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

Francesca Maio; Tieme A Helderman; Manuel Arroyo-Mateos; Miguel van der Wolf; Sjef Boeren; Marcel Prins; Harrold A van den Burg

doi:10.3389/fpls.2020.01069

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

Front Plant Sci. 2020 Jul 15:11:1069. doi: 10.3389/fpls.2020.01069. eCollection 2020.

Authors

Francesca Maio¹, Tieme A Helderman¹, Manuel Arroyo-Mateos¹, Miguel van der Wolf¹, Sjef Boeren², Marcel Prins^{1

3}, Harrold A van den Burg¹

Affiliations

¹ Molecular Plant Pathology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, Amsterdam, Netherlands.
² Laboratory of Biochemistry, Wageningen University, Wageningen, Netherlands.
³ Keygene N.V., Wageningen, Netherlands.

Abstract

Geminiviruses are plant-infecting DNA viruses that reshape the intracellular environment of their host in order to create favorable conditions for viral replication and propagation. Viral manipulation is largely mediated via interactions between viral and host proteins. Identification of this protein network helps us to understand how these viruses manipulate their host and therefore provides us potentially with novel leads for resistance against this class of pathogens, as genetic variation in the corresponding plant genes could subvert viral manipulation. Different studies have already yielded a list of host proteins that interact with one of the geminiviral proteins. Here, we use affinity purification followed by mass spectrometry (AP-MS) to further expand this list of interacting proteins, focusing on an important host (tomato) and the Replication initiator protein (Rep, AL1, C1) from Tomato yellow leaf curl virus (TYLCV). Rep is the only geminiviral protein proven to be essential for geminiviral replication and it forms an integral part of viral replisomes, a protein complex that consists of plant and viral proteins that allows for viral DNA replication. Using AP-MS, fifty-four 'high confidence' tomato proteins were identified that specifically co-purified with Rep. For two of them, an unknown EWS-like RNA-binding protein (called Geminivirus Rep interacting EWS-like protein 1 or GRIEP1) and an isoform of the THO complex subunit 4A (ALY1), we were able to confirm this interaction with Rep in planta using a second method, bimolecular fluorescence complementation (BiFC). The THO subunit 4 is part of the THO/TREX (TRanscription-EXport) complex, which controls RNA splicing and nuclear export of mRNA to the cytoplasm and is also connected to plant disease resistance. This work represents the first step towards characterization of novel host factors with a putative role in the life cycle of TYLCV and possibly other geminiviruses.

Keywords: PCNA; Rep; Tomato yellow leaf curl virus; geminivirus; interactome; proteomics; viral replication.