Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.RNA silencing, also referred to as RNA interference and posttranscriptional gene silencing, is an evolutionarily conserved mechanism that protects cells against invasive nucleic acids, such as viruses, transposons, and transgenes (19). RNA silencing is triggered by double-stranded RNA (dsRNA), effects sequence-specific degradation of cognate viral or endogenous RNA, and, at least in plants, causes de novo methylation of homologous DNA (33). In plants, silencing is increasingly viewed as an adaptive immune system targeting pathogenic RNA and DNA (28, 52). To counteract this defense system, viruses have evolved suppressor proteins (4, 6, 37) that interfere with different steps of the RNA silencing pathway (11), thus allowing efficient viral replication in a single cell and systemic spread of the infection. For example, the coat protein of Turnip crinkle virus blocks generation of small interfering RNAs (siRNAs) (38), derived from dsRNA processing by the RNase III-like enzyme Dicer at an early initiation step of silencing. p19 of tombusviruses binds siRNAs (27, 51), thereby inhibiting a downstream step involving cleavage of cognate RNA by an siRNA-guided, RNA-induced silencing complex. Movement protein P25 of Potato virus X prevents systemic spread of silencing through the vascular system (54). Potyvirus protein HC-Pro might interfere with both the initiation and spread of silencing, although the mechanism of HC-Pro action is still a matter of debate (reference 32 and references therein). Interestingly, HC-Pro and other viral suppressors not only are able to suppress RNA silencing but also can interfere with a relate...

Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virusVigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.The family Geminiviridae comprises small circular singlestranded DNA viruses that cause severe diseases in major crop plants worldwide. On the basis of genome organization, host range, and type of insect vector, the family is divided into four genera: Mastrevirus, Curtovirus, Topocuvirus, and Begomovirus (42). Members of the largest genus, Begomovirus (10), infect primarily dicotyledonous plants and are transmitted by the whitefly Bemisia tabaci. Many begomoviruses have a bipartite genome, with a DNA-A component encoding all the protein functions necessary for virus replication in a single cell while the DNA-B component provides movement functions required for systemic spread.Transcription regulation in begomoviruses has been extensively studied in both transgenic plants (13,19,23,40,48,60) and protoplast systems (8,9,13,16,17,24,46,47,49,51,52,53,60). Transcription start and poly(A) sites on both DNA-A and -B have been partially or completely mapped for African cassava mosaic virus (ACMV) (54), Tomato golden mosaic virus (TGMV) (19,36,45,50), Abutilon mosaic virus (AbMV) (14), and the monopartite Tomato leaf curl virus (TLCV) (34). However, major gaps in our understanding of the structural organization of begomovirus promoters and transcription units, and of the regulation of transcription by viral proteins, remain. Bidirectional promoters have been identified in the intergenic regions (IGR) of DNA-A and DNA-B, which share a common region (CR) of about 160 to 200 bp. The CR includes all cisacting elements required for DNA replication and a core promoter driving leftward transcription of the AC1 gene encoding the replication-associated protein (Rep) (reviewed in reference 20). AC1 also functions as a negativ...