A Plant Endophytic Bacterium Priestia megaterium StrainBP-R2 Isolated from the Halophyte Bolboschoenus planiculmis Enhances Plant Growth under Salt and Drought Stresses.

Global warming and climate change have contributed to the rise of weather extremes. Severe drought and soil salinization increase because of rising temperatures. Economically important crop production and plant growth and development are hindered when facing various abiotic stresses. Plant endophytic bacteria live inside host plants without causing visible harm and can be isolated from surface-sterilized plant tissues. Using plant endophytic bacteria to stimulate plant growth and increase environmental stress tolerance has become an alternative approach besides using the traditional breeding and genetically modifying approaches to select or create new crop types resistant to different environmental stresses. The plant endophytic bacterium, Priestia megaterium (previously known as Bacillus megaterium) strain BP-R2, was isolated from the surface-sterilized root tissues of the salt marsh halophyte Bolboschoenus planiculmis. The bacteria strain BP-R2 showed high tolerance to different sodium chloride (NaCl) concentrations and produced the auxin plant hormone, indole acetic acid (IAA), under various tested growth conditions. Inoculation of Arabidopsis and pak choi (Brassica rapa L. R. Chinensis Group) plants with the strain BP-R2 greatly enhanced different growth parameters of the host plants under normal and salt and drought stress conditions compared to that of the mock-inoculated plants. Furthermore, the hydrogen peroxide (H2O2) content, electrolyte leakage (EL), and malondialdehyde (MDA) concentration accumulated less in the BP-R2-inoculated plants than in the mock-inoculated control plants under salt and drought stresses. In summary, the plant endophytic bacterium strain BP-R2 increased host plant growth and stress tolerance to salt and drought conditions.

Downregulation of Methionine Cycle Genes MAT1A and GNMT Enriches Protein-Associated Translation Process and Worsens Hepatocellular Carcinoma Prognosis.

The major biological methyl donor, S-adenosylmethionine (adoMet) synthesis occurs mainly in the liver. Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are two key enzymes involved in the functional implications of that variation. We collected 42 RNA-seq data from paired hepatocellular carcinoma (HCC) and its adjacent normal liver tissue from the Cancer Genome Atlas (TCGA). There was no mutation found in MAT1A or GNMT RNA in the 42 HCC patients. The 11,799 genes were annotated in the RNA-Seq data, and their expression levels were used to investigate the phenotypes of low MAT1A and low GNMT by Gene Set Enrichment Analysis (GSEA). The REACTOME_TRANSLATION gene set was enriched and visualized in a heatmap along with corresponding differences in gene expression between low MAT1A versus high MAT1A and low GNMT versus high GNMT. We identified 43 genes of the REACTOME_TRANSLATION gene set that are powerful prognosis factors in HCC. The significantly predicted genes were referred into eukaryotic translation initiation (EIF3B, EIF3K), eukaryotic translation elongation (EEF1D), and ribosomal proteins (RPs). Cell models expressing various MAT1A and GNMT proved that simultaneous restoring the expression of MAT1A and GNMT decreased cell proliferation, invasion, as well as the REACTOME_TRANSLATION gene EEF1D, consistent with a better prognosis in human HCC. We demonstrated new findings that downregulation or defect in MAT1A and GNMT genes can enrich the protein-associated translation process that may account for poor HCC prognosis. This is the first study demonstrated that MAT1A and GNMT, the 2 key enzymes involved in methionine cycle, could attenuate the function of ribosome translation. We propose a potential novel mechanism by which the diminished GNMT and MAT1A expression may confer poor prognosis for HCC.

A Plant Endophytic Bacterium, Burkholderia seminalis Strain 869T2, Promotes Plant Growth in Arabidopsis, Pak Choi, Chinese Amaranth, Lettuces, and Other Vegetables.

Plant endophytic bacteria live inside host plants, can be isolated from surface-sterilized plant tissues, and are non-pathogenic. These bacteria can assist host plants in obtaining more nutrients and can improve plant growth via multiple mechanisms. Certain Gram-negative Burkholderia species, including rhizobacteria, bioremediators, and biocontrol strains, have been recognized for their plant-growth-promoting abilities, while other isolates have been identified as opportunistic plant or human pathogens. In this study, we observed the auxin production, siderophore synthesis, and phosphate solubilization abilities of B. seminalis strain 869T2. Our results demonstrated that strain 869T2 promoted growth in Arabidopsis, ching chiang pak choi, pak choi, loose-leaf lettuce, romaine lettuce, red leaf lettuce, and Chinese amaranth. Leafy vegetables inoculated with strain 869T2 were larger, heavier, and had more and larger leaves and longer and heavier roots than mock-inoculated plants. Furthermore, inoculations of strain 869T2 into hot pepper caused increased flower and fruit production, and a higher percentage of fruits turned red. Inoculation of strain 869T2 into okra plants resulted in earlier flowering and increased fruit weight. In conclusion, the plant endophytic bacterium Burkholderia seminalis 869T2 exerted positive effects on growth and production in several plant species.

Arabidopsis RAB8A, RAB8B and RAB8D Proteins Interact with Several RTNLB Proteins and are Involved in the Agrobacterium tumefaciens Infection Process.

Arabidopsis thaliana small GTP-binding proteins, AtRAB8s, associate with the endomembrane system and modulate tubulovesicular trafficking between compartments of the biosynthetic and endocytic pathways. There are five members in Arabidopsis, namely AtRAB8A-8E. Yeast two-hybrid assays, bimolecular fluorescence complementation assays and glutathione-S-transferase pull-down assays showed that RAB8A, 8B and 8D interacted with several membrane-associated reticulon-like (AtRTNLB) proteins in yeast, plant cells and in vitro. Furthermore, RAB8A, 8B and 8D proteins showed interactions with the Agrobacterium tumefaciens virulence protein, VirB2, a component of a type IV secretion system (T4SS). A. tumefaciens uses a T4SS to transfer T-DNA and Virulence proteins to plants, which causes crown gall disease in plants. The Arabidopsis rab8A, rab8B and rab8D single mutants showed decreased levels of Agrobacterium-mediated root and seedling transformation, while the RAB8A, 8B and 8D overexpression transgenic Arabidopsis plants were hypersusceptible to A. tumefaciens and Pseudomonas syringae infections. RAB8A-8E transcripts accumulated differently in roots, rosette leaves, cauline leaves, inflorescence and flowers of wild-type plants. In summary, RAB8A, 8B and 8D interacted with several RTNLB proteins and participated in A. tumefaciens and P. syringae infection processes.

Arabidopsis RETICULON-LIKE4 (RTNLB4) Protein Participates in Agrobacterium Infection and VirB2 Peptide-Induced Plant Defense Response.

Agrobacterium tumefaciens uses the type IV secretion system, which consists of VirB1-B11 and VirD4 proteins, to deliver effectors into plant cells. The effectors manipulate plant proteins to assist in T-DNA transfer, integration, and expression in plant cells. The Arabidopsis reticulon-like (RTNLB) proteins are located in the endoplasmic reticulum and are involved in endomembrane trafficking in plant cells. The rtnlb4 mutants were recalcitrant to A. tumefaciens infection, but overexpression of RTNLB4 in transgenic plants resulted in hypersusceptibility to A. tumefaciens transformation, which suggests the involvement of RTNLB4 in A. tumefaciens infection. The expression of defense-related genes, including FRK1, PR1, WRKY22, and WRKY29, were less induced in RTNLB4 overexpression (O/E) transgenic plants after A. tumefaciens elf18 peptide treatment. Pretreatment with elf18 peptide decreased Agrobacterium-mediated transient expression efficiency more in wild-type seedlings than RTNLB4 O/E transgenic plants, which suggests that the induced defense responses in RTNLB4 O/E transgenic plants might be affected after bacterial elicitor treatments. Similarly, A. tumefaciens VirB2 peptide pretreatment reduced transient T-DNA expression in wild-type seedlings to a greater extent than in RTNLB4 O/E transgenic seedlings. Furthermore, the VirB2 peptides induced FRK1, WRKY22, and WRKY29 gene expression in wild-type seedlings but not efr-1 and bak1 mutants. The induced defense-related gene expression was lower in RTNLB4 O/E transgenic plants than wild-type seedlings after VirB2 peptide treatment. These data suggest that RTNLB4 may participate in elf18 and VirB2 peptide-induced defense responses and may therefore affect the A. tumefaciens infection process.

Functional analysis of McSnRK1 (SNF1-related protein kinase 1) in regulating Na/K homeostasis in transgenic cultured cells and roots of halophyte Mesembryanthemum crystallinum.

KEY MESSAGE: Transgenic callus and roots of ice plant with altered SnRK1 function were established using Agrobacterium-mediated transformation. The role of McSnRK1 in controlling Na+ influx and Na/K ratio was demonstrated. SnRK1 kinases (SNF1-related protein kinase1) control metabolic adaptation during energy deprivation and regulate protective mechanisms against environmental stress. Yeast SNF1 activates a P-type ATPase, the Na+ exclusion pump, under glucose starvation. The involvement of plant SnRK1 in salt stress response is largely unknown. We previously identified a salt-induced McSnRK1 in the halophyte ice plant (Mesembryanthemum crystallinum). In the current study, the function of McSnRK1 in salt tolerance was analyzed in transgenic cultured cells and roots of ice plant. Ice plant callus constitutively expressed a high level of McSnRK1 and introducing the full-length McSnRK1 did not alter the Na/K ratio at 24 h after 200 mM NaCl treatment. However, interfering with McSnRK1 activity by introducing a truncate McSnRK1 to produce a dominant-negative form of McSnRK1 increased cellular Na+ accumulation and Na/K ratio. As a result, the growth of cultured cells diminished under salt treatment. Hydroponically grown ice plants with roots expressing full-length McSnRK1 had better growth and lowered Na/K ratio compared to the wild-type or vector-only plants. Roots expressing a truncate McSnRK1 had reduced growth and high Na/K ratio under 400 mM NaCl treatment. The changes in Na/K ratio in transgenic cells and whole plants demonstrated the function of SnRK1 in controlling Na+ flux and maintaining Na/K homeostasis under salinity. The Agrobacterium-mediated transformation system could be a versatile tool for functional analysis of genes involved in salt tolerance in the ice plant.

Correction: Huang, F.-C., et al. Arabidopsis RETICULON-LIKE3 (RTNLB3) and RTNLB8 Participate in Agrobacterium-Mediated Plant Transformation. Int. J. Mol. Sci. 2018, 19, 638.

The authors would like to make a correction to their published paper [...].

Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum).

BACKGROUND: Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols. RESULTS: To establish efficient transformation system of ice plants, three types of ice plant materials, hypocotyl-derived callus, aseptically-grown seedlings and pot-grown juvenile plants, were used to develop Agrobacterium-mediated transformation protocols. The highest transient transformation efficiency was with 5-day-old ice plant callus co-incubated with an Agrobacterium tumefaciens at 2.5 × 109 cells mL-1 for 48 h. The 3-day-old ice plant seedlings with root tip removed were successfully infected with A. tumefaciens or A. rhizogenes, and obtained 85% and 33-100% transient transformation rates, respectively. The transient transformation assays in ice plant callus and seedlings demonstrated that the concentrations of Agrobacteria, the durations of co-incubation time, and the plant growth stages were three important factors affecting the transient transformation efficiencies. Additionally, pot-grown juvenile plants were syringe-injected with two A. rhizogenes strains A8196 and NCPPB 1855, to establish transformed roots. After infections, ice plants were grown hydroponically and showed GUS expressions in transformed roots for 8 consecutive weeks. CONCLUSIONS: Our Agrobacterium-mediated transformation protocols utilized hypocotyl-derived callus and seedlings as plant materials, which can be easily obtained in large quantity. The average successful transient transformation rates were about 2.4-3.0% with callus and 33.3-100.0% with seedlings. We also developed a rapid and efficient protocol to generate transgenic roots by A. rhizogenes infections without laborious and challenging tissue culture techniques. This protocol to establish composite ice plant system demonstrates excellent improvements in efficiency, efficacy, and ease of use over previous ice plant transformation protocols. These Agrobacterium-mediated transformation protocols can be versatile and efficient tools for exploring gene functions at cellular and organ levels of ice plants.

Presence of an Agrobacterium-Type Tumor-Inducing Plasmid in Neorhizobium sp. NCHU2750 and the Link to Phytopathogenicity.

The genus Agrobacterium contains a group of plant-pathogenic bacteria that have been developed into an important tool for genetic transformation of eukaryotes. To further improve this biotechnology application, a better understanding of the natural genetic variation is critical. During the process of isolation and characterization of wild-type strains, we found a novel strain (i.e., NCHU2750) that resembles Agrobacterium phenotypically but exhibits high sequence divergence in several marker genes. For more comprehensive characterization of this strain, we determined its complete genome sequence for comparative analysis and performed pathogenicity assays on plants. The results demonstrated that this strain is closely related to Neorhizobium in chromosomal organization, gene content, and molecular phylogeny. However, unlike the characterized species within Neorhizobium, which all form root nodules with legume hosts and are potentially nitrogen-fixing mutualists, NCHU2750 is a gall-forming pathogen capable of infecting plant hosts across multiple families. Intriguingly, this pathogenicity phenotype could be attributed to the presence of an Agrobacterium-type tumor-inducing plasmid in the genome of NCHU2750. These findings suggest that these different lineages within the family Rhizobiaceae are capable of transitioning between ecological niches by having novel combinations of replicons. In summary, this work expanded the genomic resources available within Rhizobiaceae and provided a strong foundation for future studies of this novel lineage. With an infectivity profile that is different from several representative Agrobacterium strains, this strain may be useful for comparative analysis to better investigate the genetic determinants of host range among these bacteria.

Arabidopsis RETICULON-LIKE3 (RTNLB3) and RTNLB8 Participate in Agrobacterium-Mediated Plant Transformation.

Agrobacterium tumefaciens can genetically transform various eukaryotic cells because of the presence of a resident tumor-inducing (Ti) plasmid. During infection, a defined region of the Ti plasmid, transfer DNA (T-DNA), is transferred from bacteria into plant cells and causes plant cells to abnormally synthesize auxin and cytokinin, which results in crown gall disease. T-DNA and several virulence (Vir) proteins are secreted through a type IV secretion system (T4SS) composed of T-pilus and a transmembrane protein complex. Three members of Arabidopsis reticulon-like B (RTNLB) proteins, RTNLB1, 2, and 4, interact with VirB2, the major component of T-pilus. Here, we have identified that other RTNLB proteins, RTNLB3 and 8, interact with VirB2 in vitro. Root-based A. tumefaciens transformation assays with Arabidopsis rtnlb3, or rtnlb5-10 single mutants showed that the rtnlb8 mutant was resistant to A. tumefaciens infection. In addition, rtnlb3 and rtnlb8 mutants showed reduced transient transformation efficiency in seedlings. RTNLB3- or 8 overexpression transgenic plants showed increased susceptibility to A. tumefaciens and Pseudomonas syringae infection. RTNLB1-4 and 8 transcript levels differed in roots, rosette leaves, cauline leaves, inflorescence, flowers, and siliques of wild-type plants. Taken together, RTNLB3 and 8 may participate in A. tumefaciens infection but may have different roles in plants.

FXYD8, a Novel Regulator of Renal Na+/K+-ATPase in the Euryhaline Teleost, Tetraodon nigroviridis.

FXYD proteins are important regulators of Na+/K+-ATPase (NKA) activity in mammals. As an inhabitant of estuaries, the pufferfish (Tetraodon nigroviridis) responds to ambient salinity changes with efficient osmoregulation, including alterations in branchial, and renal NKA activities. Previous studies on teleostean FXYDs have mainly focused on the expression and potential functions of FXYD proteins in gills. The goal of the present study was to elucidate the potential role of FXYD8, a member of the fish FXYD protein family, in the modulation of NKA activity in the kidneys of this euryhaline pufferfish by using molecular, biochemical, and physiological approaches. The results demonstrate that T. nigroviridis FXYD8 (TnFXYD8) interacts with NKA in renal tubules. Meanwhile, the protein expression of renal TnFXYD8 was found to be significantly upregulated in hyperosmotic seawater-acclimated pufferfish. Moreover, overexpression of TnFXYD8 in Xenopus oocytes decreased NKA activity. Our results suggest the FXYD8 is able to modulate NKA activity through inhibitory effects upon salinity challenge. The present study further extends our understanding of the functions of FXYD proteins, the regulators of NKA, in vertebrates.

Agrobacterium-mediated plant transformation: biology and applications.

Plant genetic transformation heavily relies on the bacterial pathogen Agrobacterium tumefaciens as a powerful tool to deliver genes of interest into a host plant. Inside the plant nucleus, the transferred DNA is capable of integrating into the plant genome for inheritance to the next generation (i.e. stable transformation). Alternatively, the foreign DNA can transiently remain in the nucleus without integrating into the genome but still be transcribed to produce desirable gene products (i.e. transient transformation). From the discovery of A. tumefaciens to its wide application in plant biotechnology, numerous aspects of the interaction between A. tumefaciens and plants have been elucidated. This article aims to provide a comprehensive review of the biology and the applications of Agrobacterium-mediated plant transformation, which may be useful for both microbiologists and plant biologists who desire a better understanding of plant transformation, protein expression in plants, and plant-microbe interaction.

Dichloromethane extracts of propolis protect cell from oxygen-glucose deprivation-induced oxidative stress via reducing apoptosis.

BACKGROUND: Bee propolis, a mixture of the secretion from bee tongue gland and wax gland, was collected from the tree bud and barked by bees. The components were rich in terpenes, phenolics, and flavonoids, and had anti-cancer, anti-bacterial, anti-inflammatory, hepatoprotective, and neuroprotection abilities. However, the potential anti-oxidative stress of propolis was not well documented. This study aimed to study the protective effect of propolis on high-incident nonfatal diseases, such as stroke and cerebral infarction caused by ischemia. OBJECTIVE: Oxidative stress caused by acute stroke results in inflammation and injury followed by cell damage and apoptosis. Clarification of the anti-oxidative stress effect of propolis may contribute to stroke prevention and damage reduction. DESIGN: Propolis was separated and purified into 70% ethanol and dichloromethane extracts systematically. The fraction three (Fr.3) of dichloromethane was further separated into pinocembrin, pinobanksin, pinobanksin-3-acetate, chrysin, and galangin by chromatography. Compounds extracted from propolis were tested for cell-protection effects in an oxygen-glucose deprivation (OGD) N2a cell model. MTT assay, oxidative stress markers measurement, flow cytometry, and QPCR were used to evaluate cell viability and apoptosis. RESULTS: All compounds, especially pinocembrin and galangin, enhanced cell viability in OGD-treated N2a cells. In addition, anti-oxidative enzymes were elevated and cellular Ca(2+) was reduced. They also had extreme anti-apoptosis effects by up-regulating the expression of Bcl-2 mRNA and down-regulating caspase-3 and Bax expression. Taken together, propolis had anti-oxidative effects on stress and protected cells from damage. CONCLUSION: The anti-oxidative effect of propolis can be applied to daily food supplements and may benefit stroke patients.

Overexpression of the HspL Promotes Agrobacterium tumefaciens Virulence in Arabidopsis Under Heat Shock Conditions.

Agrobacterium tumefaciens transfers a specific DNA fragment from the resident tumor-inducing (Ti) plasmid and effector virulence (Vir) proteins to plant cells during infection. A. tumefaciens VirB1-11 and VirD4 proteins assemble as the type IV secretion system (T4SS), which mediates transfer of the T-DNA and effector Vir protein into plant cells, thus resulting in crown gall disease in plants. Previous studies revealed that an α-crystallin-type, small heat-shock protein (HspL) is a more effective VirB8 chaperone than three other small heat-shock proteins (HspC, HspAT1, and HspAT2). Additionally, HspL contributes to efficient T4SS-mediated DNA transfer and tumorigenesis under room-temperature growth. In this study, we aimed to characterize the impact of HspL on Agrobacterium-mediated transformation efficiency under heat-shock treatment. During heat shock, transient transformation efficiency and VirB8 protein accumulation were lower in the hspL deletion mutant than in the wild type. Overexpression of HspL in A. tumefaciens enhanced the transient transformation efficiency in root explants of both susceptible and recalcitrant Arabidopsis ecotypes. In addition, the reduced transient transformation efficiency during heat stress was recovered by overexpression of HspL in A. tumefaciens. HspL may help maintain VirB8 homeostasis and elevate Agrobacterium-mediated transformation efficiency under both heat-shock and nonheat-shock growth.

The Tzs protein and exogenous cytokinin affect virulence gene expression and bacterial growth of Agrobacterium tumefaciens.

The soil phytopathogen Agrobacterium tumefaciens causes crown gall disease in a wide range of plant species. The neoplastic growth at the infection sites is caused by transferring, integrating, and expressing transfer DNA (T-DNA) from A. tumefaciens into plant cells. A trans-zeatin synthesizing (tzs) gene is located in the nopaline-type tumor-inducing plasmid and causes trans-zeatin production in A. tumefaciens. Similar to known virulence (Vir) proteins that are induced by the vir gene inducer acetosyringone (AS) at acidic pH 5.5, Tzs protein is highly induced by AS under this growth condition but also constitutively expressed and moderately upregulated by AS at neutral pH 7.0. We found that the promoter activities and protein levels of several AS-induced vir genes increased in the tzs deletion mutant, a mutant with decreased tumorigenesis and transient transformation efficiencies, in Arabidopsis roots. During AS induction and infection of Arabidopsis roots, the tzs deletion mutant conferred impaired growth, which could be rescued by genetic complementation and supplementing exogenous cytokinin. Exogenous cytokinin also repressed vir promoter activities and Vir protein accumulation in both the wild-type and tzs mutant bacteria with AS induction. Thus, the tzs gene or its product, cytokinin, may be involved in regulating AS-induced vir gene expression and, therefore, affect bacterial growth and virulence during A. tumefaciens infection.

Agrobacterium-produced and exogenous cytokinin-modulated Agrobacterium-mediated plant transformation.

Agrobacterium tumefaciens is a plant pathogenic bacterium that causes neoplastic growths, called 'crown gall', via the transfer and integration of transferred DNA (T-DNA) from the bacterium into the plant genome. We characterized an acetosyringone (AS)-induced tumour-inducing (Ti) plasmid gene, tzs (trans-zeatin synthesizing), that is responsible for the synthesis of the plant hormone cytokinin in nopaline-type A. tumefaciens strains. The loss of Tzs protein expression and trans-zeatin secretions by the tzs frameshift (tzs-fs) mutant is associated with reduced tumorigenesis efficiency on white radish stems and reduced transformation efficiencies on Arabidopsis roots. Complementation of the tzs-fs mutant with a wild-type tzs gene restored wild-type levels of trans-zeatin secretions and transformation efficiencies. Exogenous application of cytokinin during infection increased the transient transformation efficiency of Arabidopsis roots infected by strains lacking Tzs, which suggests that the lower transformation efficiency resulted from the lack of Agrobacterium-produced cytokinin. Interestingly, although the tzs-fs mutant displayed reduced tumorigenesis efficiency on several tested plants, the loss of Tzs enhanced tumorigenesis efficiencies on green pepper and cowpea. These data strongly suggest that Tzs, by synthesizing trans-zeatin at early stage(s) of the infection process, modulates plant transformation efficiency by A. tumefaciens.

Branchial FXYD protein expression in response to salinity change and its interaction with Na+/K+-ATPase of the euryhaline teleost Tetraodon nigroviridis.

Na+/K+-ATPase (NKA) is a ubiquitous membrane-bound protein crucial for teleost osmoregulation. The enzyme is composed of two essential subunits, a catalytic alpha subunit and a glycosylated beta subunit which is responsible for membrane targeting of the enzyme. In mammals, seven FXYD members have been found. FXYD proteins have been identified as the regulatory protein of NKA in mammals and elasmobranchs, it is thus interesting to examine the expression and functions of FXYD protein in the euryhaline teleosts with salinity-dependent changes of gill NKA activity. The present study investigated the expression and distribution of the FXYD protein in gills of seawater (SW)- or freshwater (FW)-acclimated euryhaline pufferfish (Tetraodon nigroviridis). The full-length pufferfish FXYD gene (pFXYD) was confirmed by RT-PCR. pFXYD was found to be expressed in many organs including gills of both SW and FW pufferfish. pFXYD mRNA abundance in gills, determined by real-time PCR, was significantly higher in FW fish than in SW fish. An antiserum raised against a partial amino acid sequence of pFXYD was used for the immunoblots of gill homogenates and a major band at 13 kDa was detected. The relative amounts of pFXYD protein and mRNA in gills of SW and FW pufferfish were identical, but opposite to the expression levels of NKA. Immunofluorescent staining of frozen sections demonstrated that pFXYD was colocalized to NKA-immunoreactive cells in the gill filaments. In addition, interaction between pFXYD and NKA was demonstrated by co-immunoprecipitation. Taken together, salinity-dependent expression of pFXYD protein and NKA, as well as the evidence for their colocalization and interaction in pufferfish gills suggested that pFXYD regulates NKA activity in gills of euryhaline teleosts upon salinity challenge.

Proteomic analysis of Agrobacterium tumefaciens response to the Vir gene inducer acetosyringone.

Agrobacterium tumefaciens causes crown gall disease in a wide range of plants by transforming plants through the transfer and integration of its transferred DNA (T-DNA) into the host genome. In the present study, we used two-dimensional gel electrophoresis to examine the protein expression profiles of A. tumefaciens in response to the phenolic compound acetosyringone (AS), a known plant-released virulence (vir) gene inducer. Using mass spectrometry, we identified 11 proteins consisting of 9 known AS-induced Vir proteins and 2 newly discovered AS-induced proteins, an unknown protein Y4mC (Atu6162) and a small heat shock protein HspL (Atu3887). Further expression analysis revealed that the AS-induced expression of Y4mC and HspL is regulated by the VirA/VirG two-component system. This report presents the first proteomics study successfully identifying both known and new AS-induced proteins that are implicated in Agrobacterium virulence.

Transgenic Arabidopsis plants expressing Agrobacterium tumefaciens VirD2 protein are less susceptible to Agrobacterium transformation.

SUMMARY Agrobacterium tumefaciens causes crown gall disease on many plant species and can result in considerable economic losses. Here we report a new strategy to control crown gall disease by over-expressing Agrobacterium tumefaciens VirD2 protein in plants. Transgenic Arabidopsis plants over-expressing virD2 from constitutive or wound-inducible promoters are less susceptible to Agrobacterium-mediated transformation. Additionally, the transient introduction of an A. tumefaciens virD2 gene in tobacco BY-2 cells reduces subsequent Agrobacterium-mediated transformation.