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

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 EWSlike 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.

RNA-Seq Transcriptome Analysis Provides Candidate Genes for Resistance to Tomato Leaf Curl New Delhi Virus in Melon

“…Prior works suggest an interconnection between loss of function of translation factors and replication of the viral genomes, changes in the cell wall affecting viral movement and activation of immunity pathways ( Sanfaçon, 2015 ; Ding et al, 2018 ). Indeed, geminiviruses not only recruit plant translational components, but also enhance their replication and spread using and relocalizing host genes involved in DNA replication and transcription ( Preiss and Jeske, 2003 ; Kushwaha et al, 2017 ; Wu et al, 2019 ; Maio et al, 2020 ). The interference with this viral strategy is widely described conferring resistance to begomoviruses ( Ullah et al, 2014 ; Chakraborty and Basak, 2018 ).…”

Fine‐tuning of SUMOylation modulates drought tolerance of apple

“…To identify the interacting proteins of MdDREB2A in vivo , AP‐MASS assay was performed as described previously (Maio et al ., 2020 ) with modifications. Total proteins were extracted in leaves of GL‐3 plants with or without 2‐h dehydration treatments using extraction buffer [50 m m Tris‐HCl, pH 8.0, 150 m m NaCl, 2 m m EDTA, 1 m m DTT, 10% glycerol, 1% Triton X‐100, 1 m m phenylmethylsulfonyl fluoride (PMSF) and 1× Halt protease inhibitor cocktail].…”

Members of the ribosomal protein S6 (RPS6) family act as pro‐viral factor for tomato spotted wilt orthotospovirus infectivity in Nicotiana benthamiana

“…To quantify the transcript levels of a gene‐of‐interest or TSWV N , a total of 100 mg of systemically infected leaf tissue was collected from a 6‐week‐old N. benthamiana plant at 2 weeks after TSWV inoculation (i.e., 4 weeks after TRV agro‐inoculation to initiate VIGS). RNA isolation, transcript quantification, and data analyses were performed as described by Maio et al (2020). Gene expression data were normalized using N. benthamiana APR as a reference gene (Liu et al, 2012).…”