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

The induction of pathogen resistance with biogenic elicitors is now considered as a promising plant protection method. Elicitors obtained from fungal, bacterial, and oomycetic pathogens can be of peptide, glicoproteid, lipid, and oligosaccharide origin. The first reported protein elicitor was harpin isolated from Erwinia amylovora. Based on this elicitor, a Messenger® preparation intended to protect plants from a wide range of pathogens was developed and commercialized in USA. The MF3 protein able to induce the systemic resistance of plants to viral and fungal pathogens was isolated from the strain 197 of Pseudomonas fluorescens in the course of our earlier studies. Such a wide range of action assumed prospectivity of MF3 using as a potential plant protection remedy. The full amino acid sequence of this protein was determined, and its high homology to the FKBP-type peptidyl-prolyl cys/trans isomerases of the same bacteria was demonstrated, so the isolated protein was called Pf197_PPIase. As a rule, active centers of the majority of known elicitor proteins are localized at the most conserved domains. We supposed that the active center of Pf197_PPIase responsible for its ability to induce the resistance is localized within the most conserved sequence of this protein. The calculation of this sequence using a PROSITE bioinformational resource showed it contains sites composed of arginine and lysine and subjected to the tripsinolysis. The preparations of Pf197_PPIase were obtained from the recombinant strain of Escherichia coli BL21(DE3)+plMF3 - the super-producer of Pf197_PPIase. The treatment of Pf197_PPIase with tripsin resulted in the loss of its elicitor properties that indirectly confirmed our hypothesis about the responsibility of the studied conserved domain for the elicitor activity of the MF3 protein. A Pf_29ac oligomer consisting of 29 amino acids and corresponding to the revealed conserved region was obtained by a chemical synthesis (Pushchino affiliated branch of the Institute of bioorganic chemistry). The further experiments showed that equimolar concentrations of Pf197_PPIase and Pf_29ac induced a similar level of resistance of tobacco plants to Tobacco Mosaic Virus (TMV). These results were confirmed in biotests when necrosis were calculated on the surfaces of the inoculated tobacco leaves. The leaves were treated with preparations and incubated in wet chambers for 24 hours at 22 oC. After that the leaves were inoculated with viral suspension and incubated in wet chambers during 3-4 days at 22 oC. The obtained results permitted us to conclude the conserved domain of Pf197_PPIase alone is sufficient for the induction of the TMV resistance in tobacco. The further determination of a minimum size of the active center able to provide the elicitor activity is planned.