Isolation and Characterization of Two Protein Isoforms with Antiviral Activity from Chenopodium album L Leaves

Two antiviral proteins (AVPs) named CAP-I and CAP-II isolated and purified from the leaves of Chenopodium album cv Pusa Bathua 1 were found to inhibit tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) infection on their respective host plants. The molecular weight of both the AVPs was found to be 24 kD. They were devoid of carbohydrate moiety and were highly basic with pI ～10.2. However, they differed with respect to amino acid composition and N-terminal sequence. They also differed with respect to IC₅₀ values, and CAP-I was found to be 2.5 fold more effective than CAP-II in inhibiting viral infection.     

Cloning and expression of antiviral/ribosome-inactivating protein from <Emphasis Type="Italic">Bougainvillea</Emphasis> x<Emphasis Type="Italic">buttiana</Emphasis>

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).     

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific fashion.Small interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells.Here,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant tissue.Our results indicate that transiently expressed shRNA efficientlyinterfered with TMV infection.The interference observed is sequence-specific,and time-and site-dependent.Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated tobamovirus.In order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation.Ourresults support the view that RNAi opens the door for novel therapeutic procedures against virus diseases.We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.     

Modification of Antioxidant Status of Host Cell in Response to Bougainvillea Antiviral Proteins

Bougainvillea xbuttiana antiviral proteins (AVPs) exhibited high antioxidant activity as measured by ferric reducing / antioxidant (FRAP) power assay. These AVPs were also found to modify activities of antioxidant enzymes like superoxide dismutase, peroxidase and catalase. The activities of superoxide dismutase and peroxidase increased, while the activity of catalase decreased in Tobacco mosaic virus (TMV) infected tobacco leaves. The trend was reversed when the leaves were treated with AVP alone. However, in TMV + AVP treated leaves, the activities of all the three enzymes were found to be midway between the activities obtained with other two treatments. It is therefore, suggested that Bougainvillea AVPs might be controlling viral diseases by scavenging reactive oxygen species as well as by altering host plant cell metabolism to maintain its antioxidant status.     

Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco.

Systemic induction of pathogenesis-related (PR) proteins in tobacco, which occurs during the hypersensitive response to tobacco mosaic virus (TMV), may be caused by a minimum 10-fold systemic increase in endogenous levels of salicylic acid (SA). This rise in SA parallels PR-1 protein induction and occurs in TMV-resistant Xanthi-nc tobacco carrying the N gene, but not in TMV-susceptible (nn) tobacco. By feeding SA to excised leaves of Xanthi-nc (NN) tobacco, we have shown that the observed increase in endogenous SA levels is sufficient for the systemic induction of PR-1 proteins. TMV infection became systemic and Xanthi-nc plants failed to accumulate PR-1 proteins at 32 degrees C. This loss of hypersensitive response at high temperature was associated with an inability to accumulate SA. However, spraying leaves with SA induced PR-1 proteins at both 24 and 32 degrees C. SA is most likely exported from the primary site of infection to the uninfected tissues. A computer model predicts that SA should move rapidly in phloem. When leaves of Xanthi-nc tobacco were excised 24 hr after TMV inoculation and exudates from the cut petioles were collected, the increase in endogenous SA in TMV-inoculated leaves paralleled SA levels in exudates. Exudation and leaf accumulation of SA were proportional to TMV concentration and were higher in light than in darkness. Different components of TMV were compared for their ability to induce SA accumulation and exudation: three different aggregation states of coat protein failed to induce SA, but unencapsidated viral RNA elicited SA accumulation in leaves and phloem. These results further support the hypothesis that SA acts as an endogenous signal that triggers local and systemic induction of PR-1 proteins and, possibly, some components of systemic acquired resistance in NN tobacco.     

Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli

A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV).     

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.     

Replication in the phloem is not necessary for efficient vascular transport of tobacco mosaic tobamovirus

Plant viruses move systemically from one leaf to another via phloem. However, the viral functions needed for systemic movement are not fully elucidated. An experimental system was designed to study the effects of low temperature on the vascular transport of the tobacco mosaic tobamovirus (TMV). Vascular transport of TMV from lower inoculated leaves to upper non-inoculated leaves via a stem segment kept at low temperature (4 degrees C) was not affected. On the other hand, several experiments were performed on tobacco leaves to demonstrate that virus replication did not occur at the same temperature. The data suggest that replication of TMV in the phloem of wild-type tobacco plants is not necessary for the vascular transport of TMV, and that the virus moves with photoassimilates as suggested previously.     

Expression of tobacco mosaic virus RNA in transgenic plants

Summary Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in planta.     

Systemic Acquired Resistance Induced in Tobacco Plants by Localized Virus Infection Does Not Operate Against Challenging Viruses That Infect Systemically

Localized infections produced by tobacco necrosis virus (TNV) or tomato mosaic virus (ToMV) in White Burley tobacco induced a systemic acquired resistance in upper, uninoculated leaves. This resistance was effective against challenge infection by TNV or ToMV but not by potato virus Y, necrotic strain (PVYⁿ), tobacco mosaic virus (TMV) or tobacco rattle virus (TRV), viruses giving systemic infections. Systemic acquired resistance against TNV or ToMV was expressed as a reduction in lesion size but not in viral antigen content of the resulting necrotic local lesions. The acquisition of resistance was concurrent with an increased capacity of the resistant leaves to convert 1-aminocyclopropane-1-carboxylic acid into ethylene. Systemic acquired resistance was ineffective to contrast or minimize in whatever way the systemic challenge infection produced by PVY^N, TMV or TRV. Severity of symptoms and virus multiplication did not differ in resistant leaves from controls. This result does not allow any optimistic promise on possible application of the systemic acquired resistance against severe viral diseases of crops.     

Tobacco plants epigenetically suppressed in phenylalanine ammonia-lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia-lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL-suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis-related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL-suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL-suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild-type and PAL-suppressed tobacco, the SAR response can be transmitted from a PAL-suppressed root-stock, but SAR is not observed if the scion is PAL-suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre-existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL-suppressed plants with dichloro-isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.     

The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.     

Acquired Resistance in Hypersensitive Tobacco Operates by Limiting Cell-to-Cell Spread of Virus Infection without Affecting Virus Multiplication

Localized and systemic acquired resistance against tobacco mosaic virus (TMV) or tobacco necrosis virus was induced when local lesions were produced in leaves of Xanthi-nc tobacco by mechanical inoculation with TMV. Both types of resistance were characterized by reduction in the size of lesions produced by the challenging viruses, whereas accumulation of viral antigen in lesions was slightly increased. These results, confirming previous findings relative to other hypersensitive plant virus combinations, do not support the view that an inhibitor of virus replication operates in the resistant tissues, but indicate that both types of resistance operate only against cell-to-cell spread of virus.     

Virus-induced virus inhibitors from systemically virus-infected plants

After infection ofNicotiana tabacum cv. Samsun with tobacco mosaic virus (TMV) crude extracts from dark-green spots of upper leaves had a more strongly marked inhibitory effect upon TMV addedin vitro than crude extracts from the surrounding light-green tissue. Likewise, crude extracts from leaves ofNicotiana tabacum cv. Samsun showing recovery after infection with tobacco ringspot virus (TRV) were seen to have a marked inhibitory effect on TMV addedin vitro. The results obtained suggest that virus inhibitors are produced after virus infections not only in hypersensitive hosts but also in systemic hosts. Necrotizing processes are not an indispensable prerequisite of the production of virus-induced virus inhibitors.     

Synthesis of the tobacco mosaic virus in blocked spreading of infection

The possibility of infection of tobacco upper leaves with tobacco mosaic virus (TMV) was examined in experiments where the inoculum was imbibed through the cut stem. The inoculum used were: a) a preparation of a virus-specific informosome-like ribonucleoproteins (vRNP) isolated from TMV-infected plants; b) a TMV preparation; or c) a mixture of TMV and vRNP. Multiplication of TMV in upper leaves was observed in neither of the variants; nevertheless in the vascular tissue and/or probably in adjoining parenchymal cells, two kinds of RNA were synthesized: of mol. w. (1.1--1.3) X 10(6) and (0.6--0.8) X 10(6). These RNA were not found in healthy plants in the presence of actinomycin D. The synthesis of genomic TMV RNA is suppressed under these conditions. Thus, some kind of abortive TMV infection takes place under the condition of experimental inoculation of plants through a cut stem. Molecular hybridization with the DNA of recombinant plasmid containing a nucleotide sequence complementary to the 3'-portion of genomic TMV RNA proves that short RNAs synthesized under the abortive infection conditions are TMV-specific. The experiments with differential temperature treatment of N-gene-containing plants under abortive infection conditions suggest that necrotization is not necessarily induced by genomic TMV RNA synthesis.     

Lactones Produced through Thermal Oxidation of Higher Fatty Acids

Inhibitors of plant virus infection with systemic effects were found in the culture filtrates of Basidiomycetes such as Fomes fomentarius and Schizophyllum commune. These inhibitors were widely distributed in Agaricales and Polyporales. The inhibitors designated as BAS (Basidiomycete Antiviral Substance) were highly active against the mechanical transmission of tobacco mosaic virus (TMV). No toxic effect was observed on the host plants. BAS-F, a polysaccharide produced by F. fomentarius, almost completely inhibited infection, when BAS-F at 2 μg/ml was applied to the same surface of leaves of Xanthi-nc tobacco 24 h before TMV inoculation to the upper surface of the leaves, and 500/0 inhibition was shown when BAS-F at 10 μg/ml was applied to the under surface of leaves. BAS-F also induced systemic resistance to the non-treated leaves when it was applied to only one leaf of the plant. BAS-F also had similar effects against the infection of TMV on bell pepper and tomato plants.     

PeaT1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR-1a and PR-1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene.     

Inhibitory effect of fucoidan from brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus in tobacco leaves of two cultivars

The effect of fucoidan from the brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus (TMV) was investigated in the leaves of tobacco (Nicotiana tabacum L.) of two cultivars (Ksanti-nk and Samsun). In the leaves of cv. Ksanti-nk inoculated with a mixture of TMV preparation (2 μg/ml) and fucoidan (1 mg/ml), the number of local necrotic lesions induced by the virus decreased by more than 90% as compared with the leaves inoculated with the virus alone. In tobacco leaves of cv. Samsun, virulence and the concentration of the virus 3 days after inoculation with the same mixture of TMV and fucoidan were by 62 and 66%, respectively, lower than in the leaves inoculated with TMV alone. As the infection spread, the inhibitory effect of fucoidan decreased. When the leaves were treated with fucoidan before and after the inoculation with TMV, its antiviral activity was less pronounced than when a mixture of the virus and the polysaccharide was used as inoculum. Electron microscopic investigation of TMV mixed with fucoidan often showed agglutinated virions. The highest virulence of the mixture (TMV preparation, 12 μg/ml, plus fucoidan, 1 mg/ml) was observed upon its twofold dilution, and after that it decreased. It was concluded that, when the leaves were inoculated with the mixture of TMV and fucoidan, the latter affected not only the plant but the virus as well. Treatment of tobacco leaves, cv. Ksanti-nk, with actinomycin D (10 μg/ml) 24 h before the inoculation with TMV almost completely suppressed the effect of fucoidan, indicating that fucoidan acted at a gene level.