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Synthetic promoters from blueberry red ringspot virus (BRRV)

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Abstract

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.

Abstract

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.

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taken from transgenic flowers, (d) 3 week-old whole transgenic tobacco seedlings, (e) Root tips of above-mentioned promoters. f Transient GFP expression analysis of BR3, MBR3, FBR3 promoter along with CaMV35S(35S) and CaMV35S2(35S2) promoters by agro-infiltration of tobacco leaves and pictures taken under a UV. g Transient GFP expression analysis of above promoters measured using ImageJ software in agro-infiltrated N. tabacum Samsun NN leaves, presented with respective standard deviation and statistical significance was determined by Student’s t test (*p < 0.05; **p < 0.01; ***p < 0.001)

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Abbreviations

BRRV:

Blueberry red ringspot virus

CP:

Core promoter

UAS:

Upstream activation sequence

MMV:

Mirabilis mosaic virus

FMV:

Figwort mosaic virus

MBR3:

Chimeric promoter developed by fusing MMV (−297 to −38) and BRRV (−212 to + 160)

FBR3:

Chimeric promoter developed by fusing FMV (−249 to −54) and BRRV (−212 to + 160)

KanR :

Kanamycin resistant

KanS :

Kanamycin susceptible

X-gluc:

5-Bromo-4-chloro-3-indolyl β-d-glucopyranosiduronic acid

GUS:

β-Glucuronidase

GFP:

Green fluorescent protein

AMP:

Antimicrobial peptide

PaDef:

Persea americana Var. drymifolia defensin

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Acknowledgment

We are greatly thankful to Dr. Indu B. Maiti, KTRDC, University of Kentucky, for providing the genetic materials for this research. We sincerely thank the Director, Institute of Life Sciences, for his interest and support in this study. We are sincerely thankful to Dr. Noohi Nasim and Dr. I. Sriram Sandeep for their kind technical help and support. We also thank Mr. Abhimanyu Das for his technical assistance and support.

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  1. Division of Plant and Microbial Biotechnology, Institute of Life Sciences, NALCO Nagar Road, NALCO Square, Chandrasekharpur, Bhubaneswar, Odisha, 751023, India

    Lini Sethi, Debasish Deb, Badrinath Khadanga & Nrisingha Dey

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  1. Lini Sethi
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  2. Debasish Deb
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Correspondence to Nrisingha Dey.

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Sethi, L., Deb, D., Khadanga, B. et al. Synthetic promoters from blueberry red ringspot virus (BRRV). Planta 253, 121 (2021). https://doi.org/10.1007/s00425-021-03624-1

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