Suppression of Methylation-Mediated Transcriptional Gene Silencing by βC1-SAHH Protein Interaction during Geminivirus-Betasatellite Infection

Abstract

DNA methylation is a fundamental epigenetic modification that regulates gene expression and represses endogenous transposons and invading DNA viruses. As a counter-defense, the geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a betasatellite called DNA β. This study presents evidence that suppression of methylation-mediated TGS by the sole betasatellite-encoded protein, βC1, is crucial to the association of Tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB). We show that TYLCCNB complements Beet curly top virus (BCTV) L2- mutants deficient for methylation inhibition and TGS suppression, and that cytosine methylation levels in BCTV and TYLCCNV genomes, as well as the host genome, are substantially reduced by TYLCCNB or βC1 expression. We also demonstrate that while TYLCCNB or βC1 expression can reverse TGS, TYLCCNV by itself is ineffective. Thus its AC2/AL2 protein, known to have suppression activity in other geminiviruses, is likely a natural mutant in this respect. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed that βC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. We further demonstrate that βC1 protein inhibits SAHH activity in vitro. That βC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product, S-adenosyl methionine, may be a common viral strategy for methylation interference. We propose that inhibition of methylation and TGS by βC1 stabilizes geminivirus/betasatellite complexes.

Arabidopsis Double-Stranded RNA Binding Protein DRB3 Participates in Methylation-Mediated Defense against Geminiviruses

“…Recovery occurs when tissues arising after the establishment of a systemic infection exhibit symptom remission. Recovery additionally is characterized by reduced viral DNA levels, as documented previously for several geminivirushost combinations, including BCTV in N. benthamiana and Arabidopsis (33,36,40,50,51). Nicotiana benthamiana and Arabidopsis plants rarely recover from infection with wild-type BCTV but nearly always recover from infection with BCTV L2 Ϫ mutant virus, which lacks the L2 (also known as C2) pathogenicity factor that suppresses both PTGS and TGS and nonspecifically inhibits methylation (33,34,40,43,45).…”

“…Viral mRNAs are subject to posttranscriptional gene silencing (PTGS), and as a counterdefensive measure, geminivirus proteins suppress this cytoplasmic aspect of silencing (31,32). In addition, we have shown that RNA-directed methylation of viral chromatin leading to transcriptional gene silencing (TGS) is a potent antiviral defense which is suppressed by geminivirus-and geminivirus satellite-encoded proteins (33)(34)(35)(36). Genetic analysis has revealed that similar pathways are used to methylate geminivirus chromatin and resident transposons, suggesting that geminiviruses could serve as sensitive probes for the identification and analysis of methylation pathway components and effectors (4,33,37).…”

The role of virus-derived small interfering RNAs in RNA silencing in plants

“…Tomato yellow leaf curl China virus (TYLCCNV), a member of the geminiviruses, has a satellite RNA called DNA  (betasatellite). The latest study from Zhou's lab suggests that the pathogenicity factor C1 encoded by  satellite stabilized geminivirus/ satellite-complexes through the inhibition of methylation-mediated TGS and thereby in-duced disease symptoms [66]. At present, evidence for the role of vsiRNAs in translational repression is limited.…”

Antiviral Silencing and Suppression of Gene Silencing in Plants

“…In vitro methylated TGMV cannot replicate in protoplasts suggesting that viral genome methylation is a bona fide defense against geminiviruses (Bisaro 2006). Similarly, ßC1 suppressor of Tomato yellow leaf curl China virus (TYLCCNV) interacts and inhibits activity of S-adenosyl-homocystein-hydrolase (SAHH) that is involved in methyl-cycle and therefore indirectly affects TGS (Yang et al 2011). …”