Functional characterization of a bidirectional plant promoter from cotton leaf curl Burewala virus using an Agrobacterium-mediated transient assay

Abstract

The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional gene promoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant.

Figure 1

The 455 bp DNA sequence of the full-length transcript of the bidirectional gene promoter of the CLCuBuV coordinates from 2595–292 nucleotides in the viral genome. The putative motifs are in bold and underlined. The replication initiation site/putative stem-loop motif was composed of 35 bp consisting of ATC-motif (GGCCATCC) and GC-rich region (GGCCGCGC) around the nonanucleotide. The conserved late element (CLE) was present at position +185 was suggested to functional target for C2. The POLASIG3 motif (AATAAT) was also identified at position +242. The TATA-box for Rep promoter was found at position 65 nucleotide and TATA-box for Cp promoter was located at position 259 nucleotide in the LIR. The Box-II are predicted at position 433 in the LIR. MBS (MYB drought responsive element) was found at position 20 within the LIR. The labeling of motifs are indicated above the line. The right-side numbering shows the position of LIR in the genome.

Figure 2

(A) Genome organization of the DNA-A component of CLCuBuV. Five ORFs are shown with the arrows. V1 and V2 (coat protein) are located on the plus strand, C1 (replication associated protein), C3 (replication enhancer protein) and C4 (unnamed) are located on the complementary sense strand. The 455 bp large intergenic region (LIR) contains the potential stem-loop with nonanucleotides conserved among all geminiviruses. Sequence numbering begins at base 8 of the conserved nonamer; (B) A schematic drawing of the CLCuBuV LIR. Shown, is the invariant loop sequence with the nonanucleotide sequence (grey box) flanked by inverted repeats (short arrows); the viral protein start sites and replication associated proteins; (C) A schematic representation of both expression vectors shows the cloning of the C1 and V1 promoters; (D) Schematic representation of important features of large intergenic region (LIR) of CLCuBuV including REP-binding sites (iterons), TATA box and structure of hair pin showing nonanucleotide (underlined). The TATA box for Rep promoter was to be present at position 2660–2664 in the genome. Two directly repeats iteron motifs AAATTC were predicted at position 2709–2715; 2715–2721 present downstream to TATA box and immediate upstream to stem-loop motif .The stem-loop motif composed of 35 bp was predicted at position 2738–15 in the CLCuBuV genome.

Figure 3

Transient GUS expression in plant leaves and roots agro-inoculated with the AC1 and AV1 promoter constructs from CLCuBuV (pV1GUS1301, pC1GUS1301) in comparison with positive control CaM 35S promoter (pCAMBIA1301) and a negative promoterless control (pCAMBIA1391-Z). Error bars represent standard deviation.GUS activity measured in RFU mg⁻¹protein min⁻¹. Each treatment was carried out in three replications.

Figure 4

Occurrence of 21 enriched cis-acting regulatory DNA elements distributed in the 12 bidirectional geminivirus promoters.

Figure 5

(A) Occurrence of the top 21 CREs with a total occurrence in 12 promoters; (B) Occurrence of 32 CREs distributed in the CLCuBuV bidirectional promoter.

Figure 6

Overrepresented sequences in the bidirectional promoters of geminiviruses used in this study. Letters abbreviating the nucleotides (A, C, G, T) in the images are sized to their relative occurrence. (A) MNB1A motif (MA0053.1) p-value 3.1e-075 belongs to Zinc-coordinating class and Dof family; (B) HAP4 motif (MA0315.1) p-value 4.2e-027; (C) HAP5 motif (MA0316.1) p-value 7.33e-05; (D) NFYA motif (MA0060.1) p-value 5.3e-06; (E) PBF motif (MA0064.1) p-value 1.4e-017 belongs to Zinc-coordinating class and Dof family; (F) HAP3 motif (MA 00314.1) p-value 1.2e-07. All HAP motifs belong to Alpha Helix class and NFY CCAAT-binding family.