Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.     

Direct visualization of the RNA location in cucumber green mottle mosaic virus and tobacco mosaic virus protein disk

The location of RNA in cucumber green mottle mosaic virus and tobacco mosaic virus protein disks was visualized by a negative staining method as a narrow ring localized at a radius of 4 nm, which corresponds to the location of RNA obtained by X-ray diffraction studies of tobacco mosaic virus. The same ring-shaped stains were observed in the end views of helical rods prepared in acidic solutions from viral protein without RNA. Since such a ring-shaped image could not be observed in end views of natural particles and reconstituted particles composed of protein and RNA, the narrow ring was concluded to indicate the RNA location on the basis of X-ray analysis.     

Plant resistance to fungal diseases induced by the infection of cucumber mosaic virus attenuated by satellite RNA

Biological control agents (BCAs) composed of attenuated cucumber mosaic (CMV) and its satellite RNA for controlling CMV diseases were found to induce plant resistance to a number of fungal diseases. Tests conducted in both the field and greenhouse showed evident protective effects against fungal infections by the BCAs. Artificial inoculation with a fungal spore suspension using BCA-treated plants, satellite transgenic plants and plants infected with CMV alone indicated that the resistance to fungi was induced by the virus infection, not by the presence of satellite RNA.     

High resistance to cucumber mosaic virus conferred by satellite RNA and coat protein in transgenic commercial tobacco cultivar G-140.

A chimeric vector was constructed to express cucumber mosaic virus (CMV) satellite (Sat) RNA and coat protein (CP). Transgenic lines of tobacco cultivar G-140 expressing CP and Sat-RNA were obtained; these lines had high resistance to CMV. Fifty to 70% of the transgenic plants were symptomless 90 days after inoculation with 25-50 micrograms/ml of CMV. Resistance was about twice that conferred by the Sat-RNA or the CP gene alone in transformed plants.     

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP⁺) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP⁺ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP⁺ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.     

Replication of cucumber mosaic virus satellite RNA in vitro by an RNA-dependent RNA polymerase from virus-infected tobacco.

An RNA-dependent RNA polymerase purified from tobacco infected with cucumber mosaic virus catalyzes the synthesis of (-) and (+) strands of the viral satellite RNA, CARNA 5, but fails to replicate the satellite RNA of peanut stunt virus (PSV). The enzyme replicates the genomic RNAs of the three principal cucumoviruses CMV, PSV and tomato aspermy virus (TAV) with varying efficiencies. The specificity with which CMV RdRp replicates different sequence-unrelated RNA templates suggests that the site of their recognition requires secondary or higher level structural organization.     

The suppressor of transgene RNA silencing encoded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance

The Cucumber mosaic virus (CMV)-encoded 2b protein (Cmv2b) is a nuclear protein that suppresses transgene RNA silencing in Nicotiana benthamiana. Cmv2b is an important virulence determinant but nonessential for systemic spread in N. glutinosa, in contrast to its indispensable role for systemic infections in cucumber. Here, we report that Cmv2b became essential for systemic infections in older N. glutinosa plants or in young seedlings pretreated with salicylic acid (SA). Expression of Cmv2b from the genome of either CMV or Tobacco mosaic virus significantly reduced the inhibitory effect of SA on virus accumulation in inoculated leaves and systemic leaves. A close correlation is demonstrated between Cmv2b expression and a reduced SA-dependent induction of the alternative oxidase gene, a component of the recently proposed SA-regulated antiviral defense. These results collectively reveal a novel activity of Cmv2b in the inhibition of SA-mediated virus resistance. We used a N. tabacum line expressing a bacterial nahG transgene that degrades SA to provide evidence for a Cmv2b-sensitive antiviral defense mechanism in tobacco in which SA acts as a positive modifier but not as an essential component. We propose that SA induces virus resistance by potentiating a RNA-silencing antiviral defense that is targeted by Cmv2b.     

NRG1, a CC-NB-LRR protein, together with N, a TIR-NB-LRR protein, mediates resistance against tobacco mosaic virus

In animals and plants, innate immunity is regulated by nucleotide binding domain and leucine-rich repeat (NB-LRR) proteins that mediate pathogen recognition and that activate host-cell defense responses. Plant NB-LRR proteins, referred to as R proteins, have amino-terminal domains that contain a coiled coil (CC) or that share similarity with animal Toll and interleukin 1 receptors (TIR). To investigate R protein function, we are using the TIR-NB-LRR protein N that mediates resistance against tobacco mosaic virus (TMV) through recognition of the TMV p50 protein. Here, we describe N requirement gene 1 (NRG1), a novel N-resistance component that was identified by a virus-induced gene silencing (VIGS) screen of a cDNA library. Surprisingly, NRG1 encodes an NB-LRR type R protein that, in contrast to N, contains a CC rather than a TIR domain. Our findings support emerging evidence that many disease-resistance pathways each recruit more than a single NB-LRR protein. The results also indicate that, in addition to the previously recognized role in elicitor recognition, NB-LRR proteins may also function in downstream signaling pathways.     

Activation of Tm-22 resistance is mediated by a conserved cysteine essential for tobacco mosaic virus movement

The tomato Tm-2² gene was considered to be one of the most durable resistance genes in agriculture, protecting against viruses of the Tobamovirus genus, such as tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV). However, an emerging tobamovirus, tomato brown rugose fruit virus (ToBRFV), has overcome Tm-2², damaging tomato production worldwide. Tm-2² encodes a nucleotide-binding leucine-rich repeat (NLR) class immune receptor that recognizes its effector, the tobamovirus movement protein (MP). Previously, we found that ToBRFV MP (MP^ToBRFV) enabled the virus to overcome Tm-2²-mediated resistance. Yet, it was unknown how Tm-2² remained durable against other tobamoviruses, such as TMV and ToMV, for over 60 years. Here, we show that a conserved cysteine (C68) in the MP of TMV (MP^TMV) plays a dual role in Tm-2² activation and viral movement. Substitution of MP^ToBRFV amino acid H67 with the corresponding amino acid in MP^TMV (C68) activated Tm-2²-mediated resistance. However, replacement of C68 in TMV and ToMV disabled the infectivity of both viruses. Phylogenetic and structural prediction analysis revealed that C68 is conserved among all Solanaceae-infecting tobamoviruses except ToBRFV and localizes to a predicted jelly-roll fold common to various MPs. Cell-to-cell and subcellular movement analysis showed that C68 is required for the movement of TMV by regulating the MP interaction with the endoplasmic reticulum and targeting it to plasmodesmata. The dual role of C68 in viral movement and Tm-2² immune activation could explain how TMV was unable to overcome this resistance for such a long period.     

Inheritance of resistance to watermelon mosaic virus in the cucumber line TMG-1: tissue-specific expression and relationship to zucchini yellow mosaic virus resistance

The inbred cucumber (Cucumis sativus L.) line TMG-1 is resistant to three potyviruses:zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). The genetics of resistance to WMV and the relationship of WMV resistance to ZYMV resistance were examined. TMG-1 was crossed with WI-2757, a susceptible inbred line. F₁, F₂ and backcross progeny populations were screened for resistance to WMV and/or ZYMV. Two independently assorting factors conferred resistance to WMV. One resistance was conferred by a single recessive gene from TMG-1 (wmv-2). The second resistance was conferred by an epistatic interaction between a second recessive gene from TMG-1 (wmv-3) and either a dominant gene from WI-2757 (Wmv-4) or a third recessive gene from TMG-1 (wmv-4) located 20–30 cM from wmv-3. The two resistances exhibited tissue-specific expression. Resistance conferred by wmv-2 was expressed in the cotyledons and throughout the plant. Resistance conferred by wmv-3 + Wmv-4 (or wmv-4) was expressed only in true leaves. The gene conferring resistance to ZYMV appeared to be the same as, or tightly linked to one of the WMV resistance genes, wmv-3.     

Structure of the RNA in tobacco mosaic virus

A model of the RNA of tobacco mosaic virus has been built using computer model-building techniques. The model has good stereochemistry, and fits the electron density map of the virus obtained by fiber diffraction methods considerably better than did earlier models. The three sugar rings in the asymmetric unit all have the A (3′-endo) conformation, One of the bases is in the syn conformation, a conformation observed only rarely in nucleic acid structures.     

Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase.

A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation. Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific. The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands. TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase. A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of [alpha-32P]UTP, was genomic-length single-stranded RNA. This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA. Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed. Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein. All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme.     

Rapid production and field testing of homozygous transgenic tobacco lines with virus resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic virus

A procedure for the fast production of homozygotic transgenic plants was developed. Leaf discs of haploid tobacco plants from anther cultures were transformed with a chimaeric vector containing coat protein (CP) and satellite RNA (Sat-RNA) genes from cucumber mosaic virus (CMV). One-hundred-and-twelve Kanamycin-resistant transformed haploid plants were subjected to selection based on the expression of both CP and Sat-RNA. Eighty-nine transgenic plants expressing both genes were selected and tested for their resistance to CMV by inoculation with high concentration of CMV (200 g ml^–1). Only five plants showed no symptoms of viral infection 30 days after inoculation. These plants were then diploidized by colchicine treatment. Three homozygous diploid lines with high levels of resistance to CMV were obtained after only one generation. The three transgenic lines were further tested under field conditions. The results showed that the progenies of these transgenic lines were homozygous and were highly resistant to CMV under natural field infection and manual inoculation conditions.     

Degradation of tobacco mosaic virus movement protein by the 26S proteasome

Cell-to-cell spread of tobacco mosaic virus is facilitated by the virus-encoded 30-kDa movement protein (MP). This process involves interaction of viral proteins with host components, including the cytoskeleton and the endoplasmic reticulum (ER). During virus infection, high-molecular-weight forms of MP were detected in tobacco BY-2 protoplasts. Inhibition of the 26S proteasome by MG115 and clasto-lactacystin-beta-lactone enhanced the accumulation of high-molecular-weight forms of MP and led to increased stability of the MP. Such treatment also increased the apparent accumulation of polyubiquitinated host proteins. By fusion of MP with the jellyfish green fluorescent protein (GFP), we demonstrated that inhibition of the 26S proteasome led to accumulation of the MP-GFP fusion preferentially on the ER, particularly the perinuclear ER. We suggest that polyubiquitination of MP and subsequent degradation by the 26S proteasome may play a substantial role in regulation of virus spread by reducing the damage caused by the MP on the structure of cortical ER.     

Electron microscopy of double-stranded RNA induced by turnip yellow mosaic virus and tobacco mosaic virus

Summary Double-stranded RNA isolated by phenol extraction from turnip yellow mosaic virus-infected chinese cabbage leaves and from tobacco mosaic virus-9nfected tobacco leaves was rotary shadowed and examined in the electron microscope. The TYMV and TMV molecules are similar in appearance, having uniform width and a linear configuration similar to that previously described for double-stranded RNA and double-stranded DNA molecules. More than 99.5% of the molecules of each virus fall within the range 0.1 to 2.2 , there being a predominance of smaller molecules in both cases (TYMV mean=0.24 , TMV mean 0.42 ). The mode of the larger molecules of TYMV 1.92 and of TMV 1.8 . These values are close to the expected lengths of whole molecules, calculated from biophysical data. Apparently branched molecules were observed in preparations of both TYMV and TMV double-stranded RNA. It was found, however, that the number of such branches per unit length of RNA decreases with a decrease in density of the RNA in the fields examined.