doi: 10.15389/agrobiology.2024.3.446eng

UDC: 632.938.2:577.112.4:577.112.6

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



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,,, (✉ corresponding author);
2Federal Research Center Fundamentals of Biotechnology RAS, 33/2, Leninskii prospect, Moscow, 119071 Russia, e-mail,,

ORCID: Erokhin D.V.
Popletayeva S.B.
Sinelnikov I.G.
Shcherbakova L.A.
Shashkov I.A.Х
Dzhavakhiya V.G.
Denisenko Y.A.

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.



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