T. A. Valueva

Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms

This review analyzes the literature on various mechanisms of proteolytic enzyme inhibitors involved in plant defense against attack by phytopathogenic microorganisms. The action of proteinase inhibitors from plants upon the enzymes from pathogenic microorganisms and viruses is reviewed. Considerable attention is given to the induction of proteinase inhibitors in plants in response to the invasion of pathogens. Some aspects of application of proteinase inhibitors in biotechnology for production of transgenic plants with enhanced resistance to diseases are discussed.

Proteinase inhibitors and their function in plants: A review

The spread, classification, and properties of plant proteins capable of inhibiting proteinases have been reviewed. Data from the literature on the likely physiological functions of these inhibitors in plants are analyzed.

Kunitz-type proteinase inhibitors from intact and Phytophthora-infected potato tubers

Three protein proteolytic enzyme inhibitors with molecular masses 21, 22, and 23 kDa have been isolated from intact potato tubers (Solanum tuberosum L. cv. Istrinskii). The 21 and 22 kDa proteins denoted as PSPI‐21 and PSPI‐22, respectively, are serine proteinase inhibitors with different specificity. The 23 kDa protein denoted as PCPI‐23 is an inhibitor of plant cysteine proteinases. The PSPI‐21 molecule consists of two disulfide‐linked polypeptide chains with molecular masses of 16.5 kDa and 4.5 kDa. The PSPI‐22 and PCPI‐23 have one polypeptide chain. Their amino‐termini numbered 21–25 amino acid residues have significant homology to other plant inhibitors which are members of the soybean Kunitz inhibitor family. It is found that at least PSPI‐21 and PSPI‐22 can predominantly accumulate in potato tubers infected with Phytophthora infestans zoospores.

Primary structure of potato kunitz-type serine proteinase inhibitor.

Abstract The serine proteinase inhibitor (PSPI-21) isolated from potato tubers (Solanum tuberosum L.) comprises two protein species with pI 5.2 and 6.3, denoted as PSPI-21-5.2 and PSPI-21-6.3, respectively. They were separated by anion exchange chromatography on a Mono Q FPLC column. Both species tightly inhibit human leukocyte elastase, whereas their interaction with trypsin and chymotrypsin is substantially weaker. The sequences of both PSPI-21-5.2 and PSPI-21-6.3 were determined by analysis of overlapping peptides obtained from the oxidized or reduced and S-pyridylethylated proteins after digestion with trypsin or pepsin. Both species of PSPI-21 are composed of two chains, named chains A and B, which are linked by a disulfide bridge between Cys(146) and Cys(157). The other disulfide bridge is located within the A chains between Cys(48) and Cys(97). The amino acid sequences of the large A chains of the two forms, consisting of 150 amino acids residues each, differ in a single residue at position 52. The small chains B, containing 37 and 36 residues in PSPI-21-6.3 and PSPI-21-5.2, respectively, have nine different residues. The entire amino acid sequences of the two inhibitors show a high degree of homology to the other Kunitz-type proteinase inhibitors from plants.

Role of Protease Inhibitors in Potato Protection

Mechanical wounding or infection of potatoes with Phytophthora infestans caused an accumulation of only serine protease inhibitors in exudates of potato tubers. Among them, proteins prevailed that are structurally similar to those present in healthy tubers: a 22-kDa trypsin inhibitor, a 21-kDa serine protease inhibitor consisting of two polypeptide chains, and a 8-kDa potato chymotrypsin I inhibitor produced de novo. The accumulated proteins inhibited the growth of hyphae and germination of zoospores of P. infestans. Treatment with elicitors, jasmonic and arachidonic acids, intensified the accumulation of these inhibitors in tubers in response to the wound stress, whereas salicylic acid blocked this process. These results suggest that lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.

Participation of proteolytic enzymes in the interaction of plants with phytopathogenic microorganisms.

Different forms of participation of proteolytic enzymes in pathogenesis and plant defense are reviewed. Together with extracellular proteinases, phytopathogenic microorganisms produce specific effectors with proteolytic activity and are able to act on proteins inside the plant cell. In turn, plants use both extracellular and intracellular proteinases for defense against phytopathogenic microorganisms. Among the latter, a special role belongs to vacuolar processing enzymes (legumains), which perform the function of caspases in the plant cell.

Interaction of proteinases secreted by the fungal plant pathogen Rhizoctonia solani with natural proteinase inhibitors produced by plants

The fungal plant pathogen Rhizoctonia solani Kuhn. grown in a medium containing thermostable potato tuber proteins produced proteinases active at moderately alkaline pH values. Electrophoretic analysis in polyacrylamide gel with SDS and copolymerized gelatin showed that the extracellular proteinase complex contained four components that differed in molecular weight. Studies on the action of the exoenzymes on various synthetic substrates indicated that the culture liquid of R. solani contained mainly trypsin-like proteinases. The exoproteinase activity was virtually completely suppressed by trypsin inhibitor proteins isolated from potato tubers and seeds of various legume species. The results suggest that the extracellular proteinases produced by R. solani play a significant role in attacking plant tissue, and natural inhibitors contribute to the protection of Solanaceae and Leguminosae from this fungal pathogen.

[Trypsin-like proteinases and trypsin inhibitors in fruiting bodies of higher fungi].

The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst).

Heterologous expression, purification, and properties of a potato protein inhibitor of serine proteinases

The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The ability of 96 microbial strains degrading oil and 32 strains degrading polycyclic aromatic hydrocarbons (PAHs) to consume diesel fuel and oil at 4-6 degrees C and 24 degrees C and at elevated NaCl concentrations was studied. The temperature range, salt tolerance, ability to produce bioemulsifiers, range of substrates, and antibiotic resistance were determined. The eleven most active oil-degrading and PAH-degrading strains were genotyped by a polymerase chain reaction with BoxA1R primers and a restriction analysis of ribosomal DNA amplicons. For six strains, the degree of oil degradation at 4-6 degrees C was higher than at 24 degrees C. For the most active strains, the degree of oil degradation in liquid mineral medium ranged from 15 to 26% at 24 degrees C and from 28 to 47% at 4-6 degrees C. An artificial association of six of the strains degraded the oil by 46% at 24 degrees C.The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0–10.0). When studied by SDS-PAGE in the presence of β-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30–60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.