Infection of cowpea mesophyll protoplasts with cowpea chlorotic mottle virus (CCMV) RNA encapsulated in large liposomes

It has been demonstrated that cowpea chlorotic mottle virus RNA encapsulated in phosphatidyl serine/cholesterol reverse evaporation vesicles (REV) could infect cowpea mesophyll protoplasts under conditions known to enhance liposome-protoplast interactions. Positively charged phosphatidylcholine/stearylamine multilamellar liposomes did not deliver functional CCMV RNA despite their very high nucleic acid trapping capacity and their high affinity for protoplasts.     

Expression of bottom component RNA of cowpea mosaic virus in cowpea protoplasts

Upon inoculation of cowpea protoplasts with the bottom component of cowpea mosaic virus, at least six virus-induced proteins (with sizes of 170, 110, 87, 84, 60, and 32 kilodaltons) are synthesized, but not the capsid proteins (37 and 23 kilodaltons). These bottom-component-induced proteins were studied with respect to their genetic origin and mode of synthesis. The analyses were based on their electrophoretic peptide patterns resulting from partial digestion with Staphylococcus aureus protease V8. Comparison of the peptide patterns of the virus-induced proteins with those of the cowpea mosaic virus RNA-coded polypeptides produced in rabbit reticulocyte lysate showed that the 170- and 32-kilodalton polypeptides, which are the first viral products in cowpea mosaic virus-infected cells, were actually coded by the bottom component RNA of the virus. The 110-, 87-, and 84-kilodalton polypeptides, and possibly the 60-kilodalton polypeptide, appeared to have amino acid sequences in common with the 170-kilodalton polypeptide, demonstrating that they were virus coded as well. The results indicated that cowpea mosaic virus bottom component RNA was translated in vivo into a single 200-kilodalton polyprotein from which probably all bottom-component-specific proteins arose by three successive cleavages.     

Bromovirus movement protein genes play a crucial role in host specificity.

Monocot-adapted brome mosaic virus (BMV) and dicot-adapted cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes. Although RNAs 1 and 2 together are sufficient for RNA replication in protoplasts, systemic infection also requires RNA3, which encodes the coat protein and the nonstructural 3a movement protein. We have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by RNA3 as well as by RNA1 and/or RNA2. Here, to test their possible role in host specificity, the 3a movement protein genes were precisely exchanged between BMV and CCMV. The hybrid viruses, but not 3a deletion mutants, systemically infected Nicotiana benthamiana, a permissive host for both parental viruses. The hybrids thus retain basic competence for replication, packaging, cell-to-cell spread, and long-distance (vascular) spread. However, the hybrids failed to systemically infect either barley or cowpea, selective hosts for parental viruses. Thus, the 3a gene and/or its encoded 3a protein contributes to host specificity of both monocot- and dicot-adapted bromoviruses. Tests of inoculated cowpea leaves showed that the spread of the CCMV hybrid containing the BMV 3a gene was blocked at a very early stage of infection. Moreover, the BMV hybrid containing the CCMV 3a gene appeared to spread farther than wt BMV in inoculated cowpea leaves. Several pseudorevertants directing systemic infection in cowpea leaves were obtained from plants inoculated with the CCMV(BMV 3a) hybrid, suggesting that the number of mutations required to adapt the hybrid to dicots is small.     

The plant defense response to cucumber mosaic virus in cowpea is elicited by the viral polymerase gene and affects virus accumulation in single cells.

Resistance to infection in cowpea by strains of cucumber mosaic virus (CMV) involves a local, hypersensitive response (HR) and a localization of infection. These responses can be separated by mutation at two sites (nucleotides 1978 and 2007, in codons 631 and 641) in the CMV 2a polymerase gene. Changes to both sites of a restricted strain allow systemic infection without an HR and increase the accumulation of both the 2a protein and viral RNA in protoplasts, while changing position 1978 alone results in a systemic infection, a systemic HR, and an increase in viral RNA accumulation in protoplasts. It is suggested that the inhibition response observed in protoplasts, where an HR does not occur, leads to localization of infection in whole plants and that different plant genes are involved in eliciting the HR and the localization response.     

Age dependence of cowpea protoplasts for uptake of spermidine and infectibility by alfalfa mosaic virus

Cowpea protoplasts were prepared from plants of different ages and examined for their ability to take up polyamines and for their infectibility by alfalfa mosaic virus. A lag period of 20 h was necessary before the onset of rapid polyamine uptake; the occurrence of this rapid uptake depended on the age of the leaves used for protoplast preparation. The percentage of infection of cowpea protoplasts by alfalfa mosaic virus, and the amount of virus produced also depended on the age of the plants used for protoplast preparation. In contrast, the uptake of amino acids was rapid in all cowpea protoplasts tested.     

The cowpea mosaic virus M RNA-encoded 48-kilodalton protein is responsible for induction of tubular structures in protoplasts.

Tubular structures extending from plasmodesmata in cowpea mosaic virus (CPMV)-infected tissue have been implicated to play an important role in cell-to-cell movement of this virus. Using a cauliflower mosaic virus 35S promoter-based transient expression vector, we show that expression of only the CPMV M RNA-encoded 48-kDa protein (48K protein) in cowpea protoplasts is sufficient to induce these structures. Strikingly, expression of the 48K protein in protoplasts from a number of nonhost plant species, such as barley, Arabidopsis thaliana, and carrot, also resulted in tubular structure formation. Thus, it is not likely that the viral 48K protein, though playing a key role in cell-to-cell movement of CPMV, has a role in determining the host range of CPMV.     

Rapid detection of cowpea aphid-borne mosaic virus in cowpea seeds

Four cultivars of cowpea (Vigna unguiculata [L]. Walp.) were infected with cowpea aphid-borne mosaic virus (CABMV) by natural infection in field plots. Seeds taken from these plants were tested for the presence of the virus by ELISA and symptom observation on the plantlets grown from the seeds. A biotin/ streptavidin ELISA technique was used and found to be more sensitive than a standard ELISA protocol for detecting CABMV infection in seed. There was a good correlation between the ELISA detection of CABMV in tissue taken from single cowpea seeds and subsequent development of infected plants grown from the same seeds. The ELISA technique is reliable for selecting CABMV-free stocks of cowpea seeds.     

The nucleotide sequence of cowpea mosaic virus B RNA

The complete sequence of the bottom component RNA (B RNA) of cowpea mosaic virus (CPMV) has been determined. Restriction enzyme fragments of double-stranded cDNA were cloned in M13 and the sequence of the inserts was determined by a combination of enzymatic and chemical sequencing techniques. Additional sequence information was obtained by primed synthesis on first strand cDNA. The complete sequence deduced is 5889 nucleotides long excluding the 3' poly(A), and contains an open reading frame sufficient to code for a polypeptide of mol. wt. 207 760. The coding region is flanked by a 5' leader sequence of 206 nucleotides and a 3' non-coding region of 82 residues which does not contain a polyadenylation signal.     

Purification and some properties of a new strain of cowpea mild mottle virus in Israel*

The virus responsible for tomato pale chlorosis disease in Israel was purified from Nicotiana glutinosa plants. Purified virus contained a single stranded RN A of mol. wt 2.5 × 10⁶ and a single coat protein subunit of mol. wt 31 000. Enzyme-linked immunosorbent assay and an immunoelectron microscopy decoration test demonstrated a serological relationship with cowpea mild mottle virus (CMMV). Based on the present study and previously reported data the virus was identified as a new strain of CMMV designated CMMV/I.     

Coalescence of the sites of cowpea mosaic virus RNA replication into a cytopathic structure

Cowpea mosaic virus (CPMV) replication induces an extensive proliferation of endoplasmic reticulum (ER) membranes, leading to the formation of small membranous vesicles where viral RNA replication takes place. Using fluorescent in situ hybridization, we found that early in the infection of cowpea protoplasts, CPMV plus-strand RNA accumulates at numerous distinct subcellular sites distributed randomly throughout the cytoplasm which rapidly coalesce into a large body located in the center of the cell, often near the nucleus. The combined use of immunostaining and a green fluorescent protein ER marker revealed that during the course of an infection, CPMV RNA colocalizes with the 110-kDa viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, indicating that these sites correspond to the membranous site of viral replication. Experiments with the cytoskeleton inhibitors oryzalin and latrunculin B point to a role of actin and not tubulin in establishing the large central structure. The induction of ER membrane proliferations in CPMV-infected protoplasts did not coincide with increased levels of BiP mRNA, indicating that the unfolded-protein response is not involved in this process.     

Effect of maize plants on colonisation of cowpea plants by bean flower thrips,Megalurothrips sjostedti

The effect of non-host maize plants on colonisation of cowpea byMegalurothrips sjostedti (Trybom) (Thysanoptera; Thripidae) was investigated. There were no differences in population density and activity ofM. sjostedti on sole cowpea crop and mixed cowpea/maize crop during the colonisation phase (i.e. 10–30 days after emergence of the plants. However, subsequentlyM. sjostedti numbers were lower in the mixed than in the sole crop, suggesting that maize did not interfere with colonisation of cowpea crop by thrips. In a choice situation, higher numbers ofM. sjostedti oriented towards, and settled on, sole cowpea plants than on those mixed with maize. Olfactory tests indicated that fewer thrips oriented towards a cowpea/maize mixed odour source. When equal numbers of thrips were introduced into the centre of sole- and mixed-cropped cowpea plots, the thrips became randomly distributed in each plot. Fewer thrips were recovered from the mixture than from the sole crop. It is concluded that, although non-host plant odours do not reduce thrips colonisation they interfere with host plant utilisation.     

Interactions of Cowpea Mosaic Virus with Cowpea Protoplasts1)

The capability of cowpea mosaic virus to attach to and infect protoplasts of immune, hypersensitive, and susceptible cowpea (Vigna unguiculata) lines was examined by inoculating protoplasts with either purified virus or radioiodinated purified virus ¹²⁵I-CPMV. Systems were used in which plants were immune and protoplasts susceptible, plants were immune and protoplasts resistant, and plants and protoplasts were susceptible to CPMV. No differences were observed in the attachment of ¹²⁵I-CPMV to resistant and susceptible protoplasts. Polycations, proteins, or virus particles were added to the inoculation medium to neutralize potential nonspecific interactions between cells and virus particles. The various additives induced quantitative differences in binding of virus particles to protoplasts.     

Flow cytometric analysis of tobacco and cowpea protoplasts infected in vivo and in vitro with alfalfa mosaic virus

Immunofluorescence flow cytometry was used to study the distribution of viral antigen in protoplast populations. Protoplasts were isolated from healthy and alfalfa mosaic virus (AMV) infected tobacco leaves (designated in vivo infected). Furthermore isolated tobacco and cowpea protoplasts were infected in vitro with AMV. The FITC-conjugated antibodies could penetrate formaldehyde fixed protoplasts. The flow cytometric measurements were rapid and reproducible. Comparable immunofluorescence patterns were found for all infected samples (per sample 10⁴ protoplasts were measured). Infectious virus could only be detected in in vivo infected tobacco protoplasts and in in vitro infected cowpea protoplasts.     

Isolation of a fraction from cauliflower mosaic virus-infected protoplasts which is active in the synthesis of (+) and (-) strand viral DNA and reverse transcription of primed RNA templates

Sub-cellular fractions, isolated from cauliflower mosaic virus (CaMV)-infected turnip protoplasts, are capable of synthesising CaMV DNA in vitro on an endogenous template and of reverse transcribing oligo dT-primed cowpea mosaic virus RNA. The activity was not detected in mock-inoculated protoplasts. In vitro-labelled DNA hybridized to single-stranded M13 clones complementary to the putative origins of (-) and (+) strand CaMV DNA synthesis and to restriction endonuclease fragments encompassing more than 90% of the CaMV genome. The synthesis of (-) and (+) strand DNA appeared asymmetric. The template(s) for in vitro CaMV DNA synthesis are in a partially nuclease-resistant form. Fractions capable of in vitro CaMV DNA synthesis contained CaMV RNA both heterogeneous and as discrete species; they also contained a range of different sizes of CaMV DNA. Several lines of evidence indicate that this range of in vitro-labelled CaMV DNA, extending from 0.6kb to 8.0kb in length, represents elongating (-) strand DNA. These are discussed in relation to their role as possible replicative intermediates.     

Olive latent ringspot virus, a newly recognised virus infecting olive in Italy

A manually transmissible virus, for which the name olive latent ringspot virus (OLRV) is proposed, was isolated from a symptomless olive tree. The virus was mechanically transmitted to test plants. Purified preparations of OLRV contained three classes of isometric particle, c. 28 nm in diameter, with sedimentation coefficients of 525 (T), 975 (M) and 1325 (B) and containing 0, 30 and 43% nucleic acid respectively. At equilibrium in CsCl gradients, the buoyant densities of T and M components were 1–29 and 1–43 g/cm³ respectively, whereas B component separated into two sub-components with buoyant densities of 1–51 g/cm³ (BJ and 1–52 g/cm³ (B₂). Particle preparations contained two species of single-stranded RNA with mol. wt 1–40 times 10⁶ and 2–65 times 10⁶, both necessary for infectivity. The coat protein of OLRV, dissociated under strong denaturing conditions, separated into four components in polyacrylamide gel electrophoresis. Over 75% of the protein was found in a band with mol. wt 57 600, but all four components were recognised as oligomers of a monomeric form with mol. wt 14 300. OLRV was serologically unrelated to 26 different isometric plant viruses including 17 recognised nepoviruses. Its properties strongly indicate that it is a hitherto undescribed member of the nepovirus group.     

Effects of point mutations in the major capsid protein of beet western yellows virus on capsid formation,virus accumulation,and aphid transmission

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected approximately 90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.