Caracterización de la modificación por O-GlcNAc de la proteína de la cápside del potyvirus Plum pox virus y su relevancia par la infección viral

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.