Syringe Infiltration Research Articles

A versatile, rapid Agrobacterium-mediated transient expression system for functional genomics studies in cannabis seedling.

We have developed and optimized a rapid, versatile Agrobacterium-mediated transient expression system for cannabis seedlings that can be used in functional genomics studies of both hemp-type and drug-type cannabis. Cannabis (Cannabis sativa L.) holds great promise in the medical and food industries due to its diverse chemical composition, including specialized cannabinoids. However, the study of key genes involved in various biological processes, including secondary metabolite biosynthesis, has been hampered by the lack of efficient in vivo functional analysis methods. Here, we present a novel, short-cycle, high-efficiency transformation method for cannabis seedlings using Agrobacterium tumefaciens. We used the RUBY reporter system to monitor transformation results without the need for chemical treatments or specialized equipment. Four strains of A. tumefaciens (GV3101, EHA105, LBA4404, and AGL1) were evaluated for transformation efficiency, with LBA4404 and AGL1 showing superior performance. The versatility of the system was further demonstrated by successful transformation with GFP and GUS reporter genes. In addition, syringe infiltration was explored as an alternative to vacuum infiltration, offering simplicity and efficiency for high-throughput applications. Our method allows rapid and efficient in vivo transformation of cannabis seedlings, facilitating large-scale protein expression and high-throughput characterization studies.

First report of Pectobacterium brasiliense causing banana soft rot in Ecuador.

Banana (Musa spp.) is the most economically important crop in Ecuador, with exports representing 35% of the agricultural GDP of the country. It covers 230,000 hectares, mostly concentrated in three coastal provinces, Guayas, Los Ríos, and El Oro. Between July and September 2022, disease symptomatic banana cv. Williams plants were observed in commercial plantations located in two parishes in the province of Guayas (Naranjito and Lorenzo de Garaicoa) and one parish in the province of Santo Domingo de los Tsáchilas (La Concordia), with an incidence that ranged from 5% to 15%. Symptoms included soft rot of the pseudostem and rhizome decay, characterized by a fetid odor. Three symptomatic pseudostems from each location were collected, washed with running water to remove any debris, and dried with absorbent paper. From the lesion of each pseudostem, seven pieces of 2 cm² were taken, surface-sterilized, and macerated in 9 ml of sterile peptone water (0.1% w/v). The macerate was diluted three fold in sterile water, plated on nutrient agar, and incubated at 30°C for 24 h. Eight randomly picked colonies, with convex elevation and creamy white color, were isolated on nutrient agar. Each of the bacterial isolates was biochemically profiled by the Biolog system (Biolog Inc., USA) and identified as Pectobacterium. Three isolates, one from each parish (FP220416, FP220694, and FP220904), were selected for testing Koch's postulates and further identification. Sequences from fragments of the 16S, dnaA, gapA and gyrB genes were obtained from these isolates, following the protocols used by Dobhal et al. (2020) and Boluk et al. (2020), showing 98.1-99.0%, 98.2%, 99.7-99.8%, and 98.4-98.9% identitity, respectively, with sequences from the P. brasiliense type strain LMG_21371 (Acc. number JQOE00000000). The obtained sequences were deposited in GenBank with the following accession numbers: OR392417, OR371545,OR371546, OR727281, OR727282, and OR739074-OR739080. Using BEAST v.1.10.4 (Suchard et al.,2018), a bayesian multilocus phylogenetic tree was built with multiple sequence alignments of dnaA, gapA, ang gyrB from 22 P. brasiliense isolates and 2 P. aquaticum isolates used as outgroup. The phylogenetic analysis showed that the Ecuadorian isolates cluster with P. brasiliense BF20, isolated from Opuntia ficus-indica in México and are closely related with the type strain. Pathogenicity tests were conducted through syringe infiltration with 1 ml of 1 × 10^8 CFU ml-1 bacterial suspensions. Each of the three characterized isolates were inoculated into the pseudostems of five healthy 4-month-old banana plants of the Williams cultivar. Negative control plants were infiltrated with sterile distilled water. The plants were incubated at 25°C and 74% relative humidity. Black lesions started to appear 11 days after inoculation and 5 weeks after inoculation plants showed clear symptoms of soft rot of the pseudostem, including fetid odor associated with plant tissue decomposition. Control plants remained symptom-free. Bacteria were re-isolated only from symptomatic pseudostems and identified as P. brasiliense with specific primers Pb1F and Pb1R. Soft rot of banana caused by different enterobacteria including Dickeya zeae, Erwinia carotovora, and Erwinia chrysanthemi hasve been previously reported (Jingxin et al. 2022, Arun et al. 2012, Loganathan, et al. 2019). This is the first report of P. brasiliense causing soft rot of banana in Ecuador, the biggest exporter of the fruit in the world.

Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation.

Transcript stability is an important determinant of its abundance and, consequently, translational output. Transcript destabilisation can be rapid and is well suited for modulating the cellular response. However, it is unclear the extent to which RNA stability is altered under changing environmental conditions in plants. We previously hypothesised that recovery-induced transcript destabilisation facilitated a phenomenon of rapid recovery gene downregulation (RRGD) in Arabidopsis thaliana (Arabidopsis) following light stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis and investigate processes regulating transcript abundance and fate by quantifying changes in transcription, stability and translation before, during and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil-grown plants in combination with stress treatments. Compared with measurements in juvenile plants and cell culture, we find reduced stability across a range of transcripts encoding proteins involved in RNA binding and processing. We also observe light-induced destabilisation of transcripts, followed by their stabilisation during recovery. We propose that this destabilisation facilitates RRGD, possibly in combination with transcriptional shut-off that was confirmed for HSP101, ROF1 and GOLS1. We also show that translation remains highly dynamic over the course of light stress and recovery, with a bias towards transcript-specific increases in ribosome association, independent of changes in total transcript abundance, after 30 min of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription and stability that occurs to coordinate translation during light stress and recovery in Arabidopsis.

First report of bacterial leaf streak in sweet corn caused by Pantoea ananatis in southeast China.

Sweet corn (Zea mays convar. saccharata var. rugosa) is a popular vegetable crop in southeast China. During the spring seasons of 2018-2021, a serious outbreak of bacterial leaf streak was observed in sweet corn variety Yuetian28 in the field in Guangzhou, Guangdong Province. The disease incidence was 50%-70%. Infected leaves initially displayed long, chlorotic streaks parallel to veins at the V5-V6 stage, and then turned white or brown and dried out over the course of disease development. In severe infections, leaf lesion coalesced to form large irregular blight areas (Fig. S1A). To investigate this disease, we collected 0.5 cm2 samples of infected leaves from four plants after surface sterilization and rinsed them three times with sterile distilled water. We placed all leaf samples on nutrient agar (NA) medium and incubated them at 28℃ for 48 hours. Bright-yellowish colonies were observed near the edges of the samples. We picked the colonies and re-streaked them onto NA medium three times to obtain pure cultures. Four isolates, GZ2201, GZ2202, GZ2203, and GZ2204, were selected for further study. All isolates were gram-negative rods and were negative for oxidase, urease, nitrate reductase reactions, and gelatin liquefaction. They were positive for catalase, citrate utilization, indole production, and the Voges-Proskauer test. We sequenced the 16S rDNA, rpoB, leuS, and gyrB sequences using previously reported primers (Brady et al. 2008) and deposited the sequences in GenBank (accession nos. ON740665 to ON740668 for 16S rDNA; ON755167 to ON755170 for rpoB; ON755171 to ON755174 for leuS; and OP227136 to OP227139 for gyrB). The sequences share >98% identity with sequences from Pantoea ananatis type strain LMG2665 (GenBank JFZU01) indicating that the causal pathogen of bacteria leaf streak of sweet corn is P. ananatis (Fig. S1B). Phylogenetic analysis of gyrB, leuS, and rpoB concatenated sequence showed that the four isolates clustered with P. ananatis (Fig S2). To test the pathogenicity of the isolates of P. ananatis on the sweet corn variety Yuetian28, we inoculated plants at the V3 stage by syringe infiltration of bacterial suspension (108 CFU/ml) (Kini et al. 2020) or sterile distilled water as a negative control. Inoculated plants were placed in a growth chamber at 28 ℃, 60% relative humidity, 16-h/8-h light-dark cycle. After 7 days of incubation, chlorotic streaks resembling the original symptoms developed on inoculated plants (Fig. S1D), while control plants remained symptomless (Fig. S1C). We successfully re-isolated bacteria from the inoculated plants and confirmed their identity by sequencing of 16S rDNA, rpoB, leuS, and gyrB. P. ananatis was previously reported to cause leaf spot disease in maize grown in Argentina, Ecuador, and China (Alippi et al. 2010; Toaza et al. 2021; Cui et al. 2022). To our knowledge, this is the first report of P. ananatis causing leaf streak in sweet corn in southeast China. Further research on P. ananatis management is needed to help control disease spread.

Development and application of a virus-induced gene silencing system for functional genomics in pecan (Carya illinoinensis)

Pecan is a high-value nut tree that cultivated globally. Nowadays, in-depth studies of this species are often hampered by lacking of efficient molecular genetic tools. To facilitate functional genomics in pecan, a virus-induced gene silencing (VIGS) system based on tobacco rattle virus (TRV) was explored in this study. We used pecan magnesium chelatase subunit H (CiChlH) gene as a visual indicator to evaluate the effect of several factors including infiltration method, insert size, insert orientation, inoculation concentration, host genotype, and culture temperature on VIGS. Our results showed that the optimized conditions for pecan VIGS system were as followed: construction of TRV-based vector harboring ∼300 bp target sequence in sense or antisense orientation, adjustment of inoculation solution to a final OD600 of 0.7, use of syringe infiltration method, inoculation of ‘Pawnee’ or ‘Shaoxing’ seedlings at 2∼4 true-leaf stage with obvious shoot apical growth, and subjecting plants to a temperature cycle of 24 °C (day, 12 h)/16 °C (night, 12 h). Using the optimized system, we obtained an expected photobleaching at 4 weeks post infiltration. To further verify the stability of the system, a pecan phenylalanine ammonialyase (CiPAL) gene was used for silencing and we detected a significant reduction in total phenolics in silenced plants with respect to control seedlings, as anticipated. Altogether, the TRV-based VIGS system has been successfully established in pecan and could be used for future reverse genetic studies.

Dentapen versus traditional syringe infiltration - which LA technique is preferred by patients?

Design Single-blind randomised cross-over trial.Intervention The study compared solution deposition pain of a maxillary lateral incisor infiltration between a computer-controlled local anaesthesia delivery device (Dentapen) and traditional syringe. The Dentapen was given with a slow flow rate of 1.8 mL/162 sec and ramp-up mode, and the traditional syringe infiltration was delivered at a flow rate of 1.8 ml/60 sec. Patients were randomly assigned to a sequence to receive both interventions at two separate appointments, with each participant acting as their own control. Patients rated the pain of each intervention using a Heft-Parker visual analogue scale and completed a preference survey at the conclusion of the second appointment.Case selection A total of 130 adult patients with ASA I or II were included in the study. Criteria for exclusion were patients under 18 years of age or older than 65, allergies to local anaesthetics or sulphites, pregnant or nursing, history of significant medical conditions (ASA III or higher), taking any medications that may affect pain assessment, active pathosis at the injection site, or inability to give informed consent.Data analysis Differences in pain of solution deposition for the Dentapen and traditional infiltration techniques were analysed using paired t-tests and odds ratios. Interactions between study groups, gender and anxiety were analysed using a linear mixed-effect model with a P value <0.05.Results Solution deposition pain was significantly less (P <0.001) with the Dentapen infiltration than the traditional infiltration. The preference survey revealed that 75% of patients preferred the Dentapen infiltration over the traditional technique.Conclusions The findings of this clinical trial suggest that pain during maxillary lateral incisor infiltrations can be reduced by using the Dentapen with a slow flow rate and ramp-up mode compared with the traditional syringe technique.

Comparison of Maxillary Lateral Incisor Infiltration Pain Using the Dentapen and a Traditional Syringe: A Prospective Randomized Study

IntroductionThe anterior maxillary infiltration is one of the more painful dental injections. The Dentapen is an electronic syringe that uses computer-controlled delivery technology to administer dental local anesthesia at a slow controlled rate. The purpose of this prospective, randomized, single-blind study was to evaluate solution deposition pain of a maxillary lateral incisor infiltration using the Dentapen with the slow flow rate (1.8 mL/162 sec) and ramp-up setting compared with a traditional syringe infiltration at a flow rate of 1.8 mL/60 sec. MethodsOne hundred thirty adults were administered a maxillary lateral incisor infiltration with the Dentapen and a traditional syringe at 2 separate appointments in a single-blind manner. The infiltrations of 2% lidocaine with 1:100,000 epinephrine were given at a rate of 1.8 mL/162 sec with the ramp-up feature for the Dentapen and 1.8 mL/60 sec for the traditional infiltration. The pain of solution deposition was recorded on a visual analogue scale. At the conclusion of the study, subjects selected their preferred injection technique. The data were analyzed statistically using paired t tests, a mixed-effect model, and odds ratio. ResultsThe pain of solution deposition was significantly less for the Dentapen injection than the traditional injection (P < .001). With the Dentapen device, 16% experienced moderate pain, and for the traditional syringe, 39% experienced moderate pain. Overall, 75% of subjects preferred the Dentapen injection over the traditional injection. ConclusionsThe Dentapen, using the slow flow rate and ramp-up mode, significantly reduced the pain of solution deposition for maxillary lateral incisor infiltrations.

A Novel Sulfated Glycoprotein Elicitor Extracted from the Moroccan Green Seaweed Codium decorticatum Induces Natural Defenses in Tomato

Sulfated glycoproteins extracted for the first time from the Moroccan green seaweed Codium decorticatum were investigated for their ability to induce a natural defense metabolism in the roots and the upper leaves of tomato seedlings. The crude (AGB) and the purified fractions (AGP) were characterized chemically (Colorimetric assays) and structurally (SEC-MALS, GC-EI/MS, ATR-FTIR). The elicitor aqueous solutions (1 g/L) were applied by foliar spray and syringe infiltration into the internodal middle of 45-day-old tomato seedlings. Phenylalanine ammonia-lyase (PAL) activity, polyphenols, and lignin contents were measured in the roots and the leaves after 0 h, 12, 24, 48, and 72 h of treatment. The AGB and AGP extracts contained 37.67% and 48.38% of the total carbohydrates, respectively, and were mainly composed of galactose, glucose, arabinose, and a minor amount of xylose and rhamnose. They were characterized by an important molecular weight (Mw) &gt; of 2000 × 103 g·mol−1 and a high degree of sulfation and protein (12–23% (w/w)), indicating that the extracted polysaccharides could be an arabinogalactan-rich protein present in the cell wall of the green seaweed C. decorticatum. Both crude and purified fractions exhibited an elicitor effect by inducing the PAL activity, the accumulation of phenolic compounds and lignin contents in the roots and the leaves of tomato seedlings. These responses were systemic in both the methods used (injection and foliar spray) and were mobilized throughout tissues that are not directly treated (roots and/or leaves). Regarding the elicitor activities, AGB and AGP presented globally similar patterns, which revealed the importance of crude extracts in the stimulation of plant immunity. These results suggest the new application of sulfated glycoprotein isolated from green seaweed in agriculture as inducers of natural defenses of plants.

First Report of Bacterial Canker on Blueberry (Vaccinium corymbosum) Caused by Pseudomonas syringae pv. syringae in Serbia.

During May 2021, necrosis of young twigs and flower buds were observed on two-year-old highbush blueberry plants (Vaccinium corymbosum) cv. Draper, in a 1 hectare orchard in the municipality of Šabac, Serbia. Disease symptoms included reddish-brown to black irregularly shaped cankers developing on the shoot tips that extended downwards along the branches. In some plants, cankers surrounded the stem, causing shoot-tip dieback and necrosis of the buds. Beneath the bark, a distinct margin between diseased and healthy tissue was visible. A few weeks before symptoms development, seven freezing events with temperature from -3°C to -1°C, and five near-freezing temperatures were recorded in this area, leading to the hypothesis that symptoms were associated to the presence of ice nucleating bacteria belonging to Pseudomonas syringae. The observed disease incidence was 80%, while 10% of the plants died. Bacteria were isolated from symptomatic tissue on King's medium B (KB). After 2 to 3 days of incubation at 27°C, predominantly grey-whitish, shiny, round, convex bacterial colonies were observed on agar plates. Ten isolates producing a fluorescent pigment on KB were selected for further characterization by biochemical and molecular tests. The isolates were Gram, oxidase and arginine-dihydrolase negative, levan positive, induced hypersensitive response on tobacco leaves and showed no pectinolytic activity on potato slices. Based on the results of API 20E and API 20NE tests (BioMerieux, France), and the fact that isolates did not utilize tartrate nor had tyrosinase activity, they were preliminarly identified as Pseudomonas syrinage pv. syringae (Braun-Kiewnick and Sands 2001). Additionally, all tested isolates had ice-nucleation activity at -5°C. The syrB gene responsible for syringomycin synthesis, was amplified in all isoaltes with the specific primer pair B1/B2 (Sorensen et al. 1998). The 16S rRNA gene sequences of five selected isolates (GenBank MZ410287 to 91) showed 100% identity to P. s. pv. syringae isolated from Prunus avium in United Kingdom (GenBank CP026568) and France (GenBank LT962480). Sequences of gyrB gene (Sarkar and Guttman 2004) of two selected isolates (GenBank MZ420633 and MZ420634) showed 98,44% identity to the P. s. pv. syrinage strain isolated in France (GenBank LT962480). Pathogenicity of the isolates was confirmed on 2-year-old blueberry plants cv. Draper, by inoculating two plants per isolate. One-cm long wounds were made on branches using a scalpel and 20 µl of bacterial suspension (106 CFU/ml) was infiltrated into the tissue. The cuts were then covered with moist sterile cotton pads and wraped in parafilm for 3 days. Inoculation was also performed on two leaves per plant by needleless syringe infiltration (106 CFU/ml). Sterile distilled water was used as a negative control. Plants were maintained in greenhouse at 27°C day and 15°C night temperature. Three weeks after inoculation, the inoculated branches and leaves developed necrosis, leaves spots and cankers respectively, resembling the natural infection. Symptoms were not observed on the control plants. Bacteria were reisolated from symptomatic tissue and their identity was confirmed by amplifying the syrB gene sequence and additional biochemical tests. This is the first report of bacterial canker of highbush blueberry caused by P. s. pv. syringae in Serbia. In Europe, there was only one report on Pseudomonas spp. causing disease on blueberry leaves in Poland (Kaluzna et al. 2013). Due to market demands and export potential, blueberry production in Serbia has been rapidly increasing. In 2015, highbush blueberry was cultivated on 220 ha, while in 2020 the area increased to 1899 ha. However, under favourable environmental conditions, blueberry production might be severely affected by bacterial canker.

Transient recombinant expression of highly immunogenic CagA, VacA and NapA in Nicotiana benthamiana

Interest in the plant-based transient production of recombinant immunogenic antigens has tremendously progressed because plants are cost-effective, easily selectable, free of mammalian contamination, and support complex post-translational modifications. Nicotiana benthamiana is a convenient system for transient expression of recombinant antigens. The present study documented a platform for rapid production of Helicobacter pylori CagA, VacA and NapA antigens three days (first harvest, FH) and six days (second harvest, SH) after agro-infiltration using a syringe. In this study, CagA, VacA and NapA antigen genes from Helicobacter pylori were cloned into the binary vector pBI121 and transformed into Nicotiana benthamiana by the Agrobacterium-mediated process. Leaves of four to five weeks old Nicotiana benthamiana plants were agroinfiltrated with EHA105 subtype of Agrobacterium tumefaciens strain containing cloned CagA (pBI121-CagA), VacA (pBI121-VacA) and NapA (pBI121-NapA) constructs. The transient expression and accumulation of the recombinant genes containing CagA, VacA and NapA expression cassettes were confirmed using qRT-PCR by comparing the relative expression at FH and SH post-infiltration with the non-infiltrated (control) samples and using ELISA at 1/5 and 1/10 dilution ratios. The qRT-PCR findings showed that Agrobacterium-mediated syringe infiltration of leaves of four to five weeks old Nicotiana benthamiana plants produced significantly higher transcript levels of CagA (about 8-fold and 7-fold), VacA (38-fold and 24-fold) and NapA (7-fold and 5-fold) genes at FH and SH compared to the control sample. Besides, the maximum amount of CagA, VacA and NapA antigens were detected at the FH stage compared to the SH stage, when the antibody concentrations of the agro-infiltrated leaf extracts containing these recombinant antigens were diluted in a 1/5 ratio. This study has developed evidence to support that recombinant CagA, VacA and NapA can be transiently produced in Nicotiana benthamiana plants.

First Report of Pantoea ananatis causing leaf spot disease of maize in Ecuador.

Maize (Zea mays) is the second most cultivated grain crop in Ecuador, with growing significance as a source of fodder and food. During the rainy season (November and December) of 2018 and 2019, a disease of maize that was not previously observed in Ecuador was found at commercial fields of Misqui Sara variety, at four parishes of canton Quito (Tumbaco, Pifo, Puembo, and Checa), province of Pichincha. Infected plants, at tassel initiation, displayed symptoms of localized chlorotic streaks on leaves that expanded with time, and around a month later turned necrotic. Severely affected plants wilted and died. Symptoms appeared in lower leaves first and were later observed in upper leaves as the disease progressed. Disease incidence was between 20 and 30% in the affected plantations, with around 30% of infected plants wilting and dying, resulting in 20-25% of yield losses. Upper leaves from ten symptomatic plants, five from Puembo and five from Checa, were collected randomly. Two 0.5 cm2 pieces of leaf from each plant were excised from the margins of the necrotic lesions, surface sterilized and macerated in 9 mL of sterile peptone water. The 10-3 dilutions were plated onto nutrient agar and incubated at 28°C for 24 hours. Yellow, mucoid colonies were isolated on nutrient agar. Three isolates from Puembo and two from Checa were selected for testing Koch´s postulates and further biochemical and molecular characterization. Isolates were Gram-negative rods, oxidase negative, catalase, indol and citrate positive. Fragments of the 16S, gyrB, and rpoB loci were amplified and sequenced using the 27F/1492R (Lane, D. J., 1991), UP-1/UP-2r (Yamamoto & Harayama, 1995), and rpoBCM81-F/rpoBCM32b-R (Brady, C., et al., 2008) primer pairs, respectively. All isolates presented identical sequences for the different loci, therefore only sequences from isolate FP191505 were deposited in GenBank (GenBank accession no. MW528428-MW528430). A search of homologous sequences using BLAST resulted in identities of 99.3, 99.7, and 100 % for 16S, gyrB, and rpoB, respectively, with sequences from Pantoea ananatis type specimen LGM 2665 (Brady, C., et al., 2008; Hauben, L., et al., 1998; GenBank accession nos NR_119362.1, EF988824.1 EF988996.1), indicating that our isolates belong to this species. Pathogenicity tests were performed by syringe infiltration of bacterial suspensions. Each one of the five characterized P. ananatis isolates was inoculated in four leaves (500 ul of 1 x 108 CFU mL-1 per leave) of three healthy maize plants. Negative control plants were infiltrated with sterile distilled water. Plants were incubated at 28-30°C and 60% relative humidity for 24 hours. Later, plants were maintained in a greenhouse with 27°C/21°C day/night temperatures and observed daily. After six weeks all bacteria-inoculated plants developed symptoms of chlorosis and necrosis while the control was symptomless. Bacteria were re-isolated from symptomatic leaves and identified as P. ananatis following the same methodologies used for the initial identification. To our knowledge, this is the first report of P. ananatis causing leaf spot of maize in Ecuador.

An improved and efficient method of Agrobacterium syringe infiltration for transient transformation and its application in the elucidation of gene function in poplar

BackgroundForest trees have important economic and ecological value. As a model tree, poplar has played a significant role in elucidating the molecular mechanisms underlying tree biology. However, a lack of mutant libraries and time-consuming stable genetic transformation processes severely limit progress into the functional characterization of poplar genes. A convenient and fast transient transformation method is therefore needed to enhance progress on functional genomics in poplar.MethodsA total of 11 poplar clones were screened for amenability to syringe infiltration. Syringe infiltration was performed on the lower side of the leaves of young soil-grown plants. Transient expression was evaluated by visualizing the reporters β-glucuronidase (GUS) and green fluorescent protein (GFP). The experimental parameters of the syringe agroinfiltration were optimized based on the expression levels of the reporter luciferase (LUC). Stably transformed plants were regenerated from transiently transformed leaf explants through callus-induced organogenesis. The functions of Populus genes in secondary cell wall-thickening were characterized by visualizing lignin deposition therein after staining with basic fuchsin.ResultsWe greatly improved the transient transformation efficiency of syringe Agrobacterium infiltration in poplar through screening for a suitable poplar clone from a variety of clones and optimizing the syringe infiltration procedure. The selected poplar clone, Populus davidiana × P. bolleana, is amenable to Agrobacterium syringe infiltration, as indicated by the easy diffusion of the bacterial suspension inside the leaf tissues. Using this technique, we localized a variety of poplar proteins in specific intracellular organelles and illustrated the protein–protein and protein–DNA interactions. The transiently transformed leaves could be used to generate stably transformed plants with high efficiency through callus induction and differentiation processes. Furthermore, transdifferentiation of the protoxylem-like vessel element and ectopic secondary wall thickening were induced in the agroinfiltrated leaves via the transient overexpression of genes associated with secondary wall formation.ConclusionsThe application of P. davidiana × P. bolleana in Agrobacterium syringe infiltration provides a foundation for the rapid and high-throughput functional characterization of Populus genes in intact poplar plants, including those involved in wood formation, and provides an effective alternative to Populus stable genetic transformation.

Transformation of Amorphadiene Synthase and Antisilencing P19 Genes into Artemisia annua L. and its Effect on Antimalarial Artemisinin Production.

Purpose: The low content of artemisinin related to the biosynthetic pathway is influenced by the role of certain enzymes in the formation of artemisinin. The regulation of genes involved in artemisinin biosynthesis through genetic engineering is a choice to enhance the content. This research aims to transform ads and p19 gene as an antisilencing into Artemisia annua and to see their effects on artemisinin production. Methods: The presence of p19 and ads genes was confirmed through polymerase chain reaction (PCR) products and sequencing analysis. The plasmids, which contain ads and/or p19 genes, were transformed into Agrobacterium tumefaciens, and then inserted into leaves and hairy roots of A. annua by vacuum and syringe infiltration methods. The successful transformation was checked through the GUS histochemical test and the PCR analysis. Artemisinin levels were measured using HPLC. Results: The percentages of the blue area on leaves by using vacuum and syringe infiltration method and on hairy roots were up to 98, 92.55%, and 99.00% respectively. The ads-p19 sample contained a higher level of artemisinin (0.18%) compared to other samples. Transformed hairy root with co-transformation of ads-p19 contained 0.095% artemisinin, where no artemisinin was found in the control hairy root. The transformation of ads and p19 genes into A. annua plant has been successfully done and could enhance the artemisinin content on the transformed leaves with ads-p19 up to 2.57 folds compared to the untransformed leaves, while for p19, cotransformed and ads were up to 2.25, 1.29, and 1.14 folds respectively. Conclusion: Antisilencing p19 gene could enhance the transformation efficiency of ads and artemisinin level in A. annua.

Human Pathogen Colonization of Lettuce Dependent Upon Plant Genotype and Defense Response Activation.

Fresh produce contaminated with human pathogens may result in foodborne disease outbreaks that cause a significant number of illnesses, hospitalizations, and death episodes affecting both public health and the agribusiness every year. The ability of these pathogens to survive throughout the food production chain is remarkable. Using a genetic approach, we observed that leaf colonization by Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium 14028s) and Escherichia coli O157:H7 was significantly affected by genetic diversity of lettuce (Lactuca sativa L. and L. serriola L.). In particular, there was a significant variation among 11 lettuce genotypes in bacterial attachment, internalization, and apoplastic persistence after surface- and syringe-inoculation methods. We observed a significant correlation of the bacterial leaf internalization rate with stomatal pore traits (width and area). Moreover, bacterial apoplastic populations significantly decreased in 9 out of 11 lettuce genotypes after 10 days of surface inoculation. However, after syringe infiltration, populations of E. coli O157:H7 and S. Typhimurium 14028s showed positive, neutral, or negative net growth in a 10-day experimental period among seedlings of different lettuce types. The relative ability of the bacteria to persist in the apoplast of lettuce genotypes after syringe inoculation was minimally altered when assessed during a longer period (20 days) using 3.5- to 4-week-old plants. Interestingly, contrasting bacterial persistence in the lettuce genotypes Red Tide and Lollo Rossa was positively correlated with significant differences in the level of reactive oxygen species burst and callose deposition against S. Typhimurium 14028s and E. coli O157:H7 which are related to plant defense responses. Overall, we characterized the genetic diversity in the interaction between lettuce genotypes and enterobacteria S. Typhimurium 14028s and E. coli O157:H7 and discovered that this genetic diversity is linked to variations in plant immune responses towards these bacteria. These results provide opportunities to capitalize on plant genetics to reduce pathogen contamination of leaves.

Phosphoinositide 3-Kinase Promotes Oxidative Burst, Stomatal Closure and Plant Immunity in Bacterial Invasion.

Phosphoinositide 3-kinase (PI3K) plays a vital role in plant response to abiotic stress. However, the role of PI3K in plant immunity is largely unknown. This study showed that PI3K enhanced Arabidopsis resistance to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) and Pst DC3000 (avrRpt2). Overexpression of AtVPS34 promoted stomatal closure while PI3K inhibitors blocked that after spray inoculation. Additionally, gene expression of AtVPS34 was increased upon infection by Pst DC3000 (avrRpt2), and SA upregulated AtVPS34 gene expression in this process. Furthermore, overexpression of AtVPS34 enhanced PR gene expression after syringe infiltration with Pst DC3000 (avrRpt2), while PI3K inhibitors inhibited that. The production of hydrogen peroxide and the expression of gene encoding antioxidant enzyme were both enhanced in AtVPS34 overexpressing lines after spray inoculation or syringe infiltration with Pst DC3000 (avrRpt2). Collectively, these results unraveled a novel and broad role of PI3K in plant immunity which promoted stomatal closure and PR gene expression possibly via regulating ROS production.

Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen

Dengue is a viral disease that represents a significant threat to global public health since billions of people are now at risk of infection by this mosquito-borne virus. The implementation of extensive screening tests is indispensable to control this disease, and the Dengue virus non-structural protein 1 (NS1) is a promising antigen for the serological diagnosis of dengue fever. Plant-based systems can be a safe and cost-effective alternative for the production of dengue virus antigens. In this work, two strategies to produce the dengue NS1 protein in Nicotiana benthamiana leaves were evaluated: Targeting NS1 to five different subcellular compartments to assess the best subcellular organelle for the expression and accumulation of NS1, and the addition of elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags to NS1. The transiently expressed proteins in N. benthamiana were quantified by Western blot analysis. The NS1 fused to ELP and targeted to the ER (NS1 ELP-ER) showed the highest yield (445 mg/kg), approximately a forty-fold increase in accumulation levels compared to the non-fused protein (NS1-ER), representing the first example of transient expression of DENV NS1 in plant. We also demonstrated that NS1 ELP-ER was successfully recognized by a monoclonal anti-dengue virus NS1 glycoprotein antibody, and by sera from dengue virus-infected patients. Interestingly, it was found that transient production of NS1-ER and NS1 ELP-ER using vacuum infiltration of whole plants, which is easier to scale up, rather than syringe infiltration of leaves, greatly improved the accumulation of NS1 proteins. The generated plant made NS1, even without extensive purification, showed potential to be used for the development of the NS1 diagnostic tests in resource-limited areas where dengue is endemic.

Virus-Induced Gene Silencing of Cell Wall Genes in Flax (Linum usitatissimum).

Plants have developed defense mechanisms against viruses by using an RNA silencing-based process, which has many common features with the endogenous RNA silencing pathway used for regulating the level of transcripts derived from developmental genes. In the virus-induced gene silencing (VIGS) method, it is possible to take advantage of this mechanism by inserting a plant nucleic fragment within the viral genome to knock down the corresponding gene. This tool has been used in many species as a fast and easy reverse genetics technique in order to gain information on the role of genes with poorly understood functions. Here we describe in detail two Agrobacterium-mediated infection protocols in flax, based on a whole plant vacuum infiltration and a leaf syringe infiltration that systemically impact the transcript levels in the stem.

Investigation of &lt;i&gt;Agrobacterium&lt;/i&gt;-mediated Transient dsRNA Expression in Tobacco

The Agrobacterium tumefaciens mediated gene transfer is widely used to generate genetic transformation of plants and transient assay of temporal exogenous gene expression. Syringe infiltration system into tobacco (Nicotiana benthamiana) leaves is a powerful tool for transient expression of target protein to study protein localization, protein-protein binding and protein production. However, the protocol and technical information of transient gene expression, especially double strand RNA (dsRNA), in tobacco using Agrobacterium is not well known. Recently, dsRNA is crucial for insecticidal effect on destructive agronomic pest such as Corn rootworm. In this study, we investigated the factor influencing the dsRNA expression efficiency of syringe agro-infiltration in tobacco. To search the best combination for dsRNA transient expression in tobacco, applied two Agrobacterium cell lines and three plant vector systems. The efficiency of dsRNA expression has estimated by real-time PCR and digital PCR. As a result, pHellsgate12 vector constructs showed the most effective accumulation of dsRNA in the cell. These results indicated that the efficiency of dsRNA expression was depending on the kind of vector rather than Agrobacterium cells. In summary, the optimized combination of transient dsRNA expression system in tobacco might be useful to in vivo dsRNA expression for functional study and risk assessment of dsRNA.

Metallic syringe versus electronically assisted injection system: a comparative clinical study in children.

Local anaesthesia injection necessary for pain control in paediatric dentistry may itself be painful sometimes, partly because of the pressure felt during injection; electronically assisted injection systems were developed to address this problem. Study design: The present study is a clinical study in children that compared two types of devices for paediatric buccal infiltration anaesthesia: the aspirating syringe and an electronically assisted injection system, the Wand STA. A split mouth, randomised controlled clinical trial was conducted on 30 healthy six to eight-year-old patients (6.64 ± 0.803 years) requiring pulpotomies on two symmetrical primary maxillary molars. Each patient received the following types of anaesthesia, in separate, consecutive, randomly ordered sessions: conventional buccal infiltration by metallic aspirating syringe and buccal infiltration by computer-controlled local anaesthetic device (Single Tooth Anesthesia). Parameters assessed were: pain experienced during injection, patient's heart rate and behaviour, anaesthesia quantity required and onset time. No statistical differences were observed between the two techniques (p? 0.05) for all assessed parameters. Results suggest that computer-assisted anaesthesia may represent an alternative to conventional syringes for local buccal anaesthesia in paediatric dental treatment; comparison to other types of dental infiltration anaesthesia needs further investigation.

EASI Transformation: An Efficient Transient Expression Method for Analyzing Gene Function in Catharanthus roseus Seedlings.

The Catharanthus roseus plant is the exclusive source of the valuable anticancer terpenoid indole alkaloids, vinblastine (VB) and vincristine (VC). The recent availability of transcriptome and genome resources for C. roseus necessitates a fast and reliable method for studying gene function. In this study, we developed an Agrobacterium-mediated transient expression method to enable the functional study of genes rapidly in planta, conserving the compartmentalization observed in the VB and VC pathway. We focused on (1) improving the transformation method (syringe versus vacuum agroinfiltration) and cultivation conditions (seedling age, Agrobacterium density, and time point of maximum transgene expression), (2) improving transformation efficiency through the constitutive expression of the virulence genes and suppressing RNA silencing mechanisms, and (3) improving the vector design by incorporating introns, quantitative and qualitative reporter genes (luciferase and GUS genes), and accounting for transformation heterogeneity across the tissue using an internal control. Of all the parameters tested, vacuum infiltration of young seedlings (10-day-old, harvested 3 days post-infection) resulted in the strongest increase in transgene expression, at 18 – 57 fold higher than either vacuum or syringe infiltration of other seedling ages. Endowing the A. tumefaciens strain with the mutated VirGN54D or silencing suppressors within the same plasmid as the reporter gene further increased expression by 2 – 10 fold. For accurate measurement of promoter transactivation or activity, we included an internal control to normalize the differences in plant mass and transformation efficiency. Including the normalization gene (Renilla luciferase) on the same plasmid as the reporter gene (firefly luciferase) consistently yielded a high signal and a high correlation between RLUC and FLUC. As proof of principle, we applied this approach to investigate the regulation of the CroSTR1 promoter with the well-known activator ORCA3 and repressor ZCT1. Our method demonstrated the quantitative assessment of both the activation and repression of promoter activity in C. roseus. Our efficient Agrobacterium-mediated seedling infiltration (EASI) protocol allows highly efficient, reproducible, and homogenous transformation of C. roseus cotyledons and provides a timely tool for the community to rapidly assess the function of genes in planta, particularly for investigating how transcription factors regulate terpenoid indole alkaloid biosynthesis.