Soumitra Ghoshroy

Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell‐to‐cell movement protein

Cell‐to‐cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata, is mediated by a specialized viral movement protein (MP). In vivo studies using transgenic tobacco plants showed that MP is phosphorylated at its C‐terminus at amino acid residues Ser258, Thr261 and Ser265. When MP phosphorylation was mimicked by negatively charged amino acid substitutions, MP lost its ability to gate plasmodesmata. This effect on MP–plasmodesmata interactions was specific because other activities of MP, such as RNA binding and interaction with pectin methylesterases, were not affected. Furthermore, TMV encoding the MP mutant mimicking phosphorylation was unable to spread from cell to cell in inoculated tobacco plants. The regulatory effect of MP phosphorylation on plasmodesmal permeability was host dependent, occurring in tobacco but not in a more promiscuous Nicotiana benthamiana host. Thus, phosphorylation may represent a regulatory mechanism for controlling the TMV MP–plasmodesmata interactions in a host‐dependent fashion.

Uptake, distribution and toxicity of gold nanoparticles in tobacco (Nicotiana xanthi) seedlings

Abstract Understanding plant interactions with nanoparticles is of increasing importance for assessing their toxicity and trophic transport. The primary objective of this study was to assess uptake, biodistribution and toxicity associated with exposure of tobacco plants (Nicotiana xanthi) to gold nanoparticles (AuNPs). We employed synchrotron-based X-ray microanalysis with X-ray absorption near-edge microspectroscopy and high resolution electron microscopy to localize AuNPs within plants. Results from these experiments reveal that AuNPs entered plants through the roots and moved into the vasculature. Aggregate bodies were also detected within root cell cytoplasm. Furthermore, AuNP uptake was size selective as 3.5 nm AuNP spheres were detected in plants but 18 nm AuNPs remained agglomerated on the root outer surfaces. Finally, leaf necrosis was observed after 14 days of exposure to 3.5 nm AuNPs. Overall, results of this work show the potential for AuNPs to enter plants through size-dependent mechanisms, translocate to cells and tissues and cause biotoxicity.

Identification of an Arabidopsis thaliana mutation (vsm1) that restricts systemic movement of tobamoviruses

Following inoculation, many plant viruses spread locally from cell to cell until they reach the vascular system, through which they then move to other parts of the plant, resulting in systemic infection. To isolate host genes involved in systemic transport of plant viruses, ethyl methanesulfonate-mutagenized Arabidopsis thaliana plants were screened for significant delays in the systemic movement of turnip vein clearing virus (TCVC). One such mutant, designated vsm1 (virus systemic movement), was identified. Unlike the wild-type plants, vsm1 did not develop viral disease and did not allow the systemic spread of the virus. The local viral movement within the inoculated vsm1 leaves, however, was not affected. TVCV systemic movement within the vsm1 plants was likely blocked at the step of viral entry into the host plant vasculature from the infected leaf tissue. vsm1 plants also restricted the systemic movement of another tobamovirus but not of an unrelated carmovirus.

Movement and subcellular localization of a tobamovirus in Arabidopsis

Tobamoviruses represent a well-characterized system used to examine viral infection, whereas Arabidopsis is a choice plant for most genetic experiments. It would be useful to combine both approaches into one experimental system for virus-plant interaction. Most tobamoviruses, however, are not pathogenic in Arabidopsis. Here, we describe infection of Arabidopsis by a recently discovered crucifer-infecting turnip vein clearing tobamovirus (TVCV). Using this system, we determined patterns and kinetics of viral local and systemic movement within Arabidopsis plants. Localization studies showed that the virus infects both vegetative and reproductive plant tissues. However, there may be a transport barrier between the seed coat and the embryo which virions cannot cross, preventing seed transmission of TVCV. The ability to move both locally and systemically in Arabidopsis, causing mild and fast-developing symptoms but allowing survival and fertility of the infected plants, distinguish TVCV infection of Arabidopsis as a model system to study virus-plant interaction.

Controlled assembly of rodlike viruses with polymers.

A practical method to assemble rodlike tobacco mosaic virus and bateriophage M13 with polymers was developed, which afforded a 3D core-shell composite with morphological control.

Electrospinning fabrication, structural and mechanical characterization of rod-like virus-based composite nanofibers

Tobacco mosaic virus (TMV) was electrospun with polyvinyl alcohol (PVA) into continuous TMV–PVA composite nanofibers to form a biodegradable nonwoven fibrous mat as an extracellular matrix (ECM) mimetic. Morphological characterizations by electron microscopy showed that the addition of varying amounts of TMV resulted in homogeneous nanofibers without phase separation and did not change the diameter of the composite nanofibers. The orientation of TMV in as-spun fibers could be readily controlled and post-processing of the nonwoven TMV–PVA mat significantly improved its water resistance. In addition, tensile tests were performed on individual nanofibers, which revealed that the TMV–PVA composite nanofibers achieved a comparable Youngs modulus as PVA nanofibers. Since the modification of TMV is readily achieved via genetic or chemical methods, this process offers a facile way to incorporate a variety of functionalities into polymer nanofibers. As a demonstration of its potential as ECM mimetic, a mutant TMV containing RGD peptide was co-spun with PVA and the resulting fibrous substrates were used to promote cell growth.

Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.

Adsorption kinetics of Escherichia coli and Staphylococcus aureus on single-walled carbon nanotube aggregates

Batch adsorption studies to determine adsorption kinetics of Escherichia coli (E.coli) K12 and Staphylococcus aureus (S.aureus) SH 1000 bacterial cells on single-walled carbon nanotube aggregates were performed at two different initial concentrations. The diffusivity of E. coli cells in single-walled carbon nanotube aggregates obtained was 6.54 x 10(-9) and 8.98 x 10(-9) cm(2)/s, whereas that of S. aureus was between 1.00 x 10(-7) and 1.66 x 10(-7) cm(2)/s respectively. In addition to batch adsorption studies, electron microscopy studies were also conducted. The results suggest that diffusion kinetics of bacterial cells is concentration dependent as well as bacteria dependent. Diffusivity of S. aureus is two orders of magnitude greater than E. coli cells. This proves to be beneficial from an adsorption perspective where it is desired to filter microorganisms (water pretreatment and wastewater post treatment) and from nanotube biosensor perspective where it is desired to simultaneously capture and detect biothreat agents in a shorter span of time.

Single-Walled Carbon Nanotubes as Fluorescence Biosensors for Pathogen Recognition in Water Systems

The possibility of using single-walled carbon nanotubes (SWCNTs) aggregates as fluorescence sensors for pathogen recognition in drinking water treatment applications has been studied. Batch adsorption study is conducted to adsorb large concentrations of Staphylococcus aureus aureus SH 1000 and Escherichia coli pKV-11 on single-walled carbon nanotubes. Subsequently the immobilized bacteria are detected with confocal microscopy by coating the nanotubes with fluorescence emitting antibodies. The Freundlich adsorption equilibrium constant (k) for S.aureus and E.coli determined from batch adsorption study was found to be 9×108 and 2×108 ml/g, respectively. The visualization of bacterial cells adsorbed on fluorescently modified carbon nanotubes is also clearly seen. The results indicate that hydrophobic single-walled carbon nanotubes have excellent bacterial adsorption capacity and fluorescent detection capability. This is an important advancement in designing fluorescence biosensors for pathogen recognition in water systems.

Preservation of plant cell ultrastructure during immunolocalization of virus particles

Most immunoelectron microscopy techniques used for ultrastructural analyses of virus-infected plant tissues significantly compromise cellular membranous structures as well as overall contrast and resolution of the image. Here, we describe a protocol which avoids these flaws but retains full antigenicity of the sample. A direct comparison of the conventional and the improved electron immunostaining procedures is presented using tobacco and Arabidopsis thaliana plants infected with turnip vein clearing virus.