Effects of the expression of the bacterial YEFM-YOEBSPN chromosomal toxin-antitoxin system in Arabidopsis Thaliana / Fauziah Abu Bakar
Toxin-antitoxin (TA) systems are extensively found in bacteria as well as in archaea where they play diverse roles in important cellular functions. Bacterial TA systems usually comprise of a pair of genes encoding a stable toxin and its cognate labile antitoxin and are located in the chromosome o...
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| Format: | Thesis |
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2016
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| Online Access: | http://studentsrepo.um.edu.my/6869/1/fauziah.pdf http://studentsrepo.um.edu.my/6869/ |
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| Summary: | Toxin-antitoxin (TA) systems are extensively found in bacteria as well as in
archaea where they play diverse roles in important cellular functions. Bacterial TA
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. Chromosomally-encoded TA
systems are involved in a variety of cellular processes including as part of the global stress
response of bacteria, and as mediators of programmed cell death as well as biofilm
formation, in which the activation of the toxins usually leads to cell death or dormancy.
The genome of the human pathogen Streptococcus pneumoniae contains up to 10 putative
TA systems and among these, the yefM-yoeBSpn locus has been well studied and
demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin has been
shown to lead to cell stasis and eventually cell death in its native host cell as well as in E.
coli. Several toxins of TA systems have been heterologously expressed in eukaryotic
systems including yeasts, zebrafish, frog embryos and human cell lines where they have
been shown to be lethal. However, there has been no report on the functionality of any
bacterial TA systems in plants. In this study, a two-component 17-β-estradiol-inducible
expression system was utilized to investigate the heterologous expression of the yoeBSpn
toxin along with its cognate yefMSpn antitoxin in Arabidopsis thaliana. The coding
sequence of the yoeBSpn toxin was cloned as a translational fusion with Green Fluorescent
Protein and A. thaliana was transformed via floral dip using Agrobacterium tumefaciensmediated
transformation method. Transgenic A. thaliana were allowed to grow on
selection media until T2 generation. Induced expression of the yoeBSpn toxin-GFP fusion
transgene apparently triggered apoptosis and was lethal in A. thaliana. To investigate if
expression of the yefMSpn could mitigate the toxicity of yoeBSpn in A. thaliana, transgenic
plant carrying yefMSpn was first constructed and then cross-pollinated with transgenic
plant containing the yoeBSpn-GFP transgene. The yefMSpn × yoeBSpn-GFP hybrid
transgenic plants obtained were allowed to grow until maturity on selection media. When
co-expressed in A. thaliana, the YefMSpn antitoxin was found to neutralize the toxicity of
YoeBSpn-GFP. Interestingly, the inducible expression of both the yefMSpn antitoxin and
yoeBSpn toxin-GFP fusion transgenes in transgenic hybrid plants resulted in larger rosette
leaves, taller plants with more inflorescence stems and increased silique production. The
detailed mechanism by which co-expression of yoeBSpn-GFP and yefMSpn led to enhanced
plant growth remains to be elucidated. In their original bacterial hosts, YefMSpn forms a
tight protein complex with YoeBSpn and this TA complex binds to the operator site
overlapping the yefM-yoeBSpn promoter to repress its transcription. It is possible that the
YefM-YoeBSpn complex in A. thaliana binds to certain regions of the plant genome
leading to the enhanced growth phenotype. To our knowledge, this is the first
demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. The
functional lethality of the YoeBSpn toxin enables it to be harnessed for a potential novel
plant cell ablation system. |
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