Abstract
The antimicrobial activity of the alpha-HAIRPININ ANTIMICROBIAL PEPTIDE X (SmAMP-X gene, GenBank acc. No. HG423454.1) from Stellaria media plant has been shown in vitro. Here, we isolated the SmAMP-X gene promoter and found two genomic sequences for the promoter (designated pro-SmAMP-X and pro-SmAMP-X-Ψ2) with 83% identity in their core and proximal regions. We found that the abilities of these promoters to express the uidA reporter and the nptII selectable marker differ according to the structural organization of T-DNA in the binary vector used for plant transformation. Analysis of Agrobacterium-infiltrated Nicotiana benthamiana leaves, transgenic Arabidopsis thaliana lines, and transgenic Solanum tuberosum plants revealed that both promoters in the pCambia1381Z and pCambia2301 binary vectors generate 42–100% of the ß-glucuronidase (GUS) activity generated by the CaMV35S promoter. According to 5’-RACE (rapid amplification of cDNA ends) analysis, both plant promoters are influenced by the CaMV35S enhancer used to express selectable markers in the T-DNA region of pCambia1381Z and pCambia2301. The exclusion of CaMV35S enhancer from the T-DNA region significantly reduces the efficiency of pro-SmAMP-X-Ψ2 promoter for GUS production. Both promoters in the pCambia2300 vector without CaMV35S enhancer in the T-DNA region weakly express the nptII selectable marker in different tissues of transgenic N. tabacum plants and enable selection of transgenic cells in media with a high concentration of kanamycin. Overall, promoter sequences must be functionally validated in binary vectors lacking CaMV35S enhancer.
Similar content being viewed by others
Data availability
All data is available in this work.
References
de Beer A, Vivier MA (2008) Vv-AMP1 a ripening induced peptide from Vitis vinifera shows strong antifungal activity. BMC Plant Biol 8:75. https://doi.org/10.1186/1471-2229-8-75
Boutrot F, Meynard D, Guiderdoni E et al (2007) The Triticum aestivum non-specific lipid transfer protein (TaLtp) gene family: comparative promoter activity of six TaLtp genes in transgenic rice. Planta 225:843–862. https://doi.org/10.1007/s00425-006-0397-7
Breen S, Solomon PS, Bedon F et al (2015) Surveying the potential of secreted antimicrobial peptides to enhance plant disease resistance. Front Plant Sci 6:900. https://doi.org/10.3389/fpls.2015.00900
Broekaert WF, Cammue BPA, De Bolle MFC et al (1997) Antimicrobial peptides from plants. Crit Rev Plant Sci 16(3):297–323. https://doi.org/10.1080/07352689709701952
Báez-Magaña MD-M, López-Meza JE, Ochoa-Zarzosa A (2018) Immunomodulatory effects of thionin Thi2.1 from Arabidopsis thaliana on bovine mammaryepithelial cells. Int Immunopharmacol 57:47–54. https://doi.org/10.1016/j.intimp.2018.02.001
Efremova LN, Strelnikova SR, Gazizova GR et al (2020) A synthetic strong and constitutive promoter derived from the Stellaria media pro-SmAMP1 and pro-SmAMP2 promoters for effective transgene expression in plants. Genes 11:1407. https://doi.org/10.3390/genes11121407
Fernández JA, Moreno M, Carmona MJ et al (1993) The barley alpha-thionin promoter is rich in negative regulatory motifs and directs tissue-specific expression of a reporter gene in Tobacco. Biochim Biophys Acta 1172:346–348. https://doi.org/10.1016/0167-4781(93)90229-7
Gao AG, Hakimi SM, Mittanck CA et al (2000) Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat Biotechnol 18:1307–1310. https://doi.org/10.1038/82436
Germain H, Lachance D, Pelletier G et al (2012) The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana, indicating the conservation of signalling pathways between angiosperms and gymnosperms. J Exp Bot 63:785–795. https://doi.org/10.1093/jxb/err303
Goyal RK, Mattoo AK (2014) Multitasking antimicrobial peptides in plant development and host defense against biotic/abiotic stress. Plant Sci 228:135–149. https://doi.org/10.1016/j.plantsci.2014.05.012
Gudynaite-Savitch L, Johnson DA, Miki BL (2009) Strategies to mitigate transgene-promoter interactions. J Plant Biotechnol 7:472–485. https://doi.org/10.1111/j.1467-7652.2009.00416.x
Higo K, Ugawa Y, Iwamoto M et al (1999) Plant cis-acting regulatory DNA elements (PLACE) database. J Nucleic Acids Res 27:297–300. https://doi.org/10.1093/nar/27.1.297
Holaskova E, Galuszka P, Frebort I et al (2015) Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology. Biotechnol Adv 33:1005–1023. https://doi.org/10.1016/j.biotechadv.2015.03.007
Inui Kishi RN, Stach-Machado D, Singulani JL et al (2018) Evaluation of cytotoxicity features of antimicrobial peptides with potential to control bacterial diseases of citrus. PLoS ONE 13:e0203451. https://doi.org/10.1371/journal.pone.0203451
Jefferson RA, Burgess SM, Hirsh D (1986) Beta-glucuronidase from Escherichia coli as a gene-fusion marker. Proс Natl Acad Sci USA 83:8447–8451. https://doi.org/10.1042/bst0150017
Jung HW, Kim KD, Hwang BK (2005) Identification of pathogen-responsive regions in the promoter of a pepper lipid transfer protein gene (CALTPI) and the enhanced resistance of the CALTPI transgenic Arabidopsis against pathogen and environmental stresses. Planta 221:361–373. https://doi.org/10.1007/s00425-004-1461-9
Komakhin RA, Komakhina VV, Milyukova NA et al (2010) Transgenic tomato plants expressing recA and NLS-recA-licBM3 genes as a model for studying meiotic recombination. Russ J Genet 46:1440–1448. https://doi.org/10.1134/S1022795410120069
Komakhin RA, Vysotskii DA, Shukurov RR et al (2016) Novel strong promoter of antimicrobial peptides gene pro-SmAMP2 from chickweed (Stellaria media). BMC Biotechnol 16:43. https://doi.org/10.1186/s12896-016-0273-x
Kovalchuk N, Li M, Wittek F et al (2010) Defensin promoters as potential tools for engineering disease resistance in cereal grains. J Plant Biotechnol 8(1):47–64. https://doi.org/10.1111/j.1467-7652.2009.00465.x
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. https://doi.org/10.1093/molbev/msw054
Lazzaro BP, Zasloff M, Rolff J (2020) Antimicrobial peptides: application informed by evolution. Science 368(6490):eaau5480. https://doi.org/10.1126/science.aau5480
Lee SC, Hwang IS, Choi HW et al (2008) Involvement of the pepper antimicrobial protein CaAMP1 gene in broad spectrum disease resistance. Plant Physiol 148:1004–1020. https://doi.org/10.1104/pp.108.123836
Lescot M, Dehais P, Thijs G et al (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. J Nucleic Acids Res 30:325–327. https://doi.org/10.1093/nar/30.1.325
Li J, Hu S, Jian W et al (2021) Plant antimicrobial peptides: structures, functions, and applications. Bot Stud 62:5. https://doi.org/10.1186/s40529-021-00312-x
Li Z, Zhou M, Zhang Z et al (2011) Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium Graminearum and Rhizoctonia Cerealis. Funct Integr Genom 11:63–70. https://doi.org/10.1007/s10142-011-0211-x
Liu Y, Hua YP, Chen H et al (2021) Genome-scale identification of plant defensin (PDF) family genes and molecular characterization of their responses to diverse nutrient stresses in allotetraploid rapeseed. PeerJ 9:e12007. https://doi.org/10.7717/peerj.12007
Madzharova NV, Kazakova KA, Strelnikova SR et al (2018) Promoters pro-SmAMP1 and pro-SmAMP2 from wild plant Stellaria media for the biotechnology of dicotyledons. Russ J Plant Physiol 65:750–761. https://doi.org/10.1134/S1021443718040040
Manners JM, Penninckx IA, Vermaere K et al (1998) The promoter of the plant defensin gene PDF1.2 from arabidopsis is systemically activated by fungal pathogens and responds to methyl jasmonate but not to salicylic acid. Plant Mol Biol 38:1071–1080. https://doi.org/10.1023/a:1006070413843
Miroshnichenko D, Firsov A, Timerbaev V et al (2020) Evaluation of plant-derived promoters for constitutive and tissue-specific gene expression in potato. Plants 9:1520. https://doi.org/10.3390/plants9111520
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nguyen LT, Haney EF, Vogel HJ (2011) The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol 29:464–472. https://doi.org/10.1016/j.tibtech.2011.05.001
Pogorelko GV, Fursova OV (2008) A highly efficient miPCR method for isolating FSTs from transgenic Arabidopsis thaliana plants. J Genet 87:133–140. https://doi.org/10.1007/s12041-008-0020-8
Ramadevi R, Rao KV, Reddy VD (2011) Antimicrobial peptides and production of disease resistant transgenic plants. In: Vudem DR, Poduri NR, Khareedu VR (eds) Pests and pathogens: management strategies. CRC Press, pp 379–452
Shirasawa-Seo N, Mitsuhara I, Nakamura S et al (2002) Constitutive promoters available for transgene expression instead of CaMV 35S RNA promoter: Arabidopsis promoters of tryptophan synthase protein β subunit and phytochrome B. Plant Biotechnol 19:19–26. https://doi.org/10.5511/plantbiotechnology.19.19
Shukurov RR, Voblikova VD, Nikonorova AK et al (2012) Transformation of tobacco and Arabidopsis plants with Stellaria media genes encoding novel hevein-like peptides increases their resistance to fungal pathogens. Transgen Res 21:313–325. https://doi.org/10.1007/s11248-011-9534-6
Sidorchuk YV, Marenkova TV, Kuznetsov VV et al (2021) Peculiarities of uidA gene expression under the control of AP3 and RPT2a tissue-specific gene promoters of Arabidopsis thaliana L. in Nicotiana tabacum L. transgenic plants. Russ J Plant Physiol 68:838–848. https://doi.org/10.1134/S1021443721040178
Singer S, Cox K, Liu Z (2010) Both the constitutive cauliflower mosaic virus 35S and tissue-specific AGAMOUS enhancers activate transcription autonomously in Arabidopsis thaliana. Plant Mol Biol J 74:293–305. https://doi.org/10.1007/s11103-010-9673-9
Slavokhotova AA, Rogozhin EA (2020) Defense peptides from the α-Hairpinin family are components of plant innate immunity. Front Plant Sci 11:465. https://doi.org/10.3389/fpls.2020.00465
Slavokhotova AA, Rogozhin EA, Musolyamov AK et al (2014) Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution. Plant Mol Biol 84:189–202. https://doi.org/10.1007/s11103-013-0127-z
Stotz HU, Spence B, Wang Y (2009) A defensin from tomato with dual function in defense and development. Plant Mol Biol 71:131–143. https://doi.org/10.1007/s11103-009-9512-z
Terras FR, Eggermont K, Kovaleva V et al (1995) Small cysteine-rich antifungal proteins from radish: their role in host defense. Plant Cell 7:573–588. https://doi.org/10.1105/tpc.7.5.573
Vetchinkina EM, Komakhina VV, Vysotskii DA et al (2016) Expression of plant antimicrobial peptide pro-SmAMP2 gene increases resistance of transgenic potato plants to Alternaria and Fusarium pathogens. Russ J Genet 52:939–951. https://doi.org/10.1134/S1022795416080147
Vignutelli A, Wasternack C, Apel K et al (1998) Systemic and local induction of an Arabidopsis thionin gene by wounding and pathogens. Plant J 14:285–295. https://doi.org/10.1046/j.1365-313x.1998.00117.x
Vriens K, Peigneur S, De Coninck B et al (2016) The antifungal plant defensin AtPDF2.3 from Arabidopsis thaliana blocks potassium channels. Sci Rep 6:32121. https://doi.org/10.1038/srep32121
Vysotskii DA, Strelnikova SR, Efremova LN et al (2016) Structural and functional analysis of new plant promoter pro-SmAMP1 from Stellaria media. Russ J Plant Physiol 63:663–672. https://doi.org/10.1134/S1021443716050174
Yao J, Luo JS, Xiao Y et al (2019) The plant defensin gene AtPDF2.1 mediates ammonium metabolism by regulating glutamine synthetase activity in Arabidopsis thaliana. BMC Plant Biol 19:557. https://doi.org/10.1186/s12870-019-2183-2
Yoo SY, Bomblies K, Yoo SK et al (2005) The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta 221:523–530. https://doi.org/10.1007/s00425-004-1466-4
Zhang X, Henriques R, Lin SS et al (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1:641–646. https://doi.org/10.1038/nprot.2006.970
Zhang T, Wu A, Yue Y et al (2020) uORFs: important cis-regulatory elements in plants. Int J Mol Sci 21:6238. https://doi.org/10.3390/ijms21176238
Zheng X, Deng W, Luo K et al (2007) The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep 26:1195–1203. https://doi.org/10.1007/s00299-007-0307-x
Acknowledgements
Not applicable.
Funding
This work was supported by the Ministry of Science and Higher Education of the Russian Federation (FGUM-2022-0004).
Author information
Authors and Affiliations
Contributions
LAI and RAK designed the experiments, performed the experiments, analyzed the data, and wrote the paper. The authors read and approved the final version of the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
The authors understand the ethics disclosure statement. Any approval of ethics is not required for the work.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Ivanova, L.A., Komakhin, R.A. Efficiency of the alpha-hairpinin SmAMP-X gene promoter from Stellaria media plant depends on selection of transgenic approach. Transgenic Res 33, 1–19 (2024). https://doi.org/10.1007/s11248-023-00374-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-023-00374-6