The Great Escape: How a Plant DNA Virus Hijacks an Imprinted Host Gene to Avoid Silencing

Plant viruses are a major threat to global food security. The large family of geminiviruses has a broad pathogenic impact on economically important crops such as maize ( Zea mays ), tomato ( Solanum lycopersicum ), and cassava ( Manihot esculenta ), and causes millions of tons of losses per year

Plant viruses are a major threat to global food security. The large family of geminiviruses has a broad pathogenic impact on economically important crops such as maize (Zea mays), tomato (Solanum lycopersicum), and cassava (Manihot esculenta), and causes millions of tons of losses per year worldwide (Rojas et al., 2005). Geminiviruses possess a circular DNA genome encoding proteins necessary for viral replication and virion assembly. The plant hosts have developed antiviral defense systems such as RNA interference that ensures viral posttranscriptional gene silencing (TGS) by targeting viral transcripts, and small RNA-mediated DNA methylation that triggers TGS of the viral DNA. In an evolutionary arms race, geminiviruses have developed counter-defense systems to overcome both TGS and post-TGS through multiple viral suppressors of silencing, including Rep and C2 that are produced early during viral infection and are thus instrumental for successful viral amplification (Zhang et al., 2011;Rodríguez-Negrete et al., 2013). While the anti-silencing activity of C2 itself is well described, little is known about how C2 allows the virus to escape silencing soon after infection.
In this issue of The Plant Cell, Chen et al. (2020) used Arabidopsis (Arabidopsis thaliana) plants infected by the geminivirus Beet Severe Curly Top Virus (BSCTV) as a model to investigate the molecular mechanisms permitting early C2 expression. Using transgenic Arabidopsis lines carrying a BSCTV Rep transgene that also contained the C2 N-terminal sequence (C2 N ) under the control of the C2 promoter, the authors observed accumulation of Rep mRNA in all lines tested, but unexpectedly detected C2 N transcripts in one of the transgenic lines. The accumulation of C2 N transcripts correlated with hypomethylation at the C2 N promoter that may have allowed transcription. To illuminate the molecular determinants of C2 N transcript accumulation, the authors compared the transcriptomes of the transgenic line expressing C2 N to wild-type plants and observed ectopic transcription of the imprinted E3 ubiquitin ligase-encoding gene VARIANT IN METHYLATION5 (VIM5). Overexpression of VIM5 in another transgenic line that did not show C2 N transcription triggered C2 N transcript accumulation and correlated with decreased DNA methylation at the transgenic C2 promoter. BSCTV infection of wild-type Arabidopsis plants recapitulated the molecular patterns observed in the transgenic plants. In BSCTV-infected vim5 mutants, the C2 promoter appeared heavily methylated and viral replication was delayed. A p35S-VIM5 transgene complemented the vim5 mutation, reduced DNA methylation at the C2 promoter, and restored early viral replication. The authors concluded that upon BSCTV infection, ectopic activation of VIM5 facilitates transcription of the viral C2 gene by limiting DNA methylation at the C2 promoter.
VIM5 has E3 ubiquitin ligase activity, suggesting that VIM5 maintains hypomethylation at the C2 promoter by targeting host DNA methyltransferases for degradation by the ubiquitin 26S proteasome proteolytic pathway. VIM5 indeed interacted in vitro and in vivo with the CG BSCTV-Induced Ectopic VIM5 Expression Promotes Degradation of the Host DNA Methyltransferases and Facilitates Viral Accumulation.
BSCTV infection triggers the ectopic expression of the host endosperm-imprinted E3 ubiquitin ligase-encoding gene VIM5. VIM5 promotes 26S proteasomal degradation of the host DNA methyltransferases MET1 and CMT3. VIM5 activity results in the hypomethylation and transcription of C2, the viral suppressor of silencing that promotes early viral accumulation. (Adapted from Chen et al. [2020], Supplemental Figure 9.) [OPEN] Articles can be viewed without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.20.00631 methyltransferase MET1 and the CHG methyltransferase CMT3, but not with the CHH methyltransferase DRM2. Coinfiltration experiments demonstrated that VIM5-dependent ubiquitination of MET1 and CMT3 proteins triggered their proteasomal degradation. To assess the molecular consequences of DNA methylation at the C2 promoter during BSCTV viral accumulation, the authors compared BSCTV infection between wild-type Arabidopsis plants and met1 and cmt3 mutants, in which CG and CHG methylation, respectively, are impaired. In agreement with previous reports, they showed that loss of MET1 and CMT3 facilitate viral replication. Accordingly, met1 and cmt3 plants exhibit lower levels of DNA methylation at the C2 viral promoter than vim5 plants. The authors generated a modified BSCTV containing 13 nucleotide substitutions at CG and CHG sites in the C2 promoter, preventing MET1-and CMT3-dependent methylation. In the vim5 background, the modified BSCTV showed higher viral accumulation compared with the unmodified BSCTV, demonstrating that DNA methylation at the viral C2 promoter inhibits viral accumulation and disease development.
While the mechanism by which BSCTV infection activates VIM5 remains an open question, this study highlights another layer of geminiviral counter-defenses through post-translational interference with the host DNA methylation machinery. The resulting impaired methylation likely threatens the genome stability of the plant host, but the molecular consequences remain to be addressed.