Interplay between S-nitrosylation and SUMOylation in plant immunity
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2015-06-29Author
Skelly, Michael J.
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Post-translational protein modifications (PTM) vastly increase the complexity and
functional diversity of the proteome, to precisely regulate crucial cellular processes.
The plant immune system is composed of complex signalling networks that are
influenced by various PTMs. Activation of plant immunity is associated with a rapid
burst of nitric oxide (NO), which can covalently modify cysteine thiols within target
proteins by a process termed S-nitrosylation to form S-nitrosothiols (SNOs),
constituting a redox-based PTM. Another key PTM involved in plant immunity is
SUMOylation, an essential mechanism involving the conjugation of the small
ubiquitin-like modifier (SUMO) peptide to lysine residues within target proteins.
Although the targets and mechanisms of S-nitrosylation and SUMOylation are
becoming evident, how these key PTMs are themselves regulated remains obscure.
Work presented in this thesis reveals that during plant immune signalling, the sole
Arabidopsis thaliana SUMO conjugating enzyme, SUMO CONJUGATING
ENZYME 1 (SCE1), is S-nitrosylated at a highly conserved, but previously
uncharacterized cysteine. S-nitrosylation of SCE1 was shown to inhibit its SUMO
conjugating activity in vitro and mutational analysis revealed that the site of this
modification, Cys139, is not required for enzyme activity but rather constitutes a
redox-sensitive inhibitory switch. Generation and characterization of transgenic
Arabidopsis plants overexpressing both wild-type and mutant forms of SCE1
revealed that Cys139 is required for efficient immunity against bacterial pathogens.
Furthermore, after immune activation, S-nitrosylation of this residue inhibits global
SUMOylation of proteins. These results provide evidence of a novel means of
crosstalk between S-nitrosylation and SUMOylation in the context of plant
immunity.
The abundant cellular antioxidant, glutathione (GSH), is S-nitrosylated to form S-nitrosoglutathione
(GSNO), which is thought to constitute a stable reservoir of NO
bioactivity. In Arabidopsis, GSNO levels are controlled by the enzyme S-NITROSOGLUTATHIONE
REDUCTASE 1 (GSNOR1), which indirectly
influences the levels of protein SNOs. In this study, transgenic plants overexpressing
FLAG-epitope tagged GSNOR1 were generated in various mutant backgrounds,
including nitric oxide overproducer 1 (nox1), to further investigate the roles of
GSNOR1 and NO in plant immunity. It was shown that ectopic GSNOR1 expression
completely recovers developmental and disease susceptibility phenotypes of gsnor1,
but not nox1 mutant plants, highlighting in vivo differences between accumulation of
GSNO and free NO. Surprisingly, elevated NO levels in nox1 plants promote S-nitrosylation
of GSNOR1, inhibiting its enzymatic activity. This suggests a
previously unreported means by which NO might regulate its own bioavailability.
Further work in this study revealed that recombinant GSNOR1 can be SUMOylated
in vitro, which appeared to increase its enzymatic activity. Several potential SUMO
modification sites were identified within GSNOR1 and mutational analysis revealed
that at least one of these, Lys191, is SUMOylated. Co-immunoprecipitation
experiments revealed that transgenic GSNOR1 might be SUMOylated in vivo,
although the site(s) and biological function of SUMOylation were not identified.
Nonetheless, these results reveal another possible layer of interplay between S-nitrosylation
and SUMOylation.