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Highly infectious phenol extracts from tobacco leaves infected with cucumber mosaic virus
VIROLOGY
Highly
11,
329-338
Infectious Infected
(1960)
Phenol Extracts with Cucumber I-).\VII)
1%
from Tobacco Mosaic Virus
Leaves
~CHIJWI’;L
An unusually infectious fraction of c~11~lu~lt)t~r mosaic> virrls was cst.rwletl from inferted leaves by homogenizing the le:tvcls in :I mist urc of phenol and t et,rasodium pyrophosphute. The aqueous phascl of the homogenate W:W found to be as much as 6.6 times as infcctiorls :ts ext racls of similar Ir:tvc~ prepared without phenol. The infectious agent in the phenol extr:tct wits high1.v sensitive to inactiv:rtion by RNAase, was more stable in 67 ‘A alcohol t.han in distilled water, and had an ultraviolet absorption curve typical for nucleir arid. On t.he other hand, after ultrscentrifugation the Yupernatant, liquid contained only :Itmllt one-third of the infectivity whereas t,he pellet had about t,wo-thirdn of thus infectivit,y, indicating that more t ban OIIC size of iufeciious particle \~:ts present.
l’reutment of purified tobacco mosaic virus (‘l3lV) with cold phenol results in the separation of the virus into its component parts, protein and infectious nucleic acid (Gierer and Schramm, 1956). ‘l’his nucleit acid, however, has less than 1 ‘2 of the infecti\*ity of the original virus. Studies on it’s role in virus infect,ious are therefore difficult tSo evaluate, since freedom from protein contamination must first ho clearly demoitstrated. Recently Wecker (1959) showed that the use of phenol at 40” also liberated infectious TMV-rihonuclcic acid, and that this rihonuclcic: acid (RKA) was slightly more infect,ious than t,hat produc~rd by the cx~ltl pheuol treatment,. Infectious RNA has also been obtained directly from virus-infected plant tissues (Welkie, 1959; Engler and Schramm, 1959; Diener and Weaver, 1959) and animal tissues (Colter, 1958) treated with cold phenol. :Is in the case of the phenol treatment of purified viruses, large tosses of infectivity resulted from the phenol treatment of \-irus-iuft:ct,ed plant, and animal t,issues. However, t,hc phenol t reatmrut of tissue in-
330
SCHLEGEL
fected with cucumber mosaic virus (CMV) resulted in infectivities of 0.5-5.0 times that of buffer homogenates of the same tissue (Schlegel, 1960). The very high infectivity of phenol preparations of CMV-infected tissues is of great interest since it provides a tool for a more precise study of the role of nucleic acid in the infection process. This paper is therefore devoted to a study of some of the properties of this unusually infectious “RNA.” MATERIALS
Aru’l) METHODS
Tobacco leaves, Nicotiuna tabacum L. var. Xanthi, n.c., systemically infected with cucumber mosaic virus, were used in all experiments. Infectious “RNA” was prepared from CMV-infected tobacco leaves by methods described previously (Welkie, 1959; Schlegel, 1960). Virusinfected leaf tissueswere homogenized in four or more times their weight of a mixture of equal volumes of cold, water-saturated phenol and 1 o/L tetrasodium pyrophosphate solutions for 5-8 minutes and then centrifuged. The aqueous phase was then either diluted with 1% KtHPOb and used as inoculum immediately, or treated with phenol two more times followed by three ether extractions and two alcohol precipitations, depending upon the use to be made of the preparation. Previous work showed that “RNA” prepared by a single phenol extraction was more infectious than “RNA” given the entire purification procedure (purified “RNA”) (Schlegel, 1960). Consequently, wherever possible the former method was used. For convenience the term “RNA” has been used throughout to designate the infectious material released by the phenol treatment, since it is considered possible that the infectivity of the phenol preparations of diseased tissues is not due to RNA alone. This aspect of the problem will be covered in more detail later in the paper. Virus and “RNA” infectivities were determined by local lesion assay on the primary leaves of lo-14-day-old cowpea, Vigna sinensis (L.) Endl., plants. The inoculations were made in the laboratory where refrigeration facilities were available, since solutions had to be kept cold prior to and during the inoculations. Cowpea seedlings lo-14 days old were cut, brought into the laboratory and held with their cut stems in tap water. A single 40-watt fluorescent light 18 inches above the plants provided light during the 24448-hour period from inoculation until the lesions were counted. The infectivity of the phenol extracts was enhanced strikingly by making the inoculum dilutions with 1% K2HP04 as compared with dis-
ISFECTIOUS
PHENOL
EXTR.4CTS
FK0.M
TOR;1C(‘O
I,F:.IVES
:::3 I
tilled water or 1% tetrasodium pyrophosphat)e. Therefore, both the phenol-treat-ed homogenat,es and the buffer homogenates (Yarwood, 1952) were diluted to a final concentrut,ion of 1 ‘ii K,HP01. Dilutions were necessary in order to reduce the resulting lesions to a countable number. Inoculum dilutions of 1: 100-l :300 of t,hc original tissue ivere used throughout t,hese experiments. All inoculat~io~ were mudr using I’nrborundum and a glass spatula. The infect ivities of “RKA” prepared by honlogeniz:tt.io~~ of diseased tissues in phenol-water, phenol-phosphate buffer, phenol-pyropho~phat’c*, :III~ phenol-pyrophosphatc plus Versc11~ wer(’ compared. ‘l’hr~ phenol-pyrophosphate mixture was found to give preparut*ions of high infectivity; although t’he phenol-pyrophospha,te-Verlc preparat,ions also resulted in high infectivities, hhey did Ilot, give as reproduci bl(a results as the phenol-pyrophosphntc alone. (‘onsequcntly, the Iattcr was used t,hroughout this work. l;requent’ly t#he results of different, experiments showed considerable variability, making their interpretation difficult. As a consequence, extra care was given to maintaining uniform elrvironmentul condit,ions surrounding the tobacco leaves used for the production of CM\‘. Only the younger leaves of young greenhouse-grown tobacco plants about .i inches high were used. These leaves were harvested and brought into the laboratory where they were Cart)oruiidum-dusted and brush-inoculutcltl with CMY. CMV-infected tobacco tissues, homogenized in t,rn times their Gght of 1 7%~K~HP04, served as the inoculum. The inoculnt~ctl lcaves were washed thoroughly wit,h tap water and cwcw water was removed by blotting with paper towels. These lruv~ were floated for 4-5 days on t,ap water in a light chamber (1300 foot-candles) at 26” f 0.5”. They were then stored in the refrigerat)or at 5” until uwl (I-1 days).
The infect#ivity of the phenol-treated homogenat,es was t’ypically about 1.5Xi.G times that of the buffer homogenates (Table I). Comparisons were also made of dilution curves of the phenol extracts and buffer homogenates. The inocula were adjusted so that each resulted in npproximately the same number of lesions when inoculated to cowpea. They were then diluted serially and test’ed for infectivity. The slopes of the dilution curves obtained in these experiments were cssent,ially parallel (Fig. 1). It, was of interest t’o note that lesions appeared more quickly on cowpeas inoculated with phenol extracts than with bufier homoqennttls.
332
SCHLEGEL
TABLE
1
THC RELATIVE INFECTIVITY OF “RNA” AND BUFFER HOMOGENATES COMPARABLE TISSUE SAMPLES AS SHOWN BY THE AVERAGE NUMBER LESIONS PER LEAF ON 4 OR 5 INOCULATED COWPEA LEAVES Experiment
FROM OF
number
Inoculum”
“RNA” Tissue Ratio n Assays
homogenate “RNA”:virus were
made
1
2
3
4
331 139 2.4
113 17 6.6
540 130 4.2
340 215 1.5
at a dilut,ion
of 1: 100 of original
tissue.
This difference was only about l-2 hours, but since lesions are visible within 16-20 hours after inoculation if the plants are held above 25”, this small difference is probably significant. The ultraviolet absorption curves of these preparations were characteristic of RNA, having a 260 : 230 and 260 : 280 rnp ratio of about 2.0. These ratios are somewhat lower than those reported for the nucleic acids of purified plant viruses (Fraenkel-Conrat et al., 1957; Kaper and Steere, 1959a, b; Rushizky and Knight, 1959). This is not surprising since the “RNA” used in this study was prepared directly from infected whole tissues rather than from highly purified virus nucleoprotein and some contamination by cell components, other than nucleic acid, is to be expected. Attempts to make quantitative protein determinations on these preparations, using the method of Ramachandran and FraenkelConrat (1958) were uniformly unsuccessful owing to the formation of precipitates, probably as a result of a carry-over of interfering materials. Aqueous solutions of purified “RNA” remained infective for only l-2 hours at room temperature. Aliquots of the same preparations held in the refrigerator at 5’ retained infectivity for about 24 hours. On the other hand, aliquots of “RNA” in 67 % alcohol (second alcohol precipitation) were infective after 3 months’ storage at -18”, although there was a gradual decline in the infectivity. Similar alcohol preparations were infective for only 3-5 days when stored at 5”. A solution of “RNA” in distilled water frozen at - 18” lost its infectivity in about 1 week. Both purified “RNA” and tissue homogenate were readily inactivated by ribonuclease (RNAase); however, the “RNA” was lo-100 times more sensitive to RNAase inactivation than virus in tissue homogenates. In a typical experiment the infectivities of the ‘
11VFECTIOUS
1000
FIG.
EXTRACTS
FROM
TORi\NYf
LK4VES
. v,*, .I*).’
r
/ 1.0 I o-3
6’ItP\‘A”; prosimstcly
PHENOL
lO-2 CONCENTRATION
10-l OF INOCULUM
1. 1)ilution curves of phenol eAracts and tissue homogenates; O-----O, O-0, tissue homogenates. The inocula were adjusfd to giw nI)the same numtwr of lesions on POW~FL:~ at thr highest ~oric.~ntI,:ttiotI.
virus were bot#h adjusted to give about. 1000 lesions per leaf brfore t)hc addition of RKAsse. RKAase was then added to both preparations to give a range of enzyme concentrations varying from 1.00 t)o 0.0001 pg/ml. The mixture was then incubated at 4” for 1 hour and the infectivities of each determined. As is shown in Fig. 2, at 0.01 wg/‘ml RKAasr, only about two lesions appeared on leaves inoculat8ed with the “RNA”RNAase mixture, whereas t)here was no reduction in the number of lesions produced by the tissue homogenate-RKAase mixt,ure. Higher concentrations of RKAase further reduced the infect,ivit>y of the phenol extract, and also the virus in t,he tissue homogenates. .Uthough the “RKA” was sensitive to much smaller amounts of IZNAase than the virus in tissue homogenat,es, the slopes of t,he two curves were similar. To dekrmine the sedimentation behavior of phenol extracts, several preparations were ultracentrifuged. Phenol extracts and tissue homogenates were prepared by t.he usual methods and each was divided int,o t,wo samples. One sample of each was centrifuged for I,$>-2 hours :II 114,000 9 in a refrigerated aenkifuge and the other wa?; held in the
334
SCHLEGEL IOOO~ \ \
0.000 I FIG. different
WHOLE
0.01 0.001 RNAASE CONCENTRATION
2. The inactivation concentrations
of tissue at RNAase
VIRUS
0.10 (pg/ml)
homogenates and “RNrZ” at 4” for 1 hour.
1.0 by incubation
with
refrigerator as a control. After centrifuging, the infectivity of the supernatant liquids and controls were compared (Table 2). The results showed that from one-half to three-fourths of the infectivity of phenol preparations was found in the ultracentrifuge pellet. In spite of this large loss, the infectivity remaining in the supernatant liquid was equal to or greater than the original infectivity of the tissue homogenate. The infectivity of the tissue homogenate was completely sedimented. To learn whether the phenol extracts of purified CMV were as infectious as, or more infectious than, the virus, attempts were made to purify CMV and to extract the RNA with phenol. Unfortunately, purification of this isolate of CMV proved to be very difficult. Numerous purification procedures were tried, including the use of CMV-infected cucumber (Cucumis sativus L. var. Straight Eight) corolla tissue as a virus source (Sill and Walker, 1952), and density gradient, fluorocarbon, isoelectric point, and ammonium sulfate techniques, none of which was
“RNA” Ultracentrifuge supernatant 15w 3 I ‘1 -I!)”
Buiier
Uncentrifuged
control
Ultracentrifuge supernatant
homogenates ITncentrifugetl control
331 113 127
‘1 Cenlrifnged 90 minutes. I1 l’rtrificd “RNA,” centrifuged
2 hours.
successful. The method of ‘I’omlinson et al. (1959) was used with relatively little success, but in two trials infectious partially purified virus was obt’ained. RXA extracted from bhis material by treatment with phenol had only 0.5-0.7 ‘% of the infectivity of the partially purified \.irus. This is of t,he same order of magnitude as t)hat observed for RXA from purified TRlV (Gierer and Schramm, 1956), tobacco ringspot (Kaper and Steerc, lY5Ya), t~urnip yellow mosaic (Kaper and Steere, 1959b), and tomato bushy st,unt (Rushieky and Knight’, 1950) viruses. Further verificatiorl of these results will be recfuired before t’he infcct’ivitv of RNA from puritied CRfV can be definit~ely established.
The trent)ment of ChIV-infected tobacco leaves with phenol-pyrophosphnt,e results in preparations several times as infectious as buffer homogenates of the same amount of virus-infect,ed t,issues. This infectivity may be due to (1) infectious RNA released from intact’, or possibly incomplete, virus by the action of phenol, (2) free infectious RSA which was protected by the phenol from RNAase degradaCon during homogenizatjion and extraction, (3) partially degraded virus whose RXA was exposed m such a manner t.hat it exhibits certain RNA characteristics, and (1) whole virus carried through the entire procedure. Actually it seems probable that more than one of t’hese explanations are involved since t,he data oht)ained do not, support any single hypothesis to t’hr exelusion of the ot,hers. The data obtained from t.he ult,racentrifugation experiment,s seem to indicate the presence of infectious material of at least two different sizes,
336
SCHLEGEL
since about 65% of the infectivity was found in the ultracentrifuge pellets. The infectious agents remaining in the supernatant were not, however, sedimented after 2 hours at 144,000 g, and, therefore, are probably much smaller than the virus. The presence of different-sized particles in these phenol preparations may indicate that both RNA and partially degraded or whole virus were present. It is also possible that the phenol extracts contained highly aggregated RNA which was sedimented during centrifugation. It does not appear likely that much of the sedimented infectious material is whole virus. The properties of uncentrifuged phenol preparations differ in certain respects from those of tissue homogenates, and these differences are evidence against any hypothesis that attributes the increase in infectivity following treatment with phenol to an increase in the amount of whole virus carried through the procedure as a result of the neutralization of virus inhibitors or the dispersion of virus aggregates by phenol. The more rapid appearance of lesions on plants inoculated with “RNA” also suggests that RNA is the infectious material, since this property has been reported for tobacco mosaic virus-RNA (Schramm and Engler, 1958; Fraenkel-Conrat et al., 1958). If nucleic acid is the only infectious material present in these preparations, it would appear, at first sight, that it could not have come entirely from virus within the tissues, since the infectivity of RNA from purified CMV is low. There is, however, the possibility that the infectivity of the buffer homogenate is not a full measure of the total amount of virus present. It is well known that CMV is readily lost in the slow speed centrifuge (Tomlinson et al., 1959). It may be that only a very small fraction of the total virus present is infective and that the remainder is bound by inhibitors, other cell components, or in large noninfective virus aggregates. If treatment of CMV-infected tissues with phenol releasesall the virus-RNA present, including that from virus which is bound and noninfective, it may explain the high infectivity of the phenol extracts. If this is true, the tot,al concentration of CMV in the tissues may approach that of tobacco mosaic virus. The greater sensitivity of “RNA’” than virus to inactivation by RNAase is to be expected if RNA is primarily responsible for the infectivity observed in the phenol extracts. On the other hand, when sufficient RNAase was added, inactivation of the tissue homogenates took place at about the samerate as the inactivation of the “RNA” (Fig. 2). The inactivation of CMV in tissue homogenates by RNAase has also been shown by Badami (1959). He reported that inhibition occurred
INFECTIOUS
PHENOL
EXTRACTS
FROM
TOI3.\(‘(‘0
LE.iVES
:‘,:17
immediat’ely after the virus and RKAase were mixed and that the infectivitjy was restored when the mixture was diluted suitably. Since the ina&ivation was immediate and infectivity was regained upon dilut,ion, it was concluded that the inhibitory effect of ribonuclease upon the infect’ivity of &sue homogenates was not’ due t,o enzymatic hydrolysis. A similarity between “RKA” and the virus in tissue homogenates can bc seen by comparing dilution curves of bot,h preparations (Fig. I ). Here t,hc curves were essentially parallel. In this respect, t’he RNA preparatjiolln of C&IV-infected tobacco leaf tissues differ from RX-4 from tobacco mosaic virus since Rawden and Erie (1957) showed that, t,hr illf&ivity of the latter decreased more rapidly on dilution than did t,hnt of the \G-us. It is clear from this discussion that no definite conclusions can be drawn as to t,he precise nature and source of the highly infectious material present in phenol extracts of CMV-infecbed t’issues. This, however, does not alter the significance of the consistently higher infectivitjy obtained with phenol extracts than with buffer homogenat’es of similar tissues. Regardless of the explanation of this phenomenon, it appears very significant t’hat,, in this disease, keatment with phenol produces more rather than less infectious material.
I
338 soluble protein
SCHLEGEL
and infectious
Virology 8, 527-530. RAMACHANDRAN, L.
nucleic
acid from turnip
yellow mosaic virus.
K., and FRAENKEL-CONRAT, H. (1958). The estimation of protein contamination in ribonucleic acid. Arch. Biochem. Biophys. 74,224-228. RUSHIZKY, G. W., and KNIGHT, C. A. (1959). Ribonuclease-sensitive infectious units from tomato bushy stunt virus. Virology 8, 448-455. SCHLEGEL, D. E. (19FO). Transmission of several plant viruses by phenol-water extracts of diseased tissues. Phytopathology 60, 156-158. SCHRAMM, G., and ENGLER, R. (1958). The latent period after infection with tobacco mosaic virus and virus nucleic acid. Nature 181, 916-917. SILL, W. H., JR., and WALKER, J. C. (1952). A virus inhibitor in cucumber in relation to mosaic resistance. Phytopathology 42, 349-352. TOMLINSON, J. A., SHEPHERD, It. J., and WALKER, J. C. (1959). Purification, properties, and serology of cucumber mosaic virus. Phytopalhology 49, 293-299. WECKER, E. (1959). The extraction of infectious virus nucleic acid with hot phenol. Virology ‘I, 241-243. WELKIE, G. W. (1959). Infective ribonucleic acid from cucumber-mosaic-infected tobacco. (Abstract.) Phytopathology 49, 114. YARWOOD, C. E. (1952). The phosphate effect in plant virus inoculations. Phytopathology 42, 137-143.
Highly
11,
329-338
Infectious Infected
(1960)
Phenol Extracts with Cucumber I-).\VII)
1%
from Tobacco Mosaic Virus
Leaves
~CHIJWI’;L
An unusually infectious fraction of c~11~lu~lt)t~r mosaic> virrls was cst.rwletl from inferted leaves by homogenizing the le:tvcls in :I mist urc of phenol and t et,rasodium pyrophosphute. The aqueous phascl of the homogenate W:W found to be as much as 6.6 times as infcctiorls :ts ext racls of similar Ir:tvc~ prepared without phenol. The infectious agent in the phenol extr:tct wits high1.v sensitive to inactiv:rtion by RNAase, was more stable in 67 ‘A alcohol t.han in distilled water, and had an ultraviolet absorption curve typical for nucleir arid. On t.he other hand, after ultrscentrifugation the Yupernatant, liquid contained only :Itmllt one-third of the infectivity whereas t,he pellet had about t,wo-thirdn of thus infectivit,y, indicating that more t ban OIIC size of iufeciious particle \~:ts present.
l’reutment of purified tobacco mosaic virus (‘l3lV) with cold phenol results in the separation of the virus into its component parts, protein and infectious nucleic acid (Gierer and Schramm, 1956). ‘l’his nucleit acid, however, has less than 1 ‘2 of the infecti\*ity of the original virus. Studies on it’s role in virus infect,ious are therefore difficult tSo evaluate, since freedom from protein contamination must first ho clearly demoitstrated. Recently Wecker (1959) showed that the use of phenol at 40” also liberated infectious TMV-rihonuclcic acid, and that this rihonuclcic: acid (RKA) was slightly more infect,ious than t,hat produc~rd by the cx~ltl pheuol treatment,. Infectious RNA has also been obtained directly from virus-infected plant tissues (Welkie, 1959; Engler and Schramm, 1959; Diener and Weaver, 1959) and animal tissues (Colter, 1958) treated with cold phenol. :Is in the case of the phenol treatment of purified viruses, large tosses of infectivity resulted from the phenol treatment of \-irus-iuft:ct,ed plant, and animal t,issues. However, t,hc phenol t reatmrut of tissue in-
330
SCHLEGEL
fected with cucumber mosaic virus (CMV) resulted in infectivities of 0.5-5.0 times that of buffer homogenates of the same tissue (Schlegel, 1960). The very high infectivity of phenol preparations of CMV-infected tissues is of great interest since it provides a tool for a more precise study of the role of nucleic acid in the infection process. This paper is therefore devoted to a study of some of the properties of this unusually infectious “RNA.” MATERIALS
Aru’l) METHODS
Tobacco leaves, Nicotiuna tabacum L. var. Xanthi, n.c., systemically infected with cucumber mosaic virus, were used in all experiments. Infectious “RNA” was prepared from CMV-infected tobacco leaves by methods described previously (Welkie, 1959; Schlegel, 1960). Virusinfected leaf tissueswere homogenized in four or more times their weight of a mixture of equal volumes of cold, water-saturated phenol and 1 o/L tetrasodium pyrophosphate solutions for 5-8 minutes and then centrifuged. The aqueous phase was then either diluted with 1% KtHPOb and used as inoculum immediately, or treated with phenol two more times followed by three ether extractions and two alcohol precipitations, depending upon the use to be made of the preparation. Previous work showed that “RNA” prepared by a single phenol extraction was more infectious than “RNA” given the entire purification procedure (purified “RNA”) (Schlegel, 1960). Consequently, wherever possible the former method was used. For convenience the term “RNA” has been used throughout to designate the infectious material released by the phenol treatment, since it is considered possible that the infectivity of the phenol preparations of diseased tissues is not due to RNA alone. This aspect of the problem will be covered in more detail later in the paper. Virus and “RNA” infectivities were determined by local lesion assay on the primary leaves of lo-14-day-old cowpea, Vigna sinensis (L.) Endl., plants. The inoculations were made in the laboratory where refrigeration facilities were available, since solutions had to be kept cold prior to and during the inoculations. Cowpea seedlings lo-14 days old were cut, brought into the laboratory and held with their cut stems in tap water. A single 40-watt fluorescent light 18 inches above the plants provided light during the 24448-hour period from inoculation until the lesions were counted. The infectivity of the phenol extracts was enhanced strikingly by making the inoculum dilutions with 1% K2HP04 as compared with dis-
ISFECTIOUS
PHENOL
EXTR.4CTS
FK0.M
TOR;1C(‘O
I,F:.IVES
:::3 I
tilled water or 1% tetrasodium pyrophosphat)e. Therefore, both the phenol-treat-ed homogenat,es and the buffer homogenates (Yarwood, 1952) were diluted to a final concentrut,ion of 1 ‘ii K,HP01. Dilutions were necessary in order to reduce the resulting lesions to a countable number. Inoculum dilutions of 1: 100-l :300 of t,hc original tissue ivere used throughout t,hese experiments. All inoculat~io~ were mudr using I’nrborundum and a glass spatula. The infect ivities of “RKA” prepared by honlogeniz:tt.io~~ of diseased tissues in phenol-water, phenol-phosphate buffer, phenol-pyropho~phat’c*, :III~ phenol-pyrophosphatc plus Versc11~ wer(’ compared. ‘l’hr~ phenol-pyrophosphate mixture was found to give preparut*ions of high infectivity; although t’he phenol-pyrophospha,te-Verlc preparat,ions also resulted in high infectivities, hhey did Ilot, give as reproduci bl(a results as the phenol-pyrophosphntc alone. (‘onsequcntly, the Iattcr was used t,hroughout this work. l;requent’ly t#he results of different, experiments showed considerable variability, making their interpretation difficult. As a consequence, extra care was given to maintaining uniform elrvironmentul condit,ions surrounding the tobacco leaves used for the production of CM\‘. Only the younger leaves of young greenhouse-grown tobacco plants about .i inches high were used. These leaves were harvested and brought into the laboratory where they were Cart)oruiidum-dusted and brush-inoculutcltl with CMY. CMV-infected tobacco tissues, homogenized in t,rn times their Gght of 1 7%~K~HP04, served as the inoculum. The inoculnt~ctl lcaves were washed thoroughly wit,h tap water and cwcw water was removed by blotting with paper towels. These lruv~ were floated for 4-5 days on t,ap water in a light chamber (1300 foot-candles) at 26” f 0.5”. They were then stored in the refrigerat)or at 5” until uwl (I-1 days).
The infect#ivity of the phenol-treated homogenat,es was t’ypically about 1.5Xi.G times that of the buffer homogenates (Table I). Comparisons were also made of dilution curves of the phenol extracts and buffer homogenates. The inocula were adjusted so that each resulted in npproximately the same number of lesions when inoculated to cowpea. They were then diluted serially and test’ed for infectivity. The slopes of the dilution curves obtained in these experiments were cssent,ially parallel (Fig. 1). It, was of interest t’o note that lesions appeared more quickly on cowpeas inoculated with phenol extracts than with bufier homoqennttls.
332
SCHLEGEL
TABLE
1
THC RELATIVE INFECTIVITY OF “RNA” AND BUFFER HOMOGENATES COMPARABLE TISSUE SAMPLES AS SHOWN BY THE AVERAGE NUMBER LESIONS PER LEAF ON 4 OR 5 INOCULATED COWPEA LEAVES Experiment
FROM OF
number
Inoculum”
“RNA” Tissue Ratio n Assays
homogenate “RNA”:virus were
made
1
2
3
4
331 139 2.4
113 17 6.6
540 130 4.2
340 215 1.5
at a dilut,ion
of 1: 100 of original
tissue.
This difference was only about l-2 hours, but since lesions are visible within 16-20 hours after inoculation if the plants are held above 25”, this small difference is probably significant. The ultraviolet absorption curves of these preparations were characteristic of RNA, having a 260 : 230 and 260 : 280 rnp ratio of about 2.0. These ratios are somewhat lower than those reported for the nucleic acids of purified plant viruses (Fraenkel-Conrat et al., 1957; Kaper and Steere, 1959a, b; Rushizky and Knight, 1959). This is not surprising since the “RNA” used in this study was prepared directly from infected whole tissues rather than from highly purified virus nucleoprotein and some contamination by cell components, other than nucleic acid, is to be expected. Attempts to make quantitative protein determinations on these preparations, using the method of Ramachandran and FraenkelConrat (1958) were uniformly unsuccessful owing to the formation of precipitates, probably as a result of a carry-over of interfering materials. Aqueous solutions of purified “RNA” remained infective for only l-2 hours at room temperature. Aliquots of the same preparations held in the refrigerator at 5’ retained infectivity for about 24 hours. On the other hand, aliquots of “RNA” in 67 % alcohol (second alcohol precipitation) were infective after 3 months’ storage at -18”, although there was a gradual decline in the infectivity. Similar alcohol preparations were infective for only 3-5 days when stored at 5”. A solution of “RNA” in distilled water frozen at - 18” lost its infectivity in about 1 week. Both purified “RNA” and tissue homogenate were readily inactivated by ribonuclease (RNAase); however, the “RNA” was lo-100 times more sensitive to RNAase inactivation than virus in tissue homogenates. In a typical experiment the infectivities of the ‘
11VFECTIOUS
1000
FIG.
EXTRACTS
FROM
TORi\NYf
LK4VES
. v,*, .I*).’
r
/ 1.0 I o-3
6’ItP\‘A”; prosimstcly
PHENOL
lO-2 CONCENTRATION
10-l OF INOCULUM
1. 1)ilution curves of phenol eAracts and tissue homogenates; O-----O, O-0, tissue homogenates. The inocula were adjusfd to giw nI)the same numtwr of lesions on POW~FL:~ at thr highest ~oric.~ntI,:ttiotI.
virus were bot#h adjusted to give about. 1000 lesions per leaf brfore t)hc addition of RKAsse. RKAase was then added to both preparations to give a range of enzyme concentrations varying from 1.00 t)o 0.0001 pg/ml. The mixture was then incubated at 4” for 1 hour and the infectivities of each determined. As is shown in Fig. 2, at 0.01 wg/‘ml RKAasr, only about two lesions appeared on leaves inoculat8ed with the “RNA”RNAase mixture, whereas t)here was no reduction in the number of lesions produced by the tissue homogenate-RKAase mixt,ure. Higher concentrations of RKAase further reduced the infect,ivit>y of the phenol extract, and also the virus in t,he tissue homogenates. .Uthough the “RKA” was sensitive to much smaller amounts of IZNAase than the virus in tissue homogenat,es, the slopes of t,he two curves were similar. To dekrmine the sedimentation behavior of phenol extracts, several preparations were ultracentrifuged. Phenol extracts and tissue homogenates were prepared by t.he usual methods and each was divided int,o t,wo samples. One sample of each was centrifuged for I,$>-2 hours :II 114,000 9 in a refrigerated aenkifuge and the other wa?; held in the
334
SCHLEGEL IOOO~ \ \
0.000 I FIG. different
WHOLE
0.01 0.001 RNAASE CONCENTRATION
2. The inactivation concentrations
of tissue at RNAase
VIRUS
0.10 (pg/ml)
homogenates and “RNrZ” at 4” for 1 hour.
1.0 by incubation
with
refrigerator as a control. After centrifuging, the infectivity of the supernatant liquids and controls were compared (Table 2). The results showed that from one-half to three-fourths of the infectivity of phenol preparations was found in the ultracentrifuge pellet. In spite of this large loss, the infectivity remaining in the supernatant liquid was equal to or greater than the original infectivity of the tissue homogenate. The infectivity of the tissue homogenate was completely sedimented. To learn whether the phenol extracts of purified CMV were as infectious as, or more infectious than, the virus, attempts were made to purify CMV and to extract the RNA with phenol. Unfortunately, purification of this isolate of CMV proved to be very difficult. Numerous purification procedures were tried, including the use of CMV-infected cucumber (Cucumis sativus L. var. Straight Eight) corolla tissue as a virus source (Sill and Walker, 1952), and density gradient, fluorocarbon, isoelectric point, and ammonium sulfate techniques, none of which was
“RNA” Ultracentrifuge supernatant 15w 3 I ‘1 -I!)”
Buiier
Uncentrifuged
control
Ultracentrifuge supernatant
homogenates ITncentrifugetl control
331 113 127
‘1 Cenlrifnged 90 minutes. I1 l’rtrificd “RNA,” centrifuged
2 hours.
successful. The method of ‘I’omlinson et al. (1959) was used with relatively little success, but in two trials infectious partially purified virus was obt’ained. RXA extracted from bhis material by treatment with phenol had only 0.5-0.7 ‘% of the infectivity of the partially purified \.irus. This is of t,he same order of magnitude as t)hat observed for RXA from purified TRlV (Gierer and Schramm, 1956), tobacco ringspot (Kaper and Steerc, lY5Ya), t~urnip yellow mosaic (Kaper and Steere, 1959b), and tomato bushy st,unt (Rushieky and Knight’, 1950) viruses. Further verificatiorl of these results will be recfuired before t’he infcct’ivitv of RNA from puritied CRfV can be definit~ely established.
The trent)ment of ChIV-infected tobacco leaves with phenol-pyrophosphnt,e results in preparations several times as infectious as buffer homogenates of the same amount of virus-infect,ed t,issues. This infectivity may be due to (1) infectious RNA released from intact’, or possibly incomplete, virus by the action of phenol, (2) free infectious RSA which was protected by the phenol from RNAase degradaCon during homogenizatjion and extraction, (3) partially degraded virus whose RXA was exposed m such a manner t.hat it exhibits certain RNA characteristics, and (1) whole virus carried through the entire procedure. Actually it seems probable that more than one of t’hese explanations are involved since t,he data oht)ained do not, support any single hypothesis to t’hr exelusion of the ot,hers. The data obtained from t.he ult,racentrifugation experiment,s seem to indicate the presence of infectious material of at least two different sizes,
336
SCHLEGEL
since about 65% of the infectivity was found in the ultracentrifuge pellets. The infectious agents remaining in the supernatant were not, however, sedimented after 2 hours at 144,000 g, and, therefore, are probably much smaller than the virus. The presence of different-sized particles in these phenol preparations may indicate that both RNA and partially degraded or whole virus were present. It is also possible that the phenol extracts contained highly aggregated RNA which was sedimented during centrifugation. It does not appear likely that much of the sedimented infectious material is whole virus. The properties of uncentrifuged phenol preparations differ in certain respects from those of tissue homogenates, and these differences are evidence against any hypothesis that attributes the increase in infectivity following treatment with phenol to an increase in the amount of whole virus carried through the procedure as a result of the neutralization of virus inhibitors or the dispersion of virus aggregates by phenol. The more rapid appearance of lesions on plants inoculated with “RNA” also suggests that RNA is the infectious material, since this property has been reported for tobacco mosaic virus-RNA (Schramm and Engler, 1958; Fraenkel-Conrat et al., 1958). If nucleic acid is the only infectious material present in these preparations, it would appear, at first sight, that it could not have come entirely from virus within the tissues, since the infectivity of RNA from purified CMV is low. There is, however, the possibility that the infectivity of the buffer homogenate is not a full measure of the total amount of virus present. It is well known that CMV is readily lost in the slow speed centrifuge (Tomlinson et al., 1959). It may be that only a very small fraction of the total virus present is infective and that the remainder is bound by inhibitors, other cell components, or in large noninfective virus aggregates. If treatment of CMV-infected tissues with phenol releasesall the virus-RNA present, including that from virus which is bound and noninfective, it may explain the high infectivity of the phenol extracts. If this is true, the tot,al concentration of CMV in the tissues may approach that of tobacco mosaic virus. The greater sensitivity of “RNA’” than virus to inactivation by RNAase is to be expected if RNA is primarily responsible for the infectivity observed in the phenol extracts. On the other hand, when sufficient RNAase was added, inactivation of the tissue homogenates took place at about the samerate as the inactivation of the “RNA” (Fig. 2). The inactivation of CMV in tissue homogenates by RNAase has also been shown by Badami (1959). He reported that inhibition occurred
INFECTIOUS
PHENOL
EXTRACTS
FROM
TOI3.\(‘(‘0
LE.iVES
:‘,:17
immediat’ely after the virus and RKAase were mixed and that the infectivitjy was restored when the mixture was diluted suitably. Since the ina&ivation was immediate and infectivity was regained upon dilut,ion, it was concluded that the inhibitory effect of ribonuclease upon the infect’ivity of &sue homogenates was not’ due t,o enzymatic hydrolysis. A similarity between “RKA” and the virus in tissue homogenates can bc seen by comparing dilution curves of bot,h preparations (Fig. I ). Here t,hc curves were essentially parallel. In this respect, t’he RNA preparatjiolln of C&IV-infected tobacco leaf tissues differ from RX-4 from tobacco mosaic virus since Rawden and Erie (1957) showed that, t,hr illf&ivity of the latter decreased more rapidly on dilution than did t,hnt of the \G-us. It is clear from this discussion that no definite conclusions can be drawn as to t,he precise nature and source of the highly infectious material present in phenol extracts of CMV-infecbed t’issues. This, however, does not alter the significance of the consistently higher infectivitjy obtained with phenol extracts than with buffer homogenat’es of similar tissues. Regardless of the explanation of this phenomenon, it appears very significant t’hat,, in this disease, keatment with phenol produces more rather than less infectious material.
I
338 soluble protein
SCHLEGEL
and infectious
Virology 8, 527-530. RAMACHANDRAN, L.
nucleic
acid from turnip
yellow mosaic virus.
K., and FRAENKEL-CONRAT, H. (1958). The estimation of protein contamination in ribonucleic acid. Arch. Biochem. Biophys. 74,224-228. RUSHIZKY, G. W., and KNIGHT, C. A. (1959). Ribonuclease-sensitive infectious units from tomato bushy stunt virus. Virology 8, 448-455. SCHLEGEL, D. E. (19FO). Transmission of several plant viruses by phenol-water extracts of diseased tissues. Phytopathology 60, 156-158. SCHRAMM, G., and ENGLER, R. (1958). The latent period after infection with tobacco mosaic virus and virus nucleic acid. Nature 181, 916-917. SILL, W. H., JR., and WALKER, J. C. (1952). A virus inhibitor in cucumber in relation to mosaic resistance. Phytopathology 42, 349-352. TOMLINSON, J. A., SHEPHERD, It. J., and WALKER, J. C. (1959). Purification, properties, and serology of cucumber mosaic virus. Phytopalhology 49, 293-299. WECKER, E. (1959). The extraction of infectious virus nucleic acid with hot phenol. Virology ‘I, 241-243. WELKIE, G. W. (1959). Infective ribonucleic acid from cucumber-mosaic-infected tobacco. (Abstract.) Phytopathology 49, 114. YARWOOD, C. E. (1952). The phosphate effect in plant virus inoculations. Phytopathology 42, 137-143.