of GFP using stable gene experiments ......................................................... 133
8.3.2 Western blot analysis ...................................................................................................... 136
APPENDIX E ....................................................................................................... 138
9 NOVEL AND UNIVERSAL APPROACH TO SILENCE ALL
GEMINIVIRUSES IN PLANTS ............................................................. 138
9.1 SUMMARY ............................................................................................... 138
9.2 INTRODUCTION .................................................................................... 139
ix
9.3 MATERIALS AND METHODS ............................................................ 141
9.3.1 Vector construction ......................................................................................................... 141
9.3.2 Plant transformation ........................................................................................................ 143
9.3.3 Characterization of transgenic lines ................................................................................ 143
9.3.4 Agroinoculation .............................................................................................................. 144
9.3.5 Detection of viral genome in infected plants .................................................................. 145
9.4 RESULTS .................................................................................................. 145
9.4.1 Production of transgenic lines ......................................................................................... 145
9.4.2 Transgenic plant evaluation against infectious clones of AEV ...................................... 146
9.4.3 Testing of transgenic plants for resistance against ToLCNDV ...................................... 148
9.5 CONCLUSION ......................................................................................... 150
REFERENCES ..................................................................................................... 154
x
List of Tables
Table 2.1 Oligonucleotides/ primers used in the production of different
constructs. ............................................................................................ 33
Table 2.2 Primer sequences used for semi-quantitative and real time RT-
PCR. .................................................................................................... 44
Table 2.3 Relative quantification (expression) of GFP-transcripts derived
from transgenic leaves harboring given constructs using
comparative real time RT-PCR. .......................................................... 54
Table 2.4 Relative quantification of chloroplast RNA expression of pTR:127
and pC-ELVd-GFP using real time RT-PCR. ..................................... 58
Table 3.1 Primer sequences used for semi-quantitative PCR. ................................. 70
Table 3.2 Summary of the results of the infectivity assays ..................................... 78
Table 7.1 Sequences of the translation initiation signals in the pC-GFP
vector. ................................................................................................ 115
Table 8.1 Oligonucleotides/ primers used in the production of different
constructs with or without a hairpin structure to investigate the
IRBS. ................................................................................................. 131
xi
List of Figures
Figure 1.1 The organization of the Potexvirus genome............................................. 2
Figure 1.2 Genome organization of isolates in various geminivirus. ...................... 16
Figure 1.3 Genome organizations of begomoviruses and their associated
DNA satellites. .................................................................................... 17
Figure 2.1 Genome of Potato virus X with five open reading frames. .................... 23
Figure 2.2 A partial physical map of modified pCAMBIA1300 construct
designated as pC-GFP with 35S Promoter, GFP gene, and T-
nos terminator cassette. ....................................................................... 30
Figure 2.3 Schematic representation of constructs (A-E) in pC-GFP plasmid
previously studied in our lab. .............................................................. 31
Figure 2.4 Schematic representation of the constructs used in this study for
“RNA tractor” activity. ....................................................................... 32
Figure 2.5 Partial DNA sequence of the pTR:127 construct used in this study
as “RNA tractor”. ................................................................................ 32
Figure 2.6 Detection of PVX RNA and coat protein inside the chloroplast
using RT-PCR and western blot. ......................................................... 49
Figure 2.7 RT-PCR analyses of total and chloroplast RNAs expressed. ................ 53
Figure 2.8 Graphical representation of real-time PCR data to quantify
translocated “RNA tractor” sequence using SYBR® Green
detection method. ................................................................................ 56
Figure 2.9 Graphical representation of real-time RT-PCR data (using
SYBR® Green detection method) showing relative
translocation activity of pTR:127 compared to Eggplant latent
viroid (pCELVd-GFP). ....................................................................... 57
Figure 2.10 Mitochondria isolation and RT- PCR- analyses with
mitochondria and total RNA from transgenic tobacco plants
harboring pTR:127 construct. ............................................................. 59
Figure 3.1 Photographs of symptomatic and non-symptomatic different
Nicotiana species: ............................................................................... 76
Figure 3.2 Photographs of symptomatic and non-symptomatic different
Nicotiana species: ............................................................................... 77
Figure 3.3 PCR-mediated detection of AEV and ToLCNDV DNA extracted
from chloroplasts and leaf tissues (total DNA) of infected plants
at 35 dpi. .............................................................................................. 79
Figure 3.4 Reconstruction experiments to reject the possibility of adsorption
of virions or/and DNA during the purification of chloroplasts. ......... 81
Figure 3.5 Phase contrast and electron microscopic studies of chloroplasts. .......... 83
xii
Figure 3.6 PCR-mediated detection of AEV DNA extracted from
mitochondria and leaf tissues of N. benthamiana infected plants
at 35 dpi. .............................................................................................. 84
Figure 5.1 Schematic representation of the 3´end portion of tobacco
chloroplast 16SrRNA (290). .............................................................. 94
Figure 5.2 Details of partial DNA sequences of the pCrbcLSD-GFP
construct under the control of 35S promoter and the nopaline
synthase terminator (T-nos). ............................................................... 95
Figure 5.3 Confocal microscopic observation of Nicotiana tabacum cv.
Xanthi leaves harboring pCrbcLSD-GFP. .......................................... 95
Figure 5.4 Details of partial DNA sequences of the pCvdTCR-GFP and
pC127TCR-GFP constructs under the control of the
Cauliflower mosaic virus 35S promoter and the nopaline
synthase terminator (T-nos). ............................................................... 97
Figure 5.5 Confocal microscopic observation of GFP in N. benthamiana
leaves after 72 hr of agro-infiltration. ................................................. 98
Figure 5.6 Confocal microscopic observation of GFP in agrobacteria cells
after 48 hr. ........................................................................................... 99
Figure 5.7 Details of partial DNA sequences of the pCELVdpsbA-GFP
construct in pC-GFP under the control of the Cauliflower
mosaic virus 35S promoter and the nopaline synthase
terminator (T-nos). ............................................................................ 101
Figure 5.8 Confocal microscopic observation for GFP in transgenic tobacco
plant leaves and agrobacteria cells harboring pCELVdpsbA-
GFP construct. ................................................................................... 101
Figure 5.9 Details of partial DNA sequences of the pET-GFP construct in
pET29 under the control of T7 promoter and T7 terminator. ........... 104
Figure 5.10 Fluorescence micrograph of GFP in E. coli cells transfected with
the pET-GFP construct and induced with 0.5 mM IPTG for 16
hr. ....................................................................................................... 104
Figure 5.11 Details of partial DNA sequences of the pC127pETSD-GFP
construct in pC-GFP under the control of the Cauliflower
mosaic virus 35S promoter and the nopaline synthase
terminator (T-nos). ............................................................................ 104
Figure 5.12 Confocal microscopic observation of GFP in leaves and
agrobacteria cells harboring pC127pETSD-GFP after 72 hr. ........... 105
Figure 6.1 Details of partial DNA sequence of Eggplant latent viroid for
different constructs. ........................................................................... 108
Figure 6.2 The GFP arising from different ELVd-5´-UTR-GFP transcripts. ....... 109
Figure 7.1 Schematic representation of constructs used in this study. .................. 118
xiii
Figure 7.2 Quantitation of equivalent GFP transcript levels for all the
constructs used in this study. ............................................................. 119
Figure 7.3 Detection of green fluorescence due to GFP expression (and
translational efficiency) for each of the constructs (Panels 1-10)
after transformation into Agrobacterium and confocal
microscopy. ....................................................................................... 122
Figure 7.4 Western blots of the enhanced GFP protein (28 kDa) using anti-
GFP antiserum and alkaline phosphatase enzyme-linked
secondary antibody conjugate. .......................................................... 123
Figure 7.5 Confocal microscopic observation of GFP in N. tabacum leaves
after 72 hr of agro-infiltration with a) pC rbcL58-GFP and b)
pC-GFP constructs respectively. ....................................................... 126
Figure 8.1 Confocal microscopic observation of GFP in transgenic N.
tabacum leaves harboring constructs without and with hairpin
structure (Panels A-I). ....................................................................... 135
Figure 8.2 Western blot using anti-GFP antiserum to detect GFP (27 kDa)
expression in transgenic N. tabacum cv. Xanthi plants
harboring constructs in the presence or absence of a hairpin
structure. ............................................................................................ 136
Figure 9.1 A partial Schematic diagram of the binary construct pART27-
AEVIR used for plant transformation. .............................................. 142
Figure 9.2 PCR-verification of transgenic N. benthamiana plants harboring
pTR27-AEVIR construct. ................................................................. 146
Figure 9.3 Semi-quantitative PCR-based testing of wild-type (Wt) and
transgenic N. Benthamiana plants harboring pART27AEV-IR
construct for their resistance against AEV after three weeks of
challenging with infectious clones of AEV DNA-A and DNA-
β in A. tumefaciens strain GV3101. .................................................. 147
Figure 9.4 Infectivity of infectious clones of ToLCNDV in tobacco plants. ........ 149
Figure 9.5 Semi-quantitative PCR-based testing of wild-type and transgenic
N. Benthamiana plants harboring pART27AEV-IR construct
for their resistance against ToLCNDV after three weeks of
challenging with infectious clones of ToLCNDV (DNA-A and
DNA- B) in A. tumefaciens strain GV3101. .................................... 150
Figure 9.6 Organization of a Geminivirus replication origin. ............................... 151
xiv
List of Abbreviations
A. tumefaciens Agrobacterium tumefaciens
AbMV Abutilon mosaic virus
AEV Ageratum enation virus
AlMV Alfalfa mosaic virus
AltMV Alternanthera mosaic virus
ASBVd Avocado sunblotch viroid
BaMV Bamboo mosaic virus
BAP 6-benzylaminopurine
BCTIV Beet curly top Iran virus
BCTV Beet curly top virus
β Beta
β-ME β-mercaptoethanol
BGMV Bean golden mosaic virus
bp base pair
BSA Bovine Serum Albumin
CaMV Cauliflower Mosaic Virus
5´cap m7GpppGp
°C degree Celsius
cc cubic centimeter
CChMVd Chrysanthemum chlorotic mottle viroid
CIP Calf Intestinal Alkaline Phosphatase
cm centimeter
CNV Cucumber necrosis tombusvirus
CP capsid/coat protein
cpDNA Chloroplast deoxyribonucleic acid
Cq quantification cycle
C-sens complementary sense
CTAB Cetyl trimethylammonium bromide (hexadecyl-trimethyl-
ammonium bromide
cv cultivar
xv
ddH2O double distilled water
DEPC Diethylpyrocarbonate
DIC Differential Interference Contrast
DNase deoxyribonuclease
dNTP deoxynucleotide triphosphate
dpi days post-inoculation
dsRNA double-stranded ribonucleic acid
DTT dithiothreitol
E. coli Escherichia coli
ECSV Eragrostis curvula streakvirus
EDTA ethylenediaminetetraacetic acid
EF-G elongation factor G
EF-Tu elongation factor thermo unstable
eIF4E Eukaryotic translation initiation factor 4E
ELVd Eggplant latent viroid
ER endoplasmic reticulum
EtOH Ethanol
FdV Ferredoxin V
FIA Freund’s Incomplete Adjuvant
g gravitational constant (9.8m/s2)
g gram
GFP Green Fluorescent Protein
Gs guanosin(s)
HCl Hydrochloric acid
HC-pro helper-component protease
HEL Helicase
HELD helicase-like domain
HEPES N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid
hr hour
IF-1 or 3 Initiation Factors 1 or 3
ihp intron-containing hair-pin
Inac Inactivated
xvi
IPTG isopropyl-β-D-thiogalactopyranoside
IRBS Internal Ribosome Binding Site
IRES Internal Ribosome Entry Site
kbp kilo base pairs
kDa kiloDalton
KOH Potassium hydroxide
L Liter
LB Luria-Bertani (media)
LBA LB media with 15 g/L agar
µg microgram
µL microliter
µM micromolar
M Molar
mA milliamperes
MES 2-(N-morpholino) ethanesulfonic acid
min minutes
mM millimolar
MP movement protein
mRNA messenger ribonucleic acid
MS Murashige and Skoog
MSV Maize streak virus
MT methyltransferase
mtDNA mitochondrial deoxyribonucleic acid
NAA Naphthalene acetic acid
NaCl sodium chloride
NaOAc Sodium acetate
NbRbCS Nicotiana benthamiana ribulose-1,5-bisphosphate
carboxylase/oxygenase small sub-unit
ncRNA non-coding ribonucleic acid
NEP nuclear encoded polymerase
NIG NSP-interacting GTPase
Ni-NTA nickel-nitrilotriacetic acid
xvii
nm nanometer
NPTII neomycin phosphotransferase
NSP nuclear shuttle protein
nt nucleotide
OCS octopine synthase terminator
OD optical density
ORF open reading frame
ORI origin
PAGE polyacrylamide gel electrophoresis
PCR polymerase chain reaction
PD plasmodesmata
Pdk pyruvate dehydrogenase kinase
PEG Polyethylene glycol
PGK phosphoglycerate kinase
Pi post-inoculation
PLMVd Peach latent mosaic viroid
PMSF phenylmethylsulfonyl fluoride
POL Polymerase
PTGS Post-transcriptional gene silencing
PVP Polyvinylpyrrolidone
PVX Potato virus X
RaLC radish leaf curl
rbcL ribulose-1,5-bisphosphate carboxylase/oxygenase large sub-unit
RbCS RuBisCO small subunit
RBR retinoblastoma-related protein
RBS ribosome binding site
RCR rolling circle replication
RFU relative fluorescence unit
RNase ribonuclease
RNP ribonucleoprotein
rpm revolutions per minute
rRNA ribosomal ribonucleic acid
xviii
RT-PCR reverse transcription polymerase chain reaction
RT-qPCR reverse transcription quantitative polymerase chain reaction
RuBisCO Ribulose1, 5-bisphosphate carboxylase/oxygenase
SD Shine/Dalgarno
SDS sodium dodecyl sulphate
sec second
SEL size exclusion limit
sgRNA subgenomic RNA
siRNA small interfering RNAs
TBS Tris-buffered saline
5´-TCR 5´-translation control region
TCTV Turnip curly top virus
TE-1 1:10 dilution of 10 mM Tris-HCl (pH 8), and 1mM EDTA
TEM Transmission Electron Microscope
TGBp triple gene block protein
Tic translocon of the inner envelope membrane of the chloroplast
TIM transporter inner membrane
TIR translation initiation region
TMV Tobacco mosaic virus
T-nos Nopalin synthase terminator
Toc translocon of the outer envelope membrane of the chloroplast
ToLCNDV Tomato leaf curl New Delhi virus
TOM transporter outer membrane
ToMV Tomato mosaic tobamovirus
TPCTV Tomato pseudo-curly top virus
TPs Transit peptides
Tris Tris (hydroxymethyl)aminomethane
tRNA transfer ribonucleic acid
TRoV turnip rosette virus
TRV Tobacco rattle virus
TYLCV tomato yellow leaf curl virus
3´and 5´-UTR 3´and 5´-untranslated region
xix
V Volt
v/v volume/volume
VIGS virus-induced gene silencing
vRNA viral ribonucleic acid
V-sense viral sense
w/v weight/volume
1
CHAPTER 1
1 LITERATURE REVIEW
1.1 POTEXVIRUSES
1.1.1 Replication
Potexviruses belong to the Alphaflexiviridae, a new family of plant RNA viruses has been
extensively studied. The genomes of the genus Potexvirus contain five open reading frames
(ORFs) encoding an RNA-dependent RNA polymerase (RdRp; replicase), three overlapping
proteins, named triple gene block (TGB1-3), and the coat protein (CP) (1, 2) as shown in Figure
1.1.
2
Figure 1.1 The organization of the Potexvirus genome.
(A) The RNA-dependent RNA polymerase (RdRp, replicase) gene contains a methyltransferase domain
(MT), a helicase domain (HEL), and an RNA polymerase domain (POL). The three genes of the triple
gene block (TGB) are partially overlapped. Arrows indicate subgenomic (sg) RNAs for expression of
TGBs. (B) The organization of the three TGB genes. TGB1: The first TGB ORF encodes the TGB1
protein and has a helicase- like domain (HELD), which contains seven typical motifs of a general helicase
(I, Ia, II, III, IV, V, and VI; dark boxes). TGB2: the TGB2 protein is encoded in the second TGB ORF and
has two transmembrane domains (dark boxes). The GDx6GGxYxDG sequence is conserved inTGB2-
encoding viruses.TGB3: The TGB3 protein is encoded by the third TGB ORF and contains a
transmembrane domain (dark box). Among the TGB3-encoding potexviruses, the TGB3 gene has a
conserved C(x5) G (x6−9) C sequence (3).
These viral proteins are used either in viral replication or in movement in infected host plants (4-
7). At the early stage of infection, potexviruses, which have a (+) positive stranded RNA
genome, release viral RNA (vRNA) from the virion and synthesize the virus-encoded replicase
using host translation machinery. Replicase then forms a viral replication complex along with
host factors and subsequently synthesizes (i) minus (-) stranded vRNA from (+) vRNA and (ii)
(+) vRNA or (+) subgenomic (sg) RNA from synthesized (-) vRNA. CP and TGB1-3 proteins
3
are derived from (+) sgRNAs and are used for encapsidation and movement of their progeny (+)
vRNAs, which were synthesized from (-) vRNA as a template, into nearby uninfected cells
through the plasmodesmata (PD). In moving the progeny (+) vRNAs or virions via PD into
adjacent cells, most plant viruses use their own movement proteins. In the case of potexviruses, it
has been established that viral cell-to-cell movement requires TGB proteins and CP (3, 5, 6, 8-
11). Solovyev, et al. (6) abridged the information about TGB proteins and TGB-mediated plant
viruses. The TGB proteins have been divided into two main potex- and hordei-like TGBs groups,
based on phylogeny and on differences in the viral movement mechanism (5, 12). The potex-like
viruses form filamentous virions containing a monopartite RNA genome and depend on CP for
cell-to-cell movement, whereas hordei-like viruses are rod-shaped, consist of multipartite RNA
genomes, and do not require the CP for cell-to-cell movement (2, 5, 12, 13). Verchot-Lubicz, et
al. (5) summarized and compared the movement strategies employed by TGB proteins in potex-
like viruses and hordei-like viruses. Recently, Park, et al. (14) have described the recent findings
on the cell-to-cell movement of potexvirus vRNA and/or virions through the PD including the
intracellular trafficking and intercellular transport of vRNA. TGB1 protein is translated from
sgRNA1, whereas TGB2 and TGB3 proteins are co-translated from sgRNA2 (15). Potexvirus
TGB1 protein is encoded by the first TGB ORF and contains a helicase-like domain (HELD) and
this protein is also important for viral movement (16, 17). Potexvirus TGB1 protein also
functions as a suppressor of RNA silencing (18, 19). Potexvirus TGB2 protein is an important
membrane protein that carries two predicted transmembrane domains that interact with ER
membranes and has sequence-independent RNA-binding activity (12, 20-22). TGB3 protein,
which is translated by the third TGB ORF, is also an integral protein in ER membranes and is
important for cell-to-cell viral movement (23, 24). Studies have shown that localization of TGB2
4
and TGB3 proteins into ER is critical for viral cell-to-cell movement (20, 23). In addition, both
TGB2 and TGB3 proteins may be responsible for gating the PD (25, 26).
1.1.2 Intercellular Transport of Potexvirus
For intercellular movement of viral RNA, most plant viruses need to increase the PD size
exclusion limit and exit through the PD. Potexvirus and plant viruses, in general, pass their
vRNA through the PD as its RNP movement complex (27) or the virion form (28). Lough, et al.
(29) showed that TGB1 is an integral protein for plasmodesmal gating rather than coat protein
which is involved in RNP movement complex. Potexviruses employ a complex cell-to-cell
movement strategy with the involvement of the triple gene block (TGB) (27). TGBp1 defined as
the potexvirus movement protein, potentiates the intercellular movement of viral RNA in the
presence of TGBp2 and TGBp3 (3, 27, 29-33). Studies provide evidence that the fifth ORF of
potexvirus protein, the coat protein (CP), is also required for potexvirus cell-to-cell movement
(9, 34, 35). The TGBp1-RNA complex appears to be delivered to PD by means of vesicle
trafficking along the ER-microfilament pathway (36, 37). In this model, TGBp2 and TGBp3 are
integral membrane proteins that serve to anchor the TGBp1-RNA complex to the vesicle surface
(3, 38-40) and, following cargo delivery to PD, the TGBp2 and TGBp3 are suggested to be
recycled through the endocytic pathway (38). In a new model for cell to cell movement of PVX
vRNA at the entrances of PD at the late stage of infection, that was proposed by Tilsner, et al.
(41), vRNA processing and movement are highly compartmentalized at PD, i.e., replication
occurs at the PD so that vRNA is rapidly passed through PD and to the nearby cells instantly
after replication. In contrast to earlier models, the new model indicates that virus replication and
movement are not spatially separated within the cell. However, some concerns about interactions
between TBG proteins still need to be experimentally confirmed, i.e., how three TGB proteins
5
coordinate to facilitate vRNA transport (41) and whether other factors including host protein(s)
are required for these interactions and for vRNA transport.
1.1.3 Intracellular trafficking of viral RNA in potexviruses
After the replication of (+) vRNA, (+) vRNA is changed to the PD-transportable potexvirus
vRNA form by TGB1 protein for cell-to-cell movement through the PD. Two models (virion or a
ribonucleoprotein (RNP) movement complex containing vRNA, TGB1 protein, and CP) have
been suggested for the formation of PD-transportable potexvirus vRNA during the cell-to-cell
movement of vRNA through the PD (14). Lough, et al. (42) showed that vRNAs of potexviruses
were transported by the formation of RNP movement complex involving vRNA, TGB1 protein,
and CP rather than intact virion alone. In contrast, experimental evidence has shown that the PD-
transportable potexvirus vRNA form is partially or fully encapsidated by the CP subunit and that
the TGB1 protein is associated with the 5′ end of the CP-coated vRNA (5, 43). As the cell-to-cell
movement of potexvirus vRNA through the PD requires three TGB proteins and the CP. Studies
also indicate that potexvirus TGB1 protein requires viral CP in the RNP movement complex to
move together with their vRNA into PD (44, 45). Various host proteins might also be required
for the formation of the RNP movement complex, but how host proteins cooperate with the RNP
movement complex remains unanswered. It has been demonstrated that both TGB2 and TGB3
proteins are important membrane proteins in the ER or ER-associated vesicles located at actin
filaments (12, 46). Considering the role of TGB2 and TGB3 proteins for potexvirus vRNA
trafficking to PD, two models have been designed (5, 45). Verchot-Lubicz, et al. (5) summarized
the first model with two pathways of potexvirus vRNA trafficking to PD based on the
interactions between TGB2 and TGB3 proteins. One pathway suggests that the potexvirus RNP
movement complex is transported by TGB2-induced granular vesicles as directed by TGB3
protein (TGB2/3 granular vesicles) to PD. The first pathway, therefore, suggests that the
6
potexvirus RNP movement complex is released from membrane bound bodies by TGB3 protein
and that the released RNP movement complex then binds to the TGB2/3 granular vesicles in the
ER tubule and moves to the PD (5). The second pathway for the vRNA trafficking of potexvirus
to PD by TGB3 protein is supported by interaction and localization assays between TGB2 and
TGB3 proteins (24, 47). In the second model, the stable association of the virion cargo with the
TGB2- and TGB3-based membrane complex and recruitment of TGB1 protein to the PD has
been proposed for cell-to-cell movement of bamboo mosaic virus (48). They also found that the
stable TGB2-TGB3-virion complex associates with TGB1 protein for targeting PD and
suggested the refined model for potexvirus vRNA trafficking to PD (48).
1.1.4 Interaction between viral and chloroplast proteins
Various specific interactions are known to occur between viral and chloroplast proteins. Qiao, et
al. (49) have reported that Potato virus X coat protein (PVXCP) interacts with the precursor of
plastocyanin, a protein involved in photosynthesis, and thus is involved in the virus movement
and symptom development processes. Tomato mosaic virus coat protein (CP) interacts with a
long distance movement-related protein in tobacco, designated IP-L, and localizes at the
thylakoid membranes and it is believed to develop the chlorotic symptoms in infected plants
(50). The role of the chloroplast protein, N receptor-interacting protein 1, in the activation of
defense signaling is affected by direct interaction with both the plant N immune receptor and the
helicase domain of Tobacco mosaic virus (TMV) (51). In addition, this viral domain may also be
associated with the 33K subunit of the oxygen-evolving complex of photosystem II, as a
decrease in 33K subunit mRNA was observed after infection of Nicotiana benthamiana with
TMV (52). Similarly, an increase of Plum pox virus titer was observed after downregulation of
the psaK gene of photosystem I in infected plants (53). The HC-Pro of Potato virus Y has been
shown to interact with the chloroplast division-related factor, NtMinD in the chloroplasts of
7
infected leaves (54). The HC-Pro protein of Sugarcane mosaic virus likewise interacts with the
precursor of Ferredoxin-5 (Ferredoxin V) (FdV) in maize and affect the import of FdV into
chloroplasts, which could lead to disruption of chloroplast structure and function (55). Kong, et
al. (56) showed that silencing of PsbA, a 23-kDa oxygen-evolving complex protein, expression
increased Rice stripe virus (RSV) accumulation and disease symptom severity in infected plants,
suggesting an interaction between disease-specific protein (SP) of (RSV) and PsbA.
Additionally, accumulation of SP during RSV infection resulted in perturbation of chloroplast
structure and function. Zhao, et al. (57) showed that Tomato mosaic tobamovirus (ToMV)
movement protein (MP) interacted with the RuBisCO small subunit (RbCS) of Nicotiana
benthamiana in vitro and in vivo, as silencing of Nicotiana benthamiana RbCS
(NbRbCS) enabled ToMV to induce necrosis in infected leaves, thus suggesting
that NbRbCS plays a key role in tobamovirus movement and plant antiviral defenses.
Alternanthera mosaic virus (AltMV) TGB3 protein was localized near the chloroplast membrane
in mesophyll cells, suggestive of facilitating virus movement between different cell types (58).
Jang, et al. (59) revealed an interaction between AltMV TGB3 and Photosystem II (PSII)
oxygen-evolving complex (OEC) protein (PsbO), a nuclear-encoded major component of the
chloroplast-localized OEC of PS II, surrounding chloroplast in mesophyll cells, raising the
possibility that the interaction induces symptom development. Together, these findings show that
the mechanisms for viral movement may differ among potexviruses (60).
1.1.5 Virion and viral RNA within chloroplasts
Previously, it was shown that tobacco mosaic virus (TMV) was accumulated in the
chloroplasts of infected plants (61). However, it was later revealed that some of the isolated
virions were only one-third the length of the wild-type virus and that not all of the virus-like
particles actually contained TMV RNA (62). It was also demonstrated that the TMV coat
8
protein was able to encapsidate some chloroplastic RNAs, and encapsidation was more likely
to occur with chloroplastic transcripts than with nuclear transcripts (63). It was then proposed
that the TMV coat protein is able to encapsidate chloroplast RNA transcripts inside the
chloroplast itself, and this leads to the formation of pseudovirions within the organelle (64).
Thus, it was established that both the TMV coat protein and virus-like rods are present within
the chloroplasts of infected plants (65). Recently, Cheng, et al. (66) observed that Bamboo
mosaic virus (BaMV) viral RNA with the coordination of chloroplast phosphoglycerate kinase
localizes to chloroplasts of infected cells of Nicotiana benthamiana plant, suggesting that host
factors play a key role in targeting of viral RNA to the cellular organelles.
1.1.6 Targeting of nuclear-encoded proteins to organelles
Plant cells contain two types of endosymbiotic organelles, chloroplasts, and mitochondria, where
as a result of endosymbiotic gene transfer, the majority of their proteins are encoded in the
nucleus which post-translationally must be transported into the respective organelle after
synthesis in the cytoplasm (67-69). The most common pathway of this transport involves N-
terminal targeting signals, also known as transit peptides, which are usually cleaved off after
import into the organelle. Such signal peptides are recognized by import receptors on the
organellar outer membrane, and precursors are targeted into the organelle through translocase
complexes located on the outer and inner membranes of the organelles, such as Toc (translocon
of the outer envelope membrane of the chloroplast) and Tic (translocon of the inner envelope
membrane of the chloroplast) in the chloroplasts and Tom (transporter outer membrane) and Tim
(transporter inner membrane) in the mitochondria (70). The following translocation into the
chloroplast stroma or mitochondrial matrix, the targeting signals are cleaved off by either the
stromal processing peptidase or the mitochondrial processing peptidase, respectively. It is known
that targeting signals for mitochondria and chloroplasts are distinct from that for the endoplasmic
9
reticulum, with respect to sequence composition and predicted secondary structure. Despite
similarities observed between chloroplastic and mitochondrial targeting signals, a given protein
is targeted specifically into either mitochondria or chloroplasts (67, 70). However, a number of
proteins have been identified that exhibit dual targeting properties, i.e., they are imported into
both chloroplast and mitochondria (71, 72). In some cases, such dual targeting results from
transit peptides comprising two independent transport signals in tandem. As a result of
differential transcription, splicing and/or translation processes, either of the two signals can be
exposed at the N-terminus of the precursor protein, where it decides the target organelle. In some
cases, however, the dual attribute is due to ambiguous transit peptides, which are able to interact
with the protein transport machinery of both endosymbiotic organelles (71, 72). Recently,
Baudisch, et al. (67) have estimated the number of proteins in Arabidopsis with dual importing
attributes by a combination of extended in silico analyses and protein transport experiments.
1.1.7 mRNA-based protein targeting to different organelles
Previously, it was assumed that proteins are synthesized at random locations in the cytosol and
then imported into the different organelles using localization information in the polypeptide
sequence (73). Over the past decades, mRNAs and ribosome subunits were observed to target to
the ER membrane in the absence of translation and, hence, the signal peptide and nascent chain.
These results raised the possibility that proteins are targeted to the ER by the localization of the
mRNAs encoding them (74, 75). Supporting these possibilities, it was shown that most mRNAs
encoding mitochondrial proteins were not equally distributed in the cytoplasm but enriched in
the vicinity of mitochondria (76-78). Further studies also showed the mRNA localization in the
proximity of chloroplast and peroxisomes (79, 80). In addition to targeting the protein, this
mRNA-based targeting may also function to (i) keep out the protein from intracellular regions
where it would be toxic, (ii) overcome the requirement for other targeting mechanisms, (iii)
10
guarantee expeditious translational responses to changing abiotic or biotic conditions, (iv) allow
the regulation of the protein synthesis by cellular and extracellular stimuli that reflect demand for
the product, (v) impart economic benefits from not having to localize the many copies of a
protein translated from a single mRNA and (vi) substantiate translation sites that are secluded
from other regions under stress. Localization is specified by a cis-acting sequence in the mRNA
called a localization element or Zipcode (81). Zipcodes range from only a few nucleotides (82) to
highly complex and redundant sequences of up to 1 kb (83). These codes are most often located
within the 3'-UTR and in most cases sufficient for the localization of a reporter mRNA.
Currently, many of the 3'-UTR features leading to mRNA localization are known (84) and were
found by experiments using fluorescence microscopy or cross-linking and immunoprecipitation
(85, 86).
1.1.8 The accumulation of Avsunviroidae viroids within the chloroplasts
Viroids are single-stranded, circular RNA plant pathogens that are approximately
247-401 nucleotides in length (87). Viroids are divided into two families, the Pospiviroidae,
and the Avsunviroidae. The four members of the Avsunviroidae family are the Avocado
sunblotch viroid (ASBVd), the Peach latent mosaic viroid (PLMVd), the Chrysanthemum
chlorotic mottle viroid (CChMVd), and the Eggplant latent viroid (ELVd) (88). Viroids do not
code for any proteins and they depend on their host factors for replication (89). It has also
been shown that members of the Avsunviroidae family accumulate and replicate within the
chloroplasts of infected plants (90, 91) and that these viroids may use the nuclear-encoded
polymerase (NEP) of the chloroplast for their replication (92). Therefore, it has been proposed
that these viroids enter the chloroplast using some endogenous RNA translocation pathway,
however, the mechanism of this RNA import has yet to be described (93). Furthermore, there is
very little sequence conservation between the four members of the Avsunviroidae outside of
11
their hammerhead structures, therefore secondary structure might play a more important role in
the import of the RNA into the chloroplast (94). In a recent work, it has been shown that
Eggplant latent viroid RNA sequence acting as a 5'-UTR end mediates the specific trafficking
and accumulation of a functional foreign mRNA into N. benthamiana chloroplasts (95, 96).
1.1.9 Non-coding RNAs in plastids
As a result of relaxed transcription and translation in plastids, many transcripts may
arise from a single promoter from both strands. After their downstream processing, a number
of stable RNA species are synthesized including a distinct class of plastid-encoded non-
coding (nc) RNA, however, their role still needs to be determined in plastid gene regulation
(97). Surprisingly, strand-specific RNA sequencing has shown a large number of ncRNAs in
Arabidopsis and barley chloroplasts (98, 99). Most of these transcribed ncRNAs are antisense
to the protein-coding genes. Such antisense transcripts bind near the 3’end of the mRNA and
stabilize the target transcripts by protecting the 3’ ends from 3’ 5’exoribonulceases (99).
1.1.10 Translation in chloroplast
Chloroplasts are membrane-enclosed organelles that are characteristic of
photosynthetic plants and algae (100). Of all the organelles contained within a eukaryotic cell,
chloroplasts and mitochondria are unique because they carry some of their own genetic
information and are able to synthesize some of their own proteins (101). Chloroplasts contain
double-stranded, circular chromosomes that range in size from 120 to 160 kbp and typically
contain four segments: a large region of single copy genes, a small region of single copy genes
and 2 copies of inverted repeats (101). These genomes encode various components that are
necessary for protein syntheses such as 4 ribosomal RNAs (rRNAs), 30 transfer RNAs
(tRNAs), 21 ribosomal proteins and 4 RNA polymerase subunits. The chloroplast genome also
12
encodes proteins that are involved in photosynthesis such as 28 thylakoid proteins and the
ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (102). Although
chloroplasts possess their own genome and protein synthesis machinery, these organelles are
unable to exist autonomously outside of the eukaryotic cell. This is likely due to a considerable
relocation of genetic information from the chloroplastic genome to the host nucleus (103). This
suggests that the maintenance of the chloroplast is likely to require rigid coordination of both
transcription and translation in the nucleus as well as in the chloroplast (104). Despite
importing numerous peptides, the chloroplast also utilizes prokaryotic protein synthesis
machinery to generate many of its own proteins. Although prokaryotic protein synthesis
follows the same three steps required for eukaryotic translation (Initiation, Elongation, and
Termination), there are a few major differences. For example, while eukaryotic messenger
RNAs (mRNAs) possess 5’caps (m7GpppGp) and 3’poly-A tails, prokaryotic RNA transcripts
are missing both of these structures. Without the 5’cap, a prokaryotic ribosome identifies the
translational start site within an mRNA transcript by binding to a Shine-Dalgarno sequence
(typically GGAGG in chloroplasts) upstream of the initiator AUG (101, 105). The prokaryotic
and eukaryotic systems also differ in the sizes of their ribosomal subunits, in the number of
initiation factors involved in translation, and in the number of cistrons contained within their
mRNA transcripts (105).The chloroplast utilizes a prokaryotic system to synthesize proteins
encoded in its own genome. Prokaryotic translation can be divided into 3 stages: Initiation,
Elongation, and Termination. Protein synthesis initiates when the 16SrRNA of the 30S small
ribosomal subunit base pairs with the Shine-Dalgarno sequence upstream of the initiator AUG
in the mRNA transcript. Meanwhile, Initiation Factor 2 (IF-2) binds to a tRNA aminoacylated
with formylmethionine (tRNAfMet
) and facilitates the base pairing between this tRNA and the
start codon of the mRNA (105). Finally, the 50S large ribosomal subunit unites with the
13
previously mentioned components to complete the initiation complex. The formation of this
complex is promoted by two additional initiation factors. Initiation Factor 3 (IF-3) binds to the
30S subunit and prevents it from joining the 50S subunit when no mRNA transcript is present
and Initiation Factor 1 (IF-1) promotes the dissociation of the 70S ribosome (106). In the
second phase of protein synthesis, the peptide chain is elongated through the addition of amino
acids. First, a new amino-acyl tRNA molecule bound to an EF-Tu elongation factor enters the
ribosome, and if the correct codon-anticodon pairing is made, a molecule of Guanosine
Triphosphate (GTP) within the EF-Tu is hydrolyzed and the elongation factor dissociates from
the tRNA (105). The amino-acyl tRNA then moves into the A site of the ribosome and peptidyl
transferase catalyzes the formation of a new peptide bond between the amino acids in the A
and P sites. Next, another elongation factor, EF-G, enters the ribosome, which triggers the
hydrolysis of an attached GTP molecule. This hydrolysis than triggers a drastic change in the
conformation of the ribosome that shifts the tRNAs located in the A and P sites to the P and E
sites, respectively. The uncharged tRNA that is now located in the E site is expelled from the
ribosome and the A site is now free to accept a new amino-acyl tRNA molecule (105). The
final stage of protein synthesis is called termination and this occurs when one of the three
termination codons enters the A site of the ribosome (105). Since these codons are not
recognized by any tRNA molecule, an additional amino acid is not added. Rather, these codons
are recognized by release factors that cleave the polypeptide from the final tRNA and release
the newly synthesized protein. Release Factor 1 (RF-1) recognizes the UAA and UAG codons
while Release Factor 2 (RF-2) recognizes the UAA and UGA codons. A third Release Factor
(RF-3) promotes the release of RF-1 and RF-2 as the final step in the translation process (107).
14
1.1.11 RNA transport into mitochondria
Mitochondria of the most eukaryotic cells play an integral role in cellular processes including
respiration, oxidative phosphorylation-mediated ATP production, cellular metabolism and
apoptosis (108). These organelles carry their own genome which varies depending on species
(e.g. 17 kb in human and 367 kb in Arabidopsis thaliana), but normally encode only a limited set
of proteins (e.g. 13 in human and 32 in A. thaliana), suggesting that the most of the
mitochondrial proteins are encoded in the nucleus and translocated into the mitochondria (109).
In addition to nuclear-encoded proteins, synthesis of mitochondria-encoded proteins is essential
for organelle functions which require rRNAs and a complete set of tRNAs. Plant mitochondrial
genomes lack several tRNA genes, consequently, nuclear-encoded tRNAs are imported from the
cytosol (110). Based on genetic origin, in plant mitochondria, there are three tRNAs: 1. native
mitochondrial tRNAs coded for by the mitochondrial genome, 2. chloroplast-like tRNAs,
initially coded for by chloroplast DNA and finally inserted into the mitochondrial genome during
evolution, and 3. cytosol-like tRNAs, coded for by the nuclear genome, which are required to
import from the cytosol into the mitochondria (110). So far it is known that only noncoding
RNAs are translocated into mitochondria. The import of cytosolic 5S rRNA into mitochondria
has been demonstrated in mammals, however, its functional importance remains unanswered
(111). Additionally, two other cytosolic RNAs, the RNA component of the nuclease
mitochondrial RNA processing and the RNA component of RNase P, are imported in humans
but their existence within the mitochondria remains questionable (112, 113). In higher plants,
one-third to one-half of the mitochondrial tRNAs are encoded in the nucleus and then imported
into mitochondria (114). Of the nuclear-encoded tRNAs imported from the cytosol, tRNAs
aminoacylated with Glycine and Valine (tRNAGly and tRNAVal, respectively) have been most
thoroughly studied. Salinas, et al. (115) demonstrated that import of tRNAGly into tobacco
15
mitochondria is sequence-dependent. They found that tRNAG1y (UCC) and tRNAGly (CCC) were
detected in the cytosol and mitochondria, while tRNAGly (GCC) was only present in the cytosol.
It has been demonstrated that point mutations in the anticodon of tRNAVal abolish both
aminoacylation and import and that D- and T-domains are essential for tRNAVal import (114).
Furthermore, It has been found that both the anticodon and the D-domain regions contain
essential determinants for tRNAVal(AAC) import into plant mitochondria (114). Mitochondrial
tRNA import has been experimentally documented in several organisms including protozoa, the
yeast Saccharomyces cerevisiae, higher plants, and marsupials, however, little is known about
the mechanism of tRNA translocation across plant mitochondrial membranes. The import of a
tagged bean tRNALeu into mitochondria of transgenic potato was the first direct evidence of this
phenomenon (116). Later, it was found that in order to be imported, a nuclear-encoded tRNA
first needs to interact with mitochondrial membrane receptors which require ATP-dependent step
(s) (117). The tRNA would then pass through the transporter outer membrane (TOM) and
transporter inner membrane (TIM) complex via a still unknown mechanism.
1.2 GEMINIVIRUSES
The family Geminiviridae is comprised of plant DNA viruses that have long been known as
model systems for the elucidation of basic cellular components of the plant replication and
transport machinery (118-121). This family consists of phytopathogenic viruses with
characteristic twinned, quasi-isometric virions encapsidating genomes of circular single-stranded
(ss) DNA. These viruses replicate through an intermediate dsDNA molecule in the nuclei of
infected host plant cells and rely on the host DNA replication machinery (122). Geminiviridae is
classified into seven genera, six of which (Mastrevirus, Curtovirus, Topocuvirus, Becurtovirus,
Eragrovirus, and Turncurtovirus) consist of viruses with monopartite genomes while the seventh
one (Begomovirus) comprises of either monopartite or bipartite (Fig.2). Geminiviruses, with the
16
smallest known genome of plant-infecting viruses, replicate independently in the host cells by
using bidirectional mode of transcription from some of the overlapping genes for efficient coding
of proteins (121).
Figure 1.2 Genome organization of isolates in various geminivirus.
lineages (LIR, long intergenic region; SIR, short intergenic region; CR, common region; rep, replication-
associated protein (C1or AC1); ren, replication enhancer (C3 or AC3); trap, trans activator protein (C2 or AC2);
ss, silencing suppressor; sd, symptom determinant (C4 or AC4); cp, capsid protein V1 or AV1); mp, movement
protein V2 or BC1); reg, regulatory gene (V3); nsp, nuclear shuttle protein (BV1) (123).
17
1.2.1 Genus Begomovirus
Viruses of the genus Begomovirus consists of either monopartite (a single DNA) or bipartite
(with two DNA components: DNA-A and DNA-B) genomes (123-127). The DNA-A of bipartite
and the single component of monopartite begomoviruses contain five or six Open Reading
Frames (ORFs) while the DNA-B contains two ORFs (BV1 and BC1, in V-sense and C-sense
strand, respectively). Both DNA-A and DNA-B are approximately 2.8-3.0 kb in size.
Monopartite begomoviruses are often associated with one or smaller DNA components, about
1.4 kb in size, known as satellite DNAs (Figure 1.3).
Figure 1.3 Genome organizations of begomoviruses and their associated DNA satellites.
Lollipop, origin for rolling-circle replication; C2, possible transcriptional activator protein; C4/AC4, possible
symptom determinant; CP, coat protein; NSP, nuclear shuttle protein; AV2, anti-defence proteins; V2, movement
protein; Rep, Replication initiator protein; TrAP, transcriptional activator protein; REn, Replication enhancer
protein; MP, movement proteins; βC1, Betasatellite encoded protein (128).
Two types of satellite DNAs are known: the alpha-satellites and beta-satellites, depending upon
the organization of their DNA and their effects on the symptoms produced by the helper
18
begomovirus. Both the alpha- and betasatellites are dependent upon the helper virus for
replication and, in many cases, mitigate the symptoms produced by it (129). DNA-A and DNA-B
components in bipartite begomoviruses differ from each other, except a short sequence of ~200
nucleotides with high sequence identity that is referred to as “common region” (CR). The
genomes of monopartite (and DNA-A components of bipartite) begomoviruses are typically
∼2.8 kb in size and have genes in both orientations from a non-coding intergenic region (IR),
which contains promoter elements and the origin (ori) of virion-strand DNA replication. The
virion strand ori consists of a predicted hairpin structure containing a conserved (between
geminiviruses) nonanucleotide (TAATATTAC) sequence in the loop and repeated upstream
motifs known as “iterons”. The DNA-A component of begomoviruses consists of either five or
six ORFs in both orientations. These proteins are required for multiple functions: viral
replication; virus assembly; host gene regulation and silencing suppression; and vector
transmission. Despite the genes are named on the basis of their functions, however, their
functions can differ within the genus Begomovirus (130, 131). The virion-sense strand of most
begomoviruses encodes the following two proteins:
Coat protein (CP; V1): Coat protein is required for encapsidation, insect transmission and
movement in plants (128, 132, 133). It is also believed that CP interferes with nicking of DNA
thus limiting the viral DNA copy number during rolling circle replication (RCR) (121, 134). It
also functions as the nuclear shuttle protein (NSP) for monopartite viruses (135).
Pre-coat protein (Pre-CP; V2): A pathogenicity determinant, which is believed to involve in
virus movement in plants (121, 128) and/or acts as a suppressor of RNA silencing (134, 136). It
also contributes in the perinuclear distribution of begomoviruses by association with the
endoplasmic reticulum (ER) and cytoplasmic strands (137).
The complementary sense strand encodes four proteins:
19
Replication-associated protein (Rep; C1):
The only virus-encoded gene product required for viral DNA replication. Rep is an RCR-initiator
protein that recognizes the reiterated motifs (iterons) and nicks within the nonanucleotide
sequence to initiate replication (138, 139). It also conducts ATPase and helicase activities and
binding of retinoblastoma-related proteins (140).
Transcriptional activator protein (C2; TrAP):
This protein up-regulates the late (virion sense) genes (for bipartite begomoviruses) and also acts
as a suppressor of RNA silencing in bipartite (128, 141) as well as monopartite begomoviruses
(142). It also prevails over virus-induced hypersensitive cell death (143, 144).
Replication enhancer protein (REn; C3): It is involved in establishing an environment
conducive for optimal virus replication by interacting with host-encoded proteins (145-147).
C4 protein: The role of the C4 protein is unknown but for some viruses it is a pathogenicity
determinant and also counteracts PTGS (148-150).
As mentioned earlier, the bipartite begomovirus genome comprises of two components. Both
components are required for different functions; DNA-A component is responsible for
replication and transcription while DNA-B is required for inter- and intracellular movement of
the virus. DNA-A and DNA-B together are required for a successful systemic infection. The
DNA-B component contains two ORFs in opposite orientations encoding.
Nuclear shuttle protein (NSP; BV1): NSP is responsible for transport of viral DNA from the
nucleus into the cytoplasm (151-153).
Movement protein (MP; BC1): BC1 coordinates the movement of viral DNA across
plasmodesmata boundaries (152) and it is also responsible for viral pathogenic properties (154).
Its function is also mediated by V2 alone or in a complex with C4 (155).
20
1.2.2 Begomovirus infection
As with all other geminiviruses, which require an insect vector to be transmitted to other plants,
begomoviruses rely entirely on their arthropod vector the whitefly Bemisia tabaci for their plant-
plant transmission. The feeding of a viruliferous whitefly vector, B. tabaci, on the phloem cells
of a suitable host plant leads to the beginning of the begomovirus infection cycle. As soon as the
feeding starts, viral particles enter into the vascular system of the plant. From the cells in the
vascular system, the viral particles are transmitted to the mesophyll cells. Once these viral
particles are in the cells they become uncoated and viral DNA enters the nucleus where viral
DNA replication and transcription occur (156). For monopartite begomoviruses CP is
responsible for the transfer of viral DNA into the host cell nucleus and later into the cytoplasm.
Bipartite begomoviruses do not need CP for movement and they use NSP to act as a shuttle for
virus movement from the nucleus into the cytoplasm (151). In the nucleus, the complementary
strand is synthesized following primer synthesis to produce a dsDNA intermediate, which serves
as a template for transcription of viral proteins (157). Once the dsDNA is formed, bi-directional
transcription starts with the help of promoter sequences located in the IR. The viral transcripts
are transported into the cytoplasm for translation (133). The translated proteins enter the nucleus
to carry out replication, packaging, and movement of viral DNA. The Rep protein of the
begomovirus binds to the ori and starts RCR mode of replication. After accumulation of ssDNA
CP switch RCR and shuttles ssDNA into the cytoplasm (long distance movement of
begomovirus DNA will be discussed in detail in the preceding section). The CP starts packaging
of the viral DNA to produce virions and the virus is either transported to the next cell through
plasmodesmata or taken up by the whitefly to be transmitted to the next plant.
21
1.2.3 Long distance movement within plants
The movement of geminiviruses within host plants has been studied extensively (120, 137, 151,
155, 158-161). These viruses use the DNA replication machinery of their host to amplify their
genomes in the nuclei of infected plant cells (162). When the viral DNA reaches an optimum
level in the nucleus it is transported out of the plant cell nucleus to undergo systemic spread by
crossing plasmodesmata openings in the cell membrane. Bipartite begomoviruses are dependent
upon DNA-B encoded NSP and MP for their movement in host plants (152, 153, 156). The NSP
supports viral DNA export from the nucleus into the cytoplasm from where MP transports viral
DNA to neighboring cells via plasmodesmata (36, 163). It has been shown that βC1 of CLCuMB
can substitute the movement function of DNA-B to facilitate movement of begomovirus from the
nucleus to the cell periphery (159). Monopartite begomoviruses cross cell membranes with the
help of interaction between CP and Pre-CP (137). The CP of monopartite begomoviruses
localizes to the periphery of the nucleus and nucleolus, thus acting as a nuclear shuttle
homologous to NSP of bipartite begomoviruses. Pre-CP localizes around the nucleus and at the
cell periphery with the ER. Such a localization pattern is similar to MP of bipartite
begomoviruses, probably assigning movement function to these proteins (135, 137). The
transport of viral ssDNA from the nucleus towards plasmodesmata is facilitated by a nuclear
export signal (NES) on the CP C-terminus and NES on the Pre-CP N-terminus (132, 137).
1.2.4 Translocation of begomoviruses into chloroplast
The DNA of Abutilon mosaic virus (AbMV), a geminivirus that has a circular single-stranded
DNA genome, was isolated from intact chloroplasts (164) representing the only other example of
a geminiviral genome in chloroplasts. Chloroplasts were purified from AbMV- infected and
uninfected control Abutilon sellovianum var. marmorata plants. The single-stranded AbMV DNA
was examined in the plastids of infected plants. The possibility of adsorption of virions or DNA
22
on the external surface of intact chloroplasts was ruled out by treating them with DNase I and
protease. Furthermore, the lamellar system of plastids from AbMV-infected plants was
degenerated, suggesting that the virus affected the structure of the plastids in AbMV-infected
plants. Bhattacharyya, et al. (165) found that chloroplast structure was severely damaged with
the coinfection of Tomato leaf curl New Delhi virus DNA-A and the betasatellite which is
associated with radish leaf curl disease (RaLC), conversely, the structure of chloroplasts
remained undamaged when the host cells were infected with Tomato leaf curl New Delhi virus
DNA-A alone. Furthermore, these findings demonstrate that protein βC1 encoded for by
betasatellite is responsible for damaging the structure and is capable of targeting to chloroplasts,
suggesting that a DNA virus-encoded protein is responsible for causing structural and functional
damage to this vital organelle. With its unique origin as an endosymbiont converted into a
subcellular organelle, a chloroplast is speculated to have potential to carry tools necessary for
replication and transcription of viruses (166).
23
CHAPTER 2
2 STUDIES ON TRANSLOCATION OF RNAS FROM
CYTOSOL TO ORGANELLES
2.1 INTRODUCTION
Potato Virus X (PVX) is the type member of the Potexvirus genus and systemically infects many
species of the Solanaceae family (167). This virus is highly applicable as a model system for
exploring various aspects of its infection and how these attributes can be exploited in the
molecular biology field. This virus is very useful for genetic studies of proteins and RNA
components required for infection, isolation, and biochemical characterization of viral proteins
and replication complexes (168). It is a rod-shaped, filamentous virus that possesses a single-
stranded, ~6435-7560 nucleotide positive-sense RNA genome. This polycistronic RNA genome
is capped at the 5´ end (m7GpppGp), polyadenylated at the 3´ end, and contains five open reading
frames (ORFs) (169) as depicted in Figure 2.1.
Figure 2.1 Genome of Potato virus X with five open reading frames.
TGBp, Triple gene block protein; CP, coat protein; m7G, 7-methylguanylate cap; Poly-A, Polyadenylation.
Note: Figure not drawn to scale.
24
The first ORF encodes a 166 kDa RNA replicase (RNA-dependent RNA polymerase). The
second, third and fourth ORFs encode a 25 kDa protein, a 12 kDa protein, and an 8 kDa protein
respectively. These proteins are known as the “triple gene block” (TGB) and are thought to be
involved in the cell-to-cell movement of the virus (170). The fifth ORF encodes a capsid protein
(CP), which has also been implicated in viral intercellular movement (171). Some plant virus
proteins, in particular, the capsid protein (CP), are known to accumulate in the chloroplasts of
infected plants (172-175). The CP, as well as other viral RNAs, have been reported to translocate
into chloroplasts (172, 176).
Many studies have documented the role of transit peptides and viral proteins in targeting
a variety of viruses to specific subcellular regions of their hosts (14, 20, 45, 58, 59, 177, 178).
The majority of chloroplast proteins are translated in the cytoplasm as pre-proteins with amino-
terminal transit peptides (TPs) that direct transport to the chloroplast via specific interactions
with various components of the import machinery (172, 179). Some viruses seem to use the same
TP mechanism to direct their proteins to chloroplasts. The cucumber necrosis Tombusvirus
(CNV) uses the first 38 amino acids of the capsid protein not only to translocate the CP to the
chloroplast but also the green fluorescent protein which is in fusion with the 38 amino acid from
the CNV CP. This viral protein seems to contain the 14-3-3 chloroplast targeting motif which is
typical to most cellular proteins which are targeted to chloroplasts (177). The TGB proteins of
PVX were shown to play an important role in the cell-to-cell movement of PVX RNA during
viral infection (170). The 25 kDa protein of TGB is targeted to plasmodesmata (180) and it is
also known to modulate plasmodesmata gating by increasing the size exclusion limit (SEL) to
allow the 25 kDa protein as well as the viral RNA to move from cell-to-cell (181). TGBp3 (25
kDa protein) also plays an important role as a suppressor protein which could delay the onset of
post-transcriptional gene silencing (PTGS) in plant tissues (182). The other two proteins 12 kDa
25
and 8 kDa were also shown to be implicated in cell-to-cell movement of viral RNA (44, 183).
The CP of PVX has also been shown to play a role in viral intercellular movement by forming
ribonucleoprotein particles with the viral RNA and the 25 kDa protein; allowing the PVX RNA
to move from cell-to-cell and possibly facilitating long distance movement (42, 184-186).
However, there are currently no reports showing any role of the CP and/or TGB proteins in the
RNA transport to organelles.
Of all the organelles contained within a eukaryotic cell, chloroplasts and mitochondria
are unique because they carry their own genetic information and are able to synthesize some of
their own proteins (187). The interactions between viruses and host components underlie the
appropriate subcellular targeting of viral proteins and nucleic acids during the viral infection
cycle (188-190). The genome replication of all plus-strand RNA viruses infecting eukaryotic
cells is associated with cellular membranes (191). The membranes can be derived from the
endoplasmic reticulum (ER), other organelles of the secretory pathway, mitochondria,
chloroplasts, or from the endo-lysosomal compartment. The membrane association provides a
structural framework for replication: it fixes the RNA replication process to a spatially confined
place, increasing the local concentration of necessary components (192). After entry into host
cells, viruses usually target a specific organelle for replication. Tobacco etch virus (193),
Cowpea mosaic virus (194), Tomato ringspot virus (195), Potato virus X (196), and Tobacco
mosaic virus (197, 198) target the ER membrane; Tomato bushy stunt virus (199) and Melon
necrotic spot carmovirus (200) target the peroxisome and mitochondria, respectively; and
Turnip yellow mosaic virus (201), Turnip mosaic virus (202) and Bamboo mosaic virus (66)
associate with the chloroplast membrane. These findings suggest that members from different
viral families might be associated with the same organelle, but that members of the same family
do not necessarily target the same organelle or organellar membrane (203). Otulak, et al. (204)
26
examined the Tobacco rattle virus (TRV) not only in the membranous and vesicular ER
structures but other cell organelles (chloroplast and mitochondria) as well. This finding also
indicates that same virus has the ability to target different cellular organelles during its infectious
cycle.
Previous research conducted in our laboratory revealed the presence of PVX CP within
the chloroplasts of both PVX-infected potato plants and transgenic potato plants containing the 8
kDa and CP sequences. Further, these studies showed that the dicistronic mRNA was found not
only to be translocated to chloroplasts, but also the CP was translated by chloroplastic ribosomes
that are sensitive to chloramphenicol treatment. In addition, a Shine/Dalgarno-like (SD) sequence
was identified upstream of the CP gene. This SD sequence was probably essential for the
translation of CP by chloroplast ribosomes (205). Northern blot analyses were performed to
confirm the existence of PVX CP messenger RNA inside the chloroplasts of infected and
transgenic plants (205). Furthermore, it was also shown that a small viral sequence from the
PVX RNA is responsible for translocating the PVX RNA to chloroplasts (206). We mapped this
viral RNA sequence “RNA tractor” (located near the end of 8 kDa and the start of CP genes
including the small non-coding intergenic region) on the PVX genome by a series of mutation
and deletion experiments using pC-GFP binary vector, where PVX sequence is driven by the 35S
promoter followed by the GFP gene and the T-nos (nopaline synthase terminator). Previous data
indicate that the “RNA tractor” is composed solely of RNA sequence and there is no expressed
viral protein involved in such translocation. In this respect it may be comparable to the transport
and translocation of the RNA of viroids of the Avsunviridae, which do not code for any proteins
and they have to rely on cellular proteins (if any) for their transport, entry to chloroplast for
replication (likely using chloroplastic DNA-dependent RNA polymerase) and for exit (90).
Gomez and Pallas (95) demonstrated for the first time that an RNA sequence of Eggplant latent
27
viroid (ELVd) can be transported to chloroplast and it is also functional in this organelle. This
supports the existence of a novel signaling mechanism between the host cell and these
organelles. (95, 207). In an earlier study, it was demonstrated that the mRNA encoding the
eukaryotic translation factor 4E enters the chloroplasts. Furthermore, the localization in the
chloroplast of a heterologous GFP mRNA fused to the eIF4E RNA was also observed. However,
interestingly, the eIF4E RNA was not translated in the chloroplasts (208). mRNA localization
might facilitate the import of proteins targeted to specific organelles. This highlights a novel
host-modulated regulatory mechanism that would be potentially able to control the gene
expression and the accumulation of the nuclear-encoded proteins in chloroplasts
In a recent study, it has been shown that the Bamboo mosaic virus (BaMV) RNA could
be transported to chloroplasts by interacting with nuclear-encoded chloroplast proteins (209).
The “RNA tractor” activity described here is the first report of a virus non-coding sequence that
is capable of not only the translocation of its own sequence but also that of a foreign RNA
sequence (GFP) to chloroplasts. Presumably, any foreign RNA could be targeted to chloroplast
by this “RNA tractor” sequence. These findings suggest that the translocation of PVX RNA into
chloroplasts is dependent upon a limited region of the PVX RNA transcript. However, from this
work, many questions may be asked. Does the PVX RNA translocation depend upon the sequence
or is it a secondary structure? Is this RNA tractor activity limited to chloroplasts or
mitochondria can also be targeted? To explore these secrets, experiments are designed to
answer some of the above questions.
2.2 RESEARCH PLAN
The overall objectives of this study are:
1. Detection of PVX RNA and coat protein in chloroplasts.
28
2. To determine the smallest RNA sequence required for the translocation process observed
with PVX RNA.
3. To determine the efficiency (quantitation) of translocation of “RNA tractor” to chloroplasts.
4. To compare the translocation efficiency of “RNA tractor” (pTR:127) sequence with
Eggplant latent viroid (pCELVd-GFP; used as a positive control) to chloroplasts.
5. Finally to study the translocation of “RNA tractor” sequence to plant mitochondria.
2.3 MATERIALS AND METHODS
2.3.1 Plasmid construction and transformation
The plasmid pCAMBIA1300 (CAMBIA, Canberra, Australia), a compact binary vector (8.9-
kbp), is used in this study. A 35S:GFP:T-nos expression cassette (Gen Bank EF546437) of size
1.9-kbp was subcloned into this binary vector by HindIII and EcoRI sites in the multiple cloning
sites and designated as pC-GFP (Figure 2.2). To create pCELVd-GFP, a chimeric DNA
containing a modified Eggplant latent viroid (ELVd) sequence, (AN - HM136583) (95),
pCATvd-GFP (ELVd sequence with AT-rich sequence derived from 5´-UTR of capsid protein of
Alfalfa mosaic virus (AlMV) RNA), pCATvdmut-GFP, SD-like sequence GGAGGATTCG
within ELVd was replaced with CCTCCTAAGC, pC127TCR-GFP and pCELVdTCR-GFP
constructs containing a translation control region (TCR) (210), comprised of 58 nucleotides of
5'-UTR and 45 nucleotides from N-terminal coding region of ribulose-1,5-bisphosphate
carboxylase/oxygenase large sub-unit (rbcL) gene and pCELVdpsbA-GFP construct comprised
of ELVd sequence with 85 nucleotides of 5´-UTR of tobacco chloroplast gene psbA (211) were
synthesized and cloned in pUC57 plasmid (Bio Basic Inc.). Following digestion of pUC57
by KpnI/ BamHI and NheI or/and XbaI/BglII restrictions enzymes and gel purification (QIAquick
29
Gel Extraction Kit, QIAgen), the fragments were subcloned into a pC-GFP binary plasmid using
the respective restriction sites. The pCrbcLSD-GFP was generated by amplified 127 nucleotides
using pTR:127 as a template and RBSKpnIF and RBSBglIIR, with an extension of rbcL anti-SD-
like sequence (CCCTCCC), primers. The resulting product was cloned into the pC-GFP vector
using its KpnI/BamHI sites. To generate pCSD-GFP (construct with PhageT7 trailer sequence,
T7 translational enhance RBS, is available in pET-X-series), first GFP sequence was amplified
using GFP specific primers and resulting PCR product was cloned into the pET29 vector using
NdeI/BamHI sites, designated pET-GFP. Subsequently, GFP with an extension corresponding to
the T7 translational enhance RBS in pET vector was amplified using F-Pet BglII and R- GFP
BamHI primers with BgIII and BamHI restriction sites respectively (Table 2.1). The obtained
product after digestion and purification was introduced into the pre-existing recombinant
pTR:127 construct using its BamHI site and named pC127pETSD-GFP. The construct pC8K-
GFP, containing the sequence upstream of the ATG codon of the PVX CP gene including the 8
kDa ORF and 177 nucleotides upstream of this ORF, was generated by amplifying the product
using pre-existing recombinant pTR:8k as a template, subsequently the obtained product was
inserted into pC-GFP in its KpnI/BamHI sites. The ATG of CP ORF was fused with ATG of
GFP ORF. To generate pChp-GFP and pChp8K-GFP, a sequence expected to form a stable
hairpin was introduced in KpnI site (inac) of both pC-GFP and PC8k-GFP constructs. To create
pCAT-GFP, AT-rich sequence derived from 5´-UTR of the capsid protein of Alfalfa mosaic virus
RNA was inserted into a pC-GFP construct using its KpnI (inac) and XbaI sites. To generate
pCATvd80-GFP construct, ELVd sequence comprised of 80 nucleotides was cloned into a
pCAT-GFP construct using its XbaI/BamHI sites. These four constructs were produced by
ligating double-stranded oligonucleotides into restriction-enzyme digested plasmid DNA with
compatible ends (Table 2.1). Briefly, complementary oligonucleotides synthesized by Eurofins
30
MWG Operon (Huntsville, AL) were mixed in equimolar amounts (50 µM each), boiled and
annealed by cooling to room temperature and ligated into already restriction enzyme digested
pC-GFP vector using T4 DNA ligase (New England Biolabs, NEB) according to the
manufacturer's protocol. All those constructs which were linearized with single restriction
enzyme were also treated with Calf Intestinal Alkaline Phosphatase (CIP) (New England
Biolabs, NEB) to prevent religation of linearized plasmid DNA. The product of each ligation
reaction was used to transform E.coli DH5-alpha competent cells and Kanamycin (50 µg/mL)
resistant bacterial colonies were screened for the presence of the proper recombinant constructs.
Plasmid extraction was done using the QIAprep spin miniprep kit (Qiagen) or mini-prep method
described by Sambrook, et al. (212).
Table 2.1 shows a complete list oligonucleotides and DNA sequence used to generate the
plasmids in this study.
Figure 2.2 A partial physical map of modified pCAMBIA1300 construct designated as pC-GFP with 35S
Promoter, GFP gene, and T-nos terminator cassette.
GFP; green fluorescent protein, 35S; Cauliflower mosaic virus 35S promoter, T-nos; nopaline synthetase
terminator, Lac p; lac promoter, lac Z α; lacZ gene alpha codes for beta- galactosidase in E.coli, L; left (T-
border), R; right (T-border). Note: Figure not drawn to scale.
To determine the smallest RNA sequence required for the translocation process, a series of
deletion clones (Figure 2.3 A-E) were generated from the previous dicistronic construct
(sequence between the PVX 8 kDa and CP proteins (174).
31
Figure 2.3 Schematic representation of constructs (A-E) in pC-GFP plasmid previously studied in our lab.
To investigate the translocation of PVX RNA into chloroplasts, regions of the PVX sequence were cloned into PC-
GFP binary vector to generate transgenic tobacco plants. A) pTR:8K-CP; this construct contains both the 8 kDa
and CP genes and was produced to confirm the previously observed translocation of CP mRNA into chloroplasts. B)
pTR:8K(insG80)-CP; the pTR:8K-CP clone was digested with EcoRI and filled with a Klenow fragment; which
caused the insertion of a G in the 80th nt position of the 8 kDa gene. This produces a frame shift mutation in the
resulting protein. C) pTR:8K; In this construct, the CP gene was truncated so that only the first 13 nt remained.
PCR inserts were cloned into PC-GFP binary vector using KpnI/XbaI sites. D) pTR:224; this construct consists of
224 nt of the PVX sequence, including 201 nucleotides from the 3´ end of 8 kDa gene, 10 nt of intergenic region
and the first13 nucleotides from the CP gene. E) pTR:127; this construct contains 127 nt of the PVX sequence
(“RNA tractor”), including 104 nt from the 8 kDa gene, 10 nt of intergenic region and 13 nucleotides from the CP
gene. 35S; Cauliflower mosaic virus 35S promoter, 8k; 8 kDa gene, IR; intergenic region, CP; coat protein, GFP;
green fluorescent protein, T-nos; nopaline synthetase terminator. Note: Figures not drawn to scale.
In this study two potential constructs pTR:127 and pTR:27 are used to verify the PVX RNA
sequence responsible for translocation of not only PVX RNA but also GFP RNA from cytosol to
chloroplasts. Two constructs namely pCELVd-GFP containing Eggplant latent viroid (ELVd) (a
non-coding viroid) sequence and pC-GFP are included as positive and negative controls
respectively (Figure 2.4 A-D).
32
Figure 2.4 Schematic representation of the constructs used in this study for “RNA tractor” activity.
To confirm the trafficking of “RNA tractor” into chloroplasts, transgenic tobacco plants were generated using
these constructs. A) pC-GFP; this construct, without any PVX sequence, is included as a negative control B)
pTR:27; this construct contains only last 5 nt of 8 kDa, 10nt of intergenic region and 12 nt from the CP gene C)
pTR:127; this construct comprises 127 nt of the PVX sequence (“RNA tractor”), including 104 nt from the 8
kDa gene, 10 nt of intergenic region and 13 nt from the CP gene. D) pCELVd-GFP; in this construct a chimeric
Eggplant latent viroid (ELVd) sequence consisting of 330 nt (Accession Number - HM136583) is used for a
positive control. 35S; Cauliflower mosaic virus 35S promoter. 8k; 8 kDa gene. IR; intergenic region. CP; coat
protein. GFP; green fluorescent protein. T-nos; nopaline synthetase terminator. Note: Figures not drawn to scale.
Figure 2.5 Partial DNA sequence of the pTR:127 construct used in this study as “RNA tractor”.
KpnI and XbaI are restriction enzymes that were used in the cloning of the pTR:127 construct into the pC-GFP
binary plasmid. XbaI (Inac) indicates the inactivated XbaI site.
pTR:127 35S KpnI 8k (104 nt)
…//TATATAAGGAAGTTCATTTCATTTGGAGAGAACACGGGGACggtacccaggcCTGGAGAATCAATCACA
GTGTTGGCTTGCAAGTTAGATGCAGAAACCATCAGAGCCATTGCCGATCTCAAGCCACTCTCCGTTGA
IR (10 nt) CP (13 nt) XbaI Inac GFP T-nos
ACAGTTAAGTTTCCATTGATACTCGAAAGATGTCAGCACCAGgctagaggatccATGGTGAG//……………
33
Table 2.1 Oligonucleotides/ primers used in the production of different constructs.
Constructs
Oligo Name/
Remarks
Oligo/Primer sequence* (5'-3´)
Cloning
sites**
pTR:127 S8K-F
8K-R
AATATTGGTACCCAGGCCTGGAGAATCAATCACAGTGTTG
ACTACTGCTAGCCTGGTGCTGACATCTTTCGAGTATC
KpnI
NheI
pTR:27 27 sense
27 antisense
TAGGCCTATTGATACTCGAAAGATGTCAGCACCAT
TAGATGGTGCTGACATCTTTCGAGTATCAATAGGCCTAGATC
XbaI /
KpnI
pET-GFP F-GFPNdeI
R- GFP BamHI
AATTAACATATGGTGAGCAAGGGCG
ACGTGGATCCTTTACTTGTACAGCTCGCC
NdeI/
BamHI
pCrbcLSD-
GFP
F-RBSKpnI
R-BSBglII
ATGTACGGTACCCAGGCCTGGAGAATCAATCACAGT
AATTATAGATCTCCCTCCCTGGTGCTGACATCTTTCG
KpnI/
BglII
pC8k-GFP 12K-F
12K-R
ATCGGGTACCCTAGAAATAGTTTACCCC
CCATGGATCCTCTAGCTGGTGCTGACAT
KpnI/
BamHI
pcSD-GFP F-Pet BglII.
R- GFPBamHI
CACTCCAGATCTAATAATTTTGTTTAACTTTAAG
ACGTGGATCCTTTACTTGTACAGCTCGCC
BglII/
BamH1
pChp-GFP
pChp8k-GFP
Stem loop sense
Stem loop antisense
ACGCGCTCCCCCCGGGGGGTCGACCCCCCGGGGGGAAAGCAGTAC
TGCTTTCCCCCCGGGGGGTCGACCCCCCGGGGGGAGCGCGTGTAC
KpnI
(inac)
pCAT-GFP AT sense
AT anti sense
TTAAATCTAGCTATATAAGGAAGTTCATTTCATTTGGAGAGGGTTTTTATT
TTTAATTTTCTTTCAAATACTTCCAGGATCAGTAC
TGATCCTGGAAGTATTTGAAAGAAAATTAAAAATAAAAACCCTCTCCAA
ATGAAATGAACTTCCTTATATAGCTAGATTTAAGTAC
KpnI
pCATvd80-
GFP
80 ELVd sense
80 ELVd antisense
CTAGCACTTTAAATTCGGAGGATTCGTCCTTTAAACGTTCCTCCAAGAGT
CCCTTCCCCAAACCCTTACTTTGTAAGTGTGGTTCG
GATCCGAACCACACTTACAAAGTAAGGGTTTGGGGAAGGGACTCTTGGA
GGAACGTTTAAAGGACGAATCCTCCGAATTTAAAGTG
XbaI
(inac)/
BamHI
pCATvd-GFP Eggplant latent
viroid (ELVd)
chimeric sequence
with an At-rich
leader sequence.
DNA was
synthesized.
Sequence of only
plus strand is given.
5´GCTAGCTATATAAGGAAGTTCATTTCATTTGGAGAGGGTTTTTATTTTT
AATTTTCTTTCAAATACTTCCAGGATCGGTACCTTGGCGAAACCCCATTTC
GACCTTTCGGTCTCATCAGGGGTGGCACACACCACCCTATGGGGAGAGGT
CGTCCTCTATCTCTCCTGGAAGGCCGGAGCAATCCAAAAGAGGTACACCC
ACCCATGGGTCGGGACTTTAAATTCGGAGGATTCGTCCTTTAAACGTTCC
TCCAAGAGTCCCTTCCCCAAACCCTTACTTTGTAAGTGTGGTTCGGCGAA
TGTACCGTTTCGTCCTTTCGGACTCATCAGGGAAAGTACACACTTTCCGA
CGGTGGGTTCGTCGACACCTCTCCCCCTCCCAGGTACTATCCCCTTTCCAG
GATTTGTTCCCAGATCTAAAAAGCCTTCCATTTTCTATTTTGATTTGTAGA
AAACTAGTGTGCTTGGGAGTCCCTGATGATTAAATAAACCAAGATTTTAC
CATGGGATCC
NheI/
BamHI
pCELVd-GFP Eggplant latent
viroid (ELVd)
sequence. DNA was
synthesized. The
sequence of only plus
strand is given.
GGTACCTTGGCGAAACCCCATTTCGACCTTTCGGTCTCATCAGGGGTGGC
ACACACCACCCTATGGGGAGAGGTCGTCCTCTATCTCTCCTGGAAGGCCG
GAGCAATCCAAAAGAGGTACACCCACCCATGGGTCGGGACTTTAAATTC
GGAGGATTCGTCCTTTAAACGTTCCTCCAAGAGTCCCTTCCCCAAACCCTT
ACTTTGTAAGTGTGGTTCGGCGAATGTACCGTTTCGTCCTTTCGGACTCAT
CAGGGAAAGTACACACTTTCCGACGGTGGGTTCGTCGACACCTCTCCCCC
TCCCAGGTACTATCCCCTTTCCAGGATTTGTTCCCGGATCC
KpnI
/BamHI
34
* Underlined bold letters indicate restriction endonuclease recognition sequences.
** Restriction endonuclease recognition sequences introduced into the primers to facilitate cloning of fragments into
PC-GFP.
All these constructs were transformed into E. coli DH5α cells. The presence and accuracy of
each inserted DNA sequence in the final recombinant constructs were confirmed by DNA
sequencing (The Centre for Applied Genomics, Toronto, Canada) using the GFP-R reverse
primer Table 2.2. Subsequently, these confirmed clones were transformed into Agrobacterium
tumefaciens strain GV3101 as given in section 2.3.5.
Constructs
Oligo Name/
Remarks
Oligo/Primer sequence* (5´-3´)
Cloning
sites**
pCATvd mut-GFP SD-like
(GGAGGATTCG)
sequence is
replaced with anti-
SD-like
(CCTCCTAAGC)
sequence.
5’GCTAGCTATATAAGGAAGTTCATTTCATTTGGAGAGGGTTTTTATTTT
TAATTTTCTTTCAAATACTTCCAGGATCGGTACCTTGGCGAAACCCCATT
TCGACCTTTCGGTCTCATCAGGGGTGGCACACACCACCCTATGGGGAGA
GGTCGTCCTCTATCTCTCCTGGAAGGCCGGAGCAATCCAAAAGAGGTAC
ACCCACCCATGGGTCGGGACTTTAAATTCCCTCCTAAGCTCCTTTAAAC
GTTCCTCCAAGAGTCCCTTCCCCAAACCCTTACTTTGTAAGTGTGGTTCG
GCGAATGTACCGTTTCGTCCTTTCGGACTCATCAGGGAAAGTACACACT
TTCCGACGGTGGGTTCGTCGACACCTCTCCCCCTCCCAGGTACTATCCCC
TTTCCAGGATTTGTTCCCAGATCT
NheI/
BglII
pCELVdpsbA-
GFP
5´-UTR of psbA gene
is included for
translation in
chloroplasts.
5´GGTACCTTGGCGAAACCCCATTTCGACCTTTCGGTCTCATCAGGGGTG
GCACACACCACCCTATGGGGAGAGGTCGTCCTCTATCTCTCCTGGAAGG
CCGGAGCAATCCAAAAGAGGTACACCCACCCATGGGTCGGGACTTTAA
ATTCGGAGGATTCGTCCTTTAAACGTTCCTCCAAGAGTCCCTTCCCCAAA
CCCTTACTTTGTAAGTGTGGTTCGGCGAATGTACCGTTTCGTCCTTTCGG
ACTCATCAGGGAAAGTACACACTTTCCGACGGTGGGTTCGTCGACACCT
CTCCCCCTCCCAGGTACTATCCCCTTTCCAGGATTTGTTCCCAGATCTAA
AAAGCCTTCCATTTTCTATTTTGATTTGTAGAAAACTAGTGTGCTTGGGA
GTCCCTGATGATTAAATAAACCAAGATTTTACCATGGGATCC
KpnI/
BamHI
pC127TCR-GFP 5´-translation control
region of the rbcL
gene, comprised of
14 N-terminal amino
acids and 58 of 5´-
UTR region, was
added for translation
in the chloroplasts.
GGTACCCAGGCCTGGAGAATCAATCACAGTGTTGGCTTGCAAGTTAGAT
GCAGAAACCATCAGAGCCATTGCCGATCTCAAGCCACTCTCCGTTGAAC
AGTTAAGTTTCCATTGATACTCGAAAGATGTCAGCACCAGTCTAGAGTC
GAGTAGACCTTGTTGTTGTGAGAATTCTTAATTCATGAGTTGTAGGGAG
GGATTTATGTCACCACAAACAGAGACTAAAGCAAGTGTTGGATTCAAA
GCTAGATCT
KpnI/
BglII
pCELVdTCR-
GFP
5´-translation control
region of the rbcL
gene, comprised of
14 N-terminal amino
acids and 58 of 5´-
UTR region, was
added for translation
in the chloroplasts.
GGTACCTTGGCGAAACCCCATTTCGACCTTTCGGTCTCATCAGGGGTGG
CACACACCACCCTATGGGGAGAGGTCGTCCTCTATCTCTCCTGGAAGGC
CGGAGCAATCCAAAAGAGGTACACCCACCCATGGGTCGGGACTTTAAAT
TCGGAGGATTCGTCCTTTAAACGTTCCTCCAAGAGTCCCTTCCCCAAACC
CTTACTTTGTAAGTGTGGTTCGGCGAATGTACCGTTTCGTCCTTTCGGAC
TCATCAGGGAAAGTACACACTTTCCGACGGTGGGTTCGTCGACACCTCT
CCCCCTCCCAGGTACTATCCCCTTTCCAGGATTTGTTCCCAGATCTGTCG
AGTAGACCTTGTTGTTGTGAGAATTCTTAATTCATGAGTTGTAGGGAGG
GATTTATGTCACCACAAACAGAGACTAAAGCAAGTGTTGGATTCAAAGC
TGGATCC
KpnI/
BamHI
35
2.3.2 Heat shock transformation of E.coli
Escherichia coli (strains DH5 alpha or BL21- CodonPlus used for pET vector only) were made
competent and transformed using a calcium chloride heat shock method described by Sambrook,
et al. (212). A glycerol stock of E. coli, strain DH5 alpha, was used to inoculate 2 mL of LB
medium (1% tryptone, 0.5% bacto yeast extract and 1% sodium chloride, pH 7.5 adjusted with 1
M NaOH), and the culture was grown overnight at 37°C on a shaker (225 rpm). Two hundred
microliter of the overnight culture was added to 25 mL fresh LB media and set on a 37°C shaker
for an additional 2-3 hours (hr) until the optical density at 595 nm (OD595) was 0.4-0.6. The
culture was incubated on ice for 10-20 min and then divided into two tubes which were
centrifuged at
3,000 g at 4°C for 5 min. The supernatant was decanted and the pellet was resuspended in 5 mL
of sterilized ice-cold 50 mM calcium chloride and incubated 20 min on ice. The cells were
pelleted under the same conditions for 5 min. The supernatant was discarded and the pellet was
resuspended in 670 μL of ice-cold 100 mM calcium chloride. The tubes were chilled on ice for
30 min. Aliquots of 200 µL were used for transformation with 5 μL ligation mixture (500 ng
DNA of each undigested and digested plasmids used as positive and negative controls
respectively). After chilled on ice for 30 min, heat-shock was performed on all samples at 42°C
for 50 sec followed by chilled on ice for 2 min. Eight hundred microliter of fresh LB medium
was added to each sample and tubes shaked (225 rpm) at 37°C for 45 min. The cells were
pelleted at 10, 000 g for 1 minute at room temperate and 800 μL of the supernatant was
discarded. The pellet was resuspended in the remaining supernatant and evenly plated on LBA
plates (LB media, 15 g/L agar) with 50 μL/mL Kanamycin. The agar plates were inverted and
incubated at 37°C overnight for growth. Single colonies were selected and grown in 2 mL LB
media supplemented with 50 μL/mL Kanamycin in a 37°C shaker and plates were stored at 4°C.
36
Plasmid DNA was extracted from these cultures (see section 2.3.3). Cloning was then confirmed
by sequencing the plasmid insert using a GFP reverse primer (Table 2.1).
2.3.3 Isolation and purification of plasmid DNA from E.coli (mini-prep)
E. coli cells (store at -80°C) were used to inoculate 2 mL of LB media (1% tryptone, 0.5% bacto
yeast extract and 1% sodium chloride, pH 7.5 adjusted with 1M NaOH) supplemented with the
appropriate selection antibiotic (60 μg/mL Ampicillin for pUC 57 plasmid and 50 μg/mL
Kanamycin for constructs in pC-GFP) and the cells were cultured overnight at 37°C on an orbital
shaker (225 rpm). The plasmid DNA was extracted using a modified method as described
previously by Sambrook, et al. (212). E.coli cells were pelleted at 10, 000 g for 2 min. The
supernatant was decanted and pellets were resuspended in 100 μL of Solution I (50 mM glucose,
10 mM EDTA pH 8.0, 25 mM Tris-HCl pH 8.0) stored at 4°C and 5 mg/mL lysozyme stored at -
20°C. After a 10 min incubation period at room temperature, 200 μL of freshly prepared Solution
II (0.2 N sodium hydroxide (NaOH) and 1% sodium dodecyl sulphate (SDS)) was added to each
sample to promote lysis of bacterial cells, denaturation of cellular proteins, chromosomal DNA,
and degradation of cellular RNA. The solution was thoroughly mixed by gently inverting the
tubes and chilled on ice for 20 min followed by adding 150 µL of ice-cold neutralizing Solution
III (3M sodium acetate pH 4.8). After thoroughly mixing, samples were incubated on ice for 45
min and centrifuged at 14,000 g for 5 min at 4°C to separate precipitated proteins, lipids, and
chromosomal DNA from plasmid DNA. The supernatant with plasmid DNA of each sample was
transferred to a new tube and mixed with 0.6 volumes of isopropanol. After incubation at room
temperature for 1 hr, plasmid DNA was pelleted at 14,000 g for 15 min. Plasmid DNA pellets
were washed with 70% ethanol to remove salt followed by 95% ethanol. These pellets were air-
dried resuspended in 50 μL of TE-1 buffer (1 mM Tris-HCl pH 8.0 and 0.1 mM EDTA pH 8.0)
37
and purified by adding an equal volume of phenol (saturated with 0.1 M Tris-HCl pH 8.0). Each
sample was thoroughly mixed by vortexing and centrifuged at 14,000 g for 5 min at room
temperature. The top phase was dispensed without disturbing the interface into a new tube. Two
volumes of saturated chloroform stored at 4°C were added to each sample. After mixing, samples
were centrifuged at 14,000 g for 5 min at room temperature. The chloroform was removed and
this step was repeated. The aqueous phase containing DNA was taken into a new tube and
sodium acetate was added to a final concentration of 0.1 M along with 2.5 volumes of ice-cold
95-100% ethanol. The samples were thoroughly mixed and stored at -20°C for further
downstream applications.
2.3.4 Gel electrophoresis
DNA and RNA samples were analyzed using gel electrophoresis. TBE buffer (0.1 M Tris base,
0.5 M Boric acid, and 2 mM EDTA pH 8.0) was used for both the preparation of the agarose gel
(1-2%) as well as the running buffer. The DNA or RNA was diluted with 5 μL of TE-1 and 3 μL
loading dye (0.25% xylene cyanol, 0.25% bromophenol blue, 20% glycerol). After loading the
sample, the gel was subjected to electrophoresis at a constant current of 50 mA and voltage of
120 V. Once the bromophenol dye had migrated half-way down the gel, the gel was stained with
1% ethidium bromide and photographed under ultraviolet light (300 nm) with a transilluminator.
2.3.5 Agrobacterium transformation
A freeze-thaw method for transformation of Agrobacterium tumefaciens strain GV3101 was used
as reported previously (213) with minor modifications. A single colony of A.tumefaciens was
selected and grown in 2 mL LB medium containing 50 µg/mL Gentamycin (BioBasic) at 28°C
on a shaker at 225 rpm. Overnight cell culture was diluted with 50 mL fresh LB medium and
grown at 28°C until cells reached an optical density (OD595) of 0.4. The cells were harvested by
38
centrifugation at 3,000 g for 10 min and resuspended in 1 mL precooled 20 mM calcium chloride
and incubated on ice for 20 min. Aliquots of 100 µL were used directly for transformation with
plasmid DNA (500 ng of the respective constructs) and incubated further for 20 min on ice. In
the next step, the mixture of cells, calcium chloride, and DNA was momentarily frozen in liquid
nitrogen and then incubated at 37°C for 5 min. After dilution in 1 mL LB-medium, the cells were
incubated 3 h at 28°C with gentle shaking (150 rpm). Cells were pelleted down by a brief spin
and the supernatant was discarded while leaving behind 100 µL of the media. Cells were mixed
well with the media, plated on LB plates containing Kanamycin (100 µg /mL) and Gentamycin
(50 µg/mL) and incubated for 2 days at 28°C. Single colonies from the respective plates with the
various constructs were recovered from the plates and grown in 2 mL LB media with Kanamycin
and Gentamycin overnight at 28°C with gentle shaking. Next day, 200 µL of the culture was
transferred to 20 mL of fresh medium supplemented with the same antibiotics and 100 µM
acetosyringone and shaked for 5-6 hr at 28°C until cells reached an optical density (OD595) of
0.4-0.6. These cultures were used for plant transformation and also mixed with autoclaved
glycerol (1:1 v/v) and stored at -80°C.
2.3.6 Plant transformation
Healthy viable seeds of tobacco (Nicotiana. tabacum cv. Xanthi) were surface-sterilized by
rinsing in 10% household bleach with 0.05% tween-20 (as a surfactant) for 10 min and then
washed 3-5 times in sterile distilled water. Seeds were blotted dry on sterilized Whatman filter
paper and cultured on ½ MS medium (2.2 g/L MS salts, 15g/L sucrose, 8 g/L agar, pH 5.8
adjusted with 1M KOH) in GA-7 magenta vessels (Sigma- Aldrich). Stable Agrobacterium-
mediated transformation was performed as described by Horsch, et al. (214) with some minor
modifications. Four to five weeks old plants were used for transformation. Explants (leaf discs,
39
2-3 mm) were co-cultivated for 10-20 min with Agrobacterium cultures (prepared as mentioned
in section 2.3.5. These Agrobacterium infected explants were then blotted on sterile filter paper
and placed with the abaxial surface touching the regeneration MS1 medium (4.4 g/L MS salts,
30g/L sucrose, 2 g/L 2-(N-morpholino) ethanesulfonic acid (MES) , 1 mg/L 6-benzyl-
aminopurine (BAP), 0.4 mg/L naphthalene acetic acid (NAA), and 8 g/L agar, pH 5.8) in Petri-
dishes. After three days, transformants were selected on the regeneration MS2 medium (4.4 g/L
MS salts, 30 g/L sucrose, 2 g/L MES, 1 mg/L BAP, 0.4 mg/L NAA, 8 g/L agar, 0.4g/L
Carbenicillin and 0.02 g/L Hygromycin, pH 5.8). Following a 4-6 week culture period,
Hygromycin resistant shoots were transferred to a phytohormone-free ½ MS medium containing
0.4 g/L Carbenicillin and 0.02 g/L Hygromycin in magenta vessels. After 4-6 weeks, the
transformants were confirmed by PCR of plant chromosomal DNA and transgene expression was
verified by RT-PCR of total plant RNA using the primer sets (Table 2.2). Transgenic plants
with roots were transferred from Magenta vessels to pots containing Pro-Mix (Premier Tech,
Canada). Plastic pots were enveloped in polyethylene bags to preserve humidity for one week.
The plants were grown at 23-27°C under 16 hr light/8 hr dark condition in an insect-free
greenhouse. All the cultures were kept in the growth chamber at 23±1°C under 16 hr
photoperiods of 3000 Lux supplied with cool white fluorescent tube lights. All plant growth
regulators (filter sterilized) were added after autoclaving the media. All operations of tissue
culture and transformation were carried out in laminar airflow sterile cabinet.
2.3.7 Infection of N. tabacum cv. Xanthi with PVX and virus isolation
N. tabacum cv. Xanthi is a good propagation host for PVX. Plants (4 true-leaf-age) were lightly
dusted with Carborundum (400 mesh) and inoculated with infected leaf extracts. After 20 min,
the infected plants were washed to remove any residual inoculum. The inoculated plants were
40
kept under greenhouse conditions (23-27°C, 16 hr photoperiod). Two-three weeks post-
inoculation (pi), the virus was purified according to the method of AbouHaidar, et al. (215).
Infected leaves were homogenized in 0.1 M Tris-borate buffer, pH 7.5, 0.25% β-mercaptoethanol
(βME) (2 mL of buffer for 1 g of leaves). Subsequent steps were performed at 4°C.
Homogenized tissue was squeezed through four layers of cheesecloth with the addition of n-
butanol to the plant sap to a final concentration of 6%. The mixture was kept on ice for 45 min
with constant stirring. A low-speed centrifugation for 15 min was then performed (15,000 g) and
the supernatant was saved. The virus was precipitated from the supernatant by the addition of
polyethylene glycol (PEG M, 8,000) to a final concentration of 8% in the presence of 2% sodium
chloride (NaCl) and left at 4°C for 30 to 60 min. After centrifugation at 15,000 g for 10 min, the
pellets were resuspended in 0.1 M Tris-borate buffer, pH 7.5. The virus solution was then
centrifuged three times at 7,500 g for 5 min each. The supernatant was overlaid onto a 4 mL
cushion of 30% sucrose in 0.1 M Tris-borate buffer, pH 7.5 (w/v), in Ti 60 ultracentrifuge tubes
(Beckman). The virus was then pelleted at 86,500 g (35,000 rpm, Ti 60 rotor) for 3 hr. Pellets
were re-dissolved in Tris-borate buffer, pH 7.5 (w/v), in Ti 60 ultracentrifuge tubes (Beckman).
The virus was then pelleted at 86,500 g (35,000 rpm, Ti 60 rotor) for 3 hr. Pellets were re-
dissolved in the same buffer as above overnight at 4°C and centrifuged three times at 7,500 g for
10 min each. For further purification, the virus in the supernatant can be centrifuged in a CsCl
density gradient for 17 hr at 86,500 g at 15°C. Virus band can be collected and diluted 4 times
with 0.1 M Tris-borate acid buffer, pH 7.5. The virus can be sedimented by centrifugation at
100,000 g for 2 hr re-dissolved in the same buffer. OD readings were taken at 260 and 280 nm to
determine the purity of the virus (A260/A280 ratio of 1.2 for PVX). The concentration of the virus
was determined using an extinction coefficient (E260nm 0.1%,1 cm = 3.0 for PVX) (216). The virions
41
were utilized in a reconstruction control experiment to rule out externally adsorbed virions on
chloroplasts.
2.3.8 Extraction of viral genomic RNA
Before the isolation of RNA, purified virions were subjected to DNase I treatments according to
the manufacturer’s instructions (New England Biolabs, NEB) to remove host DNA left in the
virus. The reaction was carried out at 37°C for 30 min and the reaction was terminated by
EDTA. Treated virus solution was then subjected to a high-speed centrifugation at 86,500 g for 2
hr and the virus pellets were dissolved in diethylpyrocarbonate-treated distilled water (DEPC-
dH2O). The viral RNA was extracted from purified virions according to the methods of
AbouHaidar, et al. (215). Virions were incubated in 0.1% (w/v) SDS at 37°C for 10 min. The
RNA was extracted with 2 volumes of phenol/chloroform (1:1 v/v) at 40°C with occasional
vortexing (phenol was equilibrated with 0.1 M Tris-HCl, pH 4.0 containing 0.2% βME). The
aqueous phase was re-extracted with an equal volume of phenol/chloroform and subsequently
subjected to two chloroform extractions with 2 volumes of chloroform/isoamyl alcohol (24:1,
v/v). The RNA was precipitated by the addition of sodium acetate (NaOAc) to a final
concentration of 0.1 M and 2.5 volumes of ice-cold 95% ethanol (EtOH). The mixture was
chilled to -70°C for 15-20 min (or -20°C overnight). Viral RNA was centrifuged at 12,000 g for
30 min. RNA pellets were rinsed with 70% EtOH, vacuum-dried for 5 min, and dissolved in a
desired volume of DEPC-dH2O. The purity and concentration of the RNA were determined
according to the OD readings at 260 and 280 nm (E260nm 0.1%,1 cm =25 for RNA). The virion RNA
was utilized in a reconstruction control for chloroplastic RNA isolation as well as in a positive
control RT-PCR analyses.
42
2.3.9 Chloroplast isolation
Chloroplasts were isolated from transgenic, non-transgenic and PVX- infected plants using the
modified method (205). Each isolation step was performed at 4°C separately. Twenty grams of
leaves were harvested and homogenized with mortar and pestle in 100 mL of cold grinding
buffer (50 mM HEPES-KOH, pH 7.3, 330 mM mannitol, 0.1% BSA, 1 mM MgCl2, 1 mM
MnCl2, 2 mM Na2EDTA, 1 mM DTT). Homogenate was filtered through eight layers of
cheesecloth and the filtrate was pelleted at 500 g for 2 min to remove the plant debris. After that,
the suspension containing chloroplasts was sedimented by centrifugation at 4°C, 2,500 g for 20
min. Subsequently, each pellet containing the chloroplasts was carefully resuspended in 1 mL
grinding buffer. These chloroplasts in each sample were counted by haemocytometer and the
quantity was equally adjusted ensuring a similar sample size. Each sample was loaded on top of
a sucrose step gradient developed with 4 mL of 30%, 3 mL of 45%, and 2 mL of 60% sucrose in
grinding buffer and centrifuged at 77,140 g for 55 min. Intact chloroplasts were collected from
the interphase between 30% and 45% sucrose, washed twice with washing buffer (50 mM
HEPES-KOH, pH 8.0, and 330 mM mannitol) and resuspended in 1 mL washing buffer. The
purified chloroplasts were visualized under a light microscope to confirm their integrity. Each
suspension was incubated at 30°C with RNase A (100 µg/mL) for 40 min to ensure that no
cytoplasmic RNA associated with the chloroplasts and/or first with proteinase K (4000 µg/mL)
in case of PVX-infected plants followed by twice washing and then RNase A (100 µg/mL). After
washing, each pellet was gently resuspended in 1 mL of washing buffer and treated with
proteinase K (4000 µg/mL) for 40 min to inactivate the RNase A. These chloroplasts were
washed twice with 15 mL of washing buffer. After the final washing, the chloroplasts were used
for protein isolation or/and lysed for RNA isolation using phenol-chloroform method.
Subsequently, isolated RNAs were treated twice with DNAse1 (New England Biolabs, NEB) to
43
remove any traces of genomic DNA. After phenol/chloroform purification, the RNAs were
precipitated by the addition of sodium acetate (NaOAc) to a final concentration of 0.1 M and 2.5
volumes of ice-cold 95% EtOH and stored at -20°C. Next day RNAs were centrifuged at 12,000
g for 30 min and the pellets were rinsed with 70% EtOH, vacuum-dried for 5 min, and dissolved
in a desired volume of DEPC-dH2O. RNA concentration and the 260/280 nm absorbance ratios
were determined for purity using an ND-1000 Spectrophotometer (NanoDrop Technologies Inc.,
USA). For reconstruction experiments, purified chloroplasts (1 mL) were incubated with 5 µg of
purified PVX RNA (extracted from purified virus particles). Half of this sample was used to
extract chloroplast RNA without any treatment with RNase A and used as a positive control. The
remaining 0.5 mL sample was treated with 100 µg/mL of RNase A for 60 min at room
temperature followed by washing. Washed chloroplasts were again resuspended in 0.5 mL
washing buffer and treated with 4,000 µg/mL proteinase K for 60 min followed by three
washings.
2.3.10 cDNA synthesis and RT-PCR
Samples of DNAse-treated total and chloroplast RNAs were subjected to 200 units of M-MLV
reverse transcriptase (New England Biolabs, NEB) with the GFP187-R, 16SrRNA187-R,
Act187-R, and PVXCP-R reverse primers shown in Table 2.2. Two hundred and fifty
nanograms of RNA was used as a template for cDNA synthesis with 200 units of M-MLV
reverse transcriptase, 400 nM of each primer and 500 of mM dNTPs in each reaction of 20 µL
final volume. PCR reaction were carried out on 2 µL cDNA in a final volume of 30 µL, with
reagents provided by New England BioLabs (longTaq polymerase), in a PTC-100 thermocycler
(MJResearch). The PCR was performed at 95°C for 5 min, followed by 95°C for 50 sec, 60°C
for 50 sec, 72°C for 50 sec for 32 cycles, and 72°C for 5 min. A minus RT control was included
44
in each RT-PCR reaction to check for any possible genomic DNA contamination. All the primers
used to amplify the total and chloroplast RNAs are displayed in Table 2.2.
Table 2.2 Primer sequences used for semi-quantitative and real time RT-PCR.
2.3.11 Real-time RT-PCR
Real-time RT-PCRs were performed using a CFX96 Real-Time PCR Detection System (Bio-
Rad) with the use of a Power Sybr Green Master Mix (Applied Biosystems). Reaction mixtures
contained 10 µL of 2X SYBR Green I Master Mixture, 400 nM of each primer (GFP187F&R,
16SrRNA187 F&R, and Actin187 F&R) and 2 µL of cDNA as template, in a total volume of 20
µL. The following amplification program was used in all PCR reactions: 95ºC for 3 min, 32
cycles of 10 sec at 95°C and 30 sec at 62°C. The specificity of each amplification reaction was
verified by a dissociation curve (melting curve) analysis after the 32 cycles, by heating the
amplicon from 65°C to 95°C. No-template controls were included for each primer pair. All
treatments are performed in triplicate including a duplicate of minus RT controls. The relative
quantification of gene expression was performed using the comparative CT (threshold cycle)
method in which the amount of target (GFP), normalized to an endogenous reference (16SrRNA)
and relative to a calibrator (pTR:127), is given by the formula 2-∆∆CT (217).
Target
sequence
Forward (F) and reverse (R) primers (5´-3´) Gene
Bank
Accession
No
RT-PCR
product
size/bp
GFP GFP187-F ACGTAAACGGCCACAAGTTC
GFP187-R AAGTCGTGCTGCTTCATGTG
JQ733047 187
16SrRNA 16SrRNA-F GAAGAACCTTACCAGGGCTTGA
16SrRNA-R CAGTCTGTTCAGGGTTCCAAAC
Z00044.2 187
Actin
Act187-F AGTCCTCTTCCAGCCATCCA
Act187-R AGCCAAAGCCGTGATTTCC
U60495 187
PVX 8K S8K-F AATATTGGTACCCAGGCCTGGAGAATCAATCACAGTGTTG
8K-R ACTACTGCTAGCCTGGTGCTGACATCTTTCGAGTATC -
-
PVXCP PVXCP-R AAAATACTATGAAACTGGGGTAG - -
45
2.3.12 SDS-PAGE and western blot analysis
For preparation of total proteins from PVX-infected tobacco plants, 0.2 g of plant tissue or
enzymatic treated chloroplast was homogenized in 160 µL of protein extraction TMPDTNU
buffer (50 mM Tris, 20 mM MgCl2, 1 mM PMSF, 100 mM DTT, 2% Triton X-100, 0.5% NP-40
and 8 M urea) plus 40 µL of 5x SDS-PAGE loading dye (212). These samples were boiled at 95-
100 °C for 5 min and 40 µL of each sample was loaded onto 12% SDS-PAGE gels along with
the appropriated protein molecular weight markers (Thermo Fisher Scientific). Protein
concentrations were determined by the Bradford Protein Assay reagent kit (Bio-Rad, Hercules,
CA). Electrophoresis was performed initially at 150 V until the samples entered the separating
gel followed by 100 V until dye reached at the bottom of the gel (218). The proteins were then
transferred onto nitrocellulose membrane (0.45 nm pore size, Pall Corporation) for 1 hr in
transfer buffer using the Bio-Rad protein electrophoresis unit. The membrane containing the
transferred proteins was blocked in Tris-buffered saline (TBS buffer: 50 mM Tris and 150 mM
sodium chloride) along with 5% skimmed milk for 5 hr. Subsequently, the membrane was
incubated at 4°C overnight with mild shaking with (1:1000) Anti-PVX coat protein, raised in
Rabbit, polyclonal antibodies in TBS+3% BSA. The membrane was washed (TBS, 0.3% Tween
20) 4 times and incubated with (1:3000) Goat Anti-Rabbit IgG (H & L) Alkaline Phosphatase
(Bioshop Canada Inc) for 2 hr at room temperature with mild shaking. The membrane was
washed 3 times with TBS-T followed by a final washing with TBS. Finally, signals were
developed with alkaline phosphatase substrate solution (BCIP / NBT, Bioshop Canada Inc.)
according to the manufacturer instructions. The membranes were dried and photographed.
46
2.3.13 Isolation of intact mitochondria and enzymatic treatments
Tobacco (N. tabacum cv. Xanthi) seeds harvested from transgenic plants harboring pTR:127,
pCELVd-GFP constructs were screened on ½ MS medium supplemented with 20 μg/mL
Hygromycin in magenta vessels. Three-week-old plants were transferred to pots (1-2 plants per
pot) containing Pro-Mix (Premier Tech, Canada), and grown in a greenhouse. These pots were
covered with polyethylene bags to preserve humidity for one week. Non-transgenic tobacco
seeds were also sown in Pro-Mix and infected with PVX virus as mentioned in Section 2.3.7.
The plants were grown at 23-27°C under 16 hr light/8 hr dark condition in an insect-free
greenhouse. After 4-6 weeks mitochondria were isolated from these plants using the modified
method as described previously by Block, et al. (219). Fresh leaves (50 g), for each preparation,
were cut and homogenized in a mortar in 120 mL of extraction medium (EM) (20 mM HEPES-
Tris pH 7.6, 0.4 M sucrose, 5 mM EDTA pH 8.0, 0.6% PVP (w/v), 0.6 mM cysteine). The
extract was filtered through 8 layers of cheesecloth and centrifuged 5 min at 3500 g. The
supernatant was centrifuged at 28,000 g for 10 min to pellet organelles. The pellet was
resuspended in 120 mL EM without PVP and centrifuged at 28,000 g for 10 min and the pellet
resuspended in 2 mL of suspension buffer (SB) (10 mM MOPS-KOH pH 7.2, 0.2 M sucrose)
and loaded on a percoll gradient developed with 2 mL of 10%, 3.5 mL of 32% and 3.5 mL of
50% percoll in SB. The gradient was centrifuged at 40,000 g for 1h and the mitochondria
collected as a fuzzy yellow band between the 32% and 50% percoll stages. These mitochondria
were washed in 2 volumes of SB buffer at 85,600 g for 90 min at 4°C to remove the percoll. The
intact mitochondria isolated from PVX-infected leaves were incubated with 1/10 volume of
Proteinase K (20 mg/mL) at RT for 1 hr to ensure that no virions associated with the
mitochondria and washed twice with SB buffer and centrifuged at 28,000 g for 10 min. The
supernatant was discarded and each pellet was gently resuspended in washing buffer. These
47
proteinase K treated and other mitochondria, isolated from transgenic plants, were treated with
RNase A (100 µg/mL) (New England Biolabs, NEB) for 1 hr at RT to digest the viral RNA or
transgene transcripts adsorbed on the surface of mitochondria and washed with SB washing
buffer at 28,000 g for 10 min. Each preparation was again treated with proteinase K to inactive
RNase. After twice washing, the mitochondria were lysed with 1/10 volume of mitochondrial
lysis buffer (MLB) (10% (w/v) N-lauroylsarcosine sodium salt, 25 mM Tris-HCl pH 7.5, 20 mM
EDTA pH 8.0 and 2% βME) at 65°C for 30 min. After lowering the temperature of the sample
to room temperature, one volume of chloroform: isoamyl alcohol (24:1) was added, mixed well
and centrifuged at 14,000 × g for 10 min at room temperature. The aqueous phase was with
mixed 1 μg/μL glycogen (Thermo scientific) as a carrier for nucleic acid, 0.1 M sodium acetate
and 0.6 volume of isopropanol to precipitate the mitochondrial RNA (mtRNA) overnight at -
20ºC. The mtRNA was pelleted by centrifugation at 14000 × g for 10 min, washed with 70%
EtOH and air-dried. Finally, each pellet was dissolved in 50 μl TE buffer (10 mM Tris-HCI pH
8.0 and 1 mM EDTA). RT-PCR reactions were carried out as mentioned above in Section
2.3.10.
2.4 RESULTS
2.4.1 Detection of PVX RNA and coat protein in chloroplast
PVX RNA and coat protein were identified inside the chloroplasts of infected N.tabacum cv.
Xanthi leaf tissues by RT-PCR. To determine the PVX RNA inside the chloroplasts, RNAs were
isolated from enzymatic (Proteinase K and RNAase A) treated chloroplasts of PVX-infected
plants and reverse transcribed into cDNA using reverse primer specific to coat protein gene
followed by PCR amplification using primers specific to 8k gene sequence. These PCR results
48
confirm the existence of PVX RNA inside the chloroplasts of infected leaf tissues as shown in
Figure 2.6 A; Lane 3.
49
Figure 2.6 Detection of PVX RNA and coat protein inside the chloroplast using RT-PCR and
western blot.
A) RT-PCR analysis. Total and chloroplast RNAs from health and PVX-infected plants were
reverse transcribed into cDNAs with CP reverse primer followed by RT-PCR using forward and
reverse primers specific to 8 kDa gene sequence. An RT-PCR product of size 156 bp could be seen
in both total and chloroplasts RNAs samples isolated from PVX-infected plants (Panel a: Lanes 1
and 3 respectively). However this RT-PCR product was absent in both total and chloroplasts RNAs
samples isolated from healthy plants (Panel a: Lanes 2 and 4 respectively) which were used as
negative controls. The purity of chloroplast was confirmed using nuclear-encoded Actin gene. The
187 bp Actin product was not detected in chloroplast RNAs (Panel b: Lanes 3 and 4). A DNA
size marker (100 bp) in 100bp increments was electrophoresed in Lane L. The resulting PCR
products were analyzed on a 2% agarose gel. B) Western blot analysis of a 12.5% acrylamide
gel. PVX CP specific antisera reacted with 50 µg protein (25 kDa coat protein) extracted from
chloroplasts and leaf tissues of PVX-infected plants (Lanes 2 and 4 respectively). However,
this 25 kDa band corresponding to coat protein was absent in both samples purified from
chloroplasts and leaf tissues of healthy plants (Lanes 1 and 3 respectively). C) Reconstruction
experiments: RT-PCR analyses were performed on total and chloroplast RNAs using primers
specific to the PVX 8 kDa gene sequence (Panel a). Total RNA samples isolated from PVX-
infected (Lane 1) and healthy plants (Lane 2) were used as positive and negative controls
respectively. Chloroplast RNA isolated from healthy chloroplasts (mixed with PVX RNA) without
(Lane 3) and with (Lane 4) RNase A treatment respectively. 16SrRNA gene was included as an
internal control (Panel b).
50
To determine whether PVX CP is present in the chloroplasts, these organelles were purified and
treated with protease K to remove any externally associated cytoplasmic protein, and tested for
the presence or absence of PVX gene products. A band, 25 kDa in size, is seen only in protein
extracts from total and chloroplast proteins purified from PVX-infected plants (Figure 2.6 B;
Lanes 2 and 4) but not in the samples of total and chloroplast proteins isolated from non-
infected plants (Figure 2.6 B; Lanes 1 and 3).
2.4.2 Reconstruction control experiments
Additionally, reconstruction controls were also included to rule out that the RNA found inside
the chloroplasts did not originate from RNA adsorbed on the exterior surface of chloroplasts.
Experiments were conducted where chloroplasts from leaf tissues of healthy non-transgenic N.
tabacum plants were isolated and further purified by sucrose gradient centrifugation. Purified
chloroplast were mixed with PVX RNA and subjected to enzymatic treatments as mentioned in
materials and methods section 2.3.9. Chloroplasts from PVX-infected tobacco plants were
isolated and treated with proteinase K prior to the RNase A treatment. Another preparation of
untreated purified chloroplasts from leaf tissues of healthy non-transgenic plants was also
included as a negative control. Chloroplast RNA samples were reverse transcribed after DNase I
treatment, followed by subsequent RT-PCR reactions. It is clear from the reconstruction
experiments that viral RNA is completely degraded after RNase treatment (Figure 2.6 C; Lane
4). However, a sample without RNase A treatment showed a prominent band of viral RNA
corresponding to 8k gene (Figure 2.6 C; Lane 3). These results clearly demonstrate that PVX
RNA found in chloroplasts cannot be due a simple contamination of adsorbed RNA on the
surface of chloroplasts.
51
2.4.3 Design of constructs to confirm RNA tractor activity in chloroplasts
Previously Hefferon, et al. (205) demonstrated that potato plants transformed with clones
containing the PVX sequences of the CP and 8 kDa proteins resulted in the translocation of PVX
RNA sequences to the chloroplasts. To establish the role, if any, of either the CP and/or 8kDa
proteins in the translocation process of viral RNA from the cytosol to chloroplasts and to
determine the minimum sequence “RNA tractor” required for RNA translocation to chloroplasts,
several constructs were engineered. To establish the minimum PVX sequence (“RNA tractor”)
required for the translocation of RNA to chloroplasts, five constructs were produced in a pC-
GFP binary plasmid, where all PVX sequences are driven by the 35S promoter followed by the
GFP gene and the T-nos terminator (Figure 2.3). Successful transformation of tobacco plants
was confirmed for every construct by RT-PCR of total RNA and by DNA sequencing and
selected for further experiments. Results obtained from RT-PCR analyses with constructs (pTR:
8K-CP, pTR: 8K (insG80)-CP, pTR: 8K and pTR: 224) indicated that PVX sequence
corresponding 8 kDa RNA was present within chloroplasts of transgenic plants harboring these
constructs, even where both 8 kDa and CP proteins were disabled either independently or in
tandem (Figure 2.3). This suggests that neither protein is responsible for translocation of PVX
RNA into chloroplasts. Furthermore, to elucidate the smallest possible region that retains “RNA
tractor” activity, two more constructs, pTR:127 and pTR:27 were designed (Figure 2.3). Note
that pTR:127 construct was designed in a manner that the AUG for the GFP is not in frame with
the AUG of CP (Figure 2.5). Consequently, the GFP protein will not be translated in
chloroplasts. In addition, the viroid clone (pCELVd-GFP) containing 330 nucleotides of
Eggplant latent viroid (GenBank Accession number AN - HM136583) as a 5´-UTR followed by
the GFP gene and pC-GFP construct which contains solely the GFP gene were included as
positive and negative controls respectively (Figure 2.3).
52
2.4.4 Analyses for expression of different constructs in total cellular RNA
Total RNA from leaf tissues of plants transformed with pC-GFP, pTR:27, pTR:127 and
pCELVd-GFP constructs were subjected twice to DNaseI treatments, sequentially repeated using
deproteinization and re-precipitation steps between the successive DNAseI digestion steps, to
ensure that the RNA in the extract was entirely free of genomic DNA. A reverse transcription
reaction of each sample was performed and cDNAs were amplified by PCR using GFP187
primers specific to GFP gene sequence. Chloroplast 16SrRNA, and nuclear Actin genes were
also utilized as reference controls using 16SrRNA187 (forward and reverse primers) and
Actin187 (forward and reverse primers) respectively. The PCR products obtained demonstrated
that all samples for total RNA used expressed the same levels of RNAs (GFP, Actin, and
16SrRNA) as seen in Figure 2.7 Lanes 1, 3, 5 and 7 respectively.
53
Figure 2.7 RT-PCR analyses of total and chloroplast RNAs expressed.
RNAs were extracted from leaf tissues (total RNA) and chloroplasts of transgenic N. tabacum cv. Xanthi harboring
pTR:127, pTR:27, pC-GFP and pCELVd-GFP constructs. Chloroplast coded 16SrRNA and nuclear-coded Actin
transcripts were used as positive and negative controls respectively for chloroplast samples using gene specific primer
pairs giving product size 187 bp in each case. The resulting RT-PCR products were analyzed on 2% gel and the
expected 187 bp product was detected from A) GFP: Lanes 1,3,5 and 7 in the case of total RNA and only Lanes 2
and 8 in the case of chloroplast RNA, the 187 bp GFP product was detected. B) Actin: Lanes 1, 3, 5 and 6 in the
case of total RNAs, the 187 bp Actin product was not detected in chloroplast RNAs Lanes 2, 4, 6 and 8. C)
16SrRNA: Lanes 1 to 8, the 16SrRNA 187 bp product was detected in all samples which ensure the integrity of
RNAs.
2.4.5 Translocation of RNA transcripts driven by different constructs into
chloroplasts
RT-PCR experiments were carried out to detect the presence of PVX RNA short sequences
within the chloroplasts of transgenic plants. Chloroplast RNAs, from the same transgenic plants
used for total RNA extractions, were reverse transcribed into cDNAs and amplified by PCR.
Chloroplast 16SrRNA and nuclear Actin Tob103 genes were utilized as positive and negative
controls respectively. Results obtained show that the expected 187 bp fragment of GFP was
detected in the only pTR:127 and in the positive control of pCELVd-GFP (Figure 2.7, Lanes 2
and 8). GFP was not detected in pTR:27 and pC-GFP (Figure 2.7, Lanes 4 and 6). 16SrRNA
54
(chloroplast-encoded gene) bands with equal intensity were visible in all lanes while Actin bands
with equal intensity were only visible in total RNA samples (Figure 2.7 Lanes 1, 3, 5 and 7).
This indicated that RNAs extracted from chloroplasts were free from any contaminating
cytosolic RNAs. pTR:27 failed to be translocated to chloroplasts which indicates that the PVX
RNA tractor activity requires additional PVX sequence beyond the 27 nucleotides. On the other
hand the pTR:127 preserved this RNA tractor activity and could also translocate a foreign
mRNA (GFP mRNA) (Figure 2.7 Lane 2).
2.4.6 Quantitation of translocated RNA to chloroplasts by real-time RT-PCR
To reinforce the semi-quantitative RT-PCR data obtained, real time RT-PCR experiments were
performed. To check for the variability in expression in transgenic plants, comparative real time
RT-PCR experiments were performed with total RNA isolated from leaf tissue harvested from
selected lines of transgenic plants harboring pC-GFP, pTR:27, pTR:127 and pCELVd-GFP
constructs respectively (Table 2.3).
Table 2.3 Relative quantification (expression) of GFP-transcripts derived from transgenic leaves harboring
given constructs using comparative real time RT-PCR.
Total RNAs were extracted and normalized to the expression of endogenous reference actin gene. GFP-transcripts
driven by pC-GFP were used as a control.
(a): Threshold cycle (Ct: the number of cycles at which the fluorescence exceeds the threshold); mean values of
duplicate assays carried out with two different samples. (b): The range given is relative to control (in brackets).
ΔCt: Difference between values of reference and target (target is normalized to the reference). ΔΔCt: ΔCt of each
sample is further normalized to the control. 2-ΔΔCt: Fold change relative to control.
Total
Cta (Mean)
ΔCt
ΔΔCt
2-ΔΔCt
GFP
(Target)
Actin
(Reference)
GFP fold
difference
relative to
control
pC-GFP (Control) 18.99±0.01 23.75±0.07 -4.76±0.07 0.00±0.07 1
(0.94-1.72)b
pTR:27
18.95±0.08 24.21±0.00 -5.56±0.08 -0.8±0.08 1.7
(1.641-1.846)
pTR:127
18.90±0.01 23.40±0.02 -4.50±0.02 0.26±0.02 0.8
(0.604-1.612)
pCELVd-GFP 18.80±0.20 24.15±0.70 -5.35±0.73 -0.59±0.73 2.0
(0.907-2.49)
55
Calculated Ct values in each case were around 18.9 showing almost the same level of expression.
Furthermore, the change (in the fold) was calculated after normalizing with a reference gene
(Actin). Results shown in Table 2.3 indicate values 1.7, 0.8 and 2-fold of messenger RNA for
pTR:27, pTR:127 and pCELVd-GFP respectively, suggesting that even a lower expression of
pTR:127 could manage to translocate into chloroplasts and rules out external contamination.
To check the sub-cellular (chloroplast) localization of transcripts, RT-q PCR experiments
were performed with chloroplast RNAs isolated from transgenic plants expressing different
constructs. RNA transcripts driven by pCELVd-GFP were used as a positive control for
chloroplast translocation. Amplification plot is shown in Figure 2.8 A validated the translocation
of PVX sequence “RNA tractor”. The light green and dark green plots representing Eggplant
latent viroid (pCELVd-GFP) and RNA tractor sequence (pTR:127) respectively showed
amplification while there is no amplification for the blue and orange curve in case of pTR:27 and
pC-GFP samples respectively. PCR amplification efficiency (91.8%) was set by means of
standard curve which was set with serial 10-fold dilutions of the template PCR product (GFP
187) with 5 points (red) as shown in Figure 2.8 A. The efficiency of the PCR should be close to
100 (90-110 %) meaning doubling of the amplicon at each cycle with r2 (coefficient of
determination) values above 0.98 and the slope -3.32 (with a tenfold serial dilution the Cq or Ct
values should be separated by 3.32 cycles).
56
Figure 2.8 Graphical representation of real-time PCR data to quantify translocated “RNA tractor” sequence using
SYBR® Green detection method.
A) Quantitative PCR amplification is performed on serial 10-fold dilutions with 5 points (red color) of the template (GFP
standardized to 187bp) to establish a standard curve. Duplicate lines indicate a repeat of the same sample. Chloroplast RNA
samples; pTR:127 (dark green), pCELVd-GFP (light green), pTR:27 (Blue) and pC-GFP (orange) were amplified by RT-
qPCR along with standard (GFP 187). B) Standard curve: Cq is calculated from values in A are plotted (Y-axis) against the
log of the copy number (x-axis) of the template to establish a standard curve with an efficiency of 91.8% from the slope -
3.536 and r2 value 0.999. C) Melt curve analysis of the amplicons shows a single peak (about 88°C) displaying the negative
first derivative of temperature versus relative fluorescence units (-d (RFU)/dT) plotted against temperature. Cq:
quantification cycle. RFU: relative fluorescence unit. A standard curve was a duplicate.
As illustrated in the melting curve in Figure 2.8 C, there is a significant single sharp peak with
Tm of 88°C in each amplicon. This single peak rules out the presence of non-specific bands
which may arise due to non-specific binding of primers. These RT-qPCR results are similar to
those described for the semi-quantitative RT- PCR (Figure 2.7).
57
2.4.7 Comparison of translocation efficiency of PVX RNA tractor (pTR:127)
to Eggplant latent viroid sequence (pCELVd-GFP)
Chloroplast translocation capacity of pTR:127 was compared with that of pCELVd-GFP using
chloroplast 16SrRNA gene as an internal control (Figure 2.9 A and B).
Figure 2.9 Graphical representation of real-time RT-PCR data (using SYBR® Green detection method)
showing relative translocation activity of pTR:127 compared to Eggplant latent viroid (pCELVd-GFP).
A) Relative real time RT-PCR amplification is performed on chloroplast transcripts (GFP) isolated from pTR:127
(dark green) and pCELVd-GFP (light green) in triplicate samples. Chloroplast 16SrRNA from pTR:127 (black)
and pCELVd-GFP (purple) were used as internal controls. B) Melt curve analyses of the amplicons (GFP and
16SrRNA) in single peaks, displaying the negative first derivative of temperature versus relative fluorescence
units (- d (RFU)/dT) plotted against temperature. RFU, relative fluorescence units.
Steep curves were observed in the amplification profile suggesting that the reference gene
(16SrRNA) was expressed in all samples almost in a similar way (Figure 2.9 A). A single sharp
peak is observed in each sample which confirms the presence of a specific PCR product (Figure
2.9 B). The translocational activity of RNA tractor (pTR:127) was compared to that of Eggplant
latent viroid (pCELVd-GFP). Results obtained show that relative RNA abundance of viroid is
about 120-fold that of pTR:127 (Table 2.4).
58
Table 2.4 Relative quantification of chloroplast RNA expression of pTR:127 and pC-ELVd-GFP using real
time RT-PCR.
RNAs were extracted from purified chloroplasts and normalized to the expression of endogenous reference
16SrRNA gene. The abundance of GFP-transcripts driven by pCELVd-GFP was compared with that of GFP-
transcripts driven by pTR:127 in chloroplasts.
(a): Threshold cycle (number of cycles at which the fluorescence exceeds the threshold). Mean values of duplicate
assays carried out with two different samples. (b): The range relative to the control is given in brackets.
2.4.8 Translocation of “RNA tractor” sequence to plant mitochondria
To determine the translocation of “RNA tractor” sequence into mitochondria, RT-PCR with
mitochondrial RNA isolated and purified from transgenic plants harboring pTR:127 construct
were performed using GFP187 primers specific to GFP gene sequence. Results obtained showed
that the expected 187 bp fragment of GFP was detected in only total pTR:127, while this GFP
gene fragment was absent in mitochondrial sample (Figure 2.10, Panel a; Lanes 1 and 2
respectively).
Chloroplast
RNA
Cta (Mean)
ΔCt
ΔΔCt
2-ΔΔCt
GFP
(Target)
16SrRNA
(Ref.)
GFP fold
change
pTR:127 (control) 28.82±0.08 8.81±0.27 20.01±0.29 0±0.29 1
(0.817-1.22)b
pCELVd-GFP tested 22.34±34 9.24±0.01 13.10±0.38 -6.91±.10 120.25
(112.20-128.89)
59
Figure 2.10 Mitochondria isolation and RT- PCR- analyses with mitochondria and total RNA from
transgenic tobacco plants harboring pTR:127 construct.
A) The mitochondria were collected as a fuzzy yellow band between the 32% and 50% percoll stages after
ultracentrifugation and subjected to enzymatic treatments to remove any external contamination. B)
Mitochondrial RNA was isolated and subjected to RT-PCR after DnaseI treatment. GFP primers were used to
amplify 187 bp product from leaf tissues (total) and mitochondria (Panel a; Lane 1 and 2 respectively). Primers
for18SrRNA gene from mitochondrial genome were used to amplify 187 bp product as an internal control to
check the integrity of RNA from leaf tissues (total) and mitochondria (Panel b; Lane 1and 2 respectively). A
DNA size marker (100 bp) in 100bp increments was electrophoresed in Lane L. The resulting PCR products
were analyzed on a 2% agarose gel.
Taken together these preliminary results, it might be concluded that “RNA tractor” sequence of
PVX failed to translocate into mitochondria of pTR:127 transgenic plants.
60
2.5 DISCUSSION
Our previous studies have shown that the CP and CP RNA of PVX accumulate within
chloroplasts of plants transformed with the PVX 8 k-CP dicistronic construct (174, 205).
Although these results established the presence of PVX RNA within chloroplasts, very little was
known about the mechanism by which this RNA entered the organelle. At first, it was believed
that viral proteins (i.e. the CP and/or 8 kDa proteins) were involved in this translocation
phenomenon since they have been previously implicated in the intercellular movement of the
viral RNA (170). Results described in this study clearly demonstrated that neither protein (8 kDa
or CP) alone or together were required for the translocation of RNA into the chloroplast. Indeed,
when an out of frame mutation was introduced into the 8 k gene or when a large segment of the 8
k gene was deleted (pTR:127), the RNA was still translocated to the chloroplast. Similar results
were obtained when the CP gene was essentially deleted, with the exception of the first 13
nucleotides including the initiation codon. Construct pTR:127 was still capable of translocation
of not only its original PVX sequence but also the downstream sequence of GFP, which
constitutes a part of the same original tricistronic (8 k, CP and GFP genes respectively) transcript
and is under the control of the 35S promoter. Conversely, results obtained with the pTR:27
construct suggested that the translocation capability was abolished when 127 nucleotides
sequence was further deleted to only 27 nucleotides. This finding provides a second line of
evidence, indicating the required length of the RNA sequence must be more than 27 RNA
nucleotides to maintain the RNA tractor activity. A reconstruction control experiment (Figure
2.6 C) was also included to eliminate the possibility that the RNA we detected was attached to
the surface of the chloroplast as a result of the isolation procedure and not degraded even treated
with RNase A. The results shown in Figure 2.7 validate the purity of our chloroplast
61
preparations and rule out the possibility that cytoplasmic/nuclear RNAs may co-purify with
chloroplasts.
To calculate the relative expression level and also to reinforce the semi-quantitative
results, we have used the comparative Ct method, also known as 2-∆∆CT, which is a convenient
way to analyze the relative changes in gene expression (217, 220, 221). Comparative Ct method
assumes that the amplification efficiency of the target gene, i.e. GFP, and endogenous control,
i.e. 16SrRNA, must be the same (221). It is noteworthy that RNA expression level in the selected
transgenic lines of pTR:27 and PC-GFP is higher in comparison with RNA level in pTR:127
which further supports our findings and rules out external contamination. The chloroplast
translocation efficiency of the PVX RNA tractor was determined to be 120 X lower than that of
Eggplant latent viroid. One explanation for this major difference is the fact that unlike PVX,
eggplant latent viroid replicates inside chloroplasts. It is possible that PVX RNA targets this
organelle to escape from the host immune system.
To provide another line of evidence of the RNA tractor activity for chloroplasts and
determine that the GFP sequence is functional in the chloroplast, pTR:127 construct was
redesigned considering the translation mechanism of chloroplasts. Various strategies were
attempted to make the RNA tractor sequence functional for GFP mRNA as a reporter gene
(Appendix A). Despite the presence of chloroplastic genome sequences, required for the
translation, GFP was failed to express in the chloroplasts, however, it was observed that the GFP
was functional in the agrobacteria cells (Appendix C). Further research is required to establish
the generality of this phenomenon using RNA tractor.
Since the Eggplant latent viroid chimeric sequence was included as a positive control, for
translocation into the chloroplasts, on the basis of the previous findings by Gomez and Pallas
(95) who demonstrated that the viroid sequence acting as a 5´-UTR end mediated the trafficking
62
and accumulation of a functional foreign mRNA into the N. benthamiana chloroplasts. However,
it is not clear how and why such a chimeric viroid sequence allowed the translation in
chloroplasts. Whether the viroid sequence or/and specific structure motifs are required for the
translation of GFP in the chloroplasts. To address these questions, mutational analyses were
performed with this chimeric sequence (Appendix B). Overall these results suggest that
sequence elements and/or secondary or tertiary structural domain together may require the
translation of functional mRNA into the chloroplasts. Further experiments are required to solve
this mystery.
Previously in our lab Hefferon, et al. (174) demonstrated with transgenic plants
that the 8 kDa protein and the CP could be translated from a dicistronic construct
corresponding to the C-terminal half of the 12 kDa protein, the complete 8 kDa and CP
genes of PVX, indicating that translation of CP could take place either by internal entry
of ribosomes (IRBS) or by a termination/reinitiation mechanism. To confirm and reassess
the IRBS property of the PVX 8K region using the GFP gene as a reporter (fused with ORF of
CP of PVX) in in vivo, a stable transgene expression system was used. Western blot and
confocal studies indicated the expression of a downstream cistron (GFP) only in absence of the
hairpin in transgenic tobacco plants harboring the dicistronic construct, suggesting that that the
translation of GFP could take place by a termination/reinitiation or leaky scanning rather an
internal ribosome binding site (IRBS) mechanism (Appendix D).
Numerous positive-sense RNA viruses were shown to replicate their genomes on a
variety of membrane systems including endosomes and lysosomes, nuclear envelopes,
endoplasmic reticulum (ER), and organelles (chloroplasts, mitochondria) (see (222) for more
citations). A recent study has shown that Bamboo mosaic virus (BaMV) RNA was transported to
chloroplast by interacting with nuclear-encoded chloroplast proteins (209). Many viral proteins
63
(in particular RNA-dependent-RNA polymerases) contain hydrophobic regions which interact
with the specific cellular membrane system(s) to generate “vesicle-like” systems where the viral
replication is shown to take place. Normally such replication is carried out on the surface of
organelles and/or membrane system. Other viruses employ the normal cellular strategy to
translocate their proteins to chloroplasts. The fungus transmitted Tombusvirus cucumber necrosis
virus (CNV) employs the strategy of the signal peptide to translocate its capsid proteins to the
chloroplast. Such a targeting motif was also shown to contain 14-3-3 binding domain typical of
cellular protein translocation from cytosol to chloroplasts (177). These findings suggest that viral
and/or host proteins could be responsible for the movement of the viral genome and proteins to
the outer membrane system. In our case, however, the PVX RNA tractor system seems to
involve no viral proteins. In this respect, it may be most comparable to the transport and
translocation of the RNA of viroids and in particular, Avsunviroidae. An Eggplant latent viroid -
derived sequence (pCELVd-GFP) was used as a 5´-UTR end to mediate the import of GFP-
mRNA into chloroplasts (95). Viroids do not code for any proteins and they have to rely on
cellular proteins (if any) for their transport, entry into chloroplasts for replication and for the exit.
Some cellular proteins such as PARBP33 and PARBP35 were shown to be involved in
replication, self-cleavage (ribozyme), protection of RNA and possibly escort of this type of
viroid to the chloroplast (223). The RNA tractor described here is the first reported for a viral
small sequence that is capable of not only translocating its own sequence but also a foreign
sequence such as that of GFP into chloroplasts. Presumably, any foreign RNA could be targeted
to chloroplast by this RNA tractor. However, the exact mechanism of viroid and “RNA tractor”
translocation (and exit) to chloroplasts remains unclear.
Sequence analysis data of pTR:127 with other Potex and Carlaviruses revealed no
sequence homology which implies that our RNA tractor may be unique to PVX. The role of
64
cellular protein (s) involved in such an RNA translocation event is/are not yet established.
However, we can theorize that the RNA translocation to chloroplast may simply involve an RNA
structure which is capable of interacting with a chloroplastic membrane protein which acts in an
analogous fashion to a receptor binding process to trigger pinocytosis and/or endocytosis, thereby
allowing the internalization of the RNA to the chloroplast. RNA folding analysis of “RNA
tractor” sequence revealed the formation of a hairpin (finger-like) structure which potentially
could be involved in the attachment of the RNA tractor to the chloroplastic membrane followed
by its entry into the chloroplast.
Although this study has provided an intriguing rationale for “RNA tractor” localization to the
chloroplasts, the functional consequences or mechanism of localization remains to be determined.
Since it has been established that none of the viral proteins are involved for the movement of
PVX “RNA tractor” into chloroplasts, it is, therefore, possible that some host factors are
interacting with PVX “RNA tractor” and facilitating its transport into the chloroplasts. Further
studies are required to detect any potential host protein(s) interacting with Eggplant latent viroid
and PVX “RNA tractor” using biochemical approaches like electrophoretic mobility shift assay
(224, 225) or/ and UV cross-linking RNA/protein complexes (223, 226, 227), followed by mass
spectrometry to analyze the sequence of the purified proteins (228). Understanding the molecular
mechanisms of this RNA tractor may lead in the future to further comprehension of some of the
trafficking mechanisms of RNAs between organelles (chloroplasts and mitochondria) and
nucleus. This may also lead to understanding some aspects of gene regulation and development.
65
CHAPTER 3
3 STUDIES ON INFECTIVITY AND TRANSLOCATION OF
VIRAL DNAS FROM CYTOSOL TO ORGANELLES
3.1 INTRODUCTION
The family Geminiviridae is comprised of plant DNA viruses that have long been known as
model systems for the elucidation of basic cellular components of the plant replication and
transport machinery (118, 119, 229, 230). This family consists of phytopathogenic viruses with
characteristic twinned, quasi-isometric virions encapsidating genomes of circular single-stranded
(ss) DNA. Geminiviridae is classified into seven genera, six of which (Mastrevirus, Curtovirus,
Topocuvirus, Becurtovirus, Eragrovirus and Turncurtovirus) consist of viruses with monopartite
genomes. The seventh genus Begomovirus consists of viruses with either monopartite (a single
DNA) or bipartite (with two DNA components: DNA-A and DNA-B) genomes (123, 125, 126,
231, 232). The DNA-A of bipartite and the single component of monopartite begomoviruses
contain five or six Open Reading Frames (ORFs) while the DNA-B contains two ORFs (BV1
and BC1, in viral-sense and complementary sense strand, respectively). Both DNA-A and DNA-
B are approximately 2.8-3.0 kb in size. Monopartite begomoviruses are often associated with one
or smaller DNA components, about 1.4 kb in size, known as satellite DNAs. Two types of
satellite DNAs are known: the alpha-satellites and beta-satellites, depending upon the
organization of their DNA and their effects on the symptoms produced by the helper
begomovirus. Both the alpha- and betasatellites are dependent upon the helper virus for
replication and, in many cases, mitigate the symptoms produced by it (233). The major
symptoms caused by begomoviruses are leaf curling, stunting, and chlorosis. Geminiviruses
encode proteins that contribute to pathogenicity. These proteins differ between monopartite and
66
bipartite begomoviruses, as well between viruses within the individual groups (234, 235).
Begomoviruses infect a wide range of economically important dicotyledonous host plants and are
transmitted by the whitefly Bemisia tabaci (236, 237). Various aspects of the Family
Geminiviridae have been comprehensively reviewed (238-240).
I used tobacco and tomato plants for pathogenicity assays. Tobacco is a model plant
organism to study basic biological processes (241) and it is also a major crop species used for
studying plant disease susceptibility, which it shares with other Solanaceae plants like potato,
tomato, and pepper (242). The genus Nicotiana (family Solanaceae) has been the main focus of
research which has provided information about the host-pathogen relationship in the context of
innate immunity and defense signaling. Particularly Nicotiana benthamiana and N.tabacum (both
allotetraploid) species have been widely used as experimental hosts of plant virology studies
(243). N. benthamiana is generally susceptible to the majority of plant viruses. It is the most
widely used experimental host in plant virology mainly but not restricted to its ability to express
foreign genes, used as a virus-induced gene silencing (VIGS), as a research model for
agroinfiltration (243) and also combination of these methods to investigate signal transduction
(244) and protein trafficking (245).
The movement of geminiviruses within host plants has been studied extensively (161,
229, 243, 246-250). Geminiviruses use the DNA replication machinery of their host to amplify
their genomes in the nuclei of infected plant cells (251). This viral DNA is transported out of the
plant cell nucleus to undergo systemic spread by crossing plasmodesmatal openings in the cell
membrane. For monopartite begomoviruses coat protein with the conjunction of pre-coat is
required to cross cell membranes (249). In contrast to monopartite begomoviruses, bipartite
begomoviruses are dependent upon DNA-B encoded nuclear shuttle protein (NSP) and
movement protein (MP) for their movement in host plants (156, 246, 252). It has been revealed
67
that βC1 of beta-satellite can substitute the movement function of DNA-B to facilitate movement
of begomovirus from the nucleus to the cell periphery (248). The transport of viral ssDNA from
the nucleus towards plasmodesmata is mediated by a nuclear export signal (NES) on the coat
protein C-terminus and NES on the Pre-coat protein N-terminus (249, 250). Nuclear shuttle
protein interacts with histone H3, raising the possibility that viral DNA moves as a
minichromosome (253). An NSP-interacting GTPase (NIG) associated with the exterior of the
nuclear envelope might facilitate NSP transit into the cytosol, probably through the nuclear pore
(254). The NSP-DNA complex then moves to the cell periphery through interaction with MP.
Viral DNA might be transferred to MP through a mechanism involving NIG-catalyzed GTP
hydrolysis (255). Alternatively, NIG might facilitate the interaction of MP with an NSP-DNA
complex that moves through plasmodesmata, which provides a mechanism for movement of
viral DNA into the nucleus of the next cell. The chaperone, the nuclear-encoded and chloroplast-
targeted heat shock cognate 70 kDa protein (cpHSC70-1) was shown to interact with the
Abutilon mosaic virus (AbMV, Geminiviridae) movement protein (MP) for trafficking along
plastids and stromules into a neighboring cell or from plastids into the nucleus (256). An
involvement of plastids and stromules is assumed in the DNA-virus life cycle as well, but their
functional role needs to be determined (257).
The molecular mechanisms underlying intercellular movement of viruses have been well
studied; however studies on sub-cellular, other than the nucleus, localization of genome of these
viruses have been less explored. The key research question in this study is whether or not, viral
DNA can be translocated into chloroplasts. The study conducted to answer this question
confirms the presence of only AEV DNA-A (monopartite), conversely, ToLCNDV DNA-A was
absent in chloroplasts of the viral infected leaves. The DNA of Abutilon mosaic virus was
isolated from intact chloroplasts (164) representing the only other example of a geminiviral viral
68
genome in chloroplasts. Considering these findings on the sub-cellular localization, it is
conceivable that viruses use fundamentally different transport mechanisms within their hosts. In
this study, I also demonstrate the infectivity of monopartite and bipartite begomoviruses to
Solanum lycopersicum and different Nicotiana species to assess the effects of ploidy level on
susceptibility to begomoviruses. These findings could be useful to provide us a better
understanding of begomovirus pathogenicity and virus-host interactions.
3.2 RESEARCH PLAN
The overall objectives of this study are:
1. To determine the infectivity of monopartite (AEV) and bipartite (ToLCNDV)
begomoviruses in tomato and different Nicotiana species.
2. To study the capacity of these single-stranded DNA viruses (AEV and ToLCNDV) to
translocate their genomic DNA to chloroplasts of different Nicotiana species and tomato
plants.
3. Finally to study the translocation of AEV DNA-A genome to plant mitochondria.
3.3 MATERIALS AND METHODS
3.3.1 Plant growth conditions
Tobacco (Nicotiana alata, N. benthamiana, N. clevelandii, N. glutinosa, N. rustica, N. sylvestris,
N. tabacum cv. Xanthi, N. tabacum cv. Samsun) and tomato (Solanum lycopersicum, variety
Ultra Girl VFN) seeds were sown in Pro-Mix (Premier Tech ,Canada) and transferred to pots (1-
2 plants per pot) containing Pro-Mix when the seedlings were 3 weeks old. The plants were
grown at 23-27°C under 16 h light/8 h dark condition in an insect-free greenhouse.
69
3.3.2 Agrobacterium-mediated inoculation
Infectious clones of AEV (DNA-A and DNA-β) and ToLCNDV (DNA-A and DNA-B) were
kindly provided by the Molecular Virology Laboratory, Institute of Agricultural Sciences, The
Punjab University. These clones were transformed into competent cells of Agrobacterium
tumefaciens strain GV3101. A single colony of each infectious clone of AEV (DNA-A and
DNA-β) and ToLCNDV (DNA-A and DNA-B) in A. tumefaciens was cultured in 2 mL of LB
culture containing antibiotics Kanamycin (100 µg/mL) and Gentamycin (30 µg/mL) and grown
overnight at 28°C at 225 rpm. A large 30 mL LB media suspension was then inoculated with the
overnight culture and grown at 28°C to an optical density (OD595) of ~1.0. The cells were
harvested by centrifugation at 1200 g for 10 min and resuspended in Agrobacterium induction
medium (10 mM MgCl2, 10 mM MES pH 5.6 and 150 µM acetosyringone) to a final OD595 of
1.0 and incubate at room temperature for 4-6 hr with gentle shaking (80-100 rpm). These
cultures were pelleted again by centrifugation at 1200 g for 10 min and resuspended in 10 mM
MES buffer and adjust to OD595~0.5. At the four true leaf stage, plants were inoculated with
AEV (A + β) and ToLCNDV (A+B) using a 1 cc syringe into the abaxial surface of the leaves.
Each experiment was repeated ten times. Plants were also infiltrated with buffer alone used a
negative control. Following inoculation, plants were observed daily for the appearance of
symptoms. At 30-35 days post-inoculation (dpi) the plants were photographed and leaf samples
were harvested to isolate DNA for PCR analysis.
3.3.3 Extraction of total nucleic acids from plants and PCR
Total genomic DNA was extracted from leaf samples using modified CTAB method (258).
About 100 mg plant material were harvested from newly emerged leaves and ground in 800 μl
2×CTAB buffer (100 mM Tris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl, 2% (w/v) cetyl
70
trimethylammonium bromide (CTAB), 0.25 % polyvinylpolypyrrolidone (PVP) and 2.5% (v/v)
β-mercaptethanol) using a pestle and mortar and incubated at 65°C for 30 min. After lowering
the temperature of the sample to room temperature, a 500 μl of chloroform: isoamyl alcohol
(24:1) was added, mixed well and centrifuged at 10,000 × g for 10 min at room temperature. The
upper DNA containing phase was mixed with 0.6 volume isopropanol to precipitate the DNA.
DNA was pelleted by centrifugation at 14,000 × g for 10 min, washed with 70% ethanol and air
dried. Finally, each pellet was dissolved in 50 μl TE buffer (10 mM Tris-HCI pH 8.0/1 mM
EDTA). PCR reaction were carried out on one microgram DNA as template, determined by ND-
1000 Spectrophotometer (NanoDrop Technologies Inc., USA), in a final volume of 30 µL with
reagents provided by FroggaBio (2X PCR MasterMix) in a PTC-100 thermocycler (MJ
Research). The PCR was performed at 95°C for 5 min, followed by 95°C for 50 sec, 60°C for 50
sec, 72°C for 50 sec for 32 cycles, and 72°C for 5 min. All the primers used are displayed in
Table 3.1.
Table 3.1 Primer sequences used for semi-quantitative PCR.
Target sequence Forward (F) and reverse (R) primers (5´-3´) PCR product size
(base pairs)
Bego CP
F GAAGCGACCAGCAGATATAATC
R CATCCTGTACATCCTGGGCTT
169
AEV CP
F GCCCAGGATGTACAGGATGT
R CACAGGCCTACGATCCCTAA
283
16SrRNA
F GAAGAACCTTACCAGGGCTTGA
R CAGTCTGTTCAGGGTTCCAAAC
187
Actin (tobacco)
F AGTCCTCTTCCAGCCATCCA
R AGCCAAAGCCGTGATTTCC
187
Actin (tomato)
F GAAATAGCATAAGATGGCAGACG
R ATACCCACCATCACACCAGTAT
277
3.3.4 Isolation of intact chloroplast and enzymatic treatments
Chloroplasts were isolated from healthy and infected plants using the modified method (205).
Each isolation step was performed at 4°C separately. Five grams of leaves were harvested and
71
homogenized with mortar and pestle in 50 mL of cold grinding buffer (50 mM HEPES-KOH pH
7.3, 330 mM mannitol, 0.1% BSA, 1 mM MgCl2, 1 mM MnCl2, 2 mM Na2EDTA, and 1 mM
DTT). Homogenate was filtered through eight layers of cheesecloth and the filtrate was pelleted
at 500 g for 2 min to remove the plant debris. After that, the suspension containing chloroplasts
was sedimented by centrifugation at 2,500 g for 20 min at 4°C. Subsequently, each pellet
containing the chloroplasts was carefully resuspended in 1 mL grinding buffer. These
chloroplasts in each sample were counted by haemocytometer and the quantity was equally
adjusted ensuring a similar sample size. Each sample was loaded on top of a sucrose step
gradient developed with 4 mL of 30%, 3 mL of 45%, and 2 mL of 60% sucrose in grinding
buffer and centrifuged at 77,140 g for 55 min. Intact chloroplasts were collected from the
interphase between 30 and 45 % sucrose, washed twice with washing buffer (50 mM HEPES-
KOH pH 8.0, and 330 mM mannitol) and resuspended in 1 mL washing buffer. The purified
chloroplasts were visualized under a light microscope to confirm their integrity. Each suspension
was incubated with 1/10 volume of Proteinase K (20 mg/mL) for 1 hr at 32°C to ensure that no
virions associated with the chloroplasts and washed twice with washing buffer. The supernatant
was discarded and each pellet was gently resuspended in 1 mL of washing buffer and treated
with 20 units DNase 1 (New England Biolabs, NEB) for 1 hr at 32 °C to digest the viral DNA
adsorbed on the surface of chloroplasts. Theses chloroplasts were washed twice with 15 mL of
washing buffer. These chloroplasts were lysed with 1/10 volume of 2×CTAB at 65°C for 30 min.
After lowering the temperature of the sample to room temperature, one volume of chloroform:
isoamyl alcohol (24:1) was added, mixed well and centrifuged at 10,000 × g for 10 min at room
temperature. The aqueous phase was mixed with 1 μg/μL glycogen (Thermo scientific) as a
carrier for nucleic acid, 0.1 M sodium acetate and 0.6 volume of isopropanol to precipitate the
chloroplast DNA (cpDNA) overnight at -20ºC. The cpDNA was pelleted by centrifugation at
72
14,000 × g for 10 min, washed with 70% ethanol and air dried. Finally, each pellet was dissolved
in 50 μL TE buffer (10 mM Tris-HCI pH 8.0 /1 mM EDTA). PCR reaction were carried out on
one microgram cpDNA as mentioned in material and method section 3.3.3.
3.3.5 Light microscopy and transmission electron microscopy (TEM)
The chloroplasts were visualized under a light microscope to confirm their integrity and purity.
A drop of each sample was overlaid on a glass slide and live chloroplast imaging was performed
with a 40 × oil objective lens using a differential interference contrast (DIC) feature of a
confocal microscope (TCS SP5, Leica Microsystems). For TEM sample preparation, chloroplasts
were pelleted by centrifugation, and the pellets were resuspended 3% glutaraldehyde in 0.1M
Sorensen phosphate buffer pH 7.35 for 60 min on a shaker at room temperature and stored
overnight at 4°C. After 3x buffer washing (each 10 min), samples were post-fixed with 1%
osmium tetroxide in phosphate buffer for 60 min at room temperature. After three washing, these
samples were dehydrated for 10 min each step in a graded series of increasing concentrations of
ethanol (30%, 50%, 70%, 80%, 90%, and 100%) and infiltrated with 3:1, 1:1 and 1:3 mixtures
of ethanol: Spurr’s resin (EMS, USA) for 60 min each step. These samples were replaced with
100% Spurr’s resin and left overnight at room temp. Infiltration was continued the next day and
finally, samples were embedded in fresh 100% Spurr’s resin and polymerized at 65°C overnight.
Sections of one-micron thickness were cut with a Leica EM UC6 ultramicrotome (Leica
Microsystems Inc.) to visualize the samples under a light microscope. Ultimately ultra-thin
sections (100 nanometer) for TEM were picked up on 200 mesh Cu grids (EMS, USA) and
stained with 3% Uranyl acetate in 50% methanol for 45min, followed by Reynold’s lead citrate
for 10 min, and allowed to dry overnight at RT and examined with the Hitachi H7700
Transmission Electron Microscope.
73
3.3.6 Isolation of intact mitochondria and enzymatic treatments
Mitochondria were isolated from AEV-infected N.benthamiana leaves using the modified
method (219). Fresh leaves (50 g) were cut and homogenized in a mortar in 120 mL of extraction
medium (EM) (20 mM HEPES-Tris pH 7.6, 0.4 M sucrose, 5 mM EDTA, 0.6% PVP (w/v) and
0.6 mM cysteine). The extract was filtered through 8 layers of cheesecloth and centrifuged 5 min
at 3500 g. The supernatant was centrifuged at 28,000 g for 10 min to pellet organelles. The pellet
was resuspended in 120 mL EM without PVP and centrifuged at 28,000 g for 10 min and the
pellet resuspended in 2 mL of suspension buffer (SB) (10 mM MOPS-KOH pH 7.2 and 0.2 M
sucrose) and loaded on a percoll gradient of 10%, 32% and 50% percoll in SB. The gradient was
centrifuged at 40,000 g for 1h and the mitochondria collected as a fuzzy yellow band between
the 32% and 50% percoll stages. These mitochondria were washed in 2 volume of SB buffer at
85,600 g for 90 min at 4°C to remove the percoll. These intact mitochondria were incubated with
1/10 volume of Proteinase K (20 mg/mL) at RT for 1hr to ensure that no virions associated with
the mitochondria and washed twice with SB buffer and centrifuged at 28,000 g for 10 min. The
supernatant was discarded and each pellet was gently resuspended in washing buffer and treated
with 20 units DNaseI (New England Biolabs, NEB) for 1hr at RT to digest the viral DNA
adsorbed on the surface of mitochondria and washed twice with SB washing buffer at 28,000 g
for 10 min. After the final washing, the mitochondria were lysed with 1/10 volume of 2×CTAB
buffer at 65°C for 30 min. After lowering the temperature of the sample to room temperature,
one volume of chloroform: isoamyl alcohol (24:1) was added, mixed well and centrifuged at
14,000 × g for 10 min at room temperature. The aqueous phase was with mixed 1 μg/μL
glycogen (Thermo scientific) as a carrier for nucleic acid, 0.1 M sodium acetate and 0.6 volume
of isopropanol to precipitate the mitochondrial DNA (mtDNA) overnight at -20ºC. The mtDNA
was pelleted by centrifugation at 14,000 × g for 10 min, washed with 70% ethanol and air dried.
74
Finally, each pellet was dissolved in 50 μL TE buffer (10 mM Tris-HCI pH 8.0 /1 mM EDTA).
PCR reaction were carried out on one microgram mtDNA as a template as mentioned in material
and method section 3.3.3.
3.3.7 Isolation of virus
N.benthamiana plants, at the four true leaf stage, were inoculated with infectious clones of AEV
(A + β; 1:1) and ToLCNDV (A+B; 1:1) in Agrobacterium tumefaciens strain GV3101 using a 1
cc syringe into the abaxial surface of the leaves as mentioned above. At 2-3 weeks post-
inoculation, virions were purified as described previously (259) with the following
modifications. Infected leaves were homogenized in virus extraction buffer (EB) (0.1 M
trisodium citrate, 0.75% (w/v) sodium sulphite, 5 mM disodium EDTA, 1% (v/v) 2-
mercaptoethanol and 0.325% (w/v) L-ascorbic acid pH 7.0, adjusted with NaOH); 2 mL/g of
fresh tissue. The homogenate was made 2.5% (v/v) in Triton X-100, stirred for 16 hr at 4°C and
then squeezed through four layers of cheesecloth. The filtrate was clarified by centrifuged at
10,000 g for 15 min and the supernatant was collected and centrifuged at 91,862 g in a Beckman
Ti 60 rotor for 3 hr. The virus pellets were covered with resuspension buffer (RB) (0.01 M
trisodium citrate, 1 mM disodium EDTA with 0.05% 2-mercaptoethanol, adjust to pH 7 with
NaOH) overnight at 4°C and then resuspended. The suspension was overlaid onto a cushion of
20% (w/v) sucrose in RB buffer and centrifuged for 3 hr at 91,862 g in a Beckman Ti 60 rotor
for 3 hr. The virus solution was clarified by centrifugation three times at 15,000 g for 5 min each.
The virus can be further purified by centrifugation for 16 hr at 52,836 g through 10 to 50% (w/v)
sucrose gradients in RB buffer. These virions were stored at 4°C in the presence of 0.01 %
sodium azide for further downstream applications.
75
3.4 RESULTS
3.4.1 Infectivity Assays: Inoculation of plants with AEV and ToLCNDV DNA
clones
Infectious clones of monopartite AEV (DNA-A and DNA-β) and bipartite ToLCNDV (DNA-A
and DNA-B) begomoviruses in Agrobacterium strain of GV3101 were infiltrated into plants to
assess their ability to infect systemically. Agro-infiltrated plants were observed periodically for
the appearance of symptoms. Plants inoculated with the monopartite begomovirus AEV
remained asymptomatic. In contrast, inoculation with the bipartite begomovirus ToLCNDV
showed mild to severe characteristic symptoms in all plants at 35 days post-inoculation (Figure
3.1 and 3.2; Table 3.2).
76
Figure 3.1 Photographs of symptomatic and non-symptomatic different Nicotiana species:
N. alata (A, F, and K), N. clevelandii (B, G and L), N. rustica (C, H and M), N. sylvestris (D, I and N), and N. tabacum
(E, J, and O). Plants were inoculated with infectious clones of ToLCNDV (DNA-A and DNA-B), showed severe to no
typical symptoms (Panel 1), AEV (AEV-A and DNA-β), remained symptomless (Panel 2) and buffer only as a control
(Panel 3). Photographs were taken at 35 days post-inoculation (dpi).
77
Figure 3.2 Photographs of symptomatic and non-symptomatic different Nicotiana species:
N. Benthamiana (A, F, and K), N. glutinosa (B, G, and L), N. tabacum cv. Xanthi (C, H and M), N. tabacum
cv. Samsun (D, I and N), and Solanum lycopersicum (E, J, and O). Plants were inoculated with infectious
clones of ToLCNDV (DNA-A and DNA-B), showed typical symptoms (Panel 1), AEV (AEV-A and DNA-β),
remained symptomless (Panel 2) and buffer only as a control (Panel 3). Photographs were taken at 35 days
post-inoculation (dpi).
78
Table 3.2 Summary of the results of the infectivity assays
Species
(ploidy level,
chromosome
number)
Infectivity
of
ToLCNDV
(plants
infected
/inoculated)
Plants
PCR
positive
for
ToLCNDV
Infectivity
of
AEV
(plants
infected
/inoculated)
Plants
PCR
positive
for
AEV
Symptoms
ToLCNDV
AEV
N.benthamiana
(4x = 38)
10/10
10
10/10
10
Severe symptoms
leaf curling, thickening of
veins, stunted growth,
leaf crumple
no to very
mild
symptoms
N.glutinosa
(2x=24)
10/10
10
10/10
10
Severe symptoms
distortion of leaves,
stunted
growth, leaf crumple,
depressions on the upper
surface of the leaves
no
symptoms
N. clevelandii
(4x=48)
10/10
10
10/10
10
Severe symptoms
distortion of leaves,
stunted growth, leaf
crumple, depressions on
the upper surface of the
leaves
no
symptoms
N. sylvestris
(2x=24)
10/10
10
10/10
10
no to very mild symptoms
no
symptoms
N. rustica
(4x=48)
10/10
10
10/10
10
no to very mild symptoms
no
symptoms
N. alata
(2x=24)
10/10
10
10/10
10
no to very mild symptoms
no
symptoms
N. tabacum
cv. Samsun
(4x=48)
10/10
10
10/10
10
mild symptoms
depressions on the upper
surface
of the leaves
no
symptoms
N.tabacum
cv. Xanthi
(4x=48)
10/10
10
0/10
0
Severe symptoms
distortion of leaves,
stunted growth, leaf
crumple, depressions on
the upper surface of the
leaves
no
symptoms
N.tabacum
cv. unknown
(4x=48)
10/10
10
10/10
10
no to very mild symptoms no
symptoms
Solanum
lycopersicum
(2x=24-26)
10/10
10
10/10
10
Severe symptoms
leaf curling, thickening of
veins, stunted growth,
yellow mosaic or mottled
pattern, failure of
reproductive organs to
develop normally
no
symptoms
79
Consequently, the virus infectivity was determined by PCR (Figure 3.3). Total genomic DNA
was extracted from leaves and subjected to semi-quantitative PCR using consensus Bego CP F
and R primers which were designed to amplify a 169 base pair region of coat protein gene of
begomoviruses (Table 3.1). The expected size of coat protein gene could be amplified from
plants challenged with infectious clones of AEV and ToLCNDV whereas no band was detected
with DNA extracted from buffer treated plants (Figure 3.3, Panel C). The exception was N.
tabacum cv. Xanthi plant where only ToLCNDV was systemically infected. The DNA-A titer of
AEV was higher in N. benthamiana than other plants and in comparison to DNA-A of
ToLCNDV as well (Figure 3.3 Panel C). To validate the semi-quantitative results of AEV and
ToLCNDV, DNA samples were amplified by PCR using primers pairs for Actin gene used as an
internal control (Figure 3.3, Panel D).
Figure 3.3 PCR-mediated detection of AEV and ToLCNDV DNA extracted from chloroplasts and leaf tissues
(total DNA) of infected plants at 35 dpi.
Bego CP primers specific to the similar coat protein gene sequences of AEV and ToLCNDV were used to amplify 169
product from chloroplasts (Ch) and leaf tissues (Panels A and C respectively). 16SrRNA gene with consensus primers
was included as an internal control to check the integrity of DNA isolated from chloroplasts (Panel B). Actin genes
(tobacco and tomato, Table 2) were used as a loading control for total DNA (Panel D). A DNA size marker (100 bp)
in 100bp increments was electrophoresed in Lane L. The resulting PCR products were analyzed on a 2% agarose gel.
80
Overall, these results indicate that AEV likewise ToLCNDV can manage to replicate and spread
from the site of inoculation, however, the ability of AEV to systemically infect plants without
causing symptoms is of interest and suggests that the virus is unable to interact with factors
involved in inducing symptoms. It is also noteworthy that AEV behaves distinctly in different
cultivars of same species of N. tabacum. This virus infects the cv. Samsun but not the cv.Xanthi,
both belong to the same tabacum species, as shown in Figure 3.3.
3.4.2 Chloroplast DNA Analysis
To investigate the subcellular localization of DNA of AEV and ToLCNDV, chloroplasts were
isolated under isotonic conditions from different tobacco and tomato plants infected with AEV
and ToLCNDV infectious clones (Figure 3.2). DNA was isolated from purified chloroplasts and
subjected to PCR analysis. Results obtained demonstrated that the expected size fragment of the
coat protein (CP) gene could be amplified from DNA of chloroplasts isolated from only AEV
infected plants (Figure 3.3 Panel A, Lanes 1, 2, 3 and 4). On the contrary, no specific CP gene
bands were detected with DNA of chloroplasts extracted from ToLCNDV (Figure 3.3, Panel A,
Lanes 5, 6, 7 and 8). Chloroplast DNA of N. tabacum cv. Xanthi was used as a negative control
in case of AEV CP gene (Figure 3.3 Panel A, Lane 9). The integrity of chloroplast DNA was
confirmed with 16SrRNA gene included as an internal control (Figure 3.3 Panel B, Lanes 1-9).
Thus, these results for both ToLCNDV and AEV infectious clones suggest that the viruses
themselves exhibit different properties with respect to subcellular localization.
3.4.3 Reconstruction control experiments
Additionally, reconstruction controls were also included to rule out that the AEV DNA-A found
inside the chloroplasts did not originate from DNA adsorbed on the exterior surface of
chloroplasts. Experiments were conducted where chloroplasts from leaf tissues of healthy N.
81
benthamiana plants were isolated and purified by sucrose gradient centrifugation. These purified
chloroplasts were incubated with AEV virions, half of this sample was used to extract
chloroplast DNA without proteinase K and DNase I treatment and used as a positive control. The
remaining half sample was treated with proteinase K and DNase I followed by proteinase K with
in between washings as described in material and method section 3.3.4. Another preparation of
untreated purified chloroplasts from leaf tissues of healthy plants was also included as a negative
control. Subsequently, DNA from these chloroplasts was isolated and subjected to PCR reactions
using Bego CP F and R primer pairs.
Figure 3.4 Reconstruction experiments to reject the possibility of adsorption of virions or/and DNA during
the purification of chloroplasts.
PCR experiments were performed with (Panel A, Lanes1-4). Lanes: 1, DNA isolated from infected leaves
(AEV) used as a positive control with Bego CP primers; 2, DNA isolated from chloroplast of healthy plants
where AEV virions were mixed and used as a reconstruction experiment but without proteinase K and DNase I
treatment; 3, DNA isolated from chloroplast of healthy plants and used as a reconstruction experiment where
AEV virions were mixed and subsequently treated with proteinase K and DNase I treatment to confirm enzymatic
activity and to rule out the possibility that virions or DNA adsorbed with chloroplasts; 4, DNA of chloroplast
isolated from healthy leaves infiltrated with buffer only, used as a negative control. Panel B: 16SrNA gene was
included as an internal control to check the integrity of chloroplast DNA. Panel C: Actin gene was used as a
negative control for chloroplast DNA as shown in Lanes 2, 3 and 4 while Lane 1 band was amplified from total
DNA isolated from AEV infected leaf tissues to confirm the integrity of Actin primers. The resulting PCR
products were analyzed on a 2% agarose gel.
82
It is clear from the reconstruction experiments that sample which lacks enzymatic treatment
exhibits a prominent band of viral DNA, however, virions and DNA were completely degraded
after enzymatic treatment (Figure 3.4 Panel A, Lane 2 and 3 respectively). These results clearly
demonstrate that DNA found in chloroplasts cannot be the result of a simple contamination of
adsorbed virions or DNA on the surface of chloroplasts. This study is also consistent with the
data as seen most clearly in Figure 3.3. In addition, chloroplast 16SrRNA, and nuclear Actin
genes used as positive and negative reference controls respectively further confirmed the ability
to cleanly purify chloroplast DNA from any contaminating complete cells. The reliability of
primers and experimental conditions were confirmed with total DNA isolated from AEV infected
N. benthamiana plants (Figure 3.4, Panel A, B and C Lane 1). The efficiency of DNase I
digestion after proteinase K treatment was controlled with DNA of AEV that was added to some
plastid samples (data not shown). Overall, these results demonstrate that solely the DNA from
AEV is capable of being translocated into chloroplasts. Members from the same begomoviruses
family do not necessarily target the same organelles.
3.4.4 Microscopic studies
Chloroplast purity and intactness were further confirmed using a phase contrast microscopy.
Purified chloroplasts were visualized under a phase contrast microscopy to confirm their
integrity. Ten aliquots of each sample were examined in detail for their purity. Figure 3.5 A
shows these isolated chloroplasts are free from intact cells. In addition, electron microscopic
examination with purified chloroplasts revealed that these samples were free of other cellular
organelles (Figure 3.5 B and C). Furthermore, ultrastructure of chloroplasts from healthy and
AEV infected asymptomatic plants were studied in details. In both samples, chloroplasts
contained a dense stroma with thylakoids, starch grains, and plastoglobuli. However, the
83
chloroplast of infected plants have more plastoglobuli than those of uninfected plants, in
addition, chloroplasts of infected plants are also associated with mild damage in thylakoids and
grana as shown (Figure 3.5 C).
Figure 3.5 Phase contrast and electron microscopic studies of chloroplasts.
(A) Phase contrast photographs of chloroplasts to examine intactness after purification through sucrose gradient
centrifugation. (B) Electron micrographs of chloroplasts from healthy (C) and infected plants. Chloroplasts from
infected plants are characterized by degenerated thylakoids (circled) and more plastoglobuli (p). Bar =10µM
3.4.5 Translocation of AEV DNA in mitochondria
To determine the translocation of AEV DNA-A into mitochondria, DNA was isolated from
mitochondria of infected plant leaves and subjected to PCR using AEV CP F and R primers. The
expected size of coat protein gene could be amplified from total DNA sample whereas no band
was detected with DNA extracted from mitochondria of the infected plants (Figure 3.6, Panel A;
Lanes 1 and 2 respectively). It might be concluded from these experiments that DNA-A of AEV
failed to translocate into mitochondria of AEV infected pants.
84
Figure 3.6 PCR-mediated detection of AEV DNA extracted from mitochondria and leaf tissues of N. benthamiana
infected plants at 35 dpi.
AEV CP primers specific to the coat protein gene were used to amplify a 283bp product from AEV infected leaf tissues
and mitochondria (Panel A; Lane 1 and 2 respectively). Primers for18SrRNA from mitochondrial genome were used to
amplify 187 bp product as an internal control to check the integrity of DNA from leaf tissues and mitochondria (Panel B;
Lane 1and 2 respectively). A DNA size marker (100 bp) in 100 bp increments was electrophoresed in Lane L. The
resulting PCR products were analyzed on a 2% agarose gel.
3.5 DISCUSSION
The results presented here suggest that all the Nicotiana and tomato plants tested are susceptible
to ToLCNDV and exhibit rigorous symptoms of infection. All Nicotiana species, with the
exception of N. tabacum cv. Xanthi plants were shown to be susceptible to Ageratum enation
virus infection, however, all of these plants remained asymptomatic. This contrasts with the
results described by others (260, 261), where tobacco and tomato plants infected with AEV
Tomato isolate and AEV isolate ACL exhibited severe leaf curling, vein clearing, vein enation,
reduction in leaf lamina, dimples on upper leaf surface, chlorosis, necrosis and stunted growth
symptoms. The causes of symptom induction are multiple but always depend on the aggregation
of viral nucleic acids or proteins that interfere with the normal function of the plant and/or trigger
a symptomatic defense response (262, 263). Previously, it has been revealed that begomovirus
and curtovirus Rep proteins bind to retinoblastoma-related protein (RBR), a key regulator of the
plant cell cycle, through a unique motif. Mutation of these motifs in Rep A and Rep results in
85
milder symptoms and reduced viral DNA accumulation (264, 265). AEV used in the study is
associated with betasatellite (DNA-β). DNA-β bears a βC1 open reading frame on the
complementary sense strand, which is conserved among distinct betasatellites in terms of
position and size. Mutational analyses and constitutive expression have revealed that βC1 is a
strong pathogenicity/symptom determinant (266-268). Guo, et al. (269) exhibited more severe
symptoms and also enhanced viral DNA accumulation when Tomato yellow leaf curl Thailand
virus was inoculated in association with betasatellites. These findings are not consistent with our
observations where all plants remained symptomless when infected with infectious clones of
AEV DNA-A and DNA-β. The DNA-A molecule nucleotide sequence of infectious AEV clone
used in this study exhibited the highest levels of nucleotide sequence identity (94.1%) with the
DNA-A of AEV Tomato isolate. This asymptomatic infectivity of AEV is suggestive of the
virus’s inability to interact with factors involved in inducing symptoms. In certain cases, these
factors are considered to be involved in the miRNA pathway, which is affected by virus
pathogenicity determinants (270). In specific geminiviruses, the C4 protein is a pathogenicity
determinant and a suppressor of PTGS by binding to siRNAs (271, 272). Different host proteins
such as Shaggy-like protein kinases like SK4-1/SKK have been shown to interact with other
geminiviral C4 proteins; this interaction is required to trigger disease symptoms (273, 274) and
for C4 function to suppress gene silencing (273). These findings suggest that viral and host
factors play a key role in symptom development which are probably correlated with the viral
sequences and their mimicry to certain cellular mRNAs in plants.
The present study also shows that individual Nicotiana species even with same ploidy levels
differ in their susceptibility to begomoviruses. Species of Nicotiana vary from immune (N.
tabacum cv. Xanthi) to high susceptibility (N. benthamiana) to AEV. The susceptibility of N.
benthamiana (polyploid), N. glutinosa (diploid) and N. tabacum (polyploid) and resistance
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response of N. tabacum cv. Xanthi (polyploid) to AEV illustrates that there is no clear
relationship between infectivity and ploidy levels. Our results are consistent with those described
by others (275) who showed that there is no clear relationship between begomovirus
susceptibility/resistance and the ploidy level of Nicotiana spp. Gottula, et al. (276) also
demonstrated that there is a limited relationship between host ploidy level and virus resistance.
Interestingly, our results showed that the levels of viral DNA were lower in symptomatic
plants than those of asymptomatic plants inoculated with ToLCNDV and AEV respectively. Our
studies also exhibited a higher level of viral DNA-A in the case of N. benthamiana plants as
compared to that of other Nicotiana species and Solanum lycopersicum. Tsuda, et al. (277)
showed that the pathogenicity of pepper mild mottle virus is regulated by the RNA silencing
suppressor activity of its replication protein, and not by the levels of viral accumulation. The
virus titer does not necessarily correlate with the severity of symptoms indicating that disease
can be the result of other molecular mechanisms that underlie the onset of disease symptoms and
not general distress.
To address the geminivirus infection, an RNA silencing system which targets the
conserved region (CR) of many geminiviruses is designed to generate transgenic tobacco plants.
This system generates a 176 base pair double stranded RNA which encompasses most of the CR
region of many begomo- and geminiviruses infecting a large number of economically important
crops. This construct was tested against two begomoviruses (AEV and ToLCNDV). The
preliminary studies show a very strong reduction in virus replication in the transgenic Nicotiana
benthamiana plants (Appendix E). Molecular mechanisms involve in the resistance, as well
other molecular approaches for the development of plant resistance are also discussed (see
details in Appendix E).
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Another question that we investigated was whether genome of the begomovirus genomes
could be isolated from chloroplasts of tobacco and tomato leaves systemically infected with
these viruses. Accordingly, we have demonstrated that only DNA-A of AEV is present within
chloroplasts. Several lines of evidence are presented to allow us to conclude that Ageratum
enation virus DNA-A enters the chloroplast in vivo. A reconstruction control included in the
experiment ruled out the possibility that virions or DNA may co-purify with chloroplasts or that
they might become attached to the surface of chloroplasts as a result of the isolation procedure.
The nuclear gene encoding Actin, used as a negative control, further confirmed our ability to
cleanly purify chloroplast DNA from any contaminating complete cells. Microscopic studies
with isolated chloroplasts provide another line of evidence that these chloroplasts are free from
other cellular organelles. These results are in accordance with earlier studies on Abutilon mosaic
virus (AbMV), a begomovirus, conducted by Groning, et al. (164) who showed that AbMV DNA
was present in the plastids of AbMV-infected Abutilon sellovianum plants. Despite several
decades of research, the mechanism by which geminiviruses DNA translocates into the
chloroplast remains to be determined. It is believed that viral proteins are involved in the
intracellular movement of DNA. The transport of viral ssDNA from the nucleus towards the
plasmodesmata is facilitated by a nuclear export signal (NES) on the CP C-terminus and NES on
the Pre-CP N-terminus (249, 250). A nuclear shuttle protein is involved in the transportation of
viral DNA from the nucleus into the cytoplasm (246, 278). Host factors also play a major role in
targeting of the genome of the viruses towards different organelles. Krenz, et al. (256) showed
that a chloroplastic HSC70 from Arabidopsis interact with Abutilon mosaic virus movement
protein; an interaction that seems to be important for viral transport and symptom induction.
Cheng, et al. (209) demonstrated that chloroplast phosphoglycerate kinase is responsible for the
targeting of the bamboo mosaic virus to chloroplasts in N. benthamiana plants. Our studies also
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revealed that DNA of ToLNDV could not be isolated from chloroplasts of infected plants. These
findings suggest that members of the same family do not necessarily target the same organelles.
These studies also indicate that same virus AEV is incapable of targeting different cellular
organelles (mitochondria) during its infectious cycle. The potential underlying transport
mechanism of AEV genome into chloroplasts is not yet known but it can be hypothesized that
genomic determinants in combination with host factors may play a major role in the targeting of
nucleic acid to different organelles.
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CHAPTER 4
4 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS
4.1 GENERAL CONCLUSIONS
The subject of the current study involves pioneering research on the discovery of equivalent
RNA activity embedded in PVX genome where a part of viral RNA functions as a tractor to
transport the tagged RNA into chloroplasts. This “RNA tractor” activity is dependent upon a
limited non-coding region (127 nucleotides) of the PVX RNA transcript, located near the end of
the PVX 8 kDa gene and the start of the coat protein (CP) gene as well as the small non-coding
intergenic region. Our PVX “RNA tractor” system doesn’t seem to involve any viral proteins,
and in this regard, may be comparable to the translocation of the Eggplant latent viroid RNA.
The PVX “RNA tractor” activity described here is the first report of its kind for a virus non-
coding sequence that is capable of translocating not only its own sequence (the entire PVX RNA
and the PVX CP mRNA) but also that of a foreign RNA sequence (GFP) into chloroplasts.
Another key research question in this study is whether or not a viral DNA can be
translocated into chloroplasts. The research conducted with two begomoviruses, Ageratum
enation virus (AEV) and Tomato leaf curl New Delhi virus (ToLCNDV), answers this question
by confirming the presence of only AEV DNA-A (monopartite) in chloroplasts of viral infected
leaves. The DNA of abutilon mosaic virus was isolated from intact chloroplasts (164),
representing the only other example of a geminiviral viral genome in chloroplasts. Considering
these findings on the sub-cellular localization, it is plausible that viruses use fundamentally
different transport mechanisms within their hosts.
Since both chloroplasts and mitochondria share many structural similarities, in
particular, a double membrane and prokaryotic ribosomes (279), we postulated that the PVX
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“RNA tractor” and AEV DNA-A might be able to target the mitochondria. However, I
determined that both the PVX “RNA tractor” and AEV DNA do not target the mitochondria.
Although mitochondria and chloroplasts both considered being evolved from prokaryotic
ancestors.
4.2 FUTURE DIRECTIONS
Since it has been determined that none of the viral proteins are involved in the movement of
PVX “RNA tractor” into chloroplasts, it is predicted that host factor (s) is/are interacting with the
PVX RNA and facilitating its trafficking into the chloroplasts. Further studies are required in
order to detect whether any potential host protein(s) interact with eggplant latent viroid and the
PVX “RNA tractor,” through the use of biochemical approaches such as Electrophoretic
Mobility Shift Assay (224, 225) or/and UV cross-linking RNA/protein complexes (223, 226,
227), followed by mass spectrometry to analyze the sequence of the purified proteins (228). In
addition to this, a chloroplast localization signal within the RNA tractor/DNA sequence,
analogous to a nuclear localization signal, needs to be identified. Also, the unique structural
features (such as receptors) of chloroplast membranes involved in interaction with RNA tractors
would need to be further explored. The prime determinants of the tractor activity such as a
canonical nucleotide sequence or secondary structure within the tractor need to be addressed. It
would also be interesting to investigate whether this translocation phenomenon occurs in related
viruses. Understanding the contingent scenarios of this molecular landscape will provide us clues
into how the noncoding RNAs and pathogenic DNAs evolved, and should ultimately allow us to
characterize them. This may also lead us to understand some aspects of gene regulation,
development and help establish evolutionary relationships.
Our studies also revealed that DNA of AEV, but not of ToLNDV, could be isolated from
chloroplasts of infected plants. This finding suggests that members of the same family do not
91
necessarily target the same organelles. The potential underlying transport mechanism of the AEV
genome into chloroplasts is not yet known but it can be hypothesized that genomic determinants
in combination with viral and/or host protein(s) may be playing a major role in the targeting of
nucleic acids to chloroplasts. In addition, the relevance of the findings should be tested for
additional geminiviruses other than AEV. All plants inoculated with ToLCNDV were
systematically infected and showed characteristic symptoms. However, in the case of AEV, all
plants tested, with the exception of N. tabacum, were infected by the virus but remained
symptomless. The mechanism by which the N. tabacum cv. Xanthi conferred resistance has not
been addressed. Detailed studies of both the molecular genetics of these viruses and their hosts’
natural defense systems will result in the development of novel ways to control virus diseases in
plants. The knowledge gained from these studies will not only contribute significantly to the
elucidation of the geminiviral intra- and intercellular movement processes but will additionally
provide a better understanding of virus replication processes as well as insight with respect to
strategies designed to reduce the economic damage caused by these viruses.
Lastly, a novel idea has emerged to use plant chloroplasts as bioreactors to target and
overexpress nucleic acid and/or protein molecules in chloroplasts through the previously
mentioned RNA and DNA tractors. This could act as a viable biotechnological alternative to
bacterial and fungal fermentation or mammalian cell culture towards the industrial-scale
production of several compounds (280-283). Therefore, the identification of non-coding RNAs
and DNAs as untranslated signals capable of mediating the stable expression of foreign proteins
in chloroplasts provides an enriched conceptual basis to develop distinctive strategies for
production of biologicals, biopharmaceuticals, vaccines or drugs in bioreactors designed using
plant chloroplasts. Genetic engineering of proteins with chloroplast permeability would be
another approach in this direction.
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APPENDICES
93
APPENDIX A
5 ATTEMPTS FOR RNA TRACTOR SEQUENCE
MODIFICATION FOR GFP EXPRESSION IN
CHLOROPLASTS
5.1 INTRODUCTION
We have demonstrated that the pTR:127 has the capacity to translocate the GFP mRNA to
chloroplasts. To provide another line of evidence of RNA tractor activity in chloroplasts and
determine that the GFP sequence is functional in the chloroplast, pTR:127 construct was
redesigned considering the translation mechanism of chloroplasts. Chloroplasts are plant cellular
organelles that have their own genome and a prokaryotic-type translation machinery consisting
of 70S-type ribosomes, ~30 tRNA species, initiation/elongation factors (e.g. IF-1, EF-Tu, and
EF-G) and aminoacyl-tRNA synthetases which are highly homologous to those in prokaryotes
(284-288). In prokaryotes, translation is believed to be facilitated by mRNA-rRNA interactions
between the Shine-Dalgarno (SD) sequence upstream of the translation initiation codon and the
anti-Shine-Dalgarno sequence (ASD) at the 3´end of the small (16S) ribosomal RNA.
Chloroplast mRNAs are not capped, instead, over 90% of chloroplast genes in land plants
possess an upstream sequence similar to the bacterial SD sequence (typically GGAGG) that is
capable of binding to a complementary sequence near the 3´end of the chloroplast 16SrRNA
(289) as shown in Figure 5.1.
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Figure 5.1 Schematic representation of the 3´end portion of tobacco chloroplast 16SrRNA (290).
Chloroplast ribosome-binding sites were identified on the plastid RuBisCO large subunit (rbcL)
mRNAs. The rbcL translation initiation domain is highly conserved which contains a
prokaryotic Shine-Dalgarno (SD) like sequence (AGGGAGGGA) located 4 to 12 nucleotides
upstream of the initiation AUG codon and found to be essential for translation (291). Knowing
about translation system of chloroplasts and PVX RNA tractor sequence (127 nt) which is
enough to translocate not only its own PVX RNA sequence but also a reporter gene (GFP
mRNA) into the chloroplast, these strategies were attempted to make RNA tractor sequence
functional for GFP mRNA as a reporter gene.
5.2 Addition of SD-like sequence (pCrbcLSD-GFP)
To determine the functionality of rbcL SD-like sequence (AGGGAGGGA) and reflecting the
importance of RuBisCO (the most abundant protein in leaves, accounting for 30-50% of soluble
leaf protein in plants) the SD-like sequence was inserted to the upstream of GFP initiation codon
AUG in pTR:127 construct and named it pCrbcLSD-GFP (Figure 5.2). Note that
pTR:127rbcLSD construct was designed in such a way that the AUG for the GFP is not in frame
with the AUG of PVX CP, consequently GFP will not be functional in the cytosol.
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However, confocal microscopic observations with transgenic plant harboring pCrbcLSD-GFP
construct failed to show GFP expression inside the chloroplasts as depicted in Figure 5.3.
Figure 5.3 Confocal microscopic observation of Nicotiana tabacum cv. Xanthi leaves harboring pCrbcLSD-
GFP.
Autofluorescence of chloroplasts is shown in red. DIC: Differential interference contrast (microscopy).
From this experiment, it could be speculated that the SD sequence alone might not be able to
mediate an efficient initiation of translation but needs to be complemented with an enhancer
sequence or/and additional levels of regulation for translation in chloroplasts. According to the
previous studies, the following sequence elements of the translation initiation region (TIR)
contribute to its translation efficiency: (a) the initiation codon, which is most commonly AUG
but sometimes GUG and very rarely UUG, AUU or CUG (292-295); (b) the Shine-Dalgarno
(SD) sequence (296, 297); (c) regions upstream of the SD sequence and downstream of the
initiation codon, which are often described as enhancers of translation (297-299). Cross-linking
studies have shown that the nucleic acid-binding domain of S1 is aligned with a region of the
Figure 5.2 Details of partial DNA sequences of the pCrbcLSD-GFP construct under the control of 35S promoter
and the nopaline synthase terminator (T-nos).
SD-like sequence (AGGGAGGG) is located 6 nucleotides upstream of the initiation AUG codon of GFP for possible
translation in the chloroplast.
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mRNA upstream of the SD, suggesting that S1 may interact with 5´ parts of the TIR (300, 301).
Consistent with this observation, A/U-rich sequences in front of the SD or downstream of the
initiator codon enhance protein synthesis (302, 303). Komarova, et al. (302) demonstrated that
nine sequences were acting as translational enhancers. They are all A/U-rich and contain very
few Gs contents. Disruption of the E. coli gene coding for S1 has been reported to be lethal
(304). A decreased level of S1 protein in the cell leads to a rapid decrease in total protein
synthesis (305). Thus, it can be speculated that the SD sequence alone cannot mediate efficient
initiation of translation but has to be complemented with an enhancer sequence.
5.3 Addition of 5´-translation control region of large sub-unit RuBisCO gene
To determine whether the additional determinants along with SD sequence are required to
translate GFP mRNA in chloroplasts, 5´-translation control region of chloroplastic large sub-unit
RuBisco gene, comprise of 14 N-terminal amino acids and 59 of 5´-UTR region, is designed
based on previous studies (210). In higher plant plastids mRNA sequences in the 5´-untranslated
region (UTR) were shown to be important for translation. 5´-UTRs and cis-elements required for
efficient translation of plastid mRNAs have been characterized by both in vivo and in vitro
studies (211, 306, 307). Using in vitro system, Yukawa, et al. (308) found that mRNAs carrying
unprocessed or processed rbcL 5´-UTRs were efficiently translated at similar rates by employing
a green fluorescent protein (GFP). Transcription of the tobacco rbcL mRNA initiates at 182
nucleotides upstream of the translation initiation codon (309). The primary transcript may be
processed to create an mRNA with a 58 nucleotide 5´-UTR (310, 311). Kuroda and Maliga (210)
employed a transgenic approach to demonstrate accumulation of the neomycin
phosphotransferase (NPTII) reporter enzyme when translationally fused with 14 N-terminal
amino acids encoded in the rbcL. Fifty-nine nucleotides of upstream were used as 5´-UTR
region. N-terminal coding region and the 5´-UTR were collectively designated as the 5´-
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translation control region or 5´-TCR. Considering the importance of 5´-TCR region, two clones
pC127TCR-GFP, and pCVdTCR-GFP are designed with the 5´-TCR region (Figure 5.4).
Figure 5.4 Details of partial DNA sequences of the pCvdTCR-GFP and pC127TCR-GFP constructs under the
control of the Cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (T-nos).
Eggplant latent viroid chimeric construct pCvdTCR-GFP was included on the base of previous
findings by Gomez and Pallas (95) who demonstrated that the viroid sequence acting as a 5´-
UTR end mediated the trafficking and accumulation of a functional foreign mRNA into N.
benthamiana chloroplasts. However when the tobacco leaves were agroinfiltrated with
pCvdTCR-GFP and pC127TCR-GFP constructs it was observed that GFP is functional in
agrobacteria cells but not in chloroplasts (Figure 5.5).
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Figure 5.5 Confocal microscopic observation of GFP in N. benthamiana leaves after 72 hr of agro-
infiltration.
GFP is expressed inside the infiltrated leaves due to fluorescent bacterial cells harboring pCvdTCR-GFP (Panel
A) and pC127TCR-GFP (Panel B) constructs respectively. The signal for GFP is shown in green, the
autofluorescence of the chloroplast is shown in red.
To confirm whether this GFP expression is inside the agrobacteria cells, these bacterial
cells are analyzed as well under confocal microscopy. Consequently, a robust expression of GFP
is detected in bacterial cells as demonstrated by confocal microscopy as depicted in Figure 5.6.
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Figure 5.6 Confocal microscopic observation of GFP in agrobacteria cells after 48 hr.
GFP is expressed inside the bacteria cells harboring pCvdTCR-GFP (Panel A) and pC127TCR-GFP (Panel B)
constructs respectively in the presence of 5´TCR of rbcL which is located upstream of the GFP gene in the both
constructs.
In this context, it is important to note that the 16SrRNA of both the chloroplasts and the A.
tumefaciens share 79% nucleotide sequence homology and both have the sequence CCUCC at
their 3´ end that is complementary to the SD-like sequence GGAGG in the translation initiation
region. However, this expression in A. tumefaciens was at least 10 times less than what was
observed for the pCpETSD-GFP construct containing the highly efficient phage T7 5´-UTR
context (Figure 5.10). Previously we have shown (312) that there was no difference in GFP
expression between agrobacteria cells harboring constructs containing the entire 5´-TCR of rbcL
and only the 58 nucleotide 5´-UTR region, implying that the coding region downstream of the
AUG codon did not affect protein translation initiation in agrobacteria cells unlike that of
chloroplasts which require the entire 5´-TCR for successful protein translation (210).
Despite the presence of 5´-TCR in both pCvdTCR-GFP and pC127TCR-GFP constructs,
GFP was failed to express in the chloroplast of the infiltrated plants. One of the possibilities that
RNA tractor sequence is not translocated in the chloroplasts, it can be ruled out by the fact that
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Eggplant latent viroid (ELVd), a member of the Avsunviroidae family (a class of subviral plant
pathogens that infect, replicate and accumulate in chloroplasts), sequence definitely targets to the
chloroplast. In the case of pCvdTCR-GFP if the TCR is functional then GFP should be expressed
inside the chloroplast. So in this case, it might require some additional levels of regulation for
translation or change in secondary structure of RNA due to TCR sequence which inhibits
translation. Secondary structure formation near the 5´-end of a eukaryotic mRNA can have
negative or positive effects upon translation initiation (313).
5.4 Addition of 5´-UTR of Psb A gene for translation initiation of GFP in
chloroplast
Another attempt was carried out to translate GFP in chloroplast using 5´-UTR of psbA
chloroplast gene along with ELVd sequence used as a carrier sequence to the chloroplast. The 5´-
UTR of psbA gene previously has been successfully characterized for translation of reporter
genes both in vivo and in vitro studies (211, 308, 314, 315). Three elements within the 5´-UTR of
the chloroplast mRNA are reportedly required for translation in psbA gene. Two of them are
complementary to the 3´-terminus of chloroplast 16SrRNA (termed RBS1 and RBS2) and the
other is an AU-rich sequence (UAAAUAAA) located between RBS1 and RBS2 and is termed
the AU box. RBS1 and RBS2 are cooperatively required for efficient translation of psbA mRNA
encoding the D1 protein of photosystem II that is synthesized only in light-grown chloroplasts.
To determine translation in the chloroplast, a construct pCELVdpsbA-GFP that contained
ELVd sequence, 85 nucleotides as a 5´-UTR including RBS, AU-rich region and ATG of the
psbA gene upstream of the GFP gene was designed (Figure 5.7) and transformed in
Agrobacterium.
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Figure 5.7 Details of partial DNA sequences of the pCELVdpsbA-GFP construct in pC-GFP under the control
of the Cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (T-nos).
Confocal microscopy studies with agro-infiltrated N.tabacum leaves exhibit that there is no
expression of GFP in both chloroplasts and agrobacterium (Figure 5.7), suggesting a major
difference in the translatability of the GFP protein between the 5´ non-coding sequences of the
Figure 5.8 Confocal microscopic observation for GFP in transgenic tobacco plant leaves and agrobacteria
cells harboring pCELVdpsbA-GFP construct.
GFP is not expressed both in the chloroplasts of transgenic plants (Panel A) and inside the bacteria cells
(Panel B) in the presence 5´-UTR of chloroplastic psbA gene which is located upstream of the GFP gene
in the construct.
RuBisCO large subunit gene and that of the psbA gene, even though both are encoded by the
chloroplastic genome and are known to be involved in photosynthesis. This led me to conclude
that the presence of the SD-like sequence close to the AUG start codon and a specific 5´-UTR
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sequence are required for translatability in A. tumefaciens. The rbcL gene with the SD-like
sequence 10 nucleotides away from the AUG codon satisfies this requirement whereas the psbA
gene SD-like sequence is much farther away (40 nucleotides upstream) from the AUG codon
and, therefore, does not allow positive GFP expression. GFP expression was not observed inside
the chloroplasts, even in the plants harboring pCELVdpsbA-GFP construct it can be assumed
that it requires some additional levels of regulation for translation in the chloroplasts. It might
need the interaction of 5´and 3´ ends of chloroplast mRNA which is common in cytoplasmic
mRNAs. In eukaryotes, interactions between the two termini of cytoplasmic mRNAs stimulate
the initiation of translation. The poly (A) binding protein (PABP) bound to the 3´poly (A) tail
interacts with initiation factors bound to the 5´-UTR, thus creating a ‘closed loop’ that promotes
the recruitment of the 40S ribosomal subunit. It is generally thought that the ‘closed loop’ role is
a quality control mechanism to promote translation of full-length mRNAs rather than truncated
forms (316). Translatable chloroplast mRNAs do not contain poly (A) tails. Most of them,
similarly to prokaryotic mRNAs, contain an AU-rich 3´-UTR with a terminal inverted repeat.
The 3´-UTR inverted repeat has been shown to play a role in the processing and stabilization of
the mRNA (317). Examples of modulation of translation initiation by interactions between the
two termini of mRNA in prokaryotes (318, 319) raise the possibility that such interactions might
also exist in chloroplast mRNAs and influence their expression. Indeed, there are several reports
that support a role for the 3´-UTR in translation initiation of several mRNAs. Correct processing
of the 3´-UTR was suggested to be required for high levels of translation initiation and
polysomal association in Chlamydomonas reinhardtii cells (320). Recent results from tobacco
transformants in which the influence of the psbA UTRs on the translation of a reporter gene were
studied indicated that including the psbA 3´-UTR resulted in a three to four-fold enhancement of
translation (321). Furthermore, through high-affinity binding of regulatory proteins to C.
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reinhardtii psbA mRNA is primarily via its 5´-UTR, the 3´-UTR was shown to increase the
affinity of binding of the 5´-UTR-binding protein complex (322). In another study, deletion of
the inverted repeat of the 3´ UTR of tobacco petD mRNA led to a reduction in petD expression
beyond that expected by the decrease in mRNA accumulation alone, indicating that the 3´-UTR
might also contribute to efficient translation (317). Further research is needed to establish the
generality of this phenomenon and its importance for translation efficiency.
5.5 Addition of bacterial translation initiation region (TIR) for GFP
expression
Since the translation machinery in the chloroplast generally resembles that of prokaryotes; the
chloroplast ribosomes are closely related to the eubacterial 70S-type ribosomes, chloroplast
transcripts like prokaryotes are not m7G capped at their 5´end, and lack 3´poly (A) tails.
Furthermore, the anti-Shine-Dalgarno (SD) sequences at the 3´ends of the 16SrRNAs of
cyanobacteria and chloroplasts share high homology with the E. coli anti-SD sequence (323-
325). I decided to express GFP using in E.coli translation initiation region, comprises the
initiator codon, Shine-Dalgarno (SD) sequence and translational enhancer A/U-rich sequences.
To achieve this target, first pET: GFP-construct was generated by cloning the GFP gene into a
Kanamycin-resistant plasmid pET29 vector, containing original SD sequence (AGGAGA) and
A/U-rich region (uuuguuuaacuuuaagaAGGAGAuauacauAUG) under the control of strong
bacteriophage T7 promoter (Figure 5.9). For protein production, this recombinant plasmid was
transferred to a host containing a chromosomal copy of the gene for T7 RNA polymerase. The
addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a growing culture induces T7 RNA
polymerase, which in turn transcribes the target DNA in the plasmid. The SD sequence
(AGGAGA) helps recruit the ribosome to the mRNA (GFP) to initiate protein synthesis by
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aligning it with the codon. The expression of GFP in E. coli BL21 Codon Plus strain harboring
pET-GFP construct is very high as shown in Figure 5.10.
Figure 5.9 Details of partial DNA sequences of the pET-GFP construct in pET29 under the control of T7
promoter and T7 terminator.
Figure 5.10 Fluorescence micrograph of GFP in E. coli cells transfected with the pET-GFP construct and induced
with 0.5 mM IPTG for 16 hr.
This clearly indicates that the S/D sequence (AGGAGA) is quite functional in E. coli.
Subsequently, this cassette including A/U rich, SD sequence and GFP was inserted into pTR:127
construct and designated as pC127pETSD-GFP (Figure 5.11).
Figure 5.11 Details of partial DNA sequences of the pC127pETSD-GFP construct in pC-GFP under the
control of the Cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (T-nos).
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Figure 5.12 Confocal microscopic observation of GFP in leaves and agrobacteria cells harboring pC127pETSD-
GFP after 72 hr.
GFP is expressed inside the infiltrated leaves due to fluorescent agrobacterial cells (Panel A) which is confirmed by
observing the agrobacteria cells alone (Panel B). The signals for GFP are shown in green and the autofluorescence of
the chloroplast is shown in red.
Agroinfiltration experiments demonstrated that GFP was expressed in agrobacterial cells but not
in the chloroplast as shown in Figure 5.12. CaMV 35S promoter was regarded to be plant
specific and not active in other organisms such as bacteria, fungi or human cells. This
assumption had been proven wrong. It has also been established that the CaMV35S promoter is
not only active in plants but also in E.coli, in soil bacteria Agrobacterium rhizogenes (326),
yeast (327) and in extracts of human cancer cell lines (328). According to these results this viral
35S promoter has the ability to initiate gene expression in A. tumefaciens (Figure 5.12).
However from the Figure 5.12, it seems that the additional levels of regulation are required for
translation in chloroplasts. The chloroplast S1 protein is a nuclear-encoded protein and is much
shorter than the bacterial protein. Different RNA-binding specificities were reported for the
chloroplast S1 protein with preference to AU-rich RNA sequences that are common in the 5´-
UTR of chloroplast genes (329-332). Further research is needed to establish the generality of this
phenomenon and its importance for translation efficiency. In future experiments are required to
106
generate a construct where, beside the RNA tractor activity, sequences like SD and other
chloroplast ribosomal recognition sequences would be tested to allow translation of the GFP
reporter gene in the chloroplast.
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APPENDIX B
6 STRATEGY TO FIND OUT THE CAPACITY OF CHIMERIC
EGGPLANT LATENT VIROID SEQUENCE AS A 5´-UTR FOR
GFP EXPRESSION IN CHLOROPLASTS
Gomez and Pallas (95) reported that a chimeric DNA containing a modified Eggplant latent
viroid cDNA sequence fused as a 5´-UTR of GFP mediates not only the import of GFP mRNA
into the chloroplasts but also allows a high expression of GFP in chloroplasts. The specific
localization of the functional chimeric transcripts was demonstrated in transient expression
assays with N. benthamiana plants using confocal microscopy. This non-coding viroid,
a member of the Avsunviroidae family, is naturally transported and replicated in chloroplasts.
When a chimeric sequence of this viroid was placed in front of GFP, it resulted in a high degree
of the GFP expression (95). However, it is not clear how and why such a chimeric viroid
sequence allowed the translation in chloroplasts. Whether the viroid sequence or/and specific
structure motifs are required for translation of GFP in chloroplasts. To address these questions,
first the chimeric viroid sequence (AN -HM136583) from the Eggplant latent viroid (ELVd) was
synthesized and cloned in a binary vector pC-GFP carrying the GFP cDNA under the control of
the Cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (T-nos). The
resultant vector pCELVd-GFP contains an ELVd derived cDNA fused as a untranslated region
(UTR) to the 5´end of the GFP cDNA but without AT-rich leader sequence Figure 6.1.
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Figure 6.1 Details of partial DNA sequence of Eggplant latent viroid for different constructs.
A) pCELVd-GFP without an AT-rich leader sequence B) pCATvd-GFP, with an AT-rich leader sequence C)
pCATvdAnti-GFP, SD-like (GGAGGATTCG) sequence (red) is replaced with anti-SD-like (CCTCCTAAGC) sequence
D) and pCATvd80-GFP (an internal110 nt of the functional chimeric ELVd sequence previously shown to be sufficient
for the trafficking of functional GFP-mRNA into chloroplasts (96) is further truncated to 80 nt. All constructs are under
the control of the Cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (T-nos).
This construct was transfected into A.tumefaciens. When the functionality of this chimeric
transcript in N. benthamiana transgenic plants was analyzed by confocal microscopy, GFP
expression was either invisible or very low in the chloroplasts. However when AT- rich leader
sequence is inserted to the upstream of viroid sequence (Figure 6.1) the accumulation of GFP in
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chloroplasts is very high (Figure 6.2, panel B), suggesting that viroid sequence is fully
functional for GFP expression only in the presence of AT-rich sequence.
Figure 6.2 The GFP arising from different ELVd-5´-UTR-GFP transcripts.
Confocal microscope observation of the N. benthamiana leaves expressing GFP: Panel A) pCELVd-GFP
construct without AT-rich region at the upstream of ELVd, GFP is localized in nucleus, cytoplasm and less in
chloroplast; Panel B) pCATvd-GFP construct with AT-rich region, GFP is mostly localized in the chloroplasts;
Panel C) pCATvdAnti-GFP construct, SD-like (GGAGGATTCG) sequence is replaced with anti-SD-like
(CCTCCTAAGC) sequence, GFP is equally localized in the nucleus, cytoplasm, and chloroplasts; Panel D)
pCATvd80-GFP construct, ELVd sequence is truncated to 80 nucleotides only, GFP is localized in nucleus and
cytoplasm only. These observations were taken from agroinfiltrated leaves after 72 hr. The left Panel (top to
bottom) show the GFP fluorescence (green), middle Panel (top to bottom) indicates the autofluorescence (red) of
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chloroplasts (chlorophyll) and any overlap of GFP and chloroplast fluorescence is indicated in yellow in the
merged right Panel (top to bottom).
This AT-rich sequence is derived from the 5´-UTR region of the capsid protein of Alfalfa mosaic
virus (AIMV) and is believed one of the most efficiently translated RNAs known (333). This
sequence was shown to function as a translational enhancer in vitro (334) and in vivo (335).
Previously it has been also shown that the middle region of the chimeric vd 5´-UTR, comprised
of 110 nucleotides, is important for the expression GFP in the chloroplast, however, the
functionality of the localization is increased when it is combined with other regions (96). A Shin-
Dalgarno like sequence (GGAGGATTCG) is noticed in this middle region of the chimeric ELVd
sequence. It is hypothesized this sequence in combination with secondary or/and tertiary
structure of the central region may be playing a role in the translation of GFP in chloroplasts. To
find this, the SD-like sequence GGAGGATTCG is replaced with CCTCCTAAGC sequence and
a new construct, pCATvdAnti-GFP, is generated. When the functionality of pCATvd-GFP was
analyzed by comparing its transient expression with that of the pCATvdAnti-GFP, it was
observed in agroinfiltrated N.benthamiana plants that the GFP from the transcripts of pCATvd-
GFP was mostly localized in the chloroplasts (Figure 6.2, Panel B). However, GFP from the
transcripts of pCVdAnti-GFP was equally distributed in the nucleus, cytoplasm and the
chloroplasts (Figure 6.2, Panel C), indicating that the Shine-Dalgarno-like sequence may be
contributing more in the localization of the RNA rather an expression of GFP in chloroplasts or it
might have a dual role. In another experiment, the middle region (110 nt) was further truncated
to 80 nucleotides, still having an SD-like sequence, and inserted into pCAT-GFP to create
pCATvd80-GFP to determine its functionality for GFP expression. When the agroinfiltrated N.
benthamiana plants were examined by confocal microscopy, it was observed that the GFP
arising from the transcripts of this construct was uniformly distributed in the nucleus and
cytoplasm (Figure 6.2, Panel D) but not in the chloroplasts, suggesting this SD-like sequence
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alone is not enough for translation in chloroplasts which also confirms the requirement of its
structure motif. Another possibility is, it might have lost its translocation capacity to ship its
RNA to the chloroplasts which needs to be determined. However, it is not clear how and why
such a chimeric viroid sequence allowed the translation in chloroplasts. Whether the viroid
sequence or/and specific structure motifs are required for translation of GFP in chloroplasts.
Overall these results suggest that sequence elements and/or secondary or tertiary structural
domain together may require the translation of functional mRNA into the chloroplasts. Further
experiments are required to solve this mystery.
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APPENDIX C
7 VIRAL AND CHLOROPLASTIC SIGNALS ESSENTIAL
FOR INITIATION AND EFFICIENCY OF TRANSLATION
IN AGROBACTERIUM TUMEFACIENS
Results of this study were published (Ahmad T, Venkataraman S, Hefferon K, AbouHaidar MG.
2014.. Biochemical and biophysical research communications 452:14-20).
7.1 SUMMARY
High-level protein expression vectors using CaMV 35S promoter and highly efficient translation
initiation signals for Agrobacterium tumefaciens are relatively less explored compared to that of
Escherichia coli. In the current study, we experimentally investigated the capacity of CaMV 35S
promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and
chloroplastic translation initiation signals. GFP expression and concomitant translational
efficiency were monitored by confocal microscopy and western blot analysis. Among all of the
constructs, the highest level of translation was observed for the construct containing the phage
T7 translation initiation region followed by that with chloroplastic RuBisCO Large Subunit
(rbcL) 58-nucleotide 5´ leader region including its SD-like (GGGAGGG). Replacing the SD-like
(GGGAGGG) with non-SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL
with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide
region of rbcL serves as a translational enhancer in plants when located within 5´-UTR of the
GFP mRNA. Other constructs including those containing sequences upstream of the coat
proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI
and/or PsbA 5´-UTR sequence supported low levels of GFP expression or none at all. From these
studies, we propose high expression vectors in A. tumefaciens and /or plants which contain the
CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation
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initiation region or the rbcL 58-nucleotide 5´ leader region upstream of the gene for the protein
of interest.
7.2 INTRODUCTION
Initiation of translation in E. coli involves base pairing between a purine-rich Shine-Dalgarno
(SD) domain at the 5´ untranslated region (5´-UTR) of mRNA and the complementary anti-SD
sequence at the 3´ end of 16SrRNA (336). There are distinct sequence elements of the translation
initiation region known to contribute to its efficiency (337): the initiation codon, the Shine-
Dalgarno (SD) sequence (297, 338) as well as regions upstream of the SD sequence and
downstream of the initiation codon, described as enhancers of translation (339). The distance
between the SD sequence and the initiation triplet has a marked effect on the efficiency of
translation (340). The 6-nucleotide consensus SD AGGAGG core sequence causes the highest
level of protein synthesis.
Chloroplasts have their own translation system, which shows strong homologies to that of
prokaryotes. This is consistent with the presence of a Shine-Dalgarno (SD) sequence (GGAGG)
located within 12 nucleotides of the AUG initiation codon of many plastid genes (341).
Moreover, the sequence near the 3´ end of the plastid 16SrRNA contains a highly conserved
polypyrimidine-rich region (CCUCC) complementary to the SD sequence as in bacteria. Over
90% of higher plant chloroplast genes encoding polypeptides possess an upstream sequence
similar to the bacterial SD sequence. The spacing of these chloroplast SD-like sequences is less
conserved, ranging from -2 to -29 nucleotides (342). Translation of several chloroplast mRNAs
is also regulated in response to light as well as to some nuclear-encoded factors. In this regard, it
is interesting to study how well chloroplastic translational machinery function in Eubacteria such
as E. coli and A. tumefaciens. The transfer of T-DNA from Agrobacterium into the plant genome
represents a natural horizontal gene transfer across kingdom barriers and implicates a closer
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evolutionary relationship between Agrobacterium and plants than between any other Eubacterial
organism (such as the E. coli) and plants. The aim of the present study is to investigate the
sequence determinants responsible for efficient translation in A. tumefaciens, which on the one
hand is highly similar to E. coli in terms of its dependency on the SD sequence for the
translation, while on the other hand is also mechanistically similar to chloroplast genes such as
the large subunit of the RuBisCO in its dependence on the 5´upstream control region. Also, the
essential molecular determinants for the design of an ideal Agrobacterial expression vector are
considered.
7.3 MATERIALS AND METHODS
7.3.1 Construction of GFP expression plasmids:
The binary vector pCAMBIA1300 (CAMBIA, Canberra, Australia) was used in this study. To
create a pCTCR-GFP construct, the translation control region (TCR) (210), comprised of 58
nucleotides of 5´-UTR and 45 nucleotides from the N-terminal coding region of the rbcL gene
were synthesized and cloned in the pUC57 plasmid (Bio Basic Inc.). Following digestion of
pUC57 by KpnI/BamHI and XbaI/BglII respectively and gel purification (QIAquick Gel
Extraction Kit, QIAgen), rbcL TCR DNA fragments were subcloned into a pC-GFP binary
plasmid using the respective restriction sites. All other vectors of the pC-GFP series were
produced by ligating double-stranded oligonucleotides into restriction-enzyme digested plasmid
DNA with compatible ends (Table 7.1).
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Table 7.1 Sequences of the translation initiation signals in the pC-GFP vector.
Vector Description Oligonucleotid/DNA sequence (5´→3´)
pC T7SD-GFP
Construct with
PhageT7 trailer
sequence (T7
translational enhance
RBS) and is
available in pET-X-
series.
Sense (XbaI overhang)
ctagttaataattttgtttaactttaaGAAGGAGatatacatATGg
Antisense ( BamHI overhang)
gatccCATatgtatatCTCCTTCttaaagttaaacaaaattattaa
pC rbc58-GFP
Construct with only
58 nucleotides of 5´-
UTR of the rbcL gene.
Sense (XbaI overhang)
ctagtgtcgagtagaccttgttgttgtgagaattcttaattcatgagttgtaGGGAGGGatttATGg
Antisense (BamHI overhang)
gatccCATaaatCCCTCCCtacaactcatgaattaagaattctcacaacaacaaggtctactcgaca
PC rbc58AT-GFP Construct with 58
nucleotides of 5´UTR
of the rbcL gene
where GGGAGGG
sequence is replaced
with TTTATTT.
Sense (XbaI overhang)
ctagtgtcgagtagaccttgttgttgtgagaattcttaattcatgagttgtaTTTATTTatttATGg
Antisense (BamHI overhang)
gatccCATaaatAAATAAAtacaactcatgaattaagaattctcacaacaacaaggtctactcgaca
pC rbc33-GFP
Construct with 33
nucleotides of 5´-UTR
of the rbcL gene.
Sense (XbaI overhang ctagtaattcttaattcatgagttgtaGGGAGGGatttATGg
Antisense (BamHI overhang) gatccCATaaatCCCTCCCtacaactcatgaattaagaatta
pC rbcSD-GFP
Construct with only
SD sequence of rbcL
gene, the 5´-UTR
sequence is replaced
with non rbcL
sequence
Sense (KpnI overhang)
gtacattgaacagttaagtttccattgatactcgaaagatgtcagcaccaGGGAGGGg
Antisense (BamHI overhang)
gatccCCCTCCCtggtgctgacatctttcgagtatcaatggaaacttaactgttcaat
pC PsbA-GFP
Construct with 85
nucleotides of 5´-UTR
of PsbA gene
Sense (XbaI overhang)
ctagtaaaaagccttccattttctattttgatttgtagaaaactagtgtgcttGGGAGtcccTGATGATtaaataa
accAAGattttaccATGg
Antisense (BamHI overhang)
gatccCATggtaaaatCTTggtttatttaATCATCAgggaCTCCcaagcacactagttttctacaaatca
aaatagaaaatggaaggcttttta
pC ATP58 Construct with 58
nucleotides of 5´-UTR
of ATPI gene.
Sense (XbaI overhang)
ctagtagatggttgaatcaaaaaattttgtttaaagttcaattttttcaGAGGGCAAGGcaatATGg
Antisense (BamHI overhang
gatccCATattgCCTTGCCCTCtgaaaaaattgaactttaaacaaaattttttgattcaaccatcta
pC AT-GFP Construct with 5´-
UTR of the capsid
protein of alfalfa
mosaic virus RNA.
Sense (KpnI overhang) gtacagtttttatttttaattttctttcaaatacttccaggatctctaGAg
Antisense (BamHI overhang)
gatcCTCtagagatcctggaagtatttgaaagaaaattaaaaataaaaact
pC TCR-GFP
Construct with 58
nucleotides of 5´-UTR
and 45 nucleotides
from the N-terminal
coding region of the
rbcL gene. The
required DNA
fragment was
synthesized. The
sequence of only plus
strand is given.
ctagtgtcgagtagaccttgttgttgtgagaattcttaattcatgagttgtaGGGAGGGatttATGtcaccacaaa
cagagactaaagcaagtgttggattcaaagctg
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Italic letters indicate restriction site overhangs. Underlined capitalized bold letters indicate SD-sequences. Upper
case bold letters indicate start codons. Sequence of the sense and antisense primers used to generate the various
constructs is shown
Briefly, complementary oligonucleotides synthesized by Eurofins MWG Operon (Huntsville,
AL) were mixed in equimolar amounts (50 µM each), boiled and annealed by cooling to room
temperature and ligated into already restriction enzyme digested pC-GFP vector using T4 DNA
ligase (New England Biolabs) according to the manufacturer's protocol. The product of each
ligation reaction was used to transform E. coli DH5-alpha competent cells and Kanamycin
(50µg/mL) resistant bacterial colonies were screened for the presence of the proper recombinant
constructs. The presence and accuracy of the inserted gene within the expression cassette in the
final recombinant constructs was confirmed by DNA sequencing (The Centre for Applied
Genomics, Toronto, Canada) using the GFP-R reverse primer:5´-
AAGTCGTGCTGCTTCATGTG -3´.
7.3.2 Agrobacterium transformation
A modified freeze-thaw method for transformation of Agrobacterium tumefaciens was used as
reported previously (343). After transformation, the cells were resuspended in LB such that all
the samples contained a uniform OD595 of 1.0. From this, equal culture amounts were in turn
taken to perform the downstream RNA, confocal microscopy, and western blot analyses.
7.3.3 RNA isolation, reverse transcription and PCR
Total RNA was isolated according to a modified method described by AbouHaidar, et al. (344)
and subjected twice to DNase I treatments (New England Biolabs, NEB). A reverse transcription
reaction of each sample was performed on 1 µg of total RNA with 200 units of M-MLV reverse
transcriptase (Promega), 200 ng of GFP/16SrRNA reverse primer and 500 µM dNTPs in a final
volume of 20 µl as recommended. Primers GFP 5´-ACGTAAACGGCCACAAGTTC-3´
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(forward) and GFP 5´-AAGTCGTGCTGCTTCATGTG-3´ (reverse) were used to amplify an
187 bp of GFP gene. Primers 16SrRNA 5´-AACACATGCAAGTCGAACGC-3´ (forward) and
16SrRNA-R 5´-TAGGCCTTTACCCCACCAAC-3´ (reverse) were used to amplify a 187 bp
fragment of Agrobacterium 16SrRNA as an internal and comparative control for semi-
quantitative PCR.
7.3.4 Detection of GFP expression
7.3.4.1 Confocal microscopy
Following Agro-transformation, cell samples each containing OD595 to 1.0 were spun down and
the pellets resuspended in 10 mM MES (4-Morpholineethanesulfonic acid sodium salt) buffer,
pH 5.7. A drop of each cell culture was overlaid on a glass slide and live cell imaging was
performed on a confocal microscope (TCS SP5, Leica Microsystems) using a 100× oil objective
lens. The 488-nm laser was used for GFP imaging. Differential interference contrast (DIC)
microscopy was used for comparative studies of all the constructs. Images were analyzed by
Leica Application Suite Advanced Fluorescence (LAS AF) software.
7.3.4.2 SDS-PAGE and Western blotting
Following Agro-transformation, the cell samples, each adjusted OD595 to 1.0, were harvested by
centrifugation and protein from each pellet was extracted using in TMPDTNU (50 mM Tris, 20
mM MgCl2, 1 mM PMSF, 100 mM DTT, 2% Triton X-100, 0.5% NP-40 and 8 M urea) buffer
Equivalent protein amounts were loaded as determined by the Bradford Protein Assay reagent kit
(Bio-Rad, Hercules, CA) and Coomassie Brilliant Blue R-250 staining. SDS-PAGE and western
blot analyses were according to Sambrook, et al. (212).
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7.4 RESULTS AND DISCUSSION
GFP expression in ten pCAMBIA constructs (Fig. 1B) containing different translation initiation
contexts upstream of the GFP gene was monitored by confocal microscopy and western blot
analysis, after transformation of A. tumefaciens (GV3101 strain) with the respective constructs.
Figure 7.1 Schematic representation of constructs used in this study.
Arrows indicate the direction of transcription and translation. 35S is the CaMV 35S promoter. T-nos:
represents the transcription terminator; the box between the 35S and GFP contains the different translation
initiation contexts. GFP box is differently colored to reflect the efficiency of its expression. Dark green box for
the T7SD shows the highest expression, followed by that of the rbcL TCR and the rbcL 58 nucleotide 5´-UTR
region (light green). Boxes in light green represent marginal GFP expression while unfilled boxes show no
GFP expression. Note: Figures not drawn to scale.
All constructs uniformly contained the CaMV 35S promoter and GFP gene followed by the T-
nos terminator. This produces the same GFP transcript levels for all the constructs. The only
difference between the constructs was in the sequence of the translation context upstream of the
GFP coding sequence, which resulted in the differential GFP expression.
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7.4.1 Estimation of equal GFP transcript levels in A. tumefaciens harboring
each of the above constructs
Transcription levels of the GFP mRNA for all the constructs were measured by semi-quantitative
RT-PCR experiments using the 16SrRNA expression levels as the internal control (Figure 7.2,
Top Panel).
Figure 7.2 Quantitation of equivalent GFP transcript levels for all the constructs used in this study.
Two percentage agarose-TBE gel analysis of RT-PCR products using primers specific for the 16SrRNA of
Agrobacterium as well as primers specific for the GFP mRNA (Materials and Methods). Note the relatively
higher levels of the cDNA for 16SrRNA (Panel A) compared to that of the GFP mRNA (Panel B); also of note is
the equivalent amounts of the cDNA for the 16SrRNA in all the Lanes (Panel A) as well as equivalent amounts
of the GFP-specific cDNA in all the Lanes (Panel B), each representing the constructs used in this study. The
fractional numbers in Panel C represent the various dilutions of the RT-PCR product for the 16SrRNA. Compare
the amounts of cDNA in Panel B with those of Panel C: the amounts of the GFP cDNA is equivalent with that of
the 1/6th dilution of the RT-PCR product for the 16SrRNA.
The transcriptional efficiency of the CaMV 35S promoter was also compared to that of the
ribosomal RNA (rrn) promoter, as the latter uniformly showed similar high-level stable
expression in all the cells harboring the respective constructs. PCR reactions in the above
experiment were extended only up to 20 cycles in order to enable quantitation of the RNAs at the
log phase before cDNA synthesis reached saturation levels. We observed that the GFP mRNA
expression was uniform and the transcript levels corresponded to 1/6 dilution of the 16SrRNA in
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all the cells harboring the respective constructs (Figure 7.2, compare middle and bottom
Panels).
7.4.2 Major differences in translation initiation requirements between A.
tumefaciens and E. coli: High GFP translation levels in A. tumefaciens
under the control of phage T7 translational enhancer and RBS
Figure 7.1 shows a summary of a series of constructs with different ribosomal initiation contests.
Construct pC T7 SD-GFP which contained the phage T7 translational enhancer along with the
Shine-Dalgarno sequence (GAAGGAG) and the ribosome binding site (derived from the 5´ non-
coding region of the Novagen expression vector, pET29) upstream of the GFP coding sequence,
yielded very high levels of GFP protein (Figure 7.3, Panel 2) as observed by strong green
signals upon confocal microscopy and by western blot analysis of the expressed protein at ca.27
kDa (Figure 7.4, Lane 1, pCT7SD-GFP). Surprisingly, this construct gave very poor expression
in E. coli (data not shown) indicating major differences in the translational machinery between
these two microorganisms.
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122
Figure 7.3 Detection of green fluorescence due to GFP expression (and translational efficiency) for each of
the constructs (Panels 1-10) after transformation into Agrobacterium and confocal microscopy.
The first image of each Panel represents an image with GFP filter; the middle image that of the DIC filter; and the
last image is an overlap of the GFP over the DIC picture.
On the other hand, a construct containing solely the phage T4 SD sequence GAGG between the
CaMV 35S promoter and the ATG of the GFP gene did not express GFP in A. tumefaciens
showing that the T4 SD sequence alone was not sufficient for translation initiation in this
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organism (Figure 7.3, Panel 1 and Figure 7.4, Lane 10, pC-GFP), whereas in E. coli where the
T4 SD sequence by itself was sufficient to drive detectable GFP expression (345).
Figure 7.4 Western blots of the enhanced GFP protein (28 kDa) using anti-GFP antiserum and alkaline
phosphatase enzyme-linked secondary antibody conjugate.
Note the highest level of GFP expression for the pCT7SD-GFP construct (Lane 1), followed by that of the
pCrbcL TCR-GFP (Lane 2) and the pCrbcL58-GFP constructs (Lane 3), the latter two in equivalent amounts.
The pCrbcL33-GFP (Lane 4), pCAT-GFP (Lane 6) and pCATP58-GFP (Lane 7) constructs show faint bands
indicating marginal GFP expression. All other Lanes (Lanes 5, 8, 9 and 10) are negative for GFP expression.
7.4.3 Effect of the AT-rich sequence from the (AIMV) upstream of the GFP
gene on its translation in A. tumefaciens
AIMV CP RNA is one of the most efficiently translated RNAs known (333) and its sequence has
been shown to function as a strong translational enhancer (335). Thirty-three nucleotides
containing the 5´-UTR of the AIMV capsid protein gene with SD sequence GAGG were cloned
upstream of the GFP coding sequence and then expressed in A. tumefaciens. Data presented in
Figure 7.3, Panel 3 and Figure 7.4, Lane 6, pC AT-GFP, showed weak GFP signals as
compared to that of T7 SD construct. This result demonstrated that in A. tumefaciens, the T4 SD
sequence did not produce enhanced levels of GFP translation even though in combination with
the reportedly translationally robust AIMV CP 5´-UTR sequence.
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7.4.4 Analysis of 5´ -UTR sequences derived from some natural chloroplastic
genes on translation in A. tumefaciens.
Regulation of expression of chloroplastic genes occurs mainly at the level of translation and has
several features similar to that of prokaryotes. However, although the SD complementary
sequence of the chloroplast 16SrRNA is highly conserved between prokaryotes and plastids
(346), the putative SD sequence is poorly conserved in chloroplasts, both in terms of primary
sequence and location relative to the start codon (306, 347). Also, plastid gene expression is
controlled at the posttranscriptional level by protein factors that are encoded in the nucleus and
transported into the chloroplast (348, 349), adding a layer of complexity to chloroplast gene
expression that is not found in prokaryotes.
In order to compare the above prokaryotic translation initiation sequence context with
that of the chloroplast context, and in order to examine the evolutionary closeness of
translational regulation between A. tumefaciens and chloroplasts as against E. coli, we made
constructs with 5´ initiation contexts from different chloroplast genes and used them to examine
the extent of GFP expression in A. tumefaciens.
7.4.5 Identification of the minimal translation initiation sequence of the rbcL
gene required for high-level expression in A. tumefaciens
Ribulose1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (rbcL) is encoded by
chloroplast genome. The 5´-UTR of rbcL is highly conserved in the region - 1 to -58 and
contains an SD sequence (GGAGG) between -4 and -12 (350). When GFP was cloned
downstream of the 5´ translation initiator region of the rbcL gene that included the SD-like
sequence (GGAGG that is complementary to the CCUCC at the 3´ terminal region of the
Agrobacterium 16SrRNA), there was no detectable translation of the GFP in A. tumefaciens
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(Figure 7.3, Panel 5, Figure 7.4, Lane 5, pC rbcSD-GFP), demonstrating that the rbcL 5´
translation initiator region (GGGAGGG) by itself is not sufficient for successful translation
initiation. When the 5´ TCR (translation control region containing the 58 nucleotide 5´ leader,
the SD-like sequence and the N-terminal coding sequence for the first 14 amino acids) of the
rbcL gene that was essential for successful translation initiation in chloroplasts (210), was
introduced upstream of the ATG of the GFP gene sequence, a robust expression of GFP was
detected in Agrobacterium, as demonstrated by confocal microscopy (Figure 7.3, Panel 6) and
by immunoblot analysis (Figure 7.4, Lane 2, pC rbcLTCR-GFP). Next, we cloned just the 58
nucleotide 5´ -UTR of the rbcL gene upstream of the GFP gene and transformed it into A.
tumefaciens. Confocal microscopy (Figure 7.3, Panel 7) and western blotting (Figure 7.4, Lane
3, pCrbc58-GFP) showed that the GFP expression with this construct was equivalent to that of
the 5´ TCR. However, it was comparatively less than what was observed for the construct
containing the highly efficient phage T7 5´-UTR context (Figure 7.3, compare Panels 2 and 6,
Figure 7.4, compare Lane 1, pC T7SD-GFP and Lane 2, pC rbcLTCR-GFP). This led us to
the conclusion that just the 58 nucleotides at the rbcL 5´-UTR was sufficient to initiate efficient
translation in Agrobacteria. Furthermore, it was observed that these 58 nucleotides serve as
translational enhancers when located within 5´-untranslated mRNA leaders (Figure 7.5, a) in
plants.
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Figure 7.5 Confocal microscopic observation of GFP in N. tabacum leaves after 72 hr of agro-infiltration
with a) pC rbcL58-GFP and b) pC-GFP constructs respectively.
A truncation of the same sequence from 58 to 33 nucleotides from the 5´-terminus resulted in a
dramatic reduction of GFP translation (Figure 7.3, Panel 8, Figure 7.4, Lane 4, pC rbcL33-
GFP), showing the importance of the 58 base leader sequence for translation in A. tumefaciens.
In another experiment when SD-like (GGGAGGG) of the 58 base leader sequence was mutated
to the TTTATTT sequence, the translation was totally abolished (Figure 7.3, Panel 4, Figure
7.4, Lane 9, pC rbcL58AT-GFP), indicating that SD-like sequence and context sequence are
important for successful translation.
7.4.6 Comparison of the 5´-UTRs of both rbcL and Psb A genes for
translation initiation in A. tumefaciens
Testing translational requirements for successful protein expression in A. tumefaciens was
performed using the psbA gene that encodes the D1 protein of photosystem II. A construct that
contained 85 nucleotides as a 5´-UTR including RBS, AU-rich region and ATG of the psbA gene
(350) upstream of the GFP gene was made and transformed into Agrobacterium. Results showed
no detectable GFP expression as judged by confocal microscopy (Figure 7.3, Panel 9) and by
western blot analysis (Figure 7.4, Lane 8, pC psbA-GFP). This indicated that there is a major
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difference in the translatability of the GFP protein between the 5´ non-coding sequences of the
rbcL gene and that of the psbA gene, even though both are encoded by the chloroplastic genome
and are known to be involved in photosynthesis. The rbcL gene with the SD-like sequence 10
nucleotides away from the AUG codon (along with its 58 nucleotide 5´ leader) satisfies the
requirement for successful translation in A. tumefaciens, whereas the psbA gene SD-like
sequence is much farther away (40 nucleotides upstream) with unfavorable 5´ sequence context
and, therefore, does not allow positive GFP expression.
7.4.7 5´-UTR of the chloroplastic atp1 gene supports low GFP translation
levels in A. tumefaciens
The atpI gene, which encodes the CFo-IV subunit of the ATP synthase complex (351) is an
important chloroplastic gene, which possesses an SD-like, sequence at an ideal distance: 5
nucleotides upstream of the start codon. When the SD-like sequence along with the 58 nucleotide
5´ translational determinant of the atpI gene in chloroplasts (352), was cloned upstream of the
GFP coding sequence and expressed in A. tumefaciens, a low level of GFP expression was
observed (Figure 7.3, Panel 10; Figure 7.4, Lane 7, pC ATP58-GFP). This result shows that
recognition of the translational context in A. tumefaciens is dependent on factors other than just
the correctly positioned SD-like sequence and that the upstream sequence that works in
chloroplasts may not work in A. tumefaciens. Therefore, of all the chloroplastic constructs used
in this study, the rbcL 58 with the ideal spacing of the SD-like sequence from the initiation
codon (10 nucleotides) and the ideal upstream sequence was the most robust in supporting GFP
expression in A. tumefaciens.
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7.5 CONCLUSION
In the light of the above findings, it would be interesting to examine if there is any other
chloroplastic gene besides the rbcL gene that can be translated to the same level, if not higher
than that of the rbcL gene product in A. tumefaciens. Results from such further experiments
would enable us to make a firm conclusion on both the cis- and trans-acting factors of the
Agrobacterial translation machinery. It would also help establish the nature of the evolutionary
relationship between A. tumefaciens and the chloroplasts as much of the studies in this regard
have so far been predominantly performed using E. coli as the major Eubacterial organism.
The current study reveals unique translation initiation requirements for high-level protein
expression in A. tumefaciens. This together with the high strength 35S promoter that shows
enhanced transcription levels would enable the design of unique, robust protein expression
vectors for A. tumefaciens using binary vectors such as pCambia. This system also facilitates
transgene design for high-level expression of recombinant proteins using a binary vector in A.
tumefaciens before further downstream applications such as generation of transgenic plants and
plastid-based expression. Thus preliminarily enhanced translation in A. tumefaciens can be used
as a predictor of high-level protein synthesis in transgenic plants considering the time-consuming
nature of the latter process.
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APPENDIX D
8 ANALYSIS OF THE INTERNAL RIBOSOME BINDING
SITE (IRBS) OF PVX
8.1 BACKGROUND
In potexviruses, translation of the downstream ORFs, triple gene block, and CP, is believed to
take place from a series of capped subgenomic RNAs (sgRNAs) which are generated from the
genomic RNA. In vitro translation studies (171, 353) showed that two sgRNAs (sgRNA1 and
sgRNA2) of 2.1 and 1.4 kb were necessary for translation of the TGB while a third sgRNA of
0.9 kb (sgRNA3) was required for expression of the viral coat protein. It was also noticed that
that the 25 kDa protein was synthesized as a single translation product of the 2.1 kb subgenomic
(sg) RNA and that both the 12 kDa and 8 kDa proteins are expressed from the same 1.4 kb
sgRNA. In vitro translation studies also indicated that the CP could not be translated from
genomic RNA; rather, it could be readily translated from a smaller, subgenomic RNA encoding
the CP gene (171, 354, 355). However, in vitro studies of papaya mosaic virus, narcissus mosaic
virus and clover yellow mosaic virus exhibited that expression of the CP could take place from
genomic as well as subgenomic RNAs, possibly by means of internal initiation of translation
(356-360).
Previously in our lab Hefferon, et al. (174) demonstrated with transgenic plants that the 8
kDa protein and the CP could be translated from a dicistronic construct corresponding to the C-
terminal half of the 12 kDa protein, the complete 8 kDa and CP genes of PVX, indicating that
translation of CP could take place either by internal entry of ribosomes or by a
termination/reinitiation mechanism. Furthermore, these authors showed that expression of the
downstream cistron was persisted in protoplasts electroporated with RNA transcripts of the
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dicistronic construct, even after stable hairpin structures were placed in front of dicistronic
constructs containing either the PVX CP gene or a reporter gene as the downstream cistron. The
8 kDa protein or reporter gene was detected only in the absence of the hairpin structure. Since
CP was detected in the presence or absence of a stable hairpin structure at the 5´ terminus,
suggesting that the former model (IRBS) is more likely.
This study aimed to confirm and further investigate (reassess) the IRBS property of the
PVX 8K region using the GFP gene as a reporter (fused with ORF of CP of PVX) in in vivo
with stable transgene expression systems and to identify the precise sequence within that region
that is responsible for the internal initiation function. Western blot and confocal studies indicate
the expression of a downstream cistron (GFP) only in the absence of the hairpin in transgenic
tobacco plants harboring the dicistronic construct, suggesting that that translation of GFP could
take place by a termination/reinitiation rather internal ribosome binding site (IRBS)
mechanism.
8.2 MATERIALS AND METHODS
8.2.1 Construction of GFP expression plasmids
To test the IRBS nature of the PVX sequence, the construct pC8K-GFP, containing the sequence
upstream of the ATG codon of the PVX CP gene including the 8 kDa ORF and 177 nucleotides
upstream of this 8k ORF, was generated by amplifying the product using pre-existing
recombinant pTR:8k as a template and 12KKpnI.F/CPBamHI.R primers. To map the IRBS
sequence, pC8K-GFP was truncated to generate pC220K-GFP and pC127K-GFP constructs by
amplifying the products using a pC8K-GFP construct as a template, 8K220KpnI.F /CPBamHI.R,
and 8K127KpnI.F/CPBamHI.R primers respectively. Subsequently, the obtained products were
inserted into the pC-GFP construct in its KpnI/BamHI sites. The start codon of the CP was also
retained as part of the constructs, such that it was in frame with the GFP ORF. Furthermore to
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confirm IRBS sequence, a sequence expected to form a stable hairpin was introduced in KpnI
site of all above constructs to generate pChp8K-GFP, pChp220K-GFP, and pChp127K-GFP
constructs. This stable hairpin sequence was also inserted into pC-GFP in KpnI/XbaI sites to
create a pChp-GFP construct which is believed to block the translation of GFP completely and
used as a negative control. The hairpin was ligated into the digested constructs as mentioned in
section 2.3.1. The presence and accuracy of the inserted sequence were confirmed by DNA
sequencing (The Centre for Applied Genomics, Toronto, Canada) using the GFP-R reverse
primer: 5´- AAGTCGTGCTGCTTCATGTG -3´. Table 8.1 shows a list oligonucleotides and
DNA sequence used to generate the plasmids in this study.
Table 8.1 Oligonucleotides/ primers used in the production of different constructs with or without a hairpin
structure to investigate the IRBS.
* Underlined bold letters indicate restriction endonuclease recognition sequences.
** Restriction endonuclease recognition sequences introduced into the oligos to facilitate cloning of fragments
into PC-GFP.
8.2.2 Plant transformation for stable gene expression
Stable Agrobacterium-mediated transformation was performed as described in section 2.3.6.
Constructs Primers Oligo/Primer sequence* (5´-3´) Cloning
sites**
pC8K-GFP 12KKpnI.F
CPBamHI.R
ATCGGGTACCCTAGAAATAGTTTACCCC
CCATGGATCCTCTAGCTGGTGCTGACAT
KpnI
BamHI
pC220-GFP 220KKpnI.F
CPBamHI.R
AATATTGGTACCCAGGCCTCATATCTCAACGCAATC
ATACTAGGATCCTGGTGCTGACATCTTTCGAGTATC
KpnI
BamHI
pC127-GFP 127KKpnI
CPBamHI.R
AATATTGGTACCCAGGCCTGGAGAATCAATCACAGT
ATACTAGGATCCTGGTGCTGACATCTTTCGAGTATC
KpnI
BamHI
Hairpin For
above three
construct
Sense
Antisense
ACGCGCTCCCCCCGGGGGGTCGACCCCCCGGGGGGA
AAGCAGTAC
TGCTTTCCCCCCGGGGGGTCGACCCCCCGGGGGGAG
CGCGTGTAC
KpnI
(inac)
Hairpin for
pC-GFP
Sense
Antisense
TCGCGCTCCCCCCGGGGGGTCGACCCCCCGGGGGGA
AAGCT
CTAGAGCTTTCCCCCCGGGGGGTCGACCCCCCGGGG
GGAGCGCGAGTAC
KpnI
(inac)/
XbaI
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8.2.3 Confocal microscopy
Live cell imaging was performed on a confocal microscope (Leica TCS SP5; Leica
Microsystems) using a 40× or 63× oil immersion objective. GFP fluorescence was imaged at an
excitation wavelength of 488 nm, and the emission signal was detected between 495 and 530 nm
for GFP and between 643 and 730 nm for chlorophyll autofluorescence. Differential interference
contrast (DIC) and fluorescence images were acquired simultaneously for comparative studies of
all the constructs. Images were analyzed by Leica Application Suite Advanced Fluorescence
(LAS AF) software.
8.2.4 Western Blot
Following plant-transformation, leaf samples were grinded in the presence of liquid nitrogen
using pre-cooled pestle and mortar. Using a flame-sterilized spatula, the powder was transferred
to 1.5 mL tubes containing 160 µL of protein extraction TMPDTNU (50 mM Tris, 20 mM
MgCl2, 1 mM PMSF, 100 mM DTT, 2% Triton X-100, 0.5% NP-40 and 8 M urea) buffer plus
40 µL of 5× SDS-PAGE loading dye (212). These samples were boiled at 95-100 °C for 5 min
and 40 µL of each sample was loaded onto 12% SDS-PAGE gels along with the appropriated
protein molecular weight markers (Thermo Fisher Scientific). Protein concentrations were
determined by the Bradford Protein Assay reagent kit (Bio-Rad, Hercules, CA). Electrophoresis
was performed initially at 150 V until the samples entered the separating gel followed by 100 V
until dye reached at the bottom of the gel (218). The proteins were then transferred onto
nitrocellulose membrane (0.45 nm pore size, Pall corporation) for 1 hr in transfer buffer (212)
using the Bio-Rad protein electrophoresis unit. The membrane containing the transferred proteins
was blocked in Tris-buffered saline (TBS buffer: 50 mM Tris and 150 mM sodium chloride)
along with 5% skimmed milk for 5 hr. Subsequently, the membrane was incubated at 4°C
overnight with mild shaking with (1:1000) Anti-GFP, Rabbit IgG Fraction (Anti-GFP, IgG),
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polyclonal antibody (Invitrogen) in TBS+3% BSA. The membrane was washed (TBS, 0.3%
Tween-20) 4 times and incubated with (1:3000) Goat Anti-Rabbit IgG (H & L) alkaline
phosphatase (Bioshop) for 2 hr at room temperature with mild shaking. The membrane was
washed 3 times with TBS-T followed by a final washing with TBS. Finally, signals were
developed with alkaline phosphatase substrate solution (BCIP/NBT, Bioshop) according to the
manufacturer instructions. The membranes were dried and photographed.
8.3 RESULTS AND DISCUSSION
8.3.1 Expression of GFP using stable gene experiments
To determine the expression strategy of the GFP in dicistronic and deletion constructs where CP
ORF fused in frame to the N-terminus of the GFP ORF (CP-GFP fusion) and to better define the
mechanisms (internal ribosome binding or an alternative mechanism such as leaky scanning or
termination/reinitiation of translation) on its translation initiation, transgenic tobacco plants
harboring different constructs with and without stable hairpin structure were analyzed by western
blot and confocal microscopy. Previous studies have shown that secondary structure in the 5′
leader inhibits translation by influencing the binding of 40S ribosomal subunits to the 5′ end of
an mRNA (361-364). Kozak (362) also demonstrated the positioning effect of a hairpin in
translation, according to the author, the translation was drastically inhibited when a hairpin was
inserted within the first 12 nucleotides of the gene, however when the same hairpin was
repositioned 52 nucleotides from the 5′ end, it no longer inhibited translation. The stable hairpin
structure is placed within 25 nucleotides from the 5′ end in the present studies. The pChp-GFP
construct was included as a negative control to test the functionality of hairpin structure to stop
the translation. Confocal and western blot studies with transgenic plants show that translation of
GFP gene is completely blocked by inserting the hairpin as depicted in Figure 8.1 (Panel B, D,
F, and H) and Figure 8.2 (Lanes 2,4,5,6 and 7), confirming the stability and functionality of the
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hairpin in case of stable gene expression. In contrast, expression and accumulation of GFP can
be observed clearly in the case of transgenic plants harboring pC-GFP, pC127-GFP, pC220-GFP
and pC8K-GFP constructs without hairpin (Figure 8.1: panel A, C, E, and G).
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Figure 8.1 Confocal microscopic observation of GFP in transgenic N. tabacum leaves harboring constructs
without and with hairpin structure (Panels A-I).
The first image of each Panel represents the image with a GFP filter; the middle image that of the DIC filter; and
the last image is an overlap of GFP over the DIC picture. The red small block represents CP-GFP 'fusion protein'
includes the first few amino acids of the CP. Bar =20µM
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8.3.2 Western blot analysis
Western blot result also confirms the GFP expression in the case of transgenic plants harboring
pC8K-GFP construct as shown in Figure 8.2; Lane 1. Finally, when a 0.4 kb cDNA fragment
containing the sequence upstream of the AUG codon of the PVX CP gene was placed between
two reporter genes, expression of the downstream GFP cistron was lost, suggesting the absence
of IRBS in this PVX sequence as shown in Figure 8.1; Panel I and Figure 8.2; Lane 4.
Figure 8.2 Western blot using anti-GFP antiserum to detect GFP (27 kDa) expression in transgenic N.
tabacum cv. Xanthi plants harboring constructs in the presence or absence of a hairpin structure.
Lanes 1 and 3 contain total proteins extracted from transgenic plants with pC8K-GFP and pC-GFP constructs
respectively. Note the lower GFP expression in the case of pC8K-GFP as that of pC-GFP used as a positive
control, suggesting an alternative translation mechanism. Lane 4 consists of total protein from the plant where
PVX sequence is placed between two reporter genes, indicating non-functionality of PVX sequence as an IRBS.
Lanes 2, 5, 6 and 7 contain total proteins from transgenic plants harboring constructs with a stable hairpin,
confirming the complete blockage of GFP expression. Lane 5 protein ladder where green band depicts 25 kDa.
The present data provides an evidence for the absence of IRBS sequence which was previously
suggested by Hefferon, et al. (174). It is noteworthy that the same PVX sequence, previously
believed to be working as an IRBS sequence, is investigated. However, the current findings have
ruled out the translation of GFP by the IRBS. These results contradict the previous results (174).
Since 8 KDa was shown to be expressed by dicistron (8K-CP) (174), suggesting that translation
of the downstream cistron (CP) could be controlled by leaky scanning and/or with a
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termination/reinitiation mechanism. The expression of GFP is lower in the case of pC8K-GFP as
that of pC-GFP, suggesting the presence of an alternative mechanism(s). Leaky ribosome
scanning also contributes in translation from downstream start codons in some positive-stranded
RNA viruses and retroviruses (365, 366). Verchot, et al. (15) described an in vivo analysis of the
PVX TGB translation strategy, where they presented evidence that the 8K ORF could be
translated by leaky ribosome scanning through the 12K ORF. It is also possible the expression of
a downstream cistron (CP/GFP) is controlled by termination-reinitiation mechanism as well.
Previous studies showed that translation of the HBV polymerase gene could be controlled by
leaky scanning together with a termination-reinitiation mechanism involving an upstream
minicistron (367, 368). In the case of PVX, coat protein is a translation by sub-genomic RNA
(sgRNA3) of 0.9 kb, why it requires alternative translation mechanisms? It can be hypothesized
that it may be expressed by genomic, larger subgenomic or before sgRNAs are produced and
play some roles early in virus infection. This speculation is supported by McCormick, et al. (369)
who reported that capsid protein of bovine norovirus could be expressed as a result of translation
termination-reinitiation between ORF1 and ORF2. The alternative translation strategies may be
common in all polycistronic viruses to assist during their replication cycle for the maximum
accumulation of required proteins (174). However relative importance of these alternative
translation strategies remains to be determined.
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APPENDIX E
9 NOVEL AND UNIVERSAL APPROACH TO SILENCE ALL
GEMINIVIRUSES IN PLANTS
9.1 SUMMARY
Plant-microbe interactions have been explored for many years. In recent years, molecular
dissections of some of those interactions have been investigated, particularly, the role played by
a battery of host plant small interfering RNAs with pathogen replication. RNA interference
(RNAi) was shown to play a major role in controlling infections caused by RNA viruses. Since
the begomoviruses are DNA viruses, it was assumed that RNAi does not function against DNA
viruses. Recent studies have shown that RNAi may also function against DNA viruses (370-
374). Although the molecular mechanisms are being deciphered, the results indicate that
begomoviruses may also be targeted with an engineered RNAi system. Previous studies in this
lab were focused on the development of plant resistant to RNA viruses (375). Presently we are
focusing on geminiviruses which are known to infect a large number of economically important
plants. There are over 680 isolates of geminiviruses infecting over 200 plant species. Most of
those viruses are transmitted in the field by white flies. Geminiviruses infecting major crops like
cotton, vegetables (tomato, potato, pepper etc.) and ornamental plants cause enormous economic
losses not only in the yield of those crops but also in the quality of crops. In this study, I
demonstrate that an engineered RNAi system which targeted the conserved control region (CR)
of many geminiviruses resulted in the protection of transgenic plants from geminivirus
infections. This construct generates a 176 base pair double stranded RNA which encompasses
most of the CR region of many begomo- and geminiviruses infecting a large number of
economically important crops. This construct was tested against two begomoviruses (Ageratum
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enation virus (AEV), and Tomato leaf curl New Delhi virus (ToLCNDV) as model studies. Data
show that a very strong reduction in virus replication in transgenic Nicotiana benthamiana plants
in comparison to non-transgenic healthy control plants. Sequence alignments of our construct to
available begomovirus sequences indicate that a large number of those viruses will be protected
using this construct. Molecular mechanisms involve in the resistance, as well other molecular
approaches for the development of plant resistance will also be discussed.
9.2 INTRODUCTION
Geminiviruses have recently emerged not only as the cause of devastating diseases of important
crop plants (376) but also as a tool to study fundamental aspects of RNA interference (RNAi)
and virus-induced gene silencing (377). RNA silencing is an evolutionarily conserved
mechanism protecting cells from pathogenic RNA and DNA, which is increasingly viewed as an
adaptive immune system of plants against viruses (378). Expression of hairpin double-stranded
RNA (dsRNA) homologous to coding sequences of RNA and DNA viruses has been shown to
restrict viral infection in plants (379-381). It is assumed that long dsRNA is processed by dicer
proteins into small interfering RNAs (siRNA), which then target viral RNA for cleavage and
degradation in a sequence specific manner (382, 383). siRNAs have also been implicated in
transcriptional gene silencing (TGS) when Mette, et al. (384) found that dsRNA expression
could trigger the methylation of a cognate target promoter sequence. Sijen, et al. (385) conclude
that DNA methylation is an essential process for regulating TGS and important for reinforcing
Post-transcriptional gene silencing (PTGS). This ability has been correlated with reduced
transcription levels (386).
Geminiviruses are known to contain a conserved nine nucleotides (nonanucleotides) at
the origin of replication. The flanking sequences are involved in the recognition of cellular DNA
polymerase to the single stranded viral DNA to start the replication process and to produces a
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double-stranded circular DNA. Furthermore, this nonanucleotide sequence is also nicked by the
viral rep protein to allow the viral DNA replication through the rolling circle model (see Figure
9.6). In this study, we report a novel approach which is based on the design of a complementary
RNA sequence to bind to the origin of replication of geminiviruses and consequently blocking
their replication. Since in geminiviruses the promoter region and the origin of replication are not
normally transcribed, blocking this region of viral DNA will have a detrimental consequence on
the viral replication and consequently confers an excellent resistance of plants to geminiviruses
infection. Further, the presence of complementary RNA sequences to the non-coding region of
geminiviruses may also induce the viral DNA methylation in the promoter and origin of
replication regions which will also lead to the blocking of gene transcription and lack of viral
gene expression which also will reinforce the lack of replication and consequently improve the
resistance of plants to viral infections. This provides a novel method to engineer DNA virus
resistance in plants without targeting the coding sequence.
In order to investigate the efficacy of this approach in a stably transformed plant system,
we produced transgenic N. benthamiana expressing hairpin dsRNA homologous to the sequences
including the bidirectional promoter and common region (CR) of Ageratum enation virus (AEV)
a begomovirus of family Geminiviridae. Begomoviruses infect a wide range of economically
important dicotyledonous host plants and are transmitted by the whitefly Bemisia tabaci (236,
237). Begomoviruses consist of either monopartite (a single DNA) or bipartite (with two DNA
components: DNA-A and DNA-B) genomes (123, 125, 126, 231, 232). The DNA-A of bipartite
and the single component of monopartite begomoviruses contain five or six Open Reading
Frames (ORFs) while the DNA-B contains two ORFs (BV1 and BC1, in viral-sense and
complementary sense strand, respectively). Both DNA-A and DNA-B are approximately 2.8-3.0
kb in size. Both components are organized into divergent transcription units separated by an
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intergenic region (IR) of about 200 bp, which contain the replication origin and two divergent
promoters (133). The virus AEV consists of a monopartite circular, single-stranded DNA
genome (DNA-A) of a size 2.8 Kb enclosed in a characteristic twinned quasi-icosahedral particle
(387). In addition, AEV is also associated with a class of single-stranded DNA satellites known
as DNA β which range from 1247-1374 nucleotides in length (388). In this study, we report the
development of RNAi-based resistance to AEV (monopartite) and ToLCNDV (bipartite) through
the expression of dsRNA homologous to its viral non-coding sequence. These results expand the
potential of RNAi strategy against DNA viruses to their entire genome.
9.3 MATERIALS AND METHODS
9.3.1 Vector construction
An infectious clone of Ageratum enation virus (AEV) was used for vector construct. The 176
nucleotides fragment corresponding to the intergenic region (IR) of DNA-A of AEV for
antisense was amplified by using PCR primers (AEVKpnI 5’-
CTGACAGGTACCACTCCAATGGCATAATTGTA-3’ and AEVSalI 5’-
GACTGAGTCGACGGGACCACGAAACAATTAAG-3’) from position 2671-96 (GenBank
accession number AM261836) (including the underlined sites for KpnI and SalI respectively). A
primer pair (AEV ClaI 5’-CTGACAATCGATACTCCAATGGCATAATTGTA-3’ and
AEVNheI 5’-GACTGAGCTAGCGGGACCACGAAACAATTAAG-3’ (including the
underlined sites for ClaI and NheI respectively, was used to amplify a 176 bp fragment from the
same intergenic region for sense strand. PCR reactions were carried out in a 50 μL solution
containing 10-30 ng of DNA, 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 µM
each dNTP, 0.5 µM each primer, and 0.05 units/µL of Taq DNA polymerase (Sigma, CA,
U.S.A.). The mixture was treated at 95ºC (5 min) and subjected to 30 cycles of amplification
(95ºC for 1 min, 68ºC for 40 sec and 72ºC for 1 min), with a final elongation cycle of 10 min at
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72ºC. These sense and antisense fragments were cloned into the pHANNIBAL vector (389).
Once the assembly of the inverted repeat was completed and verified by sequencing then this
cassette (Figure 9.1) was cloned with NotI into a pART27 binary vector and named pART27-
AEVIR.
Figure 9.1 A partial Schematic diagram of the binary construct pART27-AEVIR used for plant
transformation.
A) The intergenic common region-containing promoter sequences from positions 2671-2750 and 1-96 of AEV
DNA- A (GenBank accession number AM261836) separated by a pyruvate dehydrogenase kinase (Pdk) intron in
the reverse and the forward orientations were inserted between CaMV 35S promoter and octopine synthase
terminator (OCS). The expression cassette was subcloned in the NotI site of pART27 to generate the binary
vector pART-AEVIR. B) Predicted hairpin secondary structure of the RNA transcript.
This pART27-AEVIR vector was transferred to Agrobacterium tumefaciens strain GV3101
competent cells. Recombinant colonies were selected on LB plates supplemented with 100
µg/mL Spectinomycin and 30 µg/mL Gentamycin.
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9.3.2 Plant transformation
Stable Agrobacterium-mediated transformation of N. benthamiana plants was performed by a
standard protocol (390) with some modifications. Three to four weeks old tissue cultured plants
were used for transformation. Leaf discs were co-cultivated for 10 min with 36 hr old
Agrobacterium culture incubated at 28°C in a shaker. These leaf discs were cultured on MS
medium containing 100 mg/L BAP and 0.4 mg/L NAA. After three days, transformants were
selected on MS medium containing 100 mg/L Kanamycin, 400 µg/ml Carbenicillin, 1 mg/L BAP
and 0.4 mg/L NAA. Every three weeks, the explants were subcultured to a fresh selection
medium for shoot regeneration. Developed shoots were transferred to a phytohormone-free ½
MS medium containing 300 mg/L Kanamycin, and 400 mg/L Carbenicillin for root formation.
Regenerated plants were transferred from Magenta boxes to pots and further grown under
greenhouse conditions (23-27°C, 16 hr light and 8 hr dark).
9.3.3 Characterization of transgenic lines
N. benthamiana genomic DNA of transgenic lines was extracted from leaves of tissue cultured
plants according to Kang and Yang (391). About 0.5 cm2 leaf of each tissue cultured grown plant
was put in a 1.5 mL microfuge tube. The leaf tissue was homogenized in 50 µL DNA extraction
buffer (500 mM NaCl, 100 mM Tris-HCl pH 7.5, and 50 mM EDTA pH 7.5), using a hand-
operated homogenizer (Sigma, Z35997-1) with a plastic pestle, for 15~20 sec. After an initial
homogenization, another 150 µl of DNA extraction buffer was added and homogenized with the
same homogenizer for 15~20 sec. Then, 20 µL of 20% SDS were added and vortexed for 30 sec.
Samples were incubated at 65°C for 10 min for cell lysis. An equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1) was added to the samples, mixed by vortexing for
30 sec, and then centrifuged at 10,000 g for 3 min at 4°C. The supernatant was transferred to a
fresh tube and extracted one more time with phenol/chloroform/isoamyl alcohol (25:24:1) and
144
then with chloroform alone. The supernatant was transferred to a fresh tube, and a double
volume of ethanol was added to each sample, mixed well, and the samples were incubated at -
20°C for 30 min. The samples were centrifuged at 10,000 g for 10 min at 4°C. The pellet was
washed with 70% ethanol, dried, and resuspended in sterile distilled H2O containing 20 µg/mL
DNase-free RNase A. The concentration and purity were determined from the A260/A280 ratio
using a spectrophotometer. PCR amplifications were performed as mentioned before but at 55°C
annealing temperature. Primers Pdk 5’- AACAAAGCGCAAGATCTATCA -3’ (forward) and
Ocs 5’- TAGGCGTCTCGCATATCTCA-3’ (reverse) were used to amplify a 456 bp region
including IR sense of the transgene T-DNA cassette. Primers 35S 5’-
CCACTATCCTTCGCAAGACC-3’ (forward) and Pdk 5’-
CTTCGTCTTACACATCACTTGTCA-3’ (reverse) were used to amplify a 428 bp region
including IR antisense of the transgene T-DNA cassette. The PCR products were resolved by
electrophoresis in 2.0% agarose gels. Successful transformation of transgenic plant lines was also
confirmed by chromosomal DNA sequencing.
9.3.4 Agroinoculation
A single colony of each infectious AEV clones of DNA-A and DNA-β in Agrobacterium strain
of GV3101 was cultured in 5 ml of LB culture containing antibiotics Kanamycin (100 µg/mL)
and Gentamycin (50 µg/mL) and grown overnight at 28°C at 225 rpm. A large LB media
suspension was then inoculated with the overnight culture and grown at 28°C to an OD600 of
~1.0. The cells were harvested by centrifugation at 1200 g for 10 min and resuspended in
Agrobacterium induction medium (10 mM MgCl2, 10 mM MES pH 5.6 and 150 µM
acetosyringone to a final OD595 of 1.0 and incubate at room temperature for 4-6 h with gentle
shaking (80-100 rpm). The culture was pelleted again by centrifugation at 1200g for 10 min and
resuspended in 10 mM MES buffer and adjust to OD595~0.0005. The both bacterial suspensions
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were mixed and taken in a syringe and infiltrated through the abaxial surface of two lower leaves
of different transgenic lines (T0 generation) and wild-type plants at four leaf stage. Each
experiment was repeated five times (five treatments). Five plants were also infiltrated with buffer
alone for negative control.
9.3.5 Detection of viral genome in infected plants
Total DNA was extracted from systemic leaves of infected plants of each transgenic line and
wild-type plants grown under greenhouse conditions as mentioned in section 3.3.1. One
microgram of DNA was used per PCR reaction. Primers AEVCP.F 5’-
GCCCAGGATGTACAGGATGT-3’ (forward) and AEVCP.R 5’-
CACAGGCCTACGATCCCTAA-3’ (reverse) were used to amplify a 283 bp of the coat protein
of AEV (GeneBank accession AM698011). Primers TlcvCP.F
5’CCTAGCACTGCCACTGTGAA-3’ (forward) and TlcvCP.R 5’-
CGGGATTAGAGGCGTGAGTA -3’ (reverse) were used to amplify a 232 bp of the coat
protein of ToLCNDV (GenBank accession HM134234.1). Primers Actin-F 5’-
ATCCGTGGAGAAGAGCTACG-3’ (forward) and Actin-R 5’-
TGGTACCACCACTGAGGACA-3’ (reverse) were used to amplify a 209 bp of Nicotiana
benthamiana actin gene (GeneBank accession AY179605) as an internal control for semi-
quantitative PCR.
9.4 RESULTS
9.4.1 Production of transgenic lines
Nicotiana benthamiana plants were regenerated from Kanamycin resistant embryogenic calli that
had been transformed with Agrobacterium tumefaciens GV3101 harboring the binary vector
pART27-AEVIR. All generated plants had a normal phenotype. The integration of the transgene
T-DNA cassettes has been confirmed by PCR. A simple and reproducible procedure for PCR
146
amplification of transgenes was done. Four independent transgenic lines were confirmed by PCR
(Figure 9.2). Expression of the transgene was under the control of the cauliflower mosaic virus
(CaMV) 35S promoter to produce high levels of hp-derived small interfering RNAs (siRNA) in
leaves, where virus transmission usually occurs.
Figure 9.2 PCR-verification of transgenic N. benthamiana plants harboring pTR27-AEVIR construct.
The expected 456 and 428 bp fragments for regions including IR sense and IR antisense respectively were
detected in four transgenic lines (Lanes 1-4). However, these products were absent in wild-type plant (Lane 5).
9.4.2 Transgenic plant evaluation against infectious clones of AEV
To determine the resistance against AEV virus, wild-type and transgenic N. benthamiana plants
harboring pART-AEVIR construct were infiltrated with infectious clones of AEV (DNA-A and
DNA-β) in Agrobacterium. Since the optical density value of 1 corresponds to 108 cells/mL
culture. This number (OD595~0.5 ) of bacterial cells harboring infectious clones is very high as
compared to a number of virus particles during a natural infection by white flies. At an
exceptionally high inoculum, the virus resistance mechanism in transgenic plants will certainly
be overcome. Consequently, transgenic plants will naturally produce large quantities of virus.
Serial dilutions were produced and used to infect plants. A dilution factor of 1000 fold (i.e. OD
595 = 0.0005, equivalent to about 10 cells/mL) was considered as adequate. All agroinfiltrated
plants were observed periodically for the appearance of symptoms. However, both transgenic
and non-transgenic plants showed no viral symptoms. Consequently, determination of the virus
147
quantity or viral genome produced in wild-type and transgenic plants was the method of choice
to gauge the virus resistance (see Figure 9.3). To investigate the effect of the hairpin sequence
on the accumulation of viral DNA (replication), total DNA was extracted from the uppermost
fully expanded leaf tissues of all treatments at 21 days post inoculation and 1µg of this DNA was
subjected to semi-quantitative PCR using specific primer pairs (AEV-F and AEV-R) for coat
protein to detect AEV and primers (Actin-F and Actin-R) to detect the N. benthamiana Actin
gene for internal control.
Figure 9.3 Semi-quantitative PCR-based testing of wild-type (Wt) and transgenic N. Benthamiana plants
harboring pART27AEV-IR construct for their resistance against AEV after three weeks of challenging
with infectious clones of AEV DNA-A and DNA- β in A. tumefaciens strain GV3101.
A) Primers specific to the coat protein gene (Tlcv and AEV-CR, 283bp fragment) were used to produce the PCR
amplicons: Lane 1; wild-type treated with buffer alone used as a negative control. Lanes 2 and 3; wild-type
plants infected with AEV infectious clones. Lanes 4, 5 and 6; three transgenic lines infected with AEV infectious
clones. B) Actin gene was included for internal control experiments. The resulting PCR products were analyzed
on a 2% Agarose gel.
The capsid protein gene was used to determine the amount of virus in infected plants. The
expected size of AEV coat protein fragment (283 bp) could only be amplified from plants
challenged with infectious clones of AEV whereas no bands could be detected when DNA
extracted from control. The expected 283 bp PCR product is very prominent in wild-type plants
148
(Figure 9.3; Lanes 2 and 3) that indicate high susceptibility of wild-type plants against the
virus. In transgenic plants light to the intense band could be detected depending upon the level of
resistance that indicates the various level of tolerance or resistance of transgenic lines against
AEV (Figure 9.3). To ascertain the semi-quantitative results, DNA samples from non-transgenic
and transgenic plants were amplified by PCR using primers pairs for Actin. The fragment size of
209 bp from Actin gene was detected in both transgenic and non-transgenic plants as shown in
Figure 9.3.
9.4.3 Testing of transgenic plants for resistance against ToLCNDV
Transgenic plants harboring pART27 AEVIR construct were also tested for resistance against
another begomovirus; Tomato leaf curl New Delhi virus (ToLCNDV). When wild-type and
transgenic N.benthamiana plants were challenged with infectious clones of ToLCNDV (DNA-A
and DNA-B), all wild-type plants showed symptoms of virus infection in the upper, newly
emerging leaves at 21 days post-inoculation (dpi) consisting of foliar yellowing, curling upwards
and thickening of veins (Figure 9.4; B). In contrast, transgenic plants remained symptomless or
appeared with mild symptoms (Figure 9.4; A) for first 4 weeks.
149
Figure 9.4 Infectivity of infectious clones of ToLCNDV in tobacco plants.
Symptomatic wild-type plants (B) compared to putative transgenic plants showing mild symptoms (A)
Photographs were taken at 21 days post-inoculation.
To investigate the resistance and/or tolerance effect on the accumulation of viral DNA
(replication) in transgenic plants, total DNA was extracted from non-inoculated uppermost fully
expanded leaf tissues of all treatments inoculated with either infectious clones or buffer alone
after three-week post inoculation. One microgram DNA of each plant was subjected to semi-
quantitative PCR using specific primer pairs to amplify a 232 bp of the coat protein of
ToLCNDV. The expected size of coat protein gene could only be amplified from plants
challenged with infectious clones of ToLCNDV whereas no bands were detected with DNA
extracted from buffer treated plants. The CP PCR product is in a range from sharp to faint in
different transgenic lines harboring pART27 AEV-IR construct that indicates the various level of
tolerance or resistance of transgenic lines against the virus (Figure 9.5; Panel A, Lanes 2-5).
150
Figure 9.5 Semi-quantitative PCR-based testing of wild-type and transgenic N. Benthamiana plants
harboring pART27AEV-IR construct for their resistance against ToLCNDV after three weeks of
challenging with infectious clones of ToLCNDV (DNA-A and DNA- B) in A. tumefaciens strain GV3101.
A) Primers specific to the coat protein gene (TlcvCP.F and Tlcv.R for 232 bp fragment) were used to produce the
PCR amplicons: Lane 1; wild-type treated with buffer alone used as a negative control. Lanes 2, 3 and 4; three
transgenic lines infected with TolCNDV infectious clones. B) Actin gene was included for internal control
experiments. The resulting PCR products were analyzed on a 2% Agarose gel.
To validate the semi-quantitative result, the same DNA samples from non-transgenic and
transgenic plants were amplified by PCR using primers pairs for Actin gene as an internal
control (Figure 9.5; Panel B)
9.5 CONCLUSION
From the results obtained, we can conclude that the dsRNA strategy confers a good resistance to
viral infection. A 176 bp sequence of the non-coding intergenic region (IR) from AEV infectious
clone was chosen as the blocking sequence in sense and anti-sense orientations interrupted with a
pyruvate dehydrogenase kinase (pdk) intron. The blocking sequence (seen below in Figure 9.6;
B) which spans the origin of replication (Ori) of geminiviruses contains 100% identity to the
begomovirus (AEV).
151
Figure 9.6 Organization of a Geminivirus replication origin.
A) A diagram of the tomato golden mosaic virus (TGMV) replication origin is presented by Bisaro (392). Shown are
the relative positions of Rep-binding sites, the invariant sequence (TAATATTAC), and the site where plus-strand
synthesis initiates. Sequences involved in origin recognition/specificity are also depicted (393). The location of
sequence elements that interact with the transcription machinery, including TATAA sequence, Rep and CP
transcription start sites (references cited in (394)), a putative binding site for G-box family transcription factors, and a
putative TrAP response element (the conserved late element; (395)) are also indicated. A sequence that appears to be
an additional Rep-binding site in inverted orientation has been identified by sequence analysis (395). Nucleotide
coordinates are from TGMV DNA-A. B) Blockage of the origin of replication by an antisense single stranded
complementary RNA (depicted in red) sense RNA. The internal sequence is that of geminivirus viral origin of
replication (+strand).
This blocking sequence also contains 42-100 % sequence homology to several other published
sequences of begomoviruses and expectedly to a large number of geminiviruses which are
circulating in the field but are not yet discovered and/or identified. Our blocking sequence is
designed to contain in its core region the highly conserved nonanucleotide sequence in
geminiviruses. This conserved sequence is also flanked by complementary sequences to the IR
control region for geminiviruses. We expect this blocking sequence to target the origin of
replication of all major members of begomoviruses. Because this sequence contains several
stretches of perfect homology to the origin of replication of begomoviruses, it is expected that
inhibition of the replication of these viruses will be carried out (for molecular mechanisms of
blockage, see Figure 9.6). An intron-containing hairpin (ihp) transformation construct pART27-
AEVIR was made by using the pHANNIBAL/pART27 system. The 176-bp double-stranded
RNA sequence is the target for the RNA silencing/dicer machinery which could produce 21-25
double-stranded RNA sequences. Binding of the Argonaut and other plant proteins to the double-
stranded RNA fragments result in activation of several plant defense mechanisms against the
152
invading begomoviruses. A complementary RNA sequence could be expected to target the
hairpin loop (nonanucleotides) at the origin of replication on the viral begomovirus single-
stranded (positive-sense) (Figure 9.6). An RNA-DNA hybrid is quite stable which might result
in blocking the origin of replication of the virus by the cellular polymerases (there will not be at
this stage any viral transcription from single-stranded viral DNA) and consequently, the viral
replication could be inhibited at very early stages. Binding of the single-stranded complementary
RNA may also disturb the double-stranded stem-loop of viral DNA rendering it not recognizable
by cellular polymerases. Further, the complementary RNA sequences generated by our construct
possibly activate the RNA-directed DNA methylation which targets the stem of the hairpin loop
which is double-stranded and rich in GC. Consequently, the DNA methylation of the stem of the
hairpin loop which constitutes the origin of replication could result in blocking that region of
viral DNA of being copied into a double-stranded sequence (replicative form) by cellular
enzymes. This stem is quite rich in CG dinucleotides (primary target for DNA methylation).
During viral replication, the viral “rep” protein is responsible for nicking the double-stranded
DNA at the origin of replication to allow the replication to continue through the rolling circle
model. Our complementary single-stranded sequences may be responsible for blocking the
nicking of the hairpin loop a sine qua none condition for replication by rolling circle model.
Further, the control region (which includes the nonanucleotide) could also be methylated by the
RNA-directed DNA methylation. Consequently, the rolling circle replication and the promoter
regions may be entirely methylated which leads not only to blocking replication but also
inhibition of transcription of viral essential genes (e.g. rep gene and other viral genes). Viral
double-stranded DNA is known to be covered by heterochromatin which also can be targeted by
the RNA-directed methylation. The complementary sequences of hairpin may be involved to
block the region at the stem-loop which is required in the binding of rep protein (Figure 9.6).
153
The prototype for transgenic plants resistant to geminiviruses is completed and proved to be
functional. Based on the results, three major novelties in this system are 1) the targeted control
region in Geminiviruses. 2) The universality covering ALL the Geminiviruses. 3) Inhibition of
the Rep protein of initiating the replicative cycle of Geminiviruses. In conclusion, our study
demonstrates that resistance to geminiviruses in plants can be achieved via TGS and/or PTGS by
expressing siRNA derived from non-coding viral sequences.
154
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