Tobacco Mosaic Virus-infected Plants Research Articles

Induction of resistance to Myzus persicae-nicotianae in Cucumber mosaic virus infected tobacco plants using silencing of CMV-2b gene

Aphids such as tobacco aphid Myzus persicae-nicotianae, are among the most important plant viral vectors and plant viruses encode genes to interact with their vectors. Cucumber mosaic virus (CMV) encodes 2b protein as a suppressor of plant immune and it plays a vital role in CMV accumulation and susceptibility to aphid vectors. In this study, the resistance of tobacco plants (Nicotiana tabacum) to M. p. nicotianae was evaluated by silencing of 2b in CMV-infected plants. However, the pFGC-C.h silencing gene construct was transiently expressed using Agrobacterium tumefacience, LBA 4404 in tobacco leaves, and four days later, the plants were mechanically inoculated by CMV (Kurdistan isolate), and then, 15 days post-inoculation 1 nonviruliferous aphid was placed on each leaf for evaluation of resistance to M. p. nicotianae. To evaluate the tobacco plants resistance and susceptibility to M. p. nicotianae, the number of aphids existent per tobacco leaf, life table and, demographic parameters were recorded and used as a comparison indicator. The obtained results were analyzed using the age-stage, two-sex life table. The highest number of aphids was recorded on the control CMV-infected plants, while the lowest number on CMV infected leaves expressing CMV-2b silencing construct (pFGC-C.h). The obtained data revealed the lowest rate for all of intrinsic rate of natural increase (rm) (0.246/day), the rate of reproduction (r0) (17.04 females/generation), and finite rate of increase (λ) (1.279/day), on the pFGC-C.h treatment. The maximum generation time (T) (11.834 days) was observed on (V) treatment. However, the collected data revealed induction of resistance to tobacco aphids by silencing of CMV-2b in CMV infected plants.

In planta proximity-dependent biotin identification (BioID) identifies a TMV replication co-chaperone NbSGT1 in the vicinity of 126 kDa replicase

Tobacco mosaic virus (TMV) is a positive, single-stranded RNA virus. It encodes two replicases (126 kDa and 183 kDa), a movement protein and a coat protein. These proteins interact with host proteins for successful infection. Some host proteins such as eEF1α, Tm-1, TOM1, 14–3-3 proteins directly interact with Tobamovirus replication proteins. There are host proteins in the virus replication complex which do not interact with viral replicases directly, such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. We have used Proximity-dependent biotin identification (BioID) technique to screen for transient or weak protein interactions of host proteins and viral replicase in vivo. We transiently expressed BirA* tagged TMV 126 kDa replicase in TMV infected Nicotiana benthamiana plants. Among 18 host proteins, we identified NbSGT1 as a potential target for further characterization. Silencing of NbSGT1 in N. benthamiana plants increased its susceptibility to TMV infection, and overexpression of NbSGT1 increased resistance to TMV infection. There were weak interactions between NbSGT1 and TMV replicases but no interaction between them was found in Y2H assay. It suggests that the interaction might be transient or indirect. Therefore, the BioID technique is a valuable method to identify weak or transient/indirect interaction(s) between pathogen proteins and host proteins in plants. Biological significanceTMV is a well characterized positive-strand RNA virus model for study of virus-plant host interactions. It infects >350 plant species and is one of the significant pathogens of crop loss globally. Many host proteins are involved in TMV replication complex formation. To date there are few techniques available for identifying interacting host proteins to viral proteins. There is limited knowledge on transient or non-interacting host proteins during virus infection/replication. In this study, we used agroinfiltration-mediated in planta BioID technique to identify transiently or non-interacting host proteins to viral proteins in TMV-infected N. benthamiana plants. This technique allowed us to identify potential candidate proteins in the vicinity of TMV 126 kDa replicase. We have selected NbSGT1 and its overexpression suppresses TMV replication and increase plant resistance. NbSGT1 is believed to interact transiently or indirectly with TMV replicases in the presence of Hsp90/Hsp70. BioID is a novel and powerful technique to identify transiently or indirectly interacting proteins in the study of pathogen-host interactions.

Comparative proteomics of Tobacco mosaic virus-infected Nicotiana tabacum plants identified major host proteins involved in photosystems and plant defence

Tobacco mosaic virus (TMV) is a positive single-stranded RNA virus. Its 5′ end ORF codes for the replicase proteins, namely 126 kDa and 183 kDa, respectively. These proteins interact with many host proteins to form a virus replication complex (VRC). This study aims to dissect the proteome profile of TMV-infected Nicotiana tabacum in host cellular and molecular pathways. We used the isobaric tags for relative and absolute quantification (iTRAQ) technique to analyse the differential global proteomic profile of TMV infected and mock infected plants. Out of 1897 total proteins, we identified 407 differentially abundant proteins and grouped them into three functional categories, namely metabolism, cellular processes and signalling processing. Our results showed that photosynthesis, carbon metabolism, plant defence, protein synthesis, and protein processing in the endoplasmic reticulum were significantly altered. Carbon metabolism and photosynthesis were present in very low abundance, whereas accumulation of reactive oxygen species and misfolded proteins lead to the accumulation of thioredoxin H-type 1. In conclusion, we identified several key host proteins that are involved in TMV infection/replication in N. tabacum plants. Significance of the studyTMV is one of the most widely studied plant virus. It is used as a tool to study host-virus interaction. There are several host proteins reported that facilitate VRC formation and replication of TMV. However, there is limited knowledge in the expression regulation of these host proteins upon TMV infection. This study is the first report that investigates the response of host protein expression involved in TMV infection through a quantitative proteomics technique iTRAQ, combined with LC-MS/MS analysis. We used TMV-infected Nicotiana tabacum plants to investigate the effects of TMV infection on host proteins. Our results revealed differential abundance of proteins involving various pathways in protein translation, protein processing, photosynthesis and plant defence. There was a high abundance of thioredoxin H-type 1, a protein that counters oxidative stress and accelerated regulation of fatty acid synthesis to provide additional lipid molecules for VRC formation. There was a significant reduction in abundance of psaA and psbB proteins in the photosynthetic pathways. Our results identified key candidate host proteins involved in TMV-infected N. tabacum for functional studies in future.

RNA silencing-mediated resistance is related to biotic / abiotic stresses and cellular RdRp expression in transgenic tobacco plants

The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.

Involvement of wound‐induced receptor‐like protein kinase in wound signal transduction in tobacco plants

The wound-induced receptor-like protein kinase (WRK) gene, isolated as one of the genes whose transcripts accumulated during the early period of N gene-dependent synchronized cell death in tobacco mosaic virus-infected tobacco plants, encodes a leucine-rich repeat receptor-like protein kinase, and its transcript was transiently increased 15 min after wounding. In the present study, analysis of a green fluorescent protein fusion protein indicated that WRK is localized in the plasma membrane. In transgenic tobacco plant lines with elevated or suppressed levels of WRK transcript, the wound-induced accumulation of both basic PR-1 and PR-6 transcripts was slightly enhanced or significantly suppressed respectively. The decrease in wound-induced basic PR gene expression in WRK suppressed lines was restored by jasmonic acid (JA) treatment. Furthermore, the levels of wound-induced enzymatic activation of both salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) and wound-induced accumulation of JA were reduced in the WRK suppressed lines in comparison with a control line. These results suggest that WRK functions upstream of SIPK and WIPK and regulates wound signal transduction in tobacco plants.

Changes in key enzymes of viral-RNA biosynthesis in chloroplasts from PVY and TMV infected tobacco plants

Effects of the infection with tobacco mosaic virus (TMV) and potato virus Y (PVY) on chloroplasts from susceptible tobacco plants were determined. Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), 6-phosphogluconate dehydrogenase (6PG DH), glucokinase (GK), and fructokinase (FK) activities in thylakoid/envelope and stroma fractions were studied. Slight increase in the activities of PME, PDE, G6P DH and 6PG DH of thylakoid/envelope fraction as well as of RNases, PME, PDE, G6P DH, 6PG DH, GK and FK of stroma fraction was found in chloroplasts isolated from leaf tissues infected with PVY. Infection with TMV produced higher increase in enzymes activities in chloroplasts; especially, PME, G6P DH and 6PG DH in fraction of thylakoid/envelope, and RNases, PME, PDE, G6P DH, 6PG DH, and GK in stroma fraction.

Activation of defense-related gene expression and systemic acquired resistance in cucumber mosaic virus-infected tobacco plants expressing the mammalian 2'5'oligoadenylate system.

Tobacco plants expressing the mammalian 2'5'oligoadenylate system (2-5A system) exhibit resistance to cucumber mosaic virus (CMV). Here, to characterize the molecular aspect of the resistance to CMV in 2-5A system-expressing tobaccos, the activation of defense-related genes and systemic acquired resistance (SAR) as the markers for the hypersensitive resistance (HR), were elucidated. Northern hybridization analysis indicated that the expression of four pathogenesis-related (PR) protein genes and five HR-related genes were induced in CMV-infected tobaccos expressing 2-5A system. Furthermore, the induction of SAR against Pseudomonas syringae pv. tabaci as second challenge, was observed on CMV-inoculated tobaccos expressing 2-5A system. These results suggested that the resistance to CMV in tobacco expressing 2-5A system is associated with the establishment of an HR-like response.

Ultrastructural detection of β-1,3-glucans in tobacco root tissues infected by Phytophthora parasitica var. nicotianae using a gold-complexed tobacco β-1,3-glucanase

A β-1,3-glucanase, purified to homogeneity from tobacco mosaic virus-infected tobacco plants, was successfully conjugated to colloidal gold at pH 5·5 and used for localizing β-1 ,3-glucans in tobacco root tissues infected by Phytophthora parasitica var. nicotianae. Specificity of the enzyme for β-1,3-glucans was demonstrated by competition in binding using several polysaccharides as potential substrates. Of the various sugars tested only laminarin, a polymer of β-1,3-glucans was found to inhibit the enzyme-binding activity efficiently. In addition, the abolition of labelling over isolated fungal cell walls previously treated to remove β-1,3-glucan further confirmed the strict affinity of the tobacco β-1,3-glucanase for β-1,3-glucan molecules. In healthy tobacco root tissues, β-1,3-glucans occurred as small deposits in sieve elements and were seldom detected in other tissues. However, in fungus-infected tobacco root tissues, large amounts of β-1,3-glucans (callose) were found at sites of attempted pathogen penetration such as sieve tube members, plasmodesmata, and intercellular spaces. Papillae, thought to be physical barriers formed in response to infection, contained β-1,3-glucans distributed mainly over an amorphous matrix. The occurrence of β-1,3-glucan molecules in host cell walls adjacent to fungal cells raises the question of their origin. The possibility that they are released from the fungal cell wall through the action of plant β-1,3-glucanases is discussed. The present study demonstrates the potential value of the tobacco β-1,3-glucanase (PR-N) for ultrastructural studies on β-1,3-glucans in plant biology and pathology.

Complete replication of a satellite RNA in vitro by a purified RNA-dependent RNA polymerase.

The 334 nucleotide R satellite RNA was used as a template for purified RNA-dependent RNA polymerase (RdRp) from cucumber mosaic virus-infected tobacco plants. The products of the reaction were dsRNA and positive-strand RNA of the same size as the R satellite RNA. Similar products were obtained when T7 RNA polymerase positive-strand transcripts of a cDNA clone of the satellite RNA, designed to have the same 5' and 3' ends as the satellite RNA, were used as templates. The formation of the positive strands demonstrates complete replication of the satellite RNA. A positive-strand transcript with 65 and 255 additional nucleotides at the 5' and 3' ends of the satellite RNA respectively was also utilized as a template by the RdRp, but only dsRNA was formed. However, no products could be detected when the RdRp was programmed with transcripts corresponding to the negative-strand satellite RNA, either with no additional terminal nucleotides or with 24 and 310 additional nucleotides at the 5' and 3' ends respectively.

Growth, development, feeding preference, and food consumption and utilization by tobacco hornworm on tobacco mosaic virus-infected and non-infected tobacco leaves

Plant disease may cause a variety of changes in plants which influence their suitability to insect herbivores. The reported physiological changes in tobacco due to infection by tobacco mosaic virus have the potential to influence the feeding, growth and behavior of the tobacco hornworm,Manduca sexta. However, feeding on foliage of systematically infected plants had no statistically significant effects on development time, mean pupal weight or the amount of foliage consumed by 4th instars. Although an initial preference was exhibited by larvae for healthy leaves after about 72 h no differences were observed. These results do not support the results of previous research on the influence of tobacco mosaic virus-infected plants on the tobacco hornworm.

DNA sequence analysis of a PR-1a gene from tobacco: Molecular relationship of heat shock and pathogen responses in plants

Pathogenesis-related proteins (PR-proteins) are a heterogeneous group of host encoded, low molecular weight proteins, which are induced in plants by different external stimuli such as pathogen attack or exposure of the plants to certain chemicals. Three independent bacteriophage λ clones hybridizing to PR-1 cDNA probes were isolated from a tobacco (Nicotiana tabacum cv. Wisconsin 38) genomic library. All three clones are derived from different genomic locations and contain single gene copies. DNA sequence analysis revealed that one of the genomic clones is essentially identical to the PR-1a cDNA clones described previously from tobacco mosaic virus infected tobacco plants (Nicotiana tabacum cv. Samsun NN). This clone contains 335 bp of the 5′ upstream and the entire transcribed regions. The PR-1a gene is not interrupted by intervening sequences. In the 5′ upstream region, characteristic eukaryotic transcription signals as well as putative regulatory sequences were identified. A consensus TATA box is found at position-34 and an-11 pb long imperfect direct repeat is present at position-116 and-135 with respect to the transcription initiation site. Furthermore, the sequence C-GAA-TTC-G, which differs from the consensus heat shock regulatory element only by the insertion of one extra nucleotide, is located at position-57. The presence of this element suggests a molecular relationship of heat shock and pathogen responses in plants.

Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.

Histopathology and histochemistry of anthers in virus-infected chilli plants (Capsicum annuum L. cv. Byadgi)

Summary Histopathological and histochemical changes in the anthers of chilliplants, inoculated with pepper vein banding virus (PVBV) and tobacco mosaic virus (TMV), were studied in comparison with those in the anthers of healthy plants. In the developing anthers, up to sporogenous tissue stage, the histology and histochemistry were similar in both healthy and virus-infected plants. Subsequently a conspicuous size reduction was noticed in the developing anthers and their component structures, such as pollen mother cells (PMCs), tetrads, and pollen grains in virus-infected plants. Even though starch accumulated in the wall layers and connective tissues of developing anthers in both healthy and TMV infected plants, the former contained more starch than the latter. However, there was no starch accumulation in the anther tissues of the PVBV-infected plants. The tapetal cells in virus-infected plants had relatively less of cytoplasmic RNA and proteins as compared to control. The pollen grains of healthy (control) plants had rich accumulation of starch, cytoplasmic RNA, and proteins, whereas those of virus-infected plants lacked these macromolecular substances.

The changes in metabolic utilization of galactose in tobacco mosaic virus infected tobacco plants treated with 2,4-dichlorophenoxyacetic acid

The study deals with the changes in metabolic processes of galactose breakdown in tobacco mosaic virus infected tobacco tissues treated with a 1.0 × 10−4 M solution of 2,4-D (2,4-dichlorophenoxyacetic acid). At the time of maximum virus reproduction the plants infected exhibited a considerable increase in galactokinase and galactose-1-phosphate uridyltransferase activities. The activity of both galactose metabolizing enzyme systems investigated was not affected markedly by 2,4-D, but its increase was induced preferentially by virus biosynthesis.

The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants

The 2 M LiCl-soluble RNA fraction extracted from tobacco mosaic virus (TMV)-infected tobacco plants contains, in addition to the viral replicative form of 4 × 10 6 MW, three smaller double-stranded (ds) RNA species with apparent molecular weights (estimated by polyacrylamide gel electrophoresis, using ds RNAs as markers) of 2.25, 1.1, and 0.23 × 10 6. The synthesis of all four ds RNAs is insensitive to actinomycin D. They are completely RNase insensitive at high salt concentrations and are found both in directly inoculated and in apical tissues. In tissues incubated in the presence of 3H-uridine and actinomycin D, the three small ds RNAs accounted for 6 to 11.5% of the total radioactivity incorporated into viral ds RNA. On a molar basis, however, in apical leaves the smallest ds RNA was synthesized to almost the same level as the replicative form. By molecular hybridization, the three small ds RNAs have been shown to be of viral origin, and each contains sequences represented in the 5′ end of complementary (negative strand) TMV RNA. Based on molecular weight data, none of the ds RNAs can be considered to be a ds form of the subgenomic TMV coat protein mRNA (the LMC), suggesting that it is not replicated independently. None of the small ds RNAs was found to be an endogenous product of the bound TMV RNA replicase.

A striking change in symptoms on cucumber mosaic virus-infected tobacco plants induced by a satellite RNA

The small RNA from cucumber mosaic virus (CMV) we had formerly referred to as RNA 5 was shown to have a satellite nature similar to CARNA 5 (Kaper and Tousignant, 1977, Virology 80, 186-195) and Sat-RNA (Mossop and Francki, 1978, Virology 86, 562-566). Our satellite RNA has a great deal in common with CARNA 5 and Sat-RNA in many respects, except that its replication causes a striking change in symptoms on tobacco and on some species of Nicotiana infected with CMV. Large amounts of satellite RNA were found in RNAs of naturally occurring CMV isolates which induced a unique brilliant yellow symptom on tobacco. When purified satellite RNA was inoculated onto tobacco after being mixed with CMV isolates which contained no satellite RNA and which normally produced ordinary green mosaic symptoms, all of the infected tobacco developed the brilliant yellow symptom along with some symptom attenuation. Virions isolated from those plants contained much satellite RNA. CMV could be freed from satellite RNA by local lesion transfer and selection. Satellite RNA had substantial stability in vivo and in vitro. It caused the severe necrotic symptom or ultimate death of tomato like CARNA 5 but unlike Sat-RNA.

RNA-dependent RNA polymerases in uninfected and in alfalfa mosaic virus-infected tobacco plants

Extracts of AMV-infected tobacco leaves were found to contain membrane-bound and soluble RNA-dependent RNA polymerases. Both also existed in healthy extracts. The bound enzyme was stimulated by added RNA and the soluble enzyme was highly dependent on the addition of RNA for activity. The bound enzyme was solubilized and freed from endogenous RNA template by centrifugation onto a 20% sucrose cushion. The [ 3H]RNA synthesized by the bound, soluble, and solubilized enzymes of healthy and infected extracts appeared to be largely double-stranded and of various molecular weights. Some high molecular weight RNA, similar in electrophoretic migration to the replicative forms of AMV, was obtained with bound enzyme. Hybridization competition experiments showed that the enzymes synthesized “minus”-strand AMV-RNA.

The effects of virus infection on oxygen uptake and respiratory quotient of the leaves of French bean and cowpea

Increases in oxygen uptake of leaves of French bean and cowpea were greater as the result of infection by tobacco necrosis virus which induces necrotic local lesions than by the legume strain of tobacco mosaic virus which induces systemic chlorosis. In tobacco mosaic virus-infected plants, the respiration of the systemically infected leaves was higher than that of the inoculated primary leaves when compared with the comparable healthy controls. In neither type of infection was the increased respiration rate directly related to the development of symptoms or virus multiplication. The respiratory quotient of all types of infected leaf was less than that of comparable healthy leaves, the lowest values being recorded in leaves showing necrotic local lesions.

In vitro synthesis of double-stranded RNA by an enzyme system isolated from tobacco leaves

An RNA-dependent RNA polymerase which catalyzes the polymerization of ribonucleotides into an acid-insoluble product has been isolated from tobacco leaves and partially characterized. 1. 1. The enzyme activity is found in the 30 900 × g supernatant fraction of a leaf homogenate. The reaction requires the presence of a divalent metal ion and four ribonucleoside triphosphates for activity. 2. 2. The product of the enzyme-catalyzed reaction is double-stranded RNA as evidenced by its ribonuclease resistance and sharp thermal transition at 80 °C in 30 mM Na +. It is of low molecular weight (less than 5 S) as determined by polyacrylamide gel electrophoresis. 3. 3. Addition of various species of RNA stimulates the polymerization reaction, but the added RNA does not appear to be associated with the enzyme product. It appears neither to act as a template nor as a primer. 4. 4. The enzyme can be isolated from apparently healthy plants, but is also found in tobacco mosaic virus infected plants; its specific activity increases after infection. The enzyme activity and its product are not altered by infection.

Proteins in tobacco mosaic virus-infected tobacco plants

Tobacco mosaic virus (TMV)-infected leaf tissue was exposed to 3H-leucine following a treatment with actinomycin D. Uninfected leaf tissue was similarly treated, but was labeled with 14C-leucine. Extracts of the two tissues were combined and analyzed by electrophoresis in sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. At least four polypeptides were either engendered or stimulated in diseased tissue; one of these was the coat protein. Incorporation into one component was depressed in diseased tissue.