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 encodes five double-stranded RNA binding (DRB) proteins. DRB1 and DRB2 are involved in microRNA (miRNA) biogenesis, while DRB4 functions in cytoplasmic posttranscriptional small interfering RNA (siRNA) pathways. DRB3 and DRB5 are not involved in double-stranded RNA (dsRNA) processing but assist in silencing transcripts targeted by DRB2-associated miRNAs. The goal of this study was to determine which, if any, of the DRB proteins might also participate in a nuclear siRNA pathway that leads to geminivirus genome methylation. Here, we demonstrate that DRB3 functions with Dicer-like 3 (DCL3) and Argonaute 4 (AGO4) in methylation-mediated antiviral defense. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, using an RNA-directed DNA methylation (RdDM) pathway similar to that used to suppress endogenous invasive DNAs such as transposons. Chromatin methylation inhibits virus replication and transcription, and methylation-deficient host plants are hypersusceptible to geminivirus infection. Using a panel of drb mutants, we found that drb3 plants uniquely exhibit a similar hypersensitivity and that viral genome methylation is substantially reduced in drb3 compared to wild-type plants. In addition, like dcl3 and ago4 mutants, drb3 plants fail to recover from infection and cannot accomplish the viral genome hypermethylation that is invariably observed in asymptomatic, recovered tissues. Small RNA analysis, bimolecular fluorescence complementation, and coimmunoprecipitation experiments show that DRB3 acts downstream of siRNA biogenesis and suggest that it associates with DCL3 and AGO4 in distinct subnuclear compartments. These studies reveal that in addition to its previously established role in the miRNA pathway, DRB3 also functions in antiviral RdDM. IMPORTANCEPlants use RNA-directed DNA methylation (RdDM) as an epigenetic defense against geminiviruses. RNA silencing pathways in Arabidopsis include five double-stranded RNA binding proteins (DRBs) related to Drosophila R2D2 and mammalian TRBP and PACT. While DRB proteins have defined roles in miRNA and cytoplasmic siRNA pathways, a role in nuclear RdDM was elusive. Here, we used the geminivirus system to show that DRB3 is involved in methylation-mediated antiviral defense. Beginning with a panel of Arabidopsis drb mutants, we demonstrated that drb3 plants uniquely show enhanced susceptibility to geminiviruses. Further, like dcl3 and ago4 mutants, drb3 plants fail to hypermethylate the viral genome, a requirement for host recovery. We also show that DRB3 physically interacts with the RdDM pathway components DCL3 and AGO4 in the nucleus. This work highlights the utility of geminiviruses as models for de novo RdDM and places DRB3 protein in this fundamental epigenetic pathway.

SUMMARYThe hypersensitive response (HR), a form of programmed cell death (PCD), is a tightly regulated innate immune response in plants that is hypothesized to restrict pathogen growth and disease development. Although considerable efforts have been made to understand HR PCD, it remains unknown whether the retrograde pathway from the Golgi to the endoplasmic reticulum (ER) is involved. Here we provide direct genetic evidence that two Nicotiana benthamiana homologs, ERD2a and ERD2b, function as ER luminal protein receptors and participate in HR PCD. Virus-induced gene silencing (VIGS) of ERD2a and/or ERD2b caused escape of ER-resident proteins from the ER, and resulted in plants that were more sensitive to ER stress. Silencing of ERD2b delayed HR PCD induced by the non-host pathogens Xanthomonas oryzae pv. oryzae and Pseudomonas syringae pv. tomato DC3000. However, both silencing of ERD2a and co-silencing of ERD2a and ERD2b exacerbated HR PCD. Individual and combined suppression of ERD2a and ERD2b exaggerated R gene-mediated cell death. Nevertheless, silencing of ERD2a and/or ERD2b had no detectable effects on bacterial growth. Furthermore, VIGS of several putative ligands of ERD2a/2b, including the ER quality control (ERQC) component genes BiP, CRT3 and UGGT, had different effects on HR PCD induced by different pathogens. This indicates that immunity-related cell death pathways are separate with respect to the genetic requirements for these ERQC components. These results suggest that ERD2a and ERD2b function as ER luminal protein receptors to ensure ERQC and alleviate ER stress, thus affecting HR PCD during the plant innate immune response.

Regulation of protein synthesis is critical for maintaining cellular homeostasis. In mammalian systems, translational regulatory networks have been elucidated in considerable detail. In plants, however, regulation occurs through different mechanisms that remain largely elusive. In this study, we present evidence that the Arabidopsis thaliana energy sensing kinase SnRK1, a homologue of mammalian AMP-activated kinase and yeast sucrose non-fermenting 1 (SNF1), inhibits translation by phosphorylating the cap binding proteins eIF4E and eIFiso4E. We establish that eIF4E and eIFiso4E contain two deeply conserved SnRK1 consensus target sites and that both interact with SnRK1 in vivo. We then demonstrate that SnRK1 phosphorylation inhibits the ability of Arabidopsis eIF4E and eIFiso4E to complement a yeast strain lacking endogenous eIF4E, and that inhibition correlates with repression of polysome formation. Finally, we show that SnRK1 over-expression in Nicotiana benthamiana plants reduces polysome formation, and that this effect can be counteracted by transient expression of eIF4E or mutant eIF4E containing non-phosphorylatable SnRK1 target residues, but not by a phosphomimic eIF4E. Together, these studies elucidate a novel and direct pathway for translational control in plant cells. In light of previous findings that SnRK1 conditions an innate antiviral defense and is inhibited by geminivirus pathogenicity factors, we speculate that phosphorylation of cap binding proteins may be a component of the resistance mechanism.Abbreviations AD, activation domain; ADK, adenosine kinase; AMPK, AMP-activated kinase; BD, binding domain; BiFC, bimolecular fluorescence complementation; CBE1, conserved binding of eIF4E1; DCL4, Dicer-like 4; DRB4, double-stranded RNA binding protein 4; eIF4E, eukaryotic initiation factor 4E; eIFiso4E, eukaryotic initiation factor iso4E; GPD, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-Stransferase; RFP, red fluorescent protein; rRNA, ribosomal RNA; SNF1, sucrose non-fermenting 1; SNF1, sucrose non-fermenting 1; SnRK1-KDKR, SnRK1 kinase domain inactive.; SnRK1-KD, SnRK1 kinase domain; SnRK1, SNF1-related kinase 1; TOR, target of rapamycin; YFP, yellow fluorescent protein.A. N. Bruns et al.SnRK1 phosphorylates eIF4E and eIFiso4E