A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein

Abstract

Geminiviruses are single-stranded DNA (ssDNA) viruses that infect a wide range of plants. To promote viral replication, geminiviruses manipulate the host cell cycle. The viral protein Rep is essential to reprogram the cell cycle and then initiate viral DNA replication by interacting with a plethora of nuclear host factors. Even though many protein domains of Rep have been characterized, little is known about its nuclear targeting. Here, we show that one conserved lysine in the N-terminal part of Rep is pivotal for nuclear localization of the Rep protein from Tomato yellow leaf curl virus (TYLCV), with two other lysines also contributing to its nuclear import. Previous work had identified that these residues are essential for Rep from Tomato golden mosaic virus (TGMV) to interact with the E2 SUMO-conjugating enzyme (SCE1). We here show that mutating these lysines leads to nuclear exclusion of TYLCV Rep without compromising its interaction with SCE1. Moreover, the ability of TYLCV Rep to promote viral DNA replication also depends on this highly conserved lysine independently of its role in nuclear import of Rep. Our data thus reveal that this lysine potentially has a broad role in geminivirus replication, but its role in nuclear import and SCE1 binding differs depending on the Rep protein examined. IMPORTANCE Nuclear activity of the replication initiator protein (Rep) of geminiviruses is essential for viral replication. We now define that one highly conserved lysine is important for nuclear import of Rep from three different begomoviruses. To our knowledge, this is the first time that nuclear localization has been mapped for any geminiviral Rep protein. Our data add another key function to this lysine residue, besides its roles in viral DNA replication and interaction with host factors, such as the SUMO E2-conjugating enzyme.

Identification of Tomato Proteins That Interact With Replication Initiator Protein (Rep) of the Geminivirus TYLCV

“…Moreover, the BiFC couple between Rep and this EWS-like protein resided in nuclear bodies (NBs). We reported previously that the BiFC pair Rep-SCE1 (SUMO E2 conjugating enzyme 1) also aggregates in NBs (Maio et al, 2019). Co-localization studies with marker proteins of different NBs should elucidate the nature and biological function of these different NBs.…”

“…For transient expression in N. benthamiana plants, the binary constructs were introduced in Agrobacterium tumefaciens GV3101, as previously described ( Maio et al., 2019 ). Briefly, single colonies of A. tumefaciens were grown overnight to an OD 600 of 0.8–1.5 in low salt (2.5 g/l NaCl) LB medium and resuspended in infiltration medium (1× MS [Murashige and Skoog] salts (Duchefa), 10 mM MES pH 5.6, 2% w/v sucrose, 200 μM acetosyringone) and incubated at room temperature for at least 2 h. Fully expanded leaves of 4-week old N. benthamiana plants were then syringe-infiltrated with these A. tumefaciens cell suspensions at an OD 600 = 1 for all constructs.…”

“…Two weeks post agroinoculation, one half of a fully expended systemic leaf was infiltrated with REP TYLCV -RFP, while the other half was infiltrated with KtoA triple mutant of REP TYLCV -RFP (K65A K69AK99A) at an OD 600 of 1.0 (Maio et al, 2019). This latter KtoA triple mutant can no longer replicate the 2IR-GFP cassette (negative control).…”

“…The VIGS experiment was repeated three times with a similar result. The 2IR-GFP extrachromosomical molecules (ECMs) were quantified as described previously (Maio et al, 2019). Primers for the ECMs and the internal reference gene, 25S rRNA, are given in Table S2.…”

RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus

“…Assembly of geminivirus particles also occurs in the nucleus, and large aggregates consisting of geminivirus particles have been observed in the nuclei of infected plant cells (Rushing et al, 1987). Due to the extensive reliance of geminivirus life cycle on nuclear activities, many geminivirus proteins, such as Replication initiator protein (Rep) and transcription activator protein, target to the plant nucleus (Maio et al, 2019). As the nucleolus is the critical sub-domain of a plant nucleus, it is reasonable that the nucleus-replicating geminiviruses could interact with the immediately accessible nucleolus.…”