Knockout of Tobacco Homologs of Arabidopsis Multi-Antibiotic Resistance 1 Gene Confers a Limited Resistance to Aminoglycoside Antibiotics.

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the ArabidopsisMulti-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacumMAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs-knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants' sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.

Enzyme Inhibitory, Antioxidant And Antibacterial Potentials Of Synthetic Symmetrical And Unsymmetrical Thioureas.

BACKGROUND: In this study, 2 symmetrical and 3 unsymmetrical thioureas were synthesized to evaluate their antioxidant, antibacterial, antidiabetic, and anticholinesterase potentials. METHODS: The symmetrical thioureas were synthesized in aqueous media in the presence of sunlight, using amines and CS2 as starting material. The unsymmetrical thioureas were synthesized using amines as a nucleophile to attack the phenyl isothiocyanate (electrophile). The structures of synthesized compounds were confirmed through H1 NMR. The antioxidant potential was determined using DPPH and ABTS assays. The inhibition of glucose-6-phosphatase, alpha amylase, and alpha glucosidase by synthesized compounds was used as an indication of antidiabetic potential. Anticholinesterase potential was determined from the inhibition of acetylcholinesterase and butyrylcholinesterase by the synthesized compounds. RESULTS: The highest inhibition of glucose-6-phosphatase was shown by compound V (03.12 mg of phosphate released). Alpha amylase was most potently inhibited by compound IV with IC50 value of 62 µg/mL while alpha glucosidase by compound III with IC50 value of 75 µg/mL. The enzymes, acetylcholinesterase, and butyrylcholinesterase were potently inhibited by compound III with IC50 of 63 µg/mL and 80 µg/mL respectively. Against DPPH free radical, compound IV was more potent (IC50 = 64 µg/mL) while ABTS was more potently scavenged by compound I with IC50 of 66 µg/mL. The antibacterial spectrum of synthesized compounds was determined against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Agrobacterium tumefaction and Proteus vulgaris). Compound I and compound II showed maximum activity against A. tumefaction with MIC values of 4.02 and 4.04 µg/mL respectively. Against P. vulgaris, compound V was more active (MIC = 8.94 µg/mL) while against S. aureus, compound IV was more potent with MIC of 4.03 µg/mL. CONCLUSION: From the results, it was concluded that these compounds could be used as antibacterial, antioxidant, and antidiabetic agents. However, further in vivo studies are needed to determine the toxicological effect of these compounds in living bodies. The compounds also have potential to treat neurodegenerative diseases.

Alleviation of chromium toxicity in maize by Fe fortification and chromium tolerant ACC deaminase producing plant growth promoting rhizobacteria.

Chromium (Cr) is becoming a potential pollutant with the passage of time. Higher intake of Cr does not only affect the productivity of crops, but also the quality of food produced in Cr polluted soils. In the past, foliar application of Fe is widely studied regarding their potential to alleviate Cr toxicity. However, limited information is documented regarding the combined use of PGPR and foliar Fe. Therefore, the current study was conducted to screen Cr tolerant PGPR and examine effect of foliar Fe with and without Cr tolerant PGPR under Cr toxicity (50 and 100 mg kg-1) in maize (Zea mays) production. Out of 15, two Cr tolerant PGPR were screened, identified (Agrobacterium fabrum and Leclercia adecarboxylata) and inoculated with 500 µM Fe. Results confirmed that Agrobacterium fabrum + 500 µM Fe performed significantly best in improving dry weight of roots and shoot, plant height, roots and shoot length and plant leaves in maize under Cr toxicity. A significant increase in chlorophyll a (51.5%), b (55.1%) and total (32.5%) validated the effectiveness of A. fabrum + 500 µM Fe to alleviate Cr toxicity. Improvement in intake of N (64.7%), P (70.0 and 183.3%), K (53.8% and 3.40-fold) in leaves and N (25.6 and 122.2%), P (25.6 and 122.2%), K (33.3% and 97.3%) in roots of maize at Cr50 and Cr100 confirmed that combined application of A. fabrum with 500 µM Fe is a more efficacious approach for alleviation of Cr toxicity and fortification of Fe comparative to sole foliar application of 500 µM Fe.

Interaction Analysis of Commercial Graphene Oxide Nanoparticles with Unicellular Systems and Biomolecules.

The ability of commercial monolayer graphene oxide (GO) and graphene oxide nanocolloids (GOC) to interact with different unicellular systems and biomolecules was studied by analyzing the response of human alveolar carcinoma epithelial cells, the yeast Saccharomyces cerevisiae and the bacteria Vibrio fischeri to the presence of different nanoparticle concentrations, and by studying the binding affinity of different microbial enzymes, like the α-l-rhamnosidase enzyme RhaB1 from the bacteria Lactobacillus plantarum and the AbG ß-d-glucosidase from Agrobacterium sp. (strain ATCC 21400). An analysis of cytotoxicity on human epithelial cell line A549, S. cerevisiae (colony forming units, ROS induction, genotoxicity) and V. fischeri (luminescence inhibition) cells determined the potential of both nanoparticle types to damage the selected unicellular systems. Also, the protein binding affinity of the graphene derivatives at different oxidation levels was analyzed. The reported results highlight the variability that can exist in terms of toxicological potential and binding affinity depending on the target organism or protein and the selected nanomaterial.

A Small Regulatory RNA Controls Cell Wall Biosynthesis and Antibiotic Resistance.

Small regulatory RNAs play an important role in the adaptation to changing conditions. Here, we describe a differentially expressed small regulatory RNA (sRNA) that affects various cellular processes in the plant pathogen Agrobacterium tumefaciens Using a combination of bioinformatic predictions and comparative proteomics, we identified nine targets, most of which are positively regulated by the sRNA. According to these targets, we named the sRNA PmaR for peptidoglycan biosynthesis, motility, and ampicillin resistance regulator. Agrobacterium spp. are long known to be naturally resistant to high ampicillin concentrations, and we can now explain this phenotype by the positive PmaR-mediated regulation of the beta-lactamase gene ampC Structure probing revealed a spoon-like structure of the sRNA, with a single-stranded loop that is engaged in target interaction in vivo and in vitro Several riboregulators have been implicated in antibiotic resistance mechanisms, such as uptake and efflux transporters, but PmaR represents the first example of an sRNA that directly controls the expression of an antibiotic resistance gene.IMPORTANCE The alphaproteobacterium Agrobacterium tumefaciens is able to infect various eudicots causing crown gall tumor formation. Based on its unique ability of interkingdom gene transfer, Agrobacterium serves as a crucial biotechnological tool for genetic manipulation of plant cells. The presence of hundreds of putative sRNAs in this organism suggests a considerable impact of riboregulation on A. tumefaciens physiology. Here, we characterized the biological function of the sRNA PmaR that controls various processes crucial for growth, motility, and virulence. Among the genes directly targeted by PmaR is ampC coding for a beta-lactamase that confers ampicillin resistance, suggesting that the sRNA is crucial for fitness in the competitive microbial composition of the rhizosphere.

Influence of Tween-80 on the production and structure of water-insoluble curdlan from Agrobacterium sp.

In order to explore the mechanism by which Tween-80 enhances the production of curdlan produced by Agrobacterium sp., the effects of Tween-80 on the production and structure of curdlan and Agrobacterium sp. were evaluated. Maximum curdlan production (51.94g/L) was achieved when 16g/L Tween-80 was added at the beginning of the cell growth stage. The addition of Tween-80 at higher concentration inhibited cell growth. However, the addition of 16g/L Tween-80 enhanced the production of curdlan with a looser ultrastructure, significantly weakened the envelopment of curdlan on Agrobacterium sp., altered the fine structure of cell membrane, and increased the cell membrane permeability. Moreover, the efficiency of oxygen and mass transport, respiration intensity, UTP regeneration, ATP regeneration, activity of curdlan synthetase, capacity of stress response and energy supply of Agrobacterium sp. were all greatly improved by the addition of Tween-80. These findings demonstrate the mechanisms by which Tween-80 enhances curdlan production and provide a cheap and feasible approach to weaken the envelopment of water-insoluble polysaccharides on bacteria.

The antimicrobial volatile power of the rhizospheric isolate Pseudomonas donghuensis P482.

Soil and rhizosphere bacteria produce an array of secondary metabolites including a wide range of volatile organic compounds (VOCs). These compounds play an important role in the long-distance interactions and communication between (micro)organisms. Furthermore, bacterial VOCs are involved in plant pathogens inhibition and induction of soil fungistasis and suppressivenes. In the present study, we analysed the volatile blend emitted by the rhizospheric isolate Pseudomonas donghuensis P482 and evaluated the volatile effect on the plant pathogenic fungi and bacteria as well as one oomycete. Moreover, we investigated the role of the GacS/GacA system on VOCs production in P. donghuensis P482. The results obtained demonstrated that VOCs emitted by P. donghuensis P482 have strong antifungal and antioomycete, but not antibacterial activity. The production of certain volatiles such as dimethyl sulfide, S-methyl thioacetate, methyl thiocyanate, dimethyl trisulfide, 1-undecan and HCN is depended on the GacS/GacA two-component regulatory system. Apparently, these compounds play an important role in the pathogens suppression as the gacA mutant entirely lost the ability to inhibit via volatiles the growth of tested plant pathogens.

Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis.

Tea (Camellia sinensis L.) is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir) gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS) basal salts medium supplemented with 30 g·L(-1) sucrose, 0.1 g·L(-1) l-glutamine and 5 g·L(-1) polyvinylpolypyrrolidone (PVPP) as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L(-1) sucrose). Additionally, the reporter genes ß-glucuronidase (gusA) and cyan fluorescent protein (cfp) were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties.

Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

An effective transformation protocol for Dunaliella, a ß-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 µM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 µM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

An extractive membrane biofilm reactor as alternative technology for the treatment of methyl tert-butyl ether contaminated water.

Among the strategies developed for contaminated groundwater bioremediation, those based on the use of bacteria adhering to inert supports and establishing biofilms have gained great importance in this field. Extractive membrane biofilm reactor (EMBFR) technology offers productive solutions for the removal of volatile and semi-volatile compounds. EMBFR technology is based on the use of extractive semipermeable membranes through which contaminants migrate to the biological compartment in which microorganisms with pollutant biotransformation and/or mineralization capacities can grow, forming an active biofilm on the membrane surface. The objective of this study was to assess the use of three bacterial strains (Paenibacillus sp. SH7 CECT 8558, Agrobacterium sp. MS2 CECT 8557, and Rhodococcus ruber EE6 CECT 8612), as inoculum in a lab-scale EMBFR running for 28 days under aerobic conditions to eliminate methyl tert-butyl ether (MTBE) from water samples. Three different hydraulic retention times (1, 6, and 12 h) were employed. MTBE degradation values were determined daily by a gas GC-MS technique, as well as suspended bacterial growth. The biofilm established by the bacterial strains on the semipermeable membrane was detected by Field-Emission Scanning Electron Microscopy (FESEM) at the end of each experiment. The acute toxicity of the treated effluents and biomedium was determined by Microtox© assay (EC50 ).The results achieved from the MTBE degradation, biofilm formation, and toxicity analysis indicated that bacterial strains MS2 and EE6 were the best options as selective inoculum, although further research is needed, particularly with regard to their possible use as a mixed culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1238-1245, 2016.

Enhanced cadmium accumulation and tolerance in transgenic tobacco overexpressing rice metal tolerance protein gene OsMTP1 is promising for phytoremediation.

One of the most grievous heavy metal pollutants in the environment is cadmium (Cd), which is not only responsible for the crop yield loss owing to its phytotoxicity, but also for the human health hazards as the toxic elements usually accumulate in the consumable parts of crop plants. In the present study, we aimed to isolate and functionally characterize the OsMTP1 gene from indica rice (Oryza sativa L. cv. IR64) to study its potential application for efficient phytoremediation of Cd. The 1257 bp coding DNA sequence (CDS) of OsMTP1 encodes a ∼46 kDa protein belonging to the cation diffusion facilitator (CDF) or metal tolerance/transport protein (MTP) family. The OsMTP1 transcript in rice plant was found to respond during external Cd stress. Heterologous expression of OsMTP1 in tobacco resulted in the reduction of Cd stress-induced phytotoxic effects, including growth inhibition, lipid peroxidation, and cell death. Compared to untransformed control, the transgenic tobacco plants showed enhanced vacuolar thiol content, indicating vacuolar localization of the sequestered Cd. The transgenic tobacco plants exhibited significantly higher biomass growth (2.2-2.8-folds) and hyperaccumulation of Cd (1.96-2.22-folds) compared to untransformed control under Cd exposure. The transgenic plants also showed moderate tolerance and accumulation of arsenic (As) upon exogenous As stress, signifying broad substrate specificity of OsMTP1. Together, findings of our research suggest that the transgenic tobacco plants overexpressing OsMTP1 with its hyperaccumulating activity and increased growth rate could be useful for future phytoremediation applications to clean up the Cd-contaminated soil.

Efficacy of hydrogen peroxide treatment for control of hairy root disease caused by rhizogenic agrobacteria.

AIMS: Rhizogenic Agrobacterium strains are the causative agent of hairy root disease (HRD), an increasing problem in the hydroponic cultivation of tomato and cucumber in Europe. A previous study has demonstrated that different lineages of rhizogenic agrobacteria are able to form biofilms. Although hydrogen peroxide (H2 O2 ) is a frequently used biocide in hydroponic systems, until now its effectiveness to remove rhizogenic agrobacteria has not been unequivocally demonstrated. Therefore, the main objective of this study was to assess the efficacy of H2 O2 in controlling Agrobacterium-containing biofilms. METHODS AND RESULTS: Using lab-scale experiments, we found a huge variation between different rhizogenic Agrobacterium strains in EC50 values, ranging from 18·8 to 600 ppm H2 O2 , representing the lowest and highest concentration tested respectively. Using pilot-scale experiments in which different H2 O2 concentrations were tested, treatment with 25 ppm H2 O2 was found to be ineffective. In contrast, treatment with 50 ppm significantly affected a catalase-negative Agrobacterium population, while a catalase-positive population was only marginally affected. For the catalase-positive Agrobacterium population, a treatment of 100 ppm H2 O2 was required to be effective. Finally, H2 O2 treatment of HRD in two commercial greenhouses was monitored, and showed that the H2 O2 concentration decreased considerably towards the end of the irrigation circuits. Further, a clear correlation was found between the actual concentration of H2 O2 and the incidence of HRD. CONCLUSION: We showed that H2 O2 may be effective to reduce biofilm formation by rhizogenic bacteria. Nevertheless, it was clear from our results that the required H2 O2 concentration depends on the particular Agrobacterium strain(s) present in the greenhouse. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that examined the effectiveness of H2 O2 to control HRD in hydroponic systems, and the effect of catalase activity on H2 O2 effectiveness. Our study has direct relevance for the highly intensive horticultural sector.

Simultaneous denitrification and phosphorus removal by Agrobacterium sp. LAD9 under varying oxygen concentration.

Although efficient aerobic denitrification has received increasing attention, few studies have been made on simultaneous denitrification and phosphorus removal (SDPR) under aerobic condition. In this study, SDPR by an efficient aerobic denitrifier, Agrobacterium sp. LAD9, was firstly demonstrated. High nitrate and phosphorus removal rates of 7.50 and 1.02 mg L(-1) h(-1) were achieved in wide range of O2 concentration from 5.92 to 20.02 mg L(-1). The N2O production would be inhibited as O2 concentration exceeded 11.06 mg L(-1), while the phosphorus removal efficiency would be generally improved with increasing O2 concentration. (15)N mass spectrometry revealed that nitrogen removal accorded with the typical aerobic denitrification pathway, while (31)P nuclear magnetic resonance spectroscopy ((31)P NMR) indicated the fate of phosphorus to cells, extracellular polymeric substances (EPS), and polyphosphate (poly-P) of the denitrifier. EPS acted as a reservoir of phosphorus and the transformation of poly-P was dynamic and depended on initial orthophosphate (ortho-P) content. The aerobic SDPR would greatly simplify the conventional wastewater treatment processes which required separated considerations of nitrogen and phosphorus removal.

A novel inclusion complex (ß-CD/ABP-dHC-cecropin A) with antibiotic propertiess for use as an anti-Agrobacterium additive in transgenic poplar rooting medium.

The increasing resistance of bacteria and fungi to currently available antibiotics is a major concern worldwide, leading to enormous effort to develop novel antibiotics with new modes of action.We recently reported that ABP-dHC-cecropin A exhibited strong antibacterial and antifungal activity, making it a candidate antibiotic substitute. In this study, ß-cyclodextrin (ß-CD) combined with ABP-dHC-cecropin A enhanced the physical and chemical properties of ABP-dHC-cecropin A but did not significantly decrease its antibacterial activity. Thus, ß-CD/ABP-dHC-cecropin A should be considered a novel antibacterial drug. We used ß-CD/ABP-dHC-cecropin A as an anti-Agrobacterium compound to supplementtransgenic poplar medium. Sideeffects of the inclusion complex had little impact on plantgrowth. Thus, ß-CD/ABP-dHC-cecropin A may be used as traditional antibiotics forpoplar transplantation with greater antibbacterial effects.

Agrobacterium-mediated transformation of tomato with the ICE1 transcription factor gene.

ICE1 genes play a very important role in plants in cold conditions. To improve the cold resistance of tomato, the ICE1 gene of Arabidopsis thaliana was used to construct the plant expression vector p3301-ICE1, and was overexpressed in tomato through Agrobacterium-mediated transformation. Five strains of resistant plants were obtained. PCR and half-quantitative results showed that the ICE1 gene was transferred to tomato; three strains tested positive. After low-temperature stress treatment, praline content and peroxide and catalase activities in the transgenic tomato plants were higher compared with non-transgenic controls, while malondialdehyde content was clearly lower.

Inside out: high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars.

KEY MESSAGE: Selection of pre-embryogenic callus from a core structure from mature seed-derived callus is the key for high-efficiency plant regeneration and transformation of switchgrass different cultivars. Switchgrass (Panicum virgatum L.) has been identified as a dedicated biofuel crop. For its trait improvement through biotechnological approaches, we have developed a highly efficient plant regeneration and genetic transformation protocol for both lowland and upland cultivars. We identified and separated a pre-embryogenic "core" structure from the seed-derived callus, which often leads to development of highly regenerative type II calluses. From the type II callus, plant regeneration rate of lowland cultivars Alamo and Performer reaches 95%, and upland cultivars Blackwell and Dacotah, 50 and 76%, respectively. The type II callus was also amenable for Agrobacterium-mediated transformation. Transformation efficiency of 72.8% was achieved for lowland cultivar Alamo, and 8.0% for upland cultivar Dacotah. PCR, Southern blot and GUS staining assays were performed to verify the transgenic events. High regenerative callus lines could be established in 3 months, and transgenic plants could be obtained in 2 months after Agrobacterium infection. To our knowledge, this is the first report on successful plant regeneration and recovery of transgenic plants from upland switchgrass cultivars by Agrobacterium-mediated transformation. The method presented here could be helpful in breaking through the bottleneck of regeneration and transformation of lowland and upland switchgrass cultivars and probably other recalcitrant grass crops.

Molecular structure, chemical synthesis, and antibacterial activity of ABP-dHC-cecropin A from drury (Hyphantria cunea).

The increasing resistance of bacteria and fungi to currently available antibiotics is a major concern worldwide, leading to enormous efforts to develop new antibiotics with new modes of actions. In this paper, cDNA encoding cecropin A was amplified from drury (Hyphantria cunea) (dHC) pupa fatbody total RNA using RT-PCR. The full-length dHC-cecropin A cDNA encoded a protein of 63 amino acids with a predicted 26-amino acid signal peptide and a 37-amino acid functional domain. We synthesized the antibacterial peptide (ABP) from the 37-amino acid functional domain (ABP-dHC-cecropin A), and amidated it via the C-terminus. Time-of-flight mass spectrometry showed its molecular weight to be 4058.94. The ABP-dHC-cecropin A was assessed in terms of its protein structure using bioinformatics and CD spectroscopy. The protein's secondary structure was predicted to be α-helical. In an antibacterial activity analysis, the ABP-dHC-cecropin A exhibited strong antibacterial activity against E. coli K12D31 and Agrobacterium EHA105.

Biological activities of the antiviral protein BE27 from sugar beet (Beta vulgaris L.).

MAIN CONCLUSION: The ribosome inactivating protein BE27 displays several biological activities in vitro that could result in a broad action against several types of pathogens. Beetin 27 (BE27), a ribosome-inactivating protein (RIP) from sugar beet (Beta vulgaris L.) leaves, is an antiviral protein induced by virus and signaling compounds such as hydrogen peroxide and salicylic acid. Its role as a defense protein has been attributed to its RNA polynucleotide:adenosine glycosidase activity. Here we tested other putative activities of BE27 that could have a defensive role against pathogens finding that BE27 displays rRNA N-glycosidase activity against yeast and Agrobacterium tumefaciens ribosomes, DNA polynucleotide:adenosine glycosidase activity against herring sperm DNA, and magnesium-dependent endonuclease activity against the supercoiled plasmid PUC19 (nicking activity). The nicking activity could be a consequence of an unusual conformation of the BE27 active site, similar to that of PD-L1, a RIP from Phytolacca dioica L. leaves. Additionally, BE27 possesses superoxide dismutase activity, thus being able to produce the signal compound hydrogen peroxide. BE27 is also toxic to COLO 320 cells, inducing apoptosis in these cells by either activating the caspase pathways and/or inhibiting protein synthesis. The combined effect of these biological activities could result in a broad action against several types of pathogens such as virus, bacteria, fungi or insects.

Composite potato plants with transgenic roots on non-transgenic shoots: a model system for studying gene silencing in roots.

KEY MESSAGE: Composite potato plants offer an extremely fast, effective and reliable system for studies on gene functions in roots using antisense or inverted-repeat but not sense constructs for gene inactivation. Composite plants, with transgenic roots on a non-transgenic shoot, can be obtained by shoot explant transformation with Agrobacterium rhizogenes. The aim of this study was to generate composite potato plants (Solanum tuberosum) to be used as a model system in future studies on root-pathogen interactions and gene silencing in the roots. The proportion of transgenic roots among the roots induced was high (80-100%) in the four potato cultivars tested (Albatros, Desirée, Sabina and Saturna). No wild-type adventitious roots were formed at mock inoculation site. All strains of A. rhizogenes tested induced phenotypically normal roots which, however, showed a reduced response to cytokinin as compared with non-transgenic roots. Nevertheless, both types of roots were infected to a similar high rate with the zoospores of Spongospora subterranea, a soilborne potato pathogen. The transgenic roots of composite potato plants expressed significantly higher amounts of ß-glucuronidase (GUS) than the roots of a GUS-transgenic potato line event. Silencing of the uidA transgene (GUS) was tested by inducing roots on the GUS-transgenic cv. Albatros event with strains of A. rhizogenes over-expressing either the uidA sense or antisense transcripts, or inverted-repeat or hairpin uidA RNA. The three last mentioned constructs caused 2.5-4.0 fold reduction in the uidA mRNA expression. In contrast, over-expression of uidA resulted in over 3-fold increase in the uidA mRNA and GUS expression, indicating that sense-mediated silencing (co-suppression) was not functional in roots. The results suggest that composite plants offer a useful experimental system for potato research, which has gained little previous attention.

Inhibitory and toxic effects of volatiles emitted by strains of Pseudomonas and Serratia on growth and survival of selected microorganisms, Caenorhabditis elegans, and Drosophila melanogaster.

In previous research, volatile organic compounds (VOCs) emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans). VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds--ketones (2-nonanone, 2-heptanone, 2-undecanone) and dimethyl disulfide--were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions.