Geminivirus Replication Protein Impairs SUMO Conjugation of Proliferating Cellular Nuclear Antigen at Two Acceptor Sites

Abstract

Geminiviruses are DNA viruses that replicate in nuclei of infected plant cells using the plant DNA replication machinery, including PCNA (proliferating cellular nuclear antigen), a cofactor that orchestrates genome duplication and maintenance by recruiting crucial players to replication forks. These viruses encode a multifunctional protein, Rep, which is essential for viral replication, induces the accumulation of the host replication machinery, and interacts with several host proteins, including PCNA and the SUMO E2 conjugation enzyme (SCE1). Posttranslational modification of PCNA by ubiquitin or SUMO plays an essential role in the switching of PCNA between interacting partners during DNA metabolism processes (e.g., replication, recombination, and repair, etc.). In yeast, PCNA sumoylation has been associated with DNA repair involving homologous recombination (HR). Previously, we reported that ectopic Rep expression results in very specific changes in the sumoylation pattern of plant cells. In this work, we show, using a reconstituted sumoylation system in Escherichia coli, that tomato PCNA is sumoylated at two residues, K254 and K164, and that coexpression of the geminivirus protein Rep suppresses sumoylation at these lysines. Finally, we confirm that PCNA is sumoylated in planta and that Rep also interferes with PCNA sumoylation in plant cells.IMPORTANCE SUMO adducts have a key role in regulating the activity of animal and yeast PCNA on DNA repair and replication. Our work demonstrates for the first time that sumoylation of plant PCNA occurs in plant cells and that a plant virus interferes with this modification. This work marks the importance of sumoylation in allowing viral infection and replication in plants. Moreover, it constitutes a prime example of how viral proteins interfere with posttranslational modifications of selected host factors to create a proper environment for infection.

Keywords: PCNA; Rep; SUMO; begomovirus; geminivirus; homologous recombination; sumoylation.

FIG 1

Sumoylation of tomato PCNA in a reconstituted SUMO conjugation system in bacteria is modulated by Rep. Tomato PCNA, Rep from Tomato golden mosaic virus (TGMV), E1/E2 (mammalian SAE1, SAE2, and Ubc9), and SUMO (HsSUMO1) were coexpressed in E. coli NCM631 cells and extracted. Protein extracts were blotted with the antibodies indicated (left side). Expression (+/−) of the individual components is indicated at the top of each lane. Relevant bands are labeled on the right side. The band labeled “X1” could correspond to a SUMO tetramer or, more likely, to a complex of PCNA-2×SUMO. Band “X2” corresponds to a truncated form of the Rep protein. Molecular weight markers (Mw) are indicated.

FIG 2

Rep interferes with sumoylation of only PCNA but not other proteins. (A) Tomato PCNA, Rep from TGMV, C2 from Tomato yellow leaf curl Sardinia virus (TYLCSV), E1/E2 (mammalian SAE1, SAE2, and Ubc9), and SUMO (HsSUMO1) were coexpressed in E. coli NCM631 cells and extracted. Protein extracts were blotted with the antibodies indicated (left side). Expression (+/−) of the individual components is indicated. Relevant bands are labeled on the right side. (B) Similar to panel A except that catalase 3 (CAT3) is coexpressed. Molecular weight markers (Mw) are indicated.

FIG 3

Identification of tomato PCNA SUMO acceptor sites. (A) Primary sequence of tomato PCNA with residues K164 and K254 highlighted (red and green, respectively) (top) and the predicted 3D model of the structure of a tomato PCNA trimer (middle) or a monomer (bottom). (B) Sumoylation assay of tomato PCNA single mutants K164A and K254A and double mutant K164A/K254A in bacteria while coexpressing Rep from TGMV. The assay is similar to the one described in the legend of Fig. 1. Expression (+/−) of the individual components is indicated. E1/E2 enzymes were expressed in all samples. Molecular weight markers (Mw) are indicated.

FIG 4

The Rep-SCE1 interaction is not essential to suppress PCNA sumoylation in bacteria. The assay is similar to the one described in the legend of Fig. 1 except that a Rep variant that fails to interact with SCE1 was coexpressed (K68A/K102A) (lane 4). Molecular weight markers (Mw) are indicated.

FIG 5

Identification of Rep domains involved in interference of tomato PCNA sumoylation. (A) Graphical representation of the TGMV Rep protein. The TGMV intergenic region interaction domain (DNA, residues 1 to 130), the interaction domain with itself (Rep, residues 121 to 181), the interaction domain with other viral proteins (REn, residues 101 to 180), or the interaction domain with proteins from the plant (RBR, residues 101 to 180; SCE1, residues 56 to 130; and PCNA, residues 120 to 184) are represented. Domains involved in DNA replication initiation (I, II, and III) are also indicated. Rep deletion mutants are represented by horizontal bars: dark or light gray indicates, respectively, high or low levels of accumulation of truncated Rep in the bacterial extracts (see panel C). Molecular weight markers (M_w) are indicated. (B) Sumoylation assay of tomato PCNA in the presence of wild-type or truncated versions of Rep (Rep_xx–xxx). Mammalian SAE1, SAE2, SCE1, and SUMO proteins and mutant and wild-type (Wt) versions of tomato PCNA proteins were overexpressed in E. coli NCM631 cells and extracted. Protein extracts were blotted with anti-PCNA antibody. The expression of PCNA and SUMO (+/−) is indicated. E1/E2 enzymes were expressed in all samples. Relevant bands are labeled on the right. The amount of sumoylated PCNA was quantified using ImageJ software and relativized against the amount detected in the protein extracts containing wild-type Rep. (C) Expression levels of wild-type and truncated Rep obtained by blotting bacterial extracts with an anti-Rep antibody. All protein extracts were obtained from E. coli NCM631 cells expressing mammalian SAE1, SAE2, SCE1, and SUMO and tomato PCNA. Bands with an asterisk are identified as the result of Rep protein expression. Molecular weight markers (Mw) are indicated.

FIG 6

Arabidopsis SUMO conjugation enzymes modify the same lysine residues in tomato PCNA in bacteria. (A). Similar to the assay described in the legend of Fig. 1 except that AtE1/E2 (Arabidopsis SAE1, SAE2, and SCE1) was coexpressed together with the Arabidopsis SUMO paralogues SUMO1, -2, and -3 (AtS1/2/3). Expression (+/−) of the individual components is indicated. E1/E2 enzymes were expressed in all samples. (B) Similar to panel A except that PCNA variants in which K164, K254, or both residues were mutated to Ala were coexpressed. At, AtSUMO1; Hs, HsSUMO1 (lanes 4 to 6 and 8); Wt, wild-type tomato PCNA, as used in panel A. (C) Similar to panel A except that Rep from TGMV is coexpressed (lane 4). Molecular weight markers (Mw) are indicated.

FIG 7

Rep from Tomato yellow leaf curl virus (TYLCV) compromises sumoylation of tomato PCNA in planta. (A) Flag-tagged tomato PCNA together with Arabidopsis thaliana SUMO1 were transiently expressed in Nicotiana benthamiana leaves in the presence or absence of GFP-tagged Rep from TYLCV. Total protein extracts were subjected to immunoprecipitation, and proteins that coeluted from the Flag affinity resin (IP:anti-Flag) were blotted with anti-Flag or anti-AtSUMO1 antibodies. The top panel shows the enrichment of PCNA-Flag as a monomer but also as a dimer and trimer (∼70 and 100 kDa, respectively); the anti-SUMO immunoblot reveals that in the presence of Rep, the ∼50-kDa band, corresponding to the sumoylated monomeric form of PCNA-Flag, is reduced when Rep is coexpressed. (B) Total protein extracts (input) were analyzed by immunoblotting using anti-GFP antibody to show the accumulation of Rep-GFP and by Coomassie staining to confirm equal protein loading. Molecular weight markers (Mw) are indicated.