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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.

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 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.

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.