John Watts

The coat protein of beet curly top virus is essential for infectivity

We have applied the procedure of Agrobacterium-mediated inoculation to develop a simple, efficient, and reproducible assay for the infectivity of the leafhopper-transmitted geminivirus, beet curly top virus (BCTV). This assay system was used to show that a coat protein mutant of BCTV is not infectious, but could be complemented by coagroinoculation with a second mutant bearing a lethal mutation in the complementary-sense open reading frame, C1. Furthermore, the coat protein mutant retained the ability to replicate and to produce both ssDNA and dsDNA when electroporated into Nicotiana tabacum protoplasts. We conclude that the coat protein of BCTV is essential for spread of the virus. The results are discussed in the light of results with coat protein mutants of other geminiviruses.

Mutational analysis of complementary-sense genes of African cassava mosaic virus DNA A

We have investigated the ability of African cassava mosaic virus DNA A mutants, containing disrupted complementary-sense genes, to infect Nicotiana benthamiana and to replicate in Nicotiana tabacum protoplasts. Three overlapping open reading frames (ORFs) with the capacity to encode proteins with an Mr greater than 10K (AC1, AC2 and AC3) are highly conserved between geminiviruses that infect dicotyledonous plants and one (AC4) is less well conserved. Of these, only AC1 is a prerequisite for DNA replication; disruption of this ORF rendered the DNA noninfectious in plants and prevented DNA replication in protoplasts. Disruption of ORF AC2 prevented plant infection but mutants were capable of autonomous replication and replicated DNA B in trans in protoplasts to produce DNA forms that comigrated with wild-type virus DNAs. The AC2 mutant phenotype suggests that the product of this ORF is involved in virus spread within the plant. Mutants in which ORF AC3 had been disrupted retained the ability to replicate and to infect plants systemically although symptom development was delayed and attenuated, and mutant DNA accumulated to much lower levels (10 to 20%) in comparison with wild-type infection. Typical geminate virus particles were observed in extracts of plants infected with ORF AC3 mutants indicating that this gene is not essential for coat protein synthesis or virus assembly but possibly acts by modulating virus levels in infected tissues. Disruption of ORF AC4 had no effect on infectivity or symptom development suggesting that this ORF is maintained only because it overlaps the highly conserved ORF AC1.

Size reversion of African cassava mosaic virus coat protein gene deletion mutants during infection of Nicotiana benthamiana.

Mutants of African cassava mosaic virus containing extensive deletions across the coat protein gene that remove up to one-third of the genomic component have been constructed and shown to be infectious when mechanically inoculated onto Nicotiana benthamiana by leaf abrasion. Using N. tabacum protoplasts we demonstrate that mutant pCLV.CP delta 11, containing a 712 bp deletion, is competent for replication in its deleted form. However, systemic spread of pCLV. CP delta 11 and other deletion mutants is associated with reversion of DNA 1 to a size comparable to that of the native genomic component. This contrasts with the behaviour of coat protein mutants of the closely related geminivirus tomato golden mosaic virus which maintain their deletions during spread. Appraisal of the different inoculation procedures used to introduce the mutants into plants suggests the imposition of a stringent size requirement for localized cell-to-cell spread which is relaxed for long distance spread through the vascular system.

Evidence that nucleocapsid disassembly and a later step in virus replication are inhibited in transgenic tobacco protoplasts expressing TMV coat protein

Tobacco mosaic virus (TMV)-like pseudovirus particles containing mRNA for Escherichia coli beta-glucuronidase (GUS) were electroporated into mesophyll protoplasts from control or TMV coat protein (CP)-transgenic tobacco (Nicotiana tabacum cv. Xanthi). GUS-particles were expressed 100-fold less efficiently in CP-transformed than in control protoplasts whereas unencapsidated GUS mRNA was expressed only 2.8-fold less efficiently. Lower transient expression of packaged GUS mRNA is probably due to inhibited disassembly of nucleocapsids in CP-transgenic protoplasts. Control and U1 CP-transformed protoplasts are equally susceptible to infection by cowpea strain TMV (Cc), as well as unencapsidated Cc or U1 RNA. In contrast, native or in vitro reconstituted U1 TMV particles result in 5- to 6-fold fewer infected CP-transgenic than control protoplasts. When Cc RNA was transcapsidated in U1 CP in vitro, the hybrid virions were equally infectious in both classes of protoplasts. We conclude that although compatible U1 protein-protein interactions significantly inhibit (GUS) nucleocapsid disassembly in CP-transgenic protoplasts, the endogenous CP must also interfere with a later stage of infection involving the homologous viral RNA.

Inoculation of protoplasts with viruses by electroporation.

Optimal conditions for electroporation have been determined using inoculation of brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) and its RNA into protoplasts of Nicotiana tabacum and N. plumbaginifolia. The most satisfactory medium was 0.5-0.7 M mannitol; calcium ions were toxic and other electrolytes were not helpful during electroporation. Brief pulses (ca. 10 microsec) were less destructive to the protoplasts than longer ones (ca. 10 msec) and gave high percentage infections with CCMV RNA. RNA entered the protoplasts only if present during the voltage pulse. Optimal voltage depended on the sample size, interelectrode distance, and pulse duration. A 50-nF capacitor discharging a 5- to 10-microsec pulse through a 1-ml sample in 0.7 M mannitol with a 4-mm interelectrode distance gave maximum infection with minimal protoplast damage at 2.5 kV/cm. A single pulse was sufficient; multiple pulses slightly increased infection. Electroporation of viral RNA was at least as effective as inoculation in the presence of polyethylene glycol. Positively charged BMV also infected readily but negatively charged CCMV only poorly.

Double infection of tobacco protoplasts with brome mosaic virus and cowpea chlorotic mottle virus

Mixed infections of tobacco protoplasts with Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV) have been studied. Fluorescent antibody staining showed that capsids of both viruses were made in doubly infected protoplasts but only infectious BMV could be recovered, i.e., BMV dominated in mixed infections and prevented synthesis of infectious CCMV. Healthy protoplasts could be infected after several hours in culture but the susceptibility to infection decreased with time in culture; the decrease was much faster with BMV than with CCMV, probably because the difference in electrical charge of the two viruses affects the method of entry into the protoplasts. If inoculation with the second virus was delayed there was an increasing resistance to superinfection (interference or cross-protection) so that when the delay between inoculations was more than 8 hr BMV was no longer able to infect protoplasts previously infected with CCMV and vice versa. No recombinants between BMV and CCMV were observed. Attempts to rescue a temperature-sensitive strain of CCMV with BMV under restrictive conditions (35 degrees ) were unsuccessful; dominance by BMV prevented synthesis of infectious CCMV RNA.

Plant Growth Conditions and Yield of Viable Protoplasts Isolated from Leaves of Lycopersicon esculentum and L. peruvianum

Summary Protoplasts were isolated from leaves of the cultivated tomato Lycopersicon esculentum cultivar Rheinlands Ruhm and the wild species L. peruvianum which were grown under different environmental conditions. These conditions included different calcium concentrations in the root medium, treatment with abscisic acid and kinetin, and different temperatures and humidities. No consistent differences in final yields of protoplasts were found between control and plants treated with either calcium, abscisic acid or kinetin. The final yields, however, were significantly higher in plants grown under relatively high humidity (82 % relative humidity) and low temperature (15 °C) as compared with those grown under low humidity (56 %) and high temperature (25 °C). The increase of final yields was greater in the cultivated plants. Greater proportions of spherical as well as viable protoplasts as expressed by their plating efficiency, were isolated from plants grown under the lower temperature and higher humidity. Some of the changes characterize the leaves of plants grown under the different environmental conditions are discussed.

Signals and Structures Involved in Early Interactions between Plants and Viruses or Pseudoviruses

One mechanism by which stable plant RNA virus particles might be disassembled in vivo involves recruitment of cell factors and ribosomes by a short, easily exposed portion of RNA. No cell specificity exists during virus uptake or early cotranslational disassembly. Virus-like reporter ribonucleocapsids were synthesized in vitro to study the mechanism, sequence-dependence and site of disassembly and transient gene expression without concomitant replication. “Pseudoviruses” and transcapsidated viral RNAs can elucidate the mechanism(s) of genetically engineered, coat protein-mediated cross-protection in electroporated tobacco protoplasts or plants.