Localization of Pathogenesis-Related Proteins in the Epidermis and Intercellular Spaces of Tobacco Leaves after Their Induction by Potassium Salicylate or Tobacco Mosaic Virus Infection

The localization of pathogenesis-related (PR) proteins inducedin tobacco leaves by treatment with potassium salicylate ora hypersensitive response to tobacco mosaic virus (TMV) infectionwas studied using immunochemical methods. Total PR protein levelsincreased with time after these treatments. The proportion ofPR proteins in the intercellular spaces to the total contentin the leaf discs rapidly rose in the later stage of the treatmentto about 75% on the 9th day after salicylate treatment and tomore than 80% on the 6th to 9th day after TMV inoculation. After5 days of salicylate treatment, the amounts of PR proteins inthe peeled leaf epidermis were two fold those in the mesophylltissue. Only five percent or less of the total PR proteins inthe epidermal and mesophyll tissues of salicylate-treated leaveswere detected in the isolated epidermal and mesophyll protoplasts.The sugar content in highly purified PR la, lb and lc was lessthan one mole of monosaccharide per mole of each protein. Theseresults show that the PR proteins are non-glycoproteins secretedinto the intercellular spaces. (Received January 16, 1987; Accepted July 14, 1987)     

The Extracellular Acidic and Basic Pathogenesis-Related Proteins of Soybean Induced by Viral Infection

Seventeen major host-encoded pathogenesis related (PR)-proteins have been found in intercellular fluids of necrotic virus-infected soybean leaves. None of them was present in fluids of healthy controls. By native and SDS-denaturing polyacrylamide gel electrophoresis, ten major acidic PR-proteins have been identified and classified on the basis of their molecular weight in three groups: group 1 included four proteins of 16–17 Kd; group 2, three proteins of 26 Kd; group 3, three proteins of 32 Kd. Seven PR-proteins were basic, and were classified in three groups: group 1 included three proteins of 16 Kd; group 2, one protein of 23 Kd; group 3, three proteins of 32 Kd. As found for tobacco and potato, soybean PR-protein patterns show high number of acidic and basic proteins.     

Type I J-Domain NbMIP1 Proteins Are Required for Both Tobacco Mosaic Virus Infection and Plant Innate Immunity

Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by recognizing the viral movement protein (MP). Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s) associate with tobamovirus MP, Tm-2² and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-2²-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for Tm-2²-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.     

Temperature-Dependent Induction of Salicylic Acid and Its Conjugates during the Resistance Response to Tobacco Mosaic Virus Infection

Increases in endogenous salicylic acid (SA) levels and induction of several families of pathogenesis-related genes (PR-1 through PR-5) occur during the resistance response of tobacco to tobacco mosaic virus infection. We found that at temperatures that prevent the induction of PR genes and resistance, the increases in SA levels were eliminated. The addition of exogenous SA to infected plants at these temperatures was sufficient to induce the PR genes but not the hypersensitive response. However, when the resistance response was restored by shifting infected plants to permissive temperatures, SA levels increased dramatically and preceded PR-1 gene expression and necrotic lesion formation associated with resistance. SA was also found in a conjugated form whose levels increased in parallel with the free SA levels. The majority of the conjugates appeared to be SA glucosides. The same glucoside was formed when plants were supplied with exogenous SA. These results provide further evidence that endogenous SA signals the induction of certain defense responses and suggests additional complexity in the modulation of this signal.     

Effect of 1,3;1,6-β-D-glucan on Infection of Detached Tobacco Leaves with Tobacco Mosaic Virus

Glucan preparations were obtained by transformation of laminaran from the alga Laminaria cichorioides with endo-β-1,3-glucanase from marine mollusks. These preparations, like those of other sources, have an inhibitory effect on tobacco mosaic virus (TMV) infection of detached leaves of local and systemic host tobacco plants.     

The Effects of Salicylic Acid and Tobacco Mosaic Virus Infection on the Alternative Oxidase of Tobacco

Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA. Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxidase protein, suggesting that steady-state alternative oxidase accumulates in an SA-independent manner. Infection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants. Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.     

黄瓜花叶病毒对烟草叶片和叶绿体光合活性的影响

研究了烟草（Ｎｉｃｏｔａｎａ ｔａｂａｃｕｍ Ｌ．）感染黄瓜花叶病毒后烟草植株不同部位（顶层、中层、下层）中片的光合速率和叶绿体光化学反应的变化。感病的叶片净光合速率减少,顶层叶砬少最多。病毒侵染抑制ＰＳⅡ活性比ＰＳⅠ活性更明显,特别是顶层叶。叶绿素ａ荧光测定表明还原态Ｑ库减少,ＣＯ２同化减少。从低温荧光发射光谱图变化中发现能量分配受干扰。     

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.     

Effects of Cucumber Mosaic Virus Infection on Photosynthetic Activities of Tobacco Leaves and Chloroplasts

Changes in photosynthetic activities were studied with tobacco (Nicotiana tabacum L.) leaves and chloroplasts infected by cucumber mosaic virus (CMV) at the top, middle and bottom located leaves. Net photosynthetic rate was reduced at all three positioned leaves, with the maximum reduction occurring at the top leaves (31.9% of control). The infected chloroplasts showed a reduction in electron transport rates of the whole chain electron transport, photosystem Ⅱ (PSⅡ) and photosystem Ⅰ (PSⅠ). Since the decline in the whole chain electron transport (15.6% of control, H2O→MV) closely paralleled the decline in PSⅡ activity (20.9% of control, H2O→PBQ), the inhibition of the latter was probably responsible for the overall decrease. Chlorophyll a fluorescence measurements showed a variable reduced fluorescence yield (Fv/Fo) which indicated that PSⅡ was impaired and the CO2 assimilation was disturbed by CMV infection. Fluorescence emission spectra at 77 K indicated that energy distribution between PSⅡ and PSⅠ was affected. F686/F734 of infected leaves and chloroplasts increased and the greatest increase (331.1% of control ) was found in the top leaves. These data may conclude that the infection inhibited mainly the PSⅡ activity.     

Glucose-6-Phosphate Dehydrogenase, Ribonucleases and Esterases upon Tobacco Mosaic Virus Infection and Benzothiodiazole Treatment in Tobacco

Effect of the benzothiodiazole (BTH) pre-treatment was monitored during the acute infection stage in the susceptible and the hypersensitive tobacco plants infected with the tobacco mosaic virus (TMV). Dynamic changes in the contents of chlorophyll, the total proteins, and the pathogenesis related proteins (PR-proteins), and activities of ribonucleases (RNase), phosphomonoesterase (PME), phosphodiesterase (PDE), and glucose-6-phosphate dehydrogenase (G6P DH) were studied. Neither the protein nor the chlorophyll contents were significantly changed by the TMV infection and/or the BTH treatment. The BTH pre-treatment caused a substantial reduction in the multiplication of TMV in the locally-infected leaves of the hypersensitive cultivar Xanthi-nc (to 15.1%). A lesser decrease (to 50.3%) was observed in the locally-infected leaves of susceptible cultivar Samsun. But in the systemically-infected leaves of this cultivar, only a 4-d delay in the multiplication of TMV was found. In the locally-infected leaves of both cultivars, the activities of the RNase, PME, PDE and G6P DH were sharply increased during the acute phase of TMV multiplication (when compared with the healthy plants) and the curves of these activities correlated with the multiplication curves of TMV. The BTH alone also strongly enhanced the activities of these enzymes early after application. Only low additional increases in some enzymes and even slight declines in the others were observed when the inoculation of leaves of cultivar Xanthi-nc followed the pre-treatment with the BTH. No inhibition of the enzymes was observed when the direct effect of different concentration of the BTH (1 – 1000 M) was examined in vitro during a measurement of the activity. The analysis of intercellular proteins by PAGE under native conditions shows the similar spectrum of the proteins extracted from either the BTH-treated or the TMV-infected tobacco cv. Xanthi-nc.     

An Examination of the Effect of Human α-Interferons on the Infection and Multiplication of Tobacco Mosaic Virus in Tobacco

Human α interferon had no direct effect on TMV RNA infectivity and when appiled to tobacco had no significant effect on local lesion development or virus multiplication.     

The Systemic Infection by Tobacco Mosaic Virus of Tobacco Plants Coutaining the N Gene at Temperatures Below 28°C

Tobacco plants containing the N-gene are occasionally systemically infected with tobacco mosaic virus (TMV) at tempreratures below 28°C, but contain low concentrations of virus: they often fail to set seed, and can outlive healthy control plants. Infection is thus similar to that induced when N-gene tobacco plants are grafted onto systemically infected tobacco lacking the N-gene. Shoots from systemically infected N-gene plants can induce systemic infection in other graft-inoculated N-gene plants. Stem sections of N-gene tobacco plants act as good conduits for TMV between plants lacking the N-gene, and girdling experiments suggest that virus movement probably occurs in the phloern.     

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.