Inhibition of continuous cropping obstacle of celery by chemically modified biochar: An efficient approach to decrease bioavailability of phenolic allelochemicals.

The accumulation of allelochemicals released by plants is commonly found in continuous monocropping systems. These chemicals, such as phenolic acids, were shown to be the major sources of autotoxin or pathogen accumulation in soils, leading to a direct or indirect continuous cropping obstacle. In this study, three types of agricultural residuals, i.e., rice husk, tea waste, and wood meal, were chosen as feedstocks. Biochar samples were prepared from these feedstocks to examine their abilities to remove gallic acid, a representative phenolic acid. Biochar, which was prepared from wood meal soaked with H3PO4 (1:1.5, w/w) and pyrolyzed at 400 °C (symbolized as WP400), exhibited the highest adsorption capacities of gallic acids and other phenolic acids. The mechanisms of phenolic acid removal by WP400 were evaluated via experimental and spectroscopic investigations to elucidate the notable adsorption capacity of WP400. The adsorption of gallic acids was pH-dependent and followed a pseudo-second-order kinetic model. The combination of high surface area, the existence of O-containing groups, and the enhancement of H bonds between CC groups and phenolic acids may contribute to the high adsorption capacity of WP400. In a pot experiment, we found that celery growth was promoted with the addition of 0.3% (w/w) WP400 to soils that were continuously monocropped with celery. A large decrease in the water-soluble phenolic compound by more than 40% may be responsible for the results. However, WP400 scavenged nitrate, and this study showed that the synergistic actions of WP400 and nutrients exhibited the greatest efficiencies in mitigating the continuous cropping obstacles of celery.

Organic fragments newly released from heat-treated peat soils create synergies with dissolved organic carbon to enhance Cr(VI) removal.

Surface fires occur naturally or anthropogenically and can raise the temperature at the soil surface up to 600 °C. The heat derived from the surface fire can be subsequently transferred into CO2-enriched subsoils. As a result, the chemical compositions of soil organic matter (SOM) may be altered in fire-impacted anaerobic environments, indirectly influencing the redox transformations of pollutants, such as Cr(VI). In this study, a peat soil was heated up to 600 °C with limited air flow to simulate the effects of heat on the SOM during surface fire events. Then, Cr(VI) removal, including reduction and sorption, by the heat-treated peat soils was determined in relation to changes in the soil organic components. The results showed that the amount of O-containing functional groups, -CH2/-CH3 units of aliphatic groups, and dissolved organic carbon (DOC) in the SOM gradually decreased with an increase in the heating temperature. The removal of 0.1932 mM Cr(VI) did not exhibit a consistent decline along with the changes in these soil components. The heating temperatures of 200 and 250 °C were the thresholds that led to the decomposition of temperature-sensitive soil organic components such as lignin and other labile SOM. Such newly released organic fragments synergized lignin-like substances and carboxyl groups, resulting in up to 99% removal of the initially added Cr(VI). As the heating temperatures were increased from 300 to 600 °C, Cr(VI) reduction decreased from 66% to 20%. The black carbon-like materials and/or aromatic-containing moieties were the major components responsible for Cr(VI) reduction in 600°C-treated peat soils.

Tobacco mosaic virus-directed reprogramming of auxin/indole acetic acid protein transcriptional responses enhances virus phloem loading.

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.

High performance liquid chromatography-tandem mass spectrometry method for ex vivo metabolic studies of a rhenium-labeled radiopharmaceutical for liver cancer.

The radio-isotope rhenium-labeled N-[2-(triphenylmethyl)thioethyl]-3-aza-19-ethyloxycarbonyl-3-[2-(triphenylmethyl)thioethyl] octadecanoate) ligand (188Re-MN-16ET) is a novel therapeutic agent under preclinical evaluation for hepatoma. A reversed-phase high performance liquid chromatography coupled with a tandem mass spectrometric analysis method and diode array detector (DAD) involving a T type splitter was developed to characterize this pharmaceutical in rat liver tissue solution and determine its biotransformation rate. The separation was accomplished on a C18 column (chromolith silica, 4.6 mm x 100 mm) using an acetonitrile-ammonium acetate buffer gradient as the mobile phase. The detection was achieved by DAD set at 250nm and tandem mass spectrometry using electrospray ionization in the positive ion mode. Re-MN-16ET displayed a retention time of 23.2 min and a transition ion pair corresponding to m/z677 --> 631 for multiple reaction monitoring. Its biotransformation reaction in rat liver homogenate proceeded for 90 min in a 37°C water bath. The characterization was conducted using aliquots that were extracted and concentrated from the reaction mixture for various incubation times. Re-MN-16ET exhibited a biotransformation half-life (t1/2) of 8-9 min in liver tissue solution and was almost completely exhausted after 90 min. Two of its metabolites, consisting of the Re-labeled carboxylic acid derivative, predominately, and its corresponding demetallized disulfide ligand were found in the liver homogenate, providing a metabolism pathway for the radio-pharmaceutical.

Fabrication of a decellularized liver matrix-based hepatic patch for the repair of CCl4-induced liver injury.

This article primarily introduces a new treatment for liver fibrosis/cirrhosis. We developed a hepatic patch by combining decellularized liver matrix (DLM) with the hepatocyte growth factor (HGF)/heparin-complex and evaluated its restorative efficacy. In vitro prophylactic results, the HGF/heparin-DLM patches effectively mitigated CCl4-induced hepatocyte toxicity and restored the cytotoxicity levels to the baseline levels by day 5. Furthermore, these patches restored albumin synthesis of injured hepatocytes to more than 70% of the normal levels within 5 days. In vitro therapeutic results, the urea synthesis of the injured hepatocytes reached 91% of the normal levels after 10 days of culture, indicating successful restoration of hepatic function by the HGF/heparin-DLM patches in both prophylactic and therapeutic models. In vivo results, HGF/heparin-DLM patches attached to the liver and gut exhibited a significant decrease in collagen content (4.44 times and 2.77 times, respectively) and an increase in glycogen content (1.19 times and 1.12 times, respectively) compared to the fibrosis group after 1 week, separately. In summary, liver function was restored and inflammation was inhibited through the combined effects of DLM and the HGF/heparin-complex in fibrotic liver. The newly designed hepatic patch holds promise for both in vitro and in vivo regeneration therapy and preventive health care for liver tissue engineering.

Increasing DNA damage sensitivity through corylin-mediated inhibition of homologous recombination.

BACKGROUND: DNA repair allows the survival of cancer cells. Therefore, the development of DNA repair inhibitors is a critical need for sensitizing cancers to chemoradiation. Sae2CtIP has specific functions in initiating DNA end resection, as well as coordinating cell cycle checkpoints, and it also greatly interacts with the DDR at different levels. RESULTS: In this study, we demonstrated that corylin, a potential sensitizer, causes deficiencies in DNA repair and DNA damage checkpoints in yeast cells. More specifically, corylin increases DNA damage sensitivity through the Sae2-dependent pathway and impairs the activation of Mec1-Ddc2, Rad53-p and γ-H2A. In breast cancer cells, corylin increases apoptosis and reduces proliferation following Dox treatment by inhibiting CtIP. Xenograft assays showed that treatment with corylin combined with Dox significantly reduced tumor growth in vivo. CONCLUSIONS: Our findings herein delineate the mechanisms of action of corylin in regulating DNA repair and indicate that corylin has potential long-term clinical utility as a DDR inhibitor.

Synergism of Fe and Al salts for the coagulation of dissolved organic matter: Structural developments of Fe/Al-organic matter associations.

Dissolved organic matter (DOM) is distributed ubiquitously in water bodies. Ferric ions can flocculate DOM to form stable coprecipitates; however, Al(III) may alter the structures and stability of Fe-DOM coprecipitates. This study aimed to examine the coprecipitation of Fe, Al, and DOM as well as structural developments of Fe-DOM coprecipitates in relation to changes in Fe/Al ratios and pHs. The results showed that the derived Fe/Al/DOM-coprecipitates could be classified into three categories: (1) at pH 3.0 and 4.5, the corner-sharing FeO6 octahedra associated with Fe-C bonds with Fe/(Fe + Al) ratios ≥0.5; (2) the Fe-C bonds along with single Fe octahedra having Fe/(Fe + Al) ratios of 0.25; (3) at pH 6.0, the ferrihydrite-like Fe domains associated with Fe-C bonds with Fe/(Fe + Al) ratios ≥0.5. At pH 3.0, the Fe and C stability of the coprecipitates increased with increasing Al proportions; nonetheless, pure Al-DOM coprecipitates were unstable even if they exhibited the maximum ability for DOM removal. The associations of Al-DOM complexes and/or DOM-adsorbed Al domains with external surfaces of Fe domain or Fe-DOM coprecipitates may stabilize DOM, leading to lower C solubilization at pH 4.5. Although the preferential formation of Fe/Al hydroxides decreased Fe/Al solubilization at pH 6.0, adsorption instead of coprecipitation of DOM with Fe/Al hydroxides may decrease C stabilization in the coprecipitates. Aluminum cations inhibit DOM releases from Fe/Al/DOM-coprecipitates, promoting the treatment and reuse efficiencies of wastewater and resolving water shortages. This study demonstrates that Al and solution pH greatly affect the structural changes of Fe-DOM coprecipitates and indirectly control the dynamics of Fe, Al, and C concentrations in water.

Comparative analysis of the capacity of tombusvirus P22 and P19 proteins to function as avirulence determinants in Nicotiana species.

We have used an agroinfiltration assay for a comparative study of the roles of tombusvirus P22 and P19 proteins in elicitation of hypersensitive response (HR)-like necrosis and the role of P19 in silencing suppression in Nicotiana species. The advantage of agroinfiltration rather than expression in plant virus vectors is that putative viral avirulence proteins can be evaluated in isolation, eliminating the possibility of synergistic effects with other viral proteins. We found that tombusvirus P22 and P19 proteins elicited HR-like necrosis in certain Nicotiana species but, also, that Nicotiana species could recognize subtle differences in sequence between these proteins. Furthermore, Nicotiana species that responded with systemic necrosis to virion inoculations responded to agroinfiltration of tombusvirus P19 with a very weak and delayed necrosis, indicating that the rapid HR-like necrosis was associated with putative resistance genes and a plant defense response that limited the spread of the virus. Tombusvirus P19 proteins also appeared to differ in their effectiveness as silencing suppressors; in our assay, the P19 proteins of Cymbidium ringspot virus and Tomato bushy stunt virus were stronger silencing suppressors than Cucumber necrosis virus P20. Finally, we show that agroinfiltration can be used to track the presence of putative plant resistance genes in Nicotiana species that target either tombusvirus P19 or P22.

Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless.

Endeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19/R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19/R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19/R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N. benthamiana yielded plants that expressed detectable levels of P19/R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19/R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19/R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19/R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing.

Perfusion-based microfluidic device for three-dimensional dynamic primary human hepatocyte cell culture in the absence of biological or synthetic matrices or coagulants.

We describe a perfusion-based microfluidic device for three-dimensional (3D) dynamic primary human hepatocyte cell culture. The microfluidic device was used to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality of primary human hepatocytes by restoring membrane polarity and hepatocyte transport function in vitro without the addition of biological or synthetic matrices or coagulants. A unique feature of our dynamic cell culture device is the creation of a microenvironment, without the addition of biological or synthetic matrices or coagulants, that promotes the 3D organization of hepatocytes into cord-like structures that exhibit functional membrane polarity as evidenced by the expression of gap junctions and the formation of an extended, functionally active, bile canalicular network.

Diverse and newly recognized effects associated with short interfering RNA binding site modifications on the Tomato bushy stunt virus p19 silencing suppressor.

The Tomato bushy stunt virus-encoded P19 forms dimers that bind duplex short interfering RNAs (siRNAs) to suppress RNA silencing. P19 is also involved in multiple host-specific activities, including the elicitation of symptoms, and in local and/or systemic spread. To study the correlation between those various roles and the siRNA binding by P19, predicted siRNA-interacting sites were modified. Twenty-two mutants were generated and inoculated onto Nicotiana benthamiana plants, to reveal that (i) they were all infectious, (ii) symptom differences did not correlate strictly with mutation-associated variation in P19 accumulation, and (iii) substitutions affecting a central domain of P19 generally exhibited symptoms more severe than for mutations affecting peripheral regions. Three mutants selected to represent separate phenotypic categories all displayed a substantially reduced ability to sequester siRNA. Consequently, these three mutants were compromised for systemic virus spread in P19-dependent hosts but had differential plant species-dependent effects on the symptom severity. One mutant in particular caused relatively exacerbated symptoms, exemplified by extensive morphological leaf deformations in N. benthamiana; this was especially remarkable because P19 was undetectable. Another striking feature of this mutant was that only within a few days after infection, viral RNA was cleared by silencing. One more original property was that host RNAs and proteins (notably, the P19-interactive Hin19 protein) were also susceptible to degradation in these infected N. benthamiana plants but not in spinach. In conclusion, even though siRNA binding by P19 is a key functional property, compromised siRNA sequestration can result in novel and diverse host-dependent properties.

Phosphate Removal in Relation to Structural Development of Humic Acid-Iron Coprecipitates.

Precipitation of Fe-hydroxide (FH) critically influences the sequestration of PO4 and organic matter (OM). While coatings of pre-sorbed OM block FH surfaces and decrease the PO4 adsorption capacity, little is known about how OM/Fe coprecipitation influences the PO4 adsorption. We aimed to determine the PO4 adsorption behaviors on humic acid (HA)-Fe coprecipitates in relation to surface and structural characteristics as affected by HA types and C/(C + Fe) ratios using the Fe and P X-ray absorption spectroscopy. With increasing C/(C + Fe) ratios, the indiscernible changes in the proportion of near-surface C for coprecipitates containing HA enriched in polar functional groups implied a relatively homogeneous distribution between C and Fe domains. Wherein PO4 adsorbed on FH dominated the P inventory on coprecipitates, yielding PO4 sorption properties nearly equivalent to that of pure FH. Structural disruptions of FH caused by highly associations with polar functional groups of HA enhanced the C solubilisation. While polar functional groups were limited, coprecipitates consisted of core FH with surface outgrowth of HA. Although surface-attached HA that was vulnerable to solubilisation provided alternatively sites for PO4 via ternary complex formation with Fe bridges, it also blocked FH surfaces, leading to a decrease in PO4 adsorption.

On-chip microdialysis system with flow-through sensing components.

Microdialysis probes have been used for diabetes treatment as continuous monitoring system coupled to a glucose sensor. An on-chip microdialysis system with in-line sensing electrodes is demonstrated. As a first step towards greater biosensor integration with this miniaturized microdialysis system, a stacked system with in-line sensing electrodes was developed. Impedance electrodes sputtered within the microchannels were used to determine fluid electrical resistance from a dialyzed phosphate buffered saline (PBS) solution, which characterizes solution conductivity as a function of PBS concentration. The permeability of the membrane to the salt ions was obtained as 0.246+/-0.028 microm/s (15 nm pores). Subsequently, experiments measuring PBS dialysis in the time-domain at 64.4% recovery were conducted. The PBS concentration of the reservoir was changed in both a step response and sinusoidally with an 800 s period. The subsequently measured impedance indicates that the system is able to continuously track concentration changes in the reservoir with a 210 s system response delay. Most of this delay is due to the dead volume within the tubing between the syringe pumps and the microsystem. In addition, the predicted response was modeled using linear systems theory and matches the experimental measurements (r=0.98). This system is expected to have the proper sensitivity to track physiologically relevant concentration changes of biomolecules such as glucose (which has a physiological maximum change rate of approximately 4 mg/dl min with a periodicity of 1h or greater) with minimal lag time and amplitude reduction.

Prevalence of Nontyphoidal Salmonella and Salmonella Strains with Conjugative Antimicrobial-Resistant Serovars Contaminating Animal Feed in Texas.

The objective of this study was to characterize 365 nontyphoidal Salmonella enterica isolates from animal feed. Among the 365 isolates, 78 serovars were identified. Twenty-four isolates (7.0%) were recovered from three of six medicated feed types. Three of these isolates derived from the medicated feed, Salmonella Newport, Salmonella Typhimurium var. O 5- (Copenhagen), and Salmonella Lexington var. 15+ (Manila), displayed antimicrobial resistance. Susceptibility testing revealed that only 3.0% (12) of the 365 isolates displayed resistance to any of the antimicrobial agents. These 12 isolates were recovered from unmedicated dry beef feed (n = 3), medicated dry beef feed (n = 3), cabbage culls (n = 2), animal protein products (n = 2), dry dairy cattle feed (n = 1), and fish meal (n = 1). Only Salmonella Newport and Salmonella Typhimurium var. O 5- (Copenhagen) were multidrug resistant. Both isolates possessed the IncA/C replicon and the blaCMY-2 gene associated with cephalosporin resistance. Plasmid replicons were amplified from 4 of 12 resistant isolates. Plasmids (40 kb) were Salmonella Montevideo and Salmonella Kentucky. Conjugation experiments were done using 7 of the 12 resistant isolates as donors. Only Salmonella Montevideo, possessing a plasmid and amplifying IncN, produced transconjugants. Transconjugants displayed the same antimicrobial resistance profile as did the donor isolate. Three isolates that amplified replicons corresponding to IncA/C or IncHI2 did not produce transconjugants at 30 or 37°C. The results of this study suggest that the prevalence of antimicrobial-resistant Salmonella contaminating animal feed is low in Texas. However, Salmonella was more prevalent in feed by-products; fish meal had the highest prevalence (84%) followed by animal protein products (48%). Ten of the 35 feed types had no Salmonella contamination. Further investigation is needed to understand the possible role of specific feed types in the dissemination of antimicrobial resistant bacteria.

Components of an integrated microfluidic device for continuous glucose monitoring with responsive insulin delivery.

Miniaturized medical diagnostic and treatment devices are currently being developed. Microneedles and miniaturized microdialysis systems are particularly well suited to impact diabetes treatment for continuous glucose monitoring and feedback-controlled insulin delivery. Microneedles are an attractive advanced drug delivery system used to mechanically penetrate the skin and inject insulin intradermally where it is rapidly absorbed by the capillary bed into the bloodstream. The real advantage of microneedle-enhanced drug delivery lies in the fact that drug is actively injected into a patient so the dosage may be varied with time to allow complex drug delivery profiles. The delivery is independent of the drug composition and merely relies on the subsequent drug absorption into the bloodstream. A miniaturized microdialysis probe for continuous glucose sensing has also been designed. Microdialysis is based upon controlling the mass transfer rate of glucose diffusing across a semipermeable membrane into a dialysis fluid while excluding larger molecules such as proteins. Polymer microdialysis membranes are integrated with microfluidic systems. Because of the high surface area to fluid volume ratio of miniaturized fluid channels, faster recovery of glucose to increase glucose sensing frequency is expected. This work highlights recent advances made in the design and fabrication of microneedles to make them more biocompatible and more fracture resistant in order to effectively enter the biomedical market. In addition, the design of a miniaturized microdialysis system for increased glucose sampling frequency is presented. The sensing and infusion technologies may be combined into a miniaturized "artificial pancreas" for minimally invasive feedback-controlled insulin delivery.

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants.

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing.

A synthetic peptide-acrylate surface for production of insulin-producing cells from human embryonic stem cells.

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable ß-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. Moreover, we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigel-coated surface. Thus, we provided a totally defined condition from hESC culture to insulin-producing cell differentiation, and the derived cells could be a therapeutic resource for diabetic patients in the future.

Differential requirements for Tombusvirus coat protein and P19 in plants following leaf versus root inoculation.

Traditional virus inoculation of plants involves mechanical rubbing of leaves, whereas in nature viruses like Tomato bushy stunt virus (TBSV) are often infected via the roots. A method was adapted to compare leaf versus root inoculation of Nicotiana benthamiana and tomato with transcripts of wild-type TBSV (wtTBSV), a capsid (Tcp) replacement construct expressing GFP (T-GFP), or mutants not expressing the silencing suppressor P19 (TBSVΔp19). In leaves, T-GFP remained restricted to the cells immediately adjacent to the site of inoculation, unless Tcp was expressed in trans from a Potato virus X vector; while T-GFP inoculation of roots gave green fluorescence in upper tissues in the absence of Tcp. Conversely, leaf inoculation with wtTBSV or TBSVΔp19 transcripts initiated systemic infections, while upon root inoculation this only occurred with wtTBSV, not with TBSVΔp19. Evidently the contribution of Tcp or P19 in establishing systemic infections depends on the point-of-entry of TBSV in the plants.

Host impact on the stability of a plant virus gene vector as measured by a new fluorescent local lesion passaging assay.

Viruses can be used as vectors for transient expression of proteins in plants but frequently foreign gene inserts are not maintained stably over time due to recombination events. In this study the hypothesis was that the choice of plant host affects the foreign gene retention level by a Tomato bushy stunt virus (TBSV) vector expressing green fluorescent protein (GFP). To accomplish this, a novel virus vector integrity bioassay was developed based on an old concept, whereby RNA transcripts of the TBSV-GFP vector were rub-inoculated onto leaves of test plants, and at 3 days post inoculation (dpi), these leaves were used as inoculum for passage to cowpea (Vigna unguiculata), a local lesion host. Chlorotic lesions at points of virus infection were counted on cowpea at 4dpi and then the leaves were exposed to ultraviolet light to count green fluorescent foci. These tests with seven different plant species covering five families showed that the percentage of green fluorescent lesions varied on the cowpea indicator plants in a host-dependent manner. For instance, the vector was relatively unstable in Nicotiana benthamiana, tomato, bean, and spinach, but compared to those its stability in lettuce was significantly improved (~3-fold). This host-dependent effect suggests that some plants may present a more suitable environment than others to support or maintain optimum levels of virus vector-mediated foreign gene expression.