Synthetic promoters from blueberry red ringspot virus (BRRV).

MAIN CONCLUSION: We analyzed the synthetic full-length transcript promoter of Blueberry red ringspot virus (BRRV) and developed two chimeric promoters (MBR3 and FBR3). Transcriptional activities of these chimeric promoters were found equivalent to that of the CaMV35S2 promoter. Chimeric promoters driven plant-derived PaDef protein showed high antimicrobial activities against several pathogens. Blueberry red ringspot virus (BRRV) is a pararetrovirus under the genus, Soymovirus belongs to the Caulimoviridae family. We have made a synthetic version of the BRRV-Flt promoter and analyzed its activity in detail. A 372 bp promoter fragment BR3 (- 212 to + 160) showed the strongest transcriptional activity compared with other fragments in both transient and transgenic assays; its activity was found near equivalent to that of the CaMV35S promoter. We constructed two chimeric promoters; MBR3 and FBR3 by fusing the UASs (Upstream activation sequences) of Mirabilis mosaic virus (MUAS; - 297 to - 38; 335 bp) and Figwort mosaic virus (FUAS; - 249 to - 54; 303 bp) respectively to the core promoter domain of BR3 (BR3; - 212 to + 160; 372 bp). The activities of MBR3 and FBR3 promoters were found equivalent to that of the activity of the CaMV35S2 promoter and approximately 4.0 (four) times stronger than that of the CaMV35S promoter. Histochemical and fluorometric GUS assays confirmed the above observation. The transcriptional efficacies of these recombinant promoters were tested by evaluating the antibacterial and antifungal activities of recombinant plant-derived antimicrobial peptide Persea americana var. drymifolia defensin (PaDef) driven under these promoters. Bioassays showed promising antifungal activities of the plant made PaDef against Alternaria alternata and antibacterial property against Gram-positive (S. aureus and R. fascians) and Gram-negative bacteria (E. coli and P. aeruginosa). Based upon the above results, MBR3 and FBR3 could be useful promoters for plant genetic engineering and can become useful substitutes for the widely used CaMV35S2 promoter in plant biology.

Synthetic Salicylic acid inducible recombinant promoter for translational research.

In the present study, we have developed an inter-molecularly shuffled caulimoviral promoter for protein over-expression by placing the Upstream Activation Sequence (UAS) of Figwort Mosaic Virus (FMV; -249 to -54) at the 5'-end of the Cassava Vein Mosaic Virus (CsVMV) promoter fragment 8 (CsVMV8; -215 to +166) to design a hybrid promoter; FUASCsV8CP. The FUASCsV8CP promoter exhibited approximately 2.1 and 2.0 times higher GUS-activities than that obtained from the CaMV35S promoter, in tobacco (Xanthi Brad) protoplasts and in Agroinfiltration assays respectively. Hereto, when FUASCsV8CP was assayed using transgenic tobacco plants (T2- generation), it showed 2.0 times stronger activity than CaMV35S promoter and almost equivalent activity to that of CaMV35S2 promoter. The promoter displayed Salicylic acid (SA) inducibility and hence can also be used for ensuring effective gene expression in plants under constitutive as well as specific inducible conditions. Furthermore, FUASCsV8CP was used to drive the expression of victoviral Vin gene (encoding Victoriocin) transiently in tobacco. The recombinant Victoriocin could be successfully detected by western blotting three days post infiltration. Also, the in vitro Agar-based killing zone assays employing plant-derived Victoriocin-His (obtained from transient expression of Vin) revealed enhanced antifungal activity of Victoriocin against hemi-biotrophic pathogen Phoma exigua Desm. var. exigua.

Recombinant Promoter (MUASCsV8CP) Driven Totiviral Killer Protein 4 (KP4) Imparts Resistance Against Fungal Pathogens in Transgenic Tobacco.

Development of disease-resistant plant varieties achieved by engineering anti-microbial transgenes under the control of strong promoters can suffice the inhibition of pathogen growth and simultaneously ensure enhanced crop production. For evaluating the prospect of such strong promoters, we comprehensively characterized the full-length transcript promoter of Cassava Vein Mosaic Virus (CsVMV; -565 to +166) and identified CsVMV8 (-215 to +166) as the highest expressing fragment in both transient and transgenic assays. Further, we designed a new chimeric promoter 'MUASCsV8CP' through inter-molecular hybridization among the upstream activation sequence (UAS) of Mirabilis Mosaic Virus (MMV; -297 to -38) and CsVMV8, as the core promoter (CP). The MUASCsV8CP was found to be ∼2.2 and ∼2.4 times stronger than the CsVMV8 and CaMV35S promoters, respectively, while its activity was found to be equivalent to that of the CaMV35S2 promoter. Furthermore, we generated transgenic tobacco plants expressing the totiviral 'Killer protein KP4' (KP4) under the control of the MUASCsV8CP promoter. Recombinant KP4 was found to accumulate both in the cytoplasm and apoplast of plant cells. The agar-based killing zone assays revealed enhanced resistance of plant-derived KP4 against two deuteromycetous foliar pathogenic fungi viz. Alternaria alternata and Phoma exigua var. exigua. Also, transgenic plants expressing KP4 inhibited the growth progression of these fungi and conferred significant fungal resistance in detached-leaf and whole plant assays. Taken together, we establish the potential of engineering "in-built" fungal stress-tolerance in plants by expressing KP4 under a novel chimeric caulimoviral promoter in a transgenic approach.