- Email: [email protected]
Induction of localized necrotic lesions by actinomycin D on leaves systemically infected with tobacco mosaic virus
48,601-603
VIROL0G-F
(19n)
Short uction of Localized
Necrotic Infected
Communications
Lesions by Actinomycin with Tobacco Mosaic Virus
It was reported in our previous papers that leaves from hosts which normally become systemically infected with tobacco mosaic virus (TBIV) developed local lesions if they were exposed to a brief heat or cold treatment (3, 4, 7). This paper will describe similar necrotic lesion formation on both systemic and necrotic lesion hosts induced by actinomycin D (Merck Sharp & Dohme) or chromomycin A8 (Takeda Pharmaceutical industries, Ltd., Osaka). Leaves (8-10 em long) of Nicotiana tabacum “Xanthi’ or ‘Samsun’ were inoculaOed with TMV (common strain OIM, 10 pg/ml in phosphate buffer, 0.05 M, pH 7.0) and the plants were incubated at 30” under continuous illumination throughout the experiments. One to 2 days after inoculat,ion, the inoculated leaves were detached and their petiofes were put into vials containing .25-0.50 ml of a solut,ion of 200 pg/ml actinomycin D (50-100 pg/leaf). Aft’er the aetinomycin D solution was completely absorbed through the petioles within one hour, distilled water was added to the vials. About 6-7 hours after the treatment with aetinomyein D, localized necrot’ic lesions appeared on the treated leaves, as shown in Fig, la. No lesions appeared on the healthy control leaves which were rubbed with phosphate buffer alone and then treated with actinomycin D. The lesions which formed on t’he treated leaves appeared at. first as necrotic rings and then the apparently intact tissues of the inner part of the rings gradually coliapsed. Experiments were conducted t’o determine whether a similar chemical treatment would result in lesion formation on leaves of a necrotic lesion host infected with TMV and incubated at 30”, a t’emperature at which no lesions appear and in which TMV 601 Copyright
c
I872 by Academic
Press,
Inc.
spreads systemically. Leaves of N. &&1os~~ or Nicotiana tabacum. “Xanthi nc’ were inoculat,ed with TMV, and the plants were incubated at 30” under continuous ilhnnination. One day after the start of the 30” incubation, the inoculated leaves were detached, treated with actinomycin D ah described above, and then incubated again at 30”. As shown in Figs. IF and Pd, necrotic lesions appeared 6-7 hours after the treatment. That the necrotic areas corresponded with t,he initial &es of infection was &on-n by a regular decrease in lesion number with the dilution of the inoculum, increase in lesion size as the interval between the inocuiation and the chemical treatment was increased, and also by a constant recovery of virus from lesion areas but not from nonlesion areas. No lesions appeared on untreated control leaves which were ’ after inoculaincubated const.antly at control lea,ves tion, nor on uninoculat which were rubbed with and then treated with actmomycin D. The lesions formed csn leaves of necrotic lesion. hosts by this treatment were distinctiy dark brown, whereas those in the case of systemic, hosts were faint brown at 30”. Further experiments were conducted to determine whet’her lesions would iorm if infect’ed leaves of a systemic host or a necrotic lesion host were treated with chromomycin A,. Chromomycin A8 was originally isolated from culture filtrates of Streptomgces griseus hTo. 7 (ATCC 13273) by Tatsuoka et d. (8), and was shown to inhibit DSA-dependent RNA synthesis as does actinomycin D (3, 9). The results of tin A, our experiment show that chr imuiais also similar t’o actinomycin tive activity on TXV symhesis CTable I.)One day after inoculation with TR4”1?‘,Dot-
602
SHORT
COMMUNICATIONS
FIG. 1. Localized necrotic lesions formed by actinomycin D or chromomycin &on leaves systemically infected with TMV, photographed 1 day after the treatment. a, Necrotic lesions formed on ‘Xanthi’ tobacco leaves treated with actinomycin D 1 day after inoculation; b, Necrotic lesions formed on N. glutinosa leaves treated with actinomycin D 1 day after inoculation; c, Necrotic lesions formed on N. glutinosa leaves treated with chromomycin AS 1 day after inoculation; d, Necrotic lesions formed on N. glutinosa leaves treated with actinomycin D 2 days after inoculation. In b, c, and d the leaves were treated at 30”, a temperature at which necrotic lesions normally do not appear.
SHORT TABLE
COMMUNICATIONS
1
EFPECTOFACTISOMYCIN D AND CHROMOMYCIN AS ox TMV MULTIPLX-4~x0~ AND NUCLEIC ACID CONTENT IN LEAF DISKS
Actinomycin Ghromomycin
(pg’m1)
Decrease in nucleic acid content (%)6
Increase in TMV content (70)”
10 25 100 200
13 17 15 24
15 18 16 36
(fr($$
Chemicals
D Aa
Q Actinomycin D at 50 ,&ml caused a chemical injury. * Healthy ‘Xnnthi’ tobacco leaf disks were floated on various concentrations of chemicals or distilled water at 23”. Nucleic acid content in leaf disks 3 days after incubation was estimated by the method of Schneider (5). c TMV (10 pg/ml) inoculated ‘Xanthi’ tobacco leaf disks were floated on various concentrations of chemicals or distilled water at 23”. TMV content in leaf disks 4 days after incubation was estimated by chemical assay method (6).
tached leaves of the systemic hosts (‘Xanthi’ and ‘Samsun’ tobacco) and the necrotic lesion hosts (N. glutinosa and ‘Xanthi nc’ tobacco) were treated with 0.5-1.0 ml of a solution of 500 pg/ml chromomycin A, in t’he same way as actinomycin D was applied, and then incubated at 30”. Localized necrot’ic lesions which closely resembled those produced by actinomycin D appeared on the leaves of both kinds of hosts treated with chromomycin Aa (Fig. lc). In other experiments, actinomycin D and chromomycin Aa also induced necrotic lesions on the leaves of N. ghtinosa inoculated with cucumber mosaic virus, However, rifampicin (400, 600, 1000 pg/leaf), 2-thiouracil (100, 500, 1000 pg), mitomyein C (50, 100, 200 pg), cycloheximide (IO, 50 pg), blasticidin S (0.5, 4.0 g) and chloramphenicol (200, 400 ,ug) which are inhibitors of nucleic acid or protein synthesis, did not induce localized necrotic lesions on the leaves of N. gluutinosa systemically infected with TNIV and incubat,ed at 30”.
603
Since localized necrotic lesions can be induced equally by t’reatrnent with netinomvcin D and chromomycin A, which arc i considerably different in chemical constitution, the induction of lesion formation by both these chemicals may be ascribed to their common inhibitory activity on dependent RNA synthesis. The exact, nature of the action of these chemicals, however; remains to be studied. Hirai and Wildman (1) reported systemic symptoms ou uninfected tobacco plants treated -or-ith aetinomycin D, which are similar to t8hose produced by TJBV. Apparently, me are dealing with separate aspects of the action of this chemical. ACKNOWLEDGMENTS We wish to acknowledge the valuable advice of Dr. T. T. Iida. Thanks are due also to Merk Sharp and Dohme for actinomycin D, and Takeda “barmaceutical Industries, Ltd. for chromompcin &. REFERENCES 1. HIRAI, A., and WILDMAX~ S. G., Viroloyg 3X. 721-722 (1967). 2. KAJIR~, Y., and KAXIYANA, M., Biochenz. Bsophys. Res. Gommun. 19, 433-437 (1965). 3. OH~SHI, Y., and SHIMOMURI, T.> A-nr~. Pkyfopathol. Sot. Jap. 37, 22-28 (1971). .J. Oa~srrr, Y., and &~IMOM~RA. T., Anr,. Phylapathol. Sot. Jap. 37, 211-214 (1971). 5. SCHNEIDER, W. C., J. Biol. Chem. P61., 293-393 (1945). 6. SHIMOMURA, T., Phytopathol. Z. 70, 185-196 (1971). 7. SHIMOMURA, T., and QW.ISHI, Y., Virology/ 43, 531-532 (1971). 8. TAEXJOKA, S., i%IL4L4\VA, M., ?&~.\KE, ii., KAZIT\IARA, Ii., ARAXAKI, Y., SHIKITI, M., TANABE, K., KGLADA, Y., HITOMI, R., MIY~LMOTO, M., MIZUNO, K., W.%~azr.
VIROL0G-F
(19n)
Short uction of Localized
Necrotic Infected
Communications
Lesions by Actinomycin with Tobacco Mosaic Virus
It was reported in our previous papers that leaves from hosts which normally become systemically infected with tobacco mosaic virus (TBIV) developed local lesions if they were exposed to a brief heat or cold treatment (3, 4, 7). This paper will describe similar necrotic lesion formation on both systemic and necrotic lesion hosts induced by actinomycin D (Merck Sharp & Dohme) or chromomycin A8 (Takeda Pharmaceutical industries, Ltd., Osaka). Leaves (8-10 em long) of Nicotiana tabacum “Xanthi’ or ‘Samsun’ were inoculaOed with TMV (common strain OIM, 10 pg/ml in phosphate buffer, 0.05 M, pH 7.0) and the plants were incubated at 30” under continuous illumination throughout the experiments. One to 2 days after inoculat,ion, the inoculated leaves were detached and their petiofes were put into vials containing .25-0.50 ml of a solut,ion of 200 pg/ml actinomycin D (50-100 pg/leaf). Aft’er the aetinomycin D solution was completely absorbed through the petioles within one hour, distilled water was added to the vials. About 6-7 hours after the treatment with aetinomyein D, localized necrot’ic lesions appeared on the treated leaves, as shown in Fig, la. No lesions appeared on the healthy control leaves which were rubbed with phosphate buffer alone and then treated with actinomycin D. The lesions which formed on t’he treated leaves appeared at. first as necrotic rings and then the apparently intact tissues of the inner part of the rings gradually coliapsed. Experiments were conducted t’o determine whether a similar chemical treatment would result in lesion formation on leaves of a necrotic lesion host infected with TMV and incubated at 30”, a t’emperature at which no lesions appear and in which TMV 601 Copyright
c
I872 by Academic
Press,
Inc.
spreads systemically. Leaves of N. &&1os~~ or Nicotiana tabacum. “Xanthi nc’ were inoculat,ed with TMV, and the plants were incubated at 30” under continuous ilhnnination. One day after the start of the 30” incubation, the inoculated leaves were detached, treated with actinomycin D ah described above, and then incubated again at 30”. As shown in Figs. IF and Pd, necrotic lesions appeared 6-7 hours after the treatment. That the necrotic areas corresponded with t,he initial &es of infection was &on-n by a regular decrease in lesion number with the dilution of the inoculum, increase in lesion size as the interval between the inocuiation and the chemical treatment was increased, and also by a constant recovery of virus from lesion areas but not from nonlesion areas. No lesions appeared on untreated control leaves which were ’ after inoculaincubated const.antly at control lea,ves tion, nor on uninoculat which were rubbed with and then treated with actmomycin D. The lesions formed csn leaves of necrotic lesion. hosts by this treatment were distinctiy dark brown, whereas those in the case of systemic, hosts were faint brown at 30”. Further experiments were conducted to determine whet’her lesions would iorm if infect’ed leaves of a systemic host or a necrotic lesion host were treated with chromomycin A,. Chromomycin A8 was originally isolated from culture filtrates of Streptomgces griseus hTo. 7 (ATCC 13273) by Tatsuoka et d. (8), and was shown to inhibit DSA-dependent RNA synthesis as does actinomycin D (3, 9). The results of tin A, our experiment show that chr imuiais also similar t’o actinomycin tive activity on TXV symhesis CTable I.)One day after inoculation with TR4”1?‘,Dot-
602
SHORT
COMMUNICATIONS
FIG. 1. Localized necrotic lesions formed by actinomycin D or chromomycin &on leaves systemically infected with TMV, photographed 1 day after the treatment. a, Necrotic lesions formed on ‘Xanthi’ tobacco leaves treated with actinomycin D 1 day after inoculation; b, Necrotic lesions formed on N. glutinosa leaves treated with actinomycin D 1 day after inoculation; c, Necrotic lesions formed on N. glutinosa leaves treated with chromomycin AS 1 day after inoculation; d, Necrotic lesions formed on N. glutinosa leaves treated with actinomycin D 2 days after inoculation. In b, c, and d the leaves were treated at 30”, a temperature at which necrotic lesions normally do not appear.
SHORT TABLE
COMMUNICATIONS
1
EFPECTOFACTISOMYCIN D AND CHROMOMYCIN AS ox TMV MULTIPLX-4~x0~ AND NUCLEIC ACID CONTENT IN LEAF DISKS
Actinomycin Ghromomycin
(pg’m1)
Decrease in nucleic acid content (%)6
Increase in TMV content (70)”
10 25 100 200
13 17 15 24
15 18 16 36
(fr($$
Chemicals
D Aa
Q Actinomycin D at 50 ,&ml caused a chemical injury. * Healthy ‘Xnnthi’ tobacco leaf disks were floated on various concentrations of chemicals or distilled water at 23”. Nucleic acid content in leaf disks 3 days after incubation was estimated by the method of Schneider (5). c TMV (10 pg/ml) inoculated ‘Xanthi’ tobacco leaf disks were floated on various concentrations of chemicals or distilled water at 23”. TMV content in leaf disks 4 days after incubation was estimated by chemical assay method (6).
tached leaves of the systemic hosts (‘Xanthi’ and ‘Samsun’ tobacco) and the necrotic lesion hosts (N. glutinosa and ‘Xanthi nc’ tobacco) were treated with 0.5-1.0 ml of a solution of 500 pg/ml chromomycin A, in t’he same way as actinomycin D was applied, and then incubated at 30”. Localized necrot’ic lesions which closely resembled those produced by actinomycin D appeared on the leaves of both kinds of hosts treated with chromomycin Aa (Fig. lc). In other experiments, actinomycin D and chromomycin Aa also induced necrotic lesions on the leaves of N. ghtinosa inoculated with cucumber mosaic virus, However, rifampicin (400, 600, 1000 pg/leaf), 2-thiouracil (100, 500, 1000 pg), mitomyein C (50, 100, 200 pg), cycloheximide (IO, 50 pg), blasticidin S (0.5, 4.0 g) and chloramphenicol (200, 400 ,ug) which are inhibitors of nucleic acid or protein synthesis, did not induce localized necrotic lesions on the leaves of N. gluutinosa systemically infected with TNIV and incubat,ed at 30”.
603
Since localized necrotic lesions can be induced equally by t’reatrnent with netinomvcin D and chromomycin A, which arc i considerably different in chemical constitution, the induction of lesion formation by both these chemicals may be ascribed to their common inhibitory activity on dependent RNA synthesis. The exact, nature of the action of these chemicals, however; remains to be studied. Hirai and Wildman (1) reported systemic symptoms ou uninfected tobacco plants treated -or-ith aetinomycin D, which are similar to t8hose produced by TJBV. Apparently, me are dealing with separate aspects of the action of this chemical. ACKNOWLEDGMENTS We wish to acknowledge the valuable advice of Dr. T. T. Iida. Thanks are due also to Merk Sharp and Dohme for actinomycin D, and Takeda “barmaceutical Industries, Ltd. for chromompcin &. REFERENCES 1. HIRAI, A., and WILDMAX~ S. G., Viroloyg 3X. 721-722 (1967). 2. KAJIR~, Y., and KAXIYANA, M., Biochenz. Bsophys. Res. Gommun. 19, 433-437 (1965). 3. OH~SHI, Y., and SHIMOMURI, T.> A-nr~. Pkyfopathol. Sot. Jap. 37, 22-28 (1971). .J. Oa~srrr, Y., and &~IMOM~RA. T., Anr,. Phylapathol. Sot. Jap. 37, 211-214 (1971). 5. SCHNEIDER, W. C., J. Biol. Chem. P61., 293-393 (1945). 6. SHIMOMURA, T., Phytopathol. Z. 70, 185-196 (1971). 7. SHIMOMURA, T., and QW.ISHI, Y., Virology/ 43, 531-532 (1971). 8. TAEXJOKA, S., i%IL4L4\VA, M., ?&~.\KE, ii., KAZIT\IARA, Ii., ARAXAKI, Y., SHIKITI, M., TANABE, K., KGLADA, Y., HITOMI, R., MIY~LMOTO, M., MIZUNO, K., W.%~azr.