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.     

Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non-Necrotic Alterations

The level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVY^N). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVY^O). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis-inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVY^N-infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from-virus-infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non-necrotic, interaction with tobacco leaves, like the necrosis-inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus-infected leaves are not clearly related.     

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

Summary 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.     

CUTICLE THICKNESS IN PHLOX AND RESISTANCE TO POWDERY MILDEW: AN UNRELIABLE LINE OF DEFENSE

The relationship between leaf cuticle thickness and resistance to the powdery mildew Erysiphe cichoracearum was investigated for nine feral Phlox taxa. Cuticle thickness, the first potential host resistance feature, is variable within plants, although not correlated with leaf age, and both within and among taxa. There is also considerable variation within and among taxa in resistance response to artificial inoculations with a broadly compatible E. cichoracearum strain. However, no significant correlations between cuticle thickness and resistance were observed. The implications of these results for anti-mildew defenses in both feral and cultivated hosts are discussed.     

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.     

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.     

Induction of a new Lipoxygenase Form in Cucumber Leaves by Salicylic Acid or 2,6-Dichloroisonicotinic Acid*

Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX-95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX-97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.     

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.     

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.     

Powdery mildew of tobacco (Erysiphe cichoracearum DC.)

Percentage germination, and growth of hyphae from single conidia of Erysiphe cichoracearum DC., were measured on leaf discs from topped and intact tobacco plants, grown in aerated nutrient solutions consisting of basal medium plus large or small amounts of potassium. The effect of supplying sodium was also studied. Discs were incubated on water and on 10% sucrose solution. Changes in free amino nitrogen and carbohydrate in comparable uninfected leaf discs, before and after incubation, were also measured. Potassium deficiency resulted in more free amino nitrogen and soluble carbohydrate and less insoluble carbohydrate, per cm.² of leaf. Spore germination was not greatly affected by treatments, though it was usually less on discs from potassium-deficient leaves. The pathogen grew slower on potassium-deficient leaf discs, whether they were incubated on water or on sucrose. Incubating discs from some leaves on sucrose, compared with water, gave greatly increased sugar content and less fungal growth; discs from other leaves had a much smaller increase in sugar, and hyphal length was similar to that on discs incubated on water. Sodium, when potassium was scarce, increased potassium deficiency symptoms, free amino nitrogen and sugar content, and resistance to powdery mildew.     

Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.     

Xanthine Oxidase Activity in the Susceptible and Hypersensitive Responses of Tobacco Leaves to Tobacco Mosaic Virus Infection

A superoxide-producing xanthine oxidoreductase was isolated and quantified after polyacrylamide disc gel electrophoresis of tobacco leaf extracts. The results obtained indicate that, like uricase activity, a slight increase in tobacco xanthine oxidase activity takes place in the susceptible interaction with tobacco mosaic virus (TMV). In contrast, out of three hypersensitive tobacco cultivars tested, only two showed the same slight increase m activity during the late stage of hypersensitive response.
Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] a specific and potent in vitro and in vivo inhibitor of xanthine oxidoreductase, applied to tobacco plants by root absorption, starting about 8 days before the inoculation, did not affect the hypersensitive response but weakened the hypersensitivity-linked virus localization and promoted the movement of a certain amount of TMV particles and/or virus related material from necrotic lesions which induced systemic necrotic symptoms in uninoculated leaves. However, due to the inefficacy of allopurinol in preventing necrotic lesion development, all results are consistent with the hypothesis that xanthine oxidoreductase, the first enzyme in purine oxidative degradation, plays only a secondary role during induction of primary hypersensitive cell death in TMV infected tobacco leaves.     

Studies on Induced Resistance to Tobacco Mosaic Virus, in Samsun NN Tobacco and Changes in Ribosomal Fractions

Resistance to tobacco mosaic virus (TMV) was activated by various forms of induction in Samsun NN tobacco leaves, and the intensity of the different forms was compared. Induced resistance was highest in leaf tissue between TMV inoculated stripes parallel to the mid-vein and after injection of ethylene maleic anhydride copolymer (EMA), followed by that induced in distal half leaves after inoculating the basal halves with TMV. Resistance in upper leaves following inoculation of the lower leaves with TMV was relatively low, while induction due to lesions caused by ethrel gave an intermediate degree of resistance. Estimation of resistance by size and number of local lesions was correlated with the amount of extractable virus as measured by enzyme-linked immunosorbent assay (ELISA), thus indicating that in the resistant tissue virus replication, and not only the development of necrotic local lesions, is suppressed. An increase in a specific ribosomal fraction (R₂), recovered by a two-step procedure, was observed in tissues where resistance was most intense, i.e., between TMV stripes and after EMA injection. It may be that this specific ribosomal fraction participates in maintaining the resistant state.     

Artificial elevation of glutathione contents in salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) reduces susceptibility to the powdery mildew pathogen Euoidium longipes

• The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild‐type cv. Xanthi plants, where salicylic acid (SA) contents are normal.
• Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation.
• SA‐deficient NahG tobacco was hyper‐susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA‐deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild‐type plants (i.e. enhanced basal resistance). However, expression of the SA‐mediated pathogenesis‐related gene (NtPR‐1a) did not increase significantly in GSH or OTC‐pretreated and powdery mildew‐inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH.
• Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA‐deficient tobacco.

     

The Association of "Pathogenesis-related" Proteins with Viral Induced Necrosis in Nicotiana sylvestris

The association of “pathogenesis-related” (PR) proteins with protection from superinfection, systemic acquired resistance and production of localized necrotic lesions was examined with a system using tobacco mosaic virus (TMV) and Nicotiana sylvestris. Leaves of N. sylvestris with a mosaic from earlier inoculation with a systemically infecting strain of TMV (TMV-C) and control plants were challenged with a necrotizing strain of TMV (TMV-P), RNA of TMV-P and turnip mosaic virus (TuMV). TMV-P virions produced localized necrotic lesions only in the dark green areas of the mosaic of TMV-C infected plants. Both RNA of TMV-P and TuMV produced localized necrotic lesions in both light green and dark green areas of the mosaic of TMV-C infected plants. All three challenge inocula produced localized necrotic lesions in previously uninoculated plants. Six days after challenge inoculation proteins were extracted from separated dark green and light green mosaic leaf tissue, and leaf material from control plants. Proteins were separated by electrophoresis in a 5 % polyacrylamide spacer gel and 10 % polyacrylamide running gel. PR proteins were found in tissue where localized necrotic lesions were produced as a result of challenge inoculation, but not in tissue that was not superinfected. PR proteins were not found in light green or dark green mosaic leaf tissue as a result of TMV-C inoculation. No PR proteins were evident in protein extracts from light green tissue challenged with TMV-P, although PR proteins were produced in dark green tissue, where necrosis occurred, from the same leaves. Systemic acquired resistance (reduction in size of lesions formed by a challenge inoculation) to TuMV or RNA of TMV-P and PR protein concentration was measured at various times in light green areas of mosaic leaves where dark green areas of the mosaic leaves had been inoculated with TMV-P. No quantitative or temporal relationship between the onset of resistance and PR protein production was found. It is concluded that PR proteins are a result of pathogen induced necrosis and not significantly involved in the mechanism(s) of viral induced resistance.     

辣椒抗烟草花叶病毒相关基因研究进展

病毒病是危害辣椒生产的主要病害之一。烟草花叶病毒(TMV)是最早被发现的病毒,它引起的烟草花叶病毒病是多种作物的重要病害,给辣椒等茄科作物的生产带来重大损失。文中综述了辣椒抗TMV防御反应中的相关基因及其研究进展,为明确辣椒抗TMV机理,挖掘抗病基因,选育抗病材料提供参考。     

3-Acetonyl-3-hydroxyoxindole: a new inducer of systemic acquired resistance in plants

Systemic acquired resistance (SAR) is an inducible defence mechanism which plays a central role in protecting plants from microbial pathogen attack. Guided by bioassays, a new chemical inducer of SAR was isolated from the extracts of Strobilanthes cusia and identified to be 3-acetonyl-3-hydroxyoxindole (AHO), a derivative of isatin. Tobacco plants treated with AHO exhibited enhanced resistance to tobacco mosaic virus (TMV) and to the fungal pathogen Erysiphe cichoracearum (powdery mildew), accompanied by increased levels of pathogenesis-related gene 1 ( PR-1 ) expression, salicylic acid (SA) accumulation and phenylalanine ammonia-lyase activity. To study the mode of action of AHO, its ability to induce PR-1 expression and TMV resistance in nahG transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of SA, was analysed. AHO treatment did not induce TMV resistance or PR-1 expression in nahG transgenic plants, suggesting that AHO acts upstream of SA in the SAR signalling pathway. In addition, using two-dimensional gel electrophoresis combined with mass spectrometry, five AHO-induced plant proteins were identified which were homologous to the effector proteins with which SA interacts. Our data suggest that AHO may represent a novel class of inducer that stimulates SA-mediated defence responses.     

Protein composition of tobacco leaves with antiviral resistance induced by activators of defense responses and TMV infection

In tobacco (Nicotiana tabacum L.) plants of hypersensitive cv. Samsun NN, a capability of necrosis lesion formation and protein patterns were studied after induction of antiviral resistance by defense responses activators (DRA) (arachidonic acid, ubiquinone 50, and vitamin E) and by infection with tobacco mosaic virus (TMV). DRA and TMV improved both local and systemic leaf resistance to TMV. Native protein electrophoresis demonstrated differences in the composition of leaf proteins extracted under acidic and alkaline conditions. SDS-PAGE revealed proteins accumulated during the development of systemic antiviral resistance after lower leaf treatments with DRA and of local resistance induced by pretreatment with TMV. It was shown that various DRA affected protein patterns similarly, whereas TMV infection resulted in other changes. It is supposed that different pathways function in tobacco plants during induction of systemic resistance by DRA and TMV infection.     

Sensibilité du Melon àSphaerotheca fuliginea en Fonction de L'Etage Foliaire

Effect of leaf position on the susceptibility of melon plants to artificial infection with powdery mildew, Sphaerotheca fuliginea The leaf position of melon plants seems to play a role on their susceptibility when they are artificially infected with powdery mildew Sphaerotheca fuliginea. The cotyledons are generally very susceptible, while the first leaf relatively resistant; the susceptibility again continues up to the 4th–5th leaf (but less susceptible than the cotyledons) and then after it decreases; these results can be obtained on plants in greenhouse or on detached leaves in Petri dishes. From this observation, we think that the screening of melon genotypes for resistance to powdery mildew can be evaluated neither on the cotyledons which are very susceptible nor on the first leaf which is resistant, but on the third leaf which is moderately susceptible. In fact, there is a good correlation between the reaction of the third leaf and the resistance or susceptibility of genotypes.