Dark Green Tissue Research Articles

MiRNAs are involved in regulating the formation of recovery tissues in virus infected Nicotiana tabacum.

MiRNAs play an important role in regulating plant growth and immune response. Mosaic diseases are recognized as the most important plant diseases in the world, and mosaic symptoms are recovery tissues formed by plants against virus infection. However, the mechanism of the formation of mosaic symptoms remains elusive. In this study, two typical mosaic systems consisting of Nicotiana tabacum-cucumber mosaic virus (CMV) and N. tabacum-tobacco mosaic virus (TMV) were used to investigate the relevance of miRNAs to the appearance of mosaic symptoms. The results of miRNA-seq showed that there were significant differences in miRNA abundance between dark green tissues and chlorotic tissues in mosaic leaves caused by the infection of CMV or TMV. Compared with healthy tissues, miRNA expression was significantly increased in chlorotic tissues, but slightly increased in dark green tissues. Three miRNAs, namely miR1919, miR390a, and miR6157, were identified to be strongly up-regulated in chlorotic tissues of both mosaic systems. Results of overexpressing or silencing of the three miRNAs proved that they were related to chlorophyll synthesis, auxin response, and small GTPase-mediated immunity pathway, which were corresponding to the phenotype, physiological parameters and susceptibility of the chlorotic tissues in mosaic leaves. Besides, the newly identified novel-miRNA48, novel-miRNA96 and novel-miRNA103 may also be involved in this formation of mosaic symptoms. Taken together, our results demonstrated that miR1919, miR390a and miR6157 are involved in the formation of mosaic symptoms and plant antiviral responses, providing new insight into the role of miRNAs in the formation of recovery tissue and plant immunity.

RNA-seq approach to analysis of gene expression profiles in dark green islands and light green tissues of Cucumber mosaic virus-infected Nicotiana tabacum.

Dark green islands (DGIs) surrounded by light green tissues (LGTs) are common leaf symptoms of plants that are systemically infected by various viruses that induce leaf mosaic in infected plants. The inoculation of Cucumber mosaic virus (CMV) in Nicotiana tabacum produced a commonly occurring sequence of classic patterns of DGIs and LGTs. Previous studies confirmed that there are significant differences between DGIs and LGTs in terms of physiology, biochemistry and molecular biology, but the mechanisms by which DGIs form remain unclear. To investigate the global gene expression changes that occur in these special tissues, individual differential gene expression tag libraries were constructed from three total RNA samples isolated from DGIs, LGTs and control plants (CK) and were sequenced using an Illumina HiSeqTM 2000. An analysis of differentially expressed genes (DEGs) and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. These analyses revealed the differences between DGIs, LGTs and CK. GO enrichment and KEGG pathway analyses suggested that several pathways related to photosynthesis and chlorophyll metabolism were enriched in DGIs compared to LGTs and CK. Several pathways related to apoptosis were significantly up-regulated in LGTs compared to DGIs. Additionally, we identified sets of DEGs that may be related to the formation or development of DGIs and LGTs. Our systematic analyses provide comprehensive transcriptomic information regarding DGIs and LGTs in CMV-infected N. tabacum. These data will help characterize the detailed mechanisms of DGI and LGT formation.

In vivo noninvasive detection of chlorophyll distribution in cucumber ( Cucumis sativus) leaves by indices based on hyperspectral imaging

The objective of this study was to investigate the spectral behavior of the relationship between reflectance and chlorophyll content and to develop a technique for non-destructive chlorophyll estimation and distribution in leaves using hyperspectral imaging. The hyperspectral imaging data cube of cucumber ( Cucumis sativus) leaves in the range of 450–850 nm was investigated and preprocessed. Sixty optical signatures or indices as a function of the associated reflectance ( R λ ) at the special wavelength ( λ) nm which proposed in the literatures were used to predict the total chlorophyll content in cucumber leaves. Finally, R 710/ R 760, ( R 780 − R 710)/( R 780 − R 680), ( R 750 − R 705)/( R 750 + R 705), ( R 680 − R 430)/( R 680 + R 430), R 860/( R 550 × R 708), ( R 695−705) −1 − ( R 750−800) −1, and REP-LEM (a index based on red edge position and estimated with a linear extrapolation method) were identified as optimum indices. Red-edge waveband (680–780 nm) appeared in all these optimum indices, indicating the importance of REP (red edge position) in chlorophyll estimation. When ( R 695−705) −1 − ( R 750−800) −1, the best index was applied to an independent validation set, chlorophyll content ( r = 0.8286) were reasonably well predicted, indicating model robustness. Depending on the sample, this technique enables to identify and characterize the relative content of various chlorophyll that distribution in the cucumber leaves. The map shows a relatively low level of chlorophyll at margins. Higher level can be noticed in the regions along the main veins and in some areas exhibiting dark green tissue. Our results indicate that hyperspectral imaging has considerable promise for predicting pigments in leaves and, the pigments can be detected in situ in living plant samples non-destructively.

Difference of Physiological Characters in Dark Green Islands and Yellow Leaf Tissue of Cucumber mosaic Virus (CMV)-Infected Nicotiana tabacum Leaves

Dark green islands (DGIs) are a common symptom of plants systemically infected with the mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow leaf tissue that is full of virus particles. In Cucumber mosaic virus (CMV)-infected Nicotiana tabacum leaves, the respiration and photosynthesis capabilities of DGIs and yellow leaf tissues were measured. The results showed that the cyanide-resistant respiration was enhanced in yellow leaf tissue and the photosynthesis was declined, while in DGIs they were less affected. The activities of the oxygen-scavenging enzymes catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in infected leaves were significantly higher than those in the healthy leaves, and the enzyme activities in DGIs were always lower than in the yellow leaf tissues. Reactive oxygen species (ROS) staining showed that the hydrogen peroxide content in yellow leaf tissues was apparently higher than that in DGIs, while the superoxide content was on the contrary. Formation of DGIs may be a strategy of the host plants resistance to the CMV infection.

Dark and light green tissues of tobacco leaves systemically infected with tobacco mosaic virus

There are significant changes in the structure of the upper tobacco (Nicotiana tabacum L.) leaves systemically infected with tobacco mosaic virus (TMV) especially in the light green tissue (LGT). Dark green areas (DGI) had intermediate status between healthy tissue and LGT. DGI contained significantly less infectious TMV and viral antigen than the LGT. The DGI, LGT and healthy tissues did not differ in the permeability of cell membranes and in the set of acidic pathogenesis-related (PR) proteins but the total content of PR-proteins in the healthy plants was higher than in the infected ones with the DGI being intermediate between healthy tissue and LGT. The crude leaf extracts from DGI and LGT showed less total ribonuclease activity and ribonuclease isozymes in comparison with control.

Antiviral RNA Silencing Is Restricted to the Marginal Region of the Dark Green Tissue in the Mosaic Leaves of Tomato Mosaic Virus -Infected Tobacco Plants

Mosaic is a common disease symptom caused by virus infection in plants. Mosaic leaves of Tomato mosaic virus (ToMV)-infected tobacco plants consist of yellow-green and dark green tissues that contain large and small numbers of virions, respectively. Although the involvement of RNA silencing in mosaic development has been suggested, its role in the process that results in an uneven distribution of the virus is unknown. Here, we investigated whether and where ToMV-directed RNA silencing was established in tobacco mosaic leaves. When transgenic tobaccos defective in RNA silencing were infected with ToMV, little or no dark green tissue appeared, implying the involvement of RNA silencing in mosaic development. ToMV-related small interfering RNAs were rarely detected in the dark green areas of the first mosaic leaves, and their interior portions were susceptible to infection. Thus, ToMV-directed RNA silencing was not effective there. By visualizing the cells where ToMV-directed RNA silencing was active, it was found that the effective silencing occurs only in the marginal regions of the dark green tissue ( approximately 0.5 mm in width) and along the major veins. Further, the cells in the margins were resistant against recombinant potato virus X carrying a ToMV-derived sequence. These findings demonstrate that RNA silencing against ToMV is established in the cells located at the margins of the dark green areas, restricting the expansion of yellow-green areas, and consequently defines the mosaic pattern. The mechanism of mosaic symptom development is discussed in relation to the systemic spread of the virus and RNA silencing.

A New Technique to Select Mild Strains of Cucumber mosaic virus.

An efficient technique to select a good attenuated virus to control Cucumber mosaic virus (CMV) disease was developed. Preliminary screenings were conducted to assess the virulence of virus recovered from dark-green islands and yellow tissues of mosaic leaves of Nicotiana rustica after co-inoculation with an attenuated mutant P2bR46C of CMV and its original severe isolate Pepo. All single-lesion isolates (SLIs) obtained from dark-green islands had the attenuated P2bR46C phenotype, but the SLIs from yellow tissue had either the virulent Pepo or the P2bR46C phenotype. When Pepo-infected N. rustica and tomato plants were grown at 15 or 36°C for 30 days, 17 of 288 SLIs obtained from the treated leaves elicited mosaic and dark-green spots without malformation. Dark-green tissue from each plant infected with 1 of these 17 SLIs then was used to inoculate one plant of N. rustica. All 17 plants had either very mild mosaic or no visible symptoms. One of these potential mild strains, 36R37, had an amino acid substitution on the 2b gene encoding the 2b protein. Isolate 36R37 also was highly cross-protective, and its symptom attenuation was stable for three serial host passages. After cold or heat treatment, the dark-green tissue proved to be a good source for isolating mild strains of the virus.

Pattern of N gene-mediated systemic hypersensitive response and turnover of viral replicase protein in tobacco

We examined the pattern of the N gene-mediated systemic hypersensitive response (HR), which was induced by tobacco mosaic virus upon temperature shift, and analyzed the distribution of the coat protein and the virus-encoded 126 kDa replicase protein (126 K protein) by immunoblot analysis. In the middle- and lower-positioned leaves, HR occurred in the advancing edge of the infected area, where we detected both the coat protein and the 126 K protein. In the areas between the main vein and the advancing edge of these leaves, we observed no HR and did not detect 126 K protein, though virus was present in these areas. In the upper-positioned mosaic leaves, patterns of the HR were different depending on the leaf age. In these mosaic leaves, the mechanism preventing the virus from invading dark green tissue seemed to be broken down in 8-14 days old leaves, and HR was observed only in the tissue just invaded by the virus, where we detected the 126 K protein. Those results suggested that the viral 126 K protein was present when the viral replication was taking place, and easily degraded when the amount of the virus was saturated in the cells.

Ficus rubiginosa 'variegata', a chlorophyll-deficient chimera with mosaic patterns created by cell divisions from the outer meristematic layer.

Sections leaves of Ficus rubiginosa 'Variegata' show that it is a chimera with a chlorophyll deficiency in the second layer of the leaf meristem (GWG structure). Like other Ficus species, it has a multiseriate epidermis on the adaxial and abaxial sides of the leaf, formed by periclinal cell divisions as well as anticlinal divisions. The upper and lower laminae of the leaf often exhibit small dark and light green patches of tissue overlying internal leaf tissue. The distribution of chlorophyll in transverse sections of typical leaves was determined by fluorescence microscopy. Patches of dark and light green tissue which arise in the otherwise colourless palisade and spongy mesophyll tissue in the entire leaf are due to further cell divisions arising from the bundle sheath which is associated with major vascular bundles or from the green multiseriate epidermis. Leaves produced in winter exhibit more patches of green tissue than leaves which expand in mid-summer. Many leaves produced in summer have no spotting and appear like a typical GWG chimera. There is a strong relationship between the number of patches on the adaxial side of leaves and the number on the abaxial side, showing that the cell division in upper and lower layers of leaves is strongly coordinated. In both winter and summer, there are fewer patches on the abaxial side of leaves compared with the adaxial side, indicating that periclinal and anticlinal cell divisions from the outer meristematic layer are less frequent in the lower layers of leaf tissue. Most of the patches are small (<1 mm in longest dimension) and thus the cell divisions which form them occur late in leaf development. Leaves which exhibit large patches generally have them on both sides of the leaves. In this cultivar, the outer meristematic layer appears to form vascular bundle sheaths and associated internal leaf tissue in the entire leaf lamina.

Zinc, copper and chlorophyll-derivatives in the polychaete Owenia fusiformis

An earlier study of metals in the polychaete Owenia fusiformis showed that individuals within intertidal populations were remarkably variable in terms of whole-body concentrations of zinc and copper. Four populations have now been studied, two in south Cornwall (Fal Estuary and Par Sands) and one each in east Devon (Torre Abbey Sands) and north Brittany (Grève de St Michel). Investigations of the distributions of zinc and copper within the body have demonstrated that consistently zinc is concentrated in the middle body whilst copper increases to a maximum posteriorly. Zinc is accumulated in the mid-gut cells in the form of numerous spherules, 1–2 μm in diameter: X-ray microanalysis shows these to be largely composed of zinc phosphate but containing also magnesium, calcium and iron, together with sulphur and chlorine. Viewed under the microscope the spherules are greenish due to a pigment identified by high performance liquid chromatography absorption scanning as a pheophorbide-like chlorophyll-degradation product. In contrast, copper is widely distributed throughout the body and much is deposited as small granules, 0.2–0.3 μm in diameter, in laminar groupings in hypodermal and peri-intestinal tissues. The identity of the green pigment responsible for the characteristic body colour of O. fusiformis has not been specifically determined but it appears to have a copper basis since dark-green tissues have a significantly higher copper content than those with a light-green colour. The possible role of chlorophyll degradation products in zinc and copper uptake is discussed.

The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves.

The defective chloroplasts and leaves-mutable (dcl-m) mutation of tomato was identified in a Ds mutagenesis screen. This unstable mutation affects both chloroplast development and palisade cell morphogenesis in leaves. Mutant plants are clonally variegated as a result of somatic excision of Ds and have albino leaves with green sectors. Leaf midribs and stems are light green with sectors of dark green tissue but fruit and petals are wild-type in appearance. Within dark green sectors of dcl-m leaves, palisade cells are normal, whereas in albino areas of dcl-m leaves, palisade cells do not expand to become their characteristic columnar shape. The development of chloroplasts from proplastids in albino areas is apparently blocked at an early stage. DCL was cloned using Ds as a tag and encodes a novel protein of approximately 25 kDa, containing a chloroplast transit peptide and an acidic alpha-helical region. DCL protein was imported into chloroplasts in vitro and processed to a mature form. Because of the ubiquitous expression of DCL and the proplastid-like appearance of dcl-affected plastids, the DCL protein may regulate a basic and universal function of the plastid. The novel dcl-m phenotype suggests that chloroplast development is required for correct palisade cell morphogenesis during leaf development.

Unusual Accumulations of Cauliflower Mosaic Virus in Local Lesions, Dark Green Leaf Tissue, and Roots of Infected Plants

Unusual Accumulations of Cauliflower Mosaic Virus in Local Lesions, Dark Green Leaf Tissue, and Roots of Infected Plants

Biological diversity of cauliflower mosaic virus isolates expressed in two Brassica species

Infection characteristics and symptom pathology of 39 isolates of cauliflower mosaic virus (CaMV), obtained worldwide, were compared following infection of a susceptible host (Brassica‐rapa‐rapifera‐turnip) and a tolerant host (Brassica oleracea‐gongylodes‐kohlrabi). Considerable variation in symptom expression between isolates was observed in turnip plants. Local lesions appeared between 4 and &gt;10 days post inoculation (dpi) and systemic symptoms appeared from 10 to 20 dpi depending upon the CaMV isolate. Leaf coloration effects due to systemic infection ranged from generation of dark green tissue through various patterns of vein clearing and vein banding to severe chlorosis. Plant distortions in turnip varied from mild to severe stunting, and various types of leaf wrinkling and rugosity. In contrast, symptoms caused by the same CaMV isolates in kohlrabi were restricted to older leaves and were much milder, showing vein clearing, some chlorosis, but no plant distortions. Kohlrabi plants infected by all isolates tested eventually showed recovery and became asymptomatic. One symptom character in turnip, veinlet necrosis, was dependent upon time of inoculation rather than infection by a particular isolate. Virus titre was estimated by dot‐blot analysis of CaMV DNA. The greatest difference in viral DNA amounts was observed between turnip and kohlrabi plants for all isolates. A significant decline in viral DNA occurred in kohlrabi, concomitant with plant recovery from infection. Variations in virus titre observed early in infection (10 dpi) tended to become more uniform with time. We observed no clear correlation between virus titre and symptom type and severity in turnip, although plants infected with viral isolates producing mild symptoms generally, but not always, contained less viral DNA than isolates causing more severe symptoms. The great variety of CaMV biotypes we have characterized offer considerable scope for investigating mechanisms of plant viral pathogenesis.

Mosaic disease induced by turnip yellow mosaic Tymovirus

Mosaic disease is one of the most common consequences of infection of plants by viruses. Most such mosaics include islands of dark green tissue, which are normal in appearance, contain little or no virus and are resistant to superinfection at least for a period of weeks. The mosaic pattern that develops in successive expanded leaves of larger plants is variable and unpredictable. Therefore it has not been possible to investigate the early events in leaf primordia that lead to the formation of dark green tissue. Here we describe an experimental system which should allow the application of molecular techniques to the study of the nature of dark green tissue.

Synthesis and accumulation of benzoic acid in Pinus radiata needles in response to tissue injury by dothistromin, and correlation with resistance of P. radiata families to Dothistroma pini

Within 5 days of the introduction of dothistromin into Pinus radiata needles, red band symptoms typical of those produced by Dothistroma pini appeared. When injected into needles From different full-sib families, a continuous distribution of necrotic lesion length was obtained ranging from short ( 10 mm). The response was dependent on the age of tissue and, possibly, the season. The maximum response was obtained with currently emerging (harvested in early summer), and the minimum response with 1-year-old needles (harvested in winter), Injected dothistromin was metabolized by needle cells, or photolytically degraded, to a low but persistent level (e.g. 10–20% of the initial dose) within 24 h, concomitant with the formation of a small (approx. 2–5 mm) necrotic lesion. The detectable products of dothistromin metabolism were CO 2 and oxalic acid. Based on the formation of the metabolites, a mechanism involving peroxidase catalysed oxidation of dothistromin by hydrogen peroxide is proposed. Cells adjacent to those killed by dothistromin themselves die and the initial lesion rapidly expands over 2–3 days. These adjacent cells have been shown to synthesize and accumulate benzoic acid, which was toxic to P. radiata needle mesophyll cells and highly fungistatic to D. pini, in vitro . Yellow, necrotic needle tissue adjacent to the red band lesions, containing mycelium, of natural D. pini infections also accumulated benzoic acid, which prevents immediate colonisation of that tissue by the fungus. Benzoic acid is therefore proposed as a phytoalexin of P. radiata needles. The length of the dothistromin-induced lesion was proportional to the amount of benzoic acid isolated from the dead tissue, and a linear dose-response relationship was obtained for benzoic acid injected into P. radiata needles. The dothistromin-induced lesions were usually terminated by narrow (0·1–0·5 mm) areas of dark green tissue. The cells in this tissue were highly lignified, the lignin deposited being disproportionately high in 4-hydroxyphenyl residues compared with the guaiacyl residue content. The lignin polymer also contained bound benzoic acid. The magnitude of the response of needles to dothistromin has been correlated with observed field resistance to D. pini among 7-year-old control-cross progeny comprising 50 full-sib P. radiata families. This correlation supports the hypothesis that a rapid response of P. radiata needles to dothistromin accompanied by accumulation of the phytoalexin, benzoic acid, is a mechanism of resistance of P. radiata to D. pini .

Tobacco-mosaic-virus-induced increase in abscisic-acid concentration in tobacco leaves:

The concentrations of free and bound abscisic acid (ABA and the presumed ABA glucose ester) increased three- to fourfold in leaves of White Burley tobacco (Nicotiana tabacum L.) systemically infected with tobacco mosaic virus. Infected leaves developed a distinct mosaic of light-green and dark-green areas. The largest increases in both free and bound ABA occurred in dark-green areas. In contrast, virus accumulated to a much higher concentration in light-green tissue. Free ABA in healthy leaves was contained predominantly within the chloroplasts while the majority of bound ABA was present in non-chloroplastic fractions. Chloroplasts from light-green or dark-green tissues were able to increase stromal pH on illumination by an amount similar to chloroplasts from healthy leaf. It is unlikely therefore that any virus-induced diminution of pH gradient is responsible for increased ABA accumulation. Tobacco mosaic virus infection had little effect on free ABA concentration in chloroplasts; the virus-induced increase in free ABA occurred predominantly out-side the chloroplast. The proportional distribution of bound ABA in the cell was not changed by infection. Treatment of healthy plants with ABA or water stress increased chlorophyll concentration by an amount similar to that induced by infection in dark-green areas of leaf. A role for increased ABA concentration in the development of mosaic symptoms is suggested.

The Association of “Pathogenesis‐related” Proteins with Viral Induced Necrosis in Nicotiana sylvestris

AbstractThe 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.

Variations in the Protein Content and Tobacco Mosaic Virus Contamination of an Endomembrane Fraction from Leaves of Nicotiana tabacum L. “Samsun” after Infection with Tobacco Mosaic Virus

The protein content of a fraction of smooth cytoplasmic (= SC) membranes, which was isolated from the resuspended 1,000 to 20,000 g pellet of primarily TMV-infected leaves of Nicotiana tabacum L. “Samsun” and harvested at 27% saccharose of a 10 to 50% saccharose gradient, dropped below that of the initial concentration within few days after inoculation with TMV. Thereafter it increased rapidly, with a maximum reached 8 d post infection. In contrast, the content of membrane-bound active virus increased continuously. The time course of this in-vivo-binding of TMV was different in 4 subfractions of SC membranes isolated from the resuspended pellets after centrifugation at 0 to 1,000 g, 1,000 to 7,000 g, 7,000 to 13,000 g, and 13,000 to 19,000 g. In subfraction no. 2, the contamination with TMV was greatest at 5 d after infection. However, 10 d after infection the TMV content of subfraction no. 1 exceeded that of all other subfractions. The protein content of SC membranes of leaves being secondarily infected with TMV also increased considerably, but only in light-green and not in dark-green tissue zones. Compared with the SC membranes from healthy leaves, we observed a strong binding of BSA to membranes from light-green and a weaker binding to membranes from dark-green tissue.

The presence of viral antigen in the apoplast of systemically virus-infected plants

Abstract In healthy and tobacco mosaic virus (TMV)-infected tissues infiltrated with TMV, there are quantitative and qualitative differences in virus cell wall attachment, and in the retention of virus or its coat protein. An 125 I-labeled antibody technique was combined with cell wall digestion to determine antigen content of Nicotiana silvestris cell walls from light green (LG) and dark green (DG) areas of infected leaf tissues. The values (expressed in μg of bound 125 I-labeled TMV-U 1 -IgG/mg cell wall) ranged between 1 and 1.5 for light green (LG) and from 0.2 to 0.5 for dark green (DG) tissues of systemically infected leaves. Autoradiography with 125 I-labeled anti-TMV-U 1 -Fab fragments of IgG showed that Fab fragments can penetrate cell walls of tobacco leaf tissue, since viral antigen was shown to be present in cell walls of cells sustaining virus multiplication by this method.

The specific involvement of coat protein in tobacco mosaic virus cross protection

Nicotiana sylvestris infected by strains of tobacco mosaic virus (TMV) causing mosaic can be superinfected in the dark green leaf tissue, but not light green tissue, by necrotizing strains of TMV. The dark green tissue, however, is much less susceptible than healthy tissue, to some extent, even to unrelated viruses. The RNA of necrotizing strains of TMV was relatively more infectious than intact virus on mosaic than on healthy leaves and caused lesions in both light and dark green tissues. The same relationship was found in Nicotiana longiflora and, when the protecting strain in N. sylvestris could be used as a challenge, in Capsicum baccatum. The efficiency of superinfection by RNA was not found with viruses unrelated to TMV. When bentonite at 1 mg/ml, which is known to strip protein from TMV, was included in the inoculum of intact TMV it superinfected in the same manner as RNA. RNA of a necrotizing strain of TMV, encapsidated in brome mosaic virus protein and used as a challenge, superinfected in the same manner as RNA. When encapsidated in common TMV protein, however, it behaved as native virus. Cross protection apparently results from the prevention of uncoating of related challenge virus in light green tissue of N. sylvestris. Locally inoculated N. sylvestris leaves were insusceptible to challenge RNA or intact virus when the protecting virus was increasing. After increase ceased, RNA was more infectious than intact virus.