Xanthi NN Research Articles

Analysis of the Spatial Dispersion of Tomato Brown Rugose Fruit Virus on Surfaces in a Commercial Tomato Production Site

The tomato brown rugose fruit virus (ToBRFV) causes severe damage to tomato cultivars and has international economic importance. The harmful tobamovirus is easily mechanically transmissible and highly stable. An ongoing cultivation of infected tomato plants may lead to the spread of ToBRFV in and around the production area of the infested tomato farm. We conducted a study in which we collected a representative number of swab samples from various inanimate surfaces in greenhouses, packaging halls, and shared and private accommodations. In addition, numerous fabrics, such as outer clothing, bed linen, and items used by greenhouse workers, were tested. The infectivity of ToBRFV-contaminated surfaces was tested in bioassays using Nicotiana tabacum cv. Xanthi NN and confirmed using DAS-ELISA. The proportion of ToBRFV-contaminated surfaces varied among locations, from 48.7% in greenhouses to 0% in offices with limited access to staff. Samples from shared accommodation and private accommodation were 18.4% and 3.6% ToBRFV positive, respectively. Clothing and protective items were found to be highly contaminated with ToBRFV, and even around the sleeping area, infective ToBRFV was detected in a few apartments. This study provides evidence for the first time on how and where infectious ToBRFV can be spread by humans beyond the production area. To avoid further dissemination, strict hygiene protocols are required to interrupt transmission routes.

Decontamination of Tomato Brown Rugose Fruit Virus-Contaminated Shoe Soles under Practical Conditions

Due to its global spread, easy mechanical transmissibility inside greenhouses, and detrimental effects on marketability, Tomato brown rugose fruit virus (ToBRFV) is considered to be one of the biggest threats to tomato production. Regarding such crop epidemics, it is essential to identify all conceivable transmission routes and to interrupt them with effective decontamination strategies. We analyzed the potential efficacy of reliable shoe sole cleaning in combination with a disinfection measure. For this purpose, first, a suspension test was undertaken that involved applying different disinfectants to an infected plant homogenate. This was followed by a simulated carrier test. Finally, shoe-sole decontamination was tested under practical conditions. The extent of decontamination was determined by bioassays of the infectivity of the initial load remaining after treatment. Thereby, necrotic local lesions on the susceptible indicator plant Nicotiana tabacum cv. Xanthi NN were counted. Recommendations for practical applications, based on suspension or simulated carrier tests, are limited in their applicability, since very short contact times between ToBRFV and disinfectants reduce efficacy. Under practical conditions, the approved disinfectant MENNO Florades was able to achieve complete inactivation of the virus in the disinfection mat following mechanical depletion from the shoe soles.

Cleaning of Tomato brown rugose fruit virus (ToBRFV) from Contaminated Clothing of Greenhouse Employees

The highly infectious Tomato brown rugose fruit virus (ToBRFV) is a new viral threat to tomato production worldwide. In production, the very easy mechanical transmissibility combined with the high resistance in vitro is of great concern. We tested: (i) whether household cleaning products, commercial agricultural detergents, and an authorized plant protectant are suitable for cleaning contaminated clothing, and (ii) whether infectious viruses remain in the resulting cleaning water. The evaluation of the sanitation effect was performed using bioassays, by counting ToBRFV-associated necrotic local lesions on Nicotiana tabacum cv. Xanthi NN. For this purpose, leaves were mechanically inoculated with treated fabrics and cleaning solutions which would normally be discharged to the sewer system. The detergents Fadex H+ (FH) and Menno Hortisept Clean Plus, as well as the disinfectant Menno Florades (MF), led to an almost complete removal of ToBRFV from contaminated fabrics, corresponding to a reduction in local lesions by 99.94–99.96%. In contrast, common household cleaning products (Spee ActivGel (SAG), Vanish Oxi Action Gel (VO) did not effectively remove the pathogen from the fabric, where the reduction was 45.1% and 89.7%, respectively. In particular, cleaning solutions after the use of household cleaners were highly contaminated with ToBRFV. After a 16-h treatment with the disinfectant MF, infectious ToBRFV was no longer present in VO, FH, and MF cleaning solutions, as demonstrated by extensive bioassays.

Tomato mottle mosaic virus: Characterization, resistance gene effectiveness, and quintuplex RT-PCR detection system

Tomato mottle mosaic virus (ToMMV), an economically important species of the genus Tobamovirus, causes significant loss in yield and quality of tomato fruits. Here, we identified the Shandong isolate of ToMMV (ToMMV-SD) collected from symptomatic tomato fruits in Weifang, Shandong Province of China. ToMMV-SD caused symptoms such as severe mosaic, mottling, and necrosis of tomato leaves, yellow spot and necrotic lesions on tomato fruits. The obtained full genome of ToMMV-SD was 6 399 nucleotides (accession number MW373515) and had the highest identity of 99.5% with that of isolate SC13-051 from the United States of America at the genomic level. The infectious clone of ToMMV-SD was constructed and induced clear mosaic and necrotic symptoms onto Nicotiana benthamiana leaves. Several commercial tomato cultivars, harboring Tm-22 resistance gene, and pepper cultivars, containing L resistance gene, were susceptible to ToMMV-SD. Plants of Solanum melongena (eggplant) and Brassica pekinensis (napa cabbage) showed mottling symptoms, while N. tabacum cv. Zhongyan 100 displayed latent infection. ToMMV-SD did not infect plants of N. tabacum cv. Xanthi NN, Brassica rapa ssp. chinensis (bok choy), Raphanus sativus (radish), Vigna unguiculata cv. Yuanzhong 28-2 (cowpea), or Tm-22 transgenic N. benthamiana. A quintuplex RT-PCR system differentiated ToMMV from tomato mosaic virus, tomato brown rugose fruit virus, tobacco mosaic virus, and tomato spotted wilt virus, with the threshold amount of 0.02 pg. These results highlight the threat posed by ToMMV to tomato and pepper cultivation and offer an efficient detection system for the simultaneous detection of four tobamoviruses and tomato spotted wilt virus infecting tomato plants in the field.

Glutathione Can Compensate for Salicylic Acid Deficiency in Tobacco to Maintain Resistance to Tobacco Mosaic Virus.

Earlier studies showed that the artificial elevation of endogenous glutathione (GSH) contents can markedly increase the resistance of plants against different viruses. On the other hand, salicylic acid (SA)-deficient NahG plants display enhanced susceptibility to viral infections. In the present study, the biochemical mechanisms underlying GSH-induced resistance were investigated in various tobacco biotypes displaying markedly different GSH and SA levels. The endogenous GSH levels of Nicotiana tabacum cv. Xanthi NN and N. tabacum cv. Xanthi NN NahG tobacco leaves were increased by infiltration of exogenous GSH or its synthetic precursor R-2-oxo-4-thiazolidine-carboxylic acid (OTC). Alternatively, we also used tobacco lines containing high GSH levels due to transgenes encoding critical enzymes for cysteine and GSH biosynthesis. We crossed Xanthi NN and NahG tobaccos with the GSH overproducer transgenic tobacco lines in order to obtain F1 progenies with increased levels of GSH and decreased levels of SA. We demonstrated that in SA-deficient NahG tobacco the elevation of in planta GSH and GSSG levels either by exogenous GSH or by crossing with glutathione overproducing plants confers enhanced resistance to Tobacco mosaic virus (TMV) manifested as both reduced symptoms (i.e. suppression of hypersensitive-type localized necrosis) and lower virus titers. The beneficial effects of elevated GSH on TMV resistance was markedly stronger in NahG than in Xanthi NN leaves. Infiltration of exogenous GSH and OTC or crossing with GSH overproducer tobacco lines resulted in a substantial rise of bound SA and to a lesser extent of free SA levels in tobacco, especially following TMV infection. Significant increases in expression of pathogenesis related (NtPR-1a, and NtPRB-1b), and glutathione S-transferase (NtGSTtau, and NtGSTphi) genes were evident in TMV-inoculated leaves in later stages of pathogenesis. However, the highest levels of defense gene expression were associated with SA-deficiency, rather than enhanced TMV resistance. In summary, elevated levels of glutathione in TMV-infected tobacco can compensate for SA deficiency to maintain virus resistance. Our results suggest that glutathione-induced redox changes are important components of antiviral signaling in tobacco.

Isolation and molecular characterization of an ethylene response factor NtERF1-1 in Nicotiana tabacum cv. Xanthi.

Apetala2/Ethylene Response Factors (AP2/ERF) play important roles in regulating gene expression under abiotic and biotic stress in the plant kingdom. Here, we isolated a member of the AP2/ERF transcription factors, NtERF1-1, from Nicotiana tabcum cv. Xanthi NN carrying the N gene, which is resistant to Tobacco mosaic virus (TMV). NtERF1-1 encoded a putative protein of 229 amino acids with a predicted molecular mass of 24.58 kDa. Nucleotide sequence analysis showed that NtERF1-1 contained a conserved DNA-binding domain at the N-terminal. Comparison of amino acid sequences revealed that NtERF1-1 possessed high similarities to ERFs from diverse plants. Semi-quantitative and real-time quantitative RT-PCR analyses indicated that NtERF1-1 was up-regulated following TMV infection. In addition, we speculated that NtERF1-1 might participate in the signal transduction pathway of defence response inducted by the interaction between the N gene and TMV.

Arabis mosaic virus in Grapevines in New York State.

In a limited survey of commercial vineyards and a germplasm repository in Ontario County, NY, 20 vines of Vitis sp. were tested in fall and spring 2010 to 2012 for viruses using a double-antibody sandwich (DAS)-ELISA and macroarray with oligonucleotide probes for grapevine viruses ((3) and unpublished). The plants selected for analysis included those showing atypical growth including leaf deformation, yellowing, cupping or spotting, vein clearing, shortening of internodes, and reduced vigor. Arabis mosaic virus (ArMV; genus Nepovirus, family Secoviridae) was detected in leaf tissue and wood scrapings in two vines using the DAS-ELISA with antibodies from Bioreba (Reinach, Switzerland). The ArMV positive vines were from Vitis hybrid cultivars Noah and Geisenheim 26. ArMV was also detected in these two vines using the macroarray, with hybridization observed to 24 of 32 oligonucleotide probes specific to this virus. To confirm the identification of the virus, total RNAs were extracted from leaf tissues, hybridized with random hexamers, and reverse-transcribed using MMLV reverse transcriptase (Life Technologies, Grand Island, NY). Complementary DNAs were amplified by PCR using an IQ supermix (BioRad, Hercules, CA), and two sets of generic primers for nepoviruses (1,4). Thermocycler conditions were 94°C 5 min (1×); 94°C 30 s, 50°C 30 s, and 69°C 2 min (35×), and 72°C for 5 min. The PCR products were sequenced directly. Sequences from the 340-bp products obtained from cultivars Geisenheim 26 (GenBank Accession No. HE984333) and Noah (HE984334) using the Wei et al. primers (4) had 76 to 84% sequence identity to ArMV RNA1 GenBank accessions GQ369528 and AY303786. Sequences from the 301-bp products obtained from cultivars Geisenheim 26 (HE984335) and Noah (HE984336) using the Digiaro et al. primers (1) had 87 to 91% sequence identity to ArMV RNA2 GenBank accessions AY017339 and X81814. ArMV was mechanically transmitted from Geisenheim 26 to Nicotiana tabacum cultivar Xanthi NN. Inoculation gave rise to necrotic local lesions on the inoculated leaves of five plants in each of two experiments (10 of 10 plants total). The presence of ArMV in tobacco was confirmed by DAS-ELISA. Thus, the presence of ArMV in New York grapevines has been confirmed by the detection of the coat protein antigen, virus specific oligonucleotide probes, and the sequencing of portions of both genomic RNAs. There are limited reports of ArMV in North America and in grapevine in particular (2), but with a wide host range and seed and nematode transmissibility, ArMV has the ability to become more widespread among grapevine and other crops.

Low Co-Cultivation Temperature at 20°C Resulted in the Reproducible Maximum Increase in Both the Fresh Weight Yield and Stable Expression of GUS Activity after <i>Agrobacterium tumefaciens</i>-Mediated Transformation of Tobacco Leaf Disks

The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.

Allopurinol, an inhibitor of purine catabolism, enhances susceptibility of tobacco to Tobacco mosaic virus

Tobacco (cv. Xanthi nn) plants were watered with allopurinol [4-hydroxypyrazolo (3,4-d) pyrimidine, HPP], a xanthine oxidoreductase (XOR) inhibitor, to investigate its effects on infection by Tobacco mosaic virus engineered to express the green fluorescent protein (TMV.GFP). TMV.GFP infection was monitored by examination of inoculated leaves under UV light, by confocal scanning laser microscopy and by epifluorescence microscopy. Susceptibility to TMV.GFP was enhanced in HPP-treated plants. This was seen as a statistically significant increase in numbers of infection sites per leaf and in the number of infected cells per infection site. Two hypotheses are discussed to explain the enhanced susceptibility. The inhibition exerted by HPP against XOR activity could provoke either (i) an increased adenine and guanine nucleotide pool, which could facilitate viral RNA synthesis or (ii) it could cause changes in IAA/auxin levels, which has been proposed to influence TMV susceptibility in tobacco.

Structure–function relationship between the tobamovirus TMV-Cg coat protein and the HR-like response

The tobamovirus TMV-Cg induces an HR-like response in Nicotiana tabacum cv. Xanthi nn sensitive plants lacking the N or N' resistance genes. This response has been characterized by the appearance of necrotic lesions in the inoculated leaf and viral systemic spread, although the defence pathways are activated in the plant. A previous study demonstrated that the coat protein (CP) of TMV-Cg (CPCg) was the elicitor of this HR-like response. We examined the influence of four specific amino acid substitutions on the structure of CPCg, as well as on the development of the host response. To gain insights into the structural implications of these substitutions, a set of molecular dynamic experiments was performed using comparative models of wild-type and mutant CPCg as well as the CP of the U1 strain of TMV (CPU1), which is not recognized by the plants. A P21L mutation produces severe changes in the three-dimensional structure of CPCg and is more unstable when this subunit is laterally associated in silico. This result may explain the observed incapacity of this mutant to assemble virions. Two other CPCg mutations (R46G and S54K) overcome recognition by the plant and do not induce an HR-like response. A double CPCg mutant P21L-S54K recovered its capacity to form virions and to induce an HR-like response. Our results suggest that the structural integrity of the CP proteins is important for triggering the HR-like response.

AtUTr1, a UDP-glucose/UDP-galactose Transporter from Arabidopsis thaliana, Is Located in the Endoplasmic Reticulum and Up-regulated by the Unfolded Protein Response

The folding of glycoproteins in the endoplasmic reticulum (ER) depends on a quality control mechanism mediated by the calnexin/calreticulin cycle. During this process, continuous glucose trimming and UDP-glucose-dependent re-glucosylation of unfolded glycoproteins takes place. To ensure proper folding, increases in misfolded proteins lead to up-regulation of the components involved in quality control through a process known as the unfolded protein response (UPR). Reglucosylation is catalyzed by the ER lumenal located enzyme UDP-glucose glycoprotein glucosyltransferase, but as UDP-glucose is synthesized in the cytosol, a UDP-glucose transporter is required in the calnexin/calreticulin cycle. Even though such a transporter has been hypothesized, no protein playing this role in the ER yet has been identified. Here we provide evidence that AtUTr1, a UDP-galactose/glucose transporter from Arabidopsis thaliana, responds to stimuli that trigger the UPR increasing its expression around 9-fold. The accumulation of AtUTr1 transcript is accompanied by an increase in the level of the AtUTr1 protein. Moreover, subcellular localization studies indicate that AtUTr1 is localized in the ER of plant cells. We reasoned that an impairment in AtUTr1 expression should perturb the calnexin/calreticulin cycle leading to an increase in misfolded protein and triggering the UPR. Toward that end, we analyzed an AtUTr1 insertional mutant and found an up-regulation of the ER chaperones BiP and calnexin, suggesting that these plants may be constitutively activating the UPR. Thus, we propose that in A. thaliana, AtUTr1 is the UDP-glucose transporter involved in quality control in the ER.

Expression of the crucifer-infecting TMV-Cg movement protein in tobacco plants complements in trans a TMV-U1 trafficking-deficient mutant

Tobamovirus movement proteins play a determinant role in the establishment of infections in plants, allowing the local movement of viral RNA genome through plasmodesmatas. We expressed the movement protein (MP) of the crucifer- and garlic-infecting Tobacco Mosaic Virus strain Cg (TMV-Cg) in both resistant Xanthi NN and sensitive Xanthi nn Nicotiana tabacum plants. MP-Cg function was assayed by inoculating transgenic plants with a trafficking-deficient mutant of TMV strain U1. Following infection, local necrotic lesions were developed in resistant transgenic plants, and a systemic infection was produced in sensitive tobaccos. Thus, movement function of the mutant virus was complemented in trans by MP-Cg expressed in transgenic plants, causing the same symptoms as wild-type strain. We demonstrated that the function of MP-U1 could be replaced efficiently by MP-Cg, even though these proteins share only 36% of identity. Similar hydrophobic patterns of MP-Cg and MP-U1 suggests structure and function conservations of both proteins. This work is an example of how two tobamoviruses differing in their host range help to understand viral movement mechanism during the infection.

Indexing system forTomato mosaic virus (ToMV) in commercial tomato seed lots

An indexing system for detectingTomato mosaic virus (ToMV) in commercial tomato seed lots is described. Factors associated with the procedure were analyzed and the following standard two-step working scheme is proposed: (i) mass screening by ELISA for the presence of the virus; (ii) evaluation of virus infectivity within the infested seed lots. A threshold of 10 ng ml−1 was determined for detection of purified ToMV by either ELISA or plant inoculation.Nicotiana tabacum cv. Xanthi NN was found to be a highly sensitive local lesion assay plant for the detection of ToMV. A positive ELISA threshold (1.3 times above the non-specific background) was set for seed samples taken from commercial seed lots by testing the same samples by both ELISA and a bioassay.

Identification and characterization of a novel tobacco mosaic virus resistance N gene homologue in Nicotiana tabacum plants.

Nicotiana tabacum cv. Xanthi nn plants are susceptible to infection by most tobamoviruses (TMV). However, such plants display a partial hypersensitive resistance response (HR-like response) to TMV-Cg. The genetic mechanism of the HR-like response has yet not been determined, but it may involve a gene with a function similar to that of a resistance gene, responsible for HR in resistant plants. We have cloned a gene homologous to the resistance N gene, named NH, from Nicotiana Xanthi nn plants. The coding region of NH is 5.028 base pairs (bp) long and has 82.6% nucleotide identity with the N gene. In contrast to the N gene, the NH gene lacks intron 4 and does not have sites for alternative splicing of intron 3. Analysis of its sequence revealed that NH belongs to the TIR / NSB / LRR gene class. We were able to detect stable levels of NH-transcript in Nicotiana Xanthi nn plants from 0 to 18 h post-inoculation (hpi) with TMV-Cg. Transcript levels increased slightly at 24 hpi and dropped below basal values at 48 hpi. The NH transcript was also detected in a range of resistant Nicotiana plants (N. tabacum Xanthi NN, N. glutinosa, N. glauca and N. rustica) suggesting that NH is a homologue of the N gene, rather than an allele. We have cloned and characterised the NH gene (GenBank acc. no. bankit598573 AY535010) from nn susceptible plants and postulate that this gene might be involved in the HR-like response seen in these plants.

The Commelina yellow mottle virus promoter drives companion-cell-specific gene expression in multiple organs of transgenic tobacco.

Previous work has demonstrated that some endogenous plant gene promoters are active in selective companion cells of the phloem, depending on organ types and developmental stages. Here we report that the Commelina yellow mottle virus (CoYMV) promoter is active in the companion cells of leaves, stems and roots of transgenic Nicotiana tabacum cv. Xanthi NN, using beta-glucuronidase (GUS) as a reporter. Thus, the CoYMV promoter has a broad organ specificity. This promoter can be useful in molecular studies on the functions of companion cells in many aspects of phloem biology, such as regulation of long-distance transport, macromolecular traffic, plant development and interaction with pathogens. It may also be useful in engineering crops that produce specific gene products in the companion cells to block long-distance movement of pathogens.

Antiviral activity of alginate against infection by tobacco mosaic virus

In the course of searching for antiviral substances to tobacco mosaic virus (TMV), it was found that sodium alginate (Alg) had a high inhibitory activity against TMV infection. The addition of Alg to the inoculum solution greatly reduced the number of local lesions formed on Xanthi NN tobacco leaves. The degree of inhibition increased with Alg concentration and was higher in Alg polymer of lower composition of mannuronate to guluronate ratio (M/G ratio) than higher Alg, which suggests that the strength of inhibition relates to the stiffness of polymer chain of Alg. This behavior was similar to chondroitin sulfate (Chs), However, in contrast to Chs, the infectivity of TMV in the case of Alg was enhanced at very low concentrations of Alg. The range of maximum infectivity shifted to the higher Alg concentration with increasing M/G ratios. The degree of inhibition increased with the molecular weight of Alg (M/G ratio at 0.8). The electron micrograph showed that the TMV suspension itself was almost monodisperse and that the addition of Alg caused TMV to form large raft-like aggregates. The antiviral activity of Alg on infectivity of TMV may be caused by blocking the decapsulation process of TMV protein on the cell membrane surface.

Cloning of tobacco genes that elicit the hypersensitive response.

We used a functional screening method to isolate genes whose products elicit the hypersensitive response (HR) pathway of defense against plant pathogens. A cDNA library derived from tobacco leaves undergoing the HR was cloned into a tobacco mosaic virus (TMV)-based expression vector. Infectious transcripts were generated and used to inoculate tobacco plants lacking the N resistance gene (genotype Xanthi nn). Approximately 1/1000 of the infectious transcripts produced local lesions, and may thus elicit the HR. The cDNA inserts from 50 lesion-forming clones were recovered by RT-PCR, and 12 unique clones were sequenced. Comparisons with protein databases revealed homologies to (a) ubiquitin, (b) tobacco tumor-related protein, similar to Kunitz-type trypsin inhibitors and (c) ribosomal protein S14. The remaining nine clones revealed no homology to known proteins and are thus considered novel. Five clones were able to induce the expression of PR2, a gene which is specifically activated in the tobacco HR. Northern and western blot analyses of leaves infected by the clone encoding ubiquitin strongly suggest that the infection produced a co-suppression response; the endogenous level of ubiquitin mRNA and protein in infected leaves are ca. 50% less than those found in healthy leaves. This observation supports a previous report on the involvement of the ubiquitin system in the tobacco HR [2], and validates and utility of the functional cloning method.

Identification of the TMV Replicase Sequence That Activates the N Gene-Mediated Hypersensitive Response

The N gene-mediated hypersensitive response (HR) in tobacco provides a high degree of resistance against most tobamoviruses by halting the progress of infection at the site of inoculation. A previous report indicated a role for the 126/183-kDa replicase in induction of the HR in tobacco containing the N gene (H. S. Padgett and R. N. Beachy, Plant Cell 5:577-586, 1993). Chimeric virus genomes were constructed in which the genes encoding the 126/183-kDa proteins of the HR-eliciting pathogen, tobacco mosaic virus (TMV), and the resistance breaking tobamovirus, Ob, were exchanged. Inoculation of the chimeric viruses to leaves of Nicotiana tabacum cv. Xanthi NN confirmed that either the replicase protein of TMV or its mRNA was responsible for induction of HR. An expression vector based on the Ob virus was used to express fragments of various replicase genes. With this approach, it was determined that the HR is caused by a portion of the replicase protein extending from amino acid 692 to 1116. Consistent with this result, Ob mutants that induce the HR on NN tobacco were found to carry mutations within the same portion of the replicase gene. The N gene-mediated HR is inactive at high temperatures, yet these mutants were able to overcome the HR at significantly lower temperatures than could TMV, indicating that the temperature sensitivity of the N gene response is manifested at the level of interaction between the virus and the defense response mechanism.

Antiviral activity of chondroitin sulfate against infection by tobacco mosaic virus

In the course of searching for antiviral substances against the tobacco mosaic virus (TMV), it was found that chondroitin sulfate (Chs) A-type and C-type had a high inhibitory activity against TMV infection. The addition of Chs to the inoculum solution greatly reduced the number of local lesions formed on Xanthi NN tobacco leaves. The degree of inhibition increased with Chs concentration and was higher in C-type than A-type, which suggests that the strength of inhibition relates to the stiffness of the polymer chain of Chs. For Chs C-type inhibition was independent of molecular weight where the molecular weight was between 30000 and 70000, and decreased with molecular weight below 30000. The electron micrograph showed that the TMV suspension itself was almost monodisperse and that the addition of Chs caused TMV to form large raftlike aggregates. The TMV solution became turbid after the addition of a large amount of Chs. The antiviral activity of Chs on the infectivity of TMV may be caused by blocking the decapsulation process of TMV protein on the cell membrane surface.

Invasion of minor veins of tobacco leaves inoculated with tobacco mosaic virus mutants defective in phloem-dependent movement.

To fully understand vascular transport of plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, U1mCP15-17, and SNC015, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of non-native capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas U1mCP15-17 and SNC015 do not accumulate by phloem-dependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and U1mCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP of wild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.