Kohei Tomiyama

Effect of hyphal wall components from Phytophthora infestans on protoplasts of potato tuber tissues

Abstract Effect of hyphal wall components of various races of Phytophthora infestans on protoplasts prepared from tuber tissues of potato cultivars having various resistance genes to the pathogen was investigated to know the interactions with surfaces of host cell membrane. Reaction of protoplasts to the components was characterized by rapid protoplasmic aggregation followed by exolysis of the aggregate leaving a ghost of spherical membrane behind. There was no significant difference in the physiological activities of the components from 7 races of the fungus. Significant differences in the reactivity of protoplasts from different cultivars used was found regardless of the resistance genes of the cultivars. There was a linear correlation between % of reacted protoplasts and concentration of fungal components (expressed as log) used for the treatments in each reaction system. The higher the field resistance of the cultivars used, the more reactive to components were the protoplasts prepared from them. Nearly 1 μg ml−1 of components was enough to initiate the reaction of protoplasts and more than 125 μg ml−1 elicited the reaction at maximum rate within 5 to 10 min after application. These phenomena suggested a possibility that the recognition sites of the protoplasts for the fungal components may reside in the protoplasmic membrane, resulting in occurrence of a hypersensitive reaction in potato cells. These results are discussed in connection with the host-parasite interaction in potato late blight.

Suppression of the hypersensitive response of potato tuber protoplasts to hyphal wall components by water soluble glucans isolated from Phytophthora infestans

Abstract The suppressive effect of water soluble glucans isolated from Phytophthora infestans on the hypersensitive response of potato tuber-protoplasts was investigated qualitatively and quantitatively in relation to host-parasite specificity in potato late blight. Protoplasts were prepared from nine cultivars having different resistance genes (r, R1, R3, R4 R1, R2, R1, R3, R1, R4, R2, R3 and R2, R4), and glucans were isolated from 7 races (0, 1, 3, 4, 1.2, 1.4, and 1.2.3.4). Each glucan itself did not cause any visible effect on the protoplasts. Pretreatment of protoplasts with glucans from each race for 15 min suppressed the hypersensitive reaction of protoplasts elicited by hyphal wall components isolated from P. infestans. Greater suppressive activity of the glucans was partly characteristic of the compatible relationships between protoplasts and races used as a source of glucans. There was a linear correlation between the suppressive effect of glucans and the log concentration of glucans applied. Each glucan showed some different activity in suppressing the hypersensitive response of protoplasts among compatible cultivars. These results suggested that glucans from P. infestans may be a determinant of host-parasite specificity in potato late blight as a suppressor of the effects of hyphal wall components on elicitation of the hypersensitive reaction.

The role of rishitin in localizing the invading hyphae of Phytophthora infestans in infection sites at the cut surfaces of potato tubers

Abstract After inoculating the cut surfaces of tubers and leaf petioles of potatoes with an incompatible race of Phytophthora infestans, the time that elapsed before hypersensitive cell death and initiation of rishitin formation was measured. Rishitin was produced earlier in tuber tissue inoculated 24 h after cutting than in tissue inoculated just after cutting. However, the time that elapsed from hypersensitive death of 20% of the infected cells to initiation of rishitin formation was 8 to 9 h in both types of tissues. When leaf petioles were held in air for 15 h after cutting and then inoculated with the incompatible race, the time from 20% cell death to rishitin formation was about 5·5 h. In petiolar tissue, rishitin was first detected when the growth rate of the intracellular hyphae was about to decrease. In the case of the cut and air-exposed tuber tissue infected by the incompatible race, rishitin reached a concentration of 100 μg/g fresh weight at a time when development of the lesion was ceasing (calculated assuming rishitin accumulated locally in the upper third of the slices used for chemical analysis). This concentration was sufficient to inhibit hyphal growth in vitro. Rishitin seems therefore to play an important role in the inhibition of hyphal growth and lesion development.

Effect of blasticidin S on development of potential of potato tuber cells to react hypersensitively to infection by Phytophthora infestans

Abstract It was reported previously that the hypersensitive death of surface cells of discs infected by the incompatible race 0 of Phytophthora infestans occurred late, when inoculated soon after preparation (cutting) of the discs. When other discs were inoculated 17 to 20 h or more after cutting, on the contrary, hypersensitive cell death occurred very quickly. These results indicated that the cells at the cut surface lack the ability to undergo immediate hypersensitivity, but they acquire this ability by 20 h after cutting. When tuber discs were treated with 10 parts/10 6 of blasticidin S (BcS) for 3 h beginning 3 h after cutting, the cells did not acquire the potential to react hypersensitively. This indicated that de novo protein synthesis was necessary for cells of the cut surface of tubers to acquire this potential. On the contrary, treatment with BcS had no effect on the potential, when the treatment was done after the potential had been allowed to build up after cutting. However, hypersensitive cell death in tissues which were treated with BcS 20 h after cutting was delayed when inoculation with incompatible race 0 was made 14 h after BcS treatment. Potato cells infected by compatible race 1 survived for a long time. In this case, too, cell death was greatly delayed by treatment with BcS, resulting in an increase in aerial mycelium and spores production. This suggested that de novo protein synthesis is not necessary for the establishment of a compatible relation between the host and the parasite.

Effect of infection by Phytophthora infestans on the membrane potential of potato cells

The membrane potential (Ψm) of potato cells (cultivar Rishiri) infected by incompatible and compatible races of Phytophthora infestans was measured, while the infection process of the measured cells was observed under a microscope. The passive (Ψd) and respiration dependent electrogenic (Ψp) components of Ψm were also analysed using the anoxia method. Cytoplasm often gathered around the inserted microelectrode and sometimes disturbed the measurement. However, it was shown that the experimental system was suitable for the long-term measurement of fm during the infection process. At the moment of penetration of the host cell wall by both the incompatible and compatible races, no signal such as an action potential was observed in the Tm. Infection by the incompatible race caused little change of Tm for a while after penetration, but |Ψd| then began to decrease. |ΨP| did not decrease, but rather increased and apparently compensated for the decrease in |Ψd|, resulting in little or slow reduction of |Ψm|. Later |Ψp| also decreased and Ψm began to depolarize gradually and at about one to several hours after penetration, rapid depolarization took place to reach a steady level of −45 to −55 mV before the cell content became granulated. In some instances, slow and steady depolarization was followed by cell death. Ψd and Ψp of the uninfected cell did not change for more than 10 h after the beginning of the measurement. The compatible race had no effect on either Ψp or Ψd at least within 24 h infection.

Effect of blasticidin S on hypersensitive death of potato leaf petiole cells caused by infection with an incompatible race of Phytophthora infestans

Abstract The effect of an inhibitor of protein synthesis, blasticidin S, on the infection process of an incompatible race of Phytophthora infestans was studied in cut surface cells of potato leaf petiole tissue. Ten parts/106 of blasticidin S applied to the host tissue 0·5, 1·0 and 1·5 h before inoculation did not affect the time required for penetration of the host cell wall by the parasite and the growth rate of intracellular hyphae. The time elapsing from penetration to the hypersensitive death of the infected host cell was not affected by treating the potato cells with 10 parts/106 of blasticidin S 1·5 h before inoculation. Incorporation of 3H-leucine into the acid insoluble fraction of potato petiole tissue showed that protein synthesis was strongly inhibited by the blasticidin S treatment. These results suggested that de novo protein synthesis does not play an important role in the processes leading to hypersensitive death of potato cells caused by infection with an incompatible race of P. infestans.

The rôle of potato lectin in the binding of germ tubes of Phytophthora infestans to potato cell membrane

Abstract Germinated cystospores of incompatible and compatible races of Phytophthora infestans were mixed with the cell membrane-rich fraction isolated from tubers of potato cv. Rishiri (R1 gene) in 0·02 M Tris-MES buffer at pH 7·2 in vitro. Electron microscopic observation showed that the cell membrane vesicles of the host cells were bound to hyphal surfaces of both the incompatible and compatible races, when potato lectin was added to the mixture. The binding phenomenon by potato lectin was inhibited when N, N′-diacetylchitobiow, a specific hapten of potato lectin, was added to the mixture. The binding did not occur in the absence of lectin. There was no soecificity in the activity of lectins isolated from cv. Rishiri and Irish Cobbler (r gene) to bind the cell membrane vesicles to the surface of the races of P. infestans. These results suggested that the binding of cell membranes of potato to cell wall surfaces of infecting hyphae of compatible and incompatible races of P. infestans in vivo is mediated by potato lectin. Rishiri and Irish Cobbler lectins caused agglutination of germinated cystospores of both race 0 and race 1, 2 of P. infestans to the same degree. This agglutination of germinated cystospores caused by lectin was inhibited by N, N′-diacetylchitobiose. Concanavalin A (Con A) also agglutinated the germinated cystospores of both races to the same degree. This agglutination was inhibited by α-methyl- d -mannoside and α-methyl- d -glucoside, the specific hapten of Con A. This indicates that both compatible and incompatible races of P. infestans have almost the same amounts and/or locations of binding sites for the lectins on the cell wall surfaces.

Evidence for adherence of host plasmalemma to infecting hyphae of both compatible and incompatible races of Phytophthora infestans

Abstract Aged discs of potato tuber were inoculated with incompatible race 0 and compatible race 1 of Phytophthora infestans , and then plasmolysed with hypertonic sucrose solution. Both the plasmolysed cells infected by the incompatible and compatible races showed conically shaped protoplasts, parts of which adhered to the intracellular hyphae. We named this phenomenon “tent-type plasmolysis”. Electron microscopic observation showed that many fragments of host plasmalemma adhered to the infecting hyphae, when the host plasmalemma appeared to be detached from the hyphae upon plasmolysis. These fragments adhering to the infecting hyphae seemed to be torn off from the host plasmalemma which was separated from the hyphae by plasmolysis. All these results suggested that the host plasmalemma strongly adhered to infecting hyphae of both compatible and incompatible races from the very earliest time of penetration. Tent-type plasmolyses in infected cells were also observed with petioles of potato leaves.

Effect of N, N′-diacetyl-D-chitobiose, the potato-lectin hapten and other sugars on hypersensitive reaction of potato tuber cells infected by incompatible and compatible races of Phytophthora infestans

Abstract Cut surfaces of aged disks of potato tubers were treated with 30 kinds of sugars including 3 pentoses, 8 hexoses, 6 disaccharides, 3 methyl glucosides, 3 sugar alcohols, 6 amino sugars and laminarin, and then inoculated with incompatible or compatible races of Phytophthora infestans . Among these sugars, only N, N′-diacetyl- D -chitobiose (D-chitobiose) inhibited greatly the rapid occurrence of hypersensitive death of cells infected by an incompatible race of P. infestans , when it was pre-infectionally applied to the disks for 1 h. Post-infectional treatment with D-chitobiose done 2 h after inoculation, however, had little effect on the hypersensitive cell death. D-chitobiose had little effect on the response of the potato cells to infection by a compatible race and also on the growth of intracellular hyphae. The cells, which were pre-infectionally treated with D-chitobiose and then inoculated by the incompatible race, were plasmolyzed with 0·7 m sucrose solution, and then fixed and observed using an electron microscope. The observation showed that the percentages of the infecting hyphae, to which the host plasmalemma or vesicles torn off from the plasmalemma did not adhere or adhered only a little, was 33· 3% (for 219 hyphae observed) or 4·1% (for 171 hyphae) in the cells treated with D-chitobiose or water respectively. All these results suggested that exogenously supplied D-chitobiose inhibits the binding between host plasmalemma and infecting hyphae during the period of initial contact between them, and also inhibits rapid occurrence of the hypersensitive cell death. Since D-chitobiose has been known as a specific hapten of potato-lectin, the results suggested that potato-lectin may play a role in these phenomena.

Effects of hyphal wall components of Phytophthora infestans on membrane potential of potato tuber cells

Abstract The effect of the hyphal wall component (HWC) of Phytophthora infestans on the membrane potentials (Ψm) of cells in the aged cut surface of potato tuber tissue (cv. Rishiri) were investigated. Within a few minutes of the application of HWC (3 mg ml−1), cellular depolarization occurred. Initially, depolarization was rapid. After a mean depolarization of about 25 mV, the Ψm reached an asymptotic plateau. HWC reduced the oxygen dependent electrogenic potential difference (∣Ψd∣) by about 60%, but exerted little effect on the passive potential difference (∣Ψd∣). In a later stage, ∣Ψd∣ also decreased very gradually. The extent of the HWC effect was not always the same, possibly owing to the heterogeneity of potato cells in their reactivity or accessibility to HWC. There was no difference in the activity on Ψm of HWC isolated from compatible or incompatible races. Results showed that the effect of HWC on the membrane potential of potato cells was different from that of infection by P. infestans.