PLANT BIOLOGY
ANIMAL BIOLOGY
SUBSCRIPTION
E-SUBSCRIPTION
 
MAP
MAIN PAGE

 

 

 

 

doi: 10.15389/agrobiology.2024.3.446eng

UDC: 632.938.2:577.112.4:577.112.6

Acknowledgements:
Supported financially by the Russian Scientific Foundation (project No. 22-16-00154).

 

PRODUCTION OF A CHIMERIC PROTEIN ELICITOR MF2/MF3 AND EVALUATION OF ITS PROTECTIVE ACTIVITY AGAINST TOBACCO MOSAIC VIRUS

D.V. Erokhin1, I.G. Sinelnikov2, I.A. Shashkov2, Y.А. Denisenko2,
S.B. Popletayeva1, L.A. Shcherbakova1, V.G. Dzhavakhiya1✉

1All-Russian Research Institute of Phytopathology, 5, ul. Institut, pos. Bol’shie Vyazemy, Odintsovskii Region, Moscow Province, 143050 Russia, e-mail rokhin.denis.v@gmail.com, unavil@yandex.ru, larisavniif@yahoo.com, dzhavakhiya@yahoo.com (✉ corresponding author);
2Federal Research Center Fundamentals of Biotechnology RAS, 33/2, Leninskii prospect, Moscow, 119071 Russia, e-mail sinelnikov.i@list.ru, igorshashkov@bk.ru, denisenkoyura@mail.ru

ORCID: Erokhin D.V. orcid.org/0009-0009-6471-1358
Popletayeva S.B. orcid.org/0000-0002-0341-9611
Sinelnikov I.G. orcid.org/0000-0001-6359-1125
Shcherbakova L.A. orcid.org/0000-0003-0254-379X
Shashkov I.A. orcid.org/0000-0001-6037-730Х
Dzhavakhiya V.G. orcid.org/0000-0001-8704-0512
Denisenko Y.A. orcid.org/0000-0003-2363-0374

Final revision received November 20, 2023
Accepted December 26, 2023

Preparations based on environmentally safe protein elicitors of plant resistance could be an acceptable alternative to the widely used synthetic pesticides contaminating the environment. This approach is considered to be especially relevant and promising for the crop production in the organic agriculture. Under natural conditions, such protein elicitors are decomposed to harmless natural amino acids. Applcation of preparations based on protein elicitors could significantly reduce the risk of the development of a pathogen resistance to pesticides representing now a global problem. Earlier we isolated and studied bacterial proteins MF2 and MF3 able to induce defense responses to viral and fungal pathogens in a wide range of plants. A 7.2-kDa MF2 protein was isolated from Bacillus thuringiensis. According to our data, its primary structure is highly homologous to the amino acid sequences of bacterial CspD cold shock proteins. Another bacterial protein (MF3), a 16.9-kD a FKBP-type peptidyl-prolyl cis/trans isomerase, was isolated from Pseudomonas fluorescens. Both these proteins are able to induce the resistance of potato, tomato, and tobacco plants to the tobacco mosaic virus (TMV), but their amino acid sequences, 3D structures, and the structures of active centers responsible for their eliciting properties significantly differ from each other. This made it possible to suppose that these proteins may interact with different plant receptors and trigger different defense responses in treated plants. In this case, a combined application of MF2 and MF3 could result in a more efficient plant protection than the use of each of the proteins alone.  Such an assumption was evidenced by the fact of the enhanced protective efficiency of the MF2 and MF3 mixture in the tobacco TMV pathosystem compared to the efficiency of MF2 or MF3 taken alone at a concentration twice exceeding the content of each protein in the tested mixture. These findings open the prospects of a combined use of both elicitors. However, preparation of such mixture requires production of both proteins via their heterologous expression in two different E. coli clones transformed with the corresponding encoding genes. Moreover, the corresponding procedures of isolation and purification would be required for each protein. In this work, the MF2/MF3 chimeric protein was first obtained and its protective activity in the tobacco plant (Nicotiana tabacum L.)-tobacco mosaic virus (TMV, family Virgaviridae, genus Tobamovirus, species Tobacco mosaic virus) pathosystem was demonstrated. It was also established for the first time that the protective activity of MF2/MF3 is not inferior to the activity of a mixture of individual proteins taken in equivalent concentrations. The purpose of our study was obtaining of a chimeric MF2/MF3 protein via heterologous expression in Escherichia coli and a comparison of its protective activity with that of individual MF2 or MF3 and their mixture. The chimeric MF2/MF3 protein is a structure composed of two domains, in which MF2 protein was linked to the N-terminal region of MF3 via the polyalanine linker (AAALIAA). The MF2/MF3 protein encoding sequences were constructed by the use of classic and overlap PCR. Recombinant MF2/MF3 protein was expressed by transformed E. coli Rosetta cells in the form of a 25-kDa water-soluble protein and purified to the homogeneity by immobilized metal affinity chromatography (IMAC). To evaluate the antiviral activity of the MF2/MF3 hybrid and to compare its protective effect with that of individual proteins and their mixtures, a biotest with pre-inoculation treatments of detached tobacco leaves of the necrosis-forming variety Xanthi NN, which were then infected with TMV, was used. Plants were grown in a climate chamber (PGV 36, Conviron, Canada) until the stage of 3‒5 true leaves; leaves of the 3rd or 4th tier were used in the experiments. Before inoculation, detached leaves were treated with preparations of the chimeric MF2/MF3 protein, individual proteins (MF2 or MF3), or their mixture. The preparations were applied onto one half of the leaf (5‒10-μl drops, 10 leaves per each treatment), and the corresponding controls were applied onto another half. The next day, the leaves were inoculated with TMV. In the first series of experiments, we tested the protein preparations obtained by heterologous expression for the saving the target activity. Therefore, the chimeric protein MF2/MF3, MF2, or MF3 were applied on “experimental” leaf halves, while control halves of the same leaves were treated with distilled water. In the second series of experiments aimed to compare the protective activity of the chimeric elicitor with each of the individual elicitor proteins, MF2/MF3 was applied to one half of the leaf, while the control half of the same leaf was treated with MF2 or MF3. Finally, in the third series of experiments, the protective effect of the chimeric elicitor MF2/MF3 was compared with the effect of a mixture of MF2 and MF3 via treating leaf halves with MF2/MF3 and applying a mixture of elicitor proteins to the corresponding control halves. In addition, a variant with application of distilled water to both halves of 5 leaves and the subsequent leaf inoculation by TMV was included in each series of experiments (untreated control). The obtained chimeric elicitor as well as MF2 and MF3 were shown to have a high protective activity against TMV. Moreover, the antiviral activity of MF2/MF3 exceeded ~ 1.5-fold the activity of each individual protein. A comparison of the levels of protective effects of MF2/MF3 chimeric protein with the mix of proteins showed no significant difference indicating the possibility to use apply this the recombinant elicitor instead of the MF2 and MF3 mixture. Obtaining the chimeric MF2/MF3 in one producer strain is more technologically profitable, since it does not require the separate production and purification of the individual proteins.

Keywords: plant protection, plant diseases, plant resistance, elicitor proteins, chimeric proteins, tobacco mosaic virus.

 

REFERENCES

  1. Mikaberidze A., Paveley N., Bonhoeffer S., van den Bosch F. Emergence of resistance to fungicides: the role of fungicide dose. Phytopathology, 2017, 107(5): 545-560 CrossRef
  2. Thind T.S. New insights into fungicide resistance: a growing challenge in crop protection. Indian Phytopathology, 2022, 75: 927-939 CrossRef
  3. Alengebawy A., Abdelkhalek S.T., Qureshi S.R., Wang M.-Q. Heavy metals and pesticides toxicity in agricultural soil and plants: ecological risks and human health implications. Toxics, 2021, 9(3): 42 CrossRef
  4. Wee S.Z., Aris A.Z. Ecological risk estimation of organophosphorus pesticides in riverine ecosystems. Chemosphere, 2017, 188: 575-581 CrossRef
  5. Mishra A.K., Sharma K., Misra R.S. Elicitor recognition, signal transduction and induced resistance in plants. Journal of Plant Interactions, 2012, 7(2): 95-120 CrossRef
  6. Peng D.-H., Qiu D.-W., Ruan L.-F., Zhou C.-F., Sun M. Protein elicitor PemG1 from Magnaporthe grisea induces systemic acquired resistance (SAR) in plants. Molecular Plant-Microbe Interactions, 2011, 24(10): 1239-1246 CrossRef
  7. Shcherbakova L., Odintsova T., Stakheev A., Fravel D., Zavriev S. Identification of a novel small cysteine-rich protein in the fraction from the biocontrol Fusarium oxysporum strain CS-20 that mitigates Fusarium wilt symptoms and triggers defense responses in tomato. Front. Plant Sci., 2016, 6: 1207 CrossRef
  8. Shagdarova B.T., Ilyina A.V., Lopatin S.A., Kartashov M.I., Arslanova L.R., Dzhavakhiya V.G., Varlamov V.P.  Study of the protective activity of chitosan hydrolyzate against septoria leaf blotch of wheat and brown spot of tobacco. Applied Biochemistry and Microbiology, 2018, 54: 71-75 CrossRef
  9. Wiesel L., Newton A.C., Elliott I., Booty D., Gilroy E.M., Birch P.R.J., Hein I. Molecular effects of resistance elicitors from biological origin and their potential for crop protection. Front. Plant Sci., 2014, 21(5): 655 CrossRef
  10. Albert M. Peptides as triggers of plant defence. Journal of Experimental Botany, 2013, 64(17): 5269-5279 CrossRef
  11. Erbs G., Newman M.-A. The role of lipopolysaccharide and peptidoglycan, two glycosylated bacterial microbe-associated molecular patterns (MAMPs), in plant innate immunity. Molecular Plant Pathology, 2012, 13(1): 95-104 CrossRef
  12. Newman M.-A., Sundelin T., Nielsen J.T., Erbs G. MAMP (microbe-associated molecular pattern) triggered immunity in plants. Front. Plant Sci., 2013, 4: 139 CrossRef
  13. Shcherbakova L.A., Dzhavakhiya V.G., Duan Y., Zhang J. Microbial proteins as elicitors of plant resistance to pathogens and their potential for eco-friendly crop protection in sustainable agriculture (review). Sel’skokhozyaistvennaya biologiya [Agricultural Biology],2023, 58(5): 789-820 CrossRef
  14. Qui D., Dong Y., Zhang Y., Li S., Shi F. Plant immunity inducer development and application. Molecular Plant-Microbe Interactions, 2017, 30(5): 355-360 CrossRef
  15. Mejia-Teniente L., Torres-Pacheco I., González-Chavira M.M., Ocampo-Velazquez R.V., Herrera-Ruiz G., Chapa-Oliver A.M., Guevara-González R.G. Use of elicitors as an approach for sustainable agriculture. African Journal of Biotechnology, 2010, 9(54): 9155-9162.
  16. Gómez-Gómez L., Boller T. FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Molecular Cell, 2000, 5(6): 1003-1011 CrossRef
  17. Boller T., Felix G. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual Review of Plant Biology, 2009, 60: 379-406 CrossRef
  18. Popletaeva S.B., Statsyuk N.V., Voinova T.M., Arslanova L.R., Zernov A.L., Bonartsev A.P., Bonartseva G.A., Dzhavakhiya V.G. Evaluation of eliciting activity of peptidyl prolyl cys/trans isomerase from Pseudonomas fluorescens encapsulated in sodium alginate regarding plant resistance to viral and fungal pathogens. AIMS Microbiology, 2018, 4(1): 192-208 CrossRef
  19. Wei Z.-M., Laby R.J., Zumoff C.H., Bauer D.W., He S.Y., Collmer A., Beer S.V. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science, 1992, 257(5066): 85-88 CrossRef
  20. Fontanilla J.M., Montes M., De Prado R. Induction of resistance to the pathogenic agent Botrytis cinerea in the cultivation of the tomato by means of the application of the protein “Harpin” (Messenger). Communications in Agricultural and Applied Biological Sciences, 2005 70(3): 35-40.
  21. Wang H., Yang X., Guo L., Zeng H., Qiu D. PeBL1, a novel protein elicitor from Brevibacillus laterosporus strain A60, activates defense responses and systemic resistance in Nicotiana benthamiana. Applied and Environmental Microbiology, 2015, 81(8): 2706-2716 CrossRef
  22. Zhang Y., Zhang Y., Qiu D., Zeng H., Guo L., Yang X. BcGs1, a glycoprotein from Botrytis cinerea, elicits defence response and improves disease resistance in host plants. Biochemical and Biophysical Research Communications, 2015, 457(4): 627-634 CrossRef
  23. Kulye M., Liu H., Zhang Y., Zeng H., Yang X., Qiu D. Hrip1, a novel protein elicitor from necrotrophic fungus, Alternaria tenuissima, elicits cell death, expression of defence-related genes and systemic acquired resistance in tobacco. Plant Cell Environ., 2012, 35(12): 2104-2120 CrossRef
  24. Zhang W., Yang X., Qiu D., Zeng H., Guo L., Mao J. Activator protein PeaT1 induced systemic resistance to Tobacco mosaic virus in tobacco. Acta Phytopathologica Sin., 2010, 40: 290-299.
  25. Zhang W., Yang X., Qiu D., Guo L., Zeng H., Mao J., Gao Q. Pea T1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway. Molecular Biology Reports, 2011, 38: 2549-2556 CrossRef
  26. Mao J., Liu Q., Yang X., Long C., Zhao M., Zeng H., Liu H., Yuan J., Qiu D. Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobacco. Annals of Applied Biology, 2010, 156(3): 411-420 CrossRef
  27. Kromina K.A., Dzhavakhiya V.G. Molekulyarnaya genetika, mikrobiologiya i virusologiya, 2006, 1: 31-34 (in Russ.).
  28. Shumilina D., Krämer R., Klocke E., Dzhavakhiya V. MF3 (peptidyl prolyl cis-trans isomerase of FKBP type from Pseudomonas fluorescens) — an elicitor of non-specific plant resistance against pathogens. Phytopathol. Polonica, 2006, 41: 39-49.
  29. Dzhavakhiya V.G., Voinova T.M., Shumilina D.V. Search for the active center of peptidyl-prolyl cys/trans isomerase from Pseudomonas fluorescens responsible for the induction of tobacco (Nicotiana tabacum L.) plant resistance to tobacco mosaic virus. Sel’skokhozyaistvennaya biologiya [Agricultural Biology], 2016, 51(3): 392-400 CrossRef
  30. Djavakhia V.G., Nikolaev O.N., Voinova T.M., Battchikova N.V., Korpela T., KhomutovR.M. DNA sequence of gene and amino acid sequence of protein from Bacillus thuringiensis, which induces non-specific resistance of plants to viral and fungal diseases. Journal of Russian Phytopathological Society, 2000, 1: 75-81.
  31. Horn G., Hofweber R., Kremer W., Kalbitzer H.R. Structure and function of bacterial cold shock proteins. Cell. Mol. Life Sci., 2007, 64: 1457-1470 CrossRef
  32. Skabkin M.A., Skabkina O.V., Ovchinnikov L.P. Uspekhi biologicheskoy khimii, 2004, 44: 3-52 (in Russ.).
  33. Ovchinnikov L.P., Skabkin M.A., Ruzanov P.V., Evdokimova V.M. Molekulyarnaya biologiya, 2001, 35 (4): 548-558 (in Russ.).
  34. Subin C.S., Pradeep M.A., Vijayan K.K. FKBP-type peptidyl-prolyl cis-trans isomerase from thermophilic microalga, Scenedesmus sp.: molecular characterisation and demonstration of acquired salinity and thermotolerance in E. coli by recombinant expression. Journal of Applied Phycology, 2016, 28: 3307-3315 CrossRef
  35. Chang H.-H., Lee C.-J., Chang C.-J., Jan F.-J. FKBP-type peptidyl-prolyl cis-trans isomerase interacts with the movement protein of tomato leaf curl New Delhi virus and impacts viral replication in Nicotiana benthamiana. Molecular Plant Pathology, 2022, 23(4): 561-575 CrossRef
  36. Kromina K.A., Ignatov A.N., Abdeeva I.A. Biologicheskie membrany, 2008, 25(4): 243-251 (in Russ.).
  37. Dzhavakhiya V.G., Filippov A.V., Skryabin K.G., Voinova T.M., Kouznetsova M.A., Shulga O.A., Shumilina D.V., Kromina K.A., Pridannikov M.V., Battchikova N.V., Korpela T. Proteins inducing multiple resistance of plants to phytopathogens and pests. Intern. Pat. Classification: C07K 14/21. Intern. applic. number: PCT/FI2004/000766. Intern. Filing date: 17 December 2004 (17.12.2004). Priority data: 20031880 22 December 2003. Intern. Public. number: WO 2005/061533 A1.
  38. Popletaeva S., Erokhin D., Dzhavakhiya V. Comparison of the protective activity of elicitor proteins MF2 and MF3 applied individually or in combination against tobacco mosaic virus on tobacco leaves. In: Agriculture digitalization and organic production. ADOP 2023. Smart innovation, systems and technologies. V. 362. A. Ronzhin, A. Kostyaev (eds.). Springer, Singapore, 2023: 225-234 CrossRef
  39. Urban L., Lauri F., Ben Hdech D., Aarrouf J. Prospects for increasing the efficacy of plant resistance inducers stimulating salicylic acid. Agronomy, 2022, 12(12): 3151 CrossRef
  40. Reglinski T., Havis N., Rees H.J., de Jong H. The practical role of induced resistance for crop protection. Phytopathology, 2023, 113(4): 719-731 CrossRef
  41. Walters D.R., Havis N.D., Paterson L., Taylor J., Walsh D.J., Sablou C. Control of foliar pathogens of spring barley using a combination of resistance elicitors. Frontiers in Plant Science, 2014, 5: 241 CrossRef
  42. Oxley S.J., Walters D.R. Control of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) with resistance elicitors. Crop Protection, 2012, 40: 59-62 CrossRef
  43. Aćimović S.G., Meredith C.L., Santander R.D., Khodadadi F. Proof of concept for shoot blight and fire blight canker management with postinfection spray applications of prohexadione-calcium and acibenzolar-S-methyl in apple. Plant Disease, 2021, 105(12): 4095-4105 CrossRef
  44. Anith K.N., Momol M.T., Kloepper J.W., Marois J.J., Olson S.M., Jones J.B. Efficacy of plant growth-promoting Rhizobacteria, acibenzolar-S-methyl, and soil amendment for integrated management of bacterial wilt on tomato. Plant Disease, 2004, 88(6): 669-673 CrossRef
  45. Bahadou S.A., Ouijja A., Boukhari M.A., Tahiri A. Development of field strategies for fire blight control integrating biocontrol agents and plant defense activators in Morocco. Journal of Plant Pathology, 2017, 99: 51-58.
  46. Percival G.C., Noviss K., Haynes I. Field evaluation of systemic inducing resistance chemicals at different growth stages for the control of apple (Venturia inaequalis) and pear (Venturia pirina) scab. Crop Protection, 2009, 28(8): 629-633 CrossRef
  47. Esquivel-Cervantes L.F., Tlapal-Bolaños B., Tovar-Pedraza J.M., Pérez-Hernández O., Leyva-Mir S.G., Camacho-Tapia M. Efficacy of biorational products for managing diseases of tomato in greenhouse production. Plants, 2022, 11(13): 1638 CrossRef
  48. Park K., Park J.-W., Lee S.-W., Balaraju K. Disease suppression and growth promotion in cucumbers induced by integrating PGPR agent Bacillus subtilis strain B4 and chemical elicitor ASM. Crop Protection, 2013, 54: 199-205 CrossRef

 

back

 


CONTENTS

 

 

Full article PDF (Rus)