Hydroponic Common-Bean Performance under Reduced N-Supply Level and Rhizobia Application.

This study aims to explore the possibility of a reduced application of inorganic nitrogen (N) fertiliser on the yield, yield qualities, and biological nitrogen fixation (BNF) of the hydroponic common bean (Phaseolus vulgaris L.), without compromising plant performance, by utilizing the inherent ability of this plant to symbiotically fix N2. Until the flowering stage, plants were supplied with a nutrient solution containing N-concentrations of either a, 100%, conventional standard-practice, 13.8 mM; b, 75% of the standard, 10.35 mM; or c, 50% of the standard, 6.9 mM. During the subsequent reproductive stage, inorganic-N treatments b and c were decreased to 25% of the standard, and the standard (100% level) N-application was not altered. The three different inorganic-N supply treatments were combined with two different rhizobia strains, and a control (no-inoculation) treatment, in a two-factorial experiment. The rhizobia strains applied were either the indigenous strain Rhizobium sophoriradicis PVTN21 or the commercially supplied Rhizobium tropici CIAT 899. Results showed that the 50-25% mineral-N application regime led to significant increases in nodulation, BNF, and fresh-pod yield, compared to the other treatment, with a reduced inorganic-N supply. On the other hand, the 75-25% mineral-N regime applied during the vegetative stage restricted nodulation and BNF, thus incurring significant yield losses. Both rhizobia strains stimulated nodulation and BNF. However, the BNF capacity they facilitated was suppressed as the inorganic-N input increased. In addition, strain PVTN21 was superior to CIAT 899-as 50-25% N-treated plants inoculated with the former showed a yield loss of 11%, compared to the 100%-N-treated plants. In conclusion, N-use efficiency optimises BNF, reduces mineral-N-input dependency, and therefore may reduce any consequential negative environmental consequences of mineral-N over-application.

Legume-Based Mobile Green Manure Can Increase Soil Nitrogen Availability and Yield of Organic Greenhouse Tomatoes.

Information about the availability of soil mineral nitrogen (N) in organic greenhouse tomatoes after the application of mobile green manure (MGM), and its impact on plant nutrient status and yield is scarce. Considering this knowledge gap, the effects of legume biomass from faba beans that are cultivated outdoors (FAB), or from feed-grade alfalfa pellets at two different doses (AAL = 330 g m-2; AAH = 660 g m-2) that were applied as MGM on the nutrition and yield of an organic greenhouse crop of tomatoes were evaluated. All of the MGM treatments increased the mineral N concentrations in the soil throughout the cropping period, and the total N concentration in tomato leaves when compared to the untreated control. FAB and AAH treatments had a stronger impact than AAL in all of the measured parameters. In addition, AAL, AAH, and FAB treatments increased the yield compared to the control by 19%, 33%, and 36%, respectively. The application of MGM, either as faba bean fresh biomass or as alfalfa dry pellets, in organic greenhouse tomatoes significantly increased the plant available soil N, improved N nutrition, and enhanced the fruit yield. However, the N mineralization rates after the MGM application were excessive during the initial cropping stages, followed by a marked decrease thereafter. This may impose an N deficiency during the late cropping period.

Impact of Plant Growth-Promoting Rhizobacteria Inoculation and Grafting on Tolerance of Tomato to Combined Water and Nutrient Stress Assessed via Metabolomics Analysis.

In the current study, inoculation with plant growth-promoting rhizobacteria (PGPR) and grafting were tested as possible cultural practices that may enhance resilience of tomato to stress induced by combined water and nutrient shortage. The roots of tomato grown on perlite were either inoculated or not with PGPR, applying four different treatments. These were PGPR-T1, a mix of two Enterobacter sp. strains (C1.2 and C1.5); PGPR-T2, Paenibacillus sp. strain DN1.2; PGPR-T3, Enterobacter mori strain C3.1; and PGPR-T4, Lelliottia sp. strain D2.4. PGPR-treated plants were either self-grafted or grafted onto Solanum lycopersicum cv. M82 and received either full or 50% of their standard water, nitrogen, and phosphorus needs. The vegetative biomass of plants subjected to PGPR-T1 was not reduced when plants were cultivated under combined stress, while it was reduced by stress to the rest of the PGPR treatments. However, PGPR-T3 increased considerably plant biomass of non-stressed tomato plants than did all other treatments. PGPR application had no impact on fruit biomass, while grafting onto 'M82' increased fruit production than did self-grafting. Metabolomics analysis in tomato leaves revealed that combined stress affects several metabolites, most of them already described as stress-related, including trehalose, myo-inositol, and monopalmitin. PGPR inoculation with E. mori strain C3.1 affected metabolites, which are important for plant/microbe symbiosis (myo-inositol and monopalmitin). The rootstock M82 did not affect many metabolites in plant leaves, but it clearly decreased the levels of malate and D-fructose and imposed an accumulation of oleic acid. In conclusion, PGPR are capable of increasing tomato tolerance to combined stress. However, further research is required to evaluate more strains and refine protocols for their application. Metabolites that were discovered as biomarkers could be used to accelerate the screening process for traits such as stress tolerance to abiotic and/or abiotic stresses. Finally, 'M82' is a suitable rootstock for tomato, as it is capable of increasing fruit biomass production.

Genetic characterization at the species and symbiovar level of indigenous rhizobial isolates nodulating Phaseolus vulgaris in Greece.

Phaseolus vulgaris (L.), commonly known as bean or common bean, is considered a promiscuous legume host since it forms nodules with diverse rhizobial species and symbiovars. Most of the common bean nodulating rhizobia are mainly affiliated to the genus Rhizobium, though strains belonging to Ensifer, Pararhizobium, Mesorhizobium, Bradyrhizobium, and Burkholderia have also been reported. This is the first report on the characterization of bean-nodulating rhizobia at the species and symbiovar level in Greece. The goals of this research were to isolate and characterize rhizobia nodulating local common bean genotypes grown in five different edaphoclimatic regions of Greece with no rhizobial inoculation history. The genetic diversity of the rhizobial isolates was assessed by BOX-PCR and the phylogenetic affiliation was assessed by multilocus sequence analysis (MLSA) of housekeeping and symbiosis-related genes. A total of fifty fast-growing rhizobial strains were isolated and representative isolates with distinct BOX-PCR fingerpriniting patterns were subjected to phylogenetic analysis. The strains were closely related to R. anhuiense, R. azibense, R. hidalgonense, R. sophoriradicis, and to a putative new genospecies which is provisionally named as Rhizobium sp. I. Most strains belonged to symbiovar phaseoli carrying the α-, γ-a and γ-b alleles of nodC gene, while some of them belonged to symbiovar gallicum. To the best of our knowledge, it is the first time that strains assigned to R. sophoriradicis and harbored the γ-b allele were found in European soils. All strains were able to re-nodulate their original host, indicating that they are true microsymbionts of common bean.

Impact of Legumes as a Pre-Crop on Nitrogen Nutrition and Yield in Organic Greenhouse Tomato.

An organic greenhouse crop of tomato was established in February following cultivation of cowpea (CP) or common bean (CB) for green pod production, or faba bean (FB) for green manuring. The vegetative residues of CP and CB were incorporated to the soil together with farmyard manure (FYM), prior to establishing the tomato crop. The FB plants were incorporated to the soil at anthesis together with either FYM or composted olive-mill waste (CO). Green manuring with FB resulted in higher soil mineral N levels during the subsequent tomato crop and higher tomato fruit yield when combined with FYM, compared to compost. The level of soil mineral N was the main restrictive factor for yield in organic greenhouse tomato. FB for green manuring as preceding crop to tomato increased significantly the level of soil mineral N and tomato yield compared to CB or CP aiming to produce green pods. The lowest tomato yield was obtained when the preceding crop was CB cultivated for green pod production. The soil mineral N was significantly higher when FYM was applied as base dressing compared with CO, despite the higher total N concentration in CO, pointing to slower mineralization rates of CO during tomato cultivation.

Defining the Rhizobium leguminosarum Species Complex.

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a 'natural' unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, "R. indicum" and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.

Genetic diversity and phylogeny of indigenous rhizobia nodulating faba bean (Vicia faba L.) in Greece.

The genetic diversity and phylogeny of fast-growing rhizobia isolated from root nodules of Vicia faba grown in different geographical regions of Greece were assessed. Although Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia spp. in European soils, there is no available information on native rhizobia nodulating faba bean in Greece. Seventy bacterial strains were isolated and grouped into sixteen distinct profiles based on BOX-PCR fingerprinting. The phylogenetic affiliation was further defined by sequence analysis of the rrs and multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and gyrB). Fifty-eight isolates were affiliated with recently described genospecies gsF-2, represented by R. laguerreae FB206T, whereas six isolates were closely related to gsB and two isolates might belong to gsA. Two isolates assigned to R. hidalgonense and another two non-nodulating strains could not be assigned to any validly defined species and possibly belong to a new rhizobial lineage. Interestingly, R. laguerreae strains were commonly found at all sampling sites, suggesting that they could be the main symbionts of faba beans in Greek soils. According to the phylogenies of two symbiosis-related genes (nodC and nifH), all nodulating isolates belonged to symbiovar (sv.) viciae harboring four distinct nodC gene haplotypes and they were grouped into two clades together with strains assigned to R. laguerreae and genospecies of R. leguminosarum isolated from other countries and continents. This is the first report that R. hidalgonense strains belong to sv. viciae. No correlation was observed between the nodC haplotypes, geographic origin and chromosomal background of the isolates in the study.

Monitoring Biofilm Formation and Microbial Interactions that May Occur During a Salmonella Contamination Incident across the Network of a Water Bottling Plant.

The present study aims to monitor the ability of Salmonella to colonize and compete as a member of the mixed species biofilm within key points at a water bottling plant, in case of a contamination incident with this major foodborne pathogen. To achieve this goal, bacterial communities throughout the production line were collected and their identities were investigated by microbial counts and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). These bacterial communities alone or along with constructed Salmonella enterica serovar Typhimurium (ST) fluorescence-based bioreporters were left to form a biofilm on stainless steel for 6 days at 20 °C. ST bioreporters were constructed by introducing plasmids expressing EYFP (enhanced yellow fluorescent protein) fusions of the genes csgB, csrA, sspH2, and fliD into ST 14028S. The bead vortexing-plate counting method was applied for the enumeration of the biofilm population, while the behavior of the bioreporters was evaluated by fluorescence microscopy. From a set of 16 samples that were collected from the plant, species of Citrobacter, Staphylococcus, Pseudomonas, Bacillus, and Exiguobacterium were identified. The presence of these indigenous bacteria neither inhibited nor enhanced the biofilm formation of ST in mixed bacterial communities (p > 0.05). Furthermore, the csrA-based bioreporter was shown to be induced in multispecies biofilms with Citrobacter. In conclusion, this study enhanced our knowledge of bacterial interactions occurring within a biofilm in a water bottling plant.

Image analysis driven single-cell analytics for systems microbiology.

BACKGROUND: Time-lapse microscopy is an essential tool for capturing and correlating bacterial morphology and gene expression dynamics at single-cell resolution. However state-of-the-art computational methods are limited in terms of the complexity of cell movies that they can analyze and lack of automation. The proposed Bacterial image analysis driven Single Cell Analytics (BaSCA) computational pipeline addresses these limitations thus enabling high throughput systems microbiology. RESULTS: BaSCA can segment and track multiple bacterial colonies and single-cells, as they grow and divide over time (cell segmentation and lineage tree construction) to give rise to dense communities with thousands of interacting cells in the field of view. It combines advanced image processing and machine learning methods to deliver very accurate bacterial cell segmentation and tracking (F-measure over 95%) even when processing images of imperfect quality with several overcrowded colonies in the field of view. In addition, BaSCA extracts on the fly a plethora of single-cell properties, which get organized into a database summarizing the analysis of the cell movie. We present alternative ways to analyze and visually explore the spatiotemporal evolution of single-cell properties in order to understand trends and epigenetic effects across cell generations. The robustness of BaSCA is demonstrated across different imaging modalities and microscopy types. CONCLUSIONS: BaSCA can be used to analyze accurately and efficiently cell movies both at a high resolution (single-cell level) and at a large scale (communities with many dense colonies) as needed to shed light on e.g. how bacterial community effects and epigenetic information transfer play a role on important phenomena for human health, such as biofilm formation, persisters' emergence etc. Moreover, it enables studying the role of single-cell stochasticity without losing sight of community effects that may drive it.

A novel symbiovar (aegeanense) of the genus Ensifer nodulates Vigna unguiculata.

BACKGROUND: Cowpea (Vigna unguiculata) forms nitrogen-fixing root nodules with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium, although a few studies have reported the isolation of fast-growing rhizobia under laboratory and field conditions. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, very limited information is available on cowpea rhizobia in European soils. The aim of this study was to study the genetic and phenotypic diversity of indigenous cowpea-nodulating rhizobia in Greece. RESULTS: The genetic diversity of indigenous rhizobia associated with cowpea was investigated through a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into three groups. Based on the analysis of the 16S rRNA genes, IGS and on the concatenation of six housekeeping genes (recA, glnII, gyrB, truA, thrA and SMc00019), rhizobial isolates were classified within the species Ensifer fredii. However, symbiotic gene phylogenies, based on nodC, nifH and rhcRST genes, showed that the Ensifer isolates are markedly diverged from type and reference strains of E. fredii and formed one clearly separate cluster. The E. fredii strains were able to nodulate and fix nitrogen in cowpea but not in soybean and common bean. CONCLUSION: The present study showed that cowpea is nodulated under field conditions by fast-growing rhizobia belonging to the species E. fredii. Based on the phylogenies, similarity levels of symbiotic genes and the host range, the Ensifer isolates may constitute a new symbiovar for which the name 'aegeanense' is proposed. © 2017 Society of Chemical Industry.

Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece.

Cowpea (Vigna unguiculata) is a promiscuous grain legume, capable of establishing efficient symbiosis with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, little is known about the genetic and symbiotic diversity of indigenous cowpea rhizobia in European soils. In the present study, the genetic and symbiotic diversity of indigenous rhizobia isolated from field-grown cowpea nodules in three geographically different Greek regions were studied. Forty-five authenticated strains were subjected to a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into seven groups and representative strains of each group were further analyzed. The analysis of the rrs gene showed that the strains belong to different species of the genus Bradyrhizobium. The analysis of the 16S-23S IGS region showed that the strains from each geographic region were characterized by distinct IGS types which may represent novel phylogenetic lineages, closely related to the type species of Bradyrhizobium pachyrhizi, Bradyrhizobium ferriligni and Bradyrhizobium liaoningense. MLSA analysis of three housekeeping genes (recA, glnII, and gyrB) showed the close relatedness of our strains with B. pachyrhizi PAC48T and B. liaoningense USDA 3622T and confirmed that the B. liaoningense-related isolate VUEP21 may constitute a novel species within Bradyrhizobium. Moreover, symbiotic gene phylogenies, based on nodC and nifH genes, showed that the B. pachyrhizi-related isolates belonged to symbiovar vignae, whereas the B. liaoningense-related isolates may represent a novel symbiovar.

Impact of rhizobial inoculation and reduced N supply on biomass production and biological N2 fixation in common bean grown hydroponically.

BACKGROUND: Testing rhizobial inoculation of common bean (Phaseolus vulgaris L.) in hydroponics enables accurate quantification of biological N2 fixation (BNF) and provides information about the potential of reducing inorganic N fertilizer use. In view of this background, common bean grown on pumice was inoculated with Rhizobium tropici CIAT899 (Rt) and supplied with either full-N (total nitrogen 11.2 mmol L-1 ), 1/3 of full-N or N-free nutrient solution (NS). BNF was quantified at the early pod-filling stage using the 15 N natural abundance method. RESULTS: Full-N supply to Rt-inoculated plants resulted in markedly smaller nodules than less- or zero-N supply, and no BNF. Rt inoculation of full-N-treated plants did not increase biomass and pod yield compared with non-inoculation. Restriction (1/3 of full-N) or omission of inorganic N resulted in successful nodulation and BNF (54.3 and 49.2 kg N ha-1 , corresponding to 58 and 100% of total plant N content respectively) but suppressed dry shoot biomass from 191.7 (full-N, +Rt) to 107.4 and 43.2 g per plant respectively. Nutrient cation uptake was reduced when inorganic N supply was less or omitted. CONCLUSION: Rt inoculation of hydroponic bean provides no advantage when full-N NS is supplied, while 1/3 of full-N or N-free NS suppresses plant biomass and yield, partly because the restricted NO3- supply impairs cation uptake. © 2017 Society of Chemical Industry.

The nitrogen-fixation island insertion site is conserved in diazotrophic Pseudomonas stutzeri and Pseudomonas sp. isolated from distal and close geographical regions.

The presence of nitrogen fixers within the genus Pseudomonas has been established and so far most isolated strains are phylogenetically affiliated to Pseudomonas stutzeri. A gene ortholog neighborhood analysis of the nitrogen fixation island (NFI) in four diazotrophic P. stutzeri strains and Pseudomonas azotifigens revealed that all are flanked by genes coding for cobalamin synthase (cobS) and glutathione peroxidise (gshP). The putative NFIs lack all the features characterizing a mobilizable genomic island. Nevertheless, bioinformatic analysis P. stutzeri DSM 4166 NFI demonstrated the presence of short inverted and/or direct repeats within both flanking regions. The other P. stutzeri strains carry only one set of repeats. The genetic diversity of eleven diazotrophic Pseudomonas isolates was also investigated. Multilocus sequence typing grouped nine isolates along with P. stutzeri and two isolates are grouped in a separate clade. A Rep-PCR fingerprinting analysis grouped the eleven isolates into four distinct genotypes. We also provided evidence that the putative NFI in our diazotrophic Pseudomonas isolates is flanked by cobS and gshP genes. Furthermore, we demonstrated that the putative NFI of Pseudomonas sp. Gr65 is flanked by inverted repeats identical to those found in P. stutzeri DSM 4166 and while the other P. stutzeri isolates harbor the repeats located in the intergenic region between cobS and glutaredoxin genes as in the case of P. stutzeri A1501. Taken together these data suggest that all putative NFIs of diazotrophic Pseudomonas isolates are anchored in an intergenic region between cobS and gshP genes and their flanking regions are designated by distinct repeats patterns. Moreover, the presence of almost identical NFIs in diazotrophic Pseudomonas strains isolated from distal geographical locations around the world suggested that this horizontal gene transfer event may have taken place early in the evolution.

Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria.

Plant pathogenic bacteria and rhizobia infect higher plants albeit the interactions with their hosts are principally distinct and lead to completely different phenotypic outcomes, either pathogenic or mutualistic, respectively. Bacterial protein delivery to plant host plays an essential role in determining the phenotypic outcome of plant-bacteria interactions. The involvement of type III secretion systems (T3SSs) in mediating animal- and plant-pathogen interactions was discovered in the mid-80's and is now recognized as a multiprotein nanomachine dedicated to trans-kingdom movement of effector proteins. The discovery of T3SS in bacteria with symbiotic lifestyles broadened its role beyond virulence. In most T3SS-positive bacterial pathogens, virulence is largely dependent on functional T3SSs, while in rhizobia the system is dispensable for nodulation and can affect positively or negatively the mutualistic associations with their hosts. This review focuses on recent comparative genome analyses in plant pathogens and rhizobia that uncovered similarities and variations among T3SSs in their genetic organization, regulatory networks and type III secreted proteins and discusses the evolutionary adaptations of T3SSs and type III secreted proteins that might account for the distinguishable phenotypes and host range characteristics of plant pathogens and symbionts.

Characterization of the biocontrol activity of pseudomonas fluorescens strain X reveals novel genes regulated by glucose.

Pseudomonas fluorescens strain X, a bacterial isolate from the rhizosphere of bean seedlings, has the ability to suppress damping-off caused by the oomycete Pythium ultimum. To determine the genes controlling the biocontrol activity of strain X, transposon mutagenesis, sequencing and complementation was performed. Results indicate that, biocontrol ability of this isolate is attributed to gcd gene encoding glucose dehydrogenase, genes encoding its co-enzyme pyrroloquinoline quinone (PQQ), and two genes (sup5 and sup6) which seem to be organized in a putative operon. This operon (named supX) consists of five genes, one of which encodes a non-ribosomal peptide synthase. A unique binding site for a GntR-type transcriptional factor is localized upstream of the supX putative operon. Synteny comparison of the genes in supX revealed that they are common in the genus Pseudomonas, but with a low degree of similarity. supX shows high similarity only to the mangotoxin operon of Ps. syringae pv. syringae UMAF0158. Quantitative real-time PCR analysis indicated that transcription of supX is strongly reduced in the gcd and PQQ-minus mutants of Ps. fluorescens strain X. On the contrary, transcription of supX in the wild type is enhanced by glucose and transcription levels that appear to be higher during the stationary phase. Gcd, which uses PQQ as a cofactor, catalyses the oxidation of glucose to gluconic acid, which controls the activity of the GntR family of transcriptional factors. The genes in the supX putative operon have not been implicated before in the biocontrol of plant pathogens by pseudomonads. They are involved in the biosynthesis of an antimicrobial compound by Ps. fluorescens strain X and their transcription is controlled by glucose, possibly through the activity of a GntR-type transcriptional factor binding upstream of this putative operon.

Functional characterization of NopT1 and NopT2, two type III effectors of Bradyrhizobium japonicum.

NopT1 and NopT2, putative type III effectors from the plant symbiotic bacterium Bradyrhizobium japonicum, are predicted to belong to a family of YopT/AvrPphB effectors, which are cysteine proteases. In the present study, we showed that both NopT1 and NopT2 indeed possess cysteine protease activity. When overexpressed in Escherichia coli, both NopT1 and NopT2 undergo autoproteolytic processing which is largely abolished in the presence of E-64, a papain family-specific inhibitor. Mutations of NopT1 disrupting either the catalytic triad or the putative autoproteolytic site reduce or markedly abolish the protease activity. Autocleavage likely occurs between residues K48 and M49, though another potential cleavage site is also possible. NopT1 also elicitis HR-like cell death when transiently expressed in tobacco plants and its cysteine protease activity is essential for this ability. In contrast, no macroscopic symptoms were observed for NopT2. Furthermore, mutational analysis provided evidence that NopT1 may undergo acylation inside plant cells and that this would be required for its capacity to elicit HR-like cell death in tobacco.

High level resistance against rhizomania disease by simultaneously integrating two distinct defense mechanisms.

With the aim of achieving durable resistance against rhizomania disease of sugar beet, the employment of different sources of resistance to Beet necrotic yellow vein virus was pursued. To this purpose, Nicotiana benthamiana transgenic plants that simultaneously produce dsRNA originating from a conserved region of the BNYVV replicase gene and the HrpZ(Psph) protein in a secreted form (SP/HrpZ(Psph)) were produced. The integration and expression of both transgenes as well as proper production of the harpin protein were verified in all primary transformants and selfed progeny (T1, T2). Transgenic resistance was assessed by BNYVV-challenge inoculation on T2 progeny by scoring disease symptoms and DAS-ELISA at 20 and 30 dpi. Transgenic lines possessing single transformation events for both transgenes as well as wild type plants were included in inoculation experiments. Transgenic plants were highly resistant to virus infection, whereas in some cases immunity was achieved. In all cases, the resistant phenotype of transgenic plants carrying both transgenes was superior in comparison with the ones carrying a single transgene. Collectively, our findings demonstrate, for a first time, that the combination of two entirely different resistance mechanisms provide high level resistance or even immunity against the virus. Such a novel approach is anticipated to prevent a rapid virus adaptation that could potentially lead to the emergence of isolates with resistance breaking properties.

Gene expression and biochemical characterization of Azotobacter vinelandii cyclophilins and Protein Interaction Studies of the cytoplasmic isoform with dnaK and lpxH.

The soil nitrogen-fixing bacterium Azotobacter vinelandii possesses two cyclophilins, comprising putative cytoplasmic and periplasmic isoforms, designated as AvPPIB and AvPPIA, respectively. Both recombinant cyclophilins have been purified and their peptidyl-prolyl cis/trans isomerase activity against Suc-Ala-Xaa-Pro-Phe-pNA synthetic peptides has been characterized. The substrate specificity of both cyclophilins is typical for bacterial cyclophilins, with Suc-Ala-Ala-Pro-Phe-pNA being the most rapidly catalyzed substrate. The cytoplasmic cyclophilin also displays a chaperone function in the citrate synthase thermal aggregation assay. Using real-time quantitative RT-PCR, we demonstrate that AvppiB is expressed under various physiological and growth conditions, mainly upregulated by acetate and downregulated by the stationary growth state, while AvppiA shows a tendency for downregulation under the tested conditions. Further, we identified chaperone protein dnaK and UDP-2, 3-diacylglucosamine hydrolase lpxH as probable interacting partners of AvPPIB and we demonstrate their physical interaction by coexpression studies. An increase in AvPPIB PPIase activity in the presence of AvdnaK and a decrease in the presence of AvlpxH further confirms each interaction. However, the PPIase activity does not seem to be essential for those interactions since AvPPIB active site mutants still interact with dnaK and lpxH, while their minor PPIase activity cannot be modulated by the interaction.

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet.

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.

Playing the "Harp": evolution of our understanding of hrp/hrc genes.

With the advent of recombinant DNA techniques, the field of molecular plant pathology witnessed dramatic shifts in the 1970s and 1980s. The new and conventional methodologies of bacterial molecular genetics put bacteria center stage. The discovery in the mid-1980s of the hrp/hrc gene cluster and the subsequent demonstration that it encodes a type III secretion system (T3SS) common to Gram negative bacterial phytopathogens, animal pathogens, and plant symbionts was a landmark in molecular plant pathology. Today, T3SS has earned a central role in our understanding of many fundamental aspects of bacterium-plant interactions and has contributed the important concept of interkingdom transfer of effector proteins determining race-cultivar specificity in plant-bacterium pathosystems. Recent developments in genomics, proteomics, and structural biology enable detailed and comprehensive insights into the functional architecture, evolutionary origin, and distribution of T3SS among bacterial pathogens and support current research efforts to discover novel antivirulence drugs.