V2 of tomato yellow leaf curl virus can suppress methylation-mediated transcriptional gene silencing in plants

Abstract

Tomato yellow leaf curl virus (TYLCV) is a DNA virus belonging to the genus Begomovirus. TYLCV replicates using double-stranded DNA intermediates that can become the target of plant transcriptional gene silencing (TGS). Here, we show that the V2 protein of TYLCV can suppress TGS of a transcriptionally silenced green fluorescent protein (GFP) transgene in Nicotiana benthamiana line 16-TGS. Through bisulfite sequencing and chop-PCR, we demonstrated that the TYLCV V2 can reverse GFP transgene silencing by reducing the methylation levels in the 35S promoter sequence. Both AtSN1 and MEA-ISR loci in Arabidopsis thaliana were previously reported to be strongly methylated, and we show that the methylation status of both loci was significantly reduced in TYLCV V2 transgenic Arabidopsis plants. We conclude that TYLCV can efficiently suppress TGS when it infects plants, and its V2 protein is responsible for the TGS suppression activity.Cytosine DNA methylation is a conserved epigenetic silencing mechanism modulating many important biological processes (Bird, 2002;Goll & Bestor, 2005;Zhang et al., 2006) and a defence against biotic stresses such as geminivirus infection (Bisaro, 2006;Raja et al., 2010; Yang et al., 2011). Geminiviruses (family Geminiviridae) are a group of plant-infecting viruses containing circular, singlestranded DNA (ssDNA) genomes packaged into twinned particles (Dry et al., 1993;Fauquet et al., 2008;Zhang et al., 2009;Zhou, 2013). Geminiviruses are transmitted by whiteflies or leafhoppers and can cause significant damage to agronomically important crops (Navot et al., 1991;Hanley-Bowdoin et al., 2004;Rojas et al., 2005). Geminiviruses are classified into four genera (Mastrevirus, Curtovirus, Begomovirus, and Topocuvirus) and replicate in the plant cell nucleus using double-stranded DNA (dsDNA) intermediates as their replication and transcription templates (Pilartz & Jeske, 1992Fauquet et al., 2008).Recent studies have demonstrated that plants employ an RNA-directed methylation strategy to control transcriptional gene silencing (TGS) and use it as a defence mechanism against geminivirus infection (Raja et al., 2008;Rodríguez-Negrete et al., 2009). As a counter defensive measure, geminiviruses express unique proteins to serve as TGS suppressors. For example, the AC2/AL2 proteins encoded by cabbage leaf curl virus and tomato golden mosaic virus, the C2/L2 proteins encoded by beet curly top virus and beet severe curly top virus, and the bC1 protein encoded by tomato yellow leaf curl China betasatellite (TYLCCNB) can decrease DNA methylation and suppress TGS by interfering with the methyl cycle (Buchmann et al., 2009; Yang et al., 2011;Zhang et al., 2011). A recent report indicated that geminivirus replication-associated protein (Rep, also known as C1, AL1 or AC1) could also suppress TGS by reducing the expression of plant DNA methyltransferases (Rodríguez-Negrete et al., 2013).Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus and contains a single genome component with six open r...

The AC5 protein encoded by Mungbean yellow mosaic India virus is a pathogenicity determinant that suppresses RNA silencing‐based antiviral defenses

“…Total DNA was extracted from mock‐, PVX‐, PVX‐AC5‐ and PVX‐βC1‐infected 16‐TGS plants, and bisulfite reagent was used to convert unmethylated cytosine to uracil. The 35S promoter sequence which contains eight CG, 11 CHG and 50 CHH sites was then amplified with specific primers (Wang et al ., ). The PCR products were cloned and c .…”

“…To improve the efficiency of bisulfite treatment, total DNA (2 lg) was digested with the restriction enzyme EcoRI which cuts outside the region of interest to decrease the size, followed by overnight treatment with proteinase K. Bisulfite modification was carried out using the EZ DNA Methylation Gold TM kit (Zymo Research, Orange, CA, USA) using a PCR machine. Bisulfite treatment and subsequent PCR cloning were performed as described previously (Wang et al, 2014).…”

Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant

“…Total nucleic acids were extracted from systemically infected leaves using a Hi-DNA secure plant kit (Tiangen). Chop-PCR was performed as described previously (39,68). Briefly, 1 g total nucleic acids was digested with a methylation-sensitive restriction endonuclease, HinfI, or a methylationdependent restriction endonuclease, McrBC (NEB), in a 20-l reaction mixture according to the manufacturer's recommendations.…”

Tomato plant cell death induced by inhibition of HSP90 is alleviated by Tomato yellow leaf curl virus infection

“…V1 encodes the coat protein (CP) that functions as a nuclear shuttle protein and mediates vector transmission (Priyadarshini et al, 2011). V2 encodes a protein involved in virus movement (Priyadarshini et al, 2011), in the suppression of posttranscriptional gene silencing (PTGS) (Zhang et al, 2012) and in the suppression of methylation-mediated transcriptional gene silencing (TGS) (Wang et al, 2014a). C1 encodes a replicationassociated protein (Rep) which initiates viral replication (Hanley-Bowdoin et al, 2000).…”