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Plant protoplasts may be efficiently infected with viruses. Attachment to the plasmalemma is a critical step in infection; positively charged viruses readily adhere and infect but negatively charged viruses require the presence of a polycation (e.g. poly-L-ornithine) before infection can occur. The role of the polycation appears to be twofold: to modify the charge of the virus and to damage the surface of the protoplast. Virus probably enters the protoplast during repair of damaged regions of the plasmalemma. Pinocytosis does not appear to play a significant part in inoculation.

Infection of tobacco protoplasts with RNA of tobacco mosaic virus and the bromoviruses has been studied. The quality of the RNA was very important. Ribonuclease activity was controlled by incorporating 5 mᴍ ZnCl2 in the inoculum. Up to 60% infection has been observed with bromovirus RNA but infection with TMV RNA was much less efficient. A procedure for inoculating in 40% polyethylene glycol was developed. High levels of infection were obtained with all the viruses and viral RNA examined.

The four components of the genome of cowpea chlorotic mottle virus have been prepared in a highly active and highly purified state by a method based on their resolution by polyacrylamide gel electrophoresis. Activity and purity have been confirmed by gel electrophoresis under denaturing and non-denaturing conditions, infectivity tests on whole plants, translation in an mRNA dependent rabbit reticulocyte lysate and fingerprinting of T1 ribonuclease digests after labelling with 32P at the 5′ ends of the oligonucleotide products. The quality of the RNAs obtained was superior to those previously obtained by zonal centrifugation but only comparatively small batches of whole RNA can be handled with ease.

Summary The synthesis of proteins has been studied in tobacco protoplasts infected with cowpea chlorotic mottle virus (CCMV). Three new proteins were found with mol. wt. 19000 (P1), 34000 to 36000 (P2) and 100000 (P3). Protein P2 reached a maximum steady level about 16 to 20 h after infection; it was not a precursor of P1, which is virus coat protein. P3 appeared later than the other two proteins, reached its maximum about 44 h after infection and then declined. P2 and P3 were localized in the 1000 g and 20000 g pellets of a cell homogenate, in contrast to P1 (coat) which was largely found in the supernatant fraction after centrifuging at 20000 g for 30 min. The three proteins account for about 60% of the tripartite genome of CCMV.

Summary The properties of a mutant of cowpea chlorotic mottle virus which, although infective, cannot be disassembled in 1 m-NaCl, pH 7.5, are described. The mutant coat protein contains a lysyl to arginyl replacement which affects the hydrodynamic and titration characteristics of the virus which are discussed in regard to the anomalous ionization of a carboxyl-carboxylate pair and a lysyl residue.

Summary Freshly prepared tobacco mesophyll protoplasts behave as if they contain two classes, one of which is resistant to infection but the other is susceptible and can readily be doubly infected with brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV). BMV dominates in all double infections and although both types of virus particles are produced, only those of BMV are infectious. The defective CCMV particles contain normal amounts of RNA but only RNA 3 could be detected in them. No CCMV RNA 2 could be detected in doubly infected protoplasts, showing that there was only partial replication of the genome of CCMV. The proteins encoded by CCMV RNA 3 were produced in mixed infections. It is possible that BMV may exploit the gene products of CCMV while suppressing synthesis of infectious CCMV particles. There was no evidence that BMV RNA was encapsidated in CCMV coat protein.

Summary The multiplication of a temperature-sensitive (ts) mutant of cowpea chlorotic mottle virus (CCMV) in tobacco protoplasts has been examined. The results were similar to those obtained with intact cowpea plants. No intact virus was produced above 33.5 °C, and no coat protein, either soluble or insoluble was detected in the absence of intact virus. Ribonucleic acid (RNA) of the ts mutant, however, was synthesized at 35 °C. Virus particles made at 25 °C were degraded in vivo when the infected protoplasts were subsequently cultured at 35 °C. RNA-3 of ts CCMV was apparently encapsidated much more often than RNA-3 of wild-type (wt) CCMV in doubly infected protoplasts cultured at either 25 or 35 °C.

Summary The proteins synthesized in protoplasts infected with strain V5 of brome mosaic virus have been studied. Four new proteins of mol.wt. 2, 3.5, 10 and 10.7 × 104 were observed. These account for over 90% of the virus genome. The three proteins with lowest mol.wt. corresponded closely in size to those observed in protoplasts infected with cowpea chlorotic mottle virus.

Summary The progeny from mixed infections of tobacco protoplasts with a temperature sensitive (ts) and a temperature resistant (wt) strain of cowpea chlorotic mottle virus at restrictive temperatures (35 °C) was largely (90%) ts, judged by the coat protein produced by single lesion isolates grown at 25 °C. However, although apparently ts at 25 °C, many isolates were temperature resistant at 35 °C and produced virus. Successive ‘purification’ through single lesions allowed normal ts or wt viruses to be recovered from these unusual isolates. The genomes of ts and wt appear able to co-exist in mixed infections; ts is dominant over wt at all temperatures so that most of the progeny has ts genome, but wt remains available to rescue ts under restrictive conditions. There was no evidence that the phenomenon was due to recombination.

SUMMARY The differing responses of a wide range of tobacco mosaic virus (TMV) isolates to various genes controlling resistance in tomato were not correlated with the amino acid composition of their coat proteins. Nor were they necessarily linked with ability to cause necrotic local lesions in White Burley tobacco, which is usually associated with the presence of methionine in the coat protein.

Summary The behaviour and properties of a temperature-sensitive mutant of cowpea chlorotic mottle virus are described. The mutant multiplied well at 21° but, unlike the wild-type, only slightly at 32° although considerable amounts of uncoated RNA accumulated in inoculated leaves at this temperature. The specific infectivity of the mutant was much lower than that of the wild-type virus because the largest species of encapsidated mutant RNA was almost completely degraded even in virus from plants grown at 21°. The temperature sensitivity and low specific infectivity of the mutant were related to properties of its coat protein, which was much less heat stable than that of the wild-type virus. Glutamic acid and alanine replaced lysine and valine respectively in the mutant coat protein and these replacements, in addition to affecting thermal stability, influenced the polymerization of isolated protein at pH 6.7.

SUMMARY Tomato mosaic virus derived from susceptible tomato plants (the standard virus) was cultured in resistant plants. Sap from non-inoculated leaves of resistant tomato plants infected with virus from the resistant host was more infective and contained more virus particles than leaf sap of resistant plants infected with the standard virus. Leaves of resistant tomatoes infected with virus from the resistant host also showed more obvious symptoms. Susceptible plants infected with virus from resistant plants not only showed fewer symptoms than when infected with standard virus, but samples were less infective and contained less virus up to 26 weeks, when values for infectivity were similar. This modification in activity was not reversible and was obtained with two lines of tomato having different types of resistance. Passage of virus from resistant plants through susceptible plants did not impair its ability to infect resistant plants.

SUMMARY Multiplication of tomato mosaic virus (strain of tobacco mosaic virus) in susceptible and in resistant tomato plants was studied by assaying the infectivity and virus content of sap from non-inoculated leaves on infected plants. In susceptible plants, infectivity and virus particle number increased rapidly to a maximum at about 14 days after inoculation; leaves produced subsequently contained less virus. In resistant plants, no virus was detected in non-inoculated leaves until 5 weeks after inoculation. Infectivity then gradually increased at a somewhat higher rate than did numbers of virus particles. The concentration of virus in resistant plants remained lower than in susceptible ones. At first the ratio of infectivity to particle number was larger for samples from the susceptible than the resistant line, but the ratio from the resistant line increased slowly until it eventually equalled that from the susceptible one. Virus cultured in either of two lines of tomato with different types of resistance invaded healthy plants of either line more readily than virus cultured in the susceptible variety. Resistant lines seem to select self-invasive new forms of tomato mosaic virus.

1. Sucrose was the only sugar which supported a high growth rate of the excised root clone 1 of the Dorset Marlgrass strain of red clover (Trifolium pratense L.). D-fructose, D-glucose, and maltose supported a lower level of growth. The other sugars tested were inactive as carbon sources. Additions of D-xylose, D-mannose, and D-arabinose strongly inhibited the growth of roots when simultaneously supplied with sucrose. 2. Nitrate was the most effective source of nitrogen tested. Ammonium, urea, and L-glutamine as nitrogen sources supported a lower level of growth. The optimum pH range for growth was the same whether nitrate or ammonium was used as nitrogen source. 3. The omission of any constituent from White's vitamin mixture limited growth. The most marked growth depression followed the omission of niacin. Niacin plus thiamine was more effective than any other combination of two factors. There was no evidence that pyridoxine and niacin could partially replace one another. 4. Indoleacetic acid (IAA), acco...

1. The effects of manipulative procedures on intrastrain variation in excised roots of the Dorset Marlgrass strain of red clover (Trifolium pratense L.) have been studied, and a technique established for the satisfactory maintenance of two derived excised root clones. 2. A study has been made, using roots of clone 1, of the influence of sucrose concentration of the medium on growth of roots cultured 8 days and upon survival of growth activity in individual meristems when repeatedly subcultured. Main-axis meristems have a lower optimum sucrose concentration than do lateral meristems. At the standard 2% sucrose concentration nearly all lateral roots are robust and opaque. They remain opaque and increase in diameter with increase in the sucrose concentration. At 0.75% or lower sucrose concentrations, all the lateral roots are fine and transparent. 3. Roots which develop from tips excised from these robust and fine laterals differ in growth rate, morphology, and survival of their main-axis meristems. Attempts...

SUMMARY Amino acid analyses have been made of the proteins of single-lesion isolates of five strains of tobacco mosaic virus (TMV) differentiated by Lycopersicon hosts. These hosts differed in their genetical control of resistance to TMV, and the virus strains had therefore survived specific selection pressures. Two of the five strains differed in their amino acid composition from type TMV and from all other tomato strains of TMV previously examined. Symptoms induced by the five strains in four tomato lines and in Nicotiana tabacum cvs White Burley and Kawala are described.

Cowpea chlorotic mottle virus RNA has been prepared in comparatively high yield (at least 50%) by a modified phenol extraction method. The preparation, which has high biological activity, has been resolved into four components by zonal centrifugation on a 15 - 40% (w/v) sucrose density gradient. The components obtained have been tested for biological activity against whole plants and plant protoplasts. Each of the two largest components RNA 1 and RNA 2 was by itself infective (50 - 90% of the specific infectivity of the whole genome) and produced virus-specific proteins (coat protein and P2) and RNAs (“RNA 3“ and “RNA 4”). Contamination by small proportions « 10 %) of neighbouring RNAs is presumed to be involved in this infectivity. The two smallest components were obtained in an almost pure form.