Summary: tet system in Arabidopsis

Chris Somerville CRS at ANDREW.STANFORD.EDU
Sat Nov 25 17:23:32 EST 1995


Here is a condensed version of some of the many responses to my 
question about the utility of the tet system or other bacterial 
regulatory systems in Arabidopsis.  The short answer is that it does 
not work.  I have also appended several abstracts pertaining to the 
issue.  My thanks to all who provided information and insights.  
Chris


Quite some time ago we like several others tryed to get the
tet-system to work in Arabidoposis. However, neither when the
tet-repressor gene and the p35STX-driven gene (GUS) were introduced
separately nor when they were located on the same T-DNA we could
achieve repression/induction. Peter Morris observed that the level of
tet-R expression was quite low in Arabidopsis in comparison to the
situation in tobacco. Whether this fairly low expression indeed is
the reason for the lack of repression (naively one might assume that
the smaller Arab genome would offer fewer binding sites which might
compete with the tet-op in the TX-promoter) and why it was so low we
do not know. Christiane Gatz is currently starting a new effort to
get the system working in Arabidopsis. Thomas Altmann  
altmann at mpimg-berlin-dahlem.mpg.de

We have tried to regulate the Agrobacterium T-DNA genes "ipt" and
"rolC" by the tetR/triple-op system of Christiane Gatz, but without
success untill now (both in tobacco and arabidopsis). The approach we
have taken was to put the tetR gene and the triple-op-T-DNA gene at
one construct, do transformations with this construct and try to
obtain plants that have a wild-type phenotype. In tobacco a few
"normal" looking plants were obtained, but when grown in the green
house they still showed phenotypes typical for either the "ipt" or
the "rolC" gene. Also we have tested the so called "super-repressor"
line of Gatz for its ability to silence the triple-op-GUS or the
triple-op-DT-A gene (cocultivation of tobacco protoplasts with
Agrobacterium). With the first gene we obtained a lot of blue calli
and with the second gene we obtained only a few calli (DT-A kills
cells). Presently, I am testing a tetR construct in which a NLS has
been added to the C-terminus of the protein. This has been reported
to improve the lacR/lac-op-system and hopefully it will also improve
the tetR/triple-op system. I hope to have the results in the first
months of next year. Another option may be to weaken the
35S-triple-op promoter by replacing the enhancer. Conclusion:
although some people reported that the system works in tobacco (Gatz,
Inze), it is not yet a ready to use system in arabidopsis. Hope this
info is of help Remko Offringa  OFFRINGA at rulsfb.LeidenUniv.nl

Two labs I know have not been able to express Tet repressor in
Arabidopsis to detectable level. The system apparently works well in
tobacco (as per C.Gatz in Germany and Pam Green in MSU). Lac was used
by Mike Bevan's group with some success. They published a paper in
EMBO few years back, first author is D.Shufflebottom. Hope this
helps. Dima Belostotsky DAB at cnsvax.albany.edu

According to christina gatz, the tet system does not work in
arabidopsis. she is working on this now.  the vp16-tet activation
system is coming on line as well however, the expression of this
hybrid factor is not stable yet in tobacco.  nothing for
arabidopsis.  Alan Jones alan_jones at unc.edu

I don't know if Roy Morris is on this list, but his lab has been
getting a tet system going and he told me that it has failed in
arabidopsis, apparently because the plant has some factor that
relieves the repression (ie, as if tetrcycline were always present).
But he can tell you more. His address is:
bcmorris at muccmail.missouri.edu. Hope this Helps, Tobias I. Baskin 
baskin at biosci.mbp.missouri.edu

We have used LACi::VP16 fusions in cotransfection experiments, but
so far only in transient situations. In parsley protoplasts we tried
to use IPTG, but without success in terms of regulation. Bernd
Weisshaar MPI fuer Zuechtungsforschung Abteilung Biochemie
Carl-von-Linne-Weg 10 50829 Koeln Germany e-mail:
weisshaa at mpiz-koeln.mpg.de

TITLE:           Efficiency of the tetracycline-dependent gene 
expression  system: Complete suppression and efficient 
induction of the rolB phenotype in transgenic plants.
AUTHOR:          Roeder, F T (Inst. Genet., Univ. Bielefeld, D-33051 
Bielefeld, GER); Schmuelling, T; Gatz, C
PUBLICATION:     Molecular & General Genetics. 1994 243(1):32-38.
ABSTRACT:  We have investigated the use of the
tetracycline-dependent gene expression system to regenerate and
propagate tobacco plants transformed with a gene whose product when
highly expressed interferes with regeneration and/or further
reproduction. Plants transformed with the Agrobacterium rhizogenes
rolB gene under the control of the tetracycline-dependent expression
system were phenotypically indistinguishable from wild type owing to
efficient repression of the promoter. Induction of the rolB gene
with tetracycline led to high-level expression of the rolB mRNA which
resulted in extremely stunted plants with necrotic and wrinkled
leaves that did not develop a floral meristem. Upon cessation of
tetracycline treatment healthy shoots developed even from severely
affected meristems. Data on the dose response of the rolB phenotype
as a function of tetracycline concentration demonstrate that the
tetracycline-dependent gene expression system can be used to modulate
the manifestation of a particular phenotype.

TITLE:           A chimeric transactivator allows 
tetracycline-responsive gene   expression in whole plants.
AUTHOR:          Weinmann, P (Inst. Genetik, Univ. Bielefeld, 33501 
Bielefeld,   GER); Gossen, M; Hillen, W; Bujard, H; Gatz, C
PUBLICATION:     Plant Journal. 1994 5(4):559-569.
ABSTRACT:  The chimeric transcriptional activator tTA, a fusion
between the Tn 10 encoded Tet repressor and the activation domain of
the Herpes simplex virion protein VP16, was stably expressed in
transgenic tobacco plants. It stimulates transcription of the
beta-glucuronidase (gus) gene from an artificial promoter consisting
of 7 tet operators and a TATA-box. Tetracycline, which interferes
with binding of tTA to operator DNA, reduces gus expression over
several orders of magnitude. This stringency of regulation suggests
that the system can be used to construct transgenic plants encoding a
potentially lethal gene product. Furthermore, the specific and fast
inactivation of tTA allows study of the stability of RNAs and
proteins.

TITLE:           Control of gene expression in tobacco cells using a
bacterial   operator-repressor system. AUTHOR:          Wilde, R J
(Ici Joint Lab., Univ. Leicester, University Road, Le1 7rh, Uk.);
Shufflebottom, D; Cooke, S; Jasinska, I;  Merryweather, A; Beri, R;
Brammar, W J; Bevan, M; Schuch, W PUBLICATION:     Embo Journal. 1992
 11(4):1251-1259.
 ABSTRACT:  We have investigated the efficacy of
using the Escherichia coli lac operator-repressor system to control
plant gene expression. The lacI gene was modified to allow optimal
expression in plant cells and then placed downstream of the
cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was
introduced into tobacco plants by leaf disc transformation.
Transgenic tobacco plants synthesized significant quantities of LacI
protein (up to 0.06% of total soluble protein). We have used the E.
coli .beta.-glucuronidase gene (gus) as the reporter gene by placing
it downstream of the maize chlorophyll a/b binding protein (CAB) gene
promoter. Lac operators were introduced into several positions within
the CAB promoter and operator-free plasmid was used as control.
Repression was assessed by comparing the transient expression from
CAB-operator-gus reporter constructs in protoplasts expressing lac
protein, with that in control cells not expressing the repressor.
Repression varied between 10 and 90% with different operator
positions. Transient assays were also performed in the presence of
the inducer, isopropyl-.beta.-D-thiogalactoside (IPTG). In lacI
protoplasts the presence of IPTG manifested itself in a 4.2-fold
relief of repression. The study was extended to show regulation of
expression in stable transformants. Tobacco transformants harbouring
a CAB-operator-gus reporter construct and the lacI gene were shown to
have repressed GUS levels, but in the presence of IPTG, repression
was relieved 15-fold. We conclude that the lac repressor can enter
the plant cell nucleus, find its cognate operator sequence in the
chromatin to form a repressor-operator complex and effectively block
transcription of a downstream gene. 



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