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Structural characterization of a novel full-length transcript promoter from Horseradish Latent Virus (HRLV) and its transcriptional regulation by multiple stress responsive transcription factors

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Abstract

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The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions.

Abstract

Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (− 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (− 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.

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Abbreviations

ABA:

Abscisic acid

DRE:

Dehydration-responsive element

ABRE:

ABA-responsive element

DREBs:

DRE-binding proteins

AREBs:

ABRE-binding proteins

JRE:

Jasmonic response element

ERF:

Ethylene-responsive binding factor

AP2:

APETALA2

CBF:

C-repeat binding factor

LTRE:

Low-temperature-responsive element

CRT:

C-repeat

bZIP:

Basic leucine zipper

CLSM:

Confocal Laser Scanning Microscope

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Acknowledgements

This study was supported by the funds from Department of Biotechnology, Govt. of India under Grant No. BT/HRD/35/01/05/2015 and Council of Scientific and Industrial Research (CSIR), Govt. of India under Research Grant No. 38(1438)/17/EMR-11. We thank Mr. Abhimanyu Das, Ms. Vasundhara Sundar and Mr. Amol Kanampalliwar for their technical help and support.

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  1. Ahamed Khan, Ankita Shrestha have contributed equally to this article.

Authors and Affiliations

  1. Department of Gene Function and Regulation, Department of Biotechnology, Government of India, Institute of Life Sciences, Chandrasekharpur, Bhubaneswar, Odisha, India

    Ahamed Khan, Ankita Shrestha, Kashyap Bhuyan & Nrisingha Dey

  2. Department of Molecular Plant Virology and Plant Genetic Engineering, KTRDC, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546-0236, USA

    Indu B. Maiti

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  1. Ahamed Khan
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Contributions

AK and AS equally performed the experimental work. KB contributed to the mutational stress assays. Dr. IBM initiated the project and Dr. ND coordinated the whole experimental plan and provided intellectual inputs towards completion of this project.

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Correspondence to Nrisingha Dey.

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Accession numbers

The sequence information for the following genes referred in this study can be found in the NCBI GenBank database under the following Accession Numbers: JX429923.1 (HRLV), NM_179446 (AREB1), FJ169308.1 (DREB1), X16449.1 (TGA1a).

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Khan, A., Shrestha, A., Bhuyan, K. et al. Structural characterization of a novel full-length transcript promoter from Horseradish Latent Virus (HRLV) and its transcriptional regulation by multiple stress responsive transcription factors. Plant Mol Biol 96, 179–196 (2018). https://doi.org/10.1007/s11103-017-0693-6

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