THE HISTOLOGY OF THE NECROTIC LESIONS INDUCED BY VIRUS DISEASES

About 12 hours after a leaf of Nicotiana glutinosa is inoculated with ancuba mosaic disease a band of necrotic material begins to form within the cell wall, usually between the lower epidermis and the spongy parenchyma. This band extends both laterally and towards the upper side of the leaf. At the same time, nuclear division is observed in the spongy parenchyma cells, but karyokinesis is not followed by cell division. As the necrotic mesh work extends the cells within it die and dry out. After about 3 days, the lesion consists of a meshwork of this necrotic material. The cells are all dead and the virus is isolated within the necrotic area, all interchange between the infected and healthy parts of the leaf having been prevented.
I am indebted to Fraulein Lina Cunow for assistance in making the preparations used in this study.     

Pathogenesis-related proteins induced by agroinoculation-associated cell wall weakening can be obviated by spray-on inoculation or mannitol ex vivo culture

Agroinoculation transient expression systems are commonly accompanied with elevated pathogenesis-related (PR) protein production and leaf necrosis. We identified the major PR proteins in Nicotiana benthamiana in response to agroinoculation and determined that their occurrence was mainly due to agrobacterium infection and the method of inoculation, rather than due to viral vectors overexpressing foreign proteins. A spray-on inoculation method was optimized in this research and used to obviate the leaf necrosis and PR proteins induced by agrobacterium. Subsequently, this method also increased the yield and purity of the protein-of-interest. A further investigation of PR protein induction by a necrosis-inducing protein, Jun a 1, suggested that the plant pathogenic response was related to biochemical integrity of plant cell wall, which was also confirmed by an osmo-stabilizing mannitol ex vivo culture experiment. These findings provide insight into the response of plants to agroinoculation and suggested a connection between cell wall weakening and PR protein elicitation.     

SOME EFFECTS OF VIRUS INFECTION ON LEAF WATER CONTENTS OF NICOTIANA SPECIES

Infection with tobacco mosaic virus decreases the water content which detached tobacco leaves attain when kept for 20 hr. in conditions of minimum water stress, and does so more when the plants are kept in light before inoculation than when they are kept in darkness. No such effects of infection during the first day after inoculation were obtained with tobacco leaves infected with either tobacco etch virus or potato virus X , or with Nicotiana glutinosa leaves infected with tobacco mosaic virus. These results, like those showing early effects of TMV on respiration and photosynthesis of tobacco leaves, suggest that inoculation with TMV affects deeper leaf tissues than the epidermis earlier in tobacco leaves than in other leaves, and earlier than other viruses in tobacco leaves.     

Pathogenesis-related protein b1' in plants and in cell suspension cultures of Nicotiana glutinosa Nicotiana debneyi and an amphidiploid cross (N. glutinosa×N. debneyi)

The expression of PR-protein b₁' in plants and cell suspension cultures of Nicotiana glutinosa L., Nicotiana debneyi Domin, and an amphidiploid cross of these two species, a hybrid, has been investigated. An enzyme linked immunosorbent assay has been employed to determine the concentration of PR-protein b₁' in extracts. The PR-Protein b₁' was constitutively produced in intact plants of the hybrid (around 25 μg g⁻¹ leaf tissue), while only trace amounts of the protein (< 50 ng g⁻¹ leaf tissue) were found in plants of the two parents. In suspension culture, the concentrations of PR-protein b₁' were 8, 0.4 and less than 0.1 mg l⁻¹ medium for the hybrid. N. debneyi and N. glutinosa , respectively. Only trace amounts of the protein were found in extracts from cells. Seven days after infection by tobacco mosaic virus (TMV) the concentration of PR-protein b₁' in leaves of N. glutinosa was 22.5 μg g⁻¹ leaf tissue. In N. debneyi and the hybrid a relatively limited induction of PR-protein b₁' by TMV was observed. The influence of various phenoxyacetic acids on the expression of PR-protein b₁' in the 3 cell cultures has been investigated. Cultures of N. glutinosa responded to treatments with 2,4-D and 2,4,5-T while cultures of N. debneyi and the hybrid were essentially unaffected. In the former case a concentration of 5–10 mg l⁻¹ 2,4,5-T was optimal and cells were most responsive to the treatment 4 days after subcultivation. The concentration of PR-protein b₁' in elicited cell cultures of N. glutinosa was 2 to 4 mg l⁻¹ medium.     

Leaf Disk Inoculation — a Useful Tool for Selecting Infections of Sunflower Downy Mildew at Low Inoculum Concentration, but Inappropriate to Pathotype Characterization

A modified technique for leaf disk inoculation of sunflower with zoosporangia of the downy mildew pathogen Plasmopara halstedii was developed. Infection with low concentrations of inoculum was obtained down to the level of single sporangia. No significant difference in the infection rate was seen between disks from cotyledons and true leaves. This makes leaf disk inoculation particularly suitable for infections at low sporangium density and for the investigation on plant tissue which cannot be infected with whole seedling inoculation. The technique seems, however, to be inappropriate for pathotype characterization using differential host lines.     

Purification of potato virus X and preparation of its antiserum

Potato virus X (PVX) isolated from the potato leaf and tuber samples which were collected from various fields in Damavand and Ardabil. The initial isolations of the virus were made from potato by mechanical inoculation on Gomphrena globosa L. and Chenopodium spp. that produce local lesion, and then it causes mosaic on Nicotiana spp. and Datura stramonium L. An isolate of the virus inoculated to Nicotiana glutinosa L. and it was maintained throughout the work. Sap from infected N. glutinosa was ineffective after dilution to 10-6, 10 minutes at 70 degrees and 10 weeks at room temperature. The virus was readily purified from infected leaves and the best protocol was Moreira & Jones 1980 than the other 2 methods of Fribourg 1975 and Shepard & Shalla 1972. Antisera were prepared against native, degraded proteins and micro precipitin test showed that both antisera had a 1/512 titer. Precipitin lines with D - Protein antiserum was better of the native protein antiserum in agar double diffusion test than treated with SDS. The isolate of the virus was not transmitted by none of 2 species of Cuscuta but transmitted from infected leaves to healthy plants with sap inoculation without using Carburandum. This isolate showed positive reaction with gamaglubulin in kate received from CIP centre.     

Plant disease and the regulation of enzymes involved in lignification

Tobacco varieties carrying the N gene from Nicotiana glutinosa respond to infection by Tobacco Mosaic Virus (TMV) by forming necrotic local lesions (hypersensitive reaction), thereby localizing the infection. In this study, infected mesophyll leaf tissue of N. tabacum Samsun NN was treated with the non-permeating, non-metabolizable carbohydrate mannitol. The local lesions developed under iso-osmotic conditions (0.28 M mannitol), though with a slight delay and with a reduced rate of growth, as compared to those on attached leaves. At increasing plasmolysing concentrations of mannitol, necrotization was progressively inhibited, but not completely suppressed. The leaf tissue produced tiny translucent zones, with a delay that increased between the virus inoculation and application of the plasmolytica. Activities of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and O-methyltransferase (OMT, EC 2.1.1.6) are strongly stimulated in hypersensitively reacting tobacco and were used as biochemical markers in the present study. This study was done to determine whether the inhibitory effect of plasmolysis on the elicitation of the hypersensitive response is due to a decrease in virus spread, resulting from the rupture of plasmodesmata or, at least in part, to metabolic alterations of the host cell exposed to osmotic stress. Since necrotization is normally preceded by intense virus multiplication, the inhibitory effects found for early applications (i.e., before local lesion appearance) of plasmolytica could easily be related to an inhibition of virus spread which also occurred in similarly treated leaf tissue of the systemically reacting variety Samsun. The most meaningful data were obtained from mannitol treatments performed on leaf tissue already carrying local lesions, i.e., in which the elicitor(s) and/or the factor(s) of necrotization were already operating. Under iso-osmotic conditions, we found the stimulated PAL and OMT activities characteristic of the hypersensitive response. At plasmolysing concentrations of mannitol, we observed the counteracting effects of two different mechanisms controlling the phenylpropanoid enzymes. Floating the leaf material on the liquid medium induced an ageing-like effect with a continuous increase in enzyme activities that was independent on osmotic pressure and sensitive to cycloheximide. At the same time, the stimulated enzyme activities related to hypersensitivity decreased at a rate related to osmotic pressure. Since PAL and OMT of tobacco leaves are long-lived enzymes, it is likely that the increased de novo synthesis of the enzymes was suppressed by plasmolysis while their degradation and/or inactivation was maintained or even increased. From these results it is concluded that the apparent inhibition of the hypersensitive response by plasmolysis is due to both a decrease in virus spead (artificially caused by the rupture of connections between cells) and to drastic metabolic alterations of the host cell exposed to high osmotic pressure.     

Absence of arabinan in the side chains of the pectic polysaccharides strongly associated with cell walls of Nicotiana plumbaginifolia non-organogenic callus with loosely attached constituent cells

When leaf disks from haploid plants of Nicotiana plumbaginifolia Viv. were transformed with T-DNA and cultured on shoot-inducing medium, nonorganogenic callus. designated nolac (for non-organogenic callus with loosely attached cells), appeared on approximately 7% of leaf disks. In contrast, normal callus was generated on T-DNA-transformed leaf disks from diploid plants and on non-transformed leaf disks from haploid and diploid plants. Transmission electron microscopy revealed that the middle lamellae and the cell walls of one line of mutant callus (nolac-H14) were barely stained by ruthenium red. even after demethylesterification with NaOH, whereas the entire cell wall and the middle lamella were strongly stained in normal callus. In cultures of nolac-H14 callus, the level of sugar components of pectic polysaccharides in the hemicellulose fraction was reduced and that in the culture medium was elevated, as compared with cultures of normal callus. These results indicate that pectic polysaccharides are not retained in the cell walls and middle lamellae of nolac-H14 callus. In nolac-H14, the ratio of arabinose to galactose was low in the pectic polysaccharides purified from all cell wall fractions and from the medium, in particular, in the hemicellulose fractions. The low levels of arabinofuranosyl (T-Araf, 5-Araf, 2,5-Araf, and 3,5-Araf) residues in the pectic polysaccharides of the hemicellulosic fraction of nolac-H,14 indicated that no neutral-sugar side chains, composed mainly of linear arabinan. were present in nolac-H14. Arabinose-rich pectins. which are strongly associated with cellulose-hemicellulose complexes, might play an important role in intercellular attachment in the architecture of the cell wall.     

Modified plasmodesmata inSorghum (Sorghum bicolor L Moench) leaf tissues infected by maize dwarf mosaic virus

The early acute response (EAR), a type of hypersensitive response, is defined by small chlorotic spots at the base of the youngest leaf of sorghum (Sorghum bicolor L. Moench) cultivar HOK, and usually appears within five days after inoculation with maize dwarf mosaic virus strain A (MDMV-A). These chlorotic spots become necrotic one to two days later and the leaf tissues are rapidly killed. In leaf tissues showing EAR, plasmodesmal fields contained many modified plasmodesmata of various sizes and structures within thickened cell walls. The membranous vesicles and tubules, derived from the extended terminal structures of modified plasmodesmata, were blocked by callose-like deposits in the area between the cell wall and plasmalemma. Also observed were two opposite-directed channels united via a central cavity at the middle lamella of the cell wall, one end of which was connected to the plasmalemma, but the other end sealed off to form a bulbous extension. The localized structure, an extraprotoplasmic sac containing aggregates of elongated virus-like particles associated with the modified plasmodesmata, was located between the plasmalemma and the cell wall. The sac was bound by membranes, and appeared to be sealed and completely excluded from the protoplasm. Extraprotoplasmic sacs appeared to be derived from the terminal extension of modified plasmodesmata, and these modification seem to be related to restriction of the viral spread.     

The effect of reversion and duplication of leaf disks on the accuracy of photosynthesis determination by the dry weight method

Experiments with sunflower and fodder cabbage showed that reversion of the leaf disks lower side upwards (towards the radiation source) decreased the photosynthetic rate by as much as 26% both at full (2. 10⁵ erg. cm^?2. sec.^?1) and half density of irradiation. On placing two disks one on another the photosynthetic rate was decreased in the upper disk in a similar way as it was on reversion even if it were placed in the normal position (upper side upwards). But assimilation in the lower disk was only a fraction (0–40%) of that of the upper disk and in thick leaves was within the limits of the compensation point. The sum of the increase in dry weight in both disks due to photosynthesis exceeds the increment in one normally placed disk only in thin leaves and at high density of radiation. On the basis of the differences found it is advised to discard experimental samples where a quarter of the disks were reversed during exposure. On evaluating samples in which some of the disks were duplicated in error, it is most accurate to deduct the weight of one control disk from the weight obtained. The decrease in photosynthetic rate found in reversed and duplicated disks is evidently due to the simultaneous effect of differences in the supply of CO₂ and radiation energy to the assimilating tissue and apparatus and also to differences in the photosynthetic capacity of different leaf tissues.     

黄瓜花叶病毒卫星RNA XJs1侵染性cDNA克隆构建及其生物学功能初步研究

利用RT_PCR的方法,获得了黄瓜花叶病毒卫星RNA XJs1的全长侵染性cDNA克隆pMSC20。序列分析显示,XJs1全长384nt(GenBank登录号:DQ070748),比较XJs1与具有代表性的CMV卫星RNA的序列结构表明,在XJs1核苷酸序列的325nt~350nt间,具有典型的坏死型卫星RNA保守序列。通过体外转录,将XJs1与不含卫星RNA的辅助病毒分离物CMV_AH组合接种普通烟和心叶烟并进行检测。初步研究结果表明,XJs1为一致弱卫星RNA。     

Detection of the 3a Protein of Cucumber Mosaic Virus in a Cell Wall Fraction from Infected Nicotiana clevelandii Plants

The 3a protein of cucumber mosaic virus was expressed in Escherichia coli and, after purification, used to produce an antiserum. The 3a protein was detected in a cell wall fraction obtained from infected Nicotiana clevelandii leaf tissue by immunoblotting using the 3a antiserum. The 3a protein reached a maximum level 4 days after inoculation and remained at this level for a further 8 days before slowly declining. In contrast, the virus capsid protein, detected in an 80 000 g pellet by immunoblotting using a virus particle antiserum, reached a maximum 5 days after inoculation and remained at this level for at least a further 16 days.     

Callose deposition at plasmodesmata is a critical factor in restricting the cell-to-cell movement of <Emphasis Type="Italic">Soybean mosaic virus</Emphasis>

Callose is a β-l,3-glucan with diverse roles in the viral pathogenesis of plants. It is widely believed that the deposition of callose and hypersensitive reaction (HR) are critical defence responses of host plants against viral infection. However, the sequence of these two events and their resistance mechanisms are unclear. By exploiting a point inoculation approach combined with aniline blue staining, immuno-electron microscopy and external sphincters staining with tannic acid, we systematically investigated the possible roles of callose deposition during viral infection in soybean. In the incompatible combination, callose deposition at the plasmodesmata (PD) was clearly visible at the sites of inoculation but viral RNA of coat protein (CP-RNA) was not detected by RT-PCR in the leaf above the inoculated one (the upper leaf). In the compatible combination, however, callose deposition at PD was not detected at the site of infection but the viral CP-RNA was detected by RT-PCR in the upper leaf. We also found that in the incompatible combination the fluorescence due to callose formation at the inoculation point disappeared following the injection of 2-deoxy-d-glucose (DDG, an inhibitor of callose synthesis). At same time, in the incompatible combination, necrosis was observed and the viral CP-RNA was detected by RT-PCR in the upper leaf and HR characteristics were evident at the inoculation sites. These results show that, during the defensive response of soybean to viral infection, callose deposition at PD is mainly responsible for restricting the movement of the virus between cells and it occurs prior to the HR response.     

Detection of the 3a Protein of Cucumber Mosaic Virus in a Cell Wall Fraction from Infected Nicotiana clevelandii Plants

The 3a protein of cucumber mosaic virus was expressed in Escherichia coli and, after purification, used to produce an antiserum. The 3a protein was detected in a cell wall fraction obtained from infected Nicotiana clevelandii leaf tissue by immunoblotting using the 3a antiserum. The 3a protein reached a maximum level 4 days after inoculation and remained at this level for a further 8 days before slowly declining. In contrast, the virus capsid protein, detected in an 80 000 g pellet byimmunoblotting using a virus particle antiserum, reached a maximum 5 days after inoculation and remained at this level for at least a further 16 days.     

Early trans-plasma membrane responses to Tobacco mosaic virus infection

Early trans-plasma membrane behavior after in vivo mechanical inoculation of Nicotiana tabacum with Tobacco mosaic virus (TMV) was investigated and compared to virus quantification in leaf tissues. To identify early events related to virus/host interaction, the systemic virus TMV was used to infect lower leaves and tests were carried out on upper leaves which were not directly infected. Non-invasive microelectrodes were used to estimate trans-plasma membrane electron transport and membrane potential after artificial inoculum of virus, monitoring the plant for the following 15 days. Virus infection was assessed by ELISA and quantified by quantitative RT-PCR. Collected data showed that after 2-day post-inoculation (dpi), TMV was able to modify membrane parameters: transient hyperpolarization of trans-membrane potential was observed until 10 dpi, while redox activity in infected samples was higher compared to control until end of tests. Conversely, ELISA diagnostic test was not able to reveal the virus presence in tobacco leaves until 6 dpi, while leaf symptoms were manifested after 13 dpi.     

Detection of Potato Leaf Roll Virus in Intact Sprout Disks by Enzyme-linked Immunosorbent Assay

Potato leaf roll virus (PLRV) was detected by enzyme-linked immunosorbent assay (ELISA) when intact sprout, stem or leaf tissue disks were substituted for leaf or tuber extracts as test samples. Absorbance (A₄₀₅) values increased with increasing number of disks per plate well. Readings with sprout disks were significantly higher than those with disks cut from other plant tissues or with tuber sap. A₄₀₅ values obtained by using 7 or 5 sprout disks per well were near the maximum oneobtained with leaf sap. PLRV was slightly more efficiently detected by ELISA in light sprout disks than in etiolated sprout ones. When ten out of 34 healthy tubers were replaced by PLRV-infected ones in the tuber indexing test, the diseased samples werereliably detected with 5 etiolated sprout disks per well. The sprout disk sampling technique should be useful for qualitative evaluation of PLRV infection in sprouted potato tubers without necessity to wound them and using sprouts not long enough for maceration.     

Photosynthesis and predisposition of plants to infection with certain viruses

The effects on susceptibility to infection with certain viruses of subjecting plants to various periods of darkness or reduced illumination before and after inoculation were tested. The viruses and hosts used were a tobacco necrosis virus in French bean and tobacco; tomato aucuba mosaic virus in tobacco; and tobacco mosaic and tomato bushy stunt viruses in Nicotiana glutinosa . All the virus-host combinations give necrotic local lesions, and susceptibility was measured by local lesion counts. Susceptibility was consistently increased by pre-inoculation treatments of host plants, whereas post-inoculation treatments had relatively little effect, but most often decreased susceptibility.
Short periods in the dark produced similar responses to longer periods in shade, but the different plants varied in their response to, and tolerance of, darkness. The maximum number of lesions was usually obtained with bean plants kept for only 24 hr. in the dark before inoculation, but with tobacco plants susceptibility increased with increasing time in the dark up to 5 days.
It is suggested that the successful establishment of infection occurs in two stages, the first of which is affected by. the accumulation of photosynthetic products. Whether these products confer resistance by increasing cell turgor or by reacting specifically with virus particles is unknown, but sap from plants in the light possesses no greater virus-inhibiting power than sap from plants kept in the dark.     

Uncoupling resistance from cell death in the hypersensitive response of Nicotiana species to cauliflower mosaic virus infection

Cauliflower mosaic virus strain W260 elicits a hypersensitive response (HR) in leaves of Nicotiana edwardsonii, an interspecific hybrid derived from a cross between N. glutinosa and N. clevelandii. Interestingly, we found that N. glutinosa is resistant to W260, but responds with local chlorotic lesions rather than necrotic lesions. In contrast, N. clevelandii responds to W260 with systemic cell death. The reactions of the progenitors of N. edwardsonii to W260 infection indicated that each contributed a factor toward the development of HR. In this study, we present two lines of evidence to show that the resistance and cell death that comprise the HR elicited by W260 can indeed be uncoupled. First, we showed that the non-necrotic resistance response of N. glutinosa could be converted to HR when these plants were crossed with N. clevelandii. Second, we found that cell death and resistance segregated independently in the F2 population of a cross between N. edwardsonii and N. clevelandii. We concluded that the resistance of N. edwardsonii to W260 infection was conditioned by a gene derived from N. glutinosa, whereas cell death was conditioned by a gene derived from N. clevelandii. An analysis of pathogenesis-related (PR) protein expression in response to W260 infection revealed that elicitation of PR proteins was associated with resistance rather than with the onset of cell death.     

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis

Background and Aims

Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells.

Methods

Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins.

Key Results

The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls.

Conclusions

The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.