THE S T R U C T U R E OF CELLS D U R I N G TOBACCO MOSAIC VIRUS R E P R O D U C T I O N Mesophyll Cells Containing Virus Crystals

The submicroscopic organization of mcsophyll ccUs from tobacco leaves systemically infected with tobacco mosaic virus (TMV) is described. After fixation with glutaraldchyde and osmium tctroxidc the arrangement of the TMV particles within the crystalline inclusions is well preserved. Only thc ribonuclcic acid-containing core of thc virus particles is visible in the micrographs. Besides the hexagonal virus crystals, several characteristic types of "inclusion bodies" arc definable in the cytoplasm: The so-called fluid crystals sccm to correspond to single layers of oriented TMV particles between a network of the cndoplasmic rcticulum and ribosomes. Unordcrcd groups or well oriented masses ot tubes with the diameter of the TMV capsid arc found in certain areas of the cytoplasm. A complicated inclusion body is characterized by an extensively branched and folded part of the cndoplasmic rcticulum, containing in its folds long aggregates of flexible rods. Certain parts of the cytoplasm arc filled with large, strongly electron-scattering globules, probably of lipid composition. These various cytoplasmic differentiations and the different forms of presumed virus material arc discussed in relation to late stages of TMV reproduction and virus crystal formation. I N T R O D U C T I O N In a previous paper (33) the submicroscopic organization of the nucleus and cytoplasm during successive phases of tobacco mosaic virus (TMV) reproduction was described for singly infected hairs of tobacco lcavcs. For further studics on the sites of virus multiplication and cspccially on thc synthesis as well as assembly of thc virus coat protcin in the cell, it was thought dcsirablc to look for improved fixation and embedding techniques. For this purposc various procedures have bccn tricd on leaves systemically infected with a T M V flavum strain. Uninfected leaves have been studied for comparison. Fixation with glutaraldchydc and osmium tetroxide has been found especially useful in elucidating the structure of infected cells. In this paper the organization of systemically infected mesophyll ceils with T M V inclusions, representing the final stage in T M V reproduction, is outlined M A T E R I A L S A N D M E T H O D S Twoto four-month-old plants of Nicotiana tabacum (hybr. Samsun X White Burlcy) systemically infected with a flavum strain of T M V (kindly supplied by Dr. Wittmann) were used. For comparativc studies pieces from leaves about 3 cm long with symptoms were fixcd and embedded. The following mcthod gavc thc best results: 77 on O cber 0, 2017 jcb.rress. org D ow nladed fom Small leaf pieces were fixed for 3 hours in 6.5 per cent glutaraldehyde (23) in 0.07 M phosphate buffer (pH 7.0) at 4°C. After 6 hours of washing in 0.07 M phosphate buffer, postfixation was carried out in 2 per cent buffered OsO4 solution at 4°C. The material was dehydrated by passing it through increasingly concentrated acetone solutions up to pure acetone. After 10 minutes in propylene oxide, the pieces were kept for 12 hours in a 1:1 mixture of propylene oxide and Epon, using Luft's formula (16). After embedding in Epon, polymerization was carried out for one day at room temperature, followed by one day at 38°C, and finally at 60°C. Sections were cut on an LKB ultrotome and examiced in a Siemens Elmiskop Ib after treatment with 1 per cent uranyl acetate and/or lead citrate (21). Electron microscopic magnifications were determined with the aid of a diffraction grating replica.