DISCGSSION AND PRELIMINARY REPORTS 171

9. R.INDOLPH, L. R., Stain ?“echnol. 10, 95-96 (1935).

10. DC~MAN, W. F., Biochim. Biophys. Acta 120, 212-221 (19G6).

11. GIBBOXS, I. R., Nalwe 184, 375-376 (1959). 12. C.4no, L. G., and VBN TGBERGEX, R. I'., J.

Cell. Biol. 1.5, 173-188 (1962). IS. SCHLEGEL, D. E., and DE ZOETEN, G. A., J.

Cell. Biol. ii1 press. WILLEM G. LASGEXBEKG D.tvro E. SCHLEGEL

Deparfmenf oj Plant Pathology Cniversily of California Berkeley, California 94Y20

Accepted March 6, 1967

Distribution of Tobacco Mosaic Virus in

Systemically Infected Tobacco

Leaves

Fulton (1) showed that in the mosaic pat- tern produced by various strains of t,obacco mosaic virus (TMV) in Nicotiana syluestuh Spegaz. & Comes leaves t’he dark green areas of tissue contained less virus, measured by infectivity, t’hsn did light green areas. Solberg and Bald (2) showed that in N. glauca Grah. infect’ed with TMV, virus invasion into ex- panding leaves was correlated with maturity of leaf tissue. In systemically infected Chinese cabbage (Bras&a pekinensis Rupr.) leaves showing the mosaic pattern induced by turnip yellow mosaic virus (TYMV), the dark green areas of bhe patt,ern cont’ain little or no virus, appear t,o contain normal amounts of ribo- somes and soluble proteins, and arc cyt’o- logirally normal (3, 4). The dark green areas in the mosaic pattern usually remain con stant in shape in individual leaves but increase in size from the earliest observable stage until leaf senescence. We have reinvestigated by several methods the distribution of TRIT’ in tobacco leaves showing the typical mosaic sympt,oms, and have found a basically similar situation t)o that report’ed for TYXV in Chinese cabbage.

TMV (a local type strain) was cultured in Nicotiana tabacum vars. White Burley and Warnes, in pots in t,he glasshouse. Plants were inoculated on the sixth to seventh leaf

above the cotyledon Ieaves. In both tobacco varieties under these conditions, plants be- coming systemically invaded show a regular succession of symptoms in the newly develop- ing leaves: (a) in the first and second leaves above the inoculated leaf general diffuse yel- lowing developed, with no clear mosaic pat- tern containing dark green areas. (b) in tha next one or two leaves very numerous small islands of dark green t,issue were produced. Typically in this group t,here were 110 islands of dark green tissue in the distal area of the leaf. The green islands were more numerous toward the centrc and base of t,he leaf, with larger islands of green usually concentrated in the cent,ral-basal region. (c) the next OIR or two leaves developed a pattern containing fewer and much larger dark green areas. In

this group there was a marked tendency for the dist#al portion of the leaf to be solid yellow-green, the dark green areas being confined to the lower three quarters of the lamina. (d) In subsequent leaves the dark green areas appeared more randomly over the whole leaf. Occasional leaves in this group showed yellowing 0111~7, with no islands of dark green.

The amount of virus in the dark green was much less than in the yellow-green tissue. In infectivit#y assays on half-leaves of N. glutinosa L., 6 samples of dark green tissue from different plants gave only 4-10s as many lesions as did a comparable set of samples from yellow green &sue from t,he snme leaf. Relative amount,s of TR,IV per unit fresh weight of t,issue were also estimat,cd following fractionat’ion of extracts from dark green and yellow green areas of infected leaf in sucrose density gradients (l&40 % sucrose containing 0.01 Jf KCl, 0.01 M MgC12, and 0.01 AI Tris-HCl buffer, pH 7.2 centrifuged for 2 hours at 35,000 rpm in a Spinco SW 39 rotor). In tests on leaves from four plant)s, dark green tissue contained l-6 ‘36 as much virus as yellow green tissue from the same leaf based on optical density measurements at 260 rnp of hhe TMV band in the gradients. In a test using virus precipitation end poi& with a TJIV antiserum, a heat-clarified ex- tract (10 minut,es at 54’) from dark green

172 I~ISCUSSIOX ,4X1) PRELI~MINARY REPORTS

tissue gave a visible precipitate to a dilution of 1: 4, while an extract of yellow green tissue from the same leaf precipitated to a dilution of 1:64. Portions of the same ext,racts were examined in t’he analytical ultracentrifuge. From areas under the schlieren peaks it was calculated that dark green had 9 ‘,;;, as much TMV as yellow green &sue. Infectivity tests on these extracts gave 9 local lesions per hnlf- leaf (mean of 15 leaves) for dark green and 148 for yellow green tissue.

Thus all three methods agreed in showing that dark green areas in leaves with mosaic symptoms contain much less T-llV than yellow green areas. In different’ experiment’s the concentration of virus in dark green areas varied. There was also variation in how dark the green areas were, some areas grading from fully dark green to yellow green. The amount of virus in such graded areas meas- ured by infectivit,y appeared to be correlated with the intensity of greenness. For cxamplc, in one test green t’issue gave 0.4 local lesions per half-leaf; paler green, 26; and full yel- low green, 234.

Mesophyll cells in dark green tissue may have a limited capacity for supporting TBSV replication. However, the possibilities that the virus found in dark green samples is due to contamination with yellow green tissue or t,hat it represents virus synthesized in leaf veins or epidermis have not yet been ruled out.

The platelike and hexagonal crystals seen by light microscopy as intracellular inclusions in leaves infected with TMV have been described by many workers. These inclusions are particularly readily seen in living prepa- rations in leaf hair and epidermal cells (5). WC have examined these cells from dark and yellow green areas of T&IV-infected leaf. Under our conditions of growth most leaf hair cells overlying yellow green areas showed one or more of the charact’eristic ThIV crystalline inclusions. Such cells from fully dark green areas cornained no crystalline inclusions. At macroscopically sharp junc- tions between a fully dark green arca and a yellow green area there was a zone accom- modating about 3-5 epidermal cells in which leaf hairs with and without crystalline inclu- sions were close neighbours. This junction was most easily observed in a piece of tissue

where the line of demarcation ran to the leaf margin, but it should be noted that very fre- quently a narrow zone of yellow green tissue ran along the actual leaf margin outside a dark green area.

For electron microscope examination of virus inclusion bodies small blocks of leaf tissue were fixed in glutaraldehyde, post,- fixed in osmium, rapidly dehydrated in an acet’one series, embedded in Araldite, sec- tioned, and stained with lead citrate (6). Three sets of blocks of yellow green and of fully dark green tissue from different leaves were prepared, and numerous s&ions were examined. In sections of yellow green t,iesuc we observed numerous TMV inclusion bodies of the type described by Iiolehmainen et al. (7) in both mesophyll and epidermis. Ko such inclusion bodies were seen in sections of fully dark green tissue or in t’issue from uninfected plants. We have not yet examined tissue of grade intermediate between fully dark green and yellow green.

Relative concentration of ribosomes, per unit fresh weight, in dark green and yellow tissue was estimated following sucrose density gradient fractionation of leaf extracts. In

four tests the concentrations of S3 S ribosomes in yellow green ranged from 69 to X8 % of that’ in dark green, and the concent,ration of 6s S ribosomes was 27-S2 % of that’ found in dark green tissue.

The detailed mosaic patterns typically produced by TYMV in Chinese cabbage and TMV in tobacco differ in detail in a number of respects not described here. Nevert)heless the two disease patterns appear to have several major features in common: (a) the dark green areas in the mosaic contain low levels of virus compared with bhe yellow green; (b) the dark green areas may persist for long periods in spite of the fact that they are surrounded by, and are in apparently normal cont,act8 wit’h, tissue containing a high concent’ration of virus; (c) the mosaic pat- terns containing dark green areas appear to develop only in leaves that would still con- tain some dividing cells at the time of infec- tion.

In our previous work with TYMV (3, /,) we put forward the tentative hypothesis that dark green areas of the mosaic may represent the progeny of single cells that’ were

infected at the time of cell division, and which were made resistant to TYMV by some process similar to lyeogeny in bacteria. In one respect our observat#ions wit’h TRIV tend tjo support, this view. The pat’tern of distribu- tion of t)he numerous small green islands seen in the oldest 011e or two leaves that show mosaic symptoms is very similar to the terminal st,ages in the growth pattern of the tobacco leaf described by Avery (8). In t.he oldest part of these leaves (the distal portion) there is no mosaic. Below the distal region is a zone showing numerous very small dark green islands of tissue. This zone grades into t’he cent’ral and basal region (the young- cst part of t’he lamina), where the islands of dark green are less numerous but larger than those in the intermediate zone.

On the other hand, in the ontogeny of t,he tobacco leaf as described by Avery (8), the epidermal tissues arise independently from t’he mesophyll. The crystalline inclusion bodies characteristic of TRZV appear t#o be uniformly present in leaf hair cells above yellow green areas of t,he mosaic, and absent above dark green areas. This correspondence would not be expected if the islands of cells without virus or with low concent’rat’ion of virus arose in a random way from single cells during leaf ontogeny. A critical point yet to be established is whether the virus present in t*he leaf hair cells of yellow green areas is always synthesized Ohere, or whether in some areas it migrates passively from the underlying mesophyll. Similarly, it, may be that virus is produced in some areas of epidermis overlying dark green areas and

that this virus migrates passively to the un- derlying mesophyll, reducing the concentra- Con in t,he epidermis b&w the level neces- sary to form t;r>;stalline inclusion bodies. Whatever t’he orlgm of the dark green areas, the fact, that, t)hose produced by TMV and TYMV have a number of import’ant features in common suggest that dark green areas in the mosaic patt,erns produced by rmmy

ot,her viruses may have similar properties.

iZCKXOWLElX;PENT

This work was srlpported in part by U.S.P.H.S. Grant, AI-04973.

REFERENCES

1. FULTON, Ii. IV., Ph~glopathology 41, 579-592 (1951).

2. SOLBERG, It. A., and BALD, J. G., Virology 17, 359-361 (1962).

3. CH~LCROPT, J., and MATTHEWS, R. E. F., Virology 28, 555-562 (1966).

4. REID, M. S., and ~~~~~~~~~~~~ R. E. F., Virology 28, 563-570 (1966).

5. BEBLE, H. f., Conbib. Boyce Thompson Inst. 8, 413-431 (1937).

6. REYXOLDS, E. H.? J. Cell. Hiol. 17, 208-212

(1963).

7. KOLEHMMNEN, L., ZECH, I-l., and VON WETT-

STEIN, II., J. Cell. Riol. 25, 77-97 (1965). 8. AVERY, G. S., Jlt., dnl. J. Botany 20, 565-592

(1933). P. II. ATKINSON

I?. E. F. MATTHEWS Department of Cell Biologly Unioersity of Auckland Auckland, i\iew Zealand

Accepted March 3, 1961