Hypersensitive Death Research Articles

Effect of sulfhydryl-binding compounds on hypersensitive death of potato tuber cells following infection with an incompatible race of Phytophthora infestans

Pre-infectional treatments of aged discs of potato tubers with the low molecular weight sulfhydryl compounds (SH-reagents) 2,4-dinitrophenol, 2,4-dinitrofluorobenzene, N-ethylmaleimide and p-chloromercuribenzoic acid (PCMB) and the high molecular weight SH-reagent, dextran-bound p-chloromercuribenzoic acid (PMDT), inhibited the rapid hyper-sensitive death of potato tuber cells following infection with an incompatible race of Phytophthora infestans. Cell browning was also inhibited by pre-infectional treatment with all the SH-reagents tested. If the infected discs were treated 6 h after inoculation, when most of the infected cells had died, PCMB inhibited browning but PMDT did not, which suggests that PMDT did not penetrate the tissue. The results suggest that metabolic activity may be involved in the hypersensitive death of potato cells challenged by an incompatible race of P. infestans and that some SH-reagents may inhibit such cell death by acting directly on cell membranes.

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

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.

Alteration of membrane permeability of potato-tuber tissue infected by incompatible and compatible races of Phytophthora infestans during the initial phase of infection.

Electric potential difference (EPD) between the surfaces of potato-tuber disks infected by compatible and incompatible races of Phytophthora infestans and between each of them and noninfected ones were determined during the initial period of infection. EPD began to increase almost simultaneously with host wall penetration by both the incompatible and compatible races and the increase was already larger in the former than in the latter. Electric conductivity (EC) measurements of the exudates from the infected tuber disks showed that an increase in EC was also found in both the compatible and incompatible infections. The increase in EC was larger in the incompatible combination before hypersensitive death of infected cells occurred. In both incompatible and compatible combinations the increasing rate in EC was larger when the disks were inoculated 20hr after cutting rather than just after cutting. Leakage of preabsorbed 32P from potato-tuber disks was increased by infection with both the incompatible and compatible races, and the increase was higher in the former than in the latter. All these results suggested that the permeability of cell membranes of potato tuber disks increased very soon after the infection (or at the same time as penetration occurred) when they were inoculated with either incompatible or compatible races of P. infestans. However, the rate of increase seemed to be larger in the incompatible than in the compatible combination before hypersensitive cell death of the former occurred.

The ultrastructure of M1-a-mediated resistance to powdery mildew infection in barley

M1-a-mediated resistance in barley to invasion by the CR3 race of Erysiphe graminis f. sp. hordei does not occur in every host cell with the same speed and severity. In some cells ultrastructural changes within the host cell as a result of resistance will occur within 24 h after inoculation, whereas in other cells these changes may take up to 72 h. In some cells the ultrastructural changes are so drastic that they give the appearance of a hypersensitive death of the host cell, whereas in other cells the changes are very slight. In any case, at the end of these changes the fungus ceases growth. The ultrastructural changes occur in penetrated host epidermal cells as well as non-infected adjacent epidermal and mesophyll cells.The following ultrastructural changes have been observed: (1) an electron-dense material which occurs either free in the vacuole or adhering to the tonoplast (the material is granular or in large clumps); (2) an increased electron density of the host cytoplasm and nucleus; (3) a breakdown of the tonoplast so that the cytoplasmic constituents become dispersed throughout the cell lumen; and (4) the deposition of papillar-like material in areas other than the penetration site. The first three changes take place within the host cell protoplasts and are directly attributable to the gene M1-a. These changes are typical of stress or incompatibility responses and thus M1-a appears to trigger a generalized incompatibility response in the presence of race CR3. The papillar-like material occurs outside the host cell protoplast in the same manner as the papilla and probably is not directly attributable to M1-a.

Prevention of the hypersensitive reaction of potato cells to infection with an incompatible race of Phytophthora infestans by constituents of the zoospores

The hypersensitive reaction induced in potato tuber or petiole tissue by infection by an incompatible race was prevented or delayed by prior treatment with a high molecular weight fraction obtained from supernatant of zoospore homogenates of Phytophthora infestans. Experiments were carried out with the potato varieties Rishiri (resistant gene R 1), Eniwa (resistant gene R 1), Pentland Ace (resistant gene R 3) and two races of P. infestans, race 0 and race 1. Browning of tissues induced by infection by the incompatible race was reduced by prior treatment with the high molecular weight fraction which was obtained in a void volume of a Sephadex G-25 column. The proportion of unbrowned cells infected by the incompatible fungus increased with increasing concentrations of the fraction. Hypersensitive death of cells treated with the fraction was prevented without significant effects on either the penetration of the host cells by an incompatible race of the fungus or growth of the intracellular hyphae at an early stage of infection. These protective effects of the high molecular weight fraction of zoospore homogenates seemed in part to be host specific for the cultivars and races used.

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

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/10 6 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/10 6 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 rôle in the processes leading to hypersensitive death of potato cells caused by infection with an incompatible race of P. infestans.

Resistance to anthracnose of French bean

Resistance of Phaseolus vulgaris L. to Colletotrichum lindemuthianum Sacc. et Magn. Bri. et Cav., the cause of anthracnose, was studied physiologically and by light and electron microscopy of a number of combinations of host cultivar and pathogen race giving reactions ranging from highly susceptible to highly resistant. Hypersensitive death of cells appeared to be the most important cause of resistance. Granulation of walls of killed cells did not extend beyond the middle lamella, and the pits between killed and adjacent living cells became occluded. The cytoplasm of these living cells differed in a number of ways from that of corresponding cells in uninfected plants; in particular, the volume of cytoplasm was increased about threefold, nuclei were convolated and chlorophyll was not removable by the usual solvents. There was no evidence to suggest that the capacity to respond in a hypersensitive manner was confined to specific cells in a large group of visually similar cells. Epidermal cells of different parts of the same plant or of the same plant structure such as a leaf may respond differently to infection. Cells of susceptible cultivars occasionally responded in a manner very similar to the hypertensive response characteristic of resistant cultivars. Filtrates from cultures of the pathogen also caused changes in fine structure that resembled those of hypersensitivity; activity of filtrates was non-specific. Reaction material sometimes appeared in the lumen of cells as an early response to penetration; its amount, type and its significance in resistance depended on the host-pathogen combination. Outer walls of epidermal cells appear to be significant only in the resistance of older hypocotyls in which a zone of cortical sclerenchyma may also be important in this context. The rôle in resistance of hypersensitive responses, reaction material and cell walls is discussed.

Role of Underlying Healthy Tissue in the Hypersensitive Death of a Potato Plant Cell Infected by an Incompatible Race of Phytophthora infestans

One side of potato tuber slices having different thickness was inoculated with such dense zoospore suspensions of an incompatible race of Phytophthora infestans as to infect almost all of the surface cells. The less the number of layers of underlying healthy cells, the more prolonged the time taken for the infected cells to die hypersensitively. Browning of the infected cells occurred more rapidly in thick slices than in thin slices. These results suggested that healthy cells underlying the infected cells play an important role in the hypersensitive death of the infected cells and also supply substances necessary to localize the infection.

Effect of Temperature on the Process of the Hypersensitive Death of a Potato Plant Cell Infected by an Incompatible Race of Phytophthora infestans

The process of hypersensitive death of a potato cell infected by an incompatible race of Phytophthora infestans was observed by using a temperature controlled, inverted microscope. More precise observation than in previous experiments disclosed that the process of hypersensitive death of the cells consisted of at least two phases: cessation of protoplasmic streaming and then loss of stainability of vacuole with neutral red. The infected cell still maintained stainability with neutral red and could absorb it after the protoplasmic streaming stopped. After a while, the cell lost the stainability with neutral red. The effect of temperature on this process was investigated. The time elapsed from inoculation to the penetration was longer at 20C than at 25 and 30C.By extraporating the growth curve of the intracellular hyphae to O, length of the penetration was judged to finish 10-15min before the hyphae become actually visible, so that the time required for hypersensitive cell death is longer than that estimated in prlor experiments.In 10-20% of the cut surface cells inoculated 18hr after cutting, protoplasmic streaming stopped about 30min after penetration (at 20, 25 and 30C) and in 50% of the cells protoplasmic streaming stopped about 37min (20C) 40min (25C) and 45min (30C) after penetration. The cells seemed to lose their stainability with neutral red about 30min (20C), 10min (25C) and 10min (30C) after protoplasmic streaming stopped.

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

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.

The influence of a pre-infectional heat treatment on the resistance of potato plant to Phytophthora infestans.

1. Tissues of a potato petiole and slices of potato tuber were heated at 45°-53.5°C for five minutes, and then inoculated with Phytophthora infestans. The resistance of them to P. infestans was reduced remakably.2. No influence of the heat-treatment on the time required for penetration of the fungus into the host cell, nor on the growth rate of the intracellular hypae was found by microscopical observations on the cortex cells of the petioles.3. The necrogenous reaction of potato-plant tissue to infection by incompatible race of Phytophthora infestans was delayed remarkably by pre-infectional heat-treatment. The delay of the hypersensitive death of an infected cell was ascertained by microscopical observation on the cortex cells of the petioles.4. The appearance of protoplasmic strands, in the cut-surface cell of a petiole was reduced remarkably by the heat-treatment.5. The R.Q. of the potato tuber slice was not affected by the heat-treatment, but the respiratory rate was depressed remarkably by the same treatment. The rate of respiratory depression seems to go along with that of the reduction of resistance.6. Basis was found for the supposition that the metabolic activity in the cell is closely associated with the hyper-sensitive death of infected cell.