| dc.description.abstract | The addition of residues of N-acetylglucosamine through O linkages (O-GlcNAc) to
target proteins is a post-translational modification widely distributed in eukaryotic cells. A
large number of O-GlcNAc modification sites have been mapped in mammalian proteins and
the functions of these proteins have been thoroughly studied. However in plants, targets of OGlcNAcylation
are largely uncharacterized. Arabidopsis thaliana has two O-GlcNAc
transferases (OGTs), Spindly (SPY) and Secret Agent (SEC), and previous results of the
laboratory where this PhD thesis was carried out demonstrated that the coat protein (CP) of
the potyvirus Plum pox virus (PPV) is O-GlcNAcylated and phosphorylated. In this work, we
identified that SEC, and not SPY, is the OGT responsible of the PPV CP O-GlcNAcylation.
In null sec- mutants of A. thaliana and in Nicotiana benthamiana transgenic plants in which
the SEC-like genes where silenced by RNAi, O-GlcNAcylation of PPV CP was abolished or
drastically disturbed, respectively. Virus accumulation was reduced at early times of infection
in these plants compared to wild type plants.
Site-directed mutagenesis and mass spectrometry analysis demonstrated that
threonines 19, 24, 41, 50, 53, 54 and 58 could be modified by O-GlcNAc or influenced the
modification of other residues. Further mapping by ETD MS/MS confirmed that Thr-19, Thr-
24, Thr-41, Thr-53 and Thr-54 and/or Thr-58, as well as the serine 65 are O-GlcNAcylation
targets. CP O-GlcNAcylation appeared to be completely abolished in a PPV multiple mutant
(PPV CP7-T/A) in which the seven threonines involved in O-GlcNAc modification were
replaced by alanines. The O-GlcNAcylation-deficient PPV mutant infected N. clevelandii, N.
benthamiana, and P. persica without noticeable defects. In contrast, this mutant infected
poorly A. thaliana. The host-specific relevance of O-GlcNAcylation of PPV CP is further
supported by the result of mixed infections in different host plants. O-GlcNAcylation seems
to enhance PPV CP stability in a host-specific way. Thus, CP of PPV CP7T/A was much
more unstable than that of wild type PPV in extracts of A. thaliana, whereas differences in
stability of the two CPs was less noticeable in extracts of N. clevelandii and P. persica. The
stabilities of wild type and CP7T/A CPs were quite similar in extracts of A. thaliana sec-,
suggesting that the instability of the CP7T/A CP is the result of the O-GlcNAcylation
deficiency rather than of the amino acid substitutions. All these results are in agreement with
a fine-tuning effect of CP O-GlcNAcylation on PPV infection, probably facilitating its
adaptation to different hosts and environmental conditions. | en_US |
