A Novel Prokaryotic Promoter Identified in the Genome of Some Monopartite Begomoviruses

Abstract

Geminiviruses are known to exhibit both prokaryotic and eukaryotic features in their genomes, with the ability to express their genes and even replicate in bacterial cells. We have demonstrated previously the existence of unit-length single-stranded circular DNAs of Ageratum yellow vein virus (AYVV, a species in the genus Begomovirus, family Geminiviridae) in Escherichia coli cells, which prompted our search for unknown prokaryotic functions in the begomovirus genomes. By using a promoter trapping strategy, we identified a novel prokaryotic promoter, designated AV3 promoter, in nts 762-831 of the AYVV genome. Activity assays revealed that the AV3 promoter is strong, unidirectional, and constitutive, with an endogenous downstream ribosome binding site and a translatable short open reading frame of eight amino acids. Sequence analyses suggested that the AV3 promoter might be a remnant of prokaryotic ancestors that could be related to certain promoters of bacteria from marine or freshwater environments. The discovery of the prokaryotic AV3 promoter provided further evidence for the prokaryotic origin in the evolutionary history of geminiviruses.

Characterization of the Cryptic AV3 Promoter of Ageratum Yellow Vein Virus in Prokaryotic and Eukaryotic Systems

“…AYVV-NT and tomato leaf curl virus (TLCV), which is another monopartite begomovirus, respective full-length clones, pAYVVNT and pTLCV, have been described previously [13] , [15] . The nucleotide sequence of AYVV-NT genome has been deposited in GenBank under the GenBank index (GI) number: 149193093.…”

“…For yeast assays, the pYES2/NT-C vector (Invitrogen, Life Technologies, Carlsbad, CA, USA) was modified to replace the original GAL1 promoter with two different AYVV-NT fragments comprising the same AV3 promoter region plus two downstream ORFs fused to the Cycle 3 GFP gene. Two plasmids described in our previous study, pGP762-889GFP and pGP762-1062GFP [13] , were used as templates. The primer pair AY762-SpeIF and C3GFP-KpnIR ( Table 1 ) was used to amplify the corresponding regions with GFP fusion fragments.…”

“…GFP-based promoter activity assays were performed and analyzed as described previously [16] with minor modifications. Individual constructs, pGlow-TOPO, pGP762-869 [13] (abbreviated as pAT-WT in this study), pAY-M, pGP762-869TLCV [13] (abbreviated as pTL-WT), and pTL-M, were used to transformed TOP10 E. coli . Bacteria harboring each construct were cultivated in LB broth containing 100 µg of ampicillin ml −1 at 37°C for 16 h. The cultures were then diluted 200-fold and incubated at 37°C for 4 h to reach the mid-log phase.…”

“…Each of the plasmids pBT-1634SacI, pBT-AV1, pBT-CP, pBT-Rep, pBT-TrAP, pBT-Ren, and pBT-C4 was co-transformed with the pAY-WT or p rrn B-P1 [13] , in which the rrn B P1 promoter was cloned into the GFP-reporter vector pGlow-TOPO, into the E. coli XLI-BLUE MRF' strain. Individual colonies of each treatment were selected and cultivated at 30°C overnight.…”

“…Recently, a novel cryptic prokaryotic promoter, designated AV3 promoter, was identified at positions near the 3′-terminus of coat protein (CP) open reading frame (ORF) in some monopartite begomoviruses genomes [13] . The AV3 promoter activity is similar to the well-characterized E. coli constitutive promoter of ribosomal RNA, rrn B P1 promoter [14] .…”