A.Frank Ross

Systemic acquired resistance induced by localized virus infections in plants

Abstract Inoculation of half-leaves of Nicotiana tabacum var. Samsun NN, with tobacco mosaic (TMV) virus induced a high level of resistance to TMV in the opposite half-leaves. Challenge inoculation with TMV of the resistant half-leaves 7 days after the first inoculation resulted in limited lesion formation; the lesions were consistently only one-fifth to one-third as large (in diameter) as were lesions in susceptible half-leaves, and they usually were fewer in number. Similarly, inoculation of lower leaves on a plant induced resistance in upper leaves and vice versa. Resistance was first detected in 2–3 days, reached a maximum in about 7 days, and persisted for at least 20 days. The uninoculated resistant leaves or half-leaves proved to be free of virus. The highest level of resistance developed when plants were kept at 20°C before and after the challenge inoculation. Resistance could be detected in plants kept at 29° after challenge. Complete protection was not obtained by increasing the ratio of the area first inoculated to that challenged. Resistance was induced by mixed infections by TMV and potato virus X (PVX), but leaves highly resistant to TMV alone were only slightly resistant when challenged with TMV plus PVX. Resistance was induced by TMV infection in detached leaves, in excised shoots, and in plants kept in the dark 2 days before challenge inoculation. No resistance was induced by mechanical or chemical injury or by viruses that do not cause necrosis. The resistance induced by TMV is not specific for TMV. Leaves with TMV-induced resistance were resistant not only to TMV but also to tobacco necrosis virus, to turnip mosaic virus, and to tobacco and tomato ringspot viruses. The resistance was not as effective against these viruses as against TMV, but each of these local-lesion viruses induced a resistance in Samsun NN tobacco indistinguishable from that induced by TMV.

Localized acquired resistance to plant virus infection in hypersensitive hosts

Abstract A high degree of resistance to tobacco mosaic virus (TMV) developed in a 1- to 2-mm zone surrounding each TMV-induced local lesion on the hypersensitive host, Nicotiana tabacum L. var. Samsun NN. No lesions, or at most a few tiny ones, developed in this area following subsequent challenge inoculation with TMV. The resistant zones increased in size and in degree of protection for about 6 days, a period coinciding with that during which the original lesion was enlarging. Development of the resistant zone was not affected by relative humidity or by exclusion of light before or after challenge. Resistance was not detectable at temperatures near 30°C nor in leaves also infected by potato virus X (PVX). The resistance was not virus specific; zones around a TMV lesion were also resistant to tobacco necrosis virus (TNcV), tobacco ringspot virus, and tomato ringspot virus, but not to turnip mosaic virus. TNcV, but not turnip mosaic virus, induced localized resistance to TMV. Areas around TMV or TNcV lesions are resistant to TNcV in leaves also infected by PVX. The results are interpreted as indicating that the resistance developing in cells beyond those infected eventually acts as a barrier to further cell-to-cell spread of the virus, thus localizing the virus in the lesion.

Virus multiplication in plants doubly infected by potato viruses X and Y.

Abstract The ratio of virus X in juice from tobacco plants simultaneously inoculated with potato viruses X and Y to that in juice from comparable plants inoculated only with virus X varied with the stage of the disease. Ratios up to 10:1 were obtained with leaves invaded during expansion. With leaves that expanded after the growing tip had been invaded, increases up to threefold were obtained; with inoculated leaves increases were usually twofold. There was a direct relationship between severity of symptoms and virus X concentration of doubly infected plants. In leaves invaded during expansion, the relative increase in virus X concentration of XY-infected leaves over that of X-infected leaves was greater in veinal tissue than in interveinal tissue. With singly infected leaves the concentration of virus X in juice from interveinal tissue was about six times that in juice from veinal tissue, whereas with XY-infected leaves juice from interveinal tissue contained about twice as much virus X as did juice from veinal tissue. When X-infected plants were inoculated with virus Y, there occurred increases in virus X similar to those observed in plants inoculated simultaneously with the two viruses. When Y-infected plants were inoculated with virus X, large relative increases of virus X did not occur and the usual severe symptoms did not develop. The magnitude of the increase in the concentration of virus X in juice from XY-infected leaves over that in juice from X-infected leaves varied considerably among experiments. Tests indicated that this variation was not caused by changes in either virus or in nutrition of the host plants. The variation was seasonal, with the greatest ratios occurring in the spring when greenhouse temperatures favor multiplication of virus Y during the day and that of virus X during the night. In both singly (Y) and doubly (X + Y) infected leaves, the concentration of virus Y in interveinal tissue was about twice that in veinal tissue. Neither the concentration nor distribution of virus Y was affected by the presence of virus X.

Enhancement of potato virus X synthesis in doubly infected tobacco occurs in doubly infected cells

Abstract Enhancement of potato virus X (PVX) synthesis by potato virus Y (PVY) or tobacco mosaic virus (TMV) in tobacco is the result of increased synthesis per cell, not an increase in the number of cells infected. Ultrastructural features typical of PVX-infected cells and those typical of PVY-infected cells were present together in a majority of cells examined by electron microscopy from leaves supporting enhancement. PVX was detected in over 70% of protoplasts prepared from infected leaves and stained with fluorescein-labeled PVX antibody; the proportion was the same for cells from doubly infected and singly infected leaves. In leaves infected with TMV and PVX, 70% of the cells were doubly infected. The fluorescence intensity of individual cells after treatment with fluorescein-labeled PVX antibody, a measure of relative virus concentration, was greater in cells from leaves doubly infected by PVX and PVY than in cells from leaves infected by PVX alone.

A marked synergistic interaction of potato viruses X and Y in inoculated leaves of tobacco

Abstract When the outer half of a half-leaf of Turkish tobacco (Nicotiana tabacum L.) was inoculated with potato virus X (PVX) and a similar area of the opposite half-leaf was inoculated with potato virus Y (PVY) on the same day, severe necrosis and stimulated PVX synthesis occurred along the midvein in the uninoculated area adjacent to the strip inoculated with PVY. When the inoculation with PVX was made at different intervals prior to that with PVY, the necrosis appeared progressively farther from the PVX-inoculated area as the interval between the inoculations was increased. Increasing the interval between inoculations made in the reverse sequence resulted in the necrosis appearing progressively farther from the PVY-inoculated area. Inoculation of entire tobacco leaves with a 1:1 mixture of juice from PVX-infected tobacco plants and juice from PVY-infected tobacco plants resulted in only mild synergism. When the concentration of PVY was held constant and the concentration of PVX was reduced by tenfold steps to 0.001 of that in the original mixture, symptom severity increased progressively until more than 80% of the leaf became necrotic. Despite the increase in symptom severity, juice from those leaves with the most severe symptoms contained no more than 2.5 times as much PVX as did juice from comparable leaves inoculated with the corresponding concentration of PVX without PVY. Moderate synergism in symptomatology resulted when tobacco leaves systemically invaded by PVY were inoculated with PVX, but little or no synergism occurred when PVX was the systemic virus and PVY the one applied directly. The data are interpreted as indicating that the prime requisite for the strong PVX-PVY interaction is that a cell must be invaded first by PVY and then by PVX while PVY is still undergoing active multiplication. It is suggested that infection by PVX normally induces reactions that act to limit PVX synthesis but is unable to do so in a cell in which PVY is actively multiplying.

The relationship of ethylene to formation of tobacco mosaic virus lesions in hypersensitive responding tobacco leaves with and without induced resistance

Abstract This study has investigated the possible roles of ethylene in the localization of tobacco mossaic virus (TMV) in local lesions in hypersensitive responding tobacco and also in the early cessation of lesion growth in tobacco having the type of acquired resistance that develops in leaves far removed from the primary local infections. In resistant and nonresistant tissues, the patterns of ethylene synthesis were similar except that the infection-stimulated synthesis began earlier and was quantitatively less in resistant tissues. Ethylene synthesis increased about 3 hr before lesions were visually perceptible in nonresistant leaves and 8 hr before lesion appearance in resistant leaves. In both cases, ethylene synthesis reached a maximum (about 21 nl/100 cm 2 /hr at 4 days after inoculation of nonresistant leaves and about 10 nl/100 cm 2 /hr at 3 days after inoculation of resistant leaves) coinciding with the time when lesion growth abated, and rapidly dropped to about half-maximum levels by cessation of lesion growth. In contrast, injury-stimulated ethylene increased only during necrotization, after which it returned to levels equal to those of controls. During the period of active lesion growth, infection-stimulated ethylene roughly paralleled the amount of necrosis that was quantitatively manipulated by varying dilutions of inoculum, increased by inoculating with potato virus X, or inhibited by continuously incubating inoculated plants at 32°. Lesion development was not significantly altered by inhibitors of ethylene synthesis (succinic acid-2-2′-dimethylhydrazide) or ethylene action (CO 2 ), but lesions were appreciably smaller in leaves in which the infection-stimulated ethylene was rapidly removed by reduced-pressure treatments. In experiments where light was manipulated to alter phytochrome-related ethylene synthesis, red light slightly increased the rate of virus localization whereas far red light had the opposite effect. Generally, treatments that stimulated ethylene production during lesion development stimulated lesion growth while those treatments reducing ethylene levels had the opposite effect. It was hypothesized that the mechanisms by which infection-stimulated ethylene is synthesized and the manner in which it acts may not be comparable to what occurs in uninfected plants. The suggestion is made that infection-stimulated ethylene may either promote or inhibit lesion development depending upon local concentrations, timing with respect to the infection process, and its interaction with other host-directed responses to infection.

Enhancement by potato virus Y of potato virus X synthesis in doubly infected tobacco depends on the timing of invasion by the viruses

Abstract The time required for movement of potato virus Y (PVY) and two strains of potato virus X (PVX) from an inoculated lower leaf of small tobacco plants to the fourth leaf above, where maximal enhancement of PVX synthesis occurs, was determined. PVY required 12 hr longer than one PVX strain and 6 hr longer than the other; timing was from the time of inoculation of lower leaves. The rates of movement of the two viruses were the same in singly or doubly inoculated plants. Lower leaves were then inoculated with PVX and PVY at times so that invasion of fourth leaves by the two viruses occurred at predicted intervals. Maximum enhancement of one PVX strain occurred when PVY invaded fourth leaves 12 hr before PVX; enhancement of the other PVX strain occurred when PVY invaded 6 hr after PVX. Enhancement was always less than maximal when invasion by either virus preceded invasion by the other by more than 24 hr. These results were interpreted as indicating that enhancement occurs only in cells invaded by the two viruses within a relatively short period of time and that maximum enhancement results when critical stages in the replication cycle of each virus coincide; in such cells replication of both viruses was probably occurring simultaneously.

AN EXTRACTABLE AGENT, INDUCED IN UNINFECTED TISSUES BY LOCALIZED VIRUS INFECTIONS, THAT INTERFERES WITH INFECTION BY TOBACCO MOSAIC VIRUS.

Abstract Juice extracted from uninfected apical halves of leaves of Datura stramonium previously inoculated on their basal halves with tobacco mosaic virus (TMV) or tobacco necrosis virus, a procedure known to induce resistance to TMV in the uninfected apical halves, was found consistently to interfere with infection by TMV (when mixed with it just prior to inoculation of Nicotiana glutinosa, N. tabacum var. Samsun NN, or D. stramonium ) to a much greater extent than did juice from apical halves of control leaves. In some tests, a standard TMV inoculum induced only 8% as many lesions when mixed with extracts from resistant half-leaves of Datura as it did when mixed with extracts from comparable control leaves. The difference in ability of the two juices to interfere with infection by TMV was found not to be due to differences in pH nor to inactivation of the virus in vitro . The differential effect of the two types of juice was eliminated by heating at 78°C for 15 minutes but not by heating at 68° for 15 minutes, by aging at 3° for 5 days, by dialysis, or by centrifugation at 93,000 g for 1 hour. Partial purification was effected by dialysis and use of an anion-exchange cellulose; after each step in the procedure, extracts from resistant half-leaves remained more inhibitory than did those from control leaves. Evidently, infection in basal leaf halves either induced the formation in apical halves of an interfering agent (or agents) not found in healthy leaves or stimulated the production of an inhibitor (or inhibitors) normally found in Datura leaves. Extracts from resistant half-leaves of Samsun NN tobacco were sometimes, but not always, more inhibitory than extracts from control leaves.

Effect of 2,4-dichlorophenoxyacetic acid on tobacco mosaic virus lesions in tobacco and on the fine structure of adjacent cells.

Abstract Lesions induced in Samsun NN tobacco and in Datura stramonium by tobacco mosaic virus (TMV) normally enlarge rapidly for a few days after appearance and then gradually approach a static stage of no further enlargement. Treatment of inoculated leaves with 2,4-dichlorophenoxyacetic acid (2,4-D), at concentrations slightly below toxic levels, sustained the rapid phase of lesion growth indefinitely, leading to greater than 15-fold increases in lesion area and parallel increases in virus content. Stimulation resulted from treatments made between 1 hr and 9 days after inoculation, maximum effects resulting from applications made between 1 and 4 days. The local resistance to TMV that normally develops in tissue immediately surrounding TMV lesions developed to a much lesser degree in 2,4-D-treated leaves than in untreated controls. Electron microscopy of leaves given postinoculation 2,4-D treatments revealed that the characteristic crystals commonly found in spherosomes of this normally resistant zone were lacking. Also, TMV particles, which normally are lacking in this zone, were found in various arrays. Preinoculation treatments inhibited lesion development slightly, and treatment of uninoculated leaves induced structural changes that resembled, 6 days after treatment, those characteristic of cells in the resistant zone ringing a lesion in an untreated leaf.

Comparison of local-lesion and electron-microscope particle-count methods for assay of potato virus X from plants doubly infected by potato viruses X and Y

Abstract Evidence of large increases of potato virus X in plants also infected by potato virus Y was obtained by different methods of assay. Parallel assays by means of local lesions on plants of Gomphrena globosa and by electron-microscope particle counts were in agreement. Results of both methods showed that in tobacco plants simultaneously inoculated with both viruses the virus X concentration in extracted juice of the first noninoculated leaves to develop symptoms is up to ten times as great as in juice of comparable leaves infected with virus X alone. Smaller relative increases were recorded with juice from inoculated leaves and with juice from leaves in the chronic stage of the disease. A difference in virus content of doubly and singly infected plants was apparent early in the infection process. Parallel assays by the local-lesion method and by a serological method also were in agreement.