Tobacco Necrosis Virus Infection Research Articles

Tobacco necrosis virus replication and spread in Arabidopsis thaliana ecotype Columbia: a potential system for studying plant defense reactions to symptomless virus infections

Arabidopsis thaliana is susceptible to various different pathogens (fungi, bacteria, viruses). However, the effect of Tobacco necrosis virus (TNV) infection on Arabidopsis is not well known. TNV causes local necrotic and chlorotic lesions in the Arabidopsis ecotype Wassilewskija (Vannini et al., Physiol Mol Plant Pathol 69:26–42, 2006) but virus replication and systemic spread was not determined. Our goal was to monitor reactions to TNV infection in the most widely used A. thaliana ecotype, ‘Columbia’ (Col-0) by assessing in planta virus replication and systemic spread. We have shown that although TNV does not cause visible symptoms in A. thaliana ecotype Col-0 it is capable of replicating in planta as determined by real-time RT-qPCR. TNV levels progressively increased in inoculated leaves up to 4 days after inoculation (DAI) with a further severalfold increase between 4 and 8 DAI. Back inoculation of Nicotiana benthamiana resulted in symptoms typical of TNV infection (i.e., local necrotic lesions) within a few days. Interestingly, only a very low TNV titer (i.e., less than 20 % of that found in inoculated leaves) was detectable in non-inoculated upper leaves of Arabidopsis Col-0 even in an advanced stage of pathogenesis (21 DAI). The almost complete absence of systemic spread suggested that the virus is not seed-transmissible which was confirmed by real-time RT-qPCR analysis of plants grown from seeds produced by TNV-infected individuals. The present study is the first to show that TNV can infect the most widely used A. thaliana ecotype, Col-0, providing a novel system for future research on plant defense mechanisms during symptomless virus infections.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs.

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

Transport of Salicylic Acid in Tobacco Necrosis Virus-Infected Cucumber Plants.

The transport of salicylic acid (SA) was studied in cucumber (Cucumis sativus L.) using 14C-labeled benzoic acid that was injected in the cotyledons at the time of inoculation. Primary inoculation with tobacco necrosis virus (TNV) on the cotyledons led to an induction of systemic resistance of the first primary leaf above the cotyledon against Colletotrichum lagenarium as early as 3 d after inoculation. [14C]SA was detected in the phloem or in the first leaf 2 d after TNV inoculation, whereas [14C]benzoic acid was not detected in the phloem during the first 3 d after TNV inoculation of the cotyledons, indicating phloem transport of [14C]SA from cotyledon. In leaf 1, the specific activity of [14C]SA decreased between 1.7 and 8.6 times compared with the cotyledons, indicating that, in addition to transport, leaf 1 also produced more SA. The amount of SA transported after TNV infection of the cotyledon was 9 to 160 times higher than in uninfected control plants. Thus, SA can be transported to leaf 1 before the development of systemic acquired resistance, and SA accumulation in leaf 1 results both from transport from the cotyledon and from synthesis in leaf 1.

Evidence of a two-fold mechanism responsible for the inhibition by allopurinol of the hypersensitive response induced in tobacco by tobacco necrosis virus

Allopurinol applied to tobacco plant roots interferes strongly with tobacco necrosis virus (TNV) induced hypersensitive response and the TNV infection process. The first detectable effect of allopurinol treatment was a great reduction in the hypersensitive response seen as reduced collapse and extent of necrosis than in untreated controls. This was evaluated as reduction in electrolyte leakage from lesions in the leaves. Only 4–6 days of allopurinol treatment were needed to achieve this effect. With prolonged treatment (6–8 days), there was a second effect consisting of a strong reduction in the number of necrotic lesions produced with a basipetal (tip-to-base) intensity pattern. This second aspect was related to a lack of TNV antigen production and therefore to the inhibition of the TNV infection process. To explain the metabolic basis for these phenomena, a study was pursued to establish the metabolic fate of allopurinol in leaves in different positions on the stem. The results obtained demonstrate that: (i) allopurinol which accumulated in leaves was oxidized to oxypurinol by host xanthine oxidase which was in turn inhibited, and consequently increasing amounts of xanthine accumulated in leaves; (ii) both allopurinol and oxypurinol were converted to their respective ribonucleosides. All these metabolic aspects connected with allopurinol metabolism in leaves, analogous with the reduced production of necrotic lesion had a basipetal (tip-to-base) intensity pattern. The present results are discussed considering that these two phenomena, i.e. inhibition of TNV-induced hypersensitive response and inhibition of the TNV infection process, are related to two metabolic mechanisms triggered by allopurinol treatment. The first depends on the inhibition of xanthine oxidase mediated superoxide production and the consequent general attenuation of the necrotic process. The second depends on the synthesis of both allopurinol and oxypurinol ribonucleosides which might directly or indirectly interfere with the process of TNV infection, and therefore be responsible for the reduction of the number of lesions in treated leaves.

Induction of glutathione S‐transferase activity in tobacco by tobacco necrosis virus infection and by salicylic acid

Induction of glutathione S‐transferase activity in tobacco by tobacco necrosis virus infection and by salicylic acid

Pathogen-activated induced resistance of cucumber: response of arthropod herbivores to systemically protected leaves.

Restricted (non-systemic) inoculation of cucurbits, green bean, tobacco, and other plants with certain viruses, bacteria, or fungi has been shown to induce persistent, systemic resistance to a wide range of diseases caused by diverse pathogens. The non-specificity of this response has fueled speculation that it may also affect plant suitability for arthropod herbivores, and there is limited evidence, mainly from work with tobacco, which suggests that this may indeed occur. Young cucumber plants were immunized by restricted infection of a lower leaf with tobacco necrosis virus (TNV), and upper leaves were later challenged with anthracnose fungus, Colletotrichum lagenarium, to confirm induction of systemic resistance to a different pathogen. The response of arthropod herbivores was simultaneously measured on non-infected, systemically protected leaves of the same plants. As has been reported before, immunization with TNV gave a high degree of protection from C. lagenarium, reducing the number of lesions and the area of fungal necrosis by 65-93%. However, there was no systemic effect on population growth of twospotted spider mites, Tetranychus urticae Koch, on upper leaves, nor did restricted TNV infection of leaf tissue on one side of the mid-vein systemically affect mite performance on the opposite, virus-free side of the leaf. Similarly, there were no effects on growth rate, pupal weight, or survival when fall armyworm larvae were reared on systemically protected leaves from induced plants. In free-choice tests, greenhouse whiteflies oviposited indiscriminately on induced and control plants. Feeding preference of fall armyworms was variable, but striped cucumber beetles consistently fed more on induced than on control plants. There was no increase in levels of cucurbitacins, however, in systemically-protected foliage of induced plants. These findings indicate that pathogen-activated induced resistance of cucumber is unlikely to provide significant protection from herbivory. The mechanisms and specificity of induced resistance in cucurbits apparently differ in response to induction by pathogens or herbivores.

Ethylene biosynthesis in soybean plants during the hypersensitive reaction to tobacco necrosis virus

Tobacco necrosis virus (TNV) producing necrotic local lesions in soybean primary leaves, stimulated ethylene production, ethylene-forming activity (EFA) and 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation. The inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine and Co 2+, completely prevented ethylene production by the infected leaves, while a free-radical scavenger, propyl gallate, drastically inhibited it. These results indicate that the biosynthesis of stress ethylene during the hypersensitive reaction (HR) of soybean to TNV proceeds through the methionine cycle. Ethylene inhibitors did not prevent necrosis, suggesting that ethylene does not act as a phytotoxin. Ethylene production does not appear to be involved in localizing TNV infection, because its complete inhibition did not affect lesion growth or accumulation of TNV antigen. Ethylene production, EFA and free ACC were not affected in uninoculated leaves of inoculated soybean plants, indicating that there is no systemic effect on ethylene biosynthesis during HR. In contrast, EFA was activated in analogous uninoculated leaves of inoculated cowpea and asparagus bean plants, a finding that illustrates that care must be taken in interpreting results from single models.

Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase.

An extracellular, acidic chitinase was purified to homogeneity from tobacco necrosis virus-infected leaves of Cucumis sativis. The amino acid sequences of the intact protein and of peptides isolated following endoproteinase Lys-C digestion, cyanogen bromide cleavage, and trypsin digestion were determined. Oligonucleotide probes derived from this sequence were used to isolate a cDNA clone encoding this protein. No significant homology was found between this chitinase and either the basic chitinase isolated from bean or tobacco or the chitinase isolated from Serratia marcescens; however, strong homology was found between the cucumber chitinase and a lysozyme/chitinase from Parthenocissus quinquifolia. The induction of the protein by tobacco necrosis virus infection or salicylate was found to be at the level of RNA accumulation. Genomic Southern analysis indicates that a single gene in the cucumber genome encodes this protein.

Effect of salicylate stress on the hypersensitive reaction of asparagus bean to tobacco necrosis virus

Treatments of primary leaves of asparagus bean ( Vigna sesquipedalis Fruhw.) with salicylate had different effects on resistance to local lesion development caused by tobacco necrosis virus (TNV), depending upon the concentration used for treatment. At 1 m m, salicylate slightly reduced lesion size but not virus accumulation, while at 3 m m it significantly decreased lesion size and virus accumulation. Treatments with 5 m m solutions appreciably increased both parameters, but also damaged the leaf tissue. The hypersensitive reaction of the leaves to virus inoculation was accompanied by the accumulation of five host-coded protein bands (VS 0to VS 4). However, three (or more) days after treatment with salicylate at any concentration between 1 and 5 m m only one band accumulated and this band appeared to correspond to VS 1. This finding clearly questions the role of the VS 1 protein in limiting the spread and multiplication of TNV. The leaves responded rapidly to treatment with salicylate with stomatal closure, increased K + leakage and ethylene production. These responses are generally considered to be general reactions to stress. None of them however, was correlated with any of the observed changes in TNV infection.

RNA polymerase from tobacco necrosis virus-infected and uninfected tobacco: II. Properties of the bound and soluble polymerases and the nature of their products

RNA polymerase activities in extracts of healthy and tobacco necrosis virus (TNV)-infected tobacco plants were investigated. Considerable ”bound“ enzyme activity, associated with endogenous viral RNA template, was found in the 30,000 g pellet obtained from TNV-infected plants. This activity was usually not increased by addition of RNA. In the corresponding fraction from healthy plants a much lower activity was detected which was increased when RNA was added. The conditions favoring the enzyme activity and its general properties were investigated. The RNA synthesized by this enzyme on the endogenous template, be it in the particulate state or solubilized and ammonium sulfate-fractionated, was about 90% double-stranded and of high molecular weight. It corresponds to a viral plus strand. After melting, this RNA comigrated with TNV-RNA. Thus it appears that the membrane-bound enzyme in TNV-infected plants represents a TNV replicase which is able to synthesize or finish full-length TNV-RNA on an endogenous template, the minus strand of TNV-RNA; this enzyme-template complex can be solubilized and fractionated without losing this ability. Nucleotide polymerizing activity was also detected in the 100,000 g supernatant of healthy tobacco plants and this activity was significantly increased upon TNV infection. This activity which resembled in some respects that reported by Duda et al. (1973) was greatly stimulated by added RNA. The products synthesized by this “soluble” enzyme extracted from healthy and infected plants were of similarly low molecular weight; they consisted of both single- and doubles-tranded RNAs and behaved partly as expected for fragments of minus-strand viral RNA.

The effect of light on the transmission of tobacco necrosis virus by Olpidium brassicae

The transmission of tobacco necrosis virus (TNV) to roots of lettuce ( Lactuca sativa L. ‘Triumph’) by Olpidium brassicae (Wor.) Dang. was reduced if the process occurred in the light rather than in the dark. Fewer Olpidium zoospores encysted on the roots and encysted zoospores were inactivated on the root surface if roots were exposed to light. Protoplasts of zoospores, after entry into the host cell, were not inactivated by exposure of the host plant to light, and in fact development of both Olpidium and TNV appeared to be enhanced. TNV was inactivated by light to a much lesser degree than was Olpidium, but in roots inoculated with both TNV and Olpidium infection and development of both TNV and Olpidium were similarly affected by exposure to different durations and timing of light. This supports existing evidence that TNV is dependent on Olpidium for entry into the host cell.

THE EFFECT OF LEAF WATER BALANCE ON THE SUSCEPTIBILITY OF FRENCH BEAN TO TOBACCO NECROSIS VIRUS

French beans were germinated under constant temperature, relative humidity, and day period, and were transferred to Hoagland's culture solution. Variations of the preinoculation treatment were begun at an age of 10 days for a period of 24 h. Treatments were selected which would induce turgor changes in the primary leaves.It was observed that susceptibility to tobacco necrosis virus infection was increased by preinoculation conditions of continuous darkness, high relative humidity, and low suction tension of the culture solution. Susceptibility was lowered by preinoculation conditions of continuous light, low relative humidity, and high suction tension of the culture solution.Diffusion pressure deficit, osmotic pressure, and turgor pressure measurements were made with the primary leaves at time of inoculation. Considerable agreement was noted between turgor changes and susceptibility.Preinoculation treatments such as darkening will alter susceptibility through changes in turgor pressure. It is suggested that there may be a direct effect of leaf turgor on the number of infectible sites.

Transmission of a tobacco necrosis virus by Olpidium brassicae

Olpidium brassicae (Wor.) Dang. reproduced better in the roots of lettuce ( Lactuca sativa L.) at 10–16 ° than at 22 or 27 ° and better in tobacco necrosis virus (TNV)-free than in TNV-infected roots. After exposure to Olpidium and TNV the first new TNV in roots was detected after 8 hours at 19 or 22 ° and after 12 hours at 13 °, but after 4–7 days more TNV was detected at 10, 13, or 16 ° than at 22 ° or 27 °. Roots of 3- or 7-day-old lettuce plants were more susceptible to both Olpidium and TNV than roots of 12- or 19-day-old plants. Fifteen lettuce varieties differed greatly in susceptibility to Olpidium; the roots of most varieties reacted necrotically to TNV. TNV infection decreased Olpidium reproduction more in the necrotic-reacting variety ‘Climax’ than in the nonnecrotic-reacting variety ‘Dark Green Cos.’ TNV was transmitted with as few as 58 Olpidium zoospores per milliliter; with about 10 5 zoospores per milliliter TNV was transmitted from solutions so dilute that no lesions were produced by mechanical assay on leaves of Phaseolus vulgaris L. Additional evidence supporting the hypothesis that Olpidium is the vector of TNV was provided by two kinds of experiments. Filtration experiments showed that removal of Olpidium zoospores from suspensions of zoospores plus TNV gave TNV-containing filtrates that did not transmit TNV to lettuce roots. Olpidium and TNV were found to infect the same parts of lettuce roots.

Further studies of Olpidium as a vector of tobacco necrosis virus

When a suspension of zoospores of Olpidium brassicae (Wor.) Dang. and tobacco necrosis virus (TNV) was added to seedling roots of mung bean ( Phaseolus mungo L.) and the seedlings were placed in a moist chamber 30 minutes later, infection of the roots by Olpidium and by TNV resulted. When the seedlings were placed in running tap water instead of in the moist chamber, TNV infection was increased. Infection was heavy in seedlings placed in TNV, exposed to prolonged washing, and inoculated with zoospores; apparently the zoospores transmitted TNV retained by the roots through the washing. Although zoospores do not complete root penetration until 2–3 hours after encystment, addition of TNV to roots 1–2 hours after zoospore encystment on the roots resulted in little or no TNV infection. When appropriate dilutions of TNV and antiserum were mixed and added to zoospores released into water, no virus transmission to roots resulted from the mixture, but transmission resulted when zoospores were released from roots into the virus suspension and the antiserum was added later. Zoospores released from TNV-infected roots into antiserum caused one-fifth to one-half as much TNV infection as similar zoospores released into normal serum. The addition of TNV antiserum to roots bearing zoospores that had encysted on the roots in the presence of TNV did not prevent TNV infection, although the antiserum inactivated the virus in suspension. Thus, the evidence indicates that the Olpidium zoospore acquires and harbors TNV in such a manner that antiserum cannot inactivate it. Therefore it is probably not merely a surface carriage. Unsuccessful attempts were made to transmit tobacco ringspot virus and tobacco rattle virus to Nicotiana tabacum L. ‘Xanthi’, tobacco mosaic virus and tomato bushy stunt virus to Lycopersicon esculentum Mill. ‘Bonny Best’, southern bean mosaic virus to Phaseolus vulgaris L. ‘Bountiful,’ and peach yellow bud mosaic virus to Cucumis sativus L. ‘National Pickling’, by adding combined suspensions of zoospores and the viruses to the roots.

Transmission of tobacco necrosis virus by a fungus, Olpidium brassicae

Experiments were performed to determine whether tobacco necrosis virus (TNV) entered plant roots at the same time and position as zoospores of Olpidium brassicae (Wor.) Dang. Mung bean ( Phaseolus mungo L.) seedlings were immersed in a combined suspension of Olpidium zoospores and TNV and heated 10 seconds at 50° to kill the Olpidium without inactivating the virus. TNV infection did not usually occur if the roots were heated within 2 hours, but did occur if the roots were heated at 3 or more hours after immersion. This lag of 2 to 3 hours before TNV infection corresponds with the time it takes for Olpidium to penetrate roots. However, when mung bean seedlings were placed in a suspension of Olpidium zoospores for 3 hours, heated to destroy Olpidium, and then placed in a TNV suspension, no infection occurred. Thus, the Olpidium did not appear to predispose the roots to subsequent TNV infection. Olpidium zoospores encysted on and entered the roots of 4-week-old lettuce ( Lactuca sativa L.) plants mainly within 5 mm of the root apex, and subsequent assays showed that the apical 5 mm of the root was also the zone where most TNV infection occurred. TNV infection of roots was induced only when actively swimming Olpidium zoospores were able to encyst on roots in the presence of TNV. Mild treatments, such as aging for 1 2 hour or more, heating to 35° for 10 minutes, and addition of 10 −3% CuSO 4. 5H 2O permanently inactivated the delicate zoospores, and prevented TNV infection of plants subsequently placed in the suspensions. Washings of Olpidium-infected lettuce roots, if taken during the first 2 minutes of washing, did not contain zoospores and did not induce TNV infection of mung bean seedlings. However, washings taken after 2 minutes did contain zoospores and did induce TNV infection. The amount of TNV infection was approximately proportional to the number of zoospores. The above is considered strong evidence that Olpidium zoospores transmit TNV in a vectorlike manner. In line with results with Californian strains of TNV, Olpidium zoospores transmitted a British strain of TNV to cowpea [ Vigna sinensis (Torner) Savi], mung bean, tomato ( Lycopersicon esculentum Mill.), and tobacco ( Nicotiana tabacum L.) roots. Thus, it appears likely that Olpidium transmits TNV in areas other than California.