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Research Collection
Doctoral Thesis
A study of African cassava mosaic virus gene expression for thedevelopment of virus resistance
Author(s): Frey, Petra Maria
Publication Date: 2000
Permanent Link: https://doi.org/10.3929/ethz-a-003881275
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Diss. ETH Nr. 13 557
A study of African Cassava Mosaic Virus
gene expression for the development of
virus resistance
A dissertation submitted to the
Swiss Federal Institute of Technology Zürich
foi the degree of
Doctor of Natural Sciences
presented bv
Petra Maria Frey
Dipl. Natw. ETH
bom May L. 1972
citizen of Lanfen-Stadt. BL
accepted on the recommendation of
Prof, Dr. I. Potrykus, examiner
Prof. Dr. K. Apel, co-examiner
February 2000
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to Reto
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Acknowledgements
I would like to thank all the people who have helped me to accomplish the work
described in this thesis. Without them this work would not have been possible.
I am grateful to PD Dr. Johanna Puonti-Kaerlas, for giving me the opportunity
to work in her group, for her interest and support during this project and for care¬
fully reading this manuscript.
I would like to thank Prof. Dr. Ingo Potrvkus for accepting me as a PhD student
and for his constant support, Prof. Dr. K. Apel foi officiating as co-referent and
Prof. Dr. N. Amrhcin for his help during the last year of this thesis.
Thanks to all the past anel picsent members of the Potrvkus group and especially
of the cassava group for the help and advice, for many discussions and a friendly
working atmosphere. I especially want to thank Dr. Johannes Fiittcrcr for always
being willing to help and to discuss strange results and for his encouragement durin ^
all the bad times.
Very special thanks go to Dr. Nania Schärcr-Hernandez, for sharing with me
her enthusiasm for plant viruses, foi her constant, generous help anel interest, for
carefully correcting this manuscript, for all the good times in and out of the lab, for
the Sushi and for being such a great friend. (Grrrrrrrr!)
I am very grateful to Dr. Roland Bilang and Dr. Claudio Lupi for their friendship
and for all the lively discussions about science anel many other things. They made
working here so much more fun.
I would also like to thank PD Dr. Thomas Frischmuth and Margit Engel for
conducting the virus infection experiments in Stuttgart, anel for helpful discussions.
Christoph Lehmann (Guggu) is kindly acknowledged for being my personal
ÈvTt?X~ hotline.
Finally, special thanks go to mv parents anel to Mirjam and Matthias for their
interest in my work and their support during all mv studies and to Reto for his love,
his interest in my scientific problems and for always being thcie for me.
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Contents
1 General Introduction I
1.1 Cassava (Manihot esciilenfa Crantz) 1
1.2 African cassava mosaic disease 2
1.3 African cassava mosaic virus 3
1.3.1 Genome organization 3
1.3.2 Viral replication 5
1.3.3 Gene regulation 5
1.4 Strategics for engineering virus resistance 7
1.5 Plant genetic engineering 9
1.5.1 Regeneration 9
1.5.2 Transformation 10
1.5.3 Cassava transformation anel regeneration 10
1.6 Lucifcrasc reporter genes 11
1.7 Aim of this thesis 12
2 ACMV Promoter Analysis 14
2.1 Abstract 14
2.2 Introduction 14
2.3 Materials and methods 17
2.3.1 Plant material 17
2.3.2 Bacterial strains 17
2.3.3 Construction of plasmids 17
2.3.4 Protoplast, isolation and transformation 19
2.3.5 Agrobactenvm-mediated seedling transformation of tobacco. .
20
2.3.6 DuabLuciferasclM reporter assav 20
2.3.7 Southern blot analysis 21
2.3.8 Virus infection of transgenic plants and assay of viral DNA
replication 21
2.4 Results 22
rr
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2.4.i Expression of firefly and Rcmlla lucifcrase genes under the
control of ACMV promoters 22
2.4.2 Effect of the expression of viral ORFs on the activity of the
luciferase genes under the control of ACMV promoters ....24
2.4.3 Analysis of a short and a longer form of the DNA A promoter 27
2.4.4 Production of transgenic tobacco plants containing pACMVs
and pACMVlcaeler 28
2.5 Discussion 32
3 Developing a new ACMV resistance strategy by mimicking a hy¬
persensitive reaction using the barnase and barstar ORFs 39
3.1 Abstract 39
3.2 Introduction 40
3.3 Materials and methods 43
3.3.1 Plant material 43
3.3.2 Bacterial strains 43
3.3.3 Construction of plasmids 43
3.3.4 Tobacco mesophyll protoplast isolation, transformation and
regeneration 45
3.3.5 Agrobactcrium-mcdi&tcd seedling transformation of tobacco. .
46
3.3.6 Dual-Luciferase reporter assav 46
3.3.7 Molecular analysis 47
3.3.8 Replication assays 48
3.4 Results 49
3.4.1 Expression of active barnase can be enhanced transiently in
tobacco protoplasts 49
3.4.2 Analysis of transgenic tobacco plants containing a regulatable
barnase ORF 49
3.4.3 Viral replication in transient assays is reduced in transgenic
plants 51
3.5 Discussion 52
4 Cassava regeneration and transformation 55
4.1 Abstract 55
4.2 Introduction 55
4.3 Materials and methods 57
4.3.1 Plant material 57
4.3.2 Bacterial strains and constructs 58
m
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4.3.3 Transformation 59
4.3.4 Molecular analysis of îesistant shoots 61
4.4 Results 62
4.4.1 TMS60444 transformation and regeneration via organogenesis 62
4.4.2 MCol22 transformation and regeneration via organogenesis . .63
4.4.3 Transformation and regeneration of suspension cultures....
64
4.5 Discussion 67
5 General Discussion 70
IV
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Zusammenfassung
Maniok (Cassava, Manihot escvlenta Crantz) ist eine ausdauernde, buschige Pflanze
der Familie der Euphorbia ceae. Sie ist das Grundnahrungsmittel von über 500 Mil¬
lionen Menschen und somit eine der wichtigsten Nahrungsquellcn in den Tropen.
Eine der grössten Bedrohungen der Maniok-Produktion in Afrika ist die afrikani¬
sche Cassava-Mosaik-Krankhcit. Sic wird vom afrikanischen Cassava-Mosaik-Virus
(ACMV) verursacht, einem Geminivirus aus der Gruppe der Begomovircn. Diese
Gruppe beinhaltet Viren, welche ein ein- oder zweiterliges Genom besitzen (DNA A
und DNA B) und von weissen Fliegen überfragen werden. Es gibt zur Zeit keine voll¬
kommen virus-resistenten Manioksorten, und die einzige Möglichkeit, die Pflanzen
axrf dem Feld vor einer Infektion zu schützen, besteht darin, die Überträger mit
Chemikalien zu bekämpfen. Da die traditionelle Züchtung von Maniok schwierig
ist, könnte die Gentechnik bereits existierende Projekte zur Verbesserung von Ma¬
niok ergänzen und erweitern. Mit Hilfe der Gentechnik wurde in dieser Arbeit eine
neuartige Virusresistenz, welche auf dem regulierten Promotor des ACMV basiert,
erprobt.
Das Studium der beiden bidirektionalen viralen Promotoren mit Hilfe von zwei
verschiedenen Luciferase Genen war eine Voraussetzung für weitere Experimente.
Mit Hilfe viraler DNA A wurde eine Virus Infektion transient simuliert, womit
gezeigt werden konnte, dass der Promotor des Hüllproteins (AV-Promotoi) posi¬
tiv reguliert wird (ca. 50-fach), während der Promotor des Proteins, welches bei
der Replikation eine Rolle spielt. (AC-Promotor) negativ reguliert wird (ca. 2-fach).Dies konnte sowohl in Tabak- als auch in Maniok-Protoplastcn gezeigt werden. Der
Promotor eler DNA B ist, anders reguliert: In Tabak-Protoplasten wird die Expres¬
sion sowohl in C- wie auch in V-Richtung gleich staik erhöht, während in Maniok-
Protoplastcn die Expression in C-Richtung stärker erhöht wird als in V-Richtung.
Analoge Experimente wurden auch in transgenen Tabakpflanzcn durchgeführt, wel¬
che den ACMV Promotor mit den beiden Lucifcrase-Genen enthalten. Die Resultate
der transienten Experimente konnten dabei bestätigt werden, wobei jedoch die posi¬
tive Regulation des AV Promoters in transgcnen Pflanzen weniger ausgeprägt ist.
Aufgrund dieser Resultate wurde der ORF der Barnase, einer unspezifischen
RNAse von Bacillus arnylohqvefaciens, so klonicrt, dass er vom AV-Promotor kon¬
trolliert wird; der ORF der Barstar, des spezifischen Inhibitors eler Barnase, wird
vom AC-Promotor kontrolliert. Die einzigartige Regulation des ACMV-Promotors,
welche ausschliesslich vom Virus selbst induziert werden kann, und ein fein abge¬
stimmtes Toxin-/Anti-Toxin-Systcm sollten es möglich machen, eine hypersensitive
Reaktion zu simulieren. Da die Regeneration von transgcnen Pflanzen, welche eine
v
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vollständige Kopie dieses Konstruktes enthalten, schwierig war, wurde ein kurzer
ORF vor die Barnase eingefügt. Dies bewirkt eine zusätzliche Reduktion der Trans¬
lation der giftigen Barnase. Transgene Pflanzen wurden mit Hilfe einer „Genkanone"
auf die Menge stattfindender viraler Replikation getestet. Eine Reduktion der vira¬
len Replikation konnte bei allen transgcnen Pflanzen gezeigt werden (1 - 30% der
Replikation, welche in Wildtvp Pflanzen gemessen wurde).
Als letzer Schritt wurde versucht, bereits publizierte Transformations- und R(j-
gcnerations-Protokolle an eine afrikanische Manioksorie anzupassen, um in naher
Zukunft neue ACMV-resisternte Manioksorten zu produzieren.
vi
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Summary
Cassava (Manihot esculenta Crantz) is a perennial shrub, belonging to the family
Euphorbiaceae. It is one of the most important sources of calorics produced within
the tropics and provides food for over 500 million people. Also in Africa one of
the main staples is cassava, however, its production is now seriously threatened by
the African cassava mosaic disease. It is caused by African cassava mosaic virus
(ACMV), a gcminivirus of the Beqomovrms group, which comprises viruses having
a single or bipartite genome (DNA A and DNA B) transmitted by whitefly. Until
now there is no completelv ACMV-resistant cassava variety available, furthermore,
there is no method known to protect the plants in the field from the virus, except
by trying to control the spread of the vector using agrochemicals. Since traditional
breeding of cassava is constrained, genetic engineering could complement existing
breeding programs. Using genetic engineering, the feasibility of a novel virus resis¬
tance strategy based on the regulated ACMV promoter was assessed in this thesis.
As a prerequisite for this. ACMV promoter regulation was studied with two
different lucifcrase genes under the control of the bidirectional ACMV promoter.
Using viral DNA A for transient transformation experiments to mimic a viral in¬
fection, it could be shown that the coat protein promoter (AV promoter) is up-
regulated (around 50-fold), while the promoter of the replication associated protein
(AC promoter) is down-regulated (2-fold) by DNA A in both tobacco and cassava
protoplasts. The regulation of the DNA B promoter is different, as both v- and
c-sense expression are enhanced by the DNA A in tobacco protoplasts, while in
cassava protoplasts the expression in c-sensc was enhanced more than in v-sense.
Analogous experiments were performed in transgenic tobacco plants, containing the
ACMV-promotcr-luciferasc constructs. Transient results could be confirmed, but
the tip-regulation of the AV promoter was less pronounced.
Based on these results, the barnase ORF. producing an uuspecific ribonucleasc
of Bacillus arnyloliquefaciens. was cloned under the control of the AV promoter and
the barstar ORF, producing a specific inhibitor of the barnase, was cloned under
the control of the AC promoter. The unique properties of the regulation of the
ACMV promoter and its specificity for the virus should make it possible to mimic
a hypersensitive reaction bv using this finely tuned toxin/anti-toxin system. As
regeneration of plants containing complete copies of this construct proved difficult,
a sORF was inserted downstream of the barnase to reduce its translation. Transgenic
plants were tested in a viral replication assay using particle bombardment; for all
analyzed plants reduced viral replication (1 -- 30% of the replication in wild type
plants) could be shown.
vu
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As a last, step the transferability and adaptability of published transformation
and regeneration protocols to an African cassava cultivar were assessed in order to
allow the engineering of African cassava mosaic virus resistant cassava in the near
future.
vm
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Abbreviations
2,4-D
AC promoter
ACMD
ACMV
ATP
AV promoter
BA
BC promoter
BCTV
BMS
bp
BV promoter
CaMV
CBN
CIAT
CP
DI DNA
DIG
DNA
ds DNA
FAO
EEC
GA3
GFP
GUS
IBA
UTA
kb
kDa
LIR
MES
MPB
mRNA
NAA
NSP
ORF
2,4-clichlorophenoxy-acetic acid
DNA A promoter in c-sense
African cassava mosaic disease
African cassava mosaic virus
adenosine triphosphate
DNA A promoter in v-sensc
6-Benzylaminopurine
DNA B promoter in c-sense
beet, curiv top virus
basic MS medium
basepair
DNA B promoter in v-sense
cauliflower mosaic virus
Cassava Biotechnology Network
International renter for Tropical Agriculture, Columbia
coat protein
defective interfering DNA
digoxvgcnm
deoxyribonucleic acid
double-stranded DNA
Food and Agricultural Organization of the United Nations
friable embryogénie callus
gibberellic acid
green fluorescent protein
//-glucuronidase
indole-3-butyric acid
International Institute of Tropical Agriculture, Nigeria
kilobasc, kilobasc pairs
kilo Dalfon
large intergenic region
2-(N-morpholino)ethanesulfonic acid
movement protein
messenger RNA
O'-naphthalene acetic acid
nuclear shuttle protein
open reading frame
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PCR polymerase chain reaction
PDR pathogen derived resistance
PEG Polyethylene glycole
PIG particle inflow gun
REn replication enhancer protein
Rep replication associated protein
RIP ribosome inactivating protein
RLU relative light units
RNA ribonucleic acid
RT room tempcratur
SAR scaffold attachment region
sORF short open reading frame
ss DNA single-stranded DNA
TGMV tomato golden mosaic virus
TMV tobacco mosaic viius
TrAP ri ansactivator protein
TYLCV tomato yellow leaf curl virus
WDV wheat dwar f virus
X
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Chapter 1
General Introduction
1.1 Cassava (Manihot esculenta Crantz)
Cassava (Manihot esculenta Crantz), a member of the family Euplwrbiaceae, is a
woody perennial shrub. It originated in Latin America and was introduced to Africa
in the 16th cen+ury by Portuguese traders (Gold. 1994). Today it is cultivated world¬
wide between 30° south and 30° north of the Equator, providing basic staple food for
over 500 million people. Mostly it is grown for its tuberous roots, containing starch
up to 85% of their dry weight (Cock, 1982), but also for its protein rich leaves.
Cassava has the ability to grow on infertile soiP where no other crop could exist
without costly external inputs and therefore is the cheapest available food source in
many developing countries. It can survive seasonal drought and has the advantageof a highlv flexible harvesting time, making it, an excellent famine reserve. In Africa
it is mostly grown by small-scale farmers as a subsistence crop, providing up to 60%
of their daily caloric uptake. An average of 8 t/ha of cassava roots can be produced
in a 12-month growing season in Africa (world average is 10 t/ha) and more than
80% of the harvest rs used as food (FAO. 1989).
Due to the long culture period of cassava, various insect pests and diseases
represent serious challenges to its sustainable cultivation. It has been estimated
that around 50% or more of the potential harvest is lost every year (Roca et ab,
1992) as a result of viral diseases, bacterial blight disease, mealybugs and green
mites or other pests. Furthermore, cassava transport, storage and marketing is
complicated by postharvest deterioration. Roots can be kept on growing plants
long after the first harvest, but once harvested thev must, be processed within 2-7
days (Wenham, L995). Another constraint to cassava use is its cyanogenic nature.
To prevent cyanide poisoning, cyanogenic glucosides have to be removed and using
inadequately processed cassava can have fatal consequences (Akintowa el ah, 1993).
1
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CHAPTER 1. GENERAL INTRODUCTION 2
Attempts to solve these problems bv traditional breeding of cassava plants have
had limited success. Traditional breeding in cassava is restricted due to cassava
being a highly heterozygous, allopolyploid plant with low natural fertility in many
cultivars. In addition the lack of icsistance genes in compatible germplasm further
complicates breeding improved cassava varieties. Biotechnology, therefore, could
provide an alternative approach and could complement efforts in traditional breeding
to contribute to a more profitable and sustainable agriculture in Africa by developing
improved cassava varieties.
1.2 African cassava mosaic disease
African cassava mosaic disease (ACMD), rated the most serious vector-borne plant
disease in Africa (Geddes, 1990). is a severe constraint, to cassava cultivation and
is an ideal target, for an approach combining genetic engineering and traditional
breeding. The disease is caused bv African cassava mosaic virus (ACMV), Avhich is
transmitted by whitcflies (Benri^/a tabaci) feeding on cassava leaves (Swanson and
Harrison, 1994). The viruses are able to persist in their insect vectors for many days
or even for the life of the insect, but, they do not replicate in their vectors (Shaw,
1996a). ACMV can also be transmitted during propagation via infected tools and
planting material, but, not via seed (Swanson and Harrison, 1994). Typical symp¬
toms of ACMD are the mosaic bleaching of the leaves, leaf deformation, stunted
groAvth and the loss of storage root formation. ACMD can cause local losses of
up to 80%, with losses throughout Africa reaching 36% of total cassava production
(Fargette et ab, 1988). The onlv way to protect, the plants in the field from being
infected is to control the spread of the whitcflies bv insecticides but, since whitcflies
have overlapping generations and move between cassava and weed hosts, they arc
not, an easy target (Fishpool anel Durban. 1994), Frequent spraying, which is envi¬
ronmentally unsound and too expensive for subsistence farmers, would be necessary.
Growth reduction of cultivated plants is not directly related to the yield loss due to
viral infection; this slows and complicates traditional bleeding, as evaluation of new
cultivars can only be done at the end of a growing season. To elate no completely
resistant cultivars haAT been produced by traditional breeding (Hahn et ab, L980;
Thresh and Otim-Nape, 1994), but in Uganda the use of improved cultivars and
virus-free planting material have secured and enhanced yields (Bock, 1994; Otim-
Nape et ab. 1994a; Fargette et ab. 1996: Otim-Nape et ab. 1997). Disease-frcc
planting material could increase vields Iavo to threefold (Lozano and Booth, 1974):but, due to the long groAving season of cassava, under high infection pressure, plants
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CHAPTER L GENERAL INTRODUCTION 3
might be reinfected and the benefits of disease-free plants might be short-lived.
1.3 African cassava mosaic virus
The African cassava mosaic virus (ACMV, earlier called the cassava latent virus)is a Begomovirus of the family Gemimvindae (Frischmuth and Stanley, 1991; La-
zarowitz, 1992; Bisaro, 1996). Geminiviruses have attracted considerable interest
as pathogens attacking manv economically important crop plants worldAvide. They
are transmitted by insect, vectors to a variety of monocotylcdonous and dicotyle¬
donous plants. The geminivirus family is divided into three subgroups depend¬
ing on their genome structure and host range (Bisaro, 1996; Shaw, 1996b; Palmer
and Rybicki, 1997): The leafhoppcr-transmltted Mastreviruses (formerly subgroup
I viruses) like the wheat dwaif virus (WDV). the leaf- and grasshopper-transmitted
Curtoviruses (formerly subgroup II viruses) like the beet curly top virus (BCTV)and the Avhitefly-transmitted Begomouirines (fonneily subgroup III viruses). Mas¬
treviruses and Curtoviruses have onlv a single genomic component of approximately
2.7 kb; Bcgomovirvses mav have one or two components of the same size, one of
which is dependent on the other for replication.
1.3.1 Genome organization
Only two groups of plant viruses are known to contain DNA genomes: the pararetro-
viruscs and the geminiviruses. Unlike that of the pararetroviruscs, the genome of
the geminiviruses consists of one or two covalently closed, single-stranded DNA
molecules (Stanley and Gay. 1983) denoted DNA A and B. For bipartite viruses,
both components were sliOAvn to be required for systemic infection (Stanley, 1983).
Comparison of the DNA A and B has revealed a region of approximately 200
nucleotides with almost, identical sequences. The 5"-end of this common region has
been designated as the zero map unit of DNA A and B (Stanley, 1985). This
commorr region, AAdiich is located in the large inteigcnic region (LIR), is completely
different in its sequence among the diffeieut geminivirus strains with the exception
of a sequence element, of about 30 nucleotides. This element can form a hairpin
structure consisting of a GC-rich stem and an AT-rich loop (LazaroAvitz. 1992).
This structure contains the nonamer motif TAATATTAC. which is identical in all
geminiviruses (Laufs et ab. 1995a).
The seven genes, first identified as open reading frames (ORFs) and named ac¬
cording to their location on the genome anel direction of transcription (Stanley and
Gay, L983) and now renamed according to function, have had functions ascribed to
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CHAPTER J. GENERAL INTRODUCTION 4
Figure 1.1: Genome map of ACMV DNA A and DNA B. The DNA is represented
by a thin line with the two arrowheads showing the extent of the large iutergenic region
(LIR) and the main ORFs are indicated bv thick airows. See text for details on gene
names.
them through the construction of mutants and theii analysis in infected and in trans¬
genic plants (sec Fig. [1.1]). The position of the ORFs relative to tue LIR. as well
as the polyadenvlation signals located on the opposite side of the genome (Buck,
1994), suggest that transcription occuis bidireetionally from the LIR (Townsendet al., 1985), The DNA A contains genes leqmred for encapsidation (CP or AVE
the coat protein gene), for viral replication (Rep or ACl, the replication associated
protein gene) and two regulator proteins (TrAP oi AC2, a replication transactiva tor
protein and REn or AC3, a replication enhancer protein), required for gene reg¬
ulation and for efficient replication (Townsend et ab, 1985; Etessami et ab, 1991;
Morris et, ab, 1991). The DNA B contains 1aa*o genes required for systemic movement
in the host plant (MPB or BCl. responsible for cell-to-cell movement and NSP or
BV1, a nuclear shuttle protein) and is probably responsible for symptom production
(Morris et ab. 1991; von Arnim et ab, 1993; Haley et ab, 1995; Sanclerfoot et ab,
1996; Ward et ab, 1997). A corresponding protein for AV2 has been reported in
tomato leaf curl virus but not foi ACMV (Padidam et ab, 1996). This ORF is only-
present in Old World begomoviruses (Hamilton et al., 1984). A major anel a minor
transcription start site have been mapped upstream of the coat protein and only
transcription from the major transcription start site would allow translation of the
AV2 protein (Davies et ab. 1987). Synthesis of AV2 would require ribosomes to
initiate on a very short leader of about 8 nucleotides, which might be expected to
result in a rather Ioav level of expression (Kozak, 1983). On the other hand, the coat
protein transcript, from the major transcription start site has a 160 bp untranslated
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CHAPTER 1. GENERAL INTRODUCTION b
leader, containing four AUG triplets. tAvo in phase Avith the AV2 ORF and tAvo in
phase with termination codons upstream of the coat piotein (Stanley, 1985). Of
these only the coat protein initiation codon shows the preferred A at position -3
(Kozak, 1984), which mav be important, in its selective recognition (Davies et ab,
1987). So far little is known about the regulation of CP expression using the full
length promoter,
1.3.2 Viral replication
Geminivirus DNA has been shown to leplicatc through a circular double-stranded
(ds) DNA intermediate (Ikegami et ab. 1981) via a rolling-circle mechanism (La-
zarowitz, 1992). Apart boni a single viral-encoded protein, Rep, which is required
for DNA A replication in eis and for DNA B leplication in trans, replication re¬
lics entirely on the enzymes of the host plant. Knowm DNA polymerases have no
homology with the Rep protein, but Rep mav be related to proteins responsible
for DNA replication initiation of ss DNA plasmids (Ilyina and Koonin. 1992). The
rolling-circle replication is initiated by the Rep protein, which serves as the origin
recognition protein and as a site-specific endonuclease (Laufs et ab, 1995b; Orozco
and tlanley-Boweloin. 1996). The origin of replication has been mapped to the
conserved nonamer motif (TAATATT4-AC) Avitfiin the LIR (Hcyraud et ab, 1993),
the stem-loop structure of AAdiich is essential for replication as the Rep protein can
only nick ss DNA (Fontes et ab, 1994). Affei nicking, the polymerization of viral
strand DNA docs not only require Rep protein, but also host DNA polymerases,
that normally arc only active during S-phase of the cell cycle. As geminiviruses
infect differentiated cells, they have to re-activate cells to enter cell cycle. Con¬
sidering that the Rep protein possesses an ATP binding and hydrolyzing activity
and that Rep proteins of a wheat ebvaif geminivirus have been shown to form sta¬
ble complexes with proteins of the retinoblastoma family, it, can be speculated that
geminiviruses can modulate plant cell cycle (Laufs et ab. 1995a; Collin et ab, 1996).
Once a full-length copy of the viral DNA has been produced, it is cut anel ligated
to circular ss DNA. This new ss DNA can act as a template for further replication,
after complementary-sense DNA synthesis, or can be encapsidated and transportée!
out of the cell.
1.3.3 Gene regulation
During the viral infection cvcle. different gene products will be required at different
time points and in elifierent quantities. Viral RNAs aie polvadcnvlated and initiate
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CilAPTER 1. GENERAL INTRODUCTION 6
downstream of TATA box motifs or initiator elements, which indicates that they
are transcribed by host RNA polymerase TL The regulation of transcription relies
on viral proteins as well as on host factors (Sunter and Bisaro, 1997).The replication-associated protein is not, onlv responsible for replication, the
N-tcrminus also acts as a primary regulator of its OAvn promoter. It, can reduce
complementary-sense transcription (Halev et ab. 1992; Hong and Stanley, 1995)
by binding between the TATA-box anel the transcription start site of its own gene
(Sunter et ab, 1993: Eagle and llanlev-BoAvdom, 1997: Gladfelter et ab, 1997). Re¬
duced c-sense transcription from the DNA B was observed for ACMV (Haley et ab,
1992), but not for tomato golden mosaic virus (TGMV), even though DNA A and B
promoters are similar also in TGMV. A possible explanation for this discrepancy is
the presence of a downstream transcription start site in the TGMV DNA B, which
allows the MPB gene to escape repression (Suntei et ab, 1993).TrAP is a nuclear protein (Sanderfoot and LazaroAvitz, 1995) that transactivatcs
virion-sense gene expression in the Begomovirus group (Sunter and Bisaro, 1991; Ha¬
ley et ab, 1992) at the level of tianscription (Sunter and Bisaro, 1992). In transgenic
plants containing AV promotcr-GUS fusions it could be shown, that TGMV TrAP
regulates expression through two different mechanisms: activating the promoter in
mesophyll cells and derepressing the piomotcr in phloem tissues (Sunter and Bisaro,
1997). Also TrAP of ACMV can enhance expression of transgenes under the control
of an AV promoter in transgenic plants (Hong et ab. 1996). Therefore, TrAP is able
to interact with a viral promoter integrated in the plant genome and might also
transactivate host genes during the infection caxIc. Little is known about the actual
mechanism of activation by TrAP, although it was demonstrated that it strongly
prefers binding ss DNA over ds DNA (Noris et ab, L996) and that both activities
are sequence independent. Further analysis of TrAP revealed a basic N-terminus,
an acidic C-terminus and a potential zinc-fingei motif. AAdiich might mediate DNA
binding (Sunter and Bisaro, 1992). The lack of sequence specificity is inconsistent
with TrAP's ability to specifically activate v-sense transcription. It has been spec¬
ulated that specific DNA binding requires post-tiauslational modifications or that
TrAP interacts with other proteins of the basal transcription apparatus to activate
RNA synthesis (Liu and Green, 1994: Noris et ab, 1996; Hanley-Bowdoin et ab,
1999). Also REn transactivatcs virion sense expiession at the level of transcription,
but, the effect is less pronounceel (Halev et ab. 1992). The mode of action here is
not known.
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CHAPTER 1. GENERAL INTRODUCTION 7
1.4 Strategies for engineering virus resistance
Virus infections in plants can be controlled and suppressed through genes occurring
naturally within the plant or the virus. Advances in genetic engineering have led
to the development of different strategies to combat virus infections by manipulat¬
ing these natural processes and engineering a 'pathogen derived resistance1 (PDR)
(Sanford and Johnson. 1985). The idea originated from the observation of cross-
protection: a plant inoculated with one strain mav become resistant to a second
infection by a related strain of the same virus. This Avas used as a strategy of biolog¬
ical control, in spite of the risk of Afield losses due to the mild infection or of a severe
disease produced by the interaction between the mild strain and another virus. As a
result of these observations various stiatcgies based on PDR Avere developed. Genes
that confer viral PDR include those for coat pi olefins (Powell et ab, 1986). replicases
(Carr and Zaitlin, 1993). movement proteins (Beck et ab, 1994; üuan et ab, 1997)and for different untranslated nucleic acids (Hcmenway et ab, L988; Stanley, 1990:
Stanley et ab, 1990; Frisedimuth anel Stanley. 1991: Yepes et ab, 1996; Beachy, 1997:
Bendahmanc and Groenborn. 1997). Usually thes^ strategies arc more successful
for RNA viruses than for DNA viruses. More recently, new strategics, Avhich aie
not directly pathogen relateeb have been developed, using ribozymes or ribosome
inactivating proteins (Lodge et ab. 1993: Hong et ab, 1996; Hong et ab, 1997; Yang
et ab, 1997).
The first PDR was reported in the form of a coat protcin-meeliated resistance
(PoAA'ell et ab, 1986) that protects the plant from virus infection without the risk
of yield reduction. Since then there have been a number of examples of engineered
resistance in a variety of crops like tomato, potato, cucumber, rice and against,
viruses of different families (Beachy et ab. 1990: Fitchen and Beachy, 1993). Different
mechanisms may lead to coat, protein-mediated resistance, but usually the coat
protein prevents the disassembly of the viral capsid, an early event of the viral
infection (Beachy, 1997). This mechanism is supported by the fact that mostlv
coat protein-mediated resistance can be overcome by the inoculation of plants with
naked RNA (Powell et ab. 1986). Most, publications rcpoit effective resistance to
RNA viruses, Avhilc coat, protein-mediated resistance against DNA viruses is still
rare (Kunik et ab, 1994: Smistcrra et al.. 1999).
Sequences of complete or partial viral replicase proteins can confer a high level
of resistance to the virus. Rcplicasc-mediatcd resistance was first described against
tobacco mosaic virus (TMY) in transgenic plants expiessing a fragment of the repli¬
case. Although no protein fragment could be detected, plants Avere resistant. In
this case, the active molecule seems to be the produced mRNA (Carr and Zaitlin,
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CHAPTER 1. GENERAL INTRODUCTION 8
1993). A similar approach provided resistance against a, tomato yelloAv leaf cuil
virus (TYLCV), which is a DNA geminivirus (Bendahmane and Grocnborn. 1997).The mechanism involved in replicasc-meeliated resistance has not been elucidated,
but it is speculated, that the expressed fragment interferes with viral replication. In
contrast to coat protein-mediated virus resistance, resistance can not be overcome
by inoculation with naked RNA. since the resistance is at the level of replication and
not at the level of uncoating. The replieasc-niediated resistance is quite specific.
Movement proteins enable viruses to move from cell to cell or spread SArstemically
in the plant by interacting with the cytoskcleton or plasmodesmata (Koonin et ab,
1991; Heinlein et ab, 1996: Sanderfoot and LazaroAvitz, 1996). Both in the case of
DNA and RNA viruses, mutated movement pioteins (expressed in plants can confer
variable levels of resistance against different related viruses (Beck et ah, 1994; Duan
et ab, 1997).
A variety of PDR stiatcgies are based on the expression of nucleic acids which
do not encode proteins. One of these approaches is the expression of antisense RNA
to reduce the replication of RNA and e\-en DNA AÙruses. Virus resistance in trans¬
genic model plants ranges from very Ioav to almost total inhibition of viral infection
(Hemenway et ab, 1988: Yepes et ab, 1996: Bendahmane anel Groenborn. 1997).Resistance can be clue to different mechanisms, cither to formation and subsequent
degradation of double-stranded RNA. to direct reduction of virus replication or gene
expression by high levels of antisense RNA or to gene silencing (Beachy, 1997). An¬
other approach is the use of defective interfering (DI) DNA (Stanley, 1990; Stanley
et ab. 1990; Frischmuth and Stanley. 1991: Fnschmuth et ab, 1997a). DI DNAs
can be found in plants during natural viral infection. They are partial genomes of
the virus which can redirect, replication from the viral genomes, thereby decreas¬
ing amounts of replicated viral DNA and at the same time reducing spread and
symptoms of the virus in the plants.
Other possible strategies for engineering viius resistance in plants include ribo-
zyme-mediated resistance (Yang et ab. 1997), where small RNA molecules with a
catalytic activity can specifically cleave viral RNA. Another kind of resistance is
conferred by an RNA satellite (Harrison et ab. 1987). RNA satellites do not have
any homology to the virus but thev arc replicated and encapsidated with the helpof the virus. Furthermore, the use of ribosome inactivating proteins (RIPs) has
shown to be a successful way of engineering virus resistance in plants (Lodge et ab,
1993; Hong et ab. 1996: Hong et ab. 1997). RIPs are naturally occurring plant
proteins, which have antiviral activity against a broad spectrum of plant viruses.
They catalyze the depurination of a specific adenine residue of the cucarvotic large
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CHAPTER 1. GENERAL INTRODUCTION 9
subunit, ribosomal RNA, thereby blocking translation (Taylor et ab, 1994). The
activation of RIP expression during pathogen attack and release of RIPs into the
cytosol is speculated to lead to local cell death, which prevents replication anel spread
of the virus (Bonness et ab. 1994).
Engineering ACMV resistance would complement efforts in traditional breeding
and could provide a sustainable solution securing cassava yields. There arc indi¬
cations from the model plant Nicotiana bentharmana that, genetic engineering of
ACMV resistance is feasible. A number of transgenic model plants with a variable
increase in ACMV resistance have been lcported: Constitutive expression of de¬
fective viral DNA (Stanley and lAnvnsend. 19S5; Stanley et ah, 1990), of antisense
Rep gene (Day et ab, 1991). of defective Rep piotein (Hong and StanleA-, 1996), of
defective movement proteins (von Arnim and Stanley, 1992; Duan et ab, 1997). of
coat protein (Kunik et ab, 1994) and the virus-induced expression of dianthin in
stably transformed plants (Hong et ab. 1996). This suggests that resistant cassava
cultivars using analogous strategies could be obtained in the near future.
1.5 Plant genetic engineering
In order to apply biotechnology in plants, a regeneration system with a compati¬
ble transformation system that alloAvs regeneration of transgenic plants is required.
Plant cells are considered to be totipotent, and thus able to regenerate whole plants
from a single cell. This ability is, hoAATA"er. often limited to certain tissues anel devel¬
opmental stages. Further, an efficient transformation svstem, compatible with the
regeneration system, and a system for identifying and selecting transformed cells are
necessary.
1.5.1 Regeneration
Different regeneration systems have been reported for cassava using a variety of
starting materials (Roca. 1984: Puonti-Kaerlas. 1998). One of the earliest reports is
of a meristcm culture (Kartha, 1974) that can be used to obtain planting material
free of viruses and diseases (Kartha anel Gamborg. 1975). Now meristems can also
be used to multiply improved cassava varieties by inducing multiple shoot formation
on cytokinin-containing medium (Roca. 1984: Konan et ab. 1994). The most reliable
de novo regeneration system for cassava has been through somatic embryogenesis
and cyclic somatic embivogenesis (Stamp and Henshaw, 1982; Stamp and Henshaw,
1987a; Stamp anel Henshaw. 1987b) using different auxin-containing media to induce
primary embryogenesis in various cultivars. Maturing somatic embryos can produce
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CHAPTER 1. GENERAL INTRODUCTION 10
friable embryogénie callus that can be used to establish suspension cultures (Tayloret ab, 1996). Alternatively, somatic embrvos can be germinated and the developing
cotyledons can be used for direct induction of shoot primorclia (Li et al., 1998).
1.5.2 Transformation
Gene transfer to plant cells can be obtained bv two different systems: direct gene
transfer or Agrobacterium-modràtcd gene transfei. The first plants expressing for¬
eign genes were tobacco plants transfoimed with Aqrobactcrium tumcfaciens (Baiton
et ab, 1983; Horsch et ab, 1985: DeBlock et ab, 1987). Aqrobactcrium has the unique
ability of transferring genes to eucarvotes in nature. This has been taken advan¬
tage of in genetic engineering bv using modified Aqrobactcrium, vectors to transfer
genes of interest to plant cells. Direct gene transfer methods, such as microin¬
jection, PEG treatment, calcium-phosphate precipitation or electroporafion mostly
need specific protocols to regenerate fertile plants from protoplasts (PaszkoAvskiet ab, 1984; Paszkowski et ab, 1986: Potrvkus. L991). Direct gene transfer through
bombarding small DNA coated paitides to intact cells can be used to transform
organized tissues (Klein et ab, 19S8). Under optimum conditions particles can pen¬
etrate the cell Avail and release the DNA for expiession and/or integration in the
plant genome. J. Finer et ab developed the svstem fmthcr and constructed the
Particle InfloAv Gun (PIG: (Finer et ab. 1992)). which is inexpensive anel simple
to use. Microtargeting can be particularly used to target a small, precise area of
organized tissue (Sautter et ab, 1991).As stable transformation frequencies are Ioav, different marker genes to identify
and select stably transformed cells are necessary. Usually an antibiotic resistance
gene is introduced to select for transformed cells. alloAving the cells containing the
transgenc to survive and proliferate. In some species (e.g. Dendrobivm) selection
with antibiotics mav not be possible, in this case a visual marker mav be used (Chia
et ab, 1994). ß-glucuronidase (GUS) (Jefferson. 1987), luciferasc (Ow et ab, 1986;
Mayeihofcr et ab, 1995) or green fluorescent piotein (GFP) (Sheen et ab, 1995)
may also be used to monitor transient transformation and to partially optimize
transformation conditions. While GUS assays aie lethal, both lucifcrase and GFP
expression can be studied in living material.
1.5.3 Cassava transformation and regeneration
Cassava regeneration and transient transformation have been reported using many
different, methods and taiget materials, but until recently no compatible systems
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CHAPTER 1. GENERAL INTRODUCTION U
had been reported. Now first transgenic cassava plants have been produced using
different methods for regeneration and transformation (Li et ab, L996; Schöpkc
et ab, 1996; Gonzalez et ab, 1998). One svstem makes use of friable embryogénie
callus as starting material. Hie neAV cmbrvogenic units appear to be of single cell
origin, which makes them a good target for transformation (Taylor et ab, 1996).After transformation of embryogénie suspensions, transgenic plants of cassava could
be regenerated by selection for resistance to paromomycin (Gonzalez et ab, 1998)or using the visual selection system AArith firefly lucifciase (Raemakers et ab. 1996).The other system uses cotvledon expiants as stalling material and regenerates shoots
via organogenesis. Since shoots regcneiated via organogenesis develop mostly from
the cells close to the cut edges of the cotyledons, it makes this system ideal for
Agrobacterium-mcdxated transformation. Transgenic cassava shoots can then be
regenerated using hygromvein as a seleetiw agent (Li et ab. 1996). The advantage
of this system is the short amount of time neccssaiv in in vitro culture, which reduces
the risk of soma clonal variation.
1.6 Luciferase reporter genes
Reporter genes arc rourinelv used for transient studies of promoter regulation and
gene expression (Oav et ab, 1987: Maitin et ab. 1992; Arias-Garzon and Sayrc, 1993;
Freeman et ab, 1994; Srikantha et ab, 1996; Needham et ab, 1998). Studies of
gene and promoter regulation in ACMV Avere performed using the GUS reporter
gene svstem (Zhan et ab, 1991: Halev et ab, 1992). but since then new, improved,
reporter systems have been established. One commonly used is the firefly lucifcrase
gene and since recently this can be combined with a Rcnilla lucifcrase to measure
two different reactions in a single extract.
Firefly luciferase of the common Noith American firefly Photmus pymhs is the
most commonly used of the bioluminescent reporters (Oav et ab. 1986; de Wet, et ab,
1987). The luciferase is a monomelic enzvme of 61 kDa, which catalyzes a two-
stcp oxidation reaction emitting light in the vellow to green region (550-570 nm).
ALP, oxygen and luciferin are required as substrates. In order to generate a stable
luminescence signal, instead of an initial burst of light, coenzyme A is incorporated
in the assay solution to yield maximal luminescence intensity that slowly decays
over several minutes. None of these compounds aie toxic for plant cells and assavs
can be performed in vivo. Firefly luciferase assavs aie verv sensitive, allowing the
quantification of less than JO-20 moles of enzvme (Technical manual. Promega).Rcnilla lucifcrase, of the soft coral Renilla remformis, is a monomeric enzyme of
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CHAPTER L GENERAL INTRODUCTION 12
31 kDa that catalyzes the oxidation of coelenterazine to yield coelentcramide and
blue light of 480 nm (Matthcnvs et ab, 1977; Maverhofer et ab, 1995). A limitation
of the Renilla luciferase is the presence of a Ioav level of nonenzymatic luminescence,
which reduces assay sensitivity bv about L0-fold compared with the firefly luciferase
assay.
With the Dual-Luciferase1" reporter assav it is possible to measure both lu-
ciferases individually within a single svstem. Since the firefly and the Rcnilla lu-
ciferascs are of distinct evolutionary origins and therefore have different enzyme
structures and different substrates, it is possible to discriminate between the two
reactions. The luminescence of the fiicflv lucifcrase can be quenched Avhile at the
same time the Rcnilla luciferase is actbvated bv its substrate. With these two genes,
it is now possible to measure the activity of two different promoters in a single rapid
assay with high sensitivity.
1.7 Aim of this thesis
African cassava mosaic virus disease is a major tin eat to African cassava production
despite efforts in traditional cassava breeding and genetic engineering of vims resis¬
tance in tobacco model plants. Engineering ACMV resistance is of high priority in
the genetic improvement of cassava. This will contribute to food security and sus¬
tainable agriculture in Africa and at the same time provide important, information
on both host-pathogen relationships and on virus piomoter regulation, ddic aim
of this thesis was to assess and develop ucav techniques and strategies to produce
ACMV resistant cassava.
The properties of regulation of the ACMV promoter make it possible to mimic
a hypersensitive reaction bv using a finely tuned toxin/anti-toxin system. The two
genes chosen for our system are the Bacillus amylohquefaciens barnase and its spe¬
cific inhibitor the barstar.
Tobacco Avas selected as a model plant because of the ease of its transformation
(as compared to cassava) and because it has been aheadv used in other ACMV
resistance strategies to studv ACMV promoter regulation in transiently and stably
transformed plants. For promoter regulation studies, the Dual-LuciferaselH reporter
assay was used, in order to measure the activity of Iavo different genes (v- anel c-sense
expression of the ACMV promoter) in a single rapid assay A transient assay system
using transformed cassava protoplasts Avas developed to test promoter regulation,not just in a model plant, but also in the nattual host of the virus. After the
promising results of the transient studies. ACMV-promofei -barnase constructs were
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CHAPTER 1. GENERAL INTRODUCTION 13
transformed into tobacco to assess virus resistance. Transgenic plants were tested
using a replication assay to sIioav reduced viral replication in the transgenic plants
as compared to wildtype plants.
With the recently developed cassava transformation system, ACMV resistant
cassava can be produced. The transferability and adaptability of the current cassava
transformation and regeneiation Avere assessed for an African cultivar using the
tested ACVIV-promoter-barnase constructs. As cassava is grown as many local
cultivars it is crucial to adapt transformation to as many cultivars as possible in
order to engineer ACMV resistance in local varieties of cassava.
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Chapter 2
A ^~i T\ TT T ~W~\ À Al®
ACMV Promoter Analysis
2.1 Abstract
African Cassava Mosaic Virus (ACMV) DNA A and DNA B promoters
were cloned together with the firefly luciferase and the Renilla luciferase
to study the regulation of viral genes. The relative activity of both sHes
of each promoter was tested in tobacco and cassava protoplasts and for
the DNA A promoter also in transgenic tobacco plants. Regulation of
the promoters was assessed by cotransforming individual ACMV open
reading frames (ORFs) or the complete DNA A with the luciferase con¬
structs. The transactivator protein (TrAP) activates coat protein (CP)and nuclear shuttle protein (NSP) gene expression in both cassava and
tobacco protoplasts, but not in transgenic tobacco plants, while DNA A
does activate CP expression also in transgenic plants. At the same time
DNA A reduces the expression of the replication-associated protein (Rep)but activates the expression of the movement protein (AIPB) in tobacco
and relatively more so in cassava. These results indicate that by using
this promoter, a new virus resistance strategy mimicking a hypersensitive
reaction could be designed.
2.2 Introduction
African cassava mosaic virus (ACMV) is a member of the Geminiviridae, a diverse
family of plant infectious agents characterized bv their circular single-stranded DNA
(ssDNA) cncapsidated in double icosahcdral paitides (Jlanlcy-BoAvdoin ct, ah, 1999;
LazaroAvitz, 1992; Stanley. L9S5). It, belongs to the Begomoviruses, which comprise
viruses transmitted bv Avhitefly having a mono- 01 bipartite genome (DNA A and
L4
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CHAPTER 2. ACMV PROMOTER JiNALYSIS 15
Figure 2.1: Genome map of ACMV DNA A and DNA B. The DNA is represented
by a thin line with the two arrowheads showing the extent of the large intergcnic region
(LIR) and the main viral ORFs arc indicated by thick arrows. See text, for details on gene
names.
DNA B). ACMV replicates in the nuclei of host cells through double stranded DNA
intermediates via a rolling circle mechanism and it recruits most proteins of the
replication machinery from its hosts. The coat protein and all viral functions re¬
quired for replication arc encoded on the DNA A: CP. the coat protein (ToAvnscndet ab, 1985): Rep, the replication-associated protein (Etessami et ab, 1991), TrAP
(transactivator protein) and REn (replication enhancer protein), regulator proteins
(Halev et ab, 1992; Etessami et ab. 1991). DNA A. therefore, is capable of au¬
tonomous replication and encapsidation but is unable to infect, plants systemically
(Townsend et ab, 1986). NSP (nuclear shuttle piotein) and MPB (moving protein),the two genes positioned on DNA B. provide functions for virus movement (vonArnim et ab, 1993; Haley et ab, 1995). Both genomic components, DNA A and B,
arc required for infectivity (Stanley, 1983).
The arrangement of the open reading fiâmes (ORFs) of the DNA A and B (Fig.
[2.1]) shows that, they aie expressed in a bidiiectional maimer (Townsend et ab,
1985). The ORFs on both DNA A and B aie arranged similarly with divergent
transcription units separated by a large infeigenie legion that is highly conserved
between the tAA'o DNAs (Stanley and Gav. 1983). This segment also contains a
~30 nt sequence Avith the potential to foim a stem-loop structure that is conserved
among bipartite geminiviruses and has been associated with the start of rolling circle
replication (Lazarovritz. 1992). Fragments of DNA A (nucleotides 2759 to 282) and
DNA B (nucleotides 2705 to 5S1) have been shoAvn to act as inducible promoters
with relatively Ioav basal activity (further on referred to as AC and AV promoter for
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CHAPTER 2. ACMV PROMOTER ANALYSIS 16
the DNA A in c- and v-sensc and BC and BV promoter for the DNA B promoters in
c- and v-sense) (Zhan et ab. 1991: Halev et ab. 1992). Studies of promoter regulation
have shown that, the AV, the BV and the BC promoters can be activated by TrAP
while the AC promoter is repressed bv its own gene product (Haley et, ab, 1992;
Hong and Stanley, 1995; Sunter and Bisaro. 1997). Both activation and repression
occur at the level of transcription (Sunter and Bisaro. 1992; Sunter et ab, 1993). Rep
is known to bind between the TATA-box and the transcription start site, thereby
probably hindering its own transcription by interfering Avith the RNA polymerase
(Haley et ab, 1992; Sunter et ab, 1993; Hong and Stanley, 1995). Up to elate there
is little information available on the molecular mechanisms responsible for promoter
activation by TrAP (Halev et ab. 1992; Suntei and Bisaro. 1997).
All published data on promoter regulation has been obtained from transient
expression experiments using only the u/clA (GUS) reporter gene (Jefferson et, ab,
1987). This has the disadvantage that only one promoter could be studied in each
experiment. The use of the firefly and the Rcnilla luciferase now permit the discrim¬
ination between the two reactions in a single extract and thereby make if possible to
measure t/wo different promoter acthaties in one experiment (Hannah et ab, 1998).This is due to the fact that the firefly and the Rcnilla lucifcrases are of distinct
ewjlutionary origins and therefore have differ en t enzvme structures and different
substrates. Moreover, both luciferases can be detected at Ioav levels in a fast and
simple assay.
Here we report the analysis of the activities of the ACMV promoters by quan¬
tifying transient expression of the firefly and Rcnilla lucifcrase reporter genes using
the Dual-Luciferase repoiter assay foi the first time in plant cells. We examined
the expression from both sides of the ACMV DNA A and DNA B promoters and the
regulation of these promoters by Rep and TiVP and the complete DNA A, simulat¬
ing a viral infection. To studv viral promotei activity in planta, transgenic plants
containing the luciferase constructs aatic infected Avith a geminivirus and analyzed.
The characteristics of the regulation of the bidirectional ACMV promoter indicate
that it might be a useful tool for the creation of a virus resistance strategy mimicking
hypersensitivity.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 17
2.3 Materials and methods
2.3.1 Plant material
Cassava (Manihot esculenta Crantz) Avas maintained as embryogénie suspension cul¬
tures. These Avere established from friable embryogénie calti of cultivar TMS 60444.
The suspensions were groAvn axcnicallv in 25 ml of modified liquid SH medium
(Schenk and Hildebrandt. 1972: Taylor et ab. 1996). at 30°C under continuous Ioav
light in a growth chamber (Infors Incnbatoi LIL04) on a rotary shaker (90 rpm). All
suspensions were subculturcd once a week.
Tobacco, Nicotian a tabacum L, cv. Petit Havanna Str-rl (SRI; (Maliga et ab,
1973)), was maintained as axenic shoot cultures. These were grown in vitro at 26DC
with a 16/8 h photoperiod on half-strength MS medium without growth regula¬
tors (Murashige and Skoog. 1962). SRI tobacco seedlings used for Agrobactervum-
mediated transformation were grown axcnicallv on filter paper wetted with sterile
water.
2.3.2 Bacterial strains
E. coli strain XLl-Blue was purchased from Stratagcnc (La Jolla, CA, USA). A.
turnefaciens strain GV3101, containing helper plasmid pGV2260 (Deblaere et ab,
1985) was kindly provided bv B. Tinland. Zürich.
2.3.3 Construction of plasmids
The plasmid pACMVA is a partial repeat (l.Smer) of DNA A (West Kenyan isolate
844 (Stanley and Gay, 1983)) in pBS KS- (Stratagcnc). The plasmid pACMVB is
a tandem repeat of DNA B (West Kenyan isolate 844 (Stanley anel Gay, 1983)) in
pAT153. The cloning of pACMVA and pACMVB has been described (Klinkenberget ab. 1989). Both plasmids were kindly provided by T. Fiischmuth, Stuttgart. The
ACMV ORFs Rep anel TiAP constructs (Halev et al.. 1992) were kindly provided
by B. Morris, Ncav Zealand.
All DNA manipulations were performed according to standard procedures (Ma-niatis et ab, 1982). Nucleotide numbering of the ACMV genome refcis to p.IS092
and p.JS094 (Stanley and Davies, 19S5). DNA A. DNA B and names of the open
reading frames arc according to suggested nomenclature (Davies and Stanley, 1989).In case the gene function is knoAvn. the name of the gene product is indicated, lire
bidirectional ACMV promoteis Avere produced bv PCR amplification of ACMV DNA
using primers ACMVc (GGAA -2755- GCTTTTTGACCAAGTCAATTGG -2776)
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CHAPTER 2. ACMV PROMOTER ANALYSIS 18
and ACMVs (300-GTGGTACCCACTATTGCGCACTAGC -267) for the short ver¬
sion of the DNA A promoter, primers ACMVe and ACMVl (GGGGTACC -440-
AGCCCTGATAACTGAG -425) for the longei DNA A promoter. The DNA B
promoter (Zhan et ab, 1991) was amplified with primers ACMVBL (ATCCCGT-581- CAATGTATATACTTCC -564) and ACMVBR (GTTAGGCCATGG -2265-
TTCAACACTTTGAGTA1AAGC -2286). All versions of pACMV were obtained
by introducing the ACMV piomoters as Kpnl/Munl fragments in between the tAvo
tobacco scaffold attachment regions (SAR: (BiCA-ne et ab, 1992)) in the backbone of
pGluChi (Bliffeld et ab, 1999). The fireflv lucifeiase ((Ow et ab, 1986): kindly pro¬
vided by G. Ncuhaus-Urb Basel) together Avith a nos terminator was introduced as
an Ncol/Xbal fragment under the control of the AC or the BC promoter, Avhilc the
Rcnilla luciferase from pPCV702 (MaA-erhofer et ab. 1995) AAras introduced together
with a nos terminator as a blunt-ended iimdlll fragment under the control of the
AV or BV promoter.
A short and a longer version of the DNA A promoter were cloned. The shoit
version of the promoter ends at the major transcription start, site of the coat protein
while the longer version also includes the minor transcription start site of the coat
protein. Transcription from the majoi transcription start, site of the longer version
gives rise to a 160 nt-long leader fiagment upstieam of the Renilla luciferase gene.
The plasmids were called pACMVs. pACMVleadcr and pACMVB depending on the
inserted promoter (see also Fig. [2.2] and Fig. [2.3]). Each of the ACMV promoter
constructs created bv PCR amplification AA-as sequenced to ensure that no mutations
had been introduced. As positive conti ois both the Rcnilla and firefly luciferase
were separately cloned under the control of a truncated 35S promoter and a 35S
terminator ((Odell et ab. 1985); kindly provided bv S. Brunner, Zürich) rendering
plasmids pLucpos and pRcnpos. To reduce the luciferase expression, thereby making
measurement more convenient, the truncated 35S piomoter consisted of nucleotides
1 to 93 only.
In order to transform plants with Agrobactcniim. the luciferase construct con¬
taining the short, DNA A promoter was introduced into pNCl as a complete I-Scel-
fragnicnt. pNCL is a pCambiabSOO backbone (Gambia, Australia) with a pMCSb
(MoBiTec, Germany) polvlinker containing an I-Seel site. This plasmid, pNCsh,
Avas then electroporateel (Mattanovich et ab. 1989) into GV3101 (pGV2260) result¬
ing in strain AgNCsir.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 19
SAR. ànos firefly lnc
'
~~f DNA A { rauliahic fjnns j—M-| JaJ [4—
ffhir jo'e
Figure 2.2: Schematic representation of luciferase expression cassette. Luciferase
expression cassette showing the position of the cloned ACMV promoter (the short or the
longer version of the DNA A piomotcr or the DNA B promoter) between the Renilla
and the firefly luciferase genes. Both luciferase genes carry a nos terminator. The whole
expression cassette is flanked by scaffold attachment regions (SAR). The bar underneath
the map marks the position of the ffluc probe used for Southern hybridization analysis.
2.3.4 Protoplast isolation and transformation
Tobacco mesophyll protoplasts
Mesophyll protoplasts Avere isolated fionr in vitro-giown tobacco plants. Leaves were
cut into 1 cm2 pieces and digested overnight in PCN1 (Golds et ab, 1993) contain¬
ing 1% Cellulase Onozuka RIO (Yakult Pharmaceuticals Ind. Co., Ltd., Japan),0.5% Maccrozyme RIO (Yakult Pharmaceuticals Inch Co., Ltd., Japan) and 1 drop
TweenSO (Nagy and Maliga, 1976). The protoplast suspension was filtered through
a 100 /im steel mesh sicA'c. After collecting the protoplasts by floating on PCN1
they Avere Avashed tAvice with W5 (Menczel et ab. 1981) and then incubated in the
dark at 4°C for 1-2 hours. Samples of 250,000 to 500,000 protoplasts Avere resus-
pended in transformation buffer (15 mM MgCL. 0.1% MES 0.5 M mannitol, pH 5.8)and transformed with 20 fig of DNA using PEG (Negrutiu et ab, 1987). FolloAving
transformation, protoplasts were incubated in PCN2 (Golds et ab, 1993) at RT in
the dark.
After 24 hours protoplasts Avere harvested bv centrifugal ion and resusponded in
100 fj\ passive lysis buffer for the Dual-Luciferase"' Reporter assay (Promega,Catalys AG, Switzerland). Extiacts aattc stored at -S0TJ for up to one month.
Cassava protoplasts from embryogénie suspension cultures
Approximately 1 g of LMS 60444 embryogénie suspension culture was digested in
the dark during 20 h at 2S°C on a shaken (30 rpm). The enzyme solution and
the washing solution have been described (Sofiaii et ab. 1998). The digest was
then filtered thiough a 50 //in mesh sicA^e and L0 ml washing solution Avere added
through the filter. The fihrate Avas distributed in sterile tissue culture tubes and
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CHAPTER 2. ACMV PROMOTER ANALYSIS 20
centrifugée! at 70 g (30%) for 7 min in a Hettich table centrifuge (Universal II).due supernatant was remoATcl and the pelleted protoplasts Avere Avashed twice and
resuspended in 10 ml Avashing solution. The piotoplasts were counted, stored and
transformed as described for tobacco protoplasts, llic protoplasts were incubated
at RT in the dark in TM2G (Shahim 1985) for 24 hours prior to harvesting and
resuspending in passive Ivsis buffer.
2.3.5 Agrobacterium-med'mtcd seedling transformation of
tobacco
Tobacco seedlings groAvn for 8 daA-s Avere transfoimed by vacuum infiltration as
described (Rossi ct ah. 1993) using an overnight culture of AgNCslr grown in 2
ml YEB (Vervliet et ab, 1975) containing the appropriate antibiotics. The seedlings
were cocultivated for 3 clays on solid MS medium and then washed in 10 mM MgSO |.
They were then placed on MS medium containing 0.1 //g/ml cv-naphtalene acetic
acid, 1/ig/ml benzylamino purine. 25 /yg/ml Irvgiourvcin, 500 /;g/ml cefotaxime
and 500 /ig/ml vancomycin. Seedlings wcic transferred to fresh plates evcrv Aveck.
After 7-8 weeks, shoots could be collected fioni single calli and transferred to MS
medium Avithout selection. After multiplying each plant line, rooted plantlets could
be transferred to soil or be kept as in vitro cultures.
2.3.6 Dnal-Luciferase1M reporter assay
Protoplast or leaf disc extracts Avere thawed and cell debris was collected by cen-
trifugation. Aliquots of 50 fi\ of Lucifeiase Assay Rcagentll (Promcga, Catalys
AG, SAvitzerland) Avere pro-dispensed into luminometcr tubes before adding 10 ft\of extract and mixing well with a pipette. Measurements of 5 seconds each were
performed with a luminometcr (Luinat, LB 9507. EG L G Berthold, Switzerland)Avith dual injectors. After measuring the luciferase activity, in 'relative light units'
(RLU), Stop and Glo'v Reagent Avas delivered into the tube bv automatic injection.
Measurement of the Rcnilla luminescence Avas started after a tAvo second delay. For
each experiment, background luciferase activity from protoplasts transformed with
a vector Avithout luciferase genes Avas subtracted throughout,. Protein concentration,
estimated using a Bio-Rad protein assay (Bio-Rad Laboratories, LISA) as described
(Bradford. 1976). A\*as used to normalize each measurement. All resulting values
were standardized to the positive control consisting of protoplasts transformed with
pLucpos and pRcnpos.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 21
2.3.7 Southern blot analysis
Total DNA from transgenic and untransformed control tobacco Avas extracted us¬
ing the NucleonPhytopure kit (Amcrsham Life Science. England) as described by
the manufacturer. Aliquots of fO fig DNA were digested Avith Ncol, resulting in
a single cut in the T-DNA. After electrophoresis, DNA was transferred to a pos¬
itively charged nylon membrane (Amersham Pharmacia Biotech, UK) by blotting
overnight as described (Sambrook ct ah. 1989). After a short AArashing of the mem¬
brane, the DNA AATas covalentlv bound to the membrane by exposure to UM light
(UV Stratalinker 1800. Stiatagcne). The membrane AA-as prehybiidizecl Avith 10 ml
DIG Easy HybrM (Roche Molecular Biochemicals. Switzerland). After one hour the
prehybridization solution Avas replaced Avith fresh h}bridization solution including
20 ng DIG-labeled probe and hybridization took place at 42TJ o\*ernight. DIG-
labeling of a 330 bp fragment of the luciferase gene Avas performed according to the
PCR DIG Probe Synthesis Kit " standard protocol (Roche Molecular Biochemicals,
Switzerland). After removal of unspecific hybridization and incubation in blocking
buffer, the anti-DIG antibodies Avere added. Folkrwiug Avashing and equilibration,
the DNA side of the membrane was ovorhrved Avith substrate solution (CDPstarTii,Roche Molecular Biochemicals. Switzerland). The damp membrane Avas scaled in
a plastic bag. fixed in a film cassette and exposed to X-ray film (BIOMAX MR"',
Kodak, Rochester NY, LTSA) for 1 hour at 37°C. The film Avas developed with a
developing machine (AGFA. Cuiix 60).
2.3.8 Virus infection of transgenic plants and assay of viral
DNA replication
Transgenic plants Avere groAvn in soil in a controlled growth chamber and inoculated
with A. tumcjaciens containing the viral DNA of ACMV or of Sida Goklen Mosaic
Virus (SiGMV, Colombian isolate) as described (Stanley et ab. 1990). All A. tumc¬
jaciens strains were kindly provided bv T. Frischmnlh. Stuttgart, and have been
described (Stanley et ab, 1990: Fnschmuth et ab. 1997b). Samples of leaves of three
different ages (leaf 1, leaf 3 or 4 and leaf 5 or 6: numbered from bottom to top) Avere
collected 24 days after infection. Luciferase extracts of infected and non infected
IcaA^es Avere measured.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 22
2.4 Results
2.4.1 Expression of firefly and Renilla luciferase genes un¬
der the control of ACMV promoters
The ACMV DNA A and DNA B promoters were cloned between two different lu¬
ciferase genes. This allowed the study of the actn-ation/doAvn-regulation of both
sides of the bidirectional promoteis in a single experiment. The firefly luciferase
gene was cloned under the contiol of the AC promotei (c-sense expression). Avhich
in the DNA A is the piomoter of the replication associated protein and in the DNA
B the promoter of the protein responsible for long distance movement. The Renilla
lucifcrase Avas cloned under the control of the VI promoter (v-sense expression),
which in DNA A is the promoter of the coat protein and in the DNA B the pro¬
moter of the nuclear shuttle piotein. For the DNA A promoter the Renilla lucifcrase
gene was cloned under the contiol of Iavo diffident versions of the VI promoter: a
short version, Avhich includes onlv the major tianseiiption start site at genome po¬
sition 278 (construct pACMYs). and a longer vcision (construct pACMVleadcr; sec
Fig.[2.3]), which enables tianseiiption from the major and the minor transcription
start, site (genome position 378; (Davies et al., 1987)). Transcription from the ma-
joi transcription start site of pACMVleadcr icsults in an RNA containing a L60
nt teacler sequence upsticam of the Renilla luciferase ORF, AAhich in this construct
replaces the ACMV coat protein ORF. In this leader two AUGs (at, 286 anel 305)are upstream of the initiation codon of the coat protein gene at nucleotide 446.
These additional AUGs are in frame Avith a small ORF V2. beginning 8 nucleotides
downstream of the major transcription start site and overlapping the ORF of the
coat protein, The existence of the ORF Y2 encoded protein for ACMV has not been
reported to elate, but a coiresponding protein for AY2 has been reported in tomato
leaf curl virus (Padidam et ab. 1996).
In different experiments \-atiable background levels for firefly luciferase (100 -
1000 RLUs) and Rcnilla luciferase (1500 - 30000 RLUs) were observed. Within
one experiment, these background lewels were quite constant and expression levels
Avere regarded as significant onlv when thev Avere at least, 3 times above this back¬
ground. Background levels Avere substracted from all values before standardization
against total protein content. The basal le\-els of the fireflv luciferase from pACMVs
and pACMVleadcr Avere around S000 RLUs. The Renilla luciferase from pACMVs
gave rise to values of about 50000 RLUs. while the activity bom pACMVleadcr was
higher, about 145000 RLUs. The fiieflv lucifeiase activity of pACMVB was com¬
parable to that from pACMYs. but the Renilla luciferase activity Avas on average
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CHAPTER 2. ACMV PROMOTER ANALYSIS 23
ACMV DNA A
Rep
*-i rV_
pACMVleader
- SAR - nos firefh lue
*n r^iD\\ \ renilla lue nos - SAR
pACMYs
- SAR - nos firefh lue
i—"
DNA A ' renilla lue 'nos— SAR —
ACMV DNA B
MPB DN VB NSP
pACMVB
SAR - nos fircflv lue DN-VB renilla lue nos - SAR
Figure 2.3: Constructs used for ACMV promoter studios in comparison with
original ACMV DNA. Luciferase expression cassettes compared with ACMV DNA,
showing the position of the cloned ACMV promoter (the short or the longer version of the
DNA A promoter or the DNA B promoter) between the Renilla and the firefly lucifcrase
genes. Both luciferase genes carry a nos terminator. The whole expression cassette is
flanked by scaffold attachment regions (SARs).
25 times higher (fable 2.1). In our experimental system the Renilla and firefly lu¬
ciferase genes under the control of the same promoter produced RLUs that varied by
a factor of maximum 2.5 (data not shoAvn) and similar results ha\re been reported for
mammalian cells (Promega User Manual): theicfore, similar RLUs for Renilla and
firefly luciferase reflect, similar promoter strengths. All luciferase activities measured
for any of the ACMV promoteis Avere at least 100-fold and up to 2000-fold loAvcr
than activities obtained for the same hiciferases fused to a tiuncated (-1 to -93) 35S
promotei.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 24
Table 2.1:
Basal luciferase activities of pACMVs, pACMVleader and pACMVB
pACMVs pACMVleader pACMVB
firefly Rcnilla firefly Renilla firefly Renilla,
(c-sense) (v-sense) (c-sense) (v-sense) (c-scnsc) (v-sense)
RLU 6600 48200 13000 145000 4200 1220000
Standardized ° 100% 100% 195% 300% 63% 2500%
Relative6 100% 730% 100% 1100% 100% 29000%
"Luciferase activities ol pACMVleadcr and pACMVB were standardized to luciferase acti\ities
of pACMVs.6V-scnsc Ren/Ha luciferase acthities weie standardized to the respective c-sense firefly lurifeiase
activities.
2.4.2 Effect of the expression of viral ORFs on the activity
of the luciferase genes under the control of ACMV
promoters
The constructs pACMYs or pACMVB wore cotransformed into tobacco proto¬
plasts, either with equimolar amounts of the Rep construct (ACMV Rep ORF under
the control of a 35S promotei). TrAP construct (ACMV TrAP ORF under the con¬
trol of a 35S promoter). ACMV DNA A or with carrier DNA. All experiments aa'cic
repeated 4 to 6 times and the results are summarized in Fig. [2.4].Cotransformation with the TrAP construct increased \--sense expression (Renilla
lucifcrase) of pACMVs 16-fold and c-sense expression (firefly luciferase) 10-fold; co-
transformation with the Rep construct reduced c-sense expression about, 35-fold
but did not affect v-sense expression. Cotransformation Avith the complete DNA A
resulted in an almost 50-fold increase of v-sense expression and an approximately2-fold reduction of c-sense expression. Even in the cases of loAvest c-sense expression,
firefly luciferase activity remained significantly abowe background levels, indicating
that also under these conditions, the promoter maintains a basal activity. The îeg-
ulation of the B promoter bv the TrAP construct Avas similar; the Rep construct.
hoAvcver. shoAA"ed a stronger negative effect on v-sense expression (Renilla luciferase)and a slightly weaker negative effect on c-sense expression. The most, striking dif¬
ference Avas observed foi cotransfoimation Avith the DNA A. which led to a strong
activation of c-sense (20-fold) and v-sense (16-fold) expiession from the DNA B
promoter.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 25
miiieflv UReniUa
7000
6000
>- 5000
-S 2000
pACMVs ^Icp <-TiAP +DNAA pACMVB +Rcp
constmcts used foi transformation
'-TiAP HONAA
Figure 2.4: Effect of the expression of ACMV ORFs on the activity of fire¬
fly and Renilla luciferase under the control of ACMV promoters in tobacco
protoplasts. Luciferase constructs pACMYs and pACMVB were assayed in transient
for luciferase activity in tobacco SRI protoplasts following cotransformation with the Rep
construct, the TrAP construct or the complete DNA A. A value of 100% Avas assigned (o
the activity of each promoter construct alone. Columns represent the resulting luciferase
activity as a percentage of the basât activity and are the means of four to six independent
experiments; error bars represent standard deviation.
Analogous experiments aa-cic also peifoimed with cassava protoplasts (sec Fig.
[2.5]). The results avcic similar in as far as TrAP functioned as an activator of both
c- and v-sense expression with DNA A and B promoteis; the highest increase in
activity, hoAvever, was observed Avith the DNA BC piomoter in presence of TrAP
(57-fold). Like in tobacco protoplasts. Rep reduced c-seuse expression in general and
most, stronglv again in the case of the AC promoter (35-fold). The cotransformation
with DNA A resulted in a 20-fold increase of v-sense expression (Renilla luciferase)but had no significant, effect on the c-sense expression (firefly lucifcrase). The results
using pACMVB in cassava Avere again different from those in tobacco. In cassava
the increase of c-sense expression Avas much stionger (35-fold) as compared Avith
the 6-fold increase of v-sense expression. Avhile in tobacco both luciferase activities
increased to a similar extent.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 26
lfneflv DRenilla
7000
6000
5000
4000
3000
2000
1000
127
niiiil 1-
37 127
pACMVs l-Rep -TrAP JDXAA pACMVB +Rep
constiucts used foi trans (carnation
+TrAP tDNA A
Figure 2.5: Effect of the expression of ACMV ORFs on the activity of fire¬
fly and Renilla luciferase under the control of ACMV promoters in cassava
protoplasts. Luciferase constructs pACMVs and pACMVB were assayed in transient,
for luciferase activity in cassava SRI protoplasts folloAving cotransformation with the Rep
construct, the Tr'AP construct or the complete DNA A. A value of 100% was assigned to
the activity of each promoter construct alone. Columns represent the resulting lucifcrase
activity as a percentage of the basal activity and are the means of four to six independent
experiments; error bars represent standard deviation.
Furthermore pACMVs was cotransformed Avith the TrAP and Rep constructs to¬
gether using different amounts of DNA for each construct (f or 10 /ig each, sec Fig.
2.6]). As these cxpeiiments were perfoimecl separateR-, results AArere not combined
with experiments Avhere Rep and TrAP Avere cotransfoimcd individually. Transfor¬
mation with increasing amounts of the Rep construct reduced c-sense expression,
while v-sense expiession AA-as still enhanced even if TrAP Avas cotransformed at very
Ioav amounts (l /ig TrAP vs. 10 fig Rep construct). In the case Avhere equal amounts
of both constructs are transfoimecl. the inet case of v-sense expression together with
the decrease of c-sense expression icsembles a cotiansformatiou experiment using
DNA A.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 27
[nfneflv~UReniUa I
7000
6000
>
5000
GJ
CO
4000
u
>
5~i
3000
2000
IS I—EE—I 15 79
pACMVs +RepTi\P=0 10 -SRep TiAP=I 10 Rep 1 i \P = ] 0 10 -^Rep TiAP=10 1 'Rep I iA.P=10 0
constructs used foi tiansfoiniation
Figure 2.6: Effect of the combined expression of ACMV ORFs on the activity of
firefly and Renilla luciferase under the control of ACMV promoters in tobacco
protoplasts. Lucifcrase construct pACMVs was assayed in transient for fncifcrasc activity
in tobacco SRI protoplasts following cotransformation Avith different combinations of the
Rep and TrAP constructs. A value of 100% was assigned to the activity of pACMVs
alone. Columns represent, the resulting luciferase activity as a percentage of the basal
activity and are the means of four independent experiments: error bars represent, standard
deviation.
2.4.3 Analysis of a short and a longer form of the DNA A
promoter
The constructs with iavo different segments of the ACMV DNA A promoter pACMVs
and pACMVleader Avere cotransformed into tobacco protoplasts together Avith the
complete DNA A. A summary of the experiments is shown in Fig. [2.7]. When
cotransformed -with DNA A. the increase in v-sense expression (Renilla lucifcrase)from the short, version Avas much higher (58-fold) than the increase from the longer
version of the piomoter (6-fold). The reduction of c-sense expression (firefly lu¬
ciferase) was 4- to 10-fold for both the short and longer version of the DNA A
promoter.
1000
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CHAPTER 2. ACMV PROMOTER ANALYSIS 28
'fflfiicflv URemlh
10000
9000
8000
» 7000
Î 6000
^ 5000CJ
2 4000
"~
3000
2000
1000
0
5if47
638
100 100 100 100 26T
10 1
p VCMVs p \CM\k ider pACMVs . DN A A.
constructs used foi ttanstoimation
pACMVieadei-DISAA
Figure 2.7: ACMV ORFs regulate luciferase expression from the short and
longer version of the ACMV DNA A promoter in tobacco protoplasts. pACMVs
and pACMVleader were cotransformed with the complete DNA A into tobacco protoplasts.
Luciferase activities were determined and are expressed as a percentage of the activity of
each promoter construct alone. Columns represent the mean values of four experiments:
error bars represent the standard deviation.
2.4.4 Production of transgenic tobacco plants containing
pACMVs and pACMVleader
To test whether the results from the transient expiession experiments in protoplasts
could be confirmed for stablv integrated genes, transgenic tobacco plants containing
pACMVs or pACMVleader Avere produced by Agrobactenum-modiated transforma¬
tion. Resistance to hvgromycin Avas used to select transgenic plants. The transgenic
nature of 10 independent lines Avas confirmed bv Southern blot analysis (see Fig.[2.8])
and luciferase assays of leaves. Of these 10 transgenic lines. 6 contained the short
version of the DNA A piomoter. while 4 contained the longer version of the pro¬
moter. All lines contained 2 - 10 copies of the T-DNA. Two of these lines shoAvcd
a 3 : f segregation pattern (sir 6 and sh 11). indicating a single locus integration.
All plants Avere grown as in vitro cultures as well as in the greenhouse. Avhere they
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CHAPTER 2. ACMV PROMOTER ANALYSIS 29
N b Ö,^ Y ^ ^ b <^ C\ ^
£ # S- ^ >V ^ ^ v>> ^ ^ £
bp
8576__
7427 —
6106 —„
âklls«»
4399 — --««
3639 —- s *A
2799 —
1900 —
1482 —
992 —-
Figure 2.8: Southern analysis of ACMV luciferase transgenes in tobacco. South¬
ern blot of Ycol restriction fragments hybiiclizecl to the ffluc probe.
produced seeds normalh.
Regulation of the luciferase genes under the control of the ACMV pro¬
moter in transgenic tobacco
Mesophyll protoplasts aa'cic isolated from transgenic plants and transformed with
the Rep or TrAP construct or Avith the complete DNA A Piotoplast extiacts were
analyzed with the Dual-Lucifciase'1'1 assav and standardized Avith Bradford assays,
but, the variation of the relative values betAA'cen plants and experiments Avas high. A
représenta tree experiment Avith some of the plant lines and a wildtype SRI tobacco
(cotransformed with pACMYs oi with pACMYs Imeaiized outside of the expression
units) is shoAvn in table 2.2. Suipiismgly. c-sense expression (firefly luciferase), in the
transgenic plants. was much stronger than v-sense expression (Renilla luciferase) or
at least similar (sir 9). This is in conti ast Avith transient experiments, where v-sense
expression Avas ahvavs strongci. Also in conti ast to the eailiei cotransfoimation
experiments in protoplasts, TrAP did not function as an acth*ator in protoplasts
from transgenic plants. Onh* Avhen the complete DNA V AA-as used for transforma¬
tion, an increase of v-sense expression could be found. Avhile the c-sense expression
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CHAPTER 2. ACMV PROMOTER ANALYSIS 30
Table 2.2:
Luciferase expression (in RLUs) of protoplasts from transgenic tobacco
plants after transient transformation with ACMV ORFs
conshucts used for transformation
plant line reporter gene
200000
Rep
2 10000
TrAP DNA A
sir 1 fireflv lucifcrase 260000 230000
sir 1 Rcnilla luciferase 6000 6200 7500 31000
sir 9 firefly luciferase 1870 2300 2200 2150
sir 9 Rcnilla luciferase 1150 2900 2500 UOOO
sir 11 firefly luciferase 13000 21000 22000 28000
sir 11 Rcnilla luciferase 3500 3800 4000 170000
llr 7 fireflv luciferase 16300 18000 15000 20400
llr 7 Renilla lucifcrase 3100 2800 2600 3800
llr 5b fireflv luciferase 2100 2900 2300 2700
llr 5b Ren ilia luciferase 1600 1300 930 1400
wt fireflv Incilerase a 660 88 4000 700
wt Rcnilla luciferase 7800 8900 78000 451000
wt, linear firefly luciferase h520 67 200 230
wt, linear Renilla lucifcrase 16000 L0400 16000 67500
"Wildtypc protoplasts weic cotiandoimcd A\itli pACMVs and the concsponding ACMV ORF
''Wildtype protoplasts, cotransformed \ufh tmeaiized pACMVs and the coiresponding ACMV
ORF.
remained unaffected. This incicase varied from 3- to 50-fold between different exper¬
iments. This same effect could also be obseiwcd Avhen using lincaiizcd pACMVs for
cotransfoimation studies, although heie TrAP shghtlv activated Renilla lucifetase
expression in some experiments (data not sIioami). As variation between experi¬
ments was much higher than between chfTeient lines, the degree of activation could
not be correlated to the gene copy number Also, in contiast to cotransfoimation
experiments desciibecl above, the expiession in both v- and c-sense from the longci
version of the DNA A promoter Avas not notably altered bv transformation Avith the
DNA A or the TiAP construct. When transformed with the TrAP constiuct even
a slight reduction of both lucifeiascs could be detected in piotoplasts of transgenic
plants containing the longer version of the DNA A piomoter.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 31
Infection of transgenic tobacco plants
Seeds from selfcd transgenic plants were collected, germinated and infected Avith
ACMV. Infected teaves of three different stages (leaf 1, leaf 3 or 4 and leaf 5 or
6) were analyzed for lucifcrase activity 24 days after infection, even though plants
shoAved no ACMD symptoms. Ten infected plants of five different, lines Avere tested
using uninfected plants of the same line as conti ois. Of these 50 plants onlv three
plants shoAved an activation of the Rcnilla lucifeiase expression after viral infection
(srl6-4, srl9-13 and srli 1-8). In order to assess the amount of viral DNA present.
DNA of infected plants Avas isolated and analyzed in a Southern blot. No viral DNA
could be detected in any of the plants tested. Therefoie. new plants were germinated
and infected with SiGMV (Colombian isolate). All plants sIioavccI typical mosaic
bleaching of leaves, indicating an infection rate of 100%. Tlucc infected - and three
non-infected plants of 5 different, lines were tested. All of these plants showed verv
high lucifcrase activities but Renilla activities Aveie hardly above background eAren if
the plant was infected (data not shewn). A relative activation of Renilla luciferase
activity and down-regulation of fireflv luciferase activity (2- to 5-fold) could be
observed in all tested lines.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 32
2.5 Discussion
The large intergenic region of the geminivirus genome contains c?'s-acting elements
that arc important for the regulation of vital gene expression and viral replication.
A regulatcel, bidirectional piomoter is situated Avithin this intergenic region (Stanleyand Gay, 1983; ToAvnsend et ab. 1985: Haley et, ab, 1992; Eagle and Hanley-BoAvdoin,
1997; Orozco et ah. 1998). Pievious studies on geminivirus promoter regulation in¬
vestigated mainly promotcr-GUS fusions, testing the transcriptional activity for each
direction of the promoter separately (Zhan et ab. 1991; Monis et, ab, 1991: Brough
et ab, 1992; Haley et ab. 1992: Snntei et ab. L993: Zhan et ab. 1993; Gröning et ab,
1994; Hong and Stanley, 1995: Sunter and Bisaro. 1997: Gooding et ab, 1999: Ruiz-
Medrano et ab, 1999). Owoiall. the results obtained with Begomovirus promoters
revealed a similar regulation pattern: the TrAP protein Avas found to induce v-sense
transcription while Rep doAvn-regulates c-sense transcription (Haley et ab, 1992).
However, details of basal promotei sticngth and the degree of activation or repression
by viral proteins varied considerably. It remained unclear whether these variations
reflect differences of the respective assav SA-stcms (e.g. plant matciial, incubation
time of transformed protoplasts, exact promoter sequences, etc.) or, alternatively,
differences in the replication cycle of the respective viruses. Tomato golden mosaic
virus (TGMV), for example, staits leplicating after 18 - 24 hours in protoplasts
(Brough et, ah, 1992), while in AC MA' or beef cm ly fop virus (BCTV) infected cells,
replicating DNA can only be detected 2 - 3 days after infection (Townsend et ab,
1986; Briddon et ah. 1989).
We intended to use the regulation mode of the ACMV promoter for a novel
strategy for engineered virus-inducible Alius resistance, which required a more de¬
tailed knowledge of the relative strengths of the Iaa'o promoter directions in absence
and presence of viral gene products. In older to studv simultaneously transeiiption
from both sides of the promoter (further on referred to as AC and AV promoter for
the DNA A and BC and BA" promotei for the DNA B promoters), the promoter
fragments Avere fused to the fiieflv luciferase gene in c-sense orientation (AC and
BC promoters) and to the Renilla luciferase gene in A'-scnse oiientation (AV and
BV promoters). Since several start sites have been reported (Davies et ab, 1987) for
the DNA A v-sense transcription. Ave also tested both a short promoter, covering
only the start site upstream of the AY2 gene, and a longer version of the promoter,
covering all the stait sites upstream of the coat piotein gene (see Fig.[2.3]). The
two different hiciferases can be tested in the same icaction mixture and should,
therefore, alloAv a precise compaiison of promotei activities. Using reference con¬
structs containing either one luciferase unci«- the contiol of a truncated (1-93) 35S
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CHAPTER 2. ACMV PROMOTER ANALYSIS 33
promoter, we could establish that similai amounts of light units aie produced bv
both hiciferases from these constructs, indicating that light units can be dncctlv
related to promoter activhY.
An additional advantage of the lucifeiasc reporter genes is the shorter half-life
(for Rcnilla luciferase onlv reported in mammalian cells) of both proteins (and pos¬
sibly mRNAs) compared to the piCA-iouslv used GUS reporter proteins (Thompsonct ah, 1991; Martin et, ab. 1992). This allows a bettei evaluation of gene induction
or repression.
In tobacco or cassava protoplasts transformed \vith plasmid DNA. the strongest
expression from anv of the introduced genes under the control of an ACMV pro¬
moter Avas still considerably 1oaa*ci- than the expression obtained with a truncated 35S
promoter, which again was about 3-fold weaker than from the full-length promotei
(data not shown). Thus, in our assay system, the ACMV promoters arc about, 300-
to 6000-fold weaker than the 35S CaMY piomoter. in contrast to previous reports,
which stated a 10- to 40-fold loAvcr activity of the ACMV promoter (Zhan et ab.
1991; Hong and Stanley. 199-5). The activity of a TGMV AV promoter on a replicon
was repoited to be even 60- to 90-fold stronger than that of a 35S promoter and
without replication, the activity of the FGAIA* piomoter was still comparable to
that of the 35S promoter (Biough ct ah. 1992).
In contrast to earlier reports. Avhere the basal expression from the AC promoter
was considerably stronger than from the other thiee promoters, we find that in
transient assays c-sense expression is always loAvcr than Ar-scnse expression (see table
2.1). In transgenic plants, on the other hand, the AC promoter leads to higher
expression rates than the VA" promoter, although the differences of lucifcrase activity
between separate plant lines and experiments A-aried strongb-. This could be clue to
the different availability of host factois in the whole plant, or clue to the higher
stability of the firefly luciferase compared to the Renilla, luciferase. To date nothing
is knoAvn about the half-life of Rcnilla lucifeiasc in plant cells.
For the repressing effect of the Rep and the activating effect of TrAP avc found
quantitative differences to published data. Both, the 10- to 20-fold repression of c-
sensc expression by Rep and the 10- to f5-fold stimulation of v-sense expression by
TrAP are more similar to values previously found for ITJAiA'" (Giöning et ab, 1994;
Sunter et ab, 1993) than to the much smaller values reported for ACMV (Haleyet ab. 1992; Hong and Stanley, 1995). These results ate in agreement with a model
of carlv/latc gene regulation: since Rep is needed foi replication, it Avili be expiesscd
early in the infection cycle but repressed later on. On the other hand, expression of
the coat, protein (CP protein), which will be îequired in high amounts later on in
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CHAPTER 2. ACMV PROMOTER ANALYSIS 34
the infection cycle once the replicated DNA is ready for encapsulation, is activated
by TrAP (Stanley, 1985).
Rep also repressed BC expression 2- to 3-fold, but, as expected, had no or only
small effects on v-sense expression. Surprisingly, TrAP also activated DNA A c-
sense expression by about 10-fold, a property not, previously recognized. Activation
by TrAP was also strong for the DNA B promotei in both orientations in tobacco
protoplasts, but in cassava protoplasts a high disparity of activation between the
two DNA B promoters Avas obscrwed: the activation of the BC promoter was much
higher than that of the RA* promotei. Otherwise, the two protoplast systems reacted
similarly. The regulation of the DNA B promotei in cassava is in accordance with
the role of the Pwo DNA B proteins: NSP has been reported to bind newly replicated
ssDNA and to move the DNA out of the nucleus (Sandetfoot et ai., 1996) while the
MPB forms encloplasmatic rcticulum-derived tubules which extend through the cell
wall to the next cell (Sanderfoot and LazaroAvitz. 1995: Sanderfoot et ab, L996; Ward
et ab, 1997). The nuclear shuttle piotein expressed individually, is localized in the
nucleus, however when MPB is coexprcssccl. it is relocalizecl to the cell pciipheiy
(Sanderfoot and LazaroAvitz. 1996; La/aiowitz and Beachv. 1999). The results ob¬
tained for cassava suggest a regulation of targeting NSP: early in the infection cycle
more NSP will be produced which will favor localization in the nucleus. Later, after
activation of c-sense expiession. the excess of AlPB will icdirect NSP. together with
the bound viral DNA to the periphciy of the cell, and facilitate its movement across
the cell wall, This model does not incoipoiate the encapsiclation step which, how¬
ever, is not necessarv for systemic moA-cment of ACMV in the plant (Sandcifoof, anel
LazaroAvitz, 1996); it is only necessary for the transmission via the whitcfly vector
(LazaroAvitz, 1992).
Since the regulation of the DNA B promoter is different in cassava and tobacco
it follows, that, not, onlv the viral pioteins but also cellular factors play a role in the
regulation of the ACAIA" promoters. Spccies-specihc variation in the availability of
such factors could account foi the lower activation of the BV promoter in cassava
protoplasts as compared to tobacco protoplasts. This further shows the importance
of testing the obtained results in the natural host of the virus.
To mimic a viiaf infection, reporter gene constructs Avere cotransformed Avith
the complete ACAIA* DNA A. which should express legulatorv proteins under viral
expression signals and viral control. DNA A pioved to be a more efficient inducer of
v-sense expression than TrAP and a less efficient repressor of c-sense expression than
Rep for the DNA A promoters. It appeals that the repressive effect of the DN.A A
is dominant for the AC promoter, Avhile the induch-c effect is dominant for the AV
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CHAPTER 2. ACMV PROMOTER ANALYSIS 35
promoter. This dominance was also apparent in cotransformation experiments with
different ratios of Rep anel TrAP. where a small amount of TrAP expressing plasmid
Avas sufficient to suppress the repressing effect of Rep on v-sense expression (sec
Fig. 2.6]). The strength of the activation of v-sense expression is surprising since in
the cotransformation of single genes, these genes were under the control of the 35S
promoter, which should produce much higher levels of protein. Possibly the complete
DNA A contains additional activating sequences, which cither allow more efficient
TrAP production or Avhich pioducc additional proteins or protein variants with
activation potential. Another possibility is that the replication associated protein
induces cell cycle factors that could increase the expression of the reporter gene
(Ruiz-Medrano et ab, 1999). In contrast to the differential effect, on the DNA A
promoter, both DN.A B promoters Avere strongly stimulated by DNA A, suggesting
that the stimulatoiy effect of TrAP (and/or othei proteins) is dominant for both
the BC and BV* promoters.
The longer version of the DNA A promotei was activated much less efficiently
and even considering the 3-fold higher basal activity, the final expression lewels of
Renilla luciferase (v-sense expression) remained loAvcr than tltosc from the short
promoter. According to published transciipt analyses, the major transcription start
site is located upstream of the AUG coclon of the coat protein ORF, implying that
Renilla, luciferase fusions to the downstream coat protein ORF, like in the construct,
pACMVleadcr, Avould be less efficiently translated due to the presence of upstream,
out-of-frame start codons. It aa-ouIcL therefore, be expected that expression from
the short promoter is more efficient than fiom the longer version of the promoter.
The observed lower activation of Renilla luciferase expression could be explained
by assuming that only transcription at the major start site is activated by DNA A.
According to this model, using pACMAleader moie of the longer mRNA (containingthe 160 bp leader sequence) is produced while the level of the short, mRNA, tran¬
scribed from the minor transcription site, should not be affected. Since translation
of the longer mRNA is piobablv not verv efficient due to out-of-frame start, codons
in the leader sequence upstream of the CP ORF (Fütterer and Hohn, 1992), it will
not give rise to a high level of luciferase activity. On the other hand, with pACMVs,
transcription of the leaderless mRNA will be enhanced and translation can take
place efficiently, resulting in a much higher level of luciferase activity. We have not
been able to detect the corresponding mRNAs bv RNase protection assav (resultsnot shown). This is probably due to the instability of the luciferase mRNAs. It
therefore remains unclear Avhich transcripts are pioduced Avith the different expres¬
sion constructs in our assav system.
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CHAPTER 2. ACMV PROMOTER ANALYSIS 36
For the envisaged virus resistance strategies the use of a promoter integrated
in the plant, genome is necessary. The studies performed Avith the free plasmid
in protoplasts might be close to the situation of a virus infecting a cetl, but the
regulation of a promoter integrated in the chromatin could be very different, hi
parallel experiments we compared the effects of Rep, TrAP or DNA A on reporter
expression from cotransformed or integrated copies of pACMVs or pACMVleader.
Interestingly, the absolute values of these transformation experiments are very dif¬
ferent in individual plant lines. Foi example, line sir f shows a much higher basal
c-sense expression (fuefiv luciferase) than \--sense expression (Renilla lucifeiasc) in
mesophyll protoplasts, while line sh 9 exhibits a slightly higher basal v-sense than
c-sense expression. It seems that, expression is not ontv regulated by the ACAIA'*
promoter but that also the integration site plavs a role in the regulation of gene
expression, despite the presence of SARs.
After cotransformation with DNA A the relathe increase of expression of the
wildtypc is higher than that of all the other upregulated transgenic plants. The
stronger increase of v-sense expiession in the cotransformed Avilcltype plants could be
due to a different regulation of the integrated vims promoter versus a virus promoter-
localized in a plasmid. At the same time this could piovidc an explanation for the
fact that, unlike in AvildtA-pc protoplasts, no increase of expression can be detected
after transformation with the TrAP construct. Using the longer version of the
promoter, activation of v-sense expression could be observed neither when using
TrAP nor DN.A A for cotransformation. Alreach- in the transient assay the increase
of v-sense expression from the longer promotei upon transformation Avith DN.A A
is lower than that from the short A'crsion. As this increase of v-sense expression
from pACMVs is 4-fold loAA-er in the transgenic plants as compared to the transient
assay, the increase from pACMAloader in transgenic plants is haidly above the basal
act ivitA".
During the manv repetitions performed in the course of these experiments, the
results involving transactivation of reporter activity paiticnlarlv shoAved a relatively
largc variability. Insufficient control of DNA structure cluiing the experiments could
be one reason for this variability and possibly for differences between our data and
those reported previously. Importance of DNA structure was first suggested by re¬
sults obtained with piotoplasts fiom transgenic tobacco plants haiboring the same
reporter genes as used for the tiansient assavs. DNA unvatuic has been implicated
in control of replication and possibly tianscription foi the Avheat chvaif geminivirus
(Gutierrez et ab. 1995: Gooding et ab. 1999). Avhere the expiession of a reporter
gene from introduced plasmid DNA was different from that fiom in p/ar/ia-generated
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CHAPTER 2. ACMV PROMOTER ANALYSIS 37
replicons. The authors speculate, that the effect could be clue to the activation states
of the chromatin or the different methvlation states. Such features Avould almost
certainly be different on a supereoilecl plasmid and on integrated DNA, as the latter
will probably be associated with nuclcosomes differently. To verify this, we trans¬
formed protoplasts Avith reporter DNA that was linearized outside of the expression
units. In these experiments, expression from hneai DNA was hardly activated by
TrAP protein, whereas this process was efficient with circular DNA (see table 2 2).
Also the activation of Rcnilla lucifcrase expiession with DNA A was less efficient
using linear DNA than circular DNA. It can also be speculated, that additionally a
viral protein, e.g. Rep. interacting with host transcription factors (Hanlcv-BoAvdoinet ah, 1999; Liu et ab. 1999: Ruiz-Medrano et ab, 1999), is required for maximum
virion sense expression, even though the protein itself is not an activator. In this case
transformation Avith TrAP might not be sufficient for activation when the promoter
is integrated in the genome.
As a logical step after transient assavs transgenic plants were infected with
ACMV. Unexpectedly. ACAIA* Avas not able to infect tobacco SRI plants, there¬
fore, another gcminiviius. the Sicla Golden Alosaie Virus (SiGMV) Avas used for
infection studies. Typical symptoms could be detected on infected plants after 2
to 3 Aveeks, indicating a svstcmic infection. HoAvcA'er, quantification of tuciferase
activities in different leaves of infected plants revealed that, Renilla expression Avas
only slightly aboA*e background, cwen in infected plants. Possibly, SiGMV is not
related closely enough to ACAIA* to activate its promoter properfv. The viability
of pseudorecombinants produced by reassort ment of genome components of ACMV,
Squash Leaf Cuil Alius or Tomato Golden Alosaie Ahrus implies that trans-acting
functions are interchangeable (Stanley et ab. 1985: Lazarowitz, 1991). Nevertheless,
it has been shoAvn that viruses of the Ncaa* Woild have a more limited ability to rec¬
ognize and functionally interact with DNA of other geminiviruses than viruses from
the Old World (Frischmufh et ab. 1993b). Avhich is in agi cement with our results.
An approach in which the unique properties of this promoter were exploited
has already been published (Hong ct ah. 1996). There expression of a ribosome
iuactivating protein Avas induced upon vims infection. Since the AV promoter is
slightly leakv, as shown bv using the GUS reporter system (Hong et ab. 1996).this might, lead to abnormal or reduced growth, when using a toxic gene under the
control of the AV promoter. Theiefore. in addition to using the upregulation of
the AV promotei. we propose to also use the doAvniegulation of the AC promoter:
the AC promoter could regulate the expression of the inhibitor of a toxin which
is under the control of the AA" promoter. As long as no infection occurs, more
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CHAPTER 2. ACMV PROMOTER ANALYSIS 38
inhibitor than toxin will be produced. Onlv if the cell is infected, the balance will
be shifted towards more toxin production. Consequently a hypersensitive reaction
could be mimicked thereby protecting the plants fiom viral infection. The results of
our studies on ACMV promoter regulation are of practical significance for designing
new virus resistance strategies in the future.
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Chapter 3
Developing a new ACMV
resistance strategy by mimicking a
hypersensitive reaction using the
barnase and barstar ORFs
o.JL _r\DSTrû.Cti
Barnase is an unspecific ribonriclease of Bacillus amyloliquefaciens which
has a specific inhibitor, the barstar. Here the RNase activity of the
barnase was exploited to engineer resistance to a DNA plant virus, the
African cassava mosaic virus in transgenic Nicotiana tabacum SRI. For
this purpose the barnase ORF was cloned under the ACMV virion-sense
promoter, which is f-rans-aetivated by the TrAP, a viral protein. Ad¬
ditionally, the barstar was introduced under the control of the ACAIV
complementary-sense promoter to counteract basal expression of barnase.
Upon viral infection the ratio of barnase/barstar would be expected to
shift in favor of the barnase due to the viral protein TrAP, resulting in lo¬
cal cell death before the virus can spread to adjacent cells. A replication
assay using leaves of transgenic plants compared with wildtype plants
could show a 3- to 100-fold reduction of viral replication in transgenic
leaves.
39
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ACMV Resistance 40
Figure 3.1: Genome map of ACMV DNA A and DNA B. The DNA is represented
by a thin line with the two arroAvhcads showing the exterrt of the large intergenic region
(LIR) and the main viral ORFs aie indicated by thick arrows. See text for ctetails on gene
names.
3.2 Introduction
African cassava mosaic disease (ACMD) has been rated one of the most important
and devastating diseases of cassava in Africa (Geclcles, 1990). It Avas first reported in
1884 (Warburg, 1894) and seweial serious outbieaks have been reported since then
(Thresh et ab, 1994). At present, a paiticuhulv severe epidemic of the disease is
spreading in central and eastern Africa (Gibson et ab, 1996) causing local losses of
up to 80%, while losses throughout Afiica reach 36% of total cassava production
(Fargette et ab, 1988). Cassava (Manihot cHculanla Crantz) is a major food crop in
Africa, grown for its tuberous roots containing up to 85% starch (dry weight) and for
its protein-rich leaves. It is the basic staple food foi over 200 million people in Sub-
Saharan Africa, Avhere the majorité of cassava is produced bv small-scale farmers
on a subsistence basis. Conventional breeding to control African cassaA-a mosaic
disease by inter- and intraspecific crosses A\lth plants having a natural resistance
but a limited agricultural value, is still difficult due to the Ioav fertility of many local
varieties (Hahn et ab, L9S0: Thresh and Otim-Nape, 1994). Genetic engineering is a
poAverful tool to complement these efforts bv extending the gene pool for useful gene
sources over species ban ici s and bv engineering single, desirable traits precisely.
ACA4D is caused bv a Avhitefly-transraitted vims of the family Geminwiridac:
the African cassava mosaic vims or ACAIA* (Briddon et ab. 1998). The ACMV
genome is composed of Iavo circular single-stranded DNA molecules, DNA A and B
(see Fig. [3.1]). The larger of the tAvo components. DNA A (2779 bp). encodes the
coat protein (CP or AA'l) and all viral functions required for viral replication (Rep
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ACMV Resistance 41
or ACl, replication associated protein. TrAP or AC2 and REn or AC3, regulator
proteins). DNA A is therefore capable of autonomous replication and encapsulation
(Townsend et ab, 1986), but incapable of moving from cell to cell in the host plant.
The second component, DNA B (2724 bp), encoding two proteins (MPB or BCl and
NSP or BV1) that are itrvolvcd in cell-to-cell movement in the plant (von Arnim
et ah, L993; Halev et ab, 1995). is requited for a full svstemic infection. Transcription
occurs in a bidirectional manner from the huge intergenic region (LIR). which is
highly conserved between DNA A and DN A B.
Increasing knowledge of the functions of ACMA" genes and their regulation has
led to the development of virus resistant transgenic model plants. Transgenic con¬
stitutive expression of defective vital DNA (Stanley and ToAvnscnd, 1985: Stanlev
et ah, 1990), of antisense Rep gene (Dav et ab. 1991). of defective Rep protein (Hong
and Stanley, 1996), of defective movement proteins (von Arnim and Stanley, 1992:
Duan et ah, 1997), of coat piotein (Kunik et ab. 1994) or the virus-induced expres¬
sion of dianthin in stablv transformed plants (Hong ct ah, 1996) has shown to confer
geminivirus resistance. As shown with the libosome-inactivating protein dianthin,
the regulatable ACAIA* promoter can be used for the controlled expression of highlv
toxic genes that otherwise AA'ould be lethal or lead to abnormal development of the
transgenic plants, thus limiting their cffcctrvcncss as antiviral agents (Hong et ab,
1996).
The unique properties of the regulation of the ACAIA" promoter should make it,
possible to mimic a hvpeisensitive reaction by using a finely tuned toxin/anti-toxin
system. It, is knoAvn that a basal Ioav level of tianscription fakes place from both
sides of the ACMA'* promoter (Hong et ab. 1996). Therefoie, basal expression of
the toxin has to be counteracted Avith basal expiession of the anti-toxin. Upon viral
infection of transgenic plants, expiession of the toxin under the control of the AV
promoter would be aeth-ated specifically in virus infected cells, thus overriding the
effect of the anti-toxin under the contiol of the VC piomoter.
Barnase, the ribonuclcase produced Ida' Bacillus amijloliquefacieris (Mariani et, ab,
1990; Mariani et ab. 1992), and ifs specific inhibitoi, barstar, could be applied in the
aforementioned virus resistance strategy. Due to the high toxicity of the barnase,
further doAvn-regulation of its production might prove necessary. This could be
achieved by introducing additional short open reading frames (sORF), upstream of
the barnase gene, to dcnvii-regulate its translation (Fütteret and Holm, 1992; Ruan
et ab. 1996; Alizé et ab. 1998).
AVc report here the cloning and transfoimation of such constructs into A*, iabacum,
SRI in order to analyze the influence of the regulated barnase expression on plant
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ACMV Resistance 42
development, to assess the reduction of viial replication in transgenic plants tran¬
siently and to study the effect of the tiansactiA'ation of the barnase gene upon viral
infection.
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ACMV Resistance 43
3.3 Materials and methods
3.3.1 Plant material
Tobacco, Nicoiiana tabacum L. cv. Petit Havanna Str-rl (SRI) (Maliga ct ah.
1973), was maintained as axcnic shoot, cultures. These AA'ere groAvn in, vitro at 26°C
Avith 16 h light per dav on half-strength MS medium Avithout growth regulators
(Murashige and Skoog. 1962). Tobacco seedlings used for Aqrobactcrium-mediated
transformation were giown on filter paper wetted with sterile tap Avalen- for 8 davs
prior to use.
3.3.2 Bacterial strains
E. coli strain XLl-Blue Avas purchased from Stratagcnc (La Jolla, CA, USA). A.
turnefaciens strain GV31Û1, containing helper plasmid pGA'2260 (Deblaere ct ah,
1985) was kindly provided bv B. 'Finland. Zürich.
3.3.3 Construction of plasmids
Plasmid pACMV A is a partial repeat (1.3mei) of DNA A (West Kenyan isolate
844 (Stanley and Gav. 1983)) in pBS II KS-. The cloning of the J.Smer of DNA A
has been described (Klinkenbeig et ab, 1989).
All DNA manipulations Avère pciformed according to standard procedures (Ma-
niatis et ah, 1982). Nucleotide numbering of the \CMV genome refers to p.IS092 and
pJS09f (Stanley and Davies, 1985). DNA A and names of the open reading frames
are according to suggested nomenclature (DaAdes and Stanley, 1989), unless the
function of a gene is knoAvn. In this case the name of the gene product, is indicated.
The bidirectional ACAIA' promoter Avas produced lw PCR amplification of ACMV
DNA A using primers ACAIA'c (GGAA -2755- GCTTTTTGACCAAGTCAATT-
GG -2776) and ACAlA's (300-GTGGTACCCACTATTGCGCACTAGC -267). The
ACMA* promoter was introduced as a Kpnlf Muni fragment between the two to¬
bacco scaffold attachment legions (SAR) (Brevnc et ab. 1992) in the backbone of
pGluChi (Bhffcld et ab. 1999). The barstar ORF (Hartley, 1988) was cloned under
the control of the AC promoter as an Ncol/Psll fiagment ,Avhile the barnase ORF
(Hartley. 1988) (both kindly provided by J. Fiitteicr. Zürich) Avas introduced as a
blunt-end fragment into the blunt Spel site under the contiol of the AVI promoter.
Both ORFs Avere cloned together with a 35 S teiminator. The hvgromycin resis¬
tance cassette Avas cloned as a blunt end fragment cloAvnstream of the barstar gene,
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ACMV Resistance 44
Table 3.1
Number of ORFs in different constructs, depending on oligo-nucleotide
sequence
Ncol site ß'coRA* site no. of sORFs
+ -T- one (6 aa)
y - tAA"o, overlapping
4 none
- - one (7 aa)
resulting in plasmid pACAIA'bb (sec also Fig. [3.2]). Correct cloning of inserts into
the vector was confhmccl Ida- restriction digests and sequencing.
In order to regulate barnase expression also at the translational level, vari¬
ous short ORFs Avere cloned bctAA'ccu the ACMY promoter and the barnase gene
(Ruan et ah, 1996). For the introduction of these sORFs it was necessary to sub¬
clone the Ihn dill fragment of p.ACAfA<*bb into pGN35Sluc+ (kindly provided by G.
Ncuhatis. Basel). Primers sORFJ (CGAACGGATCAACCAT(G/A)GCCGGCG-
AT(A/G)TCAGCTAGCT) and sORF2 (AGCTGA(C/T)ATCGCCGGC(T/C)AT-
GGTTCATCCGTTCGGTAC) were annealed and ligated to the Kpnl and Sad
sites of the Ihndlll fragment. This resulted in four different sORF constructs de¬
pending on AAdiich of the degenerated oligonucleotides AA-as introduced. These sORFs
could be discriminated by an AYR site (present in the primer with G in the hist
degenerate position) and an iYoRA* site (present in the primer Avith A in the second
degenerate position; see also table 3.1).
As a positive control the fireflv luciferase and the Rcnilla lucifcrase were cloned
under the control of a 35S promoter and a 35S tciminator (kindly provided by S.
Brunner, Zürich) rendering plasmid pLucpos and pRenpos respectively (see Chapter
2). To reduce the lucifeiasc activity and thereby make measurement more conve¬
nient, the 35S promoter only contained nucleotides 1 to 93. As a non-replicating
negative control, pACMA" AACl. a part of a 1.3nier of the DNA A, Avithout the
fragment containing the DNA A from bp 2732 OA'er 2779/1 until bp L868, in which
most of the Rep ORF (bp 1680 to 2756) is excised. Avas used. This plasmid Avas
kindly provided by II. Wohlwend. Zürich.
In order to transform plants Avith Agrobactci /utn. the bainase construct was intro¬
duced into pNCl as a complete I-Seel-fragment. p.NCf is a pCambial-300 backbone
with a pMCSö polylinker (MoBiTcc. Germany) containing an I-Seel site. The re¬
sulting plasmid, pNCbb. Avas then electioporatcd (Alattanovich et ab, 1989) into
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ACMV Resistance 45
« 1 ^ -Sf« t 'S g. g 5$
-jj SAR~~}|iSS HEärnäis^ }j ACMVfJBres""^ hpt fcjiä^' SAB. }—
Figure 3.2: Schematic representation of barnase expression cassette. Barnase
expression cassette shoAving the position of the cloned ACAIA7' DNA A promoter between
the barnase and the barstar ORFs. The barnase ORF contains the iutron from rice tungro
bacilliform virus; both ORFs cany a 35S terminator (35S). An hpt ORF under the control
of a 35S promoter (35SP). with a not terminator (nos') is included as a selectable marker.
The whole expression cassette is flanked by scaffold attachment regions (SAR). The bar
over the map represents the position of the barnase probe used for Sorrthern hybridization
analysis.
GV3101 (pGV2260) resulting in strain GA*bainase for agroinoculation of seedlings.
3.3.4 Tobacco mesophyll protoplast isolation, transforma¬
tion and regeneration
Mesophyll protoplasts Avere isolated from in vitro-grown tobacco plants. Leaves
were cut into 1 cm2 pieces and digested overnight in PCN1 (Golds et ab, 1993)
containing 1% Cellulase Onozuka RIO (Yakult Pharmaceuticals Inch Co.. Ltd.,
Japan), 0.5% Alacero/vme RIO (Yakult Pharmaceuticals Inch Co.. Ltd.. Japan) and
1 drop TweenSO (Nagy and Alaliga. L976). The protoplast suspension was filtered
through a 100-//m steel mesh sicA-e. After collecting the protoplasts by floating on
PCN1 they Acere AATcshcd tAvice Avith W3 (Alenc/el et ab. 1981) and then incubated
in the dark at 4°C for 1-2 hours. Samples of 250.000 to 500.000 protoplasts were
resuspended in transformation buffer (15 mAl AlgCL. 0,1% VIES 0.5 A4 mannitol,
pH 5.8) and transformed with 20 rig of DNA using PEG (Negrufiu et, ab. 1987).
PCN2 (Golds et ab. 1993) AA-as added to the transfoimcd protoplasts before thcy
AA'crc incubated at RT in the dark.
After 21 hours protoplasts Avere harvested bv centrifugation and rcsuspended in
100 fil passive lysis buffer for the Dual-Luciferasel!i reporter assay (Promega, Catalvs
AG, Switzerland). Extracts avcic stored at -80°C for up to one month.
For stable transformation, protoplasts avcic Avashed after the transformation and
rcsuspended in 0.5 ml of PCN2. Afiquots of 4.5 mf AA-aim PCN2 containing 0.6%
seaplaqtic agarose Avere caicfullv mixed with the transformed protoplasts in a 6 cm-
Falcon petri dish. Aftei solidification of the agarose, the piotoplasts Avere cultured
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ACMV Resistance 46
in the dark for 24 hours and in semi-daikness for the following Aveek. The agarose
disc was then cut in half and each half immersed into 30 ml of medium A (Caboche,
1980) containing the appropriate selective agent. Bead cultures Avere incubated on
a rotary shaker with 80 rpm until resistant colonies could be seen after 3-4 weeks.
One week later, these were placed on solid MS moipho medium (Spangcnbcrg et ab,
1990) where shoot, development took place after 2-6 Aveeks. Fully developed shoots
were transferred to MS medium Avithout hoimones. After establishment of a root
system, plantlets could be kept as stciile cultures or transplanted to soil in the
greenhouse.
3.3.5 Agrobacterium-medmtcd seedling transformation of
tobacco
Eight-day-old tobacco seedlings were transformed bv A-acuum infiltration (Rossi
ct ah, 1993) using an overnight, culture of GA'bainasc groAvn in 2 mi YEB (AVrvlietet ah, 1975) containing the appropriate antibiotics. The seedlings were cocultivated
for 3 days on solid MS medium (Alurashigc and Skoog, 1962) aid then Avashed
in 10 rail AlgSOt. They avcic then placed on MS medium containing 0.1 /ig/ml
NAA, l/ig/ml BA. 25 //g/ml hvgronrvcin. 500 fig/ml cefotaxime and 500 /ig/ml
A'ancomyein. Seedlings AA-ere tiansfcired to fresh plates cA-erv Aveek. After 7-8 Aveeks,
shoots could be collected from single calli and transferred to MS medium without
selection. After multiplying each plant line, rooted plantlets could be transferred to
soil or kept as in vitro cultures.
3.3.6 Dual-Luciferasem reporter assay
Protoplast extracts Avere tlnrwed and cell clebiis Avas collected by centrifugation. 50 /d
of Luciferase Assay Rcagentlt (Piomega. Catalvs, Switzeiiand) Avere pro-dispensed
into luminometcr tubes before adding 10 //I of extract and mixing Avell with a
pipette. Measurements Avere peifoimed Avith a luminometer (Lumat LB 9507. EG &
G Berchthold, SAvitzerland) Avith dual injectors dining 5 seconds each. After mea¬
suring the luciferase activity, in 'relative light units' (RLU). Stop and Glo Reagent
was delivered into the tube by automatic injection. Measurement of the Renilla lu¬
minescence was started after a tAvo second delay. Foi each experiment, background
luciferase activity from protoplasts transformed Avith a A'ector Avithout lucifcrase
genes was subtracted throughout. Protein concentiation. estimated using a Bio-Rad
protein assay as desciibed (Bradford. 1976), Avas used to normalize each measure¬
ment. All resulting valuer Aveie standardized to the positive control consisting of
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ACMV Resistance 47
protoplasts transformed with pLucpos and pRenpos.
3.3.7 Molecular analysis
PCR
Total DNA from transgenic tobaccos and an untiansformed control Avas extracted
using the NucleonPhytopurc kit (Amersham Life Science, England) as described by
the manufacturer. PCR reactions (Mullh and Faloona, 1987) Avere performed in
a final volume of 50 /d using J //g of plant DNA and 5 units of Taq polymerase
(QIAGEN, Hilden, Germany). Primers avcic added to a final concentration of 1/jJM
each and dNTPs (Boehiinger Mannheim. Gcimauv) Avere used at a concentration of
250 ßM. PCR cycles consisted of a 1 min clenaturation at 95 °C a 1 min annealing
at around 60 °C (depending on GC content and length of primer) and an elongation
period of 1 min at 72 °C. After 25 cycles, amplified piociucts were sepaiated by
agarose gel electrophoresis. In order to detect the bainase gene, a 600 bp fragment of
the barnase and of part of the ACAIA' promoter AA-as amplified using primers ACMVF
(5'-CCACTATATACTTACAGCCC-3") and baniaseF(5;-C ATGTTCGTCCGCTT-
TTGCC-3'). To verify the presence of the hvgromvein resistance cassette, a 550 bp
fragment was amplified with primers 35Shpt5 (5'-G AAAAGGAAGGTGGCTCC-3")and hpt3 (5YAATAGGTCAGGCTCTCGC-3').
Southern blot
Total DNA from transgenic tobaccos and an untransfoimed control was extracted
using the NucleonPhytopurc kit (Amersham Life Science, England) as described
by the manufacturer. Aliquots of 10 fig of this DNA Avere digested either Avith
RstL expected to give lise to a fiagment containing the complete barnase-ACMV
promoter-barstar (around 1.5 kb) or H//?clIII. Avhich should result in a fragment
of around 700 bp and a single cut in the T-DNA leading to the plant DNA. Af¬
ter agarose gel electrophoresis. DNA Avas transferred to a positively charged nylon
membrane (HybondN. Amersham) bv blotting overnight as described (Sambrooket ah. 1989). After a shoit Avashing of the mombiane. the DNA was coA^alently
bound bv exposure to UA* (LA* Stratalinker 1800. Stratagcnc). The membrane Avas
prehybriclized at 42°C with 10 ml DIG Easy Ha-Id1" (Roche .Molecular Biochemicals,
Switzerland). After one hour the prehybiidization solution Avas replaced Avith fresh
hybridization solution including 20 ng DIG-labeled probe and hybridization took
place at 42°C overnight. DIG-labeding of the DNA Ava-, performed according to the
PCR DIG Probe SAmthesis Kit ' standard protocol (Roche Molecular Biochemicals,
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ACMV Resistance 48
Switzerland). A 600 bp fragment of the bainase anel part of the AC.VfV promotei Avas
amplified. After remoA'at of unspecific Irvbridization and an incubation in blocking
buffer, the anti-DIG antibodies avcic added. FolloAving equilibration, the DNA side
of the membrane was OA'erlaycd Avith substrate solution (CDPstarlM, Roche Molecu¬
lar Biochemicals, Switzerland). The damp membrane AA-as sealed in a hybridization
bag, fixed in a film cassette anel exposed to X-ray film (BIOMAX AIRY Kodak,
Rochester NN", USA) for f hour at 37°C. The film Avas developed with a developing
machine (AGFA, Cmix CO).
3.3.8 Replication assays
Transforrnation of tobacco leaf discs by particle bombardment
Tavo hours before bombai ciment leaf discs of 2 cm in diameter were placed on MS
medium, pH 5.8, supplemented Avith 10% sucrose and solidified Avith 0.8% agar. Tavo
/d DNA (0.1 fig/fd) were added into a tube containing 25 /d of gold-particle suspen¬
sion (50 fig/fd) and DNA AA-as precipitated on particles using CaCL, spermidine and
ethanol (Klein et ab, 1988). Aliquots of 5 //I Avere used for bombardment. A nylon
baffle grid (500 //m mesh) AA-as placed betAA'cen the filter and the target at 10 cm
fiom the filter, in order to reduce gas impact on the thsue. The target Avas placed at
12.5 cm from the filter. The chamber Avas evacuated to 100 mbar and the particles
AArcre accelerated bv a helium jet generated bv 6-7 bar pressure for 50 milliseconds.
Tavo hours after bombardment leaf discs AA-ere transferred to MS medium Avithout
groAvth regulators containing 2% suciose and incubated in the light at 24°C for six
days.
Analysis of ACMV DNA replication
Total DNA from bombarded leaf discs Avas isolated as described (Soni and Mur¬
ray-, 1994). Aliquots of 5 fig of total DNA from each sample weic digested with
Mlul/Dpnl and Avith Mlul/Dpnl/Mbol. The restricted samples were clcctiophoresed
through 1% agarose gels and transferred to nylon membranes (Hybond N, Amer¬
sham). ACAIA* replicated DNA \vas detected by hybridization to an AVI probe
labeled with digoxigenin-tl-dUTP. Labeling, hybridization and chcmiluminescent
detection were peiformed according to the instiuctions of the manufacturer (RocheMolecular Diagnostics). p.VCAlAA. lestiicted Avith Mini, resulting in a hybridizing
fragment of 2.7 kb. Avas used as a positive contiol.
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ACMV Resistance 49
3.4 Results
3.4.1 Expression of active barnase can be enhanced tran¬
siently in tobacco protoplasts
In order to test the activity of the barnase and to assess the ACMV promoter regu¬
lation, tobacco protoplasts aatic cotransformed Avith pLucpos. pACMVbb and Avith
or Avithout complete DNA A. theiebv mimicking a viral infection. As expected.
the luciferase activity in protoplasts in which the barnase expression AA-as activated
(cotransformed Avith DNA Ah Avas much Icwcr (30% of the luciferase activity in
protoplast in AAdiich barnase was not activated) than in protoplasts cotransformed
only Avith the barnase and the luciferase constructs. Due to the high toxicity of
the barnase, it was expected, that additional doAvmegulation of barnase expression
might be necessary. Different sORFs (see table 3.1) AA-ere introduced upstream of
the barnase to reduce translation. Four different constructs containing sORFs avcic
tested. Also with these constructs a reduction of luciferase activity could be ob¬
served upon cotransfoimation AA'ith the complet" DNA A, although this reduction
was slightly less marked (45 - 50%, of the lucifcrase acthdt\- in protoplasts in Avhich
barnase was not activated).
3.4.2 Analysis of transgenic tobacco plants containing a reg¬
ulatable barnase ORF
Tobacco plants were transformed with p.ACAIA'bb via Agrobaderium-mediatcd seed¬
ling transformation. Resistant plants were analyzed bv PCR, amplifying a 600 bp
fragment of the barnase anel part of the ACAfA" piomoter or a 550 bp fragment, of the
hvgromycin resistance cassette. PCR results iCA-ealecl that even though all plants
contained the hygromycin gene, no plants contained the barnase gene. In order to
examine this partial integration of p.ACAfA*bb. plants were analyzed additionally
by Southern blot. Of the analyzed plants onlv one plant contained the complete
fragment liberated by Pstl (sbbl7), but at least 4 plants contained rearranged in¬
serts AAuth only part of the barnase gene present. Since transformation efficiency
seemed Ioav and onlv few plants could be icgeneratcd, a neAv appioach Avas tested,
transforming plants with p.ACAIA'bb containing diffeicnt sORFs. These sORFs have
been shoAvn to down-regulate gene expiession in mammalian, veast and plant cells
8- and 20-fold (Fütterer and Hohn. 1992: Ruan et ab. 1996; Afize et ab, 1998).Transgenic tobacco plants Avere pioduced via PEG-mediated protoplast transforma¬
tion. Upon analysis wit h PCR, transformed plants icgeneratcd from protopfasts
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ACMV Resistance 50
sbbl? +-f4 ++10 + -10 + -11PHPITPHPHPII
-+5 wt
kbpHPIIPHPHPII
P H P H
8.6
4-é
6.0
4.8
3.5
2.7
1.9
1.5
1.4
1.2
1.0
0.7 — m&
Figure 3.3: Southern analysis of barnase transgenes in tobacco. Southern blot
of ffmdITT (H) and Pstl (P) restriction fragments hybiiclized to the barnase probe. To¬
tal DNA of transgenic plants digested with Pstl gives rise to a fragment containing the
complete barnase-ACMV promoter-barstai fragment (around 1.5 kb), while digests with
ffindlll result in a fragment of around 700 bp and a single cut in the T-DNA leading to
the plant DNA. Names of plant lines are according to the respective sORF (see table 3.1).
were negative for amplification of the barnase gene but positive for amplification of
the hvgromycin resistance gene. When the same plants were analyzed by South¬
ern blots, most sliOAA-ed rearrangements AA'hen hybridized with the barnase probe.
Only 6 plants that contained the complete Pstl fiagment avcic found (see Fig. [3.3])in addition to sbb!7. AAdiich had been transformed earlier via Agrobacterium. The
7 plant lines were groAvn and multiplied as in vitro shoot cultures and developed
normally except for plants of line sbb!7. On older leaves, these plants developed
necrotic lesions that gradually spread over the complete leaf and also to younger
leaves. Plant growth was reduced after the first lesions were visible. 3-6 Aveeks
after subculturing.
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ACMV Resistance 51
Mini Dpnl MlulDpnlMbol
sbbl? -+5 wt sbbl7 -+5 wt
kb A AACt 0 A AACt 0 A AAC1 0 A AAC1 0 A AAC1 0 A AAC10
4.8—
3.5 —
2.7—
Figure 3.4: Leaf replication assay. Total DNA of shot leaves of plants sbb!7, - +5 and
wildtype tobacco was isolated and equal amounts Avere digested either with Mlul/Dpnl or
with Mlul/Dpnl/Mbol. Viral replication occurred only Avhen leaves were shot, with DNA
A and was reduced in transgenic plants.
3.4.3 Viral replication in transient assays is reduced in trans¬
genic plants
Plants containing the complete barnase and baistar genes and untransformed control
plants were used for replication assays to test the inhibition of viral replication in
these plants. Leaf discs avcic transformed bv particle bombardment with p.ACMV
A, pACMVAACl, a replication defective DNA A. or with a control vector Avithout
viral DNA A. Standard amounts of total DNA, isolated after 6 days, were digested
Avith Mlul/Dpnl or with Mlul/Dpnl and Mbol. Taking advantage of the mcthylation
sensitivity of Dpnl (active onlv if DNA is methylated) and Mbol (active only if DNA
is not methylated) it is possible to distinguish between input DNA (methylated) and
de-novo synthesized viral DNA (not methylated). In the restriction digest Avithout
Mbol a viral band AA-as detected only if viral replication occurred in the leaf. This
band disappeared upon additional digestion Avith Mbol, confirming the viral nature
of this DNA. Input DNA Avas completely digested in both cases. Compared to
replication in wildtype plants (100%). viral replication in leaves of plants sbbl7,
sORF-+5 and sORF-+Ll transfoimed with pACMV A was much loAver at 13%,
30% and less than 1% lespectivcly (see representative example in Fig.[3.4]). No
replication occurred in leaA-es transformed Avith p.ACA'IA* AACl or with an empty-
vector. A repetition of this expeiiment could confirm the reduced viral replication
in transgenic plants.
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ACMV Resistance 52
3.5 Discussion
Barnase, an RNase of Bacillus amyloliqucfacieiis, is able to efficiently trigger cell
death and can be specifically inhibited by the barstar in plants (Mariani et ab,
1990; Mariani et ab, L992). As expression of a dianthin gene under the control of the
regulatable ACMV AV promoter has been shown to confer ACMV resistance (Hong
et at., L996), our strategy, using the ACAIA* promoter together with the barnase and
barstar to confer virus resistance, should be feasible. The system tested here is more
advanced, since beside the barnase. undei the contiol of the ACMV AV promoter,
also the barstar, under the control of the ACAIA* AC promoter, plays an integral
role, inhibiting the Ioav level of produced bainasc in non-infected plants.
The fact that the bainasc is active and that its expression can be activated Avas
demonstrated with transient experiments in protoplasts using the lucifcrase as a re¬
porter gene. The luciferase activity in protoplasts cotransformed Avith pACMVshvg
and pLucpos was higher than that of piotoplasts cotransformed Avith pACMVshyg,
pLucpos and ACA'IA" DN.A A. indicating that barnase expression AA'as activated by
the DNA A and that the increase of barnase production did actually reduce RNA.
This could be seen for barnase constructs both with and without sORFs.
Stable transformation of tobacco plants with these constructs proved to be diffi¬
cult even though, initialh, the regeneration frequency of hvgromycin resistant plants
seemed normal. I4oweA-er, Avhen analyzed bv PCR, no fragment could be amplified
from any of the regenerated plants. Onlv Avhen analyzed bv Southern blot could
the presence of the AA1CA* piomoter and the barstar ORF be confirmed. Secondary-
structures of the ACAIA* promoter sequence and the barstar ORF with high melt¬
ing temperatures of 6LC and 58°C respectivclv, might be the reason for the false
negative results of the PCR. Since these secondary structures did not melt at the
annealing temperatures required by the primeis chosen for PCR analysis, primer
annealing and subsequent polymerization AA-ere very inefficient and no PCR product
could be detected.
Only one plant containing the complete barnase-ACMA* promoter-barstar frag¬
ment could be identified from the first 8 transformation experiments. It has been
shoAvn for tomato golden mosaic vhus that repression of v-sensc transcription is not
maintained in unorganized tissue (Sunter and Bisaro, 1997). This could explain the
Ioav yield of plants containing the complete insert: most plants containing a com¬
plete insert did not survive regeneration via organogenesis, since this comprises a
callus phase. Consequently, a reduction of barnase expression from the uninduced
promoter was nceessan-. For this purpose, four diffeient, ORFs. Avhich in another
system (Ruan et ah. 1996: Afize et ab, 1998) inhibit translation of downstream ORFs
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ACMV Resistance 53
between 8- and 20-fold, were introduced upstieam of the barnase ORF. The yield
of plants containing the complete insert AA-as slightly better in these transformation
experiments. A total of 6 plants containing the barnasc-ACMV promoter-barstar
fragment with a sORF could be regenerated, but still the amount of plants with
rearranged copies of the transformed plasmid was higher than expected. Probably,
these plants would usually regenerate slower than plants containing a normal copy
of the transformed plasmid and would not be detected in an experiment Avithout
toxic genes involved. Since in this case, a genie leading to the production of a toxin.
the barnase, was used for transfoimation. plants containing rearranged copies of
the barnase ORF might have an ach-antage and might regenerate faster and more
successfully compared Avith plants aaIUi a complete bainasc ORF. Furthermore, the
variability of transgene expression, as demonstrated bv the higher activity of Rcnilla,
luciferase (corresponding to barnase) as compared to firefly luciferase (correspond¬
ing to barstar) observed in line sir9 (sec Chapter 2), could be another explanation
for the Ioav number of plants regenerated containing an intact copy of the transgene.
If at one stage during regeneration more barnase than barstar is present, in the cell,
no plants will be able to regenerate fiom these cells.
The regeneration of plants containing an intact copy of the transgene, was sloA\rcr
when compared to wildtypc regeneiation. but all plants developed normally, except
for those of line sbb!7. The observed random pattern of brown spots spreading
on older leaves of sbbl7 seems to be due to bainasc activity. As these spots first
appear on older leaves, it could be speculated that barnase is slightly more stable
than barstar and slowlv accumulates until more barnase than barstar is present in
the cell. The death of such a single cell might also affect surrounding cells either
directly through the barnase or bv initiating a hypersensith'c response. It is also
possible, that the rclath-c kwel of barnase1 to baistai expression is not exactly the
same in all the cells of a plant, that some produce shghtlv more barnase than others,
leading to stress and consequently to a hypersensitive response. As these broAvn
spots were not observed in anv of the other tiansgenic lines, it can be assumed,
that line sbbl7 has the most fragile balance of barnase to barstar and that in ail
the other lines relatively less barnase is produced. That such a variation is likely to
occur can be concluded from results obtained Avith tiansgenic plants containing the
ACMV-luciferase construct (see Chapter 2).
The results of the replication assavs with leaves of transgenic plants compared
to AAdldtype leaves cleailv show that leplication of viral DNA is severely reduced in
transgenic plants of all tested lines. The highest i eduction of replication was seen for
plants of line sORF-+ll and sbbl7. which does suggest that the balance between
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ACMV Resistance 54
barnase and barstar is actually more fragile in these plant, lines than in the others
containing sORFs. It cannot be expected that replication of viral DNA is completely
abolished due to the barnase. As long as the barnase is activated before the virus
can spread out of the infected cell, the plant would still be resistant even if some
replication occurs. In order to find an optimum balance of a high and fast barnase
production which the plant can still support even in older leaves, more transgenic
plants need to be regenerated.
Since Nicotian a taba cum SRI plants could not be agioinoculated with ACAIA*.
even though replication in SRI protoplasts Avas shcnvii. it Avas not possible to test
whether also the spreading of the virus avouIcI be icduced or inhibited. HoAveArer,
the obseiwed resistance of plants containing the dianthin under the control of the
ACYIV promoter (Hong et ab, 1996; Hong et ab. 1997), strongly suggests that our
approach could provide resistance against ACAIA* in the future.
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Chapter 4
Cassava regeneration and
transformation
4.1 Abstract
Transforrnation and regeneration of cassava (Manihot esculanta Crantz),
an integral plant for food security in developing countries, using different
systems has been published before. These protocols are all developed for
two cassava model cultivars. Since cassava is grown as many different
land races, it is crucial to adapt these methods to various cultivars. The
adaptability of transformation systems based on Agrobacterium or par¬
ticle bombardment to an African cultivar and the changes needed in the
published protocols for regeneration of transgenic plants of this cultivar
were assessed in this work.
4.2 Introduction
Cassava (Manihot esculcnta Crantz) is a perennial shiub, belonging to the family
Evphorbiaceae. It is one of the most important sources of calories produced within
the tropics (Cock, 1982: Puonti-lvaerlas, 1998) and provides food for OA-er 500 million
people. Cassava roots can be processed into a Avide vaiiety of products for food and
inclustiial uses and since most, of the processing can be clone locally, it provides rural
jobs and income (Henry et ah, 1995). Cassava is grown because of its robustness and
its reliability as a superior producer of carbohydrates even on poor soil and for its
ability to survive seasonal diought and othei aclveisc environmental conditions (Thro
et ah, 1995). Up to 80 t/ha loots can be produced nuclei optimal conditions in a 12
month growing season, but yields arc severely reduced by insect pests and diseases,
o5
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 56
that cause up to 70 - 80% losses locally (Roca et ab. 1992). During the last decade the
interest in cassaA^a breeding has groAvn and it has been recognized that biotechnology-
might be a useful tool for genetic improvement of cassava; especially since traditional
breeding is very difficult. High heterozygosity, irregular flowering, Ioav seed set and
variable germination rates constrain the crossing of different cassava cultivars to
combine desirable traits. Genetically engineered plants could complement, these
efforts and contribute to a sustainable agricnltuie and an improved diet in the third
world countries.
Gene transfer to plant cells can be performed by tAvo different systems: di¬
rect gene transfer or Agrobacferivm-mQdiatQd gene transfer. Direct gene transfer
methods, such as microinjection, PEG ticatmcnt. calcium-phosphate precipitation
or electroporation. mostly need specific protocols to regenerate fertile plants from
protoplasts. On the other hand, cliiect gene transfer through bombarding small
DNA-coated particles to intact, cells can be used to transform organized tissues
(Klein et ah, 1988). One of the devices that Avere developed for biolistic transfor¬
mation is the Particle InfloAV Gun. Avhich is inexpensive and simple to use (Finer
et ah, 1992). Both methods have been used to transform cassava: cotyledons from
somatic embryos were transformed via Aqrobactenum, and particle bombardment (Lict ah, 1996; Legris et ah, 1998) and embiwogenic suspensions AArcrc transformed via
Agrobacterivm, and particle bombardment (Schöpke et, ab, 1996; Raemakers et ab,
1996; Gonzalez et ab, 1998).
As cassaAra is groAvn as manv different land races, it is crucial that these regener¬
ation and transformation systems can be applied to a Aviclc range of cultivars. The
transferability and adaptability to an African cassava cultivar were assessed in order
to engineer African cassava mosaic vims resistant cassava.
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 57
4.3 Materials and methods
4.3.1 Plant material
In vitro culture of cassava (Manihot esculenta Crantz)
Cassava cultivars TMS 60114 (kincllv provided by UTA) and YlCol22 (kindly pro¬
vided by CIAT) were maintained as shoot, cultures in Magenta-vessels on basic
medium BMS (MS salts and vitamins (Alurashige and Skoog, 1962) Avith 2% su¬
crose, supplemented Avith 2 //Al C11SO4 (Schopkc ct ah, 1993) and solidified with
0.7% agar, pH 5.8). The cultures Avere kept at 25 "C at an 18 h photoperiod anel
subcultured every 8 weeks, transferring the shoot apexes to fresh medium.
Suspension cultures
Embryogénie suspension cultures avcic established from fiiable embryogénie calli of
cultivar TMS 60444. The suspensions Avere grown axcnicallv in liquid SH medium
(SH salts with AIS vitamins. 6% sucrose and 12 mg/1 picloram. pH 5.8 (Schenkand Hildebrandt, 1972; Taylor et ah. 1996)) on a rotaiy shaker (80 rpm) under
continuous Ioav light in a groAvth chamber (Infors Incubator HT04). Suspensions
Avere subcultured once to twice a week depending on the desired growth rate. Every
four Aveeks the suspensions were filtered through a 1 mm2 mesh sicA^e and larger
particles were discarded. For matination. the suspensions Avere transferred to solid
maturation medium (BAIS with 4% sucrose and supplemented with 1 mg/1 picloram)and subcultured CA-erv Iaa-cd weeks as described (Racmakcrs et ah, 1996). Variations
of maturation medium consisted of BMS supplemented Avith 0.5 mg/1 picloram, 2
mg/1 picloram L mg/1 NAA or no picloram. A [attire embryos were transferred to
germination medium (BAfS supplemented Avith 1 mg/1 BA) for shoot, development.
Regeneration via organogenesis
Cassava mcristems Avere excised from shoot cultures and transferred to BMS con¬
taining 12mg/l picloram (Taylor et ah. 1993) to induce embryo development. To
remove faster groAving callus, somatic embryos AA-ere subcultured every 4 Aveeks,
therebv establishing a evelic somatic embryo system. Mature somatic embryos Avere
harvested and transferred to maturation medium (BMS supplemented Avith 0.1 mg/1
BA). Green cotyledons from mat ruing embtwos Avere cut into pieces of 1 - 3 mm2
and transferred to organogenesis medium (BAfS supplemented with 1 mg/1 BA and
0.5 mg/1 IBA). After 20 da\'s of induction, shoots or organogenic structures could
be detached and transferied to elongation medium (BAfS supplemented with 0.4
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 58
mg/1 BA). Elongated shoots Avere moved to BALS for rooting. Additional shoots
could be harvested until up to three months after induction. In order to enhance
regeneration, various organogenesis and elongation media AA-ere tested. Organogen¬
esis medium Avith different auxins (0.1 mg/1 IB A, 0.25 mg/1 IBA, 0. L mg/1 NAA or
0.25 mg/1 NAA), with different concentiations of BA and IBA (1.5 mg/1 BA with 1
mg/1 IBA, 1 mg/1 BA with 0.8 mg/1 IBA, 0.5 mg/1 BA with 0.1 rag/1 IBA and 0.5
mg/l GA.3 or 1 mg/1 BA Avithout, IBA) and organogenesis medium prepared Avith B5
salts (Gamborg ct ah. 1968) Avere tested in different experiments. Furthermore, an
elongation medium with additional GA^ at 0.1 mg/1 was tested. Recent, results aris¬
ing during the course of these experiments shoAved that AgNOs had a positive effect
on organogenesis of cassava (P. Zhang, pers. connu.), therefore, AgN03 AA-as added
routinely to organogenesis medium at a concentiation of 4 mg/1 in later experiments.
4.3.2 Bacterial strains and constructs
E. coli strain XLl-Bluc Avas purchased fiom Stiatagcnc (La Jolla, CA, USA). A.
tumejacicns strain GA*3101, containing helper plasmid pGV2260 (Dcblacre ct ah,
1985) was kindly provided bv B. "Finland. Ziiiich.
All DNA manipulations weic peiformed according to standard procedures (Ma-niatis et ah, 1982). Nucleotide numbeiing of the ACMA* genome refers to pJS092
and pJS094 (Stanley and Davies. L985). DNA A and names of the open reading
frames are according to suggested nomenclature (Davies and Stanley, 1989), unless
the gene function is known. In this case the name of the gene product is indicated.
The bidirectional ACAIA* piomoter was pioduced bv PCR amplification of ACMA'*
DNA A using primers ACAfA'c (GGAA -2755- GCTTTTTGACCAAGTCAATT-
GG -2776) and ACMVs (300-GTGGTACCCACTATTGCGCACTAGC -267). The
ACMA* promoter AA-as introduced as a Kpnl/Munl fiagment bctAvccn the two to¬
bacco scaffold attachment legions (SAR) (Brevnc et ab. 1992) in the backbone of
pGluChi (Bliffcld et ab, 1999). flic baistar ORF (Haitley, 1988) was introduced as
an Ncol/Psll fragment under the contiol of the AC I promoter, Avhile the barnase
ORF (Hartley, 1988) (both kincllv provided by .1. Fütteret. Ziiiich) Avas introduced as
a blunt-end fiagment into the blunt Spel site tinclci the control of the AVI promoter.
Both ORFs Avere introduced together with a 35S terminator. The hvgromycin resis¬
tance cassette AA-as cloned as a blunt-end fiagment downsticam of the barstar gene,
resulting in plasmid pACAlVbb (see also Fig. "4.1"). Correct cloning of inserts into
the vector Avas confirmed bv restriction digests and sequencing.
In order to transform plants with Agrobaclcrium. the barnase construct Avas intro¬
duced into pNCl as a complete l-oYel-fiagment. pNCl is a pCambial300 backbone
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 59
t-3
Hi
"a *3 ^
i< do^
~H SAlY~}[35S|JBia-nase| rjTACftlV [fil^^ hpt H^C/^^%)—
Figure 4.L: Schematic representation of barnase expression cassette. Barnase
expression cassette showing the position of the cloned ACMV DNA A promoter between
the barnase and the barstar ORFs. The barnase ORF contains the intron from rice tungro
bacilli form virus; both ORFs can a* a 35S terminator (35S). An hpt ORF under the control
of a 35S promoter(35SP), Avith a nos terminator (nos1) is included as a selectable marker.
The whole expression cassette is Hanked by scaffold attachment regions (SAR). The bar
over the map represents the fragment amplified by PCR.
Avith a pMCS5 polylinker (AfoBiTec. Germany) containing an LoYel site. The re¬
sulting plasmid, pNCbb. AA-as then elcctroporaled (Alattanovich et ab, 1989) into
GV3101 (pGV2260) resulting in strain GA'barnase for agroinoculation of seedlings.
A defective interfering (Dl) DNA (Stanley et ab. 1990; Frischmtith and Stanley,
1991), kindly proA-idecl bv T. Frischmuth. Stuttgart, Avas cloned together with a
hvgromycin resistance gene under the contiol of a 35S promoter (construct pNPf).The hvgromycin resistance cassette Avas introduced as a Sail/BamUl fragment into
the Xhol and BamYLl sites of the DI DNA construct. For construct pDIluc, the
same DI DNA Avas combined as a Sad fragment, Avith a 35S-luciferase cassette as a
Hin dill/Aalll fragment in a pSP72 vector (purchased from Promega, Catalys AG,
Switzerland).
4.3.3 Transformation
Particle bombardment
Cotyledon explants Avere harvested from secondai'}' embrwos and transferred to trans¬
formation medium (BMS Avith 10% sucrose) 18 - 2 f h prioi to particle bombardment.
Embryogénie clusters avcic collected from suspension cultures and spread on sterile
filter papers on transformation medium L8 - 24 h before transformation. The particle
infloAv gun used is a microptojectilc accelerator (Finer ct ah, 1992), AAdiich is equipped
Avith a 2-Avay solenoid valve Lucifer E121K14 instead of the described one (ASCO,Floiham Park. N.T. Red Hat II). Commeiciallv aATtilable gold-particles (Aldrich or
BioRael) were coated with DNA using a protocol modified from EMBO Advanced
Laboratory Course (Potrvkus et ab. 1993). Briefly, 2 /d DNA (0.1 //g//d) were added
into a tube containing 25 /d of gold-particle suspension (50 /yg//d) and DNA Avas
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 60
precipitated on particles using CaCR- spermidine and ethanol. The DNA-particle
solution Avas vortexed shortly before loading 4.5 /d per shot. For bombardment,
helium pressure Avas kept constant at 5 bar. the target plate was positioned at, a
distance of 15 cm and the 500 //hi mesh stopping grid at a distance of 10 cm. Some
parameters for transformation of cassa\-a cotyledons have been partially optimized
by monitoring of tiansient GLAS expiession (Legris et ab, 1998). The bombarded
cotyledons or suspensions Aveie transferred to organogenesis or SH medium respec¬
tively 4 - 26 h after shooting and hvgromvein, at a concentration of 15 or 17.5 mg/1,Avas added 3 days after bombai ciment of the cotAledons. Emerging shoots could be
transferred to BMS between 20 elavs and 3 months after transformation.
Luciferase selection of transformed suspensions was performed using a sloAV-scan
CCD camera (CCD 3200 LN/C, Astrocam Ltd, Cambridge, UK) that detects pho¬
tons, which are emitted clue to the turnover of the luciferin substrate by the lu¬
ciferase. The luciferin substrate mixture (liquid BMS supplemented Avith 1 mM
click beetle luciferin (Promega) and ImAf YTP (Kost et ab. 1995)) was applied im¬
mediately before taking images on suspensions that AA-ere spread as a thin layer on
solid SH medium. Reference images, to localize the emitted light, fiom the expiants,
were taken by exposing the chip for 100 msec to i effected light. For bioluminescence
images, the time with an open diaphiagm. AA*as L0 min. Lucifcrase detection for
selection Avas performed every 3 to 4 Aveeks. Lucifcrase positive embryonic clusters
Avere isolated and either tiansferred to fresh SH medium for further multiplication
or transferred to maturation medium and subcultured every 2 Aveeks.
Agrobacterium-mediated transformation
Strain OA'barnase was groAvn for 2 davs in liquid AB medium (Chilton et ab, 1974)
containing the appropriate antibiotics at 28 °C on a rotary shaker (280 rpm). Before
cocultivation, the bacteria aati-c diluted to OD-fa0 0.5 and preindueed for 5 hours in
liquid BMS supplemented with 200 mAI aeetosviingonc. pbl 5.2. Cotyledon expiants
from secondary embryos Avere inoculated with Aqrobactenum, for 30 min, transferred
to cocultivation medium (BAfS supplemented with 100 mM acetosyringone, 1 mg/1BA and 0.1 mg/1 IBA) and cocultured for 3 cbrvs. Expiants were washed with
liquid BA4S, blotted dry on filter paper and tiansferred to organogenesis medium
supplemented with 500 mg/1 carbenicillin and 10.5 mg/1 hvgromycin. After 20 clays
the first developing shoots aa'Cic transferied to BAIS.
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 6L
4.3.4 Molecular analysis of resistant shoots
Total DNA from regenerated shoots and a Avildtvpe control Avas extracted using the
NucleonPhytopurc kit (Amersham Life Science, England) as described by the man¬
ufacturer. PCR reactions (Alullis and Faloona, 1987) using L rtg of plant DNA, were
performed in a final volume of 50 fd using 5 units of Taq polymerase (QIAGEN,
Hilden, Germany). Primers were added to a final concentration of 1 /.dYl each and
clNTPs (Boehiinger Alannhcim, Germany) AA'erc used at a concentration of 250 pM.
PCR cycles consisted of a 1 min clenatutation period at, 95 °C, a I min annealing
period at around 60 °C (depending on GC content and length of primer) and an elon¬
gation period of 1 min at 72 TJ. Amplified products Avere separated by electrophore¬
sis through an agarose gel. Primers AA-ere designed to amplify a 600 bp fragment
of the barnase ORF and of part of the ACAIA* promoter (ACMVF (5:-CCACTA-
TATACTTACAGGCC-3') and bamaseF (5'-C.ATGTTCGTCCGCTTTTGCC-3'))and a 550 bp fragment of the lrvgromvein resistance cassette (35Shpt5 (5'-GAA-
AAGGAAGGTGGCTCC-3") and hpt3 (5'-AATAGGTCAGGCTCTCGC-3')). As
internal control primeis Q3 (5'-TGACGGAGTAACATCAATGT-3') and Q4 (3"-
TACAATGGCTTGGTCATGAC-3": kmdlv provided by S.Bohl, Zürich) were used
to amplify a 380 bp fragment of the branching enzyme (Kallak et ah, 1997).
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 62
4.4 Results
4.4.1 TMS60444 transformation and regeneration via organo¬
genesis
Somatic embryos AA-erc subcultured cwerv four weeks cither to fresh embryo induction
medium or to maturation medium. Cotvleclon expiants AA-ere harvested for particle
bombardment after 10 to 14 clavs oi as soon as cotyledons were light green and
around 5 - 10 mm2. Three clavs aftci bombai ciment with pACMVbb or pNPi,
selection using hygromvein was started at a concentration of 15 mg/1. The first
organogenic structures could be seen after I to 6 Aveeks on organogenesis medium
in about, 90% of the expiants, although these structures looked slightly different
from organogenic structures of AICA122 (-,ee Fig.[4.2]). resembling primary embryos
emerging from a green, elongated structure. When these structures Avere transferred
to elongation medium, no shoots emerged and the embiyo-like bulbs eventually dried
out. Subculturing to fresh organogenesis medium also had the same effect. None
of the tested organogenesis media induced production of any structures similar to
organogenesis of AICol22. neither did the elongation medium supplemented with
GA}, improve regeneration. A summary of some of the tested media is shown in
table 4.1. Organogenesis medium wit h onlv 0.5 mg/1 BA (instead of 1 mg/1) or
Avithout IBA produced roots on 2% of the expiants while higher concentrations of
BA (2 mg/1) produced mostly callus anel foAv organogenic structures. Root forma¬
tion Avas inhibited if expiants were kept in the daik but callus formation was further
enhanced. The most, expiants Avith organogenic stiuctures (85%) Avere observed on a
medium containing 1.5 mg/1 BA with 0.5 mg/1 IBA cultured in the light and on the
standard medium (95%). but no shoots could be regenerated. In addition, exper¬
iments with organogenesis in the daik and the different transformation conditions
tested (variable times of pre- and postplasmolvsis) did not have any positive effect
on the regeneration of shoots. At least 50 explants Avere used for each individual
treatment and out of a total of around 8000 bombarded expiants only 4 lines could
be regenerated and rooted. All four lines Avere regenerated using the standard pro¬
tocol, with two lines bombarded Avith the barnase construct and two Avith the DI
construct. Neither the presence of the barnase genc/DI DNA, nor the hvgromycin
resistance gene could be confirmed bv PCR analysis. The internal control, part of
the branching enzyme, Avas correctly amplified foi all four plants.
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CHAPTER 4 CiSSAViRFGLNERATIOA IAD 1R AN3I0RM iTION (O
Figuie 1 2 Organogenic structures of cassava cultivars TMS6Ö444 and MCol22
(A) Oiganogcmc structures on TMSbOill (ot\l(don iftcr 20 davs on organogenesis
medium (B) Organogenic stiurtuie on AlCol22 cotA l< don after 11 davs on organogenesis
medium
4.4.2 MCol22 transformation and regeneration via organo¬
genesis
Vs regeneiation of 1AIS60441 pioAecl \<i\ drffrc ult if awis decided to use eultuai
MC0I22 for huilier expeiiments Secondan somatic ombnos of AfCol22 (kmdh
provided by S Phansm Bangkok) aacic geimmated and bombaided using PIG 01
m one evpeimrent ( ">(!(.) expiants) [qiobadrniim Since non-transgeme plants legen-
eiated under selee hon a higher com entiation ot h\giom\cra awis used for selection
(17 5 nig 1) and also \g\0> AAas added loutmeh to the oiganogenesis medium
Organogenic stiuctuies could be detected as soon as 3 aaccYs after bombardment
and shoots could be hanested until up to 5 months latei Out of 6200 expiants
bombarded with p ACMA bb 7 lines ( ould be icgennated and of 2700 expiants bom¬
barded AAifh pNPI I lines < ould b< legeneiated \o shoots could be icgeneiated
(10m the» iqiobadniiiin-medxàïed tiansfoimation oxpeuitin nts Fach organogenic,
stiuctme (oiigm of one- plant Inn) produced more than one shoot (see Fig [ 1 I])
The 11 regenerating lines produced about 50 shoots m total of A\hieh about 90%
could be looted 111 BAfS Aeithei tlie bainasc» OR! nor the DI DA \ noi the h\-
gionnun resistance gene could be detected m am of the lines Ida PCR anahsis
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 64
Table 4.1:
Influence of different hormone concentrations In the organo-
genesis medium on the formation _of organogenic structures.
Hormone cone, (mg/1) % of expiants with org. structure
BA IBA light dark
1 0.5
0.5 0.5
1.5 0.5
2 0.5
1 0
"additional root, formation
The expected part of the branching enzvme a\xis correctly amplified in all lines as
an internal control. An example of a PCR with some of the shoots bombarded with
the barnase constmct is shoAvn in Fig.[4.3].
4.4.3 Transformation and regeneration of suspension cul¬
tures
Suspension cultures of cultivar TA1S6041 f Avere established from friable cmbrvogerric
callus (FEC) and maintained as described (Taylor et ah. 1996). Freshly filtered sus¬
pensions Avere used for transfoimation. spreading a thin layer of suspension on a
filter paper of 2 cm diameter on transformation medium. All suspensions were bom¬
barded using the particle infioAv gun and the construct pDIhrc. After transfoimation,
the suspension cells of one experiment (10-20 dishes) AA'crc combined and transferred
back to liquid SH medium. Three to four weeks after bombai ciment, suspensions
were spread on solid SH medium and analyzed for lucifcrase activity. A 1 cm2 region
of suspension around the luciferase positive spot Avas isolated anel cultured individ¬
ually in liquid SH medium. Only after the second round of selection these luciferase
positive foci were transferred to solid maturation medium. All tiansformecl suspen¬
sions (200 dishes in 10 experiments) had single lucifcrase positive spots 3-4 weeks
after transformation (around 10 - 20 spots per experiment), however, about 50% of
these were not positive when tested for the second time. Furthermore, around 20 -
30% of the isolated luciferase positive suspensions turned Avhitc and stopped dividingafter the luciferase assav. Theicfore. in later expeiimcnts. transformed suspensions
were tested for luciferase activity onlv 2 months after bombardment, before transfer
to maturation medium. In these cases onlv very few luciferase positive spots (1 -
95
50
85
80
10
55
60
30
50
0
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 65
Internal control Barnase
bp sA sC sD sE sH sK sL sM sN It?C) sA sC sD sE sit sK sL sMsN + II2O bp
Figure 4.3: Gel electrophoresis of PCR analysis of cassava transformed with
pACMVbb. No amplified product of the barnase could be detected in any of the tested
shoots. As an internal control, a 380 bp fragment of the branching enzyme was amplified
for all shoots. 10 ng of pACMVbb avcic used as a positive control for the barnase PCR:
sterile water served as a negative control.
2 per experiment) could be detected. Four experiments still had luciferase positive
cell clusters 4 months after transformation (2 months on maturation medium), but
none of these could be regenerated. Maturation of cmbrvos was achieved onlv in
one experiment (luc-5) but. Avhen transfeired to germination medium, these embryos
did not develop further. No improA-ement of matmation could be observed Avith the
various tested hormone concentrations in the medium. Only in maturation medium
containing 1 mg/1 NAA two embiwos Avith green cotyledons were found, but after-
transfer to germination medium both of these turned white and did not develop
further.
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CHAPTER 4, CASSAVA REGENERATION AND TRANSFORMATION 66
Figure 4.4: Regeneration of multiple shoots from organogenic structure of
MCol22 after 3 months on elongation medium.
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 67
4.5 Discussion
Production of transgenic cassava plants has been reported using both Agrobactenum
and particle bombardment (Li et ah, 1996: Raemakers et ah, 1996; Gonzalez et ah,
1998) and regeneration via organogenesis of cotvledon expiants or via germination
of embryogénie suspensions. Applying these transformation systems to different
cultivars of cassava is important as cassa\*a is gioAvn as manv land races and often
exhibits Ioav fertility. Therefore, crossing of iicav genes into focal varieties might be
difficult. The goal of this Avork AA'as to strich" the applicability of the biolistic trans¬
formation system together with the icgeneration via organogenesis to the African
cultivar TMS60444.
The regeneration frequency of cassava cult ivai TMS60444 Avas low under all the
different conditions tested and onlv fecw shoots could be regenerated and rooted.
This could be due to different, reasons. Genotype dependent variations of regen¬
eration have been reported befoie (Kartha. 1971: Kallak et ah, 1997), therefore it
is not, surprising that a protocol established for organogenesis of AfCol22 does not
apply to the organogenesis of TAIS60441. More profound adjustments would be
needed, since our results demonstrate that minor changes in hormone concentra¬
tions of the standard media could not enhance regeneration. Preliminary results
in our laboratory show a considerable effect, of AgNYly on the regeneration of this
cultivar (P. Zhang, pcrs. comm.), hoAvevcr, at the time of these experiments, these
resutts were not available. The positive effect of AgNO^ on organogenesis of oil
seed Brassica, species (Eapen and George. 1997) and on icgeneration on a variety of
plants (Songstad et ah, 1988; Chi et ah. 1990) has been reported before, although
the actual mode of action has not vet been elucidated.
Regeneration of AJCol22 proA-ecl to be mote efficient, especially in combination
with particle bombardment, which in fact enhanced the formation of organogenic
structures. The Avounding of cells by particle bombardment and the thereby trig¬
gered wound reaction could be responsible for this effect. Nevertheless, not all
organogenic structures could be regcneiated to shoots. The step between the first
small shoot primordia and the complete shoot is the bottleneck in the regeneration
via organogenesis. Therefore, optimization of the protocol should not only be con¬
centrated on the organogenesis medium, but also on the elongation medium and on
the timing of transfer of organogenic structures to elongation medium. Our results
indicate that it is crucial to use only 10- to 14-dav-old cotyledons for transformation,
since older cotyledons form less organogenic struetuies and cannot be regenerated
to shoots, and younger cotyledons are too small to be used.
Regenerated shoots of both TMS60 144 anel MCol22 were not transgenic, as
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 68
shown by PCR analysis. Transformation Avith barnase - even in combination with
barstar - can be difficult (see Chapter 3: (Mariani et ab, 1990)) due to its high
toxicity, which leads to earlv loss of transformed cells. Also Dl DNA, consisting
of part of the ACMV DNA B. might have a negative effect on plant cells. As the
presence of the hygromvein resistance gene could not be proA^en either, the regen¬
erated shoots must have escaped selection. Selection with a higher concentration
of hygromvein (17.5 mg/1) could not prevent the regeneration of untrans formed
shoots cither, even though shoot regeneiation could be efficiently suppressed in con¬
trol expiants. It, appears that transformation via particle bombardment requites
a tighter selection system, since rccoA-civ of non-transgenic plants Avas not a se¬
vere problem after Agrobacteri um-mediated transformation (Li et ab, 1996). This
problem could be partlv clue to the enhanced icgeneration observed after bombard¬
ment, or to the smaller sectors transformed bv panicle bombardment as compared
to Agrobacterium-mediatcd transformation. As shoots regenerated Ala organogene¬
sis do not, originate fiom a single cell, it is less likely that a completely transgenic
shoot primordia is formed by biohstic transformation and small transformed sectors
could be lost after partial shoot formation due to the negative effect of the barnase
gene. Since cassava is groAvn as many landraces, biolistic transformation, which is
less genotype-dependant, may still be desirable to complement transformation us¬
ing Agrobactenum,. Further method clcA-elopment will be needed and selection will
have to be optimized. A selection system using luciferase expression in addition to
antibiotic selection for monitoring transgenic shoots has been proposed. Luciferase-
based selection has the advantage that no non-tiansgcnic shoots will be regenerated,
Avithout further enhancing stress due to high antibiotic concentrations.
A luciferase-based selection system Avas applied for the regeneration of trans¬
genic plants from suspensions. Luciferase positive embryogénie clusters could still
be obseiwecl up to fom months aftei transfoimation, but, also here regeneration to
complete shoots proA'ecl to be difficult. Diffeicnt factors may have influenced the
regeneration frequency. One is the age of the suspension culture used, since voungcr
suspension cultures seem to regenerate more easily and less somaclonal variation,
due to a prolonged time in tissue culture, would be expected. Our experiments
indicate a connection between suspension age and regeneration capacity (data not
shown, P. Zhang, pers. coinm.). Furthermore, the stress caused by lucifcrase expres¬
sion, which requires ATP. might affect transformed cells negatively in comparison
to untransformed cells. These avouIcI then groAA- faster and supersede the luciferase
positive cells. Culturing smaller A-olumes of suspensions (i.e. transferring less tin-
transformed cells after the luciferase assav) is not optimal, since a certain density
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CHAPTER 4. CASSAVA REGENERATION AND TRANSFORMATION 69
of cell volume in the liquid medium is required for the survival of the suspension.
Regeneration has uoav been enhanced by using 1 mg/l NAA instead of picloram in
the maturation medium and bv transferring cotAdcdons of regenerated mature em¬
bryos to organogenesis medium, instead of germinating embryos directly at a loAver
frequency (P. Zhang, pcrs.comm.). This transformation system has been further im¬
proved by selecting with 50 mg/l hygromvein and transforming with Agrobaderium,
and is now applied routinely in our laboratory (P. Zhang, in prep.).
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Chapter 5
General Discussion
Hunger and malnutrition are among the most severe problems in many areas of the
world, particularly in Africa. At present, over 800 million people are starving every
year while the Avorld population incicascs bv 85 million people (FAO, 1998). It has
been estimated that food production has to be doubled during the next 25 years in
order to keep up with the world population gicnvth. As the land available for agricul¬
ture is declining clue to urbanization, soil erosion and salinization (Brown and Kane,
1994), the future increase in food production must, come from higher productivity
and more efficient use of the existing resources under sustainable conditions.
Among the traits to engineer in cas^rva. resistance against African cassaA-a mosaic
virus (ACAIV) has been listed as the highest pnoiity in the Cassava Biotechnology-
Network (CBN, 1995). which was confirmed again at the CBN meeting in Uganda
1996. ACA4D is the most dewastating disease in cassava, leading to total crop fail¬
ures locally and also threatening ccitain varieties with extinction. The benefits of
virus resistant cassava can onlv be calculated appioximately by using the figures of
estimated losses. Avhich e.g. in 1993 in Uganda reached LIS$ 180 million per year
(Otim-Nape et, ah. 1994b). In case of subsistence farmers, at present severely threat¬
ened in their existence Ida- acute food shortage, the cost in human lives cannot be
estimated in dollars.
The goal of this thesis was to develop and evaluate a hcav ACA4V resistance
strategy in a model system and to assess the possibility of transferring this resistance
to an African cassava cultivar. The resistance is based on a regulatable toxin/anti¬toxin system mimicking a hypersensitive reaction by using the regulated ACAIA*
promoter in combination Avifh the barnase. an RNase of Bacillus amyloliqvcjaciens.
and its specific inhibitor, the baistar (Haitlcv. 1989).
The properties of ACAIA* piomoter regulation Aveie first studied transiently in
tobacco and cassava protoplasts and in stably tiansformed tobacco as a model plant,
70
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CHAPTER 5. GENERAL DISCUSSION 71
system using two different hiciferases as reporter genes. The bidirectional promoter
of African cassava mosaic virus is regulated bv viral proteins during the infection cy¬
cle, permitting a discrimination between carlv and late genes. The following model
has been proposed: Earlv after infection the complementary-sense gene products
of Rep (replication associated protein), TrAP (transcription activator protein) and
REn (replication enhancer piotein) are produced. Rep then reduces its OAvn tran¬
scription while REn enhances replication and TrAP activates virion-sensc tianseiip¬
tion. The late genes CP (coat protein) and NSP (mo\-ement protein) arc produced
and encapsidation and spread of viral DNA begins (Frischmuth, 1999). The results
on promoter regulation piescntecl in this thesis aie essentially in agreement Avith
and confirm the presented model, but in addition clraAv a more differentiated picture
of ACMV promoter regulation. It could be shoAvn that TrAP not only activates
expression of the v-sense ORFs but also of c-sense ORFs (see Figs. [2.4] and [2.5])and that, activation of c-sense expression is inhibited by small amounts of Rep. Our
results using the complete DNA A for cotransfoimation can confirm the aforemen¬
tioned model: a doAvnregulation of c-sense expression and a strong upregulation of
v-sense expression.
Surprisingly, the regulation of the DNA B promoter in cassava proved to be
the opposite fiom that of the DNA A promotei. Avith no significant effect on v-
sense expression and an upregulation of c-sense expression. NSP has been shoAvn
to transport the DNA out of the nucleus (Sanderfoot et ab, L996) while the MPB
forms encloplasmatic retictilum-dorh-ed tubules that extend through the cell Avail
to the next cell (Ward et ab. 1997). In the case in Avhich only NSP is transiently
expressed in protoplasts, the protein can be observed solely in the nucleus, while,
when cotransformed Avith AIPB, it can be found also in the cytoplasm (Sanderfootand LazaroAvitz, 1996: Lazarcnvitz and Beachy. 1999). It, can be speculated that,
the enhanced production of movement piotein helps relocating the nuclear shuttle
protein NSP with bound viral DNA to the cvtoplasm, Avhere it can be tiansferred
to adjacent cells, thus spreading viral DNA.
On the other hand, the upregulation of A'-scnse expression is not as pronounced
in tobacco cefls, therefore, host factors, which are different in cassava and in to¬
bacco, seem to have an influence on the promotei regulation. Similar observations
of different results from infection of a model plant as compared to the natural host,
arc reported for Beet curlv top virus infecting A*, tabacinu and B. vulgaris, respec¬
tively (Frischmuth cd ab. 1993a). This illustrates the limitation of model systems
and emphazises the necessity of an assay system that actually reflects the natural
situation. All transient assavs reported here. Avere peiformed in protoplasts oiigi-
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CHAPTER 5. GENERAL DISCUSSION 72
nating either from mesophyll cells, for tobacco, or from suspension cells, for cassava.
This method is extremely convenient since it is simple, fast, and has the advantage
that the cells used are quite uniform. Nevertheless, the behavior of a whole plant
cannot be entirely infeired from the results obtained from protoplasts, Avhich arc
single cells subjected to stress dining protoplast isolation and transformation.
A logical step to folloAv up transient expiession assays is the stable integration of
reporter genes to studv promoter regulation on the whole plant level. As it Avas tech¬
nically impossible to produce transgenic cassava plants, wc again relied on tobacco
as a model plant, in spite of the limitations of this system. In cxpciintents with to¬
bacco plants containing the ACAIA'-lucifcrasc constructs, several discrepancies Avith
earlier transient expression studies could be found. First, the basal firefly lucifeiasc
expression in transgenic plants Avas higher than the expression of Renilla, luciferase,
Avhilc in transient expiession assavs in piotoplasts this relation Avas inversée!. These
results seem very promising for our intended vims resistance strategy, since this
would indicate, that expression of the bainase and barstar from the ACAIY pro¬
moter could be tolerated in transgenic plants, as more barstar than barnase would
be produced in a non-infected cell. Second, in protoplasts of tiansgenic plants, the
activation of lucifcrase expression by TrAP alone could not be observed anvmore. It
appears as if the regulation of the integrated ACAIA/* promoter did not correspond
to that of the viral promoter dining a natural infection. For our purpose of devel¬
oping a virus resistance strategy using the integrated, regulatable ACMV promoter,
however, it was necessary to sIioav that DNA A can still regulate an integrated pro¬
moter, litis was confirmed bv our results. Additionally, our results from different
plant, lines illustrate the necessity for the production of many different transgenic
lines with the same construct, since basal levels of repoiter gene expression and
subsequent upregulation \-aried strongly betAA-een separate lines. The variation in
expression could be due to different integration sites in the genome, but, also to the
number of transgencs integrated. Judging from our results, further testing of the
regulation of the ACAIA" promoter in transgenic cassava plants avouIc! be required,
although results fiom cassava protoplasts and from transgenic tobacco plants indi¬
cate that the ACMV promoter can still be regulated, even Avhen integrated in the
plant genome. Since the cassava transformation system is not routine enough yet,
production of large numbers of transgenic plants with the lucifeiasc construct Avas
not feasible during the course of this thesis.
Another important factor to consider is that the DNA structuic of the integrated
promoter is different fiom the one of an infecting virus. That this aspect has to be
considered could be sIioavu bv experiments eompaiing linear and supercoiled input,
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CHAPTER 5. GENERAL DISCUSSION 73
DNA. We could show that when linear DNA was used for cotransformation TrAP
did not, function as an activator anymoie and only Avhen the complete DNA A
Avas used for transformation, a slight increase of A'-sense expression could be found.
The structure anel context of the DNA is important in promoter regulation and
transcription (Gutierrez et ab, 1995: Frv and Farnham, 1999; Muro-Pastor et ah,
1999). It might be possible, that a certain bending of the DNA is needed in order
to bind TrAP. On the other hand. Avhen the complete DNA A is expressed, other
proteins, like the Rep or the REn. might help to icmoclel the promoter DNA. such
that TrAP can activate transcription.
It is shoAvn in transient protoplast assays, that Rep cloAvnrcgulatcs both v- and
c-sense transcription, while TrAP enhances transcription from both sides. Only
when both proteins are present, aftei cotransfoimation with the DNA A, a side
specific regulation of transcription can be obseived. This can be explained by the
fact that there is either an interaction bctAvecn Rep and TrAP or that also the REn
is necessary for viral promoter regulation. The fine tuning of this system seems to be
very delicate, as results gained bv cotransforming vaiious ratios of the Rep and TrAP
constructs Avith the p.ACAfA*s shoved a high vaiiation. Furthermore, the effect of
REn, AAdiich might still play a role in the regulation process. AA-as not systematically
studied in these experiments. It could be speculated that by enhancing replication,
the transcription rate might be altered due to a shift, in the availability of certain
viral or host factors.
The results on promoter regulation presented in this thesis show that a viral pro¬
moter integrated in the plant genome can be regulated bv the ACMV DNA A. This
may indicate that an infecting Alms could achicA-e an analogous result. Furthermore,
it could be shown that in transgenic plants, the basal level of c-sense tianscription
is higher than v-sense transcription, indicating, that in plants transformed Avith
pACMVbb, more barstar than barnase aa-ou1cI be produced, therebv neutralizing the
toxic effect of the barnase. The presented results imply that, the ACMV promoter
is usable in the intended strategy of mimicking a hypersensitive reaction.
Obviously, many parameters of the presented Alms resistance strategy cannot
be simulated using reporter genes. Therefore, tobacco Avas chosen for pACMVbb
transformation, although even this proved to be problematical. Of the first 8 trans¬
formation experiments, only one plant containing the complete insert could be re¬
generated. Possibly, in most lines the bainasc Avas expressed at higher levels than
the barstar even from an uninduced promoter and. theicfore. onlv those Avith par¬
tial copies of the barnase could be regenerated. Another possible explanation is the
different regulation or non-existent regulation of the ACAIA* piomoter at the callus
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CHAPTER 5. GENERAL DISCUSSION 74
level. For tomato golden mosaic virus it has been shown that repression of v-sense
transcription is not maintained in unorganized tissue (Sunter and Bisaro, 1997). As
transgenic tobacco plants are regenerated through a callus phase with subsequent
organogenesis, it, could be that expiants containing an intact copy of the barnase are
lost at, this stage due to uncontrolled production of barnase. The further downrcg-
ulation of barnase translation, using different shoit ORFs upstream of the barnase
ORF, enabled the regeneration of further transgenic plants. Nonetheless, regenera¬
tion frequencies were still lower than usual, and manv lines containing incomplete
barnase ORFs could be detected by Southern blot (data not shown).
Regenerated plants, containing the complete gene fiagment, Avere tested in a
transient ACMA-* replication assay bv shooting autonomously rcplicatablc ACMV
DNA A into leaf pieces. The amount of replicated viral DNA in shot Avildtype lea\*cs
and in shot transgenic leaves could then be compaied. In all tested transgenic lines,
viral replication was considerably reduced. shoAvmg that, due to the regulation of
the ACA4V promoter, barnase production is in fact enhanced. Beside presenting the
first indications that this system could generate virus resistant, plants, this assay-
can also be used to identify Eansgcnic plants which are silenced or plants in Avhich
the barnase expression cannot be upregulatecl sufficiently. Results from replication
assays, hoAvcver, do not give any indication on the kinetics of gene regulation leading
to barnase production. It cannot be concluded that plants exhibiting reduced viial
replication will actually be virus resistant in the held. For our purpose it is impoitant
to confine the virus to as few cells as possible and cause local cell death before
viral replication and viral movement fakes place. Otherwise, cell death due to
barnase expression will spread along the leaf together with the virus. It, is possible
to specifically destroy infected cells using the ACAIA* promoter before the virus can
spread to adjacent cells, as has been shown before Avith dianthin (Hong et ah, 1996;
Hong et al., 1997). Our system is shghtlv more sophisticated, however: the barnase
first has to overcome its antitoxin barstar. which alieadv is present in the cells at a
Ioav concentration. Whether the retardation of the barnase clue to the presence of
barstar is crucial cannot be deduced fiom results obtained with reporter genes or
the replication assav. Although, consideiing the high variation of responses in the
different plant lines containing the ACAIA*-lucifcrase construct, if can be expected
that some of the generated ACAlA*-barnase plants will be able to produce enough
barnase to overcome the antitoxin and tiigger cell death before the vims is able to
replicate and spread.
It is unclear, Avhy Y. tabacum SRI plants could not be infected with ACMV.
N. tabacum cvs. Samstm and Nanthi plants have been previously used in infection
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CHAPTER 5. GENERAL DISCUSSION 75
studies (Frischmuth et ah, 1993b). nevertheless, no viral DNA could be detected in
agroinoculatccl SRI tobacco plants 2 - 3 Aveeks after transformation. As Ave have
shoAA'n in the replication assays that replication of ACMV DNA A does take place
in SRI plants, it has to be assumed, that the movement of the virus is impaired
in these plants. Whether the inability of systemic movement is due to MPB or
NSP can only be speculated since both movement proteins have to interact with
components of the host cell. Also the infection Avith another geminivirus did not
provide any conclusive results. Possibly. SiGAIA*. a virus from the New World, Avas
not closely related enough to regulate the ACAIA* promoter.
A next logical step in testing Alms resistant plants, would be the infection
through the natural vector, the B. tabaci. The icsults from these experiments could
still be slightly elifierent than after agroinoculation. since then the virus is inoculated
directly in the leaf mesoptrvll cells and not through the phloem. Furthermore, the
DNA introduced through agroinoculation has to excise itself from the plant genome,
resulting directly in a double-stranded DNA, Avhile naturally introduced viral DNA
is single-stranded and transcription cannot occur directly. Replicated ACAIA'* DNA
can only be found 2-3 davs after inoculation, -while lucifcrase expression driven by
thc ACMV promoter can be measured alieadv after 21 hours. It thus appears that
gene transcription and subsequent promoter regulation occur before viral replica¬
tion. These results, hoAvevcr, Avere obtained in transient, assavs Avith double-stranded
viral DNA, and it is not clear Avhethcr the single-stranded form of naturally occur-
ring viral DNA would behave differenttw Moreover. Avhiteflies can feed in more than
one site on a cassava leaf and theiebv deposit ACAIA/* in various cells across the leaf.
Depending on hewv far the virus can spread and Iicdav fast it can replicate before local
cell death due to the barnase can prevent further spreading and replication, but also
depending on Iioav far flic induced local cell death spreads due to barnase to adjacent
cells, damage might be considerable, even if systemic infection is prevented. Once
more, it is expected, that under a huge number of tiansgenic lines, the ideal tuning
of the toxin/anti-toxin system can be found.
Provided that the transgenic plants pioducecl during the course of this thesis
are Alms resistant, the next question is Iioav easily this resistance could be overcome
in the field. The ACAIA* promotei used in this strategy is conserved between the
DNA A and DNA B. In order to inactivate the integrated promoter between the
barnase and barstar, a mutation avouIcI need to take place in the promoter of both
genomic components. Aforeover mutations in both Rep and TrAP would then be
necessary to regulate this hcav. mutated promoter but not the original promoter.
The likelihood for this is statistically minimal. Another possibility for the virus to
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CHAPTER 5. GENERAL DISCUSSION 76
overcome this resistance would be to accelerate replication and spread to adjacent
cells before the barnase can overcome the barstar and cause local cell death. It, can
be assumed, that, these faster viruses avouIcI spread through the leaf, initiating cell
death in infected cells on their way and thereby destroying the plant before they
can be spread to other plants through feeding whiteflies, thus becoming extinct in
a fast manner.
As mentioned before, it is necessary to geneiate many different transgenic ACM V-
barnase lines because the A-anation of tiansgene expression and viral transgene reg¬
ulation between different lines is high. The cassaA-a transformation svstem is still
being improved and transgenic plants cannot be produced routinely yet. Given that
the regeneration frequency of ACAW-barnasc tobaccos containing an intact bainasc
ORF Avas very Ioav (around 10% of all regenerated plants), it cannot be expected that
transgenic cassava plants containing this construct can be produced at this stage.
Not only Avould the necessary tissue culture AA-ork exceed our limits, also the molecu¬
lar analysis would be labor-intensive for cassava, as long as selection is not improved.
At present, too many non-transgenic plants can suiaIvc the selection scheme. These
constraints in cassava transformation and regeneration should be overcome in the
near future. The transfoimation system used now. has been improved, and more
transgenic plants aie being produced in our laboratory. Selection is tighter and at
the same time the regeneiation frequency could be improved. Therefore, it should
be possible to generate vims resistant cassava employing the promising strategy
presented in this thesis in the near future. These plants should then be tested not
only by agroinoculation of the vims, but also by exposure to the whrteflv vector and
later, in the field. In case the in, vitro lesults reported in this thesis can be matched
in the field, the same construct can either be introduced directly or through tradi¬
tional breeding into other local A-arictics. As soon as other mechanisms of ACMV
resistance are introduced in cassava, these different transgencs could be pyramided,
resulting in broader range of resistance and in a more reliable resistance also un¬
der high infection pressure in the field, thereby contributing to a future sustainable
cassaA-a cultivation and to food secmitv in Africa.
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Curriculum vitae
Name: Petra Alalia Frcv
Date of birth: May 1st, 1972
Place of birth: Lucerne, SAvitzerland
1979 - 1982 Primary school. Rothenburg
1982 - 1985 German SaaIss International School. Hong Kong
1985 - 1992 Kantonsschtile Reussbühl: Marina Tvpe C (nattual sciences)
1989 - 1990 Arirden Collegiate Instil ute. Alanitoba, Canada: Pligh School Giaduation
1992 - 1996 Studv of Experiment af Biology at the Depaitnicnt of Biology, ETTI Zürich
Diploma thesis at the Institute foi Plant, Sciences:
'ToAvards regeneration and transfoimation of cassava meristems"
1996 - 2000 Research work for PhD thesis at the Institute for Plant Sciences,
ETH Zürich, SAvitzeiland. in the group of Prof. I. Potrvkus