BACKGROUND
most bacteria harbour toxin-antitoxin  systems, usually comprising a pair of genes coding for a toxin and its cognate antitoxin  <cit> .
upon antitoxin degradation, the toxin induces cell stasis or death in part of the cell population leading to the early proposal of ta systems being a form of programmed cell death in prokaryotes  <cit> .
ta systems were originally discovered in bacterial plasmids, where they function in the maintenance of plasmids through the post-segregational killing of plasmid-free daughter cells  <cit> .
chromosomally-encoded ta systems were subsequently found to be also abundant in bacteria and archaea where they have been implicated in several cellular functions such as programmed cell death  <cit> , stress responses  <cit>  and in persistence and antibiotic tolerance  <cit> .
a wide variety of ta systems have been discovered in the past decade and currently, ta systems are grouped into classes i, ii, iii, iv and v according to the nature and action of the antitoxin  <cit> .
type ii ta systems have been most widely studied  <cit>  and encode a toxic protein and a relatively less stable cognate proteic antitoxin.
environmental stress conditions usually lead to the induction of endogenous proteases which results in the degradation of antitoxins, thereby releasing the toxins from the inert toxin-antitoxin complex.
the unbound toxin is now free to exert its lethal effect through different modes of action depending on the toxin type.
the majority of characterized toxins act as endoribonucleases, some toxins interfere with dna gyrase activity and thus function as an inhibitor of dna replication and transcription, while other toxins inhibit translation initiation and interfere with cell wall synthesis  <cit> .
the chromosomal ta systems from gram negative bacteria are among the most prevalent and well-studied.
some of these prokaryotic toxins have been shown to have activity when expressed in eukaryotic cells such as yeasts and have been proposed to have potential application in the control of cell growth in eukaryotic cells, especially in preventing the escape of genetically modified cells  <cit> .
the rele toxin of e. coli was demonstrated to be functional in the yeast saccharomyces cerevisiae where induction of the toxin gene in transformed yeast cells inhibited growth  <cit> .
expression of the rele toxin and the kid toxin were also shown to trigger apoptosis in a human osteosarcoma cell line  <cit>  and in hela cells  <cit> , respectively.
these findings eventually led to the development of a method using the kis-kid ta system to select for mammalian cells with a stable and high level expression of transgenes  <cit> .
another ta system that has been shown to be functional in eukaryotes is the epsilon-zeta system from plasmid psm <dig> of streptococcus pyogenes where expression of the zeta toxin in the yeast saccharomyces cerevisiae was shown to be lethal  <cit> .
up to  <dig> putative ta systems have been found through a bioinformatics search of sequenced streptococcus pneumoniae genomes  <cit> .
out of these, three have been demonstrated to be functional bona fide ta systems, namely relbe <dig>  <cit> , yefm-yoebspn  <cit>  and pezat  <cit> .
the yoeb toxin homologues have been shown to be endoribonucleases and overexpression of the yoebspn toxin led to cell death in both s. pneumoniae and e. coli  <cit> .
to our knowledge, there have been no reports on the functionality of bacterial ta systems in plants.
here, we investigate the effects of expressing the yoebspn toxin gene in arabidopsis thaliana as a model plant system using a 17-β-estradiol-inducible expression system.
ultimately, this work may lead to the development of a bio-containment strategy, which may be useful for preventing the release of unwanted genetically modified plants to the environment, for the development of male sterile plants for hybrid seed production as well as the development of a novel cell ablation system for functional studies in plants.
RESULTS
analysis of transgenic plants
a 17-β-estradiol-inducible two-component system  <cit>  was used to obtain transgenic arabidopsis thaliana for controlled expression of the yoebspn toxin gene, cloned as a translational fusion with the gfp gene in the responder vector pmdc221_yoebgfp while the camv 35s promoter was cloned into the activator vector pmdc150_35s to drive the constitutive expression of the 17-β-estradiol-responsive xve transcriptional activator .figure  <dig> vector constructs and pcr amplification from four-week-old t <dig> transgenic a. thaliana using gfp-specific primers.
schematic diagram of the plant inducible expression vectors that were constructed for this study based on  <cit> .
agarose gel electrophoresis of pcr products using gfp-specific primers; samples were total dna from plants transformed with pmdc221_yoebgfp  or with pmdc221-gfp  and showing the expected amplified product of 732 bp; lane 13: sample was total dna from a wild type arabidopsis plant ; 1 kb: 1 kb dna ladder marker .
five independent transformation experiments were conducted and  <dig> independent transgenic lines were selected for further analysis.
out of 100 t <dig> yoebspn-gfp transgenic plants that grew on the selection medium,  <dig> were randomly chosen for genomic dna preparation and pcr amplification using gfp-specific primers .
the presence of a 732 bp band indicated the presence of the yoebspn-gfp fusion transgene and, thus, the successful integration of the transgene into the arabidopsis plants.
this band was also present in the positive control plants expressing gfp alone from vector pmdc221_gfp  and was absent in the negative control  plant .
similarly, when using yoebspn-specific primers, the presence of a yoebspn-specific band was also observed in the transgenic plants .
when the leaves of the transgenic plants were examined under fluorescence confocal microscope, gfp fluorescence was detectable in all parts of the leaves 24 hours after 17-β-estradiol induction .
leaves showed gfp fluorescence at days  <dig> and  <dig> after induction, and even at days  <dig> and  <dig>  after plant death .
distortion of the newly emerging rosette leaves and necrotic symptoms in all leaves were observed from day  <dig> onwards, as shown in figure  <dig> figure  <dig> pcr confirmation of yoeb
spn toxin gene from four-week-old-t <dig> transgenic a. thaliana using yoeb-specific primers.
samples were total dna extracted from the t <dig> transgenic plants showing the expected amplified product of 255 bp.
lanes 1–2: transgenic line 1; lanes 3–4: transgenic line 2; lanes 5–6: transgenic line 3; lane -ve: dna from a wild type arabidopsis plant as negative control; 1 kb: 1 kb dna ladder marker .
arabidopsis thaliana.
plants transformed with pmdc221_yoebgfp were induced with 100 μm 17-β-estradiol at 4 weeks after selection.
no induction;  1 day after induction;  3 days after induction;  6 days after induction;  8 days after induction; and  9 days after induction.
<dig> and  <dig> represent bright field and fluorescent images, respectively.
scale bars = 500 μm.
effects of yoebspn-gfp expression on the transgenic arabidopsis thaliana after induction with 17-β-estradiol
induction of expression of the yoebspn toxin in four-week-old t <dig> generation transgenic a. thaliana with 17-β-estradiol, resulted in plant defects and tissue necrosis by 3 days after induction, followed by plant death over a period of 9 days .
the transgenic plants showed severe discolouration over time and were completely discoloured on the 9th day after induction , whereas no such abnormalities were observed in the control  transgenic a. thaliana that were sprayed with ethanol instead of 17-β-estradiol.
similar results were observed for all four transformed plant lines.
the control plants grew normally and were able to produce flowers .
likewise, transgenic plants that expressed only gfp did not show abnormalities following 17-β-estradiol induction , indicating that the plant death was due to the expression of the yoebspn toxin.figure  <dig> effects of yoeb
spn-gfp expression on arabidopsis thaliana.
the appearance of transgenic t2
a. thaliana over a time course of 9 days.
17-β-estradiol induced plants with pmdc221_yoebgfp,  mock induced  plants with pmdc221_yoebgfp and  17-β-estradiol induced pmdc221_gfp.
in rt-pcr analysis of transgenic plants, the gfp and actin transcripts were detected by the presence of amplicons of the expected sizes of 168 bp and 100 bp, respectively .
quantitative rt-pcr analysis revealed that the expression of the yoebspn toxin gene in transgenic arabidopsis increased up to 3 days following induction with 17-β-estradiol, after which it decreased .figure  <dig> transcript analysis of t <dig> transgenic a. thaliana after induction with 17-β-estradiol.
agarose gel following rt-pcr with yoeb toxin and the control actin primers from four different plants transformed with pmdc221_yoebgfp and wild type control 24 hours after induction;  expression levels of the yoeb toxin in a. thaliana plants transformed with pmdc221_yoebgfp from day  <dig> – day  <dig> after induction as determined by qrt-pcr.
each bar represents the mean and standard error for  <dig> biological replicates which were chosen randomly from 100 t <dig> transgenic plants from line  <dig>
expression of the yoebspn toxin triggers dna laddering in a. thaliana
the results so far indicated that expression of the yoebspn toxin is lethal in arabidopsis.
to investigate the possibility that the lethality of yoebspn is due to activation of apoptosis, a dna fragmentation assay was carried out on 17-β-estradiol-induced arabidopsis samples.
genomic dna was extracted from  <dig> randomly selected transgenic plants at 6 h, 12 h and 24 h after induction.
agarose gel electrophoresis of the extracted genomic dna indicated that dna fragmentation was evident at 24 h after induction where distinct oligonucleosomal dna fragments ranging from  <dig> – 200 bp were observed in all the sampled plants , suggestive of apoptosis.
these oligonucleosomal fragments were not evident in extracted genomic dna 6 h and 12 h after induction but smearing of dna was observed .
such fragmentation was also not observed in the non-induced transgenic plant where the genomic dna isolated formed a single high molecular weight band following gel electrophoresis .figure  <dig> laddering of nuclear dna from transgenic a. thaliana.
dna extracted from a. thaliana and electrophoresed for 3 h on  <dig> % agarose.
lane 1: dna from non-induced plant transformed with pmdc221_yoebgfp.
dna isolated from plants transformed with pmdc221_yoebgfp  and wild type plants  at  <dig> and 12 hours after induction.
dna isolated from plants 24 hours after induction with 17-β-estradiol; lanes 1–6: plants transformed with pmdc221_yoebgfp; lanes wt1-wt3: wild type.
m: 100 bp dna ladder ; m2:1 kb dna ladder .
arrows indicate dna laddering or fragmentation suggestive of apoptosis.
discussion
to date, three chromosomally-encoded ta systems from streptococcus pneumoniae have been studied in detail, namely relbe- <dig>  <cit> , pezat  <cit>  and yefm-yoeb  <cit> .
overproduction of yoebspn was shown to be toxic to s. pneumoniae and e. coli cells, inhibiting cell growth and reducing cell viability  <cit> .
here we report the cloning and expression of yoebspn from streptococcus pneumoniae as a yoebspn-gfp fusion transgene in the model plant arabidopsis thaliana.
as it was possible that expression of the yoebspn toxin gene would be lethal to the plant, we placed the yoebspn toxin gene construct under the control of a strictly inducible promoter.
thus, a 17-β-estradiol-inducible expression system was used in this study to observe the effects of yoebspn toxin gene expression in a. thaliana.
a. thaliana transformed with pmdc221-yoebgfp was not detectably different from the wild type in the absence of 17-β-estradiol, however, after induction, plant leaves were distorted and had severe lesions .
quantitative rt-pcr analysis confirmed that the yoebspn-gfp fusion gene was expressed from day  <dig> after induction  and rna sampling stopped at day  <dig> because all the plants were dead by day  <dig> .
the presence of fluorescence showed that the yoebspn-gfp gene was functionally expressed in the transgenic a. thaliana and that the yoebspn-gfp protein is stable in transgenic a. thaliana as the fluorescence was observed even at days  <dig> and  <dig>  after plant death .
expression of the yoebspn-gfp protein was clearly detrimental to the transgenic plants producing distorted leaves and leading to 100% death of the t <dig> generation transgenic plants by the 8th day after induction with 17-β-estradiol.
as the plants with induced expression of only gfp, from the control vector pmdc221_gfp, did not show any leaf distortion , we conclude that the symptoms and dna damage seen in plants expressing the yoebspn-gfp fusion are due to expression of the toxin.
few studies have investigated the effects of the expression of bacterial ta systems in eukaryotic cells.
one of the earliest studies of such heterologous expression reported the toxic effects of the e. coli-encoded rele toxin in yeast cells  <cit> .
these findings led to the proposal that rele could be used to restrict growth of genetically modified yeast strains to controlled environments, i.e., as a biocontainment mechanism to prevent the accidental escape of genetically modified yeasts  <cit> .
the rele toxin functions as an inhibitor of translation in its native e. coli host and also when expressed in streptococcus pneumoniae  <cit> .
rele was subsequently shown to induce apoptosis in the human osteosarcoma cell line u2os where characteristic dna laddering was observed besides morphological changes such as membrane blebbing and chromatin condensation  <cit> .
since the yoeb toxin comes from the rele superfamily of toxins  <cit> , and also functions as an inhibitor of translation in e. coli  <cit> , it was expected that this toxin would also be functional in eukaryotic cells and thus may lead to apoptosis when expressed in arabidopsis.
similarities have been shown to exist between programmed cell death in plants and apoptosis in animals  <cit> , and dna laddering associated with programmed plant cell death has been reported  <cit> .
the uniform degradation of genomic dna into oligomers of apporoximately 180–200 bp, or multiples of that, characterizes internucleosomal cleavage of dna.
such fragmentation is a biochemical hallmark of apoptosis and was originally described in animal cells  <cit> .
we detected the oligonucleosomal dna ladder in yoebspn-gfp-expressing plants 24 h after induction, suggesting that expression of yoebspn may have triggered apoptosis, possibly mimicking mechanisms that are common to both animal and plant cell death programs.
besides rele, the kid toxin from the kis-kid tas has been shown to be functional in several eukaryotic cells by inhibiting proliferation in yeast, the frog xenopus laevis and human cell lines  <cit> .
kid was demonstrated to trigger apoptosis in hela cells with widespread cell death occuring beyond three days of kid expression and total cell death after 15 days  <cit> .
thus, although the oligonucleosomal dna laderring observed in the yoebspn-gfp-expressed arabidopsis is suggestive of apoptotic cell death, previous results from the rele  <cit>  and kid  <cit>  studies are indicative of the bacterial toxin triggering apoptosis.
it was reported that the bacterial yoebspn toxin caused cell death in both s. pneumoniae and e. coli but this toxin can be neutralized by tight binding with the cognate yefmspn antitoxin  <cit> .
this indicates that regulated expression of yoebspn and yefmspn might be used to kill particular cells in a selective way.
this could be achieved by expressing these two genes under the control of promoters that are, respectively, induced and repressed in these cells, and that have the inverse behaviour in normal cells.
furthermore, rele and yoeb have been shown to have similar folding as the rnase barnase  <cit> , a protein with a characteristic microbial rnase fold which has been used in plant ablation studies  <cit> .
previously, bacillus amyloliquefaciens barnase and barstar genes were used to genetically engineer a new system of male fertility control in higher plants  <cit> .
barstar binds specifically with barnase, forming highly stable complexes that could inhibit barnase from functioning  <cit> .
we showed that the expression of yoebspn toxin is lethal to arabidopsis thaliana and this could form the platform of an inducible plant cell ablation system where it would have potential application in biotechnology such as tissue specific expression to ablate pollen formation for the development of male sterile plants for containment of transgenic plants or for hybrid seed production.
the conditional expression of the yoebspn toxin gene could be used to contain unwanted genetically-modified plants arising from accidental out-crossing events, without danger to unmodified plants.
under this condition, the presence of inducer would convert an inert toxin to an active toxin that kills the transgenic plants.
future studies can be carried out to see whether the expressed toxin can be neutralized by its cognate antitoxin.
experiments analysing the effects of co-expression of the yefm-yoebspn toxin-antitoxin gene system in arabidopsis thaliana are in progress.
CONCLUSIONS
this study shows that the s. pneumoniae-encoded yoebspn toxin is functional and lethal in arabidopsis plants and that the gene can remain in the transgenic plant genome without any adverse effects until its expression is induced by the inducer.
the results presented here mark an important milestone towards the development of a bio-containment strategy, which may be useful for preventing the release of unwanted genetically modified plants to the environment, for the development of male sterile plants for hybrid seed production as well as the development of a novel cell ablation system for functional studies in plants.

 most bacteria harbour toxin-antitoxin systems, usually comprising a pair of genes coding for a toxin and its cognate antitoxin. upon antitoxin degradation, the toxin induces cell stasis or death in part of the cell population leading to the early proposal of ta systems being a form of programmed cell death in prokaryotes. ta systems were originally discovered in bacterial plasmids, where they function in the maintenance of plasmids through the post-segregational killing of plasmid-free daughter cells. chromosomally-encoded ta systems were subsequently found to be also abundant in bacteria and archaea where they have been implicated in several cellular functions such as programmed cell death, stress responses and in persistence and antibiotic tolerance. a wide variety of ta systems have been discovered in the past decade and currently, ta systems are grouped into classes i, ii, iii, iv and v according to the nature and action of the antitoxin. type ii ta systems have been most widely studied and encode a toxic protein and a relatively less stable cognate proteic antitoxin. environmental stress conditions usually lead to the induction of endogenous proteases which results in the degradation of antitoxins, thereby releasing the toxins from the inert toxin-antitoxin complex. the unbound toxin is now free to exert its lethal effect through different modes of action depending on the toxin type. the majority of characterized toxins act as endoribonucleases, some toxins interfere with dna gyrase activity and thus function as an inhibitor of dna replication and transcription, while other toxins inhibit translation initiation and interfere with cell wall synthesis. the chromosomal ta systems from gram negative bacteria are among the most prevalent and well-studied. some of these prokaryotic toxins have been shown to have activity when expressed in eukaryotic cells such as yeasts and have been proposed to have potential application in the control of cell growth in eukaryotic cells, especially in preventing the escape of genetically modified cells. the rele toxin of e. coli was demonstrated to be functional in the yeast saccharomyces cerevisiae where induction of the toxin gene in transformed yeast cells inhibited growth. expression of the rele toxin and the kid toxin were also shown to trigger apoptosis in a human osteosarcoma cell line and in hela cells, respectively. these findings eventually led to the development of a method using the kis-kid ta system to select for mammalian cells with a stable and high level expression of transgenes. another ta system that has been shown to be functional in eukaryotes is the epsilon-zeta system from plasmid psm of streptococcus pyogenes where expression of the zeta toxin in the yeast saccharomyces cerevisiae was shown to be lethal. up to putative ta systems have been found through a bioinformatics search of sequenced streptococcus pneumoniae genomes. out of these, three have been demonstrated to be functional bona fide ta systems, namely relbe, yefm-yoebspn and pezat. the yoeb toxin homologues have been shown to be endoribonucleases and overexpression of the yoebspn toxin led to cell death in both s. pneumoniae and e. coli. to our knowledge, there have been no reports on the functionality of bacterial ta systems in plants. here, we investigate the effects of expressing the yoebspn toxin gene in arabidopsis thaliana as a model plant system using a 17-β-estradiol-inducible expression system. ultimately, this work may lead to the development of a bio-containment strategy, which may be useful for preventing the release of unwanted genetically modified plants to the environment, for the development of male sterile plants for hybrid seed production as well as the development of a novel cell ablation system for functional studies in plants.  analysis of transgenic plants a 17-β-estradiol-inducible two-component system was used to obtain transgenic arabidopsis thaliana for controlled expression of the yoebspn toxin gene, cloned as a translational fusion with the gfp gene in the responder vector pmdc221_yoebgfp while the camv 35s promoter was cloned into the activator vector pmdc150_35s to drive the constitutive expression of the 17-β-estradiol-responsive xve transcriptional activator.figure vector constructs and pcr amplification from four-week-old t transgenic a. thaliana using gfp-specific primers. schematic diagram of the plant inducible expression vectors that were constructed for this study based on. agarose gel electrophoresis of pcr products using gfp-specific primers; samples were total dna from plants transformed with pmdc221_yoebgfp or with pmdc221-gfp and showing the expected amplified product of 732 bp; lane 13: sample was total dna from a wild type arabidopsis plant; 1 kb: 1 kb dna ladder marker. five independent transformation experiments were conducted and independent transgenic lines were selected for further analysis. out of 100 t yoebspn-gfp transgenic plants that grew on the selection medium, were randomly chosen for genomic dna preparation and pcr amplification using gfp-specific primers. the presence of a 732 bp band indicated the presence of the yoebspn-gfp fusion transgene and, thus, the successful integration of the transgene into the arabidopsis plants. this band was also present in the positive control plants expressing gfp alone from vector pmdc221_gfp and was absent in the negative control plant. similarly, when using yoebspn-specific primers, the presence of a yoebspn-specific band was also observed in the transgenic plants. when the leaves of the transgenic plants were examined under fluorescence confocal microscope, gfp fluorescence was detectable in all parts of the leaves 24 hours after 17-β-estradiol induction. leaves showed gfp fluorescence at days and after induction, and even at days and after plant death. distortion of the newly emerging rosette leaves and necrotic symptoms in all leaves were observed from day onwards, as shown in figure  figure pcr confirmation of yoeb spn toxin gene from four-week-old-t transgenic a. thaliana using yoeb-specific primers. samples were total dna extracted from the t transgenic plants showing the expected amplified product of 255 bp. lanes 1–2: transgenic line 1; lanes 3–4: transgenic line 2; lanes 5–6: transgenic line 3; lane -ve: dna from a wild type arabidopsis plant as negative control; 1 kb: 1 kb dna ladder marker. arabidopsis thaliana. plants transformed with pmdc221_yoebgfp were induced with 100 μm 17-β-estradiol at 4 weeks after selection. no induction; 1 day after induction; 3 days after induction; 6 days after induction; 8 days after induction; and 9 days after induction. and represent bright field and fluorescent images, respectively. scale bars = 500 μm. effects of yoebspn-gfp expression on the transgenic arabidopsis thaliana after induction with 17-β-estradiol induction of expression of the yoebspn toxin in four-week-old t generation transgenic a. thaliana with 17-β-estradiol, resulted in plant defects and tissue necrosis by 3 days after induction, followed by plant death over a period of 9 days. the transgenic plants showed severe discolouration over time and were completely discoloured on the 9th day after induction, whereas no such abnormalities were observed in the control transgenic a. thaliana that were sprayed with ethanol instead of 17-β-estradiol. similar results were observed for all four transformed plant lines. the control plants grew normally and were able to produce flowers. likewise, transgenic plants that expressed only gfp did not show abnormalities following 17-β-estradiol induction, indicating that the plant death was due to the expression of the yoebspn toxin.figure effects of yoeb spn-gfp expression on arabidopsis thaliana. the appearance of transgenic t2 a. thaliana over a time course of 9 days. 17-β-estradiol induced plants with pmdc221_yoebgfp, mock induced plants with pmdc221_yoebgfp and 17-β-estradiol induced pmdc221_gfp. in rt-pcr analysis of transgenic plants, the gfp and actin transcripts were detected by the presence of amplicons of the expected sizes of 168 bp and 100 bp, respectively. quantitative rt-pcr analysis revealed that the expression of the yoebspn toxin gene in transgenic arabidopsis increased up to 3 days following induction with 17-β-estradiol, after which it decreased.figure transcript analysis of t transgenic a. thaliana after induction with 17-β-estradiol. agarose gel following rt-pcr with yoeb toxin and the control actin primers from four different plants transformed with pmdc221_yoebgfp and wild type control 24 hours after induction; expression levels of the yoeb toxin in a. thaliana plants transformed with pmdc221_yoebgfp from day – day after induction as determined by qrt-pcr. each bar represents the mean and standard error for biological replicates which were chosen randomly from 100 t transgenic plants from line expression of the yoebspn toxin triggers dna laddering in a. thaliana the results so far indicated that expression of the yoebspn toxin is lethal in arabidopsis. to investigate the possibility that the lethality of yoebspn is due to activation of apoptosis, a dna fragmentation assay was carried out on 17-β-estradiol-induced arabidopsis samples. genomic dna was extracted from randomly selected transgenic plants at 6 h, 12 h and 24 h after induction. agarose gel electrophoresis of the extracted genomic dna indicated that dna fragmentation was evident at 24 h after induction where distinct oligonucleosomal dna fragments ranging from – 200 bp were observed in all the sampled plants, suggestive of apoptosis. these oligonucleosomal fragments were not evident in extracted genomic dna 6 h and 12 h after induction but smearing of dna was observed. such fragmentation was also not observed in the non-induced transgenic plant where the genomic dna isolated formed a single high molecular weight band following gel electrophoresis.figure laddering of nuclear dna from transgenic a. thaliana. dna extracted from a. thaliana and electrophoresed for 3 h on % agarose. lane 1: dna from non-induced plant transformed with pmdc221_yoebgfp. dna isolated from plants transformed with pmdc221_yoebgfp and wild type plants at and 12 hours after induction. dna isolated from plants 24 hours after induction with 17-β-estradiol; lanes 1–6: plants transformed with pmdc221_yoebgfp; lanes wt1-wt3: wild type. m: 100 bp dna ladder; m2:1 kb dna ladder. arrows indicate dna laddering or fragmentation suggestive of apoptosis. discussion to date, three chromosomally-encoded ta systems from streptococcus pneumoniae have been studied in detail, namely relbe-, pezat and yefm-yoeb. overproduction of yoebspn was shown to be toxic to s. pneumoniae and e. coli cells, inhibiting cell growth and reducing cell viability. here we report the cloning and expression of yoebspn from streptococcus pneumoniae as a yoebspn-gfp fusion transgene in the model plant arabidopsis thaliana. as it was possible that expression of the yoebspn toxin gene would be lethal to the plant, we placed the yoebspn toxin gene construct under the control of a strictly inducible promoter. thus, a 17-β-estradiol-inducible expression system was used in this study to observe the effects of yoebspn toxin gene expression in a. thaliana. a. thaliana transformed with pmdc221-yoebgfp was not detectably different from the wild type in the absence of 17-β-estradiol, however, after induction, plant leaves were distorted and had severe lesions. quantitative rt-pcr analysis confirmed that the yoebspn-gfp fusion gene was expressed from day after induction and rna sampling stopped at day because all the plants were dead by day. the presence of fluorescence showed that the yoebspn-gfp gene was functionally expressed in the transgenic a. thaliana and that the yoebspn-gfp protein is stable in transgenic a. thaliana as the fluorescence was observed even at days and after plant death. expression of the yoebspn-gfp protein was clearly detrimental to the transgenic plants producing distorted leaves and leading to 100% death of the t generation transgenic plants by the 8th day after induction with 17-β-estradiol. as the plants with induced expression of only gfp, from the control vector pmdc221_gfp, did not show any leaf distortion, we conclude that the symptoms and dna damage seen in plants expressing the yoebspn-gfp fusion are due to expression of the toxin. few studies have investigated the effects of the expression of bacterial ta systems in eukaryotic cells. one of the earliest studies of such heterologous expression reported the toxic effects of the e. coli-encoded rele toxin in yeast cells. these findings led to the proposal that rele could be used to restrict growth of genetically modified yeast strains to controlled environments, i.e., as a biocontainment mechanism to prevent the accidental escape of genetically modified yeasts. the rele toxin functions as an inhibitor of translation in its native e. coli host and also when expressed in streptococcus pneumoniae. rele was subsequently shown to induce apoptosis in the human osteosarcoma cell line u2os where characteristic dna laddering was observed besides morphological changes such as membrane blebbing and chromatin condensation. since the yoeb toxin comes from the rele superfamily of toxins, and also functions as an inhibitor of translation in e. coli, it was expected that this toxin would also be functional in eukaryotic cells and thus may lead to apoptosis when expressed in arabidopsis. similarities have been shown to exist between programmed cell death in plants and apoptosis in animals, and dna laddering associated with programmed plant cell death has been reported. the uniform degradation of genomic dna into oligomers of apporoximately 180–200 bp, or multiples of that, characterizes internucleosomal cleavage of dna. such fragmentation is a biochemical hallmark of apoptosis and was originally described in animal cells. we detected the oligonucleosomal dna ladder in yoebspn-gfp-expressing plants 24 h after induction, suggesting that expression of yoebspn may have triggered apoptosis, possibly mimicking mechanisms that are common to both animal and plant cell death programs. besides rele, the kid toxin from the kis-kid tas has been shown to be functional in several eukaryotic cells by inhibiting proliferation in yeast, the frog xenopus laevis and human cell lines. kid was demonstrated to trigger apoptosis in hela cells with widespread cell death occuring beyond three days of kid expression and total cell death after 15 days. thus, although the oligonucleosomal dna laderring observed in the yoebspn-gfp-expressed arabidopsis is suggestive of apoptotic cell death, previous results from the rele and kid studies are indicative of the bacterial toxin triggering apoptosis. it was reported that the bacterial yoebspn toxin caused cell death in both s. pneumoniae and e. coli but this toxin can be neutralized by tight binding with the cognate yefmspn antitoxin. this indicates that regulated expression of yoebspn and yefmspn might be used to kill particular cells in a selective way. this could be achieved by expressing these two genes under the control of promoters that are, respectively, induced and repressed in these cells, and that have the inverse behaviour in normal cells. furthermore, rele and yoeb have been shown to have similar folding as the rnase barnase, a protein with a characteristic microbial rnase fold which has been used in plant ablation studies. previously, bacillus amyloliquefaciens barnase and barstar genes were used to genetically engineer a new system of male fertility control in higher plants. barstar binds specifically with barnase, forming highly stable complexes that could inhibit barnase from functioning. we showed that the expression of yoebspn toxin is lethal to arabidopsis thaliana and this could form the platform of an inducible plant cell ablation system where it would have potential application in biotechnology such as tissue specific expression to ablate pollen formation for the development of male sterile plants for containment of transgenic plants or for hybrid seed production. the conditional expression of the yoebspn toxin gene could be used to contain unwanted genetically-modified plants arising from accidental out-crossing events, without danger to unmodified plants. under this condition, the presence of inducer would convert an inert toxin to an active toxin that kills the transgenic plants. future studies can be carried out to see whether the expressed toxin can be neutralized by its cognate antitoxin. experiments analysing the effects of co-expression of the yefm-yoebspn toxin-antitoxin gene system in arabidopsis thaliana are in progress.  this study shows that the s. pneumoniae-encoded yoebspn toxin is functional and lethal in arabidopsis plants and that the gene can remain in the transgenic plant genome without any adverse effects until its expression is induced by the inducer. the results presented here mark an important milestone towards the development of a bio-containment strategy, which may be useful for preventing the release of unwanted genetically modified plants to the environment, for the development of male sterile plants for hybrid seed production as well as the development of a novel cell ablation system for functional studies in plants. 
moreover, we detected dna laddering in the transgenic plants at hours after yoeb induction, indicative of apoptosis. the results presented here mark an important milestone towards the development of a cell ablation based bio-containment strategy, which may be useful for functional studies and for the control of spread of transgenic plants. overexpression of the chromosomally-encoded yoeb toxin from the yefm-yoeb toxin-antitoxin locus of the gram-positive bacterium streptococcus pneumoniae has been shown to cause cell death in s. pneumoniae as well as e. coli. expression of the yoeb toxin from streptococcus pneumoniae is lethal in arabidopsis. chromosomally-encoded toxin-antitoxin systems are involved in bacterial stress responses and activation of the toxins usually leads to cell death or dormancy. examination of plants by fluorescent confocal microscopy showed gfp fluorescence in all parts of the leaves at hours after 17-β-estradiol induction, continuing up to plant death. quantitative rt-pcr analysis revealed that the expression of the yoeb toxin gene peaked at days after induction with 17-β-estradiol, coinciding with the onset of visible effects on the plants.
 bacterial toxin-antitoxin systems usually comprise of a pair of genes encoding a stable toxin and its cognate labile antitoxin and are located in the chromosome or in plasmids of several bacterial species. chromosomally-encoded toxin-antitoxin systems are involved in bacterial stress responses and activation of the toxins usually leads to cell death or dormancy. overexpression of the chromosomally-encoded yoeb toxin from the yefm-yoeb toxin-antitoxin locus of the gram-positive bacterium streptococcus pneumoniae has been shown to cause cell death in s. pneumoniae as well as e. coli.  induction of a yoeb-gfp fusion protein using a 17-β-estradiol-inducible plant expression system in arabidopsis thaliana col was lethal in all t progeny. examination of plants by fluorescent confocal microscopy showed gfp fluorescence in all parts of the leaves at hours after 17-β-estradiol induction, continuing up to plant death. quantitative rt-pcr analysis revealed that the expression of the yoeb toxin gene peaked at days after induction with 17-β-estradiol, coinciding with the onset of visible effects on the plants. moreover, we detected dna laddering in the transgenic plants at hours after yoeb induction, indicative of apoptosis.  expression of the yoeb toxin from streptococcus pneumoniae is lethal in arabidopsis. we believe this is the first report of a toxin from a bacterial toxin-antitoxin system functioning in plants. the results presented here mark an important milestone towards the development of a cell ablation based bio-containment strategy, which may be useful for functional studies and for the control of spread of transgenic plants. keywords toxin-antitoxin systembacteriaarabidopsis thaliana17-β-estradiolexpressionissue-copyright-statement© the author 2015 