The V2 protein encoded by a monopartite begomovirus is a suppressor of both post-transcriptional and transcriptional gene silencing activity

Highlights

• V2 protein encoded by Papaya leaf curl virus (PV2) is suppressor of post transcription gene silencing (PTGS).

• PV2 is also suppressor of transcription gene silencing (TGS).

• PV2 is an important factor in overcoming host RNA-silencing based defenses.

Abstract

Papaya leaf curl virus (PaLCuV) is a begomovirus (genus Begomovirus; family Geminiviridae) with a monopartite genome that is usually associated with beta- and alphasatellites in plants. Geminiviruses are DNA viruses with small circular genomes that occur as minichromosomes in the nucleus and are susceptible to post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). Transient expression of the PaLCuV V2 (PV2) protein together with the green fluorescent protein (GFP) in Nicotiana benthamiana resulted in enhanced levels of GFP fluorescence and GFP mRNA, indicative of suppression of PTGS. Expression of PV2 from a Potato virus X vector restored GFP expression in N. benthamiana plants harbouring a transcriptionally silenced GFP transgene, indicative of suppression of TGS. The results show that the PV2 protein encoded by PaLCuV has both suppressor of PTGS and TGS activity and is an important factor in overcoming host RNA-silencing based defenses.

Introduction

RNA silencing (also known as RNA interference) is involved in numerous fundamental biological processes, including defense against pathogens and mobile genetic elements such as transposons, regulation of gene expression, de novo DNA methylation and modification of chromatin (Dalakouras and Wassenegger, 2013) A characteristic of RNA silencing is the production of 21–24 nucleotide small interfering RNAs (siRNAs) that are derived from the processing of large double-stranded RNAs by the nuclease activity of RNase III-like enzymes called Dicer (Hamilton et al., 2002). RNA silencing occurs at both the transcriptional and post-transcriptional levels. Post-transcriptional gene silencing (PTGS) acts on target RNAs, including the genomes of RNA viruses, resulting in their degradation. Transcriptional gene silencing (TGS) results from the de novo methylation within promoter sequences that may induce chromatin modifications. TGS has role in maintaining genome integrity by preventing rearrangement in, for example, telomere and centromere repeats and by suppressing the activity of invasive DNAs such transposons (Pumplin and Voinnet, 2013).

The family Geminiviridae is the largest group of DNA viruses that encompasses pathogens that cause economically important diseases of food and fibre crops throughout the world (Mansoor et al., 2006; Hanley-Bowdoin et al., 2013). Geminiviruses have small circular single-stranded DNA (ssDNA) genomes that replicate in the nucleus and occur as minichromosomes (Bisaro, 2006). The family encompasses nine genera; Begomovirus, Mastrevirus, Topocuvirus, Curtovirus, Turncurtovirus, Eragrovirus, Becurtovirus, Grablovirus and Capulavirus (Zerbini et al., 2017). Of these the genus Begomovirus includes the agronomically most important viruses. Begomoviruses are exclusively transmitted by the whitefly Bemisia tabaci and infect only dicotyledonous plants in the warmer parts of the world. In the New World (NW) the vast majority of begomoviruses are bipartite, with a genome consisting of two ssDNA components known as DNA A and DNA B. Only three monopartite begomoviruses, with genomes consisting of a homolog of the DNA A component of bipartite begomoviruses, have been identified in the NW (Sánchez-Campos et al., 2013; Melgarejo et al., 2013; Macedo et al., 2017). By contrast the majority of begomoviruses in the Old World have monopartite genomes and are usually associated with small ssDNA satellites known as betasatellites and alphasatellites (Zhou, 2013).

The genomes (genomic components) of geminiviruses encode genes in both virion- and complementary-sense orientations. For monopartite begomoviruses, as well as the DNA A components of bipartite begomoviruses, the genes in the virion-sense encode the coat protein (which forms the characteristic geminate capsid and is involved in insect transmission and movement in plants) and the (A)V2 protein (which is involved in virus movement and may overcome small RNA-based host defenses) whereas the genes in the complementary-sense encode the replication-associated protein (Rep; a rolling-circle replication initiator protein), the transcriptional-activator protein (TrAP; which is involved in the upregulation of late viral gene expression, modulating host gene expression and overcoming small RNA-based host defenses), the replication enhancer protein (which is involved in creating a cellular environment that is conducive for virus replication) and the (A)C4 protein (which is involved in overcoming small RNA-based host defenses and can be a pathogenicity determinant; Sharma and Ikegami, 2008; Fondong, 2013).

Geminivirus DNA is subjected to epigenetic modifications that involve DNA methylation as well as methylation of associated histone proteins (Rodríguez-Negrete et al., 2009; Raja et al., 2010). The transcription of geminivirus DNA occurs in the nucleus and is affected by epigenetic modifications that causes silencing at the transcriptional level (Buchmann et al., 2009; Yang et al., 2011). Viral transcripts are transported to the cytoplasm where they are subjected to gene silencing at the post-transcriptional level (PTGS) (Voinnet et al., 1999). Thus, unlike plant RNA viruses that replicate in the cytoplasm, geminiviruses are the target of host defense both at the transcriptional and post-transcriptional levels. Characterization of siRNA from geminivirus infected plants has shown that they are homologous to both coding and regulatory sequences, consistent with the idea that geminiviruses are targets of both TGS and PTGS (Rodríguez-Negrete et al., 2009).

To counter RNA silencing mediated host defense geminiviruses encode suppressors of RNA silencing (Bisaro, 2006). Begomovirus-encoded proteins shown to have suppressor of PTGS activity include the V2 protein (Amin et al., 2011), TrAP (Wang et al., 2005) and the (A)C4 protein (Amin et al., 2011). Additionally the βC1 and Rep proteins encoded by the monopartite begomovirus-associated betasatellites and alphasatellites, respectively, have been shown to have suppressor of PTGS activity (Nawaz-Ul-Rehman et al., 2010; Amin et al., 2011). Those shown to have suppressor of repressive histone methylation and TGS activity include TrAP (Buchmann et al., 2009), Rep (Rodríguez-Negrete et al., 2013), the V2 protein (Wang et al., 2014) as well as the βC1 and Rep proteins of the satellites (Yang et al., 2011; Saeed et al., 2015; Abbas et al., 2017). These suppressors act at different stages in the gene-silencing pathway and by varying mechanisms (Raja et al., 2008).

The study detailed here as shown that the V2 protein encoded by the monopartite, betasatellite-associated begomovirus Papaya leaf curl virus (PaLCuV) is a suppressor of both PTGS and TGS. The significance of these findings with respect to begomoviruses overcoming host RNA-silencing based defenses is discussed.