Comparative characterization of lectins of pathogenic and saprophytic filamentous fungi Fusarium solani

. In the present study, we isolated, purified and characterized the lectin of the phytopathogenic fungus Fusarium solani 6 and compared it with the properties of the lectin of the saprophytic strain Fusarium solani 4. Electrophoretically homogeneous lectin was obtained from the mycelium of the fungus F. solani 6 by hydrophobic chromatography and gel filtration. The molecular weight of the native lectin molecule was established to be 30.0 kDa, and it was found that it consists of two identical subunits. Comparison of the physicochemical properties of the lectin of the phytopathogenic strain with the lectin of the saprophytic strain showed that the F. solani 6 lectin was a more thermostable and alkali-resistant protein. F. solani 6 lectin showed affinity for simple sugars, and F. solani 4 lectin - for glycoproteins. In contrast to the lectin of a saprophytic fungus, the treatment of pea seedling roots with F. solani lectin 6 before they were infected with the phytopathogen led to a decrease in the degree of damage to the plant root system and the prevalence of Fusarium . These results open up prospects for further study of the phytopathogen lectin and its potential application as a means of eliciting action.


Introduction
The rapid development of research in the field of glycochemistry and glycobiology in recent years has made it possible to reveal the main role of lectins in carbohydrate-protein and carbohydrate-carbohydrate interactions between cells of organisms [1], which has increased the interest of researchers in this group of proteins.Lectins are proteins and glycoproteins that have unique biological properties and are able to selectively interact with certain carbohydrate structures of tissue cells and change signals in the biological system [2. 3].They are used in various industries [4. 5] because they have a wide range of biological activity.In agriculture, lectins are used to fight against various insect pests, phytopathogens [6. 7], as plant growth stimulants [8], to create resistant transgenic plants to fungal pathogens and insects [9] and other stress factors [10].
They are found in any living system, from viruses to higher vertebrates [11], and participate in many biological processes at different levels of organization of a living organism.However, most of the research is devoted to the study of the function of plant and animal lectins; information on the properties and functions of fungal lectins in the scientific literature is very limited, despite the potential for their use in agriculture.Recently, studies conducted by foreign researchers on fungi indicate that the lectins of these organisms can play an important role in the interactions between plants and microorganisms [12.13].
Among a significant number of phytopathogens that can parasitize on a wide range of hosts from various families of higher plants, fungi of the genus Fusarium are of particular interest [14].The active phytopathogens of this genus include the species Fusarium solani, the causative agent of Fusarium blight in many agricultural and cultivated plants.Despite the available research data on the ability of fungi Fusarium solani to synthesize lectins [15][16][17], issues related to the functions of lectins in pathogenic and saprophytic fungi of this species remain unexplored.
In previous works, we studied some biochemical properties of Fusarium solani lectins [17. 18], as well as isolation, purification, and characterization of the lectin of the saprophytic strain F. solani 4 [18].From the mycelium of the fungus F. solani 4, a lectin was isolated, which is a protein with a molecular weight of 38 kDa and specificity for sialic acids, fetuin, and fibrinogen.This protein has been shown to be active over a wide range of temperatures (10-60°C) and pH (pH 6.5-8.5).
The present work is devoted to the isolation, purification and characterization of the lectin of the phytopathogenic fungus Fusarium solani 6 and its comparison with the properties of the lectin of the saprophytic strain Fusarium solani 4. It was found that the F. solani 6 lectin was a low molecular weight protein (30 kDa) and differed in physicochemical and biochemical parameters from F. solani 4 lectin.It had an increased affinity for L-arabinose, L-fucose, D-mannose and was able to reduce the intensity of damage and the prevalence of pea root rot.The data obtained give grounds to consider the lectins of this fungus as a possible elicitor of induced plant immunity.

Microorganisms and culture conditions
The fungi F. solani 4 (saprophytic strain) and F. solani 6 (phytopathogenic strain) [18. 19] from the collection of cultures of microscopic fungi of the Department of Biochemistry, Biotechnology and Pharmacology of the Institute of Fundamental Medicine and Biology of the Kazan (Volga Region) Federal University were used in the work.Fungal cultures were maintained on a PGA medium (potato-glucose agar) containing (g/l) glucose 20.0, potatoes 200.0, agar 20.0, at a temperature of 5°C.To establish the toxigenic potential, fungal strains were grown on Czapek-Dox-peptone medium [18] containing (g/l) sucrose 30.0, peptone 10.0, NaNO3 2.0, K2HPO4 1.0, MgSO4 0.5, KCl 0.5, FeSO4 0.01, at 25 °C for 12 days under stationary conditions.To obtain the F. solani 4 lectin, the fungus was cultivated in a liquid PG medium, with stirring, at 28°C for 8 days.

Toxigenic potential of F. solani strains
Determination of the toxigenic potential of F. solani was carried out on thirty male Wistar rats, body weight from 180 to 190 g, according to the method described by us earlier (adaptation time before the start of the experiment was at least 14 days) [19].Animals were divided into three equal groups.Individuals of group I, which served as control, received standard food for 28 days, animals of groups II and III received gran-ules contaminated with metabolites of the studied microscopic fungi.Animals were kept in the appropriate zoohygienic conditions, had free access to water (without re-strictions) and feed (according to daily norms).At the beginning and every 7 days of the study, the animals were weighed and their body weight was analyzed.
Animals were kept in accordance with the rules adopted by the European Convention for their Protection (86/609 EEC).

Isolation and purification of lectin from mycelium extract
To isolate the lectin of the fungus, we used an extract of its mycelium, which was obtained on the 8th day of growing the producer according to the method described by us earlier.
Lectin was isolated from the fungal mycelium extract by precipitation of proteins with 75% crystalline ammonium sulfate (V/V) for 1 day.The precipitate was separated from the supernatant by centrifugation at 10,000 g for 15 min, dissolved in a minimum volume of 20 mM Tris-HCl buffer solution, pH 7.4, and dialyzed against the same buffer in dialysis bags with a pore size of 12-14 kDa (Orange Scientific, Belgium) for 2 days at 4°C.The obtained protein solution was used for further purification of lectin.
Lectin was purified from total proteins by two successive hydrophobic chromatographies on a Phenyl Sepharose High Performance (1 ml) column and gel filtration on Sephadex G-50.Before applying the protein samples to the columns, filtration was carried out through special sterile Millex membrane filters (d 33 mm, d pore 0.45 μm, polyethylsulfone, Merck Millipore, USA).
The bound lectin was eluted with a hydrophobic sorbent using a concentration gradient from 1.5 to 0 M (NH4)2SO4 in a 20 mM Tris-HCl buffer solution, pH 7.4, at a flow rate of 0.5 ml/min.The protein was eluted from the gel filtration sorbent with 20 mM Tris-HCl buffer, pH 7.4, at a rate of 1.0 ml/min.
At each stage of isolation and purification of lectin, the protein content was analyzed by spectrophotometry and hemagglutination activity, the degree of purification and the yield of lectin were calculated.

Purity and molecular weight of lectin
The determination was carried out by one-dimensional electrophoresis in 12.5% polyacrylamide gel with sodium dodecyl sulfate and gel filtration on a Sephadex G-100 column.
Denaturing electrophoresis in PAAG was performed by the Laemmli method [18].Protein gels were stained with a silver nitrate solution containing 0.4% AgNO3, 0.2% NH3 (V/V), and 0.09% NaOH (V/V).As proteins with a known molecular weight, a commercial set of marker proteins PageRuler Unstained Protein Ladder (Fermentas, Lithuania) (10-200 kDa) was used.Gel protein band analysis was performed using Image Lab™ Software.
Gel filtration was carried out on an Econo-Column (1.5 × 50 cm, Bio-Rad, USA) filled with Sephadex G-100 (particle diameter 40.0-120.0µm).As proteins with a known molecular weight, lysozyme (14 kDa), α-chymotrypsin (26 kDa), peroxidase (34 kDa), and bovine serum albumin (70 kDa) were used.The elution of marker proteins applied to the column in a volume of 1 ml (2 mg/ml) was carried out with Tris-HCl buffer (pH 7.4) at a flow rate of 0.5 ml/min.

Temperature and pH-optimum, influence of metal ions, and carbohydrate specificity of lectin
The determination was carried out according to the method described by us earlier [18. 19].
Determination of the pH-optimum of lectin activity was carried out by protein incubation in buffer solutions with pH values from 3.0 to 11.0 (50 mM citrate buffer, pH 3.0-6.0;50 mM phosphate buffer, pH 6.0-8, 0; 50 mM Tris-HCl buffer pH 8.0-9.0;20 mM glycine-NaOH buffer, pH 9.0-11.0)at 20°C for 1 hour.After protein incubation, the protein content and hemagglutination activity were analyzed.
The temperature optimum of lectin activity was determined by protein incubation in the temperature range from 20 to 80°C for 20 minutes.After protein incubation, the protein content and hemagglutination activity were analyzed.
The influence of metal ions on the activity of lectin was carried out by its interaction with various metals.Solutions of MnCl2, CaCl2, MgCl2, ZnCl2, CoCl2, FeCl3, AlCl3, CuSO4, FeSO4 in final concentrations from 1.25 to 20 mM were used as metal salts.
The specificity of lectin to carbohydrates was determined by the method of inhibition of the direct hemagglutination reaction after the interaction of the protein with mono-and polysaccharides at concentrations of 300 mM and 5 mg/ml, respectively.

Effect of lectin on the development of Fusarium plants
The assessment was carried out according to the degree of development and prevalence of root rot disease in plants preliminarily treated with lectin prior to their infection with the pathogen [20. 21].Seeds of pea varieties "Albumen" (created at the Falenskaya breeding station of the FGBNU "Federal Agrarian Research Center of the North-East named after N.V. Rudnitsky") were provided by the FGBNU "All-Russian Research Institute of Phytopathology" (Odintsovsky district, Russia).
The study of the biological activity of lectin was carried out by treating the roots of three daily pea seedlings with protein (0.7, 0.9 and 1.1 μg/ml) for 1 day, after which, on the fifth day, the plant roots were soaked with a spore suspension of the phytopathogenic fungus F. solani 6 (titer 10 5 macroconidia/ml).On the 14th day, the damage by root rot of plants was determined, as well as the measurement of their growth and development parameters (height of the above-ground part, weight of above-ground organs, length and weight of roots).The roots of pea seedlings treated without the addition of lectin were used as a control.
The degree of development of root rot in peas was calculated by the formula: R = (Σ(a•b))•100 / N•4, where R -degree of development of the disease (%), a -number of organs or plants with a given damage score, b -defeat score; N -the total number of organs or plants, 4 -maximum damage score.
The prevalence of root rot in peas was estimated by the formula: Р = n / N•100, where P -prevalence of the disease (%), n -number of affected plants, N -total number of plants (healthy and diseased).

Lectin Activity
A suspension of native human erythrocytes (2%) of group 0 (I) for the determination of lectin activity by direct hemagglutination (HA) was obtained by the method of Lutsik et al. [18].The activity of lectins was expressed in titers and calculated according to the formula [18]: HA= 2 n-1 , where: HA -hemagglutination activity (titer, units), n -dilution or minimum protein concentration at which erythrocyte agglutination is noted.

Statistical Analyses
Statistical processing of three independent experiments was carried out using the spreadsheet package of Microsoft Office Excel 2013 (Windows, USA).Samples were evaluated for the normality of their distribution by the Shapiro-Wilk test.The significance of differences between comparable values was determined by Student's t-test for independent samples (with Bonferroni correction).Differences were considered statistically significant at p ≤ 0.05.

Toxigenic potential of F. solani strains
Previously, we showed the ability of F. solani 6 to exhibit pronounced phytopathogenic properties in relation to pea roots and the absence of this ability in F. solani 4 [18].In this study, the toxigenic potential of the studied strains of fungi was established (Table 1).*no significant differences were found between the experimental and control groups at p<0.05 In the group of rats that received food treated with the culture supernatant of F. solani 4 (group II), the decrease in their body weight on day 28 was 2.03% compared with the control (group I).However, statistically significant differences between the results of this experimental and control groups of animals were not found.In rats fed with food treated with F. solani 6 culture supernatant (group III), on day 28, their body weight decreased by 10.79% relative to the control group.

Isolation and purification of F. solani lectin 6
For a comparative characterization of the lectin of the phytopathogenic strain F. solani 6 with the isolated and characterized lectin of the saprophytic strain F. solani 4, it was necessary, first of all, to develop the conditions for its isolation and purification in a homogeneous state.Using the previously developed scheme for obtaining lectin from the F. solani 4 mycelium extract allowed us to select new parameters for the purification of the lectin of the phytopathogenic strain F. solani 6 to a homogeneous state (Table 2).First, proteins from F.  Then the lectin was purified using two successive hydrophobic chromatographies (Figures 1A).The elution of proteins from a hydrophobic sorbent with a buffer solution made it possible to separate the proteins into several components (I.II.III).Determination of the hemagglutinating activity of the components in fractions showed that only component II had lectin activity.The use of hydrophobic chromatography twice made it possible to increase the degree of purity of the resulting lectin without losing its activity (1536.0U). but it was not possible to completely purify the lectin from ballast proteins.The total protein yield was 52.6%.
At the last stage.proteins for the purification of the F. solani 6 lectin were fractionated by gel filtration on a Sephadex G-50 column (Figures 1B).As a result.it was possible to completely purify the protein from impurities and increase its specific activity by 90.3 times.
The high degree of purity of the isolated and purified F. solani 6 lectin was confirmed by the presence of one band on the electropherogram obtained by electrophoresis in PAAG in the presence of Ds-Na.The specific hemagglutinating activity of the obtained lectin was 7111.1 U/mg of protein.The calculation showed that 8.8 μg/ml of purified protein can be obtained from 1 g of dry fungal mycelium.
The developed scheme for the isolation and purification of the F. solani 6 lectin differed from the scheme for obtaining lectin from the F. solani 4 mycelium extract.Thus, the stage of lectin purification using hydrophobic chromatography differed in the spectrum of lectin proteins.While in the saprophytic strain F. solani 4 components with lectin activity were detected in the initial fractions [18], in the phytopathogenic strain lectin activity was detected in fractions 9 and 10.

Molecular weight, physicochemical and biochemical properties of lectin
The isolation of the lectin from the F. solani 6 mycelium extract in a homogeneous form made it possible to determine its molecular weight, physicochemical and biological properties.
The use of standard marker proteins with a known molecular weight (15-200 kDa) after protein electrophoresis under denaturing conditions revealed that the molecular weight of the F. solani 6 lectin was 15 ± 1.6 kDa (Figures 2A).Under native conditions of protein gel filtration on a Sephadex G-100 column, F. solani 6 lectin had a molecular weight of 30.0 ± 2.1 kDa (Figures 2B).This suggested that the F. solani 6 lectin was a dimer and consisted of two identical subunits.In a previous work [18], we found that the lectin from the mycelium of the fungus F. solani 4 was a dimer (38.0 kDa) consisting of two identical subunits, each of which had a molecular weight of 19.0 ± 2.0 kDa.
In this work, the pH-optimum activity of the F. solani 6 lectin was set at 20°C, since it was shown that the range of the optimal lectin temperature ranges from 5 to 75°C.To determine the pH-optimum of lectin activity, the preparation incubated in various buffer solutions at pH values from 3.0 to 11.0 for an hour.
In a previous work [18], we found that the F. solani 4 lectin was a thermostable (10-60°C) and alkali resistant (pH 6.5-8.5)protein.The same is true for F. solani 6 lectin was stable over a wide temperature range (5-75°C) and at alkaline pH values (pH 5.5-9.0).
The study of the effect of various metal ions on the lectin activity of the F. solani 6 protein showed that its activity was independent of the presence of metal salts in the reaction mixture.A similar pattern was noted for the F. solani 4 lectin we obtained previously [18].
A significant difference between the lectin of a phytopathogenic strain and the lectin of a saprophytic strain was found in their interaction with various carbohydrates.While the lectin of the F. solani 6 strain showed carbohydrate specificity to the monosaccharides L-arabinose, L-fucose, and D-mannose (with a minimum inhibitory concentration of 9.375 mM, 0.586 mM, and 150 mM, respectively), the lectin of the F. solani 4 strain [18] -to glycoproteins fetuin and fibrinogen (with a minimum inhibitory concentration of 0.01 and 0.08 mg / ml, respectively).

Effect of lectin on the development of Fusarium blight in pea plants
Subsequently, the ability of lectins of F. solani 4 and F. solani 6 strains as potential elicitor agents to protect pea plants from F. solani phytopathogens was evaluated.It was found that the F. solani 6 lectin reduced the intensity of damage to the plant root system and the prevalence of Fusarium (Figures 3).The most active lectin was at a concentration of 1.1 μg/ml, at which the degree of damage to the pea root system and the prevalence of Fusarium decreased by 5.4 and 2.1%, respectively, relative to the control.At a given concentration of protein, it increased the growth of aboveground and underground parts of plants, as well as the biomass of shoots and roots of seedlings.However, the observed increase in these indicators of plant productivity was not statistically significant due to the high dispersion of the data obtained.
Treatment of plants with the lectin of the saprophytic fungus F. solani 4 at a concentration of 1.1 μg/ml did not affect the length and biomass of the aboveground and underground parts of the seedlings, as well as the prevalence of Fusarium and the degree of damage to their root system.

Discussion
In this work, we isolated, purified, and characterized the lectin of the phytopathogenic fungus Fusarium solani 6 and compared it with the properties of the lectin of the saprophytic strain Fusarium solani 4, obtained by us earlier [18] from its mycelium extract.
The analysis of lectins from the mycelium of phytopathogenic and saprophytic F. solani strains showed differences between them in the schemes of their production.Differences between the two strains were also observed in the chromatographic spectra of proteins, and the extract of the phytopathogenic strain was distinguished by a relatively high content of proteins in them and a more pronounced hemagglutination activity than the extract of the saprophytic strain [18].
Previous experiments showed some relationship between the degree of phytopathogenicity of fungi and the activity of lectins in their extracts [18].This study established the presence of toxigenic properties of metabolites of the phytopathogen F. solani 6 and the absence of these properties in metabolites of the saprophyte F. solani 4.These results are consistent with the data of foreign authors on a high degree of correlation between the degree of phytopathogenicity of F. solani fungi and the ability to produce metabolites toxic to animals and humans [22].
Further comparative study of lectins of phytopathogenic and saprophytic F. solani strains revealed differences in their molecular weight, physicochemical and biochemical properties.
It was shown that despite the fact that the compared lectins were low molecular weight and dimeric proteins, the molecular masses of F. solani 6 lectin (30.0 kDa) and F. solani 4 lectin (38.0 kDa) differed.These protein molecular mass values were close in size to the lectins of fungi Arthrobotrys oligospora, Rhizoctonia solani [23], Sclerotinia sclerotiorum [24] and Sclerotium rolfsii [25] (31-37 kDa), which also had a dimeric protein structure with two identical subunits.
Both lectins functioned in a wide temperature range and mainly in alkaline pH values, which is consistent with the data obtained earlier by other foreign researchers [23. 26].However, F. solani 6 lectin remained stable over a wider range than F. solani 4 protein.
Comparison of the activity of lectins of phytopathogenic and saprophytic strains after exposure to a wide range of different metal ions did not reveal differences between them.The independence of the activity of the studied lectins from the presence of metal salts in the reaction mixture suggested the absence of these metal ions in the active site of carbohydrate binding of the protein.For the overwhelming majority of the discovered lectins of microscopic fungi, the presence of metal ions in the composition of their molecules is not strictly necessary [23].
Along with the differences noted above, differences in the carbohydrate specificity of lectins of phytopathogenic and saprophytic strains were determined.While the F. solani 6 lectin showed affinity for simple sugars, the F. solani 4 lectin showed affinity for glycoproteins.It was suggested that these carbohydrates are involved in the process of intercellular recognition when the host plant is infected with a phytopathogen.These results are consistent with the data on the specificity of most lectins of phytopathogenic F. solani strains for L-fucose monosaccharides, obtained earlier in the experiment [17].Affinity for Lfucose was found for lectins of fungi of the genera Aspergillus [26. 27], Cephalosporium [28], as well as for a lectin produced by a bacterium of the genus Azospirillum [29], which is present in the root system of higher plants and is in symbiotic relationship with them.
In connection with the discovered features of the interaction of lectins of phytopathogenic and saprophytic strains of F. solani 6 with carbohydrates and glycoproteins, we studied their effect on the development of Fusarium blight in peas.The results of foreign studies indicate that lectins of microscopic fungi can play an important role in the processes of plant pathogenesis [12. 30-32] and act as elicitor compounds, inducers of plant defense reactions that can form their resistance [12. 32].
In contrast to the lectin of a saprophytic fungus, the treatment of the roots of the sowing pea with F. solani 6 lectin prior to their infection with the phytopathogen prevented the development of root rot in plants.This suggested the possibility of lectin participation as a phytopathogen molecule "recognizing" by plants in the process of pathogenesis of pea plants.However, apart from the interaction of the lectin of the phytopathogenic fungus F. solani 6 with the above monosaccharides and its effect on the development of fusarium in peas, we still know very little about the role of this lectin protein in the pathogenesis of these plants.

Conclusions
A lectin was isolated from the mycelium of the phytopathogenic fungus F. solani 6 and purified to an electrophoretically homogeneous state.The study of the physicochemical and biochemical characteristics of the lectin of the phytopathogenic strain showed its difference from the lectin of the saprophytic strain F. solani 4. In particular, the lectin from the mycelium of F. solani 6 was characterized by affinity for simple sugars, while the lectin of F. solani 4 strain had an affinity for glycoproteins.In contrast to the saprophytic fungus lectin, F. solani 6 lectin prevented the development of root rot on the underground part of pea plants.These results open up prospects for further study of the functional role of lectin in the mycelium of this Fusarium strain, and as an elicitor drug.

Funding
The work to determine the toxigenic potential of fungal strains was supported by the Russian Science Foundation under grant № 23-26-00161.

Fig. 3 .
Fig.3.Influence of lectin on the development of root rots of pea plants (degree of development of the disease (R) and prevalence of the disease (P)) before their infection with F. solani 6 phytopathogen.

Table 1 .
Dynamics of body weight of Wistar rats that consumed food treated with culture supernatant of F. solani strains.

Table 2 .
Isolation and purification of lectin from F. solani 6 mycelium extract.
*indicate respectively differences at p ≤ 0.05 probability level