vIIloI,o(:Y 41, 459-W (1970)

Loss of Ribosomes in Nicotiana glutinosa L. Infected with

Lettuce Necrotic Yellows Virus

J. W. RANDLES AND D. F. COLEMAN

Department of Plant Pathology, Waite Agricultural Research Institde, South Australia

Accepted March 5, 1970

Changes that occur in the amounts of ribosomes and ribosomal RNA in leaves of Yicotiana glutinosa L. following systemic infection with lettuce necrotic yellows virus (LNY\-) have been studied in relation to the development of infection and the onset of symptoms. In plants grown under continuous light, 70 S chloroplast ribosomes commenced to decline in concentration within 1 day of the appearance of sympt,oms, and were not det,ected l-3 days later. Chloroplast ribosomal 1G S and 23 S RNA’s were isolated in trace amounts only 1 day after symptoms appeared, and incorporated negligible quantities of 32P; they then became undetect,able. Growth of infected leaves almost completely ceased at this stage, virus content commenced to decline, and rhloroplasts became smaller. Lower concentrations of cytoplasmic 80 S ribosomes were extracted from infected leaves after the 70 S ribosomes had declined, but this was not accompanied by a loss of cytoplasmic ribosomal18 S and 25 S RNA or a failure to incorporate 3*P. These changes were also observed in plants grown in the glass- house and in plants infected with either a mild or a severe isolate of LNYJ’.

INTRODUCTION

Lettuce necrotic yellows virus (LNYV) induces vein-clearing, curling, and chlorosis in the terminal leaves of systemically infected Nicotiana ylutinosa L. (Stubbs and Grogan, 1963). The onset of systemic symptoms is accompanied by cessation of growth. LNTV particles, distinguishable by their bacilli- form or bullet shape (Harrison and Crowley, 1965; Wolanski et al., 1967) appear in the cytoplasm of mesophyll, epidermal, leaf hair, and xylem cells, usually enclosed in mem- branes (Chambers et al., 196,5; Chambers and Francki, 1966). Degenerative changes occur in cells during the later stages of infection (Wolanski, 1969). In particular, chloroplasts show displacement and dis- organization of lamellar membranes, they become vacuolate with osmiophilic granules, and eventually lose their outer membrane.

During studies on the nucleic acid metab- olism of LNTV-infected N. qlutinosa leaves, marked changes in ribosomes and ribosomal RKA (rRKA) were observed. These changes, and their relationship to the development

of infection, onset of symptoms, and changes in chloroplast morphology, are described in this paper.

MATI~X IALS AND METHODS

Cultwe of injected plants. Nicotiana glu- tinosa plants were raised in the glasshouse to the 5-7-leaf stage, dusted with Carborun- dum, and inoculated on all expanded leaves with infective sap extracts. Matched groups of inoculated and uninoculated plants were then placed under continuous fluorescent lighting (350-450 foot-candles) at 25 f 2°C. The smallest expanded leaf w-as marked; the leaf above this! which was the first to develop systemic symptoms of infection, was used in all investigations. Tissue samples of 22-25 mg were harvested by taking one disk with a 2-mm cork borer from this leaf on each of 10 plants.

A severe strain of LNYV (SE3 isolate; Stubbs and Grogan, 1963), and a mild isolate which induced a yellow-green chloro- sis on iV. ylutinosa, were used.

Infectivity assays. Infectivity was assayed

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460 RANDLES AND COLEMAN

by macerating 22225mg samples in 0.2 ml of water and inoculating at least 12 whole leaves on 4 N. glutinosa plants with the sus- pension. Infectivity was expressed as the number of local lesions per leaf.

Ribosome assay. Ribosome concentrations were determined by homogenizing samples in a medium containing 0.01 M Tris-chloride buffer, pH 7.3, 0.02 M MgC12, and 0.003 M mercaptoethanol (Reid and Matthews, 1966), to which had been added bovine pancreatic ribonuclease (Sigma Chemical Co.) at a concentration of 2.5 pg/ml. After clarification of extracts at 1OOOg for 4 min, aliquots were layered on 1040% linear sucrose gradients. The sucrose contained 0.01 Al- Tris-chloride buffer, pH 7.3,0.005 M MgCIZ, and 0.01 M KCI. Gradients were centrifuged for 2 hours in the SW 39 rotor of a Spinco ultracentrifuge. UV absorptiop profiles of gradients were obtained at 2540A with the ISCO apparatus. Concentrations of 70 S and 80 S ribosomes were determined from the areas below the appropriate peaks in the absorption profile; the OD!$,% of ribosomes was taken to be 12.7 (Clark, 1965).

The use of 0.05 ill phosphate buffer, pH 6.5, in the extracting medium and in sucrose gradients, in place of Tris-chloride, resulted in lower concentrations of 70 S chloroplast ribosomes being isolated (possibly because Mg2+ is precipitated). Equal amounts of 80 S cytoplasmic ribosomes were extracted with either buffer system. The addition of ribo- nuclease at concentrations of 0.5, 2.5, and 12.5 pg/ml during extraction resulted in the isolation of higher concentrations of both 70 S and 80 S ribosomes

Isolation and fractionation of 32P-labelled nucleic acids. Infected and healthy plants of the same size and age were labelled by apply- ing 100-200 PC1 of high specific activity 32P (as orthophosphate in dilute HCl-Austra- lian Atomic Energy Commission) directly to the washed roots. After 5 hours under continuous light, the first systemically infected leaf and its equivalent from the healthy plant were each homogenized in equal volumes of 90% phenol containing 0.1% 8-hydroxyquinoline and 4 % sodium dodecyl sulphate. Homogenates were shaken for 45 min at 20°, centrifuged at 10,000 rpm

for 10 min, and the aqueous supernatants were reextracted a further three times with equal volumes of 90 % phenol. Nucleic acids were collected by precipitation with 75% ethanol at 0”.

Nucleic acids were fractionated by elec- trophoresis in 2.5 % polyacrylamide gels (Peacock and Dingman, 1968) using the vertical flat-sheet apparatus of Reid and Bieleski (1968). Gels were loaded with O.Ol- 0.05-ml samples of 0.25-0.50 OD2eo units. They were electrophoresed at 10 mA for 15 min, then at 20 mA for approximately 90 min until the bromophenol blue marker dye had migrated 8 cm. Gels were fixed in 5% trichloroacetic acid, washed, stained with aqueous toluidine blue, and dried on glass plates for autoradiography.

RESULTS

Development of Infection

Under continuous light, inoculated leaves developed local chlorotic lesions 3-5 days after infection. Systemic symptoms were first observed 2 days later on leaves that were 3-5 cm long.

Plants infected with the mild or severe isolates showed the same initial vein-clear- ing, leaf distortion, and yellow-green chloro- sis on the first systemically infected leaf. Three or 4 days after symptoms appeared, the characteristic differences in the severity of symptoms were observed (Fig. 1).

The time at which virus entered the first systemically infected leaf was determined by excising leaf disks at daily intervals and floating them on water under lights for at least 5 days before assaying for infectivity. Virus was found to move into the leaf on the day when local lesions appeared on inocu- lated leaves. Infectious virus was extracted between 1 and 2 days later, one day before systemic symptoms appeared. Infectivity increased to a maximum at about the time symptoms first appeared, then declined (Fig. 2). This was paralleled by a rise and fall in the concentration of an infectious UV-absorbing component which was isolated from extracts of systemically infected leaves on sucrose density gradients, and which is assumed to be LNYV virions. The sedi- mentation behavior of this component in

1:TI3OSOM15S AXI) I,NYV INFECTION xi1

FIG. 1. Xyicotiana glulinosa plallt,s 11 days after inoculation with severe (left) and mild (cerrt.re) iso- lates of I,NY\-, compared with a healt,hy platlt (right) Necrotic or chlorot ica lesions are visible OII the inoculated leaves; chlorosis, distortion, and struntilug call he see11 011 the systemically itlfcctrd leaves. The first systemirally illfccted leaf is nne above the plulched leaf.

gradients was characteristic of T,SYV (Ale- Iean and E‘rancki, 1967)) and LNYV-like particles were observed with the electron microscope.

The growth rate of the first systemically infected leaves decreased within 1 day of the appearance of symptoms. Elongation of this leaf was almost completely arrested in plants affected with the severe isolate (Fig. 2) and was greatly retarded in plants in fected with the mild isolate. The growth of the xvhole plants was similarly arrested or retarded (Fig. 1). I%tnts grown in the glass- house after inoculation behaved similarly to those grown under continuous light except that the events were extended over a longer period.

effect of Infection on Ribosome Concentrations in Leaves

The concentration of 70 S ribosomes in extracts of the first systemically infected leaves fell below that of healthy leaves within 1 day of the appearance of symptoms (Fig. 3), and they could not be detected l- 3 days later. The effect was observed in plants infected with either the mild or severe isolates of LNYV. The 80 S ribosomes showed a similar more rapid decline in concentration, compared with healthy leaves

FIG. 2. Rate of elongation of first systemically- infected (I,NY\.) and healthy (II) leaves of Sicoliana ylulinosa plants, shown in relation to infectivity. Plalrts were infected wit,l: the severe isolate.

(Fig. 3), but this lagged behind the decline in 70 S ribosome concentration in every experiment.

Examination of sap extracts in the analyt- ical ultracentrifuge 11 days after plants were inoculated confirmed that 70 S ribo- somes could not be detected in extracts of infected tissue, and that the concentration of SO S ribosomes was lower than in healthy leaves. The concentration of Fraction I protein, which is localized in chloroplasts (Lyttleton and Ts’o, 195S), was approxi- mately half that in healthy leaf; Fraction II

462 1:ANI)LISS AND COLEMAN

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1.5.

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F OS-

70 s

- HEALTHY 0-Q MILD LNYV _ O--* SEVERE LNYV

2 4 6 8 10 12 2 L 6 8 10 12

DAYS AFTER INOCULATION

FIG. 3. Changes in concentration of 70 9 and 80 S ribosomes in leaves systemically infected with mild and severe isolates of LNYV, compared with healthy leaves. Symptoms appeared 6 days aft,er plants were inoculated.

protein was at the same concentration as in healthy leaf.

In the glasshouse, the infection-induced decline in the concentration of 70 S and SO S ribosomes was extended over a longer period, with a lag of 2-3 days between the appear- ance of systemic symptoms, and the first observed decline in 70 S ribosomes.

Fractionation of High Molecular Weight T-RNA

The rRNA composition of infected leaves was examined to determine whether the loss of ribosomes was correlated with a loss of their rRNA.

Before systemic symptoms appeared (4-5 days after inoculation) healthy and infected leaves showed similar rRNA patterns (Fig. 4, a, b). All four high molecular weight rRNA species, 16 S, 18 S, 23 S, and 25 S, were detected by staining and autoradio- graph.

Seven days after inoculation, when symp- toms first appeared and the accelerated loss of 70 S ribosomes was first observed in in- fected leaves, only trace amounts of chloro- plast rRNA (16 S and 23 S) (Loening and Ingle, 1967) were detected by staining and autoradiography (Fig. 4, c, d). All four

species were isolated from healthy leaf and all incorporated 32P.

Ko chloroplast rRNA was detected in infected leaves 11 days after inoculation, but as on day 7 the 1s S and 25 S rRNA com- ponents originating from the 80 S ribosomes (Loening and Ingle, 1967) were present in approximately the same amounts and in- corporated 32P as in healthy tissue. The 16 S and 23 S rRNA’s of healthy leaf did not incorporate detectable amounts of 32P, suggesting that chloroplast ribosome syn- thesis slows or ceases in fully expanded healthy leaves.

In the glasshouse, at the time systemic symptoms were first observed, 16 S, 18 S, 23 S, and 25 S rRNA species were all detected by staining in both infected and healthy leaves. In the healthy leaves all species incorporated 32P, whereas in the infected leaves the 16 S and 23 S rRSA’s incorporated no detectable 32P even though incorporation into the 18 S and 25 S rRNA’s was as high as in healthv leaves.

No additional species of RKA mere detected in preparations from infected leaves. RNA components with approximate estimated sedimentation coefficients of 12 S and 20 S were detected in preparations which

1: IROSOXII::: ANI) I,NY\. TNFI~XTIOX 4G3

FIG. 1. I~Xeclrophorei ic separation in poly- acrylamide gels of high molecrdar weight nucleic acids isolalcd from “YP-labelled healthy and in- feclcd leaves at 5 and 7 days after illoclllalion. Approximate sedimetltation coefficient, values (in Svedherg Illrits) are give11 it, the margill. The J)NA band is above i.he 25 S l<NA. Sample a, healthy, day 5; b, infected, day 5; c, healt,hy, day 7; 11. infected, day 7. Upper patterns, stained wit,h toltlidine blrle; lower patterns, autoradiograph of staitted gels. Samples of eq11a1 tot.al radioac- tivity are compared.

contained 16 S and 23 S RNA, and they are assumed to be breakdown products of the chloroplast rRNA (Ingle, 1968).

Chloroplasts in thin sections, and chloro- plasts isolated in Honda medium (Spencer and Wildman, 1%X), were examined by light microscopy to observe whether changes in

FIG. 5. Mean length of chloroplasts in t,issue sections of healthy (II) and infected (LXY\.) leaves during the development of infection.

morphology accompanied the described changes in the concentration of chloroplast ribosomes. Chloroplasts in infected tissue were found to become smaller after systemic symptoms appeared when plants were grown either under continuous illumination (Fig. 5) or under glasshouse conditions. Similar effects wverc observed in isolated chloro- plasts. There were no differences in the shape of chloroplasts from healthy and infected plants.

The results reported in this paper indicate that infection with LNYV has a profound effect on the nucleic acid metabolism of the chloroplasts of N. glutinosa. However, the loss of chloroplast rRNA and chloroplast 70 S ribosomes which followed the appear- ance of symptoms in systemically infected leaves occurred without any apparent loss of the structural integrity of the chloro- plasts, even though they became smalkr. Chloroplasts retained their shape, and E’rac- tion I protein K:LS still detectable although in reducrd amounts, afttbr symptoms ap- peared.

The effect of infection on the 80 S ribo- somcs differed from that on the 70 S ribo- somes, since the drop in SO S ribosome concentration was not accompanied by a marked fall in either the concentration of, or 321’ incorporation into, cytoplasmic rRNA. Perhaps the 80 S ribosomes became more susceptible to degradation during extraction,

464 BANDLEH AN11 COLXMAN

either by endogenous leaf ribonuclease or the added pancreatic ribonuclease. Another possible explanation is that the rRKAs were still being synthesized and maintained in the cell, but failed to become associated into 80 S ribosomes.

Xo conclusion can be drawn from the present results as to the relevance of these changes to the replication of LNYV, or to the role of chloroplasts in virus replication. Hoxvever, because these changes were first detected aft#er multiplication of LNYV had commenced, it seems improbable that they were necessary for initial virus replication. Because these changes in ribosomes would be expected to result in impaired protein synthesis they may be associated with symptom development, the cessation of growth, and may also lead to the observed late decline in the concentration of virus in the systemically infected leaves. Hirai and Wildman (1969) recently showed that in TMV-inoculated tobacco leaves, chloroplast rRNA did not incorporate 32P during the phase of initial increase in virus, and amino acid incorporation into chloroplast ribosomes and chloroplast proteins was very small. Chloroplast ribosomes were still present, but these workers did not make observations at later times.

Some of the effects of infection with LNYV appear similar to those described with TMV, but the virtually complete loss of 70 S ribosomes appears to be an observa- tion unique to the LNYV system. It will be of interest to see whether other viruses that induce symptoms similar to those of LNYV cause changes in the rRNA and ribosomes of their host plants similar to those de- scribed here.

ACKNOWLEDGMENTS

We thank Dr. R. I. B. Francki for helpful dis- cussion and Mrs. L. Wichman for preparation of drawings. The study was supported by a grant from the Australian Research Grants Committee.

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