[Elicitor activity of chitosan and arachidonic acid: their similarity and distinction].

Two elicitors-chitosan and arachidonic acid-induced the same defense responses in potatoes, stimulating the processes of wound reparation and inducing the formation of phytoalexins, inhibitors of proteinase, and active forms of oxygen. However, chitosan induced the defense potential of plant tissues at concentrations higher than those of arachidonic acid. The protective action of chitosan was defined by two parameters, i.e., the ability to induce the immune responses in plant tissues and to exhibit a toxic effect on the pathogen development, causing late blight and seedling blight, whereas the elicitor effect of arachidonic acid depended on its ability to induce the defense potential of plant tissues only.

[Chymotrypsin and trypsin inhibitor isolated from potato tubers].

Potato Kunitz-type chymotrypsin inhibitor (PKCI-23) was isolated from potato tubers (Solanum tuberosum L., Zhukov's Jubilee breed) and purified to a homogenous state. The protein was purified by gel-filtration chromatography and ion-exchange chromatography using Sephadex G-75 and CM-Sepharose CL-6B, respectively. PKCI-23 protein has been shown to inhibit both chymotrypsin and trypsin with equal efficacy, forming equimolar complexes with these enzymes. However, much weaker inhibitory effect of PKCI-23 has been observed for Carlsberg subtilisin. The N-terminal 20 amino acid sequence of PKCI-23 has been sequenced. PKCI-23 has been shown to suppress, with different efficacy, the growth and development of pathogenic microorganisms Fusarium culmorum (Wm. G. Sm.) Sacc. and Phytophtora infestans (Mont.) de Bary that infect potato.

[Immunomodulating activity of chitosan derivatives with salicylic acid and its fragments].

A study of biological activity of the derivatives of the chitin-chitosan oligomer with salicylic acid and its fragments showed that chitosan salicylate actively protected potato tubers against Phytophthora infestans but sharply inhibited reparation of potato tissues. N-(2-Hydroxybenzyl)chitosan exhibited good protective properties but did not influence wound reparation. N-(2-Hydroxy-3-methoxybenzyl)-N-pyridox-chitosan, which contained the pyridoxal and 2-hydroxy-3-methoxy fragments, was the most efficient, stimulating both defense against late blight and wound reparation in potato tissues.

Protein trypsin inhibitor from potato tubers.

A protein of 22 kDa designated as PKTI-22 was isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and purified to homogeneity using CM-Sepharose CL-6B ion-exchange chromatography. The protein efficiently suppressed the activity of trypsin, affected chymotrypsin less, and did not affect subtilisin Carlsberg. The N-terminal sequence of PKTI-22 (20 amino acid residues) was found to be highly homologous with the amino acid sequences of the potato Kunitz-type proteinase inhibitors of group B (PKPI-B) that were aligned from the corresponding gene sequences and was identical to the sequence (from the 2nd to the 20th residue) of the recombinant protein PKPI-B10. These data together with the observed similarity of the properties of two proteins indicate that the PKTI-22 protein is encoded by the PKPI-B10 gene.

[The inhibition of potato tubers wound reparation].

The multiple washing of the wound surface of potato tubers by water adversely affected the protective properties of wound periderm. Immune inhibitor beta-1,3-beta-1,6 glucan had a property of local effect and inhibited the process of wound healing. The pentasaccharide of xyloglucan caused necrosis of potato tuber tissue and prevented the wound reparation process.

[Wound healing and induced resistance in potato tubers].

It was demonstrated that biogenic elicitors, arachidonic acid and chitosan, locally and systemically stimulated wound healing in potato tuber tissues by increasing the number of wound periderm layers, accelerating the development of cork cambium (phellogen), and inducing proteinase inhibitors. The signal molecules, jasmonic and salicylic acids, had different effects on the development of wound periderm: jasmonic acid locally and systemically stimulated potato wound healing and elevated the level of proteinase inhibitors, whereas salicylic acid did not have any effect on wound healing and even blocked the formation of proteinase inhibitors.

[Activation of elicitor defensive properties by systemic signal molecules during the interaction between potato and Phytophthora].

The elicitor arachidonic acid in combination with jasmonic acid (JA) induced a higher level of defense against the late blight agent in potato (Solanum tuberosum L.) tissues than in combination with salicylic acid (SA). On the contrary, the elicitor chitosan displayed a higher inductive effect in combination with SA as compared with JA. The optimal concentrations of tested compounds were selected for designing the compositions activating wound repair, induction of proteinase inhibitors, and resistance to the biotrophic pathogen Phytophthora infestans (Mont.) de Bary. It was demonstrated that the compositions of elicitor and systemic signal molecules provided a faster spreading of an inducing effect in the potato tissues.

[Effect of proteinaceous proteinase inhibitors from potato tubers on the growth and development of phytopathogenic microorganisms].

We studied the effect of two proteins, PSPI-21 and PKSI, on the growth and development of phytopathogenic microorganisms (Phytophthora infestans oomycete and Fusarium culmorum fungus). Both proteins were isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and served as inhibitors of serine proteinases. These proteins differed in the ability to inhibit growth of Phytophthora infestans oomycete and Fusarium culmorum fungus. PSPI-21 was the most potent in modulating the growth of oomycete mycelium. PKSI primarily affected the growth of the fungal mycelium. The proteins under study induced complete destruction of oomycete zoospores and partial destruction of fungal macroconidia. Our results suggest that these proteins are involved in the protection of potato plants from phytopathogenic microorganisms.

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.

[Role of L-phenylalanine ammonia lyase in the induced resistance and susceptibility of potato plants].

Biogenic elicitors (chitosan and its complex with salicylic acid) and an immunosuppressor (laminarin) were shown to increase the activity of L-phenylalanine ammonia lyase (EC 4.3.1.5) and protein synthesis in potato tubers. Laminarin did not decrease L-phenylalanine ammonia lyase activity. It is unlikely that the activity of this enzyme may serve as a criterion for the induced resistance.

[Exoproteinases of the oomycete Phytophthora infestans]. / Ekzoproteinazy oomitseta Phytophthora infestans.

When grown in a medium containing heat-stable potato tuber proteins, the oomycete Phytophthora infestans (Mont.) de Bary produces a set of exoproteinases active at neutral and mildly basic pH values. These extracellular proteinases have been shown by SDS-PAGE with the presence of gelatin to include at least six components differing in molecular weight. Inhibitory analysis and study of the effects of the enzymes on various synthetic substrates show that the culture liquid of P. infestans contains mainly serine proteinases specific to trypsin and subtilisin and metalloproteinases. Their activity is suppressed by proteinase-inhibitor proteins from potato tubers. It is suggested that P. infestans exoproteinases may be the metabolic target for natural proteinase inhibitors from potato.

[Effect of systemic signaling molecules on the rate of spread of the immunizing effect of elicitors over potato tissues]. / Vozdeistvie sistemnykh signal'nykh molekul na skorost' rasprostraneniia po tkaniam kartofelia immuniziruiushchego éffekta élisitorov.

Mobile systemic signaling molecules (salicylic and jasmonic acids) enhance and accelerate the spread of systemic immunizing effect of elicitors (arachidonic acid and chitosan) over potato tuber tissues (Solanum tuberosum L.).

[Role of proteinase inhibitors in potato protection]. / Rol' ingibitorov proteinaz v zashchite kartofelia.

Mechanical damage 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 the lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.

[Regulation of potato immune responses by laminarin]. / Reguliatsiia immunnykh otvetov kartofelia laminarinom.

Laminarin blocks potato immune responses by inhibiting the reaction of oversensitivity, formation of phytoalexins, wound repair, and the activity of proteinase inhibitors. It was found that laminarin exhibits antielicitor activity. Addition of salicylic acid to laminarin enhances its immunosuppressing effect, which becomes systemic.

[Induced phytophthora resistance in transgenic potato tubers]. / Indutsirovanie fitoftoroustoichivosti u klubnei transgennogo kartofelia.

Resistance of transgenic cultivars based on the expression of one or more resistance genes is sooner or later broken by pathogens whose race-producing rates are high. Thus, combining transgenesis with elicitor-induced resistance is a promising approach. The elicitor-induced resistance is based on the expression of multiple resistance genes, which can prevent the adaptation of pathogens to transgenic races, maintain the stability of cultivars, and increase their lifespan. In this work, we used transgenic potato cultivars Temp and Superior transformed with Bacillus thuringiensis delta-endotoxin gene and Lukyanovskii transformed with leukocyte alpha-interferon gene. Arachidonic acid (10(-8) M) and soluble chitosan (5 kDa, 100 micrograms/ml) were used as elicitors for tuber treatment. Our data showed that pretreatment with elicitors causes a 15-25% increase in both the systemic prolonged resistance of potato tubers to Phytophthora infestans and their ability to repair mechanical damage.

[Effect of elicitors on accumulation of protease inhibitors in injured potato tubers]. / Vliinie élisitorov na nakoplenie ingibitorov proteinaz v poranennykh klubniakh kartofelia.

The time course of accumulation and the composition of proteinase-inhibiting proteins in diffusates from potato tubers treated with elicitors such as salicylic, jasmonic, and arachidonic acids were studied. The 40-kDa reserve protein patatin and the chymotrypsin inhibitors, among which proteins of 24.6, 22.0, and 16.0 kDa were prevalent, accumulated in diffusates from potato tubers. Jasmonic and arachidonic acids activated the accumulation of the chymotrypsin inhibitors in tubers in response to the injury stress, whereas salicylic acid inhibited this process. The effects of jasmonic and arachidonic acids increased when their concentrations decreased to 10(-6) M. The data suggest an important role of the lipoxygenase metabolism in signal transduction of the anti-injury defense system in the dormant potato tubers.