Evaluation of a viral vector system, based on a defective interfering RNA of tomato bushy stunt virus, for protein expression and the induction of gene silencing [Elektronische Ressource] / Christian Naumer

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"Evaluation of a viral vector system, based on a defective interfering RNA of tomato bushy stunt virus, for protein expression and the induction of gene silencing"D i s s e r t a t i o nzur Erlangung des Grades"Doktor de rN aturwissenschaften"am Fachbereich B iologieder Johannes Gutenberg- U niversitätin M ainzChristian Naumergeb. Am 14.12.1972 in SpeyerM ainz, 2005Dekan:1.Berichterstatter:2.Berichterstatter: Tag der mündlichen Prüfung: 04. Oktober 2005T able of Contents1 Table of Contents1 T able of Contents....................................................................................................................................................I2 A bbreviations .......................................................................................................................................................V3 A bstract................................................................................................................................................................VI4 Introduction............................................................................................................................................................14.1 M olecular farming.........................................................................................................................................14.1.1 Viral vectors in molecular farming...................................................................................................

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"Evaluation of a viral vector system, based on a defective interfering
RNA of tomato bushy stunt virus, for protein expression and the
induction of gene silencing"
D i s s e r t a t i o n
zur Erlangung des Grades
"Doktor de r
N aturwissenschaften"
am Fachbereich B iologie
der Johannes Gutenberg- U niversität
in M ainz
Christian Naumer
geb. Am 14.12.1972 in Speyer
M ainz, 2005Dekan:
1.Berichterstatter:
2.Berichterstatter:
Tag der mündlichen Prüfung: 04. Oktober 2005T able of Contents
1 Table of Contents
1 T able of Contents....................................................................................................................................................I
2 A bbreviations .......................................................................................................................................................V
3 A bstract................................................................................................................................................................VI
4 Introduction............................................................................................................................................................1
4.1 M olecular farming.........................................................................................................................................1
4.1.1 Viral vectors in molecular farming.......................................................................................................3
4.2 Post transcriptional gene silencing (P T GS)..................................................................................................4
4.2.1 Virus-induced gene silencing (VIGS)..................................................................................................6
4.3 Effect of D I sequences on ge ne silencing.....................................................................................................7
4.4 Tomato bus hy stunt vi rus (T B SV)................................................................................................................8
4.4.1 Replication of TB SV............................................................................................................................9
4.4.2 D efective interfering pa rticles............................................................................................................10
4.5 Aim..............................................................................................................................................................11
5 M aterials and M ethods.........................................................................................................................................13
5.1 Chemicals....................................................................................................................................................13
5.2 Enzymes......................................................................................................................................................13
5.3 Antibodies....................................................................................................................................................13
5.4 Plants...........................................................................................................................................................13
5.5 Bacterial strains...........................................................................................................................................14
5.6 Plasmids.......................................................................................................................................................14
5.7 Cultivation media........................................................................................................................................15
5.8 Cultivation conditions of greenhouse m aterial...........................................................................................15
5.9 M olecular biology m ethods.........................................................................................................................16
5.9.1 M ini-preparation of pl asmid D N A w ith Qiaprep spin m iniprep kit .................................................16
5.9.2 M ini-preparation of plasmid DN A from Agrobacterium tumefaciens with W izard P lus DN A
purification system.......................................................................................................................................16
5.9.3 Cryopreservation of bacterial cells.....................................................................................................16
5.9.4 D etermination of D N A and RN A concentration by U V spectrometry..............................................16
5.9.5 T-tailing of pl asmid D N A ...................................................................................................................16
5.9.6 Restriction enzyme digests.................................................................................................................17
5.9.7 Agarose ge l electrophoresis................................................................................................................17
5.9.8 Agarose ge l extraction........................................................................................................................18
5.9.9 Klenow fragment “fill in” reaction.....................................................................................................18
5.9.10 Phenol/chloroform pur ification of nucleic acids..............................................................................18
5.9.11 Phosphorylation of nuc leic acids using T 4 pol ynucleotide ki nase (T 4 PN K ).................................19
5.9.12 D ephosphorylation of di gested pl asmid D N A w ith shrimp alkaline pho sphatase (SA P )...............19
5.9.13 Ligation with T4 ligase.....................................................................................................................19
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5.9.14 Production of chemically competent E. coli cells............................................................................20
5.9.15 Production of electro competent Agrobacterium tumefaciens cells.................................................20
5.9.16 Transformation of chemically competent E. coli cells.....................................................................20
5.9.17 Transformation of electro competent Agrobacterium tumefaciens cells.........................................21
5.9.18 PCR and sequencing pr imers............................................................................................................21
5.9.19 PCR w ith Taq DN A pol ymerase......................................................................................................22
5.9.20 PCR w ith Pfx DN A pol ymerase.......................................................................................................23
5.9.21 Sequencing PCR...............................................................................................................................23
5.9.22 QuickChange™ site-directed m utagenesis ki t.................................................................................24
5.9.23 RN A extraction from plants.............................................................................................................24
5.9.24 RT -P CR us ing SuperScript One-Step RT -P CR kit from Invitrogen ..............................................25
5.9.25 Northern bl ot.....................................................................................................................................25
5.9.26 In vi tro transcription of RN A ...........................................................................................................27
5.10 P rotein analysis..........................................................................................................................................27
5.10.1 Histochemical GU S staining............................................................................................................27
5.10.2 Sample pr eparation for SDS-P A GE.................................................................................................28
5.10.3 SDS-P A GE.......................................................................................................................................28
5.10.4 Coomassie staining...........................................................................................................................29
5.10.5 Western bl ot......................................................................................................................................29
5.10.6 EL ISA (enzyme linked immuno sorbent assay)...............................................................................30
5.11 Inoculation of Nicotiana benthamiana w ith RN A transcripts..................................................................31
5.12 Agrobacterium infiltration.........................................................................................................................32
5.13 Fluorescence microscopy and phot ography of GFP .................................................................................32
5.14 ClustalX alignments..................................................................................................................................32
5.15 P rotoplast isolation and transfection.........................................................................................................33
6 Results..................................................................................................................................................................35
6.1 Functionality of the T B SV replicase expressed in transgenic plants.........................................................35
6.2 Protein expression us ing the D I ve ctor system...........................................................................................36
6.2.1 Cloning strategy of the ppD I-GU S construct.....................................................................................36
6.2.2 Inoculation experiments with D I-GU S RN A transcripts...................................................................37
6.2.3 Introduction of GFP , scFv, and vi ral coat pr otein ge nes into the ve ctor ..........................................37
6.2.4 Insertion of the vi ral m ovement protein into the ve ctor.....................................................................38
6.2.5 Inoculation experiments with D I reporter ge ne RN A transcripts......................................................40
6.2.6 Northern bl ot analysis of DI-scFv/T B SV-infected plants..................................................................41
6.2.7 Protoplast transfection w ith D I RN A transcripts...............................................................................43
6.2.8 Northern bl ot analysis of protoplast RN A ..........................................................................................44
6.2.9 Construction of the pp mDI plasmid...................................................................................................45
6.2.10 Insertion of GU S, GFP , SCFV, YFP , CP and p2 2 into the ppmDI construct..................................47
6.2.11 Inoculation experiments w ith m DI RN A transcripts........................................................................48
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6.3 Induction of ge ne silencing using the DI vector system.............................................................................49
6.3.1 Cloning of pT B SVDp19 ....................................................................................................................49
6.3.2 Construction of pT B SVDDp19 ...........................................................................................................50
6.3.3 Isolation of a FtsH ge ne fragment from N. benthamiana..................................................................52
6.3.4 Insertion of the FtsH fragment into the ppD I vector..........................................................................53
6.3.5 Insertion of a phyt oene desaturase (P DS) ge ne fragment into the pp DI ve ctor.................................53
6.3.6 Construction of a GFP silencing ve ctor.............................................................................................54
6.3.7 Inoculation experiments with TR4 pl ants..........................................................................................55
6.3.8 Inoculation experiments with the GFP silencing construct................................................................55
6.3.9 Inoculation experiments with the FtsH and PDS silencing construct................................................57
6.3.10 Construction of infectious T B SV clones for A. tumefaciens infiltration experiments....................59
6.3.11 Construction of an infectious T B SVDp19 clone for A. tumefaciens infiltration experiments.........62
6.3.12 Construction of a bi nary vector for the inoculation of pl ants w ith DI-P DS....................................62
6.3.13 A. tumefaciens infiltration experiments with D I-P DS bi nary vectors on TR4 pl ants......................64
6.3.14 A. tumefaciens infiltration experiments with D I-P DS bi nary vectors..............................................64
6.3.15 A. tumefaciens infiltration experiments with D I-P DS and virus bi nary ve ctors..............................65
6.3.16 Northern bl ot analysis of TB SVDp19/DI-FtsH infected plants.......................................................66
6.3.17 RT -P CR and cloning of the D I-FtsH fragment................................................................................67
6.3.18 D ifferences between T B SVDp19 and T B SVDDp19 ........................................................................68
6.4 Protection from ge ne silencing....................................................................................................................71
6.4.1 Construction of a bi nary vector ha rboring D I-gfpE R and m DI-gfpE R.............................................71
6.4.1.1 Cloning of pB SK -mgfp5............................................................................................................71
6.4.1.2 Cloning of pU C-gfpE R..............................................................................................................71
6.4.1.3 Cloning of pB SK -gfpE R........................................................................72
6.4.1.4 Cloning of pT P CR-gfpE R..........................................................................................................74
6.4.1.5 Cloning of ppD I-gfpE R and ppm DI-gfpE R..............................................................................74
6.4.1.6 Cloning of pT P CR-DI-gfpE R and pT P CR-mDI-gfpE R...........................................................74
6.4.1.7 Cloning of pT P CR-DI-gfpE RK and pT P CR-mDI-gfpE RK ......................................................75
6.4.1.8 Transfer of DI-gfpE RK and mDI-gfpE RK constructs into 35 S promoter cassette...................76
6.4.1.9 Cloning of pG J-DI-gfpE R and pG J-mDI-gfpER.......................................................................76
6.4.2 Agroinfiltration for transient expression of GFP constructs on N. benthamiana .............................77
6.4.3 A groinfiltration for transient expression of GU S constructs on N. tabacum and GU S silenced N.
tabacum........................................................................................................................................................77
7 Discussion............................................................................................................................................................79
8 L iterature..............................................................................................................................................................92
9 A ppendix............................................................................................................................................................100
9.1 ClustalX alignment of the isolated DI-FtsH Sequences...........................................................................100
9.2 Plasmid m ap of ppD I-scFv, -mGFP and -CP ............................................................................................103
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9.3 Plasmid m ap of ppD I-p2 2 and ppD I-p2 2M ..............................................................................................104
9.4 Plasmid m ap of ppm DI.............................................................................................................................105
9.5 Plasmid m ap of ppm DI-GU S, -GFP , -scFv, -YFP , -CP , -p2 2..................................................................106
9.6 Plasmid m ap of pT B SVDp19 and pT B SVDDp19 .....................................................................................109
9.7 Plasmid m ap of ppD I-FtsH and ppD I(-)3'gf p...........................................................................................110
9.8 Plasmid m ap of ppD I-P DS(+) and ppD I-P DS(-)......................................................................................111
9.9 Plasmid m ap of ppdS and dT B SV............................................................................................................112
9.10 P lasmid map of ppzp- T B SV and ppz p-T B SVDp19 ................................................................................113
9.11 P lasmid map of ppzp- SDI-P DS(+) and ppz p- SDI-P DS(-).....................................................................114
9.12 P lasmid map of pG J-DIgfpE RK and pGJ-mDIgfpERK .........................................................................115
IVA bbreviations
2 Abbreviations
Amp ampicillin electrophoresis
AP alkaline phosphatase PBS phosphate buffered saline
PCR polymerase chain reactionAPS ammonium persulfate
Asn asparagine PDS phytoen desaturase
bp base pairs PEG polyethylenglycol
DI defective interfering particle PNK polynucletide kinase
DNA deoxyribonucleic acid PO D peroxidase
dNTP deoxynucleotide triphosphate PTGS post transkriptional gene
silencingdpi days post inoculation
EDTA ethylenediaminetetraacetic PVDF polyvinylidene fluoride
acid RNA ribonucleic acid
ELISA enzyme linked immuno sorbent rpm rounds per minute
assay SAP shrimp alkaline phosphatase
ER endoplasmatic reticulum scFv single chain antibody variable
EtO H ethanol fragment
GFP green fluorescents protein SDS sodiumdodecylsulfate
Ser serineGUS β-D-glucuronidase
TAE tris acetate EDT AHis histidine
TBE tris borate EDT AIgG immunoglobulin G
TBS tris buffered saline kb kilo bases
TBST tris buffered saline tweenkD kilo dalton
TBSV tomato bushy stunt virusMan mannitol
TEMED N ,N ,N ,N ,tetramethyl-MeO H methanol
ethyleneamineMES 2-M orpholinoethansulfonsäure
Thr threonineMS M urashige and Skoog
VIGS virus-induced gene silencingnt nucleotides
X-GlcA 5-bromo-4-chloro-3-indolyl-β-NTP nucleotide triphosphate
G-glucoronic acid O RF open reading frames
YFP yellow fluorescents protein PAGE polyacrylamide gel
VA bstract
3 Abstract
In this thesis a viral vector system was developed based on a DI-RN A , a sub-viral particle
derived from T B SV-B S3-statice. T his newly designed vector system was tested for its
applicability in protein expression and induction of gene silencing. T wo strategies were
pursued in this study. T he first strategy being the replication of the DI-RN A by a
transgenically expressed T B SV replicase and the second being the replication by a so called
helper virus. It could be demonstrated by northern blot analysis that the viral replicase,
expressed by the transgenic N. benthamiana plant line T R4 or supplied by the helper virus, is
able to replicate DI-RN A introduced into the plant cells. Various genes were inserted into
different DI constructs in order to study the vector system with regard to protein expression.
However, independent of how the replicase was provided no detectable amounts of protein
were produced in the plants. P ossible reasons for this failure are identified: the lack of
systemic movement of the DI-RN A in the transgenic T R4 plants and the occurrence of
deletions in the inserted genes in both systems. A s a consequence the two strategies were
considered unsuitable for protein expression.
T he DI-RN A vector system was able to induce silencing of transgenes as well as
endogenous genes. Several different p19 deficient helper virus constructs were made to
evaluate their silencing efficiency in combination with our DI-RN A constructs. In addition to
the various helper virus constructs different inoculation methods were studied to adapt the
vector system for a high efficiency, high throughput screening approach. However, it was
found that our vector system can not compete with other existing VIGS (virus induced gene
silencing) systems in this field.
Finally, the influence of DI sequences on mRN A stability on transient GFP and GU S
expression experiments in GFP /GU S silenced plants was evaluated. However, A. tumefaciens
infiltration with the binary vectors carrying the GFP gene did not result in GFP expression in
wild type N. benthamiana plants. T herefore, further experiments with GFP silenced plants
were not conducted. T he alternative GU S reporter gene system was found to be unsuitable for
distinguishing between expression levels of wild type plants and GU S silenced transgenic
plants. N evertheless, the results indicate a positive effect of the DI sequences on the level of
protein expression and therefore further research into this area is recommended.
VIIntroduction
4 Introduction
4.1 Molecular farming
Commercial success of plant based production processes has demonstrated that plants
expression systems possess an enormous potential for large scale production of recombinant
proteins. P lants have several advantages in terms of practical, economical and safety issues
compared to other expression systems. T here are already several efficient plant based
expression systems available and development still continues driven by those advantages.
M any different proteins have been successfully produced in plants including antibodies,
vaccines and hormones [Fischer and Emans, 2000; Giddings, 2001]. A key advantage of using
transgenic plants as “bioreactors” is the comparatively low cost of large scale production.
T his is mostly due to the fact that no expensive fermenters and peripheral equipment are
needed. Extensive research in this field is fueled by the possibility of high protein yields. For
instance, yields of 0.1 to 1 % of total soluble protein are already competitive with other
expression systems.
Conventional bioreactors have certain restrictions regarding the scale-up whereas plant
systems can be scaled up rapidly in response to the market demand.
T he largest part of the production costs are caused by downstream processing and
purification of the recombinant protein. Regardless of the protein expression system the
purification processes are similar and the costs do not vary greatly. However, several types of
recombinant proteins produced in plants can be used in an unprocessed or only partially
processed state. Since, plant material is usually unproblematic for human consumption,
recombinant vaccines can be administered by the consumption of raw fruits or vegetables.
A ccepting many advantages of plant based systems one has to acknowledge a major
drawback which is the time needed to establish a stable transgenic plant line. T ransient
expression systems, as for example Agrobacterium tumefaciens infiltration and viral protein
expression vectors try to address this problem.
T ransient expression systems commonly use three different approaches (Fig. 1); biolistic
delivery of DN A , infiltration with Agrobacteria and infection with modified viral vectors.
However, the first system using particle bombardment is not suitable for the expression of
large amounts of proteins in plants since only a small number of cells per leaf express the
protein of interest (Fig. 1).
1Introduction
G e n e X
V e c t o r s y s t e m
( A ) ( B ) ( C )
D N A a g r o i n f i l t r a t i o n v i r a l v e c t o r
P l a n t c e l l
s p o t s o n t h e l e a f w h o l e l e a f w h o l e p l a n t
Figure 1: Transient expression systems most commonly
used for the heterologous expression of proteins in plants.
(A) Biolistic introduction of DNA directly into the plant
cells. (B) Gene transfer using Agrobacterium infiltration.
(C) Protein expression using a genetically modified viral
vector. [Fischer et al., 1999]
A groinfiltration utilizes the delivery of Agrobacteria into intact leaf tissue by vacuum
infiltration or by infiltration using a needle-less syringe. T his permits the use of conventional
binary vectors. Several proteins have been successfully expressed in plants. Examples include
scFvs, a protein containing only the variable regions of the heavy and light chain of an
antibody linked by a glycine linker peptide, and full size antibodies [Vaquero et al., 1999]
which demonstrates the capability of the system to express multi-component protein
complexes. However, the Agrobacterium transient expression systems has its drawbacks
concerning the large scale production of proteins, as it is not feasible to infiltrate large
amounts of leaf material. T he lack of systemic production of the heterologous protein and the
susceptibility to gene silencing is also limiting the yield and the applicability of the system for
2