Begomoviruses are numerous and geographically widespread viruses that cause devastating diseases in many crops. Monopartite begomoviruses are frequently associated with betasatellites or alphasatellites. Both betasatellite and alphasatellite DNA genomes are approximately half the size of begomovirus DNA genomes. Betasatellites are essential for induction of typical disease symptoms. The βC1 genes encoded by the betasatellites have important roles in symptom induction, in suppression of transcriptional and posttranscriptional gene silencing, and they can affect jasmonic acid responsive genes. Host plants of begomoviruses have evolved diverse innate defense mechanisms against the βC1 protein to counter these challenges. Alphasatellites have been identified mainly in monopartite begomoviruses that associate with betasatellites and have no known contributions to pathogenesis of begomovirus-betasatellite disease complexes. Applications of current molecular tools are facilitating viral diagnosis and the discovery of novel species of geminiviruses and satellite DNAs and are also advancing our understanding of the global diversity and evolution of satellite DNAs.

We report here that all 25 isolates of Tomato yellow leaf curl China virus (TYLCCNV) collected from tobacco, tomato, or Siegesbeckia orientalis plants in different regions of Yunnan Province, China, were associated with DNAβ molecules. To investigate the biological role of DNAβ, full-length infectious clones of viral DNA and DNAβ of TYLCCNV isolate Y10 (TYLCCNV-Y10) were agroinoculated into Nicotiana benthamiana , Nicotiana glutinosa , Nicotiana. tabacum Samsun (NN or nn), tomato, and petunia plants. We found that TYLCCNV-Y10 alone could systemically infect these plants, but no symptoms were induced. TYLCCNV-Y10 DNAβ was required, in addition to TYLCCNV-Y10, for induction of leaf curl disease in these hosts. Similar to TYLCCNV-Y10, DNAβ of TYLCCNV isolate Y64 was also found to be required for induction of typical leaf curl diseases in the hosts tested. When the βC1 gene of TYLCCNV-Y10 DNAβ was mutated, the mutants failed to induce leaf curl symptoms in N. benthamiana when coinoculated with TYLCCNV-Y10. However, Southern blot hybridization analyses showed that the mutated DNAβ molecules were replicated. When N. benthamiana and N. tabacum plants were transformed with a construct containing the βC1 gene under the control of the Cauliflower mosaic virus 35S promoter, many transgenic plants developed leaf curl symptoms similar to those caused by a virus, the severity of which paralleled the level of βC1 transcripts, while transgenic plants transformed with the βC1 gene containing a stop codon after the start codon remained symptomless. Thus, expression of a βC1 gene is adequate for induction of symptoms of viral infection in the absence of virus.

Our previous results demonstrated that the DNAβ satellite (Y10β) associated with Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) is essential for induction of leaf curl symptoms in plants and that transgenic expression of its βC1 gene in Nicotiana plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the βC1 proteins of Y10β and DNAβ (Y35β) found in the Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) were studied following their expression as six-His fusion proteins in Escherichia coli . Electrophoretic mobility shift assays and UV cross-linking experiments revealed that βC1 proteins could bind both single-stranded and double-stranded DNA without size or sequence specificity. Suppression of green fluorescent protein (GFP) transgene silencing was observed with the new leaves of GFP-expressing Nicotiana benthamiana plants coinoculated by TYLCCNV-Y10 plus Y10β or by TbCSV-Y35 plus Y35β. In a patch agroinfiltration assay, the transiently expressed βC1 gene of Y10β or Y35β was able to suppress host RNA silencing activities and permitted the accumulation of high levels of GFP mRNA in the infiltrated leaf patches of GFP transgenic N. benthamiana plants. The βC1 protein of Y10β accumulated primarily in the nuclei of plant and insect cells when fused with β-glucuronidase or GFP and immunogold labeling showed that the βC1 protein is present in the nuclei of infected N. benthamiana plants. A mutant version of Y10β carrying the mutations within the putative nuclear localization sequence of the Y10 βC1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the βC1 protein is a key requirement for symptom induction and silencing suppression.

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. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, and geminiviruses respond by elaborating proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a satellite called DNA β (betasatellite), which depends on the helper virus for replication and spread within and between hosts. In return, the sole betasatellite encoded protein, βC1, encodes a pathogenicity factor that enhances viral replication and is responsible for inducing disease symptoms. Geminivirus/betasatellite complexes are common and cause significant losses of food and fiber crops. Here, we explore the molecular basis of the association between Tomato yellow leaf curl China virus (TYLCCNV) and its betasatellite (TYLCCNB). We show that TYLCCNV by itself is unable to reverse TGS. However, co-inoculation of TYLCCNB, or expression of βC1 protein, results in reduced methylation of both the helper virus and host genome, and reversal of TGS directed against a transgene and an endogenous locus. We also present evidence that βC1 accomplishes this by interacting with and inhibiting the activity of S-adenosyl homocysteine hydrolase (SAHH), an enzyme needed to maintain the methyl cycle that generates the methyltransferase co-factor S-adenosyl methionine. Thus, we propose that inhibition of methylation-mediated TGS by βC1 drives geminivirus/betasatellite association.

In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection. In plants, RNA silencing plays a key role in developmental regulation and antiviral defense. To successfully infect their hosts, plant viruses encode silencing suppressors (VSRs) as counter-defense measures. These VSRs function to disable host antiviral RNA silencing defenses through various mechanisms that are not well understood. Here we report that a host calmodulin-like protein called Nbrgs-CaM, which appears to be an endogenous suppressor of RNA silencing, plays essential roles in suppression of RNA silencing and induction of symptoms by the VSR βC1, the sole protein encoded by a geminivirus-associated DNA satellite. The Nbrgs-CaM was up-regulated by Tomato yellow leaf curl China geminivirus (TYLCCNV)-encoded VSR βC1 upon virus infection or stable expression via a transgene. Further analyses revealed that up-regulation of Nbrgs-CaM by βC1 suppressed RNA silencing likely through repressing the expression of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6). We have demonstrated that RDR6-mediated RNA silencing plays an important role in antiviral defense in Nicotiana benthamiana and confers host range restriction against TYLCCNV infection on Arabidopsis thaliana. Our study suggests that exploiting a cellular suppressor can be an efficient mechanism for viruses to counteract host RNA silencing defense response.