Effect of cycloheximide on tobacco mosaic virus synthesis in callus from hypersensitive tobacco

VIROLOGY

66,320-328

Effect

(1973)

of Cycloheximieie Callus

on Tobacco From

Hypersensitive

R. N. BEACHY’ Department

of Botany

and Plant

Mosaic

Pathology,

Virus

Synthesis

in

Tobacco

AND

H. H. MURAKISHI

Michigan

State University,

East Lansing,

Michigan

48823

Accepted June Yi 1973 Local lesions were formed on callus derived from Nicotiana tabacum cv Xanthi-nc 36-44 hr following inoculation with tobacco mosaic virus (TMV), but these had little or no inhibitory effect on virus synthesis. The greatest accumulation of [3H]uridineand [14C]leucine-labeled virus occurred 48-84 hr after inoculation. When infected callus tissue was treated with cycloheximide (1 pgjml) during the period of rapid virus accumulation, the synthesis of host protein and complete virus was reduced by 90% within 1 hr. The synthesis of TMV RNA was not affected even after 4 hr of treatment. The results suggest that cycloheximide prevented the formation of complete virus by inhibiting the synthesis of viral protein and/or by preventing the assembly of virus particles. INTRODUCTION

Cycloheximide (CHX) is a potent inhibitor of protein synthesis (McKeehan and Hardesty, 1969) in plant tissue cultures (Nude1 and Bamberger, 1971). Reports indicate that it may stimulate or inhibit tobacco mosaic virus (TMV) synthesis in inoculated leaves of Nicotia.na tabacum. Ouchi et al. (1969) reported that 1 pg CHX/ ml stimulated virus synthesis in infected leaf discs, whereas higher concentrations reduced synthesis. Takebe et a.1. (1971) and Zaitlin et al. (1968), using infected leaf protoplasts and leaf discs, respectively, reported near complete inhibition of TMV synthesis by CHX at 10-15 pg/ml. We reexamined the effect of CHX on the synthesis of TMV in tobacco tissue cultures and also studied the effects of CHX on the synthesis of TMV RNA. Tissue cultures have certain advantages over leaf discs or protoplasts used in previous work. Callus tissue can be cultured in a

metabolically active state under aseptic conditions; thus, problems caused by tissue senescence or by microbial contaminants are avoided. We used TMV and Xanthi-nc callus cultures described previously by Beachy and Murakishi (1971). The degree of virus infection in such cultures is indicated by the number of necrotic lesions produced after inoculation. The conditions for maximum efficiency of infection were determined in a preliminary study. In addition, the virus growth curve was characterized, because this had not previously been done for a hypersensitive callus culture.

1 Portion of a Ph.D. Thesis presented by the senior author to the Graduate College of Michigan State University. Partially supported by the Michigan Agricultural Experiment Station. Present address: Dept. of Plant Pathology, Cornell University, Ithaca, NY 14850. 320 Copyright -411 rights

@ 1973 by Academic Press, of reprodwtion in any form

Inc. reserved.

MATERIALS

AND

METHODS

Callus cultures. The culture medium was a combination of the media described by White (W) (1963) and Murashige and Skoog (MS) (1962). The W and MS media were prepared separately, each was brought to pH 5.9 and combined in a ratio of 3 MS/ZW. The combined medium contained 50 ml coconut water, 30 pg kinetin, 250 rg 2,4dichlarophenoxyacetic acid, and 40 rg naphthaleneacetic acid per lit’er. The medium (MS-W) was sterilized by autoclaving. Solid medium was prepared by adding agar (1 % w/v).

Stcbm pith cores (explants) of Nicotiana tabacu~z cv Xanthi-nc were removed aseptically with a cork borer, placed on solidified MS-W medium, and grown under Gro-Lux fluorcwc>nt lamps (80 fc). Callus growt,h usua,lly bocamr green-pigmented within 2 w&s and \vas maintained by monthly tran&rs on solid medium. To prepare cells for inoculation, 1 g of agar-grown callus was transfwrcd to 20 ml of liquid medium in 125.ml flasks and placed on a ro tar-y shaker (11’0 rpm). After G-9 days, l-2 ml of rcsulting wll suspension was transferred wit#h a large’ mouth pipct (3.mm opening) to fresh liquid medium. After G-9 days of additional growth, t’hc crlls had developed into small aggregates, 20-75 cellsjaggregatc, and were inoculat~cd. 1rI~,7~.s i~oculafion. A common strain of T;\I\’ \vas purified according to Knight (19G) from leaves of S. tabacum cv Havana:iX inoculattad 2-4 weeks earlier. The virus was fwcd of microorganisms by passage through an ultrafirw sintered glass filter. Suqwnsion ccl11cultures were inoculated with 1.50 ~g T>IV/ml by the vibratory tcchniquc of Murakishi et al. (1970, 1971). Cells from scwral flasks were combined on funwls linrd nith Miraclot’h filters, washed with frwh medium, and dispensed in tubes rontaining 3 ml of MS-W medium. After adding the T1\IV suspension, the tubes were vibratcbd for 10 see on a Vortex mixer (Sricwtific Products Corp., Evanston, IL), and th(l wlls wre collected on ,Iliraclothlirwd funrwls and n-ashed with fresh liquid medium. The ccl1 clumps were gently stirred to (wsur(’ randomization, transferred to filter pads roasting OII solid medium, and incubated at 24” under the Go-lux lamps (l’olcliw et al., 192). Ittcor,/,/)t.ntioi~ oj [3H]urirZirte and [‘“ClZeuritw i/r ?-Tan/h ~-UCcallus. The incorpora-

tion of radioactive metabolites into Xanthinc callus was d(ktcrmined b-y placing cell sam& into 2 ml of liquid medium containing [3H]uridirlcl (sp act 9-27 Ci/mmolc) or L-[‘4C’]lwcine (sp act 230 mCi/mmole) from Nw- England Kuclear. Cell samples were harwsted, washed with ice cold 0.25 dl sucrose’ containing 1O-4 J!1 uridine and/or lo--” -11 r,-lcucinra, and ground in a conical g1as.qhc)mogc~rrizw in 0.1 dl Tris-HCl (pH

8.0) containing 0.25 111 sucrose. 1OP ,I/ uridinc, 1OP N L-lrucinct, and 75 m:l/ ‘,,mercaptoethanol. Tht> homogonatc \~as centrifugcld at 1,000 q for 10 min to wrnow cell debris, snd aliyuots of the supernatant were precipitated at 4” in 10 ‘1: trichloroacetic acid (TCA), wllectcxd on glass fibttr filters, and washed with cold 5 ‘Z TC.2, 95 % ethanol, and chthanol-othw (1 : 1, v !v). The filter-trapped materials wr( digwtrhd at 40” for 4 hr with 0.5 ml of a solution (‘o~Itaining 20 ‘% x CS Solubilizcr (,\mcwham ;i Searle, Des l’laincs. IL), 3.75 ‘:; \vatcr, :md 76.25 % toluene. Ten milliliters of toluc~~c~based I’~~I’~l’-I-‘P~~ SOlUticJn n-as add(td, a11d samples were counted in a, Packard Tri-C’arl) or B&man 1%133 liquid scintillation counter with a counting cfiicic~ncy of 17’; for 3H, and 51 (‘c for 14C. I)oubly latwlcd samples wre corrwtcd for blccdowr using [14C]leucinc~standards prcyarcbd in an id(lrlt ical manner. To determine the efficirw~y of the lab(,ling procedure, randomly mixed callus wun~~lw were placed in liquid m&urn cont’aining 2.5 FCi [3H]uridinc/ml and 0.1 &Ji[14C’]leucine/ml. Callus samples \\-(w harvwt od after 1, :?, 6, 9, and 13 hr. and the :lc*id insoluble radioactivity was dctc~rminwl a> described above. Bot,h isotcqw w(w ir-rcorporatcd a,t a const~snt rate, u~ltlw tlicw* labeling conditions (Fig. 4). Cycloheximid(b (Sigma Chemical Co., St . Louis, JIO) leas prepared in liquid MS-W medium and strrilizcd by Jlillipow fitration. ISxtmction of labeled 7’:1L I’ Ji’011r ir/jw/ctl incubatc>d in callus. InfectcLd wlls ww

[3H]uridine (12.,j-25 FCilml) and/or [‘“(‘Ileucine (0.:5-1.0 pCi/ml) in licfuid nwdium. harvcstcd by washing \\-ith 0.2.; II sucros;(’ containing 10e4 .!!l uridinc and IO-” .l/ lcucinr. wrighed, and frozen. Tha\vc,d wlls were ground in a glass homogc~nizc~rin 0. I ,l/ Tris-HCl, pH S.O>containing 10v4 .lI uridinca, 10P3 31 lrucirw, and 7;, m:ll t’-mcrcaptoethanol, and wntrifugcbd at 1.000 q for 10 min. The supernat)ant fluid was wn t rifugc>d at 12,000 c/ for 13 min. The final sqwrnatarlt was la?-cwd on 1 ml of 30 ‘i sucrose in thck Tris buff(,r and ccntrifugcld in thr> SW 391J rotor in the Spinco Nodcl L ultr:lc~csltrifug(, at 35,000 rpm for 90 min. Thcx final ~wIl(~t

322

BEACHY

AND MURAKISHI

was resuspended in 1 ml of water and given a low-speed centrifugation, and the resultant supernatant was layered on 1040% linear sucrose gradients prepared in 0.01 M potassium phosphate buffer (pH 7.3). After centrifugation at 23,000 rpm for 90 min in the SW 25.1 rotor, gradients were fractionated with an ISCO Model D fractionator coupled to a Model UA-2 UV (254 nm) analyzer. One-milliliter fractions were collected and co-precipit.ated with 0.5 mg bovine serum albumin with 10 % TCA. Precipitates were trapped on glass fiber filters, and radioactivity was determined as described above. Radioactivity profiles of sucrose gradients from extracts of TMV-infected and noninfected cells labeled with [3H]uridine were used to determine incorporation into TMV (Fig. 1). The peak of radioactivity representing 3H-labeled TMV was found in fractions 9-11. TMV purified by the differential centrifugation procedure (Knight, 1962) sedimented to the same level under these conditions. RNA extraction and polyacrylamide gel electrophoresis. Inoculated callus was incubated in 20 &i[aH]uridine/ml, harvested by washing with the cold sucrose solution, weighed, and RNA extracted at 4” by the sodium dodecyl sulfate (SDS)-phenol procedure described by Pelcher et al. (1972). The RNA was taken up in the electrophoresis buffer (0.04 M Tris, 0.02 M sodium acetate, 0.001 M EDTA; brought to pH 7.8 with glacial acetic acid) made 5 % in sucrose. The RNA concentration of t’he solution was determined assuming an 0.1% <260nm = 30. The 2.4 % polyacrylamide gels were prepared as described by Bishop et al. (1967). Polymerized gels were allowed to stand in the electrophoresis buffer containing 0.2 % SDS (w/v) at 4” for 72 hr prior to use. The gels were pre-run for 30 min at 5 mA/gel and t’hen loaded with 15 c(g of the RNA in volumes of 15-25 ~1. Following electrophoresis for 90 mm at 5 mA/gel (10 V/cm), the gels were scanned at 260 nm in a Gilford 2400 spectrophotometer with attached gel scanner. The gels were frozen in dry ice, sliced into l-mm sections, and the radioactivity was determined as previously de-

3c 2C I(

P

e

0

i Yd n=

4

2

5

10 FRACTION

15 (ml)

20

25

FIG. 1. Radioactivity profiles of sucrose density gradients. TMV-infected and noninfected Xanthinc callus were incubated in 12.5 NCi [3H]uridine/ml of medium and extracted for TMV as described in the text. The extracts were centrifuged in lo-40% sucrose gradients, and radioactivity in l-ml fractions was determined. (O---O) noninfected, (@---a) TMV-infected.

scribed (Pelcher et al., 1972). Specific activities (cpm/pg RNA) were determined by calculating micrograms of RNA from opt’ical density profiles of gels and by establishing the counts per minute in RNA from the corresponding radioactivity profiles of the gels. RESULTS

Eficiency

of Virus Infection

The efficiency of TMV infection of Xanthi-nc callus is expressed by the numbers of lesions produced (Beachy and hlurakishi, 1971). The greatest number of lesions was produced on callus subcultured two times in liquid medium prior to inoculation; fewer lesions were obtained from callus continuously subcultured. Highly friable cell

CYCLOHEXIMIT>F:

AN11 TMV

cultures consisting mainly of single cells produced fcwr local lesions than did cultures composed of small (0.5-2 mm diam) cell aggregates with a “grainy” texture. Cult.urcs composed of grainy cell aggregates new produced by carefully regulating the amount, of cells transferred in the second liquid subculture. Sew explants were taken when th(: efficiency of infection began to drop. Tl/T

Sy,cthesis i,r Xanthi-nc

callus

Thr TRlV growth curve in Xanthi-nc callus \vas determined by measuring the incorporation of [3H]uridine (12.5 FCi) and

! ...k..j....I...1 ...’*.*’ i i ,.’..i’I ’

.*: *,:

[14C]leucine (0.5 FCi) into virus ow’r IL’-fir intervals until 144 hr after inoculation. TilIV was purified from l..? g of fwshl~harvested caells utilizing sucrow gradiwt :i11tl IIatwials (as in cfntrifugation Methods). The results arc c~xpnwscd irt counts per minute (cpm) in TXlV for wch 12-hr interval. The rates of virus synthwk and total yields of TAN from inoculat (ld Xanthi-nc callus varied slightly with cwh experimrnt, hut, in general, tlic! grcwtll curves wrn similar to thfl wrv(’ givcw in Fig. 2. Virus synthesis was first &twtc~tl 12-24 hr aftw inoculat,ion, tlw ratcL (11 synthesis increasing until (iO--7”. or 72~S-l hr. The: high rata of synt,hCsis MY~Sfollowc~l by a sharp decline (12-24 hr in duratiorr) and then by! a second peak of virus syrrt~hc& The swond burst of spnthcsis Iastcd for 1” or 24 hr and was followed by a drop ill lhcs rate of synthesis and the mnintcwanw of tlw low rate through thtl wmaind(r of t 1~. experiment . Local lesions wrc obscrwd 3-44 hr aftcbr inoculation in all experiments and (7)1itinucd to enlargcl until S4-(JB 111,(Icig. L’). Marc than 90 5’; of the cxtractc~d virus u :w produwd after lrsionx appeawd. To tlctc~~.mire whethw or not lesions nffwtc~cl virus callus containing Iwions :III~ extraction, callus without lesions wew homogcnizc~ti in the presenw of purified [3H]uridiJlr,-la~)c~11 Ye TJIV. Analysis of sucrose gradicntjs of tlrcs t’n-0 extracts showt~d that c>xtractnbilit!’ ()f virus was not affwted 11). I(3iorw in thr callus. [14CjLeurine

0

24 HOURS

48

72 AFTER

96

120

I‘

INOCULATION

FIG. 2. TMV growth curve in Xanthi-nc callus. Cells were incubated for 12 hr in 12.5 &i [Wruidine and 0.5 &i ]%]leucine/ml, and TMV was purified as described in the text. Sucrose gradients Rere nronitored at, 254 nm, and radioactivity in l-ml fractions was determined as described. Hadioactivity in TMV was determined by calculating the counts per minute above background (see Fig. 1) under the TMV optiral density peak. The upper portion of the figure indicates the diameter (mm) of the local lesions (with standard deviation) over the experimental period.

::‘,‘:;

SYNTHESIS

mrl

[3N] 1 ‘J~iflitir

The sensitivity of Xanth-rich c~alius to cycloh(~ximidc (CHX) \vas drxtcrmincld I )y placing randomly mixfhd cc>11samplw in liquid medium containing [14C]lrwinc~ (0.1 pCi/ml) and various conwntrations of CHX for 3 hr. Radioactivit!in th(b a&linsoluble fraction of thcl ~11 homogcwa tc,s leas detcrmincd. Cycloh(xximido at 1 ~8’ ml gave rioar maximum inhibition of ]‘“(‘I lrucinc incorporation (l;ig. 3) without cawing ~11 damage as determincld 1)~. microscopic examination. This wnwntration \V:IS used in all later chxpcriments ~mlvss oth(rn.iw indicatfxd.

BEACHY

324

AND MURAKISHI

21 1 .“I

CYCLOHEXIMIiE

CO&.-

jJg 61

FIG. 3. Effect of increasing concentrations of cycloheximide on protein synthesis in Xanthi-nc callus. Cells were incubated with [W]leucine (0.1 ,.&i/ml) for 3 hr, washed with cold 0.25 M sucrose containing 10m3M leucine, and homogenized in 0.1 M Tris-HCl, pH 8.0, with 0.25 M sucrose, low3 M leucine, and 75 mM 2-mercaptoethanol. The homogenate was centrifuged, and aliquots of the supernatant were precipitated with 10% TCA; the precipitates were collected on glass filters and washed with 57, TCA, ethanol, and ether. Radioactivity was determined by liquid scintillation. Percent inhibition was determined by comparing counts per minute in CHX-treated and nontreated samples. Nontreated cells incorporated 1500-2000 cpm/O.OG g (fresh wt) of cells in 4 experiments.

The effect of CHX on the incorporation of [14C]leucine and [3H]uridine into the acidinsoluble fraction was determined at time intervals after simultaneously adding the inhibitor and labeled precursors (Fig. 4). Cycloheximide gave 50 % inhibition of [3H] uridine incorporation in 12 hr and about 90 % inhibition of [l*C]leucinc within 1 hr. There was no inhibition of the incorporation of either

isotope

into the acid-soluble

fraction.

Under these conditions, CHX remained an effective inhibitor of protein synthesis for 16-20 hr. Thereafter, the rate of [‘*C]leucine incorporation

treated

approached

that

controls. The recovery h.ymena and rat cells from CHX

of the non-

of Tetrainhibition

3

6 HOURS

9

1~2

FIG. 4. Effect of cycloheximide (CHX, 1 rg/mlon incorporation of (A) [Wlleucine (0.1 pCi/ml) and (0) [3H]uridine (1.0 &i/ml into the TCA) insoluble fraction of Xanthi-nc callus. Radioactive materials and CHX were added simultaneously. See Fig. 3 for details.

was observed previously Yeh and Shils, 1969). Eject

of Cycloheximide Synthesis

(Frankel,

1970;

on Complete Virus

Forty-eight hours after inoculation, Xanthi-nc callus was treated with 20 &i [3H]uridine/ml in the presence or absence of CHX. After 2, 4, and 6 hr, 3.0 g cell samples were harvested and analyzed for 3H incorporation into complete virus. Cycloheximide reduced the rate of [3H]uridine incorporation into T&IV throughout the 6-hr period, and complete virus format’ion was inhibited by approximately 90% at the end of this time (Fig. -5). Ouchi et al. (1969) reported that the inhibition of T?iMV synthesis in leaf discs by CHX at 1 fig/ml was bipartite in nature; treatment at O-12 hr after inoculation resulted in decreased virus synthesis, and treatment beyond 12 hr caused a twofold increase in virus titer. To determine whether or not CHX inhibition of T&/IV synthesis in our system was dependent upon the time of treatment, inoculated tissue cultures were incubated in 0.4 &i [14C]leucine/ml and 12.5 #.?i [3H]uridine/ml in the presence or

CYCLOHEXIMIDE

CONTROL

AND

TMV

SYNTHESIS

X;i

14C m

3H OCONTROL 5644 :::::: C H X

0

0 4 P c

L 2 r)i

CHX

2

4

2436 36-48 4860 TREATMENT PERIOD 6

HOURS FIG. 5. Effect of cycloheximide (CHX, 1 pg/ml) on incorporation of [aH]uridine (20 &i/ml) into TMV in Santh-nc callus. At 48 hr after inoculation, cells were treated simultaneously with L3H]uridine and CHX. TMV was extracted from 3.0 g (fresh wt) of cells, and radioactivity in virus was determined as in Fig. 2.

ahsenw of CHX for 12.hr intervals at four time periods aft’er inoculation. When CHX was applied at, 24-36, 36-48, 48-60, and GO-72 hr after inoculation, t,he incorporation of both isotopes into TMV was inhibited approximately 90 ( o (Fig. 6). To det,ermine whethw or not TRIV extractibility was affwtod b\- thrb CHX treatment, callus twit urw Lvcrc placed in a s&&on of CHX or in growth medium alone, harvested, and homogenized with purified [3H]uridinclab&d T;\IV. After purification by sucrose gradient wntrifugat’ion, equal amounts of [“HITfiN were recovered from tissues given both tjrcatmmts. When inoculated callus 1~:~streated with CHX at 5 pg/ml (O-30 min after inoculation), virus synthesis and local Icsion formation were inhibited for at l(aast 84 hr. l’:$Fcf oj Cyclohexi,mide ,Synthesis

on

TMV

RNA

The results suggested that synthesis of wmplete virus was inhibit’ed by CHX (1

(HPI)

FIG. 6. Effect of cycloheximide (CHS, 1 fig 1111) on incorporation of [‘4C]leucine (1 &i;ntl, and [sH]uridine (20 rCi/ml) into TM\‘. Each sxn~plc was treated for 12 hr during 24-7’ hr post inoclll:ltion (HPI), and radioactivity in TML’ WB?: determined as described in Fig. 2.

pg/ml) t,o a degree similar to thcb inhibitiol~ of cellular protein synthesis. To d~+r~rmint: tht effect, of CHX on the synthesis of TJl\‘RXA, inoculakd wlls (at 4X hr aftw incwilation) were incubated in 20 &i ~~HJuridilw ,’ ml with and without CHX for 2. 4, and 6 hr. Thr specific activit,y (cpm,‘pg RSA) of t,he 25 S and 18 S ribosomal RNA (Lotwing and Ingle, 1967) incroawd throughout thus 6.hr period in the untwated ~~~11s (I’iy. i). Cycloheximide, honcvrr, wduccbd that r:~t,t of incorporation of lZH]uridirw into 25 S ;md 18 S ribosomal RI\‘A and c*auwd :L !I() (, reduction in their specific activitiw by :! hr of trcatmcnt. Thcrc was no inhibition ot [sH]uridine into 4-5 S RNA. Similar c~ffwts of CHX on t’hc synthesis of wllular RNA. wr(’ reported in .\‘eurosporn (Vian :III~ Davis, 1970) and in rat liver (1Iur:tm;~tsu et nl., 1970). The sJ;nt,hesis of T,1IV IZSA. expressed as spcclfic act,ivit?., \vas IN)~ affected by CHX treatment’. The spwific activit,?, of TMV RYA from thcb ClH.X treated and nontreatcld cells \v:ts approximately 2.5 times that of ribwomal IIS’A from nontwated rrlls by the t’ourtl~ holu, rlt labeling.

326

BEACHY

AND

0 CONTROL * CHX

3 P 2 ‘2 X L P U

0

;:

2

4 HOURS

6

FIG. 7. Effect of cycloheximide (CHX, 1 pg/ ml) on the incorporation of [$H]uridine in RNA of virus-infected Xanthi-nc callus. RNA was extracted from 3.0 g of cells in SDS-phenol and separated by electrophoresis on 2.4% polyacrylamide gels. Gels were scanned at 260 nm, and radioactivity in l-mm slices was determined. Counts per minute in each RNA species were determined as described in Materials and Methods. DISCUSSION

Leaves of tobacco cultivars such as Samsun NN and Xanthi-nc contain the N gene (Holmes, 1939) and react by producing necrotic local lesions after inoculation with TMV. Callus cultures from these cultivars also produce lesions following TMV infection (Beachy and Murakishi, 1971). The necrotic response to virus infection has not been demonstrated, however, in isolated cells (Jackson and Zaitlin, personal communication) nor in protoplasts (Otsuki et al., 1972) from Samsun NN and Xanthi-nc. Otsuki et al. (1972) suggested that the necrotic reaction depends upon the interaction between infected and neighboring cells. Our results support their suggestion, since the reaction occurred only in callus cultures composed of cell aggregates. The interactions could occur by plasmodesmatal

MURAKISHI

connections between cells in tobacco callus aggregates (Hartmann, 1971). Tobacco mosaic virus synthesis in Xanthinc callus occurred in two major bursts during 144 hr and was similar to TMV synthesis in callus from a systemic tobacco (Pelcher et a.Z., 1972). The necrotic reaction appeared to have little or no inhibitory effect on virus synthesis since more than 90% of the virus was synthesized after lesions were evident. In leaf systems it is well established that virus synthesis occurs in advance of the spreading necrotic lesion (Osawa and Yamaguchi, 1970; Hayashi and Matsui, 1965). Recently Otsuki et al. (1972) compared the synthesis of TMV in leaf discs of Samsun tobacco (a systemic host) and Samsun NN, and reported that the rate of virus synthesis slowed after lesions became apparent. Their data showed about a tenfold difference in extractible virus (as measured by infectivity assays) by 96 hr after inoculating the two cultivars. The possibility that the lower infectivity could have resulted from decreased virus extractibility from the local lesion host was apparently not explored. From all of these data it appears that the effects of the necrotic response on TMV synthesis in the initially infected and immediately adjacent cells cannot be specified at this time. Cycloheximide inhibits protein synthesis by preventing peptide elongation (McKeehan and Hardesty, 1969) and is effective in tobacco callus cultures (Fig. 3; Nude1 and Bamberger, 1971). We found that tobacco callus recovered from the inhibition caused by low concentrations of CHX (0.01-1.0 pg/ml) but not from higher concentrations. A similar recovery phenomenon was observed with Tetrahymena (Frankel, 1970) and rat tissues (Yeh and Shils, 1969) when low concentrations of CHX were used. The data clearly emphasize the importance of monitoring the length of time that CHX remains effective. The inhibition of complete virus synthesis by cycloheximide appeared to be similar to the inhibition of [14C]leucine incorporation in Xanthi-nc callus (compare Figs. 4 and 5). The low level of incorporation into TMV could be due to translation by chloroplast ribosomes (not effected by CHX), but this

docls not seem likely, since the addit’ion of in oiw and t,lie nature of virus coat lw,teili chloramphenicol (lo-100 pg/ml), an in- pools. hibitor of chloroplast protein synthesis (rwiewd by Boultrr et al., 1972), had no on [14C]lrucinc incorporation in rffrct The authors express their appreciation 11, I,. 141. Xant,h-nc callus (unpublished). In addi- Pelchrr for helpful discussion and assistance wit II tion, WC extracted only tract an1ount.s of t,he polyacrylanlide gel electrophoresis, and I Itank (~hlort)plast R.KA from this t.issur (data not, Drs. A. Elliugboe, I:. I’. Schefler, and M. 1.. %‘iew for critical evaluation of the manuscript This is sho\vn). Our data compare favorably with thaw for tobacco lclaf discs (Zaitlin et al., Journal Paper ?rTo. 6305 of the Micahigarr :\qi~~\llI!)tiX). Synthesis of TnIV RI\‘A, howr~vcr, tural Experiment Station. ws not affr&d by 4 hr of CHX twatment (l’ig. 7). The wsults agree with those obtairwd for swcwl animal viruses (Nob4 and BISACHI-, I<. ;I’.. and R~URXISRI, Il. 11. il!Gl:t). Local lesion formation in tobarco tisslw 1*111. I,c:vinto\\-, 19’70; E’ric~dman and Grimley, ture. Phylopalholoy~ 61, 877-878. 1969). Harrison and Crockatt (1971) and II. N., and MURAKIRHI, II. 1~1. il!l7lb). JIcCarthy rf al. (1972) reported that the Beacon, Proteins froin c~dturrd cells of Satlth-r,c, synthesis of tobacco rattle and tobacco tobacco inoclllated with tobacco mos;~ic vitals. rwcrosis viral RNAs, dctermincd by measurPhytopufhofogy 61, 884 (Abstract). ing the infwtivit\associated with phenol BISHOP, Il. II. I,., (LITBROOK, J. I:., anti PMI (;I..T,cstrarts of lwf &sues, cont,inurd in the MAN, s. (lM7). Electrophoretic srparatioli ot prwcrw of CHX. To tho best of our knowlviral nucleic alcid on polv:l~r?-lanlid(, gable, ./. cldgcl, our results wprcsent th(> first such d/al. Rid. 26, 3i’:3-887. wport for T3IV. I:ailur-c of th(> specific BOUI,TI~;R, Il., ELMS, I:. J., and >‘.~R\Y~KID, .;\. activity of T1\IV R1VA to increase during (1972). Biochemistry of protcain synthesis in plants. f2iol. Z sam(l time period, complete in 2’e(ruhU?J/e,,rr. pyrifo:forwis (+L-C: An analysis virus colltinwd to accumulate in untreated employing cyclohrsimide. Cell I’h!/sirj/. 74. IX(~,Ils (Fig. 5). l-18. Sinw TJIV coat protein is present, in I<. M., and (;RIMLICI-, 1'. XI. (l!)(iUi. FRIIXNAK, cbutrac+ from infwted Xanthi-nc callus Inhibition of arbovirus assembly hy I*~.(*Io(Beach~ and Murakishi, 197lb) and in heximide. .1. I’irol. 4, 292-299. T_\IV-infected Samsun iYN leaves (van HARRISON, B. J)., and CROCKATT, A. 8. (l!Jil). Loon and van Iiammen, 1970), it is conceivEffects of cycloheximide on the acclumulnt ioll ot ably that TMV RXA synthesized in the toharco rat t,le virus in leaf discs of .\~i~~/io~~, clcwdadii. J. GPII. l’ird. 12, 1X3-185. absonw of coat protein synthesis (CHX .J. S. (1971). Klectron microscrtpv 01’ wnsitiw) would have been encapsidated in HARTM~SN, plant tissue cultlwe cells inoculated with T1f V. r*oat protciin prwxisting in the ~11s. This ZIL J’itro 6, 373-374 (Abstract,). \ws apparc~ntl>- not the cast‘, however, since HAYASHI, T., and MATSCI, C. (19G). Fillcx SI rtl(~~ thci irworporation of [‘4C]leucin~~ and [3H]-tllre of lesion periphery produwd h?; ~~~I):IKo uridirw into compIctt> virus ww equally mosaic virus. Ph~lopatholoyy .55, X87-3W inhihitcd by CHX treatment (I‘ig. 6). HOLMP:S, F’. 0. (1938). Tnhcritanrr of reaistunw to tobacco mosaic disease in tcjbacw). /‘/I I//OInhibition of cornplcte virus formation pathology/ 28, 553%5(il. could btl cauwd by t’he inhibition of viral KNIGHT, C. A. (19(U). Tobacco mosaic vinw /I/ coat or othw protc%l synthesis, or by the “Rioc~henlical Preparations” (M. .J. (“oott, we.), inhibit~ion of virion assembly, as suggested Vol. 9, pp. 13%13G. Wiley, New York. by the failuw of coat protein present in LOKNING. T=. I.:., and INGLP:, d. (1967). I)ivrrsity 01 tlw ~11s to cwapsidate viral RKA. Furt#her RNA con~ponents in green plant tissrw. .\.rr/~r.c studicls utilizing appropriate plant-tissue(LOtdoti) 21.5, Xi3 -3(ii. T1IV -inhibitor systrms may bo useful in ~I~widatin~ tht> nnturc of T3IV asscmhlv

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heximide and chloramphenicol on the multiplication of tobacco necrosis virus. J. Gen. Viral. 17, 91-97. McK~~HAN, W., and HARDESTY, B. (1969). The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulocytes. Biochem. Biophys. Res. Comm. 36, 625630. MURAKISHI, H. H., HARTMANN, J. X., PELCHER, L. E., and BEACHY, R. N. (1970). Improved inoculation of cultured plant cells resulting in high virus titer and crystal formation. Virology 41, 365-367. MURAKISHI, H. H., HARTMANN, J. X., BEACHY, R. N., and PELCHER, L. E. (1971). Growth curve and yield of tobacco mosaic virus in tobacco callus cells. Virology 43, 62-68. MURAMATSU, M., SHIMADA, N., and HIGASHINAKAGA~A, T. (1970). Effect of cycloheximide on the nucleolar RNA synthesis in rat liver. J. Mol. Biol. 53, 91-106. MURASHIGE, T., and SKOOG, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473497. NOBEL, J., and LEVINTOU., L. (1970). Dynamics of poliovirus-specific RNA synthesis and the effects of inhibitors of virus replication. ViTology 40, 6344642. NUDEL, U., and BAMBERQER, E. S. (1971). Kinetin inhibition of 3H-uracil and i4C-leucine incorporation by tobacco cells in suspension culture. Plant Physiol. 47, 400-403. OSAWA, K., and YAMAGUCHI, A. (1970). Comparative studies of virus multiplication and quality of necrotic lesions in the inoculated leaves of some local lesion hosts to tobacco mosaic virus. Ann. Phytopath. Sot. Japan 36, 254-259. OTSUKI, Y., SHIMOMURA, T., and TAKEBE:, T. (1972). Tobacco mosaic virus multiplication and

MURAKISHI expression of the N gene in necrotic responding tobacco varieties. Virology 50, 45-50. OUCHI, S., FURUSAWA, I., and AKAI, S. (1969). Bipartite nature of the effects of cycloheximide and chloramphenicol on the multiplication of tobacco mosaic virus. Phytopath. 2.65,279-287. PELCHER, L. E., MURAKISHI, H. H., and HARTMANN, J. X. (1972). Kinetics of TMV-RNA synthesis and its correlation with virus accumulation and crystalline viral inclusion formation in tobacco tissue culture. Virology 47, 787-796. TAKEBE, I., OTSUKI, Y., and AOKI, S. (1971). In “Les Cultures de Tissus de Plantes,” pp. 503511. Editions du Centre National de la Recherche Scientifique, Paris. VAN LOON, L. C., and VAN KAMMEN, A. (1970). Polyacrylamide disc electrophoresis of the soluble leaf proteins from ivicotiana tabacum var. “Samsun” and “Samsun NN”. II. Changes in protein constitution after infection with tobacco mosaic virus. Virology 40, 199-211. VIAU, J., and DAVIS, F. F. (1970). Effect of cycloheximide on the synthesis and modification of ribosomal RNA in Neurospora crassa. Biochim. Biophys. Acta 209, 190-195. WHITE, P. R. (1963). Zn “The Cultivation of Animal and Plant Cells,” 2nd ed., pp. 57-63. Ronald Press Co., New York. YEH, S. D. J., and SHILS, M. E. (1969). Quantitative aspects of cycloheximide inhibition of amino acid incorporation. Biochem. Pharmacol. 18, 1919-1926. ZAITLIN, M., SPENCER, D., and WHITFIELD, P. R. (1968). Studies on the intracellular site of tobacco mosaic virus assembly. In “Proceedings of the International Symposium on Plant Biochemical Regulation on Viral and other Diseases or Injury” (T. Hirai, Z. Hidaka, and I. Uritani, eds.), pp. 91-103. Kyorimu Printing Co., Tokyo.