Identification of a Sequence Element Involved in AC2-Mediated Transactivation of the Pepper Huasteco Virus Coat Protein Gene

Abstract

The geminivirus AC2 gene product transactivates the expression of the coat and movement protein (CP and BV1) genes, and this effect seems to be mediated by specific although hitherto unknown cis-acting elements. In this work we examined regions from the CP and BV1 gene promoters of pepper huasteco virus (PHV) to define the sequence elements involved in regulation by AC2. Results from transient gene expression and transgenic plant assays suggest that a truncated 115-nt CP promoter is still responsive to the viral transactivator. This promoter contains three elements similar to a sequence motif termed conserved late element (CLE), which is found in the regulatory regions of many geminiviruses and that was previously suggested, on a theoretical basis, to be a potential functional target for AC2 (Argüello-Astorga et al. (1994), Virology 203, 90-100). To confirm these results, an oligonucleotide containing two CLE motifs was synthesized and characterized in gain-of-function experiments. Transient expression assays showed that this 29-nt sequence is able to confer AC2 responsiveness to heterologous promoters. A smaller oligonucleotide (16 nt) containing a single CLE also conferred this activity. In addition, when the CLE motifs were mutated in their original context (truncated 115-nt promoter), this modified promoter lost its ability to be transactivated by AC2. All these results support the involvement, at least in the case of PHV, of CLE sequences in the process of transactivation.

Promoters, Transcripts, and Regulatory Proteins of Mungbean Yellow Mosaic Geminivirus

“…A conserved late element (CLE) sequence has previously been implicated in AC2-mediated transactivation based on its conservation in begomoviruses (2) and some experimental evidence (40). Our inspection of the MYMV sequence revealed a consensus CLE (GTGGT CCC) at position 45 of DNA-B, 20 bp from CR (Fig.…”

“…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).…”

“…We have recently shown that Mungbean yellow mosaic virus-Vigna (MYMV-Vig; referred to below as MYMV) AC2 and its ACMV homolog also transactivate several plant genes (55). The molecular mechanism of transactivation remains unknown, and attempts to identify a single cis element conserved in all AC2-responsive promoters have met with little success (2,40,49). In MYMV, ACMV, and other Old World begomoviruses, the AV1 gene is overlapped at the 5Ј end by an open reading frame (ORF) of unknown function (AV2), and the mechanism of transcription and translation of these overlapping genes is not understood.…”

Suppression of RNA Silencing by a Geminivirus Nuclear Protein, AC2, Correlates with Transactivation of Host Genes

“…However, the molecular mechanism of AC2-mediated transactivation is largely unknown. Attempts to identify any conserved AC2-responsive cis element have met with little success (40,47). Moreover, sequence-nonspecific and weak binding to doublestranded DNA in vitro (21,43) suggests that AC2 may rather engage (through its Zn finger) in interaction with one or more cellular factors, which would in turn target it to different promoters.…”

Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco

“…Direct repeats of CLE-like motifs fused to minimal ()46 to +8 bp) or truncated ()90 to +1 bp) CaMV 35S promoters (CLE-46 and CLE-90, respectively) have been shown to enhance reporter gene (GUS) expression in bombarded pea and tobacco leaf tissues (Ruiz-Medrano et al, 1999). Furthermore, transformed tobacco plants harboring the CLE-90::GUS fusion present a GUS expression pattern different from that observed in CLE-lacking control transgenics ()90::GUS) (Ruiz-Medrano et al, 1999).…”