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Wound-healing as a factor in limiting the size of lesions in Nicotiana glutinosa leaves infected by the very mild strain of tobacco mosaic virus (TMV-VM)
VIROLOGY
61, 474-484 (1973)
Wound-healing Nicotiana
as a Factor glutinosa
Leaves
of Tobacco
in Limiting Infected
Mosaic
the Size by the Very
Virus
of Lesions Mild
in
Strain
(TMV-VM)’
J. H. WU Department
of Biological
Sciences, California State Polytechnic Pomona, California 91768 Accepted November
University,
Pomona,
13, 1972
In the temperature range 20-25”C, the very mild strain of tobacco mosaic virus (TMV-VM) produces necrotic lesions of limited size on leaves of N. glutinosa. The lesions stop enlarging 2-3 days after inoculation. Factors involved in this limited lesion expansion have been investigated. In immature growing leaves a lesion consists of a white halo zone surrounding a necrotic brown center; in mature leaves the necrotic brown spot is directly surrounded by green color without a white halo zone. The white halo results from cell division, similar to wound-periderm formation. The nonhalo lesions in older leaves are like TMV lesions in pinto bean primary leaves; the necrotic area is surrounded by nonnecrot,ic cells with callose deposited in their cell walls. The effectiveness of these structures (wound periderm and callosed wall) as a barrier against expansion of necrosis was tested by transferring the VM inoculated plants from 21°C t,o 30°C. When infected plants were transferred to 30°C at 0 or 24 hr after inoculation, lesion appearance was delayed for 24-30 hr compared to that incubated continuously in 21”C, and when the lesions appeared they were large and expanding. Such expanding lesions showed no wound periderm and only a small amount of callose deposition in the walls of normal cells adjacent to the necrotic cells. However, those lesions that were allowed to form for 3-4 days at 21°C remained small and nonexpanding when subsequently transferred to 30°C. Histological changes similar to that induced by VM necrotic lesions at 21°C could also be induced by simple mechanical or chemical injury. Needle punct,ures induced wound-periderm formation, whereas localized application of chemicals (such as 10 pg/ ml cycloheximide or 0.01 M HCI) induced callose deposition in walls of cells around the treated area in all ages of leaves tested. Thus, the limited expansion of VM lesions at 21°C may be attribut,ed to nonspecific wound-healing processes induced by rapid necrosis of infected cells. INTRODUCTION
Pathogenesis in viral infection can be viewed as a genetic phenomenon (Diener, 1963) in the sense that the host cells are provided with new genetic materials. Therefore, a host plant infected by a virus develops symptoms which are charact’eristic of the specific virus-host combination under specific environmental conditions. This is par1 This work GB-25003.
was
supported
by
NSF
Grant
titularly true when changes induced in infect,ed cells are studied, such as changes in the kinds of protein produced or changes in metabolic pathways. On the other hand, the symptoms observed may be entirely due to secondary reactions which are a sole response of the host genome. This was shown by Esau (1948) when she found similarities between the symptoms induced by viruses and those induced by mechanical injury. Therefore, in order to understand the physlology of symptom expression, it is also im474
Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
WOUND-HEALING
AND
TMV
LESION
SIZE
475
portant to study the general host reaction to in individual enzyme activities. Therefore, injury in cases where the virus infection re- histological changes of the host tissue were examined rather than the changes in individsults in necrosis of the infected cells. I have been interested in the so-called hy- ual enzyme activities which have been inpersensitive reactions of host plants, and the vestigated extensively (Farkas, 1968). mechanism of cellular resistance t’o virus MATERIALS AND METHODS spread in TMV-infected leaves. From extensive studies on changes in metabolism associPlants and viruses. Nicotiana glutinosa L. ated with the so-called hypersensitive reac- plants were grow-n in 4-in. pots in a greention to infection, it is generally conceded t’hat house. When the plants had six to eight inthere is a correlation between host resistance oculable leaves (2-3 months), the leaves were and changes in enzymat’ic activities, espe- inoculated with purified VM strain of tocially those related to phenolic compounds of bacco mosaic virus. The virus was suspended the infected tissues (Goodman, KirBly, and in pH 7 phosphate buffer (0.05 M) wit’h 0.5 % Zaitlin, 1967; Ladygina et al., 1971). HowCelite as abrasive and inoculated on the ever, the details of the mechanism as t’o how upper epidermis. Some of the inoculated such changes in enzymatic activities directly plants were returned to the greenhouse, while stop the spread of virus into neighbor cells others were placed in growt’h chambers at is not clear (Goodman, KiriLly, and Zaitlin, either 21 or 30°C under continuous cool lvhite 1967). Esau (1967) proposed the mechanical fluorescent light (800-1100 ft-c). For hisinterruption of cellular continuity as the di- tological study, necrotic lesions were harrect’ means of preventing virus spread from vested once each day, starting at’ 2 or 3 days cell to cell. Since viral spread from cell to cell after inoculat’ion when lesions began to apis presumably t’hrough plasmodesmata, the pear, and continued up to 10 days after severing of plasmodesmata by purely me- inoculation. chanical injury, or the plugging of plasmoMeasurement of lesion size in relation to desmata by subsequent wound-healing proc- incubation temperature. For the study of leesses such as the deposition of callose in pit sion enlargement, both at,tached and deareas (Currier, 1957) may be able to account tached leaves were used. The lesions profor the nonexpansion of virus lesions. In the duced in leaves attached to t,he plants in a pinto bean-TMV strain-U1 syst’em, it was growth chamber were marked by circles of concluded that the rapid death of infected India ink and lesion size was cstimat’cd with cells, which induces rapid callose deposition a millimeter ruler. When detached leaves in adjacent nonnecrotic cells, perhaps is the were used, the leaves were kept in high hudirect cause for limiting the necrotic lesions midity by insert’ing the petioles through holes from enlarging (Wu, Blakely, and Dimitman, in a sheet of Styrofoam which floated on 1969, Wu and Dimitman, 1970). water in a roasting pan, covered with a glass In the case of N. glutinosa, when t’he leaves plate. A constant temperature of either 21 are infected with the wild-type strain U1, t’he or 30°C was used to study the difference in necrotic local lesions that form enlarge con- lesion size and the time of appearance of tinuously with time (Rappaport and Wildnecrotic lesions. The same lesions were measman, 1957). However, when the VM strain ured at different times after inoculation for is used (Wu, Hildebrandt, and Riker, 1960), determination of the rate of lesion expansion. the glutinosa leaves produce small lesions An ocular micrometer and dissecting micro(average 0.9 mm diameter), which stop en- scope were used for measuring small lesions larging 2-3 days after inoculation. This sys- (Rappaport and Wildman, 1957). tem was examined to see if specific histologiHistochemical tests for callose and ligni$cacal changes can be correlated with the tion of the cell walls. Freehand sections wtre characteristics of lesions of limited size. I used for histochemical and anatomical studfwl that the final stage of host resistance in ies of necrotic lesions. The fluorcsccnt t’his case may be related more directly to his- method of Currier and St’rugger (1956) was tological tissue changes than to the changes used for detection of callow deposition. Solu-
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wu
ble aniline blue (0.1% dissolved in 0.1 N K,POd) was used for staining callose material and the sections were observed under a Bausch and Lomb fluorescent microscope (Wu et al., 1969). Phloroglucinol-HCl was used for staining lignified cell walls. Phloroglucinol also stains suberized walls; therefore, Sudan IV was used to confirm that a phloroglucinol-positive test was not due to suberization. Suberin is stained by Sudan IV, but lignin is not (McClure, 1960). Since chloroplast pigments interfere with observation, photography and histochemical tests, the pigments were removed by 80% alcohol before freehand sectioning. For the callose histochemical test, the leaf tissue was killed in boiling water immediately after cutting from the leaf to avoid callose induction due toia mechanical cut. Comparison of histological changes induced by TMV-VM strain and by mechanical or chemical injury. Dissecting needles were used to puncture holes similar in size to the VM lesions. Chemical necrotic spots were produced by placing lo-p1 drops of solutions of actinomycin D, cycloheximide, HCl, or NaOH on the upper epidermis. The histological and histochemical changes induced by such mechanical and chemical agents were compared with those induced by VM lesions.
tinct zones: a light necrotic center (A), a dark-brown necrotic ring (B), and a white halo (C). The diameter of the lesions mcasured to the outer edge of the brown ring varies from 0.7 mm to 1.5 mm. As described later in the text, the white halo zone is considered not to be virus-infected tissue: rather, it is the reaction of uninfected tissue in response to adjacent infected necrotic cells. In very old lesions (20-30 days), the brown rings gradually turn a lighter color similar to the central portion of the lesions. VM lesions in old leaves. In older (not enlarging) leaves, white halos are not visible; the brown rings are surrounded directly by green tissue. Histological observation of VM lesion development. Two days after inoculation, when only dark spots were visible (no white halo) cross sections of the lesions showed that only the upper epidermis and the palisade were affected. The cell walls were black, and became stained with water-soluble aniline blue
RESULTS
Macroscopic observations on the development of VM lesions on young Nicotiana glutinosa leaves. In the greenhouse, where the temperature varies from 19-35”C, the lesions in young (still enlarging) leaves begin to appear 2 days after inoculation as black spots. At 3 to 4 days after inoculation, a light-colored necrotic central portion and a brown necrotic ring can be distinguished. If transmitted light is used, a white halo is visible, which surrounds the brown ring. The lesions enlarge somewhat until the development of the white halo takes place. After white halo formation, the lesions stop enlarging, and the central area expands at the expense of the brown ring; but essentially the appearance of the lesions will not change after 4-5 days from inoculation. The appearance of a stabilized lesion is shown in Fig. 1, exhibiting three dis-
FIG. 1. A local lesion on a young N. glutinosa leaf 6 days after inoculation with TMV-VM strain (x50). Three zones can be recognized in transmitted light: light-brown necrotic center (A); dark-brown necrotic ring (B); and white halo (C). Outside of the white halo is the normal leaf tissue (U). The younger the leaf, the wider the white halo, indicating that the response of host cells to wound stimuli is leaf age dependent. The black line indicates the approximate region from which the cross section was made for Fig. 2.
WOUNl,-Hti:ALING
AND
to give a slight callow fluorescence. TI~P cells at the edge of th(l lesions included nonneerotic crlls which usually exhibited stronger flu~rc~wncc than the wnt’ral necrotic cells. Howvcr, no hypwplasia, nor lignification of tho cell walls was detc>&d. In young leaves 3-4 days after inoculation, palisade and spongy cells surrounding the necrotic cells had divided (undergone hyperplasia) as evidenced by an incraase in the number of cells. The increase in ccl1 number was accompanied by degeneration and disappearance of the chloroplasts in the cells that divided. Five days after inoculat8ion, the necrosis-induced cell division involved three to four layws of cells surrounding the necrotic brown ring and all the newly formed ccl1 walls were lignilied (positive with phloroglucinol-HCI, negat’ive with Sudan IV). The white halo zone (C) of Fig. 1 corresponds to t,his ZC~I~C: of cell division and chloroplast degoncration among palisade and spongy cells. Tho histological changes observed in the process of format,ion of the whit’c halo are no differcntJ from that, described for woundpcridorm formation by Wylie (1930-1931), and reviewed b\: Bloch (1941, 1952) and by Lip&z (1970). The cells in the white halo ZOIW which surround the brown ring can be obscrvcd more clearly in cross sections cut at ttw edge of thn l&m. Figure 2 shows the
TMV
LESION
SIZE
477
lignified cross walls formed in the palisade cells. One to three cross walls may be laid down within one long palisade cell. Nonlignificd cell walls are not clear in the picture because of the treatment with concentrated HCl in the phloroglucinol reaction. The normal size of a palisade parenchyma is indicated by a dashed outline for comparison. Lignified ccl1 walls gave dull-yellow fluorescence (natural) when viewed with the fluorcscent microscope. Lignified vessels gave a similar fluorescencr. In leaves where no white halo is observed, when the lesion is finally stabilized (3-4 days after inoculation) there is always a band of callose in the epidermal cells which surround the necrotic epidermal cells similar to Ulinfected pinto bean primary leaves (Wu and Din&man, 1970). Walls of palisade and spongy cells surrounding necrotic palisade and spongy cells also fluoresce when stained with aniline blue, indicating callose deposition throughout’ the entire cell wall of these cells. The callose-staining characteristics at the time of lesion appearance (a days after inoculation at 21°C) are similar in young and old leaves. The difference is that at the later stage, in young leaves, white halo zone format’ion commences white in older leaves no further changes in the histological struc-
Fro. 2. A leaf cross section cut tangentially to a VM lesion (cut as indicated in Fig. 1) 9 days after virus iltoculation. The section was treated with phloroglucinol-HCl. Only lignified cell walls appear dark and sharp, the nonlignified cell walls are swollen and indistinct. At the edge of the lesion, which corresponds to the white halo zone, palisade parenchyma have divided and lignified. The size of a nonliguified normal palisade cell is shown by the dotted outline in the right of the picture. The number of cross walls in each palisade cells varies from one to three. The newly formed cell waIIs were perpendicuIar to the long axis of the original cells, hut when the center of the lesion dries and shrinks, the new walls become tilt,ed toward the cent,er (arrow).
478
wu
ture take place, except enhanced callose formation in the walls of cells surrounding a lesion. Size. of VM lesions in relation to incubation temperature. The size of lesions was larger if the inoculated leaves,.either detached or attached to glutinosa plants, were incubated at a higher, temperature (Zaitlin, 1967). Four independent experiments are summarized in Figs. 3 and 4. At 2l”C, 48 hr after inoculation, 80 % of the lesions were visible, and the lesions remained small. At 3O”C, there were no lesions .visible 2 days after inoculation, but-after 3 days. 75 % of the lesions became visible, The larger lesions produced at the higher temperature were correlated with the delayed appearance of lesions and steady enlargement of lesions as a function of time. At the time of lesion appearance, the average diameter of the lesions was about three times that of the lesions formed at 21°C. The histological make up and developmental sequence of lesions incubated at 21°C was essentially similar to that described for greenhouse conditions, When lesions enlarge without limit, there:is:n.o conspicuous deposition of callose in the cell wall (Wu et al., 1969), nor is there wound-periderm forma-
1
OO
1 2 DAYS AFTER
3
4
6
6
I
INOCiJ’LATION
FLG. 3. Rate of lesion appearance on VM-inoculated N. glutinosa leaves incubated in 30” or 21°C. Lesion appearance was delayed for 24-30 hr at 30°C (lower curve) compared with 21°C (upper curve).
0
0
0'
i DAYS
.2'-3J AFTER.
Q
- ‘.
4
5
. 6
INOCItLAT’n’:
FIQ. 4. Enlargement of VM lesions as a function of time when inoculated glutinosa leaves were kept at 30°C (upper curve.) o,r 21°C (lower curve). The lesions remained about the same size when incubated in 21°C.
tion. Necrotic lesions constitute collapsed necrotic undivided cells, which take phloroglucinol stain (reddish brown), but not the typical color of lignin. Response of ‘N. gldtinosa leaves to mechaizical or chemical injury. The histological changes involving cell division and lignification (wound-pcriderm formation) in V&4infected N. glutinosa leaves may not be a specific response of leaf tissue to viral infcction, but rather may be a more nonspecific manifestatio’n of wound healing by the surrounding noninfected cells in response to death of cells within the lesion. Figure 5 shows the structures resulting from puncturing a leaf with a dissecting needle. The macroscopic symptoms arc essentially similar to the VM lesions, and the anatomical relationship between brown ring and white halo zones are similar to the VM lesions described above. As in the white halo of VIM lesions, the younger the leaf the wider the whitct halo around puncture-induced lesions. The difference between VlLf lesions and needle puncture lesions, is that ’ the needle puncture induces cell division even in the old, nonexpanding leaves. The cell division (white halo) zone in old leaves consisted of only one to two cell layers.
WOUND-HEALING
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TMV
LESION
SIZE
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FIG. 5. Wound reaction by a young 1V. gluti,nosa leaf in respo!&‘i & needle punctures. Photo made 5 days after puncturing. The histological structure is similar t,o VM-induced lesions. A: punctured hole; B: a brown ring, bruised necrotic cells; C : white halo (wound periderm; lignified cells) ; D : normal leaf tissue.
It was noted that certain chemicals, when applied as small drops to the leaf surface, would induct formation of a necrotic lesion. Thus, 10.~1 drops of 10 pg/ml cycloheximide, 0.01 IlI HCI, or 0.01 M NaOH produced small brown spots visible 24 hr after application to the leaf surface. The necrotic cells making up the brown spots were surrounded b,y cells with callosed walls in both young and old leaves. At, the final stage when the xizc: of necrot’ic spots were stabilized (3-4 days after the application of the chemicals), no wound periderm h&d developed, but callost deposition was st;rong. This response mimicked that ,produced by virus lesions in old leaves (although young leaves did not respond as they did to virus lesions, i.e., the young l(>avcs did not form wound periderm m response to the chemicals). The chemically induced necrotic lcsinnb, surrounded by callosod tolls, \v(‘r(’ similar in appearance to virus-inducc>d lrsions formed in U1- or Us-inf&cd pinto bean primary lcavns (Wu et al., 1969).
The>rcsponsc to both mechanical or chemical injury occurred fast,er at’ 30°C than at “1°C.
Thus, it is possibht, using nonviral agents, to bring about responses in the host tissue which closc~ly imitate the responses to virus infa&m. It is well known in other syst’cms that mechanical wounds may induce either \vound-pcridrrm formation (Lipetz, 1970) or
callose formation (Currier, 1957) ; and as shown here injury due to chemicals may induce callose formation. EJectiveness of wound peridem as a barrier in preventing virus front spreading. A test for the effectiveness of the white halo (wound periderm) as a barrier was made by transferring leaves having lesions with white halos (4 days after inoculation) to 32°C. If the wound periderm is effective in preventing the virus from spreading, the lesion should stay the same size. One half of each leaf in a N. glutinosa plant was inoculated with VM, the inoculated plants were placed in the greenhouse or incubated in a 22°C growth chamber. Four days after inoculat’ion, the prcsence of distinct white halos on young leaves were confirmed. At this .time, the opposite half of each leaf was inoculatfed with VM and leaves were detached and ,incubated at 32°C in high humidity. Figure 6 shows the results of such an experiment! The lesions with white halos stayed the same size but’ on the newly inoculated half leaf VIM formed large lesions which enlarged continuously. Once t’hr barrier to virus spread is established it is cffcctivc even t.hough later the environmental condition is changed t’o encourage cont’inu, ous enlargement of lesions. To discover when tht> barrier produced at 22°C becomes cffectivc, 1 inoculated leaves were transferred from 22 to 30°C after various periods of time. A similar experimental
480
FIG. 6. Nonspreading of white halo VM lesions when transferred to a higher t,emperature. The left half of the N. glutinosa leaf was inoculated with VM and incubated at 22°C 4 days before transferring to 32°C. The right half of the leaf was inoculated with VM immediately before t,ransfer to 32°C. The picture was taken after 6 days’ incubation at 32°C.
design to that described above was used except that a whole plant in a pot was used instead of isolated leaves. One half of each leaf was inoculated with VM and incubated in a 22°C growth chamber. After 1, 2, 3, and 4 days of incubation, the plants were transferred to a 30°C growth chamber. The opposite half-leaf was inoculated with VM just before the transfer ,to, 30°C. Representative leaves were photographed 6 days after incubation at 30°C and are shown in Figs. 7, 8,9, and 10. No lesions that were 3 or 4 days old enlarged after the transfer. This corresponded to the stage when 90-100 % of the lesions had appeared and most of them were with white halos (Figs. 7 and S). However, when leaves were transferred at 48 hr after inoculation, 20 % of the lesions continued to enlarge, but 80 % remained small with wound-periderm formation (Fig. 9). This corresponded to the percentage of visible necrotic lesions (80 %) at t’he time of transfer. When leaves were transferred 24 hr after inoculation, all incipient lesions (infective centers) enlarged without white halo formation. This corresponded to no visible necrotic lesions at the time of transfer (Fig. 10). These observations clearly indicated that, all the incipient lesions (infective centers) enlarged but visible necrotic lesions did not, and visible lesions which developed a wound periderm remained the same size. It can be
inferred t’hat at thr time of lesion appcarante, the stimuli causing wound reactions had already gone to neighboring cells, and transfer to 30°C did not change the reaction of the neighboring cells. An alternat’ive interpretation is t’hat the callose deposition was the primary barrier even in the young leaves where woundperiderm formation xas the most striking response to VM infection. In both young and old leaves, callose deposition is a rapid process (Currier, 1957; Wu et al., 1969), and callose can be detected as soon as the lesion appears. Such callose deposition alone may constitute an effective barrier to further VM virus spread. This inference is supported by the observation that the nonhalo lesions on mature and older leaves did not expand upon transfer to higher temperature. Slowness in spreading is characteristic of Vikf lesims. The fact that slowness in spreading is the nature of the VM strain can bc demonstrated by incubating inoculated N. glutinosa leaves in complete darkness at 30°C. Under these conditions, U1 makes large green spots (Hayashi and Matsui, 1965) as shown in Fig. 11, \+hile VM makes very small green spots instead of necrotic lesions. The uninfected areas senescc rapidly, becoming yellow, and only infected areas remained green. At 20-25”C, and in the light (under which conditions VM causes necrosis)
WOUND-HEALING
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FIGS. 7,8, 9, and 10. An experiment to find the incubation period (at 22°C) necessary for VM lesions to become unresponsive to high temperature (30°C) induction of rapid lesion expansion. Left halves of all the leaves were incubated at 22°C for various durations after VM inoculation before transfer to 30°C. Figure 7 represents 4 days; Fig. 8, 3 days; Fig. 9, 2 days; and Fig. 10, 1 day at 22°C. The right halves of all the leaves were inoculated with VM immediately before transfer to 30°C. All lesions on the right halves corrtinued to expand after the appearance of lesions. The results were photographed 6 days aft,er incubation in 30°C.
the slo~n~~ss in spreading of VRI lesions perhaps allolvs the: noninfected neighbor cells sufficient time to respond to necrosis of infected cells hy ivound-healing processes lvhich retard virus spread, causing lesions of
limited size. On the other hand, the fast spreading of U1 must be responsible for t’he unlimited cnlargomcnt of U1 necrotic lesions, since invasion of U1 is so fast there would not bc enough time for uninfected neighbor cells to undergo wound-healing reactions. DISCUSSION
The observation that TbIV st,rain VM formed small lesions of limited size (Wu et al., 1960) raised the question as to what limits the expansion of lesions since the U1 or the U, strains produced large lesions which
continued to cnlargc n-it’h time (Rappaport and Wildman, 19:i7). Although both types of lesion formation (UI and VM lesions) are generally rcfcrred to as a hypersensitive reaction, there is, however, a basic difference between the two kinds of lesions. The histological changes associated with VM lesion formation revealed that the process for limiting lesion size may be associated with well-known wound-healing mechanisms (Bloch, 1941, 1952; Wylie, 1930, 1931; Lipetz, 1970). l&au (1933) found that a virus caused abnormalities which were associated with the necrosis of vascular systems. She concluded t’hat vein clearing, vein banding, hyperplasia, lignification, and suberization are common responses to injury, and not
482
FIG. 11. Comparison of the rate of virus spread between U1 and VM strains in N. glutinosa without involvement of necrosis. UI makes large green spots, VM makes small green spots, when the detached leaves are kept at 30°C in darkness. This picture was taken 3 days after virus inoculation.
necessarily specific reactions to viruses. The present study agrees with her conclusion, that normal wound-healing processes are involved in the characteristic expression of virus symptoms. However, these nonspecific wound reactions become significant when they are considered together with the host resistance to the expansion of virus infection. It is suggested that the wound-healing process is intimately associated with the defense mechanism of the leaf tissue which inhibits virus spread from cell to cell. This conclusion was reached by the following reasoning: Since incubation of M. glutinosa at high temperature (35-36°C) after inoculation with TMV (either U, or VM) produced systemic symptoms rather than a necrotic reaction, it is obvious that the necrotic reaction (hypersensitivity) is lost at 35°C (Samuel, 1931). The necrosis of the infected cells is thus a temperature-dependent reaction. In plants incubated at 30°C the appear-
wu ance of VM lesions is delayed for 24-30 hr compared to those at 21°C and the resultant necrotic lesions expand as a function of time. The hypersensitivity of leaf cells to VM infection at 30°C is not so strong as at 21°C since death of the infected cells was delayed for 2430 hr. Thus, the so-called hypersensitive reaction is not all of the same degree, but there is a gradient depending upon the temperature during the incubation period. By the time (2-3 days after inoculation) when the VM lesions became visible at a lower temperature (2l”C), the stimuli causing wound reactions already had gone to the neighboring cells and the subsequent changes in structure (wound periderm in young leaves; callose deposition in old leaves) were effective in preventing virus from spreading even when leaves were transferred to a higher temperature (30°C). It seems that normal wound healing is the common mechanism of defense against pathogens in plant tissues, and the nonlimitation of virus spread in leaf tissue represents the exceptional case in which the wound-healing mechanism fails to operate properly. Therefore, a question arises as to what makes the TMV strain-U1 lesion expand indefinitely, and why does the wound-healing mechanism fail to operate? The continuous enlargement of the U1 lesions at normal temperatures (al-22°C) may have character in common with that of VM lesions in higher temperature (30°C). The continuous enlargement of lesions can be accounted for by the slower death of infected cells accompanied with greater rapidity of virus spread than the process of wound healing. Therefore, any factors which contribute to the rapid spread of virus and slow process of barrier formation (wound healing) induced by the necrosis of cells may contribute to the growth characteristics of expanding lesions. The relationship between the antiviral factors (Sela and Applebaum, 1962) and host resistance as revealed by the direct change in host histology is not clear. It is conceivable that the antiviral factors (AVF) are formed in infected cells as a part of the death process of virus-infected cells which decreases the virus replication and results in slower virus movement. Since the rate of virus movement
WOUND-HEALING
AND
may be a function of rate of virus synthesis, any factors which slow down virus synthesis and/or movement may aid in the woundhealing process of uninfected adjacent cells by giving these uninfected adjacent cells time to react to t,he incoming stimuli of necrotic cells. it is However, in this investigation, pointed out t’hat from a histological viewpoint it may be possible to account for the factors which limit, necrotic lesions from expanding, wit,hout invoking the substance specifically induced by virus infection. Xany physical and chemical trcatmcnts induced large necrotic lesion formation in response to virus infection (Wu, Stubbs, and Dimitman, 1968). The large lesion formation was attributed to inactivation of host resistancc by the physical and chemical treatments, \\;hil(l Still allowing virus multiplication and movement to continue (Wu et al., 1909; Simons, Isracxl, and Ross, 1972). There ma?’ bc ma,ny rn&ant factors involwd, and callosc~ dq,osition \vas thought to b(‘ one which corrt4attrd with nonenlargc>mctnt of lwions in pinto bran primary Iravcs (Wu et al., 1969). Hiruki and Tu (1972) made d&&d studies \\-ith tho potato virus :\I-kidrwy bean system using bot,h light and elect,ron micaroscopy, and have: confirmed that thci deposition of callow occurs on the \zalls of nonnwrot~ic wlls, \vhich surround the necrotic cells. Rowv~~r, Spwc’er and Kimmins (1971) using the Ul-pinto bean system were unahl~ to dctcsct’ tho prcsww of callose in their ultrast’ructural studiw. The: reason for such discrcpawy is not, clear, but the comment made bp Hiruki and Tu (1972) seems appropriate. “The apparent lack of callosc . . as dctcrmincd by c~lwtron microscopy, may wll bc due to the different area of tissue cxamincd or to the method of sampling for obscvvation as well as to the use of different t~wlmical procedures.” They (Hiruki and Tu, 1972) further suggested additional histologic~al changes (secondary Ivall thickening in cells of the seminecrotic zone) might also be involved in limitation of lesion enlargemcint. The callosed cell wall might be further modified by deposition of other chemicals swh as polyphenols. Simons et al., (197:‘) using Nicot&w~a
TMV
LESION
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483
L. ‘Samsun NN’ also reported that callosc was not detected in their electron microscopic studies. In this case too, the comment made by Hiruki and Tu (1972) mentioned above may apply. However, it is always possible that the resistance in the ‘Samsun NN’ may involve factors other than callose deposition, as suggested by the authors. tabacum
ACKNOWLEDGMENT I thank
Dr. S. 0. Wildman
for his assistance in space at U.C.L.A. A special appreciation is due to Drs. L. M. Blakely, J. G. Bald, J. E. Dimitman, and M. Zaitlin for their valuable suggestions and criticisms during the course of this study and their assistance in preparation of this manuscript.
supplying virus samples and greenhouse
REFERb;NCES l<. (1941). Wound-healing in higher plants. Bat. IZev. 7, 110-146. BLOCH, R. (1952). Wound-healing in higher plants. II. Uot. Rev. 18, 655-679. CURIIII.:R, H. (1957). Callose substance in plant cells. Awm. J. Hotany 44, 478-483. CURRII~I<, H., atld STRLJcQI*:R, S. (1956). Anilin blue and fluorescence microscopy of callose in bulb scales of Allium cepa L. Protoplasmn 45, 552-559. DII:XI~IL, 1’. 0. (1963). Physiology of virus-infected plants. ATL~U. Rev. Phytopathol. I, 197-218. Esau, K. (1933). Pat,hologic changes in the anatomy of leaves of the sugar beet, 13elrc vu/geris L., affected by Curly top. Phytopathology 23, 679712. Es.\u, K. (1948). Some anatomical aspects of plant virus disease problems. II. Bat. I$ev. 14,413-449. Esau, K. (1967). Anatomy of pla,nt virus infections. Amu. Rev. Phytopathol. 5, 45-76. F.
wu
484 marginal tissue in red kidney ogy 62, 77-85.
bean. Phylopalhol-
M. E., TAIML.~, E. A., and RUBIN, B. A. (1971). Isoenzyme composition of peroxidase and polyphenoloxidase during viral pathogenesis in tobacco. Soviet Pla)rt Physiol. (transl. 17, 773779. LIPE~I~Z, J. (1970). Wound-healing in higher plants. Id. Rev. Cgtol. 27, l-28. McC~um;, T. T. (1960). Chlorogenic acid accumulation and wound-healing in sweet potato roots. Amer. J. Rot. 47, 227-280. R.~PP.\PoI~T, I., and WILDM.\N, R. (+. (1957). A kinetic study of local lesion growth on ,Vicotint/a glu(i,/osa resulting from tobacco mosaic virus infection. Virology 4, 205-274. SAMUEL, G. (1931). Some experiments on inoculating methods with plant viruses, and 011 local lesions. Ann. Appl. Biol. 18,494-507. SELA, I. and APPLEBMJM, S. W. (1962). Occurrence of antiviral factor in virus-infected plants. Virology 17, 543-548. SIMONS, T. J., ISR.\EI+ H. W., and Ross, A. F. (1972). Effect of 2, 4-dichlorophenoxyacetir acid on tobacco mosaic virus lesions in tobacco and on the fine structure of adjacent cells. Virology 48, 502-515. SPENCEH, D. F., and KIMMINS, W. C. (1971). Ultrastructure of tobacco mosaic virus lesions LBDYGIN.~,
and surrounding tissue in phaseolus vulgaris var. Pinto. <,“a!~. J. Bot. 49, 417-421. WYLIE, I<. B. (1930). Cicatrization of foliage leaves, I. Wound responses of certain mesophytic leaves. Bot. Gaz. 90, 260-278. WYLIE, 11. B. (1931). Cicatrization of foliage leaves, II. Wound responses of certain broadleaved evergreens. Bot. Gaz. 92,279-295. Wu, J. H., BLBKELY, L. M., and DIMITMAN, J. E. (1969). Inactivation of a host resistance mechanism as an explanation for heat activation of TMV-infected bean leaves. Virology 37,658-666. J. E. (1970). Leaf strucWV, J. H., and DIMITMAN, ture and callose formation as determinants of TMV movement in bean leaves as revealed by UV irradiation studies. Virology 40,82W327. Wu, J. H., HII,DICF~R.\XD’~, A. C., and RIKER, A. J. (19GO). Virus-host relationship in plant tissue culture. Phylopathology 50, 587-594. Wu, J. H., Swsrrs, A., and I~IMITMAN, J. E. (1968). Formation of large necrotic lesions by sublethal physical and chemical treatments of Pinto bean primary leaves infected with tobacco mosaic virus (Abstr.). Phytopathology 58,1073. ZAITLIN, M. (1967). Cited as unpublished observation. “The Biorhemistry and Physiology of Infectious Plant Disease” (by (Goodman, Kirftly, and Zaitlin), p. 209, Van Nostrand, Princeton, NJ.
61, 474-484 (1973)
Wound-healing Nicotiana
as a Factor glutinosa
Leaves
of Tobacco
in Limiting Infected
Mosaic
the Size by the Very
Virus
of Lesions Mild
in
Strain
(TMV-VM)’
J. H. WU Department
of Biological
Sciences, California State Polytechnic Pomona, California 91768 Accepted November
University,
Pomona,
13, 1972
In the temperature range 20-25”C, the very mild strain of tobacco mosaic virus (TMV-VM) produces necrotic lesions of limited size on leaves of N. glutinosa. The lesions stop enlarging 2-3 days after inoculation. Factors involved in this limited lesion expansion have been investigated. In immature growing leaves a lesion consists of a white halo zone surrounding a necrotic brown center; in mature leaves the necrotic brown spot is directly surrounded by green color without a white halo zone. The white halo results from cell division, similar to wound-periderm formation. The nonhalo lesions in older leaves are like TMV lesions in pinto bean primary leaves; the necrotic area is surrounded by nonnecrot,ic cells with callose deposited in their cell walls. The effectiveness of these structures (wound periderm and callosed wall) as a barrier against expansion of necrosis was tested by transferring the VM inoculated plants from 21°C t,o 30°C. When infected plants were transferred to 30°C at 0 or 24 hr after inoculation, lesion appearance was delayed for 24-30 hr compared to that incubated continuously in 21”C, and when the lesions appeared they were large and expanding. Such expanding lesions showed no wound periderm and only a small amount of callose deposition in the walls of normal cells adjacent to the necrotic cells. However, those lesions that were allowed to form for 3-4 days at 21°C remained small and nonexpanding when subsequently transferred to 30°C. Histological changes similar to that induced by VM necrotic lesions at 21°C could also be induced by simple mechanical or chemical injury. Needle punct,ures induced wound-periderm formation, whereas localized application of chemicals (such as 10 pg/ ml cycloheximide or 0.01 M HCI) induced callose deposition in walls of cells around the treated area in all ages of leaves tested. Thus, the limited expansion of VM lesions at 21°C may be attribut,ed to nonspecific wound-healing processes induced by rapid necrosis of infected cells. INTRODUCTION
Pathogenesis in viral infection can be viewed as a genetic phenomenon (Diener, 1963) in the sense that the host cells are provided with new genetic materials. Therefore, a host plant infected by a virus develops symptoms which are charact’eristic of the specific virus-host combination under specific environmental conditions. This is par1 This work GB-25003.
was
supported
by
NSF
Grant
titularly true when changes induced in infect,ed cells are studied, such as changes in the kinds of protein produced or changes in metabolic pathways. On the other hand, the symptoms observed may be entirely due to secondary reactions which are a sole response of the host genome. This was shown by Esau (1948) when she found similarities between the symptoms induced by viruses and those induced by mechanical injury. Therefore, in order to understand the physlology of symptom expression, it is also im474
Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
WOUND-HEALING
AND
TMV
LESION
SIZE
475
portant to study the general host reaction to in individual enzyme activities. Therefore, injury in cases where the virus infection re- histological changes of the host tissue were examined rather than the changes in individsults in necrosis of the infected cells. I have been interested in the so-called hy- ual enzyme activities which have been inpersensitive reactions of host plants, and the vestigated extensively (Farkas, 1968). mechanism of cellular resistance t’o virus MATERIALS AND METHODS spread in TMV-infected leaves. From extensive studies on changes in metabolism associPlants and viruses. Nicotiana glutinosa L. ated with the so-called hypersensitive reac- plants were grow-n in 4-in. pots in a greention to infection, it is generally conceded t’hat house. When the plants had six to eight inthere is a correlation between host resistance oculable leaves (2-3 months), the leaves were and changes in enzymat’ic activities, espe- inoculated with purified VM strain of tocially those related to phenolic compounds of bacco mosaic virus. The virus was suspended the infected tissues (Goodman, KirBly, and in pH 7 phosphate buffer (0.05 M) wit’h 0.5 % Zaitlin, 1967; Ladygina et al., 1971). HowCelite as abrasive and inoculated on the ever, the details of the mechanism as t’o how upper epidermis. Some of the inoculated such changes in enzymatic activities directly plants were returned to the greenhouse, while stop the spread of virus into neighbor cells others were placed in growt’h chambers at is not clear (Goodman, KiriLly, and Zaitlin, either 21 or 30°C under continuous cool lvhite 1967). Esau (1967) proposed the mechanical fluorescent light (800-1100 ft-c). For hisinterruption of cellular continuity as the di- tological study, necrotic lesions were harrect’ means of preventing virus spread from vested once each day, starting at’ 2 or 3 days cell to cell. Since viral spread from cell to cell after inoculat’ion when lesions began to apis presumably t’hrough plasmodesmata, the pear, and continued up to 10 days after severing of plasmodesmata by purely me- inoculation. chanical injury, or the plugging of plasmoMeasurement of lesion size in relation to desmata by subsequent wound-healing proc- incubation temperature. For the study of leesses such as the deposition of callose in pit sion enlargement, both at,tached and deareas (Currier, 1957) may be able to account tached leaves were used. The lesions profor the nonexpansion of virus lesions. In the duced in leaves attached to t,he plants in a pinto bean-TMV strain-U1 syst’em, it was growth chamber were marked by circles of concluded that the rapid death of infected India ink and lesion size was cstimat’cd with cells, which induces rapid callose deposition a millimeter ruler. When detached leaves in adjacent nonnecrotic cells, perhaps is the were used, the leaves were kept in high hudirect cause for limiting the necrotic lesions midity by insert’ing the petioles through holes from enlarging (Wu, Blakely, and Dimitman, in a sheet of Styrofoam which floated on 1969, Wu and Dimitman, 1970). water in a roasting pan, covered with a glass In the case of N. glutinosa, when t’he leaves plate. A constant temperature of either 21 are infected with the wild-type strain U1, t’he or 30°C was used to study the difference in necrotic local lesions that form enlarge con- lesion size and the time of appearance of tinuously with time (Rappaport and Wildnecrotic lesions. The same lesions were measman, 1957). However, when the VM strain ured at different times after inoculation for is used (Wu, Hildebrandt, and Riker, 1960), determination of the rate of lesion expansion. the glutinosa leaves produce small lesions An ocular micrometer and dissecting micro(average 0.9 mm diameter), which stop en- scope were used for measuring small lesions larging 2-3 days after inoculation. This sys- (Rappaport and Wildman, 1957). tem was examined to see if specific histologiHistochemical tests for callose and ligni$cacal changes can be correlated with the tion of the cell walls. Freehand sections wtre characteristics of lesions of limited size. I used for histochemical and anatomical studfwl that the final stage of host resistance in ies of necrotic lesions. The fluorcsccnt t’his case may be related more directly to his- method of Currier and St’rugger (1956) was tological tissue changes than to the changes used for detection of callow deposition. Solu-
476
wu
ble aniline blue (0.1% dissolved in 0.1 N K,POd) was used for staining callose material and the sections were observed under a Bausch and Lomb fluorescent microscope (Wu et al., 1969). Phloroglucinol-HCl was used for staining lignified cell walls. Phloroglucinol also stains suberized walls; therefore, Sudan IV was used to confirm that a phloroglucinol-positive test was not due to suberization. Suberin is stained by Sudan IV, but lignin is not (McClure, 1960). Since chloroplast pigments interfere with observation, photography and histochemical tests, the pigments were removed by 80% alcohol before freehand sectioning. For the callose histochemical test, the leaf tissue was killed in boiling water immediately after cutting from the leaf to avoid callose induction due toia mechanical cut. Comparison of histological changes induced by TMV-VM strain and by mechanical or chemical injury. Dissecting needles were used to puncture holes similar in size to the VM lesions. Chemical necrotic spots were produced by placing lo-p1 drops of solutions of actinomycin D, cycloheximide, HCl, or NaOH on the upper epidermis. The histological and histochemical changes induced by such mechanical and chemical agents were compared with those induced by VM lesions.
tinct zones: a light necrotic center (A), a dark-brown necrotic ring (B), and a white halo (C). The diameter of the lesions mcasured to the outer edge of the brown ring varies from 0.7 mm to 1.5 mm. As described later in the text, the white halo zone is considered not to be virus-infected tissue: rather, it is the reaction of uninfected tissue in response to adjacent infected necrotic cells. In very old lesions (20-30 days), the brown rings gradually turn a lighter color similar to the central portion of the lesions. VM lesions in old leaves. In older (not enlarging) leaves, white halos are not visible; the brown rings are surrounded directly by green tissue. Histological observation of VM lesion development. Two days after inoculation, when only dark spots were visible (no white halo) cross sections of the lesions showed that only the upper epidermis and the palisade were affected. The cell walls were black, and became stained with water-soluble aniline blue
RESULTS
Macroscopic observations on the development of VM lesions on young Nicotiana glutinosa leaves. In the greenhouse, where the temperature varies from 19-35”C, the lesions in young (still enlarging) leaves begin to appear 2 days after inoculation as black spots. At 3 to 4 days after inoculation, a light-colored necrotic central portion and a brown necrotic ring can be distinguished. If transmitted light is used, a white halo is visible, which surrounds the brown ring. The lesions enlarge somewhat until the development of the white halo takes place. After white halo formation, the lesions stop enlarging, and the central area expands at the expense of the brown ring; but essentially the appearance of the lesions will not change after 4-5 days from inoculation. The appearance of a stabilized lesion is shown in Fig. 1, exhibiting three dis-
FIG. 1. A local lesion on a young N. glutinosa leaf 6 days after inoculation with TMV-VM strain (x50). Three zones can be recognized in transmitted light: light-brown necrotic center (A); dark-brown necrotic ring (B); and white halo (C). Outside of the white halo is the normal leaf tissue (U). The younger the leaf, the wider the white halo, indicating that the response of host cells to wound stimuli is leaf age dependent. The black line indicates the approximate region from which the cross section was made for Fig. 2.
WOUNl,-Hti:ALING
AND
to give a slight callow fluorescence. TI~P cells at the edge of th(l lesions included nonneerotic crlls which usually exhibited stronger flu~rc~wncc than the wnt’ral necrotic cells. Howvcr, no hypwplasia, nor lignification of tho cell walls was detc>&d. In young leaves 3-4 days after inoculation, palisade and spongy cells surrounding the necrotic cells had divided (undergone hyperplasia) as evidenced by an incraase in the number of cells. The increase in ccl1 number was accompanied by degeneration and disappearance of the chloroplasts in the cells that divided. Five days after inoculat8ion, the necrosis-induced cell division involved three to four layws of cells surrounding the necrotic brown ring and all the newly formed ccl1 walls were lignilied (positive with phloroglucinol-HCI, negat’ive with Sudan IV). The white halo zone (C) of Fig. 1 corresponds to t,his ZC~I~C: of cell division and chloroplast degoncration among palisade and spongy cells. Tho histological changes observed in the process of format,ion of the whit’c halo are no differcntJ from that, described for woundpcridorm formation by Wylie (1930-1931), and reviewed b\: Bloch (1941, 1952) and by Lip&z (1970). The cells in the white halo ZOIW which surround the brown ring can be obscrvcd more clearly in cross sections cut at ttw edge of thn l&m. Figure 2 shows the
TMV
LESION
SIZE
477
lignified cross walls formed in the palisade cells. One to three cross walls may be laid down within one long palisade cell. Nonlignificd cell walls are not clear in the picture because of the treatment with concentrated HCl in the phloroglucinol reaction. The normal size of a palisade parenchyma is indicated by a dashed outline for comparison. Lignified ccl1 walls gave dull-yellow fluorescence (natural) when viewed with the fluorcscent microscope. Lignified vessels gave a similar fluorescencr. In leaves where no white halo is observed, when the lesion is finally stabilized (3-4 days after inoculation) there is always a band of callose in the epidermal cells which surround the necrotic epidermal cells similar to Ulinfected pinto bean primary leaves (Wu and Din&man, 1970). Walls of palisade and spongy cells surrounding necrotic palisade and spongy cells also fluoresce when stained with aniline blue, indicating callose deposition throughout’ the entire cell wall of these cells. The callose-staining characteristics at the time of lesion appearance (a days after inoculation at 21°C) are similar in young and old leaves. The difference is that at the later stage, in young leaves, white halo zone format’ion commences white in older leaves no further changes in the histological struc-
Fro. 2. A leaf cross section cut tangentially to a VM lesion (cut as indicated in Fig. 1) 9 days after virus iltoculation. The section was treated with phloroglucinol-HCl. Only lignified cell walls appear dark and sharp, the nonlignified cell walls are swollen and indistinct. At the edge of the lesion, which corresponds to the white halo zone, palisade parenchyma have divided and lignified. The size of a nonliguified normal palisade cell is shown by the dotted outline in the right of the picture. The number of cross walls in each palisade cells varies from one to three. The newly formed cell waIIs were perpendicuIar to the long axis of the original cells, hut when the center of the lesion dries and shrinks, the new walls become tilt,ed toward the cent,er (arrow).
478
wu
ture take place, except enhanced callose formation in the walls of cells surrounding a lesion. Size. of VM lesions in relation to incubation temperature. The size of lesions was larger if the inoculated leaves,.either detached or attached to glutinosa plants, were incubated at a higher, temperature (Zaitlin, 1967). Four independent experiments are summarized in Figs. 3 and 4. At 2l”C, 48 hr after inoculation, 80 % of the lesions were visible, and the lesions remained small. At 3O”C, there were no lesions .visible 2 days after inoculation, but-after 3 days. 75 % of the lesions became visible, The larger lesions produced at the higher temperature were correlated with the delayed appearance of lesions and steady enlargement of lesions as a function of time. At the time of lesion appearance, the average diameter of the lesions was about three times that of the lesions formed at 21°C. The histological make up and developmental sequence of lesions incubated at 21°C was essentially similar to that described for greenhouse conditions, When lesions enlarge without limit, there:is:n.o conspicuous deposition of callose in the cell wall (Wu et al., 1969), nor is there wound-periderm forma-
1
OO
1 2 DAYS AFTER
3
4
6
6
I
INOCiJ’LATION
FLG. 3. Rate of lesion appearance on VM-inoculated N. glutinosa leaves incubated in 30” or 21°C. Lesion appearance was delayed for 24-30 hr at 30°C (lower curve) compared with 21°C (upper curve).
0
0
0'
i DAYS
.2'-3J AFTER.
Q
- ‘.
4
5
. 6
INOCItLAT’n’:
FIQ. 4. Enlargement of VM lesions as a function of time when inoculated glutinosa leaves were kept at 30°C (upper curve.) o,r 21°C (lower curve). The lesions remained about the same size when incubated in 21°C.
tion. Necrotic lesions constitute collapsed necrotic undivided cells, which take phloroglucinol stain (reddish brown), but not the typical color of lignin. Response of ‘N. gldtinosa leaves to mechaizical or chemical injury. The histological changes involving cell division and lignification (wound-pcriderm formation) in V&4infected N. glutinosa leaves may not be a specific response of leaf tissue to viral infcction, but rather may be a more nonspecific manifestatio’n of wound healing by the surrounding noninfected cells in response to death of cells within the lesion. Figure 5 shows the structures resulting from puncturing a leaf with a dissecting needle. The macroscopic symptoms arc essentially similar to the VM lesions, and the anatomical relationship between brown ring and white halo zones are similar to the VM lesions described above. As in the white halo of VIM lesions, the younger the leaf the wider the whitct halo around puncture-induced lesions. The difference between VlLf lesions and needle puncture lesions, is that ’ the needle puncture induces cell division even in the old, nonexpanding leaves. The cell division (white halo) zone in old leaves consisted of only one to two cell layers.
WOUND-HEALING
AND
TMV
LESION
SIZE
479
FIG. 5. Wound reaction by a young 1V. gluti,nosa leaf in respo!&‘i & needle punctures. Photo made 5 days after puncturing. The histological structure is similar t,o VM-induced lesions. A: punctured hole; B: a brown ring, bruised necrotic cells; C : white halo (wound periderm; lignified cells) ; D : normal leaf tissue.
It was noted that certain chemicals, when applied as small drops to the leaf surface, would induct formation of a necrotic lesion. Thus, 10.~1 drops of 10 pg/ml cycloheximide, 0.01 IlI HCI, or 0.01 M NaOH produced small brown spots visible 24 hr after application to the leaf surface. The necrotic cells making up the brown spots were surrounded b,y cells with callosed walls in both young and old leaves. At, the final stage when the xizc: of necrot’ic spots were stabilized (3-4 days after the application of the chemicals), no wound periderm h&d developed, but callost deposition was st;rong. This response mimicked that ,produced by virus lesions in old leaves (although young leaves did not respond as they did to virus lesions, i.e., the young l(>avcs did not form wound periderm m response to the chemicals). The chemically induced necrotic lcsinnb, surrounded by callosod tolls, \v(‘r(’ similar in appearance to virus-inducc>d lrsions formed in U1- or Us-inf&cd pinto bean primary lcavns (Wu et al., 1969).
The>rcsponsc to both mechanical or chemical injury occurred fast,er at’ 30°C than at “1°C.
Thus, it is possibht, using nonviral agents, to bring about responses in the host tissue which closc~ly imitate the responses to virus infa&m. It is well known in other syst’cms that mechanical wounds may induce either \vound-pcridrrm formation (Lipetz, 1970) or
callose formation (Currier, 1957) ; and as shown here injury due to chemicals may induce callose formation. EJectiveness of wound peridem as a barrier in preventing virus front spreading. A test for the effectiveness of the white halo (wound periderm) as a barrier was made by transferring leaves having lesions with white halos (4 days after inoculation) to 32°C. If the wound periderm is effective in preventing the virus from spreading, the lesion should stay the same size. One half of each leaf in a N. glutinosa plant was inoculated with VM, the inoculated plants were placed in the greenhouse or incubated in a 22°C growth chamber. Four days after inoculat’ion, the prcsence of distinct white halos on young leaves were confirmed. At this .time, the opposite half of each leaf was inoculatfed with VM and leaves were detached and ,incubated at 32°C in high humidity. Figure 6 shows the results of such an experiment! The lesions with white halos stayed the same size but’ on the newly inoculated half leaf VIM formed large lesions which enlarged continuously. Once t’hr barrier to virus spread is established it is cffcctivc even t.hough later the environmental condition is changed t’o encourage cont’inu, ous enlargement of lesions. To discover when tht> barrier produced at 22°C becomes cffectivc, 1 inoculated leaves were transferred from 22 to 30°C after various periods of time. A similar experimental
480
FIG. 6. Nonspreading of white halo VM lesions when transferred to a higher t,emperature. The left half of the N. glutinosa leaf was inoculated with VM and incubated at 22°C 4 days before transferring to 32°C. The right half of the leaf was inoculated with VM immediately before t,ransfer to 32°C. The picture was taken after 6 days’ incubation at 32°C.
design to that described above was used except that a whole plant in a pot was used instead of isolated leaves. One half of each leaf was inoculated with VM and incubated in a 22°C growth chamber. After 1, 2, 3, and 4 days of incubation, the plants were transferred to a 30°C growth chamber. The opposite half-leaf was inoculated with VM just before the transfer ,to, 30°C. Representative leaves were photographed 6 days after incubation at 30°C and are shown in Figs. 7, 8,9, and 10. No lesions that were 3 or 4 days old enlarged after the transfer. This corresponded to the stage when 90-100 % of the lesions had appeared and most of them were with white halos (Figs. 7 and S). However, when leaves were transferred at 48 hr after inoculation, 20 % of the lesions continued to enlarge, but 80 % remained small with wound-periderm formation (Fig. 9). This corresponded to the percentage of visible necrotic lesions (80 %) at t’he time of transfer. When leaves were transferred 24 hr after inoculation, all incipient lesions (infective centers) enlarged without white halo formation. This corresponded to no visible necrotic lesions at the time of transfer (Fig. 10). These observations clearly indicated that, all the incipient lesions (infective centers) enlarged but visible necrotic lesions did not, and visible lesions which developed a wound periderm remained the same size. It can be
inferred t’hat at thr time of lesion appcarante, the stimuli causing wound reactions had already gone to neighboring cells, and transfer to 30°C did not change the reaction of the neighboring cells. An alternat’ive interpretation is t’hat the callose deposition was the primary barrier even in the young leaves where woundperiderm formation xas the most striking response to VM infection. In both young and old leaves, callose deposition is a rapid process (Currier, 1957; Wu et al., 1969), and callose can be detected as soon as the lesion appears. Such callose deposition alone may constitute an effective barrier to further VM virus spread. This inference is supported by the observation that the nonhalo lesions on mature and older leaves did not expand upon transfer to higher temperature. Slowness in spreading is characteristic of Vikf lesims. The fact that slowness in spreading is the nature of the VM strain can bc demonstrated by incubating inoculated N. glutinosa leaves in complete darkness at 30°C. Under these conditions, U1 makes large green spots (Hayashi and Matsui, 1965) as shown in Fig. 11, \+hile VM makes very small green spots instead of necrotic lesions. The uninfected areas senescc rapidly, becoming yellow, and only infected areas remained green. At 20-25”C, and in the light (under which conditions VM causes necrosis)
WOUND-HEALING
AND
TMV
LESION
SIZE
451
FIGS. 7,8, 9, and 10. An experiment to find the incubation period (at 22°C) necessary for VM lesions to become unresponsive to high temperature (30°C) induction of rapid lesion expansion. Left halves of all the leaves were incubated at 22°C for various durations after VM inoculation before transfer to 30°C. Figure 7 represents 4 days; Fig. 8, 3 days; Fig. 9, 2 days; and Fig. 10, 1 day at 22°C. The right halves of all the leaves were inoculated with VM immediately before transfer to 30°C. All lesions on the right halves corrtinued to expand after the appearance of lesions. The results were photographed 6 days aft,er incubation in 30°C.
the slo~n~~ss in spreading of VRI lesions perhaps allolvs the: noninfected neighbor cells sufficient time to respond to necrosis of infected cells hy ivound-healing processes lvhich retard virus spread, causing lesions of
limited size. On the other hand, the fast spreading of U1 must be responsible for t’he unlimited cnlargomcnt of U1 necrotic lesions, since invasion of U1 is so fast there would not bc enough time for uninfected neighbor cells to undergo wound-healing reactions. DISCUSSION
The observation that TbIV st,rain VM formed small lesions of limited size (Wu et al., 1960) raised the question as to what limits the expansion of lesions since the U1 or the U, strains produced large lesions which
continued to cnlargc n-it’h time (Rappaport and Wildman, 19:i7). Although both types of lesion formation (UI and VM lesions) are generally rcfcrred to as a hypersensitive reaction, there is, however, a basic difference between the two kinds of lesions. The histological changes associated with VM lesion formation revealed that the process for limiting lesion size may be associated with well-known wound-healing mechanisms (Bloch, 1941, 1952; Wylie, 1930, 1931; Lipetz, 1970). l&au (1933) found that a virus caused abnormalities which were associated with the necrosis of vascular systems. She concluded t’hat vein clearing, vein banding, hyperplasia, lignification, and suberization are common responses to injury, and not
482
FIG. 11. Comparison of the rate of virus spread between U1 and VM strains in N. glutinosa without involvement of necrosis. UI makes large green spots, VM makes small green spots, when the detached leaves are kept at 30°C in darkness. This picture was taken 3 days after virus inoculation.
necessarily specific reactions to viruses. The present study agrees with her conclusion, that normal wound-healing processes are involved in the characteristic expression of virus symptoms. However, these nonspecific wound reactions become significant when they are considered together with the host resistance to the expansion of virus infection. It is suggested that the wound-healing process is intimately associated with the defense mechanism of the leaf tissue which inhibits virus spread from cell to cell. This conclusion was reached by the following reasoning: Since incubation of M. glutinosa at high temperature (35-36°C) after inoculation with TMV (either U, or VM) produced systemic symptoms rather than a necrotic reaction, it is obvious that the necrotic reaction (hypersensitivity) is lost at 35°C (Samuel, 1931). The necrosis of the infected cells is thus a temperature-dependent reaction. In plants incubated at 30°C the appear-
wu ance of VM lesions is delayed for 24-30 hr compared to those at 21°C and the resultant necrotic lesions expand as a function of time. The hypersensitivity of leaf cells to VM infection at 30°C is not so strong as at 21°C since death of the infected cells was delayed for 2430 hr. Thus, the so-called hypersensitive reaction is not all of the same degree, but there is a gradient depending upon the temperature during the incubation period. By the time (2-3 days after inoculation) when the VM lesions became visible at a lower temperature (2l”C), the stimuli causing wound reactions already had gone to the neighboring cells and the subsequent changes in structure (wound periderm in young leaves; callose deposition in old leaves) were effective in preventing virus from spreading even when leaves were transferred to a higher temperature (30°C). It seems that normal wound healing is the common mechanism of defense against pathogens in plant tissues, and the nonlimitation of virus spread in leaf tissue represents the exceptional case in which the wound-healing mechanism fails to operate properly. Therefore, a question arises as to what makes the TMV strain-U1 lesion expand indefinitely, and why does the wound-healing mechanism fail to operate? The continuous enlargement of the U1 lesions at normal temperatures (al-22°C) may have character in common with that of VM lesions in higher temperature (30°C). The continuous enlargement of lesions can be accounted for by the slower death of infected cells accompanied with greater rapidity of virus spread than the process of wound healing. Therefore, any factors which contribute to the rapid spread of virus and slow process of barrier formation (wound healing) induced by the necrosis of cells may contribute to the growth characteristics of expanding lesions. The relationship between the antiviral factors (Sela and Applebaum, 1962) and host resistance as revealed by the direct change in host histology is not clear. It is conceivable that the antiviral factors (AVF) are formed in infected cells as a part of the death process of virus-infected cells which decreases the virus replication and results in slower virus movement. Since the rate of virus movement
WOUND-HEALING
AND
may be a function of rate of virus synthesis, any factors which slow down virus synthesis and/or movement may aid in the woundhealing process of uninfected adjacent cells by giving these uninfected adjacent cells time to react to t,he incoming stimuli of necrotic cells. it is However, in this investigation, pointed out t’hat from a histological viewpoint it may be possible to account for the factors which limit, necrotic lesions from expanding, wit,hout invoking the substance specifically induced by virus infection. Xany physical and chemical trcatmcnts induced large necrotic lesion formation in response to virus infection (Wu, Stubbs, and Dimitman, 1968). The large lesion formation was attributed to inactivation of host resistancc by the physical and chemical treatments, \\;hil(l Still allowing virus multiplication and movement to continue (Wu et al., 1909; Simons, Isracxl, and Ross, 1972). There ma?’ bc ma,ny rn&ant factors involwd, and callosc~ dq,osition \vas thought to b(‘ one which corrt4attrd with nonenlargc>mctnt of lwions in pinto bran primary Iravcs (Wu et al., 1969). Hiruki and Tu (1972) made d&&d studies \\-ith tho potato virus :\I-kidrwy bean system using bot,h light and elect,ron micaroscopy, and have: confirmed that thci deposition of callow occurs on the \zalls of nonnwrot~ic wlls, \vhich surround the necrotic cells. Rowv~~r, Spwc’er and Kimmins (1971) using the Ul-pinto bean system were unahl~ to dctcsct’ tho prcsww of callose in their ultrast’ructural studiw. The: reason for such discrcpawy is not, clear, but the comment made bp Hiruki and Tu (1972) seems appropriate. “The apparent lack of callosc . . as dctcrmincd by c~lwtron microscopy, may wll bc due to the different area of tissue cxamincd or to the method of sampling for obscvvation as well as to the use of different t~wlmical procedures.” They (Hiruki and Tu, 1972) further suggested additional histologic~al changes (secondary Ivall thickening in cells of the seminecrotic zone) might also be involved in limitation of lesion enlargemcint. The callosed cell wall might be further modified by deposition of other chemicals swh as polyphenols. Simons et al., (197:‘) using Nicot&w~a
TMV
LESION
SlZE
483
L. ‘Samsun NN’ also reported that callosc was not detected in their electron microscopic studies. In this case too, the comment made by Hiruki and Tu (1972) mentioned above may apply. However, it is always possible that the resistance in the ‘Samsun NN’ may involve factors other than callose deposition, as suggested by the authors. tabacum
ACKNOWLEDGMENT I thank
Dr. S. 0. Wildman
for his assistance in space at U.C.L.A. A special appreciation is due to Drs. L. M. Blakely, J. G. Bald, J. E. Dimitman, and M. Zaitlin for their valuable suggestions and criticisms during the course of this study and their assistance in preparation of this manuscript.
supplying virus samples and greenhouse
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wu
484 marginal tissue in red kidney ogy 62, 77-85.
bean. Phylopalhol-
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