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Grafting is a common horticultural practice used to confer desirable traits between rootstock and scion, including disease resistance. To investigate graft-conferred resistance against viral diseases a novel heterografting system was developed using Nicotiana benthamiana scions grafted onto different tomato rootstocks. N. benthamiana is normally highly susceptible to tobacco mosaic virus (TMV) infection. However, specific tomato rootstock varieties were found to confer a range of resistance levels to N. benthamiana scions inoculated with TMV. Conferred resistance was associated with delays in virus accumulation and the reduction in virus spread. RNA sequencing analysis showed the enrichment of transcripts associated with disease resistance and plant stress in N. benthamiana scions grafted onto resistance-inducing tomato rootstocks. Genome sequencing of resistance- and nonresistance-conferring rootstocks was used to identify mobile tomato transcripts within N. benthamiana scions. Within resistance-induced N. benthamiana scions, enriched mobile tomato transcripts were predominantly associated with defence, stress, and abscisic acid signalling when compared to similar scions grafted onto nonresistance-inducing rootstock. Combining these findings suggests that graft-induced resistance is modulated by rootstock scion transcriptional responses and rootstock-specific mobile transcripts., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The self-assembly system of the rod-shaped tobacco mosaic virus (TMV) has been studied extensively for nanoscale applications. TMV coat protein assembly is modulated by intersubunit carboxylate groups whose electrostatic repulsion limits the assembly of virus rods without incorporating genomic RNA. To engineer assembly control into this system, we reprogrammed intersubunit carboxylate interactions to produce self-assembling coat proteins in the absence of RNA and in response to unique pH and ionic environmental conditions. Specifically, engineering a charge attraction at the intersubunit E50-D77 carboxylate group through a D77K substitution stabilized the coat proteins assembly into virus-like rods. In contrast, the reciprocal E50K modification alone did not confer virus-like rod assembly. However, a combination of R46G/E50K/E97G substitutions enabled virus-like rod assembly. Interestingly, the D77K substitution displays a unique pH-dependent assembly-disassembly profile, while the R46G/E50K/E97G substitutions confer a novel salt concentration dependency for assembly control. In addition, these unique environmentally controlled coat proteins allow for the directed assembly and disassembly of chimeric virus-like rods both in solution and on substrate-attached seed rods. Combined, these findings provide a controllable means to assemble functionally discrete virus-like rods for use in nanotechnology applications.

Bioelectronic microdevices, with spatially arranged biosynthetic machinery, can be programmed to convert raw materials to high-value products in a controlled manner. Generic methods for biofunctionalization that enable precise control over biocomponent assembly at the nano and meso scales are necessary to diversify the range and capabilities of these systems. Here, we used tobacco mosaic virus (TMV) derived virus like particles (VLPs) as 3D interfacial scaffolds for the assembly of biosynthetic enzymes onto gold electrodes. The TMV capsids are aligned in a vertical brush configuration by cysteine modifications to the capsid protein and by taking advantage of the well-known gold/cysteine affinity. This alignment enables high surface density and biosynthetic enzyme-enzyme proximity. Enzymes are covalently tethered to the capsid protein of TMV by the N- and C-terminal addition of lysine-rich assembly domains which react with surface exposed glutamine residues on the capsid surfaces; the lysine/glutamine linkages are mediated by a microbial transglutaminase (mTG). We demonstrate flexible mTG-mediated assembly of a three-enzyme biosynthetic pathway that converts S-adenosylmethionine (SAM) to autoinducer-2 (AI-2), a bacterial signal molecule that mediates quorum sensing behavior. We propose that our VLP and mTG based fabrication approach will help in the modular assembly of biological components onto microelectronic devices and that these will find utility in many applications including sensing and lab on chip devices.

Viruses are unique biological agents that infect living host cells through molecular delivery of a genomic cargo. Over the past two decades advancements in genetic engineering and bioconjugation technologies have allowed the unprecedented use of these "unfriendly" biological molecules, as nanoscopic platforms for the advancement of an array of nanotechnology applications. This mini-review focuses on providing a brief summary of key demonstrations leveraging the versatile characteristics of Tobacco mosaic virus (TMV) for molecular assembly and bio-device integration. A comprehensive discussion of genetic and chemical modification strategies along with potential limiting factors that impact the assembly of these macromolecules is presented to provide useful insights for adapting TMV as a potentially universal platform toward developing advanced nanomaterials. Additional discussions on biofabrication techniques developed in parallel to enable immobilization, alignment, and patterning of TMV-based functional particles on solid surfaces will highlight technological innovations that can be widely adapted for creating nanoscopic device components using these engineered biomacromolecules. Further exploitation in the design of molecular specificity and assembly mechanisms and the development of highly controllable and scalable TMV-device integration strategies will expand the library of nanoscale engineering tools that can be used for the further development of virus-based nanotechnology platforms., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Virus-like particles (VLPs) are genome-free protein shells assembled from virus coat proteins (CPs). The uniform and nanoscale structure of VLPs combined with their noninfectious nature have made them ideal candidates for the display of functional peptides. While the vast majority of VLPs are derived from spherical viruses, tobacco mosaic virus (TMV) produces a rod-shaped particle with a hollow central channel. However, under physiological conditions the TMV CP forms only disk-shaped macromolecules. Here, we describe the design, construction, purification, and processing of rod-shaped TMV-VLPs using a simple bacterial expression system. The robust nature of this system allows for the display of functional peptides and molecules on the outer surface of this novel VLP.

In this study we use vascular specific promoters and a translating ribosome affinity purification strategy to identify phloem associated translatome responses to infection by tobacco mosaic virus (TMV) in systemic hosts Arabidopsis thaliana ecotype Shahdara and Nicotiana benthamiana. Results demonstrate that in both hosts the number of translatome gene alterations that occurred in response to infection is at least four fold higher in phloem specific translatomes than in non-phloem translatomes. This finding indicates that phloem functions as a key responsive tissue to TMV infection. In addition, host comparisons of translatome alterations reveal both similarities and differences in phloem responses to infection, representing both conserved virus induced phloem alterations involved in promoting infection and virus spread as well as host specific alterations that reflect differences in symptom responses. Combined these results suggest phloem tissues play a disproportion role in the mediation and control of host responses to virus infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This paper reports the implementation of temporal capillary microfluidic patterns and biological nanoscaffolds in autonomous microfabrication of nanostructured symmetric electrochemical supercapacitors. A photoresist layer was first patterned on the substrate, forming a capillary microfluidics layer with two separated interdigitated microchannels. Tobacco mosaic virus (TMV) macromolecules suspended in solution are autonomously delivered into the microfluidics, and form a dense bio-nanoscaffolds layer within an hour. This TMV layer is utilized in the electroless plating and thermal oxidation for creating nanostructured NiO supercapacitor. The galvanostatic charge/discharge cycle showed a 3.6-fold increase in areal capacitance for the nanostructured electrode compared to planar structures. The rapid creation of nanostructure-textured microdevices with only simple photolithography and bionanostructure self-assembly can completely eliminate the needs for sophisticated synthesis or deposition processes. This method will contribute to rapid prototyping of wide range of nano-/micro-devices with enhanced performance.

This paper presents a comprehensive study of the self-assembly dynamics and the biosensing efficacy of Tobacco mosaic virus-like particle (TMV VLP) sensing probes using an impedimetric microsensor platform. TMV VLPs are high surface area macromolecules with nanorod structures constructed from helical arrangements of thousands of identical coat proteins. Genetically modified TMV VLPs express both surface attachment-promoting cysteine residues and FLAG-tag antibody binding peptides on their coat protein outer surfaces, making them selective biosensing probes with self-assembly capability on sensors. The VLP self-assembly dynamics were studied by the continuous monitoring of impedance changes at 100Hz using interdigitated impedimetric microsensors. Electrical impedance spectroscopy revealed VLP saturation on impedance sensor surface with the coverage of 68% in self-assembly process. The VLP-functionalized impedance sensors responded to 12ng/ml to 1.2μg/ml of target anti-FLAG IgG antibodies in the subsequent enzyme-linked immunosorbent assays (ELISA), and yielded 18-35% total impedance increases, respectively. The detection limit of the target antibody is 9.1ng/ml using the VLP-based impedimetric microsensor. These results highlight the significant potential of genetically modified VLPs as selective nanostructured probes for autonomous sensor functionalization and enhanced biosensing., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. In this study, an interaction between the replication protein of tobacco mosaic virus (TMV) and phloem-specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading in an age-dependent manner. Promoter expression studies show that in mature tissues TMV 126/183-kDa-interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CCs). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus. In situ analysis of virus spread shows that the inability to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at moving out of older plant tissues than a noninteracting virus. Similarly, CC expression and overaccumulation of a degradation-resistant Aux/IAA-interacting protein was found to inhibit TMV accumulation and phloem loading selectively in flowering plants. Transcriptional expression studies demonstrate a role for Aux/IAA-interacting proteins in the regulation of salicylic and jasmonic acid host defense responses as well as virus-specific movement factors, including pectin methylesterase, that are involved in regulating plasmodesmata size-exclusion limits and promoting virus cell-to-cell movement. Combined, these findings indicate that TMV directs the reprogramming of auxin-regulated gene expression within the vascular phloem of mature tissues as a means to enhance phloem loading and systemic spread.

This paper presents a selective differential sensing method based on diffusion modulation of the target molecules through suspended Tobacco mosaic virus-like particles modified with binding peptides for TNT sensing in solution.

A modified version of the rod-shaped Tobacco Mosaic Virus (TMV1cys) provides a robust template for the self-assembly and fabrication of high-surface-area materials for numerous applications including batteries and sensors. TMV1cys surface fabrication is facilitated by the addition of a single cysteine residue to the virus coat protein that directs the vertical attachment of the virus particle onto substrate surfaces and enables deposition of functional inorganic layers. Here we describe the production and purification of the virus, its assembly onto suitable surfaces, and coating with metallic nickel and cobalt.

Designed as a high-capacity alloy host for Na-ion chemistry, a forest of Sn nanorods with a unique core-shell structure was synthesized on viral scaffolds, which were genetically engineered to ensure a nearly vertical alignment upon self-assembly onto a metal substrate. The interdigital spaces thus formed between individual rods effectively accommodated the volume expansion and contraction of the alloy upon sodiation/desodiation, while additional carbon-coating engineered over these nanorods further suppressed Sn aggregation during extended electrochemical cycling. Due to the unique nanohierarchy of multiple functional layers, the resultant 3D nanoforest of C/Sn/Ni/TMV1cys, binder-free composite electrode already and evenly assembled on a stainless steel current collector, exhibited supreme capacity utilization and cycling stability toward Na-ion storage and release. An initial capacity of 722 mA·h (g Sn)(-1) along with 405 mA·h (g Sn)(-1) retained after 150 deep cycles demonstrates the longest-cycling nano-Sn anode material for Na-ion batteries reported in the literature to date and marks a significant performance improvement for neat Sn material as alloy host for Na-ion chemistry.

Genetic modifications of the tobacco mosaic virus (TMV) coat proteins allow for an increase in the selective deposition and controlled growth of different metals onto the surface of the virus, making it an ideal biotemplate for metal nanowire formation. In the current process, TMV2Cys is coated sequentially with multiple uniform layers of palladium metal in aqueous solution under very mild conditions. Palladium nanowires of 300 nm in length and 30-40 nm in diameter have been created with this process. Transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS) are used to characterize the thickness and uniformity of the metal surface. The TEM and SAXS results confirm that the final thickness of the palladium nanowires is controllable by varying the number of coating layers or the initial palladium concentration., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Control of the host transcriptome represents a key battleground in the interaction of plants and pathogens. Specifically, plants have evolved complex defense systems that induce profound transcriptional changes in response to pathogen attack while pathogens have evolved mechanisms to subvert or disable these defenses. Several NAC transcription factors such as ATAF2 have been linked to plant defense responses, including those targeting viruses. The replication protein of Tobacco mosaic virus (TMV) has been shown to interact with and target the degradation of ATAF2. These findings suggest that the transcriptional targets of ATAF2 are involved in defense against TMV., Results: To detect potential ATAF2 transcriptional targets, a genomic pull-down assay was utilized to identify ATAF2 promoter binding sequences. Subsequent mobility shift and DNA footprinting assays identified a 30-bp ATAF2 binding sequence. An in vivo GUS reporter system confirmed the function of the identified 30-bp binding sequence as an ATAF2 specific transcriptional activator in planta. Gel filtration studies of purified ATAF2 protein and mutagenesis studies of the 30-bp binding sequence indicate ATAF2 functions as a dimer. Computational analysis of interacting promoter sequences identified a corresponding 25-bp A/T-rich consensus sequence with repeating [GC]AAA motifs. Upon ATAF2 induction real-time qRT-PCR studies confirmed the accumulation of select gene transcripts whose promoters contain this consensus sequence., Conclusion: We report the identification of a cis-regulatory binding sequence for ATAF2. Different from other known NAC protein binding sequences, the A/T-rich ATAF2 binding motif represents a novel binding sequence for NAC family proteins. Combined this information represents a unique tool for the identification of ATAF2 target genes.

An interaction between the Tobacco mosaic virus (TMV) 126kDa replication protein and a host-encoded Rab GDP dissociation inhibitor (GDI2) was identified and investigated for its role in infection. GDI proteins are essential components of vesicle trafficking pathways. TMV infection alters the localization of GDI2 from the cytoplasm to ER-associated complexes. Partial silencing of GDI2 results in significant increases in the number of TMV infection foci observed in inoculated tissues. However, GDI2 silencing does not affect TMV accumulation at the infection site, cell-to-cell movement, or susceptibility of the host to mechanical inoculation. Furthermore, increases in the number of successful infection foci were specific to TMV and correlated with the appearance of vesicle-like rearrangements in the vacuolar membrane. Tissue infiltrations with brefeldin A, an inhibitor of vesicle trafficking, also enhanced host susceptibility to TMV. Combined these findings suggest that the 126kDa-GDI2 interaction alters vesicle trafficking to enhance the establishment of an infection., (Copyright © 2010 Elsevier Inc. All rights reserved.)

A new synthetic strategy enabling highly controlled aqueous-phase palladium crystallization on the tobacco mosaic virus (TMV) is demonstrated without the addition of external reducing agents. This low cost, solution processing method yields continuous and uniform coatings of polycrystalline palladium on TMV, creating highly uniform palladium nanowires of tens of nanometers in thickness and hundreds of nanometers in length. Our approach utilizes a palladium chloride precursor to produce metallic Pd coatings on TMV without the need for an external reducing agent. X-ray photoelectron spectroscopy and in situ transmission electron microscopy were used to confirm the reduction of the surface palladium oxide layer on the palladium metal wires during room temperature hydrogenation. This leads to metallic palladium nanowires with surfaces free of residual organics, making these structures suitable for applications in nanoscale electronics.

A novel three-dimensional Tobacco mosaic virus assembled silicon anode is reported. This electrode combines genetically modified virus templates for the production of high aspect ratio nanofeatured surfaces with electroless deposition to produce an integrated nickel current collector followed by physical vapor deposition of a silicon layer to form a high capacity silicon anode. This composite silicon anode produced high capacities (3300 mAh/g), excellent charge-discharge cycling stability (0.20% loss per cycle at 1C), and consistent rate capabilities (46.4% at 4C) between 0 and 1.5 V. The biological templated nanocomposite electrode architecture displays a nearly 10-fold increase in capacity over currently available graphite anodes with remarkable cycling stability.

Mutations disrupting helicase domain motifs of the Tobacco mosaic virus 126/183-kDa proteins were investigated for their effect on replicase function and assembly. These mutations inhibited virus replication but did not affect 126-kDa induced N gene resistance or RNAi suppression. However, in vivo expressed 126-kDa motif mutants yielded two distinct cytoplasmic phenotypes that correlated with ATPase activity. Specifically, ATPase active 126-kDa proteins produced small cytoplasmic bodies that resembled the ovoid granular-like bodies found early in virus infection while 126-kDa proteins defective in ATPase activity produced large tubule containing cytoplasmic bodies similar to those observed late in infection. Additional studies indicate that the helicase ATPase activity resides predominantly within monomer and dimer helicase forms and that motifs affecting ATPase activity induce alterations in helicase assembly. Combined these findings indicate that helicase ATPase activity modulates the progression of replicase complex assembly and maturation., (Copyright 2010 Elsevier Inc. All rights reserved.)

One major obstacle in the mineralization of metal onto biologically derived templates is the lack of fundamental information pertaining to the relationship between metal ion loading and overall metal deposition onto the biotemplate. This study focuses on Au(III) and Pd(II) biosorption on the genetically-modified model biological template Tobacco mosaic virus (TMV1Cys). Metal ion (Au(III) or Pd(II)) loading on the TMV1Cys template was measured as a function of the equilibrium concentration of Au(III) or Pd(II) ions in solution at several temperatures. In addition, the Pd(II) loading on the TMV-wild (wild-type TMV) and TMV1Cys were compared to estimate the improvement of metal ion loading by genetic modification of the biotemplate. The gold or palladium coatings on the TMV1Cys were prepared using various metal ion loadings. Results show, for a range of metal ion loadings, a positive correlation existed between the concentration of the metal ions and the coating density of the metals deposited on the virus surface., (Copyright 2009 Elsevier Inc. All rights reserved.)

This paper reports on novel methodologies for the patterning and templated synthesis of virus-structured nanomaterials in two- and three-dimensional microfabricated architectures using the Tobacco mosaic virus (TMV). The TMV is a high aspect ratio biological molecule which can be engineered to include amino acids with enhanced binding properties. These modifications facilitate self-assembly of the TMV onto various substrates and enable its use as a template for the synthesis of nanostructured materials. This work focuses on the combination of this bottom-up biologically inspired fabrication method with standard top-down micromachining processes that allow direct integration of the virus-structured materials into batch-fabricated devices. Photolithographic patterning of uncoated as well as nickel-coated TMV nanostructures has been achieved using a lift-off process in both solvent and mild basic solutions and their assembly onto three-dimensional polymer and silicon microstructures is demonstrated. In addition to these patterning techniques, in situ formation of metal oxide TMV coatings in patterned microfabricated environments is shown using atomic layer deposition directly on the nickel-coated viruses. The biofabrication 'process toolbox' presented in this work offers a simple and versatile alternative for the hierarchical patterning and incorporation of biotemplated nanomaterials into micro/nanofabrication schemes.

An interaction between the helicase domain of the Tobacco mosaic virus (TMV) 126-/183-kDa replicase protein(s) and the Arabidopsis thaliana NAC domain transcription factor ATAF2 was identified via yeast two-hybrid and in planta immunoprecipitation assays. ATAF2 is transcriptionally induced in response to TMV infection, and its overexpression significantly reduces virus accumulation. Proteasome inhibition studies suggest that ATAF2 is targeted for degradation during virus infection. The transcriptional activity of known defense-associated marker genes PR1, PR2, and PDF1.2 significantly increase within transgenic plants overexpressing ATAF2. In contrast, these marker genes have reduced transcript levels in ATAF2 knockout or repressor plant lines. Thus, ATAF2 appears to function in the regulation of host basal defense responses. In response to TMV infections, ATAF2 and PR1 display increased transcript accumulations in inoculated tissues but not in systemically infected tissues. ATAF2 and PR1 transcript levels also increase in response to salicylic acid treatment. However, the salicylic acid treatment of systemically infected tissues did not produce a similar increase in either ATAF2 or PR1 transcripts, suggesting that host defense responses are attenuated during systemic virus invasion. Similarly, noninfected ATAF2 knockout or ATAF2 repressor lines display reduced levels of PR1 transcripts when treated with salicylic acid. Taken together, these findings suggest that the replicase-ATAF2 interaction suppresses basal host defenses as a means to promote systemic virus accumulation.

The use of biological molecules as templates for the production of metal nanoparticles and wires is often limited by the stability of the bio-template and its affinity for nucleating metal deposition. In this study, Tobacco mosaic virus (TMV) was used as a model bio-template to investigate the use of silica coatings as a means to both enhance template stability and increase its affinity for metal ions. Results indicate that the unmodified TMV particle can function as a template for the growth of thin (<1 nm) silica layers. However, this thin silica shell did not enhance the stability of the template during metal deposition. To increase silica growth on the TMV template, a pretreatment with aniline was used to produce a uniform silica attractive surface. Aniline pretreated templates yielded significant silica layers of >20 nm in thickness. These silica shells conferred a high degree of stability to the TMV particle and promoted the deposition of various metal nanoparticles through conventional silica mineralization chemistries. This process provides a simple and robust method for the layering of inorganics onto a biological template.

This paper examines the interaction between a block copolymer and a virus. A poly(styrene-b-4-vinylpyridine) block copolymer was loaded with nickel, and cast from a selective solvent mixture to form a cylindrical microstructure (PS/P4VP-Ni). The nickel ions were confined within the P4VP block of the copolymer. The binding of tobacco mosaic virus (TMV) and tobacco necrosis virus on microphase-separated PS/P4VP-Ni was examined. A staining technique was developed to simultaneously visualize virus and block copolymer structure by transmission electron microscopy. Electron microscopy revealed virus particles associated with block copolymer microphase-separated domains, even after extensive washes with Tween. In contrast, virus associated with PS/P4VP block copolymers lacking Ni were readily removed by Tween. The cylinder long axis of the microstructure was oriented using a hot press and a cooled channel die for quenching, resulting in PS/P4VP cylinders that had a strong anisotropic directional preference. When exposed to flowing solutions of TMV, the PS/P4VP-Ni surface exhibited an ability to retain TMV in a partially aligned state, when the direction of flow coincided with the long axis of the PS/P4VP-Ni cylinders. These results suggest that Coulombic interactions provide a robust means for the binding of virus particles to block copolymer surfaces.

The coagulation and colloidal stability of tobacco mosaic virus (TMV) in alcohol-water-LiCl solutions were studied. Without the addition of LiCl salt, the coagulation was promoted by the increase of hydrophobicity of the alcohols that is proportional to their alkyl chain length and concentration. Addition of the LiCl salt reduced the electrostatic repulsion between TMV particles resulting in coagulation in methanol-water and ethanol-water solutions. In water-alcohol-LiCl mixture, the coagulation of TMV was driven by both the hydrophobic interaction of the solution and the screening effect of the salt simultaneously. To understand the particle-particle interaction during the coagulation, the interaction energy was calculated using DLVO theory. Considering the electrostatic repulsive energy, van der Waals attractive energy, and hydrophobic interaction energy, the total energy profiles were obtained. The experiment and model calculation results indicated that the increase of alcohol concentration would increase hydrophobic attraction energy so that the coagulation is promoted. These results provide the fundamental understanding on the coagulation of biomolecular macromolecules.

The replicase protein of Tobacco mosaic virus (TMV) disrupts the localization and stability of interacting auxin/indole acetic acid (Aux/IAA) proteins in Arabidopsis, altering auxin-mediated gene regulation and promoting disease development (M. S. Padmanabhan, S. P. Goregaoker, S. Golem, H. Shiferaw, and J. N. Culver, J. Virol. 79:2549-2558, 2005). In this study, a similar replicase-Aux/IAA interaction affecting disease development was identified in tomato. The ability of the TMV replicase to interact with Aux/IAA proteins from diverse hosts suggests that these interactions contribute to the infection process. To examine the role of this interaction in virus pathogenicity, the replication and spread of a TMV mutant with a reduced ability to interact with specific Aux/IAA proteins were examined. Within young (4- to 6-week-old) leaf tissue, there were no significant differences in the abilities of Aux/IAA-interacting or -noninteracting viruses to replicate and spread. In contrast, in mature (10- to 12-week-old) leaf tissue, the inability to interact with specific Aux/IAA proteins correlated with a significant reduction in virus accumulation. Correspondingly, interacting Aux/IAA levels are significantly higher in older tissue and the overaccumulation of a degradation-resistant Aux/IAA protein reduced virus accumulation in young leaf tissue. Combined, these findings suggest that TMV replicase-Aux/IAA interactions selectively enhance virus pathogenicity in tissues where Aux/IAA proteins accumulate. We speculate that the virus disrupts Aux/IAA functions as a means to reprogram the cellular environment of older cells to one that is more compatible for virus replication and spread.

High area nickel and cobalt surfaces were assembled using modified Tobacco mosaic virus (TMV) templates. Rod-shaped TMV templates (300 x 18 nm) engineered to encode unique cysteine residues were self-assembled onto gold patterned surfaces in a vertically oriented fashion, producing a >10-fold increase in surface area. Electroless deposition of ionic metals onto surface-assembled virus templates produced uniform metal coatings up to 40 nm in thickness. Within a nickel-zinc battery system, the incorporation of virus-assembled electrode surfaces more than doubled the total electrode capacity. When combined, these findings demonstrate that surface-assembled virus templates provide a robust platform for the fabrication of oriented high surface area materials.

Molecular imprinting is a technique that creates synthetic materials containing highly specific receptor sites that have an affinity for a target molecule. When large particles such as viruses are imprinted, special consideration must be taken to ensure the formation of complementary cavities. Factors that influence imprint formation, include uniformity of the precross-linked mixture and release of the virus template after cross-linking. In this study, tobacco mosaic virus (TMV) was used as a model virus. Polymer-virus aggregates formed when poly(allylamine hydrochloride) (PAA) was mixed with TMV at low polymer concentrations (<0.0001% w/v), but such aggregates were prevented at high polymer concentrations (>25% w/v). Various wash protocols were compared for their ability to remove the virus template from the cross-linked molecularly imprinted polymer (MIP), with sodium hydroxide (1 M) exhibiting the best performance. On the basis of these results, optimized MIPs targeted for TMV virus were synthesized, exhibiting a high affinity to TMV (imprinting factor of 2.3) and low affinity to tobacco necrosis virus, the nontarget virus.

The intrinsic ability of biological molecules to self-organize into complex structures has the potential to revolutionize methods for the assembly of nanomaterials and devices. In this work, nucleic acid hybridization was used to simultaneously assemble different Tobacco mosaic virus (TMV) nanotemplates onto a glass substrate patterned with address specific capture DNAs. To accomplish this, TMV-based nanotemplates were programmed with linker DNAs containing sequence specific addresses and hybridized directly to the capture DNAs. This assembly process proved to be a reliable, selective, and controllable means to assemble multiple TMV nanotemplates.

Molecular imprinted Polymers (MIP) targeted for Tobacco mosaic virus (TMV) have been synthesized. Batch equilibrium studies using imprinted and non-imprinted polymer hydrogels in TMV and TNV solutions were conducted to determine virus-binding capacities. TMV-imprinted hydrogels showed increased binding to TMV (8.8 mg TMV/gpolymer) compared to non-imprinted hydrogels (4.2 mg TMV/gpolymer). Furthermore, TMV-imprinted hydrogels exhibited increased binding to TMV compared to TNV, while non-imprinted hydrogels bound similar amounts of TMV or TNV. This research has demonstrated that molecular imprinting of viruses can be used to selectively induce binding of target viruses based on shape differences of their virions.

Previously, we identified a correlation between the interaction of the Tobacco mosaic virus (TMV) 126/183-kDa replicase with the auxin response regulator indole acetic acid (IAA)26/PAP1 and the development of disease symptoms. In this study, the TMV replicase protein is shown to colocalize with IAA26 in the cytoplasm and prevent its accumulation within the nucleus. Furthermore, two additional auxin (Aux)/IAA family members, IAA27 and IAA18, were found to interact with the TMV replicase and displayed alterations in their cellular localization or accumulation that corresponded with their ability to interact with the TMV replicase. In contrast, the localization and accumulation of noninteracting Aux/IAA proteins were unaffected by the presence of the viral replicase. To investigate the effects of the replicase interaction on Aux/IAA function, transgenic plants expressing a proteolysis-resistant IAA26-P108L-green fluorescent protein (GFP) protein were created. Transgenic plants accumulating IAA26-P108L-GFP displayed an abnormal developmental phenotype that included severe stunting and leaf epinasty. However, TMV infection blocked the nuclear localization of IAA26-P108L-GFP and attenuated the developmental phenotype displayed by the transgenic plants. Combined, these findings suggest that TMV-induced disease symptoms can be attributed, in part, to the ability of the viral replicase protein to disrupt the localization and subsequent function of interacting Aux/IAA proteins.

Aggregation of the biotemplates in mineralization processes is a considerable obstacle in preparing well-dispersed bio-inorganic hybrid materials. In this study, aggregation and mineralization of Tobacco mosaic virus (TMV) biotemplates were investigated as a function of the copper precursor (CuCl2) concentration. The mean hydrodynamic radius of TMV in an aqueous CuCl2 solution was determined by dynamic light scattering for the monitoring of the TMV aggregation. At CuCl2 concentration of 0.5 mM or higher, the mean hydrodynamic radius of TMV increased dramatically indicating aggregation of the TMV particles. Numerical calculations on the long-range interaction energy between parallel model TMV particles agreed with the experimental observations for the TMV aggregation. Mineralization of copper precursors on the TMV biotemplates was achieved only at the CuCl2 concentrations that induced considerable aggregation of the biotemplate. From the numerical calculations and experimental results, it was concluded that a dense copper cluster deposition cannot be achieved without aggregation of TMV templates.

Nanoscaled Pt conductors were prepared from genetically engineered Tobacco mosaic virus (TMV) templates through Pt cluster deposition on the outer surface of the TMV. Pt clusters were synthesized and deposited on the engineered TMV with surface-exposed cysteine via the in situ mineralization of hexachloroplatinate anions. This deposition was driven by the specific binding between thiols and the solid metal clusters. In addition, Pt-thiolate adducts are suggested to form on the engineered TMV in aqueous solutions that work as nucleation sites for the formation of the Pt clusters. The specific binding between Pt clusters and the engineered TMV template was investigated using UV/vis spectrophotometry and quartz crystal microbalance (QCM) analysis. The electric conductance of Pt-deposited TMV was greater than that of the uncoated TMV virion particles. This result suggests the application of metal cluster-deposited engineered TMV in future electrical devices such as rapid response sensors.

The patterning of nanoparticles represents a significant obstacle in the assembly of nanoscale materials and devices. In this report, cysteine residues were genetically engineered onto the virion surface of tobacco mosaic virus (TMV), providing attachment sites for fluorescent markers. To pattern these viruses, labeled virions were partially disassembled to expose 5' end RNA sequences and hybridized to virus-specific probe DNA linked to electrodeposited chitosan. Electron microscopy and RNAase treatments confirmed the patterned assembly of the virus templates onto the chitosan surface. These findings demonstrate that TMV nanotemplates can be dimensionally assembled via nucleic acid hybridization.

Virus-infected plants often display developmental abnormalities that include stunting, leaf curling, and the loss of apical dominance. In this study, the helicase domain of the Tobacco mosaic virus (TMV) 126- and/or 183-kDa replicase protein(s) was found to interact with the Arabidopsis Aux/IAA protein PAP1 (also named IAA26), a putative regulator of auxin response genes involved in plant development. To investigate the role of this interaction in the display of symptoms, a TMV mutant defective in the PAP1 interaction was identified. This mutant replicated and moved normally in Arabidopsis but induced attenuated developmental symptoms. Additionally, transgenic plants in which the accumulation of PAP1 mRNA was silenced exhibit symptoms like those of virus-infected plants. In uninfected tissues, ectopically expressed PAP1 accumulated and localized to the nucleus. However, in TMV-infected tissues, PAP1 failed to accumulate to significant levels and did not localize to the nucleus, suggesting that interaction with the TMV replicase protein disrupts PAP1 localization. The consequences of this interaction would affect PAP1's putative function as a transcriptional regulator of auxin response genes. This is supported by gene expression data indicating that approximately 30% of the Arabidopsis genes displaying transcriptional alterations in response to TMV contain multiple auxin response promoter elements. Combined, these data indicate that the TMV replicase protein interferes with the plant's auxin response system to induce specific disease symptoms.

In this study, mRNA profiles generated from cDNA microarrays were used to identify gene expression changes in Arabidopsis thaliana ecotype Shahdara infected with Tobacco mosaic virus (TMV). Shahdara is a susceptible TMV host, permitting rapid accumulations of virus in both inoculated and systemic tissues, accompanied by defined disease symptoms that include stunting, necrosis, and leaf curling. Gene expression profiles were monitored in whole tissues of inoculated leaves at four days postinoculation (dpi) and in systemically infected leaves at 14 dpi. Microarrays contained cDNAs representing between 8,000 and 10,000 Arabidopsis genes. Expression analysis identified 68 genes that displayed significant and consistent changes in expression levels, either up or down, in either TMV inoculated or systemically infected tissues, or both. Identified TMV-responsive genes encode a diverse array of functional proteins that include transcription factors, antioxidants, metabolic enzymes, and transporters. Thus, the TMV infection process has a significant impact on a wide array of cellular processes that likely reflect the biochemical and physiological changes involved in the development of this disease syndrome.

A protein-protein interaction within the helicase domain of the Tobacco mosaic virus (TMV) 126- and 183-kDa replicase proteins was previously implicated in virus replication (S. Goregaoker, D. Lewandowski, and J. Culver, Virology 282:320-328, 2001). To further characterize the interaction, polypeptides covering the interacting portions of the TMV helicase domain were expressed and purified. Biochemical characterizations demonstrated that the helicase domain polypeptides hydrolyzed ATP and bound both single-stranded and duplexed RNA in an ATP-controlled fashion. A TMV helicase polypeptide also was capable of unwinding duplexed RNA, confirming the predicted helicase function of the domain. Biochemically active helicase polypeptides were shown by gel filtration to form high-molecular-weight complexes. Electron microscopy studies revealed the presence of ring-like oligomers that displayed six-sided symmetry. Taken together, these data demonstrate that the TMV helicase domain interacts with itself to produce hexamer-like oligomers. Within the context of the full-length 126- and 183-kDa proteins, these findings suggest that the TMV replicase may form a similar oligomer.

A transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a beta-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein.

The structural proteins of plant viruses have evolved to self-associate into complex macromolecules that are centrally involved in virus biology. In this review, the structural and biophysical properties of the Tobacco mosaic virus (TMV) coat protein (CP) are addressed in relation to its role in host resistance and disease development. TMV CP affects the display of several specific virus and host responses, including cross-protection, systemic virus movement, hypersensitive disease resistance, and symptom development. Studies indicate that the three-dimensional structure of CP is critical to the control of these responses, either directly through specific structural motifs or indirectly via alterations in CP assembly. Thus, both the structure and assembly of the TMV CP function as determinants in the induction of disease and resistance responses.

Induction of reactive oxygen species (ROS) was observed within seconds of the addition of exogenous tobacco mosaic virus (TMV) to the outside of tobacco (Nicotiana tabacum cv Samsun NN, EN, or nn) epidermal cells. Cell death was correlated with ROS production. Infectivity of the TMV virus was not a prerequisite for this elicitation and isolated coat protein (CP) subunits could also elicit the fast oxidative burst. The rapid induction of ROS was prevented by both inhibitors of plant signal transduction and inhibitors of NAD(P)H oxidases, suggesting activation of a multi-step signal transduction pathway. Induction of intracellular ROS by TMV was detected in TMV-resistant and -susceptible tobacco cultivars isogenic for the N allele. The burst was also detected with strains of virus that either elicit (ToMV) or fail to elicit (TMV U1) N' gene-mediated responses. Hence, early ROS generation is independent or upstream of known genetic systems in tobacco that can mediate hypersensitive responses. Analysis of other viruses and TMV CP mutants showed marked differences in their ability to induce ROS showing specificity of the response. Thus, initial TMV-plant cell interactions that lead to early ROS induction occur outside the plasma membrane in an event requiring specific CP epitopes.

The Tobacco mosaic virus (TMV) 126-kDa and read-through 183-kDa replicase-associated proteins have been shown to interact [Watanabe, T., Honda, A., Iwata, A., Ueda, S., Hibi, T., Ishihama, A. (1999). J. Virol. 73, 2633-2640]. To identify and investigate the sequence required for this interaction, five segments covering different portions of the 126/183-kDa open reading frame, including the methyl-transferase, intervening region (IR), helicase-like (HEL), and polymerase domains, were screened via the yeast two-hybrid system against a library of TMV protein segments. Only one specific interaction between the HEL domain clone and a TMV library clone, IRnHEL, encoding the C-terminal half of the IR and the N-terminal portion of the HEL domain was identified. Sequence and deletion analysis revealed that the interacting clones share a region containing the helicase NTP-binding motif and that this region was essential for the interaction. To determine the functional significance of this interaction, mutants of the HEL domain segment that conferred a temperature-sensitive (ts) defect in the yeast interaction were identified and cloned into a recombinant TMV strain. Of the five selected mutants, three (V823I/S824N/V1042M, A877V, V1087I) produced a ts replication phenotype in protoplasts while the other two (A1073V, T884I) abolished TMV replication at both the permissive and the nonpermissive temperatures. An additional mutation, K839S, designed to disrupt the shared NTP-binding motif, nearly abolished the two-hybrid interaction and prevented virus replication, suggesting that NTP-binding and/or the structure of this motif is a contributing factor in the interaction. Taken together, these results provide support for an interaction between TMV replicase-associated proteins that involves specific structural features of the HEL and IR domains., (Copyright 2001 Academic Press.)

To identify host factors that regulate susceptibility to Tobacco mosaic virus (TMV), 14 Arabidopsis thaliana ecotypes were screened for their ability to support TMV systemic movement. The susceptibility phenotypes observed included one ecotype that permitted rapid TMV movement accompanied by symptoms, nine ecotypes that allowed a slower intermediate rate of systemic movement without symptoms, and four ecotypes that allowed little or no systemic TMV movement. Molecular comparisons between ecotypes representing the rapid (Shahdara), intermediate (Col-1), and slow (Tsu-1) movement phenotypes revealed a positive correlation between the ability of TMV to move cell to cell and its speed of systemic movement. Additionally, protoplasts prepared from all three ecotypes supported similar levels of TMV replication, indicating that viral replication did not account for differences in systemic movement. Furthermore, induction of the pathogenesis-related genes PR-1 and PR-5 occurred only in the highly susceptible ecotype Shahdara, demonstrating that reduced local and systemic movement in Col-1 and Tsu-1 was not due to the activation of known host defense responses. Genetic analysis of F2 progeny derived from crosses made between Shahdara and Tsu-1 or Col-1 and Tsu-1 showed the faster cell-to-cell movement phenotypes of Shahdara and Col-1 segregated as single dominant genes. In addition, the Shahdara symptom phenotype segregated independently as a single recessive gene. Taken together, these findings suggest that, within Arabidopsis ecotypes, at least two genes modulate susceptibility to TMV.

Specific sequences of the tobacco mosaic virus (TMV) RNA-dependent RNA-polymerase (RdRp) gene were investigated for their ability to confer cross-protection. Nine overlapping segments ranging from 713 to 1070 nucleotides in length and covering the methyltransferase, helicase, and polymerase (POL) domains of the TMV RdRp open reading frame were systemically expressed in Nicotiana benthamiana using a potato X virus (PVX) vector [Chapman, S., Kavanagh, T., and Baulcombe, D. C. (1992). Plant J., 1, 549-557]. PVX-infected plants were subsequently challenge inoculated with 10 microg of wild-type TMV and monitored for TMV accumulation. Mock inoculated plants and plants preinfected with the unmodified PVX vector rapidly accumulated high levels of challenge virus. In contrast, plants preinfected with PVX vectors expressing segments of the TMV RdRp open reading frame displayed either high or low levels of protection. High protection levels were observed for PVX constructs expressing segments of the TMV POL domain, whereas low protection levels were observed for PVX constructs expressing segments covering the methyltransferase and helicase domains. Frameshift mutations that blocked protein expression from RdRp segments disrupted only the high levels of protection derived from POL segments and not the low levels derived from the other segments. However, all RdRp segments conferred similarly high levels of protection against a TMV construct with restricted local movement. Thus both RNA and protein sequences in conjunction with the speed of the infecting challenge virus can affect the protection derived from the TMV RdRp gene., (Copyright 2000 Academic Press.)

To investigate the molecular role of the tobacco mosaic tobamovirus (TMV) coat protein (CP) in conferring cross-protection, a potato X potexvirus (PVX) vector (S. Chapman, Plant J. 2, 549-557, 1992) was used to systemically express a set of TMV mutant CPs in Nicotiana benthamiana prior to challenge inoculation with TMV. PVX-expressed wild-type TMV CP delayed TMV accumulation for up to 2 weeks compared to unprotected plants or plants preinfected with the unmodified PVX vector. Similar delays in TMV accumulation were obtained using TMV CPs that were deficient in virion formation but competent to assemble into helical aggregates. In contrast, TMV CPs that were incapable of helical aggregation or unable to bind viral RNA did not delay the accumulation of TMV. Furthermore, TMV CPs with enhanced intersubunit interactions that favor helical aggregation produced significantly greater delays in the accumulation of challenge TMV than obtained from the wild-type CP. Thus the capabilities of TMV CP to interact with viral RNA and self-associate in a helical fashion appear to be essential to its ability to confer protection. Taken together, these findings support a model for CP-mediated resistance in which the protecting CP recoats the challenge virus RNA as it disassembles., (Copyright 1998 Academic Press.)

Tobacco mosaic tobamovirus (TMV) coat protein (CP) mutant E50Q lacks a repulsive intersubunit carboxylate group and can effectively inhibit the disassembly of wild-type TMV (Culver et al, 1995, Virology 206,724). To investigate the ability of this mutant CP to block disassembly, a series of second-site amino acid substitutions were added to the E50Q CP. These second-site mutations were designed to disrupt specific intersubunit stabilizing interactions involving hydrophobic or polar residues, salt bridges, and CP-RNA contacts. Results showed substitutions disrupting intersubunit interactions that face the disassembling surface of the virion dramatically reduced the ability of CP E50Q to inhibit TMV disassembly. Substitutions that disrupted the CP inner loop, RNA binding capabilities, or intersubunit interactions that faced away from the disassembling surface did not dramatically interfere with CP E50Q's ability to inhibit disassembly. Taken together, these findings suggest that intersubunit interactions made by 5' terminal E50Q subunits, not associated with RNA, provide the stabilizing forces that prevent virion disassembly. The role of these stabilizing interactions in TMV disassembly and their potential use for creating disassembly inhibiting CPs are discussed.

Structural studies of tobacco mosaic tobamovirus (TMV) have identified two coat protein (CP) intersubunit carboxyl-carboxylate interactions and one CP carboxylate-RNA phosphate interaction whose electrostatic repulsion is believed to drive virion disassembly. In this study, the involvement of each interaction in the disassembly process was examined. Site-directed mutagenesis was used to replace selected negatively charged CP residues, E or D, with neutral residues, Q or N, respectively. Purified mutant CPs were assayed for their ability to inhibit wild-type TMV disassembly both in vitro and in vivo. Results indicate that the lateral carboxylate interaction made by residue E106 is much more complex than previously thought, involving three residues, E95, E97, and D109, from an adjacent subunit. Mutations at all three residues are required to inhibit disassembly significantly. Different mutant coat proteins inhibited disassembly of the wild-type virus to varying degrees. Mutant E50Q, which modified the axial intersubunit interaction, had the greatest ability to inhibit disassembly followed by mutants E95Q/E97Q/D109N and D116N, which modified the lateral and CP-RNA interactions, respectively. Within each set of interacting carboxylate groups, mutations in the face opposite the disassembling surface of the TMV virion conferred the greatest ability to inhibit disassembly. This observation is consistent with the polar nature of TMV disassembly and confirms that repulsive intersubunit interactions derived from the 5' terminal subunits provide the key controlling mechanisms for virion disassembly.

Electrostatic repulsion between carboxylate groups across subunit interfaces has for many years been recognized as important in the disassembly of simple plant viruses. In the coat protein of tobacco mosaic virus (TMV), the amino acids Glu50 and Asp77 have been proposed as examples of such carboxylate groups. Site-directed mutagenesis has been used to replace these amino acids by Gln and Asn, respectively. Increased virion stability, together with reduced infectivity and reduced capacity for long-distance transport within the host plant confirms that the negative charges on the side chains of these amino acids are involved in the disassembly of TMV. Mixing purified mutant coat proteins with wild-type virions under appropriate conditions stabilizes the virions to alkaline disassembly and reduces their infectivity. It is suggested that transgenic plants expressing such mutant coat proteins could have enhanced resistance to virus infection.

Alterations in the structure of the tobacco mosaic virus (TMV) coat protein affect the elicitation of the N' gene hypersensitive response (HR) in Nicotiana sylvestris. To investigate this structure-function relationship, amino acid substitutions with predicted structural effects were created throughout the known structure of the TMV coat protein. Substitutions that resulted in the elicitation of the HR resided within and would predictably interfere with interface regions located between adjacent subunits in ordered aggregates of coat protein. Substitutions that did not result in the elicitation of the HR were either conservative or located outside these interface regions. In vitro analysis of coat protein aggregates demonstrated HR-eliciting coat proteins to have reduced aggregate stability in comparison with non-HR-eliciting coat proteins and a correlation existed between the strength of the elicited HR and the ability of a substitution to interfere with ordered aggregate formation. This finding corresponded with the predicted structural effects of HR-eliciting substitutions. Radical substitutions that predictably disrupted coat protein tertiary structure were found to prevent HR elicitation. These findings demonstrate that structural alterations that affect the stability of coat protein quaternary structure but not tertiary structure lead to host cell recognition and HR elicitation. A model for HR elicitation is proposed, in which disassembly of coat protein aggregates exposes a host "receptor" binding site.

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.

Specific nucleotide changes in the coat protein gene of tobacco mosaic virus (TMV) have been identified as responsible for the induction of the hypersensitive reaction (HR) in Nicotiana sylvestris. Each of these nucleotide changes resulted in amino acid substitutions in the coat protein. To determine if the altered viral RNA or the altered protein acted directly to elicit the HR, the coat protein translational starts were removed from full-length cDNA clones of the HR-inducing mutant TMV 25 and the systemically infecting TMV U1 strain. Infectious transcripts of these altered genomes failed to induce HR in inoculated leaves of N. sylvestris. These free-RNA mutants moved poorly out of inoculated leaves and produced a systemic mosaic symptom 9 to 12 weeks after inoculation. Infectious viral RNA, from both mutants, was recovered from inoculated and systemic mosaic leaves. Western blot analysis of both inoculated and noninoculated leaves revealed the presence of TMV-encoded 126-kDa protein and the absence of coat protein for both mutants. This study demonstrates that the coat protein of TMV 25 is an elicitor molecule responsible for the induction of HR in N. sylvestris and that the TMV coat protein is not required for the development of systemic mosaic symptoms.