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.
Highlights
During the past twenty years, geminiviruses have emerged as one of the most destructive plant-infecting virus families (Rojas et al, 2005)
In order to identify novel host proteins that interact with Tomato yellow leaf curl virus (TYLCV) Rep, we isolated tomato protoplasts from in vitro cultured plants and transfected them with plasmid DNA to express Rep-GFP alone or in combination with FLAG-PCNA (Figure 1)
The total protein fraction was extracted from three experimental replicates and subjected to affinity purification using anti-GFP resin followed by tryptic digestion in combination with a nano LC–MS/MS analysis
Summary
During the past twenty years, geminiviruses (family Geminiviridae) have emerged as one of the most destructive plant-infecting virus families (Rojas et al, 2005). Among them were the allelic genes Ty-1 and Ty-3, which encode an RNA-dependent RNA polymerase (Verlaan et al, 2013) Introgression of these genes into tomato cultivars conferred resistance to TYLCV by increasing cytosine DNA methylation of the viral genome, acting through viral transcriptional gene silencing (Caro et al, 2015). Another example of a TYLCV resistance locus is Ty-2, which was genetically linked to the gene TYNBS1 that encodes a NB-LRR (Nucleotide-Binding domain and Leucine-Rich Repeat-containing) type plant immune receptor (Yamaguchi et al, 2018). One strategy is to screen for resistance based on natural variation in Susceptibility (S) genes (van Schie and Takken, 2014), in this particular case for host genes that are critical for geminiviruses to complete their life cycle in plant cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
