Withania somnifera (L.) Dunal: Enhanced production of withanolides and phenolic acids from hairy root culture after application of elicitors.

Withania somnifera (L.) Dunal is an important indigenous medicinal plant with extensive pharmaceutical potential. The root is the main source of major bioactive compounds of this plant species including withanolides, withanine, phenolic acids, etc. Hairy root culture (HRC) is a crucial method for low-cost production of active compounds on a large scale. Four different Agrobacterium rhizogenes strains have been used for the hairy root induction. Maximum transformation efficiency (87.34 ± 2.13%) was achieved with A4 bacterial strain-mediated transformed culture. The genetic transformation was confirmed by using specific primers of seven different genes. Seven HR (Hairy root) lines were selected after screening 29 HR lines based on their fast growth rate and high accumulation of withanolides and phenolic acids content. Two biotic and three abiotic elicitors were applied to the elite root line to trigger more accumulation of withanolides and phenolic acids. While all the elicitors effectively increased withanolides and phenolic acids production, among the five different elicitors, salicylic acid (4.14 mg l-1) induced 11.49 -fold increase in withanolides (89.07 ± 2.75 mg g-1 DW) and 5.34- fold increase in phenolic acids (83.69 ± 3.11 mg g- 1 DW) after 5 days of elicitation compared to the non-elicited culture (7.75 ± 0.63 mg g-1 DW of withanolides and 15.66 ± 0.92 mg g-1 DW of phenolic acids). These results suggest that elicitors can tremendously increase the biosynthesis of active compounds in this system; thus, the HRC of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides and phenolic acids.

Revealing the phosphate-solubilizing characteristics and mechanisms of the plant growth-promoting bacterium Agrobacterium deltaense C1.

AIMS: This study explores the phosphate (Pi)-solubilizing characteristics and mechanisms of a novel phosphate-solubilizing bacterium, Agrobacterium deltaense C1 (C1 hereafter). METHODS AND RESULTS: The growth-promoting effects of C1 were investigated by gnotobiotic experiments, and the Pi-solubilizing mechanism was revealed by extracellular metabolomics, liquid chromatography analysis, and reverse transcription quantitative polymerase chain reaction. Results showed that C1 significantly increased Arabidopsis biomass and total phosphorus (P) content under P deficiency. Under Ca3(PO4)2 condition, the presence of C1 resulted in a significant and negative correlation between available P content and medium pH changes, implying that Pi dissolution occurs through acid release. Metabolomics revealed C1's ability to release 99 organic acids, with gluconic acid (GA), citric acid, and α-ketoglutaric acid contributing 64.86%, 9.58%, and 0.94%, respectively, to Pi solubilization. These acids were significantly induced by P deficiency. Moreover, C1's Pi solubilization may remain significant even in the presence of available P, as evidenced by substantial pH reduction and high gcd gene expression. Additionally, C1 produced over 10 plant growth-promoting substances. CONCLUSIONS: C1 dissolves Pi primarily by releasing GA, which enhances plant growth under P deficiency. Notably, its Pi solubilization effect is not significantly limited by available Pi.

Agrobacterium sp. ZX09 ß-Glucan Attenuates Enterotoxigenic Escherichia coli-Induced Disruption of Intestinal Epithelium in Weaned Pigs.

To explore the protective effect of dietary ß-glucan (BGL) supplementation on intestinal epithelium exposure to enterotoxigenic Escherichia coli (ETEC), thirty-two weaned pigs were assigned to four groups. Pigs were fed with a basal diet or basal diet containing 500 mg/kg BGL, and were orally infused with ETEC or culture medium. Results showed BGL supplementation had no influence on growth performance in weaned pigs. However, BGL supplementation increased the absorption of D-xylose, and significantly decreased the serum concentrations of D-lactate and diamine oxidase (DAO) in the ETEC-challenged pigs (p < 0.05). Interestingly, BGL significantly increased the abundance of the zonula occludens-1-(ZO-1) in the jejunal epithelium upon ETEC challenge (p < 0.05). BGL supplementation also increased the number of S-phase cells and the number of sIgA-positive cells, but significantly decreased the number of total apoptotic cells in the jejunal epithelium upon ETEC challenge (p < 0.05). Moreover, BGL significantly increased the duodenal catalase (CAT) activity and the ileal total superoxide dismutase (T-SOD) activity in the ETEC-challenged pigs (p < 0.05). Importantly, BGL significantly decreased the expression levels of critical inflammation related proteins such as the tumor necrosis factor-α (TNF-α), interlukin-6 (IL-6), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) in the jejunal and ileal mucosa upon ETEC challenge (p < 0.05). BGL also elevated the propanoic acid content and the abundance of Lactobacillus and Bacillus in the colon upon ETEC challenge (p < 0.05). These results suggested BGL could alleviate the ETEC-induced intestinal epithelium injury, which may be associated with suppressed inflammation and improved intestinal immunity and antioxidant capacity, as well as the improved intestinal macrobiotic.

Plant growth promoting rhizobacteria improve growth and yield related attributes of chili under low nitrogen availability.

Nitrogen (N) is a macronutrient desired by crop plants in large quantities. However, hiking fertilizer prices need alternative N sources for reducing its requirements through appropriate management practices. Plant growth promoting rhizobacteria (PGPR) are well-known for their role in lowering N requirements of crop plants. This study assessed the impact of PGPR inoculation on growth, allometry and biochemical traits of chili under different N doses. Two PGPR, i.e., Azospirillum 'Er-20' (nitrogen fixing) and Agrobacterium 'Ca-18' (phosphorous solubilizing) were used for inoculation, while control treatment had no PGPR inoculation. Six N doses, i.e., 100, 80, 75, 70, 60 and 50% of the N required by chili were included in the study. Data relating to growth traits, biochemical attributes and yield related traits were recorded. Interaction among N doses and PGPR inoculation significantly altered all growth traits, biochemical attributes and yield related traits. The highest values of the recorded traits were observed for 100% N with and without PGPR inoculation and 75% N with PGPR inoculation. The lowest values of the recorded traits were noted for 50% N without PGPR inoculation. The PGPR inoculation improved the measured traits compared to the traits recorded noted in same N dose without PGPR inoculation. Results revealed that PGPR had the potential to lower 25% N requirement for chili. Therefore, it is recommended that PGPR must be used in chili cultivation to lower N requirements.

Hairy Roots of Scutellaria spp. (Lamiaceae) as Promising Producers of Antiviral Flavones.

We measured and studied the growth parameters and the qualitative and quantitative composition of the flavones of hairy roots of the Scutellaria genus: S. lateriflora, S. przewalskii and S. pycnoclada. Hairy roots were obtained using wild-type Agrobacterium rhizogenes A4 by co-cultivation of explants (cotyledons) in a suspension of Agrobacterium. The presence of the rol-genes was confirmed by PCR analysis. The hairy roots of the most studied plant from the Scutellaria genus, S. baicalensis, were obtained earlier and used as a reference sample. HPLC-MS showed the predominance of four main flavones (baicalin, baicalein, wogonin and wogonoside) in the methanol extracts of the studied hairy roots. In addition to the four main flavones, the other substances which are typical to the aerial part of plants were found in all the extracts: apigenin, apigetrin, scutellarin and chrysin-7-O-ß-d-glucuronide. According to the total content of flavones, the hairy roots of the studied skullcaps form the following series: S. przewalskii (33 mg/g dry weight) > S. baicalensis (17.04 mg/g dry weight) > S. pycnoclada (12.9 mg/g dry weight) > S. lateriflora (4.57 mg/g dry weight). Therefore, the most promising producer of anti-coronavirus flavones is S. przewalskii.

Application of Hairy Root Culture for Bioactive Compounds Production in Medicinal Plants.

Medicinal plants are rich sources of natural bioactive compounds used to treat many diseases. With the development of the health industry, the market demands for Chinese medicine have been rapidly increasing in recent years. However, over-utilization of herbal plants would cause serious ecological problems. Therefore, an effective approach should be developed to produce the pharmaceutically important natural drugs. Hairy root culture induced by Agrobacterium rhizogenes has been considered to be an effective tool to produce secondary metabolites that are originally biosynthesized in the roots or even in the aerial organs of mature plants. This review aims to summarize current progress on medicinal plant hairy root culture for bioactive compounds production. It presents the stimulating effects of various biotic and abiotic elicitors on the accumulation of secondary metabolites. Synergetic effects by combination of different elicitors or with other strategies are also included. Besides, the transgenic system has promising prospects to increase bioactive compounds content by introducing their biosynthetic or regulatory genes into medicinal plant hairy root. It offers great potential to further increase secondary metabolites yield by the integration of manipulating pathway genes with elicitors and other strategies. Then advances on two valuable pharmaceuticals production in the hairy root cultures are illustrated in detail. Finally, successful production of bioactive compounds by hairy root culture in bioreactors are introduced.

Genetic transfection, hairy root induction and solasodine accumulation in elicited hairy root clone of Solanum erianthum D. Don.

An efficient genetic transfection technique has been established using A4 strain of Agrobacterium rhizogenes for the first time in a medicinally valuable plant Solanum erianthum D. Don. The explants were randomly pricked with sterile needle, inoculated with bacterial suspension. The infected leaves were then washed and transferred to MS basal medium fortified with cefotaxime for hairy root induction. A maximum transformation efficiency of 72 % has been recorded after two days of co-cultivation period. The transfer of rolA and rolB genes from the bacterium to the plant genome has been confirmed in five transformed hairy rootlines by standard Polymerase Chain Reaction technique. On the basis of growth analysis and secondary metabolite study two potential rhizoclones (A4-HR-A and A4-HR-B) were selected. Rhizoclone A4-HR-A can produce highest amount of alkaloid, phenolic and flavonoid, whereas A4-HR-B was observed to be highest tannin producer. Alkaloid like solasodine, commercially important for steroidal drug synthesis, was quantified from leaf and A4-HR-A clone by an improved High Performance Liquid Chromatography method. This showed a sustainable increase (1.33 fold) in production of solasodine in hairy rootline.

Pathogen manipulation of chloroplast function triggers a light-dependent immune recognition.

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1-mediated resistance is compromised because plants produce a shorter GLYK-lacking the intact chloroplast transit peptide-that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum).

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7-10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p < 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

Establishment and optimization of a hemp (Cannabis sativa L.) agroinfiltration system for gene expression and silencing studies.

Industrial hemp (Cannabis sativa L.) is a high-yielding annual crop primarily grown for fiber, seeds, and oil. Due to the phytochemical composition of hemp, there has been an increased interest in the market for nutraceuticals and dietary supplements for human health. Recent omics analysis has led to the elucidation of hemp candidate genes involved in the syntheses of specialized metabolites. However, a detailed study of these genes has not been undertaken due to the lack of a stable transformation system. We report for the first time an agroinfiltration system in hemp utilizing vacuum infiltration, which is an alternative method to stable transformation. A combination of 0.015% Silwett L-77, 5 mM ascorbic acid, and thirty second sonication followed by a 10-minute vacuum treatment resulted in the highest ß-glucuronidase expression in the leaf, male and female flowers, stem, and root tissues. The phytoene desaturase gene was silenced with a transient hairpin RNA expression, resulting in an albino phenotype in the leaves and the male and female flowers. This agroinfiltration system would be useful for overexpression and silencing studies of target genes to regulate the yield of specialized metabolites in hemp.

Establishment of hairy root cultures by Agrobacterium rhizogenes mediated transformation of Trachyspermum ammi L. for the efficient production of thymol.

Trachyspermum ammi is an important medicinal plant that contains a bioactive compound namely thymol. In the study, T. ammi was transformed by Agrobacterium rhizogenes strains. Seedling stem explants were inoculated with A. rhizogenes strains A4, LBA 9402, ATCC 15834, and the effect of different co-cultivation media along with incorporation of acetosyringone (100 µM) was evaluated comparatively on the frequency of hairy root induction. The polymerase chain reaction using rolB and virD specific primers was served to confirm the putative transformed hairy roots. All strains established hairy root with various frequencies, among which strain ATCC 15834 was significantly the most efficient strain for hairy root induction (84.3%). Half-strength B5 medium and incorporation of acetosiryngone (100 µM) were also significantly optimal for hairy root induction. Hairy roots culture induced by ATCC 15834 using half-strength B5 liquid medium supplemented with 30 g L-1 sucrose indicated the highest accumulation of biomass (99.05 g L-1 FW and 10.95 g L-1 DW) and thymol content (11.30 mg g-1 DM) at 20 days. Nearly 4.9-fold and 5.3-fold increment of biomass and thymol accumulation was observed, respectively, at 20 days in comparison with the untransformed control roots. The results showed the high potential of T. ammi hairy roots for the biosynthesis of thymol.

Agrobacterium rhizogenes-mediated transformation of Hypericum sinaicum L. for the development of hairy roots containing hypericin

Hypericum sinaicum L. is an endangered Egyptian medicinal plant of high importance due to the presence of naphthodianthrones (hypericins), which have photodynamic properties and pharmaceutical potential. We sought to assess H. sinaicum ability to develop hairy roots that could be cultured in contained conditions in vitro and used as a source for hypericin production. We used four A. rhizogenes strains differing in their plasmids and chromosomal backgrounds to inoculate excised H. sinaicum root, stem and leaf explants to induce hairy root development. Additionally, inoculum was applied to shoots held in Rockwool cubes supporting their stand after removal of the root system. All explant types were susceptible to A. rhizogenes although stem explants responded more frequently (over 90%) than other explant types. The A4 and A4T A. rhizogenes strains were highly, and equally effective in hairy root induction on 66-72% of explants while the LBA1334 strain was the most effective in transformation of shoots. Sonication applied to explants during inoculation enhanced the frequency of hairy root development, the most effective was 60 s treatment doubling the percentage of explants with hairy roots. However, shoot transformation was the most effective approach as shoots developed hairy roots within 10 days after inoculation. Molecular analyses confirmed that the established hairy root cultures in vitro were indeed obtained due to a horizontal gene transfer from bacteria. These cultures grew fast and the hypericin content in hairy roots was about two fold higher than in H. sinaicum plants as determined by HPLC.

Production of Rare Ginsenosides Rg3 and Rh2 by Endophytic Bacteria from Panax ginseng.

The ginsenosides Rh2 and Rg3 induce tumor cell apoptosis, inhibit tumor cell proliferation, and restrain tumor invasion and metastasis. Despite Rh2 and Rg3 having versatile pharmacological activities, contents of them in natural ginseng are extremely low. To produce ginsenosides Rh2 and Rg3, the saponin-producing capacity of endophytic bacteria isolated from Panax ginseng was investigated. In this work, 81 endophytic bacteria isolates were taken from ginseng roots by tissue separation methods. Among them, strain PDA-2 showed the highest capacity to produce the rare ginsenosides; the concentrations of rare ginsenosides Rg3 and Rh2 reached 62.20 and 18.60 mg/L, respectively. On the basis of phylogenetic analysis, it was found that strain PDA-2 belongs to the genus Agrobacterium and was very close to Agrobacterium rhizogenes.

Transgene stacking in potato using the GAANTRY system.

OBJECTIVE: GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). We examined the ability of the GAANTRY Agrobacterium rhizogenes ArPORT1 '10-stack' strain to generate transgenic potato plants. RESULTS: The 28.5 kilobase 10-stack T-DNA, was introduced into Lenape potato plants with a 32% transformation efficiency. Molecular and phenotypic characterization confirmed that six of the seven tested independent transgenic lines carried the entire desired construct, demonstrating that the GAANTRY 10-stack strain can be used can be used in a tissue culture-based callus transformation method to efficiently generate transgenic potato plants. Analysis using droplet digital PCR showed that most of the characterized events carry one or two copies of the 10-stack transgenes and that 'backbone' DNA from outside of the T-DNA was absent in the transgenic plants. These results demonstrate that the GAANTRY system efficiently generates high quality transgenic potato plants with a large construct of stacked transgenes.

Targeted metabolomic profiling reveals interspecific variation in the genus Hypericum in response to biotic elicitors.

Shoot cultures of eight Hypericum species belonging to the sections Hypericum, Oligostema, Ascyreia and Webbia were evaluated for their phytochemical profiles by high-performance liquid chromatography. In total, 17 secondary metabolites assigned to the groups of anthraquinones, phloroglucinols, hydroxycinnamic acids and flavonoids were detected. Furthermore, the elicitation potential of 18 biotic factors derived from saccharides, endophytic fungi and Agrobacterium rhizogenes was examined and statistically analysed with the paired two-sample t-test and principal component analysis. The production of naphthodianthrones and emodin was predominantly stimulated by elicitors derived from Fusarium oxysporum and Trichoderma crassum, while Piriformospora indica promoted the phloroglucinols production. Among flavonoids, the aglycone amentoflavone was readily increased by several elicitors up to 15.7-fold in H. humifusum treated by potato-dextrose broth. However, the chlorogenic acid proved to be the most susceptible metabolite to elicitation, when 31.7-times increase was detected in H. maculatum shoots upon D-glucose treatment. In spite of several biotic factors have been tested, no metabolite was commonly induced in all Hypericum spp. as a response to elicitor treatments.

Sodium nitroprusside stimulated production of tropane alkaloids and antioxidant enzymes activity in hairy root culture of Hyoscyamus reticulatus L.

Hyoscyamus reticulatus L. is a herbaceous biennial belonging to the solanaceae family. Hyoscyamine and scopolamine as main tropane alkaloids accumulated in henbane are widely used in medicine to treat diseases such as parkinson's or to calm schizoid patients. Hairy roots media manipulation which uses elicitors to activate defense mechanisms is one of the main strategies for inducing secondary metabolism as well as increasing the production of valuable metabolites. Cotyledon-derived hairy root cultures were transformed by Agrobacterium rhizogenes. Sodium nitroprusside (SNP), a nitric oxide donor), was used in various concentrations (0, 50, 100, 200 and 300 µM) and exposure times (24 and 48 h). Treatment with SNP led to a significant reduction in fresh and dry weight of hairy roots, compared to control cultures. ANOVA results showed that elicitation of hairy root cultures with SNP at different concentrations and exposure times significantly affected the activity of as antioxidant enzymes such as catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). The highest hyoscyamine and scopolamine production (about 1.2-fold and 1.5-fold increases over the control) was observed at 50 and 100 µM SNP at 48 and 24 hours of exposure time, respectively. This is the first report of SNP elicitation effects on the production of tropane alkaloids in hairy root cultures.

Transformed Root Culture: From Genetic Transformation to NMR-Based Metabolomics.

Hairy root (HR) culture is considered as "green factory" for mass production of bioactive molecules with pharmaceutical relevance. As such, HR culture has an immense potential as a valuable platform to elucidate biosynthetic pathways and physiological processes, generate recombinant therapeutic proteins, assist molecular breeding, and enhance phytoremediation efforts. However, some plant species appear recalcitrant to the classical Agrobacterium rhizogenes transformation techniques. Sonication-assisted Agrobacterium-mediated transformation (SAArT) is a highly effective method to deliver bacteria to target plant tissues that includes exposure of the explants to short periods of ultrasound in the presence of the bacteria.Nuclear magnetic resonance (NMR)-based metabolomics is one of the most powerful and suitable platforms for identifying and obtaining structural information on a wide range of compounds with a high analytical precision. In terms of plant science, NMR metabolomics is used to determine the phytochemical variations of medicinal plants or commercial cultivars in certain environments and conditions, including biotic stress and plant biotic interaction, structural determination of natural products, quality control of herbal drugs or dietary supplements, and comparison of metabolite differences between plants and their respective in vitro cultures.In this chapter, we attempt to summarize our knowledge and expertise in induction of hairy roots from rare and recalcitrant plant species by SAArT technique and further methodology for extraction of secondary metabolites of moderate to high polarity and their identification by using NMR-based metabolomics.

Genomic insights into metabolic potentials of two simultaneous aerobic denitrification and phosphorus removal bacteria, Achromobacter sp. GAD3 and Agrobacterium sp. LAD9.

Bacteria capable of simultaneous aerobic denitrification and phosphorus removal (SADPR) are promising for the establishment of novel one-stage wastewater treatment systems. Nevertheless, insights into the metabolic potential of SADPR-related bacteria are limited. Here, comprehensive metabolic models of two efficient SADPR bacteria, Achromobacter sp. GAD3 and Agrobacterium sp. LAD9, were obtained for the first time by high-throughput genome sequencing. With succinate as the preferred carbon source, both strains employed a complete TCA cycle as the major carbon metabolism for potentials of various organic acids and complex carbon oxidation. Complete and truncated aerobic denitrification routes were confirmed in GAD3 and LAD9, respectively, facilitated by all the major components of the electron transfer chain via oxidative phosphorylation. Comparative genome analysis revealed distinctive ecological niches involved in denitrification among different phylogenetic clades within Achromobacter and Agrobacterium. Excellent phosphorus removal capacities were contributed by inorganic phosphate uptake, polyphosphate synthesis and phosphonate metabolism. Additionally, the physiology of GAD3/LAD9 is different from that displayed by most available polyphosphate accumulating organisms, and reveals both strains to be more versatile, carrying out potentials for diverse organics degradation and outstanding SADPR capacity within a single organism. The functional exploration of SADPR bacteria broadens their significant prospects for application in concurrent aerobic carbon and nutrient removal.

The Stringent Response Induced by Phosphate Limitation Promotes Purine Salvage in Agrobacterium fabrum.

Agrobacterium fabrum induces tumor growth in susceptible plant species. The upregulation of virulence genes that occurs when the bacterium senses plant-derived compounds is enhanced by acidic pH and limiting inorganic phosphate. Nutrient starvation may also trigger the stringent response, and purine salvage is among the pathways expected to be favored under such conditions. We show here that phosphate limitation induces the stringent response, as evidenced by production of (p)ppGpp, and that the xdhCSML operon encoding the purine salvage enzyme xanthine dehydrogenase is upregulated ∼15-fold. The xdhCSML operon is under control of the TetR family transcription factor XdhR; direct binding of ppGpp to XdhR attenuates DNA binding, and the enhanced xdhCSML expression correlates with increased cellular levels of (p)ppGpp. Xanthine dehydrogenase may also divert purines away from salvage pathways to form urate, the ligand for the transcription factor PecS, which in the plant pathogen Dickeya dadantii is a key regulator of virulence gene expression. However, urate levels remain low under conditions that produce increased levels of xdhCSML expression, and neither acidic pH nor limiting phosphate results in induction of genes under control of PecS. Instead, expression of such genes is induced only by externally supplemented urate. Taken together, our data indicate that purine salvage is favored during the stringent response induced by phosphate starvation, suggesting that control of this pathway may constitute a novel approach to modulating virulence. Because bacterial purine catabolism appears to be unaffected, as evidenced by the absence of urate accumulation, we further propose that the PecS regulon is induced by only host-derived urate.

Neutralizing immune responses induced by oligomeric H5N1-hemagglutinins from plants.

Plant-based transient expression is an alternative platform to produce hemagglutinin-based subunit vaccines. This production system provides not only fast and effective response in the context of a pandemic but also enables the supply of big volume vaccines at low cost. Crude plant extracts containing influenza hemagglutinin are considered to use as vaccine sources because of avoidance of related purification steps resulting in low cost production allowing veterinary applications. Highly immunogenic influenza hemagglutinins are urgently required to meet these pre-conditions. Here, we present a new and innovative way to generate functional H5 oligomers from avian flu hemagglutinin in planta by the specific interaction of S·Tag and S·Protein. A S·Tag was fused to H5 trimers and this construct was transiently co-expressed in planta with S·Protein-TPs which was multimerized by disulfide bonds via cysteine residues in tailpiece sequences (TP) of IgM antibody. Multimerized S·Protein-TPs serve as bridges/molecular docks to combine S·Tag-fused hemagglutinin trimers to form very large hemagglutinin H5 oligomers. H5 oligomers in the plant crude extract were highly active in hemagglutination resulting in high titers. Immunization of mice with two doses of plant crude extracts containing H5 oligomers after storage for 1 week at 4 °C caused strong immune responses and induced neutralizing specific humoral immune responses in mice. These results allow for the development of cheap influenza vaccines for veterinary application in future.