Utilization of plant-derived recombinant human ß-defensins (hBD-1 and hBD-2) for averting salmonellosis.

We describe the use of plant-made ß-defensins as effective antimicrobial substances for controlling salmonellosis, a deadly infection caused by Salmonella typhimurium (referred to further as S. typhi). Human ß-defensin-1 (hBD-1) and -2 (hBD-2) were expressed under the control of strong constitutive promoters in tobacco plants, and bio-active ß-defensins were successfully extracted. In the in vitro studies, enriched recombinant plant-derived human ß-defensin-1 (phBD-1) and -2 (phBD-2) obtained from both T1 and T2 transgenic plants showed significant antimicrobial activity against Escherichia coli and S. typhi when used individually and in various combinations. The 2:1 peptide combination of phBD-1:phBD-2 with peptides isolated from T1-and T2-generation plants reduced the growth of S. typhi by 96 and 85 %, respectively. In vivo studies employing the mouse model (Balb/c) of Salmonella infection clearly demonstrated that the administration of plant-derived defensins individually and in different combinations enhanced the mean survival time of Salmonella-infected animals. When treatment consisted of the 2:1 phBD-1:phBD-2 combination, approximately 50 % of the infected mice were still alive at 206 h post-inoculation; the lowest number of viable S. typhi was observed in the liver and spleen of infected animals. We conclude that plant-made recombinant ß-defensins (phBD-1 and phBD-2) are promising antimicrobial substances and have the potential to become additional tools against salmonellosis, particularly when used in combination.

Effective control of Salmonella infections by employing combinations of recombinant antimicrobial human ß-defensins hBD-1 and hBD-2.

We successfully produced two human ß-defensins (hBD-1 and hBD-2) in bacteria as functional peptides and tested their antibacterial activities against Salmonella enterica serovar Typhi, Escherichia coli, and Staphylococcus aureus employing both spectroscopic and viable CFU count methods. Purified peptides showed approximately 50% inhibition of the bacterial population when used individually and up to 90% when used in combination. The 50% lethal doses (LD50) of hBD-1 against S. Typhi, E. coli, and S. aureus were 0.36, 0.40, and 0.69 µg/µl, respectively, while those for hBD-2 against the same bacteria were 0.38, 0.36, and 0.66 µg/µl, respectively. Moreover, we observed that bacterium-derived antimicrobial peptides were also effective in increasing survival time and decreasing bacterial loads in the peritoneal fluid, liver, and spleen of a mouse intraperitoneally infected with S. Typhi. The 1:1 hBD-1/hBD-2 combination showed maximum effectiveness in challenging the Salmonella infection in vitro and in vivo. We also observed less tissue damage and sepsis formation in the livers of infected mice after treatment with hBD-1 and hBD-2 peptides individually or in combination. Based on these findings, we conclude that bacterium-derived recombinant ß-defensins (hBD-1 and hBD-2) are promising antimicrobial peptide (AMP)-based substances for the development of new therapeutics against typhoid fever.

Development of an intra-molecularly shuffled efficient chimeric plant promoter from plant infecting Mirabilis mosaic virus promoter sequence.

We developed an efficient chimeric promoter, MUASMSCP, with enhanced activity and salicylic acid (SA)/abscisic acid (ABA) inducibility, incorporating the upstream activation sequence (UAS) of Mirabilis mosaic virus full-length transcript (MUAS, -297 to -38) to the 5' end of Mirabilis mosaic virus sub-genomic transcript (MSCP, -306 to -125) promoter-fragment containing the TATA element. We compared the transient activity of the MUASMSCP promoter in tobacco/Arabidopsis protoplasts and in whole plant (Petunia hybrida) with the same that obtained from CaMV35S and MUAS35SCP promoters individually. The MUASMSCP promoter showed 1.1 and 1.5 times stronger GUS-activities over that obtained from MUAS35SCP and CaMV35S promoters respectively, in tobacco (Xanthi Brad) protoplasts. In transgenic tobacco (Nicotiana tabacum, var. Samsun NN), the MUASMSCP promoter showed 1.1 and 2.2 times stronger activities than MUAS35SCP and CaMV35S(2) promoters respectively. We observed a fair correlation between MUASMSCP-, MUAS35SCP- and CaMV35S(2)-driven GUS activities with the corresponding uidA-mRNA level in transgenic plants. X-gluc staining of transgenic germinating seed-sections and whole seedlings also support above findings. Protein-extracts made from tobacco protoplasts expressing GFP and human-IL-24 genes driven individually by the MUASMSCP promoter showed enhanced expression of the reporters compared to that obtained from the CaMV35S promoter. Furthermore, MUASMSCP-driven protoplast-derived human IL-24 showed enhanced cell inhibitory activity in DU-145 prostate cancer cells compared to that obtained from the CaMV35S promoter. We propose chimeric MUASMSCP promoter developed in the study could be useful for strong constitutive expression of transgenes in both plant/animal cells and it may become an efficient substitute for CaMV35S/CaMV35S(2) promoter.

Development of an efficient bi-directional promoter with tripartite enhancer employing three viral promoters.

We have developed a novel bi-directional promoter (FsFfCBD) by placing two heterogeneous core-promoters from the Figwort mosaic virus sub-genomic transcript promoter (FsCP, -69 to +31) and Cauliflower mosaic virus 35S promoter (CCP, -89 to +1) respectively on upstream (5') and downstream (3') ends of a tri-hybrid enhancer (FsEFfECE), in reverse orientation. The FsEFfECE domain encompasses three heterologous enhancer fragments from Figwort mosaic virus sub-genomic transcript promoter (FsE, 101 bp, -70 to -170), Figwort mosaic virus full-length transcript promoter (FfE, 196 bp, -249 to -54) and Cauliflower mosaic virus 35S promoter (CE, 254 bp, -343 to -90). The bi-directional nature of the FsFfCBD promoter (coupled to GFP and GUS) was established both in transient systems (onion epidermal cells and tobacco protoplasts) and transgenic plant (Nicotiana tabacum samsun NN) by monitoring the simultaneous expression of GFP and GUS employing fluorescence (for GFP) and biochemical (for GUS) based assays. In transgenic plants, the FsFfCBD promoter was found to be 6.8 and 2.5 times stronger than two parent promoters; Fs and FfC respectively. The bi-directional compound promoter FsFfCBD, composed of three heterologous enhancers with enhanced activity could become a valuable additional tool for efficient plant metabolic engineering and molecular pharming.

Development of a salicylic acid inducible minimal sub-genomic transcript promoter from Figwort mosaic virus with enhanced root- and leaf-activity using TGACG motif rearrangement.

In Figwort mosaic virus sub-genomic transcript promoter (F-Sgt), function of the TGACG-regulatory motif, was investigated in the background of artificially designed promoter sequences. The 131bp (FS, -100 to +31) long F-Sgt promoter sequence containing one TGACG motif [FS-(TGACG)] was engineered to generate a set of three modified promoter constructs: [FS-(TGACG)(2), containing one additional TGACG motif at 7 nucleotides upstream of the original one], [FS-(TGACG)(3), containing two additional TGACG motifs at 7 nucleotides upstream and two nucleotides downstream of the original one] and [FS-(TGCTG)(mu), having a mutated TGACG motif]. EMSA and foot-printing analysis confirmed binding of tobacco nuclear factors with modified TGACG motif/s. The transcription-activation of the GUS gene by the TGACG motif/s in above promoter constructs was examined in transgenic tobacco and Arabidopsis plants and observed that the transcription activation was affected by the spacing/s and number/s of the TGACG motif/s. The FS-(TGACG)(2) promoter showed strongest root-activity compared to other modified and CaMV35S promoters. Also under salicylic acid (SA) stress, the leaf-activity of the said promoter was further enhanced. All above findings were confirmed by real-time and semi-qRT PCR analysis. Taken together, these results clearly demonstrated that the TGACG motif plays an important role in inducing the root-specific expression of the F-Sgt promoter. This study advocates the importance of genetic manipulation of functional cis-motif for amending the tissue specificity of a plant promoter. SA inducible FS-(TGACG)(2) promoter with enhanced activity could be a useful candidate promoter for developing plants with enhanced crop productivity.

Development and functional analysis of novel genetic promoters using DNA shuffling, hybridization and a combination thereof.

BACKGROUND: Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications. METHODOLOGY: Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter. SIGNIFICANCE/CONCLUSION: Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants.

Development of useful recombinant promoter and its expression analysis in different plant cells using confocal laser scanning microscopy.

BACKGROUND: Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. METHODOLOGY/PRINCIPAL FINDINGS: We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, -271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. CONCLUSION AND SIGNIFICANCE: We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells.

Efficient chimeric promoters derived from full-length and sub-genomic transcript promoters of Figwort mosaic virus (FMV).

Addition of multiple repeats of the FS3 upstream activation sequence (FS3-UAS, -270 to -60) intra-molecularly to the TATA containing core-domain of the FS3 (-151 to +31) promoter resulted in 2-3-folds enhanced promoter activity. The chimeric promoter, FS3-UAS-3X with maximum activity, showed 3.31 times stronger activity in root vascular tissue compared to FS3 promoter and could be used efficiently in translational research.