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High salt concentration in soils cause osmotic and ionic stress for plants, along with oxidative stress and inhibition of growth and development, as a consequence, reducing the yield of many crops important for agriculture. Buckwheat (Fagopyrum esculentum Moench.) is classified as a salt-sensitive glycophyte plant, but salt tolerance of this culture is higher among many important food crops. The salt impact on the total levels of phenolics, anthocyanins and TBA-active products as the oxidative lipid peroxidation parameter, was determined in buckwheat plants (cv. Ukrainka) on 48, 72 h and 7th days of high NaCl exposure. Plants were grown on the Ѕ Hoagland–Arnon’s nutrient solution with/without addition of 100 mM NaCl. After 72 h of salt exposure, the TBA-active products increased in leaves and stems (130 and 204 %, respectively). Maximum of the TBA-active products was noticed at 72nd h, particularly in stems. On the 7th day of salt stress, the TBA-active products level decreased in leaves and roots, while in stems it remained at high level. Total amount of the phenolic compounds in the buckwheat roots treated with NaCl ranged from 35.55 to 64.95 µg/g DW in leaves – from 98.36 to 112.49 µg/g DW at different time points. In stems, the amount of total phenolic compounds was at the level in between 80.71 to 108.32 µg/g DW. After 48 h of NaCl impact, the total phenolic level decreased by 51 and 26 % in the roots and leaves, respectively. The same tendency was observed on the 3rd day of the experiment. Finally, on the 7th day the total phenolic content in roots approximated to the control level, however in leaves it was lower and in stems higher than in control. Content of the phenolic compounds was significantly higher in the above ground parts. An increase of the anthocyanin content during the long-term exposure to the stress was consistent with the statement regarding their secondary response to overcome an oxidative stress. Salt shock adversely affected physiological activity of the buckwheat plants whereas a decrease of the TBA-active products, and a restoring of phenolics quantity in plant organs under prolonged salt stress points to an activated adaptive response.

Manipulating the rhizosphere microbiome to enhance plant stress tolerance is an environmentally friendly technology and a renewable resource to restore degraded environments. Here we suggest a sustainable bioremediation strategy on the example of Stebnyk mine tailings storage. We consider Salicornia europaea rhizosphere community, and the ability of the phytoremediation plant Salix viminalis to recruit its beneficial microbiome to mediate the pollution stress at the Stebnyk mine tailings storage. The tailings contain large amounts of brine salts and heavy metals that contaminate the ground water and surrounding areas, changing soil biogeochemistry and causing increased erosion. The species richness of the endophytic bacterial community of S. viminalis roots was assessed based on observed OTUs, Shannon-InvSimpson, and evenness index. Our results obtained using the plant-based enrichment strategy show that biodiversity was decreased across the contamination zones and that S. europaea supplementation significantly increased the species richness. Our results also indicate that the number of dominating bacteria was not changed across zones in both S. europaea-treated and untreated bacterial populations, and that the decrease in richness was mainly caused by the low abundant bacterial OTUs. The importance of selecting the bioremediation strains that are likely to harbor a reservoir of genetic traits that aid in bioremediation function from the target environment is discussed., (© 2024. The Author(s).)

Global climate change poses challenges to land use worldwide, and we need to reconsider agricultural practices. While it is generally accepted that biodiversity can be used as a biomarker for healthy agroecosystems, we must specify what specifically composes a healthy microbiome. Therefore, understanding how holobionts function in native, harsh, and wild habitats and how rhizobacteria mediate plant and ecosystem biodiversity in the systems enables us to identify key factors for plant fitness. A systems approach to engineering microbial communities by connecting host phenotype adaptive traits would help us understand the increased fitness of holobionts supported by genetic diversity. Identification of genetic loci controlling the interaction of beneficial microbiomes will allow the integration of genomic design into crop breeding programs. Bacteria beneficial to plants have traditionally been conceived as "promoting and regulating plant growth". The future perspective for agroecosystems should be that microbiomes, via multiple cascades, define plant phenotypes and provide genetic variability for agroecosystems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Timmusk, Pall, Raz, Fetsiukh and Nevo.)

In coming decades, drought is expected to expand globally owing to increased evaporation and reduced rainfall. Understanding, predicting, and controlling crop plants' rhizosphere has the potential to manipulate its responses to environmental stress. Our plant growth-promoting rhizobacteria (PGPR) are isolated from a natural laboratory, 'The Evolution Canyon', Israel, (EC), from the wild progenitors of cereals, where they have been co-habituating with their hosts for long periods of time. The study revealed that commercial TM50 silica particles (SN) triggered the PGPR production of exopolysaccharides (EPS) containing D-glucuronate (D-GA). The increased EPS content increased the PGPR water-holding capacity (WHC) and osmotic pressure of the biofilm matrix, which led to enhanced plant biomass in drought-stressed growth environments. Light- and cryo-electron- microscopic studies showed that, in the presence of silica (SN) particles, bacterial morphology is changed, indicating that SNs are associated with significant reprogramming in bacteria. The findings encourage the development of large-scale methods for isolate formulation with natural silicas that ensure higher WHC and hyperosmolarity under field conditions. Osmotic pressure involvement of holobiont cohabitation is also discussed.

Fusarium head blight (FHB) caused by Fusarium pathogens is one of the most devastating fungal diseases of small grain cereals worldwide, substantially reducing yield quality and food safety. Its severity is increasing due to the climate change caused by weather fluctuations. Intensive research on FHB control methods has been initiated more than a decade ago. Since then, the environment has been rapidly changing at regional to global scales due to increasing anthropogenic emissions enhanced fertilizer application and substantial changes in land use. It is known that environmental factors affect both the pathogen virulence as well as plant resistance mechanisms. Changes in CO 2 concentration, temperature, and water availability can have positive, neutral, or negative effects on pathogen spread depending on the environmental optima of the pathosystem. Hence, there is a need for studies of plant-pathogen interactions in current and future environmental context. Long-term monitoring data are needed in order to understand the complex nature of plants and its microbiome interactions. We suggest an holobiotic approach, integrating plant phyllosphere microbiome research on the ecological background. This will enable the development of efficient strategies based on ecological know-how to fight Fusarium pathogens and maintain sustainable agricultural systems.

Fusarium Head Blight (FHB) caused by Fusarium graminearum pathogens constitutes a major threat to agricultural production because it frequently reduces the yield and quality of the crop. The disease severity is predicted to increase in various regions owing to climate change. Integrated management where biocontrol plays an important role has been suggested in order to fight FHB. P. polymyxa A26 is known to be an effective antagonist against F. graminearum. Deeper understanding of the mode of action of P. polymyxa A26 is needed to develop strategies for its application under natural settings in order to effectively overcome the pathogenic effects. This study aims to re-evaluate a former study and reveal whether compounds other than non-ribosomal antibiotic lipopeptides could be responsible for the antagonistic effect, despite what is often reported. Wheat seedlings were grown to maturity and the spikes infected with the pathogen under greenhouse conditions. The development of FHB infection, quantified via the disease incidence severity and 100-kernel weight, was strongly correlated (r > 0.78, p < 0.01) with the content of the polysaccharide component D-glucuronic acid in the biofilm. Furthermore, while increased inoculum density from 10 6 to 10 8 cells/ml did not affect wild type performance, a significant increase was observed with the P. polymyxa mutant deficient in nonribosomal lipopeptide synthesis. Our results show that P. polymyxa A26 biofilm extracellular polysaccharides are capable of antagonizing F. graminearum and that the uronate content of the polysaccharides is of critical importance in the antagonism.

A novel use of nanotitania (TNs) as agents in the nanointerface interaction between plants and colonization of growth promoting rhizobacteria (PGPR) is presented. The effectiveness of PGPRs is related to the effectiveness of the technology used for their formulation. TNs produced by the Captigel patented SolGel approach, characterized by the transmission and scanning electron microscopy were used for formulation of the harsh environment PGPR strains. Changes in the biomass of wheat seedlings and in the density of single and double inoculants with and without TNs were monitored during two weeks of stress induced by drought salt and by the pathogen Fusarium culmorum. We show that double inoculants with TNs can attach stably to plant roots. Regression analysis indicates that there is a positive interaction between seedling biomass and TN-treated second inoculant colonization. We conclude that TN treatment provides an effectual platform for PGPR rational application via design of root microbial community. Our studies illustrate the importance of considering natural soil nanoparticles for PGPR application and thereby may explain the generally observed inconsistent behavior of PGPRs in the field. These new advancements importantly contribute towards solving food security issues in changing climates. The model systems established here provide a basis for new PGPR nanomaterials research.

Global population increases and climate change pose a challenge to worldwide crop production. There is a need to intensify agricultural production in a sustainable manner and to find solutions to combat abiotic stress, pathogens, and pests. Plants are associated with complex microbiomes, which have an ability to promote plant growth and stress tolerance, support plant nutrition, and antagonize plant pathogens. The integration of beneficial plant-microbe and microbiome interactions may represent a promising sustainable solution to improve agricultural production. The widespread commercial use of the plant beneficial microorganisms will require a number of issues addressed. Systems approach using microscale information technology for microbiome metabolic reconstruction has potential to advance the microbial reproducible application under natural conditions.

Fusarium graminearum and F. culmorum are the causing agents of a destructive disease known as Fusarium head blight (FHB). FHB is a re-emerging disease in small grain cereals which impairs both the grain yield and the quality. Most serious consequence is the contamination of grain with Fusarium mycotoxins that are severe threat to humans and animals. Biological control has been suggested as one of the integrated management strategies to control FHB. Paenibacillus polymyxa is considered as a promising biocontrol agent due to its unique antibiotic spectrum. P. polymyxa A26 is an efficient antagonistic agent against Fusarium spp. In order to optimize strain A26 production, formulation and application strategies traits important for its compatibility need to be revealed. Here we developed a toolbox, comprising of dual culture plate assays and wheat kernel assays, including simultaneous monitoring of FHB causing pathogens, A26, and mycotoxin production. Using this system we show that, besides generally known lipopeptide antibiotic production by P. polymyxa, biofilm formation ability may play a crucial role in the case of stain A26 F. culmorum antagonism. Application of the system for effective strain selection and maintenance is discussed.

Paenibacillus polymyxa is a common soil bacterium with broad range of practical applications. An important group of secondary metabolites in P. polymyxa are non-ribosomal peptide and polyketide derived metabolites (NRPs/PKs). Modular non-ribosomal peptide synthetases catalyze main steps in the biosynthesis of the complex secondary metabolites. Here we report on the inactivation of an A26 Sfp-type 4'-phosphopantetheinyl transferase (Sfp-type PPTase). The inactivation of the gene resulted in loss of NRPs/PKs production. In contrast to the former Bacillus spp. model the mutant strain compared to wild type showed greatly enhanced biofilm formation ability. A26Δsfp biofilm promotion is directly mediated by NRPs/PKs, as exogenous addition of the wild type metabolite extracts restores its biofilm formation level. Wheat inoculation with bacteria that had lost their Sfp-type PPTase gene resulted in two times higher plant survival and about three times increased biomass under severe drought stress compared to wild type. Challenges with P. polymyxa genetic manipulation are discussed.

Water is the key resource limiting world agricultural production. Although an impressive number of research reports have been published on plant drought tolerance enhancement via genetic modifications during the last few years, progress has been slower than expected. We suggest a feasible alternative strategy by application of rhizospheric bacteria coevolved with plant roots in harsh environments over millions of years, and harboring adaptive traits improving plant fitness under biotic and abiotic stresses. We show the effect of bacterial priming on wheat drought stress tolerance enhancement, resulting in up to 78% greater plant biomass and five-fold higher survivorship under severe drought. We monitored emissions of seven stress-related volatiles from bacterially-primed drought-stressed wheat seedlings, and demonstrated that three of these volatiles are likely promising candidates for a rapid non-invasive technique to assess crop drought stress and its mitigation in early phases of stress development. We conclude that gauging stress by elicited volatiles provides an effectual platform for rapid screening of potent bacterial strains and that priming with isolates of rhizospheric bacteria from harsh environments is a promising, novel way to improve plant water use efficiency. These new advancements importantly contribute towards solving food security issues in changing climates.

Background: Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds., Methods and Principal Findings: To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega). To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes., Conclusions: We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

Background: All plants in nature harbor a diverse community of rhizosphere bacteria which can affect the plant growth. Our samples are isolated from the rhizosphere of wild barley Hordeum spontaneum at the Evolution Canyon ('EC'), Israel. The bacteria which have been living in close relationship with the plant root under the stressful conditions over millennia are likely to have developed strategies to alleviate plant stress., Methodology/principal Findings: We studied distribution of culturable bacteria in the rhizosphere of H. spontaneum and characterized the bacterial 1-aminocyclopropane-1-carboxylate deaminase (ACCd) production, biofilm production, phosphorus solubilization and halophilic behavior. We have shown that the H. spontaneum rhizosphere at the stressful South Facing Slope (SFS) harbors significantly higher population of ACCd producing biofilm forming phosphorus solubilizing osmotic stress tolerant bacteria., Conclusions/significance: The long-lived natural laboratory 'EC' facilitates the generation of theoretical testable and predictable models of biodiversity and genome evolution on the area of plant microbe interactions. It is likely that the bacteria isolated at the stressful SFS offer new opportunities for the biotechnological applications in our agro-ecological systems.

Interaction studies have suggested that the non-structural protein encoded by open reading frame 3 (ORF3) of porcine circovirus type 2 (PCV2) binds specifically to a regulator of G protein signalling (RGS) related to human RGS16 (huRGS16). The full-length clone of RGS16 was generated from porcine cells and sequence analysis revealed a close relationship to huRGS16 and murine RGS16. In vitro pull-down experiments verified an interaction between porcine RGS16 (poRGS16) and ORF3 from PCV2. Using GST-linked ORF3 proteins from three different genogroups of PCV2 and from porcine circovirus type 1 (PCV1) in the pull-down experiments indicated that there were differences in their ability to bind poRGS16. Quantitative RT-PCR demonstrated that the expression of poRGS16 mRNA could be induced by a number of cell activators including mitogens (LPS and PHA), interferon inducers (ODN 2216 and poly I : C) and the neurotransmitter norepinephrine. Immunofluorescence labelling confirmed the induced expression of poRGS16 at the protein level and suggested that the PCV2 ORF3 protein co-localized with poRGS16 in LPS-activated porcine PBMC. Furthermore, poRGS16 appeared to participate in the translocation of the ORF3 protein into the cell nucleus, suggesting that the observed interaction may play an important role in the infection biology of porcine circovirus.

A real-time PCR assay, based on Primer-Probe Energy Transfer (PriProET), was developed to improve the detection and quantification of porcine circovirus type 2 (PVC2). PCV2 is recognised as the essential infectious agent in post-weaning multisystemic wasting syndrome (PMWS) and has been associated with other disease syndromes such as porcine dermatitis and nephropathy syndrome (PDNS) and porcine respiratory disease complex (PRDC). Since circoviruses commonly occur in the pig populations and there is a correlation between the severity of the disease and the viral load in the organs and blood, it is important not only to detect PCV2 but also to determine the quantitative aspects of viral load. The PriProET real-time PCR assay described in this study was tested on various virus strains and clinical forms of PMWS in order to investigate any correlation between the clinical signs and viral loads in different organs. The data obtained in this study correlate with those described earlier; namely, the viral load in 1 ml plasma and in 500 ng tissue DNA exceeds 10(7) copies in the case of PMWS. The results indicate that the new assay provides a specific, sensitive and robust tool for the improved detection and quantification of PCV2.

The porcine circovirus type 2 (PCV2) genome encodes three major open reading frames (ORFs) encoding the replicase proteins (ORF1), the viral capsid protein (ORF2), and a protein with suggested apoptotic activity (ORF3). Previous phylogenetic analyses of complete genome sequences of PCV2 from GenBank have demonstrated 95-100% intra-group nucleotide sequence identity. However, although these isolates were readily grouped into clusters and clades, there was no correlation between the occurrence of specific PCV2 genotypes and the geographic origin or health status of the pig. In the present study, a unique dataset from a field study spanning the years pre and post the recognition of postweaning multisystemic wasting syndrome (PMWS) in Sweden was utilized. Using this dataset it was possible to discriminate three Swedish genogroups (SG1-3) of PCV2, of which SG1 was recovered from a pig on a healthy farm ten years before the first diagnosis of PMWS in Sweden. The SG1 PCV2/ORF2 gene sequence has been demonstrated to exhibit a high genetic stability over time and has subsequently only been demonstrated in samples from pigs on nondiseased farms. In contrast, SG2 was almost exclusively found on farms that had only recently broken down with PMWS whereas the SG3 genogroup predominated in pigs from PMWS-affected farms. These results further support the results obtained from earlier in vitro and in vivo experimental models and suggest the association of specific PCV2 genogroups with diseased and nondiseased pigs in the field.

Gli3 is a key regulator of development, controlling multiple patterning steps. Here we report the generation of a scFv antibody specific to the repressor domain of human Gli3. We show that this scFv retains the binding capacity of its parent anti-Gli3 monoclonal antibody derived from hybridoma clone 5E1. When expressed in mammalian cells, the anti-Gli3 scFv co-localizes with intracellular Gli3. Immunocytochemical staining of the intrabody in Gli3-positive TM4 cells shows a distinct perinuclear cytoplasmic localization. Such a scFv constitutes a useful tool for studying transcriptional regulation of the hedgehog pathway in mammals and offers a starting point for developing novel Gli-related therapeutic intrabodies.

Porcine circovirus type 2 (PCV2) is now recognized as the essential infectious component of porcine postweaning multisystemic wasting syndrome (PMWS). PMWS was first recognized in high-status, specific pathogen-free pigs in Canada in 1991 and is now an economically important disease that affects the swine industry around the world. Recently, reports of genomic studies on PCV2 viruses indicated that 2 distinctive genogroups of PCV2 exist.4,10 This report involves the results of a study on the distribution of predominant PCV2 genogroups recovered from samples taken from PMWS-affected and PMWS-nonaffected farms on the island of Ireland over a 9-year period and the results of a study on PCV2 genogroup recovery from fecal samples taken from a farm in Northern Ireland from 2003 to 2005 that was first diagnosed as PMWS positive in August 2005. The results indicate that, although at least 2 distinct genogroups of PCV2 have been circulating on pig farms on the island of Ireland, there does not appear to be a direct relationship between infection with these different genogroups of PCV2 and the development of PMWS.

GLI3 is a transcriptional effector of the developmentally important hedgehog (Hh) signaling pathway. Here we report the production of mouse monoclonal antibody (MAb) against putative repressive motif in GLI3 (GLI3pRM). BALB/c mice were immunized with purified recombinant human GLI3pRM protein, and the splenocytes from these mice were fused with myeloma cell line (SP2/0) by using standard hybridoma production techniques. Resulting hybridomas producing anti-GLI3pRM antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and isotyped. The specificity of MAb 5E1 was determined based on its activity in Western blot and immunofluorescence analyses of the human NT2/D1 cell line. The results showed that MAb 5E1 was immunoglobulin IgM/kappa, recognizing recombinant human GLI3pRM specifically. In addition, MAb 5E1 bound to the full-length (FL-GLI3) as well as a short protein (GLI3R) and did not cross-react with a similar region in GLI2. MAb 5E1 could also be used to detect the expression of Gli3 in mouse cell lines and embryonic tissues.

DNA sequences containing CpG motifs are recognized as immunomodulators in several species. Phosphodiester oligodeoxyribonucleotides (ODNs) representing sequences from the genome of porcine circovirus type 2 (PCV2) have been identified as potent inducers (ODN PCV2/5) or inhibitors (ODN PCV2/1) of alpha interferon (IFN-alpha) production by porcine peripheral blood mononuclear cells (poPBMCs) in vitro. In this study, the IFN-alpha-inducing or -inhibitory activities of specific phosphodiester ODNs were demonstrated to be dependent on their ability to form secondary structures. When a poly(G) sequence was added to a stimulatory self-complementary ODN, high levels of IFN-alpha were elicited, and the induction was not dependent on pretreatment with the transfecting agent Lipofectin. In addition, the IFN-alpha-inducing ODN required the presence of an intact CpG dinucleotide, whereas the inhibitory activity of ODN PCV2/1 was not affected by methylation or removal of the central CpG dinucleotide. Of particular significance, the IFN-alpha inhibition elicited by ODN PCV2/1 was only effective against induction stimulated by DNA control inducers and not RNA control inducers, indicating activity directed to TLR9 signaling. The PCV2 genome as a whole was demonstrated to induce IFN-alpha in cultures of poPBMCs, and the presence of immune modulatory sequences within the genome of PCV2 may, therefore, have implications with regard to the immune evasion mechanisms utilized by PCV2.

Porcine circovirus type 2 (PCV2) is an important porcine pathogen that establishes persistent subclinical infections but may, on activation, contribute to the development of post-weaning multisystemic wasting syndrome (PMWS). This disease is characterized by weight loss, respiratory or digestive disorders and enlarged lymph nodes with lymphocyte depletion. The molecular mechanisms behind the development of the disease are completely unknown. In order to clarify functions of the different viral proteins and, if possible, to connect these new findings to molecular mechanisms behind the pathogenesis or the viral life cycle, a bacterial two-hybrid screening of a porcine expression library from PK-15A cells was conducted. Using viral proteins corresponding to ORFs 1, 2, 3 and 4 as bait, a number of interactions were identified and two of them were chosen for further characterization. GST pull-down assays confirmed that viral replicase (Rep) interacted with an intermediate filament protein, similar to human syncoilin, and with the transcriptional regulator c-myc. Furthermore, interactions of the viral proteins to each other revealed an interaction between PCV2 Rep and the capsid (Cap) protein and Cap to itself.

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of this organism as a biocontrol agent has not been very efficient. In previous work we showed that this bacterium protects Arabidopsis thaliana against pathogens and abiotic stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact. 12:951-959, 1999; S. Timmusk, P. van West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28, in Mechanism of action of the plant growth promoting bacterium Paenibacillus polymyxa, 2003). Here, we studied colonization of plant roots by a natural isolate of P. polymyxa which had been tagged with a plasmid-borne gfp gene. Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly the root tip, where they formed biofilms. Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder. Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues. Studies were performed in both a gnotobiotic system and a soil system. The fact that similar observations were made in both systems suggests that colonization by this bacterium can be studied in a more defined system. Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant growth-promoting bacteria are discussed.

A cell mediated immune (CMI) response was measured in vitro to heat-killed and to paraformaldehyde fixed Renibacterium salmoninarum (Rs) in rainbow trout (Oncorhynchus mykiss) experimentally challenged with live Rs. The mitogenic response to the T lymphocyte mitogen Concanavalin A (Con A) was reduced during samplings 4 to 6 weeks after immersion, but no effect of the response to the B lymphocyte mitogen lipopolysaccharide (LPS) was detected. The subpopulation of lymphocytes, detected by the monoclonal antibody 1C2, was decreased from the 4th week to the 5th week of infection, and remained at the decreased level up to 10 weeks post immersion. The proportion of Immunoglobulin (Ig) bearing lymphocytes was not affected during the Rs infection period. The humoral antibody level to heat-stable Rs-antigens was increased up to 10 weeks after immersion but after 27 weeks was reduced to a level similar to that of the non-challenged fish. An anamnestic response was demonstrated in challenged fish, as intraperitoneal injection of heat-treated Rs bacteria into Rs challenged fish elicited a stronger humoral antibody response compared with injection into non-challenged fish.

Production of monoclonal antibodies (mAb) using genetic immunisation is a potential alternative when purified antigen is difficult to obtain, or when induction of an antibody response to a limited part of an antigen is wanted. DNA immunisation using only the constant parts of trout immunoglobulin light chains coding regions was attempted here, because mAbs against the variable (V) part of immunoglobulins do not recognise the whole repertoire of the isotype. After positive results with the light chains and establishing of a proper screening system (ELISA), generation of monoclonal antibodies against trout T cell receptor was also performed. The DNA constructs were used both for immunisation of mice and for protein expression in EBNA 293 cells. Mice were immunised with the constructs 3-5 times by intramuscular injection, with or without adjuvants during 1-3 months. Spleens of positive mice were fused with myeloma Sp2/0 cells and clones were screened by ELISA using double-screening (recombinant protein/trout cells).MAbs 46E5 (anti-IgL2C), 4F2 (anti-TCRalpha), 18B3 (anti-TCRalphaC) and 4E5 (anti-TCRalphaC) show specific binding to its antigen in Western blot, mAb 18B3 and 7H7(anti-TCRalpha) shows specific staining of trout splenocytes in flow cytometry and mAb 7H7 induces proliferation of trout peripheral blood leucocytes (PBL) in vitro.

Three rainbow trout (Oncorhynchus mykiss) light chain isotypes (L1, L2 and L3) have been identified at the cDNA level. Genomic clones have previously been obtained for L1 and L2, revealing different structures of the V/J/C clusters and divergent putative promoter regions. While the L1 putative promoter has a classical Ig promoter structure with TATA, E-box, and octamer, the L2 putative promoter lacks typical features of the Ig promoter. The L2 putative promoter region contains a kappa-Y motif, an E-box and two putative non-consensus TATA boxes. In this study the isolation of a genomic clone that encompassed a VJC cluster of the recently described L3 isotype is described. The structure of the L3 putative promoter region is very similar to that of the L1 promoter. The transcriptional activities of the promoters of the trout L1 and L2 isotypes were compared. The promoter region of the L1 isotype showed a strong and predictable B cell-specific activity. Despite the unusual structure of the L2 V gene promoter, the transcriptional activity of it was much stronger in B-cells as compared to non B-cells. Deletion analyses of L2 promoter constructs showed that the region containing the kappa-Y element is critical for the transcriptional activity of the L2 promoter. Both L1 and L2 promoters can cooperate with a B cell-specific enhancer from Atlantic cod (Gadus morhua).

cDNA studies have distinguished two isotypes of the rainbow trout (Oncorhynchus mykiss) immunoglobulin (Ig) light chain (designated L1 and L2). This study characterized genomic clones of these isotypes. L1 genes are arranged in clusters with single copies of variable (V), joining (J), and constant (C) segments. The transcriptional orientation of the V genes is opposite to that of the J and C segments, indicating that the V genes must be rearranged by inversion. L2 is also organized in clusters, consisting of two or three V, one J, and one C exon, all in the same transcriptional orientation. L1 and L2 of rainbow trout are similar to the previously identified cod and catfish clusters. Repeat sequences were found upstream of each J segment in the L2 genes, each of which includes a 16-bp sequence similar to the conserved kappa sequence motifs of mammalian Jkappa1 genes. Sequence analyses showed that the regions upstream of L1 and L2 genes have several putative cis-acting elements also present in the promoter regions of Ig genes of other organisms. Octamer motifs, a TATA box, and an E-box were found in the 5' region of an IgL1V gene. A kappa-Y element, a CCCT element, a TATA box, an E-box but no classical octamer were found in the 5' region of the IgL2 gene. Northern blot analyses showed that L1 and L2 are expressed in spleen, head kidney, excretory kidney, thymus, and heart. The expressed ratio of L1 and L2 is estimated to 85:15% in blood and lymphoid tissues.

This paper addresses changes in plant gene expression induced by inoculation with plant-growth-promoting rhizobacteria (PGPR). A gnotobiotic system was established with Arabidopsis thaliana as model plant, and isolates of Paenibacillus polymyxa as PGPR. Subsequent challenge by either the pathogen Erwinia carotovora (biotic stress) or induction of drought (abiotic stress) indicated that inoculated plants were more resistant than control plants. With RNA differential display on parallel RNA preparations from P. polymyxa-treated or untreated plants, changes in gene expression were investigated. From a small number of candidate sequences obtained by this approach, one mRNA segment showed a strong inoculation-dependent increase in abundance. The corresponding gene was identified as ERD15, previously identified to be drought stress responsive. Quantification of mRNA levels of several stress-responsive genes indicated that P. polymyxa induced mild biotic stress. This suggests that genes and/or gene classes associated with plant defenses against abiotic and biotic stress may be co-regulated. Implications of the effects of PGPR on the induction of plant defense pathways are discussed.

A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription - polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Ckappa and Clambda isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus.