Subsequently, the resin was collected by centrifugation (5,000 for 1 min
Subsequently, the resin was collected by centrifugation (5,000 for 1 min. etc.). In yeast, PCNA sumoylation has been associated with DNA repair including homologous recombination (HR). Previously, we reported that ectopic Rep expression results in very specific changes in the sumoylation pattern of herb cells. In this work, we show, using a reconstituted sumoylation system in and that Rep also interferes with PCNA sumoylation in herb 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 herb PCNA occurs in herb cells and that a herb virus interferes with this modification. This work marks the importance of sumoylation in allowing viral contamination 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 contamination. (TGMV), or monopartite genomes, like (TYLCV). Begomoviruses encode two proteins involved in viral replication: Rep (also called AL1, AC1, and C1), a multifunctional essential protein, and C3 (also called AL3, AC3, C3, and REn), which interacts with Rep and greatly enhances begomovirus DNA accumulation in host cells (4). Rep has different functions: it mediates acknowledgement of its cognate origin of replication in a geminivirus species-specific manner (5), it is required for initiation and termination of viral DNA synthesis (6,C8), and it functions as a DNA helicase (9, 10). Growing evidence strongly supports the notion that geminivirus proteins have a significant impact on a variety of host processes, including cell differentiation, cell cycle control, DNA replication, plasmodesma function, and RNA silencing (3). By these means, geminiviruses reshape their environment by co-opting cellular processes necessary for viral replication, systemic spread, and impairment of herb defenses. There are numerous mechanisms by which geminiviruses mediate their effects on the host cell, including targeting of posttranslational modification systems. Such systems play crucial roles in many cellular processes because they cause rapid changes in (i) the function of preexisting proteins, (ii) the composition of multiprotein complexes, and (iii) their subcellular localization. Their versatility in regulating protein function and cellular behavior makes them a particularly attractive target for viruses. One example of a key cellular regulatory system targeted by viruses is usually sumoylation (11, 12), a posttranslational process mainly involved in nuclear functions that modifies protein function, activity, or localization of its targets through covalent attachment of a 10-kDa ubiquitin-like polypeptide called SUMO (small ubiquitin-like modifier) (13,C15). Briefly, posttranslational modification by SUMO entails a cascade of ATP-dependent reactions that are mechanistically much like ubiquitination, including sequential activation and conjugation of SUMO. SUMO activation is usually driven by an E1 enzyme (SUMO-activating enzyme SAE1/SAE2 heterodimer), while SUMO conjugation is usually mediated by a single E2 enzyme (SUMO-conjugating enzyme VHL SCE1, also known as Ubc9 in yeast and mammals). The final transfer of SUMO from SCE1 to specific lysine residues in target proteins can occur directly or can be enhanced by SUMO ligases (14, 16). Target proteins can undergo monosumoylation of one lysine, polysumoylation (SUMO chain formation), or multisumoylation (modification of several lysines in one substrate) (17,C19). SUMO can be specifically detached from altered lysines by SUMO proteases (ubiquitin-like specific proteases; ULPs), making it Tomatidine a reversible and dynamic process (18, 20). The consequences of sumoylation on targets are very diverse, ranging from Tomatidine changes in localization to altered activity and, in some cases, stabilization of the altered protein. All of these effects are frequently the result of changes in the molecular interactions of the sumoylated proteins. Sumoylation can either mask a binding site in its target, thus inhibiting its interactions with other proteins; increase the quantity of binding sites on its target, hence facilitating the binding of molecules, such as proteins or DNA; or produce a conformational switch that modulates its activity. In plants, the characterization of the sumoylation enzymes has largely been restricted to genome carries eight full-length SUMO genes (genes), a single gene encoding the SUMO-conjugating enzyme SCE1 ((2, 3, 23,C27). In plants, Tomatidine sumoylation is important for embryonic development, organ growth, flowering transition, and hormone regulation (4, 28). In addition, SUMO also plays a key role in Tomatidine stress-associated responses to stimuli such as extreme temperatures, drought, salinity, and nutrient assimilation (5, 29, 30)..