Identification of secret agent as the O-GlcNAc transferase that participates in Plum pox virus infection

Abstract

Serine and threonine of many nuclear and cytoplasmic proteins are posttranslationally modified with O-linked N-acetylglucosamine (O-GlcNAc). This modification is made by O-linked N-acetylglucosamine transferases (OGTs). Genetic and biochemical data have demonstrated the existence of two OGTs of Arabidopsis thaliana, SECRET AGENT (SEC) and SPINDLY (SPY), with at least partly overlapping functions, but there is little information on their target proteins. The N terminus of the capsid protein (CP) of Plum pox virus (PPV) isolated from Nicotiana clevelandii is O-GlcNAc modified. We show here that O-GlcNAc modification of PPV CP also takes place in other plant hosts, N. benthamiana and Arabidopsis. PPV was able to infect the Arabidopsis OGT mutants sec-1, sec-2, and spy-3, but at early times of the infection, both rate of virus spread and accumulation were reduced in sec-1 and sec-2 relative to spy-3 and wild-type plants. By matrix-assisted laser desorption ionization-time of flight mass spectrometry, we determined that a 39-residue tryptic peptide from the N terminus of CP of PPV purified from the spy-3 mutant, but not sec-1 or sec-2, was O-GlcNAc modified, suggesting that SEC but not SPY modifies the capsid. While our results indicate that O-GlcNAc modification of PPV CP by SEC is not essential for infection, they show that the modification has a role(s) in the process.

FIG. 1.

Localization of the capsid protein coding sequence in the genome of PPV-NK-GFP. The different PPV protein products are shown in the box representing the PPV polyprotein. Dark gray and black boxes represent the N- and C-terminal regions and the core region, respectively, of the PPV CP. The location of the introduced GFP open reading frame is indicated above the map. The sequence of the N-terminal tryptic peptide from amino acids 1 through 39 is underlined.

FIG. 2.

(A to F) MALDI-TOF analysis of trypsin-digested PPV virions purified from the plants indicated in each panel. The mass/charge ratio (m/z, in Daltons) assigned to peaks that can derive from the peptide from amino acids 1 through 39, as well as their suggested modifications, are indicated. O-GlcNAc, O-GlcNAc modification; PHOS, phosphorylation; AC, acetylation; a.i., arbitrary intensity; wt, wild type.

FIG. 3.

PPV spreading in mutant and wild-type (wt) Arabidopsis plants. Plants were inoculated with PPV-NK-GFP and observed at different times postinoculation under a fluorescence microscope. A ruler with minor divisions in mm is shown beside each picture.

FIG. 4.

Level of PPV infection in mutant and wild-type (wt) Arabidopsis plants. Four or five PPV-NK-GFP-infected plants of each Arabidopsis type were collected at 12 dpi and 19 dpi. Infection was assessed in all leaves of each plant by monitoring GFP expression with a fluorescence microscope. Leaves were classified as heavily infected when virus infection occupied more than one-fourth of the leaf lamina or more than half of the leaf vasculature.

FIG. 5.

PPV accumulation in infected leaves of mutant and wild-type (wt) Arabidopsis plants. Virus amount was determined by ELISA in two pools of caulinar and two pools of rosette systemically infected leaves from five (Col-0 wt, sec-2, and spy-3) or four (WS wt and sec-1) PPV-NK-GFP-inoculated plants collected at 12 dpi (between 8 and 36 caulinar and between 15 and 28 rosette-infected leaves) and 19 dpi (between 15 and 35 caulinar and between 6 and 21 rosette-infected leaves). The graphs show the average values with their standard deviations. Differences between sec-2 and Col-0 wt at 12 dpi and between sec-1 and WS wt at 12 and 19 dpi were statistically significant (P values of 0.04, 0.01, and 0.01). Differences between sec-2 and Col-0 wt at 19 dpi and between spy-3 and Col-0 wt at 12 and 19 dpi were not statistically significant (P values of 0.5, 0.82, and 0.53).