Infection Of Tobacco Protoplasts Research Articles

Expression of thePeanut stunt virusCoat Protein Gene Is Essential and Sufficient for Production of Host-Dependent Ribbon-Like Inclusions in Infected Plants

ABSTRACT We previously have reported that infection of tobacco protoplasts or leaf tissue with the cucumovirus Peanut stunt virus (PSV) induced the production of unusual cytoplasmic ribbon-like inclusions. The formation of these novel inclusions is strain-specific, because infection of tobacco with subgroup II PSV strains, but not subgroup I strains, induced the production of inclusions. Furthermore, we have demonstrated that induction of the ribbon-like inclusions maps to PSV subgroup II RNA3, which codes for the coat protein (CP) and movement protein (MP). We have now extended these studies using chimeric constructs containing CP and MP open reading frames (ORFs) from PSV strains ER and W that belong to subgroups I and II, respectively. Additionally, recombinant Potato virus X (PVX) vectors containing translatable and untranslatable PSV CP ORF were constructed. Plants inoculated with infectious chimeric PSV or recombinant PVX transcripts were analyzed for CP expression by enzymelinked immunosorbent assay and reverse transcription-polymerase chain reaction and for inclusion production by electron microscopy. The results of these experiments indicated that translation of the CP ORF alone is essential and sufficient for inclusion production. In immunogold labeling experiments using an antiserum to PSV virions, abundant gold labeling of the inclusions was observed, suggesting that PSV CP is probably a major component of the inclusions. Because inclusion production is host specific, a host factor is likely to be involved. In addition to their diagnostic importance, these novel inclusions may also prove valuable in identifying the host factors that interact with PSV CP.

A specific cis-hairpin ribozyme facilitates infection of a TMV-based DNA vector in tobacco protoplasts

The effect of a specific cis-hairpin ribozyme on TMV-based vectors in the infection of tobacco protoplasts was studied. Vectors contained full-length TMV genome cDNA linked to a T7 promoter or a CaMV 35S promoter at the 5′-end and an NOS gene polyadenylation signal at the 3′-end. The coat protein (CP) gene was replaced with the green fluorescent protein (GFPuv) gene allowing quantification of protoplast infection. In plasmids pTMVGFPRIB (T7-driven) and pSTMVGFPRIB (CaMV 35S-driven), the cDNA fragment of the cis-hairpin ribozyme (designed to specifically cleave the transcripts immediately downstream of the 3′-terminus of TMV RNA) was inserted between the 3′-terminus of TMV genome and NOS sequence. The in vitro transcript TMVGFPRIB was three- to fivefold more infectious than the control TMVGFPNOS. Northern blot analysis indicated that the 3′-terminal non-viral sequence had been cleaved from the in vitro transcripts by the cis-hairpin ribozyme soon after in vitro transcription. pSTMVGFPRIB and pSTMVGFPNOS plasmid DNAs were, as expected, less infectious than their in vitro transcript counterparts. However, pSTMVGFPRIB was somewhat more infectious than pSTMVGFPNOS. Northern blot analysis indicated that pSTMVGFPRIB synthesized more genomic and sub-genomic RNAs in the protoplasts. The significant increase in infectivity and viral RNA synthesis is due to the specific activity of the cis-hairpin ribozyme in vivo. Therefore, the cis-hairpin ribozyme described here may improve TMV-based vectors in the expression of foreign protein in plants.

Unusual Cytoplasmic Inclusions Induced in Tobacco by Peanut Stunt Virus Subgroup II Strains Map to RNA3

ABSTRACT Infection of tobacco protoplasts or leaf tissues with peanut stunt virus (PSV) subgroup II strains induced the production of unusual cytoplasmic ribbon-like inclusions. The inclusion structures appeared as long, thin, densely staining sheets that were prevalent within the cytoplasm, accumulating most commonly near vacuoles. Numerous virions and ribosomes could be seen adjacent to the inclusion surfaces. The formation of these novel inclusions appeared to be subgroup specific, since infection of tobacco with PSV strains W and B (subgroup II), but not strains ER, V, and J (subgroup I), induced the inclusions. Furthermore, inclusion formation was shown to be host specific, because the inclusions were not detected in either of two leguminous host species infected with PSV subgroup II strains. Using tobacco protoplasts electroporated with various assortments of infectious RNA transcripts derived from cDNA clones of genomic RNAs of PSV-ER and PSV-W, we demonstrated that induction of the unusual ribbon-like inclusions maps to PSV-W (subgroup II) RNA3. This conclusion is consistent with the finding that PSV strain BV-15, a natural intraspecific reassortant that derives its RNA2 and RNA3 from a subgroup I strain, did not induce inclusion formation.

Functional Significance of Three Basic N-Terminal Amino Acids of Alfalfa Mosaic Virus Coat Protein

Infection of tobacco protoplasts with mutant alfalfa mosaic virus (AMV) RNAs indicated that three basic amino acids in the N-terminus of AMV coat protein are important for the biological activity of the coat protein in the beginning of infection. Substitution of alanines for lysines at position 14 or 17 in the coat protein resulted in a 5- or 10-fold reduction in the activity of the protein, respectively. However, substitution of alanine for arginine at position 18 entirely abolished activity. Arginine 18 was also required for the coat protein to bind to the 3′ noncoding region of the virus RNAin vitro, whereas lysine 14 or 17 was not required. Thus, these results indicate that arginine 18 is essential for the activity of the coat protein in early infection and that binding of the coat protein to AMV RNA correlates with activity.

Subgenomic RNAs mediate expression of cistrons located internally on the genomic RNA of tobacco necrosis virus strain A.

Upon infection of tobacco protoplasts, the genomic RNA of tobacco necrosis virus strain A (TNV-A) accumulates linearly in time. The accumulation patterns of the two subgenomic RNAs resemble those of endogenous mRNAs in that the peak levels are reached after several hours. The accumulation of the 1.3-kb subgenomic RNA is delayed by 1 h compared with that of the 1.6-kb subgenomic RNA, which illustrates the important role of the subgenomic RNAs in the regulation of TNV-A gene expression. The locations of the 5' nucleotides of the subgenomic RNAs reveal that the 5'-proximal cistrons of the 1.6- and 1.3-kb RNAs encode an 8-kDa protein from open reading frame (ORF) 3 and the coat protein from ORF 5, respectively. In a wheat germ translation system, a synthetic transcript resembling the 1.6-kb RNA expresses both ORFs 3 and 4. Moreover, the synthesis of the 6-kDa protein from ORF 4 depends on the translation efficiency of ORF 3, suggesting that in vivo, ORFs 3 and 4 are both expressed from the 1.6-kb RNA. The major in vitro translation product of TNV-A genomic RNA is the coat protein. We show that the region upstream of the coat protein promotes internal initiation of translation in vitro. However, this region is functionally inactive in vivo, suggesting that TNV-A genomic RNA is not important for coat protein synthesis in plants.

An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80-90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.

Infection of tobacco protoplasts with liposome-encapsulated potyviral RNA

The RNA of the potyvirus, tobacco vein mottling virus (TVMV), was encapsulated in reverse-phase evaporation vesicle liposomes containing phosphatidylserine and cholesterol. The liposomes were used to inoculate tobacco mesophyll protoplasts. Analysis of extracts of protoplasts, by enzyme-linked immunosorbent assay (ELISA), hybridization with labeled cDNA, electron microscopy and infectivity assays, indicated that significant multiplication of the virus had occurred after incubation of the protoplasts for 2–3 days. It was estimated that approx. 15% of the protoplasts had become infected.

The Effect of Charge on Infection of Tobacco Protoplasts by Bromoviruses

SUMMARY The susceptibility of freshly isolated tobacco protoplasts to infection by brome mosaic virus (BMV) fell rapidly to a low residual level over a period of 8 h in culture. In contrast, susceptibility to infection by cowpea chlorotic mottle virus (CCMV) fell much more slowly over 15 h in culture to a similar residual level. In the absence of polycations, BMV infected many more protoplasts if CCMV were also present in the inoculum even though CCMV did not infect such doubly inoculated protoplasts. Both effects are thought to be a consequence of the different electrical charges of particles of the viruses and support the view that inoculation of protoplasts depends primarily on physical interactions between virus and protoplast.

Effects of Actinomycin D on the Infection of Tobacco Protoplasts by Four Viruses

SUMMARY The addition of actinomycin D (25 µg/ml) or cordycepin (1 mm) to protoplast cultures immediately after inoculation with particles of tobacco mosaic, tobacco rattle, tobacco ringspot or potato leafroll viruses resulted in a decrease in the proportion of protoplasts becoming infected, as judged by staining with fluorescent antibody to virus particles. A delay of a few hours between the inoculation and the addition of either inhibitor largely or completely eliminated this effect. In contrast, infection was unaffected by the addition of actinomycin D when the protoplasts were inoculated with RNA preparations from tobacco mosaic or tobacco rattle viruses.

Wirkung von Virushemmstoffen auf die Infektion von Tabakprotoplasten mit dem Tabakmosaik‐Virus

Abstract Effects of virus inhibitors on the infection of tobacco protoplasts with tobacco mosaic virus Yeast extract inhibits the infection of Nicotiana glutinosa plants with tobacco mosaic virus (TMV), whereas in N. sandérae yeast extract is not effective. This phenomena was compared with the effect of yeast extract on protoplasts, and on the infection of protoplasts of both tobacco species with TMV. Additionally, skim milk and ribonuclease were included in the experiments as further inhibitors of early stages of virus infection. It was examined whether these inhibitors damage non‐inoculated protoplasts (a), and whether they affect virus infections in protoplasts as they do in cells of intact plants (b).To investigate protoplast damage by the inhibitors, conductivity measurements of protoplast suspensions containing inhibitors, and the ability of protoplasts for cell wall regeneration after treatment with the inhibitors, were used. Inhibitor concentrations which prevent virus infections in plants did not damage the protoplasts.The inhibitor effect on the course of infection was investigated by protoplast treatments before, during and after inoculation with TMV, and by addition of the substances to the culture medium. Measurements of virus content in protoplasts after cultivation revealed different results for the three inhibitors, however, there was no difference in the response of protoplasts from the two tobacco species to yeast extract. It is concluded that there are principal differences between the inhibition of plant and protoplast infections. Therefore, it is unlikely that protoplasts are a useful system for the mode of action studies on inhibitors of early stages of virus infection in plants.

The effect of protoplast concentration during inoculation on the infection of tobacco protoplasts.

When tobacco protoplasts were inoculated with tobacco rattle virus, the proportion infected was inversely related to the protoplast concentration between 0.5 X 10(5) and 4 X 10(5) per milliliter when the inocula contained phosphate or Tris-HCL buffers, but was independent of protoplast concentration when using citrate buffer. The decrease in infection obtained at high protoplast concentrations in phosphate was diminished by increasing the poly-L-ornithine (PLO) concentration. At usual PLO concentrations, inocula in phosphate produced more infection that those in citrate, but at some suboptimal PLO concentrations the position was reversed. These effects may result from differences in the reaction between PLO and the different buffer anions.

The Mechanism of Virus Entry during Infection of Tobacco Protoplasts with TMV

The Mechanism of Virus Entry during Infection of Tobacco Protoplasts with TMV

Effects of viral protein, viral RNA, and some other polyelectrolytes on infection of tobacco protoplasts by TMV

Tobacco cv. Samsun protoplasts were inoculated with TMV in the presence of different polyelectrolytes. Retention of [ 14C]TMV was determined by radioactivity of the protoplasts or by infectivity in homogenates of samples frozen immediately after inoculation and washing. Virus yield was determined by infectivity in protoplasts following incubation for 48 hr in light at 28°. Infectious TMV, noninfectious TMV, and TMV protein (100 μg/ml) enhanced virus retention. However, the virus yield for the first substance was essentially lower. When the substances were added to the incubation medium, they decreased virus yield. Bovine serum albumin and casein hydrolysate did not appreciably affect retention; yet, they also decreased virus yield. Virus RNA, whether infective or not, strongly suppressed the virus yield when present in the inoculation medium at a concentration of 1 μg/ml or more. This effect was independent of the presence of actinomycin D in the medium. Experiments with labeled TMV revealed the low virus yield to be the result of a reduction of virus retention in inoculum containing a mixture of TMV and TMV RNA. Similar effects on virus retention and yield were produced by total RNA of animal origin and by dextran sulfate. The authors assume that the three above-mentioned polyelectrolytes block virus adsorption on the plasmalemma by binding with a virus-polyornithine complex.

The Infection of Tobacco Protoplasts with a Variant of Brome Mosaic Virus

SUMMARY Neither tobacco mesophyll protoplasts nor tobacco leaves could be infected with wild type brome mosaic virus (BMV wt) or its RNA, but both were infected when inoculated with 50 µg/ml or more of a variant of BMV (BMV V5). This is about 100 × the amount required for efficient infection by cowpea chlorotic mottle virus (CCMV). Poly-l-ornithine stimulated the infection of protoplasts with BMV V5, but was not essential. BMV V5. RNA inoculum caused infection of protoplasts under conditions similar to those needed for their infection with CCMV-RNA.

The infection of tobacco protoplasts with pea enation mosaic virus.

SUMMARY Tobacco protoplasts inoculated with pea enation mosaic virus (PEMV) contained virus antigen which was detectable by fluorescent antibody staining 15 h or more after inoculation. Increase in the proportion of fluorescing protoplasts and in the intensity of fluorescence with time of culture suggested that new virus antigen was synthesized. At early stages the fluorescence was found both in the nucleus and in the cytoplasm, but by about 48 h it was more restricted to the nucleus. The uptake of [32P] into virus and into virus RNA indicated that PEMV multiplied in the protoplasts, but the yield of virus was relatively low, about 1 to 5 × 104 particles per infected protoplast. The optimum inoculation conditions for routine infection as assessed by fluorescent antibody staining were 1.5 µg/ml PEMV, 1.5 µg/ml poly-l-ornithine, and pH 4.7. Protoplasts could be infected in the absence of poly-l-ornithine at the higher PEMV concentrations. Actinomycin D added to the culture medium reduced the number of fluorescing protoplasts and the intensity of fluorescence.

Structural and autoradiographic observations of the infection of tobacco protoplasts with pea enation mosaic virus

The structural changes accompanying infection of tobacco protoplasts with pea enation mosaic virus have been examined by electron microscopy. The first visible response to infection is the appearance at about 17 h post inoculation of cytoplasmic membrane bound bodies enclosing a series of fibril-containing vesicles. At later stages of infection a proportion of these bodies appear to fuse with the nuclear membrane. Intact virus is found only within the nucleus. The fibrils within the vesicles are digestible by RNAse but not DNAse and they may be labelled using [(3)H]uridine. These results are discussed in terms of the course of infection of plant cells by viruses.

Effect of phosphate on the infection of tobacco protoplasts by tobacco rattle virus.

At pH 6.0, substituting phosphate for citrate in the inoculum greatly increased the infection of tobacco leaf protoplasts by tobacco rattle virus, but it had little effect on the yield of virus per in

Structural changes accompanying infection of tobacco protoplasts with two spherical viruses

Ultrastructural changes occurring in isolated tobacco leaf protoplasts following infection with cowpea chlorotic mottle virus and bromegrass mosaic virus variant 5, have been examined. Only the endoplasmic reticulum/nuclear membrane complex has shown a consistent series of changes in structure and distribution. Both viruses induce proliferation of the endoplasmic reticulum and its modification. The nuclear membrane gives rise to small cytoplasmic vacuoles probably containing nucleic acid by a process of blebbing involving both its inner and outer membrane. This is a prominent feature of CCMV infection. BMV5 infection is particularly characterised by the appearance of local organised arrays of assembled virus particles. The results are discussed in terms of possible nucleocytoplasmic interactions.