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 an Arabidopsis thaliana Mutant in Which the Multiplication of both Cucumber Mosaic Virus and Turnip Crinkle Virus Is Affected

During the systemic infection of plants by viruses, host factors play an important role in supporting virus multiplication. To identify and characterize the host factors involved in this process, we isolated an Arabidopsis thaliana mutant named RB663, in which accumulation of the coat protein (CP) of cucumber mosaic virus (CMV) in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in RB663 plants was controlled by a monogenic, recessive mutation designated cum2-1, which is located on chromosome III and is distinct from the previously characterized cum1 mutation. Multiplication of CMV was delayed in inoculated leaves of RB663 plants, whereas the multiplication in RB663 protoplasts was similar to that in wild-type protoplasts. This suggests that the cum2-1 mutation affects the cell-to-cell movement of CMV rather than CMV replication within a single cell. In RB663 plants, the multiplication of turnip crinkle virus (TCV) was also delayed but that of tobacco mosaic virus was not affected. As observed with CMV, the multiplication of TCV was normal in protoplasts and delayed in inoculated leaves of RB663 plants compared to that in wild-type plants. Furthermore, the phenotype of delayed TCV multiplication cosegregated with the cum2-1 mutation as far as we examined. Therefore, the cum2-1 mutation is likely to affect the cell-to-cell movement of both CMV and TCV, implying a common aspect to the mechanisms of cell-to-cell movement in these two distinct viruses.     

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.     

Glutinosone,a new antifungal sesquiterpene from Nicotiana glutinosa infected with tobacco mosaic virus

Glutinosone is an antifungal substance induced in leaves of Nicotiana glutinosa following infection with tobacco mosaic virus. From chemical, spectroscopic and biogenetic evidence it has been assigned structure (1).     

Heat Inducible Expression of a Chimeric Maize hsp70CAT Gene in Maize Protoplasts

The response of maize (Zea mays L.) protoplasts to high temperature stress was investigated. After isolation and electroporation, protoplasts were preincubated for 12 hours at 26°C then incubated for 6 hours at elevated temperatures. The pattern of polypeptides synthesized by these protoplasts during the last hour was monitored by in vivo labeling with ³⁵S-methionine. Incubation at 40° and 42°C resulted in the synthesis of polypeptides not detectable at 26°C. Introduction of a chimeric maize heat shock protein 70 promoter-chloramphenicol acetyltransferase coding region gene into protoplasts via electroporation resulted in the temperature-dependent induction of chloramphenicol acetyltransferase activity with maximal activity at 40°C. In the same protoplasts, a second chimeric gene, in which the firefly luciferase coding region was under the control of the 35S promoter from cauliflower mosaic virus, did not show an increase in expression after incubation at higher temperatures. Maize protoplasts provide a system to study molecular responses to high temperature stress.     

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

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 Arabidopsis cucumovirus multiplication 1 and 2 loci encode translation initiation factors 4E and 4G

The cum1 and cum2 mutations of Arabidopsis thaliana inhibit cucumber mosaic virus (CMV) multiplication. In cum1 and cum2 protoplasts, CMV RNA and the coat protein accumulated to wild-type levels, but the accumulation of the 3a protein of CMV, which is necessary for cell-to-cell movement of the virus, was strongly reduced compared with that in wild-type protoplasts. In cum2 protoplasts, the accumulation of turnip crinkle virus (TCV)-related RNA and proteins was also reduced. Positional cloning demonstrated that CUM1 and CUM2 encode eukaryotic translation initiation factors 4E and 4G, respectively. Unlike most cellular mRNA, the CMV RNA lacks a poly(A) tail, whereas the TCV RNA lacks both a 5′-terminal cap and a poly(A) tail. In vivo translation analyses, using chimeric luciferase mRNA carrying the terminal structures and untranslated sequences of the CMV or TCV RNA, demonstrated that these viral untranslated sequences contain elements that regulate the expression of encoded proteins positively or negatively. The cum1 and cum2 mutations had different effects on the action of these elements, suggesting that the cum1 and cum2 mutations cause inefficient production of CMV 3a protein and that the cum2 mutation affects the production of TCV-encoded proteins.     

Five new alkaloids from Aconitum apetalum (Ranunculaceae)

Twenty-one alkaloids, including five new ones, acoapetaldines A–E (1–5), were isolated from the whole plants of Aconitum apetalum. Among them, 1 is an aconitine-type diterpenoid alkaloid, 2 and 3 are aporphine alkaloids and 4 and 5 are napelline-type diterpenoid alkaloids. The structures of the new alkaloids were elucidated by spectroscopic data analysis and that of acoapetaldine D (4) was confirmed by single crystal X-ray crystallography. Acoapetaldine A (1) and aconorine (12) exhibited moderate anti-tobacco mosaic virus (anti-TMV) activity, while corydine (15) displayed moderate antimicrobial activity against Pseudomonas aeruginosa.     

The mechanism of infection of plant protoplasts by viruses

Summary The process of virus infection of protoplasts isolated from tobacco leaves has been examined by means of electron microscopy. Immediately after inoculation, virus particles appear at two types of site: trapped in complex surface lesions of the plasmalemma, or in peripheral cytoplasmic vesicles. The complex lesions are only visible after treatment of the protoplasts with inocula containing poly-l-ornithine. With infection by tobacco mosaic virus and cowpea chlorotic mottle virus, which require poly-l-ornithine, the majority of virus particles occur at lesion sites. Pea enation mosaic virus, which does not require poly-l-ornithine for infection to become established, is found predominantly and in high numbers in peripheral vesicles. The behaviour of these three viruses is discussed in terms of a probable mechanism for infection of the protoplasts.     

Isoflavones from the roots and stems of Nicotiana Tabacum and their anti-tobacco mosaic virus activities

Two new isoflavones, 7-hydroxy-6,3′,4′,5′-tetramethoxy-isoflavone (1) and 6-hydroxy-7,3′,4′,5′-tetramethoxy-isoflavone (2), together with seven known isoflavones were isolated from the roots and stems of Nicotiana tabacum. Their structures were determined by means of HRESIMS, extensive 1D and 2D NMR spectroscopic studies and chemical evidences. The anti-tobacco mosaic virus (anti-TMV) activities of the isoflavones were also evaluated. The results reveal that compound 9 shows high anti-TMV activity, compound 2 shows moderate anti-TMV activity, and compounds 1, 3–8 show weak anti-TMV activities.     

Phenolic amides from the leaves of Nicotiana tabacum and their anti-tobacco mosaic virus activities

Three new phenolic amides, tabamides A–C (1–3), together with three known phenolic amides (4–6), were isolated from the leaves of Nicotiana tabacum. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D NMR techniques. Compounds 1–6 were also tested for their anti-tobacco mosaic virus (anti-TMV) activity. The results showed that compound 1 exhibited high anti-TMV activity with inhibition rate of 38.6%, which is higher than that of positive control (ningnanmycin). The other compounds also showed potential anti-TMV activity with inhibition rates in the range of 15.3–26.5%, respectively.     

Antiviral flavones from the leaves of Nicotiana tabacum

Three new flavones, tabaflavones A–C (1–3), together with three known flavones (4–6), were isolated from the leaves of Nicotiana tabacum. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. Compounds 1–6 were evaluated for their anti-tobacco mosaic virus (anti-TMV) activities. The results showed compound 1 exhibited moderate anti-TMV activity with inhibition rate of 29.2%, which is close to that of the positive control. The other compounds also showed the anti-TMV activities with inhibition rates in the range of 14.2–20.8%.     

High-speed electro-fusion and electro-transfection of plant protoplasts by a continuous flow electro-manipulator

A continuous flow electro-manipulator available both for mass production of fused and of transfected plant protoplasts was devised using a flow chamber with gold-coated glass panel electrodes. Up to 100 ml of protoplasts suspension were treated within 20 min at the rate of approximately 1×10⁶ protoplasts / min. The yield of diheterokaryons between tobacco mesophyll and carrot root protoplasts reached approximately 10 % of total protoplasts by flow electro-fusion. More than 95 % of tobacco and cowpea mesophyll protoplasts became infected with tobacco mosaic virus RNA by flow electro-transfection.     

Three new xanthones from the stems of Garcinia oligantha and their anti-TMV activity

For more bioactive compounds, phytochemical investigations of the acetone extract of the stems of Garcinia oligantha resulted in the isolation of three new xanthones (1–3) and five known analogues (4–8). Structural elucidations of 1–3 were performed by spectral methods such as 1D and 2D (HMQC, HMBC, and ROESY) NMR spectroscopy, in addition to high resolution mass spectrometry. Compounds 1, 2, 6, 7, and 8 showed anti-tobacco mosaic virus (anti-TMV) activities with inhibition rates above 10%.     

Biosynthesis of tobacco mosaic virus RNA in tobacco protoplasts

Changes in the number of protoplasts, viability, protein and chlorophyll contents and ribonucleases activity were studied in tobacco mesophyll protoplastsin vitro inoculated with tobacco mosaic virus (TMV). The number of protoplasts slowly increased during the cultivation period and the viability decreased from 95 to 67% in the control noninoculated protoplasts, and to 55% in the infected protoplasts. 30 h after inoculation the protein and chlorophyll contents strongly decreased to 25–30% and 17–19%, respectively, in comparison with contents 3 h after inoculation. The chlorophylla/b ratio decreased from 2.11 and 2.02 to 0.79 and 0.60 in healthy and infected protoplasts, respectively. The activities of ribonucleases in protoplasts quickly decreased during experiment but they were higher in infected than in noninfected protoplasts (between 20 to 30 h after inoculation they were 132 to 146% higher than that in healthy controls). These activities corresponded to the multiplication curve of TMV.     

Three new prenylated xanthones from Comastoma pedunculatum and their anti-tobacco mosaic virus activity

Comastomaxanthones A–C (1–3), three new xanthones with a rare 2-oxoprenyl substituent, along with three known ones (4–6) were isolated from the aerial parts of Comastoma pedunculatum. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. In addition, compounds 1–6 were tested for their anti-tobacco mosaic virus activity. The results showed that all isolated compounds exhibited weak anti-TMV activity with IC₅₀ values ranging from 122.7 to 242.9 μM.     

Novel α,β-unsaturated amide derivatives bearing α-amino phosphonate moiety as potential antiviral agents

Based on flexible construction and broad bioactivity of ferulic acid, a series of novel α,β-unsaturated amide derivatives bearing α-aminophosphonate moiety were designed, synthesized and systematically evaluated for their antiviral activity. Bioassay results indicated that some compounds exhibited good antiviral activities against cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) in vivo. Especially, compound g18 showed excellent curative and protective activities against CMV, with half-maximal effective concentration (EC₅₀) values of 284.67 μg/mL and 216.30 μg/mL, which were obviously superior to that of Ningnanmycin (352.08 μg/mL and 262.53 μg/mL). Preliminary structure-activity relationships (SARs) analysis revealed that the introduction of electron-withdrawing group at the 2-position or 4-position of the aromatic ring is favorable for antiviral activity. Present work provides a promising template for development of potential inhibitor of plant virus.     

Genetic analysis of resistance to six virus diseases in a multiple virus-resistant maize inbred line

Key message

Novel and previously known resistance loci for six phylogenetically diverse viruses were tightly clustered on chromosomes 2, 3, 6 and 10 in the multiply virus-resistant maize inbred line, Oh1VI.

Abstract

Virus diseases in maize can cause severe yield reductions that threaten crop production and food supplies in some regions of the world. Genetic resistance to different viruses has been characterized in maize populations in diverse environments using different screening techniques, and resistance loci have been mapped to all maize chromosomes. The maize inbred line, Oh1VI, is resistant to at least ten viruses, including viruses in five different families. To determine the genes and inheritance mechanisms responsible for the multiple virus resistance in this line, F₁ hybrids, F₂ progeny and a recombinant inbred line (RIL) population derived from a cross of Oh1VI and the virus-susceptible inbred line Oh28 were evaluated. Progeny were screened for their responses to Maize dwarf mosaic virus, Sugarcane mosaic virus, Wheat streak mosaic virus, Maize chlorotic dwarf virus, Maize fine streak virus, and Maize mosaic virus. Depending on the virus, dominant, recessive, or additive gene effects were responsible for the resistance observed in F₁ plants. One to three gene models explained the observed segregation of resistance in the F₂ generation for all six viruses. Composite interval mapping in the RIL population identified 17 resistance QTLs associated with the six viruses. Of these, 15 were clustered in specific regions of chr. 2, 3, 6, and 10. It is unknown whether these QTL clusters contain single or multiple virus resistance genes, but the coupling phase linkage of genes conferring resistance to multiple virus diseases in this population could facilitate breeding efforts to develop multi-virus resistant crops.